Departamento de Farmacia y Tecnología Farmacéutica

Facultad de Farmacia

UNIVERSIDAD DE NAVARRA

TESIS DOCTORAL

Novel targeted polyamidoamine (PAMAM)

nanocarriers for gene delivery:

design, development and evaluation

Koldo Urbiola Pérez

Pamplona, 2014

Departamento de Farmacia y Tecnología Farmacéutica

Facultad de Farmacia

UNIVERSIDAD DE NAVARRA

TESIS DOCTORAL

Novel targeted polyamidoamine (PAMAM)

nanocarriers for gene delivery:

design, development and evaluation

Trabajo presentado por D. Koldo Urbiola Pérez para la obtención del grado

de Doctor en Farmacia

Fdo. Koldo Urbiola Pérez

Pamplona, 2014

DÑA. Mª CONCEPCIÓN TROS DE ILARDUYA APAOLAZA, Profesora

Titular de Farmacia y Tecnología Farmacéutica de la Universidad de

Navarra,

CERTIFICA:

Que el presente trabajo titulado “Novel targeted

polyamidoamine (PAMAM) nanocarriers for gene delivery:

design, development and evaluation” realizado por D. Koldo

Urbiola Pérez, para optar al grado de Doctor en Farmacia, ha sido

llevado a cabo en el Departamento de Farmacia y Tecnología

Farmacéutica de la Universidad de Navarra bajo su dirección y que,

una vez revisado, no encuentra objeciones para que sea presentado a

su lectura y defensa.

Y para que así conste, firma el presente certificado.

Fdo. Dra. Mª Concepción Tros de Ilarduya Apaolaza

Pamplona, 2014

Las investigaciones realizadas en el presente

trabajo se han financiado gracias a los proyectos de

“Aplicación de la nanotecnología al diseño y desarrollo

de nuevas formulaciones farmacéuticas para el

tratamiento del cáncer” del Departamento de Desarrollo

Rural, Industria, Empleo y Medio Ambiente del

Gobierno de Navarra y a la Línea Especial

“Nanotecnologías y liberación controlada de fármacos”

de la Universidad de Navarra.

Este trabajo ha sido llevado a cabo gracias a la

beca predoctoral concedida a D. Koldo Urbiola Pérez por

la Asociación de Amigos de la Universidad de Navarra.

Todo lo que una persona puede imaginar,

otras pueden hacerlo realidad

Julio Verne

AGRADECIMIENTOS

Puede resultar sorprendente la cantidad de personas que ayudan a

que una Tesis Doctoral llegue a buen puerto. El apoyo de todas ellas hace

que el camino sea más llevadero y el resultado más fructífero.

Probablemente, muchas de ellas no valoren lo que han aportado por

considerarlo algo natural. No obstante, quiero agradecer toda la

colaboración profesional y todas las muestras de afecto o interés recibidas a

lo largo de estos años.

En primer lugar, quiero expresar mi agradecimiento a mis padres,

Lidón y Pancho. Al fin y al cabo, son los que han estado ahí desde el

comienzo, ayudando en las decisiones y animándome a acometer las

empresas más difíciles. Muchas gracias por el cariño y apoyo incondicional.

En segundo lugar, por supuesto, a mi directora, la Dra. Conchita

Tros de Ilarduya, por haber aceptado discutir muchas de las cosas que

están incluidas en esta Tesis y dirigirme siempre con una ilusión de la que

me he contagiado. Igualmente, por esas palabras de ánimo y confianza,

pero, sobre todo, por haber sido una directora que ha puesto siempre por

delante la persona y la relación humana, especialmente durante los

períodos más difíciles.

También quiero transmitir mi más sincero agradecimiento a Dña.

Carmen Sanmartín, del Dpto. de Química Orgánica y Farmacéutica, cuya

ayuda en la fase de síntesis química ha sido imprescindible para que esta

Tesis haya podido llevarse a cabo.

A todos los profesores y personal del Departamento de Farmacia y

Tecnología Farmacéutica, Dña. María Jesús Renedo, Dña. Carmen Dios,

Dña. Maribel Calvo, D. Iñaki Fernández de Trocóniz, Dña. María Jesús

Garrido, Dña. María Blanco, Dña. Socorro Espuelas, D. Juan Manuel

Irache, D. Fernando Martínez, D. Félix Recarte, Dña. María Huici, Dña.

Mª Mar Goñi y D. Juan Luis Elizondo por su cercanía e interés a lo largo

de estos años.

A todas las personas que han formado parte del grupo de

investigación durante estos años: a Gemma Navarro, por establecer el

punto de partida de mi tesis, sin sus mails no hubiera podido resolver

algunas de mis dudas. A Cristina Aranda, por dejarme aprender con ella

durante mis primeros días en el laboratorio. A todos los alumnos internos y

de máster con los que he tenido la oportunidad de trabajar en el laboratorio:

Carlos, Natalia, Mercedes y Ainhoa. Y, por último, a Laura Blanco, por

todas esas horas en la sala de células y el animalario, pelando y pinchando

ratones, incluso cuando ya no había luz o era domingo, o ambas cosas,

aunque los resultados no fueran tan prometedores como queríamos. Su

disposición y ayuda durante este tiempo ha hecho que esta tesis sea lo que

es haciéndola posible.

A Sheyla, Fabio, Ander, Edurne, Elisa, Maite, Irene, Eneko,

Nekane, Patricia C., Patricia O., Judit, Luisa, Rebeca, Zinnia, Teresa,

Beatriz, Melissa, Nuria, Paula, Ana Margarita, Laura I., Noelia, Simón,

Yolanda, Ana G., Ana B., Inés… y todos los compañeros que han pasado

por el Departamento en algún momento. A Hugo, nuestro técnico, que con

tanta paciencia y tan bien nos soluciona cualquier inconveniente en el

laboratorio. Gracias por alegrar los días en la Universidad, creando durante

estos años un ambiente de compañerismo y de trabajo agradable y

motivador.

Lots of thanks to Prof. Dr. Ernst Wagner and Dr. Manfred Ogris for

their supervision during my stay at the Department of Pharmaceutical

Biotechnology at the Ludwig-Maximilians University and for their

understanding and patience. Many thanks to Joana Viola, Alexandra Vetter

and Elisabeth Calewaert for all the nice talks at lunch, while eating

Schnitzels. Furthermore, I would like to thank the rest of the members of the

lab for the support and the pleasant atmosphere. También, a los que

hicieron mi estancia en Múnich mucho más llevadera, acogiéndome como

uno más de la familia: Iñaki, Cristina, Leire, Unai y Julen, muchas gracias

de todo corazón.

Mi agradecimiento a todas las personas con las que he tenido la

suerte de compartir vivencias a lo largo de estos años. A Javier Cantero y

Manuel A. Escalada, el año del máster no hubiera sido el mismo. A César y

Jose, convivir en el piso fue una experiencia inigualable. Gracias por tener

siempre un rato para encontrarnos cuando pasamos por Madrid. A las

chicas de Farmacocinética que ya terminaron sus tesis (las “secretarias”): a

Elba, Arianna, María y Orlando por la jovialidad, esa tortillera que tan

buen servicio nos da aún, por enseñarme a hablar mexicano y a hacer

shushi y, sobre todo a Arianna, por la visita más inesperada durante mi

estancia. A Sara, por estar siempre dispuesta a ayudar, resolver las dudas

que han ido surgiendo, incluso cuando estabas de estancia. Gracias a todos

por lo que hemos compartido.

Muchas gracias a Cristina T. Tabar por ser algo más que una

compañera de laboratorio. Porque formar parte de los Hímsters es algo más

que trabajar juntos, también son todos esos mails y conversaciones por los

pasillos para quejarnos, consolarnos y darnos fuerza en los momentos duros

que hemos pasado. Los Jueves de Terapia han sido realmente

imprescindibles todo este último año.

A mi cuadrilla: María, Chantal, Adrián, Marta, Nadia y Lara.

Gracias por conseguir que todos los fines de semana que logramos vernos

sigamos haciendo esas pequeñas cosas que nos encantan y podamos

continuar disfrutando de las que nos unen desde hace tantos años.

Finalmente, muchas gracias a mi familia, aparte de mis padres, a mi

hermana Irache, Iker, mi sobrina Olarizu y Lara. Todos vosotros me habéis

apoyado y animado desde antes de comenzar esta tesis. Gracias por la

disposición a escuchar, incluso cuando no entendíais parte del trabajo, y por

vuestro apoyo incondicional a lo largo de los años. Muchas gracias, Lara,

por estar a mi lado y por hacer los días más felices en Pamplona y durante

la estancia, por hacer de “revisora externa” de este trabajo y discutir la

infinidad de ideas que me iban surgiendo y, sobre todo, por tu cariño,

paciencia y comprensión.

A todos, de verdad, muchas gracias.

I

ÍNDICE

ÍNDICE .................................................................................................... I

ABREVIATURAS .................................................................................. VII

RESUMEN / SUMMARY ........................................................................ 1

INTRODUCCIÓN .................................................................................... 7

1. CONCEPTO DE TERAPIA GÉNICA ......................................................... 9

2. SISTEMAS DE TRANSFERENCIA DE GENES .................................... 11

2.1. Sistemas virales ............................................................................................ 12

2.2. Sistemas no virales ...................................................................................... 14

2.2.1. Sistemas lipídicos ................................................................................. 14

2.2.2. Sistemas químicos y poliméricos: polietilenimina ........................... 15

2.2.3. Dendrímeros catiónicos: derivados de la poliamidoamina

(PAMAM) ....................................................................................................... 17

3. OPTIMIZACIÓN DE LOS VECTORES ................................................... 22

3.1. Principales obstáculos tras la administración sistémica ........................ 23

3.1.1. Distribución en el organismo ............................................................ 23

3.1.2. Entrada y transporte intracelular ....................................................... 25

3.2. Principales estrategias de optimización ................................................... 27

3.2.1. Enmascaramiento de la carga superficial ......................................... 27

3.2.1. Direccionamiento o targeting ............................................................... 28

4. TERAPIA GÉNICA Y CÁNCER ................................................................. 32

4.1. Corrección génica ....................................................................................... 33

II

4.2. Terapia génica suicida ................................................................................ 33

4.3. Inmunoterapia génica ................................................................................ 34

4.4. Bloqueo de la expresión génica: small interfering RNA (siRNA) ........... 35

5. BIBLIOGRAFÍA ............................................................................... 36

HIPÓTESIS Y OBJETIVOS .................................................................. 45

CHAPTER 1: Synthesis and characterization of a novel PAMAM-

hyaluronic acid conjugate for gene delivery ........................................... 49

ABSTRACT ........................................................................................................... 51

1. INTRODUCTION .......................................................................................... 53

2. MATERIALS AND METHODS .................................................................. 57

2.1. Materials ...................................................................................................... 57

2.2. Synthesis, purification and characterization of the PAMAM-

Hyaluronic Acid conjugate (P-HA) ................................................................ 57

2.3. Preparation of HA/PAMAM/DNA dendriplexes ............................... 58

2.4. Particle size and surface charge determinations .................................... 59

2.5. Gel retention studies .................................................................................. 59

2.6. DNase I protection assay .......................................................................... 59

3. RESULTS ........................................................................................................... 60

3.1. Synthesis and characterization of the PAMAM-HA conjugate .......... 60

3.1.1. 1H-NMR characterization .................................................................. 61

3.1.2. Elemental analysis ............................................................................... 65

3.2. Formation of HA targeted PAMAM dendriplexes ............................... 66

3.3. Particle size and zeta potential of HA containing dendriplexes .......... 66

3.4. Stability of dendriplexes against DNase I digestion .............................. 68

4. DISCUSSION ................................................................................................... 69

III

5. CONCLUSIONS............................................................................................... 73

6. REFERENCES ................................................................................................. 73

CHAPTER 2: In vitro and in vivo evaluation of a new PAMAM-

hyaluronic acid conjugate for gene delivery ............................................ 79

ABSTRACT ............................................................................................................ 81

1. INTRODUCTION ........................................................................................... 83

2. MATERIALS AND METHODS ................................................................... 85

2.1. Materials ....................................................................................................... 85

2.2. Cell culture ................................................................................................... 86

2.3. Synthesis, purification and characterization of PAMAM-Hyaluronic

Acid conjugate (P-HA) ...................................................................................... 86

2.4. Preparation of HA/PAMAM/DNA dendriplexes ................................ 87

2.5. CD44 expression ........................................................................................ 88

2.6. In vitro transfection activity ........................................................................ 88

2.7. Toxicity studies ........................................................................................... 89

2.8. In vivo biodistribution studies and intratumoral assays .......................... 89

2.9. Statistical analysis ........................................................................................ 90

3. RESULTS ........................................................................................................... 91

3.1. CD44 receptor expression ......................................................................... 91

3.2. In vitro transfection activity in B16F10 and MDA-MB-231 cells ......... 92

3.3. Specificity of targeting to the HA receptor ............................................. 93

3.4. Cytotoxicity of HA targeted dendriplexes .............................................. 95

3.5. In vivo studies ............................................................................................... 97

3.5.1. Biodistribution profile in healthy mice ............................................. 97

3.5.2. Transfection activity in tumor-bearing mice ................................... 98

4. DISCUSSION .................................................................................................... 98

IV

5. CONCLUSIONS ............................................................................................ 102

6. REFERENCES ................................................................................................103

CHAPTER 3: Evaluation of improved PAMAM-G5 based dendriplexes

targeted to the transferrin receptor......................................................... 107

ABSTRACT ......................................................................................................... 109

1. INTRODUCTION ........................................................................................ 111

2. MATERIALS AND METHODS ................................................................ 113

2.1. Materials .................................................................................................... 113

2.2. Synthesis of PAMAM-Transferrin conjugate ..................................... 114

2.3. Preparation of complexes ....................................................................... 115

2.4. Gel retention studies ................................................................................ 116

2.5. DNAse I protection assay....................................................................... 116

2.6. Size and zeta potential determination ................................................... 117

2.7. Cell culture ................................................................................................ 117

2.8. In vitro gene transfer ................................................................................. 117

2.9. Toxicity studies ......................................................................................... 118

2.10. Statistical analysis ................................................................................... 119

3. RESULTS ......................................................................................................... 119

3.1. Binding of pDNA in the complexes ..................................................... 119

3.2. Plasmid protection inside the dendriplexes .......................................... 120

3.3. Characterization of particle size and zeta potential ............................. 120

3.4. In vitro transfection activity ..................................................................... 122

3.5. Toxicity studies ......................................................................................... 124

3.6. Uptake mechanism................................................................................... 125

4. DISCUSSION ................................................................................................. 126

5. CONCLUSIONS ............................................................................................ 130

V

6. REFERENCES ............................................................................................... 131

CHAPTER 4: Novel PAMAM-PEG-Peptide conjugates for siRNA

delivery targeted to the transferrin and epidermal growth factor

receptors ................................................................................................. 137

ABSTRACT .......................................................................................................... 139

1. INTRODUCTION ......................................................................................... 141

2. MATERIALS AND METHODS ................................................................. 143

2.1. Materials ..................................................................................................... 143

2.2. Conjugate synthesis .................................................................................. 144

2.2.1. Synthesis of PAMAM-PEG-OPSS ................................................. 144

2.2.2. Conjugation of GE11 and B6 to PAMAM-PEG-OPSS ............. 145

2.2.3. Synthesis of PAMAM-PEG-Cys ..................................................... 145

2.3. Preparation of PAMAM-PEG-Peptide/siRNA and LPEI/siRNA

complexes .......................................................................................................... 146

2.4. Particle size and zeta potential measurements ..................................... 146

2.5. Ethidium bromide exclusion assay ......................................................... 146

2.6. Cell culture ................................................................................................. 147

2.7. Gene silencing capacity ............................................................................ 147

2.8. Toxicity studies ......................................................................................... 148

2.9. Statistical analysis ...................................................................................... 149

3. RESULTS ......................................................................................................... 149

3.1. Size and zeta potential determination .................................................... 149

3.2. Ethidium bromide exclusion assay ......................................................... 150

3.3. Gene silencing efficacy ............................................................................ 151

3.4. Toxicity studies ......................................................................................... 153

4. DISCUSSION .................................................................................................. 155

VI

5. CONCLUSIONS ............................................................................................ 159

6. REFERENCES ............................................................................................... 160

GENERAL DISCUSSION .................................................................... 165

CONCLUSIONES / CONCLUSIONS ................................................ 183

VII

ABREVIATURAS

1H-NMR Resonancia magnética nuclear de protón

ADA Adenosín desaminasa

ADNp / pDNA ADN plasmídico

AH / HA Ácido hialurónico

ARN/RNA Ácido ribonucleico

ARNm / mRNA ARN mensajero

bPEI Polietilenimina ramificada

CMV Citomegalovirus

DAB Butilendiamina

DLS Dispersión dinámica de la luz (Dinamic light scattering)

DMEM Medio de cultivo Dulbecco's Modified Eagle Medium

DNAse Desoxiribonucleasa

DOPE dioleoilfosfatidiletanolamina

DOTAP 1,2-dioleoiloxi-3-(trimetilamonio)propano

DOTMA N-[1-(2,3-dioleoiloxipropil)-N,N,N-trimetilamonio]

DTT Ditiotreitol

EDA Etilendiamina

EDC 1-etil-3-(3-dimetilaminopropil)carbodiimida

EDTA Ácido etilendiaminotetraacético

EF1α Factor de elongación 1α

EGF Factor de crecimiento epidermal

eGFP Proteína verde fluorescente mejorada

EGFR Receptor del factor de crecimiento epidérmico

EPR Efecto de permeabilidad y retención acentuadas

EtBr Bromuro de etidio

FBS Suero fetal bovino

FCS Suero fetal de ternera

VIII

HBG Buffer HEPES glucosado

HBS Buffer HEPES salino

HEPES Ácido N-(2-hidroxietil)piperacina-N'-2'-etanesulfónico

HSV Virus herpes simple

HSV-TK Timidín-quinasa del virus herpes simple

LPEI Polietilenimina lineal

miRNA Micro-ARN

MTT Bromuro de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolio

NMR Resonancia magnética nuclear

ODN Oligo-desoxirribonucleótidos

OPSS ω-2-piridilditio poletilenglicol α-succinimidil-éster

PAMAM Poliamidoamina

PAMAM-Gn Generación n del dendrímero de poliamidoamina

PBS Tampón fosfato salino

pCMVLuc Plásmido que codifica para la luciferasa bajo el promotor

de CMV

PDI Índice de polidispersión

PEG Polietilenglicol

PEI Polietilenimina

P-HA Conjugado PAMAM-Ácido Hialurónico

PLL Polilisina

PPI Polipropilenimina

P-Tf Conjugado PAMAM-Transferrina

RES Sistema retículo-endotelial

RISC Complejo de silenciacimiento inducido por ARN (RNA-

Induced Silencing Complex)

RLU Unidades relativas de luz

IX

SCO Splice Correction Oligonucleotides

SDS Dodecil sulfato sódico

siRNA RNA interferente de pequeño tamaño (small interfering

RNA)

siRNA-Control siRNA inespecífico

siRNA-Luc siRNA específico del ARNm de la enzima luciferasa

TBE buffer Tampón tris-bórico-EDTA

Tf Transferrina

TfR Receptor de transferrina

TNBS Ácido 2,4,6-trinitrobenceno sulfónico

ZP Potencial zeta

RESUMEN / SUMMARY

Resumen / Summary

3

RESUMEN

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

Resumen / Summary

4

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.

Resumen / Summary

5

SUMMARY

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

Resumen / Summary

6

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.

INTRODUCCIÓN

Introducción

9

1. CONCEPTO DE TERAPIA GÉNICA

La terapia génica consiste en la administración de material génico con

fines terapéuticos. Este enfoque terapéutico surgió como una nueva estrategia

para el tratamiento de enfermedades hereditarias monogénicas, en las que el

reemplazamiento de un único gen puede curar una enfermedad como la fibrosis

quística o la inmunodeficiencia combinada severa. Actualmente, la terapia

génica también está considerada como una alternativa eficaz para el tratamiento

de enfermedades adquiridas como el Síndrome de Inmunodeficiencia Adquirida

(SIDA) o el cáncer. De hecho, esta última ha sido la diana de gran parte de los

ensayos clínicos llevados a cabo, representando más de dos tercios del total

(Figura 1).

Figura 1: Indicaciones para las que se han realizado ensayos clínicos hasta la actualidad

[1].

El término de terapia génica fue introducido en 1963 por Lederberg,

pero no fue hasta 1990 cuando la Food and Drug Administration americana (FDA)

aprobó el primer estudio clínico en una paciente de 4 años diagnosticada de

Introducción

10

inmunodeficiencia combinada severa, la cual es causada por el déficit de

adenosina desaminasa (ADA)[2]. Aunque los resultados fueron positivos, su

alcance es incierto, ya que simultáneamente se le administraba un fármaco con

la enzima. A partir de ese momento, se han llevado a cabo más ensayos clínicos

evaluando gran cantidad de vectores y, al mismo tiempo, se han descrito los

efectos adversos causados. A este respecto, en 1999, un paciente murió tras

desarrollar una reacción exagerada a un vector viral y fallo multiorgánico [3]. En

un ensayo francés en el que se utilizaron retrovirus, el 40% de los niños que se

sometieron al ensayo desarrolló leucemias, ya que el uso de estos vectores

puede provocar la integración del ADN cerca de oncogenes y activarlos. Estos

datos han tenido un efecto negativo en el progreso de la terapia génica, por ello

en este momento la investigación se dirige hacia el desarrollo de nuevos

sistemas para la administración de genes, también llamados vectores, seguros y

eficaces en los que el balance beneficio/riesgo sea positivo.

A pesar de que los problemas asociados a la administración de estos

vectores han conllevado graves efectos secundarios, actualmente existen

diferentes formulaciones aceptadas para su uso en humanos. En 1998, la FDA

aprobó Vitravene®, un oligonucleótido antisentido que degrada el ARN

mensajero de dos proteínas virales. Este medicamento estaba indicado para el

tratamiento de infecciones oculares por citomegalovirus en pacientes con

SIDA. En el año 2003, la Agencia China de Alimentos y Medicamentos aceptó

el fármaco para terapia génica Gendicine®, un virus no replicativo para el

tratamiento de carcinoma escamoso de cabeza y cuello. Sin embargo, poco

después de su aprobación se discutió la eficacia del tratamiento [3]. Dos años

más tarde, en 2005, la misma agencia aprobó el uso de otro producto llamado

Oncorine®, un adenovirus replicativo que se administraba en combinación con

quimioterapia [3]. La FDA aprobó en 2004 el uso de Cerepro®, un

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medicamento basado en un adenovirus para el tratamiento de tumores

cerebrales tras la resección del tumor. En 2012 la Agencia Europea del

Medicamento recomendó la aprobación de un producto de terapia génica en la

Unión Europea para el tratamiento de la lipoproteín-lipasa con un adenovirus

asociado que codifica para esta enzima.

La gran cantidad de ensayos clínicos llevados a cabo, así como la

investigación desarrollada, demuestran el enorme potencial que se le atribuye a

esta nueva terapia. Sin embargo, la necesidad de mejorar los sistemas de

liberación de genes actuales y desarrollar nuevos vectores que potencien su

actividad, sigue siendo uno de los principales retos en esta área.

2. SISTEMAS DE TRANSFERENCIA DE GENES

Tanto el ADN como el ARN son moléculas de alto peso molecular,

polianiónicas e hidrófilas, lo que hace que su paso a través de la membrana

celular sea muy dificultoso. Además, su administración directa conlleva su

rápida eliminación del organismo como mecanismo de defensa. Por ello, es

necesario que a la hora de administrar estas moléculas exista algún tipo de

transportador o vector que ayude a la protección e internalización en la célula

del mismo, con el fin de llegar al lugar de acción en su forma activa.

Exceptuando algunas técnicas, en las que no es necesaria la utilización de un

vector (administración directa, electroporación, administración hidrodinámica o

la pistola de genes), en la mayoría de los casos la administración de ácidos

nucleicos libres conlleva un rápido aclaramiento y la desaparición del posible

efecto terapéutico. Este hecho hace que el desarrollo de nuevos sistemas para la

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12

administración segura y eficaz de este tipo de moléculas sea indispensable para

su futura aplicación a la práctica clínica.

2.1. Sistemas virales

Los virus están constituidos por un material genético (ADN o ARN)

rodeado de una cápside proteica y, en algunos casos, una cubierta lipídica. Su

capacidad natural para infectar células e introducirles su material génico con el

fin de llevar a cabo la replicación mediante el uso de la maquinaria celular, hace

que estos vectores sean altamente eficaces. A día de hoy constituyen el grupo

más estudiado, de hecho, más del 60% de los ensayos clínicos llevados a cabo

utiliza algún tipo de vector viral.

En general, estos vectores son virus modificados para bloquear su

replicación, aunque su capacidad de introducir genes en la célula ha sido

mantenida. Las principales ventajas que presentan son su alta eficacia de

transfección y la duración de la misma. En cualquier caso, hay importantes

limitaciones que condicionan su uso. En la Tabla 1 se han recogido los

principales vectores virales utilizados, así como sus ventajas e inconvenientes

más representativos [4-6].

A pesar de que los sistemas virales han demostrado ser mucho más

eficaces a la hora de conseguir altos niveles de transfección, las limitaciones que

presentan en términos de capacidad de contener ADN, su inmunogenicidad y el

riesgo de oncogénesis y mutación que conlleva la integración del material

genético en el genoma, hacen que, a día de hoy, se estén buscando otras

alternativas para la administración de material génico.

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Tabla 1. Características de los principales virus utilizados en terapia génica.

Tipo Ventajas Inconvenientes

Adenovirus

No hay inserción del ADN.

Transfectan células replicativas y quiescentes.

Transfección temporal. Muy inmunogénicos. Asociados a procesos

inflamatorios

Retrovirus Transfección a largo plazo

Se integran en el genoma. No son específicos. Sólo infecta células

replicativas.

Adenovirus asociados

No estimulan la inflamación.

Son poco inmunogénicos. Infectan células que no

están en división. Expresión duradera.

Requieren la coinfección con un adenovirus o

herpesvirus. Problemas para su

producción a gran escala. Se integran en el genoma del

huésped

Poxvirus

Transportan genes de gran tamaño.

Posibilidad de uso como vacunas.

Inducen respuesta inmune.

Lentivirus No son replicativos.

Eficaces en células que no están en división.

Se integran en el genoma. Dificultad para producir a

gran escala.

Herpes Virus

No se integran, se mantienen como episoma. Infectan células que no

están en división. Capacidad de transportar genes de gran tamaño.

Posible reversión a la cepa salvaje.

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2.2. Sistemas no virales

Los sistemas no virales los componen una serie de moléculas que son

capaces de proteger el ácido nucleico incluido durante los procesos de

biodistribución y liberarlo en el interior celular. Las principales ventajas que

presentan, frente a los sistemas virales, son la facilidad de formulación y

posibilidad de escalado, mayor tamaño del gen encapsulado y menor toxicidad e

inmunogenicidad. Estos datos han hecho que diferentes autores consideren los

vectores no virales como la mejor estrategia a seguir, aunque los niveles de

transfección que se consiguen son menores [7].

2.2.1. Sistemas lipídicos

Los sistemas lipídicos incluyen una serie de formulaciones en las que el

vector está formado por un lípido neutro, catiónico o aniónico. Principalmente,

hay dos tipos de sistemas lipídicos que se utilizan: los liposomas y las

nanopartículas lipídicas sólidas. Los liposomas son partículas coloidales

formadas por la superposición de bicapas lipídicas sobre un centro acuoso de

forma similar a la membrana celular. Las nanopartículas lipídicas sólidas son

sistemas en los que la nanopartícula está formada por una matriz de lípidos en

la que está embebido el fármaco o molécula que se quiere liberar [8].

La eficacia de los liposomas como agentes de transferencia de genes se

demostró por primera vez en la década de los 70, al comprobar la capacidad

fusogénica de estas formulaciones con la membrana celular [9, 10].

Posteriormente, en la década de los 80 se demostró que eran capaces de

transportar y liberar ADN [11]. Actualmente, existe gran cantidad de lípidos

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15

capaces de mediar una transfección eficaz, como el DOTAP, DOTMA o el

DOSMA [12] y una gama de productos comerciales optimizados para la

transfección celular como la Lipofectina®, Lipofectamina® o el Transfectam®.

De forma general, el ADN se une en la formulación mediante la

interacción electrostática entre el ácido nucleico y el lípido, comúnmente

catiónico. Esto se realiza de manera espontánea. El resultado es un complejo

llamado lipoplejo [13]. Aunque en un comienzo se planteó que la entrada y

liberación del ADN en la célula diana se realizaba por fusión de las membranas,

en la actualidad se cree que es un proceso similar a la endocitosis, mediante el

que la célula internaliza la partícula en una vesícula de endocitosis y, una vez en

el endosoma, los lípidos del sistema lipídico interaccionan con la membrana

provocando una desestabilización del lipoplejo y generando la liberación del

ADN incluido [14]. En cualquier caso, se debe tener en cuenta que las

características del lípido, la presencia de un colípido que ayude a la estabilidad y

el protocolo de formulación, pueden generar complejos de tamaño y

propiedades muy variables [15, 16].

Estas formulaciones han demostrado ser eficaces tanto in vitro como in

vivo, aunque la administración puede generar inflamación o activación del

complemento [17]. Las principales ventajas que presentan los sistemas lipídicos

frente a los otros sistemas son su fácil preparación a nivel de laboratorio y a

gran escala y su buena tolerabilidad.

2.2.2. Sistemas químicos y poliméricos: polietilenimina

Los polímeros catiónicos son, sin lugar a dudas, los vectores más

utilizados hasta el momento y considerados superiores por algunos autores [18].

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La presencia de grupos con carga positiva a pH fisiológico, de forma similar a

los lípidos catiónicos, permite una interacción electrostática con los fosfatos del

ADN o ARN, provocando su condensación y la formación de partículas que

ayudan a proteger y liberar el material génico en el interior de la célula. Algunas

de las moléculas más utilizadas son los polímeros polietilenimina, la polilisina y

los dendrímeros, así como algunos poli- y oligosacáridos catiónicos como el

quitosano o las ciclodextrinas [19, 20].

La polietilenimina (PEI) es considerada como el polímero de

referencia según varios autores. Existen diferentes tipos de PEI dependiendo

de su peso molecular y la presencia de ramificaciones o no en su estructura [21].

Su eficacia se demostró en 1995 por Behr y cols. [22] y desde entonces se ha

utilizado ampliamente. Su estructura y características han sido modificadas para

mejorar su capacidad de transfección y disminuir su toxicidad [21]. La alta

capacidad de transfección de este polímero se debe, en primer lugar, a su alta

densidad de cargas y la flexibilidad de la molécula, que le permite condensar

fragmentos de ADN o ARN muy grandes, haciéndola especialmente

interesante. En segundo lugar, otra de las características que ha hecho que la

PEI sea considerada un buen vector es su capacidad tampón, conocida como

“esponja de protones” [23]. A pH fisiológico, únicamente una de cada tres o

cuatro aminas está cargada. Tras la endocitosis de la partícula, a medida que el

lisosoma va disminuyendo el pH para destruir su contenido, el polímero

permite tamponar ese proceso provocando la disrupción del lisosoma y la

liberación del material génico. Los cambios que se generan durante este proceso

han sido puestos en entredicho y la variación del pH respecto al mecanismo

fisiológico normal parece no verse tan afectada por la presencia del polímero

[24].

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A pesar de sus ventajas, la polietilenimina genera toxicidad, tanto in vitro

como in vivo, la cual ha sido descrita en la bibliografía [25, 26]. Por ello, una de

las estrategias que se utiliza actualmente para disminuir dicha toxicidad es el

enlace de la molécula con otras estructuras aniónicas, como el polietilenglicol, o

la unión a ligandos con gran carga negativa.

2.2.3. Dendrímeros catiónicos: derivados de la poliamidoamina (PAMAM)

La palabra dendrímero proviene del griego dendron (árbol) y meros

(parte). Su principal característica es que son macromoléculas esféricas

altamente ramificadas. Este tipo de moléculas fue descrito por primera vez a

finales de los años 70 por Vögtle [27], aunque su desarrollo fue posterior. En la

actualidad hay dos grandes tipos de dendrímeros que se utilizan para la

liberación de ADN y ARN: 1) derivados de la poliamidoamina (PAMAM),

descritos por primera vez por Tomalia y cols. [28] y 2) los derivados de la poli-

propilenimina (PPI).

Todos los dendrímeros presentan una estructura característica de

“estrella densa”, en la que a partir de un centro químico o core surge un número

determinado de ramificaciones (Figura 2A). El core puede tener, dependiendo de

su estructura, un número diferente de puntos de crecimiento, a partir de los

cuales comienzan las ramificaciones. Su síntesis se realiza por repetición de un

protocolo, por lo que con cada repetición se provoca el crecimiento de la

molécula en una generación (Figura 2A). Existen dos alternativas, la más común

es la síntesis divergente (Figura 2B), desde el core hacia el exterior, y la menos

común es la convergente (Figura 2C), en la que primero se sintetizan las

ramificaciones que se ensamblan al core. Para los dendrímeros de PAMAM esta

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síntesis consiste en la adición de metacrilato seguida de una amidación del

grupo éster resultante con etilendiamina. El incremento de cada generación

provoca un aumento en el número de grupos funcionales expuestos en la

superficie al doble que la generación anterior, mientras que su masa crece

exponencialmente. Como consecuencia, se pueden diferenciar tres partes en la

estructura de los dendrímeros: el centro químico o core, las ramificaciones y los

grupos funcionales de la superficie. Estos métodos de síntesis permiten un

control exhaustivo de las características de la molécula, por lo que el índice de

polidispersión es muy bajo y el tamaño y estructura quedan bien definidos [29-

31].

Los dendrímeros de PAMAM han resultado ser un grupo de moléculas

con un gran potencial tanto en la industria química como catalizadores y en

áreas biomédicas como agentes de imagen o transportadores. Su uso en terapia

génica se debe, en primer lugar, a su capacidad de unión al ADN o ARN

mediante interacciones electrostáticas [32]. En este caso, también se consigue

una condensación y compactación de los ácidos nucleicos que dependen de la

estequiometria, la concentración de ADN, la dinámica de mezclado y las

propiedades del solvente [29]. En general, se acepta que para que todo el ADN

esté unido al PAMAM, la relación de cargas entre los grupos amino primarios y

los grupos fosfato del material génico debe ser, como mínimo, 1:1. A pesar de

ello, se ha demostrado que se necesitan relaciones de carga mayores para

conseguir complejos estables y eficientes [33]. Un factor importante a este

respecto es la generación de dendrímero utilizada: a mayor generación, mayor

capacidad de condensación [34, 35]. Se ha postulado que tras la unión del ADN

con el dendrímero se forman unas regiones fuertemente unidas alrededor de los

dendrímeros, mientras que otras hacen de nexo de unión. Se cree que la mayor

interacción que ofrecen los dendrímeros de generación más alta se debe a que

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19

éstos son capaces de formar regiones más grandes en las que el ADN está

fuertemente unido y más protegido [35, 36].

Figura 2: Esquema de la estructura de un dendrímero (A), proceso de síntesis

divergente (B) y convergente (C) [31].

El mecanismo por el que los complejos catiónicos, ya sean lipídicos o

poliméricos, son internalizados comienza por una interacción electrostática con

la membrana celular, cargada negativamente para, a continuación, ser

introducidos en la célula por diferentes rutas. Este proceso se puede ver

favorecido por la presencia de determinados componentes de la membrana y la

matriz extracelular, como las glucoproteínas. Sobre los mecanismos de

internalización que siguen los dendriplejos no hay un consenso claro. A pesar

A

B

C

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20

de que Manunta y cols. demostraron que la entrada de los dendriplejos depende

de la presencia de colesterol y está asociada a endocitosis dependiente de

clatrina [37], estudios posteriores de los mismos autores sugieren que esta

entrada puede estar también relacionada con procesos de endocitosis mediada

de caveolina, aunque depende del tipo celular [38]. Albertazzi y cols. realizaron

un estudio comparativo en dos líneas celulares con diferentes generaciones (G2,

G4 y G6) de dendrímeros de PAMAM y obtuvieron resultados similares,

confirmando que la línea celular influye en la dinámica de los procesos de

internalización [39].

Una de las características de la endocitosis mediada por clatrina y otras

vías de internalización es la transformación de las vesículas de endocitosis en

lisosomas. A lo largo de este proceso, las vesículas de internalización, o

endosomas, se van acidificando. Este cambio en el pH intravesicular hace que

el ADN administrado sea degradado. Una de las características que influye para

que los dendrímeros de PAMAM sean interesantes para su uso como vectores

no virales es su capacidad tampón, similar a la que ya se ha comentado antes

para la PEI. En este caso, las aminas secundarias y terciarias que quedan dentro

de la estructura del dendrímero, se comportan como bases débiles capaces de

tamponar el medio, provocando una deceleración en la acidificación del

endosoma, un aumento de la concentración de iones cloruro y, como

consecuencia del cambio de presión osmótica, un aumento del volumen de la

vesícula que puede llegar a causar la disrupción de la misma [23, 40]. Los

aspectos mecanísticos relacionados con el escape del endosoma han sido

revisados por otros autores recientemente [41].

Por otra parte, uno de los factores más importantes que se debe tener

en cuenta a la hora de utilizar un vector catiónico es su toxicidad ya que, debido

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a la presencia de grupos catiónicos en la superficie, puede generar una

desestabilización de la membrana y lisis celular. En cualquier caso, la membrana

queda reestablecida pasadas 2 horas tras la retirada de los dendriplejos, según

indican estudios in vitro [42]. Diferentes autores afirman que los dendriplejos de

PAMAM son menos tóxicos que los complejos formados con PEI [43-45]. De

todas formas, se debe tener en cuenta que se ha observado una relación entre la

toxicidad y la generación de los dendrímeros, siendo los de generaciones más

altas, más tóxicos, aunque también depende de la concentración a la que se

administren y el tiempo de exposición [46, 47].

La eficacia de transfección de este tipo de moléculas fue inicialmente

explorada por Haensler y Szoka [48] con buenos resultados. Uno de los puntos

importantes al que hacen referencia en esa publicación está relacionado con la

capacidad de transfección de cada generación del dendrímero. Como se ha

comentado anteriormente, la generación parece que influye en la capacidad de

compactación del ADN. De la misma forma, Haensler y Szoka señalan una

dependencia: a mayor generación, mayor capacidad de transfección para la

misma relación de cargas [42]. Navarro y cols. han publicado resultados

similares donde la transfección mediada por dendriplejos formulados con

PAMAM-G5 fueron más eficaces que los formados con G4 [33]. Con estos

datos, algunos autores afirman que las generaciones medias de los dendrímeros

de PAMAM son las más efectivas, ya que presentan una flexibilidad óptima

para la formación de los complejos [30].

La biodistribución de los dendrímeros de diferentes generaciones ha

sido estudiada, aunque estos datos no se corresponden totalmente con los

resultados obtenidos tras la administración intravenosa de complejos

PAMAM/ADN. Los dendrímeros libres (G3 y G4) son retirados rápidamente

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del torrente sanguíneo (<2% de dosis recogida en sangre tras 1 h),

acumulándose en el hígado (30-90% de la dosis tras 1 h) [47]. La modificación

de los grupos amina primarios por grupos aniónicos hace que este patrón de

biodistribución se modifique y se mantenga más tiempo en circulación (20-40%

de la dosis recogida en sangre tras 1 h). Roberts y cols. realizaron una

evaluación del perfil de biodistribución de los dendrímeros de PAMAM (G3,

G5 y G7), concluyendo que las diferentes generaciones son acumuladas en

diferentes órganos y que su excreción también varía. Además, también afirman

que sólo los dendrímeros G7 a las concentraciones más altas provocaron algún

tipo de complicación [46]. Con todo, la toxicidad de estos compuestos parece

ser baja ya que la administración repetida de 10 mg/kg por vía intravenosa

durante 10 semanas, y su observación tras 6 meses, no mostró signos claros de

toxicidad [49].

Existen datos que confirman que la distribución de los complejos

dendrímero/ADN no se corresponde con la de los dendrímeros solos. Navarro

y cols. afirman que tras la administración de complejos formulados con 60 µg

de ADN con PAMAM G4 y G5 consigue expresión principalmente en el

pulmón, siendo mayor para la G5 respecto a la G4 y sin aparecer signos de

toxicidad [33].

3. OPTIMIZACIÓN DE LOS VECTORES

Uno de los importantes desafíos a los que debe hacer frente una nueva

formulación es su administración in vivo. Tras la administración sistémica los

principales obstáculos que una formulación debe superar son 1) circular por el

torrente sanguíneo sin ser retirado del mismo por el sistema retículo-endotelial,

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2) evitar la toxicidad por interacciones inespecíficas con otros tejidos que no

sean el diana, 3) ser capaz de abandonar el torrente sanguíneo en el tejido diana

y 4) ser internalizado de forma eficiente para conseguir el efecto. La

combinación de una buena estabilidad y una buena capacidad de liberación del

ADN o ARN intacto en la célula diana es un objetivo difícil de alcanzar. Por

ello, para conseguir mejorar la biodistribución y la liberación de la carga del

complejo en el lugar indicado se han desarrollado diferentes estrategias.

3.1. Principales obstáculos tras la administración sistémica

3.1.1. Distribución en el organismo

Tras la administración intravenosa, los complejos deben superar

procesos de agregación, inestabilidad, toxicidad y retirada del torrente

sanguíneo por el sistema inmune. Estos procesos dependen, en gran medida, de

la carga superficial positiva de los vectores no virales.

En el torrente sanguíneo, las partículas entran en contacto directo con la

sangre y todos sus componentes. Uno de los fenómenos que se ha descrito para

muchos vectores no virales de naturaleza catiónica es la formación de agregados

con las proteínas séricas y los eritrocitos, lo que disminuye la carga superficial y

limita su capacidad de transfección [50]. Además, puede llegar a provocar la

desestabilización del complejo y la pérdida del ADN incorporado. La

interacción inespecífica también se puede dar con las proteínas del

complemento o algunas inmunoglobulinas [51, 52], provocando una

disminución de su actividad y un aumento de la captación por el sistema

inmune. Entre las consecuencias de esta interacción inespecífica está la

acumulación de los complejos en el pulmón, donde los capilares bloquean el

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paso de los agregados [53, 54]. A menudo, estos agregados no son estables y

permiten una recircularización posterior de los vectores hacia el resto del

organismo. Por lo tanto, la caracterización de la carga superficial y el tamaño de

las partículas deben ser estudiados, ya que mediante la modificación de estos

parámetros se puede mejorar el comportamiento in vivo de los complejos.

Un punto a resaltar es que los perfiles de expresión génica son similares

a los de distribución. A pesar de que la mayoría de los complejos son

fagocitados por el sistema retículo-endotelial, a menudo no conlleva unos

niveles altos de expresión en esas células porque el ADN internalizado es

degradado. Como ya se ha dicho, la administración de un vector producirá unos

mayores niveles de expresión génica en el pulmón, seguido del hígado y bazo,

lo que los convierte en órganos interesantes para su tratamiento. No obstante,

el tejido diana puede no estar incluido entre esos tres y, por lo tanto, ese patrón

de distribución no es favorable para conseguir transfectar otros órganos.

Para conseguir una transfección eficaz, los complejos deben alcanzar el

tejido diana, extravasarse hasta el espacio intercelular, alcanzar las células diana

e internalizarse. En el caso del tejido tumoral, este proceso se ve favorecido por

un fenómeno pasivo denominado EPR (del inglés Enhanced Permeability and

Retention Effect) [55]. Los vasos sanguíneos del tejido tumoral a menudo

presentan una permeabilidad más alta porque las fenestraciones son mayores,

además, el drenaje linfático está disminuido por lo que la retirada de los

complejos es baja. Para favorecer el paso de las nanopartículas al medio

extracelular del tumor se estima que el tamaño máximo que deben presentar es

del orden de los nanómetros (tamaño máximo del poro en el endotelio tumoral

de 380-780 nm) [56]. A pesar de ello, el alcanzar el tejido tumoral no significa

conseguir el efecto deseado, dado que el ADN tiene su lugar de acción en el

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núcleo, lo que hace que la entrada a la célula y su posterior transporte hasta la

liberación tengan gran importancia.

3.1.2. Entrada y transporte intracelular

La adhesión a la célula, una vez la partícula ha llegado a un tejido, se

lleva a cabo de forma pasiva por contacto directo o de forma activa mediante el

direccionamiento con ligandos dirigidos a un receptor o molécula de la

superficie. En el primer caso, se llevará a cabo mediante una interacción

electrostática de las partículas con la membrana celular o sus componentes, lo

que desencadenará el proceso de internalización de la misma, ya sea por

endocitosis, vía caveolas o por pinocitosis (Figura 3). Aunque en este caso se

puede llegar a conseguir una internalización efectiva de las partículas, los tejidos

en los que muestran efecto vendrán definidos por las características de la

formulación y el tejido (tamaño, carga superficial, vascularización…), es por ello

que en la actualidad se está investigando sobre el direccionamiento activo de las

partículas. La incorporación de ligando a una formulación puede mejorar su

biodistribución y su eficacia, al conseguir la expresión del gen incorporado en

un órgano o tejido concreto [57, 58].

Uno de los procesos más importantes en la liberación de ácidos

nucleicos tras la entrada a la célula es el paso por el compartimento lisosomal.

Tras la endocitosis, las vesículas endocíticas (endosomas) pueden unirse a los

lisosomas primarios lo que las termina convirtiendo en lisosomas secundarios.

Durante este proceso, destinado a la degradación del contenido endosomal, el

pH de las vesículas disminuye progresivamente. Este proceso se puede

modificar utilizando determinadas sustancias como la cloroquina o péptidos

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26

que alteran la acidificación de las vesículas. Algunos polímeros, de forma

inherente, son capaces de tamponar el medio intravesicular mediante el proceso

denominado esponja de protones, protegiendo así el ADN incorporado en la

nanopartícula del pH ácido del medio intravesicular y evitando su degradación.

Este fenómeno puede producir un aumento hasta del 140% del volumen del

endosoma provocando su rotura y la liberación de su contenido al citoplasma.

Figura 3: Esquema de las vías de internalización que puede seguir una partícula [59].

Finalmente, una vez que el ADN ha escapado del compartimento

lisosomal, debe llegar al núcleo celular para poder expresarse. La entrada al

núcleo es un proceso altamente limitado y controlado que se lleva a cabo por

pequeños poros presentes en la superficie de la membrana nuclear. Un punto

importante que ha sido descrito es que la expresión de un ADN es mayor en

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27

células en división. Esto puede ser debido a que las células pueden interiorizar

más fácilmente el ADN liberado durante el proceso de reorganización del

nuevo núcleo celular tras la mitosis.

3.2. Principales estrategias de optimización

3.2.1. Enmascaramiento de la carga superficial

Como se ha puesto en evidencia, entre los principales problemas que

acarrea el uso de sistemas catiónicos para la administración de genes, tanto in

vivo como in vitro, está el exceso de carga positiva que presentan estos sistemas.

Esto, por un parte, resulta ventajoso, ya que favorece la interacción con la

superficie celular y la posterior internalización. Sin embargo, a la hora de utilizar

una vía sistémica se produce una clara limitación debido a las interacciones

inespecíficas y sus consecuencias.

Probablemente, el polímero aniónico más utilizado para el

enmascaramiento de la carga superficial es el polietilenglicol (PEG). Este es un

polímero lineal, biocompatible, poco inmugénico, con alta solubilidad en medio

acuoso y con baja toxicidad, que ha demostrado gran utilidad en la formulación

de liposomas furtivos, capaces de evadir la captación por el sistema retículo-

endotelial y lograr una circulación larga [60]. Esta ventaja también se ha

aplicado a los complejos utilizados para terapia génica. El PEG es capaz de

mediar un bloqueo de la captación de forma similar, haciendo que la retirada de

los mismos se haga de forma más lenta, permitiendo alcanzar en mayor medida

el lugar de acción. Además, su presencia provoca una disminución de la carga

superficial, incluso hasta valores de potencial zeta negativos. A este respecto,

Ogris y cols. sintetizaron un conjugado PEI-PEG que fue capaz de mantener el

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28

tamaño de las nanopartículas en presencia de suero, mejorar la biodistribución

de las nanopartículas formadas y disminuir su toxicidad [52]. Aunque los

efectos beneficiosos del PEG están claros, su uso debe ser controlado, ya que la

inclusión de moléculas de gran tamaño puede tener influencia en la capacidad

de condensación de los complejos y en los procesos de salida del lisosoma [61].

Otros polianiones que también han demostrado ser útiles a la hora de

enmascarar la carga superficial de los vectores no virales son los

glicosaminoglucuranos de la matriz extracelular. Estos compuestos están

presentes de forma natural en el organismo por lo que son biocompatibles.

Kurosaki y cols. realizaron un cribado de algunos de estos compuestos con el

fin de estudiar su uso como agentes para mejorar la transfección [62]. En su

estudio se demuestra que la adición de estos polianiones no modifica el tamaño

de la partícula, mientras que la carga superficial disminuye de forma significativa

alcanzando valores negativos, lo que redujo la interacción de las partículas con

los eritrocitos y la formación de agregados.

3.2.2. Direccionamiento o targeting

Una de las opciones más utilizadas para aumentar la eficacia de los

vectores no virales es la incorporación de ligandos a las partículas. Tras la

administración sistémica, los vectores son distribuidos y acumulados en

diferentes órganos dependiendo de sus características fisicoquímicas y la

fisiología del tejido. La posibilidad de llegar a un tejido diana concreto de forma

específica podría reducir la aparición de efectos secundarios adversos en el resto

del organismo, lo que pone de manifiesto la importancia e interés de esta

estrategia de mejora. Aunque la llegada a determinados tejidos, como el

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29

tumoral, puede verse favorecida por la presencia de algún tipo de característica

fisiológica (EPR), la internalización de los complejos es un proceso complicado

en el que influyen muchos factores. A pesar de ello, algunos autores consideran

el direccionamiento mediante proteínas o moléculas dirigidas a receptores

expresados en la superficie de las células una estrategia interesante [63].

3.2.2.1. Ácido hialurónico y el receptor CD44

El ácido hialurónico (AH) es un glicosaminoglucurano de alto peso

molecular y uno de los principales componentes de la matriz extracelular. Es,

por lo tanto, un compuesto biodegradable, no tóxico y biocompatible. Algunos

autores han demostrado que es capaz de unirse de forma electrostática a

complejos catiónicos para terapia génica sin romper la partícula [62, 64].

Además, es capaz de enmascarar la carga superficial, conseguir un

direccionamiento de las formulaciones y mejorar la biodistribución y el perfil de

toxicidad de conjugados fármaco-AH o de nanopartículas dirigidas [65]. El

receptor de ácido hialurónico mejor conocido para su uso como diana es el

CD44, ya que está sobreexpresado en algunos tipos tumorales como el de

pulmón, mama, colon, hígado, cérvix y riñón así como en el melanoma [66, 67].

Sus funciones están relacionadas con la difusión metastásica de células

tumorales, señalización intracelular, inflamación y endocitosis [67-69].

Platt y cols. han revisado las publicaciones existentes dirigidas al CD44

y afirman que existen datos sobre liposomas dirigidos con HA en la superficie,

que resultan prometedores en varios modelos tumorales [65]. En terapia génica

el uso de este ligando ha sido limitado, aunque existen claros indicios de su

capacidad como agente de direccionamiento de vectores no virales.

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30

Principalmente, su actividad ha sido estudiada en sistemas lípidicos y

poliméricos (poliplejos con PEI) y su uso con dendrímeros de PAMAM para

liberación de genes ha sido puntual. Este aspecto se desarrollará más

profundamente en los Capítulos 1 y 2 de este trabajo.

3.2.2.2. Transferrina

La transferrina (Tf) es una de las proteínas encargadas del transporte de

hierro en el organismo y ha sido utilizada como agente de direccionamiento

para un amplio espectro de células tumorales [70]. Aunque su receptor está

expresado en prácticamente todos los tipos celulares del organismo, su

presencia está aumentada en muchas células tumorales debido al requerimiento

incrementado de hierro, necesario en el metabolismo o en la división celular

[71, 72]. Esto ha hecho que el direccionamiento hacia este receptor haya

conseguido aumentar la capacidad de transfección de sistemas no virales tanto

in vitro como in vivo con vectores poliméricos [73, 74], lipídicos [75, 76] e incluso

dendriméricos [77]. Gracias a su buena actividad, esta proteína se ha utilizado

para el direccionamiento de múltiples sistemas basados en nanopartículas en la

lucha contra el cáncer, demostrando ser un ligando y un receptor altamente

versátiles [70]. Además, ha demostrado tener una alta capacidad para

enmascarar la carga superficial positiva, mejorando la biodistribución y

aumentando la expresión del gen incluído en un modelo tumoral in vivo [74].

La utilización de la transferrina ha demostrado ser útil, pero el uso de

grandes proteínas en la formulación puede conllevar problemas durante los

procesos de síntesis y almacenaje, según afirman Nie y cols. [78]. Por ello, han

surgido nuevas moléculas capaces de unirse de forma eficaz al receptor de

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31

transferrina como el péptido B6 [79], que ha demostrado ser eficaz en el

direccionamiento de un vector adenoviral y como sustituto de la proteína entera

en poliplejos [78, 80, 81]

3.2.2.3. Factor de crecimiento epidermal (EGF)

El receptor del EGF (EGFR) está frecuentemente sobreexpresado en

diferentes cánceres como el de mama, próstata, vejiga, pulmón o hígado [82],

convirtiéndolo en una interesante diana para la administración de vectores

dirigidos. Su actividad está relacionada con procesos de diferenciación y

proliferación celular. El EGFR ha demostrado ser eficaz para el

direccionamiento de nanopartículas, liposomas y fármacos tanto in vitro como in

vivo [83, 84]. De la misma forma que para la transferrina, se han buscado otras

opciones al uso de la proteína completa, como los anticuerpos específicos

contra el EGFR. Otra de las alternativas a la proteína completa es el péptido

GE11 descrito por Li y cols. [85]. Este péptido ofrece una serie de ventajas

frente a la proteína entera, como la ausencia de activación del receptor,

mantenimiento de unos niveles constantes del EGFR en la superficie celular y

la posibilidad de producir vectores con alta densidad de ligando con buen

escalado [86]. Además, estudios realizados hasta ahora han demostrado que es

un ligando útil para el direccionamiento de los vectores en modelos de tumores

in vivo [61, 87, 88]

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32

4. TERAPIA GÉNICA Y CÁNCER

La terapia génica surgió como una alternativa al tratamiento de

enfermedades monogénicas, sin embargo actualmente se centra

preferentemente en el cáncer [89]. La alta prevalencia y gravedad de esta

enfermedad, que produjo en 2012 8,2 millones de muertes [90], hace que sea

objeto de dos de cada tres ensayos clínicos (Figura 1). Hasta ahora se han

desarrollado varias estrategias terapéuticas para la lucha contra el cáncer.

Aunque el enfoque más común es atacar directamente el tejido tumoral, existen

otras alternativas que implican la estimulación del sistema inmune que será el

que medie la eliminación del tumor. En la Figura 4 se recogen las diferentes

alternativas para el tratamiento del cáncer.

Figura 4. Vista esquemática de las principales estrategias aplicadas al tratamiento del

cáncer mediante terapia génica. [91]

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33

4.1. Corrección génica

Los primeros ensayos clínicos se realizaron con el objetivo de suplir la

falta de síntesis de la enzima adenosín-desaminada por los pacientes, ya que

causa una inmunodeficiencia grave. Según algunos autores, este enfoque con el

fin de tratar el cáncer ha tenido poca repercusión [91], sin embargo, se ha

demostrado que las células cancerosas pueden sufrir alteraciones y mutaciones

que provocan la inactivación de genes supresores de tumores o activación de

algunos oncogenes [92], lo que permite modificar la expresión de dichos genes

mediante estrategias de terapia génica para lograr la erradicación o mejora en el

pronóstico de la enfermedad.

En la actualidad, se pueden destacar dos estudios clínicos en los que se

ha intentado reestablecer la expresión de genes supresores de tumores. En uno

se utilizó el gen TUSC2 en una formulación liposomal, consiguiendo expresión

en los tumores primarios y metastásicos [93]. El otro, utilizó una formulación

liposomal dirigida con anticuerpos hacia el receptor de transferrina para la

liberación sistémica del gen p53. Esta formulación consiguió la expresión

selectiva del gen en biopsias primarias y de nódulos metastáticos, detectándose

incluso necrosis intratumoral en dos de los pacientes [94].

4.2. Terapia génica suicida

Esta estrategia conlleva la introducción en las células cancerosas de un

gen que permite la conversión de un compuesto no tóxico en una sustancia

letal. Aunque este tipo de terapia ha sido utilizada con éxito en diferentes

estudios in vitro e in vivo, su aplicación no ha conseguido la relevancia esperada

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34

[95]. Existen varios tipos de genes que se pueden utilizar para conseguir la

supresión del tumor, como el gen para la timidín quinasa del virus herpes

simple (VHS-TK) o el de la citosín desaminasa (CD) [95].

A día de hoy existen algunos ensayos clínicos como el publicado por

Voges y cols. en el que se administró una formulación liposomal catiónica con

el gen HSV-1-TK, consiguiendo beneficio terapéutico en algunos de los

pacientes con glioblastoma multiforme [96].

4.3. Inmunoterapia génica

La inmunoterapia génica hace referencia a aquellas estrategias de

tratamiento en las que la transferencia de genes busca generar una respuesta del

sistema inmune contra el cáncer. De forma innata, el cuerpo es capaz de

detectar las células cancerosas y eliminarlas, desafortunadamente, esta vigilancia

no es suficiente para evitar el desarrollo de tumores. El conocimiento de cómo

el sistema inmune es activado para reconocer de forma específica antígenos

tumorales y los mecanismos de control de la respuesta inmune que se

desencadenan, forman la base para este tipo de terapia [97].

Uno de los enfoques que han demostrado ser eficaces es el uso de

citoquinas con propiedades inmuno-moduladoras que pueden activar la

respuesta inmune antitumoral [98]. Algunas de estas citoquinas son la

interleuquina-2, el interferón α, o la interleuquina-12. Esta última ha

demostrado ser eficaz en el tratamiento de diferentes cánceres mediante terapia

génica en modelos animales [99] y en algunos ensayos clínicos [100].

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35

4.4. Bloqueo de la expresión génica: small interfering RNA (siRNA)

Hasta ahora, todas las estrategias comentadas están relacionadas con la

administración de un ADN que codifica una proteína o enzima concreta. A

pesar de que estos enfoques han demostrado ser eficaces, en los últimos años

ha ganado fuerza el bloqueo de la expresión génica. Con este fin se han

desarrollado diferentes opciones: en primer lugar está la administración de

oligodesoxiribonucleótidos (ODN) capaces de unirse de forma específica a un

ARN mensajero (ARNm) y bloquear su acción. En segundo lugar están unos

oligonucleótidos antisentido (SCO: splice correction oligonucleotides) que sirven para

el tratamiento de algunas enfermedades como la distrofia muscular de

Duchenne. Estas secuencias se unen con las moléculas de pre-ARNm en el

núcleo y las modifican para que codifiquen una enzima o proteína funcional.

Finalmente, se encuentra el ARN interferente que se desarrolló en los años 90.

Al principio se utilizaban secuencias de más de 30 pares de bases que

provocaban respuesta inmune, por lo que la estrategia evolucionó hasta

secuencias más pequeñas capaces de bloquear la expresión génica después de

los procesos de transcripción (ARN interferente de pequeño tamaño: siRNA,

small interfering RNA)

El mecanismo de acción del siRNA es claramente diferente del que

presenta el ADN. Mientras que éste debe ser liberado al citoplasma y

transportado hasta el interior del núcleo, el siRNA tiene su lugar de acción en el

citoplasma. Una vez el siRNA ha llegado al citoplasma, la hebra específica, tras

la acción de la enzima dicer, se une a un complejo llamado RISC (RNA-interfering

silencing complex) que tiene acción catalítica y destruirá el ARNm complementario

de la secuencia que lleva incorporada. Los sistemas utilizados para su

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administración son los mismos que los descritos hasta ahora para ADN,

aunque, debido al diferente tamaño y características, no siempre un mismo

vector es igual de eficaz para ambos fines [101]. Algunos de los vectores más

utilizados, así como su uso en los ensayos clínicos más relevantes y su

aplicación en el tratamiento del cáncer, han sido resumidos por Yuan y cols.

[102] y Lee y cols. [103].

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71. Daniels TR, Delgado T, Rodriguez JA, Helguera G, Penichet ML: The transferrin receptor part I: Biology and targeting with cytotoxic antibodies for the treatment of cancer. Clin Immunol 121(2), 144-158 (2006).

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83. Bunuales M, Duzgunes N, Zalba S, Garrido MJ, De Ilarduya CT: Efficient gene delivery by EGF-lipoplexes in vitro and in vivo. Nanomedicine (Lond) 6(1), 89-98 (2011).

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86. Mickler FM, Mockl L, Ruthardt N, Ogris M, Wagner E, Brauchle C: Tuning nanoparticle uptake: live-cell imaging reveals two distinct endocytosis mechanisms mediated by natural and artificial EGFR targeting ligand. Nano Lett 12(7), 3417-3423 (2012).

87. Abourbeh G, Shir A, Mishani E et al.: PolyIC GE11 polyplex inhibits EGFR-overexpressing tumors. IUBMB Life 64(4), 324-330 (2012).

88. Klutz K, Schaffert D, Willhauck MJ et al.: Epidermal growth factor receptor-targeted (131)I-therapy of liver cancer following systemic delivery of the sodium iodide symporter gene. Mol Ther 19(4), 676-685 (2011).

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91. Walther W, Schlag PM: Current status of gene therapy for cancer. Curr Opin Oncol 25(6), 659-664 (2013).

92. Vogelstein B, Kinzler KW: Cancer genes and the pathways they control. Nat Med 10(8), 789-799 (2004).

93. Lu C, Stewart DJ, Lee JJ et al.: Phase I clinical trial of systemically administered TUSC2(FUS1)-nanoparticles mediating functional gene transfer in humans. PLoS One 7(4), e34833 (2012).

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96. Voges J, Reszka R, Gossmann A et al.: Imaging-guided convection-enhanced delivery and gene therapy of glioblastoma. Ann Neurol 54(4), 479-487 (2003).

97. Ribas A, Butterfield LH, Economou JS: Genetic immunotherapy for cancer. Oncologist 5(2), 87-98 (2000).

98. Dougan M, Dranoff G: Immune therapy for cancer. Annu Rev Immunol 27, 83-117 (2009).

99. Pavlin D, Cemazar M, Sersa G, Tozon N: IL-12 based gene therapy in veterinary medicine. J Transl Med 10, 234 (2012).

100. Del Vecchio M, Bajetta E, Canova S et al.: Interleukin-12: biological properties and clinical application. Clin Cancer Res 13(16), 4677-4685 (2007).

101. Scholz C, Wagner E: Therapeutic plasmid DNA versus siRNA delivery: common and different tasks for synthetic carriers. J Control Release 161(2), 554-565 (2012).

102. Yuan X, Naguib S, Wu Z: Recent advances of siRNA delivery by nanoparticles. Expert Opin Drug Deliv 8(4), 521-536 (2011).

103. Lee JM, Yoon TJ, Cho YS: Recent developments in nanoparticle-based siRNA delivery for cancer therapy. Biomed Res Int 2013, 782041 (2013).

HIPÓTESIS Y OBJETIVOS

Hipótesis y objetivos

47

La hipótesis fundamental que centra este trabajo se basa en la

sobreexpresión de determinados receptores de membrana en diferentes células

tumorales. Esto hace que la preparación y evaluación de distintos nanovectores

“dirigidos” a esos receptores adquiera especial interés. La selectividad y

especificidad que aportarían estas formulaciones, al ser dirigidas a las células

tumorales en mayor medida respecto a las células sanas, podría contribuir al

diseño de terapias antitumorales más efectivas y con menor toxicidad asociada.

El objetivo general de este trabajo comprende el diseño y evaluación de

nuevas formulaciones, basadas en la nanotecnología, en presencia del polímero

dendrimérico PAMAM y cuatro ligandos diferentes (ácido hialurónico,

transferrina y los péptidos GE11 y B6).

Entre los objetivos específicos se encuentran:

1. Síntesis y caracterización de un nuevo conjugado PAMAM–HA.

Estudio de la capacidad de formación de nanosistemas estables en

presencia de ADN, así como de su acción protectora frente a la acción

degradadora de las nucleasas.

2. Evaluación in vitro de los dendriplejos PAMAM-HA-ADN en diferentes

líneas celulares tumorales, así como la determinación del mecanismo de

internalización.

3. Evaluación in vivo de los dendriplejos PAMAM-HA-ADN sintetizados,

tanto en animales sanos como en aquellos portadores de tumor.

4. Caracterización de los complejos PAMAM-Tf y su evaluación in vitro en

cultivos celulares.

Hipótesis y objetivos

48

5. Preparación y caracterización de nanosistemas PAMAM-PEG-GE11 y

PAMAM-PEG-B6 para la vehiculización de siRNA. Evaluación de la

eficacia de silenciamiento génico de estos sistemas en células tumorales.

6. Evaluación de la toxicidad de los nuevos vectores diseñados y

evaluados.

CHAPTER 1

SYNTHESIS AND CHARACTERIZATION OF A

NOVEL PAMAM-HYALURONIC ACID CONJUGATE

FOR GENE DELIVERY

Published in:

Journal: Nanomedicine UK (2014) (In Press)

Authors: K. Urbiola, C. Sanmartín, L. Blanco-Fernández, C. Tros de Ilarduya

CHAPTER 2

IN VITRO AND IN VIVO EVALUATION OF A

NEW PAMAM-HYALURONIC ACID CONJUGATE

FOR GENE DELIVERY

Published in:

Journal: Nanomedicine UK (In Press)

Authors: K. Urbiola, C. Sanmartín, L. Blanco-Fernández, C. Tros de Ilarduya

CHAPTER 3

EVALUATION OF IMPROVED PAMAM-G5 BASED DENDRIPLEXES TARGETED TO THE TRANSFERRIN

RECEPTOR

Chapter 3: Evaluation of improved PAMAM-G5 based dendriplexes targeted to the transferrin receptor

109

ABSTRACT

The transfection activity of non-viral vectors is highly dependent on the

delivery capacity of the carriers. In this respect, the use of the transferrin (Tf)

receptor, which is known to be overexpressed on the surface of different

cancer cells, has been used as a tool to enhance the uptake of the non-viral

systems and improve its activity. Therefore, the aim of this work has been to

evaluate the activity of a new PAMAM dendrimer – Transferrin conjugate (P-

Tf) with better gene delivery activity to cancer cells. The formulations

containing the novel P-Tf were able to bind pDNA and protect it from the

activity of DNAse I enzyme. Moreover, it formed nanoparticles with positive

surface charge, although the presence of Tf led to a decrease of the zeta

potential up to almost electroneutral values. This new vector, formulated at

N/P 6, exhibited excellent transfection efficacy in HeLa, HepG2 and CT26 cell

lines, whereas in Neuro2A no improvement was achieved. Compared to

control complexes with bPEI, targeted dendriplexes were more efficient in

HepG2 and HeLa. Cellular viability was always kept over 80% in these cell lines

with higher values than bPEI control polyplexes. The uptake via receptor-

mediated endocytosis was ensured by a competition assay, by adding an excess

of free Tf, which led to a decrease in the transfection activity of targeted

dendriplexes.

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111

1. INTRODUCTION

The use of strategies to improve the activity of non-viral vectors for

gene delivery has been one of the most important issues to be overcome in

order to enhance the application of these systems to clinical practice. One

major approach in non-viral gene therapy is based on cationic polymers, such as

polyethyleneimine (PEI), polylysine (PLL), polyamidoamine (PAMAM) or

polypropileneimine (PPI) [1, 2]. This kind of vectors are interesting since they

are easily manufactured, flexible, versatile and are able to mediate the delivery

of natural or synthetic nucleic acid of any kind and size [2-4]. As this type of

vectors have difficulty in obtaining high levels of expression, especially in the

presence of serum, some of the most used strategies to improve their activity

have included the addition of anionic molecules in order to shield the surface

charge, inclusion of new components such as penetrating peptides, activation

of the structure of dendrimers and targeting nanosystems to specific receptors

[5-8].

Regarding the last approach, one of the most used receptors for the

delivery of drugs and improving the activity of non-viral vectors is the

transferrin receptor (TfR), which has been reported to be overexpressed on the

surface of different cancer cells [9, 10]. This receptor is the principal route by

which iron is internalized into cells. It is considered an ubiquitous receptor

expressed on most active proliferating cell types and its expression is

upregulated in multiple cancer cells since iron is a basic element during the

DNA synthesis, cell division and cellular metabolism [11-13]. Its ligand,

Transferrin (Tf), presents a molecular weight of 80 kDa and it is one of the

proteins in charge of the iron transport through the body [12]. The uptake of

Tf via TfR involves a clathrin dependent endocytosis process that leads to two

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112

different intracellular pathways. One involves the recycling of the complex Tf-

TfR and the other one the lysosomal degradation. It is believed that only 5-15%

of the intracellular pathways follow the lysosomal and subsequent elimination

route [14].

Concerning the use of Tf to enhance gene delivery, in 1990, Wagner et

al. [15] introduced the term “transferrinfection” to define the transfection

procedure when Tf is included as a ligand to enhance the uptake of DNA.

Since then, the use of Tf as a ligand to enhance the delivery of drug conjugates

or nanoparticles has increased its interest and has proven to be effective, not

only for the enhancement in gene delivery and expression, but also for the

potential use as a shielding and targeting agent of polyethyleneimine containing

polyplexes and other cationic polymers after the administration in vitro and in

vivo in tumor-bearing mice [15-20]. In parallel, Xu et al. described the

administration of Tf-liposomes carrying the p53 gene that result in the

regression of a human neck and head cancer model [21]. Tros de Ilarduya et al.

published a study where Tf, electrostatically assembled, was able to enhance the

transfection activity of cationic liposomes in vitro and in vivo [22] and the ability

of that formulation to produce the regression of a CT26 tumor after the

administration of the interleukin 12 gene [23]. This ligand has also exhibited

good activity in enhancing the delivery of siRNA as Cardoso et al. reported for

Tf-lipoplexes [24, 25].

PAMAM dendrimers, described in 1986 by Tomalia et al. [26] present

exceptional chemical features for the modification of the surface groups and

have been used as imaging agents or drug carriers [27, 28]. The ability to target

the Tf receptor by these systems has been studied by Li et al. [29], who designed

targeted PAMAM-G4 dendrimers with Tf carrying tamoxifen that were able to

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113

mediate an effective transport across the blood-brain barrier and induce the

inhibition and death of glioma cells. The ability of the PAMAM dendrimers to

mediate an effective gene transfer has also been demonstrated [28, 30] but their

use as DNA or RNA carriers targeted to the Tf receptor has not been widely

examined. In this respect, Huang et al. [31, 32] studied the possibility of

targeting brain tissue through the use of lactoferrin-PAMAM and transferrin-

PAMAM conjugates, suggesting that the proposed dendriplexes can be

exploited as potential non-viral gene vectors targeting the brain via non-

invasive administration.

Therefore, the aim of this study is to evaluate the transfection activity of

a new Tf containing dendriplex for gene delivery. We propose a novel

PAMAM-Tf conjugate for gene delivery as a new carrier for pDNA delivery

that can improve the transfection efficacy of the dendriplexes used so far and

enhance the activity of the commercial available PAMAM by attaching the

targeting ligand transferrin to the formulation.

2. MATERIALS AND METHODS

2.1. Materials

Polyamidoamine dendrimer generation 5 (PAMAM) with

ethylendiamine core (MW 28,825 Da, 128 N-terminal amines) and branched

polyethylenimine (25 kDa) (bPEI), used as control, were provided by Sigma-

Aldrich (Madrid,Spain). Transferrin conjugated with PAMAM (P-Tf) was kindly

provided by Dr. Manfred Ogris (Department of Pharmacy and Pharmaceutical

Biotechnology, Ludwig-Maximilians University, Munich, Germany). The

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114

plasmid pCMVLuc (BioServe Technologies, Maryland, USA) encoding

luciferase gene was used in the transfection studies. Plasmid was amplified in E.

coli, isolated and purified using a QUIAGEN Plasmid Giga Kit (QUIAGEN,

Germany). DNA concentration and purity were measured by NanoDrop

(NanoDrop ND1000, Thermo Scientific). Agarose, Tris, boric acid and

ethylenediaminetetraacetic acid (EDTA) were provided by Sigma Aldrich

(Barcelona, Spain). The HEPES glucose buffer (pH 7.4) was prepared from D-

(+)-glucose and N-(2-hydroxyethyl) piperazine-N′-[2-ethanesulfonic acid]

(HEPES, Sigma-Aldrich). Alamar Blue Dye was purchased from Invitrogen

(Barcelona, Spain), and was used in the toxicity studies. Human transferrin for

competition assay was purchased by BD Biosciences (Bedford, Massachusetts,

USA).

2.2. Synthesis of PAMAM-Transferrin conjugate

The synthesis of the PAMAM-Transferrin conjugate was performed

similarly as described previously for Tf-PEI (25kDa) [19]. Firstly, methyl

alcohol was removed from the commercial PAMAM solution by rotary

evaporation under reduced pressure. Then, the film was hydrated with 0.25 M

sodium chloride and 20 mM HEPES. A solution of transferrin in 30 mM

sodium acetate buffer (pH 5) was cooled to 0ºC and three equivalents of

sodium periodate in 30 mM sodium acetate buffer were added. The mixture

was kept on ice for 90 min. For removal of the low molecular weight products,

gel filtration (Sephadex G-25 superfine, 30 mM sodium acetate buffer pH 5)

was performed (monitoring: UV absorption at 280 nm). The modified

transferrin solution was added to the PAMAM solution (0.25 M sodium

chloride, 20 mM HEPES) at a molar ratio of 1:1.2 and vigorously mixed at

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115

room temperature. The pH was adjusted to 7.3 by the addition of 2 M HEPES

pH 7.9. After 30 min, four portions of sodium cyanoborohydride (1 mg per 10

mg transferrin) were added at 1 h intervals. After 19 h, the salt concentration

was adjusted to 0.5 M by addition of 3 M sodium chloride. The mixture was

loaded on to cation-exchange column (MacroPrep High S 10/10, Bio-Rad,

Hercules, CA, USA) and fractioned with a salt gradient from 0.5 M to 3 M

sodium chloride (with a constant content of 20 mM HEPES pH 7.3). The

major amount of conjugated eluted between 2.1 and 3 M salt. After dialysis

against 4 L of HBS (20 mM HEPES, 150 mM NaCl, pH 7.4), the conjugate

(designated P-Tf) was obtained at a molar ratio of transferrin: PAMAM-G5 of

1:0.94. TNBS (trinitrobenzene sulfonate) assay for determination of primary

amines in PAMAM was perfomed. The amount of transferrin was determined

by absorption measurement at 280 nm. Iron was incorporated by the addition

of 1.25 µL 10 mM iron (III) citrate buffer (200 mM citrate, pH 7.3 adjusted

with sodium bicarbonate) per milligram of transferrin.

2.3. Preparation of complexes

First, methyl alcohol was removed from the commercial PAMAM

solution by rotary evaporation under reduced pressure. Then, the film was

hydrated with Buffer HEPES (10 mM) Glucose 10% w/v (pH 7.4) (BHG) to a

final concentration of 0.25 mg/mL. Plain dendriplexes were formed by

incubating equal volumes of pDNA and dendrimers at room temperature (20-

25ºC) for 15 minutes. Complexes were formulated at different N/P ratios,

which represented the relation of positive equivalents of cationic component to

negative charge equivalents of the nucleic acid. Control polyplexes formulated

with bPEI were formed using the same protocol.

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116

Targeted dendriplexes were prepared similarly to plain dendriplexes but

an amount of commercial PAMAM was substituted by the P-Tf conjugate. This

means that, commercial PAMAM was partially replaced by P-Tf and condensed

with pDNA at different N/P ratios. The degree of replacement is represented

as percentage (e.g. 25% means 1 part of P-Tf plus 3 parts of commercial

PAMAM).

2.4. Gel retention studies

For gel retention studies, complexes containing 1 µg of pDNA prepared

at different N/P ratios in BHG were electrophoresed through a 0.8% agarose

gel using TBE buffer (100 mM Tris, 90 mM boric acid, 1 mM EDTA, pH 8.4).

The gel was stained with ethidium bromide, electrophoresed for 1h, 100 mV

and visualized under UV illumination (Doc 2000, Bio-Rad, USA).

2.5. DNAse I protection assay

Dendriplexes were prepared at different N/P ratios and containing

different percentages of P-Tf at a final concentration of 100 µg of pDNA/mL

in a total volume of 20 µL. After the formation of the complexes, DNAse I (1

U/µg pDNA) was added to each sample and the mixtures were incubated at

37ºC for 1h. In order to stop the DNAse I activity and disassemble the

dendriplexes, 5 µL of EDTA 0.25 M and 5 µL of SDS 10% in water were

added. Then, the samples were electrophoresed as described in the gel retention

studies. Non-treated and digested pDNA were included as controls.

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117

2.6. Size and zeta potential determination

Particle size was measured by Dynamic Light Scattering (DLS) and the

overall charge by zeta-potential measurements, using a particle analyzer (Zeta

Nano Series; Malvern Instruments, Barcelona, Spain). Samples were prepared

as previously described in BHG at a final concentration of 10 µg pDNA/mL.

2.7. Cell culture

HepG2 (human hepatoblastoma), HeLa (human cervix carcinoma),

Neuro2A (murine neuroblastoma) and CT26 (murine colon carcinoma) cell

lines were obtained from the American Type Culture Collection (Rockville,

Maryland). Cell lines were maintained at 37ºC under 5% CO2 in Dulbecco’s

modified Eagle’s medium high glucose, supplemented with 10% (v/v) heat-

inactivated fetal bovine serum (FBS) and penicillin (100 units/mL),

streptomycin (100 µg/mL) and L-glutamine (4 mM) (Gibco BRL Life

Technologies, Barcelona, Spain). Cells were trypsiniced twice a week.

2.8. In vitro gene transfer

For each transfection, 100.000 cells were seeded in 1 mL of medium in

48-well culture plates (Iwaki, Japan) and incubated for 24 hours at 37ºC in 5%

CO2. The primary growth medium was removed and replaced with 0.3 mL of

new media and 0.2 mL of complexes formulated in BHG containing 1 µg of

pDNA per well. After 4 hours of incubation, complexes were removed and

replaced with cell culture medium containing 10% FBS. 48 hours later, cells

were washed with phosphate-buffered saline and lysed using 100 µL of

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118

Reporter Lysis Buffer (Promega, Madison, Wisconsin, USA) at room

temperature for 10 minutes, followed by two freeze-thaw cycles. The cell lysate

was centrifuged for 2 minutes at 12,000 g to pellet debris. 20 µL of the

supernatant were assayed for total luciferase activity using the Luciferase Assay

Reagent (Promega), according to the manufacturer’s protocol. A luminometer

(Sirius-2; Berthold Detection Systems, Innogenetics, Diagnóstica y Terapéutica,

Barcelona, Spain) was used to measure luciferase activity. The Bio-Rad Dc

Protein Assay (Bio-Rad Laboratories, USA), using bovine serum albumin as the

standard, was used for quantifying protein content. Data were expressed as

nanograms of luciferase (based on a standard curve for luciferase activity) per

milligram of protein.

2.9. Toxicity studies

Cell viability was quantified by Alamar Blue assay. 100,000 cells per well

were seeded and grown overnight in 48 well culture plates. Cells were

transfected as described previously. After 4 hours complexes were removed and

substituted by new fresh media. 48 hours later, media was removed and 1 mL

of 10% (v/v) Alamar Blue dye in medium supplemented with 10% FBS was

added to each well. After 2.5 hours of incubation at 37ºC, 200 µL of the

supernatant was assayed by measuring the absorbance at 570 and 600 nm. Cell

viability was calculated according to the formula:

cellscontrol

cellstreated

AA

AAViability

)(

)(100%

600570

600570

−

−

×=

Chapter 3: Evaluation of improved PAMAM-G5 based dendriplexes targeted to the transferrin receptor

119

2.10. Statistical analysis

Results are reported as the mean values ± standard deviation. Statistical

analysis was performed with SPSS 15.0 (SPSS®, Chicago, IL, USA). The

different transfection activities in vitro were compared with ANOVA (Tukey

post-hoc adjust). Differences were considered statistically significant at p<0.05.

3. RESULTS

3.1. Binding of pDNA in the complexes

The formation of complexes and the binding of dendrimers to DNA

were ensured by examining the retardation in the migration of the plasmid

DNA after agarose gel electrophoresis. Figure 1 shows that different N/P

ratios and percentages of P-Tf incubated with 1 µg of plasmid DNA resulted in

a total electrophoretic inmobilization of DNA in all the cases, showing the

binding of the plasmid inside the nanoparticles.

Figure 1. Retardation assay of plain and targeted nanoparticles containing increasing

percentages of P-Tf (0%, 16%, 25% and 50%) at different N/P ratios: N/P 2 (lanes 2,

3, 4, 5), N/P 4 (lanes 6, 7, 8, 9), N/P 6 lanes (10, 11, 12, 13). DNA was included as

control (lane 1).

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120

3.2. Plasmid protection inside the dendriplexes

To ensure the ability of targeted PAMAM-dendriplexes to protect the

complexed pDNA from the activity of DNAses, complexes were exposed to

DNAse I. Figure 2 shows that free plasmid is completely degraded by the

enzyme (lane 2) but it remains intact after the dissociation of the dendriplexes

(Lanes 3-14).

Figure 2. Protection assay. Untreated DNA (lane 1), DNA treated with DNAse I

(lane 2). Dendriplexes containing increasing percentages of P-Tf (0%, 16%, 25% and

50%) at different N/P ratios: N/P 2 (lanes 3, 4, 5, 6), N/P 4 (lanes 7, 8, 9, 10), N/P 6

(lanes 11, 12, 13, 14).

3.3. Characterization of particle size and zeta potential

Particle size and surface charge were characterized in order to study the

influence of both, the N/P ratio and the presence of the ligand transferrin

(Table 1).

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Table 1. Particle size, polydispersity index and surface charge at different N/P ratios

and increasing percentages of P-Tf (0, 16, 25 and 50%). The data are represented as

the mean ± s.d. of three independent measurements.

N/P Ratio % P-Tf Size (nm) PDI Zeta potential

(mV)

2

0 108.0 ± 0.8 0.25 8.0 ± 0.2 16 106.2 ± 0.6 0.32 6.7 ± 1.6 25 109.1 ± 1.0 0.25 6.5 ± 0.0 50 114.3 ± 0.5 0.23 3.3 ± 0.1

4

0 84.3 ± 1.3 0.27 17.5 ± 1.3 16 95.2 ± 0.4 0.18 8.3 ± 0.9 25 105.5 ± 0.6 0.18 5.8 ± 1.1 50 145.0 ± 1.6 0.25 2.2 ± 0.1

6

0 80.4 ± 0.9 0.24 22.1 ± 0.0 16 83.1 ± 0.5 0.25 11.0 ± 0.0 25 80.9 ± 0.2 0.22 11.1 ± 0.8 50 85.1 ± 1.6 0.23 8.1 ± 0.6

The DLS measurements showed particles in the nanometric range, with

good polydispersity index (PDI) values in all the cases, regardless of the

percentage of P-Tf included and the N/P ratio. A tendency to present smaller

particle size as the N/P ratio increases was shown, maintaining optimal

hydrodynamic diameters under 150 nm. The substitution of the commercial

PAMAM by the P-Tf conjugate did not produce any modification of the size

except for N/P 4, where particle size grew up to 145 nm in the case of 50% P-

Tf. All the complexes were stable and no aggregation was observed in any case.

The surface charge was positive for all the conditions assayed but the

presence of increasing percentages of P-Tf did produce a significant decrease of

the zeta potential up to almost electroneutral values for N/P 2 and 4 in the case

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of 50% P-Tf containing complexes (3.3 and 2.2 mV respectively). Zeta

potential values did also exhibit an increase as the N/P ratio grew.

3.4. In vitro transfection activity

Transfection activity of PAMAM/P-Tf/pDNA dendriplexes was

evaluated by using HeLa, HepG2, Neuro2A and CT26 cell lines (Figure 3). The

behaviour of the dendriplexes in the CT26 and HeLa cells showed a tendency

to increase luciferase activity when the percentage of P-Tf increased at any N/P

ratio. In CT26 cells at N/P 6, the dendriplexes containing 50% of P-Tf

produced the highest luciferase values, which resulted 32.5 times higher than

the plain dendriplexes (p<0.001). In HeLa cells, the substitution of 50% P-Tf in

the formulations did produce a statistically significant rise of the luciferase

activity compared to the plain dendriplexes at N/P ratios 4 and 6 (p<0.001).

The addition of 50% P-Tf to the formulation at N/P 6 ratio exhibited a

5.4 fold increase in the luciferase values (p<0.001) in HepG2 cells compared to

plain dendriplexes. The substitution of commercial PAMAM by P-Tf did not

result in any increment at N/P 2 and 4. The transfection with P-Tf in Neuro2A

cells did not show any improvement independently of the percentage of P-Tf

and the transfection activity was always significantly reduced. The increment in

the N/P ratio led to an increase in the transfection activity for each percentage

of substitution.

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Figure 3. In vitro transfection activity by plain and targeted dendriplexes containing

increasing percentages of P-Tf in CT26, HeLa, HepG2 and Neuro2A in the presence

of 10% FBS. Plain bPEI polyplexes were used as control. Data represent the mean ±

s.d. and are representative of three independent experiments.

Compared to control bPEI-polyplexes, PAMAM dendriplexes showed

higher gene expression values in HeLa, HepG2 and Neuro2A cells. In contrast,

in the CT26 cell line, control polyplexes achieved higher transfection activity

than dendriplexes.

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3.5. Toxicity studies

Alamar Blue dye was used to verify the toxicity of the new complex

(Figure 4).

Figure 4. In vitro cellular viability by plain (non-targeted) and targeted dendriplexes

containing increasing percentages of P-Tf in CT26, HeLa, HepG2 and Neuro2A in the

presence of 10% FBS. Plain bPEI polyplexes and non-treated cells were used as

control. Data represent the mean ± s.d. of three wells and are representative of three

independent experiments.

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The result confirmed that plain PAMAM dendriplexes and the mixtures

with P-Tf did not exhibit toxicity in the conditions assayed for HeLa, CT26 and

HepG2 and viability values were always over 80% (Figure 4). For Neuro2A, cell

viability drop up to 60%. Compared to bPEI polyplexes, plain and targeted

dendriplexes were less toxic in all the conditions assayed.

3.6. Uptake mechanism

In order to clarify if the uptake mechanism of P-Tf conjugates was

mediated via receptor-specific endocytosis, a competitive inhibition experiment

was performed in HeLa cells. Transfection activity in the presence of an excess

of free Tf (5 mg Tf/mL) showed a decrease of the luciferase expression values

when commercial PAMAM had been substituted by P-Tf, independently of the

percentage of substitution (p<0.05). Non-targeted dendriplexes showed similar

transfection values after blocking the transferrin receptor (Figure 5).

Figure 5. Competition assay by adding an excess of free Tf (5 mg/mL) to HeLa cells

previous to transfection of plain and targeted dendriplexes.

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4. DISCUSSION

The major disadvantages of non-viral systems are the low efficacy of

transfection and transient expression of the transgene. For this reason, progress

has been made towards the development of new strategies for gene transfer

improvement, for instance, coupling cell-binding ligands or antibodies in order

to achieve target specificity. In our case, we have formulated dendriplexes for

targeted gene transfer with increased and improved specificity to the transferrin

receptor (TfR).

The chemical synthesis of the new PAMAM-Transferrin (P-Tf)

conjugate was performed by Wolfgang Rödlt (Department of Pharmaceutical

Biotechnology, Ludwig Maximilians University, Munich, Germany) and was

carried out in a similar way to the one used by Kircheis et al. for PEI based

polyplexes [19]. This process was always performed at room temperature;

therefore, the differences generated during the subsequent experiments have to

be related to the presence of transferrin and it they are not likely to be due to

different processes that can increase the activity of PAMAM dendrimers, e.g.

activation of the structure.

Firstly, the capacity of the new conjugate P-Tf to bind to the pDNA

and form stable dendriplexes was evaluated at different N/P ratios (Figure 1).

The absence of signal compared to the naked pDNA control showed the great

capacity of these compounds, independently of the percentage of P-Tf included

and the N/P ratio, to form stable particles able to bind pDNA and avoid gel

migration. Secondly, the ability of those particles to protect the included pDNA

from the activity of DNAse I enzyme was assayed (Figure 2). In this case, the

presence of PAMAM dendrimer, even at low N/P ratios, led to the protection

of the included pDNA, while naked pDNA was completely digested. These

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data provide an interesting basis for considering the P-Tf conjugate as a

promising non-viral gene delivery carrier in vivo since, once put into circulation,

vectors are subjectes to serum inactivation and enzymatic degradation of the

complexes, causing the loss and disappearance of the pDNA and the

subsequent effect [33, 34].

After ensuring the formation of particles in the presence of pDNA, size

and surface charge were measured (Table 1). The inclusion of the P-Tf

conjugate produced nanoparticles with good features for their use as

transfecting agents. Size was always between 80 and 150 nm with excellent PDI

values below 0.3. Increasing the N/P ratio produced a drop in the particle size,

probably related to the presence of more positive charges that can enhance the

condensation of the pDNA leading to smaller hydrodynamic diameter. Zeta

potential values were always positive and increased as a function of the excess

of positive charges included in the formulation (N/P ratio). The addition of

increasing percentages of P-Tf led to a decrease of the surface charge, down to

almost electroneutral zeta potential values, in accordance with the anionic

structure of the protein and previous studies [19, 22]. It is known that

electroneutral values may generate an increase in particle size since the absence

of charge repulsion leads to the formation of bigger aggregates. This relation

between size and surface charge has been described for liposomes [35, 36].

Nevertheless, our data point out that, despite presenting a slight increase in

particle size when the highest amount of P-Tf is included, stable nanoparticles

are formed, even at low N/P ratios without presenting important differences

between targeted dendriplexes and non-targeted ones. Therefore, the

subsequent variations of the transfection activity of these dendriplexes should

be related to the presence of Tf and cannot be attributed to unequal sizes of the

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formulations. These data are in agreement with the presented results of

protection against DNAse I and pDNA immobilization (Figures 1 and 2).

The experiments performed in order to evaluate the transfection

capacity of the P-Tf conjugates showed a tendency to increase the transfection

activity except in Neuro2A. For CT26 and HepG2 cell lines, this increment was

statistically significant at N/P ratio 6 containing 50% of P-Tf (Figure 3).

Probably, this fact is related to the lower zeta potential values of targeted

particles at N/P 2 and 4 compared to the ones formulated at N/P 6. The

inclusion of anionic molecules, usually, in order to shield the surface charge, has

been reported to produce complexes with lower transfection activity, since the

drop of the surface charge generates a blockage in the non-specific adsorptive

endocytosis [37, 38]. The absence of improvement in the HepG2 cell line at

N/P 2 and 4 could also be related to the TfR data reported by Sakaguchi et al.

[39], who claims that, apparently, the expression of the TfR is low for HepG2

cells. Moreover, the comparison of the TfR internalization results between

different cell lines suggested that this cell line presents low internalization

values, which was considered an indicative of low activity of the receptor

mediated endocytosis. This implies that a large fraction of the TfR is trapped in

early endosomes of a static nature. Hence, it may be possible that, with the

lowest N/P ratios, 2 and 4, transfection could not be carried out as the

presence of Tf could not be enough for enhancing luciferase expression and

higher zeta potential values might be needed for an effective gene transfer.

HeLa cells, considered a positive cell line for TfR [39], presented a better

transfection pattern for N/P 4 and 6 and a statistically significant increment

was described when 50% of commercial PAMAM was substituted by P-Tf

conjugate. Between both ratios, differences could not be described and similar

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values were achieved. N/P 6 ratio was selected as the best condition in order to

perform the subsequent competition studies.

In contrast with previous results, where Neuro2A, a TfR positive cell

line [40], is used as a model with TfR [41], this cell line did not show any

improvement by using P-Tf conjugates. In this case, transfection activity

mediated by Tf-targeted dendriplexes was always lower than the one mediated

by the non-targeted dendriplexes, being statistically significant for all the N/P

ratios when 50% of P-Tf was included. The reason why this cell line presents

different expression patterns for the same complexes could be related to the

fact that transfection processes are highly dependent on the cell line and the

complex used [42]. Apart from that, cell lines will not only differ in their level

of TfR, but also in the presence of glycosaminoglycans, such as heparane

sulfate, which can contribute to mask the transferrin-mediated gene transfer by

the adsorptive endocytosis. Moreover, another possible reason is that the large

Tf molecule may be hampering endosomal release, which could be more

prominent in the Neuro2A cells than in the other cell lines tested. Given that,

despite Neuro2A being widely used as a well transfectable cell line and tumor

model in gene therapy, the uptake via TfR with PAMAM dendrimers might be

less advantageous. bPEI based polyplexes were included as a control.

Compared to dendriplexes, control polyplexes presented lower transfection

values in HeLa, HepG2 and Neuro2A, whereas in CT26 cells poyplexes

achieved slightly higher transfection activity.

The toxicity of the new P-Tf conjugate had not been previously studied.

As shown in Figure 4, plain and targeted dendriplexes turned out to be non-

toxic and cell viability was always maintained over 80% except for Neuro2A

cells. Branched PEI containing polyplexes were much more toxic than

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dendriplexes, especially in HeLa and Neuro2A cell lines, where the decrease

was statistically significant. The absence of acute toxicity supports the use of P-

Tf conjugate and suggests that the differences found in the transfection activity

studies must be related to the presence of TfR and the P-Tf conjugate, which is

supposed to enhance the expression of the transgene, and not with a different

viability of the cells.

Finally, in order to ensure the uptake route via receptor mediated

endocytosis, HeLa cells were incubated with an excess of free Tf, as described

for other TfR targeted complexes [41]. The result pointed out that the

transfection activity is inhibited by the presence of free Tf in the transfection

medium, which can produce a saturation and subsequent reduction of the

transfection efficacy by blocking the Tf-receptor (Figure 5).

5. CONCLUSIONS

In this work we have evaluated the ability of a new PAMAM-

Transferrin conjugate to form nanoparticles in the presence of pDNA and its

transfection activity. The results showed that the dendriplexes containing the

novel P-Tf conjugate were able to form stable particles and protect the

complexed pDNA. The particles showed nanometric size with positive surface

charge, although the inclusion of Tf led to a decrease of the zeta potential

values. The presence of P-Tf in the complexes formulated at N/P 6 produced a

statistically significant increase in the transfection activity when 50% of P-Tf

was included in CT26, HeLa and HepG2 cell lines, whereas in Neuro2A the

substitution of 50% of commercial PAMAM by P-Tf always led to a decrease in

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131

the gene expression. Moreover, the competition assay confirms that the uptake

is mediated by specific receptor-mediated endocytosis. Targeted and plain

dendriplexes exhibited low toxicity, except for Neuro2A cell line, where cell

viability fell up to 60%. The toxicity of P-Tf dendriplexes were found to be

lower compared to that of bPEI-polyplexes, frequently used as non-viral gene

delivery systems.

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CHAPTER 4

NOVEL PAMAM-PEG-PEPTIDE CONJUGATES FOR siRNA DELIVERY TARGETED TO THE TRANSFERRIN AND

EPIDERMAL GROWTH FACTOR RECEPTORS

Chapter 4: Novel PAMAM-PEG-Peptide conjugates for siRNA delivery targeted to the transferrin

and epidermal growth factor receptors.

139

ABSTRACT

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 structure via a PEG 2 kDa chain with

the aim of improving the silencing capacity of the PAMAM based dendriplexes.

The dendriplexes showed an excellent binding capacity to the siRNA with a

maximal condensation at N/P 2. The nanoparticles formed exhibited

hydrodynamic diameters below 200 nm and positive zeta potential although

PEG containing complexes showed 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 cassete. The dendriplexes containing a specific

siRNA-Luc, 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 a unspecific siRNA-Control. In all the conditions

assayed, dendriplexes were found to be no-toxic and viability was always above

75%.

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and epidermal growth factor receptors.

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1. INTRODUCTION

During last years, research has focused on the improvement of non-

viral systems for gene therapy. In order to achieve better vectors with enhanced

gene delivery activity, different ligands have been included in the formulations.

The Epidermal Growth Factor Receptor (EGFR) and Transferrin Receptor

(TfR) are two of the most used target sites for specific delivery of nucleic acids,

since they are overexpressed on the surface of tumoral cells compared with

healthy tissues and have demonstrated good activity for drug and gene delivery

[1].

The gene transfer capacity of targeted non-viral vectors and novel drug

delivery systems including specific ligands has proven to be effective in

increasing internalization and delivery of the cargo in vitro and in vivo [2].

However, the inclusion of large proteins in a formulation may result in

problems including synthesis procedures and stability [3], therefore, the

necessity of smaller synthetic ligands has increased. The peptides GE11 and B6

do not contain more than 15 aminoacids and bind specifically the EGFR and

TfR respectively. B6, described by Xia et al. in a phage display library [4], has

been used for targeting of LPEI polyplexes [3], peptide-PEG-

oligo(ethaneamino)amides [5] and other polymeric systems [6, 7]. In a similar

way, GE11 peptide was described by Li et al. [8] and subsequently used for the

targeting LPEI complexes and other polymers [9, 10] with similar results to the

ones obtained by EGF protein even in vivo [9]. Concerning the use of these

peptides with PAMAM dendrimers, the main objective has pursued the

enhancement of the uptake and transfection rate of adenovirus electrostatically

coated with PAMAM-G2-PEG-GE11 [11-13].

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and epidermal growth factor receptors.

142

The use of small interfering RNA (siRNA) has become one interesting

tool for the specific knockdown of disease-causing genes. A siRNA molecule is

constituted by, approximately, 20 pair of bases highly specific, which are able to

activate a complex cellular mechanism that leads to the destruction of a

messenger RNA (mRNA). This interaction will have as a consecuence the

disappearance of the protein codified by the specific mRNA. So far, the use of

siRNA in non-viral systems has been based on the use of cationic polymers

such as polyethyleneimine and chitosan [14, 15]. PAMAM dendrimers are

considered an interesting alternative to other cationic polymers due to its low

cytotoxicity, controlled molecular weight, high charge density and versatility

[16]. Nevertheless, its use as siRNA delivery system has been limited. Zhou et

al. reported the ability of non-degraded PAMAM dendrimers to form

nanoparticles in the presence of RNA [17] and other groups have tried to

describe the binding mechanism between PAMAM and siRNA, as well as the

importance of the N/P ratio, ionic strength of the medium, the size and the

dendrimer generation on the formation of complexes [18-20]. The activity of

this family of molecules has also been studied [21] and, as the presence of

targeting is known to improve the activity of non-viral vectors,

RNA/dendriplexes have been targeted to the CD44 receptor [22], TfR [23] and

EGFR [24]. At the same time, Tsutsumi et al. and Arima and coworkers have

extensively studied the behaviour of PAMAM dendrimer/α-cyclodextrin

conjugates [25-29] and Biswas et al. have improved the behaviour of the

PAMAM dendrimer by generating a micellar formulation of PAMAM-PEG-

DOPE/siRNA complexes [30]. The different strategies adopted for improving

the siRNA delivery activity of PAMAM/siRNA complexes has been extensively

reviewed elsewhere [31, 32].

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and epidermal growth factor receptors.

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For the first time, in this work we have evaluated the ability to form

nanoparticles in the presence of siRNA, the gene silencing capacity and

cytotoxicity of two new PAMAM conjugates. These novel carriers are coupled

to the specific peptides GE11 or B6 via a PEG 2 kDa linker, which have

previously demonstrated to be effective in improving gene delivery in other

non-viral systems.

2. MATERIALS AND METHODS

2.1. Materials

DMSO (dimethylsulfoxide purissimum), DTT (DL-dithiothreitol) and

TNBS (2,4,6-trinitrobenzenesulfonic acid solution) were obtained from

Sigma.Aldrich GmbH (Munich, Germany). Water was used as purified, de-

ionized water. NHS-PEG-OPSS (ω-2-pyridyldithio polyethylene glycol α-

succinimidylester, 2 kDa) was synthesized by Rapp Polymere GmbH

(Tübingen, Germany). Peptides GE11 (CYHWYGYTPQNVI-OH, TFA salt,

>95% purity) and B6 (GHKAKGPRK-OH, TFA salt, >95% purity) by

Biosyntan GmbH (Berlin, Germany). Linear polyethylenimine 22 kDa (LPEI)

was synthesized as described by Schaffert et al. [33] by Wolfang Rödlt

(Pharmaceutical Biotechnology, Center for System-based Drug Research,

Department of Pharmacy, Ludwig Maximilians University, Munich, Germany).

Amine terminated PAMAM dendrimer (Generation 5, 1,4-diaminobutane core,

molecular weight 28,854 Da) was purchased from Dendritic Nanotechnologies

(Mount Pleasant, MI, 48858, USA). HEPES (4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid) was obtained from Biomol GmbH (Hamburg,

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Germany). H-LCysteine fron IRIS Biotech GmbH (Marktredwitz, Germany)

and MacroPrep High S from BioRad GmbH (Munich, Germany). Dialysis was

performed with Spectra/Por membranes (molecular mass cut-off 10 kDa) from

Spectrum Laboratories Inc. (Breda, Netherlands). Cell culture 5X lysis buffer

and D-Luciferin sodium salt were purchased from Promega (Mannheim,

Germany). Cell culture media, antibiotics, L-alanine-L-glutamine and non-

essential aminoacids (NEAA) were obtained from Biochrom (Berlin,

Germany). Fetal calf serum was purchased from Gibco (Life Technologies,

Carlsbad, USA).

2.2. Conjugate synthesis

Conjugate syntheses were performed by Wolfgang Rödl

(Pharmaceutical Biotechnology, Center for System-based Drug Research,

Department of Pharmacy, Ludwig Maximilians University, Munich, Germany)

2.2.1. Synthesis of PAMAM-PEG-OPSS

Synthesis of PAMAM-PEG-OPSS was carried out in principle as

described recently for LPEI-PEG-OPSS [9] with some modifications. In brief,

PAMAM-G5 in EtOH was reacted with NHS-PEG-OPSS (2kDa) dissolved in

DMSO for 3 h under agitation at 37ºC. Thereafter, 2 M HEPES pH 7.4, 3 M

NaCl and water were added to give a final concentration of 20 mM HEPES

and 0.6 M NaCl and pH adjusted to 7.4 using hydrochloric acid. The reaction

mixture was loaded on a cation-exchange column (Macro-prep High S; 10/10;

BioRad, Munich, Germany) and fractionated with a salt gradient from 0.6 to 3

M NaCl in 20 mM HEPES, pH 7.4. The product eluted between 2 and 2.6 M

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NaCl and was dyalized overnight at 4ºC against HBS (20mM HEPES, 150 mM

NaCl, pH 7.4) with a Spectra/Por membrane (molecular mass cut-off 10 kDa).

The PAMAM content of the conjugate was determined by TNBS assay, which

is used for detection of primary amines. To ascertain the quantity of linker

coupled to the PAMAM, the amount of dithiopyridine after reduction of an

aliquot with dithiothreitol (DTT) was evaluated by spectrophotometric

measurement of released pyridine-2-thione at 343 nm. PAMAM-PEG-OPSS

was synthesized at a final molar ratio of 1:1.1.

2.2.2. Conjugation of GE11 and B6 peptides to PAMAM- PEG-OPSS

The peptides GE11 and B6 were added to PAMAM-PEG-OPSS. After

3h incubation at room temperature, the released thiopyridone was measured at

343 nm to determine the extent of peptide conjugation. Removal of unreacted

peptides was carried out on a Macroprep High S cationic-exchange column

(Bio-Scale MT 2, BioRad, Munich, Germany) as described previously. The

amount of peptide was calculated via the extinction coefficient at 280 nm. The

molar ratio of PAMAM to GE11 was 1:0.47 and 1:0.5 for B6.

2.2.3. Synthesis of PAMAM-PEG-Cys

PAMAM-PEG-Cys was synthesized by mixing one part of PAMAM-

G5-PEG-OPSS with four parts of cysteine at ambient temperature. Purification

was carried out on a gel-filtration column (Sephadex G-25; HR10/30 columns;

20 mM HEPES, pH 7.4) to remove piridine-2-thione and unreacted cysteine.

The molar ratio of PAMAM5 to Cys was 1:1.1.

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2.3. Preparation of PAMAM-PEG-Peptide/siRNA and LPEI/siRNA

complexes

In general, polyplexes were generated by condensing specific siRNA-

Luc or siRNA-Control with PAMAM or PAMAM conjugates at different N/P

ratio of PAMAM nitrogen to RNA phosphate. Therefore, PAMAM/siRNA

polyplexes were freshly prepared at a final siRNA concentration of 10 µg/ml in

HEPES-buffered glucose (BHG 5 % (w/w) glucose, 20 mM HEPES, pH 7.4)

by mixing equal volumes of BHG containing the desired amount of siRNA and

PAMAM or LPEI. Dendriplexes and polyplexes were allowed to stand for at

least 20 min at room temperature before use.

2.4. Particle size and zeta potential measurements

Particle size of dendrimer/siRNA formulations and zeta potentials were

measured by laser-light scattering using a Zetasizer Nano ZS (Malvern

Instruments, Worcestershire, U.K.). Polyplexes were prepared at a final

concentration of 10 µg siRNA/mL in BHG and measured after 30 min of

incubation.

2.5. Ethidium bromide exclusion assay

A Cary Eclipse fluorescence spectrophotometer (Varian, Germany) was

used for the quantification of Ethidium bromide (EtBr) fluorescence at the

excitation wavelength λex = 510 nm and emission wavelength λem = 590 nm.

1 ml HBG buffer containing 0.4 µg EtBr was used as blank. After addition of

10 µg siRNA-Control the solution was incubated for 3 minutes and EtBr

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fluorescence was assigned to 100%. Increasing amounts of PAMAM or

PAMAM-PEG-Peptide corresponding to indicated N/P ratios were added,

incubated for 30 seconds and EtBr fluorescence was determined in relation to

the 100% value.

2.6. Cell culture

LS174T/eGFPLuc cell line (LS174T) (colorectal adenocarcinoma) was

cultured in Roswell Park Memorial Institute 1640 medium (RPMI 1640) and

HeLa/eGFPLuc (HeLa) (cervix adenocarcinoma) cells were cultured in DMEM

(Dulbecco’s modified eagle medium). Both cell lines were stably transfected

with the eGFPLuc gene cassette. All cell culture media was supplemented with

10% fetal calf serum, 100 units/mL penicillin, 100 µg/mL streptomycin and

1% L-alanine-L-glutamine (200 mM). All cultured cells were grown at 37°C in

5% CO2 in humidified atmosphere. Cells were passed twice a week by

trypsinization.

2.7. Gene silencing capacity

HeLa and LS174T cells were cultured as described before. For each

experiment 104 cells were seeded in 96 well plates 24 h before the treatment.

Complexes containing specific siRNA-Luc or siRNA-Control were freshly

prepared at the indicated N/P ratios. 20 µL containing 200 ng of siRNA and 80

µL of medium were added to each well and incubated for 4 h. After this time,

transfection medium was removed and substituted by 100 µL of fresh culture

medium. After 48 h cells were rinsed with PBS and treated with Lysis Buffer

(Promega, Germany). 30 µL of the lysate were assayed with the Luciferase

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Assay Kit (100 µL Luciferase Assay buffer, Promega, Germany) in a Centro LB

960 plate reader luminometer (Berthold, Germany). The relative light units

(RLU) obtained were normalized and presented as the percentage of expression

compared to non-treated control cells.

2.8. Toxicity studies

HeLa and LS174T cells were seeded into 96-well plates at a density of

104 cells/well. After 24 h, culture medium was replaced with 80 µL fresh

growth medium and 20 µL of transfection complexes. 4 h after, transfection

media was replaced by fresh medium. 48 h post transfection, 10 µL MTT (5

mg/mL) were added to each well reaching a final concentration of 0.5 mg

MTT/mL. After an incubation time of 2 h, unreacted dye and medium were

removed. The purple formazan product was dissolved in 100 µL DMSO/well

and quantified by a microplate reader (Spectrafluor Plus, Teca Austria, GnbH,

Grödig, Austria) at a 590 nm with background correction at 630 nm. The

relative cell viability (%) compared to control cells containing cells treated with

HBG was calculated according to the formula:

cellscontrol

cellstreated

AA

AAViability

)(

)(100%

630590

630590

−

−

×=

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2.9. Statistical analysis

Results are reported as the mean values ± standard deviation. Statistical

analysis was performed with SPSS 15.0 (SPSS®, Chicago, IL, USA). The

different transfection activities in vitro were compared with ANOVA (Tukey

post-hoc adjust). Differences were considered statistically significant at p<0.05.

3. RESULTS

3.1. Size and zeta potential determination

Particle size and surface charge were measured in order to study the

influence of the N/P ratio and the presence of the different ligands (Table 1).

Size measurements were performed by dynamic light scattering. As

shown in Table 1, particle size was always below 200 nm, presenting excellent

hydrodynamic diameter independently of the N/P ratio used. The presence of

the ligand seemed to have little effect on the particle size. Plain and GE11

containing conjugates presented similar size, PAMAM-PEG-B6 complexes

exhibited the smallest sizes, whereas PAMAM-PEG-Cys presented increased

diameters and high polydispersity index (PDI). The other formulations showed

PDI values below 0.3.

Surface charge was always positive, with zeta potential values above 20

mV in all the cases. The formulation of the dendriplexes with PAMAM

conjugates containing a PEG 2 kDa chain produced nanoparticles with lower

zeta potential compared to plain dendriplexes, independently of the N/P ratio

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used. The presence of the targeting peptide did not influence the zeta potential

results.

Table 1. Particle size, polydispersity index and surface charge in the presence or

absence of ligands and different N/P ratios. The data are represented as the mean ±

s.d. of three measurements.

N/P Size (nm) PDI Zeta potential

(mV)

Plain 2 109.1 ± 0.8 0.15 32.1 ± 1.1 4 114.7 ± 0.7 0.16 37.6 ± 0.3 6 122.6 ± 0.9 0.17 40.0 ± 1.0

GE11 2 106.6 ± 0.3 0.09 23.0 ± 2.0 4 105.7 ± 0.8 0.17 27.4 ± 0.5 6 107.5 ± 1.1 0.23 27.7 ± 1.8

B6 2 71.2 ± 1.0 0.12 23.5 ± 0.9 4 75.5 ± 0.8 0.21 20.3 ± 0.6 6 n.d. n.d. n.d.

Cys 2 103.0 ± 3.0 0.24 21.1 ± 1.1 4 125.0 ± 29.0 0.37 25.4 ± 1.1 6 199.3 ± 19.7 0.51 26.0 ± 2.0

3.2. Ethidium bromide exclusion assay

The interaction between PAMAM dendrimers and siRNA was

evaluated by the ability of dendrimers to displace the intercalating dye, ethidium

bromide, from the siRNA. As shown in Figure 1, as the charge ratio of the

complexes increased, the relative fluorescence decreased to a maximum binding

degree at N/P ratio 2. The formulation of the dendriplexes with PEG linked

peptides did not produce any difference in the behaviour of the complexes and

similar profiles were obtained.

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Figure 1. Condensation assay. siRNA condensation measured as a decrease in

fluorescence of EtBr added to dendriplexes.

3.3. Gene silencing efficacy

In order to explore the blockage of the luciferase expression in two cell

lines stably transfected with the cassette eGFPLuc, a specific siRNA (siRNA-

Luc) was used in order to block the expression and unspecific siRNA (siRNA-

Control) was used as a control.

In HeLa cell line (Figure 2), the treatment with siRNA-Control

produced a decrease in the expression of approximately 20% for all the

dendriplexes assayed compared to non-treated cells. The inhibition mediated by

the specific siRNA-Luc carring dendriplexes, compared to control cells, was

slightly higher and statistically significant at N/P 4 and 6 ratios. This drop,

compared to siRNA-Control, represented a mean decrease of 14% of the

luciferase expression values. The inclusion of the different ligands was not able

to produce differences in any case compared to plain dendriplexes and Cys

0

20

40

60

80

100

120

0 2 4 6 8 10

% F

luo

resc

en

ce

N/P ratio

PAMAM G5 PAMAM-PEG-B6

PAMAM-PEG-GE11 PAMAM-PEG-Cys

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conjugate containing dendriplexes. LPEI polyplexes carring specific siRNA-Luc

were able to generate a statistically significant decrease in luciferase expression

values compared to non-treated cells and the siRNA-Control containing LPEI

polyplexes.

Figure 2. Luciferase silencing efficacy of non-targeted PAMAM and PAMAM-PEG-

GE11/B6/Cys dendriplexes with specific siRNA-Luc or siRNA-Control complexes

prepared at various N/P ratios. Provided data represent the percentage of expression

compared to non-treated (control) cells. Data are expressed as the mean ± SD (n=8).

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Gene silencing efficacy was also assayed in LS174 cell line (Figure 2),

where control-siRNA containing dendriplexes produce an average drop of 5%

of the luciferase expression values. The mean decrease of the siRNA-Luc

containing dendriplexes was around 20% compared to siRNA-Control. The

inclusion of targeted dendrimers did not produce any difference in the

inhibitory activity of the dendriplexes compared to non-targeted ones. LPEI

polyplexes were able to mediate an effective fall of the luciferase values,

however, comparing the effect of the specific siRNA-Luc with siRNA-Control,

the difference was not statistically significant.

3.4. Toxicity studies

For all the dendriplexes tested in this work and for all the N/P ratios,

MTT assay was performed (Figure 3). Viability values of dendriplexes were

always above 75% in HeLa and LS174T cells. LPEI polyplexes exhibited similar

viability values to those obtained for dendriplexes in HeLa, whereas in LS174

cells LPEI treatment lead to a drop of viability values below 60% compared to

non-treated cells. Between LPEI polyplexes containing specific siRNA-Luc and

siRNA-Control in HeLa cells, similar values were obtained, however, in

LS174T cells, LPEI polyplexes containing specific siRNA-Luc turned out to be

more toxic than PAMAM-dendriplexes, resulting in a drop in the viability down

to 25%.

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Figure 3. Viability of HeLa and LS174T cells. LPEI polyplexes, plain and targeted

PAMAM conjugates with specific siRNA-Luc or siRNA-Control were applied at

different N/P ratios. Non-treated cells were included as control. Data are expressed as

the mean ± SD (n=4).

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4. DISCUSSION

One of the main problems to be overcome concerning the use of non-

viral systems is the low efficiency and selectivity. The TfR is known to be

overexpressed in a variety of tumor cells, as well as the EGFR receptor.

Therefore, these receptors are an interesting target for gene therapy

improvement. As Scholz et al. claims, the presence of big proteins can lead to

problems during synthesis and storage [15]. In order to solve this drawback, the

peptides B6 and GE11, specific for the TfR and EGFR respectively, were

selected as the targeting moieties to improve the activity of the

PAMAM/siRNA dendriplexes.

The size and zeta potential measurements confirmed that the PAMAM

dendrimer and PAMAM-PEG-Peptide conjugates were able to form

nanoparticles with positive surface charge (Table 1). Apparently, significant

differences in size could not be described between the plain dendriplexes and

the targeted ones, although PAMAM-PEG-B6 containing dendriplexes

presented slightly smaller sizes, which could be related to the cationic nature of

the B6 ligand. On the other hand, PAMAM-PEG-Cys complexes showed the

highest hydrodynamic diameter and PDI. Zeta potential values exhibited a clear

drop comparing plain to targeted conjugates. The presence of the linker PEG 2

kDa is likely to be the reason of this variation since the PAMAM-PEG-Cys

conjugate containing dendriplexes, which do not contain a peptide in its

structure, do also present this tendency. In agreement with previous data, the

PEG 2 kDa chain produced a decrease in the zeta potential values but did not

lead to completely neutral charged particles in LPEI based polyplexes [3, 9].

Moreover, it is known that PEG can shield the surface charge of the complexes

[34].

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One aspect to be considered during the optimization of this kind of

complexes is the importance of the polymer flexibitiliy. In this respect, as

siRNA strand contains only up to 20-25 pairs of bases, the structure of the

polymer plays an important role during coupling and gene silencing. Kwok et al.

studied the stability of LPEI and bPEI siRNA polyplexes, concluding that

bPEI showed a better behaviour, due to its more flexible branched structure

[35]. More flexible PAMAM dendrimers have reported to improve the activity

in gene delivery [36]. This approach has been adapted and bigger initial cores of

the molecule have been selected with good results [17]. In this chapter, the

PAMAM structure, instead of the etilendiamine one used for pDNA delivery,

presents a 1,4-diaminobutane core, which can provide higher flexibility to the

dendrimer. Subsequently, to ensure the formation of a complex, in which the

RNA was tightly condensed, an ethidium bromide exclusion assay was

performed (Figure 1). The results showed that plain dendriplexes were able to

efficiently bind and condense RNA at N/P 2 and higher ratios. The presence of

the peptide or the PEG 2 kDa chain did not produce any disturbance of the

behaviour. These data agree with previous results, which reported that PEG

with this molecular weight produced a decrease of the surface charge but it did

not negatively influence the DNA condensation of LPEI-PEG-GE11/DNA

complexes [9].

The activity of the new conjugates was assayed in modified HeLa and

LS174T cell lines stably transfected with the eGFPLuc cassette (Figure 2). The

silencing effect of the unspecific siRNA used as control was negligible. When a

specific siRNA-Luc was used, the expression values fell a 14% and 20% on

average in HeLa and LS174T cells respectively compared to the unspecific

siRNA-Control. The PAMAM-PEG-Cys conjugate did not produce any

important variation of the silencing activity of the complexes compared to the

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plain dendriplexes as the silencing efficacy was in the same range. Similarly, the

conjugation with the specific peptides B6 and GE11 did not produce an

improvement of the inhibitory activity of the dendriplexes compared to non-

targeted dendriplexes. This lack of improvement, compared to plain

dendriplexes, can be partially explained by the presence of PEG, as this

molecule is known to reduce the gene delivery activity of different formulations

[34, 37]. Schafer et al. explored the possibility of targeting LPEI conjugated with

the GE11 peptide (LPEI-PEG-GE11/pDNA complexes). In U-87 MG cells,

the LPEI-PEG-GE11 conjugate was not able to produce higher transfection

values compared to non-targeted polyplexes. As they claim, the detargeting can

be related to the reduced surface charge or the influence of bulky molecules on

the endosomal release [9]. Similar results have been reported by Nie et al. using

the peptide B6 as a targeting agent in LPEI-PEG-B6/pDNA based polyplexes.

Transfection values in PC3 cells were in a similar range compared to the non-

targeted control, nevertheless, the B6 containing polyplexes were able to

enhance the cellular association [3]. The siRNA molecule has reached its place

of action once in the cytosol and an early release from the endosome is highly

desired [15]. The TfR and EGFR-mediated endocytosis are known to imply

degradation processes at some extent [38]. At this respect, Martin et al. were

able to enhance the transfection activity of B6 peptide-PEG-oligo(ethane-

amino)amides only by the presence of chloroquine or other endosomolytic

agents [5]. Therefore, if the presence of the PEG chain may block the

endosomal release, and a lytic pathway is favored by the presence of the ligand,

the proton sponge mechanism of the PAMAM dendrimer structure could not

be enough for promoting the rupture of the vesicles and the lack of

improvement in gene silencing efficacy could be partially expected.

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Despite the use of these peptides having not been explored with

PAMAM dendrimers in gene delivery, Vetter et al. studied the ability of the

GE11 peptide to enhance the transfection activity of adenovirus type 5 (Ad)

and achieve a targeted expression in EGFR positive tumors with the binding of

Ad to PAMAM-PEG-GE11 conjugates [13]. They compared the PAMAM-G5-

PEG-GE11 conjugates with the PAMAM-G2 ones and concluded that

PAMAM-G5 was less efficient in terms of targeting compared to G2. The

different molecular weight and coupling rate, as well as the necessity of a

cooperative binding of several GE11 peptides are the explanations they suggest

for this difference [13]. At this respect, the peptide:dendrimer molar ratio

should be optimized and new chemical syntheses carried out to modify this

ratio. In close relation with the amount of ligand, the presence of the receptor

can play an important role in the gene silencing process. The overexpression of

the TfR and EGFR was contrasted in the bibliography. HeLa cell line has been

reported to present high amounts of TfR in its surface [39] as well as EGFR

[40, 41], similarly LS174T cell line is considered as positive for both receptors

[42-44]. It is known that the receptor expression varies from one cell batch to

another and, hence, differences in the endocytic processes may be expected.

Moreover, it is known that each cell line brings a specific behaviour with each

complex [45]. Because of that, the presence of the specific receptor must be

ensured to correlate the behaviour of the conjugates.

As expected, the viability values of the targeted complexes was similar

to the one produced by the non-targeted dendriplexes and was always above

75% (Figure 3), which clearly agrees with previous reported data in this work

(Chapter 2, Figure 5 and Chapter 3, Figure 4). As a consequence, the silencing

effect of the treatment with siRNA-Luc dendriplexes must be mediated by the

siRNA-Luc specific inhibition. Control LPEI polyplexes were toxic in LS174T

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cells, reducing the viability values up to 25%, whereas in HeLa cells viability

was similar compared to dendriplexes.

The excellent results provided by the toxicity assays make these vectors

suitable for further improvement. Moreover, the interesting shielding effect of

the PEG chain included can partially explain the lack of improvement in the

gene silencing profile, without eradicating the possibility of using this

conjugates for in vivo purposes, since the extrapolation from in vitro results to in

vivo is still limited for these systems.

5. CONCLUSIONS

In this work two novel PAMAM-PEG(2 kDa)-Peptide conjugates

targeted to the TfR and EGFR have been evaluated. The presence of the

peptides B6 and GE11 did not produce any variation in the size and zeta

potential values, presenting always a nanometric size and positive zeta potential

values. The presence of the PEG chain produced a decrease in the surface

charge, but the condensing ability was not negatively influenced, as showed the

ethidium bromide assay. The gene silencing capacity mediated by a siRNA-

Control was negligible; while the use of a specific siRNA-Luc was able to

reduce luciferase expression a 14% and 20% on average in HeLa and LS174

cells respectively, compared to siRNA-Control. The presence of the peptides

did not improve the gene silencing activity compared to plain dendriplexes.

Toxicity studies confirmed the low toxicity of the PAMAM dendrimer

conjugates, whereas LPEI polyplexes resulted in being highly toxic in the

LS174T cell line.

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20. Jensen LB, Pavan GM, Kasimova MR et al.: Elucidating the molecular mechanism of PAMAM-siRNA dendriplex self-assembly: effect of dendrimer charge density. Int J Pharm 416(2), 410-418 (2011).

21. Liu X, Liu C, Laurini E et al.: Efficient delivery of sticky siRNA and potent gene silencing in a prostate cancer model using a generation 5 triethanolamine-core PAMAM dendrimer. Mol Pharm 9(3), 470-481 (2012).

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24. Yuan Q, Lee E, Yeudall WA, Yang H: Dendrimer-triglycine-EGF nanoparticles for tumor imaging and targeted nucleic acid and drug delivery. Oral Oncol 46(9), 698-704 (2010).

25. Tsutsumi T, Hirayama F, Uekama K, Arima H: Evaluation of polyamidoamine dendrimer/alpha-cyclodextrin conjugate (generation 3, G3) as a novel carrier for small interfering RNA (siRNA). J Control Release 119(3), 349-359 (2007).

26. Tsutsumi T, Hirayama F, Uekama K, Arima H: Potential use of polyamidoamine dendrimer/alpha-cyclodextrin conjugate (generation 3, G3) as a novel carrier for short hairpin RNA-expressing plasmid DNA. J Pharm Sci

97(8), 3022-3034 (2008).

27. Arima H, Tsutsumi T, Yoshimatsu A et al.: Inhibitory effect of siRNA complexes with polyamidoamine dendrimer/alpha-cyclodextrin conjugate (generation 3, G3) on endogenous gene expression. Eur J Pharm Sci 44(3), 375-384 (2011).

28. Arima H, Yoshimatsu A, Ikeda H et al.: Folate-PEG-appended dendrimer conjugate with alpha-cyclodextrin as a novel cancer cell-selective siRNA delivery carrier. Mol Pharm 9(9), 2591-2604 (2012).

29. Arima H, Motoyama K, Higashi T: Polyamidoamine Dendrimer Conjugates with Cyclodextrins as Novel Carriers for DNA, shRNA and siRNA. Pharmaceutics 4(1), 130-148 (2012).

30. Biswas S, Deshpande PP, Navarro G, Dodwadkar NS, Torchilin VP: Lipid modified triblock PAMAM-based nanocarriers for siRNA drug co-delivery. Biomaterials 34(4), 1289-1301 (2013).

31. Wu J, Huang W, He Z: Dendrimers as carriers for siRNA delivery and gene silencing: a review. ScientificWorldJournal 2013, 630654 (2013).

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34. Choi JH, Choi JS, H. S, S. PJ: Effect of Poly(ethylene glycol) Grafting on Polyethylenimine as a Gene Transfer Vector in vitro. Bull Korean Chem Soc 22(1), 46-52 (2001).

35. Kwok A, Hart SL: Comparative structural and functional studies of nanoparticle formulations for DNA and siRNA delivery. Nanomedicine 7(2), 210-219 (2011).

36. Tang MX, Redemann CT, Szoka FC, Jr.: In vitro gene delivery by degraded polyamidoamine dendrimers. Bioconjug Chem 7(6), 703-714 (1996).

37. Li W, Szoka FC: Lipid-based nanoparticles for nucleic acid delivery. Pharm Res

24(3), 438-449 (2007).

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45. Von Gersdorff K, Sanders NN, Vandenbroucke R, De Smedt SC, Wagner E, Ogris M: The internalization route resulting in successful gene expression depends on both cell line and polyethylenimine polyplex type. Mol Ther 14(5), 745-753 (2006).

GENERAL DISCUSSION

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Cancer figures among the leading causes of death worldwide,

accounting for 8.2 million deaths in 2012 according to the World Health

Organization [1]. At this respect, gene therapy has become one of the most

important approaches for the treatment of several acquired and inherited

diseases. Hence, this disease is, by far, one of the sickness that are currently in

gene therapy’s bull’s-eye and approximately two thirds of the clinical trials

carried out worldwide address this illness [2]. Despite the fact that viral vectors

are an interesting alternative, usually considered more effective than non-viral

systems, and have coped most of the research, the possibility of developing

excessive immune responses, activating oncogenic processes, as well as the

limited size of the cargo that can be included, have increased the investment in

synthetic alternatives. Therefore, the current necessity to develop new synthetic

carriers has, in recent years, focused the attention on looking for better non-

viral vectors with improved features, specificity and gene delivery activity.

The different carriers for non-viral gene delivery range from lipidic to

polymeric molecules, usually with positive charge, that are able to condense

nucleic acids to nanoparticles and protect the included genetic material from

the activity of nucleases and other enzymes. The main advantages lay on the

lack of immunostimulatory effects, excellent ability to include huge amounts of

DNA or RNA, good biocompatibility and low toxicity. The final goal of these

vectors is to carry the genetic material included and to deliver the cargo in the

cytoplasm or the nucleus, where it will take its effect. PAMAM dendrimers are

one type of polymeric molecules with a well-established structure. Due to its

synthetic process, the molecular weight can be exhaustively controlled and the

surface presents high amount of charged groups. The ability of PAMAM

dendrimers to mediate an efficient transfection in cultured cells and the low

toxicity, compared to other polymers used such as polyethyleneimine, have

General discussion

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been proved with good results and therefore, the perspectives for this family of

polymers are encouraging.

The modified characteristics that the tumor tissue exhibits are highly

interesting for gene delivery. It is widely known that tumoral cells usually

present a different pattern of expression of several cell components e.g. cellular

receptors [3]. Several tumor types are amenable to direct injection or local

application but, the metastatic tumor can only be reached by administering

therapeutic genes into the bloodstream and targeting the specific site [4]. This

objective can be challenging as long as the journey through the bloodstream

carries degradation processes or removal by the immune system [5]. Although

the vascular endothelium is also altered and allows an easy access to the

tumoral tissue by passive mechanisms such as the Enhanced Permeability and

Retention effect (EPR) [6], the non-viral system should be internalized before

releasing the cargo and achieving the desired pharmacological effect. Therefore,

taking this information into account, the main objective of this work has been

to achieve and evaluate improved gene delivery vectors based on the PAMAM-

G5 dendrimer targeted to different receptors overexpressed on the surface of

cancer cells: CD44, transferrin receptor (TfR) and epidermal growth factor

receptor (EGFR).

Hyaluronic acid (HA) is one of the components of the extracellular

matrix that can enhance the gene transfection efficiency via specific receptor

interaction. Nevertheless, it has not been extensively used for gene therapy and

the data existing of PAMAM and HA formulations are limited. Firstly, the

formulation of targeted PAMAM/DNA dendriplexes with HA electrostatically

attached was performed. Despite representing an easy formulation protocol,

some authors have reported that when the targeting ligand is electrostatically

General discussion

169

attached, it is not as effective as the covalently linked one in vitro [7] and for

tumor targeting in vivo [8]. Therefore, a covalently coupled alternative was

explored for HA. The synthesis protocol via a reductive amination between

oxidized HA and the primary amines of PAMAM dendrimer (Chapter 1, Figure

1) was selected as the less damaging one. The PAMAM structure can be

activated, as described by Tang et al. [9] which leads in a dendrimer with a more

flexible structure with better transfection activity for pDNA and siRNA.

Notwithstanding, this activation was avoided in order to ensure that the

improvement achieved during the subsequent experiments was related to the

presence of the ligand. In a similar way, transferrin (Chapter 3), whose receptor

is widely distributed because the cell requirement of iron is clearly increased

during the cancer malignant process [10], was coupled to the PAMAM

structure. This ligand has been widely used for the targeting of other

formulations and it has shown an inherent ability to produce shielded

complexes with good targeting ability even in vivo [8, 11]. The synthesis of the

PAMAM-PEG-Peptides used in Chapter 4 was done looking for a similar

effect. In this case, instead of the huge Tf protein, two specific peptides of the

TfR and EGFR, B6 and GE11 respectively, were used covalently attached to

the PAMAM structure by a polyethylenglycol (PEG) chain as a linker. With this

molecule, the surface charge of the dendriplexes can be shielded, which has

demonstrated to improve the biodistribution process by hindering the removal

from the bloodstream by the reticuloendothelial system (RES) [12, 13]. The

protocols used in the syntheses of these new PAMAM-Transferrin (P-Tf) and

PAMAM-PEG-Peptides conjugates were also carried out under soft conditions

in order to ensure the structure of the dendrimer and the peptides or proteins.

Size and zeta potential of the formulated nanoparticles are considered

as two important factors that govern a successful nanoparticle mediated gene

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and drug delivery. The size is a key point in the development of this kind of

vectors, as long as the transfection and biodistribution are clearly influenced by

this parameter. Owens et al. reviewed this aspect of polymeric systems,

concluding that nanoparticles below 200 nm have significantly longer

circulation time due to low uptake by the (RES) [14]. On the other hand,

formulation with bigger sizes or aggregates are known to mediate a more

efficient transfection. Usually this fact is related to an increased sedimentation

of the nanoparticles in vitro [15] and the in vivo administration is limited. The

surface charge has been widely discussed too. Nanoparticles formulated with

cationic polymers or lipids generate positively charged systems. This feature is

considered interesting for gene delivery, since a positive surface charge can

trigger unspecific interactions with the negatively charged cellular membrane

and, subsequent endocytosis processes. Nonetheless, this positive envelope can

generate unspecific interactions with negatively charged serum proteins, which

leads to removal of the nanoparticles from the bloodstream [16].

In this work, all the conjugates used were able to efficiently bind the

pDNA or siRNA at N/P 2 and higher ratios in a similar way, like the non-

targeted PAMAM dendrimer. The complexes formed turned out to be

nanometric with sizes below 200 nm for all the conjugates and in all the

conditions assayed. This result supports the excellent ability of PAMAM-G5

dendrimers to form nanoparticles reported previously [17]. Probably, the main

differences found during the characterization of these vectors were observed in

the surface charge measurements. A clear influence of the N/P ratio can be

seen regardless of the conjugate. The higher the N/P ratio, related to the excess

of positive charges added, the higher the zeta potential values were generated.

As expected, the presence of a 2 kDa PEG chain produced a decrease of the

zeta potential in the PAMAM-PEG-Peptide containing conjugates (Chapter 4,

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171

Table 1). This molecule is known to shield the surface charges of the positively

charged nanoparticles. In a similar way, a decrease was also noted in the P-Tf

containing dendriplexes (Chapter 3, Table 1). In this case, the addition of

increasing amounts of P-Tf led to more negative nanoparticles in accordance

with the data provided by Kircheis et al. [8]. The PAMAM dendriplexes

containing HA electrostatically attached (Chapter 1, Table 2) did also show this

tendency, especially when the highest amount was included. In contrast, when

the P-HA conjugate was used, this drop was not noticeable and the zeta

potential values were similar to the ones obtained in the non-targeted PAMAM

dendriplexes (Chapter 1, Table 3). The differences found regarding the surface

charge are not related to the PAMAM:ligand coupling ratio, but with the

different molecular weights of the different ligands used and the amount of

negative charges provided by each molecule.

Previously to the in vitro/in vivo evaluation of the vectors, the complete

binding of the DNA to the dendrimer was assessed with a gel electrophoresis.

In all the complexes for pDNA delivery, independently of the N/P ratio and

ligand or amount used, a total retention of the migration was detected, in

agreement with previous PAMAM/pDNA complexes data. Moreover, after

ensuring the encapsulation, a protection assay was performed. The capacity of

PAMAM dendrimers to protect the included pDNA has been previously

demonstrated, but the influence of the addition of HA or Tf as targeting was

not clear. The data provided by the degradation assays confirmed the excellent

binding and protecting characteristics of these systems to protect and maintain

the integrity of the plasmid after the disruption of the complexes. These data

provide the certainty that the vectors designed for pDNA may be efficient

carriers as the included genetic sequence will be protected.

General discussion

172

After the biophysical characterization, the logical next step in the

development of a vector is the in vitro evaluation. Thus, we assessed the

effectiveness and toxicity of the dendriplexes in various tumoral cell lines. The

results point out that the new developed conjugates were effective in the

different specific receptor overexpressing cell lines but the efficacy depends on

the cell type and amount of ligand. As shown in Chapter 2 (Figures 2 and 3),

the transfection experiments with the complexes containing HA were carried

out in B16F10, MDA-MB-231, MCF-7 and Neuro2A cell lines after studying

the presence of the hyaluronan receptor CD44 on the surface of these cells

(Chapter 2, Figure 1). Two trends were found for P-HA conjugate containing

complexes. On the one hand, the more HA included in the formulation, the

higher transfection achieved in the P-HA containing complexes in B16F10 and

MDA-MB-231 (clearly positive for CD44). In those cell lines, complexes

prepared at a N/P ratio 6 were able to mediate an effective transfection, which

was improved by the substitution of increasing percentages of P-HA. On the

other hand, MCF-7 and Neuro2A did not present any improvement, probably

because of the poor receptor expression on the surface. Electrostatically

targeted complexes did not show any improvement in transfection activity,

independently of the amount of HA included, and the transfection values were

always below the transfection level obtained with P-HA containing complexes,

probably related to the lower zeta potential values (Chapter 1, Table 2). This

fact supports the previously published data by Surace et al., who reported a

similar behaviour for HA targeted lipoplexes [7]. Therefore, the P-HA

containing dendriplexes rise as the best targeted carrier compared to

electrostatic ones for in vitro gene delivery.

The PAMAM-Tf conjugate was also evaluated in vitro in the cells CT26,

HeLa, HepG2 and Neuro2A (Chapter 3, Figure 3). In those cells, a clear

General discussion

173

influence of the N/P ratio and percentage of P-Tf was observed. In all the cell

lines, excepting Neuro2A, the highest improvement was detected at N/P 6

containing 50% of P-Tf, whereas N/P 2 and 4 were not as effective although a

tendency to increase the transfection activity was seen for CT26 and HeLa

when the substitution percentage was higher. This fact could be related to the

zeta potential values of the complexes, clearly influenced by the N/P ratio and

the presence of Tf as stated for other systems [8]. Moreover, HepG2 cell line

has been reported to have less receptors on the surface than HeLa cells [18],

which have been found to be less active in terms of internalization. These data,

and the knowledge that the highest amount of Tf is found in the dendriplexes

prepared at N/P 6, can explain the lack of enhancement at lower N/P ratios.

Although Neuro2A cells have been used as a model for Tf targeted delivery

with PEI polyplexes [8], an absence of improvement was detected in our case.

In this sense, different cell lines respond in a non-similar manner to the

treatment with different polyplexes. The reasons for the cell type dependency

observed in this study and other might be quite complex [19, 20]. It could be

that, for Neuro2A cells, adsorptive endocytosis by electrostatic interactions of

charged complexes with cell membrane heparane sulphates is masking the

transferrin-mediated gene transfer or that the presence of bulky molecules, such

as Tf, can negatively affect the endosomal release as suggested by Schaffer et al.

for PEG containing polyplexes [21]. However, the differences found could not

be attributed to different toxicities since the dendriplexes assayed turn out to be

non-toxic.

In order to clarify if the targeted complexes containing P-HA or P-Tf

conjugates were internalized by the interaction with the specific receptors, a

transfection with an excess of free ligand was performed (Chapter 2, Figure 4;

Chapter 3, Figure 5). In both experiments, transfection activity of conjugate

General discussion

174

containing complexes was blocked by the presence of the free ligand by the

saturation of the cellular receptors, whereas the non-targeted dendriplexes were

able to mediate similar transfection activity. These results verify that the uptake

is mediated by a specific mechanism.

In all the experiments for pDNA delivery, a branched PEI (bPEI)

control was used. This polymer represents one of the gold standards in gene

delivery and has been widely used [22]. In this work, although bPEI has

exhibited better transfection activity than targeted and plain dendriplexes in

some of the cell lines tested (B16F10 and CT26), which proves the excellent

activity as a transfection agent, it has been exceeded by the designed

dendriplexes. For all the cell lines tested, the viability values obtained by using

PEI-polyplexes were below the ones obtained with the PAMAM dendriplexes,

independently of the N/P ratio and conjugate (P-HA or P-Tf) used. Those data

provide an excellent framework for further studies and improvement of the

presented dendriplexes, which have demonstrated that PAMAM dendrimers

can efficiently bind to the pDNA and protect it from the enzymatic activity.

Moreover, the PAMAM structure can be easily modified in order to ensure an

enhanced transfection activity with low toxicity.

As the in vitro evaluation showed excellent results with low toxicity, the

P-HA containing dendriplexes were tested in a murine model. Firstly,

dendriplexes were applied to healthy BalbC mice, in order to obtain

information related to their biodistribution and toxicity. The formulation

protocol was optimized because the direct mixture of the components

produced big aggregates (data not shown). Therefore, dendriplexes were

prepared at a final concentration of 60 µg of pDNA/mL and then

ultracentrifuged up to the desired volume. With this protocol, the nanoparticles

General discussion

175

maintained similar characteristics to those observed in the in vitro experiments

in terms of size and zeta potential. In general, systemic application of DNA

condensed in a cationic carrier, either lipidic or polymeric, turned into gene

expression mainly located in the lung, considered as the first step organ after

intravenous administration [23, 24]. In the same way, gene expression mediated

by non-targeted dendriplexes was mainly located in the lung (Chapter 2, Figure

6). This transfection pattern dramatically changed when 50% P-HA containing

dendriplexes were applied. In this case, the lung did not show any variation of

the transfection activity, whereas it was clearly enhanced in the liver and heart.

This fact can be related to the presence of CD44 receptor in the liver

endothelium, which can mediate the enhancement as suggested by Yao et al.

[25]. The sharp increase showed in heart is not likely to be dependent on the

presence of the CD44 receptor in the tissue, which has reported to be low [24],

but with other tissue features. Nevertheless, further experiments should be

performed to clarify this aspect.

The encouraging results obtained in the in vivo evaluation suggested a

good behaviour for in vivo administration in a tumor model. Among the

different choices for cancer gene therapy, the immune activation with the

interleukin 12 (IL-12) gene has demonstrated to be effective in different animal

models reviewed by Pavlin et al. [26] and holds great promises for human

cancer treatment. It has been published that the amount of IL-12 available at

the tumor site is critical for tumor regression [27] and different authors have

decided to administer the IL-12 gene intratumorally with excellent results [11,

28]. In this sense, the murine melanoma B16F10 cells were selected and

subcutaneously injected to syngenic C57BL/6 female mice. After 7-14 days,

tumors were intratumorally injected with dendriplexes containing the optimized

pCpG-hCMV-SCEP-eFLuc plasmid (eFLuc) [29]. These experiments showed

General discussion

176

that the targeted 50% P-HA containing dendriplexes presented higher mean

transfection values compared to plain dendriplexes, although the differences

could not be considered statistically significant. A targeted formulation with

HA electrostatically assembled, with the same amount of HA included in the P-

HA conjugate, was also studied. Interestingly, this formulation was able to

equalize the transfection mediated by the covalent P-HA containing

dendriplexes. Nonetheless, the transfection activity of the electrostatically

attached HA dendriplexes has not been assayed for in vivo delivery after

intravenously administration and further studies should be performed in order

to ensure this superiority. The background provided by the in vitro experiments

and the result provided by other authors allow to consider the P-HA containing

dendriplexes the best choice for future research. At this point, the increased

transfection values achieved by the targeted P-HA conjugate should be

considered highly valuable despite the absence of significant differences. The in

vivo administration carries the unspecific interactions with plasmatic and

extracellular proteins, the presence of DNAses that can destroy the included

pDNA and the removal of the complexes by the immune system, which makes

the transfection processes highly complicated. The information obtained here

demonstrates the possibility of increasing the transfection activity with HA and

proves the possibility of treating cancer disease with a gene therapy approach,

which encourages the research and development in the future.

Although the pDNA delivery represents one of the most used

approaches in gene therapy and has been reported to be effective in the fight

against cancer disease, other strategies have also emerged in the last years. At

this respect, one of the most interesting ones is the use of small interfering

RNA (siRNA). In this case, a small double strand of RNA, which is

complementary to a specific messenger RNA (mRNA) is able to specifically

General discussion

177

bind it and trigger a complex cellular mechanism that finishes with the

disappearance of a protein [30, 31]. PAMAM dendrimer is known to be a good

pDNA delivery agent but the use with siRNA has not been so widely explored.

In Chapter 4, the possibility of targeting PAMAM-PEG(2kDa)-Peptide/siRNA

complexes to the TfR and EGFR has been evaluated. The complexes showed

excellent ability to bind the siRNA included (Chapter 4, Figure 1) at N/P ratio

2 and higher ratios and the nanoparticles formed presented sizes below 200 nm

(Chapter 4, Table 1). An interesting point is the drop in the surface charge

values of the PEG containing dendriplexes, which was expected. The silencing

effect of the dendriplexes was evaluated in two cell lines stably expressing the

eGFPLuc cassette, HeLa and LS174T (Chapter 4, Figure 2). A specific siRNA-

Luc was able to reduce the gene expression of luciferase (14% in HeLa and a

20% in LS174T cells) compared to a non-specific siRNA-Control. The

inclusion of the different ligands did not show any difference compared to

control plain dendriplexes and the different N/P tested neither. This result can

be explained by the shielding effect provided by the PEG chain. It is known

that shielding agents, like PEG, can produce complexes with low gene delivery

activity since it blocks the unspecific interaction between the positive

nanoparticle and the cellular membrane. At this respect, if the ligand cannot

perform a really effective receptor mediated endocytosis, the activity will rarely

be improved. Moreover, some authors have reported that bulky molecules can

interact with the endosomal release process, highly important to the small

siRNA molecule [21]. Although this result can be considered negative due to

the absence of improvement in the gene silencing efficacy, the low toxicity of

the dendriplexes compared to LPEI polyplexes used as control should be taken

into account. The information provided by other authors, who have used the

same ligands, points out that the improvement was not always achieved in all

General discussion

178

the cancer cells tested and was only significant in some cultures [21, 32].

Notwithstanding, the GE11 peptide has proven to be effective in vivo in the

targeting of prostate cancer cells with PEI based polyplexes targeted to the

EGFR [21]. Those data provide an excellent background for further

improvement of the formulations used for siRNA delivery even in vivo despite

the apparent lack of positive results in vitro.

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CONCLUSIONES / CONCLUSIONS

Conclusiones / Conclusions

185

CONCLUSIONES

Del trabajo realizado y desarrollado en esta Tesis Doctoral, cuyo objetivo

ha sido el diseño y evaluación de nuevas formulaciones dendriméricas dirigidas,

se pueden extraer las siguientes conclusiones:

1. Se ha sintetizado un nuevo conjugado dirigido PAMAM-ácido

hialurónico (P-AH) con una unión covalente entre el PAMAM y el ácido

hialurónico (AH) mediante una aminación reductiva. Este conjugado fue

capaz de formar nanopartículas estables en presencia de DNA y proteger

al plásmido de la degradación enzimática por endonucleasas.

2. 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. El ensayo de competición

con exceso de AH libre llevado a cabo, confirmó la captación específica

mediada por receptor.

3. Los ensayos de toxicidad de los dendriplejos dirigidos con AH mostraron

una viabilidad celular mayor del 80%. Estos complejos demostraron ser

menos tóxicos que los poliplejos de PEI.

4. Los resultados in vivo de los dendriplejos con AH mostraron un

incremento en la expresión de luciferasa en hígado y corazón de ratones

Balb-C sanos comparados con los complejos no dirigidos. Estos sistemas

fueron también capaces de transfectar de forma eficiente tumores

B16F10 inducidos en ratones C57BL/6, aunque no se encontraron

diferencias significativas en comparación con los no dirigidos.

Conclusiones / Conclusions

186

5. Los conjugados PAMAM-Tf (P-Tf) también fueron capaces de formar

nanopartículas estables con buenas capacidades para unir ADN y

proteger el plásmido de la degradación por nucleasas.

6. La evaluación in vitro de los nuevos complejos formulados con P-Tf a

N/P 6, en comparación con los no dirigidos, mostraron un incremento

en la actividad de transfección en células cancerígenas (HeLa, HepG2 y

CT26). La toxicidad fue baja, siendo la viabilidad celular mayor del 80%.

Los complejos resultaron ser menos tóxicos que los poliplejos de PEI. La

captación mediante endocitosis mediada por receptor fue comprobada

mediante un ensayo de competición.

7. Los dendriplejos formados con los conjugados PAMAM-PEG-B6 y

PAMAM-PEG-GE11 en presencia de siRNA fueron capaces de formar

nanopartículas estables y de condensar el siRNA de forma efectiva.

8. La evaluación in vitro de la capacidad de silenciamiento génico del siRNA

encapsulado en los dendiplejos PAMAM-PEG-B6 y PAMAM-PEG-

GE11, mostró que el siRNA-Luc específico fue capaz de reducir la

expresión de luciferasa en células HeLa y LS174, aunque no se

observaron diferencias significativas con los complejos no dirigidos,

probablemente debido a la presencia de PEG, el cual inhibe la

transfección. Estos complejos resultaron no ser tóxicos para las células,

especialmente en la línea celular LS174, comparados con los poliplejos de

PEI.

Conclusiones / Conclusions

187

CONCLUSIONS

The experimental work compiled in this dissertation has focused on the

design and development of new targeted dendrimer-based formulations. The

results obtained allow drawing the following conclusions:

1. A novel targeted PAMAM-hyaluronic acid conjugate has been

synthetized by covalently linking PAMAM to hyaluronic acid (HA) via

reductive amination. This conjugate was able to form stable nanoparticles

in the presence of DNA and protect the plasmid from enzymatic

degradation by nucleases.

2. 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.

3. Toxicity studies by HA-dendriplexes showed a cell viability higher than

80%. These dendriplexes showed lower toxicity than the highly used

PEI-polyplexes.

4. In vivo results by HA-dendriplexes 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 were detected compared to non-targeted ones.

Conclusiones / Conclusions

188

5. PAMAM-Tf conjugates were also able to form stable nanoparticles with

good capacity of binding DNA and protecting the plasmid from

degradation by nucleases.

6. In vitro evaluation of the new PAMAM-Tf conjugate compared to non-

targeted complexes, showed increased gene delivery to cancer cells

(HeLa, HepG2 and CT26), when complexes were formulated at N/P

ratio of 6. Toxicity was low, being cell viability higher than 80%. The

complexes were less toxic than PEI-polyplexes. The uptake via receptor-

mediated endocytosis was ensured by a competition assay.

7. Small interfering RNA (siRNA) was also formulated in targeted

complexes containing specific peptides. PAMAM-PEG-B6 and

PAMAM-PEG-GE11 dendriplexes formed stable nanoparticles able to

condense effectively siRNA.

8. In vitro evaluation of the gene silencing capacity of siRNA encapsulated

into PAMAM-PEG-B6 or PAMAM-PEG-GE11 dendriplexes showed

that specific siRNA-luc was able to reduce luciferase expression in HeLa

and LS174 cells, although no significant differences with plain-complexes

were detected, probably due to the presence of PEG, which inhibits gene

delivery. These complexes turned out to be non-toxic to the cells and

particularly in LS174 cells less toxic than PEI-polyplexes.