Indian Res. J. Genet. & Biotech. 6(3) : 505-510 (2014)
Nanoparticles encapsulated drug enhanced in vitro anticancer activity by induction of apoptosis. Sourabh Tiwari1 and Ravi Upadhyay2 1-2
Govt. P.G. College, Pipariya, Bhopal Memorial Hospital & Research Centre, Bhopal
(Received : May, 2014 : Revised : June, 2014; Accepted : July 2014) Abstract Colorectal cancer is a major cause of mortality worldwide. Chemotherapy represents a powerful therapeutic keystone in the treatment of colorectal cancer. More or less all the anticancer drugs have severe side effects on normal tissues and organs. The undesired toxicity of currently available anticancer drugs and the inefficiency of chemotherapeutic treatments have limited the optimization of drug regimens and effective chemotherapeutic procedures. Currently the field of nanomedicine allows the release of anticancer drugs by biodegradation and self-regulation of nano materials in vitro and in vivo. For that reason, a need is felt to encapsulate these drugs into better drug delivery carriers such as SLN to minimize systemic side effects. In the current study SLN was prepared by solvent injection method and built-in with 5-Fluorouracil, Irinotecan and Ca-Leucovorin. Anticarcinogenic potential of the nano-engineered formulations were investigated using cultured HT29 cells. Evaluation of anti-carcinogenic potential by Annexin-V-FITC/PI apoptosis assay and mitochondrial depolarization following dose dependendent and time course experiments treatment with SLN and native drugs described noteworthy differences, creating better prospective efficacy of nano-engineered drugs. The result indicates that the SLN is a promising controlled release carrier. A nanoparticle has the potential to overcome current chemotherapeutic obstructions in colorectal cancer treatment, because of the unique nano scale size and properties. Key words : Colorectal cancer, chemotherapy, nanomedicine, nanoparticles, apoptosis. Inroduction Colorectal cancer (CRC) is second most common malignant tumors worldwide. Most CRCs arise from sporadic adenomas, and a small number of from genetic polyposis syndromes or inflammatory bowel disease (IBD). More than 95% of colorectal cancers are adenocarcinomas, which develop from glandular tissue. The tumor arises from an adenomatous polyp. About 25% of patients with colorectal cancer present with overt metastatic disease, and metastatic disease
develops in 40 to 50% of newly diagnosed patients. Among all cases of CRC, the majorities (~ 75%) are sporadic in origin and the remainders are related to inflammatory bowel diseases or family history [Hisamuddin and Yang, 2004]. Despite the fact that a majority of patients have the entire tumor removed by surgery, as many as 50 to 60% will develop a recurrence without further treatment. Chemotherapy is a cancer treatment that uses anticancer drugs to stop the growth of cancer cells, either by destroying the cells or by stopping them from multiplying. Chemotherapy is given to reduce this chance of recurrence. Standard first-line
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therapy for patients with advanced disease includes a combination of 5-fluorouracil, leucovorin and irinotecan [Douillard et al., 2000; Saltz et al., 2000]. Regimens adding irinotecan to 5-fluorouracil and leucovorin were found to be more effective than using the fluorouracil and leucovorin alone in the patients. The mechanism of 5-fluorouracil (5-FU) is known to be due to its metabolic conversion to 5-fluorouridine59-triphosphate with subsequent incorporation into RNA, and/or the formation of 5-fluoro-29-deoxyuridine 59-monophosphate, a well-known inhibitor of thymidylate synthetase. Irinotecan is a potent inhibitor of topoisomerase I, an enzyme necessary for DNA replication and leucovorin a bio modulating agent that helps in increasing the inhibition of DNA synthesis and enhancing the antitumor effect of 5-FU [Kulig et al., 2007]. Thus majority of anticancer drugs available may work through a mechanism causing adequate damage to activate so called programmed cell death or apoptosis [Hickman, 1992]. The efficiency of conventional anticancer drugs is hampered by the following limitations: i) drug resistance at the tumour level due to physiological obstacles ii) drug resistance at the cellular level and iii) non-specific distribution, biotransformation and rapid clearance of anticancer drugs in the body [Couvreur and Vauthier, 2006]. Another important obstacle to efficient anticancer chemotherapeutic treatment is the toxicity of drug by attacking normal proliferating cells such as hair follicles, gastrointestinal tract, bone marrow and reproductive system. This leads to only a small fraction of drug dosage reaches cancer tissues. To conquer these limits, the “magic bullet” theory, which refers to a drug which goes straight to its specific target, was proposed at the beginning of the 20th century [Strebhardt and Ullrich, 2008]. In the past few decades the purpose of this model has led to the progress of an excess of colloidal systems meant at deliver the drug entirely to the diseased tissues, thus reducing systemic toxicity. Nanotechnology has the ability to overcome current chemotherapeutic drug in cancer treatment
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because of their nanoscale size ranging in size from 10-100nm in diameter. Since their growth, a number of different particle types have come out with distinct properties, all the while retaining the qualities that make them best carrier systems. Nanoparticles are characterized by successful drug encapsulation, controllable self-assembly, specificity, excellent stability and biocompatibility as a result of their own material properties [Grodzinski et al., 2006]. The anticancer agent incorporated within a core, where the drug is protected from degradation. The small size of nanoparticles helps in their avoidance of the RES, liver and spleen filtration and thus, results in longer circulation times [Pearce et al., 2012]. Poly ethylene glycol (PEG) is a hydrophilic polymer that helps in modifying the surface of nanoparticles to protect it from plasma clearance. Through enhanced permeability rate (EPR) and poor lymphatic drainage mechanism nanoparticles change the pharmacodynamic properties of that encapsulated drug, reducing systemic toxicity to healthy tissue and increasing accumulation within the tumor. This results in reducing toxicity in patients and potentially heightened antitumor efficacies. Solid lipid nanoparticles (SLNs) are new generation nanoconstructs where the liquid lipid has been replaced with solid lipid [Jabir et al., 2012]. SLNs were first described by Muller et al in 1995. SLNs are physiological lipid based nanoparticles systems that offer unique properties such as small size, biodegradable, easy preparation, higher permeability through blood brain barrier (BBB), good stability, controlled drug release activity enable drug protection and high drug loading capacity [Uner and Yener, 2007]. Recently several studies reported the use of SLN as drug delivery carriers in cancer treatment [Mulik, 2010; Kang et al., 2010]. The aim of the present study was to evaluate the apoptotic potential of native versus nanoparticle loaded neoplastic drugs on colorectal cancer cell line. The experiments were performed on the cultured human colorectal cancer cell line HT-29 using native and solid lipid nanoparticle loaded drugs. Evaluation
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Santosh Sourabh Tiwari Kumar & RaviSingh Upadhyay
of apoptosis through Annexin-V assay and mitochondrial depolarization were carried out to determine the apoptotic index. Materials and methods Reagents The culture petri-dishes were procured from BD Falcon (Rockville, MD, USA). Fetal calf serum was obtained from HyClone Labs (Logan, Utah, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) growth medium was procured from Gibco/BRL Life Technologies, Inc. (NY, USA). Fetal calf serum was obtained from HyClone Labs, Logan, Utah, USA. Antibiotic antimycotic solution was obtained from HiMedia Labs Pvt. Ltd (Mumbai, India). The cell growth supplements sodium pyruvate, nonessential amino acids and sodium bicarbonate were obtained from MP Biomedicals, Solon, USA. SLN encapsulated drug sample was kindly gifted by Mahakal Institute of Pharmacy, Ujjain. To quantify apoptosis, the Annexin-V-FITC/PI assay kit from Roche Applied Sciences, Mannheim, Germany was used. Mitochondrial depolarization was studied by Mitochondrial Membrane Potential Detection kit from BDTM Biosciences, San Diego, CA. Cell line and culture conditions The human colorectal adenocarcinoma cell line, HT29, was obtained from the National Centre for Cell Science (NCCS), Pune, India. The cells were seeded at 2 x 105 cells/60 mm culture dishes in DMEM supplemented with 10% fetal calf serum plus 1.5 g/ L sodium bicarbonate. Cultures were incubated the humidified atmosphere of 5% CO2 at 37°C according to NCCS catalogue instructions. After optimum confluency, the cells were treated with the experimental agent, free drugs and encapsulated drugs and harvested with trypsin-EDTA for use in the following experiments. Study design Studies were conducted in two sections: dose dependent and time-course kinetics (n=3). Dose dependent response of native and nano-engineered
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drugs on colorectal cell line was carried out with concentration ranging from 0.1 to 100 ìg, whereas time course experiments were done with constant concentration of 5 ìg at time intervals ranging from 3h to 24 h. Assessment of apoptotic index Measurement of apoptotic index of cultured cells was performed using Annexin-V-FITC/PI assay kit as per manufacturer’s recommendations. From each cell, forward light scatter (FSC), orthogonal light scatter (SSC), and Annexin-V-FITC and PI fluorescence were measured using Cell-Quest Software (BD-IS, USA). The gate was applied in the FSC/SSC dot plot to restrict the analysis to cultured cells only. For the gated cells, the percentages of annexin-V-FITC positive or negative or PI positive or negative cells were evaluated. In each case total 10,000 events were recorded in HI mode with 10/10 log quadrant gate. Mitochondrial Membrane Potential Detection Assay Mitochondrial membrane potential of the cells was detected by staining cells with JC-1 (5, 5’, 6, 6’-tetrachloro1,1’,3,3’-tetraethylbenzimidazolcarbocyanine iodide). The assay was carried out as per supplier’s instructions. The gate was applied in the FSC/SSC dot plot to restrict the analysis to lymphocytes only. For the gated cells, the ratio of FL1/FL2 was evaluated. In each case, a total of 10,000 events were recorded in HI mode. Results and Discussion Annexin-V-FITC/PI Assay Annexin V is a Ca2+-dependent phospholipid binding protein with high affinity for phosphatidyl serine (PS). This protein is used as a sensitive probe for PS exposure upon the outer leaflet of the cell membrane and suitable for detection of apoptotic cells. Number of apoptotic cells was incremental with increased in SLN engineered drugs time period. At 5ìg concentration, in the time-course study, a peak percentage of cells undergoing apoptosis (86.8%) were observed following 24 h after treatment (Figure 1).
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Figure 1. Apoptotic index of SLN loaded FOLFIRI drug and native drug in HT-29 cells. Time course study of Annexin V binding in HT-29 cells treated with 5 µg concentration of drugs. Mitochondrial Membrane Potential Detection Assay Membrane-permeable lipophilic cationic fluorochrome JC-1 (5, 5’, 6, 6’-tetrachloro-1, 1’, 3, 3’tetraethylb enzimidazolcarbocyanine iodide) is used as a probe of transmembrane potential (“ø).
JC-1 penetrates into cells and its fluorescence is a reflection of “ø. Maximum cells with depolarized mitochondria (loss of “ø) was found at 100 ìg concentration, that is, 78.8% (Figure 2).
Figure 2. Exposure of Native and SLN loaded FOLFIRI drug induces apoptosis in HT-29 cells. Percentage of cells showing mitochondrial depolarization following treatment with drugs at concentrations ranging from 0.1 to 100 ìg in cells after 6 h incubation period.
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Santosh Sourabh TiwariKumar & RaviSingh Upadhyay
In our study we tried to determine the apoptotic potential of the neoplastic drugs. In recent years, the combination chemotherapy drug regimens that include 5-fluorouracil, Ca-leucovorin and irinotecan (FOLFIRI) have been recommended by clinical trials as first line treatment for advanced colorectal cancer [Bae et al., 2006]. Our results are similar to those reported in literature that is 5-FU inhibits cell viability and its mechanism is inhibiting DNA synthesis through this action in cancer cells. 5-FU down regulates Bcl-2 family and induces caspase family [Yim et al., 2004]. These three core drugs are widely used as first line therapy in colorectal cancer.
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Acknowledgement The authors are grateful to Bhopal Memorial Hospital Trust, India for providing financial support. References 1. Bae S.B., Lee N.S., Kim H.J., Kim K.H., Kim H.J., Kim C.K., Lee K.T., Park S.K., Won J.H., Hong D.S. and Park H.S. (2006) A Phase II Study of Irinotecan, 5-Fluorouracil and Leucovorin for treatment in patients with previously untreated advanced colorectal cancer. Cancer Res Treat., 38(2): 72-77. 2.
Couvreur P. and Vauthier C. (2006)
Treatment of cancer using conventional formulations of FOLFIRI is found to have many
Nanotechnology: intelligent design to treat
drawbacks such as adverse side effects because conventional cytotoxic anticancer drugs do not distinguish between tumour and normal cells, and research efforts have been fixed on finding new agents that target tumour tissue. The incorporation
3. Douillard J.Y., Cunningham D. and Roth
of FOLFIRI into solid lipid nanoparticle may be potential new approach in colorectal cancer treatment. Results obtained reflect that among native and SLN engineered drugs, the SLN showed highest apoptotic potential and cytotoxicity at lower doses. The developed regimen of SLN engineered FOLFIRI overcome and improves the drawbacks and limitations of conventional drug. Conclusion The ability of anti cancer drug to bind specifically to tumor site is very poor and this consider as a major obstacle to effective anticancer treatment. A solid lipid nanoparticle suggests specific advantages of easy and rapid preparation method which is simple to scale up. This provides the basis for an efficient targeted delivery system design for 5-fluorouracil, leucovorin and irinotecan in SLN based system for colorectal cancer with possible reduction of side effects of native drugs. Nanotechnology is gaining importance in cancer as a useful approach to design drug carrier for combining multiple anti cancer drug regimens.
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