When fluorescence correlation spectroscopy (FCS) in combination with a confocal microscope is used to determine the hydrodynamic radius a of particles comparable to or larger than the linear size σ of the confocal volume of the microscope, a correction must be used that depends on the a(2)/σ(2) ratio and the distribution of the dye within the particle. Here we present the experimental validation of the theoretically predicted approximate correction necessary for appropriate measurements of the size of uniformly fluorescently labeled spheres of radius comparable to the size of the confocal volume. We also test the approximate correction formula for different ranges of the a/σ ratio and propose a simple procedure to obtain the correct nanoparticle size from such a measurement.
Polymeric nanoparticles based on poly-N-isopropylacrylamide (pNiPAM NPs) and their bio-medical applications have been widely investigated in recent years. These tunable nanoparticles are considered to be great candidates for drug delivery systems, biosensors and bioanalytical devices. Thus, the biocompatibility and toxicity of these nanoparticles is clearly a crucial issue. In this work, the cytotoxicity of thermo-responsive pNiPAM nanoparticles was studied, followed by a detailed analysis of the NPs morphology in growing cell cultures and their 3D structure. Cytotoxic examination was conducted for two cell cultures - HeLa (cervical cancer cell line) and HeK293 (human embryonic kidney cell line), employing MTT (3-4, 5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide) assay and viability tests. We used Cryo-SEM (scanning electron microscopy) and fluorescence microscopy (IN Cell Analyzer) in order to investigate the morphological structure of the polymer network. We show that pNiPAM nanoparticles do not exhibit any cytotoxicity effects on the investigated cell lines. Additionally, we report that the pNiPAM nanoparticle based scaffold promotes cell growth.
In some applications the dye distribution within fluorescently labeled nanoparticles and its stability over long periods of time are important issues. In this article we study numerically and experimentally the applicability of fluorescence correlation spectroscopy (FCS) to resolve such questions. When the size of fluorescently labeled particles is comparable to or larger than the confocal volume, the effective confocal volume seen in FCS experiments is increasing. Such an effect has already been studied for uniformly labeled spherical particles. In this work we analyze the form of the FCS correlation functions (CFs) for core-labeled and shell-labeled core-shell particles. For shell-labeled particles an additional fast decay was found both in simulations and in experiments on custom-made surface-labeled particles. Universal scaling of FCS correlation times based on the squared ratio of the labeled part radius of gyration to the Gaussian radius of the beam profile was found. Recipes based on the analysis of simulated CFs, proposed for interpretation of experimental results, were successfully applied to the FCS results on suspensions of large core-labeled and surface-labeled particles.
The biocompatibility of pNiPAM (Poly N-isopropylacrylamide) copolymers has been examined and they did not exert any cytotoxic effects. Their properties and vulnerable temperature characteristics make them candidates for use in medical applications. We synthesized a well-characterized nanoparticles-based cargo system that would effectively deliver a biological agent to human skeletal myogenic cells (SkMCs); among other aspects, a downregulating apoptotic pathway potentially responsible for poor regeneration of myocardium. We confirmed the size of the pNiPAM based spheres at around 100 nm and the nanomeric shape of nanoparticles (NP) obtained. We confirmed that 33 °C is the adequate temperature for phase transition. We performed the dynamics of cargo release. A small amount of examined protein was detected at 10 min after reaching LCTS (lower critical solution temperature). The presented results of the test with BSA (bovine serum albumin) and doxorubicin loaded into nanoparticles showed a similar release profile for both substances. SkMCs incubated with NP loaded with antiapoptotic agent, BCB (Bax channel blocker), significantly diminished cell apoptosis (p < 0.01). Moreover, the lowest apoptotic level was detected in SkMCs treated with camptothecin and simultaneously incubated with pNiPAMs loaded with BCB. Application of nanoparticles loaded with BCB or subjected to BCB alone did not, however, diminish the amount of apparently necrotic cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.