Yuhong Xu

Identification and characterization of a novel peptide ligand of epidermal growth factor receptor for targeted delivery of therapeutics

Epidermal growth factor receptor (ErbB1, EGFR) is overexpressed in a variety of human cancer cells. It has been considered as a rational target for drug delivery. To identify novel ligands with specific binding capabilities to EGFR, we screened a phage display peptide library and found an enriched phage clone encoding the amino acid sequence YHWYGYTPQNVI (designated as GE11). Competitive binding assay and Scatchard analysis revealed that GE11 peptide bound specifically and efficiently to EGFR with a dissociation constant of ∼22 nM, but with much lower mitogenic activity than with EGF. We showed that the peptides were internalized preferentially into EGFR highly expressing cells, and they accumulated in EGFR overexpressing tumor xenografts after i.v. delivery in vivo. In gene delivery studies, GE11‐conjugated polyethylenimine (PEI) vectors were less mitogenic, but still quite efficient at transfecting genes into EGFR highly expressing cells and tumor xenografts. Taken together, GE11 is a potentially safe and efficient targeting moiety for selective drug delivery systems mediated through EGFR.—Li, Z., Zhao, R., Wu, X., Sun, Y., Yao, M., Li, J., Xu, Y., Gu, J. Identification and characterization of a novel peptide ligand of epidermal growth factor receptor for targeted delivery of therapeutics. FASEB J. 19, 1978–1985 (2005)

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)

Preparation of nanobubbles for ultrasound imaging and intracelluar drug delivery.

Echogenic bubble formulations have wide applications in both disease diagnosis and therapy. In the current study, nanobubbles were prepared and the contrast agent function was evaluated in order to study the nanosized bubbles property for ultrasonic imaging. Coumarin-6 as a model drug was loaded into nanobubbles to investigate the drug delivery potential to cells. The results showed that the nanobubbles composed of 1% of Tween 80, and 3 mg/ml of lipid worked well as an ultrasonic contrast agent by presenting a contrast effect in the liver region in vivo. The drug-loaded nanobubbles could enhance drug delivery to cells significantly, and the process was analyzed by sigmoidally fitting the pharmacokinetic curve. It can be concluded that the nanobubble formulation is a promising approach for both ultrasound imaging and drug delivery enhancing.

Investigation of archaeosomes as carriers for oral delivery of peptides

Oral administration of peptide and protein drugs faces a big challenge partly due to the hostile gastrointestinal (GI) environment. Lipid-based delivery systems are attractive because they offer some protection for peptides and proteins. In this context, we prepared a special lipid-based oral delivery system: archaeosomes, made of the polar lipid fraction E (PLFE) extracted from Sulfolobus acidocaldarius, and explored its potential as an oral drug delivery vehicle. Our study demonstrates that archaeosomes have superior stability in simulated GI fluids, and enable fluorescent labeled peptides to reside for longer periods in the GI tract after oral administration. Although archaeosomes have little effect on the transport of insulin across the Caco-2 cell monolayers, the in vivo experiments indicated that archaeosomes containing insulin induced lower levels of blood glucose than a conventional liposome formulation. These data indicate that archaeosomes could be a potential carrier for effective oral delivery of peptide drugs.

Immunoprotection against influenza H5N1 virus by oral administration of enteric-coated recombinant Lactococcus lactis mini-capsules

Edible vaccines that can be made widely available and easily administered could bring great benefit to the worldwide battle against pandemic viral infections. They can be used not only for the vaccination of humans and domesticated animals, but also for wild herds and live stock which are otherwise difficult to vaccinate. In this study, we report the development of an edible mini-capsule form of live, non-persisting, recombinant Lactococcus lactis (L. lactis) vaccine against the highly virulent influenza H5N1 strain. Recombinant L. lactis-based H5N1 HA antigen expression constructs were made and shown to be able to induce higher levels of HA-specific serum IgG and fecal IgA antibody production after oral administration. The vectors were then formulated into a mini-capsule dosage form and fed to mouse. Four doses of oral administration rendered complete protection of the mouse against lethal challenges of H5N1 virus.

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.

Archaeosomes with encapsulated antigens for oral vaccine delivery

Traditional phosphodiester lipid vesicles (liposomes) are not stable and could be easily degraded in the gastrointestinal (GI) tract. We prepared a novel lipid based oral delivery system: archaeosomes, made of the polar lipid fraction E (PLFE) extracted from Sulfolobus acidocaldarius, and tested their immunogenic potentials as oral vaccine delivery vehicles. Our study showed that the archaeosomes had significant superior stability in simulated gastric and intestinal fluids, and would help fluorescent labeled antigens to reside longer time in the GI tract after oral administration. The resulted immune responses against model antigen ovalbumin (OVA) were greatly improved, eliciting substantial IgG response systemically as well as IgA response mucosally. In addition, the archaeosomes also facilitated antigen specific CD8(+) T cell proliferation. These data indicate that archaeosomes may be a potential vaccine carrier and adjuvant for effective oral immunization.

Improved antigen cross-presentation by polyethyleneimine-based nanoparticles.

Purpose In the development of therapeutic vaccines against cancer, it is important to design strategies for antigen cross-presentation to stimulate cell-mediated immune responses against tumor antigens. Methods We developed a polyethyleneimine (PEI)-based protein antigen delivery system to promote cross-presentation through the major histocompatibility complex (MHC) I pathway using ovalbumin (OVA) as a model antigen. PEIs formed nanoparticles with OVA by electrostatic interactions, as demonstrated by electrophoresis analysis, scanning electron microscopy, and photon correlation spectroscopy analysis. Results The nanoparticles were used to stimulate mouse bone marrow-derived dendritic cells in vitro and resulted in significantly more OVA257–264/MHC I complex presentation on dendritic cell surfaces. The activated dendritic cells interacted specifically with RF33.70 to stimulate interleukin-2 secretion. The cross-presentation promoting effect was more prominent in dendritic cells that had been cultured for longer periods of time (13 days). Further studies comparing the antigen presentation efficacies by other polyanionic agents, such as PLL or lysosomotropic agents, suggested that the unique “proton sponge effect” of PEI facilitated antigen escape from the endosome toward the MHC I pathway. Conclusion Such a PEI-based nanoparticle system may have the potential to be developed into an effective therapeutic vaccine delivery system.

Evaluation of Oral Immunization with Recombinant Avian Influenza Virus HA1 Displayed on the Lactococcus lactis Surface and Combined with the Mucosal Adjuvant Cholera Toxin Subunit B

ABSTRACT The development of safe and efficient avian influenza vaccines for human and animal uses is essential for preventing virulent outbreaks and pandemics worldwide. In this study, we constructed a recombinant (pgsA-HA1 gene fusion) Lactococcus lactis strain that expresses and displays the avian influenza virus HA1 antigens on its surface. The vectors were administered by oral delivery with or without the addition of cholera toxin subunit B (CTB). The resulting immune responses were analyzed, and the mice were eventually challenged with lethal doses of H5N1 viruses. Significant titers of hemagglutinin (HA)-specific serum IgG and fecal IgA were detected in the group that also received CTB. Cellular immunities were also shown in both cell proliferation and gamma interferon (IFN-γ) enzyme-linked immunospot (ELISpot) assays. Most importantly, the mice that received the L. lactis pgsA-HA1 strain combined with CTB were completely protected from lethal challenge of the H5N1 virus. These findings support the further development of L. lactis-based avian influenza virus vaccines for human and animal uses.