Biodistribution of the photosensitizer temoporfin after in vivo topical application of temoporfin-loaded invasomes in mice bearing subcutaneously implanted HT29 tumor.

Temoporfin (mTHPC) has a great potential for the topical photodynamic therapy. However, it presents a highly hydrophobic second generation photosensitizer with low percutaneous penetration. In order to use mTHPC for dermal/transdermal delivery it is necessary to employ some of the penetration enhancement methods. In this study invasomes were used as a highly effective drug nanocarrier system to enhance its skin penetration, being composed of non-hydrogenated soybean lecithin (10% w/v), ethanol (3.3%w/v), a mixture of terpenes (1% w/v of the mixture cineole:citral:d-limonene = 45:45:10 v/v) and phosphate buffer saline up to 100% w/v. A pharmacokinetic/biodistribution study was performed in mice bearing s.c. implanted human colorectal tumor HT29 upon the application of mTHPC-loaded invasomes onto the skin above the underlying tumor. The aim was to obtain the biodistribution profile of mTHPC i.e. to gain data on mTHPC-distribution in the body (tumor, treated skin, muscle, blood, liver and untreated skin) of mice after the topical application of mTHPC-loaded invasomes. The results revealed that a significant mTHPC-amount was found in treated skin already after 2 h of incubation time. As to the tumor, significant amounts were found after 12 h, while the highest mTHPC-amount was found after 24 h. This study showed that invasomes applied onto the skin may deliver mTHPC to the tumor being necessary for PDT. Since mTHPC was also found in blood and liver, transdermal mTHPC delivery was confirmed. In conclusion, mTHPC-invasomes could be used for topical PDT of cutaneous and subcutaneous lesions, however with general photoxicity induced by systemic apsorption of mTHPC lasting only for 2 weeks. Additionally, due to systemic absorption of mTHPC after invasomes application onto the skin, they could be used transdermally for the PDT treatment of diseases, which need systemic drug absorption. However, it should be emphasized that mice were used in the study, differing in the skin properties compared to human skin. Thus, additional studies should be conducted.

A physiologically-based nanocarrier biopharmaceutics model to reverse-engineer the in vivo drug release.

Over the years, a wide variety of nanomedicines has entered global markets, providing a blueprint for the emerging generics industry. They are characterized by a unique pharmacokinetic behavior difficult to explain with conventional methods. In the present approach a physiologically-based nanocarrier biopharmaceutics model has been developed. Providing a compartmental framework of the distribution and elimination of nanocarrier delivery systems, this model was applied to human clinical data of the drug products Doxil®, Myocet®, and AmBisome® as well as to the formulation prototypes Foslip® and NanoBB-1-Dox. A parameter optimization by differential evolution led to an accurate representation of the human data (AAFE < 2). For each formulation, separate half-lives for the carrier and the free drug as well as the drug release were calculated from the total drug concentration-time profile. In this context, a static in vitro set-up and the dynamic in vivo situation with a continuous infusion and accumulation of the carrier were simulated. For Doxil®, a total drug release ranging from 0.01 to 22.1% was determined. With the time of release exceeding the elimination time of the carrier, the major fraction was available for drug targeting. NanoBB-1-Dox released 76.2-77.8% of the drug into the plasma, leading to an accumulated fraction of approximately 20%. The mean residence time of encapsulated doxorubicin was 128 h for Doxil® and 0.784 h for NanoBB-1-Dox, giving the stealth liposomes more time to accumulate at the intended target site. For all other formulations, Myocet®, AmBisome®, and Foslip®, the major fraction of the dose was released into the blood plasma without being available for targeted delivery.

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.

A dialysis-based in vitro drug release assay to study dynamics of the drug-protein transfer of temoporfin liposomes.

Today, a growing number of nanotherapeutics is utilized to deliver poorly soluble compounds using the intravenous route of administration. The drug release and the direct transfer of the active pharmaceutical ingredient to serum proteins plays an important role in bioavailability and accumulation of the drug at the target site. It is closely related to the formation of a protein corona as well as the plasma protein binding of the compound. In the present study, two in vitro drug release methods, the flow-through cell and the dispersion releaser technology, were evaluated with regards to their capability to measure a time-resolved profile of the serum protein binding. In this context, the photosensitizer temoporfin and temoporfin-loaded liposomes were tested. While in the fine capillaries of the flow-through cell a rapid agglomeration of proteins occurred, the dispersion releaser technology in combination with the four-step model enabled the measurement of the transfer of drugs from liposomes to proteins. In presence of 10% of fetal calf serum approximately 20% of the model compound temoporfin were bound to serum proteins within the first 3 h. At higher serum concentration this binding remained stable for approximately 10 h.

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.

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.

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.

Photodynamic killing of Enterococcus faecalis in dentinal tubules using mTHPC incorporated in liposomes and invasomes.

OBJECTIVES: The present in vitro study investigates the antimicrobial photodynamic efficiency of the photosensitizer 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (mTHPC) incorporated in liposomes (LIP) and highly flexible invasomes (INV) on the endodontopathogenic species Enterococcus faecalis in infected dental root canals. MATERIALS AND METHODS: A total of 48 root canals were prepared mechanically to file size ISO 50 and inoculated with E. faecalis for 48 h. In the test groups, the infected root canals were subjected to aPDT with either mTHPC linked to LIP or INV. The controls were either incubated with 1 % chlorohexidine gel (CHX, positive control) or root canals were irrigated with normal saline (NaCl, negative control). After treatment all canals were mechanically enlarged (ISO 50-110), and the debris of each filing process was subjected to bacterial culture analysis. RESULTS: Both mTHPC formulations showed a significant antimicrobial effect. A bacterial reduction by up to 3.6 log-steps was ascertained for INV directly at the root canal wall. aPDT using INV (ISO 60) was more effective than CHX, which caused a decrease in only 1.2 log-steps. It was found that both liposomal mTHPC formulations were capable to suppress E. faecalis inside the dentinal tubules up to 300 µm. CONCLUSIONS: The results show that mTHPC linked to LIP and INV is capable of efficiently reducing E. faecalis in dental root canals. CLINICAL RELEVANCE: As evidenced, E. faecalis is resistant to several conventional antibacterial treatment measures. In this context, photodynamic treatment with mTHPC delivered by INV is superior to temporary dressing with 1 % CHX gel applied for 24 h.

Photodynamic antimicrobial effect of safranine O on an ex vivo periodontal biofilm.

BACKGROUND AND OBJECTIVE: The increasing resistance of oral pathogens against antibiotic measures urgently requires new therapeutic strategies. In this context, antimicrobial photodynamic therapy (aPDT) may play a crucial part in the future. The aim of the present study was to compare the antibacterial efficiency of aPDT using the photosensitizer safranine O with that of chlorhexidine (0.2% CHX) on an ex vivo biofilm. METHODS: First the antibacterial activity of both measures against planktonic cultures of Streptococcus gordonii ATCC 33399, Streptococcus mutans ATCC 25175, Fusobacterium nucleatum ATCC 10953, Aggregatibacter actinomycetemcomitans ATCC 33384 and Porphyromonas gingivalis ATCC 33277 was observed. Then a patient specific ex vivo biofilm was established from plaque and saliva samples of patients (n = 19) with chronic periodontitis. The antibacterial effects of aPDT and of 0.2% CHX were determined on the ex vivo biofilms cultivated for 24 and 72 hours. After cultivation of the treated samples on blood agar (2 days) the results were quantified by counting the colony forming units (cfu/ml). RESULTS: Photodynamic treatment with safranine O showed a distinct antibacterial effect on F. nucleatum and P. gingivalis. Whereas S. gordonii was suppressed completely by aPDT, treatment with 0.2% CHX caused only a partial reduction. In the ex vivo biofilm model (24-hour biofilm), aPDT caused a significantly higher bacterial killing than treatment with 0.2% CHX. Compared to the untreated control, there was no significant difference on the 72-hour biofilm for both methods. CONCLUSIONS: The results show that oral-pathogenic species in planktonic solution can be suppressed significantly by aPDT with safranine O. Especially for bacteria in a 24-hour ex vivo biofilm, this method is more effective than treatment with 0.2% CHX. Both antibacterial treatments did not show any significant effect on the biofilm cultivated for 72 hours.

Long-term outcomes following Foscan®-PDT of basal cell carcinomas.

BACKGROUND AND OBJECTIVE: In a previous publication we showed that mTHPC-PDT (Foscan®-PDT) is an effective treatment of basal cell carcinomas (BCCs) in "difficult to treat" locations and presented optimized treatment parameters to reduce costs and side effects. Now we present long-term results of the same study population. STUDY DESIGN/MATERIALS AND METHODS: Following PDT of a total of 460 BCCs in 117 subjects, the patients/lesions were followed-up for a mean duration of 42 (range: 2-72) months. Two patients dropped out of follow-up; 13 patients died of unrelated causes. Recurrences were treated either by repeated PDT or other established methods. RESULTS: The sustained clearance rate was 93.7% and the overall treatment success rate was 90.7%. Kaplan-Meier analysis revealed an estimated recurrence free fraction of patients at 5 years of 95.1%, 92.4%, 85.1%, and 74.0% for the four different photosensitizer dose groups (0.06-0.15, 0.05, 0.04, and 0.03 mg/kg). High-risk lesions (recurrences, thickness >3 mm) recurred more often than low-risk ones, and recurrences mostly (>50%) occurred during the first year of follow-up. CONCLUSION: Long-term outcomes of high-dose (0.06-0.15 mg/kg) and reduced-dose (0.05 mg/kg) Foscan®-PDT in "difficult to treat" BCCs compare favorably with other methods, even in high-risk lesions (recurrent and/or thick lesions). A recommended combination of treatment parameters for low-dose therapy seems to be: 0.05 mg/kg Foscan®, 24 hours drug-light interval (DLI), fluence ≥40 J/cm(2) . Prospective randomized studies are needed to look into low-dose mTHPC-PDT of BCCs in more detail and to directly compare it with other treatments.

Wheat germ agglutinin modified liposomes for the photodynamic inactivation of bacteria.

Photodynamic inactivation (PDI) of bacteria is a promising approach for combating the increasing emergence of antibiotic resistance in pathogenic bacteria. To further improve the PDI efficiency on bacteria, a bacteria-targeting liposomal formulation was investigated. A generation II photosensitizer (temoporfin) was incorporated into liposomes, followed by conjugation with a specific lectin (wheat germ agglutinin, WGA) on the liposomal surface. WGA was successfully coupled to temoporfin-loaded liposomes using an activated phospholipid containing N-hydroxylsuccinimide residue. Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa were selected to evaluate the WGA modified liposomes in terms of bacteria targeted delivery and in vitro PDI test. Fluorescence microscopy revealed that temoporfin was delivered to both kinds of bacteria, while flow cytometry demonstrated that WGA- modified liposomes delivered more temoporfin to bacteria compared to nonmodified liposomes. Consequently, the WGA- modified liposomes eradicated all MRSA and significantly enhanced the PDI of P. aeruginosa. In conclusion, the WGA- modified liposomes are a promising formulation for bacteria targeted delivery of temoporfin and for improving the PDI efficiency of temoporfin on both Gram-positive and Gram-negative bacterial cells.

Antimicrobial peptide-modified liposomes for bacteria targeted delivery of temoporfin in photodynamic antimicrobial chemotherapy.

Photodynamic antimicrobial chemotherapy (PACT) and antimicrobial peptides (AMPs) are two promising strategies to combat the increasing prevalence of antibiotic-resistant bacteria. To take advantage of these two strategies, we integrated a novel antimicrobial peptide (WLBU2) and a potent generation II photosensitizer (temoporfin) into liposomes by preparing WLBU2-modified liposomes, aiming at bacteria targeted delivery of temoporfin for PACT. WLBU2 was successfully coupled to temoporfin-loaded liposomes using a functional phospholipid. The delivery of temoporfin to bacteria was confirmed by fluorescence microscopy and flow cytometry, thus demonstrating that more temoporfin was delivered to bacteria by WLBU2-modified liposomes than by unmodified liposomes. Consequently, the WLBU2-modified liposomes eradicated all methicillin-resistant Staphylococcus aureus (MRSA) and induced a 3.3 log(10) reduction of Pseudomonas aeruginosa in the in vitro photodynamic inactivation test. These findings demonstrate that the use of AMP-modified liposomes is promising for bacteria-targeted delivery of photosensitizers and for improving the PACT efficiency against both gram-positive and gram-negative bacteria in the local infections.

Photodynamic suppression of Enterococcus faecalis using the photosensitizer mTHPC.

BACKGROUND AND OBJECTIVES: Enterococcus faecalis is frequently found in persistent endodontic infections. In this context, the antimicrobial photodynamic therapy (aPDT) could become a modern alternative to existing antibacterial treatment approaches. The aim of this study was to investigate the effect of aPDT on E. faecalis using the photosensitizer (PS) 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (mTHPC) enriched in liposomes. MATERIALS AND METHODS: Enterococcus faecalis was cultivated in Schaedler submerged culture for 24 hours, then isolated and adjusted in PBS to 10(8) cells/ml. The bacterial suspension was pipetted into a black microtitration plate and incubated for 15 minutes in the dark with mTHPC in various concentrations (10, 30, and 50 µM). The photosensitized suspensions were subjected to laser light (652 nm) at a light fluence of 100 J cm(-2) (test group A). In addition, the suspension sensitized with 50 µM mTHPC was irradiated with 25, 50, and 75 J cm(-2) (test group B). The following controls were used: non-irradiated bacterial suspension in the absence of mTHPC (C); irradiated bacterial suspension in the absence of mTHPC (D); non-irradiated bacterial suspension incubated with mTHPC (E). Dilution series (10(0)-10(-6)) were made of all groups and applied on Schaedler agar. After anerobic cultivation (4 days), the colony-forming units (CFU/ml) were determined. RESULTS: Enterococcus faecalis was suppressed completely after incubation with 50 µM mTHPC and illumination with 100 J cm(-2). Photodynamic treatment with 10 and 30 µM mTHPC caused reduction in CFU by 5.8 and 6.7 log-units. The application of an energy fluence <100 J cm(-2) resulted in a decline of antibacterial efficiency. Irradiation of the non-photosensitized solution showed no suppressing impact. Incubation of the PS without additional irradiation caused a maximal reduction in CFU by 1.5 log-units. CONCLUSION: The results show that aPDT using the PS mTHPC incorporated in liposomes could be a new approach to adjuvant treatment of endodontic infections with E. faecalis.

Development of liposomes containing ethanol for skin delivery of temoporfin: characterization and in vitro penetration studies.

The aim of this study was to develop ethanol-containing (3.3-20%, w/v) liposomes loaded with temoporfin (mTHPC), which presents a highly hydrophobic photosensitizer with low percutaneous penetration, and to investigate their skin penetration enhancing effect. Characterization parameters of liposomes were measured by photon correlation spectroscopy, lamellarity was analyzed by cryo-electron microscopy and mTHPC-content in formulations was determined spectrofotometrically. In order to assess the stability of mTHPC-liposomes at 4 and 23 degrees C, at predetermined time intervals characterization parameters and mTHPC-content were measured. The in vitro skin penetration of mTHPC was investigated using human abdominal skin mounted in Franz cells. The results indicated that mTHPC-liposomes were of a small particle size, small polydispersity index, negative surface charge, unilamellar or oligolamellar, and of a spherical or oval shape. All liposomes were stable during 12 months' storage at 4 degrees C. Increasing the amount of ethanol in mTHPC-liposomes the skin deposition of mTHPC increased also. Liposomes without ethanol delivered the lowest amount of mTHPC into the skin, while liposomes containing 20% ethanol showed the highest penetration enhancement. In conclusion, mTHPC-liposomes containing 20% ethanol could be a promising tool for delivering temoporfin to the skin, which would be beneficial for the photodynamic therapy of cutaneous malignant or non-malignant diseases.

Development of different temoporfin-loaded invasomes-novel nanocarriers of temoporfin: characterization, stability and in vitro skin penetration studies.

A previous study revealed that the invasome dispersion containing 3.3% (w/v) ethanol and 1% (w/v) of the terpene mixture (cineole:citral:d-limonene=45:45:10, v/v=standard mixture) could significantly enhance skin penetration of the highly hydrophobic photosensitizer temoporfin (mTHPC). Invasomes enhanced mTHPC-deposition in stratum corneum (SC) compared to liposomes without terpenes and conventional liposomes, and they were efficient in delivering mTHPC to deeper skin layers [J. Control. Release 127 (2008) 271-280]. The aim of this study was to develop new mTHPC-loaded invasomes in order to further enhance the drug penetration. The ratio between d-limonene, citral and cineole was varied in the standard terpene mixture and also single terpenes were used. As a result new mTHPC-loaded invasome dispersions were prepared, characterized and investigated for stability and in vitro penetration of mTHPC into abdominal human skin using Franz diffusion cells. Invasomes were of a small particle size (<150nm), high homogeneity (<0.3), mostly unilamellar and spherical, but also deformed vesicles were detected. Invasomes containing 1% (w/v) cineole provided the highest skin penetration enhancement of mTHPC, i.e. they provided high amounts of mTHPC in the SC and deeper skin layers, indicating that also incorporation of a single terpene into invasomes could provide efficient nanocarriers of mTHPC. These invasomes could be considered as a promising tool for delivering the photosensitizer mTHPC to the skin. However, in contrast to most invasomes, being effective nanocarriers of mTHPC, there were also formulations less effective than liposomes containing 3.3% (w/v) ethanol and one formulation was less efficient than conventional liposomes.

Optimization of treatment parameters for Foscan-PDT of basal cell carcinomas.

BACKGROUND AND OBJECTIVE: Basal cell carcinomas (BCCs) are the most common form of skin cancers with high and increasing incidence rates. Photodynamic therapy (PDT) with mTHPC (Foscan) has shown to be a promising treatment alternative with good cosmetic results. The current study was aimed to determine optimal treatment parameters for this indication. STUDY DESIGN/MATERIALS AND METHODS: mTHPC-PDT was performed in 117 patients with a total of 460 BCCs with diagnosis confirmed by scratch cytology. Treatment parameters were altered as follows: Foscan dose 0.03-0.15 mg/kg, drug-light interval (DLI) 1-96 hours, total energy density 20-120 J/cm(2). Outcomes were assessed 8 weeks post-PDT following WHO guidelines. RESULTS: The overall rate of complete remissions (CR) was 96.7% and the cosmetic outcome was very good. In the largest subgroup (n=80) where low-dose Foscan was applied (0.05 mg/kg mTHPC; 48 hours DLI; 50 J/cm(2) total energy density), a CR rate of 100% with a high and narrow 95% Confidence Interval of 0.955-1.000 was achieved. Smaller variations of the treatment parameters (i.e., reducing the photosensitizer dose to 0.04 mg/kg or shortening the DLI to 24 hours) yielded similarly good results. Side effects were encountered in 52 out of 133 PDT sessions. They were more common in patients who had received high drug doses (0.06-0.15 mg/kg) and comprised mostly pain and phototoxic reactions. Three patients developed severe sunburns with subsequent scarring at the injection site following bright sunlight exposure 15-19 days after photosensitizer administration. CONCLUSIONS: The presented data suggest that mTHPC-PDT with the treatment parameters mentioned above seems to be an effective treatment option for BCCs. If sensibly applied, it is well tolerated and provides mostly excellent cosmetic results. Long-term results are yet to be evaluated.

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).

Temoporfin-loaded invasomes: development, characterization and in vitro skin penetration studies.

Temoporfin (mTHPC) is a highly hydrophobic second generation photosensitizer with low percutaneous penetration. In order to enhance its percutaneous penetration it was necessary to develop a mTHPC-loaded drug carrier system for enhanced skin delivery. mTHPC-loaded invasomes were developed, characterized and investigated for the in vitro percutaneous penetration of mTHPC into abdominal human skin using Franz diffusion cells. mTHPC-loaded invasomes were prepared using non-hydrogenated soybean lecithin (10% w/v), ethanol (3.3% w/v) and a mixture of terpenes (0.5 and 1% w/v). The invasomes obtained were of a sufficiently small particle size (<150 nm) and polydispersity index (<0.3). The particle size of invasomes increased following an increase in the amount of terpenes in the invasomes. All invasomes possessed a negative surface charge. The vesicles appeared to be unilamellar and oligolamellar, spherical and oval in shape. An interesting phenomenon was the finding that with increasing the amount of terpenes, the number of deformed vesicles in the dispersion increased. In vitro skin penetration data revealed that the invasome dispersion with 1% of the mixture of terpenes showed a significantly enhanced deposition (p<0.05) of the drug in the SC compared to liposomes without terpenes and the ethanolic solution.

Efficacy of photodynamic therapy on inflammatory signs and two selected periodontopathogenic species in a beagle dog model.

BACKGROUND: Current research aims to find alternatives to conventional methods for suppressing periodontopathogenic bacteria. Photodynamic therapy (PDT) could be a suitable treatment procedure of periodontal infections. METHODS: In the present study, the PDT method was tested with two photosensitizers, chlorine e6 and BLC1010, in an experiment on beagle dogs. The animals were infected with Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn) in all subgingival areas. After infection, we observed clinical signs of gingival inflammation, including an increase of redness and bleeding on probing. Microbiological monitoring before and after treatment was performed using polymerase chain reaction (PCR). PDT was conducted with a diode laser with a wavelength of 662 nm using a power of 0.5 W and the photosensitizers. RESULTS: The PDT procedure carried out with either of the photosensitizers caused a significant reduction in the clinical inflammation signs of redness and BOP, compared to the controls (laser only and no treatment). Furthermore, PDT with chlorine e6 caused a significant reduction in P. gingivalis-infected sites, whereas there was a lack in suppression after PDT with BLC1010. F. nucleatum could hardly be reduced with chlorine e6, and only to a certain extent with BLC 1010 and laser only. In the control groups, the Pg-infected test sites did not change. CONCLUSIONS: This study demonstrated that the photodynamic therapy using photosensitizer and a 662 nm laser light source is distinctly advantageous in reducing the periodontal signs of redness and bleeding on probing. The procedure also appears to significantly suppress P. gingivalis.

Killing of periodontopathogenic bacteria by photodynamic therapy.

BACKGROUND: The aim of this study was to evaluate a new approach for killing periodontopathogenic bacteria using photodynamic therapy (PDT). METHODS: In this study, we investigated the photosensitizers chlorin e6, BLC 1010, and BLC 1014 by three different methods for their effect in PDT on the viability of periodontopathogenic bacterial species. The methods included examination of inhibition zones on agar plates, determination of colony-forming units (CFU), and the use of a bacterial viability kit. RESULTS: Using the CFU method, we were able to demonstrate that the anaerobic bacteria Porphyromonas gingivalis, Fusobacterium nucleatum, and Capnocytophaga gingivalis can be photoinactivated completely by illumination with an intensity of 5.3 J/cm2 in the presence of 10 microM chlorin e6 and 10 microM BLC 1010. With the photosensitizers chlorin e6 and BLC 1010, we were able to induce zones of inhibition on agar plates. BLC 1014 failed to produce a zone of inhibition. The results of the bacterial viability test also showed that the photosensitizer BLC 1014 provides the lowest photodynamic effect in comparison to the others. CONCLUSION: The data collected to date suggest that photodynamic therapy with chlorin e6 and BLC 1010 is advantageous for suppressing periodontopathogenic bacteria.