Maleic acid incorporated poly-(N-isopropylacrylamide) polymer nanogels for dual-responsive delivery of doxorubicin hydrochloride

Abstract

This paper discusses the fabrication and characterization of temperature sensitive polymer nanogels for stimuli responsive release of a drug, a study that specifically focuses to improve the functionality of poly-N-isopropylacrylamide (PNIPAM) nanogels in drug delivery applications. Maleic acid has been incorporated into PNIPAM nanogels towards this aim and characterized for its effectiveness in biologically relevant temperatures. The PNIPAM based nanogel was tuned for burst release of the drug at a biologically relevant temperature of 41 °C which was achieved by incorporation of 0.2 mol percent of maleic acid during the polymerization of PNIPAM. The maleic acid incorporated PNIPAM nanogels was found to show ∼50% reduction in its hydrodynamic size at 41 °C when compared to the ∼30% reduction of the hydrodynamic radius of PNIPAM nanogels at 32 °C. Apart from the raising of the LCST of the PNIPAM nanogels to higher temperature, the hydrophilicity and the negative zeta potential imparted to the nanogels by maleic acid incorporation has also resulted in improved drug loading efficiency. Electrostatic conjugation of doxorubicin hydrochloride has resulted in negligibly low release of drug at a normal physiological pH of 7.4 and temperature 37 °C but a statistically significant release at the cellular pH 4 of cancer cells and a temperature of 41 °C.

Introduction

Controlled drug delivery is a major thrust in biomedical research due to the potent advantage of targeted release of drugs, optimal drug dose, minimized side effects and reduced cost of production [1], [2], [3]. It mainly focuses on the sustained or burst release of the payload in the site of interest thereby leading to the rest of the advantages. In traditional chemotherapy, the drug is administered in such a way as to maintain the dose within the therapeutic index in the entire body, leading to the apoptosis and subsequent removal of the unhealthy cells or the pathogens. There are two complications related to this strategy: (i) drop in the therapeutic dose of the drug at short intervals and (ii) undesirable secondary responses in the body. Sustained release strategies address the first issue wherein the therapeutic level of the drug is prolonged in the body. However, the latter issue is of major concern and various targeting strategies are proposed and practiced to minimize the systemic toxicity of therapeutic agents. The scenario is intricate in cancer therapy as a high dose of the therapeutic agent needs to be maintained in the body for a long time leading to high-risk secondary responses. Targeted therapy has contributed a lot in pacifying this concern and various studies are underway to further advance these targeting mechanisms [4].

One of the promising techniques towards this aim is stimuli mediated release of drugs in the target tissue: the payload is released in response to one or multiple external stimuli like pH, temperature, ionic strength, enzymatic, magnetic, electrical or chemical [5], [6], [7]. This work mainly deals with the synthesis and characterization of temperature responsive release using a thermoresponsive system. The fundamental principle of drug expulsion in thermoresponsive release is the transformation of the drug carrier in response to variation in the surrounding temperature either through swelling/deswelling (e.g., PNIPAM, PDMA, PEGMA) or by disintegration of the carrier construct (e.g., liposomal DPPC, DSPC). The temperature at which the material shows its temperature response is referred to as the lower critical solution temperature (LCST). Thermoresponsive particles are often used in conjunction with heat inducing agents like gold nanostructures or magnetic nanoparticles. Laser irradiation or alternating magnetic field results in hyperthermia leading to carrier responses resulting in the expulsion of the payload. Sudeshna et al. [8] and Jaiswal et al. [9] report the synthesis of dendrimers and polymer nanogels, respectively for drug release by magnetic hyperthermia. The promising feature of thermoresponsive release in comparison to other stimuli responses like pH, enzymatic, chemical and ionic strength is that the release can be controlled specifically at the site as it is dependent highly on the site of irradiation/magnetic field. However, the concern in fabricating thermoresponsive delivery systems is the fact that it should exhibit its temperature response at physiologically relevant temperatures.

PNIPAM is a well documented and widespread polymer among the thermosensitive polymers used for drug delivery application. PNIPAM hydrogels shows a characteristic LCST at 32 °C [10], [11], [12]. Many strategies are employed to raise or lower this LCST. For drug delivery applications the LCST of the polymer needs to be raised and is usually achieved by incorporation of hydrophilic moieties [13], [14]. Acrylamide and methacrylamide incorporated PNIPAM nanogels are the classic examples towards this [15], [16]. Incorporation of comonomers also has the advantage of adding functional groups to an otherwise limited functionality of PNIPAM nanogels. More the functional groups present, higher is the versatility of the drug delivery system for different functionalizing agents like drug molecules [17], [18], targeting molecules [19], [20], [21] or bio-imaging molecules [22], [23], [24]. Acid containing monomers like acrylic acid, methacrylic acid copolymerized with PNIPAM had been shown to display dual response delivery with the additional response to the variation in pH conditions [25]. Gu et al. [26], and Chen et al. [27], describes fabrication and characterization of acrylic acid and polyacrylic acid incorporated PNIPAM nanogels for drug delivery application, respectively. The incorporation of the acid has provided pendant carboxylic acid functional groups which enables strong binding of the drug molecules resulting in dual responsive nanogel particles ideal for drug delivery applications. While acrylic acid incorporated PNIPAM nanogels as described by Gu et al. showed a maximum drug loading capacity of 49.5% and release characteristics of ∼96% at pH 1.2, 37 °C as compared against a ∼46% at pH 7.4, 37 °C, the polyacrylic acid incorporated PNIPAM as described by Chen et al. showed a maximum loading capacity of 120% and release characteristics of ∼60% at pH 7, 37 °C as compared to a ∼80% release at pH 4, 37 °C. In both the cases, though temperature had an effect, the release was primarily pH dependent while PNIPAM served as a polymeric backbone providing ideal structure, polydispersity and size.

Here, use of maleic acid (C₄H₄O₄) to raise the LCST of the polymer nanogels is described. Maleic acid was chosen owing to the virtue of its structure that provides two functional groups which is desirable for biomedical applications. In the present study, an anticancer drug, doxorubicin hydrochloride, was conjugated via electrostatic binding into these functional groups and drug delivery in response to both temperature and pH was obtained. The effect of the addition of maleic acid on the structure, size and swelling behavior of PNIPAM was also studied. The results were either comparable or better when compared against other acid incorporated PNIPAM nanogels.