Thesis

Continuous manufacturing of liposome formulations incorporating anti-cancer agents : exploiting novel methods in microfluidics

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Awarding institution
  • University of Strathclyde
Date of award
  • 2025
Thesis identifier
  • T17383
Person Identifier (Local)
  • 202161746
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Abstract
  • The preparation of liposomes can be challenging, costly, and time-consuming if prepared by traditional manufacturing methods. This research aimed to investigate microfluidic methods for producing liposomes that can be loaded by the pH gradient method. The liposomes formulation was prepared from a combination of 3 lipids: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N [methoxy (polyethene glycol)-2000 (ammonium sulfate)] (DSPE-PEG2000) at a w/w ratio of 3:1:1 respectively. The parameters of the microfluidic process studied included flow rate ratio, total flow rate, and the incubation time for loading the drug (using acridine orange as a surrogate initially) at 60 C° for 30, 40, 60 and 120 minutes were tested and the process optimised. The process was then applied to doxorubicin, and the particle size, PDI, and encapsulation efficiency were studied. Using this production method, liposomes with different sizes (65 nm, 80 nm and 90 nm in diameter), with a low PDI (≤ 0.25) and high entrapment efficiency (≥ 90%) were produced. These parameters were controlled by varying the flow rate ratio of lipid to aqueous phase but were not impacted by increasing production speeds. These results show the rapid production of liposomes using microfluidics. All liposome products prepared (irrespective of size) were stable for one month at 4 ⁰C with no significant change in the physicochemical properties before and after loading. This method of liposome production was further used to co-load two drugs (Doxorubicin and Disulfiram), and their toxicity was evaluated with chick embryo models with different concentrations. The results showed that all liposomes were not toxic. The result shows that the tumour development with cell lines MDA MB231 GFP was approximately 57%, soft tissue sarcoma was 40%, and U87 MG glioblastoma was 30% compared to other tumour cell lines. The in vitro dissolution was studied, and by considering the f2 similarity factors the results show a significant effect in the in vitro dissolution between the FRR 2:1 and 3:1 size (80 nm and 65 nm) also between the FRR 2:1 and 1.5:1 size (80 nm and 90 nm) but no significant difference in the dissolution between FRR 1.5:1 and 3:1 (90 nm and 65 nm). The results show significant differences in the effect on the growth of MDA MB 231 breast tumour cells between liposomes loaded with Doxorubicin size 80 nm, liposomes loaded with Doxorubicin size 65 nm and free Doxorubicin. However, in the spheroid model, there was no significant difference between loaded liposome size (90 nm and 65 nm) in reducing the growth of MDA MB 231 cell during the time of treatment (0-21 days). The results also show significant effect on the growth of MDA MB 231 breast tumour cells for all loaded liposome in all sizes (65 nm, 80 nm, 90 nm) and free Doxorubicin when compared with control for reducing the growth of tumour cell that indicant all liposomes sizes are active for treatment of breast cancer. In vivo, the biodistribution in mice for different liposome sizes (65 nm, 80 nm, 90 nm) was studied. The results show no significant difference in distribution between all liposome sizes in all organs (lung, liver, spleen, heart and kidney) and plasma. However, the result shows a higher concentration of Doxorubicin in the plasma than in the other organs. This indicates that these differences in size and manufacturing process did not impact distribution. When considering co-loaded liposomes (containing disulfiram and doxorubicin (1:1, 2:1 and 0.5:1 v/v), the results show a synergistic effect for all ratios of the combination of disulfiram with doxorubicin after 24h of treatment in MDA MB231 breast cancer cells compared to the free drug combination and one liposome combination for both drugs. In conclusion, these studies demonstrate that microfluidics can prepare a range of liposomes with controlled size and high loading. The particle sizes tested tended to make a difference in vitro but not to biodistribution in vivo. This highlights the versatility of the microfluidic production process for liposomes, which can facilitate the translation from laboratory research to larger-scale manufacturing while maintaining product consistency and quality.
Advisor / supervisor
  • Perrie, Yvonne
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