Strategies towards selective functionalization of polycationic amphiphilic cyclodextrins (paCDs): engineering novel non-viral gene carriers by tailoring self-assembling and DNA-condensing capabilities
|Director||Benito Hernández, Juan Manuel|
|Department||Universidad de Sevilla. Departamento de Química orgánica|
|Document type||PhD Thesis|
|Abstract||The use of preorganized macrocyclic scaffolds to achieve a precise alignment of functional elements has proven to be extremely useful over the years in the design of artificial receptors and ligands capable of mimicking ...
The use of preorganized macrocyclic scaffolds to achieve a precise alignment of functional elements has proven to be extremely useful over the years in the design of artificial receptors and ligands capable of mimicking the supramolecular events occurring in living organisms. The cyclic maltooligosaccharide (cyclodextrin, CD) nucleus is considered a privileged platform for these channels, as it combines biocompatibility, availability and a tubular symmetric framework with well-differentiated faces. CD-based architectures can additionally take advantage of their distinctive inclusion capabilities. Recently, CDs have found their way into the field of gene delivery, giving rise to a plethora of synthetic CD-containing carriers, such as CD-embedding or pendant polymers, polyrotaxanes, and CD-centered and coated dendrimers and dendripolymers. Although many of these systems have proven to be efficient vectors, their essentially disperse nature handicaps both investigational studies and clinical applications. The development of monodisperse CD derivatives that are capable to self-organize in the presence of nucleic acids and deliver them into cells constitutes an interesting alternative that critically depends on the availability of efficient methods to systematically manipulate the CD topology in order to attain a precise control of the presentation and orientation of functional elements and, thereby, of their supramolecular capabilities. In this Ph.D. Thesis, a molecular-diversity-oriented approach has been exploited for the preparation of well-defined polycationic amphiphilic cyclodextrins (paCDs) as gene delivery systems. The synthetic strategy takes advantage of the differential reactivity of primary versus secondary hydroxyl groups on the CD torus to regioselectively decorate each rim with cationic and lipophilic tails, respectively. The charge density, the display and the nature of the hydrophobic domain can be finely tuned by using "click chemistry" methodologies, preserving the molecular homogeneity (and architectural symmetry), thereby providing an easy-to-use tool for tight control over the hydrophilic/hydrophobic balance. The monodisperse nature of paCDs and the modularity of the synthetic scheme are particularly well suited to correlate molecular structure with self-assembling and gene delivery capabilities in the way that structure-activity relationship (SAR) studies are carried out in typical medicinal chemistry programs. Their self-assembling capabilities in aqueous environment have been investigated and a progressive decrease of the critical aggregation concentration (CAC) with increasing amphiphilicity of the CD conjugates was observed. Acid-base titration experiments revealed that in comparison to their non-amphiphilic analogs amphiphilic CD derivatives showed improved buffering capacities in the pH range from 7 to 5. This indicates that paCD-based nanoparticles may exhibit proton sponge capabilities upon acidification, which might help to promote endosomal escape after cell internalization. Gel electrophoresis and fluorescence quenching assays evidenced that paCDs self-assemble in the presence of calf thymus DNA (ctDNA) to provide stable nanoparticles (CDplexes) that fully protect nucleic acids from the environment. As characterized by DLS measurements, paCDs formed narrow populations of compact and ordered nanoparticles with ctDNA, exhibiting small hydrodynamic diameters (60-90 nm) and positive zeta (¿) potentials (40-50 mV). Fluorescence tracking of CDplex dissociation promoted by heparin showed that CDplex dissociation kinetics is drastically influenced by the hydrophilic/hydrophobic balance. Furthermore, the transfection efficiency of the CDplexes was investigated in vitro on COS-7 cells in serum-containing medium and was found to be intimately dependent on architectural features. In the following chapter of this Ph.D. Thesis, the focus has been moved to the assessment of the gene delivery potential of paCDs decorated with cyclic oligoamines as alternative polar headgroups. In particular, the goal consisted in exploiting cationic preorganized scaffolds such as cyclen and cyclam, which exhibit unique DNA-binding capabilities. With the purpose of rationalizing the role of the cationic element in gene delivery, their structure-activity relationships were investigated and compared to acyclic oligoamine-grafted paCDs. The self-assembling capabilities of cyclen/cyclam-bearing paCDs resembled those of paCDs with acyclic polycationic elements and lipophilic tails of the same length. Cyclen/cyclam-grafted paCDs exhibited improved pH buffering abilities in the range from 7 to 5. Gel electrophoresis shift assays evidenced that paCDs furnished with cyclic polyamines self-assemble in the presence of calf thymus DNA (ctDNA) to provide stable nanoparticles (CDplexes) that fully protect ctDNA from the environment. Hydrodynamic diameter and zeta (¿) potential measurements indicated that modifications of the molecular structure did influence neither particle size (80-100 nm) nor surface charge (40-60 mV) significantly. Moreover, the transfection efficiency of the CDplexes derived from paCDs decorated with cyclic oligoamines was investigated in vitro on COS-7 and HeLa cells, both in the absence and in presence of serum and was found to be similar to that of CDplexes formulated with paCDs bearing acyclic cationic headgroups. The above chapters demonstrate that fine-tuning of CD topology holds a great potential to manipulate supramolecular capabilities, including nucleic acid delivery. Unfortunately, the actual toolbox for selective CD modification is still limited. The final chapter of this Ph.D. Thesis has been devoted to the development of a conceptually novel approach to the selective functionalization of cyclodextrins. This novel strategy exploits a solid matrix to display the complementary reagent functionalities sufficiently far from each other to prevent a single CD species from reacting through more than one site. Using a "catch-and-release" process based on the Staudinger reaction, complex CD functionalization patterns could be produced in one pot and without any purification step.
|Cite||Pfluger, I. (2013). Strategies towards selective functionalization of polycationic amphiphilic cyclodextrins (paCDs): engineering novel non-viral gene carriers by tailoring self-assembling and DNA-condensing capabilities. (Tesis Doctoral Inédita). Universidad de Sevilla, Sevilla.|