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Polymeric Nanoparticles for Targeted Delivery of Bioactive Agents and Drugs
Published in Severian Dumitriu, Valentin Popa, Polymeric Biomaterials, 2020
Cesare Errico, Alberto Dessy, Anna Maria Piras, Federica Chiellini
The final concern in the incorporation of antibodies or antibody fragments (Fab) to DDS is the method of conjugation. Abs or Fabs fragments are normally conjugated either directly to nanoparticle surfaces or through linker molecules such as PEG. Conjugation of the antibody can be random or site specific. Random conjugation is commonly carried out by carbodiimide-mediated chemistry, which creates stable amide bonds between carboxylic acid groups in the nano-carrier and primary amine groups, including lysines and the N-terminus amine, of the antibody (Chapman 2002). Due to the lack of specificity of this conjugation route, antibodies can be bound to the nanocarrier in a number of ways, some of which can even block access to the binding site of the antibody (Lee et al. 1999).
Optical Nanosensors
Published in Vinod Kumar Khanna, Nanosensors, 2021
Labeling of biomolecules, such as peptides, proteins or oligonucleotides (short nucleic acid polymers), with a fluorophore requires suitable functional groups for covalent binding or for non-covalent attachment of the fluorophore. The advantage of organic dyes in this aspect is the market availability of a tool-box of functionalized dyes, in conjunction with established labeling protocols. What is the situation with QDs? There are no consensus methods for labeling biomolecules with QDs. The general principle for QD bio-functionalization is that, first, the QDs are made water dispersible and then they are bound to biomolecules. Binding is done by selecting from one of several methods, such as electrostatically, via biotin-avidin interactions, or by covalent cross-linking (e.g., carbodiimide-activated coupling between amine and carboxylic groups or maleimide-catalyzed coupling between amine and sulfhydryl groups or between aldehyde and hydrazide functions) or by binding to polyhistidine tags. Avidin is a biotin-binding protein produced in the oviducts of birds, reptiles, and amphibians, and deposited in the whites of their eggs. Biotin is a water-soluble B-complex vitamin (vitamin B7). A carbodiimide or a methane-diimine is a functional group, with formula RN=C=NR, often used to activate carboxylic acids (oxoacids having the structure RC(=O)OH) toward amide formation; amides are amine (a group of organic compounds of nitrogen, such as ethylamine, C2H5NH2, that may be considered to be ammonia derivatives) derivatives of carboxylic acids. Maleimide is an unsaturated imide (a functional group consisting of two carbonyl groups (C=O) bound to nitrogen) with the formula H2C2(CO)2NH. A polyhistidine-tag is an amino acid motif in proteins that consists of at least five histidine (His) residues, often at the N- or C-terminus of the protein.
Tissue Adhesives
Published in Chih-Chang Chu, J. Anthony von Fraunhofer, Howard P. Greisler, Wound Closure Biomaterials and Devices, 2018
It is known that carbodiimide is a coupling agent for amide bond formation between carboxyl and amino groups even in the presence of water.44 An aqueous solution of gelatin sets to a gel when a water-soluble carbodiimide (WSC) is added to the solution due to the formation of intermolecular amide bonds in the gelatin molecules having both carboxyl and amino groups. An addition of poly(L-glutamic acid) (PLGA) to the gelatin solution shortens the incipient gelation time, as shown in Figure 11.21.45 Since PLGA will be converted to acid anhydrides by an addition of WSC which functions also as an agent to produce acid anhydride from carboxyl groups, PLGA molecules with acid anhydrides will readily undergo crosslinking with gelatin molecules having amino groups to yield a hydrogel. This gelatin-PLGA hydrogel exhibited firm adhesion to the mouse skin and other soft tissues with a higher bonding strength than fibrin glue.45Table 11.9 shows a representative result of bonding strength of gelatin-PLGA hydrogel to various rabbit tissues when compared with fibrin glue. Bonding was performed using a mixture of 100 ml of aqueous solution containing 100 mg/ml gelatin, 100 mg/ml PLGA, and 35 ml of 9.6 mg/ml 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide as WSC. The bonding strength of the gelatin-PLGA adhesive is higher than that of fibrin glue, irrespective of the tissue type. The difference in the bonding strength among the various soft tissues may be explained in terms of their surface properties. For example, the presence of hydrophobic fat in tissues will reduce the wettability of the fatty soft tissues by gelatin-PLGA solution or fibrin glue, resulting in a decrease in bonding strength. In addition, fat will reduce the anchoring effect of the crosslinked gelatin-PLGA adhesive on the soft tissues and hence the bonding strength. Contrary to fibrin glue, cohesive failure inside the crosslinked gel was observed when the gel-tissue bond was intentionally broken. The bonding strength of the gelatin-PLGA hydrogel became higher with an increase in PLGA concentration in the initial solution.
Crosslinking hyaluronic acid soft-tissue fillers: current status and perspectives from an industrial point of view
Published in Expert Review of Medical Devices, 2021
Jimmy Faivre, Amos I. Pigweh, Julien Iehl, Pauline Maffert, Peter Goekjian, François Bourdon
BCDI can also be used as a bifunctional crosslinker to directly crosslink adjacent HA chains. In this technique, the carboxyl functionality on the HA main chain is utilized under mildly acidic medium to form a stable HA acylurea with the biscarbodiimide on both ends in the absence of a nucleophile (such as amines) or any other polyanionic polysaccharide other than HA itself [39]. The reaction proceeds by protonation of the carbodiimide, followed by attack of the carboxylate anion on the carbon atom of the BCDI to form an O-acylisourea intermediate. In acidic medium, the intermediate then undergoes O-to-N migration to form the more stable N-acylurea depicted in Figure 1. This reaction is carried out in the presence of a pH buffer usually between pH 4.5 and 5.5. One of the specifications of this method is keeping the pH of the medium within the required range as carbodiimides are unstable at lower pH values while the reaction rate is diminished [40]. Since the rearrangements at both ends of the biscarbodiimide are non-concerted, the O-to-N migration can be incomplete which would result in a mixture of N-acylurea at both ends, a mixture one N-acylurea and one O-acylisourea at either end, and O-acylisourea at both ends.
Analysis of hydrolytic differences of free and “polyacrylic acid (PAAc)-conjugated trypsin and chymotrypsin” by using fluorescence lifetime distributions
Published in Preparative Biochemistry & Biotechnology, 2020
Ümmügülsüm Polat, İbrahim Ethem Özyiğit, Emine Karakuş
A synthetic polymer can be equipped with a three-dimensional structure to allow a higher hierarchical arrangement structure by covalent conjugation with a protein. An immobilized reagent 1-Ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) derivative of Carbodiimides (CDI), it is commonly known as a zero-length cross-linking agent used to conjugate a carboxylate-containing molecule to an amine-containing molecule to form a stable covalent amide linkage. EDC reacts with the carboxylic acid group first and activates the carboxyl group to form an active ester O-acylisourea intermediate, which then the carbonyl group of this ester attacked by the amine compound generate an amide bond and release of an isourea (Fig. 2).
In-situ crosslinking of electrospun gelatin-carbodiimide nanofibers: fabrication, characterization, and modeling of solution parameters
Published in Chemical Engineering Communications, 2021
Maryam Hajiabbas, Iran Alemzadeh, Manouchehr Vossoughi, Amir Shamloo
Zero-length crosslinking allows researchers to immobilize protein-protein interactions without introducing a spacer arm. Carboxylates (–COOH) may be reacted to NHS or Sulfo-NHS in the presence of a carbodiimide such as EDC, resulting in a semi-stable NHS or Sulfo-NHS ester, which may then be reacted with primary amines (–NH2) to form amide crosslinks (Figure 8). Although NHS or Sulfo-NHS is not required for carbodiimide reactions, their use greatly enhances coupling efficiency (Grabarek and Gergely 1990; Staros et al. 1986). Using NHS or Sulfo-NHS makes it possible to perform a two-step reaction (Grabarek and Gergely 1990; Staros et al. 1986). Figure 8 (from Thermo Fisher Scientific) shows the mechanism of zero-length crosslinking with EDC/NHS in the presence of the carboxylate and primary amines in a protein structure to produce the stable amide bond. As, the optimum pH range for the activation of carboxyl groups using EDC is 5–6 (Grabarek and Gergely 1990; Staros et al. 1986). In this work, a small amount of acetic acid was added to reach a pH of 4 to stop crosslinking in the syringe. Also, using a higher amount of NHS (molar ratio of NHS: EDC was 2:1) leads to two-step reaction to delay the crosslinking process. Then, during electrospinning, solvent evaporation occurs, and acetic acid content in G fibers decreases. Next, by storing scaffolds in a desiccator under control humidity, EDC/NHS acted, and in-situ crosslinking in G fibers occurred. The amount of remaining free amines in the system through a ninhydrin assay was determined to measure the Degree of crosslinking. Ninhydrin readily reacts with free amines and produces a vivid purple hue upon reaction with free amine groups (Kishan et al. 2015). As depicted in Table 3, there was a small decrease in the degree of crosslinking with increasing G concentration.