Iron oxide nanocarrier-mediated combination therapy of cisplatin and

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Iron oxide nanocarrier-mediated combination therapy of cisplatin and artemisinin for combating drug resistance through highly increased toxic reactive oxygen species generation Zhiguo Gao, Yao-Jia Li, Chaoqun You, Kai Sun, Peijing An, Chen Sun, Ming-Xin Wang, Xiaoli Zhu, and Bai-Wang Sun ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00056 • Publication Date (Web): 03 Jul 2018 Downloaded from http://pubs.acs.org on July 6, 2018

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ACS Applied Bio Materials

Iron oxide nanocarrier-mediated combination therapy of cisplatin and artemisinin for combating drug resistance through highly increased toxic reactive oxygen species generation Zhiguo Gaoa, Yaojia Lia, Chaoqun Youa, Kai Suna, Peijing Ana, Chen Suna, Mingxin Wanga, Xiaoli Zhub, Baiwang Suna, * a

School of Chemistry and Chemical Engineering, Southeast University, Nanjing

210089, PR China. Fax: +86 25 52090614, Tel: +86 25 52090614, E-mail address: [email protected] b

Department of Respiratory Medicine, The Affiliated Zhongda Hospital of Southeast

University.

Abstract Combination therapy of multiple drugs through a multipronged assault as a strategy to combat cisplatin resistance shows great potential in biochemical therapy of cancer. But inherent issues such as low drug loading and poor synergistic effects of multiple drugs partly limit further application of combination therapy. Here, we synthesized a new compound ART-Chol by coupling artemisinin and cholesterol as a based material, combined

with

cyclic(Arg-Gly-Asp-D-Phe-Lys)]-poly(ethyleneglycol)

distearoylphosphatidylcholine (cRGD-PEG-DSPE) and phospholipid to form a magnetic liposome cRGD-AFePt@NPs encapsulating superparamagnetic ferric oxide nanoparticles and cisplatin for achieving high drug loading and better synergistic effect. The cRGD-AFePt@NPs could be effectively internalized and responsively release loading cargos under alternating magnetic field (AMF) irradiation due to local hyperthermia generated from magnetic nanoparticles by hysteresis loss and/or Néel relaxation. The generated Fe2+/Fe3+ from Fe3O4 NPs in the acid lysosomes motivated cisplatin and catalyzed the Fe-dependent anti-cancer drug artemisinin (ART) to 1

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generate the high toxic ROS through Fenton reaction, which greatly enhance anticancer effect of cisplatin with minimize side effects. In vitro cytotoxicity test demonstrated the cRGD-AFePt@NPs exhibited 15.17-fold lower IC50 value of free cisplatin (IC50=32.47 µM) against A549/R cells. Further flow cytometry test also showed obviously increased intracellular ROS generation and cell apoptosis rate. We highlight the potential of Fe2+/Fe3+ mediated combination therapy of cisplatin and ART for circumvent cisplatin drug resistance. Keyword; Reactive oxygen species, Magnetic liposomes. Fe3O4 nanoparticles, Artemisinin and cisplatin, Fenton reaction

1. Introduction Cancer remains to be one of most menacing diseases in which abnormal cells uncontrolled proliferate and spread to other healthy tissues1. Reactive oxygen species (ROS) act as an important messenger in cancer cell proliferation and homeostasis by reversibly oxidizing the sulfydryl-containing proteins to modify the structure and function of proteins2,3. For examples, ROS could activate signing pathway such as MAPK (mitogen-activated protein kinase), ERK (extracellular signal-regulated kinase), and JNK (c-Jun N-terminal kinase), thereby promoting the expression of Cyclin D1 gene. The activation of pathways and signaling molecules concerns the cell growth and survival4,5. However, when the intracellular ROS levels exceed the threshold value, the highly reactive ROS could oxidize any surrounding biomacromolecules rapidly such as protein, DNA, glutathione, and lipid, thereby leading to unrecoverable damage to cells and even inducing cell apoptosis6–8. Therefore, regulating the levels of intracellular ROS in tumor cells has been a novel anticancer strategy. Superparamagnetic iron oxide nanoparticles (Fe3O4 NPs) has been widely 2


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explored in targeted drug delivery due to unique superiority, such as low toxic to healthy tissues, in vivo imaging by magnetic resonance imaging (MRI), and magnetic hyperthermia through hysteresis loss and/or Néel relaxation in an alternating magnetic field (AMF)9–13. Administrating an encapsulation of Fe3O4 NPs into drug delivery system such as liposomes14–18, nanomicelle19,20, or inorganic mesoporous silica nanoparticles21–24can adequately exploit their favourable features in drug delivery. In a study by Lin et al.25, they developed a novel magnetic hybrid nanosystem for overcoming multidrug resistance of ovarian cancer through successfully delivering cisplatin and Fe3O4 NPs into the region of cancer cells. When magnetic nanoparticles substantially extravasate into cells via endocytosis, Fe3O4 NPs were metabolized inside the lysosome and the generated Fe2+/Fe3+ are transported across the endo/lysosomal membranes. Therefore, the homeostasis of ROS inside cancer cells is considerably broken through the Fenton reaction26–28, Fe2+ + H2O2 → Fe3+ + OH· + OH−, Fe3+ + H2O2 → Fe2+ + OOH· + H+. Excessive generation of ROS in the cancer cells contribute to the enhanced sensitivity of cells to chemotherapy drug and thus highly enhanced anticancer efficacy. However, the endogenous peroxide from induction of cisplatin is relatively insufficient to generate abundant ROS through Fenton reaction. As a result, exogenous sources are required to increase the concentration of intracellular peroxide. As an endoperoxide-containing sesquiterpene, artemisinin (ART) has special anti-cancer activity against breast, ovarian, and melanoma29–31. Further, in the presence of ferrous iron, the endoperoxide of ART broke and thus generated highly toxic radicals in tumor cells32,33. The generated ROS could induce mitochondrial apoptosis, thus targeting the DNA damage and repair system in traditional chemotherapeutic drug-resistant cancer cells34, but the efficiency of ART in killing 3



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cancer cells is very low in the absence of ferrous iron ions31,35–37. The poor solubility in water and iron-dependent feature greatly limit its application in the cancer treatment. Researches that Fe2+/Fe3+ mediated ART or cisplatin to increase anticancer efficacy have become a burgeoning research area30,38. However, to our best knowledge, the Fe2+/Fe3+ mediated combination therapy of cisplatin and ART to highly improve the efficiency of ROS generation for circumventing acquired cisplatin-resistance has never been reported. Hence, in present work, as showed in Fig 1, we develop a novel Fe3O4 NPs and cisplatin

dual-loaded

magnetic

liposome

based

on

cyclic(Arg-Gly-Asp-D-Phe-Lys)]-poly(ethyleneglycol) distearoylphosphatidylcholine (cRGD-PEG-DSPE),

artemisinin-cholesterol

(ART-Chol)

and

dipalmitoyl

phosphatidylcholine (DPPC). cRGD cyclic peptides are an effective target ligand and could be recognized by αvβ3 integrin receptors over-expressed on tumor cells such as A549 cells and MDA-MB-231 cells. The cisplatin and Fe3O4 nanoparticles were encapsulated into liposomes by hydrophobic interaction, while ART coupled with cholesterol served as based materials to form present liposomes for increasing ART loading capacity. The prepared cRGD-AFePt@NPs substantially extravasate into the interstitial fluid at tumor sites through enhanced permeability and retention (EPR) effects and magnetism of Fe3O4 nanoparticles. Then, the anchored cRGD cyclic peptides on the surface of liposomes are recognized for localizing liposomes into the cancer cell, under alternating magnetic field releasing cisplatin and ART and Fe3O4 nanoparticles. The Fe3O4 NPs produce the external sources of intracellular Fe2+/Fe3+ in acid lysosomes for the demands of Fenton reaction to generated a large amount of ROS, and the generated Fe2+/Fe3+ catalyzes H2O2 from induction of cisplatin and mediates cleavage of the endoperoxide bridge from ART for abundant ROS. The 4


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intracellular ROS from the above two sources could cause mitochondrial apoptosis and damage DNA and repair systems in the cisplatin-resistant A549 (A549/R) cells. The ROS production could also sensitize cisplatin, and significantly enhancing anticancer efficiency of cisplatin.

Figure 1. The schematic illustration on the formation of the magnetic liposomes cRGD-AFePt@NPs.

2. Experimental Section 2.1 Material HOOC-PEG2000-DSPE

copolymer

and

Amino-modified

cRGD

[cyclic

(Arg-Gly-Asp-D-Phe-Lys)] cyclic polypeptide was obtained from GL Biochem Co., Ltd. Cisplatin was purchased from Shandong Boyuan Pharmaceutical Co., Ltd (Jinan, Shandong, china). Iron(III) chloride hexahydrate (97%), ferric acetylacetonate (98%), phenyl ether (99%), benzyl ether (99%), 1,2-hexadecanediol (97%), oleic acid (90%), octadecylamine (97%), and 1-octadecene (95%) was obtained from Alfa Aesar. N-Acetyl-L-cysteine

(NAC),

Deferoxamine

mesylate

(DFO),

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, C18H16BrN5S, 97.5%) were used as purchased from Sigma-Aldrich. Annexin-V-FITC Apoptosis 5



Detection Kit was purchased from Bestbio Co. (Shanghai, China). Probe Assay Kit was purchased from Heliosense Biotechnologies Inc. (Xiamen, China). The rest of chemicals (analytical grade) were purchased from Sigma-Aldrich and used without further purification.

2.2 The synthesis of 8 nm Fe3O4 nanoparticles The 8 nm Fe3O4 nanoparticles were synthesized through a thermal deposition using oleic acid as the stabilizing agent following two previously modified method39,40. Typically, FeCl3·6H2O (20 mmol) and oleic acid (17 mL) were first dissolved in 100 mL methanol. 200 mL methanol solution containing 2.4 g NaOH was dropped into the above ferric salt solution under stirring vigorously. The observed brown ferric oleate was collected by centrifugation (3000 rpm, 10 min) following by washing 3 times with methanol. The obtained brown precipitate was dried under vacuum overnight at 50 °C. The brown ferric oleate (5.5 g) and oleic acid (1.5 g) were dissolved in 1-octadecene (30 mL) at 70 °C and subsequently the brown mixture solution was heated to 320 °C for 30 min under protection of nitrogen gas. The Fe3O4 nanocrystal was precipitated from the reaction mixture by adding 35 mL ethanol and the crude Fe3O4 nanoparticles were obtained by centrifugation (10,000 rpm, 15 min). The nanoparticles were further redissolved in 10 mL of hexane and purified by adding 35 mL of acetone, centrifuging at 8000 rpm for 15 min. After more than two times of washing step, nanoparticles were re-dispersed in anhydrous tetrahydrofuran (5 mg/mL). The Fe3O4 nanoparticles was characterized by X-ray diffraction analysis (XRD) (Rigaku, Japan; Rint-2100, Cu-Kα, 40 kV, 30 mA), JEM-100CXII transmission electron microscopy (TEM), and FT-IR spectrometer (Nicolet 5700, USA).

2.3 The synthesis of Art-Chol and cRGD-PEG-DSPE copolymer 6


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Under anhydrous and anaerobic conditions, 2.5 g of anhydrous piperazine and 40 mL of anhydrous dichloromethane were added into a 250 mL three-necked flask, and the mixture was stirred until completely dissolved. Then, 30 mL of anhydrous xylene and 2 g (7 mmol) of DHA were added to a 250 mL three-necked flask under the protection of Argon gas, stirred for 5 min at room temperature, and then added with 50 µL of anhydrous DMSO followed by continuous stirring for 5 min. Then, 0.65 mL of oxalyl chloride was slowly added, after the gas was generated, stirring was continued for 10 min. The mixture was slowly dropped into the flask containing the piperazine solution in dichloromethane, followed by TLC, and phosphomolybdic acid was developed. After the reaction was completed, the reaction was quenched with a mixture of 20 mL of saturated aqueous sodium carbonate solution and 20 mL of saturated brine and extracted with 40 mL of ethyl acetate three times. The organic phases were combined, dried over anhydrous sodium sulfate and the solvent was removed by spin-drying on an aqueous ammonia-treated silica gel column chromatography, V(MeOH): V(DCM) = 1: 20, as a yellow solid in 72% yield. As a result, artemisinin was coupled with piperazine and ART-PIP was obtained. In a 25 mL of flask, 0.15 g of cholesteryl chloroformate, 0.5 mL of triethylamine and 100 mL of DMF were added, and the mixture was stirred for 1 min. 0.11g of ART-PIP was added into above flask and the reaction was carried out overnight at room temperature. After completion of the reaction, the reaction was quenched with 15 mL of saturated NaHCO3 solutions. The reaction solution was extracted by DCM and the organic phase was washed twice with saturated sodium chloride solution, the organic phase was dried and the solvent was removed by spin column chromatography (EA / PE = 1/12) to yield 0.1 g of white solid in a 43% yield. The cRGD-PEG-DSPE copolymer was prepared as our previous work41,42. 7



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2.4 The preparation of magnetic liposomes The magnetic nanoparticles were prepared using solvent evaporation method following a published procedure43. 1.5 mg of DSPE-PEG-cRGD copolymer, 10 mg DPPC, 2 mg ART-Chol, and 1 mg of Fe3O4 nanoparticles was dissolved in 1 mL of THF. 600 µg cisplatin dispersed in 200 µL DMSO and THF solution then were added into 10 mL of ultrapure water under vigorous ultrasonic conditions using a Type 60 Sonic Dismembrator (Fisher Scientific). The obtained solution was sat under stream of nitrogen to allow evaporation of THF and formation of nanoparticles. The remanent was removed by three rounds of centrifugation (3000 rpm, 30 min) in centrifugal filter tubes (Vivaspin 20, 100 KDa cutoff size) and lyophilized for further tests. The lyophilized sample was characterized by FT-IR spectrometer (Nicolet 5700, USA), and UV-2600(Shimadzu, Japan), X-ray diffraction analysis (XRD) (Rigaku, Japan; Rint-2100, Cu-Kα, 40 kV, 30 mA). The cRGD-FePt@NPs without ART-Chol, cRGD-APt@NPs without Fe3O4, and AFePt@NPs without cRGD polypeptides were prepared using the same procedure except absence of additive. Rhodamine B labelled magnetic liposomes cRGD-AFeRhB@NPs were prepared by replace cisplatin with the same amount of rhodamine B and other steps are similar with the preparation of cRGD-AFePt@NPs. The successful encapsulate of characterization rhodamine B into cRGD-AFeRhB@NPs was determined using UV-2600 (Shimadzu, Japan)

2.5 Characterization of cRGD-AFePt@NPs The

amounts

of

ART-Chol,

cisplatin, and

Fe3O4 nanoparticles from

cRGD-AFePt@NPs The ART-Chol incorporated in hybrid nanoparticles was demonstrated by the 8


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changes of absorbance at maximum wavelength in nanoparticles solutions using UV-2600 (Shimadzu, Japan). The quantitative analysis with cisplatin and Fe3O4 nanoparticles was implemented using a standard inductively coupled plasma mass spectrometry (ICP-MS) procedure. Each sample was tested in triplicate. Briefly, 2 mg of cRGD-AFePt@NPs was dissolved into 10 mL deionized water followed by measured with ICP-MS to determine contents of Pt and Fe elements. Each sample was tested in triplicate. The quantitative analysis of ART-Chol was performed using UV-2600. The loading rates of cargos including cisplatin and Fe3O4 NPs were calculated by the following formula: DLC (wt %) =

( ) @

The size, Zeta potential, and morphology of several nanoparticles The size and polydispersity of cRGD-AFePt@NPs and cRGD-FePt@NPs, cRGD-APt@NPs, cRGD-NPs, AFePt@NPs were measured by dynamic light scattering (DLS). Briefly, a certain amount of nanoparticles water solution (1 mg /mL) was added into a square translucent quartz dish which was placed in the groove of the instrument. And the surface potential of several nanoparticles was determined using zeta plus zeta-potential analyzer (using Malvern Dispersion Technology Software) at room temperature. The surface morphology of cRGD-AFePt@NPs and cRGD-APt@NPs were observed using JEM-100CXII transmission electron microscopy (TEM).

The magnetic measurement of Fe3O4 NPs and cRGD-AFePt@NPs The magnetism of the Fe3O4 nanoparticles and cRGD-AFePt@NPs were measured by superconducting quantum interference device vibrating sample magnetometer (SQUID, Quantum Design MPMS). Typically, dried samples were 9



fixed in gelatin capsules which placed into transparent plastic drinking straws. The measurements were implemented under direct current magnetic fields in steady increments up to 0.8 Tesla at room temperature.

The assessment of the concentrations of Fe3+/Fe2+ in the Fenton reaction. The concentrations of Fe3+/Fe2+ in the Fenton reaction were assessed through determining the change of Fe3+/Fe2+ ratio before and after Fenton reaction using X-ray photoelectron spectroscopy (XPS) equipped with PHI 5400 ESCA System. Briefly, the 1.0 mg of cRGD-AFePt@NPs were dissolved into 10 mL of pH 5.4 PBS containing 100 µM of H2O2. After 12 h of reaction, the reaction solution was lyophilized and further analyzed using XPS.

The stability assessment The stability access of several liposomes was operated as our previous work41,44.

In vitro release of cisplatin from polymeric nanoparticles 3 mL of nanoparticles water solution including cRGD-AFePt@NPs, and cRGD-APt@NPs, was put into dialysis tubing (MW, 12 000), then submerged into 2 L of PBS buffer (pH = 7.4 or 5.0 ) as dissolution media at 37 °C in a New Brunswick Scientific C24 Incubator Shaker with or without a high-frequency magnetic field generator (HI-HEATER 5010, Daiichi High Frequency). The frequency of the alternating magnetic field was fixed to 360 kHz. At desired time intervals, a certain amount of PBS outside the dialysis tubing was removed for ICP-MS analysis, and replaced with fresh PBS. The results were obtained from triplicate measurement.

2.6 Cell culture A549 and A549/R cells were cultured in DMEM completed medium with 10 % fetal bovine serum (FBS) and 1% penicillin and 1% streptomycin. The cells were 10


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incubated under 5 % of CO2 atmosphere at 37 °C.

2.7 In vitro cellular uptake Detection of RhB-labelled cRGD-AFePt@NPs taken in by cells Intracellular cRGD-AFePt@NPs were detected using dual photon laser confocal microscope. Briefly, A549/R cells were seeded into confocal dish at a density of 1×105 cells and incubated for 24 h. Then, 20 µL of cRGD-AFePt@NPs suspension was dissolved into the 1mL of fresh completed medium, which was added into the confocal dish followed by 6 h and 12 h of incubation. At the end, the cells were washed with PBS two times, and 1µL 2,7-dichlorofluorescein diacetate (DCFH-DA) disperse 1 mL PBS was added into confocal dish containing 1mL of PBS and cells were incubated for 10min as previously described. Then, the dyes were removed by triplicate washing in PBS and cell were stained using DAPI at 2 µg mL-1 for 10min. Same steps were performed in order to clean the confocal dish, which was observed under dual photon laser confocal microscope.

Detection of intracellular liposomes in the lysosome and intracellular iron ions quantitative measurement A549/R cells were incubated using Rhodamine B labeled nanoparticles for 3 h incubation, and then the residual media were removed followed by washing three times using PBS. Then the lysosomes of cells were dyed by LysoTracker® Green DND-26. The cell nucleus was stained and the rest steps followed abovementioned procedure. A549/R cells were incubation for 6 h using cRGD-AFe@NPs and then the residual media were removed followed by washing three times with PBS. The A549/R cells were lysed by cell lysis buffer and collected by centrifugation (1000 rpm, 5min). 11



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Finally, the cells were dispersed into PBS and iron contents were determined using ICP-MS.

Qualitative analysis of the enhanced uptake efficiency of cRGD-AFePt@NPs with cRGD cyclic peptides The quantitative analysis was performed using flow cytometry. Briefly, A549/R cells were seeded into 6-wells at a density of 1 × 105 cells and continued to incubate for 24 h.

Then,

cells

were

treated

by

equal

concentration

of

RhB-labelled

cRGD-AFePt@NPs and AFePt@NPs for 12 h. the cells in the wells were digested and collected by centrifugation (1000 rpm, 5min) followed by washing with PBS and redispersing in 1 mL PBS. The samples were quickly conducted on a flow cytometer

Qualitative and quantitative analysis of the reactive oxygen species (ROS) generate in the cells For qualitatively detecting the formation of ROS, Dichlorofluorescindiacetate (DCFH-DA) was selected as a fluorescent probe for ROS that could be oxidized to the highly fluorescent dichlorofluorescein (DCF) by ROS. Typically, A549/R cells were seeded into confocal dish (1 × 105) and incubated for 1 day. Cells were treated for desirable time intervals with different concentrations of cRGD-AFePt@NPs, cRGD-FePt@NPs, cRGD-APt@NPs, and cRGD-Pt@NPs solution and further treated by DCFH-DA (10 µM) for 15 min after residual medium removed. Finally, the glass dishes were observed by CLSM. For quantitative analysis of intracellular ROS, A549/R cells were seeded into 6 wells plates at a density of 1 × 105 cells and incubated for 24 h. Then, cells were washed with PBS three times and treated by completed medium containing different concentrations of therapeutic agents for desirable time intervals. After removing 12


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unconsumed drugs, the cells in the wells were digested and collected by centrifugation (1000 rpm, 5min) followed by washing with PBS. Finally, the cells were incubated by DCFH-DA for 20 min and quickly conducted on a flow cytometer.

The evaluation of cytotoxicity The toxicity of Fe3O4 nanoparticles and cRGD-NPs were determined using MTT assay. Briefly, A549/R cells were subcultured into 96 wells plates at a density of 8000 cells each wall and cultured for 24 h. Then, different concentrations of Fe3O4 nanoparticles and cRGD-NPs without therapeutic drug were added into 96 wells plates and cells were incubated for 12 h. Then, the cells were exposed under AMF for 20 min and continued to incubate 12h. The uninternalized therapeutic agents were washed by PBS and 100 µg completed medium containing MTT reagents was added into each well. After 4 h of incubation, 150 µg DMSO replaced the completed medium and the plate was subjected to analyze the absorbance at 490 nm using a microplate reader (Thermo Scientific, Varioskan Flash, Waltham, MA, USA). All date was measured in triplicate. The toxic of cisplatin, cRGD-Pt@NPs, cRGD-APt@NPs, cRGD-FePt@NPs, and cRGD-AFePt@NPs with or without AMF exposure also were accessed using the same way as previously mentioned.

The quantitative analysis of intracellular cisplatin. The quantitative analysis of intracellular iron ions and cisplatin were performed by ICP-MS. A549/R cells were subcultured into 6 well plates at a density of 1 × 106 cells each wells and cultured for 24 h. Then, cRGD-AFePt@NPs at 10 µM cisplatin concentration in media were added into wells and incubated for 6 h. After cells were lysed and collected by centrifugation, the cells were re-dispersed by PBS followed by 13



measurement of Pt content by ICP-MS.

The evaluation of cell apoptosis. A549/R cells from the exponential phase of the culture were seeded into 6 wells plates at a density of 1 × 105 cells each well. After 24 h of incubation, the residual medium was replaced with different concentrations of therapeutic agents including (cisplatin, cisplatin plus ART, cisplatin plus ART and Fe3O4 NPs, AFePt@NPs, cRGD-AFePt@NPs, and cRGD-AFePt@NPs plus AMF) dissolved fresh completed medium with the platinum concentration in the culture medium regulated to the same value of 10 µM and incubated for 24 h. Then, the cells in the wells were trypsinized and collected with centrifugation (1000 rpm, 5 min). The cells dispersed in buffer were stained with Annexin V-FITC Apoptosis Detection Kit II (BD Pharmingen, San Jose, USA). Finally, the apoptosis kit was removed and re-dispersed into buffer followed by performing flow cytometric assay on FACS Calibur (BD Biosciences, USA).

3. Results and discussion 3.1 The synthesis and characterization of Fe3O4 nanoparticles and ART-Chol The hydrophobic and monodisperse Fe3O4 NPs nanoparticles was synthesized using a thermal decomposition method following a published work. The TEM image of Fe3O4 NPs in Fig 3A showed uniform size with 8±0.19 nm. The FT-IR image in Fig S1A determined the surface identity of Fe3O4 NPs. The signals of 580 cm-1, 1583 cm-1 and 1648 cm-1 indicated the iron-oxygen bond vibration, C=C and C=O from oleic acid, respectively. The XRD pattern of Fe3O4 NPs from Fig S1B showed the obvious peak with (331), (400), (333), and (440) assigned to each crystal faces, respectively. And the relatively broad peak also indicated the nanoscale crystallization 14


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of Fe3O4 NPs. As showed in Fig 2, The ART-Chol was synthesis via two-step chemical reaction. First, the successful attachment of piperazine to ART was characterized via FT-IR, NMR, and high-resolution MS. According to Fig S2A, the absorption peak at 3000 cm-1 and 2800-3000 cm-1 were regarded as -NH- from hydrogen bond coupling and hydrogen from skeleton of piperazine, respectively. The successful conjugation of cholesterol to ART-PIP was confirmed via the characteristic peak of 2800-3000cm-1 belonging to -C=C- and alkyl peak and of 1600-1700cm-1 from vibration peak of carbonyl. The further structure confirmation of ART-PIP and ART-Chol was from 1H NMR spectra in Fig S2B, the chemical shift of each hydrogen was labelled. In addition, in Fig S2C, the MS signal at 765.5683 Da [MW (theoretical value) = 765.57 Da] powerfully testify the structure of ART-Chol.

Figure 2. The overview of synthesis of the ART-PIP and ART-Chol. COCl:oxalyl chloride; TEA: triethyl amine.

3.2 The synthesis and characterization of magnetic liposomes 15



The cRGD-AFePt@NPs magnetic liposomes were prepared by a solvent inversion method. THF was selected as inversion because its non-polar and water miscible nature allowed for the mixture of monodisperse oleic acid coated magnetite nanoparticles and lipids. The mixture then was dropwise added into a larger volume of aqueous phase and then dialyzed against volatile THF. In this study, the cRGD-PEG2000-DSPE block copolymer, ART-Chol, and DPPC was employed for magnetic liposomes formation encapsulating Fe3O4 NPs and cisplatin. The preparation of cRGD-PEG2000-DSPE was from our previous work. According to the Fig 3C, the hydrodynamic size of cRGD-AFePt@NPs magnetic liposomes from DLS was 108nm. The PDI was 0.233 that indicated a uniform size distribution and good colloidal stability, and the TEM image (Fig 3B) indicated the prepared nanoparticles exhibited a spherical shape with a uniform size consistent with the DLS results. The drug loading capacity (DLC) of cisplatin and Fe3O4 was investigated using ICP-MS, and the results was 4.0% and 7.7%, respectively. The DLC of ART was determined by the UV-2600. According to the Fig S3, the loading ratio of ART in the magnetic liposomes reached up to 10.2%. The zeta potential of cRGD-AFePt@NPs, cRGD-APt@NPs, cRGD-FePt@NPs and AFePt@NPs were -25.6, -24.9, -40.8, -37.6 mV (Fig 3D). The results were mainly because the introduction of cRGD peptide neutralized the surface charge. And the ART-PIP on the surface of magnetic liposomes containing the tertiary amine allowed for complexing proton in the aqueous solution and then neutralized the surface charge.

16


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Figure 3. A). The TEM image of Fe3O4 NPs. Inset: The corresponding SAED pattern of Fe3O4 NPs; B). The TEM image of prepared cRGD-AFePt@NPs; C). Size distributions of cRGD-AFePt@NPs in BSA-free PBS by DLS. D). Zeta potential of cRGD-AFePt@NPs, cRGD-APt@NPs, cRGD-FePt@NPs, and AFePt@NPs. During the process of formulation, as showed in Table 1, three different concentrations of Fe3O4 nanoparticles was adjusted to determine the desirable cargo content that was loaded into the cRGD-AFePt@NPs magnetic liposomes. The DLS results indicated the dramatical increase of hydrodynamic size from 80.6 nm to 141.4 nm and PDI from 0.159 to 0.324 when Fe3O4 nanoparticles were loaded. And the effect of Fe3O4 nanoparticles concentration made a huge change in the structural morphology and size. Similarly, to obtain a favorable drug content loaded in magnetic liposomes and a good colloidal stability, the size and size distribution and drug 17



Page 18 of 38

loading ratio of magnetic liposomes prepared by different concentrations of ART-Chol were investigated. As showed in Table 1, with the gradual increase of ART-Chol drug concentration, the size and PDI of the prepared liposomes changed slightly, but the loading of Fe3O4 nanoparticles and cisplatin gradually decreased. We believed that cholesterol in the process of liposomes formation mainly was embedded into the voids of bilayer of phospholipid and thereby the admixture of cholesterol reduced the contents of hydrophobic agent such as Fe3O4 NPs or cisplatin.

Table 1. The physicochemical parameters including size, polymer dispersity index(PDI), and drug loading capacity (DLC) of cRGD-AFePt@NPs in different feed mass of additive.

Feed mass/mg

DLC (wt %), Mean size/nm

PDI

Fe3O4 NPs: ART-Chol: lipid

Fe3O4 NPs; ART; cisplatin

0: 2: 8

80.6

0.159

0; 7.54; 5.7

0.5: 2: 8

92.7

0.192

0; 6.63; 5.2

1: 2: 8

108.4

0.233

7.7; 5.12; 4.0

2: 2: 8

141.4

0.324

9.4; 2.7; 1.7

1: 0: 8

104.5

0.204

9.2; 0; 4.8

1: 1: 8

102.9

0.225

7.9; 4.25; 4.7

1: 2: 8

108.4

0.233

5.6; 5.12; 4.0

1: 3: 8

109.4

0.246

1.7; 7.69; 2.4

Further, the structural morphology, surface identity, and magnetic nature of 18


Page 19 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


cRGD-AFePt@NPs were investigated by XRD (Fig S4A), FT-IR (Fig S4B), and SQUID (Fig 4A). The FT-IR characteristic peaks of Fe3O4 NPs with 580, 1583, 1650 cm-1 were seen from cRGD-AFePt@NPs, and the crystal faces with (331), (400), (333), and (440) from XRD pattern could been also seen in both Fe3O4 NPs and cRGD-AFePt@NPs, which indicated successful encapsulation of Fe3O4 NPs into magnetic liposomes. In addition, the saturation magnetization (MS) of Fe3O4 nanoparticles and cRGD-AFePt@NPs, were 40.1 and 30.3 emu/g by SQUID, respectively, and both had little coercivity (HC) with(0.03KOe), the results also revealed the above conclusion. Sequentially, the concentrations of Fe2+/Fe3+ in the Fenton reaction were detected by XPS. The loaded Fe3O4 produced Fe2+/Fe3+ sources under weak acid environment, and the theoretical scale of Fe2+/Fe3+ is 33.3 %. As showed in Fig S5, the major component Fe3+ in Fe 2p3/2 showed enhanced rate from 61.5% to 72.9% after 12 h of reaction. The main reason may be that a part of Fe2+ was oxidated into Fe3+ through Fenton reaction, while the conversion kinetic constant of Fe3+ to Fe2+ was relatively lower. In a study by He et al., the main drawback of Fenton reaction derived from the comparatively low recovery efficiency45, which submitted to the assumption. The stability of prepared cRGD-AFePt@NPs was crucial to deliver therapeutic agents into disease site without leakage in the region of healthy tissue. As showed in Fig 4B, we monitored the variation tendency of size distribution and PDI of the cRGD-AFePt@NPs submerged into H2O, PBS, and RPMI-1640 medium dispersion medium. It could be observed that the vibration on size and PDI of cRGD-AFePt@NPs slightly increased after 5 days and the results indicated the remarkable stability of prepared magnetic liposomes in the physiological media, which ensured the discreet packaging of therapeutic agents inside liposomes. 19



Page 20 of 38

3.3 In vitro release evaluation of AMF-triggered drug release. To access efficiency of alternating magnetic field (AMF) irradiation on the drug release,

the

vitro

cisplatin

release

profiles

of

cRGD-AFePt@NPs

and

cRGD-APt@NPs under different pH conditions (pH=5.0 or pH=7.4) was observed. As showed in Fig 4C, at pH 7.4, almost 55% of loaded cisplatin was released from cRGD-AFePt@NPs exposed by AMF over a period of 24 h, whereas less than 20% of cisplatin was liberated from liposomes without treatment of AMF; At pH 5.0 (Fig 4D), more than 80% loaded cisplatin from liposomes treated by AMF was observed and only 50% of cisplatin without exposure of AMF was released after 10 h. By contrast, cRGD-APt@NPs in the absence of Fe3O4 NPs under pH 5.0 or pH 7.4 both showed similar release capacity with or without exposure of AMF. The results were mainly attributed to incompleteness of liposomes structure due to local hyperthermia generated from magnetic nanoparticles by hysteresis loss and/or Néel relaxation. The generated local heat increased the liposomes permeability and thus accumulated drug release. Meanwhile, cRGD-AFePt@NPs without exposure AMF under pH 5.5 exhibited a relatively prompt cisplatin release (>56%, within 24 h) than that liposomes under pH 7.4 (>20%, within 24 h). The week acid environment facilitated breakage of amide bonds and ester bonds from fundamental self-assembly materials such as amphiphilic polymer and phospholipid. Afterwards, the variation about the hydrodynamic size and PDI of cRGD-AFePt@NPs exposed by AMF in pH 5.0 PBS medium was also investigated. According to Fig S6, the highly enhanced PDI and the hydrodynamic size at acid environment after 12 h environment and exposure of AMF concordantly contributed to the rupture of magnetic liposomes and thereby sharply released delivery cargos. Taken together, the enhanced release of cisplatin could be achieved by the 20


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combination of lower pH- and heat-triggered behavior of cRGD-AFePt@NPs exposed by AMF at pH 5.0.

Figure 4. A). Magnetic hysteresis loop as a function of applied magnetic field measured at room temperature for Fe3O4 NPs and cRGD-AFePt@NPs; B). Variation curve of Particle size (black) and PDI (green) of cRGD-AFePt@NPs in H2O, PBS, RPMI-1640, and FBS media; The cumulative release kinetics of cRGD-AFePt@NPs and cRGD-APt@NPs in pH 7.4 PBS (C). and pH 5.0 PBS (D). at room temperature with or without AMF exposure.

3.4 The access of intracellular therapeutic agents and generated ROS in vitro To access the self-accelerating drug release efficiency and ROS generating capability of cRGD-AFePt@NPs, the confocal laser scanning microscopy (CLSM) was used to observe the specific cell sites of nanoparticles and generated ROS in our study. As showed in Fig 5, with cellular co-incubation experiments of Rhodamine B 21



Page 22 of 38

labelled magnetic liposomes, the red fluorescence was found in A549/R cells after 3 hours and the fluorescence intensity continuously strengthened with time and even gradually spread to the cytoplasm and the nucleus, indicating that the prepared cRGD-AFePt@NPs were successfully taken into the cells and gradually released loaded cargos, such as cisplatin, ART, and Fe3O4 nanoparticles. Meanwhile, the intracellular

generated

ROS

was

examined

by

fluorogenic

substrate

2,7-dichlorofluorescein diacetate (DCFH-DA) which could be oxidized to the dichlorofluorescein (DCF) with highly green fluorescent by ROS. With the extension of incubation time, the green fluorescence gradually heightened and the conspicuous green fluorescence of DCF in A549/R cells abound in entire cells after 12 h. Afterwards, we investigated the enhanced uptake efficiency of cRGD-AFePt@NPs with cRGD cyclic peptides. As showed in Fig S7, compared with the AFePt@NPs, A549/R cells treated with cRGD-AFePt@NPs after 12 h exhibited 2.25-fold fluorescence intensity, indicating the introduction of cRGD to surface of magnetic liposomes substantially increased the uptake of therapeutic agents due to high affinity between cRGD cyclic peptides and αvβ3 integrin overexpressed on the A549 cells cytomembrane

22


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Figure 5. Confocal laser scanning of RhB-labelled cRGD-AFePt@NPs labeled after 3, 6, and 12 h culture with A549/R cells. The green fluorescence comes from DCF oxidized by intracellular ROS. The red fluorescence comes from rhodamine B. The blue fluorescence comes from a nuclear dye DAPI. Scale bar: 20 µm Afterwards, since the generation of labile iron ions is crucial in the mediation of cisplatin and ART to produce abundant ROS in the cells, the labile iron ions were from the degradation products of Fe3O4 in the acid lysosomes. As showed in Fig S8, after 3 h of treatment of cRGD-Fe@NPs in the A549/R cells lines, the majority of fluorescence of cRGD-Fe@NPs (red) was localized in the lysosome(green), which revealed that the cRGD-Fe@NPs in the acid lysosome could been degraded into labile iron ions after being endocytosed. It could be concluded that cRGD-Fe@NPs were localized into cancer cells lysosome within 3 h of treatments. However, cRGD-Fe@NPs in the week acid lysosome gradually diffused into cytoplasm and after 12 h the red fluorescence was brimming in whole cells (Figure 5), indicating the prepared cRGD-AFePt@NPs could be effectively endocytose into the acid lysosome and diffused time-dependently into the cytoplasm accompanied by release of delivery 23



Page 24 of 38

cargos. Sequentially, the quantification of intracellular labile iron ions in A549 and A549/R after 6 h of incubation was performed using ICP-MS. According to Fig 6B, the concentration of labile iron ion in the A549 and A549/R cells treated by cRGD-Fe@NPs was much higher than those in the absence of treatments. The intracellular ROS augmentation capability was accessed by CLSM and flow cytometry analysis. Firstly, the co-incubated time was fixed at 6 h, and then we observed the green fluorescence intensity when cells are incubated with four different liposomes

formulation

including

cRGD-AFePt@NPs,

cRGD-APt@NPs,

cRGD-AFe@NPs, cRGD-FePt@NPs, and cRGD-Pt@NPs. As showed in Fig 6A, when the A549/R cells were treated with ART and Fe3O4 NPs loaded cRGD-AFePt@NPs, the green fluorescent intensity was obviously stronger than the treatment alone with cRGD-APt@NPs, cRGD-AFe@NPs or cRGD-FePt@NPs. It was worth noting that the incubation of only cisplatin-loaded cRGD-Pt@NPs to cells showed also weak green fluorescence due to the induction of cisplatin to NOX. Sequentially, the quantitative analysis of intracellular ROS generation was performed by flow cytometer. As showed in Fig 6C, compared with cRGD-Pt@NPs alone treating cells based by traditional DDS only encapsulating cisplatin, A549/R cells incubated

with

cRGD-AFePt@NPs,

cRGD-APt@NPs,

cRGD-AFe@NPs,or

cRGD-FePt@NPs showed a relative ROS levels of about 5.7-, 1.9-, 2.4-, and 2.8-fold after 12 h of incubation, respectively. The desired results adequately validated that the exogenous addition of intracellular Fe2+/Fe3+ concentrations combined with ART or cisplatin amplified ROS generation, while the mixture of ART and Fe2+/Fe3+ combined with cisplatin could highly enhance the generation efficiency of ROS in the cells. However, when cells were treated with cRGD-AFePt@NPs and iron chelator deferoxamine mesylate (DFO, 100 µM) under AMF, the intracellular ROS generation 24


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was completely interdict, which effectively demonstrated the absence of Fe2+/Fe3+ ion in the cells badly hamper the occurrence of Fenton reaction and further decrease the ROS production.

Figure 6. A). Evaluation of the generated ROS capacity of different formulated liposomes; Scale bar: 20 µm. B). Free iron levels in the A549 and A549/R cells after 6 h of incubation. C). Quantitative analysis of A549/R cells treated with different formulated liposomes after 6 h of incubation. Data is shown as mean ± SD (n = 3). Significance is defined as *P < 0.05, **P

Iron oxide nanocarrier-mediated combination therapy of cisplatin and

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