Synthesis, Stability, Cellular Uptake, ..
Next, we were interested in investigating the differences in cellular internalization of the polymer aggregates, nanogels and T-NGs loaded with hydrophobic dye molecules. We hypothesized that any leakiness of the nanocarriers would result in the transport of their lipophilic cargo to surrounding cellular membranes due to the hydrophobic interactions. This would permit the cellular internalization of free dye prior to that of the dye molecule’s nanocarrier. To test this, we added the polymer aggregates and nanogels, containing DiO as a hydrophobic dye, into three different cell cultures (293T, MCF-7, and HeLa cell lines). The uptake was then monitored by tracing the dye’s fluorescence using confocal microscopy (). In the case of the polymer aggregates, intense green emission inside the cells was observed within 6h across all cell lines. In contrast, the nanogels showed no emission, indicating that the nanogels are not internalized in this short time period. This result suggests that the dyes from the leaky amphiphilic polymeric aggregates might be easily moved from the carrier to cellular membrane. To confirm this, we performed in situ FRET experiments with HeLa cells using the amphiphilic aggregates in which the FRET pair DiO (green emission) and DiI (red emission) was co-encapsulated. In this case, if the polymeric aggregates were internalized through the membrane, then FRET (red emission, 585-615 nm spectral filter) would be continually observed in the cytosolic interior within a short time period. After 3h incubation, we found that green (no FRET, 505-520 nm spectral filter) fluorescence is dominant inside the cell by confocal microscopy (ex = 488 nm), indicating that the two dyes are not close together within the assembly core, but that they have released from the micelles (). This indicates that liphophilic guests loaded inside micellar aggregates can be transferred into any cells, resulting in non-specific delivery. Thus, high encapsulation stability is essential to prevent premature, non-triggered release and achieve selective delivery of the drug molecules to target cells.
Synthesis, Stability, And Cellular ..
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Synthesis, characterization, and cellular ..
Peptide was synthesized on 2-chloro-trityl chloride resin by solid phase synthesis using standard Fmoc methodology. Briefly, coupling reaction was achieved by 3 equiv. Fmoc protected amino acid, 3 equiv. HATU, and 3 equiv DIPEA in DMF and monitored by Kaiser Test. Fmoc protection group was removed by 20% piperidine in DMF. Cleavage of peptide from resin was performed in the presence of TFA/TIS/H2O/EDT mixture. Then cleavage mixture was precipitated in cold ether 5 times to afford crude peptide. Peptide was used without further purification. Yield: 80%. Peptide was characterized by 1H-NMR and mass spectrometry (see ).
Cellular internalization of ingested aSNPs by endocytosis ..
2,2’-Dithiodipyridine, 2-mercaptoethanol, polyethylene glycol monomethyl ether methacrylate (MW 450), -dithiothreitol (DTT), 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbo-cyanine perchlorate (DiI) and 3,3’-dioctadecyloxacarbocyanine perchlorate (DiO), folic acid, cyclo(Arg-Gly-Asp--Phe-Cys) peptide, 2-cyano-2-propyl benzodithioate and other conventional reagents were obtained from commercial sources and were used as received, unless otherwise mentioned. Polymers were synthesized using RAFT polymerization and then purified by precipitation. Pyridyl disulfide ethyl methacrylate (PDSEMA) was prepared using a previously reported procedure. 1H-NMR spectra were recorded on a 400 MHz Bruker NMR spectrometer using the residual proton resonance of the solvent as the internal standard. Molecular weights of the polymers were estimated by gel permeation chromatography (GPC) using PMMA standard with a refractive index detector. Dynamic light scattering (DLS) measurements were performed using a Malvern Nanozetasizer. The fluorescence spectra were obtained from a JASCO FP-6500 spectrofluorimeter. Transmission electron microscopy (TEM) images were taken from JEOL 100CX at 100 KV.
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