Notably, after the cell membrane coating, the shift of CBSA-associated positive surface charge of hybrid nanoparticles to negative confirmed the formation of a cell membrane cloaking (Figure 5ACC). was reduced up to 10 mL. Next, the retentate was gently aspirated, collected from sample chamber, and dialyzed against distilled water by using a regenerated cellulose dialysis bag of MWCO-3.5 kDa for 72 h. The total CBSA recovery was calculated about 85% of initial protein concentration. Finally, the CBSA concentrate was freeze-dried to obtain CBSA. After synthesis, the native BSA and CBSA were characterized by determining their zeta potentials by using a ZetaPlus Zeta Potential Analyzer (Brookhaven Instruments, Holtsville, NY, USA) at 25 C and SDS-PAGE assay by taking native BSA as control. 2.2.2. Isolation of Cell Plasma Membranes The cell plasma membrane of RBCs, LO2, 4T1, and A549/T was isolated by using a previously described protocol with few modifications [17]. Briefly, the RBCs were collected from whole blood of female BALB/c nude mice from the orbit of mice with the addition of 1.5 mg of EDTA per milliliter of blood for anticoagulation, while LO2, 4T1, and A549/T cells were harvested and washed with PBS three times. Next, the cells were resuspended in hypotonic lysis buffer supplemented with protease inhibitor cocktail (MedChem Express LLC, USA) on ice for 5 min. Thereafter, individual cells were homogenized using a Dounce homogenizer with a pestle. To remove the unbroken cells and cellular nuclei, we centrifuged the homogenate at 800 for 10 min at 4 C. Next, the supernatant was centrifuged at 10,000 for 15 min to remove the cell mitochondria, followed by centrifugation at 100,000 with a magnetic stirrer at room temperature. Next, PTX (6 mg) and DSF (1 mg) were dissolved into 2 mL of ethanol and continuously added at 1 mL/min. Subsequently, the extra ethanol was continuously added at the same flow rate until the solution appeared milky. Next, under dimmed light, we added 200 L of 4% glutaraldehyde and the mixture was placed for 6 h at 25 C for crosslinking. Finally, ethanol was evaporated on a rotary evaporator at 40 C under reduced pressure, followed by centrifugation at 12,000 for 15 min at 25 Penicillin V potassium salt C, and supernatant was collected to Penicillin V potassium salt determine the drug loading (DL) and entrapment efficacy (EE) of PTX and DSF, respectively. 2.2.5. Functionalization of Nanoparticle Cores The RBCs, LO2, 4T1, and A549/T cell membrane-coated hybrid nanoparticles (RBC CM-HNPs, LO2 CM-HNPs, 4T1 CM-HNPs, and A549/T CM-HNPs) were prepared by co-incubating respective cell membrane vesicles and hybrid nanoparticle cores followed by sonication and co-extrusion through a 200 nm polycarbonate membrane. Finally, RBC CM-HNPs, LO2 CM-HNPs, 4T1 CM-HNPs, and A549/T CM-HNPs (CM-HNPs) were centrifuged at 500 for 3 min to separate any precipitates formed during the extrusion process. The BSA- Penicillin V potassium salt and FITC-labelled hybrid nanoparticles were prepared by the same procedure, except BSA was used in place of CBA to prepare BSA hybrid nanoparticles (HNPs), while FITC was co-dissolved with drug solution (FITC, PTX, DSF) to prepare FITC-loaded hybrid nanoparticles (FITC-HNPs, FITC-RBCs CM-HNPs, FITC-LO2 CM-HNPs, FITC-4T1 CM-HNPs, FITC-A549/T Penicillin V potassium salt CM-HNPs). 2.2.6. Rabbit Polyclonal to MRPL2 Determination of Membrane-Associated Protein The presence of membrane-associated proteins on RBC CM, LO2 CM, 4T1 CM, and A549/T CM (cell membrane vesicles) as well as on CM-HNPs was determined by Coomassie blue staining assay. The RBC CM, LO2 CM, 4T1 CM, A549/T CM, and CM-HNPs were collected after 0, 24, and 48 h. Next, the prehomogenated cell membrane vesicles and respective CM-HNPs were lysed in radioimmunoprecipitation (RIPA) lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) with protease inhibitor solution on ice for 5 min. Next, lysates were centrifuged at 13,000 for 5 min at 4 C to collect the supernatants and subjected to BCA protein assay (Beyotime Biotechnology, Haimen, China) for total protein quantification. Thereafter, the individual supernatant was mixed with SDS loading buffer and heated up to 95 C for 5 min. A 20 g equivalent protein for each sample was added in each well of 10% SDS-PAGE in an electrophoresis chamber system (Bio-Rad Laboratories, Philadelphia, PA, USA) and run at 120 V for 1.5 h. Penicillin V potassium salt Finally, the gel was stained with Coomassie blue (Beyotime Biotechnology, Haimen, China) overnight,.