Molecular Retention Limitations for Prevascularized Subcutaneous Sites for Islet Transplantation.

Beta cell replacement therapies utilizing the subcutaneous space have inherent advantages to other sites: the potential for increased accessibility, noninvasive monitoring, and graft extraction. Site prevascularization has been developed to enhance islet survivability in the subcutaneous zone while minimizing potential foreign body immune responses. Molecular communication between the host and prevascularized implant site remains ill-defined. Poly(ethylene oxide)s (PEOs) of various hydrated radii (i.e., ∼11-62 Å) were injected into prevascularized subcutaneous sites in C57BL/6 mice, and the clearance and organ biodistribution were characterized. Prevascularization formed a barrier that confined the molecules compared with the unmodified site. Molecular clearance from the prevascularized site was inversely proportional to the molecular weight. The upper limit in molecular size for entering the vasculature to be cleared was determined to be 35 kDa MW PEO. These findings provide insight into the impact of vascularization on molecular retention at the injection site and the effect of molecular size on the mobility of hydrophilic molecules from the prevascularized site to the host. This information is necessary for optimizing the transplantation site for increasing the beta cell graft survival.

Detailing Protein-Bound Uremic Toxin Interaction Mechanisms with Human Serum Albumin in the Pursuit of Designing Competitive Binders.

Chronic kidney disease is the gradual progression of kidney dysfunction and involves numerous co-morbidities, one of the leading causes of mortality. One of the primary complications of kidney dysfunction is the accumulation of toxins in the bloodstream, particularly protein-bound uremic toxins (PBUTs), which have a high affinity for plasma proteins. The buildup of PBUTs in the blood reduces the effectiveness of conventional treatments, such as hemodialysis. Moreover, PBUTs can bind to blood plasma proteins, such as human serum albumin, alter their conformational structure, block binding sites for other valuable endogenous or exogenous substances, and exacerbate the co-existing medical conditions associated with kidney disease. The inadequacy of hemodialysis in clearing PBUTs underscores the significance of researching the binding mechanisms of these toxins with blood proteins, with a critical analysis of the methods used to obtain this information. Here, we gathered the available data on the binding of indoxyl sulfate, p-cresyl sulfate, indole 3-acetic acid, hippuric acid, 3-carboxyl-4-methyl-5-propyl-2-furan propanoic acid, and phenylacetic acid to human serum albumin and reviewed the common techniques used to investigate the thermodynamics and structure of the PBUT-albumin interaction. These findings can be critical in investigating molecules that can displace toxins on HSA and improve their clearance by standard dialysis or designing adsorbents with greater affinity for PBUTs than HSA.

The Possible Uses and Challenges of Nanomaterials in Mast Cell Research.

Mast cells are tissue-resident immune cells that are involved in inflammation and fibrosis but also serve beneficial roles, including tissue maintenance, angiogenesis, pathogen clearance, and immunoregulation. Their multifaceted response and the ability of their mediators to target multiple organs and tissues means that mast cells play important roles in numerous conditions, including asthma, atopic dermatitis, drug sensitivities, ischemic heart disease, Alzheimer disease, arthritis, irritable bowel syndrome, infections (parasites, bacteria and viruses), and cancer. As a result, mast cells have become an important target for drug discovery and diagnostic research. Recent work has focused on applying novel nanotechnologies to explore cell biology. In this brief review, we will highlight the use of nanomaterials to modify mast cell functions and will discuss the potential of these technologies as research tools for understanding mast cell biology.

Towards preventing exfoliation glaucoma by targeting and removing fibrillar aggregates associated with exfoliation syndrome.

Exfoliation syndrome presents as an accumulation of insoluble fibrillar aggregates that commonly correlates with age and causes ocular complications, most notably open-angle glaucoma. Despite advances in understanding the pathogenesis and risk factors associated with exfoliation syndrome, there has been no significant progress in curative pharmacotherapy of this disease. It is thought that the ability to target the fibrillar aggregates associated with exfoliation may offer a new therapeutic approach, facilitating their direct removal from affected tissues. Phage display techniques yielded two peptides (LPSYNLHPHVPP, IPLLNPGSMQLS) that could differentiate between exfoliative and non-affected regions of the human lens capsule. These peptides were conjugated to magnetic particles using click chemistry to investigate their ability in targeting and removing exfoliation materials from the anterior human lens capsule. The behavior of the fibrillar materials upon binding to these magnetic particles was assessed using magnetic pins and rotating magnetic fields of various strengths. Ex vivo studies showed that the magnetic particle-peptide conjugates could generate enough mechanical force to remove large aggregates of exfoliation materials from the lens capsule when exposed to a low-frequency rotating magnetic field (5000 G, 20 Hz). Biocompatibility of targeting peptides with and without conjugated magnetic particles was confirmed using MTT cell toxicity assay, live/dead cell viability assay, and DNA fragmentation studies on primary cultured human trabecular meshwork cells. This is a novel, minimally invasive, therapeutic approach for the treatment of exfoliation glaucoma via the targeting and removal of exfoliation materials that could be applied to all tissues within the anterior segment of the eye.

Effect of amino acid composition of elastin-like polypeptide nanoparticles on nonspecific protein adsorption, macrophage cell viability and phagocytosis.

Development of elastin-like polypeptide (ELP) biomaterials is widespread, but information critical for clinical deployment is limited, with biocompatibility studies focused on a narrow cross-section of ELP sequences. Macrophages can impair biomaterial systems by degrading or isolating the biomaterial and by activating additional immune functions. Their phagocytic response will reveal early immune biocompatibility of ELP nanoparticles (NPs). This study examines that response, induced by the adsorbed protein corona, as a function of ELP guest amino acid, chain length and NP diameter. The breadth of proteins adsorbed to ELP NPs varied, with valine-containing ELP NPs adsorbing fewer types of proteins than leucine-containing constructs. Particle diameter was also a factor, with smaller leucine-containing ELP NPs adsorbing the broadest range of proteins. Macrophage viability was unaffected by the ELP NPs, and their phagocytic capabilities were unimpeded except when incubated with a 500 nm valine-containing 40-mer. This NP significantly decreased the phagocytic capacity of macrophages relative to the control and to a corresponding 500 nm leucine-containing 40-mer. NP size and the proportion of opsonin to dysopsonin proteins likely influenced this outcome. These results suggest that certain combinations of ELP sequence and particle size can result in an adsorbed protein corona, which may hinder macrophage function.

Enzymatic Activity in Fractal Networks of Self-Assembling Peptides.

The tissue environment is exceptionally complex, with well-controlled biochemical communication occurring between similar and dissimilar cells as well as between these cells and local extracellular matrices (ECM). To build an artificial ECM that can directly affect regional cell populations, a designer system should allow for controlled degradation, molecular release, and reorganization as related to local cellular function. (RADA)4 self-assembling peptide (SAP) hydrogels are excellent candidates for precisely tuned ECMs, or nanoscaffolds, with several beneficial qualities. They are a class of materials with uncomplicated fabrication and potentially allow for a diverse set of release strategies for many types of bioactive ligands. Enzyme-induced degradation and release of peptide sequences, synthesized within the SAP for on-demand cell signaling, could prove impactful to a plethora of human health applications. However, the degradation products and their release kinetics from these nanoscaffolds may greatly affect the overall system. To address this, enzyme kinetics in self-assembled hydrogels were studied by tethering matrix metalloproteinase 2 (MMP-2) cleavable peptide substrates of differing activities to the C-terminus of (RADA)4. High and low activity sequences, GPQG+IASQ (CP1) and GPQG+PAGQ (CP2), were respectively chosen for tunable release. When incubated with 5 nM MMP-2, over 3 days, both CP1 and CP2 sequences showed product formation values of ∼32% and ∼9% of the original substrate, respectively. On-demand product formation was found to be dependent upon both SAP composition and enzyme concentrations and could be tuned over the course of several days and weeks. Despite the fact that the self-assembling peptides are not directly cleavable by MMP-2, the CP1 and CP2 nanoscaffold morphology was visibly degraded by the protease. This degradation yielded a lower fractal dimensions for the matrix and suggested clearance of these materials may be possible over time.

Self-assembly/disassembly hysteresis of nanoparticles composed of marginally soluble, short elastin-like polypeptides.

BACKGROUND: Elastin-like polypeptides (ELPs) are a fascinating biomaterial that has undergone copious development for a variety of therapeutic applications including as a nanoscale drug delivery vehicle. A comprehensive understanding of ELP self-assembly is lacking and this knowledge gap impedes the advancement of ELP-based biomaterials into the clinical realm. The systematic examination of leucine-containing ELPs endeavors to expand existing knowledge about fundamental assembly-disassembly behaviours. RESULTS: It was observed that these marginally soluble, short ELPs tend to behave consistently with previous observations related to assembly-related ELP phase transitions but deviated in their disassembly. It was found that chain length, concentration and overall sequence hydrophobicity may influence the irreversible formation of sub-micron particles as well as the formation of multi-micron scale, colloidally unstable aggregates. Amino acid composition affected surface charge and packing density of the particles. Particle stability upon dilution was found to vary depending upon chain length and hydrophobicity, with particles composed of longer and/or more hydrophobic ELPs being more resistant to disassembly upon isothermal dilution. CONCLUSIONS: Taken together, these results suggest marginally soluble ELPs may self-assemble but not disassemble as expected and that parameters including particle size, zeta potential and dilution resistance would benefit from widespread systematic evaluations. This information has the potential to reveal novel preparation methods capable of expanding the utility of all existing ELP-based biomaterials.

Peptides for targeting ßB2-crystallin fibrils.

Crystallins are a major family of proteins located within the lens of the eye. Cataracts are thought to be due to the formation of insoluble fibrillar aggregates, which are largely composed of proteins from the crystallin family. Today the only cataract treatment that exists is surgery and this can be difficult to access for individuals in the developing world. Development of novel pharmacotherapeutic approaches for the treatment of cataract rests on the specific targeting of these structures. ßB2-crystallin, a member of ß-crystallin family, is a large component of the crystallin proteins within the lens, and as such was used to form model fibrils in vitro. Peptides were identified, using phage display techniques, that bound to these fibrils with high affinity. Fibrillation of recombinantly expressed human ßB2-crystallin was performed in 10% (v/v) trifluoroethanol (TFE) solution (pH 2.0) at various temperatures, and its amyloid-like structure was confirmed using Thioflavin-T (ThT) assay, transmission electron microscopy (TEM), and X-ray fiber diffraction (XRFD) analysis. Affinity of identified phage-displayed peptides were analyzed using enzyme-linked immunosorbent assay (ELISA). Specific binding of a cyclic peptide (CKQFKDTTC) showed the highest affinity, which was confirmed using a competitive inhibition assay.

pH-Triggered Release of Hydrophobic Molecules from Self-Assembling Hybrid Nanoscaffolds.

Self-assembling peptide based hydrogels have a wide range of applications in the field of tissue repair and tissue regeneration. Because of its physicochemical properties, (RADA)4 has been studied as a potential platform for 3D cell culture, drug delivery, and tissue engineering. Despite some small molecule and protein release studies with this system, there is a lack of work investigating the controlled release of hydrophobic compounds (i.e., anti-inflammatory, anticancer, antibacterial drugs, etc.) that are important for many clinical therapies. Attempts to incorporate hydrophobic compounds into self-assembling matrices usually inhibited nanofiber formation, rather resulting in a peptide-drug complex or microcrystal formation. Herein, a self-assembling chitosan/carboxymethyl-ß-cyclodextrin nanoparticle system was used to load dexamethasone, which formed within a self-assembling (RADA)4 nanoscaffold matrix. Nanoparticles dispersed within the matrix were stabilized by the nanofibers within. The in vitro release of dexamethasone from the hybrid system was observed to be pH sensitive. At pH 7, release was observed for more than 8 days, with three distinct kinetic domains in the first 6 days. Data suggest that the deprotonation of chitosan at a solution pH > 6.8 leads to nanoparticle dissociation and ultimately the release of dexamethasone from the hybrid system. This system has the potential to form a multifunctional scaffold that can self-assemble with the ability to control the release of hydrophobic drugs for a wide variety of applications.

Application of isothermal titration calorimetry for characterizing thermodynamic parameters of biomolecular interactions: peptide self-assembly and protein adsorption case studies.

The complex nature of macromolecular interactions usually makes it very hard to identify the molecular-level mechanisms that ultimately dictate the result of these interactions. This is especially evident in the case of biological systems, where the complex interaction of molecules in various situations may be responsible for driving biomolecular interactions themselves but also has a broader effect at the cell and/or tissue level. This review will endeavor to further the understanding of biomolecular interactions utilizing the isothermal titration calorimetry (ITC) technique for thermodynamic characterization of two extremely important biomaterial systems, viz., peptide self-assembly and nonfouling polymer-modified surfaces. The advantages and shortcomings of this technique will be presented along with a thorough review of the recent application of ITC to these two areas. Furthermore, the controversies associated with the enthalpy-entropy compensation effect as well as thermodynamic equilibrium state for such interactions will be discussed.

Adsorption Dynamics of Uremic Toxins to Novel Modified Magnetic Nanoparticles.

Kidney dysfunction leads to the retention of metabolites in the blood compartment, some of which reach toxic levels. Uremic toxins are associated with the progression of kidney disease and other symptoms of kidney failure (i.e., nausea, itchiness, and hypertension). Toxin removal ameliorates symptoms and reduces further organ damage, but membrane-based methods are inadequate for this purpose. Engineered adsorbents may facilitate enhanced removal of retained toxins, especially those bound strongly by proteins. Poly 2-(methacryloyloxy)ethyl phosphorylcholine-co-ß-cyclodextrin (p(MPC-co-PMßCD)) coated magnetic nanoparticles are synthesized, characterized for their physicochemical properties (Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), thermogravimetric analysis(TGA), gel permeation chromatography (GPC), and transmission electron microscope (TEM), and evaluated toxin adsorption from a complex solution for the first time to quantify the effects of film chemistry and incubation time on the adsorbed toxinome (the collection of toxins). Uremic toxins are bound by even "low-fouling" polymer films themselves; providing further insight into how small molecule interactions with "low-fouling" films may affect protein-surface interactions. These results suggest a dynamic interaction between toxins and surfaces that is not driven by solution concentration alone. This knowledge will help advance the design of novel adsorbent films for clearing uremic toxins.

Understanding the effect of secondary structure on molecular interactions of poly-L-lysine with different substrates by SFA.

Nonspecific adsorption of proteins on biomaterial surfaces challenges the widespread application of engineered materials, and understanding the impact of secondary structure of proteins and peptides on their adsorption process is of both fundamental and practical importance in bioengineering. In this work, poly-L-lysine (PLL)-based α-helices and ß-sheets were chosen as a model system to investigate the effect of secondary structure on peptide interactions with substrates of various surface chemistries. Circular dichroism (CD) was used to confirm the presence of both α-helix and ß-sheet structured PLL in aqueous solutions and upon adsorption to quartz, where these secondary structures seemed to be preserved. Atomic force microscopy (AFM) imaging showed different surface patterns for adsorbed α-helix and ß-sheet PLL. Interactions between PLL of different secondary structures and various substrates (i.e., PLL, Au, mica, and poly(ethylene glycol) (PEG)) were directly measured using a surface forces apparatus (SFA). It was found that ß-sheet PLL films showed higher adsorbed layer thicknesses in general. Adhesion energies of ß-sheet versus Au and ß-sheet versus ß-sheet were considerably higher than that of α-helix versus Au and α-helix versus α-helix systems, respectively. Au and ß-sheet PLL interactions seemed to be more dependent on the salt concentration than that of α-helix, while the presence of a grafted PEG layer greatly diminished any attraction with either PLL structure. The molecular interaction mechanism of peptide in different secondary structures is discussed in terms of Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, Alexander-de Gennes (AdG) steric model and hydrogen bonding, which provides important insight into the fundamental understanding of the interaction mechanism between proteins and biomaterials.

Toward a mechanistic understanding of ionic self-complementary peptide self-assembly: role of water molecules and ions.

Ionic self-complementary peptides are considered an important class of self-assembling peptides. In particular, RADARADARADARADA (RADA4) is well-known to form a relatively regular nanofiber structure that has been primarily studied in terms of its physicochemical properties, as related to its biomedical applications. However, the molecular level interactions that are involved in promoting the self-assembly of this peptide into nanofibers have not been fully elucidated. Herein, a thermodynamic analysis of the influences of peptide chemistry upon self-assembly is discussed for RADA4, RADA4-K5, and RADA4-S5. The regular nanofiber structure of the assembled peptides makes it a good candidate for isothermal titration calorimetry (ITC) studies for determining the propensity for self-assembly, the critical assembly concentration (CAC), and the role hydration and ion content play in the assembly of these peptides. First, solutions containing only RADA4-K5 did not self-assemble; illustrating even slight alterations in the asymmetric terminal amino acid chemistry affects assembly. The CAC of the remaining self-assembling peptides was between ~0.1 and ~0.15 mM. Interestingly, we found that self-assembly was entropically driven with hydrophobic forces being the main driving force for RADA4 and hydrogen bonding for RADA4-S5. The role of water molecules and counterions in self-assembly was also highlighted: assembly of RADA4 led to desolvation of interfacial surfaces, whereas the net number of water molecules in the assembled complex increased upon RADA4-S5 self-assembly. Moreover, it was found that counterions did not seem to contribute significantly to self-assembly: a result in contrast to current concepts regarding the role of electrostatic interactions in self-assembly of RADA4-like peptides. A molecular level understanding of peptide self-assembly will allow for further engineering of peptides for a vast array of biomedical applications.

Targeting active sites of inflammation using inherent properties of tissue-resident mast cells.

Mast cells play a pivotal role in initiating and directing host's immune response. They reside in tissues that primarily interface with the external environment. Activated mast cells respond to environmental cues throughout acute and chronic inflammation through releasing immune mediators via rapid degranulation, or long-term de novo expression. Mast cell activation results in the rapid release of a variety of unique enzymes and reactive oxygen species. Furthermore, the increased density of mast cell unique receptors like mas related G protein-coupled receptor X2 also characterizes the inflamed tissues. The presence of these molecules (either released mediators or surface receptors) are particular to the sites of active inflammation, and are a result of mast cell activation. Herein, the molecular design principles for capitalizing on these novel mast cell properties is discussed with the goal of manipulating localized inflammation. STATEMENT OF SIGNIFICANCE: Mast cells are immune regulating cells that play a crucial role in both innate and adaptive immune responses. The activation of mast cells causes the release of multiple unique profiles of biomolecules, which are specific to both tissue and disease. These unique characteristics are tightly regulated and afford a localized stimulus for targeting inflammatory diseases. Herein, these important mast cell attributes are discussed in the frame of highlighting strategies for the design of bioresponsive functional materials to target regions of inflammations.

Hemocompatibility of ß-Cyclodextrin-Modified (Methacryloyloxy)ethyl Phosphorylcholine Coated Magnetic Nanoparticles.

Adsorbing toxins from the blood to augment membrane-based hemodialysis is an active area of research. Films composed of ß-cyclodextrin-co-(methacryloyloxy)ethyl phosphorylcholine (p(PMßCD-co-MPC)) with various monomer ratios were formed on magnetic nanoparticles and characterized. Surface chemistry effects on protein denaturation were evaluated and indicated that unmodified magnetic nanoparticles greatly perturbed the structure of proteins compared to coated particles. Plasma clotting assays were conducted to investigate the stability of plasma in the presence of particles, where a 2:2 monomer ratio yielded the best results for a given total surface area of particles. Total protein adsorption results revealed that modified surfaces exhibited reduced protein adsorption compared to bare particles, and pure MPC showed the lowest adsorption. Immunoblot results showed that fibrinogen, α1-antitrypsin, vitronectin, prekallikrein, antithrombin, albumin, and C3 correlated with film composition. Hemocompatibility testing with whole blood illustrated that the 1:3 ratio of CD to MPC had a negative impact on platelets, as evidenced by the increased activation, reduced response to an agonist, and reduced platelet count. Other formulations had statistically significant effects on platelet activation, but no formulation yielded apparent adverse effects on hemostasis. For the first time, p(PMßCD-co-MPC)-coated MNP were synthesized and their general hemocompatibility assessed.

Administration of selective brain hypothermia using a simple cooling device in neonatal rats.

BACKGROUND: The interruption of oxygen and blood supply to the newborn brain around the time of birth is a risk factor for hypoxic-ischemic encephalopathy and may lead to infant mortality or lifelong neurological impairments. Currently, therapeutic hypothermia, the cooling of the infant's head or entire body, is the only treatment to curb the extent of brain damage. NEW METHOD: In this study, we designed a focal brain cooling device that circulates cooled water at a steady state temperature of 19 ± 1 °C through a coil of tubing fitted onto the neonatal rat's head. We tested its ability to selectively decrease brain temperature and offer neuroprotection in a neonatal rat model of hypoxic-ischemic brain injury. RESULTS: Our method cooled the brain to 30-33 °C in conscious pups, while keeping the core body temperature approximately 3.2 °C warmer. Furthermore, the application of the cooling device to the neonatal rat model demonstrated a reduction in brain volume loss compared to pups maintained at normothermia and achieved a level of brain tissue protection the same as that of whole-body cooling. COMPARISON WITH EXISTING METHODS: Prevailing methods of selective brain hypothermia are designed for adult animal models rather than for immature animals such as the rat as a conventional model of developmental brain pathology. Contrary to existing methods, our method of cooling does not require surgical manipulation or anaesthesia. CONCLUSION: Our simple, economical, and effective method of selective brain cooling is a useful tool for rodent studies in neonatal brain injury and adaptive therapeutic interventions.

Nanothin Conformal Coating with Poly(N-vinylpyrrolidone) and Tannic Acid (PVPON/TA) Preserves Murine and Human Pancreatic Islets Function.

Beta cell replacement therapies can restore glycemic control to select individuals living with type 1 diabetes. However, the obligation of lifelong immunosuppression restricts cell therapies from replacing exogenous insulin administration. Encapsulation strategies can reduce the inherent adaptive immune response; however, few are successfully translated into clinical testing. Herein, we evaluated if the conformal coating of islets with poly(N-vinylpyrrolidone) (PVPON) and tannic acid (TA) (PVPON/TA) could preserve murine and human islet function while conferring islet allograft protection. In vitro function was evaluated using static glucose-stimulated insulin secretion, oxygen consumption rates, and islet membrane integrity. In vivo function was evaluated by transplanting human islets into diabetic immunodeficient B6.129S7-Rag1tm1Mom/J (Rag-/-) mice. The immunoprotective capacity of the PVPON/TA-coating was assessed by transplanting BALB/c islets into diabetic C57BL/6 mice. Graft function was evaluated by non-fasting blood glucose measurements and glucose tolerance testing. Both coated and non-coated murine and human islets exhibited indistinguishable in vitro potency. PVPON/TA-coated and control human islets were able to restore euglycemia post-transplant. The PVPON/TA-coating as monotherapy and adjuvant to systemic immunosuppression reduced intragraft inflammation and delayed murine allograft rejection. This study demonstrates that PVPON/TA-coated islets may be clinically relevant as they retain their in vitro and in vivo function while modulating post-transplant immune responses.

Nanostructured, self-assembling peptide K5 blocks TNF-α and PGE2 production by suppression of the AP-1/p38 pathway.

Nanostructured, self-assembling peptides hold promise for a variety of regenerative medical applications such as 3D cell culture systems, accelerated wound healing, and nerve repair. The aim of this study was to determine whether the self-assembling peptide K5 can be applied as a carrier of anti-inflammatory drugs. First, we examined whether the K5 self-assembling peptide itself can modulate various cellular inflammatory responses. We found that peptide K5 significantly suppressed the release of tumor-necrosis-factor- (TNF-) α and prostaglandin E2 (PGE2) from RAW264.7 cells and peritoneal macrophages stimulated by lipopolysaccharide (LPS). Similarly, there was inhibition of cyclooxygenase- (COX-) 2 mRNA expression assessed by real-time PCR, indicating that the inhibition is at the transcriptional level. In agreement with this finding, peptide K5 suppressed the translocation of the transcription factors activator protein (AP-1) and c-Jun and inhibited upstream inflammatory effectors including mitogen activated protein kinase (MAPK), p38, and mitogen-activated protein kinase kinase 3/6 (MKK 3/6). Whether this peptide exerts its effects via a transmembrane or cytoplasmic receptor is not clear. However, our data strongly suggest that the nanostructured, self-assembling peptide K5 may possess significant anti-inflammatory activity via suppression of the p38/AP-1 pathway.

Controlled release of functional proteins through designer self-assembling peptide nanofiber hydrogel scaffold.

The release kinetics for a variety of proteins of a wide range of molecular mass, hydrodynamic radii, and isoelectric points through a nanofiber hydrogel scaffold consisting of designer self-assembling peptides were studied by using single-molecule fluorescence correlation spectroscopy (FCS). In contrast to classical diffusion experiments, the single-molecule approach allowed for the direct determination of diffusion coefficients for lysozyme, trypsin inhibitor, BSA, and IgG both inside the hydrogel and after being released into the solution. The results of the FCS analyses and the calculated pristine in-gel diffusion coefficients were compared with the values obtained from the Stokes-Einstein equation, Fickian diffusion models, and the literature. The release kinetics suggested that protein diffusion through nanofiber hydrogels depended primarily on the size of the protein. Protein diffusivities decreased, with increasing hydrogel nanofiber density providing a means of controlling the release kinetics. Secondary and tertiary structure analyses and biological assays of the released proteins showed that encapsulation and release did not affect the protein conformation and functionality. Our results show that this biocompatible and injectable designer self-assembling peptide hydrogel system may be useful as a carrier for therapeutic proteins for sustained release applications.

Evaluating the effect of graphene oxide PEGylation on the properties of chitosan-graphene oxide nanocomposite scaffold.

In this study, graphene oxide (GO) was functionalized with polyethylene glycol (PEG) to understand the effect of PEGlayted GO on properties of chitosan-based nanocomposite scaffold. GO was synthesized according to modified Hummer's method and covalently linked to polymeric chains of PEG to produce polyethylene glycolated GO (PGO). Successful preparation of GO and PGO was confirmed by FT-IR and Raman techniques, where the chemical bonding of PEG and GO nanosheets were concluded based on PGOs' lower zeta potential compared to GO. Nanocomposite scaffolds were prepared by adding equal amounts of GO and PGO into 2% (w/v) chitosan (Cs) solutions. The highly porous scaffolds were developed by lyophilization of solutions. Incorporation of GO and PGO into chitosan scaffold network resulted in uniform and spherical pores. Modified samples offered higher porosity and density, indicating adequate scaffold structure. Improvements in the physical properties of prepared chitosan scaffolds were concluded through higher water absorption and retention values. Compressive strength measurement showed 6.33 and 5.5 times improvement respectively for Cs-GO and Cs-PGO samples compared to Cs scaffold. The Cs-GO scaffolds showed minimum susceptibility toward enzymatic degradation and higher degrees of protein adsorption (26% and 23% improvement in value of adsorbed protein respectively for Cs-GO and Cs-PGO compared to Cs scaffold) and biomineral formation on scaffold surface. Also, Cs-PGO sample showed the highest degree of cell viability and lower hemolysis than both Cs and Cs-GO scaffolds. Investigations showed that cell infiltration into scaffold porous structure was more prominent in Cs-PGO scaffolds than in Cs and Cs-GO scaffolds.