Dataset Information

Nanomotor-Enabled pH-Responsive Intracellular Delivery of Caspase-3: Toward Rapid Cell Apoptosis.

ABSTRACT: Direct and efficient intracellular delivery of enzymes to cytosol holds tremendous therapeutic potential while remaining an unmet technical challenge. Herein, an ultrasound (US)-propelled nanomotor approach and a high-pH-responsive delivery strategy are reported to overcome this challenge using caspase-3 (CASP-3) as a model enzyme. Consisting of a gold nanowire (AuNW) motor with a pH-responsive polymer coating, in which the CASP-3 is loaded, the resulting nanomotor protects the enzyme from release and deactivation prior to reaching an intracellular environment. However, upon entering a cell and exposure to the higher intracellular pH, the polymer coating is dissolved, thereby directly releasing the active CASP-3 enzyme to the cytosol and causing rapid cell apoptosis. In vitro studies using gastric cancer cells as a model cell line demonstrate that such a motion-based active delivery approach leads to remarkably high apoptosis efficiency within a significantly shorter time and with a lower amount of CASP-3 compared to other control groups not involving US-propelled nanomotors. For instance, the reported nanomotor system can achieve 80% apoptosis of human gastric adenocarcinoma cells within only 5 min, which dramatically outperforms other CASP-3 delivery approaches. These results indicate that the US-propelled nanomotors may act as a powerful vehicle for cytosolic delivery of active therapeutic proteins, which would offer an attractive means to enhance the current landscape of intracellular protein delivery and therapy. While CASP-3 is selected as a model protein in this study, the same nanomotor approach can be readily applied to a variety of different therapeutic proteins.

SUBMITTER: Esteban-Fernandez de Avila B

PROVIDER: S-EPMC5894870 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Nanomotor-Enabled pH-Responsive Intracellular Delivery of Caspase-3: Toward Rapid Cell Apoptosis.

Esteban-Fernández de Ávila Berta B Ramírez-Herrera Doris E DE Campuzano Susana S Angsantikul Pavimol P Zhang Liangfang L Wang Joseph J

ACS nano 20170505 6

Direct and efficient intracellular delivery of enzymes to cytosol holds tremendous therapeutic potential while remaining an unmet technical challenge. Herein, an ultrasound (US)-propelled nanomotor approach and a high-pH-responsive delivery strategy are reported to overcome this challenge using caspase-3 (CASP-3) as a model enzyme. Consisting of a gold nanowire (AuNW) motor with a pH-responsive polymer coating, in which the CASP-3 is loaded, the resulting nanomotor protects the enzyme from relea ...[more]

PMID: 28467853

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Project description:Purpose: The levels of reactive oxygen species (ROS) in tumor cells are much higher than that in normal cells, and rise rapidly under the influence of exogenous or endogenous inducing factors, eventually leading to the apoptosis of tumor cells. Therefore, this study prepared a dual pH/reducing-responsive poly (N-isopropylacrylamide-co-Cinnamaldehyde-co-D-?-tocopheryl polyethylene glycol 1000 succinate, PssNCT) nanogels, which employed two exogenous ROS inducers, cinnamaldehyde (CA) and D-?-tocopheryl polyethylene glycol 1000 succinate (TPGS), to selectively induce apoptosis by regulating ROS levels in tumor cells. Methods: The PssNCT nanogels were prepared by the free radical precipitation polymerization under the crosslink between pH-sensitive hydrazone and reducing-sensitive disulfide bonds, followed by the physicochemical and morphological characteristics investigations. Plasma stability, dual pH/reducing responsive degradation and in vitro release were also assessed. In cell experiments, cytotoxicity in different cells were first detected. The intracellular ROS levels and mitochondrial functions of tumor cells were then evaluated. Moreover, the apoptosis and western-blot assays were employed to verify the association between ROS levels elevation and apoptosis in tumor cells. Results: The nanogels exhibited a round-like hollow structure with the diameter smaller than 200nm. The nanogels were stable in plasma, while showed rapid degradation in acidic and reducing environments, thus achieving significant release of CA and TPGS in these media. Furthermore, the sufficient amplification of ROS signals was induced by the synergistically function of CA and TPGS on mitochondria, which resulted in the opening of the mitochondrial apoptotic pathway and enhanced cytotoxicity on MCF-7 cells. However, nanogels barely affected L929 cells owing to their lower intracellular ROS basal levels. Conclusion: The specific ROS regulation method achieved by these nanogels could be explored to selectively kill tumor cells according to the difference of ROS signals in different kinds of cells.

Project description:We previously developed a pH-responsive copper-doxorubicin (CuDox) cargo in lysolipid-based temperature-sensitive liposomes (LTSLs). The CuDox complex is released from the particle by elevated temperature; however, full release of doxorubicin from CuDox requires a reduced pH, such as that expected in lysosomes. The primary goal of this study is to evaluate the cellular uptake and intracellular trafficking of the drug-metal complex in comparison with intact liposomes and free drug. We found that the CuDox complex was efficiently internalized by mammary carcinoma cells after release from LTSLs. Intracellular doxorubicin and copper were 6-fold and 5-fold greater, respectively, after a 0.5h incubation with the released CuDox complex, as compared to incubation with intact liposomes containing the complex. Total cellular doxorubicin fluorescence was similar following CuDox and free doxorubicin incubation. Imaging and mass spectrometry assays indicated that the CuDox complex was initially internalized intact but breaks down over time within cells, with intracellular copper decreasing more rapidly than intracellular doxorubicin. Doxorubicin fluorescence was reduced when complexed with copper, and nuclear fluorescence was reduced when cells were incubated with the CuDox complex as compared with free doxorubicin. Therapeutic efficacy, which typically results from intercalation of doxorubicin with DNA, was equivalent for the CuDox complex and free doxorubicin and was superior to that of liposomal doxorubicin formulations. Taken together, the results suggest that quenched CuDox reaches the nucleus and remains efficacious. In order to design protocols for the use of these temperature-sensitive particles in cancer treatment, the timing of hyperthermia relative to drug administration must be examined. When cells were heated to 42°C prior to the addition of free doxorubicin, nuclear drug accumulation increased by 1.8-fold in cancer cells after 5h, and cytotoxicity increased 1.4-fold in both cancer and endothelial cells. Endothelial cytotoxicity was similarly augmented with mild hyperthermia applied prior to treatment with released CuDox. In summary, we find that the drug-metal complex formed in temperature-sensitive particles can be internalized by cancer and endothelial cells resulting in therapeutic efficacy that is similar to free doxorubicin, and this efficacy can be enhanced by elevated temperature.

Project description:Doxorubicin is a chemotherapeutic drug typically administered systemically which frequently leads to cardiac and hepatic toxicities. Local delivery to a tumor has a chance to mitigate some of these toxicities and can further be mitigated by including a means of tumor-specific drug release. Our laboratory has explored the use of molecular interactions to control the rate of drug release beyond that capable of diffusion alone. To this system, we added an additional affinity group (adamantane) to doxorubicin through a pH-sensitive hydrazone bond. The result was a modified doxorubicin which had an even higher affinity to our drug delivery polymer, and virtually no release in normal conditions, but showed accelerated release of drug in tumor-like low pH. Further, we show that adamantane-modified doxorubicin (adamantane-doxorubicin) and cleaved adamantane-doxorubicin showed equivalent capacity to kill human U-87 glioblastoma cells in vitro as unmodified doxorubicin. Taken together, these data demonstrate our ability to load high levels of modified chemotherapeutic drugs into our affinity-based delivery platform and deliver these drugs almost exclusively in the acidic microenvironments, such as those surrounding the tumor tissue via pH-cleavable bond while minimizing drug delivery in neutral pH tissue, with the ultimate goal of reducing systemic through better local delivery. Impact statement Doxorubicin (DOX) is especially cytotoxic to the heart, liver, kidneys, and healthy tissues surrounding the tumor microenvironment. This systemic toxicity can be partially addressed by local, tumor-specific drug delivery systems. While pH-sensitive DOX delivery systems have been developed by several other groups, many lack a prolonged and consistent release profile required to successfully treat heterogeneous tumors. Our system of a chemically modified form of DOX combined with an affinity-based cyclodextrin delivery system is capable of delivering DOX for 87 days while maintaining its the drug cytotoxicity. This finding is particularly relevant to improving cancer treatments because it enables regulated local delivery of DOX specifically to tumor tissue and allows the drug to be continuously delivered over a therapeutically relevant amount of time.

Dataset Information

Nanomotor-Enabled pH-Responsive Intracellular Delivery of Caspase-3: Toward Rapid Cell Apoptosis.

Publications

Nanomotor-Enabled pH-Responsive Intracellular Delivery of Caspase-3: Toward Rapid Cell Apoptosis.

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