Dual drug loaded liposome bearing apigenin and 5-Fluorouracil for synergistic therapeutic efficacy in colorectal cancer

https://doi.org/10.1016/j.colsurfb.2019.04.035Get rights and content

Highlights

  • Dual drug loaded liposomal nanocarrier bearing apigenin and 5-Fluorouracil designed and characterized.

  • Effect of dual drug loaded liposomes on human colorectal cancer cell lines HCT-15 and HT-29 investigated.

  • In vitro testing revealed apoptotic potential while in vivo tumor regression observed.

  • Dual drug loaded liposomal formulation can be a potential chemotherapeutic strategy.

Abstract

Multidrug-based combinatorial therapeutic regiments which target multiple pathways simultaneously are being utilized as a therapeutic strategy of choice due to reduction in toxicity profile and enhancement of therapeutic index of the individual drugs. 5-Fluorouracil is a clinically approved drug which has limited response rate in the realm of colorectal cancer amelioration, hence our study aims to improve its efficacy by aiming the simultaneous delivery of 5-Flurouracil and apigenin which is naturally occurring flavone abundantly present in fruit and vegetables through a single liposome to combat and control colorectal cancer effectively in-vitro and in-vivo. The liposomal nanocarrier bearing the anti-tumorigenic agent apigenin was designed in this study in order to improve the bioavailability of the flavone while at the same time achieve combinatorial drug regime with 5- Fluorouracil. This study reports the synthesis and production of a relatively robust dual drug-loaded liposomal formulation by modified thin film hydration method which substantially entraps both the drugs. Even though there have been reports of the combinatorial regimen involving apigenin and 5-Flurouracil our study reports the optimal molar ratio for effective synergistic therapeutic application of this drug combination to alleviate colorectal cancer. The cytotoxicity and cellular effects of individual, combinatorial free drugs and their liposomal counterparts tested against two human colon cancer cell lines revealed significantly higher cytotoxicity of the dual-drug liposomes. The dual-drug liposomes demonstrated enhanced inhibition of angiogenesis, better reduction in cell proliferation and increased apoptotic potential. Cell signaling studies indicating a significant upregulation of pAMPK and activity against downstream targets by dual drug liposomes suggested its role in the reversal of Warburg effect. The formulation was tested in a preclinical setting in nude mice tumor xenograft model and was found to have greater anti-neoplastic and anti-tumorigenic effect. The study indicated that the increased chemotherapeutic potential in vivo was due to the passive targeting achieved by the liposomal drug loaded nano-carrier and the synergistic effect of apigenin in 5-Fluorouracil treatment offers a new attractive alternative to enhance the therapeutic potency of drugs and paves way for potential clinical applications.

Introduction

Recent advances in nanotechnology focused at disease alleviation have revolutionized cancer therapeutics. The development and progress in the field of cancer nano-therapeutics has led to the emergence of a vast array of biocompatible nanoscale therapeutic vectors. At the systemic level nanoparticle drug delivery exhibit numerous advantages which include longer circulation time, enhanced pharmacokinetics and reduced side effects [1]. Liposomes constitute a major and integral part of the extensive assortment of clinically feasible nano-carriers which are showing promising preclinical results. Entrapment of hydrophilic and hydrophobic drugs is possible in a single vesicle since it possesses a hydrophilic core and hydrophobic bilayer [2,3]. Liposomes are currently becoming a therapeutic strategy of choice due to their ability to co-encapsulate multiple therapeutic agents leading to simultaneous delivery of the drugs culminating in enhanced therapeutic effects [[4], [5], [6]].

Colorectal cancer includes cancers of the colon and/or rectum and is the third most commonly diagnosed type of cancer worldwide in both women and men alike. Screening efforts or presentation of clinical symptoms by individuals lead to colorectal cancer (CRC) diagnosis [7]. Drug discovery endeavors have yielded a wide array of chemo-therapeutics like oxaliplatin, 5-Fluorouracil, irinotecan, capecitabine, vascular endothelial growth factor and epidermal growth factor receptor inhibitors which effectively combat CRC.

5-Fluorouracil (5-FU) is extensively used in treating numerous forms of cancers which includes breast and colorectal cancers. The choice of 5-FU as the first line treatment in advanced colorectal cancer yields response rates of only 10–15% which limits its use [8]. Combinatorial treatment strategies involving 5-FU and other therapeutic agents has found to improve survival as well as response in head and neck and breast cancers, however the highest therapeutics effect has been exerted in colorectal cancer only [8].

Dietary flavonoids have anti-proliferative effect and play important role in cancer chemoprevention, especially cancers of the gastrointestinal tract, due to their direct association with food (Kuo, 1996). The chemopreventive effects of bioactive phytochemicals in vitro have been reported in a wide range of colon cancer cell lines which includes LoVo, SW480, CaCo2, HCT-116 and HT-29. The source of these phytochemicals with attractive therapeutics properties with respect to colorectal cancer are varied and include black raspberry, strawberry, apple, grape seeds and garlic [9].

Recent combinatorial endeavors involving liposomal formulations to combat advanced colorectal cancer is reflective from the clinical trials of CPX-1 (Celator Pharmaceuticals, New Jersey, USA) which contains a fixed combination of irinotecan and floxuridine. In this light the focus of the present study was to develop a viable therapeutic carrier of choice with the ability to load and co-deliver effectively a myriad of drugs to combat CRC. Moreover, taking into consideration the limitations of 5-FU as the first line of treatment, a naturally occurring flavone therapeutic molecule apigenin, abundantly present in fruit and vegetables was chosen for co-delivery. The dual drug loaded liposomal nanocarrier bearing the anti-tumorigenic agent apigenin circumvents the impediment posed by the high hydrophobicity of the flavone and provided scope for improved bioavailability.

The co-administration of two or more drugs exerts its maximal efficacy by targeting different molecular pathways, thereby increasing tumor cell killing while reducing the likelihood of drug resistance frequently culminating in enhanced durability of responses in combinatorial drug treated patients. Our study reports simultaneous delivery of 5-FU and apigenin through a dual drug loaded liposome to combat and control colorectal cancer effectively in-vitro and in-vivo. Even though the combinatorial drug regimens involving 5- Fluorouracil and apigenin have been reported before in other cancers like hepatocellular carcinoma and ascitic tumor model [10,11], this manuscript indicates the effective ratio of the drugs required to achieve maximal therapeutic effect. The combination index results revealed that the treatment strategy involving co-delivery of apigenin and 5-FU is feasible and has an overall synergistic therapeutic effect. The molar ratio for effective synergistic therapeutic application of apigenin and 5-Fluorouracil has been reported in this study. In the present study we have investigated the role of 5′ AMP-activated protein kinase (AMPK) as well as cyclooxygenase-2 (COX-2) in the apoptosis induced by combinatorial treatment of apigenin and 5-FU in human colon cancer cells.

Section snippets

Materials

1, 2–Distearoyl–sn–glycero-3-phosphocholine (DSPC), Apigenin, Propidium Iodide (PI), MTT reagent [3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide] and Ribonuclease A (RNase A) were all purchased from Sigma Chemicals Co., USA. 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES) was obtained from Sisco Research Laboratories Pvt. Ltd., India. Live/Dead viability cytotoxicity assay kit was bought from Molecular Probes (Invitrogen), USA. Dulbecco’s modified Eagle’s medium

Particle size distribution, structural and compositional analysis

Table 1 shows the particle size distribution of the neat, single and dual loaded liposomes. AFM microscopy images [Fig. 1(a)–(d)] provided visual evidence and reconfirmation of the formation of mono-dispersed spherical structures of nano-dimensions with a narrow particle size distribution of liposomes which were concomitant with results obtained from DLS measurements whereby the diameters of free DSPC LUVs, apigenin loaded DSPC LUVs and 5-FU loaded DSPC LUVs were found to be 93.29 (± 0.04) nm,

Conclusions

With the advent of increased resistance to commonly used anticancer chemotherapeutic compounds and the strategy of structural modifications of parent anticancer chemotherapeutics to which resistance has been established not yielding desired results, significant and concerted effort has recently been focused on widespread testing of numerous plant extracts for deciphering their pharmaceutical and therapeutic virtues [38]. The dietary plant derived bioactive compounds are highly pertinent in

Conflict of interest

The authors declare no conflict of interest.

Author contributions

Kacoli Sen: Conception and design of study, acquisition and analysis of data, drafting of article and/or critical revision, final approval of manuscript.

Shubhadeep Banerjee: Conception and design of study, acquisition and analysis of data.

Mahitosh Mandal: Conception and design of study, drafting of article and/or critical revision, final approval of manuscript.

Ethical approval

The animals were maintained in accordance with the institute animal ethical committee (IAEC) guidelines approved by Indian Council of Medical Research (ICMR), New Delhi. The collection of blood, tissue and the animal research protocol was approved and sanctioned at National Centre for Laboratory Animal Sciences, National Institute of Nutrition, Hyderabad (P46F/IAEC/NIN/11/2012).

Acknowledgements

This work was supported by research grants from the Department of Biotechnology (DBT) [Grant no.: BT/PR13996/Med/30/309/2010] and Department of Science and Technology (DST) [Grant no.: SR/SO/BB-58/2008], Ministry of Science and Technology, Government of India. KS gratefully acknowledges University Grants Commission, India for awarding her with Senior Research Fellowship [Grant no.: F.No. 2-16/98(SA-I)]. The authors gratefully acknowledge Mr. Anjani Kumar, Director, Ministry of Environment and

References (40)

  • J.-T. Hwang et al.

    Combination of 5-fluorouracil and genistein induces apoptosis synergistically in chemo-resistant cancer cells through the modulation of AMPK and COX-2 signaling pathways

    Biochem. Biophys. Res. Commun.

    (2005)
  • K. Banerjee et al.

    Probing the potential of apigenin liposomes in enhancing bacterial membrane perturbation and integrity loss

    J. Colloid Interface Sci.

    (2015)
  • S.-M. Kuo

    Antiproliferative potency of structurally distinct dietary flavonoids on human colon cancer cells

    Cancer Lett.

    (1996)
  • M.E. Davis et al.

    Nanoparticle therapeutics: an emerging treatment modality for cancer

    Nat. Rev. Drug Discov.

    (2008)
  • K. Banerjee et al.

    Liposomes as a Drug Delivery System

    (2015)
  • R. Gowda

    Use of nanotechnology to develop multi-drug inhibitors for cancer therapy

    J. Nanomed. Nanotechnol.

    (2013)
  • K. Sen et al.

    Abstract 4594: reversal of Warburg effect by Apigenin and 5-fluorouracil loaded dual drug liposomes result in enhanced colorectal chemotherapy

    Cancer Res.

    (2014)
  • D.B. Longley et al.

    5-Fluorouracil: mechanisms of action and clinical strategies

    Nat. Rev. Cancer

    (2003)
  • S. Rajamanickam et al.

    Natural products and colon cancer: current status and future prospects

    Drug Dev. Res.

    (2008)
  • X.-Y. Hu et al.

    5-Fluorouracil combined with apigenin enhances anticancer activity through mitochondrial membrane potential (ΔΨm)-mediated apoptosis in hepatocellular carcinoma

    Clin. Exp. Pharmacol. Physiol.

    (2015)
  • Cited by (0)

    View full text