Studies On Brain-targeting Dendrimer-based Nano-scaled Gene Delivery System

Gene therapy offers a promising cure for various brain disorders.However, therapeutic genes themselves,viral vectors and unmodified non-viral vectors can not reach the brain following an intravenous administration due to the presence of blood-brain barrier(BBB).Most genes or current vectors must be given via parenchymal injections which are considered to be highly invasive and unable to deliver genes products to global areas of the brain.Non-viral vectors modified with specific proteins might enable widespread expression of exogenous genes throughout the brain following a non-invasive transvascular pathway.The main problem is the low transfection and expression efficiency.Therefore,developing a brain-targeting and efficient non-viral gene vector to enable the global gene expression in the brain is currently one of the focuses in the field of brain gene therapy.In this study,a novel efficient brain-targeting non-viral nano-scaled gene delivery system was finally constructed with means of pharmaceutics, macromolecular chemistry and biology.Polyamidoamine(PAMAM) was applied as the main macromolecular gene vector.And lactoferrin(Lf),for the first time,was investigated as a brain-targeting ligand in the design of PAMAM-based nanoparticles (NP) to the brain,using polyethyleneglycol(PEG) as a spacer.There are two important attributes of Lf-modified PAMAM-based nanoparticles (PAMAM-PEG-Lf/DNA NP).They are(1) modified with Lf to target the corresponding receptors in the brain to enhance the brain-targeting ability,(2) employing novel cationic macromolecular material,PAMAM,as the main gene vector,with high gene encapsulation abilityThe first part compared the in vitro characteristics including gene incorporation efficiency and transfection efficiency in brain capillary endothelial cells(BCECs) of several cationic vectors.The results of agarose gel electrophoresis and PicoGreen assay showed that all the prepared nanoparticles could effectively encapsulate DNA and protect themselves from the displacement of anionic substances and the digestion of DNase I.The results of fluorescent microscopy and luciferase assay indicated that PAMAM G5(the 5^th generation of PAMAM) had a reasonable gene expression. Given both the literature and our results,PAMAM G5 could balance the gene transfection efficiency and cytotoxicity,and thereafter was chosen as the main macromolecular vector in the following studies.Next,transferrin(Tf) was selected as a brain-targeting ligand,conjugated to PAMAM via bifunctional PEG,yielding PAMAM-PEG-Tf.The results of UV-vis, ¹H-NMR and SDS-PAGE demonstrated the successful synthesis of PAMAM-PEG-Tf. This vector showed a concentration-dependent manner in low concentration range and exhibited a trend of saturation in higher concentrations through fluorescent microscopy and flow cytometry.Furthermore,PAMAM-PEG-Tf showed 2.25-fold brain uptake compared to PAMAM in vivo.The PAMAM-PEG-Tf/DNA NP has a particle size around 200 nm and zeta potential around 13 mV.It is spherical and has regular shape under the examination of transmission electron microscope.The modification of Tf significantly enhanced the gene expression of PAMAM-PEG-Tf/DNA NP compared to unmodified nanoparticles in both BCECs and mouse brains.However,there exist some shortcomings of Tf as a brain-targeting ligand.For example,the Tf receptor-mediated bidirectional transcytosis of Tf through the BBB might decrease the brain accumulation of Tf-modified nanoparticles,and most of Tf receptors might be occupied by endogenous Tf under physiological conditions so to reduce the brain-targeting ability of Tf-modified nanoparticles.Consequently,it is necessary to find a novel efficient brain-targeting ligand for construction of brain gene delivery systems,then to enhance the brain-targeting ability and brain gene expression efficiency.Several lines of evidence demonstrated that Lf had higher brain transport than Tf and a mouse monoclonal antibody against rat Tf receptor(OX26),which suggested that Lf might be a potential brain-targeting ligand.Up to now,there has been little information about the characteristics of Lf receptors in the brain tissue.The third part was to characterize the Lf receptors in the BBB and brain tissue of mice.The results from confocal microscopy showed the presence of Lf receptors on the surface of BCECs and the receptor-mediated endocytosis for Lf to enter the cells.The time course studies demonstrated the saturation phenomenon of binding between Lf and its receptors.Saturation binding analyses revealed that Lf receptors exhibited two classes of binding sites in BCECs(high affinity:dissociation constant(K_d)=6.77 nM; low affinity:K_d=4815 nM) and membrane preparations of mouse brains(high affinity:K_d=10.61 nM;low affinity:K_d=2228 nM).According to the above-mentioned results and the literature,Lf has several advantages when being a brain-targeting ligand.For example,the Lf receptor-mediated unidirectional transcytosis of Lf through the BBB might increase the brain accumulation of Lf-modified nanoparticles,and most of Lf receptors might not be occupied by endogenous Lf under physiological conditions.Therefore,Lf was,for the first time,exploited as a novel brain-targeting ligand here to synthesize PAMAM-PEG-Lf,and the in vitro and in vivo characteristics of the macromolecular vector and its DNA-loaded nanoparticles were evaluated in the fourth part.This vector showed a concentration-dependent manner in low concentration range and exhibited a trend of saturation in higher concentrations through fluorescent microscopy and flow cytometry.Furthermore,at the same concentration, PAMAM-PEG-Lf showed higher cellular uptake,higher brain accumulation and lower distribution in peripheral tissues,compared to PAMAM-PEG-Tf.The PAMAM-PEG-Lf/DNA NP has a particle size around 210 nm and zeta potential around 25 mV.It is spherical and has regular shape under the examination of transmission electron microscope.Moreover,the observation of colloidal gold on the surface of PAMAM-PEG-Lf/DNA NP demonstrated the successful conjugation of several Lf on the nanoparticle.The modification of Lf significantly enhanced the gene expression of PAMAM-PEG-Lf/DNA NP compared to Tf-modified nanoparticles in both BCECs and mouse brains.The results of quantitative examination of luciferase showed that the brain gene expression of PAMAM-PEG-Lf/DNA NP was about 2.3-fold than that of PAMAM-PEG-Tf/DNA NP,when the gene expression in peripheral tissues markedly reduced.All the results provide evidence that Lf is a promising ligand for the design of gene delivery systems targeting to the brain.Because Lf was first exploited as a brain-targeting ligand and PAMAM-PEG-Lf was first synthesized in this study,the brain entry mechanisms of this vector and its DNA-loaded nanoparticles remain unclear.These researches were carried out in the fifth part.The results of cellular inhibition showed that PAMAM-PEG-Lf maintained the macromolecular features of PAMAM and the ligand-receptor binding attribute of Lf,and could be taken up by BCECs via multiple pathways including clathrin-,caveolae-dependent endocytosis and macropinocytosis.PAMAM-PEG-Lf and its DNA-loaded nanoparticles could cross the BBB mainly through a receptor-mediated manner,and reserved the adsorptive mediated pathway as well. In addition,the observation of copper chlorophyll under analytical transmission electron microscope demonstrated that PAMAM-PEG-Lf/DNA NP could cross the BBB and reach the brain parenchyma successfully.As known,an overexpression of Lf receptors was observed in both microvessels and neurons of the substantia nigra in patients with Parkinson's disease(PD).The acute and chronic rat PD models were chosen in the sixth part to evaluate the pharmacodynamics of Lf-modified nanoparticles encapsulating human glial cell line-derived neurotrophic factor gene(hGDNF).And a multiple dosing regimen was for the first time designed to compare the therapeutic effects of single and multiple dosing administrations.The results of single injection showed that PAMAM-PEG-Lf/hGDNF NP had higher brain gene expression compared to other types of nanoparticles,and this gene expression decreased with time.Multiple dosing administrations could enhance and maintain the brain gene products in a higher level.The gene expression of five dosing administrations was significantly higher that that of three dosing administrations.These results demonstrated the feasibility and effectiveness of multiple dosing administrations.In the 6-hydroxydopamine(6-OHDA)-lesioned acute PD model,multiple dosing administrations of PAMAM-PEG-Lf/hGDNF NP could markedly reduce the apomorphine-induced rotations,significantly enhance the number of dopaminergic neurons in striatum and substantia nigra,and increase the content of dopamine(DA) and its metabolites in the lesioned side of rats.In the rotenone chronic PD model, multiple dosing administrations also exhibited better therapeutic effects than single injection.Extending the administration period of PAMAM-PEG-Lf/hGDNF NP could significantly improve locomotor activity,reduce dopaminergic neuronal loss and enhance monoamine neurotransmitter levels on PD rats,which indicating more powerful neuroprotective effects of multiple dosing administrations of Lf-modifined nanoparticles.All the results clearly demonstrate that PAMAM-PEG-Lf/DNA NP is an efficient non-viral gene delivery system,and multiple dosing intravenous administrations could be used as a practical non-invasive means to enhance and maintain the higher brain gene expression of non-viral gene vectors.