Synthesis of Ni2+-functionalized polydopamine magnetic beads for facilitated purification of histidine-tagged proteins.

Facilitated purification of proteins, at a low cost and a short time, is one of the key steps in the industrial production of recombinant proteins. In the current study, polydopamine nanoparticles (PDA-NPs) are considered in the synthesis of magnetic beads for purifying recombinant proteins due to advantages such as biocompatibility/ biodegradability, easy synthesis, as well as the ability to directly chelate metal ions. They were synthesized in Tris buffer (pH: 8:5), then chelated with Fe3+(20 mg) and Ni2+ ions at concentrations of 2, 3, 5, and 7 mg/ml. Prepared nanoparticles were characterized through scanning electron microscopy (SEM), ultraviolet-visible spectroscopy (UV-vis), dynamic light scattering (DLS), Inductively Coupled Plasma (ICP), and vibrating sample magnetometer (VSM). The size distribution of the particles was reported in the narrow range of 120-140 nm and 200 to 220 nm by the SEM image and DLS analysis, respectively. The chelation of ions on the surface of the nanoparticle was confirmed by the ICP technique with a magnetization of 35.42 emu/g. The highest adsorption rate of Ni2+ ions to polydopamine was obtained at a ratio of 1.4. The SDS-PAGE and western blot analysis confirmed the purification of eGFP and Hsp40 by PDA/Fe3+/Ni2+ at 26 and 40 kDa compared to the commercial nickel column. Moreover, the concentration of purified eGFP by PDA/Fe3+/Ni2+ was reported 138.83 µg/ml by the fluorescent signals, which is almost equal to or more than the protein purified by commercial Ni-NTA column (108.28 µg/ ml). The stability of PDA/Fe3+/Ni2+ has also been evaluated by ICP-OES for 10 days, and the result suggested that PDA magnetic beads were stable. Therefore, it can be concluded that PDA/Fe3+/Ni2+ have the ability to purify recombinant proteins in one less step and shorter time.

Synthesis and characterization of Gd3+-loaded hyaluronic acid-polydopamine nanoparticles as a dual contrast agent for CT and MRI scans.

Magnetic resonance imaging and computed tomography (CT) suffer from low contrast sensitivity and potential toxicity of contrast agents. To overcome these limitations, we developed and tested a new class of dual contrast agents based on polydopamine nanoparticles (PDA-NPs) that are functionalized and targeted with hyaluronic acid (HA). These nanoparticles (NPs) are chelated with Gd3+ to provide suitable contrast. The targeted NPs were characterized through ultraviolet-visible spectroscopy (UV-vis), scanning electron microscopy (SEM), infrared Fourier transform (FTIR), and dynamic light scattering (DLS). The cytotoxicity was investigated on HEK293 cells using an MTT assay. The contrast property of synthesized Gd3+/PDA/HA was compared with Barium sulfate and Dotarem, as commercial contrast agents (CAs) for CT and MRI, respectively. The results illustrated that synthesized PDA-NPs have a spherical morphology and an average diameter of 72 nm. A distinct absorption peak around 280 nm in the UV-vis spectrum reported the self-polymerization of PDA-NPs. The HA coating on PDA-NPs was revealed through a shift in the FTIR peak of C=O from 1618 cm-1 to 1635 cm-1. The Gd3+ adsorption on PDA/HA-NPs was confirmed using an adsorption isotherm assay. The developed CA showed low in vitro toxicity (up to 158.98 µM), and created a similar contrast in MRI and CT when compared to the commercial agents. The r1 value for PDA/HA/Gd3+ (6.5 (mg/ml)-1 s-1) was more than Dotarem (5.6 (mg/ml)-1 s-1) and the results of the hemolysis test showed that at concentrations of 2, 4, 6, and 10 mg/ml, the hemolysis rate of red blood cells is very low. Additionally, the results demonstrated that PDA/HA/Gd3+ could better target the CD44+-expressing cancer cells than PDA/Gd3+. Thus, it can be concluded that lower doses of developed CA are needed to achieve similar contrast of Dotarem, and the developed CA has no safety concerns in terms of hemolysis. The stability of PDA/HA/Gd3+ has also been evaluated by ICP-OES, zeta potential, and DLS during 3 days, and the results suggested that Gd-HA NPs were stable.

Indirect optimization of staphylokinase expression level in dicistronic auto-inducible system.

Design of experiment (DOE) is a statistical approach for designing, performing, and interpreting a large set of data with the minimum number of tests. In our previous study, we developed a novel Hsp27 SILEX system for production of recombinant proteins. In the present study, we optimized indirectly the most effective factors including inoculation load, self-induction temperature, and culture media on autoinduction of staphylokinase (SAK) expression using RSM methodology and fluorometry. The expression level of SAK was assayed at different runs after 6 h incubation at 90 rpm. The results indicated all parameters significantly affect the SAK expression level (p < 0.05). The optimum expression condition was obtained with an inoculation load of 0.05, a temperature of 25 °C, and TB culture medium. The analysis of variance with a R2 value of 0.91 showed that a quadratic model well described this prediction (p < 0.05). Applying the optimized condition led to an approximately fourfold increase in the SAK expression level (from 1.3 to 5.2 µg/ml). Moreover, the recombinant protein was purified using immobilized metal affinity chromatography and the activity was also confirmed by semi-quantitative caseinolytic method.

Towards principled design of cancer nanomedicine to accelerate clinical translation.

Nanotechnology in medical applications, especially in oncology as drug delivery systems, has recently shown promising results. However, although these advances have been promising in the pre-clinical stages, the clinical translation of this technology is challenging. To create drug delivery systems with increased treatment efficacy for clinical translation, the physicochemical characteristics of nanoparticles such as size, shape, elasticity (flexibility/rigidity), surface chemistry, and surface charge can be specified to optimize efficiency for a given application. Consequently, interdisciplinary researchers have focused on producing biocompatible materials, production technologies, or new formulations for efficient loading, and high stability. The effects of design parameters can be studied in vitro, in vivo, or using computational models, with the goal of understanding how they affect nanoparticle biophysics and their interactions with cells. The present review summarizes the advances and technologies in the production and design of cancer nanomedicines to achieve clinical translation and commercialization. We also highlight existing challenges and opportunities in the field.

Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system.

BACKGROUND: Identification of high-expressing colonies is one of the main concerns in the upstream process of recombinant protein development. The common method to screen high-producing colonies is SDS-PAGE, a laborious and time-consuming process, which is based on a random and qualitative way. The current study describes the design and development of a rapid screening system composed of a dicistronic expression system containing a reporter (enhanced green fluorescent protein, eGFP), protein model (staphylokinase, SAK), and a self-inducible system containing heat shock protein 27 (Hsp27). RESULTS: Dicistronic-autoinducible system expressed eGFP and SAK successfully in 5-ml and 1-L culture volumes. High expressing colonies were identified during 6 h via fluorescent signals. In addition, the biological activity of the protein model was confirmed semi-quantitatively and quantitatively through radial caseinolytic and chromogenic methods, respectively. There was a direct correlation between eGFP fluorescent intensity and SAK activity. The correlation and linearity of expression between the two genes were respectively confirmed with Pearson correlation and linear regression. Additionally, the precision, limit of detection (LOD), and limit of quantification (LOQ) were determined. The expression of eGFP and SAK was stable during four freeze-thaw cycles. In addition, the developed protocol showed that the transformants can be inoculated directly to the culture, saving time and reducing the error-prone step of colony picking. CONCLUSION: The developed system is applicable for rapid screening of high-expressing colonies in most research laboratories. This system can be investigated for other recombinant proteins expressed in E. coli with a potential capability for automation and use at larger scales.

Comparison of E. coli based self-inducible expression systems containing different human heat shock proteins.

IPTG-inducible promoter is popularly used for the expression of recombinant proteins. However, it is not suitable at the industrial scale due to the high cost and toxicity on the producing cells. Recently, a Self-Inducible Expression (SILEX) system has developed to bypass such problems using Hsp70 as an autoinducer. Herein, the effect of other heat shock proteins on the autoinduction of green fluorescent protein (EGFP), romiplostim, and interleukin-2 was investigated. For quantitative measurements, EGFP expression was monitored after double-transformation of pET28a-EGFP and pET21a-(Hsp27/Hsp40/Hsp70) plasmids into E. coli using fluorimetry. Moreover, the expression level, bacterial growth curve, and plasmid and expression stability were compared to an IPTG- inducible system using EGFP. Statistical analysis revealed a significant difference in EGFP expression between autoinducible and IPTG-inducible systems. The expression level was higher in Hsp27 system than Hsp70/Hsp40 systems. However, the highest amount of expression was observed for the inducible system. IPTG-inducible and Hsp70 systems showed more lag-time in the bacterial growth curve than Hsp27/Hsp40 systems. A relatively stable EGFP expression was observed in SILEX systems after several freeze-thaw cycles within 90 days, while, IPTG-inducible system showed a decreasing trend compared to the newly transformed bacteria. Moreover, the inducible system showed more variation in the EGFP expression among different clones than clones obtained by SILEX systems. All designed SILEX systems successfully self-induced the expression of protein models. In conclusion, Hsp27 system could be considered as a suitable autoinducible system for protein expression due to less metabolic burden, lower variation in the expression level, suitable plasmid and expression stability, and a higher expression level.