Influence of different conjugation methods for activating antibodies on polymeric nanoparticles: Effects for polyclonal expansion of human CD8+ T cells.

Particle-based systems have become a state-of-the-art method for in vitro expanding cytotoxic T cells by tailoring their surface with activating molecules. However, commonly used methods utilize facile carbodiimide chemistry leading to uncontrolled orientation of the immobilized antibodies on the particle surface that can lead to poor binding to target cells. To address this, selective coupling strategies utilizing regioselective chemical groups such as disulfide bridges offer a simple approach. In this work we present a set of methods to investigate the effect of polymeric nanoparticles, conjugated with either regioselective- or randomly-immobilized antiCD3 and antiCD28 antibodies, on the activation potential, expansion and expression of activation markers in T cells. We show that nanoparticles with well-oriented monovalent antibodies conjugated via maleimide require fewer ligands on the surface to efficiently expand T cells compared to bivalent antibodies randomly-immobilized via carbodiimide conjugation. Analysis of the T cell expression markers reveal that the T cell phenotype can be fine-tuned by adjusting the surface density of well-oriented antibodies, while randomly immobilized antibodies showed no differences despite their ligand density. Both conjugation techniques induced cytotoxic T cells, evidenced by analyzing their Granzyme B secretion. Furthermore, antibody orientation affects the immunological synapse and T cell activation by changing the calcium influx profile upon activation. Nanoparticles with well-oriented antibodies showed lower calcium influx compared to their bivalent randomly-immobilized counterparts. These results highlight the importance of controlling the antibody density and orientation on the nanoparticle surface via controlled coupling chemistries, helping to develop improved particle-based expansion protocols to enhance T cell therapies.

Antibacterial Activity of CdTe/ZnS Quantum Dot-ß Lactum Antibiotic Conjugates.

ß-Lactum antibiotics are broad class of antibiotics which kills bacteria by inhibiting the formation of peptidoglycan that constitutes the bacterial cell wall. The resistance that develops in bacteria for antibiotics led the scientific world to think about the future aspects for modifying the way through which antibiotics are acted on the bacteria and become lethal for them. In this consequence, the potential of latest marketed antibiotics e.g. Amoxiciline (I), ceftazidim (II) have been evaluated after being conjugated with quantum dots. The surface of quantum dots has been conjugated with antibiotics by carbodiimide coupling with the help of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as conjugating agent between antibiotic and functionalized quantum dots. The antibacterial properties of QD-conjugated antibiotics have been determined by disc diffusion assay. The potency of QD-conjugated antibiotics has been estimated by determining their MIC50 for the selected strain of Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. Minimum inhibitory concentration study, minimum bactericidal concentration and growth pattern analysis revealed that QD-antibiotic conjugates showed slightly more prospective than pure native antibiotics against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria.

N-lauric-O-carboxymethyl chitosan: Synthesis, characterization and application as a pH-responsive carrier for curcumin particles.

A pH-responsive amphiphilic chitosan derivative, N-lauric-O-carboxymethyl chitosan (LA-CMCh), is synthesized. Its molecular structures are characterized by FTIR, 1H NMR, and XRD methods. The influencing factors are investigated, including the amount of lauric acid (LA), carboxymethyl chitosan (CMCh), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS), and their molar ratio, reaction time, and reaction temperature on the substitution. The degrees of substitution (DS) of the lauric groups on the -NH2 groups are calculated based on the integrated data of 1H NMR spectra. The optimum reaction condition is obtained as a reaction time of 6 h, a reaction temperature of 80 °C, and a molar ratio of lauric acid to O-carboxymethyl chitosan to N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide of 1:3:4.5:4.5, respectively. The crystallinity and initial decomposition temperature of LA-CMCh decrease, but the maximum decomposition temperature increases. The crystallinity is reduced due to the introduction of LA and the degree of hydrogen bonding among LA-CMCh molecules. LA-CMCh could self-aggregate into particles, which size and critical aggregation concentration depend on the degree of substitution and medium pH. LA-CMCh aggregates could load curcumin up to 21.70 %, and continuously release curcumin for >200 min. LA-CMCh shows nontoxicity to fibroblast HFF-1 cells and good antibacterial activity against S. aureus and E. coli, indicating that it could be used as an oil-soluble-drug carrier.

Gold nanoparticles conjugated with epidermal growth factor and gadolinium for precision delivery of contrast agents in magnetic resonance imaging.

The utilization of contrast agents in magnetic resonance imaging (MRI) has become increasingly important in clinical diagnosis. However, the low diagnostic specificity of this technique is a limiting factor for the early detection of tumors. To develop a new contrast agent with a specific target for early stage tumors, we present the synthesis and characterization of a nanocontrast composed of gold nanoparticles (AuNPs), gadopentetic acid (Gd-DTPA), and epidermal growth factor (EGF). Carbodiimide-based chemistry was utilized to modify Gd-DTPA for functionalization with AuNPs. This resulted in the formation of the Au@Gd-EGF nanocontrast. The relaxation rate (1/T1) of the nanocontrast was analyzed using MRI, and cytotoxicity was determined based on cell viability and mitochondrial activity in a human breast adenocarcinoma cell line. Fourier-transform infrared spectroscopy analysis confirmed the effectiveness of carbodiimide in the formation of the Gd-DTPA-cysteamine complex in the presence of bands at 930, 1042, 1232, 1588, and 1716 cm-1. The complexes exhibited good interactions with the AuNPs. However, the signal intensity of the Au@Gd-EGF nanocontrast was lower than that of the commercial contrast agent because the r1/r2 relaxivities of the Gd-DTPA-based contrast agents were lower than those of the gadoversetamide-based molecules. The Au@Gd-EGF nanocontrast agent exhibited good biocompatibility, low cytotoxicity, and high signal intensity in MRI with active targeted delivery, suggesting significant potential for future applications in the early diagnosis of tumors.

Impact of bridge heterology on functional parameters of ELISA for 17α-methyltestosterone.

In this study, we have developed bridge heterologous ELISA for the detection of 17α- Methyltestosterone by incorporating aromatic spacers between 17α-Methyltestosterone-3-Carboxymethyloxime and Horseradish peroxidase label through N-hydroxysuccinimide mediated carbodiimide reaction method. The immunogen 17α-Methyltestosterone-3-Carboxymethyloxime-Bovine serum albumin used to generate the antibody was also prepared by the N-hydroxysuccinimide mediated carbodiimide reaction without using any spacer. We have studied the impact of bridge/aromatic spacers on functional parameters i.e. sensitivity, affinity and ED50 of the bridge heterologous assay and compared it with homologous assay. The five combinations of bridge heterologous assay using 17α-Methyl testosterone-3-CMO-BSA antiserum and 17α-MT-3-CMO-4,4'-Diaminodiphenyl sulphide-HRP, 17α MT-3-CMO-4,4'-Oxydianiline-HRP, 17α-MT-3-CMO-Benzidine-HRP, 17α- MT-3-CMO-p-Phenylenediamine-HRP and 17α-MT-3-CMO-Dapson-HRP enzyme conjugates were evaluated. Out of these five combinations, the combination 17α-MT-3-CMO-BSA with 17α-MT-3-CMO-Benzidine-HRP showed the best results. Sensitivity, affinity and ED50 were improved and found to be 0.02 ng/mL, 0.086 × 10-8 L/mol and 2.95 ng/mL than homologous assay where Sensitivity, affinity and ED50 were 0.11 ng/mL, 0.02 × 10-8 L/mol and 5.78 ng/mL respectively. The cross-reactivity for this bridge heterologous assay combination was seen with only 4 steroids (6-hydrotestosterone- 6%, Testosterone-5.14%, Danazol-0.9% and Nandrolone-0.85%) instead of eight steroids (6-hydrotestosterone-43.75%, Testosterone-38.3%, Danazol-25.14%, Androstenediol-19.16%, Nandrolone-19%, Metandienone-5%, Androstenedione-3.52%, and 17α dimethyltestosterone-2%) as in homologous assay out of 59 structurally related steroids. Thus, the results of this study conclude that the incorporation of aromatic spacer (bridge) in enzyme conjugate has a crucial role in improving sensitivity, specificity, ED50 and affinity of the developed assay. The assay was then studied for parameters such as recovery (97.4%-108.6%), precision (Inter and Intra-assay coefficient of variation <10%), correlation coefficient (R2 = 0.96) by comparing with the commercial kit and validated by measuring levels of 17α- methyltestosterone in rat serum after administering them.

S-Protected Thiolated Chitosan versus Thiolated Chitosan as Cell Adhesive Biomaterials for Tissue Engineering.

Chitosan (Ch) and different Ch derivatives have been applied in tissue engineering (TE) because of their biocompatibility, favored mechanical properties, and cost-effectiveness. Most of them, however, lack cell adhesive properties that are crucial for TE. In this study, we aimed to design an S-protected thiolated Ch derivative exhibiting high cell adhesive properties serving as a scaffold for TE. 3-((2-Acetamido-3-methoxy-3-oxopropyl)dithio) propanoic acid was covalently attached to Ch via a carbodiimide-mediated reaction. Low-, medium-, and high-modified Chs (Ch-SS-1, Ch-SS-2, and Ch-SS-3) with 54, 107 and 140 µmol of ligand per gram of polymer, respectively, were tested. In parallel, three thiolated Chs, namely Ch-SH-1, Ch-SH-2, and Ch-SH-3, were prepared by conjugating N-acetyl cysteine to Ch at the same degree of modification to compare the effectiveness of disulfide versus thiol modification on cell adhesion. Ch-SS-1 showed better cell adhesion capability than Ch-SS-2 and Ch-SS-3. This can be explained by the more lipophilic surfaces of Ch-SS as a higher modification was made. Although Ch-SH-1, Ch-SH-2, and Ch-SH-3 were shown to be good substrates for cell adhesion, growth, and proliferation, Ch-SS polymers were superior to Ch-SH polymers in the formation of 3D cell cultures. Cryogels structured by Ch-SS-1 (SSg) resulted in homogeneous scaffolds with tunable pore size and mechanical properties by changing the mass ratio between Ch-SS-1 and heparin used as a cross-linker. SSg scaffolds possessing interconnected microporous structures showed good cell migration, adhesion, and proliferation. Therefore, Ch-SS can be used to construct tunable cryogel scaffolds that are suitable for 3D cell culture and TE.

RNA structural probing of guanine and uracil nucleotides in yeast.

RNA structure can be essential for its cellular function. Therefore, methods to investigate the structure of RNA in vivo are of great importance for understanding the role of cellular RNAs. RNA structure probing is an indirect method to asess the three-dimensional structure of RNA by analyzing the reactivity of different nucleotides to chemical modifications. Dimethyl sulfate (DMS) is a well-established compound that reports on base pairing context of adenine (A) and cytidine (C) in-vitro and in-vivo, but is not reactive to guanine (G) or uracil (U). Recently, new compounds were used to modify Gs and Us in plant, bacteria, and human cells. To complement the scope of RNA structural probing by chemical modifications in the model organism yeast, we analyze the effectiveness of guanine modification by the glyoxal family in Saccharomyces cerevisiae and Candida albicans. We show that within glyoxal family of compounds, phenylglyoxal (PGO) is the best guanine probe for structural probing in S. cerevisiae and C. albicans. Further, we show that PGO treatment does not affect the processing of different RNA species in the cell and is not toxic for the cells under the conditions we have established for RNA structural probing. We also explore the effectiveness of uracil modification by Cyclohexyl-3-(2-Morpholinoethyl) Carbodiimide metho-p-Toluenesulfonate (CMCT) in vivo and demonstrate that uracils can be modified by CMCT in S. cerevisiae in vivo. Our results provide the conditions for in vivo probing the reactivity of guanine and uracil nucleotides in RNA structures in yeast and offer a valuable tool for studying RNA structure and function in two widely used yeast model systems.

Tendon-derived matrix crosslinking techniques for electrospun multi-layered scaffolds.

Tendon tears are common and healing often occurs incompletely and by fibrosis. Tissue engineering seeks to improve repair, and one approach under investigation uses cell-seeded scaffolds containing biomimetic factors. Retention of biomimetic factors on the scaffolds is likely critical to maximize their benefit, while minimizing the risk of adverse effects, and without losing the beneficial effects of the biomimetic factors. The aim of the current study was to evaluate cross-linking methods to enhance the retention of tendon-derived matrix (TDM) on electrospun poly(ε-caprolactone) (PCL) scaffolds. We tested the effects of ultraviolet (UV) or carbodiimide (EDC:NHS:COOH) crosslinking methods to better retain TDM to the scaffolds and stimulate tendon-like matrix synthesis. Initially, we tested various crosslinking configurations of carbodiimide (2.5:1:1, 5:2:1, and 10:4:1 EDC:NHS:COOH ratios) and UV (30 s 1 J/cm2 , 60 s 1 J/cm2 , and 60 s 4 J/cm2 ) on PCL films compared to un-crosslinked TDM. We found that no crosslinking tested retained more TDM than coating alone (Kruskal-Wallis: p > .05), but that human adipose stem cells (hASCs) spread most on the 60 s 1 J/cm2 UV- and 2.5:1:1 EDC-crosslinked films (Kruskal-Wallis: p < .05). Next, we compared the effects of 60 s 1 J/cm2 UV- and 2.5:1:1 EDC-crosslinked to TDM-coated and untreated PCL scaffolds on hASC-induced tendon-like differentiation. UV-crosslinked scaffolds had greater modulus and stiffness than PCL or TDM scaffolds, and hASCs spread more on UV-crosslinked scaffolds (ANOVA: p < .05). Fourier transform infrared spectra revealed that UV- or EDC-crosslinking TDM did not affect the peaks at wavenumbers characteristic of tendon. Crosslinking TDM to electrospun scaffolds improves tendon-like matrix synthesis, providing a viable strategy for improving retention of TDM on electrospun PCL scaffolds.

Analysis of the adhesive interface of dentine treated with carbodiimide and chitosan before cementation of fiberglass posts with different resin cements.

The objective of this study is to evaluate the effect of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and chitosan (CHI) on the adhesive interface of resin cements to root dentine. Forty-five upper canines were sectioned, endodontically treated, prepared and divided into three groups according to dentine treatment (distilled water-DW, CHI 0.2% and EDC 0.5) and in three subgroups according to resin cement: RelyX ARC, Panavia F 2.0 or RelyX U200. Slices were obtained, with five slices of each third submitted to the analysis of the adaptation of the adhesive interface through scores and the perimeter with gaps in confocal laser scanning microscopy and one slice of each third later evaluated qualitatively in scanning electron microscopy. The results were analyzed using with Kruskal-Wallis and Spearman correlation tests. There was no difference in adaptation for the different resin cements (p = .438). EDC presented better adaptation when compared to the groups treated with DW and CHI (p < .001), while the CHI and DW presented similar adaptation values (p = .365). No difference was observed in the perimeter referring to the gap areas for the different resin cements (p = .510). EDC showed a lower percentage of perimeters with gaps when compared to CHI (p < .001), with the percentage of perimeter with gaps of teeth treated with CHI being lower than DW (p < .001). A positive correlation coefficient equal to 0.763 was obtained between the perimeter with gaps and the adaptation data of the adhesive interface (p < .001). EDC resulted in better adaptation of the adhesive interface and a lower percentage of perimeters with gaps compared to chitosan.

Cascade Synthesis of 5-Aminotetrazoles from Isonitriles and N,N-Dibromoarylsulfonamides via Carbodiimide Coupling with Azide.

A cascade protocol for the synthesis of aminotetrazoles have been developed by treating isonitriles, N,N-dibromoarylsulfonamides, and sodium azides in the presence of K2CO3. This metal-free process proceeds via an isolable carbodiimide intermediate at room temperature, which could further react with sodium azide and subsequently cyclizes intermolecularly to provide 5-aminotetrazoles within a short reaction time. The present protocol's remarkable achievements are the wide substrate scope, good to excellent yields, and good functional group tolerance.

The effect of carbodiimide on push-out bond strength of fiber posts and endogenous enzymatic activity.

BACKGROUND: To investigate the effect of 0.3 M 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) aqueous solution pretreatment on push-out bond strength (PBS) and matrix-metalloproteinases (MMPs) activity within radicular dentin when different post cementation strategies were employed. METHODS: One hundred and twenty monoradicular human teeth were endodontically treated and randomly divided into six groups, depending on the cementation strategy and root dentin pretreatment (n = 20): EAR: cementation with an etch-and-rinse adhesive (LuxaBond Total Etch, DMG) and resin cement (LuxaCore Z Dual, DMG); EAR/EDC: 1 min EDC pretreatment after etching + EAR; SE: cementation with a self-etch primer (Multilink Primer, Ivoclar Vivadent) and corresponding cement (Multilink Automix, Ivoclar Vivadent); SE/EDC: self-etch primer + EDC pretreatment + SE; SA: cementation with a universal self-adhesive cement (RelyX Universal, 3 M); SA/EDC: EDC pretreatment + SA. Slices were submitted to PBS test and interfacial nanoleakage evaluation 24 h after cementation or after thermocycling (40.000 cycles, 5-55 °C). To investigate the effect of EDC on MMPs activity, 4 additional first maxillary premolars per group were processed for in situ zymography analysis. Multivariate ANOVA and post hoc Tukey tests were used to analyze PBS values. The data from in situ zymography were analyzed with Kruskal-Wallis test and Dunn's pairwise multiple comparison procedures (α = 0.05). RESULTS: The variables "EDC pretreatment", "root region" and "thermocycling" significantly influenced PBS (p < 0.05), while the variable "cementation strategy" had no influence (p > 0.05). Thermocycling significantly reduced PBS in SE and SA groups (p < 0.05). EDC was effective in preserving PBS after artificial aging. EDC pretreatment significantly reduced enzymatic activity at baseline in EAR and SE groups, and in SA group after thermocycling (p < 0.05). CONCLUSIONS: The use of EDC prevents the reduction of bond-strength values after artificial aging and silences endogenous enzymatic activity within radicular dentin when different cementation strategies were employed.

Effect of Carbodiimide (EDC) on the Bond Strength Longevity of Epoxy Resin-based Endodontic Sealer to Root Dentin: An In-Vitro Study.

PURPOSE: EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) can increase dentin bonding longevity. This study aimed to evaluate the effect of final irrigation of the root canal with EDC on the bond strength (BS) longevity of an epoxy resin-based root-canal sealer. MATERIALS AND METHODS: Twenty maxillary canines were sectioned and standardized for root length at 17 mm. Roots were instrumented and distributed into 2 groups according to the final irrigation protocol: EDTA 17%+NaOCl 2.5% (C) and EDTA 17%+NaOCl 2.5%+EDC 0.5M (EDC). The canals were dried and filled with AH Plus (Dentsply Sirona). Three slices were obtained per third, and the first slice from each third was used for the immediate push-out test (i) followed by analysis of the failure pattern (n = 10); the second slice from each third was used for the push-out test after 6-month aging (A) followed by analysis of the failure pattern (n = 10); the third slice from each third was used to examine the adhesive interface under confocal laser scanning microscopy (CLSM) (n = 10). Data were analyzed with ANOVA, Fisher's exact and Kruskal-Wallis tests. RESULTS: Higher BSs were found for EDC-A (5.6 ± 1.9) than for EDC-I (3.3 ± 0.7), C-i (2.5 ± 1.0) and C-i (2.6 ± 1.0) (p = 0.0001), while C-A values were in some cases similar to C-i and in others similar to EDC-i. No statistically significant difference was observed between the thirds (p > 0.05), except for EDC-i, which showed lower BS for the cervical (2.79 ± 0.46) compared to the apical third (3.8 ± 0.5), while the middle third in some cases had values similar to those of the apical and in others to the cervical third (3.2 ± 0.7) (p = 0.032). More mixed adhesive failures were found in the cervical third, and more adhesive failures to the sealer occurred in the middle and apical thirds (p = 0.014). A significant difference was observed between treatments in terms of adaptation of the adhesive interface, with a higher percentage of good adaptation using EDC (66.7%) than using C (40%), and a lower percentage of poor adaptation with EDC (10%) compared to C (20%) (p < 0.05). CONCLUSION: Root canal irrigation with EDC increased the longevity of the adhesive interface of an epoxy resin-based root-canal sealer.

Degradation products of crosslinked silk fibroin scaffolds modulate the immune response but not cell toxicity.

Silk fibroin (SF) scaffolds have widely been used as functional materials for tissue engineering and implantation. For long-term applications, many cross-linking strategies have been developed to enhance the stability and enzymatic degradation of scaffolds. Although the biocompatibility of SF scaffolds has been investigated, less is known about the extent to which the degradation products of these scaffolds affect the host response in the long term after implantation. In this work, we first studied the effect of two different crosslinkers, namely, 1-ethyl-3-(3-dimethylaminopropyl-carbodiimide hydrochloride) (EDC) and glutaraldehyde (GA), on the topology, mechanical stability and enzymatic degradation of SF scaffolds. We found that the SF scaffolds treated with GA (GA-SF) appeared to show an increase in the sheet thickness and a higher elastic modulus when compared to that treated with EDC (EDC-SF) at a similar level of crosslinking degree. The uncrosslinked and both crosslinked SF scaffolds were completely digested by proteinase K but were not susceptible to degradation by collagenase type IV and trypsin. We next investigated the effect of the degradation of SF on the cytotoxicity, genotoxicity, and immunogenicity. The results demonstrated that the degradation products of the uncrosslinked and crosslinked SFs did not trigger cell proliferation, cell death, or genotoxicity in primary human cells, while they appeared to modulate the phenotypes of macrophages. The degradation products of GA-SF promoted pro-inflammatory phenotypes, while those from EDC-SF enhanced polarization towards anti-inflammatory macrophages. Our results demonstrated that the degradation products of SF scaffolds can mediate the immune modulation of macrophages, which can be implemented as a therapeutic strategy to control the long-term immune response during implantation.

Can Carbodiimide (EDC) and Chitosan Cross-linking Agents Effect the Longevity of Fiberglass Posts Luted with Different Types of Composite Cements to Root Dentin?

PURPOSE: To evaluate the effect of carbodiimide (EDC) and chitosan (CHI) on the enzymatic activity (EA) and bond strength (BS) of different composite cements to root dentin. MATERIALS AND METHODS: Ninety (90) maxillary canines were sectioned, standardizing the length of the roots. The roots were endodontically treated, prepared, divided into 3 groups according to dentin treatment (distilled water [DW], CHI 0.2 wt%, or EDC 0.5M), and further subdivided into 3 subgroups according to composite cement (RelyX ARC [3M Oral Care], Panavia F 2.0 [Kuraray Noritaki], or RelyX U200 [3M Oral Care]). Of the slices obtained by sectioning, the most cervical of each third were subjected to a push-out test and the most apical were subjected to in-situ zymography. Half of the slices were analyzed immediately, and the other half after 6 months. The results were analyzed with ANOVA or the chi-squared test. RESULTS: RelyX ARC showed higher BS associated with CHI, while RelyX U200 showed higher BS associated with EDC (p = 0.044). For Panavia F 2.0, the treatment did not influence BS (p > 0.05). For the cervical and middle thirds, no differences were observed between the cements, while the apical third revealed higher BS for RelyX U200 (p < 0.001). The highest percentage of adhesive-to-dentin failures was observed for Panavia F 2.0. EDC showed the lowest percentage of adhesive-to-dentin failures. According to zymographic analysis, DW and CHI showed greater fluorescence for RelyX ARC, while EDC exhibited the lowest fluorescence of all cements (p > 0.05). CONCLUSION: The different mechanisms of action of solutions for pre-treatment of intraradicular dentin yielded different results depending on the adhesive used. EDC resulted in higher bond strength and higher enzyme inhibition for RelyX U200, while the treatment with chitosan resulted in higher bond strength and lower enzymatic activity for RelyX ARC. Although EDC and chitosan treatments did not influence the bond strength for Panavia F 2.0, both resulted in higher enzyme inhibition for this composite cement.

Carbodiimide crosslinked decellularized lenticules as a drug carrier for sustained antibacterial eye treatments.

In this study, the drug-loading and antibacterial activity of carbodiimide/N-hydroxysuccinimide (EDC/NHS) crosslinked decellularized lenticules (CDLs) were evaluated. Small incision lenticule extraction derived lenticules were decellularized and modified with crosslinking concentrations of 0.00 (E/L00, non-crosslinked), 0.01 (E/L01), 0.05 (E/L05) and 0.25 mmol (E/L25) EDC per mg lenticules at 5:1 EDC/NHS ratios with non-decellularized non-crosslinked lenticules (NDLs) as controls. NDLs and EDC/NHS CDLs had similar water contents. The light transmittance percentages (400-800 nm) were 91.55 ± 1.16%, 88.68 ± 1.19%, 80.86 ± 1.94%, 85.12 ± 2.42% and 85.62 ± 2.84% for NDLs, E/L00, E/L01, E/L05 and E/L25, respectively (P< 0.01). The EDC/NHS CDLs (diameter: 6.36 ± 0.18 mm; central thickness: 117.31 ± 3.46 µm) were soaked in 3% (wt./vol.) levofloxacin (LEV) solution for 3 h. The drug release concentrations of LEV-impregnated EDC/NHS CDLs were determined by high-performance liquid chromatography. Zone inhibition (ZOI) againstStaphylococcus aureusof E/L01, E/L05 and E/L25 were superior to E/L00 CDLs (P< 0.01) and among the different crosslinked groups, E/L05 lenticules produced the largest ZOIs and their drug concentration release over 21 d was the highest. EDC/NHS crosslinking can improve the drug-loading effect and antibacterial activity of decellularized lenticules. LEV-impregnated EDC/NHS CDLs are promising drug delivery carriers.

Chemical modification of acellular fish skin as a promising biological scaffold by carbodiimide cross-linker for wound healing.

Biological matrices can be modified with cross-linkers to improve some of their characteristics as scaffolds for tissue engineering. In this study, chemical cross-linker 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was used with different ratios (5, 10, 20, 30, and 40 mM) to improve properties such as mechanical strength, denaturation temperature, and degradability of the acellular fish skin as a biological scaffold for tissue engineering applications. Morphological analysis showed that the use of cross-linker at low concentrations had no effect on the structure and textiles of the scaffold, while increasing mechanical strength, denaturation temperature, and degradation time. Cytotoxicity and cellular studies showed that the optimal cross-linker concentration did not significantly affect cell viability as well as cell adhesion. In general, utilising the carbodiimide cross-linker with the optimal ratio can improve the characteristics and function of the biological tissues such as acellular fish skin.

Insights on Chemical Crosslinking Strategies for Proteins.

Crosslinking of proteins has gained immense significance in the fabrication of biomaterials for various health care applications. Various novel chemical-based strategies are being continuously developed for intra-/inter-molecular crosslinking of proteins to create a network/matrix with desired mechanical/functional properties without imparting toxicity to the host system. Many materials that are used in biomedical and food packaging industries are prepared by chemical means of crosslinking the proteins, besides the physical or enzymatic means of crosslinking. Such chemical methods utilize the chemical compounds or crosslinkers available from natural sources or synthetically generated with the ability to form covalent/non-covalent bonds with proteins. Such linkages are possible with chemicals like carbodiimides/epoxides, while photo-induced novel chemical crosslinkers are also available. In this review, we have discussed different protein crosslinking strategies under chemical methods, along with the corresponding crosslinking reactions/conditions, material properties and significant applications.

A novel targeted iron oxide nanocarrier for inhibiting M2-type macrophages in the tumor microenvironment.

Background: Tumor-associated macrophages (TAMs) are vital to the tumor microenvironment. They are classified as antitumor M1-type or protumor M2-type macrophages. M2-type macrophages accumulate in the tumor stroma and are related to poor prognosis. Iron oxide nanoparticles are used as drug delivery vehicles because of the structure of carboxyl groups on their surface and their ability to be easily phagocytosed by macrophages. Aim: The signal transducer and activator of transcription 6 (STAT6) signaling pathway controls M2 macrophage polarization, but the STAT6 signaling pathway inhibitor AS1517499 lacks efficient targeting in vivo. Thus, our study aimed to block the polarization of TAMs to M2-type macrophages. Methods and Material: We used ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) as drug carriers coated with the STAT6 signaling pathway inhibitors AS1517499 and CD163 monoclonal antibodies to synthesize the targeted nanocomplex AS1517499-USPION-CD163 utilizing the carbodiimide method. Then, we determined its physicochemical properties, including hydrodynamic size distribution, ultrastructure, iron concentration, protein content and activity of the CD163 monoclonal antibody, AS1517499 content, and selectivity for M2-type macrophages, and its biological applications. Results: The hydrodynamic size distribution was stable (average size = 95.37 nm). Regarding biological applications, the targeted nanocomplex selectively inhibited M2-type macrophages. Conclusions: The targeted nanocomplex AS1517499-USPION-CD163 showed high selectivity for M2-type macrophages. Therefore, iron oxide nanoparticles targeting TAMs may be an effective approach to TAM therapy.

Melimine-Modified 3D-Printed Polycaprolactone Scaffolds for the Prevention of Biofilm-Related Biomaterial Infections.

Biomaterial-associated infections are one of the major causes of implant failure. These infections result from persistent bacteria that have adhered to the biomaterial surface before, during, or after surgery and have formed a biofilm on the implant's surface. It is estimated that 4 to 10% of implant surfaces are contaminated with bacteria; however, the infection rate can be as high as 30% in intensive care units in developed countries and as high as 45% in developing countries. To date, there is no clinical solution to prevent implant infection without relying on the use of high doses of antibiotics supplied systemically and/or removal of the infected device. In this study, melimine, a chimeric cationic peptide that has been tested in Phase I and II human clinical trials, was immobilized onto the surface of 3D-printed medical-grade polycaprolactone (mPCL) scaffolds via covalent binding and adsorption. X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) spectra of melimine-treated surfaces confirmed immobilization of the peptide, as well as its homogeneous distribution throughout the scaffold surface. Amino acid analysis showed that melimine covalent and noncovalent immobilization resulted in a peptide density of ∼156 and ∼533 ng/cm2, respectively. Furthermore, we demonstrated that the immobilization of melimine on mPCL scaffolds by 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide hydrochloride (EDC) coupling and noncovalent interactions resulted in a reduction of Staphylococcus aureus colonization by 78.7% and 76.0%, respectively, in comparison with the nonmodified control specimens. Particularly, the modified surfaces maintained their antibacterial properties for 3 days, which resulted in the inhibition of biofilm formation in vitro. This system offers a biomaterial strategy to effectively prevent biofilm-related infections on implant surfaces without relying on the use of prophylactic antibiotic treatment.

Microfluidic synthesis of Janus-structured QD-encoded magnetic microbeads for multiplex immunoassay.

Uniform and monodisperse quantum dot (QD)-encoded magnetic microbeads with Janus structure were produced in a microfluidic device via photopolymerization. UV light through a microscope objective was used to solidify the microbeads which showed sharp interfaces and excellent magnetic responses. QDs with different emission peaks (450 nm for blue and 640 nm for red) were mixed at different ratios to provide three spectral codes. The QD-encoded microbeads can be distinguished by analyzing their fluorescent images in HSV color space. After hydrolysis of the anhydride group in alkaline solution, protein was immobilized on microbeads via activation of carboxyl groups using (1-ethyl-3(3-dimethylaminoprophyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS). A microhole array in polydimethylsiloxane (PDMS) substrates with a specific size was fabricated to trap individual microbeads in a single microhole. The combination of Janus-structured QD-encoded magnetic microbeads and microhole arrays facilitates both flexibility, binding kinetics, sensitivity for suspension assay, and fluorescence mapping analysis for conventional biochips, thus providing a novel platform for multiplex bioanalysis. The capability of this integration for multiplex immunoassays was verified using three kinds of IgG and their corresponding anti-IgG. A detection limit of 0.07 ng/mL was achieved for human IgG, indicating practical applications in various fields.