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Link to original content: http://pubmed.ncbi.nlm.nih.gov/36619382/
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. 2022 Dec 21:10:1066887.
doi: 10.3389/fbioe.2022.1066887. eCollection 2022.

PEGylation and folic-acid functionalization of cationic lipoplexes-Improved nucleic acid transfer into cancer cells

Marco Hoffmann  1 , Sven Gerlach  1 , Christina Hoffmann  1 , Nathalie Richter  1 , Nils Hersch  1 , Agnes Csiszár  1 , Rudolf Merkel  1 , Bernd Hoffmann  1
Affiliations

PEGylation and folic-acid functionalization of cationic lipoplexes-Improved nucleic acid transfer into cancer cells

Marco Hoffmann et al. Front Bioeng Biotechnol. .

Abstract

Efficient and reliable transfer of nucleic acids for therapy applications is a major challenge. Stabilization of lipo- and polyplexes has already been successfully achieved by PEGylation. This modification reduces the interaction with serum proteins and thus prevents the lipoplexes from being cleared by the reticuloendothelial system. Problematically, this stabilization of lipoplexes simultaneously leads to reduced transfer efficiencies compared to non-PEGylated complexes. However, this reduction in transfer efficiency can be used to advantage since additional modification of PEGylated lipoplexes with functional groups enables improved selective transfer into target cells. Cancer cells overexpress folate receptors because of a significantly increased need of folate due to high cell proliferation rates. Thus, additional folate functionalization of PEGylated lipoplexes improves uptake into cancer cells. We demonstrate herein that NHS coupling chemistries can be used to modify two commercially available transfection reagents (Fuse-It-DNA and Lipofectamine® 3000) with NHS-PEG-folate for increased uptake of nucleic acids into cancer cells. Lipoplex characterization and functional analysis in cultures of cancer- and healthy cells clearly demonstrate that functionalization of PEGylated lipoplexes offers a promising method to generate efficient, stable and selective nucleic acid transfer systems.

Keywords: DNA-transfer; PEGylation; biocompatibility; functionalized lipoplexes; selective nucleic acid transfer.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Quantification of folate receptor one alpha (FolR1α) protein levels and DNA transfer into healthy and cancer cells. FolR1α was quantified by western blot in MCF-7 breast cancer cells and healthy primary human fibroblasts (HFF) (A) as well as U87 glioblastoma cells and healthy primary cortical neurons (B). The relative quantities (RQ) were adjusted to tubulin and the resulting ratios were analyzed for significance. For all cell types, transfection efficiency was determined using Fuse-It-DNA and Lipofectamine® 3000 with transfer of an eGFP encoding plasmid. Results were recorded by fluorescence microscopy (phase contrast and eGFP channel) and flow cytometry. Scale bar 200 μm. n = at least three independent experiments.
FIGURE 2
FIGURE 2
Development of in vivo applicable transfection protocols and functionalization of Fuse-It-DNA as well as Lipofectamine® 3000 with PEG-FA. To optimize manufacturer’s transfection protocols (in vitro protocols) for in vivo applications, protocols were established that require minimum volumes to transfer 1 μg of plasmid (in vivo protocol). In addition, lipoplexes were functionalized with different molarities of NHS-PEG-FA-stocks with concentrations between 0.4 to 12 mM by post modification. Analysis of transfections was performed by fluorescence microscopy and flow cytometry (A). The influence of PEG-FA modification on transfer efficiencies is shown in (B). Unmodified complexes as well as the PEG-FA modified complexes were characterized for zeta potential (C) and complex size (D). Scale bar 200 μm. n = at least three independent experiments.
FIGURE 3
FIGURE 3
Effect of pure PEGylation and additional FA-functionalization on transfer efficiencies. MCF-7 and HFF cells, were transfected with eGFP plasmid using Fuse-It DNA (in vivo protocol). In addition, this lipoplexes were further functionalized with PEG and PEG-FA at equal concentrations. Analysis was performed by fluorescence microscopy and flow cytometry (A). To demonstrate the supplemental effects of FA, relative transfection efficiencies are plotted in (B) and alterations of mean values are shown in green (positive) and red (negative). In (C), progressions of transfection efficiencies by PEG-FA with improvements in favor of cancer cells (green) and deteriorations (red) are shown. Scale bar 200 μm. n = at least three independent experiments.
FIGURE 4
FIGURE 4
Effect of pure PEGylation and additional FA-functionalization on transfer efficiencies. In U87 glioblastoma cells and primary cortical neurons, eGFP plasmid was transferred using Lipofectamine® 3000 (in vivo applicable protocol). In addition, this protocol was further modified with PEG and PEG-FA at equal concentrations. Analysis was performed by fluorescence microscopy and flow cytometry (A). To demonstrate the supplemental effects of FA, relative transfection efficiencies are plotted in (B) and alterations of mean values are shown in green (positive) and red (negative). In (C), progressions of transfection efficiencies by PEG-FA with improvements in favor of cancer cells shown in green and deteriorations in red are shown. Scale bar 200 μm. n = at least three independent experiments.
FIGURE 5
FIGURE 5
Reduction of FA binding capacity and the impact on transfer efficiencies of FA functionalized lipoplexes. To reduce the uptake capacity of FA- functionalized lipoplexes, cancer cells (MCF-7) and healthy HFF cells were pre-incubated with 1 mM FA. The resulting transfer efficiencies were analyzed by fluorescence microscopy and flow cytometry (A). The reduction in transfection efficiencies of FA-functionalized lipoplexes was characterized comparatively between unmodified in vivo protocols and PEG-FA functionalized lipoplexes (0.4–12 mM) for cancer cells (B) and healthy cells (C). Effects were normalized to the influence of free FA incubation on the in vivo protocol. Scale bar 200 μm. n = at least three independent experiments.
FIGURE 6
FIGURE 6
Reduction of FA binding capacity and the impact on transfer efficiencies of FA functionalized lipoplexes. To reduce the uptake capacity of PEG-FA functionalized lipoplexes, cancer cells (U87) and healthy primary cortical neurons were pre-incubated with 1 mM FA. The resulting transfer efficiencies were analyzed by fluorescence microscopy and flow cytometry (A). The reduction in transfection efficiencies of FA-functionalized lipoplexes was characterized comparatively between unmodified in vivo protocols and FA functionalized lipoplexes (0.4–12 mM) for cancer cells (B) and healthy cells (C) normalized to the influence of FA incubation on the in vivo protocol. Scale bar 200 μm. n = at least three independent experiments.
FIGURE 7
FIGURE 7
Therapeutic potential characterization by in vitro assays. To investigate the therapeutic potential of the functionalization developed here, a ca-Caspase3 was transferred using FA-functionalized lipoplexes ((A), (B) fusion/(D), (E) lipofection). Quantification of ca-Caspase3 activity in target and non-target cells is shown in (C) and (F). Scale bar 200 μm. n = at least three independent experiments.
FIGURE 8
FIGURE 8
Characterization of PEG-FA functionalized lipoplexes. Physicochemical characterization of FA functionalized complexes compared with unmodified lipoplex-variants are shown in (A) (particle size) and (B) (zeta potential). n = at least three independent experiments.
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