Decationized polyplexes as stable and safe carrier systems for improved biodistribution in systemic gene therapy

Abstract

Many polycation-based gene delivery vectors show high transfection in vitro, but their cationic nature generally leads to significant toxicity and poor in vivo performance which significantly hampers their clinical applicability. Unlike conventional polycation-based systems, decationized polyplexes are based on hydrophilic and neutral polymers. They are obtained by a 3-step process: charge-driven condensation followed by disulfide crosslinking stabilization and finally polyplex decationization. They consist of a disulfide-crosslinked poly(hydroxypropyl methacrylamide) (pHPMA) core stably entrapping plasmid DNA (pDNA), surrounded by a shell of poly(ethylene glycol) (PEG). In the present paper the applicability of decationized polyplexes for systemic administration was evaluated. Cy5-labeled decationized polyplexes were evaluated for stability in plasma by fluorescence single particle tracking (fSPT), which technique showed stable size distribution for 48 h unlike its cationic counterpart. Upon the incubation of the polymers used for the formation of polyplexes with HUVEC cells, MTT assay showed excellent cytocompatibility of the neutral polymers. The safety was further demonstrated by a remarkable low teratogenicity and mortality activity of the polymers in a zebrafish assay, in great contrast with their cationic counterpart. Near infrared (NIR) dye-labeled polyplexes were evaluated for biodistribution and tumor accumulation by noninvasive optical imaging when administered systemically in tumor bearing mice. Decationized polyplexes exhibited an increased circulation time and higher tumor accumulation, when compared to their cationic precursors. Histology of tumors sections showed that decationized polyplexes induced reporter transgene expression in vivo. In conclusion, decationized polyplexes are a platform for safer polymeric vectors with improved biodistribution properties when systemically administered.

Keywords: Biocompatibility; Biodistribution; EPR; Gene delivery; Nanoparticle; Polymer.

Fig. 1

¹NMR spectra of p(HPMA-co-PDTEMA-co-APMA) and p(HPMA–DMAE-co-PDTEMA-co-Cy7)-b-PEG in DMSO.

Fig. 2

GPC chromatograms (a) UV detection at 700 nm of the crude product from coupling of Cy7-NHS to p(HPMA-co-PDTEMA-co-APMA)-b-PEG, using a 2 molar equivalent excess of Cy7-NHS to primary amines in the polymer. (b) Refractive index signal (RI) and UV signal at 700 nm of the final purified p(HPMA–DMAE-co-PDTEMA-co-Cy7)-b-PEG polymer.

Fig. 3

Decationized pHP-PEG and cationic pHDP-PEG polyplexes size distribution determined by NTA in PBS at 25 °C.

Fig. 4

The size distribution of cationic pHDP-PEG and decationized pHP-PEG polyplexes as determined by fSPT after incubation in 90% (v/v) human plasma at 37 °C for 1 h and 48 h. Size distribution was also determined in 20 mM HEPES pH 7.4.

Fig. 5

Safety evaluation of decationized polyplexes (a) HUVEC cell relative viability relative upon exposure for 24 h or 72 h to decationized pHP-PEG, cationic pHDP-PEG and b-PEI from 0.1 mg/mL to 3 mg/mL. Results are expressed as mean±SD (n=4). (b) Representative images of zebrafish embryo development upon exposure to decationized pHP-PEG and to cationic pHDP-PEG at concentrations ranging from 0.1–3 mg/mL. *significant mortality, **significant developmental defects (i.e. decreased pigmentation, delayed hatching ratio and slower heartbeat). (c) zebrafish survival upon exposure to decationized pHP-PEG and cationic pHDP-PEG polymers in comparison with b-PEI (*100% mortality; n=6). hpf (hours post-fertilization).

Fig. 6

2D FRI quantification as %ID (per 100 μl) of decationized pHP-PEG and cationic pHDP-PEG Cy7-labeled polyplexes signals in (a) blood and (b) urine at different time points p.i. Results are expressed as mean±SD (n=3). *p<0.01 (t-test).

Fig. 7

Noninvasive in vivo 3D CT-FMT imaging of the biodistribution and tumor accumulation of decationized pHP-PEG and cationic pHDP-PEG Cy7-labeled polyplexes. (a) Principle of 3D CT-FMT imaging: anatomical information obtained using μCT is used to assign the Cy7 signals coming from polyplexes to a specific organ or tissue of interest. The images were obtained at 15 min, 4 h, 24 h and 48 h p.i. and show Cy7 localization mainly in liver (red) and kidney (orange). Tumor accumulation was more prominent for decationized polyplexes. (b) Quantification of the tumor accumulation and biodistribution of Cy7-labeled decationized pHP-PEG and cationic pHDP-PEG polyplexes in tumors, liver, lungs, kidney, bladder and heart, expressed as %ID per 100 mm³ tissue. Results are presented as mean±SD (n=3).

Fig. 8

Ex vivo analysis. (a) Representative ex vivo 2D FRI assessment of the tumor accumulation and biodistribution of decationized pHP-PEG and cationic pHDP-PEG Cy7-labeled polyplexes at 48 h p.i. (b) Quantification of polyplex accumulation in tumors and healthy organs. Results are expressed as mean±SD (n=3). (c–d) Fluorescence microscopy imaging (c) and quantification (d) of decationized pHP-PEG and cationic pHDP-PEG Cy7-labeled polyplexes (blue) accumulating in tumors at 48 h p.i. and inducing EGFP expression (green). Blood vessels are labeled using rhodamine-lectin (red). Results are expressed as mean±SD (n=3). *p<0.05 (t-test).

Scheme 1

Synthesis of p(HPMA-co-PDTEMA-co-APMA)-b-PEG, by free-radical polymerization of HPMA, PDTEMA and APMA using ((mPEG₅₀₀₀)₂-ABCPA) macroinitiator.

Scheme 2

Synthesis of p(HPMA–DMAE-co-PDTEMA-co-Cy7)-b-PEG, by sequential coupling of Cy7-NHS and DMAE-CI to p(HPMA-co-PDTEMA-co-APMA)-b-PEG.

Scheme 3

Route for the preparation of interchain disulfide-crosslinked decationized polyplexes, through a 3-step process: 1. charge-driven condensation with nucleic acids; 2. stabilization through disulfide crosslinking; 3. decationization of cationic pHDP-PEG polyplexes, resulting in decationized pHP-PEG polyplexes (adapted from [33]).