Carbodimide Chemistry Analysis

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The optimized PF0 encapsulated hybrid NCs formulation was achieved by a two-step process (Fig 1) using nanoprecipitation and subsequently coating with chitosan. In Step-I, the nanoprecipitation technique was used to prepare PF0 loaded BSA NCs (PF0-ANC). In this nanoprecipitation technique, acetone was used as a desolvent [40]. Varying concentrations of PF0 solutions (125µM - 375µM) and BSA solution (156.25µM – 468.75µM) (Table 1) were mixed in a glass vial. The PF0 BSA solution was then added slowly, at a controlled speed of 0.2 ml/min using syringe pump, under continuous stirring at 600 rpm and the stirring was continued overnight to allow acetone evaporation. The NCs obtained in the above step was used for the future steps.
In Step II, PF0-HNC
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The carbodimide chemistry reaction involves first the activation of the carboxyl group using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EDC and then a covalent reaction between free amino group and activated carboxyl group in PEG-EGFR and PF0-HNC respectively [42]. In brief, 10 mg of Methoxy-PEG-COOH and 5 times molar excess of the EDC was dissolved in 5 ml 0.1 M MES buffer (pH 4 ~ 5) under continuous stirring for 2 hours. The unreacted EDC was removed by performing dialysis with dialysis membrane (MW cut off 1000). Later, 5 mg of EGFR peptide was added to the above activated Methoxy-PEG-COOH to form Methoxy-PEG-EGFR. To attain conjugation of PEG-EGFR peptide on PF0-HNC, first the carboxyl group located on PEG-EGFR peptide was activated by the addition of 20% molar excess of N-hydroxysulfo-succinimide (NHS) and EDC followed by the addition of this carboxyl activated peptide to PF0-HNC to form PF0-HNC-E. The reaction was continued for 6 hr followed by the addition of 1 M glycine to the reaction mixture to quench the activated carboxyl group and terminate the reaction [43, 44]. The resultant conjugate was purified by dialysis using dialysis membrane (molecular weight cut off 15kD) against 500 ml of PBS for 4 hr to remove the free PEG-EGFR peptide, NHS, and EDC. The carboxyl-PEG-EGFR peptide (American Peptides, USA) was …show more content…
Accordingly, PPMIL cells (1 x 105) were seeded in 24-well culture plates with an appropriate medium and grown to confluence. Fluorescein isothiocyanate (FITC) labeled BSA was used to prepare the targeted and non-targeted formulations (FITC-HNC-E and FITC-HNC-PEG) for this study using the same protocol described before [53]. FITC labeled formulation was mixed and incubated with ~1×105 PPMIL cells at 4 °C for 3 h in a 24-well plate. After the incubation period, the cells were washed, trypsinized and collected for flow cytometry analysis. The cell suspension was centrifuged at 13,000 g and rinsed with DPBS twice to remove the non-bound NCs, followed by resuspending in 0.1 ml of PBS. The intensity of FITC fluorescence in the cells was detected and cells were counted by flow cytometer (Beckman Coulter Cell Lab Quanta SC flow cytometer, US) equipped with a 488 nm laser to analyze FITC labeled NCs (λex max = 490 nm, λem max = 525 nm) [54]. At least 10,000 cells were analyzed for each experiment. The flow cytometric data were presented as frequency distribution histograms. The uptake of maximum fluorescence NCs was set as 100%, and the relative uptake of different treatments was presented as a percentage. The mean cell fluorescence was normalized to that of the untreated cells. All experiments were repeated at least three

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