Josephine H. Woodhams

Improved in vivo delivery of m-THPC via pegylated liposomes for use in photodynamic therapy

Pegylated liposomal nanocarriers have been developed with the aim of achieving improved uptake of the clinical PDT photosensitiser, m-THPC, into target tissues through increased circulation time and bioavailability. This study investigates the biodistribution and PDT efficacy of m-THPC in its standard formulation (Foscan®) compared to m-THPC incorporated in liposomes with different degrees of pegylation (FosPEG 2% and FosPEG 8%), following i.v. administration to normal and tumour bearing rats. The plasma pharmacokinetics were described using a three compartmental analysis and gave elimination half lives of 90 h, 99 h and 138 h for Foscan®, FosPEG 2% and 8% respectively. The accumulation of m-THPC in tumour and normal tissues, including skin, showed that maximal tumour to skin ratios were observed at ≤ 24 h with FosPEG 2% and 8%, whilst skin photosensitivity studies showed Foscan® induces more damage compared to the liposomes at drug-light intervals of 96 and 168 h. PDT treatment at 24h post-administration (0.05 mg kg⁻¹) showed higher tumour necrosis using pegylated liposomal formulations in comparison to Foscan®, which is attributed to the higher tumour uptake and blood plasma concentrations. Clinically, this improved selectivity has the potential to reduce not only normal tissue damage, but the drug dose required and cutaneous photosensitivity.

In vitro and in vivo characterization of temoporfin-loaded PEGylated PLGA nanoparticles for use in photodynamic therapy

AIMS In this study we evaluated temoporfin-loaded polyethylene glycol (PEG) Poly-(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) as a new formulation for potential use in cancer treatment. MATERIALS & METHODS NPs were characterized for their photophysical properties, temoporfin release, cellular uptake and intracellular localization, and dark and photocytotoxicities of temoporfin by using A549, MCF10A neoT and U937 cell lines. In vivo imaging was performed on athymic nude-Foxn1 mice. RESULTS Temoporfin was highly aggregated within the NPs and the release of temoporfin monomers was faster from PEGylated PLGA NPs than from non-PEGylated ones. PEGylation significantly reduced the cellular uptake of NPs by the differentiated promonocytic U937 cells, revealing the stealth properties of the delivery system. Dark cytotoxicity of temoporfin delivered by NPs was less than that of free temoporfin in standard solution (Foscan(®), Biolitec AG [Jena, Germany]), whereas phototoxicity was not reduced. Temoporfin delivered to mice by PEGylated PLGA NPs exhibits therapeutically favorable tissue distribution. CONCLUSION These encouraging results show promise in using PEGylated PLGA NPs for improving the delivery of photosensitizers for photodynamic therapy.

The role of oxygen monitoring during photodynamic therapy and its potential for treatment dosimetry

Understanding of the biology of photodynamic therapy (PDT) has expanded tremendously over the past few years. However, in the clinical situation, it is still a challenge to match the extent of PDT effects to the extent of the disease process being treated. PDT requires drug, light and oxygen, any of which can be the limiting factor in determining efficacy at each point in a target organ. This article reviews techniques available for monitoring tissue oxygenation during PDT. Point measurements can be made using oxygen electrodes or luminescence-based optodes for direct measurements of tissue pO2, or using optical spectroscopy for measuring the oxygen saturation of haemoglobin. Imaging is considerably more complex, but may become feasible with techniques like BOLD MRI. Pre-clinical studies have shown dramatic changes in oxygenation during PDT, which vary with the photosensitizer used and the light delivery regimen. Better oxygenation throughout treatment is achieved if the light fluence rate is kept low as this reduces the rate of oxygen consumption. The relationship between tissue oxygenation and PDT effect is complex and remarkably few studies have directly correlated oxygenation changes during PDT with the final biological effect, although those that have confirm the value of maintaining good oxygenation. Real time monitoring to ensure adequate oxygenation at strategic points in target tissues during PDT is likely to be important, particularly in the image guided treatment of tumours of solid organs.

Photodynamic therapy with WST09 (Tookad): quantitative studies in normal colon and transplanted tumours

Photodynamic therapy (PDT) is attracting increasing interest for the safe destruction of localised tumours in a range of organs. However, most photosensitising drugs require a delay of hours to days between drug administration and light activation with skin photosensitivity that may last for weeks. WST09 (Tookad) is a new faster acting photosensitiser that clears within a few hours. In normal rat colon, after sensitisation with an intravenous bolus of WST09, light was delivered to a single point on the mucosa and the extent of PDT necrosis measured 3 days later. The lesion diameter was greatest with the highest dose of drug and light and the shortest drug light interval (DLI), falling rapidly with a DLI more than 5 min. In tumours transplanted subcutaneously or into the colon, the extent of necrosis only started falling with a DLI greater than 15 min, suggesting a possible window for tumour selectivity. Histological changes 3 days after PDT were essentially the same as those seen with longer acting photosensitisers. The lesion dimensions were comparable to the largest ones seen with other photosensitisers under similar experimental conditions. We conclude that WST09 is a powerful photosensitiser that produces PDT effects similar to those seen with longer acting drugs, but with the major advantages of a short DLI and rapid clearance.

Meta-tetra(hydroxyphenyl)chlorin-loaded liposomes sterically stabilised with poly(ethylene glycol) of different length and density: characterisation, in vitro cellular uptake and phototoxicity.

We studied the effects of density and thickness of PEG coating on in vitro cellular uptake, and dark- and photo-toxicity of liposomal formulations (Fospeg) of the photodynamic agent meta-tetrahydroxyphenyl chlorin (m-THPC). The cellular uptake of various Fospeg formulations was determined by flow cytometry in CCD-34Lu human normal fibroblasts and A549 lung cancer cells. Dark and light-induced cytotoxicity was measured by MTS assay after exposure to increasing concentrations of Fospeg only and followed by irradiation with red light. Intracellular localization of m-THPC delivered by Fospeg was determined by fluorescence microscopy. The studies were carried out in comparison with m-THPC delivered by the standard solvent. In the dark all Fospeg formulations were less cytotoxic than m-THPC in standard solvent (ethanol/poly(ethylene glycol 400/water; 20 : 30 : 50 by vol.) and cytotoxicity decreased by increasing PEGylation. m-THPC delivered as Fospeg was internalised by endocytosis and localised mainly in the Golgi apparatus and endoplasmic reticulum. The efficiency of cellular uptake of Fospeg was reduced by 30-40% with respect to m-THPC in standard solution causing a slight reduction of the phototoxicity but without serious impairment of the efficacy of the treatment. Our study suggests that PEGylated liposomes are promising nanocarriers for the delivery of photosensitisers for photodynamic therapy because they reduce dark cytotoxicity while preserving therapeutic efficacy.

Intracellular re-localisation by photochemical internalisation enhances the cytotoxic effect of gelonin - Quantitative studies in normal rat liver

Photochemical internalisation (PCI) is a delivery technology that employs a sub-lethal form of photodynamic therapy (PDT) in which a photosensitiser is activated by light to break down intracellular membranes and release macromolecules into the cytosol where they can be biologically active. Although PCI does enhance the PDT killing of transplanted tumours in mice after local injection of the cytotoxic agent, gelonin, the redistribution of gelonin from intracellular organelles into the cytosol has only previously been demonstrated in vitro. This study is designed to understand the factors controlling the efficacy of PCI in vivo and to document the mechanism of action. Using the photosensitiser AlS(2)Pc in studies on normal rat liver, we have demonstrated in vivo that gelonin is initially taken up into lysosomes, but can be released into the cytosol using PCI. Furthermore, PCI enhances the PDT effect after systemic administration of gelonin (volume of necrosis increased x2.5 when gelonin is given one hour before light), with the remarkably low dose of 5 microg/kg (10,000 times lower than the LD50); in the absence of light, there is no effect with 500 microg/kg. These results suggest that PCI may have a useful role to play in the site specific activation of cytotoxic agents like gelonin, given at a dose level that has no effect in the absence of light.

Targeted delivery of photosensitizers: efficacy and selectivity issues revealed by multifunctional ORMOSIL nanovectors in cellular systems

PEGylated and non-PEGylated ORMOSIL nanoparticles prepared by microemulsion condensation of vinyltriethoxy-silane (VTES) were investigated in detail for their micro-structure and ability to deliver photoactive agents. With respect to pure silica nanoparticles, organic modification substantially changes the microstructure and the surface properties. This in turn leads to a modulation of both the photophysical properties of embedded photosensitizers and the interaction of the nanoparticles with biological entities such as serum proteins. The flexibility of the synthetic procedure allows the rapid preparation and screening of multifunctional nanosystems for photodynamic therapy (PDT). Selective targeting of model cancer cells was tested by using folate, an integrin specific RGD peptide and anti-EGFR antibodies. Data suggest the interference of the stealth-conferring layer (PEG) with small targeting agents, but not with bulky antibodies. Moreover, we showed that selective photokilling of tumour cells may be limited even in the case of efficient targeting because of intrinsic transport limitations of active cellular uptake mechanisms or suboptimum localization.

Enhancing the efficacy of cytotoxic agents for cancer therapy using photochemical internalisation

Photochemical internalisation (PCI) is a technique for improving cellular delivery of certain bioactive agents which are prone to sequestration within endolysosomes. There is a wide range of agents suitable for PCI‐based delivery including toxins, oligonucleotides, genes and immunoconjugates which demonstrates the versatility of this technique. The basic mechanism of PCI involves triggering release of the agent from endolysosomes within the target cells using a photosensitiser which is selectively retained with the endolysosomal membranes. Excitation of the photosensitiser by visible light leads to disruption of the membranes via photooxidative damage thereby releasing the agent into the cytosol. This treatment enables the drugs to reach their intended subcellular target more efficiently and improves their efficacy. In this review we summarise the applications of this technique with the main emphasis placed on cancer chemotherapy.

OPTICAL MEASUREMENT OF PHOTOSENSITIZER CONCENTRATION IN VIVO

Most techniques for measuring tissue concentrations of drugs are invasive, time-consuming, and often require the removal of tissue or body fluids. Optical pharmacokinetics (OP) is a minimally invasive alternative giving an immediate result. Pulses of white light are directed at the tissue of interest using a fiber optic probe. Scattered light is detected by a second fiber immediately adjacent to the first in the same probe (separation 1.7 mm). Using the photosensitizer disulfonated aluminium phthalocyanine (AlS2Pc), OP measurements were made in phantoms and on the mouth, stomach, colon, skin, and liver of normal rats 1 and 24 h after intravenous AlS2Pc administration. AlS2Pc concentration was determined by calculating the area under the curve (AUC) in the spectral region around the peak drug absorption or measuring the height of the peak. Spectral baseline interpolation removed the need for pre-drug, control optical measurements. OP measurements correlated well with values from alkali chemical extraction (CE) of the corresponding tissues, (R2 0.87–0.97). OP measurements in the mouth also correlated with CE of less accessible internal organs (R2 0.77–0.88). In phantoms, the lowest detectable concentration was 0.1 μg/g. In vivo, results were limited by the lower accuracy in the CE measurements but were almost certainly comparable. An incidental finding was a 12–15 nm red shifted component in the spectra observed 1 h after drug administration, suggesting partitioning of the drug in different microenvironment compartments, which could prove to be of considerable interest in future studies. In conclusion, OP shows promise for real-time measurement of concentrations of drugs with suitable absorption peaks.

Efficacy of photochemical internalisation using disulfonated chlorin and porphyrin photosensitisers: An in vitro study in 2D and 3D prostate cancer models

This study shows the therapeutic outcome of Photochemical Internalisation (PCI) in prostate cancer in vitro surpasses that of Photodynamic Therapy (PDT) and could improve prostate PDT in the clinic, whilst avoiding chemotherapeutics side effects. In addition, the study assesses the potential of PCI with two different photosensitisers (TPCS2a and TPPS2a) in prostate cancer cells (human PC3 and rat MatLyLu) using standard 2D monolayer culture and 3D biomimetic model. Photosensitisers were used alone for photodynamic therapy (PDT) or with the cytotoxin saporin (PCI). TPPS2a and TPCS2a were shown to be located in discrete cytoplasmic vesicles before light treatment and redistribute into the cytosol upon light excitation. PC3 cells exhibit a higher uptake than MatLyLu cells for both photosensitisers. In the 2D model, PCI resulted in greater cell death than PDT alone in both cell lines. In 3D model, morphological changes were also observed. Saporin-based toxicity was negligible in PC3 cells, but pronounced in MatLyLu cells (IC50 = 18 nM). In conclusion, the study showed that tumour features such as tumour cell growth rate or interaction with drugs determine therapeutic conditions for optimal photochemical treatment in metastatic prostate cancer.