Tuning the Envelope Structure of Enzyme Nanoreactors for In Vivo Detoxification of Organophosphates.

Encapsulated phosphotriesterase nanoreactors show their efficacy in the prophylaxis and post-exposure treatment of poisoning by paraoxon. A new enzyme nanoreactor (E-nRs) containing an evolved multiple mutant (L72C/Y97F/Y99F/W263V/I280T) of Saccharolobus solfataricus phosphotriesterase (PTE) for in vivo detoxification of organophosphorous compounds (OP) was made. A comparison of nanoreactors made of three- and di-block copolymers was carried out. Two types of morphology nanoreactors made of di-block copolymers were prepared and characterized as spherical micelles and polymersomes with sizes of 40 nm and 100 nm, respectively. The polymer concentrations were varied from 0.1 to 0.5% (w/w) and enzyme concentrations were varied from 2.5 to 12.5 µM. In vivo experiments using E-nRs of diameter 106 nm, polydispersity 0.17, zeta-potential -8.3 mV, and loading capacity 15% showed that the detoxification efficacy against paraoxon was improved: the LD50 shift was 23.7xLD50 for prophylaxis and 8xLD50 for post-exposure treatment without behavioral alteration or functional physiological changes up to one month after injection. The pharmacokinetic profiles of i.v.-injected E-nRs made of three- and di-block copolymers were similar to the profiles of the injected free enzyme, suggesting partial enzyme encapsulation. Indeed, ELISA and Western blot analyses showed that animals developed an immune response against the enzyme. However, animals that received several injections did not develop iatrogenic symptoms.

Cytochalasin B-Induced Membrane Vesicles from TRAIL-Overexpressing Mesenchymal Stem Cells Induce Extrinsic Pathway of Apoptosis in Breast Cancer Mouse Model.

Tumor-necrosis-factor-associated apoptosis-inducing ligand (TRAIL) is one of the most promising therapeutic cytokines that selectively induce apoptosis in tumor cells. It is known that membrane vesicles (MVs) can carry the surface markers of parental cells. Therefore, MVs are of interest as a tool for cell-free cancer therapy. In this study, membrane vesicles were isolated from TRAIL-overexpressing mesenchymal stem cells using cytochalasin B treatment (CIMVs). To evaluate the antitumor effect of CIMVs-TRAIL in vivo, a breast cancer mouse model was produced. The animals were intratumorally injected with 50 µg of native CIMVs or CIMVs-TRAIL for 12 days with an interval of two days. Then, tumor growth rate, tumor necrotic area, the expression of the apoptosis-related genes CASP8, BCL-2, and BAX and the level of CASP8 protein were analyzed. A 1.8-fold increase in the CAS8 gene mRNA and a 1.7-fold increase in the CASP8 protein level were observed in the tumors injected with CIMVs-TRAIL. The expression of the anti-apoptotic BCL-2 gene in the CIMV-TRAIL group remained unchanged, while the mRNA level of the pro-apoptotic BAX gene was increased by 1.4 times, which indicated apoptosis activation in the tumor tissue. Thus, CIMVs-TRAIL were able to activate the extrinsic apoptosis pathway and induce tumor cell death in the breast cancer mouse model.

Artificial microvesicles: new perspective on healing tendon wounds.

Tendons have a limited capacity to repair both naturally and following clinical interventions. Damaged tissue often presents with structural and functional differences, adversely affecting animal performance, mobility, health and welfare. Advances in cell therapies have started to overcome some of these issues, however complications such as the formation of ectopic bone remain a complication of this technique. Regenerative medicine is therefore looking towards future therapies such as the introduction of microvesicles (MVs) derived from stem cells (SCs). The aim of the present study was to assess the characteristics of artificially derived MVs, from equine mesenchymal stem cells (MSCs), when delivered to rat tendon cells in vitro and damaged tendons in vivo. The initial stages of extracting MVs from equine MSCs and identifying and characterising the cultured tendon stem/progenitor cells (TSCs) from rat Achilles tendons were undertaken successfully. The horse MSCs, and the rat tendon cells, were both capable of differentiating in three directions: adipogenic, osteogenic and chondrogenic pathways. The artificially derived equine MVs successfully fused with the TSC membranes, and no cytotoxic or cytostimulating effects were observed. In addition, co-cultivation of TSCs with MVs lead to stimulation of cell proliferation and migration, and cytokine VEGF and Fractalkine expression levels were significantly increased. These experiments are the first to show that artificially derived MVs exhibited regeneration-stimulating effects in vitro, and that fusion of cytoplasmic membranes from diploid cell lines originating from different species was possible. Explorations in vivo showed accelerated regeneration of injury tendons after introduction of the MVs into damaged areas. The results from the studies performed indicated obvious positive modifying effects following the administration of MVs. This represents the initial successful steps required prior to translating this regenerative medicine technique into clinical trials, such as for tendon repair in injured horses.

Enzyme Nanoreactor for In Vivo Detoxification of Organophosphates.

A nanoreactor containing an evolved mutant of Saccharolobus solfataricus phosphotriesterase (L72C/Y97F/Y99F/W263V/I280T) as a catalytic bioscavenger was made for detoxification of organophosphates. This nanoreactor intended for treatment of organophosphate poisoning was studied against paraoxon (POX). Nanoreactors were low polydispersity polymersomes containing a high concentration of enzyme (20 µM). The polyethylene glycol-polypropylene sulfide membrane allowed for penetration of POX and exit of hydrolysis products. In vitro simulations under second order conditions showed that 1 µM enzyme inactivates 5 µM POX in less than 10 s. LD50-shift experiments of POX-challenged mice through intraperitoneal (i.p.) and subcutaneous (s.c.) injections showed that intravenous administration of nanoreactors (1.6 nmol enzyme) protected against 7 × LD50 i.p. in prophylaxis and 3.3 × LD50 i.p. in post-exposure treatment. For mice s.c.-challenged, LD50 shifts were more pronounced: 16.6 × LD50 in prophylaxis and 9.8 × LD50 in post-exposure treatment. Rotarod tests showed that transitory impaired neuromuscular functions of challenged mice were restored the day of experiments. No deterioration was observed in the following days and weeks. The high therapeutic index provided by prophylactic administration of enzyme nanoreactors suggests that no other drugs are needed for protection against acute POX toxicity. For post-exposure treatment, co-administration of classical drugs would certainly have beneficial effects against transient incapacitation.