Complete sequence randomness of lactate-based copolymers, LAHBs with varied lactate monomer fractions employing a series of propionyl-CoA transferases.

Previously, we synthesized an evolved version of a bio-based polylactide (PLA) on microbial platforms using our engineered lactate-polymerizing enzyme (LPE). This lactate (LA)-based copolyester, LAHB, has advantages over PLA, including improved flexibility and biodegradability, and its properties can be regulated through the LA fraction. To expand the LA-incorporation capacity and improve polymer properties, in the state of in vivo LAHB production, propionyl-CoA transferases (PCTs) that exhibited enhanced production of LA-CoA than the conventional PCTs were selected. Here, the present study has demonstrated that the LA fraction of LAHB could be altered using various PCTs. Enhanced PCT performance was achieved by balancing polymer production and cell growth. Both events are governed by the use of acetyl-CoA, a commonly shared key metabolite. This could be attributed to the different reactivities of individual PCTs towards acetyl-CoA, which serves both as a CoA donor and a leading compound in the TCA cycle. Interestingly, we found complete sequence randomness in the LAHB copolymers, independent of the LA fraction. The mechanism of LA fraction-independent sequence randomness is discussed. This new PCT-based strategy synergistically combines with the evolution of LPE to advance the LAHB project, and enables us to perform advanced applications other than LAHB production utilizing CoA-linked substrates.

Production of single cell oil by Lipomyces starkeyi from waste plant oil generated by the palm oil mill industry.

Only a few reports available about the assimilation of hydrophobic or oil-based feedstock as carbon sources by Lipomyces starkeyi. In this study, the ability of L. starkeyi to efficiently utilize free fatty acids (FFAs) and real biomass like palm acid oil (PAO) as well as crude palm kernel oil (CPKO) for growth and lipid production was investigated. PAO, CPKO, and FFAs were evaluated as sole carbon sources or in the mixed medium containing glucose. L. starkeyi was able to grow on the medium supplemented with PAO and FFAs, which contained long-chain length FAs and accumulated lipids up to 35% (w/w) of its dry cell weight. The highest lipid content and lipid concentration were achieved at 50% (w/w) and 10.1 g/L, respectively, when L. starkeyi was cultured in nitrogen-limited mineral medium (-NMM) supplemented with PAO emulsion. Hydrophobic substrate like PAO could be served as promising carbon source for L. starkeyi.

Comparative responses of flocculating and nonflocculating yeasts to cell density and chemical stress in lactic acid fermentation.

While flocculation has demonstrated its efficacy in enhancing yeast robustness and ethanol production, its potential application for lactic acid fermentation remains largely unexplored. Our study examined the differences between flocculating and nonflocculating Saccharomyces cerevisiae strains in terms of their metabolic dynamics when incorporating an exogenous lactic acid pathway, across varying cell densities and in the presence of lignocellulose-derived byproducts. Comparative gene expression profiles revealed that cultivating a nonflocculant strain at higher cell density yielded a substantial upregulation of genes associated with glycolysis, energy metabolism, and other key pathways, resulting in elevated levels of fermentation products. Meanwhile, the flocculating strain displayed an inherent ability to sustain high glycolytic activity regardless of the cell density. Moreover, our investigation revealed a significant reduction in glycolytic activity under chemical stress, potentially attributable to diminished ATP supply during the energy investment phase. Conversely, the formation of flocs in the flocculating strain conferred protection against toxic chemicals present in the medium, fostering more stable lactic acid production levels. Additionally, the distinct flocculation traits observed between the two examined strains may be attributed to variations in the nucleotide sequences of the flocculin genes and their regulators. This study uncovers the potential of flocculation for enhanced lactic acid production in yeast, offering insights into metabolic mechanisms and potential gene targets for strain improvement.

Effect of different metabolic pathways on itaconic acid production in engineered Corynebacterium glutamicum.

Itaconic acid (IA), a C5-dicarboxylic acid, is a potential bio-based building block for the polymer industry. There are three pathways for IA production from natural IA producers; however, most of the engineered strains were used for IA production by heterologous expression of cis-aconitate decarboxylase gene (cadA) from Aspergillus terreus. In this study, IA was produced by an engineered Corynebacterium glutamicum ATCC 13032 expressing two different types of genes from two distinct pathways. The first involves the mammalian immunoresponsive gene1 (Irg1) derived from Mus musculus. The second (termed here the trans-pathway) involves two genes from the natural IA producer Ustilago maydis which are aconitate-delta-isomerase (Adi1) and trans-aconitate decarboxylase (Tad1) genes. The constructed strains developing the two distinct IA production pathways: C. glutamicum ATCC 13032 pCH-Irg1opt and C. glutamicum ATCC 13032 pCH-Tad1optadi1opt were used for production of IA from different carbon sources. The results reflect the possibility for IA production from C. glutamicum expressing the trans-pathway (Adi1/Tad1 genes) and cis-pathway (Irg1 gene) other than the well-known cis-pathway that depends mainly on cadA gene from A. terreus. The developed strain expressing trans-pathway from U. maydis; however, proved to be better at IA production with high titers of 12.25, 11.34, and 11.02 g/L, and a molar yield of 0.22, 0.42, and 0.43 mol/mol from glucose, maltose, and sucrose, respectively, via fed-batch fermentation. The present study suggests that trans-pathway is better than cis-pathway for IA production in engineered C. glutamicum.

Sucrose Signaling Contributes to the Maintenance of Vascular Cambium by Inhibiting Cell Differentiation.

Plants produce sugars by photosynthesis and use them for growth and development. Sugars are transported from source-to-sink organs via the phloem in the vasculature. It is well known that vascular development is precisely controlled by plant hormones and peptide hormones. However, the role of sugars in the regulation of vascular development is poorly understood. In this study, we examined the effects of sugars on vascular cell differentiation using a vascular cell induction system named 'Vascular Cell Induction Culture System Using Arabidopsis Leaves' (VISUAL). We found that sucrose has the strongest inhibitory effect on xylem differentiation, among several types of sugars. Transcriptome analysis revealed that sucrose suppresses xylem and phloem differentiation in cambial cells. Physiological and genetic analyses suggested that sucrose might function through the BRI1-EMS-SUPPRESSOR1 transcription factor, which is the central regulator of vascular cell differentiation. Conditional overexpression of cytosolic invertase led to a decrease in the number of cambium layers due to an imbalance between cell division and differentiation. Taken together, our results suggest that sucrose potentially acts as a signal that integrates environmental conditions with the developmental program.

Enhanced production of itaconic acid from enzymatic hydrolysate of lignocellulosic biomass by recombinant Corynebacteriumglutamicum.

Itaconic acid (IA) is a value-added chemical currently produced by Aspergillus terreus from edible glucose and starch but not from inedible lignocellulosic biomass owing to the high sensitivity to fermentation inhibitors present in the hydrolysate of lignocellulosic biomass. To produce IA from lignocellulosic biomass, a gram-positive bacterium, Corynebacterium glutamicum, with a high tolerance to fermentation inhibitors was metabolically engineered to express a fusion protein composed of cis-aconitate decarboxylase from A. terreus responsible for IA formation from cis-aconitate and a maltose-binding protein (malE) from Escherichia coli. The codon-optimized cadA_malE gene was expressed in C. glutamicum ATCC 13032, and the resulting recombinant strain produced IA from glucose. IA concentration increased 4.7-fold by the deletion of the ldh gene encoding lactate dehydrogenase. With the Δldh strain HKC2029, an 18-fold higher IA production was observed from enzymatic hydrolysate of kraft pulp as a model lignocellulosic biomass than from glucose (6.15 and 0.34 g/L, respectively). The enzymatic hydrolysate of kraft pulp contained various potential fermentation inhibitors involved in furan aldehydes, benzaldehydes, benzoic acids, cinnamic acid derivatives, and aliphatic acid. Whereas cinnamic acid derivatives severely inhibited IA production, furan aldehydes, benzoic acids, and aliphatic acid improved IA production at low concentrations. The present study suggests that lignocellulosic hydrolysate contains various potential fermentation inhibitors; however, some of them can serve as enhancers for microbial fermentation likely due to the changing of redox balance in the cell.

An analysis of sugary endosperm in sorghum: Characterization of mutant phenotypes depending on alleles of the corresponding starch debranching enzyme.

Sorghum is the fifth most important cereal crop. Here we performed molecular genetic analyses of the 'SUGARY FETERITA' (SUF) variety, which shows typical sugary endosperm traits (e.g., wrinkled seeds, accumulation of soluble sugars, and distorted starch). Positional mapping indicated that the corresponding gene was located on the long arm of chromosome 7. Within the candidate region of 3.4 Mb, a sorghum ortholog for maize Su1 (SbSu) encoding a starch debranching enzyme ISA1 was found. Sequencing analysis of SbSu in SUF uncovered nonsynonymous single nucleotide polymorphisms (SNPs) in the coding region, containing substitutions of highly conserved amino acids. Complementation of the rice sugary-1 (osisa1) mutant line with the SbSu gene recovered the sugary endosperm phenotype. Additionally, analyzing mutants obtained from an EMS-induced mutant panel revealed novel alleles with phenotypes showing less severe wrinkles and higher Brix scores. These results suggested that SbSu was the corresponding gene for the sugary endosperm. Expression profiles of starch synthesis genes during the grain-filling stage demonstrated that a loss-of-function of SbSu affects the expression of most starch synthesis genes and revealed the fine-tuned gene regulation in the starch synthetic pathway in sorghum. Haplotype analysis using 187 diverse accessions from a sorghum panel revealed the haplotype of SUF showing severe phenotype had not been used among the landraces and modern varieties. Thus, weak alleles (showing sweet and less severe wrinkles), such as in the abovementioned EMS-induced mutants, are more valuable for grain sorghum breeding. Our study suggests that more moderate alleles (e.g. produced by genome editing) should be beneficial for improving grain sorghum.

Fluorophore-Decorated Mie Resonant Silicon Nanosphere for Scattering/Fluorescence Dual-Mode Imaging.

Inorganic nanoparticles with multiple functions have been attracting attention as multimodal nanoprobes in bioimaging, biomolecule detection, and medical diagnosis and treatment. A drawback of conventional metallic nanoparticle-based nanoprobes is the Ohmic losses that lead to fluorescence quenching of attached molecules and local heating under light irradiation. Here, metal-free nanoprobes capable of scattering/fluorescence dual-mode imaging are developed. The nanoprobes are composed of a silicon nanosphere core having efficient Mie scattering in the visible to near infrared range and a fluorophore doped silica shell. The dark-field scattering and photoluminescence images/spectra for nanoprobes made from different size silicon nanospheres and different kinds of fluorophores are studied by single particle spectroscopy. The fluorescence spectra are strongly modified by the Mie modes of a silicon nanosphere core. By comparing scattering and fluorescence spectra and calculated Purcell factors, the fluorescence enhancement factor is quantitatively discussed. In vitro scattering/fluorescence imaging studies on human cancer cells demonstrate that the developed nanoparticles work as scattering/fluorescence dual-mode imaging nanoprobes.

Pretreatment with dilute maleic acid enhances the enzymatic digestibility of sugarcane bagasse and oil palm empty fruit bunch fiber.

Lignocellulose is resistant to degradation and requires pretreatment before hydrolytic enzymes can release fermentable sugars. Sulfuric acid has been widely used for biomass pretreatment, but high amount of degradation products usually occurred when using this method. To enhance accessibility to cellulose, we studied the performances of several dilute organic acid pretreatments of sugarcane bagasse and oil palm empty fruit bunch fiber. The results revealed that pretreatment with maleic acid yields the highest xylose and glucose release among other organic acids. The effects of concentration, duration of heating and heating temperature were further studied. Dilute maleic acid 1 % (w/w) pretreatment at 180 °C was the key to its viability as a substitute for sulfuric acid. Moreover, maleic acid did not seem to highly promote the formation of either furfural or 5-HMF in the liquid hydrolysate after pretreatment.

Comprehensive Machine Learning Prediction of Extensive Enzymatic Reactions.

New enzyme functions exist within the increasing number of unannotated protein sequences. Novel enzyme discovery is necessary to expand the pathways that can be accessed by metabolic engineering for the biosynthesis of functional compounds. Accordingly, various machine learning models have been developed to predict enzymatic reactions. However, the ability to predict unknown reactions that are not included in the training data has not been clarified. In order to cover uncertain and unknown reactions, a wider range of reaction types must be demonstrated by the models. Here, we establish 16 expanded enzymatic reaction prediction models developed using various machine learning algorithms, including deep neural network. Improvements in prediction performances over that of our previous study indicate that the updated methods are more effective for the prediction of enzymatic reactions. Overall, the deep neural network model trained with combined substrate-enzyme-product information exhibits the highest prediction accuracy with Macro F1 scores up to 0.966 and with robust prediction of unknown enzymatic reactions that are not included in the training data. This model can predict more extensive enzymatic reactions in comparison to previously reported models. This study will facilitate the discovery of new enzymes for the production of useful substances.

Harnessing originally robust yeast for rapid lactic acid bioproduction without detoxification and neutralization.

Acidic and chemical inhibitor stresses undermine efficient lactic acid bioproduction from lignocellulosic feedstock. Requisite coping treatments, such as detoxification and neutralizing agent supplementation, can be eliminated if a strong microbial host is employed in the process. Here, we exploited an originally robust yeast, Saccharomyces cerevisiae BTCC3, as a production platform for lactic acid. This wild-type strain exhibited a rapid cell growth in the presence of various chemical inhibitors compared to laboratory and industrial strains, namely BY4741 and Ethanol-red. Pathway engineering was performed on the strain by introducing an exogenous LDH gene after disrupting the PDC1 and PDC5 genes. Facilitated by this engineered strain, high cell density cultivation could generate lactic acid with productivity at 4.80 and 3.68 g L-1 h-1 under semi-neutralized and non-neutralized conditions, respectively. Those values were relatively higher compared to other studies. Cultivation using real lignocellulosic hydrolysate was conducted to assess the performance of this engineered strain. Non-neutralized fermentation using non-detoxified hydrolysate from sugarcane bagasse as a medium could produce lactic acid at 1.69 g L-1 h-1, which was competitive to the results from other reports that still included detoxification and neutralization steps in their experiments. This strategy could make the overall lactic acid bioproduction process simpler, greener, and more cost-efficient.

3-Amino-4-hydroxybenzoic acid production from glucose and/or xylose via recombinant Streptomyces lividans.

The aromatic compound 3-amino-4-hydroxybenzoic acid (3,4-AHBA) can be employed as a raw material for high-performance industrial plastics. The aim of this study is to produce 3,4-AHBA via a recombinant Streptomyces lividans strain containing griI and griH genes derived from Streptomyces griseus using culture medium with glucose and/or xylose, which are the main components in lignocellulosic biomass. Production of 3,4-AHBA by the recombinant S. lividans strain was successful, and the productivity was affected by the kind of sugar used as an additional carbon source. Metabolic profiles revealed that L aspartate-4-semialdehyde (ASA), a precursor of 3,4-AHBA, and coenzyme NADPH were supplied in greater amounts in xylose medium than in glucose medium. Moreover, cultivation in TSB medium with a mixed sugar (glucose/xylose) was found to be effective for 3,4-AHBA production, and optimal conditions for efficient production were designed by changing the ratio of glucose to xylose. The best productivity of 2.70 g/L was achieved using a sugar mixture of 25 g/L glucose and 25 g/L xylose, which was 1.5 times higher than the result using 50 g/L glucose alone. These results suggest that Streptomyces is a suitable candidate platform for 3,4-AHBA production from lignocellulosic biomass-derived sugars under appropriate culture conditions.

Reactive oxygen species-inducing titanium peroxide nanoparticles as promising radiosensitizers for eliminating pancreatic cancer stem cells.

BACKGROUND: Despite recent advances in radiotherapy, radioresistance in patients with pancreatic cancer remains a crucial dilemma for clinical treatment. Cancer stem cells (CSCs) represent a major factor in radioresistance. Developing a potent radiosensitizer may be a novel candidate for the eradication of pancreatic CSCs. METHODS: CSCs were isolated from MIA PaCa-2 and PANC1 human pancreatic cancer cell lines. Titanium peroxide nanoparticles (TiOxNPs) were synthesized from titanium dioxide nanoparticles (TiO2NPs) and utilized as radiosensitizers when added one hour prior to radiation exposure. The antitumor activity of this novel therapeutic strategy was evaluated against well-established pancreatic CSCs model both in vitro and in vivo. RESULTS: It is shown that TiOxNPs combined with ionizing radiation exhibit anti-cancer effects on radioresistant CSCs both in vitro and in vivo. TiOxNPs exhibited a synergistic effect with radiation on pancreatic CSC-enriched spheres by downregulating self-renewal regulatory factors and CSC surface markers. Moreover, combined treatment suppressed epithelial-mesenchymal transition, migration, and invasion properties in primary and aggressive pancreatic cancer cells by reducing the expression of proteins relevant to these processes. Notably, radiosensitizing TiOxNPs suppressed the growth of pancreatic xenografts following primary or dissociating sphere MIA PaCa-2 cell implantation. It is inferred that synergy is formed by generating intolerable levels of reactive oxygen species (ROS) and inactivating the AKT signaling pathway. CONCLUSIONS: Our data suggested the use of TiOxNPs in combination with radiation may be considered an attractive therapeutic strategy to eliminate pancreatic CSCs.

The flocculant Saccharomyces cerevisiae strain gains robustness via alteration of the cell wall hydrophobicity.

When lignocellulosic biomass is utilized as a fermentative substrate to produce biochemicals, the existence of a yeast strain resistant to inhibitory chemical compounds (ICCs) released from the biomass becomes critical. To achieve the purpose, in this study, Saccharomyces yeast strains from a NBRC yeast culture collection were used for exploration and evaluated in two different media containing ICCs that mimic one another but resemble the hydrolysate of real biomass. Among them, S. cerevisiae F118 strain shows robustness upon the fermentation with unique flocculation trait that was strongly responsive to ICC stress. When this strain was cultured in the presence of ICCs, its cell wall hydrophobicity increased dramatically, and reduced significantly when the ICCs were depleted, demonstrating that cell-surface hydrophobicity can also act as an adaptive response to the ICCs. Cells from the strain with the highest cell-wall hydrophobicity displayed progressively stronger flocculation, indicating that the F118 strain is having unique robustness under ICC stress. Gene expression perturbation analysis revealed that mot3 gene encoding regulatory Mot3p from the F118 strain was expressed in response to the concentration of ICCs. This gene was found to control expression of ygp1 gene that encoding Ygp1p, one of cell wall proteins. Deep sequencing analysis revealed that the Mot3p of the F118 strain features a unique insertion and deletion of nucleotides that encode glutamine or asparagine residues, particularly in N-terminal domain, as determined by comparison to the Mot3p sequence from the S288c strain, which was employed as a control strain. Furthermore, the cell wall hydrophobicity of the S288c strain was greatly enhanced and became ICC-responsive after gene swapping with the mot3 gene from the F118 strain. The gene-swapped S288c strain fermented 6-fold faster than the wild-type strain, producing 14.5 g/L of ethanol from 30 g/L of glucose consumed within 24 h in a medium containing the ICCs. These such modifications to Mot3p in unique locations in its sequence have a potential to change the expression of a gene involved in cell wall hydrophobicity and boosted the flocculation response to ICC stress, allowing for the acquisition of extraordinary robustness.

Recent advances in lignocellulosic biomass white biotechnology for bioplastics.

Lignocellulosic biomass has great potential as an inedible feedstock for bioplastic synthesis, although its use is still limited compared to current edible feedstocks of glucose and starch. This review focuses on recent advances in the production of biopolymers and biomonomers from lignocellulosic feedstocks with downstream processing and chemical polymer syntheses. In microbial production, four routes composed of existing poly (lactic acid) and polyhydroxyalkanoates (PHAs) and the emerging biomonomers of itaconic acid and aromatic compounds were presented to review present challenges and future perspectives, focusing on the use of lignocellulosic feedstocks. Recently, advances in purification technologies decreased the number of processes and their environmental burden. Additionally, the unique structures and high-performance of emerging lignocellulose-based bioplastics have expanded the possibilities for the use of bioplastics. The sequence of processes provides insight into the emerging technologies that are needed for the practical use of bioplastics made from lignocellulosic biomass.

An integrated biorefinery strategy for the utilization of palm-oil wastes.

Each year, the palm oil industry generates a significant amount of biomass residue and effluent waste; both have been identified as significant sources of greenhouse gas (GHG) emissions. This issue poses a severe environmental challenge for the industry due to the possibility of long-term negative effects on human well-being. The palm-oil industry must invest significantly in the technology that is required to resolve these issues and to increase the industry's sustainability. However, current technologies for converting wastes such as lignocellulosic components and effluents into biochemical products are insufficient for optimal utilization. This review discusses the geographical availability of palm-oil biomass, its current utilization routes, and then recommends the development of technology for converting palm-oil biomass into value-added products through an integrated biorefinery strategy. Additionally, this review summarizes the palm oil industry's contribution to achieving sustainable development goals (SDGs) through a circular bioeconomy concept.

Enhanced production of γ-amino acid 3-amino-4-hydroxybenzoic acid by recombinant Corynebacterium glutamicum under oxygen limitation.

BACKGROUND: Bio-based aromatic compounds are of great interest to the industry, as commercial production of aromatic compounds depends exclusively on the unsustainable use of fossil resources or extraction from plant resources. γ-amino acid 3-amino-4-hydroxybenzoic acid (3,4-AHBA) serves as a precursor for thermostable bioplastics. RESULTS: Under aerobic conditions, a recombinant Corynebacterium glutamicum strain KT01 expressing griH and griI genes derived from Streptomyces griseus produced 3,4-AHBA with large amounts of amino acids as by-products. The specific productivity of 3,4-AHBA increased with decreasing levels of dissolved oxygen (DO) and was eightfold higher under oxygen limitation (DO = 0 ppm) than under aerobic conditions (DO ≥ 2.6 ppm). Metabolic profiles during 3,4-AHBA production were compared at three different DO levels (0, 2.6, and 5.3 ppm) using the DO-stat method. Results of the metabolome analysis revealed metabolic shifts in both the central metabolic pathway and amino acid metabolism at a DO of < 33% saturated oxygen. Based on this metabolome analysis, metabolic pathways were rationally designed for oxygen limitation. An ldh deletion mutant, with the loss of lactate dehydrogenase, exhibited 3.7-fold higher specific productivity of 3,4-AHBA at DO = 0 ppm as compared to the parent strain KT01 and produced 5.6 g/L 3,4-AHBA in a glucose fed-batch culture. CONCLUSIONS: Our results revealed changes in the metabolic state in response to DO concentration and provided insights into oxygen supply during fermentation and the rational design of metabolic pathways for improved production of related amino acids and their derivatives.

Constitutive cell surface expression of ZZ domain for the easy preparation of yeast-based immunosorbents.

We describe a novel expression cassette that enables efficient and constitutive expression of the ZZ domain derived from Staphylococcus aureus protein A on the yeast cell surface to easily prepare yeast-based immunosorbents. Using this expression cassette containing the PGK1 promoter, a secretion signal derived from α-factor, and a Flo1-derived anchor protein, we successfully created a yeast-based immunosorbent for human serum albumin.

Accelerated glucose metabolism in hyphae-dispersed Aspergillus oryzae is suitable for biological production.

Recently, a hyphae-dispersed type of filamentous fungus Aspergillus oryzae was constructed via genetic engineering, and industrial applications are expected due to the ease of handling and to the level of protein production properties. In this study, we constructed cellulase-expressing strains using wild-type and hyphae-dispersed strains to investigate the correlation between protein productivity and metabolism. Compared with the original strain, the hyphae-dispersed cellulase-expressing strain showed elevated cellulase activity, rapid glucose consumption, increased mycelial dry weight, an increased expression of cellulase genes, and activated respiration activity. Comparative metabolomic analysis showed fewer metabolites in the glycolysis and TCA cycles in the dispersed strains than in the original strains. These results indicate that the flux of carbohydrate metabolism in the hyphae-dispersed strains is smoother than that in the original strains. Such efficient metabolic flux would contribute to efficient energy conversion and to sufficient energy supply to anabolisms, such as mycelial growth and protein production. Our findings suggest that the hyphae-dispersed strains could be a useful host not only for protein production but also for the biological production of various chemicals such as organic acids.

Titanium oxide nano-radiosensitizers for hydrogen peroxide delivery into cancer cells.

Polyacrylic acid-modified titanium peroxide nanoparticles (PAA-TiOx NPs) are promising radiosensitizers that enhance the therapeutic effect of X-ray irradiation after local injection into tumors. However, the mechanism for this reaction has remained unclear with the exception of the involvement of hydrogen peroxide (H2O2), which is released by PAA-TiOx NPs to a liquid phase during dispersion. In the present study, a clonogenic assay was used to compare PAA-TiOx NPs with free H2O2 molecules to investigate the effect exerted on the radiosensitivity of cancer cells in vitro. A cell-free dialysis method revealed that a portion of the H2O2 adsorbed onto the PAA-TiOx NPs during synthesis could be released during a treatment regimen. The H2O2 release lasted for 7 h, which was sufficient for one radiation treatment procedure. For in vitro experiments, cultured human pancreatic cancer cells took up PAA-TiOx NPs in 10 min after administration. Interestingly, when the cells were washed with a buffer after treatment with either a PAA-TiOx NP or H2O2 solution, the intracellular H2O2 levels remained higher with PAA-TiOx NP treatment compared with the H2O2 solution treatment. Furthermore, the effects of subsequent X-ray irradiation corresponded to the intracellular H2O2 levels. These results indicate that PAA-TiOx NPs are efficient carriers of H2O2 into cancer cells and thus enhance the radiosensitivity.