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Design of antiretroviral drug-polymeric nanoparticles laden buccal films for chronic HIV therapy in paediatrics
Colloid and Interface Science Communications 27 (2018) 49–59
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Colloid and Interface Science Communications journal homepage: www.elsevier.com/locate/colcom
Design of antiretroviral drug-polymeric nanoparticles laden buccal films for chronic HIV therapy in paediatrics R. Sneha, B.N. Vedha Hari, D. Ramya Devi
T
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School of Chemical & Biotechnology, SASTRA Deemed University, Thanjavur 613401, Tamil Nadu, India
ARTICLE INFO
ABSTRACT
Keywords: Buccal film Nanoparticles Lamivudine Eudragit E100 HIV
Anti-HIV drug Lamivudine exhibit shorter half-life (2 h) and poor bioavailability (62% in paediatric patients), multiple dosing is required. To overcome the limitations, nanoparticles loaded buccal film was developed for prolonged drug release and improved efficiency. Nanoprecipitated Lamivudine-Eudragit E100 polymeric nanoparticles exhibited average particle size of 338 nm with polydispersity index 0.315. Buccal films prepared by solvent casting technique using sodium carboxy methyl cellulose and polyvinyl pyrrolidone (3:2) loaded with the nanoparticles showed initial drug release of 49% followed by sustained release to reach the maximum of 93% at the end of 8 h. Encapsulation of drug in nanoparticles and the film was confirmed through scanning electron microscopy, thermal and crystal characterization. Sustained drug release through Fickian diffusion mechanism from the matrix system was confirmed. The optimized formulation could be used to enhance therapeutic effect at reduced dose and side effects, especially for the paediatric anti-HIV therapy.
1. Introduction HIV has taken the lives of millions of people around the world making it as a one of the most serious global crisis. According to the report of Joint United Nations Programme on HIV and AIDS (UNAIDS), approximately 36.7 million people worldwide are living with HIV/ AIDS, out of which 2.1 million are children below the age of 15 years [1]. As per UNAIDS data, In India 2.1 million people are living with HIV, and among them 693,000 patients are children. The mortality of children is comparatively higher than adults because of their weak immune system and lack of proper treatment [2]. HIV infects the vital cells in the immune system such as macrophages, dendritic cells, and CD4+ T cells. The rate of virus progression varies widely between individuals and depends on many factors like age of the individual, body's ability to defend against HIV, the presence of other infections, individual's genetic inheritance, resistance to certain strains of HIV, etc. The increase in viral load leads to decline in the number of CD4+ cells leading to the loss of cell-mediated immunity, making the body more susceptible to various types of infections, which in overtime leads to Acquired Immunodeficiency Syndrome (AIDS) [1,3]. Although there is no complete cure for HIV Infection, symptoms of the disease can be reduced and the life of the individual can be prolonged by administration of different classes of antiretroviral drugs [4–6]. Most of the antiretroviral drugs available are not suitable for
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paediatric patients because of higher dosage and toxicity associated [7]. Lamivudine belongs to the class of Nucleoside Reverse Transcriptase Inhibitors (NRTIs), which is phosphorylated to active metabolites that compete for incorporation into viral DNA, thereby preventing the multiplication of viral DNA in the host. It is one of the preferred firstline medications for paediatric patients [8], based on the safety profile observed in the clinical trials. The commercially available dosage form for administration is oral tablet and oral solution (Syrup/suspension) and the dosage vary according to the age and body weight of the children, recommended as 4 mg/kg/day up to the maximum of 300 mg/ day for 35 kg [9,10]. The limitations of the drug are the low half-life of 2 h and oral bioavailability of 68% in paediatric patients [8]. To maintain the therapeutic level, multiple dose administration of Lamivudine is required that leads to overdosing and related side effects such as headache, nausea, diarrhoea, abdominal pain and skin rash, which reduces patient compliance especially in children [11,12]. The nanoparticles provide better encapsulate of drugs and could provide sustained release with reduced dose and toxicity [13–15]. The drug can directly reach the systemic circulation by direct contact with buccal mucosa which is rich in blood supply and also avoids the gastrointestinal degradation. The two major pathways for drug permeation across the buccal mucosa are the paracellular route for hydrophilic solutes and transcellular route for lipophilic solutes [16]. Mucoadhesive polymers like Polyvinyl Alcohol (PVA), Polyvinyl Pyrrolidone (PVP),
Corresponding author. E-mail address: [email protected] (D. Ramya Devi).
https://doi.org/10.1016/j.colcom.2018.10.004 Received 28 July 2018; Received in revised form 22 October 2018; Accepted 27 October 2018 2215-0382/ © 2018 Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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Sodium carboxymethylcellulose (SCMC), hydroxypropyl methylcellulose (HPMC) and sodium alginate (SA) are commonly used for the films preparation, which could be held on the surface of the buccal mucosa by interfacial force. Mucoadhesion is generally referred to the interaction between the polymer and the epithelial surface, which could improve the drug absorption and bioavailability in the system [17,18]. Buccal route drug delivery improves the absorption and maintains the therapeutic level of drug in plasma for a prolonged period of time [19]. The buccal film is mostly preferred than a buccal tablet as it is more convenient and comfortable for paediatric patients [17]. With this background and to overcome the challenges of Lamivudine therapy in children, the present work focus on development of buccal films loaded with polymeric nanoparticles of the drug that could provide burst effect followed for sustained release of the drug.
2.3. Particle Size and Zeta Potential The particle size and zeta potential of the prepared nanoparticles were analyzed using Zeta-sizer to determine size distribution and the stability of the particles. The Zeta-sizer (Nano series, Malvern, UK) uses dynamic light scattering principle that measures Brownian motion and relates to the particle size. In this, the particle is illuminated with a laser and the intensity of fluctuation in scattered light is analyzed [24]. 2.4. Entrapment Efficiency Accurately 0.5 mL of polymeric nanosuspension was centrifuged (C24 plus, REMI, India) at 12,000 rpm for 20 min to separate the pellet containing the polymeric nanoparticles of the drug and the supernatant with the unentrapped drug. The supernatant solution was suitably diluted with phosphate buffer (pH 6.8) and the absorbance was measured using UV-Spectrophotometer at 270 nm (λmax of drug) using the respective blank. The percentage of the entrapped drug was calculated using the formula, Total drug content Drug content in supernatant %Entrapment Efficiency = × 100 Total drug content
2. Materials and Methods 2.1. Materials Lamivudine was obtained as gift sample from Aurobindo Pharma Ltd., India. Eudragit E100 was obtained as gift sample from Evonik Nutrition & Care GmbH, Germany. Polymers like polyvinyl chloride, polyvinyl pyrrolidone, hydroxypropyl methyl cellulose, sodium carboxymethylcellulose, sodium alginate were purchased from SD Fine Chem. Pvt. Ltd., India. All the chemicals and reagents used were of analytical grade.
2.5. In Vitro Drug Release Study of Lamivudine Polymeric Nanoparticles The in vitro drug release studies of the developed Lamivudine polymeric nanoparticle formulations were performed using dialysis membrane method. About 1 mL of the formulation was loaded into the dialysis bag (Dialysis membrane-110, Himedia) and kept in a vial containing 10 mL of Phosphate buffer pH 6.8. The vial was kept in a beaker containing water maintained at 37 °C and placed in a magnetic stirrer at 100 rpm. Samples were collected at regular intervals for 8 h and replaced with 10 mL of buffer to maintain the perfect sink conditions. The collected samples were analyzed using UVSpectrophotometer (Evolution 201, Thermo Scientific, USA) at 270 nm [24].
2.2. Preparation of Lamivudine Loaded Eudragit E100 Nanoparticles Nanoparticles were prepared by nanoprecipitation method which is also known as interfacial deposition or solvent-displacement method [20]. The polymer selected for preparation of nanoparticles was Eudragit E100, which is a cationic hydrophobic polymer [21]. The concentration of the drug (Lamivudine) and surfactant (Pluronic F127) was kept constant and the concentration of the polymer (Eudragit E100) was varied at three different levels (Table 1). In this process, the drug was dissolved in 10 mL aqueous solution containing 0.5% Pluronic F127 as a surfactant, which could reduce the surface tension between the aqueous and organic phase and thereby stabilize the nanoparticle suspension formation while preventing the aggregation of fine particle. The mixture was placed on a magnetic stirrer at a constant stirring rate of 1500 rpm. The polymer was dissolved in 5 mL of volatile organic solvent (methanol) and was added dropwise in the aqueous solution. The solution was kept under stirring until the organic solvent was completely diffused and evaporated, resulting in the precipitation and formation of polymeric nanoparticles of the drug [22]. In order to ensure high stability of the nanoparticles and complete removal of the solvents (water and methanol) [23], the nanosuspension was lyophilized (Christ Alpha 2–4 LD Plus, Osterode Am Harz, Germany) for 48 h. The formulated nanosuspension was ultracentrifuged (C24 plus, REMI, India) at 15,000 rpm and − 4 °C for 20 min to obtain the pellet, which was re-dispersed in distilled water using cyclone mixer (Remi CM101 DX, Mumbai, India) to get uniform nanoparticles aqueous dispersion and then freeze-dried.
2.6. Characterization of Polymeric Nanoparticles 2.6.1. Fourier-Transform Infra-Red (FT-IR) Spectroscopy Drug-polymer interactions due to chemical bonding were studied using FTIR (6701F, Perkin Elmer, USA) by potassium bromated (KBr) disc/pellet technique. The infrared spectrum of the nanoparticles was compared with pure drug and pure polymers. The samples were mixed with dried and saturated potassium bromate. The discs were obtained by compressing the powder under a hydraulic pressure of 150 kg/cm2 for 5 min. The pellet was placed in the sample holder and scanned between the wave number range of 4000–400 cm−1 [24]. 2.6.2. X-Ray Diffraction (XRD) Analysis XRD Analysis was carried out to determine the crystallinity of the drug in the nanoparticle formulation by constructive interference of monochromatic X-ray. The nature of the sample was studied at room temperature using X-Ray Diffractometer (D8 Focus, Bruker, Germany) over a 2θ range at 0 °C to 80 °C with the current of 30 mA and voltage of 40 kV.
Table 1 Preparation and Physico chemical characterization of Lamivudine loaded Eudragit E100 Polymeric Nanoparticles. S. No.
Lamivudine: Eudragit E100
Average size (nm)
Poly dispersity index
Zeta potential (mV)
Encapsulation efficiency (%)
1 2 3
1: 0.5 1: 1 1: 2
147.9 228.7 338.6
0.154 0.201 0.315
+3.89 +5.47 +7.06
72.3 90 94.4
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2.6.3. Differential Scanning Calorimetry (DSC) Differential Scanning Calorimetry was used to characterize the thermal behavior of drug and the nanoparticles. The DSC thermogram (Q100, TA Instruments, USA) was obtained by automatic thermal analyzer technique, in which the sample was sealed in an aluminum pan and an empty aluminum pan was taken as reference. The sample was heated at the rate of 10 °C/min, from the range of 25 °C to 300 °C [24].
2.9. Preparation of Lamivudine Polymeric Nanoparticles Loaded Buccal Film
2.6.4. Field Emission Scanning Electron Microscopy (FESEM) The surface morphology of the drug and polymeric nanoparticles was analyzed by FESEM. The sample was loaded into the instrument and scanned by electrons liberated by field emission source. The images were taken at the magnification ranging from 10,000× to 30,000× at an accelerating voltage of 3 kV [24].
2.10. Determination of Drug Content
The required amount of polymeric nanoparticles to achieve 4 mg equivalent dose of the drug in each film (with area of 2 cm2) was added to the film-forming polymeric solution and the mixture was bath sonicated to obtain a homogeneous dispersion. The film was formed using similar mould dimensions by the previously explained solvent casting technique [19].
The drug content in the buccal film was determined by dissolving the film in 50 mL phosphate buffer pH 6.8 which was kept overnight. The solutions were then transferred to volumetric flasks and appropriate dilutions were made with phosphate buffer. Then the resulting solution was filtered with Whatman filter paper to remove undissolved polymer and then drug content was analyzed using UV–Visible spectrometer at 270 nm. The experiment was conducted in triplicate and the results represented as mean ± SD [14,19].
2.7. Preparation of Buccal Film With Mucoadhesive Polymers Buccal films were prepared using circular moulds with 3.8 cm2 area by solvent casting technique. The films were prepared using the mucoadhesive polymers like HPMC, SCMC, SA, PVA, PVP, and Carbopol 934p in various ratios, so that a total of 37 formulations were trialed experimentally (Supplementary data Table 1). The polymer was dissolved in a measured volume of water and ethanol in addition with 1% of propylene glycol as plasticizer. The polymeric solution was homogenized using bath sonicator and poured onto the moulds and then placed in a hot air oven at 55 °C for 5 to 6 h followed by air drying for an hour. The dried film was carefully removed from the mould and checked for folding endurance and stability. Formulations with better physical and thermal stability (ability to peel off from mould without brittleness, colour integrity, uniformity of thickness) and folding endurance were chosen for loading the pure drug / polymeric nanoparticles [25,26].
2.11. In Vitro Drug Release Study of Lamivudine/Polymeric Nanoparticles Loaded Buccal Film The drug release was carried out for each buccal film (containing pure Lamivudine / polymeric nanoparticles) and the amount of drug release was studied using USP type V (Paddle over disc) dissolution method. The buccal film was placed below the glass disc kept at the bottom of the vessel and the paddle was positioned above the disc [27]. About 100 mL of phosphate buffer pH 6.8 was taken as the media with constant stirring at 50 rpm and the temperature was maintained at 37 ± 2 °C. Accurately 10 mL of the samples were collected at predetermined intervals and replaced with 10 mL of fresh media to maintain the sink conditions. The collected samples were quantified for cumulative percentage of drug release by UV-Spectrophotometer at 270 nm [14,19]. The study was carried out for 8 h and the average of three trials was calculated for plotting the cumulative percentage of drug release vs. time profile.
2.8. Preparation of Pure Lamivudine Loaded Buccal Film Among the 37 trial buccal films, only 9 formulations (Table 2) with good stability and folding endurance were chosen for loading the drug. Circular moulds with radius of 0.8 cm was used for the casting method, so that each buccal films loaded with 4 mg of Lamivudine (least oral dose in children) with the area of 2 cm2 was fabricated. The total amount of drug needed for formulation of 12 films was accurately weighed and dissolved in distilled water added to the film-forming polymeric solution along with propylene glycol as the plasticizer. This solution was homogenized completely followed by bath sonication to remove air bubbles. The required volume of mixture (to obtain 4 mg dose in each film) was poured on the moulds and kept in a hot air oven at 55 °C for 5 to 6 h, followed by air drying for an hour [19]. The dried film was carefully removed from the moulds and stored in desiccator for further characterization studies.
2.12. In Vitro Drug Release Kinetics To understand the kinetics of the drug release from the formulations, the data obtained from in vitro release studies were fitted into different mathematical models such as zero order, first order, Korsmeyer-Peppas, Higuchi, Hixson-Crowell, Hopfenberg, BakerLonsdale, Weibull, Makoid-Banakar and Gompertz models using DD solver software [28]. 2.13. Characterization of Lamivudine Polymeric Nanoparticles Loaded Buccal Film The physical characteristics of the optimized buccal film loaded with the polymeric nanoparticles of Lamivudine was analyzed through swelling index, tensile strength, elongation at break and folding endurance. The swelling property of the selected buccal film was evaluated by hydration method, wherein the film was immersed in a 10 mL of distilled water and the initial weight of the dry film (W1) before immersion and the weight of swelled film (W2) after immersion was noted at periodic time intervals for 8 h. Swelling index at each time point was calculated from following equation. W W Swelling Index (%) = 2W 1 × 100 1 An Instron Universal Testing Machine (Model 2519–104, Instron Corp., USA) was used to determine the tensile strength and percentage elongation at break [29]. The folding endurance was measured manually by folding the film repeatedly at a point till it broke. All the
Table 2 Formulation and evaluation of Lamivudine loaded buccal films. S. No.
Polymer ratio in buccal film
Ratio
Drug content (%)
1 2 3 4 5 6 7 8 9
HPMC: PVP HPMC: PVA HPMC: SA HPMC: SCMC SCMC: SA SCMC: SA SCMC: PVP SCMC: PVA SCMC: PVA
3: 4: 3: 1: 4: 3: 3: 4: 3:
100 ± 2.30 86.32 ± 6.52 82.50 ± 7.83 92.01 ± 6.99 96.66 ± 2.50 87.96 ± 1.36 86.03 ± 3.01 82.58 ± 4.65 93.36 ± 5.76
1 1 2 1 1 2 2 1 2
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experiments were carried out in triplicate and the results presented as mean with standard deviation. The optimized polymeric nanoparticles loaded buccal film was subjected to analytical characterization studies like XRD, DSC, and FESEM (similar method as mentioned for the pure drug and nanoparticles) to evaluate the stability, drug-polymer interactions and morphology. The IR study of the film was performed through ATR (Attenuated total reflection) mode. In this method the film was placed on the ATR crystal and beam of infrared light was passed through it. The changes in the internally reflected IR beam when it comes in contact with the sample were measured.
flowing dry powder for improved stability and further characterization studies. 3.2. Encapsulation Efficiency in Nanoparticles at Varying Polymer Level The amount of drug entrapped within synthesized Lamivudine loaded Eudragit E100 nanoparticles were analyzed using UV visible spectrophotometer at 270 nm and shown in Table 1. The percentage encapsulation efficiency of Lamivudine in Eudragit E100 polymer ranged from 72.3 to 94.4%. Nanoparticles prepared with Lamivudine: Eudragit E100 in the ratio 1:2 showed maximum drug encapsulation, which could be due to the requirement of a higher proportion of the hydrophobic polymer for the complete entrapment of the hydrophilic drug [32]. The result could be approved, since Sadoun et al. [33] had previously reported an increase in the drug entrapment due to increase in the polymer concentration because, the increase in the organic phase viscosity promotes the formation of higher microparticles that could consequently entrap higher quantity of drug. Similar pattern of encapsulation was observed in Eudragit nanoparticle loaded with an antihypertensive drug Valsartan, wherein 1:5 ratio of drug and polymer have shown high entrapment efficiency than 1:2.5 ratio [34].
2.14. Cytotoxicity Studies of the Polymeric Nanoparticles The cytotoxicity of the selected nanoparticles formulation was evaluated using Green monkey kidney cells (Vero) cells [30] procured from the National Center for Cell Sciences (NCCS), Pune, India through MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The cells were maintained in Minimal Essential medium (MEM) supplemented with 2 mM L-glutamine and balanced salt solution (BSS) adjusted to contain 1.5 g/L Na2CO3, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 1.5 g/L glucose, 10 mM (4(2-hydroxyethyl)-1-piperazineethane sulfonic acid) (HEPES) and 10% fetal bovine serum (GIBCO, USA). Penicillin and streptomycin (100 IU/ 100 μg) were adjusted to 1 mL/L. The cells were maintained at 37 °C with 5% CO2 in a humidified CO2 incubator. The cells were grown in a 96-well plate for 48 h into 75% confluence. To make single cell suspensions, the monolayer cells were treated with trypsin-ethylene diamine tetraacetic acid (EDTA) to detach the cells. The viability of cells was counted using hemocytometer and the cell suspension was diluted using a medium containing 5% FBS to make the final density of 1 × 104 cells/mL. The medium was replaced with fresh medium containing serially diluted test samples with drug concentrations of 10, 20, 30, 40 and 50 μg/mL and the cells were further incubated for 48 h. The culture medium was removed, and 100 μL of the MTT [3-(4,5-dimethylthiozol2-yl)-3,5-diphenyl tetrazolium bromide] (Hi-Media) solution was added to each well and incubated at 37 °C for 4 h. After removal of the supernatant, 50 μL of DMSO was added to each of the wells and incubated for 10 min to solubilize the formazan crystals. The optical density was measured at 620 nm in an ELISA multiwell plate reader (Thermo Multiskan EX, USA). The OD value was used to calculate the percentage of viability using the following formula,
%Cell Viability =
3.3. In Vitro Drug Release Study of Lamivudine Polymeric Nanoparticles The in vitro drug release profile of Lamivudine polymeric nanoparticles was displayed in Fig. 1 (a). The nanoparticles containing drug: the polymer ratio 1: 0.5 showed drug release of 58% within first 15 min and reached the maximum of 87% at the end of 1.5 h. In case of the formulations with ratio 1:1 and 1:2, the initial drug release was found to be around 60%. The complete drug release of 82% was achieved at the end of 2 h in a formulation containing 1:1 ratio. Whereas, the drug release was sustained for 4 h in the formulation with ratio 1: 2 which reached the maximum of 89%. As the polymeric concentration was increased, drug release was prolonged over a time. It was due to the encapsulation of drug in a proportionately higher amount of polymer matrix and also because of low solubility of the Eudragit E100 polymer in pH 6.8 media, which reduces the dissolution of the drug resulting in a prolonged release [32]. 3.4. Drug Content of Buccal Films The drug content for the selected 9 buccal film formulations was analyzed using UV–Visible Spectrophotometer at 270 nm and the results tabulated in Table 2. The drug content ranged from 82% to 100%. The film formulation with polymer combination of HPMC and PVP in the ratio of 3:1 showed the maximum amount of drug content. As the drug was more uniformly dispersed in the hydrophilic polymer matrix film of HPMC/PVP, maximum amount of drug was loaded in this formulation [35]. The blending of HPMC with PVP in films has been reported to decrease the crystallinity of HPMC, which could also favor the loading of high amount of drug [36]. Also, significant increase in the thickness, drug content and mass of the films containing PVP has been observed by Maheswari et al. as compared to the films without PVP [37].
[A]test × 100 [A]control
where [A]test is the absorbance of the test sample and [A]control is the absorbance of the control [31]. 3. Results and Discussion 3.1. Particle Size Distribution and Stability of Lamivudine-Eudragit E100 Nanoparticles The average size and the zeta potential of the nanoparticles prepared with three different levels of Lamivudine: Eudragit E100 ratio was evaluated and reported in Table 1. The average size range was between 140 and 340 nm and Polydispersity index < 0.4 indicated the monodisperse nature of the preparation. The particle size increased gradually with increase in the concentration of the polymer (Eudragit E100). The maximum zeta potential of +7.06 was achieved by the formulation containing 1:2 ratio of drug: polymer. However, this value does not confirm the dynamic stability of the nanosuspension. Hence, lyophilization was performed to convert the nanosuspension into free-
3.5. In Vitro Drug Release Study for Lamivudine Buccal Films The in vitro drug release profile of the buccal films loaded with pure Lamivudine was displayed in Fig. 1 (b, c, d). The drug release from the film containing the polymer combination of HPMC and SA in the ratio of 3:2 showed 84.5% drug release in first 15 min and attained the maximum of 94% at the end of 3 h. The formulation containing HPMC and SCMC in the ratio of 1:1 showed 70% release initially and gradually
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Fig. 1. In vitro drug release profile in phosphate buffer pH 6.8 (a) Lamivudine polymeric nanoparticles (b) Buccal films containing HPMC and pure drug (c) Films containing SCMC and pure drug (d) Films containing SCMC with PVA and pure drug (e) Lamivudine polymeric nanoparticles loaded Buccal films (f) Comparison of optimized Buccal film formulation to the pure drug.
increased up to 87% at the end of 8 h. The formulations containing HPMC: PVA in ratio 4:1 and HPMC: PVP in ratio 3:1 showed drug release of 55% and 41% at the end of 8 h, respectively (Fig. 1 (b)). The retarded drug release from these polymeric combination buccal films was due to the increase in the concentration of HPMC. As HPMC is a swellable polymer, the polymer matrices could be eroded slowly and the drug was released only after polymer swelling [38]. There is an inverse relationship between the rate of polymer matrix swelling and drug release [39]. Therefore, as the concentration of HPMC increases, the swelling rate increases which results in the retarded drug release. The formulation with the polymeric combination of SCMC and SA in ratio 4:1 reached 100% drug release (Fig. 1 (c)) within first 15 min interval. The film containing SCMC: PVP in the ratio of 3:2 showed 77% initial drug release and reached 92% at the end of 8 h. Similarly, SCMC: SA in ratio 3:2 showed drug release of 67% initially and reached 82% at the end of 8 h. The drug release percentage of these two formulations (SCMC: SA & SCMC: PVP) in ratio 3:2 are almost similar due to the presence of an equal concentration of swelling polymer (SCMC) and
hydrophilic polymers SA & PVP. After the swelling of SCMC polymer matrix, PVP and SA which is also present in the swelling matrix may be dissolved rapidly in phosphate buffer media [40], which may create pores in the matrix. The drug release of the formulation containing a polymeric combination of SCMC and PVA in different ratios of 3:2 and 4:1 showed drug release of 70% initially (Fig. 1 (d)). At the end of 8 h the drug release of the formulation with 3:2 ratio was 88% and that of 4:1 was 79%. The slight variation in the drug release pattern was influenced by PVA concentration which could dissolve in the phosphate buffer pH 6.8 because of its hydrophilic nature [40]. The SCMC polymer swells in the media and the PVA entrapped in the swelling matrix creates pores through which the drug may be released. Higher the concentration of PVA, more the pore formation which could increase the percentage of drug release. Thus, films containing SCMC: PVA in the ratio of 3:2 showed higher drug release percentage. Based on the obtained in vitro release profile, the buccal films prepared with SCMC:PVA (3:2), SCMC:PVP (3:2) and HPMC:SA (3:2),
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Fig. 2. FTIR spectrum of (a) Pure Lamivudine (b) Pure Eudragit E100 (c) Lamivudine polymeric nanoparticles (d) Buccal Film loaded with nanoparticles.
which exhibited complete release of the drug (> 90% at the end of 8 h) were selected as optimized formulations for the incorporation of the Lamivudine polymeric nanoparticles. Due to the hydrophilic nature of the drug, the maximum amount of drug was released from the films within the first 2 to 3 h. In order to improve the bioavailability and halflife of the drug, a sustained release pattern of drug release is required. To achieve the sustained drug release profile, the optimized hydrophobic polymeric nanoparticles were incorporated into the selected films.
two formulations (SCMC & PVA, HPMC & SA) showed the maximum release of only 70% and 40% respectively at the end of 8 h. The interaction of the polymeric nanoparticles and film-forming polymers retarded the drug release in these formulations. Since the half-life of Lamivudine is only 2 h, when the high percentage drug release is reached early, the drug will be eliminated from the body in short time period. The interaction of hydrophobic polymer and hydrophilic drug in the nanoparticles reduced the solubility of the drug. The decline in solubility has reduced the dissolution rate of the drug which could provide slow drug release over a period of time. The combination of the nanoparticles and the film-forming polymers (SCMC and PVP) provided the sustained drug release profile.
3.6. In Vitro Drug Release Study of the Buccal Film Loaded With Polymeric Nanoparticles The in vitro drug release profile of the selected buccal films loaded with the optimized polymeric nanoparticles of Lamivudine was displayed in Fig. 1 (e). Films containing SCMC and PVP in ratio 3:2 loaded with polymeric nanoparticles showed drug release of 48% initially and gradually increased in a sustained manner over a period of time and reached 93% at the end of 8 h. The presence of polymeric nanoparticles showed a minimal amount of drug release initially which slowly increased over a period of time attaining the maximum release. The other
3.7. Comparison of the In Vitro Release Profile the Optimized Formulation With Pure Drug The drug release of the pure drug and the buccal film containing SCMC: PVP in ratio 3:2 loaded with polymeric nanoparticles was compared. The pure drug showed 100% drug release within 15 min whereas the optimized buccal film showed a sustained drug release from 48% to 93% at 1 h to 8 h (Fig. 1 (f)). Hence, the extended drug
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Fig. 3. XRD spectrum of (a) Pure Lamivudine (b) Pure Eudragit E100 (c) Lamivudine polymeric nanoparticles (d) Buccal Film loaded with nanoparticles.
release could be achieved in vivo, which could maintain the therapeutic concentration of drug in plasma for a prolonged period of time. This could ultimately improve the bioavailability of the drug and reduce the overall dose required for the therapy in children.
drug release followed by slow converged release to reach maximum dissolution of the drug from the buccal films was explained by the Gompertz kinetics [41]. 3.9. Characterization Studies
3.8. Mechanism of Drug Release Kinetics
3.9.1. Physical Characterization of the Buccal Film Loaded With Lamivudine Polymeric Nanoparticles The buccal film containing SCMC and PVP (3:2 ratio) loaded with polymeric nanoparticles of Lamivudine exhibited swelling index of 101.30 ± 2.2% within 15 min and reached about 136.89 ± 8.7% at 1 h. Further exposure of films in hydration media resulted in 162.09 ± 3.6% swelling index at 2 h, followed by steady state swelling within the range from 164.58 ± 5.2% to 168.01 ± 3.7% till the end of 8 h. This could be correlated with the in vitro drug release data, wherein similar trend was observed in the profile. The tensile strength of the optimized film was found to be 65.2 ± 2.1 MPa, which is reported to be strongly dependent on the size of nanoparticles and the polysaccharide polymer in the film. Literature explain that the nanoparticles could fill in gaps in the matrix film acting as reinforcing agents, which could improve the mechanical property of the film [29,36]. The folding endurance of the film was > 300 as it had not
Mechanism of drug release for the polymeric nanoparticles, buccal films, and the nanoparticles loaded buccal film formulations was obtained by fitting the drug release data in various kinetic models to find the best fit (Supplementary data Tables 2, 3 and 4). The polymeric nanoparticles, buccal films containing drug/nanoparticles followed Korsmeyer-Peppas, Weibull and Gompertz kinetic models. KorsmeyarPeppas kinetics is generally used to explain and differentiate the drug release pathway either through Fickian or non-Fickian diffusion. Since the n values were ≤0.45 in Korsmeyer-Peppas model, Fickian type of diffusion mechanism for the drug release was confirmed. The high R2 value obtained in the Weibull kinetics model proved that the drug release occurred from the matrix type of buccal drug delivery system wherein, the drug was embedded in the polymer and released slowly. Gompertz model kinetics is used to explain the influence of solubility and intermediate drug release process. The steep increase in the initial 55
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Fig. 4. DSC thermogram of (a) Pure Lamivudine (b) Pure Eudragit E100 (c) Lamivudine polymeric nanoparticles (d) Buccal Film loaded with nanoparticles.
indicating C]C bond and the peak between 600 and 700 cm−1 showed the presence of CeS bond. Major functional groups of the drug were retained with slight shifts due to the mild interaction of film-forming polymers (SCMC and PVP) and drug-loaded polymeric nanoparticles (Eudragit E100) [45,46].
shown any cracks even after folding for > 300 times, and the percentage of elongation at break was 8.2 ± 0.5% [42]. 3.9.2. Interaction of Lamivudine with Polymers The IR spectrum of the optimized nanoparticles and its buccal film were compared with the pure Lamivudine and pure Eudragit E100 polymer (Fig. 2) to check the interactions between them. The characterized IR spectra for the pure drug (Lamivudine) exhibited peaks at 3212 cmre−1 (OeH stretch), 3327 cm−1 (NeH stretch), 1650 cm−1 (C]O), 1455 cm−1 (C]C), 1497 cm−1 (C]N), 1286 cm−1 (CeO), 592 cm−1 (CeS) and 1223 cm−1 & 1181 cm−1 (CeN), thus confirming the structure of Lamivudine [43]. The polymer Eudragit E100 displayed the characteristic peaks at 3447 cm−1 (N-CH3 stretch), 2926 cm−1 (CeH stretch), 1729 cm−1 (C]O stretch), 1455 cm−1 (H-C-H bending), 1375 cm−1 (-CH3 bending) and 1160 cm−1 (CeO stretch) for the identity of its structure [44]. In case of the nanoparticle formulation, peaks were observed at 1649.75 cm−1 (C]O), 1404.1 cm−1 (C]C), 1490.83 cm−1 (C]N), 1275.74 cm−1 (CeO), 605.50 cm−1 (CeS), 3378.53 cm−1 (OeH) and 2886.2 (NeH) cm−1 retaining the major functional groups of the drug. However, mild shifts in the wave numbers were observed due to the interaction of Eudragit E100 polymer with the drug during nanoparticle formation by the nanoprecipitation process. In the nanoparticle-loaded film peaks were observed in the range of 1650 cm−1 indicating the presence of C]O, 1400–1450 cm−1
3.9.3. Crystalline Behavior of Lamivudine in Nanoparticles and Buccal Film The XRD spectrum of pure drug (Fig. 3) showed sharp intense peaks in the range of 20–31 at 2θ scale, which indicated the high crystalline nature of the drug. In case of the polymer Eudragit, the peaks confirmed its amorphous nature. The drug loaded in the nanoparticles showed blurred peaks due to the masking of crystallinity of pure drug in a high amount of amorphous polymer Eudragit E100 in the formulation containing drug: the polymer in the ratio of 1:2. Hence, the mild solid-state transition from crystalline to semi-crystalline nature was observed in the nanoparticles [47]. In the Lamivudine nanoparticle loaded buccal film more blurred peaks with less intensity was identified. Presence of film-forming polymers SCMC and PVP along with the Eudragit E100 polymer in the nanoparticles have reduced the crystallinity of the drug. 3.9.4. Modification of Thermal Stability of the Drug in Nanoparticles and Buccal Film The DSC thermogram of pure drug Lamivudine (Fig. 4) exhibited a sharp endothermic peak at 177.5 °C [48], which depicted the melting
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Fig. 5. SEM images of (a) Pure Lamuvidine (b) Lamivudine polymeric nanoparticles (c) Buccal film loaded with Lamivudine polymeric nanoparticles.
point of the pure crystalline drug. The polymer showed an endothermic curve at 61.39 °C [49] for its transition range, and the curve was shallow which represented its amorphous nature. In case of the nanoparticles, an endothermic peak was observed at 51.8 °C corresponding to the glass transition temperature of the Eudragit E100 polymer. Further, a small blunt peak was observed in the range between 250 and 300 °C. Due to the encapsulation of the drug in the amorphous polymer (Eudragit E100) matrix and their mutual mild interactions, the melting point of the drug was not observed clearly [50]. Also, the crystallinity of the drug was reduced as confirmed through XRD report. In the nanoparticle-loaded film, only blunt peaks were observed at 112.5 °C and in range 250 to 300 °C. The crystallinity of the drug was masked due to the encapsulation of the drug in the polymeric nanoparticles and the film.
The nanoparticles maintained 95% cell viability up to the concentration of 10 μg/mL. With further increase in the concentration from 20 to 50 μg/mL, there was a notable reduction in the cell viability from 86% to 62%, respectively (Fig. 6 (g)). The increase in cytotoxicity at higher concentration may be due to enhanced cell permeation of the nanoparticles because of its smaller size and effective surface area [53]. As there was an increase in toxicity beyond 10 μg/mL concentration, a lower dose of the drug could be preferred for the safe treatment, which could be achieved through controlled drug delivery system. Since the polymeric nanoparticles loaded in buccal films provided sustained release of Lamivudine to maintain the therapeutic concentration of the drug, the overall dose required for the therapy could be reduced. Hence, the high dose-related side effects could be significantly avoided, especially for the chronic anti-HIV therapy in children.
3.9.5. Morphology Changes of a Drug in Nanoparticles and Buccal Film The SEM images (Fig. 5) of pure drug displayed flakes like structure with irregular shape and size. In case of the nanoparticles loaded film, spherical nanoparticles structures were observed with the smooth surface matrix film. The nanoparticles exhibited varying size from 90 nm to 216 nm [51].
4. Conclusion The polymeric nanoparticles of Lamivudine containing drug: polymer (Eudragit E100) in the ratio of 1:2 with average particle size of 338 nm and polydispersity index of 0.315 was selected as optimized formulation because of maximum drug entrapment (94%) and prolonged drug release of 89% for 4 h was obtained. The polymeric nanoparticle was loaded in the buccal film formulation containing polymers SCMC and PVP in the ratio of 3:2 which showed in vitro drug release of 49% initially and gradually increased to achieve 93% at the end of 8 h. The presence of hydrophobic polymeric nanoparticles helped in the reduction of hydrophilicity of the drug, thereby attaining sustained drug release profile. The characterization studies like XRD, DSC, FTIR, and FESEM confirmed the mild interactions between the polymer and the drug without significant changes in the stability of the drug. Hence, polymeric nanoparticles loaded buccal film of Lamivudine
3.10. Cytotoxicity Effect of Lamivudine Polymeric Nanoparticles The viability of the cells (control Fig. 6 (a)) treated with selected formulation of Lamivudine polymeric nanoparticles at concentration of 10, 20, 30, 40 and 50 μg/mL through the MTT assay is shown in Fig. 6 (b) to Fig. 6 (f), respectively. The yellow colour tetrazolium dye used for the MTT assay was reduced by the mitochondria of live cells to purple coloured formazan product. Hence, the number of viable cells was directly proportional to the amount of formazan generated [52].
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Fig. 6. Viability of the Vero cells treated with Lamivudine polymeric nanoparticles at concentration of (a) Control (b) 10 μg/mL (c) 20 μg/mL (d) 30 μg/mL (e) 40 μg/ mL (f) 50 μg/mL (g) Cytotoxicity analysis of Lamivudine-Eudragit E100 nanoparticles (MTT assay) (n = 3).
could be used to prolong the drug release and reduce the frequency of drug administration in paediatric patients, thereby improving the patient compliance.
Conflicts of Interest
Acknowledgement
Appendix A. Supplementary data
The authors acknowledge SASTRA Deemed University, Thanjavur for providing infrastructure facilities (Central Research Facility and Funds for Research & Modernization in SASTRA) and online resources required for this research. The authors would like to thank DST-FIST programme (SR/FST/ETI331/2013), DST-SERB (ECR/2016/001856) and SASTRA Deemed University, Thanjavur, India for infrastructure support.
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.colcom.2018.10.004.
All the authors declare no conflicts of interests.
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Design of antiretroviral drug-polymeric nanoparticles laden buccal films for chronic HIV therapy in paediatrics R. Sneha, B.N. Vedha Hari, D. Ramya Devi
T
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School of Chemical & Biotechnology, SASTRA Deemed University, Thanjavur 613401, Tamil Nadu, India
ARTICLE INFO
ABSTRACT
Keywords: Buccal film Nanoparticles Lamivudine Eudragit E100 HIV
Anti-HIV drug Lamivudine exhibit shorter half-life (2 h) and poor bioavailability (62% in paediatric patients), multiple dosing is required. To overcome the limitations, nanoparticles loaded buccal film was developed for prolonged drug release and improved efficiency. Nanoprecipitated Lamivudine-Eudragit E100 polymeric nanoparticles exhibited average particle size of 338 nm with polydispersity index 0.315. Buccal films prepared by solvent casting technique using sodium carboxy methyl cellulose and polyvinyl pyrrolidone (3:2) loaded with the nanoparticles showed initial drug release of 49% followed by sustained release to reach the maximum of 93% at the end of 8 h. Encapsulation of drug in nanoparticles and the film was confirmed through scanning electron microscopy, thermal and crystal characterization. Sustained drug release through Fickian diffusion mechanism from the matrix system was confirmed. The optimized formulation could be used to enhance therapeutic effect at reduced dose and side effects, especially for the paediatric anti-HIV therapy.
1. Introduction HIV has taken the lives of millions of people around the world making it as a one of the most serious global crisis. According to the report of Joint United Nations Programme on HIV and AIDS (UNAIDS), approximately 36.7 million people worldwide are living with HIV/ AIDS, out of which 2.1 million are children below the age of 15 years [1]. As per UNAIDS data, In India 2.1 million people are living with HIV, and among them 693,000 patients are children. The mortality of children is comparatively higher than adults because of their weak immune system and lack of proper treatment [2]. HIV infects the vital cells in the immune system such as macrophages, dendritic cells, and CD4+ T cells. The rate of virus progression varies widely between individuals and depends on many factors like age of the individual, body's ability to defend against HIV, the presence of other infections, individual's genetic inheritance, resistance to certain strains of HIV, etc. The increase in viral load leads to decline in the number of CD4+ cells leading to the loss of cell-mediated immunity, making the body more susceptible to various types of infections, which in overtime leads to Acquired Immunodeficiency Syndrome (AIDS) [1,3]. Although there is no complete cure for HIV Infection, symptoms of the disease can be reduced and the life of the individual can be prolonged by administration of different classes of antiretroviral drugs [4–6]. Most of the antiretroviral drugs available are not suitable for
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paediatric patients because of higher dosage and toxicity associated [7]. Lamivudine belongs to the class of Nucleoside Reverse Transcriptase Inhibitors (NRTIs), which is phosphorylated to active metabolites that compete for incorporation into viral DNA, thereby preventing the multiplication of viral DNA in the host. It is one of the preferred firstline medications for paediatric patients [8], based on the safety profile observed in the clinical trials. The commercially available dosage form for administration is oral tablet and oral solution (Syrup/suspension) and the dosage vary according to the age and body weight of the children, recommended as 4 mg/kg/day up to the maximum of 300 mg/ day for 35 kg [9,10]. The limitations of the drug are the low half-life of 2 h and oral bioavailability of 68% in paediatric patients [8]. To maintain the therapeutic level, multiple dose administration of Lamivudine is required that leads to overdosing and related side effects such as headache, nausea, diarrhoea, abdominal pain and skin rash, which reduces patient compliance especially in children [11,12]. The nanoparticles provide better encapsulate of drugs and could provide sustained release with reduced dose and toxicity [13–15]. The drug can directly reach the systemic circulation by direct contact with buccal mucosa which is rich in blood supply and also avoids the gastrointestinal degradation. The two major pathways for drug permeation across the buccal mucosa are the paracellular route for hydrophilic solutes and transcellular route for lipophilic solutes [16]. Mucoadhesive polymers like Polyvinyl Alcohol (PVA), Polyvinyl Pyrrolidone (PVP),
Corresponding author. E-mail address: [email protected] (D. Ramya Devi).
https://doi.org/10.1016/j.colcom.2018.10.004 Received 28 July 2018; Received in revised form 22 October 2018; Accepted 27 October 2018 2215-0382/ © 2018 Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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Sodium carboxymethylcellulose (SCMC), hydroxypropyl methylcellulose (HPMC) and sodium alginate (SA) are commonly used for the films preparation, which could be held on the surface of the buccal mucosa by interfacial force. Mucoadhesion is generally referred to the interaction between the polymer and the epithelial surface, which could improve the drug absorption and bioavailability in the system [17,18]. Buccal route drug delivery improves the absorption and maintains the therapeutic level of drug in plasma for a prolonged period of time [19]. The buccal film is mostly preferred than a buccal tablet as it is more convenient and comfortable for paediatric patients [17]. With this background and to overcome the challenges of Lamivudine therapy in children, the present work focus on development of buccal films loaded with polymeric nanoparticles of the drug that could provide burst effect followed for sustained release of the drug.
2.3. Particle Size and Zeta Potential The particle size and zeta potential of the prepared nanoparticles were analyzed using Zeta-sizer to determine size distribution and the stability of the particles. The Zeta-sizer (Nano series, Malvern, UK) uses dynamic light scattering principle that measures Brownian motion and relates to the particle size. In this, the particle is illuminated with a laser and the intensity of fluctuation in scattered light is analyzed [24]. 2.4. Entrapment Efficiency Accurately 0.5 mL of polymeric nanosuspension was centrifuged (C24 plus, REMI, India) at 12,000 rpm for 20 min to separate the pellet containing the polymeric nanoparticles of the drug and the supernatant with the unentrapped drug. The supernatant solution was suitably diluted with phosphate buffer (pH 6.8) and the absorbance was measured using UV-Spectrophotometer at 270 nm (λmax of drug) using the respective blank. The percentage of the entrapped drug was calculated using the formula, Total drug content Drug content in supernatant %Entrapment Efficiency = × 100 Total drug content
2. Materials and Methods 2.1. Materials Lamivudine was obtained as gift sample from Aurobindo Pharma Ltd., India. Eudragit E100 was obtained as gift sample from Evonik Nutrition & Care GmbH, Germany. Polymers like polyvinyl chloride, polyvinyl pyrrolidone, hydroxypropyl methyl cellulose, sodium carboxymethylcellulose, sodium alginate were purchased from SD Fine Chem. Pvt. Ltd., India. All the chemicals and reagents used were of analytical grade.
2.5. In Vitro Drug Release Study of Lamivudine Polymeric Nanoparticles The in vitro drug release studies of the developed Lamivudine polymeric nanoparticle formulations were performed using dialysis membrane method. About 1 mL of the formulation was loaded into the dialysis bag (Dialysis membrane-110, Himedia) and kept in a vial containing 10 mL of Phosphate buffer pH 6.8. The vial was kept in a beaker containing water maintained at 37 °C and placed in a magnetic stirrer at 100 rpm. Samples were collected at regular intervals for 8 h and replaced with 10 mL of buffer to maintain the perfect sink conditions. The collected samples were analyzed using UVSpectrophotometer (Evolution 201, Thermo Scientific, USA) at 270 nm [24].
2.2. Preparation of Lamivudine Loaded Eudragit E100 Nanoparticles Nanoparticles were prepared by nanoprecipitation method which is also known as interfacial deposition or solvent-displacement method [20]. The polymer selected for preparation of nanoparticles was Eudragit E100, which is a cationic hydrophobic polymer [21]. The concentration of the drug (Lamivudine) and surfactant (Pluronic F127) was kept constant and the concentration of the polymer (Eudragit E100) was varied at three different levels (Table 1). In this process, the drug was dissolved in 10 mL aqueous solution containing 0.5% Pluronic F127 as a surfactant, which could reduce the surface tension between the aqueous and organic phase and thereby stabilize the nanoparticle suspension formation while preventing the aggregation of fine particle. The mixture was placed on a magnetic stirrer at a constant stirring rate of 1500 rpm. The polymer was dissolved in 5 mL of volatile organic solvent (methanol) and was added dropwise in the aqueous solution. The solution was kept under stirring until the organic solvent was completely diffused and evaporated, resulting in the precipitation and formation of polymeric nanoparticles of the drug [22]. In order to ensure high stability of the nanoparticles and complete removal of the solvents (water and methanol) [23], the nanosuspension was lyophilized (Christ Alpha 2–4 LD Plus, Osterode Am Harz, Germany) for 48 h. The formulated nanosuspension was ultracentrifuged (C24 plus, REMI, India) at 15,000 rpm and − 4 °C for 20 min to obtain the pellet, which was re-dispersed in distilled water using cyclone mixer (Remi CM101 DX, Mumbai, India) to get uniform nanoparticles aqueous dispersion and then freeze-dried.
2.6. Characterization of Polymeric Nanoparticles 2.6.1. Fourier-Transform Infra-Red (FT-IR) Spectroscopy Drug-polymer interactions due to chemical bonding were studied using FTIR (6701F, Perkin Elmer, USA) by potassium bromated (KBr) disc/pellet technique. The infrared spectrum of the nanoparticles was compared with pure drug and pure polymers. The samples were mixed with dried and saturated potassium bromate. The discs were obtained by compressing the powder under a hydraulic pressure of 150 kg/cm2 for 5 min. The pellet was placed in the sample holder and scanned between the wave number range of 4000–400 cm−1 [24]. 2.6.2. X-Ray Diffraction (XRD) Analysis XRD Analysis was carried out to determine the crystallinity of the drug in the nanoparticle formulation by constructive interference of monochromatic X-ray. The nature of the sample was studied at room temperature using X-Ray Diffractometer (D8 Focus, Bruker, Germany) over a 2θ range at 0 °C to 80 °C with the current of 30 mA and voltage of 40 kV.
Table 1 Preparation and Physico chemical characterization of Lamivudine loaded Eudragit E100 Polymeric Nanoparticles. S. No.
Lamivudine: Eudragit E100
Average size (nm)
Poly dispersity index
Zeta potential (mV)
Encapsulation efficiency (%)
1 2 3
1: 0.5 1: 1 1: 2
147.9 228.7 338.6
0.154 0.201 0.315
+3.89 +5.47 +7.06
72.3 90 94.4
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2.6.3. Differential Scanning Calorimetry (DSC) Differential Scanning Calorimetry was used to characterize the thermal behavior of drug and the nanoparticles. The DSC thermogram (Q100, TA Instruments, USA) was obtained by automatic thermal analyzer technique, in which the sample was sealed in an aluminum pan and an empty aluminum pan was taken as reference. The sample was heated at the rate of 10 °C/min, from the range of 25 °C to 300 °C [24].
2.9. Preparation of Lamivudine Polymeric Nanoparticles Loaded Buccal Film
2.6.4. Field Emission Scanning Electron Microscopy (FESEM) The surface morphology of the drug and polymeric nanoparticles was analyzed by FESEM. The sample was loaded into the instrument and scanned by electrons liberated by field emission source. The images were taken at the magnification ranging from 10,000× to 30,000× at an accelerating voltage of 3 kV [24].
2.10. Determination of Drug Content
The required amount of polymeric nanoparticles to achieve 4 mg equivalent dose of the drug in each film (with area of 2 cm2) was added to the film-forming polymeric solution and the mixture was bath sonicated to obtain a homogeneous dispersion. The film was formed using similar mould dimensions by the previously explained solvent casting technique [19].
The drug content in the buccal film was determined by dissolving the film in 50 mL phosphate buffer pH 6.8 which was kept overnight. The solutions were then transferred to volumetric flasks and appropriate dilutions were made with phosphate buffer. Then the resulting solution was filtered with Whatman filter paper to remove undissolved polymer and then drug content was analyzed using UV–Visible spectrometer at 270 nm. The experiment was conducted in triplicate and the results represented as mean ± SD [14,19].
2.7. Preparation of Buccal Film With Mucoadhesive Polymers Buccal films were prepared using circular moulds with 3.8 cm2 area by solvent casting technique. The films were prepared using the mucoadhesive polymers like HPMC, SCMC, SA, PVA, PVP, and Carbopol 934p in various ratios, so that a total of 37 formulations were trialed experimentally (Supplementary data Table 1). The polymer was dissolved in a measured volume of water and ethanol in addition with 1% of propylene glycol as plasticizer. The polymeric solution was homogenized using bath sonicator and poured onto the moulds and then placed in a hot air oven at 55 °C for 5 to 6 h followed by air drying for an hour. The dried film was carefully removed from the mould and checked for folding endurance and stability. Formulations with better physical and thermal stability (ability to peel off from mould without brittleness, colour integrity, uniformity of thickness) and folding endurance were chosen for loading the pure drug / polymeric nanoparticles [25,26].
2.11. In Vitro Drug Release Study of Lamivudine/Polymeric Nanoparticles Loaded Buccal Film The drug release was carried out for each buccal film (containing pure Lamivudine / polymeric nanoparticles) and the amount of drug release was studied using USP type V (Paddle over disc) dissolution method. The buccal film was placed below the glass disc kept at the bottom of the vessel and the paddle was positioned above the disc [27]. About 100 mL of phosphate buffer pH 6.8 was taken as the media with constant stirring at 50 rpm and the temperature was maintained at 37 ± 2 °C. Accurately 10 mL of the samples were collected at predetermined intervals and replaced with 10 mL of fresh media to maintain the sink conditions. The collected samples were quantified for cumulative percentage of drug release by UV-Spectrophotometer at 270 nm [14,19]. The study was carried out for 8 h and the average of three trials was calculated for plotting the cumulative percentage of drug release vs. time profile.
2.8. Preparation of Pure Lamivudine Loaded Buccal Film Among the 37 trial buccal films, only 9 formulations (Table 2) with good stability and folding endurance were chosen for loading the drug. Circular moulds with radius of 0.8 cm was used for the casting method, so that each buccal films loaded with 4 mg of Lamivudine (least oral dose in children) with the area of 2 cm2 was fabricated. The total amount of drug needed for formulation of 12 films was accurately weighed and dissolved in distilled water added to the film-forming polymeric solution along with propylene glycol as the plasticizer. This solution was homogenized completely followed by bath sonication to remove air bubbles. The required volume of mixture (to obtain 4 mg dose in each film) was poured on the moulds and kept in a hot air oven at 55 °C for 5 to 6 h, followed by air drying for an hour [19]. The dried film was carefully removed from the moulds and stored in desiccator for further characterization studies.
2.12. In Vitro Drug Release Kinetics To understand the kinetics of the drug release from the formulations, the data obtained from in vitro release studies were fitted into different mathematical models such as zero order, first order, Korsmeyer-Peppas, Higuchi, Hixson-Crowell, Hopfenberg, BakerLonsdale, Weibull, Makoid-Banakar and Gompertz models using DD solver software [28]. 2.13. Characterization of Lamivudine Polymeric Nanoparticles Loaded Buccal Film The physical characteristics of the optimized buccal film loaded with the polymeric nanoparticles of Lamivudine was analyzed through swelling index, tensile strength, elongation at break and folding endurance. The swelling property of the selected buccal film was evaluated by hydration method, wherein the film was immersed in a 10 mL of distilled water and the initial weight of the dry film (W1) before immersion and the weight of swelled film (W2) after immersion was noted at periodic time intervals for 8 h. Swelling index at each time point was calculated from following equation. W W Swelling Index (%) = 2W 1 × 100 1 An Instron Universal Testing Machine (Model 2519–104, Instron Corp., USA) was used to determine the tensile strength and percentage elongation at break [29]. The folding endurance was measured manually by folding the film repeatedly at a point till it broke. All the
Table 2 Formulation and evaluation of Lamivudine loaded buccal films. S. No.
Polymer ratio in buccal film
Ratio
Drug content (%)
1 2 3 4 5 6 7 8 9
HPMC: PVP HPMC: PVA HPMC: SA HPMC: SCMC SCMC: SA SCMC: SA SCMC: PVP SCMC: PVA SCMC: PVA
3: 4: 3: 1: 4: 3: 3: 4: 3:
100 ± 2.30 86.32 ± 6.52 82.50 ± 7.83 92.01 ± 6.99 96.66 ± 2.50 87.96 ± 1.36 86.03 ± 3.01 82.58 ± 4.65 93.36 ± 5.76
1 1 2 1 1 2 2 1 2
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experiments were carried out in triplicate and the results presented as mean with standard deviation. The optimized polymeric nanoparticles loaded buccal film was subjected to analytical characterization studies like XRD, DSC, and FESEM (similar method as mentioned for the pure drug and nanoparticles) to evaluate the stability, drug-polymer interactions and morphology. The IR study of the film was performed through ATR (Attenuated total reflection) mode. In this method the film was placed on the ATR crystal and beam of infrared light was passed through it. The changes in the internally reflected IR beam when it comes in contact with the sample were measured.
flowing dry powder for improved stability and further characterization studies. 3.2. Encapsulation Efficiency in Nanoparticles at Varying Polymer Level The amount of drug entrapped within synthesized Lamivudine loaded Eudragit E100 nanoparticles were analyzed using UV visible spectrophotometer at 270 nm and shown in Table 1. The percentage encapsulation efficiency of Lamivudine in Eudragit E100 polymer ranged from 72.3 to 94.4%. Nanoparticles prepared with Lamivudine: Eudragit E100 in the ratio 1:2 showed maximum drug encapsulation, which could be due to the requirement of a higher proportion of the hydrophobic polymer for the complete entrapment of the hydrophilic drug [32]. The result could be approved, since Sadoun et al. [33] had previously reported an increase in the drug entrapment due to increase in the polymer concentration because, the increase in the organic phase viscosity promotes the formation of higher microparticles that could consequently entrap higher quantity of drug. Similar pattern of encapsulation was observed in Eudragit nanoparticle loaded with an antihypertensive drug Valsartan, wherein 1:5 ratio of drug and polymer have shown high entrapment efficiency than 1:2.5 ratio [34].
2.14. Cytotoxicity Studies of the Polymeric Nanoparticles The cytotoxicity of the selected nanoparticles formulation was evaluated using Green monkey kidney cells (Vero) cells [30] procured from the National Center for Cell Sciences (NCCS), Pune, India through MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The cells were maintained in Minimal Essential medium (MEM) supplemented with 2 mM L-glutamine and balanced salt solution (BSS) adjusted to contain 1.5 g/L Na2CO3, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 1.5 g/L glucose, 10 mM (4(2-hydroxyethyl)-1-piperazineethane sulfonic acid) (HEPES) and 10% fetal bovine serum (GIBCO, USA). Penicillin and streptomycin (100 IU/ 100 μg) were adjusted to 1 mL/L. The cells were maintained at 37 °C with 5% CO2 in a humidified CO2 incubator. The cells were grown in a 96-well plate for 48 h into 75% confluence. To make single cell suspensions, the monolayer cells were treated with trypsin-ethylene diamine tetraacetic acid (EDTA) to detach the cells. The viability of cells was counted using hemocytometer and the cell suspension was diluted using a medium containing 5% FBS to make the final density of 1 × 104 cells/mL. The medium was replaced with fresh medium containing serially diluted test samples with drug concentrations of 10, 20, 30, 40 and 50 μg/mL and the cells were further incubated for 48 h. The culture medium was removed, and 100 μL of the MTT [3-(4,5-dimethylthiozol2-yl)-3,5-diphenyl tetrazolium bromide] (Hi-Media) solution was added to each well and incubated at 37 °C for 4 h. After removal of the supernatant, 50 μL of DMSO was added to each of the wells and incubated for 10 min to solubilize the formazan crystals. The optical density was measured at 620 nm in an ELISA multiwell plate reader (Thermo Multiskan EX, USA). The OD value was used to calculate the percentage of viability using the following formula,
%Cell Viability =
3.3. In Vitro Drug Release Study of Lamivudine Polymeric Nanoparticles The in vitro drug release profile of Lamivudine polymeric nanoparticles was displayed in Fig. 1 (a). The nanoparticles containing drug: the polymer ratio 1: 0.5 showed drug release of 58% within first 15 min and reached the maximum of 87% at the end of 1.5 h. In case of the formulations with ratio 1:1 and 1:2, the initial drug release was found to be around 60%. The complete drug release of 82% was achieved at the end of 2 h in a formulation containing 1:1 ratio. Whereas, the drug release was sustained for 4 h in the formulation with ratio 1: 2 which reached the maximum of 89%. As the polymeric concentration was increased, drug release was prolonged over a time. It was due to the encapsulation of drug in a proportionately higher amount of polymer matrix and also because of low solubility of the Eudragit E100 polymer in pH 6.8 media, which reduces the dissolution of the drug resulting in a prolonged release [32]. 3.4. Drug Content of Buccal Films The drug content for the selected 9 buccal film formulations was analyzed using UV–Visible Spectrophotometer at 270 nm and the results tabulated in Table 2. The drug content ranged from 82% to 100%. The film formulation with polymer combination of HPMC and PVP in the ratio of 3:1 showed the maximum amount of drug content. As the drug was more uniformly dispersed in the hydrophilic polymer matrix film of HPMC/PVP, maximum amount of drug was loaded in this formulation [35]. The blending of HPMC with PVP in films has been reported to decrease the crystallinity of HPMC, which could also favor the loading of high amount of drug [36]. Also, significant increase in the thickness, drug content and mass of the films containing PVP has been observed by Maheswari et al. as compared to the films without PVP [37].
[A]test × 100 [A]control
where [A]test is the absorbance of the test sample and [A]control is the absorbance of the control [31]. 3. Results and Discussion 3.1. Particle Size Distribution and Stability of Lamivudine-Eudragit E100 Nanoparticles The average size and the zeta potential of the nanoparticles prepared with three different levels of Lamivudine: Eudragit E100 ratio was evaluated and reported in Table 1. The average size range was between 140 and 340 nm and Polydispersity index < 0.4 indicated the monodisperse nature of the preparation. The particle size increased gradually with increase in the concentration of the polymer (Eudragit E100). The maximum zeta potential of +7.06 was achieved by the formulation containing 1:2 ratio of drug: polymer. However, this value does not confirm the dynamic stability of the nanosuspension. Hence, lyophilization was performed to convert the nanosuspension into free-
3.5. In Vitro Drug Release Study for Lamivudine Buccal Films The in vitro drug release profile of the buccal films loaded with pure Lamivudine was displayed in Fig. 1 (b, c, d). The drug release from the film containing the polymer combination of HPMC and SA in the ratio of 3:2 showed 84.5% drug release in first 15 min and attained the maximum of 94% at the end of 3 h. The formulation containing HPMC and SCMC in the ratio of 1:1 showed 70% release initially and gradually
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Fig. 1. In vitro drug release profile in phosphate buffer pH 6.8 (a) Lamivudine polymeric nanoparticles (b) Buccal films containing HPMC and pure drug (c) Films containing SCMC and pure drug (d) Films containing SCMC with PVA and pure drug (e) Lamivudine polymeric nanoparticles loaded Buccal films (f) Comparison of optimized Buccal film formulation to the pure drug.
increased up to 87% at the end of 8 h. The formulations containing HPMC: PVA in ratio 4:1 and HPMC: PVP in ratio 3:1 showed drug release of 55% and 41% at the end of 8 h, respectively (Fig. 1 (b)). The retarded drug release from these polymeric combination buccal films was due to the increase in the concentration of HPMC. As HPMC is a swellable polymer, the polymer matrices could be eroded slowly and the drug was released only after polymer swelling [38]. There is an inverse relationship between the rate of polymer matrix swelling and drug release [39]. Therefore, as the concentration of HPMC increases, the swelling rate increases which results in the retarded drug release. The formulation with the polymeric combination of SCMC and SA in ratio 4:1 reached 100% drug release (Fig. 1 (c)) within first 15 min interval. The film containing SCMC: PVP in the ratio of 3:2 showed 77% initial drug release and reached 92% at the end of 8 h. Similarly, SCMC: SA in ratio 3:2 showed drug release of 67% initially and reached 82% at the end of 8 h. The drug release percentage of these two formulations (SCMC: SA & SCMC: PVP) in ratio 3:2 are almost similar due to the presence of an equal concentration of swelling polymer (SCMC) and
hydrophilic polymers SA & PVP. After the swelling of SCMC polymer matrix, PVP and SA which is also present in the swelling matrix may be dissolved rapidly in phosphate buffer media [40], which may create pores in the matrix. The drug release of the formulation containing a polymeric combination of SCMC and PVA in different ratios of 3:2 and 4:1 showed drug release of 70% initially (Fig. 1 (d)). At the end of 8 h the drug release of the formulation with 3:2 ratio was 88% and that of 4:1 was 79%. The slight variation in the drug release pattern was influenced by PVA concentration which could dissolve in the phosphate buffer pH 6.8 because of its hydrophilic nature [40]. The SCMC polymer swells in the media and the PVA entrapped in the swelling matrix creates pores through which the drug may be released. Higher the concentration of PVA, more the pore formation which could increase the percentage of drug release. Thus, films containing SCMC: PVA in the ratio of 3:2 showed higher drug release percentage. Based on the obtained in vitro release profile, the buccal films prepared with SCMC:PVA (3:2), SCMC:PVP (3:2) and HPMC:SA (3:2),
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Fig. 2. FTIR spectrum of (a) Pure Lamivudine (b) Pure Eudragit E100 (c) Lamivudine polymeric nanoparticles (d) Buccal Film loaded with nanoparticles.
which exhibited complete release of the drug (> 90% at the end of 8 h) were selected as optimized formulations for the incorporation of the Lamivudine polymeric nanoparticles. Due to the hydrophilic nature of the drug, the maximum amount of drug was released from the films within the first 2 to 3 h. In order to improve the bioavailability and halflife of the drug, a sustained release pattern of drug release is required. To achieve the sustained drug release profile, the optimized hydrophobic polymeric nanoparticles were incorporated into the selected films.
two formulations (SCMC & PVA, HPMC & SA) showed the maximum release of only 70% and 40% respectively at the end of 8 h. The interaction of the polymeric nanoparticles and film-forming polymers retarded the drug release in these formulations. Since the half-life of Lamivudine is only 2 h, when the high percentage drug release is reached early, the drug will be eliminated from the body in short time period. The interaction of hydrophobic polymer and hydrophilic drug in the nanoparticles reduced the solubility of the drug. The decline in solubility has reduced the dissolution rate of the drug which could provide slow drug release over a period of time. The combination of the nanoparticles and the film-forming polymers (SCMC and PVP) provided the sustained drug release profile.
3.6. In Vitro Drug Release Study of the Buccal Film Loaded With Polymeric Nanoparticles The in vitro drug release profile of the selected buccal films loaded with the optimized polymeric nanoparticles of Lamivudine was displayed in Fig. 1 (e). Films containing SCMC and PVP in ratio 3:2 loaded with polymeric nanoparticles showed drug release of 48% initially and gradually increased in a sustained manner over a period of time and reached 93% at the end of 8 h. The presence of polymeric nanoparticles showed a minimal amount of drug release initially which slowly increased over a period of time attaining the maximum release. The other
3.7. Comparison of the In Vitro Release Profile the Optimized Formulation With Pure Drug The drug release of the pure drug and the buccal film containing SCMC: PVP in ratio 3:2 loaded with polymeric nanoparticles was compared. The pure drug showed 100% drug release within 15 min whereas the optimized buccal film showed a sustained drug release from 48% to 93% at 1 h to 8 h (Fig. 1 (f)). Hence, the extended drug
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Fig. 3. XRD spectrum of (a) Pure Lamivudine (b) Pure Eudragit E100 (c) Lamivudine polymeric nanoparticles (d) Buccal Film loaded with nanoparticles.
release could be achieved in vivo, which could maintain the therapeutic concentration of drug in plasma for a prolonged period of time. This could ultimately improve the bioavailability of the drug and reduce the overall dose required for the therapy in children.
drug release followed by slow converged release to reach maximum dissolution of the drug from the buccal films was explained by the Gompertz kinetics [41]. 3.9. Characterization Studies
3.8. Mechanism of Drug Release Kinetics
3.9.1. Physical Characterization of the Buccal Film Loaded With Lamivudine Polymeric Nanoparticles The buccal film containing SCMC and PVP (3:2 ratio) loaded with polymeric nanoparticles of Lamivudine exhibited swelling index of 101.30 ± 2.2% within 15 min and reached about 136.89 ± 8.7% at 1 h. Further exposure of films in hydration media resulted in 162.09 ± 3.6% swelling index at 2 h, followed by steady state swelling within the range from 164.58 ± 5.2% to 168.01 ± 3.7% till the end of 8 h. This could be correlated with the in vitro drug release data, wherein similar trend was observed in the profile. The tensile strength of the optimized film was found to be 65.2 ± 2.1 MPa, which is reported to be strongly dependent on the size of nanoparticles and the polysaccharide polymer in the film. Literature explain that the nanoparticles could fill in gaps in the matrix film acting as reinforcing agents, which could improve the mechanical property of the film [29,36]. The folding endurance of the film was > 300 as it had not
Mechanism of drug release for the polymeric nanoparticles, buccal films, and the nanoparticles loaded buccal film formulations was obtained by fitting the drug release data in various kinetic models to find the best fit (Supplementary data Tables 2, 3 and 4). The polymeric nanoparticles, buccal films containing drug/nanoparticles followed Korsmeyer-Peppas, Weibull and Gompertz kinetic models. KorsmeyarPeppas kinetics is generally used to explain and differentiate the drug release pathway either through Fickian or non-Fickian diffusion. Since the n values were ≤0.45 in Korsmeyer-Peppas model, Fickian type of diffusion mechanism for the drug release was confirmed. The high R2 value obtained in the Weibull kinetics model proved that the drug release occurred from the matrix type of buccal drug delivery system wherein, the drug was embedded in the polymer and released slowly. Gompertz model kinetics is used to explain the influence of solubility and intermediate drug release process. The steep increase in the initial 55
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Fig. 4. DSC thermogram of (a) Pure Lamivudine (b) Pure Eudragit E100 (c) Lamivudine polymeric nanoparticles (d) Buccal Film loaded with nanoparticles.
indicating C]C bond and the peak between 600 and 700 cm−1 showed the presence of CeS bond. Major functional groups of the drug were retained with slight shifts due to the mild interaction of film-forming polymers (SCMC and PVP) and drug-loaded polymeric nanoparticles (Eudragit E100) [45,46].
shown any cracks even after folding for > 300 times, and the percentage of elongation at break was 8.2 ± 0.5% [42]. 3.9.2. Interaction of Lamivudine with Polymers The IR spectrum of the optimized nanoparticles and its buccal film were compared with the pure Lamivudine and pure Eudragit E100 polymer (Fig. 2) to check the interactions between them. The characterized IR spectra for the pure drug (Lamivudine) exhibited peaks at 3212 cmre−1 (OeH stretch), 3327 cm−1 (NeH stretch), 1650 cm−1 (C]O), 1455 cm−1 (C]C), 1497 cm−1 (C]N), 1286 cm−1 (CeO), 592 cm−1 (CeS) and 1223 cm−1 & 1181 cm−1 (CeN), thus confirming the structure of Lamivudine [43]. The polymer Eudragit E100 displayed the characteristic peaks at 3447 cm−1 (N-CH3 stretch), 2926 cm−1 (CeH stretch), 1729 cm−1 (C]O stretch), 1455 cm−1 (H-C-H bending), 1375 cm−1 (-CH3 bending) and 1160 cm−1 (CeO stretch) for the identity of its structure [44]. In case of the nanoparticle formulation, peaks were observed at 1649.75 cm−1 (C]O), 1404.1 cm−1 (C]C), 1490.83 cm−1 (C]N), 1275.74 cm−1 (CeO), 605.50 cm−1 (CeS), 3378.53 cm−1 (OeH) and 2886.2 (NeH) cm−1 retaining the major functional groups of the drug. However, mild shifts in the wave numbers were observed due to the interaction of Eudragit E100 polymer with the drug during nanoparticle formation by the nanoprecipitation process. In the nanoparticle-loaded film peaks were observed in the range of 1650 cm−1 indicating the presence of C]O, 1400–1450 cm−1
3.9.3. Crystalline Behavior of Lamivudine in Nanoparticles and Buccal Film The XRD spectrum of pure drug (Fig. 3) showed sharp intense peaks in the range of 20–31 at 2θ scale, which indicated the high crystalline nature of the drug. In case of the polymer Eudragit, the peaks confirmed its amorphous nature. The drug loaded in the nanoparticles showed blurred peaks due to the masking of crystallinity of pure drug in a high amount of amorphous polymer Eudragit E100 in the formulation containing drug: the polymer in the ratio of 1:2. Hence, the mild solid-state transition from crystalline to semi-crystalline nature was observed in the nanoparticles [47]. In the Lamivudine nanoparticle loaded buccal film more blurred peaks with less intensity was identified. Presence of film-forming polymers SCMC and PVP along with the Eudragit E100 polymer in the nanoparticles have reduced the crystallinity of the drug. 3.9.4. Modification of Thermal Stability of the Drug in Nanoparticles and Buccal Film The DSC thermogram of pure drug Lamivudine (Fig. 4) exhibited a sharp endothermic peak at 177.5 °C [48], which depicted the melting
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Fig. 5. SEM images of (a) Pure Lamuvidine (b) Lamivudine polymeric nanoparticles (c) Buccal film loaded with Lamivudine polymeric nanoparticles.
point of the pure crystalline drug. The polymer showed an endothermic curve at 61.39 °C [49] for its transition range, and the curve was shallow which represented its amorphous nature. In case of the nanoparticles, an endothermic peak was observed at 51.8 °C corresponding to the glass transition temperature of the Eudragit E100 polymer. Further, a small blunt peak was observed in the range between 250 and 300 °C. Due to the encapsulation of the drug in the amorphous polymer (Eudragit E100) matrix and their mutual mild interactions, the melting point of the drug was not observed clearly [50]. Also, the crystallinity of the drug was reduced as confirmed through XRD report. In the nanoparticle-loaded film, only blunt peaks were observed at 112.5 °C and in range 250 to 300 °C. The crystallinity of the drug was masked due to the encapsulation of the drug in the polymeric nanoparticles and the film.
The nanoparticles maintained 95% cell viability up to the concentration of 10 μg/mL. With further increase in the concentration from 20 to 50 μg/mL, there was a notable reduction in the cell viability from 86% to 62%, respectively (Fig. 6 (g)). The increase in cytotoxicity at higher concentration may be due to enhanced cell permeation of the nanoparticles because of its smaller size and effective surface area [53]. As there was an increase in toxicity beyond 10 μg/mL concentration, a lower dose of the drug could be preferred for the safe treatment, which could be achieved through controlled drug delivery system. Since the polymeric nanoparticles loaded in buccal films provided sustained release of Lamivudine to maintain the therapeutic concentration of the drug, the overall dose required for the therapy could be reduced. Hence, the high dose-related side effects could be significantly avoided, especially for the chronic anti-HIV therapy in children.
3.9.5. Morphology Changes of a Drug in Nanoparticles and Buccal Film The SEM images (Fig. 5) of pure drug displayed flakes like structure with irregular shape and size. In case of the nanoparticles loaded film, spherical nanoparticles structures were observed with the smooth surface matrix film. The nanoparticles exhibited varying size from 90 nm to 216 nm [51].
4. Conclusion The polymeric nanoparticles of Lamivudine containing drug: polymer (Eudragit E100) in the ratio of 1:2 with average particle size of 338 nm and polydispersity index of 0.315 was selected as optimized formulation because of maximum drug entrapment (94%) and prolonged drug release of 89% for 4 h was obtained. The polymeric nanoparticle was loaded in the buccal film formulation containing polymers SCMC and PVP in the ratio of 3:2 which showed in vitro drug release of 49% initially and gradually increased to achieve 93% at the end of 8 h. The presence of hydrophobic polymeric nanoparticles helped in the reduction of hydrophilicity of the drug, thereby attaining sustained drug release profile. The characterization studies like XRD, DSC, FTIR, and FESEM confirmed the mild interactions between the polymer and the drug without significant changes in the stability of the drug. Hence, polymeric nanoparticles loaded buccal film of Lamivudine
3.10. Cytotoxicity Effect of Lamivudine Polymeric Nanoparticles The viability of the cells (control Fig. 6 (a)) treated with selected formulation of Lamivudine polymeric nanoparticles at concentration of 10, 20, 30, 40 and 50 μg/mL through the MTT assay is shown in Fig. 6 (b) to Fig. 6 (f), respectively. The yellow colour tetrazolium dye used for the MTT assay was reduced by the mitochondria of live cells to purple coloured formazan product. Hence, the number of viable cells was directly proportional to the amount of formazan generated [52].
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Fig. 6. Viability of the Vero cells treated with Lamivudine polymeric nanoparticles at concentration of (a) Control (b) 10 μg/mL (c) 20 μg/mL (d) 30 μg/mL (e) 40 μg/ mL (f) 50 μg/mL (g) Cytotoxicity analysis of Lamivudine-Eudragit E100 nanoparticles (MTT assay) (n = 3).
could be used to prolong the drug release and reduce the frequency of drug administration in paediatric patients, thereby improving the patient compliance.
Conflicts of Interest
Acknowledgement
Appendix A. Supplementary data
The authors acknowledge SASTRA Deemed University, Thanjavur for providing infrastructure facilities (Central Research Facility and Funds for Research & Modernization in SASTRA) and online resources required for this research. The authors would like to thank DST-FIST programme (SR/FST/ETI331/2013), DST-SERB (ECR/2016/001856) and SASTRA Deemed University, Thanjavur, India for infrastructure support.
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.colcom.2018.10.004.
All the authors declare no conflicts of interests.
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