Retinoic acid-loaded PLGA nanocarriers targeting cell cholesterol potentialize the antitumour effect of PD-L1 antibody by preventing epithelial-mesenchymal transition mediated by M2-TAM in colorectal cancer.

Tumour-associated macrophages (TAMs) often promote cancer progression through immunosuppression in the tumour microenvironment (TME). However, the signalling pathways crosstalk responsible for this mechanism remain unclear. The aim of our study was to investigate whether the interaction between TAMs and colorectal cancer cells could be down-regulated by nanoparticles (NPs) loaded with retinoic acid (RA) and coated with cholesterol (CHO), in combination with an anti-PD-L1 immune checkpoint inhibitor. Tumours were evaluated by qRT-PCR and immunohistochemistry from allographic tumour growth model. In addition, human tumours were evaluated by Tissue Microarray (TMA) and immunohistochemistry. Complementary analysis of epithelial-mesenchymal transition, cell migration, and macrophage polarisation were evaluated in vitro. We showed that the IL-10R/IL-10 axis is involved in overstimulation of the STAT3 pathway as well as downregulation of the NF-κB signalling pathway, which supports a loop of immunosuppressive cytokines that induces the M2-TAM phenotype. Furthermore, our combined findings suggest that the upregulation of STAT3/NF-κB pathways crosstalk mediated by immunosuppressive cytokines, such as IL-10/PD-L1/TGF-ß, via M2-TAMs in the TME, leads to immunosuppression and epithelial-mesenchymal-transition of the colorectal cancer for stimulating Vimentin, CXCL12 and CD163 in the primary tumours. Importantly, NPs holding RA and coated with CHO in combination with anti-PD-L1 were more efficient in blocking this signalling pathway. These results contribute to our understanding of the immunological mechanisms, especially the re-educating of TAMs, and provide a novel management strategy for aggressive colorectal cancers using anti-PD-L1-conjugated nanocarriers.

Effective breast cancer therapy based on palmitic acid-loaded PLGA nanoparticles.

Although new strategies for breast cancer treatment have yielded promising results, most drugs can lead to serious side effects when applied systemically. Doxorubicin (DOX), currently the most effective chemotherapeutic drug to treat breast cancer, is poorly selective towards tumor cells and treatment often leads to the development of drug resistance. Recent studies have indicated that several fatty acids (FAs) have beneficial effects on inhibiting tumorigenesis. The saturated FA palmitic acid (PA) showed anti-tumor activities in several types of cancer, as well as effective repolarization of M2 macrophages towards the anti-tumorigenic M1 phenotype. However, water insolubility and cellular impermeability limit the use of PA in vivo. To overcome these limitations, here, we encapsulated PA into a poly(d,l-lactic co-glycolic acid) (PLGA) nanoparticle (NP) platform, alone and in combination with DOX, to explore PA's potential as mono or combinational breast cancer therapy. Our results showed that PLGA-PA-DOX NPs and PLGA-PA NPs significantly reduced the viability and migratory capacity of breast cancer cells in vitro. In vivo studies in mice bearing mammary tumors demonstrated that PLGA-PA-NPs were as effective in reducing primary tumor growth and metastasis as NPs loaded with DOX, PA and DOX, or free DOX. At the molecular level, PLGA-PA NPs reduced the expression of genes associated with multi-drug resistance and inhibition of apoptosis, and induced apoptosis via a caspase-3-independent pathway in breast cancer cells. In addition, immunohistochemical analysis of residual tumors showed a reduction in M2 macrophage content and infiltration of leukocytes after treatment of PLGA-PA NPs and PLGA-PA-DOX NPs, suggesting immunomodulatory properties of PA in the tumor microenvironment. In conclusion, the use of PA alone or in combination with DOX may represent a promising novel strategy for the treatment of breast cancer.

STAT3/NF-κB signalling disruption in M2 tumour-associated macrophages is a major target of PLGA nanocarriers/PD-L1 antibody immunomodulatory therapy in breast cancer.

BACKGROUND AND PURPOSE: Inflammation associated with the tumour microenvironment (TME) is critical for cancer development, and immunotherapeutic strategies modulating the immune response in cancer have been crucial. In this study, a methotrexate-loaded (MTX) poly(lactic-co-glycolic acid)-based (PLGA) drug nanocarrier covered with polyethyleneimine (Pei) and hyaluronic acid (HA) was developed and combined with an PD-L1 antibody to investigate anti-cancer and immunomodulatory effects in breast cancer TME. EXPERIMENTAL APPROACH: Naked or HA-coated PeiPLGA-MTX nanoparticles (NPs) were assessed on 4T1 breast cancer cells grown in culture and in a mouse model of orthotopic tumour growth. Tumours were evaluated by qRT-PCR and immunohistochemistry. The cell death profile and cell migration were analysed in vitro in 4T1 cells. Polarization of murine macrophages (RAW cells) was also carried out. KEY RESULTS: Naked or HA-coated PeiPLGA-MTX NPs used alone or combined with PD-L1 antibody modified the tumourigenic course by TME immunomodulation, leading to reduction of primary tumour size and metastases. STAT3 and NF-κB were the major genes downregulated by NPs. In tumor-associated macrophages (TAM) such regulation switched M2 phenotype (CD163) towards M1 (CD68) and reduced levels of IL-10, TGF-ß and CCL22. Moreover, malignant cells showed overexpression of FADD, APAF-1, caspase-3 and E-cadherin, and decreased expression of Bcl-2, MDR-1, survivin, vimentin, CXCR4 and PD-L1 after treatment with NPs. CONCLUSION AND IMPLICATIONS: NPs-mediated STAT3/NF-κB signalling axis suppression disrupted crosstalk between immune and malignant cells, reducing immunosuppression and critical pro-tumour events. These findings provide a promising therapeutic approach capable of guiding the immune TME to suppress the development of breast cancer.

Phytol-Loaded Solid Lipid Nanoparticles as a Novel Anticandidal Nanobiotechnological Approach.

Phytol is a diterpene alcohol and can be found as a product of the metabolism of chlorophyll in plants. This compound has been explored as a potential antimicrobial agent, but it is insoluble in water. In this study, we describe a novel approach for an interesting anticandidal drug delivery system containing phytol. Different formulations of phytol-loaded solid lipid nanoparticles (SLN) were designed and tested using a natural lipid, 1,3-distearyl-2-oleyl-glycerol (TG1). Different compositions were considered to obtain three formulations with 1:10, 1:5, and 1:3 w/w phytol/TG1 ratios. All the formulations were prepared by emulsification solvent evaporation method and had their physicochemical properties assessed. The biocompatibility assay was performed in the HEK-293 cell line and the antifungal efficacy was demonstrated in different strains of Candida ssp., including different clinical isolates. Spherical and uniform SLN (<300 nm, PdI < 0.2) with phytol-loading efficiency >65% were achieved. Phytol-loaded SLN showed a dose-dependent cytotoxic effect in the HEK-293 cell line. The three tested formulations of phytol-loaded SLN considerably enhanced the minimal inhibitory concentration of phytol against 15 strains of Candida spp. Considering the clinical isolates, the formulations containing the highest phytol/TG1 ratios showed MICs at 100%. Thus, the feasibility and potential of phytol-loaded SLN was demonstrated in vitro, being a promising nanocarrier for phytol delivery from an anticandidal approach.

Injectable platelet rich fibrin: cell content, morphological, and protein characterization.

OBJECTIVES: The aim of the present study was to evaluate the blood cell content, morphological aspects, gene expression of type I collagen, and release of growth factors on an injectable platelet rich fibrin (i-PRF). MATERIALS AND METHODS: Blood samples were collected from 15 volunteers to prepare i-PRF samples. Peripheral blood was used as a control group. Blood clot and i-PRF samples were cultured for 10 days. The supernatant of the samples was collected for ELISA immunoassay quantification of PDGF and VEGF growth factors over periods of 1, 8, 24, 72, and 240 h. I-PRF and blood clot samples were biologically characterized using histological and immunohistochemistry analysis for IL-10, osteocalcin, and TGF-ß. Scanning electron microscopy (SEM) was used to inspect the fibrin network and distribution of blood platelets and leukocytes. Reverse transcriptase polymerase chain reaction (RT-PCR) method was used to evaluate gene expression for type I collagen. RESULTS: A higher concentration of platelets and lymphocytes was recorded in i-PRF than in peripheral blood (p < 0.05). The release of VEGF was higher in blood clot samples (1933 ± 704) than that for i-PRF (852 ± 376; p < 0.001). Immunohistochemistry showed upregulation of TGF-B, IL-10, and osteocalcin in the i-PRF group. RT-PCR showed increased type I collagen gene expression in i-PRF (p < 0.05). SEM images revealed agglomeration of platelets in some regions, while a fibrin networking was noticeable in the entire i-PRF sample. CONCLUSIONS: Injectable platelet rich fibrin becomes a good approach for soft and mineralized tissue healing considering the formation of a three-dimensional fibrin network embedding platelets, leukocytes, type I collagen, osteocalcin, and growth factors. Indeed, the injectable platelet rich fibrin can be indicated in several medical applications regarding bioactivity, simplied technique, and flowable mixing with other biomaterials. CLINICAL RELEVANCE: Morphological, cell, and protein characterization of platelet rich fibrin provides a better understanding of the clinical effects and improvement of clinical guidelines for several medical applications. Once well physicochemical and biologically characterized, the use of an injectable platelet rich fibrin can be extended to other applications in the field of orthopedics, periodontics, and implant dentistry on the repairing process of both soft and mineralized tissues.

Anti-IIa activity and antitumor properties of a hybrid heparin/heparan sulfate-like compound from Litopenaeus vannamei shrimp.

In this present study, the anti-IIa activity and the antitumor properties of a hybrid heparin/heparan sulfate-like compound (sH/HS) from Litopenaeus vannamei shrimp heads are related. In addition to inhibiting 90.7% of thrombin activity at the lowest tested concentration (0.5 µg/mL), sH/HS compound stimulated the synthesis of antithrombotic heparan sulfate by endothelial cells in a dose-dependent manner. In vitro experiments demonstrated that the molecule from shrimp displayed a potent anti-angiogenic effect, reducing over 80% of the tubular structures formation at 50 and 100 µg/mL. In addition, sH/HS compound was able to inhibit the migration of B16F10 cells at all tested concentrations without affecting the cell viability. Although the studied compound had no effect on the proliferation of such cells during a period of 24 h, it had a significant long-term anti-proliferative effect, reducing about 80% of colony formation and anchorage-independent growth at 50 and 100 µg/mL concentrations. When its effectiveness was tested in vivo, it was demonstrated that sH/HS promoted a reduction of more than 90% of tumor growth. In the context of thromboembolic disorders associated with cancer, such findings make the sH/HS compound an excellent target for studies on inhibiting of development and tumor progression, and the prevention of coagulopathies.

2,3-Di-O-sulfo glucuronic acid: An unmodified and unusual residue in a highly sulfated chondroitin sulfate from Litopenaeus vannamei.

The occurrence of a natural and unmodified highly sulfated chondroitin sulfate from Litopenaeus vannamei heads (sCS) is herein reported. Its partial digestion by Chondroitinases AC and ABC together with its electrophoretic migration profile revealed it as a highly sulfated chondroitin sulfate despite its average molecular weight being similar to CSA. Using orthogonal 1D/2D NMR experiments, the anomeric signals (δ 4.62/106.0) corresponding to unusual 2,3-di-O-Sulfo-GlcA (∼36%), U33S (δ 4.42/84.1, ∼63%) and U22S (4.12/80.1, ∼50%) substitutions were confirmed. In addition, non-sulfated GlcA (δ 4.5/106.3) linked to 4-O- (A14S, 36%) or 6-O-Sulfo (A16S, 28%) GalNAc (δ 4.64/103.5) was observed. Although the biological role of sCS in shrimp is unknown, its influence on hemostasis was also demonstrated. The sCS identification brings to light new questions about the hierarchical model of GAGs biosynthesis and contributes to the better understanding of the subtle relationship between GAGs structure and function.

Apoptosis in human liver carcinoma caused by gold nanoparticles in combination with carvedilol is mediated via modulation of MAPK/Akt/mTOR pathway and EGFR/FAAD proteins.

In cancers, apoptosis signaling pathways and cell survival and growth pathways responsible for resistance to conventional treatments, such as Pi3K/Akt/mTOR and mitogen-activated protein kinase (MAPK) become dysregulated. Recently, alternative treatments to promote tumor cell death have become important. The present study reports on the antitumor and cytoprotective action of gold nanoparticles (GNPs) and carvedilol in combination and in isolated application. Apoptosis was analyzed by FITC/propidium iodide staining flow cytometry; caspase-3, caspase-8, Bcl-2 and MAPK/ERK activity by immunofluorescence microscopy; gene expression of proteins related to cell death as Akt, mTOR, EGFR, MDR1, survivin, FADD and Apaf, by the real-time PCR; and western blot analysis for MAPK/ERK, Akt and mTOR. Oxidative stress evaluation was performed by reduced glutathione (GSH) and malondialdehyde (MDA) levels. Intracellular GNPs targets were identified by transmission electron microscopy. After exposure to a combination of GNPs (6.25 µg/ml) and carvedilol (3 µM), death as promoted by apoptosis was detected using flow cytometry, for expression of pro-apoptotic proteins FADD, caspase-3, caspase-8 and sub-regulation of anti-apoptotic MAPK/ERK, Akt, mTOR, EGFR and MDR1 resistance. Non-tumor cell cytoprotection with GSH elevation and MDA reduction levels was detected. GNPs were identified within the cell near to the nucleus when combined with carvedilol. The combination of GNP and carvedilol promoted downregulation of anti-apoptotic and drug resistance genes, over-regulation of pro-apoptotic proteins in tumor cells, as well as cytoprotection of non-tumor cells with reduction of apoptosis and oxidative stress.

A non-hemorrhagic hybrid heparin/heparan sulfate with anticoagulant potential.

The structural characterization and the anticoagulant potential of a novel heparin/heparan sulfate-like compound from the heads of Litopenaeus vannamei shrimp are described. While it is distinct from either heparin or heparan sulfate, enzymatic depolymerization and nuclear magnetic resonance spectroscopy analyses revealed that this molecule does share some structural features with heparin, such as the high degree of N- and 6-O-sulfation and minor N-acetylation, and with heparan sulfate, in the glucuronic acid content. Its ability to stabilize human antithrombin explains its significant anticoagulant activity in aPTT and Factor-Xa inhibition assays. Interestingly, in contrast to mammalian heparin, the shrimp compound displayed negligible hemorrhagic effect. Together, these findings have particular interest since they reveal a novel molecule with significant anti-Xa activity coupled with low bleeding effects which make the shrimp heparin/HS-like compound a potential alternative for mammalian heparin.