The neonatal Fc receptor for IgG (FcRn) is considered critical for the regulation of endogenous IgG and serum albumin (SA) and their circulation half-life in vivo. Both IgG and SA can bind to FcRn tightly at acidic pH but not so much at neutral pH. Here we reported a few novel single chain antibody fragments (scFv) obtained based on screening of a phage library. FnAb-8 and FnAb-12 can bind to human FcRn with higher affinities than IgG at acidic pH but similar or lower affinities than IgG at pH 7.4. Fusion proteins consisted of the therapeutic peptide, GLP-1 (Glucagon-like peptide-1) connected to the N-terminus of FnAb-8 and FnAb-12, named as G8 and G12, were shown to retain the pH-dependent binding capabilities to FcRn while also bound to the GLP-1 receptor. In vivo efficacy studies in diet induced diabetes mice confirmed the GLP-1 receptor (GLP-1R) agonist activities and sustained blood sugar lowering effect. In vivo pharmacokinetics (PK) studies were performed in nonhuman primates and FnAb-8 was found to have circulation half-life several folds longer than what have been reported for scFvs. G8 may be developed into long acting GLP-1R agonists with great potentials in clinical applications.

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.Keywords: doxorubicin; EGFR; extravasation; GE11; liposome; target delivery;

To propose a novel composite nanoemulsion formulation that contains no surfactant, but offers great stability and improved oral absorption capabilities.The nanoemulsions were prepared by dispersing the oil phase into aqueous solutions containing different amounts of the PMMA/silica composite nanoparticles. The stability was tested under extreme conditions. The structure features of the nanoemulsion droplets were investigated using Electron microscope. The in vitro drug release and in vivo drug absorption profiles after oral administration were investigated using Cyclosporin A as a model drug.The composite nanoemulsion demonstrated great stability under various disruptive conditions. Electron microscopy studies indicated the existence of internal and surface domains in the nano-droplet structure. In vitro drug release and in vivo uptake characterizations also confirmed the unique interfacial properties of such nanoemulsion structures.The novel nanoemulsion formulation may have modulated drug release profiles and alternative oral absorption mechanisms, which could offer significant advantages compared to traditional emulsion formulations.

To develop a multimodal imaging guided and triggered drug delivery system based on a novel emulsion formulation composed of iron oxide nanoparticles, nanoscopic bubbles, and oil containing drugs.Iron oxide paramagnetic nanoparticles were synthesized and modified with surface conjugation of polyethylenimide (PEI) or Bovine Serum Albumin (BSA). Both particles were used to disperse and stabilize oil in water emulsions containing coumarin-6 as the model drug. Sulfur hexafluoride was introduced into the oil phase to form nanoscopic bubbles inside the emulsions. The resulted gas containing emulsions were evaluated for their magnetic resonance (MR) and ultrasound (US) imaging properties. The drug release profile triggered by ultrasound was also examined.We have successfully prepared the highly integrated multi-component emulsion system using the surface modified iron oxide nanoparticles to stabilize the interfaces. The resulted structure had distinctive MR and US imaging properties. Upon application of ultrasound waves, the gas containing emulsion would burst and encapsulated drug could be released.The integrated emulsion formulation was multifunctional with paramagnetic, sono-responsive and drug-carrying characteristics, which may have potential applications for disease diagnosis and imaging guided drug release.

To develop a liposome formulation incorporating antigen-presenting cells (APCs) membrane microdomains with enriched epitope/MHC complexes to evaluate the activities of these liposomes (RAFTsomes) to activate T cells and prime immune responses.We isolated membrane microdomain structures that contained the epitope/MHC complexes from ovalbumin (OVA) primed dendritic cells (DCs), and reconstituted them on liposomes surface by detergent dialysis. The resulted RAFTsomes were purified by density gradient centrifugation. Their T cell activation functions were evaluated by IL-2 secreting and proliferation assays in vitro. In vivo immune responses and the protective effect against OVA expressing EG.7 tumor challenge were also examined.Membrane microdomains containing enriched epitope/MHC complexes can be reconstituted into liposomes with defined size and composition. The integrity and activities of these complexes after reconstitution were confirmed by in vitro T cell assays. OVA epitope loaded RAFTsomes injected in vivo resulted in high anti-OVA IgG production (predominantly IgG1). The immunized mice were protected from EG.7 tumor cell inoculation challenge.Based on these findings, we propose that RAFTsomes can be prepared with unique properties that may be used as an antigen delivery system for immunotherapeutic applications.

Regulation of gene expression using small interfering RNA (siRNA) is a promising strategy for treatments of numerous diseases. However, the progress towards broad application of siRNA requires the development of safe and effective vectors that target to specific cells. In this study, we developed a novel recombinant high density lipoprotein (rHDL) vector with high siRNA encapsulation efficiency.They were prepared by condensing siRNA with various commercial cationic polymers and coating the polyplex with a layer of lipids and apolipoprotein AI (apo AI). The rHDL nanoparticles were used to transfect SMMC-7721 hepatoma cells with stable luciferase expression. The uptake and intracellular trafficing of siRNA were also investigated.Characterization studies revealed these rHDL nanoparticles had similar physical properties as natural HDLs. The various rHDL formulations had high silencing efficiency (more than 70% knockdown) in hepatocytes with minimum cytotoxicity. Moreover, the uptake of rHDL by SMMC-7721 was confirmed to be mediated through the natural HDL uptake pathway.The work described here demonstrated the optimized rHDL nanoparticles may offer a promising tool for siRNA delivery to the liver.

The oligopeptide transporter PEPT1 is considered as a valuable target for prodrug design, but its 3D structure and substrate specificity of PEPT1 are not fully understood. In this study, we designed a focused dipeptide conjugated azidothymidine (AZT) library and described a convenient and efficient solid phase synthesis scheme based on click chemistry. Over 60 candidate structures containing various dipeptide sequences were obtained with high purity, and screened in a PEPT1 overexpressing cell model for their abilities to compete with the known ligand cephalexin. Some of the compounds selected to have medium or high affinity were tested for their in vivo transport in a single-pass intestinal perfusion experiment. Results showed that the designed library contained some new structure features that have high affinities toward PEPT1 and could be further explored for their application in prodrug design and development.Keywords: azidothymidine−dipeptide conjugates; click chemistry; competitive inhibition; PEPT1; single-pass intestinal perfusion

The angiopoietin–Tie2 binding and related signal transduction pathways are crucial for vascular angiogenesis, blood vessel integrity and maturation. In this study, we preformed a virtual screening of small molecules targeting to Tie2. The binding site was selected at the extracellular ligand binding region of Tie2, rather than its conventional endocellular ATP binding region. It was found that loperamide, a widely-used antidiarrhea drug, was among the top hits. The binding between loperamide and Tie2 was confirmed by surface plasmon resonance (SPR) assay. Loperamide competitively inhibited the binding of both angiopoietin1 and angiopoietin2. These results indicate that loperamide is an antagonist of angiopoietin1 and angiopoietin2.

Hyperbranched poly(amido amine) (HPAMAM), which is structurally analogous to PAMAM dendrimers, has been proposed to be an effective agent for gene delivery. The facile synthesis of HPAMAM with scalable productivity by one-pot polymerization of monomers of methyl acrylate (MA) and diethylenetriamine (DETA) has been set up previously. In this study, the HPAMAM was further modified on the terminal amino groups with phenylalanine to various degrees (HPAMAM-PHE30, PHE45, PHE60). We showed that HPAMAM and HPAMAM-PHEs were all able to form complexes with plasmid DNA (pDNA) at various mass ratios. The cytotoxicity and transfection efficiencies of these polymers were evaluated in SMMC-7721 and COS-7 cell lines. The PHE modifications affected the cell transfection efficiency significantly. The HPAMAM-PHE60 was the most efficient, with transfection activities consistently higher than the commercial transfection reagent PEI. Our study demonstrated that HPAMAM-PHEs may be good new materials for gene delivery and other applications because of its large-scale availability, economical cost, and low toxicity.

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-PEG2000-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.After fluorescein labeled PH1 liposome binding, the resulted H1299 and SPCA-1 cells associated fluorescences were much higher than those after PH2 liposome binding or mPEG liposome binding.

The synthesis of stable, low toxic, multifunctional, and water-soluble quantum dots (QDs) is of crucial importance for nanobiotechnology. An in situ anionic ring-opening polymerization strategy was successfully employed to grow multihydroxyl hyperbranched polyglycerol (HPG) from surfaces of aqueous synthesized QDs directly, affording multifunctional CdTe@HPG nanohybrids. The grafted HPG content can be adjusted from about 25 to 80 wt % by manipulating the feed ratio of glycidol monomer to QDs. The resultant CdTe@HPGs still show strong fluorescence and well water-solubility, and can conjugate functional biomolecules (e.g., amino acids) with their multiple reactive hydroxyls. Cytotoxicity measurements reveal that the CdTe@HPGs are much less toxic than the pristine QDs in human lung cancer cells SPCAI and more grafted HPG leads to less toxicity, due to the envelope of biocompatible HPG on QDs. It was found that the pristine QDs were unstable and their fluorescence decreased greatly or was even completed quenched after 24 h in SPCAI cells, whereas the QD@HPGs still exhibited strong fluorescence. This report opens the door for using in situ controlled/living polymerization to tailor QDs with biocompatible dendritic polymers readily and casts a light for obtaining robust nontoxic functionalized QDs and applying them in vitro and in vivo.

Cationic polymers including polylysine (PLL) and polyethylenimine are being widely tested as gene delivery vectors in various gene therapy applications. In many cases, the polymers were further modified by hydrophilic polymer grafting or ligand conjugation, which had been shown to greatly affect the vector stability, delivery efficiency and specificity. The characterization of modified polycation is particularly critical for quality control and vector development. Here several different separation modes using capillary electrophoresis for the analytical characterization of the modified polymers are described. PLL molecules were grafted with poly(ethylene glycol) (PEG) chain or conjugated with epidermal growth factor and analyzed under various analytical conditions. Poly(N,N′-dimethylacrylamide)-coated capillary was used to analyze the modified PLL to reduce the interaction between the samples and the capillary wall. PLLs containing different numbers of conjugated ligands were well separated with the coating method but, for PLL-g-PEG, the separation was poor under the same conditions. A method using low buffer pH and hydroxypropylmethyl cellulose additive was developed. These methods are useful to characterize various polycations and important for the quality control and application of potential gene delivery vectors.

We describe in this paper an approach to synthesize superparamagnetic iron oxide nanoparticles in the presence of polymerized lactic acid. The resulted particles consisted of clusters of iron oxide monocrystals, embedded inside the polymer chains. The composite particles synthesized in situ were highly dispersible in aqueous solution with good stability. X-ray diffraction and magnetometer data all confirmed the crystalline structure and super-paramagnetic property of the particles. They exhibited narrow size distribution with hydrodynamic diameters close to 80 nm. In addition, the particles were shown to have abundant surface carboxyl groups, which can be used to conjugate various biomolecules. Such a preparation would be especially useful for developing target specific MRI contrast agents or drug delivery vehicles.

Loperamide, an antidiarrhea drug, is a peripheral opiate agonist. Some other opiate agonists have been shown to promote cell apoptosis. In this research, we studied the apoptosis-inducing and cytotoxic activities of loperamide. MTT assay was used to determine its cytotoxicity on nine established human tumor cell lines. Cell apoptosis was detected by flow cytometry. Hypodiploid cells and cell cycles were analyzed by propidium iodide (PI) staining, while early apoptotic cells were detected by annexin V-FITC/PI staining. It was found that loperamide could inhibit the proliferation of the tested tumor cell lines. The IC50 values for SMMC7721, MCF7, SPC-A1, SKOV3-DDP, H460, HepG2, SGC7901, U2OS, and ACHN cells were 24.2 ± 2.1 μM, 23.6 ± 2.5 μM, 25.9 ± 3.1 μM, 27.1 ± 2.5 μM, 41.4 ± 2.1 μM, 23.7 ± 1.3 μM, 35.4 ± 3.5 μM, 11.8 ± 2.8 μM, and 28.5 ± 3.4 μM, respectively. Loperamide was more effective to the human osteosarcoma U2OS cells with an IC50 value of 11.8 ± 2.8 μM. Meanwhile, it could induce cell apoptosis and cause G2/M-phase cell cycle arrest. The apoptotic cells could be found when treating with loperamide for 6 h and most of them belonged to early apoptosis. In loperamide-treated cells, activation of caspase-3 was found, namely that caspase-3 was involved in the loperamide-induced apoptosis. The results of these studies indicate that loperamide is a potential antitumor agent. To our knowledge, this is the first report on antitumor activity of loperamide.Download full-size image

Fluorescence labeled small interfering RNAs (siRNAs) were loaded into lipopolyplexes modified with ApoA1 (named as rHDL) and administered by intravenous injection. The biodistribution with time of these lipopolyplexes inside the liver and among various cell types was followed using tissue sections by Confocal fluorescence microscopy. At about 0.5 h after tail vein injection at a dose of 0.408 mg/kg, very few fluorescence signals were found in the liver. But then the signals could be seen to accumulate inside hepatocytes as discrete spots and diffused signals at around 2–4 h after injection. Such a distribution and uptake pattern was significantly different from what were observed using the commercial agent Invivofectamine® 2.0 or DOTAP lipoplexes as the carriers. The differences indicated different mechanisms concerning the in vivo behavior of these carriers. The rHDL carrier system we developed was able to deliver siRNA specifically into hepatocytes while avoiding the uptake by REM cells especially the Kupffer cells. With it's low toxicity and off target effect, it may be suitable to be developed as a hepatocyte targeting delivery system for siRNA.