DOI: 10.15129/f8211834-029d-4720-8d21-2e02234eef5d
title: Data for: "Formulation and manufacturing of lymphatic targeting liposomes using microfluidics."

description: This data supports work where we demonstrate a new method to produce lymphatic targeting liposomes using microfluidics.

Figure 1. The effect of charge on liposome biodistribution. Mice were injected intramuscularly with 4 formulations, as shown in A. The rate of clearance from the injection site (B) and the popliteal lymph node (POP) (C) are shown as a percentage of injected dose. The degree of aggregation of these formulations when exposed to simulated interstitial fluid is shown in D. Results are expressed of the mean ± SD of 3 mice per group. 
Figure 2. The effect of formulation on liposome update by macrophages. The presence of phosphatidylserine in liposomes shown to increase association with THP-1 (macrophages) cells was investigated by flow cytometry. (A) Percentage of THP-1 cells positive for fluorescence surface bounded or internalized) with macrophages at 0 mins and 180 mins. (C) Fold increase of mean fluorescent intensity of THP-1 cells co-cultured with above liposome formulations. Results denote mean ± SD of 3 independent experiments. 
Figure 3 The effect of liposome size on the clearance of liposomes from the injection site to the local draining lymph node. DSPC:Chol:PS liposomes were formulated as MLV (A) and SUV (B). Mice were intramuscular (i.m.) injected with SUV and MLV liposomes and the liposomes at the injection site (C) and popliteal lymph node (PLN) (D) are shown. (E) Percentage of THP-1 cells positive for fluorescence surface bounded or internalized) with macrophages at 0 mins and 180 mins. (F) Fold increase of mean fluorescent intensity of THP-1 cells co-cultured with above liposome formulations. Results are expressed of the mean ± SD of 3 mice per group. 
Figure 4. The effect of lipid dose on lymphatic uptake in mice following intramuscular injection of liposome formulations. Increasing doses of DSPC:Chol:PS liposomes were injected and the amount of liposomes at the injection site measured and shown as (A) Amount of PS (µg) at Injection site, (B) Amount of PS (µg) at Popliteal lymph node (PLN), (C) Percent dose of DSPC:Cholesterol:PS liposomes at three different escalated doses at popliteal lymph nodes. Results are expressed of the mean ± SD of 3 mice per group
Figure 5. The effect of exploiting a biotin-avidin complex system for lymphatic targeting. Mice were intramuscularly injected with either DSPC:Chol:PS:PEG2000 (6:4:2.5 µmoles; 2 mole% PEG) or DSPC:Chol:PS:PEG2000-biotin (6:4:2.5 µmoles; 2mole% PEG-biotin). Two groups of mice received the biotin-coated liposomes, one without pre-dosing of avidin (DSPC:Chol:PS:PEG2000-biotin) and one group receiving a pre-dose of avidin (DSPC:Chol:PS:PEG200-biotin-avidin). The percentage (%) injected dose of biotinylated liposomes in mice following intramuscular injection A: Injection site, B: Popliteal lymph node (POP), C: Inguinal lymph node (ILN) and D: Mesenteric lymph node (MLN). Results are expressed of the mean ± SD of 3 mice per group. 
Figure 6. Biodistribution of lymphatic targeted liposomes and antigen (H-56) in mice. Two liposome formulations were tested (avidin was intramusculary injected 2 hours prior injection of the main formulation). Liposomes and antigen dose was monitored at the site of injection (A & E), popliteal lymph node (B & F), Inguinal lymph node (C & G) and mesenteric lymph node (D & H) were isolated and counted for % injected dose of DSPC:Chol:PS and DSPC:Chol:PS:DSPE-PEG 2000 Biotin (20 mole%) formulations respectively. Results represent the mean ± SD of three replicate batches (n = 3).
Figure S1. Antigen-specific (A) IgG1 and (B) IgG2c responses in mice immunised with liposomes with and without the biotin-avidin complex formulation. C57BL/6 mice were intramuscularly immunised with the liposomes incorporating H56 and humoural responses were analysed in blood. H56-specific IgG1 (A, B, C) and IgG2c (D, E, F) serum responses detected by ELISA on sera collected (A, D) 7, (B, E) 21 and (C, F) 49 days after i.m. immunization. Antibody titres were expressed as the reciprocal log of the highest dilution with an OD value ≥0.2 after background subtraction.
Figure S2. Cytokine production in splenocyte and lymph node culture supernatants (IL-17 and IFN-ɣ) in mice immunised with liposomes with and without the biotin-avidin complex system. C57BL/6 mice were intramuscularly immunised with H56 incorporated in the various liposome formulations and spleens were collected 3 weeks after the last immunization. Levels of IFN-ɣ in the spleen (A), popliteal lymph nodes (B), and IL-17 in the spleen (C) and lymph nodes (D) were measured. Values, expressed as picograms per milliliter, are reported as the mean value ± SD of H56-stimulated of five animals per group.