Edc nhs

The process of PDMS surface modification is consisted with three steps as follow: 1) PDMS oxidation via plasma treatment (PDMS-OH). The samples were exposed to oxygen plasma (PC150, JunSun Tech Co., Ltd) to create a hydrophilic surface. They were treated with oxygen plasma at 10⁻² Torr for 5 mins at 50W, and an oxygen flow rate of 17 sccm. 2) Aminization of PDMS substrates (PDMS-NH2). After plasma treatment, the PDMS membranes were immersed in a silane solutions of 1% by volume APTES (Ca.440140, Sigma-Aldrich, Mo) in absolute ethanol. Then, 5% of by volume DI water was added to the solution to hydrolyze the silane and allowed to react for 15 min at 75°C. The PDMS samples were washed once with 75% by volume aqueous ethanol and the three times with DI water following the silane reaction to remove residual silane compounds. The aminized PDMS membrane is denoted as PDMS-NH₂. 3) Surface grafting of laminin onto PDMS-NH2 membrane. Conjugation of laminin on PDMS-NH₂ membrane was performed by crosslinker EDC/NHS (Sigma-Aldrich, Mo). EDC/NHS (1:1 molar ratio) were added to 10 μg/ml laminin in PBS buffer to obtain a final concentration of 10mM, and allowed to react with PDMS-NH₂ membrane for 1 hr at 37°C. The PDMS membranes were then washed by DI water to remove residual reagents, and rinsed by PBS before the cell seeding.

The Bombyx mori silkworm silk was purchased from Huzhou in Zhejiang province, China. Chitosan (Molecular weight =190~310 kDa, >75% deacetylated) and EDC/NHS were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The ethanol, sodium hydroxide, hydrochloric acid, phosphate buffer saline (PBS), and anhydrous sodium carbonate were all of analytical grade and purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

Here, we describe our synthesis protocol for preparation of selenium nanoparticles (NPs) with selenium core and polyethylene glycol (PEG) surface coating. Initially, 2 μg/mL poly (ethylene glycol)-carboxylic acid functionalized (average Mw 5 kDa, Sigma- Aldrich) was directly mixed with 1800 μL of 0.1 M selenious acid 98% (Sigma-Aldrich) in 10 mL deionized (DI) water, by constant stirring. Then, 3 mL of aqueous 0.1 M ascorbic acid solution (A92902, Sigma-Aldrich) was added dropwise and the resulting solution was stirred at room temperature. After 30 min, 20 μL EDC/NHS (Sigma-Aldrich) (75 mM/30 mM, v/v, 1:1) solutions was directly added into the Se-containing solution and the mixture was stirred for 30 min. After washing by DI water, 5 μL of CD71 antibody (OX26) (Santa Cruz Biotechnology) was added into Se suspension and the mixture was stirred at room temperature for 5 h. The non-bonded OX26 antibodies were removed by centrifugation at 20,000 rpm for 60 min at 4 °C. Then, the pellets were resuspended in Milli-Q water. To evaluate the impact of NP-protein interactions on cellular uptake and survival in vitro, FITC (Fluorescein isothiocyanate) - OX26 (Santa Cruz Biotechnology) was used instead of CD71 antibody (OX26).

Heparin coating of bridges has been shown to enhance lentiviral loading and transduction from bridges in vivo.63 PLG bridges were incubated with chitosan (Sigma Aldrich, St. Louis, MO, USA; 25 μg/μl in 2% glacial acetic acid) for 10 min, followed in a N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide/N‐hydroxylsuccinimide (Sigma Aldrich) mixture dissolved in 2‐(N‐morpholino)ethanesulfonic acid (EDC/NHS in MES; 1.5:1:1 mg/mg/ml, Sigma Aldrich) for 2 hrs. To conjugate chitosan, bridges were washed 3 times with water, dried and subsequently incubated with Heparin (Sigma Aldrich; 25 μg/μl in 1M MES) for 10 min followed by EDC/NHS in MES for an additional 2 hrs for covalent conjugation. Finally, bridges were washed in water three times and dried.