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In the present investigation, we have developed vancomycin (VCM) based biodegradable poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles for oral route, with the aim of allowing an extended release of the antibiotic and an improvement of its intestinal half-life. The vancomycin-loaded nanoparticles could be prepared using W1/O/W2 double-emulsion solvent evaporation method. The prepared nanoparticles were characterized for their micromeritic properties, drug loading, particle size analysis and Zeta potential, as well as by infrared spectroscopy (IR), differential scanning calorimetry (DSC) and x-ray powder diffractometry (XRD) The in vitro release studies were performed in pH 7.4, phosphate buffer saline. Particle sizes were between 450 and 466 nm for different compositions of VCM-PLGA nanoparticles. Nanoparticles F3 (VCM/PLGA) 1:3 ratio prepared with high polymer concentration were larger. Entrapment efficiency was 38.38%-78.6%. Zeta (?) potential of the nanoparticles was negative. The FT-IR, XRPD and DSC results ruled out any chemical interaction between drug and the matrix could be justified. The release behavior of drug from nanoparticles was also investigated in order to identify correlation between the chemical composition of the polymer matrix and the drug release rates. It is possible to design a sustained drug delivery system for the prolonged release of VCM, improving intestinal permeation by matrix nanoparticles.
Key words: Vncomycin, Nanoparticles, PLGA, Intestinal permeation.
Nanoparticulate polymeric delivery systems have been investigated as a possible approach to increase the oral drug availability. Biodegradable particulate carrier systems are interest as a potential means for oral delivery to enhance drug absorption improve bioavailability, targeting of therapeutic agents to particular organ .
Vancomycin (VCM), a peptide drug, is showing a high antibacterial activity against Staphylococcus aureus and other Staphylococcus species , reported to be responsible of about 70% of postoperative endophthalmitis [3-4]. Since this drug is poorly absorbed from the gastrointestinal tract, intravenous administration has been tried. But this route has been found inadequate to achieve therapeutic levels of VCM concentration in the aqueous humor .
For oral macromolecular drug delivery, such as polymeric particulate systems using PLGA has been widely used because it is the only commercially available FDA-approved biodegradable polymer. The drug release can be controlled by the molecular weight of PLGA and polymerization ratio of lactide to glycolide. Moreover, PLGA has proven to be safe because it decomposes to lactic acid and glycolic acid in the body and is finally excreted as CO2 . PLGA is a biodegradable, bioresorbable polymer, widely used for drug formulations and medical purposes since it is nontoxic and well tolerated by the human body. Its in vivo enzymatic hydrolysis leads mainly to water and carbon dioxide. The degradation of these polyesters involves a bulk erosion process . Since the bulk erosion can accelerate the diffusion and release of drug, the drug release mechanism based on these polyesters is quite complicated . Polyesters provide alternative approaches to achieving desired release profiles and zero-order release kinetics due to their surface erosion mechanism .
Therefore, the ideal oral drug delivery carrier should be biocompatible, small enough to pass through the gastrointestinal barrier (M cells), and should have an even size distribution without physical instability, such as aggregation. Indeed, a high drug encapsulation efficiency (EE %) is required to promote the pharmacological effects of a drug .
Loading of VCM, a hydrophilic antibiotic, into PLGA microparticles can be problematic owing to its high hydrophilicity. The most used technique to encapsulate hydrophilic molecules is the double (water-in-oil-in-water, W/O/W) emulsification method, followed by solvent extraction/evaporation .The encapsulation of VCM in PLGA microspheres has been described for the ocular delivery using emulsification/spray-drying in previous works . Moreover, combination of VCM and VCM loaded PLGA (75:25) microspheres blended with human grafts were evaluated in vitro for the intention of using for bone repair and to prevent infections . Hence, alternative formulations are needed to extend the time over which VCM intestinal level remains high enough and therefore enhance the oral performance of this appropriate antibiotic. Indeed, the presence of a polymeric wall provides a protection from the gastrointestinal environment and may favor a prolonged contact with the epithelium that may be sufficient to increase the bioavailability of certain drugs.
In fact the prediction of drug absorption is very important for the design of an oral preparation.
One of the most used classic techniques in the study of intestinal absorption of compounds has been the single-pass intestinal perfusion (SPIP) model , which provides experimental conditions closer to what is faced following oral administration. This technique has lower sensitivity to pH variations because of a preserved microclimate above the epithelial cells and it maintains an intact blood supply to the intestine . Since human in vivo studies are not usually possible in the early phases of drug development, therefore, some experimental methods such as animal in vivo and ex vivo models have so far been evolved to estimate gastrointestinal absorption of drugs . Nowadays interest has grown for using in vitro and in situ methods to predict, as early as possible, in vivo absorption potential of a drug.
The goal of this study was to investigate the entrapment VCM into PLGA nanoparticles, with the aim of improving intestinal permeation.
2. Materials and Methods
Vankoï¿½ (vancomycin hydrochloride powder for injection) was obtained from Jaberabne hyan Pharmaceutical Company, Iran, Poly (D, L-lactide-co-glycolide) (PLGA) (50:50 D, L-lactide:glycolide) with average molecular weight of 12000 g/mol (Resomer RG 502), was purchased from Boehringer Ingelheim, Germany. Poly vinyl alcohol, (PVA), with molecular weight of MW 95000 (Acros Organics, Geel, Belgium) and dichloromethane, methanol, glacial acetic acid, triethanolamine, hydrochloric acid, potassium chloride, sodium chloride, sodium hydrogen phosphate (dibasic), potassium dihydrogen phosphate (Merck, Darmstadt, Germany) were used. Silastic membrane (# 10,000 Da) was provided by Biogene (Mashhad, Iran). All other materials used were of analytical or HPLC grade. Male Wistar rats were purchased from the animal house of Tabriz University of Medical Sciences. All the animals were cared for according to the rules and regulations of the Institutional Animal Ethics Committee (IAEC) guidelines of the Health Ministry, Iran.
2.2. Manufacture of procedure
VCM-loaded PLGA were prepared by the W1/O/W2 modified solvent evaporation method using different ratios of drug to polymer (1:0.5, 1: 1 and 1: 2). Briefly, 5 ml of aqueous internal phase was emulsified for 15 s in 20 ml of methylene chloride (containing 100, 200 and 300 mg PLGA) using homogenizer with 22000 rpm (figure1). This primary emulsion was poured into 25 ml of a 0.2% PVA aqueous solution while stirring using a homogenizer for 3 min under, immersed in an ice water bath, to create the water in oil-in-water emulsion. Three to four ml of NP suspension was obtained after solvent evaporation under reduced pressure (Evaporator, Heidolph, USA). Nanoparticles were separated from the bulk suspension by centrifugation (Hettich universal 320R, USA) at 22,000ï¿½ g for 20 min. The supernatant was kept for drug assay as described later and the sediment nanoparticles were collected by filtration and washed with three portions of 30 ml of water and redispersed in 3 ml of purified water before freeze-drying (Figure1). After lyophilization, the dried nanoparticles were resuspended in 2 ml of purified water shortly before preparing the composite microparticles. Blank nanoparticles (without drug) were prepared under the same conditions without drug [10-11].
2.3. Nanoparicle size and zeta potential
A laser light scattering particle size analyzer (SALD-2101, Shimadzu, Japan) was used to determine the particle size of the drug, polymer and nanoparticulate formulations. Samples were suspended in distilled water (nanoparticles and polymer) or acetone (drug) in a 1 cm cuvette and stirred continuously during the particle size analysis.
Zeta potential is electric potential in the interfacial double layer (DL) at the location of the slipping plane versus a point in the bulk fluid away from the interface . Zeta potential of VCM nanoparticles was measured with Zetasizer (Malvern instruments, England). VCM nanoparticles were diluted with deionized water before measurement. Each measurement was carried out in triplicate.
2.4. Determination of drug loading and loading efficiency
The loading efficiency of VCM in PLGA nanoparticles was determined spectrophotometrically (UV-160, Shimadzu, Japan) at 280.2 nm by measuring the amount of non-entrapped VCM in the external aqueous solution (indirect method) before freeze-drying. In the case of nanoparticles, the external aqueous solution was obtained after centrifugation of the colloidal suspension for 20 min at 22,000 ï¿½ g, A standard calibration curve was performed with the VCM solution (aqueous solution of 0.2% PVA).
The loading efficiency (%) was calculated according to the following equation:
Loading efficiency (%) = (actual drug content in nanoparticles/theoretical drug content) ï¿½ 100
The production yield of the nanoparticles was determined by calculating accurately the initial weight of the raw materials and the last weight of the polymeric particles obtained. All of the experiments were performed in triplicate.
2.5. Infrared spectroscopy, differential scanning calorimetry (DSC) and X-ray diffraction studies
The infrared (IR) spectra of powder VCM, physical mixture and the nanoparticles were recorded on an IR-spectrophotometer (Bomem Hartmann & Brann, Canada) by the KBr pellet technique.
Differential scanning calorimetry (DSC) analysis was performed using a DSC-60 calorimeter (Shimadzu, Japan). The instrument was equipped with a TA-60WS thermal analyzer, FC-60A flow controller and TA-60 software. Samples of VCM, physical mixture and agglomerates were sealed in an aluminum crucible and heated at a rate of 10 ï¿½C minï¿½1 up to 300 ï¿½C under a nitrogen atmosphere. A similar empty pan was used as the reference.
Powder X-ray diffraction patterns (XRD) of the pure drug and spherical agglomerates were obtained using an X-ray diffractometer using model (Siemens D5000, Munich, Germany) equipped with a graphite crystal monochromator (CuK?) (a voltage of 40 KV and a current of 20 mA) radiations to observe the physical state of drug in the microspheres (a voltage of 40 KV and a current of 20 mA).
The scanning rate was 6ï¿½/min over range of 5-70ï¿½ and with an interval of 0.02ï¿½.
2.6. Dissolution study
VCM dissolution patterns from freez dried nanoparticles were obtained under sinking conditions. Dissolution studies were carried out using a dialysis membrane rotating method was used for all nanoparticles formulations. A set amount of nanoparticles (100 mg drug) was added to 200 ml dissolution medium (saline phosphate buffer, pH 7.4), preheated and maintained at 37ï¿½1 ï¿½C in a water bath, then stirred at 100 rpm. Then 1 ml of suspension was withdrawn at appropriate intervals (0.5, 1, 2, 3, 4, 5, 6, 8, 12 and 24 hr) and, each sample was centrifuged at 22,000ï¿½g for 10 min. The filtrate (VCM) was replaced by 3 ml of fresh buffer. The amount of VCM in the release medium was determined by UV at279.8 nm .
2.7. Chromatographic conditions
The mobile phase for VCM was a mixture of 30% methanol and 70% of glacial acetic acid aqueous solution (0.75%) adjusted to pH 5.5, to which was added triethanolamine. The mobile phases was filtered through sintered glass filter P5 (1 micron) (ISO-Lab, Germany) and degassed in sonicator (Liarre, Italy) under vacuum. The mobile phase was pumped in isocratic mode at a flow rate of 1 ml/min at ambient temperature. The UV detection was accomplished at 430 nm (phenol red) and 254 nm (VCM) and samples of 20 ï¿½l were injected using Hamilton injector syringe (Hamilton, MICROLITREï¿½#710, Switzerland) onto the column .
2.8. Perfusion solution
The perfusion solution was prepared by dissolving 7g NaCl, 0.2g KCl, 1.44 g Na2HPO4, 0.24g KH2PO4 in one liter of distilled water. The pH of prepared phosphate buffered saline was 7.4. Preliminary experiments showed that there were no considerable adsorption of the compounds on the tubing and syringe. Samples from perfusion study were filtered and directly injected onto HPLC column and required no sample preparation prior to analysis.
2.9. In situ permeation studies
Adult male Wistar rats were obtained from the Animal Centre of the local University, Tabriz, Iran. They were housed in air conditioned quarters under a photoperiod schedule of 12 h light/12 h dark. They received standard laboratory diet and tap water, available at libitum. Wistar rats of male sex, weighing 200-250 g, were selected. The rats were restrained in a supine position on a board kept at 37ï¿½C. A small midline incision was made in the abdomen and 10 cm loops of the ileum were identified and ligated at both ends. This ileum loop was made at the end of the small intestine, just proximal to the ileo-cecal junction. The rats stabilized for 30 min after operation, and then injected as follows. Blank perfusion buffer was infused for 10 min by a syringe pump (Palmer, England) followed by perfusion of VCM by using different concentration (200, 300, 400 ?g/ml) at a flow rate of 0.2 ml/min thiopental for 80 min. The concentrations were selected based on the mechanism of drug absorption. Outlet samples were collected at appropriate interval (30, 40, 50, 60, 70, 80 min) in microtubes. The volume of sample for each time interval was 2 ml. When the experiment was completed, the length of segment was measured and the animal was euthanatized with a cardiac injection of saturated solution of sodium thiopental. Samples were stored at -20 ï¿½C until analysis .
2.10. Preparation of standard solutions
Primary stock solution was prepared in phosphate buffered saline (PBS) to obtain a concentration of 1 mg/ml of each compound. Then it was diluted to 50 ï¿½g/ml make a working solution and standards for calibration curves and quality control samples were prepared using serial dilution of working solution in PBS. The concentration range for working standard solutions was 50-1000 ï¿½g/ml. This range was selected based on the concentrations that were going to use in animal studies. Preliminary studies showed that there is no chemical interactions and stability problem in the solution for all components.
2.11. In situ absorbed of VCM-loaded PLGA nanoparticles
Adult male Wistar rats were obtained from the Animal Centre of the local University, Tabriz, Iran. They were housed in air conditioned quarters under a photoperiod schedule of 12 h light/12 h dark. They received standard laboratory diet and tap water, available at libitum. Wistar rats of male sex, weighing 250 ï¿½ 10 g, were selected.
The rats were restrained in a supine position on a board kept at 37ï¿½C. A small midline incision was made in the abdomen and 10 cm loops of the ileum were identified and ligated at both ends. This ileum loop was made at the end of the small intestine, just proximal to the ileo-cecal junction. The rats stabilized for 30 min after operation, and then injected as follows .
3.1. Micromeritics properties
This primary emulsion is then rapidly emulsified in an external aqueous solution (W2) that usually contains surfactants or stabilizers such as poly(vinyl alcohol) (PVA), leading to transit double W1/O/W2 emulsion (Figure1). Encapsulation efficiencies of VCM are reported in the Table 2. It is evident from Table 2 that the percentage encapsulation efficiency was affected by the ratio of drug: polymer. Mean diameter, production yield and encapsulation efficiency of the different nanoparticles are shown in Table2. The particle size data show that nanoparticles produced were of submicron size and of low polydispersity (Table1) which indicated a relatively narrow particle size distribution. As increase in particle size from 450 nm to 466 nm with a decrease in the theoretical drug loading was also observed (Table 2). It has also been reported that the particle size increases with increasing the content in hydrophobic polymer. A volume-based size distribution of drug, polymer, and drug loaded nanoparticles indicated a logï¿½probability distribution. The increase in drug content of the nanoparticles with increased theoretical drug loading may have resulted in the decreased particle sizes displayed (p<0.05). In this case the viscosity of inner phase of the polymer did not seem to have a great influence on the particle diameter. The average yield of 96% is in the normal range and does not indicate any unexpected loss of products. The results showed that this led to a corresponding increase in polymer content from 0.33 to 0.66% w/w; however the corresponding drug entrapment decreased from 48.8 to 12.8%.
As to the zeta potential, the larger its absolute value is, the more likely the suspension is to be stable, since the charged particles repel one another and thus overcome their natural tendency to aggregate. The zeta potential measurements showed negative charged particle surfaces, varying from ?6.7 to ?10.9 mV. All the obtained values were then acceptable and favoring a good stability. The zeta potential may then be safely discarded from the optimization step. The zeta potential of three nanosphere formulations, VCM (7.09 mV) and PLGA (-1.89 mV) are showed in Table 2. Blank nanoparticles had negative charge (-15 mV).
3.2. DSC Analysis
The DSC of VCM did not show a sharp endothermic peak (Figure 2). The physical mixture of VCM and PLGA showed nearly the same thermal behavior as the individual components, indicating that there was no interaction between the drug and the polymer in the solid state. The presence of the endothermic peak of the drug at 220-222ï¿½C, its melting point in the DSC of its PLGA-based nanoparticles suggests that the drug existed in an amorphous or disordered-crystalline phase as a molecular dispersion or a solid solution state in polymeric matrix (Figure 2).
3.3. Powder X-ray diffractometry
Comparison of the X-ray diffraction patterns of VCM (Figure 3, pattern a) and nanoparticles prepared with PLGA (Figure 3, patterns e, f and g), showed no significant reduction in the characteristic peak intensities, suggesting that the extent of VCM crystallinity was not reduced by the polymer.
3.4. FTIR Studies
The Fourier transform IR spectrum of VCM showed phenolic OH at 3257.39 cm-1, aromatic C=C stretching at 1652.7 cm?1 and C=O stretching 1503.48 cm-1. The FTIR spectra of VCM-loaded PLGA nanopartiles, physical mixture F2 (1:1), and the individual components are depicted in Figure 4. No differences in the positions of the absorption bands were observed in spectra of the VCM physical mixture with PLGA, indicating that there are no chemical interactions in the solid state between the drug and the polymer.
Figure 4b shows the minimal absorbance in the amide I region of the PLGA employed in this study. The amide I (1600-1700 cmï¿½1) and amide II (1500-1600 cmï¿½1) regions are in Figure 4b. nanoparticles F1, F2 and F3: C=O stretching band at 1675.1, 1751, 1752.1 cm-1, respectively (Figure 4 e, f, g) .
Because water absorption and secretion during the perfusion may cause errors in the calculated effective permeability (Peff) values, a non-absorbable marker to correct water flux through the intestinal wall is needed. For this purpose phenol red as a non-absorbable marker, is co-perfused with drug compound in each experiment??????????????
3.5. In vitro release study
The mean values are shown in Figure 5. The physical mixture formulation shows that 96.7% of the drug was released at the first sampling time of 30 min and 100% by 60 min. The drug release from the nanoparticles appeared to have two components with an immediate release of about 10.782-12.27% at the first sampling time of 30 min. This was followed by a slower exponential release of the remaining drug over the next 6-8 h.
The in vitro VCM release profiles from drug-loaded nanoparticles are presented in Figure 5, in comparison with the dissolution profile of physical mixture (F2 formulation). The rate of dissolution of physical mixture is quite fast: more than 96% drug is dissolved in about 30 min.
Further, Figure 5 clearly illustrates that the rate of drug release from the nanoparticles depended on the polymer concentration in the system. A similar relationship was observed between polymer content and drug release rate from prepared nanoparticles. PLGA is biodegradable and amorphous polymer and when the concentration of the polymer in the system increased the release rate of VCM increased. The difference was not also significant (p > 0.05) for 0.5 or 24 h. It is suggested that a reduced diffusion path and increased tortuosity may retarded the drug release rate from the matrix at presence of polymer matrix. Nanoparticles F3containing 1:2 (VCM/PLGA) ratio released the drug more rapidly, while those with F1 containing 1:0.5 (drug/polymer) ratio exhibited a relatively slower drug release profile. F1, F2 and F3 nanopartiles showed higher dissolution efficiency 77.97, 78.48 and 82.19%, respectively and slow dissolution. Physical mixture had higher release in comparison with microspheres (p< 0.05), (Table 3 & Figure 5). According of Table 3, the lowest DE was observed for F1 (77.97%) and dissolution efficiency of the physical mixture was 98.13% (p< 0.05). The value of t50% varies in between 2.84 (F3 formulation) to 3.73 h (F3 formulation). The results of difference factor (f2) showed that the release profile of nanoparticle formulations is the dissimilar to the release profile of physical mixture (Table 3). The in vitro release profiles were fitted on various kinetic models in order to find out the mechanism of drug release .The fit parameters to Higuchi, first-order, Peppas and zero-order equations. The rate constants were calculated from the slope of the respective plots. High correlation was observed for the first and Peppas models (Table 4).
The organic phase (O) acts as a barrier between the two aqueous compartments. To control the methylene chloride removal time and rate, we used a rotary evaporator method. Thus, the migration of the drug into the external phase (W2) is impeded. The particle formation itself is based on coacervation. The solvent is extracted from the polymer containing organic phase because of its initial diffusion into the continuous W2 phase inducing phase separation of the polymer. The organic solvent is eliminated by two steps firstly by extraction and secondly by evaporation.
In general, the solvent removal rate directly affects the encapsulation efficiency of a drug [7,] because the slow membrane formation rate may reduce the encapsulation efficiency as a result of enhancing the chances of drug molecules to be diffused out from the inner W1 phase to outer W2 water phase [7,]. Thereby, the VCM component of the control nanoparticle sample could be diffused out rapidly during the centrifuge process through the water channels of the PLGA membrane before perfect hardening of the PLGA membrane [7,]. From this result, we infer that rapid membrane formation is an important criterion preventing the leakage of VCM to the outer W2 water phase. Generally, PVA is used as a surfactant in the W1/O/W2 emulsion method to incorporate it onto the surface of PLGA particles, thereby lowering the surface tension between the PLGA surface and the W2 water phase [7,20]. However, because PVA cannot be washed away perfectly, it remains on the surface of PLGA [7,]. PVA concentration in the external water phase known to be a key factor to influence the size of nanoparticles . The low drug incorporation efficiency may be attributed to the water soluble nature of VCM hydrochloride. This led to its rapid partitioning into the aqueous phase and hence decreased entrapment into the nanoparticles during polymer deposition.The decreased drug entrapment with increasing theoretical drug loadings to an enhanced drug leakage into the aqueous phase at high loadings. Zeta potential results (Table 2) showed that drug-loaded formulations carried a negative charge, which promotes particle stability because the repulsive forces prevent aggregation with aging . It has been reprted elsewhere that the negative PLGA nanoparticles is due to the ionization of carboxylic groups of surface polymer . Drug-free PLGA nanoparticles had a positive surface charge of 7.09 mV (Table 2) which can be attributed to the presence of end carboxyl groups of the polymer on the nanoparticles . Zeta potential measurements showed slightly increases in positivity (from -7.58 mV to -3.5 mV) with an increase in theoretical drug loadings. These findings are according to what was expected, namely a decrease in the surface negativity due to interaction of carboxyl groups and the cationic drug on the particle surface. The increase in nanoparticles size with increases in the theoretical drug loading of VCM (Table 2) may possibility have influenced the surface charge of the PLGA nanoparticles. Zeta potential results (Table 2) showed that drug-loaded formulations carried a negative charge, which promotes particle stability because the repulsive forces prevent aggregation with aging . The results of this study, however, agree from those of de Chasteigner et al.,  who reported a decrease in the negative surface charge when itraconazole was loaded into polycaprolactone nanoparticles. From the data it is evident that all the formulations are almost unstable in the colloidal state. This suggests that the particles should not be stored in a liquid suspension form and rather they should be stored in a lyophilized state .
The rapid initial release of VCM was probably due the drug which was adsorbed or close to the surface of the nanoparticles and the large surface to volume ratio of nanoparticles geometry because of their size . It may also be due to the water soluble nature of VCM. The exponential delayed release may be attributed to diffusion of the dissolved drug within the PLGA core of the nanoparticle into the dissolution medium. Similar observations were reported by other researchers working on paclitatel and enalapril PLGA nanoprticles [20-21]. Loading of VCM into the nanoparticles leads to a modulation of in vitro drug release, depending on their composition. The data obtained were also put in Korsemeyer-Peppas model in order to find out n value, which describes the drug release mechanism. The n value of microspheres of different drug to polymer ratio was between 0.42-0.48, indicating that the mechanism of the drug release were diffusion controlled (Table 4).
VCM-loaded poly (lactide-co-glycolide) (PLGA) nanoparticles were successfully prepared by using W1/O/W2 double-emulsion solvent evaporation method.The results of release showed that the nanoparticles were more suatain than physical mixture. Therefore, the intestinal permeation of VCM can be improved if nanoparticle formulation of the drug is used and this allows more efficient therapy compared to formulation of VCM present in the market.
Initial emulsion (W/O1)
eDifference Factor (f1)
adissolution time for 50% fractions. bDissolution Efficiency. camount of drug release after 0.5h.d amount of drug release after 24h.
Caption of tables:
Table1. Vancomycin nanoparticles formulations prepared by double-emulsion solvent evaporation method (W1/O/W2)
Table2. Effect of drug: polymer ratio on drug loading efficiency, production yield, particle size zeta potential and polydispersity index of vancomycin nanoparticles
Table3. Comparison of various release characteristics of vancomycin from different nanoparticle formulations and physical mixture
Table4. Fitting parameters of the in vitro release data to various release kinetic models for nanoparticles
Caption of Figures:
Figure1. Schematic representation of PLGA nanoparticle preparation using the modified W1/O/W2 method.
Figure2. DSC thermogram of the VCM; PLGA; physical mixture F2; blank nanoparticles, VCM nanoparticles formulations as F1, F2 and F3.
Figure3. X-ray diffraction a) VCM; b) PLGA; c) Physical Mixture F2; d) blank nanoparticles; e) VCM : PLGA (1:0.5); f) VCM : PLGA (1:2); g) VCM : PLGA (1:2).
Figure4. FTIR spectrum; a) PLGA; b) VCM; c) Physical Mixture F2; d) blank nanoparticles; e) VCM : PLGA (1:0.5), f) VCM : PLGA (1:2), g) VCM : PLGA (1:2).
Figure5. Cumulative percent release of VCM nanoparticles prepared with different drug-to-polymer ratio and physical mixture F2.