RhoA downregulation in the murine intestinal epithelium results in chronic Wnt activation and increased tumorigenesis.

Rho GTPases are molecular switches regulating multiple cellular processes. To investigate the role of RhoA in normal intestinal physiology, we used a conditional mouse model overexpressing a dominant negative RhoA mutant (RhoAT19N) in the intestinal epithelium. Although RhoA inhibition did not cause an overt phenotype, increased levels of nuclear ß-catenin were observed in the small intestinal epithelium of RhoAT19N mice, and the overexpression of multiple Wnt target genes revealed a chronic activation of Wnt signaling. Elevated Wnt signaling in RhoAT19N mice and intestinal organoids did not affect the proliferation of intestinal epithelial cells but significantly interfered with their differentiation. Importantly, 17-month-old RhoAT19N mice showed a significant increase in the number of spontaneous intestinal tumors. Altogether, our results indicate that RhoA regulates the differentiation of intestinal epithelial cells and inhibits tumor initiation, likely through the control of Wnt signaling, a key regulator of proliferation and differentiation in the intestine.

Optimization of Statin-Loaded Delivery Nanoparticles for Treating Chronic Liver Diseases by Targeting Liver Sinusoidal Endothelial Cells.

In this study, we developed functionalized polymeric micelles (FPMs) loaded with simvastatin (FPM-Sim) as a drug delivery system to target liver sinusoidal endothelial cells (LSECs) for preserving liver function in chronic liver disease (CLD). Polymeric micelles (PMs) were functionalized by coupling peptide ligands of LSEC membrane receptors CD32b, CD36 and ITGB3. Functionalization was confirmed via spectroscopy and electron microscopy. In vitro and in vivo FPM-Sim internalization was assessed by means of flow cytometry in LSECs, hepatocytes, Kupffer and hepatic stellate cells from healthy rats. Maximum tolerated dose assays were performed in healthy mice and efficacy studies of FPM-Sim were carried out in bile duct ligation (BDL) and thioacetamide (TAA) induction rat models of cirrhosis. Functionalization with the three peptide ligands resulted in stable formulations with a greater degree of in vivo internalization in LSECs than non-functionalized PMs. Administration of FPM-Sim in BDL rats reduced toxicity relative to free simvastatin, albeit with a moderate portal-pressure-lowering effect. In a less severe model of TAA-induced cirrhosis, treatment with FPM-CD32b-Sim nanoparticles for two weeks significantly decreased portal pressure, which was associated with a reduction in liver fibrosis, lower collagen expression as well as the stimulation of nitric oxide synthesis. In conclusion, CD32b-FPM stands out as a good nanotransporter for drug delivery, targeting LSECs, key inducers of liver injury.

Nanoceria as Safe Contrast Agents for X-ray CT Imaging.

Cerium oxide nanoparticles (CeO2NPs) have exceptional catalytic properties, rendering them highly effective in removing excessive reactive oxygen species (ROS) from biological environments, which is crucial in safeguarding these environments against radiation-induced damage. Additionally, the Ce atom's high Z number makes it an ideal candidate for utilisation as an X-ray imaging contrast agent. We herein show how the injection of albumin-stabilised 5 nm CeO2NPs into mice revealed substantial enhancement in X-ray contrast, reaching up to a tenfold increase at significantly lower concentrations than commercial or other proposed contrast agents. Remarkably, these NPs exhibited prolonged residence time within the target organs. Thus, upon injection into the tail vein, they exhibited efficient uptake by the liver and spleen, with 85% of the injected dose (%ID) recovered after 7 days. In the case of intratumoral administration, 99% ID of CeO2NPs remained within the tumour throughout the 7-day observation period, allowing for observation of disease dynamics. Mass spectrometry (ICP-MS) elemental analysis confirmed X-ray CT imaging observations.

Delivery Systems in Ocular Retinopathies: The Promising Future of Intravitreal Hydrogels as Sustained-Release Scaffolds.

Slow-release delivery systems are needed to ensure long-term sustained treatments for retinal diseases such as age-related macular degeneration and diabetic retinopathy, which are currently treated with anti-angiogenic agents that require frequent intraocular injections. These can cause serious co-morbidities for the patients and are far from providing the adequate drug/protein release rates and required pharmacokinetics to sustain prolonged efficacy. This review focuses on the use of hydrogels, particularly on temperature-responsive hydrogels as delivery vehicles for the intravitreal injection of retinal therapies, their advantages and disadvantages for intraocular administration, and the current advances in their use to treat retinal diseases.

Key elements of cellular senescence involve transcriptional repression of mitotic and DNA repair genes through the p53-p16/RB-E2F-DREAM complex.

Cellular senescence is a dynamic stress response process that contributes to aging. From initiation to maintenance, senescent cells continuously undergo complex molecular changes and develop an altered transcriptome. Understanding how the molecular architecture of these cells evolve to sustain their non-proliferative state will open new therapeutic avenues to alleviate or delay the consequences of aging. Seeking to understand these molecular changes, we studied the transcriptomic profiles of endothelial replication-induced senescence and senescence induced by the inflammatory cytokine, TNF-α. We previously reported gene expressional pattern, pathways, and the mechanisms associated with upregulated genes during TNF-α induced senescence. Here, we extend our work and find downregulated gene signatures of both replicative and TNF-α senescence were highly overlapped, involving the decreased expression of several genes associated with cell cycle regulation, DNA replication, recombination, repair, chromatin structure, cellular assembly, and organization. We identified multiple targets of p53/p16-RB-E2F-DREAM that are essential for proliferation, mitotic progression, resolving DNA damage, maintaining chromatin integrity, and DNA synthesis that were repressed in senescent cells. We show that repression of multiple target genes in the p53/p16-RB-E2F-DREAM pathway collectively contributes to the stability of the senescent arrest. Our findings show that the regulatory connection between DREAM and cellular senescence may play a potential role in the aging process.

Intracellular Delivery of Anti-Kirsten Rat Sarcoma Antibodies Mediated by Polymeric Micelles Exerts Strong In Vitro and In Vivo Anti-Tumorigenic Activity in Kirsten Rat Sarcoma-Mutated Cancers.

The Kirsten rat sarcoma viral oncogene (KRAS) is one of the most well-known proto-oncogenes, frequently mutated in pancreatic and colorectal cancers, among others. We hypothesized that the intracellular delivery of anti-KRAS antibodies (KRAS-Ab) with biodegradable polymeric micelles (PM) would block the overactivation of the KRAS-associated cascades and revert the effect of its mutation. To this end, PM-containing KRAS-Ab (PM-KRAS) were obtained using Pluronic F127. The feasibility of using PM for antibody encapsulation as well as the conformational change of the polymer and its intermolecular interactions with the antibodies was studied, for the first time, using in silico modeling. In vitro, encapsulation of KRAS-Ab allowed their intracellular delivery in different pancreatic and colorectal cancer cell lines. Interestingly, PM-KRAS promoted a high proliferation impairment in regular cultures of KRAS-mutated HCT116 and MIA PaCa-2 cells, whereas the effect was neglectable in non-mutated or KRAS-independent HCT-8 and PANC-1 cancer cells, respectively. Additionally, PM-KRAS induced a remarkable inhibition of the colony formation ability in low-attachment conditions in KRAS-mutated cells. In vivo, when compared with the vehicle, the intravenous administration of PM-KRAS significantly reduced tumor volume growth in HCT116 subcutaneous tumor-bearing mice. Analysis of the KRAS-mediated cascade in cell cultures and tumor samples showed that the effect of PM-KRAS was mediated by a significant reduction of the ERK phosphorylation and a decrease in expression in the stemness-related genes. Altogether, these results unprecedently demonstrate that the delivery of KRAS-Ab mediated by PM can safely and effectively reduce the tumorigenicity and the stemness properties of KRAS-dependent cells, thus bringing up new possibilities to reach undruggable intracellular targets.

Extracellular vesicles secreted by triple-negative breast cancer stem cells trigger premetastatic niche remodeling and metastatic growth in the lungs.

Tumor secreted extracellular vesicles (EVs) are potent intercellular signaling platforms. They are responsible for the accommodation of the premetastatic niche (PMN) to support cancer cell engraftment and metastatic growth. However, complex cancer cell composition within the tumor increases also the heterogeneity among cancer secreted EVs subsets, a functional diversity that has been poorly explored. This phenomenon is particularly relevant in highly plastic and heterogenous triple-negative breast cancer (TNBC), in which a significant representation of malignant cancer stem cells (CSCs) is displayed. Herein, we selectively isolated and characterized EVs from CSC or differentiated cancer cells (DCC; EVsCSC and EVsDCC , respectively) from the MDA-MB-231 TNBC cell line. Our results showed that EVsCSC and EVsDCC contain distinct bioactive cargos and therefore elicit a differential effect on stromal cells in the TME. Specifically, EVsDCC activated secretory cancer associated fibroblasts (CAFs), triggering IL-6/IL-8 signaling and sustaining CSC phenotype maintenance. Complementarily, EVsCSC promoted the activation of α-SMA+ myofibroblastic CAFs subpopulations and increased the endothelial remodeling, enhancing the invasive potential of TNBC cells in vitro and in vivo. In addition, solely the EVsCSC mediated signaling prompted the transformation of healthy lungs into receptive niches able to support metastatic growth of breast cancer cells.

Oxidative Stress in Structural Valve Deterioration: A Longitudinal Clinical Study.

The cause of structural valve deterioration (SVD) is unclear. Therefore, we investigated oxidative stress markers in sera from patients with bioprosthetic heart valves (BHVs) and their association with SVD. Blood samples were taken from SVD (Phase A) and BHV patients during the first 24 (Phase B1) and >48 months (Phase B2) after BHV implantation to assess total antioxidant capacity (TAC), malondialdehyde (MDA), and nitrotyrosine (NT). The results show that MDA levels increased significantly 1 month after surgery in all groups but were higher at 6 months only in incipient SVD patients. NT levels increased gradually for the first 24 months after implantation in the BHV group. Patients with transcatheter aortic valve implantation (TAVI) showed even higher levels of stress markers. After >48 months, MDA and NT continued to increase in BHV patients with a further elevation after 60-72 months; however, these levels were significantly lower in the incipient and established SVD groups. In conclusion, oxidative stress may play a significant role in SVD, increasing early after BHV implantation, especially in TAVI cases, and also after 48 months' follow-up, but decreasing when SVD develops. Oxidative stress potentially represents a target of therapeutic intervention and a biomarker of BHV dysfunction.

Myosin Vb as a tumor suppressor gene in intestinal cancer.

Colorectal cancer causes >900,000 deaths every year and a deeper understanding of the molecular mechanisms underlying this disease will contribute to improve its clinical management and survival. Myosin Vb (MYO5B) regulates intracellular vesicle trafficking, and inactivation of this myosin disrupts the polarization and differentiation of intestinal epithelial cells causing microvillous inclusion disease (MVID), a rare congenital disorder characterized by intractable life-threatening diarrhea. Here, we show that the loss Myosin Vb interfered with the differentiation/polarization of colorectal cancer cells. Although modulation of Myosin Vb expression did not affect the proliferation of colon cancer cells, MYO5B inactivation increased their migration, invasion, and metastatic potential. Moreover, Myo5b inactivation in an intestine-specific knockout mouse model caused a >15-fold increase in the number of azoxymethane-initiated small intestinal tumors. Consistently, reduced expression of Myosin Vb in a cohort of 155 primary colorectal tumors was associated with shorter patient survival. In conclusion, we show here that loss of Myosin Vb reduces polarization/differentiation of colon cancer cells while enhancing their metastatic potential, demonstrating a tumor suppressor function for this myosin. Moreover, reduced expression of Myosin Vb in primary tumors identifies a subset of poor prognosis colorectal cancer patients that could benefit from more aggressive therapeutic regimens.

Selectively Targeting Breast Cancer Stem Cells by 8-Quinolinol and Niclosamide.

Cancer maintenance, metastatic dissemination and drug resistance are sustained by cancer stem cells (CSCs). Triple negative breast cancer (TNBC) is the breast cancer subtype with the highest number of CSCs and the poorest prognosis. Here, we aimed to identify potential drugs targeting CSCs to be further employed in combination with standard chemotherapy in TNBC treatment. The anti-CSC efficacy of up to 17 small drugs was tested in TNBC cell lines using cell viability assays on differentiated cancer cells and CSCs. Then, the effect of 2 selected drugs (8-quinolinol -8Q- and niclosamide -NCS-) in the cancer stemness features were evaluated using mammosphere growth, cell invasion, migration and anchorage-independent growth assays. Changes in the expression of stemness genes after 8Q or NCS treatment were also evaluated. Moreover, the potential synergism of 8Q and NCS with PTX on CSC proliferation and stemness-related signaling pathways was evaluated using TNBC cell lines, CSC-reporter sublines, and CSC-enriched mammospheres. Finally, the efficacy of NCS in combination with PTX was analyzed in vivo using an orthotopic mouse model of MDA-MB-231 cells. Among all tested drug candidates, 8Q and NCS showed remarkable specific anti-CSC activity in terms of CSC viability, migration, invasion and anchorage independent growth reduction in vitro. Moreover, specific 8Q/PTX and NCS/PTX ratios at which both drugs displayed a synergistic effect in different TNBC cell lines were identified. The sole use of PTX increased the relative presence of CSCs in TNBC cells, whereas the combination of 8Q and NCS counteracted this pro-CSC activity of PTX while significantly reducing cell viability. In vivo, the combination of NCS with PTX reduced tumor growth and limited the dissemination of the disease by reducing circulating tumor cells and the incidence of lung metastasis. The combination of 8Q and NCS with PTX at established ratios inhibits both the proliferation of differentiated cancer cells and the viability of CSCs, paving the way for more efficacious TNBC treatments.

Smart and eco-friendly N-isopropylacrylamide and cellulose hydrogels as a safe dual-drug local cancer therapy approach.

Local cancer treatment by in situ injections of thermo-responsive hydrogels (HG) offers several advantages over conventional systemic anti-cancer treatments. In this work, a biodegradable and multicompartmental HG composed of N-isopropylacrylamide, cellulose, citric acid, and ceric ammonium nitrate was developed for the controlled release of hydrophilic (doxorubicin) and hydrophobic (niclosamide) drugs. The formulation presented ideal properties regarding thermo-responsiveness, rheological behavior, drug release profile, biocompatibility, and biological activity in colon and ovarian cancer cells. Cellulose was found to retard drugs release rate, being only 4 % of doxorubicin and 30 % of niclosamide released after 1 week. This low release was sufficient to cause cell death in both cell lines. Moreover, HG demonstrated a proper injectability, in situ prevalence, and safety profile in vivo. Overall, the HG properties, together with its natural and eco-friendly composition, create a safe and efficient platform for the local treatment of non-resectable tumors or tumors requiring pre-surgical adjuvant therapy.

Fine Control of In Vivo Magnetic Hyperthermia Using Iron Oxide Nanoparticles with Different Coatings and Degree of Aggregation.

The clinical implementation of magnetic hyperthermia has experienced little progress since the first clinical trial was completed in 2005. Some of the hurdles to overcome are the reliable production of magnetic nanoparticles with controlled properties and the control of the temperature at the target tissue in vivo. Here, forty samples of iron oxide superparamagnetic nanoparticles were prepared by similar methods and thoroughly characterized in terms of size, aggregation degree, and heating response. Selected samples were intratumorally administered in animals with subcutaneous xenografts of human pancreatic cancer. In vivo experiments showed that it is possible to control the rise in temperature by modulating the field intensity during in vivo magnetic hyperthermia protocols. The procedure does not require sophisticated materials and it can be easily implemented by researchers or practitioners working in magnetic hyperthermia therapies.

Oral delivery of camptothecin-loaded multifunctional chitosan-based micelles is effective in reduce colorectal cancer.

Colorectal cancer (CRC) is a heterogeneous disease with high incidence and mortality worldwide. The efficacy of conventional CRC chemotherapy is hampered by poor drug solubility and bioavailability and suboptimal pharmacokinetic profiles. In this work, camptothecin (CPT), a potent anticancer drug, was loaded into an amphiphilic chitosan modified with PEG and oleic acid, to reduce CRC progression after oral administration. While CPT-loaded micelles presented anticancer activity against HCT116, Caco-2 and HT29 CRC cell lines in vitro, empty micelles demonstrated a safe profile when incubated with human blood cells and colorectal cancer cell lines. In a more complex 3D CRC multicellular spheroid model, CPT-loaded micelles also exhibited a significant effect on the spheroid's metabolic activity and size reduction. Remarkably, in vivo studies performed in a HCT116 xenograft model, showed a significant reduction on the tumor growth during and after treatment with CPT-loaded micelles. Moreover, in a more biological relevant in vivo model of chemically-induced CRC, orally administered CPT-loaded micelles demonstrated a significant reduction on tumor incidence and inflammation signs. The findings here reported indicate that CPT-loaded into chitosan-based micelles, by improving drug solubility, alongside its safety profile for normal tissues, may have a promising role CRC chemotherapy.

Colorectal cancer inhibition by BET inhibitor JQ1 is MYC-independent and not improved by nanoencapsulation.

Bromodomain and extraterminal domain protein inhibitors (BETi) for cancer treatment did not convince during their first clinical trials. Their epigenetic mechanism of action is still not well understood, even if MYC is generally considered as its main downstream target. In this context, we intended to assess two new nanoformulations of the BETi JQ1 for the treatment of colorectal cancer (CRC). JQ1 was encapsulated at 10 mg/mL in lipid nanocapsules (LNC) or polymeric micelles (PM), both compatible for an intravenous administration. Their effect was compared with free JQ1 on several CRC cell lines in vitro and with daily intraperitoneal cyclodextrin (CD)-loaded JQ1 on the CT26 CRC tumor model in vivo. We showed that LNC preferentially accumulated in tumor, liver, and lymph nodes. LNC-JQ1 and CD-JQ1 similarly delayed tumor growth and increased median survival from 15 to 23 or 20.5 days. JQ1 altered MYC in only two among four CRC cell lines. This MYC-independence found in CT26 was confirmed in vivo by PCR and immunohistochemistry. The main explanation of the JQ1 anticancer effect was an increase in apoptosis. The investigation of its impact on the tumor microenvironment did not show significant effects. Finally, JQ1 association with irinotecan did not synergize in vivo with JQ1 nanoformulations. In conclusion, we demonstrated that the JQ1 anticancer effect was not improved by nanoencapsulation even if their tumor delivery was probably higher. MYC inhibition was not associated to JQ1 efficacy in the case of the CT26 CRC murine model.

In Vivo Antitumor and Antimetastatic Efficacy of a Polyacetal-Based Paclitaxel Conjugate for Prostate Cancer Therapy.

Prostate cancer (PCa), one of the leading causes of cancer-related deaths, currently lacks effective treatment for advanced-stage disease. Paclitaxel (PTX) is a highly active chemotherapeutic drug and the first-line treatment for PCa; however, conventional PTX formulation causes severe hypersensitivity reactions and limits PTX use at high concentrations. In the pursuit of high molecular weight, biodegradable, and pH-responsive polymeric carriers, one conjugates PTX to a polyacetal-based nanocarrier to yield a tert-Ser-PTX polyacetal conjugate. tert-Ser-PTX conjugate provides sustained release of PTX over 2 weeks in a pH-responsive manner while also obtaining a degree of epimerization of PTX to 7-epi-PTX. Serum proteins stabilize tert-Ser-PTX, with enhanced stability in human serum versus PBS (pH 7.4). In vitro efficacy assessments in PCa cells demonstrate IC50 values above those for the free form of PTX due to the differential cell trafficking modes; however, in vivo tolerability assays demonstrate that tert-Ser-PTX significantly reduces the systemic toxicities associated with free PTX treatment. tert-Ser-PTX also effectively inhibits primary tumor growth and hematologic, lymphatic, and coelomic dissemination, as confirmed by in vivo and ex vivo bioluminescence imaging and histopathological evaluations in mice carrying orthotopic LNCaP tumors. Overall, the results suggest the application of tert-Ser-PTX as a robust antitumor/antimetastatic treatment for PCa.

Development of "on-demand" thermo-responsive hydrogels for anti-cancer drugs sustained release: Rational design, in silico prediction and in vitro validation in colon cancer models.

A rational design accurate based on the use of Statistical Design of the Experiments (DoE) and Molecular Dynamics Simulations Studies allows the prediction and the understanding of thermo-responsive hydrogels prepared regarding their gelation temperature and anti-cancer drug release rate. N-isopropylacrilamide (NIPAM) modified with specific co-monomers and crosslinkers, can be used to prepare "on-demand" thermo-responsive hydrogels with the ideal properties for clinical applications in which local sustained release of drugs is crucial. Two preferential formulations resulting from the predictive studies of DoE and In Silico methods were synthesized by radical polymerization, fully characterized, and loaded with the anticancer drug Doxorubicin (Dox). The hydrogel formulations were characterized by swelling rate, turbidity, FTIR, 1H NMR, SEM, gelation time, rheology, and biocompatibility assays. Both formulations demonstrated adequate morphologic, rheological, and biocompatibility properties; however, important differences in terms of drug retention were detected. As demonstrated by a Dox cumulative release study and posteriorly confirmed by an efficacy assay in an in vitro colorectal cancer model, the formulation composed by NIPAM and 4-penten-1-ol crosslinked with poly(ethylene glycol) diacrylate (PEGDA) (PNiPenPH) present a slow release over the time, presenting ideal properties to become and ideal depot system for the local sustained release of anticancer drugs as adjuvant therapy or in the case of non-resectable tumors.

Application of Quality by Design to the robust preparation of a liposomal GLA formulation by DELOS-susp method.

Fabry disease is a lysosomal storage disease arising from a deficiency of the enzyme α-galactosidase A (GLA). The enzyme deficiency results in an accumulation of glycolipids, which over time, leads to cardiovascular, cerebrovascular, and renal disease, ultimately leading to death in the fourth or fifth decade of life. Currently, lysosomal storage disorders are treated by enzyme replacement therapy (ERT) through the direct administration of the missing enzyme to the patients. In view of their advantages as drug delivery systems, liposomes are increasingly being researched and utilized in the pharmaceutical, food and cosmetic industries, but one of the main barriers to market is their scalability. Depressurization of an Expanded Liquid Organic Solution into aqueous solution (DELOS-susp) is a compressed fluid-based method that allows the reproducible and scalable production of nanovesicular systems with remarkable physicochemical characteristics, in terms of homogeneity, morphology, and particle size. The objective of this work was to optimize and reach a suitable formulation for in vivo preclinical studies by implementing a Quality by Design (QbD) approach, a methodology recommended by the FDA and the EMA to develop robust drug manufacturing and control methods, to the preparation of α-galactosidase-loaded nanoliposomes (nanoGLA) for the treatment of Fabry disease. Through a risk analysis and a Design of Experiments (DoE), we obtained the Design Space in which GLA concentration and lipid concentration were found as critical parameters for achieving a stable nanoformulation. This Design Space allowed the optimization of the process to produce a nanoformulation suitable for in vivo preclinical testing.

Thermo-responsive hydrogels for cancer local therapy: Challenges and state-of-art.

Despite the enormous efforts done by the scientific community in the last decades, advanced cancer is still considered an incurable disease. New formulations are continuously under investigation to improve drugs therapeutic index, i.e., increase chemotherapeutic efficacy and reduce adverse effects. In this context, hydrogels-based systems for drug local sustained/controlled release have been proposed to reduce off-target effects caused by the repeated administration of systemic/oral anticancer drugs and improve their therapeutic effectiveness. Moreover, it increases the patient welfare by reducing the number of administrations needed. Among the several types of existing hydrogels, the thermo-responsive ones, which are able to change their physical state from liquid at 25 °C to a gel at the body temperature, i.e., 37 °C, gained special attention as in situ sustained drug release depot-systems in cancer treatment. To date, several thermo-responsive hydrogels have been used for drugs and/or genetic material delivery, yielding promising results both at preclinical and clinical evaluation stages. This culminates in the market authorization of Jelmyto® for the treatment of urothelial cancer. Here are summarized and discussed the last 10 years advances regarding the application of thermo-responsive hydrogels in local cancer treatment.

Pluronic F127 micelles improve the stability and enhance the anticancer stem cell efficacy of citral in breast cancer.

Aim: Improving the stability and anti-cancer stem cell (CSC) activity of citral, a natural ALDH1A inhibitor. Materials & methods: Citral-loaded micelles (CLM) were obtained using Pluronic® F127 and its efficacy tested on the growth of four breast cancer cell lines. The impact of the CLM on the growth and functional hallmarks of breast CSCs were also evaluated using mammosphere and CSC reporter cell lines. Results: CLM improved the stability and growth inhibitory effects of citral. Importantly, CLM fully blocking the stemness features of CSCs (self-renewal, differentiation and migration) and in combination with paclitaxel CLM sensitized breast cancer cells to the chemotherapy. Conclusion: Targeting CSCs with CLM could improve the treatment of advanced breast cancer in combination with the standard chemotherapy.

Hyaluronic acid (HA)-coated naproxen-nanoparticles selectively target breast cancer stem cells through COX-independent pathways.

Cytotoxic chemotherapy continues to be the main therapeutic option for patients with metastatic breast cancer. Several studies have reported a significant association between chronic inflammation, carcinogenesis and the presence of cancer stem cells (CSC). We hypothesized that the use of non-steroidal anti-inflammatory drugs targeted to the CSC population could help reducing tumor progression and dissemination in otherwise hard to treat metastatic breast cancer. Within this study cationic naproxen (NAP)-bearing polymeric nanoparticles (NPs) were obtained by self-assembly and they were coated with hyaluronic acid (HA) via electrostatic interaction. HA-coated and uncoated NAP-bearing NPs with different sizes were produced by changing the ionic strength of the aqueous preparation solutions (i.e. 300 and 350 nm or 100 and 130 nm in diameter, respectively). HA-NPs were fully characterized in terms of physicochemical parameters and biological response in cancer cells, macrophages and endothelial cells. Our results revealed that HA-coating of NPs provided a better control in NAP release and improved their hemocompatibility, while ensuring a strong CSC-targeting in MCF-7 breast cancer cells. Furthermore, the best polymeric NPs formulation significantly (p < 0.001) reduced MCF-7 cells viability when compared to free drug (i.e. 45 ± 6% for S-HA-NPs and 87 ± 10% for free NAP) by p53-dependent induction of apoptosis; and the migration of these cell line was also significantly (p < 0.01) reduced by the nano-formulated NAP (i.e. 76.4% of open wound for S-HA-NPs and 61.6% of open wound for NAP). This increased anti-cancer activity of HA-NAP-NPs might be related to the induction of apoptosis through alterations of the GSK-3ß-related COX-independent pathway. Overall, these findings suggest that the HA-NAP-NPs have the potential to improve the treatment of advanced breast cancer by increasing the anti-proliferative effect of NAP within the CSC subpopulation.