Potential of self-organizing nanogel with acetylated chondroitin sulfate as an anti-cancer drug carrier

Abstract

In order to obtain feasibility data regarding the possibility of using chondroitin sulfate (CS) in an anti-cancer drug delivery system, CS was chemically modified by a one-step process with acetic anhydride. Although 3 samples with different degrees of acetylation were synthesized, only the sample with the highest degree of acetylation (AC-CS3) was tested as a nanogel because the others (AC-CS1 and 2) dissolved in distilled water (DW) in the test range (1 - 10 mg/ml). The AC-CS3 nanogel was characterized by fluorescence probe and dynamic light scattering (DLS) techniques. Its critical aggregation concentration (CAC) was < 2.0 × 10⁻² mg/ml at 25 °C. The partition equilibrium constant, K_v, of the nanogel (7.88 × 10⁵) was similar to that of polymeric micelles, which means that the acetyl group may act as a hydrophobic core controlling pharmacokinetic behavior. The higher surface charge value in the nanogel, above - 40 due to carboxyl and sulfate groups in CS, explains its good stability. The anticancer drug doxorubicin (DOX) loading efficiency of the AC-CS3 nanogel was also superior, at above 90%. Changes in the size of the polydispersion index (PDI) of nanogels loaded with DOX over a 3-week period were negligible. The nanogels interacted with HeLa cells and were internalized together with the entrapped drug within the cytoplasm, probably via an endocytic mechanism exploited by sugar receptors. Based on these results, the AC-CS3 nanogel is expected to prove useful as an anti-cancer drug carrier for chemotherapy.

Introduction

Over the past few decades, polymeric nanocarriers from synthetic and natural polymers such as polymeric micelles, self-assembly, and nanogels have been extensively investigated, in efforts to develop a drug delivery system with enhanced bioavailability for anti-cancer drugs, coupled with minimized side effects [1], [2], [3]. The nano-size of these carriers provides for superior cancer targeting, particularly with regard to the effect of ‘enhanced permeation and retention’ (EPR), which increases their potential for localization in tumor sites [4]. Nanocarriers are constructed of a hydrophobic core covered with a hydrophilic outershell. This structure results in unique properties, including prolonged circulation and anti-cancer drug targeting. The hydrophilic outershell also serves as a barrier against interactions with other cells, proteins, and body tissues, thus resulting in prolonged and more effective circulation of the relevant drug [5], [6]. The hydrophobic core is the primary determinant of pharmacokinetic behaviors such as drug loading efficiency and release [5], [7], [8].

Among the different types of nanocarriers, biopolymer nanogels have gained increasing attention as a possible effecctive anti-cancer drug carrier, owing principally to their excellent biocompatibility, low immunogenicity, and favorable biodegradability. In particular, nanogels composed of polycores can allow for a change in carrier shape, from a sphere to ellipse; reminiscent of an erythrocyte [9]. This property is crucial to attempts to increase the duration of blood circulation through microcapillaries. At a high shear rate, the nanogels may become elongated, further reducing the apparent viscosity of the blood. Our group reported some findings regarding self-organizing nanogels consisting of a polysaccharide conjugated to a hydrophobic moiety [8], [10], [11]. However, despite their clear potential, polysaccharides have not been generally employed at a frequency comparable to synthetic polymers because they are difficult to modify and analyze, due to solvent issues.

For this research, a biocompatible polysaccharide, chondroitin sulfate (CS), was hydrophobically modified by a simple method using acetic anhydride. CS, a member of the glycosaminoglycan (GAG) family and an acidic mucopolysaccharide, is composed of alternating units of (α-1,3)-linked glucuronic acid and (α-1,4) N-acetyl galactosamine (GalNac), with sulfate at either the 4- or 6-position of GalNac [12]. Because it is a biomaterial found in cartilage, skin, corneas, extracellular matrix (ECM), and umbilical cords, CS possesses good biocompatibility, comparable to that of hyaluronic acid. Furthermore, the higher hydrodynamic volume of CS (induced by its sulfate group) may inhibit undesirable interactions with plasma proteins and cells while circulating in the body. However, CS cannot be incorporated into a self-organizing nanogel in aqueous solution, owing to its solubility in water. Thus, it must be hydrophobically modified for nanogels if it is to be utilized as a drug delivery system. CS was investigated for possible preparation in a stable nanogel by Lee et al. [5], [7]. They synthesized poly(L-lactide) grafted CS and assessed its stability in aqueous media. The obtained CS–PLLA graft copolymers evidenced hydrolytic degradabilities higher than that of PLLA homopolymer as a result of the introduction of hydrophilic segments and branch structures. More importantly, these nanoparticles evidenced favorable properties, including low toxicity, biocompatibility, and intracellular internalization.

In this study, CS was readily modified by acetic anhydride to impart hydrophobicity. Nanogels from acetylated CS were prepared via dialysis and potential as an anti-cancer drug carrier evaluated in terms of particle size, doxorubicin (DOX) release behavior, cancer cell cytotoxicity, and cellular uptake behaviors. These evaluations involved fluorescence techniques, dynamic light scattering (DLS), tetrazolium dye MTT assay, and confocal scanning imaging. A schematic of the concept of self-organizing behavior of acetylated CS (AC-CS) is shown in Figure 1.