Smart Vitamin Micelles as Cancer Nanomedicines for Enhanced Intracellular Delivery of Doxorubicin.

Chemotherapy is one of the most effective treatments for cancer. However, intracellular delivery of many anticancer drugs is hindered by their hydrophobicity and low molecular weight. Here, we describe highly biocompatible and biodegradable amphiphilic vitamin conjugates comprising hydrophobic vitamin E and hydrophilic vitamin B labeled with dual pH and glutathione-responsive degradable linkages. Vitamin-based micelles (vitamicelles), formed by self-assembly in aqueous solutions, were optimized based on their stability after encapsulation of doxorubicin (DOX). The resulting vitamicelles have great potential as vehicles for anticancer drugs because they show excellent biocompatibility (>94% after 48 h of incubation) and rapid biodegradability (>90% after 2.5 h). Compared with free DOX, DOX-loaded vitamicelles showed a markedly enhanced anticancer effect as they released the drug rapidly and inhibited drug efflux out of cells efficiently. By exploiting these advantages, this study not only provides a promising strategy for circumventing existing challenges regarding the delivery of anticancer drugs but also extends the utility of current DOX-induced chemotherapy.

Ubiquitin-Specific Protease 29 Regulates Cdc25A-Mediated Tumorigenesis.

Cell division cycle 25A (Cdc25A) is a dual-specificity phosphatase that is overexpressed in several cancer cells and promotes tumorigenesis. In normal cells, Cdc25A expression is regulated tightly, but the changes in expression patterns in cancer cells that lead to tumorigenesis are unknown. In this study, we showed that ubiquitin-specific protease 29 (USP29) stabilized Cdc25A protein expression in cancer cell lines by protecting it from ubiquitin-mediated proteasomal degradation. The presence of USP29 effectively blocked polyubiquitination of Cdc25A and extended its half-life. CRISPR-Cas9-mediated knockdown of USP29 in HeLa cells resulted in cell cycle arrest at the G0/G1 phase. We also showed that USP29 knockdown hampered Cdc25A-mediated cell proliferation, migration, and invasion of cancer cells in vitro. Moreover, NSG nude mice transplanted with USP29-depleted cells significantly reduced the size of the tumors, whereas the reconstitution of Cdc25A in USP29-depleted cells significantly increased the tumor size. Altogether, our results implied that USP29 promoted cell cycle progression and oncogenic transformation by regulating protein turnover of Cdc25A.

Ubiquitin Specific Protease 29 Functions as an Oncogene Promoting Tumorigenesis in Colorectal Carcinoma.

Colorectal carcinoma is the third foremost cause of cancer-related deaths and accounts for 5.8% of all deaths globally. The molecular mechanisms of colon cancer progression and metastasis control are not well studied. Ubiquitin-specific protease 29 (USP29), a deubiquitinating enzyme, is involved in the occurrence and development of wide variety of cancers. However, its clinical significance and biological roles in colorectal carcinoma (CRC) remain unexplored. In this research, we observed that the rate of USP29 overexpression was higher in colon cancer patient tissues relative to its corresponding normal tissues. CRISPR-Cas9-mediated depletion of USP29 triggered DNA double strand breaks and delayed cell-cycle progression in HCT116 cells. We also demonstrated that USP29 depletion hampers the colony formation and increases apoptosis of HCT116 cells. USP29 knockdown significantly decreased CRC cell proliferation in vitro. Depletion of USP29 in HCT116 cells substantially reduced the tumor volume of mouse xenografts. In conclusion, our study shows that elevated expression of USP29 promotes malignancy in CRC, suggesting that USP29 could be a promising target for colon cancer therapy.

YM155 sensitizes HeLa cells to TRAIL-mediated apoptosis via cFLIP and survivin downregulation.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis is a safe method for the treatment of various types of cancer. However, TRAIL therapy is less effective in certain types of cancer, including cervical cancer. To address this problem, a combinatorial approach was employed to sensitize cervical cancer at low dosages. YM155, a survivin inhibitor, was used at low dosages along with TRAIL to induce apoptosis in HeLa cells. The effects of the individual treatment with TRAIL and YM155 on apoptosis were assessed by propidium iodide assay. In addition, to validate the DNA damage exhibited by the combination treatment, the phosphorylation status of γH2A histone family member X was investigated by immunofluorescence and western blot analysis. TRAIL or YM155 alone had no significant effect on DNA damage and apoptosis. However, the TRAIL/YM155 combination triggered a synergistic pro-apoptotic stimulus in HeLa cells. The mRNA and protein levels of CASP8- and FADD-like apoptosis regulator (cFLIP), death receptor 5 (DR5) and survivin were monitored using RT-PCR and western blot analysis, respectively. This combinatorial approach downregulated both mRNA and protein expression levels of cFLIP and survivin. Further experimental results suggested that the combination treatment significantly reduced cell viability, invasion and migration of HeLa cells. Overall, the present findings indicated that the low dosage of YM155 sensitized HeLa cells to TRAIL-induced apoptosis via a mechanism involving downregulation of cFLIP and survivin. The results indicated the importance of combination drug treatment and reveal an effective therapeutic alternative for TRAIL therapy in human cervical cancer.

HAUSP stabilizes Cdc25A and protects cervical cancer cells from DNA damage response.

Resistance to DNA-damaging agents is one of the main reasons for the low survival of cervical cancer patients. Previous reports have suggested that the Cdc25A oncoprotein significantly affects the level of susceptibility to DNA-damaging agents, but the molecular mechanism remains unclear. In this study, we used Western blot and flow cytometry analyses to demonstrate that the deubiquitinating enzyme HAUSP stabilizes Cdc25A protein level. Furthermore, in a co-immunoprecipitation assay, we found that HAUSP interacts with and deubiquitinates Cdc25A both exogenously and endogenously. HAUSP extends the half-life of the Cdc25A protein by circumventing turnover. HAUSP knockout in HeLa cells using the CRISPR/Cas9 system caused a significant delay in Cdc25A-mediated cell cycle progression, cell migration, and colony formation and attenuated tumor progression in a mouse xenograft model. Furthermore, HAUSP-mediated stabilization of the Cdc25A protein produced enhanced resistance to DNA-damaging agents. Overall, our study suggests that targeting Cdc25A and HAUSP could be a promising combinatorial approach to halt progression and minimize antineoplastic resistance in cervical cancer.

Dual pH- and GSH-Responsive Degradable PEGylated Graphene Quantum Dot-Based Nanoparticles for Enhanced HER2-Positive Breast Cancer Therapy.

Dual stimuli-responsive degradable carbon-based nanoparticles (DS-CNPs) conjugated with Herceptin (HER) and polyethylene glycol (PEG) have been designed for the treatment of HER2-positive breast cancer. Each component has been linked through disulfide linkages that are sensitive to glutathione in a cancer microenvironment. ß-cyclodextrin (ß-CD) on the surface of DS-CNPs formed an inclusion complex (DL-CNPs) with doxorubicin (DOX) at a high loading capacity of 5.3 ± 0.4%. In response to a high level of glutathione (GSH) and low pH in a tumor environment, DL-CNPs were rapidly degraded and released DOX in a controlled manner via disruption of host-guest inclusion. These novel DL-CNPs exhibited high cellular uptake with low toxicity, which induced the efficient inhibition of antitumor activity both in vitro and in vivo. Cell viability, confocal laser scanning microscopy, and animal studies indicate that DL-CNPs are a great platform with a synergistically enhanced antitumor effect from the dual delivery of HER and DOX in DL-CNPs.

Reductively-sheddable cationic nanocarriers for dual chemotherapy and gene therapy with enhanced release.

The development of a versatile strategy to synthesize cationic nanocarriers capable of co-delivery and enhanced release of drugs and oligonucleotides is promising for synergic dual chemotherapy and gene therapy. Herein, we report a novel cationic amphiphilic diblock copolymer having a single reduction-responsive disulfide linkage at a junction between a FDA-approved polylactide (PLA) block and a cationic methacrylate block (C-ssABP). The amphiphilic design of the C-ssABP enables the formation of cationic micellar aggregates possessing hydrophobic PLA cores, encapsulating anticancer drugs; cationic coronas, ensuring complementary complexation with negatively-charged oligonucleotides through electrostatic interactions; and disulfides at interfaces, leading to enhanced release of both encapsulated drugs and complexed oligonucleotides. The reduction-responsive intracellular trafficking results from flow cytometry, confocal laser scanning microscopy, and cell viability, as well as in vitro gene transfection assay suggest that C-ssABP offers versatility as an effective nanocarrier platform for dual chemotherapy and gene therapy.

Synthesis and reduction-responsive disassembly of PLA-based mono-cleavable micelles.

Stimuli-responsive block copolymers and their self-assembled nanostructures have been extensively studied as effective building blocks in construction of various nanomaterials in nanoscience and nanotechnology. A promising stimuli-responsive platform involves an incorportion of reduction-responsive disulfide linkages that can be cleaved to corresponding thiols when needed. Herein, we describe a novel approach utilizing a combination of ring-opening polymerization and a facile coupling reaction to synthesize a reduction-responsive triblock copolymer comprising biocompatible polylactide (PLA) and poly(ethylene glycol) (PEG) blocks, thus PEG-b(PLA-ss-PLA)-b-PEG (ssBCP). This copolymer self-assembles to form colloidally-stable mono-cleavable micelles having single disulfides in hydrophobic PLA cores surrounded with PEG coronas in aqueous solution. The reductive cleavage of the core disulfides results in changes in micelle morphologies to smaller nanostructures or larger aggregates, depending on the nature of reducing agents. In the presence of glutathione (a cellular reducing agent), the micelle size increases, which enhances the release of encapsulated anticancer drugs in vitro. For biological perspectives, the ssBCP micelles having hydrophilic PEG corona are non-cytotoxic and exhibit enhanced colloidal stability as well as non-specific interactions with proteins.

Glutathione-triggered disassembly of dual disulfide located degradable nanocarriers of polylactide-based block copolymers for rapid drug release.

Reduction-responsive degradation based on disulfide-thiol chemistry is highly desirable in the development of self-assembled block copolymer nanocarriers for multifunctional polymer-based drug delivery applications. Most conventional approaches involve the incorporation of disulfide linkages at a single location. Herein, we report a new dual disulfide located degradable polylactide (PLA)-based block copolymer (DL-ssABP) synthesized by a combination of ring opening polymerization, facile coupling reactions, and controlled radical polymerization. The amphiphilic design of the DL-ssABP enables the formation of self-assembled micelles having disulfides positioned both in the hydrophobic PLA core and at the core/corona interface. The reductive response to glutathione as a cellular trigger results in the cleavage of the disulfide linkage at the interface shedding hydrophilic coronas as well as the disulfides in the PLA core causing disintegration of PLA cores. Such dual disulfide degradation process leads to a synergistically enhanced release of encapsulated anticancer drugs in cellular environments. These results, combined with flow cytometry and confocal laser scanning microscopy (CLSM) as well as cell viability measurements, suggest that DL-ssABP offers versatility in tumor-targeting controlled/enhanced drug delivery applications.

Air-spun PLA nanofibers modified with reductively sheddable hydrophilic surfaces for vascular tissue engineering: synthesis and surface modification.

Polylactide (PLA) is a class of promising biomaterials that hold great promise for various biological and biomedical applications, particularly in the field of vascular tissue engineering where it can be used as a fibrous mesh to coat the inside of vascular prostheses. However, its hydrophobic surface providing nonspecific interactions and its limited ability to further modifications are challenges that need to be overcome. Here, the development of new air-spun PLA nanofibers modified with hydrophilic surfaces exhibiting reduction response is reported. Surface-initiated atom transfer radical polymerization allows for grafting pendant oligo(ethylene oxide)-containing polymethacrylate (POEOMA) from PLA air-spun fibers labeled with disulfide linkages. The resulting PLA-ss-POEOMA fibers exhibit enhanced thermal stability and improved surface properties, as well as thiol-responsive shedding of hydrophilic POEOMA by the cleavage of its disulfide linkages in response to reductive reactions, thus tuning the surface properties.

Recent advances in stimuli-responsive degradable block copolymer micelles: synthesis and controlled drug delivery applications.

(Bio)degradation in response to external stimuli (stimuli-responsive degradation, SRD) is a desired property in constructing novel nanostructured materials. For polymer-based multifunctional drug delivery applications, the degradation enables fast and controlled release of encapsulated therapeutic drugs from delivery vehicles in targeted cells. It also ensures the clearance of the empty device after drugs are delivered to the body. This review summarizes recent development of various strategies to the design and synthesis of self-assembled micellar aggregates based on novel amphiphilic block copolymers having different numbers of stimuli-responsive cleavable elements at various locations. These cleavable linkages including disulfide, acid-labile, and photo-cleavable linkages are incorporated into micelles, and then are cleaved in response to cellular triggers such as reductive reaction, light, and low acid. The well-designed SRD micelles have been explored as controlled/enhanced delivery vehicles of drugs and genes. For future design and development of effective stimuli-responsive degradable micelles toward tumor-targeting delivery applications in vivo, a high degree of control over degradation for tunable release of encapsulated anticancer drugs as well as bioconjugation for active tumor-targeting is required.