Optima l 80 xp ultracentrifuge

Medium was collected from hMEC, hLEC, hAEC and hPEC, and centrifuged for 20 min at 1500g to remove cell debris. 10 mL of sterile/filtered phosphatase buffer saline (PBS) was added to the media of each sample placed in ultracentrifugation tubes. Samples were centrifuged for 60 min at 20,000g at 4 °C (OptimaTM L-80 XP Ultracentrifuge Beckman Coulter). The microparticle (MPs) pellet was resuspended in sterile PBS for cell stimulation (~ 200 µL). MPs counting was verified using the Nanosight technology (Nanosight^® LM14).

Bone morrow mesenchymal stem cells (BMSCs) were isolated from the bone marrow of mouse femurs as previously described (Boregowda et al., 2016 (link)). BMSCs were identified by using osteogenic, adipogenic, and chondrogenic differentiation media to confirm their multipotent differentiation potential. BMSCs were characterized by flow cytometry after being incubated with antibodies against specific surface antigens, including CD45, CD34, CD11b, CD29, CD90, and Sca-1 (BD Biosciences. San Jose, CA, United States) according to the manufacturer’s recommendations. Final quantification was conducted with a BD FACSAria III flow cytometer (BD Biosciences). Stained BMSCs were analyzed and compared to the corresponding unstained cells. The data were analyzed using flow cytometry data analysis software (FlowJo V10, FlowJo, LLC, Ashland, OR, United States). Exosomes were isolated from BMSCs at passages 3–5 using an Optima^TM L-80XP Ultracentrifuge (Beckman Coulter). Exosome concentrations and diameters were measured using IZON qNano Nanoparticle (Zen-Bio, Inc., Research Triangle Park, North Carolina) analysis. Exosome protein and RNA concentrations were analyzed using a NanoDrop spectrophotometer (Thermo-Fisher Scientific, Waltham, MA, United States). The exosomes were visualized by transmission electron microscopy (TEM).

EV isolation from MSCs at the flask-scale was performed essentially as described previously [10 (link), 26 (link)]. Low passage number [3 (link)–6 (link)] of non-senescent frozen stocks of MSCs from the six human isolates (F_1 to F_6) were expanded in T-75 cm² plastic flasks and grown to near confluence. The cells were washed with PBS (Hyclone) and replaced with MSC serum-free media (SFM) (StemPro A103332-01, ThermoFisher Scientific, Waltham, MA, USA) at 10 mL/flask. To produce LPS-EVs, MSCs from 3 isolates (F_1-F_3) were primed with LPS as described [10 (link)]. Briefly, MSCs were either unprimed (EVs) or primed with 1.0 ug/mL of E. coli LPS O111:B4 (L4391 Sigma, St Louis, MO, USA) (LPS-EVs) in SFM for 18–24 h. The conditioned media was collected, then centrifuged at a low-speed spin (2000 × g at 4 °C for 20 min) to remove any cell debris, followed by an ultra-centrifugation (UC) step (100,000 g _avg at 4 °C for 2 h) of the supernatant using Optima™ L-80XP Ultracentrifuge and SW28 rotor (Beckman Coulter Inc. Indianapolis, IN, USA). EVs or LPS-EVs were re-suspended in 1 ml PBS (Hyclone) per 300 mL of conditioned media, aliquoted, then stored at − 80 °C.

The thermosensitive formulations were prepared by the reverse phase evaporation method [16 (link)]. Liposomes were composed of DPPC:CHOL:SA in lipid molar ratios of 95:2.5:2.5 for a total lipid concentration of 40 mmol L-1. For the preparation of CDDP-loaded liposomes (TSL-CDDP), CDDP solution (2.0 mg/mL) was prepared in 0.9% (w/v) NaCl and added to the lipid film dissolved in ethyl ether. After size calibration, the untrapped CDDP was separated from the liposomes by ultracentrifugation (Optima^® L-80XP ultracentrifuge, Beckman Coulter, Brea, CA, USA) at 44,800× g at 10 °C for 30 min. The pellet was reconstituted in 0.9% (w/v) NaCl to obtain the initial volume of the liposomal formulation. TSL and TSL-CDDP were functionalized with 4 kDa HA by electrostatic interaction according to the method described by Ravar and collaborators [17 (link)].

Exosomes were isolated from 120 mL PEs with differential centrifugations as previously described.^{[29 ]} To remove cells and debris, PE supernatants were sequentially centrifuged for 20 minutes at 1200 × g and 30 minutes at 10,000 × g. To remove particles greater than 200 nm, supernatants were filtered through 0.22 μm pore filters and rinsed with PBS using ultracentrifugation (Optima L-80XP Ultracentrifuge, Beckman Coulter, Beckman) for 1 hours at 120,000 × g twice. All the steps were carried out at 4°C. Finally, the pellets were resuspended in PBS and stored at −80°C for further use.

The phase solubility studies were carried out according to Higuchi and Connors [18 ]. Briefly, an excess amount of the drug was added to aqueous solutions containing increasing amounts, 0 to 60% (w/v) of HPβCD or HPγCD. After 24 h under magnetic stirring at 25 °C, the drug suspensions were ultracentrifugated for 1 h at 35,000 rpm (Optima L-80 XP Ultracentrifuge BECKMAN COULTER, Brea, CA, USA). Note that our operating conditions are limited to 24 h according to preliminary studies, showing that 24 h and 72 h did not change the equilibrium. The supernatant was then diluted at 1:50 in the mobile phase and analyzed by HPLC. The experiments were repeated three times for each cyclodextrin derivative.
The apparent stability constant of the drug/cyclodextrin complex (D/CD), assuming that one molecule of drug forms a complex with one molecule of cyclodextrin (K_1:1), can be calculated from the slope of the linear phase-solubility profiles and the intrinsic drug solubility in the complexation media [18 ,19 (link)] in the absence of the cyclodextrins as presented in Equation (1):
$K_{1:1}=\frac{\mathrm{Slope}}{S_0\times \left(1-\mathrm{Slope}\right)},$
The complexation efficiency (CE) can be calculated by applying the following Equation (2), which also refers to the slope of the linear phase-solubility profiles [20 (link)] and intrinsic solubility:
$\mathrm{CE}=K_{1:1}\times S_0=\frac{\mathrm{Slope}}{\left(1-\mathrm{Slope}\right)},$