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

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

Nanocomplexes of Biodegradable Anticancer Macromolecules: Prolonged Plasma Half-Life, Reduced Toxicity, and Increased Tumor Targeting.

Anticancer drug resistance is a large contributing factor to the global mortality rate of cancer patients. Anticancer macromolecules such as polymers have been recently reported to overcome this issue. Anticancer macromolecules have unselective toxicity because they are highly positively charged. Herein, an anionic biodegradable polycarbonate carrier is synthesized and utilized to form nanocomplexes with an anticancer polycarbonate via self-assembly to neutralize its positive charges. Biotin is conjugated to the anionic carrier and serves as cancer cell-targeting moiety. The nanoparticles have sizes of < 130 nm with anticancer polymer loading levels of 38-49%. Unlike the small molecular anticancer drug doxorubicin, the nanocomplexes effectively inhibit the growth of both drug-susceptible MCF7 and drug-resistant MCF7/ADR human breast cancer cell lines with low half maximal inhibitory concentration (IC50 ). The nanocomplexes increase the anticancer polymer's in vivo half-life from 1 to 6-8 h, and rapidly kill BT474 human breast cancer cells primarily via an apoptotic mechanism. The nanocomplexes significantly increase the median lethal dose (LD50 ) and reduce the injection site toxicity of the anticancer polymer. They suppress tumor growth by 32-56% without causing any damage to the liver and kidneys. These nanocomplexes may potentially be used for cancer treatment to overcome drug resistance.

Drug-free neutrally charged polypeptide nanoparticles as anticancer agents.

Cationic synthetic anticancer polymers and peptides have attracted increasing attention for advancing cancer treatment without causing drug resistance development. To circumvent in vivo instability and toxicity caused by cationic charges of the anticancer polymers/peptides, we report, for the first time, a nanoparticulate delivery system self-assembled from a negatively charged pH-sensitive polypeptide poly(ethylene glycol)-b-poly(ʟ-lysine)-graft-cyclohexene-1,2-dicarboxylic anhydride and a cationic anticancer polypeptide guanidinium-functionalized poly(ʟ-lysine) (PLL-Gua) via electrostatic interaction. The formation of nanoparticles (Gua-NPs) neutralized the positive charges of PLL-Gua. Both PLL-Gua and Gua-NPs killed cancer cells in a dose- and time-dependent manner, and induced cell death via apoptosis. Confocal microscopic studies demonstrated that PLL-Gua and Gua-NPs readily entered cancer cells, and Gua-NPs were taken up by the cells via endocytosis. Notably, Gua-NPs and PLL-Gua exhibited similar in vitro anticancer efficacy against MCF-7 and resistant MCF-7/ADR. PLL-Gua and Gua-NPs also induced similar morphological changes in MCF-7/ADR cells compared to MCF-7 cells, further indicating their ability to bypass drug resistance mechanisms in the MCF-7/ADR cells. More importantly, Gua-NPs with higher LD50 and enhanced tumor accumulation significantly inhibited tumor growth with negligible side effects in vivo. Our findings shed light on the in vivo delivery of anticancer peptides and opened a new avenue for cancer treatment.

Constituents of Morus alba var. multicaulis leaf improve lipid metabolism by activating the AMPK signaling pathway in HepG2 cells.

One new compound, 3Z-1-O-ß-D-glucopyranosyl-3-hexene-1,5-diol (1), together with 26 known isolates (2-27) were obtained from the leaf of Morus alba var. multicaulis. Among the known compounds, 7, 11, 12, 14, 15, 18, 19, 23, and 24 were firstly obtained from the Morus genus; 2-5, 8, 10, 13, and 20 were firstly isolated from M. alba. var. multlcaulis. Meanwhile, the NMR data of 20 and 23 have been reported here for the first time. Moreover, compounds 1-11, 13, 21, and 23-27 showed inhibitory effects on triglyceride (TG) accumulation in HepG2 cells. In mechanism, compound 1 could activate the phosphorylation of AMP-activated protein kinase α (AMPKα) to accelerate the ß-oxidation of fatty acids via promoting the phosphorylation of acetyl-CoA carboxylase 1 and up-regulating carnitine palmitoyl-transferase 1A. Besides, compound 1 exerted lipolysis effect by activating hormone-sensitive lipase. In brief, compound 1 might play a role by up-regulating phosphorylation of AMPKα, enhancing the fatty acid ß-oxidation and lipolysis. 27 compounds were obtained from the leaf of Morus alba var. multicaulis. Among them, 18 showed inhibitory effects on TG accumulation in HepG2 cells. Moreover, the new compound, 3Z-1-O-ß-D-glucopyranosyl-3-hexene-1,5-diol (1), was found to play a role by up-regulating phosphorylation of AMPKα, enhancing the fatty acids ß-oxidation and lipolysis.

Antimicrobial Polypeptides Capable of Membrane Translocation for Treatment of MRSA Wound Infection In Vivo.

Multidrug resistant infections are plaguing the healthcare sector over the past few decades with limited treatment options. To overcome this problem, the authors synthesize a series of novel guanidinium-functionalized polypeptides. Specifically, poly(l-lysine) (PLL) with different lengths is first synthesized by ring-opening polymerization of Nε -benzyloxycarbonyl-l-lysine-N-carboxyanhydride (Lys(Z)-NCA) followed by functionalization with a guanidinium-functional group to obtain guanidinium-functionalized PLL (PLL-Gua). To study the effect of hydrophobicity on antimicrobial activity, relatively more hydrophobic leucine-NCA monomer or hydrophobic vitamin E moiety is introduced to PLL-Gua. These polypeptides are characterized for antimicrobial activity against a panel of microbes including multidrug-resistant bacteria, and hemolytic activity. Among all the polypeptides, PLL22 -Gua is most effective against bacteria and yeast. Particularly, excellent bactericidal activity is observed against Staphylococcus aureus and MRSA. PLL22 -Gua kills bacteria mainly by membrane translocation. In addition, PLL22 -Gua kills MRSA with low resistance frequency (<3.3 × 10-8 ). In an MRSA-caused wound infection mouse model, two-day treatment (twice daily) with 10, 20, or 40 mg per kg of PLL22 -Gua shows up to 99.5% bacterial removal. Moreover, no acute dermal toxicity is observed even at a dose of 200 mg per kg. These promising results show the excellent potential of PLL22 -Gua as an antimicrobial agent against multidrug-resistant infection in vivo.

Anti-inflammatory Limonoids From Cortex Dictamni.

The root barks of perennial herb Dictamnus dasycarpus (Cortex Dictamni) were reported to be rich in anti-inflammation activity constituents, limonoids. Then, the investigation of anti-inflammation therapeutic limonoids from this plant was developed in the present study. Through the combination of various chromatographies isolation, six new limonoids, named dictamlimonol A (1), dictamlimonoside B (2), and dictamlimonols C-F (3-6), along with seven known ones (7-13), were obtained. Their structures were ascertained based on the extensive spectroscopic methods and ECD data analysis. Among them, compound 1 was the first 7,19-epoxy limonoid found in natural products. The anti-inflammatory effects of all limonoids were evaluated in lipopolysaccharide (LPS)-treated RAW 264.7 cell lines. Compounds 5, 7-11, and 13 were found to inhibit LPS-induced nitric oxide (NO) production. Moreover, dictamlimonol D (5), fraxinellone (11), and dasylactone A (13) were found to reduce the LPS-induced expressions of interleukin-6 (IL-6), tumor necrosis factor (TNF-α), inducible nitric oxide synthase (iNOS), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and cyclooxygenase-2 (COX-2) at the protein levels in a dose-dependent manner. These findings support that the administration of Cortex Dictamni may be beneficial for inflammation.

Combretastatin A4 Nanoparticles Combined with Hypoxia-Sensitive Imiquimod: A New Paradigm for the Modulation of Host Immunological Responses during Cancer Treatment.

Vascular disrupting agents (VDAs) have great potential in cancer treatment. However, in addition to their direct tumoral vascular collapse effect, VDAs activate host immunological responses, which can remarkably impair their anticancer efficacy. Here, a VDA nanomedicine, poly(l-glutamic acid)-graft-methoxy poly(ethylene glycol)/combretastatin A4 (CA4-NPs), is found to induce the intratumor infiltration of immature plasmacytoid dendritic cells (pDCs), thereby curtailing anticancer immunity. To overcome this problem, hypoxia-sensitive imiquimod (hs-IMQ) is developed, which is selectively activated into imiquimod (IMQ) in treated tumors following the catalysis of CA4-NPs-induced nitroreductase (NTR). The combination of hs-IMQ and CA4-NPs causes a 6.3-fold enhancement of active IMQ concentration in tumors, as compared to hs-IMQ treatment alone. The in situ-generated IMQ alters the tumor microenvironment from a state of immunosuppression to immune activation. Hs-IMQ achieves this effect through the conversion of immature pDCs into their active form, leading to the robust infiltration and priming of natural killer cells and cytotoxic T-lymphocytes in treated tumors. Thus, the CA4-NPs and hs-IMQ combination treatment synergistically inhibits tumor growth and metastasis in 4T1 tumor-bearing mice. This work offers new approaches to harness intratumor pDCs to reverse the immune suppression resulting from VDA treatment. These findings additionally provide a mechanistic rationale for the use of VDAs in combination with TLR agonists to trigger in situ immune activation and enhance anticancer efficacy.

Glucose and pH Dual-Responsive Nanogels for Efficient Protein Delivery.

Direct delivery of protein suffers from their in vitro and in vivo instability, immunogenicity, and a relatively short half-life within the body. To overcome these challenges, pH and glucose dual-responsive biodegradable nanogels comprised of dextran and poly(L-glutamic acid)-g-methoxy poly-(ethylene glycol)/phenyl boronic acid (PLG-g-mPEG/PBA) are designed. The cross-linked network imparted drug-loading efficacy of α-amylase up to 55.6% and hyaluronidase up to 29.1%. In vitro protein release profiles reveal that the release of protein is highly dependent on the pH or glucose concentrations, that is, less amount of protein is released at pH 7.4 or healthy blood glucose level (1 mg mL-1 glucose), while quicker release of protein occurs at pH 5.5 or diabetic blood glucose level (above 3 mg mL-1 glucose). Circular dichroism spectra show that the secondary structure of released protein is maintained compared to naive protein. Overall, the nanogels have provided a simple and effective strategy to deliver protein.

Anti-inflammatory constituents from Cortex Dictamni.

Eight new compounds named as dictamalkosides A (1), B (2), C (3), dictamphenosides A (4), B (5), C (6), D (7) and E (8), as well as 23 known ones were obtained from the 70% EtOH extract of Cortex Dictamni. Their structures were ascertained based on the spectroscopic evidences. Among the known compounds, 14, 17-23, 25-28, and 31 were isolated from Dictamnus genus for the first time; 16 and 24 were firstly isolated from this plant. And the 13C NMR data of 14 was reported here for the first time. Moreover, compounds 1-8, 12, 18-21, 27 and 31 were found to exhibit potential inhibitory effect on LPS-induced NO production at 40 µM for RAW 264.7 macrophages, which suggested alkaloids and phenolic acids might be anti-inflammation therapeutic substance in Cortex Dictamni.

Selectively Potentiating Hypoxia Levels by Combretastatin A4 Nanomedicine: Toward Highly Enhanced Hypoxia-Activated Prodrug Tirapazamine Therapy for Metastatic Tumors.

Hypoxia-activated prodrugs (HAPs) have the potential to selectively kill hypoxic cells and convert tumor hypoxia from a problem to a selective treatment advantage. However, HAPs are unsuccessful in most clinical trials owing to inadequate hypoxia within the treated tumors, as implied by a further substudy of a phase II clinical trial. Here, a novel strategy for the combination of HAPs plus vascular disrupting agent (VDA) nanomedicine for efficacious solid tumor therapy is developed. An effective VDA nanomedicine of poly(l-glutamic acid)-graft-methoxy poly(ethylene glycol)/combretastatin A4 (CA4-NPs) is prepared and can selectively enhance tumor hypoxia and boost a typical HAP tirapazamine (TPZ) therapy against metastatic 4T1 breast tumors. After treatment with the combination of TPZ plus CA4-NPs, complete tumor reduction is observed in 4T1 xenograft mice (initial tumor volume is 180 mm3 ), and significant tumor shrinkage and antimetastatic effects are observed in challenging large tumors with initial volume of 500 mm3 . The report here highlights the potential of using a combination of HAPs plus VDA nanomedicine in solid tumor therapy.

Dihydroartemisinin increases gemcitabine therapeutic efficacy in ovarian cancer by inducing reactive oxygen species.

Ovarian cancer is the major cause of death in women gynecological malignancy and gemcitabine (GEM) is commonly used in related chemotherapy. However, more than 90% GEM is catalyzed into an inactive metabolite 2'-deoxy-2',2'-difluorouridine by stromal and cellular cytidine deaminase (CDA). Dihydroartemisinin (DHA), which possesses an intramolecular endoperoxide bridge, could be activated by heme or ferrous iron to produce reactive oxygen species (ROS). The excess ROS generation will excite expression of heme oxygenase-1 and suppress CDA expression. Under low CDA expression, the inactivation of GEM is decreased in turn to exert excellent therapeutic efficiency. Herein, we first studied the ROS generation by DHA in vitro with A2780 cells by means of flow cytometry and confocal laser scanning microscopy. Furthermore, cytotoxicity assay in vitro showed that DHA + GEM had synergistic effect, with molar ratio of DHA and GEM at 10. Eventually, in A2780 ovarian cancer xenograft tumor model, DHA + GEM exhibited significant antitumor efficiency with lower blood toxicity than GEM alone. Noteworthy, the combination treatment group completely eliminated the tumors on day 14.

Eudesmane-Type Sesquiterpene Glycosides from Dictamnus dasycarpus Turcz.

Eudesmane-type sesquiterpenes have been reported to exhibit varieties of biological activities. During the process of investigating this kind of natural product from the root bark of Dictamnus dasycarpus Turcz., 13 eudesmane-type sesquiterpene glycosides including six new isolates, named as dictameudesmnosides A1 (1), A2 (2), B (3), C (4), D (5), and E (6), together with seven known ones (7-13), were obtained. Herein, their structures were determined by the analysis of physical data, spectroscopic analysis, and chemical methods. The existence of α-configuration glucose units in their structures (1-5, 8) is not very common in natural glycosidic components. Meanwhile, compounds 3-5, 7, and 9-13 displayed TG accumulation inhibitory effects on HepG2 cells.

Co-administration of genistein with doxorubicin-loaded polypeptide nanoparticles weakens the metastasis of malignant prostate cancer by amplifying oxidative damage.

Prostate cancer is a typical malignant disease with a high incidence and a poor prognosis. Doxorubicin hydrochloride (DOX·HCl) is one of the most effective agents in the treatment of prostate cancer, but severe side effects and metastasis after its treatment impose restrictions on its application. Herein, a combination of genistein (GEN) and doxorubicin-loaded polypeptide nanoparticles (DOX-NPs) is constructed for the treatment of prostate cancer. The DOX-NPs can reduce the side effects caused by free DOX·HCl and produce a relatively low level of intracellular reactive oxygen species (ROS)-induced oxidative damage, while GEN, an inhibitor of the oxidative DNA repair enzyme apurinic/apyrimidinic endonuclease1 (APE1), can further amplify the ROS-induced oxidative damage by downregulating the intracellular expression of APE1 and reducing oxidative DNA repair in the prostate cancer cells. Because high levels of ROS-induced oxidative damage can prevent the distant metastasis of tumor cells, the distant metastasis of malignant prostate cancer cells is significantly inhibited by the combination of genistein and DOX-NPs with amplified oxidative damage.

pH and redox dual-sensitive polysaccharide nanoparticles for the efficient delivery of doxorubicin.

A pH and redox dual-sensitive biodegradable polysaccharide, succinic acid-decorated dextran-g-phenylalanine ethyl ester-g-cysteine ethyl ester (Dex-SA-l-Phe-l-Cys), was synthesized to load doxorubicin hydrochloride (DOX·HCl). The DOX-loaded nanoparticles, which were prepared in aqueous solution and free of organic solvent, could spontaneously self-assemble into uniform sizes. When loading DOX·HCl, mercapto Dex-SA-l-Phe-l-Cys was oxidized into a crosslinked disulfide linkage to form pH and redox dual-sensitive nanoparticles (DOX-S-NPs). The amphiphilic polymer loaded DOX·HCl into the core through electrostatic and hydrophobic interactions, meanwhile the crosslinked disulfide bond could stabilize the drug loaded nanoparticles. As a control with similar polymer structure, succinic acid decorated dextran-g-phenylalanine ethyl ester (Dex-SA-l-Phe) was prepared to obtain pH-sensitive DOX-loaded micelles (DOX-N-NPs). The controlled pH and redox-dependent release profiles of the DOX-S-NPs in vitro were certified in different releasing mediums. Furthermore, the cellular uptake of the DOX-S-NPs was comparable with that of free DOX·HCl, determined by confocal laser scanning microscopy (CLSM) and flow cytometry. Cytotoxicity assay in vitro showed that the DOX-S-NPs and free DOX·HCl were similar in inhibiting the proliferation of non-small cell lung carcinoma A549 and breast cancer MCF-7 cell lines. DOX-S-NPs displayed similar antitumor efficiency compared with free DOX·HCl, but lower toxicity by body weight. These dual-sensitive DOX-S-NPs provide a useful strategy for anti-tumor therapy.

Bioactive Constituents Obtained from the Seeds of Lepidium apetalum Willd.

Three new compounds, apetalumosides C1 (1), D (2), and 1-thio--d-glucopyranosyl(1→1)-1-thio-α-d-glucopyranoside (3), together with twenty-two known ones (4-25) were obtained from the seeds of Lepidium apetalum Willd. Among the known isolates, 5-8, 10-13, 16-20, and 25 were obtained from the genus for the first time; 4, 14, 15, and 21-24 were isolated from the species for the first time. Meanwhile, the NMR data of 16 was first reported here. Their structures were determined by means of chemical and spectroscopic methods. On the other hand, their inhibitory effects on sodium oleate-induced triglyceride (TG) overloading in HepG2 cells were evaluated. As a result, two new compounds (1 and 2), together with known isolates 7-11, 13, 14, 16-18, 20, 21, and 25 possessed significant inhibitory effects in the cells.

Inhibitory Effects of Constituents from the Aerial Parts of Rosmarinus officinalis L. on Triglyceride Accumulation.

Sixteen flavonoids (1-16) including two new ones, named officinoflavonosides A (1) and B (2) were obtained from the aerial parts of Rosmarinus officinalis. Among the known ones, 6, 10, and 13 were isolated from the rosmarinus genus for the first time. Their structures were elucidated by chemical and spectroscopic methods. Moreover, the effects on sodium oleate-induced triglyceride accumulation (TG) in HepG2 cells of the above-mentioned compounds and 16 other isolates (17-32) reported previously to have been obtained in the plant were analyzed. Results show that eight kinds of flavonoids (compounds 1, 2, 3, 6-9 and 11) and seven kinds of other known isolates (compounds 17-20, 23, 26 and 31) possessed significant inhibitory effects on intracellular TG content in HepG2 cells. Among them, the activities of compounds 1 and 20 were comparable to that of orlistat, which suggested that these compounds in this plant might be involved in lipid metabolism.