Nanoencapsulation of Bacteriophages in Liposomes Prepared Using Microfluidic Hydrodynamic Flow Focusing

Abstract

Increasing antibiotic resistance in pathogenic microorganisms has led to renewed interest in bacteriophage therapy in both humans and animals. A "Trojan Horse" approach utilizing liposome encapsulated phages may facilitate access to phagocytic cells infected with intracellular pathogens residing therein, e.g., to treat infections caused by Mycobacterium tuberculosis, Listeria, Salmonella, and Staphylococcus sp. Additionally, liposome encapsulated phages may adhere to and diffuse within mucosa harboring resistant bacteria which are challenges in treating respiratory and gastrointestinal infections. Orally delivered phages tend to have short residence times in the gastrointestinal tract due to clinical symptoms such as diarrhea; this may be addressed through mucoadhesion of liposomes. In the present study we have evaluated the use of a microfluidic based technique for the encapsulation of bacteriophages in liposomes having mean sizes between 100 and 300 nm. Encapsulation of two model phages was undertaken, 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). The yield of encapsulated T3 phages was 10⁹ PFU/ml and for phage K was much lower at 10⁵ PFU/ml. The encapsulation yield for E. coli T3 phages was affected by aggregation of T3 phages. S. aureus phage K was found to interact with the liposome lipid bilayer resulting in large numbers of phages bound to the outside of the formed liposomes instead of being trapped inside them. We were able to inactivate the liposome bound S. aureus K phages whilst retaining the activity of the encapsulated phages in order to estimate the yield of microfluidic encapsulation of large tailed phages. Previous published studies on phage encapsulation in liposomes may have overestimated the yield of encapsulated tailed phages. This overestimation may affect the efficacy of phage dose delivered at the site of infection. Externally bound phages would be inactivated in the stomach acid resulting in low doses of phages delivered at the site of infection further downstream in the gastrointestinal tract.

Keywords: E. coli; S. aureus; bacteriophage therapy; intracellular infections; liposome fabrication; microfluidics; nanoencapsulation.

Figure 1

Schematic showing the microcapillary micromixing device for the production of liposomes. (A) 3D model of the co-axial glass capillary device. Solutions were pumped using two microfluidic pumps using a total flow rate of ~1 ml h⁻¹ and a flow rate ratio (FRR) of aqueous to organic phase (Q_a:Q_o) of 2:1. (B) The round capillary was inserted into a square capillary that delivered the aqueous phase containing phages. The internal round capillary delivered the lipid dissolved in IPA solution. (C) Once the organic phase mixes with the aqueous phase, the phospholipids experience a highly polar environment that leads to self-assembly of lipids into bilayer discs. The latter grow in the radial direction by collision with other discs or through addition of phospholipid molecules from solution. When the disc bending energy is overcome by the energy gain when the edge around the bilayer disc is removed, the planar bilayer closes upon itself resulting in a spherical structure entrapping the surrounding solution and any phages in the vicinity of the bilayer.

Figure 2

NanoSight measurements of the size distributions of liposomes formed using different DSPC:cholesterol molar ratios: (A) DSPC only (dotted line), 5:1 DSPC:cholesterol molar ratio (dashed line), and 1:1 DSPC:cholesterol molar ratio (solid line). Liposomes were prepared at room temperature (~20°C) using aqueous to organic phase volumetric flow rate ratio FRR of 2:1 and the total volumetric flow rate of ~1 ml h⁻¹. (B) Inset showing average mean sizes, error bars indicate one standard deviation. *Indicates significance (p < 0.05) using 2-sample t-test.

Figure 3

NanoSight NTA measurements of size distributions of purified T3 phage (left) and phage K (right), respectively, at 10⁷, 10⁸, 10⁹ and 10⁸, 10⁹, 10¹⁰ PFU/ml. Insets show CryoTEM images of observed phage aggregates and the schematized depiction of phage cluster sizes as a function of phage titer. Some heads on the lower CryoTEM images are transparent (empty), probably due to denaturation of DNA during encapsulation or loss of DNA during CryoTEM imaging.

Figure 4

Cryo-TEM images showing (A) encapsulation of phage K, and (B,C) interaction between phage K tails and the outer leaflet of the bilayer membrane of formed liposomes (DSPC:cholesterol molar ratio was 5:1), some tails look contracted. (A1–C1) Schematized depiction showing encapsulation of phage K within the liposome (favored outcome) and interaction of phage K with the outer surface of unilamellar liposome membrane and lipid bilayer (unfavorable outcome). (D) Chemical structure of DSPC and (E) chemical structure of teichoic acid (phosphate groups highlighted in red).

Figure 5

(A) S. aureus phage K encapsulation in DSPC:cholesterol liposomes. Phages were encapsulated at FRR of 2:1 at room temperature. Phage titer of liposome encapsulated phages prior to and after acid exposure at pH 2.75 (with and without Triton X-100 disruption). *Indicates significance (p < 0.05) using 2-sample t-test. (B) The titer of free phages in the supernatant before and after incubation with empty liposomes at 4, 25, and 37°C for 15 min. After incubation, liposomes were separated by centrifugation at 13,000 × g for 3 min and the phage titer in the supernatant was measured. The difference in the titer occurred due to phage adsorption onto the outer leaflet of bilayer membranes. The maximum phage adsorption was observed at 4°C, which caused the highest phage titer reduction in the supernatant at 4°C. The adsorption was still highly significant at 37°C. *Indicates significance (p < 0.05) using 2-sample t-test of each sample compared with controls. (C) pH stability for free (non-encapsulated) phage K exposed to pH 2.75 and free phage K incubated with pre-made empty liposomes for 15 min at 37°C and subsequently dialyzed at pH 2.75 for 60 min. Error bars represent one standard deviation.

Figure 6

E. coli T3 phage encapsulation in DSPC:cholesterol liposomes. T3 phages were encapsulated at FRR of 2:1 at room temperature. Free phages were separated by centrifugation for 10 min at 13,000 × g and re-suspended in SM buffer, the process was repeated three times to remove unencapsulated phages. Liposomes were disrupted with Triton X-100 and phage titer assessed by plaque assay. (A) Phage T3 titers following encapsulation, after removal of unencapsulated free phages (following three wash steps) and after liposome disruption with Triton X-100. (B) Phage encapsulation yield using different initial phage titers and after encapsulation, free phage removal by centrifugation (as above) and then liposome disruption with Triton X-100. *Indicates significance (p < 0.05) using 2-sample t-test.