Biomaterials (v.27, #29)

Calendar (I).

Rheological properties of concentrated aqueous injectable calcium phosphate cement slurry by Changsheng Liu; Huifang Shao; Feiyue Chen; Haiyan Zheng (5003-5013).
In this paper, the steady and dynamic rheological properties of concentrated aqueous injectable calcium phosphate cement (CPC) slurry were investigated. The results indicate that the concentrated aqueous injectable CPC showed both plastic and thixotropic behavior. As the setting process progressed, the yield stress of CPC slurry was raised, the area of the thixotropic hysteresis loop was enlarged, indicating that the strength of the net structure of the slurry had increased. The results of dynamic rheological behavior indicate that the slurry presented the structure similar to viscoelastic body and the property of shear thinning at the beginning. During the setting process, the slurry was transformed from a flocculent structure to a net structure, and the strength increased. Different factors had diverse effects on the rheological properties of the CPC slurry in the setting process, a reflection of the flowing properties (or injection), and the microstructure development of this concentrated suspension. Raising the powder-to-liquid ratio decreased the distance among the particles, increased the initial strength, and shortened the setting time. In addition, raising the temperature improved the initial strength, increased the order of reaction, and shortened the setting time, which was favorable to the setting process. The particle size of the raw material had much to do with the strength of original structure and setting time. The storage module G′ of CPC slurry during the setting process followed the rule of power law function G′=A  exp(Bt), which could be applied to forecast the setting time, and the calculated results thereafter are in agreement with the experimental data.
Keywords: Calcium phosphate cement; Rheological properties; Injection; Setting time; Shear; Hydroxyapatite;

The osseous response to silicon (Si) level (0, 0.2, 0.4, 0.8 and 1.5 wt% Si) within 5 batches of matched porosity silicate-substituted hydroxyapatite (SA) scaffold was assessed by implantation of 4.6 mm diameter cylinders in the femoral intercondylar notch of New Zealand White rabbits for periods of 1, 3, 6 and 12 weeks. Histological evaluation and histomorphometric quantification of bone ingrowth and mineral apposition rate (MAR) demonstrated the benefits to early (<1 week) bone ingrowth and repair through incorporation of Si, at all levels, in porous hydroxyapatite (HA) lattices as compared to stoichiometric (0 wt% Si) HA. The group containing 0.8 wt% Si supported significantly more bone ingrowth than all other groups at 3 and 6 weeks ( P < 0.05 ), initially through its elevated MAR between weeks 1 and 2, which was significantly higher than that of all other Si-containing groups ( P < 0.05 ). The level of silicate substitution also influenced the morphology and stability of the repair, with elevated levels of bone resorption and apposition apparent within other Si-containing groups at timepoints >3 weeks as compared to the 0 and 0.8 wt% Si groups. At 12 weeks, the net amount of bone ingrowth continued to rise in the 0, 0.8 and 1.5 wt% groups, apparently as a result of adaptive remodelling throughout the scaffold. Ingrowth levels remained highest in the 0.8 wt% Si group, was characterised by a dense trabecular morphology in the superficial region graduating to a more open network in the deep zone. These results highlight the sensitivity of healing response to Si level and suggest that an optimal response is obtained when SA is substituted with 0.8 wt% Si through its effect on the activity of both bone forming and bone resorbing cells.
Keywords: Bioactivity; Bone graft; Bone ingrowth; Calcium phosphate; Hydroxyapatite; Silicon;

Angiogenic and inflammatory response to biodegradable scaffolds in dorsal skinfold chambers of mice by Martin Rücker; Matthias W. Laschke; Dominik Junker; Carlos Carvalho; Alexander Schramm; Rolf Mülhaupt; Nils-Claudius Gellrich; Michael D. Menger (5027-5038).
For tissue engineering, scaffolds should be biocompatible and promote neovascularization. Because little is known on those specific properties, we herein studied in vivo the host angiogenic and inflammatory response after implantation of commonly used scaffold materials. Porous poly(l-lactide-co-glycolide) (PLGA) and collagen–chitosan–hydroxyapatite hydrogel scaffolds were implanted into dorsal skinfold chambers of balb/c mice. Additional animals received cortical bone as an isogeneic, biological implant, while chambers of animals without implants served as controls. Angiogenesis and neovascularization as well as leukocyte–endothelial cell interaction and microvascular permeability were analyzed over 14 day using intravital fluorescence microscopy. PLGA scaffolds showed a slight increase in leukocyte recruitment compared to controls. This was associated with an elevation of microvascular permeability, which was comparable to that observed in isogeneic bone tissue. Of interest, PLGA induced a marked angiogenic response, revealing a density of newly formed capillaries almost similar to that observed in bone implants. Histology showed infiltration of macrophages, probably indicating resorption of the biomaterial. In contrast, hydrogel scaffolds induced a severe inflammation, as indicated by an ∼15-fold increase of leukocyte–endothelial cell interaction and a marked elevation of microvascular permeability. This was associated by induction of apoptotic cell death within the surrounding tissue and a complete lack of ingrowth of newly formed microvessels. Histology confirmed adequate engraftment of PLGA and isogeneic bone but not hydrogel within the host tissue. PLGA scaffolds show a better biocompatibility than hydrogel scaffolds and promote vascular ingrowth, guaranteeing adequate engraftment within the host tissue.
Keywords: Tissue engineering; Scaffold; Biocompatibility; Microcirculation; Inflammation; Angiogenesis;

The ability of poly(anhydride-esters) composed of non-steroidal anti-inflammatory drugs that biodegrade to salicylic acid (SA) and adipic acid to prevent colonization by Pseudomonas aeruginosa and their effects on the foreign-body response were studied in vitro and in vivo, respectively. Soluble SA in bacterial medium at concentrations up to 300 mg/L did not affect the growth rate or viability of P. aeruginosa, indicating that SA does not exhibit a direct toxicity effect on the bacterium. Batch degradation rates of the salicylate-based polymer in the presence of an actively growing bacterial culture only marginally (14%) increased relative to polymer degradation rates in sterile medium. Short-term (3 h) bacterial adhesion studies in agitated batch systems indicated a 47% reduction in the rate of P. aeruginosa adhesion relative to a control polymer that does not release SA upon biodegradation. Long-term (3-day) biofilm accumulation studies indicated a dramatic reduction in biofilm formation on salicylate-based polymer versus controls. A recombinant P. aeruginosa pMHLAS, containing a fluorescent reporter gene prior to the las regulon, was employed to determine whether salicylate-based polymer prevents biofilm formation by the released SA inhibiting quorum sensing pathways. Long-term biofilm accumulation studies with P. aeruginosa pMHLAS insinuate that salicylate-based polymer prevents biofilm accumulation by inhibiting the las quorum sensing system. Furthermore, unlike control polymer, salicylate-based polymer implanted subcutaneously for a period of 4 weeks-resisted cell-mediated degradation and remained intact. Histological and immunohistochemical analysis indicated a reduction in overall encapsulation and paucity of macrophages in the area of the salicylate-based polymer implant.
Keywords: Biodegradable poly(anhydride-esters); Anti-biofilm control; Pseudomonas aeruginosa; Foreign-body response;

Inactivation of nanocrystalline C60 cytotoxicity by γ-irradiation by Aleksandra Isakovic; Zoran Markovic; Nadezda Nikolic; Biljana Todorovic-Markovic; Sanja Vranjes-Djuric; Ljubica Harhaji; Nevena Raicevic; Nebojsa Romcevic; Dana Vasiljevic-Radovic; Miroslav Dramicanin; Vladimir Trajkovic (5049-5058).
We investigated the effect of γ-irradiation on the cytotoxicity of pure C60 solubilized in water by using tetrahydrofuran (THF/n-C60 or THF/n-C60). In contrast to THF/n-C60, its γ-irradiated counterpart failed to generate oxygen radicals and cause extracellular signal-regulated kinase (ERK)-dependent necrotic cell death in various types of mammalian cells. Moreover, γ-irradiated THF/n-C60 protected cells from the oxidative stress induced by native THF/n-C60 or hydrogen peroxide. The observed biological effects were associated with γ-irradiation-mediated decomposition of THF and subsequent derivatization of the n-C60 surface. These results for the first time demonstrate γ-irradiation-mediated changes in the physico-chemical properties of THF-prepared nanocrystalline C60, resulting in a complete loss of its cytotoxic effect and its conversion to a cytoprotective agent.
Keywords: Carbon; Nanoparticle; Gamma irradiation; Cytotoxicity; Antioxidant;

Skin basement membrane and extracellular matrix proteins characterization and quantification by real time RT-PCR by Tzu-Wei Wang; Jui-Sheng Sun; Yi-Chau Huang; Hsi-Chin Wu; Li-Tin Chen; Feng-Huei Lin (5059-5068).
Three-dimensional gelatin-chondroitin 6 sulphate-hyanuronic acid (gelatin-C6S-HA) biomatrices were used as the scaffold to investigate the phenotypic and molecular expression of basement membrane (BM) and extracellular matrix (ECM) proteins in vitro. The cells were cultured in three different culture conditions: keratinocytes (K) monoculture, or dermal fibroblasts (FB) monoculture, or organotypic keratinocytes and dermal fibroblasts (K&FB) coculture model. The deposition of BM proteins and ECM proteins secreted by these two kinds of cells was quantitatively characterized by real time RT-PCR and examined by immunohistochemistry.The results showed that K expressed specific keratin and E-cadherin proteins, while type I collagen was secreted by FB. FB were shown to synthesize and deposit laminin 5, type IV collagen, and type VII collagen, whereas K dominantly produced integrin alpha 6 and integrin beta 4 as well as laminin 5. Interestingly, the integrin beta 4 was expressed neither in K monoculture nor in FB monoculture, but was seen in organotypic K&FB coculture model in the early culture stage.The histology studies revealed numerous features of epidermalization including a well organized basal layer of distinct cylindrical cells, granular and a horny layer, as well as complete BM formation. These results indicated that K and FB not only kept their phenotype when culturing on 3D scaffold, but also worked together to reconstruct dermal–epidermal basement membrane zone. In brief, our results directly provide the quantification in the expression of BM and ECM proteins by using real time RT-PCR in mRNA level and morphological appearance by immunostain in protein level.
Keywords: Keratinocyte; Fibroblast; PCR (polymerase chain reaction); Biomimetic material;

Bio-functionalized thermoresponsive interfaces facilitating cell adhesion and proliferation by Hideyuki Hatakeyama; Akihiko Kikuchi; Masayuki Yamato; Teruo Okano (5069-5078).
Bio-functionalized thermoresponsive culture interfaces co-immobilized with cell adhesive peptide, RGDS, and cell growth factor, insulin (INS), are investigated to promote initial cell adhesion and cell growth for further cell sheet engineering applications. These bio-functionalized interfaces were prepared by electron beam-induced copolymerization of N-isopropylacrylamide (IPAAm) with its carboxyl-derivatized analog, 2-carboxyisopropylacrylamide (CIPAAm), and grafting onto tissue culture polystyrene dishes, followed by immobilization of RGDS and/or INS to CIPAAm carboxyls. Adhesion and proliferation of bovine carotid artery endothelial cells (ECs) were examined on the RGDS–INS co-immobilized thermoresponsive interfaces. Immobilized RGDS facilitated initial EC adhesion on the surfaces and INS modification was demonstrated to induce EC proliferation, respectively. More pronounced EC growth was indicated by co-immobilization of appropriate amount of RGDS and INS. This may be due to synergistic effect of direct co-stimulation of adhered ECs by surface-immobilized RGDS and INS molecules. ECs grown on the RGDS–INS co-immobilized thermoresponsive interfaces can also be recovered spontaneously as viable tissue monolayers by solely reducing culture temperature. RGDS–INS co-immobilized thermoresponsive interfaces strongly supported initial EC adhesion and growth than unmodified thermoresponsive surfaces even under serum-free culture. Addition of soluble growth factors to serum-free culture medium effectively induced EC proliferation to confluency. Co-immobilization of cell adhesion peptides and growth factors on thermoresponsive surfaces should be effective for rapid preparation of intact cell sheets and their utilization to regenerative medicine.
Keywords: Poly(N-isopropylacrylamide) (PIPAAm); 2-carboxyisopropylacrylamide (CIPAAm); RGDS; Insulin; Bio-functionalized thermoresponsive interfaces; Cell sheet engineering;

Study on the effects of nylon–chitosan-blended membranes on the spheroid-forming activity of human melanocytes by Sung-Jan Lin; Wen-Chu Hsiao; Shiou-Hwa Jee; Hsin-Su Yu; Tsen-Fang Tsai; Juin-Yih Lai; Tai-Horng Young (5079-5088).
Though reported limitedly in tissue engineering, modification of cellular functions can be achieved by culturing them into multicellular spheroids. We have shown melanocytes form spheroids on chitosan surface. However, how biomaterials promote spheroid formation has never been systemically investigated. In this work, nylon, which inhibits melanocyte spheroid formation, and chitosan, which promotes melanocyte spheroid formation, are used to prepare nylon/chitosan-blended membranes. Membranes composed of pure nylon, pure chitosan and various ratios of nylon and chitosan are employed to examine their effects on spheroid formation. Melanocytes show better adhesion to nylon membranes than that to chitosan membranes. In blended membranes, as more nylon is incorporated, cell adhesion increases and the trend for spheroid formation decreases. Melanocytes can only form spheroids on membranes with poorer cell adhesion. Examining the surface of the blended membranes shows phase separation of nylon and chitosan. As nylon content increases, the nylon phase on the membrane surface increases and thereby enhances cell adhesion. The opposite trend for cell adhesion and spheroid formation substantiates our hypothesis of spheroid formation on biomaterials: a balance between cell–substrate interaction and cell–cell interaction. The decrease in cell–substrate interaction tilts the balance to a state more favorable for spheroid formation. Our work can serve as a model to investigate the relative strengths of cell–cell and cell–substrate interactions and also pave way to design blended membranes with desired physical properties while preserving the spheroid-forming activity.
Keywords: Melanocyte; Chitosan; Nylon; Blend; Spheroid; Vitiligo;

Ectopic bone formation in collagen sponge self-assembled peptide–amphiphile nanofibers hybrid scaffold in a perfusion culture bioreactor by Hossein Hosseinkhani; Mohsen Hosseinkhani; Furong Tian; Hisatoshi Kobayashi; Yasuhiko Tabata (5089-5098).
The objective of this study was to enhance ectopic bone formation in a three-dimensional (3-D) hybrid scaffold in combination with bioreactor perfusion culture system. The hybrid scaffold consists of two biomaterials, a hydrogel formed through self-assembly of peptide–amphiphile (PA) with cell suspensions in media, and a collagen sponge reinforced with poly(glycolic acid) (PGA) fiber incorporation. PA was synthesized by standard solid-phase chemistry that ends with the alkylation of the NH2 terminus of the peptide. A 3-D network of nanofibers was formed by mixing cell suspensions in media with dilute aqueous solution of PA. Scanning electron microscopy (SEM) observation revealed the formation of fibrous assemblies with an extremely high aspect ratio and high surface areas. Osteogenic differentiation of mesenchymal stem cells (MSC) in the hybrid scaffold was greatly influenced by the perfusion culture method compared with static culture method. When the osteoinduction activity of hybrid scaffold was studied following the implantation into the back subcutis of rats in terms of histological and biochemical examinations, significantly homogeneous bone formation was histologically observed throughout the hybrid scaffolds when perfusion culture was used compared with static culture method. The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of hybrid scaffolds were significantly high for the perfusion group compared with those in static culture method. We conclude that combination of MSC-seeded hybrid scaffold and the perfusion method was promising to enhance in vitro osteogenic differentiation of MSC and in vivo ectopic bone formation.
Keywords: Hybrid scaffold; Mesenchymal stem cells; Peptide amphiphile; Nanofibers; Osteogenic differentiation;

The effect of cell-based bone tissue engineering in a goat transverse process model by Moyo C. Kruyt; Clayton E. Wilson; Joost D. de Bruijn; Clemens A. van Blitterswijk; Cumhur F. Oner; Abraham J. Verbout; Wouter J.A. Dhert (5099-5106).
A disadvantage of traditional posterolateral spinal fusion models is that they are highly inefficient for screening multiple conditions. We developed a multiple-condition model that concentrates on the initial process of bone formation from the transverse process and not on a functional fusion. The effect of bone marrow stromal cells (BMSCs) in four different porous ceramic scaffolds was investigated in this setting. Polyacetal cassettes were designed to fit on the goat transverse process and house four different ceramic blocks, i.e: hydroxyapatite (HA) sintered at 1150° and 1250°; biphasic calcium phosphate (BCP) and tricalcium phosphate (TCP). Goat BMSCs (n=10) were cultured and per-operatively seeded autologeously on one of two cassettes implanted per animal. The cassettes were bilaterally mounted on the dorsum of decorticated L2-processes for 9 weeks. To asses the dynamics of bone formation, fluorochrome labels were administered and histomorphometry focused on the distribution of bone in the scaffolds. A clear difference in the extent of bone ingrowth was determined for the different scaffold types. An obvious effect of BMSC seeding was observed in three of four scaffold types, especially in scaffold regions adjacent to the overlying muscle. Generally, the BCP and TCP scaffolds showed better osteoconduction and an increased response to BMSCs administration. In conclusion the model provides a reliable and highly efficient method to study bone formation in cell-based tissue engineering. An effect of cell administration was obvious in three of the four scaffold materials.
Keywords: Tissue Engineering; Bone; Bone marrow stromal cells; Ceramics; Spine model; Goats;

Chronic performance of polyurethane catheters covalently coated with ATH complex: A rabbit jugular vein model by Petr Klement; Ying Jun Du; Leslie R. Berry; Paul Tressel; Anthony K.C. Chan (5107-5117).
Covalent complexes of antithrombin (AT) and heparin (ATH) have superb anticoagulant activity towards thrombin and factor Xa. Stability of polyurethane central venous catheters covalently modified with radiolabeled ATH was studied using a roller pump with saline or protease P-5147. Saline wash removed loosely bound ATH molecules to decrease graft density from 26 to 12 pmol/cm2. However, only slightly more ATH was removed by strong protease (from 12 to 7 pmol/cm2). To evaluate ATH-coated, heparin-coated, and uncoated catheters, a chronic rabbit jugular vein model was developed with catheters maintained for up to 30–106 days. Lumen occlusion was tested by drawing blood twice daily. Although unmodified or heparin-coated catheters occluded within 5–7 days after insertion, all ATH catheters remained patent throughout the experiment. Scanning electron microscopy (SEM) analysis of heparin and uncoated catheters revealed extensive thrombosis (lumen+mural) while ATH catheters were unaffected. Visual observation showed significant deposition of protein and cells on control and heparin-modified catheters and, to a lesser degree, on ATH-coated surfaces. SEM showed no fibrin inside or outside of ATH catheters, which remained patent in extended studies out to 106 days. Although atomic force microscopy showed ATH coatings to be rough, 6-fold higher anti-factor Xa activity likely contributed to increased patency. Our data confirm that ATH-modified catheters are stable and have superior potency compared to heparin or control catheters.
Keywords: Anticoagulant; Antithrombin; Heparin; Polyurethane; Surface grafting;

The objective of this study was to evaluate the effects of a complex of β-tricalcium phosphate (β-TCP) granules, collagen, and fibroblast growth factor-2 (FGF-2) on cortical bone repair in rabbits. Segmental bone defects of 5 mm in length were created in the middle of the tibial shaft. The defect was stabilized with a plate and screws, and was filled with 0.3 ml of a complex of β-TCP granules and 5% collagen, with or without 200 μg of recombinant human fibroblast growth factor-2 (rhFGF-2). Bone regeneration and β-TCP resorption were assessed by X-ray and micro-CT scanner. A three-point bending test was also performed. The results showed that the segmental bone defect was not only radiologically, but also mechanically healed with cortical bone 12 weeks after implantation of the complex with rhFGF-2. In contrast, after implantation of the complex without rhFGF-2, most of the defect was filled with β-TCP and only a small amount of bone formation was found. These results suggest that resorption of β-TCP is important for bone formation and may be promoted by FGF-2 in the β-TCP implantation site. In addition, the complex of β-TCP granules and collagen combined with rhFGF-2 provides a paste-like material that is easy to handle. This material may be of considerable use in the treatment of cortical bone defects.
Keywords: Bioresorption; Bone regeneration; Calcium phosphate; Collagen; Fibroblast growth factor;

Interfacial behaviour of strontium-containing hydroxyapatite cement with cancellous and cortical bone by G.X. Ni; W.W. Lu; B. Xu; K.Y. Chiu; C. Yang; Z.Y. Li; W.M. Lam; K.D.K. Luk (5127-5133).
The bone-bonding behaviors of various biomaterials have been extensively investigated. However, the precise mechanisms of bone bonding have not yet been clarified, and the differences in interfacial behaviors of biomaterial bonding with cancellous bone and cortical bone have not yet been understood. In this study, strontium-containing hydroxyapatite (Sr-HA) cement, in which 10% calcium ions were substituted by strontium, was performed in a rabbit hip replacement model. Six months later, the morphology and chemical composition of interfaces between Sr-HA cement with cancellous bone and cortical bone were evaluated by field emission scanning electron microscopy (FESEM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Remarkable differences between these two interfaces were suggested both in morphology and chemical compositions. An apatite layer was found between Sr-HA cement and cancellous bone with a thickness of about 70 μm. However, only a very thin interface (about 1 μm) was formed with cortical bone. As for the cancellous bone/cement interface, high ions intensity of Ca, P, Sr, Na, and O were confirmed by FESEM-EDX and ToF-SIMS. Differences in morphology and chemical component between these two interfaces provided convincing evidences for the proposed dissolution–precipitation coupling mechanism in the formation of biological apatite.
Keywords: Strontium-containing hydroxyapatite bone cement; Bone-bonding behaviors; Interface; Mechanism; Chemical composition;

Novel polysaccharides-based viscoelastic formulations for ophthalmic surgery: Rheological characterization by Adriana Maltese; Assunta Borzacchiello; Laura Mayol; Claudio Bucolo; Francesco Maugeri; Luigi Nicolais; Luigi Ambrosio (5134-5142).
Different formulations based on bioadhesive and biocompatible polymers, hydroxypropylmethylcellulose (HPMC), sodium hyaluronate (SH) and chitosan glutamate (CG), were prepared to be potentially used as ophthalmic viscosurgical device (OVD) during cataract surgery. Their rheological properties were analyzed in terms of flow and oscillation properties and compared to a commercially available OVD, widely employed in cataract surgery, named Viscoat®. All the formulations tested presented a pseudoplastic behavior during flow. Primary systems containing HPMC or CG and HPMC/CG binary systems behaved as viscous solution (G″>G′) over the range of oscillatory frequencies observed, while the primary systems containing SH and HPMC/SH binary formulations and showed an entangled network behavior when subjected to a sinusoidal stress. By increasing the SH concentration in the binary systems, the viscoelastic parameters, G′and G″, and zero frequency viscosity (derived from the Cross model) increased. Viscoat® presents viscoelastic parameters values lower than the corresponding values of all the binary formulations of HPMC/SH and higher than all the formulations made up of CG and HPMC.As regard to HPMC/SH binary system, the cross-over frequency decreased by increasing SH concentration in the systems and it was the highest for Viscoat® and thus the opposite occurred for the relaxation time. The rheological synergy in the binary formulations was assessed by calculating the interaction parameters which increased as a function of SH and CG concentration in the binary systems. The values of the interaction parameters of the formulations based on CG, are lower than 10 Pa indicating that they did not interact synergically while the formulations based on SH show high values of the interactions parameters (in the range from 55 to 130 Pa). This indicates that secondary bonds formation occurs between SH and HPMC.From the rheological analysis it can be concluded that the binary formulations based on CG do not possess appropriate features to be used as OVD while both the viscoelastic and the flow properties of the binary formulations made up of SH and HPMC are suitable for their application as OVD being able to maintain the ocular spaces and to be easily administrated. Moreover, thank to the adhesive properties of both components, the binary formulation should be able to interact with corneal endothelium so offering a durable protection to ocular tissue.On the basis of the rheological characterization presented in this work, we concluded that the binary system named VISC26 (HPMC at 0.8% and SH at 2.3%) represents the formulation that better fulfill the OVD requirements.
Keywords: Ophthalmic viscosurgical device (OVD); Ophthalmology; Hyaluronic acid; Chitosan; Hydroxypropylmethylcellulose; Viscoelasticity;

Gene delivery to differentiated neurotypic cells with RGD and HIV Tat peptide functionalized polymeric nanoparticles by Jung Soo Suk; Junghae Suh; Kokleong Choy; Samuel K. Lai; Jie Fu; Justin Hanes (5143-5150).
A number of neurodegenerative disorders may potentially be treated by the delivery of therapeutic genes to neurons. Nonviral gene delivery systems, however, typically provide low transfection efficiency in post-mitotic differentiated neurons. To uncover mechanistic reasons for this observation, we compared gene transfer to undifferentiated and differentiated SH-SY5Y cells using polyethylenimine (PEI)/DNA nanocomplexes. Differentiated cells exhibited substantially lower uptake of gene vectors. To overcome this bottleneck, RGD or HIV-1 Tat peptides were attached to PEI/DNA nanocomplexes via poly(ethylene glycol) (PEG) spacer molecules. Both RGD and Tat improved the cellular uptake of gene vectors and enhanced gene transfection efficiency of primary neurons up to 14-fold. RGD functionalization resulted in a statistically significant increase in vector escape from endosomes, suggesting it may improve gene delivery by more than one mechanism.
Keywords: Gene therapy; CNS diseases; Polymers; Polyethylenimine (PEI);