Nanozyme-Coated Bacteria Hitchhike on CD11b+ Immune Cells to Boost Tumor Radioimmunotherapy.

Bacterial-based delivery strategies have recently emerged as a unique research direction in the field of drug delivery. However, bacterial vectors are quickly phagocytosed by immune cells after entering the bloodstream. Taking advantage of this phenomenon, herein, this work seeks to harness the potential of immune cells to delivery micron-sized bacterial vectors, and find that inactivated bacterial can accumulate at tumor-site after intravenous injection through CD11b+ cells hitchhiking. To this end, this work then designs a gold-platinum bimetallic nanozyme coated bacterial vector (Au-Pt@VNP20009, APV). Utilizing strong tumor inflammatory response induced by low dose X-rays, this work further heightens the ability of CD11b+ immune cells to assist APV hitchhiking for tumor-targeted delivery, which can significantly relieve tumor hypoxia and immunosuppression, and inhibit tumor growth and metastasis. This work elucidates the potential mechanisms of bacterial vector targeted delivery, opening up new horizons for bacterial vector delivery strategies and clinical tumor radioimmunotherapy.

Establishment of bone-targeted nano-platform and the study of its combination with 2-deoxy-d-glucose enhanced photodynamic therapy to inhibit bone metastasis.

At present, simple anti-tumor drugs are ineffective at targeting bone tissue and are not purposed to treat patients with bone metastasis. In this study, zoledronic acid (ZOL) demonstrated excellent bone-targeting properties as a bone-targeting ligand. The metal-organic framework (MOF) known as ZIF-90 was modified with ZOL to construct a bone-targeting-based drug delivery system. Chlorin e6 (Ce6) was loaded in the bone-targeted drug delivery system and combined with 2-deoxy-D-glucose (2-DG), which successfully treated bone tumors when enhanced photodynamic therapy was applied. The Ce6@ZIF-PEG-ZOL (Ce6@ZPZ) nanoparticles were observed to have uniform morphology, a particle size of approximately 210 nm, and a potential of approximately -30.4 mV. The results of the bone-targeting experiments showed that Ce6@ZPZ exhibited a superior bone-targeted effect when compared to Ce6@ZIF-90-PEG. The Ce6@ZPZ solution was subjected to 660 nm irradiation and the resulting production of reactive oxygen species increased over time, which could be further increased when Ce6@ZPZ was used in combination with 2-DG. Their combination had a stronger inhibitory capacity against tumor cells than either 2-DG or Ce6@ZPZ alone, increasing the rate of tumor cell apoptosis. The apoptosis rate caused by HGC-27 was 61.56% when 2-DG was combined with Ce6@ZPZ. In vivo results also showed that Ce6@ZPZ combined with 2-DG maximally inhibited tumor growth and prolonged mice survival compared to the other experimental groups. Therefore, the combination of PDT and glycolytic inhibitors serves as a potential option for the treatment of cancer.

Development of the Cu/ZIF-8 MOF Acid-Sensitive Nanocatalytic Platform Capable of Chemo/Chemodynamic Therapy with Improved Anti-Tumor Efficacy.

Recently, the combination of chemotherapy and chemodynamic therapy (CDT) has become a desirable strategy in the treatment of cancer. However, a satisfactory therapeutic outcome is often difficult to achieve due to the deficiency of endogenous H2O2 and O2 in the tumor microenvironment. In this study, a CaO2@DOX@Cu/ZIF-8 nanocomposite was prepared as a novel nanocatalytic platform to enable the combination of chemotherapy and CDT in cancer cells. The anticancer drug doxorubicin hydrochloride (DOX) was loaded onto calcium peroxide (CaO2) nanoparticles (NPs) to form CaO2@DOX, which was then encapsulated in a copper zeolitic imidazole ester MOF (Cu/ZIF-8) to form CaO2@DOX@Cu/ZIF-8 NPs. In the mildly acidic tumor microenvironment, CaO2@DOX@Cu/ZIF-8 NPs rapidly disintegrated, releasing CaO2, which reacted with water to generate H2O2 and O2 in the tumor microenvironment. The ability of CaO2@DOX@Cu/ZIF-8 NPs to combine chemotherapy and CDT was assessed by conducting cytotoxicity, living dead staining, cellular uptakes, H&E staining, and TUNEL assays in vitro and in vivo. The combination of chemotherapy and CDT of CaO2@DOX@Cu/ZIF-8 NPs had a more favorable tumor suppression effect than the nanomaterial precursors, which were not capable of the combined chemotherapy/CDT.

Preparation of an amphiphilic peptide (P13) with proton sponge effect and analysis of its antitumor activity.

In order to prevent drugs from being captured and degraded by the acidic environment of organelles, such as lysosomes, after entering cells, this study designed and synthesized a novel carrier amphiphilic polypeptide (DGRHHHLLLAAAA), designated P13, for use as a tumor-targeting drug delivery vehicle. The P13 peptide was synthesized by the solid phase synthesis method, and its self-assembly behavior and drug-loading capacity in aqueous solution were studied and characterizedin vitro. Doxorubicin (DOX) was loaded by dialysis method, and P13 and DOX were mixed at a mass ratio of 6:1 to form regular rounded globules. The acid-base buffering capacity of P13 was investigated determined by acid-base titration. The results revealed that P13 had excellent acid-base buffering capacity, a critical micelle concentration value of about 0.000 21 g l-1, and the particle size of P13-Dox nanospheres was 167 nm. The drug encapsulation efficiency and drug loading capacity of micelles were 20.40 ± 1.21% and 21.25 ± 2.79%, respectively. At the concentration of 50µg ml-1of P13-DOX , the inhibition rate was 73.35%. The results of thein vivoantitumor activity assay in mice showed that P13-DOX also exhibited excellent inhibitory effect on tumor growth, compared with the tumor weight of 1.1 g in the control group, the tumor weight in the P13-DOX-treated group was only 0.26 g. Additionally, the results of hematoxylin and eosin staining of the organs showed that P13-DOX had no damaging effect on normal tissues. The novel amphiphilic peptide P13 with proton sponge effect designed and prepared in this study is expected to be a promising tumor-targeting drug carrier with excellent application potential.

Biofilm Microenvironment-Mediated MoS2 Nanoplatform with Its Photothermal/Photodynamic Synergistic Antibacterial Molecular Mechanism and Wound Healing Study.

Drug-resistant bacterial infections pose a serious threat to human public health. Biofilm formation is one of the main factors contributing to the development of bacterial resistance, characterized by a hypoxic and microacidic microenvironment. Traditional antibiotic treatments have been ineffective against multidrug-resistant (MDR) bacteria. Novel monotherapies have had little success. On the basis of the photothermal effect, molybdenum disulfide (MoS2) nanoparticles were used to link quaternized polyethylenimine (QPEI), dihydroporphyrin e6 (Ce6), and Panax notoginseng saponins (PNS) in a zeolitic imidazolate framework-8 (ZIF-8). A multifunctional nanoplatform (MQCP@ZIF-8) was constructed with dual response to pH and near-infrared light (NIR), which resulted in synergistic photothermal and photodynamic antibacterial effects. The nanoplatform exhibited a photothermal conversion efficiency of 56%. It inhibited MDR Escherichia coli (E. coli) and MDR Staphylococcus aureus (S. aureus) by more than 95% and effectively promoted wound healing in mice infected with MDR S. aureus. The nanoplatform induced the death of MDR bacteria by promoting biofilm ablation, disrupting bacterial cell membranes and intracellular DNA, and interfering with intracellular material and energy metabolism. In this study, a multifunctional nanoplatform with good antibacterial effect was developed. The molecular mechanisms of MDR bacteria were also elucidated for possible clinical application.

Synthesis of a Two-Dimensional Molybdenum Disulfide Nanosheet and Ultrasensitive Trapping of Staphylococcus Aureus for Enhanced Photothermal and Antibacterial Wound-Healing Therapy.

Photothermal therapy has been widely used in the treatment of bacterial infections. However, the short photothermal effective radius of conventional nano-photothermal agents makes it difficult to achieve effective photothermal antibacterial activity. Therefore, improving composite targeting can significantly inhibit bacterial growth. We inhibited the growth of Staphylococcus aureus (S. aureus) by using an extremely low concentration of vancomycin (Van) and applied photothermal therapy with molybdenum disulfide (MoS2). This simple method used chitosan (CS) to synthesize fluorescein 5(6)-isothiocyanate (FITC)-labeled and Van-loaded MoS2-nanosheet hydrogels (MoS2-Van-FITC@CS). After modifying the surface, an extremely low concentration of Van could inhibit bacterial growth by trapping bacteria synergistically with the photothermal effects of MoS2, while FITC labeled bacteria and chitosan hydrogels promoted wound healing. The results showed that MoS2-Van-FITC@CS nanosheets had a thickness of approximately 30 nm, indicating the successful synthesis of the nanosheets. The vitro antibacterial results showed that MoS2-Van-FITC with near-infrared irradiation significantly inhibited S. aureus growth, reaching an inhibition rate of 94.5% at nanoparticle concentrations of up to 100 µg/mL. Furthermore, MoS2-Van-FITC@CS could exert a healing effect on wounds in mice. Our results demonstrate that MoS2-Van-FITC@CS is biocompatible and can be used as a wound-healing agent.

Tumor microenvironment-responsive BSA nanocarriers for combined chemo/chemodynamic cancer therapy.

Tumor microenvironment (TME), characterized by high glutathione (GSH), high hydrogen peroxide (H2O2) and acidic pH levels, is favorable for the growth, invasion and metastasis of cancer cells. Taking advantage of the specific characteristics of tumors, TME-responsive GCBD NPs are designed to deliver nanoscale coordination polymers (NCPs, GA-Cu) and chemotherapy drugs (doxorubicin, DOX) based on bovine serum albumin (BSA) nanocarriers into cancer cells for combined chemodynamic therapy (CDT) and chemotherapy. In an acidic environment, GCBD NPs could release approximately 90% copper ions, which can not only consume overexpressed GSH to modulate the TME but can also react with endogenous H2O2 in a Fenton-like reaction to achieve the CDT effect. Meanwhile, the released DOX could enter the nucleus of tumor cells and affect their proliferation to achieve efficient chemotherapy. Both in vitro and in vivo experiments showed that GCBD NPs had good biosafety and could effectively inhibit the growth of cancer cells. GCBD NPs are promising as a biocompatible nanoplatform to exploit TME characteristics for combined chemo and chemodynamic therapy, providing a novel strategy to eradicate tumors with high efficiency and specificity.

Acid-Sensitive Nanoparticles Based on Molybdenum Disulfide for Photothermal-Chemo Therapy.

The combination of multiple treatments has recently been investigated for tumor treatment. In this study, molybdenum disulfide (MoS2) with excellent photothermal conversion performance was used as the core, and manganese dioxide (MnO2), which responds to the tumor microenvironment, was loaded on its surface by liquid deposition to form a mesoporous core-shell structure. Then, the chemotherapeutic drug Adriamycin (DOX) was loaded into the hole. To further enhance its water solubility and stability, the surface of MnO2 was modified with mPEG-NH2 to prepare the combined antitumor nanocomposite MoS2@DOX/MnO2-PEG (MDMP). The results showed that MDMP had a diameter of about 236 nm, its photothermal conversion efficiency was 33.7%, and the loading and release rates of DOX were 13 and 65%, respectively. During in vivo and in vitro studies, MDMP showed excellent antitumor activity. Under the combined treatment, the tumor cell viability rate was only 11.8%. This nanocomposite exhibits considerable potential for chemo-photothermal combined antitumor therapy.

A bone-targeting drug delivery vehicle of a metal-organic framework conjugate with zoledronate combined with photothermal therapy for tumor inhibition in cancer bone metastasis.

Chemotherapy is a conventional treatment method for metastatic bone cancer, but it has limitations, such as lower drug-targeting of bone tissues and serious side effects. Bone metastasis almost always occurs in advanced cancer, and most patients in this period have strong drug resistance, which further worsens the curative effect. To address the above-mentioned difficulties, a drug delivery platform is proposed in this paper that accomplishes the bone-targeting of drugs to efficiently inhibit tumors. First, the anti-cancer drugs 5-fluorouracil (5-Fu) and indocyanine green (ICG) were loaded into a zeolitic imidazolate framework (ZIF-90) to form 5-Fu/ICG@ZIF-90. Polyethylene glycol with zoledronic acid (ZOL) was encapsulated using 5-Fu/ICG@ZIF-90 to synthesize 5-Fu/ICG@ZIF-90-PEG-ZOL nanoparticles, which showed dimensional stability, good thermal stability, and bone-targeting ability. Second, the in vitro anti-cancer activity of the designed platform was investigated using cytotoxicity, apoptosis, live-dead staining, cell cycle, and cell ultrathin section analysis. The results indicated that the nanoparticles inhibited MCF-7 cell activity when chemotherapy was combined with PTT. Finally, H&E staining and TUNEL detection were performed in mouse organs and tumors. The nanoparticles combined with photothermal therapy (PTT) and triggered by near-infrared irradiation induce apoptosis of tumor cells in vivo, displaying a better efficacy of combined chemotherapy and photothermal therapy. Experiments conducted on the 5-Fu/ICG@ZIF-90-PEG-ZOL nanoparticles demonstrated their promising performance for cancer bone metastasis inhibition.

Gold nanorod-loaded thermosensitive liposomes facilitate the targeted release of ruthenium(II) polypyridyl complexes with anti-tumor activity.

Ruthenium(II) polypyridyl complexes (Ru) show high anti-tumor activity, but their poor solubility and low biocompatibility impede their use in anti-tumor therapy. Here,we circumvented the problem of low solubility by encapsulating the Ru in thermosensitive liposomes (LTSLs) and used gold nanorods (Au NRs) modified on the surface of the liposomes to permit the precise release of Ru at the tumor site. A facile and simple method was developed to synthesize Ru-loaded Au NR-decorated LTSL (Au@LTSL-Ru NPs). The loaded Au NRs improved the anti-tumor effect of Ru and enhanced the photothermal therapeutic properties of the nanosystem. A characterization experiment indicated that the average particle size of Au@LTSL-Ru was approximately 300 nm and that the Au NRs were successfully modified on the surface of LTSL. In thein vitroanti-tumor test, Au@LTSL-Ru and NIR significantly inhibited the proliferation of SGC-7901 cells. The IC50value of Au@LTSL-Ru + NIR was 7.1 ± 1.2µM (13µg ml-1), and the inhibition rate was greater than 90% when the concentration reached 30µg ml-1.In vivostudies revealed that Au@LTSL-Ru and NIR had a significant inhibitory effect on subcutaneous tumor tissues derived from SGC-7901 cells. Analysis of histopathology and immunocytotoxicity indicated that Au@LTSL-Ru has fewer side effects and high biocompatibility. Our results confirm that Au@LTSL-Ru can effectively inhibit tumor growth and aid the development of Ru for use in the thermal response in anti-tumor activity research.

The Effects of Luminescent CdSe Quantum Dot-Functionalized Antimicrobial Peptides Nanoparticles on Antibacterial Activity and Molecular Mechanism.

BACKGROUND: With the development of bacterial resistance, the range of effective antibiotics is increasingly becoming more limited. The effective use of nanoscale antimicrobial peptides (AP) in therapeutic and diagnostic methods is a strategy for new antibiotics. METHODS: Combining both AP and cadmium selenide (CdSe) into a composite material may result in a reagent with novel properties, such as enhanced antibacterial activity, fluorescence and favorable stability in aqueous solution. RESULTS: AP-loaded CdSe NPs (AP-CdSe NPs) showed strong antibacterial activity against multidrug-resistant (MDR) Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) in vitro and in vivo. Colony-forming unit (CFU) and minimum inhibitory concentration (MIC) assays showed that AP-CdSe NPs have highly effective antibacterial activity. The quantitative analysis of apoptosis by flow cytometry analysis further confirmed that MDR E. coli and S. aureus treated with AP-CdSe NPs had death rates of 98.76% and 99.13%, respectively. Also, AP-CdSe NPs was found to inhibit bacterial activity in an in vivo bacteremia model in mice infected with S. aureus. In addition, the antibacterial mechanism of AP-CdSe NPs was determined by RNA sequencing analysis. Gene ontology (GO) analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis revealed the molecular mechanism of the antibacterial effect of AP-CdSe NPs. Importantly, histopathology analysis, and hematological toxicity analysis indicated that AP-CdSe NPs had few side effects. CONCLUSION: These results demonstrate that AP loaded on CdSe NPs had a higher water solubility, bioavailability and antibacterial effect compared with raw AP. This study reports findings that are helpful for the design and development of antibacterial treatment strategies based on AP.

De novosynthesis of pH-responsive, self-assembled, and targeted polypeptide nano-micelles for enhanced delivery of doxorubicin.

Doxorubicin (DOX) is a commonly used anticancer drug, but it is inefficient as a therapeutic due to a lack of targeting. Peptide-tuned self-assembly of DOX offers a strategy to improve targeting for greater efficacy. In this work, we designed and prepared an amphiphilic tumor cell-targeting peptide, P14 (AAAAFFFHHHGRGD), able to encapsulate DOX by self-assembly to form tumor cell-targeting and pH-sensitive nano-micelles. The results showed a critical P14-micelle concentration of 1.758 mg l-1and an average particle size of micelles of 121.64 nm, with entrapment and drug-loading efficiencies of 28.02% ± 1.35% and 12.06% ± 0.59%, respectively. The prepared micelles can release 73.52 ± 1.27% DOX within 24 h in pH 4.5 medium, and the drug cumulative release profile of micelles can be described by the first-order model. Compared with free DOX, the micelles exhibited an increased ability to inhibit tumor cell growth and cause tumor apoptosisin vitro, with IC50values of DOX and P14-DOX micelles against human breast cancer cells (MCF-7) of 0.91 ± 0.07 and 0.75 ± 0.06µg ml-1, respectively, and cellular apoptotic rates of DOX and P14-DOX micelles of 70.3% and 42.4%, respectively. Cellular uptake experiments revealed high concentrations of micelles around and inside MCF-7 cells, demonstrating that micelles can target tumor cells. These results indicate the excellent potential for the application of this amphiphilic peptide as a carrier for small-molecule drugs and suggest a strategy for the design of effective anti-tumor drugs.

Versatile labeling of multiple radionuclides onto a nanoscale metal-organic framework for tumor imaging and radioisotope therapy.

Radionuclides for cancer theranostic have confronted problems such as limitation in real-time visualization and unsatisfactory therapeutic effect sacrificed by the nonspecific distribution. Nanoscale metal-organic frameworks (nMOFs) have been widely used in biomedical applications including cancer imaging and drug delivery. However, there have been rare reports utilizing nMOFs as a single nanoplatform to label various radionuclides for tumor imaging and radioisotope therapy (RIT). In this work, we developed polyethylene glycol (PEG) modified zirconium-based nMOFs (PCN-224) with favorable size, water solubility and biocompatibility. Interestingly, without the help of chelating agents, metal radionuclides (technetium-99 m/99mTc, lutetium-177/177Lu) could be efficiently labeled onto nMOFs via chelating with the porphyrin structure and iodine-125 (125I) via chemical substitution of hydrogen in the benzene ring. The radionuclide-labeled PCN-PEG nanoparticles all exhibit excellent radiolabeling stability in different solutions. In accordance with the fluorescence imaging of mice injected with PCN-PEG, SPECT/CT imaging illustrates strong tumor accumulation of 99mTc-PCN-PEG. Moreover, 177Lu-PCN-PEG significantly inhibited the growth of tumor without inducing any perceptible toxicity to the treated mice. Hence, the radionuclide-delivery nanoplatform based on nMOFs would provide more opportunities for precise tumor theranostics and expand the biomedical applications of MOF nanomaterials.

Investigation into isomerization reaction of phenylalanine aminomutase from Pantoea agglomerans.

Phenylalanine aminomutase (PaPAM) from Pantoea agglomerans is a member of the MIO (4-methylene-imidazol-5-one) family of enzymes, which isomerizes α-phenylalanine to ß-phenylalanine, and could be used to synthesize unnatural ß-arylalanine. However, the mechanism of isomerization reaction is not clear. To investigate the mechanism, the gene (pam), which encodes PaPAM, was first expressed in E.coli, and recombinant PaPAM was prepared using affinity chromatography. Then, 15N-(2S)-α-phenylalanine, (2S)-(3-2H2)-α-phenylalanine and (2S,3S)-[2,3-2H2]-α-phenylalanine were used as substrates to analyze the mechanism of isomerization reaction. The results of MS and NMR showed that the isomerization reaction was performed through the intramolecular exchange of NH2 with pro-3R hydrogen of α-phenylalanine. The PaPAM shuttles the α-NH2 of α-phenylalanine to ß site to replace the pro-3R hydrogen. Simultaneously, the pro-3R hydrogen is shifted to α site to produce ß-phenylalanine. Furthermore, a key residue, Phe at position 455 in the active site, was determined to control the exchange way using molecular docking and sequence alignment of MIO family enzymes. The results indicated that the key 455 Phe residue is involved in changing the binding orientation of the carboxyl group of the intermediate trans-cinnamic acid to control the NH2-H pair exchange.

One-Pot Enzymatic Synthesis of D-Arylalanines Using Phenylalanine Ammonia Lyase and L-Amino Acid Deaminase.

The phenylalanine ammonia-lyase (AvPAL) from Anabaena variabilis catalyzes the amination of substituent trans-cinnamic acid (t-CA) to produce racemic D,L-enantiomer arylalanine mixture owing to its low stereoselectivity. To produce high optically pure D-arylalanine, a modified AvPAL with high D-selectivity is expected. Based on the analyses of catalytic mechanism and structure, the Asn347 residue in the active site was proposed to control stereoselectivity. Therefore, Asn347 was mutated to construct mutant AvPAL-N347A, the stereoselectivity of AvPAL-N347A for D-enantiomer arylalanine was 2.3-fold higher than that of wild-type AvPAL (WtPAL). Furthermore, the residual L-enantiomer product in reaction solution could be converted into the D-enantiomer product through stereoselective oxidation by PmLAAD and nonselective reduction by reducing agent NH3BH3. At optimal conditions, the conversion rate of t-CA and optical purity (enantiomeric excess (eeD)) of D-phenylalanine reached 82% and exceeded 99%, respectively. The two enzymes displayed activity toward a broad range of substrate and could be used to efficiently synthesize D-arylalanine with different groups on the phenyl ring. Among these D-arylalanines, the yield of m-nitro-D-phenylalanine was highest and reached 96%, and the eeD exceeded 99%. This one-pot synthesis using AvPAL and PmLAAD has prospects for industrial application.

Preparation of a tumor-targeted drug-loading material, amphiphilic peptide P10, and analysis of its anti-tumor activity.

A new tumor-targeted drug-loading material, the amphiphilic peptide DGRGGGAAAA (P10) was designed and synthesized, and its self-assembly behavior, drug-loading effects and in vitro characteristics were studied. P10 was synthesized by solid-state synthesis and doxorubicin (DOX) was loaded via dialysis. P10 and DOX were mixed with a mass ratio of 6:1 to form regular round spheres. The interconnection between groups was analyzed spectroscopically and the sphere morphology was studied with SEM and a zeta particle size analyzer. Fluorescence spectroscopy was used to analyze the ability of P10 to form micelles and the efficiency of micelle entrapment, and the drug-loading ratio and drug release characteristics were detected. Finally, the in vitro antitumor activity of P10 was studied with HeLa cells as a model. The results showed that P10's critical micelle concentration (CMC) value and its average grain diameter were approximately 0.045 mg/L and 500 nm. The micelle entrapment ratio and drug-loading ratio were 23.011 ± 2.88 and 10.125 ± 2.62%, respectively, and the in vitro drug-releasing properties of P10 were described by the Zero-order model and the Ritger-Peppas model. Compared with DOX, P10-DOX had a higher tumor cell inhibition ratio and a dose-effect relationship with concentration. When P10-DOX's concentration was 20 µg/mL, the inhibition ratio was 44.17%. The new amphiphilic peptide designed and prepared in this study could be a tumor-targeted drug-loading material with better prospects for application. In this paper, a new tumor-targeted drug-loading material, the amphiphilic peptide DGRGGGAAAA (P10) is designed and synthesized, and its self-assembly behavior, drug-loading effects and in vitro characteristics are studied, providing a theoretical basis and design ideas for further studies and the development of targeted drug-loading materials on tumor cells.

The Soluble and Particulate Form of Alginates Positively Regulate Immune Response.

BACKGROUND: Alginate materials have been widely employed for biomedical applications ranging from wound healing to cancer treatment. However, how alginate materials affect the immune system is largely unknown. OBJECTIVE: To explore the impact of alginate materials on immune system. METHODS: The effect of three types of alginate materials, low viscosity, high viscosity and particulate alginate, were examined by both in vivo and in vitro analyses. C57BL/6J (B6) mice were treated with alginate and peripheral blood was tested by ELISA for cytokine production. Dendritic cells, macrophages and splenocytes isolated from mice were analyzed for the response to alginate treatment. Administration of alginates by intra lymph node injection (I.L.N.) yielded more potent cytokines productions than other injection routes. RESULTS: Alginate materials did not affect the viability of lymphocytes. Particulate alginate induced the most potent inflammatory reaction as determined by the production of cytokines, such as, IL-1ß, IL-8, TNF-α and IFN-γ. Low viscosity and particulate alginates are more effective than high viscosity alginates in activating dendritic cells as indicated by the expression of dendritic cells surface markers (CD80, CD86 and CD40). Similarly, the level of G-CSF was slightly higher in particulate alginate treated macrophages. CONCLUSION: Alginate materials could affect immune response through different ways, including promoting inflammatory cytokine production, and activating dendritic cells. Therefore, alginate materials, especially in particulate form, have the potential to be applied in inflammation related diseases.

One step synthesis of unnatural ß-arylalanines using mutant phenylalanine aminomutase from Taxus chinensis with high ß-regioselectivity.

Phenylalanine aminomutase (TcPAM) from Taxus chinensis catalyzes the regioselective hydroamination of trans-cinnamic acid (t-CA) to yield ß-phe. However, the final product mixture consists of both α- and ß-phe owing to low regioselectivity, which is still a challenge to synthesize highly pure ß-phe. Therefore, a modified TcPAM with high ß-selectivity is expected. Based on the catalytic mechanism and structure, two amino acid residues (Asn458 and Leu108) in active sites were identified as the key residues for controlling the regioselective hydroamination of t-CA and as promising candidates for mutagenesis to enhance ß-selectivity and decrease α-selectivity. The Asn458 and Leu108 residues were mutated to yield variant TcPAM-Asn458Phe/Leu108Glu, and the ß-selectivity was approximately 5.2-fold higher than that of wild-type TcPAM, while α-selectivity decreased to 68%, and the percentage of ß-phe in the product mixture increased from 42% to 83%. In addition, the mutant was applied to synthesize ß-arylalanines using substituent t-CA as a substrate. The regioselectivity was also affected by the substituent groups at the phenyl ring of t-CA with respect to their electronic properties and position, and the 4-methoxy and methyl substituent t-CA were transferred into ß-arylalanines. The conversion rate also exceeded 90%. In summary, the engineered TcPAM proved to be useful for one-step asymmetric amination of t-CA and its derivatives to synthesize highly pure ß-arylalanines.

Nano-graphene oxide-manganese dioxide nanocomposites for overcoming tumor hypoxia and enhancing cancer radioisotope therapy.

While radiotherapy (RT) is commonly used in clinics for cancer treatment, the therapeutic efficiency is not satisfactory owing to the existence of the hypoxic tumor microenvironment which seriously affects the efficiency of RT. Herein, we design polyethylene glycol (PEG)-modified reduced nano-graphene oxide-manganese dioxide (rGO-MnO2-PEG) nanocomposites to trigger oxygen generation from H2O2 to reduce the tumor hypoxic microenvironments. We use the radioisotope, 131I labeled rGO-MnO2-PEG nanocomposites as therapeutic agents for in vivo tumor radioisotope therapy (RIT), achieving excellent tumor killing and further enhancing the therapeutic efficiency of RIT. More importantly, the dissolution of MnO2 under acidic conditions and the redox process during the catalytic pathway of H2O2 decomposition in the cellular microenvironment direct to the production of an enormous amount of Mn2+ which has been used as a contrast agent for magnetic resonance imaging (MRI). Our proposed work provides a strategy to trigger oxygen formation via an internal stimulus to enhance imaging-guided RIT efficiency.

Recent advances in enhanced enzyme activity, thermostability and secretion by N-glycosylation regulation in yeast.

Yeast has been increasingly used as a host for the expression of enzymes. Compared to other expression systems, the yeast expression system has many advantages including its suitability for large-scale fermentation and its ability to modify enzymes. When expressed in yeast, many recombinant enzymes are N-glycosylated, and this may play an important role in their activity, thermostability and secretion. Although the mechanism underlying this process is not clear, the regulation of N-glycosylation by introducing or eliminating N-glycosylation at specific sites has developed into an important strategy for improving the production or catalytic properties of recombinant enzymes. In this review, we summarize the recent advances in understanding the effects of N-glycosylation on the expression and characteristics of recombinant enzymes, and discuss novel strategies for regulating N-glycosylation in yeast. We hope that this review will help improve the understanding of the expression and the catalytic properties of N-glycosylated proteins.