The development and evaluation of a novel hybrid PLGA nanoparticle-Pheroid® with the potential to improve tuberculosis therapy

Abstract

The development of drug delivery technologies has the potential to bring both therapeutic and commercial value to future healthcare products. Drug delivery technologies are transport vehicles that help overcome the disadvantages, such as poor bioavailability and limited aqueous solubility, associated with free drugs, and enable drugs to function to their full potential. Case in point: tuberculosis (TB) is still a major health threat in South Africa, even though anti-TB drugs are available for its treatment. These anti-TB drugs have poor pharmacokinetic (PK) properties and have to be taken for lengthy periods at high daily dosage for them to be effective. Several drug delivery systems (DDS) have been investigated to improve the current TB therapy so as to reduce dosing frequency and shorten the treatment period. However, the advancement of these systems for improved TB therapy is limited by certain drawbacks of each of these DDS. Hybrid (or combined) DDS composed of a polymeric nanoparticle (NP) core and a lipid-based outer shell have recently emerged in an effort to mitigate some limitations associated with the individual DDS. The research described here explores the combination of two delivery systems with unique properties, namely poly (DL-lactic-co-glycolic acid) (PLGA) NP and Pheroid® technology. The solid PLGA NP were combined with Pheroid® vesicles using two types of mixing approaches namely, pre-mix (the addition of preformed NP during the Pheroid® manufacturing) and post-mix (the combination of the two individual preformed systems). The particle size of the hybrid system ranged from approximately 2250 nm to 2850 nm, depending on the surface properties of the NP, while the zeta potential (ZP or ζ-potential) ranged from -19 to -25 mV, measured using laser diffraction and electrophoretic velocity methods, respectively. There was an increase in the size of the Pheroid® vesicles when combined with NP that had a positive ZP, suggesting a possible electrostatic interaction between the two systems. Further physicochemical properties of this novel hybrid system were obtained through transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM), both of which revealed possible co-localisation of the NP with the Pheroid® vesicles. The effect of the NP/Pheroid® ratio when combining the two systems showed that the stability of the hybrid system is compromised at ratios above 2.5% (w/v) NP. In vitro experiments were conducted to evaluate the effect of the hybrid system on cytotoxicity, permeability as well as intracellular uptake using the Caco-2 cell line. The use viii of high concentrations of Pheroid® in the cell culture environment has previously been shown to compromise cell viability through the prevention of nutrients and gas exchange between the culture media and cells. The real-time cell analysis (RTCA) used in this study indicated that it was imperative to dilute NP, Pheroid® and the hybrid DDS for use in Caco-2 cell permeability experiments. The appropriate dilutions that showed prolonged safety for the Caco-2 cells over 24 hours (h) period using the RTCA were confirmed to be 0.004% (v/v) for the Pheroid® vesicles and a maximum of 1% (w/v) for the NP. However, the hybrid DSS did not show any significant effect on the permeability of coumarin 6 (C6) in comparison with the individual DDS. The C6 was found to be associated with the Caco-2 cell membrane rather than taken up into the cytoplasm. An in vivo evaluation of this novel hybrid system was undertaken to investigate its potential application to address challenges in tuberculosis (TB) therapy. Three types of formulations were prepared for each of the two selected anti-TB drugs, rifampicin (RIF) and isoniazid (INH). These formulations included free drug, drug-loaded PLGA NP and drug-loaded NP–Pheroid® hybrid system. A single oral dose of each formulation was administered to healthy female BALB/c mice, and the levels of RIF and INH were measured in the plasma and selected organs at several time points to determine the effect of the hybrid delivery system on the PK of these drugs. The plasma data did not provide evidence of the NP–Pheroid® hybrid formulation on improving the PK parameters for both drugs. However, the effect of the hybrid formulation was observed in the RIF distribution to the lung tissue, where there was a significant reduction of Tmax from 11 to 4 h in comparison to the RIF NP, and to the kidney, where the half-life of RIF was significantly increased to 16 h in comparison to the 4 h by the free RIF. The hybrid system also led to an increased retention of RIF in the lungs up to a period of 5 days (d), compared to the 3 d RIF circulation from free RIF and RIF NP. In conclusion, the fabrication of the PLGA NP-Pheroid® hybrid DDS was successful, as determined through size and ζ-potential measurement. Co-localisation of the NP with the Pheroid® vesicles was demonstrated by microscopy techniques, namely, TEM and CLSM. The optimal NP/Pheroid® mixing ratio for a stable hybrid system was found to be a maximum of 2.5% (w/v). The permeability of C6 was enhanced when encapsulated in all the delivery systems: NP, Pheroid®, and NP-Pheroid®. However, C6 cell uptake was not altered when formulated in any those above-mentioned delivery systems. The NP-Pheroid® hybrid system did not alter the PK parameters of either INH or RIF in the plasma. However, the effect of the novel hybrid DDS was observed on RIF distribution to the lungs and kidney.