Actively Targeted Magnetothermally Responsive Nanocarriers

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Biological and Medical Applications of Materials and Interfaces

Actively Targeted Magnetothermally Responsive Nanocarriers/ Doxorubicin for Thermo-chemotherapy of Hepatoma Minghua Li, Li Deng, Jianbo Li, Weizhong Yuan, Xiao-Long Gao, Jiong Ni, Hong Jiang, Jiaqi Zeng, Jie Ren, and Peijun Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b14972 • Publication Date (Web): 07 Nov 2018 Downloaded from http://pubs.acs.org on November 8, 2018

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ACS Applied Materials & Interfaces

Actively Targeted Magnetothermally Responsive Nanocarriers/Doxorubicin for Thermo-chemotherapy of Hepatoma Minghua Li,† Li Deng,‡ Jianbo Li,‡ Weizhong Yuan,‡ Xiaolong Gao,† Jiong Ni,† Hong Jiang,† Jiaqi Zeng,† Jie Ren, *, ‡ Peijun Wang*, † Department of Radiology, Tongji Hospital, School of Medicine, Tongji University, Shanghai 200065,

＋

P.R. China ‡

Institute of Nano and Biopolymeric Materials, School of Materials Science and Engineering, Tongji

University , Shanghai 201804, P.R. China

Keywords magnetic nanoparticles; magnetic hyperthermia; active targeting; drug delivery; cancer combined therapy

ABSTRACT Nano-drug delivery systems modified with targeting molecules allow antitumor drugs to localize to tumor sites efficiently. We conjugated monoclonal antibody that specifically bind to CD147 protein expressed highly on hepatoma cells to magnetothermally responsive nano-carriers/doxorubicin (MTRN/DOX) synthesized from Manganese Zinc (Mn-Zn) ferrite magnetic nanoparticles (MZF-MNPs), amphiphilic and thermosensitivity copolymer drug carriers together with chemotherapy drug---DOX, which formed CD147MTRN/DOX. It could target hepatoma cells actively and improve the DOX concentration in the tumor sites. Subsequently, an external alternating magnetic field (AMF) elevated the temperature of the thermomagnetic particles, resulting in structural changes of the thermosensitive copolymer drug carriers, thereby releasing DOX. Hence, CD147MTRN/DOX could enhance the responsiveness of hepatoma cells to the pre-existing chemotherapy drugs owing to active targeting combined synergistically with thermotherapy and chemotherapy, which has more significant anticancer effects than MTRN/DOX.

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1. INTRODUCTION Because of a lack of effective tumor-targeted drug carrier systems, the specific aggregation of chemotherapy drugs in hepatoma cells is limited, which causes low drug availability and poor chemotherapeutic outcomes. Hence, the development of targeted drug carriers for hepatocarcinoma to improve the sensitivity of hepatoma cells to chemotherapy drugs is warranted. Nano-drug delivery systems modified with targeting molecules can specifically bind to the surface receptors or antigens on target cells, thereby allowing the drug to actively target a specific site followed by its subsequent release.1-12 Common active targeting molecules include antibodies, peptides/proteins, vitamins (e.g., folic acid, Vitamin A, and Vitamin B12), and carbohydrates (e.g., galactose, mannose, and hyaluronic acid). CD147 is a surface transmembrane glycoprotein that is highly expressed on hepatoma cells but not, or rarely, on normal cells. It belongs to a member of the immunoglobulin superfamily (IgSF), which regulates hepatocarinoma proliferation, invasion, metastasis, and other processes.13,14 Based on its features, CD147 has become a target for anticancer therapy. Preparation of monoclonal antibodies that specifically bind to CD147 and loading of these monoclonal antibodies into drug carriers have allowed nano-drug delivery systems to deliver drugs accurately to hepatoma cells via an active targeting approach. Numerous studies have shown that various chemotherapeutic agents have a thermal sensitization effect, i.e., which could increase sensitivity of tumor cells to certain chemotherapy drugs in the environment above the body temperature to allow them to kill cancer cells at a relatively light concentration. The mechanism on synergistic effect of thermotherapy and chemotherapy is mainly comprised of three aspects: (1) Thermotherapy produces local heating effect which can enhance the cytotoxicity of some chemotherapeutic drugs;15,16 (2) Thermotherapy increases the blood flow to the tumor site and the permeability of the tumor cell membrane, thereby improving the absorption of chemotherapy drugs by the tumor cells;17 (3) Thermotherapy could inhibit DNA polymerase-mediated DNA damage repair in tumor cells after chemotherapy. 17 These result in the development of antitumor thermochemotherapy, which has better


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curative effects compared to the chemotherapy alone.3,4,18-26 In the present study, we synthesized Manganese Zinc (Mn-Zn) ferrite magnetic nanoparticles (MZF-MNPs) instead of conventional Fe3O4 particles. MZF-MNPs is a type of magnetic sbustance made from ferrous, with premium chemical stability, magnetothermally properties and biocompatibility.27-31 Thermosensitive copolymer nano-drug carriers will unload the chemotherapeutics at the tumor site in response to a specific temperature change.2,4,18-20,32-35 The random copolymer composed of 2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA) and oligo(ethylene glycol)methacrylate (OEGMA)---P(MEO2MA-co-OEGMA) is one new kind of thermosensitive copolymer which uses changes in temperature as its stimulus. We may modify the proportions of the two constituent monomers in the copolymer mentioned above in order to control the lower critical solution temperature (LCST) . LCST is a crucial temperature for phase transition. It is adjusted to 43℃, which is the temperature that chemotherapy drug doxorubicin (DOX) begins to produce the synergistic effect of thermotherapy and chemotherapy on tumor cells.16 Moreover, when the temperature has arrived at or surpassed the LCST, the molecular chains of the copolymer will undergo hydrophilic–hydrophobic transformation. The phase transition of the copolymer regulates the release of copolymer-loading drugs at a specific site and temperature,36,37 which reduce the drug adverse effects in other tissues while improving the absorption and biological availability of the chemotherapy drugs. We

synthesized

magnetothermally

responsive

nano-carriers/doxorubicin

(MTRN/DOX) formed from MZF-MNPs, thermosensitive amphiphilic copolymer nanocarriers and DOX. MTRN/DOX organically combine the magnetothermal effect of MZF-MNPs and thermosensitivity of copolymer drug carriers. In our previous studies, the synergistic effect of thermotherapy and chemotherapy of this system showed good tumor-killing effect.31 In the present study, we conjugated this drug delivery system with an external biological targeting molecule---CD147 monoclonal antibody, to achieve active targeting, which enrich the drug delivery system in tumor tissues highly and bind to hepatoma cells specifically (Scheme 1), thereby improving the anticancer therapy and reducing associated their toxicity.



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Scheme 1. Schematic illustration to show the synthesis of CD147-MTRN/DOX, active targeting to hepatoma cells, and synchronism and enhanced synergism of thermo-chemotherapy for hepatoma.

2. EXPERIMENTAL SECTION 2.1. Synthesis and Preparation of CD147-MTRN/DOX 25

mL

of

MTRN/DOX

(2

mg/mL)

was

poured

into

20

mg

1-(3-

Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to activate for 10 minutes, followed by adding 20 mg NHS and allowing to react for 2 hours, and subsequently adding 2.5 mg CD147F(ab)'2 antibody (containing excipients) to react overnight. The free CD147F(ab)'2 antibody fragments were dialyzed in distilled water with a 100-kD dialysis bag, followed by changing water at 6 hours intervals. 2.2. Nanoparticles Accumulation in Tumors Once upto 80% confluency, both MTRN/DOX and CD147-MTRN/DOX (100 μg/mL MTRN; 50 μg/mL MZF) were put in Huh-7 and L-02 cells. We removed culture medium after 4 hour-incubation with the cells. After centrifugating and discarding of supernatant, 3% glutaraldehyde was added into the cells. After leaving the cells for 2 hours at 4°C, they were prepared into ultrathin sections to be seen under transmission electron microscopy (TEM). We incubated Huh-7 and L-02 cells for 4 hours with MTRN/DOX and CD147-MTRN /DOX (25, 50, 100 and 200 μg/mL MZF). After centrifugating and discarding of supernatant, nitric acid was added to the cells. The Fe contents of Huh-7 and L-02 cells


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were measured by inductively coupled plasma-atomic emission spectrometer (ICP-AES) (SPECTROARCOS, Spectro, Germany). For flow cytometry and fluorescence staining, the Huh-7 (Some of the Huh-7 cells were pretreated with the competitive inhibitor, CD147 monoclonal antibody) and L-02 cells were incubated for 4 hours with Cy5.5-labelled MTRN/DOX and C147-MTRN/DOX (100 μg/mL MTRN; 50 μg/mL MZF). After removal of the medium containing the drug, followed by PBS washing, some cells were tested through flow cytometry (C6, BD, USA) due to fluorescent Cy5.5, and the remaining cells were stained by DAPI. Distribution of the nanoparticles, DOX and their relationships with the nucleus were observed using laser scanning confocal microscopy (LSCM) (TCS SP5, Leica, Germany). Huh-7 cells unpretreated with competitive inhibitor were undergone Hoestch nuclear-staining. A mixture of pretreated Huh-7 cells and unpretreated ones were incubated individually using Cy5.5-labelled MTRN/DOX and C147-MTRN/DOX (100 μg/mL MTRN; 50 μg/mL MZF) for 4 hours, followed by observing targeted and nontargeted nanoparticles uptake of the two groups of cells through LSCM. Using intraperitoneally injected of 10% chloral hydrate we anesthetized the mice, and then slowly injected MTRN/DOX and CD147-MTRN/DOX (1,000 μg/mL MTRN, 500 μg/mL MZF) in the mice’s caudal veins individually. 12 and 72 hours after drug administration, the mice were euthanized, following the tumor tissues being first picked off and then fixed, next to they were stained with Prussian blue, following an observation under the microscope. The tumor tissues were digested with nitric acid, and the Fe content of the tumors were measured by ICP-AES. After individual tail vein injection of Cy5.5-labeled MTRN/DOX and CD147MTRN/DOX into the mice, the animals underwent live fluorescence imaging to observe the distribution changes of the nanoparticles over time. Then the relative fluorescence intensities of tumor sites were measured. Filter lenses that have 675 nm excitation wavelength and 694 nm emission wavelength were utilized. 2.3. In Vitro and In Vivo Anticancer Effects



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We calculated Huh-7 cells viability with different treatments using a standard MTT assay. We monitored tumor sizes of different groups to evaluate anticancer effect in vivo. Detailed experimental procedures of anticancer Evaluation in vitro and in vivo can be found in the Supporting Information

3. RESULTS AND DISCUSSION 3.1. CD147-MTRN/DOX Synthesis and Characteristics We synthesized thermosensitive amphiphilic block copolymer of 6sPCL-bP(MEO2MA-co-OEGMA) through ring-opening polymerization (ROP) and atom transfer radical polymerization (ATRP) (Figure 1A). According to our previous study,30 when MEO2MA to OEGMA ratio is 92:8, the LCST can be controlled aproximately at 43°C, which is the ideal temperature to sensitize tumor cells to chemotherapy. Subsequently, in this study we have applied 6sPCL-b-P(MEO2MA92%-co-OEGMA8%) copolymer micelles. Liquid

phase

thermal

decomposition

has

been

employed

to

make

the

MnxZn1−xFe2O4 particles (MZF-MNPs).38-40 Indeed, only when x=0.6, saturated magnetization (MS) together with specific absorption rate (SAR) of the Mn0.6Zn0.4Fe2O4 reach

maximum

levels.26

Therefore,

for

optimal

thermotherapeutic

effect,

Mn0.6Zn0.4Fe2O4 was used in the following experiments. Finally, MTRN/DOX could be created due to self-assembling with MZF-MNPs, thermosensitive amphiphilic copolymer nanocarriers and DOX.


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Figure 1. Characteritics of CD147-MTRN/DOX. (A) Synthesis of thermosensitive amphiphilic copolymer by ROP and ATRP. (B) Schematic diagram to show the synthesis of CD147-MTRN/DOX. (C) TEM image of CD147-MTRN/DOX. (D) DLS curves of CD147-MTRN/DOX. (E) Magnetic curves of CD147-MTRN/DOX; inset: the responsibility of CD147-MTRN/DOX to magnet.

CD147 is highly expressed on the surface of hepatoma cells. In this study, CD147 monoclonal antibody and MTRN/DOX were conjugated to form CD147-MTRN/DOX to improve cell-killing effect for the hepatocarcinoma of drug-loading nanoparticles via antibody-antigen-mediated active targeting. Figure 1B was a schematic diagram of the nanoparticles which the surface of the copolymer was connected with CD147 monoclonal antibody. In Figure 1C, TEM was used to observe CD147-MTRN/DOX, and encapsulated MZF-MNPs were detected inside the nanoparticles. UV-Vis indicated that the drug loading content (DLC) of DOX was 5.0%. Dynamic light scattering (DLS) has shown that nano-particle's diameter is 190nm (Figure 1D). The zeta potential of CD147-MTRN/DOX is -21.4 mV (Figure S1 of the Supporting



Information). As shown in Figure 1E, Ms of CD147-MTRN /DOX is 31.6 emu/g, meaning it has a superparamagnetic property. Also seen in the inset of Figure 1E, when there was an external magnetic field, we could see evenly dispersed nanoparticles (200 μ g/mL MTRN, 100μg/mL MZF) got together towards the direction of the magnet, which indicated that nanoparticles displayed outstanding magentic responsibility. These properties of CD147-MTRN/DOX were similar to those of MTRN/DOX in our previous study.30,31 Thus, compared with MTRN/DOX, CD147-MTRN/DOX did not significantly change their properties. 3.2. Cellular Uptake of Nanoparticles We assessed the CD147 expression in Huh-7 and L-02 cells. Immunohistochemical staining of CD147 showed the brownish yellow staining in the Huh-7 cell membranes and cytoplasms, but not in L-02 cells (Figure 2), indicating that hepatoma cells expressed CD147 highly on but the expression on normal hepatocytes are in very low level.

Figure 2. CD147 expression in Huh-7 and L-02 cells. CD147 immunohistochemical staining images of two kinds of cells.

TEM images displayed cellular uptake of black granular nanoparticles in both two kinds of cells. The uptake of CD147-MTRN/DOX particles was more significant than MTRN/DOX in Huh-7 cells, whereas no significant differences were observed in the L02 cells (Figure 3A). As MTRN contains iron, iron content in the cells could reflect the intracellular uptake of nanoparticles. By ICP-AES measurement (Figure 3B), we found that the Fe content in Huh-7 cells incubated with CD147-MTRN/DOX were much higher than those incubated with MTRN/DOX. Corresponding to TEM observation, the Fe content of L-02 cells in both groups exhibited no major differences (Figure 3C).


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Figure 3. Huh-7 and L-02 cells uptake of nanoparticles. (A) TEM images of two kinds of cells incubated with two types of nanoparticles. (B) Fe content of Huh-7 cells incubated with different MZF content of two types of nanoparticles. (C) Fe content of L-02 cells incubated with different MZF content of two types of nanoparticles. **P

Actively Targeted Magnetothermally Responsive Nanocarriers

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