Aplicaciones biomédicas de matrices poliméricas fototérmicas

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 21-02-2019Los 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)

Papilomatosis bovina: epidemiología y diversidad de papilomavirus bovinos (BPV)

Bovine papillomatosis is a cattle disease characterized by the presence of papillomas and fibropapillomas. It is caused by the infection of bovine papillomavirus (BPV), naked viruses of the family Papillomaviridae. They are epithelitropic viruses, although they have also been detected in blood, milk, urine and seminal fluid. They present high viral diversity, and up to date 13 types are recognized (BPV-1 a BPV-13). Although they were originally described in cattle, some genotypes (BPV-1 and BPV-2) have been associated with the development of papillomas in buffaloes, zebras, giraffes and yaks. Some genotypes also have been associated with the development of tumors in the gastrointestinal tract and in the urinary bladder in cattle, and with equine sarcoids. BPV infection has been described in different areas of the world, although not all genotypes have the same prevalence. Recent studies show a high incidence of multiple infections.La papilomatosis bovina es una enfermedad del ganado vacuno caracterizada por la presencia de papilomas y fibropapilomas, especialmente en piel y ubres. Está originada por la infección por papilomavirus bovinos (BPV), virus desnudos de la familia Papillomaviridae. Son virus epiteliotrópicos, aunque se ha detectado su presencia en sangre, leche, orina y semen. Presentan alta diversidad viral, reconociéndose hasta la fecha 13 tipos (BPV-1 a BPV- 13). Aunque originalmente se describieron en ganado vacuno, algunos genotipos (BPV-1 y BPV-2) se han asociado al desarrollo de papilomas en búfalos, cebras, jirafas y yaks. Algunos genotipos se han relacionado asimismo con el desarrollo de tumores en tracto gastrointestinal y cáncer de vejiga urinaria en ganado bovino y con sarcoides equinos. La infección por BPV se ha descrito en diferentes zonas del mundo, aunque no todos los genotipos presentan la misma prevalencia. Los trabajos más recientes muestran una elevada incidencia de infecciones múltiples

Polymerase chain reaction detection of avipox and avian papillomavirus in naturally infected wild birds: comparisons of blood, swab and tissue samples

Avian poxvirus (avipox) is widely reported from avian species, causing cutaneous or mucosal lesions. Mortality rates of up to 100% are recorded in some hosts. Three major avipox clades are recognized. Several diagnostic techniques have been reported, with molecular techniques used only recently. Avipox has been reported from 278 different avian species, but only 111 of these involved sequence and/or strain identification. Collecting samples from wild birds is challenging as only few wild bird individuals or species may be symptomatic. Also, sampling regimes are tightly regulated and the most efficient sampling method, whole bird collection, is ethically challenging. In this study, three alternative sampling techniques (blood, cutaneous swabs and tissue biopsies) from symptomatic wild birds were examined. Polymerase chain reaction was used to detect avipoxvirus and avian papillomavirus (which also induces cutaneous lesions in birds). Four out of 14 tissue samples were positive but all 29 blood samples and 22 swab samples were negative for papillomavirus. All 29 blood samples were negative but 6/22 swabs and 9/14 tissue samples were avipox-positive. The difference between the numbers of positives generated from tissue samples and from swabs was not significant. The difference in the avipox-positive specimens in paired swab (4/6) and tissue samples (6/6) was also not significant. These results therefore do not show the superiority of swab or tissue samples over each other. However, both swab (6/22) and tissue (8/9) samples yielded significantly more avipox-positive cases than blood samples, which are therefore not recommended for sampling these viruses.The authors thank bird ringers from Alula and Monticola, especially Alfredo Ortega and Chechu Aguirre, for help with the capture and ringing of birds, which made this project possible. Thanks to Alvaro Ramírez for samples. This research was funded by the Ministerio de Ciencia e Innovación, Spain (grant number: CGL2010-15734/BOS). R.A.J.W. was supported by the Programa Internacional de Captación de Talento (PICATA) de Moncloa Campus de Excelencia Internacional while writing the manuscript

A Narrative Review of Cell-Based Approaches for Cranial Bone Regeneration.

Current cranial repair techniques combine the use of autologous bone grafts and biomaterials. In addition to their association with harvesting morbidity, autografts are often limited by insufficient quantity of bone stock. Biomaterials lead to better outcomes, but their effectiveness is often compromised by the unpredictable lack of integration and structural failure. Bone tissue engineering offers the promising alternative of generating constructs composed of instructive biomaterials including cells or cell-secreted products, which could enhance the outcome of reconstructive treatments. This review focuses on cell-based approaches with potential to regenerate calvarial bone defects, including human studies and preclinical research. Further, we discuss strategies to deliver extracellular matrix, conditioned media and extracellular vesicles derived from cell cultures. Recent advances in 3D printing and bioprinting techniques that appear to be promising for cranial reconstruction are also discussed. Finally, we review cell-based gene therapy approaches, covering both unregulated and regulated gene switches that can create spatiotemporal patterns of transgenic therapeutic molecules. In summary, this review provides an overview of the current developments in cell-based strategies with potential to enhance the surgical armamentarium for regenerating cranial vault defects

Disruption of Proteostasis by Natural Products and Synthetic Compounds That Induce Pervasive Unfolding of Proteins: Therapeutic Implications

Exposure of many cancer cells, including multiple myeloma cells, to cytotoxic concentrations of natural products celastrol and withaferin A or synthetic compounds of the IHSF series resulted in denaturation of a luciferase reporter protein. Proteomic analysis of detergent-insoluble extract fractions from HeLa-derived cells revealed that withaferin A, IHSF058 and IHSF115 caused denaturation of 915, 722 and 991 of 5132 detected cellular proteins, respectively, of which 440 were targeted by all three compounds. Western blots showed that important fractions of these proteins, in some cases approaching half of total protein amounts, unfolded. Relatively indiscriminate covalent modification of target proteins was observed; 1178 different proteins were modified by IHSF058. Further illustrating the depth of the induced proteostasis crisis, only 13% of these proteins detectably aggregated, and 79% of the proteins that aggregated were not targets of covalent modification. Numerous proteostasis network components were modified and/or found in aggregates. Proteostasis disruption caused by the study compounds may be more profound than that mediated by proteasome inhibitors. The compounds act by a different mechanism that may be less susceptible to resistance development. Multiple myeloma cells were particularly sensitive to the compounds. Development of an additional proteostasis-disrupting therapy of multiple myeloma is suggested

List of Leporidae voucher specimens inspected for SfPV growths in the Kansas University Natural History Museum.

<p>Number of individuals inspected (N), symptomatic (S) and PCR positive (Pos) and percentage of individuals’ symptomatic (% S) and of symptomatic individuals positive by PCR (% Pos).</p><p>^a Note, though 16 <i>S</i>. <i>floridanus</i> were symptomatic, only 15 were tested. Values of % Pos for <i>S</i>. <i>floridanus</i> and Leporidae were calculated accordingly.</p><p>List of Leporidae voucher specimens inspected for SfPV growths in the Kansas University Natural History Museum.</p

Gold nanoparticles for the in situ polymerization of near-infrared responsive hydrogels based on fibrin

Non-invasiveness and relative safety of photothermal therapy, which enables local hyperthermia of target tissues using a near infrared (NIR) laser, has attracted increasing interest. Due to their biocompatibility, amenability of synthesis and functionalization, gold nanoparticles have been investigated as therapeutic photothermal agents. In this work, hollow gold nanoparticles (HGNP) were coated with poly-L-lysine through the use of COOH-Poly(ethylene glycol)-SH as a covalent linker. The functionalized HGNP, which peak their surface plasmon resonance at 800 nm, can bind thrombin. Thrombin-conjugated HGNP conduct in situ fibrin polymerization, facilitating the process of generating photothermal matrices. Interestingly, the metallic core of thrombin-loaded HGNP fragmentates at physiological temperature. During polymerization process, matrices prepared with thrombin-loaded HGNP were loaded with genetically-modified stem cells that harbour a heat-activated and ligand-dependent gene switch for regulating transgene expression. NIR laser irradiation of resulting cell constructs in the presence of ligand successfully triggered transgene expression in vitro and in vivo.This work was supported by grant PI15/01118 from Instituto de Salud Carlos III (ISCIII)-Fondos FEDER, Ministry of Economy and Competitiveness (MINECO), Spain, grants RTI2018- 095159-B-I00 and SAF2013-50364-EXP from MINECO, 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. was the re- cipient of predoctoral grant FI14/00447 from ISCIII-Fondos FEDER, MINECO. N.V. is supported by Program I2 from CA

Results of tests on <i>symptomatic</i> Sylvilagus rabbits. Includes locality and year of host collection.

<p>*indicates non-symptomatic negative control. Abbreviations: KS (Kansas), Jal (Jalisco), NE (Nebraska), N.L. (Nuevo Leon), NM (New Mexico), NV (Nevada); NT (not tested). Ratio shows the results of spectrophotometric analysis of DNA templates.</p><p>Results of tests on <i>symptomatic</i> Sylvilagus rabbits. Includes locality and year of host collection.</p