Chitosan Nanolayered Cisplatin-Loaded Lipid Nanoparticles for Enhanced Anticancer Efficacy in Cervical Cancer
© The Author(s). 2016
Received: 18 July 2016
Accepted: 21 October 2016
Published: 25 November 2016
In this study, cisplatin (CDDP)-loaded chitosan-coated solid lipid nanoparticles (SLN) was successfully formulated to treat HeLa cervical carcinoma. The formulation nanoparticles were nanosized and exhibited a controlled release of drug in physiological conditions. The blank nanoparticles exhibited an excellent biocompatibility profile indicating its suitability for cancer targeting. The incorporation of CDDP in SLN remarkably increased the cancer cell death as evident from the MTT assay. Importantly, CDDP-loaded chitosan-coated SLN (CChSLN) significantly (P < 0.05) decreased the viability of cancer cells even at low concentration. The higher cytotoxicity potential of CChSLN was attributed to the higher cellular uptake as well as the sustained drug release manner in comparison with CSLN. Consistent with the cytotoxicity assay, CChSLN showed the lowest IC50 value of 0.6125 μg/ml while CSLN presented 1.156 μg/ml. CChSLN showed a significantly higher apoptosis in cancer cells compared to that of CSLN and CDDP, which is attributed to the better internalization of nanocarriers and controlled release of anticancer drugs in the intracellular environment. Our findings suggest that this new formulation could be a promising alternative for the treatment of cervical cancers. These findings are encouraging us to continue our research, with a more extended investigation of cellular response in real time and in animal models.
KeywordsCervical cancers Cisplatin Antitumor efficacy Solid lipid nanoparticles Chitosan
Human cervical cancer is one of the popular cancers in women in reproductive age across the globe . The cervical cancer is caused by human papillomavirus (HPV). International Agency for Research on Cancer has stated the main factors responsible for the cervical cancers including viral infection, inordinate sexual behavior, depleted immune system, and smoking . Recent advances in cancer diagnosis and use of vaccine have reduced the mortality rate of cervical cancer patients; nevertheless, cancer-related death in China is still high. Chemotherapy, surgery, and radiotherapy are some of the treatment options in cervical cancers; however, none is effective in controlling the cervical cancers . Recently, two prophylactic HPV vaccines (Gardasil and Cervarix) were marketed to tackle HPV-associated cervical cancers; however, it was effective only in adult patients and failed to show any therapeutic effect against present cases. Especially, in the clinical settings, chemotherapeutic drugs play an important role in killing the cervical cancer cells [4, 5].
In this perspective, cis-dichlorodiammineplatinum(II) (cis-[PtCl2(NH3)2], cisplatin (CDDP)) is a potent anticancer agent for the treatment of various solid cancers including cervical cancers . CDDP kills the cancer cells by crosslinking the DNA which in turn results in the cellular apoptosis as the repair become unsuccessful. The potent anticancer effect of CDDP is hindered by its severe adverse effects such as nephrological and neurological toxicities . CDDP is normally associated with nephrotoxicity that results in acute and chronic morbidity, while neurotoxicity is cumulative-dose dependent. More than everything, one of the biggest worries is the immediate inactivation of CDDP in the systemic circulation that will reduce its therapeutic potency and results in unwanted side effects [8, 9]. Therefore, efforts have to be made to protect the pharmacological action of CDDP in systemic circulation and prolong its systemic circulation. Furthermore, drug has to be released in a sustained or controlled manner that will improve its anticancer effect and reduce its associated side effects.
The applications of principles of nanotechnology in cancer treatment are expected to solve all the existing problems. The drug loaded in a nanocarrier could effectively protect the exposure of chemotherapeutic drug to the extracellular environment . Moreover, physiological conditions of the diseased cancerous tissue offer many benefits to the delivery system. The physiological changes in the tumor tissue could be effectively used to passively target the drug in the tumor. The poor lymphatic drainage and increased vascular permeability in cancer cells allows the drug-loaded nanoparticle to accumulate passively in the tumor via enhanced permeation and retention (EPR) effect. Additionally, nanoparticle will offer multiple advantages such as increased stability, high loading capacity, sustained release of drugs, and minimize the drug-related side effects [11, 12].
In this work, we primarily aimed to prepare chitosan-coated SLN to effectively deliver CDDP in cervical cancers. Towards this aim, drug-loaded nanoparticle was characterized in terms of particle size, zeta potential, morphology, and release characteristics. The anticancer effect of free CDDP and CDDP-loaded SLN was investigated in HeLa cervical cancer cells.
Compritol 888 ATO and monooleate of sorbitan ethoxylated (Super refined Polysorbate 80TM; Tween 80TM) were kindly provided by Gattefossé (Saint Priest, France). Cisplatin and chitosan was purchased from Sigma-Aldrich, China. All other chemicals were of analytical grade and used as such.
Preparation of Cisplatin-Loaded Chitosan-Coated Solid Lipid Nanoparticles
The SLN was formulated by hot homogenization followed by emulsification-ultrasound method. Briefly, 100 mg of Compritol, 10 mg of lecithin, and 10 mg of CDDP were melted above 60 °C and constitutes the oil phase. The aqueous phase consists of Tween 80 (2%), and the temperature was maintained similar to that of oil phase. While maintaining the same temperature, surfactant-containing phase was added slowly to the oil phase under constant agitation. The mixture was immediately homogenized using Ultra Turrax T-25 homogenizer for 5 min. The homogenized solution was immediately subjected to high intensity probe sonication (Vibracell VCX130; Sonics, USA) for 5 min. The emulsion was immediately cooled to 4 °C to allow the SLN formation. For chitosan coating, 0.1% chitosan solution was prepared in 0.1% acetic acid. The SLN and chitosan solution was mixed and stirred for 2 h at 100 rpm. The chitosan-coated SLN was separated and lyophilized (if necessary) and stored.
Dynamic Light Scattering Analysis
The size distribution was analyzed by dynamic light scattering technique using BI-200SM, Brookhaven Instruments Corp., Holtsville, NY, USA, equipped with a 35-mM HeNe laser beam at a wavelength of 637 nm. The zeta potential of nanoparticle was analyzed using laser Doppler velocimeter (Zetasizer Nano ZS90, Malvern, UK).
Drug Encapsulation Efficiency and Drug Loading
The CDDP-entrapped nanocarriers were centrifuged at high speed (15,000 rpm), and the supernatant was collected to evaluate the amount of unloaded drugs. HPLC method was used to quantify the drug loading and entrapment efficiency. HPLC (Agilent LC 1100, Santa Clara, CA, USA) was used. The column consists of Inertsils ODS-3 column (150 4.6 μm, pore size 5 μm, GL Science Inc., Tokyo, Japan) was used.
In Vitro Drug Release
Dialysis method was used to perform the release study. Briefly, 1 ml of drug-loaded nanoparticles was packed in a dialysis membrane (Spectra/Por 6, MWCO 3000, Spectrum Laboratories, Inc., TX, USA) and both the ends were sealed. The dialysis membrane was sealed and kept in 20 ml of release buffer and in turn placed in a shaker bath at 100 rpm. At specific time point, 1 ml of release buffer was taken out and replenished with same amount of new medium. The concentration of drug in the release medium was quantified by HPLC method. The mobile phase used was methanol/water/acetonitrile (40:30:30 v/v/v) at 1 ml/min.
DMEM medium was used to grow HeLa cell which is supplemented with 10% of FBS and 1% of antibiotic mixture in an incubator at 37 °C. The cytotoxicity assay was performed by MTT protocol. Briefly, 15,000 cells/well was seeded at each well of 96-well plate and let it aside for 24 h. The old media was removed and wells containing adherent cells were washed with PBS. The cells were incubated with SLNs (0.01–100 μg/mL) for 24 h. Also, free CDDP and CDDP-loaded formulations were exposed to cancer cells and incubated for 24 h. Following 24-h incubation, the old media containing free SLN and drug-loaded formulations were carefully aspirated and the cells were washed twice with PBS. This process was carried out in order to minimize the chance of carrier or drug that is not internalized by the cells and avoid any interference of the materials on the final absorbance. The cells were then added with 10 μl of MTT (5 mg/ml) and incubated for 4 h. Then MTT solution was removed or aspirated carefully, and cells were added with 150 μl of DMSO to solubilize the formazan crystals. The well plates were then kept aside for 15 min, and absorbance was studied at 570 nm using a POLAR star microplate reader (Omega, BMG LabTech). The IC50 value was calculated from using GraphPad prism software.
The apoptosis assay was performed by Annexin V/PI staining. In brief, cells (2 × 105) were seeded in 12-well plate and left aside for 24 h. The cells were treated with respective formulations and left untouched for 24 h. The cells were washed, isolated, and treated with Annexin V and PI dye for 15 min in dark conditions. The cells were then resuspended in PBS and studied using flow cytometer (BD Biosciences, USA).
Analyses for the cytotoxicity studies were performed using one-way ANOVA followed by Tukey’s test. P value <0.05 was considered statistically significant for all analyses.
Results and Discussion
Physicochemical Characterization of CChSLN
In Vitro Drug Release Kinetics
Cellular Uptake Analysis
Biocompatibility of Blank Nanoparticles
Cytotoxicity Assay of Free CDDP and CChSLN
In conclusion, CDDP-loaded chitosan-coated SLN was successfully formulated to treat HeLa cervical carcinoma. The formulation nanoparticles were nanosized and exhibited a controlled diffusion of drug in neutral conditions. The blank nanoparticles exhibited an excellent biocompatibility profile indicating its suitability for cancer targeting. The incorporation of CDDP in SLN remarkably increased the cancer cell death as evident from the MTT assay. Importantly, CChSLN significantly decreased the viability of cancer cells even at low concentration. The higher cytotoxicity potential of CChSLN might be due to that enhanced cellular internalization and controlled release of encapsulated component in comparison with CSLN. Consistent with the cytotoxicity assay, CChSLN showed the lowest IC50 value of 0.6125 μg/ml while CSLN presented 1.156 μg/ml. CChSLN showed a significantly higher apoptosis in cancer cells compared to that of CSLN and CDDP, which is attributed to the better internalization of nanocarriers and controlled release of anticancer drugs in the intracellular environment. Our results clearly reveal that these unique formulations could be a better choice for cervical cancer treatment. The investigative findings encourage us to carry out in-depth biological and in vivo analysis of the present system.
The study was supported from the funding grant of the Second Affiliated Hospital of Chengdu Medical College, China.
YW and XM were responsible for all the experiments. XM designed and written part of the manuscripts. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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