Intracellular delivery of protein drugs with an autonomously lysing bacterial system reduces tumor growth and metastases.

Critical cancer pathways often cannot be targeted because of limited efficiency crossing cell membranes. Here we report the development of a Salmonella-based intracellular delivery system to address this challenge. We engineer genetic circuits that (1) activate the regulator flhDC to drive invasion and (2) induce lysis to release proteins into tumor cells. Released protein drugs diffuse from Salmonella containing vacuoles into the cellular cytoplasm where they interact with their therapeutic targets. Control of invasion with flhDC increases delivery over 500 times. The autonomous triggering of lysis after invasion makes the platform self-limiting and prevents drug release in healthy organs. Bacterial delivery of constitutively active caspase-3 blocks the growth of hepatocellular carcinoma and lung metastases, and increases survival in mice. This success in targeted killing of cancer cells provides critical evidence that this approach will be applicable to a wide range of protein drugs for the treatment of solid tumors.

Drug-delivering-drug platform-mediated potent protein therapeutics via a non-endo-lysosomal route.

Protein therapeutics is playing an increasingly critical role in treatment of human diseases. However, current vectors are captured by the digestive endo-lysosomal system, which results in an extremely low fraction (<2%) of protein being released in the cytoplasm. This paper reports a drug-delivering-drug platform (HA-PNPplex, 200 nm) for potent intracellular delivery of protein and combined treatment of cancer. Methods: The platform was prepared by loading functional protein on pure drug nanoparticles (PNPs) followed by hyaluronic acid coating and was characterized by dynamic light scattering, transmission electron microscopy, and gel electrophoresis. In vitro, cellular uptake, trafficking, and cytotoxicity were evaluated by flow cytometry and confocal laser microscopy. Protein expression was assayed by western blot. In vivo, blood circulation and biodistribution were studied using a fluorescence imaging system, antitumor efficacy was assessed in a caspase 3-deficient tumor model, and biocompatibility was determined by comparison of hemolytic activity and proinflammatory cytokines and tissue histology. Results: HA-PNPplex delivered the functional protein, caspase 3, to cells via bypassing endo-lysosomes and raised the caspase-3 level 6.5-fold in caspase 3-deficient cells. Promoted tumor accumulation (1.5-fold) and penetration were exhibited, demonstrating a high tumor-targeting ability of HA-PNPplex. HA-PNPplex rendered a 7-fold increase in caspase 3 in tumor and allowed for a 100% tumor growth inhibition and >60% apoptosis, implying significant antitumor activities. Conclusions: This platform gains cellular entry without entrapment in the endo-lysosomes and enables efficient intracellular protein delivery and resultant profound cancer treatment. This platform, with extremely high drug-loading, is a valuable platform for combined cancer therapy with small-molecule drugs and proteins. More importantly, this work offers a robust and safe approach for protein therapeutics and intracellular delivery of other functional peptides, as well as gene-based therapy.

Utilizing Inverse Emulsion Polymerization To Generate Responsive Nanogels for Cytosolic Protein Delivery.

Therapeutic biologics have various advantages over synthetic drugs in terms of selectivity, their catalytic nature, and, thus, therapeutic efficacy. These properties offer the potential for more effective treatments that may also overcome the undesirable side effects observed due to off-target toxicities of small molecule drugs. Unfortunately, systemic administration of biologics is challenging due to cellular penetration, renal clearance, and enzymatic degradation difficulties. A delivery vehicle that can overcome these challenges and deliver biologics to specific cellular populations has the potential for significant therapeutic impact. In this work, we describe a redox-responsive nanoparticle platform, which can encapsulate hydrophilic proteins and release them only in the presence of a reducing stimulus. We have formulated these nanoparticles using an inverse emulsion polymerization (IEP) methodology, yielding inverse nanoemulsions, or nanogels. We have demonstrated our ability to overcome the liabilities that contribute to activity loss by delivering a highly challenging cargo, functionally active caspase-3, a cysteine protease susceptible to oxidative and self-proteolytic insults, to the cytosol of HeLa cells by encapsulation inside a redox-responsive nanogel.

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

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.

Hippocampal neurogenesis protects against cocaine-primed relapse.

Accumulating evidence demonstrates a functional role for the hippocampus in mediating relapse to cocaine-seeking behavior and extinction-induced inhibition of cocaine seeking, and dentate gyrus neurogenesis in the hippocampus may have a role. Here, we tested the hypothesis that disruption of normal hippocampal activity during extinction alters relapse to cocaine-seeking behavior as a function of dentate gyrus neurogenesis. Adult rats were trained to self-administer cocaine on a fixed-ratio schedule, followed by extinction and cocaine-primed reinstatement testing. Some rats received low-frequency stimulation (LFS; 2 Hz for 25 minutes) after each extinction session in the dorsal or ventral hippocampal formation. All rats received an injection of the mitotic marker 5-bromo-2'-deoxyuridine (BrdU) to label developing dentate gyrus neurons during self-administration, as well as before or after extinction and LFS. We found that LFS during extinction did not alter extinction behavior but enhanced cocaine-primed reinstatement. Cocaine self-administration reduced levels of 24-day-old BrdU cells and dentate gyrus neurogenesis, which was normalized by extinction. LFS during extinction prevented extinction-induced normalization of dentate gyrus neurogenesis and potentiated cocaine-induced reinstatement of drug seeking. LFS inhibition of extinction-induced neurogenesis was not due to enhanced cell death, revealed by quantification of activated caspase3-labeled cells. These data suggest that LFS during extinction disrupts hippocampal networking by disrupting neurogenesis and also strengthens relapse-like behaviors. Thus, newly born dentate gyrus neurons during withdrawal and extinction learning facilitate hippocampal networking that mediates extinction-induced inhibition of cocaine seeking and may play a key role in preventing relapse.

Protein nanocapsule weaved with enzymatically degradable polymeric network.

Target proteins can be functionally encapsulated using a cocoon-like polymeric nanocapsule formed by interfacial polymerization. The nanocapsule is cross-linked by peptides that can be proteolyzed by proteases upon which the protein cargo is released. The protease-mediated degradation process can be controlled in a spatiotemporal fashion through modification of the peptide cross-linker with photolabile moieties. We demonstrate the utility of this approach through the cytoplasmic delivery of the apoptosis inducing caspase-3 to cancer cells.

Targeted therapy to the IL-2R using diphtheria toxin and caspase-3 fusion proteins modulates Treg and ameliorates inflammatory colitis.

Pathogenic lymphocytes in the enteric wall of inflammatory bowel disease patients display various abnormalities, including reduced sensitivity to apoptosis. We evaluated a therapeutic approach to elimination of cytotoxic cells, using two IL-2 fusion proteins, a diphtheria toxin (IL2-DT) and a caspase-3 (IL2-cas) conjugate. In models of acute (dextran sodium sulfate and trinitrobenzene sulfonic acid) and chronic (dextran sodium sulfate) toxic colitis, therapeutic doses of the fusion proteins improved survival and prevented colon shortening. While both chimeric proteins eradicated CD4(+)CD25(+)Foxp3(+) T cells in mesenteric LN, IL2-DT caused severe lymphopenia. In contrast, IL2-cas was equally protective and increased fractional expression of Foxp3. Similar effects of the fusion proteins were observed in healthy mice: IL2-DT caused lymphopenia and IL2-cas increased fractional expression of FoxP3. The fusion proteins induced apoptosis in CD25(+) T cells in vitro, with lower toxicity of IL2-cas to Foxp3(+) T cells. These data infer that targeted depletion of cells expressing the IL-2 receptor has therapeutic potential in models of inflammatory colitis, despite depletion of CD25(+) Treg. The IL2-cas fusion protein is particularly relevant to inflammatory bowel disease, as direct internalization of toxic moieties overcomes multiple pathways of resistance to apoptosis of colitogenic T cells.