Azido-PEG3-aminoacetic acid-NHS ester

γ-PGA-NHS ester, which was prepared using poly(γ-glutamic acid) (γ-PGA) and N-hydroxysuccinimide (NHS) as the raw materials, was synthesized to be a novel cross-linker of collagen. Fourier transform infrared spectra analysis suggested that the products displayed the characteristic absorption peak of ester. Results from nuclear magnetic resonance analysis indicated that the esterification degree of γ-PGA-NHS ester was increased with the increase of NHS. Modified collagen was prepared and characterized. The results of circular dichroism analysis indicated modified collagen retained the triple helix structure of natural collagen. Sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed that the molecular weight of collagen was increased after cross-linking. Peptide mapping of collagen suggested that cross-linked collagen possessed an enhanced resistance to trypsin degradation. Differential scanning calorimeter results showed that the denaturation temperature of collagen was improved from 68.1±0.4 to 91.2±0.5°C (p<0.05). Dynamic viscoelastic measurements demonstrated the improvement of thermal stability and reflected the exponential increase in η*. The cross-linked collagen retained porous structure and the pore size became larger as observed by scanning electron microscopy. The investigation results provided useful information to produce collagen with improved physicochemical properties, particularly the thermal stability via the use of γ-PGA-NHS ester as a biomacromolecule-based cross-linker.

The poly(γ-glutamic acid)-NHS (γ-PGA-NHS) esters were used to endow collagen with both of excellent water-solubility and thermal stability via cross-linking reaction between γ-PGA-NHS and collagen. In the present work, the effect of γ-PGA-NHS on the aggregation of collagen molecules was studied by fluorescence techniques. The fluorescence emission spectra of pyrene in collagen solutions and the intrinsic fluorescence emission spectra of collagen suggested different effects of γ-PGA-NHS on collagen molecules: inhibiting aggregation below critical aggregation concentration (CAC) and promoting aggregation above CAC. The two-dimensional (2D) fluorescence correlation spectra indicated that the intermolecular hydrogen bonding and cross-linking between γ-PGA-NHS and collagen would influence the aggregation of collagen molecules. By the ultra-sensitive differential scanning calorimeter (VP-DSC), it was found that the main denaturational transition temperature (Tm2) of modified collagen increased, while its calorimetric enthalpy changes (ΔH2) decreased compared to those of native collagen, further indicating that the modification of γ-PGA-NHS influenced the aggregation of collagen molecules. The study provide useful information for the utilizing and or the processing of water-soluble collagen in aqueous solution in the fields such as cosmetics, health care products, tissue engineering and biomedical materials, etc.

The rheological behaviors of a polyanionic collagen, fabricated using poly(γ-glutamic acid)-N-hydroxysuccinimide (γ-PGA-NHS) as a novel modifier, were investigated in this study. It was found that both of the native and modified collagen solutions were pseudoplastic fluids, as shown from the steady-shear tests. While the storage modulus and loss modulus of collagen increased with increasing the amount of γ-PGA-NHS, or with increasing the degree of esterification of γ-PGA-NHS; meanwhile, the dynamic denaturation temperature determined by dynamic temperature sweep was also increased, indicating an improved thermal stability of collagen solution modified by γ-PGA-NHS. The creep-recovery measurements showed that the resistance to deformation was enhanced for modified collagen, probably due to the cross-linking occurred between the ε-amino groups of collagen molecules and α-COOH groups of γ-PGA-NHS, as well as the electrostatic interaction and hydrogen-bond interactions between the two molecules. Furthermore, the aggregation of collagen fibers was promoted due to these interactions between collagen and γ-PGA-NHS as observed by atomic force morphology. In addition, the modified collagen exhibited good cytocompatibility as demonstrated by cell growth culturing. The obtained information was expected to give valuable clues to the design and fabrication of controlled stable collagen-based products for applications in various biomedical fields.