Y. Andrew Wang

Single Chain Epidermal Growth Factor Receptor Antibody Conjugated Nanoparticles for in vivo Tumor Targeting and Imaging

Epidermal growth factor receptor (EGFR) targeted nanoparticle are developed by conjugating a single-chain anti-EGFR antibody (ScFvEGFR) to surface functionalized quantum dots (QDs) or magnetic iron oxide (IO) nanoparticles. The results show that ScFvEGFR can be successfully conjugated to the nanoparticles, resulting in compact ScFvEGFR nanoparticles that specifically bind to and are internalized by EGFR-expressing cancer cells, thereby producing a fluorescent signal or magnetic resonance imaging (MRI) contrast. In vivo tumor targeting and uptake of the nanoparticles in human cancer cells is demonstrated after systemic delivery of ScFvEGFR-QDs or ScFvEGFR-IO nanoparticles into an orthotopic pancreatic cancer model. Therefore, ScFvEGFR nanoparticles have potential to be used as a molecular-targeted in vivo tumor imaging agent. Efficient internalization of ScFvEGFR nanoparticles into tumor cells after systemic delivery suggests that the EGFR-targeted nanoparticles can also be used for the targeted delivery of therapeutic agents.

Receptor-Targeted Nanoparticles for In vivo Imaging of Breast Cancer

Purpose: Cell-surface receptor-targeted magnetic iron oxide nanoparticles provide molecular magnetic resonance imaging contrast agents for improving specificity of the detection of human cancer. Experimental Design: The present study reports the development of a novel targeted iron oxide nanoparticle using a recombinant peptide containing the amino-terminal fragment of urokinase-type plasminogen activator (uPA) conjugated to magnetic iron oxide nanoparticles amino-terminal fragment conjugated-iron oxide (ATF-IO). This nanoparticle targets uPA receptor, which is overexpressed in breast cancer tissues. Results: ATF-IO nanoparticles are able to specifically bind to and be internalized by uPA receptor–expressing tumor cells. Systemic delivery of ATF-IO nanoparticles into mice bearing s.c. and i.p. mammary tumors leads to the accumulation of the particles in tumors, generating a strong magnetic resonance imaging contrast detectable by a clinical magnetic resonance imaging scanner at a field strength of 3 tesla. Target specificity of ATF-IO nanoparticles showed by in vivo magnetic resonance imaging is further confirmed by near-IR fluorescence imaging of the mammary tumors using near-IR dye-labeled amino-terminal fragment peptides conjugated to iron oxide nanoparticles. Furthermore, mice administered ATF-IO nanoparticles exhibit lower uptake of the particles in the liver and spleen compared with those receiving nontargeted iron oxide nanoparticles. Conclusions: Our results suggest that uPA receptor–targeted ATF-IO nanoparticles have potential as molecularly targeted, dual modality imaging agents for in vivo imaging of breast cancer.

Molecular Imaging of Pancreatic Cancer in an Animal Model Using Targeted Multifunctional Nanoparticles

BACKGROUND & AIMS Identification of a ligand/receptor system that enables functionalized nanoparticles to efficiently target pancreatic cancer holds great promise for the development of novel approaches for the detection and treatment of pancreatic cancer. Urokinase plasminogen activator receptor (uPAR), a cellular receptor that is highly expressed in pancreatic cancer and tumor stromal cells, is an excellent surface molecule for receptor-targeted imaging of pancreatic cancer using multifunctional nanoparticles. METHODS The uPAR-targeted dual-modality molecular imaging nanoparticle probe is designed and prepared by conjugating a near-infrared dye-labeled amino-terminal fragment of the receptor binding domain of urokinase plasminogen activator to the surface of functionalized magnetic iron oxide nanoparticles. RESULTS We have shown that the systemic delivery of uPAR-targeted nanoparticles leads to their selective accumulation within tumors of orthotopically xenografted human pancreatic cancer in nude mice. The uPAR-targeted nanoparticle probe binds to and is subsequently internalized by uPAR-expressing tumor cells and tumor-associated stromal cells, which facilitates the intratumoral distribution of the nanoparticles and increases the amount and retention of the nanoparticles in a tumor mass. Imaging properties of the nanoparticles enable in vivo optical and magnetic resonance imaging of uPAR-elevated pancreatic cancer lesions. CONCLUSIONS Targeting uPAR using biodegradable multifunctional nanoparticles allows for the selective delivery of the nanoparticles into primary and metastatic pancreatic cancer lesions. This novel receptor-targeted nanoparticle is a potential molecular imaging agent for the detection of pancreatic cancer.

uPAR-targeted Optical Imaging Contrasts as Theranostic Agents for Tumor Margin Detection

Complete removal of tumors by surgery is the most important prognostic factor for cancer patients with the early stage cancers. The ability to identify invasive tumor edges of the primary tumor, locally invaded small tumor lesions, and drug resistant residual tumors following neoadjuvant therapy during surgery should significantly reduce the incidence of local tumor recurrence and improve survival of cancer patients. In this study, we report that urokinase plasminogen activator (uPA) and its receptor (uPAR) are the ligand/cell surface target pair for the development of targeted optical imaging probes for enhancing imaging contrasts in the tumor border. Recombinant peptides of the amino terminal fragment (ATF) of the receptor binding domain of uPA were labeled with near infrared fluorescence (NIR) dye molecules either as peptide-imaging or peptide-conjugated nanoparticle imaging probes. Systemic delivery of the uPAR-targeted imaging probes in mice bearing orthotopic human breast or pancreatic tumor xenografts or mouse mammary tumors led to the accumulation of the probes in the tumor and stromal cells, resulting in strong signals for optical imaging of tumors and identification of tumor margins. Histological analysis showed that a high level of uPAR-targeted nanoparticles was present in the tumor edge or active tumor stroma immediately adjacent to the tumor cells. Furthermore, following targeted therapy using uPAR-targeted theranostic nanoparticles, residual tumors were detectable by optical imaging through the imaging contrasts produced by NIR-dye-labeled theranostic nanoparticles in drug resistant tumor cells. Therefore, results of our study support the potential of the development of uPAR-targeted imaging and theranostic agents for image-guided surgery.

IGF1 Receptor Targeted Theranostic Nanoparticles for Targeted and Image-Guided Therapy of Pancreatic Cancer

Overcoming resistance to chemotherapy is a major and unmet medical challenge in the treatment of pancreatic cancer. Poor drug delivery due to stromal barriers in the tumor microenvironment and aggressive tumor biology are additional impediments toward a more successful treatment of pancreatic cancer. In attempts to address these challenges, we developed IGF1 receptor (IGF1R)-directed, multifunctional theranostic nanoparticles for targeted delivery of therapeutic agents into IGF1R-expressing drug-resistant tumor cells and tumor-associated stromal cells. These nanoparticles were prepared by conjugating recombinant human IGF1 to magnetic iron oxide nanoparticles (IONPs) carrying the anthracycline doxorubicin (Dox) as the chemotherapeutic payload. Intravenously administered IGF1-IONPs exhibited excellent tumor targeting and penetration in an orthotopic patient-derived xenograft (PDX) model of pancreatic cancer featuring enriched tumor stroma and heterogeneous cancer cells. IGF1R-targeted therapy using the theranostic IGF1-IONP-Dox significantly inhibited the growth of pancreatic PDX tumors. The effects of the intratumoral nanoparticle delivery and therapeutic responses in the orthotopic pancreatic PDX tumors could be detected by magnetic resonance imaging (MRI) with IONP-induced contrasts. Histological analysis showed that IGF1R-targeted delivery of Dox significantly inhibited cell proliferation and induced apoptotic cell death of pancreatic cancer cells. Therefore, further development of IGF1R-targeted theranostic IONPs and MRI-guided cancer therapy as a precision nanomedicine may provide the basis for more effective treatment of pancreatic cancer.

Active Targeting Using HER‐2‐Affibody‐Conjugated Nanoparticles Enabled Sensitive and Specific Imaging of Orthotopic HER‐2 Positive Ovarian Tumors

Despite advances in cancer diagnosis and treatment, ovarian cancer remains one of the most fatal cancer types. The development of targeted nanoparticle imaging probes and therapeutics offers promising approaches for early detection and effective treatment of ovarian cancer. In this study, HER-2 targeted magnetic iron oxide nanoparticles (IONPs) are developed by conjugating a high affinity and small size HER-2 affibody that is labeled with a unique near infrared dye (NIR-830) to the nanoparticles. Using a clinically relevant orthotopic human ovarian tumor xenograft model, it is shown that HER-2 targeted IONPs are selectively delivered into both primary and disseminated ovarian tumors, enabling non-invasive optical and MR imaging of the tumors as small as 1 mm in the peritoneal cavity. It is determined that HER-2 targeted delivery of the IONPs is essential for specific and sensitive imaging of the HER-2 positive tumor since we are unable to detect the imaging signal in the tumors following systemic delivery of non-targeted IONPs into the mice bearing HER-2 positive SKOV3 tumors. Furthermore, imaging signals and the IONPs are not detected in HER-2 low expressing OVCAR3 tumors after systemic delivery of HER-2 targeted-IONPs. Since HER-2 is expressed in a high percentage of ovarian cancers, the HER-2 targeted dual imaging modality IONPs have potential for the development of novel targeted imaging and therapeutic nanoparticles for ovarian cancer detection, targeted drug delivery, and image-guided therapy and surgery.

T1-weighted ultrashort echo time method for positive contrast imaging of magnetic nanoparticles and cancer cells bound with the targeted nanoparticles

To obtain positive contrast based on T1 weighting from magnetic iron oxide nanoparticle (IONP) using ultrashort echo time (UTE) imaging and investigate quantitative relationship between positive contrast and the core size and concentration of IONPs.

Targeted Delivery of siRNA‐Generating DNA Nanocassettes Using Multifunctional Nanoparticles

Molecular therapy using a small interfering RNA (siRNA) has shown promise in the development of novel therapeutics. Various formulations have been used for in vivo delivery of siRNAs. However, the stability of short double-stranded RNA molecules in the blood and efficiency of siRNA delivery into target organs or tissues following systemic administration have been the major issues that limit applications of siRNA in human patients. In this study, multifunctional siRNA delivery nanoparticles are developed that combine imaging capability of nanoparticles with urokinase plasminogen activator receptor-targeted delivery of siRNA expressing DNA nanocassettes. This theranostic nanoparticle platform consists of a nanoparticle conjugated with targeting ligands and double-stranded DNA nanocassettes containing a U6 promoter and a shRNA gene for in vivo siRNA expression. Targeted delivery and gene silencing efficiency of firefly luciferase siRNA nanogenerators are demonstrated in tumor cells and in animal tumor models. Delivery of survivin siRNA expressing nanocassettes into tumor cells induces apoptotic cell death and sensitizes cells to chemotherapy drugs. The ability of expression of siRNAs from multiple nanocassettes conjugated to a single nanoparticle following receptor-mediated internalization should enhance the therapeutic effect of the siRNA-mediated cancer therapy.

Theranostic Nanoparticles Carrying Doxorubicin Attenuate Targeting Ligand Specific Antibody Responses Following Systemic Delivery

Understanding the effects of immune responses on targeted delivery of nanoparticles is important for clinical translations of new cancer imaging and therapeutic nanoparticles. In this study, we found that repeated administrations of magnetic iron oxide nanoparticles (IONPs) conjugated with mouse or human derived targeting ligands induced high levels of ligand specific antibody responses in normal and tumor bearing mice while injections of unconjugated mouse ligands were weakly immunogenic and induced a very low level of antibody response in mice. Mice that received intravenous injections of targeted and polyethylene glycol (PEG)-coated IONPs further increased the ligand specific antibody production due to differential uptake of PEG-coated nanoparticles by macrophages and dendritic cells. However, the production of ligand specific antibodies was markedly inhibited following systemic delivery of theranostic nanoparticles carrying a chemotherapy drug, doxorubicin. Targeted imaging and histological analysis revealed that lack of the ligand specific antibodies led to an increase in intratumoral delivery of targeted nanoparticles. Results of this study support the potential of further development of targeted theranostic nanoparticles for the treatment of human cancers.

Tumor Penetrating Theranostic Nanoparticles for Enhancement of Targeted and Image-guided Drug Delivery into Peritoneal Tumors following Intraperitoneal Delivery

The major obstacles in intraperitoneal (i.p.) chemotherapy of peritoneal tumors are fast absorption of drugs into the blood circulation, local and systemic toxicities, inadequate drug penetration into large tumors, and drug resistance. Targeted theranostic nanoparticles offer an opportunity to enhance the efficacy of i.p. therapy by increasing intratumoral drug delivery to overcome resistance, mediating image-guided drug delivery, and reducing systemic toxicity. Herein we report that i.p. delivery of urokinase plasminogen activator receptor (uPAR) targeted magnetic iron oxide nanoparticles (IONPs) led to intratumoral accumulation of 17% of total injected nanoparticles in an orthotopic mouse pancreatic cancer model, which was three-fold higher compared with intravenous delivery. Targeted delivery of near infrared dye labeled IONPs into orthotopic tumors could be detected by non-invasive optical and magnetic resonance imaging. Histological analysis revealed that a high level of uPAR targeted, PEGylated IONPs efficiently penetrated into both the peripheral and central tumor areas in the primary tumor as well as peritoneal metastatic tumor. Improved theranostic IONP delivery into the tumor center was not mediated by nonspecific macrophage uptake and was independent from tumor blood vessel locations. Importantly, i.p. delivery of uPAR targeted theranostic IONPs carrying chemotherapeutics, cisplatin or doxorubicin, significantly inhibited the growth of pancreatic tumors without apparent systemic toxicity. The levels of proliferating tumor cells and tumor vessels in tumors treated with the above theranostic IONPs were also markedly decreased. The detection of strong optical signals in residual tumors following i.p. therapy suggested the feasibility of image-guided surgery to remove drug-resistant tumors. Therefore, our results support the translational development of i.p. delivery of uPAR-targeted theranostic IONPs for image-guided treatment of peritoneal tumors.