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Bio-Mimetic Magnetic Nanostructures: A Theranostic Platform Targeting Lipid Metabolism and Immune Response in Lymphoma. Abhalaxmi Singh, Vikas Nandwana, Jonathan S. Rink, Soo-Ryoon Ryoo, Tzu Hung Chen, Sean David Allen, Evan A. Scott, Leo I. Gordon, C. Shad Thaxton, and Vinayak P. Dravid ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b03727 • Publication Date (Web): 05 Sep 2019 Downloaded from pubs.acs.org on September 5, 2019
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Bio-Mimetic
Magnetic
Theranostic
Platform
Metabolism
and
Nanostructures: Targeting
Immune
A
Lipid
Response
in
Lymphoma.
Abhalaxmi Singhab, Vikas Nandwanaab, Jonathan S. Rinkcd, Soo-Ryoon Ryooab, Tzu Hung Chena, Sean David Allene, Evan A. Scottdef, Leo I. Gordoncg, C. Shad Thaxtonbgh and Vinayak P. Dravid*ab.
a Department
of Materials Science & Engineering, Northwestern University,
Evanston, Illinois 60208, USA.
b
International Institute of Nanotechnology, Evanston, Illinois 60208, USA.
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c
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Department of Medicine, Division of Hematology/ Oncology, Feinberg School of
Medicine, Northwestern University, Chicago, Illinois 60611, USA
d
Simpson-Querrey Institute for Bionanotechnology, Northwestern University,
Chicago, Illinois 60611, USA. e
Interdisciplinary Biological Sciences Program, Northwestern University, Evanston,
Illinois 60208, USA.
f
Department of Biomedical Engineering, Northwestern University, Evanston, Illinois
60208, USA.
g
Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago,
Illinois 60611, USA.
h
Department of Urology, Feinberg School of Medicine, Northwestern University,
Chicago, Illinois 60611, USA.
ABSTRACT
B cell lymphoma cells depend upon cholesterol to maintain pro-proliferation and pro-survival signaling via the B cell receptor. Targeted cholesterol depletion of
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lymphoma cells is an attractive therapeutic strategy. We report here high-density lipoprotein mimicking magnetic nanostructures (HDL-MNS) that can bind to the highaffinity HDL receptor, scavenger receptor type B-1 (SR-B1), and interfere with cholesterol flux mechanisms in SR-B1 receptor positive lymphoma cells, causing cellular cholesterol depletion. In addition, the MNS core can be utilized for its ability to generate heat under an external radio frequency field. The thermal activation of MNS can lead to both innate and adaptive anti-tumor immune responses by inducing expression of heat shock proteins that lead to activation of antigen presenting cells and finally lymphocyte trafficking. In the present study, we demonstrate SR-B1 receptor mediated binding and cellular uptake of HDL-MNS and prevention of phagolysosome formation by transmission electron microscopy, fluorescence microscopy and ICP-MS analysis. The combinational therapeutics of cholesterol depletion and thermal activation significantly improves therapeutic efficacy in SR-B1 expressing lymphoma cells. HDL-MNS reduces the T2 relaxation time under magnetic resonance imaging (MRI) more effectively compared with a commercially available contrast agent, and the specificity of HDL-MNS towards the SR-B1 receptor leads to differential contrast between SR-B1 positive and negative cells suggesting its utility in
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diagnostic imaging. Overall, we have demonstrated that HDL-MNS have cell specific targeting efficiency, can modulate cholesterol efflux, can induce thermal activation mediated anti-tumor immune response and possess high contrast under MRI, making it a promising theranostic platform in lymphoma.
KEYWORDS high-density lipoprotein, magnetic nanostructure, SR-B1 receptor, thermal activation, anti-tumor immune response, lymphoma.
Non-Hodgkin’s lymphoma is one of the most common cancers diagnosed in the United States with more than 80,000 new diagnoses and 20,000 deaths per year.1,2 Diffuse large B-cell lymphoma (DLBCL) is the most common and aggressive form of NHL. The standard therapeutic regimen (R-CHOP: rituximab plus combination chemotherapy), while curative in ~60-70% of patients, still possesses significant toxicity due to off-target effects and a significant number of patients eventually become refractory to therapy and relapse.2,3 Hence, there is an acute need for innovative strategies to effectively image and treat B cell lymphoma using targeted therapeutics.
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Altered metabolism is one of the hallmarks of cancer, providing malignant cells with essential nutrients to support their unchecked proliferation.4 In DLBCL, lymphoma cells depend on cholesterol and cholesteryl esters to maintain membrane-anchored pro-survival and pro-proliferative signaling pathways, such as B cell receptor signaling. In addition to de novo cholesterol biosynthesis, lymphoma cells can satisfy their increased demand for cholesterol by taking up cholesteryl esters supplied from mature, spherical high density lipoproteins (HDLs), that bind to cells via scavenger receptor type B1 (SR-B1), which is absent in normal B cells.5,6 Therefore, development of therapeutics that can target SR-B1 and deplete cellular cholesterol holds significant promise for the next generation of B-cell lymphoma therapy. Recently, gold nanoparticle-templated HDL-like nanoparticles have been shown to engage SR-B1 and modulate cellular cholesterol homeostasis in lymphoma cells, leading to a reduction in cellular cholesterol content and an increase in lymphoma cell death both
in vitro and in vivo.3,7 Further, we have recently developed an HDL-mimicking magnetic nanostructure (HDL-MNS), using iron oxide as the core. These HDL-MNS mimic natural HDLs in their ability to engage HDL receptors and efflux cholesterol from lipid-laden macrophages.8 However, given the presence of the MNS core, the HDL-
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MNS potentially provide two other additional benefits, namely the ability to act as a T2weighted contrast agent for MR imaging, and the ability to be heated through application of an external RF field. Cancer immunotherapy is an emerging therapeutic strategy that can stimulate the generation of long-lasting, tumor antigen–specific immune responses to specifically recognize and destroy the tumor cells. In this regard, thermal therapy has drawn significant attention as an adjunct to cancer immunotherapy due to its ability to initiate immune reactivity.9,10 The anti-cancer effects of thermal therapy are mediated by two distinct pathways which are dependent upon the temperature to which the tumor tissue is exposed. Temperatures above 43℃ induce apoptosis and necrosis in the tumor cells. Sub-lethal temperatures (< 43 ℃) induce structural changes that render cells vulnerable to other therapies, such as standard chemotherapy or radiotherapy. This mild range of thermal therapy (38-42 ℃) has significant effect on tumor vascular perfusion, tumor immunogenicity, immune function, lymphocyte trafficking, cytokine activity, metabolism, and gene expression, all of which affect the tumor microenvironment.11–13 The localized heating of tumor cells can induce expression of heat shock proteins (HSPs) that act as immunogens to activate the immune system
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through the maturation of dendritic cells (DC), activation of the cytolytic and migratory capacities of natural killer (NK) cells, stimulation of antigen-dependent T cell activation and their IFN-γ secretion and the release of pro- and anti-inflammatory cytokines.14 Clinically, thermal therapy has been used in combination with radiotherapy, and patients have achieved remission in both Hodgkin’s and NHL.15 The combination of thermal therapy with other therapies has also been used in clinics during different other cancer treatments.16–19 In this regard, magnetic nanostructures hold an important position as these can be influenced under radio frequency field (RF) to generate heat. Several research groups have investigated use of iron oxide-based nanoparticles with superior magnetic properties and surface functionalization for controlled thermal activation with an external RF field.20–22 The core MNS in our formulated HDL-MNS is composed of Zn0.2Mn0.8Fe2O4 nanoparticles that have shown higher contrast enhancement as well as thermal activation properties over the typically used ironoxide nanoparticles.22 Our HDL-MNS, due to their specificity towards SR-B1 receptors, specifically target the lymphoma cells where thermal activation of these MNS can be achieved using external RF field. Additionally, early detection of aggressive lymphoma or detection of relapse or residual disease after initial treatment
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is important for successful therapy as the survival rate of NHL patients is approximately 83 % if detected early which drops to 63 % when the cancer is detected in the late stage.1 Thus, there is an acute and timely need of a diagnostic agent for early detection and staging of the NHL patients. Herein we report the development of a bio-inspired HDL-MNS as a theranostic agent that mimics some features of natural HDL in function and surface composition but contains an MNS core. We demonstrate preferential uptake of HDL-MNS by SR-B1 positive lymphoma cells, and that HDLMNS escape the endolysosomal pathway, evidenced by transmission electron microscopy and fluorescence microscopy, resulting in differential contrast between SR-B1 positive and negative cell lines. HDL-MNS demonstrated efficacy against SRB1 positive lymphoma cell lines, which was enhanced in the presence of an RF field. In addition, RF-activation of HDL-MNS induced expression of HSPs and resulted in enhanced maturation and migration of dendritic cells. Taken together, these results suggest that HDL-MNS represent a promising theranostic agent for the treatment of B-cell lymphoma.
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Results/ Discussion HDL-MNS formulation and characterization We have previously reported on the synthesis of HDL-MNS using an iron oxide core, surrounded by phospholipids and apolipoprotein A1 (ApoA1).8 Here we use a similar synthetic protocol, with a Zn-Mn doped iron oxide as the core nanoparticle to enhance the imaging and thermal activation properties. The HDL-MNS were prepared by coating hydrophobic Zn-Mn doped iron oxide with the zwitterionic phospholipid DPPC followed by ApoA1.8 The core size, hydrodynamic diameter and zeta potential were determined from TEM and DLS (Figure 1). TEM images show the entrapment of MNS core (8 nm) with a surrounding layer suggesting the lipid and apolipoprotein coating (Figure 1a). The TEM size of complete HDL-MNS was found to be around 20 nm and the hydrodynamic diameter of HDL-MNS was found to be 41 ± 19 nm with a zeta potential -6.4 ± 1 mV. The difference in TEM and DLS size is due to the fact that TEM image represents the size of HDL-MNS in a dried state whereas the hydrodynamic diameter represents the size of HDL-MNS in dispersion state in water.23
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Figure 1. Physicochemical characterization. (a) Transmission electron microscopy image of HDL-MNS. (b) Stability of HDL-MNS over a week in 10 % serum containing media.
The number of ApoA1 bound to each HDL-MNS, quantified using Alexa Fluor488 tagged ApoA1, was found to be 3.5 ApoA1 per HDL-MNS. This is comparable with the natural HDL where 2-5 copies of ApoA1 are found in the mature spherical natural HDL particles.24,25 The HDL-MNS retained stability in fetal bovine serum (FBS)-containing cell culture media over a week as there is no significant change in size over the period (Figure 1b). To determine the contrast enhancement effect, r2 relaxivity of HDL-MNS of successive dilutions in water was measured at 1.4 T with a frequency of 60 MHz. Significantly high r2 relaxivity values (407 mM−1 s−1 for HDL-MNS) were obtained,
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approximately 5 times higher than those obtained for the commercially available T2 contrast agent Ferumoxytol (80 mM−1 s−1). In previous studies, our group has reported that Zn0.2Mn0.8Fe2O4 nanostructures show strongest MR contrast effect with the r2 relaxivity of 552 mM−1 s−1 as compared to Fe3O4 nanostructures that shows r2 relaxivity of 355 mM−1 s−1.22 Also, in our previous study, the HDL-MNS having similar synthesis process but, Fe3O4 core had r2 relaxivity of 340 mM−1 s−1 mM−1.8 All these data demonstrated that use of Zn0.2Mn0.8Fe2O4 core enhances the MR contrast as compared to our previous HDL-MNS construct.
Internalization of HDL-MNS through SR-B1 receptor
Figure 2. Amount of Fe ion taken up by different cells as measured via ICP-MS. (a) A time dependent study *p
