Linezolid Loaded Biodegradable Polymeric Nanoparticles Formulation and Characterization

 

Jessy Shaji*, Monika Kumbhar

Department of Pharmaceutics, Prin. K. M. Kundnani College, of Pharmacy, Cuffe Parade, Colaba Mumbai.400005

 

ABSTRACT:

After HIV, Tuberculosis(TB) is the second lethal disease on the globe. In this present study, double emulsion solvent evaporation method was used to formulate Linezolid loaded biodegradable PLGA polymeric nanoparticles (LZD-PNPs). Poly[D,L-Lactic-co-glycolic acid] 50:50 was used as a biodegradable polymer, Tween 80 as a non-ionic surfactant and PVA was used as a co-polymer or stabilizer.Design-expert® v.11.0.5.0 software was used for optimization of LZD-PNPs. Particle size distribution and entrapment efficiency were the main two factors considered  for optimization of LZD loaded polymeric formulation. Preformulation studies and characterization of  the optimized batch were carried out by Fourier Transfer Infrared Spectroscopy (FT-IR), Differential Scanning Calorimetry (DSC) and Scanning Electron Microscopy (SEM) analysis. The mean particle size of LZD loaded PNPs optimized batch was found to be at 75nm with ±22 SD and was monodispersed. Entrapment efficiency of optimised batch was calculated by indirect method & found to be 87.64%. Physicochemical Stability of LZD loaded PNPs formulations were confirmed by zeta potential analysis and was found to be -32mv which indicates  stability of the formulation.In vitro drug release study was carried out using dialysis bag method. The  cumulative percentage drug releasewas found to be 92.34±1.37 for 24 hours.  SEM studies revealed  that formulated LZD polymeric nanoparticle have spherical morphology, smooth texture, and LZD was successfully coated by Biodegradable PLGA polymer.

 

 

 

 

 

 

 

 

INTRODUCTION:

Tuberculosis continues to have a detrimental impact on public health worldwide.^[1]Tuberculosis is a deadly infectious disease caused by Mycobacterium tuberculosis bacteria with  enormous burden on disease.^[2,3] Tuberculosis has been offset by the rampant emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis.^[4] MDR-TB’s are resistant to first-line drugs and XDR-TB  are resistant to any fluoroquinolone, in addition to any of the three second-line injectables as amikacin, capreomycin, and kanamycin.^[5] Reasons for failure of chemotherapy,  involves daily administration of multiple drug for several months, resulting in poor patient compliance, drug toxicity and emergence of drug resistance,^[6] thus there is an urgent need for new strategies to combat the disease. An alternative, more attractive strategy, is to encapsulate the drug inside biodegradable polymer to form polymeric nanoparticles (PNPs) that are selectively targeted to the cells of interest, where they can  release the drug in a sustained fashion over relatively stretched period.^[7]

 

LZD disrupts bacterial growth by inhibiting the initiation process of protein synthesis. This inhibition occurs earlier in the initiation process than other protein synthesis inhibitors. Because the site of inhibition is unique to linezolid^[8] and the inhibition of protein synthesis and disruption of bacterial synthesis is the key to eradicate TB. Subsequently from 2006, the WHO has recommended the use of linezolid in the treatment of MDR/XDR-TB with the drug now being included in many TB programmes across the world.^[8,9]  The standard dose regimen for linezolid is 400 mg or 600 mg every 12 hours for a duration of 10 to 28 days, with an intravenous or oral route of administration.^[8] All the experimental data consisting of linezolid study concluded that LZD is one of the best antibiotic which can be used for the drug-resistance disease like TB.^[8,9]

Poly(D,L-lactic-co-glycolic acid) is the most often used biodegradable and  biocompatible polymer in the design of polymeric nanoparticle formulations.^[10] In this present study PLGA is used as a polymer to coat LZD. The main purpose of this study was to reduce the dose of the drug required in the treatment of TB, reducing adverse effects related to drug therapy. The formulation of polymeric nanoparticles by emulsification solvent evaporation was the first method for the  preparation of nanoparticles.^[11]

 

 

 

 

 

 

 

EXPERIMENTAL WORK:

A)      Material

 Commercial grade Linezolid from Symed Labs Limited (Hyderabad), Poly (DL-lactide-co-glycolide) PLGA [Resomer^® RG 503H] from Evonik-Degussa (Mumbai), were gift samples. PVA was obtained from S.D. fine chemicals (Mumbai). Solvents used were dichloromethane Merck (India). Millipore water, tween 80 and all other chemicals were locally procured. All the excipients used were pharmaceutically approved complying with I.P/ B.P/USP or inhouse specification.

 

B)      Method

Method for Polymeric Nanoparticle Preparation

Linezolid loaded polymeric nanoparticles were prepared by using biodegradable PLGA polymer and for the formulation double emulsion solvent evaporation method was used as shown in Fig 1. In this method, 0.1% w/v of linezolid was dissolved in phosphate buffer saline (PBS) 7.4, followed by addition of non-ionic surfactant tween 80. Another phase contains 0.2% w/v of PLGA polymer in dichloromethane. Aqueous phase was slowly added into organic phase in a fine stream with constant stirring using Magnetic stirrer (Remi, India.) at 1200 rpm at room temperature. This w/o emulsion was stirred continuously until stable emulsion was formed.  The resulting w/o emulsion was added to 0.5% w/v PVA polymeric solution at 900 rpm to form w/o/w emulsion. Solution of these polymeric nanoparticles were  kept  overnight for stirring at 100rpm for evaporation of organic solvent. Size reduction was carried out with probe sonicator (Oscar ultrasonic) for 5min at room temperature. Linezolid loaded polymeric nanoparticles were  subjected to cold centrifugation by refrigerated table top centrifuge (Eltek RC 4100 D, India) at 11000 rpm for 45 min at 4°C. The formed linezolid polymeric nanoparticles were collected and to this 1.5% trehalose solution was added as cryoprotectant to make dry powder by using (Lobocon, U.K) freeze dryer. By using different concentration of polymer and stabilizer, formulations were prepared as shown in Table 1 to know the effect of polymer and stabilizer on PNPs.

 

Fig 1 : Method of preparation of Polymeric nanoparticle

Table 1: Formulations of PLGA Nanoparticles

 

Optimization of LZD Loaded Polymeric Nanoparticles (PNPs)

A computer-generated response surface analysis Design-expert® v.11.0.5.0 software ( stat-Ease, Inc., Minneapolis, MN) was employed to evaluate effects and interactions of the factors on the variables. The factors chosen for optimization were PLGA and PVA concentrations. The factors were selected according to the parameters affecting  preparation method. Based on the validated models, response surface plots were generated for each response to determine the effects of the preparation variables on particle size distribution and entrapment efficiency.^[12]

 

In the present work, two independent factors were used such as amount of PLGA (A) and concentration of PVA (B). The three factorial levels used werecoded as -1,0, and +1 for low, medium and high levels respectivelyin the study. Totally 9 experimental runs were suggested by the software for analysing the interaction of each level of the formulation. Mean particle size distribution (R1) and  % entrapment efficiency (R2) were considered as response factors (dependent factors). Table 2 shows the factors chosen. The significance of independent factors was determined by test for Analysis of the Variance (ANOVA) model and estimated.^[13]

 

Polynomial equation for effect of PLGA and PVA on particle size distribution  and % Entrapment efficiency is given as follows:

 

Particle size

= 89.44+ 17.17A+5.83B+0.0000AB+2.83A² -0.1667B^{2                                                                                                                                              _ _ _ _ _ _} (1)

%EE = 75.23 + 10.81A + 3.89B                       --------(2)

 

Table 2: Factors used for optimization of formulation

Differential Scanning Calorimetry (DSC) :

DSC measures enthalpy changes in samples due to differences in their physical and chemical properties as a function of temperature or time.Differences in heat flow arise when a sample absorbs, or releases heat due to thermal effects such as melting, crystallization, chemical reactions, polymorphic transitions, vaporization and many other thermal processes. DSC studies were performed using a Mettler TA STAR System. The scan rate was kept at 2°C/min in the temperature range of 30–300°C. The DSC measurements were carried out on the following samples, (A): Linezolid, (B): Physical mixture of drug with other excipients and (C): Optimized formulation.^[14] Compatibility studies between the drug and the polymer was carried out. Weighed amount of sample was sealed in Aluminium pan and purged with air at a flow rate of 20ml/min and maintained at a temperature of 50- 250°C.[4] The DSC spectrum of the pure linezolid was compared with its melting point range.^[15]

 

Fourier Transformed Infrared (FTIR) Spectroscopic Analysis:

FTIR spectra were recorded with  (Alpha-T Bruker IR instrument) IR spectrometer in the range 400–4000 cm^-1 using a resolution of 4 cm^-1 and 10 cm^-1.Sample of drug  was mixed with 12 mg of potassium bromide. The mixture was taken and compressed with less than 10-ton pressure in a hydraulic press to form a transparent pellet. The spectra was recorded  to evaluate the molecular states of the micronized linezolid and for the drug interaction and physical mixture of excipients with drug was also recorded.^[16]

 

Entrapment Efficiency:

Nanoparticles were separated from dispersion by centrifugation at 15,000  rpm for 45  min. The supernatant obtained after centrifugation was suitably diluted and analysed for free linezolid by UV–Visible spectrophotometer (Thermo  Scientific,  Evolution  300, India) at 251 nm. The percentage entrapment efficiency was calculated as ^[17,18]:

                                    [Drug _Total - Drug _Supernatant ]

% Entrapment Efficiency =--------------------------- X 100

                                         Drug _Total              -------(3)

 

Particle Size Distributions:

Particle size distribution was carried out by Particle size analyser (Nano sight NS500 version 3.1 (Malvern Instruments Ltd., Worcestershire, UK).This technique combines laser light scattering microscopy with a charge-coupled device (CCD) camera, which aids the imagining and recording of nanoparticles in solution. The NTA software enable to categorize and records discrete nanoparticles moving under Brownian motion and speed of particle is derived from Stokes-Einstein Eq. 4.^[19]

                 (2kg T)

(x, y) ² = ------------------               ----     (4)

                (3r_h л η)

                                                                                                        

Where, k_B is the Boltzmann constant and (x, y) ² is the mean squared speed of a particle at a temperature T, in a medium of viscosity η, with a hydrodynamic radius of r_h.^[19]

 NTA software also enables to precisely analyse the size distribution of monodisperse and polydisperse formulations. Sample imagining, and individual particle tracking are features that allowed thorough size distribution analysis. NTA proved to be suitable to characterize nanoparticles.^[19]

 

Zeta Potential Analysis:

The zeta potential of a system is a measure of charge stability and controls all particle-particle interactions within an emulsion. Understanding zeta potential is of critical importance in controlling dispersion and determining the stability of a nanoparticle emulsion, i.e. to what degree aggregation will occur over time. The zeta potential is the extent of the electric potential at the slip plane between the bound layer of diluent molecules surrounding the particle, and the bulk solution. This can be closely linked to the particle’s surface charge in simple systems but is also heavily dependent on the properties of the diluent solution. A higher level of zeta potential results in greater electro-static repulsion between the particles, minimising aggregation/ flocculation of samples with zeta potentials  between -30mV and +30mV, although the precise stability threshold will vary according to particle type. Zeta Potential Nanoparticle Tracking Analysis (Z-NTA) expands measurements of electrostatic potential to simultaneous reporting of nanoparticle size, light scattering intensity, fluorescence, count, and does so particle-by-particle.^[20]

 

 

 

 

Scanning Electron Microscopy (FE-SEM):

Approximately 5 μL of the LZD-PNPs  formulation was taken on to a cover slip, which in turn was mounted on a specimen tab. The samples were dried at room temperature. Then the particle size of the formulation was observed and photographed using Scanning Electron Microscope. Field Emission Scanning Electron Microscopy (FE-SEM) comprises field emission electron source which (Sigma, Carl Zeiss) deliver high resolution surface information. Scanning Electron Microscope (SEM) scan a sample with a focused electron beam and deliver images which gives information about the samples  topography and composition.^[21] In this study magnification power was kept in different range i.e. from 1000X to 100000X  operated with an acceleration voltage of 5 kV and a working distance of 9.8 mm was maintained.^[22,23]

 

In  Vitro Drug Release Studies:

The dialysis bag diffusion technique was used to study the in-vitro drug release of Linezolid loaded polymeric nanoparticles. The prepared nanoparticles were placed in the dialysis bag and immersed into 50ml of Phosphate Buffer saline pH 7.4. The entire system was kept at 37±0.5ºC with continuous magnetic stirring at 200 rpm, aliquots were withdrawn from the medium at predetermined intervals and replaced by fresh medium every time. The amount of drug dissolved was determined with UV-Spectrophotometry at 251nm.^[15]

 

RESULTS AND DISCUSSION:

1)    FTIR studies:

 As shown in Fig 2 (a),  IR spectrum, indicates that the appearance of three prominent peak viz 3360.67,1741.51 & 1672.66 cm^-1 corresponds to NH amide, C=O lactone and C=O amide, respectively.^[24] Other IR absorption peaks of Linezolid at 2977 cm-1 (=N-H stretch) , 1356.1 cm-1 (C-N stretch) , 1047.73 cm-1 (C-F stretch) , 2851.11cm-1 (-OCH3) also existed.^[25] Fig 2 (b) represent IR spectrum of physical mixture, and it shows bands at 3331.13 cm ^-1,2915.82cm^-1, 2848.81cm^{- 1}.

 

 

 

Wave number cm^-1

Fig 2(a): IR graph of Linezolid

 

Wave number cm^-1

Fig 2(b): IR graph of physical mixture

 

DSC analysis

DSC thermograms corresponding to LZD, Optimized  PLGA loaded polymeric nanoparticle formulation, are shown in Figure 3 (a and b). Pure LZD showed single endothermic peak at 181.2°C indicating crystalline nature of drug. The presence of endothermic peak of LZD along with other excipients in the thermogram of physical mixture clearly indicates the incompatibility with each other. The peak of LZD disappeared in LZD-PNPs, indicating absence of crystallinity. The disappearance of endothermic peak of LZD confirmed the entrapment of LZD in the PNPs and existence of LZD  in an amorphous  drug phase of a molecular dispersion inside the polymeric matrix of PNPs.^[26]

 

Fig 3(a): DSC graph of Linezolid

 

 

Fig: 3(b) DSC graph of optimized LZD-PNP

 

2)    Optimization of LZD loaded Polymeric Nanoparticle(PNPs)

Effect on particle size, and Entrapment efficiency

As in the polynomial equation factor 𝑋1,  is a PLGA, affects the particle size distribution n of the polymeric NPs in positive direction. As shown in Fig 4 (a)and (b) the increase in concentration of polymer increases the particle size and % entrapment efficiency of the LZD-PNPs. The  reason of increase in particle size is due to increase in polymer concentration leading to an increase in the viscosity of the organic phase leading to the formation of nanoparticles with larger size at interface at the stirring intensity. Viscosity of organic phase promotes the formation of larger size PLGA nanoparticles along with increase in the amount of drug encapsulated inside the nanoparticles.^[27] From the analysis p value less than 0.050 indicated that model terms are significant. F-value of 1073.76 implies the model is significant. Table 3 shows the results for the mean particle size distribution, % entrapment efficiency and zeta potential of F1-F9 batches.

 

 

 

(a)

b)

Fig4: 3D Response surface plot showing effect of polymer (X1) and PVA concentration (X2) on Particle size distribution  & entrapment efficiency

 

Table 3: Evaluation of PLGA loaded LZD-PNPs

Formulation code	Mean Particle size distribution (nm) ±SD	% Entrapment efficiency	Zeta potential (mv)
F1	69±55	69.1	-26
F2	95±26	72.3	-29
F3	103±46.3	78.7	-31
F4	89±56.9	75.9	-28
F5	75±22	87.64	-32
F6	53.4±22	59.37	-29
F7	119.7±46.3	80.43	-30
F8	109.2±38.9	79.9	-28
F9	47.7±26	52.12	-25

 

The positive value before the factor indicates positive effect on the particle size distribution and percentage drug entrapment. As shown in Table 4 the model was found to be significant, 𝐹-value = 1047.12; 𝑃< 0.0418. The values for predicted (0.9953) and adjusted (0.9989) and  -square values were in reasonable agreement. Analysing the polynomial equation, it was found that percentage drug entrapment is increasing with increasing values of factors 𝑋1 (polymer concentration) and 𝑋2 (co-polymer and stabilizer). The increased state of viscosity of organic phase is due to the increasing factor 𝑋1 which increase resistance to drug diffusion into the aqueous phase leading to the incorporation of more amount of drug inside NPs.^[27]

                                                                                                                                                           

 

 

Table 4: Model used in response surface analysis

	P Value	F value	R2	Predicted  R2	Adjusted R2
Particle size distribution	˂0.0001	1073.76	0.9994	0.9943	0.9985
% Entrapment Efficiency	0.0418	1047.12	0.9992	0.9953	0.9989

 

 

 

3)    Zeta potential:

Zeta potential measures the surface charge of particles which can greatly influence particle stability in nanoparticle formulation through the electrostatic repulsion between particles likewise. The zeta potential of the optimized LZD loaded PNPs was found to be -32mV indicating the formation of stable Nano dispersion due to sufficient electrostatic repulsion. ^[26]

 

4)    SEM analysis:

According to SEM results nanoparticles prepared with different amount of polymer are spherical in shape, but on increasing the polymer content  constant increase in nanoparticles diameter occurs. When the amount of PLGA was increased in organic phase, the particle diameter increased. According to the result, it can be concluded that, for this technique the PLGA polymer content in the organic phase is a significant factor because the size of nanoparticles increased as polymer concentration increase. This is due to the increasing viscosity of organic phase, resulting a low dispersibility of the PLGA solution into the aqueous phase. Fig 5 shows that FEG-SEM Linezolid loaded PNPs was effectively coated with biodegradable PLGA polymer and was found to be spherical, uniform with smooth texture. Increase in polymer concentration led to an increase in the viscous forces resisting the droplet break down by sonication. These forces oppose the shear stresses in the organic phase and the final size of particles depends on the net shear stress, which is available for droplet breakdown.^[28]

 

 

Fig 5: FEG-SEM image of LZD-PNPs

 

5)    In Vitro release studies:

Dialysis bag method was used for In Vitro release studies. Fig 6 shows the release profile of Linezolid from PLGA nanoparticles. The release of LZD from the PLGA polymer varied from 79% to 92% for 24 hours. In the in vitro release profile of the Linezolid loaded polymeric nanoparticles showed 52 – 79% drug release in a sustained manner within a period of 12 hours. Remaining quantity slowly elute from the polymer.^[10] The cumulative percentage of drug release from the optimized formulation was 92.34±1.37 for 24 hours.^[15]

 

Fig 6: Graph of  In Vitro Drug Release

 

CONCLUSION:

From these studies it can be concluded that double emulsion solvent evaporation method was successfully achieved for formulating LZD loaded polymeric nanoparticle. This method emphasizes on the formulation aspects regarding the best batch having optimum particle size  with high entrapment efficiency. Biodegradable polymer have a significant advantage in polymeric nanoparticle formulation. NTA is a useful and informative characterization software that helps in development and is mainly valuable for analysing polydisperse nanosized formulations. Design expert software is informative   useful tool in pharmaceutical technology field. ^[19]

 

ACKNOWLEDGEMENTS:

The authors are thankful to Symed Labs Limited; Hyderabad, India, for the gift sample of Linezolid, and to FIST-DST for providing FTIR facility.

 

 

 

 

 

DECLARATION OF INTEREST:

The authors report no conflicts of interest

 

REFERENCE:

1.      Cooper, A. M. Cell-Mediated Immune Responses in Tuberculosis. Annu. Rev. Immunol. 2009, 27 (1), 393–422.

2.      Nasiruddin, M.; Neyaz, M. K.; Das, S. Nanotechnology-Based Approach in Tuberculosis Treatment. Tuberc. Res. Treat. 2017, 2017 (Table 1), 1–12.

3.      Churchyard, G.; Kim, P.; Shah, N. S.; Rustomjee, R.; Gandhi, N.; Mathema, B.; Dowdy, D.; Kasmar, A.; Cardenas, V. What We Know about Tuberculosis Transmission: An Overview. J. Infect. Dis. 2017, 216 (January), S629–S635.

4.      Olaru, I. D.; Von Groote-Bidlingmaier, F.; Heyckendorf, J.; Yew, W. W.; Lange, C.; Chang, K. C. Novel Drugs against Tuberculosis: A Clinician’s Perspective. Eur. Respir. J. 2015, 45 (4), 1119–1131.

5.      Sciences, P. Design, Synthesis and Evaluation of Antimycobacterial Activity of Triazoles 1. 2011, 1–11.

6.      Labana, S.; Pandey, R.; Sharma, S.; Khuller, G. K. Chemotherapeutic Activity against Murine Tuberculosis of Once Weekly Administered Drugs (Isoniazid and Rifampicin) Encapsulated in Liposomes. Int. J. Antimicrob. Agents 2002, 20 (4), 301–304.

7.      Fenaroli, F.; Westmoreland, D.; Benjaminsen, J.; Kolstad, T.; Skjeldal, F. M.; Meijer, A. H.; Vaart, M. Van Der; Ulanova, L.; Roos, N. Nanoparticles as Drug Delivery System against Tuberculosis in Zebra Fish Embryos : Direct Visualization and Treatment. ACS Nano 2014, No. 7, 7014–7026.

8.      Ament, P. W.; Jamshed, N.; Horne, J. P. Linezolid: Its Role in the Treatment of Gram-Positive, Drug-Resistant Bacterial Infections. Am. Fam. Physician 2002, 65 (4), 663–670.

9.      Agyeman, A. A.; Ofori-Asenso, R. Efficacy and Safety Profile of Linezolid in the Treatment of Multidrug-Resistant (MDR) and Extensively Drug-Resistant (XDR) Tuberculosis: A Systematic Review and Meta-Analysis. Ann. Clin. Microbiol. Antimicrob. 2016, 15 (1), 1–17.

10.   Crucho, C. I. C.; Barros, M. T. Formulation of Functionalized PLGA Polymeric Nanoparticles for Targeted Drug Delivery. Polym. (United Kingdom) 2015, 68, 41–46.

11.   Vauthier, C.; Bouchemal, K. Methods for the Preparation and Manufacture of Polymeric Nanoparticles. Pharm. Res. 2009, 26 (5), 1025–1058.

12.   Giovagnoli, S.; Blasi, P.; Schoubben, A.; Rossi, C.; Ricci, M. Preparation of Large Porous Biodegradable Microspheres by Using a Simple Double-Emulsion Method for Capreomycin Sulfate Pulmonary Delivery. Int. J. Pharm. 2007, 333 (1–2), 103–111.

13.   Kumar, L.; Sreenivasa Reddy, M.; Managuli, R. S.; Pai K., G. Full Factorial Design for Optimization, Development and Validation of HPLC Method to Determine Valsartan in Nanoparticles. Saudi Pharm. J. 2015, 23 (5), 549–555.

14.   Castelli, F.; Puglia, C.; Sarpietro, M. G.; Rizza, L.; Bonina, F. Characterization of Indomethacin-Loaded Lipid Nanoparticles by Differential Scanning Calorimetry. Int. J. Pharm. 2005, 304 (1–2), 231–238.

15.   Sakthivel, D.; Arunachalam, G. Preparation and Characterization of Polymeric Nanoparticles Used in the Treatment of Epilepsy. 2017, 9 (3), 298–301.

16.   Sharma, P.; Sharma, P. K.; Mishra, V. B.; Bhandari, A. (2014). Preparation and characterization of isoniazid chitosan loaded nanoparticles. Journal of Drug Delivery and Therapeutics 2014, 4(5), 158-166.

17.   Fazil, M.; Md, S.; Haque, S.; Kumar, M.; Baboota, S.; Sahni, J. K.; Ali, J. Development and Evaluation of Rivastigmine Loaded Chitosan Nanoparticles for Brain Targeting. Eur. J. Pharm. Sci. 2012, 47 (1), 6–15.

18.   Dora, C. P.; Singh, S. K.; Kumar, S.; Datusalia, A. K.; Deep, A. Development and Characterization of Nanoparticles of Glibenclamide by Solvent Displacement Method. Acta Pol. Pharm. - Drug Res. 2010, 67 (3), 283–290.

19.   Filipe, V.; Hawe, A.; Jiskoot, W. Critical Evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the Measurement of Nanoparticles and Protein Aggregates. Pharm. Res. 2010, 27 (5), 796–810.

20.   Nanosight. Zeta Potential Analysis Using Z-NTA. 2013.

21.   https://www.zeiss.com/microscopy/int/products/scanning-electron-microscopes.html

22.   Singh, R. P. Preparation and Evaluation of Phytosome of LAWSONE. Int. J. Pharm. Sci. Res. 2015, 6 (12), 5217–5226. doi: 10.13040/IJPSR.0975-8232.6(12).5217-26

23.   Dhase, A. S.; Saboo, S. S. Preparation and Evaluation of Phytosomes Containing Methanolic Extract of Leaves of Aegle Marmelos (Bael). Int. J. PharmTech Res. 2015, 8 (6), 231–240.

24.   Bebawy, L. I. Stability-Indicating Methods for the Determination of Linezolid in the Presence of Its Alkaline-Induced Degradation Products. Talanta 2003, 60 (5), 945–953.

25.   Gowda D.V; Datta V; S.M; H.S ; Gupta. V. K. Formulation and Evaluation of Dry Powders Anti. Indo American Journal Of Pharm Research 2013, 3 (12).1239-1248.

26.   Gajra, B.; Patel, R. R.; Dalwadi, C. Formulation, Optimization and Characterization of Cationic Polymeric Nanoparticles of Mast Cell Stabilizing Agent Using the Box–Behnken Experimental Design. Drug Dev. Ind. Pharm. 2016, 42 (5), 747–757.

27.   Sharma, D.; Maheshwari, D.; Philip, G.; Rana, R.; Bhatia, S.; Singh, M.; Gabrani, R.; Sharma, S. K.; Ali, J.; Sharma, R. K.; et al. Formulation and Optimization of Polymeric Nanoparticles for Intranasal Delivery of Lorazepam Using Box-Behnken Design: In Vitro and in Vivo Evaluation. Biomed Res Int2014, 2014, 156010.

28.   Bohrey, S.; Chourasiya, V.; Pandey, A. Polymeric Nanoparticles Containing Diazepam: Preparation, Optimization, Characterization, in-Vitro Drug Release and Release Kinetic Study. Nano Converg. 2016, 3 (1), 3.

 

 

 

 

Received on 25.09.2018          Modified on 18.10.2018

Accepted on 26.10.2018     ©A&V Publications All right reserved