Yuanying Pei

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)

Partly PEGylated polyamidoamine dendrimer for tumor-selective targeting of doxorubicin: The effects of PEGylation degree and drug conjugation style

Partly PEGylated polyamidoamine (PAMAM) dendrimers were used as the carrier for tumor-selective targeting of the anticancer drug doxorubicin (DOX). Acid-sensitive cis-aconityl linkage or acid-insensitive succinic linkage was introduced between DOX and polymeric carriers to produce PPCD or PPSD conjugates, respectively. DOX release from PPCD conjugates followed an acid-triggered manner and increased with increasing PEGylation degree. In vitro cytotoxicity of PPCD conjugates against murine B16 melanoma cells increased with, while cellular uptake decreased with increasing PEGylation degree. PPSD conjugates released negligible drug at any tested pH condition and were less cytotoxic. Confocal laser scanning microscopy confirmed the acid-sensitive release of DOX from PPCD conjugates in the lysosomes and the entrance into nuclei. Pharmacokinetic and biodistribution studies demonstrated that increasing PEGylation degree resulted in reduced liver and splenic accumulation, longer circulation time and more tumor accumulation of the conjugates. Although PPSD conjugates showed more tumor accumulation than PPCD conjugates at the same PEGylation degree, the acid-sensitive DOX release from PPCD conjugates ensured higher concentration of free DOX in tumor and more pronounced antitumor activity. Besides, the antitumor activity of PPCD conjugates increased with increasing PEGylation degree. Overall, PPCD conjugate with the highest PEGylation would be a promising candidate for solid tumor therapy.

PEGylated PAMAM Dendrimer-Doxorubicin Conjugates: In Vitro Evaluation and In Vivo Tumor Accumulation

PurposeTo investigate the effects of PEGylation degree and drug conjugation style on the in vitro and in vivo behavior of PEGylated polyamidoamine (PAMAM) dendrimers-based drug delivery system.MethodsDoxorubicin (DOX) was conjugated to differently PEGylated PAMAM dendrimers by acid-sensitive cis-aconityl linkage and acid-insensitive succinic linkage to produce the products of PPCD and PPSD conjugates, respectively. In vitro evaluations including pH-dependent DOX release, cytotoxicity, cellular uptake, cell internalization mechanism, and intracellular localization were performed. Tumor accumulation was also visualized by in vivo fluorescence imaging.ResultsDOX release from PPCD conjugates followed an acid-triggered manner and increased with increasing PEGylation degree. In vitro cytotoxicity of PPCD conjugates against ovarian cancer (SKOV-3) cells increased, while cellular uptake decreased with increasing PEGylation degree. PPSD conjugates released negligible drug at any tested pH condition and were less cytotoxic. The conjugates were internalized by SKOV-3 cells via clathrin-mediated and adsorptive endocytosis, and were delivered to acidic lysosomes where DOX was released from PPCD conjugates and diffused into the nuclei. PPCD conjugates with highest PEGylation degree showed the highest tumor accumulation in mice inoculated with SKOV-3 cells.ConclusionThe obtained results suggested that PPCD conjugates with highest PEGylation degree would be a potential candidate for solid tumor treatment.

Efficient tumor targeting of hydroxycamptothecin loaded PEGylated niosomes modified with transferrin.

The aim of the present report was to exploit the possibility of combination of the stealth action by polyethylene glycol cyanoacrylate-co-hexadecyl cyanoacrylate (PEG-PHDCA) modified niosomes and active targeting function of transferrin (Tf) by transferrin receptor-mediated endocytosis to promote drug delivery to solid tumor following intravenous administration with hydroxycamptothecin (HCPT) as model drug. HCPT-loaded PEG-niosomes (PEG-NS) were prepared by thin-film hydration and ultrasound method; the periodate-oxidated Tf was coupled to terminal amino group of PEG to produce the active targeting vesicles with average diameters of 116 nm. The uptake of Tf-PEG-NS into KB cells was concentration and time dependent, which could be inhibited by low temperature and free Tf, indicating that the endocytosis process was energy-driven and receptor specific. Compared with HCPT injection, non-stealth niosomes and PEG-NS, Tf-PEG-NS demonstrated the strongest cytotoxicity to three carcinomatous cell lines (KB, K562 and S180 cells), the greatest intracellular uptake especially in nuclei, the highest tumor concentration and largest area under the intratumoral hydroxycamptothecin concentration curve, as well as the most powerful anti-tumor activity with the inhibition rate of 71% against S180 tumor in mice. The results showed that the transferrin modified PEGylated niosomes could be one of the promising solutions to the delivery of anti-tumor drugs to tumor.

Novel anti-tumor strategy: PEG-hydroxycamptothecin conjugate loaded transferrin-PEG-nanoparticles.

The aim of the study was to prepare transferrin modified stealth nanoparticles (Tf-PEG-NP) encapsulating poly(ethylene) glycol-hydroxycamptothecin conjugate (PEG-HCPT) and exploit the possiblility of combination of the functions of passive and active targeting by Tf-PEG-NP, as well as sustained drug release in tumor by PEGylated drug for most efficient tumor targeting and anti-tumor effects enhancement. PEG was covalently linked to the 10-hydroxyl group of HCPT to produce PEG-HCPT conjugate. The conjugate was stable, highly water soluable with the cytotoxicity similar to the parent drug. By encapsulation of the drug conjugate in active targeting, long circulating nanoparticles, we further improved its therapeutic efficacy. The prepared Tf-PEG-NP with average diameters of 110nm showed more sustained in vitro release profile. The pharmacokinetic and biodistribution studies found that Tf-PEG-NP demostrated the longest retention time in blood (8.94-fold that of PEG-HCPT), the highest tumor accumulation (9.03-fold, 3.11-fold that of PEG-HCPT and HCPT-loaded counterpart, respectively), as well as the most powerful anti-tumor activity with the inhibition rate up to 93% against S180 tumor in mice (1.85-fold, 1.23-fold that of PEG-HCPT and HCPT-loaded counterpart, respectively). Such Tf-PEG-NP loaded with PEGylated drug conjugates could be one of the promising strategies to deliver anti-tumor drugs to tumor.

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.

RGD-modified PEG–PAMAM–DOX conjugates: In vitro and in vivo studies for glioma

This work was based on our recent studies that a promising conjugate, RGD-modified PEGylated polyamidoamine (PAMAM) dendrimer with doxorubicin (DOX) conjugated by acid-sensitive cis-aconityl linkage (RGD-PPCD), could increase tumor targeting by binding with the integrin receptors overexpressed on tumor cells and control release of free DOX in weakly acidic lysosomes. To explore the application of RGD-PPCD to glioma therapy, the effects of the conjugate were further evaluated in glioma model. For comparative studies, DOX was also conjugated to PEG-PAMAM by acid-insensitive succinic linkage to produce the PPSD conjugates, which was further modified by RGD to form RGD-PPSD. In vitro cytotoxicity of the acid-sensitive conjugates against C6 cells was higher than that of the acid-insensitive ones, and further the modification of RGD enhanced the cytotoxicity of the DOX-polymer conjugates as a result of the increased cellular uptake of the RGD-modified conjugates by C6 cells. In vivo pharmacokinetics, biodistribution and antitumor activity were investigated in an orthotopic murine model of C6 glioma by i.v. administration of DOX-polymer conjugates. In comparison with DOX solution, all the conjugates showed significantly prolonged half-life and increased AUC and exhibited higher accumulation in brain tumor than normal brain tissue. Although RGD-PPCD was more than 2-fold lower tumor accumulation than RGD-PPSD, it exhibited the longest survival times among all treatment groups, and therefore, RGD-PPCD conjugate provide a desirable candidate for targeted therapy of glioma.

RGD-modified PEG-PAMAM-DOX conjugate: in vitro and in vivo targeting to both tumor neovascular endothelial cells and tumor cells.

Selective targeting of anticancer drugs to the tumor site is critical for effective cancer therapy. The cell adhesion molecule integrin α v β 3 is not readily detectable in quiescent vessels but highly expressed in angiogenic vessels and tumor cells. [ 1 ] This restricted expression profi le and good accessibility of integrin α v β 3 make it an ideal target for drug delivery purposes. [ 2 ] A number of synthetic cyclized arginine–glycine–aspartic acid sequences (RGDs) containing peptides, such as RGDyK, RGDfK, RGDfV, and RGDyV, have been identifi ed to have high affi nity with integrin α v β 3 . There has been growing interest in the synthesis and utilization of polymer–RGD conjugates for drug delivery, [ 3 ] gene delivery, [ 4 ] and imaging applications. [ 5 ]

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.