Synthesis and evaluation of pyrazinoic acid loaded polymeric nanoparticles in the treatment of Tuberculosis
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Date
2024
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University of the Western Cape
Abstract
Purpose: Tuberculosis (TB) remains a deadly infectious disease worldwide despite the availability of antibiotics. TB is caused by Mycobacterium tuberculosis (M. tuberculosis), which is primarily resident in macrophages. Pyrazinamide (PZA) is a first-line antibiotic used in combination with other anti-tuberculosis drugs to treat TB. PZA is a prodrug that is converted to pyrazinoic acid (POA), the active moiety that exerts its bactericidal effects in macrophages. Low therapeutic drug concentrations of PZA, severe side effects, and poor patient adherence have led to treatment failure and increasing cases of M. tuberculosis resistance to antibiotics, including PZA. PZA resistance is mainly caused by mutations in the pncA gene that encodes the pyrazinamidase enzyme responsible for activating PZA to POA. Therefore, administering POA is a potential solution to address the challenge of treatment of drug resistant TB. However, POA encounters barriers to absorption, due to its poor biopharmaceutical properties that reduces its available concentration for cellular uptake and overall therapeutic effect. We propose a polymeric nanoparticle (NP) based system to deliver POA. The NPs are aimed to deliver POA for a therapeutic effect against mycobacteria. This study therefore investigates the synthesis, characterization and the antimycobacterial activity of such NPs.
Methods: A double emulsion solvent evaporation method was used to synthesize water-in-oil-in-water (w/o/w) POA and PZA loaded polycaprolactone (PCL) NPs. Characterization of the NPs was performed using Dynamic Light Scattering (DLS) techniques to determine particle size, polydispersity index (PDI) and zeta potential (ZP). Particle surface and morphology were analysed using high resolution scanning electron microscopy (HR-SEM). UV spectroscopy was used to quantify POA and PZA loading. Drug release studies were performed in phosphate buffer (PBS) pH 7.4 and 0.1M hydrochloric acid (HCL) pH 1.2. Drug release data obtained was mathematically modelled for all formulations. Antibacterial assays were conducted using a PZA sensitive TB strain, Mycobacterium smegmatis (M. smegmatis) strain.
Results: POA and PZA loaded PCL NPs of varying drug : polymer ratios (1:1, 0.5:1, 0.4:1, 0.25:1) were successfully synthesized. Mean particle size were in the range of 276 ± 17.10 to 333 ± 31.3 nm for POA loaded PCL NPs and 281 ± 14.8 to 345 ± 95.70 nm for PZA loaded PCL NPs. NPs were monodisperse with the PDI ranging from 0.16 ± 0.04 to 0.22 ± 0.03 for POA loaded PCL NPs and 0.1 ± 0.01 to 0.29 ± 0.10 for PZA loaded PCL NPs. ZP values were within a range of -7.7 ± 2.2 to -18.2 ± 3.4 mV for POA loaded NPs and -13.8 ± 2.0 to -20.3 ± 5.4 mV for PZA loaded PCL NPs indicating stable NPs. NPs had a spherical morphology with fairly smooth surfaces. Encapsulation efficiency for POA NP formulation was within the range of 24.07 ± 1.10 % and 65.53 ± 6.25 % with a maximum drug loading capacity of 10.57 ± 0.37% w/w while PZA NP formulation had an encapsulation efficiency range of 30.35 ± 2.76 % and 77.91 ± 6.61 % and drug loading capacity of 12.02 ± 0.63% w/w. A biphasic Fickian diffusion release pattern of POA and PZA from PCL NPs was observed in both PBS at pH 7.4 and 0.1M HCL at pH 1.2. At the 48 h timepoint, POA released was 45.67 ± 3.68% in PBS and 24.01 ± 2.45% in 0.1M HCL and PZA released was 52.63% ± 2.10 in PBS and 45.72% ± 3.58 in 0.1M HCL indicating pH sensitivity in release and sustained release of the drugs. A concentration of 200 μM was determined as the ideal minimum inhibitory concentration (MIC) for both POA and PZA solutions as they displayed the lowest % cell viability of 19.28 ± 10.99 % and 34.02 ± 8.09 % respectively following a 24 h treatment to the M. smegmatis using a range of concentrations for in vitro antibacterial assays. After the incubation period of 24 h, POA and PZA displayed a low % cell viability of 19.28 ± 10.99 % and 34.02 ± 8.09% respectively at a concentration of 200 μM. Interestingly, in comparison to the standard drug solutions, following a 24 h incubation of the antibacterial assays, the % cell viability observed for POA and PZA-PCL NPs was 71.5 ± 7.57% and 96.9 ± 15.70%, respectively. The % cell viability at 48 h for POA and PZA PCL NPs was 74.5 ± 6.69 % and 108.41 ± 11.90 %, respectively.
Conclusions: POA and PZA loaded PCL NPs were successfully synthesized. The NPs showed controlled release suggesting the ability of the nano formulations to maintain drug concentrations over time. The findings from the antibacterial study indicated greater antimicrobial effect from NPs loaded with POA than those with PZA. These findings demonstrate that PCL NPs are a useful carrier of POA and can be used in the investigations against PZA resistant M. tuberculosis
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Keywords
M. smegmatis, Polycaprolactone, Pyrazinoic acid, Nanoparticles, Tuberculosis