RESUMO
PURPOSE: There is a plethora of studies on recombinant human bone morphogenetic protein-2 (rhBMP-2) application and delivery systems, but surprisingly few reports address the biophysical properties of the protein which are of crucial importance to develop effective delivery systems or to solve general problems related to rhBMP-2 production, purification, analysis and application. METHODS: The solubility, stability and bioactivity of rhBMP-2 obtained by renaturation of E. coli derived inclusion bodies was assessed at different pH and in different buffer systems using (dynamic) light scattering and thermal shift assays as well as intrinsic fluorescence measurements and luciferase based bioassays. RESULTS: rhBMP-2 is poorly soluble at physiological pH and higher. The presence of divalent anions further decreases the solubility even under acidic conditions. Thermal stability analyses revealed that rhBMP-2 precipitates are more stable compared to the soluble protein. Moreover, correctly folded rhBMP-2 is also bioactive as precipitated protein and precipitates readily dissolve under appropriate buffer conditions. Once properly formed rhBMP-2 also retains biological activity after temporary exposure to high concentrations of chaotropic denaturants. However, care should be taken to discriminate bioactive rhBMP-2 precipitates from misfolded rhBMP-2 aggregates, e.g. resolvability in MES buffer (pH 5) and a discrete peak in thermoshift experiments are mandatory for correctly folded rhBMP-2. CONCLUSIONS: Our analysis revealed that E. coli derived rhBMP-2 precipitates are not only bioactive but are also more stable compared to the soluble dimeric molecules. Knowledge about these unusual properties will be helpful to design improved delivery systems requiring lower amounts of rhBMP-2 in clinical applications.
Assuntos
Proteína Morfogenética Óssea 2/química , Escherichia coli/química , Fator de Crescimento Transformador beta/química , Heparina/química , Humanos , Concentração de Íons de Hidrogênio , Concentração Osmolar , Oxalatos/química , Tamanho da Partícula , Agregados Proteicos/efeitos dos fármacos , Conformação Proteica , Dobramento de Proteína/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteínas Recombinantes/química , Cloreto de Sódio/química , Solubilidade/efeitos dos fármacos , TemperaturaRESUMO
The emergence of three-dimensional (3D) printing promises a disruption in the design and on-demand fabrication of smart structures in applications ranging from functional devices to human organs. However, the scale at which 3D printing excels is within macro- and microlevels and principally lacks the spatial ordering of building blocks at nanolevels, which is vital for most multifunctional devices. Herein, we employ liquid crystal (LC) inks to bridge the gap between the nano- and microscales in a single-step 3D printing. The LC ink is prepared from mixtures of LCs of nanocellulose whiskers and large sheets of graphene oxide, which offers a highly ordered laminar organization not inherently present in the source materials. LC-mediated 3D printing imparts the fine-tuning required for the design freedom of architecturally layered systems at the nanoscale with intricate patterns within the 3D-printed constructs. This approach empowered the development of a high-performance humidity sensor composed of self-assembled lamellar organization of NC whiskers. We observed that the NC whiskers that are flat and parallel to each other in the laminar organization allow facile mass transport through the structure, demonstrating a significant improvement in the sensor performance. This work exemplifies how LC ink, implemented in a 3D printing process, can unlock the potential of individual constituents to allow macroscopic printing architectures with nanoscopic arrangements.