Novel Engineered Microgels with Amphipathic Network Structures for

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Novel Engineered Microgels with Amphipathic Network Structures for Simultaneous Tumor and Inflammation Depression Xianjing Zhou, Yanxin Qi, Zhijun Zhang, Jingjing Nie, Yubin Huang, and Bin-Yang Du ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02382 • Publication Date (Web): 09 Mar 2018 Downloaded from http://pubs.acs.org on March 9, 2018

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ACS Applied Materials & Interfaces

Novel Engineered Microgels with Amphipathic Network Structures for Simultaneous Tumor and Inflammation Depression Xianjing Zhou1,2, Yanxin Qi3*, Zhijun Zhang1, Jingjing Nie4, Yubin Huang3 & Binyang Du1* 1

MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science & Engineering, Zhejiang University, Hangzhou 310027, China 2

3

Department of Chemistry, Zhejiang Sci-Tech University, Hangzhou 310018, China

State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, China 4

Department of Chemistry, Zhejiang University, Hangzhou 310027, China

ABSTRACT: A novel engineered microgels with amphipathic network structures were designed and synthesized by copolymerizing N-isopropylacrylamide (NIPAm), 1-vinylimidazole (VIM) and 2-(cinnamoyloxy) ethyl methacrylate (CEMA) in the presence of 1, 6-dibromohexane. The engineered microgels possess hydrophilic quaternization crosslinking structures and hydrophobic crosslinking inner nanodomains, which are suitable for loading and simultaneous release of hydrophilic non-steroidal anti-inflammatory drug (NSAID), diclofenac sodium (DS), and hydrophobic antic cancer drug, doxorubicin (DOX), respectively. The engineered microgels exhibited excellent stability, low cytotoxicity and long blood circulation time, could be uptaken into the cytoplasm of cells, metabolized and excreted from the living body by kidney and liver. In vivo experiments showed that with injection of DS and DOX dual-drug loaded microgels, simultaneous antitumor treatment and inflammation depression were achieved along with high antitumor efficacy and low drug-related toxicity. Such microgels with amphipathic network structures have promising applications for combination therapy.

*

Corresponding author. E-mail: [email protected]. [email protected] 1

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KEYWORDS: microgels, amphipathic network, combination therapy, inflammation depression, antitumor treatment

INTRODUCTION Curing cancer remains a daunting challenge because of the complexity of pathology,1,2 and a single drug may not suffice.3 Recent studies indicated that combination therapy could cooperatively inhibit the proliferation of tumor cells.4-7 Loading and delivering of dual or multiple drugs by a single carrier could further maximize the merits of combination therapies.8-10 Paclitaxel (PTX) and nuclear receptor siTR3 have been co-delivered by using a tumor-targeted, redox-responsive nanovehicle to synergistically treat pancreatic cancer.11 The hybrid nanoparticles with a multiple layer-by-layer structure have been developed to simultaneously deliver three drugs, namely irinotecan, 5-ﬂuorouracil, and oxaliplatin, for cancer treatment.12 It has been found that cancer-related inflammation at tumor site frequently precedes and contributes to the tumor development.13,14 Thus, inflammation depression should become an important treatment during cancer therapy. Non-steroidal anti-inflammatory drugs (NSAIDs), including ibuprofen, aspirin, salicylic acid (SA), diclofenac, etc. have shown the ability to alter inflammation and improve moderate cachexia. Remarkably, inflammation can be attenuated by treatment with NSAIDs, which could lead to tumor regression15. Curing cancer might be benefited via combination therapy with NSAID and anti-cancer drugs simultaneously delivered. To this end, a biocompatible and stable carries system is also required to simultaneously deliver multiple drugs and pass through the physiological barriers.16 For the last few decades, polymeric nanoparticles have become one of the most promising and viable candidates for delivery of therapeutics.17-19 The polymeric nanoparticles could be engineered to have various sizes and architecture as well as surface properties, for instance, polymeric micelles, 2


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nanocapsules, microgels, and liposomes.18,20-23 The drug-loaded polymeric nanoparticles could overcome some drawbacks of using free drugs and therapeutics including confer prolonged circulation time, incorporate drugs with different solubility, enhance accumulation in the tumor sites, minimize drug side effects, improve pharmacodynamics and pharmacokinetic properties of drugs.20,24-26 Among them, microgels with three-dimensional cross-linking structures exhibit excellent swelling properties and dispersion stability in aqueous solution. By selecting the appropriate monomers, comonomers or crosslinkers, the obtained microgels could respond to various environmental stimuli, like temperature, pH and so on.

27-33

Typically, drugs can be easily

entrapped into temperature-sensitive microgels at lower temperature, and then delivered to the lesion and released at higher temperature.34,35 A class of core-shell structured hybrid microgels with the silver-gold bimetallic nanoparticle as core and the thermo-sensitive crosslinked poly(ethylene glycol) as shell has been prepared to load an anticancer drug temozolomide with high capacity and give a thermo-triggered drug release.36 A thermo- and pH- dual sensitive microgel prepared by copolymerization of N-isopropylacrylamide and acrylic acid was developed to specifically deliver anticaner drug, doxorubicin (DOX) into the tumor and endothelial cells with controlled release manner.37 Herein, we reported a novel microgel, which was engineered to give amphipathic network structures by using N-isopropylacrylamide (NIPAm) as the main monomer, 1-vinylimidazole (VIM) and 2-(cinnamoyloxy) ethyl methacrylate (CEMA) as the comonomers in the presence of 1, 6-dibromohexane, as shown in Scheme 1. The hydrophilic crosslinking network structures were obtained via quaternization crosslinking reaction between 1,6-dibromohexane and VIM.38-40 The resultant positive charged quaternized crosslinking structure of microgels was suitable for loading and sustained release of hydrophilic negative charged NSAID, diclofenac sodium (DS).40 CEMA with two reactive double bonds could act as a crosslinker to form hydrophobic crosslinking network structures, leading to the formation of hydrophobic inner nanodomains within the microgels, which could be used to load and sustained release of hydrophobic anti-cancer drug, like doxorubicin 3



(DOX). As we will discuss, the engineered microgels exhibited excellent biocompatibility, longer blood circulation time and could be metabolized and excreted from the living body by kidney and liver. Loading and simultaneous sustained release of DS and DOX with the engineered microgels were achieved. We demonstrated via an in vivo tumor model that the DS and DOX dual-drug loaded microgels were promising for combination tumor therapy via simultaneous inflammation depression and antitumor treatment along with high antitumor efficacy and low drug-related toxicity.

Scheme 1. Synthesis Route and Network Structures of Engineered Thermo-sensitive Microgels with Amphipathic Crosslinking Networks

EXPERIMENTAL SECTION Materials. 2-Hydroxyethyl methacrylate (HEMA), 4-dimethylaminepyridine (DMAP), and 1-vinylimidazole (VIM) were purchased from Aladdin. N-isopropylacrylamide (NIPAm) and 1, 6-dibromohexane were obtained from Tokyo Chemical Industry Co. Ltd. Cinnamoyl chloride, cinnamyl bromide, and doxorubicin (DOX) were supplied from J&K Chemical Ltd. 2,2'-Azobis(2-aminopropane) 2HCl

4


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(AIBA) and methylthiazoletetrazolium (MTT) were obtained from Sigma-Aldrich. Fluorescein O-methacrylate (FMA) and diclofenac sodium (DS) were obtained from Alfa Aesar and Zhengzhou Huayao Biotechnology Ltd, respectively. 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Beyotime Biotechnology. Dulbecco’s Modified Eagle’s Medium (DMEM) was obtained from GIBCO Invitrogen Corp. Fetal bovine serum (FBS) was provided by Zhejiang Tianhang Biotechnology Co., Ltd. Dimethyl sulfoxide (DMSO), sodium carbonate (Na2CO3), dichloromethane (CH2Cl2), sodium chloride (NaCl), and anhydrous magnesium sulfate (MgSO4) were purchased from Sinopharm Chemical Reagents Co., Ltd. All of the chemicals were commercially obtained and used without further purification. Standard protocols were used to prepare the buffer solution (0.01 M) of phosphate-buffered saline (PBS, pH 7.4 and 6.8) and acetic acid/sodium acetate (pH 5.0). P(NIPAm-co-VIM) linear copolymer was synthesized previously.38

Cell Lines and Animals. The human embryonic kidney 293 (HEK293) and human cervical cancer cell line HeLa were obtained from American Type Culture Collection. The murine H22 hepatocarcinoma (H22) cells were obtained from Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China. Kunming (KM) mice were provided by Jilin University (China). H22 cells (1 × 106, 0.1 mL PBS) were subcutaneously injected into the right legs of KM mice to obtain the H22 tumor-bearing mice model. All animal protocols were approved by the local institution review board and were in accordance with the Guidelines of the Committee on Animal Use and Care of Chinese Academy of Sciences.

General Characterization. Proton Nuclear Magnetic Resonance (1H NMR) spectra were measured by using a Varian Mercury Plus instrument (400 MHz). Transmission electron microscopy (TEM) observations were 5



carried out by using a JEOL JEM-1200 electron microscope. FT-IR spectra were recorded on a Vector 22 Bruker spectrometer. Dynamic light scattering (DLS) and electrophoretic light scattering (ELS) techniques were used to measure the hydrodynamic size, size distribution and Zeta potentials of microgels by using a 90 Plus Particle Size Analyzer (Brookhaven Instruments Corp.). The static light scattering (SLS) of microgels were performed by using a ALV-CGS-3 instrument at scattering angle θ of 35° to 135° with a step of 5°. For each measured temperature of DLS and SLS measurements, the microgel solutions were equilibrated for 10-15 min. UV-visible spectra were collected by using a Cary 100 UV-visible spectrophotometer (Varian Australia Pty Ltd.).

Synthesis of 2-(Cinnamoyloxy) Ethyl Methacrylate (CEMA). HEMA (1.32 g, 10 mmol), Na2CO3 (2.12 g, 20 mmol), DMAP (0.12 g, 1 mmol) and 40 mL of dichloromethane were placed into a 100 mL flask and cooled in an ice bath. A solution of cinnamoyl chloride (0.34 g, 2 mmol) in 10 mL of dichloromethane was added dropwise to the ice-cold solution under stirring. The reaction mixture was then warmed to room temperature and stirred overnight, followed by filtering and washing three times with NaCl aqueous solution (0.1 M). The organic layers were concentrated and purified by flash column chromatography using dichloromethane as eluent to give 0.23 g (44%) of 2-(cinnamoyloxy) ethyl methacrylate (CEMA) as transparent liquid. The chemical structure of CEMA was confirmed by 1H NMR (Figure S1) as: 1.96 ppm (3H, CH3), 4.46 ppm (4H, COOCH2CH2OOC), 5.60 and 6.16 ppm (2H，CH3C=CH2), 6.44 and 6.48 ppm (1H, Ar-CH=CH), 7.40 and 7.53 ppm (5H, Ar), 7.70 and 7.74 ppm (1H, Ar-CH=CH).

Synthesis of Thermo-sensitive Microgels with Amphipathic Network Structures. Surfactant free emulsion copolymerization (SFEP) was applied to prepare thermo-sensitive microgels with amphipathic network structures by using NIPAm as the main monomer, VIM and CEMA as the comonomers in the presence of 1, 6-dibromohexane. 1, 6-Dibromohexane could 6


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quaternize with VIM to form hydrophilic crosslinking network, while CEMA with two reactive double bonds acted as a crosslinker to form hydrophobic crosslinking network. A certain amount of NIPAm, VIM and 45 mL deionized water were placed into a 100 mL flask and heated to 70 oC under vigorous stirring. Nitrogen was bubbled through the solution to remove oxygen. After 30 min, the initiator solution (AIBA, 5 mg/mL, 5 mL) was added into the mixture solution to start the polymerization. 20 min later, 1 mL CEMA dichloromethane solution was added dropwise to the solution under stirring. After another hour, 1 mL dichloromethane solution of 1, 6-dibromohexane was added. The molar ratio of VIM to 1, 6-dibromohexane was fixed to be 2. The solution was then stirred for 6 h at 70 oC. After reaction, the obtained microgels were placed in a dialysis tube (molecular weight cutoff of 14 kDa) and dialyze in deionized water for 3 days. It has been previously confirmed that imidazole could be quaternized with dibromo compounds completely under these experimental conditions.38 The samples were coded as PNCQ microgels, depending on the experimental conditions, which were listed in Table 1. For example, for PNC8Q3.5 microgels, P, N, C, and Q represent poly, NIPAm, CEMA, and quaternization crosslinking, respectively. The numeric values of 8 and 3.5 mean that the molar fraction of CEMA and 1, 6-dibromohexane to NIPAm are 8% and 3.5%, respectively. The microgels with CEMA as sole crosslinker were prepared by using the similar experimental condition of PNCQ microgels without addition of 1, 6-dibromohexane and coded as PNC microgels. The copolymer P(NIPAm-co-VIMCB) (PNV25) were also synthesized by using the similar experimental condition of PNC25 microgels with 3-cinnamyl-vinyl imidazolium bromide (VIMCB) as the comonomer. VIMCB was obtained by the quaternization reaction of VIM and cinnamyl bromide.41 The number value of 25 in PNV25 represented the molar fraction of VIMCB to NIPAm is 25%.

7



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Table 1. Experimental Parameters and Results of PNCQ Microgels CEMA (mmol)

VIM (mmol)

1,6-dibromohexane (mmol)

RTEM (nm)

Rh* (nm)

Rg* (nm)

Rg/Rh*

VPTT (oC)

Zeta Potential* (mV)

PNC8Q3.5

0.16

0.14

0.07

343 ± 21

366 ± 9

272

0.74

39

(+) 29 ± 5

PNC8Q5

0.16

0.2

0.1

219 ± 9

262 ± 6

192

0.86

41

(+) 45 ± 2

0.16

0.26

0.13

216 ± 5

284 ± 8

219

0.86

43

(+) 48 ± 4

PNC10Q5

0.2

0.2

0.1

203 ± 6

258 ± 6

196

0.81

41

(+) 44 ± 4

PNC12Q5

0.24

0.2

0.1

166 ± 12

247 ± 5

160

0.72

41

(+) 44 ± 3

Sample codes

NIPAm (mmol)

PNC8Q6.5

2

* measured at 25 °C.

Synthesis of Fluorescent PNC8Q6.5-FMA Microgels. The fluorescent PNCQ microgels coded as PNC8Q6.5-FMA were synthesized via the same procedure of PNC8Q6.5 microgels by adding 2 mg of FMA as fluorescent label.

Loading of DOX in PNC8Q6.5 Microgels. Firstly, 10 mg DOX was dissolved in 1 mL dichloromethane in the presence of 10 µL triethylamine. After the substances were completely dissolved, the solution was mixed with 20 mL DMSO solution of PNC8Q6.5 microgels (100 mg) and stirred in an opened flask overnight to evaporate dichloromethane. The mixture was then purified by using dialysis in deionized water (molecular weight cutoff of 14 kDa). The microgels with loaded DOX were then coded as PNC8Q6.5-DOX.

Loading of DS in PNC8Q6.5-DOX Microgels. 5 mg DS was added into 30 mL aqueous suspension of PNC8Q6.5-DOX microgels (3.3 mg/mL) and stirred at room temperature for 3 h. The resultant microgels were purified by using dialysis in deionized water (molecular weight cutoff of 14 kDa). The microgels with loaded DOX and DS were then coded as PNC8Q6.5-DOX-DS. The encapsulation efficiency (EE) and drug loading content (DLC) were determined by 8


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measuring the UV-visible spectra of the solutions outside the dialysis tubes at the wavelength of 482 nm for DOX and 277 nm for DS, respectively. The masses of the unloaded drugs were calculated based on the standard curves calibrated with DOX and DS samples of known concentrations (Figure S2). The DLC and EE were then given as: DLC ሺwt%ሻ =

mass of drug loaded in microgels × 100% mass of drugloaded microgels

(1)

EE ሺwt%ሻ =

mass of drug loaded in microgels × 100% the initial mass of drug

(2)

The DLCs of DS and DOX in PNC8Q6.5-DOX-DS microgels were measured to be about 6.5% and 1.6%, respectively. The EEs of DS and DOX in PNC8Q6.5-DOX-DS microgels were measured to be about 64.9% and 32.4%, respectively.

In Vitro Simultaneous Release of DOX and DS. The simultaneous release of DOX and DS from PNC8Q6.5-DOX-DS was quantitatively determined. Briefly, 25 mg of PNC8Q6.5-DOX-DS microgels were dissolved in 10 mL of buffer solutions with different pH values. The microgel solutions were then added into dialysis tubes and transferred to the flask with 40 mL of the same buffer solution. The flasks were shaken at 37 oC on a shaker set at 100 rpm. At predefined time points, 2 mL of the solution outside the dialysis tube was collected, and the same volume of fresh buffer solution was replenished. The DOX and DS concentrations were measured by the UV-visible spectrophotometer and calculated based on the standard curve calibrated.

MTT and CLSM. The biocompatibility of the microgels was evaluated on HEK293 and HeLa cells through MTT assay. In brief, the cells were cultured on 96-well plates for 16 h before adding microgels with various concentrations (1-500 µg/mL). After 24 h of incubation, 20 µL of MTT (5 mg/mL) was 9



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added to each well and treated for another 4 h at 37 °C. Finally, the solutions were removed, and the MTT-formazan crystals were dissolved in 100 µL/well of DMSO and measured at 570 nm with a microplate reader (BIO-RAD 680, USA). The relative cell growth (%)was given as: relative cell viability ሺ%ሻ =

ை஽౪౛౩౪ ିை஽ౘౢ౗౤ౡ

ை஽ౙ౥౤౪౨౥ౢ ିை஽ౘౢ౗౤ౡ

× 100%

(3)

where ODtest, ODcontrol, ODblank represented the optical density values of treated cells, untreated cells, and the blank, respectively. The vitro cellular uptake of the fluorescent PNC8Q6.5-FMA-DS-DOX microgels was tested by using HeLa cells. The cells were seeded in DMEM medium for 24 h. The medium was then replaced with serum-free media consisting of PNC8Q6.5-FMA-DS-DOX microgels (10 µg/mL). After 4 h of incubation, the cells were washed with pre-warmed PBS and fixed with paraformaldehyde solution (4% w/w), followed by staining of nuclei with DAPI. Confocal laser scanning microscope (CLSM 410) was employed to image the cellular uptake of microgels. The excitation wavelengths of DAPI, FMA and DOX are 405, 486 and 543 nm, respectively.23,42

In Vivo Antitumor Efficiency. H22 tumor-bearing mice (~100 mm3 in tumor volume) were individually identified with ear tags. The body weight and initial tumor volume of the mice were also measured. The mice with tumors were then treated via intravenous and intratumoral injections, respectively. For each treatment method, animals were randomly distributed into five groups (6 mice/group), including the control group, PNC8Q6.5 group, DOX group, DS group and the PNC8Q6.5-DOX-DS group. Except for the control group, mice were injected with PNC8Q6.5 (0.012 ml/g), DOX (3 mg/kg), DS (0.00075mg/g) and PNC8Q6.5-DOX-DS (0.012 ml/g), respectively, at day 0, 3 and 5. The body weight and tumor volume of the mice were measured every other day to assess the systematic toxicities and antitumor activities of various formulations. The estimated tumor volume was calculated as follows： Tumor volume = 0.5 × the longest diameter × the square of short diameter 10


(4)

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Histopathology Analysis. At day 27 (or day 21), mice treated via intravenous injection (or intratumoral injection) were anesthetized and the chests were excised open. The tumors were removed and post-fixed in phosphate-buffered paraformaldehyde (4%) overnight, and then submerged in paraffin. Five mm thick tissue samples were sliced and stained with hematoxylin and eosin (H&E) for histopathological evaluation using microscope (Nikon TE2000U).

Excised Imaging. Tumor-bearing mice were injected with PNC8Q6.5-FMA (794 nm) via tail vein (5 mg/kg). After the injection, mice were sacrificed at 1, 12, 24, 48 and 72 h. The major organs (heart, kidney, liver, lung and spleen) and tumor were excised, washed with PBS, and visualized by a Maestro in vivo Imaging System. The excitation and emission wavelengths are 523 nm and 560 nm, respectively. Three mice were used for each time point. The commercial Maestro 2.10 Analysis software was used to analyze the resulting data.

RESULTS AND DISCUSSION PNCQ Microgels with Amphipathic Crosslinking Network Structures. The microgels with amphipathic network structures were successfully prepared and coded as PNCQ microgels, depending on the experimental conditions (Table 1 and experimental section). For example, for PNC8Q3.5 microgels, P, N, C, and Q represent poly, NIPAm, CEMA, and quaternization crosslinking, respectively. The numeric values of 8 and 3.5 mean that the molar fraction of CEMA and 1, 6-dibromohexane to NIPAm are 8% and 3.5%, respectively. Figures 1A, 1B and S3 display the typical TEM images of resultant microgels. The spherical microgels with uniform shape and a narrow size distribution were successfully obtained. The average radii were counted from the TEM images and listed in Table 1. The average radii of PNC8Q3.5, PNC8Q5, PNC8Q6.5, PNC10Q5 and PNC12Q5 microgels were about 343 ± 21 nm, 219 ± 9 nm, 216 ± 5 nm, 11



203 ± 6 nm, and 166 ± 12 nm, respectively. Interestingly, dark nanodomains with sizes of 20-40 nm could be clearly observed, which were uniformly distributed in the microgels. The size of dark inner nanodomains increased with increasing the feeding amounts of CEMA. The formation of the inner nanodomains will be discussed later.

Hydrodynamic Radius (nm)

C

400

PNC8Q3.5 PNC8Q5 PNC8Q6.5 PNC10Q5 PNC12Q5

350 300 250 200 150 100 20

25

30

35

40

45

50

55

60

65

o

Temperature ( C)

D

2.0 1.8

PNC8Q3.5 PNC8Q5 PNC8Q6.5 PNC10Q5 PNC12Q5

1.6 1.4 1.2

Rg / Rh

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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1.0 0.8

0.778

0.6 0.4 0.2 0.0 20

25

30

35

40

45

50

55

60

65

o

Temperature ( C)

Figure 1. TEM images of (A) PNC8Q3.5 and (B) PNC8Q6.5 microgels. (C, D) The evolution of hydrodynamic radius 〈Rh〉 (C) and the corresponding 〈Rg〉/〈Rh〉 ratio (D) of PNCQ microgels as a function of temperature.

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a.CEMA b.PNIPAm-co-VIM c.PNC8Q3.5

Transmittanc

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


d.PNC8Q5

e.PNC8Q6.5 f.PNC10Q5 g.PNC12Q5

980 1718

3500

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm ) Figure 2. FT-IR spectra of CEMA (a), P(NIPAm-co-VIM) (b) and PNCQ microgels, i.e. PNC8Q3.5 (c), PNC8Q5 (d), PNC8Q6.5 (e), PNC10Q5 (f) and PNC12Q5 (g), respectively.

The structure of PNCQ microgels was confirmed by FT-IR spectra and is shown in Figure 2. The signals at 1456, 1544, and 1652 cm−1 were assigned to the methyl group asymmetric bending vibrations, N-H stretching vibration and the C=O stretching vibration of PNIPAm segments, respectively.38 The characteristic absorption band at 1228 cm−1 could be ascribed to the N-C-N bond of VIM segment.43,44 The signals at 980 and 1718 cm-1 (green guidelines) could be ascribed to the C-O-C stretching vibration and C=O stretching vibration of ester bond in CEMA and PNCQ microgels, respectively.45,46 However, the above characteristic absorption bands were absent in the spectrum of P(NIPAm-co-VIM) linear copolymer. The FT-IR results indicated that the CEMA segments were successfully incorporated into the microgel networks. DLS and SLS were employed to study the thermo-sensitive characters of PNCQ microgels. Figures 1C, S4 and 1D show the temperature effect on hydrodynamic radii 〈Rh〉, radii of gyration

〈Rg〉, and the corresponding 〈Rg〉/〈Rh〉 ratios of PNCQ microgels. The radii of gyration were determined from Guinier-type plots (Figure S5).39,47 Both 〈Rh〉 and 〈Rg〉 decreased with an increase 13



of measuring temperature, indicating that PNCQ microgels were thermo-sensitive. It is worthy to note that the poly(N-isopropylacrylamide) (PNIPAm) exhibits a lower critical solution temperature (LCST, ~32 °C) in aqueous solution.48 Compared with CEMA, the degree of quaternization crosslinking had a significant effect on the change of 〈Rh〉 at different temperatures. Figure 1C also shows that the size of microgels does not vary monotonically with the amount of cross-linking agents. The microgels are formed herein by two different crosslinking mechanisms. One is quaternization crosslinking reaction between 1, 6-dibromohexane and VIM, the other is CEMA crosslinking, as shown in Scheme 1. On the one hand, the microgels with more crosslinking agents (i.e. 1,6-bromohexane or CEMA) have more compact crosslinking structure, leading to the smaller radius after swelling in the aqueous solutions. On the other hand, the more crosslinking agents themselves also contributed to the larger volume. Therefore, the size of microgels changed without regularity. Analogous to LCST, volume phase transition temperature (VPTT) is used to quantify the thermo-sensitive characters of microgels. The VPTTs of PNC8Q3.5, PNC8Q5 and PNC8Q6.5 microgels were 39, 41 and 43 °C, respectively. While for PNC8Q5, PNC10Q5 and PNC12Q5 microgels with the same degree of quaternization crosslinking, the VPTTs were the same. The crosslinking density distribution of the microgels could be characterized by 〈Rg〉/〈Rh〉.49 The values of 〈Rg〉/〈Rh〉 are 0.778 and 1 for uniform hard spheres and hollow spheres, respectively. For microgels with different crosslinking structures, 〈Rg〉/〈Rh〉 usually had the value of 0.55-0.96.31,50 Depending on the amount of crosslinking agents and solution temperature, the 〈Rg〉/〈Rh〉 values of PNCQ microgels varied from 0.62 to 0.89 (Figure 1D). For PNC8Q3.5, PNC8Q5, and PNC8Q6.5 microgels, 〈Rg〉/〈Rh〉 values increased with increasing the quaternization ratios, which led to denser shell structures of the microgels. However, for PNCQ microgels with the same quaternization ratio,

〈Rg〉/〈Rh〉 values slightly decreased with increasing CEMA (Figure 1D). A large number of hydrophobic inner nanodomains consisted of CEMA crosslinking structures uniformly distributed within the microgels, leading to the decrease of 〈Rg〉/〈Rh〉. Furthermore, 〈Rg〉/〈Rh〉 values approached 14


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to 0.778 for all of the PNCQ microgels at solution temperatures above their VPTTs. It was understandable because the PNIPAm segments became hydrophobic and water-insoluble at high temperature and the collapsed microgels had more uniform microstructures. The Zeta potential of PNCQ microgels was positive (Table 1) because of the quaternization crosslinking structures and the use of cationic initiator, AIBA, which were thus suitable for loading negative charged compounds. Formation Mechanism of the Amphiphilic Crosslinking Network Structures. The PNCQ microgels are formed by two different crosslinking mechanisms: one is quaternization crosslinking reaction between 1, 6-dibromohexane and VIM, the other is CEMA crosslinking, as shown in Scheme 1. The mechanism of quaternization crosslinking reaction has been previously reported and confirmed.38 The positively charged quaternization crosslinking provides the hydrophilic crosslinking network structures of the resultant microgels, which have been confirmed by TEM, DLS and Zeta potential results (cf. Figure 1 and Table 1). For the formation of crosslinking structures with CEMA, it was reported that the cinnamic group of CEMA would undergo [2+2] cycloaddition by UV irradiation (λ > 260 nm).51-53 Furthermore, the ester bond formed by the esterification reaction of cinnamoyl chloride with HEMA can activate the cinnamyl double bond so that it can be initiated by free radical initiator. As a result, cinnamyl double bond of CEMA is radically polymerizable so that CEMA behaves as a bifunctional crosslinker in the present work. The PNC microgels could be obtained with CEMA as the sole crosslinker without addition of 1, 6-dibromohexane, as shown in Figures 3A-D. Note that no UV irradiation was applied. As long as the amounts of CEMA were large enough (i.e. greater or equal to 15%), the PNC microgels with narrow size distribution were obtained (Figures 3A-C). However, only nanoparticles with sizes of 20-40 nm were observed when small amounts of CEMA (less than 15%) were used (Figure 3D). 15



The size and shape of these nanoparticles were similar with the dark nanodomains observed within the PNCQ microgels (cf. Figures 1 and S3), suggesting that the dark nanodomains were resulted from the copolymerization and crosslinking reaction of CEMA and NIPAm. It was worthy to note that the maximum molar fraction of CEMA was 12% for PNCQ microgels, i.e. PNC12Q5 microgels. Moreover, the PNC microgels maintained the shapes and spherical morphology when the microgels were re-dispersed in DMF, which was the good solvent for NIPAm and CEMA (Figure 3E). The PNC microgels exhibited larger swelling volumes in DMF than those in deionized water. These results indicated the existence of chemical cross-linked structures within PNC microgels. Furthermore, no signals were observed at δ=6.16 and δ=7.74 (Figure 3F and S1) from the 1H NMR spectrum of PNC25 microgels, revealing that the cinnamyl double bonds had completely reacted during the formation of PNC microgels.

16


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E Intensity Weighted (a.u.)

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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2

H H2 C C

H2 C C

O

PNC15

PNC25 CEMA

O NH

1

O 6

4 7

1

O

100 75 50 25 100 75 50 25 0 230

2

3

F

100 75 50 25

O

PNC20

8 9

5 10

a

b

4

10

PNC25 240

270

3,8,9 2

DMF H2O

300

330

360

8

7

6

Hydrodynamic Diameter (nm)

5

4

3

2

1

0

δ (ppm)

Figure 3. (A-D) TEM images of PNC microgels. (A) PNC25, (B) PNC20, (C) PNC15, and (D) PNC10. (E) The size distribution of PNC microgels measured by DLS at 25 °C in deionized water and DMF, respectively. (F) 1H NMR spectra of PNC25 microgels and CEMA. Inset of (A-D) were the corresponding size distributions of microgels counted from TEM images.

For comparison, NIPAm was copolymerized with another cinnamyl-containing comonomer, 3-cinnamyl-vinyl imidazolium bromide (VIMCB) under the same conditions. In this case, only linear copolymer P(NIPAm-co-VIMCB) (PNV25) was obtained (Figure S6). Figure S7 shows the digital photos of PNC25 and PNV25 aqueous solutions. Obviously, PNC25 aqueous solution was a 17



suspension with Tyndall effect, while PNV25 aqueous solution is a clear and transparent solution. For monomer VIMCB, the cinnamyl double bond is linked to methylene, which can’t activate the cinnamyl double bond, so that the cinnamyl double bond could not participate in the radical polymerization reaction. No crosslinking structure could be formed with VIMCB as the comonomer. Based on the above results and discussion, the amphiphilic crosslinking network structures of PNCQ microgels were made with the hydrophilic quaternization crosslinking structures and hydrophobic crosslinking nanodomains formed because of CEMA. The hydrophilic positively charged quaternization crosslinking structures are suitable for loading the hydrophilic negatively charged drug via electrostatic interaction, whereas the hydrophobic crosslinking nanodomains are suitable for loading the hydrophobic drug via hydrophobic interaction54.

In Vitro Cytotoxicity and Drug Release. The PNCQ microgels with amphiphilic crosslinking network structures could be used as carriers to load and simultaneously deliver the hydrophilic and hydrophobic drugs for combination therapy. In the present work, the hydrophobic drug, DOX, was used as the anti-cancer drug and the NSAID, DS, was chosen as hydrophilic drug to depress the inflammation during cancer therapy. The PNC8Q6.5 and PNC8Q6.5-FMA microgels were used for loading and simultaneous release of DOX and DS. As a drug carrier, we first investigated the cytotoxicity of PNCQ microgels before and after drug loading by incubating with HEK293 and HeLa cells for 24 h. The cytotoxicity of PNC8Q6.5 microgels was negligible in a broad concentration range of 1-500 µg/mL, as shown in Figures 4A and S8. After loading with the dual drugs, the PNC8Q6.5-DOX-DS and PNC8Q6.5-FMA-DOX-DS microgels showed certain cytotoxicity toward human cervical carcinoma HeLa cell but negligible cytotoxicity for HEK293 cells after incubating for 24 h. Furthermore, the loading of dual drugs did 18


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not affect the size, morphology, and dispersion stability of the PNC8Q6.5 microgels in buffer solutions (Figures 4B and S9). The hydrodynamic radii of PNC8Q6.5, PNC8Q6.5-DOX-DS and PNC8Q6.5-FMA-DOX-DS microgels in buffer solutions were 242 ± 4 nm, 239 ± 4 nm, and 241 ± 6 nm at 25 °C, and 191 ± 3 nm, 197 ± 2 nm, and 199 ± 2 nm at 37 °C, respectively, which were smaller than the corresponding values in water (cf. Figure 1). It was reasonable because the charge shielding occured with larger concentrations of salt, resulting in less swelling of microgels at higher ionic strength.55

A

1.2

B Intensity Weighted (a.u.)

1.0 0.8

Cell Viability

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


0.6

HEK293 PNC8Q6.5 PNC8Q6.5-DOX-DS PNC8Q6.5-FMA-DOX-DS

0.4 0.2

HeLa PNC8Q6.5 PNC8Q6.5-DOX-DS PNC8Q6.5-FMA-DOX-DS

0.0 1

10

25

50

100

500

100 75 50 25 100 75 50 25 100 75 50 25 0 180

PNC8Q6.5

o

25 C o 37 C

PNC8Q6.5-DOX-DS

PNC8Q6.5-FMA-DOX-DS

200

220

240

Hydrodynamic Radius (nm)

Concentration of microgels (µg/mL)

19


260


Figure 4. (A) Cell viability of HEK-293 and HeLa cells in the presence of PNC8Q6.5, PNC8Q6.5-DOX-DS and PNC8Q6.5-FMA-DOX-DS microgels with various concentrations. (B) The size distribution of PNC8Q6.5, PNC8Q6.5-DOX-DS and PNC8Q6.5-FMA-DOX-DS microgels measured by DLS at 25 °C and 37 °C in buffer solution (0.01 M). (C-F) Confocal microscopy images of HeLa cell lines incubated with PNC8Q6.5-FMA-DOX-DS microgels for 4 h. For each panel, images showed the nuclear staining by DAPI (C), PNC8Q6.5-FMA-DOX-DS microgels labeled with FMA (D) and DOX (E), and the overlapping fluorescence image of cell nucleus, PNC8Q6.5-FMA-DOX-DS microgels and bright field (F). Inset of (E) was the enlarged image.

The cellular uptaking of fluorescent-labeled PNC8Q6.5-FMA-DOX-DS microgels were then studied. For PNC8Q6.5-FMA-DOX-DS microgels, the fluorescein moieties will exhibit green fluorescence, whereas the hydrophobic drug DOX will exhibit red fluorescence under excitation. HeLa cells were cultured with PNC8Q6.5-FMA-DOX-DS microgels for 4 h at 37 °C. Confocal microscopy images showed that the PNC8Q6.5-FMA-DOX-DS microgels were internalized by HeLa cells and resided in the cytoplasm regimes, and a portion of DOX were released from the 20


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microgels and entered into the cytoplasm and nucleus during cell uptaking (Figures 4C-F). These results indicated that the PNC8Q6.5-FMA-DOX-DS microgels could be taken up into the HeLa cells via nonreceptor-mediated endocytosis56 and the drugs could be released into cells over time.

B

80

100 80

40

DS

20

pH 7.4 pH 6.8 pH 5.0

Mt/M∞ (%)

60

60

2.0

log(Mt/M∞)

A Mt/M∞ (%)

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


40 20

1.8

1.4 1.2

0

0 0

2

4

6

8

10

12

14

16

pH 7.4 pH 6.8 pH 5.0

1.6

-0.5

0.0

0.5

1.0

1.5

logt

0

2

4

6

8

10

12

14

16

Time (hour)

Time (hour)

Figure 5. Time-dependent release profiles of DS (A) and DOX (B) from PNC8Q6.5-DOX-DS microgels in buffer solutions with various pH values at 37 oC. Inset of (B) shows plots of log(Mt/M∞) against log t for DOX release from PNC8Q6.5-DOX-DS microgels.

It is known that the pH values of different tissues or organs of the living creatures might be varied. For example, in blood the pH ranges between 7.3 and 7.45, in stomach the pH is about 1.2-2.0, in small intestine the pH is about 7.0-8.0, in the extracellular tissues and intracellular lysosomes/endosomes of tumors the pH ranges from 4.5 to 6.8.57-61 pH determines the release profile of loaded drugs in different microenvironments and is a key factor in drug delivery. Therefore, pH values of 7.4, 6.8 and 5.0 were selected here to simulate various microenvironments of living creatures. The time-dependent simultaneous release profiles of DS and DOX from PNC8Q6.5-DOX-DS

microgels

at

different

pH

were

then

investigated

by

dialyzing

PNC8Q6.5-DOX-DS microgels against the buffer solutions with corresponding pH at 37 oC. As 21



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shown in Figure 5, simultaneously sustained releases of DS and DOX from PNC8Q6.5-DOX-DS microgels were clearly observed within 14 h. The cumulative release ratio of DS decreased with increasing the acidity of release environment because of the lower solubility of DS in acidic conditions.62,63 The final cumulative release ratios of DS were ca. 65%, 58% and 56% in buffer solutions with pH values of 7.4, 6.8 and 5.0, respectively. The final DOX cumulative release ratios were ca. 76%, 76% and 89% in buffer solutions with pH values of 7.4, 6.8 and 5.0, respectively. The daunosamine group of DOX was protonated and became water-soluble in an acidic environment,64 leading to the higher cumulative release ratio at pH 5.0. The release kinetics and mechanism of DS and DOX released from PNC8Q6.5-DOX-DS microgels were determined by fitting the release plots with first order and Korsmeyer-Peppas models, respectively, as follows: First order model: −ln ൬1 −

‫ܯ‬௧ ݇௥ ‫ݐ‬ ൰= 2.303 ‫ܯ‬ஶ

ሺ5ሻ

Korsmeyer-Peppas model: ‫ܯ‬௧ = ݇‫ ݐ‬௡ ‫ܯ‬ஶ ‫ܯ‬௧ log ൬ ൰ = ݊ log ‫ ݐ‬+ log ݇ ‫ܯ‬ஶ

ሺ6ሻ ሺ7ሻ

where t is the time, Mt and M∞ are the amount of the drug release at time t and after infinite time, kr and k are the rate constants, and n is the release exponent. The fitting parameters of DS and DOX released from PNC8Q6.5-DOX-DS microgels were listed in Table S1. The first-order model is suitable to fit the release of DS from PNC8Q6.5-DOX-DS microgels, which is used to describe the release of water-soluble drug.38,65 The empirical power equation, Korsmeyer-Peppas equation,66,67 was used to investigate the release kinetics and mechanism of DOX. The release exponent (n) plays an important role in Korsmeyer-Peppas model, which indicating the mechanism of drug release. When n is smaller than 0.45 or greater than 0.89, the drug release behavior is diffusion controlled or 22


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swelling controlled, respectively. when n is between 0.45 and 0.89, the drug release contains both diffusion and swelling controlled mechanism.66,67 The inset of Figure 5B shows the plots of log(Mt/M∞) against log t for DOX release from PNC8Q6.5-DOX-DS microgels. Good linearity indicates that the Korsmeyer-Peppas model is applicable to DOX release of PNC8Q6.5-DOX-DS microgels. According to Korsmeyer-Peppas equation, DOX release from PNC8Q6.5-DOX-DS microgels went through two processes. In the first 1.5 h, the values of n for DOX release in buffer solutions with pH values of 7.4, 6.8 and 5.0 are all between 0.45 and 0.89, revealing that DOX release from PNC8Q6.5-DOX-DS microgels occurred through both diffusion and swelling controlled. After 2.5 h, the values of n were much lower than 0.45, indicating that it was controlled only by diffusion. The PNC8Q6.5 microgels showed fast release rate of hydrophobic drug DOX at pH 7.4 and 6.8, which could be attributed to the thermo-responsiveness of microgels.68,69 The PNIPAm-containing microgels shrank rapidly by switching temperature, and the drugs were quickly squeezed out of the crosslinking network. The drug release behaviors of thermo-sensitive microgels may be controlled through indirect thermal modulation.68 In addition, it was reported that the thermo-sensitive microgels could also kill cancer cells physically without any drugs by temperature induced volume variations.70,71 The above results indicated that simultaneously sustained release of DS and DOX from PNC8Q6.5-DOX-DS microgels was achieved.

In Vivo Combination Therapy. The H22 tumor-bearing mice model was established to investigate the antitumor activity of the dual-drug loaded microgels, i.e. PNC8Q6.5-DOX-DS microgels. Mice were treated with PBS and different drug formulations via the intravenous and intratumoral injections, the body weight and tumor volume of mice were measured every two days. The results obtained via the intravenous 23



injections were shown in Figure 6, whereas Figure S10 shows the results of intratumoral injection. As shown in Figures 6 and S10, compared with the rapid tumor growth of PBS, DS and PNC8Q6.5 microgels treated groups, the DOX and PNC8Q6.5-DOX-DS microgel groups showed significant efficacy in inhibiting the tumor growth. It was observed that in the PNC8Q6.5-DOX-DS microgel treated group and DOX group, tumor growth was almost inhibited with no obvious tumor recrudescence during the whole treatment. Figure 6A shows the tumor volumes of PNC8Q6.5-DOX-DS microgel and free DOX treated groups via the intravenous injection were 46.89-fold and 42.81-fold smaller than that of the corresponding control group. However, the tumor volumes of PNC8Q6.5-DOX-DS microgel and free DOX treated groups via intratumoral injection were only 5.02-fold and 4.07-fold smaller than that of the corresponding control group, as shown in Figure S10A. These results indicate that the intravenous injection has better treatment efficiency than that of intratumoral injection. Furthermore, for both cases of intravenous and intratumoral injections, the tumor growth of DS treated group was slightly slower than those of Control and PNC8Q6.5 microgel treated groups, which indicated that the inflammation depression by DS had indeed impact on the tumor regression. However, sole DS treatment was not sufficient to inhibit the tumor growth. Body weight is an important physiological index to indicate the systemic toxicity. As shown in Figure 6C, obvious weight loss (about 13%) was observed in mice treated via intravenous injection with free DOX at 3 mg/kg. Obvious weight loss (about 5%) was also observed for the group of intratumoral injection with free DOX at 3 mg/kg, as shown in Figure S10C. On the contrary, the body weight of the mice treated with PNC8Q6.5 or PNC8Q6.5-DOX-DS microgels did not loss significantly, indicating the reduced systemic toxicity with microgels. The survival curve of treated mice showed that the mean survival rates of H22 model mice treated by DS, PNC8Q6.5 and PNC8Q6.5-DOX-DS microgels were much higher than those in the Control and 24


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DOX treated groups (Figures 6D and S10D), which further indicated that PNC8Q6.5-DOX-DS microgels effectively reduced the systemic toxicity of DOX. With the high antitumor efficacy and the low drug-related toxicity, the dual-drug loaded PNC8Q6.5-DOX-DS microgels are promising for tumor therapy.

Figure 6. (A) Tumor volume changes and (C) body weight changes after intravenous injection of different drug formulations in tumor bearing mice. The data are shown as mean±SD (n = 6), *p < 0.01. (B) Images of excised H22 tumors of different drug delivery strategies at day 27. No solid tumors were observed in the red circle. (D) Survival studies of mice bearing H22 hepatoma model.

H22 tumor-bearing mice were then sacrificed on day 27 (or day 21) after intravenous injection (or 25



intratumoral injection) of different drug formulations. The corresponding tumors were dissected and stained with H&E for pathology analysis. Figure 7 shows the histopathology of tumors from the Control, PNC8Q6.5 microgel, DOX, PNC8Q6.5-DOX-DS microgel and DS treated groups after intravenous injection. Images of histological changes of tumor tissues after intratumoral injection of different drug formulations for 21 days were shown in Figure S11. Generally, the cell with large nuclei of spherical or spindle shape and more chromatin is normal tumor cell. Whereas if the cell becomes vague in outline, and the chromatin is darker and pyknotic or absent outside the cellular, the cell is necrotic. As shown in Figure 7 and Figure S11, tumor cells with normal shape and more chromatin were observed in the Control group, revealing a vigorous tumor growth. The tumor tissues of DOX and PNC8Q6.5-DOX-DS microgel treated groups had larger necrosis area than that of the Control group, indicating that most tumor cells were necrotic. Furthermore, the tumor tissues of PNC8Q6.5-DOX-DS microgel and DS treated groups had litter inflammatory cells, including lymphocytes, basophils and so on, as compared with the other three groups. It meant that the presence of DS effectively reduced the inflammatory environment of tumor, which could result in tumor regression.15 The simultaneous release of DS and DOX from PNC8Q6.5-DOX-DS microgels led to the improved antitumor effects, which could be attributed to the combination therapy of tumor inhibition with DOX and inflammation depression of tumor site with DS.

Figure 7. Images of histological changes of tumor tissues after intravenous injection of different 26


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drug formulations for 27 days; Necrotic cells indicated by Red arrows; Inflammatory cells indicated Black arrows. (HE staining, ×400)

The bio-distribution of microgels in living body was further investigated by using fluorescence labeled PNC8Q6.5-FMA microgels. The distribution of microgels in solid organs of H22 tumor bearing mice at 1, 12, 24, 48 and 72 h after intravenous injection was shown in Figure 8. The strongest fluorescence was first distributed in kidney at 1-12 h post-injection and then detected mainly in liver at 24-72 h post-injection due to the hepatic uptake of PNC8Q6.5-FMA microgels, which would be ascribed to the increase of the opsonin-mediated phagocytotic uptake by Kupffer cells. These results indicated that the PNC8Q6.5-FMA microgels exhibited longer blood circulation time and they could be metabolized and excreted from the living body by kidney and liver. The long blood circulation time of PNC8Q6.5-FMA microgels might come from the excellent stability of microgels in the body environment.

Figure 8. Distribution of PNC8Q6.5-FMA microgels in solid organs of H22 tumor bearing mice at 1, 12, 24, 48 and 72 h after intravenous injection. 27



CONCLUSIONS In conclusion, microgels with amphiphilic crosslinking networks were engineered and synthesized, which consisted of hydrophilic quaternized crosslinking structures and hydrophobic crosslinking nanodomains. The obtained microgels were successfully used as biocompatible and stable carriers for loading and simultaneous delivery of the hydrophilic NSAID, DS and hydrophobic anti-cancer drug, DOX. The dual-drug loaded microgels with amphiphilic crosslinking networks were proven to be promising for combination tumor therapy via simultaneous tumor inhibition and inflammation depression along with high antitumor efficacy and low drug-related toxicity.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XX.XXXX/acsami.XXXXXXX. Additional 1H NMR, TEM images, SLS data, GPC, photo pictures, MTT, and intratumoral injection results of obtained microgels.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. [email protected] ORCID Binyang Du: 0000-0002-5693-0325 Xianjing Zhou: 0000-0001-7703-7555 Notes 28


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The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors thank the National Natural Science Foundation of China (Nos. 21674097, 21704092 and 21322406), the second level of 2016 Zhejiang Province 151 Talent Project, Science Foundation of Zhejiang Sci-Tech University (No. 16062194-Y), and Open Research Fund of State Key Laboratory of Polymer Physics and Chemistry (201601), Changchun Institute of Applied Chemistry, Chinese Academy of Sciences for financial support.

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Novel Engineered Microgels with Amphipathic Network Structures for

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