Biomineralized Metal-Organic Framework Nanoparticles Enable Intracellular Delivery and Endo-Lysosomal Release of Native Active Proteins.

Efficient delivery and endo-lysosomal release of active proteins in living cells remain a challenge in protein-based theranostics. We report a novel protein delivery platform using protein-encapsulating biomineralized metal-organic framework (MOF) nanoparticles (NPs). This platform introduces an adapted biomimetic mineralization method for facile synthesis of MOF NPs with high protein encapsulation efficiency and a new polymer coating strategy to confer the NPs with long-term stability. In vitro results show that protein-encapsulating MOF NPs have the advantages of preserving protein activity for months and protecting proteins from enzyme-mediated degradation. Live cell studies reveal that MOF NPs enable rapid cellular uptake, efficient release and escape of proteins from endo-lysosomes, and preservation of protein activity in living cells. Moreover, the developed platform is demonstrated to enable easy encapsulation of multiple proteins in single MOF NPs for efficient protein co-delivery. To our knowledge, it is the first time that protein-encapsulating MOF NPs have been developed as a generally applicable strategy for intracellular delivery of native active proteins. The developed protein-encapsulating biomineralized MOF NPs can provide a valuable platform for protein-based theranostic applications.

Nanoscale Zeolitic Imidazolate Framework-8 for Ratiometric Fluorescence Imaging of MicroRNA in Living Cells.

MicroRNAs (miRNAs) play important roles in cell differentiation, proliferation, and apoptosis and have been recognized as valuable biomarkers for clinical disease diagnosis. Here, we adopt for the first time zeolitic imidazolate framework-8 (ZIF-8) as a nanocarrier to efficiently deliver a nucleic acid probe to living cells and develop a novel ratiometric fluorescence strategy based on DNAzyme for miRNA-21 imaging. A Cy5-labeled 8-17 DNAzyme strand and a Cy3-labeled substrate strand containing a segment complementary to the target miRNA-21 first form a duplex probe, and fluorescence resonance energy transfer (FRET) takes place. After adsorption on the ZIF-8 surface and cellular uptake, the probe/ZIF-8 nanocomplex degrades in acidic endosome and releases duplex probes and Zn2+, and the latter can act as an effective cofactor for 8-17 DNAzyme. The intracellular miRNA-21 hybridizes with the complementary segment of the substrate strand and results in dissociation from the DNAzyme-substrate duplex probe after DNAzyme cleaves the substrate into two fragments, accompanied by the change in the FRET signal. The proposed method has been applied to image miRNA-21 expression levels in MCF-7, HeLa, and L02 cells with high contrast and reliability. The fluctuation of miRNA-21 expression level induced by miRNA-21 mimic or inhibitor can also be monitored through the obvious imaging color change. Taken together, the proposed method provides a powerful tool for cancer diagnosis and miRNA-associated biological study.

A graphene oxide nanosensor enables the co-delivery of aptamer and peptide probes for fluorescence imaging of a cascade reaction in apoptotic signaling.

Cytochrome c (Cyt c) and caspase-3 are the key mediators in apoptotic signaling. As is known to all, the release of Cyt c from mitochondria is a vital caspase activation pathway and defines the point of no-return in cell apoptosis. However, it has not been reported that any fluorescence imaging tools could allow simultaneous visualization of Cyt c translocation and caspase-3 activation in apoptotic cells. Here, we develop a sensitive nanosensor that holds the capability of imaging of the released Cyt c from the mitochondria and a caspase-3 activation cascade reaction in apoptotic signaling. The nanosensor is constructed by the assembly of a fluorophore (Cy5)-tagged DNA aptamer on graphene nanosheets that have been covalently immobilized with a FAM-labeled peptide. After a spatially selective delivery into the cytoplasm, the Cy5-tagged DNA aptamer assembled on the nanosensor can bind with Cyt c released from the mitochondria to the cytoplasm and dissociate from graphene, triggering a red fluorescence signal. In addition, the caspase-3 activated by the Cyt c released to the cytoplasm can cleave the FAM-labeled peptide and result in a green fluorescence output. The nanosensor exhibits rapid response, high sensitivity and selectivity for in vitro assays, and high contrast imaging of Cyt c and caspase-3 in living cells. It also provides the method for the study of the kinetic relationship between the Cyt c translocation and caspase-3 activation through simultaneous imaging of Cyt c and caspase-3. The developed nanosensor described here will be an efficient and potential platform for apoptosis research.

An intelligent nanodevice based on the synergistic effect of telomerase-triggered photodynamic therapy and gene-silencing for precise cancer cell therapy.

The development of intelligent and precise cancer therapy systems that enable accurate diagnosis and specific elimination of cancer cells while protecting normal cells to improve the safety and effectiveness of the treatment is still a challenge. Herein, we report a novel activatable nanodevice for precise cancer therapy. The nanodevice is constructed by adsorbing a DNA duplex probe onto MnO2 nanosheets. After cellular uptake, the DNA duplex probe undergoes telomerase-triggered conformation switching, resulting in a Ce6 "turn-on" signal for the identification of cancer cells. Furthermore, Deoxyribozyme (DNAzyme) is activated to catalyse the cleavage of survivin mRNA, actualizing a precise synergistic therapy in cancer cells involving photodynamic therapy and gene-silencing. The MnO2 nanosheets provide Mn2+ for the DNAzyme and relieve hypoxia to improve the efficiency of the photodynamic therapy. Live cell studies reveal that this nanodevice can diagnose cancer cells and specifically eliminate them without harming normal cells, so making the treatment safer and more effective. The developed DNA-MnO2 nanodevice provides a valuable and general platform for precise cancer therapy.

In Situ Synthesis of Ultrathin ZIF-8 Film-Coated MSNs for Codelivering Bcl 2 siRNA and Doxorubicin to Enhance Chemotherapeutic Efficacy in Drug-Resistant Cancer Cells.

Multiple drug resistance is a persistent obstacle for efficient chemotherapy of cancer. Herein, we report a novel drug delivery platform. A zeolitic imidazole framework-8 (ZIF-8) film with a few nanometer thickness was in situ synthesized on the surface of carboxylated mesoporous silica (MSN-COOH) nanoparticles (NPs) for pore blocking and efficient loading of small interfering RNAs to fabricate a pH-responsive drug delivery system. The ZIF-8 film could convert the charge of MSN-COOH from negative to positive for efficient loading of siRNA via electrostatic interactions and protect siRNA from nuclease degradation. The positively charged ZIF-8 film facilitates cellular uptake and endo-lysosome escape of the NPs. In addition, the ultrathin ZIF-8 film can decompose in the acidic endo-lysosome and trigger the intracellular release of siRNAs and chemotherapeutic drugs, leading to a significantly enhanced chemotherapeutic efficacy for multidrug-resistant cancer cells including MCF-7/ADR and SKOV-3/ADR cells as demonstrated by the confocal laser scanning microscopy image, cell viability assay, Annexin V&PI staining, and flow cytometry. This approach provides a promising strategy for pH-triggered, stimuli-responsive delivery of nucleic acid drugs and chemotherapeutic agents with remarkably enhanced chemotherapeutic efficacy.