Engineered antibiotic nanoparticles for inhaled anti-biofilm therapy

Biofilm, a sessile community of bacterial cells that is enclosed by a self-secreted matrix composed of an extracellular polymeric substance, is implicated in the chronic pulmonary infection of patients with impaired lung mucociliary clearance mechanism, such as cystic fibrosis and chronic obstructive pulmonary disease (COPD) patients. The higher tolerance of biofilms to antimicrobial agents (10−1,000 fold less susceptible to antimicrobial agents compared to its planktonic counterparts) necessitates a targeted and controlled delivery of antibiotics. To this end, engineered antibiotics nanoparticles for inhaled anti-biofilm therapy via the dry powder inhaler (DPI) platform are investigated in this dissertation. First, in vitro investigations into the effects of antibiotic release profiles afforded by the encapsulation of antibiotics in nanoparticles on E. coli biofilm susceptibility shows that a biphasic extended release profile is necessary for maximum biofilm eradication. A high initial antibiotic concentration ensures that maximum biofilm cells were killed, while the subsequent slower extended release maintained an antibiotic concentration high enough to arrest biofilm proliferation. The initial maximum killing of biofilm cells is crucial as surviving biofilm cells are less susceptible to antibiotics. Second, lipid–polymer hybrid nanoparticles which combine the advantages of (i) the biofilm targeting ability of liposomes and (ii) the structural integrity and stability of polymeric nanoparticles, has been shown to result in higher antibiotic encapsulation efficiency compared to polymeric nanoparticles alone, hence are able to provide a higher initial antibiotic concentration. Furthermore, hybrid nanoparticles have been found to demonstrate higher in vitro efficacy against biofilm of P. aeruginosa compared to non-hybrid nanoparticles. For these reasons, lipid–polymer hybrid nanoparticles can be utilized as a vehicle for inhaled anti-biofilm therapy. However, the encapsulation of antibiotics in lipid–polymer hybrid nanoparticles must be tailored to the type of antibiotic and lipid used, as experiments performed show that ionic interactions between oppositely charged drug molecules and lipids prevent nanoparticle formation. Third, a new drug-polyelectrolyte nanoplexation method developed in the present work has resulted in antibiotic nanoparticles with high drug loading (up to 80%). The simple method, which only involves the mixing of two aqueous solutions of oppositely charged antibiotic and polyelectrolyte, has also been applied in the production of amorphous nanoparticles of a sparingly soluble antibiotic. The resultant amorphous antibiotic nanoparticle exhibits (i) faster dissolution rate, (ii) enhanced saturation solubility, and (iii) higher achievable supersaturation level compared to those of the raw crystalline drug. Finally, the engineered antibiotic nanoparticles can be transformed into inhalable dry powder nano-aggregates for pulmonary delivery by means of the spray drying or spray-freeze-drying (SFD) technique, both of which have been investigated in the present work. While the spray drying method to prepare dry powder nano-aggregates is more rapid and straightforward, the SFD is more suitable for the processing of thermosensitive nanoparticles due to the avoidance of a high operating temperature. Adjuvant selection in SFD is crucial as the adjuvant dictates the resulting nanoparticle-adjuvant structures, which in turn influence the aerodynamic diameter and aqueous re-dispersibility of the dry-powder aggregates.DOCTOR OF PHILOSOPHY (SCBE

Green preparation of antibiotic nanoparticle complex as potential anti-biofilm therapeutics via self-assembly amphiphile–polyelectrolyte complexation with dextran sulfate

Nanoscale antibiotic delivery has emerged as a promising therapeutic means to treat lung biofilm infection owed to its sputum penetrating ability. Due to the high antibiotic dosage requirement in anti-biofilm therapy, the most suitable formulation for this purpose is the antibiotic nanoparticles themselves, instead of the more extensively studied antibiotic-loaded nano-carriers, which often exhibit low drug loading. The present work details the preparation and characterization of antibiotic nanoparticle complex (or nanoplex) by self-assembly amphiphile–polyelectrolyte complexation process. Ofloxacin (OFX) and levofloxacin (LEV) are used as the antibiotics with dextran sulfate (DXT) as the polyelectrolyte. The nanoplex possesses high drug loading (up to 80%) and size < 400 nm ideal for sputum penetration. Unlike existing methods to prepare drug nanoparticles, the present method is fast, energy-minimal, solvent-free, and highly efficient as manifested in nearly 100% of drug is transformed into nanoplex. The effects of drug-to-polyelectrolyte charge ratio, pH, drug, and salt concentrations on the nanoplex characteristics (i.e. size, stability, drug loading) are investigated from which the optimal preparation conditions have been identified. Higher complexation efficiency and stronger agglomeration tendency are observed for LEV nanoplex owed to its higher hydrophobicity. The antibiotics are completely released from the nanoplex in aqueous salt solution within 3 h and their antimicrobial activity is preserved upon complexation. The nanoplex is readily transformed into amorphous dry powders that remain stable after one-month storage owed to the high glass transition temperature. The antibiotic nanoplexes are highly charged enabling their subsequent functionalization for targeted delivery and controlled drug release purposes

Green amorphous nanoplex as a new supersaturating drug delivery system

The nanoscale formulation of amorphous drugs represents a highly viable supersaturating drug-delivery system for enhancing the bioavailability of poorly soluble drugs. Herein we present a new formulation of a nanoscale amorphous drug in the form of a drug–polyelectrolyte nanoparticle complex (or nanoplex), where the nanoplex is held together by the combination of a drug–polyelectrolyte electrostatic interaction and an interdrug hydrophobic interaction. The nanoplex is prepared by a truly simple, green process that involves the ambient mixing of drug and polyelectrolyte (PE) solutions in the presence of salt. Nanoplexes of poorly soluble acidic (i.e., ibuprofen and curcumin) and basic (i.e., ciprofloxacin) drugs are successfully prepared using biocompatible poly(allylamine hydrochloride) and dextran sulfate as the PE, respectively. The roles of salt, drug, and PE in nanoplex formation are examined from ternary phase diagrams of the drug–PE complex, from which the importance of the drug’s charge density and hydrophobicity, as well as the PE ionization at different pH values, is recognized. Under the optimal conditions, the three nanoplexes exhibit high drug loadings of 80–85% owing to the high drug complexation efficiency ( 90–96%), which is achieved by keeping the feed charge ratio of the drug to PE below unity (i.e., excess PE). The nanoplex sizes are 300–500 nm depending on the drug hydrophobicity. The nanoplex powders remain amorphous after 1 month of storage, indicating the high stability owed to the PE’s high glass-transition temperature. FT-IR analysis shows that functional groups of the drug are conserved upon complexation. The nanoplexes are capable of generating prolonged supersaturation upon dissolution with precipitation inhibitors. The supersaturation level depends on the saturation solubility of the native drugs, where the lower the saturation solubility, the higher the supersaturation level. The solubility of curcumin as the least-soluble drug is magnified 9-fold upon its transformation to the nanoplex, and the supersaturated condition is maintained for 5 h

Biofilm-like lactobacillus rhamnosus probiotics encapsulated in alginate and carrageenan microcapsules exhibiting enhanced thermotolerance and freeze-drying resistance

Microcapsules containing high-density biofilm-like Lactobacillus rhamnosus probiotics, in place of planktonic cells, are developed in order to enhance the cell viability upon exposures to stresses commonly encountered during food lifecycle (i.e., heating, freeze-drying, refrigerated storage, and acid). The high-density (HD) capsules are prepared by in situ cultivation of the planktonic cells in the confined space of polysaccharide-based capsules (i.e., chitosan-coated alginate and carrageenan capsules). Compared to their planktonic counterparts, the HD capsules exhibit higher freeze-drying resistance (40×) and higher thermotolerance upon prolonged wet heat exposures at 60 and 70 °C (12–8000×), but not at higher temperatures even for short exposures (i.e., 80 and 100 °C). The enhanced viability of the HD capsules, however, is not observed during the refrigerated storage and exposure to the simulated gastric juice. The alginate capsules are superior to carrageenan owed to their better cell release profile in the simulated intestinal juice and storage viability

Self-assembled amorphous drug–polyelectrolyte nanoparticle complex with enhanced dissolution rate and saturation solubility

The dissolution rate and solubility of poorly soluble drugs can be enhanced by formulating them into stable amorphous nanoparticle complex (nanoplex). For this purpose, a highly sustainable self-assembly drug–polyelectrolyte complexation process is developed, with ciprofloxacin and dextran sulfate as the drug and polyelectrolyte models, respectively. The nanoplex are prepared by mixing two aqueous salt solutions – one containing the drug and the other containing the oppositely charged polyelectrolyte. The nanoplex suspension is transformed into stable dry-powder form by freeze-drying. The effects of drug concentration, drug-to-polyelectrolyte charge ratio, and salt concentration on the complexation efficiency, yield, drug loading, and nanoplex morphology are examined. The dissolution rates and solubility of the nanoplex are characterized and compared to raw drug crystals. Nearly spherical amorphous nanoplex having fairly uniform sizes in the range of 200−400 nm and 80% drug loading are successfully produced at ⩾80% complexation efficiency and yield. The complexation efficiency is governed by the drug concentration and its ratio to the salt concentration. The nanoplex powders exhibit approximately twice higher dissolution rate and solubility than raw drug crystals and remain stable after one-month storage. Overall, amorphous nanoplex represent a promising bioavailability-enhanced formulation of poorly soluble drugs owed to their superior characteristics and ease of preparation