Development and Enhancement of Entrapment Efficiency of Isoniazid Loaded Poly--caprolactone Nanoparticle

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Der Pharmacia Lettre, 2013, 5 (4):43-50

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ISSN 0975-5071 USA CODEN: DPLEB4

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Development and enhancement of entrapment efficiency of isoniazid loaded poly-ε-caprolactone nanoparticle

Atanu Kumar Behera*1, S. Shah1 and B. B. Barik2

1Monad University, Hapur, Uttar Pradesh, India

2Jazan University, Saudi Arabia _____________________________________________________________________________________________ ABSTRACT The present study was aimed at enhancing the entrapment efficiency of highly water soluble Isoniazid (INH) nanoparticles using the biodegradable polymers, Poly-ε-caprolactone (PCL) by varying the different formulation parameters such as polymer ratio, amount of drug loading (w/w), solvent selection, electrolyte addition and pH in the formulation. The PCL loaded INH were prepared by water-in-oil-in-water double emulsion technique. The prepared nanoparticles were characterized by atomic forced microscopy (AFM), differential scanning calorimetry (DSC) and for in vitro drug release study. Fourier transform infrared spectroscopy (FTIR) study, differential scanning calorimetry analysis and in vitro release kinetics study. The percentage entrapment can be enhanced up to 63 by changing different formulation parameters. Key words: Isoniazid, Polycaprolactone, AFM, DSC, Nanoparticle _____________________________________________________________________________________________

INTRODUCTION Isoniazid is the one of the first line drug used for the treatment of tuberculosis. It is a bactericidal agent active against organisms of the genus Mycobacterium, specifically M. Tuberculosis, M. bovis and M. kansasii. Isoniazid is bactericidal to rapidly-dividing mycobacteria, but is bacteristatic if the Mycobacterium is slow growing. Isoniazid is a prodrug activated by catalase-peroxidase hemoprotein, KatG. Isoniazid inhibits InhA, a nicotinamide adenine dinucleotide (NADH) -specific enoyl-acyl carrier protein (ACP) reductase involved in fatty acid synthesis [1]. Isoniazid is readily absorbed when administered either orally or parentally. The plasma half-life of isoniazid ranges from 1-4 h, those who are fast acetylators because of genetic variations, having short half-lives. The most important drawback of the current therapy used for the treatment of tuberculosis is the frequency and amount of the drug used. The limitation is because of the instability in the biological environment and premature loss through rapid clearance and metabolism [1]. Moreover, high concentration of these agents may be toxic to healthy tissues. Thus, to enhance the therapeutic efficacy, modern drug delivery plays an important role in controlled delivery of these agents to the target site of the body at a therapeutically optimal rate and concentration. These controlled release systems are proficient to maintain optimum therapeutic drug concentration in the blood with minimum fluctuation giving predictable and reproducible release rates for a longer period of time, enhancing the duration of action of drugs with a short half-life, eliminating the side effects of frequent dosing and limiting wastage of drugs, and providing an optimized therapy and improved patient compliance [2]. All of the features are strictly related to the nature of materials that constitute the continuous matrix of the delivery system.

Atanu Kumar Behera et al Der Pharmacia Lettre, 2013, 5 (4):43-50 ______________________________________________________________________________


PCL is an important member of the aliphatic polyester family can be used in alone or combined with several polymers to formulate different formulations. It has been shown that the entrapment of hydrophilic drugs inside hydrophobic polymeric nanoparticles is a difficult task [3-5]. This is because the entrapment efficiency of the hydrophilic drug inside the hydrophobic nanoparticle is low due to the low affinity of hydrophilic drugs for hydrophobic polymers. Furthermore, the interaction between the polymer and the entrapped drug is weak and the drug has a tendency to move from the organic phase to the outer aqueous phase during nanoparticle formation [6]. The objective of this study is to encapsulate RIF inside PCL nanoparticles and to increase the encapsulation efficiency by optimizing different parameters, like amount of polymer amount of drug loaded, solvent selection, pH value of inner aqueous phase and the addition of salt to outer aqueous phase. The present study included preparation and characterization of INH loaded PCL nanoparticles.

MATERIALS AND METHODS 2.1 Materials Isoniazid was a gift sample from Macleod Pharmaceuticals, Mumbai; Poly- ε-caprolactone (PCL MW 65000 g/Mol) was purchased from Sigma Aldrich. Polyvinyl alcohol (PVA MW 88000 g/Mol), DCM, Phosphate buffer, all are of analytical grade was purchased from S.D. Fine Chem Ltd., Mumbai. 2.2 Method of preparation of PCL Nanoparticles Isoniazid (INH) loaded nanoparticles were prepared with water in oil in water double emulsion solvent evaporation method [7]. In this method INH equivalent to 5-14 % w/v was dissolved in 500 µl of phosphate buffered saline (0.01 M, pH 7.4) to form INH aqueous solution. The INH aqueous solution was emulsified in an organic phase consisting of 100-500 mg of the PCL polymer in 5 ml of organic solvent (DCM) to form primary water in oil emulsion by probe sonicator using a micro tip probe sonicator (VC 505, Vibracell Sonic, Newton, MA, USA) set as 55 W of energy output for 3 minutes over an ice bath. The emulsion was further emulsified in an aqueous PVA solution (20ml 2.5% w/v) to form water –in –oil-in-water emulsion. The emulsification was carried out using a micro tip probe sonicator (VC 505, Vibracell Sonic, Newton, MA, USA) set as 55 W of energy output for 5 minutes over an ice bath by adding the primary emulsion drop wise to the 20 ml of phosphate buffer (0.01 M, pH 7.4). The emulsion was stirred for 2 hours on a magnetic stir plate at room temperature to allow the evaporation of organic solvent. Further one hour vacuum drying was also performed to remove any residual organic solvent present. Any excess amount of PVA was removed by ultracentrifugation at 16000 rpm at 40C for 20 minutes ( Remi, India) followed by washing with double distilled water. The supernatant was collected and kept for an estimation of the amount of the drug which was not encapsulated. The recovered nanoparticulate suspension was lyophilized for two days (-800 C and < 10 mm mercury pressure, LYPHILOCK 12, Labconco, Kansas City, MO, USA) to provide the lyophilized powder for further use. 2.3 Physical characterization 2.3.1 Measurement of particle size, polydispersity index and zeta potential Particle size distribution of isoniazid loaded PCL nanoparticles was determined by a laser scanning technique using Malvern instrument after appropriate dilution with distilled water. Approximately 5 mg of dried particles was re-suspended in 1.0 ml of distilled water and the resulting solution was briefly vortexes and treated in a bath sonication for 3 min. This suspension was analyzed at an obscuration of 10-20% on a Malvern Mastersizer. The mean particle size and polydispersity index zeta potential were calculated for each formulation maintained at 250C. Polydispersity index will measure the size distribution of nanoparticles population. 2.3.2 Differential scanning calorimetry (DSC) DSC analysis was performed in order to investigate the melting and crystallization behavior of crystalline materials like PCL nanoparticles. The samples were sealed in aluminum pans and measurements were recorded using DSC instrument. The samples were heated from 25 to 2000C at a heating rate of 100C /min under nitrogen atmosphere. 2.3.3 Atomic Force Microscopic studies (AFM) The shape of Drug loaded NPs was further characterized by AFM (JPK nanowizard II, JPK instrument, Bouchestrasse, Berlin, Germany) consisting of pyramidal cantilevers with silicon probes having force constants of 0.2 N/m. Samples for AFM imaging were prepared by placing a drop of the NPs suspension (1 mg/ml) on a freshly cleaved mica sheet. After 5 minutes of incubation, the surface was gently rinsed with deionized water to remove unbound NPs. The sample was air dried at room temperature and mounted on the microscope scanner. The shape



was observed and imaged in contact mode set at a frequency of 13 kHz and scanned at a speed of 1 Hz. The images were analyzed using JPK data processing software. 2.3.4 Entrapment Efficiency The prepared PCL nanoparticles dispersion was centrifuged at 15000 rpm for 30 min at 00C using REMI cooling centrifuge. Then the supernatant is analyzed for the free drug content. The concentration of RIF in the supernatant was determined by UV–Visible spectrophotometry at 262 nm. Entrapment efficiency was using the below Equation.

Entrapment efficiency (%) = (RFMinitial – RFM supernatant) / RFMinitial × 100 2.3.5 Drug Loading The INH content of nanoparticle was determined for all the formulations by dissolving 10mg of nanoparticle in 200µL of DCM has taken in 2mL Eppendorf tubes and vortexed for 10 min. The ethanol (1800µL) was added and vortexed for another 5 min followed by centrifugation at 3000 rpm for 10 min to settle down the PCL precipitated. The supernatant containing INH was suitably diluted using ethanol and the absorbance was measured at 262nm by UV–Vis spectrophotometry and equivalent concentration was determined using the calibration curve prepared using the same proportion of solvents. The percentage (%) drug loading (DL) of the nanoparticle was calculated using the following formula.

DL (%) = WDL/WNP × 100 Where WDL is the weight of the drug in INH nanoparticles and WNP is the weight of INH nanoparticles. 2.3.6 In Vitro Drug Release In vitro release studies from NPs was determined in PBS buffer (0.01 M, pH 7.4), containing 1% Na CMC at 37oC ± 0.5oC. 50 mg of drug loaded NPs was dispersed in 15 ml of above buffer. The NP suspension was equally divided in three tubes containing 5ml each. These tubes were kept in a shaker at 37 °C and 150 RPM (Wadegati Labequip, India). At particular time intervals, these tubes were taken out from shaker and centrifuged at 13,800 RPM, 4 °C for 10 minutes (REMI 1-16K, Mumbai). The supernatants were taken out to estimate the amount of drug release, at that particular time by using a spectrophotometer (UV-1700, Schimadzu). To the residue same amount of fresh PBS (0.01 M, pH 7.4, containing 1% w/v Na CMC was added and kept in shaker for further study [8]. In vitro drug-release data were fitted to kinetic models such as zero order, first order, Higuchi equation and Korsmeyer–Peppas equation. The regression analysis of Q vs. t (zero order), log Q vs. t (first order), Q vs. square root of t (Higuchi), log%Q vs. log%t (Korsmeyer–Peppas), where Q is the amount of drug released at time t, was performed [9]. 2.3.7 Statistical analysis The results are expressed as mean ± standard deviation (SD, n = 3). The data were statistically analyzed by one-way analysis of variance using Graph Pad Instat®, version 3.05 (USA), and a significant difference was set at p < 0.05.

RESULTS AND DISCUSSION

The nanoparticle formation from double emulsion/solvent evaporation system involves preparation of oil/water (o/w) emulsions with subsequent removal of the oil phase (i.e., typically a volatile organic solvent) through evaporation. The emulsions are usually prepared by emulsifying the organic phase containing the drug, polymer and organic solvent in an aqueous solution containing emulsifier. The organic solvent diffuses out of the polymer phase and into the aqueous phase, and is then evaporated, forming drug-loaded polymeric nanoparticles. 3.1 Optimization of Solvent The polarity of the organic solvent used in the emulsion formation during the nanoparticle formulation might affect the entrapment efficiency. Therefore, nanoparticles with two different organic solvents, acetonitrile (ACN) and dichloromethane (DCM) were formulated under identical conditions. The water solubility of ACN was greater than DCM and therefore the rate of precipitation was higher than the rate with DCM. Due to the high water solubility of ACN, it had a higher diffusion rate before hardening. This was the major reason for the low entrapment efficiency in



case of ACN [10]. Thus, DCM is the more appropriate solvent to use when encapsulating hydrophilic drugs in a hydrophobic polymer [11-12]. 3.2 Optimization of Polymer and Drug Ratio For enhanced drug entrapment, varying concentrations of INH were incorporated into the aqueous medium from 25 to 70 mg in 500µl of water. At the same time the amount of polycaprolactone was also varied from 300 mg to 500 mg in 5 ml of organic solvent i.e., DCM. The percentage of drug loading varied from 25 to 70 mg corresponding to the amount of polymer 300-500mg. Different possible combination were taken and the formulation was prepared with 2.5% w/v PVA as a surfactant. The entrapment efficiency of INH in the nanoparticles was found to be highest with 45 mg in 500 µl of water and 500 mg of PCL was found to be 48% which was the highest one but at the same time the particle size was around 349 ±12.84 so the formulation NP 5 was chosen as the though the entrapment efficiency is 42 % but the size was 229± 6.37nm which was detailed in table no.1. This may be due to the saturation level of INH inside the nanoparticles after 45 mg drug loading. As the amount of drug loaded increases, a more porous polymeric matrix structure may be formed with a large number of channels and hollow spaces, through which the drug could easily escape to the outer phase thereby decreasing the content of drug inside the polymeric matrix [13]. Furthermore, owing to the increased concentration of the drug inside the polymer, a difference in osmotic pressure between the outer and inner aqueous phase results, this may cause the drug to escape from the inner aqueous phase [14]. 3.3 Optimization of pH of the inner aqueous phase (in which the drug is dissolved) In the preparation of INH nanoparticle, by changing the pH of the inner aqueous phase from 7.4 to 4 and by adding 4% w/v NaCl to the outer aqueous phase the encapsulation efficiency increased from 42 to 53% table no. 2. The pH of the water phase affects the ionization of the drug substance and, hence, the solubility. An ionic drug substance is likely to stay in the water phase, while the molecular form is more likely to be attached to the hydrophobic polymer phase, and, in this case, the drug substance is more efficiently encapsulated [15]. Based on this finding, by simply adjusting and controlling the pH value, the entrapment efficiency of INH inside nanoparticles can be increased. By changing the pH of the inner aqueous phase from 7.4 to 4 the drug is dissolved in this acidic aqueous solution and does not diffuse to the outer aqueous phase. As a result the drug may be more easily entrapped in the polymeric matrix leading to a higher encapsulation efficiency of nanoparticles [16]. 3.4 Optimization of addition of an electrolyte to the outer aqueous phase The addition of an electrolyte affects the osmotic gradient between the inner and outer aqueous phases; this may have an impact on drug entrapment. With the addition of salt, the concentration of the outer aqueous phase (PVA solution) increases and becomes hypertonic; therefore, the drug does not diffuse into the outer aqueous phase and remains in the polymeric matrix [17-18]. That result an increment of entrapment of efficiency up to 63% detailed in table no.3. 3.5 DSC Studies DSC study shows the nature of the drug encapsulated in the NPs. This analysis was performed on native PCL, native Isoniazid and Isoniazid loaded NPs. Different compounds show their characteristic peaks in DSC (figure 1). The endothermic peaks of PCL and was found approximately at 65 ºC due to glass transition temperature (Tg) of PCL. The peak of PCL was slightly shifted in drug-loaded NPs as compared to that of native PCL. The endothermic peak of native Isoniazid was found approximately at 173.12 ºC. 3.6 FTIR of INH nanoparticle The FTIR analysis was used to study the chemical modifications or changes that occurred in the polymer in the form of a band stretching or bending due to the addition of the drug during the synthesis of NPs. Fig. 2, shows the FTIR spectra of native isoniazid, void NPs .Loaded NPs. FTIR results confirmed the chemical stability of rifampicin in the nanoparticles. Native Isoniazid showed characteristic bands due to the presence of different functional groups. A stretching vibrations, while those observed at 2971.63, 2865.79cm-1 are due to the C-H stretching vibrations, 1756.59 cm-1 due to C=O stretching, 1657.20 cm-1 due to N-H bend (due to substituted amide group), 1539.39 due to C=C stretching (due to pyridine ring), 1323.47 cm-1 due to C-N stretching (due to aromatic ring), 1002.82 cm-1, 1055.17 cm-1 is due to C-N stretching (due to aliphatic) and 648 cm-1 due to =CH. The bands occurring in void NPs are almost similar to the bands in isoniazid loaded NPs in addition with some extra bands due to the presence of isoniazid. Thus, in this study the bands appearing at 3300, 3691.91, 3697.7, 3724.7, 3982.21, cm-1 for native



isoniazid are also appearing in isoniazid loaded NPs (with minor shifting) indicating the chemical stability of isoniazid inside the NPs. 3.7 AFM of Rifampicin The AFM technique was used to study the detailed morphology of NPs, as the prepared NPs are too small to be closely investigated by SEM due to its limited magnification. Hydrodynamic radii measured by dynamic light scattering were confirmed by AFM micrographs. AFM results confirmed the smooth and spherical surface of the formulated NPs along with the absence of aggregation or adhesion among NPs. 3.8 Zeta Potential. Zeta Potential of the prepared formulation found to be average of -26.94 mv which gives stability to the formulation on the storage (Fig 4). 3.9 In vitro dissolution test Figure 5 shows the in vitro release profile of Isoniazid, loaded NPs. A slow sustained release of Isoniazid due to the entrapment of drugs inside the core of nanoparticles as the surface modification increased. In the formulations highest regression co-efficient was observed in zero order equations starting from day one to tenth day (Table 6). The slower and sustained release of the drug from the beginning can be attributed to the erosion of polymeric matrix which releases the encapsulated drug [19].

Table. 1 Optimization of Polymer and Drug Ratio

Batch No.

Drug Loading in 500 µl of aq. Solvent (mg)

Polymer Amount in 5 ml DCM (mg)

pH of inner aqueous phase

Particle Size

Entrapment efficiency

NP 1 25 100 7.4 215±6.84 14.42±1.29 NP 2 25 300 7.4 239±7.51 22.53±1.41 NP 3 25 500 7.4 352±4.83 32.64±2.73 NP 4 45 100 7.4 218±6.57 24.02±3.08 NP 5 45 300 7.4 222±4.54 42.18±2.31 NP 6 45 500 7.4 349±4.95 48.54±3.19 NP 7 70 100 7.4 231±6.54 18.57±1.62 NP 8 70 300 7.4 264±3.84 28.41±3.29 NP 9 70 500 7.4 347±2.17 34.71±3.38

Table. 2 Optimization of pH of the inner aqueous phase

Batch

no. Polymer Amount in 5 ml

DCM Drug Loading in500 µl of aq.

Solvent pH of inner aqueous

phase Entrapment

efficiency NP 10 300 45 8 44.25±3.28 NP 11 300 45 9 53.47±2.84 NP 12 300 45 10 38.28±1.61 NP 13 300 45 11 34.57±2.97 NP 14 300 35 9 24.24±2.63 NP 15 300 55 9 30.57±1.86

Table. 3 Optimization of addition of an electrolyte to the outer aqueous phase

Batch

no. Polymer Amount in 5

ml DCM Drug Loading in500 µl of

aq. Solvent pH of inner

aqueous phase % Salt addition in outer

aqueous medium Entrapment

efficiency NP 16 300 45 9 1 56.48±2.41 NP 17 300 45 9 2 58.39±2.61 NP 18 300 45 9 2 61.24±2.71 NP 19 300 45 9 4 63.61±2.03 NP 20 300 45 9 5 50.47±1.97 NP 21 300 55 9 4 44.27±2.80 NP 22 300 35 9 4 32.58±1.76

Table 6. R2 value for Isoniazid Dissolution Profile

Zero Order First Order Hixon Crowell Korsmeyer Pappas Higuchi Plot R2 Value 0.984488 0.938725 0.977714 0.979929 0.961455



Figure. 1

Figure. 2



Figure. 3

Figure. 4

Figure 5



CONCLUSION

Overall, formulation NP19 which has a suitable particle size, drug entrapment efficiency and controlled release profile was the most satisfactory among all the NPs. The entrapment efficiency of the formulation can be enhanced up to 63 % having a drug loading of 11.93% by optimizing the formulation parameters and process parameters. Acknowledgment The authors are thankful to Macleod Pharmaceuticals, Mumbai, India, for the gift sample. The authors are also grateful to the Head of Department, University Department of Pharmaceutical Sciences, Utkal University, Bhubaneswar, Odisha, India for making available the research facilities used.

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Development and Enhancement of Entrapment Efficiency of Isoniazid Loaded Poly--caprolactone Nanoparticle