Regenerated cellulose

For VPA1361 immunoblotting analysis, the overnight cultured WT was diluted into an M9 medium with 0.5% or 3% NaCl and cultured at 37 °C for 5–6 h. Next, 0.04% bile salt was added to induce T3SS2 expression. The bacterial cultures were adjusted based on the OD₆₀₀ value of the WT and ΔVPA1361 strains, then the cell pellets and supernatant were collected. The supernatant protein was concentrated using protein filter columns Ultracel and Regenerated Cellulose (Millipore, Billerica, MA, USA), and the cell pellets were suspended in PBS. Loading buffer was added to each sample, and the mixture was boiled for 10 min. A 20 μL aliquot of each normalized sample was then separated on a 12% polyacrylamide gel and transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking in 10% skim milk for 2 h, the PVDF membranes were incubated with the VPA1361 antibody or RNAP antibody at a 1:1000 dilution and then incubated with goat anti-rabbit IgG (Beyotime, Shanghai, China) at a 1:2000 dilution. Finally, the blots were visualized using ECL reagent.

The peripheral blood of nude mice was collected from abdominal vein after being killed by cervical dislocation, kept into a venous blood sample collection vessel, and centrifuged at 4000 rpm per minutes for 5 min. The peripheral blood was routinely examined by MS9 automatic hematology analyzer (France), and the supernatant was analyzed for hepatic and renal function examination by automatic biochemical analyzer (Hitachi 7600, Japan). Plasma samples were deproteinized with 5% trichloroacetic acid solution and centrifuged at 10,000 rpm for 15 min at 4 °C before measurement. The supernatant fractions were filtered through regenerated cellulose (Millipore, Bedford, MA, USA). The amino acid concentrations in the plasma were measured by HPLC-electrospray ionization-mass spectrometry followed by derivatization as described previously [9 (link)].

Cells at passages 1–15 were cultured in vesicle-depleted medium (with 5% depleted FBS) for 48 h before vesicle collection. All media containing exosomes were filtered through a 0.2-μm membrane filter (regenerated cellulose, Millipore), isolated by differential centrifugation (first at 10,000 × g and subsequently at 100,000 × g)^{21 (link)}, and used for exosome analysis with the APEX platform. For exosome isolation from blood cells and platelets, blood cells were derived from blood fractionation and platelets from platelet-rich plasma. All vesicles were collected as previously described⁴⁷ . For independent quantification of exosome concentration, we used the nanoparticle tracking analysis (NTA) system (NS300, Nanosight). Exosome concentrations were adjusted to obtain ~50 vesicles in the field of view to achieve optimal counting. All NTA measurements were done with identical system settings for consistency. Note that exosome isolation was only performed for cell culture experiments. Clinical measurements were performed on native plasma samples, without needing for any vesicle isolation.

Oligonucleotide d(G₄C₂) was synthesized on K&A Laborgeraete GbR DNA/RNA Synthesizer H-8 using standard phosphoramidite chemistry in DMT-off mode. Deprotection was performed with overnight incubation in 20% aqueous ammonia at 50 °C. Then, 2 M LiCl was added before heating samples at 90 °C for 10 min. Samples were purified and concentrated using an ultra-filtration device (Merck Millipore, Watford, UK) and an ultra-filtration membrane with a cut-off mass of 1 kDa (regenerated cellulose, Millipore). The samples were lyophilized overnight and diluted in 90% H₂O and 10% ²H₂O to a final concentration of 2.0 mM per strand in the presence of 100 mM of ¹⁵NH₄Cl or 100 mM of ¹⁵NH₄Cl and 80 mM of KCl. Sample concentrations were determined by measuring absorbance at 260 nm using a Varian Cary 100 Bio UV-VIS spectrometer (Varian Inc., Palo Alto, CA, USA, Agilent Tecnologies, Santa Clara, CA, USA). The extinction coefficient was determined by the nearest-neighbor method.

Cells were cultured in vesicle-depleted medium (with 5% depleted FBS) for 48 h before vesicle collection. All media containing vesicles were filtered through a 0.2-μm membrane filter (regenerated cellulose, Millipore), isolated by differential centrifugation (first at 10,000 g and subsequently at 100,000 g), and used for molecular analysis. For independent quantification of vesicle concentration, we used the nanoparticle tracking analysis (NTA v3.3) system (NS300, Nanosight). Vesicle concentrations and diameters were adjusted to obtain ~50 vesicles in the field of view to achieve optimal counting. All NTA measurements were done with identical system settings for consistency.

A collagen matrix was prepared by electrodeposition as previously described [1 (link)]. A collagen suspension of 5 mg/mL was used. The deposition was conducted in an electrochemical cell, divided by a semipermeable barrier of regenerated cellulose (Sigma-Aldrich, St. Louis, MO, USA) into two parts, each of which contained either the cathode or anode. The collagen suspension was poured into the anode part of the cell, while distilled water was poured into the cathode part of the cell. The cathode and anode were plate electrodes, connected to a DC source with a voltage of 60 V. The SBA-EPD-obtained membranes were carefully detached from the semipermeable barrier and treated with isopropanol for 20 min, then they were dried in a laminar flow hood.

Before the nanospheres were encapsulated in an alginate microbead,
CsA released from the nanospheres was studied to develop a controlled
drug delivery system. Using a dialysis membrane with a molecular weight
cutoff of 15 kDa composed of regenerated cellulose (Sigma-Aldrich,
USA), the controlled release patterns of CsA from PLGA nanospheres
were investigated. The dialysis membrane was washed with distilled
water before use. After centrifuging the nanosphere, the nanosphere
pellet containing the entrapped drug (130 mg of cyclosporine was entrapped
in the nanospheres) was collected and dispersed in 1 mL of a released
solution containing phosphate buffer saline and 0.05% w/v of nonionic
surfactant Tween 80 at pH 7.4.^{36 (link)} The 10
mL of released medium was placed outside the membrane. The drug release
from PLGA nanospheres was studied in an incubator (Wisecube, Fuzzy
Control System, Witeg Laboratory Instruments) with a shaking speed
of 90 rpm and a temperature of 37 °C. At predetermined intervals,
1 mL of released solution was collected, and 1 mL of medium was added
to the outside of the membrane. Using HPLC (Dionex Ultimate 3000),
we determined the amount of CsA released from the nanospheres. The
amount of CsA loaded in the nanospheres was used to calculate the
CsA release (ug) in the medium, which was then determined as a percentage
of the total CsA release.^{29 (link),30 (link)}