Preparation of Curdlan sulphate - Chitosan nanoparticles as a drug carrier to target Mycobacterium smegmatis infected macrophages

Highlights

• Drug encapsulated nanoparticles were prepared for targeting infected macrophages.

• Drug release follows a Weibull model with Initial burst followed by sustained release.

• CSC NPs enter through multiple pathways like, caveolae, clathrin, macropinocytosis.

• They have superior intracellular bactericidal activity when compared to free drug.

Abstract

In this study, curdlan sulphate - chitosan nanoparticles were prepared through polyelectrolyte complexing at a mass ratio of 2:1 respectively. The curdlan was produced by fermentation with Agrobacterium sp. ATCC 31750, which was then sulphated to form the polyanionic polymer. A first-line tuberculosis drug, Rifampicin and a phytochemical, DdPinitol, were encapsulated into Curdlan Sulphate (CS) - Chitosan Nanoparticles (C) (CSC NPs) of size 205.41 ± 7.24 nm. The drug release kinetics followed a Weibull model with initial burst release (48 % Rifampicin and 27 % d-Pinitol within 6 h), followed by a sustained release. The prepared CSC: d-PIN + RIF NPs was cytocompatible and entered the M.smegmatis infected macrophages through multiple endocytic pathways including clathrin, caveolae and macropinocytosis. They showed superior bactericidal activity (2.4–2.7 fold) within 4 h when compared to free drug Rifampicin (1.6 fold). The drug encapsulated CSC: RIF suppressed the pro-inflammatory gene (TNF-α by 3.66 ± 0.19 fold) and CSC: d-PIN + RIF increased expression of the anti-inflammatory gene (IL-10 by 13.09 ± 0.47 fold). Expression of TGF- β1 gene also increased when treated with CSC: d-PIN + RIF (13.00 ± 0.19 fold) which provided the immunomodulatory activity of the encapsulated CSC NPs. Thus, curdlan sulphate - chitosan polyelectrolyte complex can be a potential nanocarrier matrix for intracellular delivery of multiple drugs.

Introduction

Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis has latently infected nearly 23 % of the world population and 10 million cases are reported every year (Organisation, 2018). Mtb sustains and replicates inside the phagosomes of macrophages through the inhibition of the bactericidal responses. These macrophages phagocytose Mtb trigger the release of pro-inflammatory cytokines, chemokines (Weiss & Schaible, 2015). The crucial problem in inhibiting mycobacteria is that they reside inside the macrophages and in granulomas and when these mycobacteria are exposed to lethal dose concentration of antibiotics, they develop drug tolerance and drug resistance mutations (Nathanson et al., 2010). Therefore, there is a need for delivery of antibiotics/adjuvant targeting the macrophages to improve the efficacy and the activity of the treatment drugs. To overcome this, natural polymer and drug-based conjugation were used to deliver it at the targeted site, which offers many advantages including controlled delivery, enhanced drug solubilization, increase in drug efficiency with improved pharmacokinetics property (Basha, TS, & Doble, 2019).

Curdlan, a bacterial polysaccharide (β -1-3-glucan) produced by organisms including Agrobacterium, Alcaligenes faecalis and Rhizobium possesses the anti-inflammatory and immunomodulatory property. It recognizes the dectin 1 receptor on the surface of macrophages (Ferwerda, Meyer‐Wentrup, Kullberg, Netea, & Adema, 2008; Kim, Ahn, & Je, 2016). Curdlan is insoluble in water and sulphation yields water-soluble curdlan sulphate (Yan, Wang, Qiu, & Wu, 2017). Chitosan which is derived from chitin is extensively used in agricultural, biomedical materials and food industry (Kumar, Muzzarelli, Muzzarelli, Sashiwa, & Domb, 2004). Active electrostatic interactions are established between the sulphate groups present in curdlan sulphate (polyanionic) and the amino group present in chitosan (polycationic) to form polyelectrolyte complex (Delair, 2011). Oppositely charged polyelectrolytes are combined without the use of any chemical covalent cross-linker to form these complexes. Numerous studies have shown delivery of drugs using nanocarriers developed with chitosan and many anionic polysaccharides like alginate (Sorasitthiyanukarn, Bhuket, Muangnoi, Rojsitthisak, & Rojsitthisak, 2019) dextran sulfate (Manchanda, Sahoo, & Majumdar, 2016), carboxymethyl cellulose (Cerchiara et al., 2016), chondroitin sulfate (Tan, Selig, & Abbaspourrad, 2018), hyaluronic acid (Lallana et al., 2017), gum arabic (Ibekwe, Oyatogun, Esan, & Oluwasegun, 2017) and pectin (Birch & Schiffman, 2014). The reason for using curdlan and chitosan is that the former is known to act as an immunomodulator, while the latter is reported to target selectively towards macrophage through the dectin-1 receptor. Curdlan is also reported to exhibit immunomodulatory properties.

In the present study, we have encapsulated d-Pinitol (a plant phytochemical) and Rifampicin (a first-line drug for treating tuberculosis) in a polyelectrolyte complex and tested. Synergistic use of both these compounds successfully against Mycobacterium smegmatis is reported in our previous publication. The current work is a continuation of our previous study (Ravindran, Chakrapani, Mitra, & Doble, 2020). In this study, curdlan sulphate - chitosan nanoparticles are prepared and d-Pinitol (D-PIN) and Rifampicin (RIF), are encapsulated and tested. d-Pinitol, has anti-inflammatory, antihyperlipidemic, cardioprotective (NUMATA et al., 1990), antioxidant (Geethan & Prince, 2008). It is used to treat lung, bladder and breast cancer (Zhan & Lou, 2007). Rifampicin was used in the present study it inhibits bacterial DNA dependent RNA synthesis by inhibiting the corresponding polymerase (Campbell et al., 2001). The study was designed to evaluate the activity of drug encapsulated CSC nanoparticles to target M. smegmatis infected macrophages. The mode of NPs uptake was determined by endocytotic inhibitors. Pro-inflammatory and anti-inflammatory gene expressions of LPS stimulated RAW 264.7 macrophages were evaluated to establish a pharmacological hypothesis for the Mtb treatment linked with inflammatory diseases.