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1.
Nat Med ; 13(4): 504-9, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17351626

ABSTRACT

We describe a new generation of protein-targeted contrast agents for multimodal imaging of the cell-surface receptors for vascular endothelial growth factor (VEGF). These receptors have a key role in angiogenesis and are important targets for drug development. Our probes are based on a single-chain recombinant VEGF expressed with a cysteine-containing tag that allows site-specific labeling with contrast agents for near-infrared fluorescence imaging, single-photon emission computed tomography or positron emission tomography. These probes retain VEGF activities in vitro and undergo selective and highly specific focal uptake into the vasculature of tumors and surrounding host tissue in vivo. The fluorescence contrast agent shows long-term persistence and co-localizes with endothelial cell markers, indicating that internalization is mediated by the receptors. We expect that multimodal imaging of VEGF receptors with these probes will be useful for clinical diagnosis and therapeutic monitoring, and will help to accelerate the development of new angiogenesis-directed drugs and treatments.


Subject(s)
Contrast Media , Diagnostic Imaging , Neovascularization, Physiologic/physiology , Receptors, Vascular Endothelial Growth Factor/metabolism , Recombinant Proteins/metabolism , Animals , Cell Line, Tumor , Mice , Mice, Inbred BALB C , Mice, SCID , Microscopy, Confocal , Microscopy, Fluorescence/methods , Positron-Emission Tomography/methods , Tomography, Emission-Computed, Single-Photon/methods
2.
Antimicrob Agents Chemother ; 51(1): 245-51, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17074791

ABSTRACT

In the course of Bacillus anthracis infection, B. anthracis lethal factor (LF) and edema factor bind to a protective antigen (PA) associated with cellular receptors ANTXR1 (TEM8) or ANTXR2 (CMG2), followed by internalization of the complex via receptor-mediated endocytosis. A new group of potential antianthrax drugs, beta-cyclodextrins, has recently been described. A member of this group, per-6-(3-aminopropylthio)-beta-cyclodextrin (AmPrbetaCD), was shown to inhibit the toxicity of LF in vitro and in vivo. In order to determine which steps in lethal factor trafficking are inhibited by AmPrbetaCD, we developed two targeted fluorescent tracers based on LFn, a catalytically inactive fragment of LF: (i) LFn site specifically labeled with the fluorescent dye AlexaFluor-594 (LFn-Al), and (ii) LFn-decorated liposomes loaded with the fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid (LFn-Lip). Both tracers retained high affinity to PA/ANTXR complexes and were readily internalized via receptor-mediated endocytosis. Using fluorescent microscopy, we found that AmPrbetaCD inhibits receptor-mediated cell uptake but not the binding of LFn-Al to PA/ANTXR complexes, suggesting that AmPrbetaCD works outside the cell. Moreover, AmPrbetaCD and LFn-Al synergistically protect RAW 264.7 cells from PA-mediated LF toxicity, confirming that AmPrbetaCD did not affect the binding of LFn-Al to receptor-associated PA. In contrast, AmPrbetaCD did not inhibit PA-mediated internalization of LFn-Lip, suggesting that multiplexing of LFn on the liposomal surface overcomes the inhibiting effects of AmPrbetaCD. Notably, internalized LFn-Al and LFn-Lip protected cells that overexpressed anthrax receptor TEM8 from PA-induced, LF-independent toxicity, suggesting an independent mechanism for PA inhibition inside the cell. These data suggest the potential for the use of beta-cyclodextrins in combination with LFn-Lip loaded with antianthrax drugs against intracellular targets.


Subject(s)
Antigens, Bacterial/metabolism , Bacillus anthracis/drug effects , Monocytes/drug effects , Peptide Fragments/pharmacology , Animals , Bacillus anthracis/growth & development , Bacillus anthracis/immunology , Bacterial Toxins/metabolism , CHO Cells , Cell Line , Cricetinae , Cricetulus , Dose-Response Relationship, Drug , Endocytosis/drug effects , Luminescent Proteins/chemical synthesis , Luminescent Proteins/chemistry , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Monocytes/cytology , Monocytes/microbiology , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Binding/drug effects , beta-Cyclodextrins/chemistry , beta-Cyclodextrins/metabolism , beta-Cyclodextrins/pharmacology
3.
Bioconjug Chem ; 17(5): 1141-50, 2006.
Article in English | MEDLINE | ID: mdl-16984121

ABSTRACT

Liposomes have been a main focus of tumor-selective boron delivery strategies in boron neutron capture therapy (BNCT), a binary method for the treatment of cancer that is based on the nuclear reaction between boron atoms and low-energy thermal neutrons. Three novel carboranyl cholesterol derivatives were prepared as lipid bilayer components for the construction of nontargeted and receptor-targeted boronated liposomes for BNCT. A major structural feature of these novel boronated cholesterol mimics is the replacement of the B and the C ring of cholesterol with a carborane cluster. Computational analyses indicated that all three boronated compounds have structural features and physicochemical properties that are very similar to those of cholesterol. One of the synthesized boronated cholesterol mimics was stably incorporated into non-, folate receptor (FR)-, and vascular endothelial growth factor receptor-2 (VEGFR-2)-targeted liposomes. No major differences were found in appearance, size distribution, and lamellarity between conventional dipalmitoylphosphatidylcholine (DPPC)/cholesterol liposomes, nontargeted, and FR-targeted liposomal formulations of this carboranyl cholesterol derivative. FR-targeted boronated liposomes were taken up extensively in FR overexpressing KB cells in vitro, and the uptake was effectively blocked in the presence of free folate. In contrast, a boronated cholesterol mimic incorporated into nontargeted liposomes showed significantly lower cellular uptake. There was no apparent in vitro cytotoxicity in FR overexpressing KB cells and VEGFR-2 overexpressing 293/KDR cells when these were incubated with boronated FR- and (VEGFR-2)-targeted liposomes, respectively, although the former accumulated extensively in KB cells and the latter effectively interacted with VEGFR-2 by causing autophosphorylation and protecting 293/KDR cells from SLT (Shiga-like toxin)-VEGF cytotoxicity.


Subject(s)
Boron Compounds/metabolism , Boron Neutron Capture Therapy/methods , Cholesterol , Liposomes/metabolism , Boron Compounds/chemistry , Cell Line , Cholesterol/chemistry , Cholesterol/metabolism , Humans , Liposomes/chemistry , Models, Molecular , Molecular Structure , Neoplasms/therapy , Particle Size , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolism
4.
Bioconjug Chem ; 17(4): 912-9, 2006.
Article in English | MEDLINE | ID: mdl-16848397

ABSTRACT

Random conjugation of therapeutic or diagnostic payloads to targeting proteins generates functionally heterogeneous products. Conjugation of payloads to an adapter that binds to a peptide tag engineered into a targeting protein provides an alternative strategy. To progress into clinical development, an adapter/docking tag system should include humanized components and be stable in circulation. We describe here an adapter/docking tag system based on mutated fragments of human RNase I that spontaneously bind to each other and form a conjugate with a disulfide bond between complimentary cysteine residues. This self-assembled "dock and lock" system utilizes the previously described fusion C-tag, a 1-15 aa fragment of human RNase I with the R4C amino acid substitution, and a newly engineered adapter protein (Ad-C), a 21-127-aa fragment of human RNase I with the V118C substitution. Two vastly different C-tagged recombinant proteins, human vascular endothelial growth factor (VEGF) and a 254-aa long N-terminal fragment of anthrax lethal factor (LFn), retain functional activities after spontaneous conjugation of Ad-C to N-terminal or C-terminal C-tag, respectively. Ad-C modified with pegylated phospolipid and inserted into the lipid membrane of drug-loaded liposomes (Doxil) retained the ability to conjugate C-tagged proteins, yielding targeted liposomes decorated with functionally active proteins. To further optimize the system, we engineered an adapter with an additional cysteine residue at position 88 for site-specific modification, conjugated it to C-tagged VEGF, and labeled with a near-infrared fluorescent dye Cy5.5, yielding a unique functionally active probe for in vivo molecular imaging. We expect that this self-assembled "dock and lock" system will provide new opportunities for using functionally active proteins for biomedical purposes.


Subject(s)
Proteins/chemistry , Animals , Base Sequence , Cloning, Molecular , DNA Primers , Mice , Mice, Inbred BALB C , Mutagenesis, Site-Directed
5.
Biomaterials ; 27(31): 5452-8, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16843524

ABSTRACT

Developing tissue engineering scaffolds with immobilized growth factors requires facile and reliable methods for the covalent attachment of functionally active proteins. We describe here a new approach to immobilize recombinant proteins based on expression of the protein of interest with a 15-aa long fusion tag (Cys-tag), which avails a free sulfhydryl group for site-specific conjugation. To validate this approach, we conjugated a single-chain vascular endothelial growth factor expressed with an N-terminal Cys-tag (scVEGF) to fibronectin (FN) using a common thiol-directed bi-functional cross-linking agent. We found that the FN-scVEGF conjugate retains VEGF activity similar to that of free scVEGF when used as a soluble ligand. Cells expressing VEGF receptor VEGFR-2 grown on plates coated with FN-scVEGF displayed morphological phenotypes similar to those observed for cells grown on FN in the presence of equivalent amounts of free scVEGF. In addition, 293/KDR cell growth stimulation was observed in the same concentration range with either immobilized or free scVEGF. The effects of immobilized scVEGF, and soluble scVEGF were blocked by NVP-AAD777-NX, a VEGF receptor tyrosine kinase inhibitor. These data indicate that site-specific immobilization via Cys-tag provides a facile and reliable method for permanent deposition of functionally active growth factors on synthetic or protein scaffolds with applications for advanced tissue engineering.


Subject(s)
Coated Materials, Biocompatible/chemistry , Crystallization/methods , Cysteine/chemistry , Kidney/cytology , Kidney/metabolism , Tissue Engineering/methods , Vascular Endothelial Growth Factor A/chemistry , Binding Sites , Cell Adhesion , Cell Line , Cell Proliferation , Cross-Linking Reagents/chemistry , Humans , Protein Binding , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolism
6.
Bioconjug Chem ; 15(5): 1021-9, 2004.
Article in English | MEDLINE | ID: mdl-15366955

ABSTRACT

High-affinity interactions of two fragments of human RNase I (1-15-aa Hu-tag and 21-125-aa HuS adapter protein) can be used for assembly of targeting drug delivery complexes. In this approach, a targeting protein is expressed as a fusion protein with a 15-aa Hu-tag, while HuS is conjugated to a drug (or a drug carrier) creating a "payload" module, which is then bound noncovalently to the Hu-tag of the targeting protein. Although this approach eliminates chemical modifications of targeting proteins, the payload modules are still constructed by random cross-linking of drugs or drug carriers to an adapter protein that might lead to functional heterogeneity of the complexes. To avoid this problem, we engineered an adapter protein HuS(N88C) with an unpaired cysteine in position 88 that can be directly modified without interference with activity of assembled targeting complexes. HuS(N88C) binds Hu-tagged annexin V with K(D) of 50 +/- 6 nM, which is comparable to that of wild-type HuS. To demonstrate the utility of HuS(N88C) for developing uniform payload modules, we constructed a HuS(N88C)-lipid conjugate and inserted it into preformed liposomes loaded with a fluorescent dye. Targeting proteins, Hu-tagged vascular endothelial growth factor or Hu-tagged annexin V, were docked to liposomes decorated with HuS, and the assembled complexes delivered liposomes selectively to target cells.


Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Drug Carriers/chemistry , Drug Carriers/metabolism , Drug Delivery Systems/methods , Ribonuclease, Pancreatic/chemistry , Ribonuclease, Pancreatic/metabolism , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Sequence , Binding Sites/genetics , Cell Line , Cell Line, Transformed , Humans , Liposomes , Molecular Sequence Data , Mutagenesis, Site-Directed , Ribonuclease, Pancreatic/genetics
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