J Korean Med Sci.  2021 Dec;36(49):e333. 10.3346/jkms.2021.36.e333.

Vimentin Targeted Nano-gene Carrier for Treatment of Renal Diseases

• Mathew AP
• Uthaman S
• Bae EH
• Lee JY
• Park IK

• KMID: 2523553
• DOI: http://doi.org/10.3346/jkms.2021.36.e333

Abstract

Background
Chronic kidney disease (CKD) is a global health problem, and there is no permanent treatment for reversing kidney failure; thus, early diagnosis and effective treatment are required. Gene therapy has outstanding potential; however, the lack of safe gene delivery vectors, a reasonable transfection rate, and kidney targeting ability limit its application. Nanoparticles can offer innovative ways to diagnose and treat kidney diseases as they facilitate targetability and therapeutic efficacy.
Methods
Herein, we developed a proximal renal tubule-targeting gene delivery system based on alternative copolymer (PS) of sorbitol and polyethyleneimine (PEI), modified with vimentin-specific chitobionic acid (CA), producing PS-conjugated CA (PSC) for targeting toward vimentin-expressing cells in the kidneys. In vitro studies were used to determine cell viability, transfection efficiency, serum influence, and specific uptake in the human proximal renal tubular epithelial cell line (HK-2). Finally, the targeting efficiency of the prepared PSC gene carriers was checked in a murine model of Alport syndrome.
Results
Our results suggested that the prepared polyplex showed low cytotoxicity, enhanced transfection efficiency, specific uptake toward HK-2 cells, and excellent targeting efficiency toward the kidneys.
Conclusion
Collectively, from these results it can be inferred that the PSC can be further evaluated as a potential gene carrier for the kidney-targeted delivery of therapeutic genes for treating diseases.

Keyword

Kidney; Polyethyleneimine; Chitobionic Acid; Targeted Delivery; HK-2 Cells

Figure

• 

  Fig. 1 Schematic representation showing (A) the preparation of PSC/pDNA nanocomplexes and (B) the mode of expression of PSC/pDNA nanocomplexes in the in vivo system.PS = SDA crosslinked polyethyleneimine, PSC = PS-conjugated chitobionic acid, CA = chitobionic acid, PCT =proximal convoluted tubule, DCT =distal convoluted tubule.

• 

  Fig. 2 Representative 1H NMR spectra of SDA, PEI600, PS, CA, and PSC, confirming the formation of PS and PSC.SDA = sorbitol diacrylate, PEI600 = polyethyleneimine 600 Da, PS = SDA crosslinked polyethyleneimine, CA = chitobionic acid, PSC = PS-conjugated chitobionic acid.

• 

  Fig. 3 Physiochemical characterizations of PSC/pDNA nanocomplexes (A) Agarose gel electrophoresis of PSC/pDNA nanocomplexes at various N/P ratios ranging from 0.1 to 20 with 1 µg of pDNA. (B) The hydrodynamic diameter and (C) zeta potential measured at various N/P ratios from 2 to 20. (D) Field emission Transmission electron microscopy image of the PSC/pDNA complex of N/P 10 (low and high magnification images).PSC = PS-conjugated chitobionic acid.

• 

  Fig. 4 Cell viability assay of (A) the polymer alone and (B) nanocomplexes at various N/P ratios in HK-2 cell lines (n = 3, error bars represent standard deviations).PEI = polyethyleneimine, PS = SDA crosslinked polyethyleneimine, PSC = PS-conjugated chitobionic acid.*P < 0.05 **P < 0.01 ***P < 0.001, ****P < 0.0001.

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  Fig. 5 Transfection efficiency of nanocomplexes at various N/P ratios in HK-2 cells (A) in the absence of serum (B) and in the presence of 10% FBS.PEI = polyethyleneimine, PS = SDA crosslinked polyethyleneimine, PSC = PS-conjugated chitobionic acid.

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  Fig. 6 Transfection efficiency of polyplexes at various N/P ratios in (A) HK-2 cells and (B) MCF-7 cells (n = 3, error bars represent standard deviations, ***P < 0.001).PS = SDA crosslinked polyethyleneimine, PSC = PS-conjugated chitobionic acid, PEI = polyethyleneimine.

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  Fig. 7 Competition assay in the presence or absence of CA in HK-2 cells (n = 3, error bars represent standard deviations, ***P < 0.001).PS = SDA crosslinked polyethyleneimine, PSC = PS-conjugated chitobionic acid, CA = chitobionic acid.

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  Fig. 8 Cellular uptake assessed in HK-2 cells after treatment with different N/P ratios for 4 hours (scale bar = 20 µm).

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  Fig. 9 (A) Ex vivo fluorescence images of isolated organs 24 hours after the intravenous injection of PSC-F675/pDNA nanocomplexes. (B) Quantification of fluorescence intensity from the organs.

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  Fig. 10 Histology examination of kidney sections by confocal microscopy showing the presence of accumulated PSC-F675/pDNA in the kidneys (A) 20× magnification (B) 40× magnification.

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