Indirubin, an Active Component of Indigo Naturalis, Exhibits Inhibitory Effects on Leukemia Cells via Targeting HSP90AA1 and PI3K/Akt Pathway.

BACKGROUND: This research intended to predict the active ingredients and key target genes of Indigo Naturalis in treating human chronic myeloid leukemia (CML) using network pharmacology and conduct the invitro verification. METHODS: The active components of Indigo Naturalis and the corresponding targets and leukemia-associated genes were gathered through public databases. The core targets and pathways of Indigo Naturalis were predicted through protein-protein interaction (PPI) network, gene ontology (GO) function, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Next, after intersecting with leukemia-related genes, the direct core target gene of Indigo Naturalis active components was identified. Subsequently, HL-60 cells were stimulated with indirubin (IND) and then examined for cell proliferation using CCK-8 assay and cell cycle, cell apoptosis, and mitochondrial membrane potential using flow cytometry. The content of apoptosis-associated proteins (Cleaved Caspase 9, Cleaved Caspase 7, Cleaved Caspase 3, and Cleaved parp) were detected using Western blot, HSP90AA1 protein, and PI3K/Akt signaling (PI3K, p-PI3K, Akt, and p-Akt) within HL-60 cells. RESULTS: A total of 9 active components of Indigo Naturalis were screened. The top 10 core target genes (TNF, PTGS2, RELA, MAPK14, IFNG, PPARG, NOS2, IKBKB, HSP90AA1, and NOS3) of Indigo Naturalis active components within the PPI network were identified. According to the KEGG enrichment analysis, these targets were associated with leukemia-related pathways (such as acute myeloid leukemia and CML). After intersecting with leukemia-related genes, it was found that IND participated in the most pairs of target information and was at the core of the target network; HSP90AA1 was the direct core gene of IND. Furthermore, the in-vitro cell experiments verified that IND could inhibit the proliferation, elicit G2/M-phase cell cycle arrest, enhance the apoptosis of HL-60 cells, reduce mitochondrial membrane potential, and promote apoptosis-related protein levels. Under IND treatment, HSP90AA1 overexpression notably promoted cell proliferation and inhibited apoptosis. Additionally, IND exerted tumor suppressor effects on leukemia cells by inhibiting HSP90AA1 expression. CONCLUSION: IND, an active component of Indigo Naturalis, could inhibit CML progression, which may be achieved via inhibiting HSP90AA1 and PI3K/Akt signaling expression levels.

Study on the molecular mechanism of dihydromyricetin in alleviating liver cirrhosis based on network pharmacology.

Dihydromyricetin (DHM) is a bioactive flavonoid extracted from Hovenia dulcis, which has various activities. In the present study, the molecular mechanism of dihydromyricetin (DHM) in relieving liver cirrhosis was investigated through network pharmacology and experimental verification. The cell model was induced by TGF-ß1 activating the human hepatic stellate cell line (HSC; LX-2). The protein levels of α-SMA, collagen I, and collagen III and pathway-related proteins within LX-2 cells were detected using Western blot. EdU staining was conducted to detect cell proliferation. Immunofluorescence staining was performed to detect the expression levels of α-SMA and collagen I. Next, the drug targets of DHM were screened from the PubChem database. The differentially expressed genes in the liver cirrhosis dataset GSE14323 were identified. The expression of the identified drug targets in LX-2 cells was verified using qRT-PCR. The results showed that TGF-ß1 treatment notably increased LX-2 cell viability, promoted cell proliferation, and elevated α-SMA, collagen I, and collagen III protein contents. DHM treatment could partially eliminate TGF-ß1 effects, as evidenced by the inhibited cell viability and proliferation and reduced α-SMA, collagen I, and collagen III contents. After network pharmacology analysis, nine differentially expressed target genes (MMP2, PDGFRB, PARP1, BCL2L2, ABCB1, TYR, CYP2E1, SQSTM1, and IL6) in liver cirrhosis were identified. According to qRT-PCR verification, DHM could inhibit the expression of MMP2, PDGFRB, PARP1, CYP2E1, SQSTM1, and IL6, and enhance ABCB1 expression levels within LX-2 cells. Moreover, DHM inhibited mTOR and MAPK signaling pathways in TGF-ß1-induced HSCs. In conclusion, DHM could inhibit HSC activation, which may be achieved via acting on MMP2, PDGFRB, PARP1, CYP2E1, SQSTM1, IL6, and ABCB1 genes and their downstream signaling pathways, including mTOR and MAPK signaling pathway.

Colorimetric immunoassay of carcinoembryonic antigen based on the glucose oxidase/MnO2 nanosheet cascade reaction with self-supplying oxygen.

The detection of carcinoembryonic antigen (CEA) has profound implications in cancer diagnostics and therapeutic monitoring. In this work, we developed a colorimetric immunoassay for the detection of CEA. This assay involves the utilization of zinc(II)-based coordination polymers (ZnCPs) as a host for integrating glucose oxidase (GOx) and anti-carcinoembryonic antigen antibody (anti-CEA), which results in the formation of a detection antibody (anti-CEA/GOx@ZnCPs). The adaptable inclusion properties of ZnCPs enable the preservation of the original catalytic behavior of GOx and antigen capture ability of anti-CEA. Consequently, the anti-CEA/GOx@ZnCPs can act as a detection antibody to facilitate the development of an immunoassay. The combination of anti-CEA/GOx@ZnCPs in the immunoassay triggers a cascade reaction involving GOx and MnO2 nanosheets, leading to the generation of an amplified colorimetric signal through self-supplying oxygen. This colorimetric immunoassay exhibits a linear response ranging from 2 to 180 ng mL-1 CEA and has a detection limit of 50 pg mL-1. The practicality of this colorimetric immunoassay in biological matrices was demonstrated by the successful determination of CEA in serum samples with good recovery and precision. We believe that this study will pave the way to rationally design multifunctional CP-based composites for a wide range of applications in bioanalysis.

Expression of Concern: Low-intensity focused ultrasound (LIFU)-activated nanodroplets as a theranostic agent for noninvasive cancer molecular imaging and drug delivery.

Expression of Concern for 'Low-intensity focused ultrasound (LIFU)-activated nanodroplets as a theranostic agent for noninvasive cancer molecular imaging and drug delivery' by Jianxin Liu et al., Biomater. Sci., 2018, 6, 2838-2849, https://doi.org/10.1039/C8BM00726H.

Ultrasound-Mediated Oxygen Delivery for Enhanced Radiotherapy with Ultrasound Imaging Guidance.

As a non-invasive therapeutic, radiotherapy (RT) has been extensively used for solid tumor treatment. However, intratumoral hypoxia leads to severe RT resistance or failure. Moreover, damage from RT to normal tissues limits the application of high doses of radiation to eliminate cancer cells. Therefore, simultaneously improving the curative efficacy while minimizing the side effects of RT is in pressing need. Hence, the purpose of this study is to use oxygen-based microbubbles (O2 @MBs) combined with ultrasound (US) targeting microbubble destruction (UTMD) technology to overcome hypoxia prior to RT, evaluate the effects of O2 @MBs on contrast-enhanced ultrasound (CEUS) imaging enhancement, investigate the optimum delivery route of O2 @MBs, and evaluate the therapeutic efficacy. In this study, O2 @MBs were injected intravenously or locally and the distribution of O2 @MBs in tumors or regions surrounding the tumors are compared by US imaging. The hypoxic status of tumors and their sensitivity to RT were investigated. Our findings suggest that O2 @MBs combined with UTMD can significantly enhance the effects of RT. In addition, the in vivo biosafety assay demonstrates good biocompatibility, indicating great potential for clinical translation.

Low-intensity focused ultrasound (LIFU)-activated nanodroplets as a theranostic agent for noninvasive cancer molecular imaging and drug delivery.

Theranostics is a new trend in the tumor research field, which involves the integration of diagnostic and therapeutic functions using imageable nanoparticles coupled with therapeutic drugs. Imaging-guided targeted delivery of therapeutics and diagnostics using nanocarriers hold great promise to minimize the side effects of conventional chemotherapy. Ultrasound microbubbles have been employed as theranostic agents over the last decade, which provide both real-time dynamic imaging for diagnosis and precise control for targeted tumor therapy. However, the intrinsic defects of microbubbles such as poor tissue penetration, short circulation time and instability hinder microbubble-based theranostic applications. In recent years, liquid-to-gas transitional perfluorocarbon nanoparticles have been developed as promising diagnostic and therapeutic nanoagents to solve the abovementioned problems. In this study, phase-changeable, folate-targeted perfluoropentane nanodroplets loaded with 10-hydroxycamptothecin (HCPT) and superparamagnetic Fe3O4 (denoted as FA-HCPT-Fe3O4-PFP NDs) are prepared and investigated for multimodal tumor imaging and targeted therapy. After intravenous administration into nude mice bearing SKOV3 ovarian cancer, FA-HCPT-Fe3O4-PFP NDs exhibit the ability to enhance MR and PA imaging. Furthermore, after the phase transition activated by low-intensity focused ultrasound (LIFU) sonication, FA-HCPT-Fe3O4-PFP NDs remarkably enhance US imaging at the tumor location. Meanwhile, the HCPT released from FA-HCPT-Fe3O4-PFP NDs during the liquid-to-gas transition provides a therapeutic effect on tumor cells with relatively low side effects to normal tissue. Therefore, the combination of LIFU and FA-HCPT-Fe3O4-PFPNDs presents an ideal modality for tumor-targeted theranostics.

Bioinspired Multifunctional Melanin-Based Nanoliposome for Photoacoustic/Magnetic Resonance Imaging-Guided Efficient Photothermal Ablation of Cancer.

Background: The construction of theranostic nanosystems with concurrently high biosafety and therapeutic performance is a challenge but has great significance for the clinical translation of nanomedicine for combating cancer. Methods: Bio-inspired melanin-based nanoliposomes (Lip-Mel) as theranostic agents were constructed for simultaneous photoacoustic (PA) imaging- and T1-weighted magnetic resonance (MR) imaging-guided photothermal ablation of tumors, which was demonstrated both in vitro and in vivo. The high biosafety of Lip-Mel was also systematically evaluated. Results: The achieved Lip-Mel nanoliposomes demonstrated their imaging capability for both PA and T1-weighted MR imaging (r1 = 0.25 mM-1·s-1) both in vitro and in vivo, providing the potential for therapeutic guidance and monitoring. Importantly, the desirable photothermal-conversion efficiency of the as-prepared Lip-Mel achieved complete eradication of tumors in breast cancer-bearing mice, exhibiting remarkable photothermal-based therapeutic performance. In particular, the efficient encapsulation of melanin into the PEGylated liposome mitigated the potential toxicity of melanin and improved the photothermal performance of the loaded melanin. Systematic in vivo biosafety evaluations demonstrated the high biocompatibility of Lip-Mel at a high dose of 100 mg/kg. Conclusion: In this work, we reported a bioinspired strategy where melanin, a natural product in the human body, is encapsulated into PEGylated nanoliposomes for efficient theranostics with high biocompatibility. This work provides a new strategy for creating desirable theranostic agents with concurrent high biocompatibility and satisfactory theranostic performance through the use of materials that totally originate from biosystems.

Drug Release from Phase-Changeable Nanodroplets Triggered by Low-Intensity Focused Ultrasound.

Background: As one of the most effective triggers with high tissue-penetrating capability and non-invasive feature, ultrasound shows great potential for controlling the drug release and enhancing the chemotherapeutic efficacy. In this study, we report, for the first time, construction of a phase-changeable drug-delivery nanosystem with programmable low-intensity focused ultrasound (LIFU) that could trigger drug-release and significantly enhance anticancer drug delivery. Methods: Liquid-gas phase-changeable perfluorocarbon (perfluoropentane) and an anticancer drug (doxorubicin) were simultaneously encapsulated in two kinds of nanodroplets. By triggering LIFU, the nanodroplets could be converted into microbubbles locally in tumor tissues for acoustic imaging and the loaded anticancer drug (doxorubicin) was released after the microbubble collapse. Based on the acoustic property of shell materials, such as shell stiffness, two types of nanodroplets (lipid-based nanodroplets and PLGA-based nanodroplets) were activated by different acoustic pressure levels. Ultrasound irradiation duration and power of LIFU were tested and selected to monitor and control the drug release from nanodroplets. Various ultrasound energies were introduced to induce the phase transition and microbubble collapse of nanodroplets in vitro (3 W/3 min for lipid nanodroplets; 8 W/3 min for PLGA nanodroplets). Results: We detected three steps in the drug-releasing profiles exhibiting the programmable patterns. Importantly, the intratumoral accumulation and distribution of the drug with LIFU exposure were significantly enhanced, and tumor proliferation was substantially inhibited. Co-delivery of two drug-loaded nanodroplets could overcome the physical barriers of tumor tissues during chemotherapy. Conclusion: Our study provides a new strategy for the efficient ultrasound-triggered chemotherapy by nanocarriers with programmable LIFU capable of achieving the on-demand drug release.

Combination of microbubbles and diagnostic ultrasound at a high mechanical index for the synergistic microwave ablation of tumours.

OBJECTIVES: To determine whether combining microbubbles (MBs) with diagnostic ultrasound (US) at a high mechanical index (MI) could enhance the microwave (MW) ablation of tumours. MATERIALS AND METHODS: Five therapeutic MW adjuvant protocols were studied: MW, MW + US, MW + US + MB, MW + US + NS (saline) and MW + MB. In 30 normal rabbit livers, the synergistic effects were evaluated via temperature, necrosis volume and histology. In 90 VX2 rabbit hepatic tumours, residual cells in the peripheral ablated tumours were examined via immunohistochemical assay and tumour growth. Additional 40 VX2 hepatic tumours were evaluated for ablation safety via blood assay and weight and for survival to 105 days. Results were compared using analysis of variance. RESULTS: Compared with the other protocols, the ablation volumes in normal rabbit livers were significantly larger using the MW + US + MB protocol (p < .001). The histological examination was consistent with more efficient ablation in that protocol. In detecting residual cells, the apoptotic index was higher, the proliferating index was lower (p < .05), tumour growth was significantly smaller (p < .001), and the rabbits of the MW + US + MB T-Group survived longer (p < .05) than those of the other groups. Additionally, no damage to the liver function or blood cells was found in any of the protocols after ablation (p < .05). CONCLUSIONS: MBs in combination with diagnostic US at a high MI showed potential synergy in the MW ablation of tumours in rabbits.

Comparison of the synergistic effect of lipid nanobubbles and SonoVue microbubbles for high intensity focused ultrasound thermal ablation of tumors.

Microbubbles (MBs) are considered as an important enhancer for high intensity focused ultrasound (HIFU) treatment of benign or malignant tumors. Recently, different sizes of gas-filled bubbles have been investigated to improve the therapeutic efficiency of HIFU thermal ablation and reduce side effects associated with ultrasound power and irradiation time. However, nanobubbles (NBs) as an ultrasound contrast agent for synergistic therapy of HIFU thermal ablation remain controversial due to their small nano-size in diameter. In this study, phospholipid-shell and gas-core NBs with a narrow size range of 500-600 nm were developed. The synergistic effect of NBs for HIFU thermal ablation was carefully studied both in excised bovine livers and in breast tumor models of rabbits, and made a critical comparison with that of commercial SonoVue microbubbles (SonoVue MBs). In addition, the pathological changes of the targeted area in tumor tissue after HIFU ablation were further investigated. Phosphate buffer saline (PBS) was used as the control. Under the same HIFU parameters, the quantitative echo intensity of B-mode ultrasound image and the volume of coagulative necrosis in lipid NBs groups were significantly higher and larger than that in PBS groups, but could not be demonstrated a difference to that in SonoVue MBs groups both ex vivo and in vivo. These results showed that the synergistic effect of lipid NBs for HIFU thermal ablation were similar with that of SonoVue MBs, and further indicate that lipid NBs could potentially become an enhancer for HIFU thermal ablation of tumors.

A Multifunctional Theranostic Nanoagent for Dual-Mode Image-Guided HIFU/Chemo- Synergistic Cancer Therapy.

High-intensity focused ultrasound (HIFU) is deemed to be a promising noninvasive therapeutic modality for cancers as well as non-neoplastic diseases. However, the accuracy of the technique in the diagnosis and treatment of tumors remains unsatisfactory. HIFU, when combined with multifunctional synergistic agents (SAs), has the potential to be of greater diagnostic and therapeutic efficacy. Here we describe a smart and multifunctional hollow mesoporous Prussian blue (HMPBs) theranostic nanoplatform, the hollow structure of which is capable of encapsulating doxorubicin (DOX) and perfluorohexane (HMPBs-DOX/PFH). In vitro and in vivo studies validated that HMPBs-DOX/PFH can be used as an amplifiable dual-mode imaging contrast agent, which can simultaneously enhance ultrasound (US) and photoacoustic (PA) imaging for guiding and monitoring tumor therapy. When exposed to HIFU, this versatile HMPBs-DOX/PFH agent could increase the cavitation effect and use lower HIFU intensity to achieve coagulative necrosis. Furthermore, it significantly accelerated the release of DOX thereby enhancing chemotherapeutic efficacy and avoiding systemic side effects of the drug. Such a novel theranostic nanoplatform is expected to integrate dual-mode guided imaging with greater therapeutic efficacy and fewer side effects and is very promising for the noninvasive synergistic tumor therapy.

Phase-Shifted PFH@PLGA/Fe3O4 Nanocapsules for MRI/US Imaging and Photothermal Therapy with near-Infrared Irradiation.

Photothermal therapy (PTT) utilizes photothermal conversion reagents to generate heat energy from absorbed light to effectively treat various malignant diseases. This approach has attracted broad and increasing interest in cancer treatment. Near-infrared (NIR)-induced PTT is particularly attractive because of its minimal absorbance by normal tissue and relatively deep tissue penetration. To improve the efficacy of PTT, we have developed nanocapsules encapsulating superparamagnetic iron oxide (Fe3O4) as synergistic agents for NIR-induced PTT. In this study, phase-shift and NIR photoabsorbing poly(lactic-co-glycolic acid) (PLGA) nanocapsules (perfluorohexane (PFH)@PLGA/Fe3O4) were fabricated for MRI/US dual-modal imaging-guided PTT. The multifunctional nanocapsules can be used not only to increase the local tumor temperature by absorbing the NIR energy but also as bimodal contrast agents for both MRI and US imaging. Such nanocapsules can be converted into microbubbles under NIR irradiation, which produces excellent contrast for US imaging and enhanced cancer ablation. We refer to the nanocapsule phase transition process induced by the infrared lamp as NIR radiation droplet vaporization (NIRDV).