Design and evaluation of redox responsive disulfide containing resveratrol loaded nanocarrier anti-cancer activity in the MDA-MB-231 cell line

Abstract

Redox-responsive resveratrol (RES)-loaded nanocarrier (R-NC) was fabricated using stearic acid-disulfide-methoxy poly (ethylene glycol) (SA-SS-mPEG) as a potential carrier for stimuli-responsive drug release. The synthesized redox responsive SA-SS-mPEG was evaluated through FTIR, ¹H NMR, DTA/TGA and XRD analysis and was further utilized to develop R-NC by nanoprecipitation method. The NC was characterized for physicochemical properties and its susceptibility to redox and intracellular acidic pH were assessed through alterations in the size of the NC and release of resveratrol from the R-NC in the pH 5 & 7.4 medium with presence or absence of glutathione (GSH). The size of the NC was observed to increase from 140 nm to 1005 nm in the presence of an excessive concentration of GSH/pH 5 medium while showing lesser changes in the absence of GSH and pH 7.4. Drug release profiling of the R-NC confirmed that the presence of GSH in the pH 5 medium influenced the rapid release of the resveratrol by 89.23 ± 1.03% in 6 h compared to the GSH presence pH 7.4 (79.83 ± 1.15% in 6 h) and GSH free pH 5 & 7.4 medium respectively 59.67 ± 0.78% & 47.22 ± 1.18% in 18 h. R-NC, probably encompassed in cytotoxicity, reduces cancer cell proliferation with apoptotic regulation. Biocompatibility of the NC was confirmed through hemolytic assay, which showed that the NC deliberated safety up to the concentration of 200 µg/mL. Overall results accentuated the suitability of the developed R-NC for stimuli gated redox and pH-responsive drug delivery in the treatment of cancer.

Introduction

Breast cancer is a leading cause of death in women and a serious global health problem. Every year more than 0.25 million women are diagnosed with breast cancer, which is 15.3% of all newly diagnosed cancers. The mortality rate of breast cancer is identified as 2.6% [1], [2]. Chemotherapy is the mainstay of breast cancer treatment to improve the survival rate and prognosis, however, it is often associated with poor bioavailability, drug resistance, higher dose requirement, and severe side effects due to off-target effects resulting in poor therapeutic success and increased mortality rate [3]. Drug delivery systems tailored for site-specific delivery of drug payload while avoiding non-specific targeting of normal tissue are crucial for success of cancer therapeutics with minimum or no side effects [3], [4]. The recent evolution of nanodrug delivery has exclusive physicochemical properties, prolonged physiological retention and controlled release of therapeutic molecules for better health benefits [5]. Nanodrug delivery also contributed to the development of stimuli-responsive drug carriers that fundamentally exploit the physiological system's intrinsic features, where the delivery system-associated biological active molecules are driven to an active/dormant state in response to favorable conditions or unfavorable micro-environment [4], [5], [6]. The stimuli-responsive nanocarrier tends to deliver the drug at certain physiological stimuli specific to disease pathophysiology, for example, pH, temperature and enzymatic concentrations, which in the absence of the stimulus abstains from the release of drug [7], [8], [9]. Notably, in breast cancer, uncontrolled growth of cells and production of excessive metabolic products lead to changes in the cancer cellular environment as acidic, high redox state and other abnormal nature [10]. In this view development of stimuli-responsive delivery favorable option to control the growth of breast cancer by site-specific delivery of molecules using the stimulus present in the cancer cells which will afford the superior therapeutic activity. Natural or synthetic polymers have been considered versatile carriers with specific stimuli-responsive functionalities. Polymers with no such functionalities also offer flexibility for stimuli-responsive linkages attached between two polymers, enabling target-specific, on-demand drug delivery [11], [12]. Chiba et al., 2020, developed doxorubicin-loaded redox responsive nanoparticles using disulfide-linked, oxidized cysteine phenylalanine functionalized with folic acid for target-specific drug delivery, which reduced the cytotoxicity in C6 and B16F10 cell lines [13]. Wang et al., 2018, developed PEGylated nanoparticles with prodrug triptolide-dithioglycolic acid-vitamin E complex and PEG2000 linoleic acid using nanoprecipitation technique which exhibited a better antitumor effect compared to the native triptolide both in vitro and in vivo [14].

The prevalence of a higher metabolic rate in the inflammatory and cancer conditions increases the redox state due to the presence of excessive glutathione (GSH) concentration compared to the normal cell. The intracellular GSH concentration (approx. 10 mM) is several folds higher when compared to the extracellular (approx. 2 µM) tumor environment. The higher redox concentration in breast cancer cells has been mentioned in several research reports, hence, the physiological gradience has been potentially utilized by the redox responsive delivery system to effectively treat aggressive breast cancer disease [15], [16].

With the above knowledge, the present study was devised to develop a resveratrol-loaded redox responsive drug delivery system designed for targeted treatment of cancers, for which FDA approved stearic acid and mPEG were used as carrier materials. Resveratrol (trans-3, 4′, 5-trihydroxystilbene) is a natural polyphenol chiefly renowned for its role in cardioprotection, cancer prevention, anti-inflammatory and antioxidant activities. Resveratrol’s role in the reduction of breast cancer proliferation by altering several cancer progressing mechanisms which were well examined by several research groups. However, the application of resveratrol remains restricted owing to its short biological half-life, high chemical instability, and rapid degradation in the physiological environment [17]. Hence, encapsulation of resveratrol into the redox responsive delivery system will be a good choice to avoid the drawbacks associated with its physiochemical properties and enhancement of its biological activity to treat breast cancer. The redox responsive functionality was achieved by attachment of disulfide bond containing cystamine dihydrochloride between the two materials and this type of reaction differs from thiol mediated disulfide-linked redox responsive drug delivery development. Thiol mediated reactions face self-oxidation during the preparation, which produces intermolecular linkages thus interfering with the formation of a suitable disulfide-linked carrier material [18], [19]. Chen et al., 2016, synthesized PEGylated hyperbranched polyphosphoester by the reaction of bis (2-hydroxyethyl) disulfide with phosphoryl chloride in the presence of mPEG which was utilized for the development of doxorubicin-loaded NC by dialysis method. The prepared NC showed redox responsive drug release with increased intracellular drug release observed in the MDA-MB-231 breast cancer line due to excessive GSH concentration [20]. Yang et al., 2017, fabricated redox responsive amphiphilic polymer by grafting reaction to the attachment of cystamine dihydrochloride between tocopherol succinate and heparin. Paclitaxel loaded redox responsive nanoparticles were prepared by the dialysis method and physicochemical properties of the nanoparticles were studied. Redox response-based size variation of the nanoparticles was observed by DLS analysis and redox triggered drug release property was examined for the nanoparticles [21].

In the present study, the disulfide-linked carrier material thus synthesized was utilized for the development of R-NC by nanoprecipitation method. The NC incubated in the pH 5 & 7.4 medium with the presence/absence of GSH were assessed for alteration in size of the NC and the release of resveratrol into the dialysate medium at different time points as an indicator of redox responsiveness. Next in vitro cytotoxicity of R-NC was studied in MDA-MB-231 breast cancer cell line followed by redox response medicated reduction of cancer progression was examined using fluorescence staining methods. Finally, the biocompatibility of the NC was examined by hemolytic assay to assess the suitability of the dosage form for therapeutic applications.