Guping Tang

Polyethylene glycol modified polyethylenimine for improved CNS gene transfer: effects of PEGylation extent

Poor solubility of polycation complexes with DNA is one drawback for their in vivo use as gene delivery systems. PEGylation often can improve the solubility of the complexes, minimize their aggregation and reduce their interaction with proteins in the physiological fluid. We investigated in vivo application of polyethylene glycol (PEG) modified polyethylenimine (PEI) for gene expression in the central nervous system. Varied numbers of linear PEG (2 kDa) were grafted to branched PEI (25 kDa) from the average number of PEG per one PEI macromolecule at 1-14.5. While higher degrees of PEG grafting did not improve gene expression, a PEI conjugate with one segment of PEG was able to mediate transgene expression in the spinal cord up to 11-fold higher than PEI homopolymer after intrathecal administration of its DNA complexes into the lumbar spinal cord subarachnoid space. Improved gene expression with this conjugate was observed as well in the brain after the lumbar injection. As assessed in in vitro studies, the PEI conjugate with a low degree of PEG grafting was able to reduce the size of polymer DNA complexes, prevent the aggregation of complexes, decrease the interactions of the complexes with serum proteins, counter the inhibition of serum to gene transfer, and enhance transfection efficiency, although not significant in affecting complex formation and reducing in vitro cell toxicity of PEI. The study provides the in vivo evidence that an appropriate degree of PEG modification is decisive in improving gene transfer mediated by PEGylated polymers.

Cyclodextrin-based host-guest supramolecular nanoparticles for delivery: from design to applications.

CONSPECTUS: Efficient assembly in host-guest interactions is crucial to supramolecular nanotechnology. Cyclodextrins (CDs), which possess a hydrophilic exterior surface and hydrophobic interior cavity on the truncated cone, improve the biocompatibility of nanodelivery systems, and hence, supramolecular approaches utilizing CDs can improve and expand the design and applications of functional delivery systems. Owing to good inclusion ability, αCD and βCD are commonly used in the design and construction of supramolecular structures. In this Account, we describe the design strategies to adopt CDs in host-guest delivery systems. Modification of CDs with polymers is popular in current research due to the potential benefits rendered by cationic protection and improved capability. While the process has only minor influence on the host characteristics of the CD cavity, the interaction between the CD and the guest moiety imparts new attributes to the nanosystems with guest-decorated functional groups such as adamantyl poly(ethylene glycol) (PEG) for coating protection, hybrid guests for conformational flexibility, and adamantyl prodrugs for drug delivery. Some specific agents form inclusion complexes with the polymerized βCDs directly and core-shell nanoparticles with hydrophobic cores and are usually created to carry insoluble drugs while the hydrophilic shells offer protection. These unique designs provide the means to practically adapt special characteristics for additional functions or co-delivery. In order to be accepted clinically, delivery systems need to possess extra functions such as controlled particle size, biodegradability, controlled release, and targeted delivery to overcome the hurdles in delivery. These features can be added to biomaterials by self-assembly of functional groups facilitated by the host-guest interactions. Size control by hybridization of switchable polymer compartments in supramolecular structures contributes to the biodistribution utility and biodegradability by incorporating the moieties with hydrolyzable connections and enhancing intracellular degradation and clearance. Controlled release by application of responsive structures like molecular gatings eased by the host-guest interaction can be triggered by the tumor microenvironment at extreme pH and temperature or by external stimuli such as light. Along with the binding selectivity and controlled release, the host-guest nanoparticles show enhanced efficacy in delivery especially to tumors. Recent developments in supramolecular co-delivery systems are described in this Account. Nanoparticles can be designed to carry adamantyl prodrugs and therapeutic nucleotides to tumors so that the released drugs and gene expression synergistically inhibit malignant tissue growth. Optimization of nanoparticle delivery systems by multifunctional transitions yields better biocompatibility and controlled response, and such novel designs will expedite in vivo applications. Hence, multifunctional CD-based host-guest supramolecular nanoparticles with co-delivery ability are expected to have many potential clinical applications.

Low molecular weight polyethylenimine cross-linked by 2-hydroxypropyl-γ-cyclodextrin coupled to peptide targeting HER2 as a gene delivery vector

Gene delivery is one of the critical steps for gene therapy. Non-viral vectors have many advantages but suffered from low gene transfection efficiency. Here, in order to develop new polymeric gene vectors with low cytotoxicity and high gene transfection efficiency, we synthesized a cationic polymer composed of low molecular weight polyethylenimine (PEI) of molecular weight of 600 Da cross-linked by 2-hydroxypropyl-gamma-cyclodextrin (HP gamma-CD) and then coupled to MC-10 oligopeptide containing a sequence of Met-Ala-Arg-Ala-Lys-Glu. The oligopeptide can target to HER2, the human epidermal growth factor receptor 2, which is often over expressed in many breast and ovary cancers. The new gene vector was expected to be able to target delivery of genes to HER2 positive cancer cells for gene therapy. The new gene vector was composed of chemically bonded HP gamma-CD, PEI (600 Da), and MC-10 peptide at a molar ratio of 1:3.3:1.2. The gene vector could condense plasmid DNA at an N/P ratio of 6 or above. The particle size of HP gamma-CD-PEI-P/DNA complexes at N/P ratios 40 was around 170-200 nm, with zeta potential of about 20 mV. The gene vector showed very low cytotoxicity, strong targeting specificity to HER2 receptor, and high efficiency of delivering DNA to target cells in vitro and in vivo with the reporter genes. The delivery of therapeutic IFN-alpha gene mediated by the new gene vector and the therapeutic efficiency were also studied in mice animal model. The animal study results showed that the new gene vector HP gamma-CD-PEI-P significantly enhanced the anti-tumor effect on tumor-bearing nude mice as compared to PEI (25 kDa), HP gamma-CD-PEI, and other controls, indicating that this new polymeric gene vector is a potential candidate for cancer gene therapy.

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.

Gene delivery to tumor cells by cationic polymeric nanovectors coupled to folic acid and the cell-penetrating peptide octaarginine

Target ligand folic acid (FA) and cell-penetrating peptide octaarginine (R8) were coupled with the gene vectors (PEI(600)-CyD, PC) composed of β-cyclodextrin (β-CyD) and low-molecular-weight polyethylenimine (PEI, Mw 600) to form nanovectors for highly efficient gene delivery to tumor cells. The resultant ternary nanocomplexes of FA-PC/R8-PC/pDNA produced excellent gene transfaction abilities in the folate receptor (FR)-positive tumor cells in vitro and in vivo. The FR-mediated endocytosis and the R8-mediated transmembrane functionality together contributed to the high transfection levels. This study provides a promising means to produce gene nanovectors for in vivo applications.

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.

Two novel non-viral gene delivery vectors: low molecular weight polyethylenimine cross-linked by (2-hydroxypropyl)-β-cyclodextrin or (2-hydroxypropyl)-γ-cyclodextrin

Two novel polymers of low molecular weight polyethylenimine cross-linked by (2-hydroxypropyl)-β-cyclodextrin or (2-hydroxypropyl)-γ-cyclodextrin showed lower cytotoxicity and higher transfection efficiency for the delivery of plasmid DNA compared with those of polyethylenimine (PEI, 25 kDa).

In vivo treatment of tumors using host-guest conjugated nanoparticles functionalized with doxorubicin and therapeutic gene pTRAIL

The combination of gene therapy and chemotherapy may increase the therapeutic efficacy in the treatment of patients. In this work, the anti-cancer drug Dox and therapeutic gene pTRAIL-loaded host-guest co-delivery system was assayed for the possibility of in vivo synergistically treating tumors. The introduced Dox could act as an auxiliary component to human tumor necrosis factor-related apoptosis-inducing ligand-encoding plasmid gene pTRAIL. Such delivery system possessed the good ability of in vivo retention of chemotherapeutic drugs, achieved good therapeutic effects in the inhibition of tumor growth and significantly prolonged the survival time of tumor-bearing mice. With the efficient ability to co-deliver drug and gene, such host-guest assembly should have great potential applications in cancer therapy.

Cationic microRNA-delivering nanovectors with bifunctional peptides for efficient treatment of PANC-1 xenograft model

Therapeutic strategies based on modulation of microRNA activity possess much promise in cancer therapy, but the in vivo delivery of microRNA to target sites and its penetration into tumor tissues remain great challenge. In this work, miR-34a-delivering therapeutic nanocomplexes with a tumor-targeting and -penetrating bifunctional CC9 peptide were proposed for efficient treatment of pancreatic cancers. In vitro study indicated that the nanoparticle-based miR-34a delivery systems could effectively facilitate cellular uptake and greatly up-regulate the mRNA level of miR-34a in PANC-1 cell lines. The up-regulation of miR-34a remarkably induced cell cycle arrest and apoptosis, suppressed the tumor cell migration and inhibited the target gene expressions such as E2F3, Bcl-2, c-myc and cyclin D1. More importantly, the in vivo systemic administration of the developed targeting miR-34a delivery systems in a pancreatic cancer model significantly inhibited tumor growth and induced cancer cell apoptosis. Such bifunctional peptide-conjugated miRNA-delivering nanocomplexes should have great potential applications in cancer 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.