Protected Graft Copolymer (PGC) in Imaging and Therapy: A Platform for the Delivery of Covalently and Non-Covalently Bound Drugs

Initially developed in 1992 as an MR imaging agent, the family of protected graft copolymers (PGC) is based on a conjugate of polylysine backbone to which methoxypoly(ethylene glycol) (MPEG) chains are covalently linked in a random fasion via N-ε-amino groups. While PGC is relatively simple in terms of its chemcial composition and structure, it has proved to be a versatile platform for in vivo drug delivery. The advantages of poly amino acid backbone grafting include multiple available linking sites for drug and adaptor molecules. The grafting of PEG chains to PGC does not compromise biodegradability and does not result in measurable toxicity or immunogenicity. In fact, the biocompatablility of PGC has resulted in its being one of the few 100% synthetic non-proteinaceous macromolecules that has suceeded in passing the initial safety phase of clinical trials. PGC is capable of long circulation times after injection into the blood stream and as such found use early on as a carrier system for delivery of paramagnetic imaging compounds for angiography. Other PGC types were later developed for use in nuclear medicine and optical imaging applications in vivo. Recent developments in PGC-based drug carrier formulations include the use of zinc as a bridge between the PGC carrier and zinc-binding proteins and re-engineering of the PGC carrier as a covalent amphiphile that is capabe of binding to hydrophobic residues of small proteins and peptides. At present, PGC-based formulations have been developed and tested in various disease models for: 1) MR imaging local blood circulation in stroke, cancer and diabetes; 2) MR and nuclear imaging of blood volume and vascular permeability in inflammation; 3) optical imaging of proteolytic activity in cancer and inflammation; 4) delivery of platinum(II) compounds for treating cancer; 5) delivery of small proteins and peptides for treating diabetes, obesity and myocardial infarction. This review summarizes the experience accumulated by various research groups that chose to use PGC as a drug delivery platform

Dual radiosensitization and anti-STAT3 anti-proliferative strategy based on delivery of gold nanoparticle - oligonucleotide nanoconstructs to head and neck cancer cells

Constitutively activated signal transducer and activator of transcription 3 (STAT3) factor is an important therapeutic target in head and neck cancer (HNC). Despite early promising results, a reliable systemic delivery system for STAT3- targeted oligonucleotide (ODN) drugs is still needed for future clinical translation of anti-STAT3 therapies. We engineered and tested a novel ODN duplex/gold nanoparticle (AuNP)-based system carrying a therapeutic STAT3 decoy (STAT3d) payload. This strategy is two-pronged because of the additive STAT3 antagonism and radiosensitizing properties of AuNP. The specificity to head and neck cancer cell surface was imparted by using a nucleolin aptamer (NUAP) that was linked to AuNP for taking the advantage of an aberrant presentation of a nuclear protein nucleolin on the cell surface. STAT3d and nucleolin aptamer constructs were independently linked to AuNPs via Au-S bonds. The synthesized AuNP constructs (AuNP-NUAP-STAT3d) exhibited internalization in cells that was quantified by using radiolabeled STAT3d. AuNP-NUAP-STAT3d showed radiosensitizing effect in human HNC FaDu cell culture experiments that resulted in an increase of cell DNA damage as determined by measuring gamma-H2AX phosphorylation levels by flow cytometry. The radiosensitization study also demonstrated that AuNP-NUAP-STAT3d as well as STAT3d alone resulted in the efficient inhibition of A431 cell proliferation. While FaDu cells did not show instant proliferation inhibition after incubating with AuNP-NUAP-STAT3d, the cell DNA damage in these cells showed nearly a 50% increase in AuNP-NUAP-STAT3d group after treating with radiation. Compared with anti-EGFR humanized antibody (Cetuximab), AuNP-NUAP-STAT3d system had an overall stronger radiosensitization effect in both A431 and FaDu cells

Protected Graft Copolymer (PGC) in Imaging and Therapy: A Platform for the Delivery of Covalently and Non-Covalently Bound Drugs

Initially developed in 1992 as an MR imaging agent, the family of protected graft copolymers (PGC) is based on a conjugate of polylysine backbone to which methoxypoly(ethylene glycol) (MPEG) chains are covalently linked in a random fasion via N-epsilon-amino groups. While PGC is relatively simple in terms of its chemcial composition and structure, it has proved to be a versatile platform for in vivo drug delivery. The advantages of poly amino acid backbone grafting include multiple available linking sites for drug and adaptor molecules. The grafting of PEG chains to PGC does not compromise biodegradability and does not result in measurable toxicity or immunogenicity. In fact, the biocompatablility of PGC has resulted in its being one of the few 100% synthetic non-proteinaceous macromolecules that has suceeded in passing the initial safety phase of clinical trials. PGC is capable of long circulation times after injection into the blood stream and as such found use early on as a carrier system for delivery of paramagnetic imaging compounds for angiography. Other PGC types were later developed for use in nuclear medicine and optical imaging applications in vivo. Recent developments in PGC-based drug carrier formulations include the use of zinc as a bridge between the PGC carrier and zinc-binding proteins and re-engineering of the PGC carrier as a covalent amphiphile that is capabe of binding to hydrophobic residues of small proteins and peptides. At present, PGC-based formulations have been developed and tested in various disease models for: 1) MR imaging local blood circulation in stroke, cancer and diabetes; 2) MR and nuclear imaging of blood volume and vascular permeability in inflammation; 3) optical imaging of proteolytic activity in cancer and inflammation; 4) delivery of platinum(II) compounds for treating cancer; 5) delivery of small proteins and peptides for treating diabetes, obesity and myocardial infarction. This review summarizes the experience accumulated by various research groups that chose to use PGC as a drug delivery platform

Gold nanoparticles stabilized with MPEG-grafted poly(l-lysine): in vitro and in vivo evaluation of a potential theranostic agent

As the number of diagnostic and therapeutic applications utilizing gold nanoparticles (AuNPs) increases, so does the need for AuNPs that are stable in vivo, biocompatible, and suitable for bioconjugation. We investigated a strategy for AuNP stabilization that uses methoxypolyethylene glycol-graft-poly(l-lysine) copolymer (MPEG-gPLL) bearing free amino groups as a stabilizing molecule. MPEG-gPLL injected into water solutions of HAuCl4 with or without trisodium citrate resulted in spherical (Zav = 36 nm), monodisperse (PDI = 0.27), weakly positively charged nanoparticles (AuNP3) with electron-dense cores (diameter: 10.4 ± 2.5 nm) and surface amino groups that were amenable to covalent modification. The AuNP3 were stable against aggregation in the presence of phosphate and serum proteins and remained dispersed after their uptake into endosomes. MPEG-gPLL-stabilized AuNP3 exhibited high uptake and very low toxicity in human endothelial cells, but showed a high dose-dependent toxicity in epithelioid cancer cells. Highly stable radioactive labeling of AuNP3 with (99m)Tc allowed imaging of AuNP3 biodistribution and revealed dose-dependent long circulation in the blood. The minor fraction of AuGNP3 was found in major organs and at sites of experimentally induced inflammation. Gold analysis showed evidence of a partial degradation of the MPEG-gPLL layer in AuNP3 particles accumulated in major organs. Radiofrequency-mediated heating of AuNP3 solutions showed that AuNP3 exhibited heating behavior consistent with 10 nm core nanoparticles. We conclude that PEG-pPLL coating of AuNPs confers stealth properties that enable these particles to exist in vivo in a nonaggregating, biocompatible state making them suitable for potential use in biomedical applications such as noninvasive radiofrequency cancer therapy

Detection of early antiangiogenic effects in human colon adenocarcinoma xenografts: in vivo changes of tumor blood volume in response to experimental VEGFR tyrosine kinase inhibitor

Antiangiogenesis is emerging as efficient strategy for targeting and potentially eliminating neoplastic tumor vessels. The main goal of this study was to establish whether absolute tumor blood volume (V(b)) change could be used as an early predictor of antiangiogenesis in ectopic and orthotopic colon carcinomas. To assess therapy-induced changes of V(b), we did comparative analysis of signal intensities in tumors and muscle using steady-state magnetic resonance imaging (MRI) assisted with an intravascular paramagnetic contrast agent [gadolinium-labeled protected graft copolymer (PGC-Gd)]. Athymic mice with implanted human MV522 tumors were treated with vascular endothelial growth factor type 2 receptor tyrosine kinase inhibitor (VEGFR2-TKI) that has been shown to inhibit VEGFR2 phosphorylation and tumor growth in vivo. Animals were imaged either after a single day or after a 1-week course of treatments. The measured V(b) in ectopic tumors was 2.5 +/- 1.5% of total tissue volume 1 week after the implantation (n = 8). Two doses of VEGFR2-TKI (25 mg/kg, p.o., b.i.d.) resulted in a decrease of V(b) to 1.3 +/- 0.3%. In orthotopic tumors, the measured V(b) was initially higher (11.9 +/- 2.0%); however, VEGFR2-TKI treatment also resulted in a statistically significant decrease of V(b). The absolute V(b) was not affected in the muscle as a result of treatments. MRI measurements were corroborated by using isotope and correlative histology experiments. Our results show that steady-state MRI is highly sensitive to early antiangiogenic effects caused by small molecule drugs

High-resolution Imaging of Myeloperoxidase Activity Sensors in Human Cerebrovascular Disease

Progress in clinical development of magnetic resonance imaging (MRI) substrate-sensors of enzymatic activity has been slow partly due to the lack of human efficacy data. We report here a strategy that may serve as a shortcut from bench to bedside. We tested ultra high-resolution 7T MRI (microMRI) of human surgical histology sections in a 3-year IRB approved, HIPAA compliant study of surgically clipped brain aneurysms. microMRI was used for assessing the efficacy of MRI substrate-sensors that detect myeloperoxidase activity in inflammation. The efficacy of Gd-5HT-DOTAGA, a novel myeloperoxidase (MPO) imaging agent synthesized by using a highly stable gadolinium (III) chelate was tested both in tissue-like phantoms and in human samples. After treating histology sections with paramagnetic MPO substrate-sensors we observed relaxation time shortening and MPO activity-dependent MR signal enhancement. An increase of normalized MR signal generated by ultra-short echo time MR sequences was corroborated by MPO activity visualization by using a fluorescent MPO substrate. The results of microMRI of MPO activity associated with aneurysmal pathology and immunohistochemistry demonstrated active involvement of neutrophils and neutrophil NETs as a result of pro-inflammatory signalling in the vascular wall and in the perivascular space of brain aneurysms

Plate capture assay of fluorescent oligonucleotide duplex reporter-transcription factor complexes

We previously developed prototype oligodeoxyribonucleotide (ODN) duplex fluorescence energy transfer (FRET) reporters for optical sensing of NF-kappaB transcription factor. We report here a plate-binding assay designed for optimizing the above reporters. Nitrilotriacetate-bearing plates were prepared by using sequential (1) aminosilylation; (2) carboxylation; (3) coupling of Nalpha,Nalpha-bis(carboxymethyl)-L-lysine or, alternatively, by replacing steps 1 and 2 by treating the glass with 3-(triethoxysilyl)propylsuccinic anhydride. FRET reporters were obtained by covalent linking of Cy5.5 (fluorescence donor) and IRdye800CW (fluorescence acceptor) to complementary ODN strands encoding a high-affinity p50 binding site. Recombinant 6 x His tagged NF-kappaB p50 was used for immobilizing the protein on glass plates via linked NTA-Ni(II) groups. Imaging and quantification of the fluorescence intensity in the wells was performed in two channels (700 and 800 nm) using a near-infrared scanning device with microscopic resolution. The fluorescence intensity of the ODN duplex reporter was detectible in the plates at the concentration of 5 pM. NF-kappaB p50-ODN reporter interaction was studied by measuring the ratio of 700 nm (donor) to 800 nm (acceptor) fluorescence intensities. Using the plate assay, we were able to measure p50-mediated interference with FRET at low density of plate binding

Molecular magnetic resonance contrast agents for the detection of cancer: past and present

Magnetic resonance imaging (MRI) is a powerful diagnostic tool with unsurpassed spatial resolution that is capable of providing detailed information about the structure and composition of tumors. The use of exogenously administered contrast agents allows compartment-specific enhancement of tumors, enabling imaging of functional blood and interstitial volumes. Current efforts are directed at enhancing the capabilities of MRI in oncology by adding contrast agents with molecular specificities to the growing armamentarium of diagnostic probes that produce signal by changing local proton relaxation times as a consequence of specific contrast agent binding to cell surface receptors or extracellular matrix components. We review herein the most notable examples, illustrating major trends in the development of specific probes for high-resolution imaging in molecular oncology

Fluorescent macromolecular sensors of enzymatic activity for in vivo imaging

Macromolecular imaging probes (or sensors) of enzymatic activity have a unique place in the armamentarium of modern optical imaging techniques. Such probes were initially developed by attaching optically silent fluorophores via enzyme-sensitive linkers to large copolymers of biocompatible poly(ethylene glycol) and poly(amino acids). In diseased tissue, where the concentration of enzymes is high, the fluorophores are freed from the macromolecular carrier and regain their initial ability to fluoresce, thus allowing in vivo optical localization of the diseased tissue. This chapter describes the design and application of these probes and their alternatives in various areas of experimental medicine and gives an overview of currently available techniques that allow imaging of animals using visible and near-infrared light

MR signal amplification for imaging of the mutant EGF receptor in orthotopic human glioma model

PURPOSE: To investigate the potential of targeted MR signal amplification strategy for imaging of EGF receptor variant III (EGFRvIII) overexpression associated with the infiltrating margin of aggressive orthotopic brain tumors. PROCEDURES: F(ab\u27)2 fragments of humanized anti-EGFRvIII monoclonal antibody (EMD72000) were linked to deglycosylated horseradish peroxidase (HRP) and glucose oxidase (GOX). Detection of the F(ab\u27)2 conjugate pair colocalization in vivo was enabled by a subsequent IV injection of a low molecular weight paramagnetic substrate of HRP, diTyr-GdDTPA. RESULTS: The delivery of the targeted fragments to the tumor was validated using SPECT/CT imaging of radiolabeled anti-EGFRvIII F(ab\u27)2 conjugates. Further, by using 3 T MRI, we observed time-dependent differences in tumor signal intensity and signal retention at the endpoint depending on whether or not the animals were pre-injected with the anti-EGFRvIII F(ab\u27)2 conjugates. CONCLUSIONS: Imaging of EGFRvIII expression in vivo was enabled by consecutive administration of targeted F(ab\u27)2 conjugates and a paramagnetic substrate resulting in a tumor-specific receptor detection with high specificity and resolution