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 ..

The TEM micrographs of thin cross-sectioned MDA-MBA-231 cells showed internalization of ..
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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.

Communication Poly(methyl vinyl ether-alt-maleic acid)-Functionalized Porous Silicon Nanoparticles for Enhanced Stability and Cellular Internalization
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AB - Hypothesis: The absence of targetability is the primary inadequacy of conventional chemotherapy. Targeted drug delivery systems are conceptualized to overcome this challenge. We have designed a targetable magnetic nanocarrier consisting of a superparamagnetic iron oxide (SPIO) core and biocompatible and biodegradable poly(sebacic anhydride). -block-methyl ether poly(ethylene glycol) (PSA-mPEG) polymer shell. The idea is that this type of carriers should facilitate the targeting of cancer cells. Experiments: PSA-mPEG was synthesized with poly-condensation and the in vitro degradation rate of the polymer was monitored by gel permeation chromatography (GPC). The magnetic nanocarriers were fabricated devoid of any surfactants and were capable of carrying high payload of hydrophobic dye. The successful encapsulation of SPIO within the polymer shell was confirmed by TEM. The results we obtained from measuring the size of SPIO loaded in polymeric NPs (SPIO-PNP) by dynamic light scattering (DLS) and iron content measurement of these particles by ICP-MS, indicate that SPIO is the most suitable carrier for cancer drug delivery applications. Findings: Measuring the hydrodynamic radii of SPIO-PNPs by DLS over one month revealed the high stability of these particles at both body and room temperature. We further investigated the cell viability and cellular uptake of SPIO-PNPs in vitro with MDA-MB-231 breast cancer cells. We found that SPIO-PNPs induce negligible toxicity within a concentration range of 1-2. μg/ml. The TEM micrographs of thin cross-sectioned MDA-MBA-231 cells showed internalization of SPIO-PNPs within size range of 150-200. nm after 24. h. This study has provided a foundation for eventually loading these nanoparticles with anti-cancer drugs for targeted cancer therapy using an external magnetic field.

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In summary, we report a simple method of synthesis of Pluronic micelle-encapsulated QDs as nanoprobes for cancer imaging. Our design was not only able to protect the QDs stable enough under physiological conditions but also can be conjugated with FA by the simple EDC chemistry conjugation technique, for targeted cancer imaging applications. The result of preliminary work with this nanoprobe appeared to be promising, which revealed the potential of this nanoprobe for application due to the negligible level of toxicity. We have confirmed the targeting of QD bioconjugates in a pancreatic tumor model using small animal imaging system.

Liposomes as a gene delivery system

In summary, we describe here a facile synthetic method for the preparation of nanogels. We show that: (i) these nanogels can be prepared in under two hours from their polymeric precursor; the reaction time includes the ligand decoration step; (ii) these nanogels exhibit high encapsulation stability of lipophilic guest molecules; (iii) the facile ligand functionalization possibility can be utilized to decorate these nanogels with cell targeting ligands; (iv) while the unfunctionalized nanogels are taken up very poorly by various cells, the ligand-decorated nanogels exhibit facilitated receptor-dependent cellular uptake, as demonstrated by selective uptake of RGD- and folic acid- decorated nanogels by cells overexpressing integrin and folate receptors; (v) functionalization of the nanogels with cell penetrating peptides caused rapid non-specific uptake by the cells, independent of the receptor; (vi) the selective internalization capability can be translated to delivering a chemotherapeutic drug molecule specifically to a specific receptor-rich cell. Overall, the reported versatile one-pot synthetic method for synthesizing the ligand-decorated nanogels, combined with the intrinsic encapsulation stability and targeting capabilities of the formed T-NGs, should open up new avenues in targeted drug delivery for crosslinked polymer nanogels.