Controlled Release of rAAV Vectors from APMA-Functionalized Contact Lenses for Corneal Gene Therapy

As an alternative to eye drops and ocular injections for gene therapy, the aim of this work was to design for the first time hydrogel contact lenses that can act as platforms for the controlled delivery of viral vectors (recombinant adeno-associated virus, rAAV) to the eye in an effective way with improved patient compliance. Hydrogels of hydroxyethyl methacrylate (HEMA) with aminopropyl methacrylamide (APMA) (H1: 40, and H2: 80 mM) or without (Hc: 0 mM) were synthesized, sterilized by steam heat (121 °C, 20 min), and then tested for gene therapy using rAAV vectors to deliver the genes to the cornea. The hydrogels showed adequate light transparency, oxygen permeability, and swelling for use as contact lenses. Loading of viral vectors (rAAV-lacZ, rAAV-RFP, or rAAV-hIGF-I) was carried out at 4 °C to maintain viral vector titer. Release in culture medium was monitored by fluorescence with Cy3-rAAV-lacZ and AAV Titration ELISA. Transduction efficacy was tested through reporter genes lacZ and RFP in human bone marrow derived mesenchymal stem cells (hMSCs). lacZ was detected with X-Gal staining and quantified with Beta-Glo®, and RFP was monitored by fluorescence. The ability of rAAV-hIGF-I-loaded hydrogels to trigger cell proliferation in hMSCs was evaluated by immunohistochemistry. Finally, the ability of rAAV-lacZ-loaded hydrogels to transduce bovine cornea was confirmed through detection with X-Gal staining of β-galactosidase expressed within the tissue

Evaluation of human umbilical vein endothelial cells growth onto heparin-modified electrospun vascular grafts

One of the main challenges of cardiovascular tissue engineering is the development of bioresorbable and compliant small-diameter vascular grafts (SDVG) for patients where autologous grafts are not an option. In this work, electrospun bilayered bioresorbable SDVG based on blends of poly(L-lactic acid) (PLLA) and segmented polyurethane (PHD) were prepared and evaluated. The inner layer of these SDVG was surface-modified with heparin, following a methodology involving PHD urethane functional groups. Heparin was selected as anticoagulant agent, and also due to its ability to promote human umbilical vein endothelial cells (HUVECs) growth and to inhibit smooth muscle cells over-proliferation, main cause of neointimal hyperplasia and restenosis. Immobilized heparin was quantified and changes in SDVG microstructure were investigated through SEM. Tensile properties of the heparin-functionalized SDVG resembled those of saphenous vein. Vascular grafts were seeded with HUVECs and cultured on a flow-perfusion bioreactor to analyze the effect of heparin on graft endothelization under simulated physiological-like conditions. The analysis of endothelial cells attachment and gene expression (Real-Time PCR) pointed out that the surface functionalization with heparin successfully promoted a stable and functional endothelial cell layer.Fil: Caracciolo, Pablo Christian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; ArgentinaFil: Diaz Rodriguez, Patricia. Universidad de Santiago de Compostela; EspañaFil: Ardao, Inés. Universidad de Santiago de Compostela; EspañaFil: Moreira, David. Universidad de Santiago de Compostela; EspañaFil: Montini Ballarin, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; ArgentinaFil: Abraham, Gustavo Abel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; ArgentinaFil: Concheiro, Angel. Universidad de Santiago de Compostela; EspañaFil: Alvarez Lorenzo, Carmen. Universidad de Santiago de Compostela; Españ

Supercritical fluid (SCF)-assisted preparation of cyclodextrin-based poly(pseudo)rotaxanes for transdermal purposes

This study aims to investigate the effect of the preparation of solid dispersions using supercritical CO2 (scCO2) on the physicochemical properties and the performance of supramolecular gels based on polymer-cyclodextrin (CD) interactions (named poly(pseudo)rotaxanes, PPR) envisaging a transdermal administration. Solid dispersions containing Soluplus®, the antihypertensive drug carvedilol (CAR), and CD (αCD or HPβCD) were prepared and characterized by HPLC, XRPD, FTIR, and DSC. PPRs prepared from solid dispersions (SCF gels) and the corresponding physical mixtures (PM gels) were analyzed regarding rheology, morphology, in vitro drug diffusion, and ex vivo drug skin permeation. The application of scCO2 led to the loss of the crystalline lattice of CAR while preserving its chemical identity. On the contrary, αCD crystals were still present in the SCF solid dispersions. SCF gels were more uniform than their corresponding PM, and the supercritical treatment resulted in changes in the rheological behavior, reducing the viscosity. CAR in vitro diffusion was significantly higher (p < 0.05) for the αCD-based SCF gel than its corresponding PM gel. Drug skin permeation showed a significant increase in drug flux from CD-based SCF gels (containing αCD or HPβCD) compared to corresponding PM gels. Additionally, the pretreatment of the skin with αCD exhibited increased CAR permeation, suggesting an interaction between αCD and the skin membrane. Results evidenced that SCF processing decisively modified the properties of the supramolecular gels, particularly those prepared with αCDOpen Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This research was partially supported by the Brazilian agency Fundação de Apoio à Pesquisa do Estado de Goiás (FAPEG). The work was supported by MCIN/AEI/10.13039/501100011033 (PID 2020-113881RB-I00), Spain, Xunta de Galicia (ED431C 2020/17), and FEDERS

Pluronic®/casein micelles for ophthalmic delivery of resveratrol: In vitro, ex vivo, and in vivo tests

Ocular health may strongly benefit from the supply of antioxidant agents that counteract free radicals and reactive oxygen species responsible for long-term eye diseases. Additionally, natural antioxidants like resveratrol can inhibit bacteria growth and restore natural microbiota. However, their use is hindered by limited solubility, fast degradation, and low ocular permeability. This work aimed to overcome these limitations by preparing single and mixed micelles of Pluronic® F127 and casein that serve as resveratrol nanocarriers. Single and mixed (0.1 % casein) micelles (0.0 to −17.0 mV; 2.4 to 32.7 nm) increased 50-fold resveratrol solubility, remained stable for one month at 4 °C, withstood fast dilution, underwent sol-to-gel transitions in the 23.9–27.1 °C range, and exhibited potent antioxidant properties. All formulations successfully passed the HET-CAM assay but showed Pluronic®-casein dose-dependent toxicity in the zebrafish embryo model. Resveratrol-loaded single and mixed micelles (10–15 mM Pluronic® F127) displayed antimicrobial activity against S. aureus and P. aeruginosa. The micelles favored resveratrol accumulation in cornea and sclera, but mixed micelles showed larger lag times and provided lower amount of resveratrol permeated through sclera. In vivo (rabbit) tests confirmed the safety of resveratrol-loaded single micelles and their capability to supply resveratrol to anterior and posterior eye segments.Funding: The work was supported by MCIN/AEI/10.13039/501100011033 [PID 2020-113881RB-I00 to A.C. and C.A.-L., and PID2020-115121GB-I00 to L.S. and A.B.-I.], Spain, Xunta de Galicia [ED431C 2020/17], and FEDER. M. Vivero-Lopez acknowledges Xunta de Galicia (Consellería de Cultura, Educación e Ordenación Universitaria) for a predoctoral research fellowship [ED481A-2019/120].S

New Advances in Biomedical Application of Polymeric Micelles

Abstract In the last decade, nanomedicine has arisen as an emergent area of medicine, which studies nanometric systems, namely polymeric micelles (PMs), that increase the solubility and the stability of the encapsulated drugs. Furthermore, their application in dermal drug delivery is also relevant. PMs present unique characteristics because of their unique core-shell architecture. They are colloidal dispersions of amphiphilic compounds, which self-assemble in an aqueous medium, giving a structure-type core-shell, with a hydrophobic core (that can encapsulate hydrophobic drugs), and a hydrophilic shell, which works as a stabilizing agent. These features offer PMs adequate steric protection and determine their hydrophilicity, charge, length, and surface density properties. Furthermore, due to their small size, PMs can be absorbed by the intestinal mucosa with the drug, and they transport the drug in the bloodstream until the therapeutic target. Moreover, PMs improve the pharmacokinetic profile of the encapsulated drug, present high load capacity, and are synthesized by a reproducible, easy, and low-cost method. In silico approaches have been explored to improve the physicochemical properties of PMs. Based on this, a computer-aided strategy was developed and validated to enable the delivery of poorly soluble drugs and established critical physicochemical parameters to maximize drug loading, formulation stability, and tumor exposure. Poly(2-oxazoline) (POx)-based PMs display unprecedented high loading concerning water-insoluble drugs and over 60 drugs have been incorporated in POx PMs. Among various stimuli, pH and temperature are the most widely studied for enhanced drug release at the site of action. Researchers are focusing on dual (pH and temperature) responsive PMs for controlled and improved drug release at the site of action. These dual responsive systems are mainly evaluated for cancer therapy as certain malignancies can cause a slight increase in temperature and a decrease in the extracellular pH around the tumor site. This review is a compilation of updated therapeutic applications of PMs, such as PMs that are based on Pluronics®, micelleplexes and Pox-based PMs in several biomedical application

Computational modeling and molecular imprinting for the development of acrylic polymers with high affinity for bile salts

This work has focused on the rational development of polymers capable of acting as traps of bile salts. Computational modeling was combined with molecular imprinting technology to obtain networks with high affinity for cholate salts in aqueous medium. The screening of a virtual library of 18 monomers, which are commonly used for imprinted networks, identified N-(3-aminopropyl)-methacrylate hydrochloride (APMA·HCl), N,N-diethylamino ethyl methacrylate (DEAEM) and ethyleneglycol methacrylate phosphate (EGMP) as suitable functional monomers with medium-to-high affinity for cholic acid. The polymers were prepared with a fix cholic acid:functional monomer mole ratio of 1:4, but with various cross- linking densities. Compared to polymers prepared without functional monomer, both imprinted and non-imprinted microparticles showed a high capability to remove sodium cholate from aqueous medium. High affinity APMA-based particles even resembled the performance of commercially available cholesterol-lowering granules. The imprinting effect was evident in most of the networks prepared, showing that computational modeling and molecular imprinting can act synergistically to improve the performance of certain polymers. Nevertheless, both the imprinted and non-imprinted networks prepared with the best monomer (APMA·HCl) identified by the modeling demonstrated such high affinity for the template that the imprinting effect was less important. The fitting of adsorption isotherms to the Freundlich model indicated that, in general, imprinting increases the population of high affinity binding sites, except when the affinity of the functional monomer for the target molecule is already very high. The cross-linking density was confirmed as a key parameter that determines the accessibility of the binding points to sodium cholate. Materials prepared with 9% mol APMA and 91% mol cross-linker showed enough affinity to achieve binding levels of up to 0.4 mmol g−1 (i.e., 170 mg g−1) under flow (1 mL min−1) of 0.2 mM sodium cholate

Electrospun Fibers of Cyclodextrins and Poly(cyclodextrins)

Cyclodextrins (CDs) can endow electrospun fibers with outstanding performance characteristics that rely on their ability to form inclusion complexes. The inclusion complexes can be blended with electrospinnable polymers or used themselves as main components of electrospun nanofibers. In general, the presence of CDs promotes drug release in aqueous media, but they may also play other roles such as protection of the drug against adverse agents during and after electrospinning, and retention of volatile fragrances or therapeutic agents to be slowly released to the environment. Moreover, fibers prepared with empty CDs appear particularly suitable for affinity separation. The interest for CD-containing nanofibers is exponentially increasing as the scope of applications is widening. The aim of this review is to provide an overview of the state-of-the-art on CD-containing electrospun mats. The information has been classified into three main sections: (i) fibers of mixtures of CDs and polymers, including polypseudorotaxanes and post-functionalization; (ii) fibers of polymer-free CDs; and (iii) fibers of CD-based polymers (namely, polycyclodextrins). Processing conditions and applications are analyzed, including possibilities of development of stimuli-responsive fibers