Protein Entrapment Research Articles

Translation stalling induced mitochondrial entrapment of ribosomal quality control related proteins offers cancer cell vulnerability.

Ribosome-associated quality control (RQC) monitors ribosomes for aberrant translation. While the role of RQC in neurodegenerative disease is beginning to be appreciated, its involvement in cancer is understudied. Here, we show a positive correlation between RQC proteins ABCE1 and ZNF598 and high-grade muscle-invasive bladder cancer. Translational stalling by the inhibitor emetine (EME) leads to increased mitochondrial localization of RQC factors including ABCE1, ZNF598, and NEMF, which are continuously imported into mitochondria facilitated by increased mitochondrial membrane potential caused by EME. This reduces the availability of these factors in the cytosol, compromising the effectiveness of RQC in handling stalled ribosomes in the cytosol and those associated with the mitochondrial outer membrane (MOM). Imported RQC factors form aggregates inside the mitochondria in a process we term stalling-induced mitochondrial stress (SIMS). ABCE1 plays a crucial role in maintaining mitochondrial health during SIMS. Notably, cancer stem cells (CSCs) exhibit increased expression of ABCE1 and consequently are more resistant to EME-induced mitochondrial dysfunction. This points to a potential mechanism of drug resistance by CSCs. Our study highlights the significance of mitochondrial entrapment of RQC factors such as ABCE1 in determining the fate of cancer cells versus CSCs. Targeting ABCE1 or other RQC factors in translational inhibition cancer therapy may help overcome drug resistance.

Stimuli-Responsive Hydrogels for Protein Delivery.

Proteins and peptides are potential therapeutic agents, but their physiochemical properties make their use as drug substances challenging. Hydrogels are hydrophilic polymeric networks that can swell and retain high amounts of water or biological fluids without being dissolved. Due to their biocompatibility, their porous structure, which enables the transport of various peptides and proteins, and their protective effect against degradation, hydrogels have gained prominence as ideal carriers for these molecules' delivery. Particularly, stimuli-responsive hydrogels exhibit physicochemical transitions in response to subtle modifications in the surrounding environment, leading to the controlled release of entrapped proteins or peptides. This review is focused on the application of these hydrogels in protein and peptide delivery, including a brief overview of therapeutic proteins and types of stimuli-responsive polymers.

Do the retinal abnormalities in X-linked juvenile retinoschisis include impaired phototransduction?

X-linked juvenile retinoschisis (XLRS), a hereditary retinal disorder primarily affecting males, is characterized by the formation of cystic spaces between the outer plexiform layer and outer nuclear layer of the retina. Mutations in the RS1 gene, which encodes the extracellular binding protein retinoschisin, are responsible for XLRS pathogenesis. While the role of retinoschisin in maintaining retinal integrity is well established, there is growing evidence suggesting compromised photoreceptor function in XLRS. To investigate the molecular pathways affected by RS1 deficiency, particularly in phototransduction, we performed electroretinographic (ERG) and proteomic analyses on retinae from Rs1 knockout mice, a model of human XLRS. The Rs1 knockout mice had reduced ERG a-wave amplitudes. Correspondingly, differential expression analysis revealed downregulation of proteins crucial for phototransduction, with Ingenuity Pathway Analysis (IPA) highlighting “phototransduction” as the most significantly downregulated biological theme. Compensatory mechanisms were also observed in the IPA, including upregulation of synaptic remodeling, inflammation, cell adhesion, and G-protein signaling. These findings strongly implicate an underrecognized role of photoreceptor dysfunction in XLRS pathology. We speculate that entrapment of mutant retinoschisin protein within photoreceptor inner segments as well as disrupted reciprocal regulation between L-type voltage-gated calcium channels and retinoschisin contribute to the dysfunction in photoreceptors.

Fabrication and Evaluation of a pH-Responsive Nanocomposite-Based Colonic Delivery System for Improving the Oral Efficacy of Liraglutide.

Oral administration of liraglutide, a protein drug, suffers from low intestinal absorption and instability in the gastrointestinal tract, resulting in low bioavailability. The present study aimed to develop a pH-responsive nanocomposite based-colonic delivery system to improve the oral efficacy of liraglutide. Nanocomplex (AC-Lira) between aminoclay and liraglutide was prepared by a spontaneous self-assembly. After surface charge reversal using citric acid, AC-Lira was coated with poly(methacrylic acid-co-methyl methacrylate) (1:2). The fabricated nanocomplex underwent various in vitro studies to characterize its physicochemical properties, drug release, and cellular transport. In vivo efficacy studies were also conducted using streptozotocin-induced diabetic rats. Both uncoated (AC-Lira) and coated nanocomplex (EAC-Lira) achieved high entrapment efficiency (> 90%) and showed a narrow size distribution. While exhibiting low drug release at pH 1.2 (approximately 30%), EAC-Lira achieved rapid and extensive drug release (~90%) at pH 7.4, displaying pH-dependent drug release. EAC-Lira showed significant size reduction and surface charge reversal during dissolution at pH 7.4, probably due to the removal of the outer coating layer. Furthermore, EAC-Lira was effective at protecting the entrapped proteins against enzymatic degradation. EAC-Lira also increased the membrane transport of liraglutide by 3.5 folds in Caco-2 cells. Owing to enhanced membrane transport and metabolic stability, EAC-Lira improved in vivo efficacy of orally administered liraglutide, significantly reducing blood glucose concentrations, intake of food and water, and body weight in type 2 diabetes rats. These results suggest EAC-Lira is a promising approach to improving the oral bioavailability and efficacy of liraglutide.

Characterization of binding by sulfonylureas with normal or modified human serum albumin using affinity microcolumns prepared by entrapment

Modification of proteins can occur during diabetes due to the formation of advanced glycation end-products (AGEs) with reactive dicarbonyls such as glyoxal (Go) and methylglyoxal (MGo). Human serum albumin (HSA) is a serum protein that binds to many drugs in blood and that is known to be modified by Go and MGo. This study examined the binding of various sulfonylurea drugs with these modified forms of HSA by using high-performance affinity microcolumns prepared by non-covalent protein entrapment. Zonal elution experiments were employed to compare the retention and overall binding constants for the drugs with Go- or MGo-modified HSA vs normal HSA. The results were compared to values from the literature, such as measured or estimated using affinity columns containing covalently immobilized HSA or biospecifically-adsorbed HSA. The entrapment-based approach provided estimates of global affinity constants within 3–5 min for most of the tested drugs and with typical precisions of ±10–23%. Each entrapped protein microcolumn was stable for over at least 60–70 injections and one month of use. The results obtained with normal HSA agreed at the 95% confidence level with global affinity constants that have been reported for the given drugs in the literature. It was found for HSA that had been modified with clinically-relevant levels of either Go or MGo that an increase in the global affinity constant of up to 2.1-fold occurred for some of the tested drugs. The information acquired in this study can be used in the future to adapt this entrapment-based approach to study and evaluate interactions between other types of drugs and normal or modified binding agents for clinical testing and biomedical research.

Uncovering tau in wasteosomes (corpora amylacea) of Alzheimer's disease patients.

Brain corpora amylacea, recently renamed as wasteosomes, are polyglucosan bodies that appear during aging and some neurodegenerative conditions. They collect waste substances and are part of a brain cleaning mechanism. For decades, studies on their composition have produced inconsistent results and the presence of tau protein in them has been controversial. In this work, we reanalyzed the presence of this protein in wasteosomes and we pointed out a methodological problem when immunolabeling. It is well known that to detect tau it is necessary to perform an antigen retrieval. However, in the case of wasteosomes, an excessive antigen retrieval with boiling dissolves their polyglucosan structure, releases the entrapped proteins and, thus, prevents their detection. After performing an adequate pre-treatment, with an intermediate time of boiling, we observed that some brain wasteosomes from patients with Alzheimer's disease (AD) contained tau, while we did not detect tau protein in those from non-AD patients. These observations pointed the different composition of wasteosomes depending on the neuropathological condition and reinforce the role of wasteosomes as waste containers.

Proteomics of fibrin amyloid microclots in long COVID/post-acute sequelae of COVID-19 (PASC) shows many entrapped pro-inflammatory molecules that may also contribute to a failed fibrinolytic system

BackgroundPost-acute sequelae of COVID-19 (PASC), also now known as long COVID, has become a major global health and economic burden. Previously, we provided evidence that there is a significant insoluble fibrin amyloid microclot load in the circulation of individuals with long COVID, and that these microclots entrap a substantial number of inflammatory molecules, including those that might prevent clot breakdown. Scientifically, the most challenging aspect of this debilitating condition is that traditional pathology tests such as a serum CRP (C-reactive protein) may not show any significant abnormal inflammatory markers, albeit these tests measure only the soluble inflammatory molecules. Elevated, or abnormal soluble biomarkers such as IL-6, D-Dimer or fibrinogen indicate an increased risk for thrombosis or a host immune response in COVID-19. The absence of biomarkers in standard pathology tests, result in a significant amount of confusion for patients and clinicians, as patients are extremely sick or even bed-ridden but with no regular identifiable reason for their disease. Biomarkers that are currently available cannot detect the molecules present in the microclots we identified and are therefore unable to confirm their presence or the mechanisms that drive their formation.MethodsHere we analysed the protein content of double-digested microclots of 99 long COVID patients and 29 healthy controls. The patients suffering from long COVID reported their symptoms through a questionnaire completed by themselves or their attending physician.ResultsOur long COVID cohort’s symptoms were found to be in line with global findings, where the most prevalent symptoms were constant fatigue (74%,) cognitive impairment (71%) and depression and anxiety (30%). Our most noteworthy findings were a reduced level of plasma Kallikrein compared to our controls, an increased level of platelet factor 4 (PF4) von Willebrand factor (VWF), and a marginally increased level of α-2 antiplasmin (α-2-AP). We also found a significant presence of antibodies entrapped inside these microclots.ConclusionOur results confirm the presence of pro-inflammatory molecules that may also contribute to a failed fibrinolysis phenomenon, which could possibly explain why individuals with long COVID suffer from chronic fatigue, dyspnoea, or cognitive impairment. In addition, significant platelet hyperactivation was noted. Hyperactivation will result in the granular content of platelets being shed into the circulation, including PF4. Overall, our results provide further evidence of both a failed fibrinolytic system in long COVID/PASC and the entrapment of many proteins whose presence might otherwise go unrecorded. These findings might have significant implications for individuals with pre-existing comorbidities, including cardiovascular disease and type 2 diabetes.

Modeling and Optimization of β-Galactosidase Entrapping in Polydimethylsiloxane-Modified Silica Composites.

Protein entrapment has multiple applications in enzymatic hydrolysis, drug delivery, etc. Here, we report the studies that successfully utilized the Box–Behnken design to model and optimize the parameters of β-galactosidase entrapment in sol–gel-derived silica composites. We have also demonstrated the influence of polymer–polydimethylsiloxane as a composite modifying agent on the activity of entrapped enzymes. We have determined how different sol-gel process parameters influence the activity of entrapped enzymes. The highest impact on β-galactosidase activity was exerted by the water:tetramethoxysilane ratio, followed by polydimethylsiloxane content. Optimized synthesis parameters have been utilized to obtain a composite with maximum β-galactosidase activity. Performed porosity studies have shown that the addition of polydimethylsiloxane increased the pore diameter. Microscopy studies demonstrated that polydimethylsiloxane-modified composites are softer and less rough. Studies of β-galactosidase activity using the o-NPG test showed statistically significant shifts in the enzyme temperature and pH profiles compared to the soluble form. An improvement in the reusability of the enzyme and a significant increase in the thermal stability was also observed. When lactose was used, a strong correlation was observed between the substrate concentration and the type of the catalyzed reaction. Moreover, we have demonstrated that the yields and rates of both lactose hydrolysis and galactooligosaccharides formation were correlated with reaction temperature and with the presence of polydimethylsiloxane. All these findings provide the opportunity for industrial use of optimized PDMS-modified silica composites in lactose elimination from dairy products, e.g., milk or whey.

Entrapment of Proteins Within Columns for High-Performance Affinity Chromatography.

Entrapment is a noncovalent immobilization method that enables a large biological binding agent, such as a protein, to be put within a support without modifying the structure of the binding agent. This chapter describes an on-column entrapment method that can be used with proteins and HPLC-grade silica to prepare columns for high-performance liquid chromatography. In this method, a protein is trapped within a dihydrazide-activated silica support by using oxidized glycogen as a capping agent. This method allows the protein to be placed within the support in a soluble form and with little or no loss of activity. The approach and reagents needed for this method are described in this chapter, along with some applications reported for columns that have been made using on-column protein entrapment.

Quality Control of Proteins Solubilized from Inclusion Bodies.

Despite substantial development of production and purification protocols for heterologous recombinant proteins, some proteins are difficult to produce or, when produced, are accumulated in inclusion bodies (IBs). Nondenaturing protocols can be used to recover the entrapped protein from these protein aggregates. In this chapter, we provide a detailed procedure to analyze the physicochemical properties of one of those proteins produced in prokaryotic expression systems. Serum amyloid A3 (SAA3) was recovered from inclusion bodies (IBs) and itssecondary structure associated to thermal stability and size was determined by circular dichroism (CD) and dynamic light scattering (DLS), respectively. These techniques were also applied to evaluate the SAA3 interaction with model membranes. These results show the importance of the structural analysis of proteins released from inclusion bodies under nondenaturing procedures, although similar approaches can be extended to any type of recombinant protein preparation.

A review on multivesicular liposomes for pharmaceutical applications: preparation, characterization, and translational challenges.

Multivesicular liposomes (MVLs) are non-concentric, lipid-based micron-sized spherical particles. The usage of MVL for sustained drug delivery has seen progression over the last decade due to successful clinical and commercial applications. It provides attractive characteristics, such as high encapsulation efficiency, variety of sizes, structural stability, and different choices for the route of administration. Drug molecules are encapsulated in internal aqueous compartments of MVL, separated by lipid bilayer septa to form polyhedral structures. The integrity of these entrapped small molecules, peptides, or proteins is maintained throughout the therapy, thus providing sustained drug release on non-vascular administration. Despite the frequent use of unilamellar liposomes, characterization of MVLs is critical due to different puzzling problems, such as real-time size evaluation, initial burst, and in vivo performance. Moreover, available regulatory guidelines on liposomal drug product development are insufficient to assure ample in vitro-in vivo behavior of MVL. This review hereby highlights the innovations pertaining to development and manufacturing procedures, drug release mechanisms, and characterization techniques. The review also summarizes the applications, challenges, and future perspectives for successfully translating the research concept to a clinically accepted delivery system. Despite the intricacies involved in the development of MVL, establishing steadfast characterization techniques and regulatory paths could pave the way to its extensive clinical use.

Hyaluronic Acid-Based Nanoparticles for Protein Delivery: Systematic Examination of Microfluidic Production Conditions.

Hyaluronic acid-based nanoparticles (HA NPs) can be used to deliver a protein cargo to cells overexpressing HA receptors such as CD44 since they combine the low toxicity of the carrier and the retention of the protein integrity with the receptor-mediated internalization. HA properties play a crucial but sometimes unclear role in managing the formation and stability of the meshwork, cell interactions, and ultimately the protein entrapment efficacy. Nowadays, microfluidic is an innovative technology that allows to overcome limits linked to the NPs production, guaranteeing reproducibility and control of individual batches. Taking advantage of this technique, in this research work, the role of HA weight average molecular weight (Mw) in NPs formation inside a microfluidic device has been specifically faced. Based on the relationship between polymer Mw and solution viscosity, a methodological approach has been proposed to ensure critical quality attributes (size of 200 nm, PDI ≤ 0.3) to NPs made by HA with different Mw (280, 540, 710 and 820 kDa). The feasibility of the protein encapsulation was demonstrated by using Myoglobin, as a model neutral protein, with an encapsulation efficiency always higher than 50%. Lastly, all NPs samples were successfully internalized by CD44-expressing cells.

Phase separation and toxicity of C9orf72 poly(PR) depends on alternate distribution of arginine.

Arg (R)-rich dipeptide repeat proteins (DPRs; poly(PR): Pro-Arg and poly(GR): Gly-Arg), encoded by a hexanucleotide expansion in the C9ORF72 gene, induce neurodegeneration in amyotrophic lateral sclerosis (ALS). Although R-rich DPRs undergo liquid-liquid phase separation (LLPS), which affects multiple biological processes, mechanisms underlying LLPS of DPRs remain elusive. Here, using in silico, in vitro, and in cellulo methods, we determined that the distribution of charged Arg residues regulates the complex coacervation with anionic peptides and nucleic acids. Proteomic analyses revealed that alternate Arg distribution in poly(PR) facilitates entrapment of proteins with acidic motifs via LLPS. Transcription, translation, and diffusion of nucleolar nucleophosmin (NPM1) were impaired by poly(PR) with an alternate charge distribution but not by poly(PR) variants with a consecutive charge distribution. We propose that the pathogenicity of R-rich DPRs is mediated by disturbance of proteins through entrapment in the phase-separated droplets via sequence-controlled multivalent protein-protein interactions.

Inhibition of Nucleocytoplasmic Export Enhances Selective Elimination of Leukemic Stem Cells in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a hematopoietic stem cell malignancy driven by BCR-ABL1 tyrosine kinase and effectively managed with tyrosine kinase inhibitors (TKIs) such as imatinib. Reactivation of BCR-ABL1 through mutations in the kinase is a common mechanism of resistance, but fails to explain clinical resistance in a significant proportion of cases, a situation referred to as BCR-ABL1-independent resistance. Additionally, most patients require continuous TKI therapy to avoid recurrence of active leukemia, suggesting that primitive, fully leukemogenic CML stem cells (LSCs) are BCR-ABL1-independent. We have previously demonstrated that BCR-ABL1-independent CML cells from patients with overt resistance require functional nucleocytoplasmic export (NCE) and are sensitive to knockdown of the NCE regulator RAN and selective inhibitors of NCE (SINEs) such as the CRM-1 inhibitor KPT-330 (selinexor, Karyopharm) [Khorashad et al. Blood. 2015;125(11):1772-81]. We hypothesize that reliance on NCE may extend to LSCs, the reservoir for persistent CML. To test this, we performed in vivo and in vitro experiments to evaluate the effects of a prototype SINE, KPT-330, on LSCs (defined as CD34+CD38-) and leukemic progenitor cells (LPCs, CD34+CD38+) isolated from newly diagnosed chronic phase CML patients and normal cord blood (CB).Treatment with KPT-330 (50 nM) reduced colony formation by CML (n=4) LPCs and LSCs to 42% and 55% of controls, compared to 78% and 96% for CB (n=3) (Table 1). When combined with imatinib (2.5 µM), KPT-330 treatment dramatically reduced colony formation by LPCs and LSCs to 8% and 2% of controls, compared to 66% and 43% for CB. Single agent KPT-330 (50 nM) had modest effects on apoptosis in LSCs and LPCs, but the combination increased apoptosis in LPCs and LSCs by 280% and 940% relative to controls, compared to 180% and 270% respectively for CB (Table 1). These data suggest that combining KPT-330 with imatinib has the most profound effects on LSCs, with a large differential compared to normal hematopoietic stem cells.To test the effect of KPT-330 on functionally defined LSCs, CML CD34+ cells were cultured ex vivo with or without KPT-330 (50 nM) and/or imatinib (2.5 µM) for 72 hours, followed by plating in long term culture-initiating cell (LTC-IC) assays (n=5) or injection into NSG mice (n=20, 5 per treatment arm). While no reduction in colonies was seen in LTC-IC experiments with single agent KPT-330 or imatinib, a 64% reduction in LTC-IC colonies was observed with combination treatment (p<0.05). Genotyping of LTC-IC colonies by fluorescent-in-situ-hybridization (FISH) revealed that BCR-ABL1+ colonies were reduced by 11% and 22% relative to controls, when treated with KPT-330 or imatinib respectively, but combination treatment reduced BCR-ABL1+ colonies by 38%, significantly lower than either single agent (p<0.05). Thus far results are available from one xenograft experiment. All treatment groups engrafted with a median of 80% human CD45+ cells in bone marrow and 54% in spleen at 12 weeks after injection. Human CD45+ cells were sorted and genotyped. The median proportion BCR-ABL1+ cells in the control was 40%, and was not significantly reduced by imatinib or KPT-330 alone. However combination treatment reduced BCR-ABL+ cells by 42% (p<0.01).To identify potential CRM1 clients regulated in LSCs, we performed a meta-analysis of published gene expression data on CML vs. normal C34+38- or quiescent cells. Candidate genes with differential expression between CML and normal were cross-referenced against putative CRM1 cargos predicted by the LocNES tool [Xu et al. Bioinformatics. 2015;31(9):1357-65]. Confocal microscopy is ongoing to validate cytoplasmic relocalization upon KPT-330 exposure for several candidates, including CD26, an established marker of LSCs that has recently been shown to enhance quiescence in hematopoietic cells [O'Leary et al. Leukemia. 2017 Apr 21;Epub ahead of print].Our data illustrates that interrupting nucleocytoplasmic export by SINEs preferentially eliminates CML LSCs and enhances their sensitivity to TKIs, suggesting that combinations of TKI and SINEs represent a clinical strategy to target BCR-ABL1 independent resistance, as well as persistent residual CML. Mechanistic studies exploring the effects of nuclear entrapment of certain NCE cargo proteins of biological significance in CML LSCs are ongoing and will be reported. [Display omitted] DisclosuresDeininger:Celgene: Research Funding; Incyte: Consultancy; Novartis: Consultancy, Research Funding; ARIAD: Consultancy; Gilead: Research Funding; Pfizer: Consultancy; BMS: Consultancy, Research Funding; Ariad Pharmaceuticals, Bristol Myers Squibb, CTI BioPharma Corp, Gilead, Incyte, Novartis, Pfizer, Celgene, Blue Print, Galena: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Triple growth factor delivery promotes functional bone regeneration following composite musculoskeletal trauma

Successful bone healing in severe trauma depends on early revascularization to restore oxygen, nutrient, growth factor, and progenitor cell supply to the injury. Therapeutic angiogenesis strategies have therefore been investigated to promote revascularization following severe bone injuries; however, results have been inconsistent. This is the first study investigating the effects of dual angiogenic growth factors (VEGF and PDGF) with low-dose bone morphogenetic protein-2 (BMP-2; 2.5 µg) on bone healing in a clinically challenging composite bone-muscle injury model. Our hydrogel-based delivery systems demonstrated a more than 90% protein entrapment efficiency and a controlled simultaneous release of three growth factors over 28 days. Co-stimulation of microvascular fragment constructs with VEGF and PDGF promoted vascular network formation in vitro compared to VEGF or PDGF alone. In an in vivo model of segmental bone and volumetric muscle loss injury, combined VEGF (5 µg) and PDGF (7.5 µg or 15 µg) delivery with a low dose of BMP-2 significantly enhanced regeneration of vascularized bone compared to BMP-2 treatment alone. Notably, the regenerated bone mechanics reached ~60% of intact bone, a value that was previously only achieved by delivery of high-dose BMP-2 (10 µg) in this injury model. Overall, sustained delivery of VEGF, PDFG, and BMP-2 is a promising strategy to promote functional vascularized bone tissue regeneration following severe composite musculoskeletal injury. Although this study is conducted in a clinically relevant composite injury model in rats using a simultaneous release strategy, future studies are necessary to test the regenerative potential of spatiotemporally controlled delivery of triple growth factors on bone healing using large animal models. Statement of significanceVolumetric muscle loss combined with delayed union or non-union bone defect causes deleterious effects on bone regeneration even with the supplementation of bone morphogenetic protein-2 (BMP-2). In this study, the controlled delivery of dual angiogenic growth factors (vascular endothelial growth factor [VEGF] + Platelet-derived growth factor [PDGF]) increases vascular growth in vitro. Co-delivering VEGF+PDGF significantly increase the bone formation efficacy of low-dose BMP-2 and improves the mechanics of regenerated bone in a challenging composite bone-muscle injury model.

Regulation of Proteins to the Cytosol Using Delivery Systems with Engineered Polymer Architecture.

Intracellular protein delivery enables selective regulation of cellular metabolism, signaling, and development through introduction of defined protein quantities into the cell. Most applications require that the delivered protein has access to the cytosol, either for protein activity or as a gateway to other organelles such as the nucleus. The vast majority of delivery vehicles employ an endosomal pathway however, and efficient release of entrapped protein cargo from the endosome remains a challenge. Recent research has made significant advances toward efficient cytosolic delivery of proteins using polymers, but the influence of polymer architecture on protein delivery is yet to be investigated. Here, we developed a family of dendronized polymers that enable systematic alterations of charge density and structure. We demonstrate that while modulation of surface functionality has a significant effect on overall delivery efficiency, the endosomal release rate can be highly regulated by manipulating polymer architecture. Notably, we show that large, multivalent structures cause slower sustained release, while rigid spherical structures result in rapid burst release.

Single Enzyme Nanoparticles with Improved Biocatalytic Activity through Protein Entrapment in a Surfactant Shell.

A polymeric corona consisting of an alkyl-glycolic acid ethoxylate (CXEOY) surfactant offers a promising approach toward endowing proteins with thermotropic phase behavior and hyperthermal activity. Typically, preparation of protein–surfactant biohybrids is performed via chemical modification of acidic residues followed by electrostatic conjugation of an anionic surfactant to encapsulate single proteins. While this procedure has been applied to a broad range of proteins, modification of acidic residues may be detrimental to function for specific enzymes. Herein, we report on the one-pot preparation of biohybrids via covalent conjugation of surfactants to accessible lysine residues. We entrap the model enzyme hen egg-white lysozyme (HEWL) in a shell of carboxyl-functionalized C12EO10 or C12EO22 surfactants. With fewer surfactants, our covalent biohybrids display similar thermotropic phase behavior to their electrostatically conjugated analogues. Through a combination of small-angle X-ray scattering and circular dichroism spectroscopy, we find that both classes of biohybrids consist of a folded single-protein core decorated by surfactants. Whilst traditional biohybrids retain densely packed surfactant coronas, our biohybrids display a less dense and heterogeneously distributed surfactant coverage located opposite to the catalytic cleft of HEWL. In solution, this surfactant coating permits 7- or 3.5-fold improvements in activity retention for biohybrids containing C12EO10 or C12EO22, respectively. The reported alternative pathway for biohybrid preparation offers a new horizon to expand upon the library of proteins for which functional biohybrid materials can be prepared. We also expect that an improved understanding of the distribution of tethered surfactants in the corona will be crucial for future structure–function investigations.

Comparative study on the physical entrapment of soy and mushroom proteins on the durability of bacterial cellulose bio\u2010leather

This study aimed to develop eco-friendly bacterial cellulose (BC) bio-leather with improved durability using plant-based proteins, namely soy protein isolate (SPI) and mushroom protein (MP), which were physically entrapped inside the BC, respectively. The amounts of the plant-based proteins were determined by evaluating the tensile strength of BC bio-leather, and were found to be 20 wt% and 50 wt% of BC for SPI and MP, respectively. The enhanced properties of mechanical strength and durability of BC bio-leather were measured in terms of changes in water resistance, tensile strength, flexibility, crease recovery, and dimensional stability. The durability of BC was improved after the entrapment of proteins, and moreover, the durability of BC entrapped with plant-based proteins was further improved by the addition of glycerol. Especially, BC entrapped with MP and glycerol had better water resistance, tensile strength, flexibility, and crease recovery compared to cowhide leather. The chemical and physical structures of BC bio-leathers were studied using Fourier transform-infrared spectroscopy, X-ray diffraction, field-emission scanning electron microscopy, and energy dispersive X-ray spectroscopy analyses. From the results, it was confirmed that BC entrapped with MP and glycerol could be a suitable leather substitute.

Electrochemical Activity of Cytochrome P450 1A2: The Relevance of O2 Control and the Natural Electron Donor

AbstractInvited for this month's cover picture is the Group of Biomarkers and Biosensors – GB2 (https://gb29.webnode.pt) at Universidade NOVA de Lisboa (Portugal) and CiiEM (Portugal). The cover picture shows the electrochemical response of the human monooxygenase enzyme, cytochrome P450 1A2 (CYP1A2), in the presence of its natural electron donor partner, cytochrome P450 oxidoreductase (CPR), highlighting the role of the interaction with the latter in the stabilization of CYP1A2 and attaining the electrochemically driven activity towards O2. The picture also emphasizes the protein entrapment matrix, a biocompatible sol‐gel film, which proved to be crucial for the achievement of interfacial electron transfer. Read the full text of the Article at 10.1002/celc.202001255.

Insight into the Microcosm of the Human Growth Hormone and Its Interactions with Polymers and Copolymers: A Molecular Dynamics Perspective

Therapeutic proteins nowadays have increasingly been applied for their considerable potential in treating a wide variety of diseases. The effectiveness and potency of native therapeutic proteins are limited by various factors (e.g., stability, blood circulation time, specificity). Over the past years, a great deal of effort has been devoted to developing safe and efficient protein delivery systems. Entrapment of protein into polymeric and copolymeric matrices is common among the different types of protein-based drug formulation. However, despite the massive efforts toward developing therapeutic protein delivery in experimental studies and industrial applications, there is relatively little data on the influence of polymers and copolymers on therapeutic proteins at the atomic and molecular levels. Herein, molecular dynamics (MD) simulations are used to study the effects of biocompatible synthetic polymers including methoxy poly(ethylene glycol) (MPEG), poly(lactic acid) (PLA), and poly(lactic acid) copolymers (poly(lactic-co-glycolic acid)) PLGA and MPEG-PLA(PELA)) on the structure and dynamics of the human growth hormone (hGH), and the results are compared with previous experimental findings. Our results indicate that the hGH conformation remains stable both in pure water and in the presence of polymers, and these results are in good agreement with previous experimental data. It is shown that the MPEG chains are self-assembled and folded into a semicrystalline structure; therefore, only a small portion of the protein interacts with the polymer. The other three polymers, however, interact well with the protein and partially cover its surface. Our findings suggest that the use of these polymers for protein encapsulation has the advantage of reducing protein aggregation and thus increasing drug serum half-life. Eventually, we anticipate that the research results will expand the current knowledge about encapsulation mechanisms and the molecular interactions between hGH and the polymers.