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The kinetic study of isotretinoin release from nanoemulsion
Accepted Manuscript Title: The kinetic study of isotretinoin release from nanoemulsion Author: Małgorzata Miastkowska Ph.D. El˙zbieta Sikora Jan Ogonowski Michał Zielina Agnieszka Łudzik PII: DOI: Reference:
S0927-7757(16)30581-7 http://dx.doi.org/doi:10.1016/j.colsurfa.2016.07.060 COLSUA 20848
To appear in:
Colloids and Surfaces A: Physicochem. Eng. Aspects
Received date: Revised date: Accepted date:
29-2-2016 1-7-2016 21-7-2016
Please cite this article as: {http://dx.doi.org/ This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The kinetic study of isotretinoin release from nanoemulsion Małgorzata Miastkowska1*[email protected], Ogonowski1, Michał Zielina2 , Agnieszka Łudzik1
Elżbieta
Sikora1,
Jan
1Faculty
of Chemical Engineering and Technology, Institute of Organic Chemistry and Technology, Cracow University of Technology, Warszawska St. 24, 31-155 Cracow, Poland 2Faculty
of Environmental Engineering, Institute of Water Supply and Environmental Protection, Cracow University of Technology, Warszawska St. 24, 31-155 Cracow, Poland
*Corresponding author
1
Highlights
Isotretinoin-loaded nanoemulsions based on coconut oil were obtained.
Isotretinoin was incorporated into nanoemulsion which showed kinetic stability at 25ºC.
Nanoemulsions, based on coconut oil, are good carriers for controlled release of isotretinoin for topical application.
Zero order kinetic model the best fitted the profile of isotretinoin release form nanoemulsion.
2
Abstract
Acne is an inflammatory dermatosis caused by excessive production of sebum, abnormal follicular keratinization and Propionibacterium acnes. Isotretinoin is a retinoid, one of known topical delivered drugs, used in acne treatment because it normalizes the keratinization process. For greatest efficacy in dermatosis treatment, vehicles which enhance delivery of the active to the skin without reaching high serum concentrations and not causing skin irritation are still requested. The aim of this work was to develop a nanoemulsion formulation as a carrier for topical delivery of isotretinoin and determine the release kinetic of the active from the nanoemulsion. O/W isotretinoin-loaded nanoemulsions (0.05 % wt.) based on coconut oil and stabilized by a Polysorbate 80 was obtained and studied as isotretinon delivery systems. The nanoemulsions were prepared by stepwise addition of water to the mixture of polysorbate/oil/the retinoid, at room temperature. The mean droplet size of the nanoemulsions measured by Dynamic Light Scattering (DLS) was around 21 nm. The drug release study of the active was carried out using the Spectra/Por Standard Regenerated Cellulose (RC) membrane, at the temperature T=32oC. The concentration of isotretinoin in the receptor solution was analyzed by UV-Vis spectroscopy method. The release was found to follow the zero order kinetic model. The obtained nanoemulsion effect on isotretinoin release therefore can be used as a vehicle for controlled drug release which resulting in prolonged dermatological action and reduce side effects. Key words: isotretinoin, nanoemulsions, kinetic model
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1. Introduction Dermatosis is defined as a disorder involving lesions or eruptions of the skin that are acute (lasting days to weeks) or chronic (lasting months to years). The inflammation of the skin happens as a result of allergy or irritation of the skin, and is caused by the release of various substances that are important in the immune system [1]. One of the most common skin dermatosis is acne vulgaris. Acne is an inflammatory caused by excessive production of sebum, abnormal follicular keratinization, colonization by a Gram-positive bacterium (i.e., Propionibacterium acnes) and local inflammation [2]. Retinoids are the main topical drugs (applied directly to the skin) used to relieve an inflammation (swelling, itching, and redness) of the skin. They are the foundation for the treatment of acne because they normalize the skin keratinization process. Moreover these actives increase the turnover of follicular epithelial cells, whereby corneocytes are shed at an accelerated rate and comedones are extruded [3]. Isotretinoin is one of the Vitamin A derivatives which exhibits several activities, in particular a capacity to decrease sebaceous gland activity, to correct the keratinisation defect in acne and to reduce the population of the bacterium Propionibacterium acnes [4]. For greatest efficacy in dermatosis treatment, a vehicle should deliver medication in an active, stable form. The methods which enhance skin permeation of the topical drugs without reaching high serum concentrations and skin irritation are still requested. In literature there are some papers describing results of isotretinoin release from nanocarriers. All of the studies confirmed that nanosystems enhanced the bioavailability of isotretinoin. Raza and co-workers [2] investigated the release of isotretinoin from NLC (Nanostructured Lipid Carriers), SLN (Solid Lipid Nanoparticles) and commercial cream. It was found that the drug was released from nanocarriers much more effective (SLN – 84.81%, NLC – 75.21%) than from the market product (43.88 %). They supposed that it can be connected with higher biocompatibility of nanoformulation components and better interaction with the skin. The receptor medium was isotonic Palitzsch Buffer containing (Tween 80 2.7 % w/w) and ethanol (20.0 % v/v) Liu et. al. [5] obtained the isotretinoin-loaded solid lipid nanoparticles (SLN) for topical delivery. The concentration of isotretinoin in formulations amounted 0.06% . Samples consist of PRECIROL ATO5 as the lipid phase. Tween 80 and lecithin act as emulsifier. High-pressure homogenization was the method of SLN preparation. It was found that SLN formulation significantly increased the cumulative amount of isotretinoin in the skin and can act as a promising carrier for this drug. Kuar and co-workers [6] prepared the encapsulated complex of isotretinoinhydroxypropyl β cyclodextrin in elastic liposomes and studied the effect of dual carrier approach on skin targeting of isotretinoin. It was found that encapsulation of isotretinoin in elastic liposomes increased its photostability, skin targeting, and decrease its skin irritation. Chavda et al. [7] obtained self-nanoemulsifying drug delivery systems composed of Transcutol P (diethylene glycol monoethyl ether ) as the solvent and solubilizer for poorly water soluble active, Tween 80 as surfactant and PEG400 as co-surfactant. The in vitro release studies revealed that nanoformulations had higher drug release profile than the isotretinoin powder. Ex vivo permeability studies confirmed that SNEDDS (SelfNanoemulsifying Drug Delivery Systems) improved the skin permeation of isotretinoin (78.89 % after 6 h) compared to drug suspension (41.27% during 6 h). There is no information in the literature concerning the study of isotretinoin release from nanoemulsion as a carrier. Nanoemulsions are one of the most promising formulations 4
used to enhance pharmaceutical bioavailability of active substances such as ibuprofen, ramipril, aspirin, aceclofenac, and many others. They are very low-viscous, kinetically stable, colloidal dispersions with the droplet size in the range of 20-200 nm. Due to high solubilization and capacity to incorporate the active substances, nanoemulsions are the effective transport systems which enhance the actives bioavailability [8]. The aim of this work was to develop a nanoemulsion formulation based on coconut oil as a carrier for isotretinoin and determine the release kinetic of the topical agent from obtained vehicle.
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2.Materials and methods 2.1.Materials The nanoemulsions based on fractionated coconut oil (Brenntag), wheat germ oil (Brenntag) and isopropyl myristate (Sigma-Aldrich) as the oil phase were obtained. Polysorbate 80 was purchased from Caesar&Lorentz GmbH. Deionized by Milli-Q® filtration water was used as the aqueous phase of the emulsions. As the topical agent an isotretinoin (Fluka Analytical) was used. The physical properties of isotretinoin are shown at the table 1.
2.2.Nanoemulsion formation Nanoemulsions were prepared using phase inversion composition (PIC) method by stepwise water addition to the mixture of the oil and surfactant, at room temperature (25oC). The ratios of surfactant : oil (S:O) were varied in following compositions: 90:10, 80:20, 70:30, 60:40 and 50:50, and pseudo-ternary phase diagram was constructed. Compositions of mixtures that formed clear and isotropic systems were determined visually. 2.3.Incorporation topical agents into nanoemulsions Because of poor solubility of isotretinoin in water its solubility in the surfactant:oil (S:O) mixture, at ratio 80:20, was prior determined. During the preparation of topical agent-loaded O/W nanoemulsion, the active substance was incorporated to the mixture of surfactant and oil and then homogenized by the use of vortex aparature. The water phase was added, drop by drop, until final formulation was formed. The concentration of isotretinoin with respect to total formulation reached 0.05% w/w. 2.4.Mean droplet diameter and polydispersity index determination Mean droplet diameter and polydispersity index were measured using Dynamic Light Scattering (DLS) method (Zetasizer Nano ZS, Malvern Instruments, Malvern, UK), at 250C. The scattering angle was 1730. The analysis was performed three times to determine mean values and standard deviation. The measurement of particle size based on scattering photons from a sample and determining the change in diffracted light intensity. 2.5.Release studies Drug release study of isotretinoin was carried out using the Spectra/Por Standard Regenerated Cellulose (RC) membrane, at the temperature T=32oC. For release experiment, about 5 g of formulation was filled in a dialysis bag and placed in thermostatic chamber with receptor solution, for 24 h. The released concentration of isotretinoin in the receptor solution was analyzed by means of UV-Vis spectroscopy (Machery-Nagel Company), at the wavelength of 350 nm, on the basis of previously prepared calibration curve. According to Kuar et al. [6], for poorly water soluble substances in the release study, some amount of surfactants were added to increase the actives solubility in receptor solution The receptor medium was composed of 1.5 % w/w Polysorabte 80 solution in phosphate buffer (pH = 7,4). 2.6. Evaluation of release kinetic.
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The release kinetics was determined by linear regression analysis of the in vitro release curves in four models: zero order (cumulative amount (%) of drug released with time), first order (log cumulative amount (%) of drug released with time), Higuchi (cumulative amount (%) of drug released with the square root of time) and KorsemeyerPeppas (log cumulative amount (%) of drug released with log time). The mathematical model that best expressed the kinetic release profile was selected based on the highest coefficient of determination (R²). 3. Results and discussion 3.1 Formulation of nanoemulsions According to low solubility of isotretinoin in natural oils [7], the mixture of Polysorbate 80: oil (coconut oil, wheat germ oil or isopropyl myristate ) combined in the 8:2 weight ratio was used as a solvent. For determining the solubility of isotretinoin in surfactant: oil mixtures, 10 mg of isotretinoin was added to a vial containing 1 ml of tested solvent. The mixture solutions were vortexed and kept at 24°C overnight. The obtained results revealed that desirable amount of isotretinoin to obtain formulation with 0.05% drug concentration [10] was dissolved only in coconut oil and isopropyl myristate. As a consequence, in the next stage of the experiment, O/W nanoemulsions based on coconut oil or isopropyl myristate were obtained. The ternary phase diagrams of the systems Water/ Polysorbate 80/ Coconut oil (fig. 1 A) and Water/Polysorbate 80/Isopropyl Myristate (fig. 1 B), at 25 ºC were constructed to determine the area of stable nanoemulsions occurrence. The obtained results showed that coconut oil-based nanoemulsions were formed at S/O ratios comprised between 90/10 and 30/70, at minimum water concentration above 60% wt. In case of isopropyl myristate-based system the nanoemulsions were obtained only at 90/10 surfactant/oil weight ratio and minimum 60% of water phase. 3.2. Characterization of nanoemulsions The stable nanoemulsions, with maximum oil concentration in the system were analyzed. The results of the nanoemulsion mean droplet size (±SD) and polydispersity index (PDI), characterized by DLS method, are shown in table 2.
The results presented at the table 2 show that in the case of coconut oil-based nanoemulsions (NE-OK-8-2-80) and NE-OK-8-2-90) increasing water concentration reduces the droplet size and polydispersity of the system. While in the case of nanoemulsions containing isopropyl myristate (NE-MI-9-1-80 and NE-MI-9-1-90) there were no significant differences in droplet size distribution after water dilution. However the polydispersity index increased slightly with increasing water content in the system.. This data are in accordance with other research group [11, 12]. During addition of water to the O/W systems there are no change in the surfactant spontaneous curvature and the droplet size distribution does not change significantly. The surfactant-to-oil weight ratio is more important factor in this selfemulsification process. Taking in to consideration the properties of prepared nanoemulsions (table 2) as a carrier for isotretinoin the formulations with the same water concentration (90 w/w%), differ in S:O ratio were chosen. Table 3 shows the properties of isotretinoin-loaded nanoemulsions, based on different kind of oils, coconut oil (NE-ISO-OK-8-2-90) and myristyl palmitate (NEISO-MI-9-1-90). 7
The data presented at the table 3 and at the figures 2 and 3 revealed that after incorporation of isotretinoin (0.05%) to selected formulations, the nanoemulsion based on isopropyl myristate becomes bimodal (fig. 2) in contrast to coconut oil-based nanoemulsion ( fig. 3).
According to the results shown at figure 2 and 3, nanoemulsion based on coconut oil was selected to the release studies, because after the incorporation of the active substance this formulation was characterized by almost unchanged size distribution and showed kinetic stability at 25ºC.
3.3. Results of isotretinoin release tests The in vitro release profiles of isotretinoin from based on coconut oil nanoemulsion (NE-ISO-OK-8-2-90) and the coconut oil (ISO-OK) as the control sample are shown in Figure 4. The amount of isotretinoin contained in the both studied vehicles was 0.05 % w/w.
The data presented at figure 4 confirmed that the nanoemulsion improved the drug release. The released amount of isotretinoin is much higher from nanoemulsion (10.39% w/w) than form the oil (1.85% w/w). This can be due the high lipophilicity of the actives (logP = 5.66, tab.1) and what goes with it, higher affinity to the oil than receptor solution. Moreover the enhanced diffusion from the nanoemulsion may be due to the large surface area of the droplets and the presence of surfactant, which reduces the interfacial tension what is in accordance with studies of Chavda and co-workers [7]. 3.4 . Kinetic study of isotretinoin release For a better understanding the efficacy of the active substance, study of their release kinetics are important. The selection of a suitable kinetic model for fitting the isotretinoin release data helps determine the release characteristics. There are number of kinetic models, which describe the overall release of drug from the vehicle. The most common mathematical models used are: zero order model (eq. 1) , first order model (eq. 2), Higuchi model (eq.3) and Korsmeyer-Peppas (eq.4) [13-15] : Qt = Q0 +K0·t (1) lnQt =lnQ0 +K1·t (2) Qt = Q0 +KH·t1/2 (3) Qt = Q0 +KHP·tn (4) where: Qt – the amount of drug released in time t, Q0 – the initial amount of drug, K0 – zero order kinetic constant, 8
K1 - first order kinetic constant, KH - Higuchi kinetic constant, KKP - Korsmeyer-Peppas release constant, n – diffusional release exponent t – time. The model selection results for investigated isotretinoin release from nanoemulsion and oil are presented at table 4.
According to the data shown at the table 4, the profile of isotretinoin release from nanoemulsion the best fit to zero order kinetic model. High linearity of the plots was achieved (R2 = 0.9919). It means that the active substance is released slowly, with the constant rate, independent of the initial drug concentration in the nanoemulsion. A steady amount of the released substances over time can minimize potential side effects because of the reduction in the frequency of drug administration. According to Anton et. al. [16] it is the ideal method of drug release in order to achieve a dermatological prolonged action. The obtained based on coconut oil and stabilized by Polysorbate 80 nanoemulsions, can be used as effective carrier for controlled release of isotretinoin. Zero-order release profiles are the direct consequences of the Fickian diffusion of the drugs through a membrane (Fick’s first law). Those data are in accordance with the results of Liu eta al. [5]. They observed that the isotretinoin in vitro permeation from SLN followed zero order release kinetics. The similar results, concerning release of drugs from nanosystems were previously reported. Elosayli et al. [17] showed that the release of nystatin from nanoemulsion-based gel followed zero order kinetic model. Also the results concerning release of theophylline from lipid nanoemulsion as coating agents [16] and indomethacin from nanoemulsions [18] showed that the zero order model best describes a release process of these drugs. In case of the release isotretinoin from the oil phase the release profiles could be best explained by Higuchi and Korsmeyer-Peppas models. Both regression lines are characterized by higher R2 values than initial (R2 > 0.98). This indicates slow diffusion of isotretinoin and low solubility of the active ingredient in the dissolution medium [19]. The results suggested that it takes times for the drug incorporated in the oil to be released [20].
4. Conclusions Isotretinoin-loaded nanoemulsions (0.05 % wt.), based on coconut oil and stabilized by Polysorbate 80 with droplet diameters around 21 nm were obtained. The results of the release studies confirmed that the nanoemulsions improved drug release. The released amount of isotretinoin was much higher from nanoemulsion (10.39 % w/w) than form the oil (1.85 % w/w). In case of obtained nanoemulsions the release profile the best fit to zero order kinetic model. It means that the nanoemulsions can be used as a vehicle for controlled isotretinoin release which resulting in prolonged dermatological action and reduce side effects.
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References: [1] B. Kanee, Clinical studies with topical fluocinolone acetonide in the treatment of various dermatoses, Can. Med. Assoc. J.;1; 1963, 88, 999-1003. [2] K. Raza, B. Singh, S. Singla, S. Wadhwa, B. Garg, S. Chhibber, O.P. Katare,;1; Nanocolloidal Carriers of Isotretinoin: Antimicrobial Activity against Propionibacterium acnes and Dermatokinetic Modeling, Mol. Pharm. 2013, 10(5), 1958-63. doi: 10.1021/mp300722f. [3] A.A. Cyrulnik, K.V. Viola, A.J. Gewirtzman, S.R. Cohen,;1; High-dose isotretinoin in acne vulgaris: improved treatment outcomes and quality of life. Int. J. Dermatol. 2012, 51(9), 1123-30. doi: 10.1111/j.1365-4632.2011.05409.x. [4] C. A. Guimarães, F. Menaa, B. Menaa, I. Lebrun, J.S. Quenca-Guillen, A.V. Vatti Auada, L.P. Mercuri, P. Ferreira, M.I. Rocha Miritello Santoro,;1; Determination of isotretinoin in pharmaceutical formulations by reversed-phase HPLC, J. Biomed. Sci. Eng. 2010, 3, 454-458. doi: 10.4236/jbise.2010.35063. [5] J. Liu, W. Hu, H. Chen, Q. Ni, Q, H. Xu, X. Yang,;1; Isotretinoin-loaded solid lipid nanoparticles with skin targeting for topical delivery, Int J Pharm., 2007, 328(2), 191-195. . [6] N. Kaur, R. Puri, S.K. Jain,;1; Drug-Cyclodextrin-Vesicles Dual Carrier Approach for Skin Targeting of Anti-acne Agent, AAPS Pharm. Sci. Tech. 2010, 11 (2), 528-537. doi: 10.1208/s12249-010-9411-2. [7] H. Chavda, J. Patel, G. Chavada,S. Dave, A. Patel,;1; Ch. Patel, Self-Nanoemulsifying Powder of Isotretinoin: Preparation and Characterization, Journal of Powder Technology, 2013 doi: 10.1155/2013/108569. [8] Jaworska M., Sikora E., Ogonowski J.,;1; Nanoemulsions. Characteristics and methods for preparation, Przem. Chem. 2014, 93 (7), 1087–1092. [9] http://www.drugbank.ca/drugs/DB00982. [10] G.K. Jensen, L.A. McGann, V. Kachevsky, T.J. Franz,;1; The negligible systemic availability of retinoids with multiple and excessive topical application of isotretinoin 0.05% gel (Isotrex) in patients with acne vulgari, J. Am. Acad. Dermatol. 1991, 24(3), 425-8. [11] P. Fernandez, V. Andre, J. Rieger, A. Kühnle, Colloids Surf. A.;1; 2004, 251, 53-58. [12] C. Solans, I. Sole,;1; Nano-emulsions: Formation by low-energy methods, Curr. Opin. Colloid Interface Sci. 2012, 17(5), 246–254. doi: 10.1016/j.cocis.2012.07.003.
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[13] S. Dash, P.N. Murthy, L. Nath, P. Chowdhury,;1; Kinetic modeling on drug release from controlled drug delivery systems, Acta Pol. Pharm. 2010, 67(3), 217–233. [14] P. Costa P., J-M. Sousa Lobo,;1; Modeling and comparison of dissolution profiles, Eur. J. Pharm. Sci. 2001, 13(2), 123-33. [15] V. Cojocaru, A.E. Ranetti, L.G. Hinescu, M. Ionescu, C. Cosmescu, A.G. Poștoarcă, L.O. Cinteză, Formulation and evaluation of in vitro release kinetics of Na3CaDTPA decorporation agent embedded in microemulsion-based gel formulation for topical delivery, FARMACIA.;1; 2015, 63 (5), 656-664. [16] N. Anton,A. de Crevoisier, S. Schmitt, T. Vandamme,;1; A new application of lipid nanoemulsions as coating agent, providing zero-order hydrophilic drug release from tablets, J. Drug Deliv. 2012. doi:10.1155/2012/271319. [17] G. H. Elosaily,;1; Formulation and in-vitro evaluation of nystatin nanoemulsion-based gel for topical delivery, J. Am. Sci. 2012, 8(12), 541-548. [18] E. Abdelaziz, M. Elmowafy, A. Salama, A. Samy, A. Kassem, M.A. Raslan, W.I. ElEraky,;1; Development and transdermal efficacy assessment of indomethacin nanoemulsion formulation, International Journal of Pharma Sciences. 2014, 4(4), 611621. . [19] L. M. Monteiro, V.F. Lione, F.A. do Carmo, L.H. do Amaral, J.H. da Silva, L. E Nasciutti, C.R. Rodrigues, H.C. Castro, V.P. de Sousa, L.M. Cabral,;1; Development and characterization of a new oral dapsone nanoemulsion system: permeability and in silico bioavailability studies, Int. J. Nanomedicine. 2012, 7, 5175–5182. [20] B. K. NANJWADE, P.J. VARIA, V.T. KADAM, T. SRICHANA, M.S. KAMBLE,;1; DEVELOPMENT AND EVALUATION OF NANOEMULSION OF REPAGLINIDE, JSM NANOTECHNOL. NANOMED. 2013, 1(2), 1016.
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Figure captions
Figure 1. O/W nanoemulsions occurrence region (2-phase) in the case of Water/ Polysorbate 80/Coconut oil system (A) and Water/Polysorbate 80/Isopropyl myristate system (B), at 25oC. Figure 2. Size distribution by intensity of nanoemulsions based on isopropyl myristate, without isotretinoin (NE-MI-9:1-90) and isotretinoin-loaded (NE-ISO-MI-9:1-90).
Figure 3. Size distribution by intensity of nanoemulsions based on coconut oil, without isotretinoin (NE-OK-8:2-90) and with isotretinoin (NE-ISO-OK-8:2-90). Figure 4. Release profiles of isotretinoin from the nanoemulsion (NE-ISO-OK-8:2-90) and coconut oil (ISO-OK).
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Table 1. Physical properties of isotretinoin [9]. Property Water solubility [mg/mL] logP Polar Surface Area [Å2] Molar mass [g/mol]
Value 0.00477 5.66 37.3 300.44
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Table 2. Properties of obtained nanoemulsions. Formulation NE-OK-8-2-80 NE-OK-8-2-90 NE-MI-9-1-80 NE-MI-9-1-90
Mean diameter (nm) ± S.D. (n=3) 40.56 ± 3 19.00 ± 0,15 11.00 ± 0,2 12.00 ± 0,5
PDI 0.700 0.258 0.203 0.268
Legend: NE-OK-8-2-80 – nanoemulsion based on coconut oil, with S:O ratio 8:2, containing 80% of water, NE-OK-8-2-90 - nanoemulsion based on coconut oil, with S:O ratio 8:2, containing 90% of water, NE-MI-9-1-80 - nanoemulsion based on isopropyl myristate, with S:O ratio 9:1, containing 80% of water, NE-MI-9-1-90 - nanoemulsion based on isopropyl myristate, with S:O ratio 9:1, containing 90% of water.
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Table 4. The kinetic model parameters fitting to the release results. Model Zero-order First order Higuchi
Korsmeyer-Peppas
Parameter ISO-OK 0.9468 6*10-7 0.8824 0.035 0.9853 0.0082 0.9857 0.0065 0.3316
R2 K0 [mg/h] R2 K1 [h-1] R2 KH [mg/h1/2] R2 KHP [h-n] n
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Formulation NE-ISO-OK-8:2-90 0.9919 4*10-6 0.9107 0.0739 0.9517 0.0603 0.9728 0.0100 0.6839
Table 3. Properties of isotretinoin-loaded nanoemulsions. Formulation NE-ISO-OK-8-2-90
Mean diameter (nm) ± S.D. (n=3) 21.00 ± 0,4
PDI 0.351
NE-ISO-MI-9-1-90
74.00 ± 14
0.951
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S0927-7757(16)30581-7 http://dx.doi.org/doi:10.1016/j.colsurfa.2016.07.060 COLSUA 20848
To appear in:
Colloids and Surfaces A: Physicochem. Eng. Aspects
Received date: Revised date: Accepted date:
29-2-2016 1-7-2016 21-7-2016
Please cite this article as: {http://dx.doi.org/ This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The kinetic study of isotretinoin release from nanoemulsion Małgorzata Miastkowska1*[email protected], Ogonowski1, Michał Zielina2 , Agnieszka Łudzik1
Elżbieta
Sikora1,
Jan
1Faculty
of Chemical Engineering and Technology, Institute of Organic Chemistry and Technology, Cracow University of Technology, Warszawska St. 24, 31-155 Cracow, Poland 2Faculty
of Environmental Engineering, Institute of Water Supply and Environmental Protection, Cracow University of Technology, Warszawska St. 24, 31-155 Cracow, Poland
*Corresponding author
1
Highlights
Isotretinoin-loaded nanoemulsions based on coconut oil were obtained.
Isotretinoin was incorporated into nanoemulsion which showed kinetic stability at 25ºC.
Nanoemulsions, based on coconut oil, are good carriers for controlled release of isotretinoin for topical application.
Zero order kinetic model the best fitted the profile of isotretinoin release form nanoemulsion.
2
Abstract
Acne is an inflammatory dermatosis caused by excessive production of sebum, abnormal follicular keratinization and Propionibacterium acnes. Isotretinoin is a retinoid, one of known topical delivered drugs, used in acne treatment because it normalizes the keratinization process. For greatest efficacy in dermatosis treatment, vehicles which enhance delivery of the active to the skin without reaching high serum concentrations and not causing skin irritation are still requested. The aim of this work was to develop a nanoemulsion formulation as a carrier for topical delivery of isotretinoin and determine the release kinetic of the active from the nanoemulsion. O/W isotretinoin-loaded nanoemulsions (0.05 % wt.) based on coconut oil and stabilized by a Polysorbate 80 was obtained and studied as isotretinon delivery systems. The nanoemulsions were prepared by stepwise addition of water to the mixture of polysorbate/oil/the retinoid, at room temperature. The mean droplet size of the nanoemulsions measured by Dynamic Light Scattering (DLS) was around 21 nm. The drug release study of the active was carried out using the Spectra/Por Standard Regenerated Cellulose (RC) membrane, at the temperature T=32oC. The concentration of isotretinoin in the receptor solution was analyzed by UV-Vis spectroscopy method. The release was found to follow the zero order kinetic model. The obtained nanoemulsion effect on isotretinoin release therefore can be used as a vehicle for controlled drug release which resulting in prolonged dermatological action and reduce side effects. Key words: isotretinoin, nanoemulsions, kinetic model
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1. Introduction Dermatosis is defined as a disorder involving lesions or eruptions of the skin that are acute (lasting days to weeks) or chronic (lasting months to years). The inflammation of the skin happens as a result of allergy or irritation of the skin, and is caused by the release of various substances that are important in the immune system [1]. One of the most common skin dermatosis is acne vulgaris. Acne is an inflammatory caused by excessive production of sebum, abnormal follicular keratinization, colonization by a Gram-positive bacterium (i.e., Propionibacterium acnes) and local inflammation [2]. Retinoids are the main topical drugs (applied directly to the skin) used to relieve an inflammation (swelling, itching, and redness) of the skin. They are the foundation for the treatment of acne because they normalize the skin keratinization process. Moreover these actives increase the turnover of follicular epithelial cells, whereby corneocytes are shed at an accelerated rate and comedones are extruded [3]. Isotretinoin is one of the Vitamin A derivatives which exhibits several activities, in particular a capacity to decrease sebaceous gland activity, to correct the keratinisation defect in acne and to reduce the population of the bacterium Propionibacterium acnes [4]. For greatest efficacy in dermatosis treatment, a vehicle should deliver medication in an active, stable form. The methods which enhance skin permeation of the topical drugs without reaching high serum concentrations and skin irritation are still requested. In literature there are some papers describing results of isotretinoin release from nanocarriers. All of the studies confirmed that nanosystems enhanced the bioavailability of isotretinoin. Raza and co-workers [2] investigated the release of isotretinoin from NLC (Nanostructured Lipid Carriers), SLN (Solid Lipid Nanoparticles) and commercial cream. It was found that the drug was released from nanocarriers much more effective (SLN – 84.81%, NLC – 75.21%) than from the market product (43.88 %). They supposed that it can be connected with higher biocompatibility of nanoformulation components and better interaction with the skin. The receptor medium was isotonic Palitzsch Buffer containing (Tween 80 2.7 % w/w) and ethanol (20.0 % v/v) Liu et. al. [5] obtained the isotretinoin-loaded solid lipid nanoparticles (SLN) for topical delivery. The concentration of isotretinoin in formulations amounted 0.06% . Samples consist of PRECIROL ATO5 as the lipid phase. Tween 80 and lecithin act as emulsifier. High-pressure homogenization was the method of SLN preparation. It was found that SLN formulation significantly increased the cumulative amount of isotretinoin in the skin and can act as a promising carrier for this drug. Kuar and co-workers [6] prepared the encapsulated complex of isotretinoinhydroxypropyl β cyclodextrin in elastic liposomes and studied the effect of dual carrier approach on skin targeting of isotretinoin. It was found that encapsulation of isotretinoin in elastic liposomes increased its photostability, skin targeting, and decrease its skin irritation. Chavda et al. [7] obtained self-nanoemulsifying drug delivery systems composed of Transcutol P (diethylene glycol monoethyl ether ) as the solvent and solubilizer for poorly water soluble active, Tween 80 as surfactant and PEG400 as co-surfactant. The in vitro release studies revealed that nanoformulations had higher drug release profile than the isotretinoin powder. Ex vivo permeability studies confirmed that SNEDDS (SelfNanoemulsifying Drug Delivery Systems) improved the skin permeation of isotretinoin (78.89 % after 6 h) compared to drug suspension (41.27% during 6 h). There is no information in the literature concerning the study of isotretinoin release from nanoemulsion as a carrier. Nanoemulsions are one of the most promising formulations 4
used to enhance pharmaceutical bioavailability of active substances such as ibuprofen, ramipril, aspirin, aceclofenac, and many others. They are very low-viscous, kinetically stable, colloidal dispersions with the droplet size in the range of 20-200 nm. Due to high solubilization and capacity to incorporate the active substances, nanoemulsions are the effective transport systems which enhance the actives bioavailability [8]. The aim of this work was to develop a nanoemulsion formulation based on coconut oil as a carrier for isotretinoin and determine the release kinetic of the topical agent from obtained vehicle.
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2.Materials and methods 2.1.Materials The nanoemulsions based on fractionated coconut oil (Brenntag), wheat germ oil (Brenntag) and isopropyl myristate (Sigma-Aldrich) as the oil phase were obtained. Polysorbate 80 was purchased from Caesar&Lorentz GmbH. Deionized by Milli-Q® filtration water was used as the aqueous phase of the emulsions. As the topical agent an isotretinoin (Fluka Analytical) was used. The physical properties of isotretinoin are shown at the table 1.
2.2.Nanoemulsion formation Nanoemulsions were prepared using phase inversion composition (PIC) method by stepwise water addition to the mixture of the oil and surfactant, at room temperature (25oC). The ratios of surfactant : oil (S:O) were varied in following compositions: 90:10, 80:20, 70:30, 60:40 and 50:50, and pseudo-ternary phase diagram was constructed. Compositions of mixtures that formed clear and isotropic systems were determined visually. 2.3.Incorporation topical agents into nanoemulsions Because of poor solubility of isotretinoin in water its solubility in the surfactant:oil (S:O) mixture, at ratio 80:20, was prior determined. During the preparation of topical agent-loaded O/W nanoemulsion, the active substance was incorporated to the mixture of surfactant and oil and then homogenized by the use of vortex aparature. The water phase was added, drop by drop, until final formulation was formed. The concentration of isotretinoin with respect to total formulation reached 0.05% w/w. 2.4.Mean droplet diameter and polydispersity index determination Mean droplet diameter and polydispersity index were measured using Dynamic Light Scattering (DLS) method (Zetasizer Nano ZS, Malvern Instruments, Malvern, UK), at 250C. The scattering angle was 1730. The analysis was performed three times to determine mean values and standard deviation. The measurement of particle size based on scattering photons from a sample and determining the change in diffracted light intensity. 2.5.Release studies Drug release study of isotretinoin was carried out using the Spectra/Por Standard Regenerated Cellulose (RC) membrane, at the temperature T=32oC. For release experiment, about 5 g of formulation was filled in a dialysis bag and placed in thermostatic chamber with receptor solution, for 24 h. The released concentration of isotretinoin in the receptor solution was analyzed by means of UV-Vis spectroscopy (Machery-Nagel Company), at the wavelength of 350 nm, on the basis of previously prepared calibration curve. According to Kuar et al. [6], for poorly water soluble substances in the release study, some amount of surfactants were added to increase the actives solubility in receptor solution The receptor medium was composed of 1.5 % w/w Polysorabte 80 solution in phosphate buffer (pH = 7,4). 2.6. Evaluation of release kinetic.
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The release kinetics was determined by linear regression analysis of the in vitro release curves in four models: zero order (cumulative amount (%) of drug released with time), first order (log cumulative amount (%) of drug released with time), Higuchi (cumulative amount (%) of drug released with the square root of time) and KorsemeyerPeppas (log cumulative amount (%) of drug released with log time). The mathematical model that best expressed the kinetic release profile was selected based on the highest coefficient of determination (R²). 3. Results and discussion 3.1 Formulation of nanoemulsions According to low solubility of isotretinoin in natural oils [7], the mixture of Polysorbate 80: oil (coconut oil, wheat germ oil or isopropyl myristate ) combined in the 8:2 weight ratio was used as a solvent. For determining the solubility of isotretinoin in surfactant: oil mixtures, 10 mg of isotretinoin was added to a vial containing 1 ml of tested solvent. The mixture solutions were vortexed and kept at 24°C overnight. The obtained results revealed that desirable amount of isotretinoin to obtain formulation with 0.05% drug concentration [10] was dissolved only in coconut oil and isopropyl myristate. As a consequence, in the next stage of the experiment, O/W nanoemulsions based on coconut oil or isopropyl myristate were obtained. The ternary phase diagrams of the systems Water/ Polysorbate 80/ Coconut oil (fig. 1 A) and Water/Polysorbate 80/Isopropyl Myristate (fig. 1 B), at 25 ºC were constructed to determine the area of stable nanoemulsions occurrence. The obtained results showed that coconut oil-based nanoemulsions were formed at S/O ratios comprised between 90/10 and 30/70, at minimum water concentration above 60% wt. In case of isopropyl myristate-based system the nanoemulsions were obtained only at 90/10 surfactant/oil weight ratio and minimum 60% of water phase. 3.2. Characterization of nanoemulsions The stable nanoemulsions, with maximum oil concentration in the system were analyzed. The results of the nanoemulsion mean droplet size (±SD) and polydispersity index (PDI), characterized by DLS method, are shown in table 2.
The results presented at the table 2 show that in the case of coconut oil-based nanoemulsions (NE-OK-8-2-80) and NE-OK-8-2-90) increasing water concentration reduces the droplet size and polydispersity of the system. While in the case of nanoemulsions containing isopropyl myristate (NE-MI-9-1-80 and NE-MI-9-1-90) there were no significant differences in droplet size distribution after water dilution. However the polydispersity index increased slightly with increasing water content in the system.. This data are in accordance with other research group [11, 12]. During addition of water to the O/W systems there are no change in the surfactant spontaneous curvature and the droplet size distribution does not change significantly. The surfactant-to-oil weight ratio is more important factor in this selfemulsification process. Taking in to consideration the properties of prepared nanoemulsions (table 2) as a carrier for isotretinoin the formulations with the same water concentration (90 w/w%), differ in S:O ratio were chosen. Table 3 shows the properties of isotretinoin-loaded nanoemulsions, based on different kind of oils, coconut oil (NE-ISO-OK-8-2-90) and myristyl palmitate (NEISO-MI-9-1-90). 7
The data presented at the table 3 and at the figures 2 and 3 revealed that after incorporation of isotretinoin (0.05%) to selected formulations, the nanoemulsion based on isopropyl myristate becomes bimodal (fig. 2) in contrast to coconut oil-based nanoemulsion ( fig. 3).
According to the results shown at figure 2 and 3, nanoemulsion based on coconut oil was selected to the release studies, because after the incorporation of the active substance this formulation was characterized by almost unchanged size distribution and showed kinetic stability at 25ºC.
3.3. Results of isotretinoin release tests The in vitro release profiles of isotretinoin from based on coconut oil nanoemulsion (NE-ISO-OK-8-2-90) and the coconut oil (ISO-OK) as the control sample are shown in Figure 4. The amount of isotretinoin contained in the both studied vehicles was 0.05 % w/w.
The data presented at figure 4 confirmed that the nanoemulsion improved the drug release. The released amount of isotretinoin is much higher from nanoemulsion (10.39% w/w) than form the oil (1.85% w/w). This can be due the high lipophilicity of the actives (logP = 5.66, tab.1) and what goes with it, higher affinity to the oil than receptor solution. Moreover the enhanced diffusion from the nanoemulsion may be due to the large surface area of the droplets and the presence of surfactant, which reduces the interfacial tension what is in accordance with studies of Chavda and co-workers [7]. 3.4 . Kinetic study of isotretinoin release For a better understanding the efficacy of the active substance, study of their release kinetics are important. The selection of a suitable kinetic model for fitting the isotretinoin release data helps determine the release characteristics. There are number of kinetic models, which describe the overall release of drug from the vehicle. The most common mathematical models used are: zero order model (eq. 1) , first order model (eq. 2), Higuchi model (eq.3) and Korsmeyer-Peppas (eq.4) [13-15] : Qt = Q0 +K0·t (1) lnQt =lnQ0 +K1·t (2) Qt = Q0 +KH·t1/2 (3) Qt = Q0 +KHP·tn (4) where: Qt – the amount of drug released in time t, Q0 – the initial amount of drug, K0 – zero order kinetic constant, 8
K1 - first order kinetic constant, KH - Higuchi kinetic constant, KKP - Korsmeyer-Peppas release constant, n – diffusional release exponent t – time. The model selection results for investigated isotretinoin release from nanoemulsion and oil are presented at table 4.
According to the data shown at the table 4, the profile of isotretinoin release from nanoemulsion the best fit to zero order kinetic model. High linearity of the plots was achieved (R2 = 0.9919). It means that the active substance is released slowly, with the constant rate, independent of the initial drug concentration in the nanoemulsion. A steady amount of the released substances over time can minimize potential side effects because of the reduction in the frequency of drug administration. According to Anton et. al. [16] it is the ideal method of drug release in order to achieve a dermatological prolonged action. The obtained based on coconut oil and stabilized by Polysorbate 80 nanoemulsions, can be used as effective carrier for controlled release of isotretinoin. Zero-order release profiles are the direct consequences of the Fickian diffusion of the drugs through a membrane (Fick’s first law). Those data are in accordance with the results of Liu eta al. [5]. They observed that the isotretinoin in vitro permeation from SLN followed zero order release kinetics. The similar results, concerning release of drugs from nanosystems were previously reported. Elosayli et al. [17] showed that the release of nystatin from nanoemulsion-based gel followed zero order kinetic model. Also the results concerning release of theophylline from lipid nanoemulsion as coating agents [16] and indomethacin from nanoemulsions [18] showed that the zero order model best describes a release process of these drugs. In case of the release isotretinoin from the oil phase the release profiles could be best explained by Higuchi and Korsmeyer-Peppas models. Both regression lines are characterized by higher R2 values than initial (R2 > 0.98). This indicates slow diffusion of isotretinoin and low solubility of the active ingredient in the dissolution medium [19]. The results suggested that it takes times for the drug incorporated in the oil to be released [20].
4. Conclusions Isotretinoin-loaded nanoemulsions (0.05 % wt.), based on coconut oil and stabilized by Polysorbate 80 with droplet diameters around 21 nm were obtained. The results of the release studies confirmed that the nanoemulsions improved drug release. The released amount of isotretinoin was much higher from nanoemulsion (10.39 % w/w) than form the oil (1.85 % w/w). In case of obtained nanoemulsions the release profile the best fit to zero order kinetic model. It means that the nanoemulsions can be used as a vehicle for controlled isotretinoin release which resulting in prolonged dermatological action and reduce side effects.
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References: [1] B. Kanee, Clinical studies with topical fluocinolone acetonide in the treatment of various dermatoses, Can. Med. Assoc. J.;1; 1963, 88, 999-1003. [2] K. Raza, B. Singh, S. Singla, S. Wadhwa, B. Garg, S. Chhibber, O.P. Katare,;1; Nanocolloidal Carriers of Isotretinoin: Antimicrobial Activity against Propionibacterium acnes and Dermatokinetic Modeling, Mol. Pharm. 2013, 10(5), 1958-63. doi: 10.1021/mp300722f. [3] A.A. Cyrulnik, K.V. Viola, A.J. Gewirtzman, S.R. Cohen,;1; High-dose isotretinoin in acne vulgaris: improved treatment outcomes and quality of life. Int. J. Dermatol. 2012, 51(9), 1123-30. doi: 10.1111/j.1365-4632.2011.05409.x. [4] C. A. Guimarães, F. Menaa, B. Menaa, I. Lebrun, J.S. Quenca-Guillen, A.V. Vatti Auada, L.P. Mercuri, P. Ferreira, M.I. Rocha Miritello Santoro,;1; Determination of isotretinoin in pharmaceutical formulations by reversed-phase HPLC, J. Biomed. Sci. Eng. 2010, 3, 454-458. doi: 10.4236/jbise.2010.35063. [5] J. Liu, W. Hu, H. Chen, Q. Ni, Q, H. Xu, X. Yang,;1; Isotretinoin-loaded solid lipid nanoparticles with skin targeting for topical delivery, Int J Pharm., 2007, 328(2), 191-195. . [6] N. Kaur, R. Puri, S.K. Jain,;1; Drug-Cyclodextrin-Vesicles Dual Carrier Approach for Skin Targeting of Anti-acne Agent, AAPS Pharm. Sci. Tech. 2010, 11 (2), 528-537. doi: 10.1208/s12249-010-9411-2. [7] H. Chavda, J. Patel, G. Chavada,S. Dave, A. Patel,;1; Ch. Patel, Self-Nanoemulsifying Powder of Isotretinoin: Preparation and Characterization, Journal of Powder Technology, 2013 doi: 10.1155/2013/108569. [8] Jaworska M., Sikora E., Ogonowski J.,;1; Nanoemulsions. Characteristics and methods for preparation, Przem. Chem. 2014, 93 (7), 1087–1092. [9] http://www.drugbank.ca/drugs/DB00982. [10] G.K. Jensen, L.A. McGann, V. Kachevsky, T.J. Franz,;1; The negligible systemic availability of retinoids with multiple and excessive topical application of isotretinoin 0.05% gel (Isotrex) in patients with acne vulgari, J. Am. Acad. Dermatol. 1991, 24(3), 425-8. [11] P. Fernandez, V. Andre, J. Rieger, A. Kühnle, Colloids Surf. A.;1; 2004, 251, 53-58. [12] C. Solans, I. Sole,;1; Nano-emulsions: Formation by low-energy methods, Curr. Opin. Colloid Interface Sci. 2012, 17(5), 246–254. doi: 10.1016/j.cocis.2012.07.003.
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[13] S. Dash, P.N. Murthy, L. Nath, P. Chowdhury,;1; Kinetic modeling on drug release from controlled drug delivery systems, Acta Pol. Pharm. 2010, 67(3), 217–233. [14] P. Costa P., J-M. Sousa Lobo,;1; Modeling and comparison of dissolution profiles, Eur. J. Pharm. Sci. 2001, 13(2), 123-33. [15] V. Cojocaru, A.E. Ranetti, L.G. Hinescu, M. Ionescu, C. Cosmescu, A.G. Poștoarcă, L.O. Cinteză, Formulation and evaluation of in vitro release kinetics of Na3CaDTPA decorporation agent embedded in microemulsion-based gel formulation for topical delivery, FARMACIA.;1; 2015, 63 (5), 656-664. [16] N. Anton,A. de Crevoisier, S. Schmitt, T. Vandamme,;1; A new application of lipid nanoemulsions as coating agent, providing zero-order hydrophilic drug release from tablets, J. Drug Deliv. 2012. doi:10.1155/2012/271319. [17] G. H. Elosaily,;1; Formulation and in-vitro evaluation of nystatin nanoemulsion-based gel for topical delivery, J. Am. Sci. 2012, 8(12), 541-548. [18] E. Abdelaziz, M. Elmowafy, A. Salama, A. Samy, A. Kassem, M.A. Raslan, W.I. ElEraky,;1; Development and transdermal efficacy assessment of indomethacin nanoemulsion formulation, International Journal of Pharma Sciences. 2014, 4(4), 611621. . [19] L. M. Monteiro, V.F. Lione, F.A. do Carmo, L.H. do Amaral, J.H. da Silva, L. E Nasciutti, C.R. Rodrigues, H.C. Castro, V.P. de Sousa, L.M. Cabral,;1; Development and characterization of a new oral dapsone nanoemulsion system: permeability and in silico bioavailability studies, Int. J. Nanomedicine. 2012, 7, 5175–5182. [20] B. K. NANJWADE, P.J. VARIA, V.T. KADAM, T. SRICHANA, M.S. KAMBLE,;1; DEVELOPMENT AND EVALUATION OF NANOEMULSION OF REPAGLINIDE, JSM NANOTECHNOL. NANOMED. 2013, 1(2), 1016.
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Figure captions
Figure 1. O/W nanoemulsions occurrence region (2-phase) in the case of Water/ Polysorbate 80/Coconut oil system (A) and Water/Polysorbate 80/Isopropyl myristate system (B), at 25oC. Figure 2. Size distribution by intensity of nanoemulsions based on isopropyl myristate, without isotretinoin (NE-MI-9:1-90) and isotretinoin-loaded (NE-ISO-MI-9:1-90).
Figure 3. Size distribution by intensity of nanoemulsions based on coconut oil, without isotretinoin (NE-OK-8:2-90) and with isotretinoin (NE-ISO-OK-8:2-90). Figure 4. Release profiles of isotretinoin from the nanoemulsion (NE-ISO-OK-8:2-90) and coconut oil (ISO-OK).
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Table 1. Physical properties of isotretinoin [9]. Property Water solubility [mg/mL] logP Polar Surface Area [Å2] Molar mass [g/mol]
Value 0.00477 5.66 37.3 300.44
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Table 2. Properties of obtained nanoemulsions. Formulation NE-OK-8-2-80 NE-OK-8-2-90 NE-MI-9-1-80 NE-MI-9-1-90
Mean diameter (nm) ± S.D. (n=3) 40.56 ± 3 19.00 ± 0,15 11.00 ± 0,2 12.00 ± 0,5
PDI 0.700 0.258 0.203 0.268
Legend: NE-OK-8-2-80 – nanoemulsion based on coconut oil, with S:O ratio 8:2, containing 80% of water, NE-OK-8-2-90 - nanoemulsion based on coconut oil, with S:O ratio 8:2, containing 90% of water, NE-MI-9-1-80 - nanoemulsion based on isopropyl myristate, with S:O ratio 9:1, containing 80% of water, NE-MI-9-1-90 - nanoemulsion based on isopropyl myristate, with S:O ratio 9:1, containing 90% of water.
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Table 4. The kinetic model parameters fitting to the release results. Model Zero-order First order Higuchi
Korsmeyer-Peppas
Parameter ISO-OK 0.9468 6*10-7 0.8824 0.035 0.9853 0.0082 0.9857 0.0065 0.3316
R2 K0 [mg/h] R2 K1 [h-1] R2 KH [mg/h1/2] R2 KHP [h-n] n
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Formulation NE-ISO-OK-8:2-90 0.9919 4*10-6 0.9107 0.0739 0.9517 0.0603 0.9728 0.0100 0.6839
Table 3. Properties of isotretinoin-loaded nanoemulsions. Formulation NE-ISO-OK-8-2-90
Mean diameter (nm) ± S.D. (n=3) 21.00 ± 0,4
PDI 0.351
NE-ISO-MI-9-1-90
74.00 ± 14
0.951
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