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.

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.

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.

Characteristics of Serum Exosomes after Burn Injury and Dermal Fibroblast Regulation by Exosomes In Vitro.

(1) Background: Exosomes (EXOs) have been considered a new target thought to be involved in and treat wound healing. More research is needed to fully understand EXO characteristics and the mechanisms of EXO-mediated wound healing, especially wound healing after burn injury. (2) Methods: All EXOs were isolated from 85 serum samples of 29 burn patients and 13 healthy individuals. We characterized the EXOs for morphology and density, serum concentration, protein level, marker expression, size distribution, and cytokine content. After a confirmation of EXO uptake by dermal fibroblasts, we also explored the functional regulation of primary human normal skin and hypertrophic scar fibroblast cell lines by the EXOs in vitro, including cell proliferation and apoptosis. (3) Results: EXOs dynamically changed their morphology, density, size, and cytokine level during wound healing in burn patients, which were correlated with burn severity and the stages of wound healing. EXOs both from burn patients and healthy individuals stimulated dermal fibroblast proliferation and apoptosis. (4) Conclusions: EXO features may be important signals that influence wound healing after burn injury; however, to understand the mechanisms by which EXOs regulates the fibroblasts in healing wounds, further studies will be required.

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.

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.

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.

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.

The last 25 years of research on bioflocculants for kaolin flocculation with recent trends and technical challenges for the future.

The generation of kaolin-containing wastewater is an inevitable consequence in a number of industries including mining, wastewater treatment, and bitumen processing. In some cases, the production of kaolin tailings waste during the production of bitumen or phosphate is as high as 3 times greater than the actual produced product. The existing inventory of nearly five billion barrels of oil sands tailings alone represents a massive storage and reclamation challenge, as well as a significant economic and environmental liability. Current reclamation options like inorganic coagulants and organic synthetic polymers may settle kaolin effectively, but may themselves pose an additional environmental hazard. Bioflocculants are an emerging alternative, given the inherent safety and biodegradability of their bio-based compositions. This review summarizes the different research attempts towards a better bioflocculant of kaolin, with a focus on the bioflocculant source, composition, and effective flocculating conditions. Bacillus bacteria were the most prevalent single species for bioflocculant production, with wastewater also hosting a large number of bioflocculant-producing microorganisms while serving as an inexpensive nutrient. Effective kaolin flocculation could be obtained over a broad range of pH values (1-12) and temperatures (5-95°C). Uronic acid and glutamic acid were predominant sugars and amino acids, respectively, in a number of effective bioflocculants, potentially due to their structural and charge similarities to effective synthetic polymers like polyacrylamide. Overall, these results demonstrate that bioflocculants can be produced from a wide range of microorganisms, can be composed of polysaccharides, protein or glycoproteins and can serve as effective treatment options for kaolin. In some cases, the next obstacle to their wide-spread application is scaling to industrially relevant volumes and their deployment strategies.

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.

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.

In Vitro Study: Synthesis and Evaluation of Fe3O4/CQD Magnetic/Fluorescent Nanocomposites for Targeted Drug Delivery, MRI, and Cancer Cell Labeling Applications.

In the present study, first, Fe3O4 nanoparticles were functionalized using glutaric acid and then composited with CQDs. Doxorubicin (DOX) drug was loaded to evaluate the performance of the nanocomposite for targeted drug delivery applications. The XRD pattern confirmed the presence of characteristic peaks of CQDs and Fe3O4. In the FTIR spectrum, the presence of carboxyl functional groups on Fe3O4/CQDs was observed; DOX (positive charge) is loaded onto Fe3O4/CQDs (negative charge) by electrostatic absorption. FESEM and AFM images showed that the particle sizes of Fe3O4 and CQDs were 23-75 and 1-3 nm, respectively. The hysteresis curves showed superparamagnetic properties for Fe3O4 and Fe3O4/CQDs (57.3 and 8.4 emu/g). The Fe3O4 hysteresis curve showed superparamagnetic properties (Ms and Mr: 57.3 emu/g and 1.46 emu/g. The loading efficiency and capacity for Fe3O4/CQDs were 93.90% and 37.2 mg DOX/g MNP, respectively. DOX release from Fe3O4/CQDs in PBS showed pH-dependent release behavior where after 70 h at pH 5 and 7.4, about 50 and 21% of DOX were released. Fluorescence images of Fe3O4/CQD-treated cells showed that Fe3O4/CQDs are capable of labeling MCF-7 and HFF cells. Also, T2-weighted MRI scans of Fe3O4/CQDs in water exhibited high r2 relaxivity (86.56 mM-1 S-1). MTT assay showed that DOX-loaded Fe3O4/CQDs are highly biocompatible in contact with HFF cells (viability = 95%), but they kill MCF-7 cancer cells (viability = 45%). Therefore, the synthesized nanocomposite can be used in MRI, targeted drug delivery, and cell labeling.

Bacteriophage carriers localize in the brain of a rat model of neonatal hypoxic-ischemic encephalopathy.

BACKGROUND: Neonatal hypoxic-ischemic encephalopathy arises from a reduction of oxygen and blood supply to the infant brain and can lead to severe brain damage and life-long disability. The damage is greatest at the irreversibly injured necrotic core, whereas the penumbra is the surrounding, potentially salvageable tissue populated with a mix of alive and dying cells. To date, there exists no method for targeting drugs to the brain damage. METHODS AND MAJOR RESULTS: Bacteriophages are viruses that propagate in bacteria but are biocompatible in humans and also amenable to genetic and chemical modification in a manner distinctive from conventional therapeutic nanoparticles. Here, a library of M13 bacteriophage was administered into a rat model of hypoxic-ischemic encephalopathy, and unique bacteriophage clones were confirmed to localize in healthy brain tissue versus the core and penumbra zones of injury. CONCLUSIONS: For the first time, there is a potential to directly deliver therapeutics to different regions of the neonatal brain injury.

Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells.

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new therapeutics. Mas related G-protein receptor-X2 (MRGPRX2) is an important receptor that regulates mast cell activation and, thus, directs the general immune response. Numerous ligands for MRGPRX2 have been identified and include endogenous peptides like PAMPs, defensins, LL-37 and other protein fragments (i.e., degraded albumin). Further identification of MRGPRX2 specific ligands requires the screening of a large number of peptides (i.e., peptide library); however, mast cells are difficult and expensive to maintain in vitro and, therefore, not economical to use for screening large numbers of molecules. The present paper demonstrates a method to design, develop, and screen a library of small peptide molecules using MRGPRX2 expressing HEK cells. This cell line is relatively easy and inexpensive to maintain and can be used for in vitro high-throughput analysis. A calcium sensitive Fura-2 fluorescent dye to mark intracellular calcium flux upon activation was used to monitor the activation. The ratio of fluorescence intensity of Fura-2 at 510 nm against excitation wavelengths of 340 and 380 nm was used to calculate calcium concentration. The peptide library used to verify this system was based on the endogenous proadrenomedullin N-terminal 12 (PAMP-12) secretagogue, which is known to bind MRGPRX2 with high specificity and affinity. Subsequent peptides were generated through amino acid truncation and alanine scanning techniques applied to PAMP-12. The method described here is simple and inexpensive yet robust for screening a large library of compounds to identify binding domains and other important parameters that play an important role in receptor activation.

Peptide-Modified Surfaces for Binding Carbamylated Proteins from Plasma.

Carbamylation of blood proteins is a common post-translational modification that occurs upon kidney dysfunction that is strongly associated with deleterious outcomes for patients treated using hemodialysis. In this study, we focused on the removal of two representative carbamylated plasma proteins, carbamylated albumin (cHSA) and fibrinogen (cFgn), through adsorption onto a surface functionalized with a specific peptide (cH2p1). Surfaces modified with poly(hydroxyethyl methacrylate) (p(HEMA)) were prepared using surface-initiated atom transfer radical polymerization (SI-ATRP) techniques and functionalized with cH2p1. cH2p1-functionalized surfaces showed selective binding toward cHSA and cFgn, compared to their native protein form, with NH-cH2p1 of superior selectivity than CO-cH2p1. The adsorption capacity of carbamylated protein on NH-cH2p1 was maintained in diluted plasma, and ultralow adsorption of native Fgn was observed. Similar to unmodified p(HEMA) surfaces, NH-cH2p1 showed a low platelet adhesion and activation, suggesting that the designed surface does not adversely affect platelets.

Identification of short peptide sequences that activate human mast cells via Mas-related G-protein coupled receptor member X2.

Peptide based therapeutics are desirable owing to their high biological specificity. However, a number of these fail in clinical testing due to an adverse inflammatory response. Mast cells play a key role in directing the host response to drugs and related products. Although the role of FcεRI receptor is well known, Mas-related G-protein coupled receptor X2 (MRGPRX2) binding of endogenous peptides, and drugs will activate mast cells independent of FcεRI. Identifying peptides that activate mast cells through MRGPRX2, and their respective activation potency, can be used to reduce the failure rate of peptide therapeutics at clinical trial. Moreover, it will allow for peptide design where mast cell activation is actually desired. It was found that FRKKW and WNKWAL are two motifs that activate human LAD2 cells similar to PAMP-12 controls. Peptide activators of MRGPRX2 could be reduced to Xa-(Y)(n ≥ 3)-Xb where: Xa is an aromatic residue; Xb is a hydrophobic residue; and Y is a minimum 3 residue long sequence, containing a minimum of one positively charged residue with the remainder being uncharged residues. Artificial peptides WKKKW and FKKKF were constructed to test this structural functionality and were similar to PAMP-12 controls. Peptides with different activation potentials were found where FRKKW = WKKKW = FKKKF > PAMP-12 = WNKWAL > YKKKY > FRKKANKWALSR = FRKKWNKAALSR > KWKWK > FRKK = WNKWA > KYKYK > NKWALSR = YKKY = WNK. These sequences should be considered when designing peptide-based therapeutics. STATEMENT OF SIGNIFICANCE: Mast cells release immune regulating molecules upon activation that direct host's immune response. MRGPRX2 receptor provides an alternate pathway for mast cell activation that is independent of FcεRI receptor. It is thought that mast cell activation through MRGPRX2 plays a critical role in high failure rates of drugs in clinical trials. Identifying peptide sequences that activate mast cells through MRGPRX2 can serve two important purposes, namely, sequences to avoid when designing peptide therapeutics, and artificial peptides with different activation potentials for mast cells. Herein, we have identified a general amino acid sequence that induces mast cell activation through MRGPRX2. Furthermore, by modulating the identified sequence, artificial peptides have been designed which activate mast cells by varying degrees for therapeutic applications.

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.

Effect of carbamylation on protein structure and adsorption to self-assembled monolayer surfaces.

Protein adsorption research has primarily focused upon the effects of surface chemistry, with almost no emphasis on how changes to proteins that occur in various disease states may influence their adsorption. One such situation occurs with chronic kidney disease where, despite hemodialysis treatment, the retention of urea within the blood compartment leads to protein carbamylation. Protein carbamylation has been shown to alter the function and structure of proteins. This work is focused on understanding how different degrees of carbamylation affect the physicochemical properties (structure, charge, water interactions) of single proteins (α-lactalbumin, albumin, and fibrinogen) and their adsorption to self-assembled monolayers. It was found that, unlike its secondary structure, the protein's tertiary structure was significantly altered upon carbamylation. Also, compared to native proteins, an increase in carbamylation lead to an increase in the negative surface charge of the protein and a weaker hydration state of the protein. In order to study the effects of different types of neutral surfaces, of different surface-water properties, on protein adsorption both bare and alkanethiol modified (-CH3 or -OH end-groups) Au surfaces with were used as model surfaces. A significant decrease in adsorbed amounts of carbamylated fibrinogen and carbamylated α-lactalbumin, but not for carbamylated albumin, relative to native proteins was observed for both surfaces; suggesting that the increase in negative surface charge is more influential on adsorption than the change in hydration that occurs throughout the protein upon carbamylation. This data suggests that protein alterations that occur due to disease states have a significant effect on the overall protein structure and these changes affect their adsorption to surfaces.

Phage-Display-Derived Peptide Specific to Carbamylated Protein.

Protein carbamylation has been linked with diseases commonly associated with patients with reduced kidney function. Carbamylated human serum albumin (cHSA), which has been proven to be nephrotoxic and associated with heart failure for chronic kidney disease (CKD) patients, was chosen for our study. Through phage display against cHSA, one specific peptide sequence (cH2-p1) was identified with higher selectivity toward cHSA over native HSA. The cH2-p1 peptide was synthesized, and its target binding was analyzed through isothermal titration calorimetry (ITC). The result showed that cH2-p1 was able to bind cHSA of different levels of carbamylation with a similar dissociation constant of ∼1.0 × 10-4 M. This peptide also showed a binding specificity to carbamylated fibrinogen (cFgn), while not binding to native Fgn at all. For better understanding of the binding mechanism of cH2-p1, competitive binding of cH2-p1 and anti-homocitrulline to cHSA was performed, and the result revealed that cH2-p1 may bind to homocitrulline residues in a similar manner to the antibody. A molecular docking study was further performed to investigate the favored binding conformation of homocitrulline residue to cH2-p1. This work demonstrates the potential of peptides as a specific binding element to carbamylated proteins.

Drug delivery platforms for neonatal brain injury.

Hypoxic-ischemic encephalopathy (HIE), initiated by the interruption of oxygenated blood supply to the brain, is a leading cause of death and lifelong disability in newborns. The pathogenesis of HIE involves a complex interplay of excitotoxicity, inflammation, and oxidative stress that results in acute to long term brain damage and functional impairments. Therapeutic hypothermia is the only approved treatment for HIE but has limited effectiveness for moderate to severe brain damage; thus, pharmacological intervention is explored as an adjunct therapy to hypothermia to further promote recovery. However, the limited bioavailability and the side-effects of systemic administration are factors that hinder the use of the candidate pharmacological agents. To overcome these barriers, therapeutic molecules may be packaged into nanoscale constructs to enable their delivery. Yet, the application of nanotechnology in infants is not well examined, and the neonatal brain presents unique challenges. Novel drug delivery platforms have the potential to magnify therapeutic effects in the damaged brain, mitigate side-effects associated with high systemic doses, and evade mechanisms that remove the drugs from circulation. Encouraging pre-clinical data demonstrates an attenuation of brain damage and increased structural and functional recovery. This review surveys the current progress in drug delivery for treating neonatal brain injury.