Carbodiimide Crosslinker Chemistry Research Articles

Enabling multi-modal imaging and photoimmunotherapy of oral cancer spheroids through an EGFR-targeted antibody conjugate

SignificanceDelineating tumor margins during surgery and eliminating residual disease is a major challenge in the treatment of oral cancers. The dual function antibody conjugate (DFAC) developed in this study can enable 3D-imaging and destroy residual microscopic disease by photoimmunotherapy in a single intra-operative setting. ApproachDFAC was synthesized by routine carbodiimide crosslinker chemistry. Multi-modal imaging (Photoacoustic and fluorescence imaging) and photoimmunotherapy was performed on oral cancer spheroids. ResultsPhotoacoustic imaging could detect tumor spheroids, even of the smallest size (0.01 mm3), while fluorescence imaging could detect only larger spheroids with volumes greater than 0.049 mm3 and treated with a higher DFAC concentration. Photoimmunotherapy showed a spheroid size dependent response, with smaller spheroids of volumes 0.018 mm3 and lower showing no recurrence. ConclusionsThe complementary imaging and treatment capabilities of DFAC could help in minimizing healthy tissue resection and enhance recurrence free survival in oral cancer patients.

An odorant-binding protein based electrical sensor to detect volatile organic compounds

Artificial olfaction systems, such as electronic nose devices (ENs), can be used in various fields, from healthcare to food industry and the environmental sector. ENs consist of an array of sensors composed of different sensing materials. Incorporating Odorant Binding Proteins (OBPs) into gas-sensing materials can increase volatile organic compounds (VOCs) recognition and selectivity. OBPs are small soluble proteins found in vertebrates and insects, responsible for VOCs solubilization and transportation. In this work, OBP3 from Rattus norvegicus was selected to develop an OBP-based electrical VOC sensor, by optimizing protein production and immobilization on sensors surface. OBP3 was successfully produced in E. coli host cells (94 mg/L) and purified with high purity (88% purification yield, 96% purity). The protein folding and thermal stability were assessed by circular dichroism (Tm=71±1ºC) whereas ligand binding activity was verified in solution by fluorescence displacement against diisopropylphenol (Kd=0.24 µM). For the immobilization of OBP3 on gold interdigitated electrodes modified with reduced graphene oxide, we explored two strategies (covalent and non-covalent), establishing a reproducible and cost-effective methodology to develop OBP3-based electrical sensors. The non-covalent immobilization of a linker to the graphene-modified surface showed improved outcomes compared to the carbodiimide crosslinking chemistry. OBP3-sensors presented selectivity towards distinct model compounds in the gas phase (diisopropylphenol, dimethylpyrazine, menthone and decanol), in correlation with the dissociation constants measured by fluorescence displacement assays in solution. As a result, this study expands the practical applications of OBPs for gas-phase sensors, showcasing their potential for enhancing VOC detection.

Unfolding Protein-Based Hapten Coupling via Thiol-Maleimide Click Chemistry: Enhanced Immunogenicity in Anti-Nicotine Vaccines Based on a Novel Conjugation Method and MPL/QS-21 Adjuvants.

Vaccines typically work by eliciting an immune response against larger antigens like polysaccharides or proteins. Small molecules like nicotine, on their own, usually cannot elicit a strong immune response. To overcome this, anti-nicotine vaccines often conjugate nicotine molecules to a carrier protein by carbodiimide crosslinking chemistry to make them polymeric and more immunogenic. The reaction is sensitive to conditions such as pH, temperature, and the concentration of reactants. Scaling up the reaction from laboratory to industrial scales while maintaining consistency and yield can be challenging. Despite various approaches, no licensed anti-nicotine vaccine has been approved so far due to the susboptimal antibody titers. Here, we report a novel approach to conjugate maleimide-modified nicotine hapten with a disulfide bond-reduced carrier protein in an organic solvent. It has two advantages compared with other approaches: (1) The protein was unfolded to make the peptide conformation more flexible and expose more conjugation sites; (2) thiol-maleimide "click" chemistry was utilized to conjugate the disulfide bond-reduced protein and maleimide-modified nicotine due to its availability, fast kinetics, and bio-orthogonality. Various nicotine conjugate vaccines were prepared via this strategy, and their immunology effects were investigated by using MPL and QS-21 as adjuvants. The in vivo study in mice showed that the nicotine-BSA conjugate vaccines induced high anti-nicotine IgG antibody titers, compared with vaccines prepared by using traditional condensation methods, indicating the success of the current strategy for further anti-nicotine or other small-molecule vaccine studies. The enhancement was more significant by using MPL and QS-21 than that of traditional aluminum adjuvants.

Expanding the toolset of fluorescent covalent staining of biological samples by labeling carboxylate and phosphate groups.

Recently, fluorescent covalent staining methods have been developed for visualization of anatomical structures in cells and tissues. Coupled with expansion microscopy, these stains revealed various ultrastructural details. However, the covalently stainable chemical groups have been limited to amines, carbohydrates, and thiols. Here, we developed procedures for covalently labeling tissues for carboxylate and phosphate groups, utilizing carbodiimide crosslinker chemistry. In porcine kidney tissues, the carboxylate and phosphate stain provides 1.8-4.8-fold higher signal intensity than those from the three existing stains. In cancer cells, such stain allows 2-8-fold more accurate identification of nucleoli than the amine stain. In expansion microscopy samples, such stain reveals a variety of sub-cellular structures in tissues when combined with the amine stain. Such stain also allows imaging of lipid-based structures in cultured cells. With these advantages, this new covalent staining method further expands the toolset for fluorescent visualization of histology.

Gold Nanoparticles Are an Immobilization Platform for Active and Stable Acetylcholinesterase: Demonstration of a General Surface Protein Functionalization Strategy.

Immobilizing enzymes onto abiological surfaces is a key step for developing protein-based technologies that can be useful for applications such as biosensors and biofuel cells. A central impediment for the advancement of this effort is a lack of generalizable strategies for functionalizing surfaces with proteins in ways that prevent unfolding, aggregation, and uncontrolled binding, requiring surface chemistries to be developed for each surface-enzyme pair of interest. In this work, we demonstrate a significant advancement toward addressing this problem using a gold nanoparticle (AuNP) as an initial scaffold for the chemical bonding of the enzyme acetylcholinesterase (AChE), forming the conjugate AuNP-AChE. This can then be placed onto chemically and structurally distinct surfaces (e.g., metals, semiconductors, plastics, etc.), thereby bypassing the need to develop surface functionalization strategies for every substrate or condition of interest. Carbodiimide crosslinker chemistry was used to bind surface lysine residues in AChE to AuNPs functionalized with ligands containing carboxylic acid tails. Using amino acid analysis, we found that on average, 3.3 ± 0.1 AChE proteins were bound per 5.22 ± 1.25 nm AuNP. We used circular dichroism spectroscopy to measure the structure of the bound protein and determined that it remained essentially unchanged after binding. Finally, we performed Michaelis-Menten kinetics to determine that the enzyme retained 18.2 ± 2.0% of its activity and maintained that activity over a period of at least three weeks after conjugation to AuNPs. We hypothesize that structural changes to the peripheral active site of AChE are responsible for the differences in activity of bound AChE and unbound AChE. This work is a proof-of-concept demonstration of a generalizable method for placing proteins onto chemically and structurally diverse substrates and materials without the need for surface functionalization strategies.

Experimental Evaluation of Quantum Dots and Antibodies Conjugation by Surface Plasmon Resonance Spectroscopy.

The application of antibody-functionalized quantum dots (QDs) in different areas has been widely described in the literature. However, a standard routine method for obtaining information on the conjugation efficiency of QDs with antibodies in terms of the interaction of the functionalized QDs with a specific antigen is still lacking. Herein, surface plasmon resonance (SPR) spectroscopy is proposed for this purpose. Gold-coated SPR sensor disks were modified with a self-assembled monolayer of 11-mercaptoundecanoic acid, and carbodiimide cross-linker chemistry was used to covalently immobilize the CD44 biomarker on the premodified surface (Au/CD44). Meanwhile, QDs functionalized with amine-derivatized polyethylene glycol (PEG) (QDs-NH2) were chosen for conjugation with antibodies because of their low non-specific adsorption on the Au/CD44 surface. Prior to conjugation, the surface binding capacity (Bmax) and equilibrium dissociation constant (KD) of the specific antibodies against CD44 (anti-CD44) were found to be 263.32 ± 2.44 m° and 1.00 × 10−7 ± 2.29 × 10−9 M, respectively. QDs-NH2 and anti-CD44 were conjugated at their initial molar ratios of 1:3, 1:5, 1:10 and 1:12. SPR measurements showed that the conjugates (QDs-anti-CD44) prepared using 1:10 and 1:12 molar ratios interacted comparably with immobilized CD44 biomarkers. The equilibrium angles in the case of 10- and 12-fold concentrations of anti-CD44 were calculated to be 60.43 ± 4.51 and 61.36 ± 4.40 m°, respectively. This could be explained by the QDs-NH2 and anti-CD44 having a similar surface loading (about four molecules per QDs-NH2) and similar hydrodynamic diameters, which were 46.63 ± 3.86 and 42.42 ± 0.80 nm for the 1:10 and 1:12 ratios, respectively. An initial QDs-NH2: anti-CD44 molar ratio of 1:10 was chosen as being optimal. SPR spectroscopy proved to be the right choice for QDs-anti-CD44 conjugation optimization, and can be used for the evaluation of conjugation efficiency for other nanostructures with various bio-recognition molecules.

Surface-engineered sulfonation of ion-selective nanofiltration membrane with robust scaling resistance for seawater desalination

Inorganic fouling is one of the most critical issues in the seawater desalination process; it significantly deteriorates the desalination performance of the current seawater reverse osmosis (SWRO) membranes. Conventional nanofiltration (NF) membranes, which act as the pretreatment medium, are unable to effectively fractionate the scaling ions, Ca 2+ and SO 4 2− , resulting in fouling transfer from SWRO to the NF membranes. To address this issue, a new class of nanofiltration membranes was proposed and designed by surface engineering a custom-made sulfonated polyamide thin-film composite. Specifically, a new functional monomer, 4-aminobenzenesulfonic acid (ABSA), was chemically grafted onto a membrane surface via carbodiimide crosslinking chemistry. Due to the intentional optimization of mean pore sizes and surface charges, the as-prepared NF membranes (named BM-ABSA) exhibited extremely low Ca 2+ rejections (6.1 ± 0.4%) and moderate SO 4 2− rejections (57.5 ± 0.1%), corresponding to a higher ion selectivity (9.5) toward seawater compared to that of the benchmark commercial NF270 membrane (1.5). More importantly, the replacement of carboxylate groups in the substrate membrane matrix with the sulfonate groups considerably lowered the probability of the Ca–COOH chelation effect, which, in turn, facilitated steady NF-pretreatment processes without significant fouling. The subsequent SWRO process, using the BM-ABSA membrane-pretreated permeate as the feed solution, also proceeded well over a short-term operation with limited gypsum fouling. Overall, this study highlighted the effective inhibition of scaling by sulfonated NF membranes in the pretreatment process, validating their potential application for robust and efficient seawater desalination. • ABSA was chemically grafted onto the surface of NF membrane through EDC/NHS coupling reaction. • BM-ABSA-5 membrane showed a high ion-ion selectivity toward Ca 2+ and SO 4 2− . • The anti-scaling mechanism of –SO 3 H groups was investigated by molecular dynamics simulation. • Gypsum scaling on NF membrane and RO membrane was both inhibited in the hybrid NF-RO process for seawater desalination.

Development of Cellulose Nanofibril/Casein-Based 3D Composite Hemostasis Scaffold for Potential Wound-Healing Application.

Excessive bleeding in traumatic hemorrhage is the primary concern for natural wound healing and the main reason for trauma deaths. The three-dimensional (3D) bioprinting of bioinks offers the desired structural complexity vital for hemostasis activity and targeted cell proliferation in rapid and controlled wound healing. However, it is challenging to develop suitable bioinks to fabricate specific 3D scaffolds desirable in wound healing. In this work, a 3D composite scaffold is designed using bioprinting technology and synergistic hemostasis mechanisms of cellulose nanofibrils (TCNFs), chitosan, and casein to control blood loss in traumatic hemorrhage. Bioinks that consist of casein bioconjugated TCNF (with a casein content of 104.5 ± 34.1 mg/g) using the carbodiimide cross-linker chemistry were subjected to bioprinting for customizable 3D scaffold fabrication. Further, the 3D composite scaffolds were in situ cross-linked using a green ionic complexation approach. The covalent conjugation among TCNF, casein, and chitosan was confirmed by Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR), X-ray photoelectron spectroscopy (XPS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and X-ray diffraction (XRD) studies. The in vitro hemostasis activity of the 3D composite scaffold was analyzed by a human thrombin-antithrombin (TAT) assay and adsorption of red blood cells (RBCs) and platelets. The 3D composite scaffold had a better swelling behavior and a faster whole blood clotting rate at each time point than the 3D TCNF scaffold and commercial cellulose-based dressings. The TAT assay demonstrated that the 3D composite scaffold could form a higher content of thrombin (663.29 pg/mL) and stable blood clot compared to a cellulosic pad (580.35 pg/mL), 3D TCNF (457.78 pg/mL), and cellulosic gauze (328.92 pg/mL), which are essential for faster blood coagulation. In addition, the 3D composite scaffold had a lower blood clotting index (23.34%) than the 3D TCNF scaffold (41.93%), suggesting higher efficiencies for RBC entrapping to induce blood clotting. The in vivo cytocompatibility was evaluated by a 3D cell culture study, and results showed that the 3D composite scaffold could promote growth and proliferation of NIH 3T3 fibroblast cells, which is vital for wound healing. Cellulase-based in vitro deconstruction of the 3D composite scaffold showed significant weight loss (80 ± 5%) compared to the lysozyme hydrolysis (22 ± 5%) after 28 days of incubation, suggesting the biodegradation potential of the composite scaffold. In conclusion, this study proposes efficient prospects to develop a 3D composite scaffold from bioprinting of TCNF-based bioinks that can accelerate blood clotting and wound healing, suggesting its potential application in reducing blood loss during traumatic hemorrhage.

Graphene Oxide Nanoparticles Modified Paper Electrode as a Biosensing Platform for Detection of the htrA Gene of O. tsutsugamushi.

The unique structural and electrochemical properties of graphene oxide (GO) make it an ideal material for the fabrication of biosensing devices. Therefore, in the present study, graphene oxide nanoparticles modified paper electrodes were used as a low-cost matrix for the development of an amperometric DNA sensor. The graphene oxide was synthesized using the modified hummers method and drop cast on a screen-printed paper electrode (SPPE) to enhance its electrochemical properties. Further, the GO/SPPE electrode was modified with a 5′NH2 labeled ssDNA probe specific to the htrA gene of Orientia tsutsugamushi using carbodiimide cross-linking chemistry. The synthesized GO was characterized using UV-Vis, FTIR, and XRD. The layer-by-layer modification of the paper electrode was monitored via FE-SEM, cyclic voltammetry, and electrochemical impedance spectroscopy (EIS). The sensor response after hybridization with single-stranded genomic DNA (ssGDNA) of O. tsutsugamushi was recorded using differential pulse voltammetry (DPV). Methylene blue (1 mM in PBS buffer, pH 7.2) was used as a hybridization indicator and [Fe(CN)6]−3/−4 (2.5 mM in PBS buffer, pH 7.2) as a redox probe during electrochemical measurements. The developed DNA sensor shows excellent sensitivity (1228.4 µA/cm2/ng) and LOD (20 pg/µL) for detection of O. tsutsugamushi GDNA using differential pulse voltammetry (DPV).

Magnetic graphene oxide as a platform for the immobilization of cellulases and xylanases: Ultrastructural characterization and assessment of lignocellulosic biomass hydrolysis

For producing second-generation ethanol (cellulosic ethanol) and other value-added bioproducts, magnetic graphene oxide (GO-MNP) was synthesized in this work and used as the immobilization support for an industrial cellulase-and xylanase-containing preparation. GO-MNP characterization by TEM, SEM and ATR-FTIR spectroscopy showed that the magnetic nanoparticles are homogeneously distributed onto the GO sheets surface. The enzymatic preparation was immobilized by means of carbodiimide cross-linking chemistry using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (NHS). The supported final biocatalyst (GO-MNP-Enz) showed high activity for the hydrolysis of pretreated sugarcane bagasse (PSB) and presented relative endoglucanase, xylanase, β-glucosidase, and β-xylosidase activities of 70%, 66%, 88%, and 70%, respectively, after 10 cycles of hydrolysis of their respective substrates. The biocatalyst also maintained approximately 50% and 80% of its efficiency for cellulose and xylan hydrolysis, respectively, being the TOF (g.g−1.h−1) the highest observed when compared with previous results reported in literature. These findings suggest that GO-MNP-Enz may be a prospective candidate for industrial applications such as second-generation ethanol production.

Synthesis and characterization of pectin-chitosan conjugate for biomedical application

BackgroundPolysaccharides are extensively used in drug delivery systems due to their ability to undergo a broad range of chemical and enzymatic reactions, forming new molecules. Among these, chitosan (CS) and pectin (PEC) are widely used for designing new conjugates and biopolymers. MethodsIn this study, we synthesized pectin-chitosan conjugate (PEC-CS) by using carbodiimide crosslinking chemistry. Pectin-N-hydroxysuccinimide ester, formed in the presence of dicyclohexylcarbodiimide under anhydrous conditions, was conjugated with chitosan by linking the free carboxyl group of PEC with the primary amino group of chitosan. ResultsFourier-transform infrared spectroscopy confirmed the formation of the PEC-CS conjugate. X-ray diffraction, differential scanning calorimetry, thermogravimetric analysis, and scanning electron microscopy analyses showed that the PEC-CS conjugate is amorphous in nature, has high thermostability than those of native polymers, and has no cytotoxicity. ConclusionOur results indicated that PEC-CS conjugate can be further developed for use in drug delivery systems.

Immobilization of nanocarriers within a porous chitosan scaffold for the sustained delivery of growth factors in bone tissue engineering applications

To guide the natural bone regeneration process, bone tissue engineering strategies rely on the development of a scaffold architecture that mimics the extracellular matrix and incorporates important extracellular signaling molecules, which promote fracture healing and bone formation pathways. Incorporation of growth factors into particles embedded within the scaffold can offer both protection of protein bioactivity and a sustained release profile. In this work, a novel method to immobilize carrier nanoparticles within scaffold pores is proposed. A biodegradable, osteoconductive, porous chitosan scaffold was fabricated via the "freeze-drying method," leading to scaffolds with a storage modulus of 8.5 kPa and 300 μm pores, in line with existing bone scaffold properties. Next, poly(methyl methacrylate-co-methacrylic acid) nanoparticles were synthesized and immobilized to the scaffold via carbodiimide-crosslinker chemistry. A fluorescent imaging study confirmed that the conventional methods of protein and nanocarrier incorporation into scaffolds can lead to over 60% diffusion out of the scaffold within the first 5 min of implantation, and total disappearance within 4 weeks. The novel method of nanocarrier immobilization to the scaffold backbone via carbodiimide-crosslinker chemistry allows full retention of particles for up to 4 weeks within the scaffold bulk, with no negative effects on the viability and proliferation of human umbilical vein endothelial cells.

Modification of polyacrylate sorbent coatings with carbodiimide crosslinker chemistry for sequence-selective DNA extraction using solid-phase microextraction.

Selective DNA extraction is immensely useful for the isolation and detection of low-abundance sequences. Oligonucleotide-modified substrates are often used to capture sequences of interest for downstream analysis. In this study, we explore the chemical modification of commercial-available polyacrylate solid-phase microextraction fibers for selective DNA analysis using carbodiimide crosslinker chemistry. Reproducible modification conditions are found and the fibers were subsequently applied for selective DNA analysis. Several experimental parameters such as stir-rate, desorption time, and buffer-type are optimized. The developed method was able to selectively extract the target DNA sequence (260 bp) in the presence of 100-fold excess interfering salmon testes DNA.

Thiol-Functionalized Membranes for Mercury Capture from Water.

Pore functionalized membranes with appropriate ion exchange/chelate groups allow toxic metal sorption under convective flow conditions. This study explores the sorption capacity of ionic mercury in a polyvinylidene fluoride-poly(acrylic acid) (PVDFs-PAA) functionalized membrane immobilized with cysteamine (MEA). Two methods of MEA immobilization to the PVDF-PAA membrane have been assessed: (i) ion exchange (IE) and (ii) carbodiimide cross-linker chemistry using 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), known as EDC/NHS coupling. The ion exchange method demonstrates that cysteamine (MEA) can be immobilized effectively on PVDF-PAA membranes without covalent attachment. The effectiveness of the MEA immobilized membranes to remove ionic mercury from the water was evaluated by passing a dissolved mercury(II) nitrate solution through the membranes. The sorption capacity of mercury for MEA immobilized membrane prepared by the IE method is 1015 mg/g PAA. On the other hand, the sorption capacity of mercury for MEA immobilized membrane prepared by EDC/NHS chemistry is 2446 mg/g PAA, indicating that membrane functionalization by EDC/NHS coupling enhanced mercury sorption 2.4 times compared to the IE method. The efficiencies of Hg removal are 94.1 ± 1.1 and 99.1 ± 0.1% for the MEA immobilized membranes prepared by IE and EDC/NHS coupling methods, respectively. These results show potential applications of MEA immobilized PVDF-PAA membranes for industrial wastewater treatment specifically from energy and mining industries to remove mercury and other toxic metals.

Off-Resonance SERS Nanoprobe-Targeted Screen of Biomarkers for Antigens Recognition of Bladder Normal and Aggressive Cancer Cells.

The discovery of different binding receptors to allow rapid and high-sensitivity detection via a noninvasive urine test has become the goal for urothelial carcinoma (UC) diagnosis and surveillance. In this study, we developed a new screening membrane receptor platform for bladder cancer cells by integrating surface-enhanced Raman spectroscopy (SERS) with 4-aminothiophenol (4-ATP)-modified AuAg nanohollows upon NIR laser excitation. AuAg nanohollows have an absorption band at ∼630 nm, and slightly off-resonance 785 nm laser excitation is used for minimal photothermal effect. Using the same carbodiimide cross-linker chemistry to conjugate anti-EGFR, transferrin (TF), 4-carboxyphenylboronic acid (CPBA), folic acid (FA), and hyaluronic acid (HA) molecules, by screening the 4-ATP SERS signals intensity, we demonstrated that the targeting efficiency with the cost-effective CPBA molecule is comparable with the conjugation of anti-EGFR antibody to aggressive T24 cancer cells (high-grade), while weak intensity 4-ATP SERS responses to targets were obtained by grade-I RT4 bladder cancer cells, NIH/3T3 fibroblast cells, and SV-HUC1 bladder normal cells. This SERS nanoprobe platform makes primary bladder carcinoma screening from in vitro to ex vivo more straightforward. Our demonstration offers exciting potential for SERS screening of specific receptors on cancer cells of different grades and facilitates new opportunities ranging from surface engineering of SERS material tags to SERS imaging-guided and targeted phototherapy of cancer cells by controlling the laser powers.

Quantitative analysis of BMP-2 derived peptide covalently grafted onto oxidized detonation nanodiamonds

In order to deploy detonation nanodiamonds (NDs) in nanomedicine and drug delivery applications, fundamental understanding of their surface chemistry and drug loading capacity is highly desirable. Herein, bone morphogenetic protein 2 (BMP-2) derived peptide with the sequence KIPKASSVPTELSAISTLYLGGC (molecular weight = 2336 g/mol) have been successfully grafted onto NDs using carbodiimide crosslinker chemistry. Initially, functional surface groups of wet- and dry-oxidized NDs were compared using infrared and mass spectroscopy. Dry-oxidized NDs exhibited the highest amount of carboxylic acid derivatives with a surface loading of 0.113 mmol/g after air annealing at 415 °C for 5 h as determined by mass spectroscopy. Compared to wet-oxidation, the dry-oxidation process showed a 1.4-fold increased amount of carbonyl compounds deduced from infrared and mass spectroscopy. Crosslinking of the carboxylic acid derivatives to the primary amines of BMP-2 derived peptide is feasible in the range of 30–96% total surface coverage of NDs. 1:1 and 5:1 ND-peptide ratios were utilized to study the surface loading of NDs using fluorescence spectroscopy. The chemisorption of BMP-2 derived peptide onto NDs reveals superior surface coverage and results in reproducible amounts of tethered bioactive molecules.

Modification of bacterial cellulose/keratin nanofibrous mats by a tragacanth gum-conjugated hydrogel for wound healing

To enhance physicomechanical properties and bioactivity of fibrous membranes for wound dressing and tissue engineering applications, novel composite scaffolds consisting of fibrous mats and thermosensitive hydrogel particles were prepared by concurrent electrospinning and electrospraying technique. The composite scaffolds were composed of keratin/bacterial cellulose fibers (150 ± 43 nm) which are hybridized with hydrogel particles (500 nm to 2 μm) based on nonionic triblock copolymers conjugated with Tragacanth gum (TG). FTIR and H-NMR studies indicated ester reactions between carboxylated copolymers and TG through carbodiimide crosslinker chemistry. The hydrogel particles were uniformly embedded into fibrous network at fiber junctions without changing its porous structure and the fiber diameter. Modification of the fibers with the hydrogel nanoparticles significantly improved the hydrophilicity (~23%), module of elasticity (~31%), tensile strength (~35%), and ductility (~23%) of the electrospun scaffold. In vitro culturing of the mats with L929 fibroblast cells determined the biocompatibility of the fibrous composite along with improved cell adhesion and proliferation.

Avidin Adsorption to Silk Fibroin Films as a Facile Method for Functionalization

Silk fibroin biomaterials are highly versatile in terms of materials formation and functionalization, with applications in tissue engineering and drug delivery, but necessitate modifications for optimized biological activity. Herein, a facile, avidin-based technique is developed to noncovalently functionalize silk materials with bioactive molecules. The ability to adsorb avidin to silk surfaces and subsequently couple biotinylated macromolecules via avidin-biotin interaction is described. This method better preserved functionality than standard covalent coupling techniques using carbodiimide cross-linking chemistry. The controlled release of avidin from the silk surface was demonstrated by altering the adsorption parameters. Application of this technique to culturing human foreskin fibroblasts (hFFs) and human mesenchymal stem cells (hMSCs) on arginine-glycine-aspartic-acid-modified (RGD-modified) silk showed increased cell growth over a seven-day period. This technique provides a facile method for the versatile functionalization of silk materials for biomedical applications including tissue engineering, drug delivery, and biological sensing.

Antimicrobial coatings on polyethylene terephthalate based on curcumin/cyclodextrin complex embedded in a multilayer polyelectrolyte architecture.

Bacterial contamination is a growing concern worldwide. The aim of this work was to develop an antimicrobial coating based on curcumin-cyclodextrin inclusion complex and using polyethylene terephthalate (PET) film as a support matrix. After a pre-treatment aimed to provide sufficient electric charge to the PET surface, it was electrostatically coated with repeated multilayers comprising alternately deposited positively-charged poly-l-lysine (PLL) and negatively-charged poly-l-glutamic acid (PLGA) and carboxymethyl-β-cyclodextrin (CMBCD). The coatings had an architecture (PLL-PLGA)6-(PLL-PLGA-PLL-CMBCD)n, with the number of repeated multilayers n varying from 5 to 20. The CMBCD molecules were either covalently cross-linked using carbodiimide crosslinker chemistry or left unbound. The surface morphology, structure and elemental composition of the coatings were analysed by scanning electron microscopy and energy dispersive x-ray spectroscopy. To impart antimicrobial properties to the coatings they were loaded with a natural phenolic compound curcumin forming inclusion complexes with β-cyclodextrin. The non-cross-linked coatings showed bactericidal activity towards Escherichia coli in the dark, and this activity was further enhanced upon illumination with white light. Curcumin was released from the non-cross-linked coatings into an aqueous medium in the form of cyclodextrin inclusion complex. After the cross-linking, the coating lost its dark antimicrobial activity but retained the photodynamic properties. Stabilized cross-linked curcumin-loaded coatings can serve a basis for developing photoactivated antimicrobial surfaces controlling bacterial contamination and spread.

Peptide Functionalized Metallic Nanoconstructs: Synthesis, Structural Characterization, and Antimicrobial Evaluation.

Biomolecule based conjugation of metallic nanoparticles has become an important area of research lately especially considering the role of such nanoconjugates in diverse applications. Herein, synthesis of cationic dipeptide conjugated gold/silver nanoparticles through the covalent approach using carbodiimide cross-linker chemistry has been reported. Owing to the exceptional optoelectronic properties of metallic nanoparticles, peptide-gold/silver nanoparticle conjugates were synthesized employing 2-mercaptopropanoic acid as a cross-linker. The conjugates were further compared with their noncovalently synthesized counterparts using ultraviolet-visible spectroscopy. It was generally observed that the conjugates synthesized using the covalent approach were more stable than the conventional noncovalent approach. The synthesized conjugates were further evaluated for their antimicrobial efficacy. The experimental findings demonstrated that peptide capped silver nanoparticles possessed relatively better antimicrobial activity than peptide capped gold nanoparticles, native peptides as well as unconjugated gold/silver nanoparticles which was also evident in time kill assay studies. The morphological effects of active compounds on Escherichia coli and Candida albicans exhibited complete disruption of the cell wall. Thus, this study is an important step toward the opening up of avenues for the applicability of the covalent approach for functionalization of metallic nanoparticles with not only short peptide based systems but also for other biomolecules in areas such as anti-infectives, drug delivery, and bioimaging.