Manipulable permeability of nanogel encapsulation on cells exerts

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Bio-interactions and Biocompatibility

Manipulable permeability of nanogel encapsulation on cells exerts protective effect against TNF-#-induced apoptosis Wenyan Li, Guohui Zhang, Teng Guan, Xiaosha Zhang, Ali Khosrozadeh, Malcolm Xing, and Jiming Kong ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00654 • Publication Date (Web): 29 Jun 2018 Downloaded from http://pubs.acs.org on July 2, 2018

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ACS Biomaterials Science & Engineering

Manipulable permeability of nanogel encapsulation on cells exerts protective effect against TNF-α-induced apoptosis Wenyan Lia,b, Guohui Zhangc, Teng Guanb, Xiaosha Zhangb, Ali Khosrozadehe, Malcolm Xingd,e,* and Jiming Kongb,c,*

a

Department of Neurosurgery, Southwest Hospital, Third Military Medical University, 30

Gaotanyan Street, Chongqing, 400038, China b

Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, MB, R3E

0J9, Canada c

Department of Forensic Medicine, Hebei North University, Zhangjiakou, 075000, China

d

Institute of Burn Research; State Key Laboratory of Trauma, Burn and Combined Injury; Key

Laboratory of Proteomics of Chongqing, Southwest Hospital, Third Military Medical University, 30 Gaotanyan Street, Chongqing, 400038, China e

Department of Mechanical Engineering, University of Manitoba, Winnipeg, MB, R3E 0J9,

Canada

Email addresses of corresponding authors: 1*

: [email protected]

2*

: [email protected]

ACS Paragon Plus Environment

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Abstract Cell encapsulation using microgel and nanogel, as a strategy of cell surface engineering, can mimic the niches of cells and organoids. The established niche which seasons cells and tissues for the controllable development underlies the superiority of encapsulation on cells. Encapsulation by layer-by-layer nanogel coating is a bottom-up simulation of extracellular matrices via nano or micro packaging of cells in a multi-scale way. We report the nanogel encapsulation on individual neuronal cell for a basic study and application of permeability tuning to regulate cells’ apoptosis. Gelatin and hyaluronic acid (HA) are applied for encapsulating PC12 cells. The permeability of encapsulation on cells can be managed by adjusting different parameters such as material concentration, layer thickness and environmental pH. Eventually, permeability of tumor necrosis factor-α (TNF-α) is controlled by tuning encapsulating parameters for blocking the interaction with TNF-receptor 1, so that cell apoptosis is inhibited. In short, nanogel encapsulation exhibits controllable permeability to different molecules and exerts screen effect on TNF-α for protection. This technique holds great potential in basic biological research and translational research, for example, the protection of transplanted cells against apoptotic factors in target areas.

Keywords: cell surface engineering, nanogel encapsulation, permeability, layer-by-layer, apoptosis

1. Introduction Cell transplantation therapy has been regarded as a hopeful therapeutic strategy for treating nervous system disorders such as stroke1. However, the general outcome of the treatment is not


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satisfactory. One of the most critical flaws is the low survival rate of transplanted cells, mostly caused by cell apoptosis. Following the cell grafting, apoptotic factors bind to their membrane receptors2-4 to take effects. For example, tumor necrosis factor-α (TNF-α) interacts with TNFreceptor 1 (TNF-R1) which is located on cell membrane5-6 to activate the downstream cascades and subsequently leads to the cleavage of caspase-8 and caspase-3 which cause the apoptosis7. Unfortunately, current treatments fail to eliminate the impairment from apoptosis effectively. Therefore, the rapidly-developing material science began to draw researchers’ attention in recent years. Cell surface engineering has been developing tremendously in the past few years. This tissue engineering strategy provides a novel approach in not only basic cell biology research, but also the translational study. For example, cell encapsulation creates a relatively isolating microenvironment for investigating the functions of certain factors8; it also provides a capsule that can be modified to endow versatile functions on cells9; it may act as a protective shell to prevent adverse factors from damaging cells inside10; it even forms functional substrate with diazidecrosslinker for promoting cell proliferation11. Among the findings, microgel and layer-bylayer (LbL) assembly are verified to establish encapsulation structure on cells for various purposes12. These microgels are usually produced from hydrogels, which can be generated uniformly and conveniently. However, the size of hydrogels is huge for cells and they will only capture cell mass instead of single cell13-14. It brings about shortcomings for conferring heterogeneous effects on each encapsulated cell. Additionally, the large size of microgel tends to cause the formation of an impermeable barrier for nutrients to penetrate, leading to disorders in the encapsulated cells. As an alternative technique, LbL encapsulation becomes popular since it establishes thin films on the cell surface to build nanogel at single-cell scale, based on the


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electrostatic adsorption between polycation and polyanion. LbL nanogel encapsulation has been widely applied in drug delivery9, 12 and cell protection15. Permeability is a critical feature of LbL nanogel since it can be influenced by many factors and will influence cellular functions in return, however, the investigation on the nanogel permeability is rarely seen. Among the few studies in recent years, techniques such as high gravity field16, tapered fluidized bed17 and CO2-induced biomineralization18 have been reported to adjust the permeability of nanogels. However, most of these techniques cannot be utilized in mammalian cells since they are hazardous to the fragile mammalian cells. Therefore, biocompatible biomaterials and proper techniques are required in mammalian cell encapsulation and investigation on the permeability. Encapsulation of mammalian cells using natural materials has been described in our previous work. For exploring the permeability of nanogel, FITC-dextran (FD) assay is performed since it is acknowledged as a biocompatible method for investigating permeability19-20. Furthermore, influence of multiple parameters in encapsulation on permeability is included, such as concentration of materials, number of layers and environmental pH. With a comprehensive understanding of permeability manipulation, nanogel encapsulation is regarded to be instrumental for improving cell survival in transplantation therapy. LbL nanogel can act as a barrier against apoptotic factors such as TNF-α, since the permeability of these molecules through nanogel will be hindered by applying certain parameters, making it possible to maintain the cellular functions. As hypothesized, nanogel encapsulation will exhibit selectivepermeability to molecules in a predictable and pre-determined approach to inhibit cell apoptosis induced by TNF-α, promoting the survival of grafted cells. In this study, we firstly established the nanogel encapsulation on PC12 cells (which are extracted from rat pheochromocytoma, often applied in neuronal cell study because of their


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toughness and neuronal features21-22) with gelatin and HA. Permeability of encapsulation by different parameters to FD with different molecular weights under different pH conditions was examined. Next, the optimized parameters of encapsulation were applied and the screen effect of TNF-α was verified for inhibiting apoptosis. The outcome indicates that further applications in basic cell biological studies and clinical translational studies can be hopefully achieved by adjusting the permeability of nanogel.

2. Materials and Methods 2.1 Materials. FITC-dextran (FD), bovine serum albumin (BSA), proximity ligation assay (PLA) kit, gelatin, dimethylsulfoxide (DMSO), Dulbecco’s phosphate buffered saline (DPBS), Duolink in Situ Red Starter Kit, Hoechst 33258 and fluorescein 5(6)-isothiocyanate (FITC) were purchased from Sigma Aldrich (Canada). Hyaluronic acid (HA) was purchased from Freda Biopharm (China). TNF-α was obtained from Peprotech. Anti-TNF-R1, TNF-α antagonist were obtained from Santa Cruz (U.S.A.) Anti-TNF-α was purchased from Abcam (Canada). Protein G magnetic beads were from Bio-rad (Canada). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from R&D Systems (U.S.A.). TUNEL kit, Bicinchoninic acid (BCA) kit, Clean-Blot IP detection kit were obtained from Thermo Scientific (Canada). 2.2 Nanogel encapsulation. As previously described12, 2×106 PC12 cells were firstly incubated in 1 ml gelatin solution in various concentrations for 10 min with gentle shaking to form the gelatin layer. The suspension was then centrifuged at 1200 rpm for 5 min to pellet the cells. The cells were washed in 5 ml of DPBS, and centrifuged again to discard the supernatant. After three times of washing, the cells were incubated in 1 ml of HA with various concentrations for 10 min


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to form the HA layer. The gelatin-HA single-cell encapsulation was repeated until desired layers of coating was achieved. 2.3 Transmission electron microscopy (TEM). TEM was performed as described previously23. Untreated PC12 cells and PC12 cells encapsulated with 8 gelatin-HA layers were firstly fixed using 0.1 M Sorensen's buffer which contained 2.5% glutaraldehyde for 1 h. Samples were washed with 5% sucrose in 0.1 M Sorensen's buffer for 5 min × 3 times and then kept in 4 oC overnight. 1% OsO4 was added to cell samples for 2 h as the post-fixation. Following the fixation, samples were dehydrated with gradient alcohol and embedded with epoxy resin. After being dried under 60 °C for 24 h, samples were cut to obtain thin sections with a microtome (318423, Reichert Nr.) and mounted. Finally, samples were incubated with 2% uranyl acetate and 1% lead citrate. Transmission electron microscope (CM-10, Philips) was used to analyze the prepared samples at 25 °C. 2.4 Atomic force microscopy (AFM). Cells seeded on cover slides were observed under the atomic force microscope (EasyScan 2, NanoSurf). All samples were scanned under the “Phase contrast” mode with 2.5 s/ line and 512 points/ line. After obtaining the images at lower magnification, an area of around 25 µm2 on cell surface was picked to scan under a higher magnification. Furthermore, average roughness value (indicated as Sa which was calculated by the software) per 1 µm2 was obtained by selecting 10 spots randomly on each cell. 2.5 Preparation of fluorescein labeled gelatin. For the preparation of FITC-labeled gelatin, 20 mg of gelatin was dissolved with 2 ml of sodium bicarbonate buffer (0.1 M). 10 mg of FITC was diluted using 1 ml of DMSO, followed by being added slowly in gelatin solution with continuous stirring. The mixture was incubated overnight in dark at room temperature. The solution was dialyzed to get rid of the unbound FITC for three days and then lyophilized.


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2.6 FD permeability investigation. PC12 cells were encapsulated with gelatin and HA with different parameters. FD with different molecular weights were then incubated with cells in medium with varying pH for 30 min. After the incubation, cell samples were examined under the fluorescence microscope (Axio Imager Z.2, Zeiss). Intensity of the fluorescence was detected on the plate reader (1420, Wallac) at excitation of 490 nm by diluting 1×104 cells with 100 µl in a 96-well plate. 2.7 Immunofluorescence. PC12 cells were seeded on cover slides and fixed in 4% paraformaldehyde (PFA) solution at 4 °C for 15 min. Cells were washed using DPBS for 5 min × 3 times and permeabilized with PBST which comprises 0.1% Triton X-100 in DPBS for 10 min. 1% BSA in PBST was added to reduce unspecific binding. Furthermore, primary antibodies which were diluted in blocking buffer were incubated with cell samples overnight at 4 °C. Followed by three times of washing, incubation of secondary antibodies was kept from light for 1h at room temperature. Cell nucleus was counter-stained with 0.12 µg/ml Hoechst 33258 after washing for three times. The primary antibodies used was as follows: Ki-67 (mouse, 1:500). Secondary antibody was the Alexa-488 chicken anti-mouse (1:1000). 2.8 PLA.TNF-α (20 ng/ml) was incubated with cells in control group and encapsulation groups for 5 min. Fixation of cells was performed by applying 4% paraformaldehyde (PFA), followed by DPBS washing for three times. Duolink blocking solution was used to block samples for 30 min under 37 °C. Next, blocking buffer was discarded and primary antibodies were added to incubate for 1 h at 37 °C. After washing, PLA probes were added to samples for 1 h at 37 °C and subsequently, cells were incubated with ligation solution for 30 min at 37 °C. Finally, samples were treated with polymerase in amplification solution followed by Hoechst staining and mounted on slides.


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2.9 Co-IP and Western blot. TNF-α (20 ng/ml) was added in control group and encapsulation groups. After 5 min of incubation, cells were washed with cold DPBS for three times, followed by being lysed in lysis buffer (150 mM NaCl, 50 mM pH 7.4 Tris-HCl, 5 mM EDTA, 0.1% NP40, and protease inhibitor cocktail) for 20 min. When the magnetic beads were rinsed with PBST containing 0.1% Tween 20 for three times, 2 µg antibodies of TNF-α and TNF-R1 were incubated with beads separately for 10 min. Cell lysate was incubated with the antibody-loaded beads, with 100 µl for each. The mixture was rotated for 1 h at room temperature. The proteinloaded beads were then magnetized and washed, followed by elution with 6×Laemmli buffer for 10 min at 70 °C. The eluent was moved to new tubes and ready for sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Input samples were extracted immediately after the lysis of cell samples and denatured for 5 min at 95 °C. Samples were loaded on SDS gel (12% acrylamide) and then performed Western blot. The primary antibodies were: TNF-α (rabbit, 1:500), TNF-R1 (mouse, 1:300). Secondary antibodies used included: horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:5000) and HRP-conjugated donkey anti-mouse (1:5000). The results were quantified with Image Studio software (LI-COR Biosciences). 2.10 Statistical analysis. Values were expressed by Means ± standard error of the mean, which was indicated by the error bar. In comparison of different groups, one-way analysis of variance (ANOVA) was used in the study. Statistical significance was accepted when P value < 0.05.

3. Results 3.1 Cell-compatible study of single-cell encapsulation with gelatin and hyaluronic acid (HA). PC12 cells, extracted from rat adrenal medulla, are used since they are acknowledged as the neuronal cell model24. Gelatin (type A) with an isoelectric point of 7.0-9.0 and HA with that


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of 2.5 are chosen as the polycation and polyanion, respectively. They are both FDA-proved natural polyelectrolytes with reputable biocompatibility and can form multilayers under neutral pH25-26. Cell viability of PC12 cells encapsulated with different parameters was detected by Hoechst/PI staining. After being coated with 0.1%, 0.5% and 1% of materials for 4, 8 and 12 layers, cells were stained with Hoechst/PI reagents. PI is known to permeate cell membrane of dead cells to bind to DNA and display red fluorescence. As illustrated in Figure 1, cell viability was not affected by encapsulation, indicating the biocompatibility of this technique on PC12 cells.


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Figure 1. Cell viability of untreated PC12 cells and PC12 cells encapsulated with different parameters by Hoechst/PI staining. A) Hoechst/PI staining of untreated PC12 cells and cells encapsulated with 0.1%, 0.5% and 1% of materials for 4, 8, and 12 layers. Cells with blue were viable ones. B) Quantification of viability of PC12 cells in different groups.

Cell counting and Ki-67 proliferation assay at different time points were applied to indicate the feasibility of the materials and encapsulation methods. In Figure 2, the increase of cell number on day 0, 3, 5 and 7 was shown. Although cells proliferated consistently in all the groups, the increase of cell number was obviously hindered in groups of 0.5% of materials 12-layer and 1% of materials encapsulation groups after day 3. Results of Ki-67 expression also conformed to the cell counting that the proliferation of cells in these groups was about 45.3 ± 6.5%, 42.4 ± 4.2%, 37.2 ± 9.4% and 35.4 ± 8.1% on day 3. On the contrary, the proliferation rates were 48.3 ± 65.3%, 51.0 ± 8.7%, 47.2 ± 9.7%, 52.7 ± 2.9%, 47.7 ± 11.0% in 0.1% of materials groups, 0.5% of materials for 4-layer and 8-layer groups. Besides, in consideration of the undifferentiated survival rates of cells in all the groups, it was addressed that gelatin and HA were biocompatible materials for single-cell encapsulation. However, the proliferation would be negatively impacted by encapsulation with high concentration of materials and excessive number of layers. As such, permeability investigation will be then performed on cells encapsulated with the parameters imposing less influences on cellular functions, for example, 0.1% of materials for 4, 8 and 12 layers; 0.5% of materials for 4 and 8 layers.


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Figure 2. Proliferation assay of untreated PC12 cells and encapsulated cells. A) Ki-67 staining of untreated PC12 cells and cells encapsulated with 0.1%, 0.5% and 1% of materials for 4, 8, and 12 layers on day 3. B) Quantification of Ki-67-positive cells from each group on day 3. The ratio of Ki-67-positive cells was calculated from five random fields of each group; (n=5). a: p

Manipulable permeability of nanogel encapsulation on cells exerts

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