Yuan Ping

Chitosan‐Functionalized Graphene Oxide as a Nanocarrier for Drug and Gene Delivery

The covalent functionalization of graphene oxide (GO) with chitosan (CS) is successfully accomplished via a facile amidation process. The CS-grafted GO (GO-CS) sheets consist of about 64 wt.% CS, which imparts them with a good aqueous solubility and biocompatibility. Additionally, the physicochemical properties of GO-CS are studied. As a novel nanocarrier, GO-CS is applied to load a water-insoluble anticancer drug, camptothecin (CPT), via π-π stacking and hydrophobic interactions. It is demonstrated that GO-CS possesses a superior loading capacity for CPT, and the GO-CS-CPT complexes show remarkably high cytotoxicity in HepG2 and HeLa cell lines compared to the pure drug. At the same time, GO-CS is also able to condense plasmid DNA into stable, nanosized complexes, and the resulting GO-CS/pDNA nanoparticles exhibit reasonable transfection efficiency in HeLa cells at certain nitrogen/phosphate ratios. Therefore, the GO-CS nanocarrier is able to load and deliver both anticancer drugs and genes.

Star-Shaped Cationic Polymers by Atom Transfer Radical Polymerization from β-Cyclodextrin Cores for Nonviral Gene Delivery

Cationic polymers with low cytotoxicity and high transfection efficiency have attracted considerable attention as nonviral carriers for gene delivery. Herein, well-defined and star-shaped CDPD consisting of beta-CD cores and P(DMAEMA) arms, and CDPDPE consisting of CDPD and P(PEGEEMA) end blocks (where CD = cyclodextrin, P(DMAEMA) = poly(2-(dimethylamino)ethyl methacrylate), P(PEGEEMA) = poly(poly(ethylene glycol)ethyl ether methacrylate)) for gene delivery were prepared via atom transfer radical polymerization (ATRP) from the bromoisobutyryl-terminated beta-CD core. The CDPD and CDPDPE exhibit good ability to condense plasmid DNA (pDNA) into 100-200 nm size nanoparticles with positive zeta potentials of 25-40 mV at nitrogen/phosphate (N/P) ratios of 10 or higher. CDPD and CDPDPE exhibit much lower cytotoxicity and higher gene transfection efficiency than high molecular weight P(DMAEMA) homopolymers. A comparison of the transfection efficiencies between CDPD and P(DMAEMA) homopolymer indicates that the unique star-shaped architecture involving the CD core can enhance the gene transfection efficiency. In addition to reducing cytotoxicity, the introduction of a biocompatible P(PEGEEMA) end block to the P(DMAEMA) arms in CDPDPE can further enhance the gene transfection efficiency.

Comb-shaped copolymers composed of hydroxypropyl cellulose backbones and cationic poly((2-dimethyl amino)ethyl methacrylate) side chains for gene delivery.

Cationic polymers have been of interest and importance as nonviral gene delivery carriers. Herein, well-defined comb-shaped cationic copolymers (HPDs) composed of long biocompatible hydroxypropyl cellulose (or HPC) backbones and short poly((2-dimethyl amino)ethyl methacrylate) (or P(DMAEMA)) side chains were prepared as gene vectors via atom transfer radical polymerization (ATRP) from the bromoisobutyryl-terminated HPC biopolymers. The P(DMAEMA) side chains of HPDs can be further partially quaternized to produce the quaternary ammonium HPDs (QHPDs). HPDs and QHPDs were assessed in vitro for nonviral gene delivery. HPDs exhibit much lower cytotoxicity and better gene transfection yield than high-molecular-weight P(DMAEMA) homopolymers. QHPDs exhibit a stronger ability to complex pDNA, due to increased surface cationic charges. Thus, the approach to well-defined comb-shaped cationic copolymers provides a versatile means for tailoring the functional structure of nonviral gene vectors to meet the requirements of strong DNA-condensing ability and high transfection capability.

FGFR-targeted gene delivery mediated by supramolecular assembly between β-cyclodextrin-crosslinked PEI and redox-sensitive PEG

A new redox-sensitive poly(ethylene glycol) (PEG)-based gene vector specially designed to target fibroblast growth factor receptors (FGFRs) was developed by host-guest supramolecular complexation. The new vector was designed as follows: 1) A host segment was consisted of β-cyclodextrin-crosslinked low molecular polyethylenimine (PEI) conjugated with MC11 peptide (MQLPLATGGGC) that can target FGFRs, being termed as MC11-PEI-β-cyclodextrin (MPC); 2) A guest segment is consisted of PEG and adamantyl group linked by a disulfide bond, the adamantyl-SS-PEG (Ad-SS-PEG); and 3) PEGylation of MPC by supramolecular complexation between MPC and Ad-SS-PEG to generate MPC/Ad-SS-PEG polycation, where the PEG chains can stabilize the DNA polyplexes extracellularly but can be readily cleavable intracellularly. It was found that the MPC/Ad-SS-PEG complexes could efficiently condense pDNA into nanoparticles around 100-200 nm, and were able to effectively stabilize polyplexes against salt- or BSA-induced aggregation. The MPC/Ad-SS-PEG polyplexes were more readily to dissociate with the aid of heparin in the presence of 5 mm DTT. In vitro gene transfection and cytotoxicity experiments in different carcinoma cell lines expressing FGFRs showed that MPC/Ad-SS-PEG could mediate significantly higher transfection efficiency than MPC complexed with adamantyl-PEG (MPC/Ad-PEG), which has no disulfide linkage and is non-PEG-detachable. Furthermore, confocal laser scanning microscopy study indicated that MPC/Ad-SS-PEG polyplexes could mediate much more efficient endosomal escape than stably shield MPC/Ad-PEG polyplexes at 12 h post-transfection. Importantly, MPC/Ad-SS-PEG was also able to efficiently mediate tumor-targeted gene delivery in the tumor-bearing mouse model after systemic injection in vivo. These results suggest that the MPC/Ad-SS-PEG systems could be a safe and efficient non-viral vector for FGFR-mediated targeted gene delivery for cancer gene therapy.

Well-Defined Poly(2-hydroxyl-3-(2-hydroxyethylamino)propyl methacrylate) Vectors with Low Toxicity and High Gene Transfection Efficiency

Successful gene delivery vectors for clinical translation should have high transfection efficiency and minimal toxicity. In this work, well-defined poly(2-hydroxyl-3-(2-hydroxyethylamino)propyl methacrylate) (PGEA) vectors with flanking cationic secondary amine and nonionic hydroxyl units were prepared via the ring-opening reaction of the pendant epoxide groups of poly(glycidyl methacrylate) with the amine moieties of ethanolamine. It was found that PGEA carriers possess very low toxicity (<10% of the toxicity of branched polyethylenimine (PEI, 25 kDa), while exhibiting surprisingly excellent transfection efficiency (higher than or comparable to that of PEI (25 kDa)) in different cell lines. A series of transfection and cytotoxicity assays revealed that PGEAs are highly promising as a new class of safe and efficient gene delivery vectors for future clinical gene therapies.

Construction of a star-shaped copolymer as a vector for FGF receptor-mediated gene delivery in vitro and in vivo.

The success of cancer gene therapy highly relies on the gene delivery vector with high transfection activity and low toxicity. In the present study, eight-armed polyethylene glycol (EAP) and low molecular weight (LMW) polyethylenimine (PEI) were used as basic units to construct the architecture of a new star-shaped EAP-PEI copolymer (EAPP). MC11, a peptide capable of selectively binding fibroblast growth factor receptor (FGFR) on tumor cell membranes, was further conjugated to EAPP to produce the vector EAPP-MC11 (EAPPM) to enhance tumor targetability. This tumor-targeting vector EAPPM was observed to retard the plasmids mobility at a nitrogen/phosphorus (N/P) ratio of 3. The vector could efficiently condense plasmids within 300 nm nanoparticles with a positive zeta potential at the N/P ratio of 20 or above. While the cytotoxicity of EAPPM polyplexes was similar to that of LMW PEI, it was significantly lower than that of PEI (25 kDa) in HepG2 and PC3 cell lines. In vitro gene transfection with pDNA mediated by EAPPM showed that the transfection efficiency increased 15 times in HepG2 cells but remained at a similar level in PC3 cells in comparison with that of EAPP. By systemic injection of EAPPM/pDNA complexes into a HepG2-bearing mice model, luciferase expression detected in lung, liver, and tumor tissues demonstrated EAPPM could deliver in a targeted manner a reporter gene into tumor tissues, where the luciferase expression of EAPPM was 4 times higher than that of EAPP and even 23 times higher than that of PEI (25 kDa). Furthermore, it was found that the systemic delivery of EAPPM/pCSK-α-interferon complexes in vivo were much more effective in inhibiting tumor growth than EAPP or PEI (25 kDa). These results clearly show that EAPPM is an efficient and safe vector for FGFR-mediated targeted gene delivery both in vitro and in vivo. With low cytotoxicity and high targetability, EAPPM may have great potential as a delivery vector for future cancer gene therapy applications.

Polyethyleneimine-grafted poly(N-3-hydroxypropyl)aspartamide as a biodegradable gene vector for efficient gene transfection

A new biodegradable cationic copolymer, α,β-poly(N-3-hydroxypropyl)-DL-aspartamide (PHPA) grafted with polyethylenimine (PEI) was synthesized as a nonviral gene delivery vector by conjugating low molecular weight (LMW) PEI onto PHPA backbone. The polycation, termed PHPA-PEI, exhibited good ability to condense plasmid DNA (pDNA) into nanoparticles of around 150 nm with positive charge at a nitrogen/phosphorus (N/P) ratio of 15. The morphology of the nanoparticles observed by atomic force microscopy (AFM) was compact and spherical. PHPA-PEI also showed strong buffering capacity over the pH range 3–10 and protected well the condensed DNA from enzymatic degradation by DNase I over a period of time. pDNA release triggered by the synergistic effect of heparin and degradation demonstrated that PHPA-PEI formulation could continuously release the pDNA over a week. Transfection with pDNA pRL-CMV encoding Renilla luciferase reporter gene (Rluc), mediated by PHPA-PEI/pDNA complexes was carried out in 3T3, HEK293 and COS7 cell lines, and compared with that mediated by PEI (25 kDa)/pDNA complexes. The results showed that PHPA-PEI was not only superior in transfectivity to PEI (25 kDa) but also showed sustained high level luciferase expression. Furthermore, PHPA-PEI exhibited much lower cytotoxicity than PEI (25 kDa) in these cell lines. Therefore, PHPA-PEI may have great potential as a gene delivery vector with low cytotoxicity and high gene transfection efficiency for future gene therapy applications.

Thermo-responsive transfection of DNA complexes with well-defined chitosan terpolymers

Serial thermo-sensitive CS(-g-PDMAEMA)-g-PNIPAM terpolymers (termed as TCS) were prepared by atom transfer radical polymerization (ATRP) and click reactions, where CS, PDMAEMA, and PNIPAM stand for chitosan, poly((2-dimethylamino)ethyl methacrylate), and poly(N-isopropylacrylamide) respectively. Their lower critical solution temperature (LCST) determined by laser light scattering (LLS) was around 32.5–35.1 °C. The incorporation of CS and PNIPAM considerably decreased the cytotoxicity of PDMAEMA. Gel electrophoresis and TEM results revealed that the association and dissociation of TCS/DNA complexes could be tuned by varying temperature. The transfection level of TCS and controls was evaluated with COS7 and HeLa cells using two different reporter genes, pRL-CMV encoding luciferase and pEGFP-N1 encoding green fluorescence protein (GFP). The transfection efficiency of one specific terpolymer (TCS4) incubated at 37 °C for 22 h, 20 °C for 2 h and 37 °C for 24 h increased 1–4 fold compared to that incubated at 37 °C for 48 h. Encouragingly, at optimum N/P ratios, the transfection efficiency of TCS was comparable or superior to that of “gold-standard” PEI (25 kDa).