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Nanoencapsulation of coenzyme Q10 and vitamin E acetate protects against UVB radiation-induced skin injury in mice
Accepted Manuscript Title: Nanoencapsulation of coenzyme Q10 and vitamin E acetate protects against UVB radiation-induced skin injury in mice Author: Nath´ali S. Pegoraro Allanna V. Barbieri Camila Camponogara Evelyne S. Brum Marila C.L. Marchiori Sara M. Oliveira Let´ıcia Cruz PII: DOI: Reference:
S0927-7765(16)30789-5 http://dx.doi.org/doi:10.1016/j.colsurfb.2016.11.013 COLSUB 8247
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
17-8-2016 3-11-2016 7-11-2016
Please cite this article as: Nath´ali S.Pegoraro, Allanna V.Barbieri, Camila Camponogara, Evelyne S.Brum, Marila C.L.Marchiori, Sara M.Oliveira, Let´ıcia Cruz, Nanoencapsulation of coenzyme Q10 and vitamin E acetate protects against UVB radiation-induced skin injury in mice, Colloids and Surfaces B: Biointerfaces http://dx.doi.org/10.1016/j.colsurfb.2016.11.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Nanoencapsulation of coenzyme Q10 and vitamin E acetate protects against UVB radiation-induced skin injury in mice Natháli S. Pegoraroa, Allanna V. Barbierib, Camila Camponogarac, Evelyne S. Brumc, Marila C. L. Marchioria, Sara M. Oliveirac, Letícia Cruza,b a
Programa de Pós-Graduação em Ciências Farmacêuticas, Centro de Ciências da Saúde,
Universidade Federal de Santa Maria, Santa Maria 97105-900, Brazil b
Departamento de Farmácia Industrial, Universidade Federal de Santa Maria, Santa Maria
97105-900, Brazil c
Programa de Pós-Graduação em Ciências Biológicas: Bioquímica Toxicológica, Centro de
Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria 97105-900, Brazil
*Corresponding author: Letícia Cruz, Departamento de Farmácia Industrial, Universidade Federal de Santa Maria, Santa Maria, 97105-900, Brazil. Phone: +55 55 32209373. Fax: +55 55 32208149. E-mail: [email protected]
Total number of pages:21 Number of tables:3 Number of figures:5 Total number of words including references: 5795
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Graphical abstract
Highlights
Semisolids were obtained by thickening nanocapsule suspensions with gellan gum; The formulations presented characteristics suitable for cutaneous administration; NCP3 decreased MPO and NAGase activity in the UVB radiation-induced ear edema model; NC increased non-protein thiol levels and decreased lipid peroxidation induced by UVB radiation; NC with Q10 and VitE reduced the UVB irradiation-induced effects on skin.
Abstract
This study aimed to investigate the feasibility of producing semisolid formulations based on nanocapsule suspensions containing the association of the coenzyme Q10 and vitamin E acetate by adding gellan gum (2%) to the suspensions. Furthermore, we studied their 2
application as an alternative for the treatment of inflammation induced by ultraviolet B (UVB) radiation. For this, an animal model of injury induced by UVB-radiation was employed. All semisolids presented pH close to 5.5, drug content above 95% and mean diameter on the nanometric range, after redispersion in water. Besides, the semisolids presented non-Newtonian flow with pseudoplastic behavior and suitable spreadability factor values. The results also showed that the semisolid containing coenzyme Q10-loaded nanocapsules with higher vitamin E acetate concentration reduced in 73±8% the UVB radiation-induced ear edema. Moreover, all formulations tested were able to reduce inflammation parameters evaluated through MPO activity and histological procedure on injured tissue and the semisolids containing the nanoencapsulated coenzyme Q10 reduced oxidative parameters assessment through the nonprotein thiols levels and lipid peroxidation. This way, the semisolids based on nanocapsules may be considered a promising approach for the treatment and prevention of skin inflammation diseases.
Keywords Coenzyme Q10, vitamin E, nanocapsules, skin, ear edema, UV radiation.
1. Introduction Skin is often exposed to several harmful sources, among them, the ultraviolet (UV) radiations. UV radiation consists of wavelengths shorter than visible light and is classified as UVA, UVB and UVC. Among these types of light, the main responsible for skin cytotoxic events is UVB radiation, which induces several pathologic changes in skin such as erythema, edema, sunburn, immune suppression and skin cancer, and it leads to changes and often to the destruction of skin structures what can cause changes in their normal function1-3. One of the major alterations caused by UV radiation leading to deleterious outcomes on the skin is the production of highly unstable products, called reactive oxygen species (ROS), that contribute to oxidizing and cause cellular damage4,5. Sunscreens are recommended for the protection against UV light-induced skin damage due to their ability to absorb, reflect or scatter UV light. However, they are limited to repair UV-mediated cutaneous damage. In this sense, there is a growing search for new alternatives, especially for formulations containing
3
antioxidant molecules capable of attenuating the effects produced by ROS, through benefic effects such as anti-inflammatory, antioxidant and DNA repair6,7,8. The combination of antioxidants may be an advantageous strategy to improve their biological effects. Among the substances with high antioxidant activity, the coenzyme Q10 (Q10) and vitamin E (VitE) play an important role in many biological systems due to their ability to decrease oxidative stress generated by ROS. Q10, also known as ubiquinone or ubidecarenone, is an essential lipophilic antioxidant for the human body because of its key role on ATP synthesis, being fundamental on mitochondrial electron transport chain9. This compound can be obtained from sources as red meat, fish and poultry or from supplementation10. Some studies showed the Q10 benefits on anti-aging therapy, skin hydration and photoprotection11-13. In its turn, VitE is an antioxidant composed by tocopherols and tocotrienols. This vitamin can be obtained from diet by the ingestion of vegetable oils, nuts and almonds14. Its antioxidant function lies on its capability to stabilize reactive species15. It was already reported the association of VitE with UV filters, based on its antioxidant properties that play an essential role on skin repair16,17. In the last years, nanocapsule suspensions have emerged as promising alternatives to deliver substances through the skin because of their ability to control drug release and to improve drug stability18. Nanocapsules are vesicular systems composed by a central core, generally an oily core, surrounded by a polymeric wall19,20. Besides, nanocapsules are obtained as liquid suspensions, which may complicate its cutaneous application. The incorporation of nanoparticle suspensions in semisolid vehicles has been reported using Carbopol® Ultrez 10 NF21, Carbopol® 94022, Pemulen TR-122 and xantham gum22. Regarding the cutaneous application, the repairing effect of a nanoencapsulated substance was recently studied on ear edema induced by UVB irradiation in mice23. In that work, it was demonstrated that a hydrogel containing nanocapsules of rice bran oil could reduce ear edema by 60±9%. Besides that, its effects on oxidative stress and inflammation was also evidenced by the reduction of protein carbonylation and NF-κB nuclear translocation biomarkers levels, demonstrating its beneficial effects on skin damage caused by UVB exposure. With the purpose of proposing a new formulation with improved skin repair properties, in this work it was demonstrated a novel association between the Q10 and VitE in nanocapsules using gellan gum, a microbial polysaccharide with gelling properties24, as a new thickening agent for nanosuspensions. 4
2. Materials and methods 2.1 Materials Coenzyme Q10 and vitamin E acetate were obtained from Mapric (São Paulo, Brazil). Poly-ε-caprolactone (PCL) (MW: 70,000-90,000), polysorbate 80 (Tween® 80) and sorbitan monooleate (Span® 80) were provided from Delaware (Porto Alegre, Brazil). Gellan gum was kindly donated by CP Kelco (Georgia, USA). Acetone was purchased from Proquimios (Rio de Janeiro, by Brazil). Anhydrous ethanol (HPLC grade) was obtained from Tedia (São Paulo, Brazil). Dimethylsulphoxide (DMSO) was purchased from Sigma Chemical Co. (St. Louis, USA). All other reagents and solvents were of analytical grade and used as received.
2.2 Methods 2.2.1 Nanocapsule suspensions preparation and semisolid formulations Nanocapsule suspensions were prepared following the interfacial deposition of preformed polymer method25. An organic phase constituted by PCL (0.100 g), Span® 80 (0.077 g), VitE (0.1 or 0.3 g - 1 or 3%, respectively), Q10 (0.010 g) and acetone (27 mL) was kept under moderate magnetic stirring at 40°C during an hour. After the complete dissolution of the components, this phase was injected into an aqueous phase containing Tween® 80 (0.077 g) and the magnetic stirring was sustained for 10 min. After this, the organic solvent and part of the water were eliminated by evaporation under reduced pressure to obtain 10 mL of final volume and 1.0 mg/mL of Q10. Formulations without Q10 were prepared as well. The formulations were abbreviated as following: NCP1 (Q10-loaded nanocapsules containing 1% VitE), NCP3 (Q10-loaded nanocapsules containing 3% VitE), NBP1 (nanocapsules without Q10 containing 1% VitE) and NBP3 (nanocapsules without Q10 containing 3% VitE). Semisolid formulations were prepared using mortar and pestle by adding 0.2 g gellan gum directly to the nanocapsule suspensions (10 mL) to obtain around 10 g of semisolids. For the semisolid formulations containing free compounds, both Q10 (1 mg/g) and VitE (3%) were solubilized in DMSO (1 mL) and added to a dispersion of gellan gum (2%) in water (9 mL). The vehicle was prepared following the same methodology, but only dispersing gellan gum in water (10 mL). All semisolids containing Q10 and/or VitE were prepared under light protection.
2.2.2 Semisolid formulations characterization 2.2.2.1 pH determination 5
pH values were evaluated by immersing a calibrated potentiometer (Model pH 21, Hanna Instruments, Brazil) in a semisolid aqueous dispersion (10%, w/v).
2.2.2.2 Mean particle size evaluation Nanocapsules mean particle size in the semisolids was measured using ZetaSizer Nano Series Malvern Instruments (UK), by photon correlation spectroscopy method, by dispersing an aliquot of the samples in ultrapure water (1:500).
2.2.2.3 Q10 content The analysis of the Q10 total content in the semisolids was performed by HPLC employing methodology described by Mattiazzi and co-workers26, with some modifications. It was a simple RP-HPLC-UV method employing the following chromatographic conditions: C18 column at room temperature, and mobile phase constituted of a mixture of anhydrous ethanol and methanol (75:25 v/v), at a flow rate of 1.0 mL/min and detection UV at 275 nm. The analytical method was specific, linear, accurate, and precise over the concentration range of 5.0 - 25.0 μg/mL. For this evaluation, an aliquot of the semisolid was solubilized in ethanol and submitted to sonication for 10 min. Then, the sample was centrifuged at 3,000 rpm (664 xg) during 10 min. Subsequently, samples were filtered through a 0.45 μm membrane and injected into HPLC system.
2.2.2.4 Spreadability determination The semisolid formulations spreadability was evaluated through the parallel plate method27,28. An aliquot of the sample was put in a central hole of a mold glass plate, that was on the scanner surface (HP Officejet, model 4500 Desktop). Then, the mold glass was carefully removed and upon the sample was placed glass plates with known weights. Each plate was placed observing an interval of 1 min to the subsequent plate, a total of 10 plates were used. One image was captured at every 1 min of interval, employing the desktop scanner and the software Image J (Version 1.49q, National Institutes of Health, USA). Both registers were used to calculate the spreading area of the captured images. The spreadability profiles were obtained by plotting the spreading area versus the cumulative weight of the plates. Finally, the spreadability factor was calculated for all formulations. This factor represents the formulation ability to expand on a smooth horizontal surface when a gram of weight is added to it under test conditions. This equation (Eq. (1)) was employed to calculate the spreadability factor: 6
Sf =
A W
in which Sf is the spreadability factor (mm2 g-1), A is the maximum spread area (mm2) after the addition of the total number of plates, and W is the total weight added (g).
2.2.2.5 Rheological behavior Rheological analyses were conducted at 25±1ºC employing viscometer (RVDV-IPRIME model, Brookfield, USA) with a RV06 spindle. For this, about 50 g of the formulations was used and submitted to a range of speed between 5-100 rpm. The data obtained was analyzed to the best fit using Bingham, Casson, Ostwald and Herschel-Bulkley models (Eq. 2 - 5)29, employing graphical model to determine rheological behavior.
(2)
𝜏 = 𝜏0 + 𝜂 𝛾
(3)
𝜏 0.5 = 𝜏00.5 + 𝜂0.5 𝛾 0.5
(4)
𝜏 = К 𝛾𝑛
(5)
𝜏 = 𝜏0 + К 𝛾 𝑛 ,
where 𝜏0 is the yield stress, 𝜂 is the viscosity, 𝑛 is the index of flow, К is the index of consistency, 𝜏 is the shear stress and 𝛾 is the shear rate29.
2.2.3 Animals Male Swiss mice (25-30 g; n total = 114) were used in all experiments. Animals were kept under controlled temperature (22 ± 2°C) on a 12 h light-dark cycle and with standard laboratory chow and water ad libitum. The animals were habituated to the experimental room at least 1 h before the experiments. All experiments were carried out between 8:00 a.m. and 5:00 p.m. The experiments were performed in accordance to the current ethical guidelines for care of laboratory animals30 and all procedures were approved by our Institutional Ethics Committee (Process number 4921060715/2015). Animals were randomly assigned to different treatment groups and all the experiments were blindly performed. The number of animals and intensity of stimuli was the minimum necessary to demonstrate the consistent effects of treatments. 7
2.2.4 UVB irradiation model The UVB source of irradiation consisted of a Philips TL40W/12 RS lamp (MedicalEindhoven, Holland) assembled 20 cm above the table where mice were placed, and which emitted a continuous light spectrum between 270 and 400 nm with a peak emission at 313 nm. UVB output (80% of the total UV irradiation) was measured using a model IL-1700 Research Radiometer (International Light, USA; calibrated by IL service staff) with a radiometer sensor for UV (SED005) and UVB (SED240). The UVB irradiation rate was 0.27 mW/cm2 and the dose used was 0.5 J/cm2. Mice were firstly anesthetized (90 mg/kg of ketamine + 3 mg/kg of xylazine) with a single intraperitoneal injection and then exposed to UVB irradiation. Only the right ear of each animal was exposed to UVB irradiation31,32.
2.2.5 Formulation administration and experimental design Swiss mice were randomly divided in nine groups with six animals each and classified as it follows: naïve (non-irradiated); untreated irradiated; treated with vehicle, treated with semisolid containing non-encapsulated Q10 and VitE (FH); treated with semisolid containing Q10-loaded nanocapsules with 1% VitE (NCP1); treated with semisolid containing nanocapsules with 1% VitE without Q10 (NBP1); treated with semisolid containing Q10loaded nanocapsules with 3% VitE (NCP3); treated with semisolid containing nanocapsules with 3% VitE without Q10 (NBP3). As positive control to treat light burning, silver sulfadiazine (3%) was employed. Mice were topically treated on the ear surface with different semisolid formulations (15 mg/ear) immediately receiving UVB irradiation33.
2.2.6 Ear edema measurement The skin photodamage was induced by UVB irradiation and the inflammatory process was assessed through ear edema formation. The edema was measured by the increase in ear thickness after the inflammatory stimuli. The ear thickness was evaluated before and 24 h after UVB irradiation using a digital micrometer (Digimess) in animals anesthetized with isofluorane34. The micrometer was applied near the tip of the ear just distal to the cartilaginous ridges. Thickness was expressed in μm. To minimize the variation, a single investigator performed the measurements throughout each experiment.
2.2.7 Leukocyte infiltration assessment 2.2.7.1. Myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAGase) activities 8
UVB irradiation-induced leukocytes migration in the skin was assessed using the MPO and NAGase activities assay as previously described35,23. MPO and NAGase enzymes activities are used as biochemical markers of the polymorphonuclear leukocyte influx (mostly neutrophil and macrophages, respectively) to the injured tissue. Twenty-four hours after UVB irradiation, mice were euthanized and the ears were removed to determine MPO and NAGase activities. Tissue samples were homogenized in acetate buffer (80 mM, pH 5.4) containing 0.5% hexadecyltrimethylammonium bromide, centrifuged and the supernatant was collected for assay. For evaluation of MPO enzyme activity, the sample was incubated with acetate buffer and 3,3’,5,5’-tetramethylbenzidine solution (18.4 mM) at 37ºC. The reaction was stopped on ice by adding acetic acid. The formed color was analyzed by a spectrophotometer at 630 nm. In sequence, to determine NAGase enzyme activity, the sample was mixed with sodium citrate buffer (50 mM, pH 4.5) and p-nitrofenil-2-acetamide-β-D-glucopyranoside (NAG 2.25 nM) and incubated at 37ºC. After incubation time, the reaction was stopped on ice by adding glycine buffer (0.2 μM, pH 10.4). The formed color was analyzed by a spectrophotometer at 405 nm. Both reactions were read on a Fisher BiotchMicrokinetics BT 2000 microplate reader. The results were expressed as the sample optical densities (OD)/mL.
2.2.7.2 Histology Separate groups of mice were used to evaluate histological changes in ear tissue 24 h after UVB-irradiation or UVB-irradiation plus semisolid formulations. Mice were euthanized and the right ear was removed and fixed in alfac solution (16:2:1 mixture of ethanol 80%, formaldehyde 40% and acetic acid). Each sample embedded in paraffin was sectioned at 5 μm and stained with hematoxylin-eosin. A representative area was selected for qualitative light microscopic analysis of the inflammatory cellular response with 20x and 40x objectives35,36. To minimize a source of bias, the investigators did not know the group that they were analyzing.
2.2.8 Oxidative stress evaluation In order to verify the effect of different semisolid formulations on the oxidative damage induced by UVB irradiation, we determined non-protein thiol levels and lipid peroxidation. After 24 h of UVB irradiation, mice were euthanized and ears were removed and homogenized with Tris/HCl buffer (50 mM, pH 7.4) and centrifuged. The supernatants were used to continue the analysis. To determine non-protein thiol (NPSH) levels, the protein supernatant was 9
precipitated with TCA 10% and centrifuged. The deproteinized supernatant was incubated with Tris/HCl (200 mM, pH 8.9) and 5-5’dithiobis- (Z-nitro-benzoic acid) (DTNB – 2.5 mM) at room temperature37. The color of the resulting solution from the reaction was measured at 405 nm with a Fisher BiotchMicrokinetics BT 2000 microplate reader. Lipid peroxidation was determined through of the measurement of lipid hydroperoxide and thiobarbituric acid reactive substances (TBARS) assay. The concentration of lipid hydroperoxide was estimated by the FOX assay. For this purpose, the tissue homogenate supernatant was incubated with FOX reagent (sulfuric acid 250 mM, xylenol orange 0.8 μM, ferrous sulfate and ammonium sulfate 3 μM and butyl hydroxytoluene 0.04 mM) at 37ºC. After, the absorbance of the solution was read at 560 nm38. Lipid hydroperoxide content (μM) was calculated using the 4.6 x 104 M/cm molar extinction coefficient. The extent of lipid peroxidation in terms of malondialdehyde (MDA) formation was measured by TBARS assay. The supernatant was incubated with TBA 0.8%, acetic acid buffer and sodium dodecyl sulfate (SDS) 8.1% at 90ºC. The solution color resulting from the reaction was measured at 535 nm with a Fisher BiotchMicrokinetics BT 2000 microplate reader. Both the lipid peroxidation by TBARS and non-protein thiol contents were corrected for mL of sample39.
2.2.9 Statistical analysis The results are presented as mean + standard error of the mean (SEM), which are reported as geometric means plus their respective 95% confidence limits. The maximum inhibitory effect (Emax) was calculated based on the response of the control groups. The statistical significance between the groups was assessed by one-way or two-way analysis of variance (ANOVA) followed by post hoc Newman-Keuls test, Bonferroni's test or Tukey's test, when appropriate. All tests were carried out using GraphPad 5.0 Software (San Diego, CA, USA).
3. Results 3.1 Semisolid formulations characterization 3.1.1 pH determination The characteristics of semisolid formulations are presented in Table 1. pH values were in the acid range, close to 5.5 for all formulations, which is compatible with skin pH and suitable
10
for cutaneous administration40,41. Semisolids containing NCP1, NBP1 and NBP3 nanocapsule suspensions presented higher pH values (p<0.05) than the vehicle.
3.1.2 Mean particle size evaluation The measures of the semisolids mean diameter after redispersion in water are shown in Table 1. Nanostructured formulations and vehicle presented mean diameter on the nanometric range. For the nanostructured ones, values of mean diameter were significantly higher for the formulations containing Q10 (NCP1 and NCP3) (p<0.05). All formulations showed significant difference when compared to the vehicle mean diameter (p<0.05).
3.1.3 Q10 content Concerning the Q10 content, the semisolids containing NCP1 and NCP3 nanocapsules and free compounds revealed Q10 content close to 100%. The presence of nanostructures or VitE concentration in the samples did not influence this parameter (p>0.05).
3.1.4 Spreadability determination In relation to spreadability evaluation (Figure 1), the values of spreadability factors were 4.61±0.83 and 4.63±0.86 mm2/g for NCP1 and NCP3 semisolids, respectively (Table 1). For NBP1 and NBP3 they were 3.79±0.35 and 3.63±0.67 mm2/g, respectively. In this case, VitE concentration or Q10 presence in nanocapsules presented no influence on the spreadability factor (p>0.05).
3.1.5 Rheological behavior Regarding semisolids rheological properties, all samples evaluated showed nonNewtonian flow with pseudoplastic behavior. The rheograms fit better to Herschel-Bulkey model (Figure 2 and Table 2). Flow index (n) and consistency index (К) were also established (Table 3).
3.2 In vivo experiments 3.2.1 Assessment of ear edema and leukocyte infiltration-UVB irradiation-induced UVB irradiation-induced ear edema model was used to assess the influence of Q10 and VitE nanoencapsulation on inflammatory parameters induced by UVB irradiation. The UVB irradiation on the ear in mice induced a marked increase in ear thickness with an Emax of 108±5 11
µm when evaluated 24 h after UVB irradiation. Upon topical treatment with semisolids, UVB irradiation-induced ear edema and inflammatory cell infiltration were effectively reduced. All semisolids tested, except the vehicle, caused a decrease of ear edema of mice with inhibitions of 33±7%, 29±3%, 66±5%, 38±9% and 73±8% for FH, NBP1, NCP1, NBP3 and NCP3, respectively. Silver sulfadiazine, used as a positive control, was able to reduce UVB irradiationinduced ear edema in 70±5% (Figure 3A). MPO and NAGase play an important role in the innate immune system and they are considered biochemical markers of the inflammatory cells infiltration in the injured tissue. Aiming to evaluate the effect of the semisolids on inflammatory cells infiltration, the MPO and NAGase activities were assessed 24 h after UVB irradiation. The irradiation with a UVB source promoted an increase of MPO and NAGase activities in untreated and vehicle-treated irradiated groups when compared to the naïve group. All semisolids tested, except the vehicle, were able to decrease the MPO activity induced by UVB irradiation with a maximum inhibition of 44±6%, 27±4%, 42±5%, 28±8% and 64±5% for FH, NBP1, NCP1, NBP3 and NCP3, respectively. On the other hand, only semisolids containing 3% VitE reduced NAGase activity with a maximum inhibition of 28±7% for NBP3 and 25±4% for NCP3. The positive control, silver sulfadiazine, decreased MPO and NAGase activities in 60±3% and 40±5%, respectively (Figures 3B and 3C). Because MPO and NAGase enzyme activities indicated the inflammatory cells infiltration in untreated and vehicle-treated animals, we carried out histological analysis to confirm the inflammatory cell infiltration. UVB irradiation promoted intense inflammatory cell infiltration (92±6 inflammatory cell per field) when compared to the naïve (9±2 inflammatory cell per field) group. The topical treatment with semisolids containing Q10-loaded nanocapsules decreased the cell infiltration (21±4 and 26±1 inflammatory cell per field to NCP1 and NCP3, respectively) when compared to the untreated UVB irradiation group (Figures 4A and 4B).
3.2.2 Oxidative stress evaluation It has been reported that UV radiation generates ROS and their production is involved in UV-induced skin inflammatory and photodamage process. To investigate the possible antioxidant effect of semisolids containing Q10-loaded nanocapsules, we evaluated the treatments ability to reduce oxidative stress parameters through non-protein thiols levels and lipid peroxidation. UVB irradiation promoted an increase in all oxidative stress parameters 12
assessed. Semisolids containing nanocapsules increased non-protein thiols levels (100% to NCP1, NBP3 and NCP3) and decreased lipid peroxidation through lipid hydroperoxide content (100% to NCP1 and NCP3) and through TBARS assay (28±4% and 100% to NCP1 and NCP3, respectively) (Figures 5A, 5B and 5C).
4. Discussion Gellan gum has advantageous physicochemical properties and have multiple applications in different industries: food, personal care, pharmacy and others. In the pharmaceutical area, the highlighted advantages of its use can be: non-toxicity, biodegradability, rapid gelation in cations presence and mucoadhesive potential, characteristics that allow its use as a component of oral, ophthalmic, nasal and other formulations. Due to these characteristics, gellan gum was chosen for the development of the semisolid formulations24. Some analyses were made aiming to prove the suitable characteristics of these formulations. pH values were around 5.5, which is compatible with skin pH range40,41. This result is in agreement with the study of Terroso and co-workers42, which developed nanohydrogels of Carbopol® 940 containing Q10 and PCL. After redispersion in water, the nanoformulations showed mean diameter values on the nanometric range, confirming the existence of the nanostructures in the semisolids. Regarding the Q10 content, all values were close to 100%, indicating that no or minimal loss occurred during the incorporation of Q10loaded nanocapsules to the semisolids. In the semisolids formulation, spreadability and rheological analysis are usually carried out. The efficacy of a topical therapy depends on its easiness to spread on the substrate43. Spreadability values of the developed semisolids were between 3.63±0.35 and 4.63±0.86 mm2/g. In other words, when the spreadability value is high the easiness to spread the substance is enhanced, and less force is required for application43. With respect to rheological properties, this evaluation can help to predict the semisolids stability and to control the influence of the components on the flow behavior of the formulations. All samples presented pseudoplastic non-Newtonian flow behavior, which is interesting for topical application because in this type of flow behavior when an external force is applied the formulation become less viscous44. These semisolids presented suitable physicochemical and flow characteristics, required from a formulation designed for topical administration.
13
The exposure to UV radiation, particularly UVB radiation (280-315 nm) causes skin photodamage, resulting in sunburn, immunosuppression and cancer. Skin damage promotes an inflammatory process which is characterized by edema formation and inflammatory cell infiltration to the injured tissue45. Moreover, UVB irradiation-induced inflammation can be mediated in affected tissue through impairment of the oxidant/antioxidant balance, which generates an increase in cellular levels of ROS leading to the damage of cellular proteins and lipids and DNA oxidation1,46. Sunscreens are recommended to prevent UV irradiation-induced skin injury. However, the use of the synthetic molecules in sunscreens can generate an allergic skin reaction which limits its long-term use. This way, there is a growing search for formulations with natural compounds as new sources of protective agents23,47. Here, we demonstrated that semisolids, except the vehicle, reduced ear edema UVB irradiation-induced, suggesting that these formulations can inhibit some skin inflammatory responses caused by UVB exposure. The inflammatory events occur once the exposure to UVB leads to the development of skin edema, which can be considered a marker of the skin inflammation2,9. Semisolids containing nanocapsules NCP1 and NCP3 were more effective in reducing ear edema than semisolids with non-encapsulated Q10 and VitE (Figure 3A). Besides, Q10 nanoencapsulation (NCP1 and NCP3) enhanced the antiedematogenic effect when compared with semisolids containing nanocapsules without Q10 (NBP1 and NBP3), suggesting that not only the nanoencapsulation process or VitE, but also Q10 presence contributed for the NCP1 and NCP3 antiedematogenic property. Similar results were found by Rigo and co-workers9, which evaluated the effects of hydrogels with nanoencapsulated rice bran oil by the same methodology and observed that this treatment prevented the UVB irradiation-induced ear edema by 60±9%. These results can be associated to the nanocapsules ability to penetrate and be situated in different layers of the skin because of their small size47. Additionally, these nanostructures can be retained in hair follicles which act as reservoirs of nanoparticles after topical administration48. UVB irradiation promotes edema and cells inflammatory recruitment to the injured tissue49. Neutrophil and macrophage tissue infiltration after UVB irradiation can be evaluated by MPO and NAGase enzymes activities. All semisolids, except the vehicle, decreased neutrophil infiltration induced by UVB irradiation and NCP1, NBP1 and NBP3 were as effective as FH in reducing cell infiltration (Figure 3B). On the other hand, NCP3 presented better effect than FH, NBP1, NCP1 and NBP3 on neutrophil infiltration, what is probably due 14
to the presence of 3% VitE in NCP3. Besides, NCP3 presented similar effect to silver sulfadiazine. Otherwise, only semisolids containing nanocapsules with 3% VitE were able to reduce NAGase activity since this formulations present higher VitE content (Figure 3C). These results demonstrate greater anti-inflammatory efficacy of semisolids containing nanocapsules than semisolid containing non-encapsulated compounds. Furthermore, ROS production UVB irradiation-induced also contributes to the development of inflammatory process, such as neutrophil and macrophage recruitment50. Semisolids containing Q10-loaded nanocapsules were significantly more effective to reduce oxidative stress parameters than NBP1 and NBP3. Moreover, NCP3 was significantly more effective to reduce lipid peroxidation in terms of MDA formation than NCP1. These results indicate that the antioxidant capacity of semisolids containing nanocapsules is due to the Q10 presence, but also the VitE content exerts important contribution. In vivo results suggest the potential of the purposed semisolids containing nanocapsules with the association of Q10 and VitE to reduce the UVB irradiation-induced effects on skin (inflammation and oxidative damage), showing an interesting formulation with anti-aging and anti-inflammatory properties.
5. Conclusion In this study, we developed for the first time semisolid formulations based on nanoparticulate systems by the simple addition of gellan gum to nanocapsule suspensions. Furthermore, we have investigated antiedematogenic, anti-inflammatory and antioxidant activities of the semisolid formulations of Q10 and VitE associated to nanocarriers and we have demonstrated their promising effects in an animal model of UVB irradiation-induced inflammation.
6. Acknowledgements The authors thank C.B. da Silva for ZetaSizer access. Authors thank CAPES and CNPq/Brazil for fellowship. There is no conflict of interest to declare.
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[45] T. Bishop, D.W. Hewson, P.K. Yip, M.S. Fahey, D. Dawbarn, A.R. Young, S.B. McMahon Characterisation of ultraviolet-B induced inflammation as a model of hyperalgesia in the rat Pain, 13 (2007) 70–82 [46] Y.M. Fonseca, C.D. Catini, F.T.M.C. Vicentini, A. Nomizo, R.F. Gerlach, M.J.V. Fonseca Protective effect of Calendula officinalis extract against UVB-induced oxidative stress in skin: evaluation of reduced glutathione levels and matrix metalloproteinase secretion J. Ethnopharmacol., 127 (2010) 596–601 [47] Y.Yin, W. Li, Y.O. Son, L. Sun, J. Lu, D. Kim, X. Wang, H. Yao, L. Wang, P. Pratheeshkumar, A.J. Hitron, J. Luo, N. Gao, X. Shi, Z. Zhang Quercitrin protects skin from UVB-induced oxidative damage Toxicol. Appl. Pharmacol., 269 (2013) 89–99 [48] J. Lademann, H. Richter, A. Teichmann, N. Otberg, U. Blume-Peytavi, J. Luengo, B. Weib, U.F. Schaefer, C. Michael-Lehr, R. Wepf, W. Sterry Nanoparticles - an efficient carrier for drug delivery into the hair follicles Eur. J. Pharm. Biopharm., 66 (2007) 159-164 [49] M. Notara, N. Refaian, G. Braun, P. Steven, F. Bock, C. Cursiefen Short-term UVB-irradiation leads to putative limbal stem cell damage and niche cellmediated upregulation of macrophage recruiting cytokines Stem Cell Res., 15 (2015) 643–654 [50] H. Hattori, K.K. Subramanian, J. Sakai, Y. Jia, Y. Li, T.F. Porter, F. Loison, B. Sarraj. A. Kasorn, H. Jo, C. Blanchard, D. Zirkle, D. McDonald, S.Y. Pai, C.N. Serhan, H.R. Luo Small-molecule screen identifies reactive oxygen species as key regulators of neutrophil chemotaxis Proc. Natl. Acad. Sci. USA, 107 (2010) 3546–3551
20
Figure Captions Figure 1 - Spreadability (mm2) of semisolid formulations of gellan gum with nanoencapsulated systems.
21
Figure 2 - Rheological behavior of the semisolid formulations of gellan gum with coenzyme Q10 and VitE non-encapsulated (free) or nanoencapsulated systems (NBP1, NBP3, NCP1, NCP3).
22
Figure 3 - Anti-inflammatory effects of semisolid formulations on UVB irradiation-induced skin injury in mice. Ear edema (A) and Myeloperoxidase (B) and NAGase (C) activities in mice submitted to UVB irradiation (0.5 J/cm2). All formulations (15 mg/ear) were immediately applied after UVB irradiation. Ear edema and cell infiltration were measured 24 h after irradiation. Each bar represents the mean + SEM (n=6-7); ###P<0.001 when compared to the naïve group. *P<0.05, **P<0.01 and ***P<0.001 when compared to the untreated group. && P<0.01 shows significant difference between semisolids containing Q10-loaded nanocapsules (NCP1 and NCP3) with semisolids containing nanocapsules without Q10 (NBP1 and NBP3). One-way ANOVA followed by post hoc Newman-Keuls test.
23
Figure 4 - Anti-inflammatory (cell infiltration) effects of semisolid formulations on UVB irradiation-induced skin injury in mice. Ear representative light microphotographic (arrows indicate polymorphonuclear cells) and quantification of polymorphonuclear cells per field (B) of the ear tissue of mice 24 h after UVB irradiation or UVB-irradiation plus semisolids. Each bar represents the mean + SEM (n=6-7); ###P<0.001 when compared to the naïve group. ***P<0.001 when compared to the untreated group. One-way ANOVA followed by post hoc Newman-Keuls test.
24
Figure 5 - Effect of semisolid formulations on stress oxidative parameters induced by UVB irradiation in mice. Non-protein thiols (NPSH) levels (A) and lipid peroxidation (B) and (C) in mice submitted to UVB irradiation (0.5 J/cm2). The oxidative stress parameters were evaluated 24 h after UVB irradiation. Each bar represents the mean + SEM (n=6-7); #P<0.05, ##P<0.01 and ###P<0.001 when compared to the naïve group. *P<0.05 and ***P<0.001 when compared to the untreated group. &P<0.05, &&P<0.01 and &&&P<0.001 show significant difference between semisolids containing Q10-loaded nanocapsules (NCP1 and NCP3) with semisolids containing nanocapsules without Q10 (NBP1 and NBP3). One-way ANOVA followed by post hoc Newman-Keuls test.
25
Table 1 - pH values, drug content, mean diameter and spreadability factors of semisolid formulations.
Formulation
pH
Coenzyme Q10 Mean content (mg/g) (nm)
Vehicle Free NCP1 NCP3 NBP1 NBP3
5.2 ± 0.06 5.3 ± 0.08 5.7 ± 0.10 5.4 ± 0.08 5.5 ± 0.13 5.5 ± 0.13
0.95 ± 0.07 1.06 ± 0.03 1.05 ± 0.06 -
283 ± 16 331 ± 15 346 ± 23 219 ± 20 145 ± 13
diameter Spreadability factor (mm2 g-1) 4.61 ± 0.83 4.63 ± 0.86 3.79 ± 0.35 3.63 ± 0.67
26
Table 2 - Regression coefficient (r) for various flow models in shear rate–shear stress curve obtained for semisolids.
Formulation NBP1 NBP3 NCP1 NCP3 Free Vehicle
Bingham 0.920 ± 0.034 0.899 ± 0.009 0.901 ± 0.019 0.885 ± 0.015 0.909 ± 0.007 0.895 ± 0.022
Casson 0.926 ± 0.029 0.940 ± 0.012 0.947 ± 0.013 0.935 ± 0.014 0.951 ± 0.007 0.939 ± 0.015
Ostwald 0.955 ± 0.052 0.984 ± 0.009 0.989 ± 0.004 0.983 ± 0.004 0.989 ± 0.003 0.985 ± 0.005
Herschel-Bulkley 0.990 ± 0.001 0.989 ± 0.006 0.994 ± 0.005 0.990 ± 0.001 0.993 ± 0.001 0.994 ± 0.003
27
Table 3 - Flow index (n) and consistency index (К) of semisolid formulations, according to Herschel-Bulkley flow model.
Formulation NBP1 NBP3 NCP1 NCP3 Free Vehicle
n 0.106 ± 0.031 0.114 ± 0.006 0.122 ± 0.010 0.115 ± 0.003 0.133 ± 0.009 0.137 ± 0.009
К 4370 ± 745 4481 ± 345 4124 ± 401 4664 ± 499 4371 ± 391 3856 ± 273
28
S0927-7765(16)30789-5 http://dx.doi.org/doi:10.1016/j.colsurfb.2016.11.013 COLSUB 8247
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
17-8-2016 3-11-2016 7-11-2016
Please cite this article as: Nath´ali S.Pegoraro, Allanna V.Barbieri, Camila Camponogara, Evelyne S.Brum, Marila C.L.Marchiori, Sara M.Oliveira, Let´ıcia Cruz, Nanoencapsulation of coenzyme Q10 and vitamin E acetate protects against UVB radiation-induced skin injury in mice, Colloids and Surfaces B: Biointerfaces http://dx.doi.org/10.1016/j.colsurfb.2016.11.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Nanoencapsulation of coenzyme Q10 and vitamin E acetate protects against UVB radiation-induced skin injury in mice Natháli S. Pegoraroa, Allanna V. Barbierib, Camila Camponogarac, Evelyne S. Brumc, Marila C. L. Marchioria, Sara M. Oliveirac, Letícia Cruza,b a
Programa de Pós-Graduação em Ciências Farmacêuticas, Centro de Ciências da Saúde,
Universidade Federal de Santa Maria, Santa Maria 97105-900, Brazil b
Departamento de Farmácia Industrial, Universidade Federal de Santa Maria, Santa Maria
97105-900, Brazil c
Programa de Pós-Graduação em Ciências Biológicas: Bioquímica Toxicológica, Centro de
Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria 97105-900, Brazil
*Corresponding author: Letícia Cruz, Departamento de Farmácia Industrial, Universidade Federal de Santa Maria, Santa Maria, 97105-900, Brazil. Phone: +55 55 32209373. Fax: +55 55 32208149. E-mail: [email protected]
Total number of pages:21 Number of tables:3 Number of figures:5 Total number of words including references: 5795
1
Graphical abstract
Highlights
Semisolids were obtained by thickening nanocapsule suspensions with gellan gum; The formulations presented characteristics suitable for cutaneous administration; NCP3 decreased MPO and NAGase activity in the UVB radiation-induced ear edema model; NC increased non-protein thiol levels and decreased lipid peroxidation induced by UVB radiation; NC with Q10 and VitE reduced the UVB irradiation-induced effects on skin.
Abstract
This study aimed to investigate the feasibility of producing semisolid formulations based on nanocapsule suspensions containing the association of the coenzyme Q10 and vitamin E acetate by adding gellan gum (2%) to the suspensions. Furthermore, we studied their 2
application as an alternative for the treatment of inflammation induced by ultraviolet B (UVB) radiation. For this, an animal model of injury induced by UVB-radiation was employed. All semisolids presented pH close to 5.5, drug content above 95% and mean diameter on the nanometric range, after redispersion in water. Besides, the semisolids presented non-Newtonian flow with pseudoplastic behavior and suitable spreadability factor values. The results also showed that the semisolid containing coenzyme Q10-loaded nanocapsules with higher vitamin E acetate concentration reduced in 73±8% the UVB radiation-induced ear edema. Moreover, all formulations tested were able to reduce inflammation parameters evaluated through MPO activity and histological procedure on injured tissue and the semisolids containing the nanoencapsulated coenzyme Q10 reduced oxidative parameters assessment through the nonprotein thiols levels and lipid peroxidation. This way, the semisolids based on nanocapsules may be considered a promising approach for the treatment and prevention of skin inflammation diseases.
Keywords Coenzyme Q10, vitamin E, nanocapsules, skin, ear edema, UV radiation.
1. Introduction Skin is often exposed to several harmful sources, among them, the ultraviolet (UV) radiations. UV radiation consists of wavelengths shorter than visible light and is classified as UVA, UVB and UVC. Among these types of light, the main responsible for skin cytotoxic events is UVB radiation, which induces several pathologic changes in skin such as erythema, edema, sunburn, immune suppression and skin cancer, and it leads to changes and often to the destruction of skin structures what can cause changes in their normal function1-3. One of the major alterations caused by UV radiation leading to deleterious outcomes on the skin is the production of highly unstable products, called reactive oxygen species (ROS), that contribute to oxidizing and cause cellular damage4,5. Sunscreens are recommended for the protection against UV light-induced skin damage due to their ability to absorb, reflect or scatter UV light. However, they are limited to repair UV-mediated cutaneous damage. In this sense, there is a growing search for new alternatives, especially for formulations containing
3
antioxidant molecules capable of attenuating the effects produced by ROS, through benefic effects such as anti-inflammatory, antioxidant and DNA repair6,7,8. The combination of antioxidants may be an advantageous strategy to improve their biological effects. Among the substances with high antioxidant activity, the coenzyme Q10 (Q10) and vitamin E (VitE) play an important role in many biological systems due to their ability to decrease oxidative stress generated by ROS. Q10, also known as ubiquinone or ubidecarenone, is an essential lipophilic antioxidant for the human body because of its key role on ATP synthesis, being fundamental on mitochondrial electron transport chain9. This compound can be obtained from sources as red meat, fish and poultry or from supplementation10. Some studies showed the Q10 benefits on anti-aging therapy, skin hydration and photoprotection11-13. In its turn, VitE is an antioxidant composed by tocopherols and tocotrienols. This vitamin can be obtained from diet by the ingestion of vegetable oils, nuts and almonds14. Its antioxidant function lies on its capability to stabilize reactive species15. It was already reported the association of VitE with UV filters, based on its antioxidant properties that play an essential role on skin repair16,17. In the last years, nanocapsule suspensions have emerged as promising alternatives to deliver substances through the skin because of their ability to control drug release and to improve drug stability18. Nanocapsules are vesicular systems composed by a central core, generally an oily core, surrounded by a polymeric wall19,20. Besides, nanocapsules are obtained as liquid suspensions, which may complicate its cutaneous application. The incorporation of nanoparticle suspensions in semisolid vehicles has been reported using Carbopol® Ultrez 10 NF21, Carbopol® 94022, Pemulen TR-122 and xantham gum22. Regarding the cutaneous application, the repairing effect of a nanoencapsulated substance was recently studied on ear edema induced by UVB irradiation in mice23. In that work, it was demonstrated that a hydrogel containing nanocapsules of rice bran oil could reduce ear edema by 60±9%. Besides that, its effects on oxidative stress and inflammation was also evidenced by the reduction of protein carbonylation and NF-κB nuclear translocation biomarkers levels, demonstrating its beneficial effects on skin damage caused by UVB exposure. With the purpose of proposing a new formulation with improved skin repair properties, in this work it was demonstrated a novel association between the Q10 and VitE in nanocapsules using gellan gum, a microbial polysaccharide with gelling properties24, as a new thickening agent for nanosuspensions. 4
2. Materials and methods 2.1 Materials Coenzyme Q10 and vitamin E acetate were obtained from Mapric (São Paulo, Brazil). Poly-ε-caprolactone (PCL) (MW: 70,000-90,000), polysorbate 80 (Tween® 80) and sorbitan monooleate (Span® 80) were provided from Delaware (Porto Alegre, Brazil). Gellan gum was kindly donated by CP Kelco (Georgia, USA). Acetone was purchased from Proquimios (Rio de Janeiro, by Brazil). Anhydrous ethanol (HPLC grade) was obtained from Tedia (São Paulo, Brazil). Dimethylsulphoxide (DMSO) was purchased from Sigma Chemical Co. (St. Louis, USA). All other reagents and solvents were of analytical grade and used as received.
2.2 Methods 2.2.1 Nanocapsule suspensions preparation and semisolid formulations Nanocapsule suspensions were prepared following the interfacial deposition of preformed polymer method25. An organic phase constituted by PCL (0.100 g), Span® 80 (0.077 g), VitE (0.1 or 0.3 g - 1 or 3%, respectively), Q10 (0.010 g) and acetone (27 mL) was kept under moderate magnetic stirring at 40°C during an hour. After the complete dissolution of the components, this phase was injected into an aqueous phase containing Tween® 80 (0.077 g) and the magnetic stirring was sustained for 10 min. After this, the organic solvent and part of the water were eliminated by evaporation under reduced pressure to obtain 10 mL of final volume and 1.0 mg/mL of Q10. Formulations without Q10 were prepared as well. The formulations were abbreviated as following: NCP1 (Q10-loaded nanocapsules containing 1% VitE), NCP3 (Q10-loaded nanocapsules containing 3% VitE), NBP1 (nanocapsules without Q10 containing 1% VitE) and NBP3 (nanocapsules without Q10 containing 3% VitE). Semisolid formulations were prepared using mortar and pestle by adding 0.2 g gellan gum directly to the nanocapsule suspensions (10 mL) to obtain around 10 g of semisolids. For the semisolid formulations containing free compounds, both Q10 (1 mg/g) and VitE (3%) were solubilized in DMSO (1 mL) and added to a dispersion of gellan gum (2%) in water (9 mL). The vehicle was prepared following the same methodology, but only dispersing gellan gum in water (10 mL). All semisolids containing Q10 and/or VitE were prepared under light protection.
2.2.2 Semisolid formulations characterization 2.2.2.1 pH determination 5
pH values were evaluated by immersing a calibrated potentiometer (Model pH 21, Hanna Instruments, Brazil) in a semisolid aqueous dispersion (10%, w/v).
2.2.2.2 Mean particle size evaluation Nanocapsules mean particle size in the semisolids was measured using ZetaSizer Nano Series Malvern Instruments (UK), by photon correlation spectroscopy method, by dispersing an aliquot of the samples in ultrapure water (1:500).
2.2.2.3 Q10 content The analysis of the Q10 total content in the semisolids was performed by HPLC employing methodology described by Mattiazzi and co-workers26, with some modifications. It was a simple RP-HPLC-UV method employing the following chromatographic conditions: C18 column at room temperature, and mobile phase constituted of a mixture of anhydrous ethanol and methanol (75:25 v/v), at a flow rate of 1.0 mL/min and detection UV at 275 nm. The analytical method was specific, linear, accurate, and precise over the concentration range of 5.0 - 25.0 μg/mL. For this evaluation, an aliquot of the semisolid was solubilized in ethanol and submitted to sonication for 10 min. Then, the sample was centrifuged at 3,000 rpm (664 xg) during 10 min. Subsequently, samples were filtered through a 0.45 μm membrane and injected into HPLC system.
2.2.2.4 Spreadability determination The semisolid formulations spreadability was evaluated through the parallel plate method27,28. An aliquot of the sample was put in a central hole of a mold glass plate, that was on the scanner surface (HP Officejet, model 4500 Desktop). Then, the mold glass was carefully removed and upon the sample was placed glass plates with known weights. Each plate was placed observing an interval of 1 min to the subsequent plate, a total of 10 plates were used. One image was captured at every 1 min of interval, employing the desktop scanner and the software Image J (Version 1.49q, National Institutes of Health, USA). Both registers were used to calculate the spreading area of the captured images. The spreadability profiles were obtained by plotting the spreading area versus the cumulative weight of the plates. Finally, the spreadability factor was calculated for all formulations. This factor represents the formulation ability to expand on a smooth horizontal surface when a gram of weight is added to it under test conditions. This equation (Eq. (1)) was employed to calculate the spreadability factor: 6
Sf =
A W
in which Sf is the spreadability factor (mm2 g-1), A is the maximum spread area (mm2) after the addition of the total number of plates, and W is the total weight added (g).
2.2.2.5 Rheological behavior Rheological analyses were conducted at 25±1ºC employing viscometer (RVDV-IPRIME model, Brookfield, USA) with a RV06 spindle. For this, about 50 g of the formulations was used and submitted to a range of speed between 5-100 rpm. The data obtained was analyzed to the best fit using Bingham, Casson, Ostwald and Herschel-Bulkley models (Eq. 2 - 5)29, employing graphical model to determine rheological behavior.
(2)
𝜏 = 𝜏0 + 𝜂 𝛾
(3)
𝜏 0.5 = 𝜏00.5 + 𝜂0.5 𝛾 0.5
(4)
𝜏 = К 𝛾𝑛
(5)
𝜏 = 𝜏0 + К 𝛾 𝑛 ,
where 𝜏0 is the yield stress, 𝜂 is the viscosity, 𝑛 is the index of flow, К is the index of consistency, 𝜏 is the shear stress and 𝛾 is the shear rate29.
2.2.3 Animals Male Swiss mice (25-30 g; n total = 114) were used in all experiments. Animals were kept under controlled temperature (22 ± 2°C) on a 12 h light-dark cycle and with standard laboratory chow and water ad libitum. The animals were habituated to the experimental room at least 1 h before the experiments. All experiments were carried out between 8:00 a.m. and 5:00 p.m. The experiments were performed in accordance to the current ethical guidelines for care of laboratory animals30 and all procedures were approved by our Institutional Ethics Committee (Process number 4921060715/2015). Animals were randomly assigned to different treatment groups and all the experiments were blindly performed. The number of animals and intensity of stimuli was the minimum necessary to demonstrate the consistent effects of treatments. 7
2.2.4 UVB irradiation model The UVB source of irradiation consisted of a Philips TL40W/12 RS lamp (MedicalEindhoven, Holland) assembled 20 cm above the table where mice were placed, and which emitted a continuous light spectrum between 270 and 400 nm with a peak emission at 313 nm. UVB output (80% of the total UV irradiation) was measured using a model IL-1700 Research Radiometer (International Light, USA; calibrated by IL service staff) with a radiometer sensor for UV (SED005) and UVB (SED240). The UVB irradiation rate was 0.27 mW/cm2 and the dose used was 0.5 J/cm2. Mice were firstly anesthetized (90 mg/kg of ketamine + 3 mg/kg of xylazine) with a single intraperitoneal injection and then exposed to UVB irradiation. Only the right ear of each animal was exposed to UVB irradiation31,32.
2.2.5 Formulation administration and experimental design Swiss mice were randomly divided in nine groups with six animals each and classified as it follows: naïve (non-irradiated); untreated irradiated; treated with vehicle, treated with semisolid containing non-encapsulated Q10 and VitE (FH); treated with semisolid containing Q10-loaded nanocapsules with 1% VitE (NCP1); treated with semisolid containing nanocapsules with 1% VitE without Q10 (NBP1); treated with semisolid containing Q10loaded nanocapsules with 3% VitE (NCP3); treated with semisolid containing nanocapsules with 3% VitE without Q10 (NBP3). As positive control to treat light burning, silver sulfadiazine (3%) was employed. Mice were topically treated on the ear surface with different semisolid formulations (15 mg/ear) immediately receiving UVB irradiation33.
2.2.6 Ear edema measurement The skin photodamage was induced by UVB irradiation and the inflammatory process was assessed through ear edema formation. The edema was measured by the increase in ear thickness after the inflammatory stimuli. The ear thickness was evaluated before and 24 h after UVB irradiation using a digital micrometer (Digimess) in animals anesthetized with isofluorane34. The micrometer was applied near the tip of the ear just distal to the cartilaginous ridges. Thickness was expressed in μm. To minimize the variation, a single investigator performed the measurements throughout each experiment.
2.2.7 Leukocyte infiltration assessment 2.2.7.1. Myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAGase) activities 8
UVB irradiation-induced leukocytes migration in the skin was assessed using the MPO and NAGase activities assay as previously described35,23. MPO and NAGase enzymes activities are used as biochemical markers of the polymorphonuclear leukocyte influx (mostly neutrophil and macrophages, respectively) to the injured tissue. Twenty-four hours after UVB irradiation, mice were euthanized and the ears were removed to determine MPO and NAGase activities. Tissue samples were homogenized in acetate buffer (80 mM, pH 5.4) containing 0.5% hexadecyltrimethylammonium bromide, centrifuged and the supernatant was collected for assay. For evaluation of MPO enzyme activity, the sample was incubated with acetate buffer and 3,3’,5,5’-tetramethylbenzidine solution (18.4 mM) at 37ºC. The reaction was stopped on ice by adding acetic acid. The formed color was analyzed by a spectrophotometer at 630 nm. In sequence, to determine NAGase enzyme activity, the sample was mixed with sodium citrate buffer (50 mM, pH 4.5) and p-nitrofenil-2-acetamide-β-D-glucopyranoside (NAG 2.25 nM) and incubated at 37ºC. After incubation time, the reaction was stopped on ice by adding glycine buffer (0.2 μM, pH 10.4). The formed color was analyzed by a spectrophotometer at 405 nm. Both reactions were read on a Fisher BiotchMicrokinetics BT 2000 microplate reader. The results were expressed as the sample optical densities (OD)/mL.
2.2.7.2 Histology Separate groups of mice were used to evaluate histological changes in ear tissue 24 h after UVB-irradiation or UVB-irradiation plus semisolid formulations. Mice were euthanized and the right ear was removed and fixed in alfac solution (16:2:1 mixture of ethanol 80%, formaldehyde 40% and acetic acid). Each sample embedded in paraffin was sectioned at 5 μm and stained with hematoxylin-eosin. A representative area was selected for qualitative light microscopic analysis of the inflammatory cellular response with 20x and 40x objectives35,36. To minimize a source of bias, the investigators did not know the group that they were analyzing.
2.2.8 Oxidative stress evaluation In order to verify the effect of different semisolid formulations on the oxidative damage induced by UVB irradiation, we determined non-protein thiol levels and lipid peroxidation. After 24 h of UVB irradiation, mice were euthanized and ears were removed and homogenized with Tris/HCl buffer (50 mM, pH 7.4) and centrifuged. The supernatants were used to continue the analysis. To determine non-protein thiol (NPSH) levels, the protein supernatant was 9
precipitated with TCA 10% and centrifuged. The deproteinized supernatant was incubated with Tris/HCl (200 mM, pH 8.9) and 5-5’dithiobis- (Z-nitro-benzoic acid) (DTNB – 2.5 mM) at room temperature37. The color of the resulting solution from the reaction was measured at 405 nm with a Fisher BiotchMicrokinetics BT 2000 microplate reader. Lipid peroxidation was determined through of the measurement of lipid hydroperoxide and thiobarbituric acid reactive substances (TBARS) assay. The concentration of lipid hydroperoxide was estimated by the FOX assay. For this purpose, the tissue homogenate supernatant was incubated with FOX reagent (sulfuric acid 250 mM, xylenol orange 0.8 μM, ferrous sulfate and ammonium sulfate 3 μM and butyl hydroxytoluene 0.04 mM) at 37ºC. After, the absorbance of the solution was read at 560 nm38. Lipid hydroperoxide content (μM) was calculated using the 4.6 x 104 M/cm molar extinction coefficient. The extent of lipid peroxidation in terms of malondialdehyde (MDA) formation was measured by TBARS assay. The supernatant was incubated with TBA 0.8%, acetic acid buffer and sodium dodecyl sulfate (SDS) 8.1% at 90ºC. The solution color resulting from the reaction was measured at 535 nm with a Fisher BiotchMicrokinetics BT 2000 microplate reader. Both the lipid peroxidation by TBARS and non-protein thiol contents were corrected for mL of sample39.
2.2.9 Statistical analysis The results are presented as mean + standard error of the mean (SEM), which are reported as geometric means plus their respective 95% confidence limits. The maximum inhibitory effect (Emax) was calculated based on the response of the control groups. The statistical significance between the groups was assessed by one-way or two-way analysis of variance (ANOVA) followed by post hoc Newman-Keuls test, Bonferroni's test or Tukey's test, when appropriate. All tests were carried out using GraphPad 5.0 Software (San Diego, CA, USA).
3. Results 3.1 Semisolid formulations characterization 3.1.1 pH determination The characteristics of semisolid formulations are presented in Table 1. pH values were in the acid range, close to 5.5 for all formulations, which is compatible with skin pH and suitable
10
for cutaneous administration40,41. Semisolids containing NCP1, NBP1 and NBP3 nanocapsule suspensions presented higher pH values (p<0.05) than the vehicle.
3.1.2 Mean particle size evaluation The measures of the semisolids mean diameter after redispersion in water are shown in Table 1. Nanostructured formulations and vehicle presented mean diameter on the nanometric range. For the nanostructured ones, values of mean diameter were significantly higher for the formulations containing Q10 (NCP1 and NCP3) (p<0.05). All formulations showed significant difference when compared to the vehicle mean diameter (p<0.05).
3.1.3 Q10 content Concerning the Q10 content, the semisolids containing NCP1 and NCP3 nanocapsules and free compounds revealed Q10 content close to 100%. The presence of nanostructures or VitE concentration in the samples did not influence this parameter (p>0.05).
3.1.4 Spreadability determination In relation to spreadability evaluation (Figure 1), the values of spreadability factors were 4.61±0.83 and 4.63±0.86 mm2/g for NCP1 and NCP3 semisolids, respectively (Table 1). For NBP1 and NBP3 they were 3.79±0.35 and 3.63±0.67 mm2/g, respectively. In this case, VitE concentration or Q10 presence in nanocapsules presented no influence on the spreadability factor (p>0.05).
3.1.5 Rheological behavior Regarding semisolids rheological properties, all samples evaluated showed nonNewtonian flow with pseudoplastic behavior. The rheograms fit better to Herschel-Bulkey model (Figure 2 and Table 2). Flow index (n) and consistency index (К) were also established (Table 3).
3.2 In vivo experiments 3.2.1 Assessment of ear edema and leukocyte infiltration-UVB irradiation-induced UVB irradiation-induced ear edema model was used to assess the influence of Q10 and VitE nanoencapsulation on inflammatory parameters induced by UVB irradiation. The UVB irradiation on the ear in mice induced a marked increase in ear thickness with an Emax of 108±5 11
µm when evaluated 24 h after UVB irradiation. Upon topical treatment with semisolids, UVB irradiation-induced ear edema and inflammatory cell infiltration were effectively reduced. All semisolids tested, except the vehicle, caused a decrease of ear edema of mice with inhibitions of 33±7%, 29±3%, 66±5%, 38±9% and 73±8% for FH, NBP1, NCP1, NBP3 and NCP3, respectively. Silver sulfadiazine, used as a positive control, was able to reduce UVB irradiationinduced ear edema in 70±5% (Figure 3A). MPO and NAGase play an important role in the innate immune system and they are considered biochemical markers of the inflammatory cells infiltration in the injured tissue. Aiming to evaluate the effect of the semisolids on inflammatory cells infiltration, the MPO and NAGase activities were assessed 24 h after UVB irradiation. The irradiation with a UVB source promoted an increase of MPO and NAGase activities in untreated and vehicle-treated irradiated groups when compared to the naïve group. All semisolids tested, except the vehicle, were able to decrease the MPO activity induced by UVB irradiation with a maximum inhibition of 44±6%, 27±4%, 42±5%, 28±8% and 64±5% for FH, NBP1, NCP1, NBP3 and NCP3, respectively. On the other hand, only semisolids containing 3% VitE reduced NAGase activity with a maximum inhibition of 28±7% for NBP3 and 25±4% for NCP3. The positive control, silver sulfadiazine, decreased MPO and NAGase activities in 60±3% and 40±5%, respectively (Figures 3B and 3C). Because MPO and NAGase enzyme activities indicated the inflammatory cells infiltration in untreated and vehicle-treated animals, we carried out histological analysis to confirm the inflammatory cell infiltration. UVB irradiation promoted intense inflammatory cell infiltration (92±6 inflammatory cell per field) when compared to the naïve (9±2 inflammatory cell per field) group. The topical treatment with semisolids containing Q10-loaded nanocapsules decreased the cell infiltration (21±4 and 26±1 inflammatory cell per field to NCP1 and NCP3, respectively) when compared to the untreated UVB irradiation group (Figures 4A and 4B).
3.2.2 Oxidative stress evaluation It has been reported that UV radiation generates ROS and their production is involved in UV-induced skin inflammatory and photodamage process. To investigate the possible antioxidant effect of semisolids containing Q10-loaded nanocapsules, we evaluated the treatments ability to reduce oxidative stress parameters through non-protein thiols levels and lipid peroxidation. UVB irradiation promoted an increase in all oxidative stress parameters 12
assessed. Semisolids containing nanocapsules increased non-protein thiols levels (100% to NCP1, NBP3 and NCP3) and decreased lipid peroxidation through lipid hydroperoxide content (100% to NCP1 and NCP3) and through TBARS assay (28±4% and 100% to NCP1 and NCP3, respectively) (Figures 5A, 5B and 5C).
4. Discussion Gellan gum has advantageous physicochemical properties and have multiple applications in different industries: food, personal care, pharmacy and others. In the pharmaceutical area, the highlighted advantages of its use can be: non-toxicity, biodegradability, rapid gelation in cations presence and mucoadhesive potential, characteristics that allow its use as a component of oral, ophthalmic, nasal and other formulations. Due to these characteristics, gellan gum was chosen for the development of the semisolid formulations24. Some analyses were made aiming to prove the suitable characteristics of these formulations. pH values were around 5.5, which is compatible with skin pH range40,41. This result is in agreement with the study of Terroso and co-workers42, which developed nanohydrogels of Carbopol® 940 containing Q10 and PCL. After redispersion in water, the nanoformulations showed mean diameter values on the nanometric range, confirming the existence of the nanostructures in the semisolids. Regarding the Q10 content, all values were close to 100%, indicating that no or minimal loss occurred during the incorporation of Q10loaded nanocapsules to the semisolids. In the semisolids formulation, spreadability and rheological analysis are usually carried out. The efficacy of a topical therapy depends on its easiness to spread on the substrate43. Spreadability values of the developed semisolids were between 3.63±0.35 and 4.63±0.86 mm2/g. In other words, when the spreadability value is high the easiness to spread the substance is enhanced, and less force is required for application43. With respect to rheological properties, this evaluation can help to predict the semisolids stability and to control the influence of the components on the flow behavior of the formulations. All samples presented pseudoplastic non-Newtonian flow behavior, which is interesting for topical application because in this type of flow behavior when an external force is applied the formulation become less viscous44. These semisolids presented suitable physicochemical and flow characteristics, required from a formulation designed for topical administration.
13
The exposure to UV radiation, particularly UVB radiation (280-315 nm) causes skin photodamage, resulting in sunburn, immunosuppression and cancer. Skin damage promotes an inflammatory process which is characterized by edema formation and inflammatory cell infiltration to the injured tissue45. Moreover, UVB irradiation-induced inflammation can be mediated in affected tissue through impairment of the oxidant/antioxidant balance, which generates an increase in cellular levels of ROS leading to the damage of cellular proteins and lipids and DNA oxidation1,46. Sunscreens are recommended to prevent UV irradiation-induced skin injury. However, the use of the synthetic molecules in sunscreens can generate an allergic skin reaction which limits its long-term use. This way, there is a growing search for formulations with natural compounds as new sources of protective agents23,47. Here, we demonstrated that semisolids, except the vehicle, reduced ear edema UVB irradiation-induced, suggesting that these formulations can inhibit some skin inflammatory responses caused by UVB exposure. The inflammatory events occur once the exposure to UVB leads to the development of skin edema, which can be considered a marker of the skin inflammation2,9. Semisolids containing nanocapsules NCP1 and NCP3 were more effective in reducing ear edema than semisolids with non-encapsulated Q10 and VitE (Figure 3A). Besides, Q10 nanoencapsulation (NCP1 and NCP3) enhanced the antiedematogenic effect when compared with semisolids containing nanocapsules without Q10 (NBP1 and NBP3), suggesting that not only the nanoencapsulation process or VitE, but also Q10 presence contributed for the NCP1 and NCP3 antiedematogenic property. Similar results were found by Rigo and co-workers9, which evaluated the effects of hydrogels with nanoencapsulated rice bran oil by the same methodology and observed that this treatment prevented the UVB irradiation-induced ear edema by 60±9%. These results can be associated to the nanocapsules ability to penetrate and be situated in different layers of the skin because of their small size47. Additionally, these nanostructures can be retained in hair follicles which act as reservoirs of nanoparticles after topical administration48. UVB irradiation promotes edema and cells inflammatory recruitment to the injured tissue49. Neutrophil and macrophage tissue infiltration after UVB irradiation can be evaluated by MPO and NAGase enzymes activities. All semisolids, except the vehicle, decreased neutrophil infiltration induced by UVB irradiation and NCP1, NBP1 and NBP3 were as effective as FH in reducing cell infiltration (Figure 3B). On the other hand, NCP3 presented better effect than FH, NBP1, NCP1 and NBP3 on neutrophil infiltration, what is probably due 14
to the presence of 3% VitE in NCP3. Besides, NCP3 presented similar effect to silver sulfadiazine. Otherwise, only semisolids containing nanocapsules with 3% VitE were able to reduce NAGase activity since this formulations present higher VitE content (Figure 3C). These results demonstrate greater anti-inflammatory efficacy of semisolids containing nanocapsules than semisolid containing non-encapsulated compounds. Furthermore, ROS production UVB irradiation-induced also contributes to the development of inflammatory process, such as neutrophil and macrophage recruitment50. Semisolids containing Q10-loaded nanocapsules were significantly more effective to reduce oxidative stress parameters than NBP1 and NBP3. Moreover, NCP3 was significantly more effective to reduce lipid peroxidation in terms of MDA formation than NCP1. These results indicate that the antioxidant capacity of semisolids containing nanocapsules is due to the Q10 presence, but also the VitE content exerts important contribution. In vivo results suggest the potential of the purposed semisolids containing nanocapsules with the association of Q10 and VitE to reduce the UVB irradiation-induced effects on skin (inflammation and oxidative damage), showing an interesting formulation with anti-aging and anti-inflammatory properties.
5. Conclusion In this study, we developed for the first time semisolid formulations based on nanoparticulate systems by the simple addition of gellan gum to nanocapsule suspensions. Furthermore, we have investigated antiedematogenic, anti-inflammatory and antioxidant activities of the semisolid formulations of Q10 and VitE associated to nanocarriers and we have demonstrated their promising effects in an animal model of UVB irradiation-induced inflammation.
6. Acknowledgements The authors thank C.B. da Silva for ZetaSizer access. Authors thank CAPES and CNPq/Brazil for fellowship. There is no conflict of interest to declare.
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Figure Captions Figure 1 - Spreadability (mm2) of semisolid formulations of gellan gum with nanoencapsulated systems.
21
Figure 2 - Rheological behavior of the semisolid formulations of gellan gum with coenzyme Q10 and VitE non-encapsulated (free) or nanoencapsulated systems (NBP1, NBP3, NCP1, NCP3).
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Figure 3 - Anti-inflammatory effects of semisolid formulations on UVB irradiation-induced skin injury in mice. Ear edema (A) and Myeloperoxidase (B) and NAGase (C) activities in mice submitted to UVB irradiation (0.5 J/cm2). All formulations (15 mg/ear) were immediately applied after UVB irradiation. Ear edema and cell infiltration were measured 24 h after irradiation. Each bar represents the mean + SEM (n=6-7); ###P<0.001 when compared to the naïve group. *P<0.05, **P<0.01 and ***P<0.001 when compared to the untreated group. && P<0.01 shows significant difference between semisolids containing Q10-loaded nanocapsules (NCP1 and NCP3) with semisolids containing nanocapsules without Q10 (NBP1 and NBP3). One-way ANOVA followed by post hoc Newman-Keuls test.
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Figure 4 - Anti-inflammatory (cell infiltration) effects of semisolid formulations on UVB irradiation-induced skin injury in mice. Ear representative light microphotographic (arrows indicate polymorphonuclear cells) and quantification of polymorphonuclear cells per field (B) of the ear tissue of mice 24 h after UVB irradiation or UVB-irradiation plus semisolids. Each bar represents the mean + SEM (n=6-7); ###P<0.001 when compared to the naïve group. ***P<0.001 when compared to the untreated group. One-way ANOVA followed by post hoc Newman-Keuls test.
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Figure 5 - Effect of semisolid formulations on stress oxidative parameters induced by UVB irradiation in mice. Non-protein thiols (NPSH) levels (A) and lipid peroxidation (B) and (C) in mice submitted to UVB irradiation (0.5 J/cm2). The oxidative stress parameters were evaluated 24 h after UVB irradiation. Each bar represents the mean + SEM (n=6-7); #P<0.05, ##P<0.01 and ###P<0.001 when compared to the naïve group. *P<0.05 and ***P<0.001 when compared to the untreated group. &P<0.05, &&P<0.01 and &&&P<0.001 show significant difference between semisolids containing Q10-loaded nanocapsules (NCP1 and NCP3) with semisolids containing nanocapsules without Q10 (NBP1 and NBP3). One-way ANOVA followed by post hoc Newman-Keuls test.
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Table 1 - pH values, drug content, mean diameter and spreadability factors of semisolid formulations.
Formulation
pH
Coenzyme Q10 Mean content (mg/g) (nm)
Vehicle Free NCP1 NCP3 NBP1 NBP3
5.2 ± 0.06 5.3 ± 0.08 5.7 ± 0.10 5.4 ± 0.08 5.5 ± 0.13 5.5 ± 0.13
0.95 ± 0.07 1.06 ± 0.03 1.05 ± 0.06 -
283 ± 16 331 ± 15 346 ± 23 219 ± 20 145 ± 13
diameter Spreadability factor (mm2 g-1) 4.61 ± 0.83 4.63 ± 0.86 3.79 ± 0.35 3.63 ± 0.67
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Table 2 - Regression coefficient (r) for various flow models in shear rate–shear stress curve obtained for semisolids.
Formulation NBP1 NBP3 NCP1 NCP3 Free Vehicle
Bingham 0.920 ± 0.034 0.899 ± 0.009 0.901 ± 0.019 0.885 ± 0.015 0.909 ± 0.007 0.895 ± 0.022
Casson 0.926 ± 0.029 0.940 ± 0.012 0.947 ± 0.013 0.935 ± 0.014 0.951 ± 0.007 0.939 ± 0.015
Ostwald 0.955 ± 0.052 0.984 ± 0.009 0.989 ± 0.004 0.983 ± 0.004 0.989 ± 0.003 0.985 ± 0.005
Herschel-Bulkley 0.990 ± 0.001 0.989 ± 0.006 0.994 ± 0.005 0.990 ± 0.001 0.993 ± 0.001 0.994 ± 0.003
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Table 3 - Flow index (n) and consistency index (К) of semisolid formulations, according to Herschel-Bulkley flow model.
Formulation NBP1 NBP3 NCP1 NCP3 Free Vehicle
n 0.106 ± 0.031 0.114 ± 0.006 0.122 ± 0.010 0.115 ± 0.003 0.133 ± 0.009 0.137 ± 0.009
К 4370 ± 745 4481 ± 345 4124 ± 401 4664 ± 499 4371 ± 391 3856 ± 273
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