Supporting information

Bioinspired butyrate-functionalized nanovehicles for targeted oral

delivery of biomacromolecular drugs

Lei Wu, Min Liu, Wei Shan, Xi Zhu, Lijia Li, Zhirong Zhang and Yuan Huang*

Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education,

West China School of Pharmacy, Sichuan University. No. 17, Block 3, Southern

Renmin Road, Chengdu 610041, P.R. China

Corresponding Author

*E-mail: huangyuan0@163.com,

Tel.: +86-28-85501617,

Fax: +86-28-85501617.

Materials and methods

1. Cell culture

HT-29-MTX-E12 (E12) cells were cultivated in 75 cm2 culture flasks using

Dulbecco's Modified Eagle Medium (DMEM, Gibco, NY, USA) containing 10% fetal

calf serum, 1% penicillin and streptomycin (100 IU/ml) and 1% non-essential amino

acid (Hyclone, UT, USA). The culture was maintained at 37 °C, 95% relative

humidity with 5% CO2. The E12 cells were seeded in 96-well plates (Corning, NY,

USA) at 1 × 104 cells/well for cytotoxicity and uptake assays. The culture condition of

Caco-2 cells was same with that of E12 cells.

2. In vitro stability study

In brief, hydrochloric acid solution (pH 1.2) was applied to mimic gastric juice.

Phosphate buffered saline (PBS) (pH 6.8) simulated the environment of duodenum

and jejunum. PBS (pH 7.4) could simulate pH condition of the distal ileum and

intercellular fluid [1]. Freshly prepared NPs were incubated under different conditions

at 37 in a shaker. At predetermined time points, samples were collected and the℃

particle sizes were measured using the DLS via Zetasizer Nano ZS90.

3. Membrane integrity of Caco-2 Cells

To investigate the influence of NPs and free butyrate on membrane integrity of Caco-

2 cells, we used lactate dehydrogenase (LDH) test kit (Beyotime Institute of

Biotechnology, China) [2]. Prior to the test, cells were plated in wells of 96-well

plates and grown overnight. After incubated with corresponding samples for 4 h, the

cells were cultured with fresh medium for another 24 h. Then the medium was

withdrawn followed by centrifugation (400 g for 5 min). Finally, the LDH leakage

was measured according to the instructions of LDH test kit.

4. Interleukin-8 (IL-8), nitric oxide synthase (NOS), reactive oxygen species

(ROS) and glutathione (GSH) detection

For quantifying the IL-8 production, enzyme-linked immunosorbent assay (ELISA)

kit (Human IL-8 ELISA kit, Jiyinmei, China) was applied [3]. After incubation with

various samples of 4 h, the cells were replenished with fresh medium for another 24 h

before detection. According to the instructions, the optical density (OD) was detected

at a wavelength of 450 nm. Values were calculated following the calibration curve and

normalized to those of control cells for IL-8 generation. In this test,

lipopolysaccharide (LPS) (100 μg/ml) used as the positive control was incubated with

cells for 24 h [4]. For NOS detection, the total nitric oxide synthetase (T-NOS)

detection kit (Nanjing Jiancheng Bioengineering Institute, China) was applied.

Samples and the positive control group were consistent with IL-8 detection [5].

According to the instructions, the OD was detected at a wavelength of 530 nm.

The ROS content within Caco-2 cells was detected by ROS test kit (Beyotime

Institute of Biotechnology, China) [6]. Caco-2 cells grown on 24-well plates were

treated with samples for 4 h followed by fresh medium for another 24 h, and dyed

with DCFH-DA (10 μmol/L) for 20 min (37 ℃). Rosup (125 μg/ml) simulated the

cells for 1 h before the staining as a positive control group. Then cell fluorescence

was quantified via flow cytometry (Ex/Em =488/525 nm). For measurement of GSH

within cells, the GSH test kit (Beyotime Institute of Biotechnology, China) were used

[7, 8]. After incubation with various samples of 4 h, the cells in 24-well plates were

replenished with fresh medium for another 24 h. Cells were then collected and the

following process was performed according to the instructions. At last, the OD was

measured at a wavelength of 412 nm.

5. Mitochondrial depolarization assay

Mitochondrial membrane potential (MMP) was detected using fluorescent dye JC-1

(Molecular Probes) [9]. After treated with PEG NPs, Bu-PEG NPs and the mixture of

free butyrate and PEG NPs (the concentration of NPs was equal to that in uptake

study) for 4 h, Caco-2 cells were cultured with fresh medium for another 24 h, which

were then collected and incubated with JC-1 (5 μg/mL) for 20 min. After washed with

fresh medium, cells were analyzed using fluorescence spectrophotometer, and the

ratio of red to green signal was calculated. Additionally, Caco-2 cells in Glass Bottom

Cell Culture Dish were observed using a confocal microscope (FV1000, Olympus,

USA) after dyed by JC-1.

6. Bioactivity of INS loaded in NPs

To study the bioactivity of insulin (INS) after drug loading and release process, Bu-

PEG NPs (INS) were allowed to release INS in PBS at 37 °C for 300 min. Then, NPs

were isolated from the released INS by ultra-centrifugation (100 kDa). BALB/c mice

(24–26 g) (n = 5 per group) were fasted overnight, and then subcutaneously

administered with the released INS (2 IU/kg) and free INS (2 IU/kg) [10]. The

measurement of blood glucose levels was coherent with that in “2.11. In vivo

pharmacological and pharmacokinetic studies”.

7. Circular dichroism

The secondary structure of INS before and after encapsulation into NPs was

determined by circular dichroism (CD). Free INS was dissolved in pure water at the

concentration of 95 μmol/L as previously reported [11, 12]. And INS released from

Bu-PEG NPs was collected and set to the same concentration. Then, samples were

recorded in 1 mm cell from 260 to 200 nm at 25 using a Jasco Spectropolarimeter℃

J-815 (Tokyo, Japan). Each spectrum represented an average of 6 successive scans

and was expressed as the mean residual ellipticity [θ]*10-3 (deg cm2 dmol-1).

8. In vivo distribution

PEG NPs and Bu-PEG NPs loaded with DiR were administered by oral gavage to

mice at same dose. Images were taken at 2, 4 and 6 h after administration via an in

vivo imaging system [13, 14]. To measure the biodistribution of NPs in the

gastrointestinal tract, BALB/c mice (24–26 g) were sacrificed at each time point to

obtain the major organs (i.e., the stomach, intestine, heart, liver, spleen, lungs and

kidneys), which were separated and washed thrice with saline. Dye accumulation was

imaged and analyzed with Perkinelmer In Vivo Imaging System. The wavelength of

DiR was set at 720 nm for excitation and 790 nm for emission.

All animals received care in compliance with relevant laws and guidelines outlined in

the Guide for the Care and Use of Laboratory Animals. All procedures were approved

by Sichuan University Animal Care and Use Committee.

9. Statistical analysis

Results are presented as mean ± SD. Differences between groups were performed

with SPSS program 16.0 by using two tail Student’s t test. Differences were

considered statistically significant if the associated p was <0.05 and highly significant

if the associated p was <0.01.

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Fig.S1 1H NMR spectrum of DSPE-PEG-butyrate and DSPE-PEG in DMSO-d6.

Figure S2. FT-IR spectrum of DSPE-PEG-butyrate and DSPE-PEG.

Fig. S3 The size of NPs at different time point when incubated with HCl (pH 1.2), PBS (pH 6.8) and PBS (pH 7.4). Error bars represent SD (n=3).

Fig. S4 The relative viability of E12 (A) and Caco-2 cells (B) after exposure to PEG NPs and Bu-PEG NPs with varying degrees of butyrate for 4 h followed by 24 h with fresh medium. Error bars represent SD (n=3).

Fig. S5 The variation of TEER values of Caco-2 cell monolayers. The TEER values were monitored during the co-incubation with NPs (8 h) and 16 h after removal of samples. Error bars represent SD (n=3).

Fig. S6 The relative cellular uptake of PEG NPs and Bu-PEG NPs with the inhibitor (butyrate) at varying concentrations. Error bars represent SD (n=3). **p<0.01, *p<0.05, #p<0.05, versus control group (no butyrate involved).

Fig.S7 (A) The relative cell viability of Caco-2 cells after exposure to free butyrate detected by MTT assay. The level of LDH (B) and GSH (C) released from Caco-2 cells after co-incubation with varying samples. Error bars represent SD (n=3).

Fig.S8 The level of IL-8 (A) and NOS (B) released from Caco-2 cells after co-incubation with varying samples. LPS was the positive control. *p<0.05, **p<0.01, versus control group. Error bars represent SD (n=3).

Fig.S9 (A) The ROS level of Caco-2 cells after being exposed to different samples for 4h. (B) The ratio of fluorescence intensity of JC-1 polymer to monomer and (C) CLSM images with JC-1 assay of Caco-2 cells after being exposed to different samples for 4h. #p<0.05, ##p<0.01, ###p<0.001, versus PEG NPs.

Fig.S10 The variation of size of NPs at different time point when incubated with HCl

(pH 1.2), PBS (pH 6.8) and PBS (pH 7.4).Error bars represent SD (n=3).

Fig.S11 Far-UV CD spectrum of free INS solution and INS released from Bu-PEG (INS) NPs.

Fig. S12 Variation of blood glucose levels of diabetic rats after oral administration of Bu-PEG NPs (INS) at dose of 25, 50 and 100 IU/kg. Error bars represent SD (n=5). #p<0.05, ##p<0.01, versus the 50 IU/kg group.