INTRODUCTION:

X-ray radiation emitted during a panoramic radiographic examination induced genotoxic effects in epithelial gingival cells that increased chromosomal damage and induced apoptosis¹. This cell damage prevented by inhibiting the oxidation process using antioxidant compounds². One of the antioxidant compounds was beta carotene, a group of carotenoid compounds which has 2 beta (b) rings and has the main function as pro-vitamin A with the highest activity³. Mucoadhesive gingiva patches containing 1.94±0.43mg/4cm² of beta carotene has been shown to prevent the side effects of panoramic radiographic radiation exposure in test animals.

However, the application of patch preparations in humans showed insignificant reduced the micronucleus as the negative effects^4,5. This is probably due to the solubility of beta carotene which is very low, thus limiting the amount of beta carotene that dispersed in the patch preparation.

Based on the shortcomings of this research, it was necessary to develop a beta carotene formulation by forming it into nanoemulsion using Rice Bran Oil (RBO), Tween 80, and PEG 400. This mixture was hereinafter referred called SNEDDS. Self-Nanoemulsifying Drug Delivery System (SNEDDS) will form nanoemulsion with water contained in the mucoadhesive patch matrix. This formulation expected to increase the amount of beta carotene dispersed in the mucoadhesive patch matrix, so the diffused beta carotene will be higher and have an anti-radiation effect during the panoramic radiographic process.

MATERIAL AND METHODS:

Chemicals and reagents:

Materials used in this study include beta carotene powder (Chem Leader Biomedical), RBO (Nature Farm), Tween 80 (PT. Brataco), PEG 400 (PT. Brataco), distilled water, acetone pro analysis (E. Merck), HPMC-E15 (Wuhan Senwayer Century Chemical Co., Ltd.), CMC Na (PT. Brataco), PVA (PT. Brataco), ethyl cellulose (PT. Brataco), membranes cellophane.

FORMULATION MUCOADHESIVE PATCH OF BETA CAROTENE NANOEMULSION:

Preparation of beta carotene SNEDDS:

SNEDDS is a mixture of RBO, Tween 80, and PEG 400 with a composition of 8%: 85%: 7% and 2,82mg beta carotene added per gram of SNEDDS. Beta carotene was dissolved in RBO and carried out 1 mixing cycle (vortex 10 minutes, incubation at 37^oC 10 minutes and sonication 10 minutes). Tween 80 and PEG 400 were added to the mixture and mixed in 3 cycles.

Beta carotene nanoemulsion droplet size:

Nanoemulsion droplet size measured using a Malvern Particle Size Analyzer (PSA) tool. Up to 50mL SNEDDS of beta carotene was added with water up to 50mL then homogeneous and taken 1mL to be put into a cuvette and analyzed using PSA.

Preparation of mucoadhesive patches:

Mucoadhesive patch preparations was made using a solvent casting technique, which is a mixture of ingredients poured into a mold and after drying cut according to the required size⁶. The basic mucoadhesive patch formula used modification of the salbutamol sulfate mucoadhesive patch formula with increasing HPMC concentrations and drug replacement with SNEDDS of beta carotene⁷. The composition of the mucoadhesive patches of beta carotene nanoemulsion shown in Table 1.

Table 1: Composition of mucoadhesive patches of beta carotene nanoemulsion

Materials	Amount
HPMC E-15 8%	15 mL
PVA 2%	10 mL
CMC Na 1%	5 mL
PEG 400	2 mL
SNEDDS	1 gram

HPMC and CMC polymers were developed with water respectively. PVA added to water and heated until dissolved. All polymers were mixed and added PEG 400 as plasticizers, stirred to obtain a homogeneous patch base. SNEDDS added to the patch base little by little while stirring. Then sonicated during 30 minutes and leave it overnight. Printing is done by pouring 3grams of the mixture into a mold with a diameter of 5cm that has been coated with an ethyl cellulose backing layer. Then leave for ± 4 days until the patch could be removed from the mold.

THE PHYSICAL CHARACTERISTICS OF A MUCOADHESIVE PATCH:

Patch weight and thickness:

This test is carried out by weighing 3 preparations with a size of 4cm² using digital balance followed by measuring the thickness of the patch using a digital caliper⁸. The results obtained are calculated average and standard deviation of 3 replications.

Patch folding endurance:

Testing is done by folding the patches repeatedly in the same position until the patches was broken. The number of folds obtained was the value of the patch resistance to bending⁹.

Swelling index (% S) and patch surface pH:

A total of 3 samples were left swelling on the agar surface for 1.5%, then stored in an incubator whose temperature was maintained at 37^o±0.20^oC. Increased patch weight was determined at 10, 20, 30, 40, 50, and 60 minutes. Swelling index (% S), calculated using equation 1.

Xt - Xo

% S= ----------------- X 100 ………………….(1)

Where, Xt was the weight of the patch after swelling at time t minutes and Xo was the weight of the patch at zero minutes¹⁰. Swelling index calculation was done 3 times replication.

Determination of beta carotene content in the patch:

Determination of the content was carried out on a patch with a size of 4cm² dissolved in 10.0mL acetone pro analysis. The solution then stirred continuously for 24 hours. Absorbed beta carotene was measured by UV-Visible spectrophotometer at a wavelength of 454nm. The average measurement results on the 3 patches are used as a measure of the content of beta carotene in the preparation.

Mucoadhesive time of patch ex vivo:

Tests were carried out using rabbit palatal mucosa. The patch placed on the rabbit palatum membrane of the epithelium and glued to the glass object and then immersed in 200mL phosphate buffer solution pH 6.8. Stirring with a speed of 50rpm to simulate the situation in the oral cavity. Mucoadhesive time calculated from the patch preparation installed until released completely⁴.

Patch released test in vivo:

This test used Franz diffusion cells with cellophane membranes as barrier membranes and phosphate buffer pH 6.8 as acceptors¹¹. Patch preparations are cut in a circle with a diameter of 2.50cm and swelling on the 1.5% agar surface for 1 minute before being installed on the top of the cellophane membrane. The test carried out by continuous stirring and the temperature conditioned at 37^oC. Sampling carried out for up to 8 hours with a certain time interval. Beta carotene levels contained in the sample solution were measured with a UV-Visible spectrophotometer at a wavelength of 453nm.

RESULT AND DISCUSSION:

Nanoemulsion droplet size:

This test was an important factor in SNEDDS formulations because the droplet size affects the rate of drug released and increased the bioavailability of the drug^12,13. Based on the test results obtained beta carotene nanoemulsion with a droplet size of 11.05±2.03nm. The droplet size was less than 200nm, so its belongs to the nanoemulsion range (10-200nm)¹⁴. A result of nanoemulsion droplet measurements was shown in Figure 1.

Fig. 1: Beta Carotene Nanoemulsion Droplet Size

The physical characteristics of a mucoadhesive patch:

The resulting patch has a flexible texture, yellow color, non-oily surface, and the backing layer stuck to the patch matrix. Visual patch results are shown in Figure 2.

Fig. 2: Visual of Mucoadhesive Patch of Beta Carotene Nanoemulsion

The results of testing of 3 pieces of 4cm² patch produced a patch with a weight of 91.0±0.2mg and a thickness of 0.18±0.01mm. These results indicated that the shrinkage of the patch formula starting from pouring into the mold to drying tends to be homogeneous, so that the weight and thickness of the patch was relatively the same.

All patches have good folding endurance and good elasticity, because it has a folding resistance of more than 300 times⁸. This ability is caused by the presence of plasticizers in the patch formula, where in this formula PEG 400 is used which is able to increase the permeability of the patch by increasing the wetting process or decreasing the crystallinity of the polymer, so the patch more elastic, flexible and not easily damaged even though folded repeatedly¹⁵.

The beta carotene nanoemulsion patch swelling index is listed in Figure 3. The mucoadhesive patch expands maximally within 10 minutes, reaching 120.07±5.28%, but subsequently the patch has decreased swelling index. In the first 10 minutes the water absorption process occurs very quickly by a hydrophilic polymer. The more water flowing between the chains, the strength between the polymer chains would decreased and the water would filled the void between the polymer chains, so that the patch would expand and eventually erosion would occurred from the polymer layer which marked by decreasing the weight of the patch¹⁶. Swelling values that are too high can cause the release of active substances to be faster and uncontrolled, the patch is more easily removed and cause discomfort to the patient¹⁰.

Fig. 3: Curve Swelling Index of Patch with Time (data is presented in mean ± SD (n=3)

This mucoadhesive patch of beta carotene nanoemulsion would later be applied to the gingival mucosa, so the surface pH of the patch must be within the pH range of salivary fluid (5.8-7.1) to avoid irritation¹⁷. pH test results on 3 patch surface after swelling have a pH of 6, so it has no potential to irritate the gingival mucosa.

Determination of beta carotene content in the patch:

In previous studied, beta carotene contained in mucoadhesive patches amounted to 1.94±0.43mg/4 cm^{2 4}, whereas in beta carotene which was formulated first into nanoemulsion, the beta carotene content in the patch becomes 14.39±0.52mg/4 cm². These results indicated an increase in the amount of beta carotene that dispersed in the mucoadhesive patch matrix. This happened because of the presence of RBO which is a beta carotene carrier component and maintained its solubility¹⁸.

Mucoadhesive testing of time patch ex vivo:

The purpose of determining a mucoadhesive time was to determine the adhesion ability of mucoadhesive patches to the mucosal surface. The patch adhesion ability could be cause by the use of HPMC polymers which are able to form gels that strongly attached to the mucosal surface for a longer time. However, based on the results obtained, the patch was able to remain attached to the mucosa for only 44.64±2.24 minutes. It was possible because of the hydrophilic polymers used in patch formulas that have high solubility in water. These properties cause hydrophilic polymers hydrated to form a gel that is easily degraded and dissolved to produce slippery slime, so that the adhesive properties in the patch would be lost¹⁹.

Patch diffusion test in vivo:

The relationship between percent beta carotene released from mucoadhesive patch preparation to time is shown in Figure 4.

Fig. 4: Beta Carotene Released Profile in Mucoadhesive Patch (Data is Presented in Mean ± SD (n=3))

The beta carotene released profile from the mucoadhesive patch matrix (Figure 4) shows the controlled beta carotene release pattern from the mucoadhesive patch matrix, and shows the drug release percentage of 19.7±1.74% within 8 hours. Controlled release associated with swelling and degradation of the polymer in the formula.

Data obtained from the beta carotene diffusion study from the mucoadhesive patch of nanoemulsion were further analyzed with different kinetic models (zero order, first order, Higuchi, and Korsmeyer-Peppas). Based on the results of the analysis obtained the Korsmeyer-Peppas model was found to be the most suitable to describe the kinetics of beta carotene release²⁰. Korsmeyer-Peppas was a mathematical model that was describes the profile of drug release from a polymer system. Previous research also reported that the Korsmeyer-Peppas model is the most suitable model to describe drug release from a polymer system^7,21. The mathematical model in the beta carotene release kinetics was shown in Table 2.

Table 2: Calculation Result of the Parameters of the Beta Carotene Release Model from the Mucoadhesive Patch

Drug release model	Parameter	Result
Zero order	K₀	0.0550 ± 0.005
	R² adj	-1.1962 ± 0.164
	AIC	132.4007 ± 3.895
	MSC	-1.4993 ± 0.099
First Order	K₁	0.0006 ± 0.000
	R² adj	-1.0035 ± 0.163
	AIC	130.6489 ± 3.694
	MSC	-1.4071 ± 0.108
Higuchi	K_H	1.0667 ± 0.105
	R² adj	0.3552 ± 0.083
	AIC	109.0468 ± 4.132
	MSC	-0.2702 ± 0.151
Korsmeyer-Peppas	K_K-P	5.6046 ± 0.540
	R² adj	0.9041 ± 0.021
	AIC	73.5823 ± 5.971
	MSC	1.5964 ± 0.225
	n	0.1998 ± 0.011

Note: Data is Presented in Mean ± SD (n=3)

K value is a drug release constant. R² adjusted states the relationship between data obtained from beta carotene diffusion studied conducted with the predicted model. The criteria used to select a model that matches the drug release kinetics was to compare model values, i.e. models that have a correlation coefficient (R² adjusted) that is getting closer to 1, lower AIC value (Akaike Information Criterion), and MSC values greater than two, then the model was stated as suitable to describe the release kinetics²². Based on the results of calculations in Table 2, it could be seen that the Korsmeyer-Peppas model was a model that described the kinetic release of beta carotene from the nanoemulsion mucoadhesive patch, because it has an R² value of (0.9041±0.021), the lowest AIC value (73.5823±5.971), and the largest MSC value (1.5964±0.225). In addition, the results of curve fitting show the closest result between observational data and prediction data (Figure 5).

The mechanism of releasing beta carotene from mucoadhesive patch preparations could be seen based on the diffusion exponent value expressed by the value of n. The Fickian mechanism release, indicated by the value of n < 0.5, while the non-Fickian release mechanism shown with 0.5 <n <1²⁰. Based on the resulted of the analysis (Table 2), the resulting n value of 0.1998±0.011 indicated that the released of beta carotene from the mucoadhesive patch followed the Fickian release mechanism.

Fig. 5: The results of the Korsmeyer-Peppas curve fitting model (data is presented in mean ± SD (n=3)

CONCLUSION:

SNEDDS formulations increased the amount of beta carotene that dispersed into the mucoadhesive patch matrix of beta carotene nanoemulsion up to 14.39±0.52 mg/4 cm² and released beta carotene across the cellophane membrane by 19.7±1.74% for 8 hours following the Korsmeyer-Peppas kinetics and the Fickian release mechanism.

ACKNOWLEDGEMENT:

Author thanks to The Ministry of Research, Technology, and Higher Education of the Republic of Indonesia 2018^th for its grant so that this research can be done.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

1. Cerqueira EMM, Meireles J, Lopes M, Rocha V, Gomes-Filho I, Trindade S, et al. Genotoxic Effects of X-rays on Keratinized Mucosa Cells During Panoramic Dental Radiography. Dento maxillo facial radiology. 2008; 37:398–403.

2. Yadav A, Kumari R, Yadav A, Mishra JP, Srivatva S, Prabha S. Antioxidants and Its Functions in Human Body - A Review. Research in Environment and Life Sciences. 2016;1328–31.

3. Gupta A, Dixit A, Sinha A, Mittal K. Extraction of Beta Carotene from Selected Dried and Fresh Samples of Vegetables. Asian Journal of Research in Chemistry. 2013 Feb 28;6(2):169–71.

4. Shantiningsih RR, Suwaldi, Mudjosemedi M, Astuti I. Formulasi Sediaan Patch Gingiva Mukoadesif Betacaroten untuk Radioprotektor Radiografi Panoramik. In: Proceedings Gadjah Mada Dentistry Scientific Conference The Comprehensive Approach For Excellent Dental Practice In Universal Health Coverage Era. Yogyakarta: Gadjah Mada; 2015. p. 64–9.

5. Shantiningsih RR, Diba SF. Efek Aplikasi Patch Gingiva Mukoadesif β-Carotene Akibat Paparan Radiografi Panoramik. Majalah Kedokteran Gigi Indonesia. 2015;1(2):186–92.

6. Tarun P, Kumar SV, Kumar TA. Drug Delivery Via the Buccal Patch – A Novel Approach. Research Journal of Pharmaceutical Dosage Forms and Technology. 2013 Jun 28;5(3):122–30.

7. Puratchikody A, Prasanth VV, Mathew ST, Kumar BA. Development and characterization of mucoadhesive patches of salbutamol sulfate for unidirectional buccal drug delivery. Acta Pharm. 2011 Jun;61(2):157–70.

8. Begum A, Sravya, Deepika, Manasa N, Uma, Tajeswari L, et al. Formulation and Evaluation of Fexofenadine Buccal Mucoadhesive Patches. Research Journal of Pharmacy and Technology. 2018;11(11):4892–8.

9. Bhilegaonkar SP, Volvoikar SG, Naik AG. Formulation and Evaluation of Bilayer Gastric Mucoadhesive Patch of Captopril. Research Journal of Pharmacy and Technology. 2018 Jun 30;11(6):2444–53.

10. Swati J, Asawaree H, Bhanudas K, Aniruddha C. Development and Evaluation of Mucoadhesive Buccal Patches of Nifedipine. Research Journal of Pharmacy and Technology. 2011;4(6):944–8.

11. Samanthula KS, Satla SR, Bairi AG. Development, In-Vitro and Ex-Vivo Evaluation of Muco-adhesive Buccal patches of Candesartan cilexetil. Research Journal of Pharmacy and Technology. 2019 Jun 30;12(6):3038–44.

12. Ahmed AA, Dash S. Application of Novel Nanoemulsion in Drug Targeting. Research Journal of Pharmacy and Technology. 2017 Aug 28;10 (8):2809–18.

13. Pagar KR, Darekar AB. Nanoemulsion: A new concept of Delivery System. Asian Journal of Research in Pharmaceutical Sciences. 2019 Mar 31;9(1):39–46.

14. Alam S, Sharma P. Stability study of clobetasol propionate loaded tea tree oil nanoemulsion as per ICH guidelines. Intern Jour Contemp Microbiol. 2016;9(11):1999–2004.

15. Namrata P, S.b G, R.b S. Formulation and Evaluation of Mucoadhesive Buccal Patch of Saxagliptin Hydrochloride. Research Journal of Pharmaceutical Dosage Forms and Technology. 2016 Dec 27;8(4):237–47.

16. Semalty M, Semalty A, Kumar G. Formulation and Characterization of Mucoadhesive Buccal Films of Glipizide. Indian J Pharm Sci. 2008;70(1):43–8.

17. Pendekal MS, Tegginamat P. Formulation and Evaluation of A Bioadhesive Patch for Buccal Delivery of Tizanidine. Acta Pharmaceutica Sinica B. 2012 Jun 1;2: 318–324.

18. Saudagar RB, Vaishnav S. Pharmaceutical Nano emulsion as a Rational Carrier for Drug Delivery. Research Journal of Pharmacy and Technology. 2016 Mar 28;9(3):298–304.

19. Hashemi M, Ramezani V, Seyedabadi M, Ranjbar AM, Jafari H, Honarvar M, et al. Formulation and Optimization of Oral Mucoadhesive Patches of Myrtus Communis by Box Behnken Design. Adv Pharm Bull. 2017 Sep;7(3):441–50.

20. Dash S, Murthy PN, Nath L, Chowdhury P. Kinetic Modeling on Drug Release from Controlled Drug Delivery Systems. Acta Pol Pharm. 2010 Jun;67(3):217–23.

21. Ikram M, Gilhotra N, Gilhotra RM. Formulation and Optimization of Mucoadhesive Buccal Patches of Losartan Potassium by Using Response Surface Methodology. Adv Biomed Res. 2015 Oct 29; 4:239.

22. Zhang Y, Huo M, Zhou J, Zou A, Li W, Yao C, et al. DDSolver: An Add-In Program for Modeling and Comparison of Drug Dissolution Profiles. AAPS J. 2010 Apr 6;12(3):263–71.

Received on 13.12.2019 Modified on 11.02.2020

Research J. Pharm. and Tech. 2020; 13(8):3849-3853.

DOI: 10.5958/0974-360X.2020.00681.2