Weilun Ke

Gene delivery targeted to the brain using an Angiopep-conjugated polyethyleneglycol-modified polyamidoamine dendrimer

Angiopep targeting to the low-density lipoprotein receptor-related protein-1 (LRP1) was identified to exhibit high transcytosis capacity and parenchymal accumulation. In this study, it was exploited as a ligand for effective brain-targeting gene delivery. Polyamidoamine dendrimers (PAMAM) were modified with angiopep through bifunctional PEG, then complexed with DNA, yielding PAMAM-PEG-Angiopep/DNA nanoparticles (NPs). The angiopep-modified NPs were observed to be internalized by brain capillary endothelial cells (BCECs) through a clathrin- and caveolae-mediated energy-depending endocytosis, also partly through marcopinocytosis. Also, the cellular uptake of the angiopep-modified NPs were competed by angiopep-2, receptor-associated protein (RAP) and lactoferrin, indicating that LRP1-mediated endocytosis may be the main mechanism of cellular internalization of angiopep-modified NPs. And the angiopep-modified NPs showed higher efficiency in crossing blood-brain barrier (BBB) than unmodified NPs in an in vitro BBB model, and accumulated in brain more in vivo. The angiopep-modified NPs also showed higher efficiency in gene expressing in brain than the unmodified NPs. In conclusion, PAMAM-PEG-Angiopep showed great potential to be applied in designing brain-targeting drug delivery system.

Brain-targeting gene delivery and cellular internalization mechanisms for modified rabies virus glycoprotein RVG29 nanoparticles

A 29 amino-acid peptide derived from the rabies virus glycoprotein (RVG29) was exploited as a ligand for efficient brain-targeting gene delivery. RVG29 was modified on polyamidoamine dendrimers (PAMAM) through bifunctional PEG, then complexed with DNA, yielding PAMAM-PEG-RVG29/DNA nanoparticles (NPs). The NPs were observed to be uptaken by brain capillary endothelial cells (BCECs) through a clathrin and caveolae mediated energy-depending endocytosis. The specific cellular uptake can be inhibited by free RVG29 and GABA but not by nicotinic acetylcholine receptor (nAchR) agonists/antagonists, indicating RVG29 probably relates to the GABA(B) receptor besides nAchR reported previously. PAMAM-PEG-RVG29/DNA NPs showed higher blood-brain barrier (BBB)-crossing efficiency than PAMAM/DNA NPs in an in vitro BBB model. In vivo imaging showed that the NPs were preferably accumulated in brain. The report gene expression of the PAMAM-PEG-RVG29/DNA NPs was observed in brain, and significantly higher than unmodified NPs. Thus, PAMAM-PEG-RVG29 provides a safe and noninvasive approach for the gene delivery across the BBB.

Efficient gene delivery targeted to the brain using a transferrin-conjugated polyethyleneglycol-modified polyamidoamine dendrimer

The blood‐brain barrier (BBB) poses great difficulties for gene delivery to the brain. To circumvent the BBB, we investigated a novel brain‐targeting gene vector based on the nanoscopic high‐branching den‐drimer, polyamidoamine (PAMAM), in vitro and in vivo. Transferrin (Tf) was selected as a brain‐targeting ligand conjugated to PAMAM via bifunctional polyethylenegly‐col (PEG), yielding PAMAM‐PEG‐Tf. UV and nuclear magnetic resonance (NMR) spectroscopy were used to evaluate the synthesis of vectors. The characteristics and biodistribution of gene vectors were evaluated by fluorescent microscopy, flow cytometry, and a radiolabeling method. The transfection efficiency of vector/DNA complexes in brain capillary endothelial cells (BCECs) was evaluated by fluorescent microscopy and determination of luciferase activity. The potency of vector/DNA complexes was evaluated by using frozen sections and measuring tissue luciferase activity in Balb/c mice after i.v. administration. UV and NMR results demonstrated the successful synthesis of PAMAM‐PEG‐Tf. This vector showed a concentration‐dependent manner in cellular uptake study and a 2.25‐fold brain uptake compared with PAMAM and PAMAM‐PEG in vivo. Transfection efficiency of PAMAM‐PEG‐Tf/DNA complex was much higher than PAMAM/DNA and PAMAM‐PEG/DNA complexes in BCECs. Results of tissue expression experiments indicated the widespread expression of an exogenous gene in mouse brain after i.v. administration. With a PAMAM/DNA weight ratio of 10:1, the brain gene expression of the PAMAM‐PEG‐Tf/DNA complex was ~2‐fold higher than that of the PAMAM/DNA and PAMAM‐PEG/ DNA complexes. These results suggested that PAMAM‐PEG‐Tf can be exploited as a potential nonviral gene vector targeting to brain via noninvasive administration.—Huang, R‐Q., Qu, Y‐H., Ke, W‐L., Zhu, J‐H., Pei, Y‐Y., Jiang, C. Efficient gene delivery targeted to the brain using a transferrin‐conjugated polyethyleneglycol‐modified polyamidoamine dendrimer. FASEB J. 21, 1117–1125 (2007)

Gene therapy using lactoferrin-modified nanoparticles in a rotenone-induced chronic Parkinson model

BACKGROUND Gene therapy is considered one of the most promising approaches to develop an effective treatment for Parkinsons disease (PD). The existence of blood-brain barrier (BBB) significantly limits its development. In this study, lactoferrin (Lf)-modified nanoparticles (NPs) were used as a potential non-viral gene vector due to its brain-targeting and BBB-crossing ability. METHODS AND RESULTS The neuroprotective effects were examined in a rotenone-induced chronic rat model of PD after treatment with NPs encapsulating human glial cell line-derived neurotrophic factor gene (hGDNF) via a regimen of multiple dosing intravenous administration. The results showed that multiple injections of Lf-modified NPs obtained higher GDNF expression and this gene expression was maintained for a longer time than the one with a single injection. Multiple dosing intravenous administration of Lf-modified NPs could significantly improve locomotor activity, reduce dopaminergic neuronal loss, and enhance monoamine neurotransmitter levels on rotenone-induced PD rats, which indicates its powerful neuroprotective effects. CONCLUSION The findings may have implications for long-term non-invasive gene therapy for neurodegenerative diseases in general.

Neuroprotection in a 6‐hydroxydopamine‐lesioned Parkinson model using lactoferrin‐modified nanoparticles

Nonviral gene therapy of chronic degenerative diseases such as Parkinsons disease (PD) is a great challenge as a result of the low tranfection efficiency of nonviral gene vectors. We previously constructed a lactoferrin (Lf)‐modified vector, which was demonstrated to be potential for brain gene delivery both in vitro and in vivo. In the present study, this type of vector was applied to load human glial cell line‐derived neurotrophic factor gene (hGDNF).

Brain-targeting mechanisms of lactoferrin-modified DNA-loaded nanoparticles

Ligand-mediated brain-targeting drug delivery is one of the focuses at present. Elucidation of exact targeting mechanisms serves to efficiently design these drug delivery systems. In our previous studies, lactoferrin (Lf) was successfully exploited as a brain-targeting ligand to modify cationic dendrimer-based nanoparticles (NPs). The mechanisms of Lf-modified NPs to the brain were systematically investigated in this study for the first time. The uptake of Lf-modified vectors and NPs by brain capillary endothelial cells (BCECs) was related to clathrin-dependent endocytosis, caveolae-mediated endocytosis, and macropinocytosis. The intracellular trafficking results showed that Lf-modified NPs could rapidly enter the acidic endolysosomal compartments within 5 mins and then partly escape within 30 mins. Both Lf-modified vectors and NPs showed higher blood–brain barrier-crossing efficiency than unmodified counterparts. All the results suggest that both receptor- and adsorptive-mediated mechanisms contribute to the cellular uptake of Lf-modified vectors and NPs. Enhanced brain-targeting delivery could be achieved through the synergistic effect of the macromolecular polymers and the ligand.

Evaluation and mechanism studies of PEGylated dendrigraft poly-L-lysines as novel gene delivery vectors

Dendrimers have attracted great interest in the field of gene delivery due to their synthetic controllability and excellent gene transfection efficiency. In this work, dendrigraft poly-L-lysines (DGLs) were evaluated as a novel gene vector for the first time. Derivatives of DGLs (generation 2 and 3) with different extents of PEGylation were successfully synthesized and used to compact pDNA as complexes. The result of gel retardation assay showed that pDNA could be effectively packed by all the vectors at a DGLs to pDNA weight ratio greater than 2. An increase in the PEGylation extent of vectors resulted in a decrease in the incorporation efficiency and cytotoxicity of complexes in 293 cells, which also decreased the zeta potential a little but did not affect the mean diameter of complexes. Higher generation of DGLs could mediate higher gene transfection in vitro. Confocal microscopy and cellular uptake inhibition studies demonstrated that caveolae-mediated process and macropinocytosis were involved in the cellular uptake of DGLs-based complexes. Also the results indicate that proper PEGylated DGLs could mediate efficient gene transfection, showing their potential as an alternate biodegradable vector in the field of nonviral gene delivery.

Lactoferrin-modified nanoparticles could mediate efficient gene delivery to the brain in vivo

Lactoferrin (Lf)-modified nanoparticles (NPs) have been demonstrated to mediate efficient expression of exogenous genes in the brain via intravenous administration. The brain-targeting properties of Lf-modified NPs were investigated in this study. In vivo imaging results showed that the accumulation of Lf-modified NPs was higher in the brain but lower in the other organs than that of unmodified counterparts. The results of analytical transmission electron microscopy showed that some Lf-modified NPs crossed the blood-brain barrier (BBB) and reached the neural tissues, while some remained within the BBB. Similar results were observed in the distribution of exogenous gene products. All the results demonstrated the successful delivery of Lf-modified NPs into the brain. Lf-modified NPs could be exploited as potential brain-targeting delivery systems for exogenous genes, especially for those encoding secretive proteins.