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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.

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Purpose:The study of the cancer secretome suggests that a fraction of the intracellular proteome could play unanticipated roles in the extracellular space during tumorigenesis. A project aimed at investigating the invasive secretome led us to study the alternative extracellular function of the nuclear protein high mobility group A1 (HMGA1) in breast cancer invasion and metastasis.Experimental Design:Antibodies against HMGA1 were tested in signaling, adhesion, migration, invasion, and metastasis assays using breast cancer cell lines and xenograft models. Fluorescence microscopy was used to determine the subcellular localization of HMGA1 in cell lines, xenograft, and patient-derived xenograft models. A cohort of triple-negative breast cancer (TNBC) patients was used to study the correlation between subcellular localization of HMGA1 and the incidence of metastasis.Results:Our data show that treatment of invasive cells with HMGA1-blocking antibodies in the extracellular space impairs their migration and invasion abilities. We also prove that extracellular HMGA1 (eHMGA1) becomes a ligand for the Advanced glycosylation end product-specific receptor (RAGE), inducing pERK signaling and increasing migration and invasion. Using the cytoplasmic localization of HMGA1 as a surrogate marker of secretion, we showed that eHMGA1 correlates with the incidence of metastasis in a cohort of TNBC patients. Furthermore, we show that HMGA1 is enriched in the cytoplasm of tumor cells at the invasive front of primary tumors and in metastatic lesions in xenograft models.Conclusions:Our results strongly suggest that eHMGA1 could become a novel drug target in metastatic TNBC and a biomarker predicting the onset of distant metastasis.

Ytterbium ferrites are being used in many promising applications, such as visible-light photocatalysis, solar cells, magnetooptic devices, electro-magnetic equipment, etc., due to their fantastic ferroelectric and ferromagnetic properties. However, despite their good magnetic and radiopaque features, the use of ytterbium ferrites as multiplatform contrast agents in magnetic resonance imaging (MRI) and X-ray computed tomography (CT) is still under-developed. This is mainly due to difficulties in obtaining stable and biocompatible aqueous colloidal dispersions of ytterbium ferrite nanoparticles. In order to overcome this limitation, this work explores an eco-friendly method to directly synthesize such dispersions by liquid-assisted pulsed laser ablation of ytterbium ferrite massive targets. First, orthorhombic bulk YbFeO3 targets were obtained by a reaction-sintering method. Then, colloidal dispersions of nanoparticles were produced directly in both distilled water and ethanol by irradiating the bulk YbFeO3 targets with high-power infrared nanosecond lasers pulses. A battery of techniques has been used to characterize the as synthesized YbFeO3 targets and colloidal dispersions of YbFe nanoparticles to determine their composition, structure, magnetic properties, X-ray attenuation potentials, and colloidal properties. Moreover, the biocompatibility of the systems was also analysed by MTT cell viability assay. Results indicated that the use of distilled water as ablation medium yields colloidal dispersions consisted mainly of paramagnetic ytterbium ferrite nanoparticles. Contrarily, the use of ethanol as solvent leads to colloidal dispersions of polycrystalline nanoparticles with both ferromagnetic and paramagnetic behaviour, due to the coexistence, in each nanoparticle, of ytterbium ferrite, ytterbium oxide, and iron oxide crystalline phases. Both colloidal dispersions exhibit also high biocompatibility and suitable X-ray attenuation properties. Moreover, they show bio-safe hydrodynamic sizes (lower than 200 nm) with acceptable overall hydrodynamic polydispersity index values (under 0.4), being stable in water for several weeks. These results pave the way for the future evaluation of Yb–Fe based nanoparticles as multiplatform contrast agents in multimodal MRI and CT imaging., This research has been funded by the Spanish Ministry of Economy and Competitiveness (MINECO) and FEDERthrough the research project MAT2015-67354R, and a MSCA-IF postdoctoral fellowship from the Marie Curie action 2014 [grant 656908-NIMBLIS] of the H2020 program of the Executive Agency for Research Manages of EU Commission. Additionally, this research has been funded throught a research project (research project ULST-NANO) of the Proyectos Integradores MdM-IMEYMAT, call 2020, as well as the researcg project PID2020-118329RB-I00 funded by Spanish MCIN/AEI/10.13039/501100011033 action. Moreover, this research was also co-financed through two regional research projects funded by the Junta de Andalucía, specifically, the research project PECART-0096-2020 (Consejería Salud y Familias. JA Spain) and the research project P20_01293 (Consejería Economía, Conocimiento, Empresas y Universidad. JA Spain). In addition, Dr. Monserrat Llaguno-Munive is grateful for the funding received through the post-doctoral grant of the Consejo Nacional de Ciencia y Tecnología from Mexico (CONACYT, no 619639). In addition, we acknowledge the received technical assistance from the SC-ICYT of the University of Cádiz and the Servicio General de Apoyo a la Investigación-SAI, Universidad de Zaragoza. We also acknowledge to the Networking Research Centre on Bioengineering, Biomaterials, and Nanomedicine (CIBERBBN) (which is financed by the Instituto de Salud Carlos III (ISCIII) with assistance from the European Regional Development Fund (ERDF)) and the ICTS “NANBIOSIS”, specifically the FVPR/U20 (http://www.nanbiosis.es/portfolio/u20-in-vivo-experimentalplatform/) for providing access to the micro-CT. Finally, we want to thank the company LASING S.L. its technical support in the development of the NANO-GLAS laser system where the LA-PLA experiments were carried out.

Colorectal cancer (CRC) is a highly prevalent disease worldwide. Patient survival is hampered by tumor relapse and the appearance of drug-resistant metastases, which are sustained by the presence of cancer stem cells (CSC). Specific delivery of anti-CSC chemotherapeutic drugs to tumors by using targeted drug delivery systems that can also target CSC sub-population might substantially improve current clinical outcomes. CD44v6 is a robust biomarker for advanced CRC and CSC, due to its functional role in tumorigenesis and cancer initiation process. Here, we show that CD44v6-targeted polymeric micelles (PM) loaded with niclosamide (NCS), a drug against CSC, is a good therapeutic strategy against colorectal CSC and circulating tumor cells (CTC) in vivo. HCT116 cells were sorted according to their CD44v6 receptor expression into CD44v6+ (high) and CDv44v6- (low) subpopulations. Accordingly, CD44v6+ cells presented stemness properties, such as overexpression of defined stemness markers (ALDH1A1, CD44v3 and CXCR4) and high capacity to form colonspheres in low attachment conditions. NCS-loaded PM functionalized with an antibody fragment against CD44v6 (Fab-CD44v6) presented adequate size, charge, and encapsulation efficiency. In addition, Fab-CD44v6 significantly increased PM internalization in CD44v6+ cells. Further, encapsulation of NCS improved its effectiveness in vitro, particularly against colonspheres, and allowed to increase its intravenous dosage in vivo by increasing the amount of NCS able to be administered without causing toxicity. Remarkably, functionalized PM accumulate in tumors and significantly reduce CTC in vivo. In conclusion, CD44v6 targeted PM meet the essential conditions to become an efficient anti-CSC therapy.

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.

Despite all the advances seen in recent years, the severe adverse effects and low specificity of conventional chemotherapy are still challenging problems regarding cancer treatment. Nanotechnology has helped to address these questions, making important contributions in the oncological field. The use of nanoparticles has allowed the improvement of the therapeutic index of several conventional drugs and facilitates the tumoral accumulation and intracellular delivery of complex biomolecules, such as genetic material. Among the wide range of nanotechnology-based drug delivery systems (nanoDDS), solid lipid nanoparticles (SLNs) have emerged as promising systems for delivering different types of cargo. Their solid lipid core, at room and body temperature, provides SLNs with higher stability than other formulations. Moreover, SLNs offer other important features, namely the possibility to perform active targeting, sustained and controlled release, and multifunctional therapy. Furthermore, with the possibility to use biocompatible and physiologic materials and easy scale-up and low-cost production methods, SLNs meet the principal requirements of an ideal nanoDDS. The present work aims to summarize the main aspects related to SLNs, including composition, production methods, and administration routes, as well as to show the most recent studies about the use of SLNs for cancer treatment.

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, we conjugated PTX to a polyacetal-based nanocarrier to yield a tert-Ser-PTX polyacetal conjugate. tert-Ser-PTX conjugate provides sustained release of PTX over two 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 vs. PBS (pH 7.4). In vitro efficacy assessments in PCa cells demonstrated IC50 values above those for the free form of PTX due to the differential cell trafficking modes; however, in vivo tolerability assays demonstrated that tert-Ser-PTX significantly reduced the systemic toxicities associated with free PTX treatment. tert-Ser-PTX also effectively inhibited 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, our results suggest the application of tert-Ser-PTX as a robust anti-tumor/antimetastatic treatment for PCa. This article is protected by copyright. All rights reserved.

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.

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.

In the present study the use of extracellular vesicles (EVs) as vehicles for therapeutic enzymes in lysosomal storage disorders was explored. EVs were isolated from mammalian cells overexpressing alpha‐galactosidase A (GLA) or N‐sulfoglucosamine sulfohydrolase (SGSH) enzymes, defective in Fabry and Sanfilippo A diseases, respectively. Direct purification of EVs from cell supernatants was found to be a simple and efficient method to obtain highly active GLA and SGSH proteins, even after EV lyophilization. Likewise, EVs carrying GLA (EV‐GLA) were rapidly uptaken and reached the lysosomes in cellular models of Fabry disease, restoring lysosomal functionality much more efficiently than the recombinant enzyme in clinical use. In vivo, EVs were well tolerated and distributed among all main organs, including the brain. DiR‐labelled EVs were localized in brain parenchyma 1 h after intra‐arterial (internal carotid artery) or intravenous (tail vein) administrations. Moreover, a single intravenous administration of EV‐GLA was able to reduce globotriaosylceramide (Gb3) substrate levels in clinically relevant tissues, such kidneys and brain. Overall, our results demonstrate that EVs from cells overexpressing lysosomal enzymes act as natural protein delivery systems, improving the activity and the efficacy of the recombinant proteins and facilitating their access to organs neglected by conventional enzyme replacement therapies., This study has been supported by ISCIII (PI18_00871 co‐founded by Fondo Europeo de Desarrollo Regional (FEDER)), and CIBER‐BBN (EXPLORE) granted to IA. Different CIBER‐BBN units of ICTS ‘NANBIOSIS’ have participated in this work (https://www.nanbiosis.es/platform-units/), more specifically the U1/Protein Production Platform for protein purification, Unit 6 for NTA analysis and TFF purification and U20/FVPR for in vivo assays.

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-3β-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., Authors would like to thank the Spanish Ministry of Science, Innovation and Universities (MAT2017-84277-R) and CIBER-BBN for the financial support of this project. CIBER-BBN is financed by the Instituto de Salud Carlos III (ISCIII) with assistance from the European Regional Development Fund (ERDF).The work was also partially funded by ISCIII (PI18_00871 co-founded by ERDF), and CIBER-BBN (EXPLORE) granted to I.A. ICTS “NANBIOSIS” has participated in this work, more specifically the U20/FVPR in the hemocompatibility assays (http://www. nanbiosis.es/portfolio/u20-in-vivo-experimental-platform/). E. Espinosa-Cano would like to thank the training program for Academic Staff (FPU15/06109) of the Spanish Ministry of Education Culture and Sport. The kind support by Alvaro González-Gomez Rosana Ramírez from the Biomaterials Group (ICTP-CSIC) and Rafael Nunez ˜ from the Center for Biological Research (CIB-CSIC), in the synthesis, cell culture and cryoTEM experiments, respectively, is greatly appreciated.

Fabry disease is a rare lysosomal storage disorder characterized by a deficiency of α-galactosidase A (GLA), a lysosomal hydrolase. The enzyme replacement therapy administering naked GLA shows several drawbacks including poor biodistribution, limited efficacy, and relatively high immunogenicity in Fabry patients. An attractive strategy to overcome these problems is the use of nanocarriers for encapsulating the enzyme. Nanoliposomes functionalized with RGD peptide have already emerged as a good platform to protect and deliver GLA to endothelial cells. However, low colloidal stability and limited enzyme entrapment efficiency could hinder the further pharmaceutical development and the clinical translation of these nanoformulations. Herein, the incorporation of the cationic miristalkonium chloride (MKC) surfactant to RGD nanovesicles is explored, comparing two different nanosystems—quatsomes and hybrid liposomes. In both systems, the positive surface charge introduced by MKC promotes electrostatic interactions between the enzyme and the nanovesicles, improving the loading capacity and colloidal stability. The presence of high MKC content in quatsomes practically abolishes GLA enzymatic activity, while low concentrations of the surfactant in hybrid liposomes stabilize the enzyme without compromising its activity. Moreover, hybrid liposomes show improved efficacy in cell cultures and a good in vitro/in vivo safety profile, ensuring their future preclinical and clinical development., This work was supported by the financial support from European Commission through the H2020 program (Smart-4-Fabry project, ID 720942). The Networking Research Centre on Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN) is financed by the Instituto de Salud Carlos III (ISCIII) with assistance from the European Regional Development Fund (ERDF). This work was also financed by the Ministerio de Ciencia e Innovación (PID2019-105622RB-I00). The work was also partially funded by ISCIII (PI18_00871 cofounded by ERDF) and CIBER-BBN (EXPLORE) granted to I.A. We also acknowledge the ICTS “NANBIOSIS”, more specifically, the support from the Protein Production Platform of CIBER-BBN/IBB, at the UAB SepBioES scientific-technical service (https://www.nanbiosis.es/portfolio/u1-protein-production-platform-ppp/), the Soft Materials Service linked to Biomaterial Processing and Nanostructuring Unit (U6) at ICMAB-CSIC (www.nanbiosis.es/portfolio/u6-biomaterial-processing-and-nanostructuring-unit/), the Synthesis of Peptide Unit (U3) at the IQAC-CSIC (www.nanbiosis.es/portfolio/u3-synthesis-of-peptides-unit/), and the In Vivo Experimental Platform of the Functional Validation & Preclinical Research (FVPR) area (http://www.nanbiosis.es/portfolio/u20-in-vivo-experimental-platform/). We also thank the denomination of the consolidated group from Generalitat de Catalunya: 2017-SGR-1439 (M.R.) and 2017-SGR-918 (J.V.). J.T.-M. thanks the financial support by the FI-AGAUR grant by the Generalitat de Catalunya, especially the Secretary of Universities and Research of the Department of Business and Knowledge of the Generalitat de Catalunya and the European Social Fund (ESF–Investing in your future) of the European Union. This work has been done in the framework of the J.T.-M. doctorate in Materials Science of the Universitat Autònoma de Barcelona. N.G.-A. is supported by a PERIS grant from the Catalan Government (SLT006/17/270). Authors acknowledge financial support from the Agencia Estatal de Investigación-Ministerio de Ciencia e Innovación through the “Severo Ochoa” Programme for Centres of Excellence in R&D (CEX2019-000917-S).

Diamond nanoparticles (nanodiamonds) can transport active drugs in cultured cells as well as in vivo. However, in the latter case, methods allowing to determine their bioavailability accurately are still lacking. Nanodiamond can be made fluorescent with a perfectly stable emission and a lifetime ten times longer than the one of tissue autofluorescence. Taking advantage of these properties, we present an automated quantification method of fluorescent nanodiamonds (FND) in histological sections of mouse organs and tumor, after systemic injection. We use a home-made time-delayed fluorescence microscope comprising a custom pulsed laser source synchronized on the master clock of a gated intensified array detector. This setup allows to obtain ultra-high-resolution images 120 Mpixels of whole mouse organs sections, with subcellular resolution and single-particle sensitivity. As a proof-of-principle experiment, we quantified the biodistribution and aggregation state of new cationic FNDs able to transport small interfering RNA inhibiting the oncogene responsible for Ewing sarcoma. Image analysis showed a low yield of nanodiamonds in the tumor after intravenous injection. Thus, for the in vivo efficacy assay we injected the nanomedicine into the tumor. We achieved a 28-fold inhibition of the oncogene. This method can readily be applied to other nanoemitters with $\approx$100 ns lifetime., Comment: Nanoscale, Royal Society of Chemistry, 2021

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., The authors acknowledge the financial support from European Commission through H2020 program of the Smart-4-Fabry project (ID 720942). We acknowledge financial support to our research from Instituto de Salud Carlos III, through “Acciones CIBER”. The Networking Research Center on Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN) is 2008–2011, financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. Authors acknowledge financial support from the Agencia Estatal de Investigación-Ministerio de Ciencia e Innovación through the “Severo Ochoa” Programme for Centres of Excellence in R & D (CEX2019-000917-S). This work was also financed by 1084 the Ministerio de Ciencia e Innovación (PID2019-105622RB-1085 I00). We also thank the denomination of the 1101 consolidated group from Generalitat de Catalunya: 2017-1102 SGR-1439 (M.R.) and 2017-SGR-918 (J.V.). We also acknowledge the ICTS “NANBIOSIS”, more specifically the support from the Protein Production Platform of CIBER-BBN/IBB, at the UAB SepBioES scientific-technical service (www.nanbiosis.es/unit/u1-proteinproduction-platform-ppp/), the Soft Materials Service linked to Biomaterial Processing and Nanostructuring Unit at ICMAB-CSIC (www.nanbiosis.es/portfolio/u6-biomaterial-processing-and-nanostructuring-unit/) and the Peptide Synthesis unit at the IQAC-CSIC (www.nanbiosis.es/portfolio/u3-synthesis-of-peptides-unit/). N. A-G. is supported by a PERIS grant from the Catalan Government (SLT006/17/270). J. T-M. is supported by a FI-AGAUR grant from the Catalan Government and the European Social Fund (ESF-Investing in your future) of the European Union. This work has been done in the framework of the JT-M. doctorate in Materials Science of the Universitat Autònoma de Barcelona. The technical assistance of Inbal Ionita, Ella Kesselman and Mingming Zhang from Technion - Israel Institute of Technology; and Ramon González from ICMAB-CSIC is acknowledged.

CD44 is a multifunctional cell surface protein involved in proliferation and differentiation, angiogenesis and signaling. The expression of CD44 is up-regulated in several types of human tumors and particularly in cancer stem cells, representing an appealing target for drug delivery in the treatment of cancer. We have explored here several protein ligands of CD44 for the construction of self-assembling modular proteins designed to bind and internalize target cells. Among five tested ligands, two of them (A5G27 and FNI/II/V) drive the formation of protein-only, ring-shaped nanoparticles of about 14 nm that efficiently bind and penetrate CD44(+) cells by an endosomal route. The potential of these newly designed nanoparticles is evaluated regarding the need of biocompatible nanostructured materials for drug delivery in CD44-linked conditions.

Lysosomal storage disorders (LSDs) are a group of rare diseases in which the defect of a lysosomal protein results in a pathogenic accumulation of nonmetabolized products within the cells. The main treatment for LSDs is enzyme replacement therapy (ERT), consisting in the exogenous administration a recombinant protein to replace the defective one. Although several diseases such as Gaucher, Fabry, and Pompe are treated following this approach, ERT is limited to LSDs without severe neuronal affectation because recombinant enzymes do not cross the blood-brain barrier. Moreover, ERT shows additional drawbacks, including enzyme low half-life, poor bioavailability, and immunogenic responses. In this scenario, nanotechnology-based drug delivery systems (DDS) have been proposed as solution to overcome these limitations and improve the efficacy of ERT. The present review summarizes distinct approaches followed by our group and collaborators on the use of DDS for restoring lysosomal enzymes in disease-affected cells. During the last decade, we have been exploring different synthetic nanoparticles, from electrolytic complexes, to liposomes and aggresomes, for the delivery of α-galactosidase A (GLA) enzyme. Studies were mainly conducted on Fabry disease models, but results can be also extrapolated to other LSDs, as well as to other diseases treated with alternative therapeutic proteins. The advantages and disadvantages of different DDS, the difficulties from working with very labile and highly glycosylated enzymes and the relevance of using appropriate targeting moieties is thoroughly discussed. Finally, the use of natural DDS, namely extracellular vesicles (EVs) is also introduced. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease Therapeutic Approaches and Drug Discovery > Nanomedicine for Cardiovascular Disease Therapeutic Approaches and Drug Discovery > Emerging Technologies.

Within tumors, Cancer Stem Cell (CSC) subpopulation has an important role in maintaining growth and dissemination while preserving high resistance against current treatments. It has been shown that, when CSCs are eliminated, the surrounding Differentiated Cancer Cells (DCCs) may reverse their phenotype and gain CSC-like features to preserve tumor progression and ensure tumor survival. This strongly suggests the existence of paracrine communication within tumor cells. It is evidenced that the molecular crosstalk is at least partly mediated by Extracellular Vesicles (EVs), which are cell-derived membranous nanoparticles that contain and transport complex molecules that can affect and modify the biological behavior of distal cells and their molecular background. This ability of directional transport of small molecules prospects EVs as natural Drug Delivery Systems (DDS). EVs present inherent homing abilities and are less immunogenic than synthetic nanoparticles, in general. Currently, strong efforts are focused into the development and improvement of EV-based DDS. Even though EV-DDS have already reached early phases in clinical trials, their clinical application is still far from commercialization since protocols for EVs loading, modification and isolation need to be standardized for large-scale production. Here, we summarized recent knowledge regarding the use of EVs as natural DDS against CSCs and cancer resistance.

Sistemes d'administració de fàrmacs; Resistència; Tractament del càncer Sistemas de administración de fármacos; Resistencia; Tratamiento del cancer Drug delivery systems; Resistance; Cancer treatment Advanced cancer is still considered an incurable disease because of its metastatic spread to distal organs and progressive gain of chemoresistance. Even though considerable treatment progress and more effective therapies have been achieved over the past years, recurrence in the long-term and undesired side effects are still the main drawbacks of current clinical protocols. Moreover, a majority of chemotherapeutic drugs are highly hydrophobic and need to be diluted in organic solvents, which cause high toxicity, in order to reach effective therapeutic dose. These limitations of conventional cancer therapies prompted the use of nanomedicine, the medical application of nanotechnology, to provide more effective and safer cancer treatment. Potential of nanomedicines to overcome resistance, ameliorate solubility, improve pharmacological profile, and reduce adverse effects of chemotherapeutical drugs is thus highly regarded. Their use in the clinical setting has increased over the last decade. Among the various existing nanosystems, nanoparticles have the ability to transform conventional medicine by reducing the adverse effects and providing a controlled release of therapeutic agents. Also, their small size facilitates the intracellular uptake. Here, we provide a closer review of clinical prospects and mechanisms of action of nanomedicines to overcome drug resistance. The significance of specific targeting towards cancer cells is debated as well.

Tumor recurrence, metastatic spread and progressive gain of chemo-resistance of advanced cancers are sustained by the presence of cancer stem cells (CSCs) within the tumor. Targeted therapies with the aim to eradicate these cells are thus highly regarded. However, often the use of new anti-cancer therapies is hampered by pharmacokinetic demands. Drug delivery through nanoparticles has great potential to increase efficacy and reduce toxicity and adverse effects. However, its production has to be based on intelligent design. Likewise, we developed polymeric nanoparticles loaded with Zileuton™, a potent inhibitor of cancer stem cells (CSCs), which was chosen based on high throughput screening. Its great potential for CSCs treatment was subsequently demonstrated in in vitro and in in vivo CSC fluorescent models. Encapsulated Zileuton™ reduces amount of CSCs within the tumor and effectively blocks the circulating tumor cells (CTCs) in the blood stream and metastatic spread.

Polyurethane-polyurea (PUUa) reactive prepolymers with adjusted hydrophobic and hydrophilic dangling chains to achieve multiwalled sub-30 nm nanoparticles are presented. The combination of an amphiphilic and a hydrophobic prepolymer at the oil-water interface creates a stratified shell by hydrophobic interactions. These novel nanostructures enhance the encapsulation stability of lipophilic compounds compared to monowalled nanostructures and facilitate the selective and ordered functionalization along the multiwalled shell with bioactive motifs. As proof of concept, PUUa nanoparticles have been engineered with disulfide bonds and an α

Structural maintenance of chromosomes protein 2 (SMC2) is a central component of the condensin complex involved in DNA supercoiling, an essential process for embryonic stem cell survival. SMC2 over-expression has been related with tumorigenesis and cancer malignancy and its inhibition is regarded as a potential therapeutic strategy even though no drugs are currently available. Here, we propose to inhibit SMC2 by intracellular delivery of specific antibodies against the SMC2 protein. This strategy aims to reduce cancer malignancy by targeting cancer stem cells (CSC), the tumoral subpopulation responsible of tumor recurrence and metastasis. In order to prevent degradation and improve cellular internalization, anti-SMC2 antibodies (Ab-SMC2) were delivered by polymeric micelles (PM) based on Pluronic&reg, F127 amphiphilic polymers. Importantly, scaffolding the Ab-SMC2 onto nanoparticles allowed its cellular internalization and highly increased its efficacy in terms of cytotoxicity and inhibition of tumorsphere formation in MDA-MB-231 and HCT116 breast and colon cancer cell lines, respectively. Moreover, in the case of the HCT116 cell line G1, cell-cycle arrest was also observed. In contrast, no effects from free Ab-SMC2 were detected in any case. Further, combination therapy of anti-SMC2 micelles with paclitaxel (PTX) and 5-Fluorouracil (5-FU) was also explored. For this, PTX and 5-FU were respectively loaded into an anti-SMC2 decorated PM. The efficacy of both encapsulated drugs was higher than their free forms in both the HCT116 and MDA-MB-231 cell lines. Remarkably, micelles loaded with Ab-SMC2 and PTX showed the highest efficacy in terms of inhibition of tumorsphere formation in HCT116 cells. Accordingly, our data clearly suggest an effective intracellular release of antibodies targeting SMC2 in these cell models and, further, strong cytotoxicity against CSC, alone and in combined treatments with Standard-of-Care drugs.

New polymeric biomaterials and nanomedicines targeting cancer cells are highly required because of their potential clinical applications. Gene therapy enjoys high popularity as advanced therapy due to its high specificity; however, clinical translation is scarce because the lack of efficient and safe delivery methods. Here we address the design and development of a new nanosized targeting system consisting on Cetuximab-conjugated micelles (PM) of Pluronic® F127 (F127) and Gelatin for efficient delivery of small interfering RNA (siRNA) into epidermal growth factor receptor (EGFR) overexpressing breast cancer cells. Chemical modification by carboxylation of F127 is required prior to conjugation with Cetuximab and PM development. PM presenting appropriate physicochemical features (

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.

Cancer stem cells (CSC); Dynamic phenotype; Epithelial-to-mesenchymal transition (EMT) Células madre cancerosas (CSC); Fenotipo dinámico; Transición epitelial a mesenquimal (EMT) Cèl·lules mare canceroses (CSC); Fenotip dinàmic; Transició epitelial a mesenquimal (EMT) Therapeutic resistance seen in aggressive forms of breast cancer remains challenging for current treatments. More than half of the patients suffer from a disease relapse, most of them with distant metastases. Cancer maintenance, resistance to therapy, and metastatic disease seem to be sustained by the presence of cancer stem cells (CSC) within a tumor. The difficulty in targeting this subpopulation derives from their dynamic interconversion process, where CSC can differentiate to non-CSC, which in turn de-differentiate into cells with CSC properties. Using fluorescent CSC models driven by the expression of ALDH1A 1(aldehyde dehydrogenase 1A1), we confirmed this dynamic phenotypic change in MDA-MB-231 breast cancer cells and to identify Serine/Threonine Kinase 2 (AKT2) as an important player in the process. To confirm the central role of AKT2, we silenced AKT2 expression via small interfering RNA and using a chemical inhibitor (CCT128930), in both CSC and non-CSC from different cancer cell lines. Our results revealed that AKT2 inhibition effectively prevents non-CSC reversion through mesenchymal to epithelial transition, reducing invasion and colony formation ability of both, non-CSC and CSC. Further, AKT2 inhibition reduced CSC survival in low attachment conditions. Interestingly, in orthotopic tumor mouse models, high expression levels of AKT2 were detected in circulating tumor cells (CTC). These findings suggest AKT2 as a promising target for future anti-cancer therapies at three important levels: (i) Epithelial-to-mesenchymal transition (EMT) reversion and maintenance of CSC subpopulation in primary tumors, (ii) reduction of CTC and the likelihood of metastatic spread, and (iii) prevention of tumor recurrence through inhibition of CSC tumorigenic and metastatic potential. This work was funded by Fondo de Investigaciones Sanitarias (FIS) from ISCIII, Spanish ministry of Economy and Competitiveness, grant PI17/02242 co-financed by The European Regional Development Fund (FEDER); AC15/00092 grant (Target4Cancer project) from Euro-NanoMed II and PENTRI project, financed by Marato TV3, and EvoNano project, funded by European Union's Horizon 2020 FET Open programme under grant agreement. No. 800983. JSR was supported by a post-doctoral grant from Asociacion Espanola Contra el Cancer (AECC).

Hydrogels (HG) have recognized benefits as drug delivery platforms for biomedical applications. Their high sensitivity to sterilization processes is however one of the greatest challenges regarding their clinical translation. Concerning infection diseases, prevention of post-operatory related infections is crucial to ensure appropriate patient recovery and good clinical outcomes. Silver nanoparticles (AgNPs) have shown good antimicrobial properties but sustained release at the right place is required. Thus, we produced and characterized thermo-sensitive HG based on Pluronic&reg, F127 loaded with AgNPs (HG-AgNPs) and their integrity and functionality after sterilization by dry-heat and autoclave methods were carefully assessed. The quality attributes of HG-AgNPs were seriously affected by dry-heat methods but not by autoclaving methods, which allowed to ensure the required sterility. Also, direct sterilization of the final HG-AgNPs product proved more effective than of the raw material, allowing simpler production procedures in non-sterile conditions. The mechanical properties were assessed in post mortem rat models and the HG-AgNPs were tested for its antimicrobial properties in vitro using extremely drug-resistant (XDR) clinical strains. The produced HG-AgNPs prove to be versatile, easy produced and cost-effective products, with activity against XDR strains and an adequate gelation time and spreadability features and optimal for in situ biomedical applications.

Despite the progress in cancer treatment over the past years advanced cancer is still an incurable disease. Special attention is pointed toward cancer stem cell (CSC)-targeted therapies, because this minor cell population is responsible for the treatment resistance, metastatic growth and tumor recurrence. The recently described CSC dynamic phenotype and interconversion model of cancer growth hamper even more the possible success of current cancer treatments in advanced cancer stages. Accordingly, CSCs can be generated through dedifferentiation processes from non-CSCs, in particular, when CSC populations are depleted after treatment. In this context, the use of targeted CSC nanomedicines should be considered as a promising tool to increase CSC sensitivity and efficacy of specific anti-CSC therapies.

The use of hybrid materials, where a matrix sustains nanoparticles controlling the release of the chemotherapeutic drug, could be beneficial for the treatment of primary tumors prior or after surgery. This localized chemotherapy would guarantee high drug concentrations at the tumor site while precluding systemic drug exposure minimizing undesirable side effects. We combined bacterial cellulose hydrogel (BC) and nanostructured lipid carriers (NLCs) including doxorubicin (Dox) as a drug model. NLCs loaded with cationic Dox (NLCs-H) or neutral Dox (NLCs-N) were fully characterized and their cell internalization and cytotoxic efficacy were evaluated in vitro against MDA-MB-231 cells. Thereafter, a fixed combination of NLCs-H and NLCs-N loaded into BC (BC-NLCs-NH) was assayed in vivo into an orthotopic breast cancer mouse model. NLCs-H showed low encapsulation efficiency (48%) and fast release of the drug while NLCs-N showed higher encapsulation (97%) and sustained drug release. Both NLCs internalized via endocytic pathway, while allowing a sustained release of the Dox, which in turn rendered IC50 values below of those of free Dox. Taking advantage of the differential drug release, a mixture of NLCs-N and NLCs-H was encapsulated into BC matrix (BC-NLCs-NH) and assayed in vivo, showing a significant reduction of tumor growth, metastasis incidence and local drug toxicities., Centro de Investigación y Desarrollo en Fermentaciones Industriales, Centro de Química Inorgánica

The growth and development of some of the male sex accessory organs such as the prostate requires the conversion of testosterone to dihydrotestosterone (DHT) by 5alpha-reductase. To provide insights into the role of testosterone versus DHT in the prostate, we studied the impact of finasteride, a potent and specific inhibitor of 5alpha-reductase, on the expression of prostatic androgen-response genes in testis-intact rats and in 7-day castrated rats. Finasteride inhibition of the conversion of testosterone to DHT was confirmed by measuring serum and intraprostatic androgens. As expected, finasteride treatment caused a reduction in the wet weight of the prostate in the testis-intact rats and inhibited the testosterone-stimulated prostatic regrowth in the 7-day castrated rats. Although finasteride treatment had little or no effect on the expression of the surveyed androgen-response genes in testis-intact rats, its administration enhanced the expression of many androgen-response genes during the testosterone-stimulated regrowth of the regressed prostate in castrated rats. These observations suggest that testosterone is more potent than DHT in stimulating the expression of many androgen-response genes in the regressed prostate. The expression of androgen-response genes is mainly prostate specific and thus is likely to be associated with androgen-dependent prostatic differentiation. Therefore, testosterone is more potent than DHT in inducing differentiation and weaker in stimulating proliferation during prostate regrowth. The fact that testosterone is a strong inducer of prostatic differentiation has potential clinical implications.