The Study Of Cationic Lipid Based Gene Delivery System Lipid-mu Peptide-DNA

Over the last two decades gene therapy has moved from preclinicaltowards clinical studies for many diseases ranging from single gene disorderssuch as cystic fibrosis and Duchenne muscular dystrophy, to more complexdiseases such as cancer and cardiovascular disorders. Gene therapy for severecombined immunodeficiency (SCID) is the most significant success story todate, but progress in many other areas has been significant. Althoughindustrial and academic interest in some gene therapy such as cystic fibrosis(CF) has reduced over the past decade, likely because the development of CFgene therapy has turned out to be more difficult, and slower, than originallyanticipated, gene therapy still retains all future promises due to theirreplaceable advantages in treatment of diseases which are inoperable and donot respond to conventional therapies.Delivery system is an important component of gene therapy. It is also animportant factor that influences the therapeutic results. This is because genesthemselves cannot enter into cells and meanwhile they are extremelyvulnerable to physiological environment. As a result delivery systems thatfunction as protectors and carriers will influence therapies directly. Most ofthe gene therapies in clinical trials used viral vectors as the delivery systems,for viral vectors have relatively higher gene delivery efficiency. However,many of these clinical trials stopped during phase I or II due to the hightoxicity and immunogenicity. Although some investigational protocolsinvolved transplanting ex vivo virus-modified cells and transferring thosecells back to circumvent the attack from immune system, the patients stillneed to endure periodical surgeries to replace old cells with new ones, not to mention the risk of infection with the surgical procedures. Fortunatelynon-viral vectors have many potential advantages compared with viralsystems, including significantly lower toxicity, immunogenicity and potentialfor oncogenicity, which increase the safety of therapies greatly. Besides,non-viral vector systems are characteristic of size independent delivery ofnucleic acids, repeat dosages, simpler quality control, and substantially easierpharmaceutical and regulatory requirements. All of these advantages makenon-viral vectors the potential strategy to realize gene therapy finally in thefuture. And the non-viral vector that can be used in systemical delivery couldbecome the direction of therapy progress as for its easy and minimallyinvasive way of administration.The prerequisites for realizing therapeutic potential of non-viral genedelivery vectors in vivo include being stable during circulation to enhance theconcentration of cargo gene materials at lesion locations and escape from theendosomes to release the cargo genes into the cytoplasm to improvetransfection effeciency. The thesis engineered and optimized the vectorsystems according to these two preconditions. Firstly, Lipid-Mu peptide-DNA(LMD) was modified with PEG lipids which were more than commonly usedfor conventional lipid-DNA lipoplexes (LD). The high PEGylation improvedthe distribution of LMD at tumor sites. Then omeprazole, a proton pumpinhibitor drug, was used to improve the in vitro and in vivo transfectionefficacy through retarding the acidification of endosomes or lysosomes. Thecontents and main results of this thesis were summarized as below.Being different from traditional lipoplexes, LMD is prepared byprecondensing plasmid DNA with mu peptide and then combining theseanionic mu:DNA (MD) complexes with extruded cationic liposomes resultingin finally cationic ternary delivery system LMD. The whole preparationprocess could be described as self-assembly which is quick and reproducible.Through the optimization based on physiochemical characterization and invitro transfection experiments, we determined the best ratio among the threecomponents of LMD (DNA:mu:cationic lipid) as1:0.6:12(mg/mg/μmol). Under this ratio, MD particles we got were102±12nm in diameter and-33±1mV in zeta potential. LMD particles had particle size of140±15nm andzeta potential30±0.3mV. Then we observed the appearance of LMD withatomic force microscope (AFM) to find that LMD were spherical particlesand had uniform size. Together with cryo-electron microscopy images ofProfessor Miller’s group, LMD particles could be characterized as ternarysystem which contains a condensed core complex in the middle andsurrounded by lipid bilayers. As a result, this structure is more stable andformulated reproducible than the traditional lipoplex and can be modifiedwith more PEG lipids in their surfaces than conventionally used (10mol%).Layers of polyethylene glycol (PEG) shielding can reduce interactionswith serum proteins to minimize opsonization and prevent large particlesforming due to the hydrophilic chains of PEG polymers. So the PEGylatedvectors can stay more stable in vivo and circulate longer than the nonmodifiedones. So PEGylated vectors are also termed as long circulating vectors. Weused post insertion method to incorporate PEG lipid into LMD particles toprepare long circulating vectors for systemic administration. By modificationwith more PEG-lipids (15-25mol%) than commonly used, the zeta potentialof LMD decreased from30mV to nearly neutral, while the size increased alittle after modification as170±30nm.Because of the Enhanced Permeability and Retention effect of solidtumor, long circulating vectors could be passively targeted to the tumor sitesof subcutaneous xenograft tumor models. LMD particles with higherPEGylation (15-25mol%) accumulated at tumor sites more than those withless or more PEGylation due to the EPR effect. We confirmed this conclusionthrough live animal optical imaging, frozen section, immunohistochemistryand quantitative real-time PCR. Using near infrared fluorescent dye Cy5.5tolabel lipid components of LMD, we studied the tumoral distribution of LMDwith different PEGylation density (0,10,15,23,25,30mol%) at differenttime points by live animal optical imaging system. The results showed thatfluorescence signals were accumulated at tumor sites quickly after intravenous administration. With higher PEG modification densities, forexample15-25mol%, Cy5.5-PEG-LMDs have stronger fluorescenceintensity in tumors. Even after five days, prominent fluorescence signalscould still be observed at tumor sites and the fuorescent signals of tumor siteswere constantly stronger than those of other organs. Further more, weextracted the fluorescence values of interested organs and tumors from theimages and normalized them with areas and total fluorescent signals. Afteranalyzing the values using noncompartment model we got the consistentconclusion. Then we used double dyes to mark gene cargoes and lipidsrespectively and observed the tumoral distribution of these LMD throughfrozen section and confocal. The results also indicated that more LMD with23mol%PEGylation accumulated at tumor sites than those with only2or10mol%PEGylation. Besides,23mol%PEGylated LMD showed intensivefluorescent signals at tumor sites with different batches of preparation oranimals. Finally, we quantitatively analyzed the distribution of LMD withdifferent PEGylation at tumor sites by real-time PCR. The same conclusionwas made: the quantities of LMD with different PEGylation at tumor siteswere25%>23%>15%>30%>10%(mol%).After optimizing the tumor distribution of PEG-LMD, we then studiedthe gene delivey ability and transfection efficiency of LMD vectors. We usedplasmid DNA pGL3as the reporter gene. After many rounds of in vitro and invivo gene transfection experiments, we found that PEGylation decreased thetransfection efficiency dramatically. We speculated that it was because afterendocytosis, the trapped vectors could not escape from endosomes/lysosomesefficiently due to the existence of PEG lipids, instead they would beeventually hydrolysed in the gradually acidifying endosome/lysosomecompartments and consequently lost the ability of transfection completely. Toimprove the endosome/lysosome escape of gene vectors, we employed for thefirst time the proton pump inhibitors (PPI) drug omeprazole and gene deliveryin combination. Base on the fact that PPI can inhibit the H+transport intocellular organelles and retard the maturation of endosomes/lysosomes compartments, we hypothesized this characteristic could enhance the vectorsescaping out of endosomes/lysosomes. The results showed that fluorescentsignals could still be observed in23%PEGylated LMD treated cells withomeprazole in the medium even six days after removal of the vectors, whileno obvious fluorescent signals could be seen in cells without omeprazole. Andthe intensity of fluorescence correlated with the concentration of PPI drug.The in vitro pDNA transfection studies showed that0.1mg/ml omeprazoleincreased the H1299cell line transfection efficiency with23%PEG-LMD by9times. And by using PEG lipids with shorter fatty acyl chains,0.1mg/mlomeprazole increased the transfection results by34times. In vivo studiesshowed that150mg/kg omeprazole enhanced the transfection of H1299xenograft tumors with23%PEG-LMD by more than3times. The studiesindicated that omeprazole could improve the in vitro and in vivo pDNAtransfections of PEGylated LMD probably because it could inhibit thedropping of pH values in endosomes or lysosomes.