INTRODUCTION:

Targeted drug delivery to definite sites is the major problem which is being faced by the researchers. The development of new colloidal carrier nanosponges has the prospective to solve these problems. Nanosponge is innovative and emerging technology which offers controlled drug delivery for topical use. Nanosponges play an important role in targeting drug delivery in a controlled manner. A wide variety of drugs can be loaded into nanosponge for targeting drug delivery. Both water soluble and insoluble that is lipophilic as well as hydrophilic drugs can be loaded into nanosponges ^[1-3].

Nanosponge drug delivery system has emerged as one of the most promising fields in life science. The development of nanosponges has become an important step toward overcoming these problems. These tiny sponges can circulate around the body till they meet the target site, stick on the surface and begins to release the drug in a controlled and anticipated way which is more efficient for given dosage. Nanosponges are smaller in size due to their small size and spongy nature they can bind poorly-soluble drugs inside their matrix and develop their bioavailability. They can be formulated for targeting drugs to specific site and prevent drug and protein degradation and prolong the drug release in a controlled manner^[3].These nano-sized colloidal carriers have been recently proposed for drug delivery, since their application can solubilize poorly water-soluble drugs and endow with prolonged release, as well as improve the drug’s bioavailability by modifying the pharmacokinetic parameters of actives.^[4,5,6]

MATERIAL:

Praziquantel (PZQ) was obtained a gift sample from Microlabs Ltd. Bengluru, India. Ethyl cellulose, Polyvinyl Alcohol, Dichloromethane was purchased from Loba Chemie PVT, LTD. Mumbai, Whey protein purchased from Ana Lab Fine chemicals, Mumbai, India.

Experimental work

Analytical study

UV-VIS Spectrophotometry Method for Praziquantel

Study of spectra and selection of analytical wavelengths

An amount equivalent to 10 mg of the reference standard PZQ was added to 100 ml volumetric flask. PZQ was dissolved in around 25 ml methanol in a 100 ml volumetric flask with vigorous shaking followed by ultrasonication for about 5 minutes. The volume was made up to the mark with the same solvent to gain standard stock solutions of concentration 100 µg/ ml. By appropriate dilution of the standard stock solution, appropriate concentrations of Praziquantel were prepared. The standard solutions were then scanned in the spectrum mode of the instrument from 400 nm to 200 nm and the spectra were observed and recorded to acquire the value of lambda max (λ_max).

Study of Beers-Lambert’s Law

Preparation of standard curves of Praziquantel in distilled Water, in acidic buffer pH 1.2, In Phosphate Buffer pH 7.4 and Phosphate pH 6.8 respectively.

Saturation Solubility Studies:

Saturation solubility of Praziquantel in various solvent i.e. distilled water, acidic buffer (pH 1.2), phosphate buffer pH 7.4 and phosphate buffer pH 6.8 were determined. An excess amount of Praziquantel was added to the conical flask restraining 20 ml of solvent and content was stirred for 48 hrs. On rotary shaker the mixture was then filtered through Whatman filter paper. The solubility of Praziquantel was determined spectrophotometrically at 210 nm.

Preformulation studies:

The drug sample of Praziquantel was analyzed for physical appearance, color, odor, solubility and test.

Melting point determination:

The melting point of Praziquantel was determined by using melting point apparatus (Veego, Model VMP-D, India).

Spectroscopic studies

Compatibility study between drug and excipients

FT-IR spectrum interpretation: ^[7]

The dry sample of Praziquantel was mixed with IR grade KBr in the ratio of 100:1. This mixture was compressed in form of a pellet by applying 10 tons of pressure in hydraulic press. The pellets were scanned over a wave number range of 4000 to 400 cm^-1in (Perkin Elmer, Spectrum BX, and USA) FTIR instrument. The spectral analysis was done, by comparing test sample spectra with the standard spectra of Praziquantel and also by comparing the absorbance peaks with standard absorbance of the functional groups.

Differential scanning calorimetry (DSC): ^[7]

Sample was accurately weighed onto aluminium pans and then hermetically sealed with aluminium lids. Thermo gram were obtained at a scanning rate of 10° C /min conducted over a temperature range of 30-300° C in the environment of liquid nitrogen (flow rate 10ml/ min). Thermo grams of the lyophilized powder samples of different preparation were studied. An empty aluminium pan was used as a reference. DSC measurements were performed at a heating rate of 5ºC/min from 25 to 250ºC using aluminium sealed pan. The sample size was 5-10mg for each measurement. During the measurement, the sample cell was purged with nitrogen gas.

Nuclear magnetic resonance: ^[8-10]

The ¹³C NMR, ¹H NMR. 2D-NMR (ROESEY, COESEY) high resolution magic angle spinning (HR-MAS) NMR techniques have becomes an important tool to study structure of cross-linked polymer. NMR technique can also be used to study the molecular structure in order to evaluate the molecular mobilities of cross-linked in nanosponges structure and examination of in interaction in complex of drug – polymer. In NMR experiments the change in chemical shift values (ð) indicates exchange of proton between the reacting species, thereby confirming the formulation of nanosponges.

Method of nanosponge preparation:^{[11-16 ]}

A series of different ratio of polymer such as ethyl cellulose, polyvinyl alcohol/whey protein as ethyl cellulose, polyvinyl alcohol/whey protein used for formulation of nanosponges. Dichloromethane was used as cross-linking agent. Drug Praziquantel and ethyl cellulose was added in the dichloromethane in first beaker. In second the polyvinyl alcohol/Whey protein was added in distilled water. The reaction mixture of first beaker was added in second beaker. This reaction mixture was stirred at 1000 RPM for 2 Hrs. Filtration process was carried out, after filtration collect the nanosponges. Freeze dried -20°C for 24 hrs and stored in ambient temperature until further use.

2³ factorial design:

2³ factorial design which consist of the Ethyl cellulose (X1), dichloromethane (X2) and stirring speed (X3) was considered as independent variable while Particle size, % cumulative drug release and drug loading was considered as dependent variables.

Evaluation and characterization

Particle Size Analysis and zeta potential: ^[17-21]

Particle size of Praziquantel loaded Nanosponge were measured by photon correlation spectroscopy using Zeta sizer (PCS3000, Malvern, English). Samples were diluted appropriately with aqueous solution containing 1% F68 and 20% sugar. Zeta potential is a measure of surface charge. It can be measured by using additional electrode in the particle size equipment. For zeta potential determination, samples of the nanosponges were diluted with 0.1 mol/L KCl and placed in the electrophoretic cell, where an electric field of about 15 V/cm was applied. The mean hydrodynamic diameter and polydispersity index of the particles were calculated using the cumulated analysis after averaging of the total measurements.

Micromeritics study ^{[17, 18]}

Angle of repose

Angle of repose of each batch was carried out by glass funnel method. Angle of repose then calculated by measuring the height of the cone of powder and radius of the circular base of powder heap. Angle of repose can be calculated by formula,

Bulk density

Bulk density of known mass of nanosponge in graduated measuring cylinder.

Bulk density = weight of nanosponges in gram/ bulk volume of nanosponges

Tapped density:

Tapped density = weight of nanosponges in gram/ volume of nanosponges after tapping.

Carr’s compressibility index

Carr’s compressibility index = (Tapped density- Bulk density)/ Tapped density x100

Hausner’s ratio

Hausners ratio = Tapped density/ Bulk density

Powder X-ray diffraction (PXRD) ^{[17, 18]}

The PXRD patterns were recorded on an x-ray diffractometer (BRUKER aXS-D8 ADVANCE). The samples were irradiated with mono-chromatized CuKa radiation and analyzed between 2-80ºC 2Ø. The patterns were collected with voltage of 30kV and current of 30mA respectively. The scanning rate (2Ø/min^-1) was set at 10 ºC/min.

Scanning electron Microscopy (SEM) ^[17,
18]

These tools are employed to evaluate the particle shape and size and to get morphological information related to the drug delivery system under investigation. SEM involves imparting conductivity to the developed particles under vacuum with a focused electron beam. Whenever moist samples are to be investigated, FESEM can be employed.

Transmission electron microscopy (TEM) ^[17,
18]

The morphology of Nanosponges was examined using an electronic transmission microscope (PHILIPS CM-200; London; operating voltages: 20-200kv Resolution: 2.4 A^o). TEM images are formed using transmitted electrons (instead of the visible light) which can produce magnification details up to 1,000,000 x with resolution better than 10 A^o. The images can be resolved over a fluorescent screen or a photographic film.

Entrapment efficiency ^{[17, 18]}

Entrapment efficiency (Ee) and Drug loading (Dl) was determined by taking a weighed quantity of Nanosponges (25mg) in a 25ml volumetric flask, sufficient quantity of 0.1N HCL was added to make volume 25ml. The suspension was shaken vigorously and then left for 24hrs at room temperature with intermittent shaking. Suspension was filtered and drug content in filtrate was determined by using UV/VIS spectrophotometer at suitable wavelength (210nm). Entrapment efficiency for each batch was calculated in terms of percent entrapment as per the following formula. Ee= (Wa-Ws/ Wa) x 100

Where Wa, Ws and WI were the weight of drug added in system, analyzed weight of drug in supernatant and weight of polymer added in the system, respectively.

In vitro Drug release study:

In vitro dissolution studies were carried out by using USP type I Apparatus. Weighed amount of drug loaded nanosponges were dispersed into semipermeable membrane (Two ends closed small permeable bag) were ends closed by clamps. This membrane was placed in dissolution apparatus containing 900 ml dissolution medium of phosphate buffer pH 6.8 maintained at 37⁰C at 50 RPM. Withdrawn at pre-determined time intervals and equal volume of fresh dissolution medium was replaced to maintain sink condition. The samples were analyzed spectroscopically at 210 nm to determine the concentration of drug present.

RESULTS AND DISCUSSION:

Analytical study

UV-VIS Spectrophotometry Method for Praziquantel

Description: The sample of Praziquantel was found to be a white crystalline powder.

Study of spectra and selection of analytical wavelengths

The UV spectrum of Praziquantel showed lambda max (λ_max) at 210 nm which complies with the value.

Study of Beers-Lambert’s Law

Calibration Curve was drawn with preparation of standard solution of Praziquantel in Acidic Buffer of pH1.2, distilled water, phosphate buffer pH 6.8, and phosphate buffer pH 7.4 respectively and it was found to be linear in the concentration range between 2-16 mcg/ml at 210.0 nm λ_max.

Saturation Solubility Study

Saturation Solubility was determined in distilled water, acidic buffer pH 1.2, phosphate buffer pH 6.8 and phosphate buffer pH 7.4 respectively. Moreover, it was found to be 11.2, 21.1, 25.93 and 16.06 mg/25ml respectively.

Preformulation studies

Spectroscopic studies

FT-IR spectrum interpretation

Compatibility study between drug and excipients

The peak shows the presence of C – H Alkenes, Alkanes, C–C Aromatic compounds, C–O Ethers, Nitro compounds, O – H Phenols in Praziquantel sample. The peak shows the presence of C – H Alkenes, Alkanes, C – C Aromatic compounds, C–O Ethers, Nitro compounds, O – H Phenols, C=O aldehydes- ketones, C=O esters, C=C aromatic hydrocarbons. The peak observed in the FTIR spectrum of Praziquantel pure drug with polymers (EC, PVA and whey protein) showed no shift and no disappearance of characteristics peak of pure drug suggesting no interaction between drug and polymer which is depicted in Figure 1.

Figure 1: FTIR spectra of Pure Praziquantel and PM

Differential scanning calorimetry (DSC)

The DSC thermogram of nanosponge formulation was showed absence of endothermic peak which gave clear evidence that there was no formation of inclusion complex. The DSC Thermogram of physical mixture was showed an endothermic peak at its melting point. The DSC thermogram of Praziquantel which shows a sharp endothermic peak at 144.07 °C corresponding to its melting point. No significant change in the position of this peak or broadening of peak in the drug and excipients mixture was observed with respect to the thermogram of pure drug. Hence, it can be concluded that the drug and excipients does not interact with each other which is depicted in Figure 2.

Figure 2: DSC Thermogram of nanosponge formulation, Pure Praziquantel, Physical mixture

Nuclear magnetic resonance

In the ¹H-NMR spectra examined for the complex of nanosponges, both negative positive small proton shifts indicated the presence of interaction in formulation/ complexes. Moreover, chemical shifts (ð 6-7) was observed for C-H (aromatic) for PZQ. As it obvious the, lowest shifts were observed for PZQ-NS complex indicated weak interactions with almost certainly negligible effect on PZQ properties. However polarizable (O-H, N-H, and N-CH) indicates the dipole- dipole interactions i.e. weak interaction was possibly seen between polymer-drug: complex which leads to the conclusion that the sharp and intense peaks of drug got disappeared in NSGs formulation. Thus it may conclude that drug has successfully encapsulated in to the core of polymer which is depicted in Figure 3.

Figure 3: NMR spectra of (A) PZQ, (B) NSGs formulation

Formulation of Nanosponges:

In Emulsification solvent diffusion (ESD) method various proportion of ethyl cellulose and polyvinyl alcohol were used in ratios. The dispersed phase containing ethyl cellulose and drug was dissolved in dichloromethane and slowly added to a definite amount of polyvinyl alcohol or whey protein in distilled water as aqueous continuous phase. The reaction mixture was stirred at various rpm for 2 hrs. Then Nanosponge formed was collected by lyophilization. Different concentration of independent variables was mentioned in the table no. 1.

Table 1: Factorial Design of Praziquantel nanosponges

STD	RUN	FACTOR 1:EC (MG)	FACTOR 2:DCM (ML)	FACTOR 3:SS (RPM)	RESPONSE 1 PS	RESPONSE 2 %CDR	RESPONSE 3 %DC
8	1	1000	20	4000	490	60	40
3	2	500	20	500	578	67	58
5	3	500	10	4000	484	73	55
6	4	1000	10	4000	367	78	62
2	5	1000	10	500	428	83	78
1	6	500	10	500	132	92.96	97
4	7	1000	20	500	529	85	85
7	8	500	20	4000	289	79	81

Figure 4: Effect of response surface plot on (A) Particle size (B) % CDR (C) DC

PXRD (Powder X-Ray Diffraction)

The sharp and intense peaks indicate the drug was found to be crystalline in nature which is depicted in Figure 6 (A). The peaks are not sharp which was found to be a broad and hen peak; this indicates the ethyl cellulose was found be an amorphous in nature. The prepared nanosponge formulation was found to be amorphous in nature which is depicted in Figure 6 (B). The amorphous nature of optimized formulation indicates that the formulations which promote the enhanced solubility thereby improve dissolution rate, diffusion and eventually which may claim for the improved bioavailability.

Figure 6: Diffraction Pattern of (A) PZQ (B) Optimized nanosponges batch (F6)

Scanning electron Microscopy (SEM)

The spherical nanometric particles in the size range of 190-220 nm. SEM analysis revealed nanosize, almost spherical particles with numerous pores on the surface which shown in the following figure.

Transmission electron Microscopy (TEM)

TEM study revealed that the particle size distribution was observed in nanosize range the maximum number of particles was found to be around 50nm which was average particles size.

Figure 7: SEM (A) NSGs TEM (B) NSGs Selected Area Electron Diffraction (SAED) (C) NSGs

From TEM examination, thus it was concluded that the particle size was found to be in nanometer size and spherical in shape.

Entrapment efficiency

The formulations were characterized, the highest Entrapment efficiency was found with 93.2% for the formulation F6 batch which was depicted in table no.3 .

Table 3: Entrapment efficiency of all formulations of NSGs

Sr. No	% Entrapment efficiency
F1	81.4
F2	86.3
F3	89.5
F4	90.0
F5	75.0
F6	93.2
F7	79.3
F8	87.2

In-vitro drug release of all formulations (% Cumulative Drug Release)

From In-vitro drug release of all Nanosponges formulations i.e (F1- F8) was found that complex showed a significant improvement in the rate of release as compared to plain drug which is depicted in Figure 8 (A). Among all the possible formulations F1 – F8 the maximum release was observed with F6 i.e 92.96 which was framed as optimized formulation. From the Figure 8 (B) it was observed that optimized lyophilized NSGs showed maximum release (92.96%) as compared to plain drug (PZQ) and Marketed tablet.

CONCLUSION:

On the basis of production yield and practicability of the method, ESD method was selected for the formulation of NSG’S. The PVA/ Whey protein and Ethyl cellulose are the best polymers for the preparations of Nanosponge. Preliminary studies were carried out with polymers to select the polymer level for optimization and further studies. Fourier Transport Infra-red (FT-IR) and Differential Scanning Calorimetry (DSC) studies revealed that there was no interaction between the drug and the polymer. Optimization technique (Factorial Design) was applied in the preparation of Praziquantel loaded Nanosponge which was indicated the given model is significant for the study. Optimization methodology helped to predict the best possible formulation. Scanning electron Microscopy (SEM) showed that the particles had almost uniform shape, spherical surface. In addition to this the particle size analysis (using Malvern zetasizer) revealed that 95.5% of the particles had a particle size around 132.1 nm which perfectly matched with the SEM. Powder X-ray Diffraction (PXRD) pattern of Nanosponge showed an amorphous nature of optimized formulation indicates that the formulation which promote the enhanced solubility and thereby improves dissolution rate, diffusion and eventually which may claim for the improved bioavailability. Conclusively, Prepared NSG’s which showed that Nanosponge may be proposed as targeted drug delivery system for Praziquantel. Hence, it was confirm that prepared nanosponge was stable. Hence, from this technique it was concluded that drug delivery of Praziquantel could be significantly increased by preparing Nanosponge. This formulation of nanosponge thus may prove to be a novel drug delivery system that one can achieve with minimal possible efforts by systemic approach of statistical design.

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of Progressive Education Society Pune for the facilities.

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Received on 01.01.2020 Modified on 11.03.2020

Research J. Pharm. and Tech. 2020; 13(9):4491-4498.

DOI: 10.5958/0974-360X.2020.00792.1

Batches	Bulk density (gm/ml)	Tapped density(gm/ml)	Hausner’s ratio	Compressibility index (%)	Angle of repose(θ)
F1	0.911±0.012	0.910±0.092	0.998±0.08	12.6±0.152	15.8±0.141
F2	0.795±0.043	0.783±0.053	0.984±0.002	15.6±0.251	13.6±0.152
F3	0.526±0.073	0.535±0.043	1.01±0.06	16.2±0.208	10.8±0.208
F4	0.955±0.011	0.986±0.093	1.03±0.101	10.7±0.106	8.36±0.035
F5	0.381±0.019	0.384±0.022	1.0±0.306	12.2±0.152	14.9±0.272
F6	0.228±0.031	0.256±0.041	1.12±0.208	13.6±0.108	16.5±0.152
F7	0.989±0.063	1.234±0.063	1.24±0.203	15.4±0.152	19.8±0.152
F8	0.782±0.021	0.86±0.081	1.09±0.302	11.2±0.251	17.4±0.264