Theranostics 2012; 2(9):916-966. doi:10.7150/thno.4571 This issue Cite

Review

Unique Diagnostic and Therapeutic Roles of Porphyrins and Phthalocyanines in Photodynamic Therapy, Imaging and Theranostics

Leanne B. Josefsen, Ross W. Boyle

Department of Chemistry, The University Of Hull, Kingston-Upon-Hull, HU6 7RX, U.K.

✉ Corresponding author: Dr. Ross W. Boyle. Tel: +44 (0)1482 466353; E-mail: R.W.Boyleac.uk

Abstract

Porphyrinic molecules have a unique theranostic role in disease therapy; they have been used to image, detect and treat different forms of diseased tissue including age-related macular degeneration and a number of different cancer types. Current focus is on the clinical imaging of tumour tissue; targeted delivery of photosensitisers and the potential of photosensitisers in multimodal biomedical theranostic nanoplatforms. The roles of porphyrinic molecules in imaging and pdt, along with research into improving their selective uptake in diseased tissue and their utility in theranostic applications are highlighted in this Review.

Keywords: tetrapyrrolic photosensitisers, phototherapy, imaging, nanoagents, theranostics

Introduction

Porphyrins

Porphyrins are a group of naturally occurring intensely coloured compounds, whose name is drawn from the Greek word porphura (purple) [1, 2]. These molecules are involved in a number of biologically important roles, including oxygen transport and photosynthesis, and have applications in a number of fields, ranging from fluorescence imaging to medicine [3-15]. Porphyrins are classified as tetrapyrrolic molecules with the heart of the skeleton a heterocyclic macrocycle, known as porphine. The fundamental porphine framework consists of four pyrrolic sub-units linked on opposing sides (α-positions, numbered 1, 4, 6, 9, 11, 14, 16 and 19, figure 1) through four methine (CH) bridges (5, 10, 15 and 20) known as the meso- carbon atoms/positions (figure 1). The resulting conjugated planar macrocycle may be substituted at the meso- and/or β-positions (2, 3, 7, 8, 12, 13, 17 and 18); if a single hydrogen atom is attached to each of the meso- and β-carbons and two of the inner nitrogen atoms (pyrrolenines) are protonated, the compound becomes known as a free-base porphine. If the meso- and/or β-hydrogens are substituted with non-hydrogen atoms or groups, the resulting compounds are known as porphyrins.

Phthalocyanines

Phthalocyanines (PCs, figure 2) are closely related to porphyrins; they are intensely coloured symmetric, aromatic macrocycles. They form coordination complexes with a wide range of metals (via the central cavity), yielding intensely coloured blue-green compounds, and have widely been used as colourants. More recently PCs have been used as photoconducting materials in laser printers and the light absorbing layer in recordable compact discs. They are also used as photosensitisers in phototherapy; as fluorescent reporters in vitro and in vivo; as non-linear optical materials; and as industrial catalysts [16-22].

PCs have an extended conjugate pathway relative to porphyrins - a benzene ring is fused to the β-positions of each of the four pyrrolic sub-units. These benzene rings act to strengthen the absorption of the chromophore at longer wavelengths (670-780nm), with respect to porphyrins. The absorption band of PCs is almost two orders of magnitude stronger than the highest Q band of haematoporphyrin (figure 3). These characteristics, along with the ability to selectively functionalise their peripheral structure, make PCs favourable photosensitiser candidates. PCs demonstrate stronger absorption of red light (than Photofrin^®), allowing more effective light penetration of tumours and have shown promise as second generation photosensitisers [7, 9, 23].

Phototherapy: History

Porphyrins and PCs have been widely investigated for use in photodynamic therapy (pdt). Pdt is used clinically in the treatment of a number of medical conditions, including age-related macular degeneration (AMD), some cancers, skin conditions and for antiviral, antimicrobial and antibacterial applications including sterilisation of blood plasma and water [3-5, 7, 14, 24-30]. The first recorded use of “phototherapy” dates back over 4000 years to the ancient Egyptians while contemporary pdt was first reported in the late 19th century by Finsen et al. [3, 4, 13-15, 24, 28, 31, 32]. Finsen was later awarded the Nobel Prize (1903) for his work in pdt. However, it was not until 1995 that a suitable photosensitiser was approved (Photofrin^®, by the Food and Drug Administration (FDA), USA) for clinical use against certain cancers [4, 5, 14, 24, 25, 27, 28, 32-34]. Pdt is minimally invasive and shows negligible toxicity thus offering advantages for both the patient and physician over traditional cancer treatments such as delicate surgery, or painful and tiring radio- and chemo-therapy. Lengthy recuperation periods are also minimised, along with minimal formation of scar tissue and disfigurement. However, pdt is not without its drawbacks - it is associated with generalised photosensitisation of cutaneous tissue, this is a major limitation in the potential efficacy of pdt [3, 24, 27, 28]. The principle behind pdt is based on a multi-stage process (figure 4) whereby (i) a photosensitiser is administered to the patient (systemically or topically) in the absence of light. When the optimum ratio of photosensitiser in diseased verses healthy tissue is achieved (ii) the photosensitiser is activated by (iii) exposure to a carefully regulated dose of light, which is shone directly onto the diseased tissue for a specified length of time. The activated photosensitiser then reacts with molecular oxygen generating reactive oxygen species (ROS) in situ, evoking a toxic response in the tissue, culminating ultimately in (iv) cell death [4, 5, 35]. The success of pdt lies in the prolonged accumulation of the photosensitiser in diseased tissue, relative to more rapid clearance from healthy tissue. The selectivity of pdt is based on the ability of the photosensitiser to preferentially accumulate in the diseased tissue and efficiently generate singlet oxygen (the cytotoxic species), inducing cell death.

Photosensitisers

A photosensitiser is a molecule which when excited by light energy can utilise the irradiating energy to induce photochemical reactions producing lethal cytotoxic agents; these ultimately result in cell death and tissue destruction (figure 5). Photosensitisers are absorbed into cells all over the body and alone are harmless, i.e. in the absence of light and oxygen they have no effect on healthy or abnormal tissue: it is only their activated forms that have a cytotoxic effect [26, 27]. Ideally, photosensitisers should be retained by diseased tissue for longer periods of time in comparison to healthy tissue; thus, carefully timed light exposure is vital to ensure photosensitiser activation only occurs once the ratio of photosensitiser in diseased tissue is greater than that present in healthy tissue; thereby minimising unwanted healthy cell damage.

The nature of pdt requires efficient localisation of a photosensitiser in target tissue in order to achieve a satisfactory response [4, 14, 25, 28, 33, 36-43]. Pdt can offer an enhanced therapeutic effect via preferential uptake of the photosensitiser by: (i) the morphology of the target tissue; (ii) specific illumination of target tissue; (iii) strategic timing of the applied light dose; (iv) topical application of the photosensitiser; and (v) chemical manipulation of the structure of the photosensitiser.

Photosensitiser localisation in tissues and cells plays a significant role in the mechanisms and efficacy of cell death crucial for effective pdt. Pdt works by inducing the formation of cytotoxic agents which readily attack neoplastic cells, a response known to be affected in vivo by the complexity of biological systems [4, 14, 25]. Any number of subcellular targets can be attacked during pdt, including mitochondria, lysosomes, plasma membranes and nuclei - the exact subcellular localisation of the photosensitiser can govern whether cell death occurs via necrosis or the preferred mode of death, apoptosis [4, 14, 25, 28, 36-43]. ROS have a short half-life and act close to their site of generation; it is therefore hypothesised that the type of photodamage incurred in irradiated photosensitiser-loaded cells depends upon the exact subcellular localisation of the photosensitiser [5, 14, 25, 28, 33, 38-42]. Different substituted photosensitisers have significantly different biodistribution patterns, thus clinical activity is to a great degree dependent on the physicochemical characteristics of the molecules used [5, 14, 25, 28, 29, 33, 38-48].

Ideal photosensitisers - although a number of different photosensitising compounds, such as methylene blue (7-(dimethylamino)-N,N-dimethyl-3H-phenothiazin-3-iminium chloride, MB), rose bengal (4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein disodium salt) and acridine (2,3-benzoquinoline) (figure 6) are known to be efficient singlet oxygen generators (and therefore potential pdt agents) the vast majority of successful pdt photosensitisers are based upon the tetrapyrrole chromophore (figure 1); in particular porphyrin, chlorin, and bacteriochlorin (BC) derivatives (figure 6). These cyclic tetrapyrroles have an inherent similarity to the naturally occurring porphyrins present in living matter - they have no or minimal dark toxicity and, over the years, tetrapyrrolic chemistry has been well researched and developed [3-5, 24, 26, 27, 38-40, 43-52].

Photochemistry

Photochemical Processes

Only when a photosensitiser is in its excited state (³Psen*) can it interact with molecular oxygen (³O₂) to produce ROS. ROS include singlet oxygen (¹O₂), hydroxyl radicals (^.OH), and superoxide (O^2-) anions and are widely accepted as the active cytotoxic agent in pdt. These toxic species interact with cellular components including unsaturated lipids; amino acid residues and nucleic acids (figure 7); with ensuing oxidative damage resulting in apoptotic or necrotic cell death. Oxidative damage is limited (due to the short lifetime of ROS) to the immediate area (approximately 20nm) surrounding the excited photosensitiser. Singlet oxygen (the predominant ROS in pdt) can only interact with molecules and structures within this radius and is known to initiate a large number of reactions with biomolecules, including amino acid residues in proteins, such as tryptophan; unsaturated lipids like cholesterol and nucleic acid bases, particularly guanosine and guanine derivatives - the latter base is more susceptible to attack by singlet oxygen [3-5, 24, 27, 36, 40, 48-50, 53-58].

Photochemical Mechanisms

When a chromophore, such as a porphyrin, absorbs a photon of electromagnetic radiation (EMR) in the form of light energy, an electron is promoted into a higher-energy molecular orbital; hence, the chromophore is elevated from the ground state (S₀) into a short-lived, electronically excited state (S_n) composed of a number of vibrational sub-levels (S_n´) (figure 8). The excited chromophore can lose energy by rapidly decaying through these sub-levels via internal conversion (IC) to populate the first excited singlet state (S₁), before quickly relaxing back to the ground state: the excited electron depopulates the excited singlet state (S₁) and return back to the ground state (S₀) by losing the absorbed energy via fluorescence (S₁ → S₀). Singlet state lifetimes of excited fluorophores are very short (τ_fl = 10^-9 - 10^-6 seconds) since transitions between the same spin states (S → S or T → T) conserve the spin multiplicity (spin) of the electron and are considered “allowed” transitions according to the Spin Selection Rules [27, 50]. Alternatively, an excited singlet state electron (S₁) can undergo spin inversion and populate the lower-energy first excited triplet state (T₁) via intersystem crossing (ISC), a spin-forbidden process, since the spin of the electron is no longer conserved (S → Τ). The excited electron can then undergo a second spin-forbidden inversion and depopulate the excited triplet state (T₁) by decaying back to the ground state (S₀) via phosphorescence (T₁ → S₀). Owing to the spin-forbidden triplet to singlet transition, the lifetime of phosphorescence (τ_P = 10^-3 - 1 second) is considerably longer than that of fluorescence.

Photosensitisers And Photochemistry

Excited state porphyrins (¹Psen^*, S_>0 or ³Psen^*, T_>0) are relatively efficient at undergoing ISC and can have a high triplet-state (quantum) yields (Φ_T 0.62 (tetraphenylporphyrin (TPP), methanol), 0.75 (TPP, liposome, D₂O) and 0.71 (tetrasulphonated TPP, D₂O) [49, 50]. The longer lifetime is sufficient to allow the excited triplet state photosensitiser to interact with the surrounding biomolecules [4, 5]. Excited triplet-state photosensitisers can react in two ways defined as Type I and Type II processes. Type I processes involve the excited triplet photosensitiser (³Psen^*, T₁) interacting with readily oxidizable or reducible substrates; whereas, Type II processes involve the interaction of the excited triplet photosensitiser (³Psen^*, T₁) with molecular oxygen (³O₂, ³Σ_g) (figure 8) [4, 5, 9, 25-29, 49-59]. The highly-reactive oxygen species (¹O₂) produced via the Type II process act near to their site of generation with a typical lifetime of approximately 40ns in biological systems [3, 5, 14, 53]. These interactions cause damage and potential destruction to cellular membranes and enzyme deactivation, culminating in cell death [35-37, 50, 53]. It is highly probable that in the presence of molecular oxygen, both Type I and II pathways play a pivotal role in disrupting both cellular mechanisms and cellular structure as a direct result of the photoirradiation of the photosensitiser molecule. Nevertheless, there is considerable evidence to suggest that the Type II photo-oxygenation process predominates in the role of cell damage, a consequence of the interaction between the irradiated photosensitiser and molecular oxygen [3, 4, 24, 40, 50, 59, 60]. It has however, been suggested that cells in vitro are partially protected against the effects of pdt by the presence of singlet oxygen scavengers, such as histidine, and that certain skin cells are somewhat resistant to pdt in the absence of molecular oxygen; further supporting the proposal that the Type II process is at the heart of photo-initiated cell death [5, 46, 59-62]. The efficiency of Type II processes is dependent upon the triplet state lifetime (τ_T) and the triplet quantum yield (Φ_T) of the photosensitiser, both parameters have been implicated in the effectiveness of a photosensitiser in phototherapeutic medicine; further supporting the distinction between Type I and Type II mechanisms. However, it is worth noting that the success of a photosensitiser is not exclusively dependent upon a Type II process taking place - there are a number of photosensitisers whose excited triplet lifetimes are too short to permit a Type II process to occur, for example, the copper metallated octaethylbenzochlorin photosensitiser (figure 9) has a triplet state lifetime of less than 20ns and is still deemed to be an efficient photodynamic agent [46].

First-Generation Photosensitisers

The first reported use of pdt in the treatment of solid tumours came in the early 20^th Century by von Tappeiner's group in Munich, Germany - patients with skin carcinomas where successfully treated with the fluorescein-based dye eosin (figure 10) [3, 25, 29, 32, 63]. Shortly after, fellow German physician Meyer-Betz reported the major stumbling block of pdt - acute cutaneous photosensitisation. After injecting himself with the photosensitiser haematoporphyrin (Hp) Meyer-Betz swiftly experienced a general skin sensitivity upon exposure to sunlight; a problem that still exists [3, 14, 24, 25, 27]. Further research into Hp and the purified derivative (HpD) in tumours resulted in the first clinically-approved photosensitiser, Photofrin^® (figure 11). Photofrin^® was approved by the Canadian Health Agency in 1993 for use against bladder cancer and later in Japan, America and parts of Europe for use against certain cancers of the oesophagus and non-small cell lung cancer [4, 5, 13, 14, 24, 25, 27, 28, 32-34]. However, Photofrin^® has well-documented problems - in addition to patients exhibiting prolonged photosensitivity, the drug itself has a weak long-wavelength absorption (630nm) [14, 25, 28, 33]. Research into improved photosensitisers saw the development of second-generation photosensitisers, including Verteporfin^® (Visudyne^®) and third-generation photosensitisers focused on targeting strategies, such as antibody-directed photosensitisers and photosensitiser-loaded nanocarriers [4, 14, 24, 26, 32, 64, 65]. Combined diagnostic and therapeutic modalities gave begun to emerge creating theranostic tools for use in identifying (imaging) and treating diseased states: are these the next generation of pdt agents [28, 66-69]? The ability to “switch on” a cytotoxic effect, and combine photosensitisers with imaging modalities such as magnetic resonance imaging (MRI) and radioimaging, makes pdt particularly attractive as the therapeutic partner in theranostic agents. The use of single agents/entities has the added benefit of ruling out variability in localisation; uptake; pharmacokinetic; and pharmacodynamic patterns of the agent (present when separate entities are used) at any stage during the theranostic treatment; stress on the body's clearance system is also minimised when a single agent rather than multiple agents are administered. A further advantage of using small molecule multimodal agents is their clearance by the renal system -avoiding toxicity associated with long-term liver retention [70].

Second-Generation Photosensitisers

Second-generation photosensitisers were developed in order to increase the power and efficiency of pdt and broaden the type of diseased tissue that could be treated; research focused on developing agents with higher absorption wavelengths. Increasing the absorption wavelength and having a higher extinction coefficient in the red/near-infrared region of the electromagnetic spectrum (EMS, 600-850nm) allows deeper penetration of illuminating light into the skin, thus increasing the depths at which tumour cells can be targeted. The range of photosensitisers approved for clinical use includes porphyrins, chlorins, texaphyrins, porphycenes, purpurins and phthalocyanines (figures 11 and 12) [2-5, 7, 9, 12, 13, 15, 23, 24, 26, 28-30, 32-34, 43-45, 48-50, 61, 64, 65].

The prodrug 5-aminolaevulinic acid (δ-aminolaevulinic acid, ALA, marketed as Levulan^®) generates the photosensitiser protoporphyrin IX (PPIX), via the haem biosynthetic pathway, when internalised by cells and is used in the imaging and treatment of superficial tumours. It has also been used as a topical treatment for dermatological conditions (psoriasis, Bowen's disease and acne). ALA derivatives are used in the clinic for imaging and treatment of basal cell carcinomas and other skin lesions (Metvix^®, a methyl ester derivative), Benvix^® (a benzyl ester derivative) and Hexvix^® (a hexyl derivative) have also been employed in the treatment of gastrointestinal cancers and in the diagnosis of bladder cancer [4, 7, 14, 24, 25, 29, 50, 71]. The ability of small molecule ALA derivatives to penetrate the skin selectively where tumours are present and both fluoresce and photosensitise the tumours via in situ production of PPIX makes them ideal theranostic agents. A benzoporphyrin derivative, monoacid ring A (BPD-MA, trade name Visudyne^®, Verteporfin^® for injection) was developed and approved for use in wet age-related macular degeneration (wAMD) and cutaneous non-melanoma skin cancer. The success of Verteporfin^® could be attributed to the red-shifted and intensified long-wavelength absorption maxima (approximately 690nm) - light can penetrate 50% deeper into the skin at this wavelength in comparison to Photofrin^® [4, 7, 14, 25, 27, 29, 72-74]. Tin etiopurpurin (Purlytin^®, figure 12), a chlorin-based photosensitiser, is a derivative of the porphyrin chromophore with a reduced exocyclic double bond and an intensified long-wavelength absorption - the tin atom chelated in the central cavity further increases the red shift (20-30nm) of the drug (650-680nm). Purlytin^® has been approved (USA) for cutaneous metastatic breast cancer, Kaposi's sarcoma in patients with AIDS and for the treatment of psoriasis and restenosis [4, 7, 14, 25, 27, 29, 50, 75, 76]. Tetra(m-hydroxyphenyl)chlorin (mTHPC, Foscan^®/Temoporfin^®, USA and Europe) has been evaluated for use in the pdt of head and neck cancers as well as gastric and pancreatic cancers, hyperplasia, field sterilisation after cancer surgery and for the control of antibiotic-resistant bacteria (USA, Europe and the Far East). Advantages of Foscan^® are the low drug and light doses required to achieve suitable photodynamic responses - Foscan^® is approximately 100 times more photoactive than Photofrin^®, although patients can remain photosensitive for up to 20 days (the length of photosensitivity varies between the second-generation photosensitisers from 1-2 days (Verteporfin) to up to 2 weeks (Purlytin) [4, 7, 14, 24, 25, 27, 29, 50, 71, 77, 78]. First-generation photosensitisers can render patients photosensitive for 90 days. Lutetium texaphyrin (Lutex^®/Lutrin^®), an expanded porphyrin with a penta-aza core, exhibits strong absorption in the 730-770nm region of the EMS, a region where tissue transparency is optimal. Lutex^® has been approved for the treatment of breast cancer and malignant melanomas (USA). Antrin^®, a Lutex^® derivative, has undergone trials for the prevention of restenosis, while Optrin^®, a second derivative, has been in trials for the management of AMD [4, 7, 14, 24, 25, 29, 50, 76, 79-82]. Texaphyrins have also been developed for use as radiosensitisers and chemosensitisers - a gadolinium complex has been investigated as a MRI contrast agent. 9-Acetoxy-2,7,12,17-tetrakis-(β-methoxyethyl)-porphycene (ATMPn^®, porphycenes are structural isomers of porphyrins), absorption maxima ~640nm has been evaluated against psoriasis vulgaris and superficial non-melanoma skin cancer (USA) [4, 7, 83-86]. Zinc phthalocyanine (CGP55847) has undergone clinical trials against squamous cell carcinomas (SCC) of the upper aerodigestive tract (Canada) [4, 7, 24, 76, 87-93]. A sulphonated aluminium PC derivative (Photosens^®, Russia) entered clinical trials against skin, breast and lung malignancies and cancer of the gastrointestinal tract [4, 7, 24, 94-96]. Sulphonation of the PC significantly increases PC solubility in polar solvents including water, circumventing the need for alternative delivery vehicles. A silicon PC complex (PC4) has been evaluated for the sterilisation of blood components (USA), and against breast, colon and ovarian cancers and gliomas [4, 7, 97-103]. A drawback of metallo-PCs can be their tendency to aggregate in aqueous buffer (pH 7.4), leading to a decrease or total loss of their photochemical activity; detergents can limit this behaviour [7, 76]. Tetraazaporphyrins, porphyrazine (PZ, figure 13) derivatives, have been evaluated against Chinese hamster lung fibroblast cells; the PZs that exhibited the greatest induction of dark toxicity include the metallated cationic complexes PdPZ⁺, CuPZ⁺, CdPZ⁺, MgPZ⁺, AlPZ⁺ and GaPZ⁺ [7, 76].

Naphthalocyanines (NCs, figure 14) are extended PC derivatives and absorb at even longer wavelengths (740-780nm) than PCs, further increasing the depth photosensitisers could effectively be used at. The absorption region of NCs makes them particularly promising agents for pdt of highly pigmented tumours, such as melanomas, which can present significant problems with the transmission of visible light. The emission from NCs in the near-IR also gives them great potential as in vivo imaging agents. However, NCs are generally less stable than their PC counterparts - they readily decompose in the presence of light and oxygen (two of the vital components for pdt), and metallo-NCs, lacking axial ligands, have a tendency to form H-aggregates in solution; the aggregates are photoinactive. Kenney et al., van Lier et al. and Wöhrle et al. have mainly dominated the research into NC based photosensitisers [4, 7, 75, 76, 91, 104-115].

Changing the peripheral functionality of the photosensitiser macrocycle can have an effect on the potential pdt efficacy of the molecule. Diamino platinum porphyrins exhibit higher anti-tumour activity, demonstrating the combined effects of the cytotoxicity of the platinum complex and the pdt activity of the porphyrin species [7, 76, 116]. Cationic PC derivatives have also shown potential - positively charged species are believed to localise in the mitochondrion - organelles key to cell survival and the site of oxidative phosphorylation [76, 78, 91, 92]. Zinc and copper cationic species have been investigated although the cationic ZnPC was found to be less photodynamically active than its neutral counterpart in vitro against V-79 cells. Water-soluble cationic porphyrins bearing nitrophenyl, aminophenyl, hydroxyphenyl, and/or pyridiniumyl functional groups exhibit varying cytotoxicity towards cancer cells in vitro, depending on the nature of the metal ion (Mn, Fe, Zn, Ni), and on the number and type of functional groups present (figure 15). The manganese pyridiniumyl derivative has shown the greatest photodynamic activity, while the nickel analogue is photoinactive [76, 92, 118]. Another metallo-porphyrin complex, the iron chelate, was found to demonstrate greater photoactivity (towards HIV and simian immunodeficiency virus in MT-4 cells) than the manganese complexes; the zinc derivative was found to be photoinactive (figure 15) [76, 117].

The hydrophilic sulphonated porphyrins and PCs (AlPorphyrin and AlPC) have been evaluated for photodynamic activity. The disulphonated analogues (with adjacent substituted sulphonated groups, figure 16) exhibited greater photodynamic activity than their di- (symmetrical), mono-, tri- and tetra-sulphonated counterparts [7, 50, 93].

Third-Generation Photosensitisers

The poor solubility of many first- and second-generation photosensitisers (in aqueous media, particularly at physiological pH) prevents their intravenous delivery directly into the bloodstream. It is advantageous if delivery models can be developed which facilitate the transportation of these potentially useful photosensitisers to target tissue/sites. Research has mainly focused on developing systems to effect greater selectivity and specificity on the photosensitiser in order to enhance cellular uptake.

Targeting Strategies

Reported hypotheses suggest the accumulation of photosensitisers in the mitochondria efficiently triggers apoptosis (preferred mode of cell death), therefore, the efficacy of a photosensitiser potentially could be improved by mitochondrial targeting. Alternatively, the cell nucleus is the cellular organelle that is most sensitive to phototherapy damage, hence nuclear damage can lead very quickly to cell death; this implicates the nucleus as a desirable photosensitiser target in pdt [14, 24, 114, 115, 119]. The intracellular localisation of a photosensitiser is not a static process and may change during irradiation, for example, photoinduced lysosomal damage can cause lysosomes to rupture releasing any lysosomally-localised photosensitiser into the cytoplasm. To date, many photosensitisers have been non-site specific drugs, i.e. they do not target specific substrates or sites in their “free-state”, highlighting the need for site-specific/selective photosensitisers and the development of targeting strategies [5, 24-26, 38, 39, 120]. One such set of examples are the photosensitiser bioconjugates which contain a receptor-targeting moiety (and a photosensitiser): a move that is in line with the “magic bullet” theory suggested in the early 20th century by Paul Ehrlich [121]. Ehrlich's notion of, “a compound which would have a specific attraction to disease-causing microorganisms by seeking them out and destroying them (whilst avoiding other organisms and having minimal undesired/harmful effects on the patient)”, could be theoretically achieved using antibody conjugates [121-123]. Antibodies work by selectively targeting complimentary biomarkers expressed on the surface of cells; tumour cells are known to over-express certain biomarkers on their surface (such as certain antigens) against which antibodies can be raised and subsequently conjugated to a photosensitiser; facilitating the directed targeting of the photosensitiser towards specific bioreceptors with high degrees of affinity and specificity, thus making antibodies ideal targeting candidates. Free photosensitisers typically achieve ratios of only 2-5:1 (tumour to normal tissue) - improving this ratio could significantly reduce the dose of photosensitiser necessary for a pdt effect to be observed [124].

Targeting Moieties

A number of small biologically active molecules have been successfully conjugated to porphyrins, for example steroids, peptides and antibodies [3, 25, 28]. Active targeting/active uptake requires that target molecules be recognised by specific intermolecular interactions and shuttled across the cell membrane by receptors. Thus, molecules may be targeted towards these receptors by appending the appropriate substrate moieties to them. Passive uptake involves diffusion at some point in the process and results from non-specific cell-molecule interactions. The lipid membrane core dictates that the more lipophilic a molecule, the lower the barrier to traversing the cell membrane, whereas amphipathic molecules normally bind at the interface or polar region and have greater barriers in crossing the membrane [125]. Antibodies have been used in a range of techniques such as: (i) antibody-directed enzyme prodrug therapy (ADEPT) [126-132]; the use of a deactivated toxin or prodrug which can be converted into its active form by an enzyme covalently bound to a suitable monoclonal antibody, thus “switching on” activity at the target cells; and (ii) antibody-directed abzyme prodrug therapy (ADAPT) [133, 134]. ADAPT is similar to ADEPT with the exception that ADAPT uses catalytic antibodies or abzymes, which can be engineered to catalyse the activation of the prodrug but minimise the immune response. Additional targeting and localising biomolecules have been employed in targeting therapy to achieve the “magic bullet” notion.

Serum Albumin

Serum albumin is the most abundant protein in humans - it is approximately ten times the total concentration of all other lipoproteins found in the blood [3, 6, 14, 24, 25, 135, 136]. Over 60% of the protein is found in interstitial fluid - fluid which surrounds cells and acts as a medium to provide nutrients to and remove waste products from the cells. Serum albumin is unique in its ability to bind, covalently or reversibly, with a large number of ligands with a high degree of affinity. It has been reported that photosensitisers possessing a high affinity for serum albumins could be efficient pdt agents; tumour cells have a higher rate of serum albumin turnover due to their increased metabolism and rate of proliferation [135]. One of the first studies into albumin binding with respect to targeted-pdt centred on the non-covalent binding (NCB) of an unsubstituted ZnPC (figure 17) to bovine serum albumin (BSA). Results were promising - tumour regression was observed (EMT-6 mouse mammary tumours on Balb/c mice and T380 human colon carcinomas on nude mice) with no hepatic toxicity. However, further tests indicated that, post-intravenous administration, the ZnPC redistributed towards the high density lipoprotein (HDL) fraction of the serum [135]. To circumvent this behaviour research concentrated on the covalent binding of photosensitisers to albumin [135]. Physically-modified albumin is targeted by scavenger receptors that are expressed in high concentrations on macrophages. The macrophages bind a broad range of different ligands and transport them to subcellular compartments; oxidised low density lipoprotein (LDL) and maleylated BSA readily bind to macrophages while native proteins do not. Estimates suggest that in several cancers greater than half of a tumours mass is of macrophage lineage giving the potential for targeting photosensitisers to the tumour by targeting macrophages. Reports suggest tumour-associated macrophages accumulate greater concentrations of photosensitiser than neighbouring tumour cells with a 9-fold increase observed for porphyrins. Results for other photosensitisers (porphyrins, PCs, chlorins) conjugated to albumin (BSA and maleylated BSA) have shown promise against a range of tumour cell-lines (human colon carcinoma HT29 tumour cells and J774 macrophage cell line from Balb/c mouse tumour cells). Albumin-conjugated photosensitisers have also shown promise in the treatment of arterial occlusion (inhibition of intimal hyperplasia and decreased restenosis post-initial disease therapy) and as agents to induce photodynamic tissue adhesion via tissue soldering [119, 135-141].

Low Density Lipoprotein Conjugates

Lipoproteins are naturally occurring particles composed of a hydrophobic lipid core (esterified cholesterol molecules), surrounded by an outer shell of polar lipids (phospholipids and unesterified cholesterol) and apoproteins (B-100 apolipoprotein). They are biocompatible, biodegradable and non-immunogenic species that serve as the main vehicle for transporting cholesterol molecules to mammalian cells [6, 119-137, 142-146]. Lipoproteins have two main roles: to solubilise highly hydrophobic lipids and regulate the passage of specific lipids into and out of particular cells and tissue. This movement occurs via a process of specific receptor binding in the plasma membrane of non-hepatic cells, internalising through endocytosis to form a vesicle within the cell: apolipoproteins control the recognition and binding of the LDL receptor and lead to receptor-mediated endocytosis [4]. They fuse with lysosomes and hydrolyse the protein component of the LDL. LDL receptors (apo B/E receptors) are overexpressed on malignant cells [142]. Cholesterol, a key component of all eukaryotic plasma membranes, is essential for the growth and viability of cells in higher organisms hence in tumour and tumour vasculature cells the LDL receptors are also overexpressed [3, 14, 25, 119, 135]. Such chemistry highlights the potential of LDL as a carrier vehicle for targeted drug delivery. Depending on the photosensitiser-LDL binding site - either within the hydrophobic core or within the matrix of the apolipoprotein outer shell - the photosensitiser-LDL conjugates are believed to target the cellular or vascular components of the tumour, further supporting the idea of targeted drug delivery [119]. Research has identified further advantages of LDL targeted drug delivery in pdt; post-irradiation, LDLs are highly oxidised - the oxidised species are cytotoxic towards endothelial cells, thus the photodynamic action of the targeted system may be increased [135]. Reported results of photosensitiser-LDL conjugates to date differ, both in terms of association of the photosensitiser with the lipoprotein and the photodynamic effect observed. 2 types of LDL binding are recognised - non-covalent and covalent [135]. Germanium (IV) octabutoxyphthalocyanine administered in vivo, via Cremophor EL, demonstrated prolonged serum retention and stronger association with LDL in comparison to liposome-delivered PCs [135]. Similar results were observed with tin etiopurpurin. Haematoporphyrin (an amphiphilic compound) was bound to human LDL and delivered to human HT1080 fibroblast cells; accumulation of the complex in the cells was identified as a result of a high affinity for LDL receptors. In comparison, a hydrophobic zinc PC-LDL complex was internalised into the cellular environment via non-specific endocytosis - the poor affinity for the LDL receptor was a result in the changes of the apolipoprotein B structure induced by complexation of the ZnPC with the LDL. Sulphonated TPPs yield different results dependent on their chemical structures; the monosulphonated and adjacently disulphonated species have been identified as strongly associating with LDLs - up to 250 molecules of the latter porphyrin-LDL complex, per LDL, resulted in unchanged LDL receptor recognition in a human hepatoma (Hep G2) cell line. In vivo studies suggest that LDLs can incorporate up to 1000 photosensitiser molecules and still be recognised as native LDLs [135]. Naturally, improved incorporation of photosensitisers into the LDL structure is anticipated to improve pdt efficiency and overall therapeutic outcome. For ZnPC, non-covalent complexation of the photosensitiser to the LDL, prior to intravenous administration, enhanced both tumour uptake and photodynamic activity of the photosensitiser in comparison to liposomes (dipalmitoylphosphatidylcholine) [135]. LDL-Hp complexes have shown different subcellular localisation properties in comparison to albumin-Hp complexes - LDL complexes exhibited selective accumulation in sites such as the mitochondria, whereas the albumin complexes have demonstrated preferential accumulation in the vascular stroma. Such differences in delivery and localisation properties could be utilised for the desired mode of tumour control: albumin delivery for extracellular tumour cell damage and LDL transport for a more direct cell death mode [135, 141]. The binding of photosensitisers to serum proteins is generally determined by photosensitiser hydrophobicity - moderately hydrophobic photosensitisers show preferential transport via albumins in the bloodstream, highly hydrophobic photosensitisers bind more predominantly with lipoproteins, specifically LDLs [146], thus there is potential to use LDL in the targeted delivery of hydrophobic and amphiphilic photosensitisers in pdt.

Epidermal Growth Factor

The epidermal growth factor (EGF) receptor is also often overexpressed in several tumour types, such as squamous cell carcinomas, highlighting the potential of photosensitiser bioconjugates with the EGF [135, 136, 147]. EGF is internalised into the cell via receptor-mediated endocytosis [147]. To date, this strategy has not been thoroughly investigated; only one research group has reported (in 1999) data on the photosensitiser-EGF model [148]. Lutsenko and colleagues focused their research on aluminium and cobalt disulphonated PCs. They observed the photosensitiser-bioconjugates displayed a greater degree of photoactivity than their non-conjugated analogues, with a 4.5-fold increase in the photocytotoxicity of the cobalt PC-EGF conjugate compared with the aluminium PC-EGF complex. Animal studies (C57B1/6 mice) on the murine melanoma cell line B16 using the CoPC-EGF model exhibited promising results - the mean life spans and survival times of the tumour-bearing mice were increased [148]. Further work needs to be undertaken on EGF conjugates to fully evaluate their potential as pdt targeting agents.

Sugars

Coupling of sugars to photosensitisers has also shown promise in the selective targeting of tumour/diseased cells [9, 125, 149, 150]. Tumour cells have high energy requirements and their proliferation is often dependent on glucose uptake - elevated glycolysis rates are observed in cancer cells in comparison to healthy cells. Glucose traverses the cell membrane via receptor mediated endocytosis [125, 149, 150]. Porphyrin-saccharide bioconjugates have demonstrated greater binding affinities for cancer cells - cancer cells overexpress glucose transporter receptors [150]. Two main types of sugar-porphyrin/PC molecules have been investigated: those where the sugar moiety is attached to the periphery of the porphyrin/PC structure through a linker group and those where the sugar is fused directly to the porphyrin macrocycle [150]. A number of research groups have looked at glycosylated porphyrins (figure 18).

A common problem with these porphyrins is the hydrolysis of the sugar component - porphyrins bearing sugars appended via O-glycoside linkages undergo hydrolysis under physiological conditions (by enzymatic and non-enzymatic acid/base reactions) and therefore have short half-lives [125]. To overcome this problem, researchers have looked at developing porphyrins-saccharide species conjugated through C- or S-glycoside linkages; synthetic yields for these compounds were often poor and led to work focusing on the improved synthesis and pdt profile of S-glycoside linked porphyrin-saccharide conjugates bearing four sugar moieties: 5,10,15,20-tetrakis(4,1'thio-glucose-2,3,5,6,-tetrafluorophenyl)porphyrin (P-Glu₄) and 5,10,15,20-tetrakis(4,1'thio-galactose-2,3,5,6,-tetrafluorophenyl)porphyrin (P-Gal₄) [125]. Tetrapentafluorophenyl porphyrins (TPPF₂₀) can be readily synthesised in large quantities (via Adler or Lindsey routes) and are commercially available, making them an interesting porphyrin of choice for research purposes. The P-Glu₄ and P-Gal₄ saccharide-porphyrin photosensitisers exhibited enhanced binding to a human breast cancer cell line over non-sugar porphyrin derivatives, such as tetra(4-methoxyphenyl)porphyrin. Results further demonstrated preferential uptake and an enhanced pdt effect for the glucose analogue (P-Glu₄) in comparison to the galactose derivative (P-Gal₄) [125]. Observations of lower photosensitiser (P-Glu₄) uptake in normal rat fibroblast (3Y1) cells than in the transformed (3Y1^v-Src) cells were reported, highlighting the potential of photosensitiser-sugar conjugates in targeted cell attack [125]. Hirohara and colleagues observed the pdt efficacy of five different glucosylated fluorophenylporphyrins: the trans-di(S-glucosylated) porphyrin demonstrated outstanding photocytotoxicity (twenty-fold higher than other S-glucosylated porphyrins tested and a three-fold increase in uptake) in HeLa cells [149]. Tomé and co-workers evaluated the antiviral activity of a number of meso-tetraarylporphyrins appended with carbohydrates. They reported elevated inhibitory effects on viral replication, against herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) in Vero cells, for porphyrins bearing fully unprotected sugar moieties on the periphery of the porphyrin molecule [151]. The fluorinated glycoporphyrin analogue demonstrated antiviral activity of 40% towards HSV-1 and 50% against HSV-2 [9, 151]. Vedachalam et al., Banfi et al. and Ferrand et al. report that diarylporphyrin sugar derivatives are more effective at inducing photodynamic cell death in human colon adenocarcinomas than the corresponding tetraarylporphyrin analogues [150, 152, 153]. A meso-bisglycosylated diarylporphyrin has been synthesised and its photodynamic potential explored. The bioconjugate was reported to localise in lysosomes, unlike other photosensitiser-sugar loaded complexes which have exhibited preferential localisation in the mitochondria or endoplasmic reticulum - the authors attributed lysosomal localisation to the sugar proportion of the bioconjugate [150]. Work to improve cellular uptake, decrease aggregation and increase hydrophilicity of PCs has been undertaken. Alvarez-Mico and colleagues have reported the synthesis of an anomeric glycoconjugate of PCs, while Lee et al. describe the synthesis and in vitro photodynamic activity of a silicon PC appended with galactose [154, 155]. Lee and colleagues observed increased solubility in most organic solvents, minimal aggregation and high photodynamic activity for the galactose-PC compound against the human HepG2 hepatocellular carcinoma cell line [154, 155]. They also observed very high quantum yields of singlet oxygen formation (F_D): 0.94, 0.79. 0.82 and 0.88 in dimethyl formamide. Analogues of Si(IV) PC conjugated through axial coordination with ligands such as polyethylene glycol (PEG) generally have singlet oxygen quantum yields in the range 0.16-0.52 [147, 155]. Riberio and colleagues report the preparation and evaluation of glycoconjugated PCs - a cyclic glucose heptamer PC [156]. They observed that the β-cyclodextrine moiety imparted a greater degree of solubility on the conjugate in water, however in aqueous solution the conjugate co-exists in its monomeric and oligomeric forms [156]. NC-saccharide conjugate chemistry is also being developed: peripherally substituted tetraglucose Zn(II) NCs have been synthesised by Iqbal and colleagues; however, solubility issues are hindering their evaluation as potential pdt agents [157]. Therefore they have begun to focus their work on incorporating more than four sugar units into the NC-glucose complex to increase the hydrophilicity of the molecule. It appears the potential pdt efficacy of saccharide-photosensitiser conjugates is dependent on the sugar moiety and the photodynamic profile of the photosensitiser; the position and nature of the attachment of sugars to photosensitisers are also important factors in their role as pdt agents.

Antibodies

The term photoimmunotherapy (PIT) is used in reference to pdt utilising photosensitiser-antibody conjugates [3, 14, 25, 65, 122-124, 142, 158-164]. A range of antibody-photosensitiser conjugates have been trialed for pdt including - (scFv) bound to hydroxyl and pyridiniumyl porphyrins [64, 65, 159, 161], and monoclonal antibody (MAb) conjugates of cationic porphyrins [64, 65, 161, 162]. The two main antibody species that have been investigated are MAb and scFv [159, 160, 163]. A number of photosensitiser-MAb conjugates have been evaluated against a range of targets including oncofoetal antigens; receptors for signal transduction pathways; and growth factor receptors. However, the conjugation of the photosensitisers to the MAbs was not efficient: MAbs were initially appended to porphyrins via activated esters and carbodiimide coupling chemistry to porphyrins bearing multiple carboxy groups and the free amines within the MAb structure, such methodology gives rise to antibody crosslinking issues and/or changes in the photophysics of the photosensitiser [159, 163]. Coupling to polymeric carriers has also been used to increase photosensitiser loading and conjugate solubility. Sutton and colleagues developed a porphyrin molecule (figure 19) that incorporated a reactive isothiocyanate (NCS) group designed to allow conjugation to biomolecules under very mild conditions, with no intermediates or by-products [165]. The synthesised porphyrin underwent bioconjugation through direct reaction of the single reactive isothiocyanato group and the primary amino group present on the side chain of lysine residues; negligible non-specific binding was observed (figure 19) [64, 65, 159].

Hudson and colleagues directly compared the efficacy of internalising conjugates with conjugates that remained surface bound. Their work focused on conjugating the water-soluble isothiocyanato porphyrin with MAb 35A7 (non-internalising), FSP 77 (internalising) and 17.1A antibodies; the bioconjugations were performed under ambient conditions with antigen binding remaining intact. In vitro results suggested 16-fold less of the 5-(4-isothiocyanatophenyl)-10,15,20-tri-(3,5-dihydroxyphenyl)porphyrin (PS1)-FSP 77 MAb conjugate was needed in comparison to the unconjugated porphyrin to yield the same inhibitory concentration (IC₅₀) in the hybrid human ovarian carcinoma (SKOv3-CEA-1B9) cell line [64]. The results reported by Hudson and colleagues added weight to those reported by Carcenac and coworkers: the use of internalising MAb-photosensitiser conjugates has advantages over the use of non-internalising MAbs or free photosensitisers [166]. In vivo Hudson et al. were able to demonstrate that the PS1-FSP 77 MAb conjugate had biodistribution values comparable with those for the unconjugated MAb in Swiss nude mice subcutaneously implanted with the appropriate antigen expressing cell lines. Hudson and colleagues further reported that the least substituted analogues of 5-(4-isothiocyanatophenyl)-10,15,20-tris-(4-N-methylpyridiniumyl)porphyrin trichloride (PS2)-MAb conjugates had tumour uptake values similar to the native unconjugated antibodies. Hudson et al. and Pardridge et al. observed that when the substitution of the MAb with the photosensitiser increased, tumour and other organ uptake decreased significantly [64, 167]. Pardridge and colleagues noted that increasing the MAb cationic charge reduced its serum half-life to as low as 5% of the unmodified native antibody [167]. Hudson and colleague's biodistribution data, for the respective conjugate systems, demonstrated retention of MAb pharmacokinetics following substitution of PS1 and to a lesser degree PS2. Tumour/normal tissue ratios (for colon carcinomas) were exceptionally high - 33.5 for PS2-35A7MAb conjugate; data from other groups for unconjugated photosensitisers report ratios ranging between 2 and 4 [64]. There have been fewer reports of hydrophilic PC-MAb conjugates in the literature. Duan and colleagues reported a ZnPC bearing alcoxy substituents conjugated to a MAb while another article describes the first coupling of AlPC(SO₂Cl)₄ to an antibody (E7) - the bioconjugate was reported to increase photocytotoxicity in human bladder carcinoma in comparison to the free photosensitiser [147, 168, 169]. Phototoxicity in the human bladder carcinoma cell line 647V was dose-dependent - at equimolar PC doses the liposomal analogue of the conjugate was 13-fold more effective. Immunofluorescence studies identified specific cell surface localisation and internalisation. Carcenac and colleagues reported results for a AlPCS4-MAb (FSP77, internalising MAb) bioconjugate selective towards ErbB2 on a SKOv3-CEA (CEA - carcinoembyronic antigen) cell line, they found that in comparison to an earlier bioconjugate they had prepared composed of a non-internalising MAb (35A7), the reported growth inhibition after a 20 hour incubation period at a dose as low as 0.04μgmol^-1 with the AlPCS4-MAb (FSP77) system was 51% in comparison to a 68% growth inhibition with the AlPCS4-MAb (35A7) conjugate under the same conditions, clearly highlighting advantage of an internalising MAb over a non-internalising MAb bioconjugate [147, 170]. Vrouenraets and colleagues describe the coupling of a AlPCS4 to MAbs (U36, E48 and 425) via a tetraglycine derivative (AlPC(SO₂N_gly)₄). They reported having trouble conjugating a second MAb to the photosensitiser due to hydrolysis of the ester groups. The PC-MAb (425) conjugate exhibited the greatest photocytotoxicity against the A431 cell line and selective tumour targeting in nude mice [170]. Vrouenraets and colleagues further report, in vitro, photocytotoxic evaluation of mTHPC and PC-MAb conjugates using five head and neck SCC cell lines; PC in its free form was ineffective but when coupled to MAbs it presented high effectiveness [147, 171]. A recent article by Mitsunga colleagues describes a PC-based photosensitiser coupled to a MAb targeted against epidermal growth factor receptors: promising results have been observed - cell death was induced immediately after irradiating target cells with near-IR light [164].

There are notable drawbacks to using MAbs - they are relatively large and bioconjugate size limits the passage of the photosensitiser-MAb into solid, deep-seated and poorly vascularised tumours. In comparison to MAbs, scFv fragments are antibody fragments that are smaller in size but since they retain the same binding specificity as the larger MAbs they penetrate tumour cells more deeply and effectively [159]. Other antibody fragments that have been investigated include Fab fragments [172]. scFv are observed to clear more effectively from the circulatory system than MAbs because of their lack of an Fc domain, they also exhibit lower kidney uptake. Staneloudi and colleagues reported the preparation of photosensitiser-scFv conjugates utilising the novel NCS porphyrin developed by Sutton et al. [159]. Neri and colleagues published research into the targeted delivery of a photosensitiser (tin(IV) chlorin e₆) by a phage-derived antibody fragment (anti-fibrogen antibody L19) in the PIT of endothelial cells in an ocular model [173]. Staneloudi and colleagues used the same two water-soluble porphyrins they had optimised in the MAb studies and colorectal tumour-specific (LAG3) scFv fragments that had been isolated from a phage antibody display library. They found that at higher molar loading concentrations (20:1 and 40:1 PS:scFv, in comparison to 5:1) the level of binding between the scFv and cell line was reduced or destroyed - suggesting that scFv were more susceptible to interference from antigen binding sites than their MAb counterparts. They reported that conjugation between the more hydrophobic PS1 and scFv was unsuccessful but conjugation between PS2 and the scFv had occurred. PS1 gave significant non-covalent binding, with excess porphyrin reportedly blocking the scFv antigen binding site. Preliminary evaluation of the PS2-scFv bioconjugates showed selective cytotoxicity towards antigen-positive cell lines [64]. A recent perspective by Bullous and colleagues reviews photosensitiser-antibody conjugates in depth [163]. Transferrin, insulin and steroid molecules coupled to photosensitisers have also shown promise as third-generation tumour-targeting systems [135, 147]. Lovell and colleagues have recently published a thorough review on the use of activatable photosensitisers as imaging and therapeutic (theranostic) agents [68]. When in its native state, a photosensitiser's imaging

and phototoxicity properties are quenched, molecular activation of the photosensitiser unquenches its photochemical capabilities, thereby facilitating the photosensitisers role as an imaging and therapeutic agent. Activation is specific to each photosensitiser type and disease and can be achieved via a number of routes including environmental factors (such as solvent hydrophobicity and pH); enzymatic means (certain enzymes are overexpressed in specific diseases); nucleic acid mechanisms; and the synthesis of photosensitisers with cleavable bonds [68].

Delivery Vehicles

The poor solubility of many photosensitisers in aqueous media prevents their intravenous delivery directly into the bloodstream. It would therefore be advantageous if a delivery model could be conceived which would allow the transportation of these (otherwise potentially useful) photosensitisers to the site of diseased tissue. Delivery vehicles have been investigated to circumvent the solubility problems of some of these photosensitisers, the main cargo-carrier systems investigated, beside the liposomes already discussed, include emulsions and nanoparticle carriers. PEG and Cremophor EL are two of the more common systems used for the emulsification process, although a number of other agents have been investigated [14, 25, 33, 34, 42, 174-181]. Cremophor, moleculsol and γ-cyclodextrin have all been compared against one another for the effective delivery of the hydrophobic porphyrin derivative tin etiopurpurin (Purlytin^®) [3, 4, 14, 24, 25, 27, 28, 33, 42]. The three emulsifying agents (administered in an aqueous ethanolic solution) all exhibited a greater degree of intracellular localisation relative to that of the “free” ethanolic solution. Results suggested that a lower intracellular concentration of photosensitiser was required to effect cell death when an emulsifying agent was used - a direct consequence of more effective photosensitiser accumulation.

Liposomal Encapsulation

Lipophilic photosensitisers may be encapsulated within the inner hydrophobic region of a liposomal delivery vehicle, while the outer hydrophilic layer (soluble in aqueous media), allows transportation of the photosensitiser towards the target site [14, 182]. Tin-etiopurpurin, benzoporphyrin derivatives and zinc PCs have all been successfully encapsulated in order to demonstrate this technique [4, 14, 42]. Liposome-associated photosensitisers have exhibited greater efficiency and selectivity towards tumour targeting (demonstrated by improved accumulation of the photosensitiser in tumour cells) compared with the same photosensitiser administered in a homogeneous (aqueous) solution [14, 24] - the improved uptake is thought to be the result of the LDL receptor mechanism discussed earlier [24, 49].

Tetrapyrrolics As Imaging Agents

Porphyrinic molecules show potential not only as photosensitisers but their unique luminescent properties make them useful imaging agents too. Porphyrins can be excited by visible light; emit in the red or near-IR regions of the EMS and are advantageous in cellular studies in comparison to those with shorter excitation wavelengths - lower wavelength light (ultra-violet light) can lead to cell damage and cellular autofluorescence can interfere with fluorescence investigations. The ideal fluorophore should also be one with a high quantum yield of fluorescence (Φ_fl.); a large Stokes shift; minimum photobleaching; and have a high affinity for the target species. Such multimodal agents have been utilised as image guidance tools and hence are useful in setting and adjusting the parameters needed during pdt [7, 28, 183-195]. Porphyrinic photosensitisers may also be radiolabelled and coupled with MRI imaging agents to provide multifunctional probes for positron emission tomography (PET) and MRI imaging in addition to having therapeutic applications [7, 28, 68, 196-199].

Fluorescence Imaging

In the laboratory fluorescence imaging can be used for studying basic pdt mechanisms; understanding pdt-tissue interactions; developing models of disease; and as a marker of therapy response [7, 28, 68, 183-200]. Fluorescence imaging agents that can be activated around 400nm are extremely useful in diagnostic imaging; the low fluence rates needed to activate the photosensitiser at this wavelength cause little or no cytotoxicity [194]. The history and applications of fluorescence imaging in relation to photodetection have been thoroughly reviewed by Ackroyd and colleagues (2001) [200].

Fluorescence Imaging In The Clinic

Bronchoscopes allow a physician to visualise mammalian airways observing the presence of any diseased tissue. Early versions of the apparatus were rigid metal tubes which required the patient to be sedated/anaesthetised in order to minimise discomfort and relax the airway reflexes. The use of these early versions carried specific risks to the patient such as scratching or tearing of the airway tissue and damaging the vocal cords. Excessive bleeding and pneumothoraxes were further potential complications encountered when biopsies were taken with the apparatus. In 1966 the Japanese physician Ikeda developed the first flexible fibre optic bronchoscope allowing better visualisation of the airways; the flexibility of the scope tip allowed a physician to see deep into the bronchi of the lungs [201, 202]. In the late 1980's Lam and colleagues (MacAulay, Palcic and Jaggi) at the British Columbia Cancer Centre in Canada invented the highly sensitive fluorescence bronchoscopy technique (LIFE-Lung) based on the natural fluorescence (autofluorescence) of bronchial tissue to aid the detection and localisation of pre-invasive and early stage lung cancers - they were awarded the Friesen-Rygiel Award for Outstanding Canadian Academic Discovery (1999) for the invention [191, 203-205]. In 2002 Lam was awarded the Gustav Killian Medal by the World Association of Bronchology for pioneering contributions in the field of early lung cancer diagnosis. The LIFE-Lung (light-induced fluorescence emission) imaging device is now used in over 150 major medical clinics worldwide. LIFE-Lung, originally marketed by Xillix, is based on the principle of naturally occurring fluorescence, i.e. when tissue is exposed to light the native fluorophore's fluoresce - no exogenous fluorophores/photosensitisers (drug-induced fluorescence (DIF)) have been administered to the patient for the (auto)fluorescence signal to appear. The traditional bronchoscopy technique relies on illuminating an area with broad spectrum (white) light and observing the reflected light; when tissue containing abnormal cells is exposed to the light, the physician is able to observe autofluorescence and identify abnormal tissue. However, this subjective approach is generally restricted to the identification of gross macroscopic changes in a lesion - white light bronchoscopy cannot show neoplastic or early neoplastic changes [185]. The optical properties of human tissue are dominated by endogenous light absorbing chromophores, which absorb in the 250-500nm spectral region of the EMS and exhibit fluorescence emission over the 300-700nm range. Complex tissue such as bronchial mucosa is made up of different cell types (epithelial, connective and vascular); total fluorescence emission is made up from each individual chromophore, each with its own unique and characteristic spectra (spectral content and intensity - fluorescence quantum yield); this composite emission is termed autofluorescence and generally covers a broad spectral band, although characteristic features tend to be lacking [185]. Autofluorescence can be enhanced by the addition of exogenous chromophores, such as photosensitisers, facilitating the monitoring of drug localisation properties; the technique is termed photodynamic diagnosis (pdd) and relies on the selective uptake and retention of photosensitisers within abnormal tissue, enhancing the image contrast between the healthy and abnormal tissues. DIF is generally greater in intensity than autofluorescence and displays characteristic luminescence features [28, 185, 188, 193, 194, 198]. Autofluorescence bronchoscopy has been commonly used in observing/imaging the lungs, bladder, ovaries, skin, brain and gastrointestinal tract [28, 185-194]. Reviews by Ethirajan et al.; Allison et al.; Moghissi et al.; and Celli et al. cover the topic in more detail - Ethirajan et al's. and Celli al's. reviews are particularly thorough [28, 183, 186, 194]. It is worthy to note Celli and colleagues refer to “pdd” as “photosensitiser fluorescence detection” (PSFD) rather than diagnosis - diagnosis could be interpreted as the ability to grade and stage tumours, as opposed to detect them; photodynamic implies generation of ROS [194]. Therefore, for technical clarity in the remainder of this review, the term PSFD will be used instead of pdd.

Lam et al's. LIFE-Lung system uses a Cd-He laser (excitation 442nm, blue light) and an optical multi-channel analyser detection system to provide real-time video imaging capabilities. When exposed to blue light normal bronchial mucosa tissue emits a green-coloured fluorescence signal; the presence of dysplasias or carcinomas is observed through a reddish/brown fluorescence signal from the abnormal tissue. The spectral differences are due to the chemical constituents of the respective tissue histology - one hypothesis is that the connective tissue beneath the epithelial of the bronchial mucosa displays a more intense fluorescence signal in comparison to the fluorescence signal from the epithelial cells, therefore when exposed to blue light, the green fluorescence emission from the connective tissue underneath the healthy epithelial dominates the observed signals. In the case of neoplastic changes in tissue, the epithelial layer is thicker and contains a lower concentration of fluorophores, hence a reduction in the intensity of the fluorescence signal observed from the connective tissue, there is also a relative shift in the spectral emission from green (healthy) to red (abnormal) [186]. The LIFE-Lung system can be used by physicians in adjunct to the conventional white light examination technique; autofluorescence is more than four times more sensitive in identifying morphological changes specific to pathological progression than the white light used in traditional bronchoscopy and can detect lesions as small as 1mm and only a few cells thick, thus a more definitive identification of lesions can be made [186, 206]. Prior to the clinical implementation of autofluorescence it was necessary for a patient to undergo a number of bronchoscopy procedures whereby multiple biopsies where taken in order to appropriately identify and localise lesions present with occult cancer [186]. Lam and colleagues report an increase in the sensitivity in identifying and localising intra-epithelial neoplastic lesions by a factor of 6.3 when autofluorescence and white light illumination are used as complimentary imaging methods in comparison to using white light alone; sensitivity was increased by a factor of 2.7 when invasive carcinomas were also present [186, 193, 207]. A number of different systems have now been developed for autofluorescence imaging all based on laser or filtered high intensity arc lamp illumination. LIFE-Lung was marketed by Xillix (Canada) in 1993; the D-Light system, marketed since 1995 by Stortz (Germany) can be manually switched between white light and blue light modes; the SAFE system (versions 1000 and 3000) marketed by Pentax (Japan) since 2006 incorporates a single action image switching and simultaneous display; the Evis Lucera Spectrum Autofluorescence by Olympus was also brought to the market in 2006 and comprises of three signals which induce autofluorescence by blue and reflected (550-610nm) light; ONCO-Life (Novadaq Technologies, Canada) also combines fluorescence and reflectance imaging; the combination of fluorescence and reflectance imaging is aimed at reducing the number of false-positives reported; it can be difficult to distinguish fluorescence emission produced from increased vascularity (associated with airway inflammation) to that produced from pre-invasive lesions [186, 189]. The quantification of the reflectance (red) and fluorescence (green) intensity signals allows the red:green ratio (R/G), termed the colour fluorescence ratio (CFR), to be determined for the area being examined [189, 191, 192]. The resulting CFR assists the physician in making a more confident and precise identification of abnormal tissue (moderate dysplasia and/or carcinoma in situ that has begun to progress) for biopsy; Lee and colleagues report a good correlation between the CFR-identified abnormal tissue and histological-verified diseased tissue [189]. Updated versions of the LIFE-lung (LIFE-Lung II) with a filtered Xe lamp to produce blue light and with two image-intensified charge-coupled device sensors to capture emitted fluorescence: one in the green region (480-520nm) and the other in the red region (≥625nm) and the Evis Lucera system (with narrow-band imaging capabilities) are now available [186, 189]. The significant and rapid improvement in fluorescence imaging technology has increased the number of body areas physicians are able to take biopsies from without the need for surgery. New technology is focusing on high definition optical systems with recording options facilitating the ability of a team of medics to view live images (not just the physician) and for the team to view the images at a later date when planning treatment regimens [185].

History Of Fluorescence Imaging

Observation of autofluorescence in tumour localisation is not a modern interest and dates back to 1924 when Policard observed the fluorescence (brown/red) of malignant tumours - characteristic Hp fluorescence was observed when rat sarcomas where illuminated with ultraviolet light, demonstrating the preferential accumulation of a photosensitiser in cancerous tissue [185, 194, 208]. These observations where supported by the studies (1940's) of Auler and Banzer and Figge and colleagues who evaluated the tumour-localising properties of several porphyrin and metallo-porphyrin compounds [209, 210]. The latter group's studies revealed that all of the porphyrin species investigated emitted localised fluorescence from tumour tissue but not from healthy tissue upon ultraviolet illumination, with the exception of lymphatic; omental; faetal; placental; and traumatised regenerating tissue. A clinical study of PSFD was undertaken in the 1950's by Figge and colleagues utilising the Hp hydrochloride salt to demarcate benign and malignant tumour tissue; fluorescence from the photosensitiser was evident in a greater percentage of malignant tumour tissue than benign tissue and the intensity of the fluorescence signal increased with increasing photosensitiser concentration, to the extent that solid tumours could be observed through intact skin [211]. DIF was first used in the clinic (Mayo Clinic, USA) in the 1960's when Lipson and colleagues used a fluorescence endoscope to view HpD differential fluorescence between tumour and healthy tissue [212, 213]. The same decade saw Sutro and colleagues report the observation of contrasting fluorescence signals from excised human breast cancer tissue (purple/red emission) to healthy surrounding tissue (green fluorescence) [214]. Further clinical studies were undertaken with HpD in the fluorescence detection of cervical and lung cancers; head and neck tumours; and a number of bladder malignancies - the one large-scale study (226 patients) conducted by Gregorie and colleagues concluded with disappointing results: only 76.3% of the patients with confirmed malignant neoplasms displayed positive tumour fluorescence [215]. The late 1970's and early 1980's witnessed a number of groups conduct detailed investigations into the uptake and pharmacokinetic profiles of HpD-based PSFD and pdt [216, 217]. Gomer and Dougherty conducted detailed tissue timing and distribution studies in healthy and malignant tissue and reported higher concentrations of HpD in tumour tissue compared to cutaneous tissue or muscles of mouse models bearing a mammary carcinoma; the highest concentrations of HpD were in the liver, kidneys, spleen and lungs [216]. Jori and colleagues reported in a similar study that relative to the HpD in the liver, only small concentrations of HpD were metabolised by tumour cells in a rat ascites hepatoma model [217]. Both groups reported that time windows (Gomer and Dougherty 24 hours, Jori and colleagues 12 hours) existed during which the higher accumulation of HpD in the tumour tissue relative to surrounding healthy tissue allowed for tumour destruction with minimal toxicity to the healthy tissue. Kessel's investigations, along with those by Unsöld and colleagues, identified the issue of HpD impurity as a limiting factor in its ability to accurately and reliably identify the boundaries of diseased tissue in detection and/or surgical guidance [218, 219]. However, Balchum et al., Hyata et al. and Cortese et al. employed HpD to induce fluorescence as a means of locating early endo-bronchial tumours in the clinic in the 1980's [220-222]. Moghissi and colleagues highlighted in their 2008 review (on fluorescence bronchoscopy) that over 200 articles had been published on fluorescence bronchoscopy (since the 1980s) and autofluorescence (since 1992) [186]. Fluorescence imaging has now become well-established and is routinely used in a number of clinical and medical applications/situations, including in the identification of neoplastic changes; localisation and topographic distribution of neoplastic lesions; fluorescence-guided local cancer therapy for airway; dermatological; bladder; and brain cancers [28, 183, 185-194]. In oncology, the use of fluorescence detection has significantly enhanced the diagnostic process and has seen a range of minimally invasive procedures and intervention methods introduced into cancer treatment and management, particularly in the identification and localisation of pre- and early cancerous lesions as well as image-guided therapy [28, 183-194].

Lung cancer is the most common cancer in men in the industrial world and the most common cause of death amongst all cancers in men and women in the UK, Europe and USA. Surgical resection is the treatment of choice for all histological varieties of lung cancer, except the small-cell type. There is a definite relationship between the stage of cancer at diagnosis and outcome in terms of survival. For patients with early stage I cancer a greater than 70% chance of a 5 year survival rate can be achieved, however, in the absence of a reliable screening programme, less than 15% of patients are diagnosed at this stage [186]. In the majority of cases, over 80% of lung cancers are inoperable at presentation, limiting treatment options and the potential of achieving a “cure” [186, 189]. Central-type lung cancer at its initial development is radiologically occult (presentation of clear chest x-rays/CT scan) [186]. Chest x-rays have previously failed to identify up to 77% CT-detected cancers [190]. Although, it may (with great difficulty) be possible to detect the disease with white light bronchoscopy, the use of blue light more readily discriminates lesions from normal tissue; these differences are more visible by a factor of 7 in comparison to white light - this was reported for all “reviewed” cases of autofluorescence versus white light bronchoscopy [186]. Autofluorescence bronchoscopy has also been successfully used for staging cancer - 9.3% of patients who were identified for surgical resection showed additional lesions in pre-surgery autofluorescence bronchoscopy [223]. Furthermore, autofluorescence bronchoscopy has shown greater reliability than sputum testing in diagnosis - sputum testing was reported to have missed 100% of the lesions in a clinical study [224]. Unfortunately, autofluorescence isn't without drawbacks - its specificity is much lower than that of white light bronchoscopy; there are recordings of false positives in autofluorescence bronchoscopy; there is difficulty distinguishing airway inflammation from pre-invasive lesions based on the visual grading of tissue fluorescence, this can result in excessive biopsies; longer procedural times; a greater risk of complications; and greater cost, it is therefore good practice to employ autofluorescence bronchoscopy as an adjunct technique to white light bronchoscopy [186, 189]. In the post-surgical surveillance and monitoring of post-pulmonary resection patients (resection performed with curative intent), 12% of a group of 25 patients and 6% of a group of 51 patients developed intra-epithelial neoplasia solely diagnosed by autofluorescence [225, 226]. When a patient is subject to monitoring, if lesions (early and recurrent) are identified and are in the early stages of the disease, it has been postulated that any further lesions found in follow-up protocols can be treated with endoluminal pdt with curative intent [186, 227, 228] - there is widespread acceptance of pdts safety and efficacy in a bronchoscopic application for broncho-pulmonary malignant neoplasia [188].

Fluorescence Imaging, Guided Resection And Pdt - A Theranostic Approach To Disease

In the early stages of lung cancer pdt can provide long term survival opportunities (potential cure); in the mid-stages, where the extent of the disease can be more accurately diagnosed with fluorescence imaging and endoscopic ultrasonography, results can match those of surgical resection [188]. Systemic photosensitiser administration, followed by bronchoscopic illumination is used to achieve cancer necrosis; a combination of imaging and pdt (theranostics) can therefore provide improved therapeutic strategies for lung cancers [188]. The combination of the modalities can also help in laboratory-based disease research; fluorescence imaging can be utilised in studying basic pdt mechanisms; understanding pdt tissue interaction; developing disease models; and as a marker to therapy response [194]. The two modalities (fluorescence imaging and pdt) work on the same basic principles - illumination and photochemistry but differ greatly in their desired outcome - fluorescence imaging has minimal/no cytotoxicity due to the low fluence rate used to generate light activation, whereas in pdt, where a cytotoxic effect is the goal of the treatment, a high fluence rate is used to generate the singlet oxygen necessary to elicit cell death [194]. Both the uniqueness and major advantage of using fluorescence imaging and pdt in combination with each other is that the same entity can act as both the imaging and therapeutic agent, the only known exception to this is TOOKAD^®, the palladium-complexed bacteriochlorophyll (figure 20); TOOKAD^® has a negligible fluorescence quantum yield [229, 230]. This emphasises the potential of fluorescence imaging in pdt for the diagnosis, guided therapy and monitoring of surgery (or in other therapies) - the therapeutic outcome in the treatment of disease can be made more robust by utilising all of the available forms of imaging and guidance techniques/technologies. Such theranostic modalities are likely to become more routinely used in the treatment and management of diseases in the future [194].

A number of different imaging systems exist which are capable of observing molecular, structural and functional parameters of the mammalian body. The type of imaging system is chosen depending on the spatial scale of the entity to be imaged and the source of contrast enhancement. For example, imaging at the molecular level, involving small molecules and proteins (ranging from 10ppm-10nm), can be achieved with spectroscopy-based techniques including MRS (magnetic resonance spectroscopy), PET and optical spectroscopy; such imaging techniques facilitate the visualisation, characterisation and quantification of biological targets and processes at a cellular level; particularly useful for the imaging of singlet oxygen [194]. Microscopy, surgical microscopy, endoscopy, ultrasound and fluoroscopy techniques can be used for imaging organelles; cells; tissue matrices; physiological ducts; and tissue layers ranging in size from 50nm-100mm and are valuable platforms in surgical-guidance, therapy monitoring and dosimetry procedures [194]. When images of body organs and the whole body are required tomographic techniques such as computerised tomography (CT) and MRI are employed [194]. Imaging at the molecular level provides the ability to monitor in vivo responses to pdt in near/real-time [194]. In many situations, the outcome of an oncological treatment is only known at a much later date when the patient is undergoing post-sampling protocols or when the disease may have progressed/recurred due to incomplete removal and/or poor treatment response; online and/or early monitoring approaches are invaluable in developing strategies to combat inadequate treatment responses - one strategy is the use of image-guided surgical-resection (achieved with autofluorescence bronchoscopy and/or DIF) [194]. Due to the propensity of photosensitisers to preferentially accumulate in neoplastic tissue their fluorescence properties makes them inherently well-suited for the selective visualisation of tumours using the fluorescence contrast between healthy and diseased tissue to demarcate the boundaries around diseased sites. The ability to accurately define the margins of a tumour is a crucial step in the optimisation of surgical resection; a cancer-free margin around the cancerous tissue that is being excised is a major predictive factor in the success of the treatment and the long-term outcome for the patient [28, 183, 194]. Particular care must be taken not to remove too much healthy tissue - this is most apparent in brain surgery, for example, if 1mm of tissue were unnecessarily excised motor skills could be detrimentally affected with severe implications to the patient's quality of life [194]. The suitability of PSFD for clinical translation has allowed its use for selective identification of cancerous lesions in a broad range of anatomical sites including the lungs, bladder, brain, skin, breast and female reproductive tract [28, 183, 185, 188, 191, 193, 194, 198]. The limitation of PSFD, particularly, in comparison to PET, MRI and CT, is the inherent surface sensitivity of the technique; PET, MRI and CT are able to provide structural details that are not achievable with PSFD - the detection sensitivity of PSFD decreases during the resection process as the volume of non-resected disease diminishes - the sensitivity of fluorescence imaging is not affected [194]. PSFD had its first widespread and successful implementation in identifying diseased tissue after Kennedy and Pottier introduced an alternative approach of enhanced endogenous protoporphyrin (PPIX) production in the haem cycle of tumours in the early 1990s [231, 232]. The strategy is based on the in situ conversion of ALA, a non-photoactivatable precursor, into PPIX, a naturally occurring photosensitising species via the cellular haem biosynthesis pathway (Figure S1/scheme 1). In the synthetic pathway iron is inserted into the PPIX cavity to form haem; the ferrochelatase enzyme that catalyses this chelation is down-regulated in many tumours and as a result iron is chelated into the PPIX cavity at a relatively low rate and is unable to compensate for the excess PPIX formed - significant accumulation of PPIX in neoplastic tissue occurs as a result following administration of exogenous ALA. The rates of ALA uptake in healthy verses neoplastic tissue are thought to be comparable; it is the differential rates of ALA conversion and resulting accumulation of PPIX that are the primary force behind favourable tumour selectivity [194, 233]. PSFD has proved a powerful tool in the detection and guided resection of bladder cancer and has been reviewed by Witjes and Douglass [194, 234]. Bladder cancer is the fourth most common malignancy in men and the industrial world; it has a high rate of recurrence and is a very costly cancer to treat and monitor [235-237]. A critical factor in predicting disease recurrence is the detection of carcinomas in situ (CIS); these flat lesions are particularly difficult to detect with white light due to the poor contrast they generate [194, 236]. Levulan^® has proved successful in the fluorescence identification of numerous bladder lesions that were not detected by white light bronchoscopy; the detections were 100% supported by histological validation [194, 238-241]. In the follow-up assessment of patients with fluorescence imaging, the superior sensitivity of the modality led to a reduced rate of early recurrence of superficial bladder cancer in comparison to white light evaluation [242]. In a phase III trial, comparing transurethral guided resection using ALA verses white light cystoscopy, 61.5% of the patients in the PPIX fluorescence endoscopy group were tumour-free at the follow-up assessment in comparison to only 40.6% in the white light group [243]. The hexylester derivative of ALA, Hexvix^®, has been used to achieve greater tissue penetration depths into the urothelial layers and a more homogeneous distribution in malignant tissue; Hexvix^® produced a stronger fluorescence intensity at a lower dose following a shorter incubation time [244]. Fluorescence-guided resection (FGR) has also been used in the treatment of brain cancer; when ALA-induced PPIX was used in a guided resection study 63% of the patients achieved complete resection through contrast enhancement. A phase III trial was terminated at the interim analysis of 270 patients, when 65% of those in the FGR group (procedure followed by standard adjuvant radiotherapy) were free of residual disease at the post-operative 6 month assessment (by MRI), compared with 36% in the white light group. It is noted that the survival curves for the patients in both groups converged after 15 months [194, 245]. Foscan^®, particularly suited to imaging and treating bulky brain tumours due to its depth of light penetration, has been evaluated by Zimmermann and colleagues for the FGR of malignant gliomas; in 138 tissue specimens from 22 of the patients, a sensitivity and specificity of 87.9% and 95.7% were achieved [194, 246]. In 10 of the 22 patients, malignancies were not identified by white light examination but were exclusively identified by PSFD [194, 246]. Foscan^® has exhibited further advantages over ALA-induced PPIX in that after several minutes of illumination it does not undergo photobleaching. To date, FGR has been invaluable in improving the extent to which tumour tissue can be removed but is now beginning to show promise in cases where a tumour cannot be completely removed because it has infiltrated functional brain tissue; the residual tumour tissue can be treated with pdt. In a standard treatment protocol, surgical resection is followed by adjuvant radiotherapy, however, when FGR is used instead of white light, the photosensitiser is already present in the tissue at the time of resection; a logical extension of the procedure would be to use pdt to selectively destroy any residual disease in the resection bed - a great example demonstrating the promise of theranostic regimens. Kostron and colleagues reported results from a study on FGR with adjuvant pdt in a group of patients (26) with malignant brain tumours using m-THPC; an increase in median survival from 3½ months to 9 months was achieved [247].

In 2007, Stepp and colleagues also reported an evaluation of FGR in combination with adjuvant pdt in the treatment of malignant glioma. When ALA was used the assessment showed healthy brain tissue was practically free from ALA [248]. The most extensive work in image-guided resection and the pdt of gliomas is by Wilson and colleagues [249-252]. Their “point and shoot” system initially used Photofrin^®, before moving on to ALA. They demonstrated that their intra-operative fluorescence imaging system not only improved tumour resection but allowed pdt of the residual tumour [249-252]. Bogaards and colleagues have investigated the impact of low light doses over extended periods of time via implanted diodes, as opposed to the conventional methodology of a single light dose at a higher fluence rate [251]. Gibbs-Strauss and colleagues have investigated measuring the fluorescence of PPIX through the cranium with the idea that more frequent screenings can take place non-invasively [253]. Reddy and colleagues have undertaken pre-clinical studies on nanoparticles with increased specificity (through a targeting peptide) for both imaging (MRI) and the pdt of brain tumours. They monitored the pharmacokinetics and distribution of the nanoparticles within a 9L rat glioma model and reported a pronounced increase in the survival rate of the animals in the targeted-nanoparticles therapy group; a survival probability of approximately 0.4 at 60 days was achieved post-treatment compared to death within 20 days for the control groups [254]. To date, ALA-induced PPIX FGR in brain tumours has been the highest impact application of PSFD.

DIF has been utilised in ovarian cancer; ALA/Hexvix^®-induced PPIX has been used to image lesions in ovarian cancer undetected by white light imaging, the intensity ratio of neoplastic to healthy tissue was approximately 4:1 (optical biopsied lesion size was as small as 1.0mm (0.3-2.5mm) and 1.5mm (0.5-2.9mm) for white light) [255, 256]. Over 70% of patients presenting with ovarian cancer have late stage symptoms, leading to the high mortality rate observed with the disease. These patients undergo surgical debulking and cycles of chemotherapy, there is also a high likelihood of disease recurrence resulting in a 5 year survival rate of 31%, this can be increased to a 93% chance of survival after 5 years when the disease is diagnosed early and has not spread to regional and distant sites [194]. Ovarian cancer presents particular challenges in its thorough imaging and diagnosis - the disseminated disease has microscopic tumour nodules implanted throughout the peritoneal cavity. These nodules can be missed during surgical debulking and escape detection by traditional laparoscopic second-look procedures; PSFD shows great potential in the more thorough imaging/detection of the microscopic diseased nodules [257-259]. In a clinical study (29 patients) using ALA-induced PPIX, the ovarian cancer nodule detection sensitivity was 92%, a huge improvement over traditional white light imaging. The same study also identified that in 13 patients where the disease had been confirmed histologically or cytologically, 4 patients had lesions that were detected only by fluorescence imaging [260]. Zhong and colleagues combined PSFD (Visudyne^®) with high-resolution fluorescence microendoscopy to detect (in vivo) nodules of a disseminated ovarian cancer in the peritoneal cavity of mice on the order of a few tens of micrometres [261]. Zhong et al. used PSFD to conduct baseline imaging to assess the disease state immediately before intra-peritoneal therapeutic radiation, Visudyne^® was then activated and treatment response assessed in follow-up imaging several days later allowing the quantitative reporting of treatment response. The data revealed that the tumour nodules continued to grow unchecked in untreated mice while in the treatment group a significant reduction in tumour volume was observed. The observation highlights the promise photosensitiser-based fluorescence shows in monitoring pdt response in outcome assessments post-therapeutic intervention [261]. Further studies are required to indicate the true value of the modality in reporting treatment outcome.

The accessibility of skin lesions and the ease of topical photosensitiser application has seen the integration of PSFD and pdt in the imaging and treatment of dermatological conditions, including SCC, basal cell carcinoma (BCC), Bowen's disease (SCC in situ), Paget's disease and the guidance of Moh's surgery [194]. Pdt (using ALA) was successfully demonstrated in the treatment of superficial BCC and SCC in 1989; Kennedy, Pottier and Pross reported that the topical application of ALA was suited to delineating malignancies of the skin due to the preferential uptake of ALA in abnormal keratin cells versus normal keratin cells, enhancing the fluorescence emission contrast of abnormal tissue [232]. Photosensitiser fluorescence imaging has been used to try and increase PPIX-based fluorescence tissue contrast in healthy skin in seven patients with nodular BCC; SCC in situ; or cutaneous T-cell lymphoma. Andersson-Engels and colleagues used a multi-channel fluorescence imaging system to collect PPIX fluorescence emission (635nm), autofluorescence (470 and 600nm) and photobleached product emission (670nm) from cancerous lesions and surrounding normal tissue pre- and post-pdt [262, 263]. The data was used to deconvolute background autofluorescence from tumour-based PPIX fluorescence on a pixel-by-pixel basis. Andersson-Engels et al. have also used fluorescence imaging to demonstrate the potential of tracking the accumulation of photodegraded products during pdt. Further studies are needed to corroborate verification of tumour margins and pdt treatment response using multi-channel PSFD. Hewett and colleagues have demonstrated the ability to monitor the kinetics of PPIX photobleaching pre-, during and post-pdt of superficial skin cancer in vivo using a multi-spectral fluorescence imaging system with an integrated excitation source - they varied rate of light delivery and fraction schedule to optimise treatment outcome [264]. The study demonstrated rapid photobleaching early in the pdt treatment, indicating light doses should be delivered at a slower rate or fractionated over the course of the treatment to achieve greater consistency in the therapeutic effect. Hewett and colleagues attempted to use PPIX-induced fluorescence contrast and autofluorescence from the surrounding tissue to more accurately delineate the lesion margins. In understanding the importance of the role of ALA-induced PPIX synthesis and distribution in abnormal versus normal tissue in PSFD, pdt and therefore treatment outcome, Martin and colleagues examined the macroscopic heterogeneities in PPIX distribution in patients (20% ALA applied topically to 16 patients) with BCC [265]. Significant heterogeneities in PPIX fluorescence patterns were observed, suggesting no preferential selectivity of the photosensitiser for BCC over healthy tissue. Martin et al. purported any macroscopic selectivity could be attributed to the increased stratum corneum surrounding the diseased tissue as well as tumour thickness effects. Their study emphasises the usefulness of PPIX-induced fluorescence in the treatment of malignant cutaneous lesions or superficial BCC but highlights that in the treatment of deeper lesions optimisation of ALA formulation and delivery is important. The need to improve selectivity and homogeneity of photosensitisers in diseased tissue has been addressed by Fritsch and colleagues [266]. They compared ALA- versus Metvix^®-induced PPIX production in human solar keratoses to surrounding healthy skin. Higher levels of total PPIX accumulation was observed in the skin keratosis cells versus healthy cells for ALA compared to Metvix^®. Selectivity of Metvix^® for skin keratoses cells over healthy cells was significantly greater than with ALA. Distribution analysis revealed that 82% of the porphyrins in skin keratoses were specifically composed of PPIX in comparison to 89% in normal skin cells when incubated with Metvix^®. PPIX represents 90% of ALA-induced porphyrin metabolites in both healthy and diseased cells. Moan and colleagues investigated the use of a second ALA derivative, Hexvix^®, in relation to ALA and Metvix^® in the treatment of dermatological conditions [267]. They found that the PPIX concentrations needed to induce half the maximum PPIX-induced fluorescence were 2% (ALA), 8% (Metvix^®) and 1% (Hexvix^®) when the pro-drugs were applied at 0.2%, 2.0% or 20% w/w, highlighting the potential of achieving a similar fluorescence outcome (PPIX concentration) for Hexvix^® at an eighth of the concentration needed by Metvix^® in healthy cutaneous tissue. Kuijpers and colleagues have evaluated the response rates of ALA and Metvix^® pdt of BCC and acne vulgaris to report that there was no differential outcome in the two photosensitiser pro-drugs [268, 269]. A greater incidence of severe side effects was reported for ALA-pdt than Metvix^®-pdt [270]. This is perhaps surprising given that there is a marked contrast in the PSFD of bladder cancer were the ALA-derivative Hexvix^® is preferred and widely accepted in the imaging protocol rather than ALA [194]. Redondo and colleagues conducted a clinical pilot study into the use of Metvix^®-induced PPIX fluorescence for the guidance of Moh's surgery in the delineation of BCC; they compared the margins identified by Metvix^®-induced PPIX PSFD; white light imaging; and histopathology evaluation [271]. The margins in the diseased versus healthy tissue in 14 out of 20 patients determined by PSFD corresponded exactly to the gold-standard histopathological evaluation (haematoxylin-eosin staining). This allowed the step-wise excision of diseased tissue until the presence of the healthy tissue was verified (histologically). In half of the patients the diseased regions determined by PSFD (and confirmed by histology) were larger than those determined by white light imaging. It was noted that PSFD did identify 3 false-negative results and 3 false-positive results; 2 of them from recent scar tissue [271].

The outcome of oral cancer in terms of survival/cure is also determined by the stage of the cancer at the time of diagnosis: a 5 year survival rate of 82% for early stage cancer, reduced to 53% for regional spread cancer and considerably lower at 23% for distant spread cancers [272]. Leuning and colleagues evaluated the use of ALA-PSFD in the detection of small lesions/early-stage cancer and observed that ALA-induced PPIX fluorescence identified a group of patients with dysplasia; carcinoma in situ; primary tumour; secondary carcinoma; and tumour branches that were not detected by white light examination [270, 273]. Subsequent studies by the group added further support to the use of combining PSFD and autofluorescence together and observed that the ability to demarcate neoplastic regions was independent of disease stage - an additional benefit of PSFD [274]. Ebihara and colleagues reported that in a hamster cheek pouch model of oral cancer fluorescence emission from PPIX can differentiate between the different stages of pre-malignancy and malignancy [275]. Duska and colleagues have reported that in cervical malignancies oral administration of ALA overcame the penetration and distribution inhomogeneity experienced with topically delivered ALA [194, 276].

Fluorescence Imaging, Site-Activatable Constructs And Pdt - A Theranostic Approach To Identify And treating Diseased Tissue

Non-specific localisation of photosensitisers often leads to sub-optimal treatment outcomes and unwanted toxicity in healthy tissue; targeting strategies and the synthesis of target-activated photosensitisers have been investigated to try and address this short-coming. Site-activated photosensitisers achieve specificity for tumour tissue by selective activation of the entity in the diseased tissue by tumour-specific agents [194]. Applications of such entities in imaging and pdt have been reviewed by Stefflova [277]. The principle of the constructs is based on energetically quenching a photosensitiser by placing two photosensitisers within close proximity to each other (self-quenching) or a dye (energy-transfer). If the chemical bonds holding the photosensitiser and quencher together are broken the photosensitiser will no longer be quenched and will become fully activated [194]. Weissleder carried out the early work in this area by developing protease-activated near-IR probes for cancer imaging [278]. The group went on to report the conjugation of a chlorin photosensitiser (C_e6) onto a polylysine backbone [279]. The photosensitisers were held in close geometry to each other in order to facilitate efficient self-quenching, preventing energy transfer between the photosensitiser and molecular oxygen. In the presence of tumour-associated enzymes (for example cathepsins), the peptide linkages of the polylysine backbone were cleaved and the “degraded” probes became highly phototoxic and fluorescent. Weissleder and colleagues went on to evaluate (in vivo) the novel theranostic construct for pdt efficacy and imaging using fluorescence molecular tomography to determine the distribution of C_e6 in tumours; the target tissue was visualised and the local drug concentration quantified before selective therapy was applied [279]. Lovell and colleagues have reported establishing the correlation between photosensitiser fluorescence quenching and singlet oxygen quenching; implying that fluorescence intensity can be used as an indicator for the status of singlet oxygen production by a photosensitiser [280]. Zheng and colleagues put this notion into practice by introducing several Förster/fluorescence resonance energy transfer (FRET)-based target-activatable constructs consisting of a photosensitiser and quencher - the authors termed the constructs “photodynamic molecular beacons” [281]; the feasibility of the construct was demonstrated using an apoptotic factor-cleavable linker - as already mentioned apoptosis is the preferred mode of cell death in pdt, thus an apoptotic factor-cleavable linkage carries implications in the molecular imaging of pdt-induced apoptosis [281]. The construct wasn't selective for tumour cells but the selectivity will undoubtedly be fully addressed in the near future. One solution would be to photodynamically activate a photosensitiser with the matrix metalloproteinase MMP-7 - an important tumour biomarker [281]. Zheng and colleagues have also demonstrated the potential of target-activatable constructs in the treatment of pathogens - a phenothiazinium-based target-activated construct has been developed against β-lactam producing phenotypes in an approach termed “see and treat” [282].

Imaging And Monitoring - Time Gated Fluorescence, PET, MPE, OCT And MR Imaging

It can be difficult to determine specific dosimetry in pdt due to the non-linear relationship between light dose, irradiation time, photosensitiser and molecular oxygen concentrations. During the administration of light (in pdt) there are a number of photophysical, metabolic and molecular changes occurring within the target tissue, each change providing a unique signal. Several techniques for measuring effective dose-response relationships online exist including measuring a photosensitiser's fluorescence and dynamic properties during photobleaching. Time-gated fluorescence imaging is one example used to detect cancerous tissue and monitor the emission of disulphonated aluminium PC (AlS₂PC) during pdt of MS-2 fibrosarcomas [283, 284]. The technique reports differences in spatial variation to response - some mice displayed enhanced fluorescence emission while others exhibited a decrease in fluorescence intensity - however, further investigations are needed to fully evaluate this technique for use in dosimetry studies [284]. The Lecomte group (Sherbrooke, Canada) observed (1999 and 2006) online changes in tumour metabolism using dynamic PET in rat and mouse mammary tumours [285, 286]. They were able to report dynamic changes in perfusion rates and metabolic activity in the pdt of tumours. PC-based photosensitisers were irradiated for 30 minutes or 2 hours during the PET protocol. Three distinct phases of cellular metabolism were identified as being related to therapeutic or biological factors. Within the first 3 minutes of a radiolabelled agent (fluoro-2-deoxy-D-glucose) being administered the imaging scans indicated tumour perfusion - the 3-15 minute time period was associated with the type of tumour death (direct cell killing or indirect cellular necrosis) occurring; tumour uptake after 15 minutes was correlated to cellular metabolism and reflected the efficacy of the photosensitiser [285, 286].

PET imaging is relatively new in pdt and is still in its infancy but shows promise in the ability to visualise real-time changes in treatment parameters allowing an optimal therapeutic outcome to be achieved. The technique can be used to image the whole body and has been used to detect hypermetabolic cancers; it has proved sensitive enough to detect cancers ranging from lung, breast, colorectal, pancreatic, head and neck and malignant lymphoma and melanoma, it has also been used in identifying unknown primary tumours in patients [28, 194]. The technique can provide quantitative drug biodistribution data - a parameter not achievable with optical imaging platforms [70]. Blood flow dynamics have been studied using fluorescence imaging, laser speckle imaging and Doppler optical computed tomography (OCT) in the prediction of vascular occlusions as a signal for photodynamic therapeutic end point [287-294]. Fluorescently-labelled antibodies (against endothelial cells CD32 and platelet aggregation CD41) in combination with a cell permeability/viability indicator have been used to image immediately before and post-6 minute illumination period of pdt performed with Verteporfin^® [295]. The ability to measure therapeutic signals in real-time during light application potentially offers the physician the ability to appropriately adjust the therapy during the treatment procedure [194]. In pdt, monitoring photosensitiser fluorescence may facilitate immediate changes to be made; whereas during PET real-time assessment cannot be made - fluor-2-deoxy-D-glucose's metabolism is a direct result of pdt and cell death may not be observed until hours or days after the treatment has finished [194].

Fluorescence imaging, PET, CT, MRI, angiography and OCT have all been used in the assessment of the therapeutic outcome of pdt [194]. The modalities image tissue before and after pdt for evidence of cell death - necrosis, apoptosis and blood vessel occlusion. MRI of the tissue has been based on contrast-enhancement MRI analysis of T₁-weighted and T₂-weighted relaxation times; apparent diffusion coefficient maps; and longitudinal changes in tumour volume [194]. There is a good spatial correlation between MRI-determined necrosis and histopathological analysis of tissue when images are obtained a short time after pdt - due to the onset of fibrosis there is little or no correlation when the images are taken after a longer period of time [194, 296-298]. Changes in vascular perfusion and permeability can be observed in as little time as 1 hour post-pdt; permeability changes are believed to be indicative of the success of pdt [299, 300]. Tumour necrosis and the absolute volume of the tumour can be monitored using radioisotopes with PET or autoradiography. Moore and colleagues reported that PET can follow tumour volume and surviving fractions with the same degree of accuracy as high-resolution MRI [301]. Angiography has routinely been used to monitor choroidal neovascularisation (CNV) from AMD in the clinic, most commonly with fluorescein but indocyanine green has been used; there have been suggestions that the texaphyrin LuTex^® may also be used [194, 302].

The drawbacks associated with fluorescence imaging have so far been limited to the depth at which it can penetrate tissue - a few hundred millimetres. For the detection of flat lesions this limitation presents no problems but restricts the use of the modality in imaging abnormal tissue at greater depths. This can be readily overcome by using a multimodal protocol whereby a second imaging platform, capable of observing structures deep inside tissue, such as OCT, is used to compliment fluorescence imaging. Imaging at deeper penetration depths may also be addressed using other non-invasive modalities including PET, MRI and multi-photon excitation (MPE) [28, 194].

MPE can be used for deep tissue imaging; visible light that results in fluorescence emission from a photosensitiser typically penetrates only a few hundred millimetres into tissue, restricting PSFD to superficial tumour identification [194]. One solution could be to use MPE where a molecule can be promoted into the same excited state as in single photon excitation through simultaneous absorption of two or more lower-energy photons, provided the sum of the energy equals the energy of the electronic transition states. Advantages of MPE in imaging in general have been reviewed and the application of MPE in pdt follows the same basic principle [303]. Lower energy near-IR photons are subject to less scattering in the tissue and have reduced tissue absorption indicating MPE can intrinsically probe deeper (centimetre depths) into a tissue sample in comparison to visible light, allowing 3-dimensional tumour margins to be precisely defined below the surface of the tissue. MPE-pdt has been investigated with HpD and Photofrin^® - a drawback is that the porphyrin photosensitisers that are used in the clinic have very low two-photon absorption cross-sections (10GM; 1GM = 1^-50cm⁴s/photon) and thus necessitate impractical light doses (6300Jcm²) to achieve a 50% cell kill [194, 304-306]. Research efforts for the application of MPE-PSFD and pdt are focusing on developing photosensitisers with enhanced two-photon excitation properties [194]. Alternatively, photosensitisers could be appended to chromophores with strong two-photon absorption properties and transfer the required energy via FRET [306-308]. MPE could potentially be used for deep measurements and 3-dimensional imaging of blood vessel oxygenation in healthy and diseased tissue. Photosensitiser triplet state lifetime (τ_T) measurements through delayed fluorescence alongside singlet oxygen phosphorescence could be used to give a more complete idea of photosensitiser dosimetry [194].

All clinically approved photosensitisers lack efficacy in the absence of molecular oxygen. Higher light does can lead to rapid consumption and depletion of local oxygen concentration; lower dose rates or fractionated light delivery is therefore more beneficial and may lead to superior treatments; this behaviour varies greatly between different photosensitisers and tissues [194]. Imaging molecular oxygen can be difficult, particularly in tumour tissue where high spatial and temporal accuracy are difficult to measure, pdt can also readily consume all available molecular oxygen if the optical fluence and photosensitiser concentration lead to high rates of molecular oxygen consumption; understanding the behaviour of tissue oxygenation from a spatial and temporal perspective may help improve the efficacy of pdt [194]. In large regions of tumours there can be a molecular oxygen pressure near to or at zero, in other regions this pressure can be quite high making the quantification of tissue oxygen concentration a difficult process to undertake [194]. While global oxygen measurements can be achieved with good repeatability (surface oxygen partial pressure (pO₂) sensors), measurement on the microscopic level where there is a transient pO₂, are much more difficult to undertake [309, 310]. OCT, an optical analogue of ultrasound, has found success as an imaging method for the online monitoring and assessment of pdt response in ophthalmology; the technique is very effective in diagnosing retinal detachment, multiple sclerosis-related eye disease, glaucoma and AMD (types I and II) [194]. OCT was first used to monitor the response of AMD and its associated neovasculature and is being studied as a tool for monitoring vascular pdt effects - pdt has been used as the first line of treatment (imaging) in over two million patients with AMD [194]. The technique typically uses light in the 850-1350nm region of the EMS; therefore OCT can be used to image tissue pre-, during, and post- pdt treatment. The contrast in OCT comes from the refractive index changes at reflective surfaces, the OCT system does not have molecular sensitivity; this hurdle can be overcome by using OCT in combination with other imaging modalities where rapid structural contrast is collected alongside fluorescence [194]. OCT can image in 3-dimensional by collecting individual images in the xy plane of the subject at different depths, making OCT volumetric imaging almost an order of magnitude faster than even video-microscopy. OCT can image beyond 1.5mm deep into tissue - a depth comparable to that of pdt-induced necrosis. The depth (axial) resolution is decoupled from the lateral resolution, allowing for subcellular millimetre resolution, hence imaging deep into a sample [194].

MRI is usually the method of choice for many imaging needs and relies on contrast-enhancing agents to improve the inherent contrast between healthy and diseased tissue; agents normally used contain paramagnetic ions - the most widely used ion is gadolinium (Gd(III)) - Gd(III)-based agents approved by the FDA are Magnavist^®, Omniscan^® and Prohance^® [7, 28]. The plasma clearance rates for these agents is typically of the order of 15 minutes or less; this limits the usable time frame for data acquisition in MRI which can take between 5 and 15 minutes [28]. Tumour selectivity of these agents is dependent upon perturbations in normal vascular permeability - small foci of metastatic tumour cells, particularly those in normal sized or minimally enlarged lymph nodes, may not be detected by current methods [28]. Improvements can be made by slowing down in vivo tumour clearance rates or by binding (selectively or non-selectively) the contrast-enhancement agents to a tumour target. The groups of Hoffman and Pandey have investigated one such solution - tumour-avid porphyrins; the gadophrin-2 conjugate has been widely studied and determined to be necrosis-avid [311]. Pandey's group (Roswell Park Cancer Institute, USA) have extensively investigated multimodal contrast agents based on photosensitiser conjugates [198, 312]. The conjugates can be utilised in pdt, as medical imaging contrast agents (PET or MRI), and/or in fluorescence imaging allowing tissue imaging (pre-, during and post-pdt) to assess treatment outcome. Work has been conducted to generate a contrast-enhancement agent which can target living tumour cells, particularly those that are actively undergoing metabolic processes [28]. One such conjugate comprises of a photosensitiser (3-vinyl-3-[1-(hexyoxy)ethyl]pyropheophorbide a (HPPH)) appended to a MRI contrast agent (Gd(III)-aminobenzyl-diethylenetriaminepentaacetic acid (DTPA)) via a tris(polyethylene glycol)monomethyl-ether linkage in a liposomal formulation. This conjugate enhanced tumour localisation (imaged by MRI) by a 10-fold lower dose than Magnevist^® while maintaining its pdt efficacy [28, 194]. A second agent developed by Pandey and his group is a PET-active photosensitiser achieved by labelling HPPH with ¹²⁴I which demonstrated 100% tumour-free progression 60 days after pdt [28, 194].

Radioimaging

Porphyrins are excellent metal chelators and very efficient at delivering radioisotopes, one example is a copper porphyrin-peptide-folate (⁶⁴Cu-PPF) compound [199]. This complex is a PET imaging agent targeted against folate receptor positive tumours and has been evaluated as a PET probe for cancer imaging. The ⁶⁴Cu-porphyrin is extremely stable and attempts at using it in tumour localisation studies were first reported in the late 1950's; the use of PET radioisotopes in identifying the localisation of brain tumours was reported in the early 1950's [313, 314]. Wilson and colleagues attempted to detect brain cancers using ⁶⁴Cu-porphyrins and PET imaging in the 1980's but had limited success due to poor tumour localisation properties and poor spatial resolution of the available PET systems [315, 316]. More recent studies by the Wilson group have focused on utilising their pyro-folate receptor targeting conjugate technology and incorporating ⁶⁴Cu into the porphyrin moiety [199]. Porphyrins are known to have a stable ⁶⁴Cu-chelating ability; ⁶⁴Cu-porphyrin has minimal toxicity; the half-life of the radioisotope and the porphyrins pharmacokinetics are compatible; and the chelation of the ⁶⁴Cu ion does not alter the biodistribution properties of the porphyrin [315]. Such a system is envisaged to become a novel-cancer-targeting PET imaging probe - facilitating the monitoring of porphyrin tumour uptake in patients undergoing pdt by pre-treatment PET imaging and facilitating the prediction and quantitative measurement of photosensitiser uptake/accumulation in tumours, hence aiding treatment planning and monitoring [199]. The same group have reported a multimodal bacteriochlorophyll theranostic probe - the optical properties of bacteriochlorophylls are significantly red-shifted, thus it is a very attractive agent for optical imaging (near-IR wavelengths provide deeper tissue penetration) [70]. Early bacteriochlorophyll pdt and imaging studies were limited due to the chromophores unstable nature, however TOOKAD^® has been developed and shown promise as a bifunctional imaging and pdt theranostic agent; it has been used as a pdt agent in the treatment of prostate cancer [70]. Wilson and colleagues extended their targeted PFF probe to create a multifunctional targeted bacteriochlorophyll probe suited to the imaging of deep-seated tumours [70]. They reported that a bacteriochlorophyll-peptide-folate moiety (BPF) better delineates tumour margins within the surgical resection bed, ensuring complete tumour resection and aids intra-operative detection of small metastatic lesions [70]. Copper can also increase the stability of bacteriochlorophyll molecules, therefore insertion of ⁶⁴Cu into the bacteriochlorophyll central cavity may further stabilise the chromophore, making ⁶⁴CuBPF an effective and highly stable PET probe. PET imaging studies have not demonstrated ideal tumour delineation, although some ⁶⁴Cu-BPF tumour accumulation was observed [70].

PET imaging with ¹⁸F-fluordeoxyglucose (FDG) has been developed to quantitatively assess local glucose metabolism - malignant tumours exhibit increased glucose metabolism, therefore, quantification of FDG uptake using PET may help to differentiate between benign and malignant tumours; determine the degree of malignancy; evaluate the effectiveness of chemo- or radio-therapy and predict treatment prognosis [317]. Drugs labelled with ¹¹C and ¹⁸F are the most commonly used PET isotopes, however, their short half-lives limits their use in studies involving MAbs and photosensitisers which have long accumulation times (hours), therefore ¹²⁴I has been deemed to be a more suitable candidate (t_½ - 4.2 days) [28]. There are only a few reports of pdt with various ¹⁸F based radiotracers (FDG, 9-(4-¹⁸F-fluoro-3-hydroxymethylbutyl) guanine (F-FHBG) and 3´-deoxy-3´-¹⁸F-fluorothymidine (FLT)) [301, 318-321]. Radiolabeling techniques with ¹²⁴I are well defined and complexes with good radiochemical purity and in good yields can be afforded; ¹²⁴I labelled photosensitisers have been utilised to monitor cellular events post-pdt in mice [322]. Pandey and colleagues have also studied ^99mTc complexes of their HPPH-photosensitiser system in rats bearing Ward colon tumours at 4 and 24 hours post-injection; it was observed that the ^99mTc half-life (6 hours) was incompatible with the HPPH (t_½ - 24 hours) accumulation time [323]. Suggestions have been made that the longer lived ¹¹¹In isotope may be more suitable (t_½ - 72 hours) [324]. The ¹¹¹In-HPPH analogue was reported to be more than 40-fold effective in vitro and 8-fold more effective in vivo with limited skin photosensitivity [324].

An alternative approach to increasing photosensitiser-tumour specificity is the use of nanotechnology - nanoparticles are particularly well-suited to incorporating multimodal theranostic entities (targeting, imaging and therapeutic), each with their own separate domain, into a single particle platform [194, 197]. One such targeted example by Reddy and colleagues has already been mentioned [254]. These nanosystems are not without drawbacks though - there are transitional issues such as transport barriers; drug uptake; and distribution hurdles that need addressing, along with studies evaluating their long-term effects.

Nanosized Carriers

Nanosized vehicles work on a similar principle to liposomes: they can be composed of an outer hydrophilic and inner hydrophobic region (capable of encapsulating hydrophobic photosensitisers within their inner matrix); thus, facilitating the transportation of photosensitisers in biological environments. They have been developed as an alternative carrier system to liposomes in an attempt to overcome a number of disadvantages associated with liposomal formulations: it has been suggested that only a small fraction of the chosen photosensitiser can be effectively incorporated into liposomes and that the limited lifetimes associated with liposomes results in their structural degradation over time [325]. It has also been hypothesised that the type of vehicle used in photosensitiser delivery can influence the distribution of the photosensitiser within the body's serum. Thus, the transport mechanisms and kinetics of the photosensitiser to the target tissue, as well as the subcellular distribution of the photosensitiser are affected. It would appear delivery via lipid-based vehicles shows enhanced binding of the porphyrin (photosensitiser) to the lipoproteins, in particular, orienting towards LDL, possibly resulting in the selective release of the photosensitiser into neoplastic tissue [14].

Nano-Delivery

There is concern however, that although the use of these nanosystems may increase the therapeutic effect observed as a result of photodynamic therapy, a number of the carrier systems may inadvertently decrease the singlet oxygen quantum yield of the encapsulated/associated photosensitiser [24]: the singlet oxygen generated by the photochemical reaction would have to diffuse out of the carrier system (singlet oxygen has a narrow radius of action), if the singlet oxygen has a reduced sphere of action it may not reach the target tissue and elicit the desired effect, thus reducing the therapeutic effect of the photosensitiser. Furthermore, if the size of the carrier system is not sufficiently small or does not fully dissolve/disperse in physiological media, the incidence/exciting light may not be effectively absorbed; the resultant light scattering may be significant, thus reducing the singlet oxygen yield.

Why Nanosized Vehicles?

Nanotechnology is a rapidly expanding and powerful discipline capable of measuring, manipulating and manufacturing materials at the atomic, molecular and supramolecular levels. This new field is bringing together scientists, engineers and physicians in an interdisciplinary manner to develop new platforms for novel complimentary imaging and diagnostic tools; thereby, propelling crucial work at the cellular and molecular levels, as well as mediating the creation of more effective therapeutic delivery platforms. Together these developments hold the potential to facilitate dramatic advances in biomedical sciences; the market alone for molecular imaging was estimated to be worth $45 billion (USA dollars) in the year 2010 [326-333]. Molecular imaging is beginning to emerge as a complimentary technique to structural and functional imaging; it allows a wider picture of the body's biological response to treatment to be captured. Molecular imaging can be used to elucidate biological mechanisms induced by pdt; imaging dynamic molecular events in vivo allows the real-time imaging of critical pdt parameters, including optimal photosensitiser delivery time, dosage and the appropriate delivery vehicle for molecular targeted therapeutic agents, therefore allowing the more effective combination of therapies in pdt, hence limiting the photosensitiser's associated intrinsic toxicity. It is also critical in studying tumour cells that have survived sub-lethal pdt and in determining if they are able to cope with further rounds of the therapy [194]. Imaging specific molecular disease targets should allow the earlier diagnosis and more thorough assessment of cancer patients [198].

Nanospecies (NS) of many different compositions and for a variety of applications have been described in the literature [66, 328-381]. These species are generally, but not exclusively, synthesised from polymers and due to their minute size are comparable to biological molecules [325, 347, 367-369]. Through the ability to fine-tune both their physical and chemical properties, a range of versatile NS can be synthesised with varying sizes, compositions and surface chemistries [66, 67, 68, 326, 332, 334, 336, 342, 344, 346, 347, 349, 356, 364, 368, 382-387]. The ability to create species with nanometre dimensions brings with it the potential to bring about life-changing advances in both science and medicine. The design and synthesis of novel NS can have a broad and dramatic effect in the fields of: biological sensing/detection/imaging; targeted diagnostics/delivery; drug delivery/disease therapy; forensic science; and in the field of engineering [66, 67, 68, 325, 326, 329-332, 336-338, 340, 344-347, 349, 351, 353, 355-357, 361-367, 380, 382, 383, 387, 389-400]. The potential to increase the knowledgebase around intricate and complex cellular mechanisms offers huge promise, particularly in the field of disease states. By gaining direct information on intracellular trafficking and molecular functions it may be possible to gain a better understanding of disease mechanisms and therefore, tailor disease management to treat the cause, rather than the symptoms, of a disease. An example of the application of NS in disease therapy is the concept of nano-sized carriers; this type of system offers the ability to more efficiently deliver therapeutic agents to the disease site: thereby, minimising the required administration dose; systemic side effects; and the toxicity commonly associated with drugs in their free state. NS of this type have been increasingly sought in the management of cancers (especially in conjunction with pdt) and for the delivery of gene-therapy agents. Typically, such strategies have been based on those investigated for photosensitiser-targeting moieties described above. Magnetic NS have also been developed and shown potential in facilitating the detection and treatment of some diseases. They have been linked to: the in vivo detection of cancer; drug delivery; for use in the photodynamic treatment of cancer; and in the delivery of insulin [332, 347, 353, 380]. Magnetic NS can act as imaging contrast (MRI) agents to: enhance the detection of diseased tissue; in targeting strategies; and in the near-IR management of tumours - reports suggest a nearly fifty percent increase in the magnetic susceptibility of these systems compared to the gadolinium chelates approved for MRI [197, 332, 333, 348, 349, 353, 355, 364, 380, 381, 383, 384, 390, 394, 398]. Furthermore, NS have recently shown some promise in the field of forensic science [400].

Synthetic Materials And Techniques

The ability to synthesise NS from a range of materials and techniques is particularly important when considering the end application and, if applicable, the route of species administration. It is desirable in imaging and therapeutic studies to control and limit the cellular uptake and intracellular localisation of the NS used. One way of achieving this is to selectively direct NS to the specific area/site of interest. This can be achieved by chemically modifying the surface of NS in a number of ways; namely, through the incorporation of a range of functional groups [66]. For example, by conjugating the NS to a target-specific molecule (such as an antibody), the system can be selectively directed towards sites of interest. Such systems can offer reduced side effects from drug delivery and surgical procedures, making them suitable for a range of different biomedical situations. Due to their size NS can be dispersed in aqueous media [66]; show low toxicity; exhibit prolonged circulation in the blood system; are biodegradable (days to months); can be readily modified to incorporate surface functionality; encapsulate various guest molecules; their size can be varied with relative ease; and polylactic acid; polyglycolic acid; and poly (lactide-co-glycolide) have been established for use in humans [325]. The types of functional groups that can be incorporated onto the NS surface are numerous and include hydroxyl (OH), sulphonate (SO₃^-), carboxylic acid (COOH) and amino (NH₂) groups, as well as bio-reactive groups. Some NS have been reported to selectively localise within specific subcellular compartments/areas, while others appear to show no specificity for one intracellular site over another. NS internalisation can be enhanced by targeting them against internalising receptors, such as a CD19 surface antigen which is expressed on a β-cell lymphoma cell-line and a folate receptor (folate-binding protein). Localisation in a specific intracellular region/subcellular compartment can be particularly important with nanosensors fabricated for studying intracellular signaling mechanisms or imaging [60]. Localisation can also be important in drug delivery systems. Tumours express a greater degree of specific ligands at their surface and nanosystems which are selectively targeted towards these ligands have been shown to accumulate at higher concentrations at the periphery of solid tumours (known as the “binding site barrier”) rather than at deeper depths; therefore, minimising diffusion of the NS into the deeper tumour regions. This effect can have a significant impact on the management of tumours and on the initial administration concentration of the drug-loaded vehicle. It is also important to bear in mind the heterogeneous nature of the tumours; certain regions of the tumour will have a greater number of ligand receptors, whilst other areas may not express these receptors at all.

Theranostics - A 21^st Century Biomedical Imaging And Therapeutic Platform

The term theranostic refers to the dual diagnostic and therapeutic utility of a compound or methodology and was first used by Funkhouser in 2002 [401]. Theranostics entities simultaneously deliver imaging and therapeutic agents within the same “dose”; the functionality of the system can be readily “switched” between imaging (diagnostic) and therapeutic platforms, facilitating the improved detection and treatment of disease. These novel entities can further offer the potential of site-specific biomedical imaging and treatment via targeted delivery. To date, theranostic research has predominantly focused on and been evaluated (in vivo) against cancer and atherosclerosis disease types.

History Of Theranostics

The research and development of third- and fourth-generation photosensitisers highlighted the potential a single multimodal (imaging and therapeutic) platform could have in the detection and treatment of disease - such systems negate the need for multiple agents to be administered individually, circumventing variability issues in the uptake; localisation; and pharmacokinetic and pharmacodynamic behaviour of the imaging/therapeutic agents in their free-states - any toxicity associated with the agents in their free-states is also lost. The intracellular biodistribution issues when non-targeted multiple entities are administered have already been noted. DIF (discussed earlier) used for the guidance of surgical resection of cancerous tissue and in setting and adjusting parameters for and during pdt is an example of a theranostic therapy successfully used in the clinic. Being able to “switch” between an imaging state and a therapeutic/cytotoxic state in a single modality lends itself particularly well to photosensitisers and imaging modalities such as fluorescence-, magnetic resonance-, radio- and nuclear imaging and treatment modalities such as photodynamic, photothermal and photoacoustic therapies, as well as hyperthermia, gene and nuclear- and chemo-therapy [402]. There are a number of platforms that are particularly suited to bringing imaging and therapeutic agents together in a single entity; these include nanoagents (nanobiayls, magnetofluorescent-, mesoporous- and photon-up-converting nanoparticles (NPs), carbon nanotubes, quantum dots (QDs) and liposome-like porphysomes - each platform with its own unique properties, advantages and disadvantages [403-411].

Nanoagents As Theranostic Platforms

The physical properties (including small size (for efficient cell uptake) and facile surface functionalisation (for appending targeting moieties)) of NPs make them attractive candidates for theranostic applications, particularly high surface-to-volume ratios (high loading potentials) and the ability to encapsulate individual entities together in the same protected environment, thereby minimalising cellular immunostimulation and toxicity of the entities in their free states towards cells. Furthermore, the ability to increase the target-to-background contrast for/in imaging applications and the local concentration of the therapeutic agent at the target site (ultimately reducing systemic toxicity) are extremely versatile properties with respect to structural modifications and the type of cargo the nanoagents can be loaded with.

A range of nanoagents are currently being studied as theranostic platforms and include nanobiayls; macrophage-avid- and mesoporous silica NPs; carbon nanotubes; and novel porphysome vesicles [403, 404, 407-409, 411]. Kelkar and Reineke have thoroughly reviewed the developments of a broad range of theranostic agents with applications in the fields of pdt, chemo-, gene-, hyperthermia and radiation therapy and MRI, nuclear and near-IR imaging strategies [402].

Nanobiayls

Theranostic gadolinium-based perfluorocarbon NPs have been developed and successfully evaluated against detecting, characterising, treating and monitoring angiogenesis in pre-clinical models of cancer and atherosclerosis [403]. However, a serious and unexpected side effect (nephrogenic systemic fibrosis (NSF)) of gadolinium blood pool agents has recently been observed amongst a number of renal and liver-transplant patients [412]. NSF is a serious complication which causes patients to develop thickening of the skin and connective tissue, and in some cases, bone fractures. NSF has been reported to progress rapidly, with reports that it can be fatal in approximately 5% of sufferers [412]. It is therefore important to consider alternative paramagnetic contrast agents for use in MRI. One of the first alternatives investigated was the lanthanide metal manganese. Manganese was chosen for its favourable efficiency in R₁ enhancement and because it is a natural cellular constituent, often a co-factor for enzymes and receptors [403, 413]. Manganese blood pool agents, such as mangafodipir trisodium, have been approved as hepatocyte-specific contrast agents with transient side-effects due to dechelation of the manganese ion from the linear chelate. Release of manganese (caused by disruption of the vesicles) allowed MR detection of sites where vesicles such as MnSO₄^3c or Mn-DPTA were non-specifically entrapped [403]. Pan and colleagues have developed theranostic targeted Mn(III)-labelled nanobiayls based on the novel Mn(III)-protoporphyrin chloride contrast agent and chemotherapeutic drugs (doxorubicin and camptotherin) for the imaging and treatment of vascular targets [403]. The nanobiayl entities are produced from the molecular self-assembly of amphiphilic branched polyethylenimine, forming inverse micelles and taking on a torodial (do-nut, “biayl”) shape; the nanobiayls have desirable tunable particle sizes and low polydispersity indices [403]. The biayl geometry drives the increased stability of the nanobiayl and presents kinetically stable, porphyrin-coupled Mn(III) complexes directly to the surrounding water environment. Pan and colleagues evaluated (in vitro) the imaging component of the nanobiayls against fibrin (a critical component of intravascular thrombosis) by targeting them towards (avidin-biotin interactions and MAbs (biotinylated fibrin-specific, NIB5F3). The resultant MR images of the clot samples showed marked contrast enhancement of the fibrin-targeted Mn(III)-nanobiayls, with no contrast improvements from the two control experiments (non-target and metal-free nanobiayls) [403]. Pan and co-workers further evaluated (in vitro) the potential of their nanobiayls as drug carrier vehicles, with the hydrophilic and hydrophobic drugs doxorubicin and camptotherin, respectively. Results demonstrated loading efficiencies of approximately 98% and 99% and drug release values of 12% and 20% over three days, supporting the group's hypothesis that nanobiayls offer great potential as biomedical theranostic platforms [403].

Macrophage-Targeted Theranostic Nanoparticles

Atherosclerotic vascular disease (myocardial infarctions and cerebrovascular accidents) is a leading cause of death worldwide. Atherogenesis is an insidious process that occurs over many years, hence there is a vital need to diagnose and treat sub-clinical and high risk atherosclerotic plaques prior to their sub-clinical manifestations [414]. Macrophages have been identified as key biological, imaging and therapeutic targets in atherosclerosis [404, 414-418]. Research has begun into the investigation of anti-macrophage therapies, including the systemic modulation of macrophage activity and localised pdt, the latter involves de-bulking of atherosclerotic plaques via angioplasty [404, 419]. The benefit of using pdt is the localised and selective destruction of the target area. A number of photosensitisers have demonstrated preferential uptake in atherosclerotic plaques, purported to be a result of the interactions between the relatively hydrophobic nature of the porphyrin and the lipid-rich core of the atheroma [420-426]; the most studied photosensitiser Antrin^® is currently in clinical trials against coronary artery disease [427, 428]. McCarthy and colleagues hypothesised that by building on the already existing field of photosensitiser-NP pdt agents, they could increase the efficacy of pdt by conjugating photosensitisers to macrophage-avid magnetofluorescent NPs [342, 350, 429-431]. McCarthy et al. had previously reported the enhanced uptake (in vivo) of a dextran-coated magnetofluorescent NP in atherosclerotic plaques, in addition to specificity of the agent for macrophages [405, 406, 432]. McCarthy and colleagues further identified the need for the magnetofluorescent NPs to be detectable by MRI and catheter-based fluorescence imaging - achieved by incorporating a magnetic core (iron oxide) and fluorescent dye (Alexa Fluor^® 750) respectively within the NP [404]. The potent clorin photosensitiser 5-(4-carboxyphenyl)-10,15,20-triphenyl-2,3-dihydroxychlorin (F_D 0.65) was appended to the surface of the iron oxide NP. There was approximately 100nm difference between the longest wavelength of absorption of the photosensitiser and Alexa Fluor^®, minimising the energy transfer between the two chromophores when the theranostic agent was excited at the therapeutic wavelength (650nm); hence minimising cell death when the intracellular distribution of the theranostic agent was being imaged. The theranostic magnetofluorescent NPs were detectable by MRI and fluorescence imaging and did not demonstrate significant dermal-localisation. The evaluation (in vitro) of the agent demonstrated that when murine macrophage cells (RAW 264.7) were illuminated at 750nm, under conditions comparable to intravital fluorescence imaging of atherosclerosis (0.8mW total power, scanning laser, 512 x 512 pixels, 3min 45s acquisition), minimal cell death was observed. The theranostic NPs were observed to be no more toxic to macrophage cells than the control photosensitiser-free NPs, however, when the system was therapeutically irradiated, dose-dependent phototoxicity was observed - complete cell killing was achieved in transformed human macrophage (U937) cells 1 hour post-incubation and irradiation (0.2mg Fe per mL, irradiation with 646nm laser) [404].

McCarthy and colleagues report that their system should enhance the pdt of various diseases including atherosclerosis and in-stent restenosis and have significant in vivo applicability with high macrophage uptake; efficient cell killing; safe monitoring of the spatial distribution of the agent in vivo by MR; and near-IR fluorescence imaging [404].

Mesoporous Silica Nanoparticles

Ohulchanskyy et al. reported the synthesis of a novel silica-based (ORMOSIL) NP which covalently incorporated a photosensitiser (iodobenzylpyropheophorbide) for the pdt of colon cancer in 2007 [433]. Zhang and colleagues also reported the use of silica in the formulation of silica-coated NPs (photon up-converting NPs) in 2007 for the theranostic imaging and pdt of breast cancer cells with the non-porphyrinic photosensitiser merocyanine 540 [408, 434]. More recently (2009), Cheng et al. have reported the synthesis of mesoporous silica (MPS) NPs comprised of highly-ordered, hexagonal pores with mean particle diameters of 70-100nm. These NPs are readily modified and taken up by breast cancer cells; offer payload protection; and low cytotoxicity towards cells. To date, MPS NPs have been functionalised for use in chemical catalysis, drug delivery, cell labelling and the controlled release of therapeutics [435-445].

The large surface areas offered by MSP NPs enable the high-loading concentration of photosensitisers per particle and the efficient delivery of large concentrations of photosensitisers, thereby reducing the energy of photoirradiation needed to achieve efficient oxidative cytotoxicity - avid cell-uptake enables significant intracellular accumulation of photosensitiser for optimal pdt [407]. Cheng and colleagues appended a phosphorescent Pd-porphyrin photosensitiser onto the nanochannel surface of a MSP NP. Pd-TPP is conventionally used for oxygen sensing and imaging - tissue oxygen distribution is measured via oxygen-dependent quenching of phosphorescence [446, 447] - the long-lived triplet state of Pd-TPP is produced with a unity quantum yield, while the concentration of singlet oxygen generated in pdt depends on the accessibility of the excited Pd-TPP to oxygen. The energy of photoirradiation needed in phosphorescence imaging is typically 10^-4 to 10^-5 times that used in pdt [446, 448], therefore the concentration of singlet oxygen generated via Pd-TPP during oxygen sensing/imaging from phosphorescence quenching is much less than that produced during pdt. Hence, when Pd-TPP is irradiated with respect to imaging energy requirements, the singlet oxygen produced has a negligible therapeutic effect. This can be readily changed by simply altering the energy parameters used to irradiate the chromophore, thus the Pd-TPP can readily be “switched” from being a phosphorescent oxygen sensing/imaging probe to a pdt agent. Cheng et al. chose MSP NPs for their facile uptake into cells via endocytosis and reported that breast cancer cells (MDA-MB-231) transfected with their pdt-MSP NPs underwent significant morphological changes and cell death post-irradiation (532nm, 1.2Jcm^-2) [407]. Cheng and colleagues hypothesise that their multimodal Pd-TPP-MSP NP is a promising platform for cancer theranostics.

Carbon Nanotubes

Advances in photoacoustic imaging and photothermal therapy techniques based on the non-radiative conversion of absorbed energy in thermal and accompanying acoustic phenomena has demonstrated great potential in various spectroscopic sensing/imaging and therapeutic applications [449]. Endogenous biomolecules and exogenous contrast agents have successfully been used in photoacoustic imaging and photothermal therapies. The exogenous contrast agents have even based on small molecule optical dyes, such as Evans blue, indocyanin green and MB, and synthetic NPs, including gold and carbon-based ones [409]; NPs offer advantages over biomolecules since they typically exhibit strong optical absorption and photostability properties [409]. Carbon nanotubes (CNTs) have demonstrated numerous biomedical applications over the years as single-walled and multi-walled nanotubes (SWNT and MWNT, respectively) [409]. The ability to absorb light over a broad range of wavelengths (UV, visible, near-IR and microwave) lends CNTs as natural contrast agents for photoacoustic and photothermal techniques [409]. Zharov and colleagues were the first to demonstrate (in vitro and in vivo) the use of CNTs as photoacoustic contrast agents in 2007. The group were able to detect circulating CNTs alone or via circulating Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) labelled with CNTs in flowing blood using a novel photoacoustic/photothermal flow cytometry system [450]. They also reported the application of two-colour photoacoustic flow cytometry (in real-time) for in vivo identification of CNTs in lymph flow [451]. de la Zerda and colleagues went on to image (photoacoustically) CNTs by molecularly targeting (using arginine-glycine-aspartic acid peptides) SWNTs to tumour neovasculature in living mice [452]. Research has continued into the use of CNTs for photoacoustic imaging, with a number of groups focusing on chemical modifications to enhance CNT photoacoustic signals [409]; the modifications include coating CNTs with organic optical dyes; gold with folates and antibodies for the molecular targeting of circulating tumour cells (CTC); and endothelial lymphatic receptors with potential lower toxicity or antibodies for targeting primary tumours [453-456]. About 90% of cancer deaths result from metastatic spread from the primary tumour; detection of CTCs is therefore a marker of metastases development and the efficiency of any therapy delivered. The sensitivity of the majority of current CTC assays is limited; de la Zerda and colleagues believe they can increase the sensitivity of detection by assessing large volumes of blood in vivo [409]. Additional applications of CNTs include the photoacoustic detection of metastasis in sentinel lymph nodes and the identification of tumour-initiating cancer stem cells in circulation among bulk CTCs [455, 457, 458]. Photoacoustic and photothermal microscopy has been reported to demonstrate promise in studying the distribution of CNTs in histological samples [459].

The photoacoustic and photothermal effects of CNTs can be transferred into therapeutic use, including pulsed photothermal nanotherapy (nanophotothermolysis) with laser-induced nano- and micro-bubbles around overheated gold NPs, leading to tumour (and bacterial) cell death, as well as for targeting bones, atherosclerotic plaques, blood lymph vessels and thrombus [458, 460-468]. The pulsed-mode in laser treatment has the potential to precisely kill individual cancer cells with a spatial accuracy of a few millimetres, sparing healthy cells, unlike continuous-wave photothermal treatment [348, 366, 460, 469]. Photoacoustic imaging and photothermal therapeutic modalities are based on similar thermal effects, enabling their facile integration into a multimodal/functional theranostic system and facilitating the navigation, optimisation and monitoring efficiency of photothermal nanotherapy [409]. The photoacoustic and photothermal effects of de la Zerda et al's. nanosystem can be amplified and the spectral peaks sharpened, increasing the sensitivity, specificity and capability of multiplex photothermal therapy by targeting multiple NPs with different optical spectra to different disease-associated biomarkers, thereby potentially offering personalised medicine [454, 470].

Several techniques have been developed to synthesis CNTs, including high-pressure carbon monoxide, chemical vapour deposition, arc discharge and laser ablation methods [471-474]. All of the atoms in SWNTs are exposed on the entities surface, thus generating very high surface areas (theoretically approximately 1300m²g^-1), facilitating efficient SWNT-optical radiation interactions [409]. SWNTs can be characterised by very broad and flat optical absorption spectra, spanning the UV, visible and near-IR regions of the EMS. SWNTs absorb the majority interacting photons, resulting in local heating of the NTs surrounding environment [450, 455, 465]. When an intense nanosecond laser pulse light is used as the incident light, the local heating leads to the emission of an ultrasound wave (the Photoacoustic Effect) [450-457, 475-478]. Semi-conducting SWNTs with small band gaps (approximately 1eV) exhibit fluorescence properties with excitation and emission wavelengths in the near-IR range [409]; different excitation-emission wavelengths can be achieved with different SWNT chiralities [479]. The majority of light that interacts with SWNTs is absorbed but a small portion of the photons are scattered, leading to a distinctive Raman scattering spectrum - these unique spectra can then be used for (in vivo) detection and imaging [480-484]. The strong optical absorption characteristics of MWNTs also makes them attractive photoacoustic and photothermal contrast agents [465]; although MWNTs have pure optical absorption, they do not have the rich optical characteristics of SWNTs [409]. Drawbacks of CNTs are their relatively low near-IR absorption coefficients (with respect to other nanoagents such as goldNPs) and the high concentration of CNTs needed for effective photoacoustic and photothermal theranostic applications [409].

Functionalisation of CNTs is important, not only to increase their specificity for their chosen targets but CNTs in their “raw” state have hydrophobic surfaces and hence are not soluble in aqueous media (such as blood) [409]. Covalent and non-covalent surface functionalisation of CNTs has been reported in the literature [409]. Since “raw” CNTs are inert entities, covalent functionalisation typically involves creating defects in the sidewalls of the NT via oxidation [485-486]. However, this does not fully address the hydrophobicity issue as the oxidised CNTs aggregate in the presence of salts; remedies such PEGylation are being explored [487-488]. A disadvantage of covalent bonds is their interference with the natural physical properties (Raman scattering and photoluminescence) of CNTs, something not apparent with non-covalent functionalisation [409]. In non-covalent functionalisation the surface the CNT is coated with amphiphilic surfactant molecules or polymers so that the hydrophobic surface of the NT is non-covalently attached to the hydrophobic tail of the surfactant. A number of investigations have utilised this π-π interaction to non-covalently bind porphyrins and pyrenes to NT surfaces [489-492]. SWNTs have also been radiolabelled with ⁶⁴Cu (for imaging tumour angiogenesis) [493]. Successfully attaching and evaluating porphyrinic-type molecules to CNTs has demonstrated the theranostic biomedical utility of these nanoagents. The CNTs and their hybrids (with increased photoacoustic and photothermal contrasts) have been evaluated for nanomedicine-related theranostics by de la Zerda and colleagues [409]; the CNT-based contrasts agents facilitated minimally invasive, highly sensitive and target-specific multiplex detection and killing of solid and metastatic tumour cells and bacterial infections in static and dynamic conditions (for example blood and lymph flow) that had previously been difficult to accomplish with traditional methods [409].

Porphysomes

An alternative organic theranostic agent is based on the novel organic self-assembled porphyrin nanovesicles, known as porphysomes; these multimodal biophotonic contrast agents have been developed by Lovell and colleagues in response to the limitations (drug-loading limited to NP surface and concerns relating to long-term safety of the inorganic modalities) posed by optically active inorganic NPs [411, 494-497]. The biocompatibility and high-loading capacity of optically active organic NPs, including liposomes, micelles and polymersomes, have been evaluated in many human theranostic applications [497]. However, these organic agents are not without their own limitations - theranostic platforms in biophotonic techniques need to intrinsically absorb light in the near-IR region of the EMS. Lovell and colleagues have investigated and evaluated porphyrin conjugates and novel biophotonic tools utilising the unique light-absorbing properties of the porphyrin chromophore. Due to the lack of stability and solubility associated with many tetrapyrroles, Lovell and colleagues fabricated liposome-like NPs self-assembled from phospholipid-porphyrin conjugates (termed porphysomes) with good loading capacities; high absorption of near-IR light; structure dependent fluorescence quenching; and excellent biocompatibility - all features lending themselves to creating a biophotonic modality with diverse ranging applications [411].

The porphysome vesicles (100nm) were formulated by supramolecular self-assembly with the sub-units composed of porphyrin-lipid conjugates generated via an acylation reaction between lysophosphatidylcholine and pyropheophorbide. The hydrophobic chromophore was substituted in place of an alkyl side chain - maintaining the amphiphilic properties of the structure. The spherical conjugate then underwent self-assembly in aqueous media with extrusion to form the porphysome vesicles. TEM images revealed that the porphysome structure was comprised of two separate monolayers of material separated a 2nm gap. The porphysome vesicle exhibited two absorption peaks (400nm and 680nm (near-IR)). Lovell and co-workers also formulated red-shifted porphysomes (760nm) utilising sub-units of a bacteriochlorophyll analogue [411]. A further porphysome, with shifted optical density bands (440nm and 670nm), was generated by inserting metal ions into the porphyrin-lipid structure, demonstrating the ability of porphysomes to form metal-chelating bilayers [411]. The ability to readily alter the photochemistry of the porphysome vesicles offers an imaging platform with potential in situations where specific operating wavelengths are required - such as when the operating wavelength(s) of given laser sources are required to be matched [411].

Porphysomes are highly self-quenched - energy that is normally dissipated to fluorescence and singlet oxygen formation is disseminated elsewhere. Upon exposure to laser illumination, thermal energy was released with an efficiency comparable to gold NPs (photothermally active inorganic NPs) - irradiation of standard liposomes generated no significant increase in the temperature of the solution [411]. Since photoacoustic signal generation is related to thermal expansion, porphysomes also generated photoacoustic signals proportional to concentration and detectable as low as 25pM (note, detection in this range was reported by Lovell et al. to be non-linear) [411]. Lovell and colleagues further reported that when a detergent is added to a solution of porphysomes (in order to disrupt the vesicles structure), an (unexpected) increase in absorption was observed while the photoacoustic signal decreased six-fold. In a control experiment with MB (a clinically used contrast agent), detergent was deemed not to have an effect on the photoacoustic signal of the dye, suggesting that the structurally-based self-quenching of porphysomes is requisite for nanoscale photoacoustic properties - a phenomenon of photoacoustic signal attenuation on detergent-induced porphysome dissociation [411].

Lovell and colleagues evaluated the capability of their nanovesicles to act as tomographic agents in vivo and observed that the local lymphatic system (of rats) become clearly detectable (photoacoustically) within 15 minutes of post-intradermal injection of porphysome vesicles - as the porphysomes drained to the lymph vessels and nodes they exhibited a strong photoacoustic signal, facilitating the visualisation of the first draining lymph node, the in-flowing lymph vessel and surrounding lymph vessels; the photoacoustic investigations (6.5ns pulse width, 10Hz laser) did not generate sufficient heat to cause damage to the surrounding tissue [411]. The fluorescence imaging capabilities of the system were assessed in mice bearing KB cell xenografts. Initially, a low fluorescence signal was observed 15 minutes post-injection, demonstrating the self-quenching of porphysomes in vivo, however, high tumour fluorescence was observed after two days as the porphysome vesicles accumulated in the tumour (the porphysomes used for fluorescence imaging were appended with a 1 molar % folate-PEG-lipid moiety for specific tumour uptake) and became unquenched [411]. The notion of porphysome quenching in vivo was more markedly illustrated when Lovell and colleagues injected detergent-disrupted porphysomes into mice and reported observing higher initial fluorescence. Lovell and co-workers concluded that their observations and findings supported their hypothesis that porphysomes are intrinsically multimodal for both photoacoustic tomography and low-background fluorescence imaging. The group also assessed the appropriate factors relevant to clinical application of their porphysomes [411]. Porphysomes had been observed to be stable for months in aqueous media but demonstrated susceptibility to enzymatic degradation. On incubation with detergents and lipase the phospholipid structure of the porphysome was cleaved; the main aromatic product was pyropheophorbide, the starting material for the porphyrin-lipid porphysome vesicle [411]. Similar to chlorophyll, pyropheophorbide is known to be enzymatically cleaved into colourless pyrroles when incubated with peroxidase and hydrogen peroxide [498]. The degradation of porphysomes was verified by Lovell and colleagues: they monitored the loss of porphyrin absorption and confirmed pyropheophorbide could be efficiently degraded by peroxidase; the group concluded that, to their knowledge, the porphysomes they had fabricated were the first examples of an enzymatically biodegradable, intrinsically optically active NP [411].

In the thorough assessment of the porphysome vesicles Lovell and co-workers carried out a preliminary study to assess the potential toxicity of the vesicles and reported that mice treated with a high dose (1000mgkg^-1) of porphysomes remained healthy over a two week period [411]. Blood tests carried out post-euthanasia suggested that the hepatic function of the mice was generally normal (with the exception of elevated bile acid and alanine transferase levels less than twice the upper range of normal). Red blood cell counts and attributes were reportedly unaffected; the porphysome did not interfere with the physiological regulation of endogenous porphyrin (haem). The reported unaffected white blood cell count suggested that the porphysome vesicles were not immunogenic at the two week time point. Post-mortem histopathological examination of the liver, spleen and kidneys indicated good health of the respective organs [411].

Recognising the utility and capability of the large aqueous porphysome core towards cargo-loading and delivery and therefore as a theranostic platform, Lovell and colleagues investigated loading the potent chemotherapeutic drug doxorubicin into the porphysome core [411]. The group determined an active-loading technique (ammonium sulphate gradient method) coupled with cholesterol (50% molar ratio, cholesterol is known to enhance the loading of compounds within phosphatidylcholine-based liposomes) achieved 90% loading of the anti-cancer drug into the porphysome core [411, 499]. The porphysomes maintained a self-quenching porphyrin bilayer. Lovell and colleagues demonstrated the biophotonic potential of their organic nanovesicles investigating them as agents for photothermal therapy. The group reported that porphysomes containing 30 molar % of cholesterol demonstrated favourable biodistribution following their systemic administration, with greater accumulation in the tumour tissue (KB tumour-bearing mice) and less accumulation in the liver, spleen and kidneys than observed with standard porphysomes. The tumour was irradiated for 1 minute post-24 hour administrated (injection) of the porphysomes and the tumour temperature monitored (via thermal camera); the tumour temperature rapidly rose to 60°C; 20°C higher than the control mice where the tumours were injected with PBS. Following treatment, mice in both groups developed eschars on the tumours; the laser-alone and porphysome-alone control groups did not. The eschars healed after two weeks and the tumours in the porphysome-laser treated (photothermal) group were reportedly destroyed. Tumours in the control group of mice continued to grow rapidly [411].

Lovell and colleagues have been able to develop and successfully evaluate a porphyrin-based nanovesicle that generates large tunable extinction coefficients, structure-dependent fluorescence self-quenching and unique photoacoustic and photothermal properties, facilitating the sensitive visualisation of lymphatic systems; the near-IR fluorescence of low-background fluorescence imaging; and following active-loading and accumulation in tumour cells, the photothermal ablation of tumours, thus demonstrating the highly promising biomedical potential of their theranostic agent [411].

Porphyrinic-Based Photodynamic Inactivation Of Bacteria

Photosensitisers are also emerging as key therapeutic agents in other pathological areas too, most notably as antimicrobial agents. There is an increasing prevalence of antibacterial resistance among a number of pathogenic bacteria strains despite the advancement of antibiotics; among the most reported multidrug resistant bacterial strains are methicillin, mupirocin and vancomycin, hence the urgent need for alternative antibacterial therapies [500, 501]. One such therapy that is showing great promise is photodynamic inactivation (PDI), also referred to as photoantimicrobial chemotherapy (PACT). PACT is similar to pdt in that a photosensitising agent, light and molecular oxygen are required to elicit an appropriate cytotoxic and thus photoinactivation process. To date, PACT has shown promise against antibiotic-resistant and antibiotic sensitive strains of methicillin-resistant S. aureus (MRSA) (strains of S. aureus are often implicated in cases of superficial and serious skin and soft tissue infections as well as infecting burns and other wounds) and has the potential to be used topically as a treatment modality for chronically infected wounds [502-515]. Biotargeting of photosensitisers in PACT has the ability to offer enhanced photodynamic tissue/cell killing in line with the increased selectivity offered by photosensitiser-bioconjugates in traditional pdt. Such photosensitiser bioconjugates have been prepared utilising the targeting ability of antibodies, a cell-penetrating peptide (TAT) and bacteriophages have been raised for PACT against S. aureus, MRSA, Pseudomonas aeruginosa (P. aeruginosa), E. coli and Porphyromonas gingivalis [511, 512]. PACT has a number of advantages over traditional antibiotics (i) PACT has a broad spectrum of action and has shown efficacy in the inactivation of many antibiotic resistant strains of pathogens (ROS are toxic to nearly all bacteria if sufficient ROS-induced damage is inflicted); (ii) PACT offers low mutagenic potential since it has been shown that bacteria cannot readily develop resistance to ROS; (iii) PACT has demonstrated greater photocytotoxicity against bacteria than mammalian cells, allowing the selective destruction of bacteria. PACT is showing potential towards local microbial infections such as skin associated bacteria; bacteria present in periodontal pockets; and the oral cavity. A number of agents have been prepared based on PACT for the treatment of acne vulgaris and infected leg ulcers [69, 500]. The NCS porphyrin developed by Sutton et al. has been used in the preparation of a PACT agent selective towards MRSA [501]. The NCS photosensitiser was coupled to a purified recombinant protein and evaluated in vitro for specific anti-MRSA cytotoxic potential [501]. Observation suggested a 66% reduction in MRSA growth in comparison with non-irradiated cells, highlighting the potential of PACT as a therapeutic model. Other photosensitiser conjugates have been prepared and evaluated. Biolitec AG has marketed a liposomal formulation of Temoporfin^® under the registered name Fospeg^®. When evaluated against S. aureus (in PACT) Fospeg^® has proven to be effective in inducing phototoxic cell death [69]. Bombelli et al. have also developed a Temoporfin^®-loaded cationic liposome with comparable bactericidal activity to the free photosensitiser [516]. Antimicrobial peptides (AMPs) are a further example of bacteria-targeting agents being investigated [69]. The cationic nature of AMPs facilitates their interaction with negatively charged lipids in the bacterial membranes of both Gram-positive and Gram-negative bacteria, while amphiphilic properties allow penetration into bacterial membranes and the formation of membrane pores, culminating in damage to the integrity of the bacteria [69]. Yang et al. have recently reported the preparation of a novel bacteria-targeting liposomal formulation utilising such targeting moieties and evaluating the conjugate against MRSA resistant (S. aureus, Gram-positive) and P. aeruginosa (Gram-negative) pathogens [69]. They were able to observe greater uptake of the targeted AMP-Temoporfin^® liposome in bacteria (in vitro) than in comparison to the unmodified liposome and the eradication of MRSA, however, to date, only a reduction of P. aeruginosa has been achievable under their investigative conditions [69]. There has been concern that some photosensitisers evaluated for PACT may have an adverse effect on neutrophils. Tanaka et al. have highlighted that optimal photosensitisers for use in PACT should destroy the target bacteria but spare neutrophils - the white blood cells that are paramount in fighting disease [517]. In the treatment of localised microbial infections PACT causes direct pathogen death but also affects host neutrophils that have been stimulated by PACT. In order to enhance the antimicrobial therapeutic efficacy of PACT, cytocidal damage to neutrophils must be minimised. Tanaka and colleagues have evaluated cytocidal effects of PACT on murine peripheral-blood neutrophils with a range of antimicrobial photosensitisers [517]. They compared Photofrin^® and Laserphyrin^® (mono-L-aspartyl chlorine e6 (NPe6), Talaporfin^®) with non-porphyrinic photosensitisers - Erythrosine B, rose bengal, crystal violet, new methylene blue, toluidine blue-O and MB. Tanaka et al. found that the toluidine blue-O and MB photosensitisers had a negligible cytocidal effect on neutrophils in comparison the porphyrin based Photofrin^® and new methylene blue photosensitisers had a strong cytocidal effect on neutrophils [517]. The group hypothesise that the degree to which different photosensitisers kill bacteria (particularly Gram-negative) varies considerably and that the effectiveness of the cytocidal effect of photosensitisers against neutrophils must be considered when designing antimicrobial PACT protocols [517]. Gallium PCs have been prepared and investigated as potential PACT agents. Mantareva et al. evaluated two cationic Ga(II)PCs (tetra-methylpyridoxyl substituted GaPC and octa-methylpyridyloxy substituted analogue) against MRSA, Enterococcus faecalis (E. faecalis, also Gram-positive), P. aeruginosa and Candida albicans (C. albicans) (a fungus) in the planktonic phase [507]. The PACT activity of the two PCs was compared to MB and a ZnPC with the same substitution pattern. Relatively high uptake concentrations were observed for the two GaPCs for the bacterial and fungal species, with the octa-substituted PC exhibiting higher uptake. Photodynamic inactivation of the pathogens was observed for all but the E. faecalis bacteria. Ragàs et al. reported the synthesis of a tri-cationic water soluble porphycene evaluated in vitro for PACT; 2,7,12-tris(α-pyridinio-p-tolyl)-17-(p-methoxymethyl)phenyl porphycene (Py₃MeO-TBPo) was tested against S. aureus 8325-4, methicillin-resistant S. aureus Xen 31, E. faecalis (ATCC29212) all Gram-positive bacteria and Acinetobacter baumannii (A. baumannii, ATCC 51393), E. coli (ATCC53868), Proteus mirabilis (ATCC51393), and P. aeruginosa (ATCC 19660) as Gram-negative bacteria. C. albicans (ATCC 18804) and Candida krusei (C. krusei, ATCC 6258) were the fungi used [510]. Ragàs et al. were able to demonstrate the high photodynamic activity of the porphycene photosensitisers against a broad spectrum of microbial cells in vitro and against infection in a third-degree burn model (in vivo) [510]. Yu et al. have investigated the synthesis of a group of novel quaternary ammonium cationic porphyrins and assessed their potential (in vitro) as PACT agents against S. aureus, E. coli, P. aeruginosa and C. albicans [505]. They observed antimicrobial activity was sensitive to the functional groups and metal ion present in the photosensitiser: activity was decreased with increasing electron withdrawing effect of the functional groups attached to the aromatic rings. The Gram-negative bacteria showed resistance to all of the synthesised porphyrins, however, two of the compounds did exhibit a degree of photoinactivation against the Gram-positive bacteria and the fungus. Research has been undertaken to observe the effect of the number of positive charges and the charge distribution in a porphyrin's structure in relation to photoinactivation of bacteria in the treatment of waste water [503]. Results suggested that cationic porphyrins bearing three and four charges are highly efficient in the photoinactivation of E. faecalis and E. coli. Meso-substituent groups on the porphyrin yield different effects on photoinactivation - the 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)-porphyrin triiodide photosensitiser achieves greater cell survival at lower light doses [503, 504]. These results demonstrate the potential PACT has as a modality for the treatment of infections that are becoming more and more resistant to conventional therapies. Huang et al. have reported on the potential of cationic BCs as photosensitisers for PACT [509]. They describe four BCs with 2, 4 or 6 quaternised ammonium groups or 2 basic amine groups and their cytocidal effect on S. aureus, E. coli and C. albicans. All four of the BCs exhibited high bactericidal activity against S. aureus and showed selectivity for bacteria over human cells (phototoxicity was assessed in human HeLa cancer cells). Increasing cationic charge on the BCs increased activity against E. coli. The only BC toxic towards C. albicans was the BC bearing the basic groups [509]. Hypothesises that PACT offers a treatment modality whereby the development of resistant strains of pathogens is minimised has been made by Giuliani et al. [506]. Preliminary investigations have been undertaken to support their theory - they have investigated three common pathogens and their potential to develop resistance to PACT photosensitisers. They targeted a novel tetracationic Zn(II)PC chloride (RLP068/Cl) against the pathogens P. aeruginosa, S. aureus and C. albicans in vitro. Results demonstrated the novel PC exhibited a concentration-dependent killing efficacy against the three pathogens associated with skin and soft tissue infections. Notably, photodynamic activity was retained against multidrug resistant strains of the pathogens evaluated. Giuliani and colleagues propose that the unique mechanism of action in PACT exhibits a low propensity for inducing resistance in the three clinically relevant human pathogens tested. Nanoparticle-photosensitiser delivery systems are being investigated as agents suitable for PACT, although to date not with porphyrin based photosensitisers [518].

Conclusion and Outlook

There is potential to increase the efficiency and effectiveness of treatments offered against disease management by using targeting modalities and drug-loaded vehicles; these strategies can be used as stand-alone tools or combined to form more potent systems (theranostics) to fight disease. Advances in porphyrinic photosensitiser chemistry over the past two decades have seen improved second-generation photosensitisers authorised for photodynamic anticancer therapy and the development of porphyrinic bioconjugates that have greatly enhanced a photosensitiser's selectivity for tumour cells; significantly reducing the acute/chronic cutaneous photosensitivity associated with first- (and second-) generation photosensitisers. Current research has focused on drug delivery methods to circumvent the need to disregard potential efficacious photosensitisers that have solubility issues. Porphyrin-based chemistry has emerged as a leader in the field of fluorescence-guided imaging and detection - photosensitiser fluorescence imaging has been increasingly used as a potent adjunct therapy in detecting cancerous tissue and in the fluorescence-guided resection of tumours - greatly increasing a patient's quality of life and survival rates. The use of porphyrinic photosensitisers as theranostic agents also is beginning to show great potential - the capability of a single entity to act as a dual imaging and pdt agent holds significant benefits in the detection and treatment of cancers and other disease models. Furthermore, imaging combined with pdt has the potential to provide patient-customised treatments. The versatility of porphyrinic moieties in the imaging of cancer and other pathological conditions is further demonstrated by their use as radiolabelled imaging probes and in the development of contrast-enhancement agents for MRI and PET. Porphyrinic photosensitisers have recently been identified as strong candidates in the promising alternative treatment model (PACT) for treating microbial infections, especially those with antibiotic resistant strains.

It can be readily observed that there is a clear unequivocal deficit in the understanding of intracellular mechanisms and a need to develop a means by which these delicate and powerful events can be further visualised and understood. An enhanced comprehension of these mechanisms, in both healthy functioning cells and stressed and diseased states, could allow scientists and physicians to develop enhanced, eloquent and sensitive investigative techniques and strategies: empowering them with the potential to detect, control and eliminate cellular damage and disease. Such a physiological breakthrough would court major benefits in not only the health of the patient but financially too. One avenue would through the creation of a multimodal imagining, detective and therapeutic system developed via an interdisciplinary approach which brings together: (i) nanotechnology; (ii) porphyrinic chemistry; (iii) optical spectroscopies; and (iv) cell biology. Such a system potentially offers the ability to answer many of the mysteries surrounding the biological labyrinth of cellular functioning and life and facilitate the better management, treatment and possible control of diseases.

Abbreviations

A. Baumannii: Acinetobacter baumannii; ADAPT: antibody-directed abzyme prodrug therapy; ADEPT: antibody-directed enzyme prodrug therapy; ALA: 5-aminolaevulinic acid/δ-aminolaevulinic acid; AMD: age-related macular degeneration; AMP: antimicrobial peptides; ATMPn: 9-acetoxy-2,7,12,17-tetrakis-(β-methoxyethyl)-porphycene; BC: bacteriochlorin; BCC: basal cell carcinoma; Benvix^®: benzyl ester derivative of ALA; BPD MA: benzoporphyrin derivative monoacid A; BPF: bacteriochlorophyll-peptide-folate; BSA: bovine serum albumin; C. albicans: Candida albicans; CFR: colour fluorescence ratio; CGP55847: a zinc phthalocyanine derivative; CIS: carcinomas in situ; C. krusei: Candida krusei; CNT: carbon nanotube; CNV: choroidal neovascularisation; CT: computerised tomography; CTC: circulating tumour cells; DIF: drug-induced fluorescence; DTPA: (Gd(III)-aminobenzyl-diethylenetriaminepentaacetic acid; Escherichia coli: E. coli; E. faecalis: Enterococcus faecalis; EGF: epidermal growth factor; EMR: electromagnetic radiation; EMS: electromagnetic spectrum; FDA: Food and Drug Administration; FDG: ¹⁸F-fluordeoxyglucose; F-FHBG: 9-(4-¹⁸F-fluoro-3-hydroxymethylbutyl) guanine; FGR: fluorescence-guided resection; FLT: 3´-deoxy-3´-¹⁸F-fluorothymidine; FRET: Förster/Fluorescence Resonance Energy Transfer; Gal: galactose; Glu: glucose; HDL: high density lipoprotein; Hexvix^®: a hexyl derivative of ALA; Hp: haematoporphyrin; HpD: haematoporphyrin derivative; HPPH: 3-vinyl-3-[1-(hexyoxy)ethyl]pyropheophorbide a; HSV: herpes simplex virus; IC: internal conversion; IC₅₀: 50% cell kill; ISC: intersystem crossing; LDL: low density lipoprotein; LIFE: light induced fluorescence emission; Lutex^®/Lutrin^®: lutetium texaphyrin; MAb: monoclonal antibody; MB: methylene blue; Metvix^®: a methyl ester derivative of ALA; MPE: multi-photon excitation; MPS: mesoporous silica; MRI: magnetic resonance imaging; MRS: magnetic resonance spectroscopy; MWNT: multi-walled nanotube; NC: naphthalocyanine; NCB: non-covalent binding; NP: nanoparticle; NS: nanospecies; NSF: nephrogenic systemic fibrosis; NT: nanotube; OCT: optical computed tomography; P. aeruginosa: Pseudomonas aeruginosa; PACT: photoantimicrobial chemotherapy; PC: phthalocyanine; PC4: a silicon PC complex; pdd: photodynamic diagnosis; PDI: photodynamic inactivation; pdt: photodynamic therapy; PEG: polyethylene glycol; PET: positron emission tomography; P-Gal₄: 5,10,15,20-tetrakis(4,1'thio-galactose-2,3,5,6,-tetrafluorophenyl)porphyrin; P. gingivalis: Porphyromonas gingivalis; P-Glu₄: 5,10,15,20-tetrakis(4,1'thio-glucose-2,3,5,6,-tetrafluorophenyl)porphyrin; PIT: photoimmunotherapy; P. mirabilis: Proteus mirabilis; PPF: porphyrin-peptide-folate; PPIX: protoporphyrin IX; PS1: 5-(4-isothiocyanatophenyl)-10,15,20-tri-(3,5-dihydroxyphenyl)-porphyrin; PS2: 5-(4-isothiocyantophenyl)-10,15,20-tris-(4-N-methylpyridiniumyl) porphyrin trichloride; psen: photosensitiser; PSFD: photosensitiser fluorescence detection; PZ: porphyrazine; QD: quantum dots; ROS: reactive oxygen species; S. aureus: Staphylococcus aureus; scFv: single chain antibody fragments; SCC: squamous cell carcinoma; scFv: single chain antibody fragments; SWNT: single-walled nanotube; THPC: tetra(m-hydroxyphenyl)chlorin; TPPF₂₀: tetrapentafluorophenyl porphyrins; TPP: tetraphenylporphyrin; wAMD: wet age-related macular degeneration; F_D: singlet oxygen quantum yield; Φ_fl: fluorescence quantum yield; Φ_T: triplet quantum yield; τ_T: triplet state lifetime.

Competing Interests

The authors have declared that no competing interest exists.

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Author contact

Corresponding author: Dr. Ross W. Boyle. Tel: +44 (0)1482 466353; E-mail: R.W.Boyleac.uk

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Received 2012-5-9
Accepted 2012-8-10
Published 2012-10-4

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