Biomaterials (v.31, #28)

Cells preferentially grow on rough substrates by Francesco Gentile; Luca Tirinato; Edmondo Battista; Filippo Causa; Carlo Liberale; Enzo M. di Fabrizio; Paolo Decuzzi (7205-7212).
Substrate nanotopography affects cell adhesion and proliferation and is fundamental to the rational design of bio-adhesives, to tissue engineering and to the development of assays for in-vitro screening. Cell behavior on rough substrates is still elusive, and the results presented in the open literature remain controversial. Here, the proliferation of cells on electrochemically etched silicon substrates with different roughness and nearly similar surface energy was studied over three days with confocal and atomic force microscopy. The surface profile of the substrates is a self-affine fractal with a roughness R a growing with the etching time from ∼2 to 100 nm and a fractal dimension D ranging between about 2 (nominally flat surface) and 2.6. For four cell types, the number of adhering cells and their proliferation rates exhibited a maximum on moderately rough (R a ∼ 10–45 nm) nearly Brownian (D ∼2.5) substrates. The observed cell behavior was satisfactorily interpreted within the theory of adhesion to randomly rough solids. These findings demonstrated the importance of nanogeometry in cell stable adhesion and growth, suggesting that moderately rough substrates with large fractal dimension could selectively boost cell proliferation.
Keywords: Nanotopography; Cell adhesion; Cell proliferation; Fractal surfaces; Rational design;

Amino acid derivative-mediated detoxification and functionalization of dual cure dental restorative material for dental pulp cell mineralization by Hajime Minamikawa; Masahiro Yamada; Fuminori Iwasa; Takeshi Ueno; Yoshiaki Deyama; Kuniaki Suzuki; Yasutaka Yawaka; Takahiro Ogawa (7213-7225).
Current dental restorative materials are only used to fill the defect of hard tissues, such as dentin and enamel, because of their cytotoxicity. Therefore, exposed dental pulp tissues in deep cavities must be first covered by a pulp capping material like calcium hydroxide to form a layer of mineralized tissue. However, this tissue mineralization is based on pathological reaction and triggers long-lasting inflammation, often causing clinical problems. This study tested the ability of N-acetyl cysteine (NAC), amino acid derivative, to reduce cytotoxicity and induce mineralized tissue conductivity in resin-modified glass ionomer (RMGI), a widely used dental restorative material having dual cure mechanism. Rat dental pulp cells were cultured on untreated or NAC-supplemented RMGI. NAC supplementation substantially increased the percentage of viable cells from 46.7 to 73.3% after 24-h incubation. Cell attachment, spreading, proliferative activity, and odontoblast-related gene and protein expressions increased significantly on NAC-supplemented RMGI. The mineralization capability of cells, which was nearly suppressed on untreated RMGI, was induced on NAC-supplemented RMGI. These improved behaviors and functions of dental pulp cells on NAC-supplemented RMGI were associated with a considerable reduction in the production of intracellular reactive oxygen species and with the increased level of intracellular glutathione reserves. These results demonstrated that NAC could detoxify and functionalize RMGIs via two different mechanisms involving in situ material detoxification and antioxidant cell protection. We believe that this study provides a new approach for developing dental restorative materials that enables mineralized tissue regeneration.
Keywords: Resin-modified glass ionomer; N-acetyl cysteine (NAC); Antioxidant; Free radical; Dental pulp cells; HEMA;

The identification of a heparin binding domain peptide from bone morphogenetic protein-4 and its role on osteogenesis by Yoon Jung Choi; Jue Yeon Lee; Jung Hyun Park; Jun Beom Park; Jin Sook Suh; Young Suk Choi; Seung Jin Lee; Chong-Pyoung Chung; Yoon Jeong Park (7226-7238).
The presence of heparin binding has been become crucial in exerting growth factor related tissue formation. Receptor-mediated osteoblastic differentiation by bone morphogenetic protein (BMP)-4 and supportive function of its heparin binding has been proposed, direct role of the heparin binding site of BMP-4 on osteogenesis has not yet been fully investigated. If the binding site itself plays role on osteogenesis, the site domain can be useful in bone formation in combination with biomaterial. Herein, we synthesized a peptide sequence corresponding to residues 15–24 of BMP-4 (HBD, RKKNPNCRRH), as potential heparin binding sequence. The HBD peptide-induced ostoegenic differentiation by activating extracellular signal-regulated kinase (ERK1/2), one of the key regulators in hMSC. Also, treatment of cultured hMSCs with heparinase blocked both HBD peptide-induced osteogenic differentiation and GAG chain detection while abolishing the increased phospho-ERK level. These results suggest that the identified heparin binding domain peptide (HBD) stimulated osteoblastic differentiation via interaction with heparin and the ERK signaling. In vivo results further demonstrated that HBD, as a form of complex with alginate gel, was able to induce bone formation in the bone defect.
Keywords: Heparin binding domain (HBD) peptide; Bone morphogenetic protein-4; Osteoblastic differentiation; ERK1/2 pathway; Bioactive material; Bone formation;

The effect of incorporation of exogenous stromal cell-derived factor-1 alpha within a knitted silk-collagen sponge scaffold on tendon regeneration by Weiliang Shen; Xiao Chen; Jialin Chen; Zi Yin; Boon Chin Heng; Weishan Chen; Hong-Wei Ouyang (7239-7249).
This study developed a bioactive knitted silk-collagen sponge scaffold by incorporation of exogenous SDF-1 alpha, to enable selective migration and homing of cells for in situ tendon regeneration. With in vitro studies, it was observed that CXCR4 gene expression and migration of bone mesenchymal stromal cells and hypo-dermal fibroblasts were more sensitive to exogenous SDF-1 alpha, while expression of tendon repair gene markers by hypo-dermal fibroblasts and Achilles tendon fibroblasts were more sensitive to exogenous SDF-1 alpha. With a rat Achilles tendon injury model, exogenous SDF-1 alpha was shown to reduce infiltration of inflammatory cells and enhance migration of fibroblast-like cells into the scaffold at 4 days and 1 week post-surgery. After 4 weeks, SDF-1 alpha treated tendon had increased expression of tendon repair gene markers and endogenous SDF-1 alpha, exhibited more physiological microstructures with larger diameter collagen fibrils, and had better biomechanical properties than the control group. Hence, our bioactive scaffold improved efficacy of tendon regeneration by increasing the recruitment of fibroblast-like cells, enhancing local endogenous SDF-1 alpha and tendon extracellular matrix production, and decreasing accumulation of inflammatory cells. Incorporation of SDF-1 alpha within a knitted silk-collagen sponge scaffold can therefore be a practical application for tendon tissue engineering.
Keywords: Tendon; Tissue engineering; Knitted silk-collagen sponge scaffold; rhSDF-1 alpha; Regeneration;

Laser assisted bioprinting of engineered tissue with high cell density and microscale organization by Bertrand Guillotin; Agnès Souquet; Sylvain Catros; Martí Duocastella; Benjamin Pippenger; Séverine Bellance; Reine Bareille; Murielle Rémy; Laurence Bordenave; Joëlle Amédée; Fabien Guillemot (7250-7256).
Over this decade, cell printing strategy has emerged as one of the promising approaches to organize cells in two and three dimensional engineered tissues. High resolution and high speed organization of cells are some of the key requirements for the successful fabrication of cell-containing two or three dimensional constructs. So far, none of the available cell printing technologies has shown an ability to concomitantly print cells at a cell-level resolution and at a kHz range speed. We have studied the effect of the viscosity of the bioink, laser energy, and laser printing speed on the resolution of cell printing. Accordingly, we demonstrate that a laser assisted cell printer can deposit cells with a microscale resolution, at a speed of 5 kHz and with computer assisted geometric control. We have successfully implemented such a cell printing precision to print miniaturized tissue like layouts with de novo high cell density and micro scale organization.
Keywords: Tissue engineering; Laser assisted bioprinting; Laser manufacturing; Rapid prototyping; Micropatterning; High cell density printing;

The aim of this study was to construct a rabbit anterior cornea replacement with an acellular porcine cornea matrix (APCM) as a scaffold. The scaffold was prepared from fresh porcine corneas which were treated with 0.5% (wt./vol.) sodium dodecyl sulfate (SDS) solution and stirred for 24 h in a 4 °C refrigeration chamber. The complete removal of corneal cells was confirmed by H&E and DAPI staining. The stroma structure and mechanical properties were well preserved. The extracts had no cytotoxicity to rabbit corneal keratocytes, epithelial and endothelial cells as determined by MTT assay. Moreover, there was no sign that an immune reaction occurred in or around the transplanted disks within 6 months of animal implantation. To construct a rabbit anterior cornea replacement, keratocytes were injected into APCM and cultured for 7 days in a dynamic culturing system, followed by culturing corneal epithelial cells on the stroma construct surface for another 7 days. The phenotype of the construct was similar to normal rabbit corneas, with high expression of cytokeratin 3 in the epithelial cell layer and expression of vimentin in the stromal cells. These results suggested that the APCM developed by using SDS might be a suitable scaffold for cornea tissue engineering.
Keywords: Cornea; Decellularization; Scaffold; Sodium dodecyl sulfate; Tissue engineering;

Self assembled bi-functional peptide hydrogels with biomineralization-directing peptides by Mustafa Gungormus; Monica Branco; Hanson Fong; Joel P. Schneider; Candan Tamerler; Mehmet Sarikaya (7266-7274).
A peptide-based hydrogel has been designed that directs the formation of hydroxyapatite. MDG1, a twenty-seven residue peptide, undergoes triggered folding to form an unsymmetrical β-hairpin that self-assembles in response to an increase in solution ionic strength to yield a mechanically rigid, self supporting hydrogel. The C-terminal portion of MDG1 contains a heptapeptide (MLPHHGA) capable of directing the mineralization process. Circular dichroism spectroscopy indicates that the peptide folds and assembles to form a hydrogel network rich in β-sheet secondary structure. Oscillatory rheology indicates that the hydrogel is mechanically rigid (G′ ˜ 2500 Pa) before mineralization. In separate experiments, mineralization was induced both biochemically and with cementoblast cells. Mineralization-domain had little effect on the mechanical rigidity of the gel. SEM and EDXS show that MDG1 gels are capable of directing the formation of hydroxapatite. Control hydrogels, prepared by peptides either lacking the mineral-directing portion or reversing its sequence, indicated that the heptapeptide is necessary and its actions are sequence specific.
Keywords: Biomineralization; Biomimetic material; Peptide; Hydrogel; Scaffold; Hydroxyapatite;

Multi-lineage differentiation of hMSCs encapsulated in thermo-reversible hydrogel using a co-culture system with differentiated cells by Ji Sun Park; Han Na Yang; Dae Gyun Woo; Hyemin Kim; Kun Na; Keun-Hong Park (7275-7287).
The micro-environment is an important factor in the differentiation of cultured stem cells for the purpose of site specific transplantation. In an attempt to optimize differentiation conditions, co-culture systems composed of both stem cells and primary cells or cell lines were used in hydrogel with in vitro and in vivo systems. Stem cells encapsulated in hydrogel, under certain conditions, can undergo increased differentiation both in vitro and in vivo; therefore, reconstruction of transplanted stem cells in a hydrogel co-culture system is important for tissue regeneration. In order to construct such a co-culture system, we attempted to create a hydrogel scaffold which could induce neo-tissue growth from the recipient bed into the material. This material would enable encapsulation of stem cells in vitro after which they could be transferred to an in vivo system utilizing nude mice. In this case, the hydrogel was implanted in the subfascial space of nude mice and excised 4 weeks later. Cross-sections of the excised samples were stained with von Kossa or safranin-O and tubular formations into the gel were observed with and tested by doppler imaging. The data showed that the hydrogel markedly induced growth of osteogenic, chondrogenic, and vascular-rich tissue into the hydrogel by 4 weeks, which surpassed that after transplantation in a co-culture system. Further, a co-culture system with differentiated cells and stem cells potentially enhanced chondrogenesis, osteogenesis, and vascularization. These findings suggest that a co-culture system with hydrogel as scaffold material for neo-tissue formation is a useful tools for multi-lineage stem cell differentiation.
Keywords: Stem cell; Co-culture; Hydrogel; Differentiation; Scaffold;

Modular enzymatically crosslinked protein polymer hydrogels for in situ gelation by Nicolynn E. Davis; Sheng Ding; Ryan E. Forster; Daniel M. Pinkas; Annelise E. Barron (7288-7297).
Biomaterials that mimic the extracellular matrix in both modularity and crosslinking chemistry have the potential to recapitulate the instructive signals that ultimately control cell fate. Toward this goal, modular protein polymer-based hydrogels were created through genetic engineering and enzymatic crosslinking. Animal derived tissue transglutaminase (tTG) and recombinant human transglutaminase (hTG) enzymes were used for coupling two classes of protein polymers containing either lysine or glutamine, which have the recognition substrates for enzymatic crosslinking evenly spaced along the protein backbone. Utilizing tTG under physiological conditions, complete crosslinking occurred within 2 min, as determined by particle tracking microrheology. Hydrogel composition impacted the elastic storage modulus of the gel over 4-fold and also influenced microstructure and degree of swelling, but did not appreciably effect degradation by plasmin. Mouse 3T3 and primary human fibroblasts were cultured in both 2- and 3-dimensions without a decrease in cell viability and displayed spreading in 2D. The properties, which are controlled through the specific nature of the protein polymer precursors, render these gels valuable for in situ therapies. Furthermore, the modular hydrogel composition allows tailoring of mechanical and physical properties for specific tissue engineering applications.
Keywords: Hydrogel; Genetic engineering; Recombinant protein; Mechanical properties;

Biomimetic hydrogels for chondrogenic differentiation of human mesenchymal stem cells to neocartilage by Shao Qiong Liu; Quan Tian; James L. Hedrick; James Hoi Po Hui; Pui Lai Rachel Ee; Yi Yan Yang (7298-7307).
In this study, a collagen mimetic peptide (CMP) containing a GFOGER sequence flanked by GPO repeat units (sequence: (GPO)4GFOGER(GPO)4GCG, CMP) was synthesized and chemically incorporated into a poly(ethylene glycol) (PEG) hydrogel through Michael addition chemistry. The PEG/collagen mimetic peptide hybrid hydrogel was used as a scaffold for encapsulation, proliferation and differentiation of human mesenchymal stem cells (hMSCs) into neocartilage/chondrocytes. Biophysical studies indicated that this peptide adopts stable triple helical conformation under simulated physiological conditions. Tetra hydroxyl PEG was functionalized to generate an acrylate group and reacted with the peptide, and hydrogels were formed in situ with the addition of cells and tetra sulfhydryl PEG via Michael addition. The effect of CMP on proliferation and chondrogenesis of hMSCs was investigated. The results demonstrated that PEG–CMP hydrogels provided a natural environment, which promoted chondrogenesis of hMSCs and enhanced secretion of cartilage specific ECM as compared to PEG hydrogels without the peptide. This was attributed to enhanced cell/matrix interactions via integrin β1/GFOGER interactions. Further, chondrogenesis was found to be affected by matrix elasticity. Soft matrix induced a greater degree of chondrogenic differentiation; however, stiff matrix had an opposite effect, inhibiting chondrogenic differentiation probably due to limited mass transport. This soft PEG/CMP hydrogel shows promise as a biomimetic scaffold that provides a desirable environment for the chondrogenic differentiation of hMSCs and is useful for the repair of cartilage defects.
Keywords: Collagen mimetic peptide; PEG hydrogel; Michael addition; Human mesenchymal stem cells; Chondrogenic differentiation;

Angiogenesis of dermal equivalent is one of the key issues for treatment of full thickness skin defects. To develop a gene-activated bilayer dermal equivalent (BDE), N,N,N-trimethyl chitosan chloride (TMC), a cationic gene delivery vector, was used to form complexes with the plasmid DNA encoding vascular endothelial growth factor-165 (VEGF-165), which was then incorporated into a collagen–chitosan/silicone membrane scaffold. To evaluate the angiogenesis property in vivo, full thickness skin defects were made on the back of pigs, into which the TMC/pDNA-VEGF complexes loaded BDE and other three control BDEs, i.e. the blank BDE, and the BDEs loaded with pDNA-VEGF and TMC/pDNA-eGFP complexes, respectively, were transplanted. Biopsy specimens were harvested at day 7, 10 and 14 after surgery for histology, immunohistochemistry, immunofluorescence, real-time quantitative PCR (RT-qPCR) and western blotting analyses. The results showed that the TMC/pDNA-VEGF group had the strongest VEGF expression in mRNA and protein levels, resulting in the highest densities of newly-formed and mature vessels. The ultra-thin skin graft was further transplanted onto the dermis regenerated by the TMC/pDNA-VEGF complexes loaded BDE at day 10 and well survived. At 112 days grafting, the healing skin had a similar structure and ∼80% tensile strength of the normal skin.
Keywords: Collagen-chitosan scaffold; Gene-activated dermal equivalent; DNA complexes; Vascular endothelial growth factor (VEGF); Angiogenesis; Incisional wounds;

A comparison of osteoclast resorption pits on bone with titanium and zirconia surfaces by Thomas Hefti; Martina Frischherz; Nicholas D. Spencer; Heike Hall; Falko Schlottig (7321-7331).
Osteoclasts resorb bone at surfaces, leaving behind pits and trails where both mineral and organic phases of bone have been dissolved. Rough surface structures are deliberately imparted to synthetic implants, in order to improve osseointegration. The aim of this study is to characterize osteoclastic resorption pits on native bone surfaces and to compare these with state-of-the-art titanium and zirconia implant surfaces. The size (i.e. length, width and depth) of resorption pits was compared to the size of surface features of sandblasted and etched titanium and zirconia surfaces. It was found that resorption pits from native bone and surface features of the sandblasted and etched titanium and zirconia surfaces were quite similar in their dimensions. Most structures showed a length between 5 and 40 μm, a width between 2 and 20 μm and a depth between 1 and 8 μm. Additionally, the wavelength-dependent surface roughness was measured, revealing an S a value of 60 nm in the resorption pits, 86 nm on zirconia and between 127 and 140 nm on titanium surfaces. The results of this study may provide some insight into structural requirements for the bone-remodeling cycle and help to improve the design of new implant surfaces for osseointegration applications.
Keywords: Osteoclast; Osseointegration; Surface roughness; Titanium; Zirconia; Bone-remodeling;

Fe3O4 nanoparticles-loaded PEG–PLA polymeric vesicles as labels for ultrasensitive immunosensors by Qin Wei; Ting Li; Gaolei Wang; He Li; Zhiyong Qian; Minghui Yang (7332-7339).
A class of immuno-labels based on poly(ethylene glycol)–poly(lactic acid) (PEG–PLA) polymeric vesicles was developed in this study. To fabricate these immune-labels, the uniform Fe3O4 nanoparticles (NPs) were loaded into the vesicles followed by conjugating secondary antibody (Ab2) onto the vesicles surface, named Ab2–PEG–PLA–Fe3O4. The resulting Ab2–PEG–PLA–Fe3O4 demonstrated high catalytic activity towards H2O2, and the sensitivity of the sandwich-type immunosensor using this label for prostate specific antigen (PSA) detection increased greatly. The immunosensor based on this label exhibited high sensitivity, wide linear range (0.005–10 ng/mL), low detection limit (2 pg/mL), good reproducibility, selectivity and stability. These labels for immunosensors may provide many potential applications for the ultrasensitive detection of different cancer biomarkers. In addition, this technique also has the potential to be extended to the loading of other interesting material for preparing various kinds of labels to meet the different requirements in immunoassays.
Keywords: Fe3O4 nanoparticles; Label; Polymeric vesicles; Prostate specific antigen; Sandwich type;

Folate-targeted supramolecular vesicular aggregates based on polyaspartyl-hydrazide copolymers for the selective delivery of antitumoral drugs by Mariano Licciardi; Donatella Paolino; Christian Celia; Gaetano Giammona; Gennara Cavallaro; Massimo Fresta (7340-7354).
Supramolecular vesicular aggregates (SVAs) have the advantage of combining the safe and biocompatible properties of colloidal vesicular carriers based on phospholipids with those of polymeric materials, i.e. polyaspartyl-hydrazide (PAHy) copolymers. To provide SVAs with a certain tumour selectivity, folate moieties were chemically conjugated to PAHy copolymers. Physicochemical properties (mean sizes, polydispersity index and zeta potential) of folate-targeted SVAs (FT-SVAs) loaded with gemcitabine were evaluated. The antiproliferative and anticancer activity of gemcitabine-loaded FT-SVAs was evaluated against two cancer cell lines, i.e. MCF-7 cells which over-express the folate receptor and the BxPC-3 cells, which do not over-express this receptor. Gemcitabine-loaded FT-SVAs showed a significantly (p < 0.001) greater and more specific in vitro anticancer activity with respect to both the free drug and the drug-loaded conventional liposomes or untargeted SVAs. Confocal microscopy, flow cytometry analysis and β-scintillation highlighted that FT-SVAs were able to interact with MCF-7 cells after just 3 h and to increase the amount internalization in cells over-expressing the folate receptor. The in vivo biodistribution and pharmacokinetic experiments showed that gemcitabine-loaded SVAs and FT-SVAs were removed from the circulatory system at a slower rate than the native drug and a prolonged gemcitabine plasma concentration was observed for up to 16 h. SVAs were accumulated mainly in the lungs, spleen and kidneys, while FT-SVAs were also up taken by brain. These interesting and stimulating results suggest the existence of a possible in vivo application of SVAs and encourage the use of folate as a targeting agent in anticancer therapy.
Keywords: Biocompatibility; Controlled drug release; Cytotoxicity; Drug delivery; Liposome; Nanocomposite;

Magnetic-nanoparticle-modified paclitaxel for targeted therapy for prostate cancer by Mu-Yi Hua; Hung-Wei Yang; Cheng-Keng Chuang; Rung-Ywan Tsai; Wen-Jauh Chen; Kun-Lung Chuang; Ying-Hsu Chang; Heng-Chang Chuang; See-Tong Pang (7355-7363).
A nontoxic drug nanocarrier containing carboxyl groups was successfully developed by mixing magnetic nanoparticles (MNPs) of Fe3O4 with the water-soluble polyaniline derivative poly[aniline-co-sodium N-(1-one-butyric acid) aniline] (SPAnNa) and doping with HCl aqueous solution to form SPAnH/MNPs shell/core. SPAnH/MNPs could be used to effectively immobilize the hydrophobic drug paclitaxel (PTX), thus enhancing the drug’s thermal stability and water solubility. Up to 302.75 μg of PTX could be immobilized per mg of SPAnH/MNPs. SPAnH/MNPs-bound-PTX (bound-PTX) was more stable than free-PTX at both 25 °C and 37 °C. Furthermore, bound-PTX was more cytotoxic to human prostate carcinoma cells (PC3 and CWR22R) than free-PTX at 37 °C, and the inhibition of cellular growth was even more pronounced when magnetic targeting was applied to the bound-PTX. These data indicate that this magnetically targeted drug delivery system provides more effective treatment of prostate cancer cells using lower therapeutic doses and thus with potentially fewer side-effects.
Keywords: Drug nanocarriers; Taxane drug; Paclitaxel; Prostate cancer; Polyaniline derivative;

Amphiphilic hyper-branched co-polymer nanoparticles for the controlled delivery of anti-tumor agents by Qinghua Miao; Dongxue Xu; Zhi Wang; Li Xu; Tiewei Wang; Yan Wu; David B. Lovejoy; Danuta S. Kalinowski; Des R. Richardson; Guangjun Nie; Yuliang Zhao (7364-7375).
In this investigation, we have designed and synthesized an amphiphilic co-polymer with hyper-branched poly(amine-ester) and polylactide (HPAE-co-PLA) to generate nanoparticles (NPs). These have been used to encapsulate a highly active hydrophobic anti-tumor agent, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT). Encapsulation in NPs was done in an effort to increase the anti-tumor activity of this agent by facilitating its delivery to tumor cells. We have also examined and optimized the formulation parameters of the NPs that alter their drug-loading capacity and their physical, chemical and biological properties. The resulting NPs exhibited high Bp4eT-loading capacity and substantial stability in aqueous solution. In vitro drug release studies demonstrated a controlled drug release profile with increased release at acidic pH. Anti-tumor proliferation assays showed that both free drug and drug-encapsulated NPs markedly inhibited tumor cell proliferation in a time- and concentration-dependent manner. Direct microscopic observation revealed that the fluorescent NPs were taken up by cells and localized, in part, in organelles consistent with lysosomes. These results demonstrate a feasible application of the amphiphilic hyper-branched co-polymer, HPAE-co-PLA, as nanocarriers for intracellular delivery of potent anti-tumor agents.
Keywords: Controlled drug release; Cytotoxicity; Drug delivery; Nanoparticle;

A novel strategy for pulmonary delivery of polymeric nanocarriers (NCs) pressurized-metered dose inhalers (pMDIs) is reported in this work. Core–shell particles consisting of a water soluble, hydrofluoroalkane(HFA)-philic biodegradable copolymer of chitosan and poly(lactic acid), and a core of poly(d,l-lactide-co-glycolide) (PLGA) NCs were prepared by a modified emulsification–diffusion methodology. Dispersions of the core–shell particles in HFA propellant revealed enhanced physical stability compared to polymeric NCs alone, and more importantly, excellent aerosol characteristics as determined by inertial impaction studies. Confocal microscopy revealed that the polymeric NCs from such core–shell particles are capable not only to be taken up by Calu-3 (airway epithelial) cells that have been infected with Chlamydia pneumoniae, an intracellular pathogen, but are also internalized within chlamydial inclusions. Our results suggest that the proposed methodology can be used as a general platform for the delivery of polymeric NCs to the respiratory tract using the inexpensive pMDIs, and that such an approach may be used to target and deliver drugs to treat chlamydial-related infections.
Keywords: Pulmonary drug delivery; Pressurized-metered-dose inhalers; Polymeric; Nanocarriers; Nanomedicine; Chlamydia pneumoniae;

Doxorubicin (DOX) is an effective chemotherapeutic against a wide range of solid tumors. However, its clinical use is limited by severe side effects such as cardiotoxicity as well as inherent and acquired drug resistance of tumors. DOX encapsulation within self-assembled polymeric micelles has the potential to decrease the systemic distribution of free drug and enhance the drug accumulation in the tumor via the enhanced permeability and retention (EPR). In this study, DOX was encapsulated in micelles composed of poly (ethylene oxide)-poly [(R)-3-hydroxybutyrate]-poly (ethylene oxide) (PEO–PHB–PEO) triblock copolymers. Micelle size, DOX loading and DOX release were characterized. To evaluate DOX activity, micelles were tested in both monolayer cell cultures and three-dimensional (3-D) multicellular spheroids (MCS) that mimic solid tumors. Antitumor activity in vivo was further studied with tumor-bearing mice. The micelles improved the efficiency of Dox penetration in 3-D MCS compared with free DOX. Efficient cell killing by Dox-micelles in both monolayer cells and 3-D MCS was also demonstrated. Finally, DOX-loaded micelles mediate efficient tumor delivery from tail vein injections to tumor-bearing mice with much less toxicity compared with free DOX.
Keywords: PEO–PHB–PEO; Polymeric micelles; Doxorubicin; Multicellular spheroid; Penetration; Antitumor activity;

The development of high quality seals for silicon patch-clamp chips by Thomas Sordel; Frédérique Kermarrec; Yann Sinquin; Isabelle Fonteille; Michel Labeau; Fabien Sauter-Starace; Catherine Pudda; François de Crécy; François Chatelain; Michel De Waard; Christophe Arnoult; Nathalie Picollet-D’hahan (7398-7410).
Planar patch-clamp is a two-dimensional variation of traditional patch-clamp. By contrast to classical glass micropipette, the seal quality of silicon patch-clamp chips (i.e. seal resistance and seal success rate) have remained poor due to the planar geometry and the nature of the substrate and thus partially obliterate the advantages related to planar patch-clamp. The characterization of physical parameters involved in seal formation is thus of major interest. In this paper, we demonstrate that the physical characterization of surfaces by a set of techniques (Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM), X-ray Photoelectron Spectroscopy (XPS), surface energy (polar and dispersive contributions), drop angles, impedance spectroscopy, combined with a statistical design of experiments (DOE)) allowed us discriminating chips that provide relevant performances for planar patch-clamp analysis. Analyses of seal quality demonstrate that dispersive interactions and micropore size are the most crucial physical parameters of chip surfaces, by contrast to surface roughness and dielectric membrane thickness. This multi-scale study combined with electrophysiological validation of chips on a diverse set of cell-types expressing various ion channels (IRK1, hERG and hNav1.5 channels) unveiled a suitable patch-clamp chip candidate. This original approach may inspire novel strategies for selecting appropriate surface parameters dedicated to biochips.
Keywords: Planar patch-clamp; Silicon chips; XPS; Impedance; Surface energy; Ion channels;