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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]

There is a growing number of protein drugs, yet their limited oral bioavailability requires that patients receive frequent, high dose injections. In situ forming implants (ISFIs) for controlled release of biotherapeutics have the potential to greatly reduce the injection frequency and improve patient compliance. However, protein release from ISFIs is a challenge due to their proclivity for instability. Specifically, factors such as the acidic microclimate within ISFIs can lead to protein aggregation and denaturation. Basic salts have been shown in PLGA microparticle and microcylinder formulations to significantly reduce protein instability by neutralizing this acidic environment. The overall objective of the study was to demonstrate that basic salts can be used with an ISFI system to neutralize the implant acidification. To this end, the basic salts MgCO3 and Mg(OH)2 were added to a protein-releasing ISFI and the effect on drug release, pH, implant swelling, implant diffusivity, and implant erosion were evaluated. Either salt added at 3 wt% neutralized the acidic environment surrounding the implants, keeping the pH at 6.64 ± 0.03 (MgCO3) and 6.46 ± 0.11 (Mg(OH)2) after 28 day compared to 3.72 ± 0.05 with no salts added. The salts initially increased solution uptake into the implants but delayed implant degradation and erosion. The 3 wt% Mg(OH)2 formulation also showed slightly improved drug release with a lower burst and increased slope. We showed that salt additives can be an effective way to modulate the pH in the ISFI environment, which can improve protein stability and ultimately improve the capacity of ISFIs for delivering pH-sensitive biomolecules. Such a platform represents a low-cost method of improving overall patient compliance and reducing the overall healthcare burden. [ABSTRACT FROM AUTHOR]

Abstract Larval therapy has been reported to be beneficial in the treatment of chronic wounds by promoting granulation tissue formation, due to its antimicrobial properties and by degrading necrotic tissue. However, the use of live maggots is problematic for patient acceptance, and thus there is a need to develop materials which can release therapeutic biomolecules derived from maggot secretions to the wound bed. Here we describe the fabrication of a novel bioactive scaffold that can be loaded with Lucilia sericata maggot alimentary excretion/secretion fluids (L. sericata maggot E/S), and which can also provide structural stability for mammalian cell-growth and migration to support wound repair. Electrospun scaffolds were prepared from a poly(caprolactone)-poly(ethylene glycol)–block copolymer (PCL-b-PEG) blended with PCL with average fibre diameters of ~ 4 μm. The scaffolds were hydrophilic and were able to support viable fibroblasts that were able to infiltrate throughout the extent of the scaffold thickness. L. sericata maggot (E/S) was subsequently adsorbed to the surface and released over 21 days with retention of the protease activity that is responsible for supporting fibroblast migration. The incorporation of L. sericata maggot E/S on the surface of the electrospun fibres of PCL-PEG/PCL fibres is a novel approach with potential for future application to support skin wound healing within a clinical setting.

Despite the increased interest in macroalgae protein and fibers, little information is available on their bioaccessibility. The application of an in vitro gastrointestinal digestion model to study the degree of disintegration and release of proteins with expressed bioactivities from wild and cultivated Palmaria palmata and Saccharina latissima was proposed in this study. Macroalgae from the Gulf of St Lawrence, Canada, were submitted to digestive transit times of 2 (oral), 60 (gastric) and 120 (duodenal) minutes. Among wild samples, P. palmata had a higher percentage of disintegration, protein release and degree of hydrolysis than S. latissima. While the least digested sample, wild S. latissima, was the sample with the highest antioxidant activity (210 μmol TE g−1), the most digested sample, cultivated P. palmata, presented the highest ability to inhibit the angiotensin-converting enzyme (ACE), reaching 32.6 ± 1.2% at 3 mg mL−1. ACE inhibitory activity increased from 1 to 3 mg mL−1, but not at 5 mg mL−1. Wild samples from both species showed an ACE inhibition around 27.5%. Data suggested that the disintegration of the samples was influenced by their soluble and insoluble fiber contents. Further information on the bioaccessibility and bioactivity of these macroalgae should consider the characterization of digestion products other than protein, as well as the effects of previous product processing. [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).

The world will face a significant protein demand in the next few decades, and due to the environmental concerns linked to animal protein, new sustainable protein sources must be found. In this regard, microalgae stand as an outstanding high-quality protein source. However, different steps are needed to separate the proteins from the microalgae biomass and other biocompounds. The protein recovery from the disrupted biomass is usually the bottleneck of the process, and it typically employs organic solvents or harsh conditions, which are both detrimental to protein stability and planet health. Different techniques and methods are applied for protein recovery from various matrices, such as precipitation, filtration, chromatography, electrophoresis, and solvent extraction. Those methods will be reviewed in this work, discussing their advantages, drawbacks, and applicability to the microalgae biorefinery process. Special attention will be paid to solvent extraction performed with ionic liquids (ILs) and deep eutectic solvents (DESs), which stand as promising solvents to perform efficient protein separations with reduced environmental costs compared to classical alternatives. Finally, several solvent recovery options will be analyzed to reuse the solvent employed and isolate the proteins from the solvent phase. [ABSTRACT FROM AUTHOR]

Statistical methods were used to provide insight into a polymer complex system composed of lysozyme and alginate to quantify the effects of such parameters as pH, and ionic composition of the mixing solution on the properties of the complexes including composition, particle diameter, and zeta potential. Various crosslinkers (calcium, barium, iron[III], and bovine serum albumin), were used with lysozyme for complex formation to investigate the effect of crosslinker charge density on protein release kinetics, modelled using ktn. Multivariate statistical analysis showed that the kinetic parameters associated with the release were, not surprisingly highly dependent on the ionic strength of the release media, with higher ionic strength leading to faster release. The release parameter k was also shown to depend on the protein properties (size, ionic strength) while n was slightly, but not statistically dependent on the charge density of the crosslinker demonstrating that the nature of the crosslinker had minimal impact on drug release. The multivariate statistical has the potential to be used for optimization of the complexes and prediction of physical properties and degradation rates. [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.

Despite the increased interest in macroalgae protein and fibers, little information is available on their bioaccessibility. The application of an in vitro gastrointestinal digestion model to study the degree of disintegration and release of proteins with expressed bioactivities from wild and cultivated Palmaria palmata and Saccharina latissima was proposed in this study. Macroalgae from the Gulf of St Lawrence, Canada, were submitted to digestive transit times of 2 (oral), 60 (gastric) and 120 (duodenal) minutes. Among wild samples, P. palmata had a higher percentage of disintegration, protein release and degree of hydrolysis than S. latissima. While the least digested sample, wild S. latissima, was the sample with the highest antioxidant activity (210 μmol TE g−1), the most digested sample, cultivated P. palmata, presented the highest ability to inhibit the angiotensin-converting enzyme (ACE), reaching 32.6 ± 1.2% at 3 mg mL−1. ACE inhibitory activity increased from 1 to 3 mg mL−1, but not at 5 mg mL−1. Wild samples from both species showed an ACE inhibition around 27.5%. Data suggested that the disintegration of the samples was influenced by their soluble and insoluble fiber contents. Further information on the bioaccessibility and bioactivity of these macroalgae should consider the characterization of digestion products other than protein, as well as the effects of previous product processing.

Developing accurate methods for the assessment of therapeutic protein release from polymer drug delivery systems (microcapsules, microspheres, nanoparticles, 3D-printed systems) is of paramount importance for new formulation development. The most straightforward method for protein release assessment is spectrophotometric analysis of the release medium surrounding the formulation. However, direct spectrophotometric analysis is inapplicable to formulations releasing interfering compounds (co-encapsulated drugs, additives) absorbing light in the same spectrum as proteins. Conventional protein release assays also require frequent release medium sampling and replacement, which reduces their accuracy. We propose a one-step method to assess protein release from core/shell microcapsules eliminating the need for sampling and allowing selective real-time protein quantitation in the release medium. To prevent spectral interferences, released protein is differentiated from interfering compounds by employing a colorimetric protein assay reagent, forming a colour complex selectively with the protein, as the release medium. To eliminate sampling, we employed a continuous flow closed loop set-up, where the release medium is constantly circulating between microcapsule-containing tank and spectrophotometer. A series of colorimetric protein assay reagents (bromocresol green, tetrabromophenol blue, eosin B, eosin Y, biuret) were evaluated in terms of their applicability as the release medium in described system. Only biuret reagent was found compatible with proposed method due to formation of color complex stable over extended period of time and low adsorption to microcapsules. Presented method allowed effective evaluation of albumin release from alginate-polyethersulfone microcapsules with accuracy equal to conventional 'sample and separate' technique. Albumin release followed first-order kinetics with plateau reached after 19 h. [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]

To study the release patterns of protein bovine serum albumin (BSA), porous poly(ε-caprolactone)-chitosan scaffolds with entrapped BSA were fabricated by using supercritical CO2 (scCO2) 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(ε-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.

Focal thermal therapy (Heat), cryosurgery (Cryo) and irreversible electroporation (IRE) are increasingly used to treat cancer. However, local recurrence and systemic spread are persistent negative outcomes. Nevertheless, emerging work with immunotherapies (i.e., checkpoint blockade or dendritic cell (DC) vaccination) in concert with focal therapies may improve outcomes. To understand the role of focal therapy in priming the immune system for immunotherapy, an in vitro model of T cell response after exposure to B16 melanoma cell lysates after lethal exposures was designed. Exposure included: Heat (50 °C, 30 min), Cryo (−80 °C, 30 min) and IRE (1250 V/cm, 99 pulses, 50 µs pulses with 1 Hz intervals). After viability assessment (CCK-8 assay), cell lysates were collected and assessed for protein release (BCA assay), protein denaturation (FTIR-spectroscopy), TRP-2 antigen release (western blot), and T cell activation (antigen-specific CD8 T cell proliferation). Results showed IRE released the most protein and antigen (TRP-2), followed by Cryo and Heat. In contrast, Cryo released the most native (not denatured) protein, compared to IRE and Heat. Finally, IRE dramatically outperformed both Cryo and Heat in T cell activation while Cryo modestly outperformed Heat. This study demonstrates that despite all focal therapies ability to destroy cells, the ‘quantity’ (i.e., amount) and ‘quality’ (i.e., molecular state) of tumor protein (including antigen) released can dramatically change the ensuing priming of the immune system. This suggests protein-based metrics whereby focal therapies can be designed to prime the immune system in concert with immunotherapies to eventually achieve improved and durable cancer treatment in vivo.

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]

Biodegradable polymer microneedles (MNs) are recognized as non-toxic, safe and stable systems for advanced drug delivery and cutaneous treatments, allowing a direct intradermal delivery and in some cases a controlled release. Most of the microneedles found in the literature are fabricated by micromolding, which is a multistep thus typically costly process. Due to industrial needs, mold-free methods represent a very intriguing approach in microneedle fabrication. Electro-drawing (ED) has been recently proposed as an alternative fast, mild temperature and one-step strategy to the mold-based techniques for the fabrication of poly(lactic-co-glycolic acid) (PLGA) biodegradable MNs. In this work, taking advantage of the flexibility of the ED technology, we engineered microneedle inner microstructure by acting on the water-in-oil (W/O) precursor emulsion formulation to tune drug release profile. Particularly, to promote a faster release of the active pharmaceutical ingredient, we substituted part of PLGA with poly(1-vinylpyrrolidone-co-vinyl acetate) (PVP/VA), as compared to the PLGA alone in the matrix material. Moreover, we introduced lecithin and maltose as emulsion stabilizers. Microneedle inner structural analysis as well as collagenase entrapment efficiency, release and activity of different emulsion formulations were compared to reach an interconnected porosity MN structure, aimed at providing an efficient protein release profile. Furthermore, MN mechanical properties were examined as well as its ability to pierce the stratum corneum on a pig skin model, while the drug diffusion from the MN body was monitored in an in vitro collagen-based dermal model at selected time points. [ABSTRACT FROM AUTHOR]

The enzymatic treatment of defatted soy flour (SF) to reduce indigestible carbohydrates can result in undesirable protein loss. Here protein loss was minimized with quantitation of the effects of ionic strength (IS), protease activity, and SF toasting. At the enzyme processing condition (25% w/v SF, 50 °C, pH 4.8, 48 hours), protein loss increased linearly with the IS in enzyme broths (EB); e.g., contacting untoasted SF with water or heat‐deactivated EB showed protein loss of 28% in water but up to 40% when IS was increased in the range of 0.04–0.19 M. Protein loss also increased with protease in EB (nondeactivated): after adjusted for IS‐related loss, approximately 10% and 25% additional protein loss occurred in EB of 73 and 490–557 U/(g SF) protease, respectively. SDS‐PAGE results showed that proteolysis was not extensive, mainly on β‐conglycinin α'/α and glycinin acidic 37‐kDa subunits; and most of the proteolytic products could be recovered by heat‐induced precipitation. SF toasting effects were studied, particularly at 2‐hours 160°C, with material balances, protein distributions, and monosaccharide yields in hydrolysates. Overall, protein loss was minimized to 5.2% and the conversion of carbohydrate to monomeric sugars increased to 89.2%. [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]

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]

Long-term delivery of growth factors and immunomodulatory agents is highly required to support the integrity of tissue in engineering constructs, e.g., formation of vasculature, and to minimize immune response in a recipient. However, for proteins with a net positive charge at the physiological pH, controlled delivery from negatively charged alginate (Alg) platforms is challenging due to electrostatic interactions that can hamper the protein release. In order to regulate such interactions between proteins and the Alg matrix, we propose to complex proteins of interest in this study - CXCL12, FGF-2, VEGF - with polyanionic heparin prior to their encapsulation into Alg microbeads of high content of α-L-guluronic acid units (high-G). This strategy effectively reduced protein interactions with Alg (as shown by model ITC and SPR experiments) and, depending on the protein type, afforded control over the protein release for at least one month. The released proteins retained their in vitro bioactivity: CXCL12 stimulated the migration of Jurkat cells, and FGF-2 and VEGF induced proliferation and maturation of HUVECs. The presence of heparin also intensified protein biological efficiency. The proposed approach for encapsulation of proteins with a positive net charge into high-G Alg hydrogels is promising for controlled long-term protein delivery under in vivo conditions.

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]

Osteoporotic fracture is one of the most common bone diseases in middle and old age, as the most serious consequence of osteoporosis. Sclerostin single-chain antibody fragments (SCL-scFv) have been proven to promote bone formation by binding to scleroprotein, a natural antagonist of the Wnt pathway, but it is difficult to rule alone due to the weak permeability and immunogenicity. Herein, we prepared poly(lactic-co-glycolic acid) microspheres as a sustained-release vehicle to prolong the activity of SCL-scFv. The morphology of microspheres were uniform and nearly sphere, loading efficiency and encapsulation efficiency of SCL-scFv microspheres were 6.28 ± 1.04% and 48.37 ± 8.11%, respectively. Approximately 90% of the SCL-scFvs were released from the microspheres over 28 days with initial burst releasing (38%) in the first 4 days. Sustained-release of active SCL-scFv from microspheres promoted bone marrow mesenchymal stem cells osteogenic differentiation in vitro and enhanced fracture healing in ovariectomized rats by improving bone mass and bone formation in the fracture region. All these findings demonstrate that the microspheres are able to simultaneously achieve localized long-term SCL-scFv controlled release and effectively promote bone formation, which provides a promising approach for osteoporotic fracture. [ABSTRACT FROM AUTHOR]

Delivery of osteoinductive factors such as bone morphogenetic protein 2 (BMP-2) has emerged as a prominent strategy to improve regeneration in bone grafting procedures. However, it remains challenging to identify a carrier that provides the requisite loading efficiency and release kinetics without compromising the mechanical properties of the bone graft. Previously, we reported on porous, polymerized high internal phase emulsion (polyHIPE) microspheres fabricated using controlled fluidics. Uniquely, this solvent-free method provides advantages over current microsphere fabrication strategies including in-line loading of growth factors to improve loading efficiency. In the current study, we utilized this platform to fabricate protein-loaded microspheres and investigated the effect of particle size (∼400 vs ∼800 μm) and pore size (∼15 vs 30 μm) on release profiles. Although there was no significant effect of these variables on the substantial burst release profile of the microspheres, the incorporation of the protein-loaded microspheres within the injectable polyHIPE resulted in a sustained release of protein from the bulk scaffold over a two-week period with minimal burst release. Bioactivity retention of encapsulated BMP-2 was confirmed first using a genetically-modified osteoblast reporter cell line. A functional assay with human mesenchymal stem cells established that the BMP-2 release from microspheres induced osteogenic differentiation. Finally, microsphere incorporation had minimal effect on the cure and compressive properties of an injectable polyHIPE bone graft. Overall, this work demonstrates that these microsphere-polyHIPE composites have strong potential to enhance bone regeneration through controlled release of BMP-2 and other growth factors. This manuscript describes a method for solvent-free fabrication of porous microspheres from high internal phase emulsions using a controlled fluids setup. The principles of emulsion templating and fluid dynamics provide exceptional control of particle size and pore architecture. In addition to the advantage of solvent-free fabrication, this method provides in-line loading of protein directly into the pores of the microspheres with high loading efficiencies. The incorporation of the protein-loaded microspheres within an injectable polyHIPE scaffold resulted in a sustained release of protein over a two-week period with minimal burst release. Retention of BMP-2 bioactivity and incorporation of microspheres with minimal effect on scaffold compressive properties highlights the potential of these new bone grafts. [ABSTRACT FROM AUTHOR]

The biomaterials design as core–shell structures opens a new door to the release of susceptible biomolecules in a controllable manner and enables to place natural biomaterials as shell layers to impart the effective biofunctional features at surfaces. In this study, core–shell designed scaffolds were prepared using coaxial electrospinning with hybrid of gelatin (GT)/polycaprolactone (PCL) at different weight ratios as their shell and protein solution as their core, followed by cross‐linking to impart controllable release rates, tunable mechanical properties, and enhanced cytocompatibility. SEM, FM, and TEM confirmed the successful production of uniform core–shell nanofibers and homogeneous protein distribution. Results showed that an increase in GT proportion in the shell resulted in a decrease in fiber diameter, an increase of Young's modulus, and an intense burst release of BSA 0.2% which could be controlled through cross‐linking. The mechanical tests revealed that the GT/PCL combining and cross‐linking improved mechanical properties which correlated with an increase in spreading and proliferation of HUVECs. A slight burst release was also detected from BSA 0.05% and EGF encapsulated GT73P‐cross‐linked scaffold which demonstrated their applicability for a controlled release of dilute proteins. We were able to successfully incorporate two types of protein with different concentrations without supporting polymer into the GT shell to provide scaffolds possessing tunable mechanical properties and controllable release rates through blending with PCL at different ratios and/or cross‐linking. These findings are promising to promote delivery systems of angiogenic growth factors that are needed a sustained release with different rates at each angiogenesis stage. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2019. [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]

Growth factors are powerful molecules that regulate cellular growth, proliferation, healing, and cellular differentiation. A delivery matrix that incorporates growth factors with high loading efficiencies, controls their release, and maintains bioactivity would be a powerful tool for regenerative medicine. Alginate has several unique properties that make it an excellent platform for the delivery of proteins. Mild gelling conditions can minimize the risk of protein denaturation; moreover, alginate can serve as protection from degradation until protein release. Various modifications have been proposed to tune alginate binding and release proteins, simultaneously adjusting alginate degradability, mechanical stiffness, swelling, gelation properties and cell affinity. The primary objective of this article is to review the literature related to recent advances in the application of alginate matrices in protein delivery in regenerative medicine. A special emphasis is put on the relevance of delivery of growth factors and chemokine. [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]

Self-assembly of natural polymers constitute a powerful route for the development of functional materials. In particular, layer-by-layer (LBL) assembly constitutes a versatile technique for the nanostructuration of biobased polymers into multilayer films. Gelatin has gained much attention for its abundance, biodegradability, and excellent gel-forming properties. However, gelatin gels melt at low temperature, thus limiting its practical application. With respect to the above considerations, here, we explored the potential application of gelatin gels as a matrix for protein delivery at physiological temperature. A model protein, bovine serum albumin (BSA), was encapsulated within gelatin gels and then coated with a different number of bilayers of alginate and chitosan (10, 25, 50) in order to modify the diffusion barrier. The coated gel samples were analyzed by means of Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) and confocal Raman spectroscopy, and it was found that the multilayer coatings onto polymer film were interpenetrated to some extent within the gelatin. The obtained results inferred that the coating of gelatin gels with polysaccharide multilayer film increased the thermal stability of gelatin gels and modulated the BSA release. Finally, the influence of a number of bilayers onto the drug release mechanism was determined. The Ritger-Peppas model was found to be the most accurate to describe the diffusion mechanism.