A novel Granzyme B nanoparticle delivery system simulates immune cell functions for suppression of solid tumors.

Cell-based immunotherapy for the treatment of hematologic malignancies, such as leukemia and lymphoma, has seen much success and played an increasingly important role in clinical studies. Nevertheless, the efficacy of immunotherapy in solid tumors still needs improvements due to the immunosuppressive properties of tumor cells and the microenvironment. To overcome these limitations, we prepared a novel tumor-targeting delivery system based on the underlying mechanism of immune-targeted cell death that encapsulated granzyme B protein within a porous polymeric nanocapsule. Methods: A cell-penetrating peptide TAT was attached onto granzyme B (GrB) to enhance its transmembrane transport efficiency and potency to induce cell apoptosis. The endocytosis and internalization pathways of GrB-TAT (GrB-T) were analyzed in comparison with perforin by confocal microscopy and flow cytometry. Furthermore, the positively charged GrB-T was wrapped into nanoparticles by p-2-methacryloyloxy ethyl phosphorylcholine (PMPC)-modified HA (hyaluronic acid). The nanoparticles (called TCiGNPs) were characterized in terms of zeta potential and by transmission electron microscopy (TEM). The in vitro anti-tumor effects of GrB-T were examined by cell apoptosis assay and Western blotting analysis. The in vivo anti-tumor therapeutic efficacy of TCiGNPs was evaluated in a mouse tumor model. Results: The TAT peptide could play a role similar to perforin to mediate direct transmembrane transfer of GrB and improve GrB-induced cell apoptosis. The TCiGNPs were successfully synthesized and accumulated in the solid tumor through enhanced permeability and retention (EPR) effect. In the tumor microenvironment, TCiGNPs could be degraded by hyaluronidase and triggered the release of GrB-T. The TAT peptide enabled the translocation of GrB across the plasma membrane to induce tumor cell apoptosis in vivo.Conclusion: We successfully developed a granzyme B delivery system with a GrB-T core and a PMPC/HA shell that simulated CTL/NK cell-mediated cancer immunotherapy mechanism. The GrB delivery system holds great promise for cancer treatment analogous to the CTL/NK cell-induced immunotherapy.

Cardiovascular effects of Sp-CTx, a cytolysin from the scorpionfish (Scorpaena plumieri) venom.

Fish venom cytolysins are multifunctional proteins that in addition to their cytolytic/hemolytic effects display neurotoxic, cardiotoxic and inflammatory activities, being described as "protein lethal factors". A pore-forming cytolysin called Sp-CTx (Scorpaena plumieriCytolytic Toxin) has been recently purified from the venom of the scorpionfish Scorpaena plumieri. It is a glycoprotein with dimeric constitution, comprising subunits of approximately 65 kDa. Previous studies have revealed that this toxin has a vasorelaxant activity that appears to involve the L-arginine-nitric oxide synthase pathway; however its cardiovascular effects have not been fully comprehended. The present study examined the cardiovascular effects of Sp-CTx in vivo and in vitro. In anesthetized rats Sp-CTx (70 µg/kg i.v) produced a biphasic response which consisted of an initial systolic and diastolic pressure increase followed by a sustained decrease of these parameters and the heart rate. In isolated rats hearts Sp-CTx (10(-9) to 5 × 10(-6) M) produced concentration-dependent and transient ventricular positive inotropic effect and vasoconstriction response on coronary bed. In papillary muscle, Sp-CTx (10(-7) M) also produced an increase in contractile isometric force, which was attenuated by the catecholamine releasing agent tyramine (100 µM) and the ß-adrenergic antagonist propranolol (10 µM). On isolated ventricular cardiomyocytes Sp-CTx (1 nM) increased the L-type Ca(2+) current density. The results show that Sp-CTx induces disorders in the cardiovascular system through increase of sarcolemmal calcium influx, which in turn is partially caused by the release of endogenous noradrenaline.

A cleavable cytolysin-neuropeptide Y bioconjugate enables specific drug delivery and demonstrates intracellular mode of action.

Myxobacterial tubulysins are promising chemotherapeutics inhibiting microtubule polymerization, however, high unspecific toxicity so far prevents their application in therapy. For selective cancer cell targeting, here the coupling of a synthetic cytolysin to the hY1-receptor preferring peptide [F(7),P(34)]-neuropeptide Y (NPY) using a labile disulfide linker is described. Since hY1-receptors are overexpressed in breast tumors and internalize rapidly, this system has high potential as peptide-drug shuttle system. Molecular characterization of the cytolysin-[F(7),P(34)]-NPY bioconjugate revealed potent receptor activation and receptor-selective internalization, while viability studies verified toxicity. Triple SILAC studies comparing free cytolysin with the bioconjugate demonstrated an intracellular mechanism of action regardless of the delivery pathway. Treatments resulted in a regulation of proteins implemented in cell cycle arrest confirming the tubulysin-like effect of the cytolysin. Thus, the cytolysin-peptide bioconjugate fused by a cleavable linker enables a receptor-specific delivery as well as a potent intracellular drug-release with high cytotoxic activity.

Mycobacterium tuberculosis-specific CD8+ T cells require perforin to kill target cells and provide protection in vivo.

Optimal immunity to Mycobacterium tuberculosis (Mtb) infection requires CD8(+) T cells, and several current Mtb vaccine candidates are being engineered to elicit enhanced CD8(+) T cell responses. However, the function of these T cells and the mechanism by which they provide protection is still unknown. We have previously shown that CD8(+) T cells specific for the mycobacterial Ags CFP10 and TB10.4 accumulate in the lungs of mice following Mtb infection and have cytolytic activity in vivo. In this study, we determine which cytolytic pathways are used by these CD8(+) T cells during Mtb infection. We find that Mtb-specific CD8(+) T cells lacking perforin have reduced cytolytic capacity in vivo. In the absence of perforin, the residual cytolytic activity is CD95 and TNFR dependent. This is particularly true in Mtb-infected lung tissue where disruption of both perforin and CD95 eliminates target cell lysis. Moreover, adoptive transfer of immune CD8(+) T cells isolated from wild-type, but not perforin-deficient mice, protect recipient mice from Mtb infection. We conclude that CD8(+) T cells elicited following Mtb infection use several cytolytic pathways in a hierarchical and compensatory manner dominated by perforin-mediated cytolysis. Finally, although several cytolytic pathways are available, adoptively transferred Mtb-specific CD8(+) T cells require perforin-mediated cytolysis to protect animals from infection. These data show that CD8(+) T cell-mediated protection during Mtb infection requires more than the secretion of IFN-gamma and specifically defines the CD8(+) cytolytic mechanisms utilized and required in vivo.