Tros-de-Ilarduya, C. (Conchita)
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- P-glycoprotein silencing with siRNA delivered by DOPEmodified PEI overcomes doxorubicin resistance in breast cancer cells(Future Medicine, 2012) Navarro, G. (Gemma); Biswas, S. (Swati); Essex, S. (Sean); Tros-de-Ilarduya, C. (Conchita); Torchilin, V.P. (Vladimir P.); Sawant, R.R. (Rupa R.)AIMS: Multidrug resistance (MDR) mediated by overexpression of drug efflux transporters such as P-glycoprotein (P-gp), is a major problem, limiting successful chemotherapy of breast cancer. The use of siRNA to inhibit P-gp expression in MDR tumors is an attractive strategy to improve the effectiveness of anticancer drugs. METHOD: We have synthesized a novel conjugate between a phospholipid (dioleoylphosphatidylethanolamine) and polyethylenimine (PEI) for siRNA delivery, for the purpose of silencing P-gp to overcome doxorubicin resistance in MCF-7 human breast cancer cells. RESULTS: The dioleoylphosphatidylethanolamine-PEI conjugate enhanced the transfection efficacy of low-molecular-weight PEI, which was otherwise totally ineffective. In addition, the polyethylene glycol/lipid coating of the new complexes gave rise to small micelle-like nanoparticles with improved biocompatibility properties. Both coated and noncoated formulations delivered P-gp-specific siRNA to MDR cells. DISCUSSION: The combination of doxorubicin and P-gp silencing formulations led to a twofold increase of doxorubicin uptake and a significant improvement of the therapeutic effect of doxorubicin in resistant cells.
- Multidisciplinary approach to the transfection of plasmid DNA by a nonviral nanocarrier based on a gemini-bolaamphiphilic hybrid lipid(American Chemical Society, 2018) García-Río, L. (Luis); Aicart, E. (Emilio); Martínez-Negro, M. (María); Tros-de-Ilarduya, C. (Conchita); Junquera, E. (Elena); Domenech, O. (Oscar); Guerrero-Martínez, A. (Andrés)A multidisciplinary strategy, including both biochemical and biophysical studies, was proposed here to evaluate the potential of lipid nanoaggregates consisting of a mixture of a gemini−bolaamphiphilic lipid (C6C22C6) and the well-known helper lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) to transfect plasmid DNA into living cells in an efficient and safe way. For that purpose, several experimental techniques were employed, such as zeta potential (phase analysis light scattering methodology), agarose gel electrophoresis (pDNA compaction and pDNA protection assays), small-angle X-ray scattering, cryo-transmission electron microscopy, atomic force microscopy, fluorescence-assisted cell sorting, luminometry, and cytotoxicity assays. The results revealed that the cationic lipid and plasmid offer only 70 and 30% of their nominal positive ( = + +q 2.0 nom,C C C 6 22 6 ) and negative charges ( = − −q 2/bp nom,pDNA ), respectively. Upon mixing with DOPE, they form lipoplexes that self-aggregate in typical multilamellar Lα lyotropic liquid-crystal nanostructures with sizes in the range of 100−200 nm and low polydispersities, very suitably fitted to remain in the bloodstream and cross the cell membrane. Interestingly, these nanoaggregates were able to compact, protect (from the degrading effect of DNase I), and transfect two DNA plasmids (pEGFP-C3, encoding the green fluorescent protein, and pCMV-Luc, encoding luciferase) into COS-7 cells, with an efficiency equal or even superior to that of the universal control Lipo2000*, as long as the effective +/− charge ratio was maintained higher than 1 but reasonably close to electroneutrality. Moreover, this transfection process was not cytotoxic because the viability of COS-7 cells remained at high levels, greater than 80%. All of these features make the C6C22C6/DOPE nanosystem an optimal nonviral gene nanocarrier in vitro and a potentially interesting candidate for future in vivo experiments.
- Trehalose-based siamese twin amphiphiles with tunable self-assembling, DNA nanocomplexing and gene delivery properties(Royal Society of Chemistry, 2019) Tros-de-Ilarduya, C. (Conchita); Ortiz-Mellet, C. (Carmen); Rodríguez-Lavado, J. (Julio); Carbajo-Gordillo, A.I. (Ana I.); García-Fernández, J.M. (José M.); Jiménez-Blanco, J.L. (José Luis); Di-Giorgio, C. (Christophe); Vélaz-Rivas, I. (Icíar); Benito, J.M. (Juan M.)An original family of multivalent vectors encompassing gemini and facial amphiphilicity, namely cationic Siamese twin surfactants, has been prepared fromthe disaccharide trehalose; molecular engineering lets us modulate the self-assembling properties and the topology of the nanocomplexes with plasmid DNA for efficient gene delivery in vitro and in vivo.
- Application of different methods to formulate PEG-liposomes of oxaliplatin: Evaluation in vitro and in vivo(Elsevier, 2012) Tros-de-Ilarduya, C. (Conchita); Troconiz, I.F. (Iñaki F.); Zalba, S. (Sara); Navarro-Blasco, I. (Iñigo); Garrido, M.J. (María Jesús)In this work the film method (FM), reverse-phase evaporation (REV) and the heating method (HM) were applied to prepare PEG-coated liposomes of oxaliplatin with natural neutral and cationic lipids, respectively. The formulations developed with the three methods, showed similar physicochemical characteristics, except in the loading of oxaliplatin, which was statistically lower (P<0.05) using the HM. The incorporation of a semi-synthetic lipid in the formulation developed by FM, provided liposomes with a particle size of 115 nm associated to the lowest polydispersity index and the highest drug loading, 35%, compared to the other two lipids, suggesting an increase of the membrane stability. That stability was also evaluated according to the presence of cholesterol, the impact of the temperature, and the application of different cryoprotectans during the lyophilization. The results indicated long-term stability of the developed formulation, because after its intravenous in-vivo administration to HT-29 tumor bearing mice was able to induce an inhibition of tumor growth statistically higher (P < 0.05) than the inhibition caused by the free drug. In conclusion, the FM was the simplest method in comparison with REV and HM to develop in vivo stable and efficient PEG-coated liposomes of oxaliplatin with a loading higher than those reported for REV.
- A Non-Viral Plasmid DNA Delivery System Consisting on a Lysine-Derived Cationic Lipid Mixed with a Fusogenic Lipid(MDPI AG, 2019) Sánchez-Arribas, N. (Natalia); Aicart, E. (Emilio); Martínez-Negro, M. (María); Tros-de-Ilarduya, C. (Conchita); Junquera, E. (Elena); Mendicuti, F. (Francisco); Guerrero-Martínez, A. (Andrés); Moyá, M.L. (María Luisa)The insertion of biocompatible amino acid moieties in non-viral gene nanocarriers is an attractive approach that has been recently gaining interest. In this work, a cationic lipid, consisting of a lysine-derived moiety linked to a C12 chain (LYCl) was combined with a common fusogenic helper lipid (DOPE) and evaluated as a potential vehicle to transfect two plasmid DNAs (encoding green fluorescent protein GFP and luciferase) into COS-7 cells. A multidisciplinary approach has been followed: (i) biophysical characterization based on zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), and cryo-transmission electronic microscopy (cryo-TEM); (ii) biological studies by fluorescence assisted cell sorting (FACS), luminometry, and cytotoxicity experiments; and (iii) a computational study of the formation of lipid bilayers and their subsequent stabilization with DNA. The results indicate that LYCl/DOPE nanocarriers are capable of compacting the pDNAs and protecting them efficiently against DNase I degradation, by forming Lα lyotropic liquid crystal phases, with an average size of ~200 nm and low polydispersity that facilitate the cellular uptake process. The computational results confirmed that the LYCl/DOPE lipid bilayers are stable and also capable of stabilizing DNA fragments via lipoplex formation, with dimensions consistent with experimental values. The optimum formulations (found at 20% of LYCl content) were able to complete the transfection process efficiently and with high cell viabilities, even improving the outcomes of the positive control Lipo2000*.
- A gemini cationic lipid with histidine residues as a novel lipid-based gene nanocarrier: a biophysical and biochemical study(MDPI AG, 2018) Aicart, E. (Emilio); Martínez-Negro, M. (María); Tros-de-Ilarduya, C. (Conchita); Junquera, E. (Elena); Tentori, P.M. (Paolo M.); Pérez, L. (Lourdes); Pinazo, A. (Aurora); Blanco-Fernández, L. (Laura)This work reports the synthesis of a novel gemini cationic lipid that incorporates two histidine-type head groups (C3(C16His)2). Mixed with a helper lipid 1,2-dioleoyl-sn-glycero3-phosphatidyl ethanol amine (DOPE), it was used to transfect three different types of plasmid DNA: one encoding the green fluorescence protein (pEGFP-C3), one encoding a luciferase (pCMV-Luc), and a therapeutic anti-tumoral agent encoding interleukin-12 (pCMV-IL12). Complementary biophysical experiments (zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), and fluorescence anisotropy) and biological studies (FACS, luminometry, and cytotoxicity) of these C3(C16His)2/DOPE-pDNA lipoplexes provided vast insight into their outcomes as gene carriers. They were found to efficiently compact and protect pDNA against DNase I degradation by forming nanoaggregates of 120–290 nm in size, which were further characterized as very fluidic lamellar structures based in a sandwich-type phase, with alternating layers of mixed lipids and an aqueous monolayer where the pDNA and counterions are located. The optimum formulations of these nanoaggregates were able to transfect the pDNAs into COS-7 and HeLa cells with high cell viability, comparable or superior to that of the standard Lipo2000*. The vast amount of information collected from the in vitro studies points to this histidine-based lipid nanocarrier as a potentially interesting candidate for future in vivo studies investigating specific gene therapies.
- Novel PAMAM-PEG-Peptide Conjugates for siRNA Delivery Targeted to the Transferrin and Epidermal Growth Factor Receptors(MDPI AG, 2018) Tros-de-Ilarduya, C. (Conchita); Rödl, W. (Wolfgang); Wagner, E. (Ernst); Ogris, M. (Manfred); Urbiola-Perez, K. (Koldo); Blanco-Fernández, L. (Laura)The transferrin (TfR) and epidermal growth factor receptors (EGFR) are known to be overexpressed on the surface of a wide variety of tumor cells. Therefore, the peptides B6 (TfR specific) and GE11 (targeted to the EGFR) were linked to the PAMAM (polyamidoamine) structure via a polyethylenglycol (PEG) 2 kDa chain with the aim of improving the silencing capacity of the PAMAM-based dendriplexes. The complexes showed an excellent binding capacity to the siRNA with a maximal condensation at nitrogen/phosphate (N/P) 2. The nanoparticles formed exhibited hydrodynamic diameters below 200 nm. The zeta potential was always positive, despite the complexes containing the PEG chain in the structure showing a drop of the values due to the shielding effect. The gene silencing capacity was assayed in HeLa and LS174T cells stably transfected with the eGFPLuc cassette. The dendriplexes containing a specific anti luciferase siRNA, assayed at different N/P ratios, were able to mediate a mean decrease of the luciferase expression values of 14% for HeLa and 20% in LS174T cells, compared to an unspecific siRNA-control. (p < 0.05). In all the conditions assayed, dendriplexes resulted to be non-toxic and viability was always above 75%.
- Strategies for cancer gene-delivery improvement by non-viral vectors(Elsevier, 2021) Tros-de-Ilarduya, C. (Conchita); Santana-Armas, M.L. (María L.)Lack of selectivity together with severe side effects in conventional cancer treatment have afforded the devel- opment of new strategies based on gene therapy. Nowadays, gene therapy is employed through both viral and non-viral vectors. In spite of the high transfection activity of viral vectors, some drawbacks have pointed out to non-viral vectors as a safer alternative. To overcome low efficiency as well as other issues associated with the use of non-viral vectors, complexes formed by lipids and polymers with DNA, named lipoplexes and polyplexes respectively, have been modified in order to improve its features. Suitability of cancer gene therapy also requires the capacity to distinguish between normal and tumoral cells. This requirement has been solved by the addition of specific ligands that enable receptor binding and subsequent endocytosis. In this article we review the most recent approaches in structure modification of non-viral vectors through different methods comprising conjugation, addition of helper lipids or changes in design and synthesis as well as the strategy based on exploiting receptors that are usually overexpressed in malignancies. Such improvements confer specificity, efficient gene delivery, condensation, protection of DNA and low levels of toxicity avoiding off-target effects which turn into a potential tool to treat cancer.