Predicting human pharmacokinetics of liposomal temoporfin using a hybrid in silico model.

Over the years, the performance of the liposomal formulations of temoporfin, Foslip® and Fospeg®, was investigated in a broad array of cell-based assays and preclinical animal models. So far, little attention has been paid to the influence of drug release and liposomal stability on the plasma concentration-time profile. The drug release is a key attribute which impacts product quality and the in vivo efficacy of nanocarrier formulations. In the present approach, the in vitro drug release and the drug-protein transfer of Foslip® and Fospeg® was determined using the dispersion releaser technology. To analyze the stability of both formulations in physiological fluids, nanoparticle tracking analysis was applied. A comparable drug release behavior and a high physical stability with a vesicle size of approximately 92 ± 2 nm for Foslip® and at 111 ± 5 nm for Fospeg® were measured. The development of a novel hybrid in silico model resulted in an optimal representation of the in vivo data. Based on the information available for previous formulations, the model enabled a prediction of the performance of Foslip® in humans. To verify the simulations, plasma concentration-time profiles of a phase I clinical trial were used. An absolute average fold error of 1.4 was achieved. Moreover, a deconvolution of the pharmacokinetic profile into different fractions relevant for the in vivo efficacy and safety was achieved. While the total plasma concentration reached a cmax of 2298 ng/mL after 0.72 h, the monomolecular drug accounted for a small fraction of the photosensitizer with a cmax of 321 ng/mL only.

Advanced in silico modeling explains pharmacokinetics and biodistribution of temoporfin nanocrystals in humans.

Foscan®, a formulation comprising temoporfin dissolved in a mixture of ethanol and propylene glycol, has been approved in Europe for palliative photodynamic therapy of squamous cell carcinoma of the head and neck. During clinical and preclinical studies it was observed that considering the administration route, the drug presents a rather atypical plasma profile as plasma concentration peaks delayed. Possible explanations, as for example the formation of a drug depot or aggregation after intravenous administration, are discussed in current literature. In the present study an advanced in silico model was developed and evaluated for the detailed description of Foscan® pharmacokinetics. Therefore, in vitro release data obtained from experiments with the dispersion releaser technology investigating dissolution pressures of various release media on the drug as well as in vivo data obtained from a clinical study were included into the in silico models. Furthermore, precipitation experiments were performed in presence of biorelevant media and precipitates were analyzed by nanoparticle tracking analysis. Size analysis and particle fraction were also incorporated in this model and a sensitivity analysis was performed. An optimal description of the in vivo situation based on in vitro release and particle characterization data was achieved, as demonstrated by an absolute average fold error of 1.21. This in vitro-in vivo correlation provides an explanation for the pharmacokinetics of Foscan® in humans.

Current state of the nanoscale delivery systems for temoporfin-based photodynamic therapy: Advanced delivery strategies.

Enthusiasm for photodynamic therapy (PDT) as a promising technique to eradicate various cancers has increased exponentially in recent decades. The majority of clinically approved photosensitizers are hydrophobic in nature, thus, the effective delivery of photosensitizers at the targeted site is the main hurdle associated with PDT. Temoporfin (mTHPC, medicinal product name: Foscan®), is one of the most potent clinically approved photosensitizers, is not an exception. Successful temoporfin-PDT requires nanoscale delivery systems for selective delivery of photosensitizer. Over the last 25 years, the number of papers on nanoplatforms developed for mTHPC delivery such as conjugates, host-guest inclusion complexes, lipid-and polymer-based nanoparticles and carbon nanotubes is burgeoning. However, none of them appeared to be "ultimate". The present review offers the description of different challenges and achievements in nanoparticle-based mTHPC delivery focusing on the synergetic combination of various nano-platforms to improve temoporfin delivery at all stages of biodistribution. Furthermore, the association of different nanoparticles in one nanoplatform might be considered as an advanced strategy allowing the combination of several treatment modalities.

Investigations of the influence of liposome composition on vesicle stability and drug transfer in human plasma: a transfer study.

Liposomal delivery constitutes a promising approach for i.v. administration of temoporfin (mTHPC) because lipid membranes can host these drug molecules. This study investigates the transfer and release of mTHPC to plasma proteins and stability of various liposomal formulations. To this end, we employed traces of radioactive markers and studied the effects of fatty acid chain length and the degree of saturation in the lipophilic tail, addition of cholesterol and PEGylation of the membrane surface and different drug-to-lipid ratios (DLRs). Liposomes were incubated in human plasma for various incubation times. Drawn samples were separated by asymmetrical flow field-flow fractionation (AF4). Drug was recovered in four fractions identified as albumin, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and liposomes. Our results suggest that mTHPC fits best into fluid, unmodified bilayers when the drug-to-lipid ratio is low. Membrane rigidification as well as the presence of cholesterol and PEGyated lipids reduced the ability of the membrane to accommodate the drug but simultaneously improved the vesicle stability in plasma. Both mechanisms jointly affect the total degree of mTHPC release. We analyzed our data using a kinetic model that suggests the drug to be associated with the host membrane in two distinct states of which only one interacts directly with the plasma compartment.

Photodynamic therapy in fibrosarcoma BALB/c animal model: Observation of the rebound effect.

In vivo spectrofluorometric analysis during photodynamic therapy (PDT) is a fundamental tool to obtain information about drug bleaching kinetics. Using a portable spectrofluorometer with an excitation source emitting at 400nm wavelength and a spectral analyzer ranging from 500nm to 800nm, the evolution of the meta-tetra(hydroxyphenyl) chlorin (m-THPC) photosensitizer fluorescence spectrum at the tumoral tissue of BALB/c murines with fibrosarcoma located at their flank was followed up. Ex vivo fluorescence measurements of the tumor and skin were also performed with the aim of better characterizing the in vivo signal at different parts of the tumor. PDT was performed employing a LED 637nm light source. Fluorescence at different parts of the tumor and at the tail and armpit of mice was measured immediately after injection and followed daily. The average fluorescence intensity in the tumor reached a maximum after 24-72h. Subsequently, illuminations 24, 48, 72 and 96h post-injection were performed, and the fluorescence was measured immediately before and after each illumination. Eventually, 24h post-illumination, the fluorescence at certain parts of the tumor increased in comparison with that measured immediately after illumination. This effect, named "rebound effect", was due to the new local accumulation of the drug, and was used to perform a second illumination on some mice to increase the amount of photodynamic reaction and significantly improve the PDT outcome. These results are encouraging to optimize PDT in the proposed animal model, thinking about the possible translation to humans.

The alteration of temoporfin distribution in multicellular tumor spheroids by ß-cyclodextrins.

To be effective anticancer drugs must penetrate tissue efficiently, reaching all target population of cancer cells in a concentration sufficient to exert a therapeutic effect. This study aimed to investigate the ability of methyl-ß-cyclodextrin (Me-ß-CD) and 2-hydroxypropyl-ß-cyclodextrin (Hp-ß-CD) to alter the penetration and diffusion of temoporfin (mTHPC) in HT29 multicellular tumor spheroids. mTHPC had а nonhomogenous distribution only on the periphery of spheroids. The presence of ß-CDs significantly altered the distribution of mTHPC consisting in the increase of both the depth of photosensitizer penetration and accumulation in HT29 spheroids. We suggest that this improvement is related to the nanoshuttle mechanism of ß-CD action, when ß-CDs facilitate mTHPC transportation to the cells in the inner layers of spheroids. As a result of mTHPC distribution improvement, ß-CDs enhance mTHPC photosensitizing activity towards HT29 multicellular tumor spheroids. The observed effects strongly depend on the type of ß-CD. Thus, varying the type of ß-CD we can finely tune the possibility of using mTHPC for diagnostic (delimitation of tumor margins) or therapeutic purposes.

Macrophage selective photodynamic therapy by meta-tetra(hydroxyphenyl)chlorin loaded polymeric micelles: A possible treatment for cardiovascular diseases.

Selective elimination of macrophages by photodynamic therapy (PDT) is a new and promising therapeutic modality for the reduction of atherosclerotic plaques. m-Tetra(hydroxyphenyl)chlorin (mTHPC, or Temoporfin) may be suitable as photosensitizer for this application, as it is currently used in the clinic for cancer PDT. In the present study, mTHPC was encapsulated in polymeric micelles based on benzyl-poly(ε-caprolactone)-b-methoxy poly(ethylene glycol) (Ben-PCL-mPEG) using a film hydration method, with loading capacity of 17%. Because of higher lipase activity in RAW264.7 macrophages than in C166 endothelial cells, the former cells degraded the polymers faster, resulting in faster photosensitizer release and higher in vitro photocytotoxicity of mTHPC-loaded micelles in those macrophages. However, we observed release of mTHPC from the micelles in 30min in blood plasma in vitro which explains the observed similar in vivo pharmacokinetics of the mTHPC micellar formulation and free mTHPC. Therefore, we could not translate the beneficial macrophage selectivity from in vitro to in vivo. Nevertheless, we observed accumulation of mTHPC in atherosclerotic lesions of mice aorta's which is probably the result of binding to lipoproteins upon release from the micelles. Therefore, future experiments will be dedicated to increase the stability and thus allow accumulation of intact mTHPC-loaded Ben-PCL-mPEG micelles to macrophages of atherosclerotic lesions.

Intradermal drug delivery by nanogel-peptide conjugates; specific and efficient transport of temoporfin.

Nanogels offer many unique features rendering them as very attractive candidates for drug delivery. However, for their applications the loading capacity and specific encapsulation, in particular for hydrophobic drugs, in a complex media are two critical factors. In this work, we report for the first time on the preparation of nanogel-peptide conjugates with the ability of specific encapsulation of temoporfin (m-THPC). The peptide was selected based on combinatorial means and it was conjugated to polyglycerol as the nanogel precursor. We observed that the loading capacity of nanogels improved 16 times upon peptide conjugation. Skin penetrations tests in barrier deficient skin showed that nanogel-peptide conjugates enhance the penetration of m-THPC in the viable skin layers efficiently. This study indicates that nanogel-peptide conjugates could be used as unique carriers with high loading capacity for hydrophobic compounds, which provides the basis for the design of advanced topical drug delivery systems.

Inclusion complexation with ß-cyclodextrin derivatives alters photodynamic activity and biodistribution of meta-tetra(hydroxyphenyl)chlorin.

Application of meta-tetra(hydroxyphenyl)chorin (mTHPC) one of the most effective photosensitizer (PS) in photodynamic therapy of solid tumors encounters several complications resulting from its insolubility in aqueous medium. To improve its solubility and pharmacokinetic properties, two modified ß-cyclodextrins (ß-CDs) methyl-ß-cyclodextrin (M-ß-CD) and 2-hydroxypropyl-ß-cyclodextrin (Hp-ß-CD) were proposed. The aim of this work was to evaluate the effect of ß-CDs on mTHPC behavior at various stages of its distribution in vitro and in vivo. For this purpose, we have studied the influence of the ß-CDs on mTHPC binding to the serum proteins, its accumulation, distribution and photodynamic efficiency in HT29 cells. In addition, the processes of mTHPC biodistribution in HT29 tumor bearing mice after intravenous injection of PS alone or with the ß-CDs were compared. Interaction of mTHPC with studied ß-CDs leads to the formation of inclusion complexes that completely abolishes its aggregation after introduction into serum. It was demonstrated that the ß-CDs have a concentration-dependent effect on the process of mTHPC distribution in blood serum. At high concentrations, ß-CDs can form inclusion complexes with mTHPC in the blood that can have a significant impact on PS distribution out of the vascular system in solid tissues. Besides, the ß-CDs increase diffusion movement of mTHPC molecules that can significantly accelerate the delivery of PS to the targets cells and tissues. In vivo study confirms the fact that the use of ß-CDs allows to modify mTHPC distribution processes in tumor bearing animals that is reflected in the decreased level of PS accumulation in skin and muscles, as well as in the increased PS accumulation in tumor. Further studies are underway to verify the optimal protocols of mTHPC/ß-CD formulation for photodynamic therapy.

Bridging laboratory and large scale production: preparation and in vitro-evaluation of photosensitizer-loaded nanocarrier devices for targeted drug delivery.

PURPOSE: Industrial production of nanosized drug delivery devices is still an obstacle to the commercialization of nanomedicines. This study encompasses the development of nanoparticles for peroral application in photodynamic therapy, optimization according to the selected product specifications, and the translation into a continuous flow process. METHODS: Polymeric nanoparticles were prepared by nanoprecipitation of Eudragit® RS 100 in presence and in absence of glycofurol. The photosensitizer temoporfin has been encapsulated into these carrier devices. Process parameters were optimized by means of a Design of Experiments approach and nanoparticles with optimal characteristics were manufactured by using microreactor technology. The efficacy was determined by means of cell culture models in A-253 cells. RESULTS: Physicochemical properties of nanoparticles achieved by nanoprecipitation from ethanolic solutions were superior to those obtained from a method based upon glycofurol. Nanoencapsulation of temoporfin into the matrix significantly reduced toxicity of this compound, while the efficacy was maintained. The release profiles assured a sustained release at the site of action. Finally, the transfer to continuous flow technology was achieved. CONCLUSION: By adjusting all process parameters, a potent formulation for application in the GI tract was obtained. The essential steps of process development and scale-up were part of this formulation development.

Pharmacokinetic and biodistribution study following systemic administration of Fospeg®--a Pegylated liposomal mTHPC formulation in a murine model.

Fospeg® is a newly developed photosensitizer formulation based on meso-tetra(hydroxyphenyl)chlorin (mTHPC), with hydrophilic liposomes to carry the hydrophobic photosensitizer to the target tissue. In this study the pharmacokinetics and biodistribution of Fospeg® were investigated by high performance liquid chromatography at various times (0.5-18 hours) following systemic i.v. administration. As a model an experimental HT29 colon tumor in NMRI nu/nu mice was employed. Our study indicates a higher plasma peak concentration, a longer circulation time and a better tumor-to-skin ratio than those of Foslip®, another liposomal mTHPC formulation. Data from ex vivo tissue fluorescence and reflectance imaging exhibit good correlation with chemical extraction. Our results have shown that optical imaging provides the potential for fluorophore quantification in biological tissues.

Cytotoxic efficacy of photodynamic therapy in osteosarcoma cells in vitro.

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment. Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response(1). Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)(2). The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis. In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Localization of liposomal mTHPC formulations within normal epithelium, dysplastic tissue, and carcinoma of oral epithelium in the 4NQO-carcinogenesis rat model.

BACKGROUND AND OBJECTIVE: Foslip and Fospeg are liposomal formulations of the photosensitizer mTHPC (Foscan), which is used for photodynamic therapy (PDT) of malignancies. Literature suggests that liposomal mTHPC formulations have better properties and increased tumor uptake compared to Foscan. To investigate this, we used the 4NQO-induced carcinogen model to compare the localization of the different mTHPC formulations within normal, precancerous, and cancerous tissue. In contrast to xenograft models, the 4NQO model closely mimics the carcinogenesis of human oral dysplasia. MATERIALS AND METHODS: Fifty-four rats drank water with the carcinogen 4NQO. When oral examination revealed tumor, the rats received 0.15 mg/kg mTHPC (Foscan, Foslip, or Fospeg). At 2, 4, 8, 24, 48, or 96 hours after injection the rats were sacrificed. Oral tissue was sectioned for HE slides and for fluorescence confocal microscopy. The HE slides were scored on the severity of dysplasia by the epithelial atypia index (EAI). The calibrated fluorescence intensity per formulation or time point was correlated to EAI. RESULTS: Fospeg showed higher mTHPC fluorescence in normal and tumor tissue compared to both Foscan and Foslip. Significant differences in fluorescence between tumor and normal tissue were found for all formulations. However, at 4, 8, and 24 hours only Fospeg showed a significant difference. The Pearson's correlation between EAI and mTHPC fluorescence proved weak for all formulations. CONCLUSION: In our induced carcinogenesis model, Fospeg exhibited a tendency for higher fluorescence in normal and tumor tissue compared to Foslip and Foscan. In contrast to Foscan and Foslip, Fospeg showed significantly higher fluorescence in tumor versus normal tissue at earlier time points, suggesting a possible clinical benefit compared to Foscan. Low correlation between grade of dysplasia and mTHPC fluorescence was found.

Photodynamic therapy with conventional and PEGylated liposomal formulations of mTHPC (temoporfin): comparison of treatment efficacy and distribution characteristics in vivo.

A major challenge in the application of a nanoparticle-based drug delivery system for anticancer agents is the knowledge of the critical properties that influence their in vivo behavior and the therapeutic performance of the drug. The effect of a liposomal formulation, as an example of a widely-used delivery system, on all aspects of the drug delivery process, including the drug's behavior in blood and in the tumor, has to be considered when optimizing treatment with liposomal drugs, but that is rarely done. This article presents a comparison of conventional (Foslip®) and polyethylene glycosylated (Fospeg®) liposomal formulations of temoporfin (meta-tetra[hydroxyphenyl]chlorin) in tumor-grafted mice, with a set of comparison parameters not reported before in one model. Foslip® and Fospeg® pharmacokinetics, drug release, liposome stability, tumor uptake, and intratumoral distribution are evaluated, and their influence on the efficacy of the photodynamic treatment at different light-drug intervals is discussed. The use of whole-tumor multiphoton fluorescence macroscopy imaging is reported for visualization of the in vivo intratumoral distribution of the photosensitizer. The combination of enhanced permeability and retention-based tumor accumulation, stability in the circulation, and release properties leads to a higher efficacy of the treatment with Fospeg® compared to Foslip®. A significant advantage of Fospeg® lies in a major decrease in the light-drug interval, while preserving treatment efficacy.

Optimal treatment opportunity for mTHPC-mediated photodynamic therapy of liver cancer.

Photodynamic therapy (PDT) has been clinically used for liver cancer. The pharmacokinetics of a photosensitizer needs to be monitored so that PDT can be performed at the most favorable time and with the proper dose to increase the cure rate. As mTHPC is a fluorescent compound, we investigate its pharmacokinetics, distribution, and elimination in the rat orthotropic liver cancer model in order to confirm an optimal treatment opportunity of liver cancer PDT. After intravenous administration at a single dose of 300 µg/kg, mTHPC was extracted from tissue homogenates or plasma. Then, mTHPC concentrations were assessed by fluorescence spectroscopy and the data were processed with PK-GRAPH pharmacokinetic procedure. The plasma concentration-time profile of mTHPC showed a short distribution half-life (T½α = 0.082 h) and a relatively longer elimination half-life (T½ß = 28.23 h), which quite fitted with a two-compartment model. The results of mTHPC tissue distributions showed that the highest drug accumulation was in tumor tissue, and successively decreased in liver, heart, spleen, muscle, and skin tissues. The drug distribution ratio of tumor to normal tissue reached the peak at 24 h after mTHPC administration. mTHPC was eliminated at a suitable rate in rat orthotropic liver cancer model, and there was no long-term accumulation of mTHPC in rat tissues. For PDT of orthotropic liver cancer, 24 h after mTHPC intravenous injection may be the optimal treatment time point, which might provide higher clinical efficacy and reduce side effects.

Comparative characterization of the cellular uptake and photodynamic efficiency of Foscan® and Fospeg in a human prostate cancer cell line.

BACKGROUND: m-THPC (Foscan(®)) is one of the most potent second generation photosensitizers used in photodynamic therapy, photoactivated at higher wavelengths (652 nm). However, its strongly hydrophobic nature causes aggregation of the molecules and prevents its unbiased bioavailability in the biological media, resulting in lower accumulation in the tumor cells. Several strategies have been adopted to improve the photodynamic characteristics of the photosensitizer. Among them, very promising seems to be the encapsulation of the molecule into liposomes, due to the superior properties of liposomes as drug carriers. METHODS: In this paper the photodynamic characteristics of the PEGylated liposomal formulation of m-THPC, Fospeg, using the human prostate cancer cell line LNCaP, as an in vitro model, were investigated. In addition the spectral characteristics, cellular uptake and localization, dark and light induced cytotoxicity and photodynamic efficacy of Foscan(®) and Fospeg were compared. RESULTS: Fospeg, compared with Foscan, showed higher intracellular uptake at any concentration and incubation time. Regarding PDT efficacy, Fospeg produced more severe cytotoxicity than Foscan(®) at any concentration and energy dose. Using Fospeg, the lowest concentration (0.22 µM) and energy dose (180 mJ/cm(2)) was adequate to result in the death of 50% of the cells 24h post PDT while an approximately 10 times higher Foscan(®) concentration (1.8 µM) was needed to result in the same cytotoxicity. CONCLUSIONS: The use of the PEGylated liposomal formulation of m-THPC resulted in the improvement of its intracellular uptake and the enhancement of its photodynamic activity. Fospeg, compared to Foscan(®), proved to be a more advantageous photosensitizer for photodynamic therapy.

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.

Fluorescence localization and kinetics of mTHPC and liposomal formulations of mTHPC in the window-chamber tumor model.

BACKGROUND AND OBJECTIVE: Foslip® and Fospeg® are liposomal formulations of the photosensitizer mTHPC, intended for use in Photodynamic Therapy (PDT) of malignancies. Foslip consists of mTHPC encapsulated in conventional liposomes, Fospeg consists of mTHPC encapsulated in pegylated liposomes. Possible differences in tumor fluorescence and vasculature kinetics between Foslip, Fospeg, and Foscan® were studied using the rat window-chamber model. MATERIAL AND METHODS: In 18 rats a dorsal skin fold window chamber was installed and a mammary carcinoma was transplanted in the subcutaneous tissue. The dosage used for intravenous injection was 0.15 mg/kg mTHPC for each formulation. At seven time-points after injection (5 minutes to 96 hours) fluorescence images were made with a CCD. The achieved mTHPC fluorescence images were corrected for tissue optical properties and autofluorescence by the ratio fluorescence imaging technique of Kascakova et al. Fluorescence intensities of three different regions of interest (ROI) were assessed; tumor tissue, vasculature, and surrounding connective tissue. RESULTS: The three mTHPC formulations showed marked differences in their fluorescence kinetic profile. After injection, vascular mTHPC fluorescence increased for Foslip and Fospeg but decreased for Foscan. Maximum tumor fluorescence is reached at 8 hours for Fospeg and at 24 hours for Foscan and Foslip with overall higher fluorescence for both liposomal formulations. Foscan showed no significant difference in fluorescence intensity between surrounding tissue and tumor tissue (selectivity). However, Fospeg showed a trend toward tumor selectivity at early time points, while Foslip reached a significant difference (P < 0.05) at these time points. CONCLUSIONS: Our results showed marked differences in fluorescence intensities of Fospeg, Foslip, and Foscan, which suggest overall higher bioavailability for the liposomal formulations. Pegylated liposomes seemed most promising for future application; as Fospeg showed highest tumor fluorescence at the earlier time points.

Skin penetration and deposition of carboxyfluorescein and temoporfin from different lipid vesicular systems: In vitro study with finite and infinite dosage application.

The aim of the present research is to evaluate the influence of different lipid vesicular systems as well as the effect of application mode on skin penetration and deposition behaviors of carboxyfluorescein (hydrophilic model drug) and temoporfin (lipophilic model drug). All of the lipid vesicular systems, including conventional liposomes, invasomes and ethosomes, were prepared by film hydration method and characterized for particle size distribution, ζ-potential, vesicular shape and surface morphology, in vitro human skin penetration and skin deposition. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) defined that all of lipid vesicles had almost spherical structures with low polydispersity (PDI < 0.2) and nanometric size range (z-average no more than 150 nm). In addition, all lipid vesicular systems exhibited a negative zeta potential. In vitro skin penetration and deposition experiments demonstrated that, in the case of CF with finite dose application (10 µl/cm²) and infinite dose application (160 µl/cm²), lipid vesicular systems, especially ethosomes and invasomes, compared with non-vesicular systems, can significantly improve the delivery of hydrophilic drug such as carboxyfluorescein into skin deep layers or across the skin. While in the case of mTHPC with finite and infinite dose application, most of drug accumulation was observed in the skin superficial layer for both lipid vesicular systems and non-vesicular systems. The results also revealed that the factors influencing the drug skin distribution concern the physicochemical characteristics of the drug, the choice of the vehicle formulation and the application mode applied.

In vivo quantification of photosensitizer fluorescence in the skin-fold observation chamber using dual-wavelength excitation and NIR imaging.

A major challenge in biomedical optics is the accurate quantification of in vivo fluorescence images. Fluorescence imaging is often used to determine the pharmacokinetics of photosensitizers used for photodynamic therapy. Often, however, this type of imaging does not take into account differences in and changes to tissue volume and optical properties of the tissue under interrogation. To address this problem, a ratiometric quantification method was developed and applied to monitor photosensitizer meso-tetra(hydroxyphenyl) chlorin (mTHPC) pharmacokinetics in the rat skin-fold observation chamber. The method employs a combination of dual-wavelength excitation and dual-wavelength detection. Excitation and detection wavelengths were selected in the NIR region. One excitation wavelength was chosen to be at the Q band of mTHPC, whereas the second excitation wavelength was close to its absorption minimum. Two fluorescence emission bands were used; one at the secondary fluorescence maximum of mTHPC centered on 720 nm, and one in a region of tissue autofluorescence. The first excitation wavelength was used to excite the mTHPC and autofluorescence and the second to excite only autofluorescence, so that this could be subtracted. Subsequently, the autofluorescence-corrected mTHPC image was divided by the autofluorescence signal to correct for variations in tissue optical properties. This correction algorithm in principle results in a linear relation between the corrected fluorescence and photosensitizer concentration. The limitations of the presented method and comparison with previously published and validated techniques are discussed.