ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Cell Polarity/drug effects , Light , Mesoporphyrins/chemistry , Nanoparticles/toxicity , Cell Differentiation/drug effects , Cell Differentiation/radiation effects , Cell Line , Cell Polarity/radiation effects , Humans , Macrophages/cytology , Macrophages/drug effects , Macrophages/metabolism , Nanoparticles/chemistry , Phenotype , Photosensitizing Agents/chemistry , Photosensitizing Agents/pharmacologyABSTRACT
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.
Subject(s)
Glycerol/administration & dosage , Metalloporphyrins/chemistry , Photochemotherapy , Photosensitizing Agents/administration & dosage , Polymers/administration & dosage , Acids/chemistry , Anti-Bacterial Agents/pharmacology , Cell Line, Tumor , Chromatography, Thin Layer , Copper/chemistry , Cycloaddition Reaction , Flow Cytometry , Humans , Hydrogen-Ion Concentration , Microscopy, Confocal , Oxidation-Reduction , Spectrophotometry, Ultraviolet , Staphylococcus aureus/drug effectsABSTRACT
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.
Subject(s)
Bone Neoplasms/drug therapy , Mesoporphyrins/therapeutic use , Osteosarcoma/drug therapy , Photochemotherapy , Animals , Apoptosis , Bone Neoplasms/metabolism , Cell Line, Tumor , Dose-Response Relationship, Drug , Female , Humans , Immune System , Liposomes/chemistry , Mice , Mice, Inbred BALB C , Mice, SCID , Microscopy, Confocal , Neoplasm Metastasis , Neoplasm Transplantation , Osteosarcoma/metabolism , Photosensitizing Agents/therapeutic use , Tibia/pathologyABSTRACT
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.
Subject(s)
Emulsions/therapeutic use , Liposomes/therapeutic use , Nanostructures/therapeutic use , Photochemotherapy/methods , Photosensitizing Agents/therapeutic use , Animals , Body Weight/drug effects , Emulsions/pharmacology , Female , Liposomes/chemistry , Liposomes/pharmacology , Mice , Mice, Nude , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathology , Photosensitizing Agents/pharmacologyABSTRACT
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.
Subject(s)
Boron Compounds/chemistry , Chemistry Techniques, Synthetic/methods , Photosensitizing Agents/chemical synthesis , Porphyrins/chemical synthesis , Pyrroles/chemistry , Tetrapyrroles/chemistry , Amines/chemical synthesis , Amines/chemistry , Benzaldehydes/chemical synthesis , Benzaldehydes/chemistry , Boron Compounds/chemical synthesis , Cell Line, Tumor , Halogenation , Humans , Models, Molecular , Neoplasms/drug therapy , Photochemotherapy , Photosensitizing Agents/chemistry , Photosensitizing Agents/pharmacology , Porphyrins/chemistry , Porphyrins/pharmacology , Pyrroles/chemical synthesis , Tetrapyrroles/chemical synthesisABSTRACT
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.
Subject(s)
Mesoporphyrins/pharmacokinetics , Photosensitizing Agents/pharmacokinetics , beta-Cyclodextrins/pharmacology , 2-Hydroxypropyl-beta-cyclodextrin , Animals , Blood Proteins/metabolism , Female , HT29 Cells , Humans , Kidney/metabolism , Liver/metabolism , Mesoporphyrins/pharmacology , Mesoporphyrins/therapeutic use , Mice , Muscles/metabolism , Neoplasms/drug therapy , Neoplasms/metabolism , Photochemotherapy , Photosensitizing Agents/pharmacology , Photosensitizing Agents/therapeutic use , Skin/metabolismABSTRACT
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.
Subject(s)
Drug Carriers/chemistry , Nanostructures/chemistry , Photochemotherapy , Photosensitizing Agents/chemistry , Administration, Intravenous , Animals , Cell Line , Cell Survival/drug effects , Chemistry, Pharmaceutical , Drug Compounding , Drug Design , Drug Industry/methods , Humans , Mesoporphyrins/chemistry , Mice , Microfluidic Analytical Techniques , Particle Size , Photosensitizing Agents/pharmacokinetics , Photosensitizing Agents/toxicity , Polyesters/chemistry , Polyethylene Glycols/chemistry , Tissue DistributionABSTRACT
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.
Subject(s)
Calcium Phosphates/therapeutic use , Fluorescence , Infrared Rays , Molecular Imaging/methods , Nanoparticles/therapeutic use , Neoplasms/drug therapy , Photochemotherapy , Animals , Apoptosis/drug effects , Cell Line, Tumor , Endocytosis/drug effects , Female , Humans , Light , Luminescent Measurements , Mice, Nude , Nanoparticles/ultrastructure , Neoplasms/blood supply , Neoplasms/pathology , Neovascularization, Pathologic/drug therapy , Oligopeptides/pharmacology , Scattering, Radiation , Tissue Distribution/drug effectsABSTRACT
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.
Subject(s)
Delayed-Action Preparations/chemistry , Mesoporphyrins/administration & dosage , Nanoparticles/chemistry , Photosensitizing Agents/administration & dosage , Polymethacrylic Acids/chemistry , Cell Line , Drug Delivery Systems , Humans , Mesoporphyrins/pharmacokinetics , Photosensitizing Agents/pharmacokinetics , Polyethylene Glycols/chemistryABSTRACT
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.
Subject(s)
Mesoporphyrins/administration & dosage , Mesoporphyrins/pharmacokinetics , Photosensitizing Agents/chemistry , Photosensitizing Agents/pharmacokinetics , Polyethylene Glycols/chemistry , Animals , Chemistry, Pharmaceutical , HT29 Cells , Humans , Liposomes , Mesoporphyrins/blood , Mesoporphyrins/chemistry , Mice , Optical Imaging , Photosensitizing Agents/administration & dosage , Photosensitizing Agents/blood , Tissue DistributionABSTRACT
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.
ABSTRACT
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.
Subject(s)
Liposomes/chemistry , Mesoporphyrins/administration & dosage , Mesoporphyrins/pharmacokinetics , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolism , Photosensitizing Agents/therapeutic use , Polyethylene Glycols/chemistry , Animals , HT29 Cells , Humans , Light , Mice , Nanocapsules/chemistry , Nanocapsules/ultrastructure , Neoplasms, Experimental/pathology , Particle Size , Photochemotherapy/methods , Photosensitizing Agents/pharmacokinetics , Tissue Distribution , Treatment OutcomeABSTRACT
In our study we wanted to elucidate a time frame for in vivo optical imaging of the therapeutic efficacy of photodynamic therapy (PDT) by using a multiplexed imaging approach for detecting apoptosis and vascularization. The internalization of the photosensitizer Foslip(®) into tongue-squamous epithelium carcinoma cells (CAL-27) was examined in vitro and in vivo. For detecting apoptosis, annexin V was covalently coupled to the near-infrared dye DY-734 and the spectroscopic properties and binding affinity to apoptotic CAL-27 cells were elucidated. CAL-27 tumor bearing mice were treated with PDT and injected 2 days and 2 weeks thereafter with DY-734-annexin V. PDT-induced changes in tumor vascularization were detected with the contrast agent IRDye(®) 800CW RGD up to 3 weeks after PDT. A perinuclear enrichment of Foslip(®) could be seen in vitro which was reflected in an accumulation in CAL-27 tumors in vivo. The DY-734-annexin V (coupling efficiency 30-50%) revealed a high binding affinity to apoptotic compared to non-apoptotic cells (17.2% vs. 1.2%) with a KD-value of 20 nm. After PDT-treatment, the probe showed a significantly higher (p <0.05) contrast in tumors at 2 days compared to 2 weeks after therapy (2-8 h post injection). A reduction of the vascularization could be detected after PDT especially in the central tumor areas. To detect the therapeutic efficacy of PDT, a multiplexed imaging approach is necessary. A detection of apoptotic cells is possible just shortly after therapy, whereas at later time points the efficacy can be verified by investigating the vascularization.
Subject(s)
Annexin A5/metabolism , Photochemotherapy/methods , Animals , Apoptosis/genetics , Apoptosis/physiology , Cell Line, Tumor , Female , Mice , Microscopy, FluorescenceABSTRACT
BACKGROUND: Collagen matrices as substitution for connective tissue are known to promote wound healing. Photodynamic therapy has been anecdotally associated with improved wound healing and reduced scarring. The present study investigates the impact of collagen based scaffolding material, embedded with a liposomal formulation of meta-tetra (hydroxyphenyl) chlorin (mTHPC, Foslip(®)) and photodynamic therapy on wound healing in mice. METHODS: After incision in the neck region, two different types of collagen material, previously incubated with Foslip(®) at different concentrations, were implanted followed by illumination at 652nm (10J/cm(2), 100mW/cm(2)). Mice were imaged daily up to two weeks, whereafter excision was performed and pathological analysis. RESULTS: Scab detachment was observed at day seven for controls whereas it occurred as early as three days for PDT at the lowest concentrations. In the latter conditions, final matrix remodelling could be observed as evidenced by elastin neosynthesis. CONCLUSIONS: Topical application of low dose Foslip(®) in a collagen matrix followed by illumination considerably accelerates wound healing.
Subject(s)
Mesoporphyrins/therapeutic use , Photochemotherapy/methods , Wound Healing/drug effects , Wound Healing/radiation effects , Wounds, Penetrating/drug therapy , Wounds, Penetrating/pathology , Animals , Female , Mice , Mice, Nude , Photosensitizing Agents/therapeutic use , Treatment OutcomeABSTRACT
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.
Subject(s)
Antineoplastic Agents/chemical synthesis , Drug Design , Mesoporphyrins/chemistry , Photochemotherapy , Photosensitizing Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Drug Evaluation, Preclinical , HT29 Cells , Humans , Hydroxylation , Mesoporphyrins/pharmacology , Molecular Structure , Osmium , Photosensitizing Agents/chemistry , Photosensitizing Agents/pharmacology , Porphyrins/chemistry , Structure-Activity RelationshipABSTRACT
Accurate quantification of photosensitizers is in many cases a critical issue in photodynamic therapy. As a noninvasive and sensitive tool, fluorescence imaging has attracted particular interest for quantification in pre-clinical research. However, due to the absorption of excitation and emission light by turbid media, such as biological tissue, the detected fluorescence signal does not have a simple and unique dependence on the fluorophore concentration for different tissues, but depends in a complex way on other parameters as well. For this reason, little has been done on drug quantification in vivo by the fluorescence imaging technique. In this paper we present a novel approach to compensate for the light absorption in homogeneous turbid media both for the excitation and emission light, utilizing time-resolved fluorescence white Monte Carlo simulations combined with the Beer-Lambert law. This method shows that the corrected fluorescence intensity is almost proportional to the absolute fluorophore concentration. The results on controllable tissue phantoms and murine tissues are presented and show good correlations between the evaluated fluorescence intensities after the light-absorption correction and absolute fluorophore concentrations. These results suggest that the technique potentially provides the means to quantify the fluorophore concentration from fluorescence images.
Subject(s)
Monte Carlo Method , Photosensitizing Agents/chemistry , Signal Processing, Computer-Assisted , Spectrometry, Fluorescence/methods , Absorption , Algorithms , Animals , Chromatography, High Pressure Liquid , Computer Simulation , Drug Monitoring , Female , Fluorescent Dyes/analysis , Fluorescent Dyes/chemistry , Fluorescent Dyes/pharmacokinetics , Mice , Mice, Nude , Models, Biological , Phantoms, Imaging , Photochemotherapy , Photosensitizing Agents/analysis , Photosensitizing Agents/pharmacokinetics , Rhodamines/analysis , Rhodamines/chemistry , Rhodamines/pharmacokinetics , Tissue DistributionABSTRACT
In the case of cutaneous malignant or non-malignant diseases, topical photodynamic therapy (PDT) with a temoporfin (mTHPC)-containing formulation would be advantageous. Unfortunately, mTHPC is a highly hydrophobic drug with low percutaneous absorption and novel mTHPC-loaded invasomes for enhanced skin delivery were developed. The purpose of this study was to investigate photodynamic efficacy of mTHPC-loaded invasomes in vitro in two cell lines, i.e. the human colorectal tumour cell line HT29 and the epidermoid tumour cell line A431. Invasomes are vesicles containing besides phospholipids a mixture of terpenes or only one terpene and ethanol. Dark toxicity, phototoxicity and intracellular localization of mTHPC were studied. Laser scanning microscopy indicated perinuclear localization of mTHPC. Results revealed that mTHPC-invasomes and mTHPC-ethanolic solution used at a 2µM mTHPC-concentration and photoirradiation at 20J/cm(2) were able to reduce survival of HT29 cells and especially of A431 cells, being more sensitive to PDT. In contrast to HT29 cells, where there was not a significant difference between cytotoxicity of mTHPC-ethanolic solution and mTHPC-invasomes, in A431 cells mTHPC-invasomes were more cytotoxic. Survival of about 16% of A431 cells treated with mTHPC-invasomes is very promising, since it demonstrates invasomes' potential to be used in topical PDT of cutaneous malignant diseases.
Subject(s)
Colorectal Neoplasms/drug therapy , Liposomes/chemistry , Mesoporphyrins/administration & dosage , Photochemotherapy , Photosensitizing Agents/administration & dosage , Skin Neoplasms/drug therapy , Cell Line, Tumor , Ethanol/chemistry , Humans , Light , Mesoporphyrins/toxicity , Microscopy, Confocal , Photosensitizing Agents/toxicityABSTRACT
PURPOSE: The present study investigates the efficacy of compartmental targeting in xenografted tumors treated by meta-tetra(hydroxyphenyl)chlorin (mTHPC)-mediated photodynamic therapy (PDT). The therapeutic efficacy was, furthermore, related to a regional photoinduced distribution of apoptosis and an mTHPC biodistribution profile. METHODS AND MATERIALS: Mice bearing EMT6 tumors were subjected to a single irradiation (10 J/cm(2)) of red laser light (652 nm) at different intervals after a single- (0.3 mg/kg or 0.15 mg/kg) or double-intravenous (2 × 0.15 mg/kg) injection(s) of mTHPC. Efficiency of the treatment was evaluated by monitoring tumor regrowth. mTHPC pharmacokinetics were assessed by high-performance liquid chromatography analysis of excised organs. The regional distribution of apoptosis in tumor sections was investigated with a newly developed colabelling immunohistochemistry technique. RESULTS: A fractionated double-injection protocol of mTHPC with 24-h and 3-h drug-light intervals (DLI) yielded 100% tumor cure, with tumors presenting a massive apoptosis of neoplastic cells along with a distortion of vessels. The best efficiency for a single injection (0.3 mg/kg) was about 54% tumor cure and corresponded to a DLI of 3 h. At this DLI, tumors showed apoptosis of endothelial cells in residual vessels. Concentrations of mTHPC observed in plasma and tumor for the fractionated injection were not statistically different and were less than the total drug dose in each compartment. CONCLUSIONS: The present work suggests that clinical PDT protocols with mTHPC could be greatly improved by fractionation of the drug administration. Time points should be chosen based on the intratumoral spatiotemporal drug distribution.
Subject(s)
Apoptosis/drug effects , Mesoporphyrins/therapeutic use , Neoplasms/drug therapy , Photochemotherapy/methods , Photosensitizing Agents/therapeutic use , Animals , Caspase 3/analysis , Caspase 3/immunology , Collagen Type IV/analysis , Collagen Type IV/immunology , Female , Immunohistochemistry/methods , Mesoporphyrins/pharmacokinetics , Mice , Mice, Inbred BALB C , Necrosis , Neoplasm Proteins/analysis , Neoplasm Proteins/immunology , Neoplasms/metabolism , Neoplasms/pathology , Photosensitizing Agents/pharmacokinetics , Tissue Distribution , Xenograft Model Antitumor Assays/methodsABSTRACT
In order to increase topical delivery of temoporfin (mTHPC), a highly hydrophobic photosensitizer with low percutaneous penetration, neutral, anionic and cationic flexible liposomes (i.e. flexosomes) were prepared and investigated for their penetration enhancing ability. The in vitro skin penetration study was performed using human abdominal skin mounted in Franz diffusion cells. Besides the effect of surface charge of flexosomes on skin penetration of mTHPC, also its effect on physical properties (particle size, polydispersity index, lamellarity) and physicochemical stability of vesicles was investigated. Photon-correlation spectroscopy revealed that vesicles had after preparation a small particle size and low polydispersity index, while cryo-electron microscopy confirmed that these vesicles were mostly unilamellar and of a spherical shape. Regarding stability, contrasting to anionic flexosomes showing lack of long-term stability, neutral and cationic flexosomes were stable during 9 months storage at 4 degrees C. As to the penetration enhancing ability, cationic flexosomes possessed the highest, i.e. they delivered the highest mTHPC-amount to stratum corneum and deeper skin layers compared to conventional liposomes, neutral and anionic flexosomes. In conclusion, mTHPC-loaded cationic flexosomes could be a promising tool for delivering mTHPC to the skin, which would be beneficial for the photodynamic therapy of cutaneous malignant or non-malignant diseases.