Mesoporous Organosilica Nanoparticles to Fight Intracellular Staphylococcal Aureus Infections in Macrophages.

Intracellular bacteria are inaccessible and highly tolerant to antibiotics, hence are a major contributor to the global challenge of antibiotic resistance and recalcitrant clinical infections. This, in tandem with stagnant antibacterial discovery, highlights an unmet need for new delivery technologies to treat intracellular infections more effectively. Here, we compare the uptake, delivery, and efficacy of rifampicin (Rif)-loaded mesoporous silica nanoparticles (MSN) and organo-modified (ethylene-bridged) MSN (MON) as an antibiotic treatment against small colony variants (SCV) Staphylococcus aureus (SA) in murine macrophages (RAW 264.7). Macrophage uptake of MON was five-fold that of equivalent sized MSN and without significant cytotoxicity on human embryonic kidney cells (HEK 293T) or RAW 264.7 cells. MON also facilitated increased Rif loading with sustained release, and seven-fold increased Rif delivery to infected macrophages. The combined effects of increased uptake and intracellular delivery of Rif by MON reduced the colony forming units of intracellular SCV-SA 28 times and 65 times compared to MSN-Rif and non-encapsulated Rif, respectively (at a dose of 5 µg/mL). Conclusively, the organic framework of MON offers significant advantages and opportunities over MSN for the treatment of intracellular infections.

Inulin-lipid hybrid (ILH) microparticles promote pH-triggered release of rifampicin within infected macrophages.

Intracellular bacteria serve as a problematic source of infection due to their ability to evade biological immune responses and the inability for conventional antibiotics to efficiently penetrate cellular membranes. Subsequently, new treatment approaches are urgently required to effectively eradicate intracellular pathogens residing within immune cells (e.g. macrophages). In this study, the poorly soluble and poorly permeable antibiotic, rifampicin, was re-purposed via micro-encapsulation within inulin-lipid hybrid (ILH) particles for the treatment of macrophages infected with small colony variants of Staphylococcus aureus (SCV S. aureus). Rifampicin-encapsulated ILH (Rif-ILH) microparticles were synthesized by spray drying a lipid nano-emulsion, with inulin dissolved throughout the aqueous phase and rifampicin pre-loaded within the lipid phase. Rif-ILH were strategically designed and engineered with pH-responsive properties to promote lysosomal drug release upon cellular internalization, while preventing premature rifampicin release in plasma-simulating media. The pH-responsiveness of Rif-ILH was controlled by the acid-mediated hydrolysis of the inulin coating, where exposure to acidic media simulating the lysosomal environment of macrophages triggered hydrolysis of the oligofructose chain and the subsequent diffusion of rifampicin from Rif-ILH. This pH-provoked release mechanism, as well as the ability for ILH microparticles to be more readily internalized by macrophages, was found to be influential in triggering a 2.9-fold increase in intracellular rifampicin concentration within infected macrophages, compared to the pure drug. The subsequent increase in exposure of intracellular pathogens to rifampicin leads to a ~ 2-log improvement in antibacterial activity for Rif-ILH, at a rifampicin dose of 2.5 µg/mL. Thus, the reduction in viability of intracellular SCV S. aureus, in the absence of cellular toxicity, is indicative of ILH microparticles serving as a unique approach for the safe and efficacious delivery of antibiotics to phagocytic cells for the treatment of intracellular infections.

Enhancing the Cellular Uptake and Antibacterial Activity of Rifampicin through Encapsulation in Mesoporous Silica Nanoparticles.

An urgent demand exists for the development of novel delivery systems that efficiently transport antibacterial agents across cellular membranes for the eradication of intracellular pathogens. In this study, the clinically relevant poorly water-soluble antibiotic, rifampicin, was confined within mesoporous silica nanoparticles (MSN) to investigate their ability to serve as an efficacious nanocarrier system against small colony variants of Staphylococcus aureus (SCV S. aureus) hosted within Caco-2 cells. The surface chemistry and particle size of MSN were varied through modifications during synthesis, where 40 nm particles with high silanol group densities promoted enhanced cellular uptake. Extensive biophysical analysis was performed, using quartz crystal microbalance with dissipation (QCM-D) and total internal reflection fluorescence (TIRF) microscopy, to elucidate the mechanism of MSN adsorption onto semi-native supported lipid bilayers (snSLB) and, thus, uncover potential cellular uptake mechanisms of MSN into Caco-2 cells. Such studies revealed that MSN with reduced silanol group densities were prone to greater particle aggregation on snSLB, which was expected to restrict endocytosis. MSN adsorption and uptake into Caco-2 cells correlated well with antibacterial efficacy against SCV S. aureus, with 40 nm hydrophilic particles triggering a ~2.5-log greater reduction in colony forming units, compared to the pure rifampicin. Thus, this study provides evidence for the potential to design silica nanocarrier systems with controlled surface chemistries that can be used to re-sensitise intracellular bacteria to antibiotics by delivering them to the site of infection.

Poly(lactic-co-glycolic) Acid-Lipid Hybrid Microparticles Enhance the Intracellular Uptake and Antibacterial Activity of Rifampicin.

An urgent demand exists for the development of effective carrier systems that systematically enhance the cellular uptake and localization of antibiotic drugs for the treatment of intracellular pathogens. Commercially available antibiotics suffer from poor cellular penetration, restricting their efficacy against pathogens hosted and protected within phagocytic cells. In this study, the potency of the antibiotic rifampicin against intracellular small colony variants of Staphylococcus aureus was improved through encapsulation within a strategically engineered cell-penetrant delivery system, composed of lipid nanoparticles encapsulated within a poly(lactic-co-glycolic) acid (PLGA) nanoparticle matrix. PLGA-lipid hybrid (PLH) microparticles were synthesized through the process of spray drying, whereby rifampicin was loaded within both the polymer and lipid phases, to create a nanoparticle-in-microparticle system capable of efficient redispersion in aqueous biorelevant media and with programmable release kinetics. The ability of PLH particles to disintegrate into nanoscale agglomerates of the precursor nanoparticles was shown to be instrumental in optimizing rifampicin uptake in RAW264.7 macrophages, with a 7.2- and 1.6-fold increase in cellular uptake, when compared to the pure drug and PLGA microparticles (of an equivalent initial particle size), respectively. The enhanced phagocytosis and extended drug release mechanism (under the acidic macrophage environment) associated with PLH particles induced a 2.5-log reduction in colony forming units compared to initial colonies at 2.50 µg/mL rifampicin dose. Thus, the ability of PLH particles to reduce the intracellular viability of S. aureus, without demonstrating significant cellular toxicity, satisfies the requirements necessary for the safe and efficacious delivery of antibiotics to macrophages for the treatment of intracellular infections.

Engineering PLGA-Lipid Hybrid Microparticles for Enhanced Macrophage Uptake.

Strategies to improve the uptake of particulate delivery systems to macrophages are required for the advancement of therapeutic solutions to a range of disease states, including human immunodeficiency virus (HIV), tuberculosis, and cystic fibrosis. In this study, poly(lactic-co-glycolic) acid (PLGA) nanoparticles were combined with lipid nanoparticles, via the process of spray drying, to overcome the physiochemical limitations associated with the individual precursor systems. The hybrid nanoparticle-in-microparticle structure was investigated for its ability to redisperse in aqueous media and, subsequently, enhance particle uptake into RAW 267.4 macrophages. Moreover, the surface charge of PLGA-lipid hybrid (PLH) microparticles was varied by combining positively and negatively charged PLGA nanoparticles with negatively charged lipid nanoparticles, in an attempt to elucidate the impact of surface charge on intracellular internalization within macrophages. Anionic PLH particles were shown to increase the particle uptake 3.1 times more than the cationic PLH particles, which was established to be due to the ability of the negatively charged particles to redisperse in aqueous media into the precursor lipid and PLGA nanoparticles, due to repulsive electrostatic interactions, while positively charged particles remained as micron-sized agglomerates during redispersion. Importantly, the macrophage uptake of anionic PLH microparticles was 2.1- and 4.7-fold greater than the positively and negatively charged precursor PLGA nanoparticles, which highlights the superiority of the hybrid structure to induce endocytic pathways for intracellular internalization. These findings provide understanding for the uptake of particles by phagocytic cells and therefore guide the rational development of next-generation nanocarriers that aim to deliver encapsulated cargo to macrophages.