Hyperbranched Polyglycerol Loaded with (Zinc-)Porphyrins: Photosensitizer Release Under Reductive and Acidic Conditions for Improved Photodynamic Therapy.

An adaptable approach toward cleavable nanoparticle carrier systems for photodynamic therapy (PDT) is presented, comprising a biocompatible carrier loaded with multiple photosensitizer (PS) molecules related to the clinically employed PS Temoporfin, two linkers cleavable under different triggers and glyco-targeting with mannose. A synthetic pathway to stimuli responsive hyperbranched polyglycerol (hPG) porphyrin conjugates via the copper(I)-catalyzed 1,3-dipolar cycloaddition (CuAAC) or the strain-promoted alkyne-azide cycloaddition (SPAAC) has been developed. The PS 10,15,20-tris(3-hydroxyphenyl)-5-(2,3,4,5,6-pentafluorophenyl)porphyrin was functionalized with disulfide containing cystamine and acid-labile benzacetal linkers. Conjugates with reductively and pH labile linkers were thus obtained. Cleavage of the active PS agents from the polymer carrier is shown in several different release studies. The uptake of the conjugates into the cells is demonstrated via confocal laser scanning microscopy (CLSM) and flow cytometry. Finally, the antitumor and antibacterial phototoxicity of selected conjugates has been assessed in four different tumor cell lines and in cultures of the bacterium Staphylococcus aureus. The conjugates exhibited phototoxicity in several tumor cell lines in which conjugates with reductively cleavable linkers were more efficient compared to conjugates with acid-cleavable linkers. For S. aureus, strong phototoxicity was observed for a combination of the reductively cleavable and the pH labile linker and likewise for the cleavable conjugate with mannose targeting groups. The results thus suggest that the conjugates have potential for antitumor as well as antibacterial PDT.

Foscan and foslip based photodynamic therapy in osteosarcoma in vitro and in intratibial mouse models.

Current osteosarcoma therapies cause severe treatment-related side effects and chemoresistance, and have low success rates. Consequently, alternative treatment options are urgently needed. Photodynamic therapy (PDT) is a minimally invasive, local therapy with proven clinical efficacy for a variety of tumor types. PDT is cytotoxic, provokes anti-vascular effects and stimulates tumor cell targeting mechanisms of the immune system and, consequently, has potential as a novel therapy for osteosarcoma patients. This study investigated the uptake and the dark- and phototoxicity and cytotoxic mechanisms of the photosensitizer (PS) 5,10,15,20-tetrakis(meta-hydroxyphenyl) chlorine (mTHPC, Foscan) and a liposomal mTHPC formulation (Foslip) in the human 143B and a mouse K7M2-derived osteosaroma cell line (K7M2L2) in vitro. Second, the tumor- and metastasis-suppressive efficacies of mTHPC formulations based PDT and associated mechanisms in intratibial, metastasizing osteosarcoma mouse models (143B/SCID and syngeneic K7M2L2/BALB/c) were studied. The uptake of Foscan and Foslip in vitro was time- and dose-dependent and resulted in mTHPC and light dose-dependent phototoxicity associated with apoptosis. In vivo, the uptake of both i.v. administered mTHPC formulations was higher in tumor than in healthy control tissue. PDT caused significant (Foscan p < 0.05, Foslip p < 0.001) tumor growth inhibition in both models. A significant (Foscan p < 0.001, Foslip p < 0.001) immune system-dependent suppression of lung metastasis was only observed in the K7M2L2/BALB/c model and was associated with a marked infiltration of T-lymphocytes at the primary tumor site. In conclusion, mTHPC-based PDT is effective in clinically relevant experimental osteosarcoma and suppresses lung metastasis in immunocompetent mice with beneficial effects of the liposomal mTHPC formulation Foslip.

Nucleophilic Aromatic Substitution on Pentafluorophenyl-Substituted Dipyrranes and Tetrapyrroles as a Route to Multifunctionalized Chromophores for Potential Application in Photodynamic Therapy.

The application of porphyrinoids in biomedical fields, such as photodynamic therapy (PDT), requires the introduction of functional groups to tune their solubility for the biological environment and to allow a coupling to other active moieties or carrier systems. A valuable motif in this regard is the pentafluorophenyl (PFP) substituent, which can easily undergo a regiospecific nucleophilic replacement (SN Ar) of its para-fluorine atom by a number of nucleophiles. Here, it is shown that, instead of amino-substitution on the final porphyrinoid or BODIPY (boron dipyrromethene), the precursor 5-(PFP)-dipyrrane can be modified with amines (or alcohols). These dipyrranes were transformed into amino-substituted BODIPYs. Condensation of these dipyrranes with aldehydes gave access to trans-A2 B2 -porphyrins and trans-A2 B-corroles. By using pentafluorobenzaldehyde, it was possible to introduce another para-fluorine atom, which enabled the synthesis of multifunctionalized tetrapyrroles. Furthermore, alkoxy- and amino-substituted dipyrranes were applied to the synthesis of A3 B3 -hexaphyrins. The polar porphyrins that were prepared by using this method exhibited in vitro PDT activity against several tumor cell lines.

Lipid nanoemulsions and liposomes improve photodynamic treatment efficacy and tolerance in CAL-33 tumor bearing nude mice.

BACKGROUND: Photodynamic therapy (PDT) as promising alternative to conventional cancer treatments works by irradiation of a photosensitizer (PS) with light, which creates reactive oxygen species and singlet oxygen (1O2), that damage the tumor. However, a routine use is hindered by the PS's poor water solubility and extended cutaneous photosensitivity of patients after treatment. In our study we sought to overcome these limitations by encapsulation of the PS m-tetrahydroxyphenylchlorin (mTHPC) into a biocompatible nanoemulsion (Lipidots). RESULTS: In CAL-33 tumor bearing nude mice we compared the Lipidots to the existing liposomal mTHPC nanoformulation Foslip and the approved mTHPC formulation Foscan. We established biodistribution profiles via fluorescence measurements in vivo and high performance liquid chromatography (HPLC) analysis. All formulations accumulated in the tumors and we could determine the optimum treatment time point for each substance (8 h for mTHPC, 24 h for Foslip and 72 h for the Lipidots). We used two different light doses (10  and 20 J/cm2) and evaluated immediate PDT effects 48 h after treatment and long term effects 14 days later. We also analyzed tumors by histological analysis and performing reverse transcription real-time PCR with RNA extracts. Concerning tumor destruction Foslip was superior to Lipidots and Foscan while with regard to tolerance and side effects Lipidots were giving the best results. CONCLUSIONS: We could demonstrate in our study that nanoformulations are superior to the free PS mTHPC. The development of a potent nanoformulation is of major importance because the free PS is related to several issues such as poor bioavailability, solubility and increased photosensibility of patients. We could show in this study that Foslip is very potent in destroying the tumors itself. However, because the Lipidots' biocompatibility is outstanding and superior to the liposomes we plan to carry out further investigations and protocol optimization. Both nanoformulations show great potential to revolutionize PDT in the future.

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.

Synthesis of ß-functionalized temoporfin derivatives for an application in photodynamic therapy.

The synthesis of novel ß-functionalized derivatives of the clinically used photosensitizer Temoporfin has been achieved by nucleophilic addition reactions to a corresponding diketo chlorin. The ß-substituted dihydroxychlorin products exhibit a strong absorption in the red spectral region, a high singlet oxygen quantum yield, and were found to be highly effective in in vitro assays against HT-29 tumor cells.

Uredinorubellins and caeruleoramularin, photodynamically active anthraquinone derivatives produced by two species of the genus ramularia.

Both the phytopathogenic fungus Ramularia collo-cygni and the hyperparasite R. uredinicola biosynthesize a number of red and yellow anthraquinone derivatives called rubellins. The new compounds uredinorubellins I and II, which were isolated from R. uredinicola, contribute to understanding the biosynthesis pathway that leads from simple anthraquinones to the rubellins. In addition, we isolated for the first time such simple compounds as chrysophanol and helminthsporin from both Ramularia species. A blue compound isolated from the mycelium of R. collo-cygni was revealed to be a unique 9,4-anthracenedione derivative. Structure elucidation by (1)H and (13)C NMR of the new but unstable compound caeruleoramularin was possible only by feeding the fungus different labeled (13)C acetates. The photodynamic activity of the uredinorubellins was comparable to rubellin D, whereas chrysophanol and caeruleoramularin did not display such activity.

Topical application of temoporfin-loaded invasomes for photodynamic therapy of subcutaneously implanted tumours in mice: a pilot study.

Temoporfin (mTHPC) represents a very potent second-generation synthetic photosensitizer. It has shown to be effective in the photodynamic therapy of early or recurrent oral carcinomas, in the palliative treatment of refractory oral carcinomas and in the treatment of primary non-melanomatous tumours of the skin of the head and neck. Until now for all positive findings an intravenous application of the photosensitizer was mandatory. In the case of cutaneous malignant or non-malignant diseases a topical application of the drug onto the site of the disease followed by illumination, would be advantageous. Unfortunately, mTHPC is a highly hydrophobic drug with a low percutaneous absorption. The purpose of this experiment was to investigate the photodynamic efficacy of novel mTHPC-loaded invasomes after their topical application onto the skin of mice bearing the subcutaneously implanted human colorectal tumour HT29 followed by photoirradiation. Invasomes are vesicles containing in addition to phospholipids a mixture of terpenes (cineole, citral and d-limonene) or only one terpene (citral) and ethanol, as penetration enhancers. This was a pilot study since until now no data are available about the efficacy of mTHPC in the photodynamic therapy of HT29 tumours after its topical application. The aim of this experiment was to investigate whether a mTHPC-loaded invasome formulation can reduce tumour size by photodynamic therapy or at least to find a formulation slowing down tumour growth compared to the control group (mice without any treatment). The groups of mice treated with mTHPC-invasomes containing 1% of the terpene mixture prior to photoirradiation showed a significantly smaller (p<0.05) tumour increase compared to control groups (mice without any treatment and mice only photoirradiated).

Photodynamic therapy with intratumoral administration of Lipid-Based mTHPC in a model of breast cancer recurrence.

BACKGROUND AND OBJECTIVES: Generalized skin sensitization is a main drawback of photodynamic therapy with systemic administration of photosensitizers. We have evaluated the potential use of an intratumoral injection of a liposomal formulation of mTHPC (Foslip) in a mouse model of local recurrence of breast cancer. MATERIALS AND METHODS: Mice were directly injected into the tumor (IT) with 25 microl of a Foslip suspension (0.15 mg/ml) and illumination (652 nm, 20 J/cm(2)) was performed at different time points with pathological assessment after 48 hours. In a parallel mice series plasma samples were obtained at different endpoints after IT Foslip injection for HPLC analysis and the tumors were subjected in toto to macrofluorescence imaging. Fluorescence polarization measurements were conducted in vitro to estimate the rate of sensitizer redistribution from liposomes. RESULTS: Optimal, albeit partial, cure rates were obtained at 24 hours post-sensitizer and uninistration. Inhomogeneous and weak fluorescence was observed at early time points and became maximal at 24 hours. Plasma levels of mTHPC increased until 15 hours. Fluorescence polarization measurements showed a slow sensitizer transfer from liposomes to model membranes. DISCUSSION AND CONCLUSION: The weak intratumoral fluorescence at early time points could be explained by concentration quenching within the liposomes as evidenced from fluorescence polarization studies. Progressive mTHPC redistribution from liposomes and its further incorporation into tumor tissue resulted in fluorescence build-up over time with a maximum at 24 hours post-injection. This correlates perfectly with the best therapeutic effect at this time point. The absence of total cure can be attributed to inhomogeneous photosensitizer distribution. mTHPC is reabsorbed into the blood stream but the total administered amount is much reduced as opposed to systemic administration so that repeated PDT sessions might be favorable in terms of side effects and tumor response.

Pre-/post-functionalization in dipyrrin metal complexes - antitumor and antibacterial activity of their glycosylated derivatives.

A post-functionalization route to tris(dipyrrinato) metal complexes is presented giving access to a range of new complexes relevant in the context of medicinal inorganic chemistry. A pentafluorophenyl group in the meso-position of the dipyrrin ligand serves as an anchor for the connection with alcohols and thiocarbohydrates. The photochemotherapeutic activity of the complexes has been assessed in cellular assays with tumor cell lines and against the Gram-positive bacterium S. aureus. Finally, it is shown that this post-functionalization is also applicable to other dipyrrinato metal complexes.

Photodynamic activity of Temoporfin nanoparticles induces a shift to the M1-like phenotype in M2-polarized macrophages.

The monocyte/macrophage cell lineage reveals an enormous plasticity, which is required for tissue homeostasis, but is also undermined in various disease states, leading to a functional involvement of macrophages in major human diseases such as atherosclerosis and cancer. We recently generated in vivo evidence that crystalline, nonfluorescent nanoparticles of the hydrophobic porphyrin-related photosensitizer Aluminum phthalocyanine are selectively dissolved and thus may be used for specific fluorescent labelling of rejected, but not of accepted xenotransplants. This led us to hypothesize that nanoparticles made of planar photosensitizers such as porphyrins and chlorins were preferentially taken up and dissolved by macrophages, which was verified by in vitro studies. Here, using an in vitro system for macrophage differentiation/polarization of the human monocyte THP-1 cell line, we demonstrate differential uptake/dissolution of Temoporfin-derived nanoparticles in polarized macrophages, which resulted in differential photosensitivity. More importantly, low dose photodynamic sensitization using Temoporfin nanoparticles can be used to trigger M1 re-polarization of THP-1 cells previously polarized to the M2 state. Thus, sublethal photodynamic treatment using Temoporfin nanoparticles might be applied to induce a phenotypic shift of tumor-associated macrophages for the correction of an immunosuppressive microenvironment in the treatment of cancer, which may synergize with immune checkpoint inhibition.

Fluorescence and absorption assessment of a lipid mTHPC formulation following topical application in a non-melanotic skin tumor model.

Although the benefits of topical sensitizer administration have been confirmed for photodynamic therapy (PDT), ALA-induced protoporphyrin IX is the only sensitizer clinically used with this administration route. Unfortunately, ALA-PDT results in poor treatment response for thicker lesions. Here, selectivity and depth distribution of the highly potent sensitizer meso-tetra(hydroxyphenyl)chlorin (mTHPC), supplied in a novel liposome formulation was investigated following topical administration for 4 and 6 h in a murine skin tumor model. Extraction data indicated an average [+/- standard deviation (SD)] mTHPC concentration within lesions of 6.0(+/-3.1) ngmg tissue with no significant difference (p<0.05) between 4- and 6-h application times and undetectable levels of generalized photosensitivity. Absorption spectroscopy and chemical extraction both indicated a significant selectivity between lesion and normal surrounding skin at 4 and 6 h, whereas the more sensitive fluorescence imaging setup revealed significant selectivity only for the 4-h application time. Absorption data showed a significant correlation with extraction, whereas the results from the fluorescence imaging setup did not correlate with the other methods. Our results indicate that this sensitizer formulation and administration path could be interesting for topical mTHPC-PDT, decreasing the effects of extended skin photosensitivity associated with systemic mTHPC administration.

Tumor selectivity at short times following systemic administration of a liposomal temoporfin formulation in a murine tumor model.

Meso-tetra(hydroxyphenyl)chlorin (mTHPC) (INN: Temoporfin) is one of the most potent photodynamically active substances in clinical use. Treatment protocols for Temoporfin-mediated photodynamic therapy often rely on drug-light intervals of several days in order for the photosensitizer to accumulate within the target tissue, though tumor selectivity is limited. Here, the mTHPC localization was studied at 2-8 h following systemic administration of a liposomal Temoporfin formulation (0.15 mg kg(-1) b.w.) in HT29 human colon adenocarcinoma in NMRI nu/nu mice. Photosensitizer distribution within tumor and internal organs was investigated by means of high performance liquid chromatography following chemical extraction, as well as in situ fluorescence imaging and point-monitoring fluorescence spectroscopy. For tumor tissue, the Temoporfin concentrations at 4 h (0.16+/-0.024 ng mg(-1)) and 8 h (0.18+/-0.064 ng mg(-1)) were significantly higher than at 2 h (0.08+/-0.026 ng mg(-1)). The average tumor-to-muscle and the tumor-to-skin selectivity were 6.6 and 2, respectively, and did not vary significantly with time after photosensitizer injection. In plasma, the Temoporfin concentration was low (0.07+/-0.07 ng mg(-1)) and showed no significant variation with time. Our results indicate a rapid biodistribution and clearance from the bloodstream. Within the same type of organ, data from both fluorescence methods generally exhibited a significant correlation with the extraction results.

Fluorescence monitoring of a topically applied liposomal Temoporfin formulation and photodynamic therapy of nonpigmented skin malignancies.

Meso-tetra(hydroxyphenyl)chlorin (mTHPC) (INN: Temoporfin) is a potent photodynamically active substance in clinical use today. Usually, the substance is given systemically and a known drawback with this administration route is a prolonged skin light sensitization. For the first time to our knowledge, a liposomal Temoporfin gel formulation for topical application was studied in connection with photodynamic therapy (PDT) of nonpigmented skin malignancies in humans. Intervals of 4 hr between drug administration and light irradiation were used. Sensitizer distribution within tumor and surrounding normal skin was investigated by means of point monitoring and imaging fluorescence spectroscopy before, during, and after PDT, showing high tumor selectivity. Furthermore, the bleaching of Temoporfin was studied during the PDT procedure by monitoring the fluorescence following excitation by using a therapeutic light. A 30-35% light-induced photometabolization was shown. No pain occurred during or after treatment. It was also observed that the treated area did not show any swollen tissue or reddening, as is often seen in PDT using topical delta-aminolevulinic acid. On controlling the patients one week after treatment, healing progress was observed in several patients and no complications were registered.

Photodynamic therapy of feline cutaneous squamous cell carcinoma using a newly developed liposomal photosensitizer: preliminary results concerning drug safety and efficacy.

BACKGROUND: Squamous cell carcinomas are common skin tumors in cats. We investigated photodynamic therapy (PDT) using a new liposomal photosensitizer as a minimally invasive, effective treatment that can be easily performed while achieving good cosmetic results. AIM: The goal of this study was to assess and describe possible toxicities using a liposomal formulation of the photosensitizer meta-(Tetrahydroxyphenyl)Chlorin (m-THPC) and investigate if favorable pharmacokinetics translate into favorable tumor response and control. ANIMALS: Eighteen client-owned cats with 20 spontaneous cutaneous squamous cell carcinomas were included in the study. METHODS: PDT was performed using a new, liposomal formulation of the photosensitizer. Toxicity, tumor response, and tumor control were evaluated retrospectively. RESULTS: No general adverse effects were observed in cats treated with the new liposomal formulation. Mild local toxicity such as erythema and edema were seen in 15% of the patients. All cats responded to therapy, with a complete response rate of 100%. The overall 1-year control rate was 75%. The tumor recurrence rate was 20% with a median time to recurrence of 172.25 +/-87.1) days. CONCLUSIONS AND CLINICAL IMPORTANCE: A new liposomal photosensitizer was successfully used for squamous cell carcinoma in cats and was well tolerated. There were no systemic adverse effects observed with the liposomal formulation. The favorable pharmacokinetics of the liposomal drug resulted in a favorable tumor response.

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.

Multifunctional calcium phosphate nanoparticles for combining near-infrared fluorescence imaging and photodynamic therapy.

Photodynamic therapy (PDT) of tumors causes skin photosensitivity as a result of unspecific accumulation behavior of the photosensitizers. PDT of tumors was improved by calcium phosphate nanoparticles conjugated with (i) Temoporfin as a photosensitizer, (ii) the RGDfK peptide for favored tumor targeting and (iii) the fluorescent dye molecule DY682-NHS for enabling near-infrared fluorescence (NIRF) optical imaging in vivo. The nanoparticles were characterized with regard to size, spectroscopic properties and uptake into CAL-27 cells. The nanoparticles had a hydrodynamic diameter of approximately 200 nm and a zeta potential of around +22mV. Their biodistribution at 24h after injection was investigated via NIRF optical imaging. After treating tumor-bearing CAL-27 mice with nanoparticle-PDT, the therapeutic efficacy was assessed by a fluorescent DY-734-annexin V probe at 2 days and 2 weeks after treatment to detect apoptosis. Additionally, the contrast agent IRDye® 800CW RGD was used to assess tumor vascularization (up to 4 weeks after PDT). After nanoparticle-PDT in mice, apoptosis in the tumor was detected after 2 days. Decreases in tumor vascularization and tumor volume were detected in the next few days. Calcium phosphate nanoparticles can be used as multifunctional tools for NIRF optical imaging, PDT and tumor targeting as they exhibited a high therapeutic efficacy, being capable of inducing apoptosis and destroying tumor vascularization.

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.

Nanocarriers for photodynamic therapy-rational formulation design and medium-scale manufacture.

The development and manufacture of novel nanocarriers for drug delivery has proved challenging with regards to scale-up and pharmaceutical quality. Polymeric nanocarriers composed of poly(lactic-co-glycolic acid)-b-poly(ethylene glycol) (PLGA-PEG) were prepared and the photosensitizer meso-tetrakis(3-hydroxyphenyl) chlorin (mTHPC) was effectively encapsulated. Furthermore, the interplay of various process and formulation parameters and their impact on the most important product specifications were investigated by using a factorial design and a central composite design in a microfluidic manufacturing process. These nanoparticles for intravenous administration with a size of 97 ± 0.13 nm, narrow size distribution, and an encapsulation efficiency of more than 80% were produced at high throughput. In vitro stability and in vitro drug release testing were applied for quality control purposes. Finally, the toxicity of the photosensitizer was tested in vitro. The cytotoxicity was successfully reduced while the efficacy of the formulation was maintained. First observations using in vivo imaging suggest effective distribution of the nanocarrier system after injection into rodents. Thus, further in vivo testing of the beneficial effects of nanoencapsulation into the matrix system and its formulation will be considered for the delivery of mTHPC to tumor tissues during photodynamic therapy.

Factors affecting the selectivity of nanoparticle-based photoinduced damage in free and xenografted chorioallantoïc membrane model.

BACKGROUND: Photodynamic therapy (PDT) is a minimally invasive treatment modality for selective destruction of tumours. Critical anatomical structures, like blood vessels in close proximity to the tumour, could be harmed during PDT. PURPOSE: This study aims to discriminate the photoinduced response of normal and cancerous tissues to photodamage induced by liposomal formulations of meta-tetra(hydroxyphenyl)chlorin (mTHPC). METHODS: Normal vascular and cancerous tissues were represented, respectively, by free and xenografted in vivo model of chick chorioallantoïc membrane (CAM). Eggs received an intravenous administration of plain (Foslip®) or stabilised formulations (Fospeg®). Drug release and liposome destruction were, respectively, determined by photoinduced quenching and nanoparticle tracking analysis. PDT was performed at different drug-light intervals (DLI) with further assessment of photothrombic activity, tumoritropism and photoinduced necrosis. RESULTS: Compared to Foslip®, Fospeg® demonstrated significantly higher stability, slower drug release, better tumoricidal effect and lower damage to the normal vasculature at already 1 h DLI. DISCUSSION: This work suggests that nanoparticle-based PDT selectivity could be optimised by analyzing the photoinduced damage of healthy and tumour tissues. CONCLUSION: In fine, Fospeg® appeared to be the ideal candidate in clinical context due to its potential to destroy tumours and reduce vascular damage to normal tissues at short DLI.