The present study shows a novel proposal for the topical delivery of a photosensitizer, namely a new drug belonging to the chlorin class, for use in Photodynamic Therapy (PDT). Nanoparticles of lyotropic liquid crystals characterized as hexagonal phase and loaded with the chlorin derivative were developed. These systems were characterized by spectrofluorimetric, dynamic light scattering, and small angle X-ray diffraction (SAXRD) analyses. In vitro and in vivo penetration studies were performed using animal model membranes. The studied system remained stable during turbidity experiments, and it was demonstrated that it has a size particle of 161 Â± 4 nm and a polidispersivity index of 0.175 Â± 0.027, which are adequate for the desired application. Furthermore, studies of SAXRD studies proved that the liquid crystalline structure remained stable after drug loading. Gel chromatography assay evidenced that the encapsulation rate was higher than 50%. In vitro and in vivo skin retention studies confirmed that a larger amount of drug was retained by the nanodispersion, compared to the control formulation. Fluorescence microscopic study also demonstrated a higher biodistribution of the chlorin derivative in the skin layers. Takes together, the results show the potential of the nanodispersion for the delivery of the photosensitizer into the skin, which is crucial condition for successful topical PDT.
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Photodynamic therapy (PDT) is an emerging technology for the important therapeutic options concerning the management of neoplasic and non-neoplasic diseases (1, 2). PDT is based on the administration of a photosensitizing drug (PS) and its selective retention in the malignant tissue (3, 4). The PS can be administrated systemically, locally, or topically (5). The last step in PDT is the activation of the PS by application of light at a wavelength that matches the PS absorption spectrum (6). Photophysical reactions take place in this situation, which in turn results in cell death due to the production of free radicals and/or reactive oxygen species, especially singlet oxigen (1O2) (7, 8).
The major limitation of topical PDT is the poor penetration of PSs through biological barriers, like the skin. A series of PSs are under study in PDT experiments; however, most of them are relatively hydrophobic and have low capacity of accumulation in the target tissue. Recently, studies have focused on the development of different strategies to overcome these difficulties, including the use of nanocarriers (9), liposomes (10), ethosomes (11), invasomes (12), liquid crystals (13) and magnetic nanoparticles (14), among others, for the delivery of PSs and their precursors to the target tissue.
Lyotropic liquid crystals combine the properties of a crystalline solid with those of an isotropic liquid (15). Several research groups have been dedicated to the study of these systems, which are excellent vehicles for a variety of drugs due to their ideal structural properties, once they can accommodate the drug within both their aqueous and lipid domains (16, 17).
Reverse hexagonal phase composed of monoolein has been shown to increase topical drug delivery (18, 19). Carr et. al. (20) have studied these systems and concluded that some components of Myverol (commercial monoolein) could enhance the passive penetration of nicotine into the human stratum corneum, thus demonstrating the effectiveness of this vehicle for transdermal drug delivery. Liquid crystalline phases containing monoolein increased the skin delivery of cyclosporine A, both as a bulk phase (21) and as a nanodispersed formulation (18). Further studies have shown that therapy with topical vitamin K could be improved by using monoolein-based systems (19).
In the present study, we propose a delivery system for PDT based on a liquid crystal nanodispersion, aiming at improving the delivery of chlorin derivatives to the skin. To this end, a nanodispersion of reverse hexagonal phase loaded with the photosensitizer was developed and its in vitro and in vivo topical applications were evaluated.
MATERIALS AND METHODS
Materials Chlorin derivatives (Figure 1) were synthesized according to a procedure described in the literature (22) and were used as a diastereomeric mixture. Briefly, these compounds were obtained through a Diels-Alder reaction where the reaction in the ring A of protoporphyrin IX dimethyl ester (PpIXDME) yielded chlorin A, whilst the reaction in ring B of PpIXDME yielded chlorin B. Since these chlorin derivatives are very similar in terms of polarity, we decided to use them as a diastereomeric mixture (chlorin A + chlorin B) in all studies, and only one purification step was accomplished, in order to remove the by-products. The mixture chlorine A + chlorine B is referred to as PS in the following sections.
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Monoolein (Myverol 18-99Â®) was purchased from Quest International (USA); oleic acid (OA) and octanol were obtained from Sigma-Aldrich (Sao Paulo, Brazil); methanol was acquired from Bdick & Jackson (B & J ACS / HPLC Certified Solvent). Sephadex LH20 was provided by GE Healthcare. Water was purified using a Millipore milli-Q water system (Millipore Corporation).
Analytical methodology for chlorin quantification The PS was assayed by spectrofluorometry using a Fluorolog 3 Triax 550 (Edson, NJ, USA) apparatus working, at 400 and 670 nm of excitation (ï¬exc) and emission (ï¬em), respectively, using a bandwidth of 1 nm (22, 23). A methanolic solution of chlorin (25 mg/mL) was initially prepared, from which serial dilutions were made, in order to obtain methanolic standard solutions with concentrations ranging from 0.05 to 0.3 Î¼g/mL. An analytical method was developed and evaluated with respect to linearity, precision, accuracy, limits of detection (LOD), and lower limit of quantification (LLOQ). The LOD was the basis of signal-to-noise ratios (S/N) of 3:1, and the LLOQ was considered as the minimum concentration at which chlorin was quantified with acceptable linearity r â‰¥ 0.99 (S/N 10:1). Precision and accuracy determinations were performed for both intra-day and inter-day measurements by means of repeated analysis of three chlorin concentrations determined on the same day and on three different days, respectively (24). The stability of the samples containing the PS was also assessed. To this end, PS solutions of different concentrations were solubilized in either methanol or phosphate buffer pH 7.2 containing polyssorbate 80 at 2%, and evaluated at 12 and 120 h after preparation by spectrofluorometry. The experiments were performed in triplicate. Porcine ear skin was used as a model skin for the in vitro studies. Therefore, it was necessary to evaluate whether porcine ear skin would affect the PS fluorescence spectrum. So, the outer skin of pig ears was removed from recently killed animals with the aid of a forceps and a scalpel, followed by removal of the fatty tissue that remained in the skin. Skin sections were dermatomed to 500 Î¼m (Dermaton, Nouvag, Switzerland). An area of about 1.44 cm2 skin was measured and then fragmented into small pieces. The fragments were dipped into drug methanolic solution, mixed in a Turrax instrument for 1 min, and then subjected to ultrasonic bath for 24 min. The samples were centrifuged for 10 min at 1901 g, and the supernatant was analyzed by spectrofluorometry in the same conditions as those employed for the PS method.
Preparation of the formulations The hexagonal liquid crystalline phase nanodispersion was prepared using a previously published method (18). Briefly, the PS (1 mg) was dissolved in 0.3 g of the lipid phase (OA:MO mixture at a 2:8 ratio), and 2.7 g of the citrate buffer (pH 6) containing 1.5% Poloxamer 407 was then added. The system was allowed to equilibrate at room temperature for 24 h and was then vortex-mixed for 2 min and sonicated in ice-bath at 10 kHz for 2 min. The obtained mixture was centrifuged at 1901 g for 10 min and then filtered using a 0.8 Î¼m porous membrane. The control formulation was consisted of a PS in polyethylene glycol (0.3 mg/g) obtained by vortex-mixing the PS in this solvent for 5 min at 3.000 rpm, followed by centrifugation at 1901 g for 10 min.
Physicochemical characterization of the nanodispersion
a) Polarized light microscopy The texture of the reverse hexagonal liquid crystalline phase was observed under an optical microscope Axioplan 2 (Zeiss, Germany) equipped with a polarizing filter and coupled to a digital camera Axiocan HRc (Zeiss, Germany) equipped with a video system and automatic image acquisition. The observations were accomplished at 25 °C and 37 °C by using a hotplate model THMSG 600 (Link, England) coupled to the polarized light microscope.
b) Turbidimetric analysis The samples were analyzed at 25 Â°C and the values of apparent absorbance at 410 nm were obtained on a spectrophotometer (FentoScan, Brazil). Non-loaded and PS-loaded hexagonal liquid crystalline phase nanodispersions were diluted in citrate buffer pH 6.0 (1:50) and then analyzed for 72 h. The diluted samples were packed and stored in a quartz cuvette of 0.5 cm light path, to prevent disruption of the system during reading due to sample manipulation. The experiments were performed in triplicate.
c) Dynamic light scattering The particle size and polydispersity index (Pdl) of the hexagonal liquid crystalline phase nanodispersion were analyzed by the light scattering method (DLS) in a Zetasizer 3000 HSA apparatus (Malvern Instruments) using 10mW HeNe laser operating at 633 nm with an incidence detection angle of 90°, at 25 Â°C. Measurements of the position within the cuvette were automatically determined by the software. The samples (n= 3) were prepared by four different processes: (i) Nanodispersions without PS, processed by centrifugation at 1901 g for 10 min and filtration using a 0.8 Î¼m porous membrane; (ii) Nanodispersions containing PS and processed by centrifugation at 1901 g for 10 min; (iii) Nanodispersions containing PS and processed by filtration using a 3 Î¼m porous membrane; (iv) Nanodispersion containing PS chlorin and processed in the same as (i).
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d) Small angle X-ray diffraction (SAXRD) To characterize the liquid crystalline structure of the dispersed particles, small-angle synchrotron radiation X-ray diffraction (SAXRD) measurements were performed at the Brazilian Synchrotron Light Laboratory (LNLS) in Campinas, SP, Brazil, using the D12A-SAXS beam line. The selected wavelength was 0.1488 nm. Curves of scattered intensities curves were recorded using a two-dimensional, position-sensitive MARCCD detector (Rayonix LLC, Evanston, IL, USA) located at 803.3 mm from the sample, and an ionization detector was responsible for monitoring the intensity of the incident beam. The measurements were carried out with an exposure time of 5 min, at 25 °C. The data were corrected by detector homogeneity, incident beam intensity, sample absorption, and dark noise and blank (buffer solution containing Poloxamer) subtraction. Non-loaded and PS-loaded hexagonal phase nanodipersions were analyzed.
e) Analysis of the encapsulation degree by gel chromatography Non-encapsulated PS was removed from the hexagonal liquid crystalline phase nanodispersion by size exclusion chromatography using a Sephadex LH-20 column (3.5 cm diameter, measuring 17 cm after packaging of the column). The samples (500 ÂµL) were placed on the Sephadex column and eluted using purified water as the mobile phase. The eluted fractions were monitored by turbidity measurements at 410 nm using a FEMTO 800XI spectrophotometer (Sao Paulo, Brazil). Around 30 fractions were collected, 1 mL aliquots of each fraction were lyophilized, to remove water. After the lyophilization process, the samples were solubilized in 3 mL of methanol and assayed for the PS by spectrofluorimetry (ï¬exc= 400 nm, ï¬em= 670 nm).
In vitro skin retention studies Skin from the outer portion of porcine ears was dissected with the aid of a scalpel and then dermatomed (~500 Âµm). The skin was stored at -20Â°C and used within one month. For the experiments, the skin sections were placed on a Franz cell diffusion (diffusion area of 0.79 cm2) with stratum corneum (SC) facing the donor compartment, which was filled with 100 Î¼L of hexagonal phase nanodipersions or control formulation (n= 6 for each preparation). The receiver compartment was filled with 3 mL of 100 mM phosphate buffer pH 7.2 (Â± 0.2) containing 2% of polyssorbate 80, to improve PS solubility in the receptor medium. The system was kept at 37 Â°C by means of a thermostatic water bath. After the time of experiment (4, 8, or 12 h), the system was turned off, the skin surfaces were removed and washed with distilled water, and the excess water was removed with a piece of cotton. PS penetration into the skin layers was assessed as described before (21). In order to separate SC and epidermis plus dermis (E+D), the tape stripping process was applied using 15 tapes (DurexÂ® 3M), considering that the first one was discarded and the following one was dipped in a Falcon tube containing 5 mL of methanol. These were vortex-mixed and bath-sonicated for 2 and 30 min, respectively. The samples were filtered using a 0.45 Î¼m porous membrane. Next, the remaining skin tissue was cut into small pieces and immersed into 5 mL methanol, vortex-mixed for 1 min, bath-sonicated for 30 min, and then filtered in the same way as mentioned above. Both methanolic samples were assessed by spectrofluorimetry (ï¬exc= 400 nm, ï¬em= 670 nm), in order to quantify the PS concentration. The recovery rate imposed by the method had been previously tested using small pieces of porcine ear skin measuring 1.44 cm2, to which 30 ÂµL PS methanolic solution at 12.5 Âµg/mL were added. The same procedure was applied in the absence of the skin, and the PS concentrations were assessed and compared by spectrofluorimetry.
In vivo skin retention studies These studies were conducted according to the methodology standardized by De ROSA et al. (2003) (25) and LOPES et al. (2006) (21). The experiment employed hairless mice (male/female six to eight weeks old) strain HRS/J, obtained from Jackson Laboratories (Bar Harbor, ME, USA), housed at controlled temperature (24-26Â°C) and daily 12:12h light/dark cycles with food and water ad libitum. The in vivo protocol was approved by the University of São Paulo Animal Committee on Care and Handling (Authorization number 10.1.160.53.0). Animals were separated into 2 groups (6 animals per group), namely the control group and the hexagonal liquid crystalline phase nanodispersion group. PS topical applications were performed on the back of healthy hairless mice using 150 Î¼L of the formulations. After 8 h, the mice were euthanized by rising carbon dioxide vapor, and the topical application area in the skin was dissected for PS quantification. The PS present in the skin was then extracted as described for in vitro skin retention and assessed by spectrofluorimetry (Î»exc= 400 nm, Î»em= 670 nm).
Fluorescence microscopy Cross sections of skin samples obtained at the end of the in vivo experiments were embedded in a Tissue-TeKÂ® matrix (O.C.T compound) and frozen at -17 Â°C, for visualization of the skin penetration of PS by fluorescence microscopy. Then, the samples were cut into vertical slices of 30 Âµm thickness with the aid of a cryostat (HM550, Microm, Heidelberg, Germany) and observed under a fluorescence microscope (AxiosKop 2 plus, Carl Zeiss, Germany) operating with 395 and 440 nm band-pass excitation and emission filter, respectively. The obtained images were recorded using a digital camera device (AxioCam HR, Carl Zeiss, Germany).
Statistical analyses In vitro and in vivo studies were analyzed using non-parametric tests (Student T- Test). A 0.05 level of probability was taken as the level of significance (Pï€¼0.05).
RESULTS AND DISCUSSION
Firstly, the UV spectrum of the chlorin derivative methanolic solution was recorded and it showed that this PS has a maximum absorption peak at 400 nm (the Soret band). So this was chosen as the excitation wavelength, which is consistent with previous research using compounds belonging to the chlorin class (22, 23). The fluorescence response was used as a quantification method. Good linearity was obtained in the concentration range from 0.08 - 0.4 Âµg/mL (r= 0.99). LOD and LLOQ were determined as 6 and 12 ng/mL, respectively. Additionally, intra and inter-day precision assays demonstrated that variation coefficients were lower than 5%. Accuracy was higher than 94% in the inter-day assay. The samples containing PS in either methanolic solution or phosphate buffer showed adequate stability, showing that sample solvent does not significantly change the stability of the analyte during the whole experiment period (26).
Physicochemical characterization of the nanodispersion
a) Polarized light microscopy Polarized light microscopy of liquid crystalline hexagonal phases containing PS or not revealed a fan-like texture, typical of hexagonal phase systems (Figures 2A, 2C, and 2D), indicating that the presence of chlorin derivative and Poloxamer did not change the characteristics of the systems. In addition, it was observed that the increase in temperature from 25 °C to 37 °C did not destabilize the structure of the system (Figure 2B). After dispersion of the hexagonal phase by sonication, the system resulted in a milky and low-viscosity formulation (Figure 2E), as already reported previously (19). The stability of the system was maintained after 30 days of sample preparation.
b) Turbidimetric analysis Turbidimetry can be used to characterize particle size distribution and verify the stability of colloidal suspensions (27).
Figure 3 shows the stability of the hexagonal liquid crystalline phase nanodispersion containing PS or not, during a period of 72 h. The presence of PS in the nanodispersion leads to smaller variation in the absorbance at 410 nm compared to the nanodispersion without PS, which may indicate that PS increases system stability due to formation of a more rigid structure.
c) Light scattering:
Table 1 summarizes the results for particle size of hexagonal liquid crystalline phase nanodispersions obtained by different processes. The preparation method consisting of a combination of filtration and centrifugation (groups 1 and 4) resulted in smaller particle size, about 160 nm. The filtration process alone was not able to promote standardization of appropriate particle size (group 3), while the centrifugation process (group 2) gave rise to slightly larger particles compared to groups 1 and 4. There were no significant differences between groups 1 and 4 in terms of mean particle size and polidispersity, so the addition of PS did not affect particle size when the hexagonal liquid crystalline phase nanodispersion was submitted to centrifugation and filtration, as already described. According to Lopes et al. (2006), the sonication process in the case of the hexagonal phase consisting of monoolein-water-poloxamer leads to the formation of hexagonal phase nanodispersion, with particles with diameter around 200 nm. The systems obtained herein displayed a profile similar to those obtained with incorporation of other drugs (18, 19). The poloxamer 407 copolymer was applied in the hexagonal liquid crystalline phase nanodispersion, and its concentration may alter the particle size, as already demonstrated by others (28, 29). The range of particle size obtained in this study is in agreement with previous studies of Nakano et. al. 2001 (29), where different concentrations of poloxamer in monoolein provided particle sizes ranging from 160 to 270 nm.
d) Small angle X-ray diffraction (SAXRD) Â The SAXRD profile of the liquid crystalline phases revealed three diffraction peaks (ratio 1 / 1, âˆš 3, âˆš 4), consistent with the hexagonal phase structure (30) and agreeing with previous works (18). The lattice parameter (a) and the full width at half maximum (FWHM) of the nanodispersions were determined, to investigate the effect of PS addition on the hexagonal structure of the particles.
Figure 4 shows that the "a" value for the samples containing the drug (a = 5.860 Â± 0.004) is smaller than that observed in the absence of PS (a = 6.150 Â± 0.001).
This fact probably suggests that the drug is located within the hydrophobic layer of the nanoparticle, which can cause an approximation of the hydrophilic layer, thereby decreasing the "a" values.
The FWHM values reflect the extension of the disorder caused by the addition of molecules to the liquid crystalline structure. Its increase reflects a less ordered structure, while its reduction indicates a more ordered organization. The FWHM of the sample containing PS (0.098) is higher than that of the blank system (0.064). Therefore, the drug provoked disorder in the structure, but it did not destroy the periodic micellar arrangement.
e) Analysis of the encapsulation degree by gel chromatography Several methods have been employed to separate the free form of drugs including ion exchange chromatography, ultrafiltration, and size exclusion chromatography (31, 32). Size exclusion chromatography is the most widely used because of its simplicity, reproducibility, and its applicability to different kinds of preparations (33). In the present study, the hexagonal liquid crystalline phase nanodispersion containing PS gave rise to eluted fractions containing PS in both the encapsulated and free form. The nanodispersion was eluted from the column with water between fractions 7 and 16. Macroscopically, these fractions were whitish, besides presenting high values of absorbance at 410 nm. When it contained the PS, the nanodispersion was eluted in the same fractions, whereas the non-encapsulated PS was eluted from the column by methanol between fractions 8 and 10. Assay of these fractions by spectrofluorimetry showed an encapsulation degree of 52.10% (Â± 2.82) for PS.
In vitro skin retention studies The penetration properties are determined by the OECD Guideline TG 428: Skin Absorption: in vitro Method (34), which allows the use of porcine skin for penetration studies. Porcine ear skin is considered an excellent skin model, because the histological characteristics of the porcine ear and human skins have been reported to be very similar in terms of epidermal thickness and composition, pelage density, epidermal lipid biochemistry, and general morphology (35), making it a practicable alternative to human skin. An excellent correlation between permeation data using porcine ear skin in vitro and human skin in vivo has been demonstrated by various researchers (36, 37).
Figure 5 presents the results of skin penetration in SC and E+D of PS in the hexagonal liquid crystalline phase nanodispersion and control formulation. The nanodispersion was able to promote a significantly increase (p ï€¼ 0.001) in the amount of PS retained in the skin at all the studied times (Figures 5A and 5B). PS retention in E + D increased over time; after 4 and 12 h post application, it became about five and seven times greater compared to the control, respectively. This result could be explained by the characteristics of the lipids present in the formulation (monoolein and oleic acid), since they can affect the integrity of the skin barrier (18, 19, 21, 38, 39). The use of lipid colloidal dispersions for topical and percutaneous drugs administration of drugs has been studied (40, 41) and some works have described that the composition and particle size of dispersion systems can influence the skin penetration depth of encapsulated drugs (42, 43).
In the present work, we can refer the significant enhancement effect of the hexagonal liquid crystalline phase nanodispersion with respect to PS delivery to the skin layers in both situations, i.e., the penetration enhancement effect of the employed lipids and the facilitated penetration of the nanostructures, which can increase the interaction between the formulation and PS in the skin.
The obtained in vitro results in this research are in accordance with the ones reported by Lopes et. al. 2006 and 2007, who demonstrated increased skin retention of the lipophilic cyclosporine A using liquid crystalline nanodispersion systems (18, 19). This shows that liquid crystalline phases containing monoolein are promising for application in a variety of treatments that depends on drug delivery to the viable epidermis. It should be noted that the PS recovery rate from the skin using the extraction method was greater than 94%, which was good for spectrofluorimetric quantification. PS concentrations were not detected in the receptor phase (phosphate buffer containing 2% polyssorbate 80) by the spectrofluorometric assay used here.
In vivo skin retention studies The in vivo skin retention of PS was assayed after 8 h of topical application. The hexagonal liquid crystalline phase nanodispersion significantly improved PS skin penetration compared to control formulation. While the control formulation enabled 1.09 Â± 0.48% of applied dose /cm2 retention in the skin, the nanodispersion developed here was responsible for rates equal to 2.72 Â± 0.21% of applied dose /cm2 (p<0.05). This result is similar to that obtained in studies by our research group using cyclosporine A incorporated into this hexagonal phase nanodispersion (21).
Considering the differences in the cutaneous permeation between different model membranes used in the in vitro and in vivo studies, we can assume that both penetration studies confirm there is an increase in PS concentration retained in the skin when hexagonal liquid crystalline phase nanodispersion was the carrier.
When the control formulation composed of polyethylene glycol and PS was topically applied, the resulting fluorescence was predominantly present in the SC, and a weak fluorescence could be observed in only some regions of the epidermis (Figure 6B). Nevertheless, treatment of mouse skin with the hexagonal liquid crystalline phase nanodispersion containing PS resulted in increased fluorescence in the SC, viable epidermis, and even in some regions of the dermis, as seen in Figure 6D. Figures 6A and 6C correspond to the same skin pieces as those represented in Figures 6B and 6D, respectively, but without the use of fluorescence methods, so it is possible to see the skin layers better. Although the ability of polyethylene glycol to increase the skin penetration of some compounds is known (39), the use of the hexagonal liquid crystalline phase nanodispersion resulted in greater PS skin penetration into deeper skin layers, which is advantageous for the effectiveness of PDT. As previously observed by Lopes et al. (18) for cyclosporine A using fluorescence microscopy, PS incorporation into the nanodispersion led to enhanced skin penetration of this work. The present work has demonstrated a formulation strategy for the delivery a chlorin derivative to the skin for PDT purposes. The nanodispersed hexagonal liquid crystalline phase improved the PS uptake by skin layers were neoplasic and non-neoplasic lesions are present, being a promising formulation for further pre-clinical and clinical trials, which are very important to confirm PDT as an alternative treatment.