Jinliang Peng

Peptide ligand-mediated liposome distribution and targeting to EGFR expressing tumor in vivo

Epidermal growth factor receptor (EGFR) is an important anti-cancer therapy target that is applicable to many cancer types. We had previously reported the screening and discovery of a novel peptide ligand against EGFR named GE11. It was shown to bind to EGFR competitively with EGF and mediate gene delivery to cancer cells with high-EGFR expression. In this study, we conjugated GE11 on to liposome surface and examined their binding and distribution to EGFR expressing cancer cells in vitro and in vivo using fluorescence imaging techniques. GE11 liposomes were found to bind specifically and efficiently to EGFR high-expressing cancer cells. In vivo in H1299 xenograft mouse model, GE11 liposomes also extravasated and accumulated into the tumor site preferentially, and demonstrated better targeting and drug delivery capacities.

Novel peptide ligand directs liposomes toward EGF-R high-expressing cancer cells in vitro and in vivo

Epidermal growth factor receptor (EGF‐R) is an important target in anticancer therapy. Here we report how a novel EGF‐R peptide ligand (D4:Leu‐Ala‐Arg‐Leu‐Leu‐Thr) is identified using a computer‐aided design approach from a virtual peptide library of putative EGF‐R binding peptides by screening against the EGF‐R X‐ray crystal structure in silico and in vitro. The selected peptide is conjugated with a polyethylene glycol (PEG) lipid, and the lipid moiety of the peptide‐ PEG‐lipid conjugate is inserted into liposome membranes by a postmodification process. D4 peptide‐ conjugated liposomes are found to bind to and enter cells by endocytosis specifically and efficiently in vitro in a process apparently mediated by EGF‐R high‐expressing cancer cells (H1299). In vivo, the D4 peptide‐ conjugated liposomes are found to accumulate in EGF‐ R‐expressing xenograft tumor tissues up to 80 h after intravenous delivery, in marked contrast to controls. These results demonstrate how structure‐based peptide design can be an efficient approach to identify highly novel binding ligands against important receptors. These data could have important consequences for the development of peptide‐directed drug delivery systems with engineered specificities and prolonged times of action.— Song, S., Liu, D., Peng, J., Deng, H., Guo, Y., Xu, L. X., Miller, A. D., Xu, Y. Novel peptide ligand directs liposomes toward EGF‐R high‐expressing cancer cells in vitro and in vivo. FASEBJ. 23, 1396–1404 (2009)

A synthetic peptide mediated active targeting of cisplatin liposomes to Tie2 expressing cells

Tie2 receptor is a receptor tyrosine kinase that plays important roles in vascular angiogenesis, and also highly expressed by a number of cancer cells. In this study, we reported an active targeting liposome system directed by a novel peptide ligand PH1 that can improve drug efficacies specifically to Tie2 expressing cells. The PH1 peptide (TMGFTAPRFPHY) was selected by phage display library screening combined with surface plasmon resonance binding assays. It was covalently conjugated to the distal end of DSPE-PEG(2000)-Maleimide lipid and loaded onto liposome membranes as the targeting ligand. These PH1-PEG-liposomes containing the anticancer drug cisplatin were showed to bind tightly to Tie2 positive cells, mediate active endocytosis of the drug containing liposomes, and result in much higher cell specific cytoxicities than mPEG coated liposomes. They can be used not only to target vascular endothelial cells for anti-angiogenesis effects, but also to improve drug delivery and release in Tie2 expressing cancer cells. Such liposome formulation may be developed into a very useful agent for metronomic chemotherapy.

Effects of Surface Displayed Targeting Ligand GE11 on Liposome Distribution and Extravasation in Tumor

Targeting ligands displayed on liposome surface had been used to mediate specific interactions and drug delivery to target cells. However, they also affect liposome distribution in vivo, as well as the tissue extravasation processes after IV injection. In this study, we incorporated an EGFR targeting peptide GE11 on liposome surfaces in addition to PEG at different densities and evaluated their targeting properties and antitumor effects. We found that the densities of surface ligand and PEG were critical to target cell binding in vitro as well as pharmacokinetic profiles in vivo. The inclusion of GE11-PEG-DSPE and PEG-DSPE at 2% and 4% mol ratios in the liposome formulation mediated a rapid accumulation of liposomes within 1 h after IV injection in the tumor tissues surrounding neovascular structures. This is in addition to the EPR effect that was most prominently described for surface PEG modified liposomes. Therefore, despite the fact that the distribution of liposomes into interior tumor tissues was still limited by diffusion, GE11 targeted doxorubicin loaded liposomes showed significantly better antitumor activity in tumor bearing mice as a result of the fast active-targeting efficiency. We anticipate these understandings can benefit further optimization of targeted drug delivery systems for improving efficacy in vivo.

Impact of PEGylation on biodistribution and tumor accumulation of Lipid-Mu peptide-DNA.

For gene-based therapeutic approaches that require transfection of specific cell targets in vivo, it is important to design the delivery system to have optimized tissue distribution. Surface PEGylation of liposomes and polymer nanoparticles has been shown to lead to prolonged blood circulation and also preferential accumulation in tumor sites resulting from the enhanced permeability and retention (EPR) effect. The aim of this study was to investigate the effect of different surface PEGylation densities on resulted biodistribution profiles of Lipid-Mu peptide-DNA (LMD) nanoparticles. LMD particles containing the near infrared fluorescent lipid dye, Cy5.5-DSPE, were injected intravenously, and whole-body fluorescence images of the live animal were recorded. Analysis of these time series of images indicated that LMDs with different surface PEG2000 densities had distinctively different biodistribution and tumor accumulation profiles. LMDs containing approximately 15–25% of surface PEG2000 were shown to have the highest accumulation and longest residence time in tumor. LMD distribution and pharmacokinetic profiles in other organs were also observed to be different and were analyzed.

Targeting and liposomal drug delivery to CD40L expressing T cells for treatment of autoimmune diseases.

CD40L is considered as an important target for the treatment of autoimmune diseases. There have been many efforts devoted to the development of antibodies and other molecules to disrupt CD40/CD40L interaction for therapeutic benefits. In this study, we designed a CD40L specific peptide ligand - A25 based on CD40L crystal structure and molecular docking studies. Its binding affinity and specificity to CD40L were confirmed by Surface Plasmon Resonance (SPR) measurements. The peptide A25 was then conjugated on the surface of liposomes and shown to be able to mediate specific liposomal drug delivery to CD40L+ cells. Loaded with the cytostatic drug methotrexate (MTX), the A25 modified liposome could significantly reduce the CD40L+ cell ratios in the experimental autoimmune encephalomyelitis (EAE) mice, resulting in great improvement in clinical scores. Since CD40L+ cells are involved in the pathological development of many auto-immune diseases, A25 conjugated drug targeting systems may be useful for developing therapies that are more efficacies and with less side effects.

Short noncoding DNA fragment improve efficiencies of in vivo electroporation-mediated gene transfer.

A major obstacle to the application of gene therapy methods in experimental and clinical practice is the lack of safe and efficient gene delivery systems. Electroporation has been shown to an effective physical delivery method. A variety of factors have been shown to affect the electroporation‐mediated gene delivery efficiency. In the present study, we assessed the usefulness of noncoding short‐fragment DNA (sf‐DNA) for facilitating electroporation‐mediated gene transfer.

Examining the Interactome of Huperzine A by Magnetic Biopanning

Huperzine A is a bioactive compound derived from traditional Chinese medicine plant Qian Ceng Ta (Huperzia serrata), and was found to have multiple neuroprotective effects. In addition to being a potent acetylcholinesterase inhibitor, it was thought to act through other mechanisms such as antioxidation, antiapoptosis, etc. However, the molecular targets involved with these mechanisms were not identified. In this study, we attempted to exam the interactome of Huperzine A using a cDNA phage display library and also mammalian brain tissue extracts. The drugs were chemically linked on the surface of magnetic particles and the interactive phages or proteins were collected and analyzed. Among the various cDNA expressing phages selected, one was identified to encode the mitochondria NADH dehydrogenase subunit 1. Specific bindings between the drug and the target phages and target proteins were confirmed. Another enriched phage clone was identified as mitochondria ATP synthase, which was also panned out from the proteome of mouse brain tissue lysate. These data indicated the possible involvement of mitochondrial respiratory chain matrix enzymes in Huperzine As pharmacological effects. Such involvement had been suggested by previous studies based on enzyme activity changes. Our data supported the new mechanism. Overall we demonstrated the feasibility of using magnetic biopanning as a simple and viable method for investigating the complex molecular mechanisms of bioactive molecules.

Inhibitory effect of Ca 2+ on in vivo gene transfer by electroporation

AbstractAim:To investigate the specific effects of Ca2+ on transgene expression during electroporation-mediated gene transfer in mice.Methods:Skeletal muscle and skin were subjected to in vivo electroporation with a luciferase reporter plasmid, with or without Ca2+ and various other ions.Results:For in vivo electroporation, the presence of just 10 mmol/L Ca2+ in the DNA solution drastically reduced the resulting transgene expression, to less than 5% of control values. Only Ca2+, not other ions, caused inhibition, and the effect was not tissue specific. More surprisingly, even when Ca2+ ions were delivered by electroporation before or after DNA administration, similar effects were still observed.Conclusion:The inhibitory effect of Ca2+ on in vivo gene transfer by electroporation is specific, ie, the inhibitory effect may be related to the cell membrane properties after electroporation and the subsequent resealing event.

Multiwalled carbon nanotube-modified poly(d,l-lactide-co-glycolide) scaffolds for dendritic cell load

Poly(D,L-lactide-co-glycolide) (PLGA) is widely used in a variety of tissue engineering and drug delivery applications due to its biodegradability and biocompatibility. But PLGA surfaces are usually hydrophobic which limited the loading and seeding capacities for cells, especially semiadherent immune cells. In this paper we described an attempt to improve the hydrophilicity and surface architecture for accommodating dendritic cells (DCs) that are widely used as professional antigen presenting cells in immune therapy of cancer and other diseases. The 3D porous PLGA scaffold was made by solvent casting/salt leaching of PLGA blended with surface functionalized multiwalled carbon nanotubes (F-MWCNTs). The incorporation and dispersion of F-MWCNT in the scaffold structures resulted in not only improved surface hydrophilicity but also nanoscale surface structure that would provide a preferable microenvironment for DCs attachment. We think such a scaffold material may be more desirable for immune cell delivery for immunotherapy.