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. 2021 Mar 23;11(3):825.
doi: 10.3390/nano11030825.

Advances in the Formulation and Assembly of Non-Cationic Lipid Nanoparticles for the Medical Application of Gene Therapeutics

Richard K Fisher 3rd  1 , Phillip C West  1 , Samuel I Mattern-Schain  2 , Michael D Best  2 , Stacy S Kirkpatrick  1 , Raymond A Dieter 3rd  1 , Joshua D Arnold  1 , Michael R Buckley  1 , Michael M McNally  1 , Michael B Freeman  1 , Oscar H Grandas  1 , Deidra J H Mountain  1
Affiliations

Advances in the Formulation and Assembly of Non-Cationic Lipid Nanoparticles for the Medical Application of Gene Therapeutics

Richard K Fisher 3rd et al. Nanomaterials (Basel). .

Abstract

Lipid nanoparticles have become increasingly popular delivery platforms in the field of gene therapy, but bench-to-bedside success has been limited. Many liposomal gene vectors are comprised of synthetic cationic lipids, which are associated with lipid-induced cytotoxicity and immunogenicity. Natural, non-cationic PEGylated liposomes (PLPs) demonstrate favorable biocompatibility profiles but are not considered viable gene delivery vehicles due to inefficient nucleic acid loading and reduced cellular uptake. PLPs can be modified with cell-penetrating peptides (CPPs) to enhance the intracellular delivery of liposomal cargo but encapsulate leakage upon CPP-PLP assembly is problematic. Here, we aimed to identify parameters that overcome these performance barriers by incorporating nucleic acid condensers during CPP-PLP assembly and screening variable ethanol injection parameters for optimization. CPP-PLPs were formed with R8-amphiphiles via pre-insertion, post-insertion and post-conjugation techniques and liposomes were characterized for size, surface charge, homogeneity, siRNA encapsulation efficiency and retention and cell associative properties. Herein we demonstrate that pre-insertion of stearylated R8 into PLPs is an efficient method to produce non-cationic CPP-PLPs and we provide additional assembly parameter specifications for a modified ethanol injection technique that is optimized for siRNA encapsulation/retention and enhanced cell association. This assembly technique could provide improved clinical translation of liposomal based gene therapy applications.

Keywords: cell penetrating peptides; cellular uptake; gene therapy; lipid nanoparticles; non-cationic liposomes; non-viral gene delivery; siRNA encapsulation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Mass spectroscopy confirmed R8 peptide conjugation to DSPE-PEGdbco via azide-alkyne cycloaddition reaction (i.e., click chemistry).
Figure 2
Figure 2
Incorporation of STR-R8 via pre-insertion resulted in the highest siRNA EE% above all.
Figure 3
Figure 3
Ca2+-mediated EtOH injection of R8-PLPs resulted in (A) increased siRNA retention and EE% in (B) 50–60 nm liposome samples with a (C) homogeneous size distribution.
Figure 3
Figure 3
Ca2+-mediated EtOH injection of R8-PLPs resulted in (A) increased siRNA retention and EE% in (B) 50–60 nm liposome samples with a (C) homogeneous size distribution.
Figure 4
Figure 4
(A) Increasing lipid:siRNA (wt-to-wt) ratios enhanced R8-PLP siRNA EE% while (B) gel exclusion assays ran on unpurified liposome samples served as physical confirmation of increasing siRNA EE%.
Figure 4
Figure 4
(A) Increasing lipid:siRNA (wt-to-wt) ratios enhanced R8-PLP siRNA EE% while (B) gel exclusion assays ran on unpurified liposome samples served as physical confirmation of increasing siRNA EE%.
Figure 5
Figure 5
(A) Assay development indicated treatment with 100 ug/mL heparin was the minimum concentration required for effective removal of all outer-associated siRNA. (B) Heparin displacement assays indicated siRNA complexation to R8-PLP surface (panel 1), total EE% (panel 2), and siRNA entrapment within the R8-PLP core (panels 3 and 4), in a lipid:siRNA dependent manner.
Figure 5
Figure 5
(A) Assay development indicated treatment with 100 ug/mL heparin was the minimum concentration required for effective removal of all outer-associated siRNA. (B) Heparin displacement assays indicated siRNA complexation to R8-PLP surface (panel 1), total EE% (panel 2), and siRNA entrapment within the R8-PLP core (panels 3 and 4), in a lipid:siRNA dependent manner.
Figure 6
Figure 6
(A) Assay development indicated treatment with 0.5 ug/mL RNase A enzyme was the minimum concentration sufficient for complete siRNA degradation. (B) RNase stability assays indicated that R8-PLP nanoparticles sufficiently protected encapsulated and complexed siRNA when exposed to nuclease digestion.
Figure 6
Figure 6
(A) Assay development indicated treatment with 0.5 ug/mL RNase A enzyme was the minimum concentration sufficient for complete siRNA degradation. (B) RNase stability assays indicated that R8-PLP nanoparticles sufficiently protected encapsulated and complexed siRNA when exposed to nuclease digestion.
Figure 7
Figure 7
Slower injection rates during R8-PLP assembly (A) had no significant effect in siRNA EE%, but resulted in (B) increased mean diameter and (C) lower PDIs.
Figure 7
Figure 7
Slower injection rates during R8-PLP assembly (A) had no significant effect in siRNA EE%, but resulted in (B) increased mean diameter and (C) lower PDIs.
Figure 8
Figure 8
STEM images of R8-PLPs.
Figure 9
Figure 9
R8-PLP nanoparticles demonstrated enhanced cell association in vitro.
Figure 10
Figure 10
R8-PLP nanoparticles demonstrated negligible cytotoxic effects in vitro under (A) qualitative and (B) quantitative analyses.
See this image and copyright information in PMC

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