Caco-2 cells were seeded at a density of 250,000/well in 24 well plates. Porcine gastric mucin type II (Sigma) was dissolved in Caco-2 culture media at 37 °C at a range of 2–5% (w/v). 500 uL of the mucin/culture media solution was pipetted on top of the Caco-2 cells. LNPs formulated with varying mol% of PEG (1.5–10) were delivered on top of the mucin to the Caco-2 cells for 24 hours at a dose of 100 nM.
Porcine gastric mucin type 2
Manufactured by Merck Group
Sourced in United States, United Kingdom
Porcine gastric mucin type II is a purified glycoprotein derived from porcine stomach tissue. It is a high-molecular-weight compound that exhibits gel-forming and lubricating properties.
Automatically generated - may contain errors
Lab products found in correlation
Potassium phosphate, by Merck Group (3 mentions)
Potassium citrate, by Merck Group (3 mentions)
Uric acid sodium salt, by Merck Group (3 mentions)
Kolliphor p407, by BASF (2 mentions)
Ammonium nitrate, by Merck Group (2 mentions)
Potassium chloride (kcl), by Merck Group (2 mentions)
Lactic acid sodium salt, by Merck Group (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
Urea, by Merck Group (2 mentions)
Minimum essential medium (mem), by Merck Group (1 mentions)
Benecel e10m, by Ashland (1 mentions)
Carbopol 974p nf, by Lubrizol (1 mentions)
Hydroxypropylbetacyclodextrin, by Roquette (1 mentions)
Sodium chloride (nacl), by Fresenius (1 mentions)
Potassium chloride (kcl), by Fresenius (1 mentions)
Protanal lf 10 60 ft, by FMC Biopolymer (1 mentions)
Urea, by Thermo Fisher Scientific (1 mentions)
Sodium citrate dihydrate, by Thermo Fisher Scientific (1 mentions)
Agar, by Merck Group (1 mentions)
Sodium dl lactate, by Merck Group (1 mentions)
20 protocols using porcine gastric mucin type 2
1
Mucin-Mediated LNP Delivery to Caco-2 Cells
2
In Vitro Antiviral Screening of Mucoadhesive Formulations
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The 18β-glycyrrhetinic (GA) 97%, dexpanthenol, benzalkonium chloride, and porcine gastric mucin (type II) were purchased from Sigma-Aldrich (St. Louis, MO, USA); Kelcogel® CG-LA (deacetylated gellan gum, DGG) was kindly provided by Azelis (Istanbul, Türkiye); VANZAN® NF (xanthan gum) was kindly provided by Vanderbilt Minerals, LLC (Norwalk, CT, USA); Blanose™ (sodium carboxymethylcellulose, Na-CMC) and Benecel™ E10M (hydroxypropyl methylcellulose, HPMC) were kindly gifted by Ashland (Istanbul, Türkiye), Carbopol® 974P NF was provided as a gift from Lubrizol (Wickliffe, OH, USA); and methanol ≥ 99.9% for liquid chromatography and ortho-phosphoric acid 85% were supplied by Isolab Chemicals (Istanbul, Turkey). Fetal bovine serum (FBS), Dulbecco’s phosphate buffered saline (DPBS), antibiotic-antimycotic solution (100×), ribavirin and minimum essential medium (MEM) were purchased from Sigma-Aldrich (St. Louis, MO, USA); 0.25% Trypsin-EDTA (1×) solution was provided from (Diagnovum, Ebsdorfergrund, Germany); Trypan blue dye was purchased from NutriCulture (Skelmersdale, UK); and the XTT [2,3-Bis- (2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide] kit was obtained from Biological Industries Ltd. (Kibbutz Beit Haemek, Israel). All other chemicals and reagents used in the study were of analytical grade.
3
Mucoadhesive Dexamethasone Acetate Formulation
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Dexamethasone acetate was purchased from Aventis (Gentilly, France). Kolliphor-P407 was from BASF (Mississauga, Canada). Hydroxy-propyl-beta-cyclodextrin was purchased from Roquette (Paris, France). Mucoadhesive agents used in the experiments were xanthan gum (Satiaxane from Cargill, Puteaux, France), carbomer (Carbopol C971P from Lubrizol, Brussels, Belgium) and sodium alginate (Protanal LF 10/60 FT from FMC Biopolymer, Cork, Ireland). Porcine gastric mucin type II was from Sigma Aldrich (Saint-Louis, MO, USA). All the experiments were performed using sterile water from Fresenius Kabi (Sévres, France).
For the HBSS medium preparation, the chemical products used were calcium chloride × 2H2O, potassium chloride, potassium hydrogen phosphate anhydrate, sodium chloride, disodium dihydrogen phosphate × H2O, sodium hydrogen carbonate, magnesium chloride × 6H2O and magnesium sulfate × 7H2O. All these products were purchased from Sigma Aldrich (Saint-Louis, MO, USA).
For the HBSS medium preparation, the chemical products used were calcium chloride × 2H2O, potassium chloride, potassium hydrogen phosphate anhydrate, sodium chloride, disodium dihydrogen phosphate × H2O, sodium hydrogen carbonate, magnesium chloride × 6H2O and magnesium sulfate × 7H2O. All these products were purchased from Sigma Aldrich (Saint-Louis, MO, USA).
4
Bacterial Culture and Simulated Digestion
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For bacterial culture preparation, MRS broth was obtained from Becton Dickinson and Company (Sparks, MD, USA). Agar, L -cysteine hydrochloric acid, and sodium chloride (NaCl) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Glycerol and sodium citrate dihydrate was purchased from Fisher Scientific (Fair Lawn, NJ, USA).
For encapsulation experiments, sodium alginate (TICA-algin HG 400 powder) was donated by TIC Gums (White Marsh, ML, USA). Calcium chloride hexahydrate, chitosan (medium molecular weight) was obtained from Sigma-Aldrich. Glacial acetic acid was purchased from Fisher Scientific.
For simulated digestion, ammonium nitrate, bile extract porcine, lipase from porcine pancreas type II, pepsin from porcine gastric mucosa, porcine gastric mucin type II, potassium chloride, potassium citrate, potassium phosphate, sodiumDL -lactate, sodium hydroxide (NaOH), and uric acid sodium salt were also purchased from Sigma-Aldrich. Hydrochloric acid (HCl), phosphate buffer saline (PBS), and urea were purchased from Fisher Scientific.
For encapsulation experiments, sodium alginate (TICA-algin HG 400 powder) was donated by TIC Gums (White Marsh, ML, USA). Calcium chloride hexahydrate, chitosan (medium molecular weight) was obtained from Sigma-Aldrich. Glacial acetic acid was purchased from Fisher Scientific.
For simulated digestion, ammonium nitrate, bile extract porcine, lipase from porcine pancreas type II, pepsin from porcine gastric mucosa, porcine gastric mucin type II, potassium chloride, potassium citrate, potassium phosphate, sodium
5
S. mutans Biofilm Growth and Characterization
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S. mutans UA159 (ATCC 700610) biofilms were grown for 72 h on glass microscope slides at 37°C and 5% CO2 in brain heart infusion (Sigma-Aldrich, St. Louis, MO) supplemented with 2% (wt/vol) sucrose (Sigma-Aldrich) and 1% (wt/vol) porcine gastric mucin (type II; Sigma-Aldrich). After the growth period, the biofilm-covered slides were gently rinsed in 1% (wt/vol) phosphate-buffered saline solution (Sigma-Aldrich) and placed in petri dishes (49 ). Biofilm thickness was determined by fixing untreated samples with 4% (wt/vol) paraformaldehyde and staining with Syto 63 (Thermo Fisher Scientific, UK). Subsequently, three random confocal images were taken on three independent replicate biofilm slides, with a thickness of 51.8 ± 4.9 μm measured using COMSTAT software; thus, we used a biofilm thickness (Lb) of 55 μm in the simulations.
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6
Mucoadhesive Drug Delivery Systems
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Suppocire BP (SBP) pellets (Gattefossé Ltd., Saint-Priest, France), Methocel E4M HPMC (Colorcon Ltd., Dartford, United Kingdom), Kolliphor RH40 (KP) (BASF ChemTrade GmbH, Ludwigshafen, Germany), amoxicillin (AMX) (Antibióticos de León S.L.U., León, Spain), cetostearyl alcohol (CA), metronidazole (MZ), zinc gluconate (ZnGlu) (Ph. Eur. 8., Hungaropharma Plc., Budapest, Hungary), and zinc hyaluronate (ZnHA) (Richter Gedeon Ltd., Budapest, Hungary) were used to create the delivery systems.
For the mucoadhesion and drug dissolution tests, a phosphate-buffered saline (PBS) solution was prepared by dissolving 8 g/dm3 NaCl, 0.2 g/dm3 KCl, 1.44 g/dm−3 Na2HPO4 · 2 H2O and 0.12 g/dm3 KH2PO4 (Ph. Eur. 8., Hungaropharma Plc., Budapest, Hungary) in distilled water. The pH was adjusted to 7.4 by adding an adequate amount of 0.1 M HCl. The mucin (porcine gastric mucin type II, Sigma-Aldrich, Saint Louis, MO, USA) was used in the form of an 8 w/w% suspension.
For the mucoadhesion and drug dissolution tests, a phosphate-buffered saline (PBS) solution was prepared by dissolving 8 g/dm3 NaCl, 0.2 g/dm3 KCl, 1.44 g/dm−3 Na2HPO4 · 2 H2O and 0.12 g/dm3 KH2PO4 (Ph. Eur. 8., Hungaropharma Plc., Budapest, Hungary) in distilled water. The pH was adjusted to 7.4 by adding an adequate amount of 0.1 M HCl. The mucin (porcine gastric mucin type II, Sigma-Aldrich, Saint Louis, MO, USA) was used in the form of an 8 w/w% suspension.
7
Lectin-Based Nanoparticle Targeting Mucin
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Trimyristin (Dynasan 114), was donated by Cremer® (Hamburg, Germany). Egg lecithin E80 was purchased by Lipoid® (Ludwigshafen, Germany). The material used as a conjugated polymer (CN-PPV), Nile red, wheat germ agglutinin conjugated with fluorescein (WGA-FITC), surfactant (PEG-660 stearate), castor oil (CO), Dulbecco’s Modified Eagle’s Medium (DMEM), quercetin (QU), porcine gastric mucin (type II), rat red cell suspension, N-acetylglucosamine, PBS-BSA solution, antibody anti-WGA, anti-rabbit IgG produced in goat, dialysis bag MWCO 10000, and Ortho-Phenylene-Diamine were purchased by Sigma-Aldrich (Saint Louis, MO, EUA). The modified N-Acetyl-b-D-glucosaminyl-PEG900-stearate conjugate (C18PEG900GlcNAc) was synthesized by Alexandre G. Dal Bó [10 (link),11 (link),23 (link)]. WGA lectins were extracted by Luciano Pinto. The other reagents used were of analytical grade.
8
Quantitative ELISA for WGA Lectin
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To detect the WGA lectin and verify its functionality after the preparation of the formulation, an ELISA test was performed [24 (link)]. Briefly, a 96-well plate was precoated with 0.1% porcine gastric mucin (Type II, Sigma-Aldrich, Saint Louis, MO, USA) overnight. A PBS-BSA solution (0.1%) blocked nonspecific bonds. To perform a calibration curve, protein WGA diluted in PBS (15–1000 ng/mL) and samples (NLC and NLC-WGA) were added to the plate and incubated for 1 h. After washing the plate three times, the primary antibody (anti-WGA 1:2000) was added and incubated for 1 h. Afterward, the PBS was used to wash the plate, and the antibody conjugated with peroxidase (anti-rabbit IgG produced in goat 1:5000) was added. The substrate used for peroxidase activity detection was Ortho-Phenylene-Diamine (OPD). After 30 min, the addition of sulfuric acid (2.5 M) solution stopped the reaction, and the resulting absorbance was read on a spectrophotometer at 490 nm.
9
Ketamine-Loaded Poloxamer-Alginate Hydrogels
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(S)-ketamine hydrochloride (SK) was purchased from Seqens (Lahr/Schwarzwald, BW, Germany). Three batches of poloxamer 407 (P407, Kolliphor 407) were obtained from BASF (Geismar, LA, USA). “Low-viscosity” alginates were used (viscosity < 100 mPa·s at 20 °C for a 1% alginate solution). Three grades of sodium alginate, based on the proportions of 1,4 β-D-mannuronic acid (M) and 1,4 α-L-guluronic acid (G) residues, were bought from FMC Biopolymer (Cork, Ireland): Protanal LF 10/60 (PRO), Manucol DH (MAN), and Keltone LVCR (KEL). Water used here was sterile water, Versylène®, obtained from Fresenius Kabi (Sevres, France). Sodium chloride (NaCl), potassium dihydrogen phosphate (KH2PO4), and sodium bicarbonate (NaHCO3) were purchased from Cooper (Melun, France). Potassium chloride (KCl) and di-potassium hydrogen phosphate (K2HPO4) were obtained from Kirsch Pharma (Salzgitter, Germany). Calcium chloride (CaCl2, 2H2O) was purchased from VWR International (Fontenay-sous-Bois, France). The porcine gastric mucin type II was purchased from Sigma-Aldrich (St. Louis, MO, USA).
10
Artificial Saliva Composition and Preparation
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Artificial saliva was freshly prepared daily, its composition was adapted from Minekus et al., (2014) containing sodium chloride (1.126 g L -1 ), potassium phosphate (0.504 g L -1 ), sodium bicarbonate (1.142 g L -1 ), magnesium chloride hexahydrate (0.03 g L -1 ), ammonium carbonate (0.006 g L -1 ), calcium chloride dihydrate (0.221 g L -1 ), 3 g L -1 of mucin (porcine gastric mucin type II, Sigma Aldrich), and αamylase (8.7 g L -1 ) (A317, type VI-B from porcine pancreas, Sigma Aldrich) was added 10 minutes prior using the artificial saliva.
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