Biomaterials (v.32, #9)

Tissue response to poly(l-lactic acid)-based blend with phospholipid polymer for biodegradable cardiovascular stents by Hyung Il Kim; Kazuhiko Ishihara; Seungbok Lee; Ji-Hun Seo; Hye Young Kim; Dongwhan Suh; Min Uk Kim; Tomohiro Konno; Madoka Takai; Jeong-Sun Seo (2241-2247).
A temporary cardiovascular stent device by bioabsorbable materials might reduce late stent thrombosis. A water-soluble amphiphilic phospholipid polymer bearing phosphorylcholine groups (PMB30W) was blended with a high-molecular-weight poly(l-lactic acid) (PLLA) to reduce unfavorable tissue responses at the surface. The PLLA implants and the polymer blend (PLLA/PMB30W) implants were inserted into subcutaneous tissues of rats, the infrarenal aorta of rats, and the internal carotid arteries of rabbits. After 6 months subcutaneous implantation, the PLLA/PMB30W maintained high density of phosphorylcholine groups on the surface without a significant bioabsorption. After intravascular implantation, the cross-sectional areas of polymer tubing with diameters less than 1.6 mm were histomorphometrically measured. Compared to the PLLA tubing, the PLLA/PMB30W tubing significantly reduced the thrombus formation during 30 d of implantation. Human peripheral blood mononuclear cells were cultured on the PLLA and the PLLA/PMB30W to compare inflammatory reactions. Enzyme-linked immunosorbent assay quantified substantially decreased proinflammatory cytokines in the case of the PLLA/PMB30W. They were almost the same level as the negative controls. Thus, we conclude that the phosphorylcholine groups could reduce tissue responses significantly both in vivo and in vitro, and the PLLA/PMB30W is a promising material for preparing temporary cardiovascular stent devices.
Keywords: Polymer blend; poly(lactic acid); Phosphorylcholine group; Tissue compatibility; Cardiovascular stent;

A model for biodegradation of composite materials made of polyesters and tricalcium phosphates by Jingzhe Pan; Xiaoxiao Han; Wenjuan Niu; Ruth E. Cameron (2248-2255).
A saturation behaviour has been observed when incorporating tricalcium phosphate (TCP) in various polyesters to control the degradation rate. This paper presents an understanding of this behaviour using a mathematical model. The coupled process of hydrolysis reaction of the ester bonds, acid dissociation of the carboxylic end groups, dissolution of the calcium phosphates and buffering reactions by the dissolved phosphate ions is modelled together using a set of differential equations. Two non-dimensional groups of the material and chemical parameters are indentified which control the degradation rate of the composites. An effectiveness map is established to show the conditions under which incorporating TCP into polyesters is effective, saturated or ineffective. Comparisons are made between the model predictions and existing experimental data in the literature. The map provides a useful tool to guide the design of polyester/TCP composites for tissue engineering and orthopaedic fixation applications.
Keywords: Tricalcium phosphates; Biodegradation; Biodegradable polymers; Modelling; Composites;

Modulus-driven differentiation of marrow stromal cells in 3D scaffolds that is independent of myosin-based cytoskeletal tension by Sapun H. Parekh; Kaushik Chatterjee; Sheng Lin-Gibson; Nicole M. Moore; Marcus T. Cicerone; Marian F. Young; Carl G. Simon (2256-2264).
Proliferation and differentiation of cells are known to be influenced by the physical properties of the extracellular environment. Previous studies examining biophysics underlying cell response to matrix stiffness utilized a two-dimensional (2D) culture format, which is not representative of the three-dimensional (3D) tissue environment in vivo. We report on the effect of 3D matrix modulus on human bone marrow stromal cell (hBMSC) differentiation. hBMSCs underwent osteogenic differentiation in poly(ethylene glycol) hydrogels of all modulus (300-fold modulus range, from 0.2 kPa to 59 kPa) in the absence of osteogenic differentiation supplements. This osteogenic differentiation was modulus-dependent and was enhanced in stiffer gels. Osteogenesis in these matrices required integrin-protein ligation since osteogenesis was inhibited by soluble Arginine-Glycine-Aspartate-Serine peptide, which blocks integrin receptors. Immunostained images revealed lack of well-defined actin filaments and microtubules in the encapsulated cells. Disruption of mechanosensing elements downstream of integrin binding that have been identified from 2D culture such as actin filaments, myosin II contraction, and RhoA kinase did not abrogate hBMSC material-driven osteogenic differentiation in 3D. These data show that increased hydrogel modulus enhanced osteogenic differentiation of hBMSCs in 3D scaffolds but that hBMSCs did not use the same mechanosensing pathways that have been identified in 2D culture.
Keywords: Three-dimensional culture; Bone marrow stromal cell; Cytoskeleton; Hydrogels; Matrix modulus;

A sandwich model for engineering cartilage with acellular cartilage sheets and chondrocytes by Yi Yi Gong; Ji Xin Xue; Wen Jie Zhang; Guang Dong Zhou; Wei Liu; Yilin Cao (2265-2273).
Acellular cartilage can provide a native extracellular matrix for cartilage engineering. However, it is difficult for cells to migrate into acellular cartilage because of its non-porous structure. The aim of this study is to establish a sandwich model for engineering cartilage with acellular cartilage sheets and chondrocytes. Cartilage from adult pig ear was cut into a circular cylinder with a diameter of approximately 6 mm and freeze-sectioned at thicknesses of 10 μm and 30 μm. The sheets were then decellularized and lyophilized. Chondrocytes isolated from newborn pig ear were expanded for 2 passages. The acellular sheets and chondrocytes were then stacked layer-by-layer, in a sandwich model, and cultured in dishes. After 4 weeks of cultivation, the constructs were then either maintained in culture for another 12 weeks or implanted subcutaneously in nude mouse. Histological analysis showed that cells were completely removed from cartilage sheets after decellularization. By re-seeding cells and stacking 20 layers of sheets together, a cylinder-shaped cell sheet was achieved. Cartilage-like tissues formed after 4 weeks of culture. Histological analyses showed the formation of cartilage with a typical lacunar structure. Cartilage formation was more efficient with 10 μm-thick sheets than with 30 μm sheets. Mature cartilage was achieved after 12 weeks of implantation, which was demonstrated by histology and confirmed by Safranin O, Toluidine blue and anti-type II collagen antibody staining. Furthermore, we achieved cartilage with a designed shape by pre-shaping the sheets prior to implantation. These results indicate that the sandwich model could be a useful model for engineering cartilage in vitro and in vivo.
Keywords: Cartilage; Chondrocyte; ECM; Cartilage tissue engineering;

Multipotent neural precursor/stem cells (NPCs) are a major transplant population with key properties to promote repair in several neuropathological conditions. Magnetic nanoparticle (MNP)-based vector systems, in turn, offer a combination of key benefits for cell therapies including (i) safety (ii) delivery of therapeutic biomolecules (DNA/siRNA) enhanceable by ‘magnetofection’ approaches (iii) magnetic cell targeting of MNP-labelled cells to injury sites and (iv) non-invasive imaging of MNP-labelled transplant populations for cell tracking. However, the applications of the versatile MNP platform for NPC transplantation therapies have received limited attention so far. We have evaluated the potential of MNP vectors for gene transfer to NPCs using a neurosphere culture model system; we also assessed repeat transfection (“multifection”) and repeat transfection plus applied magnetic field (“magneto-multifection”) strategies [to enhance transfection efficiency]. We show for the first time that MNPs can safely mediate single/combinatorial gene delivery to NPCs. Multifection approaches significantly enhanced transfection with negligible toxicity; no adverse effects were observed on stem cell proliferation/differentiation. “Multifected” NPCs survived and differentiated in 3D neural tissue arrays post-transplantation. Our findings demonstrate that MNPs offer a simple and robust alternative to the viral vector systems currently used widely to transfect neural stem cells in neurobiology/neural transplantation research.
Keywords: Nanoparticle; Magnetism; Neural cell; Stem cell; Transplantation; Genetic engineering;

Dynamic cell behavior on shape memory polymer substrates by Kevin A. Davis; Kelly A. Burke; Patrick T. Mather; James H. Henderson (2285-2293).
Cell culture substrates of defined topography have emerged as powerful tools with which to investigate cell mechanobiology, but current technologies only allow passive control of substrate properties. Here we present a thermo-responsive cell culture system that uses shape memory polymer (SMP) substrates that are programmed to change surface topography during cell culture. Our hypothesis was that a shape-memory-activated change in substrate topography could be used to control cell behavior. To test this hypothesis, we embossed an initially flat SMP substrate to produce a temporary topography of parallel micron-scale grooves. After plating cells on the substrate, we triggered shape memory activation using a change in temperature tailored to be compatible with mammalian cell culture, thereby causing topographic transformation back to the original flat surface. We found that the programmed erasure of substrate topography caused a decrease in cell alignment as evidenced by an increase in angular dispersion with corresponding remodeling of the actin cytoskeleton. Cell viability remained greater than 95% before and after topography change and temperature increase. These results demonstrate control of cell behavior through shape-memory-activated topographic changes and introduce the use of active cell culture SMP substrates for investigation of mechanotransduction, cell biomechanical function, and cell soft-matter physics.
Keywords: Cell culture; Shape memory; Thermally responsive material; Surface topography;

Pre-osteoblast infiltration and differentiation in highly porous apatite-coated PLLA electrospun scaffolds by Bryce M. Whited; Jon R. Whitney; Matthias C. Hofmann; Yong Xu; Marissa N. Rylander (2294-2304).
Electrospun polymer/apatite composite scaffolds are promising candidates as functional bone substitutes because of their ability to allow pre-osteoblast attachment, proliferation, and differentiation. However these structures usually lack an adequate pore size to permit sufficient cell migration and colonization of the scaffold. To overcome this limitation, we developed an apatite-coated electrospun PLLA scaffold with varying pore size and porosity by utilizing a three-step water-soluble PEO fiber inclusion, dissolution, and mineralization process. The temporal and spatial dynamics of cell migration into the scaffolds were quantified to determine the effects of enhanced pore size and porosity on cell infiltration. MC3T3-E1 pre-osteoblast migration into the scaffolds was found to be a function of both initial PEO content and time. Scaffolds with greater initial PEO content (50% and 75% PEO) had drastically accelerated cell infiltration in addition to enhanced cell distribution throughout the scaffold when compared to scaffolds with lower PEO content (0% and 25% PEO). Furthermore, scaffolds with an apatite substrate significantly upregulated MC3T3-E1 alkaline phosphatase activity, osteocalcin content, and cell-mediated mineralization as compared to PLLA alone. These findings suggest that such a scaffold enhances pre-osteoblast infiltration, colonization, and maturation in vitro and may lead to overall improved bone formation when implanted in vivo.
Keywords: Electrospinning; Scaffold; Tissue engineering; Osteoblast; Bone;

Hydrophobic polyhydroxyalkanoate (PHA) scaffolds made of a copolyester of 3-hydroxybutyrate-co-hydroxyhexanoate (PHBHHx) were coated with a fusion protein PHA granule binding protein PhaP fused with RGD peptide (PhaP-RGD). Human bone marrow mesenchymal stem cells (hBMSCs) were inoculated on/in the scaffolds for formation of articular cartilages derived from chondrogenic differentiation of hBMSCs for cartilage tissue engineering. PhaP-RGD coating led to more homogeneous spread of cells, better cell adhesion, proliferation and chondrogenic differentiation in the scaffolds compared with those of PhaP coated or uncoated scaffolds immerging in serum minus chondrogenic induction medium. In addition, more extracellular matrices were produced by the differentiated cells over a period of 14 days on/in the PhaP-RGD coated scaffolds evidenced by scanning electron microscopy imaging, enhanced expression of chondrocyte specific genes including SOX-9, aggrecan and type II collagen, suggesting the positive effect of RGD on extracellular matrix production. Furthermore, cartilage-specific extracellular substances sulphated glycosaminoglycans (sGAG) and total collagen content found on/in the PhaP-RGD coated scaffolds were significantly more compared with that produced by the control and PhaP only coated scaffolds. Homogeneously distributed chondrocytes-like cells forming cartilage-like matrices were observed on/in the PhaP-RGD coated scaffolds after 3 weeks. The results suggested that PhaP-RGD coated PHBHHx scaffold promoted chondrogenic differentiation of hBMSCs and could support cartilage tissue engineering.
Keywords: PHB; Polyhydroxyalkanoates; PhaP; RGD; Bone marrow mesenchymal stem cells; Chondrogenic differentiation;

Active targeting and safety profile of PEG-modified adenovirus conjugated with herceptin by Pyung-Hwan Kim; Joo-Hyuk Sohn; Joung-Woo Choi; Yukyung Jung; Sung Wan Kim; Seungjoo Haam; Chae-Ok Yun (2314-2326).
PEGylation of adenovirus (Ad) increases plasma retention and reduces immunogenicity, but decreases the accessibility of virus particles to target cells. We tested whether PEGylated Ad conjugated to Herceptin (Ad-PEG-HER) can be used to treat Her2/neu-positive cells in vitro and in vivo to demonstrate the therapeutic feasibility of this Ad formulation. Ad-PEG-HER transduced Her2/neu-overexpressing cancer cells through a specific interaction between Herceptin and Her2/neu. Ad-PEG-HER treatment resulted in higher plasma retention and lower neutralizing antibody and IL-6 production than naked Ad. This formulation was extended to generate a Her2/neu-targeted, PEGylated oncolytic Ad (DWP418-PEG-HER). DWP418-PEG-HER specifically killed Her2/neu-positive cells and performed better than non-targeted and naked Ad in vivo. DWP418-PEG-HER showed a 1010-fold increase in the liver to tumor biodistribution compared with naked Ad. Immunohistochemical staining confirmed accumulation of Ad E1A in tumors. These data suggest that targeted gene therapy with the PEGylated Ad conjugated with Herceptin might shed a light on its therapeutic application for metastatic cancer in the future.
Keywords: Cancer gene therapy; Adenovirus; Ad conjugated bioreducible polymer; Hybrid vector; Active targeting;

Tumor transfection after systemic injection of DNA lipid nanocapsules by Marie Morille; Catherine Passirani; Sandrine Dufort; Guillaume Bastiat; Bruno Pitard; Jean-Luc Coll; Jean-Pierre Benoit (2327-2333).
With the goal of generating an efficient vector for systemic gene delivery, a new kind of nanocarrier consisting of lipid nanocapsules encapsulating DOTAP/DOPE lipoplexes (DNA LNCs) was pegylated by the post-insertion of amphiphilic and flexible polymers. The aim of this surface modification was to create a long-circulating vector, able to circulate in the blood stream and efficient in transfecting tumoral cells after passive targeting by enhanced permeability and retention effect (EPR effect). PEG conformation, electrostatic features, and hydrophylicity are known to be important factors able to influence the pharmacokinetic behaviour of vectors. In this context, the surface structure characteristics of the newly pegylated DNA LNCs were studied by measuring electrophoretic mobility as a function of ionic strength in order to establish a correlation between surface properties and in vivo performance of the vector. Finally, thanks to this PEGylation, gene expression was measured up to 84-fold higher in tumor compared to other tested organs after intravenous injection. The present results indicate that PEGylated DNA LNCs are promising carriers for an efficient cancer gene therapy.
Keywords: Intravenous injection; Gene delivery; Non-viral vector; Pegylation-nanoparticles;

64Cu loaded liposomes as positron emission tomography imaging agents by Anncatrine L. Petersen; Tina Binderup; Palle Rasmussen; Jonas R. Henriksen; Dennis R. Elema; Andreas Kjær; Thomas L. Andresen (2334-2341).
We have developed a highly efficient method for utilizing liposomes as imaging agents for positron emission tomography (PET) giving high resolution images and allowing direct quantification of tissue distribution and blood clearance. Our approach is based on remote loading of a copper-radionuclide (64Cu) using a new ionophore, 2-hydroxyquinoline, to carry 64Cu(II) across the membrane of preformed liposomes and deliver it to an encapsulated copper-chelator. Using this ionophore we achieved very efficient loading (95.5 ± 1.6%) and retention stability (>99%), which makes the 64Cu-liposomes highly applicable as PET imaging agents. We show the utility of the 64Cu-liposomes for quantitative in vivo imaging of healthy and tumor-bearing mice using PET. This remote loading method is a powerful tool for characterizing the in vivo performance of liposome based nanomedicine, and has great potential in diagnostic and therapeutic applications.
Keywords: Liposome; Diagnostics; Positron emission tomography (PET); Tumor accumulation; Remote loading;

Cytotoxicity of, and innate immune response to, size-controlled polypyrrole nanoparticles in mammalian cells by Sojin Kim; Wan-Kyu Oh; Yoon Seon Jeong; Jin-Yong Hong; Bo-Ram Cho; Ji-Sook Hahn; Jyongsik Jang (2342-2350).
Monodisperse polypyrrole (PPy) nanoparticles with five different diameters (20, 40, 60, 80, and 100 nm) were fabricated via chemical oxidation polymerization in order to evaluate size-dependent cytotoxicity. The cellular uptake of PPy nanoparticles in human lung fibroblasts (IMR90) and mouse alveolar macrophages (J774A.1) was observed by transmission electron microscopy. The nanoparticles were internalized into the IMR90 via endocytosis. In the J774A.1, the nanoparticles were entered via phagocytosis and endocytosis. Endocytosed nanoparticles were transported to lysosome via endosome-network. The cytotoxicity and innate immune response of PPy-treated cells were systematically investigated by viability assay, oxidative stress, apoptosis/necrosis, and expression of costimulatory molecules. The viability, oxidative stress, and apoptosis/necrosis of PPy-treated cells revealed size- and dose-dependency. Because of phagocytosis, PPy treatment had more adverse effects on the J774A.1 than the IMR90. Innate immune response of PPy-treated macrophages was measured by the expression of costimulatory molecules on surface of the cells. The expression of costimulatory molecules involved in Th1 response (CD40 and CD80) was lightly up-regulated and the other costimulatory molecule related in Th2 response (CD86) was less expressed than a negative control. These findings may provide better nanotoxicological information of polymer nanomaterials, and support the further development of PPy nanoparticles in bioelectronic applications.
Keywords: Nanoparticle; Polypyrrole; Uptake; Cytotoxicity; Immune response; Size dependence;

DNA assembly and re-assembly activated by cationic comb-type copolymer by Rui Moriyama; Naohiko Shimada; Arihiro Kano; Atsushi Maruyama (2351-2358).
Guanine-rich oligonucleotides, such as TG4T and TG5T, assemble into a tetramolecular quadruplexes with layers of G-quartets stabilized by coordination to monovalent cations. Association rates of the quadruplexes are extremely slow, likely owing to electrostatic repulsion among the four strands. We have shown that comb-type copolymers with a polycation backbone and abundant hydrophilic graft chains form water-soluble polyelectrolyte complexes with DNA and promote DNA hybridization. Here, we report the effect of cationic comb-type copolymers on the kinetics of tetramolecular quadruplex formation. The copolymer significantly increased the association rate of tetramolecular quadruplexes without altering kinetic effects of metal cations in quadruplex formation. Dissociation rates of the quadruplexes were also accelerated by the copolymer suggesting that the copolymer has chaperone-like activity that reduces the energy barriers associated with dissociation and re-assembly of base pairs. This hypothesis was further supported by the observation that the copolymer activated the strand exchange reaction between the quadruplex and a constituting single-stranded.
Keywords: Cationic comb-type copolymer; G-quadruplex; Interpolyelectrolyte complex; Strand exchange reaction;

Dual gene targeted multimeric siRNA for combinatorial gene silencing by Soo Hyeon Lee; Hyejung Mok; Sungduk Jo; Cheol Am Hong; Tae Gwan Park (2359-2368).
Simultaneous silencing of multiple up-regulated genes is an attractive and viable strategy to treat many incurable diseases including cancer. Herein we report that multimerized siRNA conjugate composed of two different siRNA sequences in the same backbone shows more efficient inhibition of the two corresponding target genes at one time than physically mixed multimerized siRNA conjugates. Two model siRNAs against VEGF and GFP gene were chemically crosslinked via cleavable and noncleavable linkages for the preparation of dual gene targeted multimeric siRNA conjugates (DGT multi-siRNA). Cleavable DGT multi-siRNA with reducible disulfide linkages exhibited significantly higher gene silencing efficiencies at mRNA and protein expression levels than noncleavable DGT multi-siRNA, the physical mixture of naked siRNA, and that of single gene targeted multimeric siRNA (SGT multi-siRNA) with eliciting negligible immune response. DGT multi-siRNAs against two therapeutic siRNAs, anti-survivin and anti-bcl-2 targeted siRNA, also showed greatly enhanced apoptotic effect. This approach for concurrent suppression of combinatorial therapeutic target genes using cleavable multimeric siRNA structure can be potentially used for improved therapeutic efficacy.
Keywords: Dual gene targeting; Multimerization; siRNA; co-RNAi; Gene delivery;

The role of PEG architecture and molecular weight in the gene transfection performance of PEGylated poly(dimethylaminoethyl methacrylate) based cationic polymers by Shrinivas Venkataraman; Wei Lin Ong; Zhan Yuin Ong; Say Chye Joachim Loo; Pui Lai Rachel Ee; Yi Yan Yang (2369-2378).
In this study, we report the synthesis of well-defined model PEGylated poly(dimethylaminoethyl methacrylate) based cationic polymers composed of different PEG architecture with controlled PEG and nitrogen content via reversible addition-fragmentation chain transfer (RAFT) polymerization, and study the effects of PEG architecture and polymer molecular weight on gene delivery and cytotoxicity. Investigation of the physico-chemical interactions of these model cationic polymers with DNA demonstrated that all these polymers effectively complexed with DNA, and PEG topology did not significantly affect the abilities of the polymers to complex and release DNA. However the size and zeta potential of the complexes were found to be influenced by PEG architecture. The polymers with the block-like configurations formed nanosized DNA complexes. In contrast, considerably higher molecular weight was necessary for the copolymer with the statistical configuration of short PEG chains to form such a small complex. Cell line-dependent influence of PEG architecture on cellular uptake, gene expression efficiency and cell viability of the polymer-DNA complexes was observed. The diblock copolymer-DNA complexes induced higher gene expression than the brush-like block copolymer-DNA complexes, and the statistical copolymer-DNA complexes mediated much lower gene expression than the block-like copolymers-DNA complexes. Increasing the molecular weight of statistical polymer to some extent improved gene expression efficiency. The statistical copolymer was less cytotoxic as compared to the block-like copolymers. These findings provide important insights into the effect of PEGylation nature on gene expression, which will be useful for the design of PEGylated gene delivery polymers.
Keywords: Cationic polymers; PEGylation; Reversible addition-fragmentation chain transfer (RAFT) polymerization; Macromolecular architecture; Gene transfection;

Oral pharmacokinetics of the anti-HIV efavirenz encapsulated within polymeric micelles by Diego A. Chiappetta; Christian Hocht; Carlos Taira; Alejandro Sosnik (2379-2387).
Aiming to improve the pediatric pharmacotherapy of the human immunodeficiency virus (HIV) infection, our group has recently developed a concentrated formulation of the first-line antiretroviral efavirenz by means of encapsulation within polymeric micelles. The aqueous solubility of the drug was increased more than 8400 times (up to 34 mg/mL) and preliminary preclinical data suggested the significantly greater oral bioavailability with respect to an extemporaneous suspension and an oleous solution (similar to the only “commercially available” pediatric formulation). As the preamble to a bioequivalence trial to evaluate the micellar system in adult healthy volunteers, the present work investigated the effect of parameters such as dose per body weight and drug concentration on the oral pharmacokinetics of the drug. The non-linear pharmacokinetics of the drug was confirmed for all the formulations. Despite the drug concentration and dose, micelles consistently resulted in significantly greater absorption rates, PK parameters increasing up to 3-fold. For example, C max values increased from 687, 1789 and 2657 ng/mL for the oily system to 1145, 2856 and 7056 ng/mL for the micellar one, for EFV doses between 20 and 80 mg/kg. Data clearly showed that the smaller the micellar size, the higher the bioavailability attained. The effect of micellar size was also assessed. In addition, a comparison between in vitro dissolution rates of EFV for the different micelles and AUC values suggested that micelles releasing faster in vitro lead to a less pronounced absorption in vivo. These findings would suggest the involvement of additional absorption mechanisms.
Keywords: Pediatric HIV/AIDS pharmacotherapy; Pure and mixed micelles; Efavirenz-loaded poloxamer and poloxamine micelles; Improved oral pharmacokinetics;

Polymer-related off-target effects in non-viral siRNA delivery by Olivia M. Merkel; Andrea Beyerle; Benedikt M. Beckmann; Mengyao Zheng; Roland K. Hartmann; Tobias Stöger; Thomas H. Kissel (2388-2398).
Since off-target effects in non-viral siRNA delivery are quite common but not well understood, in this study various polymer-related effects observed in transfection studies were described and their mechanisms of toxicity were investigated. A variety of stably luciferase-expressing cell lines was compared concerning polymer-mediated effects after transfection with polyplexes of siRNA and poly(ethylene imine) (PEI) or poly(ethylene glycol)-grafted PEI (PEG-PEI). Cell viability, LDH release, gene expression profiles of apoptosis-related genes and promoter activation were investigated. Interestingly, PEG-PEI, which is generally better tolerated than PEI, was found to activate apoptosis in a cell line- and concentration-dependent manner. While both polymers showed sigmoidal dose-response of cell viability in L929 cells (IC50(PEI) = 6 μg/ml, IC50(PEG-PEI) = 11 μg/ml), H1299/Luc cells exhibited biphasic dose-response for PEG-PEI and stronger apoptosis at 2 μg/ml than at 20 μg/ml PEG-PEI, as shown in TUNEL assays. Gene expression profiling confirmed that H1299/Luc cells underwent apoptosis via thousand-fold activation of TNF receptor-associated factors. Additionally, it was demonstrated that NFkB-mediated CMV promoter activation in stably transfected cells can lead to increased target gene levels after transfection instead of siRNA-mediated knockdown. With these results, polymeric vectors were shown not to be inert substances. Therefore, alterations in gene expression caused by the delivery agent must be known to correctly interpret gene-silencing experiments, to understand the mechanisms of off-target effects, and most of all to further develop vectors with reduced side effects. Taking these observations into account, one established cell line was eventually identified to be suitable for RNAi experiments. As shown by these experiments, materials that have been used for many years can elicit unexpected off-target effects. Therefore, non-viral vectors must be screened for several levels of toxicity to make them promising candidates.
Keywords: siRNA; Off-target effects; Polymers; Non-viral delivery; Polymer genomics;

Targeted delivery of chlorotoxin-modified DNA-loaded nanoparticles to glioma via intravenous administration by Rongqin Huang; Weilun Ke; Liang Han; Jianfeng Li; Shuhuan Liu; Chen Jiang (2399-2406).
Gene therapy offers great potential for brain glioma. However, therapeutic genes could not reach glioma spontaneously. A glioma-targeting gene delivery system is highly desired to transfer exogenous genes throughout the tumor focus. In this study, the nanoscopic high-branching dendrimer, polyamidoamine (PAMAM), was selected as the main vector. Chlorotoxin (CTX), which has been demonstrated to bind specifically to receptor expressed in glioma, was exploited as the targeting ligand to conjugate PAMAM via bifunctional polyethyleneglycol (PEG), yielding PAMAM–PEG–CTX. The cellular uptake of CTX itself was observed apparently in C6 glioma cells, almost not in 293 cells. The modification of CTX could significantly increase the cellular uptake of vectors and the DNA-loaded nanoparticles (NPs) in C6 cells. The in vivo distribution of PAMAM–PEG–CTX/DNA NPs in the brain was higher than that of PAMAM/DNA NPs and PAMAM–PEG/DNA NPs. Furthermore, the gene expression of PAMAM–PEG–CTX/DNA NPs was higher and broader in glioma than that of unmodified and PEG-modified counterparts. The TUNEL analysis showed a more wide-extended apoptosis in the CTX-modified group, compared to other groups including commercial temozolomide group. The median survival time of CTX-modified group and temozolomide group was 59.5 and 49 days, respectively, significantly longer than that of other groups. The results suggested that CTX could be exploited as a special glioma-targeting ligand, and PAMAM–PEG–CTX/DNA NPs is a potential non-viral delivery system for gene therapy of glioma via intravenous administration.
Keywords: Chlorotoxin; Polyamidoamine; Glioma-targeting; Gene therapy;

Delivery of basic fibroblast growth factor with a pH-responsive, injectable hydrogel to improve angiogenesis in infarcted myocardium by Jessica C. Garbern; Elina Minami; Patrick S. Stayton; Charles E. Murry (2407-2416).
A pH- and temperature-responsive, injectable hydrogel has been designed to take advantage of the acidic microenvironment of ischemic myocardium. This system can improve therapeutic angiogenesis methods by providing spatio-temporal control of angiogenic growth factor delivery. The pH- and temperature-responsive random copolymer, poly(N-isopropylacrylamide-co-propylacrylic acid-co-butyl acrylate) (p[NIPAAm-co-PAA-co-BA]), was synthesized by reversible addition fragmentation chain transfer polymerization. This polymer was a liquid at pH 7.4 and 37 °C but formed a physical gel at pH 6.8 and 37 °C. Retention of biotinylated basic fibroblast growth factor (bFGF) between 0 and 7 days after injection into infarcted rat myocardium was 10-fold higher with hydrogel delivery versus saline. Following 28 days of treatment in vivo, capillary and arteriolar densities were increased 30–40% by polymer + bFGF treatment versus saline + bFGF or polymer-only controls. Treatment with polymer + bFGF for 28 days resulted in a 2-fold improvement in relative blood flow to the infarct region versus day 0, whereas saline + bFGF or polymer-only had no effect. Fractional shortening determined by echocardiography was significantly higher following treatment with polymer + bFGF (30 ± 1.4%) versus saline (25 ± 1.2%) and polymer alone (25 ± 1.8%). By responding to local changes in pH- and temperature in an animal model of ischemia, this hydrogel system provided sustained, local delivery of bFGF, improved angiogenesis, and achieved therapeutic effects in regional blood flow and cardiac function.
Keywords: Angiogenesis; Hydrogel; Thermally-responsive material; Fibroblast growth factor; Controlled drug release; Copolymer;

The effect of biophysical attributes of the ocular trabecular meshwork associated with glaucoma on the cell response to therapeutic agents by Clayton T. McKee; Joshua A. Wood; Nihar M. Shah; Marion E. Fischer; Christopher M. Reilly; Christopher J. Murphy; Paul Russell (2417-2423).
Glaucoma is a devastating neurodegenerative disease, which can lead to vision loss and is associated with irreversible damage to retinal ganglion cells. Although the mechanism of disease onset remains unknown, we have recently demonstrated that the stiffness of the ocular trabecular meshwork (HTM) increases dramatically in human donor eyes with a history of glaucoma. Here we report that polyacrylamide hydrogels, which mimic the compliant conditions of normal and glaucomatous HTM, profoundly modulate cytoskeletal dynamics and the elastic modulus of the overlying HTM cells. Substratum compliance also modulates HTM cell response to Latrunculin-B, a cytoskeletal disrupting agent currently in human clinical trials for the treatment of glaucoma. Additionally, we observed a compliance-dependent rebound effect of Latrunculin-B with an unexpected increase in HTM cell elastic modulus being observed upon withdrawal of the drug. The results predict that cytoskeletal disrupting drugs may be more potent in advanced stages of glaucoma.
Keywords: Young’s modulus; Biophysical attributes; Latrunculin; AFM; Glaucoma; Extracellular matrix;

Nanoparticle based delivery of hypoxia-regulated VEGF transgene system combined with myoblast engraftment for myocardial repair by Lei Ye; Wei Zhang; Li-Ping Su; Husnain K. Haider; Kian-Keong Poh; Mary J. Galupo; Geronica Songco; Ruo-Wen Ge; Huay-Cheem Tan; Eugene K.W. Sim (2424-2431).
A regulated promoter system to control gene expression is desirable for safe and efficacious over-expression of therapeutic transgene. Combined with skeletal myoblast (SkMs), we report the efficacy of hypoxia-regulated VEGF gene delivery for myocardial repair during acute myocardial infarction (AMI). A hypoxia-regulated VEGF plasmid (pHRE-VEGF) was developed. After optimization, ∼30% SkMs were transfected using polyethyleneimine (PEI) nanoparticles. The peak VEGF expression was higher in pHRE-VEGF transfected SkMs (VEGFSkMs) under hypoxia (151.34 ± 8.59 ng/ml) than that with normoxia (16.92 ± 2.74 ng/ml). The efficacy of hypoxia-regulated gene expression system was assessed in a rabbit model of AMI. The animals were grouped to receive basal M199 without cells (group-1) or containing non-transfected SkMs (group-2) or VEGFSkMs (group-3). In group-4, VEGFSkMs were injected into normal heart to serve as normoxia control. Improved SkM survival was observed in group-3 and -4 (p < 0.05 vs group-2) at day-3 and 7 after transplantation. Blood vessel density was 20.1 ± 1.3 in group-3 which was significantly higher than any other groups (p < 0.05) at 2 weeks after treatment. Improved blood flow (ml/min/g) in the left ventricle (LV) anterior wall was observed in group-3 (1.28 ± 0.09, p < 0.05) as compared with group-1 (0.76 ± 0.05) and group-2 (0.96 ± 0.06), and similar to group-4 (1.26 ± 0.05). LV ejection fraction was best preserved in group-3 (58.4 ± 1.75%) which was insignificantly different from group-4 (61.1 ± 1.8%), and group-2 (52.8 ± 1.4%), but significantly improved compared with group-1 (44.7 ± 2.2%, p < 0.05). The study demonstrates that nanoparticle based delivery of hypoxia-regulated VEGF transgene combined with SkMs during AMI effectively preserves LV regional blood flow and contractile function of the heart.
Keywords: Skeletal myoblast; Hypoxia; VEGF; Myocardial infarction; Cardiac repair;

Reconfiguring polylysine architectures for controlling polyplex binding and non-viral transfection by Sangram S. Parelkar; Delphine Chan-Seng; Todd Emrick (2432-2444).
Poly(l-lysine) (PLL) is a cationic polyelectrolyte of interest for many applications, including in therapeutic biology for DNA complexation and transfection. Several non-lysine based polycations have been shown to afford more efficient transfection in live cells than has been achieved with PLL. We find that reconfiguring polylysine into short oligolysine grafts, strung from a hydrophobic polymer backbone, gives transfection reagents greatly superior to PLL, despite having the identical cationic functional groups (i.e., exclusively primary amines). Altering the oligolysine graft length modulates DNA-polymer interactions and transfection efficiency, while incorporating the PKKKRKV heptapeptide (the Simian virus SV40 large T-antigen nuclear localization sequence) pendent groups onto the polymer backbone led to even greater transfection efficiency over the oligolysine-grafted structures. Protein expression levels obtained with these novel polymer transfection reagents were higher than, or comparable to, expression seen in the cases of JetPEI™, FuGENE® 6 and Lipofectamine™ 2000, the later being notorious for cytotoxicity that accompanies high transfection efficiency. The relative strength of the polymer-DNA complex is key to the transfection performance, as judged by serum stability and PicoGreen analysis. Moreover, polyplexes formed from our graft copolymer structures exhibit low cytotoxicity, contributing to the therapeutic promise of these novel reagents.
Keywords: Gene therapy; Ring-opening metathesis polymerization (ROMP); Poly(cyclooctene-g-oligopeptide); Nuclear localization signal (NLS); Polylysine (PLL);