Developing and evaluating in vitro effect of pegylated liposomal doxorubicin on human cancer cells

The stability experiments results PEGylated liposomal doxorubicin on A549 and HT29 cell lines was effected when the exposure time is over 48h.The was observed after 72 hours for both c

Journal of Chemical and Pharmaceutical Research, 2015, 7(3):2239-2243

Research Article CODEN(USA) : JCPRC5 ISSN : 0975-7384

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*

National University, Hanoi, 144 Xuan Thuy, Cau Giay, Ha Noi, Vietnam

Vietnam

University, Hanoi, 144 Xuan Thuy, Cau Giay, Ha Noi, Vietnam

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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

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

PEGylated liposomal doxorubicin on A549 and HT29 cell lines was effected when the exposure time is over 48h.The

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

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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

drug-delivery 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,

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

1 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-2H-tetrazolium 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® 4 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)

2 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 và 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 ± 2 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 1 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 8 - 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   =mo − m

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 3 months under a sealed condition The mean particle size, polydispersity index, zeta potential and drug entrapment efficiency were determined after 1 week, 1 month, 2 month and 3 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 4 h After 4 hours, the content of each cell was removed and the converted dye was solubilized in 200 µL of DMSO Plates were shaken for20 min 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 <0.05)

RESULTS AND DISCUSSION Characterization of PEGylated liposomal doxorubicin

We have analyzed some physical properties of the obtained PEGylated liposomal doxorubicin including mean particle size, polydispersity index (PDI), zeta potential, and preliminary stability during 3 months The results were shown in Table 1 and Figure 1

Table 1 Physical characters of PEGylated liposomal doxorubicin

0.055±0.012 167.85± 3.68 0.957± 0.023 -27.5±3.57 0.0408

Figure 1: Graph of size particles of PEGylated liposomal doxorubicin

One important parameter is the polydispersity index, which is a measure of the width of the particle size distribution Values of polydispersity index arein the range from 0 to 1 Polydispersity index less than 0.1 are typically referred to

as "monodisperse" The liposome preparations are required to have a particle-size mean smaller than 200 nm and PDI less than 0,2 Liposomes with small particle size (< 200 nm) can increase the accumulation of drug in the tumor

by augmented permeability and retention effect [7]

As we observed in Table 1, the liposome preparations are quite homogeneous dispersions that have particle-size mean are small in the range from150-170 nm and polydispersity index less than 0.1 Compared to PEGylated liposomal without DOX there is not significant differences The entrapment efficiency of drug in the liposomes is over than 95%

Study on the stability of liposome suspension finds out that liposomes tend to fuse with one another to form larger particles, which have less the surface energy Consequently, the liposome size distribution becomes polydisperse Several factors influencethe stability of liposome suspension that includes mean particle size, surface of particles, surface charge and the environment around particle such as viscosity, electrolyte, etc One index used to evaluate the stability of liposome is zeta potential value The zeta potential is the overall charge a particle acquires in a particular medium The destination of the liposomes in vivo could be predicted by using the information of the zeta potential

of a liposome preparation The high absolute value of zeta potential indicates high electric charge on the surface of the drug-loaded liposomes, which repel strong forces between particles to prevent aggregation of the liposomes in solution Zeta potential of liposomes has values negative because of the presence of terminal carboxylic groups in the lipids [8] Many studies show that if the particles possessthe absolute valueof zeta potential greater than 30 mV, then the suspension is highly stable and capable to prevent the aggregation of particles If zeta potential values are in the range of 20-30 mV, the suspension is relatively stable And in case of zeta potential values are below than 10

mV, the suspension has poor stability The PEGylated liposomal doxorubicin have zeta potential values -27.5 ±3.57

mV (Table 1) that means they are considered relatively stable The data of the stability study of PEGylated liposomal doxorubicinwere showed in table 2

Table 2: Stability of PEGylated liposomal doxorubicin during study

Time Zeta PDI Z average (d.nm) Concentration of doxorubicin(mg/ml) EE (%)

Recent preparation -24.2± 1.44 0.065±0.027 166.26± 2.52 2.04±0.058 >95

As we observed in Table 2, the PEGylated liposomal doxorubicin stored at 2-8oC during 3 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 IC 50

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) After 24h Cell viability (%) After 48h After 72h

1 92.45 ± 18.04 53.4 ± 11.06 29.47 ± 7.47

10 81.62 ± 6.25 49.15± 3.33 29.09 ± 8.73

100 74.93 ± 13.02 25.04± 12.38 13.96 ± 5.07

According to the MTT assay, samples with IC50<20µg/ml is considered active.Thus, after 48 hours, the PEGylated liposomal doxorubicin begin to exert cytotoxic effects and the effect is strongest after 72 hours Recently, Cheng et

al also reported that IC50 of PEG-liposome containing doxorubicin in-vitro on cell line A549 was 3.35 µg/ml at 48 hours [11]

With longer exposure time, the cytotoxicity effect was increasing IC50 at 72 hours (0.7 µg/ml) was smaller, almost

15 times more than IC50 at 48 hours (9.97 µg/ml) These data demonstrated that PEGylated liposomal doxorubicin is capable of releasing prolonged and increasing circulation time in blood In previous reports, Ye et al showed the cytotoxicity of different liposome products containing doxorubicin ranged from IC50 2.7 to 4.9 µmol/Lin A549 cells with 48 hours incubation [12] As compared to those authors, our PEGylated liposomal doxorubicin with IC50 9.97 µg/ml (equivalence to 17.19 µmol/L) in A549 cells with 48 hours incubation was less cytotoxicity In this preliminary study our data showed that PEGylated liposomal doxorubicin has cytotoxicity effective with A549 cells The mechanism involved in this cytotoxicity needs to be studied in depth in in-vivo experiments

Moreover, we studied the cytotoxicity effects of the PEGylated liposomal doxorubicin through inhibition of protein synthesis on HT-29 colon adenocarcinoma The results were shown in Table 4

Table 4: Cytotoxicity effect on the cell line HT 29

Concentration (µg/ml) After 24h Cell viability (%) After 48h After 72h

1 103.9 ± 14.69 88.52± 11.03 41.52± 3.44

10 75.91± 10.94 61.17± 4.48 39.85± 5.97

100 71.13± 11.48 49.7± 6.04 19.75± 2.69

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 3 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

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