Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Apr 7;16(4):507.
doi: 10.3390/pharmaceutics16040507.

Effect of Micromixer Design on Lipid Nanocarriers Manufacturing for the Delivery of Proteins and Nucleic Acids

Enrica Chiesa  1 , Alessandro Caimi  2 , Marco Bellotti  2 , Alessia Giglio  1 , Bice Conti  1 , Rossella Dorati  1 , Ferdinando Auricchio  2 , Ida Genta  1
Affiliations

Effect of Micromixer Design on Lipid Nanocarriers Manufacturing for the Delivery of Proteins and Nucleic Acids

Enrica Chiesa et al. Pharmaceutics. .

Abstract

Lipid-based nanocarriers have emerged as helpful tools to deliver sensible biomolecules such as proteins and oligonucleotides. To have a fast and robust microfluidic-based nanoparticle synthesis method, the setup of versatile equipment should allow for the rapid transfer to scale cost-effectively while ensuring tunable, precise and reproducible nanoparticle attributes. The present work aims to assess the effect of different micromixer geometries on the manufacturing of lipid nanocarriers taking into account the influence on the mixing efficiency by changing the fluid-fluid interface and indeed the mass transfer. Since the geometry of the adopted micromixer varies from those already published, a Design of Experiment (DoE) was necessary to identify the operating (total flow, flow rate ratio) and formulation (lipid concentration, lipid molar ratios) parameters affecting the nanocarrier quality. The suitable application of the platform was investigated by producing neutral, stealth and cationic liposomes, using DaunoXome®, Myocet®, Onivyde® and Onpattro® as the benchmark. The effect of condensing lipid (DOTAP, 3-10-20 mol%), coating lipids (DSPE-PEG550 and DSPE-PEG2000), as well as structural lipids (DSPC, eggPC) was pointed out. A very satisfactory encapsulation efficiency, always higher than 70%, was successfully obtained for model biomolecules (myoglobin, short and long nucleic acids).

Keywords: lipid nanocarriers; lipid nanoparticles; liposomes; manufacture; microfluidics; nucleic acids; proteins.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Graphical representation of the different microfluidic chips with details regarding the different geometries of the ridges.
Figure 2
Figure 2
Screening DoE data elaboration for liposome size: (A) Pareto chart and (B) main effect plot.
Figure 3
Figure 3
Response surface plot for neutral liposome size. (A) Predicted size in relation to TFR and FRR, lipids concentration was constant at 2.19 mM. (B) Predicted size in relation to FFR and lipids concentration [Lipids]. TFR was 8 mL/min.
Figure 4
Figure 4
(A) Effect of the organic solvent on the liposome formation: produced vesicles were characterized in terms of size and PDI. Ethanol (red bar), methanol (blue bar) and isopropanol (green bar) were used. Sidak’s multiple comparison tests revealed statistical significance for * p-value < 0.05 and ** p-value < 0.01. (B) TEM image of liposomes produced by using ethanol and FRR of 4. (C) TEM image of liposomes produced by using ethanol and FRR of 6. Scale bar of 200 nm.
Figure 5
Figure 5
Effect of formulation variables on liposome size and size distribution. (A) Effect of the Chol concentration by using a lipid concentration of 1.1 mM and 8 mL/min TFR, three FRRs were tested: 9 (red bar), 6 (blue bar) and 4 (green bar). ** p-value < 0.01. (B) Effect of the Chol concentration by using a lipid concentration of 2.19 mM and 8 mL/min TFR, 3 FRRs were tested: 9 (red bar), 6 (blue bar) and 4 (green bar). * p-value < 0.05, ** p-value < 0.01, **** p-value < 0.0001. (C) Impact of eggPC as structural lipid (eggPC:Chol 77:23 mol%); 2 lipid concentrations were tested: 1.10 and 2.19 mM.
Figure 6
Figure 6
Effect of formulation variables on liposomes size and size distribution: (A) Production of nanocarrier made of DSPC, Chol, PEGylated lipid (DSPE-PEG550 or DSPE-PEG550) at the molar ratio 77:20:3 mol%. Lipid concentration was 1.1 mL and stealth liposomes were prepared at 8 mL/min TFR and 6 (red bar) or 4 (blue bar) FRR. (B) Production of cationic liposomes made of DSPC, Chol and DOTAP (3%, 10% and 20 mol%) starting for a lipid solution of 1.10 mM. Operating parameters: TFR of 8 mL/min and FRR of 6 (red bar) or 4 (blue bar). (C) Impact of the aqueous buffer on liposomes (77:23 mol% DSPC:Chol). Operating parameters: TFR of 8 mL/min and FRR of 4. (D) Impact of the aqueous buffer on the production of cationic liposomes (77:20:3 mol% DSPC:Chol: DOTAP). Operating parameters: TFR of 8 mL/min and FRR of 6. *, **, *** mean p-value < 0.05, <0.001, <0.0001, respectively.
Figure 7
Figure 7
CAD design of the two different micromixers adopted in this study (first row); visualization of the tangential projection of the velocity magnitude vectors onto the plane defined by Section A (second row); visualization of the tangential projection of the velocity magnitude onto the plane defined by Section B (third row).
Figure 8
Figure 8
Effect of the channel geometry on the liposomes physical features of (A) liposomes made of DSPC and Chol at the molar ratio of 77:23 mol% and (B) liposomes made of DSPC and Chol at the molar ratio of 65:35 mol%. FRR was changed to 9 (red bar), 6 (blue bar) and 4 (green bar), while lipid concentration and TFR were kept constant, namely 1.1 mM and 8 mL/min. ANOVA test revealed differences for * p-value < 0.05 and ** p-value < 0.01.
Figure 9
Figure 9
Liposome size and size distribution before (start) and after (final) dialysis of (A) neutral liposomes (DSPC:Chol 77:23 mol%) and (B) stealth liposomes (3 mol% DSPE PEG550). FRR was set at 6 (blue bar) and 4 (green bar), while lipid concentration and TFR were kept constant, namely 1.1 mM and 8 mL/min. ANOVA test revealed differences for * p-value < 0.05 and **** p-value < 0.0001.
Figure 10
Figure 10
Size and size distribution before (start) and after (final) dialysis of cationic liposomes with different concentrations of DOTAP: (A) 3 mol%, (B) 10 mol% and (C) 20 mol%. FRR was set at 6 (blue bar) and 4 (green bar), while lipid concentration and TFR were kept constant, namely 1.1 mM and 8 mL/min. ANOVA test revealed differences for * p-value < 0.05 and *** p-value < 0.001.
Figure 11
Figure 11
Effect of Myo encapsulation into stealth liposomes (DSPC:Chol:DSPE-PEG550 77:20:3 mol%): size and size distribution. FRR was set at 6 (blue bar) and 4 (green bar) while lipid concentration and TFR were kept constant, namely 1.10 mM and 8 mL/min. ANOVA test revealed differences for **** p-value < 0.0001.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Vargason A., Anselmo A., Mitragotri S. The evolution of commercial drug delivery technologies. Nat. Biomed. Eng. 2021;5:951–967. doi: 10.1038/s41551-021-00698-w. - DOI - PubMed
    1. Durán-Lobato M., López-Estévez A.M., Cordeiro A.S., Dacoba T.G., Crecente-Campo J., Torres D., Alonso M.J. Nanotechnologies for the delivery of biologicals: Historical perspective and current landscape. Adv. Drug Deliv. Rev. 2021;176:113899. doi: 10.1016/j.addr.2021.113899. - DOI - PubMed
    1. Mitchell M.J., Billingsley M.M., Haley R.M., Wechsler M.E., Peppas N.A., Langer R. Engineering precision nanoparticles for drug delivery. Nat. Rev. Drug Discov. 2021;20:101–124. doi: 10.1038/s41573-020-0090-8. - DOI - PMC - PubMed
    1. Farokhzad O.C., Langer R. Impact of nanotechnology on drug delivery. ACS Nano. 2009;3:16–20. doi: 10.1021/nn900002m. - DOI - PubMed
    1. Colonna C., Dorati R., Conti B., Caliceti P., Genta I. Sub-unit vaccine against S. aureus-mediated infections: Set-up of nanosized polymeric adjuvant. Int. J. Pharm. 2013;452:390–401. doi: 10.1016/j.ijpharm.2013.05.037. - DOI - PubMed

LinkOut - more resources

-