Estudio de las condiciones para la síntesis de la hidroxiapatita y el β-fosfato tricálcico

El propósito de este estudio es encontrar las condiciones de síntesis reproducibles para obtener una HAP y un β-TCP, de fase única, con una pureza >99% y, de tamaño de partícula entre 20 y 60 nm; debido a la falta de congruencia en los procedimientos. El estudio se desarrolló, por el método de co-precipitación, con el control y la variación de las condiciones de reacción, de añejamiento y de cristalización. El seguimiento de los resultados se hizo por IR-TF y DRX. Los resultados para el β-TCP, mostraron la fase bifásica formada por β-TCP, con una cantidad mínima de HAP. En el caso de la HAP se estableció una metodología reproducible, para una pureza mayor al 99%, un tamaño 31.86 nm; similar a 35.54 nm a una, de tipo bovino. La HAP funcional sintetizada se usará en el diseño de andamios bioactivos mediante la impresión 3D para regeneración del tejido óseo.The aim of this study is obtain reproducible synthesis conditions for the HAP and the β-TCP, as unique phase, with a purity >99% and particle size of 20-60 nm; due to incongruence in procedures. The study was performed with the co-precipitation method, varying the conditions of reaction, aging and crystallization. The XRD and FT-IR were used to follow the procedures. The synthesis results of β-TCP showed a biphasic phase formed with β-TCP and a minimum amount of HAP. In the case of the HAP, a reproducible methodology was stablished, with a purity >99% and size of 31.86 nm, similar to one bovine of 35.54 nm. The HAP functional synthesized will be used in the design of bioactive scaffolds by printing 3D for bone tissue regeneration

PEI-NIR Heptamethine Cyanine Nanotheranostics for Tumor Targeted Gene Delivery

Polymer-based nanotheranostics are appealing tools for cancer treatment and diagnosis in the fast-growing field of nanomedicine. A straightforward preparation of novel engineered PEI-based nanotheranostics incorporating NIR fluorescence heptamethine cyanine dyes (NIRF-HC) to enable them with tumor targeted gene delivery capabilities is reported. Branched PEI-2 kDa (b2kPEI) is conjugated with IR-780 and IR-783 dyes by both covalent and noncovalent simple preparative methodologies varying their stoichiometry ratio. The as-prepared set of PEI-NIR-HC nanocarriers are assayed <i>in vitro</i> and <i>in vivo</i> to evaluate their gene transfection efficiency, cellular uptake, cytotoxicity, internalization and trafficking mechanisms, subcellular distribution, and tumor specific gene delivery. The results show the validity of the approach particularly for one of the covalent IR783-b2kPEI conjugates that exhibit an enhanced tumor uptake, probably mediated by organic anion transporting peptides, and favorable intracellular transport to the nucleus. The compound behaves as an efficient nanotheranostic transfection agent in NSG mice bearing melanoma G361 xenographs with concomitant imaging signal and gene concentration in the targeted tumor. By this way, advanced nanotheranostics with multifunctional capabilities (gene delivery, tumor-specific targeting, and NIR fluorescence imaging) are generated in which the NIRF-HC dye component accounts for simultaneous targeting and diagnostics, avoiding additional incorporation of additional tumor-specific targeting bioligands

Polyelectrolyte Complexes of Low Molecular Weight PEI and Citric Acid as Efficient and Nontoxic Vectors for in Vitro and in Vivo Gene Delivery

Gene transfection mediated by the cationic polymer polyethylenimine (PEI) is considered a standard methodology. However, while highly branched PEIs form smaller polyplexes with DNA that exhibit high transfection efficiencies, they have significant cell toxicity. Conversely, low molecular weight PEIs (LMW-PEIs) with favorable cytotoxicity profiles display minimum transfection activities as a result of inadequate DNA complexation and protection. To solve this paradox, a novel polyelectrolyte complex was prepared by the ionic cross-linking of branched 1.8 kDa PEI with citric acid (CA). This system synergistically exploits the good cytotoxicity profile exhibited by LMW-PEI with the high transfection efficiencies shown by highly branched and high molecular weight PEIs. The polyectrolyte complex (1.8 kDa-PEI@CA) was obtained by a simple synthetic protocol based on the microwave irradiation of a solution of 1.8 kDa PEI and CA. Upon complexation with DNA, intrinsic properties of the resulting particles (size and surface charge) were measured and their ability to form stable polyplexes was determined. Compared with unmodified PEIs the new complexes behave as efficient gene vectors and showed enhanced DNA binding capability associated with facilitated intracellular DNA release and enhanced DNA protection from endonuclease degradation. In addition, while transfection values for LMW-PEIs are almost null, transfection efficiencies of the new reagent range from 2.5- to 3.8-fold to those of Lipofectamine 2000 and 25 kDa PEI in several cell lines in culture such as CHO-k1, FTO2B hepatomas, L6 myoblasts, or NRK cells, simultaneously showing a negligible toxicity. Furthermore, the 1.8 kDa-PEI@CA polyelectrolyte complexes retained the capability to transfect eukaryotic cells in the presence of serum and exhibited the capability to promote in vivo transfection in mouse (as an animal model) with an enhanced efficiency compared to 25 kDa PEI. Results support the polyelectrolyte complex of LMW-PEI and CA as promising generic nonviral gene carriers