DESC:RELATED APPLICATION
The present application claims the benefit of priority to Indian Provisional Patent Application no. 202041007562 filed on Feb 21, 2020 and the entire provisional specification.
FIELD OF INVENTION
The invention relates to design and development of nanoformulation of Osimertinib mesylate PLGA nanoparticles functionalized with various macrophage targeting ligands in the management of lung cancer. Particularly invention is related to the composition of Osimertinib mesylate PLGA nanoparticles. More particularly, the invention relates to composition of Osimertinib mesylate PLGA nanoparticles, their preparation and use in the treatment of cancer.
BACKGROUND OF THE INVENTION
Osimertinib mesylate, chemically N-(2-{[2-(dimethylamino)ethyl](methyl)amino}-4-methoxy-5-{[4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl]amino}phenyl)prop-2-enamide; methane sulfonic acid.
Osimertinib (OSM) is an oral, third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) drug developed by AstraZeneca Pharmaceuticals. Its use is indicated for the treatment of metastatic non-small cell lung cancer (NSCLC) in cases where tumor EGFR expression is positive for the T790M mutation as detected by FDA-approved testing and which has progressed following therapy with a first-generation EGFR tyrosine kinase inhibitor. Approximately 10% of patients with NSCLC have a rapid and clinically effective response to EGFR-TKIs due to the presence of specific activating EGFR mutations within the tumor cells (Drug Bank).

Bharali et.al discloses evaluation of PLGA tetra-iodo thyroacetic acid nanoparticles both in-vitro and in-vivo for the treatment of drug-resistant breast cancer. In-vivo PLGA nanoparticles resulted in a three to five-fold inhibition of tumor weight.
Acharya et.al discloses various biomedical applications of PLGA. PLGA nanoparticles have wide applications combining targeting, imaging, diagnostics and therapy.
Zhang et.al discloses up-to-date review of osimertinib along with its structure, mechanisms, and pharmacokinetics, summarizing clinical trials and making recommendations of osimertinib for patients with non-small-cell lung cancer (NSCLC).
WO2017117070 reports N-(2-{2-dimethylaminoethyl-methylamino}-4-methoxy-5-{[4-(1-methylindol-3-yl)pyrimidin-2-yl]amino}phenyl)prop-2-enamide with one or more deuterium-substitutions at strategic positions, that are epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) and are useful for treating various forms of cancer, e.g., non-small cell lung cancer (NSCLC). The report further discloses synthesis and in-vitro evaluation of the synthesized compounds.
WO2019183523 relates to bi-functional compounds that can be used as modulators of targeted ubiquitination. The invention discloses a compounds that contain on one end a VHL ligand moiety that binds to the VHL ubiquitin protein ligase, E3, and on the other end a moiety that binds a target protein, such as EGFR, such that degradation of the target protein/polypeptide is effectuated.
One of the major concerns associated with use of OSM mesylate and other TKIs is development of acquired resistance against the therapy, typically occurring within 8-12 months of beginning of treatment. Hence, there is a need to develop a delivery system which could demonstrate higher efficacy at lower doses to eliminate/delay acquired resistance. Nanoparticle technology is one of the strategies to eliminate acquired resistance with small dose and enhanced efficacy.
Nanomedicine particularly nanoparticles has recently gained immense potential for treatment of lung cancer. Targeted nano drug delivery approaches for cancer therapeutics have shown a steep rise over the past few decades.
To overcome the limitations of the conventional drug delivery methods, targeted nano-platform is required. It is aimed in designing and developing novel biodegradable PLGA nanoparticles functionalized with different ligands for the delivery of OSM mesylate with the aim to target and enhances its cytotoxic effect on human lung cancerous cells thereby minimizing the nonspecific tissue distribution related side effects.
Thus the inventors of the present invention have successfully addressed the existing drawbacks and formulated a nanoformulation of OSM mesylate PLGA nanoparticles with ligands.
OBJECTS OF THE INVENTION
Accordingly, it is an object of the present invention to design and develop a composition of OSM mesylate PLGA nanoparticles functionalized with various macrophage targeting ligands in the management of cancer, more particular lung cancer.
It is another object of the present invention to provide a composition of OSM mesylate PLGA nanoparticles with higher efficacy at lower doses to eliminate/delay acquired resistance in cancer treatment.
It is a further object of the present invention to provide a composition of OSM mesylate PLGA nanoparticles with ligand and process of preparation thereof.
SUMMARY OF THE INVENTION
The present invention related to design and development of composition of OSM mesylate PLGA nanoparticles functionalized with various macrophage targeting ligands in the management of lung cancer. The present invention related to targeted delivery of OSM mesylate which could demonstrate higher efficacy at lower doses to eliminate or delay acquired resistance. The present invention related to the OSM mesylate loaded PLGA nanoparticulate composition.
The present further invention related to active targeting delivery system comprising Osimertinib mesylate (OSM) PLGA nanoparticles functionalized with ligand.
The present invention further related to OSM mesylate loaded PLGA nanoparticulate composition comprising an effective amount of Osimertinib mesylate (OSM); poly D-L-lactic-co-glycolic acid (PLGA); a solvent system for preparation of polymeric nanoparticle consisted of an organic phase and aqueous phase; ligand and a surfactant.
The present invention further related to process of preparation of Osimertinib mesylate loaded PLGA nanoparticles.
The invention also related to a method of preparing OSM mesylate PLGA nanoparticulate system for treatment of cancer.
Moreover the invention also relates to a method of evaluation of active targeting PLGA nanoparticulate system of OSM mesylate.
DESCRIPTION OF THE DRAWINGS:
Figure 1: Represents diagrammatic representation of process for preparation of OSM mesylate PLGA nanoparticles.
Figure 2: Represents particle size of blank PLGA nanoparticles.
Figure 3 A: Represents the mean particle size of OSM PLGA NPs
Figure 3 B: Represents the mean particle size of CS OSM PLGA NPs
Figure 4 A: Represents Zeta potential of OSM mesylate PLGA nanoparticles.
Figure 4B: Represents Zeta potential of CS OSM PLGA NPs.
Figure 5: Represents FT-IR analysis of formulations
Figure 6: Represents in-vitro efficacy against lung cancer cell line
Figure 7: Represents cellular internalization in A459 cell line after drug and formulation treatment. Data were represented as FITC, DAPI and merge image in the respective Group 1, 2 and 3.
Figure 8A: Represents the alteration in the production of ROS and mitochondrial membrane potential on the treatment of drug and drug with formulation in A459 cell line.
Figure 8B and 8C: Represents tubular graph represent changes in the ROS and mitochondrial membrane production respectively after drug and formulation treatment.
DETAILED DESCRIPTION OF THE INVENTION:
In describing the embodiment of the invention, specific terminology is chosen for the sake of clarity. However, it is not intended that the invention be limited to the specific terms so selected and it is to be understood that such specific terms include all technical equivalents that operate in a similar manner to accomplish a similar purpose. As used herein, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there is one and only one of the elements. The disclosure of numerical ranges should be understood as referring to each discrete point within the range, inclusive of endpoints, unless otherwise noted.
The therapeutically effective amount administered to the subject, e.g., non-human mammal, particularly a bovine animal, in the context of the present invention should be sufficient to affect a therapeutic or prophylactic response in the patient over a reasonable time frame. The dose can be readily determined using methods that are well known in the art. One skilled in the art will recognize that the specific dosage level for any particular patient will depend upon a variety of potentially therapeutically relevant factors.
The terms abbreviated and used interchangeably in the specification are as described. Osimeritinib can also be read as OSM. Poly D,L-lactic-co-glycolic acid can also be read as PLGA. Nanoparticle can also be read as NP/NPs. Chitosan can also be read as CS.
The term Ligand defined as a polymer such as mono, di or poly saccharides polymer, selected from but not limited chitosan and derivatives, hyaluronic acid or 1,3 beta glucan.
According to one aspect the present invention provides an active targeting delivery system comprising Osimertinib mesylate (OSM) PLGA nanoparticles functionalized with ligand.
According to an embodiment, the present invention particularly provides the PLGA nanoparticulate system of OSM mesylate with ligand and its manufacturing process thereof.
According to another embodiment, the present invention particularly provides the ligand targeted nanoparticle composition comprising OSM mesylate; PLGA and atleast one solvent, atleast one aqueous phase, atleast one organic phase, atleast one ligand, atleast one surfactant.
According to an embodiment, the present invention particularly related to the nanoparticle composition comprising:
(a) an effective amount of Osimertinib mesylate (OSM) is in the range of (60%) i.e, 15 mg in 25 ml of formulation;
(b) Poly (DL-lactic-co-glycolic acid) PLGA;
(c) a solvent system for preparation of polymeric nanoparticle comprised of an organic phase and aqueous phase;
(d) targeting ligand is in the range of 0.05% to 0.5% and
(e) a surfactant is in the range of 0.1 to 2%.
According to another embodiment, composition described hereinabove further comprises one or more pharmaceutically acceptable components.
According to another embodiment, pharmaceutically acceptable components is/are selected from the group consisting of carriers, polymers, surfactants, stabilizers, wetting agents, emulsifiers, antioxidants, pH influencing agents, disintegrants, recrystallization agents, fluxing agents, preservatives, solvents, salts fillers, binders, foamers, defoamers, lubricants, adsorbents for adjusting the osmotic pressure and buffers.
According to a further embodiment, organic phase organic phase is selected from the group consisting of methanol, ethanol, propanol, acetone, acetonitrile or mixture thereof.
According to a further embodiment of the present invention the drug (OSM mesylate) to polymer (PLGA) ratios are 1D:3P,1D:1.5P, 1D:1P, 1.5D:1P, 2D:1P, 3D:1P,1D:2P and 1D:4P.
According to an embodiment of the invention the suitable surfactant is but not limited to PVA (Poly vinyl alcohol) or Tween 80. The PVA is present in the amount ranging from 0.1% to 2% and Tween 80 is present in the amount ranging from 0.1% to 0.5%.
According to an embodiment, targeting ligand is selected from but not limited to mono-saccharide polymer, di-saccharide polymer or polysaccharide polymer; preferably chitosan or chitosan derivatives; more preferably chitosan (CS). CS is used as targeting ligand for the fabrication around OSM PLGA nanoparticles to obtain CS OSM PLGA NPs for achieving active tumor targeting. The chitosan is present in the amount ranging from 0.05% to 0.5%.

According to an embodiment, the present invention relates to a process of preparation of OSM mesylate PLGA nanoparticles comprising the following steps:
Preparing organic phase by dissolving the OSM mesylate and PLGA in various drug to polymer ratios in solvents;
Transferring the organic phase to an aqueous phase containing a surfactant;
Evaporating the organic phase by stirring the mixture of step b and collecting aqueous formulation containing the nanoparticles
Centrifuging the formulation of step c;
Washing the nanoparticle with water and lyophilized to obtained OSM mesylate PLGA nanoparticle.
According to a further embodiment, the solvents used to dissolve OSM mesylate include but are not limited to (C1-C4 alcohol) such as methanol, ethanol or propanol.
According to a further embodiment, the solvents used to dissolve PLGA include but are not limited to acetone, acetonitrile or mixture thereof, preferably acetone.
According to a further embodiment, the solvent used to dissolve PLGA is acetone; preferably about 1ml of acetone; more preferably 1ml of acetone.
According to a further embodiment, the organic phase was added in drop wise manner in to the aqueous phase containing surfactant and stirring continuously with for 3 hour.
According to a further embodiment, the aqueous formulations containing the nanoparticles were centrifuged at 40,000 rpm at 20°C for 60 min by using ultra centrifuge.
According to an embodiment the present invention relates to a nanoprecipitation method for preparation of the OSM mesylate PLGA nanoparticles.
According to another aspect of invention provides the process of preparation of ligand targeted OSM mesylate PLGA nanoparticle comprises the following steps:
preparing organic phase by dissolving the OSM mesylate and PLGA in various drug to polymer ratios in solvents;
transferring the organic phase to an aqueous phase containing a surfactant;
evaporating the organic phase by stirring the mixture of step b and collecting aqueous formulation containing the nanoparticles;
adding the targeting ligand solution drop wise to the solution of step d (i.e. aqueous solution of OSM mesylate PLGA nanoparticle) and stirring for 30 min;
centrifuging the solution of step (d), washing the nanoparticle with water and dry to obtained ligand targeted OSM mesylate PLGA.
According to another embodiment, in the step (d) stirring continue upto 30min with stirring speed of 300 rpm.
According to another embodiment, ratio of the nanoparticle to targeting ligand is 4:1.
According to another embodiment, the present invention relates to Fabrication/preparation of OSM mesylate PLGA nanoparticle with active targeting ligand using ionic gelation method.
According to another aspect of invention provides method of treatment of the cancer by administering to the subject and therapeutically effect amount of composition as described herein above.
According to another aspect of invention provides pharmaceutical composition as described herein above, to be administered using pharmaceutically acceptable dosage form.
Other embodiments of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosed embodiments. The following examples should be considered as exemplary only.
Experimental
General process of preparation of OSM mesylate PLGA nanoparticle system by nano-precipitation method (Figure 1)
Step a: OSM mesylate (15 mg) was dissolved in a mixture of methanol and acetonitrile (1:1);
Step b: PLGA(15 mg) was dissolved in acetone;
Step c: The organic phase along with OSM mesylate and PLGA was vortexed at room temperature;
Step d: Thus obtained solution from step c, was added in dropwise manner to the aqueous phase containing Tween 80 (0.4% w/v) followed by continuous stirring with for 3 hours;
Step e: The organic phase was evaporated and the final volume of the aqueous formulation was collected which contains the nanoparticles;
Step f: The nanoparticles thus formed were then centrifuged at 40,000 rpm, 20°C, for 60 min by using ultra centrifuge.
Step g: Drug loaded PLGA nanoparticles were collected and washed with water and then lyophilized and store until use for further evaluation.
Table 1: Optimization parameters for the preparation of OSM mesylate PLGA nanoparticles
Surfactant
Type Conc.
of
Surfactant
(w/v) % Organic phase Ratio of organic phase to aqueous phase Stirring speed
(rpm) Stirring time
(Hr) Drug to polymer concentration ratio
PVA 0.1%, 0.25%,
0.4% , 0.5%,
0.75 %,
1 %,
1.5 % & 2%) Methanol,
acetone &
acetonitrile
1:3.5

1:12.5
1200 2
3
4 F5DT (1D:3P)
F10DT(1D:1.5P)
F15DT(1D:1P)
F22.5DT(1.5D:1P)
F30DT (2D:1P)
F45DT (3D:1P)
F22.5PT (1D:1.5P)
F30PT (1D:2P)
F60PT (1D:4P)
Tween 80 0.1%,
0.2 %,
0.3 %,
0.4% & 0.5%

General process of fabrication of OSM mesylate PLGA nanoparticles with chitosan
Step a: OSM mesylate (15 mg) was dissolved in a mixture of methanol and acetonitrile (1:1);
Step b: PLGA(15 mg) was dissolved in acetone;
Step c: The organic phase with OSM mesylate and PLGA was vortexed at room temperature;
Step d: Thus obtained solution from step c, was added in dropwise manner to the aqueous phase containing Tween 80 (0.4% w/v) followed by continuous stirring with for 3 hours;
Step e: The organic phase was evaporated and the final volume of the aqueous formulation was collected which contains the nanoparticles;
Step f: collect the aqueous formulation containing drug loaded nanoparticles & incubate with chitosan solution to obtained CS OSM PLGA NPs; and
Step g: CS OSM PLGA NP are further centrifuged, washed and collected.
Evaluation:
1. Mean Particle size: The z-average diameters of NPs were determined by Malvern Zetasizer Instrument. The sample was diluted with distilled water and then the sample was introduced in Malvern Zetasizer ZS 90 to determine particle size and poly dispersity index. The mean particle size of blank PLGA NPs were determined to be 94.025 ± 0.970 nm (Figure 2). The mean particle size of OSM PLGA NPs were determined to be 127.0 ± 6.34 nm (Figure 3A). Due to drug loading, the mean particle size was greatly enhanced in OSM PLGA NPs when compared to blank PLGA NPs.
The 0.5% chitosan concentration is optimized for fabricating it onto the OSM PLGA NPs. The OSM PLGA nanoparticles mean particle size was significantly enhanced, once the chitosan coating was conjugated on to them. Further upon fabrication, the mean particle size was greatly increased in CS OSM PLGA NPs when compared to OSM PLGA NPs as shown in the table 2. The CS OSM PLGA NPs mean particle size was reported as 186.3± 6.35 nm as shown in the figure 3B.
2. Zeta potential: The zeta potential shows the level of repulsion between adjacent; likewise charged particles in scattering and its value can be identified to determine the stability of nanoparticles. At the point when potential is low, attraction exceeds repulsion and the scattering breaks and the sample flocculate. Nanoparticles with high zeta potential (negative or positive) are electrically balanced out while nanoparticles with low zeta potentials have a tendency to coagulate or flocculate. Zeta potential of the nanoparticles was also measured on Zetasizer. The blank PLGA NPs of has zeta potential -11.48 ± 2.88 mv. The negative zeta potential of PLGA nanoparticles is due to carboxyl end groups in PLGA. The OSM PLGA NPs of the present invention has Zeta potential +12.5 ± 5.10mv (Figure 4A). The positive zeta potential of OSM PLGA NPs might be due to equimolar ratio of drug to polymer concentration, because of that some of the drug OSM molecules will get adsorbed on PLGA surface. Due to the amine groups of OSM on PLGA nanoparticles, zeta potential of optimized formulation shows positive value.
Zeta potential of optimized CS OSM PLGA NPs is reported as 23± 7.88 (as shown in the figure 4B). The positive zeta potential of CS OSM PLGA NPs is due to presence of amine groups of chitosan which is coated onto the OSM PLGA NPs. As the PLGA is hydrophobic in nature, it gets associated with chitosan due to ionic adsorption process forming CS OSM PLGA NPs.

3. Drug loading & Drug entrapment efficiency
Drug loading capacity is the ratio of amount of drug added to the total amount of drug & polymer in the formulation.
To evaluate the drug entrapment efficiency (DEE), the drug loaded nanoparticles were centrifuged at 1,00,000 rpm at 20°C for 60 min by utilizing ultracentrifuge (Thermo scientific sorvall). The supernatant was decanted and pellet was separated. Drug entrapment efficiency was evaluated by direct method (pellet method). The final pellet thus so formed by ultra-centrifugation process was used for further extracting the drug and analyzed by HPLC (waters 2695).
DEE (%) = (Amount of drug entrapped)/(Total amount ofdrug added)
Drug loading capacity of optimised formulation was reported as 50.1±1.19.
Entrapment efficiency of optimised formulation OSMPLGA NPs was determined by direct pellet method. DEE (%) of OSM PLGA NPs was reported as 86.6 ± 2.12%.
Table 2. Data of mean particle size, PDI, zeta potential & entrapment efficiency of blank PLGA NPs, OSM PLGA NPs, CS OSM PLGA NPs
Formulation code Particle size (nm) PDI Zeta potential (mv) DEE (%)
Blank PLGA NPs 94.025±0.97 0.22±0.04 -11.48±2.88 -

OSM PLGA NPs 121.0 ±6.34 nm 0.162± 0.018 12.5 ± 0.75 mv 86.6 ± 2.12%

CS OSM PLGA NPs 186.3± 6.35 nm 0.387± 0.03 21.1 ± 7.63 mv -

4. FT-1R analysis
FT-IR analysis was done to find the drug excipient compatibility in the optimized formulation. From the data obtained in the figure 5, drug and the compounds used in the formulation displayed the characteristic bands indicating their purity.
From the spectrum of PLGA, a sharp characteristic intense band was observed at the wave number 1747.24 cm-1 which is attributed to the stretching vibrations of the carbonyl group. The other following characteristic bands that were observed at the wave numbers 3002.7 cm-1 is due to the stretching vibrations of OH groups. C-O stretching is observed at 1047 cm-1 and C-H Bend at 1452 cm-1.
From the spectrum of OSM drug, a sharp characteristic intense band was observed at the wave number 1576.31 cm-1 which is attributed to the bending vibrations of the NH group. The other following characteristic bands that were observed at the wave numbers 3242.7 cm-1 is due to the stretching vibrations of NH groups. C=N stretching is observed at 1671.2cm-1.
From the spectrum of chitosan, a characteristic broad band was observed at the wave number 3291 cm-1 & 1589 cm-1 are attributed due to the stretching and bending vibrations of the NH group respectively. A sharp characteristic intense band was observed at the wave number 1725.38cm-1 due to C=O (carbonyl group) of amide bond. The bands that were observed at the wave numbers 2925.2 cm-1 is due to the stretching vibrations of CH group of CH3. The signal at 1218 cm-1 was attributed due to the bending vibrations of hydroxyl groups present that attached to the saccharide portion in chitosan. C-O stretching is observed at 1032.42 cm-1
Physical mixture contains OSM, PLGA & chitosan. From the spectrum of physical mixture, it was observed that the characteristic peaks of OSM, PLGA & chitosan were retained indicating there were no interactions between them displaying the compatibility. From the spectrum of CS OSM PLGA NPs it was observed that characteristic drug peaks were not appeared indicating that drug was encapsulated in the chitosan coated PLGA nanoparticle and the chitosan was conjugated around the OSM PLGA nanoparticle surface which is proved by the characteristic bands of chitosan in the final formulation such as NH stretching of amine groups at a wave number 3327 cm-1.
5. Cell viability assay: In-vitro cytotoxic activity of developed formulation
A549 cells were maintained in RPMI-1640 with the supplementation of 10% FBS and 1% of antibiotics (Streptomycin & Penicillin). Cells were incubated in humidifier incubator at and 5% CO2 and 37°C temperature. Cell cytotoxicity assay was performed and quantified through MTT assay. 1 × 105 cells/well were seeded in 96-well flat-bottomed tissue culture plates and incubated for 24 hrs at 5% CO2 and 37°C temperature in incubator followed by cells were treated with various concentration (400, 200, 100, 50, 25, 12.5, 6.25, 3.12 µg) of OSM drug, OSM PLGANPs and CS OSM PLGA NPs and incubated for 24 hrs. After incubation cells were treated with MTT solution and incubated for 4 hrs followed by media removed and formazan complex was dissolved in dimethyl sulfoxide. This formazan complex salt absorbed at 570 nm using multimode reader and calculates IC50 value.
Cytotoxicity of pure drug, OSM PLGA NPs and CS OSM PLGA NPs were evaluated by MTT assay. Result of MTT assay revealed that IC50 of OSM and OSM PLGA NPs and CS PLGA OSM NPs were showed 12.6±1.45 µg/ml, 8.6±0.61 µg/ml and 3.6±0.37 µg/ml. The chitosan coated OSM bearing PLGA nanoparticle showing higher in-vitro efficacy (as shown in the figure 6) against lung cancer cell line in compared to free OSM and uncoated PLGA OSM nanoparticle, thereby explicating active tumor targeting is achieved.
6. Cellular internalization assessment
Cellular uptake effect of developed OSM PLGA NPs and ligand coated CS OSM PLGA NPs were measured by using cellular internalization assay. A549 cells were grown on poly-L-lysine coated cover slips in the 6 well culture plates until they become confluent. Cells were treated with plain FITC (Fluorescein isothiocyanate) solution (group1), PLGA-FITC-NPs (group 2) and CS-FITC-PLGA-NPs (group 3) followed by incubation for 24 h. Fixation was done by immersing in 4% paraformaldehyde in PBS for 20 min. The cover slip fixed with mounted media containing DAPI (4',6-diamidino-2-phenylindole) for nuclear staining of cells. Cells were investigated with an LEICA inverted confocal microscope for DAPI staining at excitation and emission filters were 350 nm and 470 nm respectively. Whereas FITC were investigated at excitation and emission filters were 494 nm and 518 nm respectively., Result of cellular internalization significantly increased in PLGA-FITC-NPs (group 2) and CS -FITC PLGA NPs (group 3) as compared to FITC (group1) as shown in the figure 7. This study shows that as the FITC is impermeable into the cells, very less green fluorescence is obtained in group 1. Whereas group 2 contains PLGA nanoparticles which are of nanoscale, so that they can easily penetrate into the cells and hence increase in the green flourescence. Among all three groups, the group 3 shows enhanced cellular uptake due to the targeting ligand chitosan, hence intense fluorescence is observed and same case of DAPI nuclear staining. From the figure 6, it was revealed that composite of DAPI & FITC fluorescence is higher in case of group 2 and group 3 due to the nanoparticle size and targeting moiety attached to the nanoparticle respectively. The chitosan coated PLGA nanoparticles (CS-FITC-PLGA-NPs) showing higher cellular internalization in comparison to the uncoated PLGA FITC nanoparticles and free FITC solution. This study proves that active targeting strategy shows enhanced cellular uptake which in turn might exhibits enhanced cytotoxicity.
7. Mitochondrial Potential (??m) and Mitochondrial Superoxide Anion (O-2)
Mitochondrial potential and Superoxide anion (O-2) were analyzed by JC-1 staining. A549 cells were allowed to grown in 12 well plates followed by incubation for 24 h. Cells were treated with OSM drug, PLGA-OSM-NPs and CS-OSM-PLGA-NPs in DMEM media without FBS. Cells were washed with plane medium followed by incubation of cells with cationic JC-1 (5 µM) dye staining for 15 minutes. Fluorescent intensity in cells were analyzed by Nikon Eclipse inverted fluorescent microscope, (Japan) at ×200 magnification
The effect of OSM, OSM PLGA NPS and CS OSM PLGA NPS treatment on mitochondrial potential was evaluated through cationic JC-1 dye. The JC-1 dye can enter the mitochondria and indicates the mitochondrial membrane potential either by existing in the nature of monomers or red JC-aggregates. This study reveals that (figure 8A & 8C) group 2 & group 3 shows decreased JC-aggregates & reduced red fluorescence intensity when compared to free OSM drug.
8. ROS quantification
DCFDA (2',7'-Dichlorodihydrofluorescein diacetate) staining was performed to quantify the ROS level in A549 cell lines (3.5 × 105cells/well) in DMEM (Dulbecco's Modified Eagle Medium) medium with 10% FBS supplement and allowed to adhere for 24 h. Cells were treated with OSM drug, OSM PLGA NPs and CS OSM PLGA NPs in FBS free medium and incubated in humidified incubator for 24 hrs. After incubation media was removed and cells were incubated in DCFDA staining for 20 minutes followed by washing of cells with 1X PBS. Fluorescent intensity was analyzed by Nikon Eclipse inverted fluorescent microscope, (Japan) at ×200 magnification.
The effect of OSM (group1), OSM PLGA NPs (group 2), and CS OSM PLGA NPs (group 3), treatment on the production of ROS was evaluated through DCFDA staining. From the figure 8A & 8B, it was shown that the production of ROS was significantly enhanced in OSM PLGA NPS (**p<0.01) treatment group when compared with OSM treated group. Similarly, the production of ROS was very greatly increased (***p<0.001) in CS OSM PLGA NPS treated group when compared to OSM treated group & OSM PLGA NPs. This study reveals that group 2 & group 3 shows enhanced ROS production which was observed by the DCFDA staining intensity. The more the ROS production, more is the cytotoxic to A549 cancer cells because high concentration of ROS causes cancer cell apoptosis. The chitosan coated OSM PLGA NPs showing more cytotoxicity due to increased production of ROS.

,CLAIMS:WE CLAIM
1. A ligand conjugated nanoparticle composition comprising:
(a) an effective amount of Osimertinib mesylate (OSM);
(b) Poly (DL-lactic-co-glycolic acid) PLGA;
(c) solvent system consisting of an organic phase and aqueous phase;
(d) targeting ligand and
(e) surfactant.
2. The ligand conjugated nanoparticle composition as claimed in claim 1, wherein the organic phase is selected from the group consisting of methanol, ethanol, propanol, acetone, acetonitrile or mixture thereof.
3. The ligand conjugated nanoparticle composition as claimed in claim 1, wherein the OSM mesylate to PLGA ratios are 1D:3P, 1D:1.5P, 1D:1P, 1.5D:1P, 2D:1P, 3D:1P,1D:2P or 1D:4P.
4. The ligand conjugated nanoparticle composition as claimed in claim 1, wherein targeting ligand is selected from mono-saccharide polymer, di-saccharide polymer or polysaccharide polymer; preferably chitosan or chitosan derivatives; more preferably chitosan.
5. The ligand conjugated nanoparticle composition as claimed in claim 1, wherein surfactant is selected from a group comprising of PVA (Poly vinyl alcohol) or tween 80.
6. The ligand conjugated nanoparticle composition as claimed in claim 1, wherein composition further comprises one or more further pharmaceutically acceptable components.
7. A process of preparation of ligand conjugated OSM mesylate PLGA nanoparticle comprises the following steps:
a) preparing organic phase by dissolving the OSM mesylate and PLGA in various drug to polymer ratios in solvents;
b) transferring the organic phase to an aqueous phase containing a surfactant;
c) evaporating the organic phase by stirring the mixture of step b and collecting aqueous formulation containing the nanoparticles;
d) adding the targeting ligand solution drop wise to the solution of step c (i.e. aqueous solution of OSM mesylate PLGA nanoparticle) and continuing stirring;
e) centrifuging the solution of step (d); and
f) washing the nanoparticle with water and drying to obtained ligand targeted OSM mesylate PLGA;
8. The process as claimed in claim 7, wherein the solvent used to dissolve OSM mesylate is selected from the group consisting of methanol, ethanol or propanol.
9. The process as claimed in claim 7, wherein the solvent used to dissolve PLGA is acetone.
10. The process as claimed in claim 7, wherein the nanoparticle to targeting ligand ratio is 4:1.

Dated this on 20th day of February, 2021