Materials Research Express PAPER Magnetic field-enhanced cellular uptake of doxorubicin loaded magnetic nanoparticles for tumor treatment To cite this article: Indu Venugopal et al 2016 Mater. Res. Express 3 095010 Manuscript version: Accepted Manuscript Accepted Manuscript is “the version of the article accepted for publication including all changes made as a result of the peer review process, and which may also include the addition to the article by IOP Publishing of a header, an article ID, a cover sheet and/or an ‘Accepted Manuscript’ watermark, but excluding any other editing, typesetting or other changes made by IOP Publishing and/or its licensors” This Accepted Manuscript is © © 2016 IOP Publishing Ltd. During the embargo period (the 12 month period from the publication of the Version of Record of this article), the Accepted Manuscript is fully protected by copyright and cannot be reused or reposted elsewhere. 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All third party content is fully copyright protected, unless specifically stated otherwise in the figure caption in the Version of Record. View the article online for updates and enhancements. This content was downloaded from IP address 170.106.35.229 on 26/09/2021 at 14:27 Page 1 of 23 CONFIDENTIAL - AUTHOR SUBMITTED MANUSCRIPT MRX-101958.R2 1 2 3 4 Magnetic field-enhanced cellular uptake of doxorubicin-loaded magnetic 5 nanoparticles for tumor treatment 6 7 Indu Venugopal1, Sebastian Pernal1, Alexandra Duproz1, Jeromy Bentley2, Herbert Engelhard3, Andreas 8 1 9 Linninger * 10 11 1. University of Illinois at Chicago, Department of Bioengineering, Laboratory for Product and 12 Process Design, 851 S. Morgan St. - 218 SEO, Chicago, IL 60607-7000 13 2. NSF-RET fellow at University of Illinois at Chicago from Naperville Central High School 440 14 Aurora Ave, Naperville, IL 60540 15 3. University of Illinois College of Medicine, Department of Neurosurgery, 912 South Woods St, 16 Chicago, IL 60612 17 18 * Corresponding author (email: [email protected]) 19 20 21 22 23 1.0 Abstract 24 Cancer remains the second most common cause of death in the US, accounting for nearly 1 out of every 25 26 4 deaths. In recent years, several varieties of nanoparticles (NPs) have been synthesized with the intent 27 28 of being utilized as tumor drug delivery vehicles. We have produced superparamagnetic, gold-coated 29 magnetite (Fe3O4@Au) NPs and loaded them with the chemotherapeutic drug doxorubicin (DOX) for 30 31 magnetic drug targeting of tumors. The synthetic strategy uses the food thickening agent gellan gum 32 (Phytagel) as a negatively charged shell around the Fe O @Au NP onto which the positively charged 33 3 4 34 DOX molecules are loaded via electrostatic attraction. The resulting DOX-loaded magnetic nanoparticles 35 36 (DOX-MNPs) were characterized using transmission electron microscopy (TEM), energy dispersive x- 37 ray spectroscopy (EDS), superconducting quantum interference device (SQUID) magnetometry, surface 38 39 area electron diffraction (SAED), zeta potential (ZP) measurements, fourier transform infrared 40 41 spectroscopy (FTIR) as well as UV/Vis and fluorescence spectroscopy. Cytotoxicity of the DOX-MNPs 42 was demonstrated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay 43 44 on C6 glioma cells. Cellular uptake of DOX-MNPs was enhanced with magnetic fields, which was 45 quantitatively determined using flow cytometry. This improved uptake also led to greater tumor cell 46 47 death, which was measured using MTT assay. These magnetic drug targeting results are promising for a 48 49 new therapy for cancer. 50 51 52 53 54 55 56 57 58 59 60 CONFIDENTIAL - AUTHOR SUBMITTED MANUSCRIPT MRX-101958.R2 Page 2 of 23 1 2 3 4 2.0 Introduction 5 6 Cancer is the second most common cause of death in the US, exceeded only by heart disease. Cancer 7 8 research has been successful as in decreasing in overall death rates between 1991 and 2010, by about 20% 9 1 10 in both men and women . The cancer treatment protocol for most cancers requires chemotherapy. 11 Unfortunately most chemotherapeutic agents exhibit poor specificity in reaching tumor tissue in addition 12 13 to dose-limiting toxicity. As a result, many patients experience adverse side effects due to the 14 administered chemotherapy treatments. 15 16 17 New drug delivery strategies are being developed to improve efficacy of cancer patient treatments, while 18 minimizing systemic toxicity. One promising treatment involves using NPs as a means of targeting 19 20 cytotoxic drugs to specific tumor cells and tissue, thereby limiting systemic exposure. Typically in the use 21 22 of NPs for the purpose of drug therapy, the drug is dissolved, entrapped, encapsulated, or attached to the 23 NPs. The major goals in the design of NPs for drug delivery include: (i) control of particle size, (ii) 24 25 variation of surface properties, and (iii) ability to release of pharmacologically active agents to achieve 26 the site-specification of the drug at the therapeutically optimal rate and dose regimen. Novel NP-based 27 28 applications for cancer treatments are being explored because of their ability to (i) decrease toxicity and 29 30 the occurrence of adverse reactions, (ii) utilize drug more effectively, (iii) control the rate and site of drug 31 release, (iv) provide a more predictable drug delivery system2–4 32 33 34 Currently, there are two different types of targeting utilized by scientists studying NP drug delivery 35 systems for cancer. Passive targeting exploits the enhanced permeability and retention (EPR) effect that 36 37 is defined by leaky vasculature around tumors caused by tumor-induced angiogenesis with incomplete 38 endothelial cell junctions, resulting in the accumulation of the NPs at the tumor site5. This passive 39 40 targeting strategy has been shown to be an efficient method to reduce the toxic side effects and increase 41 6,7 42 the therapeutic index of drug molecules . Active targeting usually involves attaching ligands on the 43 surface of NPs, which bind to over-expressed receptors on tumor cells. Targeting specific surface receptor 44 45 causes selective uptake of NPs by the cancer cells8. Active targeting may also involve utilizing physical 46 47 stimuli for actively localizing NPs to the target site, including temperature, pH, electric charge, light, 48 sound and magnetism9,10. Our research aims at utilizing magnetism as a physical stimulus, for efficient 49 50 targeting of target specific cells. 51 52 Magnetic Drug Targeting (MDT) involves binding tumor drugs to biocompatible magnetic nanoparticles 53 54 (MNPs), which can be guided to specific tumor sites via magnetic fields. The magnetic field can be 55 controlled by magnets placed outside the body and/or by using magnetizable implants inside the body11. 56 57 58 59 60 2 Page 3 of 23 CONFIDENTIAL - AUTHOR SUBMITTED MANUSCRIPT MRX-101958.R2 1 2 3 NPs composed of magnetite (Fe3O4) are biocompatible and biodegradable. The iron in these particles are 4 5 absorbed into the body’s iron stores during metabolism12. 6 7 Synthesis of magnetic microparticles and NPs for the delivery of chemotherapeutics has evolved since the 8 9 1970s. Zimmerman and Pilwat in 1976 studied the delivery of cytotoxic drugs using magnetic 10 erythrocytes13. The targeting of magnetic albumin microspheres encapsulating the anticancer drug (DOX) 11 12 in animals was studied by Widder et al14. Engelhard and Petruska studied the movement of magnetic 13 15 14 microparticles in rat CSF and brain in 1992 . Hafeli et al synthesized biodegradable polylactide 15 microspheres that incorporated magnetite and the beta radiation-emitter Y-90 for radiotherapy targeting in 16 17 199416, and successfully applied it to subcutaneous tumors17. However, these studies employed 18 19 micrometer-sized magnetic particles. Use of MNPs was described for the first time by Lubbe et al in 20 199918. MNPs loaded with epirubicin were administered intra-arterially in a phase-1 clinical trial for 21 22 patients with advanced cancers in 199618. Several groups and start-up companies have synthesized 23 magnetic vectors and shown potential applications of MNPs. These include MNPs used in MRI19, 24 25 magnetic fluid hyperthermia20,21, cell sorting and targeting22,23, bioseparation24, enzyme immobilization25, 26 26,27 28,29 27 immunoassays , and gene transfection and detection systems . 28 29 Different varieties of MNPs have been coated with a “shell” of either organic or inorganic nature30 to 30 31 exploit specific properties. NP coatings often regulate the solubility, hydrophilic or hydrophobic 32 properties, stability, and the targeting ability of the particles. For example, Yang et al synthesized a poly 33 34 [N-(1-one-butyric acid]) aniline shells around Fe3O4 cores that have been further conjugated with 1,3- 35 bis(2-chloroethyl)-1-nitrosourea (BCNU)31. Ito et al have synthesized antibody-conjugated 36 37 magnetoliposomes which can used as carriers to introduce magnetite NPs into target cells, since the 38 32 39 negatively-charged cell surface interacts with the positively-charged surface of the NPs .