Solid Lipid Nanoparticles Containing Asiaticoside: Development of Topical Delivery Formulation

INTRODUCTION

The wound healing is a complex process consists of four phases namely hemostasis, inflammation, proliferation, and tissue remodeling or resolution, involving systematic, coordinated and balanced activity of inflammatory, vascular, connective tissue and epithelial cells.¹ Wound healing process results in the contraction and closure of wound tissue and restoration of a functional barrier.^2,3 Repair of injured tissues proceeds in the three interrelated dynamic and overlapping phases including the inflammation, granulation and remodelling. Wound treatments ideally accelerate healing and reduce scar complications.However, scarless healing is difficult in adult tissues. Accordingly, increasing attention has been directed to screening and development of products that are effective in both accelerating healing of wounds and thereby preventing scars.⁵ Because of anatomical and physiological characteristics of the skin, some ingredients may lack the desired activity topically and new drug carrier systems developed to modify the permeation/penetration.⁶

Wound dressings belong to biomaterials group and they provide suitable micro-environment and promote wound healing.⁷ Among the wound dressings that have been developed, special focus has been given to hydrogel based wound healing products because of their special properties which make them meet the requirements of ideal wound dressings.⁸ Hydrogel dressings entrap good amount of water which make them to have soft consistency nature resembling the natural living tissue which is different than any other class of artificial materials developed. It has been reported in literature that polysaccharide hydrogels are ideal for developing strong, flexible, biocompatible, and economical hydrogel dressings.⁹ Hydrogel wound dressings have threedimensional polymeric networks which entrap water and these dressings are available either in sheet form or as a viscous gel. Because of their nature, hydrogel based wound dressings allow permeation of both gases and water vapour which is essential for healing of wounds. This property of hydrogels dressings helps in maintaining a moist and hydrated environment.^10,11 Chitosan has been the material of choice among the polysaccharides used for hydrogels owing to biocompatibility, biodegradability and low to absent toxicity nature of chitosan.¹² In addition, chitosan has antibacterial, haemostatic mucoadhesive properties^13,14 and a wound healing accelerator properties.¹⁵

Because chitosan has poor solubility, water soluble O-carboxymethyl chitosan derivative (CMCh), has been used in various fields of drug delivery. The properties of carboxymethyl chitosan that has attracted a lot of attention are not its good solubility in water, biocompatibility, large hydrodynamic volume, good ability to form films, high viscosity and low toxicity.^16-18

Solid lipid-based nanoparticles are commonly used in topical application¹⁹ and at room temperature or at body temperature they are solid structures. Among the many nanovesicles, SLNs have attracted increasing focus as a more alternative dosage form to liposomes, niosomes and polymeric nanoparticles. SLNs have desired tolerability, stability, scaling-up feasibility, and can incorporate both hydrophobic and hydrophilic drugs.²⁰ SLNs can be used on damaged or inflamed skin as they are composed of non-irritant and nontoxic lipids. Moreover, researchers reported that SLNs exhibit good epidermal-targeting effects.²¹ Drugs like Nitrofurazone²², Metformin²³, Psoralen²⁴, Tretinoin²⁵ have been incorporated by SLNs for topical delivery. The SLN dispersions cannot be used as such for topical applications because of their low viscosity. To overcome this, SLNs are dispersed into hydrogel like semi-solid systems so as to increase the ease of application owing to better consistency and also to improve the stability of incorporated nanoparticles.

Currently, many medicinal plants have been used for various applications in health care system.²⁶ Among many plants studied for medicinal activity one plant studied widely is Centella asiatica. The plant has been used for various applications in traditional medicine to wound healing, better mental clarity, and to treat leprosy and psoriasis.²⁷ Centella asiatica main components are saponincontaining triterpene acids and their sugar esters. Centella asiatica contains asiatic acid, madecassic acid and asiaticosides which aretriterpene acids and their sugar esters are the important therapeutic compounds present in the plant.²⁸

The present work aims to develop solid lipid nanoparticles (SLN) containing Asiaticoside with glycerol tristearateas encapsulating matrix in hydrogel of O-carboxymethyl chitosan and oxidised sodium alginate for topical wound application.

Materials and methods

Materials

Chitosan (Deacetylation content of 88.2%) was purchased from Himedia, India. glycerol tristearate, sodium alginate were purchased from Himedia, India. Monochloroacetic acid, acetic acid, isopropanol, sodium periodate, ethylene glycol, sodium chloride, sodium hydroxide and methanol were all were purchased from Himedia, India and used without further purification. Asiaticoside was gift sample from S.A. Herbal Bioactives LLP, Mumbai.

Preparation of SLNs²⁹

SLNs were prepared by the method reported in literature. In 5 ml of ethanol, Glycerol tristearate and Asiaticoside were dissolved. The poloxamer 188 in water was placed on a magnetic stirrer, and the Glycerol tristearate and Asiaticoside solution was added drop wise into the water phase containing poloxamer kept at 80 °C under a stirrer of speed 1000 rpm. The mixture was stirred for 1 h and the emulsion obtained was cooled to solidify the nanoparticles by adding ice-cold water. The solidified nanoparticles were separated by centrifuging at 3000 rpm to remove free unentrapped Asiaticoside, and then centrifuged again at 6,000 rpm for 30 m at 4 °C. This was done to remove the free drug. The resultant solid mass was stored for further studies. The amount of lipid, drug and surfactant were varied in the preparation of nanoparticles (Table 1).

Evaluation of SLNs

Particle Size Analysis³⁰

The particle size and polydispersivity index of the optimized formulation was measured by dynamic light scattering (DLS Horiba scientific SZ-100 series) after equilibration for 2 m. The cell temperature was 25 °C; and the scattering angle was 90°.

Zeta Potential³¹

Zetasizer (Malvern Instruments Ltd, Worcs, UK) equipped with the Malvern PCS software (version 1.27) was used for Zeta potential measurement. A required amount of the sample was added to 5 ml of deionized water and placed into the electrophoretic cell and a potential of ±150 was applied. Sample analysis was done in triplicate.

Scanning Electron Microscopy (SEM)³²

The surface morphology of SLN containing Asiaticoside was subjected to scanning electron microscopic studies (SEM). The SLN for SEM were fixed on an aluminium stub using twosided carbon tape and sputter-coated with a gold/palladium mixture (60:40) under vacuum in an argon atmosphere using a sputter current of 40 mA. The SLN were imaged using Scanning Electron Microscope.

(%) Entrapment Efficiency³³

The prepared SLN dispersion was centrifuged to remove the unentrapped Asiaticoside at 3000 rpm. The prepared SLN were dissolved in methanol (10 ml) and subjected to vortexing to extract the Asiaticoside from the lipid. The mixture was subjected to centrifugation at 6,000 rpm for 10 m. The clear supernatant liquid obtained was diluted with methanol (15 ml), and drug content (denoted as M1) was measured by UV assay.

The amount of Asiaticoside entrapped in the Nanoparticles was estimated by centrifugation. Briefly, SLN dispersion was centrifuged at 6,000 rpm for 30 m at 4 °C to remove the free Asiaticoside drug in the aqueous water. After centrifugation, the obtained mass was dissolved in methanol (10 ml) and then vortexed to extract the drug from the lipid. This mixture was then centrifuged at 6,000 rpm for 10 min. The clear supernatant liquid was diluted with methanol (15 ml), and drug content (denoted as M2) was measured by UV assay.³⁴

EE(%)=M2/M1 ×100

Preparation of Carboxymethyl chitosan from Chitosan³⁵

The following method was adopted to prepare carboxymethyl chitosan. Chitosan (5 g), sodium hydroxide (6.75 g) and solvent isopropanol (50 ml) were mixed in a flask to swell and alkalize at 30 °C for 1 h. To this the monochloroacetic acid (15 g) solution in isopropanol was then added to the reaction mixture slowly drop by drop for 30 m and reaction was allowed to take place for 4 h at 55 °C. At the end of 4 h the reaction was stopped and isopropanol was discarded. Ethyl alcohol (80%) was then added the preparation mixture was filtered and subjected to rinsing with 80% ethyl alcohol to desalt and dewater and then vacuum dried at 50 °C.

Characterization of Carboxymethyl Chitosan

Fourier Transform Infrared (FTIR) and Nuclear Magnetic Resonance (NMR) Study36

The FTIR spectra of chitosan and the Carboxymethyl chitosan were recorded on (Agilent Cary 630) by the KBr pellet method in the wavelength region between 4000 to 400 cm-1.

NMR study was used to confirm the formation of Carboxymethyl chitosan. Carboxymethyl chitosan was characterized by ¹³C NMR (Bruker AVANCE, FT-NMR spectrophotometer, deuterium oxide solvent).

Oxidized alginate Preparation³⁷

Oxidized sodium alginate (OA) was prepared according to a previously reported method with some modification. 1% (w/v) sodium alginate (0.8 g, 4.04 mmoluronate) in ethanol was prepared and mixed with aqueous solution of sodium periodate at room temperature. To have OA with a different oxidation degree the molar ratio of 0.1, 0.3, and 0.5 (sodium periodate/urinate) was selected. The mixture were the mixed for 6 h in dark to avoid any undesirable reactions. Ethylene glycol was added to the system to stop oxidation reaction and stirred for 30 m. The prescribed amount of sodium chloride and ethanol were added while the system was under stirring. After 15 m, the precipitates were collected by a centrifuge (2000 rpm) and re-dissolved in distilled water (100 ml). To this sodium chloride and ethanol were added and after 15 m stirring, the precipitates were isolated by a centrifuge, dried by vacuum, and kept in a refrigerator prior to use

Preparation of Asiaticoside loaded Carboxymethyl chitosan hydrogel³⁸

SLNs loaded Carboxymethyl chitosan and oxidised alginate hydrogel was prepared by a simple mixing method. Briefly, a calculated amount of SLNs containing Asiaticoside was premixed with carboxymethyl chitosan aqueous solution (40 mg/ml), followed by addition of oxidized alginate aqueous solution (40 mg/ml) to incubate at 37 °C for 5-10 m to obtain loaded Asiaticoside hydrogel.

Texture Analysis³⁹

A Texture Analyser TA.XT Plus (Stable Micro Systems Ltd., Surrey, UK) was used to measure the texture properties of the hydrogels. Approximately 50 ml of the gel formulation was filled in a standard beaker (100 ml), thereby avoiding the introduction of air into the sample and assuring generation of a smooth upper surface. A 40-mm (diameter) disk was compressed and redrawn. Three samples were analysed at room temperature for each formulation.

Water Vapor Transmission⁴⁰

One gram of hydrogel sample was taken on a cloth and placed over cap on the mouth of a flask with a diameter of about 26 mm filled with 20 ml of distilled water. The flask was placed in a constant temperature-humidity chamber for 72 h (37 ºC at 75% RH). The mass loss of water from the bottle was used to calculate water vapour transmission rate (WVTR). The WVTR (g/m2/h) of each sample was determined by using the following equation.

WVTR(g/m2/h) = M₀ - M₁/ 72*A 10⁶

Where A is the flask mouth area (mm2 ), M0 and M1 are the combined mass of flask and hydrogel cap before and after placing in the chamber, respectively.

Rate of Evaporation of Water⁴¹

For these measurements, exact amount of hydrogel sample (1 gm, in triplicate) was kept at 37 ºC and 75% RH. After 72 h the weight was noted. Weight percentage was found out by the equation:

Weight remaining (%) = W_t/ W₀ * 100

where W0 and Wt are initial weight and weight after time ‘t’ respectively.

In vitro Asiaticoside Release Studies⁴²

Dialysis bag method was adopted to study the drug release. A dialysis bag retains SLN and SLNgel but permits the transfer of soluble Asiaticoside molecules into the release medium. Briefly, a dialysis bag (molecular cut-off of 8000–14,000, Himedia, India) was soaked in the release medium 12 h prior to the study. The release medium was 1 mM phosphate-buffered saline (PBS, pH 7.4). The SLN (containing 5 mg of Asiaticoside) and SLN-gel were placed in dialysis bags, which were placed in centrifugal tubes containing 30 ml of the dissolution medium. The release container was capped kept on a shaking box at 37 °C and 75 rpm. 2 ml sample was withdrawn from the dissolution contained at predetermine intervals of time and replaced with 2 ml of fresh release medium. The released Asiaticoside was estimated by uv-visible spectrophotometer (Shimadzu 1801) at 277 nm.

The Asiaticoside based SLN or SLN-hydrogel was fit using five kinetic mathematical models namely zero-order model, first-order model, Higuchi model, Hixson–Crowell model, Korsmeyer–Peppas model. The determination coefficient (R² ) was the indicator for measuring fitting level of the data.

RESULTS AND DISCUSSION

Particle size Zeta potential and SEM

Particle size and Polydispersivity index is usually used to characterize the nanoparticles, because it facilitates understanding of dispersion and aggregation.

Polydispersivity index (PDI) has been used as an indicator for stability and uniformity of nanoparticles. The nanoparticle size distribution is indicated by the PDI value. Larger PDI values indicate wider range of particle sizes and smaller PDI indicate that the sample contains evenly sized particles have lower PDI values. PDI values greater than 1 indicate that the sample contain nanoparticles of varying size distribution which may affect stability due to large particles or aggregates that could because of slow sedimentation. Particle size and PDI is efefcted to certain extent by surfactant concentration.

Particle size and PDI decreased with increasing SC. Formulation N2 showed polydispersivity index less than 1 and the particle size 155 nm to 340 nm (Figure 1).

Zeta potential was determined by Zetasizer and all the formulations showed a negative value.Zeta potential value of +30 mv or -30 mv have better stability and less chances of aggregation of particles.Formulation N2 have a Zeta potential value of -36.8 mV indicating good stability (Figure 2).

SEM was carried out to analyse surface morphology and the particles were found to be having smooth surface and round in shape (Figure 3).

Fourier Transform Infrared (FTIR) and Nuclear Magnetic Resonance (NMR) Study

The conversion of chitosan to carboxymethyl chitosan was determined by ¹³C NMR and FT-IR and the results showed that chitosan was successfully converted into CMC. FTIR spectra of CS shows important characteristic absorption bands at 3440 cm^-1 (O-H and N-H stretch), 1666 cm^-1 (stretching of C=O amide group), 1598 cm^-1 (angular 10 deformation of the N-H bonds of the amino groups), 1385 cm^-1 (symmetric angular deformation of CH3), 1158 cm^-1 (asymmetric bridge-O-stretch) and 1085 cm^-1 (skeletal vibration involving the C-O stretch). The FTIR spectrum of CMCs showed an intense band at 1600 cm^-1 and a moderate band at 1411 cm1(symmetric and asymmetric axial deformations of COO-), respectively, which indicate that the carboxymethyl groups to chitosan was introduced successfully (Figure 4 & 5).

Solid-state NMR has always been an important technique for analysing the molecular structure and dynamics of biological solids. Thus, the solid-state ¹³C NMR technique has been more and more often adopted to characterize the structures of chitosan and its derivatives. To identify the structures of the chitosan derivatives, solidstate ¹³C NMR was performed. The ¹³C NMR of chitosan was performed and compared with the spectra of CMC. ¹³C NMR of chitosan showed peaks at 177.9 ppm and at 25 ppm which can be assigned to the carbonyl carbon of –COCH3 and the methyl carbon (–CH3), respectively. These signals are less intense than the other signals. The signal at 101.3 ppm is assigned to the hydrogen bonded to carbon of chitosan and the signals in 59.6 ppm, 73.1 ppm, 81.1 ppm, 78.6 ppm, and 64 ppm are assigned to carbons of glucopyranose. The ¹³C NMR of showed the signal shifted from 101.3 ppm to 105.9 ppm (because carboxymethyl substituents show electron-withdrawing effect). The signals at 60.1 ppm, 73.8 ppm, 73.2 ppm, 82.2 ppm 78.2 ppm, and 63.9 ppm are split and shifted in relation to those detected in the spectrum of the parent chitosan. The signals were observed at 180.7 ppm (carbonyl carbons of carboxymethyl groups), 177.9 ppm (corresponds to the carbonyl carbon of –COCH3 of the parent chitosan). The signals at 53 and 57.4 ppm are attributed to methylene groups (–CH2), carbons respectively. Whereas CMC showed no signal at 53 ppm and at 58.4 ppm a weak signal was observed that can be assigned to the methylene (–CH2) bonded to the amino group (– NH). These information was taken as evidencethat carboxymethylation occurred at both -OH as well as in the amino groups of chitosan (Figure 6 & 7).

Texture analysis, Water vapor transmission and Rate of evaporation of water

The Texture analysis method settings, including speed rate and distance (depth of the insertion), were chosen according to hydrogel type. Replicates in three were analysed at room temperature for each formulation, providing the same conditions for each measurement. The hardness of the hydrogel formulation is indicated by the maximum force.

The water evaporation from a wound in an ideal dressing would be controlled at an optimal rate. For normal skin, the rate of water evaporation is 204 g/m² per day, and 279 g/m² per day for a first- degree burn, 5138 g/m² per day for a granulating wound. Both excessive dehydration as well as buildup of exudate should be prevented by the wound dressing. A water evaporation rate of 2500 g/m² per day is recommended which would provide sufficient moisture in the wound so as to prevent wound dehydration. The water loss from hydrogels according published literatures enables the hydrogel to take up from the wound the exudates and oedema fluid because of the active upward-directed process when used in exudating wounds (Table 4).

In vitro Asiaticoside Release Studies

Figure 8 shows that the SLNs and SLN-hydrogel could release Asiaticoside in a sustained for upto 8 h. At 8 h, the release percentages of the SLNs and SLN-gel were approximately 92% and 88%, respectively. For SLN, the R² values of the Higuchi, first-order, Korsmeyer–Peppas, Hixson–Crowell model and zero-order were 0.991, 0.9931, 0.9126, 0.9785 and 0.9177.

The best linearity was found in the First order plot (R² = 0.9931), indicating that the drug release from the SLN system was dependent on the amount drug remaining to be released. For SLNhydrogel, the R² values of the Higuchi, first-order, Korsmeyer–Peppas, Hixson–Crowell model and zero-order were 0.9768, 0.976, 0.927, 0.9916 and 0.9613, respectively. The best linearity was found in the Hixson–Crowell model (R² = 0.9916), suggesting that the drug release from the SLNhydrogel system was related to the change in surface area and diameter of the particles affecting the release of drug from SLN-hydrogel.

CONCLUSIONS

Asiaticoside was incorporated into SLNs through the reported solvent evaporation method. The prepared SLNs had spherical structure, nanometer range, good encapsulation efficiency and sustained release profile in vitro. Asiaticoside based SLNs were incorporated into the Carboxymethyl chitosan-oxidized alginate hydrogel, which could maintain the nanostructure and sustained release of Asiaticoside-based SLNs. Further in vivo studies are to be done to evaluate the wound healing efficiency of the prepared Asiaticosidebased SLNs-hydrogel with commonly used antibiotic.

ACKNOWLEDGEMENTS

This research was supported by advanced research grant by Rajiv Gandhi University of Health Sciences, Bangalore through Order No. RGU: RGU/ADV.RES/GRANTS/059/2016-17 dated: 30.01.2017.