Development Of A Sirna-nanoparticle Homing Aerosol

Objective and backgroundRNA interference (RNAi) is a natural cellular process that regulates gene expression by a highly precise mechanism of sequence-directed gene silencing at the stage of translation by degrading specific messenger RNAs and blocking translation. It has shown more potential than conventional gene-targeting strategies. However, the poor cellular uptake of synthetic small interfering RNA (siRNA) is a major impediment for their clinical use due to its instability, inefficient cell entry, and poor pharmacokinetic profile. Various delivery vectors have thus been developed in order to circumvent these problems. From among the gene vectors that have been studied, non-viral vectors have attracted more and more attention in comparison to viral vectors, although viral vectors have been proven to yield higher transfection efficiency in most cell lines. This is attributed to the advantages of non-viral vectors such as ease of synthesis, low immune response against the vector and unrestricted gene materials size in addition to potential benefits in terms of safety.In recent years, chitosan-based carriers are one of the non-viral vectors that have gained increasing interest for its beneficial qualities such as low toxicity, low immunogenicity, as well as an excellent biodegradability and biocompatibility. For the treatment of asthma, we previously developed a 12-alkylated chitosan nanoparticle vector that was able to deliver ECE-siRNA into airway epithelial tissues of the OVA-challenged mice, leading to down regulation of the synthesis of ET-1 to some extent. Today's question remains the delivery of siRNAs. The 12-alkylated chitosan nanoparticle vector cannot be used in human therapy and its delivery efficiency is still the most important obstacle for the siRNA-based treatment.It has been found that the arginine-induced perturbation is preferentially ascribed to the presence of the guanidinium group in Arg, indicating that modification of chitosan with guanidinium groups would optimize its delivery efficiency for siRNA. The development of methods for specific delivery of genes into target tissues is also an critical issue for the further progress of gene therapy. Introduction of targeting ligands to nonviral vector. resulted in increased gene expression and allowed to direct transfection complexes more specifically to selected cell types in order to reduce undesired side-effects in non-target cells. The beta2-adrenergic(β2-AR) receptor is found predominantly in bronchial smooth muscle and lung tissue.β2-AR agonists may serve as a suitable targeting ligand to improve receptor-mediated gene delivery. In addition, topical delivery of the drug directly to the site of action offers the advantages of enhanced drug delivery to anatomical target site with maximum therapeutic efficacy but the minimal adverse side effect.. This has led to the widespread use of inhalation therapy in lung diseases. In present study, we developed a targeting gene carrier by covalent linkage of salbutamol to the guanidinylated chitosan(Gua-chitosan).. The effect of ultrasonic aerosol on the vector/nucleic acid complexes was also studied for topical delivery of siRNA in treatment of respiratory diseases.MethodsPart one1. Characteristics of Gua-chitosanParticle size and its distribution of the Gua-chitosan/pDNA complexes were measured by transmission electron microscopy(TEM). The water-solubility of Gua-chitosan was detected under various PH conditions. Gel retardation was evaluated for the pDNA-packaged by Gua-chitosan. The cytotoxicity of Gua-chitosan nanoparticles was tested in HEK 293 cells using CCK-8 assay.2. Transfection efficiency mediated by Gua-chitosanHEK29 cells transfected with Gua-chitosan, encapsulating the pEGFP as a reporter were observed under a fluorescence microscope. Transfection efficiency of the complexes and the mean fluorescence intensity of individual cell were further evaluated by flow cytometry. The PET plasmid was labeled with fluorescein Yoyo1 for in vitro and in vivo assessment of the transport of siRNAs across the cell membrane by Gua-chitosan nanoparticle.Part two Specific modification of Gua-chitosanSal-Gua-chitosan was synthesized by covalent linkage of salbutamol to the Gua-chitosan using epichlorohydrin. 1H NMR spectroscopy was performed on investigation of the rate of substitution of salbutamol. The transfection efficiencies of Gua-chitosan and Sal-Gua-chitosan were detected. in both HEK 293 cells and bronchial epithelial cells (16HBE).Part three Effects of Ultrasonication on the transfection efficiency mediated by the Gua-chitosanThe protection of pDNA by chitosan against ultrasonication was characterized by agarose gel retardation experiment. Transfection efficiency of the Gua-chitosan/pDNA complexes was evaluated by flow cytometry. prior to their treatment of ultrasonic aerosol.. The effects of ultrasonication on the particle diameter and size distribution of Gua-chitosan nanoparticle were determined using TEM.ResultsPart one1. In TEM images, Gua-chitosan/pDNA complexes assumed spherical particles with diameter ranging from 20nm to 70nm. Chitosan modified by guanidinium group is able to be dissolved in neutral water . In contrast, the natural chitosan is water soluble only at PH below 4.0. We show that the Gua-chitosan can effectively condense DNA in spite of a weaker DNA-binding strength relative to chitosan. Cytotoxicity Assay demonstrate that Gua-chitosan has no any negative impact on the cell proliferation.2. Expression efficiency for GFP mediated by the Gua-chitosan is greatly enhanced compared with that of chitosan-mediated transfection. The highest transfection efficiency of the both is observed 72 hours after the application of vector/pDNA complexes. The mean fluorescence intensity of GFP is higher in the cells transfected with pEGFP-Gua-chitosan nanoparticle than that in those transfected with pEGFP-chitosan. nanoparticle. The fluorescence microscope shows that Gua-chitosan vector has the capability to deliver much more Yoyo1-labled pDNA into the cells, compared to the natural chitosan. This was not only observed in vitro but also in mice submitted to intracheal instillation of the complex.Part twoOn average, about 100 glucosamine units in Gua-chitosan are successfully conjugated with 5 salbutamol molecules. The salbutamol modification of Gua-chitosan increases the expression efficiency of pEGFP in HEK293 cells that expressβ2-AR, but not in 16HBE cells withoutβ2-AR expression.Part threeThe pDNA encapsulated in Gua-chitosan is perfectly protected from a shearing force generated by ultrasonic aerosol. It is found that the Gua-chitosan nanoparticle treated by ultrasonic aerosol results in an elevated transfection efficiency. The particle sizes of Gua-chitosan and Gua-chitosan/pDNA were both increased, accompanied by a narrowing of size distribution after ultrasonication.Conclusion1. Guanidinylation modification greatly improve water-solubility of chitosan vector under the physiological PH conditiion, which is important for its clinical use. Guanidinylation modification of the chitosan accelerates the transport of -pDNA across the cell memberance by the nanoparticle vector, leading to an increase of transfection efficiency.2. Theβ2-AR agonist, salbutamol is coupled to the Gua-chitosan. This made it a homing device that increases nanoparticle's internalization by the ligand-receptor interactions.3. The Gua-chitosna protects the DNA from being sheared by ultrasonication. On the other hand, ultrasonic aerosol increases the transfection efficiency of the Gua-chitosan by change of diameter and size distribution. We suggest that ultrasonic aerosol is an available approach for the delivery of gene vector complexes.