Protein Nanogels with Temperature-Induced Reversible Structures

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Protein Nanogels with Temperature-Induced Reversible Structures and Redox Responsiveness Yue Zhang, Jiamin Zhang, Cheng Xing, Mingming Zhang, Lianyong Wang, and Hanying Zhao ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00490 • Publication Date (Web): 12 Oct 2016 Downloaded from http://pubs.acs.org on October 19, 2016

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ACS Biomaterials Science & Engineering

Protein Nanogels with Temperature-Induced Reversible Structures and Redox Responsiveness Yue Zhang,†‡ Jiamin Zhang,§Cheng Xing,§Mingming Zhang,⁄⁄* Lianyong Wang,§* and Hanying Zhao†‡* †.

Key Laboratory of Functional Polymer Materials, Ministry of Education, College of Chemistry, Nankai University, Tianjin 300071, China ‡.

Collaborative Innovation Center of Chemical Science and Engineering, Nankai University, Tianjin 300071, China

§.

The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, China

⁄⁄

Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300192, China

ACS Paragon Plus Environment

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ABSTRACT: There are many natural examples of smart structures that are able to change conformations and functionalities responding to the external stimuli. The responsiveness is directly related to their unique structures. In the design of new materials it is crucial to endow these materials with the capabilities to change structures and functionalities under the external stimuli. In this research, virus-mimicking protein nanogels with temperature-induced reversible structures and redox responsiveness are synthesized by crosslinking a thermally responsive polymer

poly(di(ethyleneglycol)

methyl

ether

methacrylate-co-2-(2-pyridyldisulfide)

ethylmethacrylate) with reduced bovine serum albumin (BSA) molecules through thiol-disulfide exchange reaction. The lower critical solution temperature (LCST) and sizes of the nanogels can be controlled by controlling the reaction conditions. The nanogels are able to change their structures responding to the temperature change. Below the LCST, BSA molecules are embedded inside the nanogels and protected by the polymer chains. Above the LCST, polymer chains collapse forming the cores and BSA moves to the shells to stabilize the nanogels. The disulfide-crosslinked nanogels are dissociated in the presence of glutathione. In vitro cytotoxicity assays and cell uptake assays demonstrate that the nanogels show low toxicity towards 3T3, 293T and MCF-7 cells and can be internalized into the MCF-7 cells. The nanogels will find applications in protein delivery.

KEYWORDS: bovine serum albumin, nanogels, thiol-disulfide exchange reaction, core-shell structure


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Introduction In recent years, protein delivery has attracted widespread attention for providing a general method of transporting therapeutic proteins to specific cells or organs in living organisms.1-8 Various types of nanoparticles including micelles,9,10 vesicles11,12 and nanogels13-20 have been utilized as carriers to deliver biologically active proteins and protect the proteins from aggregation and inactivation. Among the delivery nanoparticles, nanogels, a type of nanosized network structures, have many advantages. For example, in comparison with other nanoscaled self-assemblies, nanogels are more stable and have longer blood circulation time. Therefore, different methods including physical entrapment16-19 and noncovalent interaction20 have been developed to incorporate proteins into nanogels. However, previous researches usually make use of the nanogels simply as vehicles; environmental stimuli, such as pH or temperature change, only induce the swelling or shrinking of the nanogels, but do not result in structural or functional changes significantly.21,22 In contrast to the synthetic nanoparticles, natural nanoparticles such as virus are endowed with “smart” structures, which can change their conformations responding to the external stimuli. For example, adeno-associated virus (AAV) is a nonenveloped and nonpathogenic human virus with a size of 25 nm, whose capsid is able to undergo dynamic conformational change upon exposure to the external stimuli. Specifically, upon limited heat shock, the N termini of VP1 and VP2 of AAV, which are inside the capsid lumens before heating, become exposed to the medium. Such conformational change is also found for many other parvoviruses and this property plays a major role in infectivity. 23 There has been an approach to reprogramming AAV to enable the surface exposure of metal binding motifs upon heating.24 Nevertheless, the source of such virus is


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limited and the bio-modification of the structures is difficult. Reprogramming virus provides an attractive way but the cost is much higher as compared with the synthetic polymeric systems. In this research, we report the fabrication of a new type of polymer-protein biohybrid nanogels, which are capable of undergoing conformational change upon heating. Bovine serum albumin (BSA), abundant in plasma and possessing well-studied structure, was used as a model protein. The nanogel was synthesized by crosslinking poly(di(ethyleneglycol) methyl ether methacrylateco-2-(2-pyridyldisulfide) ethylmethacrylate) (Poly(DEGMA-co-PDSM)) with reduced BSA. The random copolymer is temperature responsive and the crosslinking reaction is conducted at a temperature below the lower critical solution temperature (LCST) of the polymer. The synthesis of the polymer is shown in Scheme 1a. The synthesized nanogel is also temperature responsive. Below the critical temperature of the nanogel, reduced BSA is entrapped inside the network structures. However, at a temperature above the critical temperature, BSA molecules move to the surface (Scheme 1b). The synthesized nanogels with capability of structural change in responding to the thermal stimulus can be regarded as a mimic of virus capsid. Another feature of the nanogels synthesized in this research is the introduction of cleavable covalent bonds into network structures. In previous researches, the crosslinking reaction led to irreversible conjugation of proteins to polymer scaffolds which is not suitable for releasable protein delivery.25,26 Physical encapsulation of proteins or noncovalent incorporation of proteins into nanogels

27,28

may suffer the leakage or incomplete protection of proteins. In this research,

cleavable disulfide bonds are employed to connect proteins to polymer scaffolds so that protein molecules can be released under reduced conditions.29,30 Considering the protection of protein functionality and the potential applications of nanogels in vivo, the synthesis of nanogels is conducted in phosphate buffer (PB) without any surfactant or


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organic solvent. The multifunctional nanogels can be synthesized by simply mixing polymer and BSA solutions, which offers possibility for mass production. In this research, the cytotoxicity and cellular uptake are investigated to evaluate the potential applications of the nanogels in protein delivery.

Scheme 1. (a) Outline for the synthesis of random copolymers with pendant pyridyldisulfide groups, and (b) schematic illustration of the synthesis of protein nanogels and the structural transition of the biohybrid nanogels above the lower critical solution temperature (LCST).

Experimental Section Materials: Methacryloyl chloride (TCI, 80%) and 2-mercaptoethanol (Tianjin Guangfu Fine Chemical Company, 98%) were distilled under reduced pressure before use. Di(ethylene glycol) methyl ether methacrylate (DEGMA, Aldrich, 95%) was purified by passing through a basic aluminum oxide column. 2,2'-Azobis(isobutyronitrile) (AIBN, Guo Yao Chemical Company, 98%) was purified by recrystallization from ethanol. Fluorescein isothiocyanate (FITC, Aldrich, 95%), 2,2'-dipyridyl disulfide (DPDS, Alfa Asear, 98%), tris(2-carboxyethyl)phosphine


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hydrochloride (TCEP, Aldrich, 98%), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, Alfa Asear, 99%), hexylamine (Aladdin, 99%), 4-nitrophenyl acetate (NPA, Alfa Asear, 98%), bovine serum albumin (BSA, Genview, 96%) and 4',6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, CA) were used as received. Dulbecco’s modified Eagle Medium (DMEM) was obtained from Gibco Invitrogen and used as received. 4-(Cyanopentanoic acid)-4-dithiobenzoate (CPADB), was synthesized according to the procedure described in previous literature. 31 All the solvents used in the chemical reactions and polymerizations were distilled before use. Characterization: 1

H NMR spectra were recorded on a Varian UNITY-plus 400 spectrometer. The apparent

molecular weight (Mn) and the molecular weight distribution (Mw/Mn) of the polymers were determined on a gel permeation chromatograph (GPC) equipped with a Hitachi L-2130 HPLC pump, a Hitachi L-2350 column oven operated at 40 °C, three Varian PL columns with 1000K100K (100 000 Å), 100K-10K (10 000 Å), and 100-10K (1000 Å) molecular ranges, and a Hitachi L-2490 refractive index detector. DMF was used as eluent at a flow rate of 1.0 mL/min. The apparent molecular weight was calculated based on PMMA standards. TEM images were obtained on a Tecnai G2 F20 electron microscope at an operating voltage of 200 kV. Formvar and carbon sequentially coated copper EM grids were used in the TEM observations. To prepare the specimen of nanogels below the transformation temperature, the diluted nanogel solutions were deposited on the copper grids at 10 ºC, and dried at 10 ºC. To prepare the TEM specimens of nanogels above the critical temperature, the temperature in the preparation of the specimens was controlled at 50 ºC. The dynamic light scattering and Zeta-potential measurements of nanogels were performed on a Malvern Zetasizer Nano-ZS equipped with a 10 mW He-Ne laser (633 nm) at an angle of 90°. The results were analyzed in CONTIN mode. Z-average sizes (Dh,z)


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were calculated according to the Stokes−Einstein equation, and poly-dispersities were obtained via the cumulant analysis. UV-visible absorption spectra were collected on a Shimadzu UV-2450 spectrometer. Circular dichroism (CD) measurements were carried out on a JASCO J-715 CD instrument to determine the secondary structures of the proteins. The fluorescent images were obtained on a senior upright microscope (Zeiss Axio Imager Z1). Synthesis of pyridyldisulfide ethyl methacrylate (PDSM) PDSM monomer was synthesized according to a previous method.32 Hydroxyethylpyridyl disulfide was synthesized by adding a solution of 2-mercaptoethanol (1.40 g, 18.0 mmol) in 16 mL of methanol to a mixture of DPDS (6.00 g, 27.2 mmol), glacial acetic acid (0.55 mL) and methanol (38.0 mL), and the solution was stirred at room temperature overnight. The crude product was purified by passing through a silica gel column. The yield was 67.2%. 1H NMR spectrum of hydroxyethylpyridyl disulfide is shown in Figure S1. 1H NMR: (400 MHz, CDCl3, δ) ppm: 8.54 (m, 1H, aromatic proton ortho-N), 7.60 (m, 1H, aromatic proton meta-N), 7.42 (m, 1H, aromatic proton para-N), 7.17 (m, 1H, aromatic proton, ortho-disulfide linkage), 3.83 (t, 2H, -S-S-CH2CH2OH), 2.97 (t, 2H, -S-S-CH2CH2OH). Hydroxyethylpyridyl disulfide (2.19 g, 11.7 mmol), triethylamine (2.30 mL, 16.0 mmol) and dichloromethane (15.9 mL) were added into a flask and cooled in an ice-bath. Methacryloyl chloride (1.56 mL, 16.0 mmol) in 10 mL of dichloromethane was added dropwisely into the flask with continuous stirring. After the addition, the solution was stirred at room temperature for 24 h and was filtered to remove the solids. The filtrate was washed with NaCl solution and distilled water subsequently, and dried over anhydrous MgSO4. The solution was filtered and concentrated on a rotary evaporator. The crude product was purified by passing through a silica gel column. Yield: 51.7%. 1H NMR spectrum of PDSM is shown in Figure S2. 1H NMR (400


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MHz, CDCl3, δ) ppm: 8.50 (m, 1H, aromatic proton ortho-N), 7.70 (m, 1H, aromatic proton meta-N), 7.64 (m, 1H, aromatic proton para-N), 7.11 (m, 1H, aromatic proton, ortho-disulfide linkage), 6.13 (d, 1H, vinylic proton, cis-ester), 5.60 (d, 1H, vinylic proton,trans-ester) 4.41 (t, 2H, -S-S-CH2CH2O-), 3.10 (t, 2H, -S-S-CH2CH2O-), 1.94 (s, 3H, -CH3). Synthesis of random copolymer poly(di(ethylene glycol) methyl ether methacrylate-copyridyldisulfide ethylmethacrylate) (poly(DEGMA-co-PDSM)) Poly(DEGMA-co-PDSM) copolymer was synthesized by the reversible addition-fragmentation chain transfer (RAFT) polymerization. Briefly, DEGMA (1.20 mL, 6.51 mmol), CPADB (9.0 mg, 0.033 mmol), PDSM (55.3 mg, 0.218 mmol) and AIBN (0.9 mg, 0.005 mmol) were dissolved in 1.0 mL of DMF in a Schlenk flask. The mixture was degassed by three freezepump-thaw cycles, and stirred at 65 ºC for 8h. The reaction was stopped by exposure to air. The copolymer was precipitated in diethyl ether for three times and dried under reduced pressure. The number-average molecular weight and the dispersity of the polymer as determined by GPC are 10.0 kDa and 1.18, respectively. The GPC trace of the copolymer is shown in Figure S3. Removal of RAFT CTA at the ends of poly(DEGMA-co-PDSM) chains with hexylamine in the presence of DPDS Poly(DEGMA-co-PDSM) (50 mg, 0.0025 mmol) was dissolved in 1.5 mL of DMF with DPDS (5.5 mg, 0.025 mmol). The mixture was degassed by one freeze-pump-thaw cycle and hexylamine (7.6 mg, 0.075 mmol) was added into the mixture under argon atmosphere. After 5h reaction, the mixture was concentrated on a rotary evaporator, and the polymer was precipitated in diethyl ether for three times and dried under reduced pressure. The number-average molecular weight and the dispersity of the polymer are 9.88 kDa and 1.24, respectively. Preparation of Reduced BSA


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BSA (100 mg, 0.00150 mmol) was dissolved in 10 mL of phosphate buffer (pH=8.0) and the solution was bubbled with argon gas for 30 min. TCEP (4.3 mg, 0.020 mmol) was added into the solution under argon atmosphere. The reaction was conducted at room temperature for 4h, and after the reaction the solution was dialyzed against deionized water under argon atmosphere to remove TCEP. Reduced BSA was obtained after freeze-drying. In order to determine the average number of thiol groups on a reduced BSA molecule, BSA (6.0 mg, 9.0E-5 mmol) was dissolved in 2.5 mL of phosphate buffer (pH=8), and 50 µL of DTNB solution (4 mg/mL) was added into the solution. After 15 min stirring, the mixture was scanned on a UV-vis spectrometer. UV-vis results indicated that there were about six thiol groups on a reduced BSA molecule. Synthesis of nanogels Nanogels with different crosslinking degrees were synthesized at different polymer concentrations. Briefly, different volumes of BSA solutions (0.25 mM) were added dropwisely into the degassed polymer solutions under argon atmosphere at 10 ºC. The reaction was monitored by UV-vis. Preparation of FITC-labeled nanogels FITC (2.0 mg, 0.0051mmol) was dissolved in 0.51 mL of DMF. FITC solution (10 µL), phosphate buffer (pH=8, 0.8 mL) and nanogel solution (99.0 µL) were added into a Schlenk flask and the mixture was stirred for 4h at room temperature. After the reaction, the solution was dialyzed against phosphate buffer (pH=7) to remove the excess FITC. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)


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SDS-PAGE was performed with 14% polyacrylamide gels. Electrophoresis was carried out at 80 V voltage 16 mA current for 3.5 h. Staining was accomplished by using Coomassie Brilliant Blue R-250 solution. In vitro cytotoxicity assays In vitro cell viabilities of the three nanogel samples on MCF-7 cancer cells and nanogel H-1 on 3T3 and 293T cells were evaluated. Cells were seeded in 96-well plates at a density of 5.0×103 cells/well and cultured at 37 ºC under 5% CO2 for 24 h. The nanogel solutions were diluted with medium to reach specific concentrations ranging from 10 to 200 µg/mL. The diluted nanogel solutions with different concentrations were added into 96-well plates (100 µL/well). After 48 h incubation, the culture media were removed and 10 µL of CCK-8 and 90 µL of fresh medium were added to each well. After 30 min, the absorbance values of the solutions were measured by a multifunctional microplate reader (ELISA, Thermo Variskan Flash) and the cell viabilities were calculated using the following formula:

cell viability(%) =

A (sample) - A (blank)） × 100% A（(control) — -A (blank )

Where A(Sample) and A(Control) represent the absorbance of CCK-8 regents determined for cells treated with different samples and for control cells (untreated), respectively, and A(Blank) is the absorbance of CCK-8 regents without cells. Cell uptake assay MCF-7 cells were used to evaluate the cell uptake. The suspensions of MCF-7 cells (2.5×105 cells/well) were seeded on cover glasses (Microscope, USA) in 6-well plates and cultured for 24 h in DMEM containing 10% fetal bovine serum and penicillin/streptomycin at 37 ºC in 5% CO2 medium. After that, 2 mL solutions of DMEM medium with FITC-labeled nanogels (50 µg/mL) were added into the plates and cultured for 6 h. The cells were ﬁxed with 4% formaldehyde for


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30 min at 37 ºC and washed with PBS for three times. Cell nuclei were stained with DAPI. Cell uptake was observed on a senior upright microscope.

Results and Discussion Random copolymer of DEGMA and PDSM, poly(DEGMA-co-PDSM), was prepared by RAFT polymerization. It is well known that PEG analogues composed of multiple oligo(ethylene glycol) segments are biocompatible and their solubility in water depends on the repeating unit number of ethylene glycol on the side chains.33 The oligo(ethylene glycol) methyl ether methacrylate monomer with two ethylene glycol repeating units was employed in this research, and PDSM was used as comonomer. Pyridyldisulfide groups on PDSM are able to quantitatively react with thiol groups on the reduced BSA resulting in the formation of disulfide bonds and the production of pyridine-2-thione. In the meanwhile, the amount of pyridine-2-thione produced in the reaction can be quantified by UV-vis measurement. It has been reported that PDSM homopolymer could react with various mercapto-compounds including mercaptopropionic acid, 4-mercapto-1-butanol,

11-mercaptoundecanol,

and

reduced

L-glutathione.32

RAFT

polymerization of DEGMA and PDSM yielded well-defined poly(DEGMA-co-PDSM) random copolymer. As shown in Figure S3, GPC trace of poly(DEGMA-co-PDSM) shows symmetric and monomodal elution peak, and the apparent number-average molecular weight and the dispersity of the polymer are 10.0 kDa and 1.18, respectively. The 1H NMR spectrum and signal assignments for the random copolymer are presented in Figure S4. Based on 1H NMR result the average repeating unit numbers of DEGMA and PDSM on a polymer chain are 102 and 4, respectively. The randomcopolymer is assigned as poly(DEGMA102-co-PDSM4). The dithiobenzoate moieties at the ends of poly(DEGMA102-co-PDSM4) were reduced to thiol groups by aminolysis and subsequently the thiol end-groups were capped with 2,2’-


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dipyridyldisulfide (DPDS). After the two-step reactions, pyridyldisulfide (PDS) groups terminated polymer chains were obtained.34 The resulting polymer is denominated as poly(DEGMA102-co-PDSM4)-s-s-py. 1H NMR spectrum of the polymer is shown in Figure 1. After the reaction, the signal at δ=7.5 ppm corresponding to RAFT end-groups disappeared and a group of new peaks at δ=7.1, 7.7, 8.4 ppm representing the terminal PDS group were observed. The integration of the signals indicates that the terminal RAFT agents were converted completely into PDS groups. The polymers before and after the removal of RAFT agents were also analyzed by UV-vis and GPC. UV-vis spectra of poly(DEGMA102-co-PDSM4), poly(DEGMA102-co-PDSM4)-s-s-py and RAFT agent 4-(cyanopentanoic acid)-4-dithiobenzoate (CPADB) are shown in Figure 2a. The UV spectrum of poly(DEGMA102-co-PDSM4) possesses a shoulder peak at 305 nm due to the presence of terminal RAFT agents. However, after the reaction with DPDS no shoulder peak can be observed at 305 nm and only absorption at 272 nm assigning to the pyridyl groups is observed. The GPC traces of poly(DEGMA102-co-PDSM4) and poly(DEGMA102-co-PDSM4)-s-s-py are shown in Figure 2b. The apparent molecular weight and the dispersity of poly(DEGMA102-co-PDSM4)-s-s-py are 9.88 kDa and 1.24, respectively. In comparison to poly(DEGMA102-co-PDSM4), GPC trace of poly(DEGMA102-co-PDSM4)-s-s-py moves slightly to the low molecular weight part due to the change of the end groups. The narrow distribution of the GPC trace demonstrates that no intermolecular reactions occurred in the reactions.


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Figure 1. 1H NMR spectrum of poly(DEGMA102-co-PDSM4)-s-s-py obtained in CDCl3.


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Figure 2. (a) UV-vis spectra of poly(DEGMA102-co-PDSM4) (curve 1), poly(DEGMA102-coPDSM4)-s-s-py (curve 2) and RAFT agent CPADB (curve 3). (b) GPC traces of poly(DEGMA102-co-PDSM4) (solid line) and poly(DEGMA102-co- PDSM4)-s-s-py (dash line).

Homopolymer PDEGMA exhibits a LCST at 26 ºC.35 The introduction of pyridyl groups to the polymer exerts significant influence on the critical temperature. The change of the scattering intensity as a function of the temperature measured by dynamic light scattering (DLS) at 90° was used to determine the LCST of the random copolymer (Figure 3a). At a temperature below 18 ºC, the low scattering intensity suggests the random copolymer is fully dissolved in water. Above 18 º

C the scattering intensity increases rapidly with temperature, indicating the collapse of the

polymer chains. The LCST of the copolymer defined as the inflection point of the curve was determined to be around 18 ºC.36 BSA contains only one free cysteine (Cys-34) which is usually partially oxidized resulting in about 50% cysteine available,37 so reduced BSA was employed as cross-linkers to fabricate nanogels. Disulfide bonds on BSA were cleaved with TCEP, and as determined by 5,5'dithiobis(2-nitrobenzoic acid) (DTNB) one reduced BSA molecule carries 6 thiol groups on average. It is well known that disulfide bond plays an important role in maintaining the secondary structure of a protein molecule, so far-UV circular dichroism (CD) measurements on the native and the reduced BSA were performed to determine the effect of the reaction on the change of the secondary structure. In the α-helix structure of BSA, a negative band near 220 nm is observed and a transition at 190 nm is split into a negative band near 208 nm and a positive peak near 192 nm.38,39 The CD spectra of native BSA and reduced BSA are presented in Figure S5, where the positive peak at 192 nm and the two negative peaks at 208 and 220 nm are


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observed. The CD spectrum of the reduced BSA is very similar to that of the native BSA, which demonstrates that the reduced BSA preserves most of the structure integrity. In order to prepare nanogels, poly(DEGMA102-co-PDSM4)-s-s-py was dissolved in PB (pH=8) at 10 ºC to ensure the complete dissolution of the polymer. Thiol-disulfide exchange reactions between poly(DEGMA102-co-PDSM4)-s-s-py and the reduced BSA lead to the formation of nanogels. Reaction conditions and structural parameters of three different nanogels are listed in Table 1. The three nanogels were prepared at different initial concentrations or different polymer/protein weight ratios. The degrees of the exchange reactions can be calculated by monitoring the absorption of pyridine-2-thione produced in the reactions.40 Our calculation results indicated that the exchange reactions were complete. The UV-vis spectra of the three nanogel solutions are shown in Figure S6. BSA with an isoelectric point at 4.7 carries negative charges in neutral water. In aqueous solution at pH=7.0, the ζ-potential of BSA was measured to be around -13.7 mV. The ζ-potentials of H-1, H-0.6 and H-1-0.5 nanogels measured at 25 ºC were -17.0，-17.9 and -17.7 mV, which demonstrated the loading of BSA molecules inside the neutral PDEGMA nanogels. Table 1. Preparation conditions and structural parameters of three different nanogels Sample a

H-1

H-0.6

Poly(DEGMA102-co-PDSM4)-s-s-py [mg]

4.0

4.0

4.0

Reduced BSA [mg]

6.7

6.7

4.2

Volume of PB [mL]

1.0

0.6

1.0

Dh,z at 10°C [nm]

164

143

209

PDIs of nanogels b

0.242

0.303

0.202

ζ-Potential [mV]

-17.0

-17.9

-17.7

H-1-0.5


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α-helix content of BSA [%] a

55

54

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The nanogels were prepared by adding different volumes of BSA solutions (0.25 mM) into

the degassed polymer solutions under argon atmosphere at 10 ºC. b PDIs were obtained by DLS.

In a previous research, Dong and coworkers reported that LCST of a PDEGMA microgel is lower than its linear analogue.41 In order to determine the LCSTs of the nanogels cross-linked with BSA molecules, the changes of the scattering intensities of the nanogels with temperature were recorded. As shown in Figure 3a, the scattering intensities of H-1 and H-0.6 increase rapidly with temperature and LCSTs are determined at 35 and 31 ºC, respectively. OʼReilly and coworkers prepared bioconjugates of poly[(oligo ethylene glycol) methyl ether methacrylate] (POEGMA) and green fluorescent protein, and found that LCSTs of the bioconjugates were higher than that of POEGMA.42 They attributed the temperature shift to the protein molecules serving as hydrophilic end groups. In this research, the bonding of the reduced BSA to poly(DEGMA102-co-PDSM4)-s-s-py scaffolds increases the hydrophilicity of the polymer, leading to higher LCSTs. Because H-1 nanogel has almost the same composition as H-0.6, the LCSTs of the two nanogels are close. In H-1-0.5, there are less BSA molecules in the networks, the LCST of H-1-0.5 is determined to be at 23 ºC (Fig. 3a), which is lower than those of H-1 and H-0.6. To investigate the effect of the preparation conditions on the size of the nanogels, H-1, H-0.6 and H-1-0.5 were measured by DLS at a temperature below the LCSTs of the three nanogels. Figure 3b compares the correlation coefficient vs time, where the mean decay time is related to the average size of the nanogels. It is clear that H-0.6 relax more rapidly than H-1, and H-1 relax more rapidly than H-1-0.5, which indicate that among the three nanogels, H-1-0.5 has the biggest size and H-1 has the smallest size. As shown in Figure 3c, all the three nanogels possess


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unimodal size distributions. The Z-average sizes (Dh,z) and the polydispersity indices of H-1, H0.6 and H-1-0.5 are 164.5 nm, 0.242, 143.6 nm, 0.303 and 209.0nm, 0.202, respectively. In the synthesis of H-1 and H-0.6, the weight ratios of poly(DEGMA102-co-PDSM4)-s-s-py/BSA are the same, but the initial concentrations of the polymer and BSA are different. At a higher initial concentration, BSA molecules are fixed in the network structures very quickly due to the rapid thiol-disulfide exchange reaction, forming compact nanogel structures. So the Z-average size of H-0.6 is smaller than H-1. H-1-0.5 prepared at low BSA content has a very loose structure resulting in the biggest size among the three nanogels.


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Figure 3. (a) Scattering light intensities of poly(DEGMA102-co-PDSM4)-s-s-py aqueous solution at a concentration of 0.3 mg/mL (■), H-1 (□), H-0.6 (▲) and H-1-0.5 (△) nanogels as a function of temperature, (b) Correlation functions of H-1, H-0.6 and H-1-0.5 nanogels measured by DLS at 10℃.(c) Intensity-weighted size distributions of H-1, H-0.6 and H-1-0.5 nanogels measured at 10℃.


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The influence of temperature on the morphologies of the nanogels was investigated on a transmission electron microscope (TEM). Before TEM measurements, specimens were stained in OsO4 atmosphere and BSA molecules in the nanogels were stained. Figure 4a shows a TEM image of H-1 prepared at 10 ºC, where spherical nanogels with sizes in the range from 140 to 300 nm are observed. A magnified TEM image is shown in Figure 4b. In the image nanogels with dark cores and grey shells can be observed, which indicates that BSA molecules are not evenly distributed in the nanogels. There are more BSA molecules in the cores and less BSA in the shells. In the synthesis of the nanogels, BSA molecules with six free thiol groups were fixed in the network structures very quickly, and further reactions resulted in the grafting of polymer chains to the BSA molecules in the cores and the formation of the polymer shells around the nanogels. The process is illustrated in Scheme 2.

Scheme 2. Schematic illustration of the formation of nanogels with core-shell-like structures.

Figure 4c,d show two TEM images of H-1 prepared at 50 ºC, a temperature well above the LCST of the nanogels. In contrast to the nanogels prepared at 10 ºC, inversed core-shell structures with BSA in the shells are observed. At a temperature above the phase transition temperature of poly(DEGMA102-co-PDSM4), the polymer chains collapse forming the cores of the nanogels, while water-soluble BSA molecules migrate to the outer layer to stabilize the


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nanogels forming the dark shells around the collapsed PDEGMA cores. Such core-shell structures could also be found for H-0.6 and H-1-0.5 at 50 ºC (Figure S7). Based on TEM results, the sizes of H-1 nanogels prepared at 50 ºC are in the range from 40 to 130 nm, which are smaller than the nanogels prepared at 10 ºC due to the collapse of the polymer chains.

Figure 4. TEM images of nanogel H-1 at 10 ºC (a,b) and 50 ºC (c,d).

In this research, ζ-potential measurements of nanogels at 10 ºC and 50 ºC were also conducted to give further insight to the structure change. The ζ-potential of H-0.6 measured at 50 ºC is -24.6 mV, which is 6.7 mV lower than the nanogel measured at 10 ºC. However, when the temperature is raised back to 10 ºC, the ζ-potential reached -18 mV, very close to the initial value. The switching cycles of ζ-potential are shown in Figure 5. After each cycle, the ζ-potential of the nanogel remains relatively constant. At 10 °C, BSA molecules locate in the inner part of the nanogel with ζ-potential at about -18 mV. Upon heating to 50 °C, BSA molecules move to the


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shells of the nanogel particles, resulting in a change of surface charges and a decrease in ζpotential from around -18 mV to -24 mV. This result demonstrates that the morphology transition of the nanogels is reversible.

Figure 5: ζ-Potential values of nanogel H-0.6 during thermal cycling between 10 °C and 50 °C.

To study the conformation change of BSA in the fabrication of the nanogels, the secondary structures of BSA in the nanogels were determined by CD spectroscopy. The CD spectra of native BSA and the three nanogels are shown in Figure 6a. The CD curve of the native BSA is similar to those of BSA in the nanogels. The calculated α-helix contents of native BSA, H-1, H0.6 and H-1-0.5 are 57%, 55%, 54% and 52%, respectively, which indicates that BSA molecules basically maintain their conformations after the formation of the nanogels. It is reported that


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conformational integrity is necessary for preservation of the esterase-like activity of BSA. 37, 43 The change of protein conformation leads to a change of orientation of 4-nitrophenyl acetate (NPA) binding site relative to the reactive tyrosine residues. 44 There are several researches about the reactions between polymer-modified BSA and NPA to determine the esterase-like activity and the conformational change of the modified BSA.

45-48

Herein, the hydrolysis of NPA by

native BSA and BSA in the nanogels was tested to determine the esterase-like activity of the nanogels. The esterase-like activities of BSA in the nanogels were obtained by measuring the absorbances of 4-nitrophenolate anion at 405 nm on an UV-vis spectrometer, and the esteraselike activity of native BSA was set as 100%. Our results indicate that the esterase-like activities of BSA in all the three nanogels are about 90% (Figure 6b). Therefore, it can be concluded that BSA in the nanogels prepared under different conditions maintain most of their structures, which is in accordance with the CD results. .


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Figure 6. (a) CD spectra of native BSA, H-1, H-0.6 and H-1-0.5; (b) esterase-like activities of native BSA, reduced BSA, H-1, H-0.6 and H-1-0.5. The activities were measured in buffer solution, and hydrolysis of 4-nitrophenyl acetate in the buffer solution (PB) is also shown in (b).

The disulfides are readily reduced to thiols within a reducing environment resulting in the cleavage of the disulfide bonds.49,50 In addition to the thermal-response provided by PDEGMA chains, the nanogels crosslinked by disulfide bonds are dissociated upon exposure to reductive environments. Herein, the effect of reductant on the size of nanogels is investigated. Figure 7


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shows DLS curves of H-1 after treatment in glutathione solution for different time. The sizes of the native nanogels are in the range from 70 to 400 nm. After 1, 3 and 21 h treatments in glutathione solution at low concentration (44 µM), the size ranges are 35-800 nm, 36-1050 nm and 20-800 nm, which indicates that DLS curve gets broader with treatment time (Figure 7a). Glutathione is able to cleave the disulfide bonds in the nanogels, resulting in the dissociation of the nanogels; on the other hand, thiol groups produced in the cleavage of the disulfides can be oxidized back into disulfides,41 which leads to the association of the nanogels. So the size distribution of the nanogels becomes broader after treatment with glutathione. Figure 7b shows DLS curves of H-1 after treatment in glutathione solution at a high concentration (10 mM) for different time. Upon treatment with glutathione at a high concentration, the nanogels have a bimodal distribution. After 20 min, two peaks, one at about 70 nm and the other at about 280 nm, can be observed. The appearance of the peak at 70 nm is attributed to the cleavage of the disulfides and the dissociation of the nanogels. The oxidation-induced association of the nanogels is responsible for the shift of the scattering peak to the bigger size part. With an increase in the treatment time, more nanogels are dissociated and the scattering peak corresponding to the smaller particles gets stronger. In the meanwhile, nanogels with thiols on the surface are associated due to the oxidation of the thiols, which is responsible for the enhanced scattering intensity of the large gel particles.


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Figure 7. (a) DLS results of H-1 nanogels after treatment in glutathione solution at a low concentration (44 µM) for different time, (b) DLS results of H-1 nanogels after treatment in glutathione solution at a high concentration (10 mM) for different time.


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The biohybrid nanogels synthesized in this research have temperature- and redoxresponsiveness. At a low temperature, protein molecules stay inside the nanogels and are protected by the polymer chains. Above the critical temperature, the polymer chains collapse forming the cores of the nanogels and the protein molecules move to the shells of the nanogels. The protein covered nanogels are able to dissociate with glutathione. When internalized by cells, the protein-covered nanogels will dissociate with glutathione in the cells and the protein molecules in the nanogels are released. The release of BSA after cleavage of the disulfide bonds of the nanogels was demonstrated by SDS-PAGE results (Figure S8). After the reduction of disulfide bonds, the SDS-PAGE analysis of nanogel H-1 showed a light band at 66 kDa, demonstrating that BSA molecules were released after cleavage of the disulfide bonds. Herein, the cytotoxicity and cellular uptake are investigated to evaluate the potential application of the nanogels as “smart” delivery carriers.


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Figure 8. (a) Cytotoxicity assays of H-1, H-0.6 and H-1-0.5 incubated with MCF-7 cells for 48 h at different concentrations. (b) Cytotoxicity assays of H-1 incubated with 3T3, 293T and MCF-7 cells respectively for 48h at different concentrations.

The cytotoxicity of the nanogels towards MCF-7 cells was examined by CCK-8 assay. After incubated with nanogels for 48 h, most cell viabilities are still greater than 90% even at the nanogels concentration up to 200 µg/mL (Figure 8a), which demonstrate that all the three nanogels are essentially low toxicity towards MCF-7 cells. Moreover, the cytotoxicities of nanogel H-1 towards normal cells 3T3 and 293T cells were also conducted. As shown in Figure 8b, H-1 shows low toxicity to 3T3 and 293T cells at the concentrations up to 200 µg/mL. To investigate the cellular uptake and intracellular distribution of the nanogels, fluorescein isothiocyanate (FITC) was reacted with H-1 to label BSA. MCF-7 cells were incubated with FITC-labeled nanogels (H-1-FITC) for 6 h and observed on a fluorescence microscope. Figure


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9a shows a fluorescence image of MCF-7 cells with FITC-labeled nanogels, Figure 9b shows an image of the cells whose nuclei were stained with 4',6- diamidino-2-phenylindole (DAPI), and Figure 9c represents an overlay image of (a) and (b). As shown in Fig. 9a,c, strong fluorescence of nanogel can be observed in the cytoplasm of MCF-7 cells, which indicates the uptake of the FITC-labeled nanogels into the cells. The efficient uptake of H-1 is attributed to the specific structure of the nanogels with BSA molecules in the coronae. It has been previously reported that the uptake of nanoparticles with protein coronas is completed by receptor-mediated endocytosis process arising from cellular recognition of proteins.51,

52

BSA has at least four known cell

surface receptors that can independently bind and be endocytosed into cells,53 so BSA on the coronae of H-1 nanogels facilitate the uptake of nanoparticles via receptor-mediated endocytosis process. Examining the fluorescence images very carefully (Fig. 9a,c), we can find that the nanogels associate inside the MCF-7 cells. Typical aggregates are indicated by arrows in Fig. 9a,c. We speculate that similar to the previous DLS experiments (Fig. 7), the oxidation of the thiols produced in the cleavage of the disulfides by glutathione is responsible for the association of the nanogels inside the MCF-7 cells.


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Figure 9. Fluorescence micrographs of MCF-7 cells incubated with H-1-FITC. (a) cells with FITC-labeled nanogels, (b) cells with DAPI-stained cell nuclei , (c) overlays of (a) and (b).

Conclusion In conclusion, we have synthesized “smart” nanogels by using thermoresponsive polymers as scaffolds and BSA as crosslinkers. The nanogels are able to reversibly switch the structures and adjust the relative position of BSA molecules. At a temperature below LCST of the nanogels, protein molecules stay inside the nanogels and are protected by the polymer chains. Above the critical temperature, the polymer chains collapse forming the cores of the nanogels and the protein molecules move to the surfaces. BSA molecules in the nanogels maintain most of the structure integrity and bioactivity. Moreover, after treatments in glutathione solution the nanogels undergo dissociation and association due to the cleavage of the disulfides and the


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oxidization of thiols on the surface. The nanogels with cytocompatibility and high loading efficiency can be used as a new platform for protein delivery. The unique reversible structure of the nanogels in response to the temperature change makes virus-imitating vehicle possible. It is expected that pharmaceutical proteins can be included in the nanogel system to offer biomedical applications. Such researches are being conducted in this laboratory now.

ASSOCIATED CONTENT Supporting

Information.1H

NMR

spectrum

of

hydroxyethylpyridyl

disulfide

and

pyridyldisulfide ethylmethacrylate, GPC trace and 1H NMR spectrum of poly(DEGMA102-coPDSM4) random copolymer, CD spectra of native BSA and the reduced BSA, UV-Vis spectra of diluted H-1, H-0.6, and H-1-0.5 nanogel solution after two-day cross-linking reaction. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author E-mail: [email protected]; [email protected]; [email protected] ACKNOWLEDGMENT This project was supported by the National Natural Science Foundation of China (NSFC) under contracts 21174073, 51473079, the National Basic Research Program of China (973 Program, 2012CB821500), and Tianjin Research Program of Application Foundation and Advanced Technology (no.13JCYBJC25200). REFERENCES


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Protein Nanogels with Temperature-Induced Reversible Structures and Redox Responsiveness

Yue Zhang, Jiamin Zhang, Cheng Xing, Mingming Zhang, Lianyong Wang and Hanying Zhao


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Protein Nanogels with Temperature-Induced Reversible Structures

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