Biomaterials (v.31, #23)

Live cell-based sensor cells by Akiyoshi Taniguchi (5911-5915).
Biosensors utilizing live cells have attracted a great deal of attention because of their high specificity and sensitivity to their targets. We established cytotoxic sensor cells that were transfected with GFP plasmids derived from HSP70B′ and BTG2 promoters. These sensor cells have significant advantages in both speed and sensitivity for the detection of cytotoxicity. We created a live cell-based sensorchip fabricated with microfluidic channels. Fluorescence in the sensor cells increased in a CdCl2 dose-dependent manner in the microfluidic channels. With this system, cytotoxic reagents can be quantitatively detected in a quick, sensitive, and high-throughput manner. The combination of sensor cells and microfluidic systems will provide an important basis for the development of μ-TAS technology that can be applied to numerous fields such as biomaterials, nanotoxicology, environmental assessment, and drug screening.
Keywords: Cell signaling; Cell viability; Gene expression; Genetic engineering;

The development of electrically conductive polycaprolactone fumarate–polypyrrole composite materials for nerve regeneration by M. Brett Runge; Mahrokh Dadsetan; Jonas Baltrusaitis; Andrew M. Knight; Terry Ruesink; Eric A. Lazcano; Lichun Lu; Anthony J. Windebank; Michael J. Yaszemski (5916-5926).
Electrically conductive polymer composites composed of polycaprolactone fumarate and polypyrrole (PCLF–PPy) have been developed for nerve regeneration applications. Here we report the synthesis and characterization of PCLF–PPy and in vitro studies showing PCLF–PPy materials support both PC12 cell and dorsal root ganglia (DRG) neurite extension. PCLF–PPy composite materials were synthesized by polymerizing pyrrole in preformed PCLF scaffolds (Mn 7,000 or 18,000 g mol−1) resulting in interpenetrating networks of PCLF–PPy. Chemical compositions and thermal properties were characterized by ATR-FTIR, XPS, DSC, and TGA. PCLF–PPy materials were synthesized with five different anions (naphthalene-2-sulfonic acid sodium salt (NSA), dodecylbenzenesulfonic acid sodium salt (DBSA), dioctyl sulfosuccinate sodium salt (DOSS), potassium iodide (I), and lysine) to investigate effects on electrical conductivity and to optimize chemical composition for cellular compatibility. PCLF–PPy materials have variable electrical conductivity up to 6 mS cm−1 with bulk compositions ranging from 5 to 13.5 percent polypyrrole. AFM and SEM characterization show microstructures with a root mean squared (RMS) roughness of 1195 nm and nanostructures with RMS roughness of 8 nm. In vitro studies using PC12 cells and DRG show PCLF–PPy materials synthesized with NSA or DBSA support cell attachment, proliferation, neurite extension, and are promising materials for future studies involving electrical stimulation.
Keywords: Electrically conductive; Polypyrrole; Nerve; PCLF;

Modified polyelectrolyte complex fibrous scaffold as a matrix for 3D cell culture by Benjamin C.U. Tai; Andrew C.A. Wan; Jackie Y. Ying (5927-5935).
The paradigm of scaffold tissue engineering relies on the provision of an appropriate environment for cell growth, which includes both structural support and the presentation of cellular signals. In terms of biosignal presentation, fibrous scaffolds by interfacial polyelectrolyte complexation (IPC) offer a clear advantage over other scaffold types as IPC scaffolds are formed using an aqueous-based, room-temperature process compatible with the incorporation of biological molecules. This paper establishes two primary methods for the chemical and biochemical modification of these scaffolds: (i) physical entrapment of the bioactive component, and (ii) covalent binding of the bioactive component. For the first method, extracellular matrix (ECM) proteins, collagen, fibronectin and laminin were drawn into the IPC fiber. For the second method, the cell adhesion peptide, RGD, was chemically conjugated to a thiol-active maleimidylated form of the scaffold. Immobilization of the bioactive components was characterized by confocal fluorescence microscopy, scanning electron microscopy (SEM) and BCA protein assay. The ECM proteins were distributed throughout the bulk and surface of the fiber. The ratio of covalently bound to physisorbed RGD was ∼ 2:3. The performance of the various scaffolds as a matrix to maintain the differentiated function of primary hepatocytes showed that albumin levels in the supernatant were in the order of RGD-modified scaffold > collagen Type I-modified scaffold > fibronectin- or laminin-modified scaffold > unmodified scaffold > plate, while no clear trend in urea production could be discerned. Thus, IPC scaffolds offered a promising platform for the presentation of signals to cells, in this case, to influence their differentiated function.
Keywords: ECM (extracellular matrix); RGD peptide; Scaffold; Non-woven fabric; Hepatocyte; Cell culture;

Cultivating hepatocytes on printed arrays of HGF and BMP7 to characterize protective effects of these growth factors during in vitro alcohol injury by Caroline N. Jones; Nazgul Tuleuova; Ji Youn Lee; Erlan Ramanculov; A. Hari Reddi; Mark A. Zern; Alexander Revzin (5936-5944).
The goal of the present study was to investigate hepato-protective effects of growth factor (GF) arrays during alcohol injury. Hepatocyte growth factor (HGF) and bone morphogenetic protein (BMP)7 were mixed with collagen (I) and robotically printed onto standard glass slides to create arrays of 500 μm diameter spots. Primary rat hepatocytes were seeded on top of the arrays forming clusters corresponding in size to the underlying protein spots. Cell arrays were then injured in culture by exposure to 100 mm ethanol for 48 h. Hepatocytes residing on GF spots were found to have less apoptosis then cells cultured on collagen-only spots. Least apoptosis (0.3% as estimated by TUNEL assay) was observed on HGF/BMP7/collagen spots whereas most apoptosis (17.3%) was seen on collagen-only arrays. Interestingly, the extent of alcohol-induced apoptosis in hepatocytes varied based on the concentration of printed GF. In addition to preventing apoptosis, printed GFs contributed to maintenance of epithelial phenotype during alcohol injury as evidenced by higher levels of E-cadherin expression in HGF-protected hepatocytes. Importantly, GF microarrays could be used to investigate heterotypic interactions in the context of liver injury. To highlight this, stellate cells – nonparenchymal liver cells involved in fibrosis – were added to hepatocytes residing on arrays of either HGF/collagen or collagen-only spots. Exposure of these cocultures to ethanol followed by RT-PCR analysis revealed that stellate cells residing alongside HGF-protected hepatocytes were significantly less activated (less fibrotic) compared to controls. Overall, our results demonstrate that GF microarray format can be used to screen anti-fibrotic and anti-apoptotic effects of growth factors as well as to investigate how signals delivered to a specific cell type modulate heterotypic cellular interactions.
Keywords: Protein microarrays; Micropatterned cocultures; Hepatocytes; Growth factor microarrays; Liver injury;

Biomimetic hybrid scaffolds for engineering human tooth-ligament interfaces by Chan Ho Park; Hector F. Rios; Qiming Jin; Megan E. Bland; Colleen L. Flanagan; Scott J. Hollister; William V. Giannobile (5945-5952).
A major clinical challenge in the reconstruction of large oral and craniofacial defects is the neogenesis of osseous and ligamentous interfacial structures. Currently, oral regenerative medicine strategies are unpredictable for repair of tooth-supporting tissues destroyed as a consequence of trauma, chronic infection or surgical resection. Here, we demonstrate multi-scale computational design and fabrication of composite hybrid polymeric scaffolds for targeted cell transplantation of genetically modified human cells for the formation of human tooth dentin-ligament-bone complexes in vivo. The newly-formed tissues demonstrate the interfacial generation of parallel- and obliquely-oriented fibers that grow and traverse within the polycaprolactone (PCL)-poly(glycolic acid) (PGA) designed constructs forming tooth cementum-like tissue, ligament, and bone structures. This approach offers potential for the clinical implementation of customized periodontal scaffolds that may enable regeneration of multi-tissue interfaces required for oral, dental and craniofacial engineering applications.
Keywords: Tissue engineering; Rapid prototyping; Periodontal disease; Interfacial tissue formation; Regenerative medicine; Composite scaffolds;

A composite construct comprising of bone marrow mesenchymal stem cells (BMSCs), plasmid DNA encoding transforming growth factor-β1 (pDNA-TGF-β1), fibrin gel and poly (lactide-co-glycolide) (PLGA) sponge was designed and employed to repair articular cartilage defects. To improve the gene transfection efficiency, a cationized chitosan derivative N,N,N-trimethyl chitosan chloride (TMC) was employed as the vector. The TMC/DNA complexes had a transfection efficiency of 9% to BMSCs and showed heterogeneous TGF-β1 expression in a 10-day culture period in vitro. In vivo culture of the composite constructs was performed by implantation into full-thickness cartilage defects of New Zealand white rabbit joints, using the constructs absence of pDNA-TGF-β1 or BMSCs as controls. Heterogeneous expression of TGF-β1 in vivo was detected at 4 weeks, but its level was decreased in comparison with that of 2 weeks. After implantation for 12 weeks, the cartilage defects were successfully repaired by the composite constructs of the experimental group, and the neo-cartilage integrated well with its surrounding tissue and subchondral bone. Immunohistochemical and glycosaminoglycans (GAGs) staining confirmed the similar amount and distribution of collagen type II and GAGs in the regenerated cartilage as that of hyaline cartilage. The cartilage special genes expressed in the neo-tissue were closer to those of the normal cartilage. An overall score of 2.83 was obtained according to Wakitani’s standard. By contrast, only part of the defects was repaired by the pDNA-TGF-β1 absence constructs, and no cartilage repair but fibrous tissue was found for the BMSCs absence constructs. Therefore, combination of the PLGA sponge/fibrin gel scaffold with BMSCs and gene therapy is an effective method to restore cartilage defects and may have a great potential for practical applications in the near future.
Keywords: Gene therapy; Cartilage regeneration; BMSCs; PLGA sponge; Fibrin gel; TGF-β1;

The use of superporous Ac-CGGASIKVAVS-OH-modified PHEMA scaffolds to promote cell adhesion and the differentiation of human fetal neural precursors by Šárka Kubinová; Daniel Horák; Nataliya Kozubenko; Václav Vaněček; Vladimír Proks; Jack Price; Graham Cocks; Eva Syková (5966-5975).
Modifications of poly(2-hydroxyethyl methacrylate) (PHEMA) with laminin-derived Ac-CGGASIKVAVS-OH peptide sequences have been developed to construct scaffolds that promote cell adhesion and neural differentiation. Radical copolymerization of 2-hydroxyethyl methacrylate with 2-aminoethyl methacrylate (AEMA) and ethylene dimethacrylate in the presence of ammonium oxalate crystals resulted in the formation of superporous P(HEMA-AEMA) hydrogels. They were reacted with γ-thiobutyrolactone to yield 2-(4-sulfanylbutanamido)ethyl methacrylate (P(HEMA-AEMA)-SH) unit. The Ac-CGGASIKVAVS-OH peptide was immobilized to the sulfhydryl groups of P(HEMA-AEMA)-SH by 2,2′-dithiodipyridine linking reagent via 2-[4-(2-pyridyldisulfanyl)butanamido]ethyl methacrylate (P(HEMA-AEMA)-TPy). The adhesion and morphology of rat mesenchymal stem cells were investigated on the Ac-CGGASIKVAVS-OH-modified P(HEMA-AEMA) as well as on PHEMA, P(HEMA-AEMA)-SH and P(HEMA-AEMA)-TPy hydrogels. Superporous Ac-CGGASIKVAVS-OH-modified PHEMA scaffolds significantly increased the number of attached cells and their growth area on the hydrogel surface in the absence and in the presence of serum in the culture medium. Additionally, the Ac-CGGASIKVAVS-OH peptide supported the attachment, proliferation, differentiation and process spreading of human fetal neural stem cells during the first two weeks of expansion and contributed to the formation of a high percentage of more mature neural cells after four weeks of expansion. The Ac-CGGASIKVAVS-OH modification of superporous P(HEMA-AEMA) hydrogels improves cell adhesive properties and promotes neural stem cell differentiation.
Keywords: Cell adhesion; Laminin; Mesenchymal stem cell; Neural cell; PolyHEMA;

Effects of squalane-dissolved fullerene-C60 (Sql-fullerene) on macrophage activation and adipose conversion with oxidative stress were studied using an inflammatory adipose-tissue equivalent (ATE) and OP9 mouse stromal preadipocyte-U937 lymphoma cell co-culture systems. Differentiation of OP9 cells was initiated by insulin-rich serum replacement (SR) as an adipogenic stimulant, and then followed by accumulation of intracellular lipid droplets and reactive oxygen species (ROS), both of which were significantly inhibited by Sql-fullerene. In the OP9-U937 cell co-culture system, U937 cells rapidly differentiated to macrophage-like cells during SR-induced adipogenesis in OP9 cells. The ROS accumulation was in the co-culture more marked than in OP9 cells alone, suggesting that the interaction between adipocytes and monocytes/macrophages promotes inflammatory responses. Sql-fullerene significantly inhibited macrophage activation and low-grade adipogenesis in the OP9-U937 co-culture system. We developed a three-dimensional inflammatory adipose-tissue model “ATE” consisting of, characteristically, U937 cells in the culture-wells, and, in addition, mounted a culture insert containing OP9 cells-populated collagen gel. ATE is enabled with suitable stimulation to represent the pathology of inflammatory disorders, such as macrophage infiltration in adipose tissue. Five-day culturing of ATE in SR medium occurred U937 macrophage migration and intracellular oil-droplet accumulation that were significantly inhibited by Sql-fullerene. Our results suggest that Sql-fullerene might be explored as a potential medicine for the treatment of metabolic syndrome or other obesity-related disorders.
Keywords: Nanoparticle; Co-culture; Adipose-tissue engineering; Inflammation;

Amyloid hydrogel derived from curly protein fibrils of α-synuclein by Ghibom Bhak; Soonkoo Lee; Jae Woo Park; Sunghyun Cho; Seung R. Paik (5986-5995).
Elucidation of molecular assembly mechanism of protein-based suprastructure formation is pivotal to develop biomaterials. A single amyloidogenic protein of α-synuclein turned into two morphologically distinctive amyloid fibrils – ‘curly’ (CAF) vs. ‘straight’ (SAF) – depending on its fibrillation processes. Mutually exclusive production of CAF and SAF was achieved with either centrifugal membrane filtration of the preformed oligomeric species of α-synuclein or agitated incubation of its monomeric form, representing amyloidogeneses via double-concerted and nucleation-dependent fibrillation model, respectively. Differences in secondary structures of CAF and SAF have been suggested to be responsible for their morphological uniqueness with structural flexibility and mechanical strength. Both polymorphs exerted the self-propagation property, demonstrating that their characteristic morphologies were inherited for two consecutive generations to daughter and granddaughter fibrils through the seed-dependent fibrillation procedure. Accumulation of CAF produced amyloid hydrogel composed of fine nano-scaled three-dimensional protein fibrillar network. The hydrogel made of daughter CAF was demonstrated to be a suitable nanomatrix for enzyme entrapment, which protected the entrapped enzyme of horseradish peroxidase from loss of activity due to multiple catalyses and heat treatment. The nano-scaled fibrillar network of CAF, therefore, could exhibit a full potential to be further applied in the promising areas of nanobiotechnology including tissue engineering, drug delivery, nanofiltration and biosensor development.
Keywords: Self-assembly; Amyloidogenesis; Hydrogel; Protein nanofibrils; Nanomatrix;

Autophagy and oxidative stress associated with gold nanoparticles by Jasmine J. Li; Deny Hartono; Choon-Nam Ong; Boon-Huat Bay; Lin-Yue L. Yung (5996-6003).
Elemental metal nanoparticles like cadmium and silver are known to cause oxidative stress and are also highly toxic. Yet for gold nanoparticles (AuNPs), it is not well established whether these particles are biologically toxic. Here we show that AuNPs, which were taken up by MRC-5 human lung fibroblasts in vitro, induce autophagy concomitant with oxidative stress. We also observed formation of autophagosomes together with the uptake of AuNPs in the lung fibroblasts as well as upregulation of autophagy proteins, microtubule-associated protein 1 light chain 3 (MAP-LC3) and autophagy gene 7 (ATG 7) in treated samples. AuNP treated cells also generated significantly more lipid hydroperoxides (p-value < 0.05), a positive indication of lipid peroxidation. Verification with western blot analysis for malondialdehyde (MDA) protein adducts confirmed the presence of oxidative damage. In addition, AuNP treatment also induced upregulation of antioxidants, stress response genes and protein expression. Exposure to AuNPs is a potential source of oxidative stress in human lung fibroblasts and autophagy may be a cellular defence mechanism against oxidative stress toxicity.
Keywords: Gold nanoparticles; Oxidative stress; Autophagy; Autophagosomes; Lung fibroblasts;

Bone regeneration mediated by biomimetic mineralization of a nanofiber matrix by Alvaro Mata; Yanbiao Geng; Karl J. Henrikson; Conrado Aparicio; Stuart R. Stock; Robert L. Satcher; Samuel I. Stupp (6004-6012).
Rapid bone regeneration within a three-dimensional defect without the use of bone grafts, exogenous growth factors, or cells remains a major challenge. We report here on the use of self-assembling peptide nanostructured gels to promote bone regeneration that have the capacity to mineralize in biomimetic fashion. The main molecular design was the use of phosphoserine residues in the sequence of a peptide amphiphile known to nucleate hydroxyapatite crystals on the surfaces of nanofibers. We tested the system in a rat femoral critical-size defect by placing pre-assembled nanofiber gels in a 5 mm gap and analyzed bone formation with micro-computed tomography and histology. We found within 4 weeks significantly higher bone formation relative to controls lacking phosphorylated residues and comparable bone formation to that observed in animals treated with a clinically used allogenic bone matrix.
Keywords: Peptide amphiphiles; Bone scaffolds; Bone regeneration; Biomineralization; Regenerative medicine; Biomaterials;

SiRNA-loaded multi-shell nanoparticles incorporated into a multilayered film as a reservoir for gene silencing by Xin Zhang; Anna Kovtun; Carlos Mendoza-Palomares; Mustapha Oulad-Abdelghani; Florence Fioretti; Simon Rinckenbach; Didier Mainard; Matthias Epple; Nadia Benkirane-Jessel (6013-6018).
In this study, we presented a new type of coating based on polyelectrolyte multilayers containing sequentially adsorbed active shRNA calcium phosphate nanoparticles for locally defined and temporarily variable gene silencing. Therefore, we investigated multi-shell calcium phosphate-shRNA nanoparticles embedded into a polyelectrolyte multilayer for gene silencing. As model system, we synthesized triple-shell calcium phosphate-shRNA nanoparticles (NP) and prepared polyelectrolyte multilayers films made of nanoparticles and poly-(l-lysine) (PLL). The biological activities of these polyelectrolyte multilayers films were tested by the production of osteopontin and osteocalcin in the human osteoblasts (HOb) which were cultivated on the PEM films. This new strategy can be used to efficiently control the bone formation and could be applicable in tissue engineering.
Keywords: Calcium phosphate; Nanoparticles; Polyelectrolyte membrane films; Gene silencing; Surface modification;

The effectiveness of the controlled release of gentamicin from polyelectrolyte multilayers in the treatment of Staphylococcus aureus infection in a rabbit bone model by Joshua S. Moskowitz; Michael R. Blaisse; Raymond E. Samuel; Hu-Ping Hsu; Mitchel B. Harris; Scott D. Martin; Jean C. Lee; Myron Spector; Paula T. Hammond (6019-6030).
While the infection rate of orthopedic implants is low, the required treatment, which can involve six weeks of antibiotic therapy and two additional surgical operations, is life threatening and expensive, and thus motivates the development of a one-stage re-implantation procedure. Polyelectrolyte multilayers incorporating gentamicin were fabricated using the layer-by-layer deposition process for use as a device coating to address an existing bone infection in a direct implant exchange operation. The films eluted about 70% of their payload in vitro during the first three days and subsequently continued to release drug for more than four additional weeks, reaching a total average release of over 550 μg/cm2. The coatings were demonstrated to be bactericidal against Staphylococcus aureus, and degradation products were generally nontoxic towards MC3T3-E1 murine preosteoblasts. Film-coated titanium implants were compared to uncoated implants in an in vivo S. aureus bone infection model. After a direct exchange procedure, the antimicrobial-coated devices yielded bone homogenates with a significantly lower degree of infection than uncoated devices at both day four (p < 0.004) and day seven (p < 0.03). This study has demonstrated that a self-assembled ultrathin film coating is capable of effectively treating an experimental bone infection in vivo and lays the foundation for development of a multi-therapeutic film for optimized, synergistic treatment of pain, infection, and osteomyelitis.
Keywords: Antibacterial; Animal model; Bone; Controlled drug release; Layer-by-layer; Coating;

Tyrosine-based rivastigmine-loaded organogels in the treatment of Alzheimer’s disease by Guillaume Bastiat; François Plourde; Aude Motulsky; Alexandra Furtos; Yvan Dumont; Rémi Quirion; Gregor Fuhrmann; Jean-Christophe Leroux (6031-6038).
Organogels can be prepared by immobilizing an organic phase into a three-dimensional network coming from the self-assembly of a low molecular weight gelator molecule. In this work, an injectable subcutaneous organogel system based on safflower oil and a modified-tyrosine organogelator was evaluated in vivo for the delivery of rivastigmine, an acetylcholinesterase (AChE) inhibitor used in the treatment of Alzheimer’s disease. Different implant formulations were injected and the plasmatic drug concentration was assayed for up to 35 days. In parallel, the inhibition of AChE in different brain sections and the biocompatibility of the implants were monitored. The pharmacokinetic profiles were found to be influenced by the gel composition, injected dose and volume of the implant. The sustained delivery of rivastigmine was accompanied by a significant prolonged inhibition of AChE in the hippocampus, a brain structure involved in memory. The implant induced only a minimal to mild chronic inflammation and fibrosis, which was comparable to poly(d,l-lactide-co-glycolide) in situ-forming implants. These findings suggest that tyrosine-based organogels could represent an alternative approach to current formulations for the sustained delivery of cholinesterase inhibitors.
Keywords: Organogel; Rivastigmine; Alzheimer’s disease; Pharmacokinetic; Biocompatibility; AChE inhibition;

Gradient cross-linked biodegradable polyelectrolyte nanocapsules for intracellular protein drug delivery by Shujun Shu; Xinge Zhang; Zhongming Wu; Zhen Wang; Chaoxing Li (6039-6049).
Gradient shell cross-linked hollow polyelectrolyte nanocapsules composed of cysteamine conjugated chitosan and dextran sulfate were prepared by layer-by-layer adsorption on β-cyclodextrin (β-CD) functionalized silica spheres followed by cross-linking thiols and removal of silica core. This disulfide bond gradient cross-linked nanocapsules combined reduction and pH sensitive. Gradually increased from the inside to the outside of the cross-linking degree, one purpose is to ensure that cross-linking disulfide bond after reduction cleavage still has pH sensitive, on the other hand is to avoid cross-linked contraction of internal damage the crystal and bioactivity of protein drugs. Disulfide cross-linked nanocapsules were used to enhance the physical stability against acidic pH conditions compared to the un-cross-linked ones. Bovine serum albumin, as a model protein drug, was loaded inside nanocapsules. The disulfide bond cross-linked nanocapsules are intended to remain more stable in physiological pH and decrease the loss of protein drugs caused by the gastric cavity, and can release the drugs in the intracellular environment after glutathione reduction.
Keywords: Gradient cross-linked; Polyelectrolyte; Layer by layer technique; Reduction sensitive; Intracellular drug delivery;

Sustained steroid release in pulmonary inflammation model by Harry Karmouty-Quintana; Faleh Tamimi; Toby K. McGovern; Liam M. Grover; James G. Martin; Jake E. Barralet (6050-6059).
There is a need for particles which exhibit controlled release of therapeutic agents delivered via the inhalational route, for tissue specific applications such as anti-cancer, bronchodilators and antiviral agents as well as drugs for systemic action. The aim of this study was to assess the acute toxicity, distribution and capacity of the microspheres to exhibit controlled release properties in an in vivo model of airway inflammation. Calcium pyrophosphate nanofibrous microspheres were loaded with dexamethasone phosphate (Dex-P); the profile of drug release was studied in vitro and validated in vivo. Unloaded microspheres were administered intra-tracheally (i.t.) to rats to assess the tissue reaction. The anti-inflammatory properties of the Dex-P loaded microspheres against an inflammatory agent (compound 48/80), were evaluated in vivo. Unloaded microspheres did not cause an inflammatory response when given at doses below 3 mg, and appeared to be eliminated through mucus clearance mechanisms. Microspheres loaded with Dex-P but not Dex-P alone, were capable of inhibiting eosinophil and total inflammatory cell increases in bronchoalveolar lavage fluid for 42 h following a single application. These observations demonstrated that calcium pyrophosphate nanofibrous microspheres displayed in vivo controlled release properties, were well tolerated and did not accumulate in the lung.
Keywords: Calcium pyrophosphate; Microsphere; Controlled-release; Anti-inflammatory; Inhalation drug delivery;

Traffic of poly(lactic acid) nanoparticulate vaccine vehicle from intestinal mucus to sub-epithelial immune competent cells by Charlotte Primard; Nicolas Rochereau; Elsa Luciani; Christian Genin; Thierry Delair; Stéphane Paul; Bernard Verrier (6060-6068).
Mucosal immunization is designed to induce strong immune responses at portal of pathogen entry. Unfortunately, mechanisms underlying the fate of the vaccine vector co-administered with antigens are still partially uncovered and limit further development of mucosal vaccines. Hence, poly(lactic acid) (PLA) nanoparticles being a versatile vaccine vehicle, we have analyzed the fate of these PLA nanoparticles during their uptake at intestinal mucosal sites, both in vivo and ex vivo, to decipher the mechanisms involved during this process. We first designed specific fluorescent PLA nanoparticles exhibiting strong colloidal stability after encapsulation of either 6-coumarin or CellTrace BODIPY® before monitoring their transport through mucosa in the mouse ligated ileal loop model. The journey of the particles appears to follow a three-step process. Most particles are first entrapped in the mucus. Then, crossing of the epithelial barrier takes place exclusively through M-cells, leading to an accumulation in Peyer’s patches (PP). Lastly, we noticed specific interaction of these PLA nanoparticles with underlying B cells and dendritic cells (DCs) of PP. Furthermore, we could document that DCs engulfing some nanoparticles could exhibit a TLR8+ specific expression. Specific targeting of these two cell types strongly supports the use of PLA nanoparticles as a vaccine delivery system for oral use. Indeed, following oral gavage of mice with PLA nanoparticles, we were able to observe the same biodistribution patterns, indicating that these nanoparticles specifically reach immune target required for oral immunization.
Keywords: Nanoparticles; Poly(lactic acid); Intestinal mucosa; Oral delivery; Traffic; Dendritic cells;

Artificial oxygen carrier based on polysaccharides–poly(alkylcyanoacrylates) nanoparticle templates by Cédric Chauvierre; Romila Manchanda; Denis Labarre; Christine Vauthier; Michael C. Marden; Liliane Leclerc (6069-6074).
Biomimetic nanoparticles based on polysaccharides–poly(alkylcyanoacrylates) copolymers were initially developed in view of drug delivery. Core-shell nanoparticles covered with a sufficiently long brush of polysaccharides were shown to be very low complement activators and have the potential for long circulation times in the bloodstream. Such nanoparticles bearing haemoglobin were envisaged as potential red cell substitutes. Different core-shell nanoparticles with a brush shell made of dextran, dextran-sulphate, or heparin were prepared and haemoglobin (Hb) could be adsorbed on their surface. Benzene tetracarboxylic acid (BTCA) was used as a coupling agent for Hb to dextran-coated nanoparticles; the Hb loading capacity of the dextran nanoparticles showed a 9.3 fold increased. The coupled Hb maintained the allosteric properties of free Hb. While modification of nanoparticles by BTCA slightly increased complement activation, the further addition of Hb totally reversed this effect providing Hb-loaded nanoparticles with a very low level of complement activation. Such nanoparticles could be a suitable alternative to haemoglobin solutions in the development of a blood substitute.
Keywords: Nanoparticles; Poly(alkylcyanoacrylates); Polysaccharides; Benzene-1,2,4,5-tetracarboxylic acid; Haemoglobin; Oxygen carrier;

Targeted minicircle DNA delivery using folate–poly(ethylene glycol)–polyethylenimine as non-viral carrier by Chao Zhang; Shijuan Gao; Wei Jiang; Song Lin; Fusheng Du; Zichen Li; Wenlin Huang (6075-6086).
Targeted gene delivery systems have attracted great attention due to their potential in directing the therapeutic genes to the target cells. However, due to their low efficiency, most of the successful applications of polymeric vectors have been focused on genes which can achieve robust expression. Minicircle DNA (mcDNA) is a powerful candidate in terms of improving gene expression and prolonging the lifespan of gene expression. In this study, we have combined folate/poly(ethylene glycol) modified polyethylenimine and mcDNA as a new tumor gene delivery system. We found that folate-labeled polyplexes were homogenous, with a size ranging from 60 to 85 nm. mcDNA increased folate-labeled vector based gene expression 2–8 fold in folate receptor-positive cells. Results of folic acid competition assay indicated that mcDNA mediated by folate-labeled vector were internalized into cells through receptor-mediated endocytosis. The investigation of the endocytosis pathway of the polyplexes showed that a large portion of them escaped from endo/lysosome and the polyplexes were associated before being separated in the nucleus. Furthermore, in vivo optical imaging and luciferase assays demonstrated that systemic delivery of the folate-labeled polyplexes resulted in preferential accumulation of transgenes in folate receptor-positive tumors, and mcDNA mediated approach achieved 2.3 fold higher gene expressions in tumors than conventional plasmid. Cytotoxicity assays showed that PEG-shielding of the polyplexes reduced the toxicity of PEI.
Keywords: Folate–PEG–PEI; Receptor-mediated gene delivery; Minicircle DNA; Tumor targeting;

An optical biosensing platform for proteinase activity using gold nanoparticles by Yao-Chen Chuang; Jung-Chun Li; Sz-Hau Chen; Ting-Yu Liu; Ching-Han Kuo; Wei-Ting Huang; Chih-Sheng Lin (6087-6095).
The surface plasmon resonance (SPR) wavelength of colloidal gold nanoparticles (AuNPs) can vary when the AuNPs aggregate, have different sizes or shapes, or are modified with chemical molecules. In this study, an optical biosensing platform for a proteinase activity assay was established based on the SPR property of AuNPs. The 13-nm AuNPs were modified with gelatin (AuNPs-gelatin) as a proteinase substrate and subsequently modified with 6-mercaptohexan-1-ol (MCH) (AuNPs/MCH-gelatin). After proteinase (trypsin or gelatinase) digestion, the AuNPs lose shelter, and MCH increases the attractive force between the modified AuNPs. Therefore, the AuNPs gradually move closer to each other, resulting in AuNPs aggregation. The AuNPs aggregation can be monitored by the red shift of surface plasmon absorption and a visible color change of the AuNPs is from red to blue. Such a color change can be observed with the naked eye. For detection, the absorption ratio, A 625/A 525, of the reacted AuNPs solution can be used to estimate quantitatively the proteinase activity. A linear correlation has been established with trypsin activity at concentrations from 1.25 × 10−1 to 1.25 × 102 U and matrix metalloproteinase-2 activity at concentrations from 50 ng/mL to 600 ng/mL.
Keywords: 6-Mercaptohexan-1-ol; Gold nanoparticles; Matrix metalloproteinase; Optical biosensor; Proteinase;

Biopolymer mediated trehalose uptake for enhanced erythrocyte cryosurvival by Andrew L. Lynch; Rongjun Chen; Paul J. Dominowski; Evgenyi Y. Shalaev; Robert J. Yancey; Nigel K.H. Slater (6096-6103).
A biopolymer has been shown to facilitate efficient delivery of trehalose, a bioprotectant normally impermeable to the phospholipid bilayer, into ovine erythrocytes. Cellular uptake of trehalose was found to be dependent on polymer pendant amino acid type and degree of grafting, polymer concentration, pH, external trehalose concentration, incubation temperature and time. Optimization of these parameters yielded an intracellular trehalose concentration of 123 ± 16 mm and concomitant improvement of erythrocyte cryosurvival of up to 20.4 ± 5.6%. Intracellular trehalose was shown to impart cellular osmoprotection up to an external osmolarity of 230 mOsm and increased osmotic sensitivity above this threshold. Biopolymer mediated membrane permeability was shown to be rapidly and completely reversible via washing with phosphate buffered saline.
Keywords: Biopolymer; Blood; Cryopreservation; Trehalose; Cell viability;

Menisci are one of the most commonly injured parts of the knee with a limited healing potential. This study focuses on fabrication and characterization of biomimetic surfaces for meniscal tissue engineering. To achieve this, a combination of hyaluronic acid/chitosan (HA/CH) mutilayers with covalently immobilized major extracellular matrix (ECM) components of native meniscus, namely collagen I/II (COL.I/II) and chondroitin-6-sulfate (C6S) was employed. Adsorption of the biomolecules was monitored using a quartz crystal microbalance with dissipation (QCM-D) and fourier transform-surface plasmon resonance (FT-SPR). Immobilization of the biomolecules onto HA/CH mutilayers was achieved by sequential adsorption, with optimum binding at a molar ratio of 1.4:1 (COL.I/II: C6S). After adding COL.I/II, the layers became relatively more rigid and large aggregates were evident. The effects of the modified surfaces on cell proliferation, gene expression and proteoglycan production of rat meniscal cells were examined. Quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) analysis showed that primary meniscal cells dedifferentiated rapidly after one passage in monolayer culture. This process could be reversed by culturing the cells on C6S surfaces, as indicated by increases in both collagen II and aggrecan gene expression, as well as proteoglycan production. Cells with abundant lipid vacuoles were evident on all the surfaces over an extended culture period. The results demonstrate the feasibility of C6S surfaces to avoid the dedifferentiation that normally occurs during monolayer expansion of meniscal cells.
Keywords: Meniscal tissue engineering; Surface modification; Biomimetic materials; Chondroitin sulfate; Collagen; Dedifferentiation;