A DNA/Upconversion Nanoparticle Complex Enables Controlled Co-Delivery of CRISPR-Cas9 and Photodynamic Agents for Synergistic Cancer Therapy.

Photodynamic therapy (PDT) depends on the light-irradiated exciting of photosensitizer (PS) to generate reactive oxygen species (ROS), which faces challenges and limitations in hypoxia and antioxidant response of cancer cells, and limited tissue-penetration of light. Herein, a multifunctional DNA/upconversion nanoparticles (UCNPs) complex is developed which enables controlled co-delivery of CRISPR-Cas9, hemin, and protoporphyrin (PP) for synergistic PDT. An ultralong single-stranded DNA (ssDNA) is prepared via rolling circle amplification (RCA), which contains recognition sequences of single guide RNA (sgRNA) for loading Cas9 ribonucleoprotein (RNP), G-quadruplex sequences for loading hemin and PP, and linker sequences for combining UCNP. Cas9 RNP cleaves the antioxidant regulator nuclear factor E2-related factor 2 (Nrf2), improving the sensitivity of cancer cells to ROS, and enhancing the synergistic PDT effect. The G-quadruplex/hemin DNAzyme mimicks horseradish peroxidase (HRP) to catalyze the endogenous H2O2 to O2, overcoming hypoxia condition in tumors. The introduced UCNP converts NIR irradiation with deep tissue penetration to light with shorter wavelength, exciting PP to transform the abundant O2 to 1O2. The integration of gene editing and PDT allows substantial accumulation of 1O2 in cancer cells for enhanced cell apoptosis, and this synergistic PDT has shown remarkable therapeutic efficacy in a breast cancer mouse model.

Telomerase-Mediated Self-Assembly of DNA Network in Cancer Cells Enabling Mitochondrial Interference.

The precise control of the artificially induced reactions inside living cells is emerging as an effective strategy for the regulation of cell functions. Nevertheless, the manipulation of the assembly of exogenous molecules into artificial architectures in response to intracellular-specific signals remains a grand challenge. Herein, we achieve the precise self-assembly of deoxyribonucleic acid (DNA) network inside cancer cells, specifically responding to telomerase, and realize effective mitochondrial interference and the consequent regulation of cellular behaviors. Two functional DNA modules were designed: a mitochondria-targeting branched DNA and a telomerase-responsive linear DNA. Upon uptake by cancer cells, the telomerase primer in linear DNA responded to telomerase, and a strand displacement reaction was triggered by the reverse transcription of telomerase, thus releasing a linker DNA from the linear DNA. The linker DNA afterward hybridized with the branched DNA to form a DNA network on mitochondria. The DNA network interfered with the function of mitochondria, realizing the apoptosis of cancer cells. This system was further administered in a nude mouse tumor model, showing remarkable suppression of tumor growth. We envision that the telomerase-mediated intracellular self-assembly of the DNA network provides a promising route for cancer therapy.

Cascade dynamic assembly/disassembly of DNA nanoframework enabling the controlled delivery of CRISPR-Cas9 system.

CRISPR-Cas9 has been explored as a therapeutic agent for down-regulating target genes; the controlled delivery of Cas9 ribonucleoprotein (RNP) is essential for therapeutic efficacy and remains a challenge. Here, we report cascade dynamic assembly/disassembly of DNA nanoframework (NF) that enables the controlled delivery of Cas9 RNP. NF was prepared with acrylamide-modified DNA that initiated cascade hybridization chain reaction (HCR). Through an HCR, single-guide RNA was incorporated to NF; simultaneously, the internal space of NF was expanded, facilitating the loading of Cas9 protein. NF was designed with hydrophilic acylamino and hydrophobic isopropyl, allowing dynamic swelling and aggregation. The responsive release of Cas9 RNP was realized by introducing disulfide bond-containing N,N-bis(acryloyl)cystamine that was specifically in response to glutathione of cancer cells, triggering the complete disassembly of NF. In vitro and in vivo investigations demonstrated the high gene editing efficiency in cancer cells, the hypotoxicity in normal cells, and notable antitumor efficacy in a breast cancer mouse model.

Lipid-, Inorganic-, Polymer-, and DNA-Based Nanocarriers for Delivery of the CRISPR/Cas9 system.

The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (CRISPR/Cas9) system has been widely explored for the precise manipulation of target DNA and has enabled efficient genomic editing in cells. Recently, CRISPR/Cas9 has shown promising potential in biomedical applications, including disease treatment, transcriptional regulation and genome-wide screening. Despite these exciting achievements, efficient and controlled delivery of the CRISPR/Cas9 system has remained a critical obstacle to its further application. Herein, we elaborate on the three delivery forms of the CRISPR/Cas9 system, and discuss the composition, advantages and limitations of these forms. Then we provide a comprehensive overview of the carriers of the system, and focus on the nonviral nanocarriers in chemical methods that facilitate efficient and controlled delivery of the CRISPR/Cas9 system. Finally, we discuss the challenges and prospects of the delivery methods of the CRISPR/Cas9 system in depth, and propose strategies to address the intracellular and extracellular barriers to delivery in clinical applications.

Lysosome Interference Enabled by Proton-Driven Dynamic Assembly of DNA Nanoframeworks inside Cells.

Coupling materials chemistry systems to biological processes is a promising way to rationally modulate lysosomal functions. A proton-driven dynamic assembly of a DNA nanoframework inside cells coupled with the lysosome-mediated endocytosis pathways/lysosomal maturation, gives the rational modulation of lysosomal functions, which we term "lysosome interference". Through lysosome-mediated endocytosis, the DNA nanoframework with acid-responsive semi-i-motif enters the lysosome and assembles into an aggregate in a process triggered by lysosomal acidity. The aggregate is suitable for long-term retention. The consumption of protons resulted in lysosomal acidity reduction and hydrolase activity attenuation, thus hindering the degradation of nucleic acid drugs in the lysosome and improving gene silencing effects. This study shows a new way to achieve lysosome interference by coupling the subcellular microenvironment with a precisely programmable assembly system.

Synthesis and Catalytic Property of Ribonucleoside-Derived Carbon Dots.

Exploring appropriate precursors has been proposed to be a promising strategy for the creation of artificial enzymes that are emerging as alternatives of natural enzymes. Herein, inspired by the catalytic activities of ribose nucleic acid, using ribonucleosides as precursors including adenosine, guanosine, cytidine, and uridine, respectively, four carbonic aggregates, namely, carbon dots (A-CDs, G-CDs, C-CDs, and U-CDs) to mimic artificial enzymes are synthesized. All the CDs show a planar graphene-like structure and thus can intercalatively bind with DNA double helix. Different from the other three CDs, the uridine-derived U-CDs exhibit unique catalytic property, which can mediate the topological transformation of DNA from supercoiled to nicked open-circular conformation. U-CDs can catalyze oxidation of O2 to generate singlet oxygen 1 O2 via a Haber-Weiss reaction, and consequently mediate oxidative cleavage of phosphate backbone in DNA and release the torsional energy stored in supercoiled DNA. Explorations reveal that the unique highly active oxygenated species, namely, quinone groups that are on the edge of U-CDs, play a key role in the catalytic production of 1 O2 . This work represents a new insight that using natural biomolecules in living systems as precursors can create new species beyond life.

Dynamic Assembly of DNA Nanostructures in Living Cells for Mitochondrial Interference.

Constructing artificial dynamic architectures inside cells to rationally interfere with organelles is emerging as an efficient strategy to regulate the behaviors and fate of cells, thus providing new routes for therapeutics. Herein, we develop an intracellular K+-mediating dynamic assembly of DNA tetrahedrons inside cells, which realizes efficient mitochondrial interference and consequent regulation on the energy metabolism of living cells. In the designer DNA tetrahedron, one vertex was modified with triphenylphosphine (TPP) for mitochondrial targeting, and the other three vertexes were tethered with guanine-rich sequences that could realize K+-mediating formation of intermolecular G-quadruplexes, which consequently led to the assembly of DNA tetrahedrons to form aggregates in the cytoplasm. The DNA aggregates specially targeted mitochondria and served as a polyanionic barrier for substance communication, thus generating a significant inhibition effect on the aerobic respiration function of mitochondria and the associated glycolysis process, which consequently reduced the production of intracellular adenosine triphosphate (ATP). The lack of ATP impeded the formation of lamellipodium that was essential for the movement of cells, consequently resulting in a significant inhibitory effect on cell migration. Remarkably, the migration capacity was suppressed by as high as 50% for cancer cells. This work provides a new strategy for the manipulation of organelles via the endogenous molecule-mediating dynamic assembly of exogenous artificial architectures inside living cells, which is envisioned to have great potential in precise biomedicine.

A Proton-Activatable DNA-Based Nanosystem Enables Co-Delivery of CRISPR/Cas9 and DNAzyme for Combined Gene Therapy.

CRISPR/Cas9 is emerging as a platform for gene therapeutics, and the treatment efficiency is expected to be enhanced by combination with other therapeutic agents. Herein, we report a proton-activatable DNA-based nanosystem that enables co-delivery of Cas9/sgRNA and DNAzyme for the combined gene therapy of cancer. Ultra-long ssDNA chains, which contained the recognition sequences of sgRNA in Cas9/sgRNA, DNAzyme sequence and HhaI enzyme cleavage site, were synthesized as the scaffold of the nanosystem. The DNAzyme cofactor Mn2+ was used to compress DNA chains to form nanoparticles and acid-degradable polymer-coated HhaI enzymes were assembled on the surface of nanoparticles. In response to protons in lysosome, the polymer coating was decomposed and HhaI enzyme was consequently exposed to recognize and cut off the cleavage sites, thus triggering the release of Cas9/sgRNA and DNAzyme to regulate gene expressions to achieve a high therapeutic efficacy of breast cancer.