Ionic α-helical polypeptides toward nonviral gene delivery.

The advent of polymeric materials has significantly promoted the development and rapid growth of various technologies in biomedical applications, such as tissue engineering and controlled drug and gene delivery. Water-soluble polypeptides bearing functional side chains and adopting stable secondary structures are a new class of functional polymeric materials of potentially broad applications in medicine and biotechnology. In this article, we summarize our recent effort on the design and synthesis of the water-soluble α-helical ionic polypeptides originally developed in our laboratory and highlight their applications in cell membrane penetration and nonviral gene/small interfering RNA (siRNA) delivery.

Maximizing gene delivery efficiencies of cationic helical polypeptides via balanced membrane penetration and cellular targeting.

The application of non-viral gene delivery vectors is often accompanied with the poor correlation between transfection efficiency and the safety profiles of vectors. Vectors with high transfection efficiencies often suffer from high toxicities, making it unlikely to improve their efficiencies by increasing the DNA dosage. In the current study, we developed a ternary complex system which consisted of a highly membrane-active cationic helical polypeptide (PVBLG-8), a low-toxic, membrane-inactive cationic helical polypeptide (PVBLG-7) capable of mediating mannose receptor targeting, and DNA. The PVBLG-7 moiety notably enhanced the cellular uptake and transfection efficiency of PVBLG-8 in a variety of mannose receptor-expressing cell types (HeLa, COS-7, and Raw 264.7), while it did not compromise the membrane permeability of PVBLG-8 or bring additional cytotoxicities. Because of the simplicity and adjustability of the self-assembly approach, optimal formulations of the ternary complexes with a proper balance between membrane activity and targeting capability were easily identified in each specific cell type. The optimal ternary complexes displayed desired cell tolerability and markedly outperformed the PVBLG-8/DNA binary complexes as well as commercial reagent Lipofectamine™ 2000 in terms of transfection efficiency. This study therefore provides an effective and facile strategy to overcome the efficiency-toxicity poor correlation of non-viral vectors, which contributes insights into the design strategy of effective and safe non-viral gene delivery vectors.

Cationic, helical polypeptide-based gene delivery for IMR-90 fibroblasts and human embryonic stem cells.

Diblock copolymers consisting of poly(ethylene glycol)-block-poly(γ-4-(((2-(piperidin-1-yl)ethyl)amino)methyl)benzyl-L-glutamate) (PEG-b-PVBLG-8) were synthesized and evaluated for their ability to mediate gene delivery in hard-to-transfect cells like IMR-90 human fetal lung fibroblasts and human embryonic stem cells (hESCs). The PEG-b-PVBLG-8 contained a membrane-disruptive, cationic, helical polypeptide block (PVBLG-8) for complexing with DNA and a hydrophilic PEG block to improve the biocompatibility of the gene delivery vehicle. The incorporation of PEG effectively reduced the toxicity of the helical PVBLG-8 block without dramatically compromising the polymer's ability to destabilize membranes or form complexes with DNA. PEG-b-PVBLG-8 copolymers with low (n = 76) and high (n = 287) degrees of polymerization (n) of the PVBLG-8 block were synthesized and evaluated for gene delivery. PEG-b-PVBLG-8 diblock polymers with a high degree of polymerization have a greater transfection efficiency and lower toxicity in IMR-90 cells than the commercial reagent Lipofectamine 2000. The usefulness of PEG-b-PVBLG-8 was further demonstrated via the successful transfection of hESCs without a measured loss in cell pluripotency markers.

Cell-laden hydrogels in integrated microfluidic devices for long-term cell culture and tubulogenesis assays.

A hydrogel biochip combining microfluidic mixing and orthogonal supplementation strategies is developed and validated to allow facile generation of libraries of optically transparent 3D culture microenvironments. Live, on-chip tracing of embryonic stem cell differentiation and endothelial cell tubulogenesis confirms that the platform can be used to both create communities of discrete 3D microenvironments as well as to locally monitor subsequent divergent responses at both single cell and multi-cell scales.

Non-viral gene delivery via membrane-penetrating, mannose-targeting supramolecular self-assembled nanocomplexes.

Supramolecular self-assembled nanocomplexes (SSANs) capable of mannose receptor-mediated endocytosis and permeable to cellular and endosomal membranes are developed via the assembly of multiple rationally designed, function-specific materials. As a unique non-viral gene delivery vector, SSANs outperform commercial transfection reagents, including LPF2000, PEI, and jetPEI, by up to 2 orders of magnitude.

Size-dependent tumor penetration and in vivo efficacy of monodisperse drug-silica nanoconjugates.

The size of a nanomedicine strongly correlates with its biodistribution, tissue penetration, and cell uptake. However, there is limited understanding how the size of nanomedicine impacts the overall antitumor efficacy. We designed and synthesized camptothecin-silica nanoconjugates (Cpt-NCs) with monodisperse particle sizes of 50 and 200 nm, two representative sizes commonly used in drug delivery, and evaluated their antitumor efficacy in murine tumor models. Our studies revealed that the 50 nm Cpt-NC showed higher anticancer efficacy than the larger analogue, due presumably to its faster cellular internalization and more efficient tumor accumulation and penetration. Our findings suggest that nanomedicine with smaller sizes holds great promise for improved cancer therapy.

A cell-penetrating helical polymer for siRNA delivery to mammalian cells.

Cell-penetrating peptides (CPPs) are routinely used for intracellular delivery of a variety of cargo, including drugs, genes, and short interfering RNA (siRNA). Most CPPs are active only upon exposure to acidic environments inside of late endosomes, thereby facilitating the endosomal escape of internalized vectors. Here, we describe the generation of a synthetic polypeptide--PVBLG(n)-8--that is able to adopt a helical structure independent of pH. Like other CPPs, the helical structure of PVBLG(n)-8 allows the polypeptide to destabilize membranes. However, since the helix is stable at all physiologically relevant pH values between pH 2 and pH 7.4, the membrane permeation properties of PVBLG(n)-8 are irreversible. Given its pH-insensitive activity, our results suggest that PVBLG(n)-8 is able to facilitate efficient siRNA delivery by causing pore formation in the cell membranes through which either free or complexed siRNA is able to diffuse. This nonspecific form of entry into the cell cytosol may prove useful when trying to deliver siRNA to cells which have proven to be difficult to transfect.

The effects of PVP(Fe(III)) catalyst on polymer molecular weight and gene delivery via biodegradable cross-linked polyethylenimine.

PURPOSE: Crosslinked, degradable derivatives of low-molecular-weight polyethylenimine (PEI) are relatively efficient and non-cytotoxic gene delivery agents. To further investigate these promising materials, a new synthetic approach was developed using a poly(4-vinylpyridine)-supported Fe(III) catalyst (PVP(Fe(III))) that provides more facile synthesis and enhanced control of polymer molecular weight. METHODS: Biodegradable polymers (D.PEI) comprising 800-Da PEI crosslinked with 1,6-hexanediol diacrylate and exhibiting molecular weights of 1.2, 6.2, and 48 kDa were synthesized utilizing the PVP(Fe(III)) catalyst. D.PEI/DNA polyplexes were characterized using gel retardation, ethidium bromide exclusion, heparan sulfate displacement, and dynamic light scattering. In vitro transfection, cellular uptake, and cytotoxicity of the polyplexes were tested in human cervical cancer cells (HeLa) and human breast cancer cells (MDA-MB-231). RESULTS: D.PEIs tightly complexed plasmid DNA and formed 320- to 440-nm diameter polyplexes, similar to those comprising non-degradable, 25-kDa, branched PEI. D.PEI polyplexes mediated 2- to 5-fold increased gene delivery efficacy compared to 25-kDa PEI and exhibited 20% lower cytotoxicity in HeLa and no toxicity in MDA-MB-231. In addition, 2- to 7-fold improved cellular uptake of DNA was achieved with D.PEI polyplexes. CONCLUSIONS: PVP(Fe(III)) catalyst provided a more controlled synthesis of D.PEIs, and these materials demonstrated improved in vitro transfection efficacy and reduced cytotoxicity .

Polymeric Nanomedicines Based on Poly(lactide) and Poly(lactide-co-glycolide).

Small molecule chemotherapeutics often have undesired physiochemical and pharmacological properties, such as low solubility, severe side effect and narrow therapeutic index. To address these challenges, polymeric nanomedicine drug delivery technology has been routinely employed, in particular with the use of biodegradable and biocompatible polyesters, such as poly(lactide) (PLA) and poly(lactide-co-glycolide) (PLGA). Here we review the development and use of PLA and PLGA for the delivery of chemotherapeutic agents in the forms of polymer-drug conjugates and nanoconjugates.

Ring-Opening Polymerization of γ-(4-Vinylbenzyl)-(L)-Glutamate N-Carboxyanhydride for the Synthesis of Functional Polypeptides.

Introducing various pendant functional groups and building blocks of interest to polypeptides in a highly efficient, controlled manner is crucial to access polypeptide materials with desired structures and functions. In this study, we synthesized γ-(4-vinylbenzyl)-(L)-glutamate N-carboxyanhydride (VB-Glu-NCA), which was readily obtained and purified in large quantity. VB-Glu-NCA monomer was subsequently used for the synthesis of polypeptides containing conjugation-amenable, pendant vinyl functional groups. Controlled, living polymerizations of VB-Glu-NCA were achieved by using hexamethyldisilazane (HMDS) as the initiator, catalytic amounts of 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) as the co-catalyst, and nitrobenzene as the inhibitor of radical-induced side reactions on the vinyl group of VB-Glu-NCA. The resulting poly(γ-(4-vinylbenzyl)-(L)-glutamate) (PVBLG) gave rise to polypeptides containing pendant functional groups or moieties through various vinyl chemistries.

Hydrophobically modified dendrons: developing structure-activity relationships for DNA binding and gene transfection.

This paper develops a structure-activity relationship understanding of the way in which surfactant-like dendrons with hydrophilic spermine surface groups and a variety of lipophilic units at their focal points can self-assemble and subsequently bind to DNA with high affinity. The choice of functional group at the focal point of the dendron and the high tunability of the molecular structure have a very significant impact on DNA binding. Mesoscale modeling of the mode of dendron self-assembly provides a direct insight into how the mode of self-assembly exerts its effect on the DNA binding process. In particular, the hydrophobic unit controls the number of dendrons in the self-assembled micellar structures, and hence their diameters and surface charge density. The DNA binding affinity correlates with the surface charge density of the dendron aggregates. Furthermore, these structure-activity effects can also be extended to cellular gene delivery, as surface charge density plays a role in controlling the extent of endosomal escape. It is reported that higher generation dendrons, although binding DNA less strongly than the self-assembling lower generation dendrons, are more effective for transfection. The impact of the lipophilic group at the focal point is less significant for the DNA binding ability of these larger dendrons, which is predominantly controlled by the spermine surface groups, but it does modify the levels of gene transfection. Significant synergistic effects on gene delivery were observed when employing combinations of the dendrons and polyethyleneimine (PEI, 25 kDa), with transfection becoming possible at low loading levels where the two components would not transfect individually, giving practically useful levels of gene delivery.

Multiplexed supramolecular self-assembly for non-viral gene delivery.

Recently, there has been success in applying a semi-rational approach to non-viral gene delivery vector development using a combinatorial/parallel synthesis approach to construct libraries of materials with unique molecular structures. In this approach, it is hoped that the random incorporation of various hydrophobic and hydrophilic domains in the library will yield candidates with the appropriate balance of DNA binding strength and endosomolytic properties to yield efficient gene delivery. Herein we describe a library approach to gene delivery vector development that relies on the supramolecular self-assembly of individual components instead of chemical reaction. Each component in the described system is capable of performing a single and well-defined purpose--DNA binding (dioleylspermine), membrane permeation (oligoarginine) or targeting (folic acid). A combination of electrostatic attraction and the hydrophobic effect is used to bring the individual groups together to form nanoscale complexes with DNA. Because the components responsible for DNA binding, membrane permeation and targeting are separate, it is possible to alter the balance between hydrophilic and hydrophobic groups by varying the relative amounts in the final formulation. By doing so, we can readily identify cell-specific formulations that have greater transfection efficiency than the individual components and have superior transfection efficiency to lipofectamine 2000 under similar conditions.

Synergistic effects on gene delivery--co-formulation of small disulfide-linked dendritic polycations with Lipofectamine 2000.

This paper describes the application of gene delivery vectors based on connecting together two well-defined low-generation poly(L-lysine) (PLL) dendrons using a disulfide-containing linker unit. We report that the transfection ability of these vectors in their own right is relatively low, because the low-generation number limits the endosomal buffering capacity. Importantly, however, we demonstrate that when applied in combination with Lipofectamine 2000, a vector from the cationic lipid family, these small cationic additives significantly enhance the levels of gene delivery (up to four-fold). Notably, the cationic additives have no effect on the levels of transfection observed with a cationic polymer, such as DEAE dextran. We therefore argue that the synergistic effects observed with Lipofectamine 2000 arise as a result of combining the delivery advantages of two different classes of vector within a single formulation, with our dendritic additives providing a degree of pH buffering within the endosome. As such, the data we present indicate that small dendritic structures, although previously largely overlooked for gene delivery owing to their inability to transfect in their own right, may actually be useful well-defined additives to well-established vector systems in order to enhance the gene delivery payload.

Efficient polyethylenimine-mediated gene delivery proceeds via a caveolar pathway in HeLa cells.

Most in vivo gene therapies will require cell-specific targeting. Although vector targeting through ligand attachment has met with success in generating gene delivery particles that are capable of specific cellular interactions, little attention has been given to the possible effects of such ligands on subsequent intracellular processing. In this study, we examine the impact of targeting two distinct endocytic routes-the caveolar and clathrin pathways-on polyethylenimine-mediated gene delivery in HeLa cells. Targeting complexes to the caveolar pathway with folic acid and the clathrin pathway with transferrin yields enhanced gene delivery relative to unmodified polyethylenimine. Colocalization studies with caveolin-1 and clathrin heavy chain indicate that the ligands successfully deliver their cargo to the intended pathways. However, inhibition of only the caveolar pathway-whether through the use of small molecule drugs or RNA interference-reduces gene delivery efficiency, suggesting that successful polyethylenimine-mediated gene delivery proceeds via a caveolar pathway in HeLa cells. Transfections in the presence of chloroquine and pH tracking studies reveal that a contributing factor to the success of the caveolar pathway is avoidance of lysosomes. Collectively, these data demonstrate that uptake mechanism and subsequent endocytic processing are important design parameters for gene delivery materials.

The effect of chitosan on the migration of neutrophil-like HL60 cells, mediated by IL-8.

Research interest in chitosan stems in part from the demonstrated wound healing properties. The benefits of chitosan as a therapeutic agent appear to be paradoxical because chitosan also elicits neutrophil infiltration indicative of an inflammatory response. While the affinity between chitosan and neutrophils has been well documented, the underlying mechanism is unclear. To our knowledge, no studies have investigated the consequences of chitosan-neutrophil interaction to explain neutrophil migration. To that end, transwell migration assays to chitosan of varying extent of acetylation were conducted using a differentiated model cell line (HL60-PMN) in order to assess the effect of chitosan chemistry and the resultant physical properties such as charge and hydrophobicity on neutrophil migration. As chitosan N-acetylation increased, neutrophil migration increased and chitosan became less positively charged and more hydrophobic. Moreover, HL60-PMN cells secreted the potent neutrophil chemokine IL-8, also known as CXCL8, when exposed to chitosan and IL-8 levels increased with N-acetylation, and migration was inhibited by anti-IL-8 antibodies. Collectively these results suggest that chitosan-neutrophil interaction is encouraged by material properties, results in IL-8 secretion, and causes migration of neutrophils to chitosan. The implication is that the wound healing properties of chitosan may be enhanced through the attenuation of overabundant neutrophils, and thus the inflammatory response, simply by changing chitosan N-acetylation.

Synergistic effects in gene delivery-a structure-activity approach to the optimisation of hybrid dendritic-lipidic transfection agents.

Novel gene delivery agents based on combining cholesterol units with spermine-functionalised dendrons exhibit enhanced transfection ability-we report significant synergistic effects in mixed (hybrid) systems which combine aspects of both main classes of synthetic vectors, i.e., cationic polymers and lipids.

DNA nanoparticles encapsulated in 3D tissue-engineered scaffolds enhance osteogenic differentiation of mesenchymal stem cells.

In this study, we enhanced the expression of a plasmid DNA in mesenchymal stem cells (MSC) by the combination of three-dimensional (3D) tissue-engineered scaffold and nonviral gene carrier. To function as an enhanced delivery of plasmid DNA, acetic anhydride was reacted with polyethylenimine (PEI) to acetylate 80% of the primary and 20% of the secondary amines (PEI-Ac(80)). This acetylated PEI has been demonstrated to show enhanced gene-delivery efficiency over unmodified PEI. Collagen sponges reinforced by incorporating of poly(glycolic acid) (PGA) fibers were used as the scaffold material. DNA nanoparticles formed through simple mixing of plasmid DNA encoding bone morphogenetic protein-2 (BMP-2) and PEI-Ac(80) solutions were encapsulated within these scaffolds. MSC were seeded into each scaffold and cultured for several weeks. Within these scaffolds, the level of BMP-2 expression by transfected MSC was significantly enhanced compared to MSC transfected by DNA nanoparticles in solution (in 2D tissue culture plates). Homogeneous bone formation was histologically observed throughout the sponges seeded with transfected MSC by using DNA nanoparticles after subcutaneous implantation into the back of rats. The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of sponges seeded with transfected MSC by using DNA nanoparticles were significantly higher when compared with those seeded with other agents.