Silver Nanoparticles Covered with pH-Sensitive Camptothecin

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Silver Nanoparticles Covered with pH-Sensitive CamptothecinLoaded Polymer Prodrugs: Switchable Fluorescence “Off” or “On” and Drug Delivery Dynamics in Living Cells Liang Qiu, Jiawei Li, Chun-Yan Hong, and Cai-Yuan Pan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b14070 • Publication Date (Web): 31 Oct 2017 Downloaded from http://pubs.acs.org on October 31, 2017

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ACS Applied Materials & Interfaces

Silver

Nanoparticles

Camptothecin-Loaded

Covered Polymer

with

pH-Sensitive

Prodrugs:

Switchable

Fluorescence “Off” or “On” and Drug Delivery Dynamics in Living Cells Liang Qiu†‡, Jia-Wei Li‡, Chun-Yan Hong*, ‡, Cai-Yuan Pan*, ‡

†Institute of Biophysics, Hebei University of Technology, Tianjin 300401, P. R. China

‡CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, China.

KEYWORDS: Amphiphilic copolymer, drug release, fluorescence, pH-sensitive, silver nanoparticles.

ABSTRACT: A unique drug delivery system, in which silver nanoparticles (AgNPs) are covered with camptothecin (CPT)-based polymer prodrug, has been developed, and the polymer prodrug, in which the CPT is linked to the polymer side-chains via an acid-labile β–thiopropionate bond, is prepared

by

RAFT

polymerization.

For

poly(2-(2-hydroxyethoxy)ethyl

methacrylate-co-

methacryloyloxy-3-thiahexanoyl-camptothecin)@AgNPs [P(HEO2MA-co-MACPT)@AgNPs] The polymer thickness on the AgNPs surface is around 5.9 nm (TGA method). In vitro tests in buffer solutions at pH=7.4 reveal that fluorescence of the CPT in the hybrid nanoparticles is quenched due 1

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to the nanoparticle surface energy transfer (NSET) effect, but under acidic condition, the CPT fluorescence is gradually recovered with gradual release of the CPT molecules from the hybrid nanoparticles through cleavage of the acid-labile bond. The NSET “on” and “off” is induced by the CPT-AgNPs distance change. This unique property makes it possible to track the CPT delivery and releasing process from the hybrid nanoparticles in the living cells in a real time manner. The internalization and intracellular releasing tests of the hybrid nanoparticles in the HeLa cells demonstrate that the lysosome containing the hybrid nanoparticles displays CPT blue fluorescence due to releasing of the CPT under acidic condition and the drug releasing kinetics shows fluorescence increase of the released CPT with incubation time. The cytotoxicity of hybrid nanoparticles is dependent on activity of the acid-labile bond. Therefore, this is a potential efficient drug delivery system in cancer therapy and a useful approach to study the mechanism of release process in the cells.

1. INTRODUCTION

Owing to limitation of the small molecular drug, e.g. poor solubility, bad blood circulation and side effects, a number of drug delivery vehicles, such as polymeric nanoparticles (NPs), organic/inorganic hybrid NPs have been developed to improve the limitation.1-5 When the drug delivery vehicles transmit the drugs from external body to niduses, they have to suffer a series of ordeals. One ordeal is the release process of drug from the drug delivery vehicles. Most of studies involve ordeals of drug release from the drug-loaded carriers in vitro, however, real time release of the drug in the living cells under the environmental stimuli

2


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cannot be observed because the drug-loaded carriers and the released drug display the same fluorescence, leading to identification of the drug-loaded carriers and the released drug impossible.6,7 Only after this problem is solved, it is possible simultaneously to track the location and movement of the drug-loaded carriers and the status of the drug (loading or release) in the living cells, and then we can understand the precise mechanism behind the drug release from the drug carriers.

Similar to the fluorescence resonance energy transfer (FRET), the NSET, in which an electronically excited “donor” molecule (such as fluorescent molecule) transfers its excitation energy to the nanoparticle surface, is a unique spectral phenomenon that had various potential applications,8-10 and the efficiency of energy transfer is highly sensitive to the donor-acceptor distance,11-13 so, the energy transfer process is highly sensitive to the donor-acceptor distance. When an anticancer drug (such as doxorubicin, camptothecin and paclitaxel, etc.), which can emit fluorescence, is close enough to the nanoparticles through an organic linker, the drug fluorescence may be quenched via induced dipole-induced dipole interaction. When the drug release process can be triggered by internal or external stimuli, such as pH,14-17 light,18-20 redox,21-26 and temperature,27,28 the drug-loaded carriers in the tumor cells will release the drug leading to recovery of the drug fluorescence, then the drug status (loading or release) can be observed by confocal laser scanning microscopy and the drug release kinetics can be obtained based on change of the fluorescence intensity. Because pH value of the tumor cells is lower than the normal cells, the pH-triggered drug release process is suitable to switch the NSET “OFF” and “ON”, thus it is possible to study the drug status and release kinetics of drug. One strategy for achieving pH-triggered drug release is conjugation of the drug onto the 3



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polymer side-chain via an acid-cleavable bond including acetal, hydrazones, and imines.29-31 Compare

to

cleavage

rates

of

the

aforementioned

acid-sensitive

linkages,

the

β–thiopropionate is hydrolyzed in acidic solution at relatively slow rate, and controlling drug release in a sustained manner is expected to achieve.32-33 Therefore, the acid-labile β-thiopropionate is used to link fluorescent drug to the polymer backbone in this study.

Silver nanoparticles (AgNPs) are well-known antibacterial agent with high surface area and high reactivity,34,35 and have been extensively used in medical devices, clothing, cosmetic and pharmaceutical products.36,37Although the AgNPs demonstrate biocompatibility, facile surface modification, and adequate cell penetration,38 relative to the gold nanoparticles, their application in the nanoparticle-based drug delivery is less studied.35,39 In addition, the AgNPs with unique optical properties have been used in biomedical applications.40 However, use of the AgNPs-based NSET effect for studying the drug release process in the living cells has not been reported based on our knowledge. In this article, we design a new drug delivery system as shown in Scheme 1. The dithiobenzoate group-terminated polymer chains, to which a well-known

anticancer

drug,

camptothecin

(CPT)

is

linked

via

an

acid-labile

β–thiopropionate, are attached to the surface of AgNPs through dithiobenzoate-Ag interaction. Because the polymer backbone restricts the distance between CPT and AgNPs, the CPT fluorescence is quenched due to the NSET effect and this state is called as NSET “on” or fluorescence “off” in the following description. In the acidic environment, the CPT molecule is released from the drug delivery system due to cleavage of the β–thiopropionate bond, leading to appearance of the CPT fluorescence, which is called as fluorescence “on” or NSET “off”. Thus, the drug releasing behavior in the living cells can be studied based on the 4


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fluorescence change.

Scheme 1. Schematic illustration of the polymer prodrugs-covered Ag nanoparticle (A) and intracellular release process of CPT (B). The fluorescence of CPT is quenched when it is linked to the polymer chain on the surface of AgNPs (NSET “ON” or Fluorescence “OFF”), while the CPT fluorescence is recovered (NSET “OFF” or fluorescence “ON”) when it is released with response to cellular acid conditions.

2. EXPERIMENTAL SECTION

2.1. Material. Camptothecin (CPT), acryloyl chloride, triethylamine, 2-mercaptoethanol, methacryloyl

chloride,

hydroxyethyl

methacrylate

(HEMA),

triphosgene

and 5



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4-dimethylaminopyridine (DMAP) were purchased from Sigma-Aldrich and used as received. 2,2'-Azobis(2-methylpropionitrile) (AIBN) was obtained from Acros and recrystallized in 95% ethanol. Dichloromethane (DCM) was distilled over CaH2 and tetrahydrofuran (THF) was distilled over sodium shavings. Diethylene glycol, diethyl ether, ethyl acetate, petroleum ether, and all other reagents were purchased from Sinopharm Chemical Reagent Co. Ltd. and used as received. Water was de-ionized with a Milli-QSP reagent water system (Millipore) to a specific resistivity of 18.4 MΩ•cm. Nuclear fast red was purchased from TCI (Shanghai) Development Co., Ltd. Fetal bovine serum (FBS), dulbecco’s modified Eagle’s medium (DMEM) were obtained from Gibco Inc and used as received. Bovine serum albumin (BSA) was purchased from Invitrogen. 4-Cyanopentanoic acid dithiobenzoate (CPADB) was prepared according to our previously reported procedure.41

2.2. Characterizations. All nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV400 NMR spectrometer (resonance frequency of 400 MHz for 1H) operated in the Fourier transform mode. DMSO-d6 or CDCl3 was used as solvent. Transmission electron microscopy (TEM) observations were conducted on JEM-100SX TEM operating at 100 kV. To prepare TEM samples, 5.0 µL of a dilute copolymer solution was placed onto a carbon-coated copper grid and dried under ambient conditions. Dynamic light scattering (DLS) measurements were carried out on a DynaPro light scattering instrument (DynaPro-99E) at 25 oC with 824.3 nm laser, and the data were analyzed with DYNAMICS V6 software. Molecular weight and Mw/Mn were determined on a Waters 150C gel permeation chromatography (GPC) equipped with three ultrastyragel columns in series (500, 103 and 104 Å) and a Waters 2414 differential refractive index (RI) detector at 30 °C. Monodispersed polystyrene standards were used in the calibration of Mn, Mw, and Mw/Mn, and dimethyl formamide (DMF) was used as eluent at a flow rate of 1.0 mL/min. The UV-Vis absorption spectra were 6


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acquired on a Shimadzu UV-2401PC UV-Vis spectrophotometer. The fluorescence spectra were acquired on a HIT F4600 fluorescence spectrophotometer. The inductively coupled plasma (ICP) optical emission spectrum was acquired on a Perkin Elmer OPTIMA7000 DV Inductively Coupled Plasma Atomic Emission Spectrometer. Thermogravimetric Analysis (TGA) was performed on a Shimadzu DTG-60H Thermogravimetric Analyzer.

2.3. Synthesis of methacryloyloxy-3-thiahexanoyl-CPT (MACPT). MACPT was prepared according to our previous report,33 and characterization data are as follows. 1H NMR (400 MHz, CDCl3) δ 8.40 (s, 1H), 8.22 (d, J = 8.5 Hz, 1H), 7.95 (d, J = 8.1 Hz, 1H), 7.84 (t, J = 7.7 Hz, 1H), 7.66 (dd, J = 16.0, 8.8 Hz, 1H), 7.31 (s, 1H), 6.08 (s, 1H), 5.70 (dd, J = 17.3, 6.5 Hz, 1H), 5.55 (s, 1H), 5.42 (dd, J = 17.2, 8.6 Hz, 1H), 5.29 (s, 2H), 4.29 (t, J = 6.8 Hz, 2H), 3.06 – 2.67 (m, 6H), 2.45 – 2.05 (m, 3H), 1.91 (s, 3H), 1.10 – 0.88 (m, 3H). 13C NMR (100 MHz, CDCl3) δ 169.71 (s), 166.33 (s), 166.06 (s), 156.34 (s), 151.28 (s), 147.79 (s), 145.21 (s), 144.83 (s), 135.01 (s), 130.23 (s), 129.69 (s), 128.54 (s), 127.50 (s), 127.37 – 126.84 (m), 126.05 (s), 125.53 (d, J = 6.9 Hz), 124.87 (s), 119.11 (s), 95.36 (s), 95.14 (s), 76.07 (s), 75.75 (s), 75.30 (s), 66.94 (s), 66.05 (s), 62.82 (s), 56.31 (s), 48.98 (s), 33.60 (s), 30.75 (s), 29.67 (s), 28.67 (s), 25.95 (s), 25.35 (s), 24.59 (s), 17.25 (s), 6.59 (s).

2.4. Synthesis of 2-(CPT-carbonyl)oxy)ethyl methacrylate (MACCPT). The synthesis of MACCPT is as follows. CPT (1.0 g, 2.87 mmol) and DMAP (1.1 g, 8.65 mmol) were dissolved in dry chloroform (100 mL) under argon atmosphere. After 30 min, triphosgene was added until the mixture became clear. HEMA (0.48 g, 3.7 mmol) in 15 mL dry chloroform was added dropwise via a constant pressure funnel. The reaction mixture was stirred overnight, after evaporating all the solvents, the residues were diluted with THF. The crude product was purified by column 7



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chromatography (ethyl acetate : DCM = 1/3) to give MACCPT as a yellow solid powder (2.92 g, yield: 86%). 1H NMR (400 MHz, CDCl3) δ 8.40 (s, 1H), 8.23 (t, J = 9.8 Hz, 1H), 7.94 (d, J = 8.1 Hz, 1H), 7.84 (t, J = 7.7 Hz, 1H), 7.67 (t, J = 7.4 Hz, 1H), 7.34 (s, 1H), 6.11 (s, 1H), 5.69 (d, J = 17.3 Hz, 1H), 5.52 (d, J = 12.9 Hz, 1H), 5.46 – 5.33 (m, 1H), 5.26 (d, J = 20.1 Hz, 2H), 2.44 – 2.05 (m, 2H), 1.87 (s, 3H), 0.99 (t, J = 7.5 Hz, 3H).13C NMR (100MHz, CDCl3) δ 169.71 (s), 166.33 (s), 166.06 (s), 156.34 (s), 151.28 (s), 147.79 (s), 145.21 (s), 144.83 (s), 135.01 (s), 130.23 (s), 129.69 (s), 128.54 (s), 127.50 (s), 127.37 – 126.84 (m), 126.05 (s), 125.53 (d, J = 6.9 Hz), 124.87 (s), 119.11 (s), 95.36 (s), 95.14 (s), 76.33 (d, J = 11.8 Hz), 76.07 (s), 75.75 (s), 75.30 (s), 66.94 (s), 66.05 (s), 62.82 (s), 56.31 (s), 48.98 (s), 33.60 (s), 30.75 (s), 29.67 (s), 28.67 (s), 25.95 (s), 25.35 (s), 24.59 (s), 17.25 (s), 6.59 (s).

2.5. Synthesis of 2-(2-hydroxyethoxy)ethyl methacrylate (HEO2MA). HEO2MA was prepared as follows. Diethylene glycol (5.3 g, 0.05 mol) and triethylamine (3.03 g, 0.03 mol) were dissolved in 150 mL dry DCM in a reactor. Then methacryloyl chloride (2.6 g, 0.025 mol) was dropwise added into a mixture in the reactor immersed in an ice-water bath. After the addition was completed, the reaction mixture was stirred at room temperature overnight. After filtration and evaporation of all the solvents, the residues were diluted with DCM and washed thrice with water and brine, respectively. The organic layer was dried over anhydrous MgSO4, filtered, concentrated and finally purified by silica gel column chromatograph using ethyl acetate/DCM (1/4, v/v) as the eluent, affording HEO2MA as a colorless liquid (4.76 g, yield: 60%). 1H NMR (300 MHz, CDCl3) δ = 6.12 (s, 1H), 5.71 – 5.45 (m, 1H), 4.46 – 4.17 (m, 2H), 3.79 – 3.66 (m, 4H), 3.65 – 3.54 (m, 2H), 2.22 (s, 1H), 1.93 (s, 3H).

8


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2.6. Synthesis of silver nanoparticles (AgNPs). AgNPs was prepared by Lee-Meisel method33 with slight modification. A typical procedure is as following: 20 mL of citrate solution (1%, w/v) and 75 mL of water were added into a round bottom flask and the mixture was heated to 70 oC for 15 min. Into this mixture, 2 mL of AgNO3 solution (1%, w/v) was introduced, followed by quick addition of 2 mL of freshly prepared NaBH4 solution (0.1%, w/v). The reaction solution was kept at 70 oC under vigorously stirring for 2 h and then cooled to room temperature. Water was added to bring the volume of dispersion to 100 mL and then the AgNPs were obtained.

2.7. Determination of the AgNPs concentration. The concentration of AgNPs was determined by inductively coupled plasma optical emission spectrometer. The detail procedure is as following: 200 µL of AgNPs solution was added into 5 mL aqua regia, the reaction mixture was stirred for 2 h until the solution became clear, and then the water was removed under vacuum condition. The residue was dissolved in 2 mL ultra-pure water, and the obtained product was measured with ICP spectrometer. According to the standard curve of Ag+, the concentration of AgNPs is 11.26 mg/L (10 nM).

2.8. Preparation

of

PHEO2MA.

PHEO2MA

was

synthesized

by

reversible

addition-fragmentation transfer (RAFT) polymerization. Typical process is as following: HEO2MA (5.22 g, 0.03 mol), AIBN (5 mg, 0.03 mmol) and RAFT agent CPADB (84 mg, 0.3 mmol) were added into 25 mL ampoule which contained 6 g of DMSO. After three freeze-pump-thaw cycles, the ampoule was sealed, and then the sealed ampoule was placed in oil bath at 70oC. After 7 h, the ampoule was quenched by inserting it into liquid nitrogen to terminate the polymerization. The reaction solution was precipitated into an excess of diethyl ether, and the dissolution-precipitation 9



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cycle was repeated for three times. The final product was dried in a vacuum oven overnight at room temperature; 5 g of a pink solid powder was obtained in 94% of yield (determined by gravimetric method).

2.9. Preparation of P(HEO2MA-co-MACPT). The acid-responsive polymer prodrug was prepared as follows. HEO2MA (5.22 g, 0.03 mol), MACPT (822 mg, 1.5 mmol), AIBN (5 mg, 0.03 mmol), CPADB (84 mg, 0.3 mmol) were added into 25 mL ampoule which contained 6 g of DMSO, after three freeze-pump-thaw cycles, the ampoule was sealed and the sealed ampoule was placed in oil bath at 70oC. After 7 h polymerization, the reaction mixture was quenched by liquid nitrogen to terminate the polymerization. The reaction solution was precipitated into an excess of diethyl ether, and the dissolution-precipitation cycles were repeated for three times. The final product was dried in a vacuum oven overnight at room temperature; and 5.5 g of pink solid powder was obtained in 90% of yield (determined by gravimetric method).

2.10.

Preparation of P(HEO2MA-co-MACCPT). The acid-unresponsive polymer prodrug

was prepared using the same preparation procedure of P(HEO2MA-co-MACPT). HEO2MA (5.22 g, 0.03 mol), MACCPT (756 mg, 1.5 mmol), AIBN (5 mg, 0.03 mmol) and CPADB (84 mg, 0.3 mmol) were added into 25 mL ampoule containing 6 g of DMSO, after three freeze-pump-thaw cycles, the ampoule was sealed and then the sealed reactor was placed into oil bath at 70oC. After 7 h, the copolymer was precipitated by adding the reaction mixture into an excess of diethyl ether, and the dissolution-precipitation cycles were repeated for three times. The final product was dried in a vacuum oven overnight at room temperature, and 5.3 g of pink solid powder was obtained in 88% of yield (determined by gravimetric method). 10


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2.11.

Acid stability study. The acidic stability of these polymers was measured by thin−layer

chromatography (TLC) and the procedure is as follows. The CPT, P(HEO2MA-co-MACPT) and P(HEO2MA-co-MACCPT) were respectively dissolved in PBS solution at pH = 5.0 or 7.4. After stirring for 6 h, each solution was dealt with lyophilisation. The obtained products were respectively dissolved in CHCl3, and then all the polymer solutions were tested by thin−layer chromatography with eluent of CH3OH/CH2Cl2 (1/10, v/v).

2.12.

Drug loading content (DLC). The DLC was respectively measured by UV/Vis

quantitative analysis and the proton NMR method. The weight concentration of CPT in the polymer prodrug was estimated by the standard curve, which was obtained by plotting the UV absorbance at 365 nm with different weight concentrations of the DOX in aqueous solutions. This value was also calculated based on the integral values of CPT proton signals at δ = 7.65-9.30 and the proton signal of HEO2MA units at δ = 4.14 (d). The DLC was calculated according to the following equation:

DLC (%) = (weight of loaded drug/weight of the polymer) ×100%

2.13.

Preparation of the hybrid nanoparticles. A typical process is as follows. The

PHEO2MA solution (10 µM) was mixed with sodium citrate-stabilized AgNPs (10 nM) in ultra-pure water at pH 7.4. The mixture was stirred in dark at 25oC for 48 h. Subsequently, the hybrid nanoparticles were purified by centrifuged at speed of 15000 rpm for 15 min, and the precipitate was collected for removal of the unreacted polymer. Similar procedures were conducted for preparation of the P(HEO2MA-co-MACPT)@AgNPs and the P(HEO2MA-co-MACCPT)@AgNPs.

2.14.

Release of the CPT from the hybrid nanoparticles. The testing procedures of CPT 11



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releasing are described as follows. The P(HEO2MA-co-MACPT)@AgNPs in PBS solution (2 mL) at pH 7.4 was transferred into a dialysis bag (MWCO 1000). Then the dialysis bag was immersed in 60 mL of buffer solutions with different pH values (pH 7.4, 6, 5) at 37oC while gently stirring. The dialysis solution (2.0 mL) was taken at a predetermined time interval for determining the amount of released drug by ultraviolet quantitative analysis (UV absorbance at 365 nm was measured), and the same amount (2.0 mL) of fresh buffer solution was added into the dialysis bag. After tested for 3 days, and the buffer solutions were collected. Finally, the hydrolyzed product was dried by lyophilization for verifying its structure. The same procedure was employed for testing CPT releasing from the P(HEO2MA-co-MACCPT)@AgNPs.

2.15.

Internalization and Intracellular CPT releasing of the hybrid nanoparticles. A

confocal laser scanning microscopy (CLSM) was used to test internalization of the P(HEO2MA-co-MACPT)@AgNPs and their intracellular CPT releasing. The HeLa cells were cultured in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) for 24 h at 37oC under an atmosphere with 5% CO2 in a 96-well plate, then changed to freshly prepared DMEM with P(HEO2MA-co-MACPT)@AgNPs solution, which are respectively equivalent to CPT concentration of 1 µg/mL. After treated for regular times (4 h, 8 h, 12 h), the culture medium was removed, after being rinsed with PBS for two times, the cells were fixed with formaldehyde. After the cells were stained with nuclear fast red (red), the cells were rinsed with PBS buffer and then were observed on a CLSM (Leica TCP SP5) at 430 nm (Ex= 405 nm). In order to investigate the releasing location of the hybrid nanoparticles, a CLSM was used to test the releasing location of the hybrid nanoparticles in cells. The HeLa cells were cultured in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) 12


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for 24 h at 37oC under an atmosphere with 5% CO2 in a 96-well plate, then changed to freshly prepared DMEM with P(HEO2MA-co-MACPT)@AgNPs solution, which are respectively equivalent to CPT concentration of 0.2 µg/mL. After treated for regular times (1 h, 4 h, 7 h, 12 h, 24 h), the culture medium was removed, after being rinsed with PBS two times, the cells were fixed with formaldehyde. After the cells were stained with Lyso Tracker green DND (green), the cells were rinsed with PBS buffer and then were observed on a CLSM (Leica TCP SP5) at 430 nm (Ex= 405 nm)

2.16.

Toxicity test. In vitro cytotoxicity was examined by MTT assay. HeLa cells were

seeded in a 96-well plate with an initial density of 10000 cells per well in 100 mL of complete DMEM supplemented with 10% FBS at 37oC under an atmosphere with 5% CO2. The plate with different concentrations of the polymer or the hybrid nanoparticels was used and the plate without the polymers was used as the control. After incubation for 48 h, MTT reagent (in 20 mL of PBS buffer, 5 mg/mL) was added to each well, and the cells were further incubated with 5% CO2 for 4 h at 37 oC. The culture medium in each well was removed and replaced by 100 mL of DMSO. The plate was gently agitated for 15 min and the optical densities (OD) were recorded at a wavelength of 490 nm using a Thermo Electron MK3 spectrophotometer.

3. RESULTS AND DISCUSSION 3.1. Preparation of AgNPs covered with polymer prodrug. The drug delivery system includes two parts (see Scheme 1A), one is AgNPs, other is P(HEO2MA-co-MACPT). The latter was prepared by RAFT polymerization with feed molar ratio of CPADB/HEO2MA/MACPT=1/100/5 13



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Page 14 of 36

using CPADB as RAFT agent. The CPT-contained monomer, MACPT was synthesized according to our previous report (Scheme S1),33 and its structure was verified by 1H NMR spectrum (Figure S1B). In the copolymer, the CPT is chemically linked to the polymer chains via β–thiopropionate bond because this bond is cleaved in acidic solution at relatively slow rate, so controlled release of the drug in a sustained manner is expected. In order to illustrate the contribution of this bond to the fluorescence “on”, another CPT-contained monomer without acid-responsive bond, MACCPT was synthesized as shown in Scheme S1d, its 1H NMR spectrum in Figure S1C supports its structure. The poly(HEO2MA)

and

P(HEO2MA-co-MACCPT)

were

successfully

prepared

by

RAFT

polymerization of HEO2MA or HEO2MA and MACCPT in the presence of CPADB, respectively, and their structures were verified by 1H NMR spectra as shown in Figure S2. Their GPC curves with Mn and Mw/Mn are shown in Figure S3, their number-average molecular weights based on the 1H NMR method [Mn(NMR)], Mn(GPC) and Mw/Mn are listed in Table S1. Because size of the nanoparticles significantly influences their cell uptake and antitumor efficacies,42 we synthesized the polymers with the nearly same molecular weights and the drug loading capacity (DLC). The DLCs were calculated based on integral values of the aromatic proton signals at δ = 7.65 ~ 9.30 and the ester methylene proton signal at δ = 4.14 and also were measured by UV/Vis quantitative analysis (λex = 365 nm), the standard curve shown in Figure S4 was used for calculation of CPT content in the polymer prodrugs. The results listed in Table S1 reveal that the molecular weights and DLCs of two polymer prodrugs are close.

14


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c

20.5 27.2

D=51 nm PDI=0.195

36

47.7 63.1 83.6

Size (nm)

111

d

D=60 nm PDI=0.224

147 20.3 27.5 39.7 51.1 64.2 82.2 110

145 195

Size (nm)

Figure 1. Size and morphology characterizations of the AgNPs. TEM images (a, b) and DLS curves (c, d) of the citric acid-stabilized AgNPs (a,c) and the P(HEO2MA-co-MACPT)@ AgNPs (b, d).

A series of studies have demonstrated that the size, morphology and surface functionality are the key factors accounting for AgNP-induced toxicity and biologic responses.43,44 In this study, we firstly consider influence of the AgNP size on its UV-Vis spectrum and matching of the AgNPs absorption spectrum with CPT fluorescent spectrum, the AgNPs with diameter (D) of around 50 nm were prepared based on the reported method with slight modification,45 their characterizations are shown in Figure S5. TEM image of the resultant AgNPs in Figure 1a illustrates that the AgNPs are in quasi-spherical shape, and their diameter (D) and the PDI are respectively 51 nm and 0.195, which 15



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Page 16 of 36

was measured by DLS (Figure 1c). Such AgNPs were used in the following studies.

Because the RAFT group (typical dithioesters and trithiocarbonates) at the end of polymer chains showed strong interaction with noble metals,46 and all the three polymers, P(HEO2MA-co-MACPT), P(HEO2MA-co-MACCPT) and P(HEO2MA) used in this study have the terminal dithio group, so, the hybrid AgNPs were respectively prepared just by mixing the hydrophilic polymers with AgNPs in neutral aqueous solution for 48 h. After centrifugation of the resultant mixtures for complete removal of the free polymers, the pure P(HEO2MA-co-MACPT)@AgNPs,

P(HEO2MA-co-MACCPT)@AgNPs

and

P(HEO2MA)@AgNPs were obtained, respectively. To verify whether the polymers are coated on the surface of AgNPs, a series of characterizations were carried out. TEM image in Figure 1B obviously shows a polymer layer on the surface of AgNPs.

FT-IR spectrum of the

P(HEO2MA-co-MACPT)@AgNPs in Figure 2A(2) reveals characteristic absorption bands of the P(HEO2MA-co-MACPT): the ester carbonyl bond at 1725 cm-1 and the C-O ester bond at 1168 cm-1, demonstrating that the P(HEO2MA-co-MACPT) is attached to the surface of AgNPs. When we compare FT-IR spectra of the P(HEO2MA-co-MACPT)@AgNPs with its precursor copolymer in inserting spectra of the Figure 2A, we can see obvious change of the C=S absorption band of the RAFT group, which is induced by interaction between the terminal dithiobenzoate and the AgNPs. The FT-IR spectra of other two polymer-coated AgNPs, P(HEO2MA)@AgNPs and P(HEO2MA-co-MACPT)@AgNPs display almost the same phenomenon as shown in Figure S6.

16


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A

1

3

-1

1077 cm 1200 1150 1100 1050

-1

1168 cm

-1

1725 cm

P(HEO2MA-co-MACPT)@AgNPs

1

-1

3

1067 cm 1200 1150 1100 1050

0

-1

1730 cm

3500

3000

2500

2000

1500

1000

300

400

500 600 700 Wavelength (nm)

-1

Wavenumber (cm ) 100

C

96

92

9.10%

88

800

900

0.6

a 600

0.4

b 300

0.2

0 200

400

o

600

1.0 0.8

84 0

900

D

1200

Fluorescence [a.u]

Weight loss (%)

B

AgNPs

2

Absorbance

2

P(HEO2MA-co-MACPT)

Absorbance

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


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300

400

500

600

700

800

0.0 900

Wavelength (nm)

Temperature ( C)

Figure 2. Characterizations of P(HEO2MA-co-MACPT) covered on the surface of AgNPs. (A) FT-IR spectra of the citric acid-stabilized AgNPs (1), P(HEO2MA-co-MACPT)@AgNPs (2) and P(HEO2MA-co-MACPT)

(3);

(B)

UV-Vis

spectra;

(C)

TGA

curve

of

the

P(HEO2MA-co-MACPT)@AgNPs and (D) spectral overlap of CPT emission spectrum and the AgNPs absorption spectrum. Other strong evidence for successfully coating of the P(HEO2MA-co-MACPT) on the AgNPs is the UV-Vis absorption spectra of the hybrid nanoparticles and their precursor. As shown in Figure 2B, the former exhibits two strong absorption peak at λ = 429 and 359 nm, which are respectively ascribed to spectra of the AgNPs and the P(HEO2MA-co-MACPT). For P(HEO2MA-co-MACCPT)@AgNPs and their precursor, the same result was obtained as shown in Figure S7. All these results support successfully preparation of the hybrid 17



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nanoparticles.

3.1. NSET Effect and Drug release from the hybrid AgNPs. As aforementioned, the NSET efficiency is strongly dependent on the distance between the fluorophore and the nanoparticle, which should be less than 10 nm,12 so, it is necessary to estimate thickness of the polymer layer covered on the surface of AgNPs. The TGA was used to measure the thickness of polymer on the surface of AgNPs, and the result is shown in Figure 2C and Figure S8. Assume that the AgNPs are spherical, thicknesses of the polymers on the AgNP surface and the number of CPT molecules on each AgNP can be calculated based on the polymer weight loss, the densities of Ag (10.53g/cm3) and polymers (1.20 g/cm3), the polymer thicknesses of the PHEO2MA@AgNPs, P(HEO2MA-co-MACPT)@AgNPs, and P(HEO2MA-co-MACCPT)@AgNPs are 5.7, 5.9 and 4.5 nm, respectively, which are close to their corresponding DLS values (6.0, 4.5 and 5.0 nm). All these are less than 10 nm and meet the requirements of NSET effect. In addition, another prerequisite for the NSET effect is good overlap between the fluorescence spectrum of chromophore and the absorption spectrum of nanoparticles,11 As shown in Figure 2D, the CPT fluorescence spectrum at 440 nm is fully overlapped with the absorption spectrum of AgNPs (D = ~50 nm) in the range of 325~700 nm. Thus, the NSET effect between CPT and AgNPs in this drug delivery system (P(HEO2MA-co-MACPT)@AgNPs) can be realized. Because the NSET effect comes from the interaction between CPT and AgNP, the number of CPT molecules on each AgNP (NCPT) is important parameter for understanding the NSET effect. Assume that the AgNPs are spherical, the NCPT can be calculated and is 1.16 × 104 based on weight loss of the P(HEO2MA-co-MACPT)@AgNPs and the CPT content in the P(HEO2MA-co-MACPT). 18


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Figure 3. Acid-responsive property. (A) Acidic cleavage of the β-thiopropionate bond tested by thin−layer chromatography (CH3OH/CH2Cl2 = 1/10, v/v) after incubation of the free CPT (sample 1), the P(HEO2MA-co-MACPT) solution at pH 7.4 (sample 2) and pH 5.0 (sample 3); P(HEO2MA-co-MACCPT) solution at pH 7.4 (sample 4) and at pH 5.0 (sample 5) for 6 h; (B) In vitro CPT release profiles of the P(HEO2MA-co-MACPT)@AgNPs (a) and P(HEO2MA-co-MACCPT)@AgNPs (b) after their incubation in buffer solutions (pH=7.4, 6.0, 5.0) for different time; (C) Schematic illustration of β-thiopropionate bond cleaving under acidic condition. To study internalization and intracellular CPT releasing of the hybrid nanoparticles, the fluorescence of CPT should be recovered with release of CPT from the hybrid nanoparticels, as we mentioned in the introduction. The β-thiopropionate linkage is known to be hydrolyzed in acidic solution, which is driven by generation of a partial positive charge on the ester 19



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carbonyl carbon owing to inductive effect of sulfur atom (Figure 3C) and its cleaving behavior in acidic aqueous solutions was studied in our previous report.32,33 In order to demonstrate contribution of this bond cleavage to recovery of the CPT fluorescence, the release of CPT from the P(HEO2MA-co-MACPT) was investigated, and the results are shown in Figure 3A and 3B. When the thin-layer chromatography (TLC) was used to study stability of the P(HEO2MA-co-MACPT) and the P(HEO2MA-co-MACCPT), as shown in Figure 3A, after they were incubated with a solution at pH 7.4 for 6 h, only one single point stays at original point, indicating no CPT release from the copolymers. However, when the pH of solution is changed to 5.0, for the P(HEO2MA-co-MACT), we can see one point at Rf = 0.65, which is the same with the Rf of free CPT, indicating release of the CPT from the polymer. But for the P(HEO2MA-co-MACCPT), only one single point stays at the original point (sample 5) due to acidic stability of the carbonate linkage in the MACCPT. For understanding the CPT release behavior from the hybrid AgNPs, the P(HEO2MA-co-MACPT)@AgNPs and the P(HEO2MA-co-MACCPT)@AgNPs were respectively incubated with aqueous solutions at pH 5.0, 6.0 and 7.4, the fluorescence spectra of the released CPT are recorded at predetermined time intervals, and the CPT releasing profiles are shown in Figure 3B. When the P(HEO2MA-co-MACPT)@AgNPs are treated in PBS solution at pH 7.4, only a few amount (about 5 wt%) of the CPT is released because the β-thiopropionate linkage is stable at neutral condition.33 However, when the acidity of PBS solution is lowered to pH=6.0 or to pH=5.0, the released CPT gradually increases with the incubation time, and the released rates of CPT increase when the solution pHs decrease from 6.0 to 5.0 (Figure 3B). Similar to the results of TLC analysis, When the P(HEO2MA-co-MACCPT)@AgNPs are incubated in PBS 20


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solutions at pH=7.4, 6.0 and 5.0, almost no CPT is released from the hybrid AgNPs because the carbonate, by which the CPT is linked to the PMACCPT chains, is stable in the neutral and weakly acidic solutions (pH=6.0 and 5.0).

Figure 4. Fluorescence quenching of the hybrid AgNPs. (A) Florescence spectra of the AgNPs, the copolymer and the hybrid AgNPs. (B) Fluorescent intensity change of the P(HEO2MA-co-MACPT) (10 µM) with different concentrations of added AgNPs. (C) Dependence of F0/F-1 on the concentration of AgNPs, F0 and F represent the fluorescence intensities of CPT in the absence and the presence of AgNPs. λex=365 nm. Photoexcitation of the CPT at λex=365 nm yields a strong blue fluorescence peaking at around 440 nm (Figure 4A).47 In order to identify whether the hybrid AgNPs display NSET effect, the fluorescence was used to follow the coordination reaction of CPT with AgNPs, as shown in Figure 4B, the intensity fluorescence of CPT decreases with increase of the added AgNPs because with addition of the AgNPs, more and more P(HEO2MA-co-MACPT) chains are attached to the AgNPs, leading to increase of the fluorescence quenching. When concentration of the added AgNPs reaches to 2500 pM, about 96% of the CPT fluorescence is quenched (Figure S9). The Stern-Volmer quenching constant (KSV) can be calculated on the basis of eq 1, 21



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F0/F=1 + KSV[AgNPs]

Page 22 of 36

(1)

F0 and F are the fluorescence intensities of CPT at λex=365 nm in the absence and the presence of AgNPs, respectively. [AgNPs] is the concentration of AgNPs.9,11 Figure 4C displays linear relationship of the F0/F-1 with the AgNPs concentration, and the Stern-Volmer quenching constant (KSV) is derived from the slope in Figure 4C and is 1.95 × 109 M-1, which is close to the value (1.27×109 M-1) obtained from the coumarin 153-AuNPs (D=46 nm) system.11 This KSV value is quite high in comparison with the normal small molecule quenching processes, so, the fluorescence quenching cannot be ascribed to simple diffusion controlled collision quenching.11 In addition, the emission spectrum of CPT is completely overlapped with the UV-Vis absorption spectrum of AgNPs (Figure 2D), thus, it is reasonable to attribute the fluorescence quenching to an efficient non-radiative energy transfer from CPT to the AgNPs.

Figure 5. Principle of NSET “on” and “off” for the P(HEO2MA-co-MACPT)@AgNPs. Fluorescence spectra of the hybrid AgNPs after incubation in PBS solution at pH=7.4 (A) and

22


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at pH=5.0 (B) for different time; Schematic diagram of NSET “on” (C) and NSET “off” (D) for the P(HEO2MA-co-MACPT)@AgNPs. From practical applications, recovery of the CPT fluorescence from the hybrid AgNPs is also an important issue, this is expected to achieve by releasing the CPT from the hybrid nanoparticles, resulted in increase of the CPT-AgNP distance. To understand the relationship between the released CPT and the fluorescence recovery, the fluorescence of hybrid nanoparticles

under

different

conditions

was

studied.

When

the

P(HEO2MA-co-MACPT)@AgNPs were respectively treated with PBS solution at pH=7.4 for 48 h and 96 h, their fluorescence was measured. The results in Figure 5A reveal very weak CPT fluorescence, and no obvious increase of the fluorescence was observed with increase of the incubation time, this is ascribed to almost no release of the CPT from the copolymers because the β-thiopropionate linkage is stable at neutral solution. When the hybrid nanoparticles are irradiated under UV light at λex=365 nm, the CPT is photoexcited, and then energy transfer from the excited CPT to the AgNP occurs, which leads to quenching of the CPT fluorescence as shown in Figure 5C. However, when the hybrid nanoparticles were incubated in the PBS solution at pH=5.0, the CPT fluorescence was gradually recovered with the incubation time from 6 h to 96 h as shown in Figure 5B, which is due to gradual release of the CPT molecules from the copolymer covered on the AgNPs via cleavage of the β-thiopropionate linkage, and subsequent diffusion of the released CPT from the surface to solution leads to significant increase of the released CPT-AgNP distance, resulting in the NSET “off” or fluorescence “on” (Figure 5D). 3.2. NSET effect in a living cell. Compared to the AuNPs, use of the AgNPs as drug 23



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Page 24 of 36

nanocarrier is less studied,39,47 and most of studies focus on the toxicities, antibacterial activity,34,36-38 and their potential applications as antibacterial, optoelectronic, catalytic materials. Because the CPT-AgNPs system displays the NSET effect, it is reasonable to study whether NSET effect of the hybrid AgNPs occurs in a living cell, the confocal laser scanning microscope (CLSM) was used to study cellular uptake assay. After the HeLa cells were incubated

with

DMEM

medium

respectively

containing

free

CPT,

or

the

P(HEO2MA-co-MACPT) or its hybrid AgNPs, or the P(HEO2MA-co-MACCPT) or its hybrid AgNPs solution for 4, 8, and 12 h, and then stained with nuclear fast red (red), the fluorescence images of the HeLa cells were acquired under UV irradiation at λex= 405 or 510 nm, respectively, and the merged image of these two images obtained is shown in Figure 6A. The kinetics of cellular uptake for free CPT and two copolymer nanoparticles and the kinetics of CPT release for the two hybrid AgNPs were evaluated by quantification from the CLSM analysis (Figure 6A), and the results are shown in Figure 6B.

24


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Figure 6. Drug release from the hybrid AgNPs in the living HeLa cells. A: Confocal laser scanning microscope (CLSM) images of the HeLa cells incubated with free CPT (a), P(HEO2MA-co-MACPT)@AgNPs

(b),

P(HEO2MA-co-MACCPT)@AgNPs

(c),

P(HEO2MA-co-MACPT) (d), P(HEO2MA-co-MACCPT) (e) for 4h, 8h and 12 h. The dose of CPT is 1 µg/mL in the cell culture. All images are merged images of the two images obtained respectively under irradiation at λex = 405 nm and 510 nm. The cells were counterstained with nuclear fast red (red). B: Relationship between normalized fluorescence intensities of the HeLa cells with incubation time, the scale bar is 20 µm

As

shown

in

Figure

3A

and

3B,

CPT

cannot

be

released

from

the

P(HEO2MA-co-MACCPT)@AgNPs or its precursor polymer because the CPT is linked to the polymer chains via acid-stable carbonate linkage, the same phenomenon was observed in the living cells. After incubated with the P(HEO2MA-co-MACCPT)@AgNPs, the HeLa cells display only red fluorescence (Figure 6Ac), but the HeLa cells obtained after incubated with the P(HEO2MA-co-MACCPT) exhibit CPT blue fluorescence beside the red fluorescence as shown in Figure 6Ae because the CPT is tightly bonded to the polymer chains, leading to NSET “on” and fluorescence “off” of the hybrid AgNPs, but for its precursor polymer, no NSET effect occurs, its CPT in the cells emits strong blue fluorescence. Similar to the Figure 5B, which reveals that the CPT fluorescence increases gradually with increase of the CPT released from the P(HEO2MA-co-MACPT)@AgNPs, the same hybrid AgNPs in the HeLa cells also show this result. As shown in Figure 6Ab, the HeLa cells incubated with the hybrid AgNPs for 4 h display very weak CPT blue fluorescence, while for incubation of the HeLa cells with their precursor copolymer at the same condition for the same incubation time, the 25



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Page 26 of 36

cells exhibit stronger blue fluorescence (Figure 6Ad) because both the released CPT and the CPT bonded in the polymer chains have contribution to the blue fluorescence in the latter case, but in the former case, only the released CPT molecules emit the blue fluorescence. When the incubation time prolongs to 8 h, and then to 12 h, the CPT blue fluorescence is increasingly recovered as shown in Figure 6Ab and 6B because more and more CPT molecules are gradually released from the hybrid AgNPs with incubation time, these released CPT molecules lead to NSET “off” and fluorescence “on”. The CPT release profiles of two hybrid AgNPs and their precursor copolymers prodrug in the living cells are shown in Figure 6B, the result of P(HEO2MA-co-MACPT)@AgNPs reveals almost linear relationship of the released CPT fluorescence intensities with incubation time, which is similar to in vitro CPT release profile before 12 h shown in Figure 3B. Whatever the drug release conducts in the living cells or in the buffer solution, the CPT release rates are relatively slow because the β-thiopropionate bond is cleaved at slower rate in comparison with the acetal bond in acidic condition.32 The above results demonstrate that with the aid of NSET effect, it is possible to trace the delivery and release of an antitumor drug CPT from the hybrid AgNPs in real time at a single living cell level.

26


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Figure 7. CLSM images of the HeLa cells treated with P(HEO2MA-co-MACPT)@AgNPs (A) or P(HEO2MA-co-MACPT) (B), and stained with Lyso Tracker green DND (green) at 37 °C for 1, 4, 7, 12 and 24 h. The images from left to right display CPT (blue), Lyso Tracker green DND (green), and a merge of the two images. All the nano-object solutions contain 0.2 µg/mL of the CPT, the scale bar is 20 µm

For understanding drug release mechanism of the P(HEO2MA-co-MACPT)@AgNPs induced by pH change in the living cells, the CLSM investigation was further conducted using a lysosome tracker fluorescent dye (green) that can specifically stain the cellular lysosomes, and the CLSM images of P(HEO2MA-co-MACPT)@AgNPs and their precursor 27



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Page 28 of 36

copolymer are respectively shown in Figure 7A and 7B. Figures S10 and S11 are CLSM images of the P(HEO2MA-co-MACCPT)@AgNPs and their precursor copolymer. The kinetic profiles of drug release or cellular uptake, which are constructed based on qualification from the CLSM analysis, are shown in Figure S12, and the results in Figure S12 are similar to Figure 6B. Figure 7A reveals the localization of CPT molecules within the lysosomes over time. After 1 h of incubation with P(HEO2MA-co-MACPT)@AgNPs equivalent to CPT concentration of 0.2 µg/mL, almost no CPT blue fluorescence is observed as shown in Figure 7A-1h, which is ascribed to the hybrid nanoparticles not entering into the lysosome, leading to the NSET “on”. After 4 h of incubation, the CPT blue fluorescence appears and we can observe co-localization of the CPT blue fluorescence with the green fluorescence of the labeled lysosomes, which indicates translocation of the hybrid nanoparticles into the lysosomes, and subsequently, the CPT molecules are released from the hybrid nanoparticles under the acidic microenvironment within the lysosomes, resulted in the NEST “off” and the fluorescence “on”. With prolonging incubation time, the intensity of CPT blue fluorescence increases gradually and the CPT blue fluorescence appears to be scattered throughout the entire cytoplasm rather than being localized within the green fluorescence lysosome regions. This could be attributed to the lysosomal release of CPT molecules and their subsequent diffusion into the cytoplasm. For the P(HEO2MA-co-MACCPT)@AgNPs, almost no CPT blue fluorescence is observed before incubation of 12 h, and even at 24 h incubation, only very weak blue fluorescence in the HeLa cells can be seen as shown in Figure S10 because the carbonate bond between the CPT and the polymer chains is acid-stable. Therefore, the acid-labile linkage between the CPT and the polymer chains is a 28


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key for recovery of the CPT fluorescence. However, tracing entrance of the hybrid nanoparticles into the cells is very difficult as shown in Figure 7A owing to their NSET effect. As a reference, when the HeLa cells were incubated with their precursor copolymers, P(HEO2MA-co-MACPT) and P(HEO2MA-co-MACCPT) for different time, and their CLSM images are respectively shown in Figure 7B and S11. Both kinetic profiles of the cellular uptake are almost the same (Figure S12), the CPT blue fluorescence appears on the cell membrane and inside the cytoplasm but not in green stained lysosomes after 1 h of incubation with the precursor copolymers (Figure 7B and Figure S11). With increasing of the incubation time, the blue fluorescence from the CPT molecules becomes stronger (Figure S12), and the CPT blue fluorescence is increasingly co-localized with the green fluorescence of the labeled lysosomes.

3.4.Cytotoxicity of the hybrid nanoparticles. Cytotoxicity of the hybrid nanoparticles is an important issue for their application in drug delivery systems,39 and their cytotoxicity is evaluated by MTT assay. When the HeLa cells are respectively incubated with a series of concentrations of the copolymers, the AgNPs and the hybrid nanoparticles for 48 h, their relative cell viability were measured and the results are shown in Figure 8. We can see that both of the P(HEO2MA) and P(HEO2MA)@AgNP are of low toxicity to the HeLa cells at the concentrations from 10 to 500 µg/mL (Figure 8A and 8C), the AgNPs are also low toxic at the tested concentrations in the range of 0.11 to 5.63 µg/mL (Figure 8B). However, after the HeLa cells are incubated with P(HEO2MA-co-MACPT)@AgNPs, the relative cell viability is quite

different

from

P(HEO2MA)@AgNPs.

that As

of

shown

the in

P(HEO2MA-co-MACCPT)@AgNPs Figure

8D,

with

the

concentration

and of 29



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Page 30 of 36

P(HEO2MA-co-MACPT)@AgNPs increasing from 0.15 to 80 µg/mL, the cell viabilities decrease from 98% to 8%, and their IC50 value, which is the drug dose at median lethal dose, is 4.33 µg/mL, which is higher than IC50 ( 2.48 µg/mL) of the free CPT. This is reasonable because the uptake rate of free CPT by the HeLa cells is faster than the release rate of CPT from the hybrid nanoparticles (Figure 6B). For the P(HEO2MA-co-MACCPT)@AgNPs, the result in Figure 8D reveals slight decrease of the cell viability with concentration increase of the hybrid nanoparticles, indicating very low cytotoxicity of this hybrid nanoparticles because the CPT molecules are still encapsulated in the hybrid nanoparticles due to acid-stable carbonate bond, this is consistent with the drug release kinetics in the living cells in Figure 6B, that is, in this case, the NSET “on” and the fluorescence “off” continue to 24 h incubation.

30


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Figure 8. Relative cell viability of HeLa cells evaluated by MTT assay after incubation with the pH=7.4 solutions of (A) P(HEO2MA); (B) AgNPs; (C) P(HEO2MA)@AgNPs; and (D) free CPT, P(HEO2MA-co-MACPT)@AgNPs, P(HEO2MA-co-MACCPT)@AgNPs, at their different concentrations. Incubating temperature: 37 °C; time: 48h.

4. CONCLUSIONS

In summary, the CPT-AgNPs system displays NSET effect, and this effect is strongly dependent on the CPT-AgNPs distance. By changing the distance, switching the NSET “on” or fluorescence “off” to the NSET “off” or fluorescence “on” can be achieved. This property can be used to track the CPT delivery and releasing process from the hybrid nanoparticles in a living cell. Therefore, a unique drug delivery system, in which AgNPs are covered with the CPT-contained polymer prodrug, has been developed. The in vitro tests of P(HEO2MA-co-MACPT)@AgNPs in buffer solutions reveal that fluorescence of the CPT in the hybrid nanoparticles is quenched in the neutral condition due to the NSET effect, but under acidic condition, the CPT fluorescence is recovered gradually over time owing to CPT releasing from the hybrid nanoparticles through cleavage of the acid-labile bond. The internalization and intracellular releasing of the P(HEO2MA-co-MACPT)@AgNPs in the living cells achieve the same results with that obtained from tests in buffer solutions. The cytotoxicity of hybrid nanoparticles is dependent on activity of the acid-labile bond. Therefore, this is a potential efficient drug delivery system in cancer therapy and a useful approach to investigate the mechanism of drug release in the living cells.

ASSOCIATED CONTENT

31



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Page 32 of 36

Supporting Information. 1H NMR spectra of monomers, HEO2MA, MACPT, MACCPT (Figure S1) and the polymers, PHEO2MA, P(HEO2MA-co-MACPT), P(HEO2MA-co-MACCPT) (Figure S2); GPC traces (Figure S3) for synthesis of polymers; standard curve for calculation of CPT content (Figure S4); TEM image (Figure S5A), FT-IR (Figure S6), UV-Vis absorption (Figure S5B, Figure S7A), fluorescence (Figure S7B, Figure S9), TGA curves (Figure S8), CLSM images (Figure S10 and S11) and kinetics of cellular uptake or drug release (Figure S12) are included in the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected], [email protected]

ACKNOWLEDGMENT The research support from the National Natural Science Foundation of China (No. 21525420 and 21374107).

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Table of Content

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Silver Nanoparticles Covered with pH-Sensitive Camptothecin

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