1. INTRODUCTION:

Acne is supposed to be a trivial, self-limiting, cosmetic disorder which is mostly not very serious concern during its initial stage but on continued progression it results in reduced self-esteem, anxiety, social withdrawal as well as depression of suffering individual¹. The pathophysiology of acne principally has four key factors-abnormal differentiation and proliferation of keratinocytes, enhanced sebum production, hyperproliferation of Propionibacterium acnes, and an inflammatory response initiated by bacterial antigens and cytokines². Initial eruption of comedones, papules, pustules, inflammatory lesion and at advance stages cystic nodes if not treated in time lead to permanent mars as well as blemishes in affected skin area.³^,⁴

Retinoids are being utilized since a long time in treatment of acne due to their undisputed efficacy but poor patient compliance, associated dermatitis, and photosensitization of application area have continuously been a major limitation in treatment of acne by retinoids.⁵^,⁶ Adapalene is newer third generation retinoid having biological properties of retinoids (tretinoin, isotretinoin etc.) but have unique physiological profile like higher lipophilicity, superior photochemical stability and better patient tolerability⁷.

Vit- E is highly lipid soluble that circulate in lipoprotein and in cellular membranes it protects various biological membranes, lipoproteins, fatty acid and other important biological macromolecules from peroxidation reaction and from UV damage⁸. It has been reported to reduce inflammation and irritation caused due to peroxidation of sebaceous fluid caused due to follicular leakage because Vit- E effectively blocks peroxidation of lipids⁹. In addition, reactive species as well as various free radicals generated in skin due to excessive exposure to sunlight are efficiently neutralized by Vit-E thus protecting skin against UV radiation damage and photo-aging¹⁰. So the co administration of Vit-E in prepared gel may enhance therapeutic efficiency of principal drug adapalene as well as will have additional ameliorative effects on skin.¹¹^,¹²

A combination preparation of ADP 0.1% in the form of NLCs and Vit-E was developed by modified melt dispersion ultrasonication method. The customized formulation is designed to deliver active ingredients with selective permeation in affected pilosebaceous region and minimized systemic penetration. Further the formulation is designed in aqueous gel based vehicle to impart moisturizing attributes to skin and better compliance to patient.

Nanostructured lipid carriers are spatially imperfect nanosized carriers comprising of both solid and liquid lipids which are solid both at room and body temperature.¹³ Due to plenty of imperfections in internal structure of NLCs drug loading capacity is enhanced and extrusion of drug during storage is reduced¹⁴. These carriers presents some promising advantages compared to conventional formulations, such as higher entrapment efficiency, minimal systemic permeation of drug, lesser skin irritation, better dermal localization of drug and prolonged duration of action.¹²^,¹⁵^,¹⁶

The aforementioned study was carried out to assess efficacy and safety of combination novel NLC based gel of 0.1% adapalene and Vit-E as compared to conventional formulation in patient suffering from acne vulgaris. Enrichment of adapalene gel with Vit-E may alleviate proinflammatory action of affected skin during acne. Findings of our work were in line as expected for superior clinical efficacy than conventional topical treatments.

2. MATERIALS AND METHODS:

The ADP was supplied as a kind gift by Glenmark Pharmaceuticals Ltd. (Nasik, India). Phospholipid (90 NG) was a generous gift from Lipoid, Ludwigshafen, Germany. Glyceryl tristearin, oleic acid, and Vit-E were availed from sigma Aldrich Germany. Carbapol 980, triton X 100, pluronic F68 were purchased from Hi media Ltd. Mumbai. Filtered and deionized water was used throughout the study.

2.1 Preparation of ADP loaded NLCs (NLCs-A):

Modified Melt dispersion ultrasonication method was used to prepare the NLCs. In brief the Glyceryl tristearin, oleic acid, soya lecithin and cholesterol were intensely blended for 10 minutes. Then this blended mixture of components was subjected to melting at 78±2℃. In this lipid melt drug ADP was added with gentle stirring to form clear visible lipid phase. Now aqueous phase containing 1% tween 80 dissolved in distilled water, was prepared. After that this oil phase was slowly added to aqueous phase with continuous stirring at 1000 rpm keeping a temperature at 75±2℃ for 40 minutes to form micro emulsion. Now the prepared microemulsion was diluted with cold water (4-5℃) and further subjected to mechanical stirring to form dispersion of NLCs such that final concentration of ADP in NLCs dispersion is maintained 0.1% w/w.¹⁷

2.2 Formulation of NLC based gel:

Carbopol 980 was optimized for different concentration. After optimizing gel qualities in terms of viscosity, elegancy and spreadability 1.0% concentration of carbapol was selected for preparation of final formulation. In respective concentration carbapol and above prepared NLCs were mixed with continuous stirring at 1000rpm (Remi Instruments, Mumbai, India). Process was carried out until NLCs were completely dispersed in gelling agent. Now 0.05% of vit E was added to this gel containing NLCs of adapalene with continuous blending of gel mixture. Then in a separate beaker 0.1g if BHT was mixed with 1g Benzyl alcohal to prepare antioixdant preservative solution, this prepared BHT solution was now added in above prepared gel mixture. After formulation of pharmaceutically acceptable gel it was neutralized with 0.05%w/w concentration of Triethanolamine and pH of final gel was adjusted to 6.5.¹⁸

3. Characterization of NLCs dispersion:

Average particle size, polydispersity index (PI) and zeta potential were analyzed by Nano ZS90 Zetasizer (Malvern Instruments Corp, U.K) at temperature 25±0.1ºC keeping a fixed angle of 90º to incident beam based on the principle of photon correlation spectroscopy. Samples were properly dispersed and diluted with double distilled water to ensure proper light scattering with in instrument sensitivity range.

3.1 Transmission electron microscopy (TEM):

Size and surface morphology of prepared NLCs were characterized by TEM using Philips CM 10 electron microscope at an accelerating voltage of 200KV. After suitable dilution and proper sonication of NLCs dispersion, a drop of sample was placed over coated copper grid. After drying of sample photomicrographs were captured at suitable magnification.¹⁹

3.2 Entrapment efficiency:

As drug release characteristics of any drug are directly influenced by amount of drug entrapped in carrier, so measurement of entrapment efficiency of carrier system becomes of prime importance²⁰. After separation of free drug and any lipids, amount of drug entrapped in per unit weight of nanostructured carriers was analyzed. The above prepared NLCs-A dispersion was placed in to dialyzing bags with MWCO 10 kDa. Now this was extensively dialyzed in a magnetic stirrer at 75rpm against double distilled water maintaining sink conditions during the experiment, this results in removal of any of unentrapped drug from the formulation. Now after this samples were collected in suitable vials and were diluted suitably with mixture of dimethylformamide and methanol. Now to quantify amount of ADP isocratic HPLC analysis was carried out using Merck RP-8 column²¹. This column was appropriately degassed and saturated with mobile phase solvent mixture of acetonitrile and water (65:35) and pH was maintained to 2.5 with phosphoric acid, this degassing is done in ultrasonic bath. 20µL volume of all samples was used for injection and absorption wave length of 320nm was used for estimation. Now drug entrapment efficiency of NLCs was calculated using the formula:

Amount of added drug – amount of unentrapped drug

Entrapment = -------------------------------------------------- Χ 100

efficiency (%) Amount of added drug

Table 2: Particle size, zeta potential and polydispersity index of formulation

NLC based formulation code	Particle size (nm)	Polydispersity index	Zeta potential (mv)	Entrappment efficiency
NLC1	545.4	0.678	-37.40	66.32%
NLC2	654.6	0.566	-19.80	68.90
NLC3	219.8	0.263	-23.85	87.4%
NLC4	513.5	0.478	-44.30	83.35%
NLC5	477.6	0.676	-19.50	74.45%
NLC6	432.1	0.545	-34.45	81.20%
NLC7	932.3	0.609	-24.55	86.0%
NLC8	1765.4	0.889	-44.60	78.15%
NLC9	889.7	0.976	-31.40	81.10%

3.3. In-vitro drug release:

Concept of diffusion of drug across semipermeable membrane was utilized to study diffusion of drug through NLCs-A. Here diffusion of entrapped drug through dialysis membrane (MWCO 10-12Kda) was carried out²². After treatment of dialysis membrane, 10 ml NLCs-A dispersion free from any of unentrapped drug was filled in treated dialysis membrane and kept tied at both ends. Now this drug filled closed dialysis bag was suspended in a beaker containing 25ml of solvent mixture of DMF and methanol (1:50). Whole assembly was kept continuously stirred under magnetic stirrer at 1000rpm and maintained at temperature of 32±1℃. At regular interval 1 ml sample was withdrawn and sink conditions were maintained by replenishing fresh solvent mixture simultaneously. Quantification of amount of Adapalene was performed by previously reported HPLC method.²³

3.4. In -vitro skin permeation study:

This study was carried out in male Wistar rats. All experimental protocol were approved by institutional animal ethical committee of Bhagyoday tirth pharmacy college, Sagar M.P.India (Ref. letter No.0604/BTPC/ 2021-22/12). During all the experimental work, CPCSEA guidelines were appropriately followed.

Properly shaved skin of abdominal region of rat was excised and properly inspected for it’s integrity by lamp inspection method. After being assured about integrity of excised skin it was thoroughly washed with saline and any type of subcutaneous tissues below the skin were removed by chopping²⁴. Now this prepared skin was mounted on modified vertical franz diffusion cell in the way outer skin epidermis was facing donor segment and inner layer of dermis was facing towards receiver compartment. This receiver compartment was filled with solvent mixture PBS buffer (pH 5.6) and methanol (with little DMF) in a ratio of 1:5 and whole unit was kept stirred in circulating water bath at 500rpm maintaining temperature of 32±0.5ºC. And the whole assembly with samples is allowed to equilibrate for nearly 1hour¹². Now plain drug solution, NLC- A and NLC-A-Vit-E gel (each with quantitatively equivalent amount of adapalene to NLC-A) were gently applied over skin mounted on donor compartment of FD cell. As the time elapses drug starts permeating from donor compartment to receiver compartment. Now at regular fixed time interval, 1ml of sample from receiver compartment was collected and simultaneously receiver compartment was supplied with fresh solvent mixture to maintain sink conditions and analyzed for quantity of drug permeated across layers of skin. Before analytical estimation, drug samples were filtered through 0.22µm cellulose membrane filters and cumulative amount of drug permeated was analyzed each time. The study protocol was continued for 8 hours.

3.5. In-vivo skin distribution study:

After finishing skin permeation, in vitro skin distribution was carried out, to carry forward more in depth analysis. Skin samples were carefully removed and gently scraped to retrieve most of the adherent formulation. Now a lint free cloth soaked in deionized water was used to clean thoroughly skin pieces. After repeated washing for 3 times they were dried properly and now epidermis and dermis layers were manually separated. Now these dried skin layers were chopped individually in to small pieces and subjected to maceration with solvent mixture of DMF: methanol (1:50) to carry out complete extraction of retained drug. Resultant solution was filtered with 0.22 micron membrane filter and quantitatively analyzed for estimation of permeated amount of drug in various skin layers.

4. Stability study of NLC-A, NLC-A-Vit-E gel:

Stability study of prepared formulation was studied by observation of different parameters like change in crystalline behavior, development of any type of obnoxious odors, change in original color of gel formulation, appearance of any type of precipitate or any other visible as well as disagreeable changes. So for studying stability of above developed formulation we have done study of formulation at normal as well as stressed climatic conditions of temperature and humidity and performed accelerated stability studies in this regard. Study was carried out for 3 month at refrigerated temperature RF (5±3℃), room temperature conditions (25±2℃ and relative humidity RH 60±5%) and elevated temperature and humidity (40±2℃ and relative humidity RH 75±5%) and in these conditions drug assay, particle size, zeta potential, change in texture of formulation etc. were observed.

5. Skin irritation study:

The prepared formulations were tested for presence of any type of skin irritation in albino mice weighing 20-25 gram. Before starting experimentation mice were cautiously shaved to remove hairs. Now these animals were kept in observation. And two groups of animal which were further subdivided in two groups were prepared. The following formulation was applied to back of albino rats

1. Placebo gel

2. NLC based gel

3. Marketed gel of drug

4. Control (No treatment)

0.25g of each gel was applied over 2cm²area of animal skin and gel was gently spreaded over entire application area. Any type of visible changes in skin of animal like redness (erythema), swelling, were carefully observed over entire application area. Results of this experiment are given in table no. 2.²⁵

Table 3. Primary irritation indexing of each formulation after 24, 48, 72 hrs

Formulation	Irritation score and Time of application of individual formulation
Formulation	24 hrs	48 hrs	72 hrs
NLC based gel	0	0	0
Marketed gel	0	0	1
Placebo gel	0	0	0

Statistical analysis of data:

All the statistical analysis was completed by using analysis of variance (ANOVA) with Tukey–Kramer multiple comparison post-tests using Graph Pad InSta t™ software (GraphPad Software Inc.,San Diego, California).

5. RESULT AND DISCUSSION:

5.1 Preparation of NLCs:

The NLCs of adapalene were prepared by melt dispersion ultrasonication technique and concentration of solid lipid and liquid lipids were optimized by using varying concentration of these lipids in gradual manner. Upon varying quantity of lipids properties of NLCs are significantly affected which is given in table. 1 and 2.

We have observed that on increasing quantity of solid lipid size of carrier increases. During our experimental study we found that at a specific ratio of solid lipid and oleic acid (2:1) we found nanocarriers (NLC3) of size 219.8. nm which we have considered as optimized formulation. It was also found that in formulation code NLC 8 when GTS:oleic acid ratio is 10:3 that particle size in this formulation is suddenly very large (1765.4 nm) which could be due to higher solid lipid concentration as well as due to relatively higher concentration of oleic acid. From the particle size study of different formulations in various ratios of GTS and oleic, it was found that the formulation's particle size was smaller at higher concentrations of oleic acid. This could be because higher oleic acid concentrations result in reduced viscosities of NLCs, which in turn results in smaller and smoother surface NLCs.

The term polydispersity describe the degree of non-uniformity of size distribution in the formulation. It is a dimensionless index and scaled from two parameters to fit to the correlation data, it is dimensionless index whose value range from 0.05(highly monodisperse) to 0.7(highly polydispersity) although numerical value of PDI may range from 0 to 1. For nanostructured system PDI value below 0.3 is acceptable which indicates quite homogeneous population of carrier molecule in formulation²⁶. In our study we found PDI of our optimized formulation 0.263 which is lesser than to 0.30 which indicate good homogeneity of prepared nanostructured carriers. Electrokinetic potential or zeta potential reflects the surface charge over colloidal particles, and to know physical stability of prepared nanostructured carriers zeta potential was measured. It is measured by knowing electrophoretic mobility of colloidal carriers in a zeta sizer ZS 90 (Malvern Instruments, UK)²⁷. Generally zeta potential values ranging between -30 to +30mv is considered sufficient to offer enough repulsive interaction to obtain good physical stability of colloidal particles. We found zeta potential in the range of -19 to -44mv and zeta potential of our optimized formulation was -23.85mv which indicates good physical stability of prepared nanostructured vesicles.

5.2 TEM analysis:

Morphological and topographical information of colloidal carrier is studied by transmission electron microscopy. TEM analysis of our formulation justifies spherical, uniform and nearly monodisperse particles without any visible drug crystals.

Fig.1: TEM image of nanostructured carriers

Thus from study of particle size, zeta potential and TEM, it can be expected that prepared nanostructured carriers can effectively penetrate to desired skin layers.

5.3 Entrapment efficiency:

Entrapment efficiency is measure of quantity of drug encapsulated inside carrier system and it depends on various factors like concentration of polymer, solubility of drug in polymer, rate of removal of solvent and many other factors. In our study we have found entrapment efficiency ranging from 66 to 88% and the highest entrapment efficiency was reported in our optimized formulation NLC3. Although formulations NLC 7, NLC8 and NLC9 have higher lipid content than optimized formulation NLC3 but still their entrapment efficiency is lower it suggest that at a particular optimized ratio highest entrapment efficiency is achieved.

5.4 In- vitro estimation of drug release:

In-vitro drug release study was carried out by using dialysis bag. We have done drug release study in our optimized formulation NLC3 at temperature 32±0.5℃. The initial release profile of drug is like conventional formulation in which fast drug release pattern is observed after that sustained drug release pattern is obtained through NLC based gel which is desirable in any controlled release formulation. The initial faster drug release pattern is due to surface adsorbed drug or due to drug molecules present in surface of vesicles and sustained release pattern in longer duration has resulted due to slower release of drug encapsulated inside vesicles. The drug release pattern is presented in figure 2

Figure: 2 Drug release pattern through NLC-A-Vit-E gel

5.5. Skin permeation and bio distribution study:

To know the permeation of drug skin permeation study was carried out in mice abdomen skin. Skin hairs were removed by surgical blade and any skin was thoroughly washed and cleaned with normal saline. Any fat material adhered to skin was removed gently by isopropanol soaked cotton. Now these cleaned dehaired skin samples were stored at -20℃ in deep freezer to preserve them. ²⁸

Skin permeation study was performed on Franz diffusion cell in 8 hour study design. Here marketed gel of ADP 0.1% was used as reference to study the skin permeation efficiency of NLC-A and NLC-A-Vit-E gel. After 8 hour of study design we have found that there was no significant change in amount of adapalene from NLC-A and NLC –A-Vit-E gel while the amount of adapalene from marketed gel was higher as reported in figure 3. So it was observed that significantly lower amount of ADP has penetrated through skin from NLC based gel as compared to conventional marketed gels. The higher amount of adapalene form convention gel in receptor compartment was due to absence of any type of carrier system in gel matrix so drug penetrates from hydrophilic gel matrix in higher amount while in NLC based gel relatively lower concentration of drug was reported in receptor compartment because these lipidic carrier molecules prevent unrestricted permeation of drug in receiver compartment which is of utmost need to restrict systemic permeation of drug.

So on the basis of above observations it can be positively expected that these NLC based gel will surely minimize the systemic uptake of adapalene and will localize the drug in epidermal and subepidermal region which is quite needed in these type of disease control.

Figure 3. Skin permeation and biodistribution study of adapalene in different skin layer and receptor

6. CONCLUSION:-

So NLC –A-Vit-E gel was successfully developed with optimized process variables, characterized and evaluated for its therapeutic and pharmaceutical performance in terms of invitro drug release, skin permeation, biodistribution and skin irritation potential and performance was compared with marketed gel. The developed optimized formulation have efficiently showed improved epidermal and subepidermal localization while simultaneously reduced systemic permeation when compared with marketed formulations. Simultaneous adjuvant effects of Vit-E will definitely protect adapalene from photooxidation and antioxidant effects of Vit-E will surely ameliorate other ance associated inflammatory response and skin damage. These lipid nanocarriers enhance drug to accumulate and saturate them self in epidermal and subepidermal region while free permeation to systemic circulation is prohibited due to encapsulation of drug inside lipid vesicles this is the major outcome of this work in disease management. So this novel NLC based gel comprising of adapalene along with Vit-E will surely be beneficial in acne therapeutics. Further continued research will open new avenues in future. So in concluding remarks it can be said that finding of this work suggest that NLC can be explored as next generation carrier in topical delivery of adapalene and coadministration of antioxidant Vit-E in gel will surely have additive therapeutic as well as adjuvant effects to treat acne vulgaris.

REFERENCES:

1. Degitz K. Placzek M. Borelli C. Plewig G. Pathophysiology of acne. Journal of German Socienty of Dermatology. 2007; 5(4): 316–323. https://doi.org/10.1111/j.1610-0387.2007.06274.x

2. Irby C E. Yentzer B A. and Feldman S R. (2008). A review of adapalene in the treatment of acne vulgaris. The Journal of Adolescent Health : Official Publication of the Society for Adolescent Medicine. 2008; 43(5): 421-424. https://doi.org/10.1016/j.jadohealth.2008.06.005

3. Baghel S. Gidwani B. Kaur CD. Novel Drug Delivery Systems of Herbal Constituents Used in Acne. Asian Journal of Research in Pharmaceutical Sciences, 2017; 7(2): 57–67. https://doi.org/10.5958/2231-5659.2017.00009.1

4. Gharsan M AL. Sreeharsha N. Therapeutics of microneedling for skin repair. Asian Journal of Research in Pharmaceutical Sciences. 2022; 12(3): 199–204. https://doi.org/ 10.52711/2231-5659.2022.00035

5. Nasorallahi SA. Koohestani F. Naeimifar A. Samadi A. Vatanara A. et al Preparation and evaluation of adapalene nanostructured lipid carriers for targeted drug delivery in acne. Dermatologic Therapy, 2021; 34(2): 1–10. https://doi.org/10.1111/dth.14777

6. Umalkar D. Saudagar RB. Patel D.Rajesh K S. (2014). Formulation and Evaluation of Transferosomal gel of Isotretinoin for severe Acne. Research Journal of Topical and Cosmetic Sciences. 2014; 5(2): 39–45.

7. Bernard B A. Adapalene, a new chemical entity with retinoid activity. Skin Pharmacology : The Official Journal of the Skin Pharmacology Society, 1993; 6(1): 61–69. https://doi.org/10.1159/000211165

8. Aktürk AS. Güzel S. Bulca S. Demirsoy EO. Bayramgürler D. Bilen N. and Kıran, R. Effects of isotretinoin on serum vitamin E levels in patients with acne. International Journal of Dermatology, 2013; 52(3): 363–366. https://doi.org/10.1111/j.1365-4632.2012.05676.

9. Azulay M M. Bagatin, E. (2009). Cosmeceuticals vitamins. Clinics in Dermatology. 2009; 27(5): 469–474. https://doi.org/10.1016/j.clindermatol.2009.05.010

10. Keen M A. Hassan I. Vitamin E in dermatology. Indian Dermatology Online Journal. 2016; 7(4): https://journals.lww.com/idoj/Fulltext/2016/07040/Vitamin_E_in_dermatology.18.aspx

11. Pappas A. Liakou A. Zouboulis C C. Nutrition and skin. Reviews in Endocrine and Metabolic Disorders. 2016; 17(3): 443–448. https://doi.org/10.1007/s11154-016-9374-z

12. Jain AK. Jain A. Garg N K. Agarwal A., Jain, A. Jain S A Tyagi R K. Jain, R K. Agrawal H. Agrawal GP. et al Adapalene loaded solid lipid nanoparticles gel: an effective approach for acne treatment. Colloids and Surfaces. B, Biointerfaces. 2014; 121: 222–229. https://doi.org/10.1016/j.colsurfb.2014.05.041

13. Erawati T. Hariyadi D M. Rosita. N.Purwanti T. Comparison of the Release rate, Penetration and Physical stability of p-Methoxycinamic acid (PMCA) in Nanostructured Lipid Carriers (NLC), Solid Lipid Nanoparticle (SLN) and Nanoemulsion (NE) systems Research Journal of Pharmacy and Technology. 2021; 14(11): 5719–5724. https://doi.org/10.52711/0974-360X.2021.00994

14. Das S. Chaudhury A. Recent advances in lipid nanoparticle formulations with solid matrix for oral drug delivery. AAPS PharmSciTech. 2011; 12(1): 62–76. https://doi.org/10.1208/s12249-010-9563-0

15. Dsouza J I. More H N. Topical Anti-Inflammatory Gels of Fluocinolone Acetonide Entrapped in Eudragit Based Microsponge Delivery System. Research Journal of Pharmacy and Technology. 2008; 1(4): 502–506.

16. Madhumitha V. Sangeetha S. Transfersomes: A Novel Vesicular Drug Delivery System for Enhanced Permeation through Skin. Research Journal of Pharmacy and Technology 2020; 13(5): 2493–2501. https://doi.org/10.5958/0974-360X.2020.00445.X

17. Khurana S. Bedi P MS. Jain NK. Development of Nanostructured Lipid Carriers (NLC) for controlled delivery of mefenamic acid. Int Jouranl of Biomedical Nanoscience and Nanotechnology. 2012; 2(3-4): 232–250. https://doi.org/10.1504/IJBNN.2012.051218

18. Joshi M. Patravale V. Formulation and evaluation of nanostructured lipid carrier (NLC)-based Gel of Valdecoxib. Drug Development and Industrial Pharmacy 2006; 32: 911–918. https://doi.org/ 10.1080/03639040600814676.

19. Zhao X. Yang C. Yang K. Li K. Hu H. Chen D. Preparation and characterization of nanostructured lipid carriers loaded traditional Chinese medicine, zedoary turmeric oil. Drug Development and Industrial Pharmacy 2010; 36 (7): 773–780. https://doi.org/ 10.3109/03639040903485716

20. Ridolfi D M. Marcato P D. Justo G. Z. Cordi L. Machado D. Durán N. Chitosan-solid lipid nanoparticles as carriers for topical delivery of tretinoin. Colloids and Surfaces B: Biointerfaces. 2012; 9: 36–40. https://doi.org/10.1016/j.colsurfb.2011.11.051

21. Martins L A. Meneghini LZ. Junqueira C A. Ceni D C. Bergold A M. A Simple HPLC-DAD Method for Determination of Adapalene in Topical Gel Formulation. Journal of Chromatographic Science. 2011; 49(10): 796–800. https://doi.org/ 10.1093/chrsci/49.10.796

22. Ramezanli T. Bozena B. Kohn M. Development and Characterization of a Topical Gel Formulation of Adapalene-TyroSpheres and Assessment of Its Clinical Efficacy. Molecular Pharmaceutics 2018; 15(9): 3813–3822.https://doi.org/10.1021/acs.molpharmaceut.8b00318

23. Kumara G. Patrudub T B. Raoa MV B. Raoa T N. A Novel Method Development and Validation for Related Substances of Adapalene in Bulk Drug Product by HPLC. Research Journal of Pharmacy and Technology 2016; 9(12): 2234–2240. https://doi.org/10.5958/0974-360X.2016.00451.0

24. Fang JY. Fang CL. Liu CH. Su YH. Lipid nanoparticles as vehicles for topical psoralen delivery: solid lipid nanoparticles (SLN) versus nanostructured lipid carriers (NLC). European Journal of Pharmaceutics and Biopharmaceutics. 2008;  70(2): 633–640. https://doi.org/10.1016/j.ejpb.2008.05.008

25. Czernielewski J.Michel S. Bouclier M. Baker M. and Hensby, C. (2001). Adapalene biochemistry and the evolution of a new topical retinoid for treatment of acne. Journal of the European Academy of Dermatology and Venereology. 2001; 15(SUPPL. 3): 5–12. https://doi.org/10.1046/j.0926-9959.2001.00006.x

26. Danaei M. Dehghankhold M. Ataei S. Hasanzadeh Davarani F. Javanmard R.et al Impact of Particle Size and Polydispersity Index on the Clinical Applications of Lipidic Nanocarrier Systems. Pharmaceutics. 2018; 10(2): https://doi.org/10.3390/pharmaceutics10020057

27. Obeidat W M. Schwabe K. Müller R H. Keck C M. Preservation of nanostructured lipid carriers (NLC). European Journal of Pharmaceutics and Biopharmaceutics. 2010; 76(1): 56–67. https://doi.org/https://doi.org/10.1016/j.ejpb.2010.05.001

28. Kumar R. Singh B. Bakshi G. Katare O P. Development of liposomal systems of finasteride for topical applications: design, characterization, and in vitro evaluation. Pharmaceutical Development and Technology. 2007; 12(6): 591–601. https://doi.org/10.1080/10837450701481181

Received on 27.08.2022 Modified on 18.10.2022

Research J. Pharm. and Tech 2023; 16(11):5067-5073.

DOI: 10.52711/0974-360X.2023.00821

	Ingredient quantity is expressed in mg
Ingredient	NLC1	NLC2	NLC3	NLC4	NLC5	NLC6	NLC7	NLC8	NLC9
Adapalene	100	100	100	100	100	100	100	100	100
Vit-E	50	50	50	50	50	50	50	50	50
Glyceryl tristearin	400	400	400	300	300	300	500	500	500
Oleic Acid	100	150	200	100	150	200	100	150	200
Cholestrol	0.020	0.020	0.020	0.020	0.020	0.020	0.020	0.020	0.020
Soya lecithin	0.025	0.025	0.025	0.025	0.025	0.025	0.025	0.025	0.025
Tween 80	1.5	1.5	1.5	1.5	1.5	1.5	1.5	1.5	1.5
Triton X100	8	8	8	8	8	8	8	8	8