The antimicrobial activity against Escherichia coli, Methicilin-susceptible Staphylococcus aureus (MSSA), and Candida albicans was determined by estimating the minimum inhibitory concentration (MIC) and minimal bactericidal/fungicidal concentration (MBC/MFC). To assess the biocompatibility of chitosan/α-Ag2WO4 composites in a range involving MIC and MBC/MFC on keratinocytes cells (NOK-si), an alamarBlue™ assay and an MTT assay were carried out. The SARS-CoV-2 virucidal effects was analyzed in Vero E6 cells through viral titer quantified in cell culture supernatant by PFU/mL assay. Our results showed a very similar antimicrobial activity of chitosan/α-Ag2WO4 3.3 and 6.6, with the last one demonstrating a slightly better action against MSSA. The chitosan/α-Ag2WO4 9.9 showed a wide range of antimicrobial activity (0.49–31.25 µg/mL). The cytotoxicity outcomes by alamarBlue™ revealed that the concentrations of interest (MIC and MBC/MFC) were considered non-cytotoxic to all composites after 72 h of exposure. The Chitosan/α-Ag2WO4 (CS6.6/α-Ag2WO4) composite reduced the SARS-CoV-2 viral titer quantification up to 80% of the controls. Then, our results suggest that these composites are highly efficient materials to kill bacteria , in addition to inactivating SARS-CoV-2 by contact, through ROS production.In the current COVID-19 pandemic, the next generation of innovative materials with enhanced anti-SARS-CoV-2 activity is urgently needed to prevent the spread of this virus within the community. Herein, we report the synthesis of chitosan/α-Ag Despite the development of several vaccines and extensive vaccination programs, there is an urgent need to discover novel materials and strategies to combat and prevent the spread of viral infections for dealing with current and future pandemics. In this unprecedented scenario, materials capable of killing pathogens  are highly desirable in applications requiring a protective barrier against contamination, transmission, and proliferation5.Currently, the worldwide emergence and rapid evolution of the COVID-19 infectious disease induced by the SARS-CoV-2 virus is an ever-growing global crisis2CrO47, the three polymorphs of Ag2WO49, Ag3PO411, α-AgVO312 and β-Ag2MoO413. To provide a deeper understanding and establish a correlation among morphology, surface energy and biocidal activity, first principles calculations were conducted at the density functional theory (DFT) level to complement and rationalize the experimental findings14. In addition, the formation of Ag nanoparticles (AgNPs), on α-Ag2WO4 induced by femtosecond (fs) laser irradiation has been proved. It was found that the as-synthetized composite displays a 32-fold improvement in the bactericidal properties in relation to α-Ag2WO416. On the other hand, very recently different materials with antiviral and bactericidal properties were reported: (i) a SiO2-Ag composite as a highly virucidal material to be applied in polycotton fiber surfaces for the prevention of viral proliferation and transmission of SARS-CoV-217, (ii) immobilized SiO2-Ag composites in a polymeric matrix (ethyl vinyl acetate) with highly antibacterial activity towards Escherichia coli and SARS-CoV-218, and (iii) nanostructured aluminum Al 6063 alloy surfaces to inactivate the SARS-CoV-219. These works open up an innovative path for the design and application of adequate technologies to the next generation of antiviral surfaces in order to combat SARS-CoV-2.In this frenetic race against COVID-19, our research group has contributed significantly by developing new technologies based on the synthesis of potent biocidal materials using substantial cumulative knowledge rapidly translated to various multi-tasking applications, such as personal protective equipment  and devices for disinfection of surfaces/surroundings. In particular, different complex silver-based oxides with potent antibacterial and antifungal activities have been presented, such as Ag20. High-intensity pulsed laser of a colloidal dispersion of nano or microparticles can provoke size reduction21 by Coulomb explosion and/or particle surface melting depending on the pulse duration and intensity25. This methodology is more advantageous than wet chemical methods because when used to modify or functionalize materials for biomedical applications, it avoids the presence of additional substances capable of decreasing the materials biocompatibility.Fs laser irradiation provides unique opportunities to investigate and control the behavior of materials under strong electronic, thermal, and mechanical non-equilibrium conditions2), and hydroxy (–OH) groups, which are favorable to various chemical modifications and hybridization. So far, CS has many important advantages, such as ease of synthesis, low cost, non-toxicity, high biocompatibility and biodegradability26, as well as inherent antibacterial, antifungal, adsorbent29 and bioactivity properties30. Due to such features, CS and its composites have been widely used as drug release agents31, skin substitutes32, vaccine delivery agents33, antibacterial agents37, wound dressings39, and wastewater treatment agents40. Moreover, it is well known that CS is an efficient host for stabilizing metal oxides, such as TiO2 so as to enhance the mechanical, physical, and biological properties of the CS biopolymer44, as the TiO2 agglomeration would mask its ability to be used in technological applications.Chitosan (CS) is a natural polysaccharide with alkaline substances extracted from chitin shells of crustaceans. Its structure contains lots of free amino  the synthesis of this composite; (ii) the characterization of the photochemical properties and exploration of the potential cooperative effects resulting from the combination of the two mentioned functional entities; and finally (iii) the evaluation of the antimicrobial  and SARS-CoV-2 antiviral activities of the synthesized CS/α-Ag2WO4 composite. The α-Ag2WO4 was synthesized by the coprecipitation method and dispersed in three CS concentrations . The suspension formed was then irradiated by fs laser. The as-obtained composites were denoted as CS3.3/α-Ag2WO4, CS6.6/α-Ag2WO4 and CS9.9/α-Ag2WO4, respectively.Taking into account the above considerations, the motivation for this work is derived from our previous studies. Considering that CS is capable of stabilizing Ag nanoparticles formed during fs laser irradiation, herein we selected an appropriate processing route to obtain CS/α-Ag2WO4 was synthesized by the coprecipitation method as previously reported47 . Figure 2WO4 microcrystal, confirming its orthorhombic structure according to the inorganic crystal structure database (ICSD)—card no. 416548. This figure also presents the XRD peaks related to the semi-crystalline structure of the CS polymer, showing intense peaks located at 11.4°, 21.0° and 22.5°. Figure 2WO4 composites irradiated by fs laser, where a characteristic XRD pattern of an amorphous phase related to the CS polymer and peaks assigned to the α-Ag2WO4 orthorhombic structure can be observed. According to Khan et al.49, the crystallinity of the CS polymer is ascribed to the number of –NH2 and –OH groups in the structure. These groups form strong intra and intermolecular hydrogen bonds, leading to a certain regularity of the CS structure, thus resulting in the appearance of crystalline regions. The intensity of the CS XRD peaks decrease for the CS/α-Ag2WO4 composites  crystallographic plane.The α-Agtes Fig. B, which 2WO4 composites. Figure −1) cannot be observed, indicating no degradation of the CS polymer after fs laser irradiation49. For the CS/α-Ag2WO4 composites, the CS polymer bands present lower intensity. In addition, bands ascribed to [WO42−] and [AgOx]  can be considered constituent clusters of the α-Ag2WO4 structure  was carried out to evaluate the vibrational modes of the as-synthesized CS/α-Agure Fig. .2WO4 composites, where it can be seen that the CS polymers with α-Ag2WO4 microcrystals are dispersed under and over the film. Several α-Ag2WO4 irregular rod-like structures with hexagonal face of several sizes can be observed for all samples51, besides a covering around each microcrystal formed by the CS polymer. In the CS film, it is possible to note a ribbing forming root-type structures and different morphologies, such as pointed spear-like structures, which differ in shape depending on the CS solution concentration. While for the CS3.3/α-Ag2WO4 composite roughness layers can be observed on the surface 58.Figure res Fig. . These s2WO4 composites. The particles formed on the surface of the CS polymer are composed of larger α-Ag2WO4 NPs and smaller AgNPs, as proved by HR-TEM and EDS analyses. According to Murugadoss et al.59, the AgNPs are stabilized due to the excess of –NH2 and –OH groups present in the CS polymer chain , (031) and (231) planes with interplanar distances of 3.45, 3.31 and 2.82 Å, respectively  and (240) with interplanar distances of 2.95 and 2.62 Å, respectively , (311) and (240) with interplanar distances of 3.08, 2.98 and 2.62 Å, respectively , (101) and (102) planes with interplanar distances of 2.50, 2.42 and 2.23 Å, respectively, according to JCPDS database (PDF87-598)  medium, the CS polymer reacts with H+ ions to produce protonated CS with –NH3+ functional groups. Thus, the CS polymer provides free electrons to reduce Ag+ to Ag062, which was also demonstrated for Cu and Au62, resulting in the formation of α-Ag2WO4 NPs and AgNPs with near-spherical morphologies due to fs laser irradiation.The presence of the –OH and –NH2WO4 composites. The literature describes that pure CS (capping agent), as well as AA (used to dissolve CS), has inhibitory activity against different species of microorganisms64. However, it was verified that when used in low quantity these vehicles did not present antimicrobial activity.Initially, no statistical difference between the data obtained from vehicles (CS and AA) and the control (CT) without treatment (data not shown) was observed, which means that the vehicles did not interfere with the viability of the microorganisms tested and that the activity observed was solely due to fs laser irradiation of the CS/α-Ag15 showed that α-Ag2WO4 irradiated by fs laser can increase its biocidal activity when compared to the α-Ag2WO4. In this work, it was observed that the antimicrobial activity was dependent on the CS concentration. The MIC was determined by visual inspection (Table 2WO4 and CS6.6/α-Ag2WO4 composites presented similar activity (MIC ranging from 0.49 to 1.95 µg/mL) for all microorganisms tested, while the CS9.9/α-Ag2WO4 composite increased onefold against the microorganisms S. aureus and C. albicans . The MFC/MBC were determined for the activity capable of inhibiting 99.9%. Therefore, it was possible to observe a concentration increase in relation to the MIC necessary to reach more than 90% inhibition. This increase was represented as “fold change” in Table In a previous study, our research group2WO4 composites irradiated by fs laser were more effective against Gram-negative (E. coli) than Gram-positive (S. aureus) bacteria and the yeast . This difference can be explained by the structure of the cell wall, which in the Gram-positive bacteria and the yeast is composed of peptidoglycan and teichoic acid, bringing more stability to the cell wall. Studies suggest that silver ions are able to attach to the membrane surface of the microorganism, leading to membrane disruption and increasing its permeability. As a result, they could enter cells, condensing DNA and reacting with proteins. Moreover, thiol groups, which are responsible for enzyme activity, are inactivated by reacting with silver65.As a consequence, all CS/α-AgE. coli, a Gram-negative bacteriuma. Its membrane is composed of lipopolysaccharides (LPS) containing phosphate and pyrophosphate groups that make the cell surface negatively charged. Additionally, as CS is a cationic polymer it facilitates the binding of ions to the membrane, causing the microorganism inactivation, as previously described55. In another study with E. coli, researchers reported that silver ions trigger the separation of DNA strands and weaken the link between protein and DNA, thus altering vital processes for the microorganism66. Therefore, the CS6.6/α-Ag2WO4 composite was considered to have the best antimicrobial activity, as it exhibited low concentrations for the microbicidal effect and still presented a little difference between the MIC and MFC/MBC values .Accordingly, a greater efficacy was also observed for the 2WO4 composites irradiated by fs laser with the aim of developing an agent with antimicrobial activity for biomedical application. According to the literature, CS solutions exhibit toxicity depending on the dose and synthesis method67. Jena et al.68 observed that the cytotoxicity of CS with AgNPs (CS-AgNPs) is dose-dependent and that the cell viability decreases as the concentration increases. The same cell behavior was observed in this study with CS/α-Ag2WO4 composites irradiated by fs laser. However, the authors did not find any decrease in cell viability when evaluating only CS68, which may be explained by the CS synthesis method.In this section we evaluate the cytotoxicity of CS/α-AgH-tetrazolium bromide (MTT) and Alamar Blue assay. In the analysis by MTT assay -2,5-diphenyl-2say Fig. , it was say Fig. A,D,G,J asay Fig. G, and C5say Fig. J. After say Fig. B,E,H,K asay Fig. C,F,I,L, 2WO4 composites was carried out at different concentrations , as well as for CS3.3/α-Ag2WO4 (3.9–0.49 µg/mL). We can then conclude that the CS6.6/α-Ag2WO4 composite presented the best non-cytotoxic profile since the vehicle CS6.6 did not show cytotoxicity for the last 5 dilutions (C3–C7), corresponding to 7.81–0.49 µg/mL concentrations of the CS6.6/α-Ag2WO4 composite, which also presented no cytotoxicity.The cytotoxicity profile analysis by MTT assay of the CS/α-Agons Fig. . The resons Fig. A,D,G allite Fig. A, which ite Fig. C,F,I theIn contrast, when the cytotoxicity profile of the CS and AA vehicles was evaluated by Alamar Blue assay Fig. , we did 2WO4 composites was analyzed at different concentrations by the Alamar Blue assay . Despite the fact that the authors evaluated a different cell line, the CS concentration apparently had an influence on the biological activity. For the CS3.3/α-Ag2WO4 composite, after 48 and 72 h of contact , this is not a determinant for the loss of cell viability.Although in both methods the reagents are metabolized by mitochondrial enzymes and present in the cytoplasm, it is known that the MTT reagent is more metabolized by mitochondrial enzymes than the Alamar blue assay, thus providing important information on the influence of the compound on the cell2WO4 composite was selected for SARS-CoV-2 antiviral assays due to its greatest efficiency in the inhibition of bacterial and fungal growth, as well as its best non-cytotoxic profile.The 4.0 µg/mL concentration of the CS6.6/α-Ag2WO4 composite against virus inactivation  to study the effect of the CS6.6/α-Agion Fig. . SARS-Co2WO4 treatment, several changes were observed in the cytoplasm, such as proliferation of vesicles, vacuoles, numerous structures with concentric membranes (myelin figures) and changes in mitochondria , SARS-CoV-2 particles were not detected in the cells within 1 h of infection.Cells analyzed 1 h after infection with SARS-CoV-2 and treated with PBS presented morphological alterations in cytoplasm, such as numerous vacuoles, myelin figures and mitochondria alterations. The formation of syncytia (a large cell-like structure formed by joining two or more cells) was also noted Fig. A,B. In cons Fig. E,F. Rega2WO4 composite, vacuoles, vesicles, numerous myelin figures and rough endoplasmic reticulum thickening , causing DNA damage, and consequently producing changes in its conformation. As a result, the aforementioned composites absorb the incident photons, and the electrons h•) are generated in the VB. Moreover, the presence of AgNPs increases the population of h• in the VB causes a strong interaction with the H2O molecule, leading to the formation of •OH and H+. Simultaneously, the O2 molecule is converted into •O2− in the CB of the semiconductor due to the reaction with 2− renders the •O2H radical. It is reported that the oxidative stress is caused by imbalances in the production and elimination of ROS, resulting in biocidal activity18. It also prevents the vital function of the cell, affecting the viability, proliferation and redox status of various cell types41, thus destabilizing cell wall and membrane, and consequently leading to, cell death27.The proposed biocidal mechanism of the CS/α-Ag2WO4 composites generated by femtosecond laser irradiation. This material is very efficient to eliminate bacteria  and SARS-CoV-2 by contact. This study offers a general strategy to construct biocide materials. The biomimetic function of CS/α-Ag2WO4 composites in defeating COVID-19 transmission is promising. However, further studies are still necessary for developing new technologies based on the functionalization of this composite applied on protective materials and communal objects  to reduce both disease transmission and fear of touching objects.The rapidly spreading outbreak of COVID-19 has challenged the world’s healthcare sector over the last year. Thus, it has become crucial to trap and eradicate SARS-CoV-2 by using new materials. In this work, we reported the synthesis of chitosan/α-Ag2WO4 microcrystal was prepared by the coprecipitation (CP) method as previously described46. The procedure is described as follows: 2 × 10−3 mol of silver(I) nitrate  was dissolved in 50 mL of deionized water at 80 °C under magnetic stirring and then this solution was added in 50 ml of 1 × 10−3 mol of sodium tungstate (VI) dehydrated  previously dissolved at the same temperature. The suspension formed remained under constant magnetic stirring for 30 min. The resulting powders were washed several times with deionized water and dried in an oven at 70 °C.The α-Ag84. Briefly, 0.33, 0.66 and 0.99 g of the CS  were dissolved in 100 ml of 0.5% (v/v) acetic acid (AA) under constant stirring for 24 h at 25 °C, getting 3.3, 6.6 and 9.9 g/L concentration of CS solution.The experimental procedure employed for the synthesis of CS was obtained according to Ref.2WO4 composite was obtained according to Ferreira et al. and Ancona et al.62. Briefly, 400 mg of α-Ag2WO4 microcrystal was dispersed in 15 mL of CS solution, under constant stirring to complete homogenized the dispersion, getting 26.670 µg/mL concentration. To perform the femtosecond laser irradiation, a Femtosecond Ti: sapphire laser  of 30 fs full width at half maximum (FWHM) pulse duration emitting at the central wavelength of 800 nm, with a repetition rate of 1 kHz was employed. From the different setups usually used for laser irradiation, as the batch and a flow jet configuration85, the batch configuration was selected due to the simplicity of the technique and the lack of a requisite for a pumping device. In the batch processing the dispersed CS/α-Ag2WO4 composites was contained in a glass cell, the laser beam was focused perpendicular to the surface and during irradiation a magnetic stirrer was used to expedite the movement and prevent gravitational settling. The setup is shown in Fig. 2 criteria, mean power of 150 mW and irradiation during 2 h were found to be the optimum parameters to complete the CS/α-Ag2WO4 processing.The CS/α-Ag2WO4 microcrystals and CS/α-Ag2WO4 composites were structurally characterized by XRD patterns using a D/Max-2000PC diffractometer Rigaku (Japan) with Cu Ka radiation (λ = 1.5406 Å) in the 2θ range from 10° to 110° in the normal routine with a scanning velocity of 2°/min. Fourier-transform infrared (FTIR) spectra were obtained on a Bruker spectrometer (Vertex 70) equipped with a detector of triglycine sulfate doped with L-alanine and deuterium (DLaTGS). The spectra were collected in the spectral range from 50 to 4000 cm−1 with a nominal resolution of 4 cm-1. The shapes and sizes of the α-Ag2WO4 microcrystals and α-Ag2WO4/CS composites were observed with an FE-SEM Inspect F50  operated at 5 kV. The bifunctional nanoparticles were characterized by High-resolution transmission electron microscopy (HRTEM) using a JEM 2100F TEM/STEM micro-scope operating at 200 kV.The α-Ag2WO4 composites irradiated solutions at 26.670 µg/mL were diluted in RPMI-1640 medium supplemented (with 25 mM HEPES (Sigma) and 2 g/L sodium bicarbonate (Synth) pH 7.0 ± 0.2)86 at final concentration of 2.000 µg/mL. A serial dilution 1:2 was made in flat bottom 96-well plates. A concentration range of 1.000–0.49 µg/mL was obtained after adding a 100 µL volume of inoculum to each well.Previous to each assay, the CS/α-Ag2WO4 composites irradiated by fs laser were evaluated by experiments using two strains of bacteria (Gram-positive and Gram-negative) and a strain of fungi. The bacteria strains of Escherichia coli  and Methicillin-susceptible Staphylococcus aureus , and the yeast strain Candida albicans  were maintained frozen at − 80 °C until its use. E. coli and S. aureus were plated on Brain Heart Infusion (BHI) agar, and C. albicans on Sabouraud Dextrose Agar (SDA) for 24 h previously to pre-inoculum. It was made a prior overnight pre-inoculum in Tryptic Soy Broth (TSB) to bacterial strains, and Yeast Nitrogen Base medium (YNB) with 100 mM glucose to yeast strain. The inoculums were prepared diluting the pre-inoculum in 1:30 to E. coli, 1:20 to S. aureus and C. albicans, in their respective media. They were incubated at 37 °C under static conditions until microorganisms reached the mid-log phase (according to growth curve pre-established). The inoculums were centrifuged  and the pellet was washed twice with phosphate-buffered saline , and resuspended in RPMI-1640 at initial volume. All three microorganisms were diluted in RPMI-1640 to obtain the final concentration 1 × 103 to 5 × 103 CFU/mL for C. albicans, and 5 × 105 CFU/mL for S. aureus and E. coli, according to the protocols standardized by CLSI .The antimicrobial activity of CS/α-AgS. aureus, E. coli and C. albicans directly into a 96-well plate containing CS/α-Ag2WO4 composites irradiated by fs laser, in final concentration of 1000–0.49 µg/mL, for 24 h at 37 °C. The MIC was determined by visual inspection, where it was considered the lower concentration without visual growth. The MBC/MFC values were determined by cell recovery in an agar culture medium. For that, the MIC and 5 concentrations higher than the MIC were submitted to tenfold serial dilution in PBS. Each dilution was plated by microtip methodology on petri dishes with specific agar medium, and the plates were incubated at 37 °C, overnight. Counting the number of colonies was carried out in the lowest possible dilution. The data were converted to Log10 (UFC/mL) and converted in inhibition index related to control without treatment. The literature describes MFC/MBC as the minimum concentration of the antimicrobial agent capable of killing 99.9% of the number of colonies (CFU/mL) or reducing 3 units of Log10 in relation to the untreated control90. As control were used microorganisms in standard culture (CT), and the vehicles (CS and AA) at the same concentrations of experimental groups . This experiment was performed in quadruplicate and in two different occasions (n = 8).The minimal inhibitory concentration (MIC) and the minimal bactericidal/fungicidal concentration (MBC/MFC) against planktonic cells were determined using a broth microdilution method, as described by the Clinical and Laboratory Standards Institute (CLSI), documents M27-A3 and M7-A6. MIC and MBC/MFC were determined by incubating of 2WO4 composites irradiated by fs laser, the NOK-si cell line , kindly provided by Professor Carlos Rossa Junior 91 was used. NOK-si was grown in Dulbecco's Modified Eagle's Medium (DMEM) medium with 4.5 g/L glucose (Sigma-Aldrich), supplemented with 2.0 mM of L-glutamine (Lonza), 1% of antibiotic/antimycotic solution (Sigma-Aldrich) and 10% fetal bovine serum  in 75 cm2 bottle (Kasvi) at 37 °C, 5% CO2. After cells reach 80% confluence, they were washed with PBS, detached from the apparatus with trypsin solution (0.05% trypsin /0.53 mmol/L EDTA) (Sigma-Aldrich) and centrifuged at 400 × g for 5 min. The cell pellet was resuspended in culture medium, and viable cells index was verified by trypan blue methodology (1:1/v:v) (Sigma-Aldrich) using the automatic Countess II FL counter (LifeTechnologies). Calculations were performed to plate 5 × 103 cells/well in a 96-well plate. The plates were incubated at 37 °C, 5% CO2. After cells reach 60% confluence, the cells were washed once with PBS and treated with 200 µL of CS/α-Ag2WO4 composites irradiated by fs laser . The plates were incubated for 24, 48 and 72 h at 37 °C, 5% CO2. The data obtained for MTT and Alamar Blue assay (followed described) were converted to viability index in relation to control without treatment. The experimental controls were wells with cell monolayer without treatment (CT), culture medium without cell monolayer (sterility test), and vehicles (CS and AA) to evaluate their interference in the cell viability. From the volume of vehicle used in the first concentration of the experimental solution, 6 serial 1:2 dilutions were made in culture medium (C1–C7), with C1 being the initial one, and C7 with the highest dilution. The tests were performed in quadruplicate and on three independent occasions (n = 12).To evaluate the cytotoxicity of CS/α-AgH-tetrazolium bromide; 1.2 mg/mL; Sigma-Aldrich) in RPMI-1640 medium without phenol red (Sigma-Aldrich). The plate was incubated at 37 °C, 5% CO2. After 4 h, the supernatant was discarded and 100 µL/well of isopropanol (Synth) were added to solubilize the formazan crystals. Each well was homogenized, and the plate was submitted to analysis in a spectrophotometer at 540 nm. This protocol was performed at 24, 48 and 72 h of incubation.This assay assesses the rate of viable cells by mitochondrial activity  by quantifying tetrazolium salt reduction to formazan crystals, which occurs mainly by succinic dehydrogenase enzymes in mitochondrial fraction. After the incubation time , the supernatant was removed and it was added 100 µL/well of MTT solution -2,5-diphenyl-22WO4 composites, 20 µL of the alamar blue solution was added to each well. The plate was incubated for 4 h at 37 °C, 5% CO2 and the analysis in a spectrophotometer at 540 nm (600 nm reference filter) for 24 h was performed. The same plate was incubated at 37 °C, 5% CO2 to perform the analysis of times 48 and 72 h, since resazurin is a non-invasive and stable probe.In this assay, the viability rate was quantified by the metabolic activity of viable cells. The reagent has the compound resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide/LifeTechnologies) which is a non-fluorescent blue dye. This is reduced by reductase enzymes, present in the cytosol and mitochondria, to a highly fluorescent pink dye, resorufin. After 20 h of the cells being challenged with the CS/α-AgShapiro–Wilk and Levene’s test were performed to test data distribution and homogeneity. Based on normal and heteroskedastic distribution, statistical comparisons were performed by one-way analysis of variance (ANOVA) with Welch correction, followed by Games Howell Post Hoc, using BM SPSS Statistics program (version 23). All data are plotted as the mean + standard deviation (SD) and p < 0.05 was considered statistically significant.4 TCID50 of the virus were incubated for 10 min with CS6.6/α-Ag2WO4 (4.0 µg/mL) composite at 37 °C using a protocol that was previously described92 with some adaptations. The CS6.6 solution, in the same concentration of CS6.6/α-Ag2WO4 composite, and PBS were used as control groups. After this step, each virus inactivated solution was divided equally in 2 parts to analyze virucidal effect 1 and 24 h post infection (hpi) in cell culture. This assay was repeated 3 times. All procedures related to virus culture were handled at biosafety level 3 (BSL3) multiuser facilities, according to World Health Organization (WHO) guidelines.Firstly, with the aim of neutralizing SARS-CoV-2 , 3.0 × 102WO4 inactivate SARS-CoV-2 activity, Vero-E6 cells monolayers were incubated with viral inactivated solutions and the virus in supernatants were analyzed 1 and 24 hpi. For this purpose, Vero-E6 cells (1.5 × 106) previously seeded, maintained in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% Fetal Bovine Serum  in cell culture 24 cm2 flasks, were incubated with each viral inactivated solution in multiplicity of infection (MOI) 0.01 for 1 h at 37 °C and 5% CO2 . Then, the supernatants were harvested and the cell monolayers washed twice with PBS and physically removed from the flasks for processing for transmission electron microscopy (TEM) analysis, or incubated with DMEM/HEPES/2% FBS for 24 h, before harvesting the supernatant and preparation to TEM. The supernatants were stored at − 70 °C for posterior virus titration and RNA quantification by Plaque Forming Units (PFU/mL) and qRT-PCR (number of copies/mL), respectively94.To study the capacity of CS6.6/α-Ag5 cells/well) were seeded into 24-well culture plates (flat bottom) and grown for 24 h at 37 °C in 5% CO2. These cells were inoculated with 300 µL of infected cells supernatants dilutions (10−1 to 10−4). After 1 h at 37 °C in 5% CO2, the medium was changed to 500 µL of a solution containing DMEM-High glucose 1X, 1.8% carboxymethylcellulose and 2% FBS. 72 h post infection, cytopathic effects (CPE) were observed on optical microscope and cells fixed with 10% formalin. After 3 h, this solution was removed and monolayers stained with 0.04% crystal violet for Plaque Forming Units counting93. The molecular detection of viral RNA levels was performed as described before94. Primers, probes, and cycling conditions recommended by the Centers for Disease Control and Prevention (CDC) protocol were used to detect the SARS-CoV-2 envelope gene (E)95. Cell supernatants were used for viral RNA quantification by real time qRT-PCR and were expressed in number of copies of virus RNA per mL. Concurrently to viral RNA amplification, standard curves were plotted with different numbers of copies per cycle threshold (Ct). The standard curve method was used in comparison with the viral gene to obtain the relative quantification of the viral RNA in supernatants96.For PFU assay, monolayers of Vero-E6 cells (1082. Ultrathin sections (50–70 nm) were obtained from the resin blocks. The sections were picked up using copper grids, stained with uranyl acetate and lead citrate83, and observed using Hitachi HT 7800 transmission electron microscope.For TEM analyses the Vero-E6 cells suspensions were fixed in 2.5% glutaraldehyde in sodium cacodilate buffer , post-fixed in 1% buffered osmium tetroxide, dehydrated in acetone, embedded in epoxy resin and polymerised at 60 °C over the course of three days97. The one-way ANOVA with Tukey post-test was performed to determine the significance between the different experimental groups . The graphics were plotted by the OriginLab Pro 2021 program. Results are presented as the mean of 3 independent experiments ± standard deviation (SD) with a confidence interval of 95%, and significant p values were represented as d for < 0.005 and e for < 0.05.The data were analyzed using Past programDetailed information about the XRD, FTIR, FE-SEM, TEM, and cytotoxicity discussion are described in the Electronic Supporting Information.Supplementary Information.


UGT1A1 and SLCO1B1, and hyperbilirubinemia in Thai neonates. One hundred thirty seven neonates were recruited from Division of Clinical Chemistry, Ramathibodi Hospital. UGT1A1*28 and *6 were determined by pyrosequencing whereas, SLCO1B1 388A > G and 521 T > C genetic variants were determined by TaqMan® real-time polymerase chain reaction. Neonates carrying with homozygous (AA) and heterozygous (GA) variants in UGT1A1*6 were significantly related to hyperbilirubinemia development compared with wild type . To the combined of UGT1A1, total bilirubin levels in homozygous variant were higher significantly than heterozygous variant and wild type . Moreover, SLCO1B1 combination was significant differences between the hyperbilirubinemia and the control group (P = 0.041). SLCO1B1 521 T > C variant provide protection for Thai neonatal hyperbilirubinemia (P = 0.041). There are no significant differences in UGT1A1*28 and SLCO1B1 388A > G for the different severity of hyperbilirubinemia. The combined UGT1A1*28 and *6 polymorphism is a strong risk factor for the development of severe hyperbilirubinemia in Thai neonates. Therefore, we suggest neonates with this gene should be closely observed to avoid higher severities of bilirubin.Hyperbilirubinemia is the main mechanism that causes neonatal jaundice, and genetics is one of the risk factors of hyperbilirubinemia. Therefore, this study aims to explore the correlation between two genes,  Neonatal hyperbilirubinemia, one of the most common clinical problems in newborns, occurs up to 60% of healthy full-term newborns . In geneThe causes of neonatal jaundice can be due to the elevation of bilirubin, which is caused by the increased production or inability to metabolize and excrete it. This can be due to the immature liver of the newborn. Another cause is due to the decreased bilirubin uptake and conjugation due to the deficiency of the process of serum albumin binding to bilirubin, and carrying it to the liver . HoweverUGT1A1) which provides instructions for making enzymes called UDP-glucuronosyltransferases. This form conjugated bilirubin, which is soluble, and in turn can be excreted in the duodenum. Once inside, normal gut flora deconjugate bilirubin and convert it into urobilinogen. It is mostly oxidized by intestinal bacteria, and converted to stercobilin, which is then excreted through stool. The rest is then reabsorbed into the bloodstream as a part of the enterohepatic circulation . One-way ANOVA was performed according to genetic groups and total bilirubin levels (mg/dl). All statistical analyses were performed by using SPSS version 21.0 . A P-value < 0.05 was considered to be statistically significant.Hardy–Weinberg equilibrium was assessed using Fisher’s exact and chi-square test for All methods aforementioned above were carried out in accordance with relevant guidelines and regulations.P = 0.013). However, there were no obvious differences between gender, birth weight, and gestational age, with a P-value of 0.763, 0.352 and 0.442, respectively.A total of 137 neonates were enrolled into the study. Sixty-seven neonates were classified into the hyperbilirubinemia group and 70 neonates were control group. Table SLCO1B1 and UGT1A1 variants are shown in Table SLCO1B1 388A > G, 521 T > C, UGT1A1*28 and *6 were 0.79, 0.13, 0.17, and 0.13, respectively. Genotyping of SLCO1B1 388A > G was firstly mentioned. Homozygous variant (GG) was the most abundant, showing 84 (61.3%) and followed by 48 (35.0%) in heterozygous variant (AG) and 5 (3.6%) in wild type (AA). For SLCO1B1 521 T > C, TT, TC, and CC were also measured, and the genotype frequencies were 105 (76.6%), 29 (21.2%) and 3 (2.2%), respectively. The next gene explored was UGT1A1*28. The genotypes of TA6/TA6, TA6/TA7, and TA7/TA7 were 90 (65.7%), 46 (33.6%) and 1 (0.7%), respectively. The genotyping of UGT1A1*6211G > A, was GG, GA, and AA, the frequencies are 107 (78.1%), 25 (18.2%) and 5 (3.6%) respectively.The analysis of the genotype and allele frequency of SLCO1B1, the frequency values for normal, intermediate and low function were as follows; 105 (76.6%), 29 (21.2%) and 3 (2.2%), respectively. Lastly, combined UGT1A1 frequencies were measured. The wild type, heterozygous, and homozygous variant were genotyped to 66 (48.2%), 59 (43.1%) and 12 (8.8%) respectively.Regarding combined SLCO1B1 521 T > C variant showed significantly a low risk of neonatal hyperbilirubinemia in neonates (P = 0.041). The combined of SLCO1B1 was significantly related to severe hyperbilirubinemia (P = 0.041). In this study found that all neonates carrying homozygous variant in UGT1A1*6 had high development of hyperbilirubinemia . Moreover, UGT1A1 combination was significantly increases the risk of hyperbilirubinemia (P = 0.005).Table UGT1A1 had a significantly increased of total bilirubin levels when compared with heterozygous variant and wild type . As shown in Fig. SLCO1B1 with low , intermediate , and normal  function and the total bilirubin (mg/dL) measured. There was no significant association between combined SLCO1B1 and total bilirubin levels. However, our results shown that neonates with low function had a decreasing trend in total bilirubin levels compared with intermediate and normal function. The average of total bilirubin in low, intermediate and normal function as follow: 11.0 ± 3.0 mg/dL, 12.2 ± 5.0 mg/dL, and 13.7 ± 4.9 mg/dL, respectively.Similar to Fig. SLCO1B1 and UGT1A1 variants were investigated in Thai neonates. Our results showed that combined UGT1A1*28 and *6 is a high-risk factor for developing neonatal hyperbilirubinemia. We were the first study to conduct on combined UGT1A1 variants and its effects on Thai neonatal hyperbilirubinemia. Regarding SLCO1B1, a trend was evident, as the total bilirubin levels were a decreasing in low function compared with intermediate and normal function.In this study, the correlation between hyperbilirubinemia and two genes, UGT1A1*28 (A(TA)7TAA) is a variant allele that is commonly found in African-Americans , and less in Asian populations  [UGT1A1*28 is not a risk factor for developing neonatal hyperbilirubinemia. Similarly, to a meta-analysis by Li H, et al. [UGT1A1*28 might not be associated with the risk of neonatal hyperbilirubinemia.equency) , 20. It , et al. , concludUGT1A1*6211G > A, another SNP variant, was explored. The results from Prachukthum S, et al. [UGT1A1*6 was an important risk factor for developing jaundice in infants. It found that infants who were carrying homozygous (AA) and heterozygous (GA) variants were more susceptible to develop hyperbilirubinemia when compared with wildtype (GG). Yanagi T, et al. [UGT1A1*6 is a risk factor for prolonged unconjugated hyperbilirubinemia in Japanese preterm infants. Moreover, Nguyen TT, et al. [UGT1A1*6 were significantly classified into the hyperbilirubinemia group., et al. , reporte, et al. , showed , et al. , revealeUGT1A1*28 and *6, it showed that these two genes were strongly significantly associated with hyperbilirubinemia (P = 0.005). The total bilirubin levels in the homozygous variant were significantly higher compared with heterozygous variant and wildtype.For our results regarding combined SLCO1B1. This gene provides instructions for making a protein, OATP1B1, an influx transporter responsible for the transportation of compounds in the bloodstream to the liver [SLCO1B1 388 A > G in this study was 0.79, similarly to the Han Chinese population having an allele frequency of 0.64 reported by Liu et al. [SLCO1B1 gene was associated with infant hyperbilirubinemia in Chinese. However, the data from our study indicates that there were no statistically significant differences in risk factor of neonatal hyperbilirubinemia and SLCO1B1 388 A > G variant. Similar to Amandito R, et al. [SLCO1B1 388 A > G and hyperbilirubinemia in newborns. In SLCO1B1 521 T > C variant, there was a significant correlation between hyperbilirubinemia and SLCO1B1 521 T > C (P = 0.041). Similarity to a systematic review with meta-analysis of Liu J et al. [SLCO1B1 521 T > C variant protective factor against hyperbilirubinemia in Chinese neonates.The second gene studied was he liver . The allu et al. . Moreoveu et al. , found t, et al. , demonstJ et al. , reporteSLCO1B1, there was a decreasing trend of total bilirubin levels in normal , intermediate , low  function respectively. The relationship between low function neonates, having lower total bilirubin levels than that of intermediate and normal function, was evident.Regarding combined P = 0.013). The finding was consistent with Bratton S et al. [In the present study, total bilirubin levels were significantly between case and control groups. In case group, mean of total bilirubin levels were 18.8 ± 2.6 mg/dL, which had higher total bilirubin levels than control group (10.7 ± 3.5 mg/dL). The total bilirubin levels in control group showed slightly high levels in Thai neonates. There was a nutrition significance to be noted in this study. The results showed that there was a correlation between nutrition and neonatal hyperbilirubinemia in Thai neonates (S et al. , statingSLCO1B1 and UGT1A1, so other genes related with hyperbilirubinemia could be investigated in further studies. Since this was a retrospective study, some clinical data were also missed, including nutrition.In addition, some limitations of this study were regarding small sample size, which does not represent the whole population. Large sample size could be studied in further study. Our study was also limited to only two genes, UGT1A1*28 and *6 polymorphism was a strong risk factor for hyperbilirubinemia in Thai neonates. Therefore, we suggest neonates with this gene should be closely observed to avoid higher severities of bilirubin.The combined


This novel integrative approach identified enhanced capacity for H2S production in radiation resistant cells and extracted a key relationship between intracellular redox state and cholesterol metabolism; experimental validation of this relationship highlights the importance of redox state in cellular metabolism and response to radiation.Redox metabolism is increasingly investigated in cancer as driving regulator of tumor progression, response to therapies and long-term patients’ quality of life. Well-established cancer therapies, such as radiotherapy, either directly impact redox metabolism or have redox-dependent mechanisms of action defining their clinical efficacy. However, the ability to integrate redox information across signaling and metabolic networks to facilitate discovery and broader investigation of redox-regulated pathways in cancer remains a key unmet need limiting the advancement of new cancer therapies. To overcome this challenge, we developed a new constraint-based computational method (COSM Ionizing radiation is widely used to treat cancer, and more than 50% of cancer patients receive either definitive or adjuvant radiation therapy . Howeverro) method based on multi-omics and targeted data  needed to generate reducing equivalents in the form of NADPH and nucleotide building blocks for the repair of radiation-induced DNA damage. It also highlighted new processes such as H2S production and redox regulation of cholesterol metabolism defining the radiation response phenotype. Further investigation of mitochondrial 1-C metabolism focusing on the NADPH-producing methylene tetrahydrofolate dehydrogenase 2 (MTHFD2), has led to the discovery of MTHFD2-dependent steroidogenesis in rSCC-61 cells, a pathway previously thought to be present only in tumors of classical steroidogenic tissues . Steroidogenesis could be an important target for the treatment of non-adrenal cancers that may hijack this pathway to both lower the intracellular cholesterol and to produce metabolites such as cortisol, which support tumor growth and metastasis    was used to determine the distribution of proteins subcellular locations  approaches to infer cell-specific signaling pathways and predict the drug treatment effects .To infer the rSCC-61 specific pathway network based on the generic pathway map constructed, we minimized the differences between measurements and the simulated values, and the complexity of the network’s topology structure by developing a MIP-based approach to optimize such multi-objective functions , 17. MIPro formulation was solved with the MATLAB optimization toolbox Gurobi 9.0 , and the glutathione reductase (GR) antioxidant systems, and (4) cholesterol metabolism  increased Akt2 and decreased Akt1/3 activities driving glucose uptake and activation of glycogen synthase kinase (GSK3) α/β signaling, (2) glycolytic flux diverted into PPP leading to an increase in precursors for nucleic acid biosynthesis and increased nicotinamide adenine dinucleotide phosphate (NADPH) production, (3) suppression of Hro network analysis H with MTHFD2 contributing 34% and 24% in rSCC-61 and SCC-61, respectively (Methotrexate (MTX), an inhibitor of 1-C metabolism enzyme dihydrofolate reductase (DHFR/DHFRL1) is FDA-approved for the palliative care of HNSCC patients and often investigated in combination with other treatments to improve therapeutic efficacy . DHFR ca+ ratio are intrinsically connected to the levels and the balance of GSH and GSSG in cells  being expressed at higher level in rSCC-61 compared with SCC-61 cells and cystathionine γ-lyase (CSE) having the complementary profile with higher level in SCC-61 cells relative to rSCC-61 cells  is a major regulator of cellular cholesterol efflux, while the low-density lipoprotein receptor (LDLR) controls cholesterol uptake. Gene expression data showed a decreased level of both ABCA1 and LDLR proteins in rSCC-61 cells  and 24-OH oxysterol, which are both significantly higher in rSCC-61 (As the COSM2O2 and lipid rafts in SCC-61 cells in previous studies (2O2 in the cytosol and mitochondria resulting in increased DNA double strand breaks as indicated by imaging with γH2AX (tert-butyl hydroperoxide (tBHP), associated with higher intracellular cholesterol  using multi-omics and complementary data , 5 mM ammonium formate, pH 10), Solvent B ; 0% B for 2 min; 0-10% B in 5 min; 10- 27% B in 34 min; 27- 31% B in 4 min; 31- 39% B in 4 min; 39-60% in 7 min; 60% B for 8 min; 60-100% B in 2 min; 100% B for 4 min; 100-0% B in 1 min; 0% B for 1 min]. Eluted peptides fractions (1 mL/tube) were collected using Water Fraction Collector III. The 36 eluted fractions were combined from the beginning, middle, and end of the run to generate 12 fractions  and then dried using a SpeedVac Savant™ SPD1010 (Thermo).http://www.uniprot.org/). Data files (.raw format) were searched against the downloaded database using the following parameters: Enzyme: trypsin; Max. Missed Cleavage Sites: 2; Search Mode: MS/MS ion search with decoy database search included; Precursor Mass Tolerance: 10 ppm, Fragment Mass Tolerance: 0.6 Da; Target False Discovery Rate (FDR): 0.01.Dried peptide extracts were dissolved in 0.1% formic acid, 5% ACN, and separated by nanoLC (Dionex Ultimate 3000) equipped with a Nano Trap Column, Acclaim PepMap100  and an Acclaim PepMap RSLC nanocolumn  (Thermo Scientific). The samples (10 µL) were injected and separated on the nanocolumn at a flow rate of 300 nL/min using the following gradient: Solvent A: 95% water, 5% ACN, 0.1% formic acid; Solvent B: 20% water, 80% ACN, 0.1% formic acid); 2–10 min: 2–7% B; 10– 130 min: 7–50% B; 130–132 min: 50–85% B; 132–147 min: 85% B; 147-150 min: 85-2% B; 150–160 min, 2% B. LTQ Orbitrap Velos Pro (Thermo Scientific) was used for MS/MS analysis. The sample was ionized in the nanospray source equipped with stainless steel emitter (Thermo Scientific). The spray voltage was set to 1.5 kV and the temperature of the heated capillary was set to 200°C. Full scan MS survey spectra in profile mode were acquired in the Orbitrap. In positive ion mode by alternating full-scan MS 10 most intense peaks in the ion trap with dynamic exclusion enabled. These most intense peptide ions were fragmented by collision-induced dissociation (CID) using a normalized collision energy of 35.0. The resolution was 60,000. The lock mass (371.101240) option was enabled for survey scans to improve mass accuracy. Data were acquired using the XCalibur v.2.1 software. Protein identification and quantitation was performed using Proteome Discoverer v 1.4 and human protein database downloaded in FASTA format from UniProt (8/sample) were first extracted with 500 mL of mixture solvent (methanol:chloroform:water 2.5:1:1), followed by addition of 500 mL of methanol. The supernatant was transferred to gas chromatography (GC) sampling vials and vacuum dried at room temperature (RT). The residue was then derivatized using a two-step procedure. First, 80 μL methoxyamine (15 mg/mL in pyridine) was added to each vial and incubated for 90 min at 30°C. This was followed by addition of 80 μL BSTFA trifluoroacetamide)  to initiate the derivatization reaction. The samples were heated for 60 min at 70°C and 1-μL aliquot of the derivatized solution was injected in splitless mode into an Agilent 7890N gas chromatograph coupled with a Pegasus HT time-of-flight mass spectrometer . Separation was achieved on a Rxi-5MS capillary column , with helium as carrier gas at a constant flow rate of 1.0 mL/min. The temperature of injection, transfer interface, and ion source were set to 260°C, 260°C, and 210°C, respectively. The GC temperature programming was set to 80°C for 2 min, followed by 10°C/min oven temperature ramps to 140°C, 4°C/min to 210°C, 10°C/min to 240°C, and 25°C/min to 290°C, and a final 4.5 min maintenance at 290°C. Electron impact ionization (70 eV) at full scan mode (m/z 40-600) was used, with an acquisition rate of 20 spectra/second in the TOF MS setting. The acquired MS files from GC/TOF MS analysis were analyzed by ChromaTOF software . Statistic component analysis provided the aligned data sheet containing sample information, unique mass, and the area of unique mass of each sample. Internal standards and any known artificial peaks, such as peaks caused by noise, column bleed and BSTFA derivatization procedure, were removed from the data set. The resulting data was normalized to sum of all peaks prior to statistical analysis . The normalized data was mean centered and unit variance scaled during chemometric data analysis in the SIMCA-p 12.0 Software package . Compound identification was performed by comparing the mass fragments with NIST 11 Standard mass spectral databases in NIST MS search 2.0  software with a similarity of more than 70%.Cells  and probed for the indicated proteins after overnight incubation with the corresponding primary antibodies diluted in 5% BSA in Tris Buffered Saline (TBS)-Tween20 buffer followed by incubation with respective HRP-conjugated secondary antibodies. The western blots were developed using Western Lightning Plus-ECL reagents followed by exposure to autoradiography film (Blue Ultra Autorad Film from GeneMate). The protein ratio in rSCC-61 vs. SCC-61 was quantified using ImageJ.100 µg of total protein was loaded per each sample lane of 1 mm 10% gel and 5 ng of human cystathionine beta-synthase (CBS) and 30 ng of human cystathionase (CSE) were loaded as standards. Electrophoresis was run at 150V. Proteins were transferred to PVDF membrane during 90 min procedure at 25 V using Invitrogen XCell II Blot Module. After transfer the membrane was incubated in milk tris-buffered saline (TBS-T (tween-20) 40 min at RT to block unspecific binding and cut at the level of 50 kD. Upper part of the membrane was treated with anti-CBS, and lower part with anti-CSE antibodies overnight at 4°C. Both antibodies were chicken IgY . Then membranes were washed out of primary antibodies and incubated with secondary antibodies  diluted 1:250,000 for 1 h at RT. Then secondary antibodies were washed out, membrane was treated with Clarity Western ECL Substrate (Bio Rad) and exposed to film. After film development the membranes were stained with Ponceau S as recommended by Invitrogen for equal loading and transfer control.6 cells were grown in a 60 mm culture dish. Cells were washed 3X with ice-cold PBS, then 0.5 mL of PBS was added to the culture plates on ice. Cells were detached by scraping and collected in a 1.5 mL centrifuge tube. An aliquot of the cell suspension in PBS was mixed with an equal volume of metaphosphoric acid solution  and vortexed gently. The precipitated proteins were separated by centrifugation at 13,000 x g for 10 min at 4°C. The supernatant was transferred into a 1.5 mL centrifuge tube, iodoacetic acid was added at a final concentration of 7 mM to alkylate free thiols, the pH was adjusted to 7 - 8 with saturated K2CO3 and the reaction was allowed to proceed for 1 h in the dark at RT. Finally, an equal volume of 2,4-dinitrofluorobenzene solution (1.5% v/v in absolute ethanol) was added to the mixture, then vortexed, and the reaction was allowed to proceed for at least 4 h in the dark at RT. The N-dinitrophenyl derivatives of GSH and GSSG were separated by HPLC on a Waters μBondapak NH2 column  at a flow rate of 1 mL/min. The mobile phase consisted of solvent A (4:1 methanol/water mixture) and solvent B, which were prepared by mixing 154 g of ammonium acetate in 122 mL of water and 378 mL of glacial acetic acid and adding 500 mL of the resulting solution to 1,000 mL of solvent A. The following elution conditions were used: from 0 – 10 min isocratic 25% solvent B; from 10 – 30 min linear gradient 25 – 100% solvent B; from 30 – 34 min 100% solvent B; from 34 – 36 min 100 – 25% solvent B; and finally, 36 – 45 min 25% solvent B. Prior to injection, the column was equilibrated with 25% solution B for 10 min. Elution of metabolites was monitored by their absorbance at 355 nm. Under these conditions, GSH and GSSG exhibit retention times of 26 and 29 min, respectively. The concentration of individual thiols is determined by comparing the integrated peak areas with independently generated calibration curves for each compound. The concentration of each metabolite was normalized to the protein concentration measured by the Protein Assay Dye Reagent (Bio-Rad) in the corresponding sample. The detection limit of this method is ~ 20 pmol.To measure the concentration of intracellular GSH and GSSG, 2 – 4 x 102S production capacity, 400 μL of cell lysate was mixed with L-cysteine (Sigma)  and D,L-homocysteine (Sigma)  in a total volume of 500 μL and incubated under anaerobic conditions for 20 min at 37°C. Accumulation of H2S was measured using GC equipped with sulfur chemiluminescence detector (SCD 355) (Agilent) as described . To measure the rate of Hescribed . Cell lyS=e−αD− βD2 using SigmaPlot v.12 software (rSCC-61 and SCC-61 cells were grown to 60% confluence. Cells were then trypsinized and resuspended in complete medium (DMEM/F12 + 10% FBS and 0.5% PenStrep (Gibco)), and plated (300 cells/well) into six well culture dishes. Following overnight incubation at 37°C, each 6-well plate was treated with either (i) 1 μM sulfasalazine (SAS) in media, (ii) media without cysteine, or (iii) 0 – 10 μM methotrexate and allowed to incubate further for 12 days for the colony formation. For other clonogenic assays, first the rSCC-61 cells were grown to 60% confluence followed by transfection with MTHFD2 shRNA (50 nM) or control shRNA (50 nM) (Santa Cruz Biotechnology) using Lipofectamine 2000 (ThermoFisher Scientific) according to manufacturer’s protocol. After 48 h, transfected cells were trypsinized, resuspended in complete medium (DMEM/F12 + 10% FBS and 0.5% PenStrep), and selected using puromycin. After selection, the cells were plated (300 cells/well) into six well culture dishes and incubated overnight. Cells were then subjected to (i) 2 Gy irradiation, (ii) 10 μM methotrexate, (iii) 1.7 μM CB83, or (iv) 0.1 μM methotrexate combined with 1.7 μM CB83 and incubated for an additional 12 days for the colony formation. Once formed, the colonies were fixed in methanol and acetic acid (7:1) solution and stained with 0.5% crystal violet (Fisher Scientific). The colonies containing more than 50 cells were counted. The surviving fraction of the treated cells was normalized using the plating efficiencies of their corresponding untreated control. The data were fitted to the multi-target and linear quadratic formulae, where survival (S) was related to dose (D) by the expression software .4 cells/mL. After overnight incubation at 37°C, cells were treated with 1 mM MβCD and 1 mM MβCD-Cholesterol complex for 24 h followed by 2 Gy radiation for 48 h. After incubation, 0.125 mL MTT (5 mg/mL in PBS) was added to each well and plates were further incubated for 4 h at 37°C. At the end of incubation, 0.5 mL solubilization solution was added to each well and the absorbance was recorded at 570 nm.To assess the effects of lipid raft activation on response to radiation in rSCC-61 cells, the cells were trypsinized, resuspended in complete DMEM/F12 medium and seeded in 24-well plates at a density of 5 x 106 million SCC-61 or rSCC-61 cells using the procedures described next. For free cholesterol analysis, each cell pellet was extracted with 1 mL of ice-cold methanol to which 20 μL of oxysterol internal standard mixture  and 30 μL of [13C2]-cholesterol (1 μg/μL) was added and then mixed by vortexing. Extracts were incubated on ice for 1 h before being centrifuged at 18,000 x g. The supernatant was removed and then stored at -20°C. Free cholesterol was measured in a 1:10 dilution of the extract in methanol. For total cholesterol analysis, total cholesterol was measured after saponification of 100 μL of the initial cold methanol extraction. The 100 μL subjected to saponification was first evaporated under nitrogen until dryness and reconstituted in 1 mL of ethanol to which 100 μL of 50% KOH was added. Each sample was then capped, flushed with argon, and incubated at 60°C for 1 hour. After incubation, 1 mL of water and 3 mL of hexane were added and the samples were mixed by vortexing. These samples were then centrifuged for 5 min at 3000 x g to separate the layers. The hexane layers were then removed and evaporated to dryness under nitrogen before being reconstituted in 1 mL of methanol. For oxysterol analysis, samples were evaporated under nitrogen and re-dissolved in methanol followed by vortexing and addition of 100 μL of aqueous 50% KOH (w/w). Samples were vortexed and incubated at RT for 2 h with vortexing every 30 min. Next, a 3:1 addition of toluene and distilled H2O were added to the sample followed by vortexing and 4000 rpm centrifugation for 5 min. The upper phase was evaporated under nitrogen and re-dissolved in toluene and vortexed thoroughly. Isolute 100 mg silica SPE-cartridges  were preconditioned with hexane and samples were loaded onto column in toluene. Columns were washed with 1 mL hexane, 8 mL hexane:isopropanol (99.5:0.5) and then the oxysterol fraction was eluted with 5 mL hexane:isopropanol (70:30). The analysis was performed on a Shimadzu UHPLC-MS/MS equipped with 2 LC-30AD pumps, a SIL-30AC autosampler, a CBM-20A communications bus module, a DGU-20A5R degassing unit, a CTO-30A column oven, and an 8050 triple-quadrupole mass spectrometer operated with a DUIS source. The separation was conducted with a Kinetex C8 column  under isocratic conditions with a mobile phase combination of 5% A (0.1% formic acid in water) and 95% B (0.1% formic acid in methanol). A flow rate of 0.5 mL/minute was used with a sample injection volume of 20 μL. Ionization occurred in the DUIS source with the following conditions: nebulizing gas flow of 2 L/min, heating gas flow of 10 L/min, interface temperature of 330C, DL temperature of 250C, heat block temperature of 400C, and a drying gas flow of 10 L/min. Cholesterol and [13C2]-cholesterol were analyzed with two m/z transitions each, 369.3/161.2 and 369.3/95.1 for cholesterol and 371.2/163.2 and 371.2/177.1 for [13C2]-cholesterol.Cells were grown to ~70% confluence in complete DMEM/F12 media. Cholesterol and oxysterols were extracted from 1 x 105 cells in 0.5 mL complete media  and allowed to adhere before labeling with 2 µCi/mL [3H]cholesterol (PerkinElmer) in DMEM/F12 containing 0.5% PenStrep and 2.5% FBS for 24 h. Cells were washed with 0.5 mL 1X DMEM (Gibco) containing 14 mM HEPES followed by 20 h equilibration in DMEM/F12 with or without 0.30 mM adenosine 3’,5’-cyclic monophosphate (cAMP). Cells were washed again with 0.5 mL 1X DMEM containing 14 mM HEPES and subsequently incubated with 0.5 mL MEM-HEPES (Gibco) supplemented with or without 0.15 mM cAMP and 30 μg/mL apolipoprotein A1 (APOA1) for 4 h. Following 4 h incubation, 0.5 mL of media was removed and 0.1 mL of media combined with 5 mL Bio-Safe II scintillation fluid (Research Products Int.) to be counted. Each well was then washed 2X with 0.5 mL cold saline buffer followed by extracting lipids with 1 mL isopropanol overnight at RT on rotator. The lipid extract was dried under N2 and resuspended in 0.5 mL isopropanol. Then 0.1 mL of lipid extract was combined with 5 mL Bio-Safe II scintillation fluid (Research Products Int.) to be counted. 3H-radioactivity was counted in medium and cellular lipid extract and cholesterol efflux was calculated as the percentage of 3H-radioactivity in medium/(3H-radioactivity in medium + 3H-radioactivity in the cellular lipid extract) * 100%.SCC-61 and rSCC-61 cells were plated 2.0 x 104 cells/well of shMTHFD2 knockdown radiation resistant rSCC-61 cells and shRNA control rSCC-61 cells were plated in Microtek chambered slide and were cultured in DMEM/F12 media containing 10% FBS and 1% penicillin/streptomycin at 37°C using a 5% CO2 incubator overnight. Next day, cells were treated with 10 μM Methotrexate for 2 hr at 37°C. After completion of incubation period, cells were washed with ice cold medium and incubated with 1 μg/mL of AlexaFluor594 conjugated CT-B for 15 min on ice. Further, the cells were washed twice with ice cold Dulbecco’s phosphate-buffered saline (DPBS) and fixed with 4% formaldehyde for 20 min and permeablized in 0.1% Triton X-100 for 10 min, washed, and mounted using Fluoromount (Sigma) for imaging. Imaging was performed using a Zeiss LSM710 confocal microscope and a 40X objective. The images were processed using the LSM image browser. Total cortisol was determined in SCC-61, rSCC-61 and MTHFD2 KD rSCC-61 cells by using 96 well plate ELISA kit, commercially available in Cayman Chemicals, USA .2x10Data are presented as mean ± SEM. All statistical analyses were performed using SigmaPlot v.12. All data are from three independent experiments unless stated otherwise. Statistical significance of differences was evaluated with the student’s t test unless stated otherwise. Significance was accepted at the level of p < 0.05.https://github.com/JakeJiUThealth/MIP_V1.0 and https://github.com/kemplab/COSMro-FBA-modeling.The data presented in the study are deposited in the ProteomeXchange PRIDE repository, accession number PXD029053. Source codes for the COSMro and FBA analysis are available at GitHub: Conceptualization CF and AT. Methodology CF, XZ, ZJ, JP, RB, MK. Investigation JM, ND-B, HW, KS, EL, VV, JL, C-CK. Writing-original manuscript JM, ZJ, CF. Writing – editing, all authors. Supervision CF, XZ, MK, RB, JP. Resources RB, JP. Funding, MK, CF, XZ. All authors contributed to the article and approved the submitted version.


Cyclic arginyl-glycyl-aspartic acid peptide (cRGD) peptides show a high affinity towards αVβ3 integrin, a receptor overexpressed in many cancers. We aimed to combine the versatility of ultrasmall gold nanoparticles (usGNP) with the target selectivity of cRGD peptide for the directed delivery of a cytotoxic payload in a novel design. usGNPs were synthesized with a modified Brust-Schiffrin method and functionalized via amide coupling and ligand exchange and their uptake, intracellular trafficking, and toxicity were characterized. Our cRGD functionalized usGNPs demonstrated increased cellular uptake by αVβ3 integrin expressing cells, are internalized via clathrin-dependent endocytosis, accumulated in the lysosomes, and when loaded with mertansine led to increased cytotoxicity. Targeting via cRGD functionalization provides a mechanism to improve the efficacy, tolerability, and retention of therapeutic GNPs. Gold nanoparticles (GNPs) provide a useful scaffold to which a variety of different ligands may be attached to imbue desirable properties such as improved solubility, stability, stealth, delivering a therapeutic payload or targeting the GNP to a specific cell type or location through passive mechanisms (by enhanced permeability and retention effect) or active delivery (using a ligand specific to a tissue or a cell type) ,2. TargeComposed of an inert gold core, GNPs are not expected to have cytotoxicity; consequently, in vivo toxicity data of GNPs is limited ,16,17. uUltrasmall GNPs offer several advantages compared to GNPs with sizes over 10 nm. usGNPs have an improved surface/volume ratio, which allows the immobilization of larger amounts of payload at the same dose of gold. They have improved penetration and retention in tumors and fast clearance in normal tissues ,28. SimiTM)   equipped with a digital camera of type F-216 . Wider field-of-view images at X150k magnification were assembled with the spotscan utility of the TVIPS EMMENU4 software from a beam shift based 4 × 4 images matrix. Analyses were performed at CIC BiomaGUNE . Data processing was performed using ImageJ .Size (hydrodynamic diameter) measurements were performed with Zetasizer Nano-ZS . Size was expressed by volume (%) as an average of three measurements taken at 25 °C at a 173-degree scattering angle in a plastic cuvette .3. Size was expressed by surface mode. Sizing analysis was performed using a CPS DC24000UHR disc centrifuge  as described previously . Eleven M KCN in 0.1 M KOH (solvent D2O) after the removal of H2O by freeze-drying. Particles were incubated at 80 °C for 6 h with strong agitation to prevent pelleting (950 rpm). A transparent solution with no pellet indicated complete etching, which was visually checked. Experiments were performed at 298 K on a AVANCE III 500 spectrometer  at CIC BiomaGUNE  . Data processing was performed using MestReNova 10.0.2 .The GNP amount equivalent to 10 mg of Au was incubated with 600 µL of 0.3 M KCN and 0.01 M KOH and H2O up to 190 μL. Mixing was carried out by vortexing. Particles were incubated at 80 °C for 10 min with strong agitation to prevent pelleting (950 rpm). It was considered to have complete etching when, visually, a transparent solution with no pellet was attained. To the etched solution, 10 μL of 0.05 M TCEP that was made from a commercial, neutral 0.5 M solution  was added. The GNP amount equivalent to 350 μg of Au was incubated with 15 μL of 0.3 2O; B: 0.1% formic acid in acetonitrile. Elution conditions: 0–0.5 min, 5% B isocratic; 0.5–6 min, linear gradient 5–98% B; 6–7 min, 98% B isocratic; washing and reconditioning of the column. The flow rate was 0.350 mL/min and the injection volume was 5 µL. The system operated at 35 °C. The Corona Veo RS Evaporation temperature was set at 35 °C; Power function: 1.0; Data collection Rate: 2 Hz; Signal Filter: 3.6 s. ESI-MS analysis was performed in the positive ion mode. Nitrogen was used as a desolvation gas. The ESI parameters of the source were: capillary temperature of 150 °C, the source heater temperature was held at 45 °C, and a potential of 3.8 kV was used on the capillary for positive ion mode. MS spectra, within the m/z range of 150–2000 amu, were obtained at 35 V cone voltage.UltiMate 3000 Rapid Separation Liquid Chromatography system (Thermo Fisher Scientific) comprising a dual gradient standard pump, Corona Veo RS CAD detector (Chromeleon 7.0 software) in line with LCQ Fleet Ion trap Mass Spectrometer detector  was used for all experiments. Separation steps were performed on an Acquity UPLC BEH C18 column, 130 Å,  and an Acquity UPLC BEH C18 VanGuard precolumn, 130 Å,  . Solvents used as mobile phase were A: 0.1% formic acid in HM TCEP with 50% (v/v) DMSO/H2O up to 120 µL. Mixing was carried out by vortexing. Particles were incubated at 80 °C for 1 h with strong agitation to prevent pelleting (950 rpm). 1260 Infinity system (OpenLab CDS software) in line with 6120 Single Quadrupole mass spectrometer was used for all experiments (Agilent). Separations were performed on an Ascentis Express Peptide C-18 octadecyl phase column  and an Ascentis Express C18 octadecyl phase guard column . Solvents used as mobile phase were A: 0.1% trifluoroacetic acid in H2O; B: 0.1% trifluoroacetic acid in acetonitrile for LC-DAD; A: 0.1% acetic acid in H2O; B: 0.1% acetic acid in acetonitrile for LC-MS. Elution conditions: 0–2 min, 20% B isocratic; 2–8 min, linear gradient 20–100% B; 8–9 min, 100% B isocratic; 9–10 min linear gradient 100–20% B; 10–12 min, 20% B isocratic; washing and reconditioning of the column. The GNP amount equivalent to 12 µg of Au was incubated with 60 µL of 0.5 The flow rate was 1 mL/min and the injection volume was 10 µL. The system operated at 35 °C. ESI-MS analysis was performed in the positive ion mode. M EZ-Link NHS-PEG4-Biotin (Thermo Fisher Scientific) in PBS at room temperature for 1 h. The reaction was stopped by purification on a Zeba spin 7 kDa MWCO desalting column (Thermo Fisher Scientific) following the manufacturer’s protocol  FBS) EMEM containing usGNPs at 1 × 108 particles per cell (ppc) and incubated for 60 min as normal or at 4 °C. Competition experiments were preincubated with 500 µM free cRGD-peptide in 300 µL/well low serum EMEM for 60 min, then the usGNP solution was added at 1 × 108 ppc in a 100 µL volume  and incubated for 60 min. Cells were plated, at 2 × 10g for 5 min. The supernatant was discarded, and the pellet was washed twice with 1 mL PBS, then once with 1 mL 0.2 M acetic acid (pH 2.8) in 0.5 M NaCl at 4 °C, to remove surface-bound material. Samples were centrifuged at 200× g for 5 min and washed a further time with 1 mL PBS and stored at −20 °C prior to analysis by ICP-MS.Post-treatment with usGNPs, the cells were scraped from the plates, transferred to 2 mL tubes, and centrifuged at 200× w/v) tetramethylammonium hydroxide  solution containing 0.2% (v/v) Triton ×100 (Sigma-Aldrich) under agitation for approximately 30 min. A total of 1.55 mL of lysate was transferred into a new tube and topped up with 1 mL 3% (w/v) TMAH. Internal standard solution (2.45 mL of 4 ppb iridium (Sigma-Aldrich), 3% (v/v) HCl in H2O) was added to the samples and the resulting solution was measured on a NexION 300× ICP-MS instrument (PerkinElmer) and quantitated using a calibration curve . Alternatively, usGNP uptake was measured using inductively coupled plasma—atomic emission spectroscopy  using the iCAP 7400 ICP-AES Analyzer (Thermo Fisher Scientific)  vs. response curve using Prism 9  [Cells were plated at 2 × 10CA, USA) .5 in wells of a 24-well plate with glass coverslips, incubated overnight then treated as indicated. The medium was removed and the coverslips were rinsed with PBS twice. Cells were fixed on ice for 10 min with 4% paraformaldehyde in 250 mM HEPES pH 7.4 (both from Sigma-Aldrich). For detecting intracellular antigens cells were permeabilized with 0.1% Triton X-100 (Thermo Fisher Scientific) in PBS for 20 min on ice. Coverslips were then incubated for 1 h at 37 °C in a blocking buffer . Coverslips were then placed in a humidity chamber and incubated overnight at 4 °C with 15 µL of primary antibody solution in blocking buffer diluted as follows: mouse anti-human integrin β3 primary antibody, 1:200 ; rabbit anti-EEA1, 1:200 ; rabbit anti-RAB7, 1:100 ; rabbit anti-RAB11, 1:100 ; rabbit anti-RCAS1, 1:100 . Coverslips were washed 4× for 10 min with PBS with gentle rocking, then were subsequently incubated with 100 µL of Alexa Fluor 488 conjugated goat anti-mouse secondary antibody  or Alexa Fluor 488 conjugated goat anti-rabbit secondary antibody  at 37 °C for 1 hr. The coverslips were washed 4× with PBS as above, then with ultrapure water, dried, and mounted onto microscope slides using Vectashield® mounting medium containing DAPI . Imaging was performed on a TCS SP8 laser scanning microscope with an HC PL APO 63×/1.40 Oil CS2 objective . Image acquisition and processing parameters were kept identical between samples from the same experiment to ensure comparability.Cells were seeded at 1 × 104 per well in a 6-well plate and incubated for 24 h. Cells were detached using 0.5% trypsin (Thermo Fisher Scientific), suspended in 2 mL of PBS then collected in a conical centrifuge tube. Samples were centrifuged at 220× g for 3 min, then suspended in 1 mL PBS + 0.3% BSA (Sigma-Aldrich). All samples were filtered using a 100 µm Corning cell strainer (Thermo Fisher Scientific). Cells were counted using TC20TM Automated Cell Counter . Cell density was set to 1 × 106, and 25 µL of cell suspension was then incubated with 25 µL FITC pre-conjugated primary antibodies  or isotype control antibodies  for 30 min at room temperature in U bottom 96 well plates. Samples were centrifuged at 260× g for 2 min. Cells were then washed three times in 100 µL PBS + 0.3% BSA, then resuspended in 200 µL PBS + 0.3% BSA. The samples were transferred into a FACS tube with 200 µL FACS sheath fluid. Cells were analyzed by flow cytometry using a FACSVerse flow cytometer . A minimum of 10,000 cells were analyzed per sample. Data were collected and analyzed using FACSuite and FlowJo software packages . Cells were seeded at 1 × 104 cells per cm2, incubated overnight then treated as indicated in the figure legends with chlorpromazine-hydrochloride (Sigma-Aldrich), genistein (Sigma-Aldrich), or nocodazole (Sigma-Aldrich). Live cell imaging was performed on a TCS SP8 laser scanning confocal microscope with an HC PL APO 63×/1.40 Oil CS2 objective. Cells were stained as indicated in the figure legends with BODIPY™ FL C5-Lactosylceramide BSA complex (Thermo Fisher Scientific) or LysoTracker® Red (Thermo Fisher Scientific). Multiple dyes were imaged with sequential acquisition settings to ensure a clear signal from single dyes without bleed-through into other acquisition channels.Cells were seeded in 35 mm µ-dish (ibidi) or Nunc Lab-Tek II 8-well coverglass (Thermo Fisher Scientific) vessels at 2.85 × 102) and an oligoethylene glycol α-Thio-ω-(propionic acid) octa(ethylene glycol) (PEG(8)COOH). Two-nanometer core GNPs functionalized with the same mixed corona and the SIKVAV peptide have been previously demonstrated to have the ability to target cancer cells through α6β1 integrins [290@Au2000. The base particle obtained through the modified Brust-Schiffrin synthesis: 145(PEG(8)COOH)145@Au2000 was then functionalized using two strategies: post-functionalization and ligand exchange. Post-functionalization was performed by amidation of the carboxyl-terminal moiety of the oligoethylene glycol ligand with amine derivatives of cRGD, cRAD (similar structure but with lower affinity to αVβ3 integrin) [In order to characterize the uptake of ultrasmall gold nanoparticles functionalized with cRGD and to prove that they can deliver cytotoxic cargo to cells, plasmonic GNPs with 4 nm cores were synthesized using a modified aqueous Brust-Schiffrin method ,50,51. Tntegrins . A similntegrins . Carbohyntegrins . Polyethntegrins ,56. Theyntegrins . The terntegrins , the 4 nntegrin) ,60, and ntegrin) ,62. Ligantegrin) ,63 . Consequently, two different versions were synthetized: HighcRGD-usGNP and LowcRGD-usGNP with approximately 60 and 20 cRGD units per usGNP, respectively. The same method was applied to obtain HighcRAD-usGNP and LowcRAD-usGNP. The base usGNPs and LowcRGD-usGNPs were functionalized with approximately 20 DM1 per GNP. 1–2 Sulfo-Cyanine5 amine was added to the usGNPs for fluorescent studies (Cy5-usGNP)  .Different analytical techniques were employed to characterize the core and overall size of the usGNPs. UV-Vis spectroscopy displayed a surface plasmon band (SPR) with a local maximum of around 520 nm a. Transm1H NMR, 1H NMR and LC analyses were performed after core etching and ligand release using a solution of potassium cyanide in potassium hydroxide (KCN/KOH) [1H NMR and LC analyses were then fitted in the previously mentioned model of Vergara et al. [2 and PEG(8)COOH in the corona of the Base usGNP was measured by both 1H NMR and LC-CAD. Equally, both techniques validated the high (60 per GNP) and low (20 per GNP) loading of the peptides. Sulfo-Cyanine5 amine loading was only measured by LC-CAD (1–2 Fluorophores per GNP). DM1 quantification in mg/mL was converted into equivalents per GNP (20 DM1 per GNP). The results presented above suggest a significant improvement on our previous GNP design based on a 2 nm core that can carry eight SIKVAV ligands per 200 gold atoms [Proton Nuclear Magnetic Resonance 1H NMR,  and LiquKCN/KOH) ,65. For KCN/KOH) ,67. Absoa et al.  with a tld atoms . The sliHighcRGD-usGNP and control base usGNP were used to assess the specificity of binding to the immobilized αVβ3 integrin. HighcRGD-usGNP showed robust binding with a Kd of 29.2 ± 3.2 pM (p < 0.01) and a Hill coefficient (h) of 1.11 ± 0.25 (p < 0.05). In the absence of cRGD, the binding of 1 nM control base usGNP to the immobilized αVβ3 integrin was unmeasurable, suggesting the absence of non-specific binding to αVβ3 integrin of any of the components of the base particle . As expected, the LowcRGD-usGNP had a weaker affinity dropping approximately 100-fold (d of 3.2 ± 0.4 nM (p < 0.001) and h of 0.44 ± 0.03 (p < 0.001). The negative cooperative binding highlighted by the Hill coefficient <1 is likely due to electrostatic repulsion between nanoparticles densely bound to the sensor, which becomes apparent at high usGNP concentrations. Importantly, the data on LowcRGD-usGNPs suggest that high-affinity binding (3.2 nM) is still possible with the lower cRGD loading, therefore leaving room for the conjugation of a drug payload.αVβ3 integrin binding and release of cRGD functionalized usGNPs were measured with biolayer interferometry . First, particle a. The ve100-fold b with a 2+ dependent as evidenced by the absence of binding in the presence of 10 mM EDTA  similar to that found for the base usGNP (~105 usGNPs per cell) confirming the target specificity . HEK-293 cells express the αV integrin subunit, which forms the RGD binding integrin heterodimers pairing with a β subunit [HighcRGD-usGNP treated clone 5 cells, nevertheless, we observed no unusual cell death of the untreated CD61-HEK-293 cells.The dependency of uptake on αVβ3 integrin expression by host cells was established using the verified cell line pairs and experiments using confocal microscopy and gold content by inductively coupled plasma atomic emission spectroscopy (ICP-AES). As expected, the uptake of cRGD-loaded usGNPs by the U-87 MG cell line was visible after 1 h, while no uptake was seen by the U-251 MG cell line c,d. Surp subunit . Of thes subunit . RGD bin subunit , which c subunit . These iThese data strongly indicate that the uptake of usGNPs is cRGD-dependent, possibly through an endocytic mechanism mediated by binding to the αVβ3 integrin.Integrin-mediated endocytosis occurs via several distinct pathways . In ordeHighcRGD-usGNPs and LaCer together for 2 h. Uptake of HighcRGD-usGNPs was inhibited by CPH but not by genistein, while uptake of the LaCer control was inhibited by genistein and not by CPH  amiloride (EIPA) was also found [To assess which endocytic pathway was responsible for the internalization of usGNPs, U-87 MG cells were pre-treated with CPH, genistein, and nocodazole, a microtubule poison, and inhibitor of intracellular trafficking , or leftt by CPH a–d. Nocond LaCer e, similaso found . An uptaso found  and was so found , which hHighcRGD-usGNP were followed in live cell imaging in U-87 MG cells labeled with LysoTracker® Red 15 min before microscopy. The presence of usGNPs in lysosomes, indicated by co-localization of the LysoTracker® Red and Cy5 signals, became obvious after 30 min of incubation and reached a maximum after 120 min  for free DM1, to 30.1 nM (95% CI: 25–36.3 nM) equivalent concentration for DM1-loaded usGNP . However, for the αVβ3 integrin expressing U-87 MG cell line  to 5 nM (95% CI: 4.2–5.9 nM) DM1 equivalent concentration, which is comparable to that of free DM1 in Hep3B cells and only moderately higher than the IC50 of free DM1  in U-87 MG cells. These data indicate that the presence of the cRGD peptide can be used to selectively target a DM1-loaded usGNP for cells expressing the αVβ3 integrin, providing a mechanism for the selective killing of cells and reducing off-target effects. Lastly, we tested the ability of our functionalized usGNPs to deliver cytotoxic payload preferentially to αVβ3 expressing cells. Cytotoxicity of DM1 functionalized base usGNPs and ed usGNP a. Functiell line b, cRGD fTargeted delivery of cytotoxic drugs increases the therapeutic window by improving biodistribution, preventing off-target effects, or reducing drug degradation and elimination. Ultrasmall gold nanoparticles offer an improved surface-to-volume ratio that allows the immobilization of a larger number of different ligands at the same dose of gold, which presents an ideal platform for multiple functionalizations. Our strategy to link cRGD sidechains to the usGNP core via PEG(8)COOH lead to the successful binding of the functionalized usGNP to αVβ3 integrin both in vitro and in cell culture. This binding was selective and specifically proven by competition with free cRGD, functionalization with the non-interacting cRAD peptide, or using cell lines that do not express the αVβ3 integrin. The potential for multiple functionalization was shown by coupling fluorescent moieties to the usGNPs via the same PEG(8)COOH bridge, which permitted direct visualization of the usGNPs with confocal microscopy and monitoring their uptake and intracellular localization. Loading the maytansinoid drug DM1 onto the cRGD-usGNPs led to an improvement in selective toxicity on cells that express αVβ3 integrin and proved the potential of multi-functionalized usGNPs in improving the therapeutic window.