Biomaterials (v.31, #29)

A series of naphthalimide derivatives as intra and extracellular pH sensors by Yanqing Tian; Fengyu Su; Warner Weber; Vivek Nandakumar; Bradley R. Shumway; Yuguang Jin; Xianfeng Zhou; Mark R. Holl; Roger H. Johnson; Deirdre R. Meldrum (7411-7422).
A series of new naphthalimide derivatives were synthesized and studied. Three of the materials (SM1, SM2, and SM3) possess methacrylate(s) moieties as pH sensor monomers, enabling these compounds to be polymerized with other monomers for thin film preparation for extracellular pH sensing. Herein, poly(2-hydroxyethyl methacrylate)-co-poly(acrylamide) (PHEMA-co-PAM) was chosen as the polymer matrix. Structure influences on pH responses and pK a values were studied. The film P3 composed of the sensing moiety SM3 has a pK a close to the usual biological environmental pH of ∼7. It was used as an extracellular pH sensor to monitor pH change during the metabolism of prokaryotic Escherichia coli (E. coil). On the other hand, the three sensor monomers are new intracellular biomarkers to sense lysosomes of eukaryotic cells since (1) their pK a values are in a range of 5.9–6.8; (2) their emission intensities at acidic conditions (such as at pH 5) are much stronger than those at a neutral condition of pH 7; (3) lysosomes range in size from 0.1 to 1.2 μm in diameter with pH ranging from 4.5 to 5.0, which is much more acidic than the pH value of the cytoplasm (usually with a pH value of ∼7.2); and (4) the acidity of lysosomes enables a protonation of the amino groups of the pH probes making the sensors emit brightly in acidic organelles by inhibiting the photo-induced electron transfer from the amino groups to the fluorophores. Lysosome sensing was demonstrated using live human brain glioblastoma U87MG cell line, human cervical cancer HeLa cell line, and human esophagus premalignant CP-A and CP-D cell lines by observations of small acidic spherical organelles (lysosomes) and significant colocalizations (82–95%) of the sensors with a commercially available lysosome-selective staining probe LysoTracker Red® under confocal fluorescence microscopy.
Keywords: Optical sensors; pH sensor; Naphthalimide derivatives; Lysosome sensor; Intracellular pH sensing; Extracellular sensing;

A systematic investigation was performed on regulating materials properties and cell behavior using hybrid networks composed of amorphous poly(propylene fumarate) (PPF) and three poly(ɛ-caprolactone) diacrylates (PCLDAs) with variance in crystallinity and melting temperature. Through controlling both crosslinking density and crystallinity in the photo-crosslinked PPF/PCLDA blends, mechanical properties could be tuned efficiently in a wide range. For PCLDA synthesized from a low-molecular weight PCL diol precursor with a low crystallinity and a low melting point, crosslinks could completely suppress crystalline domains over the composition range in the PPF/PCLDA networks. Consequently, tensile, shear, torsional, and compression moduli all increased with the composition of PPF or the crosslinking density continuously for amorphous PPF/PCLDA networks. For PCLDAs synthesized using two PCL diols with higher molecular weights, crystallinity remained for the PCLDA compositions between ∼80% and 100%. Minimum moduli and tensile stress at break were found at the lowest required composition of PPF for suppressing crystallinity. Surface physicochemical properties and morphology of the crosslinked blend disks have been characterized and their capabilities of adsorbing proteins from cell culture medium have been determined. Using both mouse MC3T3-E1 cells and rat Schwann cell precursor line (SpL201) cells, cell responses to these polymer networks such as cell adhesion, spreading, and proliferation were found to be dramatically distinct on different polymer networks and demonstrated non-monotonic or parabolic dependence on the network composition, coincident with the composition dependence of the mechanical properties.
Keywords: Poly(propylene fumarate) (PPF); Poly(ɛ-caprolactone) diacrylate (PCLDA); Photo-crosslinking; Substrate stiffness; Cell–biomaterial interaction;

Comb-type grafted poly(N-isopropylacrylamide) gel modified surfaces for rapid detachment of cell sheet by Zhonglan Tang; Yoshikatsu Akiyama; Masayuki Yamato; Teruo Okano (7435-7443).
A comb-type grafted poly(N-isopropylacrylamide) (PIPAAm) gel modified surface was newly developed for providing a rapid cell sheet recovery for tissue engineering. PIPAAm macromonomer was prepared by the etherification reaction of the hydroxyl terminal moieties of PIPAAm with acryloyl chloride, followed by the radical telomerization reaction of N-isopropylacrylamide (IPAAm) monomer using 2-mercaptoethanol as a chain transfer agent. Solution containing IPAAm monomer and PIPAAm macromonomer was spread on the surface of tissue culture polystyrene (TCPS), and then the surface was subjected to electron beam irradiation for grafting the monomer and macromonomer on the surfaces, resulting in comb-type grafted PIPAAm gel modified TCPS (GG-TCPS). Besides the difference of the amount of the modified PIPAAm, no distinct difference was found between the properties of GG-TCPSs and normal-type PIPAAm gel modified TCPS (NG-TCPS) through XPS, AFM and a contact angle measurement. At 37 °C, bovine aortic endothelial cells (BAECs) were well adhered and spread on GG-TCPS as well as NG-TCPS regardless of the macromonomer concentration. By lowering temperature to 20 °C, BAECs detached themselves more rapidly from GG-TCPS compared with NG-TCPS. Upon lowering temperature, the grafted polymer was speculated to accelerate the hydration of modified PIPAAm gel, resulting in a rapid cell sheet detachment.
Keywords: Macromonomer; Comb-type grafted poly(N-isopropylacrylamide) gel; Temperature-responsive cell culture surface; Cell sheet; Tissue engineering;

The modulation of dendritic cell integrin binding and activation by RGD-peptide density gradient substrates by Abhinav P. Acharya; Natalia V. Dolgova; Nicole M. Moore; Chang-Qing Xia; Michael J. Clare-Salzler; Matthew L. Becker; Nathan D. Gallant; Benjamin G. Keselowsky (7444-7454).
Dendritic cells (DCs) are central regulators of the immune system that operate in both innate and adaptive branches of immunity. Activation of DC by numerous factors, such as danger signals, has been well established. However, modulation of DC functions through adhesion-based cues has only begun to be characterized. In this work, DCs were cultured on surfaces presenting a uniform gradient of the integrin-targeting RGD peptide generated using the recently established “universal gradient substrate for click biofunctionalization” methodology. Surface expression of activation markers (costimulatory molecule CD86 and stimulatory molecule MHC-II) and production of cytokines IL-10 and IL-12p40 of adherent DCs was quantified in situ. Additionally, bound α V integrin was quantified in situ using a biochemical crosslinking/extraction method. Our findings demonstrate that DCs upregulated CD86, MHC-II, IL-10, IL-12p40 and αV integrin binding as a function of RGD surface density, with production of IL-12p40 being the marker most sensitive to RGD surface density. Surface expression of activation markers demonstrated moderate correlation with αV integrin binding, while cytokine production was highly correlated with αV integrin binding. This work demonstrates the utility of the surface density gradient platform as a high-throughput method to investigate RGD density-dependent DC adhesive responses. Furthermore, this quantitative analysis of DC integrin-based activation represents a first of its type, helping to establish the field of adhesion-based modulation of DCs as a general mechanism that has previously not been defined, and informs the rational design of biomimetic biomaterials for immunomodulation.
Keywords: Dendritic cell; Cell adhesion; Biomimetic; RGD peptide; High-throughput; Immune response;

Rapid construction of mechanically- confined multi- cellular structures using dendrimeric intercellular linker by Xuejun Mo; Qiushi Li; Lena Wai Yi Lui; Baixue Zheng; Chiang Huen Kang; Bramasta Nugraha; Zhilian Yue; Rui Rui Jia; Hong Xia Fu; Deepak Choudhury; Talha Arooz; Jie Yan; Chwee Teck Lim; Shali Shen; Choon Hong Tan; Hanry Yu (7455-7467).
Tissue constructs that mimic the in vivo cell–cell and cell–matrix interactions are especially useful for applications involving the cell- dense and matrix- poor internal organs. Rapid and precise arrangement of cells into functional tissue constructs remains a challenge in tissue engineering. We demonstrate rapid assembly of C3A cells into multi- cell structures using a dendrimeric intercellular linker. The linker is composed of oleyl- polyethylene glycol (PEG) derivatives conjugated to a 16 arms- polypropylenimine hexadecaamine (DAB) dendrimer. The positively charged multivalent dendrimer concentrates the linker onto the negatively charged cell surface to facilitate efficient insertion of the hydrophobic oleyl groups into the cellular membrane. Bringing linker- treated cells into close proximity to each other via mechanical means such as centrifugation and micromanipulation enables their rapid assembly into multi- cellular structures within minutes. The cells exhibit high levels of viability, proliferation, three- dimensional (3D) cell morphology and other functions in the constructs. We constructed defined multi- cellular structures such as rings, sheets or branching rods that can serve as potential tissue building blocks to be further assembled into complex 3D tissue constructs for biomedical applications.
Keywords: Rapid dendrimer; Oleyl poly (ethylene glycol) (oleyl-PEG); Cell- ECM interaction; Multicellular structures; Defined cellular structures; Tissue engineering;

The non-covalent decoration of self-assembling protein fibers by Zahra N. Mahmoud; Daniel J. Grundy; Kevin J. Channon; Derek N. Woolfson (7468-7474).
The design of self-assembling fibers presents challenges in basic science, and has potential for developing materials for applications in areas such as tissue engineering. A contemporary issue in the field is the construction of multi-component, functionalized systems. Previously, we have developed peptide-based fibers, the SAF system, that comprises two complementary peptides, which affords considerable control over assembly and morphology. Here we present a straightforward route to functionalizing the SAFs with small molecules and, subsequently, other moieties. This is achieved via non-covalent recruitment of charged peptide tags, which offers advantages such as further control, reversibility, and future prospects for developing recombinant tags. We demonstrate the concept by appending fluorescent labels and biotin (and thence gold nanoparticles) to the peptides, and visualising the resulting decorated SAFs by light and electron microscopy. The peptide tags bind in the nm–μm range, and show specificity compared with control peptides, and for the SAFs over similar α-helix-based peptide fibers.
Keywords: Biomimetic material; Peptide; Protein; Self-assembly; TEM (transmission electron microscopy);

Functional skeletal muscle formation with a biologic scaffold by Jolene E. Valentin; Neill J. Turner; Thomas W. Gilbert; Stephen F. Badylak (7475-7484).
Biologic scaffolds composed of extracellular matrix (ECM) have been used to reinforce or replace damaged or missing musculotendinous tissues in both preclinical studies and in human clinical applications. However, most studies have focused upon morphologic endpoints and few studies have assessed the in-situ functionality of newly formed tissue; especially new skeletal muscle tissue. The objective of the present study was to determine both the in-situ tetanic contractile response and histomorphologic characteristics of skeletal muscle tissue reconstructed using one of four test articles in a rodent abdominal wall model: 1) porcine small intestinal submucosa (SIS)-ECM; 2) carbodiimide-crosslinked porcine SIS-ECM; 3) autologous tissue; or 4) polypropylene mesh. Six months after surgery, the remodeled SIS-ECM showed almost complete replacement by islands and sheets of skeletal muscle, which generated a similar maximal contractile force to native tissue but with greater resistance to fatigue. The autologous tissue graft was replaced by a mixture of collagenous connective tissue, adipose tissue with fewer islands of skeletal muscle compared to SIS-ECM and a similar fatigue resistance to native muscle. Carbodiimide-crosslinked SIS-ECM and polypropylene mesh were characterized by a chronic inflammatory response and produced little or no measurable tetanic force. The findings of this study show that non-crosslinked xenogeneic SIS scaffolds and autologous tissue are associated with the restoration of functional skeletal muscle with histomorphologic characteristics that resemble native muscle.
Keywords: Muscle; SIS (small intestine submucosa); Xenotransplantation; ECM (extracellular matrix);

We investigated the inflammatory response to, and the osteoinductive efficacies of, four polymers (collagen, Ethisorb™, PLGA and Polyactive®) that bore either an adsorbed (fast-release kinetics) or a calcium-phosphate-coating-incorporated (slow-release kinetics) depot of BMP-2. Titanium-plate-supported discs of each polymer (n = 6 per group) were implanted at an ectopic (subcutaneous) ossification site in rats (n = 48). Five weeks later, they were retrieved for a histomorphometric analysis of the volumes of ectopic bone and foreign-body giant cells (a gauge of inflammatory reactivity), and the degree of polymer degradation. For each polymer, the osteoinductive efficacy of BMP-2 was higher when it was incorporated into a coating than when it was directly adsorbed onto the material. This mode of BMP-2 carriage was consistently associated with an attenuation of the inflammatory response. For coated materials, the volume density of foreign-body giant cells was inversely correlated with the volume density of bone (r 2 = 0.96), and the volume density of bone was directly proportional to the surface-area density of the polymer (r 2 = 0.97). Following coating degradation, other competitive factors, such as the biocompatibility and the biodegradability of the polymer itself, came into play.
Keywords: Biocompatibility; Calcium-phosphate coating; Controlled drug release; Foreign-body giant cells; Inflammation; Osteogenesis;

Modified Gellan Gum hydrogels with tunable physical and mechanical properties by Daniela F. Coutinho; Shilpa V. Sant; Hyeongho Shin; João T. Oliveira; Manuela E. Gomes; Nuno M. Neves; Ali Khademhosseini; Rui L. Reis (7494-7502).
Gellan Gum (GG) has been recently proposed for tissue engineering applications. GG hydrogels are produced by physical crosslinking methods induced by temperature variation or by the presence of divalent cations. However, physical crosslinking methods may yield hydrogels that become weaker in physiological conditions due to the exchange of divalent cations by monovalent ones. Hence, this work presents a new class of GG hydrogels crosslinkable by both physical and chemical mechanisms. Methacrylate groups were incorporated in the GG chain, leading to the production of a methacrylated Gellan Gum (MeGG) hydrogel with highly tunable physical and mechanical properties. The chemical modification was confirmed by proton nuclear magnetic resonance (1H NMR) and Fourier transform infrared spectroscopy (FTIR-ATR). The mechanical properties of the developed hydrogel networks, with Young’s modulus values between 0.15 and 148 kPa, showed to be tuned by the different crosslinking mechanisms used. The in vitro swelling kinetics and hydrolytic degradation rate were dependent on the crosslinking mechanisms used to form the hydrogels. Three-dimensional (3D) encapsulation of NIH-3T3 fibroblast cells in MeGG networks demonstrated in vitro biocompatibility confirmed by high cell survival. Given the highly tunable mechanical and degradation properties of MeGG, it may be applicable for a wide range of tissue engineering approaches.
Keywords: Methacrylated Gellan Gum; Physical and chemical crosslinking; Biodegradable hydrogel; Mechanical properties; Tissue engineering;

Effects of RGDS sequence genetically interfused in the silk fibroin light chain protein on chondrocyte adhesion and cartilage synthesis by Yusuke Kambe; Koji Yamamoto; Katsura Kojima; Yasushi Tamada; Naohide Tomita (7503-7511).
Initial chondrocyte–silk fibroin interactions are implicated in chondrogenesis when using fibroin as a scaffold for chondrocytes. Here, we focused on integrin-mediated cell–scaffold adhesion and prepared cell adhesive fibroin in which a tandem repeat of the Arg-Gly-Asp-Ser (RGDS) sequence was genetically interfused in the fibroin light chain (L-chain) (L-RGDS×2 fibroin). We investigated the effects of the sequence on chondrocyte adhesion and cartilage synthesis, in comparison to the effects of fibronectin. As the physicochemical surface properties (e.g., wettability and ζ potential) of the fibroin substrate were not affected by the modification, specific cell adhesion to the RGDS predominately changed the chondrocyte adhesive state. This suggestion was also supported by the competitive inhibition of chondrocyte attachment to the L-RGDS×2 fibroin substrate with soluble RGD peptides in the medium. Unlike fibronectin, the expression of RGDS in the fibroin L-chain had no effect on chondrocyte spreading area but enhanced mRNA expression levels of integrins α5 and β1, and aggrecan at 12 h after seeding. Although both the sequence and fibronectin increased cell adhesive force, chondrocytes grown on the fibroin substrate exhibited a peak in the force with time in culture. These results suggested that moderate chondrocyte adhesion to fibroin induced by the RGDS sequence was able to maintain the chondrogenic phenotype and, from the histology findings, the sequence could facilitate chondrogenesis.
Keywords: Cell adhesion; Chondrocyte; Scaffold; Silk; RGD peptide;

The use of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) scaffolds for tarsal repair in eyelid reconstruction in the rat by Jing Zhou; Si-Wu Peng; Yuan-Yuan Wang; Song-Bin Zheng; Yang Wang; Guo-Qiang Chen (7512-7518).
Tarsal repair is an important part for eyelid reconstruction. Presently traditional clinic treatments do not produce satisfactory repair effects. The key is to find a proper tarsal repair material. Microbial poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) was studied for application as tarsal substitute in this study. PHBHHx scaffolds were implanted into tarsal defects of Sprague-Dawley rats. Eyelid samples of implanted materials and blank defect controls were collected for histological examination at weekly intervals post surgery. Results were compared among PHBHHx scaffolds, commercial acellular dermal matrices (ADM) and blank defect controls. Both PHBHHx scaffolds and ADM provided satisfactory repair results compared with the blank controls even though the implanted PHBHHx scaffolds showed a 2 weeks inflammation. Fibrous encapsulation and scaffold degradation were observed for the PHBHHx implants. Combined with its strong, elastic mechanical properties, the tissue compatible and biodegradable PHBHHx was proven to be a suitable candidate for tarsal repair.
Keywords: PHB; Polyhydroxyalkanoates; PHBHHx; Tarsal substitutes; Eyelid reconstruction;

Osteoclastogenesis in peripheral blood mononuclear cell cultures of periprosthetic osteolysis patients and the phenotype of T cells localized in periprosthetic tissues by Ilaria Roato; Davide Caldo; Lucia D’Amico; Patrizia D’Amelio; Laura Godio; Salvatore Patanè; Franco Astore; Guido Grappiolo; Maurizio Boggio; Roberto Scagnelli; Luigi Molfetta; Riccardo Ferracini (7519-7525).
Arthroplasty is a very successful medical procedure. Failures depend on aseptic loosening caused by periprosthetic osteolysis, where T cells have a contradictory role. We analyzed osteoclastogenesis in peripheral blood mononuclear cell (PBMC) cultures of periprosthetic osteolysis patients and the phenotype of T cells localized in periprosthetic tissues. We enrolled 45 subjects with periprosthetic osteolysis (15), stable prosthesis (15) and healthy controls (15). We performed PBMC cultures to study osteoclastogenesis. Osteoclasts and T cell phenotype were examined by immunohistochemistry, immunofluorescence and flow citometry. Periprosthetic osteolysis patients showed spontaneous osteoclastogenesis, which was inhibited by RANK-Fc and T cell depletion. In periprosthetic osteolysis patients’ PBMC cultures, CD4 and CD8 T cells increased and CD8 T cells did not express CD25. In periprosthetic tissues T cells were close to osteoclasts, suggesting their interaction. Local CD8 T cells showed a regulatory phenotype, expressing CD25 and FoxP3, while CD4 T cells did not express activation markers. Our data suggest that, in an early stage of periprosthetic osteolysis, T cells may promote osteoclastogenesis, whereas subsequently osteoclasts activate FoxP3/CD8 T cells, which inhibit CD4 effector T cells. This mechanism may explain the previous finding of non-active T cells in periprosthetic tissues.
Keywords: Periprosthetic osteolysis; T cell; Osteoclast; Spontaneous osteoclastogenesis; Arthroplasty;

The cellular responses of Escherichia coli to visible light photocatalysis were characterized by chemical, optical, electron-beam, and surface-force techniques, to elucidate the mechanisms of photocatalytic inactivation of E. coli on PdO/TiON fiber. The characterization techniques included chemical assays, fluorescence microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM). Fluorescence microscopy using the Live/Dead BacLight™ kit indicates that the photocatalytic treatment resulted in severe membrane damage to the E. coli cells. SEM, AFM and TEM revealed drastic defects in the morphology and internal sub-structure of the bacterial cells after the treatments. Combining data from our previous reports on the antimicrobial properties of visible-light-activated PdO/TiON photocatalyst, the present results point to oxidative attack from the exterior to the interior of the bacteria by hydroxyl radicals as the primary mechanism of photocatalytic inactivation.
Keywords: Antimicrobial; Titanium oxide; Cytotoxicity; SEM; TEM; Nanoindentation;

The application of complex multiple forklike ZnO nanostructures to rapid and ultrahigh sensitive hydrogen peroxide biosensors by Zao Yang; XiaoLing Zong; Zhizhen Ye; Binghui Zhao; QingLing Wang; Ping Wang (7534-7541).
A new complex multiple forklike ZnO nanostructure was fabricated by citric acid assisted annealing process. The complex multiple forklike ZnO nanostructure is composed of several nanorods. These nanorods grow from a thin platelet base and are parallel to each other to form complex multiple forklike ZnO nanostructure. The widthes of thin platelet bases range from 60 to 230 nm. The diameters of the nanorods range from 16 to 60 nm, and their lengths are 0.5 μ∼ 1.3μ. FT-IR spectrum, room-temperature PL, and UV–Vis absorption spectra are also discussed. A possible growth mechanism is proposed. Complex multiple forklike ZnO nanostructure was first used to construct a novel enzymatic biosensor based on the ZnO/CHIT inorganic–organic composite film. Well-studied horseradish peroxidase (HRP) was chosen as a model enzyme. The as-prepared biosensor displays ultrahigh sensitivity, quick response time, low detection limit and small apparent Michaelis–Menten constant. These results show that the Complex multiple forklike ZnO nanostructure is a promising material to construct enzyme biosensors.
Keywords: Electrochemistry; Crystal growth; Biosensor; Enzyme;

The importance of endo-lysosomal escape with lipid nanocapsules for drug subcellular bioavailability by Archibald Paillard; François Hindré; Caroline Vignes-Colombeix; Jean-Pierre Benoit; Emmanuel Garcion (7542-7554).
To establish the therapeutic relevance of new nanocarriers, rationalization of knowledge on their interactions with biological structures is essential. In the present study, we have investigated endocytosis and intracellular trafficking of lipid nanocapsules (LNCs) in rat glioma cells. Radiolabelled and fluorescent LNCs were synthesized by using a phase inversion process that follows the formation of an oil/water microemulsion containing triglycerides, lecithins and a non-ionic surfactant, the hydroxystearate of poly(ethylene glycol) (HS-PEG). Our data revealed that LNCs were rapidly accumulated within cells (from 2 min exposure) through active and saturating mechanisms involving endogenous cholesterol with a major contribution of clathrin/caveolae-independent pathways. Although initially present in endosomes, LNCs can bypass the endo-lysosomal compartment with only 10% of the cell-internalized fraction found in isolated lysosomes after 2 h exposure. As demonstrated by use of lysosomal probes, LNCs reverted lysosome integrity similarly to V-ATPase inhibitors and in a size-dependent fashion with best efficiency for small nanoparticles. When loaded with paclitaxel, smallest LNCs also triggered the best cell death activity. Those LNC properties are ascribed to the proportion of HS-PEG they provided to the cell. They are important to consider toward the development of nanomedicines that use drugs sensitive to lysosomal degradation or that need to reach extra endo-lysosomal targets.
Keywords: Nanoparticles; Cancer therapy; Intracellular drug delivery; Subcellular trafficking; Surfactant;

We report a class of core–shell structured hybrid nanogels to demonstrate the conception of integrating the functional building blocks into a single nanoparticle system for simultaneously optical temperature-sensing, cancer cell targeting, fluorescence imaging, and combined chemo-photothermal treatment. The hybrid nanogels were constructed by coating the Ag–Au bimetallic NP core with a thermo-responsive nonlinear poly(ethylene glycol) (PEG)-based hydrogel as shell, and semi-interpenetrating the targeting ligands of hyaluronic acid chains into the surface networks of gel shell. The Ag–Au NP core can emit strong visible fluorescence for imaging of mouse melanoma B16F10 cells. The reversible thermo-responsive volume phase transition of the nonlinear PEG-based gel shell cannot only modify the physicochemical environment of the Ag–Au NP core to manipulate the fluorescence intensity for sensing the environmental temperature change, but also provide a high loading capacity for a model anticancer drug temozolomide and offer a thermo-triggered drug release. The drug release can be induced by both the heat generated by external NIR irradiation and the temperature increase of local environmental media. The ability of the hybrid nanogels to combine the local specific chemotherapy with external NIR photothermal treatment significantly improves the therapeutic efficacy due to a synergistic effect.
Keywords: Poly(ethylene glycol); Hybrid nanogel; Ag–Au bimetallic nanoparticle; Optical temperature-sensing; Tumor cell imaging; Drug delivery;

The in vivo performance of plasmonic nanobubbles as cell theranostic agents in zebrafish hosting prostate cancer xenografts by Daniel S. Wagner; Nikki A. Delk; Ekaterina Y. Lukianova-Hleb; Jason H. Hafner; Mary C. Farach-Carson; Dmitri O. Lapotko (7567-7574).
Cell theranostics is a new approach that unites diagnosis, therapy and confirmation (guidance) of the results of therapy in one single process at cell level, thus principally improving both the rapidity and precision of treatment. The ideal theranostic agent will support all three of the above functions in vivo with cellular resolution, allowing individual assessment of disease state and the elimination of diseased cells while leaving healthy cells intact. We have developed and evaluated plasmonic nanobubbles (PNBs) as an in vivo tunable theranostic cellular agent in zebrafish hosting prostate cancer xenografts. PNBs were selectively generated around gold nanoparticles in cancer cells in the zebrafish with short single laser pulses. By varying the energy of the laser pulse, we dynamically tuned the PNB size in a theranostic sequence of two PNBs: an initial small PNB detected a cancer cell through optical scattering, followed by a second bigger PNB, which mechanically ablated this cell without damage to surrounding tissue, while its optical scattering confirmed the destruction of the cell. Thus PNBs supported the diagnosis and guided ablation of individual human cancer cells in a living organism without damage to the host.
Keywords: Theranostics; Cell; Plasmonic nanobubble; Gold nanoparticle; Laser; Cancer;

The highly efficient delivery of exogenous proteins into cells mediated by biodegradable chimaeric polymersomes by Guijing Liu; Shoubao Ma; Shaoke Li; Ru Cheng; Fenghua Meng; Haiyan Liu; Zhiyuan Zhong (7575-7585).
Biodegradable chimaeric polymersomes based on asymmetric PEG-PCL-PDEA triblock copolymers were prepared and investigated for delivery of exogenous proteins into cells. PEG-PCL-PDEA copolymers with M n PEG = 5 kg/mol, M n PCL = 18.2 kg/mol, and short PDEA blocks ranging from 1.1, 2.7 to 4.1 kg/mol (denoted as copolymer 1, 2 and 3, respectively) were obtained by controlled reversible addition-fragmentation chain transfer (RAFT) polymerization. The direct hydration of copolymer thin films in MES buffer (pH 5.3) yielded uniform polymersomes with sizes of 130–175 nm. These polymersomes had close to neutral zeta potentials (−2 ∼ +2.7 mV) at pH 7.4. The polymersomal structures were confirmed by confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and catalytic activity experiment on 3,3′,3″-phosphinidyne(trisbenzenesulfonic acid)-loaded polymersomes. MTT assays showed that these polymersomes were non-toxic up to a concentration of 0.5 mg/mL. These chimaeric polymersomes, in particular polymersome 2, showed remarkably high protein loading efficiencies and loading contents for bovine serum albumin (BSA), cytochrome C (CC), lysozyme (Lys), ovalbumin (OVA) and immunoglobulin G (IgG). The encapsulation of proteins did not significantly alter the polymersome size distributions and zeta potentials. The protein release studies showed that both BSA and CC were released in a controlled manner. Importantly, the released CC fully maintained its activity. Notably, CLSM studies showed that FITC-CC loaded polymersomes efficiently delivered and released proteins into the cytoplasm of RAW 264.7 cells. Moreover, these chimaeric polymersomes were able to simultaneously load and transport proteins and doxorubicin into the cytoplasm as well as the cell nucleus. We are convinced that these biodegradable chimaeric polymersomes have great potentials in protein therapy.
Keywords: Biodegradable; Polymersomes; Protein delivery; Drug delivery; Intracellular release; Cancer therapy;

Synergic effects of crypt-like topography and ECM proteins on intestinal cell behavior in collagen based membranes by Lin Wang; Shashi K. Murthy; Gilda A. Barabino; Rebecca L. Carrier (7586-7598).
The basement membrane of small intestinal epithelium possesses complex topography at multiple scales ranging from the mesoscale to nanoscale. Specifically, intestinal crypt-villus units are comprised of hundred-micron-scale well-like invaginations and finger-like projections; intestinal cell phenotype is related to location on this crypt-villus unit. A biomimetic intestinal cell culture system composed of type I collagen based permeable cell culture membranes incorporating both micron-scale intestinal crypt-like topography and nanometer scale topography was fabricated. Membranes were pre-incubated with either laminin (Ln) or fibronectin (Fn), inoculated with intestinal epithelial Caco-2 cells and cultured for 1–21 days to study the relative significance of influence of crypt-like topography and biomimetic substrate chemistry on cell phenotype. Crypt-like topography inhibited Caco-2 differentiation during early culture, as evidenced by slower cell spreading and lower brush border enzyme activity. For example, alanine aminopeptidase activity was lower on Ln-coated patterned collagen (∼3.4 ± 0.24 mU/mg) compared to flat collagen (10.84 ± 0.55 mU/mg) at day 7. Caco-2 cultured on Fn-coated collagen started to spread earlier (1 day vs 3 days) and formed longer protrusions than on Ln-coated collagen. Pre-coating of Ln enhanced cell differentiation, as the maximum activity of a cell differentiation marker (alkaline phosphatase) was 2–3 times higher than on Fn-coated collagen, and maintained differentiated phenotype in long term (up to 21 days) culture. In general, compared to substrate topography, coating with ECM protein had more prominent and longer effect on cell behavior. Crypt-like topography affected Caco-2 spreading and differentiation during early culture, however the effect diminished as culture progressed. This information will benefit intestinal tissue engineering scaffold design, and modification of in vitro intestinal cell models.
Keywords: Surface topography; Epithelial cell; Collagen; Laminin; Fibronectin;