Oral Excretion Kinetics of Food-Additive Silicon Dioxides and Their Effect on In Vivo Macrophage Activation.

A food additive, silicon dioxide (SiO2) is commonly used in the food industry as an anti-caking agent. The presence of nanoparticles (NPs) in commercial food-grade SiO2 has raised concerns regarding their potential toxicity related to nano size. While recent studies have demonstrated the oral absorption and tissue distribution of food-additive SiO2 particles, limited information is available about their excretion behaviors and potential impact on macrophage activation. In this study, the excretion kinetics of two differently manufactured (fumed and precipitated) SiO2 particles were evaluated following repeated oral administration to rats for 28 d. The excretion fate of their intact particles, decomposed forms, or ionic forms was investigated in feces and urine, respectively. Monocyte uptake, Kupffer cell activation, and cytokine release were assessed after the oral administration of SiO2 particles. Additionally, their intracellular fates were determined in Raw 264.7 cells. The results revealed that the majority of SiO2 particles were not absorbed but directly excreted via feces in intact particle forms. Only a small portion of SiO2 was eliminated via urine, predominantly in the form of bioconverted silicic acid and slightly decomposed ionic forms. SiO2 particles were mainly present in particle forms inside cells, followed by ionic and silicic acid forms, indicating their slow conversion into silicic acid after cellular uptake. No effects of the manufacturing method were observed on excretion and fates. Moreover, no in vivo monocyte uptake, Kupffer cell polarization, or cytokine release were induced by orally administered SiO2 particles. These finding contribute to understanding the oral toxicokinetics of food-additive SiO2 and provide valuable insights into its potential toxicity.

Convolution Neural Network with Laser-Induced Breakdown Spectroscopy as a Monitoring Tool for Laser Cleaning Process.

In this study, eight different painted stainless steel 304L specimens were laser-cleaned using different process parameters, such as laser power, scan speed, and the number of repetitions. Laser-induced breakdown spectroscopy (LIBS) was adopted as the monitoring tool for laser cleaning. Identification of LIBS spectra with similar chemical compositions is challenging. A convolutional neural network (CNN)-based deep learning method was developed for accurate and rapid analysis of LIBS spectra. By applying the LIBS-coupled CNN method, the classification CNN model accuracy of laser-cleaned specimens was 94.55%. Moreover, the LIBS spectrum analysis time was 0.09 s. The results verified the possibility of using the LIBS-coupled CNN method as an in-line tool for the laser cleaning process.

Food Additive Zinc Oxide Nanoparticles: Dissolution, Interaction, Fate, Cytotoxicity, and Oral Toxicity.

Food additive zinc oxide (ZnO) nanoparticles (NPs) are widely used as a Zn supplement in the food and agriculture industries. However, ZnO NPs are directly added to complex food-matrices and orally taken through the gastrointestinal (GI) tract where diverse matrices are present. Hence, the dissolution properties, interactions with bio- or food-matrices, and the ionic/particle fates of ZnO NPs in foods and under physiological conditions can be critical factors to understand and predict the biological responses and oral toxicity of ZnO NPs. In this review, the solubility of ZnO NPs associated with their fate in foods and the GI fluids, the qualitative and quantitative determination on the interactions between ZnO NPs and bio- or food-matrices, the approaches for the fate determination of ZnO NPs, and the interaction effects on the cytotoxicity and oral toxicity of ZnO NPs are discussed. This information will be useful for a wide range of ZnO applications in the food industry at safe levels.

Oral Toxicokinetics, Tissue Distribution, and 28-Day Oral Toxicity of Two Differently Manufactured Food Additive Silicon Dioxides.

(1) Background: Synthetic amorphous silica (SAS) is widely used as a food additive and contains nano-sized particles. SAS can be produced by fumed and precipitated methods, which may possess different physiochemical properties, toxicokinetics, and oral toxicity. (2) Methods: The toxicokinetics of fumed SAS and precipitated SAS were evaluated following a single-dose oral administration in rats. The tissue distribution and fate of both SAS particles were assessed after repeated oral administration in rats for 28 d, followed by recovery period for 90 d. Their 28-d repeated oral toxicity was also evaluated. (3) Results: Precipitated SAS showed higher oral absorption than fumed SAS, but the oral absorption of both SAS particles was low (<4%), even at 2000 mg/kg. Our tissue-distribution study revealed that both SAS particles, at a high dose (2000 mg/kg), were accumulated in the liver after repeated administration for 28 d, but the increased concentrations returned to normal levels at 29 d, the first day of the recovery period. A higher distribution level of precipitated SAS than fumed SAS and decomposed particle fates of both SAS particles were found in the liver at 28 d. No significant toxicological findings were observed after 28-d oral administration, suggesting their low oral toxicity. (4) Conclusions: Different manufacturing methods of SAS can, therefore, affect its oral toxicokinetics and tissue distribution, but not oral toxicity.

Non-back-reflecting polygon scanner with applications in surface cleaning.

Polygon mirror scanners are attracting considerable interest owing to their rapid speed and large scanning area. Here, we focused on the back-reflection effect of the polygon scanner. A new polygon scanner system was designed based on a geometric analysis. The final equations for the design express the position of the laser beam source having the largest scanning length without the reflected beam traveling back to the fiber. The proposed system performed a raster scan on an area. Additionally, a paint stripping experiment was conducted to demonstrate the potential use of our scanner in commercial laser cleaning applications.

Particle Size and Biological Fate of ZnO Do Not Cause Acute Toxicity, but Affect Toxicokinetics and Gene Expression Profiles in the Rat Livers after Oral Administration.

Zinc oxide (ZnO) particles have been used as dietary supplements because zinc is an essential trace element for humans. Along with the rapid development of nanotechnology, the use of ZnO nanoparticles (NPs) is increasing in the food industry, but their oral toxicity potential still remains to be answered. In this study, the effects of particle size and biological fate of ZnO on acute toxicity, toxicokinetics, and gene expression profiles in the livers were investigated after oral administration of ZnO NPs (N-ZnO), bulk-sized ZnO (B-ZnO) or Zn ions in rats. The plasma concentration-time profiles after a single-dose oral administration of ZnOs differed depending on particle/ionic forms and particle size, showing high absorption of Zn ions, followed by N-ZnO and B-ZnO, although in vivo solubility did not differ from particle size. No significant acute toxicity was found after oral administration of ZnOs for 14 days in rats. However, transcriptomic responses in the livers were differently affected, showing that metabolic process and metal biding were up-regulated by Zn ions and N-ZnO, respectively, which were not pronounced in the liver treated with B-ZnO. These findings will be useful to predict the potential oral toxicity of ZnO NPs and further mechanistic and long-term exposure studies are required to assume their safety.

Determination of Two Differently Manufactured Silicon Dioxide Nanoparticles by Cloud Point Extraction Approach in Intestinal Cells, Intestinal Barriers and Tissues.

Food additive amorphous silicon dioxide (SiO2) particles are manufactured by two different methods-precipitated and fumed procedures-which can induce different physicochemical properties and biological fates. In this study, precipitated and fumed SiO2 particles were characterized in terms of constituent particle size, hydrodynamic diameter, zeta potential, surface area, and solubility. Their fates in intestinal cells, intestinal barriers, and tissues after oral administration in rats were determined by optimizing Triton X-114-based cloud point extraction (CPE). The results demonstrate that the constituent particle sizes of precipitated and fumed SiO2 particles were similar, but their aggregate states differed from biofluid types, which also affect dissolution properties. Significantly higher cellular uptake, intestinal transport amount, and tissue accumulation of precipitated SiO2 than of fumed SiO2 was found. The intracellular fates of both types of particles in intestinal cells were primarily particle forms, but slowly decomposed into ions during intestinal transport and after distribution in the liver, and completely dissolved in the bloodstream and kidneys. These findings will provide crucial information for understanding and predicting the potential toxicity of food additive SiO2 after oral intake.

Fate Determination of ZnO in Commercial Foods and Human Intestinal Cells.

(1) Background: Zinc oxide (ZnO) particles are widely used as zinc (Zn) fortifiers, because Zn is essential for various cellular functions. Nanotechnology developments may lead to production of nano-sized ZnO, although nanoparticles (NPs) are not intended to be used as food additives. Current regulations do not specify the size distribution of NPs. Moreover, ZnO is easily dissolved into Zn ions under acidic conditions. However, the fate of ZnO in commercial foods or during intestinal transit is still poorly understood. (2) Methods: We established surfactant-based cloud point extraction (CPE) for ZnO NP detection as intact particle forms using pristine ZnO-NP-spiked powdered or liquid foods. The fate determination and dissolution characterization of ZnO were carried out in commercial foods and human intestinal cells using in vitro intestinal transport and ex vivo small intestine absorption models. (3) Results: The results demonstrated that the CPE can effectively separate ZnO particles and Zn ions in food matrices and cells. The major fate of ZnO in powdered foods was in particle form, in contrast to its ionic fate in liquid beverages. The fate of ZnO was closely related to the extent of its dissolution in food or biomatrices. ZnO NPs were internalized into cells in both particle and ion form, but dissolved into ions with time, probably forming a Zn-ligand complex. ZnO was transported through intestinal barriers and absorbed in the small intestine primarily as Zn ions, but a small amount of ZnO was absorbed as particles. (4) Conclusion: The fate of ZnO is highly dependent on food matrix type, showing particle and ionic fates in powdered foods and liquid beverages, respectively. The major intracellular and intestinal absorption fates of ZnO NPs were Zn ions, but a small portion of ZnO particle fate was also observed after intestinal transit. These findings suggest that the toxicity of ZnO is mainly related to the Zn ion, but potential toxicity resulting from ZnO particles cannot be completely excluded.

Protein Food Matrix⁻ZnO Nanoparticle Interactions Affect Protein Conformation, but May not Be Biological Responses.

Because of their nutritional value, zinc oxide (ZnO) nanoparticles (NPs) are applied as a dietary source of zinc, by direct addition to complex, multiple-component food matrices. The thereby occurring interactions of NPs with food matrices may have biological or toxic effects. In particular, NP interactions with food protein can lead to structural deformation of the latter, potentially changing its digestive efficiency and gastrointestinal absorption. In this study, interactions between ZnO NPs and a representative complex protein food matrix, skim milk, were compared with those between NPs and individual components of this food matrix (i.e., protein, saccharide, and mineral). The effects of the interactions on biological responses were investigated in terms of cytotoxicity, cellular uptake, intestinal transport, structural deformation for proteins, and digestive efficiency. The results demonstrated that the physicochemical properties of ZnO NPs were strongly influenced by the protein matrix type, leading to an increased dispersion stability in the complex protein matrix. However, these interactions did not affect cell proliferation, membrane damage, cellular uptake, intestinal transportation, or protein digestive efficiency, although a slight conformational change of proteins was observed in the presence of ZnO NPs. In conclusion, no toxic effects were observed, suggesting the safety of NPs when added to complex food matrices.

Effects of Interactions between ZnO Nanoparticles and Saccharides on Biological Responses.

Zinc oxide (ZnO) nanoparticles (NPs) are widely used as a Zn supplement, because Zn plays a role in many cellular and immune functions but public concern about their potentially undesirable effects on the human body is growing. When NPs are added in food matrices, interactions between NPs and food components occur, which can affect biological systems. In this study, interactions between ZnO NPs and saccharides were investigated by measuring changes in hydrodynamic radius, zeta potential and solubility and by quantifying amounts of adsorbed saccharides on NPs; acacia honey, sugar mixtures (containing equivalent amounts of fructose, glucose, sucrose and maltose) and monosaccharide solutions were used as model compounds. Biological responses of NPs dispersed in different saccharides were also evaluated in human intestinal cells and rats in terms of cytotoxicity, cellular uptake, intestinal transport and oral absorption. The results demonstrate that the hydrodynamic radii and zeta potentials of NPs were highly affected by saccharides. In addition, trace nutrients influenced NP/saccharide interactions and interactive effects between saccharides on the interactions were found. NPs in all saccharides increased inhibition of cell proliferation and enhanced cellular uptake. Oral absorption of NPs was highly enhanced by 5% glucose, which is in-line with intestinal transport result. These findings show that ZnO NPs interact with saccharides and these interactions affects biological responses.

Chondrocyte-derived extracellular matrix suppresses pathogenesis of human pterygium epithelial cells by blocking the NF-κB signaling pathways.

PURPOSE: We previously have reported that chondrocyte-derived extracellular matrix (CDECM) suppresses the growth of pterygium in athymic nude mice. The aim of this study is to demonstrate the effect of CDECM on the pterygium epithelial cells and molecular signaling pathways in human primary pterygium epithelial cells (hPECs). METHODS: Human conjunctival epithelial cells (hConECs) were used for identification of the effect of CDECM on normal conjunctiva. The effects of CDECM on proliferation were measured with the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfenyl)-2H-tetrazolium (MTS) assay. Cell migration was evaluated according to the scratch wound closure assay and the Transwell invasion assay. Pterygium-related angiogenesis, inflammation, and extracellular matrix remodeling were analyzed with immunoblot and enzyme-linked immunosorbent assay (ELISA). The level of oxidative stress was detected with 2',7'-dichlorofluorescein diacetate (DCFH-DA). Protein kinase signaling was also analyzed with immunoblot. RESULTS: CDECM did not show cytotoxicity until 1 mg/ml in the hConECs and hPECs. Cell migration and invasion were markedly reduced by treatment of 1 mg/ml CDECM in the hPECs to 34% of the control, but not in the hConECs. CDECM significantly downregulated matrix metallopeptidase 9 (MMP-9) and fibronectin and upregulated tissue inhibitor of metalloprotease 1 (TIMP-1) and -2 in the hPECs. Angiogenic factors, such as vascular endothelial growth factor (VEGF), antivascular cellular adhesion molecule 1 (VCAM-1), and cluster of differentiation 31 (CD31), and proinflammatory factors, including tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (Cox2), interleukin 6 (IL-6), and prostaglandin E2 (PGE2), were dramatically reduced by CDECM in the hPECs. Furthermore, CDECM significantly inhibited the generation of intracellular reactive oxygen species and the expression of NADPH oxidase subunits, Nox2 and p47phox. CDECM induced nuclear factor erythroid-2 related factor 2 (Nrf2) mediated-antioxidant enzyme heme oxygenase-1 (HO-1). CDECM also suppressed nuclear factor-kappa B (NF-κB) activation and the phosphorylation of p38 mitogen-activated protein kinase (MAPK), protein kinase C alpha (PKCα), and PKCθ. CONCLUSIONS: CDECM was markedly effective in pathogenesis of hPECs. CDECM-suppressed migration of hPECs resulted from the inhibition of NF-κB activation and the improvement of Nrf2 induction by blocking the p38 MAPK and PKC signaling pathways.

Bioavailability of Silica, Titanium Dioxide, and Zinc Oxide Nanoparticles in Rats.

Inorganic nanoparticles have been widely applied to various industrial fields and biological applications. However, the question as to whether nanoparticles are more efficiently absorbed into the systemic circulation than bulk-sized materials remains to be unclear. In the present study, the physico-chemical and dissolution properties of the most extensively developed inorganic nanoparticles, such as silica (SiO2), titanium dioxide (TiO2), and zinc oxide (ZnO), were analyzed, as compared with bulk-sized particles. Furthermore, the bioavailability of nanoparticles versus their bulk counterparts was evaluated in rats after a single oral administration and intravenous injection, respectively. The results demonstrated that all bulk materials had slightly higher crystallinity than nanoparticles, however, their dissolution properties were not affected by particle size. No significant difference in oral absorption and bioavailability of both SiO2 and TiO2 was found between nano- and bulk-sized materials, while bulk ZnO particles were more bioavailable in the body than ZnO nanoparticles. These finding will provide critical information to apply nanoparticles with high efficiency as well as to predict their toxicity potential.

Novel control of plasma expansion direction aimed at very low pressure laser-induced plasma spectroscopy.

A plasma confinement approach has been applied to enhance the signal intensity of laser-induced plasma in low pressure conditions down to 10(-2) torr. Detection of plasma emission spectrum is a daunting task at low pressure due to the low electron density and the short persistence time of plasma that undergoes a rapid expansion. Here we devised a spatial confinement setup that increases the electron density at various range of low pressures. A confining window is placed above the sample surface to control the direction of the expanding plasma aimed at optimizing the efficiency of the low pressure detection. More ions, atoms, and molecules can reach the detector by a direction-controlled confinement of an otherwise freely expanding plasma. The spectral intensities of neutral atoms increased up to 4 times with a single laser pulse by the proposed confining method at 1 torr. The signal of doubly ionized carbon atom which was detectable only at low pressure is also enhanced 4 times. The results of this study provide an important guideline for strengthening the otherwise weak signals at low pressure by controlling the plasma expansion direction.

Inactivation of the virulence factors from 2,3-butanediol-producing Klebsiella pneumoniae.

The microbiological production of 2,3-butanediol (2,3-BDO) has attracted considerable attention as an alternative way to produce high-value chemicals from renewable sources. Among the number of 2,3-BDO-producing microorganisms, Klebsiella pneumoniae has been studied most extensively and is known to produce large quantity of 2,3-BDO from a range of substrates. On the other hand, the pathogenic characteristics of the bacteria have limited its industrial applications. In this study, two major virulence traits, outer core LPS and fimbriae, were removed through homologous recombination from 2,3-BDO-producing K. pneumoniae 2242 to expand its uses to the industrial scale. The K. pneumoniae 2242 ∆wabG mutant strain was found to have an impaired capsule, which significantly reduced its ability to bind to the mucous layer and evade the phagocytic activity of macrophage. The association with the human ileocecal epithelial cell, HCT-8, and the bladder epithelial cell, T-24, was also reduced dramatically in the K. pneumoniae 2242 ∆fimA mutant strain that was devoid of fimbriae. However, the growth rate and production yield for 2,3-BDO were unaffected. The K. pneumoniae strains developed in this study, which are devoid of the major virulence factors, have a high potential for the efficient and sustainable production of 2,3-BDO.

Polymer coated CaAl-layered double hydroxide nanomaterials for potential calcium supplement.

We have successfully prepared layered double hydroxide (LDH) nanomaterials containing calcium and aluminum ions in the framework (CaAl-LDH). The surface of CaAl-LDH was coated with enteric polymer, Eudragit®L 100 in order to protect nanomaterials from fast dissolution under gastric condition of pH 1.2. The X-ray diffraction patterns, Fourier transform infrared spectroscopy, scanning electron and transmission electron microscopy revealed that the pristine LDH was well prepared having hydrocalumite structure, and that the polymer effectively coated the surface of LDH without disturbing structure. From thermal analysis, it was determined that only a small amount (less than 1%) of polymer was coated on the LDH surface. Metal dissolution from LDH nanomaterials was significantly reduced upon Eudragit®L 100 coating at pH 1.2, 6.8 and 7.4, which simulates gastric, enteric and plasma conditions, respectively, and the dissolution effect was the most suppressed at pH 1.2. The LDH nanomaterials did not exhibit any significant cytotoxicity up to 1000 µg/mL and intracellular calcium concentration significantly increased in LDH-treated human intestinal cells. Pharmacokinetic study demonstrated absorption efficiency of Eudragit®L 100 coated LDH following oral administration to rats. Moreover, the LDH nanomaterials did not cause acute toxic effect in vivo. All the results suggest the great potential of CaAl-LDH nanomaterials as a calcium supplement.

Ligand-based magnetic extraction and safety assessment of zinc oxide nanoparticles in food products.

Zinc oxide (ZnO) is a zinc supplement widely used in health products and is approved by the FDA as Generally Regarded as Safe (GRAS). However, concerns have arisen regarding the potential health effects of nanoscale ZnO, as its reactivity differs from that of its bulk form. This has led to the need for an efficient method to extract ZnO from food products without altering its physicochemical properties, where conventional methods have proven to be inadequate. This study introduces an innovative approach using starch magnetic particles (SMPs) functionalized with a 12-amino acid peptide modified with five lysines (ZBP), that has specific affinity to ZnO. ZBP@SMPs effectively and rapidly extract intact ZnO from food products, achieving recovery efficiencies ranging from 60% to 90%, all while maintaining its morphology and crystallinity. The diameter of ZnO particles recovered from six commercial food products ranged from 25 to 500 nm, with 33% falling below 100 nm, highlighting the need for a size-dependent toxicity study. However, cytotoxicity assessment on human intestinal Caco-2 cells shows all ZnO samples affects cell proliferation and membrane integrity in a dose-dependent manner due to partial dissolution. This study contributes to understanding the safety of ZnO-containing food products and highlights potential health implications associated with their consumption.

Food Additive Solvents Increase the Dispersion, Solubility, and Cytotoxicity of ZnO Nanoparticles.

Zinc oxide (ZnO) nanoparticles (NPs) are utilized as a zinc (Zn) fortifier in processed foods where diverse food additives can be present. Among them, additive solvents may strongly interact with ZnO NPs by changing the dispersion stability in food matrices, which may affect physico-chemical and dissolution properties as well as the cytotoxicity of ZnO NPs. In this study, ZnO NP interactions with representative additive solvents (methanol, glycerin, and propylene glycol) were investigated by measuring the hydrodynamic diameters, solubility, and crystallinity of ZnO NPs. The effects of these interactions on cytotoxicity, cellular uptake, and intestinal transport were also evaluated in human intestinal cells and using in vitro human intestinal transport models. The results revealed that the hydrodynamic diameters of ZnO NPs in glycerin or propylene glycol, but not in methanol, were significantly reduced, which is probably related to their high dispersion and increased solubility under these conditions. These interactions also caused high cell proliferation inhibition, membrane damage, reactive oxygen (ROS) generation, cellular uptake, and intestinal transport. However, the crystal structure of ZnO NPs was not affected by the presence of additive solvents. These findings suggest that the interactions between ZnO NPs and additive solvents could increase the dispersion and solubility of ZnO NPs, consequently leading to small hydrodynamic diameters and different biological responses.

Fate Determination and Characterization of Food Additive Silicon Dioxide and Titanium Dioxide in Commercial Foods.

BACKGROUND: Silicon dioxide (SiO2) and titanium dioxide (TiO2) are ones of the most widely used food additives as an anti-caking and a coloring agent, respectively, in the food industry. Understanding particle, aggregate, or ionic fates of two additives in commercial products is of importance to predict their potential toxicity. METHODS: Triton X-114 (TX-114)-based cloud point extraction (CPE) methods for two additives were optimized in food matrices. Their particle or ionic fates in various commercial foods were determined by the CPE, and the physico-chemical properties of separated particles were further characterized. RESULTS: SiO2 and TiO2 were primarily present as particle forms without changes in constituent particle size, size distribution, and crystalline phase. The maximum solubilities of SiO2 and TiO2 were 5.5% and 0.9%, respectively, depending on food matrix type, supporting their major particle fates in complex food matrices. CONCLUSIONS: These findings will provide basic information about the fates and safety aspects of SiO2 and TiO2 additives in commercial processed foods.

NOAEL cancer therapy: a tumor targetable docetaxel-inorganic polymer nanohybrid prevents drug-induced neutropenia.

To date, cancer therapies largely consist of five pillars: surgery, radiation, chemotherapy, targeted therapy, and immunotherapy. Still, researchers are trying to innovate the current cancer therapies to pursue an ideal one without side effects. For developing such a therapy, we designed a chemically well-defined route to a PEG- and docetaxel (DTX)-conjugated inorganic polymer, polyphosphazene, named "polytaxel (PTX)" with a prolonged blood circulation time and tumor localization. Here, we conducted the proof-of-concept study of the ideal therapy in orthotopic and xenograft pancreatic cancer models. We found that the average tumor inhibition rates of PTX were similar to those of DTX without any DTX toxicity-related side effects, such as neutropenia and weight loss. In conclusion, PTX met the requirements of an ideal anticancer drug with high anticancer efficacy and 100% survival rate. PTX is expected to replace any existing anticancer therapies in clinical practice.

Safety and therapeutic effects of personalized transcranial direct current stimulation based on electrical field simulation for prolonged disorders of consciousness: study protocol for a multi-center, double-blind, randomized controlled trial.

Background: Disorders of consciousness (DOC) resulting from acquired brain injury (ABI) increase the mortality rate of patients, complicate rehabilitation, and increase the physical and economic burden that DOC imposes on patients and their families. Thus, treatment to promote early awakening from DOC is vital. Transcranial direct current stimulation (tDCS) has shown great potential for promoting neuro-electrochemical activity. However, previous tDCS studies did not consider structural damage or head and brain lesions, so the applicability of the results to all DOC patients was limited. In this study, to establish a patient-specific tDCS treatment plan considering the brain lesions of and damage sustained by DOC patients, we considered the electric field calculated by a the "finite electric" three-dimensional brain model based on magnetic resonance images. This protocol was developed to aid tDCS treatment of actual patients, and to verify its safety and effectiveness. Methods/design: Twenty-four patients with DOC after ABI will be enrolled in this cross-over trial. All participants will receive typical rehabilitation combined with sham tDCS and typical rehabilitation plus personalized tDCS (P-tDCS). Each interventional period will last 2 weeks (30 min/day, 5 days/week). The primary outcome [score on the Korean version of the Coma Recovery Scale-Revised (K-CRS-R)] will be assessed at baseline and the end of the first day of the intervention. Secondary outcomes (K-CRS-R at 1 week and 2 weeks after experimental session and quantitative EEG changes quantitative electroencephalography changes) will be measured at baseline and the end of week 4. Adverse events will be recorded during each treatment session. Discussion: For patients with neurological disorders, tDCS has served as a painless, non-invasive, easily applied, and effective therapy for several decades, and there is some evidence that it can improve the level of consciousness of patients with DOC. However, variability in the effects on consciousness among subjects have been reported and personalized strategies are lacking. This protocol is for a randomized controlled trial designed to validate the effectiveness and safety of P-tDCS combined with typical rehabilitation for DOC. Clinical trial registration: https://cris.nih.go.kr, identifier KCT0007157.