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In the biomaterials field, one major issue with hydrogels (HGs) loaded with active therapeutics is the spontaneous leaking of the cargo occurring rapidly, usually over several hours. However, biological processes involved in regenerative medicine would require to have a sustained drug delivery lasting over weeks/months or be triggered only when a specific biochemical stimulus (enzymatic, cellular, or antimicrobial) is applied. In this work, this challenge is addressed by demonstrating that the spontaneous protein release from an agarose HG (used as model HG) can be partially or totally blocked by the incorporation of stellate mesoporous silica (STMS) nanoparticles (NPs, ≈150 ± 28 nm size, 15 nm pore) within the HG. It is shown here that the porous silica NPs act as sub‐micrometer size reservoirs ensuring precise level retention simply by playing on the amount of STMS embedded in the HG. Further, this effect is shown for various proteins demonstrating the versatility of this concept. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer (CRC) is one of the most commonly diagnosed cancers, posing a serious public health challenge that necessitates the development of new therapeutics, therapies, and prevention methods. Among the various therapeutic approaches, interventions involving lactic acid bacteria (LAB) as probiotics and postbiotics have emerged as promising candidates for treating and preventing CRC. While human-isolated LAB strains are considered highly favorable, those sourced from environmental reservoirs such as dairy and fermented foods are also being recognized as potential sources for future therapeutics. Results: In this study, we present a novel and therapeutically promising strain, Lactococcus lactis ssp. lactis Lc4, isolated from dairy sources. Lc4 demonstrated the ability to release the cytostatic agent - arginine deiminase (ADI) - into the post-cultivation supernatant when cultured under conditions mimicking the human gut environment. Released arginine deiminase was able to significantly reduce the growth of HT-29 and HCT116 cells due to the depletion of arginine, which led to decreased levels of c-Myc, reduced phosphorylation of p70-S6 kinase, and cell cycle arrest. The ADI release and cytostatic properties were strain-dependent, as was evident from comparison to other L. lactis ssp. lactis strains. Conclusion: For the first time, we unveil the anti-proliferative properties of the L. lactis cell-free supernatant (CFS), which are independent of bacteriocins or other small molecules. We demonstrate that ADI, derived from a dairy-Generally Recognized As Safe (GRAS) strain of L. lactis, exhibits anti-proliferative activity on cell lines with different levels of argininosuccinate synthetase 1 (ASS1) expression. A unique feature of the Lc4 strain is also its capability to release ADI into the extracellular space. Taken together, we showcase L. lactis ADI and the Lc4 strain as promising, potential therapeutic agents with broad applicability. [ABSTRACT FROM AUTHOR]

Glycosylphosphatidylinositol (GPI)-anchored proteins (APs) are anchored at the outer leaflet of plasma membranes (PMs) of all eukaryotic organisms studied so far by covalent linkage to a highly conserved glycolipid rather than a transmembrane domain. Since their first description, experimental data have been accumulating for the capability of GPI-APs to be released from PMs into the surrounding milieu. It became evident that this release results in distinct arrangements of GPI-APs which are compatible with the aqueous milieu upon loss of their GPI anchor by (proteolytic or lipolytic) cleavage or in the course of shielding of the full-length GPI anchor by incorporation into extracellular vesicles, lipoprotein-like particles and (lyso)phospholipid- and cholesterol-harboring micelle-like complexes or by association with GPI-binding proteins or/and other full-length GPI-APs. In mammalian organisms, the (patho)physiological roles of the released GPI-APs in the extracellular environment, such as blood and tissue cells, depend on the molecular mechanisms of their release as well as the cell types and tissues involved, and are controlled by their removal from circulation. This is accomplished by endocytic uptake by liver cells and/or degradation by GPI-specific phospholipase D in order to bypass potential unwanted effects of the released GPI-APs or their transfer from the releasing donor to acceptor cells (which will be reviewed in a forthcoming manuscript). [ABSTRACT FROM AUTHOR]

Using hydrogels to control the long-term release of protein remains challenging, especially for in-situ forming formulations. The uncontrollable burst release in the initial phase, the halted release in the subsequent phase, and the undesired drug dumping at the late stage are some obstacles hydrogel-based depots commonly encounter. In this study, we report hydrolyzable dextran-based hydrogels crosslinked by Michael addition to demonstrate a systematic solution to solve these problems. First, the polymer concentration was used as the critical parameter to control the proportion of releasable versus physically trapped protein molecules in the initial hydrogel meshwork. Subsequently, the dynamic change of the hydrogel meshwork was modulated by the crosslinking density and the cleavage rate of ester linkers. To this end, we designed and synthesized a series of ester linkers with hydrolytic half-life ranging from 4 h to 4 months and incorporate them into the hydrogel. Controlled release was demonstrated for model proteins varied in size, including lysozyme (14 kDa), bovine serum albumin (66 kDa), immunoglobulin G (150 kDa), and bevacizumab (149 kDa). In particular, sustained release of IgG ranging from 10 days to 8 months was achieved. Lastly, a tunable multi-phase release profile was made feasible by incorporating multiple ester linkers into one hydrogel formulation. The linker's half-life determined each phase's release duration, and the linkers' mixing ratio determined the corresponding release fraction. The reported hydrogel design engenders a versatile platform to address the needs for long-term and readily adjustable protein release for biomedical applications. [Display omitted] [ABSTRACT FROM AUTHOR]

Biomacromolecules based injectable and self-healing hydrogels possessing high mechanical properties have widespread potential in biomedical field. However, dynamic features are usually inversely proportional to toughness. It is challenging to simultaneously endow these properties to the dynamic hydrogels. Here, we fabricated an injectable nanocomposite hydrogel (CS-NPs@OSA-l-Gtn) stimultaneously possessing excellent autonomous self-healing performance and high mechanical strength by doping chitosan nanoparticles (CS-NPs) into dynamic polymer networks of oxidized sodium alginate (OSA) and gelatin (Gtn) in the presence of borax. The synergistic effect of the multiple reversible interactions combining dynamic covalent bonds (i.e., imine bond and borate ester bond) and noncovalent interactions (i.e., electrostatic interaction and hydrogen bond) provide effective energy dissipation to endure high fatigue resistance and cyclic loading. The dynamic hydrogel exhibited excellent mechanical properties like maximum 2.43 MPa compressive strength, 493.91 % fracture strain, and 89.54 kJ/m3 toughness. Moreover, the integrated hydrogel after injection and self-healing could withstand 150 successive compressive cycles. Besides, the bovine serum albumin embedded in CS-NPs could be sustainably released from the nanocomposite hydrogel for 12 days. This study proposes a novel strategy to synthesize an injectable and self-healing hydrogel combined with excellent mechanical properties for designing high-strength natural carriers with sustained protein delivery. [Display omitted] [ABSTRACT FROM AUTHOR]

Starch is one of the most frequently preferred natural polymers in hydrogel synthesis. Herein, we combined two strategies of associating brittle and ductile networks in a structure and incorporating inorganic particles into the polymeric gel to design mechanically enhanced nanocomposite double network (DN) starch gels. For the first time in the literature, nanocomposite starch gels (s-NC) were designed by cross-linking starch chains with 8-armed glycidyl-polyhedral oligomeric silsesquioxane (g-POSS) units. Fourier Transform Infrared Spectroscopy and Energy Dispersive X-Ray Spectroscopy analyses have proven that g-POSS is included in the gel structure and is homogeneously distributed throughout the network. More stable d-NC-DMA and d-NC-VP gels were obtained by incorporating N,N -dimethylacrylamide (DMA), or 1-vinyl-2-pyrrolidinone (VP) units, respectively, into g-POSS-linked starch gels, and the reaction kinetics were followed in situ. In SEM images, it was observed that d-NC-DMA had smaller pores and thicker pore walls compared to s-NC and d-NC-VP starch gels, and its mechanical strength was shown to be much superior by rheological tests, compression, and tensile analyses. In addition to increasing the mechanical strength of the gels, the potential of starch in protein release applications using amylase sensitivity has been demonstrated in vitro experiments using the model protein BSA. [ABSTRACT FROM AUTHOR]

Functional materials that can recognize the tumor microenvironment, characterized by acidic or reducing conditions, are needed for the designing of drug delivery carriers for cancer treatment. Hydrogels are potential protein drug carriers because they contain a large amount of water and stimuli-responsive functions can easily be introduced in them. However, it is difficult to introduce multi-stimuli-responsive functions and degradability at the same time. Here, we synthesized thermo- and pH-responsive hydrogels via a coupling reaction between poly(ethylene glycol) diglycidyl ether (PEGDE) and cystamine (CA). The prepared hydrogels showed lower critical solution temperature-type thermoresponsive behavior and pH-responsive swelling changes due to the protonation of secondary and/or tertiary amino groups arising from the crosslinking agent CA. Under reducing conditions, the hydrogels were degraded via the thiol exchange reaction in the presence of dithiothreitol or glutathione. The loading and release properties of FITC-labeled model proteins from the hydrogels were investigated. The loaded amount of the protein increased with decreasing molecular weight or hydrodynamic radius, which is based on the size of the network structure of the hydrogels. Notably, loaded proteins in the hydrogels were released only under reducing conditions, which mimic the tumor microenvironment. Thus, the prepared multi-responsive degradable hydrogels are expected to be used as functional drug delivery carriers for cancer treatment. Unlabelled Image [ABSTRACT FROM AUTHOR]

In mammals, interleukin 21 (IL-21) is a broad pleiotropic cytokine that plays critical roles in the development of several inflammatory and autoimmune diseases. In fish, functional information of Il-21 is limited, and its role in immune response is largely unknown. In the present study, we cloned a coding sequence of grass carp (Ctenopharyngodon idella) il21 gene (gcil21). To characterize the release patterns and biological activity of gcIl-21, we prepared recombinant gcIl-21 (rgcIl-21) and obtained the polyclonal antibody with gcIl-21 specificity. Western blotting analysis showed that in grass carp head kidney leukocytes (HKLs), gcIl-21 was undetected in culture supernatant of untreated cells but drastically induced by heat-killed Aeromonas hydrophila (A. hydrophila), uncovering the release features of gcIl-21 and its possible involvement in immune response. Subsequent functional experiments revealed that rgcIl-21 did not affect the mRNA expression of grass carp il1b and tgfb , but induced a strong expression of grass carp il10 , and to a lesser extent of grass carp tnfa in HKLs, suggesting a dominant effect of gcIl-21 in modulating Il-10 signaling as seen in rainbow trout and mammals. Furthermore, in vivo studies showed that intraperitoneal injection of rgcIl-21 was able to increase the survival rate of grass carp infected with live A. hydrophila , and reduce the pathological responses caused by the same pathogenic bacteria in head kidney and intestine. Taken together, these results for the first time revealed the close relationship of fish Il-21 production and function with inflammatory responses, and highlighted its anti-bacterial and anti-inflammatory ability, thereby providing a new insight into host defense mechanisms in fish. • Recombinant gcIl-21 (rgcIl-21) is prepared by using prokaryotic expression system. • Bacterial challenge drastically induces gcIl-21 release from head kidney leukocytes. • RgcIl-21 markedly induces il10 expression in head kidney leukocytes. • RgcIl-21 is able to increase the survival rate of bacteria-infected grass carp. • RgcIl-21 can improve bacteria-induced pathological responses in vivo. [ABSTRACT FROM AUTHOR]

PEG is the gold standard polymer for pharmaceutical applications, however it lacks degradability. Degradation under physiologically relevant pH as present in endolysosomes, cancerous and inflammatory tissues is crucial for many areas. The authors present anionic ring‐opening copolymerization of ethylene oxide with 3,4‐epoxy‐1‐butene (EPB) and subsequent modification to introduce acid‐degradable vinyl ether groups as well as methacrylate (MA) units, enabling radical cross‐linking. Copolymers with different molar ratios of EPB, molecular weights (Mn) up to 10 000 g mol−1 and narrow dispersities (Đ<1.05) were prepared. Both the P(EG‐co‐isoEPB)MA copolymer and the hydrogels showed pH‐dependent, rapid hydrolysis at pH 5–6 and long‐term storage stability at neutral pH (pH 7.4). By designing the degree of polymerization and content of degradable vinyl ether groups, the release time of an entrapped protein OVA‐Alexa488 can be tailored from a few hours to several days (hydrolysis half‐life time t1/2 at pH 5: 13 h to 51 h). [ABSTRACT FROM AUTHOR]

Platelet-derived growth factor-BB (PDGF-BB) is a promising molecule with a range of medical applications. However, the very rapid delivery of this growth factor may render treatment ineffective. A delivery system that is able to prolong the release rate is therefore required. In this study, a composite was fabricated from gelatin type IV and an aptamer. The aptamer worked as a binding ligand for PDGF-BB and was shown to slow the release rate. An inverted-tube test showed that the phase transition temperature of the gelatin increased in line with the gelatin concentration and in the presence of the coupling reagents N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide. Binding functionality of the aptamer was confirmed by surface plasmon resonance spectrometry. Rheological measurements indicated that the incorporation had no effect on the mechanical properties of the composite. The degradation of the composite and the kinetic release of the target proteins were investigated. The gelatin was shown to gradually dissolve in the release medium. The rate of PDGF-BB release from the aptamer-gelatin composite was significantly slower than from the native gelatin. This novel composite has potential applications in a range of PDGF-BB-related treatments. [ABSTRACT FROM AUTHOR]

In the context of sustainability transition to effectively utilize renewable biopolymers in building functional materials, we report a dynamic reversible nanocomposite hydrogel via thiolated galactoglucomannan (GGMSH) and TEMPO-oxidized cellulose nanofibrils (T-CNF) by deploying a UV-triggered thiol-disulfide exchange reaction. By adjusting the compositional content of GGMSH, the viscoelastic properties of the hydrogel precursors are tuneable and the crosslinking degree of disulfide bonds in these hydrogels dictates their mechanical properties upon UV irradiation. The nanocomposite hydrogels of GGMSH/T-CNF demonstrate robust self-healing properties, attributed to the dynamically reversible disulfide bonds and strong hydrogen bonds between T-CNF and GGMSH. For the hydrogel system of GGMSH/T-CNF/bovine serum albumin (BSA), the swelling behaviour of nanocomposite hydrogels largely dominated the release of BSA out of the hydrogels, suppressing the effect of redox-responsive stimuli with the addition of glutathione. Still, the cytotoxicity test proved a low cytotoxicity of the nanocomposite hydrogel and provided promising reference value to exploit this class of natural polymer-based hydrogel systems in biomedical applications. • Redox-responsive hydrogel is constructed with UV-triggered thiol-disulfide bonds in binary nanocomposite of T-CNF and GGMSH. • Bovine serum albumin (BSA) as an interactive nanofiller improved the mechanical characteristics of nanocomposite hydrogel. • GGMSH/T-CNF/BSA hydrogel possesses fast self-healing properties thanks to the thiol-disulfide exchange reaction. • GGMSH/T-CNF/BSA hydrogel supports an immediate release of laden protein and showed good cytocompatibility. [ABSTRACT FROM AUTHOR]

Endocrine dysregulation in the presence of environmental chemical risk factors is a global adverse health concern. The aim of this investigation was to explore the structural changes and binding affinity of thyroxine (T4) binding protein (TBG) upon interaction with SiO 2 particles as the second largest mineral in the Earth's crust and one of the most important constituents of rock, soil, and dust. Therefore, the interaction of TBG with SiO 2 particles was assessed by fluorescence quenching, molecular docking, ANS and synchronous fluorescence, and far-UV CD analyses. Also, the release of TBG from human hepatoblastoma cell line, Hep G2, was assessed by ELISA assay. The results displayed that the value of stoichiometry of binding site (n) of TBG for T4 was approximately equal to one, which was reduced to 0.36 in the presence of SiO 2 particles. Also, the binding affinity (K b) values revealed that the binding affinity between T4 and TBG was strong (97.90 × 105 L/mol), while the presence of SiO 2 particles resulted in the calculation of a K b around 0.00159 × 105 L/mol, which was significantly lower than that of the absence of SiO 2 particles. This data was also verified by molecular docking analyses which indicated that SiO 2 particles interacted with the T4 binding pocket of TBG. Moreover, further studies exhibited that although the equimolar concentration of T4 to TBG resulted in the superior stability of TBG-T4 complex relative to free TBG, the presence of SiO 2 particles with the same concentration led to denaturation of the secondary structure of TBG. Furthermore, it was seen that the amount of released TBG in the cell culture medium of Hep G2 was about 2.21 ng/mL protein, whereas this amount in SiO 2 particles-treated cell group was significantly reduced to 1.71 ng/mL protein (* P < 0.05). In conclusion, this study implies that SiO 2 particles show the potential to result in inhibition of TBG release, TBG denaturation, and interfere with TBG binding affinity which may lead to dysregulation of the thyroid hormone transport and associated signaling pathways. [ABSTRACT FROM AUTHOR]

Silica nanomaterials have been proposed as promising candidates for the delivery of drugs, including purified proteins, thus a systematic investigation on the role of silica surface porosity, for optimal protein loading and release, represents an urgent need. Herein, we report the studies of the loading and releasing processes of different volume-proteins, namely Myoglobin, Bovine Serum Albumin and Factor VIII (FVIII), performed using three porous silica samples. To this aim, silica nanoparticles are prepared through modified sol-gel methods, in which suitable templating and swelling agents are used to enlarge the surface pores from few nm to 25 nm, but retaining the average dimensions of the nanoparticles (80 –90 nm). TEM imaging and Zeta-Potential measurements are used to characterize the silica samples. In this study, the loading process is carried out at pH values close to the isoelectric point of each protein, and the amount of adsorbed protein is quantified and values in the range of μg protein per mg silica are obtained. Then, the release profiles are monitored at physiological conditions; for all systems, the release of the proteins is detected, although different behavior are observed. The release profiles are analyzed through the power law equation and through a modular exponential function to achieve information on the mechanism and on kinetics of the release profile. The data show the release from silica particles with small pores follows a diffusion-controlled mechanism, while the kinetic analysis enables to distinguish two-components associated to superficially adsorbed protein and to the portion of protein allocated inside the pores. On the other hand, the particles characterized by the largest pore size (ca. 25 nm) display non-Fickian release profile and a mono-exponential dependency, indicating a mono-modal distribution of the proteins on the silica matrix. Moreover, the conformation or the biological activity of the released proteins has been evaluated through in-vitro methods and the data demonstrate the excellent quality of the recovered proteins. The loading and release data were rationalized in terms of mesoporous structures of the silica nanoparticles in relation to the protein dimensions. Image 1 • Sol-gel procedures allow to tune the pores of silica nanoparticles for protein delivery. • Myoglobin, Bovine serum albumin, and Factor VIII are employed as model-proteins. • Loading and release data are obtained for the proteins on three silica samples. • The silica porosity discriminates the mechanism and the kinetics of protein release. • The released proteins preserve their conformation and their biological activity. [ABSTRACT FROM AUTHOR]

To study the release patterns of protein bovine serum albumin (BSA), porous poly(e-caprolactone)-chitosan scaffolds with entrapped BSA were fabricated by using supercritical CO2 for its potential use in tissue engineering applications. An emulsion, consisting of a polymer-solvent solution and buffer protein solution was saturated with scCO2 at 12 MPa and 37 °C and then rapidly depressurized through a release valve causing bubble nucleation and precipitation of the composite material. The controlled total protein release from biodegradable poly(e-caprolactone) with 5% chitosan (w/w) scaffolds was assessed by Bradford protein assay. After 16 to 20 days of protein release testing, 58.8% of the protein was released from composite with PCL (Mw = 10,000 g/mol) and 43.9% from composite with PCL (Mw = 60,000 g/mol). Preliminary studies for characterization of the prepared composite biomaterials using FTIR spectra, ESEM photo analysis and DSC analysis have been carried out. [ABSTRACT FROM AUTHOR]

• Dextran sulphate (DSS) forms nano- and microdomains in silk fibroin (SF) films. • DSS domains act as active porogens upon incubation in aqueous media. • The presence of DSS induces the formation of beta sheet crystals in SF. • Protein release kinetics can be tuned by selecting different SF/DSS compositions. • SF/DSS blends represent a flexible system for sustained protein delivery. Microporous films are structures with attractive features as high porosity and high intrinsic surface area. Unfortunately, most biocompatible materials useful for the preparation of such microporous films are suboptimal for drug delivery due to poor processability and/or drug encapsulation properties. Silk fibroin (SF) is a biocompatible protein with good drug release properties and suitable characteristics for pharmaceutical processing. Dextran sulphate (DSS) is an anionic polysaccharide with the capacity to interact and stabilize many proteins. In the current study, we evaluate the effect of DSS on the physical properties of SF/DSS blend films, how it affects the molecular and microscopic structure of the system and its capacity for providing controlled release of a model protein. Using this system, micro- and nanostructured films could be prepared through a green, water-only process. It was found that DSS acted both as a modifier of SF secondary structure and as a functional porogen, where the size of nano- and micropores varied with the blending ratios. High ratios showed higher swelling, porosity and crystallinity, resulting in modified protein release kinetics, as compared to pure SF films. Considering both their mild preparation method and their physical and pharmaceutical properties, SF/DSS films stand out as ideal systems for sustained protein delivery applications. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Novel acid-labile trehalose-based benzylidene diacetal crosslinkers were synthesized. • Various substitutions on aromatic ring allows for tunable acidic hydrolysis of crosslinkers. • Diacetal crosslinkers were used to manufacture hydrolytically degradable acrylamide-type hydrogels. • Choice of crosslinker structure and main monomer type affects degradation and protein release at around physiological pHs. Abstract Five acid-labile diacetal crosslinkers containing disaccharide trehalose were synthesized. Effects of different substituents on aromatic ring on hydrolysis kinetics were compared using time-dependent 1H NMR spectroscopy. A model protein (bovine serum albumin, BSA) was physically entrapped within bulk acrylamide-type hydrogels during redox-initiated free-radical polymerization. Under acidic conditions, hydrogel degradation occurred via cleavage of diacetal crosslinks to aqueous-soluble polymeric chains and free trehalose. The relationship between hydrogel degradation and protein release at around physiological pHs was studied. It was shown that selecting an appropriate crosslinker structure and main monomer type allows the tailoring of protein release profiles. [ABSTRACT FROM AUTHOR]

Understanding the structure, functionalities and biology of functional amyloids is an issue of emerging interest. Inclusion bodies, namely protein clusters formed in recombinant bacteria during protein production processes, have emerged as unanticipated, highly tunable models for the scrutiny of the physiology and architecture of functional amyloids. Based on an amyloidal skeleton combined with varying amounts of native or native-like protein forms, bacterial inclusion bodies exhibit an unusual arrangement that confers mechanical stability, biological activity and conditional protein release, being thus exploitable as versatile biomaterials. The applicability of inclusion bodies in biotechnology as enriched sources of protein and reusable catalysts, and in biomedicine as biocompatible topographies, nanopills or mimetics of endocrine secretory granules has been largely validated. Beyond these uses, the dissection of how recombinant bacteria manage the aggregation of functional protein species into structures of highly variable complexity offers insights about unsuspected connections between protein quality (conformational status compatible with functionality) and cell physiology. [ABSTRACT FROM AUTHOR]

Abstract Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease. Mutations in the gene encoding copper/zinc superoxide dismutase-1 (SOD1) are responsible for most familiar cases, but the role of mutant SOD1 protein dysfunction in non-cell autonomous neurodegeneration, especially in relation to microglial activation, is still unclear. Here, we focused our study on microglial cells, which release SOD1 also through exosomes. We observed that in rat primary microglia the overexpression of the most-common SOD1 mutations linked to fALS (G93A and A4V) leads to SOD1 intracellular accumulation, which correlates to autophagy dysfunction and microglial activation. In primary contact co-cultures, fALS mutant SOD1 overexpression by microglial cells appears to be neurotoxic by itself. Treatment with the autophagy-inducer trehalose reduced mutant SOD1 accumulation in microglial cells, decreased microglial activation and abrogated neurotoxicity in the co-culture model. These data suggest that i) the alteration of the autophagic pathway due to mutant SOD1 overexpression is involved in microglial activation and neurotoxicity; ii) the induction of autophagy with trehalose reduces microglial SOD1 accumulation through proteasome degradation and activation, leading to neuroprotection. Our results provide a novel contribution towards better understanding key cellular mechanisms in non-cell autonomous ALS neurodegeneration. Highlights • Microglial cells release SOD1 also through vesicles. • Mutant SOD1 leads to autophagy impairment and intracellular accumulation in microglia. • Microglial cells overexpressing mutant SOD1 become activated and neurotoxic. • The autophagy inducer trehalose counteracts the effects of mutant SOD1 overexpression in microglial cells. [ABSTRACT FROM AUTHOR]

The efficacy of a simple laboratory method for cell disruption based on the glass bead stirring, sonication, osmotic shock, freezing and grinding, or use of solvents and detergents was assessed in this study, via measurements of the release of total protein and L-asparaginase activity. Three different microbial sources of L-asparaginase were used: Escherichia coli BL21 (DE3), Leucosporidium muscorum, and Aspergillus terreus (CCT 7693). This study adjusted and identified the best procedure for each kind of microorganism. Sonication and glass bead stirring led to obtaining filamentous fungus cell-free extracts containing high concentrations of soluble proteins and specific activity; however, sonication was the best since it obtained 4.61 ± 0.12 IU mg−1 after 3 min of operation time. Mechanical methods were also the most effective for yeast cell disruption, but sonication was the technique which yielded a higher efficiency releasing 7.3 IUtotal compared to glass bead stirring releasing 2.7 IUtotal at the same operation time. For bacterium, sonication proved to be the best procedure due to getting the highest specific activity (9.01 IU mg−1) and total enzyme activity (61.7 IU). The data presented lead to conclude that the mechanical methods appeared to be the most effective for the disintegration of the all microbial cells studies. This is the first report related to the experimental comparison of L-ASNase extraction procedures from different microorganisms, which can also be used for extracting periplasm located enzymes from other organisms. [ABSTRACT FROM AUTHOR]

To enhance the properties of pure poly(acrylamide) hydrogel, diprotic maleic acid co-monomer was included into the reaction mixture during hydrogel synthesis. These hydrogels have been shown important swelling in distilled water and deswelling in acidic solution. In present work, we based on these considerations to study the deswelling behaviour of poly(acrylamide- co-maleic acid) hydrogels in different media (air, polyethylene glycol 200 (PEG200), acidic solution). Hydrogels of poly(acrylamide) and its copolymers with maleic acid were prepared by copolymerization and chemical crosslinking with methylene bisacrylamide. The obtained hydrogels have shown substantial mass swelling in distilled water and shrinking in acidic solution and PEG200. Results showed that the PEG200 was a better solvent for hydrogel deswelling and possibly will be used for gel drying. The swelling and deswelling kinetics were described by second- ordered and first-ordered models, respectively. BSA desorption in PEG200 was also studied. [ABSTRACT FROM AUTHOR]

Pathologies affecting the optic nerve and the retina are one of the major causes of blindness. These diseases include age-related macular degeneration (AMD), diabetic retinopathy (DR) and glaucoma, among others. Also, there are genetic disorders that affect the retina causing visual impairment. The prevalence of neurodegenerative diseases of the posterior segment is increased as most of them are related with the elderly. Even with the access to different treatments, there are some challenges in managing patients suffering retinal diseases. One of them is the need for frequent interventions. Also, an unpredictable response to therapy has suggested that different pathways may be playing a role in the development of these diseases. The management of these pathologies requires the development of controlled drug delivery systems able to slow the progression of the disease without the need of frequent invasive interventions, typically related with endophthalmitis, retinal detachment, ocular hypertension, cataract, inflammation, and floaters, among other. Biodegradable microspheres are able to encapsulate low molecular weight substances and large molecules such as biotechnological products. Over the last years, a large variety of active substances has been encapsulated in microspheres with the intention of providing neuroprotection of the optic nerve and the retina. The purpose of the present review is to describe the use of microspheres in chronic neurodegenerative diseases affecting the retina and the optic nerve. The advantage of microencapsulation of low molecular weight drugs as well as therapeutic peptides and proteins to be used as neuroprotective strategy is discussed. Also, a new use of the microspheres in the development of animal models of neurodegeneration of the posterior segment is described. [ABSTRACT FROM AUTHOR]

The recently introduced functionalized calcium carbonate (FCC), a porous microparticle with a nano-structured, lamellar surface, shows promising properties in the field of oral drug delivery. In this work, FCC was loaded with biomolecules e.g. lysozyme (Lys) and bovine serum albumin (BSA) in order to investigate its suitability to deliver protein based drugs. Loading efficiency for our model proteins was >90% and enzyme activity was preserved as demonstrated by Michaelis-Menten enzyme kinetic experiments. Circular dichroism analysis confirmed, that neither the structure of both model substances, nor the activity of Lys was affected by the loading process or the interaction with the surface of FCC. Electron microscopy (SEM) and mercury porosimetry were indicative of protein deposition on the particle surface as well as within the particle pores. Release properties were investigated in a customized flow cell, which simulates the conditions in the oral cavity. Depending on the isoelectric point of the investigated proteins, complete release was obtained within 1.5 h. This work shows, that FCC is a suitable pharmaceutical excipient for delivery of proteins. [ABSTRACT FROM AUTHOR]

Background: Bioactive glass has attracted substantial interest in orthopedics, but it has been less explored as a drug carrier. This study investigated the bovine serum albumin (BSA) release from bioactive 13-93B0 and 13-93B3 glasses. Methods: Glass disks (13-93B0 and 13-93B3; n = 5) were loaded with 4 mg of BSA and coated under different chitosan-coating conditions. The amount of BSA released in phosphate-buffered saline (PBS) was evaluated, and a degradation study was performed to find out the weight loss and pH of PBS. Secondary structures of BSA on 13-93B0 were characterized by Fourier transform infrared (FTIR) spectroscopy. Results: One hundred percent protein release occurred by 24 hours for all 13-93B3 groups. However, chitosan coating delayed 100% release up to 72 hours in 13-93B0 groups. The 13-93B3 glass showed higher degradation rates than 13-93B0 regardless of chitosan-coating status. Multilayer and sandwich chitosan coatings further delayed BSA release from 13-93B0. FTIR analysis revealed that α-helical structure was the highest among all groups and significantly higher in the 2% sandwich chitosan coating group (32.0% ± 2.1%), compared with uncoated and 4% chitosan groups. Conclusions: Chitosan coating can delay the burst release of BSA from 13-93B0 glass and be a potential coating on bioactive glass for drug delivery purposes. [ABSTRACT FROM AUTHOR]

A series of thermoresponsive hydrogels containing covalently incorporated trehalose, the well-known protein stabilizing disaccharide, was synthesized with the aim to obtain bioprotective carriers for protein release. Smart materials with trehalose present only in cross-links or both in cross-links and as pending moieties, were fabricated by redox-initiated radical copolymerization, with N -isopropylacrylamide used as the main monomer. In order to modify thermoresponsive properties, hydrogels containing more hydrophilic comonomers, acrylamide or N -(2-hydroxyethyl)acrylamide, were also obtained. The susceptibility of the cross-linker to undergo acid-catalyzed hydrolysis was used to disintegrate the polymer network and estimate the composition of the synthesized materials. The properties of hydrogels, which include: swelling capacity, thermoresponsive behavior, rheological characteristic, internal microstructure and the rate of acid-catalyzed degradation, were found to be dependent on both trehalose as well as hydrophilic comonomers. BSA and β-Galactosidase were chosen as model proteins for the release study from obtained hydrogels. The release profiles were shown to vary significantly depending on hydrogel form, the polymer network composition and temperature. [ABSTRACT FROM AUTHOR]

Poly(ethylene carbonate) (PEC) is a unique biomaterial showing significant potential for controlled drug delivery applications. The current study investigated the impact of the molecular weight on the biological performance of drug-loaded PEC films. Following the preparation and thorough physicochemical characterization of diverse PEC (molecular weights: 85, 110, 133, 174 and 196 kDa), the degradation and drug release behavior of rifampicin- and bovine serum albumin-loaded PEC films was investigated in vitro (in the presence and absence of cholesterol esterase), in cell culture (RAW264.7 macrophages) and in vivo (subcutaneous implantation in rats). All investigated samples degraded by means of surface erosion (mass loss, but constant molecular weight), which was accompanied by a predictable, erosion-controlled drug release pattern. Accordingly, the obtained in vitro degradation half-lives correlated well with the observed in vitro half-times of drug delivery ( R 2 = 0.96). Here, the PEC of the highest molecular weight resulted in the fastest degradation/drug release. When incubated with macrophages or implanted in animals, the degradation rate of PEC films superimposed the results of in vitro incubations with cholesterol esterase. Interestingly, SEM analysis indicated a distinct surface erosion process for enzyme-, macrophage- and in vivo -treated polymer films in a molecular weight-dependent manner. Overall, the molecular weight of surface-eroding PEC was identified as an essential parameter to control the spatial and temporal on-demand degradation and drug release from the employed delivery system. [ABSTRACT FROM AUTHOR]

The disintegration of three industry relevant algae ( Chlorella vulgaris , Neochloris oleoabundans and Tetraselmis suecica ) was studied in a lab scale bead mill at different bead sizes (0.3–1 mm). Cell disintegration, proteins and carbohydrates released into the water phase followed a first order kinetics. The process is selective towards proteins over carbohydrates during early stages of milling. In general, smaller beads led to higher kinetic rates, with a minimum specific energy consumption of ⩽0.47 kWh kg DW −1 for 0.3 mm beads. After analysis of the stress parameters (stress number and stress intensity), it appears that optimal disintegration and energy usage for all strains occurs in the 0.3–0.4 mm range. During the course of bead milling, the native structure of the marker protein Rubisco was retained, confirming the mildness of the disruption process. [ABSTRACT FROM AUTHOR]

In this study, the formation and structural features of distinct bovine serum albumin (BSA)−loaded cationic-functionalized poly (lactic acid) (PLA) nanoparticles were established and monitored for potential use in the medical and biotechnological applications. The PLA nanoparticles (NPs) produced by low energy solvent displacement were functionalized with the cationic agent hyperbranched polyethylenimine (PEI) for the protein adsorption. The interaction of the BSA with NPs was monitored using zeta potential, dynamic light scattering, atomic force microscopy (AFM) images, Fourier transform infrared spectroscopy and gel electrophoresis for assuring stable colloidal dispersions with mean diameter ranging of 100 to 190 nm, and high protein loading efficiency (PLE > 80%). The cationic NPs containing PLA:BSA ratio of 1:1 w/w stabilized with sorbitan monooleate and poly (vinyl alcohol) exhibited PLE% of 83.2 ± 3.3 and 76.2 ± 0.3 respectively. The slow protein release in these two formulations was diffusion-controlled dependent on BSA/PLA ratio, (Mt/M∞)/t = Kp t − 0.5 , with release rate constant of Kp = 0.127 and Kp = 0.110 respectively. The desired biocompability was dependent on PEI/PLA ratio. However, only the formulations stabilized with poly (vinyl alcohol) remained stable over than seven weeks. The data discussed in this approach demonstrated a scaffold of a low energy method for producing small−sized, spherical, stable, and cationic functionalized biodegradable and biocompatible nanoparticles as promising nanocarriers for proteins or negatively charged molecules from the biotechnology source. [ABSTRACT FROM AUTHOR]

The modification of bioresorbable polyester surfaces in order to alter their biointeractions presents an important problem in biomedical polymer science. In this study, the covalent modification of the surface of poly(lactic acid)-based (PLA-based) films with poly(acryl amide) and sodium alginate hydrogels was performed to change the non-specific polyester interaction with proteins and cells, as well as to make possible the covalent attachment of low-molecular weight ligands and to control protein release. The effect of such modification on the film surface properties was studied. Parameters such as swelling, water contact angle, surface area, and binding capacity of low-molecular weight substances were evaluated and compared. The comparative study of adsorption of model protein (BSA) on the surface of non-modified and modified films was investigated and the protein release was evaluated. Cell viability on the surface of hydrogel-coated films was also tested. The developed approach could be applied for the modification of PLA-based scaffolds for tissue engineering and will be further studied for molecular-imprinting of biomolecules on the surface of polyester-based materials for control of biointeractions. [ABSTRACT FROM AUTHOR]

A novel type of drug-delivery scaffold based on poly(ε-caprolactone) (PCL) and zein blends was prepared by improved unidirectional freeze-drying. Scaffolds with tube-like pore structure and high porosity, up to 89%, were obtained by adjusting the concentration of the PCL and zein solutions. Characters of the prepared scaffolds, such as microstructural, porosity, and compressive strength, were evaluated. The hydrophilicity and the degradability of the composite films were investigated in contact with phosphate buffer saline (PBS). It was found that the presence of zein accelerates the degradation rate of the scaffolds in the period time of investigation (28 days). The results showed an acceptable way for controlling the in vitro degradation behavior of PCL composite scaffolds by adapting the concentration of zein. In vitro protein release and degradation results revealed that the absolute weight loss of the PCL/zein scaffolds exhibited an increasing trend by increasing the amount of zein concentration in the scaffolds. The drug delivery capability of the scaffolds was tested using tetracycline hydrochloride (TCH). Sustained release of the drug was obtained, and it was found that the proportion of zein in the scaffold had a great impact on the drug release kinetics. The results demonstrated the potential of the PCL/zein biocomposite scaffolds as a suitable candidate in tissue engineering strategies for bone defect treatment. [ABSTRACT FROM AUTHOR]

We report a microfluidic approach for one-step fabrication of polyelectrolyte microcapsules in aqueous conditions. Using two immiscible aqueous polymer solutions, we generate transient water-in-water-in-water double emulsion droplets and use them as templates to fabricate polyelectrolyte microcapsules. The capsule shell is formed by the complexation of oppositely charged polyelectrolytes at the immiscible interface. We find that attractive electrostatic interactions can significantly prolong the release of charged molecules. Moreover, we demonstrate the application of these microcapsules in encapsulation and release of proteins without impairing their biological activities. Our platform should benefit a wide range of applications that require encapsulation and sustained release of molecules in aqueous environments. [ABSTRACT FROM AUTHOR]

Cellulose based hydrogels are important due to their biocompatibility, non-toxicity and natural origin. In this work, a new set of pH, temperature and redox responsive hydrogels were prepared from carboxymethylcellulose (CMC) and poly( N -isopropylacrylamide). Copolymeric (CP) hydrogels were synthesized by copolymerizing N -isopropylacrylamide (NIPA) and methacrylated carboxymethylcellulose, semi-interpenetrating network (SIPN) hydrogels were prepared by polymerizing NIPA in presence of CMC. Two types of cross-linkers were used viz. N , N′ -methylenebisacrylamide (BIS) and N , N ′-bis(acryloyl)cystamine (CBA), a redox sensitive cross-linker. The structures of the hydrogels were characterized by FTIR and SEM studies. The CP hydrogels were found to be more porous than corresponding SIPNs which resulted in higher swelling for the CP hydrogels. Swelling for both the hydrogels were found to increase with CMC content. While the swelling of SIPN hydrogels showed discontinuous temperature dependency, CP hydrogels showed gradual decrease in water retention values with increase in temperature. CBA cross-linked hydrogels showed higher swelling in comparison to BIS cross-linked hydrogels. Additionally, lysozyme was loaded in the hydrogels and its in vitro release was studied in various pH, temperature and in presence of a reducing agent, glutathione (GSH). The release rate was found to be maximum at lower temperature, lower pH and in presence of GSH. [ABSTRACT FROM AUTHOR]

Assembly of biopolymers into microgels is an elegant strategy for bioencapsulation with various potential biomedical applications. Such biocompatible and biodegradable microassemblies are developed not only to protect the encapsulated molecule but also to ensure its sustained local delivery. The present study describes the fabrication of microassemblies from a marine HE800 exopolysaccharide (EPS), which displays a glycosaminoglycan (GAG)-like structure and biological properties. HE800 EPS was assembled, through physical cross-linking with divalent ions, into microgel particles and microfibers using microfluidics. The microparticle morphology was highly affected by the polysaccharide concentration and its molecular weight. A model protein, namely Bovine Serum Albumin (BSA) was subsequently encapsulated within HE800 microparticles in one-step process using microfluidics. The protein release was tuned by the microparticle morphology with a lower protein amount released from the most homogeneous structures. Our findings demonstrate the high potential of HE800 EPS based microassemblies as innovative protein microcarriers for further biomedical applications. [ABSTRACT FROM AUTHOR]

Element-substituted hydroxyapatite (HA) based nanocomposites have become a promising therapeutic material for improving bone defect repair. Selenium substituted HA nanoparticles can both induce apoptosis of bone tumor cells and enhance osteointegration. However, the effect of selenite ions on the proteins in combination with the HA nanoparticles remains to be elucidated. Here, we investigated the influence of selenium doping concentration on the loading and release of lysozyme (LSM) as a model protein drug. The selenium substituted HA–LSM composites with different doping concentrations were synthesized and characterized. The subsequent delivery of lysozyme was studied in a phosphate buffer solution (PBS). We found that selenium substituted HA–LSM composites with Se:P = 10% showed the highest amount of lysozyme loading (41.7%), whereas the amount of lysozyme loaded in undoped HA nanoparticles was the lowest (34.1%). The doped selenium interacts with lysozyme molecules, which leads to the increase of β-sheet and unordered, and the decrease of self-association, α-helix and β-turns in protein structures. Moreover, selenium addition significantly slows the protein release from HA–LSM composites. The composites with Se:P = 10% release lysozyme at the slightly slower rate among the samples with different Se doping concentrations. It also shows that the released lysozyme retains most of its enzymatic activity. [ABSTRACT FROM AUTHOR]

Hydroxyapatite (HA) mineral tubes developed through the replication of polymer nanofiber mesh were studied for loading and delivery of biological proteins. With varying heat-treatment temperature (500, 800 and 1100 °C), the physicochemical properties of the HA mineral tubes differed substantially, i.e., while crystallinity and crystallite size increased the surface area decreased with increase in the temperature. All mineral tubes retained negative surface charges (around −10 mV) at physiological conditions. Cytochrome C (cyt C), used as a model protein of being positively-charged, was adsorbed well onto the HA mineral tubes, with adsorption being saturated within a few hours. The adsorption amount was significantly higher in the case treated at lower temperature. The cyt C showed a similar release pattern from the HA mineral tubes, with an initial rapid release for a few hours followed by a reduced rate over ∼24 h. Results indicated the HA mineral tubes might be tailored to properly load and delivery biological molecules for bone repair. [ABSTRACT FROM AUTHOR]

Significant research has focused on investigating the potential of hydrogels in various applications and, in particular, in medicine. Specifically, hydrogels that are biodegradable lend promise to many therapeutic and biosensing applications. Endonucleases are critical for mechanisms of DNA repair. However, they are also known to be overexpressed in cancer and to be present in wounds with bacterial contamination. In this work, we set out to demonstrate the preparation of DNA-enabled hydrogels that could be degraded by nucleases. Specifically, hydrogels were prepared through the reaction of dibenzocyclooctyne-functionalized multi-arm poly(ethylene glycol) with azide-functionalized single-stranded DNA in aqueous solutions via copper-free click chemistry. Through the use of this method, biodegradable hydrogels were formed at room temperature in buffered saline solutions that mimic physiological conditions, avoiding possible harmful effects associated with other polymerization techniques that can be detrimental to cells or other bioactive molecules. The degradation of these DNA-cross-linked hydrogels upon exposure to the model endonucleases Benzonase®and DNase I was studied. In addition, the ability of the hydrogels to act as depots for encapsulation and nuclease-controlled release of a model protein was demonstrated. This model has the potential to be tailored and expanded upon for use in a variety of applications where mild hydrogel preparation techniques and controlled material degradation are necessary including in drug delivery and wound healing systems. [ABSTRACT FROM PUBLISHER]

Poly(lactic- co -glycolic acid) (PLGA) microspheres and PLGA/alginate composite microspheres were prepared by a novel double emulsion and solvent evaporation technique and loaded with bovine serum albumin (BSA) or rabbit anti-laminin antibody protein. The addition of alginate and the use of a surfactant during microsphere preparation increased the encapsulation efficiency and reduced the initial burst release of hydrophilic BSA. Confocal laser scanning microcopy (CLSM) of BSA-loaded PLGA/alginate composite microspheres showed that PLGA, alginate, and BSA were distributed throughout the depths of microspheres; no core/shell structure was observed. Scanning electron microscopy revealed that PLGA microspheres erode and degrade more quickly than PLGA/alginate composite microspheres. When loaded with anti-laminin antibody, the function of released antibody was well preserved in both PLGA and PLGA/alginate composite microspheres. The biocompatibility of PLGA and PLGA/alginate microspheres were examined using four types of cultured cell lines, representing different tissue types. Cell survival was variably affected by the inclusion of alginate in composite microspheres, possibly due to the sensitivity of different cell types to excess calcium that may be released from the calcium cross-linked alginate. [ABSTRACT FROM AUTHOR]

The objectives of this study were to investigate the adsorption behavior of lysozyme (LSZ) onto magnesium aluminum silicate (MAS) at various pHs and to characterize the LSZ–MAS microparticles obtained from the molecular interaction between LSZ and MAS. The results showed that LSZ could be bound onto the MAS layers at different pHs, leading to the formation of LSZ–MAS microparticles. The higher preparation pH permitted greater adsorption affinity but a lower adsorption capacity of LSZ onto MAS. LSZ could interact with MAS via hydrogen bonds and electrostatic forces, resulting in the formation of intercalated nanocomposites. The particle size, %LSZ adsorbed, and LSZ release rate of LSZ–MAS microparticles increased when the LSZ–MAS ratio was increased. The secondary structure of LSZ bound onto the MAS layers in microparticles prepared at various pHs was altered compared with that of native LSZ. Moreover, the LSZ extracted from microparticles prepared at pH 4 showed an obvious change in the tertiary structure, leading to a decrease in the biological activity of the LSZ released. These findings suggested that LSZ can strongly interact with MAS to form microparticles that may potentially be used as delivery systems for sustained protein release. [ABSTRACT FROM AUTHOR]

Purpose This study describes how protein release from polymer matrices correlate with simple measurements on the intrinsic viscosity of the polymer solutions used for casting the matrices and calculations of the solubility parameters of polymers and solvents used. Method Matrices of poly( dl -lactide-co-glycolide) (PLGA) were cast with bovine serum albumin (BSA) as a model drug using different solvents (acetone, dichloromethane, ethanol and water). The amount of released protein from the different matrices was correlated with the Hildebrand and Hansen solubility parameters of the solvents, and the intrinsic viscosity of the polymer solutions. Matrix microstructure was investigated by transmission and scanning electron microscopy (TEM and SEM). Polycaprolactone (PCL) matrices were used in a similar way to support the results for PLGA matrices. Results The maximum amount of BSA released and the release profile from PLGA matrices varied depending on the solvent used for casting. The maximum amount of released BSA decreased with higher intrinsic viscosity, and increased with solubility parameter difference between the solvent and polymer used. The solvent used also had an effect on the matrix microstructure as determined by TEM and SEM. Similar results were obtained for the PCL polymer systems. Conclusions The smaller the difference in the solubility parameter between the polymer and the solvent used for casting a polymer matrix, the lower will be the maximum protein release. This is because of the presence of smaller pore sizes in the cast matrix if a solvent with a solubility parameter close to the one of the polymer is used. Likewise, the intrinsic viscosity of the polymer solution increases as solubility parameter differences decrease, thus, simple measurements of intrinsic viscosity and solubility parameter difference, allow the prediction of protein release profiles. [ABSTRACT FROM AUTHOR]

Recently alginate-based tissue repair scaffolds fabricated using 3D printing techniques have been extensively examined for use in tissue engineering applications. However, their physical and mechanical properties are unfavorable for many tissue engineering applications because these properties are poorly controlled during the fabrication process. Some improvement of alginate gel properties can be realized by addition of hyaluronic acid (HA), and this may also improve the ability of cells to interact with the gel. Here, we report improvement of the physical properties of alginate–HA gel scaffolds by the addition of the polycation polyethyleneimine (PEI) during the fabrication process in order to stabilize alginate molecular structure through the formation of a polyelectrolyte complex. We find that PEI has a significant beneficial influence on alginate–HA scaffold physical properties, including a reduction in the degree of gel swelling, a reduction in scaffold degradation rate, and an increase in the Young’s modulus of the gel. Further study shows that fabrication of alginate–HA gels with PEI increases the encapsulation efficiency of bovine serum albumin, a model protein, and reduces the subsequent initial protein release rate. However, it was also found that survival of Schwann cells or ATDC-5 chondrogenic cells encapsulated during the scaffold fabrication process was modestly reduced with increasing PEI concentration. This study illustrates that the use of PEI during scaffold fabrication by plotting can provide an effective means to control alginate-based scaffold properties for tissue engineering applications, but that the many effects of PEI must be balanced for optimal outcomes in different situations. [ABSTRACT FROM PUBLISHER]

The study was aimed at evaluating the effect of electrospun scaffold containing TGF‐β1 on promoting human mesenchymal stem cells (MSCs) differentiation towards a nucleus pulposus‐like phenotype under hypoxia. Two kinds of nanofibrous scaffolds containing TGF‐β1 were fabricated using uniaxial electrospinning (Group I) and coaxial electrospinning (Group II). Human MSCs were seeded on both kinds of scaffolds and cultured in a hypoxia chamber (2% O2), and then the scaffolds were characterised. Cell proliferation and differentiation were also evaluated after 3 weeks of cell culture. Results showed that both kinds of scaffolds shared similar diameter distributions and protein release. However, Group I scaffolds were more hydrophilic than that of Group II. Both kinds of scaffolds induced the MSCs to differentiate towards the nucleus pulposus‐type phenotype in vitro. In addition, the expression of nucleus pulposus‐associated genes (aggrecan, type II collagen, HIF‐1α and Sox‐9) in Group I increased more than that of Group II. These results indicate that electrospinning nanofibrous scaffolds containing TGF‐β1 supports the differentiation of MSCs towards the pulposus‐like phenotype in a hypoxia chamber, which would be a more appropriate choice for nucleus pulposus regeneration. [ABSTRACT FROM AUTHOR]

In this work, a hydrogelation method was used for the development of protein-loaded xanthan biopolymer particles. The gelation of the aqueous polymer droplets was accomplished in a mixture of calcium chloride and aluminium chloride solution. The particles were less than 900 μm in diameter and appeared spherical under scanning electron microscope. The particles retained a maximum of 90% of its initial load and released the protein molecules over an extended period in phosphate buffer saline solution (pH 7.4). The protein release was <13% in acidic medium (pH 1.2). The protein release was interrelated with pH-dependent swelling of the particles. The polymer relaxation phenomenon predominated over simple diffusion mechanism in anomalous protein release process as the ratio of di- and trivalent metal ions was decreased. No protein–biopolymer interaction was evident by FTIR spectroscopy. The divalent and trivalent metal ion cross-linked xanthan particles showed immense potential in controlled oral delivery of macromolecules. [ABSTRACT FROM AUTHOR]

Hydrogel beads as microcarriers could have many applications in biotechnology. However, bead formation by noncovalent cross-linking to achieve high cell compatibility by avoiding chemical reactions remains challenging because of rapid gelation rates and/or low stability. Here we report the preparation of homogeneous, tunable, and robust hydrogel beads from peptide-polyethylene glycol conjugates and oligosaccharides under mild, cell-compatible conditions using a noncovalent crosslinking mechanism. Large proteins can be released from beads easily. Further noncovalent modification allows for bead labeling and functionalization with various compounds. High survival rates of embedded cells were achieved under standard cell culture conditions and after freezing the beads, demonstrating its suitability for encapsulating and conserving cells. Hydrogel beads as functional system have been realized by generating protein-producing microcarriers with embedded eGFP-secreting insect cells. [ABSTRACT FROM AUTHOR]

ABSTRACT We investigated the interfacial properties of poly(vinyl alcohol) carrying UV-crosslinkable pendant quaternized stilbazole (styrylpyridinium), PVA-SbQ. The extent and dynamics of PVA-SbQ cyclodimerization reactions and crosslinking induced by UV irradiation were monitored in situ and in real time by quartz crystal microgravimetry (QCM). Sensograms reflecting time-dependent changes in density and viscoelasticity of crosslinking films followed a Boltzmann sigmoidal model, depending on precursor film composition and irradiation power. The shifts in QCM frequency and energy dissipation upon PVA-SbQ cyclodimerization correlated with three photo-crosslinking phases involving soft-to-rigid transitions, namely, induction (initiation), main crosslinking (interaction), and termination. PVA-SbQ films crosslinked to different degree were used as protein carriers and a slower release profile was determined for the films that underwent more extensive crosslinking. Overall, this study demonstrates for the first time the dynamics of PVA-SbQ crosslinking and its impact in system viscoelasticity and protein release. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2015, 53, 345-355 [ABSTRACT FROM AUTHOR]

Affinity-based controlled release modulates the delivery of protein or small molecule therapeutics through transient dissociation/association. To understand which parameters can be used to tune release, we used a mathematical model based on simple binding kinetics. A comprehensive asymptotic analysis revealed three characteristic regimes for therapeutic release from affinity-based systems. These regimes can be controlled by diffusion or unbinding kinetics, and can exhibit release over either a single stage or two stages. This analysis fundamentally changes the way we think of controlling release from affinity-based systems and thereby explains some of the discrepancies in the literature on which parameters influence affinity-based release. The rate of protein release from affinity-based systems is determined by the balance of diffusion of the therapeutic agent through the hydrogel and the dissociation kinetics of the affinity pair. Equations for tuning protein release rate by altering the strength ( K D ) of the affinity interaction, the concentration of binding ligand in the system, the rate of dissociation ( k off ) of the complex, and the hydrogel size and geometry, are provided. We validated our model by collapsing the model simulations and the experimental data from a recently described affinity release system, to a single master curve. Importantly, this mathematical analysis can be applied to any single species affinity-based system to determine the parameters required for a desired release profile. [ABSTRACT FROM AUTHOR]

Nonwoven scaffolds consisting of poly-ε-caprolactone (PCL), poly(lactic- co -glycolic acid) (PLGA) and polidocanol (PD), and loaded with lysozyme crystals were prepared by electrospinning. The composition of the matrix was varied and the effect of PD content in binary mixtures, and of PD and PLGA content in ternary mixtures regarding processability, fiber morphology, water sorption, swelling and drug release was investigated. Binary PCL/PD blend nonwovens showed a PD-dependent increase in swelling of up to 30% and of lysozyme burst release of up to 45% associated with changes of the fiber morphology. Furthermore, addition of free PD to the release medium resulted in a significant increase of lysozyme burst release from pure PCL nonwovens from approximately 2–35%. Using ternary PCL/PD/PLGA blends, matrix degradation could be significantly improved over PCL/PD blends, resulting in a biphasic release of lysozyme with constant release over 9 weeks, followed by constant release with a reduced rate over additional 4 weeks. Based on these results, protein release from PCL scaffolds is improved by blending with PD due to improved lysozyme desorption from the polymer surface and PD-dependent matrix swelling. [ABSTRACT FROM AUTHOR]

Objectives In this study, a novel biomimetic calcium phosphate bone substitute ( Bio Ca P) is introduced as a dual-drug release system with two drug/protein delivery modes: protein is incorporated into (i) the interior of Bio Ca P (an internal depot); and (ii) a superficial calcium phosphate coating on Bio Ca P (a surface-coated depot). Our aim is to investigate each of the two delivery modes of Bio Ca P. Our hypotheses are that (i) both of the drug delivery modes, in in vitro as well as in vivo environment, can achieve a sustained cell-mediated protein release; and (ii) Bio Ca P with these two delivery modes with incorporated bone morphogenetic protein-2 ( BMP-2) promotes bone formation. Materials and Methods Tablets of Bio Ca P were prepared with different carrying modes using bovine serum albumin ( BSA) as model protein. The release of this protein was analysed ( n = 6 per group). Granules of Bio Ca P with different carrying modes of BMP-2 were implanted subcutaneously in rats ( n = 6 animals per group). Samples were collected after 5 weeks for histomorphometric analysis. Results In vitro data showed that the internal and surface-coated depots of BSA resulted in a sustained osteoclast-mediated release, while the adsorbed BSA was rapidly released, and this release was not affected by osteoclasts. In vivo data showed that the volume densities of bone, bone marrow, and blood vessels were significantly higher in samples where BMP-2 was incorporated internally or in the coating compared with granules with adsorbed growth factor. Osteoclast-like cells were associated with the granules, and resorption lacunae were frequently observed. Conclusion It is shown that different modes of incorporation of BMP-2 on and in BioCaP granules have a beneficial effect on the formation of ectopic bone. This dual-drug release system makes BioCaP granule a promising tool for delivering multiple therapeutic agents for different clinical applications. [ABSTRACT FROM AUTHOR]

Hydrogels were prepared from guar gum (GG) via esterification with 1,2,3,4-butanetetracarboxylic dianhydride (BTCA). Detailed spectroscopic analysis using FTIR and solid-state NMR revealed that an increase in the BTCA feed amount in the preparation mixture led to an increased degree of crosslinking, which affected the swelling behavior and rheological properties of the hydrogels. The hydrogels exhibited enzyme degradability, and after incubation with β-mannanase and α-galactosidase, 30-57% of the hydrogels were degraded. In addition, the hydrogels adsorbed bovine serum albumin and hen egg white lysozyme thorough electrostatic and hydrophobic interactions. The protein-adsorbed GG hydrogels exhibited a slow and steady release of the proteins over a 24 h period in buffer solutions after a fast release of proteins in the first hour. As such, GG hydrogels are expected to be efficient drug delivery carriers for protein-based drugs. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the effect of polyethylene glycol (PEG) in nanoparticles based on blends of hydroxylated aliphatic polyester, poly(D,L-lactic-co-glycolic-co-hydroxymethyl glycolic acid) (PLGHMGA) and PEG-PLGHMGA block copolymers on their degradation and release behavior. Methods: Protein-loaded nanoparticles were prepared with blends of varying ratios of PEG-PLGHMGA (molecular weight of PEG 2,000 and 5,000 Da) and PLGHMGA, by a double emulsion method with or without using poly(vinyl alcohol) (PVA) as surfactant. Bovine serum albumin and lysozyme were used as model proteins. Results: PEGylated particles prepared without PVA had a zeta potential ranging from ~ −3 to ~−35 mV and size ranging from ~200 to ~600 nm that were significantly dependent on the content and type of PEG-block copolymer. The encapsulation efficiency of the two proteins however was very low (<30%) and the particles rapidly released their content in a few days. In contrast, all formulations prepared with PVA showed almost similar particle properties (size: ~250 nm, zeta potential: ~−1 mV), while loading efficiency for both model proteins was rather high (80-90%). Unexpectedly, independent of the type of formulation, the nanoparticles had nearly the same release and degradation characteristics. NMR analysis showed almost a complete removal of PEG in 5 days which explains these marginal differences. Conclusions: Protein release and particle degradation are not substantially influenced by the content of PEG, likely because of the fast shedding of the PEG blocks. These PEG shedding particles are interesting system for intracellular delivery of drugs. [ABSTRACT FROM AUTHOR]

Highlights: [•] A thiolated chitosan-4-thio-butylamidine (CS-TBA) conjugate was synthesized. [•] A novel hydrogel was prepared based on CS-TBA/hydroxyapatite/beta-glycerophosphate. [•] The novel hydrogel system is water soluble and thermo-sensitive. [•] The hydrogel shows potential applications in drug delivery and tissue engineering. [Copyright &y& Elsevier]