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. 2021 Jul;10(13):e2100125.
doi: 10.1002/adhm.202100125. Epub 2021 Jun 4.

Influence of the Modulation of the Protein Corona on Gene Expression Using Polyethylenimine (PEI) Polyplexes as Delivery Vehicle

Dingcheng Zhu  1   2   3 , Huijie Yan  1   2 , Zhuxian Zhou  1 , Jianbin Tang  1 , Xiangrui Liu  1 , Raimo Hartmann  4 , Wolfgang J Parak  2   5 , Youqing Shen  1 , Neus Feliu  2   6
Affiliations

Influence of the Modulation of the Protein Corona on Gene Expression Using Polyethylenimine (PEI) Polyplexes as Delivery Vehicle

Dingcheng Zhu et al. Adv Healthc Mater. 2021 Jul.

Abstract

The protein corona can significantly modulate the physicochemical properties and gene delivery of polyethylenimine (PEI)/DNA complexes (polyplexes). The effects of the protein corona on the transfection have been well studied in terms of averaged gene expression in a whole cell population. Such evaluation methods give excellent and reliable statistics, but they in general provide the final transfection efficiency without reflecting the dynamic process of gene expression. In this regard the influence of bovine serum albumin (BSA) on the gene expression of PEI polyplexes also on a single cell level via live imaging is analyzed. The results reveal that although the BSA corona causes difference in the overall gene expression and mRNA transcription, the gene expression behavior on the level of individual cell is similar, including the mitosis-dependent expression, distributions of onset time, expression pattern in two daughter cells, and expression kinetics in successfully transfected cells. Comparison of single cell and ensemble data on whole cell cultures indicate that the protein corona does not alter the transfection process after nuclear entry, including cell division, polyplex dissociation, and protein expression. Its influence on other steps of in vitro gene delivery before nuclear entry shall render the difference in the overall transfection.

Keywords: gene delivery; protein corona; single cell tracking.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Cellular uptake, luciferase expression, and mRNA transcription. a) Association of P 1/1/0 and P 1/1/4 polyplexes prepared from Cy5‐labelled DNA (DNACy5) to HeLa cells in terms of Cy5 fluorescence I DNA‐Cy5 per cell as measured by flow cytometer. Cells were incubated with polyplexes at 1.4 µg mL−1 DNA (containing 40 ng mL−1 DNACy5) in serum free culture medium for t = 0.5–6 h and measured immediately. b) Luciferase expression of cells after exposure to polyplexes prepared from luciferase‐encoding plasmid (pLuci) for different incubation times t. Cells were incubated with polyplexes at 1.4 µg mL−1 pLuci in serum free medium for t = 0.5–6 h, followed by further culture in fresh 10% FBS supplemented cell culture medium without polyplexes for t′ = 24 h. The luciferase luminescence I Luci from cells was detected by a luminescence detection system. Luminescence is given in relative luminescence units (RLU) per amount of total proteins in RLU/mg. c) Luciferase expression I Luci after different culture time t′. Cells were incubated with polyplexes at 1.4 µg mL−1 DNA in serum free medium for t = 3 h, followed by further culture in fresh 10% FBS supplemented cell culture medium for t″ = 3–24 h. d) mRNA transcription upon exposure of cells to polyplexes at 1.4 µg mL−1 DNA in serum free medium for t = 3 h, followed by further culture in fresh 10% FBS supplemented cell culture medium for t' = 2–24 h. mRNA transcription is displayed in terms of the relative luciferase mRNA transcription number normalized to the house keeping gene GADPH, that is, detected number of luciferase mRNA/detected number of GADPH mRNA.
Figure 2
Figure 2
Dual delivery of pre‐mixed and post‐mixed P 1/1/0 and P 1/1/4 polyplexes to HeLa cells using enhanced green fluorescent protein (eGFP)‐encoding plasmid (peGFP) and red fluorescent protein (RFP)‐encoding plasmid (pRFP) as reporting genes. Cells were incubated with polyplexes at 1.4 µg mL−1 DNA in serum free cell culture medium for t = 3 h, followed by further culture in fresh 10% FBS supplemented cell culture medium without polyplexes for t′ = 24 h. The cells were imaged by confocal laser scanning microscopy (CLSM), and the fraction of fluorescent cells was calculated. The transfection efficiency P expression is thus given in terms of percentage of cells expressing eGFP or RFP. a) Only one type of plasmid (i.e., peGFP or pRFP) was added to the respective cell cultures. b) Distributions of eGFP fluorescence intensity (denoted as I eGFP) transfected by P 1/1/4 or P 1/1/0 using peGFP. N cell is the total amount of cells measured from the images. Insert images are cells expressing eGFP. Scale bars represent 50 µm. c,d) Dual delivery of peGFP and pRFP using pre‐mixed or post‐mixed polyplexes. For pre‐mixed polyplexes the two plasmids (i.e., peGFP or pRFP) were mixed first, and PEI was added later. For post‐mixed polyplexes, polyplexes containing one type of plasmid were prepared first, and the two types of polyplexes carrying different plasmids were mixed later. In each polyplex particle, pre‐mixed polyplexes contained two types of plasmids, whereas post‐mixed polyplexes contained only one type of plasmid. The two plasmids were mixed at 1:1 mass ratio. c) Percentage of cells expressing only eGFP, only RFP, and both in successfully transfected cells. d) Images of eGFP (shown in green) and RFP (red) expressing cells measured by CLSM. Cells expressing both proteins are shown in yellow. The scale bars represent 50 µm.
Figure 3
Figure 3
Time lapse images of eGFP expression in HeLa cells upon exposure to P 1/1/0 and P 1/1/4 polyplexes. Cells were incubated with polyplexes at 1.4 µg mL−1 peGFP in serum free medium for t = 3 h. Afterwards, cells were cultured in fresh 10% FBS supplemented cell culture medium and were imaged by CLSM every 10 min (the time point t′ = 0 refers to the start of imaging). a) Representative real‐time tracking of eGFP expression after P 1/1/0 transfection. The red outline indicates a non‐dividing cell. The green, yellow, blue, and pink outlines indicate cells that had divided during observation. The expressed eGFP signal is shown in the green fluorescence channel. The scale bars represent 50 µm. Cell tracking is shown in the lower panel. Here, the cell trajectories are shown in white. Daughter cells are indicated with the same number as their respective parent cells. b) Kinetics of eGFP expression (in terms of eGFP fluorescence intensity I eGFP) as calculated from the cells shown in (a). Circles indicate the time when cells started to divide. c) Statistics of non‐dividing and dividing cells expressing eGFP. N eGFP is the number of observed cells expressing eGFP, which had or had not divided during the observation period. d,e) eGFP expression of 21 pairs of daughter cells at the end of observation (t′ = 24 h) after transfection by d) P 1/1/0 and e) P 1/1/4 (for each pair of fluorescence the intensity of one daughter cell is shown in green, of the other daughter cell in red). f) Z‐stack images of P 1/1/0 polyplex distributions in the nuclei of two daughter cells just after mitosis. Cells were incubated with P 1/1/0 polyplexes prepared from DNACy5. Circles indicate DNA that was in the nucleus, as evidenced by orthogonal views (Figure S1.3.6.2, Supporting Information). The DNACy5 fluorescence is shown in red. The nuclei were stained with Hoechst 33342 and can be seen in the blue fluorescence channel. The scale bars represent 50 µm.
Figure 4
Figure 4
Onset and mitosis time of cells transfected by P 1/1/0 and P 1/1/4 polyplexes. The onset time t onset is the time which cells took to initiate eGFP expression post mitosis. The mitosis time t mitosis is the duration from the beginning of observation at t′ = 0 to the start of mitosis. Cells were incubated with polyplexes in serum free cell culture medium at 1.4 µg mL−1 peGFP for t = 3 h, followed by culture in fresh 10% FBS supplemented cell culture medium and were then monitored by time‐lapse CLSM. a,b) Distributions of onset time in a) P 1/1/0 and b) P 1/1/4 transfected cells. N eGFP (t onset) is the number of cells with respective onset time t onset. c,d) Correlation between onset and mitosis time in c) P 1/1/0 and d) P 1/1/4 transfected cells.
Figure 5
Figure 5
eGFP expression kinetics of cells transfected by P 1/1/0 and P 1/1/4 polyplexes. Cells were incubated with polyplexes in serum free cell culture medium at 1.4 µg mL−1 peGFP for t = 3 h, followed by culture in fresh 10% FBS supplemented cell culture medium and were then monitored by time‐lapse CLSM. a,b) The cumulative number of eGFP expressing cells N eGFP over time t′ upon transfection by a) P 1/1/0 and b) P 1/1/4 polyplexes. c–f) Expression kinetics in c,d) non‐dividing and e,f) dividing sub‐populations. Red curves indicate expression the kinetics of cells which stopped expression after reaching the maximum level. Green curves indicate the expression kinetics of cells which continuously expressed eGFP.
Figure 6
Figure 6
Dissociation and nuclear entry of P 1/1/0 and P 1/1/4 polyplexes in eGFP expressing cells. a,b) Polyplexes were prepared from fluorescence labeled PEIRBITC and DNACy5. HeLa cells were incubated with a) P 1/1/0 and b) P 1/1/4 polyplexes at 1.4 µg mL−1 DNACy5 in serum free culture medium for t = 3 h, followed by further culture in fresh 10% FBS supplemented cell culture medium without polyplexes for t′ = 24 h. In the fluorescence images PEIRBITC is shown in cyan, DNACy5 is shown in red, eGFP is shown in green, and nuclei stained with Hoechst 33342 are shown in the blue fluorescence channel. The scale bars represent 20 µm. c) Overlap degree of PEIRBITC (cyan) and DNACy5 (red) calculated by pixel intensity. Red bars show Manders' coefficient m 1 (DNACy5), the percentage of red fluorescent pixels overlapping with cyan fluorescent pixels. Cyan bars show Manders' coefficient m 2 (PEIRBITC), the percentage of cyan fluorescent pixels overlapping with red fluorescent pixels. N = 21 and 18 cells were analyzed for P 1/1/0 and P 1/1/4, respectively. d,e) Orthogonal views of intranuclear condensed d) P 1/1/0 and e) P 1/1/4 polyplexes in eGFP expressing cells after t′ = 24 h. The scale bars represent 20 µm. Additional data are shown Figure S1.4.3.1, Supporting Information.
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