Targeted gene delivery by new folate-polycationic amphiphilic cyclodextrin-DNA nanocomplexes in vitro and in vivo

Aim Development and evaluation of a new targeted gene delivery system by first preforming self-assembled nanocomplexes from a polycationic amphiphilic cyclodextrin (paCD) and pDNA and then decorating the surface of the nanoparticles with folic acid (FA). Experimental section The cyclodextrin derivative (T2) is a tetradecacationic structure incorporating 14 primary amino groups and 7 thioureido groups at the primary face of a cyclomaltoheptaose (β-CD) core and 14 hexanoyl chains at the secondary face. Results and conclusions T2 complexed and protected pDNA (luciferase-encoding plasmid DNA, pCMVLuc) and efficiently mediated transfection in vitro and in vivo with no associated toxicity. The combination of folic acid with CDplexes afforded ternary nanocomplexes (Fol-CDplexes) that enhanced significantly the transfection activity of pCMVLuc in human cervix adenocarcinoma HeLa cells, especially when formulated with 1 μg FA/μg DNA. The observed transfection enhancement was associated to specific folate receptor (FR)-mediated internalization of Fol-CDplexes, as corroborated by employing a receptor-deficient cell line (HepG2) and an excess of free folic acid. The in vivo studies, including luciferase reporter gene expression and biodistribution, indicated that 24 h after intravenous administration of the T2-pDNA nanocomplexes, transfection takes part mainly in the liver and partially in the lung. Interestingly, the corresponding Fol-CDplexes lead to an increase in the transfection activity in the lung and the liver compared to non-targeted CDplexes. Folate-CDplexes developed in this study have improved transfection efficiency and although various methods have been used for the preparation of ligand–DNA-complexes, covalent binding is usually needed and insoluble aggregates are formed unless the concentration of the components is minimized. However, the complexes developed by first time in this work were prepared by simple mixing. The synthetic nature of this formulation provides the potential of flexibility in terms of composition and the capability of inexpensive and large-scale production of the complexes. These nanovectors may be an adequate alternative to viral vectors for gene therapy in the future.Ministerio de Economía y Competitividad, CTQ2010 - 15848, SAF2010 - 1567

Novel PAMAM-PEG-Peptide Conjugates for siRNA Delivery Targeted to the Transferrin and Epidermal Growth Factor Receptors

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%

Novel PAMAM-PEG-Peptide Conjugates for siRNA Delivery Targeted to the Transferrin and Epidermal Growth Factor Receptors

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 &lt; 0.05). In all the conditions assayed, dendriplexes resulted to be non-toxic and viability was always above 75%

Estudio de la violencia en la pareja de jóvenes estudiantes de ESPA y propuestas educativas para su prevención

Young partner violence arises as a social problem which can be eradicated by applying preventive actions from the educative system. Therefore, the objective of this work is to evaluate that violence in Adult Secondary Education with the aim of designing a preventive proposal for that kind of student. The sample was composed by 26 students of Adult Secondary Education of both sexes who, voluntarily, answer VERA questionnaire. This is a validated tool which fits the features of the sample and the objectives of this work. The results showed that the students present similar characteristics compared to the same age population in terms of partner violence: bidirectionality, normalization and difficult for the perception in a relationship. Nevertheless, some of the results reveal differences and suggest that the cited features more frequently appear in this group than in the wider population of teenagers and young population studied previously by other authors. The information obtained, stablish the base for proposing initiatives that can be carried out by the faculty or the tutor in order to prevent partner violence in this educational level. The suggested activities are based on the pedagogic criteria of this level and the specific features of the student, considering the information obtained in the previous study of the sample in that designing.La violencia en la pareja joven es una realidad en la sociedad que se intenta erradicar desde el sistema educativo con medidas preventivas. Este trabajo tiene como objetivo conocer y analizar esa violencia en la educación secundaria para adultos, a fin de fundamentar una propuesta preventiva dirigida a ellos. Como pasos previos al estudio, se esbozan las características educativas de la educación de adultos y se enmarca teóricamente este tipo de violencia. Para el estudio se ha contado con una muestra de 26 alumnos de dicho nivel educativo de ambos sexos a los que se les ha aplicado el cuestionario VERA (Violencia ejercida, recibida y percibida por adolescentes y jóvenes). Éste es un instrumento psicométricamente validado e idóneo para esta muestra y la investigación. En los resultados se observa un colectivo que presenta características similares al resto de población de su edad en lo que se refiere a la violencia en la pareja (bidireccionalidad de la misma, normalización y dificultad para percibirla en su relación). Sin embargo, también se aprecian resultados que diferencian al grupo e indican que las citadas características se dan con más frecuencia que la recogida por estudios dirigidos a poblaciones más amplias de jóvenes y adolescentes. La anterior información sirve para proponer iniciativas que podrían ser llevadas a cabo por el tutor para prevenir la violencia en esta etapa educativa. Estas actividades propuestas parten de los criterios pedagógicos de este nivel, se adecuan a la edad y experiencia vital de los alumnos y se centran en las carencias principales que ha detectado el estudio anterior

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

Polycationic amphiphilic cyclodextrin-based nanoparticles for therapeutic gene delivery

In this study, a set of polycationic amphiphilic cyclodextrins featuring self-assembling capabilities in the presence of nucleic acids have been evaluated as therapeutic gene vectors for in vivo purposesPeer Reviewe

Novel PAMAM-PEG-Peptide Conjugates for siRNA Delivery Targeted to the Transferrin and Epidermal Growth Factor Receptors

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%

Synthesis of Cationic Carbosilane Dendrimers via Click Chemistry and Their Use as Effective Carriers for DNA Transfection into Cancerous Cells

New amine-terminated carbosilane dendrimers have been prepared by a Huisgen cycloaddition (“click chemistry” reaction) of azide-terminated carbosilane dendrimers with two different propargyl amines. The corresponding cationic derivatives with peripheral ammonium groups were obtained by subsequent addition of MeI. Quaternized dendrimers are soluble and stable in water or other protic solvents for long time periods, and have been studied as nonviral vectors for the transfection of DNA to cancer cells. In this study DNA-dendrimeric nanoparticles (dendriplexes) formulated with two different families of cationic carbosilane dendrimers (family 1 (G1, G2 and G3) and family 2 (G1, G2)) were characterized and evaluated for their ability to transfect cells <i>in vitro</i> and <i>in vivo</i>. Dendriplex derived from second generation dendrimer of family 1 (F1G2 5/1 (+/−)) increased the efficiency of plasmid-mediated gene transfer in HepG2 cells as compared to naked DNA and the commercial control dendrimer. Also, intravenously administered dendriplex F1G3 20/1 (+/−) is superior in terms of gene transfer efficiency <i>in vivo</i>