shell nanospheres for pH and near-infrared-light induced release of 5-fluorouracil and chemo-photothermal therapy

Accepted Manuscript Title: Facile synthesis of gold nanorods/hydrogels core/shell nanospheres for pH and near-infrared-light induced release of 5-fluorouracil and chemo- photothermal therapy Author: Hui Jin Xifeng Liu Rijun Gui Zonghua Wang PII: DOI: Reference:

S0927-7765(15)00133-2 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.02.049 COLSUB 6940

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

10-12-2014 29-1-2015 26-2-2015

Please cite this article as: H. Jin, X. Liu, R. Gui, Z. Wang, Facile synthesis of gold nanorods/hydrogels core/shell nanospheres for pH and near-infrared-light induced release of 5-fluorouracil and chemo- photothermal therapy, Colloids and Surfaces B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.02.049 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Facile synthesis of gold nanorods/hydrogels core/shell nanospheres for

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pH and near-infrared-light induced release of 5-fluorouracil and chemo-

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photothermal therapy

Hui Jina, Xifeng Liub, Rijun Guia,*, Zonghua Wanga,*

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College of Chemical Science and Engineering, Collaborative Innovation Center for Marine Biomass Fiber, Shandong Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, the State Key Laboratory

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Growing Base of Fiber Materials and Modern Textiles, Qingdao University, Shandong 266071, PR China b

Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN 55905, USA

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* Corresponding author. Tel./fax: +86 532 85950873.

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E-mail addresses: [email protected] (R. Gui); [email protected] (Z. Wang) 1

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ABSTRACT We described a facile synthesis of pH and near-infrared (NIR) light dual-sensitive core/shell hybrid nanospheres,

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consisting of gold nanorods (GNR) as the core and poly(N-isopropylacrylamide-co-methacrylic acid) as the shell,

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p(NIPAM-MAA). The resultant GNR/p(NIPAM-MAA) nanospheres showed a core/shell structure, with an average

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diameter of ~110 nm and a strong longitudinal surface plasmon band at NIR region. Due to the photothermal effect

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of GNR and pH/thermal-sensitive volume transition of p(NIPAM-MAA) hydrogels, the nanospheres with loading

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of 5-fluorouracil (5-FU) by electrostatic interactions were developed as a smart carrier for pH- and photothermal-

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induced release of 5-FU. Experimental results testified that the cumulative release of 5-FU from nanospheres was

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markedly increased in a mild acidic medium. Moreover, a NIR light (808 nm) irradiation triggered a greater and

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faster release of 5-FU, which was further testified by relevant results from in vitro cytotoxicity assay, in vivo tumor

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growth inhibition and histological images of ex vivo tumor sections. These results revealed significant applications

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of GNR/p(NIPAM-MAA) nanospheres in controlled release of anticancer agents and photothermal ablation therapy

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of tumor tissues, accompanied by synergistic effect of chem-photothermal therapy.

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Keywords: gold nanorods; nanospheres; photothermal effect; drug release; 5-fluorouracil

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1. Introduction As a biologically important class of base analogues, 5-fluorouracil (5-FU) is one of time-honored chemotherapy

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drugs and has been widely utilized in cancer treatment [1,2]. 5-FU is considered as an efficient anticancer drug due

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to its inhibition capacity to the synthesis of DNA through competitively inhibiting thymidylate synthetase (which is

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the targeted enzyme for drugs) [2]. To improve anticancer activity of 5-FU, the development of efficient methods to

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understanding the action of 5-FU is the essential purpose, and is still a significant and challenging work. Intractable

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problems such as drug resistance, systemic toxicity and low drug retention in tumor tissues result in some limitation,

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which would restrain the clinical utilizations of 5-FU [3,4]. As established, pyrimidine analogues (e.g., 5-FU) can

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interfere with thymidylate synthesis and have a broad spectrum activity against solid tumors [5]. Previous efforts

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indicated that the probable limitations of 5-FU in clinical utilization are attributed to its short biological half-life,

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and incomplete and nonuniform oral absorption from its rapid metabolism by dihydropyramidine dehydrogenase

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(non-selective action against normal cells) [6-8]. To reduce 5-FU associated side-effects, to improve its therapeutic

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efficacy and to prolong its circulation time, advanced carrier systems of 5-FU should be flexibly designed through

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embedding it into functionalized carriers [9].

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In biomedicine, 5-FU plays an important role due to its excellent activity in cancer chemotherapy. Functionalized

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carriers of 5-FU have been previously engineered with the aim of reducing the absorption of 5-FU by the body and

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thus minimizing its toxic effects. Utilizing a reverse microemulsion method, Zhu et al. prepared chitosan-capping

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magnetic nanoparticles that were further researched as the carriers of 5-FU [10]. Rejinold et al. adopted an ionic-

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linking strategy to prepare biodegradable, thermal-sensitive chitosan-graft-poly(N-vinylcaprolactam) polymers that

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were developed towards the 5-FU carriers [11]. Li et al. reported a pH-sensitive (±)-α-tocopherol-5-FU adduct with

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antioxidant (vitamin E) and anticancer properties (5-FU), which could be released in an acidic medium (pH 1.0-3.0)

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[12]. Since the first detailed researches on 5-FU as an anticancer agent for drug delivery in 1985, uracil derivatives 3

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as antineoplastic agents were reported in succession [9]. Especially, as one of the standard drugs, 5-FU has been

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worldwide applied in chemotherapeutic regimens for metastatic colorectal cancers, also applied to the treatment of

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solid tumors from breast, stomach, rectum and pancreas [13-16]. Although previous reports have referred to 5-FU

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carriers, relatively complex preparation procedures and uncontrolled or spontaneous 5-FU release partly restrain the

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efficiency of carriers. So far, to develop efficient carriers of 5-FU (e.g. light-triggered nano-switch for controllable

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drug release) for biomedical applications still remains a significant and urgent task.

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Herein, we described a facile self-assembled strategy to synthesize poly(N-isopropylacrylamide-co-methacrylic

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acid), i.e. p(NIPAM-MAA) capping gold nanorods (GNR) core/shell hybrid nanospheres (Scheme 1), abbreviated

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as GNR/p(NIPAM-MAA). Under electrostatic interactions, 5-FU was absorbed into networks of p(NIPAM-MAA)

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hydrogels to fabricate 5-FU loaded nanospheres as 5-FU carriers. The release of 5-FU loaded in carriers could be

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effectively controlled by the pH-change in acidic microenvironment and photothermal effect triggered by NIR light

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irradiation. Relevant experiments were regularly conducted to evaluate release efficiency of 5-FU from the carriers,

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including in vitro cytotoxicity assays, in vivo tumor growth inhibition and histological images of ex vivo tumor

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sections. The GNR/p(NIPAM-MAA) nanospheres could be further developed as a smart nanosystem that integrates

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multiple capabilities, serving as significant therapeutic and diagnostic agents for cancer treatment.

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2. Experimental section

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2.1. Preparation of GNR/p(NIPAM-MAA)

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GNR and p(NIPMA-MAA) hydrogels were prepared using modified versions of the reported methods [17-19],

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and relevant synthetic details are available in the Supplementary data (Part S1 and Part S2). For the preparation of

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GNR/p(NIPAM-MAA), typically, 0.5 mg mL-1 of aqueous suspension was prepared by dispersing freshly prepared

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p(NIPMA-MAA) in 10 mM of phosphate buffered saline (PBS, pH 7.4, 15 mM of NaCl solution) under sonication,

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and GNR was adjusted to 0.5 mg mL-1 (5 mL) with deionized water. At room temperature, 5 mL p(NIPMA-MAA) 4

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solution was dropwise added to 5 mL GNR solution under vigorous stirring for 1 h. After centrifugation to remove

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excess polyelectrolyte, the obtained sediment was collected by drying in vacuum at room temperature, and then re-

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dispersed in 10 mL of PBS (10 mM, pH 7.4). The final products of p(NIPMA-MAA) coated GNR were named as

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GNR/p(NIPMA-MAA) nanospheres, showing characterization techniques in the Supplementary data (Part S3).

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2.2. Sensitivities of pH and photothermal

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GNR/p(NIPMA-MAA) dispersed in water was adjusted to 0.05, 0.1, 0.2 and 0.5 mg mL-1, respectively. Aqueous

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suspension (2 mL) of GNR/p(NIPMA-MAA) with each increased concentration was stored in vials at 37 oC. These

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vials were irradiated for 0-5 min with an 808 nm laser (2 W cm-2, 0.6 cm2 laser area) in sequence. The temperature

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increase from laser irradiation-induced photothermal effect was captured with a thermocouple. As a reference, PBS

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solution (10 mM) was irradiated under identical conditions, and the increase of temperature was measured in the

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period of 0-5 min. In addition, 0.5 mg mL-1 of GNR/p(NIPMA-MAA) aqueous suspension in PBS with the each

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increased pH (4.0, 5.5, 6.5 and 7.4) was irradiated with an 808 nm laser for 0-5 min. The changes of hydrodynamic

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sizes were measured by DLS. Hydrodynamic sizes and zeta potentials of GNR/p(NIPMA-MAA) dispersed in PBS

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with pH 3-12 were measured respectively.

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2.3. Loading and release of 5-FU

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GNR/p(NIPMA-MAA) nanospheres (20 mg) were ultrasonically dispersed in 25 mL of 5-FU solution (0.05-1.0

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mg mL-1, in 10 mM of PBS at pH7.4) to form mixed solution, which was continuously stirred until the achievement

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of stable 5-FU concentration. The resulting aqueous suspension was centrifuged and twice washed with water to

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remove excess unbound or surface-absorbed 5-FU. The mass of 5-FU loaded in nanospheres was calculated by

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subtracting the mass of 5-FU in supernatant from the total mass of 5-FU in initial solution (Mtotal-5-FU). The mass of

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free 5-FU (Mfree-5-FU) was determined by UV-vis spectrophotometer at 267 nm (based on Lambert-Beer law). The

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“Mtotal-nanospheres” represents the dried weight of nanospheres. The loading capacity (LC) and efficiency (LE) of 5-FU 5

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in nanospheres were calculated by the following equations;

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LE5-FU (%) = 100 × (Mtotal-5-FU — Mfree-5-FU) / Mtotal-5-FU

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LC5-FU (%) = 100 × (Mtotal-5-FU — Mfree-5-FU) / Mtotal-nanospheres

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To study pH- and photothermal-dependent release of 5-FU, 20 mg of 5-FU loaded nanospheres in 20 mL of PBS

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(10 mM, pH 7.4, 6.5 and 5.5) were transferred into a dialysis tube (MWCO 5 ×104) that was then shaken gently in

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water bath at room temperature. At a defined release time (0-12 h), 1 mL of sample in each mixture solution was

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withdrawn and analyzed by UV-vis spectroscopy at 267 nm. The sample solution was put back into the mixture

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solution after each sampling characterization to maintain a constant volume. Each experiment of 5-FU release was

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performed in triplicate, and each result was averaged. The release of 5-FU was investigated without and with 60s

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irradiation (using an 808 nm laser).

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2.4. In vitro cytotoxicity assay

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The cytotoxicity of free 5-FU and 5-FU loaded GNR/p(NIPMA-MAA) nanosphere carriers to L929 cells (mouse

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fibroblast cells) was assessed. These cells were cultured as subconfluent monolayers on 25 cm2 cell culture plates

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with vent caps in 1× minimum essential α medium, supplemented with 10% of fetal bovine serum in a humidified

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incubator containing 5% of CO2 at 37 oC. After grown to subconfluence, these cells were dissociated from surface

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with trypsin solution (0.25%), and aliquots (100 μL) were seeded (1×104 cells) in a 96-well plate. After incubation

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for 24 h at 37 oC, the medium was replaced with 10 μL of serum-free DMEM containing 0.1 or 0.5 mg mL-1 of

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added substances (5-FU or “carrier+5-FU” incubated in PBS at pH 7.4, 6.5 and 5.5). Treated cells were incubated

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for 24 h at 37 oC in the dark, and then irradiated for 60s with an 808 nm laser. When no increased cell damage was

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observed, 20 μL MTT reagent (0.5 mg mL-1) was substituted for culture medium, followed by incubation for 2 h.

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MTT medium was removed and 150 μL DMSO was added to each well to dissolve formazan crystals. Absorbance

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(A) of each well (colored solution) was recorded at 570 nm. Cell viabilities were quantitated using a standard MTT

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assay. All cytotoxicity experiments were performed in triplicate, and each result was expressed as the average of

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repeated measurements. Cell viability was calculated by the equation;

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Cell viability (%) = 100 × (Atest cells) / (Acontrol cells)

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2.5. Biodistribution in vivo

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All researches on cells and animals were performed in compliance with relevant laws and institutional guidelines,

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and the institutional committees have approved our experiments. Tumor-bearing nude mice were each injected with

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GNR/p(NIPMA-MAA) aqueous suspension (0.25 mg mL-1, 0.2 mL) intravenously. After 6 or 24 h uptake, blood

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samples (10 μL) were taken from tail vein. Percentage of injected dose per gram of tissue (ID%/g) was calculated.

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The mice were sacrificed carefully, and various organics and tissues including blood, heart, liver, spleen, kidney,

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lung, stomach, intestine, muscle, bone, brain and tumor were removed, weighed and dissolved in digest solutions

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(VHNO3/VHClO4, 4/1). These samples were heated to ~220 oC (maintaining 1 h) and then added to 2 mL HClO4 for

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continue heating. When a clear solution appeared, the reaction was stopped and the solution was cooled down to

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room temperature. Each of the solutions was diluted by deionized water to 10 mL, and analyzed by inductively

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coupled plasmon-mass spectrometry (ICP-MS) to determine the content of Au in each sample.

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2.6. Photothermal ablation therapy

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For the photothermal ablation (PTA) therapy, 5-FU loaded GNR/p(NIPMA-MAA) nanosphere (0.25 mg mL-1,

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“carrier+5-FU”) aqueous suspensions (in 10 mM of PBS) were prepared, and alone PBS solution was used as the

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control. Before experiments, twelve combined immunodeficient mice were inoculated subcutaneously with PC-3

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cells (2×106) for 21 days. PBS solution and “carrier+5-FU” were intravenously injected into the mice, respectively

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used for the control and treatment groups. After 1 h incubation, the tumor in mice from the treatment group was

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irradiated for 5 min with an 808 nm laser (2 W cm-2 of output power density), while the control wasn’t irradiated.

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The mice from both groups were killed and tumors were removed, followed by embedding tumors in paraffin and 7

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cryosectioned into 4 μm slices. The slices were stained with hematoxylin/eosin (H&E) and detected by an inverted

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fluorescence microscope. Histological images of tumor slices were captured with an attached digital camera.

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3. Results and discussion

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3.1. Preparation and characterization of nanospheres

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CTAB-stabilized GNR was prepared by a seed-mediated growth method [17,18]. Thermal and pH dual-sensitive

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p(NIPAM-MAA) hydrogels were prepared by the copolymerization reaction of NIPAM and MAA monomers in

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aqueous solution [19-21]. Under electrostatic interactions, a facile fabrication occurred [22]. The surface positively

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charged (CTAB) GNR was successively absorbed into the networks of negatively charged (MAA) hydrogels to

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form GNR/p(NIPAM-MAA) core/shell hybrid nanospheres. These nanospheres were designed to diffuse by tumor

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neovasculature and to be sequestered and retained in tumors via the enhanced permeability and retention effect.

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Absorption spectra in Fig. 1a showed that GNR displayed a weak transverse plasmon band at ~515 nm and a

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strong longitudinal surface plasmon band at ~860 nm. After coating of p(NIPAM-MAA), a dramatic red-shift in the

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longitudinal band from ~860 to 875 nm was observed, with the change in slope and the broadening of peaks. Both

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changes were ascribed to the presence of p(NIPAM-MAA) hydrogels, which contributed the swamping of plasmon

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bands [23,24]. In Fig. 1b, TEM images of GNR/p(NIPAM-MAA) indicated that the dark GNR substances were

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coated with the grey hydrogel shells. The average length and width of GNR were 56.5 ± 2.3 nm and 15.2 ± 1.8 nm,

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respectively (Fig. S1a in the Supplementary data). The whole nanospheres of GNR/p(NIPAM-MAA) exhibited a

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clear oval-shaped core/shell structure with an average length size of ~110 nm, accompanied by ~25 nm thickness of

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p(NIPAM-MAA) hydrogel shells (implying ~50 nm of hydrogels in the whole size (Fig. S1b in the Supplementary

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data). The average hydrodynamic diameter of nanospheres measured by DLS was ~195 nm at 25 oC, and there was

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a low diameter polydispersity index of 0.06 [25]. The diameter from DLS was larger than that from TEM due to the

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hydrate layers of hydrogels in aqueous environments [26-28], revealing the presence of an outer layer of polymer

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shells in nanospheres. In Fig. 1c, the peaks around 1377 and 1389 cm-1 are ascribed to the deformation of two methyls in isopropyl. The

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peak at 1459 cm-1 is from the asymmetric bending vibration of methyl. Typical absorption bands at 1643 and 1538

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cm-1 denote the stretching and bending vibration of amide. The peaks at 2873 and 2969 cm-1 are assigned to the

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symmetric and asymmetric stretching of methyl. Typical peak at 1729 cm-1 represents the carboxyl stretching bond

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of MAA units. For GNR/p(NIPAM-MAA), a sharp peak below 600 cm-1 stands for characteristic absorption peak

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of inorganic substances (GNR) [29,30]. These results revealed the occurrence of copolymerization of NIPAM and

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MAA, and the presence of GNR in nanospheres. Fig. 1d depicted the results from TGA analysis. For GNR, only

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~12% of weight loss was detected in the entire heating process (25~1000 oC), indicating a high thermal-stability of

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GNR. For GNR/p(NIPAM-MAA), ~11% of weight loss was measured at 90 oC, denoting the loss of residual water

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in nanopsheres. There is ~83% of weight loss when heating from 200 to 525 oC. It may be due to the decomposition

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of p(NIPAM-MAA) in nanospheres [19]. In the range of 525-1000 oC, no observable weight loss was detected and

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the residual weight of GNR is ~5.2%, consisting with the measured result (~5.6%, Au element) from ICP-MS.

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3.2. Photothermal- and pH-sensitivity of nanospheres

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The continuous exposure of GNR/p(NIPMA-MAA) aqueous suspension to NIR light (808 nm) induced a rapid

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elevation of microenvironment temperature, which is a key feature of gold-based nanomaterials for controlled drug

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release and photothermal therapy of solid tumors [17,18,31-40]. Under a laser irradiation (2 W cm-2), no marked

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temperature change was detected when PBS as the control was exposed to NIR light (in Fig. 2a). By contrast,

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aqueous suspension of nanospheres at increased concentrations yielded obvious temperature elevations. After 300s

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irradiation, the temperature of nanosphere aqueous suspensions (0.05, 0.1, 0.2 and 0.5 mg mL-1) was elevated to

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35.0, 40.4, 43.1 and 46.8 oC, respectively. These confirmed the excellent photothermal efficiency of nanospheres.

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At a concentration of equal or more than 0.2 mg mL-1, the nanospheres can be easily heated up above 42 oC, which 9

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is sufficient for hyperthermia treatment to kill tumor cells [41]. Thus, these nanospheres could be developed as an

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efficient NIR-light absorber for photothermal tumor therapy. Upon irradiation for different times (0-300s), hydrodynamic diameters of nanospheres showed marked decrease

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due to photothermal effect of GNR (as the core in nanospheres), as depicted in Fig. 2b. As established, PNIPAM is

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a classical thermosensitive polymer and has a lower critical solution temperature (LCST) of 32 oC in water. Above

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the LCST, PNIPAM networks will undergo an abrupt collapse to form hydrophobic nanospheres, exhibiting a coil-

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globule volume phase transition [19,27,28]. After introduction of MAA into PNIPAM networks, thermo- and pH-

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sensitive p(NIPAM-MAA) hydrogels could be achieved [19-21]. In Fig. 2b, the LCST of GNR/p(NIPAM-MAA) at

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pH7.4 and 6.5 (after 120s continuous irradiation, the diameter at LCST is one half of that measured at room

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temperature) is ~40 oC (see Fig. 2a), slightly higher than physiological temperature. Hence, the drugs loaded into

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GNR/p(NIPAM-MAA) would not be immediately released as soon as nanospheres are introduced in body. The

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nanospheres could be applied to controlled drug release for real (in vivo) biomedical applications.

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At a lower pH, –COOH from MAA in hydrogel shells of nanospheres is protonated, and relevant zeta potential is

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lower than that at a higher pH (in Fig. 3). Upon the increase of pH, the degree of protonation reduces and –COOH

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gradually translates to –COO-. Afterward, the electrostatic repulsion and hydrophilia of –COO- induce an increase

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in swelling diameter or volume [19,42]. At a higher pH (7.4), the nanospheres presented a larger swelling diameter

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(as a carrier for drug loading) in comparison with a lower pH (6.5, 5.5 or 4.0), but they showed a smaller shrinking

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degree and a higher LCST. Generally, a higher shrinking degree and a lower LCST of nanospheres are essential for

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triggered drug release. The nanospheres (loading drugs at pH 7.4) might be triggered to release more drugs inside

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tumor tissues due to the acidic microenvironment in tumors (e.g. pH 5.5 and 6.5). Under NIR-light irradiation, the

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cumulative release of loaded drugs would be much greater due to the photothermal effect of GNR in nanospheres.

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These results denoted the photothermal- and pH-sensitivity of GNR/p(NIPMA-MAA) nanospheres, implying their

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promising applications especially as multifunctional nanocarriers for controllable drug release and photothermal

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therapy of tumors.

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3.3. Drug loading and photothermal/pH-induced release 5-FU was loaded into GNR/p(NIPMA-MAA) nanospheres to form 5-FU loaded-nanospheres. Under electrostatic

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interactions, 5-FU with positively charged amine groups in solution was successively absorbed into the networks of

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negatively charged (MAA) hydrogels, realizing the loading of 5-FU into GNR/p(NIPAM-MAA) nanospheres. After

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loading of 5-FU, the nanospheres-based drug carrier system was fabricated. In Fig. 4a, these nanospheres were

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incubated in 5-FU solution of different concentrations. The loading capacity (LC) and loading efficiency (LE) were

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markedly affected by 5-FU concentration. Both LC (1.6-52.5%) and LE (16.4-66.5%) gradually increased with

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each increase of 5-FU (0.05-1.0 mg mL-1), implying that 5-FU loading into nanospheres was dependent on 5-FU

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concentration. In general, 5-FU molecule consists of acidic phenolichydroxyl and alkaline amino groups [9-12],

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which can interact with carboxyl groups of MAA in hydrogel shells of nanospheres to form intermolecular

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complexes by electrostatic interactions.

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At room temperature, 5-FU release responses from 5-FU loaded-nanosphere carriers in PBS were investigated.

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In Fig. 4b, the pH of carrier aqueous suspension showed an obvious influence on 5-FU release. A rapid cumulative

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release in acidic conditions (pH 6.5 or 5.5) was observed, compared to pH 7.4. After 12 h release, the cumulative

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amounts of 5-FU reached to 7.9% (pH 7.4), 32.7% (pH 6.5) and 71.5% (pH 5.5), respectively. Although the release

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time was extended to 24 h and 48 h, the released amount of 5-FU was not increased dramatically (Fig. S2 in the

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Supplementary data). Those results are ascribed to the pH-sensitive “swelling-shrinking” of GNR/p(NIPMA-MAA)

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nanospheres. At a lower pH, –COOH of MAA is protonated. The hydrogels become shrinking to form hydrophobic

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shells of nanospheres [19]. Consequently, 5-FU and water molecules were excluded from hydrogel networks, so

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resulting in 5-FU release. For the nanosphere carrier incubated in PBS (pH 6.5), the release of 5-FU was estimated

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by NIR-light irradiation. In Fig. 4c, the concentration of released 5-FU markedly increased in the first release

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process upon 60s irradiation, and then a similar variation remained unchanged in following release circles. These

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regular release profiles were due to the photothermal effect of GNR in nanospheres because NIR-light irradiation

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triggered an elevated temperature of the nanosphere aqueous suspension [17,38]. The elevated temperature would

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cause a greater shrinking of hydrogel shells (from thermosensitive PNIPAM), compared with the sample without

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irradiation. Thus, NIR-light irradiation triggered 5-FU loaded-nanosphere carriers to release more 5-FU. Consistent

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results were obtained from relevant experiments upon the carriers at pH 5.5 and 7.4 (Fig. 4d).

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Based upon the above results, 5-FU loaded-nanospheres showed photothermal/pH-dependent release of 5-FU. A

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mild acidic medium (pH <7.4) and NIR-light (808 nm) irradiation were highly efficient for 5-FU release. The

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photothermal effect of GNR in nanospheres caused “shrinking-collapsing” of p(NIPMA-MAA) hydrogels (thermo-

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dependent squeezing effect of PNIPAM), followed by 5-FU release. Due to the enhanced permeability and retention

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effect, 5-FU loaded-nanospheres could reach targeted tumor tissues after cell uptake. The shrinking of hydrogels (as

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a shell of nanosphere) would be performed inside lysosomes and endosomes (with mild acidic microenvironments),

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which facilitate the subsequent release of 5-FU. Upon irradiation, the release rate would be accelerated in tumor

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cells or tissues because of photothermal-induced shrinking of hydrogels. On this account, 5-FU loaded-nanosphere

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carriers probably accomplish the release and accumulation of 5-FU in tumor targets, and perform photothermal

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ablation therapy.

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3.4. In vitro cytotoxicity of nanosphere carriers

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To evaluate combined cytotoxicity from the nanosphere carriers and photothermal effect, viabilities of L929 cells

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incubated with free 5-FU and 5-FU loaded-nanospheres (carrier+5-FU) of different concentrations, without or with

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NIR-light irradiation were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)

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assay. Different buffers were selected as simulated physiological conditions (e.g. blood circulation or extracellular 12

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fluids: pH 7.4; tumor extracellular: pH 6.5; intracellular compartments: pH 5.5) [20,43]. In Fig. 5, the cells treated

2

with “carrier+5-FU” showed a higher cell viability when compared with free 5-FU, revealing the high efficiency of

3

nanospheres as drug carriers for pH-regulated release of 5-FU. For “carrier+5-FU” dispersed in PBS without or

4

with irradiation, corresponding cell viabilities decreased along with the decrease of pH (from 7.4 to 5.5), which was

5

due to an increased amount of 5-FU that induced high cytotoxicity. In Fig. 4, the effects of pH on the release of

6

5-FU loaded in nanospheres had been illustrated. The microenvironment with a lower pH facilitated a more release

7

of 5-FU. The released 5-FU still retained high anticancer activity, and thus could serve as chemotherapeutic drugs

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for tumor therapy. Upon “carrier+5-FU” treated cells exposing to NIR-light irradiation, a distinct decrease of cell

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viability was observed in comparison with the group without irradiation, which should be ascribed to photothermal-

10

induced 5-FU release and hyperthermia treatment of tumor cells. The photothermal effect of GNR in nanospheres

11

that induced an accelerated release of 5-FU had been illustrated (Fig. 4c-d). In the experimental results (Fig. 2a),

12

microenvironment temperature of the nanosphere aqueous suspension reached ~47 oC after 5 min of continuous

13

irradiation, which is sufficient for the killing of tumor cells and photothermal ablation therapy of tumor tissues

14

[17,18,36,38]. In addition, under the identical conditions as L929 cells, in vitro cytotoxicity assays of free 5-FU and

15

“carrier+5-FU” were conducted in normal human cells (human hepatocytes L02 cells). As a result (Fig. S3 in the

16

Supplementary data), similar changes of L02 cell viability were observed when compared with L929 cells, but L02

17

cell viabilities were slightly higher than L929 cells. It suggested that “carrier+5-FU” has a higher growth inhibition

18

to tumor cells than normal cells.

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The combination of chemotherapy (5-FU) and photothermal therapy (GNR) of “carrier+5-FU” is expected to

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significantly increase the possibility of cell killing, making it a promising proposal for tumor therapy. Permeability

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of tumor vessels and sensitivity of tumor cells toward chemotherapeutics would be improved by the photothermal-

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induced hyperthermia treatment greatly, showing the promise to enhance drug efficacy. Moreover, photothermal 13

Page 13 of 24

therapy may accomplish instant and triggered drug release from nanospheres, achieving the highly effective drug

2

concentration in tumor sites. In comparison with other types of hyperthermia (e.g. radiofrequency or microwave

3

ablation, requiring interstitial needle or antenna insertion) [36], NIR-light irradiation as the source of hyperthermia

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is noninvasive and can be applied extracorporally, showing unique priorities for tumor treatment.

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3.5. Biodistribution of nanosphere carriers in vivo

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The biodistribution of GNR/p(NIPMA-MAA) nanospheres was studied to reveal the retention of nanospheres in

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mouse organs. The tumor-bearing mice were sacrificed after injection of 0.2 mL nanosphere solutions (0.25 mg

8

mL-1) for 6 and 24 h. Various organs and tissues were collected and the concentration of nanospheres was measured

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by ICP-MS. In Fig. 6, the nanospheres were widely distributed in different organs and tissues. The liver and spleen

10

retained higher concentrations of nanospheres than others (after 24 h p.i.). The retention of nanospheres in most of

11

organs and tissues (especially in blood, a fast blood clearance) decreased after injection. The accumulation in liver

12

and spleen could be ascribed to the initial high uptake of nanospheres due to reticuloendothelial system nature and

13

the slow metabolic process. A slight increase of nanosphere concentrations in intestine and kidney (after p.i. from 6

14

to 24 h) was observed, which may account for the excretion of nanospheres by biliary pathway. The accumulation

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of nanospheres in tumor should be due to the non-specific enhanced permeability and retention effect [17,21,44].

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3.6. Photothermal ablation therapy of tumor cells

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As known, hyperthermic therapy (PTA) denotes the use of heat (40-45 oC) to damage tumor cells [37,43,46]. The

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body temperature is ~37 oC, after the injection of aqueous suspension of “carrier+5-FU”, human tumor tissues even

19

if covered by 0-1 mm thick skin could be easily heated to 45 oC within 5 min under an 808 nm laser irradiation,

20

inducing efficient death of tumor cells [17,18,36]. The “carrier+5-FU” as photothermal agents was evaluated by the

21

PTA of mouse tumors. Twelve combined immunodeficient mice were inoculated subcutaneously with PC-3 cells

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(2×106) for 21 days. When the tumor inside mice had grown to 5-10 mm in diameter, the mice were allocated into 14

Page 14 of 24

the treatment and control groups. The mice were respectively injected 0.1 mL of “carrier+5-FU” and PBS with or

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without 5 min irradiation. For the mice injected with PBS, the tumor surface temperature remained below ~27 oC,

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and only <1 oC of temperature increase was detected after irradiation. For “carrier+5-FU” injected mice, the tumor

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surface temperature rapidly increased to 47 oC after 5 min continuous irradiation.

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The volume changes of tumors after treatment were measured and plotted as a function of time. In Fig. 7, tumors

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showed a rapid growth in control groups (tumor-bearing mice with injection of PBS or “carrier+5-FU”). The mice

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treated with 5-FU demonstrated a slow growth of tumor volume, compared with the control groups. However, these

8

results revealed that the tumors treated with PBS, “carrier+5-FU” or 5-FU did not be eliminated (or reduce tumor

9

volume). Under NIR irradiation (808 nm, 5 min continuous irradiation at selected days), the volume of tumors

10

treated with “carrier+5-FU” displayed a remarkable decrease, realizing PTA. The tumor growth was significantly

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inhibited in the first week (Fig. S4a in the Supplementary data). These above results illustrated the synergistic effect,

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combining photothermal- and chem-therapy in tumor growth inhibition by “carrier+5-FU”.

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These tumors treated with nanosphere carriers, 5-FU or “carrier+5-FU” were removed, embedded in paraffin and

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cryosectioned into 4 μm slices that were then stained with H&E. The histological examination of tumor slices was

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performed (Fig. S4b in the Supplementary data). In control groups (carriers or 5-FU treated tumors), no marked

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differences regarding the diameter and shape of tumor cells could be observed without or with irradiation, which

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denoted that the single photothermal- (carriers) or chem-therapy (5-FU) is insufficient to kill tumor cells. Nuclear

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modification or necrosis of tumor cells was detected after irradiation, revealing a low heat energy converted (from

19

808 nm laser) by carriers and tissues, accompanying with a negligible elevation of temperature (< 1 oC) in tumor.

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Nevertheless, “carrier+5-FU” treated tumors with irradiation showed distinct degenerative changes of coagulative

21

necrosis of karyolysis. These results demonstrated that in vivo tumor cells can be efficiently destroyed by a higher

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temperature (47 oC) due to the photothermal effect of GNR in nanospheres. Therefore, “carrier+5-FU” has a great

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Page 15 of 24

potential as an efficient photothermal agent for PTA in specific in vivo tumor targets. In addition to thermo- and

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pH-sensitive release of 5-FU, “carrier+5-FU” combining chemo-photothermal therapy would significantly improve

3

its biomedical applications.

4

4. Conclusions

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In summary, a facile strategy has been developed to synthesize pH-/NIR light-sensitive GNR/p(NIPAM-MAA)

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core/shell hybrid nanospheres by electrostatic interactions. The nanospheres were elaborately designed by utilizing

7

GNR as the core, which was facilely adsorbed into the networks of pH- and thermo-sensitive p(NIPAM-MAA)

8

hydrogels as the shell. GNR in nanospheres could effectively absorb and convert NIR-light to heat upon irradiation

9

with a NIR laser. Hydrogel shells realized the pH-/thermo-induced shrinking of polymer chains, thereby enabling

10

the release of preloaded drugs (5-FU). Experimental results denoted the feasibility and advantages of 5-FU loaded

11

nanospheres for pH-/photothermal-induced release of 5-FU. Furthermore, 5-FU loaded-nanospheres could achieve

12

the synergistic effect of chemo-photothermal therapy, which markedly enhanced the efficacy of killing tumor cells

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and ablation therapy of tumors. These facile, efficient and multifunctional nanospheres would be very promising,

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greatly promoting the development of controllable nanocarrier systems for biomedical applications.

15

Acknowledgements

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This work was financially supported by the National Natural Science Foundation of China (21475071, 21405086,

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21275082 and 21203228), the Natural Science Foundation of Shandong (ZR2014BQ001 and BS2014YY009), the

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National Key Basic Research Development Program of China (2012CB722705), the Taishan Scholar Program of

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Shandong and the Natural Science Foundation of Qingdao (13-1-4-128-jch and 13-1-4-202-jch).

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FIGURES

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Scheme 1. Schematic illustration of the preparation process and drug release behavior of GNR/p(NIPAM-MAA)

6

core/shell nanospheres.

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Fig. 1. (a) Absorption spectra of GNR and GNR/p(NIPMA-MAA) nanospheres. (b) TEM images of

9

GNR/p(NIPMA-MAA). (c) FTIR spectra of p(NIPMA-MAA) and GNR/p(NIPMA-MAA). (d) TGA curves of

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GNR and GNR/p(NIPMA-MAA). 19

Page 19 of 24

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Fig. 2. (a) Temperature elevation from GNR/p(NIPMA-MAA) nanosphere aqueous suspensions with different

3

concentrations as a function of irradiation time with an 808 nm laser, and PBS was used as the control. (b) Effects

4

of irradiation time and pH on hydrodynamic diameters of nanospheres.

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Fig. 3. Effects of pH on hydrodynamic diameter and zeta potential of GNR/p(NIPMA-MAA) nanospheres.

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Fig. 4. (a) Effects of 5-FU concentration on LC and LE of 5-FU into GNR/p(NIPMA-MAA) nanospheres.

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Cumulative 5-FU release profiles from 5-FU loaded nanospheres: (b) incubated in PBS at 37 oC, (c) incubated in

4

PBS (pH 6.5) without or with NIR-light irradiation, (d) incubated in PBS before and after irradiation.

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Fig. 5. In vitro cell viabilities after incubation with different dosages of free 5-FU and 5-FU loaded-nanosphere

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carriers dispersed in 10 mM of PBS with different pHs, without or with an 808 nm laser irradiation for 5 min. Inset:

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**P < 0.01 (n = 3), compared with the two groups treated with 5-FU and “carrier+5-FU” (without irradiation). 21

Page 21 of 24

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Fig. 6. Biodistribution of GNR/p(NIPMA-MAA) nanospheres at 6 h and 24 h after injection in tumor-bearing mice.

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Fig. 7. Changes of tumor volume relative to that at 0 day (V0) are plotted over time, incubated with PBS, 5-FU or

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carrier+5-FU under irradiation or not.

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Highlights

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> A facile synthesis of pH-/near infrared (NIR) light-sensitive core/shell hybrid nanospheres was reported.

3

> The nanospheres with unique advantages were developed toward a smart carrier for controlled drug release of

4

5-fluorouracil.

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> Experiments versified the synergistic effect of chem-photothermal therapy of nanospheres.

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Page 24 of 24