Supplementary Information
In-situ grafting MPEG on the surface of cell-loaded microcapsules
for protein repellency
Xiaocen Liu, Hongguo Xie*, Huizhen Zheng, Ying Ren, Shujun Wang, Mingqian Tan,
Guangwei Sun, Bing Wang, Weiting Yu, and Xiaojun Ma
Laboratory of Biomedical Materials Engineering, Dalian Institute of Chemical Physics, Chinese
Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China
Correspondence to: xiehg@dicp.ac.cn
Experimental Methods
1
Materials
MPEG-NH2 and HOOC-MPEG-NH2 with Mn=2000 g mol-1 was purchased from Shanghai
Yare Biotech, Inc. Sodium alginate with medium viscosity (100 cp) was purchased from Qingdao
Crystal Salt Bioscience and Technology Corporation (Qingdao, China). Chitosan with molecular
weights of 60 kDa and DD (degree of deacetylation) of 90% was degraded from the raw material
(Yuhuan Ocean Biomaterials Corporation, Zhejiang, China) in our laboratory. Gamma globulin
(IgG, Bovine Blood) and fibrinogen (Fgn) were purchased from Sigma-Aldrich Chemical Co. and
used without any pretreatment. Fluorescein isothiocyanate (FITC) was purchased from Acros
Organics Co. (4-(1, 2, 4, 5-tetrazin-3-yl)-phenyl)-methana was purchased from Sigma-Aldrich
Chemical Co. All the other reagents or chemicals were commercially available and of analytical
grade.
2 Synthesis of graft copolymers and the modified microcapsules
2.1 Synthesis of MPEG-g-NB
Alginate chemistry was performed in 1.0 wt% alginate solutions in 0.1M MES buffer at pH
6.5 and NaCl concentration 0.3M for 24h. Sulfo-NHS was dissolved in the alginate solutions at a
ratio of 1:1 to EDC, and EDC was next added as a 120% percentage of uronic acids available for
reaction. The BAT at a molar ratio of 1:1 to uronic acids was added after 0.5h and acted for 24h.
The alginate product was purified by dialysis (3500 MWCO) against ddH2O for three days and
lyophilized until dried.
2.2 Synthesis of MPEG-g-NB
Norbornene carboxylic acid was stirred for three hours with 1.1 equivalents of disuccinimidyl
carbonate in acetontrile. After reaction, solvent were removed by rotary evaporation and crude
norbornene succinimidyl ester with N-hydroxysuccinimide side product was recovered.
Commercially purchased MPEG-NH2 in dilute water was stirred at room temperature with a 5
molar excess of succinimidyl ester and a 30 molar excess of crude norbornene succinimidyl ester.
The MPEG-NB was then washed by ultrafiltration for 2 days and lyophilized until dried.
2.3 Preparation of ABCPN hydrogel microcapsules
The multilayer hydrogel microcapsules with MPEG modification in situ were prepared by the
method developed by our group and the “click” reaction. Copolymer alginate-g-BAT was
dissolved in a 0.9% (w/v) NaCl solution with a concentration of 1.5% (w/v). Then the solution
was filtered over microfilters and stored at 4oC overnight to promote deaeration. The alginate
beads were prepared by extruding the solution through a 0.5 mm needle dropwise into a 1.1% (w/v)
CaCl2 solution with an electrostatic droplet generator (YD-04, Dalian Institute of Chemical
Physics, Chinese Academy of Sciences, China). After calcification for 30 min, the beads were
transfered into a 0.5% (w/v) chitosan solution (0.1 M sodium acetate/ 0.2 M acetic acid buffer
solution (pH 4.2)) for 15 min, followed by being rinsed with saline to clear away excess chitosan
and got the ABC microcapsules. After that, the modification procedure was carried out by
incubating the ABC microcapsules in a 0.5% (w/v) MPEG-NB solution for 20 min to obtain
ABCPN microcapsules in which the click reaction took place and MPEG chains covalently bound
to the surface of the microcapsules. AC microcapsules as control group were prepared according
to the same process as ABC, and the differences were that the alginate was pure and the MPEG
modification wasn’t carried through. The diameter of AC and ABCPN microcapsules prepared in
this method was about 400µm.
3 Characterization of copolymers and microcapsules
3.1 Fourier transform infrared (FTIR) characterization of copolymers and microcapsules
Fourier transform infrared (FT-IR) spectra of the copolymer Alg-g-BAT and MPEG-NB were
recorded by a Bruker Vector 22 FTIR spectrometer with KBr pressed disks on using 4 cm-1
resolution and 32 scans. For the modified microcapsules, they had been lyophilized before pressed
disks with KBr.
3.2 Characterization of MPEG-NB distribution in ABCPN microcapsules
1g of HOOC-MPEG-NH2 was prepared into HOOC-MPEG-NB using the method mentioned
in 2.2. Then the sample was dissolved in PBS (0.01 M KH2PO4, 0.2 mM Na2HPO4, 0.15 M NaCl)
to give approximately 0.5 mM carboxylic groups. EDC and Sulfo-NHS were added as 120% and
100% percentage of carboxylic groups of HOOC-MPEG-NH2, respectively. The solution was kept
at room temperature with stirring for 0.5 h. Fluoresceinamine was added as a concentration of 0.02
mM to the solution, and the reaction mixture was stirred at room temperature for 24 h. Then the
solution were transferred to dialysis membrane (MWCO 500, Medicell), purified by dialysis
against ultrapure water at room temperature for 3 days and lyophilized. All the steps during the
labeling procedure must include protection from light. Confocal laser scanning microscopy
(CLSM, Leica TCS-SP2, Germany), equipped with an inverted microscope (Leica, DMIRE2,
Germany) and laser sources blue (Ar 488 nm/5 mW), was employed to characterize the
distribution of fluoresceinamine-labeled MPEG-NB in ABCPN microcapsules. The ABC
microcapsules (0.1 mL) were incubated in the 1 mL AF-MPEG-NB solution (0.5%, w/v) which
was shaken at room temperature for 20 min, and then underwent CLSM scanning.
4 Measurement of the IgG permeability of ABCPN microcapsules
20mL of IgG solution (in NaHCO3/Na2CO3 buffer (pH 9.0)) mixed with 1mL of a solution of
FITC (1mg/ml). The reaction was carried out for 2h in the dark at room temperature. The
FITC-IgG in the solution was purified by elution through a Sephadex G-25 column with
Na2HPO4/KH2PO4 buffer solution (pH 7.4). The permeability of the modified microcapsules was
investigated with CLSM. All of the samples were analyzed in the fluorescence mode with
appropriate laser wavelength (488 nm excitation and 530 emission). The microcapsules (0.1 mL)
were incubated in the 0.3 mL FITC-IgG solution (Na2HPO4/KH2PO4 buffer solution, pH 7.4),
which was shaken at 37oC for 2 h, and then underwent CLSM scanning.
5 Measurement of protein adsorption
Protein adsorption measurement was carried through according to the Bradford method. IgG
and Fgn were used as model proteins for evaluating the protein-resistant characteristics of ABCPN
microcapsules. The microcapsules (0.1mL) were incubated with 0.3mL IgG or Fgn solution
(Na2HPO4/KH2PO4, pH 7.4) respectively with a concentration of 1.0 mg/mL at 37oC for 2h. The
protein adsorbed amount was determined by measuring the difference in protein concentration of
the centrifuged supernatant before and after the adsorption process. The calculated mass per area
(q, µg/cm2) of the protein adsorbed on the different microcapsules was obtained by using the
equation
q
(C0  C f ) V
S
Where C0 and Cf are the initial protein concentration and the protein concentration after the
adsorption process, respectively, V is the total volume of the solution (0.3mL), and S is the total
sphere surface area of the microcapsules. The reported data were mean values of triplicate samples
for each kind of microcapsules.
6 Measurement of biocompatibility of the ABCPN microcapsules
6.1 CCK8 assay
Alg-g-BAT sample solutions of different concentrations (100, 1000, 5000, 10000, 15000
µg/mL) were prepared in saline. L929 cells were used to evaluate the cell toxicity of the
Alg-g-BAT copolymer. Cell viability was determined with the CCK8 cell viability assay (Dojindo
Lab, Kumamoto, Japan) on the basis of the manufacturer’s specification. RPMI 1640 medium was
used as a blank control sample. During the procedure, 200 µL cell suspension with a cell density
of 5×104 cells/mL was pipette into a 96-well plate and cultivated at 37oC with 5% CO2 for 24 h.
After this, the culture medium was discarded, and then 180 µL RPMI 1640 medium and 20 µL
Alg-g-BAT sample solution was added into the wells, meanwhile 200 µL RPMI 1640 medium was
used as negative control and 200 µL Triton X-100 was positive control. The plate was then
cultivated at 37oC with 5% CO2 for 24 h and 48 h. Then the culture medium was discarded, and
180 µL RPMI 1640 medium and 20 µL CCK8 solution was added into each well. After 2 h
incubation at 37oC with 5% CO2, the absorbance of each well was examined at 450 nm (determine
wavelength) and 630 nm (reference wavelength) on a Labsystems Dragon Wellscan MK3
microplate reader, which DMSO was used as plank control. Three replicates were used for each
sample to obtain a mean value. The cell viability was calculated as the following equation
OD=OD450  OD630
Cell  viability (%)  (ODsample / ODblank ) 100%
where ODsample is the optical density of the Alg-g-BAT sample or the control sample, and ODblank
is the optical density of the blank control sample.
6.2 Preparation of ABCPN microencapsulated cells and microencapsulated cell culture
HepG2 cell were harvested and resuspended in 1.5% (w/v) filtersterilized sodium
alginate-g-BAT solution to achieve a final cell density of 2×106 / mL alginate. Further, the
cell-loaded microcapsules were prepared using the similar method as mentioned in 2.3.
Microencapsulated HepG2 cells were cultured in a growth medium consisting of MEM medium
supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution, maintained at
37oC in a humidified 5% CO2 atmosphere.
6.3 Live/dead viability assay
Qualitative analysis of live and dead cells within the encapsulated cell clusters was
determined by live/dead viability assay according to the specification provided by the
manufacturer. Microencapsulated HepG2 cells were incubated with dual-color dyes (2 µm ED-1
and 1µm calcein AM, Sigma, USA) at 37oC for 2 h. Then, the samples were scanned with CLSM.
7 Statistical analysis
Data were expressed as the mean ± the standard error of the mean. Statistical comparisons
were performed using one-way analysis of variance (ANOVA). p values smaller than 0.05 were
considered to indicate statistical significance.