Ana Belén Jódar-Reyes

Role of wettability and nanoroughness on interactions between osteoblast and modified silicon surfaces.

Development of new biomaterials is a constant in regenerative medicine. A biomaterials surface properties, such as wettability, roughness, surface energy, surface charge, chemical functionalities and composition, are determinants of cell adhesion and subsequent tissue behavior. Thus, the main aim of this study was to analyze the correlation between changes in wettability without topographical variation and the response of osteoblast-like cells. For this purpose oxidized silicon surfaces were methylated to different degrees. Additionally, the influence of nanoroughness, and the subsequent effect of hysteresis on cell behavior, was also analyzed. In this case oxidized silicon pieces were etched with caustic solutions to produce different degrees of nanoroughness. Axisymmetric drop-shape analysis and atomic force microscopy confirmed that the proposed surface treatments increased the nanometer roughness and/or the water contact angles. MG-63 osteoblast-like cells were cultured on the altered surfaces to study proliferation, and for ultrastructural analysis and immunocytochemical characterization. Increasing the nanometer surface roughness or water contact angle enhanced osteoblast behavior in terms of cell morphology, proliferation and immunophenotype, the effect provoked by methylation being more significant than that caused by nanoroughness.

Interactions between Pluronics (F127 and F68) and Bile Salts (NaTDC) in the Aqueous Phase and the Interface of Oil-in-Water Emulsions

Pluronics are being introduced in food research in order to delay lipid digestion, with the length of hydrophilic and hydrophobic chains playing an important role in the rate of such a process. Since bile salts play a crucial role in the lipid digestion process, the aim of this work is to analyze the interactions between Pluronic F127 or F68 and the bile salt NaTDC when the latter is added at physiological concentrations. These interactions are studied at the Pluronic-covered oil-water interface and in the aqueous phase of Pluronic-stabilized emulsions. This work has been carried out with techniques such as differential scanning calorimetry, interfacial tension, dilatational rheology, and scanning electron microscopy. As a result, Pluronic F127 was shown to be more resistant to displacement by bile salt than F68 at the oil-water interface due to the larger steric hindrance and interfacial coverage provided. In addition, Pluronics have the ability to compete for the oil-water interface and interact in the bulk with the bile salt. Concretely, Pluronic F127 seems to interact with more molecules of bile salt in the bulk, thus hindering their adsorption onto the oil-water interface. As a conclusion, Pluronic F127 affects to a larger extent the ability of bile salt to promote the further cascade of lipolysis in the presence of lipase owing to a combination of interfacial and bulk events.

Bone Regeneration from PLGA Micro-Nanoparticles

Poly-lactic-co-glycolic acid (PLGA) is one of the most widely used synthetic polymers for development of delivery systems for drugs and therapeutic biomolecules and as component of tissue engineering applications. Its properties and versatility allow it to be a reference polymer in manufacturing of nano- and microparticles to encapsulate and deliver a wide variety of hydrophobic and hydrophilic molecules. It additionally facilitates and extends its use to encapsulate biomolecules such as proteins or nucleic acids that can be released in a controlled way. This review focuses on the use of nano/microparticles of PLGA as a delivery system of one of the most commonly used growth factors in bone tissue engineering, the bone morphogenetic protein 2 (BMP2). Thus, all the needed requirements to reach a controlled delivery of BMP2 using PLGA particles as a main component have been examined. The problems and solutions for the adequate development of this system with a great potential in cell differentiation and proliferation processes under a bone regenerative point of view are discussed.

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.

Colloidal stability and “in vitro” antitumor targeting ability of lipid nanocapsules coated by folate–chitosan conjugates

In this study, the synthesis and characterization of lipid nanocapsules coated with folate–chitosan conjugates at varying folate concentrations are reported; these nanocapsules have a potential application as anticancer drug carriers. The main goal of this study was to evaluate (a) the colloidal stability of the particles and (b) their cell targeting. A classical colloidal characterization of the nanocapsules was carried out by analyzing size, electrokinetic charge, and stability in different saline solutions, including cell culture media. At neutral pH, the stability was improved by the presence of folate due to electrical interactions. In addition, folate modulated the hydrophilic/phobic nature of the surface, which became critical to keep the systems stable (or not) under physiological saline conditions due to the action of short-range repulsive hydration forces. The cellular uptake of our nanocapsules was evaluated by working with four tumor cell lines. Both fluorescent analyses with particles colored by Nile Red, and antitumor activity of our systems loaded with docetaxel, demonstrated that the folate-mediated internalization of the particles in the cancer cells was improved when the nanocapsules were coated by folate–chitosan conjugates.

The role of hydrophobic alkyl chains in the physicochemical properties of poly(β-amino ester)/DNA complexes

Two degradable poly(β-amino ester)s with an average molecular weight of 2kDa, referred to as B1 and B2, have been synthesized to be tested as non-viral gene delivery systems. B2 polymer exhibits two additional non-polar ethyl groups at both ends. This paper describes the influence of that subtle difference on the compaction ability and temporal stability of the complexes formed with plasmid DNA. Our results suggest that the inclusion of those small hydrophobic fragments into the polycation backbone improves its suitability as synthetic DNA carrier. The improvement is related to the formation and physicochemical properties of the complexes. B2 polyplexes were more stable, the polymer hydrolysis was slowed down and plasmid DNA was better protected which was translated into better transfection efficiencies. Although still not totally understood, the role played by hydrophobic forces is ubiquitous in chemical, biological and physical systems, and they must be considered to design future polymers for gene delivery.

Interaction of surfactant and protein at the O/W interface and its effect on colloidal and biological properties of polymeric nanocarriers

HYPOTHESIS The use of polymer-based surfactants in the double-emulsion (water/oil/water, W/O/W) solvent-evaporation technique is becoming a widespread strategy for preparing biocompatible and biodegradable polymeric nanoparticles (NPs) loaded with biomolecules of interest in biomedicine, or biotechnology. This approach enhances the stability of the NPs, reduces their size and recognition by the mononuclear phagocytic system, and protects the encapsulated biomolecule against losing biological activity. Different protocols to add the surfactant during the synthesis lead to different NP colloidal properties and biological activity. EXPERIMENTS We develop an in vitro model to mimic the first step of the W/O/W NP synthesis method, which enables us to analyze the surfactant-biomolecule interaction at the O/W interface. We compare the interfacial properties when the surfactant is added from the aqueous or the organic phase, and the effect of pH of the biomolecule solution. We work with a widely used biocompatible surfactant (Pluronic F68), and lysozyme, reported as a protein model. FINDINGS The surfactant, when added from the water phase, displaces the protein from the interface, hence protecting the biomolecule. This could explain the improved colloidal stability of NPs, and the higher biological activity of the lysozyme released from nanoparticles found with the counterpart preparation.

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 in vitro and in vivo 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.

Complexation and release of DNA in polyplexes formed with reducible linear poly(β-amino esters).

Designing nanocarriers for gene delivery is a multidisciplinary challenge that involves not only DNA condensation with biocompatible polymers, but also DNA-release processes. Once the genetic material is introduced into the cell, the rupture of degradable bonds permits the unpacking and release of the load. In this work, a dual-degradable polycation - composed by a linear poly(β-amino ester) chain in which ester and disulfide bonds coexist - has been used to condense a DNA plasmid. The goal was to reinforce the spontaneous hydrolysis of the ester groups with the intracellular break-up of the disulfide bonds, since these reducible bonds are degraded in the reductive intracellular environment. For a comparative study, two poly(β-amino ester) molecules differing only in the presence (or absence) of some SS bonds have been tested. DNA condensation, physico-chemical characterization of the polyplexes formed, and degradation studies have been carried out at pH 5 and pH 7. The acidic conditions gave the best nanoparticles, due to a better solubilization of both polymers and to a higher stability of the ester bonds. Despite the synthesis and storage of polyplexes were much more appropriate at pH 5, transfection efficiency in HeLa cells was similar irrespective the original pH used. Only in those polyplexes formed at low polymer:DNA ratios (i.e. 5 and 10 (w/w)) was transfection more effective when the plasmid was condensed at an acidic pH. With regard to the DNA-release efficiency in the intracellular medium, degradation of the polymers was practically governed by the rapid hydrolysis of the ester groups, this spontaneous and rapid process masking, unfortunately, any potential contribution associated with the breakup of the disulfide bonds.