Divergent Responses of Hydrophilic CdSe and CdSe@CdS Core-Shell Nanocrystals in Apoptosis and In Vitro Cancer Cell Imaging: A Comparative Analysis.

With their distinctive core-shell design, core-shell nanocrystals have drawn interest in catalysis, medicinal research, and nanotechnology. These nanocrystals have a variety of characteristics and possible uses. The application of core-shell nanocrystals offers significant potential in increasing diagnostic and therapeutic approaches for cancer research in apoptosis and in vitro cancer cell imaging. In the present study, we investigated the fluorescence behavior of hydrophilic CdSe (core-only) and CdSe@CdS (core-shell) nanocrystals (NCs) and their potential in cancer cell imaging. The addition of a CdS coating to CdSe NCs increased the fluorescence intensity tenfold. The successful fabrication of core-shell CdSe@CdS nanocrystals was proven by a larger particle size (evaluated via DLS and TEM) and their XRD pattern and surface morphology compared to CdSe (core-only) NCs. When these NCs were used for bioimaging in MCF-7 and HEK-293 cell lines, they demonstrated excellent cellular uptake due to higher fluorescence intensity within cancerous cells than normal cells. Comparative cytotoxicity studies revealed that CdSe NCs were more toxic to all three cell lines (HEK-293, MCF-7, and HeLa) than CdSe@CdS core-shell structures. Furthermore, a decrease in mitochondrial membrane potential and intracellular ROS production supported NCs inducing oxidative stress, which led to apoptosis via the mitochondria-mediated pathway. Increased cytochrome c levels, regulation of pro-apoptotic gene expression (e.g., p53, Bax), and down-regulation of Bcl-2 all suggested cellular apoptosis occurred via the intrinsic pathway. Significantly, at an equivalent dose of core-shell NCs, core-only NCs induced more oxidative stress, resulting in increased apoptosis. These findings shed light on the role of a CdS surface coating in reducing free radical release, decreasing cytotoxicity, and improving fluorescence, advancing the field of cell imaging.

The Effect of Curcumin-Loaded Glucan Nanoparticles on Immune Cells: Size as a Critical Quality Attribute.

Curcumin is known for its multiple health benefits, largely due to its antioxidant and anti-inflammatory properties. It has been extensively studied as a therapeutic agent, however, it does not have good clinical efficacy due to its poor water solubility and bioavailability. Despite accepting the encapsulation of this compound in polymeric particles as one of the most promising strategies to increase its therapeutic value, these nanoparticles have fallen short of expectations due to a lack of assessment of their possible adverse effects on the immune system. Therefore, in this work, we report on a new method to encapsulate curcumin into glucan nanoparticles and their effects on cells of the immune system were evaluated. Two different-sized curcumin-loaded glucan NPs (GluCur 100 and GluCur 380) were produced, each with an encapsulation efficiency close to 100%, and were characterized regarding their size distribution, surface properties, and morphology. The results revealed the greatest hemolytic effect and cytotoxicity for the smallest particles (100 nm) tested in human PBMCs and RAW 264.7 cells. Although GluCur 380 NPs showed a weaker ROS production, they were able to inhibit the production of NO by macrophages. Furthermore, we found that the coagulation time was not affected by both sized-particles as well as platelet function. Additionally, both nanoparticles induced lymphocyte proliferation and TNF-α secretion by Mo-DCs. In conclusion, this report emphasizes the importance of the immunotoxicity assessment and how this is dependent on the intrinsic properties of nanomaterials, hopefully contributing to increasing the safety of nanomedicines.

Fabricating versatile cell supports from nano- and micro-sized graphene oxide flakes.

Micro-sized structures made from graphene oxide (GO) attract high interest for their extensive use in tissue engineering. The fabrication and cytotoxicity of 3D graphene-based scaffolds so far have not been extensively discussed with relation to the flake sizes used. In this work we considered GO flakes of two different lognormal size distributions (GO: 4.9 ± 3.8 µm and GO 1 h: 151.6 ± 1.9 nm) as model flakes for fabrication of 3D graphene-based cell culture supports: paper (i.e. 3D layered film structure) and reduced graphene oxide (rGO) microfiber using hydrothermal methods. We then used two model cell lines of neuronal origin (SH-SY5Y and HEK-293) to study subsequent scaffolds surface-cells interactions. In particular, the adhesion of HEK cells to the formed structures was much higher than for SH-SY5Y cells, as evidenced by various atomic force, electron and optical microscopy techniques. Formed rGO microfibers had more desired nano-topography (surface roughness) for cell adhesion and growth than simple GO paper, making it ideal scaffold for neural tissue engineering. This work provides insights into the fundamental rules for fabrication of graphene oxide-based cell supports and their subsequently differing interactions with malignant and non-malignant cells.

Size-dependent apoptotic activity of gold nanoparticles on osteosarcoma cells correlated with SERS signal.

In the last decade, gold nanoparticles have emerged as promising agents for in vitro bio-sensing and in vivo cancer theranostics. However, different investigations have reported widely varying cytotoxicity and uptake efficiency of gold nanoparticles depending upon their size. Therefore, more extensive studies are needed to standardize these biological effects as a function of size on a particular cell line. In addition, to obtain robust confirmation on the correlation of a size to biological effect, thorough mechanistic study must also be performed. In this study, the size dependent biological activities of gold nanoparticles on osteosarcoma cells is investigated towards exploring their potential theranostic application in bone cancer, for which very scarce literature reports are available. Tris-assisted citrate based method was optimized to synthesize stable gold naoparticles of 40-60 nm sizes. Nanoparticles were characterized through UV-Vis spectroscopy, field emission scanning electron microscope (FESEM) and dynamic light scattering (DLS). Increasing concentrations of gold nanoparticles (AuNPs) of 46 nm size, enhanced the rate of reactive oxygen species (ROS)-induced apoptosis in MG63 cells by disrupting their mitochondrial membrane potential. Considerably higher cell death was observed for 46 and 60 nm AuNPs compared to 38 nm at all concentrations of 200, 400 and 800 ng/mL. Further, molecular signatures of cellular apoptosis under nanoparticle treatment were optically assessed through surface enhanced Raman scattering (SERS). A significant Raman enhancement in cancer cells under treatment of larger gold nanoparticles (46 and 60 nm) at fixed wavelength of 785 nm and laser power of 8.0 mW was evident. In corroboration with molecular biology techniques, SERS observation confirmed the size-dependent apoptotic phenomena in osteosarcoma cells under treatment of gold nanoparticles. Study demonstrates a facile, non-active targeting approach for detection of size-dependent AuNP-induced apoptosis in osteosarcoma cells through label-free SERS method.

A reliable approach for assessing size-dependent effects of silica nanoparticles on cellular internalization behavior and cytotoxic mechanisms.

BACKGROUND: The size of nanoparticles is considered to influence their toxicity, as smaller-sized nanoparticles should more easily penetrate the cell and exert toxic effects. However, conflicting results and unstandardized methodology have resulted in controversy of these size-dependent effects. Here, we introduce a unique approach to study such size-dependent effects of nanoparticles and present evidence that reliably supports this general assumption along with elucidation of the underlying cytotoxic mechanism. METHODS: We prepared and physically characterized size-controlled (20-50 nm) monodispersed silica nanoparticles (SNPs) in aqueous suspensions. Then, a variety of biochemical assessments are used for evaluating the cytotoxic mechanisms. RESULTS: SNP treatment in three cell lines decreased cell viability and migration ability, while ROS production increased in dose- and size-dependent manners, with SNPs <30 nm showing the greatest effects. 30- and 40-nm SNPs were observed similar to these biological activities of 20- and 50-nm, respectively. Under the conventionally used serum-free conditions, both 20-nm and 50-nm SNPs at the IC50 values (75.2 and 175.2 µg/mL) induced apoptosis and necrosis in HepG2 cells, whereas necrosis was more rapid with the smaller SNPs. Inhibiting endocytosis impeded the internalization of the 50-nm but not the 20-nm SNPs. However, agglomeration following serum exposure increased the size of the 20-nm SNPs to approximately 50 nm, preventing their internalization and cell membrane damage without necrosis. Thus, 20-nm and 50-nm SNPs show different modes of cellular uptake, with smaller SNPs capable of trafficking into the cells in an endocytosis-independent manner. This approach of using non-overlapping size classes of SNPs under the same dose, along with serum-induced agglomeration analysis clarifies this long-standing question about the safety of small SNPs. CONCLUSION: Our results highlight the need to revise safety guidelines to account for this demonstrated size-dependent cytotoxicity under serum-free conditions, which may be similar to the microenvironment after tissue penetration.

Poly(D,L-Lactic Acid) Nanoparticle Size Reduction Increases Its Immunotoxicity.

Polylactic acid (PLA), a biodegradable and biocompatible polymer produced from renewable resources, has been widely used as a nanoparticulate platform for antigen and drug delivery. Despite generally regarded as safe, its immunotoxicological profile, when used as a polymeric nanoparticle (NP), is not well-documented. Thus, this study intends to address this gap, by evaluating the toxicity of two different sized PLA NPs (PLAA NPs and PLAB NPs), produced by two nanoprecipitation methods and extensively characterized regarding their physicochemical properties in in vitro experimental conditions. After production, PLAA NPs mean diameter (187.9 ± 36.9 nm) was superior to PLAB NPs (109.1 ± 10.4 nm). Interestingly, when in RPMI medium, both presented similar mean size (around 100 nm) and neutral zeta potential, possibly explaining the similarity between their cytotoxicity profile in PBMCs. On the other hand, in DMEM medium, PLAA NPs presented smaller mean diameter (75.3 ± 9.8 nm) when compared to PLAB NPs (161.9 ± 8.2 nm), which may explain its higher toxicity in RAW 264.7. Likewise, PLAA NPs induced a higher dose-dependent ROS production. Irrespective of size differences, none of the PLA NPs presented an inflammatory potential (NO production) or a hemolytic activity in human blood. The results herein presented suggest the hypothesis, to be tested in the future, that PLA NPs presenting a smaller sized population possess increased cytotoxicity. Furthermore, this study emphasizes the importance of interpreting results based on adequate physicochemical characterization of nanoformulations in biological medium. As observed, small differences in size triggered by the dispersion in cell culture medium can have repercussions on toxicity, and if not correctly evaluated can lead to misinterpretations, and subsequent ambiguous conclusions.

Gold Nanoparticles of Diameter 13 nm Induce Apoptosis in Rabbit Articular Chondrocytes.

Gold nanoparticles (AuNPs) have been widely used in biomedical science including antiarthritic agents, drug loading, and photothermal therapy. In this report, we studied the effects of AuNPs with diameters of 3, 13, and 45 nm, respectively, on rabbit articular chondrocytes. AuNPs were capped with citrate and their diameter and zeta potential were measured by dynamic light scattering (DLS). Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assay after the rabbit articular chondrocytes were pre-incubated with 3, 13, and 45 nm AuNPs, respectively, for 24 h. Flow cytometry (FCM) analysis with annexin V/propidium iodide (PI) double staining and fluorescence imaging with Hoechst 33258 staining were used to determine the fashion of AuNPs-induced chondrocyte death. Further, 13 nm AuNPs (2 nM) significantly induced chondrocyte death accompanying apoptotic characteristics including mitochondrial damage, externalization of phosphatidylserine and nuclear concentration. However, 3 nm AuNPs (2 nM) and 45 nm (0.02 nM) AuNPs did not induce cytotoxicity in chondrocytes. Although 13 nm AuNPs (2 nM) increased the intracellular reactive oxygen species (ROS) level, pretreatment with Nacetyl cysteine (NAC), a ROS scavenger, did not prevent the cytotoxicity induced by 13 nm AuNPs, indicating that 13 nm AuNPs (2 nM) induced ROS-independent apoptosis in chondrocytes. These results demonstrate the size-dependent cytotoxicity of AuNPs in chondrocytes, which must be seriously considered when using AuNPs for treatment of osteoarthritis (OA).