CRISPR/Cas9 system is a powerful toolbox for gene editing. However, the low delivery efficiency is still a big hurdle impeding its applications. Herein, we report a strategy to deliver Cas9-sgPlk-1 plasmids (CP) by a multifunctional vehicle for tumor therapy. We condensed CPs on TAT peptide-modified Au nanoparticles (AuNPs/CP, ACP) via electrostatic interactions, and coated lipids (DOTAP, DOPE, cholesterol, PEG2000-DSPE) on the ACP to form lipid-encapsulated, AuNPs-condensed CP (LACP). LACP can enter tumor cells and release CP into the cytosol by laser-triggered thermo-effects of the AuNPs; the CP can enter nuclei by TAT guidance, enabling effective knock-outs of target gene (Plk-1) of tumor (melanoma) and inhibition of the tumor both in vitro and in vivo. This AuNPs-condensed, lipid-encapsulated, and laser-controlled delivery system provides a versatile method for high efficiency CRISPR/Cas9 delivery and targeted gene editing for treatment of a wide spectrum of diseases.

CRISPR/Cas9 system is apowerfultoolbox for gene editing. However,the low delivery efficiency is still abig hurdle impeding its applications.H erein, we report as trategy to deliver Cas9-sgPlk-1 plasmids (CP) by am ultifunctional vehicle for tumor therapy. We condensed CPs on TAT peptide-modified Au nanoparticles (AuNPs/CP,A CP) via electrostatic interactions,a nd coated lipids (DOTAP, DOPE, cholesterol, PEG2000-DSPE) on the ACPt of orm lipidencapsulated, AuNPs-condensed CP (LACP). LACP can enter tumor cells and release CP into the cytosol by lasertriggered thermo-effects of the AuNPs;the CP can enter nuclei by TATguidance,enabling effective knock-outs of target gene (Plk-1) of tumor (melanoma) and inhibition of the tumor both in vitro and in vivo.T his AuNPs-condensed, lipid-encapsulated, and laser-controlled delivery system provides aversatile method for high efficiency CRISPR/Cas9 delivery and targeted gene editing for treatment of aw ide spectrum of diseases.

The present study investigated the genetic etiology and possible immunological pathogenesis of recurrent spontaneous abortion by analyzing chromosome abnormalities, and the balance between T helper 17 (Th17) and regulatory T (Treg) cells. A total of 54 patients with recurrent spontaneous abortion were selected. The villus and decidual tissues, and peripheral venous blood were collected from each patient. Villus chromosome analysis was performed by high-throughput gene sequencing. Flow cytometry was used to detect Th17 and Treg cells in patients without chromosome abnormalities (n=30) and the control group (normal pregnancy; n=32). Immunoglobulin (IG) combined with human chorionic gonadotropin hormone (HCG) treatment was given to patients without chromosome abnormalities (n=30). Changes in the expression levels of Th17 and Treg cells before and after treatment were compared with patients with successful pregnancy (n=18). Before treatment, compared with the control group, the proportion of Th17 cells in peripheral blood and decidual tissue was increased and the proportion of Treg cells decreased. After treatment, compared with patients before treatment, the proportion of Th17 cells decreased and Treg cells increased, and the Th17 and Treg cells balance was reversed with a biased towards Treg cells. The present results suggested that the Th17 and Treg cell immune imbalance may be an important immune factor in recurrent spontaneous abortion. IG combined with HCG therapy may improve pregnancy outcomes by reversing the imbalance between Th17 and Treg cells.

In addition to genomic signaling, Estrogen receptor alpha (ERα) is associated with cell proliferation and survival through extranuclear signaling contributing to endocrine therapy (ET) resistance. However, the relationship between extranuclear ERα and ET resistance has not been extensively studied. We sought to measure extranuclear ERα expression by immunohistochemistry using phosphor-integrated dots (IHC-PIDs) and to assess its predictive value for ET resistance. After quantitative detection of ERα by IHC-PIDs in vitro, we developed “the nearest-neighbor method” to calculate the extranuclear ERα. Furthermore, tissue sections from 65 patients with HR+/HER2- BC were examined by IHC-PIDs, and the total ERα, nuclear ERα, extranuclear ERα PIDs score, and ratio of extranuclear-to-nuclear ERα (ENR) were measured using the novel method. We demonstrate that quantification of ERα using IHC-PIDs exhibited strong correlations to real-time qRT-PCR (r2 = 0.94) and flow cytometry (r2 = 0.98). High ERα ENR was significantly associated with poor overall survival (p = 0.048) and disease-free survival (DFS) (p = 0.007). Multivariate analysis revealed that the ERα ENR was an independent prognostic factor for DFS [hazard ratio, 3.8; 95% CI, 1.4–11.8; p = 0.006]. Our automated measurement has high accuracy to localize and assess extranuclear ERα. A high ERα ENR in HR+/HER2− BC indicates decreased likelihood of benefiting from ET.