pNiPAM nanoparticles suspensions as crowded complex model systems: synthesis, characterization and properties

Improvements in controlled polymer synthesis and characterization methods have lead scientists to investigate new materials that would not only improve old techniques, but also open way for new approaches in several industrial and bio-medical applications. Poly-N-isopropylacrylamide (pNiPAM) is one example among this new class of materials. Being respondent to variations in temperature this ‘intelligent’ polymer is considered as a model system that is a candidate for biomaterials, drug delivery systems, biosensors, bioanalytical devices or bio-scaffolds for cell cultures. Wide range of potential applications arises mostly from nanometric size and tunable properties. Various types of pNiPAM nanoparticles with its lower critical solution temperature (LCST), around natural human body temperature (at 33oC) were synthesized and characterized in terms of size and morphology. Additionally mechanical properties of pNiPAM microgel with oscillation rheology in the absence and in the presence of linear pNiPAM chains additives was studied. Next an efficient way for chemical labeling of the polymeric nanoparticles with a fluorescent dye was established. PNiPAM nanoparticles were suitable for fluorescent techniques which allowed using them as model systems to study diffusion process and micro/macro viscosity effect in crowded complex systems. A correction procedure to study the size of relatively large, uniformly labeled nanoparticles in FCS experiment was proposed. Finally, conducted cytotoxicity studies, not only proved the lack of toxic effect but additionally demonstrated a bioscaffold based growth promotion effect on cell cultures.Postęp w stosowaniu polimerowych materiałów koncentruje uwagę naukowców na polimerach wykazujących właściwości konkurencyjne w stosunku do tradycyjnych materiałów. Polimery tworzące struktury w skali nano stanowią obiecujące narzędzie o szerokim spektrum zastosowań jako biomateriały, biosensory, systemy dostarczania leków czy bio-podłoża do hodowli komórkowych. Jednym z takich polimerów jest pNiPAM (poly-N-isoporpylacrylamid), który dzięki temperaturze przejścia fazowego około naturalnej temperatury ciała ludzkiego (33oC) zasługuje na szczególna uwagę. Syntetyzowano różnego rodzaju nanocząsteki polimeru pNiPAM oraz scharakteryzowano pod katem rozmiaru i morfologii. Dodatkowo w celu określenia właściwości mechanicznych przeprowadzono oscylacyjne badania reologiczne samego mikrożelu i w obecności liniowych łańcuchów polimeru pNiPAM. Funkcjonalizacja nanocząstek za pomocą chemicznego wprowadzenia fluorescencyjnie barwnika w strukturę nanocząstek pNiPAM, umożliwiła ich zastosowanie w technikach fluorescencyjnych. Umożliwiło to użycie nanocząstek pNiPAM jako modelowego systemu do badania dyfuzji w gęstych kładach złożonych oraz do badań nad micro/macro lepkością. Zaproponowano także poprawkę do procedury pomiaru wielkości, relatywnie dużych, jednolicie wyznakowanych nanocząstek przy użyciu techniki FCS. Dodatkowo ze względu szeroką gammę zastosowań w biologii i biotechnologii przeprowadzono badania nad cytotoksycznością otrzymanych nanocząstek. Stwierdzono brak efektu cytotoksyczności i ponadto zaobserwowano przyspieszenie wzrostu komórek w obecności nanocząstek pNiPAM.This work was supported by the International PhD Projects Program (”The PhD in Nanoscience and Nanotechnology”) Foundation for polish Science operated within the Innovative Economy Operational Programme (IE OP) 2007-2013 within European Regional Fund

Size of submicrometer particles measured by FCS: Correction of the confocal volume

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 a2/σ2 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

pNiPAM-Nanoparticle-Based Antiapoptotic Approach for Pro-Regenerative Capacity of Skeletal Myogenic Cells

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

Cytotoxicity of thermo-responsive polymeric nanoparticles based on N-isopropylacrylamide for potential application as a bioscaffold

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

Metabolic syndrome is associated with similar long-term prognosis in non-obese and obese patients. An analysis of 45 615 patients from the nationwide LIPIDOGRAM 2004-2015 cohort studies

Aims We aimed to evaluate the association between metabolic syndrome (MetS) and long-term all-cause mortality. Methods The LIPIDOGRAM studies were carried out in the primary care in Poland in 2004, 2006 and 2015. MetS was diagnosed based on the National Cholesterol Education Program, Adult Treatment Panel III (NCEP/ATP III) and Joint Interim Statement (JIS) criteria. The cohort was divided into four groups: non-obese patients without MetS, obese patients without MetS, non-obese patients with MetS and obese patients with MetS. Differences in all-cause mortality was analyzed using Kaplan-Meier and Cox regression analyses. Results 45,615 participants were enrolled (mean age 56.3, standard deviation: 11.8 years; 61.7% female). MetS was diagnosed in 14,202 (31%) by NCEP/ATP III criteria, and 17,216 (37.7%) by JIS criteria. Follow-up was available for 44,620 (97.8%, median duration 15.3 years) patients. MetS was associated with increased mortality risk among the obese (hazard ratio, HR: 1.88 [95% CI, 1.79-1.99] and HR: 1.93 [95% CI 1.82-2.04], according to NCEP/ATP III and JIS criteria, respectively) and non-obese individuals (HR: 2.11 [95% CI 1.85-2.40] and 1.7 [95% CI, 1.56-1.85] according to NCEP/ATP III and JIS criteria respectively). Obese patients without MetS had a higher mortality risk than non-obese patients without MetS (HR: 1.16 [95% CI 1.10-1.23] and HR: 1.22 [95%CI 1.15-1.30], respectively in subgroups with NCEP/ATP III and JIS criteria applied). Conclusions MetS is associated with increased all-cause mortality risk in non-obese and obese patients. In patients without MetS obesity remains significantly associated with mortality. The concept of metabolically healthy obesity should be revised