Preliminary Study On The Ability Of Targert Liposomes To Pass Through The Blood-brain Barriers

Gene therapy is attempted to apply in the treatment of CNS diseases along with the development of molecular biology and biology engineering technology. Recently, many research of CNS gene therapy have been undergone in animal and clinical trial. However, scientists were troubled in the question that how to transport these gene drugs through the blood brain barrier (BBB). It includes three aspects, (i) how to choose the vector that carries gene drug; (ii) how does the vector transport gene drug through the BBB; (iii) which is the most efficient and secured route of administration. Nowadays, the vectors that carry gene drugs include viral and nonviral vectors. Viral vectors with the virtue of high transfection efficiency have been widely used in many animal trials. But there are still numerous disadvantages of viral vectors that have been used for gene therapy. Following viral delivery in vivo immune responses are generated to expressed viral proteins that subsequently kill the target cells required to produce the therapeutic gene product. In addition, an innate humoral immune response can be produced to certain viral vectors due to previous exposure to the naturally occurring virus. Some viral vectors integrate randomly into the host chromosome and could cause activation of proto-oncogenes resulting in tumor formation. Clearance of viral vectors delivered systemically by complement activation can also occur or these vectors can be inactivated upon re-administration by the humoral immune response. And the most commonly used nonviral transport system was liposome.The existence of BBB precludes both viral and nonviral vector to transport gene drugs to target cells in brain. The production of target limopsomes solves this problem. Circulating insulin, transportrin, the insulin-like growth factors and leptin, all have peptide-specific receptor transporters within the brain capillary endothelium that allow the circulating peptides or plasma proteins to gain access to brain. An endogenous peptide (e.g. transferrin) or a receptor-specific peptidomimetic monoclonal antibody (mAb) that crosses the endotheliumvia a receptor-mediated transport (RMT) systemcould be used to ferry across the BBB any attached drug.This research adds transferrin to the surface of pegylated liposomes, which enables the complex to transport through BBB. The plasmid pEGFP-C1 was capsulated into this liposome. Then, we injected the liposome plasmid complex into rats through five different routes, including inracranial, intraventrical, intrathecal, intra-arterial and intravenous ways. Then we executed the rats 48 hours after administration and inspect green fluorescence under fluorescence microscope. The purpose of this research is to test that the Tf target limposomes can pass through the BBB, to compare the level of gene expression among different routes of administration and to probe a safe and efficient route of administration for gene therapy.The results demonstrated that the green fluorescent protein was dramatically expressed in the brain tissue near both lateral ventricles and the third ventricle in the ventricle injection group. The level of green fluorescence decreased as the distance increased form ependyma. In the intracranial injection group, the green fluorescence was inspected in the brain tissue near the pin hole. The level of green fluorescence decreased as the distance increased form pin hole. In the intrathecal injection group, the scale of GFP expression was similar as that in the intraventricle injection group, but the intensity of fluorescence was not as strong as that in the intraventricle injection group. In intra-arterial and intravenous injection group, the GFP was widely expressed in the brain tissue; however, the intensity was greatly decreased. The result clearly showed us that the level of gene expression was highest in intracranial and intraventricle injection group, a bit lower in the intrathecal injection group and the lowest in the intra-arterial and intravenous injection group. So we seems to draw a conclusion that intracranial and intraventricle were the most efficient way for gene expression. However, these two ways can cause great complications and obviously not accepted by patients. It may cause severe brain trauma and cerebral hemorrhage. In addition, the drug cannot rapidly diffuse after intracranial administration which is likely to cause intracranial hypertension and brain hernia. There may be the reasons that two rats died 24 hours after administration and two died 48 hours after administration. Apart from this, we found that in the intraventricle injection group, the levels of green fluorescence obviously decreased as the distance from ependyma increased. The entire cerebrospinal ?uid (CSF) volume of the human brain, about 140 mL, is completely turned over every 4–5 hours. This bulk ?ow of ?uid out of the CSF ?ow tracts is rapid compared with the slow rate of solute diffusion into the brain. Consequently, drug concentration in brain parenchyma decreases exponentially within mm from the ependymal surface. Diffusion decreases exponentially with the diffusion distance. Consequently, drug delivered to the brain with the intracranial implantation approach does not move significantly into brain from the depot site. Many other reasons may include low dosed and short administration schedule. Even if the low level of gene expression in intra-arterial and intravenous injection group, the fact of gene expression proved that the Tf target liposome has the ability to pass through the BBB. If we increase the doses or prolong the detection time, the level of gene expression may increase.In this research, we can make the following conclusions.(i) The pegylated Tf target liposome can pass the BBB by the activation of receptor mediated transport system, which makes report gene was transported from blood circulation to brain.(ii) The intraventricle and intra-arterial routes of administration can transport more report gene to brain compare with other ways, but they cannot be widely used in clinical due to their low security.(iii) The level of gene expression in intrathecal injection group was a bit lower than that in intraventricle and intracranial injection group but much higher than intra-arterial and intravenous injection group. It is considered to be a relatively safe and effective route of administration.(iv) The level of gene expression in intravascular injection group was relatively low, however, it is safer, easily to re-administrate and more likely to be accepted by patients, which makes it be an optional route of administration.