3-1. Instruments
Absorption spectra of AuNPs and AuNPs-conjugates were analyzed by UV-Vis
spectrophotometer (SpectraMax 190; Molecular Devices Corporation, Sunnydale, CA, USA).
Absorbance values of protein assay were recorded at 595 nm using a UV-Vis
spectrophotometer (Molecular Devices Corporation). The sizes of AuNPs-conjugates were measured by dynamic light scattering, DLS (Brookhaven Instruments Corporation, Holtsville, NY, USA). The fluorescence signals of AuNPs/peptide-FITC were analyzed by fluorescence spectrophotometer (F-2700; Hitachi, Tokyo, Japan). Enzyme activities in plasma/serum were analyzed by Fujifilm clinical chemistry analyzer (FUJI DRI-CHEM 3500; Fujifilm
Corporation; Tokyo, Japan). The gel electrophoresis analyses were performed with horizontal electrophoresis system (Mini-Sub Cell GT; Biorad, Corston, UK). The Pocket-sized pH meter used for AuNPs pH detection was purchased from ISFETCOM (S2K299; Saitama, Japan).
Microscope equipped with a high-resolution video camera (BX51; Olympus, Tokyo, Japan).
GeneQuant 1300 Spectrophotometer for enzyme activity assays (GE Healthcare Bio-Science, UK).
3-2. Chemicals
All chemicals were of analytical grade and were used without further purification.
Sodium citrate (C6H5Na3O7 • 2H2O), calcium chloride (CaCl2), Triton X-100, hydrogen tetrachloroaurate (III) (HAuCl4 • 3H2O), Brij™ 35 solution 30% (w/v), Proteinase K (#2308), Chymotrypsin (#C4129), bovine serum albumin (#A2153), polyethylene glycol (#P2139, Mw 8000), DL-dithiothreitol (#D5545), dithizone (#D5130), collagenase type I (#C9891) were
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obtained from Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade acetonitrile (ACN), were obtained from Merck (Darmstadt, Germany). Tris-HCl, dulbecco’s phosphate buffer saline (DPBS), were purchased from Invitrogen (San Diego, LA, USA).Sodium chloride was purchased from USB (Cleveland, OH, USA). Nanopure water was obtained by passing
twice-distilled water through a Milli-Q system (18 MΩ• cm; Millipore, Bedford, MA, USA).
3-3. Peptide substrates
The peptide substrates used in the protease activated fluorescence self- assembly AuNPs platform have the following sequence: FITC-Acp-GPLGLAG(Hyp)C. The peptide substrate was conjugated with Acp as spacer and fluorescein isothiocyanate (FITC) as fluorophore at N-terminal end. The peptide-FITC was synthesized commercially by Genesis Biotech (Taipei, Taiwan). The peptide-FITC could be highly recognized and digested by proteinase K but lower specificity to chymotrypsin. Proteinase K would cleave at the carboxyl side of aliphatic, aromatic or hydrophobic residues [Ebeling et al., 1974]. The peptide substrates were
dissolved in ACN solvent (sterilized water: methanol: ACN = 1:2:4) and were preserved in -20 ºC. There are three peptide substrates were designed and conducted in this study as shown in Table 3-1.
3-4. Preparation of the self-assembly AuNPs fluorescence probe
3-4-1. Synthesis of 15 nm AuNPs
AuNPs were prepared by citrate reduction method according to the reported procedure [Wang et al., 2010; Wright et al., 2005]. Colloidal AuNPs of size 15 nm was prepared as follows: 50 mL of 1mM chloroauric acid solution was heated with oil bath
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till boiling followed by the addition of 5 mL of 38.8 mM sodium citrate solution. The color of the solution would turn from yellow to color less to black. When the color changed to wine red, the heating was stopped, while the stirring was continued till the solution was cooled to room temperature. The product of 15 nm AuNPs solution was preserved at 4°C. The size and concentration of AuNPs are confirmed by absorption spectra by UV-spectrophotometer;
besides, the sizes of AuNPs are further conformed by DLS.
3-4-2. AuNPs salt stress assays
Citrate-capped AuNPs or AuNPs conjugates (90 μL) and further diluted with 100μL sterilized water was prepared. In the stabilizers increase stability experiment, the sterilized water would be replaced by stabilizer solution. The salt stress assays were performed as mixing diluted AuNP or AuNPs conjugates with 10 μL of different concentration of NaCl (100 to 3,000 mM). The mixture was incubated for 30 min at room temperature. Then the total volume of 200 μL mixture was loaded in micro plate for UV-Vis absorption
measurement from 400 to 700 nm wavelengths within 1nm interval.
3-4-3. AuNPs pH stress assays
Citrate-capped AuNPs were adjusted to different pH with 1 M HCl or 1 M NaOH. Then the 100 μL of pH adjusted AuNPs were diluted with 100 μL sterilized water and the pH were confirmed as pH 4 to 11 . The mixture was incubated for 30 min at room temperature. Then the total volume of 200 μL mixture was loaded in micro plate for UV-Vis absorption
measurement from 400 to 700 nm wavelengths within 1nm interval.
3-4-4. Modification of AuNPs probes
The peptide substrate was modified with fluorophore, FITC. The peptide includes cysteine (Cys) in the C-terminal containing contains a thiol group (-SH) which could
conjugate on to AuNP by Au-S bond. The pH of AuNPs was needed to be adjusted, according
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to the pI of the peptide, and we used 1M HCl or 1M NaOH to adjust AuNPs to desired pH.
The probes were synthesized as following: the prepared AuNPs was adjusted with sterilized water to OD=1, which is 980 μL of 2.5 nM AuNPs (15 nm). AuNPs were mixed with 10 μL of 1 mg/mL peptide-FITC and 10 μL of 0.01 M phosphate buffer containing 0.1% SDS and 0.3 M NaCl, and then the mixture were shaken for 12 hr, 40 rpm at room temperature. The probes (AuNPs/peptide-FITC) were purified by two rounds of centrifugation. After first
centrifugation (10,000 rpm, for 20 min), the supernatant was carefully removed and added 500 μL 2% (wt/wt) PEG in sterilized water. After second centrifugation (11,000 rpm, for 20 min), the supernatant was removed and added 1000 μL 1% (wt/wt) BSA in sterilized water or only sterilized water upon to different probes.
3-4-5. Effect of peptide substrate charges on AuNPs probes
The effect of peptide substrates charges on AuNPs stability was investigated. The pI of peptide substrate is predicted by online tools — Peptide Property Calculator, Genscript.
Under different pH of solutions, the peptide substrates own maybe positive, neutral or negative charge as shown in Table 3-1. Therefore, adjusting AuNPs solution to different pH (pH 4 to 11) would make peptide substrates own different charges during modification.
Moreover, after the purification, the change of environment was also considered. The sediment of different modification AuNPs probe was suspended with various pH of TTC buffer (pH 4 to 11). The spectra change of AuNPs probes under different peptide charges were analyzed with UV-Vis absorption spectra. The aggregation level (A625/A525) is used to evaluate the effect of peptide charges.
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3-5. Evaluation of size and morphology change in AuNPs probe 3-5-1. UV-Vis spectra determination
UV-Vis spectra of AuNPs were recorded by a spectrophotometer (Molecular Devices Corporation, Sunnydale, CA, USA). A total of 200 μL of AuNPs or AuNPs conjugates was used in this analysis and the absorption wavelength was set from 400 to 700 nm within 1 nm interval.
3-5-2. Dynamic light scattering determination
Dynamic light scattering (DLS), sometimes referred to as Quasi-Elastic Light Scattering (QELS). It is a non-invasive, well-established technique for measuring the size and size distribution of molecules and particles typically in the submicron region, and with the latest technology lower than 1 nm. Usually, samples as particles, emulsions or molecules, which have been dispersed or dissolved in a liquid could apply in DLS. The Brownian motion of particles or molecules in suspension causes laser light to be scattered at different intensities.
Analysis of these intensity fluctuations yields the velocity of the Brownian motion and hence the particle size could be determined using the Stokes-Einstein relationship [Pylaev et al., 2011].
The AuNPs samples were treated with four-fold dilution using sterilized water and then filled into the light scattering cuvette. Light scattering experiments were performed using the BI-200SM Goniometer (Brookhaven Instruments Corporation, Holtsville, NY, USA) at a temperature of 20°C. The laser wavelength was 532 nm, and measurements were conducted at an angle of 90°C. The DLS data were analyzed by Brookhaven Instruments-Dynamic Light Scattering software.
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3-5-3. Transmission electron microscopy analysis
AuNPs are examined by TEM (JEM-1230; JEOL Co. Ltd, Tokyo, Japan) operated at 100kV and equipped with CCD camera. Samples were prepared by 1 mL AuNPs (Absorbance peak at 519 nm, ~1. 1011 NPs/mL) in distilled water. To prepare TEM samples, drops of the resulting solutions put on hydrophobic surface and floated the carbon coated copper TEM grids (200-mesh) on it. The grids were allowed to absorb AuNPs for 1 min, and then damp with distilled water, finally blotted off to remove excess solution. TEM images of AuNPs analyzed by software Image J and over 120 particles were involved.
3-5-4. Zeta-potential analysis
The samples were 5 fold diluted in deionized water and subjected to size analysis by Zetasizer Nano (Malvern Instruments, Worchestershire, UK) using disposable solvent resistant micro cuvette (ZEN0040) at room temperature.
3-5-5. Gel electrophoresis of AuNPs
Gel electrophoresis analysis modified from Hanauer’ protocol was used to confirm the change of AuNPs after modification and proteinase digestion [Hanauer et al., 2007]. The morphology change could be observed from visible red band change by AuNPs’ color and the change of UV-light excited fluorescence band. Agarose gels (1.5%) were used and prepared with 0.5X TBE buffer. All sample loading with 35% glycerol for increasing density as the ratio of 7:1. The citrate-capped AuNPs should mix with very small amount of 10% SDS by 1 μL; otherwise, citrate-capped AuNPs can’t move toward positive electrode. The gels were run in a horizontal electrophoresis system for 30 min at 110 V in 0.5X TBE buffer. Gel images were taken by a digital camera under white light and UV-light; besides, the images might were processed with small linear contrast adjustments in order to obtain a true representation of the visual gel appearance.
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3-6. Fluorescence assays of proteases activated AuNPs probe 3-6-1. Conjugation of peptide substrates to AuNPs
The conjugations of peptide substrate to AuNPs were discussed. During the modification, the peptide substrates were existed either on the AuNPs or in the discarded supernatant.
Dithiothreitol (DTT)-based displacement is widely utilized for separating ligands from their AuNP conjugates. DTT will reactively displace the ligands from surface sites thereby enabling quantification because it is much more reactive toward AuNP compared with most ligands of interest [Tsai et al., 2012]. In this study, the conjugation of peptide substrate with the designed fluorophore could be quantified by fluorescence intensity in every part of possible existences of peptide substrates. DDT displacement experiment was conducted as following: prepared 50 mg/mL DTT (dissolved in pH 6.5 PB buffer, 0.1 M) and then add 500 μL DTT to suspend AuNP probe sediment (0.625 nM) for 12 hr incubation at room
temperature. The total loading peptide substrates fluorescence was defined after proper dilution and acquired a linear correlation between peptide-FITC concentration and fluorescence intensity. The first discarded supernatant during purification steps and the supernatant of DTT displacement also processed same procedure to acquire the linear correlations. All dilution buffers were used in this part of experiments was TTC buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl and 0.05% Brij 35, pH 8). The florescence intensity at 515 nm was recorded with an excitation wavelength of 495 nm.
3-6-2. Proteinase activity assay by AuNPs probes
For the proteinase activity assay, protease was diluted with TTC buffer and added into AuNPs probe (125 μL) to comprise 250 μL of mixture, and incubated at 37°C. The
concentrations of protease and incubation time depended on the need of experiment design.
All of the solutions were analyzed with fluorescence spectrophotometer (Hitachi F-2700),
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which recorded the fluorescence intensity at 515 nm was also recorded with an excitation wavelength of 495 nm. Delta fluorescence intensity is the difference in released FITC fluorescence between proteases activated AuNPs/peptide-FITC probe and non-activated control group.
3-6-3. Effect of stabilizers to proteinase sensitivity to AuNPs probes
For the purpose of improving stability of the AuNPs probe, the stabilizers such as PEG and BSA were used. The question is that do these stabilizers affect the proteinase work? The first experiment is to confirm the shorty immersed in PEG solution affection. AuNPs probe washed with different concentration of PEG (0.5 ~ 5% (w/w)) and suspended with 0.1% BSA (w/w) were prepared. The second experiment is to confirm the BSA as stabilizer affection.
AuNPs probe washed with 2% PEG (w/w) and suspended with different concentration of BSA (0.1 ~ 7% (w/w)). After discarding the supernatants, AuNPs probe was suspend by adding 1 mL different concentration of BSA. Then the processed AuNPs probes were conducted to protease activity assay.
3-7. Effect of peptide design to proteinase sensitivity to AuNPs probes
Table 3-1 records the sequences of each peptide substrate and notified it’s pI value, which is predicted by online tools- Peptide Property Calculator, Genscript. The cleavage positions for each protease also record with the counting from N-terminal and cleavage occurs at the right side of the marked amino acid. The cleavage sites are predicted by PeptideCutter tool, ExPASy.
In this study, there are three peptide substrates conducted for activating AuNP probe fluorescence. The activating process is same as protease assay mentioned above. It is clear that, these three peptide substrates have the same cleavage sites for both proteinase K and
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chymotrypsin digestion. According to different sequences of peptide substrates, the needs of modification conditions are different because of their charges. Different peptide substrates modified AuNP probes would be named as the rule of “ pH of modification A/ no. of
sequences peptide-FITC”. For example: .4A/1466p-FITC, which is modifying AuNPs with sequence “GPLGLAG(Hyp)C” at pH 7.4.
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Table 3-1. Peptide sequences and their cleavage sites respective to proteases and charges at function of pH
No. FITC-Acp-peptide Proteinase K Chymotrypsin pI
peptide charge at pH
4.0 5.6 7.4 10.0 11.0
1466 GPLGLAG(Hyp)C 3,5,6 3,5 5.3 +0.4 0 -1 -2 -2
1477 GPLGLARGGGGGC 3,5,6 3,5 7.8 +1.3 +1 +0.2 -1 -1.2
1482 GPLGLARDDDDDC 3,5,6 3,5 3.6 -2 -4 -5 -6 -6
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3-8. Animal experiments 3-8-1. Animals
Female C57BL/6 mice, purchased from the National Laboratory Animal Center (NLAC, Taipei, Taiwan), were housed at the Laboratory Animal Center, National Chiao Tung
University, under standard conditions. Female mice of 6 to 8 weeks old were used in this study. All experimental procedures were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of National Chiao Tung University. Every effort was made to minimize the suffering of the animals and the number of animals used.
3-8-2. Sample collection
Body weights of each mouse were recorded. Mice were sacrificed with CO2 inhalation.
Blood was collected by direct cardiac puncture and mixed with heparin to acquire plasma after centrifugation (13,000 rpm, 10 min at 4°C). The small intestine was divided into duodenum, jejunum (equally divided into two parts) and ileum (equally divided into two parts), following the demarcation set by Shang [Shang et al., 2009]. The intestinal contents of each segment were washed with 0.5 mL DPBS except 1 mL for duodenum and collected the washed fluid. The intestinal washed fluid was treated with centrifugation (13,000 rpm, 10 min at 4°C) and collected supernatants. Pancreas tissue was homogenized with 1 mL PRO-PREP protein extraction solution (iNtRON Biotechnology, Seongnam, South Korea). Homogenates were centrifuged at 13,000 rpm for 10 min at 4°C.
All the supernatants were collected and stored at -80°C until further assay. The protein concentration of the supernatant was measured with a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA).
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3-8-3. Fasting/Feeding experiment
For intestinal chymotrypsin distribution experiments, there are six mice were used and random divided into two group. All mice were fast for 12 hr, and the feeding group was further feeding for 1 hr after fasting. Then these mice were scarified and collected plasma, intestinal fluid and pancreas.
For fecal chymotrypsin time courses experiments, there are four mice were used. All mice were treated with 3 hr fasting and 5 hr feeding, and during this process the feces were collected. All feces were recorded the weight and were preserved in -20°C before analysis, after then the feces were conducted into protein extraction process. The extraction process was dissolved one feces (10 ~ 25 mg/per feces) into 0.5 mL fecal protein extraction buffer for 5 min with vortex followed by certification (10,000 rpm, 1 min) and the supernatant was collected.
3-8-4. Cerulein-induced acute pancreatitis mouse model
AP was induced by 4 doses (first dose at the zero hour and the following administrations with interval of 2 hr for 3 times) of intraperitoneal injection of cerulein (200 μg/kg, dissolved in 0.9% saline solution). All mice were randomly divided into the following three groups (n = 4 for each group). The control subjects were equally treated with 0.9% NaCl (10 μL/mg/2hr for 4 injections). The mice were sacrificed at 8, 10 or 24 hr after the first administration of saline or cerulein. Mice were fasted for 6 hr before sacrifice. Then the plasma, duodenal fluid, and pancreas were collected.
For fecal chymotrypsin time courses of AP model experiments, the mice were divided into two groups. All mice were treated with 12 hr fasting before injections started. AP (n = 4) was induced by 4 doses cerulein (200 μg/kg/2hr), and the control subjects (n = 3) were equally treated with 0.9% NaCl (10 μL/mg/2hr). All feces were treated with the lysis process
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as mentioned above.
3-8-5. Amylase and lipase assays
Plasma levels of amylase and lipase have been routinely used to estimate the severity of pancreatitis. These levels were measured at 8, 10, or 24 hr after the first dose of cerulein administration by using Fujifilm clinical chemistry analyzer. The sample of plasma should be proper diluted 50 folds for amylase assays and for 10 folds in lipase assays. The dilution buffer is PB (0.1M, pH 7.4).
3-8-6. Islet isolation from mouse pancreas
In this study, the islet isolation from mouse pancreas and staining procedure were modified from Li’s protocol [Li et al., 2009]. Dithizone (DTZ) was used to staining isolated pancreas. Stock DTZ solution was prepared as following procedure: 25 mg DTZ was added in 0.6 mL 95% ethanol and few drops of ammonium hydroxide were added for completely dissolving. After then the solution would turn bright orange solution. DTZ solution was prepared as following: adding 0.6 mL stock DTZ solution to volumetric flask and adding DPBS to final 50 mL. Adjust DTZ solution to pH 7.4 with 1M HCl.
The isolated pancreas was digested with 1mL collagenase type I (0.5 mg/mL in DPBS) for 2 hr at 37°C. The solution would be exchanged one time during digestion and poured with 1 mL collagenase type I again for 2 hr incubation. Finally, the mixture processed with
centrifugation (1,500 rpm, 5 min). Then poured the tissue to petri-dishes and stained with prepared DTZ solution. After 2 min staining, hand-pick the isolated islets to microscope slide and observed under microscope (BX 51; Olympus, Tokyo, Japan) and digitized using an attached MagnaFireTM imaging system (Olypus, Tokyo, Japan).
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3-9. BTEE assay
The chymotrypsin activity is determined by measuring an increase in absorbance at 256 nm resulting from the hydrolysis of benzoyl-L-tyrosine ethyl ester (BTEE). Prepare buffer TC:
0.08 M Tris⋅HCl buffer, pH 7.8 containing 0.1 M CaCl2; and 0.00107 M BTEE in 50% w/w methanol. Alpha-chymotrypsin was dissolved in 1 mg/mL in 0.001 N HCl for stock and was diluted in 1 mM HCl with 2 mM CaCl2 to 10 ~ 30 μg/ml for assay. Reaction mixture contains 450 μL TC buffer and 420 μL BTEE solution, and then add 30 μL α-chymotrypsin with different concentrations. Incubate in spectrophotometer (GeneQuant 1300) at 25°C for 4 ~ 5 min to achieve temperature equilibrium. Calculate ΔA256/min from the initial linear portion of the curve.
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