Elucidating the Functional Mechanism of PTK7 in Cancer Development through Spatial Assembly Analysis Using Super Resolution Imaging.

Protein tyrosine kinase-7 (PTK7) has been reported as a vital participant in the Wnt signaling pathway, influencing tumorigenesis and metastasis. However, their specific roles in the mechanisms underlying cancer development and progression remain elusive. Here, using direct stochastic optical reconstruction microscopy (dSTORM) with aptamer-probe labeling, we first revealed that a weakening clustering distribution of PTK7 on the basal membranes happened as cellular migration increased during cancer progression. This correspondence was further supported by a diminished aggregated state of PTK7 caused by direct enhancement of cell migration. By comparing the alterations in PTK7 distribution with activation or inhibition of specific Wnt signaling pathway, we speculated that PTK7 could modulate cell migration by participating in the interplay between canonical Wnt (in MCF7 cells) and noncanonical Wnt signals (in MDA-MB-231 cells). Furthermore, we discovered that the spatial distribution morphology of PTK7 was also subject to the hydrolysis ability and activation state of the related hydrolase Matrix metallopeptidase14 (MMP14). This function-related specific assembly of PTK7 reveals a clear relationship between PTK7 and cancer. Meanwhile, potential molecular interactions predicted by the apparent assembly morphology can promote a deep understanding of the functional mechanism of PTK7 in cancer progress.

Photoinduced Electron Transfer-Based Glutathione-Sensing Theranostic Nanoprodrug with Self-Tracking and Real-Time Drug Release Monitoring for Cancer Treatment.

The rapid development of nanomedicine has considerably advanced precision therapy for cancer treatment. Superior to traditional chemotherapy, emerging theranostic nanoprodrugs can effectively realize inherent self-tracking, targeted drug delivery, stimuli-triggered drug release, and reduced systemic toxicity of chemotherapeutic drugs. However, theranostic nanoprodrugs with real-time drug release monitoring have remained rare so far. In this work, we developed a new glutathione-responsive theranostic nanoprodrug with a high drug-loading content of 59.4 wt % and an average nanoscale size of 46 nm, consisting of the anticancer drug paclitaxel and a fluorescent imaging probe with a high fluorescence quantum yield, which are linked by a disulfide-based glutathione-sensitive self-immolating linker. The strong fluorescence emission of the fluorophore enables efficacious self-tracking and sensitive fluorescence "ON-OFF" glutathione sensing. Upon encountering high-level glutathione in cancer cells, the disulfide bond is cleaved, and the resulting linker halves spontaneously collapse into cyclic small molecules at the same pace, leading to the simultaneous release of the therapeutic drug and the fluorescence-OFF imaging probe. Thereby, the drug release process is efficiently monitored by the fluorescence change in the nanoprodrug. The nanoprodrugs exerted high cytotoxicity toward various cancer cells, especially for A549 and HEK-293 cells, in which the nanoprodrugs generated better therapeutic effects than free paclitaxel. Our work demonstrated a new modality of smart theranostic nanoprodrugs for precise cancer therapy.

Characterizing the function-related specific assembly pattern of matrix metalloproteinase-14 by dSTORM imaging.

As transmembrane proteolytic enzyme, matrix metalloproteinase-14 (MMP14) regulates cell migration and cancer metastasis, but how it works at the single molecule level is unclear. Molecular localization is closely related to its function, and revealing its spatial assemble details is thus helpful to understand bio-function. Here, we apply aptamer probe and dSTORM to characterize MMP14 distribution. With demonstrating labeling properties of the probe, we investigate the specific distributed pattern of MMP14 on various cell membranes with different migratory capacities, and find that MMP14 mostly aggregate in clustering state, which becomes more significant with enhancing its hydrolysis efficiency on high-migratory cells. Lots of MMP14 are revealed to be co-localized with its substrate PTK7, and this colocalization decreases with weakening cell migration, suggesting that MMP14 may coordinate cell migration by altering its spatial relationship with substrate proteins. This work will promote a deep understanding of the roles of MMP14 in cell migration and cancer metastasis.

Esterase-activatable and GSH-responsive Triptolide Nano-prodrug for the Eradication of Pancreatic Cancer.

Triptolide (TPL) is a small molecule isolated from a traditional Chinese herb Tripterygium wilfordii Hook F and shows excellent anticancer effect for pancreatic cancer cells. However, the poor water solubility and severe liver toxicity of TPL hindered its clinical application. In this study, TPL was covalently conjugated to a polymer and entrapped inside the core of the TPL nanogel (nTPL) to protect it from premature leakage during blood circulation. With the help of lactobionic acid (LBA), nTPL-LBA could selectively target the tumors in an orthotopic pancreatic cancer mouse model. TPL could be subsequently released intracellularly in its original form due to the presence of elevated intracellular esterase and GSH, and eventually kills cancer cells. nTPL-LBA treatment reduced tumor burden by 99% while not introducing TPL associated liver and kidney toxicities. Most importantly, more than half of the nTPL-LBA treated animals were tumor-free, suggesting that nTPL-LBA is an effective approach in eradicating pancreatic cancer.

Nanogel-facilitated Protein Intracellular Specific Degradation through Trim-Away.

Recently discovered "Trim-Away" mechanism opens a new window for fast and selective degradation of endogenous proteins. However, the in vivo and clinical application of this approach is stuck by the requirement of special skills and equipment needed for the intracellular delivery of antibodies. Hereby, an antibody conjugated polymer nanogel system, Nano-ERASER, for intracellular delivery and release of antibody, and degradation of a specific endogenous protein has been developed. After being delivered into cells, the antibody is released and forms complex with its target protein, and subsequently binds to the Fc receptor of TRIM21. The resulted complex of target protein/antibody/TRIM21 is then degraded by the proteasome. The efficacy of Nano-ERASER has been validated by depleting GFP protein in a GFP expressing cell line. Furthermore, Nano-ERASER successfully degrades COPZ1, a vital protein for cancer cells, and kills those cells while sparing normal cells. Benefit from its convenience and targeted delivery merit, Nano-ERASER technique is promising in providing a reliable tool for endogenous protein function study as well as paves the way for novel antibody-based Trim-Away therapeutic modalities for cancer and other diseases.

Temperature and pH Dual Responsive Nanogels of Modified Sodium Alginate and NIPAM for Berberine Loading and Release.

pH- and temperature-sensitive nanogels (NGs) were prepared from sodium alginate (SA) and N-isopropylacrylamide (NIPAM), as the sensitivity at pH 5.5 and 31 °C. SA was pH-modified with glutamic acid (Glu) and ethylenediamine (EDA). The products Glu-SA (Glu-modified SA) and EGSA (EDA- and Glu-modified SA) were characterized by ninhydrin color reaction, infrared spectroscopy, and zeta potential, and the best reactant ratio was selected. Moreover, temperature-sensitive, pH-sensitive EGSA-NGs possessing a semi-interpenetrating network structure were prepared by radical polymerization using N-isopropylacrylamide. The morphology of EGSA-NGs was characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM). The cytotoxicity test shows the low cytotoxicity and high biocompatibility of the NGs. The newly prepared NGs were also subjected to pH-sensitive temperature-sensitive in vitro drug-loading and drug-release experiments. The pH-sensitive and temperature-sensitive experiments showed that the particle size of EGSA-NGs was reduced at pH 5.5 and above 31 °C. The drug-loading and drug-release experiments also confirmed this finding, indicating that the newly synthesized NGs could release the drug according to the environmental changes. Therefore, the material has potential application value in solid tumor targeted therapy.

Carrier-Free Nanoassembly of Curcumin-Erlotinib Conjugate for Cancer Targeted Therapy.

Anticancer drug-loaded nanoparticles have been explored extensively to decrease side effects while improving their therapeutic efficacy. However, due to the low drug loading content, premature drug release, nonstandardized carrier structure, and difficulty in predicting the fate of the carrier, only a few nanomedicines have been approved for clincial use. Herein, a carrier-free nanoparticle based on the self-assembly of the curcumin-erlotinib conjugate (EPC) is developed. The EPC nanoassembly exhibits more potent cell killing, better antimigration, and anti-invasion effects for BxPC-3 pancreatic cancer cells than the combination of free curcumin and erlotinib. Furthermore, benefiting from both passive and active tumor targeting effect, EPC nanoassembly can effectively accumulate in the tumor tissue in a xenograft pancreatic tumor mouse model. Consequently, EPC effectively reduces the growth of pancreatic tumors and extends the median survival time of the tumor-bearing mice from 22 to 68 days. In addition, no systemic toxicity is detected in the mice receiving EPC treatment. Attributed to the uniformity of the curcumin-erlotinib conjugate and easiness of scaling up, it is expected that the EPC can be translated into a powerful tool in fighting against pancreatic cancer and other epidermal growth factor receptor positive cancers.

Heterotargeted Nanococktail with Traceless Linkers for Eradicating Cancer.

Clinical application of drug cocktails for cancer therapy is limited by their severe systemic toxicity. To solve a catch-22 dilemma between safety and efficacy for drug cocktails, a hetero-targeted nano-cocktail (PPPDMA) with traceless linkers has been developed. In the PPPDMA nanogel, a hetero-targeting strategy is employed to improve its tumor selective targeting efficacy by overcoming the cancer cell mono-ligand density limitation. Benefit from its glutathione and reactive oxygen species responsiveness, the loaded paclitaxel and doxorubicin can be quickly and tracelessly released into the cytoplasm in their original form, which bestows PPPDMA nanogels the capability to overwhelm the processing capacity of cancer cell's P-glycoprotein efflux pump allows, and ultimately kill them without inducing side effects. The PPPDMA treatment reduced its tumor burden over 99% (in tumor weight) and 96% (in tumor number). Most importantly, no detectable tumor in more than half of the PPPDMA treated mice. We conclude that traceless linker and hetero-targeted nano-cocktail strategy could be a safe and effective approach for cancer treatment.

Programmable Peptide-Cross-Linked Nucleic Acid Nanocapsules as a Modular Platform for Enzyme Specific Cargo Release.

Herein we describe a modular assembly strategy for photo-cross-linking peptides into nucleic acid functionalized nanocapsules. The peptides embedded within the nanocapsules form discrete nanoscale populations capable of gating the release of molecular and nanoscale cargo using enzyme-substrate recognition as a triggered release mechanism. Using photocatalyzed thiol-yne chemistry, different peptide cross-linkers were effectively incorporated into the nanocapsules and screened against different proteases to test for degradation specificity both in vitro and in cell culture. By using a combination of fluorescence assays, confocal and TEM microscopy, the particles were shown to be highly specific for their enzyme targets, even between enzymes of similar protease classes. The rapid and modular nature of the assembly strategy has the potential to be applied to both intracellular and extracellular biosensing and drug delivery applications.

Fluorenyl-Loaded Quatsome Nanostructured Fluorescent Probes.

Delivery of hydrophobic materials in biological systems, for example, contrast agents or drugs, is an obdurate challenge, severely restricting the use of materials with otherwise advantageous properties. The synthesis and characterization of a highly stable and water-soluble nanovesicle, referred to as a quatsome (QS, vesicle prepared from cholesterol and amphiphilic quaternary amines), that allowed the nanostructuration of a nonwater soluble fluorene-based probe are reported. Photophysical properties of fluorenyl-quatsome nanovesicles were investigated via ultraviolet-visible absorption and fluorescence spectroscopy in various solvents. Colloidal stability and morphology of the nanostructured fluorescent probes were studied via cryogenic transmission electronic microscopy, revealing a "patchy" quatsome vascular morphology. As an example of the utility of these fluorescent nanoprobes, examination of cellular distribution was evaluated in HCT 116 (an epithelial colorectal carcinoma cell line) and COS-7 (an African green monkey kidney cell line) cell lines, demonstrating the selective localization of C-QS and M-QS vesicles in lysosomes with high Pearson's colocalization coefficient, where C-QS and M-QS refer to quatsomes prepared with hexadecyltrimethylammonium bromide or tetradecyldimethylbenzylammonium chloride, respectively. Further experiments demonstrated their use in time-dependent lysosomal tracking.