Strategies for cancer gene-delivery improvement by non-viral vectors

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

Application of different methods to formulate PEG-liposomes of oxaliplatin: Evaluation in vitro and in vivo

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

P-glycoprotein silencing with siRNA delivered by DOPEmodified PEI overcomes doxorubicin resistance in breast cancer cells

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

A gemini cationic lipid with histidine residues as a novel lipid-based gene nanocarrier: a biophysical and biochemical study

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

A Non-Viral Plasmid DNA Delivery System Consisting on a Lysine-Derived Cationic Lipid Mixed with a Fusogenic Lipid

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*

Trehalose-based siamese twin amphiphiles with tunable self-assembling, DNA nanocomplexing and gene delivery properties

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

Novel targeted polyamidoamine (PAMAM) nanocarriers for gene delivery: design, development and evaluation

In this work, different targeted formulations have been designed and evaluated in order to improve gene delivery to cancer cells by non-viral vectors in vitro and in vivo. All the nanosystems are based on the dendrimeric carrier PAMAM, to which four different ligands (hyaluronic acid, transferrin, B6 and GE11 peptides) have been attached, in order to evaluate the targeting capacity of each nanocarrier. First, a novel PAMAM-hyaluronic acid conjugate has been synthetized by an amine bond formation between PAMAM and oxydized hyaluronic acid. This conjugate was able to form nanoparticles in the presence of pDNA and exhibited excellent capacity to effectively bind pDNA and protect it from enzymatic degradation by nucleases. In vitro evaluation of PAMAM-hyaluronic acid dendriplexes showed an increase in transfection activity in MDA-MB231 and B16F10 cells compared to non-targeted complexes. A competition study with an excess of free HA confirmed the uptake via specific receptor-mediated mechanism. Toxicity studies showed a good cell viability and lower toxicity than the highly used PEI-polyplexes. In vivo results showed an increase in luciferase expression in the liver and heart of Balb-C mice compared to non-targeted complexes. These systems were also able to transfect efficiently B16F10 tumors in C57BL/6 tumor-bearing mice, although no significant differences compared to non-targeted ones were detected. Secondly, PAMAM-Transferrin conjugates were prepared and evaluated. In vitro evaluation of this new PAMAM-Transferrin conjugate demonstrated increased gene delivery to cancer cells (HeLa, HepG2 and CT26) when complexes were formulated at N/P ratio of 6. Toxicity was lower than PEI-polyplexes. The uptake via receptor-mediated endocytosis was ensured by a competition assay. Finally, B6 and GE11 peptides were studied as targeting ligands in these systems. Small interfering RNA (siRNA) was formulated in targeted complexes containing each peptide in the presence of PEG (2 kDa). PAMAM-PEG-B6 and PAMAM-PEG-GE11 dendriplexes formed stable nanoparticles, able to condense siRNA effectively. In vitro evaluation of the gene silencing capacity of siRNA encapsulated into PAMAM-PEG-B6 or PAMAM-PEG-GE11 complexes showed that specific siRNA-Luc was able to reduce luciferase expression in HeLa and LS174 cells, without leading to toxicity effects.En este trabajo se han diseñado y evaluado diferentes formulaciones dirigidas con el objetivo de mejorar la liberación de genes en células cancerígenas in vitro e in vivo. Todos los nanosistemas están basados en el dendrímero catiónico PAMAM, al cual se han añadido cuatro ligandos diferentes: ácido hialurónico (AH), transferrina (Tf) y los péptidos B6 y GE11, para evaluar la capacidad de direccionamiento de cada vector. En primer lugar se sintetizó un nuevo conjugado PAMAM-ácido hialurónico (P-AH) mediante la formación de un enlace amina entre el PAMAM y el ácido hialurónico oxidado. Este conjugado fue capaz de formar partículas en presencia de ADN plasmídico (ADNp), mostró una excelente capacidad para la unión efectiva del ADNp y de protegerlo de la degradación por nucleasas. La evaluación in vitro de los complejos P-AH mostró un incremento de la actividad de transfección en células MDA-MB-231 y B16F10 en comparación con los complejos no dirigidos. Además, mediante un ensayo de competición en presencia de un exceso de AH libre, se confirmó la captación mediante un mecanismo mediado por receptor específico. Los estudios de toxicidad mostraron una buena viabilidad celular y menos toxicidad que los poliplejos de PEI. Los resultados in vivo señalaron un incremento de la expresión de la luciferasa en el hígado y corazón de ratones Balb-C comparados con los complejos no dirigidos. Este sistema fue también capaz de transfectar de forma eficiente tumores B16F10 inducidos en ratones C57BL/6, aunque no se observaron diferencias significativas en comparación con los complejos no dirigidos. En segundo lugar, se prepararon y evaluaron unos complejos formulados con diferentes porcentajes del conjugado PAMAM-Tf. La evaluación in vitro de estas nuevas formulaciones dirigidas, preparadas a N/P ratio 6, mostró un incremento en la transfección en células cancerígenas (HeLa, HepG2 y CT26). La toxicidad fue menor que la de los poliplejos de PEI y la captación por endocitosis mediada por receptor fue comprobada mediante un ensayo de competición. Finalmente, se estudiaron los péptidos B6 y GE11 como ligandos de direccionamiento para la liberación de siRNA en presencia de PEG (2 kDa). Los dendriplejos formulados con los conjugados PAMAM-PEG-B6 y PAMAM-PEG-GE11 formaron nanopartículas estables, capaces de condensar de forma efectiva el siRNA. La evaluación in vitro de la capacidad de silenciamiento génico del siRNA encapsulado dentro de los complejos dirigidos con los péptidos B6 y GE11, mostró que el siRNA-Luc específico era capaz de reducir la expresión de luciferasa en células HeLa y LS174, sin dar lugar a efectos tóxicos

Novel targeted polyamidoamine (PAMAM) nanocarriers for gene delivery: design, development and evaluation

In this work, different targeted formulations have been designed and evaluated in order to improve gene delivery to cancer cells by non-viral vectors in vitro and in vivo. All the nanosystems are based on the dendrimeric carrier PAMAM, to which four different ligands (hyaluronic acid, transferrin, B6 and GE11 peptides) have been attached, in order to evaluate the targeting capacity of each nanocarrier. First, a novel PAMAM-hyaluronic acid conjugate has been synthetized by an amine bond formation between PAMAM and oxydized hyaluronic acid. This conjugate was able to form nanoparticles in the presence of pDNA and exhibited excellent capacity to effectively bind pDNA and protect it from enzymatic degradation by nucleases. In vitro evaluation of PAMAM-hyaluronic acid dendriplexes showed an increase in transfection activity in MDA-MB231 and B16F10 cells compared to non-targeted complexes. A competition study with an excess of free HA confirmed the uptake via specific receptor-mediated mechanism. Toxicity studies showed a good cell viability and lower toxicity than the highly used PEI-polyplexes. In vivo results showed an increase in luciferase expression in the liver and heart of Balb-C mice compared to non-targeted complexes. These systems were also able to transfect efficiently B16F10 tumors in C57BL/6 tumor-bearing mice, although no significant differences compared to non-targeted ones were detected. Secondly, PAMAM-Transferrin conjugates were prepared and evaluated. In vitro evaluation of this new PAMAM-Transferrin conjugate demonstrated increased gene delivery to cancer cells (HeLa, HepG2 and CT26) when complexes were formulated at N/P ratio of 6. Toxicity was lower than PEI-polyplexes. The uptake via receptor-mediated endocytosis was ensured by a competition assay. Finally, B6 and GE11 peptides were studied as targeting ligands in these systems. Small interfering RNA (siRNA) was formulated in targeted complexes containing each peptide in the presence of PEG (2 kDa). PAMAM-PEG-B6 and PAMAM-PEG-GE11 dendriplexes formed stable nanoparticles, able to condense siRNA effectively. In vitro evaluation of the gene silencing capacity of siRNA encapsulated into PAMAM-PEG-B6 or PAMAM-PEG-GE11 complexes showed that specific siRNA-Luc was able to reduce luciferase expression in HeLa and LS174 cells, without leading to toxicity effects.En este trabajo se han diseñado y evaluado diferentes formulaciones dirigidas con el objetivo de mejorar la liberación de genes en células cancerígenas in vitro e in vivo. Todos los nanosistemas están basados en el dendrímero catiónico PAMAM, al cual se han añadido cuatro ligandos diferentes: ácido hialurónico (AH), transferrina (Tf) y los péptidos B6 y GE11, para evaluar la capacidad de direccionamiento de cada vector. En primer lugar se sintetizó un nuevo conjugado PAMAM-ácido hialurónico (P-AH) mediante la formación de un enlace amina entre el PAMAM y el ácido hialurónico oxidado. Este conjugado fue capaz de formar partículas en presencia de ADN plasmídico (ADNp), mostró una excelente capacidad para la unión efectiva del ADNp y de protegerlo de la degradación por nucleasas. La evaluación in vitro de los complejos P-AH mostró un incremento de la actividad de transfección en células MDA-MB-231 y B16F10 en comparación con los complejos no dirigidos. Además, mediante un ensayo de competición en presencia de un exceso de AH libre, se confirmó la captación mediante un mecanismo mediado por receptor específico. Los estudios de toxicidad mostraron una buena viabilidad celular y menos toxicidad que los poliplejos de PEI. Los resultados in vivo señalaron un incremento de la expresión de la luciferasa en el hígado y corazón de ratones Balb-C comparados con los complejos no dirigidos. Este sistema fue también capaz de transfectar de forma eficiente tumores B16F10 inducidos en ratones C57BL/6, aunque no se observaron diferencias significativas en comparación con los complejos no dirigidos. En segundo lugar, se prepararon y evaluaron unos complejos formulados con diferentes porcentajes del conjugado PAMAM-Tf. La evaluación in vitro de estas nuevas formulaciones dirigidas, preparadas a N/P ratio 6, mostró un incremento en la transfección en células cancerígenas (HeLa, HepG2 y CT26). La toxicidad fue menor que la de los poliplejos de PEI y la captación por endocitosis mediada por receptor fue comprobada mediante un ensayo de competición. Finalmente, se estudiaron los péptidos B6 y GE11 como ligandos de direccionamiento para la liberación de siRNA en presencia de PEG (2 kDa). Los dendriplejos formulados con los conjugados PAMAM-PEG-B6 y PAMAM-PEG-GE11 formaron nanopartículas estables, capaces de condensar de forma efectiva el siRNA. La evaluación in vitro de la capacidad de silenciamiento génico del siRNA encapsulado dentro de los complejos dirigidos con los péptidos B6 y GE11, mostró que el siRNA-Luc específico era capaz de reducir la expresión de luciferasa en células HeLa y LS174, sin dar lugar a efectos tóxicos

Strategies for cancer gene-delivery improvement by non-viral vectors

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