Near-Infrared Responsive Bimetallic Nanovesicles for Enhanced

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Near-Infrared Responsive Bimetallic Nanovesicles for Enhanced Synergistic Chemo-Photothermal Therapy Liyao Luo, Hongyu He, Chunhui Li, Yaqian He, Zining Hao, Shuai Wang, Qianqian Zhao, Zhiwei Liu, and Dawei Gao ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01534 • Publication Date (Web): 08 Feb 2019 Downloaded from http://pubs.acs.org on February 8, 2019

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ACS Biomaterials Science & Engineering

Near-Infrared Responsive Bimetallic Nanovesicles for Enhanced Synergistic Chemo-Photothermal Therapy Liyao Luo1,3, Hongyu He1, Chunhui Li1, Yaqian He1, Zining Hao1,3, Shuai Wang1, Qianqian Zhao1, Zhiwei Liu1, Dawei Gao1,2*

1Applying

Chemistry Key Lab of Hebei Province, Department of Bioengineer,

Yanshan University, No.438 Hebei Street, Qinhuangdao, 066004, China. 2State

Key Laboratory of Metastable Materials Science and Technology, Yanshan University, No.438 Hebei Street, Qinhuangdao 066004, P. R. China.

3Hebei

Province Asparagus Industry Technology Research Institute, No.438 Hebei Street, Qinhuangdao, 066004, China.

*Corresponding author: Prof. Dawei Gao, Tel: (+86)13930338376. E-mail: [email protected]

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Abstract Limited therapeutic effects and obvious side effects are two critical problems affecting tumor therapy. Herein, we designed an ingenious nanocarrier, platinum/gold bimetallic nanoshell coated triptolide liposomes (Pt@Au-TP-Lips), to achieve enhanced chemo-photothermal therapy against cancers. Compared to conventional gold nanoflower structures, the platinum/gold bimetallic (Pt@Au) core-shells exhibited broader near-infrared (NIR) absorption due to the ultra-strong plasmonic coupling effect. With NIR light irradiation, the Pt@Au nanostructure could efficiently and sustainably convert light energy into substantial heat. The ultrahigh photothermal conversion efficiency (56.5%) of Pt@Au-TP-Lips was significantly higher than that of gold nanoflowers (35.7%). Specially, hyperthermia could induce the phase change of liposome membrane to accelerate the release of triptolide (TP), meanwhile, it could ablate tumor cells directly and facilitate cellular uptake of drug for enhancing chemotherapy. More importantly, owing to cooperation of TP and platinum, the Pt@Au-TP-Lips exhibited significant tumor growth suppression with a high inhibitory rate of 90.7%, achieving superior chemo-photothermal combination therapy. This work provides new insight into the development of cooperative theranostic agent for oncotherapy. Keyword: synergistic chemo-photothermal therapy, platinum/gold bimetallic nanoshells, triptolide, ultrahigh photothermal conversion efficiency, tumor


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Introduction Over the past decades, cancer has been a major health threat in the worldwide1. Since conventional therapeutics often causes severe side effect and low therapeutic efficiency, the development of combining chemotherapy with other therapies has currently been proposed to improve the outcomes of cancer treatment2-3. Photothermal therapy (PTT) is an emerging treatment regimen that depends on hyperthermia generated by light-absorbing materials transforming NIR light energy into heat4. It has drawn extensive attention due to its noninvasive, highly specific spatial-temporal selectivity, and controllable treatment features5. Unfortunately, as heat distribution cannot be so uniform in whole tumor tissues, PTT alone is unable to ablate tumor cells6. Recent advances have shown that the combination of PTT with chemotherapy could achieve a complementary effect to improve therapeutic efficiency and reduce side effect of chemotherapy7-9. Up to date, a host of photothermal agents, including gold nanoparticles10, carbon-based materials11-12, metal sulfide nanoparticles13, and semiconductor nanoparticles14-15, have been widely developed for PTT. In particular, gold-based nanomaterials have been selected as state-of-the-art efficient agents for PTT owing to the localized surface plasmon resonance (LSPR), minimum biological toxicity and chemical inertness in the past decade16-18. Quite several classes of nanomaterials (e.g., gold nanorods19-20, nanoshells21, nanocages22, nanostars23) that display strong absorbance and significant photothermal effect in the NIR region have been widely explored. Inspired by gold nanostructures, more and more interests have been directed to incorporate a second metal into gold colloidal platforms to achieve greater plasmonic modifiability or multifunctionality24. In the case of bimetallic alloyed colloids, the dielectric constant will be distinct from either of the individual metals, which manipulate the plasmonic properties and further improve optical performance of colloids25. A bimetallic Ag@AuNPs is proved to be a more biostable and efficient plasmonic hybrid photothermal agent than the single metal nanoparticles26. Additionally, composition of bimetallic alloyed colloids will give the gold platforms



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multifunctionality. A core-shell Pd@Au nanoplates are endowed with new functions of PA imaging and CT imaging, not just PTT emerged by Au nanostructures27. Platinum nanomaterial is an excellent nanomaterials that possess various outstanding

performances

such

as

catalytic

activity,

contrast

agents

and

radiosensitizers28. Especially, with a higher absorption/scattering ratio, platinum nanoparticles (PtNPs) exhibits an excellent photothermal effect. In addition, PtNPs have been proved to induction of apoptosis and cell cycle arrest. Pioneering works have shown that PtNPs (>1 nm) have cytotoxic effect on cancer cell lines, possibly resulting from the formation of Pt ions that disrupt DNA structures29-30. Due to the unique properties, when platinum as a supplementary agent mingles with other metals, the surprising synergetic effects are emerged. For instance, FePt core-shell nanoparticles showed excellent electro catalytical activity owing to the electronic and structural effects between bimetallic core-shell structure31. PtRu bimetallic nanocomplexes exhibit an excellent photothermal effect through the combination of Pt and Ru. The possible reasons might be the bimetallic nanocomplexes couple the LSPR properties of Pt and Ru to a maximum degree, accordingly enhance the absorbance of the incident electromagnetic wave and heighten the photothermal effect over monometallic nanostructures32. As the above, many researches have indicated that the introduction of Pt with other metals enhance the original performance or endow a new property33-35. So, based on these advantages, we attempt to incorporate Pt with Au to obtain a new platform and exploit its properties for antitumor application. In the study, we firstly presented a novel Pt@Au-TP-Lips for an enhanced chemo-photothermal therapy with a highly efficient and environmentally friendly method. The strategy used to construct the nanosystem was illustrated in Scheme 1. TP, as a natural antitumor drug, was loaded in the liposomes to develop drug bioavailability and prolong systemic circulation time. Due to the unique LSPR properties of the bimetallic nanoparticles, Pt@Au-TP-Lips exhibited higher photothermal conversion efficiency than that of gold nanoflower coated TP liposomes (AuNF-TP-Lips), which made it an excellent agent for PTT. Upon NIR irradiation,


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Pt@Au-TP-Lips could rapidly covert light energy into heat and trigger the on-demand release of drugs in spatial and temporal. Moreover, with the advantage of platinum antitumor ability, the drug delivery system reduced dosage of TP and achieves a better therapeutic effect. Simultaneously, the in vitro and in vivo anti-tumor studies demonstrated a superior inhibitory effect on tumor growth in the Pt@Au-TP-Lips by chemo-photothermal therapy than that treated by single treatment.

Materials&Methods Materials, Instrument characterization and cell culture were presented in supporting information. Methods Preparation of TP-Lips In this work, TP-Lips were prepared by an ethanol injection method according to our previous research36. Briefly, 1.3mg of TP, 6mg of cholesterol and 50mg of soya lecithin were completely dissolved in 4mL of ethyl alcohol as the lipid phase. 10mL of phosphate-buffered saline (PBS, pH6.5) containing 10μL Tween-80 and 4.5mg of polyethylene glycol was heated to 43°C as the aqueous phase. Then the lipid phase solution was added drop wise into the aqueous phase under continuously stirring for 1h to volatilize the ethyl alcohol and obtain TP-Lips. Subsequently, 5.5mg of glutathione (GSH) dissolved in deionized water was added dropwise into TP-Lips to further modify. Finally, the suspension was purified through dialyzing to remove the free TP and GSH. Preparation of AuNF-TP-Lips AuNF-TP-Lips were synthesized following the seeds growth method4. In a typical preparation, gold seeds were prepared by using NaBH4 to reduce Auric chloride (AuCl3) in aqueous phase. Then the synthesized gold seeds were mixed with the modified TP-Lips (1:1 in volume ratio) and reacted for 20 h to obtain gold seeds-attached TP-liposomes (AuNPs-TP-Lips). For further growth of gold nanoflowers, 15mg of potassium carbonate (K2CO3) was fully dissolved in 10 mL of deionized water. Then 20mM AuCl3 was diluted into 2mM with the K2CO3 solution.



The transparent mixture initially appeared yellow and slowly became colorless after 30minutes, indicating the formation of gold hydroxide. Subsequently, the gold hydroxide (500 μL) and fresh hydroxylamine hydrochloride (50μL, 100 mM) was added into the solution of AuNPs-TP-Lips and continuously incubated under moderate stirring for 20 h to harvest AuNF-TP-Lips. Preparation of Pt@Au-TP-Lips AuNF-TP-Lips were further mixed with 300μL chloroplatinic acid (H2PtCl4) (1 mM) and 40 μL ascorbic acid (60 mM). The mixture was shaken vigorously and incubated overnight at room temperature. The color of the solution changed from glaucous to dark gray, suggesting the formation of Pt@Au-TP-Lips. Finally, a purified Pt@Au-TP-Lips solution was achieved by centrifugation at 8000 rpm for 20 min. Photothermal Experiments of Pt@Au-TP-Lips To explore the photothermal effect of Pt@Au-TP-Lips, 500 μL aqueous solutions of AuNF-TP-Lips and Pt@Au-TP-Lips were exposed to 808 nm laser at a power density of 2 W cm-2 for 10 min. The thermographic images were recorded by an IR thermal camera showing the temperature changes of the solution at selected time intervals (1 min). 500μL PBS was irradiated at the same condition as negative control. Controllable release of drugs switched by NIR Laser The release of drugs from the Pt@Au-TP-Lips was performed using a dialysis method against PBS (pH7.4) at 37°C. Briefly, 1mL Pt@Au-TP-Lips were placed in dialysis bags (3500D) and maintained in 37°C bath with constant stirring. At the time periods of 2, 6, 12 h, the 808 nm laser irradiation (1.5 W cm-2, 3 min) was performed for the laser groups. The samples (1 mL) with or without NIR irradiation were taken out from the dialysis bags at designated times (1, 2, 4, 6, 8, 12, 24h). The concentration of TP in the supernatant was determined using a spectrophotometer. To analyze Pt release, the samples were soaked in aqua regia overnight, centrifuged and measured using inductively coupled plasma mass spectrometry (ICP-MS) to obtain the Pt concents of the samples.


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Cell uptake To demonstrate the efficient cellular uptake of nanoparticles, coumarin-6 (Cou6) with green fluorescence signal was encapsulated in the liposomes to trace location of the nanoparticles in the cells. Briefly, Hela cells were placed into 6-well plates at a density of 3×105 cells per well and allowed to adhere prior to addition of samples. Then the cells were respectively treated with free Cou6, Pt@Au-Cou6-Lips and Pt@Au-Cou6-Lips+Laser (1.5W cm-2, 3 min) for 1 h, 4 h, 8 h, respectively. After the above treatment, the cells were adequately rinsed with PBS to remove the residual free nanoparticles. Subsequently, the cells were fixed with 4% paraformaldehyde solution for 10 min and washed again with PBS, and the fluorescence imaging were obtained by inverted fluorescence microscope (LWD300-38LF, China) to exhibit the cell uptake of the nanoparticles. The quantitatively analysis of intracellular Pt@Au-Cou6-Lips was measured by flow cytometry (FCM, BD FACSCalibur, USA). In vitro anti-tumor effect of Pt@Au-TP-Lips In this work, the anti-tumor ability of Pt@Au-TP-Lips was assessed by measuring the cell viability using the standard 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Briefly, Hela cells were seeded in 96-well plates at 5.0×104 cells per well in 100μL Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS and cultured for 24h, 100μL of TP, platinum/gold bimetallic

nanoshell

coated

liposomes

(Pt@Au-Lips),

Pt@Au-TP-Lips

and

Pt@Au-TP-Lips+Laser were added the wells separately at various TP or Pt concentrations and cultured for 4h. An 808nm laser (1.5 W cm-2) was utilized to irradiate the cells treated with Pt@Au-TP-Lips+Laser for 3 min. The gold nanoflower coated liposomes (AuNF-Lips) + Laser group was given the same treatment as a control of sole photothermal therapy. After the cells continued culturing for 20h, the medium was discarded, and replaced with 200μL MTT in each well for sequentially culturing 4h. Subsequently, the MTT solution was removed, and 150 µL of dimethyl sulfoxide was added to each well to dissolve the blue formazan crystals produced by live cells. The cell viability was determined at 490 nm absorbance using a micro-plate reader (SpectraMax M2e). Additionally, the cytotoxicity of formulations was also



assessed using the standard protocol of fluorescence staining of live cells by 3,6-Diacetoxyfluoran (FDA). In vivo chemo-photothermal combination treatment of tumor All animal experiments were performed under the Statute of Experimental Animal Ethics Committee of Department of Bioengineering by Yanshan University. The U14 tumor models were generated by subcutaneous injection of 2 × 106 cells in 200μL saline into the right forelimb armpit of Female Kunming mice (20 ± 2g, Vitalriver Laboratory Animal Center). When the tumor volume reached ≈100 mm3, the tumor bearing mice were randomly separated to 6 groups (n = 5): (a) control, (b) free TP (1.4mg kg-1 of TP), (c) AuNF-Lips+Laser (808nm, 1.5W cm-2, 3min), (d) Pt@Au-Lips, (e) Pt@Au-TP-Lips (1.4mg kg-1 of TP), (f) Pt@Au-TP-Lips+Laser (1.4mg kg-1 of TP, 808nm, 1.5W cm-2, 3min). Each dose was given every 2 day by intratumor injection. 200 μL Pt@Au-TP-Lips were intratumorally injected to the tumor bearing mice with a fixed Au dose at 60 μg/mL (n=3). At different time points post administration (3h, 6h, 24h, 3day and 7days), the mice were sacrificed. The major organs (heart, liver, spleen, lung, kidney) and tumors as indicated were harvested for Au content measurements by atomic absorption spectrophotometer (AA-6800, shimadzu, Japan). The data were expressed as Au mass per gram of tissue (µg/g). The tumor temperature changes of mice were monitored by an infrared thermal imaging camera. The tumor sizes were measured every other day with a digital caliper and calculated as volume using the formula of wildth2×length/2. Body weight of the animals was also measured during the experiment. After the mice were anaesthetized by ether, their blood was drawn from the eye socket 14 days post-injection. Serum biochemical parameters, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactic dehydrogenase (LDH) and blood urea nitrogen (BUN) indexes, were examined. One day after the last injection, the animals were sacrificed under anesthetic, and the major organs were sectioned, and hematoxylin-eosin staining (H&E) stained. Statistical Analysis


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Quantitative data were presented as a mean value with its standard deviation indicated (mean ± SD). Statistical significance (*P

Near-Infrared Responsive Bimetallic Nanovesicles for Enhanced

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