[Establishment of a quantitative method for GC analysis of polyoxyethylene (35) castor oil in microemulsion extracts].

With the continuous exploration of microemulsions as solvents for traditional Chinese medicine extraction, polyoxyethy-lene(35) castor oil(CrEL), a commonly used surfactant, is being utilized by researchers. However, the problem of detecting residues of this surfactant in microemulsion extracts has greatly hampered the further development of microemulsion solvents. Based on the chemical structures of the components in CrEL and the content determination method of castor oil in the 2020 edition of the Chinese Pharmacopoeia(Vol. Ⅳ), this study employed gas chromatography(GC) and single-factor experiments to optimize the preparation method of methyl ricinoleate from CrEL. The conversion coefficient between the two was validated, and the optimal sample preparation method was used to process microemulsion extracts of Zexie Decoction from three batches. The content of methyl ricinoleate generated was determined, and the content of CrEL in the microemulsion extracts of Zexie Decoction was calculated using the above conversion coefficient. The results showed that the optimal preparation method for CrEL was determined. Specifically, 10 mL of 1 mol·L~(-1) KOH-methanol solution was heated at 60 ℃ for 15 min in a water bath. Subsequently, 10 mL of boron trifluoride etherate-methanol(1∶3) solution was heated at 60 ℃ for 15 min in a water bath, followed by extraction with n-hexane twice. CrEL could stably produce 20.84% methyl ricinoleate. According to this conversion coefficient, the average mass concentration of CrEL in the three batches of Zexie Decoction microemulsion extracts was 11.94 mg·mL~(-1), which was not significantly different from the CrEL mass concentration of 11.57 mg·mL~(-1) during microemulsion formulation, indicating that the established content determination method of this study was highly accurate, sensitive, and repeatable. It can be used for subsequent research on microemulsion extracts of Zexie Decoction and provide a reference for quality control of other drug formulations containing CrEL.

The novel use of lauromacrogol: A respective study of ultrasound-guided sclerosant injection for cesarean scar pregnancy.

AIM: To introduce the novel use of lauromacrogol for cesarean scar pregnancy (CSP), and to compare the clinical efficacy and safety of curettage combined with ultrasound-guided sclerosant injection (USI) and curettage following uterine artery embolization (UAE) in the treatment of CSP. METHODS: CSP patients undergoing curettage combined with USI (n = 72) from December 2014 to May 2020 were compared to patient with curettage following UAE (n = 72).The basic clinical findings and clinical outcomes were reviewed between the two groups. RESULTS: For USI group, 69 patients underwent successful treatment (95.8% success rate), while the number of cured patients for the UAE group was 70 (97.2% success rate). Differences between USI group and UAE group in intraoperative blood loss (10.0 [10.0-20.0] vs. 10.0 [10.0-20.0] mL) and time for serum ß human chorionic gonadotropin (ß-hCG) to reduce to normal (28.0 [21.0-40.0] vs. 28.0 [21.0-35.0] days) were not statistically significant. The hospital stay for USI group was significantly shorter than that for UAE group (4.0 [4.0-6.0] vs. 6.0 [5.0-7.0] days, respectively). Statistically significant decreases were noted in hospitalization expenses and adverse events in USI group, compared to UAE group. There was no difference in live birth rate between the two groups with fertility intentions during the follow-up. CONCLUSION: For treatment of CSP, curettage combined with USI yielded clinical results comparable to those of curettage following UAE. Curettage combined with USI was associated with lower hospitalization expenses, shorter hospital stay and less complications, and it merited an effective and safe treatment for CSP.

High power femtosecond semiconductor lasers based on saw-toothed master-oscillator power-amplifier system with compressed ASE.

High power femtosecond semiconductor laser based on saw-toothed taper mode-locked laser and amplifier was demonstrated with compressed amplified spontaneous emission (ASE). The external-cavity mode-locked taper laser generated the clean optical pulses without any sub-pulse components. A semiconductor optical amplifier (SOA) with tilted taper waveguide and saw-toothed edge reduced evidently the ASE background. The saw-tooth microstructures were optimized and it was found that the saw-tooth of right-right angled triangle showed the best effect. The ratio of the maximum intensity to background radiation was increased by 21.9% and the power was increased by 30.5% due to the saw-tooth microstructure in the SOA. The pulse duration of 495 fs and a peak power over 1.5 kW with repetition rate of 579 MHz were realized after a double-pass grating compressor.

Site-Specific Conjugation of Polymers to Proteins.

Conjugation of polymer to protein has been widely employed in therapeutics, medicine, biotechnology, and enzymatic catalysis. The synergistic effect benefits both counterparts and potentially overcomes their inherent limitations. This article reviews the strategies for the site-specific synthesis of well-defined protein-polymer conjugates, aiming to provide a toolbox for the community. First, it is essential to set a definite reactive site on the protein because the position of the reaction site can directly influence the reaction activity and the bioactivity of the protein after modification. The origins of the specific functional groups on protein include the utilization of the unique natural amino acid, mutagenesis to introduce a sole reactive amino acid, chemical modification, noncanonical amino acid incorporation, and enzyme-mediated introduction of functional groups. Second, the main conjugation methods, i.e., "grafting to" and "grafting from" methods, are summarized and compared with each other. In the "grafting to" method, a comprehensive investigation on the reactions used to attach an end functional polymer chain to a protein is conducted according to the position of the target site and its nature. In the "grafting from" method, a comparison between the commonly used controlled polymerization, i.e., atom transfer radical polymerization (ATRP) and reversible addition-fragmentation transfer (RAFT), is surveyed. Further, a special case where a noncovalent bond is adopted to link the protein and polymer together is investigated due to its high specificity and reversibility, typically biotin-(strept)avidin-based interactions and metal-mediated conjugation. Finally, applications of protein-polymer conjugates in drug delivery, biomedicine, biosensor, and the disease-related protein self-assembly are illustrated. This precise review on the conjugation of polymer chain to protein to form well-defined protein-polymer conjugates summarizes the representative strategies and may provide useful cues in the areas of biotechnology, therapeutic drugs, and biomedicine.

Quantitative Study of the Oligomerization of Yeast Prion Sup35NM Proteins.

The fibrillation of misfolded neurodegenerative disease-related proteins has been extensively studied over the past few decades, but the initial oligomerization has been rarely examined even though some recent evidence indicated that small protein oligomers are more neurotoxic than long protein fibers. It is rather difficult to study the initial oligomerization (nucleation) because most experimental methods, such as the ß-sheet-related fluorescence assay and X-ray diffraction, are unable to detect small structureless protein oligomers. In this study, we have successfully developed a method to link a short thermally sensitive poly(N-isopropylacrylamide) (PNIPAM) chain to a model protein, Sup35NM, at a specific 31st residue site (Sup35NM-31m-PNIPAM) via the efficient thiol-ene Michael addition reaction. The oligomerization was studied by a combination of laser light scattering, the thioflavin T assay, and transmission electron microscopy. We found that the lag phase of Sup35NM was delayed from 12 to >24 h under the physiological condition after the PNIPAM linkage. The oligomerization and fibrillation constants decreased from 5.0 × 10-3 to 1.5 × 10-3 h-1 and from 3.0 × 10-2 to 1.8 × 10-2 µM-1 h-1, respectively, presumably because of the steric hindrance introduced by the PNIPAM chain. Moreover, after initiating the oligomerization, we found that the oligomer distribution in the first 6 h repeatedly and quantitatively follows the Smoluchowski coagulation model. Our study paves the way for controllably and quantitatively studying the oligomerization kinetics of amyloidogenic proteins. In addition, by investigating the effects of different small molecules on the oligomerization kinetics, we should be able to screen potential drugs to slow the development of neurodegenerative diseases.

Upregulation of MHC-I and downregulation of PD-L1 expression by doxorubicin and deferasirox codelivered liposomal nanoparticles for chemoimmunotherapy of melanoma.

Tumor immunotherapy is a promising strategy to activate the immune system and eliminate tumors. Major histocompatibility complex I (MHC-I) is usually applied to potentiate antigen presentation, but it is associated with upregulation of programmed death ligand 1 (PD-L1) expression, which is unfavorable for activation of immune responses. Moreover, poor permeability of various therapeutic antibodies results in the limited immune response rates of most patients. It is necessary to develop combined small molecule drug delivery systems for simultaneous upregulation of MHC-I expression and downregulation of PD-L1 expression, promoting effective tumor treatment. A moderate dose of doxorubicin hydrochloride (DOX) can induce upregulation of MHC-I expression, while deferasirox (DFX) can inhibit the PI3K-Akt pathway, which potentially downregulates PD-L1 expression. In the present study, we designed a pH-sensitive liposome to incorporate DOX in the hydrophilic cavity and embed DFX in the hydrophobic shell, forming a dual delivery system (DOX-DFXL). In a B16F10 melanoma-bearing mouse model, DOX and DFX were released in acidic tumor microenvironment, which further lead to enhanced antigen presentation and infiltration of T cells into tumor tissues as a result of tumor remission. This codelivery system holds great potential for clinical applications of tumor immunotherapy.

Functional 2D Iron-Based Nanosheets for Synergistic Immunotherapy, Phototherapy, and Chemotherapy of Tumor.

Immunotherapy efficacy has been limited by tumor-associated macrophages (TAMs), which are the most abundant immune regulatory cells infiltrating around tumor tissues. The repolarization of pro-tumor M2 TAMs to anti-tumor M1 TAMs is a very promising immunotherapeutic strategy for cancer therapy. In this manuscript, multifunctional 2D iron-based nanosheets (FeNSs) are synthesized via a simple hydrothermal method for the first time, which not only possess photothermal and photodynamic properties, but also can repolarize TAMs from M2 to M1. After modifying with polyethylene glycol and loading with bioreductive prodrug banoxantrone (AQ4N), abbreviated as AP FeNSs, it can effectively repolarize TAMs from M2 to M1 and deliver AQ4N to tumor microenvironment (TME). Moreover, the repolarized M1 TAMs overexpress inducible nitric oxide synthase, which can convert nontoxic AQ4N to cytotoxic AQ4 under hypoxic TME, enabling immunomodulation-activated chemotherapy. A series of in vitro and in vivo results corroborate that AP FeNSs effectively exert photothermal and photodynamic effects and repolarize M2 TAMs to M1 TAMs, releasing inflammatory factors and activating the chemotherapeutic effect, thereby realizing synergistic tumor therapy.

Immunomodulation of Tumor Microenvironment by Arginine-Loaded Iron Oxide Nanoparticles for Gaseous Immunotherapy.

Tumor-associated macrophages (TAMs) of M2 phenotype have mediated the immunosuppression in a tumor microenvironment, facilitating the escape of tumor cells from immunosurveillance. Reprograming the immunosuppressive M2 TAMs to immunostimulatory M1 phenotype can activate the antitumor immune responses for cancer immunotherapy. Herein, hollow iron oxide (Fe3O4) nanoparticles (NPs) were employed to reprogram M2 TAMs toward M1 TAMs, aiming to release proinflammatory cytokines and recruit T cells to kill tumor cells. After loaded with l-arginine (l-Arg) and sealed with poly(acrylic acid) (PAA), hollow Fe3O4 NPs were fabricated into LPFe3O4 NPs, which could release l-Arg based on pH-responsive PAA and produce nitric oxide (NO) with the help of nitric oxide synthase (iNOS) overexpressed by M1 TAMs, as a result of additional tumor elimination for gas therapy. In vitro and in vivo studies demonstrate that LPFe3O4 NPs could effectively reprogram M2 to M1 macrophages, activating T cells, releasing TNF-α, and producing high levels of NO, leading to synergistic tumor therapy.

Quantitative study of effects of free cationic chains on gene transfection in different intracellular stages.

Previously, we revealed that in the application of using cationic polymer chains, polyethylenimine (PEI), to condense anionic plasmid DNA chains (pDNA) to form the DNA/polymer polyplexes, after all the pDNAs are complexed with PEI, further added PEIs exist individual chains and free in the solution mixture. It is those uncomplexed polycation chains that dramatically promote the gene transfection. In the current study, we studied how those free cationic chains with different lengths and topologies affect the intracellular trafficking of the polyplexes, the translocation of pDNA through the nuclear membrane, the transcription of pDNA to mRNA and the translocation of mRNA from nucleus to cytosol in HepG2 cells by using a combination of the three-dimensional confocal microscope and TaqMan real-time PCR. We found that free branched PEI chains with a molar mass of 25,000g/mol and a total concentration of 1.8×10(-6)g/mL promote the overall gene transfection efficiency by a factor of ~500 times. Our results quantitatively reveal that free chains help little in the cellular uptake, but clearly reduce the lysosomal entrapment of those internalized polyplexes (2-3 folds); assist the translocation of pDNA through nuclear membrane after it is released from the polyplexes in the cytosol (~5 folds); enhance the pDNA-to-mRNA transcription efficiency (~4 folds); and facilitate the nucleus-to-cytosol translocation of mRNA (7-8 folds). The total enhancement of those steps agrees well with the overall efficiency, demonstrating, for the first time, how free cationic polymer chains quantitatively promote the gene transfection in each step in the intracellular space.