Microfluidic encapsulation of bacteriophages in nanoliposomes and macrophage intracellular trafficking studies

Bacteriophages are viruses able to infect and kill their bacterial host upon replication within it. They are thought to be the most abundant organism on earth, since their population exceed the one of bacteria (about 1030) by a factor of 10. In many cases, the host range where a given bacteriophage is active is so narrow that it is usually specific to one species of bacteria. In the early 1920s, phage were used to treat bacterial infections in eastern Europe and spread to western companies that began to commercialize typhoid and urinary-tract infection therapies. Soon after, because of penicillin discovery, researchers abandoned this therapy for antibiotic treatment, mostly due to phage susceptibility to the immune system and other unknown behaviours. Due to the overuse and misuse of antibiotic drugs, antibiotic resistance fast become a serious problem of global concern, currently with no real solution, which represents a threat to humanity leading to a post-antibiotic era if no counter-measures and alternative strategies are undertaken. In the last decade, bacteriophage application for the treatment of bacterial infections has re-gained huge attention. However, phages delivered in buffer solutions are suboptimal, since they are not shielded from host defences that inexorably hinder phage activity. Therefore, for effective in vivo applications, e.g. crossing the acidic environment of the stomach or infecting intracellular pathogens, phages need to be delivered to the site of infection, for example via encapsulation. Encapsulation in targeted delivery systems will help phages to reach the target with high specificity, minimizing losses, undesired adverse side effects, and limiting the likelihood of emerging “super bacteria”. In the last decades, a considerable variety of stimuli-responsive micro/nanocarriers have been designed. Such systems show a transition to the supramolecular structure or in their chemical structure in response to given stimuli. A wise choice of the stimulus that triggers the variation allows releasing of their cargo in a spatial- and temporal- controlled fashion. The stimuli can be either external or internal and most commonly are thermal variation, pH, enzyme concentration, redox potential, magnetic field, ultrasound intensity and photo sensitivity. In this thesis, we study the encapsulation of bacteriophages in lipid carriers able to deliver and release the cargo in a controlled manner. Liposomes are biodegradable non-toxic micro/nano vesicles composed of a lipid bilayer, which encloses an aqueous core that offer many advantages in medicinal applications. It was demonstrated that phage encapsulation in liposomes improves stability. Indeed, when inside the aqueous core of a vesicle, the thermodynamics of the system is advantageous for phage storage. In this doctoral thesis we have assessed the encapsulation of two model phages, an Escherichia coli T3 podovirus (size ~65 nm) and a myovirus Staphylococcus aureus phage K (capsid head ~80 nm and phage tail length ~200 nm). Encapsulation in liposomes was carried out by producing the vesicles by alcohol injection method in a capillary microfluidic device, in the presence of the cargo. We generated liposomes having mean sizes between 100–300 nm and the encapsulation yield of T3 phages was 109 PFU ml-1 and for phage K was much lower at 105 PFU ml-1. The encapsulation efficiency was affected by the aggregation state of the virions. Furthermore, we discovered that phage K is able to interact with the lipid bilayer resulting in phages bound to the outer membrane of the liposomes instead of being encapsulated inside them. We utilised the encapsulated phages to determine whether 1) they are internalized by human macrophages in higher number compared to free phages and whether 2) they are able to reach the cytosol were intracellular pathogens reside. We discovered that, regardless the formulation of liposomes, encapsulated phages are intracellularized more efficiently in comparison to free phages. However, following uptake, liposomes and encapsulated phages are trafficked to the lysosome, where they are irreversibly digested. Therefore, we envisioned a different approach: a smaller antimicrobial agent than phages, namely a phage lysin, was co-encapsulated with a hemolysin able to make pores in the endosomal membrane. We observed that the co-encapsulated of the two proteins effectively escaped lysosomal degradation and defeated intracellular S. aureus. We expect this approach to be easily extended to phages and we suggest further perspectives and concrete future experiments. In this doctoral thesis, we made significant advances in the encapsulation and intracellular delivery of phages for antimicrobial treatment, and we obtain promising results that pave the way to the actual utilization of phages for medical treatment of infections, especially of antibiotic resistant ones.