Photoinduced Mild Hyperthermia and Synergistic Chemotherapy by

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Photoinduced mild hyperthermia and synergistic chemotherapy by onepot synthesized docetaxel-loaded PLGA/Polypyrrole Nanocomposites Jie Yuan, Jialu Liu, Qi Song, Dan Wang, Wensheng Xie, Hao Yan, Junfeng Zhou, Yen Wei, Xiaodan Sun, and Lingyun Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b07669 • Publication Date (Web): 26 Aug 2016 Downloaded from http://pubs.acs.org on August 27, 2016

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ACS Applied Materials & Interfaces

Photoinduced Mild Hyperthermia and Synergistic Chemotherapy by One-pot Synthesized DocetaxelLoaded PLGA/Polypyrrole Nanocomposites Jie Yuan,

†, ‡

Jialu Liu,

Junfeng Zhou,

†

†, ‡

†, ‡

Qi Song, §

†, ‡

Dan Wang,

Yen Wei, Xiaodan Sun*

, †, ‡ 

†, ‡

Wensheng Xie,

and Lingyun Zhao*

†, ‡

Hao Yan,

†, ‡

, †, ‡

State key laboratory of new ceramics and fine processing, School of materials Science and Engineering, Tsinghua University, Beijing 100084, P. R. China

‡

Key Laboratory of Advanced Materials of Ministry of Education of China, School of Materials Science and Engineering, Tsinghua University, Beijing 100084, P. R. China

§

Department of Chemistry, Center for Frontier Polymer Research, Tsinghua University, Beijing 100084, P.R. China

ACS Paragon Plus Environment

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ABSTRACT Mild hyperthermia has shown great advantages when combined with chemotherapy. The development of multifunctional platform for integration of mild hyperthermia capability into a drug loading system is a key issue for cancer multimodality treatment application. Herein, a facile one-pot in situ fabrication protocol of docetaxel-loaded PLGA/Polypyrrole nanocomposites was developed. While the PLGA nanoparticles allow efficient drug loading, the Polypyrrole (PPy) nano-bulges embedded within the surface of PLGA nanoparticles, formed by in situ pyrrole polymerization without introducing other template agents, can act as ideal mediators for photoinduced mild hyperthermia. Physio-chemical characterizations of the asprepared nanocomposites, including structure, morphology, photothermal effects and in vitro drug release profile were systematically investigated. Further on, 2-Deoxy-glucose (2-DG) terminated polyethylene glycol (PEG) was anchored onto the surface of the nanocomposites to endow the nano-platform with targeting ability to tumor cells, which resulted in a 17-fold increase of nanoparticles (NPs) internalization within human breast cancer cells (MCF-7) as competed with PEG modified nanocomposites. Mild hyperthermia can be successfully mediated by the nano-platform and the temperature can be conveniently controlled by carefully modulating the PPy contents within the nanocomposites or the laser power density. Importantly, we have demonstrated that MCF-7 cells, which are markedly resistant to heating treatment of traditional waterbath hyperthermia, became sensitive to the PLGA/PPy nanocomposites mediated photothermal therapy under the same mild temperature hyperhtermia. Moreover, docetaxel-loaded PLGA/PPy nanocomposites induced mild hyperthermia can strongly enhance drug cytotoxicity to MCF-7 cells. Under the same thermal dose, photoinduced hyperthermia can convert the interaction between hyperthermia and drug treatment from interference to synergism.


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This is the first report on the one pot synthesis of PLGA/PPy nanocomposites by in-situ pyrrole polymerization, and such multifunctional nano-platform is demonstrated as a highly potential agent for photoinduced mild hyperthermia and enhanced chemotherapy. KEYWORDS: PLGA/PPy nanocomposites, mild temperature hyperthermia, NIR thermotherapy, docetaxel, multifunctional nano-platform

1. Introduction

Since it was demonstrated that heat had a selective cytotoxicity on hypoxic cells in 1974 by Dr. Eugene Robinson in 19741, hyperthermia has attracted tremendous attention for its great potential in cancer therapy, especially when combined with other therapeutic strategies such as chemotherapy2–9, radiotherapy10–12, immunotherapy13, etc. Particularly, photothermal therapy (PTT), which was based on metallic14,15 or polymeric nanoparticles16,17, semiconducting nanotubes18,19 or graphenes20 to convert the near infrared light (NIR) into heat, has been being developed as a mini-invasive and effective hyperthermia protocol and currently, a great deal of nano-platforms have been designed for combining PTT with chemotherapy2–4,7,21,22. Among all the nanostructures mentioned above for PTT, polypyrrole (PPy) is considered as an ideal heatinductive material of high photothermal conversion efficiency (45% at 808nm)16, good photothermal stability17 and good biocompatibility23,24. PPy with various nanostructures, such as nanoparticles (NPs)16,17, nanocapsules25, nanocomposites15,26–30 have been successfully designed and synthesized for tumor thermal treatment. On the other hand, poly (lactic-co-glycolic acid) (PLGA) is a widely applied bio-polymer as nanomatrix for drugs, plasmid DNA, proteins and peptides due to its excellent biocompatability31. Sustainable chemotherapy has been achieved by loading drugs in biodegradable PLGA and drug release can last over 30 days32. For above


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mentioned advantages of these two materials, it is wise to combine PPy and PLGA into a singlenanoplatform for cancer chemo-thermo therapy. However, the rather inert chemical property and lack of functional groups of PPy makes it difficult to integrate PPy with other polymers, especially the incorporation with PLGA as the former is hydrophilic while PLGA is hydrophobic. So far the only PLGA/PPy composite structure was reported by Sung et al33, who fabricated a system of hollow microspheres composed of a shell of PLGA and an aqueous core comprised of antibiotic and PPy NPs. Such PLGA/PPy hybrid was micro-sized and several steps were incorporated during the preparation. Therefore, other facile synthesis protocols for PLGA/PPy nanocomposites should be developed for the combined PTT-chemotherapy cancer nanotechnology application. Currently, in spite that numerous studies about PTT have been published to prove its excellent anti-cancer effect, more investigation should be carried out to demonstrate the unique advantages of PTT. Normally for PTT studies, tumor ablation (heating temperature >60oC) was the most adopted hyperthermia treatment. It is unambiguously to understand that the higher temperature of the hyperthermia treatment, the more significant anti-tumor effect can be induced. However, high temperature in hyperthermia can make surrounding tissues exposed to toxic temperature and high-energy irradiation thus leads to cell necrosis, which is characterized by cell membrane damage and damage-associated molecular patterns (DAMPs) release rather than apoptosis34. The unwanted release of DAMPs will cause detrimental inflammatory responses and thus be very deleterious. On the other hand, mild temperature hyperthermia (43oC~46oC) has been reported to have favorable results of increased vascular permeability35,36, increased blood flow37, hyperthermic chemo-sensitization38 and restrained drug resistance22 when combined with chemotherapy. More importantly, mild temperature can benefit the structural and functional


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stability of the drug molecule when the combined hyperthermia and chemotherapy were adopted, as most of the anti-cancer drugs are required to store at low temperature for the consideration of the drug thermal stability. Except for the choice of temperature, the difference in heating model may strongly affect the therapeutic effect of hyperthermia. By adopting breast cancer stem cells (BCSCs) as a model cell-line, S.V. Torti et al39 demonstrated that BCSCs, which are significantly resistant to traditional hyperthermia treatment (waterbath heating), become highly sensitive to nanotube-mediated PTT as their results showed that carbon nanotube-mediated PTT was more cytotoxic than waterbath hyperthermia at equivalent exposure times and treatment temperature. Their inspiring finding indicates that heat-resistance of cancer cells can be overcome through the use of PTT based thermotherapy.Docetaxel is an effective anti-cancer drug that can work synergistically with hyperthermia. Due to its poor aqueous solubility, hepatic metabolism and severe side effects, effective and promising drug-releasing fomulations, such as fullerene40, micells41, multi-walled carbon nanotubes42 and many multi-functional platforms43 have been developed. In the current study, a facile in situ fabrication protocol of docetaxel-loaded PLGA/PPy composite NPs (PPNPs) was developed through one-step synthesis. 2-Deoxy-glucose terminated polyethylene glycol (PEG) was anchored to endow targeting ability, which resulted in a 17-fold increase of NPs internalization within human breast cancer cells (MCF-7). MCF-7 cells, which are markedly resistant to heating treatment of traditional waterbath hyperthermia, become sensitive to the PPNPs mediated PTT under the same mild temperature hyperthermia. Importantly, docetaxel-loaded PPNPs induced mild hyperthermia can strongly enhance drug cytotoxicity to MCF-7 cells while traditional waterbath hyperthermia will interfere at 43oC. Our findings have proven that the choice of mild hyperthermia heating mode may lead to different


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interaction between hyperthermia and chemotherapy, and the 2-DG-PPNPs can act as a potential nanoplatform for mild photothermal enhanced chemotherapy. This is the first report on the onepot synthesis of PLGA/PPy nanocomposites by in-situ pyrrole polymerization, and such multifunctional nano-platform is demonstrated as a highly potential agent for photoinduced mild hyperthermia and enhanced chemotherapy.

2. Experimental Section 2.1.

Materials

Pyrrole (Py), dichloromethane (DCM), FeCl36H2O and ethylenediamine were purchased from Sinopharm Chemical Reagent Co., Ltd (Beijing, China). Docetaxel (DTX, anhydrous, 99.56% purity) was from Shanghai Jinhe Bio-Technology Co. Ltd (Shanghai, China). Rhodamine B was obtained from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). Carboxyl group terminated Poly(lactic-co-glycolic acid) (PLGA, 50:50) was acquired from Lakeshore Biomaterials (Birmingham, AL, USA). Polyvinyl alcohol (PVA) and phosphate buffered saline (pH 7.4) were from Sigma Aldrich (St. Louise, MO, USA). N-(3Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide (NHS) were purchased from International Laboratory (San Francisco, CA, USA). Glucose PEG NHS Ester (GLUC-NHS-10K, MW 10,000) and PEG NHS Ester (mPEG-SCM, MW 10,000) were provided by Jenkem Technology (Beijing, China) and YareBio (Shanghai, China), respectively. 2-Morpholinoethanesulfonic Acid (MES) was obtained from Tokyo Chemical Industry (Tokyo, Japan). Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640 medium (RPMI 1640), Fetal bovine serum (FBS), 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), Calcein-AM, Propidium iodide (PI), penicillin-streptomycin (PS) and trypsin were bought from Gibco Life Technologies (Beijing, China). Cell proliferation and


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cytotoxicity assay kit, (MTT assay) and Lacate dehydrogenase assay kit (LDH activity assay) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). All materials were used without further purification.

2.2.

One-pot synthesis of docetaxel loaded PPNPs

Docetaxel loaded PPNPs were prepared by combining solvent extraction/evaporation method32 and polymerization of pyrrole in water-soluble polymer/metal cation system44. In brief, 8mL of dichloromethane (DCM) solution of pyrrole (pyrrole : PLGA weight ratio=1:5, 2:5 and 3:5) and PLGA (14mg/mL) was poured into 120mL aqueous phase containing polyvinyl alcohol (PVA, 5mg/mL). The mixed solution was emulsified using a microchip probe sonicator (400W, 90 seconds, JY92- , Scientz Biotechnology Co., Ltd, China). The resulting oil-in-water emulsion was then stirred at room temperature over night by a magnetic stirrer for DCM evaporation. Next, FeCl36H2O powder (2.0g) was added into the emulsion system for pyrrole polymerization in 4 hours. The final product (PPNPs) was collected by centrifuge (12,000 rpm, 10min, 10oC, himac CR 21F, HITACHI, Japan) and washed with Millipore water for 3 times. For the preparation of drug loaded or fluorescence labeled PPNPs, docetaxel (DTX) or Rhodamine B (RhB) was added into the DCM solution with PLGA and pyrrole together. Pure PLGA NPs were prepared by the solvent extraction/evaporation method32. Briefly, 110mg PLGA in 8mL DCM was added into 120mL PVA aqueous solution (5mg/mL) and sonicated by probe sonication (400W, 90 seconds). The obtained emulsion was then stirred overnight. The final product was centrifuged (12,000 rpm, 10min, 10oC) and washed with Millipore water for 3 times. PPy NPs were synthesized in water-soluble polymer/metal cation system44. Briefly, 15.55g FeCl36H2O was added into 250mL PVA solution (2.5mg/mL) and stirred for an hour. Then,


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1.73mL of pyrrole was injected into the solution. The reaction took four hours. The final product was washed with Millipore water by centrifuge (20,000 rpm, 30min, 10 oC) for 4 times.

2.3.

Surface modification of PPNPs

2-DG terminated PEG was attached to the surface of PPNPs to form 2-DG-PPNPs by EDC/NHS method. 2-DG-PEG NHS Ester, which has an active carboxyl terminal was used to react with free amino group on ethylenediamine on the surface of PPNPs modified with ethylenediamine. In brief, PPNPs (30mg) were resuspended in MES buffer saline (pH 5.5) and carboxyl groups on PPNPs were activated by adding EDC (0.1mg, excessive amount) and NHS (0.1mg, excessive amount) into the solution. The reaction took one hour at room temperature and activated NPs were centrifuged and washed three times by MES buffer saline. Afterwards, the NPs were resuspended in PBS (pH 7.4). Excess ethylenediamine (20µL, excessive amount) was added in order to modify NPs surface with amino groups, and unreacted ethylenediamine was removed by centrifuge. The modified NPs were washed with PBS for three times and resuspended again. At last, 10mg GLUC-NHS-10K, which has an active carboxyl terminal were introduced into the solution to react with the amino groups on ethylenediamine to immobilize 2DG on nanoparticle surfaces, forming the final sample 2-DG-PPNPs. The same procedure was applied to synthesize the untargeting group (PEG-PPNPs) by replacing GLUC-NHS-10K with mPEG-SCM.

2.4.

Characterization of synthesized nanoparticles

The morphology of the synthesized NPs was observed by scanning electron microscopy (SEM, MERLIN, ZEISS, German) and Transmission electron microscopy (TEM, HT7700, HITACHI,


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Japan). Fourier transform infrared spectroscopy (FTIR, Bruker, HORIBA, Japan) was adopted to verify the formation of PPy within the NPs. Particle size and size distribution were measured by the dynamic light scattering (DLS, SZ-100, HORIBA, Japan). The proportion of PPy within PPNPs was calculated according to the thermogravimetric analysis (TGA, X70, NETZSCH, German) of PPNPs, PLGA nanoparticles and PPy nanoparticles. Glass transition temperature of PPNPs was investigated by differential scanning calorimeter (DSC, X70, NETZSCH, German). Surface chemistry of NPs was analyzed by X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, ThermoFisher Scientific, USA). To explore the photothermal effect of PPNPs, 1mL particle aqueous suspensions at various concentrations were exposed under the irradiation of 808nm laser (GSCLS-05-7W00, Daheng Group. Inc. China) with different power densities for 5min. The temperature increase was recorded by an ultra-fine thermocouple fiber. The photothermal stability was carried out by repeatedly exposing PPNPs to 808nm lasers.

2.5.

Drug Release

DTX loading ratio was determined by high performance liquid chromatography (HPLC, 1290BinaryPump, Agilent Technologies, USA) with absorption peak at 227 nm. A reverse-phase column (Eclipse XDB-C18, 4.6 x 250 mm, 5 mm) was used. A designated amount of freezedried PPNPs(DTX) was dissolved in 1mL DCM to break the PLGA matrix. After DCM evaporation, 3 mL mobile phase (47% acetonitrile in water in volume ratio) was added to dissolve the extracted drugs. The solution was then centrifuged for HPLC analysis. The drug loading ratio is calculated as the weight of the drug loaded in the NPs divided by the total weight of the NPs. The drug encapsulation efficiency (EE) is calculated as the weight of the drug


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encapsulated in the nanoparticles divided by the weight of the drug dissolved in oil phase during PPNPs(DTX) synthesis. In vitro drug release profiles were determined in triplicate by HPLC. In brief, 6 mg freezedried PPNPs(DTX) was dispersed in 6 mL PBS (pH 7.4). The solution was divided into 6 groups for 1mL each before they were placed in water shaking bath at 37oC for a month. Three of them were exposed under 808nm at a power density of 0.9W/cm2 every 24h for 4 times. After a designated period of time, the solution was centrifuged and the supernatant was collected for HPLC analysis to determine the DTX content in the supernatant. The HPLC analysis procedure is the same as above.

2.6.

Cell culture, in vitro cytotoxicity and cell uptake

Human breast cancer cells MCF-7 and mouse fibroblast cells L929 were acquired from American type culture collection (ATCC). MCF-7 cells were cultured in DMEM supplemented with 10% FBS and 1% PS. L929 cells were maintained in PRMI1640 with 10% FBS and 1% PS. All cells were cultured in humidified incubators under 37oC with 5% CO2. The cytotoxicity of the synthesized blank nanoparticles (without drugs) was investigated with mouse fibroblast cells L929. L929 cells were seeded in 96-well plate at a concentration of 5000 cells/well and incubated with different concentration of 2-DG-PPNPs containing cultural medium for 24h. After incubation, cell viability was tested by MTT assay. RhB labeled 2-DG-PPNPs and PEG-PPNPs were suspended in cell culture medium at a concentration of 250 µg/mL. MCF-7 cells were cultured in 6-well plates for 24 hours, before the medium was replaced by the NPs containing medium or fresh medium (control). After coincubation for one hour, the culture medium was removed and cells were washed thrice by PBS.


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Cell nuclei were stained by DAPI following the protocol provided by supplier. The cell uptake was visualized by a confocal laser scanning microscope (CLSM, TCS SP5, Leica Microsystem, German). The pictures were acquired with same operation parameters (including exposure time, excitation intensity, etc.) so as to make the results comparable. The average fluorescence intensity of the cells was calculated by imageJ 1.47v software.

2.7.

Treatment of MCF-7 cells by 2-DG-PPNPs mediated PTT and waterbath heating

Cytotoxic effects of heating treatment through waterbath and photo-induction mediated by 2DG-PPNPs were explored at the same thermal dose. 43oC and 46oC were chosen as appropriate temperatures for mild hyperthermia because such temperatures will not transfer toxic thermal dose to surrounding tissues and drug efficacy can be enhanced. The temperature variation model of 2-DG-PPNPs culture medium solution at a concentration of 250µg/mL in 96-well plate exposed to 808nm laser was investigated so as to better control the photo-induced mild hyperthermia. All the cells were incubated in medium with 250µg/mL 2-DG-PPNPs for 24h. Then the cells were treated with different heating mode for 20 minutes. For photo-induced hyperthermia, the cells will be exposed under NIR and the appropriate laser densities to maintain the medium at 43oC and 46oC were 0.9W/cm2 and 1.5W/cm2 respectively. For waterbath mild hyperthermia, the 96-well plate were kept in 43 oC and 46 oC waterbath for 20 minutes. The thermal dose during hyperthermia was calculated and converted to an equivalent number of cumulative equivalent minutes at 43oC (CEM43, a standard thermal isoeffect dose45,46). After heating treatment, the culture media were removed and the cells were washed by PBS twice to remove the remaining NPs and damage-associated molecular patterns (DAMPs) induced by membrane integrity loss34. New fresh culture medium with 5% FBS were added and the cells were then incubated for another 24h. The LDH activity was tested as a


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reflection of DAMPs release into the medium indicating the number of cells undergoing secondary necrosis from apoptosis34. MTT assay was applied for cell viability test. The live and dead staining of cells were carried out with calcein acetoxymethyl ester (calcein AM) and propidium iodide (PI), and the fluorescent pictures were collected by a fluorescence microscope (Leica, Germany).

2.8.

Enhanced chemotherapy by mild hyperthermia in vitro

The photoinduced hyperthermia was combined with chemotherapy by adopting docetaxelloaded 2-DG-PPNPs as mediator. In brief, MCF-7 cells were seeded in 96-well plate at a concentration of 5000 cells per well and incubated with a culture medium containing 250µg/mL docetaxel-loaded 2-DG-PPNPs for 24h. The cells were divided into three groups, namely, PTT with drug, waterbath heating with drug and drug treatment only. Similar to section 2.7, cells in PTT groups received laser with power densities of 0.9W/cm2 and 1.5W/cm2 while the cells in waterbath groups were kept in 43 oC and 46 oC, respectively. Cells incubated docetaxel loaded 2-DG-PPNPs were maintained in 37 oC waterbath for 20 min as the drug treatment only group. The cells were incubated for another 24h after treatment and the cell viability was measured by MTT assay. 3. Results and discussion 3.1.

Preparation and characterization of PPNPs

PPNPs of different PPy contents were successfully prepared and the schematic illustration of the synthetic route to 2-DG-PPNPs was demonstrated in Figure 1. Solvent evaporation method was applied to form PLGA NPs with pyrrole monomers embedded within the NPs. The monomers could be polymerized into PPy nano-bulges by Fe3+. PPNPs with different PPy


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contents were prepared by adjusting the pyrrole : PLGA weight ratio in DCM at 1:5, 2:5 and 3:5 respectively. The morphology of PPNPs with different PPy contents was shown in Figure 2a, b. When the PPy content increases from 1:5 to 3:5, their PPy weight ratio are 27.7%, 40.5% and 52.21% respectively (calculation process is provided in supporting information). As observed in the TEM and SEM images, the surfaces of the NPs are rough and embedded with bulges. When the PPy content increases, the bulges will gradually cover the surface completely. TEM pictures of pure PLGA NPs and PPy NPs (see Figure S1) shows that PLGA NPs have smooth surface while PPy is rough, thus the bulges may be caused by PPy twisted within PLGA NPs. The FTIR spectra were shown in Figure 2c. The characteristic peaks at 1758cm-1 in both PLGA NPs and PPNPs represent the stretching vibration of C=O, which are not existing with PPy NPs. Peaks at 1621cm-1 and 1637cm-1 in all three NPs are attributed to O-H bending vibration. The O-H bending vibration observed on PPy NPs may be due to the residual PVA. Peaks at 1300cm-1 and 1281cm-1 of PPy NPs and PPNPs can be contributed by C-N conjugated stretching. Peaks at 726cm-1 and 1089cm-1 in PPy NPs and PPNPs are assigned for N-H wagging vibrations and C-N stretching vibrations. All information about peak positions can be referred to the publications47,48. As it is evident to notice that the as-synthesized PPNPs possess the characteristic peaks of both PPy and PLGA NPs, the nanocomposite feature of PPNPs composed of PLGA and PPy can thus be approved. In addition, TGA and DSC spectra of PPNPs shown in Figure 2d, e indicate a more stable PPy state than pure PPy NPs. PPy in the pure PPy NPs formulation goes through a gradual thermal decomposition during TGA test when temperature increases because PPy chains with different molecule weight decompose at different temperature. However, PPy in PPNPs are quite stable before PLGA decomposition. It is also confirmed by DSC that no visible heat flow change was


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observed in PPNPs. The peaks around 51oC are corresponding to the phase change of PLGA and the temperature is PLGA’s glass-transition temperature Tg. The continuous increase heat flow of PPy NPs is caused by continuous thermal decomposition of PPy molecules. PPy in PPNPs remains quite stable as no heat flow increase is observed. The results corresponded with that of TGA. The stability of PPy in PPNPs may be due to the protection of PPy by PLGA chains, caused by the intertwist of PPy chains with PLGA chains. The intertwist is supposed to come from the dissolved PLGA in pyrrole liquid drops before polymerization as demonstrated in Figure 1. This unique interaction between PLGA and PPy makes these two imcompatible materials combine into a new composite.

3.2.

Photothermal effects of PPNPs

To demonstrate the photothermal application of PPNPs for mild hyperthermia, 1 mL of PPNPs aqueous suspensions were exposed to 808nm laser and the heating inductive profiles of PPNPs with different PPy contents were examined (Figure S2). Since PPNPs with PPy:PLGA=1:5 can generate enough heat for mild hyperthermia, it is thus chosen for following experiments. The heating profiles of PPNPs suspensions at different concentrations (0.25, 0.5, 1.0, 2.0 mg/mL) and PLGA NPs suspension (1.0mg/mL) exposed to 1.0W/cm2 laser were shown in Figure 3a. As PLGA NPs aqueous suspensions almost did not show any temperature increase under the same conditions, PPy is definitely the only contributor to the temperature rise. The higher PPNPs concentrations led to higher temperature increase. Plot of temperature variation after the suspensions exposed to laser for 5min versus suspension concentration was drawn in Figure 3b. The temperature variation went up from 13.0oC to 28.8oC as the suspension concentration increased from 0.25mg/mL to 2.0mg/mL and the trend of temperature increase slowed down


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when concentration increased. Except for PPNPs content, power density of the laser can also affect the temperature increase, which is quite obvious that the higher power density, the higher temperature increase can be obtained at constant PPNPs concentration as shown in Figure 3c. Therefore, it would be convenient to control the hyperthermia temperature by adjusting the concentration of PPNPs or laser power density or both. Meanwhile, photo-stability of PPNPs was testified by repeated exposure to the NIR. It turns out to be very stable under laser irradiation since no appreciable variation decrease was observed after successive cycles of an onand-off laser. In conclusion, the novel PPNPs possess the ideal heat conversion ability and can serve as a good mild hyperthermia agent.

3.3.

Drug release profile

Docetaxel loaded PPNPs were successfully prepared by the modified solvent evaporation (single emulsion) method with drug encapsulation efficiency (EE) and drug loading ratio of 55.2% and 4.6% respectively (calculation details were presented in the supporting information). As illustrated by Figure 4, our PPNPs have been proven to be sustainable and controllable formulation for drug release. For the control group, after initial burst of docetaxel (28.5% in the first 24 hours), the profile demonstrates a linear release profile that was approximately 10% release rate every 24 hours at the beginning three days and then the release rate slowed down and lasted for about one month. NIR exposure of the formulation can promote the drug release, suggesting heat-stimulus responsive release behavior of the PPNPs. In detail, The exposure group was given 0.9W/cm2 irradiation for 20 minutes every 24 hours for 4 times. When the irradiation was provided, the release amount was approximately 15% every 24 hours (50% release increase compared with the control group). Then, the release rate became quite stable in


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the later 20 days (approximately 6% every 5 days). After 30 days, the NIR exposure group enjoyed a complete release (100%) while the control group had a release ratio of 88.5%. The DTX release in PPNPs without heating fits first order release(R2=0.9925). The release kinetics details are provided in Supporting information.

3.4.

Surface modification of 2-DG

2-DG was immobilized onto the PPNPs’ surface to endow the tumor targeting ability to the assynthesized NPs. 2-DG has been exploited as a targeting molecule for tumor cells with overexpressed glucose transporters 1 (GLUT1) such as MCF-7 cells49–51. The surface of the 2DG-PPNPs was analyzed by XPS. The C1s spectra of PPNPs and 2-DG-PPNPs were compared to reveal change of the surface chemistries in Figure 5a, b. The peaks of CO-O and O-C-CO represent PLGA in the NPs while C-O-C/C-OH peaks come from residual PVA and immobilized PEG chain with 2-DG head. The peak area ratio of C-O-C/C-OH to CO-O increased from 0.978 to 2.01 after 2-DG modification. This might be a proof of the successful anchor of 2-DG onto the PPNPs. In order to confirm the immobilization, the hydration radius change was monitored as the immobilization also cause a noticeable change to the hydration radius of the NPs. Dynamic light scattering (DLS) was exploited to measure the hydration radii and the results were shown in Figure 5c. The hydration radius increased 200nm from 100-500nm to 300-700nm after modification of 2-DG terminated PEG chain. However, the modification can hardly be noticed in TEM pictures (Figure S4) where only some blurry shadow was observed in higher magnification picture. In general, the capping was confirmed by TEM and DLS results.

3.5.

Cytotoxicity and cell uptake of 2-DG-PPNPs


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The cytocompatibility of 2-DG-PPNPs at different concentrations were evaluated by coincubating the particles with mouse fibroblast cells (L929). The cell viability was evaluated by MTT assay and the results (Figure S5) demonstrated that the 2-DG-PPNPs possess negligible cytotoxicity. Confocal laser scanning microscopy (CLSM) was employed to investigate the cellular uptake of the Rhodamine B loaded PPNPs by human breast cancer cells (MCF-7) and the images are shown in Figure 6. Cell nuclei were stained by DAPI and PPNPs internalized within the MCF-7 cells can be quantified by the luminance intensity of RhB. Cells as control group were co-incubated with PPNPs modified with PEG only. As it is evident to observe that compared with the rather dim fluorescence of the control group (cells incubated with PEGPPNPs), much stronger fluorescent signal were noticed in the cells co-incubated with 2DGPPNPs. This observation can confirm the cellular uptake efficacy by MCF-7 cells for 2-DGPPNPs is superior to that for PEG-PPNPs. The average intensities were further calculated by imageJ (calculation process in supporting information) and inserted as red bars. 2-DG modification resulted in 17-fold increase in average fluorescence intensity. It is thus can be concluded that 2-DG can greatly increase PPNPs endocytosis by MCF-7 cells, which can be explained through the Warburg effect that tumor cells frequently exhibit increase glucose consumption than normal cells49. The surface coating of PPNPs by glucose analogue may have particular attraction to MCF-7 cells and therefore the targeting ability of 2-DG-PPNPs to tumor cells can be confirmed. Moreover, the significant internalization of 2-DG-PPNPS within the tumor cells provides a substantial support for the feasibility of intra-cellular hyperthermia and chemotherapy, as will be discussed later.

3.6.

2-DG-PPNPs mediated intra-cellular mild hyperthermia


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In order to demonstrate the unique advantage of PPNPs mediated PTT, effects of hyperthermia treatment of MCF-7 cells by different heating modes (PPNPs mediated PTT and waterbath heating) under same thermal dose were compared and the results are shown in Figure 7. For a better control of the thermal dose, the thermal dose of the heating procedures was calculated according to CEM43 (supporting information). As indicated by Figure 7a, the temperature rise of PTT can be strictly controlled and kept identically the same with that of waterbath heating. However, under the same thermal dose (43oC or 46oC for 20min) for both heating modes, there exists distinguished differences in the cell viability. As shown in Figure 7b, MCF-7 cells are rather heat-resistant by waterbath heating as the cell viability maintained 93.5% under heating treatment of 46oC for 20min. However, PTT mediated by PPNPs would result in a significant decrease of cell viability (66.1%) with the same thermal dose, which suggested the heatresistance of MCF-7 could be reversed by such hyperthermia protocol. As it has been demonstrated that cellular membrane is actually thermal-insulated by nature, water heating may be not an effective approach. On the other hand, the particular attraction of the 2-DG-PPNPs to the MCF-7 cells can greatly promote the cellular uptake and the internalized PPNPs within the cells may be acted as “hot-source” upon exposure under the NIR. Such intra-cellular heating mode may have more direct heating effect and can achieve the same treatment outcome at a much lower and safer temperature as is shown that the viability of waterbath treating group at 46 o

C is 93.5% while PPNPs heating at 43 oC (83.3%) is even better.The live and dead staining were

shown in Figure 8. It is obvious that more cells were killed by 2-DG-PPNPs mediated PTT than waterbath heating after exposed to the same thermal dose. This finding is consistent with the quantitative MTT assay in Figure 7b.


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Mild temperature is normally considered to induce cell apoptosis, while higher temperature is reported to lead to more cell necrosis. Cell necrosis is characterized by cell membrane damage, and DAMPs will release right away under this condition 34. On the other hand, apoptotic cells will undergo secondary necrosis in vitro and release DAMPs afterwards as shown in Figure 7c. Therefore, when the cells were washed twice by PBS after heat treatment, DAMPs from necrosis would be washed off and DAMPs in the newly changed medium 24 hours later would come from cells undergoing seconderay necrosis from apotosis. So, it is possible to distinguish whether the cells are undergoing apoptosis by testing the DAMPs in the newly changed medium. To confirm the effect of mild hyperthermia induced by NPs, the lactic dehydrogenase (LDH), which is a kind of DAMPs was examined in the changed media. As we expected, LDH in the medium that has been treated by 2-DG-PPNPs induced mild hyperthermia has a much higher activity (49.4) than that by waterbath heating (32.5) (Figure 7d). This indicates that large amount of cells were undergoing apoptosis and later secondary necrosis when they were treated by 2-DG-PPNPs induced mild hyperthermia. Therefore, necrosis caused by high temperature thermal therapy was reduced. Detrimental inflammatory and immunogenic responses caused by such unwanted DAMPs release will be alleviated. Mild hyperthermia induced by 2-DG-PPNPs can result in apoptosis and lead to a safer treatment. Till now, the novel 2-DG-PPNPs have shown effective potential in mild hyperthermia for its controllable, high efficient, less deleterious characteristics.

3.7.

Enhanced chemotherapy by photoinduced mild hyperthermia in vitro

Based on the inspiring results of the mild hyperthermia experiments that MCF-7 cells, who markedly resist heating treatment of traditional waterbath hyperthermia become sensitive to the PPNPs mediated PTT under the same mild temperature, we further on studied the enhanced chemotherapy ability of mild temperature hyperthermia of both heating modes. The heating


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procedures were in consistent with former experiments and the results were presented in Figure 9. In comparison to the cell viability (87.7%) of MCF-7 cells under docetaxel treatment only, waterbath heating at 43 oC induced no extra cytotoxic effect on the cells but the cell viability increased a little bit higher (90.4%). However, 43oC PTT had a significant enhancement effect as the cell viability dropped to 49.4%. 46 oC hyperthermia under both modes led to remarkable decrease in cell viability as compared to docetaxel treatment only, 44.9% for thermochemotherapy by waterbath heating and 23.3% by PTT. Still, more significant enhancement effect happened to PTT. With [A], [B] and [A + B] representing the percentage cell viability for treatments of docetaxel, heat and the combination treatment of docetaxel with heat, respectively, the combined effects were defined as follows8: synergistic, [A + B]

Photoinduced Mild Hyperthermia and Synergistic Chemotherapy by

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