Evaluation of vancomycin incorporated to improve the appropriate properties

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Screening of the Process and Formulation Variables for Preparation Vancomycin Hydrochloride with Eudragit RS100-Nanoparticles for Oral Delivery

Screening of the Process and Formulation Variables for Preparation and Enhancement Intestinal Permeation and Vancomycin Hydrochloride with Eudragit RS100-Nanoparticles for Oral Delivery in Rats

Development of Vancomycin Hydrochloride with Eudragit RS100-Nanoparticles: Physicochemical Characterization and Sustained particles for Oral Delivery

Development of Vancomycin Hydrochloride with Eudragit RS100-Nanoparticles: Physicochemical Characterization and Enhancement Intestinal Permeation for Oral Delivery in Rats

Abstract

The purpose of this work was to evaluate of vancomycin (VCM) incorporated to improve the appropriate physicochemical properties, using water-in-oil-in-water (W/O/W) multiple emulsion. Nanoparticles were formed by using W1/O/W2 double-emulsion solvent evaporation method using Eudragit RS100 as a retardant material. The prepared nanoparticles were characterized for their micromeritic properties, drug content, loading efficiency, production yield, particle size analysis and Zeta potential. Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and X-ray powder diffractometry were used to characterize nanoparticles. The in vitro release studies were performed in pH 7.4. The results showed that physicochemical properties were affected by drug to polymer ratio. The results showed that nanoscale size particles ranging from 362 to 499 nm were achieved. The highest entrapment efficiency 94.76% was obtained when the ratio of drug to polymer was 1:3. Zeta (?) potential of the nanoparticles was fairly positive. The FT-IR, XRPD and DSC results ruled out any chemical interaction between VCM and Eudragit RS. FT-IR spectroscopy demonstrated no detectable interactions between the drug and polymer in molecular level.. In vitro release study showed two phases: an initial burst for 0.5 hours followed by a very slow release pattern during a period of 24 h. The results showed that, generally, an increase in the ratio of drug: polymer (1:2) resulted in a reduction in the release rate of the drug which may be attributed to the hydrophobic nature of the polymer. It was shown that the drug: polymer ratio, stirring rate, time of stirring, surfactant concentration, dispersing medium and organic solvent influenced the drug loading, particle size and drug release behavior of the formed nanoparticles. The in vitro release profile could be modified by changing various processing and formulation parameters to give a controlled release of drug from the nanoparticles. The release of VCM was influenced by the drug to polymer ratio and particle size and was found to be diffusion controlled. The best-fit release kinetic was achieved with Peppas model. In conclusion, the VCM nanoparticle preparations showed appropriate physicochemical, which can be useful for oral administrations.

Key words: Vncomycin, Nanoparticles, Eudragit RS100, physicochemical, properties.

INTRODUCTION

Vancomycin (VCM) is a glycopeptide antibiotic that inhibits bacterial cell wall synthesis at an earlier stage than the beta lactam antibiotic. Because the oral absorption of VCM is minimal, it is usually given i.v (1). VCM used for the treatment of infections caused by methicillin-resistant staphylococci. It has a molecular weight of approximately 1500 Da, water soluble and poorly absorbed from the gastrointestinal (GI) tract (2). The oral absorption of highly polar and macromolecular drugs is frequently limited by poor intestinal wall permeability. Some physicochemical properties that have been associated with poor membrane permeability are low octanol/aqueous partitioning, the presence of strongly charged functional groups, high molecular weight, a substantial number of hydrogen-bonding functional groups and high polar surface area (3). Many therapeutic compounds such as antibiotics and peptide and protein drugs require the use of some kind of absorption enhancer to obtain reasonable plasma concentrations. By loading antibiotics into the nanoparticles, one can expect improved delivery to infected cells. Nanoparticles are the carriers developed for these logistic targeting strategies and are colloidal in nature, biodegradable and similar in behavior to intracellular pathogens. These colloidal carriers, when administered intravenously, are rapidly taken up by the cells of the mononuclear phagocyte system, the very cells which may constitute a sanctuary for intracellular bacteria (4-5) Therefore, the entrapment of antibiotics within nanoparticles has been proposed for the treatment of intracellular infections (4). The encapsulation of VCM in liposomes, microspheres-human bone grafts blend and nanoparticles Eudragit RS 100 has been described in previous works (1-3). Moreover, VCM-PLGA loaded nanoparticles may show a better bioavailability than the free drug (3).

Eudragit RS 100 is a polymer commonly used for the preparation of controlled-release oral pharmaceutical dosage forms. Eudragit RS100 contains different amounts of quaternary ammonium groups ranging from 4.5 6.8% and is a neutral copolymer of poly (chlorotrimethyl-ammonioethyl methacrylate). As Eudragit RS 100 is insoluble at physiological pH values, therefore it has been used as a good polymer for the preparation of pH-independent sustained-release formulations of drugs (6). Various non-biodegradable polymers with good biocompatibility such as Eudragit and ethyl cellulose were used in the preparation of microspheres. Polymethyl methacrylate microspheres were extensively used as bone cement materials in antibiotic releasing agents for bone infection and bone tumors (6).

This is a major limitation to the encapsulation of hydrophilic compounds such as peptides and proteins. A more common way is to use a double emulsion technique in which an aqueous solution of the hydrophilic compound is first emulsified in organic solution of the polymer. The primary emulsion is then poured into a large volume of water with or without surfactant. The double emulsion technique has fairly good encapsulation efficiency for hydrophilic compounds; however, particle size is usually larger than with single emulsion technique (7).

Double-emulsion solvent extraction/evaporation technique is the most commonly used method to encapsulate hydrophilic drugs, especially protein and glycoprotein drugs, into polymeric microspheres (8). 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 the present investigation, VCM was incorporated in Eudragit RS100 nanoparticles, with the aim of improving physicochemical properties.

MATERIAL & METHODS

MATERIALS

Vanko (vancomycin hydrochloride powder for injection) was obtained from Jaberabne hyan Pharmaceutical Company, Iran, Edragit RS 100 (R hm Pharma GMBh, Weiterstadt, 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.

Methods

Preparation of Nanoparticles

VCM-loaded Eudragit RS100 were prepared by the W1/O/W2 solvent evaporation method using different ratios of drug to polymer (1:1, 1: 2 and 1: 3). Briefly, 5 ml of aqueous internal phase was emulsified for 15 s in 20 ml of methylene chloride (containing 100, 200 and 300 mg Eudragit RS100) using homogenizer with 22000 rpm. This primary emulsion was poured into 25 ml of a 0.2% PVA aqueous solution while stirring using a homogenizer for 3 min, 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 22,000 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 were redispersed in 5 ml of purified water before freeze-drying. Blank nanoparticles (without drug) were prepared under the same conditions without drug (9,10).

Micromeritic Properties

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 measurement

Zeta (?) potential measurements of diluted samples were made with a ZetaSizer (Malvern Instruments Ltd., Malvern, UK). Zeta potential values obtained from ZetaSizer were average values from twenty measurements made on the same sample. Initial measurements on several samples of the same kind showed that this number is sufficient to give a representative average value. VCM nanoparticles were diluted with deionizer water before measurement.

Loading Efficiency and Production Yield (%)

The drug concentration in polymeric particles 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 g, A standard calibration curve was performed with the VCM solution (aqueous solution of 0.1% PVA).

The loading efficiency (%) was calculated according to the following equation:

Loading efficiency (%) = (actual drug content in nanoparticles/theoretical drug content) 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.

X-ray Powder Diffraction

The X-ray powder diffraction (XRPD) of the drug, Eudragit RS100, nanoparticles and blank-nanoparticles were recorded using an automated X-ray diffractometer (Siemens D5000, Munich, Germany), using nickel-filtered CuK? radiation (a voltage of 40 KV and a current of 20 mA). The scanning rate was 6 /min over range of 5-70 and with an interval of 0.02 .

Differential Scanning Calorimetry (DSC)

A differential scanning calorimeter (Shimadzu, Kyoto, Japan) was used to identify any changes in the thermal behavior of VCM nanoparticles compared to original materials. Samples (5 mg weighed to a precision of 0.005 mg) were placed in aluminum pans and the lids were crimped using a Shimadzu crimper. Samples were heated ranging 25-300 C at a scanning rate of 10 C/min in aluminum pans.

Fourier-Transform Infrared Spectroscopy (FT-IR)

The spectra of intact VCM, Eudragit RS100, nanoparticles and blank nanoparticles were obtained

(range 450-4000 cm-1) by using KBr disk technique and were recorded on a Bomem Quebec, Hartmann & Brann (Canada) spectrometer. After obtaining sharp peaks with reasonable intensity, the spectrum was saved for further analysis at 1 cm-1 resolution.

In vitro Release Study

VCM dissolution patterns from freeze dried nanoparticles were obtained under sinking conditions. Dissolution studies were carried out using a dialysis bag rotating method was used for all nanoparticles formulations. A set amount of nanoparticles (20 mg drug) was added to 200 ml dissolution medium (phosphate buffered saline, pH 7.4), preheated and maintained at 37 1 C 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 22,000 g 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 at 279.8 nm (11).

Release characteristics and Kinetics studies

In order to have better comparison between different formulations dissolution efficiency (DE), t50% (dissolution time for 50% fractions of drug); and difference factor, f1 (used to compare multipoint dissolution profiles) were calculated and the results are listed in Table 4 (12). Dissolution efficiency (DE) to characterize drug release profiles we used the DE parameter (13), defined as the area under the dissolution curve up to a certain time t, expressed as a percentage of the area of the rectangle arising from 100% dissolution in the same time. The areas under the curve (AUC) were calculated for each dissolution profile by the trapezoidal rule. DE can be calculated by the following:

DE =

Where y is the drug percent dissolved at time t. In this paper, all dissolution efficiencies

were obtained with t equal to 1440min.The in vitro release profiles of different nanoparticle formulations were compared with physical mixture formulation using difference factor (f1), as defined by the following (12):

f1= {[? t=1n |Rt-Tt|] / [? t=1n Rt]} 100

Where n is the number of time points at which % dissolved was determined, Rt is the % dissolved of one formulation at a given time point and Tt is the % dissolved of the formulation to be compared at the same time point. The difference factor fits the result between 0 and 15, when the test and reference profiles are identical and approaches above 15 as the dissimilarity increases.

Data obtained from in vitro release studies were fitted to various kinetic equations to find out the mechanism of drug release from the Eudragit RS100 nanoparticles. The kinetic models used were:

Qt = k0t (zero-order equation)

ln Qt = ln Q0 k1.t (first-order equation)

Qt = K. S.?t = kH. ?t (Higuchi equation based on Fickian diffusion)

Where, Q is the amount of drug release in time t, Q0 is the initial amount of drug in the nanoparticles, S is the surface area of the nanoparticle and k0, k1 and kH are rate constant of zero order, first order and Higuchi rate equation, respectively. In addition to these basic release models, there are several other models as well. One of them is Peppas and Korsemeyer equation (power law).

Mt/M?= k.tn

Where Mt is the amount of drug release at time t and M? is the amount release at time t = ?, thus Mt/M? is the fraction of drug released at time t, k is the kinetic constant, and n is the diffusion exponent which can be used to characterize both mechanism for both solvent penetration and drug release. Determination the correlation coefficient assessed fitness of the data into various kinetic models. The rate constants, for respective models were also calculated from slope (13, 14).

Optimization of the VCM nanoparticles

Effect of stirring rate and time of stirring

Various batches of the selected formulation (F2) were made, but the stirring rate was the only parameter that was varied between 22000, 24000 and 26000 rpm. Also, while keeping the other parameter constant, time of homogenizer stirring was changed range 1.5, 3 and 4.5 min. After drying, the weighed batch of nanoparticles was subjected to drug content, loading efficiency, particle size and drug release tests.

Influence of formulation variables

The influence of process variables on nanoparticle formation, micromeritics and drug release characteristics was investigated on formulation F2. These variables included the emulsifier concentration (0.1, 0.2 and 0.4%) and volume of organic solvent (15, 20 and 25 ml) and dispersing medium (15, 25 and 35 ml from 0.2% PVA).

RESULTS AND DISCUSSION

A W/O/W multiple emulsion solvent evaporation/extraction method is mostly used for the encapsulation of water-soluble drug and therefore was the method of choice for the water-soluble VCM drug. Droplets of the polymer in organic solution were added to a flowing solution of poly(vinyl alcohol) (PVA, as stabilizer)/water solution; at the end of the flowing procedure uniform-sized beads were collected (15). In the nanoparticles prepared by evaporation method, the amount of drug entrapped in microspheres was lower than the theoretical value. This indicates that some free drug crystals were lost in the process of encapsulation. Figure1 shows that the manufacturing process allows the encapsulation of VCM within nanoparticles. Blank nanoparticles (B, Figure1) are totally dissolved in methylene chloride and produce a clear solution. Figure1 clearly displays increasing of turbidity in methylene chloride of nanoparticles according of increasing of polymer concentration (C, D and E). As the ratio of drug to polymer increases the amount of free drug lost decreases (Table 2) so that at the ratio of drug to polymer 1:3 the amount of drug entrapment was 23.69% which was very close to the theoretical value (25%).

The encapsulation efficiency of the drug depended on the solubility of the drug in the solvent and continuous phase. Youan & etal. have been reported Similar observation (16). Important prerequisites for high encapsulation efficiencies by the W/O/W method are: (1) the insolubility of the drug in the internal from the external aqueous phase, and (2) the fine dispersion of the aqueous drug solution into the organic polymer solution to form a W/O emulsion (17). VCM is insoluble in organic solvents used to dissolve the polymer (methylene chloride) and thus cannot partition from the internal into the external aqueous phase via diffusion through the organic polymer solution. In order to obtain a fine dispersion, the aqueous VCM solution was added to the organic. Entrapment efficiency of polypeptides was increased by enhancing the viscosity builders (18). Despite the hydrosolubility of VCM, favoring the leakage of the drug into the external aqueous phase, entrapment efficiencies were rather high (19). It is assumed that VCM is localized at the interfaces (either internal water in oil or external oil in water). Therefore a significant amount of drug is supposed to be adsorbed at the outer surface. In addition, the removal of the organic solvent under reduced pressure favors its fast evaporation followed by the polymer precipitation, thus reducing the migration of the drug to the external phase. Indeed, the faster the solvent evaporation, the higher the encapsulation efficiency (19). One possible explanation could concern the increase of the primary emulsion viscosity due to the different VCM concentrations studied which could reduce the leakage of the drug towards the external aqueous phase (20).

Generally, increasing the polymer to drug ratio increased the production yield, when the ratio of polymer-drug increased from 1:1 to 1:3, the production yield was increased (p>0.05). Size of microspheres was found to be increased with the increase in the concentration of polymer (Table2). It can be attributed to that fact that with the higher diffusion rate of non-solvent to polymer solution the smaller size of microcapsules is easily obtained (19,20). A volume-based size distribution of drug, polymer, and drug loaded nanoparticles indicated a log probability distribution. Mean particle size of original VCM and Eudragit RS100 was 675 40.78 nm and 395.52 1.68 nm, respectively. Mean particle size of F3 was 499 110 nm. The data describing the particle sizes of the nanoparticles are given in Tables 2. As it can be seen, the particles are increased with increasing polymer amount. It has already been reported that particle size is proportional to the viscosity of dispersed phase (21-24). In fact viscosity of dispersed phase was increased from F1 (1:1) to F3 (1:3). When the viscosity of the dispersed phase of these formulations was investigated it was found that particle sizes of nanoparticles were directly proportional to the apparent viscosity of dispersed phase. The results showed that the apparent viscosities of the different drug: polymer ratios (1:1, 1:2 and 1:3) were 13, 16 and 18.8 mPa.S respectively. When the dispersed phase with higher viscosity was poured into the dispersion medium, bigger droplets were formed with larger mean particle size.

The zeta potential of three nanosphere formulations, VCM (7.09 mV) and Eudragit RS100 (-3.32 mV) are showed in Table 2. Blank nanoparticles had positive charge (15.7 mV). Drug-loaded nanoparticle indicated positive charge, because VCM is cationic drug and is changed the charge of nanoparticles. Addition of a cationic substance can lead to the flocculation of yeast cells thus forming macroscopic flocs (25,26). Flocculation occurs by two main mechanisms (a) formation of macromolecular bridges between the particles, and (b) surface and charge reduction due to the adsorption of highly charged polyelectrolytes on oppositely charged particles (26). In other words, zeta potential is the potential difference the dispersion medium and stationary layer of fluid attached to the dispersed particle. A value of potential zeta (positive) can be taken as the arbitrary value that separates low-charged surfaces from highly-charged surfaces. The significance of zeta potential is that its value can be related to the stability of colloidal dispersion. The zeta potential indicates the degree of repulsion between adjacent, similarly charged particles in dispersion. For molecules and particles that are small enough, a high zeta potential will confer stability, i.e. the solution or dispersion will resist aggregation (21,26).

The physical state of the VCM in the Eudragit RS 100 was studied by classical techniques such as DSC,

PXRD and FTIR. DSC analyses were carried out for VCM, Eudragit RS 100, blank nanoparticles, and all nanoparticles formulations (Figure 2). Any abrupt or drastic change in the thermal behavior of rather the drug or polymer may indicate a possible drug-polymer interaction (10). The endothermic peak of the pure drug was observed at about 82.10 C (Figure 2) and Eudragit RS showed an amorphous state. Indeed, in the thermogram of the nanoparticles containing Eudragit RS, there was endothermic peak at 219.92 C which corresponds to the melting point of drug in the

nanoparticles. In the DSC curve of physical mixture of drug and polymer, and formulations F1, F2 and F3, the characteristic peaks of drug were observed. The result showed that there is no incompatibility between drug and polymers. nanoparticles production process did not change the nature of drug in nanoparticles. It is obvious from DSC thermograms that drug may have been dispersed in crystalline or amorphous form or dissolved in the polymeric matrix during formation of the nanoparticles.

The X-ray diffraction patterns of pure drug and Eudragi RS showed that the pure drug and are crystalline in nature (Figure 3). XRPD is a powerful tool to identify any changes in crystallinity of drug; hence, polymorphism. XRPD of all chemicals and nanoparticles are shown in Figure 3. As shown in Figure 3, Eudragit RS100 is a typical form of amorphous materials, whereas the pure drug showed the diffractographic profile of a crystalline material. When the nanoparticles were prepared with different polymer/drug ratios (F1, F2 and F3) it is clear that the nanoparticles with lower polymer concentration showed similar peaks as the blank nanoparticles. At high concentration of polymer and low concentration of drug some of the distinctive peaks for VCM are detectable but with very low intensity due to the presence of lower concentration of drug in the sample in comparison with pure VCM sample. This confirms the results obtained from DSC experiments.

In order to investigate any changes in molecular levels of VCM during the nanoencapsulation process, FT-IR spectrum was obtained for all formulations (e.g. pure VCM and polymer) and their spectra were shown in Figure 4. FTIR spectra are assigned as follows: (1) VCM: phenolic OH at 3257.39 cm-1, aromatic C=C stretching at 1652.7 cm?1 and C=O stretching 1503.48 cm-1; (2) Eudragit RS: C=O stretching band at 1734.2 cm -1; (3) nanoparticles F1, F2 and F3: C=O stretching band at 1731.2, 1733.7, 1733.96 cm-1, respectively (Figure 4). After VCM was encapsulated into the nanoparticles, the characteristic peaks for VCM showed by the stronger intensity peaks for of matrix materials [30]. On the other hand, the C=O stretching bands of VCM in polymeric systems (at 1503.48 cm-1) were merged, thus leading to a peak shifting from 1731.2 cm-1 to 1733.96 cm-1.

The bands observed for nanoparticles spectrum showed similar distinctive peaks but with smaller intensity due to low concentration of drug in nanoparticles. This suggests that there was no new chemical bond formed between these functional groups in the drug and the polymer after preparing the nanoparticles and the results confirmed that the drug is physically dispersed in the polymer (27).

The in vitro release profiles of VCM from nanoparticles exhibited in Figure 5, also initial burst effect, which may be due to the presence of some drug particles on the surface of the nanoparticles. In most cases, a biphasic dissolution profile was observed at pH 7.4: the initial rapid drug leakage generally ended very early and for the remaining time, nearly linear behavior was observed. It showed that the first portion of the curves is due to VCM dissolution, which starts immediately after the beginning of the test for the portion of drug on the surface of nanoparticles. After such a phase, two phenomena can combine in enhancing in the diffusion of the remaining dispersed drug into the bulk phase as well as the formation of pores within the matrix due to the initial drug dissolution; particle wetting and swelling which enhances the permeability of the polymer to the drug (Figure 5) (28). The results indicated that some factors such as drug-polymer ratio governed the drug release from these nanoparticles. Drug release rates were decreased with increasing amounts of polymer in the formulation (Figure 5). Higher level of VCM corresponding to lower level of the polymer in the formulation resulted in an increase in the drug release rate (F1). As more drugs are released from the nanoparticles, more channels are probably produced, contributing to faster drug release rates. However, Figure 5 shows that the burst effect is lower when the drug to polymer ratio is 1:3 (F3) compared with other formulations. In the formulation F3, an increase of the internal phase viscosity due to the different polymer concentrations could reduce the leakage of the drug towards the external aqueous phase and decrease the burst effect (to compare with F1 and F2).

VCM nanoparticles of each formulation displayed an immediate and important initial drug release in the first 30 min (9.95-12.30%), followed by an 84-87% during 24 h which was obtained (Table 4, Figure 5). This immediate high release may be due to the small diameter of nanoparticles leading to a large exchange surface and probably to a more porous structure owing to the solvent evaporation method, favoring the release of the encapsulated drug (29). Indeed, it has been already demonstrated that the slow precipitation of nanoparticles after solvent evaporation leads to more porous particles compared to the fast polymer precipitation obtained after solvent extraction (21).

However, reduce the initial burst effect can be described not only by the rather hydrophilic properties of VCM prefers its diffuse towards the surrounding dissolution in aqueous media, but also to the high encapsulation ratio of VCM nanoparticles (10,21). The presence of Eudragit RS100 in the matrix of nanoparticles conferred a slower and more progressive release of VCM during the time of the experiment (10). Therefore, any mechanism which is able to restrict this diffusion of VCM towards water would be easily observed. Indeed, due to the slow diffusion of water into the lipophilic Eudragit RS100 matrix (8,10,21).

F1, F2 and F3 nanopartiles showed higher dissolution efficiency 81.44, 76.25 and 66.37%, respectively and slow dissolution. Physical mixture had higher release in comparison with microspheres (p< 0.05), (Table 5 & Figure 5). According of Table 5, the lowest DE was observed for F3 (66.37%) and dissolution efficiency of the physical mixture was 98.03% (p< 0.05). The value of t50% varies in between 2.24 (F2 formulation) to 4.85 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 5).

The effect of stirring rate and time of stirring on the physical characteristics of the nanoparticles is shown for formulation F2. The results of stirring rate and time of stirring on the mean particle size of nanoparticles, drug entrapment and production yield are listed in Table 5. The results showed that increasing the stirring rate and time of stirring decreased the production yield and increased the drug content, loading efficiency (p>0.05) and particle size (p<0.05).

The change of stirring speed of the secondary emulsification process also influenced the characteristics of nanoparticles as shown in Table 5. The results that increasing the concentration of PVA decreased the production yield (p>0.05) and increased the drug content, loading efficiency and particle size (p<0.05).

The volume of processing medium (outer phase, W2) significantly influenced the particle size of the nanoparticles (Table 5). As the volume of processing medium increased from 15 ml to 35 ml, the entrapment particle size significantly decreased from 430 to 526 nm (comparing F2-10 and F2-12) (p<0.05). When the concentration of organic solvent (methylene chloride) increased, production yield and drug entrapment increased (p>0.05), also loading efficiency and the mean particle size of nanoparticles increased (comparing formulations F2-13, F2-14 and F2-15 in Table 5). According to the Table 5, when organic solvent concentration increased, size of nanoparticles in F2-13 and F2-15 (respectively 15 and 35 ml solvent) was larger than F2-14.

The in vitro release profiles were fitted on various kinetic models in order to find out the mechanism of drug release15. 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 Peppas model. 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 0.51-0.91, indicating that the mechanism of the drug release were diffusion and erosion controlled.

Conclusion

VCM nanoparticles were prepared using double emulsion (W/O1/O2) solvent evaporation method. Drug: polymer ratio, stirring speed, time of stirring, emulsifier, dispersing medium and organic solvent influenced the characteristics of the nanoparticles. The entrapment efficiency was high for all formulations. The encapsulation efficiency was less influenced with changing the stirring speed and time of stirring the second emulsification process, emulsifier concentration, dispersing medium concentration and organic solvent.

It was observed that at higher drug concentration, the mean particle size of the nanoparticles is high but increasing the stirring speed and emulsifier content, resulted in smaller mean particle size of nanoparticles.

High correlation was observed for the Peppas model. The data obtained were also put in Korsemeyer-Peppas model in order to find out n value, which describes the drug release mechanism [Ref 22]. The n value of nanoparticles of different drug to polymer ratio was between 0< n<0.5, indicating that the mechanism of the drug release was diffusion controlled. It was suggested that mechanism of drug release from nanoparticles was diffusion controlled. [Ref 33,34].

Acknowledgment

The financial support from biotechnology center, Tabriz University of Medical Sciences, is greatly acknowledged.

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