Preparation , Characterization and In-Vitro Cytotoxic Study of Doxorubicin PLGA Nanoparticles

Article Information Received 4 Sept 2013 Received in revised form 17 Nov 2013 Accepted 18 November 2013 Abstract In the current study we had developed and characterized Poly (lactic-co-glycolide) acid nanoparticles for anticancer drug Doxorubicin and further studied in-vitro cytotoxic effect of free and encapsulated nanoparticle drug delivery system. PLGA-drug nanoparticles were prepared by oil-water emulsion solvent evaporation method. The morphology of drug loaded nanoparticles was analyzed using a scanning electron microscope. The drug loaded nanoparticles were evaluated for cell cytotoxicity by sulforhodamine B assay. Images of formulation indicate the surface morphology of formulation. The in vitro cytotoxicity results showed Doxorubicin NPs to be active against A431 and NCI-H322 cell line compared to Doxorubicin free drug. The IC50 values were found to be 1.73 μM with A431 cell line whereas 1.64 μM for NCI-H322 cell line. This indicates that Doxorubicin NPs have more pronounced activity towards A431cell line.


Introduction
Cancer is the second largest health problem of the world after cardiovascular diseases in both developed and developing countries.
According to the World Health Organization 7.6 million people died of cancer worldwide in the year 2007, which accounts for 13 percent of all the deaths and the incidences, are expected to increase by 12 million deaths in 2030.As per the reports of 1 the most commonly diagnosed cancers worldwide are lung (1.35million), breast (1.15 million) and colorectal (1 million), and the most causes of deaths due to cancers are lung cancer (1.18 million deaths), stomach cancer (700,000 deaths) and liver cancer (598,000 deaths).Lung cancer is the leading cause of cancer -related death in the western world and the mortality rate is increasing in Asia.Approximately 85% of all primary lung cancers are Non small cell lung cancers (NSCLCs) which are classified into 3 major histological types: adenocarcinoma (approx 40% of lung cancer usually found in periphery of lungs), squamous (25-30% of lung cancers, hilar region of the lung near bronchus cell carcinoma and large cell carcinoma (approx 10-15 of lung cancers may appear in any part of lung).High copy number of EGFR has been detected in approximately 30% of NSCLC patients using FISH, and is reportedly associated with better to TKI therapy 2 .
Nanoparticles are solid, colloidal particles of macromolecular substances that vary in size from 10 nm to 1000 nm 3 .Nanoparticulate drug delievery system may offer plenty of advantage over conventional dosage forms which include improved, reduced toxicity, enhanced biodistribution and improved patient compliance 4 .These targeting capabilities of nanomedicines are influenced by particle size, surface charge, surface modification, and hydrophobicity.Among these, the size and size distributions of nanoparticles are important to determine their interaction with the cell membrane and their penetration across the physiological drug barriers.The size of nanoparticles for crossing different biological barriers is dependent on the tissue, target site and circulation 5 .For the cellular internalization of the nanoparticles, surface charge is important in determining whether the nanoparticles would cluster in blood flow or would adhere to, or interact with oppositely charged cells membrane.Cationic surface charge is desirable as it promotes interaction of the nanoparticles with the cells and hence increases the rate and extent of internalization 6 .Nanoparticles provide a range of new opportunities to increase the targeting of currently approved diagnostic and therapeutic agents to cancer.Nanoparticles (NPs) carrying a chemotherapeutic can reduce the undesirable distribution of such agents.Nanoparticles integrated with the therapeutic agents can either freely release these agents or undergo their own decomposition for the release to occur.

Advances in nanotechnology have
Improved in targeting can lead not only to increased efficiency of these agents but also to increase signal-to-noise ratio for diagnostics and better efficacy to toxicity ratios for therapeutics.Nanoparticles can access specific sites by avoiding the systemic clearance by the RES system.In the current study we had developed & characterized Poly (lactic-co-glycolide) acid (PLGA) nanoparticles for anticancer drug Doxorubicin and further studied in-vitro cytotoxic effect of free and encapsulated nanoparticle drug delivery system.

Materials
The cell lines were obtained from National Center for Cell Science, Ganeshkhind, Pune-4111007 (India).

Preparation of drug loaded nanoparticles
PLGA-Doxorubicin nanoparticles (NPs) were prepared by oil-water (o/w) emulsion solvent evaporation method.First, PLGA (25 mg) and Doxorubicin (1.25 mg) were dissolved in 2.5 ml of Dichloromethane (DCM).Emulsion (o/w) was made by adding 2.5 ml of DCM containing PLGA polymers and drug drop wise using needle and syringe, into 25 ml of 1% polyvinyl alcohol (PVA) solution using a micro-tip probe sonicator set at 55 W of energy output for 2 min over an ice bath.The emulsion was stirred overnight at room temperature to allow evaporation of organic solvent and formation of NPs.Finally nanoparticles were recovered by ultracentrifugation at 40,000 rpm for 20 min at 4 °C, washed twice with water to remove excess PVA.The nanoparticle suspension was then lyophilized for 2 days (-47 °C and < 10 µm mercury pressure) to obtained the powdered NPs for further use.

Scanning electron microscopy (SEM) analysis
The morphology of drug loaded nanoparticles was analyzed using a SEM, scanning electron microscope.Samples were prepared from dilutions in distilled water of particle suspensions and dropped onto stubs.After air drying, particle were coated with a thin layer of gold and then examined by scanning electron microscopy.

Cell cytotoxicity evaluation by (sulforhodamine B) SRB assay
SRB assay is a rapid, sensitive and inexpensive method for measuring the cytotoxic potential of test substances, based on the cellular protein content of adhered suspension cultures in 96 well plates.This method is suitable for ordinary laboratory purposes and for large-scale applications like high throughput in vitro screening in anticancer drug development.
The desired human cancer cell line i.e.NCI-H322 (Lung) and A431 (Epidermal) were grown in tissue culture flasks at 37 °C, in an atmosphere of 5% CO2 and 90% relative humidity in complete growth medium to obtain enough number of cells.The cells were harvested by treating with trypsin-EDTA, and the cell density was adjusted to 10,000 cells/100 µl in the cell suspension.The 100 µl of cell suspension was added to each well of 96 well plate with the help of handy-step; and incubate the plates at 37 o C, in an atmosphere of 5% CO2 and 90% relative humidity for 24 hours.After 24 hours the 100 µl of test sample and free drug sample of various concentrations (0.1 µM, 0.5 µM, 1.0 µM and 2.5 µM) were added to the wells of 96 well plates.The plates were taken out from the incubator after 48 hours of adding test samples.To stop the reaction, gently add 50 µl of chilled 50% trichloroaceticacid (TCA) to each well of the plate, making final concentration of 10%; incubate the plates at 4C for one hour to fix the cells attached to bottom of the wells.The plates were air-dried after washing it 5-6 times with distilled water.The 100 µl of SRB dye (0.4% in 1%acetic acid) was added to each well of the plate and leave the plates at room temperature for 30 minutes, and wash the plates with 1% acetic acid after 30 minutes.After air drying of plates, add 100 µl of Tris buffer (10.5 M) to each well, and shake the plates for 10-15 min on a mechanical shaker.The optical density was recorded with ELISA reader at 540 nm wavelength [8][9][10][11][12]

Statistics analysis
The data were reported as mean values ± standard deviation (SEM).
Values representing the concentrations of investigated test sample and free drug sample that cause 50% of inhibition (IC50) were determined by the linear regression analysis.

Determination of cellular cytotoxicity by SRB assay
SRB assay was performed to determine the in-vitro cytotoxicity against adherent cell lines (Table 1).SRB assay was based on the uptake of the negatively charged pink aminoxanthine dye, SRB by basic amino acids in the plasma membrane of the cells.
The in vitro cytotoxicity results showed Doxorubicin NPs to be active against both above used cell lines compared to Doxorubicin free drug (Table 1).In order to have a better insight into the comparative data the IC50 values (the drug concentration at which 50% of growth inhibition takes place) of Doxorubicin NPs against these two cell lines was calculated.The IC50 values were found to opened up ample opportunities in controlled drug delivery and novel combination strategies.Nanoscale particles exhibit prolonged systemic circulation lifetime, sustained drug release kinetics, and better tumor accumulations through both UK Journal of Pharmaceutical and Biosciences Available at www.ukjpb.comParashar et al.Doxorubicin PLGA Nanoparticles UK J Pharm & Biosci, 2013: 1(1); 49 passive and active mechanisms 3 .Nanomedicines of the dreadful diseases like cancer, AIDS, diabetes, malaria, prion disease and tuberculosis are in different trial phase for the testing and some of them are commercialized.Nanomedicine formulation depends on the choice of suitable polymeric system having maximum encapsulation 7 .

Parashar
et al.Doxorubicin PLGA Nanoparticles UK J Pharm & Biosci, 2013: 1(1); 50 SEM was performed for particles much smaller than 1 μm to be measured.Images of formulation indicate the surface morphology of formulation (Fig 1).

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73 µM with A431 cell line (Fig 2) whereas 1.64 µM for NCI-H322 cell line (Fig 3).This indicates that Doxorubicin NPs have more pronounced activity A431cell line.The above studies lead to a conclusion that, Doxorubicin PLGA NPs are better formulation than free Doxorubicin as supported by the IC50 value.

Fig 2 :Fig 3 :
Fig 2: IC50 value of Doxorubicin PLGA NPs in A431 cell line by SRB assay