Development of novel, biocompatible, polyester amines for microglia-targeting gene delivery

Abstract

Recent progress in personalized medicine and gene delivery has created exciting opportunities in therapeutics for central nervous system (CNS) disorders. Despite the interest in gene-based therapies, successful delivery of nucleic acids for treatment of CNS disorders faces major challenges. Here we report the facile synthesis of a novel, biodegradable, microglia-targeting polyester amine (PEA) carrier based on hydrophilic triethylene glycol dimethacrylate (TG) and low-molecular weight polyethylenimine (LMW-PEI). This nanocarrier, TG-branched PEI (TGP), successfully condensed double-stranded DNA into a size smaller than 200 nm. TGP nanoplexes were nontoxic in primary mixed glial cells and showed elevated transfection efficiency compared with PEI-25K and lipofector-EZ. After intrathecal and intracranial administration, PEA nanoplexes delivered genes specifically to microglia in the spinal cord and brain, respectively, proposing TGP as a novel microglia-specific gene delivery nanocarrier. The microglia-specific targeting of the TGP nanocarrier offers a new therapeutic strategy to modulate CNS disorders involving aberrant microglia activation while minimizing off-target side effects.

Scheme 1. Schematic illustration of microglia-targeting TGP/DNA nanoplexes. Novel TGP/DNA nanoplexes can be uptaken specifically by microglia, demonstrating their potential as a therapeutic nanocarrier of DNA or siRNA for CNS diseases.

Fig. 1. Schematic illustration of TGP synthesis.

Fig. 2. Physicochemical characterization of TGP nanoplexes. (A) and (B) Size and zeta-potential of TGP/pGL3 were analyzed using DLS at N/P ratios of 5, 10, 20, and 30. (C) TEM image showing the morphology of TGP/pGL3 nanoplexes stained with 2% aqueous uranyl acetate (scale bar: 200 nm). (D) Gel retardation assay of TGP/pGL3 nanoplexes prepared at various N/P ratios from 5 to 30.

Fig. 3. Effects on cell viability and transfection efficiency of TGP/pGL3 in primary mixed glia. (A) Cell viability was determined after incubation with TGP/pGL3 at N/P ratios from 5 to 30 for 24 h by MTS assay. PEI-25K/pGL3 was used as a control. (B) Transfection efficiency of the TGP/pGL3 nanoplex in primary mixed glia was evaluated by luciferase assay. Cells were transfected with TG/pGL3 with N/P ratios of 5, 10, 20, and 30; after 24 h, luciferase activity was measured. (C) Effect of bafilomycin A1 on the proton sponge effect of TGP/pGL3 and PEI/pGL3 nanoplexes. (n = 3, error bar represents standard deviation; *p <0.1, **p <0.05, ***p <0.01, one-way ANOVA compared to that of PEI/pGL3 nanoplexes and control (NT)).

Fig. 4. Uptake kinetics and DNA delivery of TGP in primary pure microglia. Primary pure microglial cells were transfected with Alexa488-TGP/pGL3 or TGP/Alexa546-pGL3 nanoplexes. After 5 and 24 h of transfection, live cells were stained with Hoechst (blue).

Fig. 5. Uptake kinetics and efficiency of Alexa488-TGP in primary mixed glia. (A) Primary mixed glial cells were transfected with 0.5 μg of Alexa488-TGP/pGL3. After 5 and 24 h of transfection, cells were immunostained for Iba-1 or GFAP. (B) Primary glial cells (5 × 10⁵ cells per well in 6-well plate) were transfected with 0.5 μg of Alexa488-TGP/pGL3, stained with APC-conjugated anti-CD11b and PE-conjugated anti-ACSA-2, and analyzed with a flow cytometer (n = 3 per each group). Representative data (mean ± SEM) are shown (*p <0.05, **p <0.01, ***p <0.001).

Fig. 6. Spinal cord regional delivery of Alexa488-TGP/pGL3. Mice received 0.1 μg of Alexa488-TGP/pGL3 by i.t. injection. (A) After one day, lumbar 4–6 spinal cord sections were stained with Iba-1, GFAP, and MAP-2 antibodies. Alexa488 signals were detected in Iba-1 positive cells (arrows). (B) After 24 h of Alexa488-TGP/pGL3 i.t. injection, cells of four regions of the spinal cord were isolated. Cells were stained with APC-conjugated anti-CD11b, PE-conjugated anti-ACSA-2, and anti-Thy-1.2 antibodies and analyzed using flow cytometry. (C) Representative histogram. Alexa488 signals were detected by flow cytometry and Alexa488-positive cells were gated. (D) Quantification graphs of Alexa488-positive cell population and mean fluorescence intensity. Representative data (mean ± SEM) are shown (**p < 0.01, ***p < 0.001).

Fig. 7. Brain microglia-specific delivery of Alexa488-TGP/pGL3. 0.1 μg of Alexa488-TGP/pGL3 was injected intracranially into the hippocampus (coordination ML −1.5 mm, AP +2.18 mm, DV +1.76 mm). (A) Brain slices were stained with Iba-1, GFAP, and NeuN antibodies. Alexa488 signals were detected in Iba-1-positive cells. Scale bar 50 μm. (B) 24 h after Alexa488-TGP/pGL3 nanoplex administration, intact hippocampus cells were isolated and stained with APC-conjugated anti-CD11b, PE-conjugated anti-ACSA-2, and anti-Thy-1.2 antibodies and analyzed using flow cytometry. The Alexa488-positive population was gated for each cell type. Data are expressed as mean ± SEM (n = 3 per each group, *p <0.05).