Development of a low toxicity, effective pDNA vector based on noncovalent assembly of bioresponsive amino-β-cyclodextrin:adamantane-poly(vinyl alcohol)-poly(ethylene glycol) transfection complexes

Abstract

A host:guest-derived gene delivery vector has been developed, based on the self-assembly of cationic β-CD derivatives with a poly(vinyl alcohol) (MW 27 kDa) (PVA) main chain polymer bearing poly(ethylene glycol) (MW 750) (PEG) or MW 2000 PEG and acid-labile adamantane-modified (Ad) grafts through an acid-sensitive benzylidene acetal linkage. These components were investigated for their ability to promote supramolecular complex formation with pDNA using two different assembly schemes, involving either precomplexation of the pendent Ad-PVA-PEG polymer with the cationic β-CD derivatives before pDNA condensation (method A) or pDNA condensation with the cationic β-CD derivatives prior to addition of Ad-PVA-PEG to engage host:guest complexation (method B). The pendent polymers were observed to degrade under acidic conditions while remaining intact for more than 5 days at pH 7. HeLa cell culture data show that these materials have 10(3)-fold lower cytotoxicities than 25 kDa bPEI while maintaining transfection efficiencies that are superior to those observed for this benchmark cationic polymer transfection reagent when the method A assembly scheme is employed. These findings suggest that degradable cationic polymer constructs employing multivalent host:guest interactions may be an effective and low-toxicity vehicle for delivering nucleic acid cargo to target cells.

Figure 1

Structures of amino-β-CDs, Ad-PVA-PEG₇₅₀ and Ad-PVA-PEG₂₀₀₀.

Figure 2

400 MHz ¹H NMR spectra of Ad-PVA-PEG in the absence (A), in the presence (B) of β-CD; Ad-PVA in the absence (C), and presence (D) of β-CD in D₂O at 20 °C, and Ad-PVA with β-CD at pH = 7 (E) and pH = 4 (F) in D₂O at 20 °C.

Figure 3

(A) Zeta potential measurements of pDNA:amino-β-CD complexes and pDNA:bPEI complexes. DLS Measurements of (B) pDNA:1:Ad-PVA-PEG₇₅₀ and pDNA:3:Ad-PVA-PEG₇₅₀ complexes formulated by Method A and (C) pDNA:3 and pDNA:3:Ad-PVA-PEG₇₅₀ complexes formulated by Method B.

Figure 4

AFM images of (A) Ad-PVA-PEG₇₅₀ and (B) 1:1 β-CD:Ad-PVA-PEG₇₅₀. Images of pDNA:3:Ad-PVA-PEG₇₅₀ prepared by Method B at (C) N:P = 2 and (D) N:P = 6. The insets illustrate the possible structures in these images. The samples were prepared by adding a drop of solution to the mica surface and then slowly evaporating the sample at 25 °C overnight.

Figure 5

Cytotoxicities of 1, 2, 3, Ad-PVA-PEG₇₅₀ and 1:Ad-PVA-PEG₇₅₀ host:guest complexes in HeLa cells using 25kD bPEI as a control. The cells were treated with increasing concentrations of amino-β-CDs, 1:Ad-PVA-PEG₇₅₀ complexes, and bPEI for 24 h in serum-free media before analysis by MTT assay.

Figure 6

In vitro transfection efficiencies of amino-β-CD host:guest complexes with (A) Ad-PVA-PEG₇₅₀ and mhGFP pDNA (B) Ad-PVA-PEG₂₀₀₀ and mhGFP pDNA in HeLa cells; with 25kD bPEI (control) considered as 100% transfection efficiency. Fluorescence microscope images of transfected HeLa cells (C) bPEI 25k, (D) 3:Ad-PVA-PEG₇₅₀ using Method A at N:P = 20 in serum-free media using GFP gene as a reporter gene; scale bar: 100μm.