Folic acid-modified biocompatible Pullulan/Poly(acrylic acid) nanogels for targeted delivery to MCF-7 cancer cells

Polymeric biomaterials are increasingly being explored as anticancer drug delivery systems (DDS). Delivery of a therapeutic agent using DDS to desired targets occurs through two mechanisms; passive and active targeting. Passive targeting is applicable where DDS can only pass through the tumor tissue due to its size via the enhanced permeability and retention (EPR) effect of the tumor vasculature and thus accumulate at the tumor site [1], [2]. This occurs when the size of DDS is below 100 nm and therefore they are able to escape from mononuclear phagocyte system, which results in extended plasma half‐time of the nanoparticle [3]. In “active targeting”, site-specific ligands are interacted/conjugated with DDS and therefore therapeutic agents can be delivered to specific cells without affecting normal tissues. This targeting mechanism is mostly used in cancer therapy to suppress the serious side effects of conventional chemotherapy such as systemic toxicity, nonspecific interactions, and multidrug resistance [4], [5]. Nanosized DDS specifically designed for this purpose are mainly functionalized by molecular targeting agents such as antibodies, aptamers, peptides, folic acid (FA), etc [5], [6], [7], [8].

Among nanosized polymeric carriers, nanogels are increasingly being investigated for anticancer DDS. Nanogels are three-dimensional crosslinked polymer networks that offer a large surface area and high mechanical strength and can entrap and release therapeutics without any burst release. In addition, the drug release capacity of polymeric nanogels can be improved by incorporating functional groups such as -COOH, -SO3H, -NH2, etc. with the aim of imparting stimuli-responsive property (pH, temperature, magnetic, etc.) to their structure [9], [10], [11]. Folate receptor (FR) is a membrane-bound protein on the cell surface, and it is well known that FR is frequently overexpressed on the surface of certain cancer cells, including ovary, kidney, breast and brain, but exhibits limited expression on normal cells. Therefore, targeting the folate receptor using folic acid ligands is a promising approach for tumor-specific targeting and delivery of nanosized DDS to tumor cells [12], [13], [14]. Bovine serum albumin (BSA) is a plasma protein and it is negatively charged under weakly alkaline conditions [15]. It can serve as a carrier and stabilizing agent for FA [12], [13], [14]. FA-BSA conjugates can be easily prepared by activating the carboxylic acid groups of FA with [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide] (EDC), and activated FA readily reacts with lysine side chains of BSA [16], [17], [12], [18]. Thus, nanosized DDS functionalized with BSA-FA may exhibit several advantages such as improved water solubility, good biocompatibility, low toxicity, high biodegradability and prolonged circulation in the blood owing to BSA, as well as targeting property thanks to FA fragments [6], [16], [18].

Nanocarriers can be constructed from synthetic polymers or natural polysaccharides such as, chitin, chitosan, carrageenan, alginate, pullulan, etc [19]. Pullulan offers several advantageous properties like biodegradability, non-immunogenicity, non-toxicity and non-mutagenicity. Owing to these, pullulan and its derivatives are being explored for various diagnostic and treatment applications such as tissue engineering, wound healing, drug and gene delivery, and in tissue imaging [20], [21], [22], [23], [24], [19]. For drug delivery applications, pullulan itself suffers from having a short plasma half-life and thus accumulation in the liver [25], [26]. Therefore, several pullulan derivatives such as cholesterol bearing pullulan[27], carboxymethyl pullulan [28], pullulan acetate [29], and folate modified pullulan [30], [31], [29] were reported as DDS either in the form of nanogel, nanoparticle or microsphere [32], [22].

One of the most effective strategies for cancer treatment is to use pH-sensitive DDS that encounter physical or chemical changes in acidic tumor extracellular conditions with pH ranging from 4.5 to 5.5, eminently different from the normal tissue with a pH of 7.4 [33], [34]. pH responsive pullulan derivatives have been obtained by several routes, such as partial carboxymethylation of pullulan [28], conjugation with urocanic acid [35] and modification with maleic anhydride [31]. However, besides the pH- responsive property, there is still a lack and need of the development of novel nanocarriers that exhibit good biocompatibility and tumor-targeting ability, as well as high loading capacity.

In this work, pH responsivity was incorporated to pullulan (Pull) by using poly(acrylic acid) (PAA) having pKa value of 4.5 [36]. PAA/Pull nanogels (NGs) with a fixed spherical shape were obtained in a facile and green approach by irradiating PAA-Pull self-assemblies with gamma radiation in an aqueous environment. PAA/Pull NGs were modified with FA-conjugated BSA to formulate a drug carrier system (PAA/[email protected]–FA NGs) with folate receptors. The nanogels were then loaded with doxorubicin (DOX), an anti-cancer drug that is widely prescribed and highly effective in the treatment of various types of cancer. DOX is the most active agent for breast cancer, which is one of the leading causes of death in women [37]. Thus, in this study, [email protected]/[email protected]–FA NGs with enhanced biocompatibility, targeting ability and pH responsive property are proposed to provide a novel targeted carrier for cancer treatment. The DOX release profiles of the nanogels were examined as a function of pH. In-vitro tumor cell targeting of the NGs was investigated by MTT assay and DAPI staining on MCF-7 cells and compared to the L929 cell line.

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