Low-Frequency Ultrasound Effects on Intracellular Barriers in Nonviral Gene Delivery Processes

Gene therapy, the expression in cells of genetic material with therapeutic activity, has emerged as a promising approach for the treatment or prevention of human diseases. At the present time, major somatic gene-transfer approaches employ either viral or nonviral vectors. Nonviral vectors are less efficient at introducing and maintaining foreign gene expression, but have the profound advantage of being nonpathogenic and nonimmunogenic. In this study, we aimed to develop an efficient nonviral gene delivery system in which low-frequency ultrasound (LFUS) was applied to enhance gene expression of polyplexes formed with poly(2-dimethylaminoethyl methacrylate) and plasmid encoding for green fluorescent protein. Ultrasound (US), and in particular LFUS, can cause temporary membrane permeabilization and thereby enhance drug and gene entrance into viable cells. We evaluated possible additional favorable effects of LFUS on the polyplex transfection process, such as overcoming intracellular barriers. We found that pDMAEMA protected the plasmid DNA from ultrasonic degradation. Atomic force microscopy analysis also confirmed that the LFUS did not change the polyplexes’ morphology. We also attained an insight into the structure of polyplexes during LFUS exposure and found that LFUS induced a temporary partial detachment between the polymer chains and the plasmid. In addition, LFUS application on ovarian carcinoma cells transfected with the polyplexes induced a 27% enhancement in transfection efficiency. Based on these results, we propose that LFUS enhances the decomplexation of the polyplexes, and therefore, can be used to optimize transfection efficiency.     

Hyperbranched polymeric "star vectors" for effective DNA or siRNA delivery

Although gene therapy offers an attractive strategy for treating inherited disorders, current techniques using viral and nonviral delivery systems have not yielded many successful results in clinical trials. Viral vectors such as retroviruses, lentiviruses, and adenoviruses deliver genes efficiently; however, the possibility of negative outcomes from viral transformation cannot be completely ruled out. In contrast, various types of nonviral vectors are attracting considerable attention because they are easier to handle and induce weak immune responses. Cationic polymers, such as polyethylenimine (PEI) and poly(N,N-dimethylaminopropyl acrylamide) (PDMAPAAm), can generate nanoparticles through the formation of polyion complexes, "polyplexes" with DNA. These nonviral systems offer many advantages over viral systems. The primary obstacle to implementing these cationic polymers in an effective gene therapy remains their comparatively inefficient gene transfection in vivo. We describe four strategies for the development of hyperbranched star vectors (SVs) for enhancing DNA or siRNA delivery. The molecular design was performed by living radical polymerization in which the chain length can be controlled by photoirradiation and solution conditions, including concentrations of the monomer or iniferter (a molecule that serves as a combination of initiator, transfer agent, and terminator). The branch composition is controlled by the types of monomers that are added stepwise. In our first strategy, we prepared a series of only cationic PDMAPAAm-based SVs with no branches or 3, 4, or 6 branching numbers. These SVs could form polyion complexes (polyplexes) by mixing with DNA only in aqueous solution. The relative gene expression activity of the delivered DNA increased according to the degree of branching. In addition, increasing the molecular weight of SVs and narrowing their polydispersity index (PDI) improved their activity. For targeting DNA delivery to the specific cells, we modified the SV with ligands. Interestingly, the SV could adsorb the RGD peptide, making gene transfer possible in endothelial cells which are usually refractory to such treatments. The peptide was added to the polyplex solution without covalent derivatization to the SV. The introduction of additional branching by cross-linking using iniferter-induced coupling reactions further improved gene transfection activity. After block copolymerization of PDMAPAAm-based SVs with a nonionic monomer (DMAAm), the blocked SVs (BSVs) produced polyplexes with DNA that had excellent colloidal stability for 1 month, leading to efficient in vitro and in vivo gene delivery. Moreover, BSVs served as carriers for siRNA delivery. BSVs enhanced siRNA-mediated gene silencing in mouse liver and lung. As an alternative approach, we developed a novel gene transfection method in which the polyplexes were kept in contact with their deposition surface by thermoresponsive blocking of the SV. This strategy was more effective than reverse transfection and the conventional transfection methods in solution.     

Characterization and Optimization of Chitosan-Coated Polybutylcyanoacrylate Nanoparticles for the Transfection-Guided Neural Differentiation of Mouse Induced Pluripotent Stem Cells

Gene transfection is a valuable tool for analyzing gene regulation and function, and providing an avenue for the genetic engineering of cells for therapeutic purposes. Though efficient, the potential concerns over viral vectors for gene transfection has led to research in non-viral alternatives. Cationic polyplexes such as those synthesized from chitosan offer distinct advantages such as enhanced polyplex stability, cellular uptake, endo-lysosomal escape, and release, but are limited by the poor solubility and viscosity of chitosan. In this study, the easily synthesized biocompatible and biodegradable polymeric polysorbate 80 polybutylcyanoacrylate nanoparticles (PS80 PBCA NP) are utilized as the backbone for surface modification with chitosan, in order to address the synthetic issues faced when using chitosan alone as a carrier. Plasmid DNA (pDNA) containing the brain-derived neurotrophic factor (BDNF) gene coupled to a hypoxia-responsive element and the cytomegalovirus promotor gene was selected as the genetic cargo for the in vitro transfection-guided neural-lineage specification of mouse induced pluripotent stem cells (iPSCs), which were assessed by immunofluorescence staining. The chitosan-coated PS80 PBCA NP/BDNF pDNA polyplex measured 163.8 ± 1.8 nm and zeta potential measured −34.8 ± 1.8 mV with 0.01% (w/v) high molecular weight chitosan (HMWC); the pDNA loading efficiency reached 90% at a nanoparticle to pDNA weight ratio of 15, which also corresponded to enhanced polyplex stability on the DNA stability assay. The HMWC-PS80 PBCA NP/BDNF pDNA polyplex was non-toxic to mouse iPSCs for up to 80 μg/mL (weight ratio = 40) and enhanced the expression of BDNF when compared with PS80 PBCA NP/BDNF pDNA polyplex. Evidence for neural-lineage specification of mouse iPSCs was observed by an increased expression of nestin, neurofilament heavy polypeptide, and beta III tubulin, and the effects appeared superior when transfection was performed with the chitosan-coated formulation. This study illustrates the versatility of the PS80 PBCA NP and that surface decoration with chitosan enabled this delivery platform to be used for the transfection-guided differentiation of mouse iPSCs.     

Guanidinylation: A simple way to fabricate cell penetrating peptide analogue‐modified chitosan vector for enhanced gene delivery

In view of the analogous transmembrane function to cell penetrating peptides, guanidine group was incorporated into chitosan by chemical modification to enhance the transfection performance of chitosan vectors. Guanidinylated chitosan (GCS) was shown to be well soluble in neutral aqueous solution. The interaction between GCS with plasmid DNA was characterized by agarose retardation experiment and ethidium bromide displacement assay. GCS formed more stable complexes with DNA under physiological pH than chitosan. The transfection efficiency of GCS was evaluated employing COS‐7 cell line—GCS polyplexes demonstrated higher transfection efficiency and lower cytotoxicity relative to chitosan. The optimum efficiency of GCS was achieved in the vicinity of the critical complexing ratio. The results of flow cytometry indicated that guanidinylation promoted an eightfold increase in the cell uptake. The study revealed that guanidinylated chitosan is a promising candidate as an effective nonviral vector for in vivo gene delivery. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Incorporation of Polycaprolactone to Cyclodextrin‐Based Nanocarrier for Potent Gene Delivery

Stability of polyplex and safety are key factors to achieve stable gene transfection and high transfection efficiency. In this report, a star‐like amphiphilic biocompatible cyclodextrin‐poly(ε‐caprolactone)‐poly(2‐(dimethylamino) ethyl methacrylate), β‐CD‐g‐(PCL‐b‐PDMAEMA) _x copolymer, consisting of biocompatible cyclodextrin core, biodegradable and stable poly(ε‐caprolactone) PCL segments, cationic and hydrophilic PDMAEMA blocks, is synthesized to achieve high efficiency of gene transfection with enhanced stability, due to the micelle formation by hydrophobic PCL segments. In comparison with polyethylenimine (PEI‐25k), a golden standard for nonviral vector gene delivery, this copolymer shows higher encapsulated plasmid desoxyribose nucleic acid (pDNA) ability and the persistence of transgene expression. More interestingly, this gene delivery platform by β‐CD‐g‐(PCL‐b‐PDMAEMA) _x shows lower toxicity but better gene transfection efficiency at low N/P ratios, indicating high potential in gene therapy applications.     

Development of Covalent Chitosan-Polyethylenimine Derivatives as Gene Delivery Vehicle: Synthesis,Characterization, and Evaluation

There is an increasing interest in cationic polymers as important constituents of non-viral gene delivery vectors. In the present study, we developed a versatile synthetic route for the production of covalent polymeric conjugates consisting of water-soluble depolymerized chitosan (dCS; M_W 6–9 kDa) and low molecular weight polyethylenimine (PEI; 2.5 kDa linear, 1.8 kDa branched). dCS-PEI derivatives were evaluated based on their physicochemical properties, including purity, covalent bonding, solubility in aqueous media, ability for DNA condensation, and colloidal stability of the resulting polyplexes. They were complexed with non-integrating DNA vectors coding for reporter genes by simple admixing and assessed in vitro using liver-derived HuH-7 cells for their transfection efficiency and cytotoxicity. Using a rational screening cascade, a lead compound was selected (dCS-Suc-LPEI-14) displaying the best balance of biocompatibility, cytotoxicity, and transfection efficiency. Scale-up and in vivo evaluation in wild-type mice allowed for a direct comparison with a commercially available non-viral delivery vector (in vivo-jetPEI). Hepatic expression of the reporter gene luciferase resulted in liver-specific bioluminescence, upon intrabiliary infusion of the chitosan-based polyplexes, which exceeded the signal of the in vivo jetPEI reference formulation by a factor of 10. We conclude that the novel chitosan-derivative dCS-Suc-LPEI-14 shows promise and potential as an efficient polymeric conjugate for non-viral in vivo gene therapy.     

PEG-based Polyplex Design for Gene and Nucleotide Delivery

Gene delivery is a multiple-step process which depends on the ability of a gene carrier to overcome several biological barriers and safely deliver a transgene to its target cells. Polymeric gene delivery systems, e. g., polyplexes, have emerged as a safe alternative to viral vectors. Poly(ethylene glycol) (PEG) conjugation is a common modification approach to provide polyplexes with prolonged circulation time and reduced toxicity, and to allow their accumulation in tumor tissue through the enhanced permeability and retention (EPR) effect. This review describes physicochemical properties related to the biological activity of PEG-based polyplexes, and approaches undertaken to promote a rational design for their in vivo applications.     

Clostridium neurotoxin fragments as potential targeting moieties for liposomal gene delivery to the CNS

Targeted transfection of the CNS with synthetic, nonviral vectors represents a huge technical challenge. The approach explored here attempts to combine self-assembly ABCD nanoparticles (Kostarelos and Miller, Chem. Soc. Rev. 2005, 34, 970), with the potential of Clostridium neurotoxin fragments to effect receptor-mediated transfection of neuronal cells. Cationic liposome-plasmid DNA complexes were first modified with a PEG stealth layer, before the addition of C-terminal fragments of tetanus toxin (TH(C)), botulinum toxin (BH(C)) or the truncated C-terminal domain of TH(C) as biological "targeting" ligands. First-generation nanoparticles were identified for the transfection of two neuronal cell lines (human SH-5YSY and rat/mouse hybrid N18-RE105); control studies were also performed with HeLa cells. ABCD nanoparticle transfections of the neuronal cell lines were up to 30-fold higher than corresponding control transfections with nanoparticles that lacked the protein ligand. We also demonstrate apparent receptor-mediated uptake by means of competition-binding and real-time confocal experiments. By contrast, nanoparticle transfection of HeLa cells appeared to involve alternative nonspecific enhanced cellular uptake mechanism(s). Receptor-mediated and nonspecific mechanisms appear to be in competition, potentially harming the capacity of receptor-mediated delivery to effect proper targeted delivery of nucleic acids to cells ex vivo and in vivo.     

Serum Compatible Spermine-Based Cationic Lipids with Nonidentical Hydrocarbon Tails Mediate High Transfection Efficiency

Cationic lipids are widely used as nonviral synthetic vectors for gene delivery as a safer alternative to viral vectors. In this work, a library of L-shaped spermine-based cationic lipids with identical and nonidentical hydrophobic chains having variable carbon lengths (from C10 to C18) was designed and synthesized. These lipids were characterized and the structure-activity relationships of these compounds were determined for DNA binding and transfection ability when formulated as cationic liposomes. The liposomes were then used successfully for the transfection of HEK293T, HeLa, PC3, H460, HepG2, SH-SY5Y and Calu’3 cell lines. The transfection efficiency of lipids with nonidentical hydrocarbon chains was greater than the identical analogue. These reagents exhibited superior efficiency to the commercial reagent, Lipofectamine3000, under both serum-free and 10–40 % serum conditions in HEK293T, HeLa and H460 cell lines. The lipids were not toxic to the tested cell line. The results suggest that L-shaped spermine-based cationic lipids with nonidentical hydrocarbon tails could serve as efficient and safe nonviral vector gene carriers in further in vivo studies.     

DNA delivery systems based on copolymers of poly (2-methyl-2-oxazoline) and polyethyleneimine: Effect of polyoxazoline moieties on the endo-lysosomal escape

Poly(2-methyl-2-oxazoline)-polyethylenimine (PMeOx-co-PEI) copolymers differing by degree of polymerization (DP = 50 and 200) and PEI content (from 37 to 99 mol%) were synthesized by living cationic ring-opening polymerization of 2-methyl-2-oxazoline, followed by partial hydrolysis. Upon mixing with DNA in a wide range of N/P ratios, they formed well-defined polyplex particles of small size (typically below 100 nm) and narrow size distribution. The polyplexes demonstrated good colloidal stability and very low in vitro cytotoxicity. The copolymers exhibited buffering capacity of over 50% relative to that of the reference PEI implying effective endo-lysosomal escape of the polyplexes. Increased cellular internalization of both PCR fragments and plasmid DNA, attributable to the strongly positive ζ potential and small size of the polyplexes, was observed. In spite of these favorable prerequisites, the transfection efficiency was low (below 20% relative to the control PEI) and was attributed to retarded swelling of the polyplex particles, endo-lysosomal rupture, and DNA release.     

Recent advances in nonviral vectors for gene delivery

Gene therapy has long been regarded a promising treatment for many diseases, whether acquired (such as AIDS or cancer) or inherited through a genetic disorder. A drug based on a nucleic acid, however, must be delivered to the interior of the target cell while surviving an array of biological defenses honed by evolution. Successful gene therapy is thus dependent on the development of an efficient delivery vector. Researchers have pursued two major vehicles for gene delivery: viral and nonviral (synthetic) vectors. Although viral vectors currently offer greater efficiency, nonviral vectors, which are typically based on cationic lipids or polymers, are preferred because of safety concerns with viral vectors. So far, nonviral vectors can readily transfect cells in culture, but efficient nanomedicines remain far removed from the clinic. Overcoming the obstacles associated with nonviral vectors to improve the delivery efficiency and therapeutic effect of nucleic acids is thus an active area of current research. The difficulties are manifold, including the strong interaction of cationic delivery vehicles with blood components, uptake by the reticuloendothelial system (RES), toxicity, and managing the targeting ability of the carriers with respect to the cells of interest. Modifying the surface with poly(ethylene glycol), that is, PEGylation, is the predominant method used to reduce the binding of plasma proteins to nonviral vectors and minimize clearance by the RES after intravenous administration. Nanoparticles that are not rapidly cleared from the circulation accumulate in the tumors because of the enhanced permeability and retention effect, and the targeting ligands attached to the distal end of the PEGylated components allow binding to the receptors on the target cell surface. Neutral and anionic liposomes have been also developed for systemic delivery of nucleic acids in experimental animal models. Other approaches include (i) designing and synthesizing novel cationic lipids and polymers, (ii) chemically coupling the nucleic acid to peptides, targeting ligands, polymers, or environmentally sensitive moieties, and (iii) utilizing inorganic nanoparticles in nucleic acid delivery. Recently, the different classes of nonviral vectors appear to be converging, and the ability to combine features of different classes of nonviral vectors in a single strategy has emerged. With the strengths of several approaches working in concert, more hurdles associated with efficient nucleic acid delivery might therefore be overcome. In this Account, we focus on these novel nonviral vectors, which are classified as multifunctional hybrid nucleic acid vectors, novel membrane/core nanoparticles for nucleic acid delivery, and ultrasound-responsive nucleic acid vectors. We highlight systemic delivery studies and consider the future prospects for nucleic acid delivery. A better understanding of the fate of the nanoparticles inside the cell and of the interactions between the parts of hybrid particles should lead to a delivery system suitable for clinical use. We also underscore the value of sustained release of a nucleic acid in this endeavor; making vectors targeted to cells with sustained release in vivo should provide an interesting research challenge.     

Improved cell viability of linear polyethylenimine through gamma-cyclodextrin inclusion for effective gene delivery

A polypseudorotaxane consisting of a linear polyethylenimine with Mn of 22,000 (LPEI22k) and gamma-cyclodextrins (gamma-CDs; LPEI22k/gamma-CD) has been examined as a gene carrier. The polyplex formation with luciferase-encoding plasmid DNA (pDNA), intracellular trafficking of polyplex, cytotoxicity, and transfection efficiency were evaluated by various characteristic methods. LPEI22k/gamma-CD formed a pDNA polyplex at higher N/P ratios than LPEI22k; this suggests that the gamma-CD threading sterically interfered with the polyplex formation. In addition, the zeta potentials of the polyplex significantly decreased due to the reduction in charge density of LPEI22k caused by gamma-CD threading. The cellular uptake of pDNA in the LPEI22k/gamma-CD polyplex was enhanced by free gamma-CDs released from the polyplex that might accelerate the cellular uptake through enhanced membrane affinity. LPEI22k/gamma-CD significantly increased cell viability even at high N/P ratios, and the polyplex showed high transfection efficacy. The low cytotoxicity and high gene expression of LPEI22k/gamma-CD are advantageous to polyplex administration in vivo.     

Polycationic star polymers with hyperbranched cores for gene delivery

Core-shell type stars synthesized via atom transfer radical polymerization were used for the delivery of nucleic acids. The interior of the stars consisted of hyperbranched poly(arylene oxindole), while the arms were composed of poly(N,N-dimethylaminoethyl methacrylate). The length of the star arms varied in degree of polymerization (DP) from 14 to 98. The hydrodynamic radius of the structures measured in water indicated the presence of small aggregates, while isolated stars ranging in size from 14 to 29 nm were seen in organic solvent. The phase transition temperatures of the stars in water, measured in basic conditions, were shifted to lower values with increasing DP of the arms. Stable polyplexes of stars with plasmid DNA were formed. Their size varied from 300 nm to 400 nm, depending upon the DP of arms. The zeta potential of the polyplexes was positive, which facilitated their cellular uptake. The DP of the arms influenced the transfection efficiency of HT-1080 cells, demonstrating that stars are promising candidates for synthetic gene vectors.     

Kanamycin A-derived cationic lipids as vectors for gene transfection

Cationic lipids nowadays constitute a promising alternative to recombinant viruses for gene transfer. We have recently explored the transfection potential of a new class of lipids based upon the use of aminoglycosides as cationic polar headgroups. The encouraging results obtained with a first cholesterol derivative of kanamycin A prompted us to investigate this family of vectors further, by modulating the constituent structural units of the cationic lipid. For this study, we have investigated the transfection properties of a series of new derivatives based on a kanamycin A scaffold. The results primarily confirm that aminoglycoside-based lipids are efficient vectors for gene transfection both in vitro and in vivo (mouse airways). Furthermore, a combination of transfection and physicochemical data revealed that some modifications of the constitutive subunits of kanamycin A-based vectors were associated with substantial changes in their transfection properties.     

PEGylated block copolymers containing tertiary amine side-chains cleavable via acid-labile ortho ester linkages for pH-triggered release of DNA

A new type of acid-labile cationic copolymer consisting of a hydrophilic poly(ethylene glycol) (PEG) block and a polymethacrylamide block bearing tertiary amines linked by acid-labile ortho ester rings in side chains (PAOE), with defined chain length, had been synthesized via RAFT polymerization. The copolymers could efficiently bind and condense plasmid DNA at neutral pH into narrowly dispersed nano-scale polyplexes. The hydrolysis of ortho ester group in the side-chains of PAOE followed a distinct exocyclic mechanism and the rate of hydrolysis was much accelerated at mildly acidic pH, resulting in the accelerated disruption of polyplexes and the release of intact plasmid DNA. The three polymers were not toxic to cultured COS-7 cells as measured by MTT assay. As expected, PEG segments of the PEG-b-PAOE copolymers prevented nonspecific transfection of COS-7 cells. Once conjugated to a targeting ligand to enhance cell-specific entry, PEG-b-PAOE with its pH-triggered DNA release properties may achieve efficient intracellular delivery of DNA or other nucleic acid therapeutics.     

A pH and Redox Dual Responsive 4-Arm Poly(ethylene glycol)-block-poly(disulfide histamine) Copolymer for Non-Viral Gene Transfection in Vitro and in Vivo

A novel 4-arm poly(ethylene glycol)-b-poly(disulfide histamine) copolymer was synthesized by Michael addition reaction of poly(ethylene glycol) (PEG) vinyl sulfone and amine-capped poly(disulfide histamine) oligomer, being denoted as 4-arm PEG-SSPHIS. This copolymer was able to condense DNA into nanoscale polyplexes (<200 nm in average diameter) with almost neutral surface charge (+(5–10) mV). Besides, these polyplexes were colloidal stable within 4 h in HEPES buffer saline at pH 7.4 (physiological environment), but rapidly dissociated to liberate DNA in the presence of 10 mM glutathione (intracellular reducing environment). The polyplexes also revealed pH-responsive surface charges which markedly increased with reducing pH values from 7.4–6.3 (tumor microenvironment). In vitro transfection experiments showed that polyplexes of 4-arm PEG-SSPHIS were capable of exerting enhanced transfection efficacy in MCF-7 and HepG2 cancer cells under acidic conditions (pH 6.3–7.0). Moreover, intravenous administration of the polyplexes to nude mice bearing HepG2-tumor yielded high transgene expression largely in tumor rather other normal organs. Importantly, this copolymer and its polyplexes had low cytotoxicity against the cells in vitro and caused no death of the mice. The results of this study indicate that 4-arm PEG-SSPHIS has high potential as a dual responsive gene delivery vector for cancer gene therapy.     

Before and after endosomal escape: roles of stimuli-converting siRNA/polymer interactions in determining gene silencing efficiency

Silencing the expression of a target gene by RNA interference (RNAi) shows promise as a potentially revolutionizing strategy for manipulating biological (pathological) pathways at the translational level. However, the lack of reliable, efficient, versatile, and safe means for the delivery of small interfering RNA (siRNA) molecules, which are large in molecular weight, negatively charged, and subject to degradation, has impeded their use in basic research and therapy. Polyplexes of siRNA and polymers are the predominant mode of siRNA delivery, but innovative synthetic strategies are needed to further evolve them to generate the desired biological and therapeutic effects. This Account focuses on the design of polymeric vehicles for siRNA delivery based on an understanding of the molecular interactions between siRNA and cationic polymers. Ideal siRNA/polymer polyplexes should address an inherent design dilemma for successful gene silencing: (1) Cationic polymers must form tight complexes with siRNA via attractive electrostatic interactions during circulation and cellular internalization and (2) siRNA must dissociate from its cationic carrier in the cytoplasm before they are loaded into RNA-induced silencing complex (RISC) and initiate gene silencing. The physicochemical properties of polymers, which dictate their molecular affinity to siRNA, can be programmed to be altered by intracellular stimuli, such as acidic pH in the endosome and cytosolic reducers, subsequently inducing the siRNA/polymer polyplex to disassemble. Specific design goals include the reduction of the cationic density and the molecular weight, the loss of branched structure, and changes in the hydrophilicity/hydrophobicity of the polymeric siRNA carriers, via acid-responsive degradation and protonation processes within the endosome and glutathione (GSH)-mediated reduction in the cytoplasm, possibly in combination with gradual stimuli-independent hydrolysis. Acetals/ketals are acid-cleavable linkages that have been incorporated into polymeric materials for stimuli-responsive gene and drug delivery. Tailoring the ketalization ratio and the molecular weight of ketalized branched PEI (K-BPEI) offers molecular control of the intracellular trafficking of siRNA/polymer polyplexes and, therefore, the gene silencing efficiency. The ketalization of linear PEI (K-LPEI) enhances gene silencing in vitro and in vivo by improving siRNA complexation with the polymer during circulation and cellular internalization, supplementing proton buffering efficiency of the polymer in the endosome, and facilitating siRNA dissociation from the polymer in the cytoplasm, in a serum-resistant manner. Spermine polymerization via ketalization and esterification for multistep intracellular degradations provides an additional polymeric platform for improved siRNA delivery and highly biocompatible gene silencing. The chemistry presented in this Account will help lay the foundation for the development of innovative and strategic approaches that advance RNAi technology.     

A combinatorial polymer library approach yields insight into nonviral gene delivery

The potential of gene therapy to benefit human health is tremendous because almost all human diseases have a genetic component, from untreatable monogenic disorders to cancer and heart disease. Unfortunately, a method for gene therapy that is both effective and safe has remained elusive. It has been said that "there are only three problems in gene therapy - delivery, delivery, and delivery." (quote from I. M. Verma in Jaroff, L. TIME, 1999; Jan 11). This Account describes an alternative strategy to viral gene delivery: the design of biodegradable polymers that are able to deliver DNA like a synthetic virus. Using high-throughput synthesis and screening techniques, we have created libraries of over 2000 structurally unique poly(β-amino esters) (PBAEs). PBAEs are formed by the conjugate addition of amines to diacrylates. These biomaterials are promising for nonviral gene delivery due to their ability to condense plasmid DNA into small and stable nanoparticles and their ability to promote cellular uptake and endosomal escape. Our laboratory has iteratively improved PBAE nanoparticles through polymer end modifications and nanoparticle coatings. Lead PBAEs have high gene delivery efficacy and low cytotoxicity both in vitro and in vivo. Certain polymer structural characteristics are important for effective gene delivery. The best PBAEs are linear polymers of ~10 kDa that contain hydroxyl side chains and primary amine end groups. These polymers bind DNA to form nanoparticles that are small (<200 nm) and stable and have near-neutral ζ potential in the presence of serum-containing media. Lead PBAEs also contain tertiary amines that can buffer the low pH environment of endosomes and facilitate escape of polymer/DNA particles into the cytoplasm. Diamine end-modified 1,4-butanediol diacrylate-co-5-amino-1-pentanol polymers (C32) bind DNA more tightly and form smaller nanoparticles than other PBAEs. These nanoparticles also have higher cellular uptake and the best gene expression of all gene delivery polymers in the library. These polymers are more effective for gene delivery than top commercially available nonviral vectors including jet-PEI and Lipofectamine 2000 and are comparable to adenovirus for in vitro gene delivery to human primary cells. In vivo, these PBAE/DNA particles are promising as cancer therapeutics. This Account summarizes the results of our laboratory in using a combinatorial polymer library approach to elucidate polymer structure/function relationships and enable the development of polymeric gene delivery nanoparticles with viral-like efficacy.     

Polycondensed Peptide Carriers Modified with Cyclic RGD Ligand for Targeted Suicide Gene Delivery to Uterine Fibroid Cells

Suicide gene therapy was suggested as a possible strategy for the treatment of uterine fibroids (UFs), which are the most common benign tumors inwomen of reproductive age. For successful suicide gene therapy, DNAtherapeutics should be specifically delivered to UF cells. Peptide carriers are promising non-viral gene delivery systems that can be easily modified with ligands and other biomolecules to overcome DNA transfer barriers. Here we designed polycondensed peptide carriers modified with a cyclic RGD moiety for targeted DNA delivery to UF cells. Molecular weights of the resultant polymers were determined, and inclusion of the ligand was confirmed by MALDI-TOF. The physicochemical properties of the polyplexes, as well as cellular DNA transport, toxicity, and transfection efficiency were studied, and the specificity of αvβ3 integrin-expressing cell transfection was proved. The modification with the ligand resulted in a three-fold increase of transfection efficiency. Modeling of the suicide gene therapy by transferring the HSV-TK suicide gene to primary cells obtained from myomatous nodes of uterine leiomyoma patients was carried out. We observed up to a 2.3-fold decrease in proliferative activity after ganciclovir treatment of the transfected cells. Pro- and anti-apoptotic gene expression analysis confirmed our findings that the developed polyplexes stimulate UF cell death in a suicide-specific manner.     

Amphiphilic poly[(propylene glycol)-block-(2-methyl-2-oxazoline)] copolymers for gene transfer in skeletal muscle

Amphiphilic triblock copolymers such as poly(ethylene glycol-b-propylene glycol-b-ethylene glycol) PE6400 (PEG(13)-PPG(30)-PEG(13)) have been recently shown to promote gene transfer in muscle. Herein we investigated the effect of a chemical change of the PEG moiety on the transfection activity of these compounds. We synthesized new amphiphilic copolymers in which the PEG end blocks are replaced by more hydrophilic poly(2-methyl-2-oxazoline) (PMeOxz) chains of various lengths. The resulting triblock PMeOxz-PPG-PMeOxz compounds were characterized by NMR, SEC, TGA, and DSC techniques and assayed for in vivo muscle gene transfer. The results confirm both the block structure and the monomer unit composition (DP(PG)/DP(MeOxz)) of the new PPG(34)-PMeOxz(41) and PPG(34)-PMeOxz(21) triblock copolymers. Furthermore, in vivo experiments show that these copolymers are able to significantly increase DNA transfection efficiency, despite the fact that their chemical nature and hydrophilic character are different from the poloxamers. Overall, these results show that the capacity to enhance DNA transfection in skeletal muscle is not restricted to PEG-PPG-PEG arrangements.