Tf-targeted Liposomes Cross The Blood-brain Barrier To Transduction Of Vegf Gene Therapy In Rats With Ischemic Stroke

Stroke ranks second after ischemic heart disease as a cause of death worldwide. The incidence of stroke varies among countries and increases exponentially with age. Initially after arterial occlusion, a central core of very low perfusion is surrounded by an area of dysfunction caused by metabolic and ionic disturbances but in which structural integrity is preserved (the ischemic penumbra). Depending on the rate of residual blood flow and the duration of ischemia, the penumbra will eventually be incorporated into the infarct if reperfusion is not achieved. Although thrombolysis and controlled hypothermia are an effective treatment in the acute stage, an effective treatment has not yet been established to enhance both neuroprotection and angiogenesis for the ischemic brain. Vascular endothelial growth factor (VEGF), an angiogenic growth factor, is considered a potentially useful therapeutic agent to attenuate ischemic brain injury. Previous studies have confirmed that VEGF may confer neuroprotection, promote neurogenesis and cerebral angiogenesis, accordingly improve histological and functional outcome from stroke through multiple mechanisms.The Blood-Brain Barrier (BBB) protects the central nervous system (CNS) from potentially harmful exogenous and endogenous molecules. Due to the poor permeability of the BBB, gene therapy drugs are difficult to cross it. In addition, most current gene vectors do not cross the BBB after an intra-venous(i.v.) administration and must be given via craniotomy or intracerebral injection, which are considered to be highly invasive and unable to deliver exogenous genes to global area of brain. Given the evident side effects of viral vectors, the goal of brain gene targeting technology pays more attention to the efficient, noninvasive and non-viral gene therapy of the brain.SubjectPartⅠ Preparing a kind of effective non-viral transduction vector, which can deliver exogenous gene into the brain by trans-blood brain barrier.PartⅡComparison the expression in brains and peripheral tissues of different dosage PLs, Tf-pCNV-PLs and Tf-pGFAP-PLs, which are injected from the tail vein.PartⅢComparison the therapeutic effects of Tf-VEGF-PLs and VEGF-PLs to the cerebral ischemic rats.Material and methodPartⅠBettering the synthetic process of liposomes by adjusting different proportion of materials, temperature of rotary evaporation and ultrasonic sound. We detected the encapsulation rate and Tf coupling rate by kit. Tf-pCMV-PLs were injected into rats via tail vein, and detected the expression of LacZ gene by histochmical staining to identify whether Tf-pCMV-PLs had delivered exogene into brain by crossing the BBB.PartⅡPLs low-dosage group (80μg pCMV-LacZ encapsuled), PLs high-dosage group (300μg pCMV-LacZ encapsuled), Tf-pCMV-PLs group (80μg pCMV-LacZ encapsuled), Tf-pGFAP-PLs group (80μg pGFAP-LacZ encapsuled) and blank liposomes were injected into rats via tail vein. Twenty-four hours after injection, RT-PCR was proceeded to detect the transcription of LacZ gene in the brain and peripheral tissues. Forty-eight hours after injection, the activity ofβ-galactosidase was detected in the brain and peripheral tissues by theβ-galactosidase detection kit. The distribution of the expression of LacZ gene was observed by histochemical staining and the toxic effect of liposomes was evaluated by HE staining. PartⅢTwo days after pMCAO,152 rats were distributed into 3 groups randomly, and Tf-VEGF-PLs, VEGF-PLs and saline were injected. Tf-LacZ-PLs was also injected into pMCAO rats as a marker to verify VEGF had been delivered into brain. Twenty-four hours after injection, RT-PCR was proceeded to detect the transcription of VEGF gene in the brain. Forty-eight hours after injection, Western blotting was proceeded to detect the expression of VEGF protein. On the day 1,7,14 and 21, neurological function of pMCAO rats was evaluated according to the scale of mNSS. On the day 21 after cerebral ischemia, brain ischemia area was calculated by the staining of TTC. MVD was counted by injection of 5% FITC-dextran under the fluorescence microscope to compare the number of angiogenesis. On the day 21 after cerebral ischemia, LCD was used to detect the blood flow of brain among groups.ResultPartⅠThe best proportion of synthesizing liposomes was following: phosphatides/cholesterol was 1:1, lipid/drug was 100:1, oil phrase was dichlormethane, oil/water was 4:1, temperature of rotary evaporation was 30℃, ultrasonic temperature was 10℃and duration was 5 min,10% trehalose was added to improve the stability of liposomes. The encapsulation of liposomes was 87.24%, Tf coupling rate was 69%. After the injection of Tf-pCMV-PLs into rats, an extensive expression of LacZ could be observed in the brain and peripheral tissues.PartⅡIn rat brains,24 h after injection, LacZ mRNA could be detected among PLs high-dosage group, Tf-pCMV-PLs group and Tf-pGFAP-PLs group. However, in peripheral tissues, including heart, liver, spleen, kidney and lung, only Tf-pGFAP-PLs could avoid the expression of LacZ gene. The result of histochemical staining demonstrated, Tf-pGFAP-PLs could achieve an extensive expression of LacZ gene in the brain, and the result of HE staining verified Tf-pGFAP-PLs did no harm to the brain and peripheral tissues.PartⅢThe results of RT-PCR and Western blotting demonstrated, VEGF mRNA and VEGF protein in Tf-VEGF-PLs group were significantly higher than that in VEGF-PLs group and saline control group. After 21 days of cerebral ischemia, mNSS in Tf-VEGF-PLs group was significantly increased and the ischemic area was significantly reduced. In Tf-VEGF-PLs group, the number of MVD was significantly higher than that in control group, and increased micro vessels also improve the local brain blood flow.ConclusionPartⅠTf-pCMV-PLs could be successfully prepared by the work of bettering producing technology and could trans-over the BBB to deliver exogene into the brain. This achievement made a strong foundation to realize the gene therapy of intracranial disease. Meanwhile the promoter of exogene was needed to be further refined in order to reduce the non-specific expression in peripheral tissues.PartⅡTf-pGFAP-PLs could realize the extensive brain-specific expression of exogene in the brain by trans-over the BBB, avoiding the non-specific expression in peripheral tissues. Tf-pGFAP-PLs was an excellent type of carrier for exogene entering into the brain.PartⅢTf-VEGF-PLs could increase the expression of VEGF in cerebral ischemic rat, significantly improve the neurological function, accelerate angiogenesis, increase local brain blood flow and reduce the area of cerebral ischemia. Tf-VEGF-PLs was a effective way to cure brain ischemia.