Aptamer/Peptide-Functionalized Nanoprobe for Detecting Multiple miRNAs in Circulating Malignant Cells to Study Tumor Heterogeneity.

Identification of diverse biomarkers in heterogenic circulating malignant cells (CMCs) such as circulating tumor cells (CTCs) and circulating tumor endothelial cells (CTECs) has crucial significance in tumor diagnosis. However, it remains a substantial challenge to achieve in situ detection of multiple miRNA markers in living cells in blood. Herein, we demonstrate that an aptamer/peptide-functionalized vector can deliver molecular beacons into targeted living CMCs in peripheral blood of patients for in situ detection of multiple cancer biomarkers, including miRNA-21 (miR-21) and miRNA-221 (miR-221). Based on miR-21 and miR-221 levels, heterogenic CMCs are identified for both nondistant metastatic and distant metastatic cancer patients. CMCs from nondistant metastatic and distant metastatic cancer patients exhibit similar miR-21 levels, while the miR-221 level in CMCs of the distant metastatic cancer patient is higher than that of the nondistant metastatic cancer patient. With the capability to realize precise probing of multiple intracellular biomarkers in living CMCs at the single-cell resolution, the nanoprobe can reveal the tumor heterogeneity and provide useful information for diagnosis and prognosis. The nanoprobe we developed would accelerate the progress toward noninvasive precise cancer diagnosis.

A Multifunctional Delivery System for Remodulating Cell Behaviors of Circulating Malignant Cells to Prevent Cell Fusion.

Cell fusion plays a critical role in cancer progression and metastasis. However, effective modulation of the cell fusion behavior and timely evaluation on the cell fusion to provide accurate information for personalized therapy are facing challenges. Here, it demonstrates that the cancer cell fusion behavior can be efficiently modulated and precisely detected through employing a multifunctional delivery vector to realize cancer targeting delivery of a genome editing plasmid and a molecular beacon-based AND logic gate. The multifunctional delivery vector decorated by AS1411 conjugated hyaluronic acid and NLS-GE11 peptide conjugated hyaluronic acid can specifically target circulating malignant cells (CMCs) of cancer patients to deliver the genome editing plasmid for epidermal growth factor receptor (EGFR) knockout. The cell fusion between CMCs and endothelial cells can be detected by the AND logic gate delivered by the multifunctional vector. After EGFR knockout, the edited CMCs exhibit dramatically inhibited cell fusion capability, while unedited CMCs can easily fuse with human umbilical vein endothelial cells (HUVEC) to form hybrid cells. This study provides a new therapeutic strategy for preventing cancer progression and a reliable tool for evaluating cancer cell fusion for precise personalized therapy.

A Multiple Targeting Nanoprobe for Identifying Cancer Metastatic Sites Based on Detection of Various mRNAs in Circulating Tumor Cells.

Identification of cancer metastatic sites is of importance for adjusting therapeutic interventions and treatment choice. However, identifying the location of metastatic lesions with easy accessibility and high safety is challenging. Here we demonstrate that cancer metastatic sites can be accurately detected by a triple targeting nanoprobe. Through coencapsulating molecular beacons probing a cancer biomarker (CXCR4 mRNA), a lung metastatic biomarker (CTSC mRNA), and a bone metastatic biomarker (JAG1 mRNA), the nanoprobe decorated by SYL3C conjugated hyaluronic acid and ICAM-1 specific aptamer conjugated hyaluronic acid can target diverse phenotyped circulating tumor cells (CTCs) during epithelial-mesenchymal and mesenchymal-epithelial transitions in whole blood for sensitive probing. The detection of CTCs from cancer patients shows that the nanoprobe can provide accurate information to distinguish different cancer metastasis statuses including nonmetastasis, lung metastasis, and bone metastasis. This study proposes an efficient screening tool for identifying the location of distant metastatic lesions via facile blood biopsy.

Multi-Targeting Nano-Systems Targeting Heterogeneous Cancer Cells for Therapeutics and Biomarker Detection.

Cancer heterogeneity plays a vital part in cancer resistance and metastasis. To provide a reliable approach to exert a therapy action and evaluate its efficiency in heterogeneous cancer cells, a multiple targeting delivery vector composed of histone encapsulating the therapeutic or diagnostic agent, hyaluronic acid targeting CD44 overexpressed in stem tumor cells, SYL3C aptamer targeting epithelial cell adhesion molecule (EpCAM) overexpressed in epithelial cancer cells, and CL4 aptamer targeting epidermal growth factor receptor (EGFR) overexpressed in mesenchymal cancer cells, is developed. The vector can efficiently target different cancer cells and circulating tumor cells (CTCs) in the peripheral blood of patients for mucin 1 (MUC1) knockout. Furthermore, the multiple targeting vector can be used to co-encapsulate three types of molecular beacons for probing various mRNA biomarkers at single-cell resolution after genome editing. This study provides an efficient approach for exerting therapeutic actions in heterogeneous cancer cells and assessing the therapeutic efficacy by detection of cancer biomarkers via liquid biopsy.

Lymph-Node-Targeted Drug Delivery for Effective Immunomodulation to Prolong the Long-Term Survival After Heart Transplantation.

The chronic rejection responses and side effects of the systematic administration of immunosuppressants are the main obstacles to heart allograft and patient survival. The development of xenotransplantation also urgently requires more efficient immune regulation strategies. Herein, it is demonstrated that lymph-node (LN)-targeted drug delivery can realize LN-specific immunomodulation with attenuated immune suppression on distant peripheral immune organs to effectively prolong long-term survival after heart transplantation in a chronic murine heart transplantation model. A chemokine C-C motif ligand 21 (CCL21) specific aptamer for LN targeting is decorated onto the surface of the hybrid nanoparticular delivery vector mainly composed of CaCO3 /CaP/heparin. The targeting delivery system can dramatically enhance accumulation of the loaded immunosuppressant, fingolimod hydrochloride (FTY720), in draining lymph nodes (dLNs) for inducing powerful immune suppression. By promoting the generation of endogenous regulatory T cells (Tregs ) and decreasing the proportion of effector T cells (Teffs ) in dLNs after heart transplantation, the LN-targeting strategy can effectively regulate local immune responses instead of systemic immunity, which reduces the incidence of long-term complications. This study provides an efficient strategy to improve the survival rate after organ transplantation by precise and localized immunoregulation with minimized side effects of immunosuppression.

Precise Detection on Cell-Cell Fusion by a Facile Molecular Beacon-Based Method.

Cell-cell fusion studies provide an experimental platform for evaluating disease progression and investigating cell infection. However, to realize sensitive and quantitative detection on cell-cell fusion is still a challenge. Herein, we report a facile molecular beacon (MB)-based method for precise detection on cell-cell fusion. By transfection of the spike protein (S protein) and enhanced green fluorescent protein (EGFP) in HEK 293 cells, the virus-mimicking fusogenic effector cells 293-S-EGFP cells were constructed to interact with target cells. Before mixing the effector cells with the target cells, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in 293-S-EGFP cells was silenced, and the MB for GAPDH mRNA detection was delivered into the GAPDH silenced 293-S-EGFP cells. Once cell-cell fusion occurred, MB migrated from the GAPDH silenced effector cells to the target cells and hybridized with GAPDH mRNA in the target cells to induce fluorescence emission. The cell-cell fusion can be easily visualized and quantitated by fluorescence microscopy and flow cytometry. The fluorescence intensity is strongly dependent on the number of fused target cells. This MB-based method can easily identify the differences in the cell fusions for various target cells with different angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) expression levels, resulting in dramatically different fluorescence intensities in fused target cells. Our study provides a convenient and efficient quantitative detection approach to study cell-cell fusion.

Codelivery of HBx-siRNA and Plasmid Encoding IL-12 for Inhibition of Hepatitis B Virus and Reactivation of Antiviral Immunity.

Chronic hepatitis B is a critical cause of many serious liver diseases such as hepatocellular carcinoma (HCC). The main challenges in hepatitis B treatment include the rebound of hepatitis B virus (HBV)-related antigen levels after drug withdrawal and the immunosuppression caused by the virus. Herein, we demonstrate that the HBV-related antigen can be effectively inhibited and antiviral immunity can be successfully reactivated through codelivery of the small interfering RNA (siRNA) targeting HBV X protein (HBx) and the plasmid encoding interleukin 12 (pIL-12) to hepatocytes and immune cells. After being treated by the siRNA/pIL-12 codelivery system, HBx mRNA and hepatitis B surface antigen (HBsAg) are dramatically reduced in HepG2.215 cells. More importantly, the downregulated CD47 and programmed death ligand 1 (PD-L1) and the upregulated interferon-ß promoter stimulator-1 (IPS-1), retinoic acid-inducible gene-1 (RIG-1), CD80, and human leukocyte antigen-1 (HLA-1) in treated HepG2.215 cells indicate that the immunosuppression is reversed by the codelivery system. Furthermore, the codelivery system results in inhibition of extracellular regulated protein kinases (ERK) and phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) pathways, as well as downregulation of B-cell lymphoma-2 (Bcl-2) and upregulation of p53, implying its potential in preventing the progression of HBV-induced HCC. In addition, J774A.1 macrophages treated by the codelivery system were successfully differentiated into the M1 phenotype and expressed enhanced cytokines with anti-hepatitis B effects such as interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α). Therefore, we believe that codelivery of siRNA and pIL-12 can effectively inhibit hepatitis B virus, reverse virus-induced immunosuppression, reactivate antiviral immunity, and hinder the progression of HBV-induced hepatocellular carcinoma. This investigation provides a promising approach for the synergistic treatment of HBV infection.

Detection of mRNAs of Ribosomal Protein L15 and E-Cadherin in Living Circulating Tumor Cells at Single Cell Resolution To Study Tumor Heterogeneity.

To study the heterogeneity of circulating tumor cells (CTCs) is of crucial importance to analyze cancer progression and metastasis. However, in situ detection of highly heterogeneous CTCs in peripheral blood still faces an elusive challenge. Here, we show direct detection of two metastasis-related mRNAs of diverse CTCs in whole blood by a triple-targeting nanoprobe. In the nanoprobe, two kinds of molecular beacons, MB1 to detect RPL15 mRNA and MB2 to detect E-cadherin (E-cad) mRNA, are loaded in a highly efficient delivery vector decorated with EpCAM-targeted SYL3C, EGFR-targeted CL4, and CD44-targeted hyaluronic acid chains to specifically deliver MB1/MB2 into epithelial, mesenchymal, and stem CTCs in unprocessed peripheral blood. The numbers of RPL15+ and E-cad+ CTCs are positively correlated with the metastasis stages of cancer patients. This study provides an effective strategy to realize direct observation on diverse metastasis-related genes in living CTCs with different phenotypes to provide accurate information on cancer heterogeneity and metastasis.

Tumor-triggered targeting ammonium bicarbonate liposomes for tumor multimodal therapy.

Tumor-triggered targeting ammonium bicarbonate (TTABC) liposomes were proposed to improve the uptake of ammonium bicarbonate (ABC) liposomes in tumor cells and retain their long circulation in vivo in our previous study. However, it must be solved how to precisely release the loaded drugs of the TTABC liposomes into tumor cells. In addition, synergistic multimodal therapy could result in better tumor treatment outcomes than monomodal chemotherapy. In the research, we prepared indocyanine green (ICG) and doxorubicin (DOX) encapsulated TTABC liposomes (ICG&DOX@TTABC) to achieve near-infrared (NIR) light-controlled chemo/photothermal/photodynamic multimodal therapy guided by fluorescence and photothermal imaging. In vitro and vivo studies show that ICG&DOX@TTABC can specifically accumulate in tumor tissues, effectively transform NIR light into local thermo-therapy, and have excellent anti-tumor ability without obvious side effects. ICG&DOX@TTABC could be promising for fluorescence and photothermal imaging-guided chemo/photothermal/photodynamic tumor treatment.

Functional Tumor Targeting Nano-Systems for Reprogramming Circulating Tumor Cells with In Situ Evaluation on Therapeutic Efficiency at the Single-Cell Level.

Tumor heterogeneity is primarily responsible for treatment resistance and cancer relapses. Being critically important to address this issue, the timely evaluation of the appropriateness of therapeutic actions at the single-cell level is still facing challenges. By using multi-functionalized nano-systems with the delivery vector composed of histone for plasmids loading, hyaluronic acid for tumor targeting, and a fusion peptide for C-X-C motif chemokine receptor 4 (CXCR4） targeting as well as nuclear localization, the reprogramming of circulating tumor cells (CTCs) with in situ detection on biomarkers at the single-cell level is realized. By efficient co-delivery of the genome editing plasmid for CXCR4 knockout and molecular beacons for detection of upregulated mRNA biomarkers into CTCs in unprocessed whole blood, the therapeutic outcomes of genome editing at the single-cell level can be in situ evaluated. The single-cell analysis shows that CXCR4 in CTCs of cancer patients is efficiently downregulated, resulting in upregulated anticancer biomarkers such as p53 and p21. The study provides a facile strategy for in-depth profiling of cancer cell responses to therapeutic actions at single-cell resolution to evaluate the outcomes of treatments timely and conveniently.

A targeting delivery system for effective genome editing in leukemia cells to reverse malignancy.

Therapy resistance associated with relapse is a main cause of death in acute myeloid leukemia (AML). To address this issue, a dual-targeting CRISPR-Cas9 genome editing nanosystem was constructed for CXCR4 knockout to reverse the malignancy of leukemia cells. The surface of the dual-targeting nanosystem is composed of MUC1 specific aptamer incorporated alginate (MUC1 aptamer-alginate) and T22-NLS peptide with T22 sequence targeting CXCR4; the core of the nanosystem consists of protamine complexed with CRISPR-Cas9 plasmid. The in vitro study shows that the nanosystem mediated genome editing induces cell apoptosis, cell cycle arrest, as well as inhibited cell migration and adhesion in edited THP-1 cells after CXCR4 knockout. Further, the unprocessed peripheral blood from acute myeloid leukemia (AML) patients was directly used to carry out ex vivo study. The results show the genome editing nanosystem can effectively knock out CXCR4 in leukemia cells, leading to attenuated CXCR4 protein as studied by antibody labeling and reduced CXCR4 mRNA as probed by a molecular beacon delivery system. In addition to developing a promising delivery vector for gene therapy on AML, this study also provides an effective strategy to evaluate the therapeutic efficiency of particular treatments by peripheral blood-based ex vivo studies.

In Situ Detection of Nanotoxicity in Living Cells Based on Multiple miRNAs Probed by a Peptide Functionalized Nanoprobe.

The potential toxicity of nanoparticles, especially for clinically applicable ones, has become a critical concern. Technologies that can in situ-evaluate the toxicity of nanoparticles with high sensitivity are urgently needed. In this study, a facile strategy was developed for sensitive detection on the nanotoxicity of nanoparticles with low toxicity or a low dose. A functional nanoprobe loaded with molecular beacons was constructed to realize in situ evaluation of the nanotoxicity through probing multiple miRNAs in nanoparticle-exposed living cells. Being composed of protamine complexed with molecular beacons for miRNA detection and decorated by TAT and KALA peptides, the dual-peptide functionalized nanoprobe can efficiently deliver molecular beacons into living cells to realize the real-time monitoring of early biomarkers (miR-21 and miR-221) to evaluate nanotoxicity. Using mesoporous silica nanoparticles (MSNs) with different surface modifications as typical representatives of low toxic nanoparticles, we demonstrate that our nanoprobe can sensitively detect miRNA changes in cells under diverse exposure conditions, that is, MSN-NH2 exhibits the strongest capability to upregulate miR-21 and miR-221, and the upregulation is exposure dose- and time-dependent. Our approach is much more sensitive as compared with conventional methods to study cytotoxicity such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell morphology observation, and reactive oxygen species (ROS) assay. This study paves a path for effective and facile nanotoxicity evaluation and provides insights into the biological impacts of MSNs.

Direct detection of intracellular miRNA in living circulating tumor cells by tumor targeting nanoprobe in peripheral blood.

Molecular analysis of circulating tumor cells (CTCs) is of critical significance for the non-invasive early detection of tumors. However, in situ detection of intracellular nucleic acids of CTCs in whole blood still remains challenge. By using a highly efficient tumor targeting nanoprobe, we realize in situ detection of microRNA-21 (miR-21) of living CTCs in unprocessed whole blood. In the nanoprobe, a catalytic hairpin assembly (CHA) system is complexed with protamine sulfate (PS), and then decorated by SYL3C conjugated hyaluronic acid (SHA) and hyaluronic acid (HA). The CHA system can be specifically delivered into living CTCs in whole blood, followed by hybridization between the CHA system and intracellular miR-21 in CTCs to induce strong fluorescence emission. After isolation of CTCs by membrane filtration, CTCs of cancer patients can be directly visualized by a fluorescence microscope for miR-21 detection at a single-cell level. Our study provides an efficient strategy to realize in situ genomic analysis of living CTCs in whole blood.

An Albumin-Based Therapeutic Nanosystem for Photosensitizer/Protein Co-Delivery to Realize Synergistic Cancer Therapy.

Oxygen-dependent photodynamic therapy (PDT) is hindered by the limited availability of endogenous oxygen in solid tumors and low tumor accumulation of photosensitizers. Herein, we developed a biocompatible cancer-targeted therapeutic nanosystem based on cRGD conjugated bovine serum albumin (CBSA) co-loaded with a photosensitizer (chlorin e6, Ce6) and a therapeutic protein (cytochrome c, Cytc) for synergistic photodynamic and protein therapy. The nanosystem (Ce6/Cytc@CBSA) can target αVß3 integrin overexpressed cancer cells to improve tumor accumulation due to incorporation of cRGD. In the intracellular environment, Ce6 is released to produce toxic singlet oxygen upon near-infrared irradiation. At the same time, the therapeutic protein, Cytc, can induce programmed cell death by activating the downstream caspase pathway. Most importantly, Cytc with the catalase-like activity accelerates O2 generation by decomposing excess H2O2 in cancer cells, thereby relieving the PDT-induced hypoxia to enhance therapeutic efficacy. Both in vitro and in vivo studies reveal the significantly improved antitumor effects of the combined photodynamic/protein therapy, indicating that Ce6/Cytc@CBSA shows great potential in synergetic cancer treatments.

Aptamer/Peptide-Functionalized Genome-Editing System for Effective Immune Restoration through Reversal of PD-L1-Mediated Cancer Immunosuppression.

Effective reversal of tumor immunosuppression is of critical importance in cancer therapy. A multifunctional delivery vector that can effectively deliver CRISPR-Cas9 plasmid for ß-catenin knockout to reverse tumor immunosuppression is constructed. The multi-functionalized delivery vector is decorated with aptamer-conjugated hyaluronic acid and peptide-conjugated hyaluronic acid to combine the tumor cell/nuclear targeting function of AS1411 with the cell penetrating/nuclear translocation function of TAT-NLS. Due to the significantly enhanced plasmid enrichment in malignant cell nuclei, the genome editing system can induce effective ß-catenin knockout and suppress Wnt/ß-catenin pathway, resulting in notably downregulated proteins involved in tumor progression and immunosuppression. Programmed death-ligand 1 (PD-L1) downregulation in edited tumor cells not only releases the PD-1/PD-L1 brake to improve the cancer killing capability of CD8+ T cells, but also enhances antitumor immune responses of immune cells. This provides a facile strategy to reverse tumor immunosuppression and to restore immunosurveillance and activate anti-tumor immunity.

Self-Assembled Plasmid Delivery System for PPM1D Knockout to Reverse Tumor Malignancy.

Gene delivery vectors possess critical roles in effective genome editing. In this study, a multiple functional vector for encapsulating CRISPR/Cas9 plasmid was designed to knock out PPM1D gene and prevent cancer malignancy. The plasmid was complexed with a KALA peptide with the capability of endosomal escape and histones for nuclear transportation and then decorated by hyaluronic acid (HA) and AS1411-incorporated hyaluronic acid (AHA) targeting CD44 and nucleolin overexpressed in cancer cells to form AHA/HA/KALA/histone/plasmid nanoparticles. The constructed multifunctional plasmid delivery system with the cancer targeting specificity can realize efficient genome editing for PPM1D knockout and thus dramatically downregulate PPM1D expression in targeted malignant cells. More importantly, PPM1D knockout results in upregulation of p21 and p-p38 as well as downregulation of cyclin D1, MMP9, CYR61, and vimentin. The edited cancer cells exhibit suppressed proliferation, migration, and invasion, indicating the successful reversal of tumor malignancy.

Multifunctional Albumin-Based Delivery System Generated by Programmed Assembly for Tumor-Targeted Multimodal Therapy and Imaging.

To enhance the treatment efficiency in tumor therapy, we developed a tumor-targeting protein-based delivery system, DOX&ICG@BSA-KALA/Apt, to efficiently integrate multimodal therapy with tumor imaging and realize synchronous photodynamic therapy/photothermal therapy/chemotherapy. In the delivery system, a chemotherapeutic drug (doxorubicin, DOX) and an optotheranostic agent (indocyanine green, ICG) were co-loaded in bovine serum albumin (BSA) via a hydrophobic-interaction-induced self-assembly to form stable DOX&ICG@BSA nanoparticles. After the decoration of a surface layer composed of a tumor-targeting aptamer (AS1411) and a cell-penetrating peptide (KALA), the obtained DOX&ICG@BSA-KALA/Apt nanoparticles exhibit a significantly improved multimodal cancer therapeutic efficiency due to the enhanced cancer cellular uptake mediated by AS1411 and KALA. In vitro and in vivo studies show that the multimodal theranostic system can efficiently inhibit tumor growth. In addition, the near-infrared fluorescent/photothermal dual-mode imaging enables accurate visualization of the therapeutic action in tumor sites. This study provides a facile strategy to construct self-assembled multimodal theranostic systems, and the functional protein-based theranostic system prepared holds great promise in multimodal cancer therapeutics.

Peptide and Aptamer Decorated Delivery System for Targeting Delivery of Cas9/sgRNA Plasmid To Mediate Antitumor Genome Editing.

A multiple-functionalized targeting delivery system was prepared by self-assembly for efficient delivery of Cas9/sgRNA plasmids to targeted tumor cell nuclei. The Cas9/sgRNA plasmids were compacted by protamine in the presence of calcium ions to form nanosized cores, which were further decorated by peptide and aptamer conjugated alginate derivatives. With the help of the nuclear location signal peptide and AS1411 aptamer with specific affinity for nucleolin in the tumor cell membrane and nuclei, the delivery vector can specifically deliver the plasmid to the nuclei of tumorous cells for knocking out the protein tyrosine kinase 2 (PTK2) gene to down-regulate focal adhesion kinase (FAK). The tumor cell apoptosis induced by genome editing is mitochondrial-dependent. In addition, FAK knockout results in negative regulation on the PI3K/AKT signaling pathway. Meanwhile, favorable modulation on various proteins involved in tumor progression can be realized by genome editing. The enhanced E-cadherin and decreased MMPs, vimentin, and VEGF imply the desirable effects of genome editing on suppression of tumor development. Wound healing and transwell assays confirm that the genome editing system can suppress tumor invasion and metastasis in edited cells efficiently. The investigation provides a facile and effective strategy to fabricate multiple-functionalized delivery vectors for genome editing.