1,721,127 research outputs found
Thermosensitive hydrogels for 3D bioprinting of cartilage constructs
No full textTissue engineering (TE) aims to regenerate damaged tissues by the combined use of biomaterials and cells, often in presence of bioactive molecules, such as growth factors. Particularly for tissues with poor regenerative capacity, such as articular cartilage, TE approaches may lead to promising treatments. Articular cartilage is a connective tissue responsible for absorbing and distributing the load acting on the joint, to the undelaying bone. The limited regenerative capacity of articular cartilage is due to the absence of blood vessels in this tissue and consequent low cellular activity. Currently, an optimal treatment for cartilage defects does not exist. Therefore, TE strategies are exploited to obtain cell-laden scaffolds able to provide an initial mechanical support that over time degrades, while new tissue is formed by the embedded cells. The aim of this thesis was to develop biomechanically advanced hydrogel constructs for articular cartilage regeneration. Chapter 1 provides a general introduction of the anatomy and the physiology of articular cartilage, as well as the current clinically relevant techniques for its repair. Considering the limitations of present treatments, TE strategies for cartilage regeneration are presented as a promising alternative. Moreover, three-dimensional (3D) bioprinting is presented as a novel and versatile technology for the accurate design and generation of cartilage constructs. The experimental “core” of this thesis describes a step-by-step development of thermosensitive and photo-crosslinkable hydrogels based on partially methacrylated poly[N-(2-hydroxypropyl)methacrylamide mono-dilactate]/polyethylene glycol triblock copolymers (pHPMAlac-PEG) and chemically modified polysaccharides, i.e. methacrylated chondroitin sulfate (CSMA) or hyaluronic acid (HAMA), for 3D bioprinting of cartilage constructs. In Chapter 2, 3 and 4 an extensive and comprehensive in vitro characterization of this material is reported. The synthesis of the polymeric building blocks is achieved with a full control over polymer characteristics. Further, these building blocks are used to fabricate hydrogels with favorable rheological behavior for 3D bioprinting application and able to support cartilage-like tissue formation by embedded chondrocytes. Chapter 5 describes an introductory study on the possibility to enrich pHPMAlac-PEG hydrogels with protein-laden microgels for a controlled, in situ release of proteins. Chapter 6 describes the 3D bioprinting of pHPMAlac-PEG/HAMA hydrogels in combination with a polycaprolactone (PCL)-based reinforcement for the development of cartilage composite constructs with relevant stiffness and high chondrogenic potential. In Chapter 7, we report about the ectopic implantation of pHPMAlac-PEG/HAMA hydrogels in small animal (murine) and large animal (equine) models, as well as the orthotopic implantation in a large animal (porcine) model, as a work up for the future orthotopic implantation in a highly challenging equine model. Importantly, pHPMAlac-PEG/HAMA hydrogels showed adequate biocompatibility in the tested species and locations. Nevertheless, variability in hydrogel stability and resistance between different implantation sites and among the different species highlights the need for further optimization before an orthotopic, long-term screening in horses is undertaken
Polymeric microspheres for local delivery of proteins by administration under the kidney capsule
No full textAcute kidney injury and chronic kidney disease are conditions characterized by the inability of the kidneys to adequately filter waste products from the blood. The most common treatment remains renal replacement therapy (dialysis and renal transplantation), which unfortunately is associated with increased mortality rates. This thesis investigates a relatively novel approach for the treatment of kidney related diseases, by using polymeric microspheres loaded with a therapeutic protein. These microspheres were injected under the kidney capsule as a novel administration method to deliver drugs locally to the kidneys. Microspheres were prepared with poly(D,L-lactic-co-hydroxymethyl glycolic acid) (PLHMGA) copolymer using the membrane emulsification method in order to achieve uniformly dispersed spheres. PLHMGA microspheres were first tested for their safety after subcutaneous and subcapsular renal injection. Next, near-infrared labelled albumin (NIR-BSA) was encapsulated into PLHMGA microspheres followed by an injection under the kidney capsule in order to determine the release rate and the redistribution of the protein. In the last step of this thesis, the effect of the formulation conditions was tested after encapsulation of a therapeutic protein, erythropoietin (EPO), into PLHMGA microspheres. These microspheres were prepared with solid-in-oil-in-water (s/o/w) and solid-in-oil-in-oil (s/o/o) methods. The release of EPO and subsequent stability was tested with ELISA and Western blot. PLHMGA microspheres showed good biocompatibility properties and can be safely used in vivo as a drug delivery system. Further, PLHMGA microspheres had an average diameter of 35 µm and were degraded within 28 days post injection. A continuous release of around 90% of NIR-BSA loading was observed from the injected depot within a period of 2 weeks. Mainly the intact protein was released from the injected depot. The released NIR-BSA was further metabolized in the liver and subsequently the degradation products were cleared by the kidneys. This shows that the protein from the depot is mainly located at the injection site in the kidney. The encapsulation of EPO into PLHMGA microspheres resulted in low amount of dimmers (2-11%). However, when subjected to in vitro release studies, only a burst release of EPO was observed and no further release was detected with ELISA, which could be explained that upon hydration of microspheres during in vitro release studies, EPO can undergo a moisture-induced covalent aggregation resulting in the formation of dimers, trimers and higher-order oligomers. However, there is evidence in the literature that these higher order structures of EPO are pharmacologically active. In conclusion, locally administered PLHMGA microspheres injected under the kidney capsule are characterized by a 2-week release period of an encapsulated protein, making them an attractive delivery system. The subcapsular renal injection of this delivery system has the potential to reduce side effects of therapeutic proteins by increasing their presence at the site of injection and decreasing it elsewhere in the body
Decationized polyplexes for targeted delivery of nucleic acids: from carrier design to in vivo evaluation
No full textGene therapy is considered a promising treatment for current intractable diseases. However, the clinical applicability of gene therapy is highly dependent on the development of safe and efficient gene delivery vectors. So far, viral vectors have been used for clinical applications, but due to severe risks associated with viruses, cationic polymers have been evaluated as alternatives to viral vectors. Cationic polymers can easily form nanosized particles with plasmid DNA (pDNA), named polyplexes, via electrostatic interactions and possess an enormous chemical and structural flexibility. Polycation-based vectors have demonstrated high efficiency in vitro, however, they induce severe toxicity and possess suboptimal efficiencies in vivo, mainly due to their cationic nature, which significantly hampers their clinical applicability. With our work we have developed an alternative to conventional polycation-based polyplexes: decationized polyplexes. Unlike the cationic polymer based systems, decationized polyplexes are formed by hydrophilic and neutral polymers and can be obtained by an innovative 3-step process: polyplex formation by electrostatic interaction between pDNA and a polycationic precursor, structure stabilization by disulfide crosslinking, and finally removal of cationic charge - decationization. Structurally, decationized polyplexes consist of a disulfide-crosslinked poly(hydroxypropyl methacrylamide) (pHPMA) core stably entrapping plasmid DNA (pDNA), surrounded by a shell of poly(ethylene glycol) (PEG). Retention of pDNA in the nanoparticles is exclusively based on physical entrapment given by the disulfide crosslinks, which provides an intracellularly triggered release profile, since disulfides are cleaved under the higher reducing environment present inside the cells. Through our study decationized polyplexes have demonstrated important advantages when compared to their cationic counterparts, such as much lower degree of nonspecific uptake and high degree cell specific uptake when decorated with targeting moieties as demonstrated by several cell uptake studies. Furthermore, in vitro studies showed lower cellular toxicity and in vivo nanotoxicity studies using a zebrafish model showed remarkable lower teratogenicity and mortality profile from decationized polyplexes. Stability evaluation in biological fluids a high stability for prolonged periods was found. Finally, in an in vivo biodistribution study, using tumor bearing mice, decationized polyplexes have shown greater retention in blood circulation and higher target tissue (tumor) accumulation. Given their important advantages, decationized polyplexes were also investigated and optimized for small interfering RNA (siRNA) delivery purposes, showing that decationized polyplexes can be used as a platform for different gene delivery modalities. In conclusion, decationzed polyplexes have demonstrated to be an important contribution for the development of safer polymeric gene delivery systems especially for targeted therapies. Importantly, the requirements for decationized polyplexes optimization have been identified, that will be the focus of future studies to further improve transfection efficiency and in vivo performance
RGD-based strategies for selective delivery of therapeutics and imaging agents to the tumour vasculature
No full textDuring the past decade, RGD-peptides have become a popular tool for the targeting of drugs and imaging agents to αvβ3- integrin expressing tumour vasculature. RGD-peptides have been introduced by recombinant means into therapeutic proteins and viruses. Chemical means have been applied to couple RGD-peptides and RGD-mimetics to liposomes, polymers, peptides, small molecule drugs and radiotracers. Some of these products show impressive results in preclinical animal models and a RGD targeted radiotracer has already successfully been tested in humans for the visualization of αvβ3-integrin, which demonstrates the feasibility of this approach. This review will summarize the structural requirements for RGD-peptides and RGD-mimetics as ligands for αvβ3. We will show how they have been introduced in the various types of constructs by chemical and recombinant techniques. The importance of multivalent RGD-constructs for high affinity binding and internalization will be highlighted. Furthermore the in vitro and in vivo efficacy of RGD-targeted therapeutics and diagnostics reported in recent years will be reviewed. © 2005 Elsevier Ltd. All rights reserved
Bioengineered riboflavin in nanotechnology
No full textRiboflavin (RF) is an essential water-soluble vitamin with unique biological and physicochemical properties such as transporterspecific cell internalization, implication in redox reactions, fluorescence and photosensitizing. Due to these features RF attracted researchers in various fields from targeted drug delivery and tissue engineering to optoelectronics and biosensors. In this review we will give a brief reminder of RF chemistry, its optical, photosensitizing properties, RF transporter systems and its role in pathologies. We will point a special attention on the recent findings concerning RF applications in nanotechnologies such as RF functionalized nanoparticles, polymers, biomolecules, carbon nanotubes, hydrogels and implants for tissue engineering
Aggregation of different amino acid conjugates of cholic acid in aqueous solution
No full textA series of cholic acid derivatives was synthesized with a variety of alpha-amino acids coupled via an amide bond. These conjugates form aggregates in aqueous solutions. Cryo-transmission electron microscopy (TEM) images show the presence of small micelles with a size of about 5 nm for all compounds. Critical micelle concentration (CMC) values vary slightly with the molecular structure. A small decrease in CMC was found upon increasing the hydrophobicity of the amino acid. The CMC of the tyrosine conjugate showed a small pH-effect, increasing at high pH. (C) 2003 Elsevier Science B.V. All rights reserved
Synthesis of thioether derivatives of quinazoline-4-one-2-thione and evaluation of their antiplatelet aggregation activity
No full textA series of 2-(arylmethylthio)-3-phenylquinazolin-4-one derivatives have been synthesized and their antiplatelet aggregation activities were assessed against ADP and arachidonic acid-induced platelet aggregation in human plasma. Among the tested thioethers, derivative 2, 3, 5 and 16 were the most potent compounds with satisfactory IC50 for inhibition of platelet aggregation induced by ADP. Analysis of global physicochemical parameters shows some correlations between activities and molecular volume and also surface area of the studied derivatives
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