Cultured meat platform developed through the structuring of edible microcarrier-derived microtissues with oleogel-based fat substitute.

With the increasing global demand for meat, cultured meat technologies are emerging, offering more sustainable solutions that aim to evade a future shortage of meat. Here, we demonstrate a cultured meat platform composed of edible microcarriers and an oleogel-based fat substitute. Scalable expansion of bovine mesenchymal stem cells on edible chitosan-collagen microcarriers is optimized to generate cellularized microtissues. In parallel, an oleogel system incorporated with plant protein is developed as a fat substitute, which is comparable to beef fat in appearance and texture. Combining the cellularized microtissues with the developed fat substitute, two types of cultured meat prototypes are introduced: layered cultured meat and burger-like cultured meat. While the layered prototype benefits enhanced stiffness, the burger-like prototype has a marbling meat-like appearance and a softer texture. Overall, this platform and the established technological basis may contribute to the development of different cultured meat products and promote their commercial production.

Mode of Action of RZL-012, a New Fat-Reducing Molecule.

BACKGROUND: RZL-012 (5-[3,6-dibromo-9H-carbazol-9-yl]-N,N,N-trimethylpentan-1-aminium chloride) is a novel investigational drug injected subcutaneously into fat tissues in patients with fat-related disorders (Dercum disease) or subjects seeking aesthetic changes. OBJECTIVE: Preclinical studies were undertaken to understand RZL-012's mechanism of action. MATERIALS AND METHODS: The effects of RZL-012 were tested in vitro by measuring adipocyte cell killing, membrane integrity, cytosolic calcium, and mitochondrial membrane potential (MMP). In vivo studies in pigs evaluated RZL-012's adipocyte killing effect and measured pig fat thickness in the injected areas. RESULTS: RZL-012 triggered adipocyte cell killing with IC50 values ranging from 25 to 106 µM. RZL-012 demonstrated initial effects on membrane integrity and calcium levels with delayed alterations in MMP. Incubation of RZL-012 with nanoghosts increased membrane permeability, culminating in full membrane destruction. Analysis of injected areas in pigs revealed liponecrosis 24 hours after dosing followed by an inflammatory response and formation of fibrotic tissue. Three months after dosing, an 18% reduction in mean fat thickness was observed in RZL-012 treated pigs. CONCLUSION: RZL-012 destroys adipocytes by directly disrupting cell membrane integrity. Replacement of dead fat tissue by fibrotic tissue enables healing and causes contraction of the injected area. These effects are translated into significant reduction in fat tissue volume.

Evaluation of eliciting activity of peptidil prolyl cys/trans isomerase from Pseudonomas fluorescens encapsulated in sodium alginate regarding plant resistance to viral and fungal pahogens.

Use of chemical pesticides poses a threat for environment and human health, so green technologies of crop protection are of high demand. Some microbial proteins able to activate plant defense mechanisms and prevent the development of resistance in plant pathogens, may be good alternative to chemicals, but practical use of such elicitors is limited due to need to protect them against adverse environment prior their delivery to target receptors of plant cells. In this study we examined a possibility to encapsulate heat resistant FKBP-type peptidyl prolyl cis-trans isomerase (PPIase) from Pseudomonas fluorescens, which possesses a significant eliciting activity in relation to a range of plant pathogens, in sodium alginate microparticles and evaluated the stability of resulted complex under long-term UV irradiation and in the presence of proteinase K, as well as its eliciting activity in three different "plant-pathogen" models comparing to that of free PPIase. The obtained PPIase-containing microparticles consisted of 70% of sodium alginate, 20% of bovine serum albumin, and 10% of PPIase. In contrast to free PPIase, which lost its eliciting properties after 8-h UV treatment, encapsulated PPIase kept its eliciting ability unchanged; after being exposed to proteinase K, its eliciting ability twice exceeded that of free PPIase. Using "tobacco-TMV", "tobacco-Alternaria longipes", and "wheat-Stagonospora nodorum" model systems, we showed that encapsulation process did not influence on the eliciting activity of PPIase. In the case of the "wheat-S. nodorum" model system, we also observed a significant eliciting activity of alginate-albumin complex and almost doubled activity of encapsulated PPIase as compared to the free PPIase. As far as we know, this is the first observation of a synergistic interaction between alginate and other compound possessing any bioactive properties. The results of the study show some prospects for a PPIase use in agriculture.

New Poly(3-hydroxybutyrate) Microparticles with Paclitaxel Sustained Release for Intraperitoneal Administration.

BACKGROUND: Poly(hydroxyalkanoates) (PHA) have recently attracted increasing attention due to their biodegradability and high biocompatibility, which makes them suitable for the development of new prolong drug formulations. OBJECTIVE: This study was conducted to develop new prolong paclitaxel (PTX) formulation based on poly(3- hydroxybutyrate) (PHB) microparticles. METHOD: PHB microparticles loaded with antitumor cytostatic drug PTX were obtained by spray-drying method using Nano Spray Dryer B-90. The PTX release kinetics in vitro from PHB microparticles and their cytotoxity on murine hepatoma cell line MH-22a were studied. Microparticles antitumor activity in vivo was studied using intraperitoneally (i.p.) transplanted tumor models: murine Lewis lung carcinoma and xenografts of human breast cancer RMG1. RESULTS: Uniform PTX release from PHB-microparticles during 2 months was observed. PTX-loaded PHB microparticles have demonstrated a significant antitumor activity versus pure drug both in vitro in murine hepatoma cells and in vivo when administered i.p. to mice with murine Lewis lung carcinoma and xenografts of human breast cancer RMG1. CONCLUSION: The developed technique of PTX sustained delivery from PHB-microparticles has therapeutic potential as prolong anticancer drug formulation.

Preclinical Toxicity of Paclitaxel Biopolymer Formulation.

BACKGROUND: Poly(hydroxyalkanoates) (PHA) have recently attracted increasing attention due to their biodegradability and high biocompatibility, which makes them suitable for the development of new prolong drug formulations. OBJECTIVE: A preclinical toxicology study of paclitaxel biopolymer formulation (PBF) (paclitaxel-loaded poly(3- hydroxybutyrate) (PHB) microparticles) was done in order to assess its safety and to forecast side and toxic effects in a clinical study on patients. METHOD: PHB microparticles loaded with antitumor cytostatic drug PTX were obtained by spray-drying method using Nano Spray Dryer B-90. The comprehensive study of cytotoxicity (on bone marrow stem cells), acute and chronic toxicity, allergenic and pyrogenic properties, histological investigation (in mice, rats and rabbits) of obtained PBF was carried out. RESULTS: The acute toxicity study showed that PBF is much less toxic in equivalent PTX-content doses than PTX in conventional formulation when administered intraperitoneally to mice and rats. However, the chronic toxicity study showed that at intraperitoneal administration PBF has distinct cumulative properties and toxic effects that prevent PBF from clinical testing in current composition. CONCLUSION: Thus, the PBF as a prolong drug needs to correct its parameters for further drug formulation development.

Cell attachment on poly(3-hydroxybutyrate)-poly(ethylene glycol) copolymer produced by Azotobacter chroococcum 7B.

BACKGROUND: The improvement of biomedical properties, e.g. biocompatibility, of poly(3-hydroxyalkanoates) (PHAs) by copolymerization is a promising trend in bioengineering. We used strain Azotobacter chroococcum 7B, an effective producer of PHAs, for biosynthesis of not only poly(3-hydroxybutyrate) (PHB) and its main copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV), but also alternative copolymer, poly(3-hydroxybutyrate)-poly(ethylene glycol) (PHB-PEG). RESULTS: In biosynthesis we used sucrose as the primary carbon source and valeric acid or poly(ethylene glycol) 300 (PEG 300) as additional carbon sources. The chemical structure of PHB-PEG and PHB-HV was confirmed by 1H nuclear-magnetic resonance (1H NMR) analysis. The physico-chemical properties (molecular weight, crystallinity, hydrophilicity, surface energy) and surface morphology of films from PHB copolymers were studied. To study copolymers biocompatibility in vitro the protein adsorption and COS-1 fibroblasts growth on biopolymer films by XTT assay were analyzed. Both copolymers had changed physico-chemical properties compared to PHB homopolymer: PHB-HV and PHB-PEG had less crystallinity than PHB; PHB-HV was more hydrophobic than PHB in contrast to PHB-PEG appeared to have greater hydrophilicity than PHB; whereas the morphology of polymer films did not differ significantly. The protein adsorption to PHB-PEG was greater and more uniform than to PHB and PHB-PEG copolymer promoted better growth of COS-1 fibroblasts compared with PHB homopolymer. CONCLUSIONS: Thus, despite low EG-monomers content in bacterial origin PHB-PEG copolymer, this polymer demonstrated significant improvement in biocompatibility in contrast to PHB and PHB-HV copolymers, which may be coupled with increased protein adsorption and hydrophilicity of PEG-containing copolymer.

The terpolymer produced by Azotobacter chroococcum 7B: effect of surface properties on cell attachment.

The copolymerization of poly(3-hydroxybutyrate) (PHB) is a promising trend in bioengineering to improve biomedical properties, e.g. biocompatibility, of this biodegradable polymer. We used strain Azotobacter chroococcum 7B, an effective producer of PHB, for biosynthesis of not only homopolymer and its main copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV), but also novel terpolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-poly(ethylene glycol) (PHB-HV-PEG), using sucrose as the primary carbon source and valeric acid and poly(ethylene glycol) 300 (PEG 300) as additional carbon sources. The chemical structure of PHB-HV-PEG was confirmed by (1)H nuclear-magnetic resonance analysis. The physico-chemical properties (molecular weight, crystallinity, hydrophilicity, surface energy) of produced biopolymer, the protein adsorption to the terpolymer, and cell growth on biopolymer films were studied. Despite of low EG-monomers content in bacterial-origin PHB-HV-PEG polymer, the terpolymer demonstrated significant improvement in biocompatibility in vitro in contrast to PHB and PHB-HV polymers, which may be coupled with increased protein adsorption, hydrophilicity and surface roughness of PEG-containing copolymer.