Multistimulative Nanogels with Enhanced Thermosensitivity for

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Multi-stimulative nanogels with enhanced thermosensitivity for intracellular therapeutic delivery Ping Ji, Bingjie Zhou, Yuan Zhan, Yifeng Wang, Yuhong Zhang, Yulin Li, and Peixin He ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 26 Oct 2017 Downloaded from http://pubs.acs.org on October 26, 2017

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ACS Applied Materials & Interfaces

Multi-stimulative nanogels with enhanced thermosensitivity for intracellular therapeutic delivery Ping Ji,†Bingjie Zhou,‡Yuan Zhan,† Yifeng Wang,‡Yuhong Zhang,† Yulin Li,* †,‡,§ and Peixin He*† †

Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Key

Laboratory for the Synthesis and Application of Organic Functional Molecules, Ministry of Education, College of Chemistry and Chemical Engineering, Hubei University, Wuhan 430062, People’s Republic of China. ‡

The State Key Laboratory of Bioreactor Engineering and Key Laboratory for Ultrafine

Materials of Ministry of Education, Key Laboratory for Ultrafine Materials of Ministry of Education, Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, People’s Republic of China. §

CQM – Centro de Química da Madeira, MMRG, Universidade da Madeira, Campus Universitário da Penteada, 9020-105 Funchal, Portugal. E-mail:[email protected] (Yulin Li); [email protected] (Peixin He)

ACS Paragon Plus Environment

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KEYWORDS:stimulative nanogels; alginate;

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poly(N-isopropylacrylamide); temperature-

induced cell death; drug delivery

ABSTRACT:The flexibility and hydrophilicity of nanogels suggest their potential for the creation of nanocarriers with good colloidal stability and stimulative ability. In the present study, biocompatible AGP and AGPA nanogels with triple stimulative properties (thermosensitivity, pH sensitivity and redox sensitivity) were prepared by incorporating poly(N-isopropylacrylamide) (PNIPAM) or poly(N-isopropylacrylamide-co-acrylate acid) (P(NIPAM-AA)) into alginate (AG) emulsion nanodrops followed by fixation with a disulfide-containing molecule (cystamine), respectively. Compared to AG/PNIPAM(AGP) nanogels, AG/P(NIPAM-AA) (AGPA) nanogels exhibited more sensitive volumetric expansion by switching the temperature from 40 to 25 °C under physiological medium. This expansion occurs because P(NIPAM-AA) with –COOH groups can be fixed inside the nanogels via chemical bonding with Cys, while PNIPAM was encapsulated in the nanogels through simple physical interactions with the AG matrix. AGPA nanogels carrying an anticancer drug tend to easily enter cells upon heating, while exerting toxicity through a cold shock and reverse thermally induced release of an anticancer drug. Upon internalization inside cells, the nanogels use the reducible and acidic intracellular environments to effectively release the drug to the nucleus to impart anticancer activity. These results demonstrate that multifunctional nanogels may be used as a general platform for therapeutic delivery. INTRODUCTION Nanotechnology has provided promising approaches to fabricate various nanocarriers (e.g., liposomes, dendrimers, inorganic nanoparticles, and nano-scaled hydrogels (nanogels)) for therapeutic delivery, which can be used for the treatment of various diseases, especially


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cancers.1-3Among these materials, nanogels have attracted increasing attention because of their biocompatibility, controllable architecture and easy functionalization.4 Their similarity to water in Hamaker constants confers nanogels with better colloidal stability5,6 and more effective cell uptake ability compared to conventional carriers (e.g., cationic liposomes of much less stability),7potentially resulting in improvedtherapeutic bioavailability and safetyin vivo.8 Tumors exhibit enhanced permeability and retention (EPR) effects, which permit the passive uptake ofnanovectors into tumors in the absence of any targeting ligands.9 Compared with normal tissues, the extracellular microenvironment of solid tumors is acidic (pH 6.5-6.9),which is caused by excessive lactic acid due to the inefficient conversion of glucose to energy.10Once taken up by cancer cells, the nanogels will be internalized into endo-lysosomal compartments with pH values of approximately 5.0.11Similarly, the cells present strong reductive situations in their interior (with a glutathione (GSH) tripeptide concentration approximately one thousandfold larger than the exterior of the cell).12,13 Additionally, tumor tissues are richer in GSH compared to normal biological tissues.14 Considering the tumor microenvironment, pH/reductive nanogels enabled the enhanced delivery of the doxorubicin (Dox) drug.15-18Thus, the pH sensitivity and redox sensitivity suggest that these particles can deliver therapeutic agents in different ways upon variation of the pH and/or redox conditions, which may induce their swelling/shrinking and/or bond cleavage, resulting in an improvement in the partial or complete dissolution of nanogels. Generally, stimulative nanogels can be divided into chemical-sensitive nanogels and physical-sensitive nanogels according to their sensitivity to different types of external stimuli in a physical and/or physical manner.19,20 Nanogels can be endowed with pH sensitivity through combining with compositions sensitive to chemical reactions (e.g., acidicinduced bond cleavability) and/or physiological differences (e.g., acidic protonation resulting in


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nanogel swelling).17Thus, redox-sensitive crosslinks (e.g., disulfide-containing molecules) can be incorporated into nanogels to acquire redox sensitivity.21,22,23However, thermo-sensitive nondegradable nanogels exhibit the ability to undergo temperature-induced volume variations, which can physically kill cancer cells in the absence of any anticancer drug.24,25However, most of the nanogels are fabricated from synthetic polymers, which often lack biological cues23 and thus confer a limited improvement of the anticancer bioactivity of Dox (in vitro cytotoxicity less than or equivalent to that of free Dox).17,21-23 In the present study, we constructed a new type of naturallyoccurring polymer-based nanogel, which displays thermo-, pH- and redox-responsiveness. Alginate (AG), a kind of natural polymer which has been approved by the U.S. Food and Drug Administration (FDA), was selected as a model biopolymer because it has good biocompatibility and degradability.4,26,44 A biocompatible poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAM-AA)) was in situ incorporated into the AG nanogels (AG/P(NIPAM-AA)) using a disulfide-containing cystamine as a crosslinker via a double emulsion method.45 The nanogels displayed thermo/pH/redox-triple stimulative properties, which are important factors to design intelligent drug delivery systems.18,27-30First, the thermosensitive P(NIPAM-AA) polymer endows these molecules with thermosensitivity for the effective internalization of these nanogels into cells under physiological conditions (37 °C)41, and upon internalization inside cells, the nanogels themselves induce cancer cell death by an abrupt volume expansion upon switchable temperature variation. Furthermore, the nanogels are able to load doxorubicin with high encapsulation efficiency (98%) and effectively deliver Dox in intracellular environments where acidic pH values (pH 5.0–6.5) and high reducible (redox) environments accelerate Dox release aroundthe nucleus. The Dox release can be further triggered by the remote manipulation of the temperature of the nanogels. The triple stimulative nanogels


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synergistically circumvent the drug resistance of Dox, resulting in enhanced anticancer cytotoxicity (Scheme 1).

Scheme 1. Thermo-, pH- and redoxresponsive nanogels allowed for easy internalizationat biological temperatures (with smaller nanosize), while exerting toxicity through cold shock. The triple (thermo-, pH- and reduction-) stimuli-accelerated drug release property enables the effective intracellular delivery of the drug by nanogels to improve the anticancer bioactivity of the drug. EXPERIMENTAL SECTION Materials. Cystamine dihydrochloride (Cys) was obtained from Fluka. 1-Ethyl-3-(3dimethylamino propyl) carbodiiamide hydrochloride (EDC) was obtained from J&K Chemical


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Ltd. All other materials were purchased from Sigma. A549 cells originated from the cell bank of the Chinese Academy of Sciences, Shanghai, China. Preparation and characterization of the nanogels. N-isopropylacrylamide (NIPAM) (2260 mg) or the mixture of NIPAM and acrylic acid (AA) (2420 mg, 9:1 by molar ratio) was dissolved in 20 mL ultrapure (UP) water, and 24 mg of tetramethylethylenediamine (TEMED) in 0.24 mL of UP water was added thereafter followed by 20 min N2 purge at 400 rpm stirring in an ice bath. Subsequently, 30 mg of APS in 0.60 mL of UP water was added and purged with N2 for another 10 min in an ice water bath followed by reaction at room temperature under N2 for 5 h. The samples were purified against hot water and lyophilized to obtain the final PNIPAM or P(NIPAM-AA) polymer.Thepolymerswere characterized by Nuclear Magnetic Resonance (NMR)analysis ona Bruker NMR spectrometer (600 MHz Avance II+). The molecular weight of the synthesized polymers was analyzed via Gel Permeation Chromatography (GPC) using PLgel MIXED-C columns (4.6mm*250mm). N,N-dimethylformamide (DMF) was selected as the mobile phase with a flow rate of 0.350 mL/min at 50 °C. A poly(methyl methacrylate) standard was used for calibration. Elemental analysisof the materials was performed on a Vario Micro Cube instrument. Alginate nanogels were prepared using an emulsion method according to a previous study with several modifications.32 Briefly, 11 mg of EDC in 0.5 mL of H2O was added dropwisely into 2 g of aqueous solutions of AG/P(NIPAM-AA) (2/x (wt%), x = 0 and 0.5) under 400 rpm magnetic stirring (3 h, room temperature) followed by an initial emulsification in 8 mLof 2.5 wt% dioctyl sulfosuccinate sodium salt(AOT) solution in dichloromethane (DCM) and a second emulsification in 30 gof polyvinyl alcohol(PVA)aqueous solution (2 wt%). After mixing for 10 min, the mixture was treated with 2 mL of water containing 110 mg of Cys overnight to remove


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the organic residue, which was purified by a 3-cycle centrifuge/water wash to generate AG and AG/P(NIPAM-AA)

nanogels.

TheAG/PNIPAMnanogels

were

prepared

in

a

similar

manner,except using PNIPAM instead of PNIPAM-AA. The AG, AG/PNIPAM, and AG/P(NIPAM-AA) nanogels are abbreviated as AG, AGP, and AGPA in the present study.The nanogels underwent elemental analysis (Vario Micro Cube instrument) for further compositional evaluation. For drug loading, 4 mL of water containing 4 mg of Dox was mixed with 50 mg of the nanogels (AG, AGP and AGPA) under magnetic stirring for 12 h. The mixture was centrifuged, and the unloaded drug amount was calculated by measuring the UV peak at 490 nm on a UV-Vis spectrometer (Lambda 2, Perkin Elmer), which can be used to evaluate the drug encapsulation efficiency. The precipitate was lyophilized to generate Dox-loaded nanogels. AG, AGP and AGPA nanogels loaded with Dox are abbreviated as AG-Dox, AGP-Dox and AGPA-Dox in thepresent study. The hydrodynamic sizes and surface charges were determined by Dynamic Light Scattering (DLS) analysis on a Zetasizer (Nano ZS, Malvern Instruments). In vitro drug delivery behaviors. Approximately 0.2 mg of AG-Dox, AGP-Dox and AGPADox samples in phosphate-buffered saline (PBS, 2 mL) was incubated in the absence and presence of 5 and 10 mMD,L-dithiothreitol (DTT). To assess their synergistic effect, the drug release behaviors of AGPA-Dox nanogels were investigated under different pH values and a constant intracellular redox condition (DTT, 10 mM) or different temperatures under intracellular redox conditions and different pH values.In a specific period, the mixture wascentrifuged, and the absorption peak of the supernatant was observed on a microplate reader (model Victor3 1420, PerkinElmer) (λex=480 nm, λem=560 nm) to calculate the amount of drug


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release. At each measurement, 0.1 mL of fresh PBS was refreshed. The cumulative release (Cr) of doxorubicin against time was obtained according to the equation: Cr = 100 * Abst/Abstot

(1)

where Abst and Abstotare the cumulative amount of drug released at time t and total drug contained in the nanogels used for drug release, respectively. In vitro biological study.A549 cells (a human lung carcinoma cell line) were incubated with Dulbecco’s modified eagle medium (DMEM) and 10% fetal bovine serum in flasks at 37 °C under a humidified condition with 5% CO2. At 80% confluence, the cells were collected via trypsinization. The cells (5 x 105/well) were cultured in a 96-well plate containing 100 µL of DMEM for 1 day before substituting with 200 µL of fresh DMEM media containing free Dox, AG-Dox, AGP-Dox and AGPA-Dox nanogels with the same Dox amount. Subsequently, the cells were cultured for another 48 h at 37 °C or under temperature switching (3 cooling-heating cycles between 25 (15 min each cycle) and 40 °C (1 h each cycle)) prior to the cytotoxicity study. AG, AGP and AGPA nanogels in DMEM (pH 7.4) at different concentrations underwent the same treatment to study biocompatibility. Finally, the cells in each well were incubated with 30 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) solution for 4 h at 37 °C and then replaced with 200 µL of DMSO for UV measurement at 492 nm for the cell viability analysis (ODtest/ODcontrol×100%). To determine how nanogels can be internalized in cells, the cells were incubated for 24 h and subsequently seeded onto a Φ20-mm cell plate (4 x 104/well in 1 mL DMEM). After incubation for 1 day, the cell media were substituted with fresh media containing Dox alone, AG-Dox, AGP-Dox and AGPA-Dox nanogels (all the Dox concentrations used were 3.5 µM) and cultured for 24 h at biological temperature. The cells underwent glutaraldehyde fixation and 4’6-


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diamidino-2-phenylindole (DAPI) staining followed by observation on a Confocal Laser Scanning Microscope (CLSM, A1R Nikon, Japan). Hemolysis test. To assess the potential of the nanogels for application in vivo (e.g., intravenous injection), the hemocompatibility of these nanoparticles was evaluated through a hemolysis approach.39,40 Briefly, 2 mL of fresh rat blood was added in a heparin-containing anticoagulant tube, which was diluted 10-fold using PBS (pH 7.4, containing 0.89% NaCl). After washing/centrifugation 3 times, the precipitated red blood cells were diluted with PBS to obtain a 2% cell suspension. Next, the cell suspensions were treated with water (positive control), PBS (negative control), and AGPA solution at different concentrations from 0 to 5.00 mg/mL. After 2 h incubation at 37 °C, the mixtures underwent centrifugation, and the absorbance values of the supernatants at 540 nm were subsequently measured on a UV-Vis spectrophotometer. The hemolysis rate of each sample was calculated according to the following formula: Hemolysis Rate (%) = (Asample- ANegative)/(APositive- ANegative) × 100 where Asample is the absorbance value at 540 nm for the supernatant of the experimental group, APositiveand ANegative are the absorbance values of the supernatant of the positive and negative control groups at 540 nm, respectively. Each sample was tested in triplicate.

RESULTS AND DISCUSSION Development of AGPA-Dox nanogels. P(NIPAM-AA) and PNIPAM were prepared through a free radical polymerization method.The 1H NMR spectra of PNIPAM and P(NIPAM-AA) were confirmed in CDCl3 and D2O, respectively. As shown in Figure S1a, the absorption peaks at δ = 4.0 ppm, 2.1~2.6 ppm, 1.5~2.1 ppm and 0.7~1.5 ppm are attributed to the hydrogen in the tertiary carbon in the isopropyl group, the polymer main hydrogen in the tertiary carbon in the


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chain, the hydrogen in the methylene group (–CH2–) of the polymer backbone, and the hydrogen on two methyl groups in the isopropyl group, respectively.33 In addition, a weak broad peak between 5 and 7.6 ppm may derive from the hydrogen on the amide. These results indicate that the polymer generated characteristic 1H NMR peaks at 1.1 ppm (–CH3), 3.8 ppm (–CH–), as well as 1.5 and 2.0 ppm, corresponding to methylene and methine protons, respectively (Figure S1b).13CNMR spectroscopy measurements showed peaks at 170.9 ppm (–COOH) and 168.7 ppm (–CO–), indicating that AA blocks had been incorporated into PNIPAM to form the P(NIPAMAA) copolymer(Figure S1c).33 Based on GPC analysis, PNIPAM and P(NIPAM-AA) had an averaged molecular weight of 51.6 and 62.6 kDa with polydispersity index values of 1.57 and 3.94, respectively (Table S1). Elemental analysis (EA) indicated that the molar ratio between AA and NIPAM in P(NIPAM-AA) wasapproximately1:21, smaller than their feed ratio (1:9). The smaller feed ration may be observed because AA has more reaction activity than NIPAM, which may result in the formation of PAA homopolymers. During purification by hot water treatment, hydrophilic PAA can be removed from P(NIPAM-AA), which is hydrophobic above its lower critical solution temperature.42AG, AGP and AGPA nanogels were prepared via an emulsion method using cystamine (Cys) as a crosslinker (to introduce redox sensitive bond, Scheme 1 and S1).31Based on elemental analysis, the encapsulation efficiency of PNIPAM and P(NIPAM-AA) in the nanogels was 73% and 99%, respectively. This differenceis because the PNIPAM wasintroduced into the nanogels through physical interaction;thus, a portion of these particles may be removedduring the purification processowing to its water solubility, whereas P(NIPAMAA) can be fixed inside the nanogels via its chemical bonding with Cys (Table S2). The less PNIPAM percentage in AGP may be one reason why AGPA were more thermosensitive than AGP. Based on the sulfur content in the nanogels via elemental analysis, the molar amount of


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crosslinker in 1 g ofnanogels can be calculated, which were 0.307 and 0.225 mmol per 1 g AGP or AGPA, respectively. The hydrodynamic sizes and surface charges of the nanogels were measured via Dynamic Light Scattering. AG, AGP and AGPA nanogels showed sizes of 253 ± 4,264 ± 3 and 258 ± 2 nm at 37 °C, respectively. The introduction of PNIPAM into the nanogels increased the ζpotential from −42 ± 11 mV (for AG nanogels) to −37 ± 5 mV (for AGP nanogels) under physiological conditions, likely because PNIPAM is less negative than AG.33AGPA nanogelsexhibited a more negative surface charge than both AG and AGP nanogels, which may be associated with the presence of anionic AA composition in the nanogels. Interestingly, AGPA nanogels exhibited a significant volume transition by switching temperature from 25 to 37 °C (347 ± 9 nm at 25 °C and 258 ± 2 nm at 37 °C), while the corresponding AG and AGP nanogels displayed a negligible size change at different temperatures (Figure 1).

400 AG AGP AGPA

350

Size (d,nm)

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


300

250

200 20

30

40

50

Temperature,°C Figure1.Hydrodynamic sizevariationsof AG, AGP and AGPA nanogels at different temperatures. For the therapeutic delivery study, doxorubicin (a popular anticancer drug)15-18 was mixed


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with the AG, AGP and AGPA nanogels in water to obtain drug-loaded nanogels (AG-Dox, AGPDox and AGPA-Dox) via electrostatic interaction. As shown in Table 1, all of the Dox-loaded nanogels presented higher ζ-potentials than the Dox-free nanogels, suggesting the successful loading of the cationic Dox drug into the nanogels. The Dox-loaded nanogels had larger sizes (175 ± 5 nm for AG-Dox, 171 ± 24 nm for AGP-Dox and 184 ± 11 nm for AGPA-Dox) thandidthe corresponding blank nanogels. The increase in both the size and surface charge of the Dox-loaded nanogels indicated that thecationic Dox had been encapsulated into the nanogels. According to UV quantitative analysis, the Dox encapsulation efficiency values in the AG-Dox, AGP-Dox and AGPA-Dox nanogels were 86.9 ± 0.2, 89.0 ± 0.4 and 94.5 ± 0.3%, respectively. Notably,AGPA nanogels are expected to be more stable than AGP and AGnanogels under physiological conditions because ζ-potentials in the range of ± 30 mV are considered necessary for charge stabilization.33In addition, the increase of ζ-potential of the AGPA nanogels is expected to accelerate the intracellular uptake in cells that present a negatively charged surface.34 Table 1.Physical properties of the nanogels. Sample

Size, nma

Zeta, mVa

AG

130±58

-42±11

AG-Dox

175±27

-39±7

AGP

143±61

- 37±5

AGP-Dox

171±24

-33±1

AGPA

132±15

-45±3

AGPA-Dox

184±11

-15±2

EE, %b

LC, %c

86.9±0.2

7.9±1.0

89.0±0.4

8.1±0.9

94.5±0.3

8.6±0.7

a

Experiments were performed in phosphate-buffered saline (pH 7.4). Encapsulation efficiency (EE) = 100*Wt/W0, W0andWtare the drugs that have been used and those that have been encapsulated, respectively.

b


12

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c

Loading capacity (LC) = 100*Wt/W, Wt stands for the drugs that have been loaded into the nanogels, andWindicates the total nanogel weight.

a)

b)

c)

d)

Figure2. Morphological properties of the freeze-dried AGP nanogels prepared at (a) 25 °C and (b) 37 °C, and AGPA nanogels prepared at (c) 25 °C and (d) 37 °C, respectively. Direct visualization of AGP and AGPA nanogels at different temperatures (25 and 37 °C) was performed on a scanning electron microscope (SEM) to assess their morphologies. As shown in Figure 2, all nanogels had spheric shapes with narrower dispersed distribution. Although AGP and AGPA nanogels had similar sizes at biological temperature (AGP: AGP: 81 ± 2 nm, AGPA: 77 ± 3 nm), these particles showed a significant difference in size at 25 °C (93 ± 3 nm, AGPA:


13


103 ± 1 nm). Upon temperature variation from 37 to 25 °C, the size of the AGPA nanogels increased 1.3-fold their original size, while AGP nanogels increased 1.1-fold their original size. SEM results also indicated that AGPA nanogels were more thermosensitive in size change than AGP nanogels, consistent with the hydrodynamic results. These results are similar to thoseobtained for their hydrodynamic size. Notably, all of the nanogels displayed smaller SEM sizes than their DLS sizes, since the samples for SEM observation were investigated in a dry state, while the DLS size was measured under aqueous solution.

100

a)

)

b

Cumulative Release ,％

Cumulative Release, ％

100 80 60

**

40

AG-Dox AGP-Dox AGPA-DOX Free Dox

20 0

**

60 40

2

4

6

8

10

AGPA-Dox_pH 7.4 AGPA-Dox_ pH 6.5 AGPA-Dox_ pH 5.0

20 0

0

***

80

0

2

4

100

100

80

* *

40 AGPA-Dox_0 mM DTT AGPA-Dox_5 mM DTT AGPA-Dox_10 mM DTT

20 0

0

2

4

6

Time, h

8

10


c)

60

6

8

10

Time,* h

Time, h


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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d)

80 ***

60

***

40

AGPA-Dox_25 °C AGPA-Dox_37 °C AGPA-Dox_42 °C

20 0

0

2

4

6

8

10

Time, h

Figure 3.Drug release profiles of Dox from a) AG-Dox,AGP-Dox and AGPA-Dox nanogels; AGPA-Dox nanogels, b) at different pH values (7.4, 6.5 and 5.0),c) treatment without and with


14

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DTT (5 and 10 mM) under physiological conditions at 37 °C and d) from AGPA-Dox nanogels in PBS buffer at variable temperatures (25, 37 and 42 °C) (*P﹤0.05, **P﹤0.01, ***P﹤0.001). Drug release behaviors.The drug release behaviors of the nanogels were analyzed in PBS at pH 7.4.Although Dox can be released from all the nanogels in a sustainable manner, the AGPADox nanogels showed higher cumulative releaseefficiencies than the AGP-Dox and AG-Dox nanogels. For example, AGPA-Dox nanogels exhibited a sustainable Dox release for up to10 h (up to 68 ± 1%), while the Dox release from AGP-Dox and AG-Dox nanogels terminated at10 h (AG-Dox:50 ± 2%; AGP-Dox: 51 ± 2%). The higher release efficiency of AGPA-Dox nanogels may be associated with their higher themosensitivity to significantly cause nanogel shrinking at 37 °C, which induced more Dox drug release from the nanogels.29 To study the pH sensitivity of the nanogels, their therapeutic delivery properties under acidic conditions, mimicking solid tumors (pH 6.5) and endo/lysosomal microenvironments (pH 5.0), were examined.35,36The AGPA-Dox nanogels displayed an acidic-accelerated drug release behavior, especially after 2.0 h (Figure 3b). For example, the cumulative release values of the nanogels at 10 h were 68 ± 1% (pH 7.4), 79 ± 3% (pH 6.5), and 94 ± 2% (pH 5.0), which may favor an enhanced drug release efficiency in acidic tumor tissues and endo/lysosomes to improve therapeutic anticancer bioactivity.35,36 Since both tumor tissues and various intracellular compartments present reductive microenvironments, the drug release properties of the nanogels were investigated under treatment with D, L-dithiothreitol (DTT) at different concentrations.12,13 The results indicated that the treatment of AGPA-Dox nanogels with 5 and 10 mM DTT significantly increasedcumulative Dox release at 6 h (73± 1 and 78 ± 2%) compared to that under


15


physiological conditions (pH 7.4). At 10 h, the nanogels exhibited a final cumulative release of 68 ± 1, 80 ± 2 and 88 ± 1%, respectively. To determine how temperature influences therapeutic delivery, the temperature dependence of Dox release from the AGPA nanogels was studied at 25, 37 and 42 °C in PBS at pH 7.4. Dox was released faster from AGPA-Dox at both physiological (37 °C) and higher temperatures (42 °C) compared to lower temperature (25 °C) (Figure 3d). The final drug release amounts of the nanogels were 46 ± 1%,68 ± 2%and 83 ± 2% at 25, 37 and 42 °C, respectively. The thermoresponsiveness of Dox release can be ascribed to the rapid shrinkage of the nanogels during temperature switching between 25, 37 and 42 °C (Figure 1). The thermosensitive properties of the PNIPAM-contained nanogels may enable drug release controllability through an indirect thermal modulation.

100

a)

80 60

*** *

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b)

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10 mM DTT+ 25°C+ pH7.4 10 mM DTT+ 25°C+ pH5.0 10 mM DTT+ 37°C+ pH7.4 10 mM DTT+ 37°C+ pH5.0 10 mM DTT+ 42°C+ pH7.4 10 mM DTT+ 42°C+ pH5.0

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Figure 4. In vitro drug release profiles of Dox a) from AGPA-Dox treated with 0 mM DTT at pH 7.4 or 10 mM DTT at different pH values (7.4, 6.5 and 5.0) under 25 °C, b) from AGPA-Dox at different pH values (7.4 and 5.0) under 10 mM DTT and different temperatures (25, 37 and 42 °C).

To explore their synergistic effects, the drug release behaviors of AGPA-Dox nanogels were


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investigated under neutral and acidic conditions at different temperatures (25, 37 and 42 °C) in the absence and presence of 10 mM DTT, respectively. As shown in Figure 4, under the same reductive conditions, either a decrease of pH values or increase in temperature significantly enhanced Dox release. For example, the final cumulative release (10 h) of the nanogels at 10 mM DTT, pH 7.4 and 25 °C was ~45%, while almost all Dox (~98%) was released at 10 mM DTT, pH 5.0 and 42 °C. Therefore, the AGPA-Dox nanogels presented an obvious redox-sensitive release ability, which together with the pH sensitivity may synergistically promote anticancer drug release tumor targeting, as well as intracellular delivery properties. Biological study. The bioactivity of Dox-loaded nanogels was quantitatively analyzed against A549 cells (a human lung carcinoma cell line) via MTT assay. As shown in Figure S2, both AGDox (IC50: 1.56 µM), AGP-Dox nanogels (IC50: 1.30 µM) and AGPA-Dox (IC50: 1.10 µM)significantly enhanced antiproliferation toward A549 cells compared to free Dox drug (IC50: 1.89 µM),likely due to its drug resistance.37 Because the blank nanogels were completely biocompatible, the bioactivity should come from the doxorubicin released from the AGPA-Dox nanogels. To demonstrate the effect of thermal reverse volume transition on the cell viability, A549 cells underwent 3 subsequent cooling-heating cycles between 25 (15 min each cycle) and 40 °C (1 h each cycle). As shown in Figure 5, the switchable temperature treatment obviously increased the cytotoxicity of the AGPA nanogels in A549 cells, likely resulting from the damage of endosomal vesicles (approximately200 nm) within cells because of their reverse thermal-induced volume expansion.24,25In contrast, the same treatment with the non-thermosensitive AG nanogels produced a negligible effect on the cytotoxicity. More importantly, such thermal treatment enhanced the anticancer bioactivity of both AGPA and AGPA-Dox nanogels compared to AGP


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and AGP-Dox. This effect can be attributed to the accelerated Dox release and coldinduced swelling behaviors promoted by higher thermal perturbation of the former with more thermosensitivity compared to the latter (Figure 1, 2 and 3). The combinative merits of AGPADox nanogels may enable their effective intracellular delivery for biomedical applications.

Figure 5. Cell viability of A549 cells cultured for 48 h withAG-Dox,AGP-Dox and AGPA-Dox nanogels (containing the same Dox amount of 0.5 µM), and blank nanogels, which had the same amount of Dox-loaded nanogels (± standard deviation, n=3, *P﹤0.05, **P﹤0.01), with (marked as “changing ToC”) and without (marked as “W/o changing ToC”) temperature variation treatment. To study how the drug can be taken up by cancer cells,38 the fluorescentintensity of the Dox drug accumulated in A549 cells was imaged on a Confocal Laser Scanning Microscope (CLSM). As shown in Figure 6, the AGPA-Dox nanogels presented an obvious higher reddish colorcompared to the Dox, AG-Dox and AGP-Doxnanogels. The thermosensitive nanogels exhibited higher ζ-potential and a smaller size under physiological conditions (37 °C), which


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may entice cells to endocytose these nanoparticles, compared to the corresponding more negatively charged AGP-Dox and AG-Dox. The enhanced cell uptake ability as well as high bioactivity of the nanogels may be associated with their high intracellular internalization and multi-stimulative functions in intracellular drug delivery.34

Figure 6. Optical and fluorescentimages of A549 cells incubated for 24 h with the Dox drug as well as the Dox-loaded nanogels, carrying the same Dox concentration (3.5 µM). Hemolysis study. Since hemocompatibility is an important factor for biomedical applications in vivo, the hemolysis rate of nanogels was evaluated by hemolysis experiments. As shown in Figure 7, the hemolysis rate of nanogels was low (less than 5%), even at an AGPA concentration


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of up to 5.00 mg/mL, which was within the allowable range of national standards (no more than 5%).43 These results demonstrate that the nanogels exhibit good blood compatibility, suggesting their potential for in vivo intravenous injection.

Figure 7. Hemolysis behaviors of red blood cells treated with water (positive control), PBS (negative control), and AGPA solution at concentration varying from 0 to 5.00 mg/mL (mean ±SD, n=3). The inset is a photograph of the corresponding samples. CONCLUSIONS In this study, we introduced an approach to developing thermo-, pH- and redox-responsive nanogels with good cytobiocompatibility for anticancer drug delivery. The drug-loaded nanogels are capable of inducing cancer cells towards endocytosis under physiological conditions


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(biological temperature), and these nanoparticles can produce apoptotic activity in the cells via abrupt volume expansion and an accompanying thermo-induced Dox release through a reverse cooling-heating switching treatment. Furthermore, inside cells and triggered by intracellular cellmimicking conditions (the intracellular reducible environments and acidic conditions in the endo/lysosomes), the drugs in the nanogels can be effectively intracellularly delivered to kill cancer cells. The intelligent drug delivery properties of the nanogels, together with their remote controllability (thermosensitivity) and good cytocompatibility, may enable the design of novel nanomedcines for intracellular drug delivery to improve their therapeutic bioactivity.

ASSOCIATED CONTENT Supporting Information The NMR spectra of PNIPAM and P(NIPAM-AA); Schematic illustration of the formation and drug loading of AGPA nanogels; Cytotoxicity of free Dox, AG-Dox, AGP-Dox and AGPA-Dox nanogels and AG, AGP and AGPA nanogels; The molecular weight characterization of PNIPAM and P(NIPAM-AA); The elemental analysis of AGP and AGPA nanogels.

ACKNOWLEDGMENTS The present study was financially supported by grants from the Shanghai Municipal Natural Science Foundation (15ZR1408500), the Shanghai International Cooperation Program (15520721200), and the Key Laboratory for the Synthesis and Application of Organic Functional Molecules, Ministry of Education (2014-KL-004).

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Multistimulative Nanogels with Enhanced Thermosensitivity for

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