Redox-responsive degradable prodrug nanogels for intracellular drug delivery by crosslinking of amine-functionalized poly(N-vinylpyrrolidone) copolymers

Accepted Manuscript Redox-Responsive Degradable Prodrug Nanogels for Intracellular Drug Delivery by Crosslinking of Amine-functionalized Poly(N-vinylpyrrolidone) Copolymers Huan Peng, Xiaobin Huang, Andrea Melle, Marcel Karperien, Andrij Pich PII: DOI: Reference:

S0021-9797(19)30061-X https://doi.org/10.1016/j.jcis.2019.01.049 YJCIS 24538

To appear in:

Journal of Colloid and Interface Science

Received Date: Revised Date: Accepted Date:

21 November 2018 9 January 2019 11 January 2019

Please cite this article as: H. Peng, X. Huang, A. Melle, M. Karperien, A. Pich, Redox-Responsive Degradable Prodrug Nanogels for Intracellular Drug Delivery by Crosslinking of Amine-functionalized Poly(Nvinylpyrrolidone) Copolymers, Journal of Colloid and Interface Science (2019), doi: https://doi.org/10.1016/j.jcis. 2019.01.049

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Redox-Responsive Degradable Prodrug Nanogels for Intracellular Drug Delivery by Crosslinking of Amine-functionalized Poly(Nvinylpyrrolidone) Copolymers

Huan Peng1,3†, Xiaobin Huang2†, Andrea Melle1,3, Marcel Karperien2, Andrij Pich1,3*

1

Functional and Interactive Polymers, Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Forckenbeckstraße 50, D-52074 Aachen, Germany

2

Developmental BioEngineering, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede 7500 AE, The Netherlands

3

DWI-Leibniz Institute for Interactive Materials e.V., Forckenbeckstraße 50, D-52074 Aachen, Germany

Corresponding author: Andrij Pich Email: [email protected] Address: Forckenbeckstr. 50, D-52056 Aachen Phone: +49 (0)241/80-23310

Redox-Responsive Degradable Prodrug Nanogels for Intracellular Drug Delivery by Crosslinking of Amine-functionalized Poly(N-vinylpyrrolidone) Copolymers Huan Peng1,3†, Xiaobin Huang2†, Andrea Melle1,3, Marcel Karperien2, Andrij Pich1,3* 1

2

3

†

Functional and Interactive Polymers, Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Forckenbeckstraße 50, D-52074 Aachen, Germany Developmental BioEngineering, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede 7500 AE, The Netherlands DWI-Leibniz Institute for Interactive Materials e.V., Forckenbeckstraße 50, D-52074 Aachen, Germany

These authors contributed equally to this work.

Graphic Abstract

Abstract Hypothesis: Facile approaches for the development of new tailored drug carriers are of high importance for the controlled administration of drugs. Herein we report a method for the synthesis of water-soluble reactive copolymers with well-defined architectures for fabrication of redox-sensitive degradable prodrug nanogels for intracellular drug release. Experiments: Primary amine-functionalized statistical copolymers were obtained by hydrolysis of poly(N-vinylpyrrolidone-co-N-vinylformamide) copolymers which were 1

synthesized

via

Reversible

Addition−Fragmentation

chain-Transfer

(RAFT)

polymerization. Redox-sensitive degradable nanogels with varying crosslinking densities were synthesized with a redox-sensitive cross-linker. Doxorubicin (DOX) was loaded to form prodrug nanogels (DNG) with hydrodynamic radius from 142 nm to 240 nm. Findings: The nanogels demonstrated slower degradation and retarded drug release rate with increased crosslinking density in the presence of 10 mM reduced glutathione (GSH) at 37℃. The in vitro release studies revealed that maximum 85% DOX was released in 24 h under a reductive environment. Intracellular drug release profiles in HeLa cells indicated that the DOX delivery rate was tunable via varying crosslinking density of the nanogels. Cell viability assay demonstrated that the blank nanogels were biocompatible in wide concentrations up to 0.5 mg/mL while the DOX-loaded nanogels displayed medium antitumor activity with IC50 (half-maximal inhibitory concentration) of 1.80 μg/mL, 2.57 μg/mL, 3.01 μg/mL for DNG5, DNG10 and DNG15 respectively. Keywords: Redox-responsive, degradable, nanogel, prodrug, drug delivery, tunable crosslinking density 1. Introduction Chemotherapy is one of the major bulwarks for cancer treatment in current medical research while the chemotherapeutic drugs may cause adverse effects to human body due to unspecific targeting.1, 2, 3 Therefore, administering a pharmaceutical compound with a drug delivery system in the form of encapsulation or conjugation is of vital significance to achieve a therapeutic effect without affecting normal tissues. Despite extensive

research

on

drug

delivery

platforms

including

nanotubes,4,5

(pro)liposomes,6,7 dendrimers,8,9 polymersomes and micelles,10-13 problems associated with low efficacy, high toxicity, instability and clearance by the reticuloendothelial system (RES) remain unsolved. Nanogels are physically or chemically crosslinked 3D polymeric network colloids, which arouse tremendous research interests as drug nanocarrier in the past decades.14,15 Nanogels have overwhelming advantages over the other systems including but not limited to excellent biocompatibility, easy drug 2

loading ability, abundant stimuli-response including pH, temperature, light, similar Hamaker constants as water and good stability in biological fluids. 16-20 Notably, the flexible and soft nanogel colloids could be more easily to penetrate into soft tissues compared with rigid nanoparticles.21 Furthermore, compared with traditional nanocarriers, nanogels were more efficiently taken up by cells with enhanced bioavailability of in vivo therapeutics.22, 23 The flexibility and high molecular weight of nanogels result in prolonged circulation time in the blood stream. 24 Principally, nanogels as drug delivery vehicle should be degradable after releasing therapeutics without causing accumulation and side effects in normal organs. Moreover, nanogels could be functionalized with special ligands to recognize target tumor cells, rendering more accurate targeting drug release.25-27 Generally, nanogels were synthesized in two major approaches including crosslinking of prepolymers and polymerization of monomers in the presence of crosslinking agents. Although the latter method could render nanogels with homogeneous particle size,28 the harsh conditions including high temperature or ultraviolet (UV) irradiation may lead to unfavorable results for therapeutics encapsulation.29,30 The main strategy for drug loading in such nanogels was chemical post-modification or utilization of electrostatic interactions. Haag et al. reported dual-responsive prodrug nanogels based on dendritic polyglycerols, in which doxorubicin was conjugated to the biodegradable nanogel matrix via an acid-labile hydrazone linker.31 Jiang et al. developed a bio-reducible heparin-based nanogel drug delivery system.32 The electrostatic interactions between the positively charged drug molecules and negatively charged nanogels resulted in encapsulation efficiency as high as 90%. Our group reported thermal and reduction dual-responsive prodrug nanogels whose reactive succinimide groups were mainly located in the nanogel shell, considerably increasing their accessibility for doxorubicin conjugation.33 Nevertheless, nanogels synthesized from reactive prepolymers via crosslinking reaction in mild conditions provide a facile approach for drug loading. The big advantage of such systems is that after nanogel degradation in vivo polymer chains with defined molecular weight are formed and the chain length can be programmed at the polymerization step. If these polymer chains 3

possess molecular weight less than 20.000 g/mol, they can be easily removed from the body through the kidneys. Amphiphilic prepolymers are known to form nano-assemblies in solution, providing a versatile platform to cage drugs by simply locking the drug-assembly complexation. Thayumanvan at al. reported nanogel system as drug carrier by crosslinking of random copolymers with pyridyl disulfide side groups in aqueous solution.34-36 The nanogel size could be adjustable via polymer concentration and utilizing the thermo-sensitivity of the copolymers. Hawker et al reported a facile approach for nanogel synthesis utilizing click chemistry via amidation of acrylic acid groups from polymer micelles,37 which showed promising application in controlled drug delivery. We reported a protein delivery platform based on copolymers with pyridyl disulfide side chains, which allowed highly efficient enzyme encapsulation and reversible modulation of enzyme activity. 38 As precursors of nanogels for therapeutics delivery in human body, the polymers should at least fulfill several fundamental requirements: (a) controlled architecture and excellent biocompatibility; (b) good water solubility to improve the solubility of hydrophobic drugs after loading; (c) reactive with crosslinking agent at mild conditions. Therefore, the structure design and synthesis approach of the reactive copolymers is critically important. Although thermal and redox dual-responsive lactam-based prodrug nanogels were reported in our previous work,33 the precipitation polymerization approach may lead to self-crosslinking of the prepolymer chains. This will make the nanogels difficult to degrade completely under reduction environment. Additionally, there is risk that the nanogels may be difficult to be excreted through the kidneys due to the large molecular weights of the nanogels caused by free radical polymerization. To face such challenges, many scientists tried to develop drug carrier based on prepolymers with controlled architectures. Fuoco et al. prepared pyridyl disulfide groups functionalized poly-(lactide)s polymers.39 Redox-responsive nanogels can be prepared by crosslinking with poly(ethylene glycol) dithiol. Nile red could be encapsulated in the nanoparticles and then released in the presence of glutathione at cellular concentration. Zhang et al. developed redox responsive dextrin (Dex) nanogel system equipped with 4

the FDA-approved CXCR4 antagonist AMD3100.40 The Dex nanogel (DNG) was constructed in aqueous medium by self-cross-linking of thiolated Dex, which entrapped the drug molecules during the crosslinking process. Although molecules encapsulated in the 3D polymer network by crosslinking of the well-designed copolymers can somehow overcome the obstacles, leakage may cause serious unspecific release. Therefore, a wiser strategy could be chemically conjugation of the drug molecules into the nanogels during crosslinking of reactive copolymers and releasing the drug molecules in redox condition. In

the

present

paper,

a

series

of

water-soluble

statistical

poly(N-vinylpyrrolidone-co-N-vinylformamide) copolymers were synthesized via RAFT polymerization. Obtained copolymers were hydrolyzed in alkaline conditions to obtain primary amine-functionalized reactive copolymers. The copolymers and doxorubicin (DOX) were covalently conjugated with a redox-responsive cross-linker in water-in-oil emulsion to form prodrug nanogels via Michael addition chemistry. The in vitro reduction triggered DOX release and intracellular drug transport properties in nanogels with different crosslinking densities were studied. And the influence of crosslinking density on nanogel degradation property, drug loading, release kinetics and antitumor activity were systematically investigated. Compared with other stimuli (temperature, 41 light,42 etc.) for controlling the drug release, the present redox-responsive prodrug nanogel helps tumor-specific drug delivery, considering that the intracellular GSH concentration in tumor cells can be at least fourfold higher than that in the normal cells. 43 Different from our previous report, we use new reactive amphiphilic copolymers with chemically conjugated drug for the nanogel design. We demonstrate that the control of the copolymer molecular weight allows adjustment of the crosslink density and degradation rate of nanogels, what provides additional tool to control the prodrug distribution in biological systems.

5

Scheme 1. Prodrug nanogels synthesis based on amine-functionalized polymer, integration into cells and intracellular drug release triggered by reduction environment.

2. Experimental Section Materials. Methyl 2-bromopropionate (Acros, 99%, Germany), potassium ethyl xanthogenate (Aldrich, 98%, Germany), anisole (Aldrich, anhydrous, 99.7%, Germany), dichloromethane (DCM, Aldrich, anhydrous, 99.8%, Germany), acetone (Aldrich, 99.5%, Germany), ethanol (Aldrich, anhydrous, 99.5%, Germany) diethyl ether (VWR, 99.9%, Germany), dithiodiglycolic acid (Aldrich, 98%, Germany), poly(ethylene glycol) methacrylate (PEGMA, Mn~360 Da, Aldrich, Germany), N,N′-dicyclohexylcarbodiimide 4-(dimethylamino)pyridine

(DCC,

(DMAP,

Aldrich,

Aldrich, 99%,

99%, Germany),

Germany), doxorubicin

hydrochloride (DOX, Aldrich, 98%, Germany), glutathione reduced (GSH, Aldrich, 98%, Germany), deuterium oxide (D2O, 99.7 %, Deutero GmbH, 99.8%, Germany), 6

chloroform-d1(CDCl3, Deutero GmbH, 99.8%, Germany) and dialysis tubes (molecular weight cutoff (MWCO) 12 kD~14 kD, 500 Da, Carl Roth, Germany) were used as received.

N-vinylpyrrolidone

(VP,

Aldrich,

99%,

Germany)

and

N-vinylformamide (NVF, Aldrich, 98%, Germany) were distilled before use. 2, 2'-azobis(2-methylpropionitrile) (AIBN, Aldrich, 98%, Germany) was recrystallized in ethanol before use. Deionized water was used for all experiments. Measurements. Nuclear Magnetic Resonance (NMR). 1H NMR and

13

C NMR spectra were

recorded on a Bruker DPX-400 FT NMR spectrometer at 400 MHz. Gel Permeation Chromatography (GPC). GPC was performed on a combined GPC system with a high performance liquid chromatography pump (Bischoff HPLC), a Jasco 2035-plus RI detector and four MZ-DVB gel columns (30 Å, 100 Å and 2 × 3000 Å) in series at 30 Å. A solution of DMF containing 1.0 g LiBr L-1 was used as eluent at a flow rate of 1.0 mL min-1. The molecular weights were calculated using a polystyrene (PS) calibration. Fourier Transform Infrared Spectroscopy (FTIR). FTIR spectra (resolution 4 cm−1) were recorded using a Nicolet NEXUS 670 Fourier Transform IR spectrometer. Samples were prepared onto silica plates at room temperature. For each spectrum more than 200 scans were averaged to enhance the signal-to-noise ratio. Raman Spectroscopy. Raman Spectra were recorded on Bruker RFS 100/S spectrometer. The laser used was Nd: YAG at 1064 nm wavelength at a power of 250 mW. On average 1000 scans were taken at a resolution of 4cm-1. For sample holding aluminum pans of 2 mm before were used. Software used for data processing was OPIS 4.0. UV-Visible Spectroscopy (UV-Vis) Study. The UV-Vis spectra were recorded on a CARY 100 Bio UV-Visible spectrophotometer (Agilent, USA). UV quartz cuvettes with 10 mm across were used for measurements. Dynamic Light Scattering (DLS). DLS measurements were performed with a commercial laser light scattering spectrometer (ALV/DLS/SLS-5000) equipped with an ALV-5000/EPP multiple digital time correlator and laser goniometry system 7

ALV/CGS-8F S/N 025 with a helium−neon laser (Uniphase 1145P, output power of 22 mW and wavelength of 632.8 nm) as a light source. The measurements were performed in triplicate. Confocal Microscopy Study. The fluorescence images were recorded on a Leica SP8 confocal microscope (Leica, Germany). Cryo-Field Emission Scanning Electron Microscopy. The cryo-scanning electron microscopy (Cryo-FESEM) images were taken with a HITACHI S-4800 instrument in a cryo-mode at voltages 1–15 kV. Around 10 μL nanogel solution was put into the sample holder and immediately frozen with boiling liquid nitrogen. The frozen sample was then transferred to the high vacuum cryo-unit chamber and cut by a sharp knife to fracture the sample in the aim of getting images of the surface of the inner structure. The sample was sublimated for 3 mins in order to remove humidity. The sample was then transferred to the observation chamber for measurement. Synthesis of Poly(N-vinylpyrrolidone-co-N-vinylformamide) Copolymers. The copolymers were synthesized according to our previous report. 44, 45 A 25 mL Schlenk flask

was

charged

with

2

g

N-vinylpyrrolidone

and

0.041

g

methyl

2-(ethoxycarbonothioylthio) propanoate (Supporting Information) in 3 mL anisole. The solution was degassed by five freeze-pump-thaw cycles. Two small vials containing 0.067 g NVF in 1 mL anisole and 0.0058 g AIBN in 0.2 mL anisole respectively were purged with dry nitrogen for 1 h. The Schlenk flask was heated to 60℃ before the AIBN solution was added with an airtight syringe. The NVF solution was fed continuously with a syringe pump in 5 h. The reaction was stopped immediately by rapidly immerging into liquid nitrogen after 48 h. The copolymers were precipitated in excess amount of cold diethyl ether and further purified by washing with diethyl ether. The copolymers were dried in vacuum at 40℃ and characterized by 1H NMR, FTIR, Raman and GPC. Here the copolymers with NVF feeding molar ratio of 5%, 10% and 15% were named as CP5, CP10 and CP15 respectively. 1

H NMR (in D2O): δ[ppm]=7.93-8.08 (-NHCHO), 3.65-3.80 (-CHNHCHO,

-CHNCO- in backbone, -OCH3, -OCH2CH3 in end group), 3.33 (-NCH2CH2-), 8

2.32-2.45 (-NCOCH2CH2-, -CH2CHNH- in backbone, -CHCH3 in end group), 2.04 (-NCH2CH2CH2-), 1.60-1.75 (-NCHCH2- in backbone, -OCH2CH3, -CHCH3 in end group); FTIR (on silica plate): 2954, 2870 cm-1 (-CH2-), 1660 cm-1 (-NC=OCH2-, -NHHC=O), 1538 cm-1 (-NHCHO), 830 cm-1[-(CH2CH2CH2)n-]. Raman: 2976, 2866 cm-1(-CH2-), 1665 cm-1(-NC=OCH2-, -NHHC=O), 1542 cm-1 (-NHCHO), 737 cm-1 [-(CH2CH2CH2)n-]. Hydrolysis of Copolymers. The hydrolysis process was carried out under alkaline condition following previous report.44 3g CP5 and a molar equivalent amount of NaOH (relative to the NVF ratio) in 25 mL H2O were stirred in a two-necked round bottom flask equipped with a reflux condenser under dry nitrogen. The solution was purged with nitrogen for 1 h and heated to 70℃ afterwards. The reaction proceeded for 36 h to insure complete hydrolysis of –CHO groups into –NH2 groups. The samples were precipitated in excess amount of cold acetone and further purified via dialysis to remove residual NaOH and dried with lyophilization. The dry samples were sent for 1H NMR, FTIR and GPC analysis. The hydrolyzed copolymers were named as HCP5, HCP10 and HCP15 respectively. 1

H NMR (in D2O): δ[ppm]= 3.66-3.79 (-CHNHCHO, -CHNCO- in backbone, -OCH3,

-OCH2CH3

in end group), 3.33 (-NCH2CH2-), 2.32-2.45 (-NCOCH2CH2-,

-CH2CHNH- in backbone, -CHCH3 in end group), 2.03 (-NCH2CH2CH2-), 1.61-1.75 (-NCHCH2- in backbone, -OCH2CH3, -CHCH3 in end group); FTIR (on silica plate): 3373, 3284 cm-1 (-NH2), 2954, 2870 cm-1 (-CH2-), 1660 cm-1(-NC=OCH2-), 1540-1560 cm-1 (-NHCHO), 830 cm-1[-(CH2CH2CH2)n-]. Synthesis of Degradable Cross-linker. The crosslinking agent (CL) was synthesized according to typical procedure of Steglich esterification. 46 To a stirred solution of 1.82 g (10 mmol) dithiodiglycolic acid in 25 mL anhydrous DCM in ice bath, 7.92 g (22 mmol) PEGMA, 0.122 g (1 mmol) DMAP and 4.53 g DCC (22 mmol) was added to the reaction mixture and stirred at 0℃ for 10 mins. Then the reaction proceeded at room temperature overnight. The precipitated urea was filtered off and the filtrate was placed in the freezer at -20℃ for 24 h. The precipitates were filtered and the DCM was removed under vacuum. The crude sample was largely diluted with deionized 9

water and the precipitates were removed with centrifugation and redispersion cycles. The supernatant was purified with extensive dialysis (MWCO 500 Da) in deionized water and dried with lyophilization.

1

H NMR (in D2O): δ[ppm]=6.16, 5.73

(H2C=CCH3-), 3.75-3.43 (-OCH2CH2-), 3.52-3.65 (-SSCH2CO-), 1.93 (-CCH3). Synthesis of Nanogels and DOX Loading. Nanogels were synthesized by direct crosslinking reaction between the primary amine groups of the copolymers and the vinyl groups of cross-linkers in water-in-oil emulsion through Michael addition chemistry.47, 48 Typically, 400 mg HCP5 and 10 mg cross-linker were dissolved in 1 mL aqueous solution (pH 10). The organic phase containing 0.28 g span80 in 12 mL toluene was placed in a 25 mL vial in ice bath. The aqueous phase was added into the organic phase and the mixture solution was immediately sonicated with a Branson Sonifier W450 (Terra Universal. Inc., USA) with a 1/4// horn at duty cycle of 40% and amplitude of 360 W for 10 min. Then the emulsion was transferred to a degassed flask and stirred at room temperature overnight. The obtained nanogels were separated and cleaned by centrifugation (12000 rpm, 16℃, 40 min) and redispersion cycles, and further purified with extensive dialysis (MWCO 12000 Da-14000 Da) in deionized water. The DOX-loaded nanogels were synthesized in a similar process with 2 mg additional DOX initially dissolving in the aqueous phase, reaction and dialysis proceeding in dark. The loading amount of DOX was determined by UV-Vis photometer. The drug loading capacity and drug loading efficiency were calculated according to the following Equations:

Here Md is the weight of DOX in the nanogels, Mn is the weight of nanogel while Mf is the weight of feeding DOX. Nanogel Degradability Study and Triggered Release of DOX. The degradability of the nanogels were investigated in 10 mM GSH solution via DLS at 37℃. The hydrodynamic radii of nanogels at different time intervals were monitored by DLS measurements. The release profiles of DOX from nanogels were determined in 10

phosphate buffer (PB) solution (100 mM, pH 7.4) in the presence or absence of 10 mM GSH at 37 ℃. Two aliquots of well-dispersed nanogel suspensions were transferred into dialysis tubes (MWCO 12000-14000 Da). The two dialysis tubes were immersed into 25 mL PB buffer at 37℃ in dark sealed bottles. 3 mL release medium was taken out for UV-Vis measurement periodically and replenished with same amount of fresh medium. The experiments were performed in triplicate, and repeated three times with similar results. Cellular Uptake of Nanogels and Intracellular Release of DOX. HeLa cells were seeded in a 96-well microtiter plate using Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) and incubated at 37℃ and 5% CO2. After 24 h, free DOX or DOX loaded nanogels (10 μg DOX/mL) were added and after incubation of 2 h, 10 h and 24 h the culture medium was discarded and washed with PBS buffer for 3 times. The cells were fixed with 4% paraformaldehyde for 30 mins, washed with PBS buffer for 3 times, afterwards permeabilized by PBST (0.5% TritonX-100 in PBS) for 15 mins and further

washed

with

PBS

buffer.

The

cell

nuclei

were

stained

with

4′,6-diamidino-2-phenylindole (DAPI) and fluorescence images were taken with BD pathway 435 confocal microscope (BD Biosciences). Presto Blue Cell Viability Assay. The HeLa cells were incubated in a 96-well plate (1×104 cells/well) in DMEM medium with 10% FBS and 1% P/S in 37℃ and 5% CO2. After 24 h, the medium was removed and replenished by 180 μL of fresh DMEM medium and 20 μL samples of polymeric nanogel or DOX loaded nanogel suspensions in phosphate buffer (10 mM, pH 7.4). Cells cultured in same DEMEM medium were used as controls. The medium was replaced by 90 μL fresh medium and mixed with 10 μL PrestoBlue after cells incubation for another 48 h. Fluorescence signals were measured in VICTOR™ X3 Multilabel Plate Reader (Perkin Elmer) after cells were incubated in 37℃ for 30 min. The cell viability was expressed as a percentage relative to the control cells.

11

3. Results and Discussion 3.1 Chemical Structure of Reactive Copolymers Statistical

copolymers

poly(N-vinylpyrrolidone-co-N-vinylformamide)

with

well-controlled chemical compositions and narrow polydispersity (M w/M n) were synthesized via RAFT polymerization.44 As shown in Figure 1A, the signals at around 8.01 ppm from the –CHO group of the NVF components progressively increase. It is further observed from the FTIR spectra in Figure 2A that the peak intensities at around 1540 cm-1 are enhanced accordingly, which can be assigned to the bending or the amide groups from NVF. The NVF amounts in CP5, CP10 and CP15 are calculated as 4.7%, 9.6% and 14.2% respectively by comparing the intervals at 8.01 ppm and 3.33 ppm in 1H NMR. Characterization data from GPC (Figure S4) indicates that the copolymers encompass similar molecular weights and narrow polydispersities. The –CHO groups were subsequently hydrolyzed into primary amine groups in the alkaline conditions. As demonstrated in the 1H NMR spectra Figure 1, the signals at around 8.01 ppm (–CHO group) disappeared after hydrolysis while the other signals were unchanged. This suggests that the –NHCHO groups are completely transferred into –NH2 groups while the poly(N-vinylpyrrolidone) parts remain still stable. Although the active protons from the primary amine groups were not detectable in 1

HNMR due to hydrogen–deuterium exchange with D2O, their characteristic signals

were clearly observed in the FTIR spectra in Figure 2B. The double bands at 3300-3500 cm-1 are from N-H stretching of primary groups, confirming existence of –NH2 groups. Furthermore, one can see the intensities of the double bands as well as the shoulders (insert image in Figure 2B) at 1520-1600 cm-1 increased progressively, suggesting that the ratios primary amine groups were elevated accordingly. It is worth to note that these shoulders, assigning to the in-plane-bending of the N-H bond, are largely overlapped with the –C=O groups (at around 1660 cm-1) from the poly(N-vinylpyrrolidone) components. As displayed in the GPC traces in Figure S4, the molecular weights of HCP copolymers were slightly decreased due to remove of the –CHO groups in alkaline conditions. The properties of CP/HCP copolymers were summarized in Table S1. Therefore, it is safe to conclude that the HCP copolymers are 12

functionalized with primary amine groups whose ratios are similar to those of the NVF components in corresponding CP copolymers. Therefore, based on these copolymers, functional nanogels with different crosslinking densities can be obtained.

Figure 1. 1HNMR spectra of copolymers CP5/CP10/CP15 (A) and the hydrolyzed copolymer HCP5/HCP10/HCP15(B).

Figure 2. FTIR spectra of copolymers CP5/CP10/CP15 (A) and the hydrolyzed copolymers HCP5/HCP10/HCP15 (B).

3.2. Synthesis of Nanogels The redox-sensitive nanogels were prepared by direct crosslinking of the amine groups and vinyl groups via the typical Michael-addition reaction in water-in-oil emulsion. Nanogels based on HCP5, HCP10 and HCP15 were obtained and named as NG5, NG10 and NG15 respectively. The cryo-FESEM images in Figure 3 reveal that 13

the nanogels are homogenous with average diameters of 397 nm, 319 nm and 214 nm for NG5, NG10 and NG15 respectively. The DLS results in Figure 3D are well consistent with the data with corresponding hydrodynamic radius of 210 nm, 171 nm and 128 nm respectively. The relatively smaller values from cryo-FESEM measurements may be attributed to the partial volume loss during the frozen process. The particle size could be tunable via adjusting the crosslinking density, higher crosslinking density leading to smaller particle size. To have a deeper insight of nanogels structures, FTIR spectroscopy was employed. Compared the FTIR spectra of HCP, NG and the cross-linker (CL) in Figure 2 and Figure 4, new peaks at 1740 cm-1 and 1140 cm-1 were observed, which could be assigned to the –C=O (from the ester) and –C–O– (from the –CH2O– repeating units) bonds from the cross-linker respectively. Meanwhile the characteristic double bands of the N-H stretching from –NH2 groups disappeared in the spectra of the nanogels, suggesting full conversion of the primary amine groups. Notable, the progressively enhanced peak intensity at 1740 cm-1 (from the ester of the cross-linker) confirms increased crosslinking density from NG5 to NG15.

14

Figure 3. A, B,C) Cryo-FESEM images of NG5(A), NG10(B) and NG15(C), the scale bar is 3μm; D) particle size distribution of NG5, NG10 and NG15 measured by DLS.

Figure 4. A) FTIR spectra of cross-linker (CL), NG5, NG10 and NG15; the black rectangle indicates the characteristic double bands of the N-H stretching from –NH2 groups disappeared in the spectra of the nanogels; B) enlarged part of A from 1800 cm-1 to 650 cm-1; the red rectangle suggests increased crosslinking density from NG5 to NG15 indicated from progressively enhanced peak intensity at 1740 cm-1. 15

3.3. Redox-Triggered Degradation of Nanogels The redox-sensitive property of the polymeric nanogels was investigated via DLS by monitoring particle size change in 10 mM GSH PB buffer solution at 37℃. The results show that the nanogels were rapidly destabilized by GSH in 1h and further degraded into small aggregates in 24 h (Figure 5). It was reported that hydrophilic nanogels disassociated rapidly when incubated in reduction conditions while nanogels composed of amphiphilic components would form agglomerates first.49 Zhong et al. demonstrated crosslinked colloids swelled significantly and then degraded into unimers upon dilution to lower concentration. 50 It is reasonable that the present nanogels dissociated directly after treating with GSH since the precursors of the nanogels were hydrophilic. In comparison of the graphs in Figure 5B, NG5 degraded in a more rapid speed with a much sharper decrease of particle size in 1 h than NG10 and NG15. This could be ascribed to the much lower crosslinking density of the nanogel. The particle size observed after 24 h incubation could be due to small core particles formed by intra-chain crosslinking. The nanogels can be completely degraded into clear and soluble macromolecules solution in several days based on the crosslinking density. Presumably, the degradation of the developed nanogel systems could be efficient and tunable under intracellular-mimicking reducing conditions. To further confirm that the nanogels were degraded into polymer chains, the degradants of the nanogels were characterized by GPC. As displayed in Figure 6 and Table S1, the molecular weights of the degradants of the nanogels are quite close to those of the copolymers. The slight larger molecular weight of the degradants may be attributed to the degraded crosslinkers which were covalently connected to the copolymers. Therefore, it is safe to indicate that the nanogel colloids were fully degraded into polymer chains in the reduction environment.

16

Figure 5. A, B) Particle size distribution of NG5 (A, B), NG10 (A), NG15 (A) in presence of 10 mM GSH solution at different time intervals measured via DLS. The particle sizes of NG5, NG10 and NG15 are normalized to 1 nm at 0 h in Figure 5B.

Figure 6. GPC traces of copolymer HCP5 (black), HCP10 (red), HCP15 (blue) and degradants of NG5 (magenta), NG10 (olive) and NG15 (navy).

3.4. Loading and Triggered Release of DOX. DOX was loaded into the nanogels by covalently binding to one of the arms of the redox-sensitive cross-linkers forming prodrug polymers, which were introduced into the nanogels in the crosslinking process. The prodrug nanogels were named as DNG5, 17

DNG10 and DNG15 respectively. Estimated from the Henderson-Hasselbalch equation, approximately 98% of the amino groups of DOX are protonated at pH 10 due to a pKa value of approximately 8.2,51, 52 rendering significant involvement of DOX in the crosslinking reaction. This contributes to satisfied drug loading capacities of 10.2%, 13.1% and 17.7% as well as relatively high drug loading efficiency of 51.4%, 54.7% and 57.9% for DNG5, DNG10 and DNG15 respectively. It indicates that the loading capacity increased slightly with the amine group content, which is reasonable considering that the primary amine groups serve as local sites for anchoring the prodrug polymers (the DOX-cross-linker compounds). The FTIR spectra of DOX, NG10 and DNG10 in Figure 7 proved successful loading of DOX, as suggested by the characteristic peak at 1245 cm-1 and 1065 cm-1, which could be assigned to the –C–O– of the aromatic ether and the out-of-plane bending of the aromatic ring from the quinol group from DOX respectively. As observed from the FTIR spectra of DNG5/DNG10/DNG15 in Figure S6, it is clear that the signal intensities from DOX at 1245 cm-1 and 1065 cm-1 increased from DNG5 to DNG15. It further evidences that the loading amount of DOX increases with the primary amine groups from the copolymer precursors. The confocal microscopy images in Figure 8A and Figure S7 further confirm successful loading of DOX as the fluorescence of the colloids originates from the loaded drug. The hydrodynamic radius decreased with the increase of crosslinking density with 240 nm, 197 nm, and 142 nm from DNG5, DNG10 and DNG15 respectively as shown in Figure S7. The DOX loaded nanogels are larger than corresponding blank nanogels probably due to the steric hindrance influence from DOX.

18

Figure 7. A, B) FTIR spectra of NG10, DOX and DNG10; B is the enlarged part of A from 1800 cm-1 to 650 cm-1; the black (1245 cm-1) and red arrows (1065 cm-1) point to the characteristic signals from DOX.

Figure 8. A) Confocal microscopy images of DNG15 (overlay of bright field (Figure S7C) and fluorescence field (Figure S7C), the scale bar is 5 μm; D) DOX release profiles of DNG5 (black squares), DNG10 (red circles) and DNG15 (blue triangles) in PB (100 mM, pH 7.4) at 37℃ in the presence or absence of GSH. (the results are average values of experiments performed in triplicate; CGSH=10 mM).

The in vitro drug release was studied in PB (100 mM, pH 7.4) at 37℃ with or without 10 mM GSH. As observed from Figure 8B, only maximum around 13% DOX was released from DNG5 in 24 h in the absence of GSH, which could be attributed to unstable property of the disulfide bond from oxidation or other possible side reaction 19

in the atmosphere. It should be noted that the drug release rate from DNG5, DNG10 and DNG15 decreased progressively, as evidenced by the release profiles. It is assumed that the disulfide bonds were protected in higher crosslinked nanogels, making them more stable when exposing in atmosphere. In contrast, the drug release in reduction conditions was rapid and efficient. Approximately 85%, 72% and 63% DOX were released in 24 h from DNG5, DNG10 and DNG15, respectively. It is worth noting that the cumulative release can reach to 100% after several days of incubation when the prodrug nanogels completely degraded. The significantly enhanced release speed is mainly due to the efficient cleavage of the crosslinking network of nanogels. Interestingly, the drug release rates could be tunable via crosslinking density of the nanogels. It is much easier for GSH molecules to access the disulfide bonds in lower crosslinked nanogel, which contributes to more efficient release of DOX in DNG5 and DNG10 than DNG15. These results suggest that nanogel drug carriers are quite stable in physiological conditions and the triggered drug release by intracellular-mimicking reduction conditions proceeds in a progressive and controlled manner, tunable by the crosslinking densities of the nanogels, indicating promising application in biomedical fields.

3.5. Intracellular Drug Release and Anti-Tumor Activity of DOX-Loaded Nanogels. The cellular internalization and drug release behavior of the DOX-loaded nanogels were investigated in HeLa cells. As demonstrated in Figure 9, strong fluorescence around perinucleus regions as well as very weak signals in nucleus could be observed following 2 h incubation with DNG5, indicating efficient drug release in the cells. Interestingly, the DOX fluorescence intensity observed in cells incubated with DNG10 and DNG15 in 2 h gradually decrease and particularly for DNG 15 the fluorescence is almost around the perinucleus area (Figure S8 and Figure S9). It suggests that DOX was released in relatively slower rates in DNG10 and especially in DNG15, which is in good accordance with the in vitro drug release results. However, merged fluorescence from DOX and DAPI is observed in the nucleus in Figure 9B 20

after 10 h incubation, revealing that DOX from DNG5 was completely delivered into the nucleus. Correspondingly, considerable amount of DOX molecules from DNG10 entered into the nucleus while still much fluorescence of DOX from DNG15 were observed around the perinucleus regions following 10 h incubation (Figure S8B and Figure S9B), indicating the intracellular drug release rate could be tunable via adjusting the crosslinking density of the drug carrier. After 24 h incubation, all the three systems were occupied with strong DOX fluorescence in the nucleus region, which is similar to cells incubated with free DOX for 2 h (Figure 9D), suggesting the drug was released in an efficient and controllable manner. The preliminary experiments display that in addition to release the drug molecules in a controllable manner with nanogel hosts, the release profiles at intracellular conditions can be tunable via the crosslinking densities. This may find promising applications in cancer treatment utilizing the gradient microenvironment in tumor cells.

21

Figure 9. Intracellular distributions of DOX in HeLa cells treated with DNG5 and free DOX for 2 h, 10 h and 24 h. A) DNG5, 2 h, B) DNG5, 10 h, C) DNG5, 24 h, D) free DOX, 2h. For each panel, the images from left to right show cell nuclei stained by DAPI (blue), DOX fluorescence in cells (red) and overlays of both images. The scale bars correspond to 100 μm in all the images. 22

The antitumor activity of the nanogels was investigated by cell viability assays in HeLa cells. As shown in Figure 10, the free nanogels display good biocompatibility in wide nanogel concentrations from 0.025 mg/mL to 0.5 mg/mL with cell viability higher than 96%. However, the DOX-loaded nanogels show medium antitumor activity toward HeLa cells with IC50 (the half maximal inhibitory concentration) of 1.80 μg/mL, 2.57 μg/mL and 3.01 μg/mL for DNG5, DNG10 and DNG15 respectively, which is higher than that of free DOX (0.59 μg/mL) and in good accordance with the intracellular drug release study. These results suggest that the nanogels are biocompatible with tunable antitumor activity. It is worth to note that the hydrophilic nanogels could be further functionalized with ligands such as aptamer, peptide and antibody fragment to enhance antitumor efficacy and specificity.

Figure 10. A) Cytotoxicity of DNG5, DNG10 and DNG15 nanogels to HeLa cells following 48 h incubation. B) Viabilities of HeLa cells following 48 h incubation with DNG5, DNG10, DNG15 nanogels and free DOX as a function of DOX dosages. All the data presented are average values of experiments in triplicate.

4. Conclusion In the present work, primary amine-functionalized statistical copolymers were obtained by hydrolysis of poly(N-vinylpyrrolidone-co-N-vinylformamide) polymers which

were

prepared

via

RAFT

polymerization

with

methyl

2-(ethoxycarbonothioylthio) propanoate as chain transfer agent and anisole as solvent. Biocompatible redox sensitive nanogels with different crosslinking densities were 23

prepared in water-in-oil emulsions utilizing the –NH2 groups of the reactive building blocks and the vinyl groups of the degradable cross-linkers via Michael addition chemistry. DOX covalently anchored with one of the cross-linker arms to form redox-sensitive prodrug nanogels with different crosslinking densities. The rates of nanogel degradation and DOX release decreased with the increase of crosslinking density at pH 7.4 in the presence of 10 mM GSH. The DOX-loaded nanogels have demonstrated adjustable antitumor activity to HeLa cells with progressively increased IC50 (half-maximal inhibitory concentration) from DNG5, DNG10 to DNG15. The intracellular drug release rate could be controllable via adjusting the crosslinking density of the nanogels, lower crosslinking density leading to higher release rate. The experiment data suggested that the crosslinking density of the nanogels plays an important role in tuning the release kinetics and nanogel degradability at intracellular conditions. The strategy presented here avoids self-crosslinking of prepolymers in conventional precipitation polymerization. We use new reactive amphiphilic copolymers for nanogel design to covalently conjugate drug molecules. Thus, this approach overcomes serious leakage problem caused by other encapsulation approaches.53 Additionally, we find that the amount of reactive amine groups in copolymer structure allows adjustment of the crosslink density and degradation rate of nanogels, which provides additional tool to control the prodrug distribution in biological systems. Decoration the nanogels with functional ligands to improve specific targetability and further investigation of drug delivery process in vivo will be our future research direction.

5. Associated Content Supporting Information Chain transfer agent synthetic route, 1H NMR, FTIR, polymer composition and characterization data as well as intracellular drug release study.

6. Acknowledgment. 24

A.P. thanks Volkswagen Foundation and Deutsche Forschungsgemeinschaft (DFG) with Collaborative Research Center SFB 985 „Functional Microgels and Microgel Systems“ for financial support.

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