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Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 21-02-2019, Los sistemas de liberación de agentes terapéuticos son tecnologías diseñadas para procurar su administración controlada y dirigida en el órgano o tejido necesitado de terapia. En el presente trabajo se han desarrollado hidrogeles fototérmicos como base de sistemas de liberación controlada de agentes terapéuticos. Para ello, se han combinado matrices poliméricas de diferente naturaleza con nanopartículas metálicas que presentan actividad fototérmica en el infrarrojo cercano (NIR). La inclusión de liposomas termosensibles cargados con un fármaco antitumoral en matrices de fibrina fototérmica resultó en lipogeles capaces de responder a la energía de un láser NIR mediante la liberación del fármaco encapsulado, cuya bioactividad se comprobó tratando cultivos de células de carcinoma cervical humano. Los niveles de fármaco liberado desde el sistema son dependientes del tipo de nanopartícula y su concentración, la potencia láser y el régimen de irradiación utilizado. El sistema de liberación se optimizó añadiendo colesterol a la composición de los liposomas termosensibles, lo que disminuyó las fugas de fármaco a temperatura fisiológica e incrementó la liberación de fármaco tras tratamiento térmico. La tecnología de matrices fototérmicas fue asimismo empleada para la obtención de andamiajes que pueden controlar la expresión transgénica mediante su respuesta a energía NIR, lo que permitiría definir patrones espaciotemporales de biodisponibilidad de productos génicos terapéuticos. Para ello, se incluyeron nanopartículas fototérmicas en matrices poliméricas basadas en fibrina, recombinámeros de elastina (ELR) y criogeles de un copolímero de 2-hidroxietilmetacrilato y ácido acrílico. Estas matrices resultaron ser biocompatibles y, en respuesta a NIR, generaron un incremento local de temperatura suficiente para activar en las células que contienen circuitos génicos inducibles por calor y dependientes de un ligando de bajo peso molecular. La incorporación de células troncales multipotentes en hidrogeles de fibrina que incluyen nanopartículas fototérmicas basadas en sulfuro de cobre (CuSNP) resultó en un incremento de la actividad metabólica, supervivencia y actividad fibrinolítica celular, así como en cambios en el transcriptoma relacionados con la respuesta angiogénica. Las células endoteliales incluidas en estos hidrogeles potenciaron la formación de estructuras pseudocapilares y la remodelación de la matriz de fibrina. La implantación a largo plazo de matrices de fibrina que incluyen CuSNP indujo una respuesta angiogénica que facilita su integración en los tejidos del hospedador mientras que la implantación de matrices de ELR y criogeles fototérmicos resulta en una baja respuesta inflamatoria y una degradación del biomaterial muy limitada., Drug delivery systems are tools designed to administer therapeutic agents in a controlled manner to specific organs or tissues in need of therapy. In this work, we have developed photothermal hydrogels as platforms for controlled delivery of therapeutic agents. To this aim, we have included metallic nanoparticles having photothermal activity at near-infrared (NIR) wavelengths in polymeric matrices. Incorporation of thermosensitive liposomes loaded with an antitumoral drug within photothermal fibrin matrix resulted in NIR-responsive lipogels that released their liposomal cargo after NIR irradiation. Released drug maintained their bioactivity as tested in cultures of human cervical carcinoma cells. Levels of drug released from the constructs were dependent on the type and concentration of NIR nanotransducers loaded in the lipogel, the intensity of deposited electromagnetic energy and the irradiation regime. The developed drug delivery platform was improved by the incorporation of cholesterol in thermosensitive liposomes formulation, which lessened leakiness of the liposomal cargo at physiological temperature and increased drug release after thermal treatment. Photothermal matrices technology was also used to prepare NIR responsive scaffolds that are able to control transgenic expression, allowing to achieve spatiotemporal control over the bioavailability of therapeutic gene products. Photothermal nanoparticles were included within polymeric fibrin matrices, elastin like recombinamers (ELR) and cryogels of hydroxyethyl methacrylate and acrylic acid copolymers. These matrices were biocompatible and transduced efficiently NIR energy into heat to activate transgene expression in cells harboring a gene switch triggered by heat and dependent on a low molecular weight ligand. Multipotent stem cells incorporated within fibrin hydrogels that include photothermal nanoparticles based in copper sulfide (CuSNP) increased metabolic activity, survival rate and fibrinolytic activity and displayed changes in their transcriptome related to angiogenic response. Endothelial cells entrapped within these matrices formed pseudocapillary structures and remodeled protein matrix. Long term implantation of CuSNP fibrin matrices induced an angiogenic response that facilitates its integration into the host tissue while implantation of photothermic ELR matrix and cryogels was characterized by low inflammation response and limited biomaterial degradation., Este trabajo se ha llevado a cabo mediante financiación concedida por los siguientes organismos: Instituto de Salud Carlos III (Ministerio de Ciencia, Innovación y Universidades, PI15/01118), Ministerio de Ciencia, Innovación y Universidades (SAF2013-50364-EXP), Comunidad Autónoma de Madrid (S2013/MIT-2862), Centro de Investigaciones Biomédicas en Red en Biomateriales, Bioingeniería y Nanomedicina CIBER-BBN y la Fundación para la Investigación Biomédica del Hospital Universitario La Paz (Convocatoria de ayudas financiadas por Roche Farma).

CuS nanoparticles (CuSNP) are degradable, readily prepared, inexpensive to produce and efficiently cleared from the body. In this work, we explored the feasibility of CuSNP to function as degradable near infrared (NIR) nanotransducers within fibrin-based cellular scaffolds. To prepare NIR-responsive CuSNP hydrogels, fibrinogen was dissolved in cell culture medium and supplemented with aqueous dispersions of CuSNP. Fibrinogen polymerization was catalyzed by the addition of thrombin. In some experiments, HUVEC, C3H/10T1/2 or C3H/10T1/2-fLuc cells, that harbor a heat-activated and rapamycin-dependent gene switch for regulating the expression of firefly luciferase transgene, were incorporated to the sol phase of the hydrogel. For in vivo experiments, hydrogels were injected subcutaneously in the back of adult C3H/HeN mice. Upon NIR irradiation, CuSNP hydrogels allowed heat-inducible and rapamycin-dependent transgene expression in cells contained therein, in vitro and in vivo. C3H/10T1/2 cells cultured in CuSNP hydrogels increased metabolic activity, survival rate and fibrinolytic activity, which correlated with changes at the transcriptome level. Media conditioned by CuSNP hydrogels increased viability of HUVEC which formed pseudocapillary structures and remodeled protein matrix when entrapped within these hydrogels. After long-term implantation, the skin patches that covered the CuSNP hydrogels showed increased capillary density which was not detected in mice implanted with matrices lacking CuSNP. In summary, NIR-responsive scaffolds harboring CuSNP offer compelling features in the tissue engineering field, as degradable implants with enhanced integration capacity in host tissues that can provide remote controlled deployment of therapeutic gene products. Statement of Significance: Hydrogels composed of fibrin embedding copper sulfide nanoparticles (CuSNP) efficiently convert incident near infrared (NIR) energy into heat and can function as cellular scaffolding. NIR laser irradiation of CuSNP hydrogels can be employed to remotely induce spatiotemporal patterns of transgene expression in genetically engineered multipotent stem cells. CuSNP incorporation in hydrogel architecture accelerates the cell-mediated degradation of the fibrin matrix and induces pro-angiogenic responses that may facilitate the integration of these NIR-responsive scaffolds in host tissues. CuSNP hydrogels that harbor cells capable of controlled expression of therapeutic gene products may be well suited for tissue engineering as they are biodegradable, enhance implant vascularization and can be used to deploy growth factors in a desired spatiotemporal fashion., The authors thank F. Urbano and C. Aguado (Laboratorio de Microscopía Electrónica de Transmisión, Universidad Autónomade Madrid) (TEM analyses) as well as J. García and P. Botias (Unidad de Genómica, Universidad Complutense de Madrid/Parque Científico de Madrid) (microarray analyses) for their assistance. We are also greatly indebted to L. Muñoz (CIBER-BBN and La Paz University Hospital-IdiPAZ) for excellent technical support with zymography assays. This work was supported by grant PI15/01118 from Instituto de Salud Carlos III (ISCIII)-Fondos FEDER, Ministry of Economy and Competitiveness (MINECO), Spain, grant SAF2013- 50364-EXP from MINECO, grant S2013/MIT-2862 from Comunidad Autónoma de Madrid (CAM), grant Roche-IdiPAZ from the intramural funding program of Foundation for Biomedical Research of La Paz University Hospital-IdiPAZ, grant ERC-2013-CoG-614715 (NANOHEDONISM) from ERC Consolidator Grant program and by HSF Pharmaceuticals S.A. C.E-D. is the recipient of predoctoral grant FI14/00447 from ISCIII-Fondos FEDER, MINECO. S.S.-C and N.V. were supported by grant PEJ15/BIO/AI-0250 and Program I2, respectively, from CAM.

Here we report a composite system based on fibrin hydrogels that incorporate in their structure near-infrared (NIR) responsive nanomaterials and thermosensitive liposomes (TSL). Polymerized fibrin networks entrap simultaneously gold-based nanoparticles (NPs) capable of transducing NIR photon energy into heat, and lysolipid-incorporated TSL (LTSL) loaded with doxorubicin hydrochloride (DOX). NIR irradiation of the resulting hydrogels (referred to as “lipogels”) with 808 nm laser light increased the temperature of the illuminated areas, leading to the release of the liposomal cargo. Levels of DOX that release from the “smart” composites were dependent on the concentration of NIR nanotransducers loaded in the lipogel, the intensity of the electromagnetic energy deposited and the irradiation regime. Released DOX retained its bioactivity, as shown in cultures of epithelial carcinoma cells. Finally, the developed drug delivery platform was refined by using NIR-photoabsorbers based on copper sulfide NPs to generate completely biodegradable composites as well as through the incorporation of cholesterol (Ch) in LTSL formulation, which lessens leakiness of the liposomal cargo at physiological temperature. This remotely controlled system may suit well for those therapies that require precise control over the dose of delivered drug in a defined spatiotemporal framework. Statement of Significance Hydrogels composed of fibrin embedding nanoparticles responsive to near infrared (NIR) energy and thermosensitive liposomes loaded with doxorubicin hydrochloride (DOX), were prepared by in situ polymerization. NIR-light irradiation of these constructs, referred to as “NIR responsive lipogels”, results in the controlled release of DOX to the surrounding medium. This technology may use fully degradable components and can preserve the bioactivity of liposomal cargo after remote triggering to finely regulate the dose and bioavailability of delivered payloads. NIR responsive lipogels technology overcomes the limitations of drug release systems based on the combination of liposomes and degradable polymeric materials, which in many cases lead to insufficient release at therapy onset or to overdose during high degradation period.