Stimuli-responsive biohybrid nanogels with self-immolative linkers for protein protection and traceless release

Colloids and Surfaces B: Biointerfaces 184 (2019) 110526

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Stimuli-responsive biohybrid nanogels with self-immolative linkers for protein protection and traceless release ⁎

Yahui Guoa,b, Yue Zhanga,b, , Zhanghao Niua,b, Yongfang Yanga,b, a b

T

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School of Chemical Engineering and Technology, Hebei University of Technology, Tianjin 300130, China Hebei Key laboratory of Functional Polymers, Tianjin 300130, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Nanogel Self - immolative linker Protein protection Traceless release

Nanogels have been applied in protein delivery due to the nanoscale sizes and the crosslinked structures. However, the release of protein molecules from the nanogels without damages to the structures and functionalities is quite a challenging research subject. In this research, responsive self-immolative linker dithioethyl carbamate bond is introduced to connect protein and polymer in the nanogel so that traceless release of protein occurs upon addition of glutathione (GSH) or dithiothreitol (DTT). Thermoresponsive polymer poly(di(ethylene glycol) methyl ether methacrylate-co-2-(2-(2-hydroxyethyl) disulfanyl) ethyl methacrylate) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization, and was modified with 4-nitrophenyl chloroformate yielding polymer chains with pendant dithioethyl carbonate groups. The dithioethyl carbonate groups were reacted with amine groups of lipases resulting in the formation of dithioethyl carbamate bonds. Meanwhile, biohybrid nanogels were prepared by crosslinking the polymer chains with lipases. The immobilized lipase in the nanogels exhibited enhanced heat and acid resistance. Once the nanogels were treated with GSH or DTT, lipase could be released with no residual groups and most of its bioactivity was recovered.

1. Introduction In recent years, protein delivery has drawn intensive attention and various protein carriers including inorganic nanoparticles, liposomes, vesicles and nanogels have been developed to protect proteins from denaturation and to reduce the toxicity and immune side-effects caused by proteins during the delivery [1–4]. Among the reported protein carriers, nanogels exhibit distinguished properties due to their crosslinked structures and nanoscale sizes [5–8] and are proved to be effective in protein delivery with preservation of activity [9–11]. Liu and coworkers reported the encapsulation of single enzyme in nanogels. With the protection of nanogels, horseradish peroxidase maintained most of its activity against high temperature and organic solvent [9]. Akiyoshi and coworkers prepared cholesterol-bearing pullulan nanogels with embedded lipase. The enzyme showed enhanced thermostability [10]. Many different routes have been applied to load proteins into nanogels, [12] including physical entrapment [13–16], noncovalent interactions [17–19], and covalent bonds [20,21]. Physical entrapment and noncovalent interactions are usually preferred ways for they are easy to achieve and the approaches exert less influence on the protein bioactivity. However, leakage of protein is possible due to the lack of

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strong interaction between proteins and matrix. Covalent bonds strengthen the linkage between proteins and nanogels, but often result in release difficulty. Cleavable covalent bonds, such as disulfide bonds, have been introduced to build nanogels as protein carriers to realize triggered release. But residual groups are often left on the released protein molecules, which may result in activity loss and unexpected immune response [22–24]. Therefore, it is important to choose a suitable covalent linker which can be cleaved in response to external stimuli and leaves no residual chemical groups on the protein. To meet this request, the self-immolative linker is a good choice for firm loading and traceless release of protein. Actually, the traceless release of payloads has been studied thoroughly in the field of small molecule drug delivery. The researches of polymer prodrugs involve conjugation of drugs to polymer scaffolds and their traceless release [25–27]. Various self-immolative bonds have been applied in the preparation of prodrugs [28–30] and some of them are suitable for the bioconjugation of proteins to polymers. As for the self-immolative bonds, we prefer to construct polymer-protein nanogels with redox-responsive dithioethyl carbamate linkers, which can be prepared by reactions between amine groups of proteins and dithioethyl carbonate groups of polymers. Once the disulfide bonds are reduced, the formed thiol groups will react with carbamate groups and

Corresponding authors at: School of Chemical Engineering and Technology, Hebei University of Technology, Tianjin 300130, China. E-mail addresses: [email protected] (Y. Zhang), [email protected] (Y. Yang).

https://doi.org/10.1016/j.colsurfb.2019.110526 Received 29 July 2019; Received in revised form 17 September 2019; Accepted 23 September 2019 Available online 27 September 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.

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Scheme 1. (a) Synthetic route of polymer P(DEGMA-co-HODMA-co-NPC) and schematic illustration of synthesis of nanogels and traceless release of lipases. (b) Illustration of reaction between lipase and the polymer P(DEGMA-co-HODMA-co-NPC) and traceless release of lipase from the polymer scaffold.

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(COOCH2CH2, t, 2H), 2.92 (CH2CH2OH, t, 2H), 1.98 (CH2 = CCH3, s, 3H).

the amine groups will be recovered [31–35]. DeSimone and coworkers synthesized protein particles by crosslinking bovine serum albumin (BSA) with dithio-bis(ethyl 1H-imidazole-1-carboxylate). The amine groups of BSA reacted with the crosslinkers forming dithioethyl carbamate bonds, and the reduction of disulfide bonds led to the recovery of amine groups [31]. Wang and coworkers used similar linkers for reversible protein PEGylation. In their approach disulfide group was replaced by thioester, and glutathione (GSH) could trigger the traceless release of protein with activity recovered [32]. Herein, we report the preparation of thermo- and redox-responsive polymer-protein nanogels which can undergo traceless release of protein. Lipase was used as a model protein and the nanogels were synthesized by crosslinking thermoresponsive polymer poly(di(ethylene glycol) methyl ether methacrylate-co-2-(2-(2-hydroxyethyl) disulfanyl) ethyl methacrylate-co-2-(2-(2-((4-nitrophenoxy) carbonyloxy)ethyl) disulfanyl) ethyl methacrylate) (P(DEGMA-co-HODMA-co-NPC)) with lipase (Scheme 1a). The reaction between amine groups of lipases and dithioethyl carbonate groups on polymer chains leads to the formation of dithioethyl carbamate bonds, which can be reduced by GSH or dithiothreitol (DTT) to release lipases (Scheme 1b). The sizes and thermoresponsive behaviors of nanogels depend on the feed ratio of lipase and polymer. Moreover, when the lipase is incorporated into the nanogel, amine groups are consumed resulting in activity loss. When the lipase is released from the nanogel, the amine groups are recovered as well as the activity. The temporary inactivation of protein in nanogels is favorable for avoiding unwanted effects during delivery [36]. In addition to the application in protein delivery and activity control, the nanogels are also able to protect lipase from high temperature and acid due to the preservation of lipase structure by the crosslinked structure of nanogels. The cytotoxicity of nanogels has been evaluated by NIH 3T3 and 4T1 cells, respectively. And it has been proved that the nanogels have low toxicity to the two different kinds of cells.

2.3. Synthesis of random copolymer poly(di(ethylene glycol) methyl ether methacrylate-co-2-(2-(2-hydroxyethyl) disulfanyl) ethyl methacrylate) (P (DEGMA-co-HODMA)) by reversible addition-fragmentation chain transfer (RAFT) polymerization HODMA (0.400 g, 0.00180 mol), DEGMA (3.37 g, 0.0179 mol), CPADB (28.0 mg, 0.100 mmol) and AIBN (2.2 mg, 0.013 mmol) were dissolved in 7.50 mL 1,4-dioxane in a Schlenk flask. The solution was degassed by three freeze –pump -thaw cycles, and the polymerization was conducted at 60 °C for 8 h. To stop the reaction, the solution was exposed to the air. The copolymer was purified by being precipitated in cold diethyl ether (0 °C) twice and dried in a vacuum oven. The number-average molecular weight (Mn) and dispersity (Mw/Mn) measured by gel permeation chromatograph (GPC) are 20.8 kDa and 1.11, respectively. 2.4. Modification of random copolymer P(DEGMA-co-HODMA) with 4nitrophenyl chloroformate P(DEGMA-co-HODMA) (0.170 g, 0.00654 mmol) and TEA (0.10 mL, 0.72 mmol) were dissolved in 5.0 mL dichloromethane in a 50 mL round-bottomed flask. 4-Nitrophenyl chloroformate (60.0 mg, 0.299 mmol) in 5.0 mL dichloromethane was added dropwisely at 0 °C. The reaction was conducted at room temperature for 24 h. After that, the polymer was purified by being precipitated in cold diethyl ether (0 °C) twice and dried in a vacuum oven. The modified polymer is referred to as P(DEGMA-co-HODMA-co-NPC). The Mn and dispersity measured by GPC are 23.0 kDa and 1.16, respectively. Yield: 82%. 2.5. Preparation of nanogels

2. Materials and methods

Lipase (5.0 mg for N-1 and 2.5 mg for N-0.5) was dissolved in 0.9 mL phosphate buffer (PB, pH = 8.0) and mixed with 0.1 mL DMF solution of P(DEGMA-co-HODMA-co-NPC) (5.0 mg) at 8 °C. After 0.5 h reaction, the mixture was dialyzed against PB (pH = 8.0) in an ice-bath to remove DMF. The final concentration of nanogel N-1 and N-0.5 were adjusted to 2.5 and 2.0 mg/mL, respectively.

2.1. Materials Di(ethylene glycol) methyl ether methacrylate (DEGMA, Aldrich, 95%) was purified by passing through an aluminum oxide column. 2,2′Azobis(isobutyronitrile) (AIBN, Guo Yao Chemical Company, 98%) was recrystalized from ethanol. Lipase (Sigma-Aldrich), methacryloyl chloride (TCI, 80%), 4-nitrophenyl chloroformate (Alfa Aesar, 97%), 2hydroxyethyl disulfide (Alfa Aesar, 90%), DL-dithiothreitol (Sigma, 99%), glutathione reduced (Heowns, 97%), 4-nitrophenyl palmitate (Heowns, 98%) were used as received. 4-(Cyanopentanoic acid)-4-dithiobenzoate (CPADB) was prepared according to the previous research [37]. All solvents were purified by distillation before use.

2.6. Determination of encapsulation efficacy and loading efficiency of nanogel The nanogel solution was centrifugated and washed by PBS (pH = 7.4) for three times. The supernatant was collected and the concentration of lipase in the supernatant was measured using BCA (bicinchoninic acid) according to a previous report [39]. The encapsulation efficacy and loading efficiency are calculated following the formula:

2.2. Synthesis of monomer 2-(2-(2-hydroxyethyl) disulfanyl) ethyl methacrylate (HODMA) HODMA was prepared according to literature [38]. Hydroxyethyl disulfide (6.17 g, 0.0400 mol) was dissolved in 50 mL THF in a 250 mL round-bottomed flask. Triethylamine (TEA, 2.90 mL, 0.0209 mol) was added into the flask and the flask was cooled in an ice-bath for 20 min. Methacryloyl chloride (1.05 g, 0.0100 mol) dissolved in 8 mL THF was added dropwisely. After addition, the mixture was stirred in the icebath for 2 h and then at room temperature for 48 h. The mixture was filtered and then concentrated by a rotary evaporator. After that, the solution was diluted by 100 mL dichloromethane and washed with 2% HCl solution, saturated NaHCO3 solution and water, successively. The purification of the crude product was carried out on a silica gel column (hexane: dichloromethane: ethanol = 70:10:3). Yield: 69.0%. The 1H NMR spectrum of HODMA (Fig. S1) is shown in supporting information. 1 H NMR δ (400 MHz, CDCl3, TMS, ppm): 5.62, 6.16 (CH2 = CCH3, s, 2H), 4.46 (COOCH2, t, 2H), 3.92 (CH2CH2OH, t, 2H), 3.00

Encapsulation efficacy wt%=

total lipase added − unencapsulated lipase total lipase added × 100

Loading efficiency wt%=

amount of encapsulated lipase × 100 amount of polymer

2.7. Measurement of lower critical solution temperatures (LCSTs) of polymers and nanogels The LCSTs of polymers and nanogels were determined by measuring transmittance of solution (for polymer 0.4 mg/mL, for nanogel 0.4 mg/ mL) at 800 nm using UV–vis spectrometer. It took 5 min for sample solution to be kept at each temperature before measurement. 3

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2.8. Measurement of amine groups with fluorescamine as the fluorogenic reagent

measured using a multifunctional ELISA plate reader (Thermo Varioskan Flash) at 450 nm. The cell viability (%) was calculated according to the following formula:

Sample solution (1 mL, PB pH = 9.0) was mixed with 0.1 mL acetone solution of fluorescamine (2 mM) and the reaction was conducted at 25 °C for 20 min. After that, the fluorescence emission spectra of native lipase, DTT treated lipase, N-1 and DTT treated N-1 were measured by fluorescence spectrometer, respectively. The concentration of lipase and DTT treated lipase solution was 0.06 mg/mL. As to the N-1 and DTT treated N-1 solution, both of them contained 0.06 mg/mL lipase.

cell viability (%) =

× 100%

Where A (Sample) represents the absorbance of CCK-8 measured for cells with different samples and A (Control) stands for control cells (untreated). A (Blank) represents the absorbance of CCK-8 without cells. All experiments were carried out in quintuplicate. 2.12. Characterization

2.9. Measurement of lipase activity

A Varian UNITY-plus spectrometer (400 M) was used for 1H NMR measurements. The apparent molecular weight (Mn) and the dispersity (Mw/Mn) of the polymers were measured on a gel permeation chromatograph (GPC) with a HPLC pump (Hitachi L-2130), a column oven (Hitachi L-2350) operated at 40 °C, three PL columns (Varian) with 1000–100 K (100,000 Å), 100–10 K (10,000 Å), and 100–10 K (1000 Å) molecular weight ranges, and a refractive index detector (Hitachi L2490). The mobile phase was DMF and the flow rate was 1.0 mL/min. The Mn was calculated based on PMMA standards. Transmission electron microscopy (TEM) results were measured on an electron microscope (Tecnai G2 F20) with 200 kV operating voltage. Formvar and carbon coated Cu grids were used to prepare the TEM samples. To prepare the TEM samples of nanogels below the critical temperature, the nanogel solutions were dropped on the copper grids at 8 °C, and then dried at 8 °C. To prepare the samples beyond the critical temperature, the temperature was controlled at 30 °C during the preparation of the samples. The hydrodynamic sizes and Zeta-potential values of nanogels were obtained on a Malvern Zetasizer Nano-ZS equipped with a 633 nm He–Ne laser at 90°. The measurements of UV–vis transmittance and absorption spectra were performed on a Shimadzu UV-2450 spectrophotometer. Fluorescence spectra were measured on a Shimadzu RF-5301PC fluorescence spectrophotometer. The excitation and emission slits were 5 nm and 3 nm. The excitation wavelength was set at 390 nm.

The enzymatic activity of native lipase and nanogels were measured according to a previous report [40]. Basically, p-nitrophenyl palmitate (pNPP) (4.0 mg, 0.011 mmol) was dissolved in 1.0 mL acetonitrile as a substrate solution. Sample solution (1.0 mL, pH = 8.0, containing 1 mg lipase) was prepared and incubated in a water bath at 37 °C. The reaction was started by mixing 40 μL pNPP solution with the sample solution. The reaction was monitored by measuring the UV absorbance of mixture at 401 nm at different times. To determine the effect of temperature on activities of lipase and nanogels, the lipase or nanogel solution was heated to 50 °C for 30 min and then cooled to 37 °C. And the activities were measured at 37 °C. To investigate the effect of pH on activities of lipase and nanogel, the lipase or nanogel solution was firstly dialyzed against PB (pH = 2.0) for 1 h, and then dialyzed against PB (pH = 8.0). To detect the activity of lipase released from the nanogel N-1, N-1 was reacted with DTT in PB (pH = 8.0) for 5 h and then dialyzed against PB (pH = 8.0) to remove unreacted DTT. 2.10. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) SDS-PAGE was performed using polyacrylamide gels with 12% crosslinking degree. Electrophoresis was performed at 68 V voltages and 16 mA current for 3 h. Coomassie Brilliant Blue R-250 solution was used for staining. The samples measured were prepared according to the following table (Table 1).

3. Results and discussion The random copolymer P(DEGMA-co-HODMA) was synthesized by RAFT polymerization with CPADB as the chain transfer agent and AIBN as the initiator. After purification, the polymer was characterized by GPC and 1H NMR. GPC trace (Fig. 1a) indicates that P(DEGMA-coHODMA) has narrow molecular weight distribution. According to the GPC result, the apparent molecular weight Mn and dispersity are 20.8 kDa and 1.11, respectively. The 1H NMR spectrum of P(DEGMAco-HODMA) is shown in Fig. 1b. Based on the 1H NMR result, the average unit numbers of DEGMA and HODMA are 130 and 7, respectively. The polymer is denominated as P(DEGMA130-co-HODMA 7). Polymers prepared by various oligo(ethylene glycol) methyl ether methacrylate monomers are widely used and deeply investigated due to their biocompatibility and temperature responsiveness. Homopolymer of DEGMA exhibits lower critical solution temperature (LCST) at 26 °C [41,42]. The temperature responsiveness behavior of P(DEGMA130-coHODMA7) was determined by measuring the transmittance of the solution at different temperatures with 800 nm light [43]. It can be observed in Fig. 1c that the transmittance is basically maintained at 100% at lower temperature indicating that P(DEGMA130-co-HODMA7) is well dissolved in water. The transmittance of the polymer solution starts to decrease at about 17 °C, which suggests the increase of scattering light caused by the collapse of polymer chains in the solution. The transmittance reaches 23.8% at about 21 °C. The LCST defined as the inflection point of the curve is determined at around 17 °C [44]. Compared to the homopolymer, the decrease of LCST can be attributed to

2.11. In vitro cytotoxicity assays The cytotoxicity of polymer was evaluated by a CCK-8 assay. 4T1 cells and NIH 3T3 cells were seeded in 96-well plates with a density of 5000 cells per well. The cells were cultured in DMEM medium, supplemented with 10% FBS at 37 °C, under 5% CO2. After incubation for 24 h, the culture media were replaced with fresh media containing polymer at different concentrations. The media were removed after 24 h, and then 10 μL of CCK-8 reagents (Dojindo Laboratories) and 90 μL of fresh media were added. After 30 min, the plates were gently shaken for 2 min to dissolve formazan crystals. The absorbance was Table 1 Preparation recipe of sample solutions for SDS-PAGE.a

Volume of sample solution (μL)b Volume of DTT solution (μL)c Volume of bromophenol blue solution (μL)

A(Sample) − A(Blank ) A(Control) − A(Blank )

Native lipase + DTT

Nanogel

Nanogel + DTT

20 40 10

20 0 10

20 40 10

For each sample, 20 μL solution was taken out for SDS-PAGE. Both the concentration of native lipase solution and the concentration of lipase in the nanogel solution were 1.0 mg/mL. c The concentration of DTT solution was 1.0 mol/L. a

b

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Fig. 1. (a) GPC curves of P(DEGMA130-co-HODMA7) (solid line) and P(DEGMA130-co-HODMA-co-NPC6) (dash line). (b) 1H NMR spectrum of P(DEGMA130-coHODMA7). (c) Transmittance trend against temperature of P(DEGMA130-co-HODMA7) (solid line) and P(DEGMA130-co-HODMA-co-NPC6) (dash line) in water. (d) 1H NMR spectrum of P(DEGMA130-co-HODMA-co-NPC6).

referred as P(DEGMA130-co-HODMA-co-NPC6). As shown in Fig. 1a, compared to the GPC curve of P(DEGMA130-co-HODMA7) the curve of P (DEGMA130-co-HODMA-co-NPC6) remains low dispersity and shifts to high molecular weight regime after the formation of p-nitrophenylcarbonate groups. The Mn and dispersity measured by GPC are 23.0 kDa and 1.16, respectively. The LCST of P(DEGMA130-coHODMA-co-NPC6) is determined at about 8 °C (Fig. 1c), which is lower than the LCST of P(DEGMA130-co-HODMA7). The decrease in LCST is attributed to the coupling of hydrophobic p-nitrophenylcarbonate groups to the polymer chains. Nanogels were prepared by mixing the solution of lipase and P (DEGMA130-co-HODMA-co-NPC6) at 8 °C. The temperature was lower than the LCST of the polymer in the mixed solution to avoid the collapse of polymer (Fig. S2). The structural parameters of nanogels are listed in Table 2. The nanogels were prepared by a reaction between amine groups on lipases and p-nitrophenyl carbonate groups on P(DEGMA130-coHODMA-co-NPC6), in which carbamate linkers formed (Scheme 1b). This reaction has been used for protein PEGylation and proved to be efficient in polymer bioconjugation [46]. Each lipase molecule has seven amine groups and acts as a crosslinker in the formation of the nanogels. The molar feed ratios of p-nitrophenylcarbonate groups to amine groups were controlled at 1:1 and 2:1 in the synthesis of N-1 and N-0.5, respectively. Determined by the UV absorbance of 4-nitrophenol, the conversions of p-nitrophenylcarbonate groups of polymers are 63.0% and 33.9% after 0.5 h reaction. The encapsulation efficacy and loading efficiency of N-1 are 60.0% and 50.9%. Attributed to the use of

Table 2 Structural parameters of nanogels. Sample name a

Hydrodynamic diameter (Dh) at 8 °C [nm] Polydispersity index (PDI) of nanogelsa Zeta potential at 8 °C [mV]a,b

N-1

N-0.5

191.0 ± 3.9 0.247 ± 0.052 −29.7 ± 3.8

238.4 ± 2.3 0.130 ± 0.069 −33.2 ± 3.3

a The average value and standard deviation listed in the table were calculated from results of three independent measurements. b The pH value of nanogel solution used for measurement of Zeta potential was 7.0.

the introduction of a less hydrophilic monomer HODMA. The influence of comonomer on LCST of PDEGMA has been reported in previous researches. Copolymerization with hydrophilic monomers leads to LCST increase while introduction of hydrophobic monomers results in a decrease in LCST [45]. The copolymer P(DEGMA130-co-HODMA7) was further reacted with 4-nitrophenyl chloroformate affording a polymer with pendant p-nitrophenylcarbonate groups. On 1H NMR spectrum (Fig. 1d), peaks at 7.40 and 8.30 ppm assigned to the protons on the p-nitrophenylcarbonate groups and a peak at 4.60 ppm assigned to methylene group next to the carbonate group appear after the reaction, while the peak at 3.89 ppm corresponding to methylene group next to the hydroxyl group of HODMA basically disappears. It can be calculated from the 1H NMR result that averagely there are 6 p-nitrophenylcarbonate groups on each polymer chain. And the polymer is 5

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Fig. 2. (a) Number-weighted size distributions of N-1 (solid line) and N-0.5 (dash line) at 8 °C. (b) Number-weighted size distributions of N-1 at 8 °C (solid line) and 30 °C (dash line). (c) Transmittance percentage of N-1 (solid line) and N-0.5 solution (dash line) as a function of temperature.

Fig. 3. TEM images of N-1 at 8 °C (a, b) and 30 °C (c, d). The scale bars of image a and c are 200 and 500 nm, respectively. And the scale bars of image b and d are 100 nm. All the samples were stained at OsO4 atmosphere.

crosslinker content have loose structures. The isoelectric point of lipase is about 5.2 and the average Zeta potential of it in aqueous solution (pH = 7) is −22.1 mV. The average Zeta potential of N-1 and N-0.5 are −29.7 mV and −33.2 mV, which demonstrates the incorporation of lipase in nanogels. Previously it has been reported that the polymer-protein conjugates composed of thermoresponsive polymers and proteins are responsive to temperature change [47]. The thermoresponsiveness of nanogel N-1

protein molecules as crosslinkers, the loading efficiency is relatively high. The dynamic light scattering (DLS) results of N-1 and N-0.5 are shown in Fig. 2a. The average hydrodynamic diameters and PDIs of N-1 and N-0.5 are 191.0 ± 3.9 nm, 0.247 ± 0.052 and 238.4 ± 2.3 nm, 0.130 ± 0.069, respectively. It can be proved by DLS results that nanogels have been successfully synthesized by crosslinking P(DEGMA130co-HODMA-co-NPC6) with lipase. Compared with N-1, N-0.5 with less lipase has larger size, which indicates that nanogels prepared at low 6

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Fig. 4. (a) Number-weighted size distributions of N-1 after treatment in GSH solution for different times. (b) TEM image of N-1 after treatment of GSH solution. (c) SDS-PAGE results of lipase and N-1. Lane 1: markers; Lane 2: lipase + DTT; Lane 3: N-1; Lane 4: N-1 + DTT. (d) Fluorescence emission spectra of native lipase solution (solid line), DTT treated lipase (dash line), N-1 (dash dot line) and DTT treated N-1 (dot line) after reaction with fluorescamine.

Fig. 5. (a) Relative activity statistics of lipase, N-1 and released lipase after different treatment. (b) Cytotoxicity assays of N-1 incubated with NIH 3T3 cells and 4T1 cells for 24 h at different concentrations.

grids at 8 °C and 30 °C, respectively. After drying, the copper grids were stained under OsO4 atmosphere and the lipases were stained. Fig. 3a and b shows the TEM images of N-1 at 8 °C, in which it can be observed that spherical nanogels are synthesized with lipase evenly distributed in the nanogels. Based on the TEM results, the size of N-1 is 150.5 ± 27.7 nm, which is in accordance with the DLS result. As shown in Fig. 3c and d, when the temperature is above the LCST, the spherical nanogels still can be observed with lipase in them. Compared with the nanogels at 8 °C, the size decreases to 64.0 ± 14.7 nm at 30 °C and the morphology becomes irregular because of the shrinkage of the nanogels. The nanogels are designed to be a platform for protein delivery and the dissociation of the nanogels and the release of lipases were studied. In the nanogels, lipases are connected to the polymer chains through redox-responsive disulfide bonds. Treating nanogels with GSH or DTT leads to the release of lipases and the formation of thiol groups. The thiol groups can further react with carbamate groups resulting in the recovery of amine groups of lipases (Scheme 1b). The dissociation of nanogels was monitored by DLS. N-1 was treated in 50 mM GSH solution at 37 °C and the hydrodynamic diameter was measured at different

and N-0.5 were detected by measuring the transmittance trends of the solution against temperature. It can be observed from the resulting curves (Fig. 2c) that the transmittance decreases dramatically at 17–28 °C for N-1 and 11–15 °C for N-0.5. Due to the incorporation of relatively hydrophilic lipase into nanogels, the LCSTs of N-1 and N-0.5 are higher than the LCST of the polymer P(DEGMA130-co-HODMA-coNPC6). The LCST of N-1 is higher than that of N-0.5 because of more loading of lipase in N-1. The increase in LCST after conjugation of hydrophilic proteins to thermoresponsive polymers was also reported by other researchers [48]. In order to study the influence of temperature on the nanogel, the size and morphology of N-1 at 8 °C and 30 °C were measured by DLS and TEM. The average hydrodynamic diameter of N-1 at 30 °C in Fig. 2b is 133.3 ± 3.1 nm (PDI = 0.176 ± 0.038), which is smaller than N-1 at 8 °C. The size decrease can be attributed to the collapse of polymer scaffolds caused by the temperature change. When the temperature is above the LCST, polymer chains of the nanogel collapse inducing shrinkage of the nanogel particles. The size change can be also demonstrated by TEM. The TEM samples of N-1 below and above the LCST were prepared by dropping diluted N-1 solution to the copper

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reaches 97.1%, which indicates that the lipase embedded in the nanostructures keeps its activity. The reason why the lipase can be preserved in N-1 is that the crosslinking structure of the nanogel and close encapsulation of lipase with thermoresponsive polymer at 50 °C (higher than the LCST of the nanogel) maintain lipase structure to some extent, so that the maintenance of lipase activity can be achieved. And once the nanogel dissociates by cleavage of the disulfide bonds, the lipase can recover its activity. There are a few reports on taking advantage of crosslinked structure to protect protein from high temperature or organic solvent [9,10]. Similar results can be observed when N-1 is treated with PB (pH = 2.0). After dialyzing the native lipase or N-1 against PB (pH = 2.0) at 37 °C for 1 h and then against PB (pH = 8.0) successively, the native lipase shows no activity while the lipase in N-1 still owns 42.2% of its activity. Once the lipase is released by treating N1 with DTT in PB (pH = 8.0) at 37 °C, its activity increases to 86.9%. The results prove that the immobilization of lipase in N-1 results in protection of lipase against acid condition. It can be concluded that N-1 is able to protect lipase from heat and acid conditions and to release lipase with most of its activity maintained. The nanogel is designed as a protein carrier and it is necessary to test its in vitro cell viability. The cytotoxicity of N-1 was evaluated by CCK-8 assays using NIH 3T3 cells and 4T1 cells, respectively. The NIH 3T3 cells and 4T1 cells were chosen to evaluate the cytotoxicity of N-1 towards normal and cancer cells. N-1 solutions with concentrations ranging from 0 to 200 μg/mL were incubated with the cells for 24 h and the results are shown in Fig. 5b. It can be observed that the cell viabilities of both the NIH 3T3 cells and 4T1 cells decrease slightly with the increase of N-1 concentration. But the cell viabilities are still above 95% even at the concentration up to 200 μg/mL. The results obtained lead to the conclusion that the nanogel N-1 has essentially low toxicity to NIH 3T3 cells and 4T1 cells.

time intervals. The average hydrodynamic diameters at 1, 3, 6, and 9 h are 181.7 ± 4.2, 147.8 ± 2.1, 112.0 ± 2.8 and 44.3 ± 2.4 nm, respectively. As shown in Fig. 4a, the average hydrodynamic diameter of N-1 decreases with time demonstrating the dissociation of nanogels. With the reduction of disulfide bonds by GSH, polymer and lipase are released. The dissociation of N-1 is also proved by the TEM result. As shown in Fig. 4b, fragments with irregular shapes instead of spherical nanogels can be observed in the TEM image. Besides, the release of lipases from nanogels was characterized by SDS-PAGE (Fig. 4c). The DTT treated lipase yields a band at about 66 kDa (lane 2) and the nanogel N-1 without DTT treatment owns too large size to run on the gel and no band is observed (lane 3), which also indicates that there is no free lipase in the nanogel solution. After treatment of N-1 with DTT, disulfide bonds connecting polymers and lipases are cleaved and a clear band corresponding to the released lipase can be observed in lane 4. In the introduction section, it has been emphasized that the choice of dithioethyl carbamate linkers between polymer scaffolds and lipases leads to the recovery of amine groups of lipases after cleavage of disulfide bonds. Therefore, the amount of amine groups of native lipase, DTT treated lipase, N-1 and DTT treated N-1 were evaluated by fluorescence spectrometer with fluorescamine as the fluorogenic reagent [49]. The fluorescence intensity at maximum emission wavelength is proportional to concentration of amine groups. We adjusted the concentrations of lipase in the sample solution of native lipase, DTT treated lipase, N-1 and DTT treated N-1 to be the same (0.06 mg/mL) so that the fluorescence intensity at maximum emission wavelength (480 nm) was proportional to the concentration of residual amine groups on lipase. It can be observed in Fig. 4d that the fluorescence intensity decreases after formation of the nanogel. The fluorescence intensity of N-1 at 480 nm is only 39.9% compared to that of the native lipase, which indicates 60.1% of amine groups of lipase are consumed by reaction with polymer. This result agrees with the conversion calculated by 4nitrophenol. And after N-1 is treated with DTT, the fluorescence intensity at 480 nm increases to 96.5% of intensity of native lipase solution, which demonstrates the recovery of amine groups. The fluorescence of DTT treated lipase was also measured as a control. It can be observed that the fluorescence intensity of DTT treated lipase is as same as the native lipase, which demonstrates that DTT treatment does not increase the amount of available amine groups of the native lipase. Therefore, the increase of fluorescence intensity is attributed to the recovery of amine groups from the dissociation of the nanogel and the release of lipase. One of our major concerns is the maintenance of protein activity in the design of an effective protein carrier. In the nanogels, polymer chains and proteins are connected by disulfide bonds and carbamate groups. Upon cleavage of the disulfide bonds, the generated thiol groups react with carbamate groups leading to the recovery of consumed amine groups and release of protein without residual groups. To investigate the effect of formation and dissociation of nanogels on lipase activity, hydrolysis of p-nitrophenyl palmitate (pNPP) in the presence of native lipase, N-1, and DTT treated N-1 were measured by UV–vis spectrometer (Fig. S3). The activity of native lipase measured in PB (pH = 8.0) at 37 °C is set as 100%. It can be observed in Fig. 5a that the activity of lipase decreases to 45.2% after the formation of nanogels, which is attributed to the reaction of amine groups on lipase with polymer. Upon treatment of nanogels with DTT, lipases are released from the nanogels and the activity is recovered, which demonstrates that the nanogel can release lipase with its activity maintained. Since protein is fragile and sensitive to conditions such as temperature and pH value, a carrier should protect protein molecules from unfavorable conditions. The protections of lipase by N-1 from heat and acid condition were investigated respectively and the results are summarized in Fig. 5a. After heating to 50 °C for 30 min and then cooling to 37 °C, native lipase loses 70% of its activity while the lipase in N-1 still keeps 54.5% of its activity. Interestingly, after heating and cooling process N-1 is dissociated by DTT and the activity of the released lipase

4. Conclusions In conclusion, a thermo- and redox-responsive protein-hybrid nanogel has been synthesized by crosslinking thermoresponsive polymer P (DEGMA130-co-HODMA-co-NPC6) with lipases. The size and LCST of the nanogel can be adjusted by changing the feed ratio of the polymer and lipase. Below the LCST, the nanogels are spherical with lipases evenly dispersed inside them. And when the temperature is above the LCST, the size of nanogels decreases due to the shrinkage of the nanogels. Reduced by GSH or DTT, nanogels can dissociate and release lipases without any residual groups. Loading lipases into nanogels can protect them from high temperature and low pH conditions. The released lipase still maintains most of its activity. The nanogel exhibits low cytotoxicity and can be used as an effective protein carrier. Declaration of Competing Interest None. Acknowledgement We appreciate Prof. Hanying Zhao’s kind help on this research. This work was supported by the National Natural Science Foundation of China (NSFC) [grant number 51603061]. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.110526. References [1] M. Ray, Y.W. Lee, F. Scaletti, R. Yu, V.M. Rotello, Intracellular delivery of proteins by nanocarriers, Nanomedicine 12 (2017) 941–952.

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