Recombinant human gelatin (MW 89.8 kDa) was purchased from Neosilk (Hiroshima, Japan). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was obtained from Dojindo Laboratories (Kumamoto, Japan). Ethylenediamine was purchased from Tokyo Chemical Industry Co, Limited (Tokyo, Japan) and ovalbumin (OVA), fluorescein isothiocyanate-labeled OVA (FITC-OVA), chlorpromazine, nystatin and cytochalasin D were obtained from Sigma-Aldrich (St Louis, MO). 4′,6-Diamidino-2-phenylindole (DAPI) and Vectashield mounting medium were purchased from Vector Laboratories (Burlingame, CA). LysoTracker™ Red DND-99 was obtained from Invitrogen (Carlsbad, CA). OVA-DQ™ was purchased from Molecular Probes (Eugene, OR). Cholesterol, hexamethylene diisocyanate, toluene (dry), pyridine (dry), triethylamine (TEA), hexane, dimethylsulfoxide (DMSO) (dehydrated), dichloromethane, 4% paraformaldehyde in phosphate-buffered saline (PBS), pyrene, and genistein were obtained from Wako Pure Chemical Industries, Ltd (Osaka, Japan).
The immature dendritic cell line DC 2.4 was kindly provided by Dr Kenji Kono (Osaka Prefecture University, Osaka, Japan) with the permission of Dr KL Rock (Harvard Medical School, Boston, MA). The cells were cultured at 37°C in RPMI-1640 medium with l-glutamine (Gibco BRL, Invitrogen) and supplemented with 10% fetal bovine serum (FBS), 100 μM nonessential amino acids, 50 μM 2-mercaptoethanol, and antibiotics.
Six- to 10-week-old female C57BL/6NCrSlc mice (Japan SLC Inc, Shizuoka, Japan) were maintained at the animal facilities of RIKEN Brain Science Institute (Saitama, Japan) according to the guidelines of the institution. The mice were kept in an air-conditioned room and fed standard laboratory food and water ad libitum. All experiments were approved by the RIKEN Animal Experiments Committee.
Synthesis of cholesterol derivatives
Cholesteryl N-6-(isocyanatohexyl carbamate) or cholesterol isocyanate (Ch-I) was synthesized as reported previously.6
Briefly, cholesterol (7.80 g, 20.12 mmol) was reacted with 1,6-hexyldiisocyanate (64 mL, 0.39 mol) in 200 mL of dry toluene containing 15 mL of pyridine at 80°C for 48 hours. The reaction was monitored using thin-layer chromatography (TLC). After the removal of solvent in vacuo, 500 mL of hexane was added to the residue and kept overnight at −20°C. The precipitates thus obtained were then separated and dried in vacuo to give a yield of 3.88 g (49.74%). Proton nuclear magnetic resonance (1
H NMR; 400 MHz, CDCl3,
25°C): δ = 0.60 (s, 3H, cholesterol-18 H3
), 0.78–2.30 (m, 40H, cholesterol), 1.30–1.56 (m, 8H), 3.09 (m, 2H), 3.22 (t,3J
(H,H) = 6.8 Hz, 2H), 4.42–4.51 (b, 1H, cholesterol), and 5.30 (m, 1H, cholesterol).
Cholesteryl N-6-(3-(2-aminoethyl)ureido)hexyl carbamate or amino-modified cholesterol (Ch-A) was synthesized as follows. A solution of Ch-I (200 mg, 0.360 mmol) in dichloromethane (10 mL) synthesized in the previous step was added dropwise to a vigorously stirred solution of ethylenediamine (0.481 mL, 7.2 mmol) in an excess of dichloromethane (25 mL) at room temperature (RT). The disappearance of cholesteryl N-(6-isocyanatohexyl)carbamate was monitored using TLC. The solvent was removed in vacuo and the compound was extracted using a 1:1 chloroform-to-water ratio giving a yield of 144.57 mg (72.2%). 1H NMR (400 MHz, [D6] DMSO, 25°C, TMS): δ = 0.65 (s, 3H, cholesterol), 0.84–2.33 (m, 40H, cholesterol), 1.41–1.55 (m, 8H), 1.22–1.23 (m, 2H), 2.92–3.00 (m, 8H), 4.27–4.32 (m, 1H, cholesterol), 5.34 (b, 1H, cholesterol), 5.80–5.85 (m, 2H), 7.03 (m, 1H). HR-ESI-MS: m/z calculated for (C37H66N4O3) ([M–H]−) 614.5134; found, 614.5208.
Syntheses of cholesterol-modified gelatins
aCMG was synthesized from Ch-I and rhG as follows. rhG (100 mg, 0.0273 mmol, corresponding to the lysine moieties and N-terminal) was dissolved in DMSO (15 mL) and reacted with Ch-I (7.56 mg, 0.0136 mmol) in DMSO (5 mL) containing TEA (1 mL, 7.16 mmol) at 50°C. The reaction was monitored using TLC until the disappearance of Ch-I was confirmed (developed using chloroform). It was then dialyzed against distilled water and freeze-dried to give a yield of 66 mg (66%).
cCMG was synthesized using an EDC-coupling method wherein Ch-A (23.22 mg, 0.0378 mmol) was reacted with rhG (100 mg, 0.0756 mmol, corresponding to the Asp and Glu moieties and the C-terminal) in 15 mL DMSO (Ch-A and rhG were dissolved separately in DMSO). To this mixture, an EDC-HCl solution (144.89 mg, 0.756 mmol), prepared freshly in 8 mL of DMSO, was added to the rhG and Ch-A mixture to obtain a 10-fold excess in the amounts of Asp and Glu. It was then allowed to react at room temperature overnight and dialyzed against distilled water to remove solvent and excess reagents including any byproducts such as isourea formed during the reaction, and lyophilized. Yield was 56 mg (56%).
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)
The conjugation of cholesterol with rhG was confirmed by MALDI-TOF/MS. All mass measurements were performed using a microflex MALDI-TOF mass spectrometer (Bruker Daltonics Inc, Billerica, MA).
Micelle formation and determination of critical micelle concentration (CMC)
Polymeric micelles were prepared by hydration of aCMG or cCMG in distilled water or PBS; they dispersed easily to give a uniform suspension on vortexing. The CMC values were estimated by fluorescence spectroscopy using pyrene as the fluorescence probe as described previously.19
Briefly, the concentration of pyrene was kept constant at 0.6 μM while the concentrations of the polymeric micelles were varied from 1 × 10−7
to 1 mg/mL. The fluorescence spectra were recorded using a fluorescence spectrophotometer (JASCO FP-6500; Jasco Corp, Tokyo, Japan) with an excitation wavelength of 320 nm. Fluorescent light emissions were monitored at 372 and 383 nm. The CMC was estimated as the cross-point when extrapolating the intensity ratio I372
at low- and high-concentration regions.
Scanning electron microscopy (SEM)
The morphology of the polymeric micelles was analyzed by SEM (JEM-6330; JEOL Instruments, Tokyo, Japan). SEM measurements were carried out at an accelerating voltage of 15 kV. The samples were prepared by dropping 10 μL of the micelle solution (1 mg/mL) onto a carbon seal attached to a metallic specimen holder, followed by air-drying for a day. The samples were then sputter-coated with gold before observation.
The cytotoxicity of the polymeric micelles was determined using a WST cell viability assay. DC 2.4 cells were seeded in a 96-well plate at a concentration of 2 × 104 cells/well and cultured in complete medium for 24 hours at 37°C under 5% humidified CO2. The culture medium was then replaced with fresh complete medium containing aCMG, cCMG, or rhG at predetermined concentrations and incubated under the same conditions for 24 hours. Then the cells were washed twice with PBS, and complete medium was added to the cells. Aliquots of 10 μL of cell counting kit-8 solution (CCK-8; Dojindo Laboratories) were then added to the medium and shaken for 30 seconds to mix the solution thoroughly and then incubated at 37°C for 30 minutes. The absorbance of each well was measured using a microplate reader (Model-680; Bio-Rad, Hercules, CA) at 450 nm. The percentage of cell viability was calculated from the absorbance values.
Preparation of polymeric micelles and FITC-OVA complexes
To prepare the polymeric micelle and protein complexes, aqueous solutions of FITC-OVA and polymeric micelles were mixed, vortexed gently, and incubated at RT for 30 minutes. Native rhG served as a control because it does not form a complex with OVA.
Size and zeta (ζ)-potential measurements
The mean hydrodynamic size and polydispersity index of rhG, aCMG, and cCMG were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK). Measurements were carried out at 25°C. The ζ-potential (in mV) was also determined using the Zetasizer Nano ZS. The size and ζ-potential are reported as the mean ± standard deviation (SD) of 14 subruns.
Cellular uptake efficiency of polymeric micelles measured by flow cytometry
Dual-color fluorescence-activated cell sorting (FACS) analysis was performed to quantify the uptake of FITC-OVA by the polymeric micelles in DC 2.4 cells. The cells were seeded at a concentration of 1 × 106, cultured in RPMI-1640 medium supplemented with 10% FBS and incubated with the samples for 1 hour at 4°C and 37°C. The cells were then washed with PBS and collected in PBS containing 0.25 μg/mL of 7-amino-actinomycin D (7-AAD) to stain the dead cells. Uptake of FITC-OVA was analyzed using a BD FACSCalibur (Becton-Dickinson, Tokyo, Japan). Results are expressed as the mean fluorescence intensity. Specific uptake was assessed by subtracting the mean fluorescence intensity of the cells incubated at 4°C from that recorded at 37°C.
For fluorescence microscopy, the DC 2.4 cells were seeded at a concentration of 1 × 105 cells and cultured for 24 hours in a 35-mm glass-bottomed dish containing RPMI-1640 medium supplemented with or without 10% FBS. For analyzing the cell uptake and subcellular location of FITC-OVA, free FITC-OVA (15 μg) or test samples (80 μg) complexed with FITC-OVA (15 μg) were used. For evaluating antigen processing, cCMG (80 μg) complexed with OVA-DQ™ (15 μg) was added gently to the cells and incubated at 37°C. For cellular uptake and antigen processing, after incubation for the indicated time periods, the cells were washed with PBS and fixed with 4% paraformaldehyde for 30 minutes at room temperature, after which the nuclei were stained with DAPI. For the subcellular localization of FITC-OVA, after incubation for the required time periods, LysoTracker Red™ (50 nM) was added 20 minutes before fixation. The cells were observed using a fluorescence microscope (Carl Zeiss, Göttingen, Germany).
To investigate the mechanism of uptake of the polymeric micelles, DC 2.4 cells were seeded at a concentration of 1 × 105 cells in a 35 mm culture dish and cultured for 48 hours. The cells were then incubated with four endocytosis inhibitors: nystatin (25 μg/mL), genistein (300 μM), chlorpromazine (15 μg/mL) and cytochalasin D (10 μg/mL) for 1 hour at 37°C, after which cCMG (80 μg) complexed with FITC-OVA (20 μg) was added and incubated for 4 hours. Cells incubated with only cCMG complexed with FITC-OVA served as controls. The cells were washed twice with PBS, collected in 1% bovine serum albumin (BSA) in PBS and the inhibition of FITC-OVA fluorescence was analyzed by flow cytometry (Cytomics™ FC500; Beckman-Coulter, Miami, FL). Results are expressed as the percentage of fluorescent positive cells.
Immunization and isolation of blood samples and spleens
To test the efficiency of in vivo antigen delivery, 6-week-old female C57BL/6 mice were administered with 3.3 mg/kg of free OVA or polymeric micelles complexed with OVA on days 0 and 14 by intraperitoneal injection. Blood samples were collected from the tail vein on days 7 and 21 for antibody titration. On day 28, whole blood samples were collected by heart puncture with the animal under deep diethyl ether anesthesia and the spleens were harvested before allowing the mouse to succumb. The spleens were prepared individually as single-cell suspensions in 3 mL RPMI 1640 supplemented with l-glutamine, 10% FBS and 100 μg/mL each of penicillin, streptomycin, and gentamycin. The erythrocytes were removed by suspension in a lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, and 0.1 mM EDTA-2Na). Splenocytes were washed with complete RPMI medium and live cells counted using a trypan blue exclusion method.
Specific antibody titers against OVA were determined by performing an endpoint enzyme-linked immunosorbent assay (ELISA). NUNC MaxiSorp 96-well ELISA plates (Nalge Nunc International, Roskilde, Denmark) were coated with 5 μg/mL of OVA in 0.1 M NaHCO3 (Nacalai Tesque, Kyoto, Japan), incubated overnight at 4°C and washed with PBS–Tween (PBS-T). The plates were blocked with 3% BSA in PBS for 1 hour at room temperature and washed with PBS-T. After serial dilution in 1% BSA in PBS beginning at a 40-fold dilution, serum samples (100 μL) were added into the wells and the plates were incubated for 1 hour at room temperature. The plates were washed with PBS-T and 100 μL of peroxidase-conjugated goat anti-mouse IgG (heavy chain- and light chain-specific, diluted 32000-fold in 1% BSA in PBS; Jackson ImmunoResearch Laboratories Inc, West Grove, WA) was added to each well. The plates were incubated for 1 hour at room temperature and then washed with PBS-T. Finally, colorimetric signals were generated using 1 Step Ultra TMB-ELISA solution (Pierce Biotechnology Inc, Rockford, IL). The reaction was stopped with 1 N H2SO4 after 15 minutes and the absorbance at 450 nm was measured using a Wallac ARVO counter (Perkin Elmer, Norwalk, CT). The antibody titer was determined as the highest dilution below the cutoff value indicated by the mean optical density value of the background ±2 SD.
All the animal experimental data are expressed as the mean ± SD. Multiple comparisons were performed using one-way analysis of variance (ANOVA) with post hoc analysis followed by Bonferroni’s test.