Targeting of M2 macrophages with IL-13-functionalized liposomal prednisolone inhibits melanoma angiogenesis in vivo.

The intricate cooperation between cancer cells and nontumor stromal cells within melanoma microenvironment (MME) enables tumor progression and metastasis. We previously demonstrated that the interplay between tumor-associated macrophages (TAMs) and melanoma cells can be disrupted by using long-circulating liposomes (LCLs) encapsulating prednisolone phosphate (PLP) (LCL-PLP) that inhibited tumor angiogenesis coordinated by TAMs. In this study, our goal was to improve LCL specificity for protumor macrophages (M2-like (i.e., TAMs) macrophages) and to induce a more precise accumulation at tumor site by loading PLP into IL-13-conjugated liposomes (IL-13-LCL-PLP), since IL-13 receptor is overexpressed in this type of macrophages. The IL-13-LCL-PLP liposomal formulation was obtained by covalent attachment of thiolated IL-13 to maleimide-functionalized LCL-PLP. C57BL/6 mice bearing B16.F10 s.c melanoma tumors were used to investigate the antitumor action of LCL-PLP and IL-13-LCL-PLP. Our results showed that IL-13-LCL-PLP formulation remained stable in biological fluids after 24h and it was preferentially taken up by M2 polarized macrophages. IL-13-LCL-PLP induced strong tumor growth inhibition compared to nonfunctionalized LCL-PLP at the same dose, by altering TAMs-mediated angiogenesis and oxidative stress, limiting resistance to apoptosis and invasive features in MME. These findings suggest IL-13-LCL-PLP might become a promising delivery platform for chemotherapeutic agents in melanoma.

Active Tumor-Targeting Nano-formulations Containing Simvastatin and Doxorubicin Inhibit Melanoma Growth and Angiogenesis.

Primary melanoma aggressiveness is determined by rapid selection and growth of cellular clones resistant to conventional treatments, resulting in metastasis and recurrence. In addition, a reprogrammed tumor-immune microenvironment supports melanoma progression and response to therapy. There is an urgent need to develop selective and specific drug delivery strategies for modulating the interaction between cancer cells and immune cells within the tumor microenvironment. This study proposes a novel combination therapy consisting of sequential administration of simvastatin incorporated in IL-13-functionalized long-circulating liposomes (IL-13-LCL-SIM) and doxorubicin encapsulated into PEG-coated extracellular vesicles (PEG-EV-DOX) to selectively target both tumor-associated macrophages and melanoma cells. To this end, IL-13 was conjugated to LCL-SIM which was obtained via the lipid film hydration method. EVs enriched from melanoma cells were passively loaded with doxorubicin. The cellular uptake of rhodamine-tagged nano-particles and the antiproliferative potential of the treatments by using the ELISA BrdU-colorimetric immunoassay were investigated in vitro. Subsequently, the therapeutic agents were administered i.v in B16.F10 melanoma-bearing mice, and tumor size was monitored during treatment. The molecular mechanisms of antitumor activity were investigated using angiogenic and inflammatory protein arrays and western blot analysis of invasion (HIF-1) and apoptosis markers (Bcl-xL and Bax). Quantification of oxidative stress marker malondialdehyde (MDA) was determined by HPLC. Immunohistochemical staining of angiogenic markers CD31 and VEGF and of pan-macrophage marker F4/80 was performed to validate our findings. The in vitro data showed that IL-13-functionalized LCL were preferentially taken up by tumor-associated macrophages and indicated that sequential administration of IL-13-LCL-SIM and PEG-EV-DOX had the strongest antiproliferative effect on tumor cells co-cultured with tumor-associated macrophages (TAMs). Accordingly, strong inhibition of tumor growth in the group treated with the sequential combination therapy was reported in vivo. Our data suggested that the antitumor action of the combined treatment was exerted through strong inhibition of several pro-angiogenic factors (VEGF, bFGF, and CD31) and oxidative stress-induced upregulation of pro-apoptotic protein Bax. This novel drug delivery strategy based on combined active targeting of both cancer cells and immune cells was able to induce a potent antitumor effect by disruption of the reciprocal interactions between TAMs and melanoma cells.

Trojan horse treatment based on PEG-coated extracellular vesicles to deliver doxorubicin to melanoma in vitro and in vivo.

Tailoring extracellular vesicles (EVs) as targeted drug delivery systems to enhance the therapeutic efficacy showed superior advantage over liposomal therapies. Herein, we developed a novel nanotool for targeting B16.F10 murine melanoma, based on EVs stabilized with Polyethylene glycol (PEG) and loaded with doxorubicin (DOX). Small EVs were efficiently enriched from melanoma cells cultured under metabolic stress by ultrafiltration coupled with size exclusion chromatography (UF-SEC) and characterized by size, morphology, and proteome. To reduce their clearance in vivo, EVs were PEGylated and passively loaded with DOX (PEG-EV-DOX). Our data suggested that the low PEG coverage of EVs might still favor EV surface protein interactions with target proteins from intratumor cells, ensuring their use as "Trojan horses" to deliver DOX to the tumor tissue. Moreover, our results showed a superior antitumor activity of PEG-EV-DOX in B16.F10 murine melanoma models in vivo compared to that exerted by clinically applied liposomal DOX in the same tumor model. The PEG-EV-DOX administration in vivo reduced NF-κB activation and increased BAX expression, suggesting better prognosis of EV-based therapy than liposomal DOX treatment. Collectively, our results highlight the promising potential of EVs as optimal tools for systemic delivery of DOX to solid tumors.

Inverse-orthostasis may induce elevation of blood pressure due to sympathetic activation.

Microgravity and simulated microgravity may cause cardiovascular deconditioning, but mechanisms of instantaneous responses to inverse-orthostasis are not studied. Hence, we investigated transient and steady state cardiovascular changes by combining the tilt technique with cardiovascular telemetry. Normotensive and NO-deprived hypertensive Wistar rats were used to analyze responses of mean arterial blood pressure, heart rate, contractility, spontaneous baroreflex sensitivity (sBRS), and autonomic balance. Inverse-orthostasis tests were carried out by 45 degrees head-down tilting (repeated 3 x 5 mins "R", or sustained for 120 mins "S"). In normotensive rats, horizontal control blood pressure was R111.3 +/- 1.7/S110.4 +/- 2.3 mm Hg and heart rate was R385.2 +/- 5.9/S371.1 +/- 6.1 BPM. Head-down tilt induced an increase in blood pressure by R5.9/S10.6 mm Hg, while heart rate, contractility, sBRS, and autonomic balance did not change. The hypertensive response was sustained, could be prevented by prazosin (10 mg/kgbw), and augmented by subanesthetic doses of chloralose (26 and 43 mg/kgbw). In NO-suppressed hypertension, control blood pressure and heart rate were R132.4 +/- 2.9/S130.0 +/- 4.1 mm Hg and R339.2 +/- 7.9/S307.2 +/- 23.6 BPM, respectively. Head-down tilt further increased blood pressure by R5.1/S10.5 mm Hg. These data demonstrate that conscious rats respond to inverse-orthostasis by sustained elevation of blood pressure independent of NO synthesis. This response is neither due to increased contractility and altered sBRS, nor due to non-specific stress, but probably due to sympathetic activation elicited by gravity-related reflexes, which increase peripheral resistance.

Experimental orthostasis elicits sustained hypertension, which can be prevented by sympathetic blockade in the rat.

Incidence of orthostatic hypertension is estimated at 5% but is even more prevalent in borderline hypertension and autonomic neuropathies. The aim of this study was to develop a potential model to investigate orthostatic hypertension. We used normotensive and hypertensive Wistar rats to analyze responses and diurnal variations of arterial blood pressure, heart rate, temperature, and locomotor activity by telemetry. Orthostatic tests were carried out during 45 degrees head-up tilt (R, repeated 3 times for 5 minutes; or S, sustained for 120 minutes). Hypertension was induced by blockade of nitric oxide synthesis. In normotensives, horizontal control blood pressure was R115.4 +/- 1.4/S113.7 +/- 1.6 mm Hg and heart rate R386.4 +/- 7.0/S377.9 +/- 8.8 bpm. Head-up tilt increased blood pressure by R4.5/S8.4 mm Hg, including a 3.8 mm Hg hydrostatic component. The sustained hypertensive response was prevented by prazosin (10 mg/kgbw) and augmented by a subanesthetic dose of chloralose (26 mg/kgbw). In NO-deprived hypertension, horizontal control blood pressure and heart rate were R138.4 +/- 2.6/S140.3 +/- 2.7 mm Hg and R342.1 +/- 12.0/S346.0 +/- 8.3 bpm, respectively. Tilt increased blood pressure further by R4.2/S9.4 mm Hg. In both normo- and hypertensives, variables exhibited similar diurnal rhythms except for nighttime locomotor activity, reduced from 3.7 +/- 0.4 to 2.8 +/- 0.3 counts/s. These data demonstrate that conscious rats respond to sustained orthostasis with hypertension, probably as a result of increased sympathetic output. Decreasing stress using a subanesthetic dose of chloralose increased this response, reducing the inhibitory effect on hypertensive responses.

Impaired humoral immune response against mycobacterial 65-kDa heat shock protein (HSP65) in patients with inflammatory bowel disease.

Since only scarce data are available on the immune response against heat shock proteins (HSP) in inflammatory bowel disease (IBD), we have measured with an ELISA method serum levels of IgG, IgA, and IgM antibodies to mycobacterial HSP65 and human HSP60 in 66 patients with Crohn's disease (CD), 42 patients with ulceratiVe colitis (UC), and 126 age-and gender-matched healthy controls. Serum concentration [median (25th-75th percentiles) of IgG anti-HSP65 antibodies was substantially lower in patients with either CD (P < 0.01) or UC (P < 0.001) than in healthy controls, while no difference was found in the levels of anti-HSP60 antibodies. Low anti-HSP65 antibody levels were measured in patients with active CD and in both active and inactive UC, and only in IBD patients with no extraintestinal manifestations. In conclusion, our present findings indicate that an abnormal immune response to bacterial HSP65 or some epitopes of the protein may contribute to the dysregulation of host defenses against certain intestinal bacteria.