Biomaterials (v.32, #13)

A self-assembling hydrophobically modified chitosan capable of reversible hemostatic action by Matthew B. Dowling; Rakesh Kumar; Mark A. Keibler; John R. Hess; Grant V. Bochicchio; Srinivasa R. Raghavan (3351-3357).
Blood loss at the site of a wound in mammals is curtailed by the rapid formation of a hemostatic plug, i.e., a self-assembled network of the protein, fibrin that locally transforms liquid blood into a gelled clot. Here, we report an amphiphilic biopolymer that exhibits a similar ability to rapidly gel blood; moreover, the self-assembly underlying the gelation readily allows for reversibility back into the liquid state via introduction of a sugar-based supramolecule. The biopolymer is a hydrophobically modified (hm) derivative of the polysaccharide, chitosan. When hm-chitosan is contacted with heparinized human blood, it rapidly transforms the liquid into an elastic gel. In contrast, the native chitosan (without hydrophobes) does not gel blood. Gelation occurs because the hydrophobes on hm-chitosan insert into the membranes of blood cells and thereby connect the cells into a sample-spanning network. Gelation is reversed by the addition of α-cyclodextrin, a supramolecule having an inner hydrophobic pocket: polymer hydrophobes unbind from blood cells and embed within the cyclodextrins, thereby disrupting the cell network. We believe that hm-chitosan has the potential to serve as an effective, yet low-cost hemostatic dressing for use by trauma centers and the military. Preliminary tests with small and large animal injury models show its increased efficacy at achieving hemostasis – e.g., a 90% reduction in bleeding time over controls for femoral vein transections in a rat model.
Keywords: Chitosan; Hemostasis; Self-assembly; Cyclodextrin; Amphiphilic biopolymer;

Transparent, tough collagen laminates prepared by oriented flow casting, multi-cyclic vitrification and chemical cross-linking by Yuji Tanaka; Koichi Baba; Thomas J. Duncan; Akira Kubota; Toru Asahi; Andrew J. Quantock; Masayuki Yamato; Teruo Okano; Kohji Nishida (3358-3366).
The lamellar architecture found in many natural fibrous tissues has a significant bearing on their specific functions. However, current engineered tissues have simultaneously no realistic structures and no adequate functions. This study demonstrates a two-step process for obtaining structurally mimicking laminates in natural fibrous tissues with good optical and mechanical characters from purified-clinically-safe collagen molecules. Stacked lamella structures can be created by repeating flow casting, with the controlling parallel/orthogonal directionalities of each thin single-layer (2–5 μm in thickness). The transparency of laminates is successfully improved by a unique multi-cyclic vitrification with chemical cross-linking. The directionalities of optical and mechanical functions in laminates are strongly related with the preferential collagen alignments in the laminates. The tensile strength of laminates is extremely higher than any other engineered materials as well as native cornea, which exhibit an orthogonal laminated collagen structure and a good optical transmission.
Keywords: Collagen structure; Biomimetic material; Fibrous tissue; Biofilm; Soft tissue biomechanics; Cornea;

Failure of Aβ(1-40) amyloid fibrils under tensile loading by Raffaella Paparcone; Markus J. Buehler (3367-3374).
Amyloid fibrils and plaques are detected in the brain tissue of patients affected by Alzheimer’s disease, but have also been found as part of normal physiological processes such as bacterial adhesion. Due to their highly organized structures, amyloid proteins have also been used for the development of nanomaterials, for a variety of applications including biomaterials for tissue engineering, nanolectronics, or optical devices. Past research on amyloid fibrils resulted in advances in identifying their mechanical properties, revealing a remarkable stiffness. However, the failure mechanism under tensile loading has not been elucidated yet, despite its importance for the understanding of key mechanical properties of amyloid fibrils and plaques as well as the growth and aggregation of amyloids into long fibers and plaques. Here we report a molecular level analysis of failure of amyloids under uniaxial tensile loading. Our molecular modeling results demonstrate that amyloid fibrils are extremely stiff with a Young’s modulus in the range of 18–30 GPa, in good agreement with previous experimental and computational findings. The most important contribution of our study is our finding that amyloid fibrils fail at relatively small strains of 2.5%–4%, and at stress levels in the range of 1.02 to 0.64 GPa, in good agreement with experimental findings. Notably, we find that the strength properties of amyloid fibrils are extremely length dependent, and that longer amyloid fibrils show drastically smaller failure strains and failure stresses. As a result, longer fibrils in excess of hundreds of nanometers to micrometers have a greatly enhanced propensity towards spontaneous fragmentation and failure. We use a combination of simulation results and simple theoretical models to define critical fibril lengths where distinct failure mechanisms dominate.
Keywords: Amyloid fibril; Mechanical properties; Failure; Length scale; Elasticity; Nanomechanics;

Keratin films for ocular surface reconstruction by Stephan Reichl; Maria Borrelli; Gerd Geerling (3375-3386).
Human amniotic membrane (AM) is frequently used as a substrate for ocular surface reconstruction. Its disadvantages (e.g., reduced transparency and biomechanical strength, heterogeneity depending on donor) create the need for standardized alternatives. Keratin from hair or wool has been proposed as an appropriate material for producing films or cell cultivation scaffolds. The current study was performed to develop transparent, stable and transferable films based on human hair keratin that support cellular adhesion and proliferation. The films were engineered by a multi-step procedure including keratin extraction, neutral and alkaline dialysis, drying and a curing process. Keratin films were investigated by SDS-PAGE, SEM and X-ray analyses. Furthermore, swelling and water absorption of the films were studied, as were tensile strength and light transmission (UV/VIS). Finally, the growth behavior of corneal epithelial cells on the keratin films and AM was estimated in proliferation studies. In addition, we assessed the seeding efficiency and cell detachment behavior during trypsinization. The film-forming process resulted in transparent films composed of nanoparticulate keratin structures. The film characteristics could be varied by changing the protein composition, adding softening agents or varying the curing temperature and duration. Based on these findings, an optimized protocol was developed. The films showed improved light transmission and biomechanical strength in comparison to AM. Furthermore, cell behavior on the films was similar to that found on AM. We conclude that keratin films may represent a new, promising alternative for ocular surface reconstruction.
Keywords: Hair keratin; Film; Amniotic membrane; Ocular surface; Cell culture;

Mechanical properties and in vivo behavior of a biodegradable synthetic polymer microfiber–extracellular matrix hydrogel biohybrid scaffold by Yi Hong; Alexander Huber; Keisuke Takanari; Nicholas J. Amoroso; Ryotaro Hashizume; Stephen F. Badylak; William R. Wagner (3387-3394).
A biohybrid composite consisting of extracellular matrix (ECM) gel from porcine dermal tissue and biodegradable elastomeric fibers was generated and evaluated for soft tissue applications. ECM gel possesses attractive biocompatibility and bioactivity with weak mechanical properties and rapid degradation, while electrospun biodegradable poly(ester urethane)urea (PEUU) has good mechanical properties but limited cellular infiltration and tissue integration. A concurrent gel electrospray/polymer electrospinning method was employed to create ECM gel/PEUU fiber composites with attractive mechanical properties, including high flexibility and strength. Electron microscopy revealed a structure of interconnected fibrous layers embedded in ECM gel. Tensile mechanical properties could be tuned by altering the PEUU/ECM weight ratio. Scaffold tensile strengths for PEUU/ECM ratios of 67/33, 72/28 and 80/20 ranged from 80 to 187 kPa in the longitudinal axis (parallel to the collecting mandrel axis) and 41–91 kPa in the circumferential axis with 645–938% breaking strains. The 72/28 biohybrid composite and a control scaffold generated from electrospun PEUU alone were implanted into Lewis rats, replacing a full-thickness abdominal wall defect. At 4 wk, no infection or herniation was found at the implant site. Histological staining showed extensive cellular infiltration into the biohybrid scaffold with the newly developed tissue well integrated with the native periphery, while minimal cellular ingress into the electrospun PEUU scaffold was observed. Mechanical testing of explanted constructs showed evidence of substantial remodeling, with composite scaffolds adopting properties more comparable to the native abdominal wall. The described elastic biohybrid material imparts features of ECM gel bioactivity with PEUU strength and handling to provide a promising composite biomaterial for soft tissue repair and replacement.
Keywords: Electrospinning; Electrospray; Polyurethane; Extracellular matrix; Hydrogel;

The effects of combined micron-/submicron-scale surface roughness and nanoscale features on cell proliferation and differentiation by Rolando A. Gittens; Taylor McLachlan; Rene Olivares-Navarrete; Ye Cai; Simon Berner; Rina Tannenbaum; Zvi Schwartz; Kenneth H. Sandhage; Barbara D. Boyan (3395-3403).
Titanium (Ti) osseointegration is critical for the success of dental and orthopedic implants. Previous studies have shown that surface roughness at the micro- and submicro-scales promotes osseointegration by enhancing osteoblast differentiation and local factor production. Only relatively recently have the effects of nanoscale roughness on cell response been considered. The aim of the present study was to develop a simple and scalable surface modification treatment that introduces nanoscale features to the surfaces of Ti substrates without greatly affecting other surface features, and to determine the effects of such superimposed nano-features on the differentiation and local factor production of osteoblasts. A simple oxidation treatment was developed for generating controlled nanoscale topographies on Ti surfaces, while retaining the starting micro-/submicro-scale roughness. Such nano-modified surfaces also possessed similar elemental compositions, and exhibited similar contact angles, as the original surfaces, but possessed a different surface crystal structure. MG63 cells were seeded on machined (PT), nano-modified PT (NMPT), sandblasted/acid-etched (SLA), and nano-modified SLA (NMSLA) Ti disks. The results suggested that the introduction of such nanoscale structures in combination with micro-/submicro-scale roughness improves osteoblast differentiation and local factor production, which, in turn, indicates the potential for improved implant osseointegration in vivo.
Keywords: Nanotopography; Titanium oxide; Surface roughness; Bone; Implant; Osteoblasts;

Cell affinity for bFGF immobilized heparin-containing poly(lactide-co-glycolide) scaffolds by Hong Shen; Xixue Hu; Fei Yang; Jianzhong Bei; Shenguo Wang (3404-3412).
In order to effectively and uniformly immobilize basic fibroblast growth factor (bFGF) to thick PLGA scaffold, the heparin-conjugated PLGA (H-PLGA) was synthesized at the first by reaction between heparin and a low molecular weight PLGA. Then heparin-containing PLGA (H-PLGA/PLGA) scaffold was fabricated by blending the H-PLGA with a high molecular weight PLGA. Finally, bFGF was immobilized on the H-PLGA/PLGA scaffold mainly by static electricity action between them. The effect of H-PLGA content on bFGF binding efficiency of the H-PLGA/PLGA scaffolds was investigated. It was found that bFGF binding efficiency increased with increasing H-PLGA content. The bound bFGF can release in vitro slowly from the H-PLGA/PLGA scaffolds and last over two weeks. The released bFGF has still preserved its bioactivity. The attachment and growth of mouse 3T3 fibroblasts on the H-PLGA/PLGA scaffolds were better than that on the PLGA scaffold, however bFGF immobilized H-PLGA/PLGA scaffolds showed much better cell affinity. Therefore, the method to use the H-PLGA/PLGA scaffold for immobilizing bFGF is not only effective for slow delivering bFGF with bioactivity, but also can be used for fabricating thick scaffold where bFGF could be combined and uniformly distributed.
Keywords: bFGF; Immobilization; Slow release; Heparin; PLGA; Scaffold;

Engineering spatial control of multiple differentiation fates within a stem cell population by Elmer D.F. Ker; Bur Chu; Julie A. Phillippi; Burhan Gharaibeh; Johnny Huard; Lee E. Weiss; Phil G. Campbell (3413-3422).
The capability to engineer microenvironmental cues to direct a stem cell population toward multiple fates, simultaneously, in spatially defined regions is important for understanding the maintenance and repair of multi-tissue units. We have previously developed an inkjet-based bioprinter to create patterns of solid-phase growth factors (GFs) immobilized to an extracellular matrix (ECM) substrate, and applied this approach to drive muscle-derived stem cells toward osteoblasts ‘on-pattern’ and myocytes ‘off-pattern’ simultaneously. Here this technology is extended to spatially control osteoblast, tenocyte and myocyte differentiation simultaneously. Utilizing immunofluorescence staining to identify tendon-promoting GFs, fibroblast growth factor-2 (FGF-2) was shown to upregulate the tendon marker Scleraxis (Scx) in C3H10T1/2 mesenchymal fibroblasts, C2C12 myoblasts and primary muscle-derived stem cells, while downregulating the myofibroblast marker α-smooth muscle actin (α-SMA). Quantitative PCR studies indicated that FGF-2 may direct stem cells toward a tendon fate via the Ets family members of transcription factors such as pea3 and erm. Neighboring patterns of FGF-2 and bone morphogenetic protein-2 (BMP-2) printed onto a single fibrin-coated coverslip upregulated Scx and the osteoblast marker ALP, respectively, while non-printed regions showed spontaneous myotube differentiation. This work illustrates spatial control of multi-phenotype differentiation and may have potential in the regeneration of multi-tissue units.
Keywords: Bone; BMP (bone morphogenetic protein); ECM (extracellular matrix); Fibroblast growth factor; Muscle; Tendon;

The performance of laminin-containing cryogel scaffolds in neural tissue regeneration by Marcin Jurga; Maria B. Dainiak; Anna Sarnowska; Anna Jablonska; Anuj Tripathi; Fatima M. Plieva; Irina N. Savina; Lukasz Strojek; Hans Jungvid; Ashok Kumar; Barbara Lukomska; Krystyna Domanska-Janik; Nico Forraz; Colin P. McGuckin (3423-3434).
Currently, there are no effective therapies to restore lost brain neurons, although rapid progress in stem cell biology and biomaterials development provides new tools for regeneration of central nervous system. Here we describe neurogenic properties of bioactive scaffolds generated by cryogelation of dextran or gelatin linked to laminin – the main component of brain extracellular matrix. We showed that such scaffolds promoted differentiation of human cord blood-derived stem cells into artificial neural tissue in vitro. Our experiments revealed that optimal range of scaffolds’ pore size for neural tissue engineering was 80–100 microns. We found that scaffold seeded with undifferentiated, but not neutrally committed stem cells, gave optimal cell adhesion and proliferation in “niche”-like structures. Subsequent differentiation resulted in generation of mature 3D networks of neurons (MAP2+) and glia (S100beta+) cells. We showed that cryogel scaffolds could be transplanted into the brain tissue or organotypic hippocampal slices in a rat models. The scaffolds did not induced inflammation mediated by microglial cells (ED1-, Ox43-, Iba1-) and prevented formation of glial scar (GFAP-). Contrary, laminin-rich scaffolds attracted infiltration of host’s neuroblasts (NF200+, Nestin+) indicating high neuroregeneration properties.
Keywords: Bioactivity; Biocompatibility; Transplantation; Cross-linking; Nerve tissue engineering; Stem cell;

The effect of surface charge on in vivo biodistribution of PEG-oligocholic acid based micellar nanoparticles by Kai Xiao; Yuanpei Li; Juntao Luo; Joyce S. Lee; Wenwu Xiao; Abby M. Gonik; Rinki G. Agarwal; Kit S. Lam (3435-3446).
To systematically elucidate the effect of surface charge on the cellular uptake and in vivo fate of PEG-oligocholic acid based micellar nanoparticles (NPs), the distal PEG termini of monomeric PEG-oligocholic acid dendrimers (telodendrimers) are each derivatized with different number (n = 0, 1, 3 and 6) of anionic aspartic acids (negative charge) or cationic lysines (positive charge). Under aqueous condition, these telodendrimers self-assemble to form a series of micellar NPs with various surface charges, but with similar particle sizes. NPs with high surface charge, either positive or negative, were taken up more efficiently by RAW 264.7 murine macrophages after opsonization in fresh mouse serum. Mechanistic studies of cellular uptake of NPs indicated that several distinct endocytic pathways (e.g., clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis) were involved in the cellular uptake process. After their cellular uptake, the majority of NPs were found to localize in the lysosome. Positively charged NPs exhibited dose-dependent hemolytic activities and cytotoxicities against RAW 264.7 cells proportional to the positive surface charge densities; whereas negatively charged NPs did not show obvious hemolytic and cytotoxic properties. In vivo biodistribution studies demonstrated that undesirable liver uptake was very high for highly positively or negatively charged NPs, which is likely due to active phagocytosis by macrophages (Kupffer cells) in the liver. In contrast, liver uptake was very low but tumor uptake was very high when the surface charge of NPs was slightly negative. Based on these studies, we can conclude that slightly negative charge may be introduced to the NPs surface to reduce the undesirable clearance by the reticuloendothelial system (RES) such as liver, improve the blood compatibility, thus deliver the anti-cancer drugs more efficiently to the tumor sites.
Keywords: Surface charge; Nanoparticles; Cellular uptake; Macrophage; Biodistribution; Drug delivery;

Photosensitizer-conjugated magnetic nanoparticles for in vivo simultaneous magnetofluorescent imaging and targeting therapy by Peng Huang; Zhiming Li; Jing Lin; Dapeng Yang; Guo Gao; Cheng Xu; Le Bao; Chunlei Zhang; Kan Wang; Hua Song; Hengyao Hu; Daxiang Cui (3447-3458).
A major challenge in nanotechnology and nanomedicine is to integrate tumor targeting, imaging, and selective therapy functions into a small single nanoparticle (<50 nm). Herein, photosensitizer-conjugated magnetic nanoparticles with ∼20 nm in diameter were strategically designed and prepared for gastric cancer imaging and therapy. The second generation photosensitizer chlorin e6 (Ce6) was covalently anchored on the surface of magnetic nanoparticles with silane coupling agent. We found that the covalently incorporated Ce6 molecules retained their spectroscopic and functional properties for near-infrared (NIR) fluorescence imaging and photodynamic therapy (PDT), and the core magnetic nanoparticles offered the functions of magnetically guided drug delivery and magnetic resonance imaging (MRI). The as-prepared single particle platform is suitable for simultaneous targeting PDT and in vivo dual-mode NIR fluorescence imaging and MRI of nude mice loaded with gastric cancer or other tumors.
Keywords: Magnetic nanoparticle; Photodynamic therapy; Magnetic resonance imaging; Near-infrared fluorescence imaging; Chlorin e6; Gastric cancer;

The promotion of siRNA delivery to breast cancer overexpressing epidermal growth factor receptor through anti-EGFR antibody conjugation by immunoliposomes by Jie Gao; Wei Liu; Yu Xia; Wei Li; Jing Sun; Huaiwen Chen; Bohua Li; Dapeng Zhang; Weizhu Qian; Yanchun Meng; Li Deng; Hao Wang; Jianming Chen; Yajun Guo (3459-3470).
The LPD (liposome-polycation-DNA complex) is an effective nanovector for systemically small interfering RNA (siRNA) delivery which was well characterized previously. However, little effort was spend on the development of targeted LPD conjugated with tumor specific antibody (TLPD) which would be potent in promoting siRNA delivery in tumor. Here, we prepared TLPD through a self-assembling process followed by anti-EGFR antibody conjugation. The effect of antibody type, conjugation strategy and amount on the physicochemical and biological properties of TLPD was investigated. We obtained optimized TLPD conjugated with anti-EGFR Fab’ by conventional conjugation (TLPD-FCC), which possessed a small size around 150 nm and superior in vitro stability. Compared with nontargeted LPD (NTLPD), TLPD-FCC showed significantly enhanced binding affinity and luciferase gene silencing activity in EGFR overexpressing MDA-MB-231 breast cancer cells in vitro. Moreover, the in vivo accumulation of TLPD-FCC was obviously higher than that of NTLPD in MDA-MB-231 tumor 24 h post intravenous injection. The promoted uptake of TLPD-FCC in MDA-MB-231 tumor was further confirmed by confocal microscopy. Notably, three intravenous injections of siRNA in TLPD-FCC significantly silenced luciferase expression by ∼20%, whereas NTLPD showed little effect. All these results suggested that our TLPD-FCC have a great potential in delivering siRNA to EGFR overexpressing breast cancers.
Keywords: Antibody; EGFR; Gene silencing; PEG; siRNA delivery;

Gene delivery system based on highly specific recognition of surface-vimentin with N-acetylglucosamine immobilized polyethylenimine by Sun-Jung Kim; Hirohiko Ise; Mitsuaki Goto; Kenta Komura; Chong-Su Cho; Toshihiro Akaike (3471-3480).
Gene and drug-delivery systems that use immobilization of carbohydrates are useful for the specific targeting of lectin-expressing tissues. Here, we report that N-acetylglucosamine (GlcNAc) with polyethylenimine (GlcNAc-PEI) specifically interacted with vimentin-expressing cells such as 293FT and HeLa cells. Recently, the intermediate filaments vimentin and desmin have been reported to have GlcNAc-binding lectin-like properties on the cell surface. Therefore, GlcNAc-conjugated agents can be targeted to vimentin- and desmin-expressing cells and tissues. Vimentin-expressing 293FT and HeLa cells were efficiently transfected with green fluorescent protein and luciferase genes by using GlcNAc-PEI; the expression of these genes in vimentin-knockdown cells were low. Confocal microscopic analysis showed that GlcNAc-PEI complexes interacted with vimentin on the cell surface of HeLa cells. These results demonstrate that GlcNAc-PEI/DNA complexes were specifically taken up by 293FT and HeLa cells via vimentin. We suggest that this gene-delivery system could be used to target various vimentin-expressing cells such as fibroblasts and tumor cells.
Keywords: N-Acetylglucosamine; Vimentin; Polyethlylenimine; Gene delivery;

Co-encapsulation of magnetic nanoparticles and doxorubicin into biodegradable microcarriers for deep tissue targeting by vascular MRI navigation by Pierre Pouponneau; Jean-Christophe Leroux; Gilles Soulez; Louis Gaboury; Sylvain Martel (3481-3486).
Magnetic tumor targeting with external magnets is a promising method to increase the delivery of cytotoxic agents to tumor cells while reducing side effects. However, this approach suffers from intrinsic limitations, such as the inability to target areas within deep tissues, due mainly to a strong decrease of the magnetic field magnitude away from the magnets. Magnetic resonance navigation (MRN) involving the endovascular steering of therapeutic magnetic microcarriers (TMMC) represents a clinically viable alternative to reach deep tissues. MRN is achieved with an upgraded magnetic resonance imaging (MRI) scanner. In this proof-of-concept preclinical study, the preparation and steering of TMMC which were designed by taking into consideration the constraints of MRN and liver chemoembolization are reported. TMMC were biodegradable microparticles loaded with iron-cobalt nanoparticles and doxorubicin (DOX). These particles displayed high saturation magnetization (Ms = 72 emu g−1), MRI tracking compatibility (strong contrast on T2∗-weighted images), appropriate size for the blood vessel embolization (∼50 μm), and sustained release of DOX (over several days). The TMMC were successfully steered in vitro and in vivo in the rabbit model. In vivo targeting of the right or left liver lobes was achieved by MRN through the hepatic artery located 4 cm beneath the skin. Parameters such as flow velocity, TMMC release site in the artery, magnetic gradient and TMMC properties, affected the steering efficiency. These data illustrate the potential of MRN to improve drug targeting in deep tissues.
Keywords: Magnetism; Nanoparticle; Microencapsulation; MRI (magnetic resonance imaging); Drug delivery; Liver;

Tunable physiologic interactions of adhesion molecules for inflamed cell-selective drug delivery by Sungkwon Kang; Taehyun Park; Xiaoyue Chen; Greg Dickens; Brian Lee; Kevin Lu; Nikolai Rakhilin; Susan Daniel; Moonsoo M. Jin (3487-3498).
Dysregulated inflammation contributes to the pathogenesis of various diseases. Therapeutic efficacy of anti-inflammatory agents, however, falls short against resilient inflammatory responses, whereas long-term and high-dose systemic administration can cause adverse side effects. Site-directed drug delivery systems would thus render more effective and safer treatments by increasing local dosage and minimizing toxicity. Nonetheless, achieving clinically effective targeted delivery to inflammatory sites has been difficult due to diverse cellular players involved in immunity and endogenous targets being expressed at basal levels. Here we exploit a physiological molecular interaction between intercellular adhesion molecule (ICAM)-1 and lymphocyte function associated antigen (LFA)-1 to deliver a potent anti-inflammatory drug, celastrol, specifically and comprehensively to inflamed cells. We found that affinity and avidity adjusted inserted (I) domain, the major binding site of LFA-1, on liposome surface enhanced the specificity toward lipopolysaccharides (LPS)-treated or inflamed endothelial cells (HMEC-1) and monocytes (THP-1) via ICAM-1 overexpression, reflecting inherent affinity and avidity modulation of these molecules in physiology. Targeted delivery of celastrol protected cells from recurring LPS challenges, suppressing pro-inflammatory responses and inflammation-induced cell proliferation. Targeted delivery also blocked THP-1 adhesion to inflamed HMEC-1, forming barriers to immune cell accumulation and to aggravating inflammatory signals. Our results demonstrate affinity and avidity of targeting moieties on nanoparticles as important design parameters to ensure specificity and avoid toxicities. We anticipate that such tunable physiologic interactions could be used for designing effective drug carriers for in vivo applications and contribute to treating a range of immune and inflammatory diseases.
Keywords: Integrin; ICAM-1; Avidity; Celastrol; Inflammation; Drug delivery;

Hierarchical nanoengineered surfaces for enhanced cytoadhesion and drug delivery by Kathleen E. Fischer; Ganesh Nagaraj; R. Hugh Daniels; Esther Li; Verne E. Cowles; Jennifer L. Miller; Mark D. Bunger; Tejal A. Desai (3499-3506).
Delivering therapeutics to mucosal tissues such as the nasal and gastrointestinal tracts is highly desirable due to ease of access and dense vasculature. However, the mucus layer effectively captures and removes most therapeutic macromolecules and devices. In previous work, we have shown that nanoengineered microparticles (NEMPs) adhere through the mucus layer, exhibiting up to 1000 times the pull-off force of an unmodified microsphere, and showing greater adhesion than some chemical targeting means. In this paper, we demonstrate that nanotopography improves device adhesion in vivo, increasing retention time up to ten-fold over unmodified devices. Moreover, we observe considerable adhesion in several cell lines using an in vitro shear flow model, indicating that this approach is promising for numerous tissues. We then demonstrate that nanowire-mediated adhesion is highly robust to variation in nanowire surface charge and cellular structure and function, and we characterize particle loading and elution. We present a form of cytoadhesion that utilizes the physical interaction of nanoengineered surfaces with subcellular structures to produce a robust and versatile cytoadhesive for drug delivery. These nanoscale adhesive mechanisms are also relevant to fields such as tissue engineering and wound healing because they likely affect stem cell differentiation, cell remodeling, migration, etc.
Keywords: Actin; Adhesion; Cell adhesion; Drug delivery; Nanotopography; Surface Topography;

The intracellular plasmid DNA localization of cationic reducible cholesterol-disulfide lipids by Ruilong Sheng; Ting Luo; Yingdan Zhu; Hui Li; Jingjing Sun; Shengdian Chen; Wenyan Sun; Amin Cao (3507-3519).
Stimuli-responsive biomaterials derived from natural products toward efficient drug/gene delivery have been attracting increasing attention in the past decade. In this work, we first designed and prepared a new series of cholesterol-disulfide lipids, namely CHOSS-N, CHOSS-N+, CHOSS-Lys and CHOSS-4N bearing cholesterol and a variety of headgroups via disulfide and carbonate bond linkages, and their molecular structures were characterized by NMR and ESI-MS. Furthermore, plasmid DNA binding affinity for these new CHOSS lipids was separately examined by ethidium bromide displacement and agarose-gel retardant assay. Average diameter sizes and surface potentials of the CHOSS/pDNA lipoplex particles prepared under various N/P charge ratios were analyzed by dynamic laser light scattering (DLS). Under 10 mm dithiothreitol (DTT), stability and disassembly of the CHOSS/pDNA lipoplex nanoparticles were investigated by agarose-gel retardant assay and atomic force microscopy (AFM). Employing a COS-7 cell line, cell viability was examined for the prepared CHOSS lipids and their pDNA lipoplexes with branched PEI-25k as the reference. Finally, COS-7 cell gene transfection efficacies with these CHOSS lipids as potential delivery vectors were investigated by luciferase and EGFP transfection assay in the absence and presence of serum, and intracellular uptake capability, trafficking and cellular localization of Cy3-labeled pEGFP-N1 DNA were studied with a flow cytometer and fluorescent microscopy with Lipofectamine™ 2000 as the control. The results demonstrated low cytotoxicity, strong pDNA binding affinity and high transgenetic efficacy for new prepared CHOSS lipids, and particularly high intracellular uptake capability and specific cellular localization of pDNA at the periphery of cell nuclei were for the first time interestingly observed for the CHOSS lipid delivery carriers. In general, these may pave a new way to utilize cholesterol, amino acids and other functional natural products to prepare efficient gene/drug delivery carriers with simple structure and low cytotoxicity.Display Omitted
Keywords: Gene delivery; Lipids; Cholesterol; Disulfide; Localization;

Functional peptides that transfer biomaterials, such as semiconductor quantum dots (QDs), into cells in biomaterial research have been developed in recent years. Delivery of QDs conjugated with cell-penetrating peptides (CPPs) into cells by the endocytic pathway was problematic in biomedical applications because of lysosomal trapping. Here, we demonstrate that histidine- and arginine-rich CPPs (HR9 peptides) stably and noncovalently combined with QDs are able to enter into cells in an extremely short period (4 min). Interrupting both F-actin polymerization and active transport did not inhibit the entry of HR9/QD complexes into cells, indicating that HR9 penetrates cell membrane directly. Subcellular colocalization studies indicated that QDs delivered by HR9 stay in cytosol without any organelle capture. Dimethyl sulphoxide, ethanol and oleic acid, but not pyrenebutyrate, enhanced HR9-mediated intracellular delivery of QDs by promoting the direct membrane translocation pathway. HR9 and HR9/QDs were not cytotoxic. These findings suggest that HR9 could be an efficient carrier to deliver drugs without interfering with their therapeutic activity.
Keywords: Cell-penetrating peptides (CPPs); Cellular internalization; Protein transduction domains (PTDs); Quantum dots (QDs); Semiconductor;

Improved biological half-life and anti-tumor activity of TNF-related apoptosis-inducing ligand (TRAIL) using PEG-exposed nanoparticles by Sung Mook Lim; Tae Hyung Kim; Hai Hua Jiang; Chan Woong Park; Seulki Lee; Xiaoyuan Chen; Kang Choon Lee (3538-3546).
TRAIL has received considerable attention as a potential anti-cancer agent due to its specific ability to target tumors. However, recombinant TRAIL has several limitations, such as, its short biological half-life, its inherent instability, and its potential hepatotoxicity. In this study, we developed a sustained release nanoparticle formulation of TRAIL and investigated its therapeutic effects in tumor-bearing mice. TRAIL-loaded nanoparticles (NPs) were prepared by mixing PEGylated heparin (PEG-HE), poly-l-lysine (PLL), and TRAIL. NPs prepared by the ionic interaction between polymer and TRAIL showed uniform spherical structures of diameter 213.3 ± 9.7 nm and a surface charge of 5.33 ± 1.2 mV. An in vitro study of the bioactivity of TRAIL in NPs showed that TRAIL-loaded PEG-HE/PLL NPs (TRAIL-PEG-NPs) were slightly less cytotoxic than TRAIL in vitro. To investigate pharmacokinetic parameters, TRAIL and TRAIL-PEG-NPs were intravenously injected into SD rats. The PEG-NP-based formulation demonstrated a 28.3 fold greater half-life than TRAIL alone. To evaluate the anti-tumor effect, TRAIL, TRAIL-loaded HE/PLL NPs (TRAIL-NPs), and TRAIL-PEG-NPs were intravenously injected into HCT-116 tumor-bearing BALB/c athymic mice. The TRAIL-PEG-NP formulation efficiently suppressed tumor growth (>70%), and histological findings confirmed that NPs induced significant tumor cell apoptosis without inducing liver toxicity. The PEG-exposed NP fabrication method applied in this study could be widely applied to protein and peptide delivery systems.
Keywords: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL); Polyethylene glycol (PEG); Nanoparticle; protein/peptide drug delivery;