Wenlian Zhu

PAMAM dendrimer‐based contrast agents for MR imaging of Her‐2/neu receptors by a three‐step pretargeting approach

Pretargeting of receptors is a useful approach in molecular imaging and therapy to reduce background noise or toxicity and enhance selectivity. In this study a three‐step pretargeting approach that includes a biotinylated antibody, avidin/streptavidin, and a biotinylated imaging agent is described. A PAMAM dendrimer generation 4 (G4D)‐based MRI T1 agent biotin‐G4D‐DTPA‐Gd (bG4D‐Gd) and its sister compound with remaining free surface amine groups blocked by succinic anhydride to reduce positive charges (bG4D‐Gd‐SA) were synthesized. Limited selective enhancement in MRI was observed in a Her‐2/neu mouse tumor xenograft by this three‐step pretargeting approach that includes biotinylated trastuzumab, avidin and bG4D‐Gd, or bG4D‐Gd‐SA. However, these dendrimer‐based MRI agents with molecular weight around 29 kD reached and remained in the tumor through the enhanced permeability and retention effect. Prolonged and extensive accumulation of both bG4D‐Gd and b‐G4‐Gd‐SA in the kidneys was also observed. Magn Reson Med, 2008.

Magnetic resonance imaging contrast of iron oxide nanoparticles developed for hyperthermia is dominated by iron content

Abstract Purpose: Magnetic iron oxide nanoparticles (MNPs) are used as contrast agents for magnetic resonance imaging (MRI) and hyperthermia for cancer treatment. The relationship between MRI signal intensity and cellular iron concentration for many new formulations, particularly MNPs having magnetic properties designed for heating in hyperthermia, is lacking. In this study, we examine the correlation between MRI T2 relaxation time and iron content in cancer cells loaded with various MNP formulations. Materials and methods: Human prostate carcinoma DU-145 cells were loaded with starch-coated bionised nanoferrite (BNF), iron oxide (Nanomag® D-SPIO), Feridex™, and dextran-coated Johns Hopkins University (JHU) particles at a target concentration of 50 pg Fe/cell using poly-D-lysine transfection reagent. T2-weighted MRI of serial dilutions of these labelled cells was performed at 9.4 T and iron content quantification was performed using inductively coupled plasma mass spectrometry (ICP-MS). Clonogenic assay was used to characterise cytotoxicity. Results: No cytotoxicity was observed at twice the target intracellular iron concentration (∼100 pg Fe/cell). ICP-MS revealed highest iron uptake efficiency with BNF and JHU particles, followed by Feridex and Nanomag-D-SPIO, respectively. Imaging data showed a linear correlation between increased intracellular iron concentration and decreased T2 times, with no apparent correlation among MNP magnetic properties. Conclusions: This study demonstrates that for the range of nanoparticle concentrations internalised by cancer cells the signal intensity of T2-weighted MRI correlates closely with absolute iron concentration associated with the cells. This correlation may benefit applications for cell-based cancer imaging and therapy including nanoparticle-mediated drug delivery and hyperthermia.

Controlled internalization of Her-2/ neu receptors by cross-linking for targeted delivery.

Receptor mediated internalization is a crucial step for targeted intracellular delivery of therapeutic and imaging agents. It was recently demonstrated that trastuzumab, an FDA approved humanized monoclonal antibody against Her-2/neu tyrosine kinase receptor, did not induce endocytosis of the internalization resistant Her-2/neu receptor. Here we report that accelerated internalization of trastuzumab can be induced by cross-linking the cell membrane bound antibody-receptor complex with an avidin/streptavidin-biotin system. We demonstrated that internalization was achieved both in vitro and in vivo in Her-2/neu expressing human breast cancer cell lines (BT-474, SK-BR-3, and AU-565) and that repetitive labeling cycles further amplified the loading of cargo molecules within the targeted cells. No trastuzumab binding and internalization was observed in Her-2/neu negative MDA-MB-231 cells, whereas weak membrane binding and negligible internalization were detected in MCF-7 cells with low expression level of Her-2/neu receptor. The method was used to noninvasively image Her-2/neu receptors in isolated cells and in a preclinical breast cancer model with MRI. The controlled internalization of Her-2/neu receptors can potentially enhance intracellular delivery of drugs and imaging probes, and improve imaging sensitivity and selectivity as well as therapeutic efficacy, through antibody-directed binding and internalization using a pretargeting approach.

Bioorthogonal, two-component delivery systems based on antibody and drug-loaded nanocarriers for enhanced internalization of nanotherapeutics

Nanocarriers play an important role in targeted cancer chemotherapy. The optimal nanocarrier delivery system should provide efficient and highly specific recognition of the target cells and rapid internalization of the therapeutic cargo to reduce systemic toxicity as well as to increase the cytotoxicity to cancer cells. To this end, we developed a two-step, two-component targeted delivery system based on antibody and drug-loaded nanocarrier that uses bioorthogonal click reactions for specific internalization of nanotherapeutics. The pretargeting component, anti-HER2 humanized monoclonal antibody, trastuzumab, functionalized with azide groups labels cancer cells that overexpress HER2 surface receptors. The drug carrier component, dibenzylcyclooctyne substituted albumin conjugated with paclitaxel, reacts specifically with the pretargeting component. These two components form cross-linked clusters on the cell surface, which facilitates the internalization of the complex. This strategy demonstrated substantial cellular internalization of clusters consisted of HER2 receptors, modified trastuzumab and paclitaxel-loaded albumin nanocarriers, and subsequent significant cytotoxicity in HER2-positive BT-474 breast cancer cells. Our results show high efficacy of this strategy for targeted nanotherapeutics. We foresee to broaden the applications of this strategy using agents such as radionuclides, toxins, and interfering RNA.

Imaging transplanted stem cells in real time using an MRI dual-contrast method

Stem cell therapies are currently being investigated for the repair of brain injuries. Although exogenous stem cell labelling with superparamagnetic iron oxide nanoparticles (SPIONs) prior to transplantation provides a means to noninvasively monitor stem cell transplantation by magnetic resonance imaging (MRI), monitoring cell death is still a challenge. Here, we investigate the feasibility of using an MRI dual-contrast technique to detect cell delivery, cell migration and cell death after stem cell transplantation. Human mesenchymal stem cells were dual labelled with SPIONs and gadolinium-based chelates (GdDTPA). The viability, proliferation rate, and differentiation potential of the labelled cells were then evaluated. The feasibility of this MRI technique to distinguish between live and dead cells was next evaluated using MRI phantoms, and in vivo using both immune-competent and immune-deficient mice, following the induction of brain injury in the mice. All results were validated with bioluminescence imaging. In live cells, a negative (T2/T2*) MRI contrast predominates, and is used to track cell delivery and cell migration. Upon cell death, a diffused positive (T1) MRI contrast is generated in the vicinity of the dead cells, and serves as an imaging marker for cell death. Ultimately, this technique could be used to manage stem cell therapies.

Heterogeneity of Tumor Vasculature and Antiangiogenic Intervention: Insights from MR Angiography and DCE-MRI

Purpose Solid tumor vasculature is highly heterogeneous, which presents challenges to antiangiogenic intervention as well as the evaluation of its therapeutic efficacy. The aim of this study is to evaluate the spatial tumor vascular changes due to bevacizumab/paclitaxel therapy using a combination approach of MR angiography and DCE-MRI method. Experimental Design Tumor vasculature of MCF-7 breast tumor mouse xenografts was studied by a combination of MR angiography and DCE-MRI with albumin-Gd-DTPA. Tumor macroscopic vasculature was extracted from the early enhanced images. Tumor microvascular parameters were obtained from the pharmacokinetic modeling of the DCE-MRI data. A spatial analysis of the microvascular parameters based on the macroscopic vasculature was used to evaluate the changes of the heterogeneous vasculature induced by a 12 day bevacizumab/paclitaxel treatment in mice bearing MCF-7 breast tumor. Results Macroscopic vessels that feed the tumors were not affected by the bevacizumab/paclitaxel combination therapy. A higher portion of the tumors was within close proximity of these macroscopic vessels after the treatment, concomitant with tumor growth retardation. There was a significant decrease in microvascular permeability and vascular volume in the tumor regions near these vessels. Conclusion Bevacizumab/paclitaxel combination therapy did not block the blood supply to the MCF-7 breast tumor. Such finding is consistent with the modest survival benefits of adding bevacizumab to current treatment regimens for some types of cancers.

Biocompatible blood pool MRI contrast agents based on hyaluronan

Biocompatible gadolinium blood pool contrast agents based on a biopolymer, hyaluronan, were investigated for magnetic resonance angiography application. Hyaluronan, a non-sulfated linear glucosaminoglycan composed of 2000-25,000 repeating disaccharide subunits of D-glucuronic acid and N-acetylglucosamine with molecular weight up to 20 MDa, is a major component of the extracellular matrix. Two gadolinium contrast agents based on 16 and 74 kDa hyaluronan were synthesized, both with R(1) relaxivity around 5 mM(-1)  s(-1) per gadolinium at 9.4 T at 25°C. These two hyaluronan based agents show significant enhancement of the vasculature for an extended period of time. Initial excretion was primarily through the renal system. Later uptake was observed in the stomach and lower gastrointestinal tract. Macromolecular hyaluronan-based gadolinium agents have a high clinical translation potential as hyaluronan is already approved by FDA for a variety of medical applications.

Invasive Breast Cancer Preferably and Predominantly Occurs at the Interface Between Fibroglandular and Adipose Tissue.

Micro‐Abstract The position of invasive breast tumor in relation to fibroglandular and adipose tissue was assessed in this retrospective dynamic contrast‐enhanced magnetic resonance imaging investigation of 294 patients. These tumors were found to occur predominantly at the interface between fibroglandular and adipose tissue. Background: Increasing evidence suggests adipocyte involvement in malignant breast tumor invasive front or margin. The aim of this study was to evaluate the location of invasive breast tumors in relation to fibroglandular and adipose tissue by dynamic contrast‐enhanced magnetic resonance imaging (DCE‐MRI). Patients and Methods: Pretreatment breast DCE‐MRI images of 294 patients with biopsy‐proven invasive breast cancer from 2008 to 2014 were studied. Invasive breast tumors were visualized as enhanced lesions in the postcontrast subtraction images. Positive identification of biopsy‐confirmed invasive breast tumors on DCE‐MRI images was achieved by correlation of findings from breast MRI and pathology reports. Tumor location in relation to fibroglandular and adipose tissue was investigated using precontrast T1‐weighted MRI images. Results: Of 294 patients, 291 had DCE‐MRI discernable invasive breast tumors located at the interface between fibroglandular and adipose tissues, regardless of the tumor size, type, receptor status, or breast composition. Conclusion: Invasive breast cancer preferably and predominantly occurs adjacent to breast adipose tissue.

Association between breast cancer, breast density, and body adiposity evaluated by MRI

ObjectiveDespite the lack of reliable methods with which to measure breast density from 2D mammograms, numerous studies have demonstrated a positive association between breast cancer and breast density. The goal of this study was to study the association between breast cancer and body adiposity, as well as breast density quantitatively assessed from 3D MRI breast images.MethodsBreast density was calculated from 3D T1-weighted MRI images. The thickness of the upper abdominal adipose layer was used as a surrogate marker for body adiposity. We evaluated the correlation between breast density, age, body adiposity, and breast cancer.ResultsBreast density was calculated for 410 patients with unilateral invasive breast cancer, 73 patients with ductal carcinoma in situ (DCIS), and 361 controls without breast cancer. Breast density was inversely related to age and the thickness of the upper abdominal adipose layer. Breast cancer was only positively associated with body adiposity and age.ConclusionAge and body adiposity are predictive of breast density. Breast cancer was not associated with breast density; however, it was associated with the thickness of the upper abdominal adipose layer, a surrogate marker for body adiposity. Our results based on a limited number of patients warrant further investigations.Key points• MRI breast density is negatively associated with body adiposity.• MRI breast density is negatively associated with age.• Breast cancer is positively associated with body adiposity.• Breast Cancer is not associated with MRI breast density.

Abstract 4572: Bioorthogonal, two-component drug delivery in HER2(+) breast cancer mouse models

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The HER2 receptors are overexpressed in ∼25% of breast cancer patients and characterized by poor internalization capability. The humanized monoclonal antibody, trastuzumab (Tz), is used as a first-line treatment for HER2(+) breast cancers. However, approximately 50% of patients with HER2(+) disease do not benefit from trastuzumab or the disease becomes refractory to the drug. We have demonstrated a target-specific intracellular delivery of therapeutics using new two-step/two-component drug delivery system driven by click chemistry that results in the in situ complexation and rapid internalization. Two delivery strategies based on azide (Az)/dibenzylcyclooctyne (DBCO), (Click-1) and trans-cyclooctene (TCO)/tertrazine (Tt), (Click-2) bioorthogonal click reactions were tested in human HER2(+) breast cancer mouse models. Trastuzumab was functionalized with Az or TCO groups and labeled with NIR CF-680 dye, to prepare Tz(Az/TCO)a(CF-680)b as the pretargeting component. Albumin substituted with paclitaxel (Px), functionalized with DBCO or Tt, and labeled with NIR CF-750 dye to prepare Alb(DBCO/Tt)x(Px)y(CF-750)z was used as the second delivery component. BT-474 HER2(+) breast cancer models were grown in athymic nude mice. Five groups of mice (a) untreated/PBS (b) first-component without Az, control for Click-1 (c) treated by Click-1 (d) first-component without TCO, control for Click-2, and (e) treated by Click-2 were used for the study. On day 0 mice were imaged with in vivo Xenogen optical imaging system and injected with the first pretargeting component followed by the second drug delivery component after 6h. Similarly, the second dose was administered on day 7. Mice were imaged at days 0 and 7 after administration of therapy to confirm the accumulation of components in tumors. Tumor volumes were measured every forth day up to day 20. After 21 days mice were sacrificed and tumors were extracted and analyzed by H&E staining for possible necrosis. Accumulation of the pretargeting and delivery components were observed in mice treated under Click-1 and Click-2 conditions compared to the corresponding controls and untreated mice. However, the accumulation was significantly higher in Click-2 group in comparison to all other groups. In addition, both pretargeting and delivery components were retained in the tumor for much longer time in Click-2 group than in other groups. We observed a tumor shrinking and significant decrease of tumor growth rate in Click-2 group. The results were confirmed by H&E immunohistochemistry analysis showing significant necrotic area in the Click-2 treated groups. Citation Format: Sudath Hapuarachchige, Wenlian Zhu, Yoshinori Kato, Dmitri Artemov. Bioorthogonal, two-component drug delivery in HER2(+) breast cancer mouse models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4572. doi:10.1158/1538-7445.AM2014-4572