Design, Optimization and in vitro Characterization of Dasatinib loaded PLGA Nano carrier for Targeted cancer therapy: A Preliminary Evaluation

 

Shyam S Kumar1*, Dr. G. Gopalakrishnan2, Dr. N. L. Gowrishankar1

1Department of Pharmaceutics, Prime College of Pharmacy, Kerala.

2Department of Pharmacy, Annamalai University, Tamilnadu.

*Corresponding Author E-mail: shyamchennath@gmail.com

 

ABSTRACT:

Objective: Drug nanoparticles offer a versatile platform for enhancing the dissolution rate and bioavailability of poorly water soluble drugs The present study was aimed to design and develop dasatinib (DAS) loaded Poly lactide co glycolic acid (PLGA) to enhance the dissolution rate and to study the effect of formulation variables for the BCS class II drug dasatinib for the treatment of chronic myeloid leukemia. Methods: The DAS loaded Nps were prepared by using modified double emulsion solvent evaporation method (DESE) using different stabilizers, the formulated Nps were characterized for particle size, zeta potential, Poly Dispersity Index, Surface morphology, Drug entrapment and Invitro drug release. Results: The DAS loaded NP s showed the lowest particles size of 123 nm and zeta potential of – results of Pluronic F68 loaded NP showed the lowest particle size of – and highest zeta potential of --. Surface morphology of NPs with DMAB showed distinct smooth spherical particles with the size range of 50nm. Morphology of Pluronic F68 formulated NPs showed the high degree of aggregation. In vitro drug release showed up to 24hrs in a sustained manner. Conclusion:      The result of our study indicates the use of PLGA as a sustained release polymer and using DMAB as a stabilizer for better stable formulation.

 

KEYWORDS: Polymeric nanoparticles, cancer targeting, poorly soluble drug, Double emulsion solvent evaporation.

 

 


INTRODUCTION:

Noncommunicable diseases (NCDs) are now responsible for the majority of global deaths and cancer is expected to rank as the leading cause of death and the single most important barrier to increasing life expectancy in every country of the world in the 21st century. The unsatisfactory outcome of drug treatment is because of not only the drugs’ low efficacy to stop the tumor growth and progression, but also the serious side effects, drug resistance and cancer relapse, especially for chemotherapy drugs.1 However, the growing evidence shows that the molecularly targeted therapeutics may face the similar issues as other anticancer drugs, such as poor solubility, low bioavailability, insufficient tumor specificity, and drug resistance.2

 

Chronic myeloid leukemia (CML) is a common type of cancer of white blood cells that affect both blood and bone marrow with a rising morbidity.3 Dasatinib (DAS), a small molecule tyrosine kinase inhibitor, can effectively fight against CML and ALL by inhibiting the activity of both Src and BCR-ABL tyrosine kinases in leukemia cells4,5. However, DAS treatment has been reported to cause serious hematologic and non-hematologic adverse effects due to its interaction with non-disease-related processes and cells, which often leads to a dose reduction or treatment discontinuation in clinic6. Peripheral edema and pleural effusion are the common non-hematologic side effects occurred during DAS treatment, which is likely caused by endothelial hyper permeability7–9. Polymer based NPs are commonly used to improve drug bioavailability and/or reduce drug associated side effects10. Poly lactide-co-glycolide (PLGA) is a Polymer that has been commercialized for a variety of drug delivery systems and is frequently used in the design of biocompatible NPs11 PLGA is approved by the Food and Drug Administration as a biodegradable polymer that degrades to the Nontoxic tricarboxylic acid cycle intermediates, lactic acid and glycolic acid12 NPs showed various advantages including the increased drug solubility and stability, improved Pharmacokinetics, enhanced permeability and retention (EPR) effect-mediated tumor targeting, and capability of further engineering to impart various functionalities.

 

The purpose of this study is to develop, optimize and characterize DAS loaded polymeric nanoparticles with the primary objective to enhance the dissolution rate and to investigate the effect of stabilizers on particle size, zeta potential, drug entrapment, morphology, in vitro drug release and stability.

 

MATERIALS AND METHODS:

Dasatinib is gift sample from MSN laboratories, Hyderabad Poly Lactic Glycolic Acid (PLGA, 50:50), Pluronic F-68 and DMAB was purchased from Sigma Aldrich, USA. The other ingredients used were of analytical grade

 

METHODOLOGY:

Preparation of dasatinib nanoparticle:

Dasatinib nanoparticles were prepared using a modified double-emulsion (water-in-oil-in-water) solvent evaporation technique13 briefly; polymer was dissolved in organic solvent containing. Dasatinib was dissolved distilled water containing stabilizers, and then emulsified in the polymer solution through homogenization. The primary W/O emulsion was further added to external water with homogenization (3 min) to achieve the stable double emulsion (W/O/W). The resulting emulsion was dropped gradually into the aqueous solution with different stabilizers (Pluronic F68, and DMAB) under steady stirring to make the nanoparticles solidify. The residual organic solvents were evaporated and the nanoparticles suspending in emulsion were collected by ultracentrifugation at 16000rpm and washed with distilled water three times. Finally, the products were dried by lyophilization and stored at 4C.

 

CHARACTERIZATION OF PREPARED NANOPARTICLES:

Particle Size Distribution and Zeta Potentail:

The mean particle size and zeta potential of prepared dasatinib loaded PLGA nanoparticles was determined using Zetasizer ZS 90 (Malvern Instruments, UK.) based on dynamic light scattering technique operating with 532 nm laser at an angle of 90° in 10m diameter cell at 25ºC. All the measurements were performed in triplicate and the results are reported in terms of mean diameter ± SD 14

 

               

Transmission electron microscopy (TEM):

The surface morphology of the dasatinib loaded PLGA nanoparticles were determined by using high resolution transmission electron microscopy (HRTEM). A drop of Nano suspension was placed on copper grid (3.0mm.200 mesh) and allowed to dry for 30min then the analysis was performed at 80kv using JOEL JEM 2100, (Tokyo Japan). In addition to the morphology of nanoparticles physical state of dasatinib inside the PLGA nanoparticles was analyzed by using TEM selected area diffraction pattern.

 

Drug Entrapment studies:

Dasatinib loaded PLGA nanoparticles were separated from aqueous phase by ultracentrifugation (Eppendrof, U.S.A) at 13000 rpm and 4°C for 40 minutes. The supernatants were collected and evaluated for drug by UV. The entrapment efficiency (EE) was calculated according to the following equation

 

       Total amount of drug added – Amount of Free drug

Entrapment = ––––––––––––––––––––––––––––––––––––––– x 100

efficiency (%)               Total amount of Drug Added                        

 

Drug loading determination:

The drug loading of lyophilized dasatinib nanoparticles was determined. Briefly the weighed quantity of (50mg) of freeze-dried nanoparticles was dissolved in acetonitrile and the drug concentration was estimated by ultraviolet spectroscopy at 220nm

 

Drug loading was estimated using the following equation

 

                   Amount of drug in nanopartic les

Drug loading (%) = –––––––––––––––––––––––––––––––––––– × 100

                    Amount of drug used in formulation

 

In vitro drug release studies and Kinetics:

In vitro release studies were performed using dialysis sac method15. Freeze dried dasatinib nanoparticles (equivalent to 2mg of DAS) were suspended in phosphate buffer pH 7.4 and placed in a dialysis membrane (molecular weight cut off 10,000–12,000Da) with its ends closed using membrane clips. The dialysis membrane was then placed in a beaker containing phosphate buffer pH 7.4 maintained at 37ºC with continuous magnetic stirring. At specified time intervals of 1, 2, 4, 8, 12 and 24 hours 2ml of aliquots were withdrawn from the medium and replaced with the equal volume fresh phosphate buffer. The concentration of dasatinib was estimated spectrophotometrically at 220 nm.

 

RESULT AND DISCUSSION:

To investigate the interaction between pure dasatinib and PLGA. FTIR analysis was taken into consideration. The functional groups with corresponding peaks of dasatinib, PLGA and physical mixture was interpreted. The principal peaks of DAS showed N-H stretching at 3418, O-H at 3200, C-O 1620, C-C, C-N at 1582 and C-H at 1513 cm (-1). FT-IR spectra of PLGA showed O-H deformation at 1277, methyl group C-H stretching at 1456 and Carbonyl stretch from the (C=O) 1758 cm (-1). From the results obtained it was observed that there was no extra peaks or disappearance of existing peaks in the spectrum of physical mixture which indicates no significant physical or chemical interaction between DAS and polymers.

 

Preparation of NPs by standard DESE method:

DAS loaded PLGA nanoparticles were prepared by standard DESE technique and characterized for particle size, encapsulation efficiency and zeta potential it showed in Table No.1. The drug: polymer ratio 1:1 was used for the preliminary characterization. The particle size distribution of the nanoparticles produced by DESE presented in Figure No.1 shows uniform distribution with a narrow size range. Whereas the zeta potential of nanoparticles obtained by DESE method showed -13mv indicating incipient instability of the nanoparticles and with low encapsulation of only 50% in nanoparticles prepared by standards method.

 

Table: 1 Particle size, zeta potential and Drug encapsulation of nanoparticle prepared by standard DESE method

S. No

Method

Particle size(nm)

Zeta Potential (mV)

Drug encapsulation (%)

1

Standard Double emulsion solvent evaporation

 192±10nm

 -13±3.4

 50±2.5

 

Preparation of DAS NPs by modified DESE method:

The modification was performed in the DESE the organic phase by replacing 100% DCM in standard DESE with 50% (v/v) mixture of DCM and EA and 100% EA. In this study the mixture of water immiscible solvent (DCM) and partially water miscible solvent (EA) was used the particle size slightly deceased from the standard method. Though the solubility of DCM in the aqueous phase is little, but the vapour pressure is high, so DCM can diffuse quickly in to the aqueous phase and vaporized it leads to the fast precipitation of polymer without splitting of drug to the aqueous phase resulting in increased entrapment efficiency from 50 to 80% but when considering the toxicity DCM is more toxic than EA (According to class III and II according to the ICH specifications)17-19. When 100% EA a partially water miscible solvent was used it diffused freely through the aqueous phase creating phase transformations and the aggregation of polymer takes place in the region of each emulsion droplets to forms several NP. The encapsulation efficiency of DAS in NPs formulated using EA showed the encapsulation of 80% which was 1.5 folds higher when compared with 50% (v/v) DCM/EA. Probably due to the partial miscibility of EA in water enabled a slight mutual solubility of the organic and aqueous phase and the temporary phase was created in which the polymer and RT were dissolved in EA. The particle size, zeta and encapsulation efficiency were showed in Table: 2.

 

Table: 2 Particle size, zeta potential and Drug encapsulation of nanoparticle prepared by Modified DESE method

S. No

Modified Phase

DESE Modif ication

Particle size(nm)

Zeta Potential (mV)

Drug encap sulation (%)

1

Organic Phase

50% (v/v) DCM: EA

75±8nm

 -16±2.4

60±3.4

100% EA

125 ±6nm

 -19±1.4

80±2.8

 

Optimization of DAS loaded NPs:

Based on the preliminary screening for the suitable method for nanoparticle preparation. The modified DESE method is selected for the further optimization for drug: polymer ratio and to find the best stabilizer to enhance the stability of the nanoparticles, the formulations were prepared with different drug: polymer ratio from 1:1 to 1:4 with different types of stabilizers showed in Table: 3.


 

Table 3: Compositions, size, PDI and zeta potential of DAS loaded nanoparticles

S. No

Formulation code

Stabilizer

Drug: polymer Ratio

Particle size(nm)

PDI

Zeta Potential (mV)

1

F1

Pluronic F-68

1:1

210±12

0.090±0.04

-6.64±1.2

2

F2

Pluronic F-68

1:2

275±7

0.120±0.08

-9.56±0.9

3

F3

Pluronic F-68

1:3

325±5

0.151±0.034

-11.85±1.8

4

F4

Pluronic F-68

1:4

385±10

0.201±0.004

-13.78±1.6

5

F5

DMAB

1:1

160±6

0.148±0.065

11.55±2.2

6

F6

DMAB

1:2

165±9

0.189±0.045

18.75±1.4

7

F7

DMAB

1:3

240±8

0.204±0.023

28.10±2.6

8

F8

DMAB

1:4

290 ±5

0.225±0.054

22.86±3.1

 


 

Characterization of prepared DAS NPs:

Particle size and zeta potential:

Particle size of prepared DAS loaded NPs and was determined using Dynamic light scattering method and obtained results are summarized in Table 3. Results indicates that the NPs prepared with stabilizer Pluronic F-68 has particle size range from 210±12 to 385nm±10 it was observed that the size of the nanoparticles has a direct relationship with the PLGA concentration ,this might be due to the increase in the viscosity of the dispersed phase which leads to reduction in net shear stress and gives the bigger nanoparticles, PLGA solution cannot rapidly disperse into the aqueous phase as the viscosity of the polymer is increasing and it result larger particle size with high PDI values. Further our study showed the less negative zeta potential value was influenced by the nature of the nonionic surfactants of Pluronic F-68. The presence of residual Pluronic F-68 on the nanoparticle surface has been found to mask charged groups existing on the surface of Pluronic F-68 formulated nanoparticle20,21. Thus, residual Pluronic F-68 may effectively create a shield between the nanoparticle and its surrounding medium, resulting in lower zeta potential measurements that still maintain higher levels of entrapment A second possibility is the correlation between zeta potential and nanoparticle stability. Zeta potential measurements closer to zero represents a high degree of non-stability with a weak surface charge surrounding the NP

 

The NPs prepared with DMAB as a stabilizer showed the particle size range of 123±6 to 340±5nm the same increasing trend of particle size with increase in PLGA concentration was observed but in case of surface charge it showed the positive charge, Based on the previously reported data that the positive charge on nanoparticles plays a significant role in determining the interactions with the cell membrane and more likely to provide significant influence on the cell as compared with the neutral or negatively charge NPs due to the attractive electrostatic interactions with the negatively charged cell membranes.22,23

 

The resulting negative zeta potential leads to an electrostatic repulsion between the particle system and biological membranes with the consequence of a relatively low cellular uptake or transport over cellular barriers. DMAB is a quaternary ammonium compound with a smaller critical micelle concentration in comparison to Pluronic F-68, in consequence smaller and positively charged nanoparticles result. The lowered particle diameter of about <300nm as well as shift to a positive zeta potential leads to higher particle uptake in comparison to Pluronic F-68 stabilized nanoparticles as already described in literature

 

Effect of stabilizer concentration on NP characteristics:

DAS encapsulated PLGA NPs were prepared using DMAB and Pluronic F-8 as a stabilizer. The use of DMAB and Pluronic F-68 resulted in formation of DAS loaded PLGA NPs with the surface nature that displayed positive and negative charge respectively. Because of the cationic properties of DMAB NPs formulated with the addition of DMAB showed positive surface                charge.

 

DMAB formulated DAS loaded NPs reached a peak zeta potential in the range of 11.55 to +28.1.6mV. The anionic characteristics of Pluronic F-68 led to formation of NPs with slight negative charge in the range of -6.64 to -13.78mV

 

Surface Morphology:

Surface morphology of prepared NPs was observed Using HRTEM. Fig:1 indicate that the DMAB formulated NPs have a distinct, spherical shape composed of a dense core with DAS loaded NPs with the particle size range of 50nm

 

Fig:1 Transmission electron micrographs of DAS loaded DMAB stabilized Nps

 

Drug Loading and Drug entrapment:

The drug loading and entrapment efficiency of NPs prepared by modified DESE method using pluronic F-68 and DMAB as stabilizers. The formulation containing Pluronic F-8 as stabilizer showed high drug loading and entrapment efficiency of 16.15mg and to 80.75± 0.55% respectively. All formulation with DMAB stabilizer demonstrated significant changes in the drug loading and the entrapment efficiency of 17.26mg and 86.3% respectively the increasing trend of drug loading and entrapment was observed with increasing in DMAB concentration in this study, DMAB was shown to effectively increase zeta potential, reduce particle size, and facilitate drug entrapment when compared to Pluronic F-68 based formulations. As such, DMAB based NP morphology was visualized and confirmed under transmission electron microscopy with further variable analysis carried out using DMAB formulations

 

In vitro Drug release:

In vitro release studies were performed on two different stabilizer concentrations for both DMAB and Pluronic –F-68. The in vitro release of both DMAB and Pluronic F-68 formulated DAS loaded NPs are given in Figs. 6 and 7 The comparison of the percentage drug release values obtained with the different nanoparticle stabilizer compositions at specific sampling time’ s revealed the difference in both stabilizer concentration groups. DMAB formulations at 0.1% showed an initial significant increase in drug release in comparisons to 0.1% Pluronic F-68 based formulations during the initial 4 hr time frame (Fig. 9). After 24 hrs., total drug release was similar with a cumulative release of over 80% achieved for both groups (Fig. 9). The drug release of NPs formulated with 0.25% Pluronic F-68 showed a similar pattern of initial release of DAS in comparison to DMAB formulation (Fig. 10). Both formulations experienced greater than 40% release during the first hour of the study. However, after the first initial hour, cumulative release began to increase significantly in DMAB formulated groups at each successive time point DMAB based formulations reached an average cumulative release percentage of 88%, while pluronic F-68 formulation reached an average cumulative release of 73%

 

 

Fig: 6 In vitro drug release study with various concentration of pluronic F-68

 

 

Fig: 7 In vitro drug release study with various concentration of DMAB stabilizer

 

In vitro Drug Release Kinetics:

In vitro release data were fitted in to release kinetic model and computed using DD solver, which is an excel plugin module (24,25) and the resultant data were fitted to the Korsmeyer-Peppas exponential equation to establish the mechanism of drug release. The ‘n’ values were in range of 0.232 to 0.293 with r2values ranged from 0.9917 to 0.9983 which suggest that drug release follows fickian diffusion controlled mechanism.

 

CONCLUSION:

In this research we have successfully developed DAS loaded PLGA NPs using pluronic F-68 and DMAB as stabilizer using modified double emulsion solvent evaporation method. The effect of stabilizer concentration with respect to particle size and zeta potential was evaluated. The results of this study showed that the use of DMAB as stabilizer lead to the formulation of NPs with lowest size and better positive zeta potential when compared with Pluronic F-68. Based on this preliminary data the optimized formula extended for In vivo studies to ensure the drug uptake.

 

ACKNOWLEDGMENT:

Authors are highly thankful to Prime College of Pharmacy, Kerala, India and Annamalai University, Tamilnadu for the providing the facilities to carry out this research article.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

ABBREVIATIONS:

DAS –Dasatinib

DCM- Dichloromethane

EA –Ethyl acetate

PLGA- Poly lactide co glycolic acid

DMAB- Didodecyldimethylammonium bromide

DESE –Double emulsion solvent Evaporation

 

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Received on 17.02.2020            Modified on 20.05.2020

Accepted on 30.07.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(4):2095-2100.

DOI: 10.52711/0974-360X.2021.00371