Materials
PSS-[2-(3,4-epoxycyclohexyl)ethyl]-heptaisobutyl substituted (POSS), 3-Aminophenylboronic acid monohydrate (APBA), dimethyl sulfoxide (DMSO), insulin human (100 mg) (cold shipment required), hydrazine monohydrate, ammonia solution, polyethylene glycol (Mw =200, PEG), tetrahydrofuran (THF), and sodium cyanoborohydride were purchased from Sigma Aldrich (Seoul, Korea) and used as received.
Synthesis of POSS-APBA
Synthesis of POSS-APBA is carried out in three steps. First 10 mg POSS and 1.67 mg APBA is put in 9 ml THF and stirred at 50℃ for 3 hrs in an oil bath to make a homogeneous solution. To this solution 50㎕ of hydrazine monohydrate catalyst and 50㎕ of ammonia solution are slowly added and the mixture allowed to react for 1 hr., then cooled to room temperature. Finally the POSS-APBA solution was placed in a vacuum oven at 45℃ to remove the THF solvent and obtain powders. The prepared POSS-APBA powder is stored in a dessicator at room temperature.
Synthesis of PEG-Insulin
To introduce diol groups to insulin, 8 mg insulin and 100 mg PEG were dissolved in 10 ml DMSO to which 0.1 ~ 0.2 % sodium cyanoborohydride catalyst was added and stirred for 1 hr. then stabilized at room temperature for 1 hr.
Preparation of POSS-APBA@Insulin
The preparation of POSS-APBA@Insulin is carried out in two steps, synthesis and solvent exchange. In the synthesis step, 11mg of prepared POSS-APBA powder and the PEG-Insulin DMSO solution are stirred for 2 hours and then stabilized for 30 minutes. The stabilized solution in DMSO is changed to an aqueous solution in the solvent exchange step by dialysis. The stabilized solution is put in a dialysis tube with an MWCO size of 100-500D, and the tube is immersed in a water bath filled with distilled water for dialysis. The distilled water in the tank is replaced with fresh distilled water at 10, 20, 30 min., 1, 3, 6, 12, and 24 hr. to increase the dialysis efficiency. After dialysis, the POSS-APBA@Insulin solution is stored in a refrigerator at 3℃.
Characterization
Fourier-transform infrared spectroscopy (FT-IR)
Fourier transforms infrared (FT-IR) spectra analysis was obtained on a Perkin Elmer Spectrum II, for the confirmation of synthesis and the structural analysis of the sample. The FTIR spectra were obtained from KBr pellets. The KBr pellets were made by mixing KBr powder and the sample at a ratio of about 100:1, using agate induction so that it is well dispersed, and then making a pellet by applying pressure on a pellet molding apparatus. The sample was analyzed in the range of 4000 ~ 400cm-1 to confirm the presence of characteristic peaks of POSS-APBA and the changes in peak positions and shapes.
1H-Nuclear magnetic resonance spectrometer (1H-NMR)
1H-NMR analysis was performed for structural analysis of the synthesized material. To completely remove the solvent from the POSS-APBA, POSS-APBA@Insulin, and POSS-APBA@glucose samples, samples were dried in a vacuum oven at 90°C for 24 hr. The samples were dissolved in deuterated chloroform at a concentration of 0.02g/0.7ml and analyzed.
X-ray photoelectron spectroscopy (XPS)
XPS analyses was carried out with microfocus monochromatic X-ray source: Al-Kα (1486.6 eV), energy resolution(Ag 3d5/2): ≤ 0.5 eV, sensitivity : 4,000,000 cps, ultimate vacuum : < 5.0 × 10-9 mbar, X-ray spot size: 10㎛ ~ 400㎛, analyzer type: double-focusing, hemispherical analyzer with 128-channel detector, depth profiling: MAGCIS dual mode ion source.
Thermogravimetric analysis (TGA)
Thermal stability analysis of POSS, APBA and synthesized POSS-APBA was carried out using TGA N-1000. The weight of the samples were 6.41 mg for POSS, 5.55 mg for APBA, and 7.41 mg for POSS-APBA. The samples were heated from 20°C to 700°C at a rate of 10°C/min.
Scanning Electron Microscope (SEM) & Transmission Electron Microscope (TEM)
The three-dimensional structure and shape of the particles were confirmed with SEM, S-5200 and TEM, JEM-1011. The samples were prepared by dropping POSS-APBA and POSS-APBA@Insulin, POSS-APBA@glucose nano particles stored in distilled water on carbon tape, drying the tape in a vacuum oven at 80℃ for 24 hours, and then coating the carbon tape for SEM observation. Transmission electron microscope (TEM) images were obtained using a JEM-1011, Philips CM200 transmission electron microscope operating at 40 to 100 kV.
Zeta potential (ζ)
The zeta potential was measured to determine the variation in the stability of POSS-APBA and POSS-APBA@Insulin at different pH. The zeta potential measurement was carried out on a ELSZ-2000S (Otsuka, Japan) equipment with the samples dispersed in distilled water where the pH was controlled with 0.1N HCl and 0.1N NaOH. Zeta-potential is difficult to measure directly, and the usual route is to acquire this data indirectly from the measurement of the electrophoretic mobility (particle velocity divided by the electric field strength) under an applied electrical field according to Henry's equation (eqn (1)):
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where, U/E is the electrophoretic mobility (m2 s−1 V−1), ζ is the zeta-potential (V), ε is the solvent dielectric permittivity (or constant) (kg m V−2 s−2), η is the viscosity (kg m−1 s−1), particle size to the Debye length, 1/κ. Thus, κa ≫ 1 indicates that the particle radius (a) is large compared to 1/κ (1/κ is ∼10 nm for 1 mM aqueous salt solutions).20
Zeta sizer
Zeta sizer Nano ZS90 (Malvern Instruments, Worcestershire, UK) was used to measure the particle size and behavior of POSS-APBA and POSS-APBA@Insulin under various pH conditions and sugar concentrations. Zeta-size was measured with Zeta sizer Nano ZS90 (Malvern instruments, Worcestershire, UK), and each sample was dispersed in distilled water. When measuring the Zeta-size, hydrochloric acid and sodium hydroxide were added to the distilled water solution to adjust the pH to 2, 4, 6, 7, 8, and 10. The measurements were carried out at low, middle and high glucose concentrations.
In vitro drug loading efficiency
To determine the loading efficiency of insulin in POSS-APBA NPs, the amount of free insulin in supernatants was assayed using UV-vis spectrophotometry. The drug loading efficiency was calculated using the equations below. Insulin entrapment efficiency (EE) and loading capacity (LC) were calculated using the following equations :
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)
In vitro drug release experiment
In vitro insulin release experiments were carried out. In order to simulate the in vivo conditions the temperature of 400 ml PBS in the oil bath was set at 36.5°C. 40 ml of the refrigerator stored POSS-APBA@insulin solution is put in a dialysis tube of MWCO 8000 and stabilized at room temperature. The charged dialysis tube was put in the PBS solution simulating in vivo condition. As a control, six 1.5 ml aliquots were taken every 10 min. for 1 hr. without adding glucose to the PBS solution and evaluated for insulin release. The PBS solutions of different glucose concentrations, 1.0 mg/ml, 1.5 mg/ml and 2.0 mg/ml was achieved by adding 400 mg, 600 mg and 800 mg glucose to the blank PBS solution and the insulin release at the different glucose concentrations was evaluated from 1.5 ml aliquots taken every 10 min. for 1 hr. The amount and concentration of the glucose in the PBS solutions were kept constant by replenishing with the same amount of the respective glucose containing PBS solutions. The insulin release was evaluated from the absorbance at 275 nm of the UV-Vis spectra taken in the 200-700 nm range.
Reagents and Antibodies
Anti-α-tubulin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), anti-Caspase 3 and anti-Caspase 9 were purchased from Cell Signaling Technology (Danvers, MA). HRP-tagged anti-rabbit and anti-mouse were purchased from Enzo Life Science (Farmingdale, NY). MTT reagent was purchased from Sigma Aldrich (St. Louis, MO).
Cell Lines and Culture Condition
The HeLa cell was purchased from the American Type Culture Collection (ATCC) and maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Human dermal fibroblast (HDF) cell was purchased from ATCC and supplemented with 10% FBS and 1% penicillin/streptomycin. These cells were maintained in humidified atmosphere containing 5% CO2 at 37°C.
Cell Viability Assay
Cells were seeded in 96-well culture plate and incubated in culture medium until 80% confluence. The cells were treated with various concentrations of samples for 24 hr, and incubated with MTT reagent for 2 hr. Blue formazan crystals were solubilized in DMSO, and formazan levels were determined at 570 nm using an Infinite M200 PRO plate reader (Tecan Group Ltd., Männedorf, Switzerland).
Immunoblotting
Cells were washed with cold PBS, and then lysed in the triton lysis buffer containing protease and phosphatase inhibitors. After incubation for 30 min on ice, lysates were centrifuged at 13,000 rpm for 10 min at 4°C, and supernatants were collected. Lysates were separated on 8–15% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (GE Healthcare Life Sciences, Chicago, Illinois). Membranes were blocked in 5% skim-milk for 1 h and incubated with primary antibodies overnight at 4°C. Membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hr, and then visually evaluated by using the ECL detection kit (Biomax Co., Ltd, Seoul, Korea).21