Biomaterials (v.32, #33)

Inhibition of the transport of HIV in vitro using a pH-responsive synthetic mucin-like polymer system by Alamelu Mahalingam; Julie I. Jay; Kristofer Langheinrich; Shetha Shukair; Mike D. McRaven; Lisa C. Rohan; Betsy C. Herold; Thomas J. Hope; Patrick F. Kiser (8343-8355).
In conjunction with the routine role of delivering the active ingredient, carefully designed drug delivery vehicles can also provide ancillary functions that augment the overall efficacy of the system. Inspired by the ability of the cervicovaginal mucus to impede the movement of HIV virions at acidic pH, we have engineered a pH-responsive synthetic polymer that shows improved barrier properties over the naturally occurring cervicovaginal mucus by inhibiting viral transport at both acidic and neutral pH. The pH-responsive synthetic mucin-like polymer is constructed with phenylboronic acid (PBA) and salicylhydroxamic acid (SHA), each individually copolymerized with a 2-hydroxypropyl methacrylamide (pHPMA) polymer backbone. At pH 4.8, the crosslinked polymers form a transient network with a characteristic relaxation time of 0.9 s and elastic modulus of 11 Pa. On addition of semen, the polymers form a densely crosslinked elastic network with a characteristic relaxation time greater than 60 s and elastic modulus of 1800 Pa. Interactions between the PBA-SHA crosslinked polymers and mucin at acidic pH showed a significant increase in elastic modulus and crosslink lifetime (p < 0.05). A transport assay revealed that migration of HIV and cells was significantly impeded by the polymer network at pH ≥ 4.8 with a diffusion coefficient of 1.60 x 10−4 μm2/s for HIV. Additionally, these crosslinked polymers did not induce symptoms of toxicity or irritation in either human vaginal explants or a mouse model. In summary, the pH-responsive crosslinked polymer system reported here holds promise as a class of microbicide delivery vehicle that could inhibit the transport of virions from semen to the target tissue and, thereby, contribute to the overall activity of the microbicide formulation.
Keywords: HIV; pH-responsive; Biomimetic; Crosslinked polymer;

Use of autologous blood-derived endothelial progenitor cells at point-of-care to protect against implant thrombosis in a large animal model by Alexandra E. Jantzen; Whitney O. Lane; Shawn M. Gage; Ryan M. Jamiolkowski; Justin M. Haseltine; Lauren J. Galinat; Fu-Hsiung Lin; Jeffrey H. Lawson; George A. Truskey; Hardean E. Achneck (8356-8363).
Titanium (Ti) is commonly utilized in many cardiovascular devices, e.g. as a component of Nitinol stents, intra- and extracorporeal mechanical circulatory assist devices, but is associated with the risk of thromboemboli formation. We propose to solve this problem by lining the Ti blood-contacting surfaces with autologous peripheral blood-derived late outgrowth endothelial progenitor cells (EPCs) after having previously demonstrated that these EPCs adhere to and grow on Ti under physiological shear stresses and functionally adapt to their environment under flow conditions ex vivo. Autologous fluorescently-labeled porcine EPCs were seeded at the point-of-care in the operating room onto Ti tubes for 30 min and implanted into the pro-thrombotic environment of the inferior vena cava of swine (n = 8). After 3 days, Ti tubes were explanted, disassembled, and the blood-contacting surface was imaged. A blinded analysis found all 4 cell-seeded implants to be free of clot, whereas 4 controls without EPCs were either entirely occluded or partially thrombosed. Pre-labeled EPCs had spread and were present on all 4 cell-seeded implants while no endothelial cells were observed on control implants. These results suggest that late outgrowth autologous EPCs represent a promising source of lining Ti implants to reduce thrombosis in vivo.
Keywords: Biocompatibility; Thrombosis; Titanium; Stent; Endothelium; Progenitor cell;

The association of matrix Gla protein isomers with calcification in capsules surrounding silicone breast implants by Larry W. Hunter; John C. Lieske; Nho V. Tran; Virginia M. Miller (8364-8373).
Implanted silicone medical prostheses induce a dynamic sequence of histologic events in adjacent tissue resulting in the formation of a fibrotic peri-prosthetic capsule. In some cases, capsular calcification occurs, requiring surgical intervention. In this study we investigated capsules from silicone gel-filled breast prostheses to test the hypothesis that this calcification might be regulated by the small vitamin K-dependent protein, matrix Gla protein (MGP), a potent inhibitor of arterial calcification, or by Fetuin-A, a hepatocyte-derived glycoprotein also implicated as a regulator of pathologic calcification. Immunolocalization studies of explanted capsular tissue, using conformation-specific antibodies, identified the mineralization-protective γ-carboxylated MGP isomer (cMGP) within cells of uncalcified capsules, whereas the non-functional undercarboxylated isomer (uMGP) was typically absent. Both were upregulated in calcific capsules and co-localized with mineral plaque and adjacent fibers. Synovial-like metaplasia was present in one uncalcified capsule in which MGP species were differentially localized within the pseudosynovium. Fetuin-A was localized to cells within uncalcified capsules and to mineral deposits within calcific capsules. The osteoinductive cytokine bone morphogenic protein-2 localized to collagen fibers in uncalcified capsules. These findings demonstrate that MGP, in its vitamin K-activated conformer, may represent a pharmacological target to sustain the health of the peri-prosthetic tissue which encapsulates silicone breast implants as well as other implanted silicone medical devices.
Keywords: Foreign-body response; Calcification; Biocompatibility; Hydroxyapatite; Silicone; Nanoparticle;

Effects of pico-to-nanometer-thin TiO2 coating on the biological properties of microroughened titanium by Yoshihiko Sugita; Ken Ishizaki; Fuminori Iwasa; Takeshi Ueno; Hajime Minamikawa; Masahiro Yamada; Takeo Suzuki; Takahiro Ogawa (8374-8384).
The independent, genuine role of surface chemistry in the biological properties of titanium is unknown. Although microtopography has been established as a standard surface feature in osseous titanium implants, unfavorable behavior and reactions of osteogenic cells are still observed on the surfaces. To further enhance the biological properties of microfeatured titanium surfaces, this study tested the hypotheses that (1) the surface chemistry of microroughened titanium surfaces can be controllably varied by coating with a very thin layer of TiO2, without altering the existing topographical and roughness features; and (2) the change in the surface chemistry affects the biological properties of the titanium substrates. Using a slow-rate sputter deposition of molten TiO2 nanoparticles, acid-etched microroughened titanium surfaces were coated with a TiO2 layer of 300-pm to 6.3-nm thickness that increased the surface oxygen levels without altering the existing microtopography. The attachment, spreading behavior, and proliferation of osteoblasts, which are considered to be significantly impaired on microroughened surfaces compared with relatively smooth surfaces, were considerably increased on TiO2-coated microroughened surfaces. The rate of osteoblastic differentiation was represented by the increased levels of alkaline phosphatase activity and mineral deposition as well as by the upregulated expression of bone-related genes. These biological effects were exponentially correlated with the thickness of TiO2 and surface oxygen percentage, implying that even a picometer-thin TiO2 coating is effective in rapidly increasing the biological property of titanium followed by an additional mild increase or plateau induced by a nanometer-thick coating. These data suggest that a super-thin TiO2 coating of pico-to-nanometer thickness enhances the biological properties of the proven microroughened titanium surfaces by controllably and exclusively modulating their surface chemistry while preserving the existing surface morphology. The improvements in proliferation and differentiation of osteoblasts attained by this chemical modification is of great significance, providing a new insight into how to develop new implant surfaces for better osseointegration, based on the established microtopographic surfaces.
Keywords: Bone-implant integration; Osseointegration; Sputter deposition; Molten Ti; Microtopography;

Cytotoxicity of silica nanoparticles through exocytosis of von Willebrand factor and necrotic cell death in primary human endothelial cells by Alexander T. Bauer; Elwira A. Strozyk; Christian Gorzelanny; Christoph Westerhausen; Anna Desch; Matthias F. Schneider; Stefan W. Schneider (8385-8393).
Nanoparticle-induced endothelial cell (EC) dysfunction, due to the induction of inflammation and/or the activation of the coagulation system, is associated with pulmonary and ischemic cardiovascular diseases. Although it is contigent on several mechanisms, involving formation of reactive oxygen species and inflammatory cytokines such as interleukin (IL)-6 and 8, the involvement of the coagulation system is not well understood. The results of toxicity assays using the tetrazolium reduction (MTT) and lactate dehydrogenase (LDH) release showed that silica NP-induced cytotoxicity depends on the size and the dose of applied NP. Moreover, propidium iodide (PI) stainings and caspase 3/7 assays identified increased necrosis in ECs. Exposing human umbilical vein endothelial cells (HUVECs) to SiO2 NP with diameters of 304 nm and 310 nm led to significant increase of Weibel-Palade body (WPB) exocytosis, associated with the release of von Willebrand factor (VWF) and the formation of ultralarge fibers (ULVWF). High resolution microscopy techniques revealed that internalization and perinuclear localization of perylene-labeled NP with a size of 310 nm affect not only viability, but also cell migration and proliferation. In conclusion, our data indicate that NP-induced activation and dysfunction of ECs is reflected by release of VWF and necrotic cell death.
Keywords: AFM(atomic force microscopy); Coagulation; Endothelium; Nanoparticle; Silica; VWF(von Willibrand factor);

The pivotal role of fibrocytes and mast cells in mediating fibrotic reactions to biomaterials by Paul T. Thevenot; David W. Baker; Hong Weng; Man-Wu Sun; Liping Tang (8394-8403).
Almost all biomaterial implants are surrounded by a fibrotic capsule, although the mechanism of biomaterial-mediated fibrotic reactions is mostly unclear. To search for the types of cells responsible for triggering the tissue responses, we used poly-L glycolic acid polymers capable of releasing various reagents. We first identified that CD45+/Collagen 1+ fibrocytes are recruited and resided within the fibrotic capsule at the implant interface. Interestingly, we found that the recruitment of fibrocytes and the extent of fibrotic tissue formation (collagen type I production) were substantially enhanced and reduced by the localized release of compound 48/80 and cromolyn, respectively. Since it is well established that compound 48/80 and cromolyn alter mast cell reactions, we hypothesized that mast cells are responsible for triggering fibrocyte recruitment and subsequent fibrotic capsule formation surrounding biomaterial implants. To directly test this hypothesis, similar studies were carried out using mast cell deficient mice, WBB6F1/J-KitW /KitW-v /, and their congenic controls. Indeed, mast cell deficient mice prompted substantially less fibrocyte and myofibroblast responses in comparison to C57 wild type mice controls. Most interestingly, subcutaneous mast cell reconstitution of WBB6F1/J-KitW /KitW-v /J mice almost completely restored the fibrocyte response in comparison to the C57 wild type response. These results indicate that the initial biomaterial interaction resulting in the stimulation of mast cells and degranulation byproducts not only stimulates the inflammatory cascade but significantly alters the downstream fibrocyte response and degree of fibrosis.
Keywords: Fibrosis; Inflammation; Foreign body response; Cell activation; Fibroblast; Mast cell;

We investigated the in vitro effects of the molecular weight (MW) of hyaluronic acid (HA) on the maintenance of the pluripotency and proliferation of murine embryonic stem (ES) cells. High (1000 kDa) or low (4–8 kDa) MW HA was derivatized using an ultraviolet-reactive compound, 4-azidoaniline, and the derivative was immobilized onto cell culture cover slips. Murine ES cells were cultured on these HA surfaces for 5 days. High-MW HA interacted with murine ES cells via CD44, whereas low-MW HA interacted with these cells mostly via CD168. ES cells grown on both high- and low-MW HA appeared undifferentiated after 3 days. However, more cells adhered, proliferated, and exhibited greater amounts of phospho-p42/44 mitogen-activated-protein-kinase on low- compared with high-MW HA. Expression of Oct-3/4 and phosphorylation of STAT3 were enhanced by ES cells on low-MW HA, not on high-MW HA. After release from HA, cells cultured on low-MW HA in the presence of differentiating medium showed enhanced expression of α-SMA or CD31 compared with cells cultured on high-MW HA. It was concluded that low-MW HA substrates were effective in maintaining murine ES cells in a viable and undifferentiated state, which favors their use in the propagation of ES cells for tissue engineering.
Keywords: Hyaluronic acid; Micropatterning; Tissue engineering; Embryonic stem cell; Pluripotency; Proliferation;

Effect of alginate encapsulation on the cellular transcriptome of human islets by Vijayaganapathy Vaithilingam; Nayeem Quayum; Mugdha V. Joglekar; Jan Jensen; Anandwardhan A. Hardikar; Jose Oberholzer; Gilles J. Guillemin; Bernard E. Tuch (8416-8425).
Encapsulation of human islets may prevent their immune rejection when transplanted into diabetic recipients. To assist in understanding why clinical outcomes with encapsulated islets were not ideal, we examined the effect of encapsulation on their global gene (mRNA) and selected miRNAs (non-coding (nc)RNA) expression. For functional studies, encapsulated islets were transplanted into peritoneal cavity of diabetic NOD-SCID mice. Genomics analysis and transplantation studies demonstrate that islet origin and isolation centres are a major source of variation in islet quality. In contrast, tissue culture and the encapsulation process had only a minimal effect, and did not affect islet viability. Microarray analysis showed that as few as 29 genes were up-regulated and 2 genes down-regulated (cut-off threshold 0.1) by encapsulation. Ingenuity analysis showed that up-regulated genes were involved mostly in inflammation, especially chemotaxis, and vascularisation. However, protein expression of these factors was not altered by encapsulation, raising doubts about the biosignificance of the gene changes. Encapsulation had no effect on levels of islet miRNAs. In vivo studies indicate differences among the centres in the quality of the islets isolated. We conclude that microencapsulation of human islets with barium alginate has little effect on their transcriptome.
Keywords: Cell encapsulation; Diabetes; Gene expression; Hydrogel; Microencapsulation; Transplantation;

Cryopreservation of tissue-engineered epithelial sheets in trehalose by Fanfan Chen; Wenjie Zhang; Wei Wu; Yuqing Jin; Lian Cen; James D. Kretlow; Weicheng Gao; Zhenpeng Dai; Jiaming Wang; Guangdong Zhou; Wei Liu; Lei Cui; Yilin Cao (8426-8435).
Tissue-engineered epidermal membranes are useful for clinical wound healing. To facilitate these products in the clinic, optimized storage methods need to be developed. We studied the efficiency of extracellular trehalose at various concentrations for cryopreserving human tissue-engineered epidermal membranes compared with that of dimethyl-sulfoxide (DMSO) used by most organ banks for cryopreserving skin grafts and artificial skin substitutes. Keratinocyte (KC) viability, proliferation and marker expression following cryopreservation in trehalose were examined with similar results to those using DMSO. Trehalose concentration (0.4m) was optimized according to the described cellular activities following cryopreservation. Artificial epidermal substitutes were then cryopreserved in trehalose at the optimized concentration. Cell viability, growth factor secretion and wound healing properties of cryopreserved artificial epidermal substitutes using nude mice were examined and compared with those of DMSO cryopreservation. Cryopreservation with trehalose enhanced human KC viability in suspension and artificial skin substitutes. In addition, trehalose cryopreservation provided fast recovery of EGF and TGF-β1 secretion by KCs after thawing. When transplanted into nude mice, trehalose-cryopreserved artificial skin repaired skin defects in a similar manner to that of a non-cryopreserved control. Moreover, trehalose-cryopreserved artificial skin resulted in engraftment and wound closure that was significantly enhanced compared with that of DMSO-cryopreserved epidermal membranes. The results indicate that the use of trehalose improves cryopreservation of tissue-engineered epithelial sheets.
Keywords: Tissue-engineered skin; Keratinocyte; Trehalose; Cryopreservation; Wound healing;

Changes of chondrocyte expression profiles in human MSC aggregates in the presence of PEG microspheres and TGF-β3 by Soumya Ravindran; Jacob L. Roam; Peter K. Nguyen; Thomas M. Hering; Donald L. Elbert; Audrey McAlinden (8436-8445).
Biomaterial microparticles are commonly utilized as growth factor delivery vehicles to induce chondrogenic differentiation of mesenchymal stem/stromal cells (MSCs). To address whether the presence of microparticles could themselves affect differentiation of MSCs, a 3D co-aggregate system was developed containing an equal volume of human primary bone marrow-derived MSCs and non-degradable RGD-conjugated poly(ethylene glycol) microspheres (PEG-μs). Following TGF-β3 induction, differences in cell phenotype, gene expression and protein localization patterns were found when compared to MSC aggregate cultures devoid of PEG-μs. An outer fibrous layer always found in differentiated MSC aggregate cultures was not formed in the presence of PEG-μs. Type II collagen protein was synthesized by cells in both culture systems, although increased levels of the long (embryonic) procollagen isoforms were found in MSC/PEG-μs aggregates. Ubiquitous deposition of type I and type X collagen proteins was found in MSC/PEG-μs cultures while the expression patterns of these collagens was restricted to specific areas in MSC aggregates. These findings show that MSCs respond differently to TGF-β3 when in a PEG-μs environment due to effects of cell dilution, altered growth factor diffusion and/or cellular interactions with the microspheres. Although not all of the expression patterns pointed toward improved chondrogenic differentiation in the MSC/PEG-μs cultures, the surprisingly large impact of the microparticles themselves should be considered when designing drug delivery/scaffold strategies.
Keywords: Mesenchymal stem cell; Polyethylene; Microsphere; Cartilage tissue engineering; TGF-β; Chondrocyte;

Core–shell cell bodies composed of human cbMSCs and HUVECs for functional vasculogenesis by Wen-Yu Lee; Hung-Wen Tsai; Jen-Hao Chiang; Shiaw-Min Hwang; Ding-Yuan Chen; Li-Wen Hsu; Yi-Wen Hung; Yen Chang; Hsing-Wen Sung (8446-8455).
Rapid induction and creation of functional vascular networks is essential for the success of treating ischemic tissues. The formation of mature and functional vascular networks requires the cooperation of endothelial cells (ECs) and perivascular cells. In the study, we used a thermo-responsive hydrogel system to fabricate core–shell cell bodies composed of cord-blood mesenchymal stem cells (cbMSCs) and human umbilical vascular ECs (HUVECs) for functional vasculogenesis. When seeded on Matrigel, the shelled HUVECs attempted to interact and communicate vigorously with the cored cbMSCs initially. Subsequently, HUVECs migrated out and formed tubular structures; cbMSCs were observed to coalesce around the HUVEC-derived tubes. With time progressing, the tubular networks continued to expand without regression, indicating that cbMSCs might function as perivascular cells to stabilize the nascent networks. In the in vivo study, cbMSC/HUVEC bodies were embedded in Matrigel and implanted subcutaneously in nude mice. At day 7, visible blood-filled vessels were clearly identified within the implant containing cbMSC/HUVEC bodies, indicating that the formed vessels anastomosed with the host vasculature. The cored cbMSCs were stained positive for smooth muscle actin, suggesting that they underwent smooth muscle differentiation and formed microvessels with the shelled HUVECs, as the role of perivascular cells. These data confirm that the formation of mature vessels requires heterotypic cooperation of HUVECs and MSCs. This study provides a new strategy for therapeutic vasculogenesis, by showing the feasibility of using cbMSC/HUVEC bodies to create functional vascular networks.
Keywords: Vascularization; Tissue regeneration; Cell therapy; Tube formation; Vascular network;

Single nonfouling hydrogels with mechanical and chemical functionality gradients by Louisa R. Carr; Jordan E. Krause; Jean-Rene Ella-Menye; Shaoyi Jiang (8456-8461).
Hydrogels are widely studied as tissue engineering scaffolds, but the biological tissues they are designed to mimic are often complex tissues with non-uniform chemical and mechanical profiles. This work reports a new strategy to create hydrogels composed of a continuous sheet of a single nonfouling but functionalizable material with mechanical and/or chemical functionality gradients. By using different combinations of functionalizable or nonfunctionalizable versions of nonfouling carboxybetaine methacrylate (CBMA) and carboxybetaine dimethacrylate crosslinker (CBMAX), various hydrogels with gradients of crosslinking densities and/or functionalizable groups can be created. In this work, we demonstrate this concept with two nonfouling hydrogels, both with a mechanical gradient: one with uniform functionalizability and the other with a gradient in chemical functionalizability. With this versatile system, hydrogels with built-in gradient profiles of various types can be controlled at will for a given application.
Keywords: Hydrogel; Mechanical properties; Crosslinking; Ligament replacement;

The effect of hydration on molecular chain mobility and the viscoelastic behavior of resilin-mimetic protein-based hydrogels by My Y. Truong; Naba K. Dutta; Namita R. Choudhury; Misook Kim; Christopher M. Elvin; Kate M. Nairn; Anita J. Hill (8462-8473).
The outstanding rubber-like elasticity of resilin and resilin-mimetic proteins depends critically on the level of hydration. In this investigation, water vapor sorption and the role of hydration on the molecular chain dynamics and viscoelastic properties of resilin-mimetic protein, rec1-resilin is investigated in detail. The dynamic and equilibrium swelling behavior of the crosslinked protein hydrogels with different crosslink density are reported under various controlled environments. We propose three different stages of hydration; involving non-crystallizable water, followed by condensation or clustering of water around the already hydrated sites, and finally crystallizable water. The kinetics of water sorption for this engineering protein is observed to be comparable to hydrophilic polymers with a diffusion coefficient in the range of 10−7 cm2 s−1. From the comparison between the absorption and desorption isotherms at a constant water activity, it has been observed that rec1-resilin exhibits sorption hysteresis only for the tightly bound water. Investigation of molecular mobility using differential scanning calorimetry, indicates that dehydrated crosslinked rec1-resilin is brittle with a glass transition temperature (Tg ) of >180 °C, which dramatically decreases with increasing hydration; and above a critical level of hydration rec1-resilin exhibits rubber-like elasticity. Nanoindentation studies show that even with little hydration (<10%), the mechanical properties of rec1-resilin gels change dramatically. Rheological investigations confirm that the equilibrium-swollen crosslinked rec1-resilin hydrogel exhibits outstanding elasticity and resilience of ∼92%, which exceeds that of any other synthetic polymer and biopolymer hydrogels.
Keywords: Biomimetic protein; Rec1-resilin; Rheological properties; Hydrogel; Sorption isotherm; Hydration;

The inhibition of infection by wound pathogens on scaffold in tissue-forming process using N-acetyl cysteine by Masahiro Yamada; Kazuyuki Ishihara; Takahiro Ogawa; Kaoru Sakurai (8474-8485).
Prevention of local infection from wound pathogens such as Staphylococci and Streptococci is crucial for tissue regeneration. N-acetyl cysteine (NAC), an anti-oxidant amino acid derivative, has anti-microbial potential against various species. This in vitro study evaluated whether NAC prevented bacterial infection of gingival fibroblasts and osteoblasts on a scaffold. N-acetyl cysteine delayed growth of Staphylococcus aureus and Streptococcus pyogenes cultured in brain heart infusion (BHI) broth for 12 h in an almost dose-dependent manner (2.5, 5.0 or 10.0 mm). The number of rat gingival fibroblasts on collagen scaffolds with bacterial co-incubation was less than 30% of that in cultures without bacterial co-incubation at day 7. However, pre-addition of NAC to the scaffold yielded a number comparable with that in culture without bacteria. Fibroblasts on the scaffold with bacterial co-incubation were small, rounded and filled with bacteria and reactive oxygen species. Pre-addition of NAC, however, resulted in fibroblasts similar to those observed in culture without bacterial co-incubation. N-acetyl cysteine completely prevented devastating suppression of alkaline–phosphatase activity and extracellular matrix mineralization in osteoblastic culture on scaffolds with bacterial co-incubation. These results indicate that NAC can functionalize a scaffold with anti-infective capabilities, thus assisting healing of soft and hard tissues.
Keywords: Anti-oxidant; Bone regeneration; Streptococci; Multi-functionalization; Staphylococci; Tissue-engineering;

In Vitro enzymatic degradation of poly (glycerol sebacate)-based materials by Shu-Ling Liang; Xue-Yuan Yang; Xi-Ya Fang; Wayne D. Cook; George A. Thouas; Qi-Zhi Chen (8486-8496).
Enzymatic degradation is a major feature of polyester implants in vivo. An in vitro experimental protocol that can simulate and predict the in vivo enzymatic degradation kinetics of implants is of importance not only to our understanding of the scientific issue, but also to the well-being of animals. In this study, we explored the enzymatic degradation of PGS-based materials in vitro, in tissue culture medium or a buffer solution at the pH optima and under static or cyclic mechanical-loading conditions, in the presence of defined concentrations of an esterase. Surprisingly, it was found that the in vitro enzymatic degradation rates of the PGS-based materials were higher in the tissue culture medium than in the buffered solution at the optimum pH 8. The in vitro enzymatic degradation rate of PGS-based biomaterials crosslinked at 125 °C for 2 days was approximately 0.6–0.9 mm/month in tissue culture medium, which falls within the range of in vivo degradation rates (0.2–1.5 mm/month) of PGS crosslinked at similar conditions. Enzymatic degradation was also further enhanced in relation to mechanical deformation. Hence, in vitro enzymatic degradation of PGS materials conducted in tissue culture medium under appropriate enzymatic conditions can quantitatively capture the features of in vivo degradation of PGS-based materials and can be used to indicate effective strategies for tuning the degradation rates of this material system prior to animal model testing.
Keywords: Enzymatic degradation; Esterase; Poly(glycerol sebacate); In vitro;

Vascular and micro-environmental influences on MSC-coral hydroxyapatite construct-based bone tissue engineering by Lei Cai; Qian Wang; Congmin Gu; Jingguo Wu; Jian Wang; Ning Kang; Jiewei Hu; Fang Xie; Li Yan; Xia Liu; Yilin Cao; Ran Xiao (8497-8505).
Bone tissue engineering (BTE) has been demonstrated an effective approach to generate bone tissue and repair bone defect in ectopic and orthotopic sites. The strategy of using a prevascularized tissue-engineered bone grafts (TEBG) fabricated ectopically to repair bone defects, which is called live bone graft surgery, has not been reported. And the quantitative advantages of vascularization and osteogenic environment in promoting engineered bone formation have not been defined yet. In the current study we generated a tissue engineered bone flap with a vascular pedicle of saphenous arteriovenous in which an organized vascular network was observed after 4 weeks implantation, and followed by a successful repaire of fibular defect in beagle dogs. Besides, after a 9 months long term observation of engineered bone formation in ectopic and orthotopic sites, four CHA (coral hydroxyapatite) scaffold groups were evaluated by CT (computed tomography) analysis. By the comparison of bone formation and scaffold degradation between different groups, the influences of vascularization and micro-environment on tissue engineered bone were quantitatively analyzed. The results showed that in the first 3 months vascularization improved engineered bone formation by 2 times of non-vascular group and bone defect micro-environment improved it by 3 times of ectopic group, and the CHA-scaffold degradation was accelerated as well.
Keywords: Bone tissue engineering; Vascular pedicle; Micro-environment; CT evaluation;

The regenerative treatment of large osseous defects remains a formidable challenge in orthopedic surgery today. In the present study, we have synthesized biodegradable calcium/magnesium-doped silica-based scaffolds with hierarchically macro/mesoporous structure (CMMS), and incorporated recombinant human bone morphogenetic protein-2 (rhBMP-2) into the scaffolds to obtain a hybrid system for osteogenic factor delivery in the functional repair of bone defects. The developed CMMS/rhBMP-2 scaffolds presented interconnected porous network, macropores (200–500 μm) and mesopores (5.7 nm), as well as good bioactivity and biocompatibility and proper degradation rate. Combined with the capacity to deliver ions and growth factors, the CMMS/rhBMP-2 scaffolds significantly promoted the in vitro osteogenic differentiation of bone marrow stromal cells (bMSCs), as evidenced by the enhanced expression of Runx-2, osteopontin, osteocalcin and bone sialoprotein, and induced the ectopic bone formation in the thigh muscle pouches of mice. We further assessed the in vivo effects of CMMS/rhBMP-2 scaffolds in a rabbit femur cavity defect model by using synchrotron radiation-based μCT (SRμCT) imaging and histological analysis, indicating that the CMMS/rhBMP-2 scaffolds resulted in more bone regeneration compared to that observed with the CMMS scaffolds without rhBMP-2. Moreover, scaffolds with or without rhBMP-2 underwent gradual resorption and replacement with bone and almost disappeared at 12 weeks, while the dense CMMS/rhBMP-2 material showed slower degradation rate and promoted the least extensive neo-bone formation. This study suggested that the hybrid CMMS/rhBMP-2 scaffolds system demonstrates promise for bone regeneration in clinical case of large bone defects.
Keywords: Mesoporous silica; Scaffolds; rhBMP-2; Bone regeneration; Synchrotron radiation;

Identification of a peptide that interacts with Nestin protein expressed in brain cancer stem cells by Samuel Beck; Xun Jin; Jinlong Yin; Sung-Hak Kim; Nam-Kyung Lee; Se-Yeong Oh; Xiong Jin; Min-Kook Kim; Eun-Bae Kim; Jee-Soo Son; Sung-Chan Kim; Do-Hyun Nam; Se-Hyuk Kim; Sang-Kee Kang; Hyunggee Kim; Yun-Jaie Choi (8518-8528).
Glioma stem cells (GSCs) are presumably major culprits for brain tumor initiation, progression, and recurrence after conventional therapies. Thus, selective targeting and eradication of GSCs may provide a promising and effective therapeutic approach. Here, we isolated a GSC-targeting (GSCT) peptide that demonstrated selective binding affinity for many undifferentiated GSCs using in vitro phage display technology. This GSCT peptide binds to isotypes of Nestin proteins specifically expressed in GSCs, enabling it to target Nestin-positive cells in human glioblastoma tissues. In human glioblastoma tissue specimens, the fluorescence-conjugated GSCT peptide could visualize putative GSC populations, showing its possible use as a diagnostic agent. GSCT peptide is also internalized into undifferentiated GSCs specifically in vitro, and moreover, intravenously injected GSCT peptide effectively penetrated into tissues, specifically accumulated in gliomas that arise from subcutaneous and orthotopic implantation, and predominantly targeted Nestin-positive cells in these tumors. Thus, our GSCT peptide may be useful for the development of more promising therapeutic and diagnostic modalities that target GSCs in brain tumors.
Keywords: Peptide; Adhesion molecule; Phage display; Glioma stem cells; Brain tumors;

PEGylated TNF-related apoptosis-inducing ligand (TRAIL) for effective tumor combination therapy by Hai Hua Jiang; Tae Hyung Kim; Seulki Lee; Xiaoyuan Chen; Yu Seok Youn; Kang Choon Lee (8529-8537).
Although PEGylated TNF-related apoptosis-inducing ligand (PEG-TRAIL) has good tumor cell specificity and stability, its therapeutic potential is restricted by the development of tumor cell resistance. The purpose of this study was to develop an effective combination therapy with sustained biological activity based on microspheres. Doxorubicin (DOX), PEG-TRAIL, and DOX plus PEG-TRAIL (dual agent) were microencapsulated into poly (lactic-co-glycolic acid) (PLGA) microspheres using a double-emulsion solvent extraction method. Prepared dual agent microspheres showed the encapsulation efficiency 69.4 ± 2.3 for DOX and 87.7 ± 2.9% for PEG-TRAIL. Potential anti-tumor efficacy of this system was investigated in vitro and in vivo in a human colon cancer (HCT116) and in a human prostate cancer (PC-3). DOX and PEG-TRAIL release from dual agent microspheres were biologically active and significantly inhibited the TRAIL-sensitive HCT116 and resistant PC-3 cells in vitro. Dual agent microspheres simultaneous delivery of DOX and PEG-TRAIL was superior to all other DOX or PEG-TRAIL microspheres in vivo. A single local injection of PLGA microspheres loaded with low amounts of DOX, PEG-TRAIL, or dual agent resulted in 14.8, 30.2, and 63.6% reductions in HCT116 tumor volume and 20.4, 14.2, and 67.7% reductions in PC-3 tumor volume at 35 days. Our findings show that dual agent microspheres offer a promising means of delivering DOX and PEG-TRAIL to tumor sites.
Keywords: PEG-TRAIL; Doxorubicin; Sequential delivery; Microspheres; Combination therapy;

The intracellular uptake of CD95 modified paclitaxel-loaded poly(lactic-co-glycolic acid) microparticles by Davidson D. Ateh; Veronica H. Leinster; Sally R. Lambert; Afsha Shah; Ayub Khan; Hazel J. Walklin; Jennifer V. Johnstone; Nader I. Ibrahim; Mustafa M. Kadam; Zain Malik; Míriam Gironès; Gert J. Veldhuis; Gary Warnes; Silvia Marino; Iain A. McNeish; Joanne E. Martin (8538-8547).
The CD95/CD95L receptor-ligand system is mainly recognised in the induction of apoptosis. However, it has also been shown that CD95L is over-expressed in many cancer types where it modulates immune-evasion and together with its receptor CD95 promotes tumour growth. Here, we show that CD95 surface modification of relatively large microparticles >0.5 μm in diameter, including those made from biodegradable polylactic-co-glycolic acid (PLGA), enhances intracellular uptake by a range of CD95L expressing cells in a process akin to phagocytosis. Using this approach we describe the intracellular uptake of microparticles and agent delivery in neurons, medulloblastoma, breast and ovarian cancer cells in vitro. CD95 modified paclitaxel-loaded PLGA microparticles are shown to be significantly more effective compared to conventional paclitaxel therapy (Taxol) at the same dose in subcutaneous medulloblastoma (∗∗∗P < 0.0001) and orthotopic ovarian cancer xenograft models where a >65-fold reduction in tumour bioluminescence was measured after treatment (∗P = 0.012). This drug delivery platform represents a new way of manipulating the normally advantageous tumour CD95L over-expression towards a therapeutic strategy. CD95 functionalised drug carriers could contribute to the improved function of cytotoxics in cancer, potentially increasing drug targeting and efficacy whilst reducing toxicity.
Keywords: CD95; CD95L; Paclitaxel; Polylactic-co-glycolic acid; Phagocytosis; Cancer;

Folate-targeted nanoparticle delivery of chemo- and radiotherapeutics for the treatment of ovarian cancer peritoneal metastasis by Michael E. Werner; Shrirang Karve; Rohit Sukumar; Natalie D. Cummings; Jonathan A. Copp; Ronald C. Chen; Tian Zhang; Andrew Z. Wang (8548-8554).
Peritoneal metastasis is a major cause of morbidity and mortality in ovarian cancer. While intraperitoneal chemotherapy and radiotherapy have shown favorable clinical results, both are limited by their non-targeted nature. We aimed to develop a biologically targeted nanoparticle therapeutic for the treatment of ovarian cancer peritoneal metastasis. Folate-targeted nanoparticles encapsulating chemotherapy and/or radiotherapy were engineered. Paclitaxel (Ptxl) was used as the chemotherapeutic and yittrium-90 (90Y) was employed as the therapeutic radioisotope. Folate was utilized as the targeting ligand as most ovarian cancers overexpress the folate receptor. Nanoparticle characterization studies showed monodispersed particles with controlled Ptxl release. Folate targeting ligand mediated the uptake of NPs into tumor cells. In vitro efficacy studies demonstrated folate-targeted NPs containing chemoradiotherapy was the most effective therapeutic compared to folate-targeted NPs containing a single therapeutic or any non-targeted NP therapeutics. In vivo efficacy studies using an ovarian peritoneal metastasis model showed that folate-targeted NP therapeutics were significantly more effective than non-targeted NP therapeutics. Among the folate-targeted therapeutics, the NP containing chemoradiotherapy appeared to be the most effective. Our results suggest that folate-targeted nanoparticles containing chemoradiotherapy have the potential as a treatment for ovarian peritoneal metastasis.
Keywords: Chemotherapy; Nanoparticle; Nanomedicine; Chemoradiotherapy; Molecular targeted nanoparticles; Folate-targeted nanoparticles;

Synergistic effect of chemo-photothermal therapy using PEGylated graphene oxide by Wen Zhang; Zhouyi Guo; Deqiu Huang; Zhiming Liu; Xi Guo; Huiqing Zhong (8555-8561).
Graphene has shown great potential both in photothermal therapy and drug delivery. Herein, we developed doxorubicin-loaded PEGylated nanographene oxide (NGO-PEG-DOX) to facilitate combined chemotherapy and photothermal therapy in one system. In this work, we studied the ablation of tumor both in vivo and in vitro by the combination of photothermal therapy and chemotherapy using this functional graphene oxide. The ability of the NGO-PEG-DOX nanoparticle to combine the local specific chemotherapy with external near-infrared (NIR) photothermal therapy significantly improved the therapeutic efficacy of cancer treatment. Compared with chemotherapy or photothermal therapy alone, the combined treatment demonstrated a synergistic effect, resulting in higher therapeutic efficacy. Furthermore, lower systematic toxicity of NGO-PEG-DOX than DOX was proved by the pathologic examination of main organs in our toxicity study.
Keywords: Photothermal therapy; Chemotherapy; Graphene oxide; Drug delivery; Synergistic effect;

Dendronized iron oxide nanoparticles for multimodal imaging by Giuseppe Lamanna; Marie Kueny-Stotz; Hind Mamlouk-Chaouachi; Cynthia Ghobril; Brice Basly; Annabelle Bertin; Imen Miladi; Claire Billotey; Geneviève Pourroy; Sylvie Begin-Colin; Delphine Felder-Flesch (8562-8573).
The synthesis of small-size dendrons and their grafting at the surface of iron oxide nanoparticles were achieved with the double objective to obtain a good colloidal stability with a mean hydrodynamic diameter smaller than 100 nm and to ensure the possibility of tuning the organic coating characteristics including morphology, functionalities, physico-chemical properties, grafting of fluorescent or targeting molecules. Magnetic resonance and fluorescence imaging are then demonstrated to be simultaneously possible using such versatile superparamagnetic iron oxide nanocrystals covered by a dendritic shell displaying either carboxylate or ammonium groups at their periphery which could be further labelled with a fluorescent dye. The grafting conditions of these functionalized dendrons at the surface of SPIO NPs synthesized by co-precipitation have been optimized as a function of the nature of the peripheral functional group. The colloidal stability has been investigated in water and osmolar media, and in vitro and in vivo MRI and optical imaging measurements have been performed showing encouraging biodistribution.
Keywords: Dendrimers; Iron oxide nanoparticles; Phosphonates; MRI contrast agent; Biodistribution; Multimodal imaging; Vectorization;

Triazacryptand-based fluorescent sensors for extracellular and intracellular K+ sensing by Xianfeng Zhou; Fengyu Su; Weimin Gao; Yanqing Tian; Cody Youngbull; Roger H. Johnson; Deirdre R. Meldrum (8574-8583).
A 4-amino-naphthalimide derived fluorophore with a triazacryptand moiety ligand was synthesized as a potassium ion (K+) sensor (KS1). This sensor is a monomer possessing a polymerizable vinyl group. By taking advantage of the polymerizable characteristics of the vinyl group, KS1 was polymerized with 2-hydroxyethyl methacrylate (HEMA) and acrylamide (AM) to form K+ sensing films for extracellular sensing. The sensitivity of the films to potassium ions can be further tuned through the adjustment of the HEMA and AM weight ratios as well as introduction of positive or negative charge-containing segments. KS1 and its poly(2-hydroxyethyl methacrylate)-co-poly(acrylamide) (PHEMA-co-PAM) thin films show high selectivity for K+ over competing sodium ions (Na+) at physiological concentrations. Extracellular sensing was demonstrated using a KS1-conjugated PHEMA-co-PAM thin film to measure the K+ efflux of Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) stimulated by lysozyme. Meanwhile, KS1 itself permeates human glioblastoma U87MG and human esophagus premalignant CP-A cell lines. KS1 was used to monitor K+ efflux stimulated by adenosine-5′-triphosphate (ATP), amphotericin, and a mixture of nigericin, bumetanide and ouabain, demonstrating application of this material as an intracellular potassium ion sensor.
Keywords: Potassium ion sensor; Amino-naphthalimide; Intracellular sensing; Extracellular sensing; Fluorescent probe;

In this article, we demonstrate the use of graphene–mesoporous silica–gold NP hybrids (GSGHs) as an enhanced element of the integrated sensing platform for the ultra-sensitive and selective detection of DNA by using strand-displacement DNA polymerization and parallel-motif DNA triplex system as dual amplifications. We find that the present new sensing strategy based on GSGHs is able to detect target DNA with a fairly high detection sensitivity of 10 fm through the hybridization of duplex DNA to the acceptor DNA for the formation of parallel-motif DNA triplex on the multilayer film (containing GSGHs and redox probe) modified functional interface, and even has a good capability to investigate the single nucleotide polymorphisms (SNPs). The detection limit for target DNA is about two orders of magnitude lower than that of graphene-based DNA electrochemical impedance spectroscopy (EIS) sensor (6.6 pm), four orders of magnitude lower than those of graphene-based DNA sensors coupled with fluorescent assay (100 pm and 1 nm) and five orders of magnitude lower than those of field effect transistor (FET)-based assays (1 nm and 2 nm). Most importantly, our present sensing system can also be facilely achieved in the ITO electrode array, which is of paramount importance for possible multiplex analysis in lab-on-chip.
Keywords: Graphene; Gold nanoparticle; Layer-by-layer assembly; DNA sensor; Lab-on-chip;

Strategies for the nanoencapsulation of hydrophilic molecules in polymer-based nanoparticles by Sandy Vrignaud; Jean-Pierre Benoit; Patrick Saulnier (8593-8604).
Hydrophilic drug delivery still remains a challenge; this either being attributed to the fragility and poor cellular penetration of macromolecules, or to the unsuitable pharmacokinetics and toxicity of small drugs, for instance anticancer agents. By offering more favourable pharmacokinetics and protection of the drug, encapsulation in polymer nanoparticles constitutes an attractive possibility to overcome these problems. This review provides an overview of the strategies that have been developed for encapsulating hydrophilic molecules in polymer-containing nanoparticles, e.g. nanospheres and nanocapsules. Polymer nanospheres are loaded either by drug entrapment (by pH modification, use of reverse micelles or the addition of a polyanion) and generally produce a poor level of entrapment efficiency, or molecule sorption onto the nanosphere surface (by pH modification, use of high drug concentration, or ion-pair formation) with the drawbacks of a less-well protected drug from degradation and a faster drug release. Another strategy consists of the use of aqueous-core nanocapsules, generally surrounded by a thin polymer layer, in which hydrophilic molecules are directly solubilised in internal water, and are thus entrapped within the nanocapsule core, assuring drug protection and sustained release. Nanocapsules require less polymer compared to nanospheres; on the other hand, when the drug is entrapped, it has to be added before or during the formulation process, and is thus likely to be degraded. Overall, drug encapsulation in polymer nanoparticles provides a better pharmacokinetic profile and bioavailability, enhanced anticancer activity, reduced drug toxicity and modified drug distribution as compared to free drugs.
Keywords: Nanoparticle; Polymer; Hydrophilic drug; Aqueous-core nanocapsule; Macromolecule;

The use of nitroxide radical-containing nanoparticles coupled with piperine to protect neuroblastoma SH-SY5Y cells from Aβ-induced oxidative stress by Pennapa Chonpathompikunlert; Toru Yoshitomi; Junkyu Han; Hiroko Isoda; Yukio Nagasaki (8605-8612).
The antioxidant effect and potential mechanism of nitroxide radical-containing nanoparticles (RNPs) coupled with piperine (PI) were investigated in human neuroblastoma SH-SY5Y cells. The effects of RNP/PI on SH-SY5Y cell lines was determined by WST assay for cell viability, nitroblue tetrazolium and deoxyribose assay for reactive oxygen species generation, ELISA assay for reactive oxygen species products and apoptotic cell death, and biochemical techniques for catalase and glutathione peroxidase activity. The RNP/PI significantly reduced the reactive oxygen species level and reactive oxygen species products compared with those of cells treated with RNPs alone. The RNP/PI treatment enhanced catalase and glutathione peroxidase activity. The combination of RNP/PI has been found to have an augmented antioxidant effect on an Alzheimer’s model in vitro. The mechanism of the protective effect of this combination therapy was correlated in this study with its ability to reduce the generation of reactive oxygen species and prevent apoptosis via scavenging enzyme action pathways.
Keywords: Antioxidant; Copolymer; Free radical; In vitro test; Nanoparticle; Neural cell;

The association of autophagy with polyethylenimine-induced cytotoxity in nephritic and hepatic cell lines by Xiaoling Gao; Lei Yao; Qiangxiang Song; Liang Zhu; Zheng Xia; Huimin Xia; Xinguo Jiang; Jun Chen; Hongzhuan Chen (8613-8625).
Polyethylenimine (PEI) is one of the most effective and widely used cationic macromolecules in experimental gene transfer/therapy protocols. However, the further clinical application of PEI is largely impeded by its cytotoxicity. Here we performed a fundamental investigation on the mechanism of PEI-induced cytotoxicity in both hepatic and nephritic cell lines. It was demonstrated that besides necrosis and apoptosis, autophagy was apparently associated with PEI-induced cytotoxicity and contributed to aggravated cell damage. Specifically, at the early stage (3 h) of PEI-induced cytotoxicity, autophagy was mainly correlated with lysosome damage, but in the later phase (after a 24-h recovery), autophagy was mainly related with mitochondrial injury. Modulation of Rab5, Rab7 expression and inhibition of clathrin-mediated endocytosis pathway significantly affected the formation of autophagosome, which suggested that the endolysosome transport pathway especially the clathrin-mediated endocytosis at least partly facilitated PEI-induced autophagy. As PEI-induced autophagy played a causative role in its cytotoxicity, it’s highly recommended to design PEI-based gene-carriers that could avoid the endolysosome transport pathway.
Keywords: Biocompatibility; Polyethylenimine; Autophagy; Necrosis; Apoptosis; Cytotoxicity;

In the present study we compare LPEI and BPEI characteristics related to DNA condensation and their role as free polycation chains in gene transfection. Using radioactive 32P labeled DNA, we investigated the effect of free PEI chains on the cellular uptake of polyplexes. Our investigations show different properties of BPEI and LPEI polyplexes in condensation and de-condensation processes as well as in cellular uptake, which was tightly correlated with transfection efficiency. In agreement with earlier reports we find all DNA to be condensed at N/P = 3. Further added PEI chains remain free in solution. We found that both the cellular uptake and gene transfection of BPEI polyplexes is much more efficient than LPEI polyplexes at a low N/P ratio of 3 (i.e., without free PEI chains). When N/P is high (10, with 7 portions of free PEI), the LPEI and BPEI polyplexes have similar transfection efficiency even though the cellular uptake of the LPEI polyplexes is significantly lower. In addition, we found that addition of free short or long PEI chains (2.5 and 25 kDa) leads to a comparable gene transfection efficiency.
Keywords: Polyplex; Gene transfection; Polyethylenimine; PEI; Polycations; Non-viral vectors;

Intracellular delivery of paclitaxel using oil-free, shell cross-linked HSA – Multi-armed PEG nanocapsules by Jeong Yu Lee; Ki Hyun Bae; Jee Seon Kim; Yoon Sung Nam; Tae Gwan Park (8635-8644).
Various approaches to increase the solubility of water-insoluble anti-cancer drugs in aqueous formulations have been undertaken with the aim of treating solid tumors through intravenous drug administration. Nanoscale drug carriers are particularly attractive for cancer therapy because of their passive targeting effect to enhance the therapeutic efficacy of drugs. Here we introduce an oil-free, shell cross-linked nanocapsule as an efficient intracellular delivery system for paclitaxel. The nanocapsules are prepared by emulsifying amine-reactive six-arm-branched polyethylene glycol (PEG) in dichloromethane into aqueous solution of human serum albumin (HSA), followed by cross-linking at the organic/aqueous interface. Paclitaxel is successfully incorporated into the HSA/PEG nanocapsules having a spherical shape with an average diameter of about 280 nm. In several types of cells, the surface modification of nanocapsules with a cell-penetrating peptide, Hph1, greatly facilitates cellular uptake and apoptosis-inducing effects of paclitaxel. Furthermore, the targeted anti-tumor activities of the paclitaxel-loaded nanocapsules in a mouse tumor model suggest that the shell cross-linked nanocapsules are very promising oil-free nanoscale delivery vehicles for water-insoluble anti-cancer agents.
Keywords: Paclitaxel; Drug delivery system; Human serum albumin; Nanocapsule; Cell-penetrating peptide;

Conatumumab (AMG 655) coated nanoparticles for targeted pro-apoptotic drug delivery by Francois Fay; Kirsty M. McLaughlin; Donna M. Small; Dean A. Fennell; Patrick G. Johnston; Daniel B. Longley; Christopher J. Scott (8645-8653).
Colloidal nanoparticle drug delivery systems have attracted much interest for their ability to enable effective formulation and delivery of therapeutic agents. The selective delivery of these nanoparticles to the disease site can be enhanced by coating the surface of the nanoparticles with targeting moieties, such as antibodies. In this current work, we demonstrate that antibodies on the surface of the particles can also elicit key biological effects. Specifically, we demonstrate the induction of apoptosis in colorectal HCT116 cancer cells using PLGA nanoparticles coated with Conatumumab (AMG 655) death receptor 5-specific antibodies (DR5-NP). We show that DR5-NP preferentially target DR5-expressing cells and present a sufficient density of antibody paratopes to induce apoptosis via DR5, unlike free AMG 655 or non-targeted control nanoparticles. We also demonstrate that DR5-targeted nanoparticles encapsulating the cytotoxic drug camptothecin are effectively targeted to the tumour cells, thereby producing enhanced cytotoxic effects through simultaneous drug delivery and apoptosis induction. These results demonstrate that antibodies on nanoparticulate surfaces can be exploited for dual modes of action to enhance the therapeutic utility of the modality.
Keywords: DR5; c-FLIP; Nanoparticles; Cancer; Caspase pathway; Conatumumab;

Magnetically enhanced adeno-associated viral vector delivery for human neural stem cell infection by Eunmi Kim; Ji-Seon Oh; Ik-Sung Ahn; Kook In Park; Jae-Hyung Jang (8654-8662).
Gene therapy technology is a powerful tool to elucidate the molecular cues that precisely regulate stem cell fates, but developing safe vehicles or mechanisms that are capable of delivering genes to stem cells with high efficiency remains a challenge. In this study, we developed a magnetically guided adeno-associated virus (AAV) delivery system for gene delivery to human neural stem cells (hNSCs). Magnetically guided AAV delivery resulted in rapid accumulation of vectors on target cells followed by forced penetration of the vectors across the plasma membrane, ultimately leading to fast and efficient cellular transduction. To combine AAV vectors with the magnetically guided delivery, AAV was genetically modified to display hexa-histidine (6xHis) on the physically exposed loop of the AAV2 capsid (6xHis AAV), which interacted with nickel ions chelated on NTA-biotin conjugated to streptavidin-coated superparamagnetic iron oxide nanoparticles (NiStNPs). NiStNP-mediated 6xHis AAV delivery under magnetic fields led to significantly enhanced cellular transduction in a non-permissive cell type (i.e., hNSCs). In addition, this delivery method reduced the viral exposure times required to induce a high level of transduction by as much as to 2–10 min of hNSC infection, thus demonstrating the great potential of magnetically guided AAV delivery for numerous gene therapy and stem cell applications.
Keywords: Adeno-associated virus; Superparamagnetic iron oxide nanoparticle; Hexa-histidine; Gene delivery;

Liposomes for HIV prophylaxis by Nikita K. Malavia; David Zurakowski; Avi Schroeder; Amy M. Princiotto; Anna R. Laury; Hila E. Barash; Joseph Sodroski; Robert Langer; Navid Madani; Daniel S. Kohane (8663-8668).
There are approximately 33.4 million adults living with HIV worldwide of which an estimated 15.7 million are women. Although there has been enormous progress in the therapy of HIV/AIDS, treatment is not curative. Prevention is therefore of paramount importance, but vaccine-based and microbicidal approaches are still in their infancy. Since women acquire the virus largely through sexual intercourse, we developed liposomal systems potentially suitable for intra-vaginal use to prevent HIV-1 infection. We formulated liposomes from a range of naturally-occurring and synthetic lipids with varying physicochemical properties, and tested their ability to inhibit infection of transformed cells that express receptors specific to the virus. We identified formulations with the most favorable balance between decreasing HIV infection and causing cytotoxicity (i.e. therapeutic index). The therapeutic index improved with increasing cardiolipin content, and degree of unsaturation. Tissue reaction to these formulations was benign after intra-vaginal instillation in an in vivo female mouse model. These results support the potential use of cardiolipin-based liposomes enriched with synthetic lipids as microbicides for the prevention of HIV infection in women.
Keywords: Liposome; HIV; AIDS; Prophylaxis; Cardiolipin;

A cascade targeting strategy for brain neuroglial cells employing nanoparticles modified with angiopep-2 peptide and EGFP-EGF1 protein by Gao Huile; Pan Shuaiqi; Yang Zhi; Cao Shijie; Chen Chen; Jiang Xinguo; Shen Shun; Pang Zhiqing; Hu Yu (8669-8675).
Targeted drug delivery to selected brain cell types is a crucial step in enhancing therapeutic effects while limiting side effects in non-target cells. Here we report on the development and evaluation of a new cascade targeting delivery system that employs PEG-PCL nanoparticles modified with both an angiopep-2 peptide and a EGFP-EGF1 protein for precise targeting of brain neuroglial cells. Angiopep-2 penetrates the blood–brain barrier and EGFP-EGF1 binds neuroglial cells, providing the system with two stages of targeting. In vitro studies demonstrated that both bEnd.3 cells and neuroglial cells had a higher uptake of angiopep-2 and EGFP-EGF1 conjugated nanoparticles (AENP) as compared to unmodified nanoparticles. Ex vivo imaging showed that AENP had higher accumulation in the brain over unmodified nanoparticles and EGFP-EGF1 modified nanoparticles. Fluorescent in situ hybridization of brain slides demonstrated that AENP co-localized with neuroglial cells. Transmission electron microscopy further showed that AENP could target and enter neuroglial cells. This newly developed cascade targeting delivery system that precisely targets neuroglial cells has great potential in the diagnosis and treatment of neuroglial related diseases. Replacing EGFP-EGF1 and angiopep-2 with other ligands may extend the utility of the system to diagnose and treat organ diseases beyond brain
Keywords: Cascade targeting strategy; Brain neuroglial cells; EGFP-EGF1; Angiopep-2; Nanoparticles;

Enhanced osseointegration of titanium implant through the local delivery of transcription factor SATB2 by S.G. Yan; J. Zhang; Q.S. Tu; J.H. Ye; E. Luo; M. Schuler; M.S. Kim; T. Griffin; J. Zhao; X.J. Duan; D.J. Cochran; D. Murray; P.S. Yang; J. Chen (8676-8683).
Titanium implants are widely used in dentistry and orthopedic surgery. Nevertheless, bone regeneration around the implant is a relatively slow process, after placement. This study assessed whether SATB2 can enhance osseointegration of a titanium implant. To determine the effect of SATB2 in implant integration, two different viruses encoding SATB2 (PBABE-Satb2 virus or RCAS-Satb2 virus) were locally administered to the bone defect prior to titanium implant placement in our established transgenic TVA mice. Seven and 21 days post implantation, the femurs were isolated for quantitative real-time RT-PCR, H&E staining, immunohistochemical (IHC) staining, and microcomputed tomography (microCT) analysis. Quantitative real-time RT-PCR results demonstrated that the in vivo overexpression of SATB2 enhanced expression levels of potent osteogenic transcription factors and bone matrix proteins. We also found that 21 days after implantation, there were no significant differences in the expression levels of SATB2, Osx, Runx2, COLI, OC, and BSP between the RCAS-Satb2 group and the RCAS group. Histological analysis showed that SATB2 overexpression significantly enhanced new bone formation and bone-to-implant contact after implantation. IHC staining analysis revealed that forced expression of SATB2 increased the number of BSP-positive cells surrounding the implant. MicroCT analysis demonstrated that in vivo overexpression of SATB2 significantly increased the density of the newly formed bone surrounding the implant. These results conclude that in vivo overexpression of SATB2 significantly accelerates osseointegration of titanium implants and SATB2 can serve as a potent molecule in promoting tissue regeneration.
Keywords: Implant; SATB2; Osseointegration; TVA mice;

We present a strategy to conjugate TGF-β1 into fibrin hydrogels to mimic the in vivo presentation of the growth factor in a 3D context. To this end, we engineered fusion proteins between TGF-β1 and a bi-functional peptide composed of a Factor XIII domain and a plasmin cleavage site. In another version the protease cleavage site was omitted to examine whether the growth factor that could not be released from the scaffold by cells had different effects on tissue constructs. The optimal insertion site which yielded correctly processed, functional protein was found between the latency associated peptide and mature TGF-β1 domains. In solution the fusion proteins exhibited similar biological activity as native TGF-β1 as evidenced by inhibition of cell proliferation and promoter activity assays. Immunoprecipitation experiments demonstrated that the fusion TGF-β1 protein bound to fibrinogen in a Factor XIII dependent manner and could be released from the peptide by the action of plasmin. In contrast to bolus delivery, immobilized TGF-β1 induced sustained signaling in fibrin-embedded cells for several days as evidenced by Smad2 phosphorylation. Prolonged pathway activation correlated with enhanced contractile function of vascular constructs prepared from hair follicle mesenchymal stem cells or bone marrow derived smooth muscle cells. Our results suggest that fibrin-immobilized TGF-β1 may be used to enhance the local microenvironment and improve the function of engineered tissues in vitro and potentially also after implantation in vivo where growth factor delivery faces overwhelming challenges.
Keywords: Biomimetic growth factor delivery; Peptide domains; 3D matrix for protein presentation; Vascular tissue engineering; Fibrin hydrogels; Stem cells;

Inflammatory responses to pulmonary application of PEI-based siRNA nanocarriers in mice by Andrea Beyerle; Andrea Braun; Atrayee Banerjee; Nuran Ercal; Oliver Eickelberg; Thomas H. Kissel; Tobias Stoeger (8694-8701).
Polymeric non-viral vector systems for pulmonary application of siRNA are promising carriers, but have failed to enter clinical trials because of safety and efficiency problems. Therefore, improving their transfection efficiency, as well as their toxicological profile, is the subject of intensive research efforts. Six different poly(ethylene imine) (PEI)-based nanocarriers, with hydrophilic and hydrophobic PEG modifications, were toxicologically evaluated for pulmonary application in mice. Nanocarriers were intratracheally instilled to determine their toxicological profile, with particular focus on the inflammatory response in the lungs. Nanocarriers from both groups caused an acute inflammatory response in the lungs, albeit with different resolution kinetics and cytotoxicity. Hydrophobic modifications caused a severe inflammatory response with increased epithelial barrier permeability, accompanied by an acute antioxidant response. Hydrophilic modifications, with high PEG-grafting degrees, induced less proinflammatory effects without depleting macrophages and disrupting the epithelial/endothelial barrier in the lungs, while showing only a minor oxidative stress response. For pulmonary applications, local proinflammatory effects should be optimized by further development of nanocarriers with highly grafted PEG–PEI-based carriers or Jeffamine-modified hydrophobic PEI modifications.
Keywords: PEI; siRNA; Toxicity; Lung inflammation; Oxidative stress; Epithelial permeability;

Computational design principles for bioactive dendrimer based constructs as antagonists of the TLR4-MD-2-LPS complex by Teresa Barata; Ian Teo; Sanjiv Lalwani; Eric Simanek; Mire Zloh; Sunil Shaunak (8702-8711).
The cell surface interaction between bacterial lipopolysaccharide (LPS), Toll-like receptor 4 (TLR4) and MD-2 is central to bacterial sepsis syndromes and wound healing. We have shown that a generation (G) 3.5 polyamidoamine (PAMAM) dendrimer that was partially glycosylated with glucosamine inhibits TLR4-MD-2-LPS induced inflammation in a rabbit model of tissue scaring. However, it was a mixture of closely related chemical species because of the polydispersity of the starting PAMAM dendrimer. Generation 2 triazine dendrimers with single chemical entity material status are available at low cost and at the kilogram scale. PAMAM dendrimer can be synthetically grafted onto this triazine core dendrimer to make new triazine-PAMAM hybrid dendrimers. This led us to examine whether molecular modelling methods could be used to identify the key structural design principles for a bioactive lead molecule that could be synthesized and biologically evaluated. We describe our computer aided molecular studies of several dendrimer based constructs and the key design principles identified. Our approach should be more broadly applicable to the biologically focused, rational and accelerated design of molecules for other TLR receptors. They could be useful for treating infectious, inflammatory and malignant diseases.
Keywords: Dendrimer; Triazine & polyamidoamine; TLR4-MD-2-LPS complex; Cytokines;

Effects of chitosan-nanoparticle-mediated tight junction opening on the oral absorption of endotoxins by Kiran Sonaje; Kun-Ju Lin; Michael T. Tseng; Shiaw-Pyng Wey; Fang-Yi Su; Er-Yuan Chuang; Chia-Wei Hsu; Chiung-Tong Chen; Hsing-Wen Sung (8712-8721).
Recently, we reported a pH-responsive nanoparticle (NP) system shelled with chitosan (CS), which could effectively increase the oral absorption of insulin and produce a hypoglycemic effect, presumably due to the CS-mediated tight junction (TJ) opening. It has been often questioned whether CS can also enhance the absorption of endotoxins present in the small intestine. To address this concern, we studied the effect of CS NPs on the absorption of lipopolysaccharide (LPS), the most commonly found toxin in the gastrointestinal tract. To follow their biodistribution by the single-photon emission computed tomography/computed tomography, LPS and insulin were labeled with 99mTc-pertechnetate (99mTc-LPS) and 123iodine (123I-insulin), respectively. The 99mTc-LPS was ingested 1 h prior to the administration of the 123I-insulin-loaded NPs to mimic the physiological conditions. The confocal and TEM micrographs show that the orally administered CS NPs were able to adhere and infiltrate through the mucus layer, approach the epithelial cells and mediate to open their TJs. The radioactivity associated with LPS was mainly restricted to the gastrointestinal tract, whereas 123I-insulin started to appear in the urinary bladder at 3 h post administration. This observation indicates that the insulin-loaded in CS NPs can traverse across the intestinal epithelium and enter the systemic circulation, whereas LPS was unable to do so, probably because of the charge repulsion between the anionic LPS in the form of micelles and the negatively charged mucus layer. Our in vivo toxicity study further confirms that the enhancement of paracellular permeation by CS NPs did not promote the absorption of LPS. These results suggest that CS NPs can be used as a safe carrier for oral delivery of protein drugs.
Keywords: Chitosan; Tight junction; Endotoxin; Insulin; Paracellular transport;

Target specific hyaluronic acid–interferon alpha conjugate for the treatment of hepatitis C virus infection by Jeong-A. Yang; Kitae Park; Hyuntae Jung; Hyemin Kim; Sung Woo Hong; Seung Kew Yoon; Sei Kwang Hahn (8722-8729).
Interferon alpha (IFNα) conjugated with polyethylene glycol (PEG) has been widely used for the treatment of hepatitis C virus (HCV) infection as a once-a-week injection formulation. However, the PEGylated IFNα has a low efficacy of ca. 39% and a side effect after repeated injections possibly due to the non-specific delivery with PEGylation. In this work, target specific long-acting hyaluronic acid–interferon alpha (HA–IFNα) conjugate was successfully developed for the treatment of HCV infection. HA–IFNα conjugate was synthesized by coupling reaction between aldehyde modified HA and the N-terminal group of IFNα. The IFNα content could be controlled in the range of 2–9 molecules per single HA chain with a bioconjugation efficiency higher than 95%. According to in vitro anti-proliferation assay using Daudi cells, HA–IFNα conjugate showed a comparable biological activity to PEG-Intron. In vivo real-time bioimaging confirmed the target specific delivery of near-infrared fluorescence (NIRF) dye labeled HA–IFNα conjugate to the liver in mice. In addition, pharmacokinetic analysis revealed the enhanced residence time longer than 4 days. After tail-vein injection, HA–IFNα conjugate induced ca. 60% higher expression of 2′,5′-oligoadenylate synthetase 1 (OAS 1) for innate immune responses to viral infection in the murine liver tissues than IFNα and PEG-Intron.
Keywords: Hyaluronic acid; Interferon alpha; Conjugate; Targeted delivery; Hepatitis C virus;

Structural contributions of blocked or grafted poly(2-dimethylaminoethyl methacrylate) on PEGylated polycaprolactone nanoparticles in siRNA delivery by Daoshu Lin; Yuanyu Huang; Qian Jiang; Wendi Zhang; Xinye Yue; Shutao Guo; Ping Xiao; Quan Du; Jinfeng Xing; Liandong Deng; Zicai Liang; Anjie Dong (8730-8742).
The multiformity in polymer structure and conformation design provides a great potential in improving the gene silencing efficiency of siRNA by polymer vectors. In order to provide information on the polymer design for siRNA delivery, the structural contributions of blocked or grafted poly(2-dimethylaminoethyl methacrylate) on PEGylated polycaprolactone nanoparticles (NPs) in siRNA delivery were studied. Herein, two kinds of self-assembly nanoparticles (NPs) formed by amphiphilic cationic polymers, methoxy poly(ethylene glycol)-block-polycaprolactone-block-poly(2-dimethylaminoethyl methacrylate) (mPEG-PCL-b-PDMAEMA, PECbD) and methoxy poly(ethylene glycol)-block-(polycaprolactone-graft-poly(2-dimethylaminoethyl methacrylate)) (mPEG-PCL-g-PDMAEMA, PECgD), were used to deliver siRNA for in vitro and in vivo studies. The physiochemical properties including size and zeta potential of PECbD NPs/siRNA and PECgD NPs/siRNA complexes were characterized. In vitro cytotoxicity, cellular uptake and siRNA knockdown efficiency were evaluated in HeLa-Luc cells. The endosome escape and intracellular distribution of PECbD NPs/siRNA and PECgD NPs/siRNA in HeLa-Luc cells were also observed. In vivo polymer mediated siRNA delivery and the complexes distribution in isolated organs were studied using mice and tumor-bearing mice. At the same total degree of polymerization (DP) of DMAEMA, PECgD NPs/siRNA complexes possessed higher zeta potentials than PECbD NPs/siRNA complexes (at the same N/P ratio), which may be the reason that PECgD NPs/siRNA complexes can deliver more siRNA into the cytoplasm and lead to higher in vitro luciferase and lamin A/C silencing efficiency than PECbD NPs/siRNA complexes. The in vivo imaging measurement and histochemical analysis also confirmed that siRNA could be delivered to lungs, livers, pancreas and HeLa-Luc tumors more efficiently by PECgD NPs than PECbD NPs. Meanwhile, the PDMAEMA chains of PECgD could be shortened which provides benefits for clearing. Therefore, PECgD NPs have great potential to be used as efficient non-viral carriers for in vivo siRNA delivery.
Keywords: Non-viral carriers; PDMAEMA; PEG; siRNA delivery; Cationic polymer nanoparticles;