1. INTRODUCTION:

The terminology “Nanoencapsulation or Nanoparticles” of a drug indicates the particle size of the dosages forms ranging between 1-1000nm in diameter¹. They are generally solid or sub-micronized particles engaged to carry both biodegradable and non-biodegradable matter².

Nanotechnology is a multifaceted route that can be applied in a different scope of science and technology like chemistry, biophysics, biotechnology, and medical sciences³. The polymeric nanoparticles (NPs) are widely acceptable for Biomedicine for their wide surface area to the volume ratio and biocompatibility. Thus, this dosage form can enhance the absorption and bioavailability of otherwise poorly bio-available drugs⁴. Poor bioavailability of drugs generally depends on factors like poor solubility, low permeability through the GI tract, the reduced absorption rate in the GI lumen, and also pre-systemic eradication. Presently, most of the newly introduced drugs in the pharmaceutical field are non-polar hence suffer problems related to poor bioavailability thereby invites additional problems with dose variation and toxicity. Accordingly, the judicial use of nanotechnology is believed to be of much importance ⁵.

For improvement of therapeutic effect polymeric binding is well known, which can further be beneficial if this drug can be delivered through a nanocarrier system⁶. In a modern world, hypertension is a serious burden affecting the major population of this world. Further hypertension is of high occurrence and an associated risk factor for cardiovascular disease resulting in millions of human deaths⁷. Therefore a novel drug delivery system loaded with a combination of an antihypertensive with a biodegradable polymer is expected to address the above-mentioned health hazard by enhancing patient compliance with improved efficacy, reduced dose frequency, and a more acceptable toxicity profile.

In the current investigation, Eprosartan Mesylate (EM) has been selected as an antihypertensive agent which is chemically narrated as a nonbiphenyl, nontetrazole and a non-monomethane sulfonate salt of "(E)-2-butyl-1-(p-carboxybenzyl)-α-2thienylmethylimid-azole-5-acrylic acid" (Mol wt. 520.62) with a distinctive molecular structure⁸. Moreover, this agent is a newly introduced ARBs (angiotensin receptor blocker) with added beneficial clinical claims over other members of a similar group of drugs⁹. Eprosartan is a purely competitive antagonist despite other non-competitive kinetics of ABRs and it chemically-distinct from the other ABRs. Importantly, dual blockage of angiotensin-II receptors both pre and post-synoptically induced by eprosartan and therefore it crosses the hematoencephalic barrier that justifiably beneficial on its antihypertensive effects. The sympathetic outflow was restricted by eprosartan whereas the losartan, valsartan, or irbesartan can’t do this. This variation proves that eprosartan is a more potent prejunctional angiotensin-II antagonist that increases noradrenaline release¹⁰. However, amidst all such pharmacological benefits, the drug EM is categorized as BCS-II class drugs, indicating very poor bioavailability (i.e., 13% by oral absorption) which is believed due to the reduced aqueous solubility of the mesylate salt form of eprosartan ¹¹.

Therefore, a biodegradable polymeric (PLGA) nanoparticle drug delivery system could be one of the most optimal approaches to enhance the bioavailability of this poorly bioavailable and poorly water-soluble drug taken, where the permeability through the intestinal wall is expected to increase owing to reduced particle size contributing to magnified surface area for absorption and dissolution rate¹². Poly (lactic-co-glycolic acid) (PLGA) is one of the most successfully used biodegradable polymer because of its hydrolysis conduct to metabolite monomers that are lactic acid and glycolic acid. They can improve the pharmacokinetic and pharmacodynamics profiles of many challenging therapeutic agents also. Because of its many advantages, PLGA is approved by the US FDA and European Medicine Agency (EMA) in various drug delivery systems in humans¹³.

Hence, many alternative nanocarrier techniques to improve the bioavailability of this drug and also benifites of PLGA polymer to improve pharmacokinetics of poorly water soluble drug were already reported, but this research work aims to formulate a nanoparticle for EM by using biodegradable polymer poly-lactic-co-glycolic acid (PLGA) and Poly-vinyl alcohol (PVA) as a stabilizing agent, which is more advantageous^6,7. Dichloromethane and Ethanol have been used as solvents engaging the ‘Solvent evaporation double emulsion technique’. Like conventional methods for standardization of nanoparticles FT-IR, DSC, XRD, and SEM were performed, and finally, the in-vitro study was conducted to justify the theoretical enhancement of the release of these drugs from the formulated nanocarrier system against conventional dosage form ¹⁴.

2. MATERIALS AND METHODS:

2.1 Materials:

Eprosartan mesylate was procured from Finetech Industry Limited, Hong Kong, China. The biodegradable acid terminated Poly (D, L-lactide-co-glycolide) 50:50 Resomer® RG 504 H (PLGA) Polymer (Mw 38,000- 54,000) and polyvinyl alcohol (PVA) (Mol Wt. 85,000-1,24,000) were obtained from Sigma-Aldrich (Germany) and S D Fine Chem. Ltd., Kolkata, India respectively. The analytical grade reagents such as methanol (MeOH) and acetonitrile (ACN) were purchased from Honeywell, Germany. Ethanol (EtOH) and dichloromethane (DCM) were procured from Merck KGaA, Germany, and Merck Specialities Private Limited, Mumbai, India respectively. The phosphate buffer reagents, Tween 80 solvent, and deionized Milli-Q water (Merck Millipore) obtained from TAAB Biostudy Services, Jadavpur, Kolkata,India. The Dialysis Membrane bag (internal diameter 14.3mm and MW cut of 12,000-14,000 Da) was obtained from Bioequivalence Study Centre, Department of Pharmaceutical Technology, Jadavpur University, Kolkata,India.

2.2 Methods:

2.2.1 Preparation of Eprosartan mesylate loaded nanoparticles (EM-NPs):

EM-NPs were prepared following the double emulsion solvent evaporation technique¹⁵. The variable parameters like the amount of drug, percentage of PVA concentration, and homogenization speeds were mentioned in Table1 for formulations of NPs. The solubility study was done by LC-MS/MS in the early phase. At the beginning of this development EM along with PLGA (50mg) dissolved in the oil phase of the emulsion such as the mixture of two solvents like dichloromethane and ethanol (8:2, 2ml). The oil phase was homogenized by a high-speed homogenizer (IKA T10 basic Turrax Ultra homogenizer) for 7 minutes with adding the PVA solution (1ml) dropwise to generate the primary water-in-oil (w/o) emulsion. Further, the w/o emulsion was delicately added into the second continuous phase as 60ml of 0.5% PVA solution and it homogenized for 20 minutes to produce water-in-oil-in-water (w/o/w) double emulsion. Thus, leave it overnight upon the magnetic stirrer (200rpm) to evaporate the organic solvent. To separate nanoparticles which free from excessive PVA which is hazardous for systemic circulation and free drug molecules was centrifuged at 16000 rpm at cold centrifuged (4⁰C) (3K30 Sigma Lab Centrifuge, Merrington Hall Farm, Shrewsbury, UK) for 30 min and collected NPs palette wash thrice with de-ionized Milli-Q water then collected in a petri dish and kept it for -20⁰C overnight. Finally, the frozen NPs were lyophilized at -60⁰C for 8 hours (Laboratory Freeze Dryer, Instrumentation India Ltd., Kolkata, India) and preserved in a deep freezer (-20⁰C) for future experiments.

2.2.2 Physicochemical Characterization of NPs:

2.2.2.1 Measurement of Particle Size and Zeta Potential using DLS:

Zeta potential and size distribution were obtained at room temperature (⁓25⁰C) with the Zetasizer Nano ZS (Malvern Instruments, UK). The Zetasizer nano uses a 4mW He-Ne Laser of wavelength 632.8nm. The detector was positioned at scattering angle 173⁰. The detection scattered light was sent to the signal processing correlator¹⁶. A small amount of nanoparticle dispersed properly in de-ionized milli-Q water and prepared an NPs suspension. Before leaving it at quartz cell the NPs suspension was sonicated for 1min ¹⁷.

2.2.2.2 Fourier-Transform Infrared Spectroscopy (FTIR):

FTIR spectroscopy is widely used to study for the detection of functional groups by bond vibration of pure compounds like eprosartan mesylate, PLGA, physical mixtures drug with excipients, and freeze-dried NPs ¹⁸. The samples were analyzed for the evaluation for their intermolecular interaction by applying FTIR spectrophotometer (Bruker alpha, Germany) and the spectra of the drug and excipients were taken in the wavelength 400-4000 cm^-1, in an inert atmosphere¹⁹.

2.2.2.3 Differential Scanning Calorimetry (DSC):

DSC technique was employed to determine physicochemical compatibility and thermal stability of eprosartan, PLGA polymer, their physical mixture, and nanoparticles. DSC from Jade DSC, Perkin Elmer, Japan was used for the experiment. Accurately weighed the samples 2.0mg to 4.5mg were heated up under an inert environment of nitrogen gas flow 20.0ml/min at a scanning rate of 10⁰C/min over a temperature range of 25⁰C to 350⁰C²⁰.

2.2.2.4 X-ray Diffractometry (XRD):

X-ray diffraction technique was used to identify the physical state (crystalline and amorphous) of the compound in free and embedded form. The XRD analysis of EM API, PLGA, and dried EM-PLGA-NPs was recorded using a Bruker-AXS diffractometer (Physics department of Jadavpur University) at room temperature (~30⁰C). The dried samples were analyzed by a 2ɵ scanning range of 5⁰to 80⁰ with 30kV voltage and 30mA current respectively. In XRD, CuK_α(1.54 Å) radiation is used for irradiating the sample²¹.

2.2.2.5 Liquid Chromatography-Mass Spectrometry Analysis (LC-MS/MS):

Eprosartan mesylate API was dissolved into HPLC grade dimethyl sulfoxide (DMSO), then it was diluted into HPLC grade Methanol and injected through the mass syringe method into the mass spectrometer for Q1 scan and compared with extracted eprosartan mesylate from NPs. The drug loading, encapsulation efficiency, and in-vitro kinetics release of nanoparticles were quantified by LC-MS/MS method ²².

2.2.2.6 Surface morphological study by Scanning Electron Microscope (SEM):

The morphological measurement of eprosartan loaded nanoparticles by using the scanning electron microscope (SEM, EVO 18, special edition, Zeiss)²³. The dried powdered form of NPs formulation placed by carbon adhesive of aluminum dias on the surface of microscope for a morphological study like shape and size of nanoparticles, viewed under the high-resolution SEM, by using acceleration voltage 15kV and Quorum Q150T ES under a low-pressure high vacuum atmosphere a tiny film of gold dappled over the sample ²⁴.

2.2.3 Assessment of Encapsulation Efficiency (%EE) and Drug Loading percentage (%DL):

To determination the percentage of drug loading and encapsulation efficiency of EM-PLGA-NPs by extracting from PLGA polymer matrix by using a developed and validated method in LC-MS/MS facility (API 2000, Absciex). The nanoparticles (3mg) were dissolved in an organic solvent (DCM) for separation of the polymer which was bounded with the drug in NPs. Therefore, adding 1.5ml of methanol and centrifuge the sample at 12,000 rpm at 4⁰C for 10 minutes to separate the drug from NPs. After that, the supernatant was collected and dried under an inert N2 atmosphere using the water bath facility having 45⁰C temperature. The 200µl reconstituted dried sample (acetonitrile: water: 50:50 v/v) was transferred into the auto-sampler vials for further quantification. The combination of mobile phase was 0.1% formic acid in water (Pump A) and 0.1% formic acid in acetonitrile (Pump B) with C18 Phenomenex kinetex column (50nm×3.0nm, 5μm) use for the quantification of encapsulation efficiency (%EE) (Eq.1) and percentage of drug loading (%DL) (Eq.2) of EM-PLGA loaded NPs²⁵. The following equations respectively mentioned below which were used for further calculation ²³. The yield percentage of freeze-dried nanoparticles determined by Eq.3 given below ²⁰.

Actual drug loaded in nanoparticles, mg

Encapsulation = --------------------------------------------------------- × 100

Efficiency (%EE) Theoretical drug loaded in nanoparticles, mg

(Eq.1)

Weight of drug in nanoparticles

Drug Loading = -------------------------------------------------------- × 100

(%DL) Weight of nanoparticle used for analysis……. (Eq.2)

Dried nanoparticles obtained, mg

Yield Percentage = -------------------------------------------------------×100

The entire amount of drug and polymer used, mg

(Eq.3)

2.2.4 In-vitro drug release and kinetic study:

The in vitro dissolution study of EM-PLGA-NPs was carried on by membrane diffusion technique. The NPs formulation was pre-weighted (40mg) equivalent to 400 mg EM tablet was implanted into the dialysis membrane suspended in buffer medium containing surfactant Tween 80(0.1%) solution as a polymeric carrier for increasing solubility and dissolution rate of the drug²⁶. The sealed dialysis membrane bag dispersed into the 200ml of phosphate buffer saline (PBS) medium at 7.4pH under the sink condition USP Type I Basket. The study was maintained at 37±5⁰C under the influence of continuous shaking at 75rpm ²⁷. The sample (5ml) was withdrawn and replaced with the same volume of buffer solution into the receptor compartment at a predetermined time interval (0, 1, 2, 4, 6, 8, 12, 24, 48, 72, 192, 360hrs) to maintained equilibrium^28,29. Therefore, the collected samples were centrifuged at 12,000rpm for 10min and the supernatant samples were extracted and injected into the LC-MS/MS for analysis by the previously validated method.

To evaluate the in-vitro drug release study by using the drug release data from different mathematical models such as zero order, first order, Higuchi and Korsmeyer-Peppas model. Based on the highest correlation coefficient (R2) values the best kinetic models are selected. Rate constants for zero-order kinetics (K₀), first-order kinetics (K₁), Higuchi model (K_H) and release exponent (n) for Korsmeyer–Peppas model were calculated by putting the data in the corresponding equations to evaluate the drug release kinetics ²⁰.

3. RESULTS AND DISCUSSION:

3.1 Drug loaded polymer nanoparticles:

The preparation of drug-polymer loaded uniform and spherical nanoparticles by solubility modifier double emulsion (w/o/w) solvent evaporation technique was of challenging research. The eprosartan mesylate was an excellent and newer antihypertensive drug, but due to its water insolubility and low bioavailability, it has very limited therapeutic applications. Therefore, the biodegradable poly (D, L-lactic-co-glycolic acid) (PLGA) polymeric coating and double emulsion solvent evaporation technique enhance the solubility and bioavailability of this drug to its targeted environment [30]. On the other hand, a well-known hydrophilic polymer polyvinyl alcohol (PVA), as a stabilizing agent was played an important role in the preparation of nanoparticles. For the preparation of stable primary emulsion (w/o), the variable concentration of emulsifier (PVA) shown in Table1 which made the particle size uniform, gives the mirror effect on secondary emulsion (w/o/w)³¹. The increasing concentration of PVA was directly related to the particle size due to the improvement of the emulsification process ³². The hydrophilic –OH group of PVA which is the nonpolar vinyl part lowers the surface tension between non-aqueous and aqueous parts, thus stabilizes the primary emulsion ²⁰. The major challenging part of this study was the selection of solvent for solubilized the drug. Dichloromethane was used as a major solvent in this emulsification-solvent evaporation technique, but EM has a solubility problem with this solvent. EM was a highly hydrophobic and selectively lipophilic drug ³². EM also has instability in heat and light and solubility 0.08mg/ml in an acidic environment, it exhibits pH-dependent aqueous solubility with a high pKa value of 5.30 and log P_e value ~5.78 ⁷. Drug loading in nanoparticles was affected by the relative distribution of the drug into the polymeric matrix was depend on the solid phase solubility of the drug. The polymer (PLGA 50:50) was easily dissolved into DCM but EM has a solubility in ethanol, therefore the mixed solvent system DCM: ethanol (8:2) was found to be extremely effective to dissolved polymer and drug both for the stable primary emulsion to develop nanoparticle formulation. At the beginning of the formulation, necessary knowledge regarding potential variables and proper methodology was accumulated from previously published literature³³. Percentage of PVA and homogenization speed is the major influencing parameters that are directly proportional to the particle size and drug encapsulation capacity which was shown in Table1. More homogenization speed applies more stress on the viscous dispersed phase and breaks into smaller droplets to form smaller particles. Additionally, to optimize the formulation, prepared the nanoparticle with the increasing amount of drug that affects encapsulation efficiency and drug loading percentage. Both percentages of DL and EE were increased initially but after a certain amount of drug-loaded in the polymer matrix, they were saturated. Finally, the sensitivity associated with the temperature maintained at 20⁰C throughout the emulsification process and a slow solvent evaporation process was carried out for a minimum of 12hrs in the darkroom to avoid chemical degradation of drug and formation of nanodroplets.

Table 1: Various compositions and yield percentages of prepared formulations:

Formulation Code	Amount of Drug (mg)	Amount of PLGA (mg)	Percentage of PVA concentration (%)	Homogenization speed (rpm)	Percentage of drug loading (%DL)	Percentage of encapsulation efficiency (%EE)	Yield percentage (%)
NPF1	5	50	1.0%	15,000	0.071%	12.91%	36.60%
NPF2	10	50	1.5%	17,000	19.74%	24.32%	47.20%
NPF3	10	50	2.5%	20,000	17.28%	35.22%	50.72%
NPF4	10	50	3%	25,000	34.40%	42.44%	53.60%
NPF5	25	50	3%	25,000	67.62%	72.62%	75.00%
NPF6	50	50	5%	25,000	82.32%	90.28%	80.20%

3.2 Evaluation of Encapsulation Efficiency and Drug Loading Percentage:

The rising amount of drug was reflected in the rising amount of drug loading capacity and encapsulation efficiency in this study. Hence, the saturation point of the drug: polymer ratio (1:1) is shown in table 1. The percentage of drug loading (%DL) and encapsulation efficiency (%EE) was tabulated in Table 1. Therefore, the NPF6 has the highest drug loading and encapsulation efficiencies like 82.32% and 90.28% respectively as compared with the other formulations. The Yield Percentage of prepared formulation varies from 36.60% to 80.20% which depends on the rising amount of drug in this experiment. (Table1).

3.3 Evaluation of particle size and zeta potential:

The two major characteristics of nanoparticle formulation were particle size and polydispersity index (PDI). In this present study, the average particle size and PDI of the different formulations varied between 136nm to 380nm range and 0.231 to 0.588 respectively which was shown in Table 2 and fig 4c. The polydispersity index described the distribution pattern of the particle size of the formulations. The PDI value was found to be higher than near 1 or more than 0.5 which means the wider range of particles were present in the formulation and they were not identically uniform, while evenly distributed particle size indicates the lower PDI values (less than 0.5) [34]. Preparation of nanoparticles with PLGA by employing DCM as a solvent system and PVA as a stabilizing agent were produced particle size 60-200nm range. J. Prasad Raoa, Kurt E. Geckeler were concluded in their article that a high concentration of stabilizing agent (3%, w/v, or higher) was guaranteed a good emulsification process, thus conduct to smaller particles, with a satisfactory polydispersity index [35]. Therefore, the NPF6 in this study achieved the smallest particle size with satisfactory polydispersity index.

The Zeta potential is the electrostatic potential that presents at the surface of the particle. It defined the stability of colloids³⁶. The ZP is a charge which is acquired by the particles in a colloidal medium, and the value indicates the probability of physical stability of nanoparticle dispersion. If the particles have huge positive or negative charges like ≥+30mV to ≤ -30mV, then they will avoid rapid agglomeration in a liquid state and the system will be considered as stable for a longer period of time³⁷. The ZP of the NPs formulations in this study ranged from -10.4mV to -27.3mV (Table 2, fig 4d). The experimental data suggest that the nanoparticle formulations were safely preserved in lyophilized form and reconstituted before use.

Table 2: Particle size, PDI, and zeta potential of different formulations

Formulation code	Mean particle size (nm)*	Polydispersity Index	Zeta Potential (mV)*
NPF1	380.80±2.6	0.535	-24.8±0.21
NPF2	359.10±3.9	0.588	-16.9±0.41
NPF3	298.50±2.8	0.382	-22.50±0.51
NPF4	282.50±1.6	0.571	-10.4±0.22
NPF5	182.60±2.8	0.231	-27.3±0.52
NPF6	136.00±1.34	0.356	-10.8±0.31

*Shown data mean, ±standard deviation (n=3)

3.4 Evaluation of Solid-State characterization:

3.4.1 FTIR Spectroscopy:

Among the various formulation characterization studies, the FTIR spectroscopy was analyzed the chemical structural similarity of drug and excipients with the physical mixture and formulated nanoparticles³⁸. The IR spectrum was measured in a range of 4000 to 400 cm^-1 and they were represented in fig 1. Pure Eprosartan Mesylate (fig1a) was showed characteristics spectrum at 1691.92 cm^-1 for C=N stretching, 1648.99 cm^-1 was denoted C=C stretching, 1422.56 cm^-1 due to aromatic C-C group of aromatic rings respectively. Also 1217.68 cm^-1 due to the C=O bond of the carbonyl group and 1160.15 cm^-1 due to the C-N vibration of the aromatic ring. Additionally, the spectra at 1112.45 cm^-1, 1044.31 cm^-1, and 925.38 cm^-1 were assigned to the OH group, 841.88 cm^-1 observed for the COOH group of the benzene ring, 692.32 cm^-1 due to trans RCH=CHR stretching and 639.54 cm^-1 denoted C-H bending. The spectrum of PLGA (fig1b) showed the characteristic peak at 2930.73 cm^-1for C-H bond, 1759.69 cm^-1 for C=O stretching of carbonyl group, 1424.33 cm^-1 for C-H stretching of a methyl group, 1131.01 cm^-1, and 1093 cm^-1 for C-O stretching vibration³⁴. The physical mixture(fig1d) showed the major peaks at 3352.04 cm^-1 for O-H stretching from intra and intermolecular hydrogen bond and 2971.31 cm^-1for stretching of C-H bond from an alkyl group of polyvinyl alcohol (PVA) was absent in Fig 1c that confirmed the surfactant was successfully removed from nanoformulation. The typical characteristic spectrums of EM and PLGA also present in the physical mixture and optimized nanoformulation. Here is none of such significant shift in the characteristic peaks of excipients and drug that denote, there was no such intermolecular interaction between them. The Missing spectrum of the drug in NPs indicates that the nanoparticles were completely encapsulated and there was no such free drug molecule present on the surface of it.

3.4.2 DSC Study:

Differential scanning calorimetric (DSC) study was performed to investigate the physical state and integrity of the drug and polymer in the NPs because this feature was given influence on in-vitro and in-vivo release [39]. In this present study, the drug and surfactant used in various ratios, and also used two types of solvent. Hence, to determine an undesirable interaction between excipients and drug, regarding this formulation, there was performed DSC, XRD, and LC-MS/MS analysis. The DSC thermogram of pure EM, PLGA polymer, physical mixture of excipients, and EM-PLGA loaded NPs (NPF6) were shown in fig 2. The thermogram of pure EM (fig 2a) gives a sharp endothermic peak at 251.85⁰C correlates to the melting point -temperature (T_g) 54.11⁰C, which is the characteristic of its amorphous structure. The low intensity of characteristics melting point peak of EM was found in the thermogram of EM-PLGA-NPs (fig 2d) which is explain the amorphous structure of encapsulated drugs. The thermogram of formulated NPs having a peak at 54.77⁰C (T_g of PLGA), which is specified none of the interaction happened between EM and PLGA during nanoformulation development. The decreasing rate of enthalpy (∆H) in the DSC thermogram is denoted the reduction of crystalline structure EM after formulating the EM-PLGA-NPs.

Figure 1: Fourier-transform infrared (FTIR) spectra of (a) EM, (b) poly (D, L-lactic-co-glycolide) (PLGA) and (c) optimized formulation EM-PLGA-NPF6, (d) physical mixture.

Figure 2. DSC thermograms of (a) EM, (b) PLGA

Figure 3. XRD studies of (a) EM, (b) PLGA,

(c) physical mixture and (d) TLM-PLGA NPF6. (c) physical mixture and (d) EM-PLGA-NPF6.

3.4.3 XRD study and LC-MS/MS study:

Solid matter consists of two types of states like amorphous and crystalline. The X-ray diffraction analysis (XRD) determined the physical state of the compounds which may affect on in-vitro and in-vivo release characteristics of the drug from nanoparticles ⁴⁰. The X-ray diffractogram of pure EM showed the diffraction peaks at 7.36⁰, 12.74⁰, 17.2⁰, 18.56⁰, 19.18⁰, 20.32⁰, and 24.52⁰ in fig 3a, which presented the crystalline nature of EM. On other hand less intense diffraction peak of EM was shown in the physical mixture of EM-PLGA (fig 3c) due to the EM crystalline structure undergoes the amorphous structure of PLGA. The PLGA showed (fig 3b) the regular baseline due to its amorphous nature. Finally, the absence of a similar diffraction peak of pure EM in EM-PLGA-NPs (fig 3d), that indicates EM was bounded by the amorphous solid PLGA polymer.

Additionally, EM was scanned and quantified by LC-MS/MS before and after nanoparticle formulation. The mass syringe scan of Q1 and Q3 in positive polarity denoted that the total mass of Eprosartan (M+H) and its product-ion also. Eprosartan Mesylate having a total mass of 520.62g/mol, thus when it soluble in DMSO or another solvent the weak bond between the eprosartan and mesylate group was broke down and eprosartan has a molecular weight of 424.515g/mol [41]. The Q1 and Q3 mass of the standard and extracted sample of EM from NPs were found to be the same, that was m/z 425.10→ 207.1 respectively. The quantification of drug carried out by the multi-reaction monitoring method (MRM) of LC-MS/MS, the peak intensity of the reference standard of EM (pure drug) was approximately similar concerning to the extracted samples of NPs (data not shown). These results indicated that EM has no chemical interaction with excipients in NPs.

3.4.4 Evaluation of Surface morphology study:

The morphological characteristics of EM loaded PLGA nanoparticles were examined with a scanning electron microscope (SEM). At different magnification levels, the SEM micrographs indicated that EM-PLGA loaded nanoparticles were clearly near monodispersed and spherical with none of the aggregation. Thus, the homogeneous particle size between 100-200nm is shown in fig 4 (a) and (b). The smooth surface of NPs played a factoring role in the uniform sustained release of drugs from nanoparticles. The zeta potential and particle size of formulated nanoparticles were discussed earlier and shown in fig 4.

Figure 4. SEM images of EM-PLGA-NPF6 (a) magnification at 25KX (b) magnification at 10KX and (c) particle size distributions and (d) zeta potential.

3.5 Evaluation of In-Vitro release study:

Time-controlled drug delivery could be achieved through polymeric drug delivery systems; using the widely accepted biodegradable polymer PLGA which is used in this study, therefore controlled release one of the most important characteristics of polymeric NPs [42]. The dissolution or in-vitro study of NPs is an important property that influences the mode of action of the therapeutic agent. Generally assumed that solubility may increase as particle size decreases, on the other hand, the molecular weight of the polymer is one of the key factors affecting drug release⁴³. According to the literature acid terminated PLGA (PLGA 50:50) undergoes bulk degradation through random hydrolytic scission of its backbone ester linkage throughout the matrix and more swelling due to water uptake. The acidic (lactic acid and glycolic acid) monomers and oligomers thus formed further catalyze and the degradation of the parent polymer slowly, this phenomenon is also known as autocatalysis⁴⁴. Thus, a steady and sustained release of the drug occurred by the diffusion of the drug from the polymeric core of the NPs. Consequently, due to a long degradation period of PLGA 50:50 as 102days, the EM-PLGA-NPs has observed a prolonged release profile (360 hours) in phosphate buffer saline medium (pH 7.4) which mimicking blood serum [45]. In-vitro release study of EM tablet (400mg) and EM loaded PLGA nanoparticle was carried out a comparative study for the establishment of a better therapeutic window which is shown in fig 5. EM-PLGA-NPs indicated a prolonged controlled release profile as a comparison with marketed EM tablets.

Table 3: Results of curve fitting of the in vitro release of EM from the optimized formulation.

Model	R²value	Release exponent (n)
Korsmeyer–Peppas model	0.8979	0.3328
Higuchi Model	0.8587
Zero-order	0.6585
First-order	0.3855

The EM encapsulated nanoformulation showed the biphasic drug release profile in an in-vitro release study. Initially, burst release occurred within 12 hours then followed by a slow controlled release shown in fig 5a. Due to the presence of the drug to the outer surface of the nanocarrier, occurred burst release, then the remaining drug occurred prolong diffusion from the core of the polymer matrix [20]. Therefore, after 360hrs or 15days of release study, the cumulative percentage of drug release observed, that NPF6 was 82.03%. NPF6 was comparatively better than other formulations.

Figure 5: In vitro release profile of (a) EM–PLGA-NPF6 and (b) EM tablet.

Further, the release kinetics of the drug was evaluated by fitting the cumulative drug release data in various models. The zero-order kinetic model (% cumulative drug release vs. time plot), the first-order kinetic model (log % of cumulative drug release vs. time), the Higuchi model (% cumulative drug release vs. square root of time), the Korsmeyer-Pappas model (log% of cumulative drug release vs. log time) ⁴⁶. The curve fitting in-vitro release data tabulated in table 3, according to the basis of a high correlation coefficient. Therefore, based on the highest correlation (R2) coefficient values (R2 = 0.8979) (table 3), was summarized that the Korsmeyer-Peppas model was the best-fitted model in respect of other release kinetic models of optimized nanoformulation. In Korsmeyer–Peppas model the optimized release of EM from EM-PLGA loaded NPs was followed Fickian diffusion (n=0.3328) because the Fickian diffusion-controlled drug release followed n = ≤0.43 ²⁰. Thus, the release pattern and results showed that EM release from polymeric nanoparticles by a diffusion-controlled mechanism.

4. CONCLUSION:

Nanoencapsulation is an important candidate for the delivery of the therapeutic agent in the pharmaceutical industry. Therefore, based on our study we concluded that eprosartan Mesylate a new member of the ARB group which was successfully formulated by double-emulsion solvent evaporation technique and evaluated by various characterizations and in-vitro release study. The novelty of this development work is, nanocarrier of poorly water-soluble drug EM by using biodegradable acid terminated PLGA 50:50 to boost up the therapeutic window of the drug as compared to the conventional marketed oral dosage form. PLGA loaded nanocarriers have shown many advantages for drug delivery and they can improve drug stability and protected from degradation. They can improve the potency of therapeutic level by the controlled release of drug from stable nanoparticle formulation which is already confirmed by the in-vitro release study of this developmental work. To the best of our knowledge, this developmental study of nanocarrier along with EM, loaded in the biodegradable polymer PLGA has not been reported earlier. The controlled properties of the double emulsion solvent evaporation technique like the amount of polymer, percentage of the stabilizing agent (PVA), solvent ratio, homogenization speed was explored briefly to prepare the nanoformulations. The optimized nanoparticles have high encapsulation efficiency, a low range of average particle size, and a high range of zeta potential. In conclusion, this study confirms that not only the dissolution of eprosartan mesylate improved but also the sustained-release form of nano pharmaceutical dosage form (15days) improved the therapeutic efficacy of this antihypertensive agent. Thus, there is a scope to extend further the investigations to in-vivo studies. Therefore, this experiment clearly illustrates the assurance of novel treatment by this nanoparticle in future days.

5. CONFLICTS OF INTEREST:

There are no conflicts to announce.

6. ACKNOWLEDGMENT:

The authors appreciatively acknowledge to UGC for providing financial support by ‘Rajiv Gandhi National Fellowship (UGC-RGNF) for SC/ST Candidate for The Year 2013-14’, to conduct the study and thankful to the Bioequivalence Study Centre, Jadavpur University and TAAB Biostudy Services, Jadavpur, Kolkata, India for providing essential facilities and instruments. We are also greatful to the Centre for Research in Nanoscience and Nanotechnology, Calcutta University, Kolkata, India for providing facilities to carry out the SEM and the Department of Physics, Jadavpur University, Kolkata, India for providing facilities to carry out zeta seizer. We would like to express our special thanks to Dr. Animesh Ghosh, Associate Professor, Pharmaceutical Sciences and Tech, Birla Institute of Technology, Mesra, India for providing the DSC facility.

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Received on 05.02.2021 Modified on 10.03.2021

Research J. Pharm. and Tech 2022; 15(1):103-112.

DOI: 10.52711/0974-360X.2022.00018