TY - JOUR
AU - Chorilli, Marlus
AB - Abstract The present study reports the performance of the pigment hypericin (HYP)-loaded poloxamer-based mucoadhesive in situ gelling liquid crystalline precursor system (LCPS) for the treatment of vulvovaginal candidiasis (VVC) in mice. LCPS composed of 40% of ethoxylated and propoxylated cetyl alcohol, 30% of oleic acid and cholesterol (7:1), 30% of a dispersion of 16% poloxamer 407 and 0.05% of HYP (HYP-LCPS) was prepared and characterized by polarized light microscopy (PLM), small-angle X-ray scattering (SAXS) and ex vivo permeation and retention studies across vaginal porcine mucosa were performed. In addition, the antifungal properties of the HYP-LCPS were evaluated in a murine in vivo model; for this, infected C57BL female mice groups were treated with both HYP in solution and HYP-LCPS, and after 6 days colony forming unit (CFU)/ml count was performed. PLM and SAXS confirmed that HYP-LCPS is a microemulsion situated in boundary transition region confirming its action as an LCPS. When in contact with simulated vaginal fluid, HYP-LCPS became rigid and exhibited maltase crosses and bragg peaks characteristics of lamellar phase. Ex vivo permeation and retention studies showed that HYP-LCPS provides a localized treatment on the superficial layers of porcine vaginal mucosa. HYP-LCPS induced a significant reduction in the number of CFU/ml in the mice; thus this formulation indicated it is as effective as a commercial dosage form. It was concluded that LCPS maintains the biological activity of HYP and provides an adequate drug delivery system for this lipophilic molecule at the vaginal mucosa, being a promising option in cases of VVC. liquid crystalline system, hypericin, vulvovaginal candidiasis, murine model Introduction Vulvovaginal candidiasis (VVC) is a disorder induced by species of the genus Candida in the female lower genital tract.1 It is estimated that 75% of the female population will be affected by this disease at least once in their lifetime.2 VVC can be categorized as uncomplicated or complicated depending on the response to treatment and characteristics of infection episodes, being rated according to severity, etiology, risk factors and frequency.3 Uncomplicated cases are mild or moderate infections caused by Candida albicans that occur sporadically and treatment is usually short-lived. Complicated cases include severe and recurrent cases, which commonly affect women present risk factors, such as immunosuppression, pregnancy, diabetes mellitus4 and use of antibiotics and oral-contraceptives.5 Recurrent VVC is associated with the occurrence of at least three episodes within a period of 1 year. Azole antifungal drugs are widely used for the treatment of both categories of VVC.6 Despite the increasing prevalence of cases provoked by non-albicans species,7 Candida albicans is responsible for 85–90% of infection cases4 and the majority of recurrent episodes of VVC.8 Candida albicans pathogenicity is associated with the presence of virulence factors (i.e., adherence, dimorphisms, enzymes secretion and cell surface composition9) that increase or facilitate the germination of fungal cells, which consequently would facilitate tissue invasion.10 In addition, combined with these biological mechanisms, the great availability of over-the-counter azole agents, self-administration and prolonged treatments contribute to the development of drug resistance.6,11 Therefore, the discovery of new antifungal agents is increasingly necessary. Some studies showed that hypericin (HYP) exhibits valuable antifungal properties, including against resistant pathogens.12–14 HYP is a secondary metabolite15 with an aromatic quinoid structure16 that naturally occurs in the leaves of Hipericum plants and in the tegument of some coccid insects from Australia.17 However, HYP is highly hydrophobic pigment, which limits its release in biological media,18,19 such as the vaginal mucus layer. Our research group has been developing liquid crystalline precursor systems (LCPS) in order to optimize the delivery and efficacy of various lipophilic drugs. LCPS are easy handling liquid formulations that, when in contact with bodily fluids, become highly viscous mucoadhesive liquid crystalline systems (LCS). These systems have been enabling specially the application of hydrophobic natural drugs within cavities where traditional dosage forms are diluted and present short residence times due to constant mucus turnover, such as the oral, nasal and vaginal mucosae.20–23 Beyond allowing the local release of drugs at the intended site, LCS increase the solubility of drugs, prevent their degradation and control their release rate. Polymers have been added to the aqueous phase of liquid crystalline phases in order to increase their mucoadhesive properties.24 Poloxamers are amphiphilic block copolymers that have monomeric subunits with different solubilities. At high concentrations, copolymer molecules also have properties similar to surfactants and tend to form aggregates in solution. Therefore, in addition to contributing to the formation of LCS, it is believed that the entanglement between the poloxamer 407 ethoxylated aggregates and mucin glycoproteins chains increases their retention time on mucosal surfaces.25,26 In our previous paper,25 we discussed about an HYP-loaded in situ gelling lamellar liquid crystalline phase for vaginal administration. It was observed that the formulation exhibited great rheological, mechanical and mucoadhesive properties as well as controlled release when it was diluted with different concentrations of simulated vaginal fluid. The aim of this present work was to evaluate the permeability and retention of HYP in the vaginal mucosa as well as the influence of this poloxamer-based LCPS on the biological activity of the HYP against Candida albicans in an in vivo murine model. Materials and methods Materials The non-ionic surfactants PPG-5-CETETH-20 (Procetyl® AWS) and Polysorbate 80 (Tween™ 80) were obtained from Croda, São Paulo, Brazil. Oleic acid was purchased from Synth, Brazil. Poloxamer 407, Dimethyl sulfoxide (DMSO) and cholesterol were obtained from Sigma-Aldrich, North Rhine-Westphalia, Germany. HYP (98.74% purity) was purchased from Apichem Technology, Hangzhou, China. Ultrapure water (Millipore Milli-Q Plus) was used in the preparation of all samples. Preparation of the formulations The selection and preparation of the HYP-loaded and unloaded samples were performed according to our previous work.25 All formulations contained 30% of oleic acid and cholesterol in a 7 : 1 ratio, 40% PPG-5-CETETH-20 and 30% of a Poloxamer 407 dispersion at 16%. The unloaded sample (F) was prepared by homogenizing cholesterol,PPG-5-CETETH-20, and oleic acid under magnetic stirring at 300 rpm (Fisatom®, Brazil) for 24 h. Afterwards, the oily phase was combined with the Poloxamer 407 dispersion, and both phases were blended manually at the room temperature. The HYP-loaded LCPS (HYP-F) was prepared by solubilizing HYP in the oily phase of F for other additional 5 h under magnetic stirring at 300 rpm (Fisatom®, Brazil). Then, the pigment-containing oil phase was transferred to the poloxamer dispersion, and after homogenization, the final formulation remained at rest for at least 24 h. The HYP-loaded sample was prepared at 0.05%, since it was previously observed this concentration provided a great cellular uptake.27 Six other samples were prepared by diluting F and HYP-F with simulated vaginal fluid (SVF). SVF volumes corresponding to 30, 50 and 100% of the total weight of F were incorporated. The unloaded diluted samples were identified as F30, F50 and F100, respectively. Diluted samples containing HYP were identified as HYP-F30, HYP-F50 and HYP-F100, respectively. SVF was also prepared according to our previous work.25 Characterization of the formulations Polarized light microscopy A small fraction of each sample was analyzed under a polarized light microscope (Olympus America, USA), according to methodology used by Calixto et al.28 Polarized microscope images were obtained at room temperature (25 ± 0.5°C) and at 10× magnification. Small-angle x-ray scattering Small-angle x-ray scattering (SAXS) analysis was carried out according to de Araújo et al.25 at the National Synchrotron Light Laboratory (LNLS, Brazil) on line SAXS1. SAXS measurements were performed using a sample-to-detector distance of approximately 1.5 m and incident wavelength λ of 1.488 Å. Scattering patterns were obtained for 45 s at 37°C, and interpreted according to the obtained scattering vectors, q. Ex vivo permeation and retention studies Preparation of porcine vaginal mucosa Porcine vaginas were obtained from the slaughterhouse at the University of São Paulo located in Pirassununga (Sao Paulo, Brazil). The porcine vaginas were transported and stored at the laboratory in saline solution and on ice for up to 6 h after the animal death. Subcutaneous fat and connective tissue were removed using anatomical scissors and a scalpel from fresh porcine vaginas. Then, the vaginal mucosa was wrapped and preserved at −4°C before use. The vaginal mucosa was cleaned with saline solution and kept in SVF for 20 min before the permeation experiments. Permeation experiments Ex vivo permeation study of HYP-F, diluted HYP-loaded samples and HYP solubilized in the receiver solution at 0.25% was performed using Franz's diffusion cells (Microette, Chatsworth, Los Angeles, CA, USA) with a receiver compartment with 7 ml in volume and permeation area of 1.77 cm2. The receiver solution was composed of 0.01 M pH 5 sodium phosphate buffer and 3% polysorbate 80 at 37°C under 100 rpm stirring. Afterwards, the mucosa was arranged between the donor and receptor compartments, and clamped. After being weighed, 300 mg of the formulations were transferred to the dosing ring disposed in the Franz's diffusion cell donor compartment. The samples (1.5 ml) were collected from the receiving compartment after 5 , 10 , 30 , 60 , 120 , 240 , 360 , 480 and 720 min. The collected samples were filtered (0.22 μM PVDF filter) and HYP was quantified by HPLC. Retention experiments The extraction of HYP retained in the porcine vaginal mucosa was carried out according to the method adapted from Mazzarino et al.29 After the 12 h experiment, the vaginal mucosa were removed from the donor compartment of Franz cells. The area of the vaginal mucosa exposed to the formulation was lightly cleaned with methanol, cut and pricked with scissors. The obtained fragments were transferred to 15 ml centrifuge tubes (Falcon, BD®, USA), in which 4 ml of methanol were added and homogenized by Ultra-Turrax® (IKA®, Staufen, Germany) at 10 000 rpm for 5 min, until the total mucosa tearing. Then, the tubes were taken to an ultrasonic bath for 30 min. At the end, the tubes were centrifuged for 10 min at 3500 rpm and the HYP of the supernatant was also quantified by HPLC. HPLC analytical method HYP was quantified according to analytical method validated for methanol according to Kamal et al.30 Briefly, chromatographic determinations were carried out on a Perkin Elmer liquid chromatograph. A C18 Luna® Phenomenex (5 μm; 250 × 4.6 mm) column was used as stationary phase, and an isocratic mobile phase consisting of 54 : 36 : 10 (v/v/v) acetonitrile, methanol and 10 mM ammonium acetate buffer (pH 5.0) at 1 ml/min were employed. UV-VIS detection set at 590 nm was used to determine HYP concentration. The linearity of the method was observed at the concentration range of 0.1–100 μg/ml. HYP-loaded formulations activity against Candida albicans in murine model of VVC Strain and culture condition Candida albicans (ATCC10231) inoculum, obtained from the collection of mycological strains of the Laboratory of Physiology of Microorganisms of the Department of Biological Sciences of the Faculty of Pharmaceutical Sciences of Araraquara (UNESP/FCFAR), under the responsibility of Professor Taís Maria Bauab. The strain (ATCC10231) was standardized for the inoculum used in the study, following the Manual Clinical and Laboratory Standards Institute,31 and the methodology of Gabrielson et al.,32 with adaptations. They were grown by 48 h at 37°C ± 0.5°C in Sabouraud Dextrose Broth and adjusted to a scale of 0.5 McFarland (5 × 106 CFU/ml). The concentration was confirmed by spectrophotometric reading (530 nm) and counted in the Neubauer Chamber.33 Murine model of VVC Animal experimentation was conducted following the recommendations established by the Guide for the Care and Use of Laboratory Animals34 and the Brazilian Committee for Animal Experimentation. This in vivo study was approved by the animal experimentation ethics committee of the School of Pharmaceutical Sciences, São Paulo State University (CEUA protocol, number 44/2018), obeying international laws. The in vivo activity of the system containing HYP was developed based on the study by Rodero et al.23 with modifications. Groups of 8 − 10 weeks female mice (C57BL/6) weighing 20 − 25 g were used. The animals remained on 12 h light/dark cycle acclimatized cages. Water and food were freely available throughout the experiment. Initially, subcutaneous application of 100 μl of estradiol (2 mg/ml-sesame oil) was performed to induce the pseudoestrus state in the animal, later this procedure was performed twice a week for maintaining hormonal status. After 48 h of estradiol application, the mice were infected (day 0) with an intravaginal application of 20 μl of C. albicans (ATCC10231) suspension in Phosphate-buffered saline (PBS) 0.2 M pH 7.4 (2.5 × 108 strains/ml). Vaginal lavages were collected with PBS (280 μL) after 2, 4 and 6 days of infection. After collection, vaginal lavages were diluted 1 : 10 in PBS, and 100 μl of the suspension was plated in Sabouraud Dextrose Broth and kept at 37 ± 0.5°C for 48 h, then the CFU/ml count was performed. The groups were treated on days 3, and 5 after infection, twice a day with 20 μl of each sample. Each experimental group contained five animals, and these were named negative control (infected without any treatment), positive group (Bio-vagin® ointment, which contains an association of benzoylmetronidazole, nystatin and benzalkonium chloride), HYP solution, F and HYP-F. The sample size calculation was applied based on the ANOVA test to be performed, considering the number of treatments of 5, the test power of 0.8 and alpha of 0.05, obtaining n = 4. To guarantee the efficiency of the test as well as ensuring the minimum ‘n’ in face of possible adversity, n = 5 was established (BioEstat vs 5.3). Two-way ANOVA and Tukey's post hoc tests were performed to calculate the significance (P < 0.05) between the ‘Control -’ group and the other tested groups using Prism 5.01, GraphPad Software Inc., USA. Results and discussion F and HYP-F showed a dark field when observed under PLM, which is indicative of isotropic microemulsions, as illustrated in the Figure 1. However, SAXS data presented in Table 1 did not confirm the PLM data. F and HYP-F exhibited a large peak in q = 0.538 and q = 0.55, respectively; but the presence of a small shoulder in q = 0.9765 for F and 1.048 for HYP-F, resulting a ratio practically equal to 1 : 2 in relation to the first peak, might be associated to the presence of a small fraction of lamellar phase in the formulations. Figure 1. Open in new tabDownload slide Polarized Light Microscopy images of the unloaded samples F (A), F30 (B), F50 (C) and F100 (D) and HYP-loaded samples HYP-F (E), HYP-F30 (F), HYP-F50(G) and HYP-F100(H) at room temperature and at 10× magnification. Black arrows are signalizing maltese crosses and oily streaks, which indicate the formation of lamellar mesophase. Figure 1. Open in new tabDownload slide Polarized Light Microscopy images of the unloaded samples F (A), F30 (B), F50 (C) and F100 (D) and HYP-loaded samples HYP-F (E), HYP-F30 (F), HYP-F50(G) and HYP-F100(H) at room temperature and at 10× magnification. Black arrows are signalizing maltese crosses and oily streaks, which indicate the formation of lamellar mesophase. Table 1. Bragg peak ratios for unloaded samples (F, F30, F50, F100) and HYP-loaded samples (HYP-F, HYP-F30, HYP-F50, HYP-F100). Sample . q1 . q2 . q3 . q4 . Ratio . System . F 0.538 0.9765 – – ∼1 : 2 Lα + L F30 0.3788 0.7076 1.42 2.109 1 : 2 : 3 Lα + L F50 0.366 0.6342 1.280 1.9188 1 : 2 : 3 Lα + L F100 0.3422 0.55 1.1273 1.6707 1 : 2 : 3 Lα + L HYP-F 0.55 1.048 – – ∼1 : 2 Lα + L HYP-F30 0.3788 0.7076 1.4115 2.1217 1 : 2 : 3 Lα + L HYP-50 0.3788 0.6709 1.342 2.011 1 : 2 : 3 Lα + L HYP-F100 0.3544 0.55 1.0974 1.6574 1 : 2 : 3 Lα + L Sample . q1 . q2 . q3 . q4 . Ratio . System . F 0.538 0.9765 – – ∼1 : 2 Lα + L F30 0.3788 0.7076 1.42 2.109 1 : 2 : 3 Lα + L F50 0.366 0.6342 1.280 1.9188 1 : 2 : 3 Lα + L F100 0.3422 0.55 1.1273 1.6707 1 : 2 : 3 Lα + L HYP-F 0.55 1.048 – – ∼1 : 2 Lα + L HYP-F30 0.3788 0.7076 1.4115 2.1217 1 : 2 : 3 Lα + L HYP-50 0.3788 0.6709 1.342 2.011 1 : 2 : 3 Lα + L HYP-F100 0.3544 0.55 1.0974 1.6574 1 : 2 : 3 Lα + L Open in new tab Table 1. Bragg peak ratios for unloaded samples (F, F30, F50, F100) and HYP-loaded samples (HYP-F, HYP-F30, HYP-F50, HYP-F100). Sample . q1 . q2 . q3 . q4 . Ratio . System . F 0.538 0.9765 – – ∼1 : 2 Lα + L F30 0.3788 0.7076 1.42 2.109 1 : 2 : 3 Lα + L F50 0.366 0.6342 1.280 1.9188 1 : 2 : 3 Lα + L F100 0.3422 0.55 1.1273 1.6707 1 : 2 : 3 Lα + L HYP-F 0.55 1.048 – – ∼1 : 2 Lα + L HYP-F30 0.3788 0.7076 1.4115 2.1217 1 : 2 : 3 Lα + L HYP-50 0.3788 0.6709 1.342 2.011 1 : 2 : 3 Lα + L HYP-F100 0.3544 0.55 1.0974 1.6574 1 : 2 : 3 Lα + L Sample . q1 . q2 . q3 . q4 . Ratio . System . F 0.538 0.9765 – – ∼1 : 2 Lα + L F30 0.3788 0.7076 1.42 2.109 1 : 2 : 3 Lα + L F50 0.366 0.6342 1.280 1.9188 1 : 2 : 3 Lα + L F100 0.3422 0.55 1.1273 1.6707 1 : 2 : 3 Lα + L HYP-F 0.55 1.048 – – ∼1 : 2 Lα + L HYP-F30 0.3788 0.7076 1.4115 2.1217 1 : 2 : 3 Lα + L HYP-50 0.3788 0.6709 1.342 2.011 1 : 2 : 3 Lα + L HYP-F100 0.3544 0.55 1.0974 1.6574 1 : 2 : 3 Lα + L Open in new tab Frank et al.35 observed that the incorporation of amphiphilic copolymers led to an increase in the efficiency of equal ratios of oil and water. However, at high copolymer concentration, a lamellar LCS might coexist with a microemulsion. In addition, this mixture of phases indicates that F is located close to a boundary region and that small volumes of fluid would be necessary to cause phase transition. According to Kunieda and Shinoda,36 the presence of lamellar LCS in formulations that combine non-ionic block copolymers and surfactants indicate the formation of highly efficient microemulsions. Regardless of the presence of HYP, the dilution of the systems F and HYP-F leads to the formation of lamellar LCS indicated by the presence of Maltese crosses and oily streaks under PLM (Figure 1). The addition of increasing VSF in F and HYP-F led to small displacements of the first peaks to lower q values, and the emergence of extra peaks reflections with a 1 : 2 : 3 ratio. This behavior shows that the particles of the isotropic phase started to coexist with a greater amount of lamellar LCS. In addition, the lamellar LCS were formed even after large dilutions. Therefore, these analyses indicate that a microemulsion, a fluid and stable drug delivery system, is able to gelify in situ and becomes a liquid crystalline phase after being diluted with the vaginal fluid. HYP was not detected by the chromatographic method during the 12 h of permeation experiment in any sample, even LCPS containing high concentration of oleic acid, a penetration-enhancer element and poloxamer 407, a copolymer known to increase the diffusion and targeting of drugs.37 Although the use of a porcine model is advantageous, because it is possible to predict the permeability of drugs in the human vagina,38 permeation studies show that the physicochemical properties of the drugs, beyond many factors such as vaginal fluid amount and composition and hormonal cyclic alterations, influence the permeability of active substances.39 Neutral low molecular weight and lipophilic drugs have higher diffusion rates at human vagina.40 HYP is a lipophilic and low molecular weight pigment,41 however it is negatively charged (pka = 2)42 at the vaginal pH, which might reduce its permeability. On the other hand, as shown in Figure 2, the retention of HYP in vaginal mucosa occurred proportionally to the controlled release profile of LCPS, as previously studied.25 The HYP solution showed greater retention in the mucosa, which might be related to the greater availability of the substance in contact with the mucosa throughout the experiment. However, the concentration would probably be lower considering the low retention time of the solutions in the vaginal cavity. On the other hand, HYP-F, HYP-F30, HYP-F50 and HYP-F100 exhibited lower HYP retention in the porcine mucosa, because the drug diffuses from a complex matrix before reaching the target tissue. We suggest the greater affinity of HYP for the oily components of the formulations would probably limit the diffusion of HYP to deep layers of the porcine mucosa, favoring its retention and the local action. Figure 2. Open in new tabDownload slide HYP retention (μg/ml) in porcine mucosa after 12 h. When compared to the free pigment (HYP), HYP-F and diluted samples (HYP-F30, HYP-F50 and HYP-F100) presented a significant reduction of the HYP retention in the mucosa due to the composition of the formulation and concentration of HYP and penetration enhancer component. Figure 2. Open in new tabDownload slide HYP retention (μg/ml) in porcine mucosa after 12 h. When compared to the free pigment (HYP), HYP-F and diluted samples (HYP-F30, HYP-F50 and HYP-F100) presented a significant reduction of the HYP retention in the mucosa due to the composition of the formulation and concentration of HYP and penetration enhancer component. Lopes et al.43 concluded that the concentration of penetration enhancers significantly influences the permeability of drugs. They observed that LCS that contained up to 10% monoolein, a penetration enhancer structurally similar to oleic acid, induced an increase in the permeability of cyclosporine A, which caused the peptide to reach the bloodstream. However, concentrations above 20% caused the retention of cyclosporine A in the tissue. Thus, they believe that the high affinity of the peptide for monoolein, which remains partitioned in the most superficial layers of the skin, would be mainly responsible for the retention of the drug in the epithelial layer. Boiy et al.44 observed that the penetration of HYP into skin layers occurs according to its dissolution and affinity for the drug delivery system. They observed that the high affinity of HYP for certain vehicles resulted in poor retention of the pigment in the epithelial layer. Similar results were obtained in vehicles where HYP was poorly soluble due to the formation of high molecular weight aggregates.45–47 Therefore, we conclude the mass transference of HYP occurs from a more concentrated site or reservoir to a less concentrated one and remains localized in the epithelium due to the greater tendency of HYP to accumulate in lipidic moieties and due to its ionized form at the vaginal pH after being released from the bilayers of lamellar LPS. This behavior indicates that this in situ mucoadhesive LCPS provides a long-term local treatment47 and prevents the emergence of systemic adverse reactions.48 A local release provides a greater fraction of drug at infected tissues and decreases drug toxicity on heathy cells.49 In addition, this effect is mainly desired to avoid skin photosensitization caused by HYP in people exposed to sunlight.50 The in vivo study was conducted for 6 days and yeast colonies were counted every 48 h, on 4 and 6 days after infection. As observed in Figure 3, the animals of the negative control group remained infected by C. albicans during all the experiment, a similar profile observed for the group treated with DMSO solution used to solubilize HYP. Figure 3. Open in new tabDownload slide Comparison of the reduction in colony-forming units between the negative control group and the other tested groups (positive control group, F, HYP solution, HYP solution, HYP-F), after 2, 4 and 6 days post-infection. “*” Indication of significant difference between the negative control and the other groups under test (P < 0.05). Figure 3. Open in new tabDownload slide Comparison of the reduction in colony-forming units between the negative control group and the other tested groups (positive control group, F, HYP solution, HYP solution, HYP-F), after 2, 4 and 6 days post-infection. “*” Indication of significant difference between the negative control and the other groups under test (P < 0.05). Statistical analysis was used in order to compare the negative control group and the other groups tested on their respective days of analysis. Thus, the positive control group and the other treatment groups showed a significant reduction in Log10 CFU after the first and second days of treatment, days 4 and 6, respectively. Comparing the reduction of the positive control, it is observed that the HYP demonstrated efficacy similar to the commercial presentation, both in solution and in the LCPS, demonstrating the potential of this biomolecule for the treatment of VVC. The system also showed a reduction in CFU, it is suggested that the reduction promoted by the LCPS occurred due to the impediment in the permanence of the strain. However, the LCPS did not change the effectiveness of HYP, since the HYP-LCPS group remained with the results comparable to the positive control and to HYP solution. This in turn demonstrates the potential of the system as a vehicle for vaginal application, corroborating with the data obtained by the ex vivo retention test and with studies that used the liquid crystalline system for the same purpose.21,51 Thus, the incorporation of HYP in an LCPS proved to be a promising alternative to treat VVC, since the biomolecule showed results similar to the commercialized drug and the formulation did not interfere in its activity, making possible the vaginal application of HYP and increasing your adherence at the target location. In this work, the in situ gelling HYP-LCPS controlled the retention of HYP on vaginal mucosa and efficiently reduced the viability of C. albicans colonies. We concluded that this LCPS provides a local long-term treatment of vaginal infections and it could also be employed for the administration of other various drugs in the vaginal cavity. It is important to emphasize that due to the increasing number of CVVs caused by non-albicans species, the evaluation of this system against such species becomes an interesting object for future studies. Acknowledgements We thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Grants ##2019/10261-2, 16/11198-4), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Programa de Apoio ao Desenvolvimento Científico (PADC). This study was also financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. Declaration of interest The authors report no conflict of interest. The authors alone are responsible for the content and the writing of the paper. References 1. White DJ , Vanthuyne A. Vulvovaginal candidiasis . Sex Transm Infections . 2006 ; 82 : iv28 – iv30 . Google Scholar OpenURL Placeholder Text WorldCat 2. Talaei Z , Sheikhbahaei S, Ostadi V et al. 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TI - In vivo study of hypericin-loaded poloxamer-based mucoadhesive in situ gelling liquid crystalline precursor system in a mice model of vulvovaginal candidiasis
JF - Journal of Medical and Veterinary Mycology
DO - 10.1093/mmy/myab006
DA - 2021-02-24
UR - https://www.deepdyve.com/lp/oxford-university-press/in-vivo-study-of-hypericin-loaded-poloxamer-based-mucoadhesive-in-situ-mQV78wjNOB
SP - 1
EP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -