Size and Surface Properties of Functionalized Organosilica Particles Impact Cell-Particle Interactions Including Mitochondrial Activity.

Understanding of the interactions between macrophages and multifunctional nanoparticles is important for development of novel macrophage-based immunotherapies. Here, we investigated the effects of fluorescent thiol-organosilica particle size and surface properties on cell-particle interactions, including mitochondrial activity, using the mouse macrophage cell line J774A.1. Three different sizes of thiol-organosilica particles (150, 400, and 680 nm in diameter) containing fluorescein (OS/F150, OS/F400, and OS/F680) and particles surface functionalized with polyethylenimine (PEI) (OS/F150PEI, OS/F400PEI, and OS/F680PEI) were prepared. Flow cytometric analysis, time-lapse imaging, and single-cell analysis of particle uptake and mitochondrial activity of J774A.1 cells demonstrated variations in uptake and kinetics depending on the particle size and surface as well as on each individual cell. Cells treated with OS/F150 and OS/F150PEI showed higher uptake and mitochondrial activity than those treated with other particles. The interaction between endosomes and mitochondria was observed using 3D fluorescent imaging and was characterized by the involvement of iron transport into mitochondria by iron-containing proteins adsorbed on the particle surface. Scanning electron microscopy of the cells treated with the particles revealed alterations in cell membrane morphology, depending on particle size and surface. We performed correlative light and electron microscopy combined with time-lapse and 3D imaging to develop an integrated correlation analysis of particle uptake, mitochondrial activity, and cell membrane morphology in single macrophages. These cell-specific characteristics of macrophages against functional particles and their evaluation methods are crucial for understanding the immunological functions of individual macrophages and developing novel immunotherapies.

Cellular Distribution and Intracellular Localization of Different Sizes of Fluorescent Thiol-Organosilica Particles in Mouse Lungs.

We investigated the distribution of intratracheally administered thiol-organosilica (thiol-OS) particles in mouse lungs. Toward this end, single doses of thiol-OS particles containing fluorescein (140 nm in diameter) (F140) and rhodamine B (Rh) (Rh160, Rh280, Rh420, Rh640, and Rh1630 with diameters of 160, 280, 420, 640, and 1630 nm, respectively) were administered. After 24 h, fluorescence imaging revealed homogeneous fluorescence with a patchier pattern on the lung surface and no difference among the six particle sizes. Simultaneous dual administration of Rh and F140 particles did not reveal any size-dependent differences in the lung surface fluorescence. Fluorescence microscopy of the lung sections revealed a similar tissue distribution in the fluorescent areas of Rhs and F140. Some fluorescent areas showed one type of particle fluorescence or only one fluorescence. Cellular distribution of particles was observed in bronchoalveolar lavage cells and lung sections under a high magnification, and correlative light and electron microscopy revealed large cells with fluorescence corresponding to both particle types and small cells with fluorescence of individual particle types, indicating a cell-subset-dependent particle size effect. Rh280, Rh420, and Rh640 exhibited significant size effects and were taken up by alveolar macrophages. Extracellular particles were observed, indicating that saturation exceeded the particle dose threshold in the alveoli. F140 taken up by small and large macrophages colocalized with CD68, CD11c, and CD11b and correlated with CD11c. The size effect, intracellular localization, and extracellular distribution of particles provide insights into lung and systemic drug delivery.

Size effect of fluorescent thiol-organosilica particles on their distribution in the mouse spleen.

We investigated the distribution of intravenously administered thiol-organosilica particle (thiol-OS) in the spleen to evaluate their size effect in mice. A single administration of particles of thiol-OS containing rhodamine B (Rh) (90, 280, 340, 450, 630, 1110, 1670, and 3030 nm in diameter) was performed. After 24 h, we conducted a combination analysis using histological studies by fluorescent microscopy and quantitative inductively coupled plasma optical emission spectrometry (ICP-OES), which revealed no clear correlation between the particle size and spleen uptake of particle weight and number per tissue weight, and the injection dose. Moreover, Rh with 450 nm diameter (Rh450) showed the highest uptake, and Rh with 340 nm diameter (Rh340) showed the lowest uptake. Histologically, large fluorescent areas in the marginal zone (MZ) and red pulp (RP) of the spleen were observed for all particle sizes, but less in the follicle of white pulp. Using combination analysis using the particle weights of ICP-OES and the fluorescent area, we compared the distributions of each particle in each region. Rh450 had the largest accumulated weight in the MZ and RP. Particles larger than Rh450 showed negative correlations between their sizes and accumulated weight in the MZ and RP. Simultaneous dual administration of particles using Rhs and thiol-OS containing fluorescein (90 nm in diameter) showed the size-dependent difference in cellular distribution and intracellular localization. Immunohistochemical staining against macrophage markers, CD169, and F4/80 showed various colocalization patterns with macrophages that uptook particles, indicating differences in particle uptake in each macrophage may have novel significance.

Effects of Au States in Thiol-Organosilica Nanoparticles on Enzyme-like Activity for X-ray Sensitizer Application: Focus on Reactive Oxygen Species Generation in Radiotherapy.

In radiotherapy, the use of Au nanoparticles (Au NPs) has been proposed to enhance cell damage by X-ray irradiation. Although the role of Au in radiotherapy is not fully understood, the catalytic activity of Au has been actively studied in the industrial field. Moreover, owing to their enzyme-like activity and high biocompatibility in vitro and in vivo, Au NPs present significant potential for biological applications. In this study, we incorporated different Au states both on the surface and embedded in thiol-organosilica (thiol-OS/Au series) to investigate the efficiency of anticancer cell activity of Au in radiotherapy. The thiol-OS/Au series comprised different Au(I)/Au(0) ratios and Au NPs, and different sizes of Au NPs were embedded in thiol-OS/Au. These thiol-OS/Au series samples were evaluated for enzyme-like activities in reactive oxygen species (ROS) generation by X-ray irradiation. Thiol-OS/Au embedded with small Au NPs (AC600/thiol-OS/Au) exhibited peroxidase (POD)-like activity under acidic conditions. This POD-like activity improved ROS generation and cytotoxicity under X-ray irradiation. Furthermore, AC600/thiol-OS/Au exhibited catalase (CAT)-like activity under basic conditions and showed no cytotoxicity toward nonirradiated cells. These results revealed the efficiency of functionalizing with small Au NPs that possess pH-controlled POD- and CAT-like activity as a radiosensitizer. We compared the suitability of using Au with different states to obtain the thiol-OS/Au series samples for application as radiosensitizers. The findings of this study will aid the design of efficacious strategies for the Au nanostructure-based radiotherapy of cancer cells.

Analysis of cell-nanoparticle interactions and imaging of in vitro labeled cells showing barcorded endosomes using fluorescent thiol-organosilica nanoparticles surface-functionalized with polyethyleneimine.

Biomedical imaging using cell labeling is an important technique to visualize cell dynamics in the body. To label cells, thiol-organosilica nanoparticles (thiol-OS) containing fluorescein (thiol-OS/Flu) and rhodamine B (thiol-OS/Rho) were surface-functionalized with polyethyleneimine (PEI) (OS/Flu-PEI and OS/Rho-PEI) with 4 molecular weights (MWs). We hypothesized PEI structures such as brush, bent brush, bent lie-down, and coiled types on the surface depending on MWs based on dynamic light scattering and thermal gravimetric analyses. The labeling efficacy of OS/Flu-PEIs was dependent on the PEI MW and the cell type. A dual-particle administration study using thiol-OS and OS-PEIs revealed differential endosomal sorting of the particles depending on the surface of the NPs. The endosomes in the labeled cells using OS/Flu-PEI and thiol-OS/Rho revealed various patterns of fluorescence termed barcoded endosomes. The cells labeled with OS-PEI in vitro were administrated to mice intraperitoneally after in situ labeling of peritoneal cells using thiol-OS/Rho. The in vitro labeled cells were detected and identified in cell aggregates in vivo seamlessly. The labeled cells with barcoded endosomes were also identified in cell aggregates. Biomedical imaging of in vitro OS-PEI-labeled cells combined with in situ labeled cells showed high potential for observation of cell dynamics.

Preparation of Fetal Bovine Serum-Copper Phosphate Hybrid Particles under Cell Culture Conditions for Cancer Cell Treatment.

Fetal bovine serum (FBS) particles, which mainly consist of bovine serum albumin, have the potential for biological and medical applications as drug carriers. The coacervation of albumin is a common technique for preparing albumin-based particles. The replacement of salt with novel metal salts such as Cu is an affordable way to embed the metal ion in the albumin-based particles. Further, increased Cu distribution is prevalent in many cancers. Here, we prepared adhesive cell-like FBS-copper phosphate hybrid particles [FBS-Cu3(PO4)2], which exhibited toxicity toward cancer cells, with a narrow size distribution under cell culture conditions for preventing tumor progression. FBS-Cu3(PO4)2 showed peroxidase-like activity. In addition, FBS-Cu3(PO4)2 was successfully loaded with rhodamine B and conjugated with fluorescein isothiocyanate as models of drugs by coincubation. Thus, we designed a simple preparation method for optimizing FBS-Cu3(PO4)2 synthesis under cell culture conditions. FBS-Cu3(PO4)2 has significant potential as an efficient reactive oxygen species generator and drug-delivery agent against cancer cells. Furthermore, the RhoB-loaded FBS-Cu3(PO4)2 successfully interacted with 4T1 mouse mammary tumor cells and were confirmed to exhibit toxicity.

Surface Functionalization of Organosilica Nanoparticles With Au Nanoparticles Inhibits Cell Proliferation and Induces Cell Death in 4T1 Mouse Mammary Tumor Cells for DNA and Mitochondrial-Synergized Damage in Radiotherapy.

Radiotherapy is one of the most effective cancer treatments. Au nanoparticles (NPs) are one of the most used X-ray sensitizing materials however the effective small sub-nm size of Au NPs used for X-ray sensitizers is disadvantageous for cellular uptake. Here, we propose the surface functionalization of organosilica NPs (OS) with Au NPs (OS/Au), which combined the 100 nm size of OS and the sub-nm size of Au NPs, and synthesized effective Au materials as an X-ray sensitizer. The X-ray sensitizing potential for 4T1 mouse mammary tumor cells was revealed using a multifaceted evaluation combined with a fluorescence microscopic cell imaging assay. The number of polyethyleneimine (PEI)-modified OS (OS/PEI) and OS/Au (OS/Au/PEI) uptake per 4T1 mouse mammary tumor cell was the same; however, 4T1 cells treated with OS/Au/PEI exhibited significant inhibition of cell proliferation and increases in cell death by X-ray irradiation at 8Gy. The non-apoptotic death of OS/Au/PEI-treated 4T1 cells was increased by DNA and mitochondrial-synergized damage increase and showed potential applications in radiotherapy.

Development of Non-Porous Silica Nanoparticles towards Cancer Photo-Theranostics.

Nanoparticles have demonstrated several advantages for biomedical applications, including for the development of multifunctional agents as innovative medicine. Silica nanoparticles hold a special position among the various types of functional nanoparticles, due to their unique structural and functional properties. The recent development of silica nanoparticles has led to a new trend in light-based nanomedicines. The application of light provides many advantages for in vivo imaging and therapy of certain diseases, including cancer. Mesoporous and non-porous silica nanoparticles have high potential for light-based nanomedicine. Each silica nanoparticle has a unique structure, which incorporates various functions to utilize optical properties. Such advantages enable silica nanoparticles to perform powerful and advanced optical imaging, from the in vivo level to the nano and micro levels, using not only visible light but also near-infrared light. Furthermore, applications such as photodynamic therapy, in which a lesion site is specifically irradiated with light to treat it, have also been advancing. Silica nanoparticles have shown the potential to play important roles in the integration of light-based diagnostics and therapeutics, termed "photo-theranostics". Here, we review the recent development and progress of non-porous silica nanoparticles toward cancer "photo-theranostics".