Biomaterials (v.32, #6)

Nanocapsules incorporating IgG Fc-binding domain derived from Staphylococcus aureus protein A for displaying IgGs on immunosensor chips by Masumi Iijima; Hiroyasu Kadoya; Satoko Hatahira; Shingo Hiramatsu; Giman Jung; Aaron Martin; John Quinn; Joohee Jung; Seong-Yun Jeong; Eun Kyung Choi; Takeshi Arakawa; Fumiyo Hinako; Masanobu Kusunoki; Nobuo Yoshimoto; Tomoaki Niimi; Katsuyuki Tanizawa; Shun’ichi Kuroda (1455-1464).
To enhance the sensitivities and antigen-binding capacities of immunosensors, oriented immobilization of antibodies on the surface of the sensor chip is critical, but to date, this has not been adequately achieved. We describe a way of adsorbing immunoglobulin (Ig) proteins onto 32-nm bio-nanocapsules (BNCs) through IgG Fc-binding domains derived from Staphylococcus aureus protein A (ZZ-BNC). This arrangement permits ∼60 molecules of mouse total IgG bind to ZZ-BNC and all the IgG Fv regions to be displayed outwardly for the effective binding of antigens. ZZ-BNCs adsorbed onto the gold surface of the sensor chip of the quartz crystal microbalance (QCM) could markedly enhance the sensitivity and antigen-binding capacity of the chip. On the sensor chip of surface plasmon resonance (SPR), antibodies on the ZZ-BNCs showed higher affinities to each antigen than those on protein A. The BNC-coated sensor chip is very stable, and should prove useful for various immunosensor applications due to oriented immobilization of antibodies.
Keywords: Biosensor; Gold; Nanoparticle; Scaffold; Self assembly; Surface modification;

Characterization of membrane materials and membrane coatings for bioreactor units of bioartificial kidneys by Ming Ni; Jeremy C.M. Teo; Mohammed Shahrudin bin Ibrahim; Kangyi Zhang; Farah Tasnim; Pei-Yong Chow; Daniele Zink; Jackie Y. Ying (1465-1476).
The bioreactor unit of bioartificial kidneys contains porous membranes seeded with renal cells. For clinical applications, it is mandatory that human primary renal proximal tubule cells (HPTCs) form differentiated epithelia on the membranes. Here, we show that HPTCs do not grow and survive on a variety of polymeric membrane materials. This applies also to membranes consisting of polysulfone/polyvinylpyrrolidone (PSF/PVP), which have been used in the bioreactor unit of bioartificial kidneys after coating with an extracellular matrix (ECM). Our data reveal that coating with just an ECM does not sufficiently improve HPTC performance on non-HPTC-compatible membrane materials. On the other hand, we have characterized the effects of a variety of surface treatments and coatings, and found that double coating with 3,4-dihydroxy-l-phenylalanine and an ECM markedly improves HPTC performance and results in the formation of differentiated epithelia on PSF/PVP membranes. We have also synthesized alternative membrane materials, and characterized membranes consisting of polysulfone and Fullcure™. We found that these membranes sustain proper HPTC performance without the need for surface treatments or coatings. Together, our data reveal that the materials that have been previously applied in bioartificial kidneys are not suitable for applications with HPTCs. This study elucidates the types of membrane materials and coatings that are favorable for the bioreactor unit of bioartificial kidneys.
Keywords: Biocompatibility; Bioreactor; Epithelial cell; Membrane; Polysulphone; Surface treatment;

High-throughput generation of hydrogel microbeads with varying elasticity for cell encapsulation by Alexander Kumachev; Jesse Greener; Ethan Tumarkin; Erika Eiser; Peter W. Zandstra; Eugenia Kumacheva (1477-1483).
Elasticity of cellular microenvironments strongly influences cell motility, phagocytosis, growth and differentiation. Currently, the relationship between the cell behaviour and matrix stiffness is being studied for cells seeded on planar substrates, however in three-dimensional (3D) microenvironments cells may experience mechanical signalling that is distinct from that on a two-dimensional matrix. We report a microfluidic approach for high-throughput generation of 3D microenvironments with different elasticity for studies of cell fate. The generation of agarose microgels with different elastic moduli was achieved by (i) introducing into a microfluidic droplet generator two streams of agarose solutions, one with a high concentration of agarose and the other one with a low concentration of agarose, at varying relative volumetric flow rate ratios of the two streams, and (ii) on-chip gelation of the precursor droplets. At 37 °C, the method enabled a ∼35-fold variation of the shear elastic modulus of the agarose gels. The application of the method was demonstrated by encapsulating two mouse embryonic stem cell lines within the agarose microgels. This work establishes a foundation for the high-throughput generation of combinatorial microenvironments with different mechanical properties for cell studies.
Keywords: Hydrogel; Microfluidics; Microencapsulation; Mechanical properties; Stem cell;

Exploiting bacterial peptide display technology to engineer biomaterials for neural stem cell culture by Lauren E. Little; Karen Y. Dane; Patrick S. Daugherty; Kevin E. Healy; David V. Schaffer (1484-1494).
Stem cells are often cultured on substrates that present extracellular matrix (ECM) proteins; however, the heterogeneous and poorly defined nature of ECM proteins presents challenges both for basic biological investigation of cell-matrix investigations and translational applications of stem cells. Therefore, fully synthetic, defined materials conjugated with bioactive ligands, such as adhesive peptides, are preferable for stem cell biology and engineering. However, identifying novel ligands that engage cellular receptors can be challenging, and we have thus developed a high throughput approach to identify new adhesive ligands. We selected an unbiased bacterial peptide display library for the ability to bind adult neural stem cells (NSCs), and 44 bacterial clones expressing peptides were identified and found to bind to NSCs with high avidity. Of these clones, four contained RGD motifs commonly found in integrin binding domains, and three exhibited homology to ECM proteins. Three peptide clones were chosen for further analysis, and their synthetic analogs were adsorbed on tissue culture polystyrene (TCPS) or grafted onto an interpenetrating polymer network (IPN) for cell culture. These three peptides were found to support neural stem cell self-renewal in defined medium as well as multi-lineage differentiation. Therefore, bacterial peptide display offers unique advantages to isolate bioactive peptides from large, unbiased libraries for applications in biomaterials engineering.
Keywords: Biomimetic material; ECM; Peptide; Stem cell;

In this study, hMSCs encapsulated in a fibrin hydrogel containing heparinized NPs loaded with TGF-β3 (100 ng/ml), or TGF-β3 (100 ng/ml) alone, were subjected to growth factor release and denaturation tests at one, two and four weeks in in vitro culture systems. Additionally, stem cell differentiation was assessed via RT-PCR, real-time quantitative PCR (qPCR), histology, and immunohistochemical assays. In the in vivo studies with nude mouse, when transplanted into nude mice, hMSCs embedded in fibrin hydrogels survived and proliferated more readily in those samples containing TGF-β3-loaded NPs, or TGF-β3 alone, compared to those containing only NPs or the fibrin hydrogel alone. Additionally, RT-PCR, real-time qPCR, histology, Western blotting, and immunohistochemistry analyses revealed that chondrocyte-specific extracellular matrix (ECM) genes and their proteins were expressed at high levels by hMSCs embedded in hydrogels containing TGF-β3-loaded NPs. Finally, the results observed in the rabbit animal model treated with hMSCs embedded in a fibrin hydrogel containing TGF-β3-loaded NPs were also evaluated by the RT-PCR, real-time qPCR, histology, Western blotting, and immunohistochemistry analyses. The in vitro and in vivo results indicated that transplanted hMSCs together with TGF-β3 may constitute a clinically efficient method for the regeneration of hyaline articular cartilage.
Keywords: Fibrin; Sustained release; Nanoparticles; hMSC; Animal model;

The influence of biological motifs and dynamic mechanical stimulation in hydrogel scaffold systems on the phenotype of chondrocytes by Taly P. Appelman; Joseph Mizrahi; Jennifer H. Elisseeff; Dror Seliktar (1508-1516).
Primary bovine chondrocytes and PEG-based hydrogels were used to investigate the effects of scaffold composition and architecture on the cellular response to large dynamic compressive strain stimulation. Proteins and proteoglycans were conjugated to functionalized poly(ethylene glycol) (PEG) and immobilized in PEG hydrogels to create bio-synthetic scaffolds. Second passage articular chondrocytes were encapsulated into four different scaffold compositions: PEG-Proteoglycan (PP), PEG-Fibrinogen (PF), PEG-Albumin (PA), and PEG only and subjected to 15% dynamic compressive strain at 1-Hz frequency. Cellular response was evaluated in terms of cell number, glycosaminoglycans (GAGs), collagen type II and collagen type I accumulation in the constructs following 24 h and 28 days of stimulated and static culture. Stimulation of the constructs resulted in an increase in the cell number in all scaffolds, with no statistical difference measured among them. Dynamic stimulation of PP, PF, PA and PEG constructs resulted in a respective increase in the GAGs by 33%, 53.4%, 240.5%, and 284.5%, compared to their static controls. The permissive PEG and PA scaffolds showed a significantly larger relative increase in the GAGs in comparison to the other scaffolds tested. Collagen type II content in the PF, PA and PEG constructs increased by 78%, 1266% and 896% respectively, compared to their static controls. Permissive constructs showed a significantly larger relative increase and final absolute values of GAGs and type II collagen, compared to the PF constructs. Immunostaining for collagen type I, an indicator for chondrocyte de-differentiation, indicated that stimulation inhibited its production. Correlation maps between scaffold properties highlighted the major differences between permissive and instructive scaffolds. These results support the hypothesis that both compressive strain and scaffold bioactivity have an important effect on the chondrocyte metabolic response to mechanical stimulation, and that the 3-D environment surrounding chondrocytes can actively participate in translating mechanical stimulation to the resident cells.
Keywords: Cartilage tissue engineering; Bioreactor; Hydrogel; Poly(ethylene glycol); Mechanotransduction;

The effect of elastin on chondrocyte adhesion and proliferation on poly (ɛ-caprolactone)/elastin composites by Nasim Annabi; Ali Fathi; Suzanne M. Mithieux; Penny Martens; Anthony S. Weiss; Fariba Dehghani (1517-1525).
The aim of this study was to demonstrate the effect of elastin on chondrocyte adhesion and proliferation within the structure of poly (ɛ-caprolactone) (PCL)/elastin composites. The homogenous 3D structure composites were constructed by using high pressure CO2 in two stages. Porous PCL structures with average pore sizes of 540 ± 21 μm and a high degree of interconnectivity were produced using gas foaming/salt leaching. The PCL scaffolds were then impregnated with elastin and cross-linked with glutaraldehyde (GA) under high pressure CO2. The effects of elastin and cross-linker concentrations on the characteristics of composites were investigated. Increasing the elastin concentration from 25 mg/ml to 100 mg/ml elevated the amount of cross-linked elastin inside the macropores of PCL. Fourier transform infrared (FTIR) analysis showed that elastin was homogeneously distributed throughout the 3D structure of all composites. The weight gain of composites increased 2-fold from 15.8 ± 0.3 to 38.3 ± 0.7 (w/w) % by increasing the elastin concentration from 25 mg/ml to 50 mg/ml and approached a plateau above this concentration. The presence of elastin within the pores of PCL improved the water uptake properties of PCL scaffolds; the water uptake ratio of PCL was enhanced 100-fold from 0.030 ± 0.005 g liquid/g polymer to 11.80 ± 0.01 g liquid/g polymer, when the elastin solution concentration was 50 mg/ml. These composites exhibited lower compressive modulus and energy loss compared to pure PCL scaffolds due to their higher water content and elasticity. In vitro studies show that these composites can support primary articular cartilage chondrocyte adhesion and proliferation within the 3D structures. These results demonstrate the potential of using PCL/elastin composites for cartilage repair.
Keywords: Composite scaffold; Poly (ɛ-caprolactone); Elastin; Chondrocyte;

In vitro generation of an osteochondral interface from mesenchymal stem cell–collagen microspheres by Hiu-wa Cheng; Keith D.K. Luk; Kenneth M.C. Cheung; Barbara P. Chan (1526-1535).
Creating biological interfaces between mechanically dissimilar tissues is a key challenge in complex tissue engineering. An osteochondral interface is essential in preventing mechanical failure and maintaining normal function of cartilage. Despite tremendous efforts in developing osteochondral plugs, formation of the osteochondral interface with proper zonal organization has not yet been reported. Here, we present a mesenchymal stem cell–collagen microsphere-based approach for complex tissue engineering and demonstrate in vitro formation of a stem cell-derived osteochondral interface with calcified cartilage interface separating a non-calcified cartilage layer and an underlying bone layer. Cells at the interface region are hypertrophic chondrocytes while the extracellular matrix in this region contains collagen type II and X, calcium deposits and vertically running fibers. The simultaneous presence of appropriate medium and configuration during co-culture is necessary for the interface formation.
Keywords: Complex tissue engineering; Microencapsulation; Mesenchymal stem cells; Collagen microspheres; Osteochondral interface; Calcified cartilage;

Long-term in vitro human pancreatic islet culture using three-dimensional microfabricated scaffolds by Jamal T. Daoud; Maria S. Petropavlovskaia; Jason M. Patapas; Christian E. Degrandpré; Robert W. DiRaddo; Lawrence Rosenberg; Maryam Tabrizian (1536-1542).
Human pancreatic islet in vitro culture is very challenging and requires the presence of various extra cellular matrix (ECM) components in a three-dimensional environment, which provides mechanical and biological support. The development of such an environment is vital in providing favourable conditions to preserve human islets in long-term culture. In this study, we investigated the effects of human islet culture within various three-dimensional environments; collagen I gel, collagen I gel supplemented with ECM components fibronectin and collagen IV, and microfabricated scaffold with ECM-supplemented gel. The cultured human islets were analyzed for functionality, gene expression and hormone content following long-term in vitro culture. It was clear the incorporation of ECM components within the three-dimensional support improved prolonged culture. However, long-term and highly uniform human islet culture within a microfabricated scaffold, with controlled pore structures, coupled with the presence of ECM components, displayed an insulin release profile similar to freshly isolated islets, yielding a stimulation index of ∼1.8. Moreover, gene expression was markedly increased for all pancreatic genes, giving a ∼50-fold elevation of insulin gene expression with respect to suspension culture. The distribution and presence of pancreatic hormones was also highly elevated. These findings provide a platform for the long-term maintenance and preservation of human pancreatic islets in vitro.
Keywords: ECM(extracellular matrix); Microfabricated biodegradable scaffold; Long-term culture of human islets; Islet functionality;

High-density collagen gel tubes as a matrix for primary human bladder smooth muscle cells by Lionel A. Micol; Michael Ananta; Eva-Maria Engelhardt; Vivek C. Mudera; Robert A. Brown; Jeffrey A. Hubbell; Peter Frey (1543-1548).
Tissue-engineered grafts for the urinary tract are being investigated for the potential treatment of several urologic diseases. These grafts, predominantly tubular-shaped, usually require in vitro culture prior to implantation to allow cell engraftment on initially cell-free scaffolds. We have developed a method to produce tubular-shaped collagen scaffolds based on plastic compression. Our approach produces a ready cell-seeded graft that does not need further in vitro culture prior to implantation. The tubular collagen scaffolds were in particular investigated for their structural, mechanical and biological properties. The resulting construct showed an especially high collagen density, and was characterized by favorable mechanical properties assessed by axial extension and radial dilation. Young modulus in particular was greater than non-compressed collagen tubes. Seeding densities affected proliferation rate of primary human bladder smooth muscle cells. An optimal seeding density of 106 cells per construct resulted in a 25-fold increase in Alamar blue-based fluorescence after 2 wk in culture. These high-density collagen gel tubes, ready seeded with smooth muscle cells could be further seeded with urothelial cells, drastically shortening the production time of graft for urinary tract regeneration.
Keywords: Bladder tissue engineering; Cell proliferation; Collagen; Mechanical properties; Smooth muscle cell; Urinary tract;

Co-electrospun dual scaffolding system with potential for muscle–tendon junction tissue engineering by Mitchell R. Ladd; Sang Jin Lee; Joel D. Stitzel; Anthony Atala; James J. Yoo (1549-1559).
Tissue engineering has had successes developing single tissue types, but there is a need for methods that will allow development of composite tissues. For instance, muscle–tendon junctions (MTJ) require a seamless interface to allow force transfer from muscle to tendon. One challenge in engineering MTJs is designing a continuous scaffold suitable for both tissue types. We aimed to create a dual scaffold that exhibits regional mechanical property differences that mimic the trends seen in native MTJ. Poly(ε-caprolactone)/collagen and poly(l-lactide)/collagen were co-electrospun onto opposite ends of a mandrel to create a scaffold with 3 regions. Scaffolds were characterized with scanning electron microscopy, tensile testing (uniaxial, cyclic, and video strain), for cytocompatibility using MTS, and seeded with C2C12 myoblasts and NIH3T3 fibroblasts. Native porcine diaphragm MTJs were also analyzed with video strain for comparison. Integrated scaffolds were created with fiber diameters from 452–549 nm. Scaffolds exhibited regional variations in mechanical properties with moduli from 4.490–27.62 MPa and generally withstood cyclic testing, although with hysteresis. Video analysis showed scaffold strain profiles exhibited similar trends to native MTJ. The scaffolds were cytocompatible and accommodated cell attachment and myotube formation. The properties engineered into these scaffolds make them attractive candidates for tissue engineering of MTJs.
Keywords: Electrospinning; Mechanical properties; Muscle; Tendon; Strain profile; Composite tissue engineering;

The therapeutic potential of human multipotent mesenchymal stromal cells combined with pharmacologically active microcarriers transplanted in hemi-parkinsonian rats by Gaëtan J.-R. Delcroix; Elisa Garbayo; Laurence Sindji; Olivier Thomas; Claire Vanpouille-Box; Paul C. Schiller; Claudia N. Montero-Menei (1560-1573).
Multipotent mesenchymal stromal cells (MSCs) raise great interest for brain cell therapy due to their ease of isolation from bone marrow, their immunomodulatory and tissue repair capacities, their ability to differentiate into neuronal-like cells and to secrete a variety of growth factors and chemokines. In this study, we assessed the effects of a subpopulation of human MSCs, the marrow-isolated adult multilineage inducible (MIAMI) cells, combined with pharmacologically active microcarriers (PAMs) in a rat model of Parkinson’s disease (PD). PAMs are biodegradable and non-cytotoxic poly(lactic-co-glycolic acid) microspheres, coated by a biomimetic surface and releasing a therapeutic protein, which acts on the cells conveyed on their surface and on their microenvironment. In this study, PAMs were coated with laminin and designed to release neurotrophin 3 (NT3), which stimulate the neuronal-like differentiation of MIAMI cells and promote neuronal survival. After adhesion of dopaminergic-induced (DI)-MIAMI cells to PAMs in vitro, the complexes were grafted in the partially dopaminergic-deafferented striatum of rats which led to a strong reduction of the amphetamine-induced rotational behavior together with the protection/repair of the nigrostriatal pathway. These effects were correlated with the increased survival of DI-MIAMI cells that secreted a wide range of growth factors and chemokines. Moreover, the observed increased expression of tyrosine hydroxylase by cells transplanted with PAMs may contribute to this functional recovery.
Keywords: Laminin; Mesenchymal stromal cells; Tissue engineering; Neurotrophin 3; Parkinson’s disease; Pharmacologically active microcarriers;

A bioactive self-assembled membrane to promote angiogenesis by Lesley W. Chow; Ronit Bitton; Matthew J. Webber; Daniel Carvajal; Kenneth R. Shull; Arun K. Sharma; Samuel I. Stupp (1574-1582).
We report here on a bioactive hierarchically structured membrane formed by self-assembly. The membrane is formed with hyaluronic acid and peptide amphiphiles with binding affinity for heparin, and its hierarchical structure contains both an amorphous zone and a layer of fibrils oriented perpendicular to the membrane plane. The design of bioactivity is based on the potential ability to bind and slowly release heparin-binding growth factors. Human mesenchymal stem cells (hMSCs) seeded on these membranes attached and remained viable. Basic fibroblast growth factor (FGF2) and vascular endothelial growth factor (VEGF) were incorporated within the membrane structure prior to self-assembly and released into media over a prolonged period of time (14 days). Using the chicken chorioallantoic membrane (CAM) assay, we also found that these membranes induced a significant and rapid enhancement of angiogenesis relative to controls.
Keywords: Self-assembly; Angiogenesis; Heparin; Hyaluronic acid; Growth factors; Membrane;

Cell infiltration and growth in a low density, uncompressed three-dimensional electrospun nanofibrous scaffold by Bryan A. Blakeney; Ajay Tambralli; Joel M. Anderson; Adinarayana Andukuri; Dong-Jin Lim; Derrick R. Dean; Ho-Wook Jun (1583-1590).
A limiting factor of traditional electrospinning is that the electrospun scaffolds consist entirely of tightly packed nanofiber layers that only provide a superficial porous structure due to the sheet-like assembly process. This unavoidable characteristic hinders cell infiltration and growth throughout the nanofibrous scaffolds. Numerous strategies have been tried to overcome this challenge, including the incorporation of nanoparticles, using larger microfibers, or removing embedded salt or water-soluble fibers to increase porosity. However, these methods still produce sheet-like nanofibrous scaffolds, failing to create a porous three-dimensional scaffold with good structural integrity. Thus, we have developed a three-dimensional cotton ball-like electrospun scaffold that consists of an accumulation of nanofibers in a low density and uncompressed manner. Instead of a traditional flat-plate collector, a grounded spherical dish and an array of needle-like probes were used to create a Focused, Low density, Uncompressed nanoFiber (FLUF) mesh scaffold. Scanning electron microscopy showed that the cotton ball-like scaffold consisted of electrospun nanofibers with a similar diameter but larger pores and less-dense structure compared to the traditional electrospun scaffolds. In addition, laser confocal microscopy demonstrated an open porosity and loosely packed structure throughout the depth of the cotton ball-like scaffold, contrasting the superficially porous and tightly packed structure of the traditional electrospun scaffold. Cells seeded on the cotton ball-like scaffold infiltrated into the scaffold after 7 days of growth, compared to no penetrating growth for the traditional electrospun scaffold. Quantitative analysis showed approximately a 40% higher growth rate for cells on the cotton ball-like scaffold over a 7 day period, possibly due to the increased space for in-growth within the three-dimensional scaffolds. Overall, this method assembles a nanofibrous scaffold that is more advantageous for highly porous interconnectivity and demonstrates great potential for tackling current challenges of electrospun scaffolds.
Keywords: Scaffold; Biomimetic material; Extracellular matrix (ECM); Nanofibers; Tissue engineering;

Stimulation of cell growth and resistance to apoptosis in vascular smooth muscle cells on a chondroitin sulfate/epidermal growth factor coating by Cindy Charbonneau; Benoît Liberelle; Marie-Josée Hébert; Gregory De Crescenzo; Sophie Lerouge (1591-1600).
Deficient healing after endovascular aneurysm repair is thought to be related to the pro-apoptotic environment in abdominal aortic aneurysms and inertness of the graft materials. A bioactive coating containing both chondroitin sulfate (CS) and epidermal growth factor (EGF) was developed in order to increase the growth and resistance to apoptosis of vascular smooth muscle cells (VSMC) on biomaterials surfaces. CS and EGF were covalently grafted using carbodiimide chemistry and the coating was characterized and optimized using ellipsometry, static contact angle and ToF-SIMS. Its potential to improve cell adhesion, growth and resistance to apoptosis was assessed in vitro with rat aortic VSMC. Results showed that CS and EGF immobilization allowed for the creation of a uniform coating that increased cell adhesion, growth and resistance to apoptosis in serum-free medium. Overall, CS and EGF possess great potential as bioactive anti-apoptotic mediators for vascular repair.
Keywords: Apoptosis; Bioactive coating; Chondroitin sulfate; Endovascular aneurysm repair; Epidermal growth factor; Vascular smooth muscle cells;

Gold nanorods (GNRs) have been widely used for bio-imaging. However, GNRs assisted optical in vivo deep tissue imaging is severely restricted due to signal attenuation, low contrast, complex process or low real-timing. To overcome these problems, we functionalized GNRs with both near-infrared (NIR) fluorescence and surface enhanced Raman scattering (SERS) and utilized these co-functionalized GNRs for purely optical in vivo imaging of live mice. Our proposed technology has the combined advantages of high real-timing, high imaging contrast and deep detection ability. The distribution and excretion of intravenously injected GNRs in deep tissues of live mice were observed in vivo for the first time through purely optical imaging. We also demonstrated successfully in vivo biomedical applications of the co-functionalized GNRs to sentinel lymph node (SLN) mapping and tumor targeting of mice. The present technology has great future potentials for disease diagnosis and clinical therapies.
Keywords: Gold nanorods; Near-infrared; Fluorescence; Surface enhanced Raman scattering; Purely optical; In vivo imaging;

Direct evidence for catalase and peroxidase activities of ferritin–platinum nanoparticles by Jia Fan; Jun-Jie Yin; Bo Ning; Xiaochun Wu; Ye Hu; Mauro Ferrari; Gregory J. Anderson; Jingyan Wei; Yuliang Zhao; Guangjun Nie (1611-1618).
Using apoferritin (apoFt) as a nucleation substrate, we have successfully synthesized 1–2 nm platinum nanoparticles (Pt–Ft) which are highly stable. By directly measuring the products of Pt–Ft-catalyzed reactions, we showed, with no doubt, Pt–Ft possesses both catalase and peroxidase activities. With hydrogen peroxide as substrate, we observed oxygen gas bubbles were generated from hydrogen peroxide decomposed by Pt–Ft; the generation of oxygen gas strongly supports Pt–Ft reacts as catalase, other than peroxidase. While with organic dyes and hydrogen peroxide as substrates, distinctive color products were formed catalyzed by Pt–Ft, which indicates a peroxidase-like activity. Interestingly, these biomimetic properties showed differential response to pH and temperature for different reaction substrates. Pt–Ft showed a significant increase in catalase activity with increasing pH and temperature. The HRP-like activity of Pt–Ft was optimal at physiological temperature and slightly acidic conditions. Our current study demonstrates that Pt–Ft possesses both catalase and peroxidase activities for different substrates under different conditions.
Keywords: Biomimetic enzymes; Catalase activities; ESR; Ferritin; Nanoparticles;

A serum-resistant polyamidoamine-based polypeptide dendrimer for gene transfection by H.M. Wu; S.R. Pan; M.W. Chen; Y. Wu; C. Wang; Y.T. Wen; X. Zeng; C.B. Wu (1619-1634).
A serum tolerant polycation gene vector, G2 PAMAM-PGlu-G1 PAMAMs (ALA), was designed, synthesized, characterized and evaluated. A honeycomb-like molecular structure model for mechanistic explanation of ALA was postulated and discussed. Designed as a star-shaped polyamidoamine (PAMAM)-based polypeptide dendrimer through peptide bond linkages, ALA was with non-toxic low generation G2 PAMAM (G2) as its central core, polyglutamate (PGlu)s as its star-shaped backbone branches and G1 PAMAM (G1)s as its branch grafts and peripheral terminals. IR, 1H NMR demonstrated its successful combination. As a gene carrier, ALA exhibited good DNA binding and condensation capacity with particle size (∼87 nm for N/P 40, ∼170 nm for N/P 30) and ζ-potential (∼16 mV for N/P 30–40), negligible cytotoxicity, exciting serum tolerant capacity and significant serum-promoted (serum-containing 56.6%>serum-free 32.7%), cell line dependent (Hek 293 > Bel 7402 > Hela), incubation period dependent (38 h > 18 h > 12 h > 9 h > 4 h > 2 h > 1 h) and sustained (peak transfection appeared at 30 h incubation) transfection efficiency. The presence of serum had not only no inhibition on, but also prominent promotion to, the transfection activity of ALA. All above features differentiated ALA clearly from most other serum-inhibitive nonviral gene carriers, and proved ALA the promising and challenging potential efficient gene vector for practical clinical application.Display Omitted
Keywords: Polyamidoamine; Poly amino acid; Polycation; Serum-resistant; Gene transfer; Surface modification;

Curcumin-decorated nanoliposomes with very high affinity for amyloid-β1-42 peptide by Spyridon Mourtas; Mara Canovi; Cristiano Zona; Dario Aurilia; Anna Niarakis; Barbara La Ferla; Mario Salmona; Francesco Nicotra; Marco Gobbi; Sophia G. Antimisiaris (1635-1645).
Amyloid β (Aβ) aggregates are considered as possible targets for therapy and/or diagnosis of Alzheimer disease (AD). It has been previously shown that curcumin targets Aβ plaques and interferes with their formation, suggesting a potential role for prevention or treatment of AD. Herein, a click chemistry method was used to generate nanoliposomes decorated with a curcumin derivative, designed to maintain the planar structure required for interaction with Aβ, as directly confirmed by Surface Plasmon Resonance experiments. Another type of liposomes was formed starting from curcumin-phospholipid conjugate, in which the planar structure of curcumin is disrupted. Both types of generated curcumin-decorated vesicles had mean diameters in the nano range (131–207 nm) and slightly negative ζ-potential values according to their lipid composition, and were stable for periods up to 20 days. They also demonstrated high integrity during incubation in presence of plasma proteins. Surface Plasmon Resonance experiments, measuring the binding of flowing liposomes to immobilized Aβ1-42, indicated that the liposomes exposing the curcumin derivative (maintaining the planarity) have extremely high affinity for Aβ1-42 fibrils (1–5 nM), likely because of the occurrence of multivalent interactions, whereas those exposing non-planar curcumin did not bind to Aβ1-42. In summary, we describe here the preparation and characterization of new nanoparticles with a very high affinity for Aβ1-42 fibrils, to be exploited as vectors for the targeted delivery of new diagnostic and therapeutic molecules for AD.
Keywords: Alzheimer disease (AD); Amyloid beta (Aβ); Curcumin; Click chemistry; Structure; Liposomes;

Protein adsorption and complement activation for di-block copolymer nanoparticles by Christine Vauthier; Bjorn Persson; Peter Lindner; Bernard Cabane (1646-1656).
Four types of nanoparticles with core-diffuse shell structures have been synthesized through self-assembly of PICBA-Dextran block copolymers. These nanoparticles are designed to carry pharmaceutically active molecules into the human body through injection into the blood stream. In this work, we have determined how the characteristics of the diffuse shell influence the adsorption of three types of proteins: Bovine Serum Albumin (BSA), fibrinogen, and a protein from the complement system that triggers recognition and elimination by macrophages. We have determined the structural characteristics of the diffuse shells using Nuclear Magnetic Resonance (NMR), Small Angle Neutron Scattering (SANS) and Quasi-Elastic Light Scattering (QELS). We have measured the adsorption of Bovine Serum Albumin (BSA) through Immunodiffusion methods, and found that it adsorbed in substantial amounts even when the distance between dextran chains at the core-diffuse shell interface is quite short. We have observed the aggregation of the nanoparticles induced by fibrinogen, and found that it was prevented when the density of dextran chains protruding from the core surface was sufficiently high. Finally we have measured the activation of the complement system by the nanoparticles, and found that it was also limited by the surface density of dextran chains that protrude from the core and by their mesh size within the diffuse shell.
Keywords: Core-diffuse shell nanoparticles; Mesh size; BSA; Fibrinogen; C3; Adsorption isotherm;

Single and repeated dose toxicity of mesoporous hollow silica nanoparticles in intravenously exposed mice by Tianlong Liu; Linlin Li; Xu Teng; Xinglu Huang; Huiyu Liu; Dong Chen; Jun Ren; Junqi He; Fangqiong Tang (1657-1668).
Mesoporous hollow silica nanoparticles (MHSNs) are emerging as one of the new and promising nanomaterials for biomedical applications, but the biocompatibility of MHSNs in vivo has received little attention. In the present study, the systematic single and repeated dose toxicity, biodistribution and clearance of MHSNs in vivo were demonstrated after intravenous injection in mice. For single dose toxicity, lethal dose 50 (LD50) of 110 nm MHSNs was higher than 1000 mg/kg. Further repeated dose toxicity studies indicated no death was observed when mice were exposed to MHSNs at 20, 40 and 80 mg/kg by continuous intravenous administration for 14 days. These results suggest low toxicity of MHSNs when intravenous injection at single dose or repeated administrations. ICP–OES and TEM results show that the MHSNs mainly accumulate in mononuclear phagocytic cells in liver and spleen. In addition, these particles could be excreted from the body and the entire clearance time of the particles should be over 4 weeks. These findings would be useful for future development of nanotechnology-based drug delivery system and other biomedical applications.
Keywords: Mesoporous hollow silica; Biocompatibility; Biodistribution; Clearance; Repeated dose toxicity;

Co-axial electrospun fibers can offer both topographical and biochemical cues for tissue engineering applications. In this study, we demonstrate the sustained treatment of hemophilia through a non-viral, tissue engineering approach facilitated by growth factor-releasing co-axial electrospun fibers. FVIII-producing skeletal myotubes were first engineered on aligned electrospun fibers in vitro, followed by implantation in hemophilic mice with or without a layer of core-shell electrospun fibers designed to provide sustained delivery of angiogenic or lymphangiogenic growth factors, which serves to stimulate the lymphatic or vascular systems to enhance the FVIII transport from the implant site into systemic circulation. Upon subcutaneous implantation into hemophilic mice, the construct seamlessly integrated with the host tissue within one month, and specifically induced either vascular or lymphatic network infiltration in accordance with the growth factors released from the electrospun fibers. Engineered constructs that induced angiogenesis resulted in sustained elevation of plasma FVIII and significantly reduced blood coagulation time for at least 2-months. Biomaterials-assisted functional tissue engineering was shown in this study to offer protein replacement therapy for a genetic disorder such as hemophilia.
Keywords: Hemophilia; Skeletal muscle engineering; Angiogenesis; Lymphangiogenesis; Electrospinning; Protein replacement therapy;

Construction of surfactant-like tetra-tail amphiphilic peptide with RGD ligand for encapsulation of porphyrin for photodynamic therapy by Jing-Xiao Chen; Hui-Yuan Wang; Cao Li; Kai Han; Xian-Zheng Zhang; Ren-Xi Zhuo (1678-1684).
A surfactant-like tetra-tail amphiphilic peptide, [(C18)2K]2KR8GRGDS was designed and synthesized for targeted drug delivery. The resulting peptide–amphiphile, consisting of four hydrophobic aliphatic tails and a hydrophilic peptide head group, was able to self-assemble into nanosized micelles in aqueous medium at low concentration. Ibuprofen and doxorubicin (DOX) was loaded into peptide micelles as model hydrophobic drugs respectively, and the sustained release behavior was observed. Due to the incorporation of targeted arginine-glycine-aspartic acid (RGD) sequences and cell penetrating peptide (CPP) residue octaarginine (R8), the micelles could be recognized specifically by cancer cells, as well as transport through the cell membrane efficiently. The observation of laser-scanning confocal microscopy confirmed effective cellular uptaking of porphyrin-loaded peptide micelles. Furthermore, the porphyrin-loaded micelles exhibited low dark toxicity and high phototoxicity against cancer cells, indicating the powerful potential for effective photodynamic therapy. Combined with the low cytotoxicity of the peptide against both HeLa and 293T cell lines, the surfactant-like peptide developed in this study may be promising in clinical application for targeted drug delivery.
Keywords: Amphiphilic peptide; Micelle; RGD ligand; Photodynamic therapy;

Highly porous large poly(lactic-co-glycolic acid) microspheres adsorbed with palmityl-acylated exendin-4 as a long-acting inhalation system for treating diabetes by Hyunuk Kim; Hongil Park; Juho Lee; Tae Hyung Kim; Eun Seong Lee; Kyung Taek Oh; Kang Choon Lee; Yu Seok Youn (1685-1693).
A porous large poly(lactic-co-glycolic acid) (PLGA) microspheres (MS) adsorbed with palmityl-acylated exendin-4 (Ex4-C16) was devised as an inhalation delivery system. The porous MS was prepared by a single o/w emulsification/solvent evaporation method using extractable Pluronic F68/F127, and its fabrication and formulation conditions were carefully optimized. Results show that the prepared MS was in the appropriate size range for inhalation and contained many surfaces and internal pores meaning low aerodynamic density. Ex4-C16 was more efficiently adsorbed onto porous PLGA MSs than native exendin-4, and an approximately 5% loading of Ex4-C16 onto this porous MS (RG504H) was achieved. This optimized porous MS was found to be efficiently deposited throughout the entire lungs of mice including alveoli region. Furthermore, this porous MS adsorbed with Ex4-C16 (approx. 100 μg/mouse) displayed much protracted hypoglycemic efficacy in non-fasted type 2 diabetic db/db mice. Porous PLGA MS with adsorbed Ex4-C16 showed the dual-advantages of (i) sustained release and acceptable drug-loading due to strong hydrophobic interaction and (ii) longer in vivo pulmonary hypoglycemic duration due to albumin-binding by the palmityl group. We consider that this new prototype of porous PLGA MS has considerable pharmaceutical potential as a type 2 anti-diabetic inhalation treatment.
Keywords: Inhalation; Pulmonary delivery; Porous microspheres; Albumin-binding; Palmityl-acylated exendin-4; Hydrophobic interaction;

Polyethylenimine-grafted copolymer of poly(l-lysine) and poly(ethylene glycol) for gene delivery by Jian Dai; Seyin Zou; Yuanyuan Pei; Du Cheng; Hua Ai; Xintao Shuai (1694-1705).
A major challenge in gene therapy is the development of effective gene delivery vectors with low toxicity. In the present study, linear poly(ethylenimine) (lPEI) with low molecular weight was grafted onto the block copolymer (PPL) of poly(l-lysine) (PLL) and poly(ethylene glycol)(PEG), yielding a ternary copolymer PEG-b-PLL-g-lPEI (PPI) for gene delivery. In such molecular design, PLL, lPEI and PEG blocks were expected to render the vector biodegradability, proton buffering capacity, low cationic toxicity and potentially long circulation in vivo, respectively. Given proper control of molecular composition, the copolymers demonstrated lower cytotoxicity, proton buffering capacity, ability to condense pDNA and mediate effective gene transfection in various cell lines. With folate as an exemplary targeting ligand, the FA-PPI/pDNA complex showed much higher transgene activity than its nontargeting counterpart for both reporter and therapeutic genes in folate receptor(FR)-positive cells. FA-PPI mediated effective transfection of the TNF-related apoptosis-inducing ligand gene (TRAIL) in human hepatoma Bel 7402 cells, leading to cell apoptosis and great suppression of cell viability. Our results indicate that the copolymers might be a promising vector combining low cytotoxicity, biodegradability, and high gene transfection efficiency.
Keywords: Gene therapy; Poly(l-lysine); Linear polyethylenimine; Folate targeting; TRAIL gene;

Direct and indirect effects of functionalised fluorescence-labelled nanoparticles on human osteoclast formation and activity by Andrea Tautzenberger; Ludwika Kreja; Anke Zeller; Steffen Lorenz; Hubert Schrezenmeier; Volker Mailänder; Katharina Landfester; Anita Ignatius (1706-1714).
Recently, it was demonstrated that phosphonate-functionalised nanoparticles were successfully taken up by mesenchymal stem cells without influencing their viability and differentiation capacity, suggesting that they may provide a promising basis for the development of nanoparticles for drug delivery or cell labelling. The present study aimed to investigate the effects of these nanoparticles on osteoclast formation and activity as well as on the inflammatory response of osteoclasts and osteoblasts. The intracellular uptake of the particles by human osteoclasts and osteoblasts was demonstrated by confocal laser scanning microscopy, transmission electron microscopy and fluorescence microscopy. The expression of tartrate-resistant acid phosphatase, carboanhydrase II, cathepsin K, calcitonin receptor and osteoclast-specific vacuolar proton pump subunit TCIRG1 as well as actin ring formation were not significantly altered in osteoclasts by particle treatment, as demonstrated by cytochemical staining and immunostaining. Active calcium phosphate resorption by osteoclasts was also not significantly influenced by the particles. The expression and secretion of pro-inflammatory cytokines (IL-6, IL-1β) by osteoclasts and osteoblasts and the expression of osteoclast-regulating genes (M-CSF, OPG, RANKL) in osteoblasts were similarly not significantly affected. In conclusion, phosphonate-functionalised nanoparticles did not affect osteoclast formation and activity either directly or indirectly, suggesting that they could provide a promising tool for the development of particle-based treatments for anti-resorptive therapies.
Keywords: Nanoparticle; Osteoclast; Multi-nucleated cell; Resorption activity; Osteoblast;

The immunogenicity of a vaccine formulation is closely related to the effective internalization by the innate immune cells that provide prolonged and simultaneous delivery of antigen and adjuvant to relevant antigen presenting cells. Endosome associated TLR9 recognizes microbial unmethylated CpG DNA. Clinical applications of TLR9 ligands are significantly hampered due to their pre-mature in vivo digestion and rapid clearance. Liposome encapsulation is a powerful tool to increase in vivo stability as well as enhancing internalization of its cargo to relevant immune cells. The present study established that encapsulating CpG motifs in different liposomes having different physicochemical properties altered not only encapsulation efficiency, but also the release and delivery rates that ultimately impacted in vitro and ex-vivo cytokine production rates and types. Moreover, different liposomes encapsulating CpG ODN significantly increased Th1-biased cytokines and chemokines gene transcripts Additional studies demonstrated that co-stimulatory and surface marker molecules significantly upregulated upon liposome/CpG injection. Finally, co-encapsulating model antigen ovalbumin with CpG ODN adjuvant in nanoliposomes profoundly augmented Th1 and cell mediated anti-Ova specific immune response. Collectively, this work established an unappreciated immunoregulatory property of nanoliposomes mediating immunity against protein antigen and could be harnessed to design more effective therapeutic vaccines or stand alone immunoprotective agents targeting infectious diseases, as well as cancer or allergy.
Keywords: Liposomes; Vaccine; Immune response; CpG DNA; TLR; Nanotechnology;

Hexon-specific PEGylated adenovirus vectors utilizing avidin-biotin interaction by Emi Suzuki-Kouyama; Kazufumi Katayama; Fuminori Sakurai; Tomoko Yamaguchi; Shinnosuke Kurachi; Kenji Kawabata; Shinsaku Nakagawa; Hiroyuki Mizuguchi (1724-1730).
PEGylation of recombinant adenovirus (Ad) vectors is a promising approach for not only evasion from neutralizing anti-Ad antibodies and uptake by phagocytic cells, but also prolongation of the blood retention time of Ad vectors after systemic administration. However, the conventional PEGylation leads to significant reduction in the transduction activity of Ad vectors, probably because PEG is nonspecifically conjugated to the Ad capsid protein and inhibits the binding of Ad vectors to the primary receptor, coxsackievirus-adenovirus receptor (CAR). In order to PEGylate an Ad vector without significant reduction in the transduction activity, the biotin-binding peptide (BAP) was inserted into the hypervariable region (HVR) 5 of the hexon, which is not involved in the binding to CAR, and PEG was then specifically conjugated to the hexon HVR5 via avidin-biotin interaction. In vitro transduction experiments demonstrated that the hexon-specific PEGylation did not cause an apparent reduction in the transduction efficiency of the Ad vector, although the insertion of the BAP into the HVR5 itself reduced the transduction efficiency by 50-fold, compared with the conventional Ad vector, in the absence of anti-Ad serum. In the presence of anti-Ad serum, the transduction with the Ad vector with the BAP in the hexon HVR5 was significantly blocked; however, anti-Ad serum only slightly inhibited the transduction with the hexon-specifically PEGylated Ad vector (Ad-BAP/Bio/Avi/Bio-PEG-L2). Intravenous administration of Ad-BAP/Bio/Avi/Bio-PEG-L2 resulted in prolonged blood retention, significant reduction in the transduction in the liver, and accumulation in the tumor; however, unexpectedly, the transduction efficiency of Ad-BAP/Bio/Avi/Bio-PEG-L2 in the tumor was almost at the background level.
Keywords: Gene therapy; Drug delivery; Polyethylene; Gene expression; Surface modification;

Effects of particle size on toll-like receptor 9-mediated cytokine profiles by Helen C. Chen; Bingbing Sun; Kenny K. Tran; Hong Shen (1731-1737).
Biomaterials interface with toll-like receptor (TLR) 9-mediated innate immunity in a wide range of medical applications, such as tissue implants and drug delivery systems. The stimulation of TLR9 can lead to two different signaling pathways, resulting in the generation of proinflammatory cytokines (i.e. IL-6) and/or type I interferons (IFNs, i.e. IFN-α). These two categories of cytokines differentially influence both innate and adaptive immunity. Although particle size is known to be a critical parameter of biomaterials, its role in TLR9-mediated cytokine profiles is not clear. Here, we examined how the size of biomaterials impacted cytokine profiles by using polystyrene particles of defined sizes as model carriers for TLR9 agonists (CpG oligonucleotides (CpG ODNs)). CpG ODNs bound to nano- to submicro- particles stimulated the production of both IL-6 and IFN-α, while those bound to micro particles resulted in IL-6 secretions only. The differential TLR9-mediated cytokine profiles were attributed to the pH of endosomes that particles trafficked to. The magnitude of IFN-α production was highly sensitive to the change in endosomal pH in comparison to that of IL-6. Our results define two critical design variables, size and the ability to modulate endosomal pH, for the engineering of biomaterials that potentially interface with TLR9-mediated innate immunity. The fine control of these two variables will allow us to fully exploit the beneficial facets of TLR9-mediated innate immunity while minimizing undesirable side effects.
Keywords: Non-viral DNA delivery; Toll-like receptor; Vaccine; Immunotherapy; Tissue engineering;

A reduction-sensitive linear cationic click polymer (RCP) was specially designed for the efficient gene delivery to overcome multidrug resistance (MDR) by RNA interference to silence the expression of P-glycoprotein (P-gp). RCP was synthesized via the “click chemistry” with disulfide bonds, amide–triazole moieties and secondary amine groups in the main chain. RCP could efficiently condense pDNA into nanoparticles (RCPNs) around 150 nm. Polyplex dissociation was observed in the presence of 2.5 mm DTT due to the cleavage of disulfide bonds, which indicated the efficient DNA release under the reduction condition. In vitro transfection and cytotoxicity experiments against human breast cancer MCF-7 cells and drug-resistant MCF-7/ADR cells showed that RCPNs could bring about higher transfection efficiency with much lower cytotoxicity than PEI/DNA nanoparticles. RCPNs loaded with plasmid iMDR1-pDNA could inhibit P-gp expression, increase adriamycin (ADR) accumulation and enhance cytotoxicity of ADR against MCF-7/ADR cells. Combination of RCPNs and ADR could suppress the tumor growth more efficiently than using ADR only on mouse xenograft model bearing ADR resistant human breast cancer. These results suggested that this RCP could be a potential, safe and efficient non-viral vector for reversing MDR.
Keywords: Click chemistry; RNA interference; Disulfide bond; P-glycoprotein; Multidrug resistance;

The labeling of cationic iron oxide nanoparticle-resistant hepatocellular carcinoma cells using targeted magnetoliposomes by Stefaan J.H. Soenen; Alain R. Brisson; Eveline Jonckheere; Nele Nuytten; Sisareuth Tan; Uwe Himmelreich; Marcel De Cuyper (1748-1758).
The in vitro labeling of cultured cells with nanomaterials is a frequent practice but the efficiency, specificity and cytotoxicity of labeling specific cell types using targeted nanoparticles has only rarely been investigated. In the present work, functionalized anionic lipid-coated iron oxide cores (magnetoliposomes (MLs)) bearing galactose moieties were used for the specific labeling of asialoglycoprotein receptor 1 (ASGPR-1)-expressing HepG2 cells. The optimal number of galactose moieties per particle (±26) was determined and uptake efficiency was compared with galactose-lacking anionic and cationic MLs. Using a blocking assay with free galactose, electron microscopy and co-cultures of HepG2 and non-ASGPR-1 expressing C17.2 cells, the specificity of the particles for the ASGPR-1 receptor was demonstrated. The intracellular localization of the galactose-bearing MLs was further verified by confocal microscopy. The non-toxic ML concentration was determined to be 400 μg Fe/ml. Finally, the use of these MLs for visualization of labelled cells by magnetic resonance imaging (MRI) was demonstrated. The data show a high uptake and specificity of the galactose-bearing MLs, whereas the cationic MLs remain primarily surface-associated. Thus, targeted MLs offer a successful alternative for cell labeling when cationic particles fail to be efficiently internalized.
Keywords: (Iron oxide) Nanoparticle; Magnetoliposome; Cytotoxicity; Cell labeling; Biomedical materials;

Controlled insulin release from glucose-sensitive self-assembled multilayer films based on 21-arm star polymer by Xingyu Chen; Wei Wu; Zhizhang Guo; Jianyu Xin; Jianshu Li (1759-1766).
A glucose-sensitive multilayer film was fabricated by the layer-by-layer (LbL) assembly method with positively charged 21-arm poly[2-(dimethylamino)ethyl methacrylate] (star PDMAEMA) and negatively charged insulin and glucose oxidase (GOD) in the form of {(Star PDMAEMA/Insulin)4 + (Star PDMAEMA/GOD)4 + Star PDMAEMA}. The multilayer film shows an on-off regulation of insulin release in response to stepwise glucose challenge in vitro. It is found that the unique structure of star PDMAEMA and interdiffusion of charged insulin are the main factors to control the on-off status of the film. Reversible surface morphology transitions of the multilayer film were also observed, revealing a phase separation and large-scale reorganization process. Furthermore, the multilayer film could continuously release enough insulin in vivo after being subcutaneously implanted in streptocozotin-induced diabetic rats and reduce the blood glucose level for at least two weeks. It is indicated that such system may have substantial potential as a glucose-sensitive carrier for insulin due to its distinct mechanism.
Keywords: Controlled release; Insulin; Multilayer films; Layer-by-layer assembly; 21-Arm star polymer; Animal experiments;