Application of liposomes for antimicrobial photodynamic therapy
As more and more antibiotic-resistant organisms are emerging continuously, the development of new antibiotics falls behind the evolution of antibiotic-resistance. Thus there is an urgent need to search for alternative antibacterial drugs. Nowadays, antimicrobial photodynamic therapy (APDT) has emerged as an efficacious modality to treat various kinds of microbial infections. Meanwhile, liposomes are shown to be an attractive drug delivery system in the treatment of infections and may improve the APDT efficiency. Therefore, the aims of this study are to develop bacteria-targeting liposomes to further improve APDT, and to develop a high-throughput method for screening a large number of photosensitizer-loaded liposomal formulations. In publication 1 and 2, a generation II photosensitizer (PS), temoporfin, was incorporated into liposomes for APDT, afterwards two bacteria-targeting ligands, the antimicrobial peptide WLBU2 and the lectin Wheat Germ Agglutinin (WGA) were successfully coupled to the surface of temoporfin-loaded liposomes, respectively, using an aminogroup-reactive functional lipid: NHS-PEG2000-DSPE. The delivery of temoporfin to Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (P. aeruginosa) was confirmed by fluorescence microscopy and flow cytometry, thus demonstrating that more temoporfin was delivered to bacteria by the modified liposomes than by unmodified liposomes. Consequently, both of the two bacteria-targeting liposomes eradicated all MRSA and enhanced the photodynamic inactivation of P. aeruginosa in the in vitro photodynamic inactivation test. In particular, WLBU2 seems to be a better bacteria-targeting ligand than WGA. These results demonstrate that the strategy of using bacteria-targeting liposomes is promising for improving the APDT efficiency against both gram-positive and gram-negative bacteria in the local infections. To speed up the screening process of liposomal formulations and develop a method suitable for large-scale production of liposomes, a novel strategy for the fast and convenient high-throughput screening of liposomal formulations was developed in Publication 3, utilizing the automation of the ethanol injection method. This strategy was illustrated by the preparation and screening of the liposomal formulation library of temoporfin. To optimize the formulations, different parameters were investigated, including lipid types, lipid concentration, the ratio of ethanol to aqueous solution, the ratio of drug to lipid and the addition of functional phospholipids. Numerous formulations (261 samples) were screened quickly in a high-throughput way. The factors affecting the properties of liposomes were investigated step-by-step, where liposomes were prepared and characterized automatically, making it easy and fast to optimize the liposomal formulations of temoporfin. The obtained optimized liposomes were unilamellar spheres with a diameter of about 50 nm, and were very stable for over 20 weeks. What’s more, this high-throughput method is also applicable for preparing bacteria-targeting liposomes of different compositions, showing many advantages over the conventional methods. All the results demonstrate that this high-throughput screening strategy is fast, automated, materially efficient, labor-saving, time-saving, economic, facile, and highly reproducible. This approach is promising for the development of new formulations to enhance APDT; due to the nature of the process, the approach is readily amenable to scale-up of production. In conclusion, bacteria-targeting liposomes are useful drug delivery system for APDT, and the high-throughput method will facilitate the search for more suitable liposomal formulations. These PS-loaded liposomal formulations have potential clinical applications for the treatment of microbial infections.