Hypoxia Reversion by Low-Immunogenic Ultra-Acid-Sensitive Comicelles of Protein-Polymer Conjugates Sensitizes Tumors to Photodynamic Therapy.

Hypoxia is characteristic of the tumor microenvironment, which is correlated with resistance to photodynamic therapy (PDT), radiotherapy, chemotherapy, and immunotherapy. Catalase is potentially useful to catalyze the conversion of endogenous H2O2 to O2 for hypoxia reversion. However, the efficient delivery of catalase into the hypoxia regions of tumors is a huge challenge. Here, we report the self-assembly of ultra-acid-sensitive polymer conjugates of catalase and albumin into nanomicelles that are responsive to the acidic tumor microenvironment. The immunogenicity of catalase is mitigated by the presence of albumin, which reduces the cross-linking of catalase with B cell receptors, resulting in improved pharmacokinetics. The ultra acid sensitivity of the nanomicelles makes it possible to efficiently escape the lysosomal degradation after endocytosis and permeate into the interior of tumors to reverse hypoxia in vitro and in vivo. In mice bearing triple-negative breast cancer, the nanomicelles loaded with a photosensitizer effectively accumulate and penetrate into the whole tumors to generate a sufficient amount of O2 to reverse hypoxia, leading to enhanced efficacy of PDT without detectable side effects. These findings provide a general strategy of self-assembly to design low-immunogenic ultra-acid-sensitive comicelles of protein-polymer conjugates to reverse tumor hypoxia, which sensitizes tumors to PDT.

A Superlong-Acting Growth Hormone-Polypeptide Fusion for Growth Hormone Deficiency Treatment.

Recombinant human growth hormone (rhGH) is clinically used to treat growth hormone deficiency (GHD). However, daily administration of rhGH is required due to its poor stability and short blood circulation, which causes pains and burdens as well as inconvenience to patients. In this study, a method for genetically fusing rhGH to a thermosensitive polymer of elastin-like polypeptide (ELP) is reported, using which the rhGH-ELP thermosensitive fusion protein can be purified by the thermosensitivity of ELP instead of chromatography. The ELP fusion not only drastically improves the stability of rhGH, but also enables the in situ formation of a sustained-release depot of rhGH-ELP upon subcutaneous (SC) injection, which exhibits gentle release with a platform-to-trough fluctuation in blood and a very long circulatory half-life of 594.6 h. In contrast, rhGH exhibits a peak-to-trough fluctuation in blood with a very short circulatory half-life of 0.7 h. As a result, a single subcutaneous injection of rhGH-ELP can consecutively promote the linear growth of rats and the development of major tissues and organs over 3 weeks without obvious side effects, whereas rhGH is required to be injected daily to achieve similar therapeutic results.

Spatiotemporally-Programmed Dual-Acid-Sensitive Nanoworms of Albumin-Poly(tertiary amine)-Doxorubicin Conjugates for Enhanced Cancer Chemotherapy.

Nanomedicines are potentially useful for targeted cancer chemotherapy; however, it is difficult to design nanomedicines with controllable structures and functions to overcome a series of biological and pathological barriers to efficiently kill cancer cells in vivo. Here, this work reports in situ growth of dual-acid-sensitive poly(tertiary amine)-doxorubicin conjugates from albumin to form dual-acid-sensitive albumin-poly(tertiary amine)-doxorubicin conjugates that self-assemble into nanospheres and nanoworms in a controlled manner. Both nanospheres and nanoworms rapidly dissociate into positively-charged unimers at pH < 6.9 and quickly releases the conjugated drug of doxorubicin at pH < 5.6, leading to enhanced penetration in tumor cell spheroids as well as improved uptake and cytotoxicity to tumor cells at pH < 6.9. Notably, nanoworms are less taken up by endothelial cells than nanospheres and doxorubicin, leading to improved pharmacokinetics. In a mouse model of triple negative breast cancer, nanoworms accumulate and penetrate into tumors more efficiently than nanospheres and doxorubicin, leading to enhanced tumor accumulation and penetration. As a result, nanoworms outperform nanospheres and doxorubicin in suppressing tumor growth and elongating the animal survival time, without observed side effects. These findings demonstrate that intelligent nanoworms with spatiotemporally programmed dual-acid-sensitive properties are promising as next-generation nanomedicines for targeted cancer chemotherapy.

Gut Microbiota Control the Bioavailability and Metabolism of Organoarsenicals of Seaweeds in Mice after Oral Ingestion.

Edible seaweed consumption is an essential route of human exposure to complex organoarsenicals, including arsenosugars and arsenosugar phospholipids. However, the effects of gut microbiota on the metabolism and bioavailability of arsenosugars in vivo are unknown. Herein, two nori and two kelp samples with phosphate arsenosugar and sulfonate arsenosugar, respectively, as the predominant arsenic species, were administered to normal mice and gut microbiota-disrupted mice treated with the broad-spectrum antibiotic cefoperazone for 4 weeks. Following exposure, the community structures of the gut microbiota, total arsenic concentrations, and arsenic species in excreta and tissues were analyzed. Total arsenic excreted in feces and urine did not differ significantly between normal and antibiotic-treated mice fed with kelp samples. However, the total urinary arsenic of normal mice fed with nori samples was significantly higher (p < 0.05) (urinary arsenic excretion factor, 34-38 vs 5-7%), and the fecal total arsenic was significantly lower than in antibiotic-treated mice. Arsenic speciation analysis revealed that most phosphate arsenosugars in nori were converted to arsenobetaine (53.5-74.5%) when passing through the gastrointestinal tract, whereas a large portion of sulfonate arsenosugar in kelp was resistant to speciation changes and was excreted in feces intact (64.1-64.5%). Normal mice exhibited greater oral bioavailability of phosphate arsenosugar from nori than sulfonate arsenosugar from kelp (34-38 vs 6-9%). Our work provides insights into organoarsenical metabolism and their bioavailability in the mammalian gut.

Active-targeting long-acting protein-glycopolymer conjugates for selective cancer therapy.

Non-fouling polymers are effective in improving the pharmacokinetics of therapeutic proteins, but short of biological functions for tumor targeting. In contrast, glycopolymers are biologically active, but usually have poor pharmacokinetics. To address this dilemma, herein we report in situ growth of glucose- and oligo(ethylene glycol)-containing copolymers at the C-terminal site of interferon alpha, an antitumor and antivirus biological drug, to generate C-terminal interferon alpha-glycopolymer conjugates with tunable glucose contents. The in vitro activity and in vivo circulatory half-life of these conjugates were found to decrease with the increase of glucose content, which can be ascribed to complement activation by the glycopolymers. Additionally, the cancer cell endocytosis of the conjugates was observed to maximize at a critical glucose content due to the tradeoff between complement activation and glucose transporter recognition by the glycopolymers. As a result, in mice bearing ovarian cancers with overexpressed glucose transporter 1, the conjugates with optimized glucose contents were identified to possess improved cancer-targeting ability, enhanced anticancer immunity and efficacy, and increased animal survival rate. These findings provided a promising strategy for screening protein-glycopolymer conjugates with optimized glucose contents for selective cancer therapy.

Cytokine conjugates to elastin-like polypeptides.

Cytokines are a group of pleiotropic proteins which are crucial for various biological processes and useful as therapeutics. However, they usually suffer from the poor stability, extreme short circulation half-life, difficulty in high-yield and large-scale production and side effects, which greatly restricts their applications. Over the past decades, conjugation of cytokines with elastin-like polypeptides (ELPs), a type of promising biomaterials, have showed great potential in solving these challenges due to ELP's thermal responsiveness, excellent biocompatibility and biodegradability, non-immunogenicity, and ease of design and control at the genetic level. This review presents recent progress in the design and production of a variety of ELP conjugated cytokines for extended circulation, enhanced stability, increased soluble protein expression, simplified purification, improved drug delivery, and controlled release. Notably, the unique thermoresponsive properties of cytokine-ELP conjugates make it possible to self-assemble into micelles with drastically extended circulatory half-life for targeted delivery or to in situ form drug depots for topical administration and controlled release. The challenges and issues in the emerging field are further discussed and the future directions are pointed out at the end of this review.

Self-Activated Cascade Biocatalysis of Glucose Oxidase-Polycation-Iron Nanoconjugates Augments Cancer Immunotherapy.

Biocatalytic therapy by reactive-oxygen-species-generating enzymes not only kills cancer cells directly but also stimulates an anticancer immune response and inverses the immunosuppressive microenvironment of a variety of solid tumors, which is potentially beneficial to overcoming the limitations of cancer immunotherapy. Herein, we report the in situ growth of polycation chains from glucose oxidase to generate glucose oxidase-polycation conjugates, which can be used as a template for the in situ reduction of ferrous ions into iron nanoparticles to yield glucose oxidase-polycation-iron nanoconjugates. The nanoconjugates exhibit enhanced cellular uptake and cancer retention as well as self-activated cascade biocatalysis that consumes glucose and generates highly toxic hydroxyl radicals, leading to enhanced starvation-like and chemodynamic cancer therapy. The cancer treatment with the nanoconjugates efficiently triggers the program of immunogenic cell death for enhanced immune checkpoint blockade therapy. The synergy of self-activated cascade biocatalysis and immune checkpoint blockade not only eradicates primary cancers but also inhibits the progression of distant cancers, which leads to the abscopal effect on cancers. Our findings provide a method for the in situ synthesis of self-activated cascade nano-biocatalysts for cascade biocatalysis-enhanced immunotherapy of cancer.

Scalable Purification of Plasmid DNA Nanoparticles by Tangential Flow Filtration for Systemic Delivery.

Plasmid DNA (pDNA) nanoparticles synthesized by complexation with linear polyethylenimine (lPEI) are one of the most effective non-viral gene delivery vehicles. However, the lack of scalable and reproducible production methods and the high toxicity have hindered their clinical translation. Previously, we have developed a scalable flash nanocomplexation (FNC) technique to formulate pDNA/lPEI nanoparticles using a continuous flow process. Here, we report a tangential flow filtration (TFF)-based scalable purification method to reduce the uncomplexed lPEI concentration in the nanoparticle formulation and improve its biocompatibility. The optimized procedures achieved a 60% reduction of the uncomplexed lPEI with preservation of the nanoparticle size and morphology. Both in vitro and in vivo studies showed that the purified nanoparticles significantly reduced toxicity while maintaining transfection efficiency. TFF also allows for gradual exchange of solvents to isotonic solutions and further concentrating the nanoparticles for injection. Combining FNC production and TFF purification, we validated the purified pDNA/lPEI nanoparticles for future clinical translation of this gene nanomedicine.

Construction of hierarchical 2D/2D Ti3C2/MoS2 nanocomposites for high-efficiency solar steam generation.

Solar steam generation has been considered one of the most promising approaches for dealing with the energy and freshwater resource crises in recent years. However, achieving high efficiency in photo-thermal conversion remains a considerable challenge. Here, a series of hierarchical Ti3C2/MoS2 nanocomposites were designed for steam generation by a hydrothermal method. When the mass fraction of MoS2 reached 65 wt% (TM-3), the Ti3C2/MoS2 nanocomposite presented a strong broad-band light absorption of 92.4% from the UV to NIR region because of the accordion-like layered structure. The evaporation rate and solar-thermal conversion efficiency of the TM-3 with as-fabricated evaporator could reach 1.36 kg·m-2·h-1 and 87.2% under 1 kW/m2, due to the excellent light absorption ability of TM-3 and the low thermal energy loss (8.8%) of the evaporator. Meanwhile, TM-3 permits the evaporator to have remarkable cycle stability because of its hydrophobic properties. Moreover, TM-3 showed excellent seawater desalination and wastewater treatment abilities. Thus, the excellent light absorption ability, photo-thermal conversion efficiency, and stability of the overall system suggested that these nanocomposites show great potential applications in synergetic solar desalination and sewage treatment.

Kinetic Control in Assembly of Plasmid DNA/Polycation Complex Nanoparticles.

Polyelectrolyte complex (PEC) nanoparticles assembled from plasmid DNA (pDNA) and polycations such as linear polyethylenimine (lPEI) represent a major nonviral delivery vehicle for gene therapy tested thus far. Efforts to control the size, shape, and surface properties of pDNA/polycation nanoparticles have been primarily focused on fine-tuning the molecular structures of the polycationic carriers and on assembly conditions such as medium polarity, pH, and temperature. However, reproducible production of these nanoparticles hinges on the ability to control the assembly kinetics, given the nonequilibrium nature of the assembly process and nanoparticle composition. Here we adopt a kinetically controlled mixing process, termed flash nanocomplexation (FNC), that accelerates the mixing of pDNA solution with polycation lPEI solution to match the PEC assembly kinetics through turbulent mixing in a microchamber. This achieves explicit control of the kinetic conditions for pDNA/lPEI nanoparticle assembly, as demonstrated by the tunability of nanoparticle size, composition, and pDNA payload. Through a combined experimental and simulation approach, we prepared pDNA/lPEI nanoparticles having an average of 1.3 to 21.8 copies of pDNA per nanoparticle and average size of 35 to 130 nm in a more uniform and scalable manner than bulk mixing methods. Using these nanoparticles with defined compositions and sizes, we showed the correlation of pDNA payload and nanoparticle formulation composition with the transfection efficiencies and toxicity in vivo. These nanoparticles exhibited long-term stability at -20 °C for at least 9 months in a lyophilized formulation, validating scalable manufacture of an off-the-shelf nanoparticle product with well-defined characteristics as a gene medicine.

Multi-element fingerprinting as a tool in origin authentication of four east China marine species.

The contents of 25 elements in 4 types of commercial marine species from the East China Sea were determined by inductively coupled plasma mass spectrometry and atomic absorption spectrometry. The elemental composition was used to differentiate marine species according to geographical origin by multivariate statistical analysis. The results showed that principal component analysis could distinguish samples from different areas and reveal the elements which played the most important role in origin diversity. The established models by partial least squares discriminant analysis (PLS-DA) and by probabilistic neural network (PNN) can both precisely predict the origin of the marine species. Further study indicated that PLS-DA and PNN were efficacious in regional discrimination. The models from these 2 statistical methods, with an accuracy of 97.92% and 100%, respectively, could both distinguish samples from different areas without the need for species differentiation.

Graphene based pipette tip solid phase extraction of marine toxins in shellfish muscle followed by UPLC-MS/MS analysis.

Graphene is a novel carbonic material with great potentials for the use as sorbent due to its ultrahigh surface area. Herein, we report the use of graphene as sorbent in solid-phase extraction (SPE) using pipette tip as cartridge namely GPT-SPE, together with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), for the analysis of lipophilic marine toxins (LMTs), including yessotoxins (YTX), okadaic acid (OA), dinophysistoxin-1 (DTX1), gymnodimine (GYM), spirolides-1 (SPX1), pectenotoxin-2 (PTX2) and azaspiracid-1 (AZA1) in shellfish. The GPT-SPE procedure was optimized and the performance of graphene was fully validated. Results with high-sensitivity and good reproducibility was obtained and compared with that of other sorbents like C18 silica, multi-walled carbon nanotubes (MWCNTs), commercial Oasis HLB, and Strata-X for the extraction of LMTs, which showed superiority and advantages of graphene, such as good recoveries, stability and compatibility with various solvents. In order to exhibit the potentials of graphene as an excellent sorbent material, 67 mussel samples from six coastal cities of China were analyzed. OA was found to be the dominant contaminant, while YTX was also detected with low level.

Pipette tip solid-phase extraction and ultra-performance liquid chromatography/mass spectrometry based rapid analysis of picrosides from Picrorhiza scrophulariiflora.

Pipette tip solid-phase extraction (PT-SPE) is a technique popular in sample preparation of biological fluids and protein hydrolysates. In this study, we developed a microtechnic using a pipette tip packed with C18 as sorbent for extraction and purification of bioactive compounds, picroside-I, II and III, in crude herbal extracts from Picrorhiza scrophulariiflora (P. scrophulariiflora). Compared to conventional SPE, PT-SPE is fast, easy to operate, and the tools are very accessible (pipette tip and tube, without expensive SPE set-up). Moreover, it is also cost-effective because significant amount of sorbent and solvents can be saved. The eluate was analyzed by ultra-performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS). Afterwards, the method was fully validated and the results demonstrated that the PT-SPE-UPLC-MS/MS method is an excellent technique for analysis of the herbal medicine. Finally, this PT-SPE-UPLC-MS/MS strategy was successfully applied to analyze the crude extracts from P. scrophulariiflora samples within 10min (2min for PT-SPE and 8min for UPLC), 3.5mL solvents (including water, 0.3mL for PT-SPE and 3.2mL for UPLC), and 2mg C18 sorbent for each sample. We believe this method to be very practical and, in particular, to be suitable for widespread herbal medicine analysis.

Shotgun lipidomics strategy for fast analysis of phospholipids in fisheries waste and its potential in species differentiation.

An efficient shotgun lipidomics strategy was established and optimized for fast phospholipid profiling of viscera from three fish species: Lateolabrax japonicas, Ctenopharyngodon idellus, and Carassius auratus. This strategy relies on direct infusion of total lipid extracts into a tandem mass spectrometer without additional separation of the individual molecular species. Four classes of phospholipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS), were analyzed, and at least 81 molecular species of phospholipids were identified, including 34 species of PC, 24 species of PE, 12 species of PS, and 11 species of PI, in both positive- and negative-ion electrospray ionization mode. The results show that fish viscera, which are traditionally discarded as fisheries wastes, are nutritional in phospholipids with total contents of the four detected phospholipid classes ranging from 1.52 to 3.29 mg/g in the three tested fish species. Regardless of the tested fish species, PC and PE are the dominant phospholipid classes, followed by PI and PS. Furthermore, principal component analysis (PCA) was applied to normalize the relative amounts of the identified phospholipid species. The results demonstrate that PS 18:0/22:6, PI 18:0/20:4, and PI 18:0/20:5 were the main contributors of cumulative value and could be used as an indicator for fish species differentiation. This shotgun lipidomics method was >10 times faster than traditional methods, because no chromatographic separation was needed. The successful application of this strategy paves the way for full utilization of traditionally discarded fisheries wastes and provides an alternative means for fish species differentiation.