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Ana Cepero,1– 3,* Mónica Jiménez-Carretero,4,* Ylenia Jabalera,4 Lidia Gago,1– 3 Cristina Luque,1– 3 Laura Cabeza,1– 3 Consolación Melguizo,1– 3 Concepcion Jimenez-Lopez,4 José Prados1– 3 1Institute of Biopathology and Regenerative Medicine (IBIMER), Center of Biomedical Research (CIBM), University of Granada, Granada, 18100, Spain; 2Department of Anatomy and Embryology, Faculty of Medicine, University of Granada, Granada, 18071, Spain; 3Biosanitary Research Institute ibs.GRANADA, Granada, 18012, Spain; 4Department of Microbiology, Sciences School, University of Granada, Granada, 18002, Spain*These authors contributed equally to this workCorrespondence: Laura Cabeza; Concepción Jimenez-Lopez, Email lautea@ugr.es; cjl@ugr.esPurpose: The lack of specificity of conventional chemotherapy is one of the main difficulties to be solved in cancer therapy. Biomimetic magnetoliposomes are successful chemotherapy controlled-release systems, hyperthermia, and active targeting agents by functionalization of their surface with monoclonal antibodies. The membrane receptor Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) stands out as colorectal cancer (CRC) biomarker and appears to be related to treatment resistance and the development of metastasis. The aim of this study was to assess the effectiveness and safety of LGR5-targeted biomimetic magnetoliposomes loaded with oxaliplatin (OXA) or 5-fluorouracil (5-FU) in the selective treatment of CRC and their possible application in hyperthermia.Methods: Synthesis, characterization and determination of heating capacity of magnetoliposomes transporting OXA or 5-FU (with and without LGR5 functionalization) were conducted. In vitro antitumoral activity was assayed in multiple colorectal cell lines at different times of exposition. In addition to this, cell internalization was studied by Prussian Blue staining, flow cytometry and fluorescence microscopy. In vivo acute toxicity of magnetoliposomes was performed to evaluate iron-related toxicity.Results: OXA and 5-FU loaded magnetoliposomes functionalized with LGR5 antibody showed higher cellular uptake than non-targeted nanoformulation with a reduction of the percentage of proliferation in colon cancer cell lines up to 3.2-fold of the IC50 value compared to that of free drug. The differences between non-targeted and targeted nanoformulations were more evident after short exposure times (4 and 8 hours). Interestingly, assays in the MC38 transduced cells with reduced LGR5 expression (MC38-L(-)), showed lower cell internalization of LGR5-targeted magnetoliposomes compared to non-transduced MC38 cell line. In addition, magnetoliposomes showed an in vitro favorable heating response under magnetic excitation and great iron-related biocompatibility data in vivo.Conclusion: Drug-loaded magnetoliposomes functionalized with anti-LGR5 antibodies could be a promising CRC treatment strategy for LGR5+ targeted chemotherapy, magnetic hyperthermia, and both in combination. Keywords: colorectal neoplasms, magnetoliposome, LGR5, oxaliplatin, 5-fluorouracil, magnetic hyperthermia

Francisco Quiñonero,1,2,* Belén Parra-Torrejón,3,* Gloria B Ramírez-Rodríguez,3 Victor Garcés,3 José M Delgado-López,3 Cristina Jiménez-Luna,1,2,4 Gloria Perazzoli,1,2,4 Consolación Melguizo,1,2,4 Jose Prados,1,2,4 Raul Ortíz1,2,4 1Institute of Biopathology and Regenerative Medicine (IBIMER), Center of Biomedical Research (CIBM), University of Granada, Granada, 18100, Spain; 2Instituto de Investigación Biosanitaria de Granada (ibs.GRANADA), Granada, 18014, Spain; 3Department of Inorganic Chemistry, Faculty of Science, University of Granada, Granada, 18071, Spain; 4Department of Anatomy and Embryology, Faculty of Medicine, University of Granada, Granada, 18071, Spain*These authors contributed equally to this workCorrespondence: José M Delgado-López, Department of Inorganic Chemistry, Faculty of Science, University of Granada, Granada, 18071, Spain, Email jmdl@ugr.esIntroduction: Pancreatic cancer (PC) shows a very poor response to current treatments. Development of drug resistance is one of the causes of the therapy failure, being PARP1 (poly ADP-ribose polymerase 1) a relevant protein in the resistance mechanism. In this work, we have functionalized calcium phosphate-based nanoparticles (NPs) with Olaparib (OLA, a PARP-1 inhibitor) in combination with ascorbic acid (AA), a pro-oxidative agent, to enhance their individual effects.Methods: Amorphous Calcium Phosphate (ACP) NPs were synthesized through a biomimetic approach and then functionalized with OLA and AA (NP-ACP-OLA-AA). After evaluation of the loading capacity and release kinetic, cytotoxicity, cell migration, immunofluorescence, and gene expression assays were performed using pancreatic tumor cell lines. In vivo studies were carried out on tumors derived from the PANC-1 line in NOD SCID gamma (NSG) mice.Results: NP-ACP-OLA-AA was loaded with 13%wt of OLA (75% loading efficiency) and 1% of AA, respectively. The resulting dual nanosystem exhibited a gradual release of OLA and AA, being the latter protected from degradation in solution. This ensured the simultaneous availability of OLA and AA for a longer period, at least, during the entire time of in vitro cell experiments (72 hours). In vitro studies indicated that NP-ACP-OLA-AA showed the best cytotoxic effect outperforming that of the free OLA and a higher genotoxicity and apoptosis-mediated cytotoxic effect in human pancreatic ductal adenocarcinoma cell line. Interestingly, the in vivo assays using immunosuppressed mice with PANC-1-induced tumors revealed that NP-ACP-OLA-AA produced a higher tumor volume reduction (59.1%) compared to free OLA (28.3%) and increased the mice survival.Conclusion: Calcium phosphate NPs, a highly biocompatible and biodegradable system, were an ideal vector for the OLA and AA co-treatment in PC, inducing significant therapeutic benefits relative to free OLA, including cytotoxicity, induction of apoptosis, inhibition of cell migration, tumor growth, and survival.Graphical Abstract: Keywords: nanomedicine, calcium phosphate nanocarriers, Olaparib, ascorbic acid, synergistic effect, pancreatic cancer

Ana Cepero,1– 3,* Cristina Luque,1– 3,* Laura Cabeza,1– 3 Gloria Perazzoli,1,2 Francisco Quiñonero,1– 3 Cristina Mesas,1– 3 Consolación Melguizo,1– 3 Jose Prados1– 3 1Institute of Biopathology and Regenerative Medicine (IBIMER), Center of Biomedical Research (CIBM), University of Granada, Granada, 18100, Spain; 2Department of Anatomy and Embryology, Faculty of Medicine, University of Granada, Granada, 18071, Spain; 3Biosanitary Institute of Granada (ibs.GRANADA), SAS-University of Granada, Granada, 18014, Spain*These authors contributed equally to this workCorrespondence: Consolación Melguizo, Institute of Biopathology and Regenerative Medicine (IBIMER), Center of Biomedical Research (CIBM), University of Granada, Granada, 18100, Spain, Tel +34-958-249833, Email melguizo@ugr.esAbstract: The failure of chemotherapeutic treatment in colorectal cancer (CRC), the second most mortal cancer worldwide, is associated with several drug limitations, such as non-selective distribution, short half-life, and development of multiple resistances. One of the most promising strategies in CRC therapy is the development of delivery systems based on nanomaterials that can transport antitumor agents to the tumor site more efficiently, increasing accumulation within the tumor and thus the antitumor effect. In addition to taking advantage of the increased permeability and retention effect (EPR) of solid tumors, these nanoformulations can be conjugated with monoclonal antibodies that recognize molecular markers that are specifically over-expressed on CRC cells. Active targeting of nanoformulations reduces the adverse effects associated with the cytotoxic activity of drugs in healthy tissues, which will be of interest for improving the quality of life of cancer patients in the future. This review focuses on in vitro and in vivo studies of drug delivery nanoformulations functionalized with monoclonal antibodies for targeted therapy of CRC.Keywords: nanoformulation, colon carcinoma, monoclonal antibody, 5-fluorouracil, targeted therapy

Consolación Melguizo1,2,* Laura Cabeza,1,* Jose Prados,1,2 Raúl Ortiz,1,3 Octavio Caba,1,3 Ana R Rama,1,3 Ángel V Delgado,4 José L Arias1,2,5 1Institute of Biopathology and Regenerative Medicine (IBIMER), Biomedical Research Center, 2Biosanitary Institute of Granada (IBS Granada), SAS Universidad de Granada, Granada, 3Department of Health Science, University of Jaén, Jaén, 4Department of Applied Physics, 5Department of Pharmacy and Pharmaceutical Technology, University of Granada, Granada, Spain *These authors contributed equally to this work Abstract: Doxorubicin (Dox) is widely used for the combined chemotherapy of solid tumors. However, the use of these drug associations in lung cancer has low antitumor efficacy. To improve its efficacious delivery and activity in lung adenocarcinoma cells, we developed a biodegradable and noncytotoxic nanoplatform based on biodegradable poly(butylcyanoacrylate) (PBCA). The reproducible formulation method was based on an anionic polymerization process of the PBCA monomer, with the antitumor drug being entrapped within the nanoparticle (NP) matrix during its formation. Improved drug-entrapment efficiencies and sustained (biphasic) drug-release properties were made possible by taking advantage of the synthesis conditions (drug, monomer, and surfactant-agent concentrations). Dox-loaded NPs significantly enhanced cellular uptake of the drug in the A549 and LL/2 lung cancer cell lines, leading to a significant improvement of the drug’s antitumoral activity. In vivo studies demonstrated that Dox-loaded NPs clearly reduced tumor volumes and increased mouse-survival rates compared to the free drug. These results demonstrated that PBCA NPs may be used to optimize the antitumor activity of Dox, thus exhibiting a potential application in chemotherapy against lung adenocarcinoma. Keywords: lung cancer, cancer chemotherapy, PBCA, polymeric nanoparticles, drug carrier

Laura Cabeza,1,* Raúl Ortiz,1,2,* José L Arias,3 Jose Prados,1,4 Maria Adolfina Ruiz Martínez,3 José M Entrena,5,6 Raquel Luque,7 Consolación Melguizo1,4 1Institute of Biopathology and Regenerative Medicine (IBIMER), Granada, Spain; 2Department of Health Science, University of Jaén, Jaén, Spain; 3Department of Pharmacy and Pharmaceutical Technology, University of Granada, Granada, Spain; 4Biosanitary Institute of Granada (ibs GRANADA), SAS-Universidad de Granada, Granada, Spain; 5Institute of Neuroscience, Biomedical Research Center, 6Animal Behavior Research Unit, Scientific Instrumentation Center, University of Granada, Armilla, Granada, Spain; 7Service of Medical Oncology, Virgen de las Nieves Hospital, Granada, Spain *These authors contributed equally to this work Abstract: The use of doxorubicin (DOX), one of the most effective antitumor molecules in the treatment of metastatic breast cancer, is limited by its low tumor selectivity and its severe side effects. Colloidal carriers based on biodegradable poly(butylcyanoacrylate) nanoparticles (PBCA NPs) may enhance DOX antitumor activity against breast cancer cells, thus allowing a reduction of the effective dose required for antitumor activity and consequently the level of associated toxicity. DOX loading onto PBCA NPs was investigated in this work via both drug entrapment and surface adsorption. Cytotoxicity assays with DOX-loaded NPs were performed in vitro using breast tumor cell lines (MCF-7 human and E0771 mouse cancer cells), and in vivo evaluating antitumor activity in immunocompetent C57BL/6 mice. The entrapment method yielded greater drug loading values and a controlled drug release profile. Neither in vitro nor in vivo cytotoxicity was observed for blank NPs. The 50% inhibitory concentration (IC50) of DOX-loaded PBCA NPs was significantly lower for MCF-7 and E0771 cancer cells (4 and 15 times, respectively) compared with free DOX. Furthermore, DOX-loaded PBCA NPs produced a tumor growth inhibition that was 40% greater than that observed with free DOX, thus reducing DOX toxicity during treatment. These results suggest that DOX-loaded PBCA NPs have great potential for improving the efficacy of DOX therapy against advanced breast cancers. Keywords: biodegradable polymer, carcinoma, cytotoxicity, chemotherapeutic drug, drug delivery, nanopolymer

Lucía Martín-Banderas,1 Josefa Álvarez-Fuentes,1 Matilde Durán-Lobato,1 José Prados,2 Consolación Melguizo,2 Mercedes Fernández-Arévalo,1 Mª Ángeles Holgado11Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Seville, Seville, Spain; 2Institute of Biopathology and Regenerative Medicine (IBIMER), School of Medicine, University of Granada, Granada, SpainAbstract: CB13 (1-Naphthalenyl[4-(pentyloxy)-1-naphthalenyl]methanone)-loaded poly(lactic-co-glycolic acid nanoparticles (NPs) were produced by nanoprecipitation and tested for their in vitro release behavior and in vitro cytotoxicity assays. The effects of several formulation parameters such as polymer type, surfactant concentration, and initial drug amount were studied. NPs had a particle size 90–300 nm in diameter. Results obtained show that the main influence on particle size was the type of polymer employed during the particle production: the greater the hydrophobicity, the smaller the particle size. In terms of encapsulation efficiency (%), high values were achieved (~68%–90%) for all formulations prepared due to the poor solubility of CB13 in the external aqueous phase. Moreover, an inverse relationship between release rate and NP size was found. On the other hand, low molecular weight and low lactide content resulted in a less hydrophobic polymer with increased rates of water absorption, hydrolysis, and erosion. NPs showed no cytotoxicity and may be considered to be appropriate for drug-delivery purposes.Keywords: neuropathic pain, CB13, nanoprecipitation

Raúl Ortiz1,3, José Prados1, Consolación Melguizo1, José L Arias2, M Adolfina Ruiz2, Pablo J Álvarez1, Octavio Caba1,3, Raquel Luque4, Ana Segura5, Antonia Aránega11Institute of Biopathology and Regenerative Medicine (IBIMER), 2Department of Pharmacy and Pharmaceutical Technology, University of Granada, Granada, Spain; 3Department of Health Science, University of Jaén, Jaén, Spain; 4Service of Medical Oncology, Virgen de las Nieves Hospital, Granada, Spain; 5CSIC-Estacion Experimental del Zaidin, Department of Environmental Protection, Granada, SpainAbstract: This work aimed to develop a new therapeutic approach to increase the efficacy of 5-fluorouracil (5-FU) in the treatment of advanced or recurrent colon cancer. 5-FU-loaded biodegradable poly(ε-caprolactone) nanoparticles (PCL NPs) were combined with the cytotoxic suicide gene E (combined therapy). The SW480 human cancer cell line was used to assay the combined therapeutic strategy. This cell line was established from a primary adenocarcinoma of the colon and is characterized by an intrinsically high resistance to apoptosis that correlates with its resistance to 5-FU. 5-FU was absorbed into the matrix of the PCL NPs during synthesis using the interfacial polymer disposition method. The antitumor activity of gene E from the phage ΦX174 was tested by generating a stable clone (SW480/12/E). In addition, the localization of E protein and its activity in mitochondria were analyzed. We found that the incorporation of 5-FU into PCL NPs (which show no cytotoxicity alone), significantly improved the drug's anticancer activity, reducing the proliferation rate of colon cancer cells by up to 40-fold when compared with the nonincorporated drug alone. Furthermore, E gene expression sensitized colon cancer cells to the cytotoxic action of the 5-FU-based nanomedicine. Our findings demonstrate that despite the inherent resistance of SW480 to apoptosis, E gene activity is mediated by an apoptotic phenomenon that includes modulation of caspase-9 and caspase-3 expression and intense mitochondrial damage. Finally, a strongly synergistic antiproliferative effect was observed in colon cancer cells when E gene expression was combined with the activity of the 5-FU-loaded PCL NPs, thereby indicating the potential therapeutic value of the combined therapy.Keywords: colon cancer, combined therapy, 5-fluorouracil, gene therapy, E gene, poly (ε-caprolactone)

Objective: Vascular calcification contributes to cardiovascular disease on dialysis patients. Arterial mineral content is modified but not well defined. We aim to define what is the concentration of calcium, magnesium and phosphorus in the epigastric artery of adult dialysis patients undergoing renal transplantation. Methods: All renal allograft recipients who underwent surgery at our centre between May 2003 and December 2005 and consented to be taken small samples of epigastric artery were included in our cross-sectional study. Histological, radiological and spectrometric methods were used to measure vascular calcification, deposits and concentrations of calcium, phosphorus and magnesium in epigastric artery, which were correlated with clinical and biochemical characteristics. Mineral vascular content was compared with corresponding samples from cadaveric renal donors free from renal disease (control group). Results: Calcium and magnesium concentrations in epigastric artery were much higher in recipients (n = 100) than in donors (n = 30). Histologically confirmed calcifications were more frequent in recipients. Calcium and magnesium content in epigastric artery were correlated directly with recipient age, pre-transplant serum P and Ca × P product. A high content of calcium and magnesium in this artery was observed in recipients with media and intimal calcification. Multivariate logistic regression showed that dialysis vintage > 3.5 years and calcium concentration in epigastric artery ≥ 4500 mg/kg wet weight were independent predictors of histological calcification. Conclusion: Excess mineral deposition is observed in the epigastric artery of dialysis patients, where the recipient's age, serum P, Ca × P product and time on dialysis play a decisive role. [ABSTRACT FROM AUTHOR]