Development and evaluation of plumbagin loaded chitin hydrogel for the treatment of skin cancer

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Abstract

Skin cancer represents one of the most common cancers worldwide. Current treatment involves conventional surgery and oral or intravenous administration of immunotherapeutic and chemotherapeutic drugs, which is associated with systemic side effects. To minimize the side effects, we developed a α-chitin polysaccharide-based hydrogel containing anticancer drug plumbagin (0.3%). Plumbagin loaded α-chitin hydrogel (PCHG) were characterized chemically and physically. The in vitro release of plumbagin from the PCHG showed a slow and sustained release of 33.8% in 6 days. In vitro porcine skin permeation assays demonstrated skin permeation of plumbagin from PCHG was 35.05 ± 0.73% after 48 h. The skin permeation of plumbagin from PCHG was further enhanced to 49.86 ± 9.32% when permeation enhancers 1% tween 80 and 1% polyethylene glycol was added into the PCHG. The in vitro anticancer activity of PCHG containing 1% tween 80 and 1% polyethylene glycol was further validated against melanoma A375 cell line.

Introduction

The incidence of skin cancers has been increasing over the past decades. Most common skin cancers are keratinocyte non-melanoma (squamous and basal cell carcinoma) and melanoma cancers [[1], [2], [3]]. Currently, 20,00,000 to 30,00,000 non-melanoma and 1,32,000 melanoma skin cancers occur globally each year [3]. Ultraviolet radiation, particularly UV-B light is the major risk factor in the development of skin cancers [4,5]. DNA damage, loss of activity of tumor suppressor genes such as p53 and activation of oncogenes triggers the formation of cancer cells. Current treatment options, which are frequently used for skin cancer, includes and are limited to chemotherapy, radiotherapy and surgery (Moh's surgery) or combination therapy, of which, chemotherapy is most effective [3,6,7]. Although, numerous chemotherapeutic drugs are available, adverse side effects (vomiting and nausea, fatigue, low blood cell counts, diarrhoea, kidney damage and partial or total loss of hearing etc) and drug resistance are the major disadvantages [8].

Topical delivery of drugs proves to be advantageous over systematic delivery due to minimal first pass metabolism, non-invasiveness, avoidance of adverse gastro-intestinal environment and the ability to provide prolonged and controlled drug delivery [[9], [10], [11]]. Topical delivery is non-invasive; achieves increased local drug concentration; minimizes systemic side effects; and easy to apply. However, commercial topical creams such as 5-FU, that treats pre-cancerous and tumorous skin growth are reported to cause erythema with skin irritation, dryness, burning and adverse reactions (dryness, burning sensation and redness) at the site of application [12]. Therefore, development of a skin friendly topical anti-cancer drug delivery system is highly desirable. Anti-cancer compounds isolated and extracted from natural resources may have good therapeutic efficacy, minimal side effects and lesser toxicity when compared to existing chemotherapeutics.

In this study, a chitosan based hydrogel system containing plumbagin was developed for the treatment of skin cancer. Plumbagin (Chemical name: 5-hydroxy-2-methyl-1,4-naphthoquinone) is a naphthoquinone from the plant Plumbago species. It is a well known anticancer agent which induces caspase-8 and 9 dependent apoptosis involving mitochondria through ROS generation in lymphocytes [[13], [14], [15]]. UV-radiation induced development of squamous cell carcinoma was shown to get inhibited by plumbagin [16]. The in vitro anticancer activities of plumbagin have been shown against a several cancer cell lines including breast, lung, ovarian, colon, oesophageal squamous cell, acute promyelocytic leukemia, prostate, pancreatic cancer, and skin cancer. Plumbagin modulates genes involved in angiogenesis including nuclear factor-kappaB (NF-κB), AKT/mTOR, STAT-3, VEGF and TGF. Plumbagin induces ROS mediated cell cycle arrest (p53,p21, cdkc, cyclin A and D) and apoptosis. Further studies also demonstrates plumbagin effectively induces paraptosis by inducing extensive cytoplasmic vacuolation and cell death in cervical cancer (HeLa), triple negative breast cancer (MDA-MB-231) and non-small lung cancer cells (A549) [[17], [18], [19], [20], [21]].

Due to its broad spectrum anti-cancer activity plumbagin was selected for the development of topical hydrogel against skin cancer. Plumbagin was loaded into chitin polymer to develop a topical gel. Chitin is a natural biological macromolecule polymer and one of the most abundant polysaccharide in nature that are present in the outer shell of crab, shrimp, insects and fungal cell walls [22,23]. Chitin is biodegradable in nature and a β-1,4 linked cationic polymer of N-acetyl glucosamine residues.

To enhance the penetration of plumbagin through the skin, co-enhancers such as alcohols (polyethylene glycol), surfactants (Tween 80), fatty acids (oleic acid) were added to the plumbagin loaded α-chitin hydrogel [24,25]. In this study, plumbagin loaded α-chitin hydrogel containing co-enhancers was developed in order to improve transdermal delivery of plumbagin for non-invasive topical treatment of skin cancer and evaluated its in-vitro anti-cancer activity.

Section snippets

Materials

Human melanoma cell line (A375) and non-melanoma skin cancer cell lines (A431) were received as gift from Dr. Ruby John Anto, Rajiv Gandhi Centre for Biotechnology (RGCB), Thiruvananthapuram, India. Dulbecco's Minimum Essential Medium (DMEM) were procured from Lonza, India. Penicillin-streptomycin solution, l-Glutamine and Alamar Blue reagents were purchased from Invitrogen, USA and 10% fetal bovine serum (FBS) was purchased from Gibco, India. α-chitin (degree of acetylation 72.4%, molecular

In-vitro anticancer activity of plumbagin

As a first step, we characterized the cytotoxic effects of plumbagin against A375 (melanoma) and A431 (non-melanoma skin cancer) cell lines. To estimate the IC50 value, of plumbagin, cells were treated in a 96-well plate with varying doses of plumbagin and cell viability was measured after 48 h using Alamar Blue assay (Fig. 2A, B, C). As illustrated, in Fig. 2B cell viability decreased gradual from lower to higher drug concentrations of plumbagin after 48 h of incubation for both A431 and

Conclusions

Topical administration of anticancer drugs is an important strategy for preventing systemic exposure, thereby decreasing the chances for unwanted side effects with high patient compliance. However, physiological barriers of the skin limit the efficiency of topical drug delivery from the skin to the tumor. Hence, we developed a topical formulation of plumbagin with enhanced skin permeation ability for topical skin cancer treatment. Presence of 1% Tween-80 and 1% PEG act as better co-enhancers

Author contributions

Akanksha Nair was responsible for acquisition of data and analysis; Sreeja C. Nair for intellectual inputs; Asoke Banerji for providing plumbagin and intellectual inputs; Raja Biswas for funding acquisition, conceptualization, data curation, formal analysis and writing of manuscript, Ullas Mony for study concept and design, supervision of the study, analysis and interpretation of data, manuscript writing and intellectual inputs.

Declaration of competing interest

The authors have declared no conflict of interest.

Acknowledgement

This work is financially supported by a Department of Biotechnology, Government of India (BT/PR15777/TRM/120/81/2015) to RB. All authors are grateful to Centre for Nanosciences and Molecular Medicine, Amrita Vishwa Vidyapeetham for infrastructural support. We thank Mrs Aparna Viswanathan Ammanath and Prof. R. Jayakumar, Centre for Nanosciences and Molecular Medicine, Amrita Vishwa Vidyapeetham for their valuable suggestions and help during the preparation of PCHG and PCHG-TP.

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