Design Of Polyethylenimine Based DNA Vaccine Delivery System And Evaluation On Its Immunological Response Following Airway Vaccination

In the 21 century, human still face with threaten of infection disease. Airway vaccine delivery embraces appealing characteristics for new immunization strategies against invading pathogens. Airway vaccination can result in prevention of pathogen entry into the systemic circulation by local immune responses or in induction of systemic immunity and prevention of infection spread. DNA vaccine could induce both humeral and cellular immune response. DNA vaccines hold many distinct advantages in comparison to recombinant proteins or inactivated pathogens: (1) The production is easy and similar between different plasmids, making up-scaling more simple and economical. (2) The DNA is stable at higher temperature, a property increasing shelf-life and facilitating transport and distribution of such vaccines. So DNA airway vaccinations have been widely used for the treatment or prevention of infection disease. However these studies have on its first stage, for the lack of high efficiency, low toxicity vector. Viral vector are widely used since the molecular biology of recombinant viruses is well understand and they give high level of DNA uptake and expression. However the major disadvantages of viral vectors for gene transfer include a lack of cell type specify, a restriction in the size of DNA that can be used, safety considerations, host immune response and high costs. The light of these concerns, non-viral gene delivery has emerged as a promising alternative. However, the delivery system is still limited by relatively low transfection efficiency and short duration of gene expression compared to viral transfection systems. In this study, three types of polyethylenimine (PEI) based novel non-viral gene delivery systems were designed to overcome two obstacles of gene expression: extracellular and intracellular obstacle. Also, the immunity effect was evaluated by using Mycobacterium tuberculosis Ag85b as the destination gene by nasal administration.In the first part, Mycobacterium tuberculosis Ag85b gene was cloned into the pcDNA3.0 vector to construct recombinant plasmid pcDNA85b. In order to enhance its immunogenicity and expression, tissue plasminogen activator (tPA) signal sequence was inserted into the plasmid. Construction was confirmed by PCR, enzymedigestion and DNA sequencing.The second part evaluated PEGylation of PEI as non-viral gene delivery system. Poor solubility of PEI/DNA complex is one drawback for its in vivo use. PEGylation can improve the solubility of the complex, minimize their aggregation and reduce their interaction with proteins in the physiological fluid. Varied numbers of PEG was grafted to branched PEI (25 kDa) from the average number of PEG per one PEI macromolecule at 1, 2, 5, 10, 20. Agarose gel electrophoresis and picogreen assay were used to evaluate the PEG effect on PEI/DNA binding. To investigate the cytotoxicity of the complexes, MTT assay was performed on A549 cells. The results suggested that the PEGylated PEI showed low binding to DNA. In vitro and in vivo transfection experiment showed that the transfection activity was reduced by the increase of degrees of PEG grafting. A PEI conjugate with one or two segment of PEG was able to mediate high transfection effect comparable to PEI/DNA by carefully adjust the N/P ratio. Transfection activity of PEI-PEG-1/DNA (N/P=9) and PEI-PEG-2/DNA (N/P=12) on A549 cell line was comparable to PEI/DNA (N/P=6). The in vivo transfection activity of PEI-PEG-1/DNA (N/P=9) and PEI-PEG-2/DNA (N/P=12) complex were 2.35 fold and 2.29 fold respectively as the PEI/DNA (N/P=9) complex.The third part described the PEI derivatives with terminally galactose-grafted PEG for specific gene targeting to the membrane lectin at the surface of the airway epithelial. A synthesis method of conjugating PEI derivatives with terminal galactose-grafted PEG was developed by using a bifunction PEG derivative containing both maleimide (MAL) and N-hydroxysuccinimidyl (NHS) ester groups(MAL-PEG-NHS). The NHS groups of MAL-PEG-NHS were selectively conjugated to amine groups of p-aminophenyl-β-D-galactopyranoside. Then the MAL groups were completely conjugated with the primary amine groups of PEI. Gel retardation and loading efficiency assay showed that when PEI was modified with 0.5%, 1%, 5%(mol/mol) galactose, the binding effect to DNA was reduced. However the binding effect could be improved by increase the N/P ratio and the cytotoxicity of the complexes were also greatly reduced. In 16HBE140 cell lines, luciferase activity with 1% Gala-PEG-PEI/DNA at an N/P of 36 was 3.1 fold that of PEI/DNA at an N/P ratio of 6. In mouse fibroblasts (NIH 3T3) that had no membrane lectin, the transfection efficiency with 1% Gala-PEG-PEI/DNA dramatically decrease to 0.06 fold that with PEI/DNA complex. These results suggested that the transfectionefficiency was improved by receptor-mediate endocytosis. In vivo data showed that 1% Gala-PEG-PEI/DNA complexes at an N/P ratio of 24, 36, 48 was 3.0, 11.6, 6.7 fold that of PEI/DNA complex, respectively.Poor escape of non-viral gene vectors from the endosomal compartment after cellular uptake and inefficient transfection into the nucleus substantially limit transfection efficiency. The fourth part, the arginin-rich motif of the HIV Tat protein was used to overcome these obstacles. First, the Tat protein was conjugated to mPEG-Maleimide to improve its solubility when binding to DNA, then the Tat/DNA complex was modified by PEGylated PEL The loading efficiency determination illustrated that Tat protein would not affect the DNA binding to PEL In vitro and vivo transfection experiments demonstrated that the Tat protein could improve transfection activity. The transfection activity on A549 cell line of PEI-PEG-1/PEG-Tat-1/DNA at an N/P ratio of 9 was 2.81 fold that of PEI-PEG-1/DNA, in vivo transfection activity was 2.01 fold that of latter. However, the method of modification should be optimized, so as to achieve high tansfection efficiency.The fifth part evaluated the immune response of the modified PEI/DNA complex (the one was non-modified PEI, the other three were the vectors that had achieved high transfection efficiency in mouse) following intranasal administration. The humeral (including mucosa immunity) and cellular immune response were also assayed. The results revealed that the modified three types of gene vector could procure comparable systemic IgG, local IgA, specific lymphocyte proliferation and IFN- γ. The results also proved that these three types of vectors could efficiently bind to DNA and express destination protein. Gala-PEG-PEI/pcDNA85btPA and PEI-PEG- 1/PEG-Tat/pcDNA85btPA complex achieved significance high immune response compared with PEI/pcDNA85btPA and PEI-PEG-1/pcDNA85btPA complex. High mucosal IgA levels were obtained in PEI-PEG-1/PEG-Tat/pcDNA85btPA group. It has great potentialities to induce mucosal immunity and provide immunoprotection against airway viral infection.The sixth part of this paper was to investigate the physiological compatibility of modified PEI/DNA complex. The pathological changes of lung tissue of modified PEI/DNA group were significantly lower than those of PEI/DNA group which indicates modified PEI/DNA had good physiological compatibility.Rizatriptan is a selective 5-HT_1B/1D receptor agonist that directly and selectively constricts intracranial, extracerebial blood vessels and inhibits the release of sensory neuropeptides from perivascular nerves to prevent neurogenic vasodilation and extravasation in the dual matter. Conventional oral tablets of rizatriptan (Maxalt^TM) are effective and convenient for many patients with migraine. However, oral administration of a conventional tablet may not be ideal for all patients. Swallowing a conventional tablet and water may exacerbate migraine-associated nausea and vomiting. Another limitation of oral treatment of oral treatment is that migraine-associated gastrointestinal disburdens (vomiting, inhibition of gastric motility, and delayed gastric emptying) may affect the absorption of orally administered drugs. Development of a formulation that is rapidly absorbed and has fewer side effects may help overcome these obstacles.Intranasal drug delivery system (INDDS) is one of the most heated research topics in the recent years with the advantages of rapid absorption, low first-pass effect and good compliance. And this route is also found to be useful to directly delivery drug to the brain. All these merits of INDDS inspire us to introduce it into the clinical application of central-acted RIZA to treat migraine. Therefore, the aim of this paper is mainly to develop a nasal spray dosage form of RIZA, to evaluate its pharmacokinetic behavior as well as its brain-targeting characteristics, so as to provide a more effective formulation for clinical RIZA application.Firstly, experiments were carried out to find out the optimal formula for preparing stable RIZA nasal spray. Besides, a specific and convenient HPLC method was established to determine the content of RIZA and related substance in nasal preparation. The pH, drug content of each spray, and related substance etc. were also tested for control the quality of nasal spray. Preliminary stability experiments were done in order to determine the expiration date.Secondly, the safety of RIZA nasal spray was studied. The optical microscope observation of in-situ toad palate model and scanning electron micrograph of rat nasalmucosa during multiple RIZA nasal administration for seven days showed that RIZA nasal spray had no toxicity to mucosa-ciliary movement and morphology of nasal mucosa.Thirdly, the relative bioavailability of RIZA nasal spray following intranasal administration compared with oral administration was evaluated. HPLC method with fluorescence detection was developed to determine RIZA concentrations in rabbit plasma. The method was proved to be accurate, precise, sensitive and selective. RIZA plasma concentrations following intranasal administration of nasal spray and oral administration of tablet as reference were determined in 16 healthy volunteers. The nasal preparation was bioequivalent to tablet and the mean relative bioavailability was 96.4% ±15.7%. The t_max obtained by intranasal administration was significantly quicker than the corresponding values of oral administration which indicated that nasal absorption of RIZA potentially might result in faster headache relief compared with oral formulation.Finally, an HPLC assay for the determination of RIZA concentration in rat plasma and CSF were established and validated. The pharmacokinetic profiles of RIZA in plasma and CSF in rats were evaluated following oral, intranasal, intravenous administration. The DTI index (drug targeting index) was used to evaluate the brain drug delivery of RIZA following intranasal administration. The results suggested that the DTI was 1.45, which means a fraction of the RIZA dose could be transported directly from the nasal cavity into CNS.