Ribonuclease A-polymer conjugates via in situ growth for cancer treatment.

Efficient delivery of therapeutic proteins is a critical aspect for protein-based cancer treatment. Herein, an in situ growth approach was employed to prepare ribonuclease A (RNase A)-polymer conjugates by incorporating a cationic polymer, poly(N,N'-dimethylamino-2-ethyl methacrylate) (P(DMAEMA)), and a hydrophobic polymer, poly(N-isopropylacrylamide) (P(NIPAM)), through atom transfer radical polymerization (ATRP). The synthesized RNase A-polymer conjugates (namely R-P(D-b-N)) could preserve the integrity of RNase A and exhibit a unique combination of cationic and hydrophobic properties, leading to enhanced intracellular delivery efficiency. The successful delivery of RNase A by R-P(D-b-N) conjugates effectively triggered the cell apoptosis through the mitochondria-dependent signaling pathway to achieve the anti-proliferative response. Additionally, the conjugates could inhibit cell migration and thus possess the potential for the suppression of tumor metastasis. Overall, our findings highlight that the introduction of cationic and hydrophobic moieties via ATRP provides a versatile platform for the intracellular delivery of therapeutic proteins, offering a new avenue for treating diverse diseases.

A quaternary ammonium-based nanosystem enables delivery of CRISPR/Cas9 for cancer therapy.

Genome editing mediated by CRISPR/Cas9 is an attractive weapon for cancer therapy. However, in vivo delivery of CRISPR/Cas9 components to achieve therapeutic efficiency is still challenging. Herein, a quaternary ammonium-functionalized poly(L-lysine) and a cholesterol-modified PEG (QNP) were self-assembled with a negatively charged CRISPR Cas9/sgRNA ribonucleoprotein (RNP) to form a ternary complex (QNP/RNP). Such a delivery system of QNP exhibited multiplex genome editing capabilities in vitro (e.g., the GFP gene and the PLK1 gene). In addition, QNP/RNPPLK1 containing PLK1 sgRNA led to 30.99% of genome editing efficiency in MCF-7 cells with negligible cytotoxicity of the carrier. QNP/RNPPLK1, which was capable of simultaneously inhibiting cell proliferation, mediating cell cycle arrest and downregulating expression of PLK1, held great in vitro therapeutic efficiency. Moreover, QNP/RNPPLK1 exhibited outstanding accumulation in tumors and high biocompatibility in vivo. In an MCF-7 xenograft animal model, QNP/RNPPLK1 showed excellent anti-tumor efficacy and achieved 17.75% indels ratio. This work showcases the successful delivery of CRISPR Cas9/sgRNA RNP with enhanced genome editing efficiency and provides a potential on-demand strategy for cancer therapy.

(De)carboxylation mechanisms of heteroaromatic substrates catalyzed by prenylated FMN-dependent UbiD decarboxylases: An in-silico study.

The UbiD enzymes are proposed to catalyze reversible (de)carboxylation reaction of unsaturated carboxylic acids using prenylated flavin mononucleotide (prFMN) as a cofactor. This positions UbiD enzymes as promising candidates for converting CO2 into valuable chemicals. However, their industrial-scale biotransformation is currently constrained by low conversion rates attributed to thermodynamic limitations. To enhance the carboxylation activity of UbiD enzymes, a molecular-level understanding of the (de)carboxylation mechanisms is necessary. In this study, we investigated the reaction mechanisms of heteroaromatic substrates catalyzed by PtHmfF, PaHudA, and AnlnD enzymes using molecular dynamics (MD) simulations and free energy calculations. Our extensive mechanistic study elucidates the mechanisms involved in the formation of the initial prFMN-substrate intermediate. Specifically, we observed nucleophilic attack during decarboxylation, while carboxylation reactions involving furoic acid, pyrrole, and indole tend to favor a 1,3-dipolar cycloaddition mechanism. Furthermore, we identified proton transfer as the rate-limiting step in the carboxylation reaction. In addition, we considered the perspectives of reaction energies and electron transfer to understand the distinct mechanisms underlying decarboxylation and carboxylation. Our calculated free energies are consistent with available experimental kinetics data. Finally, we explored how different rotamers of catalytic residues influence the efficiency of the initial intermediate formation.

Fisetin inhibits Salmonella Typhimurium type III secretion system regulator HilD and reduces pathology in vivo.

IMPORTANCE: Salmonella spp. remains a major worldwide health concern that causes significant morbidity and mortality in both humans and animals. The spread of antimicrobial resistant strains has declined the efficacy of conventional chemotherapy. Thus, novel anti-infection drugs or strategies are needed. Anti-virulence strategy represents one of the promising means for the treatment of bacterial infections. In this study, we found that the natural compound fisetin could inhibit Salmonella invasion of host cells by targeting SPI-1 regulation. Fisetin treatment impaired the interaction of the regulatory protein HilD with the promoters of its target genes, thereby suppressing the expression of T3SS-1 effectors as well as structural proteins. Moreover, fisetin treatment could reduce pathology in the Salmonella murine infection model. Collectively, our results suggest that fisetin may serve as a promising lead compound for the development of anti-Salmonella drugs.

The Isolation, Identification and Immobilization Method of Three Novel Enzymes with Diosgenin-Producing Activity Derived from an Aspergillus flavus.

Diosgenin is an important raw material used in the synthesis of steroid drugs, and it is widely used in the pharmaceutical industry. The traditional method of producing diosgenin is through using raw materials provided via the plant Dioscorea zingiberensis C. H. Wright (DZW), which is subsequently industrially hydrolyzed using a high quantity of hydrochloric and sulfuric acids at temperatures ranging from 70 °C to 175 °C. This process results in a significant amount of unmanageable wastewater, creates issues of severe environmental pollution and consumes high quantities of energy. As an alternative, the enzymolysis of DZW to produce diosgenin is an environmentally and friendly method with wide-ranging prospects for its application. However, there are still only a few enzymes that are suitable for production on an industrial scale. In this study, three new key enzymes, E1, E2, and E3, with a high conversion stability of diosgenin, were isolated and identified using an enzyme-linked-substrate autography strategy. HPLC-MS/MS identification showed that E1, a 134.45 kDa protein with 1019 amino acids (AAs), is a zinc-dependent protein similar to the M16 family. E2, a 97.89 kDa protein with 910 AAs, is a type of endo-ß-1,3-glucanase. E3, a 51.6 kDa protein with 476 AAs, is a type of Xaa-Pro aminopeptidase. In addition, the method to immobilize these proteins was optimized, and stability was achieved. The results show that the optimal immobilization parameters are 3.5% sodium alginate, 3.45% calcium chloride concentration, 1.4 h fixed time, and pH 8.8; and the recovery rate of enzyme activity can reach 43.98%. A level of 70.3% relative enzyme activity can be obtained after employing six cycles of the optimized technology. Compared with free enzymes, immobilized enzymes have improved stability, acid and alkaline resistance and reusability, which are conducive to large-scale industrial production.

Repurposing rabeprazole sodium as an anti-Clostridium perfringens drug by inhibiting perfringolysin O.

AIMS: Clostridium perfringens infections affect food safety, human health, and the development of the poultry feed industry. Anti-virulence is an alternative strategy to develop new drug. Perfringolysin O (PFO) is an exotoxin of C. perfringens that has been demonstrated to play critical roles in the pathogenesis of this organism, promising it an attractive target to explore drugs to combat C. perfringens infection. METHODS AND RESULTS: Based on an activity-based screening, we identified six PFO inhibitors from the Food and Drug Administration (FDA)-approved drug library, among which rabeprazole sodium (RS) showed an optimal inhibitory effect with an IC50 of 1.82 ± 0.746 µg ml-1. The GLY57, ASP58, SER190, SER193-194, ASN199, GLU204, ASN377, THR379, and ALA200 in PFO interacted with RS during binding based on an energy analysis and H-bond analysis. This interaction blocked the oligomer formation of PFO, thereby inhibiting its cytotoxicity. RS treatment significantly increased the survival rate and alleviated pathological damage in C. perfringens or PFO-treated Galleria mellonella. CONCLUSIONS: RS could potentially be used as a candidate drug for treating C. perfringens infection.

Exosome-mediated delivery of superoxide dismutase for anti-aging studies in Caenorhabditis elegans.

Aging is a dynamic and progressive process mediated by reactive oxygen species (ROS), and the antioxidant enzyme superoxide dismutase (SOD) can effectively scavenge ROS to extend longevity. However, the instability and impermeability of native enzyme limit its in vivo biomedical application. Currently, exosome as protein carriers attracts considerable attention in the disease treatment owing to low immunogenicity and high stability. Herein, SOD was encapsulated into exosomes via mechanical extrusion with saponin permeabilization to obtain SOD-loaded EXO (SOD@EXO). SOD@EXO with a hydrodynamic diameter of 101.7 ± 5.6 nm could scavenge excessive ROS and protect the cells from oxidative damage induced by 1-methyl-4-phenylpyridine. Compared with native SOD, SOD@EXO significantly extended the lifespan of N2 wild-type Caenorhabditis elegans under normal conditions. Moreover, SOD@EXO improved the resistance against heat and oxidative stress, leading to notable survival ratio under these hostile conditions. Overall, the exosome-mediated delivery of SOD could reduce ROS level and delay aging in C. elegans model, thereby providing potential strategies to treat ROS-related diseases in future.

Fluorinated polyamidoamine dendrimer-mediated miR-23b delivery for the treatment of experimental rheumatoid arthritis in rats.

In rheumatoid arthritis (RA), insufficient apoptosis of macrophages and excessive generation of pro-inflammatory cytokines are intimately connected, accelerating the development of disease. Here, a fluorinated polyamidoamine dendrimer (FP) is used to deliver miR-23b to reduce inflammation by triggering the apoptosis of as well as inhibiting the inflammatory response in macrophages. Following the intravenous injection of FP/miR-23b nanoparticles in experimental RA models, the nanoparticles show therapeutic efficacy with inhibition of inflammatory response, reduced bone and cartilage erosion, suppression of synoviocyte infiltration and the recovery of mobility. Moreover, the nanoparticles accumulate in the inflamed joint and are non-specifically captured by synoviocytes, leading to the restoration of miR-23b expression in the synovium. The miR-23b nanoparticles target Tab2, Tab3 and Ikka to regulate the activation of NF-κB pathway in the hyperplastic synovium, thereby promoting anti-inflammatory and anti-proliferative responses. Additionally, the intravenous administration of FP/miR-23b nanoparticles do not induce obvious systemic toxicity. Overall, our work demonstrates that the combination of apoptosis induction and inflammatory inhibition could be a promising approach in the treatment of RA and possibly other autoimmune diseases.

Gram-scale preparation of quercetin supramolecular nanoribbons for intestinal inflammatory diseases by oral administration.

Gastrointestinal (GI) tract, which possesses the largest surface area of mucosa in the body, is easily suffered from inflammatory damages under the exposure of external stimulations. Excessive reactive oxygen species (ROS) production and continuous oxidative stress in intestines can elicit local mucosal injury, accelerate mucosal ulceration, and amplify the inflammatory response. Thereby, antioxidant therapy is a potential strategy against intestinal inflammatory diseases. Herein, we demonstrate the gram-scale preparation of quercetin supramolecular nanoribbons (SNRs) by using free quercetin molecules as the sole building block for preventing and treating intestinal inflammatory diseases. Unlike current clinical medicines, which mainly confront with poor response and severe adverse effects via bloodstream delivery, our quercetin SNRs possess an excellent antioxidant activity in the harsh environments of GI tract, a relative long retention time in GI tract, an admirable metabolism in GI tract without burdening other organs, and a specific adhesion to the inflamed intestinal epithelium via electrostatic interactions. These advantages strongly guarantee the applications of quercetin SNRs as oral medicines for intestinal inflammatory diseases. After establishing the models of intestinal inflammatory diseases caused by irradiation and drug stimulations, our quercetin SNRs exhibit the promising protective and therapeutic effects for radiation-induced acute enteritis and dextran sulfate sodium (DSS)-induced acute colitis. Because the super easy and fast preparation procedure and the nearly 100% loading capacity of quercetin SNRs, the current work provides a supramolecular nanomedicine with great clinical translation potential against intestinal inflammatory diseases.

Construction of Fusion Protein with Carbohydrate-Binding Module and Leaf-Branch Compost Cutinase to Enhance the Degradation Efficiency of Polyethylene Terephthalate.

Poly(ethylene terephthalate) (PET) is a manufactured plastic broadly available, whereas improper disposal of PET waste has become a serious burden on the environment. Leaf-branch compost cutinase (LCC) is one of the most powerful and promising PET hydrolases, and its mutant LCCICCG shows high catalytic activity and excellent thermal stability. However, low binding affinity with PET has been found to dramatically limit its further industrial application. Herein, TrCBM and CfCBM were rationally selected from the CAZy database to construct fusion proteins with LCCICCG, and mechanistic studies revealed that these two domains could bind with PET favorably via polar amino acids. The optimal temperatures of LCCICCG-TrCBM and CfCBM-LCCICCG were measured to be 70 and 80 °C, respectively. Moreover, these two fusion proteins exhibited favorable thermal stability, maintaining 53.1% and 48.8% of initial activity after the incubation at 90 °C for 300 min. Compared with LCCICCG, the binding affinity of LCCICCG-TrCBM and CfCBM-LCCICCG for PET has been improved by 1.4- and 1.3-fold, respectively, and meanwhile their degradation efficiency on PET films was enhanced by 3.7% and 24.2%. Overall, this study demonstrated that the strategy of constructing fusion proteins is practical and prospective to facilitate the enzymatic PET degradation ability.

Oral Administration of Therapeutic Enzyme Capsule for the Management of Inflammatory Bowel Disease.

Background: Oral administration of proteins/peptides is challenging in clinical application due to their instability and susceptibility in the gastrointestinal tract. Materials and Methods: The in situ polymerization on the surface of enzymes was used to encapsulate antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)) in polymeric shells, and the reactive oxygen species (ROS) scavenging ability was monitored based on DCFH-DA probe using flow cytometry and confocal laser scanning microscopy. The mRNA expression level of pro-inflammatory factors was assessed by real-time qPCR, using lipopolysaccharide-induced RAW264.7 cells as a model. Finally, the enzyme capsules were orally administered for the treatment of inflammatory bowel disease using dextran sodium sulfate (DSS)-induced colitis mice as a model, based on the evaluation of the disease-associated index, ROS level and pro-inflammatory cytokines' expression. Results: The enzyme capsules could effectively scavenge the intracellular reactive oxygen species (ROS) through the cascade catalysis of SOD and CAT, and thus protect the cells from ROS-induced oxidative damage. Meanwhile, the enzyme capsules could inhibit the secretion of pro-inflammatory cytokines from macrophages, thereby achieving favorable anti-inflammation effect. Oral administration of enzyme capsules could facilitate the accumulation of enzymes in the inflamed colon tissues of DSS-induced colitis mice. Moreover, the oral delivery of enzyme capsules could effectively alleviate the symptoms associated with colitis, attributing to the excellent ROS scavenging ability and the inhibition of pro-inflammatory cytokines' level. Conclusion: In summary, our findings provided a promising approach to construct enzyme-based nano-formulations with favorable therapeutic efficacy and biocompatibility, exhibiting great potential in the treatment of gastrointestinal diseases in an oral administration manner.

An Atom-Economic Enzymatic Cascade Catalysis for High-Throughput RAFT Synthesis of Ultrahigh Molecular Weight Polymers.

High-throughput synthesis of well-defined, ultrahigh molecular weight (UHMW) polymers by green approaches is highly desirable but remains unexplored. We report the creation of an atom-economic enzymatic cascade catalysis, consisting of formate oxidase (FOx) and horseradish peroxidase (HRP), that enables high-throughput reversible addition-fragmentation chain transfer (RAFT) synthesis of UHMW polymers at volumes down to 50 µL. FOx transforms formic acid, a C1 substrate, and oxygen to CO2 and H2 O2 , respectively. CO2 can escape from solution while H2 O2 is harnessed in situ by HRP to generate radicals from acetylacetone for RAFT polymerization, leaving no waste accumulation in solution. Oxygen-tolerant RAFT polymerization using enzymatic cascade redox cycles was successfully performed in vials and 96-well plates to produce libraries of well-defined UHMW polymers, and represents the first example of high-throughput synthesis method of such materials at extremely low volumes.

Superoxide dismutase-embedded metal-organic frameworks via biomimetic mineralization for the treatment of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is characterized by chronic and spontaneous inflammation in the gastrointestinal tract, and has been associated with an improved level of reactive oxygen species (ROS). Herein, superoxide dismutase (SOD) was encapsulated into zeolitic imidazolate framework-zni (ZIF-zni) to construct a nanocomposite termed SOD@ZIF-zni through biomimetic mineralization, which was then used as a formulation for the IBD treatment. The SOD@ZIF-zni composite could efficiently suppress the level of ROS and pro-inflammatory cytokines, using the colorectal cancer cell line SW480 as a model. Oral administration of the SOD@ZIF-zni composite could relieve the oxidative stress and inhibit the release of pro-inflammatory cytokines in the inflamed colonic tissues, leading to the alleviation of colitis in dextran sulfate sodium-induced colitis mice. Overall, the favorable therapeutic efficacy and biocompatibility of SOD@ZIF-zni gave it potential to be used as a safe and effective formulation for IBD treatment in the future.

Genome editing of PD-L1 mediated by nucleobase-modified polyamidoamine for cancer immunotherapy.

Immune checkpoint blockade therapy against programmed death protein-1 and its ligand (PD-1/PD-L1) has been accepted as a promising approach to activate the immune system's anti-tumor response. Although small interfering RNA (siRNA) or antibodies can block the PD-1/PD-L1 pathway, the effect of this blockade is temporary and reversible. Here, we developed a nano-delivery system to achieve permanent disruption of the PD-L1 gene based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) gene editing technology. In this system, the CRISPR/Cas9 plasmid was delivered into melanoma B16F10 cells using a nucleobase-modified polyamidoamine (PAMAM) derivative namely AP-PAMAM, which was constructed through the modification with 2-amino-6-chloropurine. Meanwhile, the carrier could efficiently facilitate the endosomal escape of CRISPR/Cas9 plasmid and thereby inhibit PD-L1 expression in cancer cells. Moreover, the intravenous injection of AP-PAMAM/plasmid nanoparticles could recruit and activate CD8+ T cells at the tumor site, promoting the secretion of cytokines and the killing of tumor cells. Overall, this nano-delivery system for genome editing provided a promising strategy to block the PD-1/PD-L1 pathway and obtain effective tumor immunotherapy.

Phenylboronic Acid-Modified Polyamidoamine Mediated the Transfection of Polo-Like Kinase-1 siRNA to Achieve an Anti-Tumor Efficacy.

BACKGROUND: The construction of tumor-targeting carriers with favorable transfection efficiency was of great significance to achieve the tumor gene therapy. The phenylboronic acid-modified polyamidoamine (namely PP) was employed as a carrier for the delivery of Polo-like kinase-1 siRNA (siPlk-1), inducing an obvious anti-tumor response. MATERIALS AND METHODS: The interaction between PP and siPlk-1 was evaluated by gel retardation assay. The transfection efficiency and tumor-targeting ability were analyzed by flow cytometry and confocal laser scanning microscopy, using hepatocarcinoma cell line HepG2 as a model. The anti-proliferation effect of PP/siPlk-1 and related mechanism were studied using the strategies of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell apoptosis and cell cycle arrest. The anti-migration effect induced by PP/siPlk-1 delivery was assayed by wound healing and Transwell migration techniques. Finally, quantitative real-time PCR and Western blotting were performed to measure the expression level of Plk-1 and other key targets. RESULTS: The derivative PP could achieve the condensation of siPlk-1 into stable nanoparticles at nitrogen/phosphate groups ratio (N/P ratio) of >3.0, and it could facilitate the transfection of siPk-1 in a phenylboronic acid-dependent manner. The PP/siPlk-1 nanoparticles exhibited obvious anti-proliferation effect owing to the gene silence of Plk-1, which was identified to be associated with the cell apoptosis and cell cycle arrest at G2 phase. Meanwhile, PP/siPlk-1 transfection could efficiently suppress the migration and invasion of tumor cells. CONCLUSION: The derivative PP has been demonstrated to be an ideal tumor-targeting carrier for the delivery of Plk-1 siRNA, exhibiting great potential in the gene therapy of malignant tumors.

Immobilized enzymes in inorganic hybrid nanoflowers for biocatalytic and biosensing applications.

Enzyme immobilization has been accepted as a powerful technique to solve the drawbacks of free enzymes such as limited activity, stability and recyclability under harsh conditions. Different from the conventional immobilization methods, enzyme immobilization in inorganic hybrid nanoflowers was executed in a biomimetic mineralization manner with the advantages of mild reaction conditions, and thus it was beneficial to obtain ideal biocatalysts with superior characteristics. The key factors influencing the formation of enzyme-based inorganic hybrid nanoflowers were elucidated to obtain a deeper insight into the mechanism for achieving unique morphology and improved properties of immobilized enzymes. To date, immobilized enzymes in inorganic hybrid nanoflowers have been successfully applied in biocatalysis for preparing medical intermediates, biodiesel and biomedical polymers, and solving the environmental or food industrial issues such as the degradation of toxic dyes, pollutants and allergenic proteins. Moreover, they could be used in the development of various biosensors, which provide a promising platform to detect toxic substances in the environment or biomarkers associated with various diseases. We hope that this review will promote the fundamental research and wide applications of immobilized enzymes in inorganic hybrid nanoflowers for expanding biocatalysis and biosensing.

Lipoic Acid-Modified Oligoethyleneimine-Mediated miR-34a Delivery to Achieve the Anti-Tumor Efficacy.

MiR-34a, an important tumor suppressor, has been demonstrated to possess great potential in tumor gene therapy. To achieve the upregulation of miR-34a expression level, an oligoethyleneimine (OEI) derivative was constructed and employed as the carrier through the modification with lipoic acid (LA), namely LA-OEI. In contrast to OEI, the derivative LA-OEI exhibited superior transfection efficiency measured by confocal laser scanning microscopy and flow cytometry, owing to rapid cargo release in the disulfide bond-based reduction sensitive pattern. The anti-proliferation and anti-migration effects were tested after the miR-34a transfection to evaluate the anti-tumor response, using human cervical carcinoma cell line HeLa as a model. The delivery of LA-OEI/miR-34a nanoparticles could achieve obvious anti-proliferative effect caused by the induction of cell apoptosis and cell cycle arrest at G1 phase. In addition, it could inhibit the migration of tumor cells via the downregulation of MMP-9 and Notch-1 level. Overall, the LA-OEI-mediated miR-34a delivery was potential to be used as an effective way in the tumor gene therapy.

Urate oxidase loaded in PCN-222(Fe) with peroxidase-like activity for colorimetric detection of uric acid.

In the past two decades, the number of reports on the construction of uric acid (UA) sensors has increased dramatically, as it is a vital factor in the diagnosis of physiological functions and diseases. Among these sensors, cascade colorimetric detection based on peroxidase mimics has received great attention owing to the advantages of easier operation and more intuitive results. Herein, we report a simple UA detection method via the integration of urate oxidase (UOx) and PCN-222(Fe) with peroxidase-like activity, in which UOx is immobilized in PCN-222(Fe) by physical adsorption. UOx could catalyze the UA oxidation to produce H2O2, and then PCN-222(Fe) catalyzed the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2 to generate a blue product with a maximum absorbance at 652 nm. Under optimal conditions, the increasing absorbance at 652 nm was proportional to the UA concentration. The linear range for UA detection was 10-800 µM with a limit of detection of 3.5 µM, and the cascade system has been successfully utilised for the detection of UA in human serum samples to demonstrate the accuracy of the proposed method. In conclusion, the cascade catalytic system based on the immobilization of UOx in PCN-222(Fe) has the potential to be used as a rapid and sensitive sensor for UA detection.