Andrew Y. Wang

Targeted magnetic iron oxide nanoparticles for tumor imaging and therapy

Magnetic iron oxide (IO) nanoparticles with a long blood retention time, biodegradability and low toxicity have emerged as one of the primary nanomaterials for biomedical applications in vitro and in vivo. IO nanoparticles have a large surface area and can be engineered to provide a large number of functional groups for cross-linking to tumor-targeting ligands such as monoclonal antibodies, peptides, or small molecules for diagnostic imaging or delivery of therapeutic agents. IO nanoparticles possess unique paramagnetic properties, which and generate significant susceptibility effects resulting in strong T2 and T2* contrast, as well as T1 effects at very low concentrations for magnetic resonance imaging (MRI), which is widely used for clinical oncology imaging. We review recent advances in the development of targeted IO nanoparticles for tumor imaging and therapy.

Development of multifunctional nanoparticles for targeted drug delivery and noninvasive imaging of therapeutic effect.

Nanotechnology is a multidisciplinary scientific field undergoing explosive development. Nanometer-sized particles offer novel structural, optical and electronic properties that are not attainable with individual molecules or bulk solids. Advances in nanomedicine can be made by engineering biodegradable nanoparticles such as magnetic iron oxide nanoparticles, polymers, dendrimers and liposomes that are capable of targeted delivery of both imaging agents and anticancer drugs. This leads toward the concept and possibility of personalized medicine for the potential of early detection of cancer lesions, determination of molecular signatures of the tumor by noninvasive imaging and, most importantly, molecular targeted cancer therapy. Increasing evidence suggests that the nanoparticles, whose surface contains a targeting molecule that binds to receptors highly expressed in tumor cells, can serve as cancer image contrast agents to increase sensitivity and specificity in tumor detection. In comparison with other small molecule contrast agents, the advantage of using nanoparticles is their large surface area and the possibility of surface modifications for further conjugation or encapsulation of large amounts of therapeutic agents. Targeted nanoparticles ferry large doses of therapeutic agents into malignant cells while sparing the normal healthy cells. Such multifunctional nanodevices hold the promise of significant improvement of current clinical management of cancer patients. This review explores the development of nanoparticles for enabling and improving the targeted delivery of therapeutic agents, the potential of nanomedicine, and the development of novel and more effective diagnostic and screening techniques to extend the limits of molecular diagnostics providing point-of-care diagnosis and more personalized medicine.

Immunochromatographic Assay for Ultrasensitive Detection of Aflatoxin B1 in Maize by Highly Luminescent Quantum Dot Beads

Highly luminescent quantum dot beads (QBs) were synthesized by encapsulating CdSe/ZnS and used for the first time as immunochromatographic assay (ICA) signal amplification probe for ultrasensitive detection of aflatoxin B1 (AFB1) in maize. The challenges to using high brightness QBs as probes for ICA are smooth flow of QBs and nonspecific binding on nitrocellulose (NC) membrane, which are overcome by unique polymer encapsulation of quantum dots (QDs) and surface blocking method. Under optimal conditions, the QB-based ICA (QB-ICA) sensor exhibited dynamic linear detection of AFB1 in maize extract from 5 to 60 pg mL–1, with a median inhibitory concentration (IC50) of 13.87 ± 0.16 pg mL–1, that is significantly (39-fold) lower than those of the QD as a signal probe (IC50 = 0.54 ± 0.06 ng mL–1). The limit of detection (LOD) for AFB1 using QB-ICA sensor was 0.42 pg mL–1 in maize extract, which is approximately 2 orders of magnitude better than those of previously reported gold nanoparticle based immunochromatographic assay (AuNP-ICA) and is even comparable with or better than the conventional enzyme-linked immunosorbent assay (ELISA) method. The performance and practicability of our QB-ICA sensor were validated with a commercial ELISA kit and further confirmed with liquid chromatography tandem mass spectrometry (LC–MS/MS). Given its efficient signal amplification performance, the proposed QB-ICA offers great potential for rapid, sensitive, and cost-effective quantitative detection of analytes in food safety monitoring.

Casein-coated Iron Oxide Nanoparticles for High MRI Contrast Enhancement and Efficient Cell Targeting

Surface properties, as well as inherent physicochemical properties, of the engineered nanomaterials play important roles in their interactions with the biological systems, which eventually affect their efficiency in diagnostic and therapeutic applications. Here we report a new class of MRI contrast agent based on milk casein protein-coated iron oxide nanoparticles (CNIOs) with a core size of 15 nm and hydrodynamic diameter ~30 nm. These CNIOs exhibited excellent water-solubility, colloidal stability, and biocompatibility. Importantly, CNIOs exhibited prominent T2 enhancing capability with a transverse relaxivity r2 of 273 mM(-1) s(-1) at 3 tesla. The transverse relaxivity is ~2.5-fold higher than that of iron oxide nanoparticles with the same core but an amphiphilic polymer coating. CNIOs showed pH-responsive properties, formed loose and soluble aggregates near the pI (pH ~4.0). The aggregates could be dissociated reversibly when the solution pH was adjusted away from the pI. The transverse relaxation property and MRI contrast enhancing effect of CNIOs remained unchanged in the pH range of 2.0-8.0. Further functionalization of CNIOs can be achieved via surface modification of the protein coating. Bioaffinitive ligands, such as a single chain fragment from the antibody of epidermal growth factor receptor (ScFvEGFR), could be readily conjugated onto the protein coating, enabling specific targeting to MDA-MB-231 breast cancer cells overexpressing EGFR. T2-weighted MRI of mice intravenously administered with CNIOs demonstrated strong contrast enhancement in the liver and spleen. These favorable properties suggest CNIOs as a class of biomarker targeted magnetic nanoparticles for MRI contrast enhancement and related biomedical applications.

Anti-HER2 antibody and ScFvEGFR-conjugated antifouling magnetic iron oxide nanoparticles for targeting and magnetic resonance imaging of breast cancer

Antifouling magnetic iron oxide nanoparticles (IONPs) coated with block copolymer poly(ethylene oxide)-block-poly(γ-methacryloxypropyltrimethoxysilane) (PEO-b-PγMPS) were investigated for improving cell targeting by reducing nonspecific uptake. Conjugation of a HER2 antibody, Herceptin®, or a single chain fragment (ScFv) of antibody against epidermal growth factor receptor (ScFvEGFR) to PEO-b-PγMPS-coated IONPs resulted in HER2-targeted or EGFR-targeted IONPs (anti-HER2-IONPs or ScFvEGFR-IONPs). The anti-HER2-IONPs bound specifically to SK-BR-3, a HER2-overexpressing breast cancer cell line, but not to MDA-MB-231, a HER2-underexpressing cell line. On the other hand, the ScFvEGFR-IONPs showed strong reactivity with MDA-MB-231, an EGFR-positive human breast cancer cell line, but not with MDA-MB-453, an EGFR-negative human breast cancer cell line. Transmission electron microscopy revealed internalization of the receptor-targeted nanoparticles by the targeted cancer cells. In addition, both antibody-conjugated and non-antibody-conjugated IONPs showed reduced nonspecific uptake by RAW264.7 mouse macrophages in vitro. The developed IONPs showed a long blood circulation time (serum half-life 11.6 hours) in mice and low accumulation in both the liver and spleen. At 24 hours after systemic administration of ScFvEGFR-IONPs into mice bearing EGFR-positive breast cancer 4T1 mouse mammary tumors, magnetic resonance imaging revealed signal reduction in the tumor as a result of the accumulation of the targeted IONPs.

Quantum dot-based, quantitative, and multiplexed assay for tissue staining

The excellent optical properties of quantum dots (QDs), such as high brightness, high photostability, continuous absorption, and narrow emission bandwidth, make them ideal as optical labels to develop QD-based immunohistofluorescence (IHF) imaging for multiplexing cancer biomarker detection on formalin-fixed and paraffin-embedded (FFPE) tissues. IHF is very important for the prediction of a patients response to cancer chemotherapy or radiotherapy. QD-based IHF faces several challenges that differ from those encountered by organic dye based IHF for clinical assays. The current work addresses some of these issues. Initially, the chemical stability of QDs and organic dyes were compared. The results showed that QDs were stable for at least 5 months on FFPE tissue, whereas organic dyes were photobleached shortly after exposure to light. Various staining methods were also studied. QD fluorescence intensity on the tissue stained with primary antibody (Ab, p16, survivin, EF1α) conjugated QDs from our company was comparable to the signal from a commercially available method in which the tissue was stained with a primary p16 Ab and a QD-labeled secondary goat anti mouse Ab respectively. Finally, the effect of the amount of Ab conjugated to QD on tissue imaging was also studied. There was no significant increase in the QD fluorescence signal on tissues when the Ab:QD ratio increased from 5 to 30. In addition, protein G was tested as an adaptor protein to link Ab to QDs for IHF staining. However, the proper blocking of the protein G on QDs was necessary to reduce crosstalk. The biomarker quantification in QD-based IHF was validated by conventional Western blot and immunohistochemistry. The results contained herein demonstrate a promising application of QDs in multiplex detection and quantification of biomarkers.

Improving sensitivity and specificity of capturing and detecting targeted cancer cells with anti-biofouling polymer coated magnetic iron oxide nanoparticles

Detecting circulating tumor cells (CTCs) with high sensitivity and specificity is critical to management of metastatic cancers. Although immuno-magnetic technology for in vitro detection of CTCs has shown promising potential for clinical applications, the biofouling effect, i.e., non-specific adhesion of biomolecules and non-cancerous cells in complex biological samples to the surface of a device/probe, can reduce the sensitivity and specificity of cell detection. Reported herein is the application of anti-biofouling polyethylene glycol-block-allyl glycidyl ether copolymer (PEG-b-AGE) coated iron oxide nanoparticles (IONPs) to improve the separation of targeted tumor cells from aqueous phase in an external magnetic field. PEG-b-AGE coated IONPs conjugated with transferrin (Tf) exhibited significant anti-biofouling properties against non-specific protein adsorption and off-target cell uptake, thus substantially enhancing the ability to target and separate transferrin receptor (TfR) over-expressed D556 medulloblastoma cells. Tf conjugated PEG-b-AGE coated IONPs exhibited a high capture rate of targeted tumor cells (D556 medulloblastoma cell) in cell media (58.7±6.4%) when separating 100 targeted tumor cells from 1×105 non-targeted cells and 41 targeted tumor cells from 100 D556 medulloblastoma cells spiked into 1mL blood. It is demonstrated that developed nanoparticle has higher efficiency in capturing targeted cells than widely used micron-sized particles (i.e., Dynabeads®).

Functionalized milk-protein-coated magnetic nanoparticles for MRI-monitored targeted therapy of pancreatic cancer

Engineered nanocarriers have emerged as a promising platform for cancer therapy. However, the therapeutic efficacy is limited by low drug loading efficiency, poor passive targeting to tumors, and severe systemic side effects. Herein, we report a new class of nanoconstructs based on milk protein (casein)-coated magnetic iron oxide (CNIO) nanoparticles for targeted and image-guided pancreatic cancer treatment. The tumor-targeting amino-terminal fragment (ATF) of urokinase plasminogen activator and the antitumor drug cisplatin (CDDP) were engineered on this nanoplatform. High drug loading (~25 wt%) and sustained release at physiological conditions were achieved through the exchange and encapsulation strategy. These ATF-CNIO-CDDP nanoparticles demonstrated actively targeted delivery of CDDP to orthotopic pancreatic tumors in mice. The effective accumulation and distribution of ATF-CNIO-CDDP was evidenced by magnetic resonance imaging, based on the T2-weighted contrast resulting from the specific accumulation of ATF-CNIO-CDDP in the tumor. Actively targeted delivery of ATF-CNIO-CDDP led to improved therapeutic efficacy in comparison with free CDDP and nontargeted CNIO-CDDP treatment. Meanwhile, less systemic side effects were observed in the nanocarrier-treated groups than that in the group treated with free CDDP. Hematoxylin and Eosin and Sirius Red staining of tumor sections revealed the possible disruption of stroma during the treatment with ATF-CNIO-CDDP. Overall, our results suggest that ATF-CNIO-CDDP can be an effective theranostic platform for active targeting-enhanced and image-guided cancer treatment while simultaneously reducing the systemic toxicity.

Abstract POSTER-THER-1436: Image-guided and targeted therapy of advanced ovarian cancer using theranostic nanoparticles

About 80% of ovarian cancer patients are diagnosed at the advanced stage due to lack of a specific symptom for early detection. The major clinical challenges in the treatment of advanced ovarian cancer are inability of complete removal of peritoneal tumor metastases by debulking surgery, and drug resistant recurrent tumors developed in the majority of the patients. To address those clinical challenges, we have developed HER2/neu targeted magnetic iron oxide nanoparticles (IONPs) that efficiently deliver therapeutic and imaging agents into HER2 expressing ovarian cancer cells. Amphiphilic polymer coated 10 nm core size IONPs, without or with PEG modification, were conjugated with near infrared dye (NIR-830) labeled HER2 affibody or HER2 affitoxin, which is a fusing protein containing a HER2 affibody for targeting and a modified PE38 toxin as a therapeutic agent. A chemotherapy drug cisplatin, commonly used for ovarian cancer therapy, was further conjugated to carboxyl groups on the polymer coating of IONP via a coordinate bond. First, we demonstrated targeted drug delivery, and specificity and sensitivity of non-invasive tumor imaging by optical and MR imaging following intravenous (i.v.) or intraperitoneal (i.p.) delivery of the targeted theranostic IONPs in an orthotopic SKOV3 human ovarian cancer xenograft model with a high level of HER2/neu expression. Significant inhibition of the growth of primary ovarian tumor, and peritoneal and lung metastases was found after i.v. or i.p. delivery of cisplatin using 0.5 to 5 mg/Kg of cisplatin equivalent doses of HER2 affibody- or HER2 affitoxin-IONP in the tumor bearing mice once every 5 days for five injections. Furthermore, i.p. delivery of HER2 targeted theranostic IONP-cisplatin showed significantly better responses compared with i.v. delivery of the same nanoparticles. HER2 affitoxin-IONP-cisplatin treated mouse group showed enhanced anti-tumor effect compared to HER affibody-IONP-cisplatin treated mouse group. For both delivery approaches, we detected stronger tumor growth inhibitory effect on peritoneal metastatic tumors compared to the response in the primary ovarian tumors. Interestingly, we observed marked differences in the response to HER2 targeted therapy in different tumor bearing mice in the same group, and in different tumors in the same mouse. We also found that non-invasive optical imaging and MRI can be used to monitor IONP-drug delivery into tumors and to detect good responder or poor responder tumors during the treatment. Therefore, the HER2 targeted and image-guided treatment approach using theranostic IONPs has the potential for further development of a new biomarker targeted therapy for significantly improving survival of the ovarian cancer patients Citation Format: Lily Yang, Minati Satpathy, Liya Wang, Rafal Zielinski, Weiping Qian, Malgorzata Lipowska, Jacek Capala, Andrew Wang, Hui Mao. Image-guided and targeted therapy of advanced ovarian cancer using theranostic nanoparticles [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1436.