INTRODUCTION:

Transdermal drug delivery system (TDDS) of drug through skin provides a convenient route of administration for a variety of clinical indications.^1,2 It’s also can reduce the side effects associated with its oral therapy.³However, active ingredients that are difficult to dissolve in water generally have limitations in penetration into the skin.⁴

The skin is very effective as a selective penetration barrier. The stratum corneum provides the greatest resistance to penetration, and it is the rate-limiting step in percutaneous absorption.^5,6,7 To overcome this, nano delivery systems are developed using various forms and types of lipids, namely the nanostructured lipid carrier (NLC) system using a combination of solid lipids and liquid lipids, solid lipid nanoparticles (SLN) system uses solid lipids only and nanoemulsion (NE) system uses liquid lipids only.^8,9,10 Various forms and types of lipids used in the nano delivery system can affect the characteristics and ability of penetration of active ingredients into the skin resulting in differences in effectiveness.^11,12,13 In this research a combination of beeswax-cacao oleum and virgin coconut oil (VCO) was used as a constituent component of nanocarriers for p-methoxycinnamic acid (PMCA) which has anti-inflammatory effects and is difficult to dissolve in water. ⁹ For all systems formed the release test, deep penetration test into rat skin, and physical stability test using the centrifugation method and the real time method was performed.

MATERIAL AND METHODS:

Research Material:

The materials used in this study if not stated other have the purity degree of Pharmaceutical Grade. The materials used were p-methoxycinnamic acid (Sigma Aldrich), beeswax (PT Kurniajaya Multisentosa), cacao oleum (Research Center for Coffee and Cocoa, Jember - Indonesia), VCO, Span 80 (Sigma Aldrich), Tween 80 (Sigma Aldrich), Propylene glycol (E Merck), Sodium benzoate (PT Brataco), Sodium acetate and Glacial acetic acid pro analysis (E.Merck).

Experimental animals:

The experimental animals used in this research were male Wistar rats aged 8-10 weeks and weighing 150-250 grams. ^[14] The number of animals tested was calculated by the Federer equation (1):

Note:

n = number of experimental animals needed for each treatment group

t = number of treatment groups (t = 3x2)

Number of experimental animals needed for each treatment group:

(6-1) (n-1) ≥ 15

5n-5 ≥ 15

n ≥ 4

Total number of experimental animals (N) = 6 x 4 = 24

The use of experimental animals in this research has met the ethical requirements by the Animal Care and Use Committee, Faculty of Veterinary Medicine, Airlangga University with certificate No: 2.KE.056.04.2019

Sample Preparation:

The sample formulas of NLC-PMCA, SLN-PMCA and NE-PMCA can be seen in Table 1.⁸

Table 1: Concentration of Materials in NLC-PMCA, SLN-PMCA and NE-PMCA

Material	Concentration (% b/v)
Material	NLC-PMCA	SLN-PMCA	NE-PMCA
PMCA	0.2	0.2	0.2
Cacao Oleum	2.97	4.95	-
Beeswax	0.99	1.65	-
VCO	2.64	-	1.32
Tween 80	5.74	4.39	18.66
Span 80	14.76	16.11	1.92
Propylen Glycol	3.5	3.5	3.5
Sodium Benzoate	0.1	0.1	0.1
Acetate Buffer pH 4.2 ± 0.2	Up to 100	Up to 100	Up to 100

NLC-PMCA Preparation:

The NLC-PMCA system was made firstly by melting beeswax at 70^oC, then add cacao oleum and leave until all the cacao oleum melts. The PMCA dissolved in VCO was added to the beeswax-cacao oleum mixture (lipid phase). Then the mixture of Tween 80 and Span 80 that has been heated at 70°C is added to the lipid phase. Sodium benzoate, propylene glycol, and acetate buffer pH 4.2 ± 0.02, as the water phase is heated at 72°C. The water phase is dispersed slowly into the oil phase while stirring with Ultra-Turax High Shear Homogenizer at 5000 rpm, then stirring is continued by increasing the speed to 16,000rpm for 2 minutes. The cooling process is carried out by decreasing the stirring speed to 500 rpm using a magnetic stirrer until it reaches room temperature (25^oC).

SLN-PMCA Preparation:

SLN-PMCA system is made by first melting beeswax at 70^oC. After beeswax melts, cacao oleum is added to it and let it melt, then PMCA was added to the beeswax-oleum cacao mixture and it was stirred at 200 rpm for 2 minutes. Tween 80 and Span 80 are heated at 70°C, then added to the oil phase and it stirrer at 200 rpm for 2 minutes. Sodium benzoate, propylene glycol, and acetate buffer pH 4.2 ± 0.02 as the water phase heated at 72°C, then slowly dispersed into the oil phase while stirring with an Ultra-Turax High Shear Homogenizer at 5000 rpm until the entire water phase mixed. Then the stirring speed is increased to 16,000rpm for 3 minutes. In the cooling stage the stirring is carried out using a magnetic stirrer with a speed of 500rpm for 7 minutes, then the speed is increased to 700rpm for 8 minutes until it reaches room temperature (25°C).

Nanoemulsion-PMCA Preparation:

Span 80, tween 80, PMCA dissolved in VCO, sodium benzoate and propylene glycol are mixed one by one until homogeneous in a 50ml beaker glass. Each addition of material is stirred using a magnetic stirrer at 600 rpm each for 5 minutes. Next, add acetate buffer solution pH 4.2±0.2 slowly. Then stir with a magnetic stirrer at 1000rpm for 10 minutes until homogeneous. Then shaker for 30 minutes and form a nanoemulsion system (transparent appearance).

Characteristics measurement of NLC-PMCA, SLN-PMCA and NE-PMCA:

Measurement of the system characteristics includes organoleptic observations, pH value, viscosity, particle size and polydispersity index (PI), as well as zetapotential conducted on NE, SLN and NLC systems, while the melting point and entrapping efficiency are only carried out on SLN and NLC systems.

Release test PMCA from the system:

Release test of PMCA from the system was done by Franz Diffusion cell and cellophane membrane that has been soaked with release media for ± 12 hours. Shortly before use, the membrane is drained until no water drips. Release media using phosphate buffer pH 6.0 ± 0.05 were placed in the receptor compartment, 2 ml of preparation sample was inserted into the donor compartment. The experimental temperature was adjusted and maintained at 37 ± 0.5 °C, stirred at 100 rpm. A number of samples (1 ml) were taken at intervals of 0, 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, 360, 480, 600, 720, and 1440 minutes and immediately replaced with phosphate media buffer pH 6, 0.5 as much as the volume taken (1 ml). Next, the samples were analyzed with a UV-Vis spectrophotometer. PMCA concentrations in the sample were calculated using a standard curve regression equation. To calculate the dilution factor, the correction was carried out at the concentration measured using the Wursters’ equation (2) as follows:

Note:
Cn = true concentration after correction (ppm)

C'n = unreadable concentration (the result of absorbance value calculation sample read on a spectrophotometer) (ppm)

Cs = concentration read from the previous sample

a = volume of sample taken

b = volume of receptor media

Penetration test PMCA trough skin abdomen of rat:

Rat that had met the inclusion criteria were anesthetized with ketamine (20mg/kg BW) intramuscularly. Rat hair on the abdomen is shaved using mechanical hair clippers. Approximately 50mg of the sample containing 0.007% of Nile red, then applied to the skin abdomen of rat, an area of 2x3 cm². After 30 minutes or 2 hours the sample was applied, the residue was cleaned and then the rats were sacrificed by cervical dislocation. Then histological preparations were made using cryotome (Tissue-Tek Cryo3, Sakura) at a temperature of -59ºC with an area of 1x4 cm² and a thickness of 5μm. Histological preparations were then observed with a fluorescent microscope at 42x magnification.

Physical Stability Test:

The stability test of the preparation was carried out by the real time method for 14 days and centrifugation test for 4 cycles, each cycle for 30 minutes.

RESULT AND DISCUTION:

The characterization of systems:

The result of NLC-PMCA, SLN-PMCA and NE-PMCA characterizations can be seen in Table 2, Table 3 and Figure 1. Statistical test results with the one-way ANOVA method (α = 0.05) on the characteristic data in Table 2, it is known that SLN-PMCA has the largest particle size while NE-PMCA has the smallest droplet size. The Polydispersity Index (PI) values of the three samples in the range 0.3 - 0.5 indicate a moderate size distribution. Polydispersity index (PI) <0.5 shows homogeneous particle size distribution and no particle aggregation.¹⁶ The results of the measurement of pH values are known that the three samples have a pH range between 4.2 - 4.5 which is in the range of skin pH values that is between 4 - 6.5, making it safe for topical use. The viscosity value of the system is known that the viscosity of NLC-PMCA is smaller than SLN-PMCA but greater than NE-PMCA. An increased concentration of liquid lipids decreases the system viscosity. In the manufacturing process, a decrease in viscosity is followed by a decrease in the mass of the dispersion phase where the dispersion energy required is smaller so that a small particle size can be obtained.¹⁷The results of the melting temperature testing of NLC-PMCA and SLN-PMCA can be seen in Table 3 and Figure 1 shows that NLC-PMCA has a lower melting temperature than the SLN-PMCA melting temperature, so it is predicted to be easier to release the active ingredients. The result of zetapotential examination is known that NLC-PMCA has the highest zetapotential value. Zetapotential value > 25mV indicates the system is more stable.¹⁸From the potential zeta data, it is known that NLC-PMCA and SLN-PMCA have a stable system, while NE-PMCA has an unstable system. This is due to the presence of steric additives, including surfactant tween 80. Adsorption of steric substances will reduce the potential zeta value and cause coagulation in the system so the system becomes unstable.¹⁹

Figure 1: Thermogram Differential Scanning Calorimeters of PMCA (A), Beeswax (B), Oleum Cacao (C), NLC-PMCA (D) and SLN-PMCA (E).

Table 2: The characteristics of NLC-PMCA, SLN-PMCA and NE-PMCA

System	Particle/droplet size (nm)	Polydispersity Index (PI)	pH	Viscosity (cPs)
NLC-PMCA	423.6 ± 33.6	0.360 ± 0.019	4.43 ± 0.03	30.03 ± 6.29
SLN-PMCA	830.7 ± 71.3	0.412 ± 0.042	4.19 ± 0.01	93.77 ± 6.11
NE-PMCA	57.1 ± 1.6	0.393 ± 0.035	4.44 ± 0.06	3.43 ± 0.16

The data was the average of three replications

PMCA release test results:

PMCA release test results from the NLC-PMCA, SLN-PMCA and NE-PMCA systems can be seen in Figure 2. The results of the release rate calculation and statistical analysis using the one-way ANOVA method obtained the rate of PMCA release from the NLC-PMCA system was 0.2210±0.0089µg/cm²/minute faster than the SLN-PMCA system was 0.1972 ± 0.0145µg/cm²/minute but slower than NE-PMCA was 0.4690±0.0228 µg/cm²/minute. It's can be caused by the viscosity of NLC-PMCA lower than SLN-PMCA and greater than NE-PMCA. The higher viscosity value of the system makes particles movement more difficult so that the release rate of active ingredients smaller.²⁰ Another factor influencing the release of PMCA from the system is the particle size in the system, the small particle size results in a greater release rate.^[21]

Figure 2: Profile of PMCA releases from the NLC-PMCA, SLN-PMCA and NE-PMCA systems

NLC-PMCA, SLN-PMCA and NE-PMCA Penetration Test Results:

The results of PMCA penetration depth testing in the NLC-PMCA, SLN-PMCA and NE-PMCA systems on rat skin can be seen in Figure 3 and Table 4. The results of statistical tests on the depth of PMCA penetration using the one-way ANOVA method (α = 0.05) revealed the depth of PMCA penetration in the NLC-PMCA system is deeper than SLN-PMCA but shallower than NE-PMCA. The rate of drug release can be contributed to high permeability coefficient and affects higher penetration.^22,23,24 Increased drug release results in greater availability of drugs that are ready to pass through the skin barrier, so that the diffusion coefficient of the drug in the stratum corneum becomes greater which results in increased drug penetration. Viscosity is also related to the speed of drug penetration. Preparations with good viscosity can easily spread to the surface of the skin causing increased absorption of the drug into the skin.^25,26

Table 4: The results of PMCA penetration depth tests in the NLC-PMCA, SLN-PMCA and NE-PMCA systems in male Wistar rat skin

System	PMCA Penetration Depth (µm)
System	30 minutes	2 hours
NE-PMCA	141.43 ± 106.4	1832.5 ± 92.8
SLN-PMCA	703.3 ± 117.2	945.6 ± 140.4
NLC-PMCA	1173.0 ± 37.8	1268.8 ± 111.9

Figure 3: Histology photos of PMCA penetration in the NE-PMCA system (A = 30 minutes; A'= 2 hours), SLN-PMCA (B = 30 minutes; B' = 2 hours) and NLC-PMCA (C = 30 minutes; C '= 2 hours) into rat skin.

Stability test results with centrifugation and real time methods:

The results of the stability tests of the NLC-PMCA, SLN-PMCA and NE-PMCA systems with centrifugation and real time methods can be seen in Figure 4,5,6,7 and Table 5.

Figure 4: Photograph of centrifugation test results on the NLC-PMCA system for 4 cycles (one cycle = 30 minutes). Separation occurred since the 3^rd cycle

Based on the centrifugation test, the NLC-PMCA system undergoes separation starting from the third cycle its after centrifugation for 1.5 hours, the SLN-PMCA system undergoes separation starting from cycle 1 its after centrifugation for 30 minutes and the NE-PMCA system undergoes separation starting at the third cycle after centrifugation for 1.5 hours. Based on real time test for 14 days the NLC-PMCA system did not changes in color, odor and consistency (thickness), the SLN-PMCA system did not change color and odor but the consistency became thicker, whereas in NE-PMCA it did not changes in odor and thickness but the color becomes turbid. This can be caused by the potential zeta of the NE-PMCA system is less than 25 mV which results in more easy aggregation and coalesence ^[19]. So it can be concluded that NLC-PMCA is more stable than the other two systems.

Figure 5: Photograph of centrifugation test result on the SLN-PMCA system for 4 cycles (one cycle = 30 minutes). Separation occurred since the 1^st cycle

Figure 6: Photograph of centrifugation test result on the NE-PMCA system for 4 cycles (one cycle = 30 minutes). Turbidity occurred since the 3^rdcycle.

Figure 7: Photograph of real time test results on the NLC-PMCA system (A = day 0; A'= day 14), SLN-PMCA system (B = day 0; B' = day 14) and the system NE-PMCA (C = day 0; C' = day 14).

Table 5: Organoleptic examination results in real time stability tests on the NLC-PMCA, SLN-PMCA, and NE-PMCA systems.

System	Parameter	Day 0	Day 14
NLC- PMCA	Colour	white	white
	Smell	smelled of wax	smelled of wax
	Thickness	viscous	viscous
SLN- PMCA	Colour	white	white
	Smell	smelled of wax	smelled of wax
	Thickness	viscous	more viscous
NE-PMCA	Colour	clear yellowish	yellowy turbid
	Smell	smelled of tween 80	smelled of tween 80
	Thickness	likuid	likuid

CONCLUSION:

NLC-PMCA has better release rate and depth of penetration than SLN-PMCA even though it is lower than NE-PMCA, but NLC-PMCA has better physical stability than SLN-PMCA and NE-PMCA.

ACKNOWLEDGEMENT:

This research was funded by DIKTI, and thanks to our students Bastiana Rizka Rachmawati and Theresia Chandra Jaya Atmadja, under the guidance of the authors who helped carried out this research.

ETHICAL CLEARENCE:

All animal experiments were conducted with the permission from Animal Care and Use Committee (ACUC) of Veterinary Faculty, Airlangga University, Surabaya-Indonesia. (Certificate No; 2.KE.056.04.2019).

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

1. Vidyasagar Patidar, et al. Penetration Enhancement Techniques for Transdermal Drug Delivery System. Research J. Pharm. and Tech. 2(1): Jan.-Mar. 2009; Page 28-33.

2. Vaseeha Banu T.S., Sandhya K.V., K.N. Jayaveera. Approaches and Current Trends of Transdermal Drug Delivery System- A Review. Research J. Pharma. Dosage Forms and Tech. 2013; 5(4): 177-190.

3. Sweta S, et al. Transdermal Drug Delivery Systems: A Review. Research J. Pharm. and Tech. 5(6): June 2012; Page 757-763.

4. Wei He , et al. Nanoemulsion-Templated Shell-Crosslinked Nanocapsules as Drug Delivery Systems. International Journal of Pharmaceutics, 2013; 445(1-2): 69-78.

5. Saikumar Y, et al. A. Role of Penetration Enhancers in Transdermal Drug Delivery System. Research J. Pharma. Dosage Forms and Tech. 2012; 4(6): 300-308

6. Rakesh K Sindhu, Mansi Chitkara, Gagandeep Kaur, Preeti Jaiswal, Ashutosh Kalra, Inderbir Singh, Pornsak Sriamornsak. Skin Penetration Enhancer’s in Transdermal Drug Delivery Systems. Research J. Pharm. and Tech. 2017; 10(6): 1809-1815. doi: 10.5958/0974-360X.2017.00319.5

7. Pande SD, et al. Effect of Some Penetration Enhancers on In-vitro Permeation of Glibenclamide. Research J. Pharm. and Tech. 2010; 3(1); Page 79-81.

8. Bahari LAS and Hamishehkar H. The Impact of Variables on Particle Size of Solid Lipid Nanoparticles and Nanostructured Lipid Carriers; A Comparative Literature Review. Adv. Pharm. Bull. 2016; 6(2):143–151. doi:10.15171/apb.2016.021

9. Erawati T, Hendradi E, Soeratri W. Praformulation study of p-methoxycinnamic acid (PMCA) nanoemulsion using vegetable oils (soybean oil, corn oil, VCO). Int. J Pharm. Pharm. Sci. 2014; 6(2): 99-101

10. Tamidji, F., Shahedi, M, Varshosaz, J. and Nasirpour, A. Nanostructured lipid carriers (NLC): a potential delivery system for bioactive food molecules, Innov. Food Sci. Emerg, Tecnology. 2013; 19: 29-43

11. Bornare AS, Saudagar RB. Nanostructured Lipid Carrier (NLC): A Modern Approach for Transdermal Drug Delivery. Research J. Pharm. and Tech. 2017; 10(8): 2784-2792. doi: 10.5958/0974-360X.2017.00493.0

12. Ribeiro, L. N. M., Breitkreitz, M. C., Guilherme, V. A., Gustavo, H. R., Couto, V. M., Castro, S.R., Paula, E. De. Natural lipids-based NLC containing lidocaine: from pre-formulation to in vivo studies. European Journal of Pharmaceutical Sciences. 2017; 106:102–112.

13. Erawati, T. et al. Characteristics and Stability of Nanostructured Lipid Carrier (NLC) Aleurites Moluccana Seed Oil (AMS oil) Using Various Combinations of Beeswax and Oleum Cacao. International Journal of Drug Delivery Technology. 2019; 9(1): 94–97.

14. Tristiana Erawati M., Widji Soeratri, Noorma Rosita. Skin Penetration of Ubiquinone (Co-Q10) In Nanoemulsion Delivery System Using Virgin Coconut Oil (Vco), International Journal of Drug Delivery Technology. 2018; 8(2): 77-79

15. Tristiana Erawati, et al. The Anti-inflammatory Activity of p-methoxycinnamic acid (PMCA) in the Nanostructured lipid carrier (NLC) system using combinations of solid lipid, beeswax-oleum cacao and liquid lipid, Virgin Coconut oil (VCO). Research J. Pharm. and Tech. 2019, 12(8): 3619-3625. DOI: 10.5958/0974-360X.2019.00617.6

16. Wei Keat Ng, et al. Thymoquinone-loaded Nanostructured Lipid Carrier exhibited cytotoxicity towards breast cancer cell lines (MDA-MB-231 and MCF-7) and cervical cancer cell lines (HeLa and SiHa). BioMed Research Internatuinal. 2015. Article ID 263131 | 10 pages |. https://doi.org/10.1155/2015/263131

17. Loo, C.H., Basri, M., Ismail, R., Lau, H.L.N., Tejo, B.A., Kanthimathi, M.S., Hassan, H.A., and Choo, Y.M. Effect of compositions in nanostructured lipid carriers (NLC) on skin hydration and occlusion. International Journal of Nanomedicine. 2013; 8: 13–22.

18. Uner, M. Preparation, Characterization and Physico-Chemical Properties of Solid Lipid Nanoparticles (SLN) and Nanostructured Lipid Carriers (NLC): Their Benefits as Colloidal Drug Carrier Systems. Pharmazie. 2006; 6: 375–386.

19. Gohla, S., Ma, K. and Mu, R. H. Solid Lipid Nanoparticles (SLN) for Controlled Drug Delivery: A Review of the State of the Art, European Journal of Pharmaceutics and Biopharmaceutics. 2000; 50: 161-177.

20. Sinko, P.J., & Singh, Y. Martin's Physical Pharmacy and Pharmaceutical Science-Physical Chemical and Biopharmaceutical Principle in The Pharmaceutical Science 6th Edition. Philadelphia: Lippincott Williams & Wilkins, a Wolters Kluwer business. 2011.

21. Muller, R. and Wissing S. Solid lipid nanoparticles as carrier for suncreens: in vitro release and in vivo skin penetration. Journal of controlled release. 2002; 81: 225-233.

22. Umekar MJ, et al. Formulation Development and Evaluation of Transdermal Drug Delivery System of Antihypertensive Drug. Research J. Pharm. and Tech. 2010; 3 (3): 753-757.

23. Shikha BC. Comparative Analysis of Penetration Enhancer on Transdermal Drug Delivery of Antifertility Drugs. Research J. Pharm. and Tech 2020; 13(3): 1457-1462. doi: 10.5958/0974-360X.2020.00266.8

24. Farsiya F, et al. Formulation and Evaluation of Matrix-Type Transdermal Delivery System of Ondansetron Hydrochloride Using Solvent Casting Technique. Research J. Pharm. and Tech. 2011; 4(5); 806-814.

25. Shinde, G., Rajesh, K.S., Prajapati, N., Murthy, R.S.R. Formulation, development and characterization of Nanostructured Lipi Carrier (NLC) loaded gel for psoriasis. Scholar Research Library. 2013; 13-25.

26. Trommer, H., and Neubert, R.H.H. Overcoming the Stratum Corneum the Modulation of Skin Penetration. Skin Pharmacology and Physiology. 2006; 19: 106-121.

Received on 07.09.2020 Modified on 21.11.2020

Research J. Pharm. and Tech 2021; 14(11):5719-5724.

DOI: 10.52711/0974-360X.2021.00994