Novel Organoid Culture System for Improved Safety Assessment of Nanomaterials.

Over the past few decades, the increased application of nanomaterials has raised questions regarding their safety and possible toxic effects. Organoids have been suggested as promising tools, offering efficient assays for nanomaterial-induced toxicity evaluation. However, organoid systems have some limitations, such as size heterogeneity and poor penetration of nanoparticles because of the extracellular matrix, which is necessary for organoid culture. Here, we developed a novel system for the improved safety assessment of nanomaterials by establishing a 3D floating organoid paradigm. In addition to overcoming the limitations of two-dimensional systems including the lack of in vitro-in vivo cross-talk, our method provides multiple benefits as compared with conventional organoid systems that rely on an extracellular matrix for culture. Organoids cultured using our method exhibited relatively uniform sizing and structural integrity and were more conducive to the internalization of nanoparticles. Our floating culture system will accelerate the research and development of safe nanomaterials.

Toxicity of orally administered food-grade titanium dioxide nanoparticles.

This year, France banned the application of titanium dioxide nanoparticles as a food additive (hereafter, E171) based on the insufficient oral toxicity data. Here, we investigated the subchronic toxic responses of E171 (0, 10, 100, and 1,000 mg/kg) and tried to elucidate the possible toxic mechanism using AGS cells, a human stomach epithelial cell line. There were no dose-related changes in the Organisation for Economic Cooperation and Development test guideline-related endpoints. Meanwhile, E171 deeply penetrated cells lining the stomach tissues of rats, and the IgM and granulocyte-macrophage colony-stimulating factor levels were significantly lower in the blood from rats exposed to E171 compared with the control. The colonic antioxidant protein level decreased with increasing Ti accumulation. Additionally, after 24-h exposure, E171 located in the perinuclear region of AGS cells and affected expression of endoplasmic reticulum stress-related proteins. However, cell death was not observed up to the used maximum concentration. A gene profile analysis also showed that immune response-related microRNAs were most strongly affected by E171 exposure. Collectively, we concluded that the NOAEL of E171 for 90 days repeated oral administration is between 100 and 1,000 mg/kg for both male and female rats. Additionally, further study is needed to clarify the possible carcinogenesis following the chronic accumulation in the colon.

Oral toxicity of titanium dioxide P25 at repeated dose 28-day and 90-day in rats.

BACKGROUND: Nanotechnology is indispensable to many different applications. Although nanoparticles have been widely used in, for example, cosmetics, sunscreen, food packaging, and medications, they may pose human safety risks associated with nanotoxicity. Thus, toxicity testing of nanoparticles is essential to assess the relative health risks associated with consumer exposure. METHODS: In this study, we identified the NOAEL (no observed adverse effect level) of the agglomerated/aggregated TiO2 P25 (approximately 180 nm) administered at repeated doses to Sprague-Dawley (SD) rats for 28 and 90 days. Ten of the 15 animals were necropsied for toxicity evaluation after the repeated-dose 90-day study, and the remaining five animals were allowed to recover for 28 days. The agglomerated/aggregated TiO2 P25 dose levels used included 250 mg kg- 1 d- 1 (low), 500 mg kg- 1 d- 1 (medium), and 1000 mg kg- 1 d- 1 (high), and their effects were compared with those of the vehicle control. During the treatment period, the animals were observed for mortality, clinical signs (detailed daily and weekly clinical observations), functional observation battery, weekly body weight, and food and water consumption and were also subjected to ophthalmological examination and urinalysis. After termination of the repeated-dose 28-day, 90-day, and recovery studies, clinical pathology (hematology, blood coagulation time, and serum biochemistry), necropsy (organ weights and gross findings), and histopathological examinations were performed. RESULTS: No systemic toxicological effects were associated with the agglomerated/aggregated TiO2 P25 during the repeated-dose 28-day, 90-day, and recovery studies in SD rats. Therefore, the NOAEL of the agglomerated/aggregated TiO2 P25 was identified as 1000 mg kg- 1 d- 1, and the substance was not detected in the target organs. CONCLUSION: Subacute and subchronic oral administration of the agglomerated/aggregated TiO2 P25 was unlikely to cause side effects or toxic reactions in rats.

Injectable and Pathogen-Mimicking Hydrogels for Enhanced Protective Immunity against Emerging and Highly Pathogenic Influenza Virus.

Seasonal emerging infectious diseases such as influenza A impose substantial risk and need new translational strategies to achieve active immunomodulation. Here, a novel injectable pathogen-mimicking hydrogel (iPMH) that can enhance both cellular and humoral immune responses is suggested. By the help of poly(γ-glutamic acid) that has abundant carboxylate groups and dispersion helper function, hydrophobic immunostimulatory 3-O-desacyl-4'-monophosphoryl lipid A (MPLA) molecules and viral antigens (PR8, W150) can be successfully combined as pathogen-mimicking adjuvants. Polyelectrolyte complex between the poly(γ-glutamic acid)-based adjuvants and collagens generate in situ gel-forming hydrogel at physiological temperature. When the iPMH are immunized, they act as a pathogen-mimicking (MPLA, H1N1, H5N1) immune priming center and a depot for continuous stimulation of immune system, resulting in the induction of high levels (8.5 times higher) of antigen-specific IgG titers in the sera of mice and the increased number of IFN-γ-producing cells (7.3 times higher) compared with those in the groups immunized with antigen plus clinically used aluminum gels. Following the intranasal infection of the mouse adapted virus (emerging infectious 2009 H1N1 and highly pathogenic 2006 H5N1) at 50 times the 50% lethal dose, the mice immunized with viral antigens plus iPMH exhibit 100% protective immunity against lethal virus challenge.

Self-assembled polyelectrolyte nanoparticles as fluorophore-free contrast agents for multicolor optical imaging.

In this work, we describe the fabrication of self-assembled polyelectrolyte nanoparticles that provide a multicolor optical imaging modality. Poly(γ-glutamic acid)(γ-PGA) formed self-assembled nanoparticles through electrostatic interactions with two different cationic polymers: poly(L-lysine)(PLL) and chitosan. The self-assembled γ-PGA/PLL and γ-PGA/chitosan nanoparticles were crosslinked by glutaraldehyde. Crosslinking of the ionic self-assembled nanoparticles with glutaraldehyde not only stabilized the nanoparticles but also generated a strong autofluorescence signal. Fluorescent Schiff base bonds (C=N) and double bonds (C=C) were generated simultaneously by crosslinking of the amine moiety of the cationic polyelectrolytes with monomeric glutaraldehyde or with polymeric glutaraldehyde. The unique optical properties of the nanoparticles that resulted from the crosslinking by glutaraldehyde were analyzed using UV/Vis and fluorescence spectroscopy. We observed that the fluorescence intensity of the nanoparticles could be regulated by adjusting the crosslinker concentration and the reaction time. The nanoparticles also exhibited high performance in the labeling and monitoring of therapeutic immune cells (macrophages and dendritic cells). These self-assembled nanoparticles are expected to be a promising multicolor optical imaging contrast agent for the labeling, detection, and monitoring of cells.

Multivalent Polymer Nanocomplex Targeting Endosomal Receptor of Immune Cells for Enhanced Antitumor and Systemic Memory Response.

We have designed and synthesized linear polymer-based nanoconjugates and nanocomplexes bearing multivalent immunostimulatory ligands and also demonstrated that the synthetic multivalent nanocomplexes led to an enhanced stimulation of immune cells in vitro and antitumor and systemic immune memory response in vivo. We have developed hyaluronic acid (HA)-based multivalent nanoconjugates and nanocomplexes for enhanced immunostimulation through the combination of multivalent immune adjuvants with CpG ODNs (as a TLR9 ligand) and cationic poly(L-lysine) (PLL; for the enhancement of cellular uptake). The multivalent HA-CpG nanoconjugate efficiently stimulated the antigen-presenting cells and the multivalent PLL/HA-CpG nanocomplex also led to an enhanced cellular uptake as well as continuous stimulation of endosomal TLR9. The mice vaccinated with dendritic cells treated with the multivalent nanocomplex exhibited tumor growth inhibition as well as a strong antitumor memory response.

Influence of albumin concentration on surface characteristics and cellular responses in the pre-incubation of multi-walled carbon nanotubes.

Reliable characterization of protein coronas (PCs) that form when nanomaterials are introduced into biological fluids is a critical step in the development of safe and efficient nanomedicine. We observed that bovine serum albumin (BSA)-coated multi-walled carbon nanotubes (MWCNTs) do not induce cytotoxicity, but have different cellular uptake rates depending on the BSA pretreatment concentration. To determine how these slight differences affect A549 cell responses and intracellular changes, we conducted spectroscopic (circular dichroism and Fourier-transform infrared) and spectrometric (nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry) analyses. The various characterization techniques conducted in this study reveal the following. (i) The composition ratio of PCs on MWCNTs differs depending on the BSA concentration. (ii) Analysis of the secondary structure of the proteins revealed that the α-helix structure increased with increasing BSA concentration. (iii) Proteomic analysis showed that different biological pathways were activated at levels higher and lower than 5 mg mL-1. Such combined spectroscopic and spectrometric approaches provide an integrated understanding of PC composition as well as how nano/bio-interface states are linked to cellular-level responses. Our results can support reliable and practical applications of nanomedicine development.

Safety assessment and gastrointestinal retention of orally administered cerium oxide nanoparticles in rats.

Cerium oxide nanoparticles (CeO2 NPs, NM-212) are well-known for their catalytic properties and antioxidant potential, and have many applications in various industries, drug delivery, and cosmetic formulations. CeO2 NPs exhibit strong antimicrobial activity and can be used to efficiently remove pathogens from different environments. However, knowledge of the toxicological evaluation of CeO2 NPs is too limited to support their safe use. In this study, CeO2 NPs were orally administered to Sprague Dawley rats for 13 weeks at the doses of 0, 10, 100, and 1000 mg/kg bw/day, followed by a four week recovery period. The hematology values for the absolute and relative reticulocyte counts in male rats treated with 1000 mg/kg bw/day CeO2 NPs were lower than those in control rats. The clinical chemistry values for sodium and chloride in the treated male rat groups (100 and 1000 mg/kg/day) and total protein and calcium in the treated female rat groups (100 mg/kg/day) were higher than those in the control groups. However, these changes were not consistent in both sexes, and no abnormalities were found in the corresponding pathological findings. The results showed no adverse effects on any of the parameters assessed. CeO2 NPs accumulated in the jejunum, colon, and stomach wall of rats administered 1000 mg/kg CeO2 NPs for 90 days. However, these changes were not abnormal in the corresponding histopathological and immunohistochemical examinations. Therefore, 1000 mg/kg bw/day may be considered the "no observed adverse effect level" of CeO2 NPs (NM-212) in male and female SD rats under the present experimental conditions.

Uptake and toxicity of cerium dioxide nanoparticles with different aspect ratio.

Cerium dioxide nanoparticles (CeONPs) have been extensively applied in research for future energy development due to two common oxidation states on their surface. Considering that shape (aspect ratio) is a key determinant of NPs-induced toxicity, we compared the toxicity of hexagonal (H)- and rod-shaped (R)-CeONPs in mice. At 24 h after pharyngeal aspiration, both types of CeONPs recruited surrounding immune cells (monocytes and neutrophils) into the lung, and R-CeONPs induced a more severe pulmonary inflammatory response compared with H-CeONPs. To identify an indicator to predict pulmonary inflammatory responses at the cellular level, we also investigated their responses in alveolar macrophage cells. At 24 h after treatment, both types of CeONPs were mainly located within the vacuoles (partially, in the lysosome) in the cytoplasm. Mitochondrial damage, intracellular calcium accumulation, and increased NO production were observed in cells exposed to both types of CeONPs, ultimately resulting in a decrease in cell viability. More interestingly, both types of CeONPs formed multinucleated giant cells. Meanwhile, contrary to when suspended in deionized water, R-CeONPs were strongly aggregated with a negative charge in cell culture media, whereas H-CeONPs were relatively well-dispersed with a positive charge. R-CeONPs-induced lysosomal extension was also recovered by premix with negatively charged DNA, and even NPs suspended in cell culture media without cells were detected under the FACS system, suggesting interference by protein corona. Therefore, we suggest that shape (aspect ratio) is an important factor determining inhaled NPs-induced pathology and that the effect of the surface charge and protein corona should be carefully considered in interpreting results derived from in vitro tests. Furthermore, we propose that the relationship between the formation of multinucleated giant cells and the inflammatory response of inhaled CeONPs should be further studied.

Comparative Study on Nanotoxicity in Human Primary and Cancer Cells.

Nanomaterial toxicity tests using normal and cancer cells may yield markedly different results. Here, nanomaterial toxicity between cancer and primary human cells was compared to determine the basic cell line selection criteria for nanomaterial toxicity analyses. Specifically, we exposed two cancer (A549 and HepG2) and two normal cell lines (NHBE and HH) cell lines to SiO2 nanoparticles (NPs) and evaluated the cytotoxicity (MTS assay), cell death mode, and intracellular NP retention. MTS assay results revealed higher sensitivity of HH cells to SiO2 NPs than HepG2 cells, while no difference was observed between NHBE and A549 cells. In addition, SiO2 NPs primarily induced necrosis in all the cell lines. Moreover, we evaluated NP accumulation by treating the cell lines with fluorescein-isothiocyanate-labeled SiO2 NPs. After 48 h of treatment, less than 10% of A549 and HepG2 cells and more than 30% of NHBE and HH cells contained the labeled NPs. Collectively, our results suggest that cell viability, death mode, and intracellular compound accumulation could be assessed using cancer cells. However, the outcomes of certain investigations, such as intracellular NP retention, may differ between cancer and normal cells.

Photocatalytic activity of nanoparticles: the development of the standardized measurement for physiological conditions.

Recently a new International Standard for testing nanomaterial photocatalytic activity under physiological conditions was issued by Technical Committee 229 (Nanotechnologies) of the International Organization for Standardization (ISO 20814:2019 Nanotechnologies-Testing the photocatalytic activity of nanoparticles for NADH oxidation). The document offers a robust, high throughput photocatalytic assay using a bio-compatible indicator nicotinamide amide dinucleotide (NAD) and provides a screening tool to gauge nanomaterial potency for phototoxicity. This paper describes the measurement principles behind this assay, the scope of the standard and its validation through an interlaboratory comparison study using a traceable standard reference material (SRM 1898).

Titanium Dioxide Induces Apoptosis under UVA Irradiation via the Generation of Lysosomal Membrane Permeabilization-Dependent Reactive Oxygen Species in HaCat Cells.

Titanium dioxide nanoparticles (TiO2 NPs) have wide commercial applications, owing to their small size; however, the biosafety of TiO2 NPs should be evaluated further. In this study, we aimed to investigate the cytotoxicity of TiO2 NPs in the presence and absence of ultraviolet A (UVA) irradiation in human keratinocyte HaCaT cells. TiO2 NPs did not significantly affect cell viability in the absence of UVA irradiation. Nonetheless, UVA-irradiated TiO2 NPs induced caspase-dependent apoptosis of HaCaT cells. Exposure of HaCaT cells to TiO2 NPs and UVA resulted in reactive oxygen species (ROS) generation and lysosomal membrane permeabilization (LMP); both effects were not observed in the absence of UVA irradiation. An analysis of the relationship between LMP and ROS, using CA-074 as a cathepsin inhibitor or NAC as an antioxidant, showed that LMP stimulates ROS generation under these conditions. These results imply that LMP-dependent oxidative stress plays a critical role in the UVA phototoxicity of TiO2 NPs in HaCaT cells.

Real-time heart rate monitoring system for cardiotoxicity assessment of Daphnia magna using high-speed digital holographic microscopy.

Machine vision techniques for monitoring heart rates in aquatic bioassays have been applied to cardiotoxicity assessment. However, the requisite large data sizes and long calculation times make long-term observations of heart rates difficult. In this study, we developed a real-time heart rate monitoring system for individual Daphnia magna in a water chamber mounter that immobilizes their movement in 100 mL media. Heart rates are calculated from real-time, time-resolved relative phase information from digital holograms acquired with a 200 fps camera and parallel computation using a graphics processing unit. With this system, we monitored the real-time changes in the heart rates of individual D. magna specimens exposed to H2O2 as a positive control for reactive oxygen species (ROS) levels in an aquatic environment for 10 h, a period long enough to observe dynamic heart rate responses to the mounting process and exposure and to establish heart rate trends. An additional group analysis was conducted to compare to conventional cardiotoxicity assessment, with results of both assessments showing that the heart rates reduced according to ROS level by H2O2 exposure concentration. Notably, the results of our real-time dynamic heart rate monitoring in aquatic conditions indicated that establishing a relaxation heart rate before measurements could improve the accuracy of toxicity assessment. It is believed that the system developed in this study, achieving the simultaneous measurement, analysis, and display of reconstructed results, will find wide application in other aquatic bioassays.

Global Proteomics to Study Silica Nanoparticle-Induced Cytotoxicity and Its Mechanisms in HepG2 Cells.

Silica nanoparticles (SiO2 NPs) are commonly used in medical and pharmaceutical fields. Research into the cytotoxicity and overall proteomic changes occurring during initial exposure to SiO2 NPs is limited. We investigated the mechanism of toxicity in human liver cells according to exposure time [0, 4, 10, and 16 h (h)] to SiO2 NPs through proteomic analysis using mass spectrometry. SiO2 NP-induced cytotoxicity through various pathways in HepG2 cells. Interestingly, when cells were exposed to SiO2 NPs for 4 h, the morphology of the cells remained intact, while the expression of proteins involved in mRNA splicing, cell cycle, and mitochondrial function was significantly downregulated. These results show that the toxicity of the nanoparticles affects protein expression even if there is no change in cell morphology at the beginning of exposure to SiO2 NPs. The levels of reactive oxygen species changed significantly after 10 h of exposure to SiO2 NPs, and the expression of proteins associated with oxidative phosphorylation, as well as the immune system, was upregulated. Eventually, these changes in protein expression induced HepG2 cell death. This study provides insights into cytotoxicity evaluation at early stages of exposure to SiO2 NPs through in vitro experiments.

Interference of engineered nanomaterials in flow cytometry: A case study.

Nanotechnology is regarded as the enabling technology of the 21st century. However, only a relatively small number of nano-enabled medical and healthcare products finally made their way to the market. There are several reasons why such innovative approaches fail in translation, with one key factor being the uncertainty surrounding their safety assessment. Although well described, interference reactions of engineered nanomaterials (ENM) with classical cytotoxicity assays remain a major source of uncertainty. Flow cytometry is a powerful, widely used, in vitro technique. Its readout is based on the detection of refracted laser light and fluorescence signals. It is therefore susceptible to ENM interference. Here we investigated possible interferences of ENM in the Annexin V/propidium iodide (PI) assay, which quantifies apoptotic and necrotic cell populations by flow cytometry. Two case studies were conducted using either silica or gold nanoparticles differing in size, specific surface area and surface chemistry. Both ENM types were found to cause distinct interference reactions at realistic concentrations. Silica particles induced false-positive signals; however only in the absence of a protein corona and in conjunction with a particular fluorophore combination (FITC/PI). In contrast, gold particles led to complex quenching effects which were only marginally influenced by the presence of proteins and occurred for both fluorophore combinations analyzed. We present a versatile spike-in approach which is applicable to all ENM and cell types. It further allows for the identification of a broad range of different interference phenomena, thereby increasing the reliability and quality of flow cytometry and ENM hazard assessment.

Photothermal-modulated drug delivery and magnetic relaxation based on collagen/poly(γ-glutamic acid) hydrogel.

Injectable and stimuli-responsive hydrogels have attracted attention in molecular imaging and drug delivery because encapsulated diagnostic or therapeutic components in the hydrogel can be used to image or change the microenvironment of the injection site by controlling various stimuli such as enzymes, temperature, pH, and photonic energy. In this study, we developed a novel injectable and photoresponsive composite hydrogel composed of anticancer drugs, imaging contrast agents, bio-derived collagen, and multifaceted anionic polypeptide, poly (γ-glutamic acid) (γ-PGA). By the introduction of γ-PGA, the intrinsic temperature-dependent phase transition behavior of collagen was modified to a low viscous sol state at room temperature and nonflowing gel state around body temperature. The modified temperature-dependent phase transition behavior of collagen/γ-PGA hydrogels was also evaluated after loading of near-infrared (NIR) fluorophore, indocyanine green (ICG), which could transform absorbed NIR photonic energy into thermal energy. By taking advantage of the abundant carboxylate groups in γ-PGA, cationic-charged doxorubicin (Dox) and hydrophobic MnFe2O4 magnetic nanoparticles were also incorporated successfully into the collagen/γ-PGA hydrogels. By illumination of NIR light on the collagen/γ-PGA/Dox/ICG/MnFe2O4 hydrogels, the release kinetics of Dox and magnetic relaxation of MnFe2O4 nanoparticles could be modulated. The experimental results suggest that the novel injectable and NIR-responsive collagen/γ-PGA hydrogels developed in this study can be used as a theranostic platform after loading of various molecular imaging probes and therapeutic components.

Infection-mimicking poly(γ-glutamic acid) as adjuvant material for effective anti-tumor immune response.

Bio-derived low molecular weight poly(γ-glutamic acid) (γ-PGA) was suggested as a novel adjuvant material for use in cancer vaccines. When the infection-mimicking γ-PGA was immunized with ovalbumin (OVA) as a model antigen, increase in the dendritic cell (DC)-mediated functions such as activation, maturation, antigen uptake, migration to lymph nodes, and priming of lymphocytes, which included cross-presentation, was observed. These DC-mediated functions were found to be facilitated by γ-PGA in a dose-dependent manner, with stimulation of toll-like receptor 4 (TLR4) being one of the underlying mechanisms. The in vivo efficacy of γ-PGA was tested in a mouse tumor model where both arms of adaptive immunity (humoral and cell-mediated) were found to be significantly enhanced in the presence of γ-PGA, indicating efficient priming of B and T cells. Moreover, immunization of mice with γ-PGA followed by EG7-OVA tumor challenge led to dramatic inhibition of tumor growth. After 71 days, the cured mice were rechallenged with tumor cells at a distant site in order to test the memory effect. No tumor growth was observed, which indicates the presence of a systemic, long-lasting immune response. Based on these results, low molecular weight γ-PGA is expected to have tremendous potential for applications in cancer immunotherapy.

Sequential delivery of an anticancer drug and combined immunomodulatory nanoparticles for efficient chemoimmunotherapy.

Chemoimmunotherapy combines chemotherapy based on anticancer drugs with immunotherapy based on immune activators to eliminate or inhibit the growth of cancer cells. In this study, water-insoluble paclitaxel (PTX) was dispersed in water using hyaluronic acid (HA) to generate a tumor-associated antigen in the tumor microenvironment. Cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) were used to enhance the T helper (Th) 1 immune response. However, CpG ODNs also induced the secretion of interleukin-10 (IL-10) that reduces the Th1 response and enhances the T helper 2 (Th2) response. Therefore, RNA interference was used to downregulate IL-10 secretion from bone marrow-derived den-dritic cells (BMDCs). For the combined immunomodulation of BMDCs, we fabricated two types of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) containing CpG ODNs to activate BMDCs via Toll-like receptor 9 (CpG ODN-encapsulated PLGA NPs, PCNs) or a small interfering RNA to silence IL-10 (IL-10 small interfering RNA-encapsulated PLGA NPs, PINs). Treatment of BMDCs with both types of PLGA NPs increased the Th1/Th2 cytokine (IL-12/IL-10) expression ratio, which is important for the effective induction of an antitumor immune response. After primary injection with the HA/PTX complex, the tumor-associated antigen was generated and taken up by tumor-recruited BMDCs. After a secondary injection with immunomodulating PCNs and PINs, the BMDCs became activated and migrated to the tumor-draining lymph nodes. As a result, the combination of chemotherapy using the HA/PTX complex and immunotherapy using PCNs and PINs not only efficiently inhibited tumor growth but also increased the animal survival rate. Taken together, our results suggest that the sequential treatment of cancer cells with a chemotherapeutic agent and immunomodulatory nanomaterials represents a promising strategy for efficient cancer therapy.

Programmed nanoparticles for combined immunomodulation, antigen presentation and tracking of immunotherapeutic cells.

We report programmed nanoparticles (pNPs) that can tailor the immunotherapeutic function of primary bone marrow-derived dendritic cells (BMDCs) by ex vivo combined immunomodulation and track the in vivo migration of them after injection into body. Because DCs are the most effective antigen-presenting cells (APCs) that are able to present the antigens to T cells that contribute to tumor rejection, the maturation and monitoring of therapeutic DCs are essential for the efficient cancer immunotherapy. For combined immunomodulation of DCs, poly (lactic-co-glycolic acid) (PLGA) NPs containing both small interfering RNA (siRNA) for the knock-down of immune-suppressor gene (signal transducer and activator of transcription-3, STAT3) of DCs and an immune response modifier (imiquimod, R837) for the activation of DCs through the toll-like receptor 7 (TLR7) were developed. To deliver tumor antigen-specific information to DCs ex vivo and track the migration of DCs in vivo, another type of PLGA NPs containing tumor model antigen (ovalbumin, OVA) and near-infrared (NIR) fluorophores (indocyanine green, ICG) were also fabricated. These pNPs were taken up efficiently by DCs and various cytokines were expressed in matured DCs. DCs treated with pNPs also efficiently presented antigen-peptide to CD8 OVA 1.3 T cells through cross-presentation. Immunization of mice with these pNPs-treated DCs induced OVA-specific cytotoxic T lymphocytes (CTL) activity against the EG7-OVA tumor model and inhibited tumor growth efficiently. In addition, the migration of PLGA NPs-treated DCs to lymph nodes was monitored by NIR imaging technique. These multifunctional pNPs represent a promising technology for the combined immunomodulation and antigen-specific tumor therapy.