Development of extended-voyaging anti-oxidant Linked Amphiphilic Polymeric Nanomicelles for Anti-Tuberculosis Drug Delivery

Abstract

Amphiphilic chitosan-graft-poly(caprolactone)/(ferulic acid) (CS-g-PCL/FA) multi-co-polymers were fabricated by microwave-assisted ring opening polymerization followed by an 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC)-mediated coupling reaction and characterized by Fourier transformed infrared (FTIR) spectroscopy. Graft copolymers self-assembled into nanomicelles, and were able to incorporate rifampicin (RF) into their hydrophobic inner cores. X-ray diffraction (XRD) patterns were applied to characterize the crystal structures of graft polymers and the effects of RF on micelle morphology. Empty and RF-loaded CS-g-PCL/FA nanomicelles underwent swelling and degradation in acidic pH conditions. Scanning electron microscopy, transmission electron microscopy, and dynamic light scattering revealed that the self-assembled, RF-loaded micelles were spherical, with an average size of 100–210 nm. An in vitro study conducted at 37 °C demonstrated that RF and FA release from micelles at pH 5.3 was much faster than that at pH 7.4. The RF and FA release was significantly accelerated by switching to an acidic pH, owing to swelling of the micelles at lower pH values caused by the rapid degradation of ester and amide bonds present in the micelles. Fluorescence micrographs revealed successful entry of the polymeric micelles into A549cell lines. Thus, graft polymeric micelles have promising potential for delivery of hydrophobic antitubercular drugs and may improve therapeutic approaches for tuberculosis.

Introduction

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis and mainly affects the lungs, is one of the world’s most lethal communicable diseases (Amarnath Praphakar et al., 2016, Hasegawa et al., 2007, Choonara et al., 2011). Several antitubercular agents are used to control TB. Among these, the first line antitubercular agent, rifampicin (RF), which inhibits DNA-dependent RNA polymerase, is the most commonly used agent. In addition, trans-cinnamic acid derivatives have a wide range of therapeutic uses, including antitumor (Mishra et al., 2013), antioxidant (Sawant et al., 2014), and antibacterial activity (Torchilin et al., 2003). Cinnamic acid derivatives are also valid leads for use in the design and synthesis of M. tuberculosis inhibitors. They have good minimal inhibitory concentration (MIC) values and are extremely effective against M. tuberculosis (Park et al., 2001). Ferulic acid (4-hydroxy-3-methoxy cinnamic acid) is a cinnamic acid derivative which shows good antioxidant activity due to the presence of phenolic nucleus and extended conjugation (Graf, 1992). There are several effective drug regimens for TB; however, the disease still represents a major public health challenge primarily owing to the ease of transmission and the development of drug resistance (Ishida et al., 2001, Yang et al., 2009, Hu et al., 2008, Hu et al., 2012). Furthermore, the oral intake of RF results unwanted side effects such as hepatotoxicity, and the efficiency of anti-TB drugs can be hindered by poor solubility, drug permeability, and biodegradation (Liu et al., 2016, Hirota et al., 2010).

Amphiphilic polymeric micelles form the basis of novel nano-drug delivery systems and have been exploited for the oral delivery of poorly water-soluble drugs (Luo et al., 2016). Because of their amphiphilic nature, they self-assemble into nanoscale spherical structures with a hydrophobic inner core and a hydrophilic outer core in aqueous media (Koup et al., 1986). The hydrophobic inner core can incorporate a large amount of hydrophobic drugs, while the hydrophilic outer core can stabilize the micelles by reducing their interaction with serum proteins and tissues. They offer a simple approach for clinical treatment with reduced side effects, selective targeting, prolonged circulation time, and improved bioavailability and solubility of hydrophobic drugs (Zheng et al., 2016, Murugaraj Jeyaraj et al., 2016). Some drug moieties can be attached to the hydrophilic outer core of polymeric micelles, thereby enhancing therapeutic activity at the target site (Chen et al., 2003). To date, a number of surface modified polymeric micelles have been described, including those modified with antibodies (Chiu et al., 2010, Yang et al., 2011), transferrin (Adeleke et al., 2016, Dufes et al., 2004), and peptides (Aziz et al., 2006).

Chitosan shows good biocompatibility, biodegradability, mucoadhesivity, and non-cellular uptake, thereby making it an excellent backbone for drug delivery devices (Bezerra et al., 2006, Chung and Shin, 2007). In addition, chitosan itself possesses antibacterial activity (Naz et al., 2006). Despite these favorable characteristics, unmodified chitosan is a poor candidate for clinical use due to its pH-dependent solubility (Rajan and Raj, 2013). Therefore, chemical modification of chitosan (with either hydrophobic or hydrophilic moieties) is often used in order to overcome these drawbacks (Lepeltier et al., 2015, Yoder et al., 2004, Goyal et al., 2015, Kaur et al., 2016, Gelperina et al., 2005, Azarmi et al., 2008).

In the present work, we designed an amphiphilic polymer based on chitosan-grafted polycaprolactone for the dual delivery of FA and RF. First, chitosan was modified with caprolactone via microwave-assisted ring opening polymerization, and ferulic acid was conjugated via its carboxylate group onto an amino group of the chitosan backbone. Then, RF was encapsulated into the hydrophobic inner core of self-assembled CS-g-PCL/FA micelles. The physicochemical characteristics of CS-g-PCL/FA such as drug loading efficiency, swelling, critical micelle concentration (CMC), particle size, zeta potential, biodegradation, and in vitro drug release performance were studied. Characterization of antimicrobial activity, cytotoxicity and cellular uptake was performed. To investigate cytotoxicity, and cellular uptake, A549 cells was chosen as model cell line due to the presence of lysozyme to demonstrate the drug delivery.