High Molecular Weight Gelatin Research Articles

Cold setting of gelatin–antioxidant peptides composite hydrogels using a new psychrophilic recombinant transglutaminase (rTGase)

This study used a new psychrophilic transglutaminase (TGase) to catalyze the formation of protein and peptide hydrogels by forming isopeptide bonds. The psychrophilic recombinant TGase (rTGase) was overexpressed in E. coli and purified using a Ni-Sepharose column followed by stepwise dialysis to recover the cold-active enzyme (specific activity of 1.63 U/mg). The rTGase catalyzed the formation of cold-set hydrogels at 4 °C, which were comprised of high molecular weight gelatin and antioxidant peptides. The antioxidant bioactivity of the cold-set gelatin–peptides composite hydrogels was significantly improved with rTGase at 4 °C for 10 days. To understand the mechanism of the psychrophilic rTGase on the enhanced bioactivity of cross-linked hydrogels, in silico molecular docking studies were performed to assess the binding of antioxidant peptides into the rTGase active pocket, as well as to analyze the interactions of Gln and Lys residues in the antioxidant peptides with amino acid residues in the rTGase catalytic site. The formation of covalent cross-linkages in the gelatin–gelatin, gelatin–peptide and peptide–peptide matrices, and possible structures of composite hydrogels formed by rTGase catalysis in cold conditions, were deduced. The new psychrophilic rTGase enabled stabilization of the antioxidant bioactivity of protein- and peptide-containing cold-set hydrogels formed at low temperatures.

The Effect of Gelatin Molecular Weight on Tendon Lubrication Utilizing an Extrasynovialized Turkey Flexor Tendon Model.

Flexor tendon injuries are common hand injuries among the military population often resulting in functional impairment. Flexor tendon gliding friction has been linked to adhesion formation, especially with the use of extrasynovial grafts. Carbodiimide-derivatized hyaluronic acid with gelatin (cd-HA-gelatin) can reduce gliding friction of the tendon graft; however, the effects of gelatin molecular weight (MW) have not been studied. The turkey model has been shown to better match humans, but extrasynovial tendons are unavailable. The purpose of this study was to (1) manually roughen turkey flexor digitorum profundus (FDP) tendons to simulate extrasynovial tendons and (2) investigate the effects of gelatin MW on tendon friction. The third digit of (n = 48) turkeys were dissected with the proximal pulley, and FDP tendon and the flexor superficialis tendon were preserved. Digits were randomly assigned into four groups: one saline control and three cd-HA-gelatin-treated groups of varying gelatin MW. Flexor digitorum profundus tendon friction was measured at its original condition, serving as a baseline. Tendons were roughened using a custom rig, and tendon friction was measured again. All four groups received treatment and gliding friction was measured every 100 cycles to a total of 1,000 cycles. Tendon friction significantly increased (P < 0.05) after roughening. Friction in the saline control group increased steadily over repeated cycles, whereas friction of all gelatin-treated tendons decreased significantly compared with the saline control group (P < 0.05), maintaining low friction to 1,000 cycles representing human tendons. There was no significant difference found between gelatin-treated groups. We have developed a method to roughen synovial FDP tendons to create extrasynovial-like tendons for lubrication material evaluations. Cd-HA-gelatin effectively reduces tendon friction in this model. Our data suggest medium or low MW gelatin may provide a better reduction in friction compared with high MW gelatin.

An in vitro system to investigate IOL: Lens capsule interaction

Posterior capsule opacification (PCO) is the most common complication associated with intraocular lens (IOL) implantation. Unfortunately, current in vitro models cannot be used to assess the potential of PCO due to their failure to simulate the posterior curvature of the lens capsule (LC) and IOL, a factor known to affect PCO pathogenesis in clinic. To overcome such a challenge, a new system to study IOL: LC interaction and potentially predict PCO was developed in this effort. It is believed that the interactions between an IOL and the lens capsule may influence the extent of PCO formation. Specifically, strong adhesion force between an IOL and the LC may impede lens epithelial cell migration and proliferation and thus reduce PCO formation. To assess the adhesion force between an IOL and LC, a new in vitro model was established with simulated LC and a custom-designed micro-force tester. A method to fabricate simulated LCs was developed by imprinting IOLs onto molten gelatin to create simulated three dimensional (3D) LCs with curvature resembling the bag-like structure that collapses on the IOL post implantation. By pushing the LC mold vertically downward, while measuring the change in position of the bending bar with respect to its start position, the adhesion force between the IOLs and LCs was measured.An in vitro system that can measure the adhesion force reproducibly between an IOL and LC with a resolution of ~1 μN was established in this study. During system optimization, the 10% high molecular weight gelatin produced the best LC with the highest IOL: LC adhesion force with all test lenses that were fabricated from acrylic foldable, polymethylmethacrylate (PMMA) and silicone materials. Test IOLs exerted different adhesion force with the 3D simulated LCs in the following sequence: acrylic foldable IOL > silicone IOL > PMMA IOL. These results are in good agreement with the clinical observations associated with PCO performance of IOLs made of the same materials. This novel in vitro system can provide valuable insight on the IOL: LC interplay and its relationship to clinical PCO outcomes.

Towards the production of monodisperse gelatin nanoparticles by modified one step desolvation technique

The aim of the present study was to prepare gelatin nanoparticles (GNPs) using modified one step desolvation method to obtain small, uniformly sized, and spherical GNPs. The modifications involved the preparation of high molecular weight gelatin (HMWG) by fractionation of gelatin. HMWG was freeze-dried to be used in a specified concentration for preparation of GNPs with controllable properties. Furthermore, HMWG was cationized by dissolving it in acidified deionized water at pH value less than its isoelectric point. Factorial design analysis was utilized to investigate the effect of different preparation variables (viz. HMWG concentration, pH and ratio of HMWG solution volume to non-solvent volume) on particle size, polydispersity index (PDI), zeta potential and yield of GNPs. Results revealed the formation of monodisperse GNPs with small particle size, low PDI values (< 0.2) and high yield. FT-IR spectroscopy and differential scanning calorimetry studies revealed that the conformational structure of HMWG chains was altered within GNPs matrices. The suggested modifications introduced on the one-step desolvation method enable to prepare successfully monodispersed GNPs with narrow size distribution and high particle yield.

PVA-gelatin hydrogels formed using combined theta-gel and cryo-gel fabrication techniques

Poly(vinyl alcohol) (PVA) is a synthetic, biocompatible polymer that has been widely studied for use in bioengineered tissue scaffolds due to its relatively high strength, creep resistance, water retention, and porous structure. However, PVA hydrogels traditionally exhibit low percent elongation and energy dissipation. PVA material and mechanical properties can be fine-tuned by controlling the physical, non-covalent crosslinks during hydrogel formation through various techniques; PVA scaffolds were modified with gelatin, a natural collagen derivative also capable of forming reversible hydrogen bonds. Blending in gelatin and poly(ethylene glycol) (PEG) with PVA prior to solidification formed a highly organized hydrogel with improved toughness and dynamic elasticity. Theta-gels were formed from the solidification of warm solutions and the phase separation of high molecular weight gelatin and PVA from a low molecular PEG porogen upon cooling. While PVA-gelatin hydrogels can be synthesized in this manner, the hydrogels exhibited low toughness with increased elasticity. Thus, theta-gels were additionally processed using cryo-gel fabrication techniques, which involved freezing theta-gels, lyophilizing and re-hydrating. The result was a stronger, more resilient material. We hypothesized that the increased formation of physical hydrogen bonds between the PVA and gelatin allowed for the combination of a stiffer material with energy dissipation characteristics. Rheological data suggested significant changes in the storage moduli of the new PVA-gelatin theta-cryo-gels. Elastic modulus, strain to failure, hysteresis and resilience were studied through uniaxial tension and dynamic mechanical analysis in compression.

Abstract 4663: Lysosomal targeting from a gelatin-doxorubicin conjugate produces evidence of both nuclear and lysosomal cytotoxcity pathways in mcf-7 breast cancer cells

Abstract Background: Intra-lysosomal targeting has been investigated for the treatment of cancer but details of the cytotoxicity pathways remain unclear. A high molecular weight gelatin - doxorubicin conjugate (GDox) has been synthesized to target acidic lysosomes for intra-lysosomal drug release. The purpose of this investigation was to explore GDox feasibility for drug delivery and to make a preliminary determination of a cytotoxic pathway. Methods: GDox was synthesized in formamide using the carbodiimide, EDC. Drug load was determined spectroscopically, drug release at pH 4.8 was determined by HPLC, and molecular weight and its reduction in FBS was determined by HPSEC. Cellular localization was determined for free Dox and GDox at 10 µM or its equivalent Dox concentration using fluorescence microscopy with appropriate subcellular stains. Three to four independent trials (n=3-4) were examined with 200 to 700 cells per time point. Cytotoxicity was determing by the MTT procedure (n=3). Viabilities, growth inhibition profiles and their IC50 values were calculated with GraphPad Prism 7. The nucleus content of free Dox or Dox released from GDox after incubation was determined by UHPLC using fluorescent detection (n=3-4). Results: A typical GDox contained 5.6% w/w Dox, had a molecular weight of ~ 160 kDa, and released 58+2.3% Dox at pH 4.8 representing lysosomal pH. After incubation in FBS at 37°C for 24 hr the low molecular weight species increased a relatively small 16%. Fluorescent images with LysoTracker Green confirmed GDox localization to the lysosome which was detectable by 2 hr but substantial in swollen lysosomes by 24 and 48 hrs. No released Dox was observed in the nucleus, but a slight red nucleus haze at later times suggested this possibility. Cell debri from GDox was not observed from free Dox. As expected, free Dox rapidly accumulated in the nucleus by 2 hr. Viability determinations at 0.1, 1, and 10 µM for 2 to 48 hr showed no effect from either agent before 24 hr. By 48 hr at 10 µM, the viability of Dox (30+12%) and GDox (36+13%) were indistinguishable (p=0.68). However, by 72 hr, IC50 values varied 10-fold at 0.094 and 0.96 µM, respectively. The nucleus drug content after incubation at 10 µM for 2 to 48 hr was 0.27+0.07 to 1.39+0.6 µg/106 cells (p&amp;lt0.01) for free Dox, and 0.014+0.01 to 0.11+0.06 µg/106 cells (p&amp;lt0.01) for released Dox. Conclusions: Taken together these results suggest good feasibility of GDox lysosomal targeting and Dox release in MCF7 breast cancer cells. Of note, after 48 hr incubation GDox produced the same cytotoxicity as the free drug but with 13-fold less Dox in the nucleus. This striking nucleus difference and the substantial lysosomal GDox content suggest a lysosomal pathway of GDox cytotoxicity in addition to a nuclear pathway. Support: NIH/NCI R15CA135421 and the Agnes Varis Trust for Women's Leadership and Health. Citation Format: Mohammed Alvi, Bayan Eshmawi, Rachel Nicoletto, Hyun Kate Kim, Christopher Cammarata, Clyde Ofner. Lysosomal targeting from a gelatin-doxorubicin conjugate produces evidence of both nuclear and lysosomal cytotoxcity pathways in mcf-7 breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4663.

Molecular structural properties of extracted gelatin from Yak skin as analysed based on molecular weight

ABSTRACTThis paper dealt with the physicochemical, functional and antioxidative properties of Yak skin gelatins with various molecular weights. The gelatin was extracted by alkaline process, and different molecular weight gelatin was achieved by ultrafiltration with different molecular weight cut membrane. The SDS-PAGE showed the molecular weight of the yak skin gelatin. The UV-Vis absorption turned out that the high molecular weight (HMW) of yak skin gelatin had a higher maximum absorption peaks. The FT-IR indicated the change of gelatin structures with the change of the molecular weight. No matter the HMW or the low molecular weight (LMW), the Yak skin gelatin had a high denaturation temperature (Td), and it was higher than most of the other mammals and marine biological gelatin. The HMW gelatin had higher imino acids contents and lower oxidation resistance, foamability and emulsibility compared to the LMW gelatin. These findings showed that a certain molecular weight range of Yak skin gelatin has great potential for application as an alternative to commercial gelatin due to its good antioxidative, foamability emulsibility and thermotolerance.

Macro- vs. micromolecular stabilisation of W/O/W-emulsions

Water-in-Oil-in-Water (W/O/W) double emulsions (DEs) were prepared with high molecular weight gelatin as hydrophilic emulsifier and gelling agent of the external water phase and their stability was compared to DEs prepared with the hydrophilic emulsifier polysorbate 80. The Water-in-Oil (W/O)-emulsions were prepared with corn oil and polyglycerol polyricinoleate (PGPR) as the lipophilic emulsifier. Tartrazine, a synthetic dye, was added to the inner water phase and the encapsulation efficiency of the marker was measured. A gelled DE was successfully obtained and its rheological properties were possible to adjust without affecting the DE stability to a large degree. During a 90-days stability study, DEs stabilised by gelatin showed a high initial encapsulation yield (98.5–96.5%), with no further release of tartrazine measured after the first day.Release of tartrazine was observed from the DEs prepared with polysorbate 80 during the first days after preparation, whereafter no further release was observed for the remaining 90 days period. Contrary to the DEs stabilised by gelatin, the encapsulation efficiency of tartrazine was highly dependent on the concentration of PGPR. By addition of a counter solute (NaCl) into the external water phase, osmotically adjusted DEs (Δ −0.14, −0.07, 0.00 and 0.14 Osm) were prepared for the polysorbate 80 stabilised W/O/W-emulsion. Higher encapsulation yields were obtained with increasing amounts of NaCl, with the highest amount of tartrazine retained for the hyperosmotic DE (Δ 0.14 Osm) formulation.

Radiocarbon dating of VIRI bone samples using ultrafiltration

Ultrafiltration can effectively remove low-molecular-weight (LMW) contaminants from bone gelatin to extract high-molecular-weight (HMW) proteins that are derived from original bone collagen, though it cannot remove HMW collagen crosslinked with humic acids. Therefore, ultrafiltration is often used to obtain more accurate 14C dates of bones. However, ultrafiltration may introduce new contaminants to bone gelatins, mainly from ultrafilters used. To study the effects of ultrafiltration on 14C age, we analyzed the C/N ratio, δ13CPDB and δ15NAIR values, and 14C ages of acid-soluble bone collagen obtained by decalcification, gelatin extracted from acid-insoluble bone collagen, and the HMW gelatin and LMW fractions produced during ultrafiltration of the extracted gelatin. Bone samples from the Fifth International Radiocarbon Intercomparison (VIRI) were used: VIRI-E (mammoth), -F (horse), -G (human), and -I (whale).In this study, carbon and nitrogen content and gelatin yields were used to evaluate collagen preservation in the VIRI bone samples. Radiocarbon ages, δ13CPDB and δ15NAIR values of unfiltered and HMW gelatins were obtained and compared with the published consensus values. The LMW fraction was found to exhibit different values from those of the other fractions, indicating the possible presence of extraneous contamination. The Vivaspin™ 6 ultrafilters used in this study were analyzed and radiocarbon dated both before and after cleaning. We present evidence to suggest that LMW fraction contaminants could be derived from the ultrafilters rather than humic substances. Excessively long ultrafiltration time was suspected to have contaminated the bone samples with material from the ultrafilter, because those samples exhibited older 14C ages than did those filtered for shorter durations. The results in this study indicate that 14C ages of unfiltered gelatin extracted from well-preserved bones can be sufficiently accurate, and that care should be taken not to contaminate bone gelatin during ultrafiltration. Additional studies investigating bones with different degrees of degradation are needed to determine the effectiveness of ultrafiltration for accurate 14C dating when the collagen is not so well preserved.

Injectable, thermo-reversible and complex coacervate combination gels for protein drug delivery

Injectable and thermo-reversible physical combination gels were formed in aqueous solution by preparing complex coacervate with two oppositely charged biomacromolecules that composed of negatively charged chondroitin 6-sulfate and positively charged high molecular weight gelatin type A and co-formulating with a negative, thermo-sensitive polysaccharide, methylcellulose containing a salting-out salt, ammonium sulfate. The combination of complex coacervation and a thermo-reversible gel demonstrated synergistic effects on the complex coacervate formation the release rates of model proteins and in situ gel depot formation. Gels indicated sustained release patterns of the protein over 25 days with minimal initial bursts. Optimized novel in situ gel depot systems containing dual advantages of complex coacervation and temperature responsiveness demonstrated a potential for efficient protein drug delivery in terms of high protein loading, sustained protein release, ease of administration, an aqueous environment without toxic organic solvents, and a simple fabrication method.

Red cell aggregation induced by a high molecular weight gelatin plasma substitute.

In the course of clinical investigation of a new higher molecular weight plasma substitute made of gelatin (MW 60,000), severe side reactions were observed in 2 patients. As the erythrocyte sedimentation rate was markedly raised in both patients after the gelatin infusion, it was thought that these reactions might result from the effect of the gelatin solution on red cell aggregation. The effect of gelatin solutions on red cell aggregation was studied <i>in vitro </i>on human blood and <i>in vivo </i>in dogs using the erythrocyte sedimentation rate and an optical density method to assess its degree. It was shown by all methods that the high molecular weight gelatin fraction induced marked red cell aggregation and it has been concluded that the present gelatin solution cannot be recommended for use in clinical practice.

The depolymerising action of pepsin on collagen molecular weights of the component polypeptide chains

Molecular weight studies have been carried out on the polypeptide chains released by thermal denaturation of collagen pretreated with pepsin. Tropocollagen yields a component, α∗, with molecular weight approx. 95 000, this component has a slightly lower weight than α. Pepsin digestion of polymerised collagen fibrils releases some α∗ and some higher-molecular weight components. The ease with which α∗ is formed from these fibrils is greatest in collagen from young tissues and becomes progressively more difficult with age. Since aged collagen after pepsin digestion, yields up to 50% hot soluble gelatin and a readily detectable amount of tyrosyl peptides3, it is suggested that during maturation covalent cross-linkages are introduced, some of which are resistant to pepsin digestion and these latter prevent the solubilisation of single α∗ components. Even though many cross-linkages may be broken, sufficient remain which are resistant to pepsin resulting in an insoluble residue and high-molecular weight gelatin being formed.