Juan Parra

Anticancer and antiangiogenic activity of surfactant-free nanoparticles based on self-assembled polymeric derivatives of vitamin E: Structure-activity relationship

α-Tocopheryl succinate (α-TOS) is a well-known mitochondrially targeted anticancer compound, however, it is highly hydrophobic and toxic. In order to improve its activity and reduce its toxicity, new surfactant-free biologically active nanoparticles (NP) were synthesized. A methacrylic derivative of α-TOS (MTOS) was prepared and incorporated in amphiphilic pseudoblock copolymers when copolymerized with N-vinylpyrrolidone (VP) by free radical polymerization (poly(VP-co-MTOS)). The selected poly(VP-co-MTOS) copolymers formed surfactant-free NP by nanoprecipitation with sizes between 96 and 220 nm and narrow size distribution, and the in vitro biological activity was tested. In order to understand the structure-activity relationship three other methacrylic monomers were synthesized and characterized: MVE did not have the succinate group, SPHY did not have the chromanol ring, and MPHY did not have both the succinate group and the chromanol ring. The corresponding families of copolymers (poly(VP-co-MVE), poly(VP-co-SPHY), and poly(VP-co-MPHY)) were synthesized and characterized, and their biological activity was compared to poly(VP-co-MTOS). Both poly(VP-co-MTOS) and poly(VP-co-MVE) presented triple action: reduced cell viability of cancer cells with little or no harm to normal cells (anticancer), reduced viability of proliferating endothelial cells with little or no harm to quiescent endothelial cells (antiangiogenic), and efficiently encapsulated hydrophobic molecules (nanocarrier). The anticancer and antiangiogenic activity of the synthesized copolymers is demonstrated as the active compound (vitamin E or α-tocopheryl succinate) do not need to be cleaved to trigger the biological action targeting ubiquinone binding sites of complex II. Poly(VP-co-SPHY) and poly(VP-co-MPHY) also formed surfactant-free NP that were also endocyted by the assayed cells; however, these NP did not selectively reduce cell viability of cancer cells. Therefore, the chromanol ring of the vitamin E analogues has an important role in the biological activity of the copolymers. Moreover, when succinate moiety is substituted and vitamin E is directly linked to the macromolecular chain through an ester bond, the biological activity is maintained.

Low polydispersity (N-ethyl pyrrolidine methacrylamide-co-1-vinylimidazole) linear oligomers for gene therapy applications.

Nonviral methods for gene delivery are becoming ever more prevalent along with the need to design new vectors that are highly effective, stable in biological fluids, inexpensive, and facile to produce. Here, we synthesize our previously reported monomer N-ethyl pyrrolidine methacrylamide (EPA) and evaluate its effectiveness in gene vector applications when copolymerized with 1-vinylimidazole (VI). A range of these novel linear cationic copolymers were synthesized via free radical polymerization with low molecular weights (oligomers) and low polydispersities showing two pK(a) values as the two co-monomers are cationic. DNA-polymer polyplexes had average sizes between 100 and 250nm and zeta-potentials between 10 and 25mV, and a strong dependence of composition on the size on the zeta-potential was observed. The cytotoxicity of the homopolymers, oligomers, and polyplexes toward human fibroblasts and 3T3 mouse fibroblasts was evaluated using the MTT and AlamarBlue™ assays, proving that formulations could be made with toxicity as low as low molecular weight linear poly (dimethylaminoethyl methacrylate) (PDMAEMA). The transfection capability of the polyplexes measured using the G-luciferase marker gene far superseded PDMAEMA when evaluated in biological conditions. Furthermore, blood compatibility studies showed that these new oligomers exhibit no significant hemolysis or platelet activation above PBS controls. These new EPA based oligomers with low toxicity and ease of scalability show high transfection abilities in serum conditions, and blood compatibility showing its potential for systemic gene delivery applications.

Bioactive polymeric systems with platelet antiaggregating activity for the coating of vascular devices.

The preparation, characterization, and analysis of physicochemical and biological properties of a new bioactive polymer system, based on a copolymer of an acrylic derivative of triflusal (a molecule with chemical structure related to aspirin with antiaggregating activity for platelets) is described and evaluated as thin bioactive coating for vascular grafts and coronary stents. The acrylic monomer derived from triflusal (THEMA) provides random copolymers when it is polymerized with butyl acrylate (BA), according to their reactivity ratios, r(THEMA) = 1.05 and r(BA) = 0.33. The copolymer THBA70, containing a molar composition f(THEMA) = 0.45 and f(BA) = 0.55 presents the optimal properties of stability, flexibility, and adhesion, with a T(g) = 21 ± 2 °C, to be applied as bioactive and biostable coatings for vascular grafts and coronary stents. Thin films of this copolymer system present an excellent biocompatibility and a good inherent antiaggregant activity for platelets.

Development of advanced biantibiotic loaded bone cement spacers for arthroplasty associated infections

The incidence increase of infections in patients with hip or knee implants with resistant pathogens (mainly some S. coagulase-negative and gram positive bacteria) demands advanced antibiotic loaded formulations. In this paper, we report the design of new biantibiotic acrylic bone cements for in situ delivery. They include a last generation antibiotic (daptomycin or linezolid) in combination with vancomycin and are performed based on a novel modification of the Palacos R® acrylic bone cement, which is based on two components, a liquid (methyl methacrylate) and a solid (polymeric phase). Hence, the solid component of the experimental formulations include 45wt% of microparticles of poly(D,L-lactic-co-glycolic) acid, 55wt% of poly(methyl methacrylate) beads and supplements (10wt-% each) of antibiotics. These formulations provide a selective and excellent control of the local release of antibiotics during a long time period (up to 2 months), avoiding systemic dissemination. The antimicrobial activity of the advanced spacers tested against S. aureus shows that single doses would be enough for the control of the infection. In vitro biocompatibility of cements on human osteoblasts is ensured. This paper is mainly focused on the preparation and characterization of cements and the studies of elution kinetics and bactericidal effects. Developed formulations are proposed as spacers for the treatment of infected arthroplasties, but also, they could be applied in other antibiotic devices to treat relevant bone-related infection diseases.

Mitochondrially targeted nanoparticles for the selective treatment of Head and Neck Squamous Cell Carcinoma

The aim of this work is the preparation of an active nanovehicle for the effective administration of α-tocopheryl succinate (α-TOS). α-TOS is loaded in the core of nanoparticles (NPs) based on amphiphilic pseudo-block copolymers of N-vinyl pyrrolidone and a methacrylic derivative of α-TOS. These well-defined spherical NPs have sizes below 165 nm and high encapsulation efficiencies. In vitro activity of NPs is tested in hypopharynx squamous carcinoma (FaDu) cells and nonmalignant epithelial cells, demonstrating that the presence of additional α-TOS significantly enhances its antiproliferative activity; however, a range of selective concentrations is observed. These NPs induce apoptosis of FaDu cells by activating the mitochondria death pathway (via caspase-9). Both loaded and unloaded NPs act via complex II and produce high levels of reactive oxygen species that trigger apoptosis. Additionally, these NPs effectively suppress the vascular endothelial growth factor (VEGF) expression of human umbilical vein endothelial cells (HUVECs). These results open the possibility to use this promising nanoformulation as an α-TOS delivery system for the effective cancer treatment, effectively resolving the current limitations of free α-TOS administration.

In vitro study of the proliferation and growth of human fetal osteoblasts on Mg and Si co‐substituted tricalcium phosphate ceramics

The objective of this work was to study the feasibility of the solid state sintering, a conventional ceramic processing method, to obtain Mg and Si co-substituted tricalcium phosphate bioceramics and composites containing diopside. A series of new Ca3 (PO4 )2 based ceramics has been prepared from attrition milled mixtures of synthetic Ca3 (PO4 )2 and CaMg(SiO3 )2 powders, isostatically pressed and sintered at 1250-1300°C. Materials containing 0, 1, and 5 wt % of CaMg(SiO3 )2 were constituted by β + α - Ca3 (PO4 )2 solid solutions while the material containing 60 wt % of CaMg(SiO3 )2 was a constituted by β- Ca3 (PO4 )2 and CaMg(SiO3 )2 . The biological responses of the developed ceramics were studied in vitro using human fetal osteoblast cultures. Culture times ranged from 1 to 21 days. The new family of materials promotes the adhesion and proliferation of human osteoblasts cultured onto their surface forming a monolayer and showing a normal morphology. The results of the MTT and Alamar Blue assays showed that the soluble components extracted from the Mg/Si- co-substituted Ca3 (PO4 )2 and the Ca3 (PO4 )2 -CaMg(SiO3 )2 composite were noncytotoxic. The specimens with diopside exhibited a better in vitro behavior which is attributed to the release of Si and Mg ions to the culture medium, enhancing the activity of cells.

Preparation of Targeting Vehicles for The Delivery of N-Bisphosphonates

Bisphosphonates (BP) are drugs currently administered orally to treat diseases characterised by an excessive bone resorption. Alternative and more efficient delivery routes and more potent compounds are being investigated. Three implantable delivery systems, which allow the controlled release of therapeutic agents from the device core, are examined in this paper. (4- (aminomethyl) benzene) bisphosphonic acid (ABBP) was incorporated on Ca8.8Na0.8(PO4)4.8(CO3)1.2(OH)0.4F1.6 particles by refluxing the powder in a 60 mmol suspension in acetone at 60°C for 5 hours. 4-aminophenyl acetic bisphosphonate monosodium salt (APBP) and 1- H-indole-3-acetic bisphosphonate monosodium (IBP) were loaded on Ca10(PO4)6(OH)1F1 ceramic bodies by stirring the ceramic bodies in 0.04M BP solutions. Injectable acrylic cements based on self-curing formulations of methyl methacrylate (MMA) and vitamin E were loaded with APBP and IBP. The incorporation of ABBP was confirmed by MAS-NMR spectroscopy. Modified powder shows two different phosphorous environments, the first one at 2.91 ppm can be assigned to the apatite base and the second one at 18.0 ppm has to be attributed to the phosphonic group of the ABBP. The IBP addition on ceramic surfaces did not decrease the number of osteoclast colonies and appeared to improve the performance of the HA as a surface for osteoblast culture. A therapeutic dosage of APBP and IBP can be achieved from acrylic cements that showed lack of toxicity and an increased cellular activity and proliferation.