Hierarchically Structured Hydroxyapatite Particles Facilitate the Enhanced Integration and Selective Anti-Tumor Effects of Amphiphilic Prodrug for Osteosarcoma Therapy.

Efficient delivery of cargo into target cells is a formidable challenge in modern medicine. Despite the great promise of biomimetic hydroxyapatite (HA) particles in tissue engineering, their potential applications in bone tumor therapy, particularly their structure-function relationships in cargo delivery to target cells, have not yet been well explored. In this study, biomimetic multifunctional composite microparticles (Bm-cMPs) are developed by integrating an amphiphilic prodrug of curcumin with hierarchically structured HA microspheres (Hs-hMPs). Then, the effects of the hierarchical structure of vehicles on the integration and delivery of cargo as well as the anti-osteosarcoma (OS) effect of the composite are determined. Different hierarchical structures of the vehicles strongly influence the self-assembly behavior of the prodrug. The flake-like crystals of Hs-hMPs enable the highest loading capacity and enhance the stability of the cargo. Compared to the normal cells, OS cells exhibit 3.56-times better uptake of flake-like Hs-hMPs, facilitating the selective anti-tumor effect of the prodrug. Moreover, Bm-cMPs suppress tumor growth and metastasis by promoting apoptosis and inhibiting cell proliferation and tumor vascularization. The findings shed light on the potential application of Bm-cMPs and suggest a feasible strategy for developing an effective targeted therapy platform using hierarchically structured minerals for OS treatment.

Nitric oxide-containing supramolecular polypeptide nanomedicine based on [2]biphenyl-extended-pillar[6]arenes for drug resistance reversal.

A kind of supramolecular polypeptide nanomedicine (BPC/DOX-ICG) was constructed with an anionic water-soluble [2]biphenyl-extended-pillar[6]arene (AWBpP6), and pyridinium-terminal- and S-nitrosothiol (SNO)-modified polypeptide (PPNC) via host-guest interactions to co-deliver doxorubicin (DOX) and indocyanine green (ICG) for drug resistance reversal. Upon near-infrared (NIR) irradiation, the NO generation could down-regulate the P-glycoprotein (P-gp) expression level to reverse multidrug resistance (MDR). Subsequently, the resulting reverse MDR could sensitize the free DOX and assist photothermal therapy (PTT) to enhance the tumoricidal potential. This supramolecular polypeptide nanomedicine provides an effective strategy for the multimodal synergistic therapies of photothermal therapy, NO generation therapy, and chemotherapy (i.e., PTT-NO-CT) to overcome MDR.

Multivalent Polypeptide and Tannic Acid Cooperatively Iron-Coordinated Nanohybrids for Synergistic Cancer Photothermal Ferroptosis Therapy.

Owing to having a unique mechanism to kill cancer cells via the membrane accumulation of lipid peroxide (LPO) and the downregulation of glutathione peroxidase-4 (GPX-4), the ferroptosis therapy (FT) of tumors based on the Fenton reaction of iron nanoparticles has been receiving much attention in the past decade; however, there are some hurdles including the uncontrollable release of iron ions, slower kinetics of the intracellular Fenton reaction, and poor efficacy of FT that need to be overcome. Considering cooperative coordination of a multivalent thiol-pendant polypeptide ligand with iron ions, we put forward a facile strategy for constructing the iron-coordinated nanohybrid of methacryloyloxyethyl phosphorylcholine-grafted polycysteine/iron ions/tannic acid (i.e., PCFT), which could deliver a higher concentration of iron ions into cells. The dynamic and unsaturated coordination in PCFT is favorable for the intracellular stimuli-triggered release and fast Fenton reaction to realize efficient FT, while its intrinsic photothermia would boost the Fenton reaction to induce a synergistic effect between FT and photothermal therapy (PTT). Both immunofluorescence analyses of reactive oxygen species (ROS) and LPO confirmed that the intracellular Fenton reaction resulted in efficient FT, during which process the photothermia greatly boosted ferroptosis, and the Western blot assay corroborated that the expression level of GPX-4 was downregulated by FT and highly degraded by the photothermia to induce synergistic PTT-FT in vitro. Excitingly, by a single intravenous dose of PCFT plus one NIR irradiation, in vivo PTT-FT treatment completely eradicated 4T1 tumors without skin scar and tumor recurrence for 16 days, demonstrating prominent antitumor efficacy, as evidenced by the GPX-4, H&E, and TUNEL assays.

Polymeric photothermal agents for cancer therapy: recent progress and clinical potential.

Over the past decades, near infrared light (NIR)-sensitive photothermal agents (PTAs) that can efficiently absorb light and generate heat have been investigated worldwide for cancer photothermal therapy (PTT) and the combination treatments, which have some peculiar advantages including spatiotemporal targeting, the ability-to-reverse multidrug resistance, the immunity-stimulating function, and the synergistic effect in combination treatments. In this review, we first focus on emerging melanin-like polymers and coordination polyphenol polymer-based PTAs that hold transition potential because of their facile synthesis and good biocompatibility/biodegradability. We briefly introduce polymeric PTAs for emerging NIR-II (1000-1700 nm) PTT in deep tumors to overcome shallow penetration depth and threshold irradiation intensity of NIR-I (700-900 nm). Then we discuss polymeric PTAs for combination PTT treatments with photodynamic therapy (PDT), ferroptosis therapy (ferrotherapy), and immunotherapy, which are intensively studied for achieving anticancer synergistic effects. Finally, we discuss those polymeric PTAs for reversing cancer multidrug resistance and for mild/low-temperature PTT (43 °C ≤ T < 50 °C) in contrast to conventional high-temperature PTT (>50 °C). The polymeric PTA-based PTT and the combination treatments are still being developed in the early stage and need much more effort before potential clinical transitions and applications.

Accelerating gene function discovery by rapid phenotyping of fatty acid composition and oil content of single transgenic T1 Arabidopsis and camelina seeds.

Arabidopsis is wildly used as a model plant and camelina is increasingly used for oilseed research and applications. Although the Arabidopsis genome has been sequenced for two decades, the functions of many lipid-related genes and their regulators have not been well characterized. Improvements in the efficiency and accuracy of gene investigations are key to effective discovery of gene function and downstream bioengineering of plant oil quantity and quality. In this study, a visible marker was used to quickly identify transgenic T1 seeds and a method has been developed to phenotype fatty acid compositions and oil content of single T1 seeds. A whole seed direct transmethylation method was first optimized with multiple seeds and incubation at 85°C for 2 hours in a transmethylation solvent (5% H2SO4 in methanol with 30% toluene cosolvent) is recommended. Based on this method, a single Arabidopsis seed mini-transmethylation (SAST) method has been established in a 1.5 ml GC sample vial with 200 µl transmethylation solvent. Characteristics of the method were evaluated and it was used to phenotype transgenic T1 seeds expressing AtFAD2 or RcWRI1. Our results indicate that fatty acid composition of T1 individual seeds are consistent with those of pools of multiple seeds from higher generations. However, oil content per individual seed varied substantially and therefore pooling five seeds is recommended for phenotyping oil content of T1 seeds. Additionally, a whole camelina single-seed direct transmethylation was evaluated and results confirm its feasibility. The suitability of partial seed analysis of camelina was investigated but variation in composition of different seed tissues limits this approach.

WRINKLED1 homologs highly and functionally express in oil-rich endosperms of oat and castor.

Oat (Avena sativa) and castor (Ricinus communis) accumulate a large amount of lipids in their endosperms, however the molecular mechanism remains unknown. In this study, differences in oil regulators between oat and wheat (Triticum aestivum) as well as common features between oat and castor were tested by analyzing their transcriptomes with further q-PCR analysis. Results indicated that WRINKLED1 (WRI1) homologs and their target genes highly expressed in the endosperms of oat and castor, but not in the starchy endosperms of wheat. Expression pattern of WRI1s was in agreement with that of oil accumulation. Three AsWRI1s (AsWRI1a, AsWRI1b and AsWRI1c) and one RcWRI1 were identified in the endosperms of oat and castor, respectively. AsWRI1c lacks VYL motif, which is different from the other three WRI1s. Expressions of these four WRI1s all complemented the phenotypes of Arabidopsis wri1-1 mutant. Overexpression of these WRI1s in Arabidopsis and tobacco BY2 cells increased oil contents of seeds and total fatty acids of the cells, respectively. Moreover, this overexpression also resulted in up-regulations of WRI1 target genes, such as PKp-ß1. Taken together, our results suggest that high and functional expression of WRI1 play a key role in the oil-rich endosperms and the VYL motif is dispensable for WRI1 function.

NIR-Responsive Polypeptide Nanocomposite Generates NO Gas, Mild Photothermia, and Chemotherapy to Reverse Multidrug-Resistant Cancer.

Multidrug resistance (MDR) of cancers that results from overexpression of a P-glycoprotein (P-gp) transporter mainly causes chemotherapy (CT) failure and hinders clinical transitions of current polypeptide nanomedicines. Herein, a novel polypeptide nanocomposite PNOC-PDA that integrates heat-sensitive NO gas delivery and photothermal conversion attributes can overcome MDR and maximize CT; meanwhile the optimized CT and intracellular high-concentration NO gas can assist a mild photothermal therapy (PTT) to eradicate cancer cells. The triple therapies produced a superior and synergistic effect on MDR-reversal and killing MCF-7/ADR in vitro, and the P-gp expression level was downregulated to 46%, as confirmed by means of MTT, Western blot, flow cytometry, and confocal laser scanning microscopy. Significantly, by using one intravenous injection of PNOC-PDA/DOX and a single near-infrared irradiation, the triple therapies of mild PTT, NO gas therapy, and CT achieved complete MCF-7/ADR tumor ablation without skin damage, scarring, and tumor recurrence within 30 days. This work provides a versatile method for the fabrication of NIR-responsive polypeptide nanocomposite with intrinsic photothermal conversion and NO-releasing attributes, opening up a new avenue for reversing MDR in tumors.

[Preparation and osteogenic properties of poly ( L-lactic acid)/lecithin porous scaffolds with open pore structure].

Objective: To investigate the preparation and osteogenic properties of poly ( L-lactic acid)(PLLA)/lecithin porous scaffolds with open pore structure. Methods: PLLA/lecithin porous scaffolds with different lecithin contents (0, 5%, 10%, 20%, 30%, 40%, 50%) were prepared by thermally induced phase separation (groups A, B, C, D, E, F, and G, respectively). Scanning electron microscopy (SEM) was used to observe the surface morphology of the scaffolds. Wide-angle X-ray diffraction (XRD) and differential scanning calorimetry (DSC) were used to detect the crystallinity of the scaffolds. The water uptake ability of the scaffolds was measured. The cell growth and viability of bone marrow mesenchymal stem cells (BMSCs) of mouse on each scaffold was assessed by cell counting kit 8 (CCK-8) method. The osteogenic differentiation ability of BMSCs on each scaffold was evaluated by alkaline phosphatase (ALP) activity. Finally, a critical-size rat calvarial bone defect model was used to evaluate the osteogenesis of the scaffolds in vivo. Micro-CT was used to reconstruct the three-dimensional model of the defect area, and the bone volume and bone mineral density were quantitatively analyzed. Results: SEM results showed that the lecithin could slightly reduce the pore size; when lecithin content was 50%, platelet-like structure could be observed on the scaffolds. Wide angle XRD and DSC showed that the crystallinity of scaffolds gradually decreased with the increase of lecithin content. The water uptake ability test showed that the hydrophilicity of scaffolds increased with the increase of lecithin content. CCK-8 assay showed that cell activity gradually increased with the increase of culture time. After 7 days of culture, the absorbance ( A) value of groups C, D, E, and F were significantly higher than that of groups A, B, and G ( P<0.05), but no significant difference was found among groups C, D, E, and F ( P>0.05). After 14 days of osteogenic induction, with the increase of lecithin content, there was a significant difference in ALP activity of each group. The ALP activity in groups D, E, F, and G were significantly higher than that in groups A, B, and C ( P<0.05). In vivo, the results of Micro-CT examination and bone volume and bone mineral density showed that the scaffolds with 30% lecithin had the best repairing effect. Conclusion: Prepared by thermally induced phase separation, the cytocompatibility, osteogenic differentiation, and bone repair ability of the PLLA/lecithin porous scaffold is obviously better than that of pure PLLA scaffold. PLLA/lecithin porous scaffold with suitable lecithin content is a promising scaffold material for bone tissue engineering.

Biopolymer-Drug Conjugate Nanotheranostics for Multimodal Imaging-Guided Synergistic Cancer Photothermal-Chemotherapy.

Some of the biomedical polymer-drug conjugates are being translated into clinical trials; however, they intrinsically lack photothermal and multi-imaging capabilities, hindering them from imaging-guided precision cancer therapy and complete tumor regression. We introduce a new concept of all-in-one biopolymer-drug conjugate nanotheranostics and prepare a kind of intracellular pH-sensitive polydopamine-doxorubicin (DOX) conjugate nanoparticles (PDCNs) under mild conditions. Significantly, this strategy integrates polymeric prodrug-induced chemotherapy (CT), near-infrared (NIR) light-mediated photothermal therapy (PT), and triple modalities including DOX self-fluorescence, photothermal, and photoacoustic (PA) imaging into one conjugate nanoparticle. The PDCNs present excellent photothermal property, dual stimuli-triggered drug release behavior, and about 12.4-fold blood circulation time compared to free DOX. Small animal fluorescent imaging technique confirms that PDCNs have preferential tumor accumulation effect in vivo, giving a 12.8-fold DOX higher than the control at 12 h postinjection. Upon NIR laser irradiation (5 min, 808 nm, and 2 W·cm-2), the PDCN-mediated photothermal effect can quickly elevate the tumor over 50 °C, exhibiting good photothermal and PA imaging functions, of which the PA amplitude is 3.6-fold greater than the control. In vitro and in vivo assays persuasively verify that intravenous photothermal-CT of PDCNs produces synergistic antitumor activity compared to single PT or CT, achieving complete tumor ablation during the evaluation period.

Effects of total flavones from Dendranthema morifolium on vasocontraction and proliferation of vascular smooth muscle cells.

Pharmacological studies have shown that the active components in Dendranthema morifolium exhibit protective effects against ischemia/reperfusion injury; however, its pharmacological action on blood vessels has not yet been investigated. The purpose of the present study was to assess the effects of the total flavones extracted from D. morifolium (Ramat.) Tzvel. cv. Hangju (FDM) on the vasocontraction and proliferation of vascular smooth muscle cells (VSMCs). The tension of rat thoracic aortic rings was measured using a mechanical force transducer attached to a recording system. FDM induced a dose­dependent relaxation of rings with endothelium pre­contracted by either phenylephrine (PE; 10(­6) mol/l) or a high concentration of potassium chloride (KCl; 60 mmol/l). FDM did not significantly affect the vasorelaxant effects on mechanically removed endothelium. In endothelium­denuded aortic rings depolarized by 60 mmol/l KCl, FDM inhibited the contraction induced by Ca2+. FDM reduced the transient contraction caused by PE in a Ca2+­free solution, but did not affect the contraction induced by phorbol ester. Furthermore, FDM inhibited the proliferation of VSMCs with or without growth stimulation by insulin. In conclusion, that the vasorelaxation induced by FDM in rat aortic rings is not dependent on the endothelium but is mediated via a reduction of the influx of extracellular Ca2+ through the voltage­dependent and receptor­operated channels and via the inhibition of the release of intracellular Ca2+ in VSMCs. The anti­proliferative activity of FDM suggests that it may be beneficial in inhibiting atherosclerosis.

[Evaluation of nutrient release profiles from polymer coated fertilizers using Fourier transform mid-infrared photoacoustic spectroscopy].

The acrylate-like materials were used to develop the polymer coated controlled release fertilizer, the nutrients release profiles were determined, meanwhile the Fourier transform mid-infrared photoacoustic spectra of the coatings were recorded and characterized; GRNN model was used to predict the nutrients release profiles using the principal components of the mid-infrared photoacoustic spectra as input. Results showed that the GRNN model could fast and effectively predict the nutrient release profiles, and the predicted calibration coefficients were more than 0.93; on the whole, the prediction errors (RMSE) were influenced by the profiling depth of the spectra, the average prediction error was 10.28%, and the spectra from the surface depth resulted in a lowest prediction error with 7.14%. Therefore, coupled with GRNN modeling, Fourier transform mid-infrared photoacoustic spectroscopy can be used as an alternative new technique in the fast and accurate prediction of nutrient release from polymer coated fertilizer.

Magnesium Lithospermate B Protects Cardiomyocytes from Ischemic Injury Via Inhibition of TAB1-p38 Apoptosis Signaling.

Danshen has been used in traditional Chinese medicine for hundreds of years to treat cardiovascular diseases. However, its precise cardioprotective components and the underlying mechanism are still unclear. In the present study, we demonstrated that in a rat model of acute myocardial infarction, the treatment with magnesium lithospermate B (MLB), the representative component of phenolic acids in Danshen, significantly reduced the infarct size and the blood lactate dehydrogenase level. In contrast, tanshinone IIA, the representative component of lipophilic tanshinones in Danshen, had no such protective effects. Moreover, in the simulated ischemia cell model, MLB treatment considerably increased the cell viability and reduced the sub-G1 population and the apoptotic nuclei, indicating its anti-apoptotic effect. Further mechanism study revealed that the ischemia-induced p38 phosphorylation was abolished by MLB treatment. Interestingly, MLB specifically inhibited the TGFß-activated protein kinase 1-binding protein 1 (TAB1) mediated p38 phosphorylation through disrupting the interaction between TAB1 and p38, but it did not affect the mitogen-activated protein kinase 3/6 mediated p38 phosphorylation. In conclusion, the present study identifies MLB as an active component of Danshen in protecting cardiomyocytes from ischemic injury through specific inhibition of TAB1-p38 apoptosis signaling. These results indicate TAB1-p38 interaction as a putative drug target in treating ischemic heart diseases.

[Study of inhibitory effect of extracts from Actinidia arguta on human carcinoma of esophagus cells].

OBJECTIVE: To study the inhibitory effect of extracts from Actinidia arguta by n-butyl alcohol on human carcinoma of esophagus cells (Eca-109) and Its mechanism. METHODS: MTT colonmetric assay was used to examine the growth inhibitory of concentration-effect (1 microg/ml, 10 microg/ml,100 microg/ml) and time-effect (24 h, 48 h, 72 h) of extracts from Actinidia arguta by n-butyl alcohol on Eca-109 cells. TUNEL test were performed to observe the apoptosis induced by the extracts (1 microg/ml, 10 microg/ml, 100 microg/ml) on Eca-109 cells. RESULTS: The inhibitory effect of the extracts on Eca-109 cells increased in a dose-time Manner and the highest rate of inhibition was 87.2%. The extracts could significantly induce apoptosis of Eca-109 cells, but in control group, apoptosis wasn't observed. CONCLUSION: The extracts from Actinidia arguta by n-butyl alcohol have good inhibitory effect on Eca-109 cells.

Neuroprotective effects of Hypericum perforatum on trauma induced by hydrogen peroxide in PC12 cells.

The standard extracts of Hypericum perforatum L. (SEHP), a well-known medicinal plant, are used for the treatment of depression, exhibited upgrading and significant protective effects on the trauma of PC12 cells induced by 200 microM H2O2 in a dose-dependent manner within 24-hour treatment. Cell viability was assessed by the MTT method, and in situ cellular hydrogen peroxide (H2O2)-induced oxidative stress was examined by measurement of reactive oxygen species (ROS) formation using CDCFH procedures. Intra- and extra-cellular ROS levels decreased significantly to 71.9% and 50.0% of the control at a moderate concentration of 20 microg/ml, respectively, suggesting that SEHP could easily enter the cells and play important roles in reducing ROS levels. Our results were proved by detection of DNA fragmentation and inspection of cell morphology of PC12 cells. SEHP can obviously block DNA fragmentation and prevent the cells from shrinking and turning round of H2O2-induced apoptosis in PC12 cells at concentrations of 10 approximately 100 microg/ml. This data suggests SEHP may be a candidate for application in neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease.

Enhancement of fibrinolytic activity of bovine aortic endothelial cells by ginsenoside Rb2.

AIM: The effect of ginsenoside Rb2 purified from Panax ginseng on fibrinolytic activity of bovine aortic endothelial cells (BAEC) was investigated. METHODS: Cellular plasminogen activator (PA) level of the lysates was measured by the chromogenic substrate S-2403. Fibrin underlay technique was carried out to observe fibrinolysis by growing endothelial cells in the culture medium. Cell viability was then determined by measurement of the activity of mitochondrial dehydrogenase. The ability of Rb2 of potentiating cellular PA activity was investigated by measuring the amounts of PA and PA inhibitor-1 (PAI-1) in the culture medium using zymography and reverse zymography. Changes in the expression of urokinase-type PA (uPA), uPA receptor, and PAI-1 mRNA in BAEC after treatment with Rb2 were analyzed by Northern blot. RESULTS: Rb2 enhanced cellular PA activity in a concentration-and time-dependent manner. Treatment of BAEC with Rb2 10 mg/L for 9 h resulted in a 3.5-fold increase of PA activity without a marked cytotoxic effect, as shown by LDH levels in culture. Increased PA levels caused the increase in surface plasmin levels as observed by fibrin underlay technique. Rb2 greatly or moderately increased the amount of urokinase-type PA (uPA) or its inhibitor (PAI-1), present in the culture medium, whereas saponin did not influence mRNA levels of uPA, its surface receptor, and PAI-1, suggesting that Rb2 may stimulate the secretion of uPA without enhancing its gene expression. The enhancement of PA levels by retinoic acid alone, a stimulator of PA synthesis, was potentiated by the simultaneous addition of ginsenoside Rb2 1 mg/L. Therefore, Rb2 might exert a strong synergism in the synthesis of cellular PA in BAEC. CONCLUSION: Ginsenoside Rb2 enhanced the PA activity levels in BAEC as well as the surface plasmin activity of BAEC. Rb2 may stimulate the secretion of uPA without enhancing the gene expression of uPA, uPA receptor (uPAR), and PAI-1.

[Protective effects of Cleistocalyx operculatus on lipid peroxidation and trauma of neuronal cells].

OBJECTIVE: To observe the protective effects of Cleistocalyx operculatus on lipid peroxidation in rat liver microsomes and on the trauma of PC12 cells induced by H2O2. METHOD: The mouse liver homogenate lipid peroxidation assay and PC12 Cell culture and Cell viability (MTT assay) were applied. RESULT: Cleistocalyx operculatus showed strong protective effects on lipid peroxidation in rat liver microsomes in a dose-dependent manner and exhibited potent protective effects on the trauma of PC12 cells induced by H2O2 (200 micromol x L(-1)) when the concentration reached 1.00 g x L(-1). CONCLUSION: Cleistocalyx operculatus may be used as antioxidant to prevent or delay the pathogenesis of neural cell diseases.