An electron-rich molybdenum-molybdenum quintuple bond spanned by one lithium atom.

Take five: A unique quintuply bonded dimolybdenum complex [Mo(2)(µ-Li){µ-HC(N-2,6-Et(2)C(6)H(3))(2)}(3)] (see picture) was synthesized and characterized. The Mo-Mo interaction includes an unexpected bridging Li(+) ion. Calculations indicate the bridging Li(+) ion does not perturb the Mo-Mo bond length (2.0612(4) Å), but results in a relatively small effective Mo-Mo bond order of 3.67.

Targeted delivery of chlorotoxin-modified DNA-loaded nanoparticles to glioma via intravenous administration.

Gene therapy offers great potential for brain glioma. However, therapeutic genes could not reach glioma spontaneously. A glioma-targeting gene delivery system is highly desired to transfer exogenous genes throughout the tumor focus. In this study, the nanoscopic high-branching dendrimer, polyamidoamine (PAMAM), was selected as the main vector. Chlorotoxin (CTX), which has been demonstrated to bind specifically to receptor expressed in glioma, was exploited as the targeting ligand to conjugate PAMAM via bifunctional polyethyleneglycol (PEG), yielding PAMAM-PEG-CTX. The cellular uptake of CTX itself was observed apparently in C6 glioma cells, almost not in 293 cells. The modification of CTX could significantly increase the cellular uptake of vectors and the DNA-loaded nanoparticles (NPs) in C6 cells. The in vivo distribution of PAMAM-PEG-CTX/DNA NPs in the brain was higher than that of PAMAM/DNA NPs and PAMAM-PEG/DNA NPs. Furthermore, the gene expression of PAMAM-PEG-CTX/DNA NPs was higher and broader in glioma than that of unmodified and PEG-modified counterparts. The TUNEL analysis showed a more wide-extended apoptosis in the CTX-modified group, compared to other groups including commercial temozolomide group. The median survival time of CTX-modified group and temozolomide group was 59.5 and 49 days, respectively, significantly longer than that of other groups. The results suggested that CTX could be exploited as a special glioma-targeting ligand, and PAMAM-PEG-CTX/DNA NPs is a potential non-viral delivery system for gene therapy of glioma via intravenous administration.

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.

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 Parkinson's 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.

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.

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 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.

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

BACKGROUND: Nonviral gene therapy of chronic degenerative diseases such as Parkinson's 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). METHODS: A rat PD model was constructed by the unilateral lesion of striatum using 6-hydroxydopamine (6-OHDA). Lf-modified nanoparticles (NPs) were prepared and characterized. Neuroprotective effects of Lf-modified NPs were examined in the 6-OHDA-lesioned PD model via a regimen of multiple dosing intravenous administrations. RESULTS: The size of Lf-modified NPs was 196 +/- 10.1 nm, whereas the zeta potential value was 29.35 +/- 3.27 mV. Lf-modified NPs could protect themselves from heparin displacement and DNase digestion. The results of the neuroprotective evaluation show that increasing the number of injections of Lf-modified NPs loading hGDNF improved locomotor activity, reduced dopaminergic neuronal loss and enhanced monoamine neurotransmitter levels in PD rats. Five injections of Lf-modified NPs loading hGDNF exhibited much more powerful neuroprotection than a single injection, indicating the effectiveness and feasibility of multiple dosing administrations. The results of toxicity tests demonstrated that the dosage of NPs used in the present study was safe enough for brain gene delivery. CONCLUSIONS: The findings obtained in the present study suggest that Lf-modified NPs could be developed for potential nonviral gene therapy of chronic brain disorders.

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.

Enhanced oral bioavailability of doxorubicin in a dendrimer drug delivery system.

PAMAM dendrimers can permeate across intestinal epithelial barriers suggesting their potential as oral drug carriers. In the present study, we have developed a drug-PAMAM complex for oral administration. The loading of a model drug, doxorubicin into PAMAM, the cellular uptake and pharmacokinetics of the doxorubicin-PAMAM complex were studied. As the results, the cellular uptake of doxorubicin in Caco-2 cells treated with the doxorubicin-PAMAM complex was increased significantly with an increase in concentration and time, as compared to that treated with free doxorubicin. And the transport efficiency of the doxorubicin-PAMAM complex from the mucosal side to the serosal side was 4-7 times higher than that of free doxorubicin in different segments of small intestines of rat. The doxorubicin-PAMAM complex led to the bioavailability that was more than 200-fold higher than that of free doxorubicin after oral administration. These results indicate that PAMAM dendrimer is a promising novel carrier to enhance the oral bioavailability of drug, especially for the P-glycoprotein (P-gp) substrates.

The use of lactoferrin as a ligand for targeting the polyamidoamine-based gene delivery system to the brain.

Development of an efficient gene vector is a key-limiting factor of brain gene therapy. In this study, lactoferrin (Lf), for the first time, was investigated as a brain-targeting ligand in the design of polyamidoamine (PAMAM)-based non-viral gene vector to the brain. Using polyethyleneglycol (PEG) as a spacer, PAMAM-PEG-Lf was successfully synthesized. This vector showed a concentration-dependent manner in the uptake in brain capillary endothelial cells (BCECs). The brain uptake of PAMAM-PEG-Lf was 2.2-fold compared to that of PAMAM-PEG-transferrin (Tf) in vivo. The transfection efficiency of PAMAM-PEG-Lf/DNA complex was higher than that of PAMAM-PEG-Tf/DNA complex in vitro and in vivo. The results of frozen sections showed the widespread expression of an exogenous gene in mouse brain via intravenous administration. With a PAMAM/DNA weight ratio of 10:1, the brain gene expression of the PAMAM-PEG-Lf/DNA complex was about 2.3-fold when compared to that of the PAMAM-PEG-Tf/DNA complex. These results provide evidence that PAMAM-PEG-Lf can be exploited as a potential non-viral gene vector targeting to the brain via noninvasive administration. Lf is a promising ligand for the design of gene delivery systems targeting to the brain.

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 dendrimer, polyamidoamine (PAMAM), in vitro and in vivo. Transferrin (Tf) was selected as a brain-targeting ligand conjugated to PAMAM via bifunctional polyethyleneglycol (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 approximately 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.

Characterization of lactoferrin receptor in brain endothelial capillary cells and mouse brain.

We present, herein, the evidence for lactoferrin (Lf) binding sites in brain endothelial capillary cells (BCECs) and mouse brain. 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. Saturation binding analyses revealed that Lf receptors exhibited two classes of binding sites in BCECs (high affinity: dissociation constant (K (d)) = 6.77 nM, binding site density (B (max)) = 10.3 fmol bound/mug protein; low affinity: K (d) = 4815 nM, B (max) = 1190 fmol bound/mug protein) and membrane preparations of mouse brain (high affinity: K (d) = 10.61 nM, B (max) = 410 fmol bound/mug protein; low affinity: K (d) = 2228 nM, B (max) = 51641 fmol bound/mug protein). The distribution study indicated the effective uptake of (125)I-Lf in brain after intravenous administration. The present study provides experimental evidence for the application of Lf as a novel ligand for brain targeting.