Encapsulation of Artemisia scoparia extract in chitosan-myristate nanogel with enhanced cytotoxicity and apoptosis against hepatocellular carcinoma cell line (Huh-7)

https://doi.org/10.1016/j.indcrop.2020.112790Get rights and content

Highlights

  • Chitosan-Myristate Nanogel (CS-MA) encapsulating A. scoparia was synthesized.

  • Anti-cancer effect of CS-MA nanogel encapsulating A. scoparia was investigated against Huh-7 cell line.

  • High apoptosis was induced in Huh-7 cancer cells.

  • Pro-apoptotic genes were up-regulated and anti-apoptotic genes down-regulated.

Abstract

In the current study, encapsulation by chitosan (CS)-myristic acid (MA) nanogel was used to enhance the cytotoxic and apoptosis activities of Artemisia scoparia extract against Hepato-carcinoma cell line (Huh-7). The synthesized CS-MA nanogel had uniform size distribution below 100 nm with the spherical structure. Subsequently, cytotoxicity of free A. scopari extract, CS-MA nanogel and CS-MA nanogel-encapsulated extract was evaluated on Huh-7 cell line using MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Finally, the apoptotic effects of CS-MA nanogel-encapsulated extract was evaluated using Real-time PCR and flow cytometry techniques. The results of MTT assay showed that the CS-MA nanogel-encapsulated extract was more effective against Huh-7 cell line comparing to the free extract. In addition, CS-MA nanogel-encapsulated extract induced the apoptosis in Huh-7 cell line with up-regulation of Bax, and miR192 genes along with the suppression of Bcl-2, MMP2, MMP9 and cyclin D1 (P < 0.01). Moreover, the flow cytometric analysis of cell cycle distribution of Huh-7 cells showed enhanced sub-G1 peaks by the CS-MA nanogel-encapsulated extract treatment, which is indicative of apoptosis pathway. In conclusion, A. scoparia extract in the form of CS-MA nanogel-encapsulated can be used as an effective tool to enhance cytotoxicity against Huh-7 cell line and could be a promising candidate for hepato-carcinoma therapy.

Introduction

Hepatocellular carcinoma, also known as hepatic cancer, is one of the common cancers in men that leads to many deaths each year (Ramai et al., 2019). In 2018, a total of 841,000 cases of hepatocellular carcinoma with 263,000 deaths were reported worldwide (Le et al., 2019). Currently, surgical operation, chemo-therapeutic and ionizing radiation are the common strategies to treat hepatocellular carcinoma which have low efficacy and high toxicity on normal cells (Cai et al., 2019). In recent years, the natural products such as herbal extracts have attracted the attention of many researchers as alternative anti-cancer agents (Zhai et al., 2019). The herbal extracts are composed of various phytochemicals, many of which are considered as bioactive agents (Huynh et al., 2017). Moreover, herbal extracts contain secondary metabolites with anti-microbial, anti-cancer, anti-inflammatory and anti-oxidant effects (Seca and Pinto, 2018). Generally, there are more than 20,000 species of Artemisia worldwide. Some of the Artemisia species are used for the therapeutic applications, such as sedative factors, cough relief, anti-bacterial and anti-tumor agents, as well as for the malaria therapy (Moulavi et al., 2019). Among 20,000 Artemisia species, 34 species have been found in Iran. A. scoparia belongs to the Asteraceae family which has a high content of secondary metabolites such as flavonoids, terpenoids, and tannins (Naghavi et al., 2014).

The formulation of herbal extracts have gained huge attention to prevent evaporation, degradation of the active components and controlled release of the extract (Iannone et al., 2017). One of these formulation systems is the use of nanoscale delivery systems, which have shown increased biological activities of the extracts due to passive cell absorption strategy (Ong et al., 2017). Among nanomaterials for encapsulation, nanogels are highly suggested due to their high capacity of loading, high stability and release features (Kavetsou et al., 2019). From a structural point of view, the nanogels are three-dimensional compounds which are composed of interconnected polymer chains via self-assembly phenomenon (Abdel-Rashid et al., 2019). Nanogels can be synthesized from biodegradable synthetic materials. Among them some natural polymers such as chitosan (CS) have been used as suitable carrier for several therapeutic agents due to its low cost (Palaniraj et al., 2019). The most important characteristics of CS as a carrier system are biodegradability, biocompatibility, availability, safety, low immunogenicity and cationic charge (Chellappan et al., 2019). It has been shown in different studies that CS-based nanogels are used in tissue engineering, drug delivery and as a carrier for many macromolecules such as plant-derived molecules (Perez-Alvarez et al., 2019). Glucosamine units of CS have provided the hydroxyl and amino groups as the reactive sites for the cross-linking reactions (Ashrafi et al., 2019). The cross-linkers can modify the properties of CS hydrogels and cross-linked hydrogels can absorb a huge amount of different kinds of biological fluids or water (Li et al., 2019). Myristic acid (MA) is a non-toxic alternative cross-linker (C14H28O2) which is favorable to prepare CS nanogel (Nazem et al., 2016). In this study, the CS- myristate nanogel (CS-MA) was designed and synthesized as carrier for A. scoparia extract and its potential anti-cancer activity was evaluated against hepato-carcinoma cell line (Huh-7). CS- MA nanogel is composed of CS as backbone and MA as hydrophobic moiety which are self-assembled in aqueous environments. In this study, the extract of Artemisia scoparia, an Iranian indigenous plant species, was used for the first time for encapsulation studies.

Section snippets

Materials

Chitosan (CS) was purchased from Sigma (USA). The 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and myristic acid (MA) were obtained from Fluka (USA). Acetic acid, Tween 80, glycerol, ethanol and MTT dye were purchased from Merck (Germany). Artemisia scoparia (Herbarium NO. 1319) aerial parts dry powder and Hepato-carcinoma cell line (Huh-7, IBRC C10082) were purchased from Iranian Biological Resource Center. RPMI-1640 medium, fetal bovine serum (FBS) and trypsin-EDTA were purchased from

Characterization of CS-MA nanogel

The CS-MA nanogel was prepared by self-aggregation process through the modification of CS molecules by the hydrophobic MA. As a result, the free amine groups of CS were linked to the carboxylic groups of MA by EDC (Fig. 1).

The structural characterization obtained by FTIR spectroscopy for MA, CS, CS-MA, CS-MA-encapsulated extract and free extract are given in Fig. 2A–E, respectively. In Fig. 2A, the peaks observed at 3412 and 3469 cm−1 belonged to Osingle bondH stretch, confirming the presence of alcoholic

Conclusion

In the current study, the CS-MA was synthesized using self-assembly process and it was characterized by FTIR, SEM and TEM. The mean diameter of the CS-MA nanogel was below 100 nm with uniform size distribution and spherical structure. The CS-MA nanogel system was then used as an encapsulating material for Artemisia extract in order to increase the cytotoxicity of the extract. Based on our findings, it is concluded that the CS-MA-encapsulated extract induced the apoptosis in Huh-7 cell line. To

CRediT authorship contribution statement

Sara Mosafer Haghighi: Data curation, Formal analysis, Methodology. Farzaneh Tafvizi: Methodology, Project administration, Data curation, Supervision, Writing - review & editing. Amir Mirzaie: Formal analysis, Methodology, Writing - original draft.

Declaration of Competing Interest

The authors declare that they have no conflict of interest.

Acknowledgements

The authors would like to acknowledge the laboratory of Islamic Azad University.

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