Reversible photo-controlled release of bovine serum albumin by azobenzene-containing cellulose nanofibrils-based hydrogel

  • Lin DaiEmail author
  • Jinshun Lu
  • Fangong Kong
  • Kefeng Liu
  • Huige Wei
  • Chuanling SiEmail author
Original Research


Development of biomass-based and some other sustainable biomedical materials is a key subject and effective approach to biomass high-value utilization. In this work, we used cellulose resources to develop a photoresponse hydrogel (PR-gel) by integrating 4arm-PEG and azobenzene into cellulose nanofibrils (CNFs). This novel PR-gel exhibited good mechanical strength (storage modulus over 103 Pa), structure stability, reversible recoverability between sustained step strain of 1% and 1000%, and excellent biocompatibility. Under UV irradiation (λ = 365 nm, 10 mW/cm2, 10 min), the azobenzene cross-linker in PR-gel as photoswitch can cause the trans-cis isomerism and a softening effect of the hydrogel, thus realized the photo-controlled release of bovine serum albumin (BSA) (5-fold higher release rate under UV light irradiation). This work provided a new approach to design cellulose-based photoresponsive hydrogels. It is also can expand the application of cellulose-based hydrogels and some other sustainable materials in the biomedical field.

Graphical abstract

Cellulose nanofibrils-4arm-polyethylene glycol-azobenzene (PR-gel) exhibited good mechanical strength, reversible recoverability, excellent biocompatibility, and reversible photoresponse for protein release.


Cellulose nanofibrils Hydrogel Azobenzene Photoresponse Protein release 

1 Introduction

Cellulose is one of the most abundant renewable materials, while is full of possibilities in agricultural and forestry waste. Over the years, a new kind of cellulose-based nanomaterial, cellulose nanofibrils (CNFs), was produced and developed. Benefiting from their good characteristics, such as high aspect ratio of nanosized, impressive biocompatibility, outstanding hydrophilicity, and tunable surface chemistries, CNFs can be a kind of fascinating building blocks for various composite materials [1, 2, 3, 4]. In addition, CNFs normally are viscous and shear thinning hydrogel-like materials at a very low concentration. Therefore, they are also becoming an appealing series of biomaterials for drug delivery and wound healing [5, 6]. Although some remarkable works on CNFs-based hydrogels have been reported, the poor mechanical properties of CNFs are still hard to reach the standard of carrier materials. On the other hand, with the improvement of precision medicine, proteins and drugs should be released controllable and tailored for patients’ needs. Therefore, it is necessary to develop novel CNFs-based hydrogels with functional and stimuli-responsive cross-linker for controlled release and enhancing its mechanical properties simultaneously.

Here, we took a complementary approach to modify CNFs by hydrophilic cross-linker and photosensitive molecules, thus to achieve dynamic and reversibly tunable properties with light. Azobenzene (Azo) and its derivatives, a series of the most known photosensitive compounds, are characterized more stable trans isomerism to the less stable cis isomerism upon irradiation with UV or visible light to yield a photostationary composition that is wavelength [7, 8]. The introduction of Azo is one of the most simple and effective approaches for a reversible and light-based chemistry to control the mechanics of the hydrogel. There are no free radicals from the materials during the photoreaction proceeds of Azo. Moreover, compared with other stimuli, light is low energy consumption, environment friendly, and noninvasive stimulus.

In this work, we successfully fabricated the reinforced and reversible photoresponsive CNFs-based hydrogel with Azo-containing cross-links to control the release of bovine serum albumin (BSA) with light irradiation (365 nm). The CNFs-based photoresponsive hydrogel, CNFs-4arm-polyethylene glycol-azobenzene (PR-gel), was synthesized from conjugating 4arm-polyethylene glycol (4arm-PEG) onto CNFs, and then, cross-linking with azobenzoic acid. This strategy leads to a dynamic CNFs-based hydrogel and photo-controlled release of BSA in which the changes in network structures by photo-induced isomerization of Azo. This photoresponsive CNF-based hydrogel will be a great promising carrier for protein and drug delivery. And this feasible route also presents a good example of green material design for sustainable development.

2 Experimental section

2.1 Materials

Cellulose nanofibril (CNFs, length ~ 10 μm, width ~ 15 nm, 2 wt%) suspensions were kindly provided by Tianjin Wood Elf Biological Technology Co., Ltd. (Tianjin, China). The CNFs suspension (5 g, 2 wt%) was solvent exchanged from water to acetone and then to tetrahydrofuran (THF) by using high-speed centrifugation (10,000 rpm at room temperature for 15 min) [9]. 4arm-polyethylene glycol (4arm-PEG, MW = 10 kDa, 3arm-amine, HCl Salt, 1arm-carboxyl) was purchased from Beijing JenKem Technology Co., Ltd. Azobenzene-4,4-dicarboxylic acid (purity > 95.0%) was obtained from Shanghai Aladdin Reagent Co., Ltd. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl, 98.5%) and N-hydroxysuccinimide (NHS, 98%) as catalysts for amide reaction were purchased from Shanghai Macklin Biochemical Co., Ltd. 4-Dimethylaminopyridine (DMAP) was purchased from Adamas Reagent Co., Ltd. Dimethylformamide (DMF) and tetrahydrofuran (THF) were purchased from Tianjin Jindongtianzheng Precision Chemical Reagent Factory. Acetone purchased from Tianjin Fengchuan Reagent Technology Co., Ltd. All the reagents were used as obtained without further purification.

2.2 Synthesis of CNFs-4arm-polyethylene glycol

The 4arm-PEG powder (0.36 g, 0.036 mmol) and CNFs (20 mg) were added in 10 mL THF under the nitrogen atmosphere. After completely dispersing of CNFs, the solution was immersed into an ice-water bath, and DMAP (63.5 mg, 0.52 mmol) and EDC·HCl (78.5 mg, 0.51 mmol) were added. The mixture was stirred at 0 °C for 1 h and then transferred to room temperature for about 30 h. Finally, the white precipitate products were washed and purified by tetrahydrofuran and ether solvent successively.

2.3 Synthesis of azobenzene-di-NHS ester

The substance was prepared as the previously reported method with minor modifications [10]. Simply, azobenzoic acid ((E)-4,4′-(diazene-1,2-diyl) dibenzoic acid) (0.216 g, 0.8 mmol) was dissolved in 25 mL dry dimethylformamide (DMF) at 60 °C. DMAP (0.03 g, 0.245 mmol), NHS (0.234 g, 2 mmol), and EDC·HCl (0.302 g, 1.58 mmol) were then added sequentially to the solution. The reaction was stirred at room temperature for 24 h. A little amount of deionized water was added to terminate the reaction, and the precipitation was obtained by vacuum pump filtration for three times. The light red powder product was obtained by vacuum drying at 25 °C.

2.4 Synthesis of CNFs-4arm-polyethylene glycol-azobenzene photoresponsive hydrogel

The hydrogel was synthesized following previously reported methods with minor modifications [10]. Briefly, CNFs-4arm-polyethylene glycol (CNFs-4arm-PEG) (50 mg) and azobenzene-di-NHS ester (Azo-NHS) (15 mg) were dissolved in deionized water (2 mL) and dry DMF (2 mL), respectively. Then, the two solutions were mixed together at a 1:1 volume ratio and vortexed for 30 min. The droplets disappeared after 2 h at room temperature, and di-NHS ester of azobenzoic acid (15 mg) was dissolved in dry DMF (2 mL) to make a solution, respectively. The two solutions were mixed together at a 1:1 volume ratio and vortexed for 30 min. The droplets were left in a constant temperature and humidity for 2 h at room temperature and then cured in a biochemical incubator at 37 °C for 16 h. The photoresponsive hydrogel (PR-gel) was obtained as an orange hydrogel.

2.5 Characterization

The synthetic products and hydrogel samples were dissolved in dimethyl sulfoxide-d6, (DMSO-d6, 99.9 atom %D, Sigma-Aldrich). The spectra were obtained on a (Bruker, Av 400MHz) NMR spectrometer. X-ray diffraction (XRD) patterns of thin slices were obtained by using the Cu Kα X-ray beam on a D8 Discover X-ray diffractometer (JEOL, Japan) at 40 kV and 30 mA. The spacing of MMT layers in xerogel is calculated from Bragg’s law: 2dsin θ = . Where λ is the wavelength of the incident beam, 2θ is the angle between the incident and scattered X-ray wavevectors, and n is the interference order. For SEM analysis, a JEOM JSM-IT300LV operating in high vacuum was operated. An acceleration voltage of 10.0 kV was chosen for recording the images. The hydrogel samples were first freeze-dried, cut for cross-section by a razor, and then coated with a thin layer of gold in order to avoid charging effects. Thermal gravimetric analysis (TGA) of the samples was carried out on a TGA Q500 (TA Instruments) using a temperature range from 100 to 500 °C at 10 °C/min in a nitrogen atmosphere with a flow rate of 100.0 mL/min. Absorbance spectra of the samples were conducted using Ultraviolet-Visible Spectrophotometer (UV-2550, Shimadzu, Japan) with a wavelength range of 300 to 500 nm.

2.6 Rheology measurements

The dynamic rheological properties were tested on the rheometer (MARS 60, HAAKE, Germany). Parallel plate geometry with a plate diameter of 10 mm was used. All tests except for gelation test were conducted immediately after transferring the parallel plate and the temperature of the test is set to room temperature (25 °C). The oscillating amplitude sweep of gels was tested for strain (γ) ranges from 0.1 to 100% and frequency was maintained at 6.283 rad/s (1 Hz). The oscillating frequency sweep of gels was deformed at a stress within the linear viscoelastic region by varying the frequency from 1 to 628 rad/s (0.16 to 100 Hz) at a strain of 1% to quantify the viscoelasticity of the hydrogel while monitoring the storage modulus (G′) and the loss modulus (G″). The step strain sweep tests were performed at a fixed angular frequency (6.28 rad/s). Amplitude oscillatory strains were switched from small strain (1.0% for 120s ) to subsequent large strain (1000% for 60 s) with 180 s for every strain interval.

2.7 BSA release under UV light exposure

The lyophilized PR-gel was immersed into a BSA solution (125 mg/mL), and the mixtures were left overnight in a refrigerator at 4 °C. This BSA-loaded PR-gel was stored in an enclosed transparent plastic cuvette in the fridge prior to drug release experiment. Two milliliter of PBS solution (pH 7.4, 0.1 M) was added to this BSA-loaded PR-gel, followed by incubation of the sample at 37 °C to mimic the body temperature. After 10 min, the sample was exposed to 365 nm UV light (10 mW/cm2). The exposure time was set to 10 min for every UV light exposure (“ON”). After about 10 min of “OFF” interval (blue light, 400–500 nm, 10 mW/cm2), the visible light exposure was repeated (3 times for total 70 min). The concentration of released BSA was quantified with a UV/vis spectrometry (UV2500, Shimadzu, Japan) at 280 nm. The release rates (ron or roff, in mg .ml−1 .min−1) during the presence of visible or UV light exposure can be calculated by the following equation:
$$ {r}_{\mathrm{on}\ 1\mathrm{st}}=\frac{C_{\mathrm{on}\ 1\mathrm{st}\ \mathrm{end}}-{C}_{\mathrm{on}\ 1\mathrm{st}\ \mathrm{start}}}{t_{\mathrm{on}\ 1\mathrm{st}\ \mathrm{end}}-{t}_{\mathrm{on}\ 1\mathrm{st}\ \mathrm{start}}} $$
where C and t represent the concentration and time at the start and the end of photomodulation. For instance, Con 1st start and Con 1st end are BSA concentration in the PBS solution at the start (ton 1st start, 11 min) and the end (ton 1st end, 20 min) of the UV light exposure, respectively.

2.8 Biocompatibility

The cell compatibility of PR-gel and BSA-loaded PR-gel was evaluated by the cell cytotoxicity in vitro using a Cell Counting Kit-8 (CCK-8) assay. Briefly, 1.0 mL of Dulbecco’s modified Eagle’s medium (DMEM) was added to a 96-well plate, and then 75 μL of LLC cells (3 × 103 cells/well) were seed separately. The culture plates were incubated at 37 °C with 5% CO2/95% air humidified atmosphere for 24 h. Then different samples were added and immersed the cell culture and incubated for 24 h, 48 h, and 72 h, respectively. Pure DMEM treatment was used as the control group (100% viability). After that, 20 μL of CCK-8 solution was added and continued to incubate for 1 h. The absorbance of different samples at 450 nm was detected using an infinite M200 microplate spectrophotometer.

2.9 Statistical analysis

All experiments in this study were performed at least three times, and the data were expressed as mean ± standard deviation (SD).

3 Result and discussion

The PR-gel was fabricated in three-steps by syntheses of Azo-NHS, CNFs-4arm-PEG, and PR-gel. Briefly, the photosensitive cross-linker (Azo-NHS) and modified CNFs-based hydrogel (CNFs-4arm-PEG) were synthesized by acylation reaction of azobenzoic acid and conjugation between 4arm-PEG and CNFs, respectively. Then, Azo-NHS was mixed with CNFs-4arm-PEG and bridged between PEG side chains by cross-linking reaction (Fig. 1). The obtained PR-gel has good hydrophilicity, excellent biocompatibility, enhanced mechanical properties, and photoresponsiveness.
Fig. 1

a Structures of polymers 4arm-PEG and the synthetic procedure of Azo-NHS. b Schematic illustration of the preparation procedures of PR-gel

3.1 Synthesis and characterization of PR-gel

Figure 2 a shows the 1H-NMR spectra of 4arm-PEG and CNFs-4arm-PEG, where the signals at 3.51 ppm (4nH, −(CH2CH2O)n–), 3.60 ppm (2H, –CH2O–), and 3.68 ppm (2H, –CH2COOH) were attributed to the methylene, amino terminal methylene, and carboxyl terminal methylene proton of 4arm-PEG, respectively. For CNFs-4arm-PEG, the characteristic peaks of cellulose were obtained at 3.27 ppm, 3.30 ppm, 3.35 ppm, and 3.43 ppm. Due to the formation of the ester bond, the carboxyl terminal methylene proton peak δ 3.68 (2H, –CH2COOH) of 4arm-PEG moved to δ 3.76 (2H, –CH2COO–), and the proton peak δ 3.35 at C6 of cellulose moved to δ 3.84. This result was confirmed as the successful synthesis of CNFs-4arm-PEG.
Fig. 2

a1H-NMR spectra of 4arm-PEG and CNFs-4arm-PEG in DMSO-d6. b FT-IR. c TGA. d XRD spectra of CNFs, CNFs-4arm-PEG, and PR-gel

FT-IR measurement was used to further characterize the structure of PR-gel. As shown in Fig. 2b, the characteristic peaks at 3407 cm−1 and 665 cm−1 corresponded to the stretching vibration and deformation vibration of hydroxyl groups, respectively. The peaks at 2900 cm−1 correspond to the symmetric stretching vibration of methylene on cellulose [11], which indicated that the cellulose structure was retained in all three samples. The weak peak at 1732 cm−1 of carbonyl groups in CNFs-4arm-PEG could be due to the steric hindrance effect between cellulose and PEG chains, while the sharp peak observed at 1735 cm−1 in PR-gel could be attributed to the superimposed effect of the carbonyl groups stretching vibration in amide bonds. Differentiation of peak strength resulted from the molar ratio of carboxyl groups to amino groups was 3:1 on 4arm-PEG chains, and relatively high reactivity for the small molecular structure of Azo-NHS. In addition, the peak at 1307 cm−1 corresponds to C–N stretching vibration of azobenzene skeleton, which clarified the target products were synthesized successfully.

Environmental temperature is an important factor in the application of hydrogels. Good thermal stabilities of hydrogels can ensure the constant structures and compositions within a certain temperature range. Here, TGA was used to study the thermal stability of PR-gel. As shown in Fig. 2c, all samples have experienced slight processes of weight loss around 100 °C, which corresponded to the evaporation of adsorbed water. It is easy to find that the apparent thermal weight loss decomposes in a narrow temperature range of CNFs at 350 °C, which could be due to the typical rapid decomposition of the polymer chains [12]. While the thermal stability of CNFs-4arm-PEG was dropped down to 267 °C with the addition of 4arm-PEG, which could be due to the decreasing proportion of CNFs in the hydrogel, and resulted in the decrease of the degree of crystallinity. Compared with CNFs and CNFs-4arm-PEG, PR-gel has moderate thermal stability (weight loss ~ 87% at 500 °C, the maximum decomposition temperature at 261 °C). This phenomenon was probably attributed to the chemical cross-linking of the sample. As the framework of a cross-linking network in PR-gel was capable of improving the thermal stability to such hydrogel and preventing damage to PR-gel structure caused by high temperature environment.

Generally, CNFs possess a high aspect ratio (containing interconnected amorphous and crystalline I regions) and can form strong physical entanglements and networks in composites. These features make the CNFs-based hydrogels much stronger than in the case when the network is formed only because of weak hydrogen bonds between water and fibrils [13]. Compared with 4arm-PEG, the CNFs cross-linked in the hydrogel network has significant advantages, including a certain proportion of crystalline regions on nanosized molecular chains, which provided PR-gel with excellent mechanical strength. In order to explore the completeness of the skeleton in PR-gel, the crystal structure of samples was tested. As shown in Fig. 2d, the characteristic peaks at 15.9° and 22.6° corresponded to the I101 diffraction intensity and the I002 largest diffraction intensity of crystal plane in the natural cellulose I crystal structure, respectively [14]. The above results indicated that the skeletons of CNFs in PR-gel network were preserved well.

3.2 Photoresponse of PR-gel

As depicted in Fig. 3a, the trans to cis transition of Azo in PR-gel network could be regulated by UV and visible light. Due to two characteristic bands of Azo structure about shift depending on the substituents of the phenyl rings, the photochemistry isomerization can be regulated by the UV region (∼ 320–360 nm) that corresponds to the π−π* transition and the visible region (∼ 420–450 nm) that corresponds to the n−π* transition [15]. The UV-visible absorption spectra of PR-gel were tested to reveal photochemistry isomerization mechanism. As shown in Fig. 3b, PR-gel displayed an absorbance maximum and minimum related to the structure of Azo at 330 nm and 465 nm under three different conditions, which was respective characteristic of the π−π* and n−π* transition of Azo structure in the trans and cis state. Excess absorption wavelength for n−π* transition in 465 nm could be attributed to the red shift of absorption band, due to the influence of substituents on benzene rings [16]. Subsequently, PR-gel was executed immediately after visible light or UV irradiation at 365 nm (20 mW/cm2, 15 min). The peaks at 330 nm showed different degrees of reduction corresponding to the transition from trans-form (π−π*) to cis-form (n−π*). Compared with pre-irradiation (dark condition), the absorbance values of the sample with visible light and UV irradiation declined by 2.0% and 5.5%, respectively. These results indicated that (i) trans-Azo structure in the PR-gel was dominant before irradiation; (ii) UV irradiation was the critical factor to induce photoisomerization transition. These phenomena were due to the more polar nature of the cis isomer, and thus the whole network appeared a certain degree of hydrophilic plasticity and softening effect in PR-gel. On the contrary, the relatively nonpolar trans isomer of Azo was consistent with stiff of the initial state, which was difficult to achieve large strains under the same shear rate [17].
Fig. 3

a Schematic illustration of photochemistry isomerization transition of PR-gel. b UV-visible absorption spectra of PR-gel under dark, visible, and UV light irradiation. c UV-visible absorption spectra of PR-gel in periodic transformation by UV-visible irradiation. Rheological properties for PR-gel of oscillation amplitude sweep (d), oscillating frequency sweep (e), and continuous step strain tests (f)

In order to demonstrate the reversible photoresponse, PR-gel was fully swelled in deionized water for 48 h. Subsequently, the maximum absorption values of PR-gel at 330 nm were recorded by UV-visible absorption spectra. As depicted in Fig. 3c, the PR-gel presented alternate high and low absorption values, corresponding to visible and UV light irradiation of every 15 min, respectively. The absorbance of periodic transformation by UV-vis irradiation indicated the reversible and dynamic photochemistry isomerization (trans-cis isomerism) of azobenzene in PR-gel. Consequently, this property would act as photoswitch to endow hydrogels with photoresponsiveness and help to construct drug or proteins delivery for photo-controlled release.

3.3 Mechanics of PR-gel

Rheological tests have been applied to demonstrate the structural stability and viscoelasticity of hydrogels. As shown in Fig. 3d, whether under visible or UV light conditions, the storage modulus (G′) of PR-gel was always higher than the loss modulus (G″) in the small strain scope, which indicated that PR-gel has stable cross-linking and elastic network structure [17, 18]. When strain enhancement beyond a certain value (near 10% strain), the G′ values of the PR-gel decreased sharply and lower than G″ values. This phenomenon could be attributed to the collapse of the linear viscoelastic region causing the gel-sol transition of the sample. It is worth noting that UV irradiation (365 nm, 15 min) has a certain effect on the viscoelasticity of the PR-gel. Decreased average G′ values of the hydrogel up to 81 Pa (approximately 7% less than initial modulus) under the oscillating shear strain of 1%, which demonstrated that photochemical isomerization to the cis-form of Azo led to a measure of softening effect in the PR-gel.

The samples were further subjected to frequency sweeps with a constant strain of 1%. Figure 3 e clearly showed that the G′ values were always higher than G″, accompanied by gradually increasing during the process. The G′ and G″ values increased slightly with the increasing of the frequency from 1 to 92 rad/s (0.16 to 15 Hz), which presented a typical gel-like behavior of a soft viscoelastic and chemical cross-linked material [19, 20]. In addition, the higher values of G″ than that of G′ belong to a typical characteristic of the physical cross-linking hydrogel, which due to disassembly of macromolecular chains and physical chain entanglement occurred in the disintegration process of gel resulted from high frequency shear. This result was highly consistent with previous oscillation amplitude sweep results.

In order to further investigate the mechanical strength of PR-gel, especially for its mechanical recovery properties after a large amplitude oscillatory deformation, the continuous step strain measurements are shown in Fig. 3f. The PR-gels were subjected to an alternating amplitude oscillatory shear strain (1%) and (1000%) at a frequency of 6.283 rad/s (1 Hz). At this condition, the two modulus remained stable at different strain, and the G′ values were always much greater than the G″ values as well as both modulus remained invariant with time, which indicated the solid-like and elastic nature of PR-gels [21]. This result was consistent with our previous expectation that PR-gel could resist external forces and maintained its structural stability in rapid high and low strain transform.

In addition, from the above results, all the G′ and G″ of PR-gel with UV irradiation (365 nm, 15 min) exhibited lower values than that of the control group (visible light exposure). This decrease in modulus could be attributed to isomerization of the cross-linker to the cis state. The planar trans and twisted cis conformation may lead to the “stiff” state and “soft” state, respectively.

3.4 Morphology observation

The structure and morphology of PR-gel were closely related to the protein loading and release. As shown in Fig. 4, it was easy to find the network structure with porous morphology and relatively structured size distribution (30~100 μm), which proved that this structure has been successfully designed and fabricated. Generally, three-dimensional channels and porous structure in hydrogels could be used as carriers to accommodate small molecules substance such as drugs and proteins. Due to the surfaces of CNFs have abundant free hydroxyl groups, the CNFs-4arm-PEG holds a large capacity for water adsorption and creates a hydrophilic microenvironment, which contributed to improve the affinity between the hydrogel and some hydrophilic proteins [22].
Fig. 4

a, b The topography for SEM of PR-gel at different magnification. c The photograph of PR-gel

3.5 Release behavior and mechanism of BSA

Herein, PR-gel was used for loading and controlled release of BSA under UV exposure. UV light irrigation led to the structural changes of the hydrogel matrix. As a result, PR-gel underwent reversible softening and thus facilitated the diffusion of BSA from this drug depot to the environment (Fig. 5a). Modulated burst release is very feasible to keep the dosage within its therapeutic window, especially for some protein with nonlinear pharmacokinetics. UV (365 nm) and visible light (400–500 nm), a compatible wavelength range with live cells, were used to study the photo-controlled effect of the PR-gel on the release rate of BSA.
Fig. 5

a Schematic illustration of loading and controlled release of BSA of PR-gel by UV-vis light. b The cell viability test of PR-gel and BSA-loaded PR-gel in consecutive 72 h. c The test of concentration and release rate of BSA-loaded PR-gel

The concentration of released BSA in the PBS solution (pH 7.4, 37 °C) was monitored in real time. As depicted in Fig. 5c, the release rate (ron) of BSA increased rapidly when the BSA-loaded PR-gel system was treated by UV light for 10 min (ON stage). When the system was exposed to visible light, the release rate of the BSA (roff) reduced 5-fold. These results could be attributed to the changes in network structures, which were controlled by the photo-induced isomerization of Azo. The release rate of the BSA under the UV light should be due to the leakage or passive diffusion of the protein from the hydrogel. Two subsequent ON-OFF stage cycles of the protein were also demonstrated with reproducible ron>roff. A reduction in the release rate of ron 2nd and ron 3rd during the second and third ON-OFF cycle, respectively, could be attributed to the more amount of BSA release from the surface of the BSA-loaded PR-gel system during the first cycle.

We maintained that the enhanced loading and release of BSA under the visible and UV treatment, respectively, could be due to the trans-to-cis photoisomerization. The CNFs-4arm-PEG cross-linked Azo-NHS with a hydrophobic benzene ring structure, which could maintain the hydrophilic-hydrophobic equilibrium in PR-gel. Therefore, this stable porous network structure exhibited characteristics similar to that of zwitterionic membranes, namely, keeping proteins at specific distances or making the protein contact with the surface without conformation change [23]. In addition, porous structure with Azo units played an important role in the photoswitched trans and cis forms, corresponding to different dipole moments and surface wettability, respectively. The more hydrophobic surface with trans isomerism could adsorb more proteins, and the less hydrophobic surface with cis form could only absorb a few proteins [24].

3.6 Biosafety evaluation of PR-gel

To confirm that PR-gel was suitable for the nontoxic delivery, the biocompatibility of the sample was evaluated by the CCK-8 assay. Figure 5 b showed that around 100% cell survivals were observed from the blank PR-gel, which indicated the excellent biocompatibility of PR-gel carrier. Around 110% cell survival of the BSA-loaded PR-gel could be due to the release of the nutrient for cells BSA.

4 Conclusions

In summary, we have successfully prepared photoresponse hydrogel (PR-gel) by integrating 4arm-PEG and azobenzene into cellulose nanofibrils (CNFs). The developed PR-gel has been proved to possess good mechanical strength, structural stability, and reversible recoverability. Benefiting from the introduction of azobenzene cross-linker, the PR-gel also exhibited reversible dynamic photochemistry isomerization transition originated from trans-cis isomerism of azobenzene, and thereby caused structure transformation and softening effect to its network, which could realize controllable BSA release by UV light irradiation. Good biocompatibility of the PR-gel was confirmed by the cell viability assay. We believe that this work will provide new insights into protein release vectors and cellulose-based stimulus-responsive hydrogels, which can help to expand the application fields of cellulose-based and some other sustainable materials.


Funding information

This work was financially supported by the Young Elite Scientists Sponsorship Program by Tianjin (TJSQNTJ-2017-19) and Natural Science Foundation of Tianjin (17JCQNJC05200).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Tianjin Key Laboratory of Pulp and PaperTianjin University of Science and TechnologyTianjinPeople’s Republic of China
  2. 2.State Key Laboratory of Biobased Material and Green Papermaking, Key Lab of Paper Science and Technology of Ministry of EducationQilu University of Technology (Shandong Academy of Sciences)JinanPeople’s Republic of China
  3. 3.College of Chemical Engineering and Materials ScienceTianjin University of Science and TechnologyTianjinPeople’s Republic of China

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