Formulation and Toxicity Evaluation of Nanostructured Lipid Carriers for the Treatment of Acne

Background: Acne vulgarise is an inflammatory disease involving the pathological alteration of the sebaceous glands of the body. It is not a life-threatening disease but has a great influence on lifestyle. Topical combination therapy of vitamin A and antibacterial drugs is an effective treatment for acne. Materials and Methods: The current work investigates the nanostructure lipid and repeat dose toxicity study for histopathology and haematological examinations of organs. Conclusion: Prepared NLC formulation was aimed at epidermal targeting. Based on obtained results it is concluded that developed lipid-based nanocarrier system of selected drugs showed the targeting potential for effective acne treatment.


INTRODUCTION
Acne vulgarise is the most common, prevalent skin disease. A significant number of the population is affected by acne. It is not lifethreatening but influences quality of life [1][2][3]. Acne patients exhibit low self-esteem owing to the diverse lesions on their face, chest and back. A common type of bacteria Propionibacterium (P) acnes contributes to acne by causing inflammation. It is an inflammation of sebaceous follicles, characterized by seborrhoea, papules, comedones, nodules, pimples and pustules. Factors responsible for acne are sebum, hormones, follicular differentiation, bacteria Propionibacterium acnes, inflammation and nutrition [4,5].
Clindamycin phosphate (2mg/10 ml) is more water-soluble than tretinoin (< 0.1g/100mL). This work focuses on the incorporation of Clindamycin phosphate and Tretinoin into colloidal nanostructured lipid carrier formulation followed by characterization for morphology, entrapment efficiency, permeation, in vivo activity and toxicity potential. In vivo activities tested on Wistar rats. Dermal toxicity studies outcome is outlined in this article to characterize the skin irritation, local and systemic toxicity potential after dermal administration and evaluated as a better potential for acne treatment [34][35][36].

Screening of Drug in Liquid Lipids and Surfactants
Drug solubility in selected liquid lipids (jojoba oil, oleic acid, olive oil, medium chain triglycerides) and surfactants (Cremophore EL, Span 20, Span 80, PEG 200) was determined. The vials were filled with an excess quantity of drug and 2 ml of liquid lipid and surfactant. It was stirred to achieve equilibrium for 24 h at 25 0 C. Vials were then centrifuged using ultracentrifuge at 10,000 rpm, 30 min. The clear supernatant was collected and drugs were quantitated by U.V. Spectrophotometer. Tretinoin was determined using solvent methanol with suitable dilutions and analyzed at 339 nm whereas Clindamycin was evaluated using methanolic sodium hydroxide 0.1 N (1:1) at 215 nm after suitable dilutions [37,38].

Screening of Solid Lipids
Solid lipid solubility (stearic acid, Cetyl palmitate, Emulcier 61, Shea butter) was done by adding both drugs (5 mg) in a wide-mouth, amber coloured, screw-capped bottle. The lipids were heated till melting. This molten lipid was added in a small portion. The addition of lipid is continued until no drug particle is seen visually. Lipid melt was quantified for Tretinoin by dissolving in methanol and for clindamycin phosphate in methanolic sodium hydroxide 0.1 N by using UV Spectrophotometer [39][40][41].

Preparation and Evaluation of NLCs
High-speed homogenization-sonication technique: Based on solubility screening of both the drugs in liquid, solid lipids and surfactants oleic acid, shea butter, Emulcier 61, Cremophor EL and Span 20 were selected for formulation preparation. Various batches with different proportions of both the lipids, surfactant and cosurfactant were prepared to select a suitable combination to get a stable batch.
Selected lipids were weighed and melted. Tretinoin was added to the hot, liquified lipid phase. Clindamycin was dissolved in water (aqueous phase) and heated to emulsification temperature same to the oil phase. Both aqueous and lipid phase were mixed at a similar temperature around 60-70 0 C using a magnetic stirrer, 2000 rpm, 10 min to prepare a stable primary emulsion and then with a high-speed homogenizer at 60,000 rpm, 20 min. This mixture was further processed by probe sonicator, 20 min to get NLCs. Obtained NLC formulation was of thick, creamy consistency. The prepared formulation was stored in an amber colour bottle for further evaluation [42][43].
Morphological evaluation: Particle size, zeta potential, polydispersity were evaluated using a Horiba particle size analyzer zeta sizer. Before size determination, suitable dilutions were with double distilled water. The diluted sample was evaluated at a fixed scattered angle of 173 0 and 25 0 C temperature [44][45][46]. % Entrapment Efficiency (EE): % EE of prepared NLC was calculated by centrifugation. NLC formulation was diluted with equal parts of water and centrifuged at high-speed at 15,000 rpm, 30 min with a refrigerated centrifuge. The clear supernatant was separated and quantified for tretinoin by dissolving supernatant in methanol by U.V. Spectrophotometer at 339 nm and in methanolic sodium hydroxide 0.1 N at 215 nm for clindamycin [47]. % EE =(Drug added in the formulation -Unentrapped drug/ Drug added in the formulation) X 100 Drug permeation: A drug permeation was estimated using Franz diffusion assembly (12 ml capacity, 3.14 cm 2 area). Rat skin (Wistar rats, about 150-200 g) obtained from the college animal house. Hairs were removed using a hair trimmer and epilator cream and used as a diffusion membrane for study. The doner compartment consisted of 0.5 g of prepared NLCs formulation. The receptor chamber was filled with pH 7.4 phosphate buffer saline (PBS 7.4). The experiments were performed at temperature 37±0.5 0 C, 50 rpm. At pre-decided time interval of 0.5 h, 1 h, 2 h, 4 h, and 6 h, 0.5 ml aliquot from the receptor chamber was collected and replenished with an equal volume of media. Samples were analysed by spectrophotometer and the drug permeation profile was plotted to compared with marketed formulation [48].

Experimental animals:
Wistar rat (200-250 g) species were used for in vivo anti-acne model and toxicity evaluation. The animals were acclimatized to laboratory conditions (25 0 C, 35-60% humidity).

In vivo Anti-acne Activity
Acne induction and treatment: A rat model was used to reflect the inflammation and acne genesis by injecting a culture of P. acnes. The lyophilized culture of bacteria P. acne was procured from the Indian Institute of Microbial Technology (IMTECH), Chandigarh, India. Induction of acne was done by injecting heatkilled bacterial culture (60 0 C for 30 min) of concentration 10 -2 CFU / ml, 20μl was injected to the right ear pinna subcutaneously for 4 days, once a day. Left ear pinna was without induction and considered as a control for comparison. Acne induction was identified by the presence of redness, inflammation and acne.
Acne on the right ear pinna. After induction animal ears were treated with the test formulation, marketed formulation and placebo formulation. Once a day 0.5 g of the formulation was applied to the affected area and observed for healing and any other changes [49].  [50].

In vivo
Repeat dose toxicity study: Repeat dose toxicity test was performed according to the OECD guideline 410. Animals were divided into two groups treatment and placebo control consisting of 10 animals (5 females, 5 males). Animals were shaved before testing at the dorsal area. About 10 % of the body surface area was shaved for the application of the test substance. The test was performed at a limit dose of 1000 mg/kg body weight. The test substance was applied daily to the skin to the test and placebo control group for 6 h for a day for 28 days. The formulation was retained at a place by a nonadhesive bandage. The rat's weight were noted and visually observed for mortality, morbidity, general behaviour same as the acute study. For histopathological evaluation animals were necropsied. Skin, liver, kidneyand heart were isolated and stored in 10% formalin and processed for histopathology [51].

Screening of Drug in Lipids and Surfactants
Both drug solubility in selected solid, liquid lipids and surfactants is predicted in Fig. 1

Preparation of NLC
The solubility of tretinoin was observed more in Emulcier 61, shea butter, oleic acids and trial batches were observed to be more stable in presence of Span 20 and Cremophor. Table 1 represents the composition of a finalized batch obtained after various trials. The lipids were selected depending upon drug solubility and miscibility with each other. Prepared trial batches were evaluated for room temperature stability and particle size. The selected batch showed maximum stability and less particle size as compared to other prepared batches prepared by high speed homogenisation [54,55].

Characterization of NLCs
Morphological measurements: Particle size was found around 283 nm and zeta potential was -37.9 mv, polydispersity index 0.43 indicating a nano-size formulation with a stable behaviour. Fig. 3 represents the SEM image of NLC formulation. The SEM size was observed less than 100 nm size and of uniform spherical shape. From this data it is depicted prepared batch is of particle size (up to 200 nm) with good homogeneity, stable range zeta potential, thus represents a stable NLC system [56].  [57].

Drug permeation:
The present study is focused on the evaluation of topical target potential of the prepared system using Franz diffusion cell in PBS 7.4 at 37 0 C, 50 rpm using cellophane membrane. The amount of both drugs permeated from prepared NLC formulation was determined and compared with marketed formulation for 6 hr is shown in Fig. 4. A higher release is observed for both the drugs of the hydrogel-based marketed formulation. The initial slow release may be due to un-entrapped drug of aqueous phase and some amount of drug adsorb on the lipid carrier surface. Tretinoin release is less as entrapment efficiency in lipid core is more as compared to Clindamycin which also suggests that release prolongation is due to entrapment in lipid core. The drug permeation for NLC is found less than marketed hydroalcoholic gel formulation. Less permeation of the drug through the skin for lipid-based formulation indicated more drug retention into the skin layer and less permeated across the skin. Retention of NLCs by the skin is due occlusive property and less diffusion through the skin which is desirable for site effective treatment. NLC based drug formulation is design for controlling the drug release as well as skin targeting [58,59].

In vivo anti-acne rat model
An inflammatory acne mouse model was used to evaluate an anti-acne activity of the prepared NLC formulation. Ear pinnae were observed daily for sign of acne and inflammation. After two to three days of induction micro-comedones and inflammation at injected left ear pinna were seen and compared to the uninfected right ear. Infection was treated with the marketed formulation and prepared NLC formulation once a day 0.5 g. Inflammation and acne signs disappeared after treatment with prepared NLCs formulation in two days where it required 3 days for the standard treatment group and required five days for the control group. Antiacne in vivo rat model images of rat ear pinna of a group A formulation test group, B standard treated group are shown in Fig. 5. Animals were infected by injecting heat-killed P.acnes culture into the animal ear pinna. Injected P.acnes induced the granulomatous inflammation followed by the formation of micro-comedones which is about mild to moderate type of acne. Smooth healing was observed for the test group as some scars were visible on the standard treatment group. The prepared formulation showed faster healing compared to the standard group. In vivo results suggested that prepared NLCs effectively treated bacterial infection and healed ear pinna better than marketed preparation [60]. Acute toxicity: In acute dermal toxicity dose, 2000 mg/kg dose was found to be toxicologically insignificant. The treatment group did not show any toxicity sign on the surface of the skin at 24 and 48 h, 72 h after patch removal till 14 day, and the reaction was graded as "0" as per the Draize test score for erythema and oedema. No clinical signs of toxicity, mortality and changes in body weights were observed in treated animals. The treatment and placebo group did not show any changes in conduct, skin impacts, breathing, food, water consumption. No considerable weight loss was observed for all the animals [61].
Subchronic toxicity: Observed parameters for general signs are summarised in Table 2.
Observed changes in toxicity study are noted in Fig. 6.
Haematological and histopathological evaluation: All the erythro and leuko parameters are shown in Table 3. Microscopic structural changes of the organ liver, kidneys and skin is shown in Fig. 6.
General signs: No harmful signs or mortality were seen in test animals. Animals did not show any critical change of conduct, skin impacts, breathing, food and water consumption, postural irregularities. The physical appearance of skin, fur and eyes was normal. The bodyweight of the rats was found to be more. This indicated that applying the formulation on the skin had no effect on the growth. A proper intake of nutrients and water was observed.   Histopathology examination: It shows in considerable variations in control and treatment groups. Hence, it is suggested that the formulation is non-toxic. Histo-pathological evaluation of organs indicated no structural damage to the selected organs.

Treatment female group
Liver histology revealed no alteration in portal and central vein, bile duct and hepatic artery in the control and treated rats. There was no fibrosis, necrosis, inflammation, or local fatty degeneration in the liver hepatocytes. The kidney displayed no adverse effects. No morphological alteration was observed in cardiac muscles, arteries and veins. The microscopic examination of the skin of rats did not indicate any changes in the layers of the skin compared to the control group [62].

CONCLUSION
Both hydrophilic and lipophilic drug-loaded NLCs were prepared with Span 20, Oleic acid, Emuicier 61, Shea butter and Cremophor EL using homogenization-sonication method. The NLCs have more target potential to the topical part of the skin. NLCs were found to reduce the irritation potential and more efficacious in treatment for in vivo anti-acne study results. The results of the toxicity study suggest that formulation is with no death or no signs of toxicity at 2000 mg/kg (acute study) and 1000 mg/kg (repeat dose toxicity study), thus proving safety for use. The organ histology revealed no changes in both the groups and was considered safe since they did not induce dermal toxicity, irritation and sensitization. The results of the presented study suggest NLCs as improved nanocarriers for Tretinoin and clindamycin formulation as they offer many advantages over marketed gel.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
The experimental protocols were approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and the Institutional Animal Ethics Committee (IAEC) of the institute (Protocol no: KMKMP/IAE/081718).

ACKNOWLEDGEMENT
The authors are thankful to the Department of Pharmaceutics of Prin. K. M. Kundnani College of Pharmacy, for laboratory support for the conduct of the work.