The materials used in this research included UA at a purity of ≥ 90% (Sigma-Aldrich, Tokyo, Japan), cholesterol (Wako Pure Chemical Industries, Ltd., Osaka, Japan), Span® 60 (Wako Pure Chemical Industries, Ltd., Osaka, Japan), 19 centipoise (cps) chitosan (Biotech, Cirebon, Indonesia), coumarin-6 (J&K Scientific, Beijing, China), chloroform (Merck, Darmstadt, Germany), methanol (Merck, Darmstadt, Germany), sodium chloride (Merck, Darmstadt, Germany), hydrochloric acid (Merck, Darmstadt, Germany), N-nitrosodiethylamine (Sigma-Aldrich, Tokyo, Japan), and acetonitrile (Sigma-Aldrich, Tokyo, Japan). If not stated otherwise, the reagents and materials used were of non-technical grade.
Coumarin 6
Manufactured by J&K Scientific
Sourced in China
Coumarin-6 is a fluorescent dye commonly used in scientific research. It has an excitation wavelength of 450 nm and an emission wavelength of 504 nm. Coumarin-6 is frequently utilized as a fluorescent tracer or marker in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Span 60, by Fujifilm (1 mentions)
Cholesterol, by Fujifilm (1 mentions)
N nitrosodiethylamine, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Chloroform, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Trypsin edta 0.25, by Thermo Fisher Scientific (1 mentions)
Sephadex g 50, by GE Healthcare (1 mentions)
Plga resomer rg 503, by Evonik (1 mentions)
4 6 diamidino 2 phenylindole dapi staining solution, by Beyotime (1 mentions)
Antifade mounting medium, by Beyotime (1 mentions)
Sodium chloroacetate, by Merck Group (1 mentions)
Malic acid, by Merck Group (1 mentions)
Methanol, by Tedia (1 mentions)
9 protocols using coumarin 6
1
Lipid-Based Nanoparticle Formulation
2
Confocal Imaging of Wetting Ridge Dynamics
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The confocal images of the wetting ridge were captured with Zeiss LSM 880 laser confocal microscope at Z-stacking mode. The fluorescent Coumarin-6 (J & K Scientific) was dissolved in the dimethyl silicone (12 cst) by ultrasonic dispersion for 12 h, and dimethyl silicone served as the dispersion solution of ferrofluid.
3
Comprehensive Lipid Nanocarrier Synthesis
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Soybean phospholipids (SPC), Cholesterol(CHO), DSPE-mPEG2000 and DSPE-PEG2000-Mal were all purchased from Shanghai Advanced Vehicle Technology L. T. D. Co. (Shanghai, China). PCM and TAT peptide with terminal cysteine (WLSEAGPVVTVRALRGTGSW-Cys and AYGRKKRRQRRR-Cys) were synthesized by GL Biochem Ltd. (Shanghai, China). Coumarin-6 was purchased from J&K Scientific LTD. (Beijing, China). Sephadex G-50 was purchased from Pharmacia Biotech (Uppsala, Sweden). 4′, 6-Diamidino-2-phenylindole (DAPI) was purchased from Beyotime Biotechnology Company, Limited. (Nantong, China). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Xiongben, Japan).
Cell culture flasks, plates and centrifuge tubes were purchased from Corning Incorporation (Corning, New York, NY). Dulbecco’s-modified Eagle’s medium (DMEM, high glucose), fetal bovine serum (FBS), trypsin-EDTA (0.25%) and penicillin–streptomycin were purchased from Gibco (MA, USA). All the other reagents and chemicals of analytical grade were purchased from Chongqing Zhuonuo Biotechnology Ltd. (Chongqing, China).
Kunming mice (15 ± 2 g) and two-day-old Sprague-Dawley rats were purchased from Experiment Animal Center of Chongqing Medical University. All animal procedures and use of laboratory animals for this study were approved by the Experiment Animal Administrative Committee of Chongqing Medical University.
Cell culture flasks, plates and centrifuge tubes were purchased from Corning Incorporation (Corning, New York, NY). Dulbecco’s-modified Eagle’s medium (DMEM, high glucose), fetal bovine serum (FBS), trypsin-EDTA (0.25%) and penicillin–streptomycin were purchased from Gibco (MA, USA). All the other reagents and chemicals of analytical grade were purchased from Chongqing Zhuonuo Biotechnology Ltd. (Chongqing, China).
Kunming mice (15 ± 2 g) and two-day-old Sprague-Dawley rats were purchased from Experiment Animal Center of Chongqing Medical University. All animal procedures and use of laboratory animals for this study were approved by the Experiment Animal Administrative Committee of Chongqing Medical University.
4
Nanoparticle Formulation Development
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SPCs were purchased from the American Jiaji Company (Minnesota, MN, USA). CHO and DSPE-PEG2000-Mal were purchased from Shanghai Advanced Vehicle Technology L.T.D.Co (Shanghai, China). PCM and TAT peptide with terminal cysteine (WLSEAGPVVTVRALRGTGSW-Cys and AYGRKKRRQRRR-Cys) were synthesized by GL Biochem Ltd (Shanghai, China). Coumarin-6 and coumarin-7 were purchased from J&K Scientific LTD (Beijing, China). Penicillin–streptomycin was purchased from Gibco (California, USA).
5
Pig Intestinal Mucus Collection
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Porcine original mucus were obtained from intestine of freshly slaughtered pigs and mucus was gently scraped from the intestinal wall (Shenyang, Liaoning). In order to minimize the influence of mucus variability on the result, mucus sample was collected and stored at −20 °C before use to make sure the mucus sample used for each compound was from the same source [11] (link), [12] (link). Poloxamer 407 and 188 (P407, P188) were obtained as gifts from BASF (Germany). SDS was bought from Biotopped Co., Ltd.(China). PLGA (Resomer® RG 503) was purchased from Evonik Industries (Germany). Poly(vinyl alcohol) (PVA 205) was obtained from Kuraray China Co., Ltd.(China). Coumarin 6 were purchased from J&K chemical Ltd.(China). Tween 80 was obtained from Tianjin Bodi Chemical Co., Ltd. (Tianjin, China). DAPI (4′,6-diamidino-2-phenylindole) staining solution and antifade mounting medium were bought from Beyotime Institute of Biotechnology (China).
6
Chitosan-Based Ophthalmic Formulation
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Chitosan (CS, viscosity average molecular weight 30 kDa, degree of N-deacetylation 75–85%) was purchased from Jinan Haidebei Marine Bioengineering Co., Ltd. (China) and was used as received. Sodium chloroacetate, α-cyclodextrin (α-CD), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and malic acid were purchased from Sigma-Aldrich (St Louis, MO, USA) and used without further purification. Cyanine 5 (Cy5) NHS ester was purchased from Lumiprobe Corporation (Hallandale Beach, FL, USA). Coumarin 6 (C6) was purchased from J&K Scientific Ltd. (Beijing, China). Dialysis bags (MW cutoff: 3.5 and 14 kDa) were purchased from Shanghai Green Bird Technology Development Co., Ltd. (Shanghai, China), and stored in 1 mM ethylenediaminetetraacetic acid aqueous solution prior to use. Econazole nitrate (ECZ·HNO3) was purchased from Hefei Bomei Biotechnology Co., Ltd. (China), and ECZ base was prepared from ECZ nitrate as previously described (Pedersen et al., 1998 ). Methanol [high-performance liquid chromatography (HPLC) grade] was purchased from Tedia Company (Fairfield, CT, USA). Injection water was used to prepare the eye drop solution. All other reagents were of analytical grade and were used as received.
7
Synthesis and Characterization of Multifunctional Nanoparticles
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PTX was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Maleimide-[(polyethyleneglycol)2000]-carboxylic acid (Mal-PEG2000-COOH) and Methoxy-[(polyethyleneglycol)2000]- carboxylic acid (mPEG2000-COOH) were brought from AVT Shanghai Pharmaceutical Tech Co., Ltd. (Shanghai, China). The polypeptide dimer (CWQPDTAHHWATL)2K was custom-synthesized by Tanshui-Tech (Guangzhou, China). 2-Methylimidazole was purchased from Beijing Huabozhan Bioanalytical Technology Co., Ltd. (Beijing, China). Zinc nitrate hexahydrate was bought from Lanzhou yellow river institute of Zinc and Magnesium Nanomaterials (Lanzhou, China). Coumarin 6 (C6) was purchased from J&K Scientific Co., Ltd. (Beijing, China). Trypsin, RPMI 1640 medium and phosphate buffer saline (PBS) were purchased from Thermo Fisher Scientific Co. (Beijing, China). Cell counting kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan). All reagents were analytical grade and used without further purification.
8
Formulation and Evaluation of PUE-Loaded Nanoparticles
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1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (PEG2000-PE) was acquired from Lipoid GmbH (Ludwigshafen, Germany). PUE and its injection were purchased from Shandong Fangming Pharmaceutical Group Co., Ltd (Shandong, China). Coumarin-6 (C6) and isoprenaline (ISO) were purchased from J&K Chemical Ltd (Shanghai, china) and 4'6-diamidino-2-phenylindole (DAPI) was provided by Beyotime Biotech (Jiangsu, China). H9c2 cells derived from rat myocardium were purchased from the American Type Culture Collection (Manassas, VA). Cell cultures were maintained in a humidified atmosphere of 5% CO2 at 37 °C. All other chemicals and reagents were analytical grade or chromatography grade.
9
Cellular Uptake of C6@PEG-PE Micelles
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For cellular uptake studies, Coumarin-6 (C6) (J&K Chemical Ltd, Shanghai, China) was loaded into PEG-PE using the method for preparation of BTM-loaded micelles as described above to formulate C6@PEG-PE micelles. CT26 cells were seeded into 12-well culture plates at 1.0×105 cells per well and cultured for 24 h. Cells were then incubated with C6@PEG-PE micelles or C6 solution at an equivalent C6 concentration (0.1 mg/L). After 1 h, 2 h or 4 h of incubation, cells were washed with PBS (pH 7.4) three times to eliminate residual C6, followed by fixing with paraformaldehyde (PFA) and staining with 4ʹ6-diamidino-2-phenylindole (DAPI, Beyotime, China). C6 and DAPI showed green and blue colorations, respectively. Cellular internalization of C6@PEG-PE micelles in CT26 cells was observed using Axio Vert A1 fluorescence microscope and then analyzed by using ZEN software (Carl Zeiss, Germany). Also, to investigate the efficiency of cellular uptake, the fluorescence intensity of CT26 cells was analyzed 1 h, 2 h or 4 h post-incubation using flow cytometry (BD, USA).
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