Self-Assembly or Coassembly of Multiresponsive Histidine-Containing Elastin-Like Polypeptide Block Copolymers.

In this study a histidine containing elastin-like polypeptide (ELP) diblock copolymer is described with multiresponsive assembly behavior. Self-assembly into micelles is examined by two methods. First, the self-assembly is triggered by the addition of divalent metal ions, with Zn2+ being the most suitable one. Increasing the Zn2+ concentration stabilizes the nanoparticles over a large temperature window (4-45 °C). This diblock exhibits furthermore pH-responsiveness, and particles disassemble under mildly acidic conditions. Second, the coassembly of this ELP with a diblock ELP is examined, which is not responsive to pH and metal ions. Coassembly is triggered by heating the ELPs quickly above the transition temperature of the less hydrophobic block, which results in stable nanoparticles without the need to add metal ions. This novel ELP system offers a versatile modular nanocarrier platform that can respond to different stimuli and can be tuned effectively.

Nanoparticles based on natural, engineered or synthetic proteins and polypeptides for drug delivery applications.

Medicine formulations at the nanoscale, referred to as nanomedicines, have managed to overcome key challenges encountered during the development of new medical treatments and entered clinical practice, but considerable improvement in terms of local efficacy and reduced toxicity still need to be achieved. Currently, the fourth-generation of nanomedicines is being developed, employing biocompatible nanocarriers that are targeted, multifunctional, and stimuli-responsive. Proteins and polypeptides can fit the standards of an efficient nanovector because of their biodegradability, intrinsic bioactivity, chemical reactivity, stimuli-responsiveness, and ability to participate in complex supramolecular assemblies. These biomacromolecules can be obtained from natural resources, produced in heterologous hosts, or chemically synthesized, allowing for different designs to access suitable carriers for a variety of drugs. To enhance targeting or therapeutic functionality, additional chemical modifications can be applied. This review demonstrates the potential of polypeptide and protein materials for the design of drug delivery nanocarriers with a special focus on their preclinical evaluation in vitro and in vivo.

Compartmentalized cross-linked enzymatic nano-aggregates (c-CLEnA) for efficient in-flow biocatalysis.

Nano-sized enzyme aggregates, which preserve their catalytic activity are of great interest for flow processes, as these catalytic species show minimal diffusional issues, and are still sizeable enough to be effectively separated from the formed product. The realization of such catalysts is however far from trivial. The stable formation of a micro-to millimeter-sized enzyme aggregate is feasible via the formation of a cross-linked enzyme aggregate (CLEA); however, such a process leads to a rather broad size distribution, which is not always compatible with microflow conditions. Here, we present the design of a compartmentalized templated CLEA (c-CLEnA), inside the nano-cavity of bowl-shaped polymer vesicles, coined stomatocytes. Due to the enzyme preorganization and concentration in the cavity, cross-linking could be performed with substantially lower amount of cross-linking agents, which was highly beneficial for the residual enzyme activity. Our methodology is generally applicable, as demonstrated by using two different cross-linkers (glutaraldehyde and genipin). Moreover, c-CLEnA nanoreactors were designed with Candida antarctica Lipase B (CalB) and Porcine Liver Esterase (PLE), as well as a mixture of glucose oxidase (GOx) and horseradish peroxidase (HRP). Interestingly, when genipin was used as cross-linker, all enzymes preserved their initial activity. Furthermore, as proof of principle, we demonstrated the successful implementation of different c-CLEnAs in a flow reactor in which the c-CLEnA nanoreactors retained their full catalytic function even after ten runs. Such a c-CLEnA nanoreactor represents a significant step forward in the area of in-flow biocatalysis.

Erythrocyte Membrane Modified Janus Polymeric Motors for Thrombus Therapy.

We report the construction of erythrocyte membrane-cloaked Janus polymeric motors (EM-JPMs) which are propelled by near-infrared (NIR) laser irradiation and are successfully applied in thrombus ablation. Chitosan (a natural polysaccharide with positive charge, CHI) and heparin (glycosaminoglycan with negative charge, Hep) were selected as wall materials to construct biodegradable and biocompatible capsules through the layer-by-layer self-assembly technique. By partially coating the capsule with a gold (Au) layer through sputter coating, a NIR-responsive Janus structure was obtained. Due to the asymmetric distribution of Au, a local thermal gradient was generated upon NIR irradiation, resulting in the movement of the JPMs through the self-thermophoresis effect. The reversible "on/off" motion of the JPMs and their motile behavior were easily tuned by the incident NIR laser intensity. After biointerfacing the Janus capsules with an erythrocyte membrane, the EM-JPMs displayed red blood cell related properties, which enabled them to move efficiently in relevant biological environments (cell culture, serum, and blood). Furthermore, this therapeutic platform exhibited excellent performance in ablation of thrombus through photothermal therapy. As man-made micromotors, these biohybrid EM-JPMs hold great promise of navigating in vivo for active delivery while overcoming the drawbacks of existing synthetic therapeutic platforms. We expect that this biohybrid motor has considerable potential to be widely used in the biomedical field.

Expression of synthetic human tropoelastin (hTE) protein in Nicotiana tabacum.

Plant molecular farming (PMF) is an important growing prospective approach in plant biotechnology; it includes production of recombinant pharmaceutical and industrial proteins in large quantities from engineered plants. Elastin is a major protein component of tissues that require elasticity, it helps keep skin smooth as it stretches to allow normal. Elastin is used as a raw material for the cosmetic industry. In this work, we aimed to use plant as a bioreactor for the expression and production of the full human tropoelastin protein. Agrobacterium- mediated transient expression system into Nicotiana tabacum using syringe agroinfiltration was used to provide fast and convenient way to produce recombinant proteins with greater expression overall the plant leaf. This study aimed to establish an efficient and rapid system for transiently expression and production of human recombinant tropoelastin protein in transgenic N. tabacum plants. Modified elastin (ELN) gene was biosynthesized and cloned into pCambia1390 vector to be used into N. tabacum agroinfilteration. Optimization of codon usage for the human tropoelastin gene, without changing the primary structure of the protein was carried out to ensure high expression in tobacco plants. The obtained data proved that the 5(th) day post-infiltration is the optimum interval to obtain the maximum production of our recombinant protein. Southern blot analysis was able to detect 2175 bp fragment length representing the ELN orf (open reding frame). On the other hand, ELN -expression within plant's tissue was visualized by RT-PCR during the period 3-10 days post agroinfiltration. At the protein level, western and ELISA confirmed the expression of recombinant tropoelastin protein. Western blot analysis detected the tropoelastin protein as parent band at ∼70 kDa from freshly extracted protein, while two degraded bands of ∼55 and ∼45 kDa, representing a pattern of tropoelastin were appeared with frozen samples. This study showed that biosynthetic ELN gene was successfully expressed into N. tabacum leaves using agroinfiltration technique.