Arginine-Modified Polymers Facilitate Poly (Lactide-Co-Glycolide)-Based Nanoparticle Gene Delivery to Primary Human Astrocytes

Figures & data

Table 1 Cell/Donor Information

Cell Culture Type	Donor Number	Gestational Age	Sex	Passage/Week in Culture	Expt./Fig. #
Human Embryonic Kidney 293	N/A	N/A	N/A	P42	Figure 1A
Primary Human Astrocytes	#1	105 d	Male	P5	Figures 1A and 3A–C
	#2	117 d	Male	P5	Figure 1B
	#3	96 d	Female	P3	Figure 2A and B
	#4	122 d	Female	P2	Figure 2A and B
	#5	105 d	Female	P4	Figure 2A and B
	#6	125 d	Female	P4	Figure 2A and B
Primary Human Neurons	#1	57 d	Female	W3	Figure 2C
	#2	76 d	Male	W4	Figure 2C
	#3	77 d	Female	W3	Figure 2C

Figure 1 PLGA/pDNA-NPs were taken up by astrocytes but failed to facilitate exogenous gene expression. Luciferase reporter pDNA encapsulated in PLGA-NPs was used to assess PLGA-mediated gene delivery (A). HEKs or primary human astrocytes were treated with 120 µg PLGA/pDNA-NPs for 2, 3, and 6 days. Luciferase reporter assay was used to verify exogenous gene expression. Data are graphed as mean ± SD from a representative experiment with duplicated measurements from triplicate wells. Two additional experiments illustrated the same trend. (B) Primary human astrocytes were treated with YOYO-pDNA encapsulated in PLGA-NPs (60 or 120 µg) and imaged at 6, 24, 48 and 96 hours using fluorescent microscopy to gauge PLGA-NP-mediated pDNA delivery to the cytoplasm.

Figure 2 Co-transfection of PLGA/pDNA-NPs with A₅P₁₀ established an efficient and biocompatible mechanism for astrocyte gene therapy. Primary human astrocytes (A and B) or neurons (C) were treated with luciferase pDNA encapsulated in PLGA-NPs (60 or 120 µg) alone or with A₅P₁₀ polymer (1 or 2 µg) for 48 hours. (A) Luciferase activity was quantified using luciferase expression assay. The x-axis was broken to indicate the scale in luciferase activity, ie, bottom (0–1 RLU x 10⁴) and the top (10–200 RLU x 10⁴). Statistical significance was determined via two-way ANOVA and Tukey’s post hoc test. (B and C) Cytotoxicity was measured by LDH assay and normalized to control for each donor. Data represent mean ± SEM in 3–4 donors. Statistical significance was determined via one-way ANOVA followed by Tukey’s post hoc (**p<0.01, ***p<0.001).

Table 2 NP Characterization

Construct	Size (nm)	Polydispersity Index	Zeta Potential (mV)
A5P10 alone	155.8 ± 6.2	0.28 ± 0.05	7.72 ± 2.93
PLGA/pDNA-NPs	331.6 ± 20.5	0.39 ± 0.04	−2.59 ± 0.26
PLGA/pDNA + A5P10 (120:1)	361.6 ± 22.6	0.44 ± 0.03	−1.67 ± 0.12
PLGA/pDNA + A5P10 (60:1)	342.4 ± 6.3	0.38 ± 0.02	−1.09 ± 0.06
PLGA/pDNA + A5P10 (30:1)	344.5 ± 7.0	0.42 ± 0.03	−0.69 ± 0.40

Figure 3 A₅P₁₀ increased PLGA/pDNA-NP-mediated uptake, intracellular trafficking, and nuclear delivery of pDNA. Primary human astrocytes were treated with PLGA/pDNA-NPs (60 or 120 µg) alone or in combination with 2 µg A₅P₁₀ for 24 hours. A₅P₁₀ coupled with YOYO-pDNA was used as a positive control. Confocal images (A) and colocalization analysis (B) were achieved using YOYO-pDNA and Hoechst nuclear dye. White arrows indicate YOYO and Hoechst colocalization. Scatter regions 1 and 2 depict pixels for YOYO and Hoechst channels, respectively, and scatter region 3 determines the pixels colocalizing between the two channels. (C) Graphs report mean ± SD for calculated total YOYO pixels divided by total pixels per image and (D) weighted colocalization coefficients in relation to total Hoechst pixels per image from a minimum of 8–10 images of triplicate wells per condition. Statistical significance was determined via one-way ANOVA followed by Tukey’s post hoc test (**p<0.01, ***p<0.001).

Figure 4 Obstacles for gene delivery to CNS. A successful gene delivery system to the CNS must achieve optimal trafficking and survival in the periphery including stability in circulation and evasion of the immune system. A critical dilemma for these constructs is bypassing the BBB in order to reach the tissue or cell of interest. Ensuring specificity is also of heightened importance for gene therapy applications. Once targeting is accomplished, delivery systems must overcome a variety of obstacles for intracellular trafficking including uptake, endolysosomal escape, nuclear entry, DNA release, and finally, expression of the therapeutic target. Moreover, systemic biocompatibility is an essential objective. Our report demonstrates (solid lines) PLGA/pDNA+A₅P₁₀ was able to facilitate enhanced uptake and intracellular trafficking (Figure 3C), nuclear entry of pDNA (Figure 3D), and exogenous gene expression (Figure 2A) while maintaining biocompatibility in astrocytes and neurons (Figure 2B and C). To translate this system for successful gene therapy, additional investigations are warranted to determine the potential of PLGA/pDNA+A₅P₁₀ to overcome the remaining barriers and to further elucidate the intracellular mechanisms mediating PLGA/pDNA+A₅P₁₀ optimization (dashed lines). (Created with BioRender.com).