The present study showed a novel proposal to delivery topically a photosensitizer (a new drug of chlorin class) for Photodynamic Therapy (PDT). Nanoparticles of lyotropic liquid crystals characterized as hexagonal phase loading chlorin derivative were developed. The system were characterized by spectrofluorimentric, dynamic light scattering and small angle X-ray diffraction (SAXRD). In vitro and in vivo penetration studies were performed using animal model membranes. The system studied was stable during the experiments of turbidity and demonstrated size particle of 161 Â± 4 nm and a polidispersivity index of 0.175 Â± 0.027, which are adequate for this application. Furthermore, studies of SAXRD proved that the liquid crystalline structure remained stable after drug loading. Gel chromatography assay showed an encapsulation rate higher than 50%. In vitro and in vivo skin retention studies showed a higher amount of drug retained when the nanodispersion was used, compared to control formulation. Fluorescence microscopic study also demonstrated a higher biodistribution of the chlorine derivative in the skin layers. The results showed the potential of the nanodispersion in delivering the photosensitizer into the skin, a crucial condition for the success of PDT topical.
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Photodynamic therapy (PDT) is an emerging technology for the important therapeutic options in management of neoplastic and non-neoplastic 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 of PDT is the activation of PS by application of a light at a wavelength matching its absorption spectrum (6). In this situation, photophysical reactions take place, resulting at the cell death due to the production of free radicals and/or reactive oxygen species, especially oxygen singlets (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 target tissue. Recently, studies have been focused on the development of different strategies to overcome these difficulties, including nanocarriers to delivery PSs and their precursors (9), liposomes (10) ethosomes (11), invasomes (12), liquid crystals (13) and magnetic nanoparticles (14), among others.
Lyotropic liquid crystals combine the properties of crystalline solid and an isotropic liquid (15). Several research groups are dedicated to the study of these systems since they are excellent vehicles for a variety of drugs due to their ideal structure properties that can accommodate the drug within both aqueous and lipid domains (16, 17).
Reverse hexagonal phase composed by monoolein has been shown to increase the topical delivery of drugs (18, 19). Carr and cols (20) have studied these systems and concluded that some components of Myverol (commercial monoolein) could enhance the passive penetration of nicotine in the human stratum corneum demonstrating the effectiveness of this vehicle in delivering drugs transdermally Liquid crystalline phases containing monoolein increased the skin delivery of Cyclosporin A, both as a bulk phase (21) and as a nanodispersed formulation (18). Further studies (19) showed that therapy with topical vitamin K could be improved by the use of monoolein-based systems.
In the present study, we proposed a delivery system for PDT based in liquid crystal nanodispersion aiming to improve the skin delivery of a chlorine derivative. For that we developed PS-loaded nanodispersion of reverse hexagonal phase and evaluated its in vitro and in vivo topical application.
MATERIALS AND METHODS
Materials PS chlorin derivatives (Figures 1) were synthesized Â according to described in the literature (22) and were used such as diastereomeric mixture. These compounds could be obtained through the Diels-Alder reaction where the reaction in the ring A of protoporphyrin IX dimethyl ester (PPIXDME) yielded chlorin A and the reaction in the 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 diatereomeric mixture (chlorin A + chlorin B) in all studies, performing only a purification in order to remove the by-products. Monoolein (Myverol 18-99Â®) was purchased from Quest International (USA), oleic acid (OA) and octanol was obtained from Sigma-Aldrich (Sao Paulo, Brazil), methanol from Bdick & Jackson (B & J ACS / HPLC Certified Solvent). Sephadex LH20 was purchased by GE Healthcare. Water was purified using a Millipore milli-Q water system (Millipore Corporation).
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Analytical methodology for chlorin quantification Chlorine derivative was assayed spectrofluorimetrically using a Fluorolog 3 Triax 550 (Tokyo, Japan) at 400 and 670 nm of excitation and emission, respectively, using bandwidth 1 nm (22, 23). It was initially prepared a methanolic solution of chlorin (25 mg/mL), from which serial dilutions were performed in order to obtain methanolic standard solutions of 0.05 to 0.3 Î¼g/mL. Analytical method was developed and evaluated about linearity, precision, accuracy, limits of detection (LOD) and lower limit of quantification (LLOQ). LOD was the basis of signal-to-noise ratios (S/N) of 3:1 and 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 were performed on both intra-day and inter-day measurements by repeated analysis of three chlorin concentrations determined in the same day and three different days, respectively (24). The stability of samples containing the PS was also assessed. Thus, solutions of the PS in different concentrations were solubility in both, methanol and phosphate buffer pH 7.2 plus polissorbate 80 at 2% and evaluated at 12 and 120 h after preparation by spectrofluorimetry. The experiments were performed in triplicate. Ear porcine skin was used as a model skin of in vitro studies, because of this was necessary to evaluate the role of porcine ear skin in the band of fluorescence of the PS. For this, the outside skin of the ears from animals recently killed was removed with the aid of forceps and scalpel, followed by the removal of fatty tissue remaining in the skin. Sections of skin were dermatomed to 500 Î¼m (Dermaton, Nouvag, Switzerland). It was measured an area of about 1.44 cm2 of skin and then fragmented in small pieces which were then subjected to homogenization Turrax equipment. Fragments were dipped into drug methanolic solution and mixed in vortex 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 spectrofluorimetry in the same conditions those for the PS method.
Preparation of the formulations Hexagonal liquid crystalline phase nanodispersion was prepared based in the method early published (18). Briefly, the PS (1 mg) was dissolved in 0.3g of lipid phase (OA:MO mixture at 2:8) and 2.7 g of the citrate buffer (pH 6) containing 1.5% Poloxamer 407 was added. The system was allowed to equilibrate at room temperature for 24 h. Then, it was vortex-mixed for 2 min and sonicated in ice-bath at 10 kHz for 2 min. The mixture obtained was centrifuged at 1901 g for 10 min and then filtered using a 0.8 Î¼m membrane porous. The control formulation was a solution of PS in polyethylene glycol (0.3 mg/g) obtained by mixing during 5 min in vortex at 3.000 rpm and then centrifuged at 1901 g for 10 min.
Physicochemical characterization of the nanodispersion
Polarized light microscopy The texture of reverse hexagonal liquid crystalline phase was observed by optical microscope Axioplan 2 (Zeiss, Germany) equipped with a polarizing filter and coupled to a digital camera Axiocan HRc (Zeiss, Germany) with a video system and automatic image acquisition. The observations were done at 25 °C and 37 °C by using a hotplate model THMSG 600 (Link, England) coupled to the polarized light microscope.
Turbidimetric analysis The samples were analyzed at 25 Â° C and the values obtained of apparent absorbance 410 nm on spectrophotometer (FentoScan, Brazil). RHPNs with and without PS were diluted in citrate buffer pH 6.0 (1:50) and then analyzed during 72 h. The diluted samples were packed and stored in a quartz cuvette of fluorimeter of 1 cm light path to prevent disruption of the system during reading due to sample manipulation. The experiments were performed in triplicate.
Dynamic light scattering The particle size and polidispersity index (Pdl) of hexagonal liquid crystalline phase nanodispersion were analyzed by light scattering method (DLS) in Zetasizer 3000 HSA (Malvern Instruments) using 10mW HeNe laser operating at 633 nm with incidence detection angle of 90° at 25 Â°C. Measurements position within the cuvette was automatically determined by the software. The samples (n = 3) were obtained by four different processes: (i) Nanodispersions without PS, processed by centrifugation at 1901 g for 10 min and filtration using a 0.8 Î¼m membrane porous; (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 membrane porous; (iv) Nanodispersion containing PS chlorin and processed in the same way of first group.
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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), Campinas, SP, Brazil, using the D12A-SAXS beam line. The selected wavelength was 0.1488 nm and the focus was located at the detection plane. The scattered intensities curves were recorded using a two-dimensional, position-sensitive MARCCD detector located at 803.3 mm from the sample and ionization detector was responsible for monitoring the intensity of the incident beam. The measurements were carried out with 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. Unloaded and PS-loaded hexagonal phase nanodipersions were analyzed.
Analysis of encapsulation degree by gel chromatography No-encapsulated PS was removed from the hexagonal liquid crystalline phase nanodispersion by size exclusion chromatography using a Sephadex LH-20 column (3.5 diameter, measuring 17 cm after packaging of the column). 500 ÂµL of the samples were placed on the Sephadex column and eluted using purified water as 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 and aliquots of 1 mL of each fraction were lyophilized to remove water. After the lyophilization process, the samples were solubilized in 3 mL of methanol and assayed for PS by spectrofluorimetry (Î»exc= 400nm, Î»em= 670nm).
In vitro skin retention studies Skin from the outside 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 skins were placed on a Franz cell diffusion (diffusion area of 0.79 cm2) with stratum corneum (SC) facing the donor compartment where 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 polissorbate 80 to improve the PS solubility in the receptor medium. The system was kept at 37Â°C by a thermostatic water bath. After time of experiment (4, 8 and 12 h) the system was turned off and skin surfaces removed. They were washed with distilled water and the excess of water was removed with a piece of cotton. The penetration of PS in the skin layers was assessed as described by Lopes and cols (21). In order to separate SC and epidermis plus dermis (E+D), tape stripping process was applied using 15 tapes (DurexÂ® 3M), considering that the first one was discarded and the following dipped in a Falcon tube containing 5 mL of methanol. These were vortex-mixed and bath-sonicated during 2 and 30 min, respectively. The samples were filtered using a 0.45 Î¼m membrane porous. After this, the remaining tissue of skin was cut in small pieces and immersed in 5 mL of methanol, vortex-mixed during 1 min and bath sonicated for 30 min, and then filtrated in the same way of mentioned. Both methanolic samples were assessed by spectrofluorimetry (Î»exc= 400 nm, Î»em= 670 nm) in order to quantify the concentration of PS. The recovery rate imposed by the method was previously tested using small pieces of porcine ear skin (containing an area of 1.44 cm2) where were added 30 ÂµL of methanolic solution of PS at 12.5 Âµg/mL. 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 ROSA et al. (2003) and LOPES et. al. (2006) (25, 21). The experiment applied 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 previous approved by University of São Paulo Animal Care and Use Committee (Authorization number 10.1.160.53.0). Animals were separated into 2 groups (6 animals per group), such as control group and hexagonal liquid crystalline phase nanodispersion group. The topical applications of PS were performed on the back of hairless healthy mice using 150 Î¼L of the formulations . After 8 h, the hairless mice were euthanized by carbon dioxide vapor and the topical application area of the skin was dissected to 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 in vivo experiments were embedded in a Tissue-TeKÂ® matriz (O.C.T compound) and frozen at -17 Â°C to visualize the skin penetration of PS by fluorescence microscopy. Then the samples were sectioned on vertical slices at 30 Âµm thickness using a cryostat (HM550, Microm, Heidelberg, Germany) and observed by fluorescent microscope (AxiosKop 2 plus, Carl Zeiss, Germany) operating with 395 and 440 nm band-pass excitation and emission filter, respectively. The images obtained were recorded using a device digital camera (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, we determined the UV spectrum of methanolic chlorin derivative solution. The spectrum showed that this PS showed a maximum peak of absorption at 400 nm (the Soret band) and this was choose as the excitation wavelength, which is consistent with previous research using chlorin class compounds (22, 23). Fluorescence response was used as quantification method. It was obtained good linearity in the concentration range from 0.08 to 0.4 Âµg/mL (r= 0.9982). LOD and LLOQ were determined as 6 and 12 ng/mL, respectively. Also, intra and inter-day precision showed variation coefficients lower than 5%. Accuracy was higher than 94% in the inter-day assay. The samples containing PS either in methanolic solution or phosphate buffer showed adequate stability, ensuring sample solvent does not change significantly the stability of the analyte during the whole experiments period (26).
Physicochemical characterization of the nanodispersion
Polarized light microscopy Polarized light microscopy of liquid crystalline hexagonal phases containing or not PS revealed a fan-like texture, typical of hexagonal phase systems (Figures 2A, C and D), indicating that the presence of chlorine derivative and Poloxamer did not change the characteristics of the systems. In addition, we could observe that the increase in temperature of 25 °C to 37 °C did not disestablished the structure of the system (Figure 2B). The stability of the system was also observed after 30 days of the sample preparation. After the dispersion of hexagonal phase using sonication process, the system resulted in a milky and low-viscosity formulation (Figure 2E), as already reported before (19).
Turbidimetric analysis Turbidimetry can be used for the characterization of particle size distribution and for verification of the stability of colloidal suspensions (27).
Figure 3 shows the stability of the hexagonal liquid crystalline phase nanodispersion containing or not PS during 72 h. The presence of PS in the nanodispersion lead to less variation in the absorbance at 410 nm when compared that one without it, which may indicate that PS increases the system stability due to formation of a more rigid structure.
Table 2 shows the results of particle size of hexagonal liquid crystalline phase nanodispersion obtained by different processes. The method composed by combination of filtration and centrifugation process of the sample (group 1 and 4) showed smaller particle size about 160 nm. The filtration process alone was not able to promote the standardization of appropriate particle size (group 3) while that using centrifugation process (group 2), particles became a bit larger than those of groups 1 and 4. No significance difference about the mean particle size and polidispersity were observed between groups 1 and 4, thus the addition of PS did not modify the particle size when the hexagonal liquid crystalline phase nanodispersion was submitted to centrifugation and filtration as already described. Accordingly, Lopes et al.2006, the sonication process in hexagonal phase of monoolein-water-poloxamer results in the formation of hexagonal phase nanodispersion, with particles around 200 nm of diameter. The systems obtained in this research showed a similar profile of those obtained with incorporation of another drugs (19, 18). 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 conformance with previous studies of Nakano et. al. 2001 (29), where different concentrations of poloxamer in monoolein provide particles size ranging from 160 to 270 nm.
Small angle X-ray diffraction (SAXRD) Â The profile of SAXRD liquid crystalline phases revealed three diffraction peaks (ratio 1 / 1, âˆš 3, âˆš 4), consistent with a hexagonal phase structure (30) and agreeing with previous studies (18). The lattice parameter (a) and the full width at half maximum (FWHM) of the nanodispersions were determined to investigate the effect of the addition of the drug on the hexagonal structure of the particles.
It can be observed in Figure 4 that the "a" of the samples containing the drug (a = 5.86 Â± 0.004) is smaller than that one observed in the absence of PS (a = 6.15 Â± 0.001).
This fact probably suggests that the drug is within hydrophobic layer of the nanoparticle, which can cause an approximation of the hydrophilic layer, decreasing "a" values.
The FWHM values reflect the extension of the disorder caused by the addition of molecules in the liquid crystalline structure. Its increase reflects a less ordered structure while its reduction reflects a more ordered one. Considering the values of FWHM were 0.064 and 0.098 for blank and sample containing PS, respectively, it can be said that the presence of the drug disordered the system, without losing the crystalline structure.
Analysis of 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 it is applicable to different kind of preparations. In the present study, the hexagonal liquid crystalline phase nanodispersion containing PS showed eluted fractions containing PS in both encapsulated and free form. The nanodispersion was eluted from the column with water between the fractions 7 and 16. These fractions were presented macroscopically whitish, besides presenting high values of absorbance at 410 nm. When containing the PS, the nanodispersion was eluted in the same fractions, whereas the no-encapsulated PS chlorin was eluted from the column by methanol between the fractionsâ€¦â€¦â€¦... The assay of these fractions by spectrofluorimetry showed an encapsulation degree of 52.10% (Â± 2.82) for the PS.
In vitro skin retention studies The penetration properties are determined by the OECD Guideline TG 428: Skin Absorption: in vitro Method (33), which allows the use of porcine skin for penetration studies. Porcine ear skin is considered an excellent skin model, because the histological characteristics of 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 (34), making it a practicable alternative for human skin. An excellent correlation between permeation data using porcine ear skin in vitro and human skin in vivo have been demonstrated by various researchers (35, 36).
Figure 5 presents the results of skin penetration in SC and E+D of PS from hexagonal liquid crystalline phase nanodispersion and control formulation. The nanodispersion was able to promote a significantly increase (p ï€¼ 0.001) of PS retained into skin at all times studies (Figures 5A and B). PS retention in E + D increased over time; after 4 and 12 h post application it became about five and seven times greater than control, respectively. This result could be explained because the characteristics of the lipids presents if the formulation (monoolein and oleic acid) that can affect the integrity of the skin barrier(19, 18, 21,37, 38) The use of lipid colloidal dispersions for topical and percutaneous drugs administration has been studied (39, 40). and some studies showed that the composition and the particle size of dispersion systems can influence the skin depth penetration of encapsulated drugs (41, 42).
In the present work, we can refer the significant enhancement effect of hexagonal liquid crystalline phase nanodispersion in delivering PS into the skin layers to both situation, such as the penetration enhance effect of the lipids used and the facilitate penetration of the nanostructures, which can increase the interaction of the formulation and PS in the skin.
The in vitro results obtained in this research are in accordance with the ones performed by Lopes et al. 2006 and 2007, such demonstrated an increased the skin retention of the lipophilic Cyclosporine A using liquid crystalline nanodispersion systems (18, 19), showing that liquid crystalline phases containing monoolein are promising for application in a variety of treatments that depends on the delivery of the drug to the viable epidermis. It should be noted that recovery skin rate of PS using the extraction method was greater than 94% and this was appropriated to quantification spectrofluorimetry. PS concentrations were not detected in the receptor phase (phosphate buffer containing 2% of polissorbate 80) by the spectrofluorometric assay used.
In vivo skin retention studies The in vivo skin retention of PS chlorin was performed after 8 h post topical application. Hexagonal liquid crystalline phase nanodispersion efficiently enhanced PS skin penetration when compared to control formulation. While with control formulation 1.09 Â± 0.48% of dose applied/cm2 became retained into skin, RHPN was responsible for rates of 2.72 Â± 0.21% of dose applied/cm2 (p<0.05). This result is similar to that obtained in studies of our research group with cyclosporine A incorporated to this hexagonal phase nanodispersion (21).
Considering the differences in the cutaneous permeation between different model membranes used in vitro and in vivo studies, we can assume that both in vitro and in vivo penetration studies confirm the increase of PS concentration retained into skin when hexagonal liquid crystalline phase nanodispersion was the carrier.
When control formulation composed by polyethylene glycol and PS was topically applied, resulting fluorescence was predominantly present in SC and only in some regions of the epidermis a weak fluorescence can be observed (Figure 6B). Moreover, treatment of the mice 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 could be seen in Figure 6D. Figures 6A and 6C correspond to the same skin pieces that in 6B and 6D figures, respectively, but without the use of fluorescence methods, so it is possible to see better the skin layers. Although the ability of polyethylene glycol to increase the skin penetration of some compounds is known (38), the use of the hexagonal liquid crystalline phase nanodispersion resulted in greater skin penetration of PS in deeper skin layers, what is advantageous for the effectiveness of PDT. Like was previously seen by Lopes et al. (18) for Cyclosporin A using fluorescence microscopy, incorporation of the drug in the nanodispersion results in an increase in the skin penetration of the drug. In that work, author incorporated Cyclosporin A in an hexagonal phase nanodispersion and this formulation was effective in improving the topical delivery of the peptide without causing skin irritation, a comum problem for this kind of drug.
Acknowledgments: This work was supported by "Fundação de Amparo à Pesquisa do Estado de São Paulo" (FAPESP, Brazil, project # 04/09465-7) and "Conselho Nacional de Pesquisa" (CNPq, Brazil). R. Petrilli was the recipient of a CNPq fellowship (process # 109984/2007-2).