Victor C. Yang

The artificial peroxidase activity of magnetic iron oxide nanoparticles and its application to glucose detection

Aside from their superparamagnetic properties exploited in clinical magnetic resonance imaging (MRI), it was recently discovered that magnetic, iron oxide nanoparticles could function as an artificial, inorganic peroxidase. In this paper, we studied the impact of coating on the peroxidase activity of these nanoparticles. Nanoparticles with six different coating structures were synthesized and characterized by FTIR, TGA, TEM, size, zeta potential, and SQUID; and evaluated for peroxidase activity. Catalysis was found to follow Michaelis-Menten kinetics and peroxidase activity varied with respect to electrostatic affinity between nanoparticles and substrates, evidenced by differences in determined kinetic parameters. Glucose detection was selected as a model system because glucose could be indirectly measured from the release of hydrogen peroxide after its oxidation. Nanoparticles with high peroxidase activity exhibited higher sensitivity toward glucose, showing a larger linear slope when compared with those of low activity. A significantly improved linear correlation and detection limit of measured glucose could be readily obtained by manipulating the nanoparticle coating. Our findings suggest that iron oxide nanoparticles can be tailor-made to possess improved peroxidase-like activity. Such enhancements could further widen nanoparticle scope in glucose detection and extend its peroxidase functionality to other biomedical applications.

Magnetic brain tumor targeting and biodistribution of long-circulating PEG-modified, cross-linked starch coated iron oxide nanoparticles

Magnetic iron oxide nanoparticles (MNPs) have been studied to circumvent the limitations of status-quo brain tumor therapy and can be targeted by applying an external magnetic field to lesions. To address the pharmacokinetic shortcomings of MNPs that can limit targeting efficiency, we recently reported a long-circulating polyethylene glycol modified, cross-linked starch MNP (PEG-MNP) suitable for magnetic targeting. Using a rat model, this work explores the biodistribution patterns of PEG-MNPs in organs of elimination (liver, spleen, lung, and kidney) and shows proof-of-concept that enhanced magnetic brain tumor targeting can be achieved due to the relatively long circulation lifetime of the nanoparticles. Reductions in liver (∼12-fold) and spleen (∼2.5-fold) PEG-MNP concentrations at 1h compared to parent starch-coated MNPs (D) confirm plasma pharmacokinetics observed previously. While liver concentrations of PEG-MNPs remained considerably lower than those observed for D at 1h through 60 h, spleen values continue to increase and are markedly higher at later time points--a trend also observed with histology. Limited to no distribution of PEG-MNPs was visualized in lung or kidney throughout the 60 h course evaluated. Enhanced, selective magnetic brain tumor targeting (t = 1 h) of PEG-MNPs (12 mg Fe/kg) was confirmed in 9L-glioma tumors, with up to 1.0% injected dose/g tissue nanoparticle delivery achieved--a 15-fold improvement over targeted D (0.07% injected dose/g tissue). MRI and histological analyses visually confirmed enhanced targeting and also suggest a limited contribution of passive mechanisms to tissue retention of nanoparticles. Our results are exciting and justify both further development of PEG-MNP as a drug delivery platform and concurrent optimization of the magnetic brain tumor targeting strategy utilized.

L-Asparaginase encapsulated intact erythrocytes for treatment of acute lymphoblastic leukemia (ALL)

As a primary drug for the treatment of acute lymphoblastic leukemia (ALL), encapsulation of L-asparaginase (ASNase) into red blood cells (RBC) has been popular to circumvent immunogenicity from the exogenous protein. Unlike existing methods that perturbs RBC membranes, we introduce a novel method of RBC-incorporation of proteins using the membrane-translocating low molecular weight protamine (LMWP). Confocal study of fluorescence-labeled LMWP-ovalbumin, as a model protein conjugate, has shown significant fluorescence inside RBCs. Surface morphology by scanning electron microscopy of the RBCs loaded with LMWP-ASNase was indistinguishable with normal RBCs. These drug loaded RBCs also closely resembled the profile of the native erythrocytes in terms of osmotic fragility, oxygen dissociation and hematological parameters. The in vivo half-life of enzyme activity after administering 8 units of RBC/LMWP-ASNase in DBA/2 mice was prolonged to 4.5+/-0.5 days whereas that of RBCs loaded with ASNase via a hypotonic method was 2.4+/-0.7 days. Furthermore, the mean survival time of DBA/2 mice bearing mouse lymphoma cell L5178Y was improved by approximately 44% compared to the saline control group after treatment with the RBC loaded enzymes. From these data, an innovative, novel method for encapsulating proteins into intact and fully functional erythrocytes was established for potential treatment of ALL.

Substantiating In Vivo Magnetic Brain Tumor Targeting of Cationic Iron Oxide Nanocarriers via Adsorptive Surface Masking

Cationic magnetic nanoparticles are attractive as potential vehicles for tumor drug delivery due to their favorable interactions with both the tumor milieu and the therapeutic cargo. However, systemic delivery of these nanoparticles to the tumor site is compromised by their rapid plasma clearance. We developed a simple method for in vivo protection of cationic nanocarriers, using non-covalent surface masking with a conjugate of low molecular weight heparin and polyethylene glycol. Surface masking resulted in a 11-fold increase in plasma AUC and a 2-fold increase in the magnetic capture of systemically injected nanoparticles in orthotopic rodent brain tumors. Overall, the described methodology could expand the prospective applications for cationic magnetic nanoparticles in magnetically mediated gene/drug delivery.

High antiangiogenic and low anticoagulant efficacy of orally active low molecular weight heparin derivatives

Heparin, an anticoagulant that is widely used clinically, is also known to bind to several kinds of proteins through electrostatic interactions because of its polyanionic character. These interactions are mediated by the physicochemical properties of heparin such as sequence composition, sulfation patterns, charge distribution, overall charge density, and molecular size. Although this electrostatic character mediates its binding to many proteins related with tumor progression, thereby providing its antiangiogenic property, the administration of heparin for treating cancer is limited in clinical applications due to several drawbacks, such as its low oral absorption, unsatisfactory therapeutic effects, and strong anticoagulant activity which induces hemorrhaging. Here, we evaluated novel, orally active, low molecular weight heparin (LMWH) derivatives (LHD) conjugated with deoxycholic acid (DOCA) that show reduced anticoagulant activity and enhanced antiangiogenic activity. The chemical conjugate of LMWH and DOCA was synthesized by conjugating the amine group of N-deoxycholylethylamine (EtDOCA) with the carboxylic groups of heparin at various DOCA conjugation ratios. The LMWH-DOCA conjugate series (LHD1, LHD1.5, LHD2, and LHD4) were further formulated with poloxamer 407 as a solubilizer for oral administration. An in vitro endothelial tubular formation and in vivo Matrigel plug assay were performed to verify the antiangiogenic potential of LHD. Finally, we evaluated tumor growth inhibition of oral LHD administration in a SCC7 model as well as in A549 human cancer cell lines in a mouse xenograft model. Increasing DOCA conjugation ratios showed decreased anticoagulant activity, eventually to zero. LHD could block angiogenesis in the tubular formation assay and the Matrigel plug assay. In particular, oral administration of LHD4, which has 4 molecules of DOCA per mole of LMWH, inhibited tumor growth in SCC7 mice model as well as A549 mice xenograft model. LHD4 was orally absorbable, showed minimal anticoagulant activity and inhibits tumor growth via antiangiogenesis. These findings demonstrate the therapeutic potential of LHD4 as a new oral anti-cancer drug.

Efficient labeling of mesenchymal stem cells using cell permeable magnetic nanoparticles.

For the purpose of successfully monitoring labeled cells, optimum labeling efficiency without any side effect is a prerequisite. Magnetic cellular imaging is a new and growing field that allows the visualization of implanted cells in vivo. Herein, superparamagnetic iron oxide (SPIO) nanoparticles were conjugated with a non-toxic protein transduction domain (PTD), identified by the authors and termed low molecular weight protamine (LMWP), to generate efficient and non-toxic cell labeling tools. The cells labeled with LMWP-SPIO presented the highest iron content compared to those labeled with naked SPIO and the complex of SPIO with poly-L-lysine, which is currently used as a transfection agent. In addition to the iron content assay, Prussian staining and confocal observation demonstrated the highest intracellular LMWP-SPIO presence, and the labeling procedure did not alter the cell differentiation capacity of mesenchymal stem cells. Taken together, cell permeable magnetic nanoparticles conjugated with LMWP can be suggested as labeling tools for efficient magnetic imaging of transplanted cells.

Magnetically-enabled and MR-monitored selective brain tumor protein delivery in rats via magnetic nanocarriers

The delivery of bioactive proteins to tumors is associated with many difficulties that have impeded clinical translation of these promising therapeutics. Herein we present an approach, including (1) use of magnetically-responsive and MRI-visible nanoparticles as drug carriers, (2) topography-optimized intra-arterial magnetic targeting, (3) MRI-guided subject alignment within the magnetic field, and (4) surface modification of the protein drug with membrane-permeable polyethyleneimine (PEI), to prevail over the obstacles in protein delivery. Applying these methodologies, we demonstrated the delivery of a significant quantity of β-galactosidase selectively into brain tumors of glioma-bearing rats, while limiting the exposure of normal brain regions. Clinical viability of the technologies utilized, and the ability to deliver proteins at high nanomolar-range tumor concentrations, sufficient to completely eradicate a tumor lesion with existing picomolar-potency protein toxins, renders the prospect of enabling protein-based cancer therapy extremely promising.

PEGylation of bacterial cocaine esterase for protection against protease digestion and immunogenicity.

Enhancing cocaine metabolism by administration of cocaine esterase (CocE) has been considered as a promising treatment strategy for cocaine overdose and addiction, as CocE is the most efficient native enzyme yet identified for metabolizing the naturally occurring cocaine. A major obstacle to the clinical application of CocE, however, lies in its thermo-instability, rapid degradation by circulating proteases, and potential immunogenicity. PEGylation, namely by modifying a protein or peptide compound via attachment of polyethylene glycol (PEG) chains, has been proven to overcome such problems and was therefore exploited in this CocE investigation. The PEG-CocE conjugates prepared in this study showed a purity of greater than 93.5%. Attachment of PEG to CocE apparently inhibited the binding of anti-CocE antibodies to the conjugate, as demonstrated by the enzyme-linked immunosorbent assay (ELISA) assay. In addition, PEGylation yielded protection to CocE against thermal degradation and protease digestion. Furthermore, preliminary in vivo results suggested that, similarly to native CocE, the PEG-CocE conjugates were able to protect animals from cocaine-induced toxic effects. Overall, this study provides evidence that the PEGylation may serve as a tool to prolong CocE functionality in the circulation and reduce its potential immunogenicity.

Development and validation of an enzyme linked immunosorbent assay for the quantification of carcinoembryonic antigen in mouse plasma.

Abstract Carcinoembryonic antigen (CEA) is a tumor associated antigen that is over-expressed in colorectal cancer and several other cancers of the gastrointestinal system. An enzyme linked immunosorbent assay was developed to determine CEA concentrations in mouse plasma. The assay was validated over the standard curve range of 1–20 ng/mL. The intra-assay recoveries ranged from 93–104% with associated percent coefficients of variation (CV%) ranging between 2.5–12.8%. The inter-assay recoveries were in the range of 98.4–105% and their CV% values were between 4.77–10.1%. The assay was used to detect the presence of circulating CEA in the LS174T adenocarcinoma xenograft model and to study the pharmacokinetics of recombinant CEA in athymic mice.

Magnetically targeted nanoparticles for brain tumor therapy: what does the future hold?

CNS cancers account for approximately 142,000 deaths annually – with brain tumor patients typically surviving up to just 12 months after diagnosis [1]. Today’s ‘standard of care’ therapy for brain tumors includes surgical tumor resection coupled with adjuvant radiation and/ or small-molecule chemotherapy treatments. Despite advancements in each of these intervention routes, overall patient survival rates have generally remained unchanged for decades. The limitations of status quo treatments can generally be attributed to the challenge of providing remissive therapy and concurrently avoiding off-target damage to healthy tissues. Clearly, the development of new technologies is required to realize a cure for brain tumors. To that end, many ongoing investigations have been focused toward achieving more selective, more therapeutic tumor drug action.