Available online www.jocpr.com Journal of Chemical and Pharmaceutical Research, 2015, 7(3):2239-2243 Research Article ISSN : 0975-7384 CODEN(USA) : JCPRC5 Developing and evaluating in vitro effect of pegylated liposomal doxorubicin on human cancer cells Hue Pham Thi Minh1, Linh Le Phương1, Lam Nguyen Van1, Hai Nguyen Thanh2, Son Ho Anh3, Toan Nguyen Linh3 and Tung Bui Thanh4* Department of Pharmaceutics, Hanoi University of Pharmacy, 15 Lê Thánh Tông, Hoan Kiem, Ha Noi, Vietnam Department of Pharmaceutics and Pharmaceutical Technology, School of Medicine and Pharmacy, Vietnam National University, Hanoi, 144 Xuan Thuy, Cau Giay, Ha Noi, Vietnam Department of Pathophysiology, Vietnam Military Medical University, 160 Phung Hung, Ha Đong, Ha Noi, Vietnam Department of Pharmacology and Clinical Pharmacy, School of Medicine and Pharmacy, Vietnam National University, Hanoi, 144 Xuan Thuy, Cau Giay, Ha Noi, Vietnam _ ABSTRACT A PEGylated liposomal formulation of doxoburicin has been developed and evaluated with the purpose of reducing toxicity and improving the tumor-targeting efficacy of doxorubicin.PEGylated liposomal doxorubicin was prepared by the lipid film hydration technique using hydrogenated soybean phosphatidylcholine (HSPC) combined with cholesterol and DSPE-PEG2000 Biological activity and toxicity were tested on A549 and HT 29 cancer cell lines by the MTT method As the result, the obtained PEGylated liposomal doxorubicin using the lipid film hydration method had an entrapment efficiency of the drug more than 95% The formulated liposomes were found to be relatively uniform in size (167.8± 3.6 nm) with a negative zeta potential (−27.5 ± 3.5 mV) The stability experiments results indicated that PEGylated liposomal doxorubicinwas stable for at least months at 2-8oC The cytotoxic effect of PEGylated liposomal doxorubicin on A549 and HT29 cell lines was effected when the exposure time is over 48h.The A549 cell line was found more sensitive than the HT 29 line to PEGylated liposomal doxorubicin The lowest IC50 was observed after 72 hours for both cell lines These results indicated that PEGylated liposomal doxorubicin was valued to develop as a practical formulation for the tumor-targeting efficacy Future work will be needed to evaluate the antitumor efficacy of doxoburicin liposome formulation in vivo Keywords: PEGylated; liposome doxorubicin; MTT; A549; HT29 _ INTRODUCTION Doxorubicin (DOX), an anthracycline antitumor antibiotic, is the most active agents used in the treatment of some cancer diseases including hematological malignancies, breast carcinoma Doxorubicin binds to DNA-associated enzymes, intercalate with DNA base pairs, and target multiple molecule to produce a range of cytotoxic effects [1] However, doxorubicin is limited in the clinical use because of its toxicity properties, especially cardiomyopathy, leading to congestive heart failure and myelosuppression [2] Nowadays,, using targeted therapy would be one of the best choices today to reduce its toxicity while increasing therapeutic efficacy Liposomes, phospholipid bilayer vesicles, are one of successful approach using drug carriers to alter the pharmacokinetics and biodistribution of drug Until now, liposomes are the most advanced of the particulate drug carriers and considered as a mainstream drugdelivery system Liposomes can trap principle active, avoid decomposition of the entrapped combinations, and release the entrapped at designated targets [3] Liposomes, with biphasic character, can act as carriers for both lipophilic and hydrophilic drugs Highly hydrophilic drugs, such as doxoburicin, are located exclusively in the aqueous compartment of liposomes However, one of the major limitations of liposome isrecognized by opsonins, 2239 Tung Bui Thanh et al J Chem Pharm Res., 2015, 7(3):2239-2243 and rapidly degrades, decreased half-life time circulation in blood To overcome this limitation, liposomes are coated with inert, biocompatible polymers, such as polyethylene glycol (PEG) PEG forms a protective layer over the liposome surface and PEGylated liposomal has long circulation time and provides slow release of an encapsulated drug [4] PEGylated liposomes have been demonstratedto improve the deliveryof encapsulated doxorubicin in previous studies PEGylated liposomes are less extensively taken up by cells of the reticuloendothelial system and have a reduced tendency to leak drug while in circulation [5] The pharmacokinetics parameter of PEGylated liposomes are characterized by a longer circulating half-life, slower plasma clearance and a smaller volume of distribution compared with conventional liposomal The present study was carried out to develop a PEGylated liposomal doxorubicin formulation withthe purpose of reducing toxicity and improving the tumor-targeting efficacy of doxorubicin DOX-loaded liposomes were prepared by the lipid film hydration technique The entrapment efficiency and the stability were analyzed Furthermore, in vitro cytotoxic effect of PEGylated liposomal doxorubicin on human cancer cell lines was also studied EXPERIMENTAL SECTION Reagent and instruments Reagent: Doxorubicin hydrochloride, hydrogenated soybean phosphatidylcholine (HSPC) (Lipoid), DSPE-(PEG)2000-COOH, cholesterol, chloroform,sodium hydroxide, ammonium sulfate, potassium dihydrogen phosphate, disodium hydrogen phosphate, phosphoric acid, triton X 100 (polyethylene glycol), 3-[4,5-dimehyl-2-thiazolyl]-2,5-diphenyl-2Htetrazolium bromide (MTT) All other reagents and solvents used to meet requirements for pharmaceutical and analytical grade The human lung non-small cancer cell line (A549 cells) and HT 29 colon cancer cell linewere purchased from company ATCC, USA.Cells were grown in Eagle's minimum essential medium (ATCC, USA)containing 10 % Fetal Bovine Serum and 1%streptomycin-penicillin Instruments: The evaporation system Rovapor R-210; Spectra/Por® Dialysis Membrane, MWCO: 12,000 – 14,000 Daltons;Analyzer size system Zetasizer ZS90; Ultrasound Machines; UV-VIS Spectrophotometer; pH meter; magnetic stirrer and other common tools; Clean room Systems for cell culture; cell culture cabinets, probe optics system 96-well plate, flat-bottomed 96-well plate (South Korea) Methods Methods of PEGylated liposomal doxorubicin preparations: Using the method of hydration of a thin lipid film: Bangham method - Weight and dissolve phospholipids: HSPC, cholesterol DSPE-PEG2000 (3:1:1 w/w) inchloroform Then, the organic solvent was removed by evaporation using the Rovapor R-210 system at 50oC for 12 h to form the dry lipidic film on the flask wall The lipid film is thoroughly dried to remove residual organic solvent by placing the flask on a vacuum pump overnight Finally, hydrate the thin lipid film by adding a buffer citrate or ammonium sulfate solution pH 3.5-4.0 at 60oC for 2h - Reduce liposome size by extrude suspension through polycarbonate filters by size decreases from 1µm to 100nm, maintaining temperature at 60oC - Change the external buffer environment of liposome with buffer Hespes pH 7.5 using tangential flow filtration Weight exactly an amount of doxorubicin hydrochloride, add to the suspension of liposome, stir with 80 rpm for 30 min, temperature maintaining at 50 ± oC - Filter product through membrane filter 0.2 µm and then packed into 10 ml glass closed with rubber and aluminum cap, keep in a refrigerator from 8-10oC Liposome size, distributionand Zeta potential: using the method of dynamic light scattering (DLS) with instrument Zetasizer ZS90 Dilute suspension of liposome 200 times with deionized water [6] Quantification of doxorubicin: using a spectrophotometric method with wavelength 481 nm Entrapment efficiency: Add mL of PEGylated liposomal doxorubicin suspensioninto dialysis bag and hang the bag in an Erlenmeyer flask containing 100 ml of phosphate buffer pH 4.0 Maintain system at temperature - 10oC for 12 hours Take the solution from outside of the dialysis bag and measure optical density at 233 nm wavelength The percentage entrapment efficiency of the drug was calculated by: % Entrapment efficiency = 2240 mo − m x 100% mo Tung Bui Thanh et al J Chem Pharm Res., 2015, 7(3):2239-2243 m, mo: amount of doxorubicin diffused through the dialysis membrane and amountof doxorubicin initial Stability studies: The PEGylated liposomal doxorubicin was stored at 2-8oC for months under a sealed condition The mean particle size, polydispersity index, zeta potential and drug entrapment efficiency were determined after week, month, month and month In vitro cytotoxicity assay The cytotoxicity of PEGylated liposomal doxorubicin against human lung non-small cancer cell line (A549 cells) and HT 29 colon cancer cell line was determined by using the MTTdye reduction assay Briefly, 1.0 × 104 cells/well was seeded in 96-well flat-bottom tissue-culture plates The cells were incubated at 37oC in a 5% CO2 incubator for 24 h, during which cells were attached and resumed to grow PEGylated liposomal doxorubicin were diluted with culture media to make various concentrations of doxorubicin, andwere added in in the 96-well flat-bottom tissue culture plate (200 µL each) Control wells were treated with equivalent volumes of doxoburicin-free media After 24 h, 48h or 72 h, the supernatant was removed, the solution of MTT in PBS (pH 7.4) and culture medium (100µL each) was added to each well and incubated for h After hours, the content of each cell was removed and the converted dye was solubilized in 200 µL of DMSO Plates were shaken for20 and absorbance was read at 570 nm using the microplate reader (which brand) The IC50 values (concentration resulting in 50% growth inhibition) of doxoburicin were graphically calculated from concentration-effect curves, considering the optical density of the control well as100% Data Analysis Data were analyzed statistically by one-wayanalysis of variance (ANOVA) using SigmaPlot 10.0 program (Systat Software Inc)and by the Student’s t-test (level of significance for p 95 >95 >95 93.78±1.34 93.09±2.77 As we observed in Table 2, the PEGylated liposomal doxorubicin stored at 2-8oC during months had no significant change in the mean particle size, zeta potential and drug entrapment efficiency Those data again confirmed our liposomes are stable in that condition Cytotoxicity effect and IC50 To investigate the effective activity of the liposomes preparation, we studied the cytotoxicity effects of PEGylated liposomal doxorubicin in non-small cell lung cancer A549 and HT-29 colon adenocarcinoma cell Liposomes can interact with the target cells in many different ways Among the many factors that affect the nature of such liposome–cell interactions, they include the composition, size and charge, the presence of targetingmolecules on the liposome surface Those factors play important role in determining drug bioavailability to cells and cytotoxic effects [9] Furthermore, the cytotoxicity of PEGylated liposomal is well-known related to the steric effect of PEG chains due to PEG chains may delay internalization of the drug in the cells by masking the negative liposomal charge [10] Many studies have shown that exposure time was closely related to cytotoxicity In this study, the PEGylated liposomal doxorubicin was exposed for three different times (24h, 48 h and 72h) As data showed in Table 3, the cytotoxic effect of PEGylated liposomal doxorubicin against A549 cells was effectively when the exposure time is over 48h Table 3: Cytotoxicity effect on the cell line A549 Concentration (µg/ml) 10 100 IC50 (µg/ml) After 24h 92.45 ± 18.04 81.62 ± 6.25 74.93 ± 13.02 >100 Cell viability (%) After 48h After 72h 53.4 ± 11.06 29.47 ± 7.47 49.15± 3.33 29.09 ± 8.73 25.04± 12.38 13.96 ± 5.07 9,97 0,7 According to the MTT assay, samples with IC50100 Cell viability (%) After 48h After 72h 88.52± 11.03 41.52± 3.44 61.17± 4.48 39.85± 5.97 49.7± 6.04 19.75± 2.69 99.9 0.86 After the first 24 hours,the PEGylated liposomal doxorubicin almost have no cytotoxiceffect After 48 hours, liposome exerted its weak cytotoxicity and after 72 hours it it was most effective with IC50 0.86 µg/ml.The product has an IC50 after 48 hours very high, 99.9 µg/ml, showed a slow-release drug in culture medium and cause cell death very late Some authors publish the liposomes carrying doxorubicin have IC50 2.77 µg/ml on HT 29 cell after 48 hours of exposure time [13] Our preliminary investigation showed that HT 29 cell lines responds well tothe PEGylated liposomal doxorubicin, is a prerequisite for the latter in-vivo testing CONCLUSION In conclusion, the long-circulating PEGylated liposomal doxorubicin by hydration of a thin lipid film technique was prepared successfully The PEGylated liposomes were showed stable at 2-8oC for at least months We have assessed the cytotoxicity effect of PEGylated liposomal doxorubicinthrough determining IC50 of in vitro on A549 and HT29 cell lines Our data have showncytotoxicity effect depended on exposure time and PEGylated liposomal doxorubicin has cytotoxicity effect stronger on A549 cells than HT29 cells Future work will be needed to evaluate the antitumor efficacy of our doxoburicin liposome formulation in vivo in different conditions Acknowledgements The research was supported by Vietnam national project KC10.14/11-15.Also we would like to thank VNU-VSL (Vietnam National University, HaNoi-VNU-Scientist Links) for support to submit the manuscript REFERENCES [1] O Tacar, P Sriamornsak, and C R Dass, J Pharm Pharmacol, 2013, 65 (2), 157-70 [2] L Harris, G Batist, R Belt, D Rovira, R Navari, N Azarnia, L Welles, E Winer, and T D S Group, Cancer, 2002, 94 (1),25-36 [3] P Sapra, and T M Allen, Prog Lipid Res, 2003,42 (5), 439-62 [4] J M Harris, and R B Chess, Nat Rev Drug Discov, 2003, (3), 214-21 [5] A Gabizon, and F Martin, Drugs, 1997, 54 (4), 15-21 [6] K A Edwards, and A J Baeumner,Talanta, 2006, 68 (5), 1432-41 [7] J N Moreira, R Gaspar, and T M Allen, Biochim Biophys Acta, 2001,v1515 (2), 167-76 [8] J O Eloy, M Claro de Souza, R Petrilli, J P A Barcellos, R J Lee, and J M Marchetti, Colloids and Surfaces B: Biointerfaces, 2014, 123 (0), 345-363 [9] J A Kamps, Methods Mol Biol, 2010, 606,199-207 [10] M C Woodle, Chem Phys Lipids, 1993, 64 (1-3),249-62 [11] L Cheng, F Z Huang, L F Cheng, Y Q Zhu, Q Hu, L Li, L Wei, and D W Chen, Int J Nanomedicine, 2014,9, 921-35 [12] W L Ye, J B Du, B L Zhang, R Na, Y F Song, Q B Mei, M G Zhao, and S Y Zhou, PLoS One, 2014, (5), e97358 [13] J Lin, Y Yu, S Shigdar, D Z Fang, J R Du, M Q Wei, A Danks, K Liu, and W Duan, PLoS One, 2012, (11), e49277 2243 ... cytotoxicity effect of PEGylated liposomal doxorubicinthrough determining IC50 of in vitro on A549 and HT29 cell lines Our data have showncytotoxicity effect depended on exposure time and PEGylated liposomal. .. cytotoxicity of PEGylated liposomal doxorubicin against human lung non-small cancer cell line (A549 cells) and HT 29 colon cancer cell line was determined by using the MTTdye reduction assay Briefly,... preliminary investigation showed that HT 29 cell lines responds well tothe PEGylated liposomal doxorubicin, is a prerequisite for the latter in- vivo testing CONCLUSION In conclusion, the long-circulating