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The high hydrophobicity and aggregation phenomena exhibited by the photosensitizer zinc phthalocyanine tetrasulfonate (ZnPcSO4) make it difficult for this compound to penetrate the skin and reduce the compound's photodynamic efficacy. A microemulsion (ME) was developed in order to increase the skin penetration of ZnPcSO4 while avoiding its aggregation. Ternary phase diagrams composed of surfactants (Span® 80/Tween® 80), canola oil and a propylene glycol (PG)/water mixture (3:1) were constructed as a basis for choosing an adequate ME preparation. Rheological, electrical conductivity, dynamic light scattering and zeta potential studies were carried out in order to characterize the ME formulations. Monomerization of ZnPcSO4 in the ME was determined photometrically and fluorometrically. In vitro skin penetration and retention of the compound in the skin were measured using porcine ear skin mounted on a diffusion cell apparatus. The in vivo accumulation after 6 h of ZnPcSO4 application was determined fluorometrically in hairless mice skin. A confocal laser scanning microscopy technique was also used to investigate ZnPcSO4 skin penetration. The ME obtained was found to be of type W/O with an average z size of approximately 20 nm. Spectroscopic techniques confirmed that the ME was able to monomerize ZnPcSO4. In vitro experiments showed increased ZnPcSO4 penetration in the stratum corneum (SC) and in epidermis without stratum corneum, with dermis [E+D] of 33.0- and 28.0-fold for the ME preparation compared to the control. Experimental retention in vivo confirmed that when the ME was used as carrier, ZnPcSO4 concentrations in the SC and [E+D] were about 1.6- and 5.6-fold higher, respectively, than controls. Visualization of ZnPcSO4 skin penetration by confocal laser scanning microscopy confirmed that the ME increased the skin penetration of this photosensitizer.
Photodynamic therapy (PDT) represents an advantageous strategy for the treatment of non-melanoma skin cancers (1). Typically, topical application of a photosensitizer (PS) is followed by application of a laser, resulting in the destruction of tumor cells by a complex cascade of chemical, biological and physiological reactions that occur after the formation of highly reactive singlet oxygen (1O2), which forms upon activation of the PS by light (2). The most frequently used PS is the prodrug 5-ALA, a precursor of protoporphyrin IX (an endogenous PS) (1). However, the low extinction coefficient absorption of protoporphyrin IX when excited by red light at 630 nm has led to the development of new PSs (1). Among then, the phthalocyanines have attracted much interest because of their many advantages compared to 5-ALA (3), which include: i) selective retention in tumor tissue; ii) ease of synthesis; iii) resistance to chemical and photochemical degradation; iv) long lifetime in the photoexcited triplet state (fundamental for reactive oxygen production); and v) low dark toxicity (4-6).
The water-soluble phthalocyanine derivative zinc phthalocyanine tetrasulfonate (ZnPcSO4) (Figure 1) has the appropriate photobiological characteristics for PDT (7). However, its high molecular weight (898.15) results in poor penetration through the stratum corneum (SC) (8); thus, reaching malignant cells in the epidermis at effective concentrations can become a problem. Furthermore, self-aggregation resulting from the large hydrophobic skeleton of ZnPcSO4 can occur in most vehicles, due to a strong tendency of the compound to form dimers, especially in aqueous media. Self-aggregation of phthalocyanines reduces their efficiency to produce reactive oxygen species (9, 10).
In view of these facts, the development of new delivery systems that can efficiently deliver ZnPcSO4 in its non-aggregated form to viable epidermis could enable its clinical use for topical PDT. To date, few studies have focused on the development and evaluation of novel vehicles for topical delivery of this class of PS (11, 12). The goal of this study was to develop and evaluate novel microemulsions for topical delivery of ZnPcSO4.
Microemulsions were chosen as the delivery system for ZnPcSO4 due to their ability to increase the skin penetration of other hydrophilic drugs (13, 14). Because the size of microemulsion (ME) aggregates is typically less than 150 nm, they are able to increase the skin penetration of drugs due to promotion of high thermodynamic activity and/or to a potential penetration enhancer effect of the individual constituents (surfactants, oils and water) (13). In this study, we investigated the production of MEs composed of canola oil, polysorbate 80 and sorbitan mono-oleate and the ability of such systems to improve the in vitro and in vivo delivery of ZnPcSO4 to skin.
2. MATERIALS AND METHODS
High purity ZnPcSO4 was purchased from Frontier Scientific. Polysorbate 80, HLB = 15.0; sorbitan mono-oleate, HLB = 4.3; propylene glycol (PG) and polyethylene glycol (PEG; technical grade) were purchased from Sigma. Canola oil of food grade was obtained at a local supermarket. Dimethyl sulfoxide (DMSO; analytical grade) was purchased from Merck (Germany). Technical-grade cetilpyridinium chloride was purchased from the local market. Water was purified by double distillation and deionized using the Millipore Milli-Q® Water System (Millipore Corporation, Bedford, USA). All substances were used without further purification.
2.2. Analytical methodology for ZnPcSO4
ZnPcSO4 was assayed spectrofluorometrically using a Flurolog 3, Jobin-Yvon (France) Spex spectrofluorometer at 610 nm excitation and 650 nm emission (bandwidth 0.5 nm). A ZnPcSO4 standard curve was constructed as reference. The assay results were linear between 0.004 and 0.5 mg/mL (r = 0.999).
2.3. Formulation preparation and characterization
2.3.1. Pseudo-ternary phase diagrams
A titration method was employed for constructing phase diagrams of emulsions with different aqueous phase compositions. Mixtures of sorbitan mono-oleate and polysorbate 80 (3:1) were weighed in a dark brown, screw-cap glass vial, mixed using a magnetic bar at 1,500 rpm on a magnetic stirring plate for 1 h and subsequently stored overnight at room temperature. Canola oil was then added at ratios ranging from 9:1 to 1:9 to different vials. A small amount of aqueous phase (pure water or a PG/water mixture (3:1, w/w) was also added to the vials, followed by vortexing for 2-3 min and incubation at room temperature for 5 days. The resulting mixture was then evaluated by visual and polarized light microscopy (Carl Zeiss, Germany) observations.
2.3.2. ME preparation
Based on the phase diagrams, an ME was selected for the incorporation of ZnPcSO4. The ME was prepared by adding the aqueous phase, which was composed of PG/water (3:1, w/w), to a mixture of sorbitan mono-oleate and polysorbate 80 at 3:1 (w/w) and canola oil. The formulation was vortexed at 2,500 rpm for 3 minutes at 25°C. To prepare the drug-loaded ME, 27 µL of a stock solution containing 500 Î¼g/mL of ZnPcSO4 in DMSO was added to the oily phase (surfactants + canola oil) followed by the aqueous phase. The final concentration of ZnPcSO4 in the ME was 6.7 µg/mL.
2.3.3. Physicochemical characterization of ME
The viscosity of unloaded and drug-loaded ME (ZnPcSO4 at 6.7 µg/mL) was determined at 25°C using a controlled rate Brookfield DV-III Rheometer having a SC4-18 spindle and a shear rate in the range of 0-13 s-1 at a speed between 80 0-10 rpm. The measurements were performed for both up and down-curves. Data of the shear cycle were fitted to the power-law model using the rheometer software.
The electrical conductivity of unloaded ME and of ME loaded with ZnPcSO4 at 6.7 µg/mL was measured using a conductivity meter, model CD-20. For conductivity measurements, MEs were prepared in 0.01 M aqueous sodium chloride instead of distilled water.
Dynamic light scattering and Zeta potential
The unloaded and drug-loaded ME solutions (the latter contained 6.7 µg/mL ZnPcSO4) were subjected to light scattering measurements at 25°C using a Zetasizer Nano system ZS (Malvern Instruments, UK) containing a 4 mW He-Ne laser system operating at a wavelength of 633 nm and incorporating non-invasive backscatter optics (NIBS). Measurements were made at a detection angle of 173°; the measurement position within the cuvette was automatically determined by the software. Twelve measurements were carried out for each sample. The refractive index for unloaded and drug-loaded ME was set at 1.464. Measurements of the particle electrophoretic mobility were carried out using the same instrument. The Zetasizer Nano Series uses a combination of laser Doppler velocimetry and phase analysis light scattering (PALS) in a patented technique called M3-PALS. Samples were diluted in 10 mM NaCl. Twenty-two measurements were carried out with each sample.
2.3.4. ZnPcSO4 monomerization and stability in the ME formulation
Aggregation and stability of ZnPcSO4 in the ME were checked spectrophotometrically using a UV-VIS HITACHI-U-300 spectrophotometer and spectrofluorometrically using a Fluorolog 3 Spex Jobin-Yvon (France) Spex spectrofluorometer (excitation, 610 nm; emission, 650 nm; bandwidth, 0.5 nm). To verify aggregation, measurements were performed directly using a flat cuvette and were compared to aggregated and non-aggregated solutions of ZnPcSO4 in water and DMSO, respectively. Chemical stability of ZnPcSO4 incorporated in the ME and stored at room temperature (25oC) over a three-month period was verified. To quantify ZnPcSO4, the ME was diluted 1:10 (v/v) with DMSO, vortexed, centrifuged at 704.0 x g for 10 minutes to phase separate the DMSO, and assayed at 670 nm.
2.4. In vitro skin penetration studies
The skin penetration and transdermal delivery of ZnPcSO4 (n = 5) were assessed in an in vitro model of porcine ear skin obtained from a local slaughterhouse. This model skin was chosen because it has physiological, biochemical and histological similarities to human skin (15, 16). The skin from the outer surface of a freshly excised porcine ear was carefully dissected, dermatomed (~ 500 µm thickness), stored at -20°C and used within one month. It was mounted in a diffusion cell (diffusion area of 0.78 cm2), with the SC facing the donor compartment and the dermis facing the receptor medium; 0.3 g of either the ME or the control formulation (drug solution in polyethylene glycol) containing 6.7 µg/mL ZnPcSO4 were applied to the donor compartment. The control formulation was chosen because it was the only vehicle tested that permitted the solubilization and monomerization of the PS. The receptor compartment (3 mL) was filled with phosphate buffer (100 mM, pH 7.4) containing cetilpyridinium chloride at 30 mM (a concentration above the critical micellar concentration) to avoid ZnPcSO4 aggregation. To confirm the disaggregated state, the absorption spectrum of ZnPcSO4 in the phosphate buffer containing cetilpyridinium chloride was compared to the spectrum in a monomerized medium (DMSO). Both showed a non-coalescent Q-band with maximum absorption above 660 nm. Throughout the experiment, the receptor medium was kept constantly stirred and at 37±0.5° C by a water jacket. Samples were removed at regular intervals for up to 12 h to assay ZnPcSO4.
At the end of the experiment, the skin surfaces were gently washed with distilled water to remove any excess formulation and carefully wiped with tissue paper. The stratum corneum (SC) was separated from viable epidermis and dermis ([E + D]) using the tape-stripping technique, with 14 pieces of adhesive tape (Scotch Book Tape, 3M, St. Paul, MN); the tapes containing the SC were immersed in 4 mL of DMSO, vortex-stirred for 2 min and bath-sonicated for 30 min (40 kHz, continuous mode). The DMSO phase was filtered through a 0.45 µm membrane and the resulting filtrate was fluorimetrically assayed for ZnPcSO4. The remaining tissue, [E without SC+D], was cut into small pieces, vortex-mixed for 2 min in 4 mL of DMSO, sonicated for 30 seconds (70 mA) using an ultrasonic probe, and bath-sonicated (40 KHz, continuous mode) for 30 min. The resulting mixture was filtered through a 0.45 µm membrane and ZnPcSO4 in the filtrate was fluorimetrically assayed. Skin and formulation components did not interfere with the assay. The results were expressed as ZnPcSO4 (µg)/skin area (cm2). All procedures were performed under subdued light.
2.5. In vivo skin penetration studies
The experiment was carried out on six- to eight-week-old female hairless mice (strain HRS/J, Jackson Laboratories, Bar Harbor, ME, USA) housed at 24-26 °C and exposed to daily 12:12-h light/dark cycles (lights on at 6 a.m.), with free access to standard mouse chow and tap water. To reduce the stress associated with the experimental procedure, animals were handled daily for one week prior to experimentation. They were euthanized by carbon dioxide vapor. Protocols used were in conformance with the guidelines of the University of São Paulo Animal Care and Use Committee (Authorization number: 06.1.114.53.4) and the "Principles of Laboratory Animal Care" (NIH publication #85-23, revised in 1985).
Hairless mice were chosen as models in order to facilitate the topical application of the ME and because studies aimed at visualizing the topical penetration of PSs in vivo are usually performed with murice animals (nude Balb/c and hairless mice) (17-19).
Two hundred microliters of ME or control formulation (polyethylene glycol), containing 6.7 µg/mL ZnPcSO4 were applied for 6 h on the dorsal region of the mice over an area of 1.3 cm2. After euthanasia, the skin area where the formulation had been applied was carefully dissected; ZnPcSO4 was extracted as described for the in vitro experiments and spectrofluorometrically assayed
2.6. Confocal scanning laser microscopy (CSLM)
Cross-sections of skin samples obtained from in vivo experiments were embedded in a matrix, frozen at -17°C and sectioned at 60 mm thickness. The measuring system consisted of a Leica TCS SP 5 confocal unit (Leica, Heidelberg, Germany) equipped with Helium/Neon laser and mounted on a Leica DMIRE 2 inverted microscope (Leica, Heidelberg) with an HCPL Fluotar Leica lens (20X magnification) immersion objective in its oil position. For excitation of the label, the 633-nm laser line was used and fluorescence emission above 650 nm was detected. The depth of the optical sectioning was 0.5 mm below the cutting surface.
2.7. Statistical analyses
Results are reported as means ± SD. Data were statistically analyzed using nonparametric tests. The Mann-Whitney test was used to compare two experimental groups. The level of significance was set at p < 0.05.
3. RESULTS AND DISCUSSION
3.1. Formulation preparation and characterization
3.1.1. Pseudo-ternary phase diagrams
Pseudo-ternary phase diagrams composed of the surfactants sorbitan mono-oleate (low HLB surfactant), polysorbate 80 (high HLB surfactant), canola oil, (termed the oily phase), PG and water were constructed to show the relationship between composition and phase behavior of samples. The regions described in Figures 2A and 2B are based on microscopic visualization of each formulation after titration with the aqueous phase. When the formulation was not homogeneous, each phase was characterized separately.
The phase diagram in Fig. 2A shows the relationship between the phase behavior and the concentration of polysorbate 80, sorbitan mono-oleate, canola oil and water. Only two systems characterized as MEs (fluid, liquid isotropic phases) were observed near the surfactant vertex characterized by high surfactant (72% and 81%) and low water (10%) contents. The liquid isotropic phases consisted of surfactants, canola oil and water, 72-81%, 9-18% and 10%, respectively. Systems composed of two phases are noted in the phase diagram as isotropic liquid + birefringent.
The addition of PG to the aqueous phase (PG/water, 3:1) expanded the region corresponding to the liquid isotropic phases to 18-81%, 7-64%, and 10-30% of surfactants, canola oil and aqueous phase, respectively (Figure 2B). As can be observed, the liquid isotropic phases are located along the surfactant/oil axes, consistent with other reports (20-23). In addition, some of the isotropic liquid + emulsion became isotropic liquid following the addition of PG to the aqueous phase. PG acted as co-surfactant, lowering the interfacial tension and allowing the formation of isotropic phases with smaller amounts of surfactant (<81%) and more canola oil (>20%) and aqueous phase (>10%), as shown in the phase diagram in Figure 2B.
The addition of hydrophilic substances to systems composed of surfactant/oil/water favors the formation of MEs. The presence of substances such as glycerol and sorbitol in the aqueous phase influence the optimal head group area of the surfactants by altering the aqueous solubility of this region of the molecule. Due to these effects, water-soluble hydrophilic substances have been used as aids for ME formation (24).
Based on these results, an ME composed of 38% canola oil, 47% mixed surfactants and 15% PG/water was chosen for further studies.
3.1.2. Physicochemical characterization and stability of microemulsion containing ZnPcSO4
After selecting an ME formulation, ZnPcSO4 in DMSO was incorporated so that a final concentration of 6.7 µg/mL was obtained. The ME composed of 38% canola oil, 47% mixed surfactants and 15% PG/water was the only emulsion in the phase diagram shown in Figure 2B that maintained its physical stability after incorporating ZnPcSO4. The addition of ZnPcSO4 did not alter the physical stability of the ME after centrifugation or after three months storage at room temperature. Microscopic visualization during this period showed that the phase remained isotropic.
Table I shows the results of the physicochemical characterization of unloaded and drug-loaded ME. Due to the dynamic nature and small size of surfactant aggregates in the MEs (typically less than 100 nm in diameter), direct examination of ME structure is difficult, and indirect measurement techniques, such as electrical conductivity and rheological studies, are usually employed to obtain basic information about the internal structure (25, 26).
The low conductivity values (<1 µÎ©/cm) obtained and the low concentration of the dispersed phase indicate that the ME was of type W/O. The addition of ZnPcSO4 did not significantly (p < 0.05) alter its conductivity. The ME was characterized as a viscous liquid, and rheological measurement indicated Newtonian flow behavior both for unloaded and drug-loaded MEs. In this case, considering the flow behavior index (n) as equal to 1, the consistency index (k) is the same as the absolute viscosity (µ). This behavior was not significantly (p < 0.05) changed by the addition of ZnPcSO4.
Unloaded and drug-loaded MEs were characterized by a small particle size (15.7 nm and 20.8 nm, respectively) and very narrow size distribution, as determined by cumulative light scattering analysis. Similarly, both MEs also constituted homogenous populations with respect to surface charge properties. Low polydispersity (p < 0.2) was indicative of homogeneous (monodispersed) particles; the addition of ZnPcSO4 did not significantly (p < 0.05) alter polydispersity. The Z average mean (nm) was slightly, but not significantly (p < 0.05), increased to 20.8 nm following addition of ZnPcSO4, indicating that the drug, due to its hydrophilic nature, was incorporated in the dispersed phase. Zeta potential analysis yielded a negative value (-22.8 mV); the addition of PS did not change the surface electrical charge properties of the ME droplets.
Constantinides and Scarlat (27) reported physicochemical characteristics relating to viscosity, conductance, particle size and polydispersity similar to those reported here for a W/O ME composed of long-chain triglycerides, non-ionic surfactants and water.
3.1.3. ZnPcSO4 monomerization and stability in the ME
Because an effective delivery system for PDT must deliver the PS to the therapeutic site in its monomeric form, we investigated whether aggregation of ZnPcSO4 occurred in the selected ME. Because the absorption and emission spectra profiles of PSs in different solvents provide important information regarding aggregated or monomeric states of the dye molecules, we evaluated the absorption and emission spectra profiles of the drug in ME, water (aggregating medium) and DMSO (non-aggregating medium) at identical concentrations (6.7 µg/mL) (Figure 3).
The absorption and emission spectra of the aggregated state are characterized, respectively, by broad Q and Soret bands and weak fluorescence emission (7). This profile was observed in the absorption (Figure 3A) and emission (Figure 3B) spectra of ZnPcSO4 incorporated in water. On the other hand, the spectrum of ZnPcSO4 incorporated in DMSO displayed narrow, red-shifted absorption and strong fluorescence emission, corresponding to the typical profile of the non-aggregated ZnPcSO4 (7). Spectra of ZnPcSO4 incorporated in the ME displayed a narrow and Q-band shift to the red (66 nm) and a strong fluorescence emission at Î»max = 705 nm. The observation of a Q-band shift to the red and a strong fluorescence band strongly suggests that the PS is mainly in the monomer form when incorporated in the ME, a desirable and effective condition for a PS in PDT.
The chemical stability and aggregation of ZnPcSO4 incorporated into the ME were checked directly by fluorescence emission, using a flat cuvette as mentioned in Section 2. The data for absorbance and fluorescence emission spectra displayed in Figures 3C and 3D were obtained from the drug-loaded ME over a 90 day period. Figure 3C shows a slight change in resolution over this time, verified by the broadening of the Q-band along the red region (650-700 nm) and the display of a broad absorption peak on the 90th day. The reduction in the absorption spectrum resolution is a consequence of the slow formation of ZnPcSO4 dimers and higher aggregates. However, the PS maintained its fluorescence intensity (Figure 3D) during this period, indicating fluorescence stability. Chemical stability of the PS in ME remained unchanged during the 90 day experiment.
Large amounts of surfactant (47%) in the ME improved the solubility of PS, keeping most ZnPcSO4 molecules incorporated into the developed ME in monomeric form. Consequently, the spectral properties of the incorporated PS did not change significantly during the period studied. Therefore, even in the presence of a small amount of aggregation that developed during storage for 90 days (absorbance spectra in Figure 3C), ZnPcSO4 still shows good photophysical properties (emission spectra in Figure 3D) and adequate chemical stability to render it efficiently usable as a PS for PDT. The results also suggest that the ME developed was effective in protecting the PS against chemical degradation.
3.2. In vitro and in vivo skin penetration studies
In addition to delivering PS in its monomeric form, an effective delivery system for PDT must also deliver the PS into viable layers of the skin. The efficacy of PDT in treating precancerous lesions and non-melanoma skin cancers through the topical application of PSs, especially those of the second-generation, is mainly related to the capacity of the vehicle in which the compound is applied to improve the penetration of monomers across the SC so that they reach malignant cells present in the viable layers of the epidermis. Therefore, the development of delivery systems that improve localization and homogeneous distribution of PSs in the epidermis is desired.
The SC, the most external skin layer, acts as a lipophilic barrier that limits the penetration of substances; for this reason, several studies have focused on the discovery of methods and formulations that improve the penetration of drugs into the skin (28). It is generally known that hydrophilic and high molecular weight drugs do not penetrate the SC; as a consequence, they need to be delivered in a topical formulation that permits skin penetration. To overcome this problem, the ability of the ME to improve ZnPcSO4 penetration through the SC barrier was studied and its penetration-enhancing effect was compared to that of a control formulation (PEG). This control was chosen because it was the only vehicle tested that permitted the solubilization and monomerization of the PS for proper application.
Figure 4A shows that the ME significantly enhanced (p < 0.05) in vitro penetration of ZnPcSO4 in the SC compared to the control formulation up to 9 h post-application. After this period, drug concentration was maintained, possibly due to SC saturation. Maximal ZnPcSO4 concentrations in SC when the drug was incorporated in the control or in ME were, respectively, 0.017 ± 0.007 µg/cm2 and 0.562 ± 0.145 µg/cm2, a 33-fold increase in the case of the ME.
The in vitro penetration of ZnPcSO4 in [E+D] (Figure 4B) from ME was significantly superior (p < 0.05) to control only at 9 h post-application (Figure 4B). Maximal concentrations of ZnPcSO4 found in the [E+D] skin layer were 0.004 ± 0.010 µg/cm2 and 0.124 ± 0.032 µg/cm2, respectively, for release from control or ME, again showing increased uptake (28-fold) in the case of ME.
The relatively long lag time for significant ZnPcSO4 penetration into the [E+D] (9 h) may be explained by the intrinsic ME structure, through which W/O droplets allow only slow drug release. The diffusion of the PS solubilized in the internal aqueous phase may have been delayed by the external oil phase prior to reaching the skin (29). This finding is in accordance with previous reports (30), which showed a sustained release profile for a hydrophilic drug incorporated into the internal phase of a W/O ME.
The results obtained in the in vitro penetration tests after 6 h application of the ME showed ZnPcSO4 retention of approximately 0.25 Î¼g/cm2 and 0.01 Î¼g/cm2 in the SC and [E+D], respectively. Similar and lower concentrations in the SC and [E+D] were obtained when testing in vitro the skin penetration of Termoporfin, a hydrophobic PS, in common and ultradeformable liposomes (11).
The presence of ZnPcSO4 in the receptor phase was not detected in the spectrofluorometric assay. This result can be considered as a positive effect promoted by the delivery system, since the aim of the topical PDT is to increase PS penetration through the SC into viable layers of the skin while avoiding systemic absorption that can cause a generalized photosensitization of the patient. The fact that ZnPcSO4 did not reach the receptor phase in the in vitro experiment can be explained by two possible effects: i) the low aqueous phase content (15.0%) of the ME was not sufficient for ZnPcSO4 to freely diffuse through the skin, a concept postulated by Osborne et al. (31); ii) the lack of dermal microcirculation in the in vitro experiment might have hampered the diffusion of phthalocyanine through lower layers into the receptor phase (16).
Figure 5 shows the skin penetration profile of ZnPcSO4 in hairless mice in vivo. ME significantly increased (p < 0.05) the topical delivery of ZnPcSO4 both in the SC and the [E+D] compared to control. When control formulation was applied to the animals' skin, the maximal drug concentrations in the SC and [E+D] were 0.053 ± 0.004 µg/cm2 and 0.020 ± 0.003 µg/cm2, respectively. When incorporated in ME, ZnPcSO4 concentrations in these layers were, respectively, about 1.6- and 5.6-fold higher.
There are some differences in the in vivo and in vitro results obtained after 6 h application of the ME formulation. The amount of ZnPcSO4 in total skin (SC plus [E+D]) was similar (about 0.2 Î¼g/cm2) in both in vitro and in vivo experiments, but the percentage of ZnPcSO4 in each skin layer was different. In vivo, the percentages of ZnPcSO4 in the SC and [E+D] were, respectively, about 43.0 and 57.0% of the total. In contrast, the percentages of ZnPcSO4 found in the SC and [E+D] in the in vitro experiment were 97.3% and 2.6%, respectively. The higher percentage of ZnPcSO4 deposition in the [E+D] found in the in vivo experiment can be explained as a consequence of the thickness of hairless mouse SC, which permitted a higher penetration of the PS through this skin layer, compared to the porcine skin used in the in vitro experiment (32). The higher delivery in deeper skin layers is a promising result in terms of clinical approaches for PDT using PS drugs or their precursors.
After topical application of aluminum phthalocyanine chloride solution containing DMSO as a penetration enhancer, complete tumor remission in 60% of mice was verified (33). After 6 h of application to normal and tumoral mice skin, the author reported a PS recovery of 0.3 ng/mg of tissue in normal skin; this recovery was 40 times less than in tumoral tissue. By comparison, in normal mouse skin (SC plus [E+D]) we obtained a ZnPcSO4 recovery of 1.2 ng/mg of tissue after 6 h application, four times higher than reported in the cited study. In another study, a skin recovery of 8% of the total dose of PS applied at 5 h after topical application of a gel formulation containing a phthalocyanine derivative was verified in mice (34). This recovery remained unchanged even if the incubation time was extended to 8 h. By comparison, the ME tested in our study permitted 15% recovery of the applied PS dose after 6 h of topical application. The higher skin recovery for ZnPcSO4 obtained in our study can be attributed, among other factors, to the capacity of the ME to improve cutaneous retention of the PS.
3.5. Confocal scanning laser microscopy (CSLM)
The bio-distribution of the PS into the skin layers is also crucial for the success of topical PDT. Confocal laser scanning microscopy (CLSM) permits visualization of the intracellular localization of the fluorescent PS. The technique can be applied with high accuracy in detailed studies of weakly fluorescence fluorochromes, even under conditions when they are rapidly photobleached (35). In the present study, CLSM was used to verify the extent of ZnPcSO4 penetration after in vivo treatment.
Figure 6 shows confocal images of mechanical sections of control and treated hairless mouse skin. Figure 6A shows a histological image of hairless mouse skin; the confocal image of untreated skin (Figure 6B) shows some self-fluorescence of the skin and hair follicles in the excited wavelength (633 nm). Skin that was treated with vehicle only (PEG or ME) showed similar behavior (Figure 6C-D). Increases of blue fluorescence in the skin treated with ZnPcSO4 incorporated into PEG (Figure 6E) and into the ME (Figure 6F) compared to controls (Figure 6B-D) were observed. When the drug was applied in PEG, the homogeneous blue fluorescence was most intense in the SC layer. When ZnPcSO4 was incorporated into the ME, blue fluorescence was homogeneously distributed in the tissue, but was more intense than when applied in control vehicle, and was primarily seen in the viable epidermis and dermis, the target skin layers for topical PDT. These results correlate well with those of the in vivo penetration study.
The results from the in vitro and in vivo experiments confirm the penetration-enhancing effect of ME compared to controls. This effect may be a consequence of the ME composition, which has high surfactant (47.0%), canola oil (38.0%) and PG (11.25%) content. Canola oil is a vegetable oil rich in unsaturated fatty acid (mainly oleic) esters in the form of triglycerides. Some patents report the use of fatty acid esters alone or in combination with other penetration enhancers (ethanol, n-methyl-pyrrolidone) as transdermal enhancers for many drugs, such as ibuprofen, 1,4-dihydropyridine and estrogen (36). Surfactants are amphiphilic molecules that are often added to formulations in order to solubilize lipophilic active ingredients. Nonionic surfactants generally have low chronic toxicity and many have been shown to enhance the flux of drugs through biological membranes (37). PG, also a penetration enhancer, permeates well through human SC and consequently can alter the thermodynamic activity of the drug in the vehicle, in turn modifying the diffusional driving force of solvent partition into the tissue, facilitating uptake of the drug into skin. It has been noted that some minor disturbances may occur in intercellular lipid packing within the SC bilayers (37). It is suggested that the components (surfactants, oil and solvent) of such transdermal enhancers reduce the resistance of the SC lipid bilayer to penetration and alter the solvent properties of SC, favoring ZnPcSO4 partitioning and diffusion into the skin.
Other studies showed that MEs composed of non-ionic surfactants (sorbitan mono-oleate/polysorbate 80), the fatty acid ester isopropyl myristate and water were able to increase the topical delivery in vitro of hydrophilic (20) and lipophilic (22) drugs in human and pig skin. The authors claimed that the components of the MEs were responsible for the penetration-enhancing effect. An increased deposition of hydrophilic drug in the SC and epidermis was achieved through the addition of cholesterol, a penetration enhancer, to the W/O ME, which favored the partition of the drug into hydrophilic domains through the polar head groups of ceramide. In the case of the lipophilic drug, W/O and O/W MEs with varying percentages of water, oil and surfactant were able to increase or reduce topical penetration through the skin. In our recent study, the skin penetration profile of a lipophilic drug, quercetin, incorporated into a ME with similar composition was evaluated in vitro and in vivo in pig and hairless mice skin (38). The results showed higher skin penetration in both experiments when the drug was incorporated in the ME compared to the control formulation.
The solubilization of ZnPcSO4 in the monomer state and its stability when incorporated in the W/O ME that were proposed in this study have been demonstrated. In addition, the ME enabled skin delivery and homogeneous biodistribution of the drug in the target tissue of topical PDT. These findings represent a unique contribution to the field of topical delivery of phthalocyanine drugs in PDT, and suggest the potential advantages of using this vehicle in future preclinical and clinical studies that employ PDT to treat precancerous and cancerous skin diseases.