Data for "Scale-independent microfluidic production of cationic liposomal adjuvants and development of enhanced lymphatic targeting strategies."

This data supports work looking at the development of liposomal adjuvants using microfluidics and their targeting.

Abstract: Cationic liposomes prepared from dimethyldioctadecylammonium bromide (DDAB) and trehalose 6,6′-dibehenate (TDB) are strong liposomal adjuvants. As with many liposome formulations, within the laboratory DDAB:TDB is commonly prepared by the thin film method which is difficult to scale and gives high batch-to-batch variability. In contrast, controllable technologies such as microfluidics offer robust, continuous and scale-independent production. Therefore, within this study, we have developed a microfluidic production method for cationic liposomal adjuvants that is scale-independent and produces liposomal adjuvants with analogous biodistribution and immunogenicity compared to those produced by the small-scale lipid hydration method. Subsequently, we further developed the DDAB:TDB adjuvant system to include a lymphatic targeting strategy using microfluidics. By exploiting a biotin-avidin complexation strategy, we were able to manipulate the pharmacokinetic profile and enhance targeting and retention of DDAB:TDB and antigen within the lymph nodes. Interestingly, re-directing these cationic liposomal adjuvants did not translate into notably improved vaccine efficacy.

All data is collated and presented within Microsoft Excel.

Figure 1. Establishing the operating parameters for DDAB:TDB using microfluidics. The effect of lipid concentration on the physicochemical characteristics of the DDAB:TDB adjuvant formulations manufactured using microfluidics. DDAB:TDB liposomes were prepared at TFR 10 mL/min, FRR 3:1 and varying the initial concentration from 0.3 to 24 mg/mL and (A) Particle size, (B) PDI and (C) Zeta potential measured. The impact of the different parameters adopted during microfluidics formulation of DDAB:TDB liposomes was tested and (D) particle size (bars) and PDI (open circles) and (E) zeta potential of the DDAB:TDB liposomes manufactured by using microfluidics. Results represent the mean ± SD from at least 3 independent experiments.

Figure 2. Comparing production methods for liposomal adjuvants. Physicochemical characteristics (A), particle size and PDI, (B) ZP, (C) H56 antigen loading and (D) lipid recovery of the DDAB:TDB liposomes produced either using the LH method or microfluidics at different flow rate ratios (1:1, 3:1 and 5:1). Results represent the mean ± SD of at least 3 independent batches. Ag = H56 antigen; -Ag represents without H56 antigen, +Ag represents with H56 antigen.

Figure 3. Movement from the injection site of DDAB:TDB liposomal adjuvants their associated antigen produced by LH and MF. Percentage of (A) liposomes and (B) antigen retained at the site of injection. Dual labelling of liposomes and antigen by incorporating either 3H-lipid or 125I-antigen was used for the detection of the liposomes and antigen respectively at different time points. Liposomes were manufactured using either the LH method or microfluidics (FRR 1:1, 3:1 and 5:1). Results represent the mean of 3 mice ± standard deviation.

Figure 4. Percentage of liposomes (A,B,C) and antigen (D,E,F) detected at the local lymph node (Inguinal (A, D), popliteal (B, E) and mesenteric (C, F)) after intramuscular injection. Dual labelling of liposomes and antigen by incorporating either 3H-lipid or 125I-antigen was used for the detection of the liposomes and antigen respectively at different time points. Liposomes were manufactured using either the LH method or microfluidics (FRR 1:1, 3:1 and 5:1). Results represent the mean of 3 mice ± standard deviation.

Figure 5. Physicochemical characteristic comparison of the DDAB:TDB formulations using LH and microfluidics and DDAB:TDB liposomes incorporating a DSPE-PEG(2000) biotin. (A) Particle size (bars) and PDI (dots); (B) H56 antigen loading (bars) and ZP (triangles). Results represent the mean ± SD from at least 3 independent experiments.

Figure 6. Movement from the injection site of DDAB:TDB-biotin liposomes and their associated antigen with or without previous administration of avidin. Percentage of (A) liposomes and (B) antigen retained at the site of injection. Dual labelling of liposomes and antigen by incorporating either 3H-lipid or 125I-antigen was used for the detection of the liposomes and antigen respectively at different time points. Liposomes were manufactured using either the LH method (DDAB:TDB LH) or microfluidics (DDAB:TDB-biotin). Results represent the mean of 3 mice ± standard deviation. Significant differences are shown as *p<0.05, **p<0.01, ***p<0.001.

Figure 7. Percentage of biotin-DDAB:TDB liposomes (A,B,C) and antigen (D,E,F) detected at the local lymph node (Inguinal (A, D), popliteal (B, E) and mesenteric (C, F)) after intramuscular injection with or without previous administration of avidin. Dual labelling of liposomes and antigen by incorporating either 3H-lipid or 125I-antigen was used for the detection of the liposomes and antigen respectively at different time points. Liposomes were manufactured using either the LH method (DDAB:TDB LH) or microfluidics (DDAB:TDB-biotin). Results represent the mean of 3 mice ± standard deviation. Significant differences are shown as *p<0.05, **p<0.01, ***p<0.001.

Figure 8. Antigen-specific IgG1 and IgG2c responses after intramuscular immunization using various DDAB:TDB liposomal adjuvants. Five C57BL/6 mice were intramuscularly immunized with H56 combined with different adjuvants and humoral response was analysed in blood. H56-specific IgG1 and IgG2c serum response detected by ELISA on sera collected on days 7, 21 and 49 after i.m. immunization. Antibody titers were expressed as the reciprocal of the highest dilution with an OD value ≥0.1 after background subtraction and responses from each of the 5 mice are individually plotted for each time point. Significant differences at the end of the study are shown as *p<0.05, **p<0.01, ***p<0.001.


Figure 9. Cytokine production in splenocyte and popliteal lymph node (POP) culture supernatants: IL-5, IL-17 and IFN-ɣ. C57BL/6 mice were intramuscularly immunized with H56 combined with different and spleens were collected 3 weeks after the last immunization. Values, expressed as picograms per milliliter, are reported as the mean value ± SD of H56-stimulated of five animals per group. Significant differences are shown as *p<0.05, **p<0.01, ***p<0.001.

Figure 10. Cytokine production at the injection site (SOI). (A) IFN-ɣ (B) IL-5 and (C) IL-17. C57BL/6 mice were intramuscularly immunized with H56 combined with different adjuvants and site of injections were excised 3 weeks after the last immunization. Values, expressed as picograms per miligram, are reported as the mean value ± SD of H56-stimulated of five animals per group. Significant differences are shown as *p<0.05, **p<0.01, ***p<0.001.