Ultrasmall Ferrite Nanoparticles Synthesized via Dynamic Simultaneous Thermal Decomposition for High-Performance and Multifunctional T1 Magnetic Resonance Imaging Contrast Agent.

Large-scale synthesis of monodisperse ultrasmall metal ferrite nanoparticles as well as understanding the correlations between chemical composition and MR signal enhancement is critical for developing next-generation, ultrasensitive T1 magnetic resonance imaging (MRI) nanoprobes. Herein, taking ultrasmall MnFe2O4 nanoparticles (UMFNPs) as a model system, we report a general dynamic simultaneous thermal decomposition (DSTD) strategy for controllable synthesis of monodisperse ultrasmall metal ferrite nanoparticles with sizes smaller than 4 nm. The comparison study revealed that the DSTD using the iron-eruciate paired with a metal-oleate precursor enabled a nucleation-doping process, which is crucial for particle size and distribution control of ultrasmall metal ferrite nanoparticles. The principle of DSTD synthesis has been further confirmed by synthesizing NiFe2O4 and CoFe2O4 nanoparticles with well-controlled sizes of ∼3 nm. More significantly, the success in DSTD synthesis allows us to tune both MR and biochemical properties of magnetic iron oxide nanoprobes by adjusting their chemical composition. Beneficial from the Mn2+ dopant, the synthesized UMFNPs exhibited the highest r1 relaxivity (up to 8.43 mM-1 s-1) among the ferrite nanoparticles with similar sizes reported so far and demonstrated a multifunctional T1 MR nanoprobe for in vivo high-resolution blood pool and liver-specific MRI simultaneously. Our study provides a general strategy to synthesize ultrasmall multicomponent magnetic nanoparticles, which offers possibilities for the chemical design of a highly sensitive ultrasmall magnetic nanoparticle based T1 MRI probe for various clinical diagnosis applications.

The efficiency of magnetic hyperthermia and in vivo histocompatibility for human-like collagen protein-coated magnetic nanoparticles.

Magnetic hyperthermia is a promising technique for the minimally invasive elimination of solid tumors. In this study, uniform magnetite nanoparticles (MNPs) with different particle sizes were used as a model system to investigate the size and surface effects of human-like collagen protein-coated MNPs (HLC-MNPs) on specific absorption rate and biocompatibility. It was found that these HLC-MNPs possess rapid heating capacity upon alternating magnetic field exposure compared to that of MNPs without HLC coating, irrespective of the size of MNPs. The significant enhancement of specific absorption rate is favorable for larger sized nanoparticles. Such behavior is attributed to the reduced aggregation and increased stability of the HLC-MNPs. By coating HLC on the surface of certain sized MNPs, a significant increase in cell viability (up to 2.5-fold) can be achieved. After subcutaneous injection of HLC-MNPs into the back of Kunming mice, it was observed that the inflammatory reaction hardly occurred in the injection site. However, there was a significant presence of phagocytes and endocytosis after the injection of nonconjugated counterparts. The overall strategy to fabricate HLC-MNPs can serve as a general guideline to address the current challenges in clinical magnetic hyperthermia, improved biocompatibility, and enhanced heating characteristics through protein coating.

The selective roles of chaperone systems on over-expression of human-like collagen in recombinant Escherichia coli.

Human-like collagen (HLC) is a novel biomedical material with promising applications. Usually, insoluble HLC was formed due to over-expression. In order to improve the production of soluble HLC, the effective chaperone proteins and their mediation roles on HLC were clarified. Trigger factor (TF) pathway with low specificity and high binding affinity to nascent chains could increase soluble HLC expression; GroEL-GroES could increase the expression level of HLC by assisting the correct folding of HLC and increase mRNA level of the gene coding for HLC by enhancing mRNA stability. DnaK chaperone system did not work positively on soluble HLC due to the unbalanced ratio of DnaK:DnaJ:GrpE, especially too high GrpE significantly inhibited DnaK-mediated refolding. The production of soluble HLC with co-expression of exogenous TF and GroEL-GroES was increased by 35.3 % in comparison with the highest value 0.26 g/L reported previously.

Preparation of a low-cost minimal medium for engineered Escherichia coli with high yield of human-like collagen II.

In order to reduce the production cost of human-like collagen (HLC), a minimal medium was introduced. On the base of Design of experiments (DOE), especially Plackett-Burman design and central composite design, a modified minimal medium that could give a high yield of HLC was developed. The optimum minimal medium for engineered E. coli BL21 ΔptsG contained 6.11g/L of glucose, 5.82g/L of (NH(4))(2)SO(4), 1.80´10(-4)g/L of thiamine and 3.00´10(-2)L of trace element solution, the other ingredients were same to that in M9 medium. And the HLC production of ptsG mutant reached to 0.26g/L in this optimized minimal medium, which approached to 0.27g/L produced by the strain without deleting ptsG gene in an optimized complex medium.

Enhancing human-like collagen accumulation by deleting the major glucose transporter ptsG in recombinant Escherichia coli BL21.

Collagen has been proven to be a valuable biomedical material for many medical applications. Human-like collagen (HLC) is a novel important biomedical material with diverse medical applications. In this work, recombinant Escherichia coli BL21 3.7 ∆ptsG was constructed, the characters of ptsG mutant strain were analyzed, and real-time quantitative polymerase chain reaction (PCR) was applied to investigate the effect of ptsG gene deletion on the transcriptional level of the phosphotransferase system (PTS) genes responsible for glucose transport. The HLC production and cell growth ability were 1.33- and 1.24-fold higher than those of its parent strain in the fermentation medium, respectively, and 1.16- and 1.17-fold in the modified minimal medium individually. The acetate accumulation decreased by 42%-56% compared to its parent strain in the fermentation medium, and 70%-87% in the modified minimal medium. The results of RT-qPCR showed that the transcriptional level of crr, ptsH, ptsI, and blgF in ptsG mutant all decreased dramatically, which inferred a decrease in the glucose uptake rate, but the transcriptional level of FruB and manX increased slightly, which demonstrated the activation of fructose- and mannose-specific transport pathways in the ptsG mutant. This study demonstrates that ptsG deletion is an effective strategy to reduce acetate accumulation and increase biomass and HLC production.

Improving the production of human-like collagen by pulse-feeding glucose during the fed-batch culture of recombinant Escherichia coli.

To increase the target protein production and reduce acetic acid accumulation during fed-batch cultivation of recombinant Escherichia coli BL21 in a 30-L bioreactor, 12 different models of pulse feeding were performed to evaluate the effect of pulse feeding at different cultivation phases and pulse frequency on cell growth, acetic acid accumulation, and human-like collagen (HLC) synthesis. The results showed that the acetate concentration was kept at a low level (below 0.5 g/L) in all cases when pulse feeding was introduced before induction, whereas the pulse frequency affected cytoactivity significantly through cell growth rate, oxygen uptake rate, carbon dioxide evolution rate, and the synthesis of the target protein. The final biomass and HLC reached 75.46 and 7.26 g/L, respectively, in the model of 8-Sec feedings per 188 Sec. After induction, the pulse frequency had a great effect on HLC synthesis after high-temperature induction; low frequency was adverse to microorganisms. The model of 3-Sec feeding per 27 Sec was best and resulted in the highest biomass and HLC production. Compared to the pseudo-exponential feeding, pulse feeding reduced acetic acid accumulation effectively.

A genetic algorithm for the optimization of the thermoinduction protocol for high-level production of recombinant human-like collagen from Escherichia coli.

Production of recombinant human-like collagen (RHLC) by thermoinduction of recombinant Escherichia coli BL 21 during high cell density cultivation was investigated in a 30 L bioreactor. The effects of induction temperature (T), pH, and carbon-to-nitrogen molar ratio of the nutrient medium (C/N) were examined. The optimal thermoinduction protocol for RHLC production was determined by using a model coupling genetic algorithm and artificial neural networks. The optimal operating conditions were as follows: maintenance of induction temperature at 42°C for 3 H and then at 39.4°C until the end, induction pH at 7.03, and C/N at 4.8 (mol/mol). The theoretical maximum concentration of RHLC was 12.5 g/L, whereas the experimental value was 12.1 g/L under the optimal induction conditions.

Medium optimization based on the metabolic-flux spectrum of recombinant Escherichia coli for high expression of human-like collagen II.

Recombinant Escherichia coli BL21 was used to produce human-like collagen II in fed-batch cultivation. By performing MFA (metabolic-flux analysis), the carbon/nitrogen molar ratios in both the batch and feeding media were optimized for high-level production of human-like collagen II. Three carbon/nitrogen molar ratios in both the batch and feeding media were used in the present study, and the MFA results showed that the optimal carbon/nitrogen molar ratios for the batch and feeding media were 2.36:1 and 5.12:1 respectively, yielding the highest dry-cell density (67.2 g/l dry cell weight) and human-like collagen production (10.8 g/l).

Oxygen transfer rate control in the production of human-like collagen by recombinant Escherichia coli.

The effects of different methods for elevating the OTR (oxygen transfer rate) during foreign gene expression and the cell growth of recombinant Escherichia coli BL21 were investigated. Two strategies were introduced to control DO (dissolved oxygen) levels in the fermentation broth: (i) increasing fermentor pressure and (ii) supplying oxygen-enriched air. These two methods were compared with the glucose feedback model, which acted as the control. By adopting a fed-batch method of cultivation, the cell yield coefficient (YX/S), accumulation of acetic acid and volumetric product yield (Yp) were measured or estimated. Adoption of these two methods led to an improvement in the OTR. The cell density and volumetric product yield in the cultivation controlled by increasing the fermentor pressure reached 77 g x l(-1) (dry cell weight) and 14 g x l(-1) respectively, which were much higher than those obtained with the strategy of supplying oxygen-enriched air (48 and 6 g x l(-1) respectively) and in the control (46 and 7 g x l(-1) respectively). The results indicate that increasing fermentor pressure is an effective way to enhance the OTR and recombinant protein (human-like collagen) productivity.

Breakthrough model of recombinant human-like collagen in immobilized metal affinity chromatography.

The adsorption of recombinant human-like collagen by metal chelate media was investigated in a batch reactor and in a fixed-bed column. The adsorption equilibrium and kinetics had been studied by batch adsorption experiments. Equilibrium parameters and protein diffusivities were estimated by matching the models with the experimental data. Using the parameters of equilibrium and kinetics, various models, such as axial diffusion model, linear driving force model, and constant pattern model, were used to simulate the breakthrough curves on the columns. As a result, the most suitable isotherm was the Langmuir-Freundlich model, and the ionic strength had no effect on the adsorption capacity of chelate media. In addition, the pore diffusion model fitted very well to the kinetic data. The pore diffusivities decreased with increasing the initial protein concentration, however had little change with the ionic strength. The results also indicated that the models predict breakthrough curves reasonably well to the experimental data, especially at low initial protein concentration (0.3 mg ml(-1)) and low flow rate (34 cm h(-1)). By the results, we optimized the experimental conditions of a chromatographic process using immobilized metal affinity chromatography to purify recombinant human-like collagen.

Analysis of metabolic flux in Escherichia coli expressing human-like collagen in fed-batch culture.

Metabolic flux distributions of recombinant Escherichia coli BL21 expressing human-like collagen were determined by means of a stoichiometric network and metabolic balancing. At the batch growth stage, the fluxes of the pentose phosphate pathway were higher than the fluxes of the fed-batch growth phase and the production stage. After the temperature was increased, there was a substantially elevated energy demand for synthesizing human-like collagen and heat-shock proteins, which resulted in changes in metabolic fluxes. The activities of the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid cycle were significantly enhanced, leading to a reduction in the fluxes of the pentose phosphate pathway and other anabolic pathways. The temperature upshift also caused an increase in NADPH production by isocitrate dehydrogenase in the tricarboxylic acid cycle. The metabolic model predicted the involvement of a transhydrogenase that generates additional NADH from NADPH, thereby increasing ATP regeneration in the respiratory chain. These data indicated that the maintenance energy for cellular activity increased with the increase in biomass in fed-batch culture, and that cell growth and synthesis of human-like collagen could clearly represent the changes in metabolic fluxes. At the production stage, more NADPH was used to synthesize human-like collagen than for maintaining cellular activity, cell growth, and cell propagation.