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...
| Main Authors: | , |
|---|---|
| Format: | info:eu-repo/semantics/doctoralThesis |
| Language: | eng |
| Published: |
2016
|
| Subjects: | |
| Online Access: | https://hdl.handle.net/10171/40600 |
| _version_ | 1793400480048611328 |
|---|---|
| author | Urbiola-Perez, K. (Koldo) Tros-de-Ilarduya, C. (Conchita) |
| author_facet | Urbiola-Perez, K. (Koldo) Tros-de-Ilarduya, C. (Conchita) |
| author_sort | Urbiola-Perez, K. (Koldo) |
| collection | DSpace |
| description | 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. |
| format | info:eu-repo/semantics/doctoralThesis |
| id | oai:dadun.unav.edu:10171-40600 |
| institution | Universidad de Navarra |
| language | eng |
| publishDate | 2016 |
| record_format | dspace |
| spelling | oai:dadun.unav.edu:10171-406002020-03-03T11:09:03Z Novel targeted polyamidoamine (PAMAM) nanocarriers for gene delivery: design, development and evaluation Urbiola-Perez, K. (Koldo) Tros-de-Ilarduya, C. (Conchita) Células cancerígenas Materias Investigacion::Ciencias de la vida 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. 2016-05-11T10:19:55Z 2016-05-11T10:19:55Z 2014 2014-06-11 info:eu-repo/semantics/doctoralThesis https://hdl.handle.net/10171/40600 eng info:eu-repo/semantics/openAccess application/pdf |
| spellingShingle | Células cancerígenas Materias Investigacion::Ciencias de la vida Urbiola-Perez, K. (Koldo) Tros-de-Ilarduya, C. (Conchita) Novel targeted polyamidoamine (PAMAM) nanocarriers for gene delivery: design, development and evaluation |
| title | Novel targeted polyamidoamine (PAMAM) nanocarriers for gene delivery: design, development and evaluation |
| title_full | Novel targeted polyamidoamine (PAMAM) nanocarriers for gene delivery: design, development and evaluation |
| title_fullStr | Novel targeted polyamidoamine (PAMAM) nanocarriers for gene delivery: design, development and evaluation |
| title_full_unstemmed | Novel targeted polyamidoamine (PAMAM) nanocarriers for gene delivery: design, development and evaluation |
| title_short | Novel targeted polyamidoamine (PAMAM) nanocarriers for gene delivery: design, development and evaluation |
| title_sort | novel targeted polyamidoamine (pamam) nanocarriers for gene delivery: design, development and evaluation |
| topic | Células cancerígenas Materias Investigacion::Ciencias de la vida |
| url | https://hdl.handle.net/10171/40600 |
| work_keys_str_mv | AT urbiolaperezkkoldo noveltargetedpolyamidoaminepamamnanocarriersforgenedeliverydesigndevelopmentandevaluation AT trosdeilarduyacconchita noveltargetedpolyamidoaminepamamnanocarriersforgenedeliverydesigndevelopmentandevaluation |