Reagents and Materials
1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol sodium salt (DMPG) were purchased from Avanti Polar Lipid Inc. (Alabaster, AL, USA). Cholesterol (Chol), α-tocopherol, bovine serum albumin (BSA) fluorescein isothiocyanate conjugate (FITC-BSA), ammonium persulfate, pepsin, pancreatin, and Remicade® (IFX) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cy™7 Mono NHS Ester was purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). BCA Protein Assay Kit and PageRuler Prestained Protein Ladders were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Sodium dodecyl sulfate (SDS) was purchased from Kracker Scientific (NY, USA). Coomassie-brilliant blue R-250 staining solution, 30% acrylamide/Bis solution (29:1), TEMED, 4× Laemmli sample buffer, 1.5 M Tris-HCl buffer and 0.5 M Tris-HCl buffer were purchased from Bio-Rad Laboratories (Hercules, CA, USA.). Magnesium chloride hexahydrate (98%) and other inorganic salts were purchased from Junsei Chemical Co. (Tokyo, Japan). Eudragit® S100 was kindly donated by Evonik Korea (Seoul, Korea).
Preparation of liposomes
DMPC, Chol, DMPG, and α-tocopherol were mixed at a molar ratio of 26:10:2:2 in tertiary butyl alcohol. The mixtures were frozen at -80°C overnight followed by freeze-drying (EYELA FDU-1200, Tokyo, Japan) for 24 h. The lipid cakes (total 40 μmol of lipid mixture) obtained were hydrated with 1 mL of IFX solution, which was obtained by dissolving Remicade powder in distilled water (10 mg/mL), or FITC-BSA solution in phosphate-buffered saline (pH 7.4). The hydrated liposome dispersions were vortexed and sonicated for 1 h at 24°C of DMPC by using an ultrasonic cleaning bath (Branson®, 3510-DTH Ultrasonic Cleaner, Danbury, CT, USA). To obtain a liposomal dispersion with increased homogeneity and reduced particle size, additional sonication was carried out by using a cell disruptor (Bioruptor®, UCD-200T, Cosmo Bio Co., Tokyo, Japan) as previously described . Liposome dispersions were then subjected to freeze-thawing for five cycles of 5 min incubation at -180°C and 15 min incubation at 37°C to improve the protein encapsulation. To remove non-encapsulated proteins, liposomes were collected by centrifugation at 43,000 × g for 1 h at 4°C. The resulting pellets were re-suspended in the original volume of phosphate-buffered saline (PBS). The prepared liposomes were stored at 4°C until use.
Coating of liposomes
The 3-aminopropyl-functionalized magnesium phyllosilicate (aminoclay) was synthesized by following a method described previously. Before coating, the bulk aminoclay powder was dispersed in water, followed by ultrasonication for 10 min, for the exfoliation of the aminoclay. Aminoclay-coated liposomes (AC-L) were obtained by spontaneous assembly of positively charged aminoclay on negatively charged liposomal surfaces. Briefly, equal volumes of liposome dispersions pre-diluted with distilled water to give a lipid concentration at 10 mg/mL were added drop-wise to exfoliated aminoclay dispersion (10 mg/mL) to give a lipid/clay weight ratio of 1:1. The mixture was incubated at 25°C for 30 min with stirring and then centrifuged at 15,000 × g for 7 min at 4°C. The resulting aminoclay-liposome pellets were re-suspended in 1 mL of PBS.
For further coating of clay-liposomes with Eudragit S-100, an equal volume of clay-liposome dispersions pre-diluted to a concentration of 2.5 mg lipid per mL was added drop-wise to 0.1% Eudragit S100 solution in PBS. The mixture was incubated at 4°C for 30 min with stirring and then centrifuged at 15,000 × g for 7 min at 4°C. The resultant Eudragit S100-coated aminoclay-liposome (EAC-L) pellets were re-dispersed in the original volume of PBS.
Physicochemical characterization of liposomes
The mean particle size and polydispersity index of nanocomposite carriers were measured by dynamic light scattering using a fiber-optics particle analyzer (FPAR-1000, Otsuka Electronics, Osaka, Japan) as described in our earlier studies. Particle size analysis data were assessed using the CONTIN program provided by the manufacturer. Zeta potential (the electrical potential at the shear plane of the nanoparticle) was measured using a Zetasizer Nano ZSP (Malvern, UK). Samples were diluted 50-fold with deionized water before measuring to reach the analytical measurement range. Default instrument settings and automatic analysis were used for all measurements. Each measurement was carried out in duplicate.
The diameter and morphology of liposomes were imaged by negative-stain transmission electron microscopy. Liposome samples were 50-fold diluted with PBS solution and dropped on a 200-mesh copper grid coated with carbon and negatively stained with 2% uranyl acetate for 1 min. Excess stain was removed and the samples were allowed to air-dry completely. Dried samples were examined using a Tecnai G2 Spirit (FEI Company, Hillsboro, OR, USA) operating at 120 keV.
The encapsulated concentration of IFX and FITC-BSA were determined by BCA protein assay or by measuring the fluorescence of FITC (excitation 490 nm, emission 525 nm) with a fluorescence spectrometer (FS-2, Scinco Ltd., Seoul, Korea) after disrupting the liposome dispersions with an equal volume of 10% SDS (fluorescence assay) or ethanol (BCA assay). Standard curves pre-constructed with serial dilutions of FITC-BSA with ethanol were used to convert fluorescence to FITC-BSA concentration. The stability of BSA in the nanocomposite carriers was investigated using circular dichroism spectroscopy. Gastrointestinal stability of liposomes and protein stability analysis are described in the Supplementary Information.
Eight-week-old C57BL/6 mice were kept under standard conditions at 21-22°C under 12 h light/dark cycle and allowed to acclimate for a week before starting the experiment. Body weight and physical activity were monitored daily.
Distribution of nanocomposite carriers in dextran sulfate sodium (DSS) colitis mice
To assess the biodistribution of nanocomposite carriers after oral administration, Cyanine-7 (Cy7)-labeled nanocomposite carriers were prepared. Briefly, two μg of Cy7 was dissolved with 40 μmol of DMPC:Chol:DMPG:α-tocopherol (26:10:2:2) mixture in tertiary butyl alcohol. The liposomes were prepared from the mixture as described above, except that the un-entrapped Cy7 was separated from the liposomes by dialysis. Each liposome dispersion was adjusted to 0.44 μg/mL before oral administration.
Colitis was induced in mice by oral administration of 1.5% (wt/vol) DSS (36-50 KD molecular weight, MP Biomedicals, Solon, OH, USA) for 5 days in drinking water. After fasting for 12 h, Cy7.0-L, Cy7-AC-L, and Cy7-EAC-L were orally administered to mice with DSS-induced colitis at a dose of 20 mg/kg in a volume of 100 μL PBS. Control mice maintained normal drinking water for 5 days and then Cy7.0-EAC-L was administrated orally at the same dose. Mice were sacrificed 7 h after administration of Cy7.0-labeled nanocomposite carriers. Cy7.0-labeled nanocomposite carriers were visualized (Cy7: excitation 750 nm, emission 773 nm) and analyzed by using an in vivo image analyzer (Caliper IVIS Lumina II, Caliper Life Science, USA).
DSS-induced colitis and therapeutic effect of nanocomposite carriers
To test the therapeutic effect of nanocomposite carriers, colitis was induced in mice by administrering 1.5% DSS in their drinking water for 7 days. Mice were randomly divided into five different groups: DSS only (DSS-treated control group), DSS-treated with L (L group), DSS-treated with AC-L (AC-L group), DSS-treated with EAC-L (EAC-L group), DSS-treated with intraperitoneal IFX (IP-IFX group), and DSS-treated with per-oral IFX (PO-IFX group). Mice in the L, AC-L, and EAC-L groups were orally administered nanocomposite carriers for 9 days, from day 0. The dose of nanocomposite carriers (10 mg/kg for all groups) was predetermined to have an optimal therapeutic effect. Mice received 4 mg/kg of IFX in 200 μL PBS daily by oral or intraperitoneal administration for nine consecutive days. The dose of IFX was determined to have an optimal therapeutic effect based on existing studies [48, 49]. Drinking water was replaced with pure water and maintained for 2 days. Changes in body weight, stool consistency, and presence of blood in the stool or at the anus were measured daily throughout the study period. Mice were sacrificed at day 12; spleen and colons were collected to assess the therapeutic efficacy of the nanocomposite carriers. Disease activity index (DAI) was evaluated using the summed score of three factors (weight loss, stool consistency, and bleeding) .
DSS-induced colitis and the therapeutic effect of nanocomposite carriers-IFX conjugates
To test the therapeutic effect of nanocomposite carriers loaded with IFX, colitis was induced in mice via the addition of 1.5% DSS to drinking water for 7 days. Drinking water was then replaced with pure water for 4 days. All mice were sacrificed on day 11. Mice were orally administered, 200 μL PBS, IFX-L (10 mg/kg), AC-IFX-L (10 mg/kg), EAC-IFX-L (10 mg/kg) or PO-IFX (10 mg/kg), once daily, from day 0 to day 8 (total 9 days). The dose of nanocomposite carriers and IFX was predetermined to have an optimal therapeutic effect [48, 49]. Flow cytometry, qRT-PCR, analysis of TNF-α by enzyme-linked immunosorbent assay (ELISA), and western blotting are described in the Supplementary Information.
Colon tissues were fixed in 10% neutral formalin and then embedded in paraffin. Tissues were stained by hematoxylin and eosin and periodic acid Schiff (PAS) staining. The severity of colitis was scored as described previously . Goblet cell staining was scored from 0 to 3 (3, minimal, <20%; 2, mild, 21–35%; 1, moderate, 36–50%; 0, marked, >50%).
Goblet cell counting
Colonic tissue sections were processed with PAS staining. Stained goblet cells were counted per crypt. Maximum 52 and minimum 20 fully conserved crypts on each section were examined, and the average numbers were marked as a representative goblet cell count of each section.
The data were reported as mean and standard deviation. Comparison among groups was done by performing Student’s t-tests or one-way analysis of variance (ANOVA) followed by Dunnett post-test using GraphPad Prism software (La Jolla, CA, USA). Results with a p value < 0.05 were reported as statistically significant.