Amicon ultra 0.5 ml

To evaluate the effect of molecular charge on rhGH, a permeation study investigating iontophoresis and microneedle treatment was conducted using two different buffers (PBS, pH 7.4 and citrate buffer, pH 4.0) to prepare a liquid rhGH formulation (isoelectric point = 5.27) with two different charges. Buffer exchange was performed using a centrifugal filter device (Amicon Ultra 0.5 mL, Millipore, Burlington, MA, USA) with a 3000-Da molecular weight cut-off. Because rhGH is negatively charged at pH 7.4 and positively charged at pH 4.0, formulations were delivered under cathodal and anodal iontophoresis, respectively. A constant current of 0.5 mA/cm² was applied for 4 h through an Ag/AgCl electrode to both groups.
Furthermore, to evaluate the effect of current density on rhGH permeation, the permeation study on anodal iontophoresis and microneedle treatment was conducted using three different current densities. Current densities of 0.125, 0.25, or 0.5 mA/cm² were applied for 4 h through Ag/AgCl electrodes.

Enzymatic reactions were analysed by denaturing PAGE [4–8% polyacrylamide (acrylamide/N, N′-methylenebisacrylamide, 19:1), 7.5 M urea, 25% (v/v) formamide, 89 mM Tris, 89 mM boric acid, 2 mM EDTA]. The RNA samples were mixed with equal amount of 2× formamide loading solution [80% (v/v) formamide, 10 mM EDTA pH 8.0, 0.1 mg/mL xylene cyanol FF, 0.1 mg/mL bromophenol blue], and heated at 90 °C for 3 min before being loaded on the gel. The gels were electrophoresed and stained with SYBR Green II (Lonza) and visualised on a BioRad ChemiDoc XRS+ System (BioRad, Hercules, CA, USA), Luminescent Image Analyser LAS 4000 (Fujifilm, Tokyo, Japan) or FAS-IV Imaging System (Nippon Genetics, Tokyo, Japan). Low Range ssRNA Ladder (New England Biolabs, Ipswich, MA, USA) was used as the size marker. RNAs were also purified by preparative denaturing PAGE. The RNA bands were visualised by UV shadowing, and crushed and extracted with water. The extracts were desalted by centrifugation with Amicon Ultra-0.5 mL or Amicon Ultra-4 mL centrifugal filter unit (Millipore, Billerica, MA, USA), according to the manufacturer’s instructions. The RNAs were then precipitated with sodium acetate (pH 5.2) and 2-propanol.

VISCODERM® Skinkò E formulation was concentrated on a polyethylene sulfate membrane with a nominal molecular weight limit (NMWL) of 3 kDa (Amicon Ultra 0.5 mL Centrifugal filter: Ultracel 3 kDa, Millipore). The concentration was diafiltered using 2 volumes of HPW (High Purified Water). Successively, the ultrafiltration (UF) retentate on the 3 kDa centricon tube was recovered and eventually diluted for the hydrodynamic characterization by SEC-TDA; whereas, the permeate 3 kDa was analyzed by uronic acid assay (E.P. ed 5.0: 2.2.25). This last assay quantified the residual HA in the permeate and diafiltered sample that accounted for about the 25% w/w of the initial HA amount. Therefore, the permeate 3 kDa contained all the Skinkò E components and a residual 1,84 mg/mL of HA. This was diluted 1:2, tested as for Skinkò E, and thus, the final concentration of HA in the cell's plate was 3,2 mg/mL for the complete formulation and only 0,92 mg/mL for the permeation. In this case, the HA effect should be reduced with respect to the other compounds present in the formulation.

Silver nitrate (AgNO₃) was purchased from Kojima Chemicals Co., Ltd. Polyvinylpyrrolidone powder (PVP, average M.W. ~ 29,000), sodium citrate (trisodium salt: dehydrate) (Na₃CA, ≥ 99.5%), sodium borohydride (NaBH₄, ≥ 98.0%), L-homocysteine (Hcy), L-cysteine (Cys), L-glutathione reduced (GSH), L-lysine (Lys), L-tyrosine (Tyr), L-glutamic acid (Glu), L-asparagine (Asn), L-phenylalanine (Phe), L-methionine (Met), glycine (Gly), L-alanine (Ala), L-histidine (His), L-leucine (Leu), and L-tryptophan (Trp), and human serum (from human male AB plasma) were purchased from Sigma-Aldrich. Hydrogen peroxide (H₂O_2, 35%) was purchased from Junsei Chemical Co., Ltd. Centrifugal filters (Amicon Ultra-0.5 mL, Ultracel -3 K) were obtained from Millipore. All reagents were used without further purification. The ultrapure water was prepared from a Millipore Q-Gard water purification system (Milli-Q) with a filter membrane of 0.22 µm, which was used for the preparation of aqueous solutions of all the reagents.

Exosomes were isolated from the cell culture media and serum with total exosome isolation reagent (Thermo Fisher Scientific, MA, USA). The concentration of exosomal proteins was quantified with a BCA Protein Assay Kit (Beyotime, Nanjing, China). The purified exosomes were incubated with 4 μM of PKH67 (MIDI67, Sigma ‐Aldrich, MO, USA) and excess dye was removed using a 100‐kDa filter (Amicon Ultra‐0.5 mL, Millipore, Darmstadt, Germany). These nuclei were then labeled with 4’,6‐diamidino‐2‐phenylindole (DAPI) according to the manufacturer's guidelines (Beyotime).

Hydrolysed forms of the β-lactam antibiotics (ampicillin, cephalexin, and amoxicillin) were prepared by dissolving the drug in KPi buffer (100 mM, pH 7) to form stock solutions (10 mM). To 1 mL of antibiotic stock solution was added 100 μL β-lactamase blend (β-lactamase I (60–1500 IU/mL) and β-lactamase II (6–15 IU/mL)) in KPi buffer (50 mM, pH 7). The solutions were then vortexed and incubated for 48 h at 37 °C. β-lactamase enzymes were then removed from the solution using a centrifugal filter (Amicon^® Ultra 0.5 mL, Millipore (UK) Limited, Watford, UK) with a 30 KDa cut-off and the hydrolysed β-lactam recovered. Hydrolysis of the β-lactam was confirmed by high resolution mass spectrometry and ¹H NMR.
For use in the assay, serial dilutions of the stock concentration of the hydrolysed β-lactam antibiotics (10 mM) were prepared in KPi buffer (100 mM) at the required pH between 1 nM and 10 mM.