This study evaluated the shape memory and tensile property of 3D-printed sinusoidal sample/nylon composite for various thickness and cycles. Sinusoidal pattern of five thicknesses: 0.2 mm, 0.4 mm, 0.6 mm, 0.8 mm, and 1.0 mm were 3D-printed on nylon fabric by the fused deposition modeling (FDM) 3D printer using shape memory thermoplastic polyurethane (SMTPU). Afterward, shape memory and tensile property was investigated up to 50 shape memory cycles. The study found that 3D-printed sinusoidal sample/nylon composite had a 100% shape recovery ratio for various thicknesses up to 50 cycles. The average shape recovery rate gradually decreased from 3.0°/s to 0.7°/s whereas the response time gradually increased with the increase of a 3D-printed pattern thickness. The stress and initial modulus gradually increased with the increase of the cycle’s number. Thus, the shape memory property had a similar tendency for various cycles whereas the tensile property gradually increased with the increase of the cycle number. Moreover, this study demonstrated that this 3D-printed sinusoidal sample/nylon composite can go through more than 50 cycles without losing its tensile or shape memory property. This 3D-printed sinusoidal sample/nylon composite has vast potential as smart, reinforced, and protective clothing that requires complex three-dimensional shapes.  The deformed shape becomes permanent at temperature below (Tg) and the applied stress is stored inside. Upon reheating, the SMP releases its stored force and recovers its original shape [The shape memory polymer (SMP) belong to a class of very smart materials, which have the ability to remember and recover their original shape when an appropriate stimulus such as temperature, humidity, pH, light, magnetic field, and electric field is applied. Temperature-responsive shape memory materials can be easily deformed with external force by heating over its glass transition temperature , stereolithography (SLA), digital light processing (DLP), selective laser sintering (SLS), etc. Among them, FDM is the simplest and most inexpensive technology where the polymeric filament is melted by a nozzle and deposited on the printing bed to create a 3D object ,8,9. RecIn our former studies, 3D-printed auxetic sinusoidal pattern/nylon composite was obtained. Afterward, the mechanical property of this composite fabric and Poisson’s ratio of 3D-printed auxetic pattern was studied . Our latThe filament used in this study was shape memory polymer thermoplastic polyurethane (below SMTPU), which was purchased from SMP Technologies Inc. . The selected SMTPU filament was thermal-responsive SMP with a glass transition temperature of 55 °C and the melting temperature was 205 ~ 215 °C. The diameter of the filament was 1.75 mm and transparent in color. The selected fabric was 100% nylon plain woven fabric with a 0.1-mm thickness. The numbers of yarns per cm were 37 × 31 for warp and weft, respectively, and the fineness of yarn was 9.5 tex × 9.5 tex, respectively, for warp and weft. The 3D-printing was carried out using the fused deposition modeling (below FDM) 3D printer . The 3D-printer was equipped with a nozzle 0.4 mm in diameter.At first, a conceptual 2D sinusoidal design was developed with a repeating unit of 20 mm × 20 mm. This repeating unit was continuously combined and transformed to a sinusoidal pattern of 150 mm × 150 mm. This sinusoidal pattern was given a 3D form by Autodesk 123D as stl file. This stl file was later transferred to a 3D printable G-code file of various thicknesses such as 0.2 mm, 0.4 mm, 0.6 mm, 0.8 mm, and 1.0 mm by Cubicreator 3.1.2.3D printable G-code file was transferred to the 3D-printer and then 3D-printing of sinusoidal pattern with various thicknesses was carried out on nylon fabric using the SMTPU filament. During 3D-printing, the temperature of the nozzle was set to 230 °C and bed temperature was set to room temperature. Printing speed and filling were 50 mm/s and 100%, respectively. The layer height was 0.2 mm. The process of 3D-printing the sinusoidal sample with various thicknesses on nylon fabric is shown in g because it had higher intensity than a peak at 100 °C did [Heating-cooling process of 3D-printed sinusoidal sample/nylon composite with various thicknesses and various cycles is shown in 0 °C did . This trSurface morphology of a 3D-printed sinusoidal sample/nylon composite with various thicknesses was carried out using fabric image analysis microscopy . The surface images of the samples were taken with ×6.5 and ×23.5 magnifications. r) of 3D-printed sinusoidal sample/nylon composite with various thicknesses and various cycles was calculated up to 50 cycles based on Equation (1). The test was carried out three times and the mean value was taken.r = shape recovery ratio, Ɛ = angle at STEP-2 or temporary shape, Ɛrec = angle at STEP-3 or after recovery.For measuring the shape recovery ratio, a rectangular sample of 75 mm (length) × 25 mm (width) and various 3D-printed sinusoidal pattern thicknesses such as 0.2 mm, 0.4 mm, 0.6 mm, 0.8 mm, and 1.0 mm were obtained. At first, the angle of STEP-1 was measured using a fabric crease recovery tester. Then, the specimen was heated inside an oven  and changed the shape to 90°, which was STEP-2. After cooling down, the sample was reheated and, after recovery, the angle was measured again by a fabric crease recovery tester at STEP-3. The shape recovery ratio (Rt) of 3D-printed sinusoidal sample/nylon composite with various thicknesses and various cycles was obtained up to 50 cycles using the following formula (2).t = shape recovery rate, Ɛ = angle at STEP-2 or temporary shape, Ɛrec = angle at STEP-3 or after recovery, t = total time taken for recovery.The shape recovery process of a 3D-printed sinusoidal sample/nylon composite during reheating inside the oven  was recorded using a video camera . Total time taken by a sample for recovery was measured from the video. A protractor was placed on the back of the sample inside the oven to mark the starting and ending point. The total recovery angle was also found from that video. The sample size was 75 mm (length) × 25 mm (width) and various 3D-printed sinusoidal pattern thicknesses such as 0.2 mm, 0.4 mm, 0.6 mm, 0.8 mm, and 1.0 mm. The shape recovery rate . The sample size was 75 mm (length) × 25 mm (width) and various 3D-printed sinusoidal pattern thicknesses such as 0.2 mm, 0.4 mm, 0.6 mm, 0.8 mm, and 1.0 mm. During the test, the maintained clamp length and extension speed was 25 mm and 50 mm/min, respectively. The test was recorded using a video camera . The stress-strain curve for 50 cycles was obtained. The recorded videos, the sample appearance before and after the tensile test, and the stress-strain curves were analyzed to obtain the tensile behaviour.Tensile properties of a 3D-printed sinusoidal sample/nylon composite with various thicknesses and various cycles up to 50 cycles were obtained from the tensile test done by a constant rate of extension machines. Based on the stress-strain curve, stress, strain, and initial modulus were obtained for 50 cycles. The test was conducted three times and the mean value was used.g), the soft segment gains kinetic energy transforms from a glassy state to a rubber state and becomes easy to deform. Under an external force, SMTPU can be deformed to any shape. By cooling, the deformed shape becomes permanent due to the restricted mobility of the soft segment. The external force is stored inside. Upon reheating, the SMTPU releases its stored force and recovers its original shape [al shape . In this work, a 3D-printed sinusoidal sample/nylon composite of various thicknesses were flat and 0° at STEP-1 or at the original shape. The specimens were then heated 70 °C for 5 min and deformed to 90°. This deformed shape or STEP-2 was fixed after cooling down to room temperature. After reheating at 70 °C for 5 min, all the specimens came back to flat form or 0° again at STEP-3. As a result, the shape recovery ratio for cycle-1 of all the specimens: 0.2SM/NF, 0.4SM/NF, 0.6SM/NF, 0.8SM/NF, and 1.0SM/NF were 100%. Then STEP-3 of cycle-1 was considered as the STEP-1 of cycle-2. In cycle-2, the samples were heated. The shape was changed and reheated following the same condition as cycle-1. In cycle-2, the shape recovery ratio also found 100%. Then, the same procedure was carried on up to 50 cycles. In each cycle, the specimens recovered back to 0° or flat shape. Hence, in each cycle, the shape recovery ratio was found to be 100%. A visual demonstration of shape change at three STEPs and a corresponding shape recovery ratio of the 3D-printed sinusoidal sample/nylon composite with various thicknesses and various cycles are presented in g and achieve the rubbery state. Therefore, it can quickly release the stored force and recover quickly [The shape recovery rate of a 3D-printed sinusoidal sample/nylon composite with various thicknesses and various cycles for 50 cycles is presented in  quickly . The resMoreover, it can also be seen from g. Once the specimen reaches Tg, it starts to recover by releasing stored force. However, at the initial stage, the release of the stored force is slow due to the heavy friction among molecules. The recovery is slow at the initial stage. At the final stage, the shape recovery rate became slow once again because most of the stored force had been released with little of it remaining [emaining ,25.During the first cycle, the specimen took 7 s, 16 s, 29 s, 42 s, and 45 s, respectively, for 0.2SM/NF, 0.4SM/NF, 0.6SM/NF, 0.8SM/NF, and 1.0SM/NF for recovering 0° to 10° angle. Then 0.2SM/NF, 0.4SM/NF, 0.6SM/NF, 0.8SM/NF, and 1.0SM/NF took 16 s, 18 s, 23 s, 30 s, and 33 s, respectively, for recovering 10° to 80°. Lastly, 0.2SM/NF, 0.4SM/NF, 0.6SM/NF, 0.8SM/NF, and 1.0SM/NF took 7 s, 6 s, 13 s, 28 s, and 32 s, respectively, for 80° to 90° recovery. It was noticed that all the specimens took less time for recovering a maximum portion from 10° to 80° than time taken by the combination of the initial and final 20° recovery. Comparing between initial and final 10°, it was also noticed that the final stages took less time than initial stages did. The same tendency was found for a 3D-printed sinusoidal sample/nylon composite with various thicknesses and various cycles up to 50 cycles. During the 50th cycle, initially, the time taken by 0.2SM/NF, 0.4SM/NF, 0.6SM/NF, 0.8SM/NF, and 1.0SM/NF were 11 s, 18 s, 27 s, 35 s, and 42 s, respectively, for recovering a 0° to 10° angle. Afterward, 0.2SM/NF, 0.4SM/NF, 0.6SM/NF, 0.8SM/NF, and 1.0SM/NF took 16 s, 20 s, 23 s, 40 s, and 41 s, respectively, for recovering 10° to 80°. Lastly, 8 s, 7 s, 15 s, 40 s, and 47 s were taken by 0.2SM/NF, 0.4SM/NF, 0.6SM/NF, 0.8SM/NF, and 1.0SM/NF, respectively, for recovering 80° to 90°. Therefore, during the recovery maximum, recovery took place during the middle stage, which was followed by the final stage and the initial stage. This tendency remained similar for various cycles from cycle-1 to cycle-50. However, for 0.2SM/NF, the differences of the recovery angle on time looks like a straight line. From 0.4SM/NF, the differences of the recovery angle on the time graph started turning into an ‘S’ shape and the ‘S’ shape became more prominent as the thickness of the 3D-printed sinusoidal pattern increased. This is because, when the 3D-printed sinusoidal pattern thickness was increased, the initial and final stages became longer by taking more time for some recovery. Thus, this study confirmed that shape recovery of a 3D-printed sinusoidal sample/nylon composite with various thicknesses happened with three stages and this tendency remained similar for various cycles. However, when the thickness of a 3D-printed sinusoidal pattern was increased, the initial and final stages took more time and three stages became more prominent.g). As a result, it took more time to start recovery [Differences of response time on thickness for a 3D-printed sinusoidal sample/nylon composite with various thicknesses are shown in recovery . This stDuring the tensile test, three samples were tested three times and mean value was taken. However, in this case, the appearance of one sample is presented to make the presentation concise and precise. Tensile behavior showed the nature of failure of 3D-printed sinusoidal sample/nylon composites during the tensile test. As shown in This research has shown the viability of producing a 3D-printed sinusoidal sample/nylon composite with various thicknesses by FDM 3D-printing technology using the SMTPU filament. However, when using this technology as a reliable method, it was a prerequisite to fully understand the shape memory property and tensile property for numerous shape memory cycles. The 3D-printed sinusoidal sample/nylon composite showed a 100% shape recovery ratio for various thicknesses and various cycles up to 50 cycles. The shape recovery rate gradually decreased with the increase of the 3D-pattern thickness. The average shape recovery rates were 3.0°/s, 2.0°/s, 1.4°/s, 0.8°/s, and 0.7°/s for 0.2SM/NF, 0.4SM/NF, 0.6SM/NF, 0.8SM/NF, and 1.0SM/NF, respectively, for 50 cycles. During the shape recovery process, most of the recovery happened rapidly in the middle stage, but the recovery rate was slower at the beginning and ending stages. Response time and thickness confirmed that a thicker sample took more time and a thinner sample took less time to respond or undergo start recovery. The response time for 0.2SM/NF, 0.4SM/NF, 0.6SM/NF, 0.8SM/NF, and 1.0SM/NF were found to be 1.8 ± 0.7 s, 6.8 ± 1.5 s, 15.6 ± 4.0 s, 32.9 ± 7.0 s, and 37.1 ± 4.2 s, respectively. Thus, the shape memory property of a 3D-printed sinusoidal sample/nylon composite had a similar tendency for various cycles. To assess the effect of the cycle number, the tensile property was investigated. Tensile behaviour indicated that the stress-strain phenomenon during the tensile test was a three-stage process. During the tensile test, due to the lower strength and elongation property of nylon fabric, it got separated from the sinusoidal pattern first. This was followed by the breakage of nylon fabric and then the failure of a 3D-printed sinusoidal pattern took place. The stress and initial modulus of the 3D-printed sinusoidal sample/nylon composite increased with the rise of the number of shape memory cycles. Thus, the tensile property increased with the rise of the shape memory cycle number. Therefore, combing the advantages of this excellent shape memory property and good mechanical properties, such as a 3D-printed sinusoidal sample/nylon composite have a great potential application for creating smart textile, smart garments, smart, and protective clothing that requires repetitive shape changing. Since the usage of the shape memory polymer allows the object to obtain any shape at an elevated temperature and became fixed after cooling. This 3D-printed sinusoidal sample/nylon composite could be used as protective clothing in curvy parts of our body such as knee, elbow, etc., which requires a three-dimensional shape.


Its GLP-1 secretory activity was approximately four times higher than the positive control (10 mM glutamine). The <3 kD fraction contributed significantly to the anti-hyperglycemic activity of Protamex-derived hydrolysates, and this activity was stable after simulated digestion. Our results suggest that green crab hydrolysates obtained by Protamex treatment have the potential for type 2 diabetes management and could be incorporated in food products as a health-promoting ingredient.The predation and burrowing activity of invasive green crabs have had detrimental effects on important marine resources and habitats. Our objective is to develop bioactive hydrolysates by enzymatic proteolysis of underutilized green crab. Mechanically separated mince was hydrolyzed with Alcalase, Protamex, Flavourzyme, and Papain (1%) for 60 min. Subsequently, the hydrolysates were introduced to a simulated gastrointestinal digestion model. Selected samples were fractionated by ultrafiltration, and their anti-hyperglycemic effects including α-glucosidase, α-amylase, and dipeptidyl peptidase-IV (DPP-IV) inhibitory activities and glucagon-like 1 (GLP-1) secretory activity were evaluated. The Protamex treatment showed the highest α-glucosidase inhibitory activity (IC In recent years, consumer interest in sustainability and healthy lifestyles has significantly increased, leading to higher demand for nutritious and environmentally-friendly food products among consumers and the food industry. The development of natural, bioactive food ingredients from currently underutilized food resources may positively contribute to satisfying this demand. Nutritional and bioactive food ingredients include proteins and peptides, polyunsaturated fatty acids (PUFAs), phytochemicals, fibers, probiotics, and prebiotics . Marine Carcinus maenas) are an invasive species well established in the U.S. that negatively influence fisheries, aquaculture, and marine habitats due to their high resilience, voracious predation, and strong burrowing activity [European green crabs  and docosahexaenoic acid (DHA) . Green cType 2 diabetes mellitus (T2DM) is one of the most prevalent chronic diseases in the world and ranks in the top 10 diseases causing the most deaths worldwide (WHO) . T2DM anGreen crabs (carapace width: 40–85 mm and weight: 20–155 g) were harvested in 2018 on the Back River in Georgetown, Maine, USA. All of the chemicals and reagents that were used in this study were supplied by Fisher Scientific , Thermo Fisher Scientific , Bio-Rad Laboratories , and Sigma-Aldrich  unless otherwise described. Alcalase 2.4 L (AL), Protamex (PR), Flavourzyme (FL), and Papain (PA) were purchased from Sigma-Aldrich . Rat intestinal acetone powders of α-glucosidase (EC 3.2.1.20), porcine pancreatic α-amylase (EC 3.2.1.1), pepsin from porcine gastric mucosa , pancreatin from porcine pancreas , p-nitrophenyl-β-D-glucopyranoside (p-NPG), and Gly-Pro-p-nitroanilide were purchased from Sigma-Aldrich . Porcine kidney dipeptidyl peptidase-IV (DPP-IV) (E.C. 3.4.14.5) and glucagon-like 1 (GLP-1) ELISA kit were purchased from EMD Millipore . Cell culture materials including penicillin-streptomycin (0.1g/L), Dulbecco’s modified Eagle’s medium (DMEM) containing low glucose, Glutamax, trypsin/EDTA (10×), and Poly D-lysine were obtained from Gibco Life Technologies . Fetal bovine serum (FBS) was purchased from Atlanta Biologicals . Bovine serum albumin, acarbose, sitagliptin, and glutamine were obtained from Sigma-Aldrich .3/100 in2 oxygen transmission rate, 80 microns, UltraSource, Kansas, MO, USA). The crab mince was blast-frozen at −30 °C for 1 h and then stored at 20 °C until further use  to separate meat and shell streams, and then the minced meat was vacuum sealed in plastic bags  ratio, then homogenized for 1 min in a Waring blender at maximum speed. The enzymatic hydrolysis was conducted at the optimum pH and temperature of each enzyme including Alcalase , Protamex , Flavourzyme , and Papain . Protein content was determined based on total nitrogen content as analyzed by a combustion analyzer . Protein content was determined using a protein conversion factor of 6.25. Enzyme was added to the homogenate based on substrate (protein) content (1:100 = E:S), and the mixture was hydrolyzed for 60 min. During hydrolysis, the pH of the mixture was maintained using 6 N HCl or 6 N NaOH and temperature was monitored. After hydrolysis, the mixture was heated at 85–90 °C for 10 min to inactivate the enzyme and then immediately cooled on ice. For the crab mince control (CMC), mince was homogenized with water but not subsequently treated with the enzyme. Subsampling was performed to evaluate the degree of hydrolysis, and then the mixtures were centrifuged at 19,722× g for 15 min at 4 °C, and the supernatants were collected. All of the treatments were processed in triplicate. The collected supernatants were blast-frozen at −30 °C for 1 h, then freeze-dried  at −30 to 25 °C under 250 mT for 10 days All lyophilized supernatants were stored at −80 °C until further use.Enzymatic hydrolysis using commercially available proteases was conducted based on a modified protocol from Beaulieu et al. . The raww/v). After centrifugation at 1100× g for 10 min, 4 mL of supernatant was collected. Subsequently, the supernatant was diluted to 50 mL with deionized water. Four mL of OPA reagent were mixed with 400 µL of solubilized sample/standard (0.5 mg/mL serine) and the mixture was incubated at room temperature for 2 min. Absorbance was measured at 340 nm and the degree of hydrolysis was calculated based on the following three equations:Degree of hydrolysis was determined following the O-phthalaldehyde (OPA) method ,29. OPA W = weight of sample (g);P = protein (g/100 g sample);V1 = volume of supernatant (4 mL);V2 = volume of SDS solution (20 mL);0.05 = sample volume in L.h = number of hydrolyzed peptide bonds;α = 1.00, β = 0.40 mequiv/g protein;total) of fish: 8.6 mequiv/g protein.Total number of peptide bonds (hw/v), then pH was adjusted to pH 2 using 6 N HCl prior to heating to 37 °C. Pepsin was added to the sample mixture (4:100 = E:S protein/peptide content) and stirred at 37 °C for 60 min. Then, pH was adjusted to 7.5 using 6 N NaOH. Subsequently, pancreatin (4:100 = E:S protein/peptide content) was added for the intestinal digestion phase and the mixture was stirred at 37 °C for 2 h. To inactivate the digestive enzymes, the mixture was heated at 85–90 °C for 10 min. The mixture was freeze-dried  at −30 to 25 °C under 250 mT for 10 days and then stored at −80 °C until further use.Simulated gastrointestinal digestion of the control and crab mince hydrolysates was performed according to González-Montoya et al. . The samv/v) glycerol, 0.01% bromophenol blue, and 5% β-mercaptoethanol, then heated in a 90–95 °C water bath for 5 min [w/v) SDS) in a Bio-Rad Mini-PROTEAN III Cell . The amount that was loaded to the wells was adjusted to 1 mg for the hydrolysates, 0.5 mg for the control, and 10 µL of a protein molecular weight standard . Separated proteins were fixed in a gel fixing solution containing 50% (v/v) ethanol and 10% (v/v) acetic acid then washed with a solution consisting of 50% (v/v) methanol and 10% (v/v) acetic acid. Following washing, gel staining was performed using Coomassie blue R-250 solution at room temperature for 3 h with gentle agitation. , then the gel was destained with washing solution until the background color was removed.Molecular weight distribution was determined by SDS-PAGE. All of the samples were dissolved in a Laemmli sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 25%  for the first fraction, then centrifuged at 3234× g until the volume of retentate reached 250 µL. After collecting the retentate, the <30 kD filtrate was transferred to a 10 kD MWCO filter device for the second fraction. After centrifugation at 3234× g, 250 µL of retentate was collected. The <10 kD filtrate was added to the 3 kD MWCO filter device for the third fractionation and was centrifuged at 3234× g until the retentate volume reached 250 µL. Both the retentate and the <3 kD fraction were collected and all the hydrolysate fractions were stored at −80 °C until used for further assays.Green crab hydrolysates were fractionated using ultrafiltration based on molecular weight. A volume of 15 mL of solubilized sample (50 mg/mL) was added into a 30 kD molecular weight cut-off (MWCO) filter device  and 100 µL of enzyme solution were added in a 96 well plate then incubated at 37 °C for 10 min. Then, 50 µL of 5 mM p-NPG solution in 0.1 M phosphate buffer (pH 6.9 with 0.9% NaCl) was added and the mixture was incubated at 37 °C for 30 min. The absorbance was measured at 405 nm by a microplate reader  and compared with a control containing 50 µL of 0.1 M sodium phosphate buffer in place of the sample. The α-glucosidase inhibitory activity was calculated as follows:Rat intestine α-glucosidase inhibition assay was conducted according to the protocol in the Worthington Enzyme Manual with modifications  and KwonThe α-amylase inhibitory activity was evaluated following a modified version of the assay described by the Worthington Enzyme Manual  and KwonThe DPP-IV inhibitory activity was determined using a method from Li-Chan et al. . DPP-IV v/v) fetal bovine serum, 100 U/mL penicillin-streptomycin, and 2 mM L-glutamine in a 75 cm2 culture flask at 5% CO2 and 37 °C and GLUTag cells at 80% confluency were regularly trypsinized for subculture.Murine GLUTag cells that are widely used for the stimulation of GLP-1 secretion were used in this study. GLUTag cells were gifted from Dr. D.J. Drucker  and cultured in Dulbecco’s Modified Eagle Medium  containing 5.5 mM glucose (pH 7.4) supplemented with 10%  was added to the cells and incubated for 24 h. Then, the mixture was replaced with 100 µL of 0.5 mg/mL MTT reagent and incubated at 37 °C and 5% CO2 for 4 h. The MTT reagent was removed, then the precipitate was solubilized in 100 µL of DMSO. The absorbance was measured at 540 nm by a microplate reader  and compared with a control containing 20 µL of ultrapure water in place of the sample solution. The cell viability was calculated as follows:Viability of the GLUTag cells was evaluated using the MTT -2,5-diphenyl tetrazolium bromide) assay. The surface of 96-well culture plates was coated with poly-D-lysine solution. A density of 2 × 105 in 24-well culture plates coated with poly-D-lysine and grown for 48 h in a 37 °C and 5% CO2 incubator until the confluency reached 80%. The cells were washed with phosphate-buffered saline (PBS), then 1 mL of Kreb Ringer Buffer (KRB) containing 115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM HEPES, and 1M NaHCO3 supplemented with 1% bovine serum albumin  and 1 mM glucose (pH 7.4) was added. After pre-incubation at 37 °C and 5% CO2 for 45 min, the KRB was replaced with 1 mL of appropriate KRB test solutions containing sample or 10 mM L-glutamine (positive control). All the KRB test solutions were prepared with supplementation of 1% BSA and 2 mM glucose (pH 7.4). The culture plates were incubated at 37 °C and 5% CO2 for 2 h, then the mixture was collected. After centrifugation at 4 °C and 800× g for 5 min, the supernatant was collected and stored at −80 °C until further evaluation of GLP-1 concentration. Total GLP-1 concentration was determined based on the manufacturer’s instructions in a commercial ELISA kit . The GLP-1 secretory activity was expressed as a percentage (%) of the negative control (KRB buffer).GLP-1 secretory activity was evaluated by an assay modified from Ojo et al. . GLUTag t-test with a significance value of p < 0.05 . Correlations (p < 0.05) between the degree of hydrolysis (DH) and each biofunctional activity were analyzed through the Pearson coefficient .The enzymatic hydrolysis process using each of the four commercial proteases was replicated three times and all of the assays were conducted in triplicate on each sample replicate. Statistical differences among the means of each treatment were evaluated using one-way analysis of variance (ANOVA) followed by Tukey’s HSD post hoc test and paired The degree of hydrolysis (DH) was determined by the OPA method with serine as a standard. DH was significantly increased after enzymatic hydrolysis for 60 min. DH of green crab hydrolysates ranged from 15.8% to 18.4%, while DH of the CMC (crab mince control) was 6.8%. Among the treatments, AL showed the highest DH (18.4% ± 0.4%) followed by the PR (17.1% ± 0.2%), FL (16.5% ± 1.2%), and PA (15.8% ± 0.3%) treatments, respectively .The size distribution of proteins and peptides in the CMC and hydrolysates was determined using SDS-PAGE a. Signif50 values of both α-glucosidase and α-amylase were determined to evaluate the efficacy of the inhibition. Acarbose was used as a positive control. Acarbose had IC50 values of 0.027 mg/mL and 0.84 mg/mL for α-glucosidase and α-glucosidase inhibitory activity, respectively. The α-glucosidase inhibitory activity of the CMC was significantly improved by the PR (IC50 1.38 ± 0.19 mg/mL) and PA (IC50 5.56 ± 0.19 mg/mL) treatments while the FL (IC50 20.24 ± 0.19 mg/mL) treatment showed a decrease in α-glucosidase inhibitory activity compared to the CMC (IC50 8.54 ± 0.50 mg/mL) (50 1.49 ± 0.08 mg/mL) and PA (IC50 5.31 ± 0.51 mg/mL) treatments. However, the inhibitory activity of the FL-derived hydrolysate was significantly improved after the simulated digestion (IC50 10.61 ± 1.90 mg/mL). The green crab hydrolysates obtained from PR showed the highest α-glucosidase inhibitory activity before and after simulated digestion and the activity was approximately 4–14 times higher compared to other enzyme treatments and CMC based on IC50 values.The inhibitory activity of rat intestinal α-glucosidase and porcine pancreatic α-glucosidase was assessed to evaluate the potential anti-hyperglycemic effect of the CMC and crab hydrolysates. IC0 mg/mL) a. Simula50 9.35 ± 0.42 mg/mL) exhibited the highest α-amylase inhibitory activity followed by PR (IC50 11.02 ± 0.69 mg/mL), FL (IC50 11.12 ± 0.37 mg/mL), AL (IC50 12.53 ± 0.85 mg/mL), and the CMC (IC50 16.49 ± 0.41 mg/mL), respectively (50 10.55 ± 0.68 mg/mL) and its hydrolysates treated with AL (IC50 10.11 ± 0.34 mg/mL), PR (IC50 9.93 ± 0.82 mg/mL), and FL (IC50 9.31 ± 0.37 mg/mL), however, the inhibitory activity of PA-derived hydrolysate was not affected by simulated digestion. After simulated digestion, the α-amylase inhibitory activity among the treatments was not significantly different.The porcine pancreatic α-amylase inhibitory activity of the CMC was statistically increased by enzymatic hydrolysis using commercial enzymes. Among the treatments, PA  was significantly improved by the enzymatic hydrolysis, and the DPP-IV inhibitory activity IC50 values of hydrolysates ranged from 0.56 mg/mL to 0.72 mg/mL . SitagliThe GLP-1 secretory activity was measured at a sample concentration of 0.5 mg/mL. The cytotoxicity of the CMC and hydrolysates was determined using the MTT assay before evaluating their GLP-1 secretory activity. As a result, there was no observed cytotoxicity on GLUTag cells at 0.125, 0.25, and 0.5 mg/mL . The GLP50 values could only be calculated for the <3 kD fraction since the highest inhibitory activity of the >3 kD fractions was less than 50%. The PR treatment (IC50 1.75 ± 0.24 mg/mL) resulted in the highest α-glucosidase inhibitory effect followed by PA, CMC, and FL.Based on the positive results of their α-glucosidase and GLP-1 secretory activities, PR, FL, and PA were selected for subsequent fractionation. 50 values of all the <3 kD fractions ranged from 8.36 to 10.88 mg/mL and were not significantly different among the treatments.The α-amylase inhibition of all the fractions was evaluated at 4.2 mg/mL . The <3 50 values, the <3 kD fraction  and 3–10 kD fraction  within each sample showed the highest DPP-IV inhibitory activity while the > 30 kD fraction  exhibited the lowest DPP-IV inhibitory activity within enzyme treatments. The DPP-IV inhibitory activity of all the fractions was improved after the commercial enzyme treatments; however, there was no significant difference among the treatments.The DPP-IV inhibitory ability of the fractions obtained from the fractionation of the ±CMC and hydrolysates is shown in After the fractionation, the total GLP-1 level induced by the CMC and crab hydrolysates (0.5 mg/mL) was evaluated using GLUTag cells. The GLP-1 secretory activity of fractions was improved by the enzymatic hydrolysis except for the >30 kD fraction of PA-derived hydrolysate and <3 kD fractions of FL and PA-derived hydrolysates . GlutamiIn recent years, as interest in healthy lifestyles has increased, the demand for healthy diets, natural food ingredients, and bioactive compounds has intensified in consumers as well as the food industry. Proteins and peptides are being widely used as nutraceutical food ingredients in food products and dietary supplements to enhance human health and product value. Many bioactive peptides having antioxidant, anti-hypertensive, and anti-hyperglycemic effects have been derived from enzymatic hydrolysis of protein sources including meat, dairy, fish, and their byproducts ,37,38,39The bioactive properties of peptides obtained by enzymatic hydrolysis are influenced by (1) the primary amino acid sequence of the protein source, (2) the specificity of the proteases applied, and (3) process parameters such as DH, hydrolysis duration, and enzyme:substrate ratio. As a primary protein source, underutilized green crab was used, which contains ~80% protein in its meat . Manual In addition to the increase in DH, the molecular weight distribution of the CMC and hydrolysates before the simulated digestion also confirms that the enzymatic process was successful, and that different products were generated based on the commercial protease applied . Althougp > 0.05) with DH. The IC50 values of the control and the hydrolysates were strongly correlated with the <3 kD fractions , which indicates that the peptides in the <3 kD fraction are key contributors to the α-glucosidase inhibitory activity. The α-glucosidase inhibitory activity of PR-treated sample (IC50 1.38 ± 0.19 mg/mL) was remarkably higher than not only other treatments and the control but also the hydrolysates of sardine muscle (IC50 48.7 mg/mL), whey protein isolate (IC50 4.5 mg/mL), and edible insects (IC50 > 2.0 mg/mL) including mealworm larvae, crickets, and silkworm pupae [Pancreatic α-amylase and intestinal α-glucosidase are the two key enzymes involved in starch digestion, resulting in an increase in blood glucose levels. Therefore, the inhibition of both carbohydrases can be an indicator of the potential anti-hyperglycemic effect . Accordirm pupae ,45. In mrm pupae ,45,46. I50 <1 mg/mL) leads to the presence of non-digested polysaccharides in the large intestine, which causes side-effects including severe stomach pain, constipation, and diarrhea [The enzymatic hydrolysis by commercial enzymes, and subsequently by digestive enzymes, improved the α-amylase inhibitory activity of the green crab proteins. The inhibitory activity was most likely primarily due to the <3 kD fractions  since thdiarrhea . Therefodiarrhea .p > 0.05) correlated with DH. Recent studies on DPP-IV inhibitors derived from food sources reported that peptides containing Pro, Ala, and Gly at the P1-position and/or Trp at the N-terminal might have effective DPP-IV inhibitory activity [50 values to the hydrolysates before the simulated digestion (50 0.56–1.5 mg/mL) was significantly lower than the medication sitagliptin (IC50 43.7 ng/mL) and other dairy protein and salmon byproduct hydrolysates (IC50 < 100 µg/mL) [DPP-IV is an enzyme that rapidly metabolizes incretins such as active GLP-1 and gastric inhibitory polypeptide (GIP) hormones. These incretins are important since the hormones help in blood glucose control by insulin secretion . DPP-IV activity . The typactivity . The <3 igestion . Thus, o0 µg/mL) ,38.GLP-1 hormone has the ability to reduce blood sugar levels by enhancing the secretion of insulin; however, it has a half-life of only ~1.5 min because of rapid inactivation by DPP-IV . The GLPEnzymatic hydrolysis was successfully applied to mechanically separated green crab mince to generate a potential anti-hyperglycemic food ingredient. Our results suggest that the anti-hyperglycemic effects of green crab hydrolysates were dependent on the type of protease applied, and not on the degree of hydrolysis. Among the proteases evaluated, Protamex generated products having the highest α-glucosidase inhibitory activity. Furthermore, the fractionation study indicated that the <3 kD peptides primarily contributed to the bioactivities of the hydrolysates. Importantly for their prospective use in novel food products, the bioactivities of the hydrolysates were stable after the simulated gastrointestinal digestion. In conclusion, Protamex was the most effective protease for obtaining anti-hyperglycemic hydrolysates from green crab mince, and the development of bioactive hydrolysates may be a viable route for developing value-added food ingredients from this underutilized marine resource. However, substantial further research including identifying the bioactive compounds in the hydrolysates, evaluating their stability in a food model, and conducting in vivo assessment of their effectiveness is required for their future commercial application in health-promoting foods.


Recent documentation shows that a curcumin-induced growth arrest of renal cell carcinoma (RCC) cells can be amplified by visible light. This study was designed to investigate whether this strategy may also contribute to blocking metastatic progression of RCC. Low dosed curcumin  was applied to A498, Caki1, or KTCTL-26 cells for 1 h, followed by exposure to visible light for 5 min . Adhesion to human vascular endothelial cells or immobilized collagen was then evaluated. The influence of curcumin on chemotaxis and migration was also investigated, as well as curcumin induced alterations of α and β integrin expression. Curcumin without light exposure or light exposure without curcumin induced no alterations, whereas curcumin plus light significantly inhibited RCC adhesion, migration, and chemotaxis. This was associated with a distinct reduction of α3, α5, β1, and β3 integrins in all cell lines. Separate blocking of each of these integrin subtypes led to significant modification of tumor cell adhesion and chemotactic behavior. Combining low dosed curcumin with light considerably suppressed RCC binding activity and chemotactic movement and was associated with lowered integrin α and β subtypes. Therefore, curcumin combined with visible light holds promise for inhibiting metastatic processes in RCC. An estimated 18.1 million new patients worldwide were diagnosed with cancer in 2018, and of these, 9.6 million people died . MetastaThe natural compound curcumin  is a comHowever, due to poor water solubility, low absorption, rapid metabolism and elimination, curcumin has low bioavailability, hampering its clinical use ,20. To iThe present study continues a previous investigation on an RCC cell model demonstrating that exposing curcumin treated cells to visible light considerably enhances curcumin’s potential to suppress tumor growth and proliferation . Since mCurcumin uptake studies were carried out using 4 µg/mL curcumin instead of 0.2 µg/mL. This was necessary, since the fluorescence intensity of 0.2 µg/mL curcumin was too low to provide high quality images for confocal microscopy and optimum fluorescence detection by FACS analysis. Using 4 µg/mL, curcumin was rapidly incorporated into the cells following administration. In A498 cells, maximum fluorescence intensity was noted after 50 min, whereas a plateau phase was reached after 40 min in Caki1 and already after 20 min in KTCTL-26 cells A. Confoc2, controls versus curcuminLight) was: 62.8 ± 5.3 versus 20.6 ± 4.1 for A498; 40.6 ± 7.0 versus 9.2 ± 2.0 for Caki1; 40.6 ± 6.7 versus 14.4 ± 3.4 for KTCTL-26. Light exposure alone or curcumin alone had no more effect on adhesion than the addition of cell medium as a control. A similar response was seen in the binding behavior to immobilized collagen. Neither curcumin nor light exposure alone led to significant alterations of A498, Caki1, or KTCTL-26 cell binding, compared to the untreated controls. Combined use of curcumin and light was associated with a distinct attenuation in the tumor cell attachment rate, with maximum effects exerted on Caki1 cells , while α1 was suppressed in A498 (−48.7 ± 2.4%) and KTCTL-26 cells (−37.0 ± 2.2%). α2 (−22.1 ± 1.8%) and α6 (−29.1 ± 1.8%) were exclusively down-regulated on KTCTL-26  and diminished A498 and KTCTL-26 chemotaxis . Blocking α5 down-regulated A498 and KTCTL-26 adhesion  and chemotaxis , whereas chemotaxis of Caki1 was enhanced (+33.7 ± 4.1%). Surface blocking of β1 suppressed adhesion of all cell lines in the order Caki1 (−89.7 ± 6.7%) > A498 (−57.1 ± 1.0%) > KTCTL-26 (−21.3 ± 3.6%), and diminished Caki1 chemotaxis (−19.7 ± 3.3%). β3 blockade down-regulated KTCTL-26 adhesion (−12.3 ± 1.0%) and A498 chemotaxis (−23.5 ± 1.9%).2O2 generation [Exposing RCC cells to visible light significantly enhanced curcumin’s potential to block adhesion and migration. While low dosed curcumin alone induced no alterations in tumor-endothelial or tumor-matrix interaction, the combination of curcumin plus light did. Photodynamic properties of curcumin are well documented, though the mechanistic background is not fully understood. In general, irradiation of a photodynamic molecule with a particular wavelength shifts electrons to higher energy orbitals. This singlet state is unstable, and the electrons return to their ground state by emitting light or heat. However, changes in electron spin can also shift a photodynamic molecule to the triplet state, which then triggers two reactions. The Type 1 reaction produces free radicals and, due to an interaction with oxygen, reactive oxygen species (ROS). The Type 2 reaction results in singlet oxygen that can interact with specific intracellular molecules . Whetherneration . Bruzellneration . In our neration . The pheneration . Niu et neration .Light seems to exert its effect in the initial attachment phase. A strong benefit of adding light to a low curcumin concentration (0.2 µg/mL) was also evident in the tumor cell-matrix attachment, where curcuminLight  down-regulated the binding of all cell lines to immobilized collagen. Herman et al. have demonstrated adhesion blocking effects of curcumin on prostate cancer cells [Low-dosed curcumin (0.2 µg/mL) combined with light profoundly blocked RCC cell adhesion to HUVEC, while even 5 μM of free curcumin without light could not alter the binding of prostate cancer cells to HUVEC . In the er cells  and otheer cells , skin [3er cells , or breaer cells  to extraLight, but not 0.2 µg/mL curcumin alone, profoundly reduced motile crawling of all three RCC cell lines. The invasion blocking effect of curcumin is important, since once metastasized, cancer is difficult to treat, and the extent of metastasis rather than the primary cancer determines survival. Therefore, application of curcuminLight might be an innovative concept to accompany established RCC treatment protocols. The relevance of curcumin alone to act on tumor cell invasion has already been shown on other tumor entities, whereby curcumin concentrations of 10 µM, 15 µM, 50 µM, or higher have been applied to stop invasion of gastric [The benefit of visual light on curcumin’s bioavailability was also seen in regard to chemotaxis and migration. 0.2 µg/mL curcumin gastric , breast  gastric , prostat gastric , or hepa gastric . OngoingLight on adhesion and chemotaxis, it is notable that curcuminLight considerably blocked adhesion of Caki1, whereas KTCTL-26 adhesion was only slightly suppressed. In contrast, migration properties of KTCTL-26 were suppressed to a maximum, migration of Caki1 was suppressed moderately. Due to these differences, lowered migration does not seem to exclusively be just a consequence of a reduced attachment rate. Rather, curcuminLight is involved in both the regulation of the mechanical tumor cell-matrix contact and modulation of cytoskeletal structures. Indeed, curcumin has been shown in tumor cell models to disorganize the architecture of actin microfilaments, leading to destabilization and a decrease in F-actin polymerization [When interpreting the influence of curcuminrization ,42. Dharrization . There irization .Light, but not in the presence of curcumin alone. Of all integrin members evaluated, surface expression of four subtypes were modified in the same manner in all cell lines; α3, α5, β1, β3 were all down-regulated by curcuminLight. Since the intracellular α3 protein content was neither reduced in Caki1, nor in KTCTL-26 and A498 cells, α3 might be shed from the surface without intracellular alteration. Shedding may also be relevant for α5 (KTCTL-26) and β1 (KTCTL-26). It is hypothesized that the β3 protein increase in Caki1 is caused by curcuminLight inducing a translocation from the cell surface to the cytoplasm.Beyond intracellular components, membrane proteins expressed on the cell surface are also relevant for controlling cell movement. Alterations of the integrin α- and β-expression pattern have been closely associated with altered metastatic activity . TherefoThe relevance of integrins in tumor progression is not completely understood. Integrin α3 is thought to be closely associated with the capacity of RCC for local and distant spread . The samLight, was also prominently involved in regulating adhesion in this cell line. The integrin α5 subtype, the major regulator of KTCTL-26 adhesion, was also considerably suppressed by curcuminLight in KTCTL-26 cells. α3 integrin served as a dominant element in reducing KTCTL-26 chemotaxis. The same integrin was also considerably suppressed by curcuminLight. These data could indicate that metastatic tumor progression is controlled by different integrin members, depending on the tumor differentiation status, and that these specific integrins act as main targets for curcuminLight. This is, however, speculative. A498 chemotaxis depended equally well on α3, α5, and β3. It must also be considered that quantitative alteration of the integrin surface expression, but not activity, was evaluated. Whether integrin loss is associated with a similar loss of activity cannot be judged.Integrin β1 also plays an important role in the development of RCC tumors and advanced RCC with metastasis , the obsLight evoked α5 inhibition would be expected to contribute to increased motile behavior, which was not the case. Rather, chemotaxis of Caki1 was considerably blocked by curcuminLight. The extent to which α5 was diminished in Caki1 by curcuminLight was only −20%, compared to an α5 reduction in A498 (−60%) and KTCTL-26 cells (−40%). Speculatively, the moderate alteration of Caki1’s α5 surface level by curcuminLight is of minor relevance for adhesion and migration. Counter regulation should be considered, and this α5 behavior in Caki1 may point to resistance induction.Suppressed integrin α3 was associated with inhibited adhesion in A498 and Caki1 cells but not in KTCTL-26 cells. Chemotaxis was inhibited in A498 and KTCTL-26, but not in Caki1 cells. Suppression of β3 exclusively prevented A498 adhesion and KTCTL-26 chemotaxis. Blockade of α5 coupled to an increased chemotaxis rate of Caki1 is paradoxic and difficult to explain, since curcuminLight is shown here to act on a set of integrin receptors which, in combination, profoundly blocks metastatic progression in vitro. This indicates that the complex process of metastasis is not controlled by only one particular integrin subtype. Rather, several integrins seem to be regulatory elements driving the invasion cascade forward. Consequently, blocking a set of relevant integrin members, as curcuminLight did, might be more effective than blocking just a single integrin.Aside from the paradoxical role of α5 in Caki1 cells, curcuminThe therapeutic potential of curcumin is also reflected by its deactivation of FAK. FAK serves as a prominent linker molecule, connecting integrin related signaling with pro-mitogenic and pro-migratory pathways including the Ras-ERK and PI3K/AKT pathway . PerformThe technical aspect of curcumin–light application has been addressed. Introducing an optical fiber into RCC tumors in mice with subsequent laser illumination of the vascular-acting photosensitizer WST11 at 750 nm or multispectrally at 700–800 nm has been shown to induce significant necrosis in RCC tissue . Kroeze 2 incubator. Subcultures from passages 5–30 were selected for experimental use.Renal carcinoma Caki1 and KTCTL-26 cell lines, both derived from a clear cell renal cell carcinoma and von Hippel-Lindau (VHL) positive, were purchased from LGC Promochem . A498 cells with disrupted VHL function were derived from Cell Lines Service . The tumor cells were grown and subcultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 1% Glutamax , 2% HEPES (2-(4-(2-Hydroxyethyl)-1-piperazinyl)-ethansulfonsäure) buffer and 1% penicillin/streptomycin , at 37 °C in a humidified 5% COHuman umbilical vein endothelial cells (HUVEC), isolated from human umbilical veins, were grown in Medium 199 , 10% FCS, 10% pooled human serum, 20 μg/mL endothelial cell growth factor , 0.1% heparin, 100 ng/mL gentamycin and 20 mM HEPES-buffer. Subcultures from passages 2 to 6 were selected for experimental use.Light; 10 × 40 W lamps, distance 45 cm, emission spectrum: 400–550 nm) using a Waldmann UV 801AL system  [Curcumin  was stored at −20 °C and diluted prior to use in cell culture medium to a final concentration of 0.2 µg/mL (0.54 µM). 4 µg/mL curcumin was used to provide high quality images for confocal microscopy and optimum fluorescence detection by FACS analysis. Cells were treated with curcumin for 1 h and then exposed to visible light for 5 min with 5500 lx (curcuminGermany) . To prev4 RCC cells were plated on 6-well multiplates  and, when grown to sub-confluency, incubated with 4 µg/mL curcumin for different time periods ranging from 10 to 60 min at 37 °C. Thereafter, the tumor cells were detached, washed three times with PBS (Ca2+ and Mg2+) and subsequently added to FACS-buffer  at 0.5 × 105 cells/mL. Fluorescence intensity  of curcumin exposed versus non-exposed cells was then measured by a FACS Canto  at an absorption of 485 nm and emission of 514 nm.5 × 10To evaluate intracellular localization of curcumin, tumor cells were incubated with 4 µg/mL curcumin for 60 min, washed with PBS, fixed in cold (−20 °C) methanol/acetone (50/50 v/v) and then washed with blocking buffer (0.5% BSA in PBS). To prevent photobleaching of curcumin, tumor cells were embedded in Vectashield mounting medium including DAPI , and viewed using a confocal laser scanning microscope  with a plan-neofluar × 63/1.3 oil immersion objective.6 cells were added to the HUVEC monolayer. After 0.5, 1, or 2 h, non-adherent tumor cells were washed off using warmed (37 °C) PBS+ (Ca2+ and Mg2+). The remaining cells were fixed with 1% glutaraldehyde. Adherent tumor cells were then counted in five different observation fields of a defined size (5 × 0.25 mm2) using a phase contrast microscope and the mean cellular adhesion rate was calculated.To evaluate tumor cell adhesion, HUVEC were transferred to 6-well multiplates in complete HUVEC medium. Once the cells had reached confluence, A498, Caki-1, or KTCTL-26 cells were detached from the culture flasks by accutase treatment , and 0.5 × 106 tumor cells were then added to each well and allowed to attach for 60 min at 37 °C. Subsequently, non-adherent tumor cells were washed off, the remaining adherent cells were fixed with 1% glutaraldehyde and counted microscopically. The mean cellular adhesion rate, defined by adherent cellscoated well - adherent cellsbackground, was calculated from five different observation fields (5 × 0.25 mm2).Six-well plates were coated with collagen G  overnight at 4 °C. Plastic dishes served as background control. Subsequently, plates were incubated for one h with 1% BSA in PBS to block nonspecific cell adhesion. 0.5 × 106 A498, Caki1, or KTCTL-26 cells/mL were placed in the upper chamber in serum-free medium. The lower chamber contained 10% serum. To evaluate cell migration, Transwell chambers were pre-coated with collagen (400 µg/mL) and tumor cells then added. After 24 h incubation, the upper surface of the Transwell membrane was gently wiped with a cotton swab to remove cells that had not migrated. Cells that had moved to the lower surface of the membrane were stained using hematoxylin and counted microscopically. The mean chemotaxis and migration rate were then calculated from five different observation fields (5 × 0.25 mm2).Serum induced chemotactic movement was examined using 6-well Transwell chambers  with 8 µm pores. 0.5 × 104 cells/scan) and expressed as mean fluorescence units (MFU). Mouse IgG1-PE , IgG2a-PE , and rat IgG2b-PE  were used as isotype controls.Tumor cells were washed in blocking solution  and then incubated for 60 min at 4 °C with phycoerythrin (PE)-conjugated monoclonal antibodies directed against the following integrin subtypes: anti-α1 , anti-α2 , anti-α3 , anti-α4 , anti-α5 , anti-α6 , anti-β1 , anti-β3  or anti-β4  . Integrin expression of tumor cells was then measured using a FACScan  channel histogram analysis; 1 × 10To investigate integrin content, tumor cell lysates were applied to a 7–12% polyacrylamide gel (depending on the protein size) and electrophoresed for about 90 min at 100 V. The protein was then transferred to nitrocellulose membranes. After blocking with non-fat dry milk for 1 h, the membranes were incubated overnight with the following antibodies: integrin α1 , integrin α2 , integrin α3 , integrin α4 , integrin α5 , integrin α6 , and integrin β1 , integrin β3  and integrin β4  . HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG  served as secondary antibodies. Additionally, integrin-related signaling was explored by anti-integrin-linked kinase (ILK) , anti-focal adhesion kinase (FAK) , and anti-p-specific FAK  antibodies . HRP-conjugated goat-anti-mouse IgG  served as the secondary antibody. The membranes were briefly incubated with ECL detection reagent  to visualize the proteins and then analyzed with the Fusion FX7 system . β-actin  served as the internal control.Gimp 2.8 software was used to perform pixel density analysis of the protein bands. The ratio of protein intensity/β-actin intensity was calculated and expressed as percentage difference, related to controls set to 100%.To determine whether the integrins α3, α5, β1, and β3 impact metastatic spread, A498, Caki-1, or KTCTL-26 cells were incubated for 60 min with 10 µg/mL function-blocking anti-integrin α3 (clone P1B5), anti-integrin α5 (clone P1D6), anti-integrin β1 (clone 6S6), or anti-integrin β3 (clone B3A) mouse mAb . Controls were incubated with cell culture medium alone. Subsequently, tumor cell adhesion to immobilized collagen, as well as chemotaxis, was evaluated as described above.Curcumin uptake, adhesion, chemotaxis, and migration experiments were performed six times, and statistical significance was determined with the Wilcoxon–Mann-Whitney-U-test. Western bloting was done three times and statistics evaluated by t-test. Values are means ± SD. Differences were considered statistically significant at a p-value less than 0.05.Since it is technically feasible to apply visible light during tumor resection, combining curcumin application with visible light could enhance the RCC treatment protocol, and compensate for the low bioavailability and rapid degradation of curcumin. The data presented here indicate a curcumin uptake within 1 h. This time window should be considered during surgery, e.g., by infusing curcumin 1 h prior to light application in future RCC in vivo models.


These complexes were found to exhibit moderate antitumor activity to different types of human cancers, including A549 (human lung carcinoma), MCF-7 (breast cancer), HeLa , and HepG2 (human hepatocellular carcinoma) cell lines, but displayed low toxicity to human normal cell lines BEAS-2B  when compared with that of cisplatin. Further studies revealed that these complexes could induce apoptosis in A549 cells, including activating caspase family proteins and poly (ADP-ribose) polymerase (PARP), reducing Bcl-2/Bax and Bcl-xl/Bad ratio, enhancing cellular reactive oxygen species (ROS) accumulation, triggering DNA damage, decreasing mitochondrial membrane potential (MMP), and leading cytochrome c release from mitochondria. Notably, complex Ru-1 showed low toxicity to developing zebrafish embryos. The obtained results suggest that these new synthetic complexes have the potential to be developed as low-toxicity agents for lung cancer treatment.Two new Ru(II) complexes containing O, O-chelated ligands, Ru(dip) It is well-established that even small changes in the geometric structures of metal-based complexes could dramatically affect their biological activities, such as cyclometalated Ru(II) complexes and noncyclometalated Ru(II) complexes , 29. Fole, Ru-2) . The new3, DOG, Sucrose and Nystatin were purchased from Sigma-Aldrich . A549 (human lung carcinoma), HepG2 (human hepatocellular carcinoma), MCF-7 (breast cancer), HeLa , and BEAS-2B  cells were purchased from American Type Culture Collection . Cisplatin was purchased from Acros. Ruthenium standard solution was purchased from Aladdin Chemistry Co. . Cell Mitochondria Isolation Kit was purchased from Beyotime . Antibodies were purchased from Cell Signaling Technology Company. Comet assay reagent kit was purchased from Trevigen .Ultrapure MilliQ water was used in all experiments. DMSO, MTT, PBS, JC-1, DCFH-DA, PI, Hoechst 33342, Annexin V-FITC Apoptosis Detection Kit, QuantiPro™ BCA Assay Kit, ECL™ Start Western Blotting Detection Reagent, and endocytosis inhibitors including NaN−1 region. 1H NMR spectra were run on a Bruker AVANCE 400 spectrometer (400 MHz). UV-Vis spectra were recorded on a Perkin–Elmer Lambda-850 spectrophotometer . Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was performed by NEXION-300X . Flow cytometry was performed by an EPICS XL-MCL . Fluorescence microscopy observation was performed by Ti-E . Microplate was read by Tecan Infinite M200 pro. Protein bands were visualized using ChemiDocTM XRS Imaging System . Statistical analysis was performed using SPSS statistical software, version 17 .Microanalyses were carried out with a Perkin-Elmer 240Q elemental analyzer. Electrospray ionization mass spectrometry (ESI-MS) were recorded on Agilent LC-MS6430B Spectrometer. Fourier Transform infrared (FTIR) spectra were recorded on a Shimadzu Fourier Transform Infrared Spectrophotometer IRT racer-100 in the 4000–400 cmRu-1 was synthesized by reference to the procedure of complex [Ru(phen)2(SA)] according to our previous report [cis-[Ru(dip)2Cl2] in place of cis-[Ru(phen)2Cl2]. Firstly, a mixture of cis-[Ru(dip)2Cl2]·2H2O , o-hydroxybenzoic acid , and sodium hydroxide  were dissolved in ethanol and water mixed solution , and then, the mixture at 78°C for 11 h under the protection of argon was reflowed and a clear red solution appeared. Secondly, the solution was cooled to room temperature and concentrated by rotary evaporator at 30°C. Finally, the precipitate was dried in vacuum and purified by chromatography over alumina (200 mesh) using ethanol/water  as an eluent, and complex Ru-1 was obtained. Yield: 81.8%. 1H NMR  δ 9.71 , 9.57 , 8.20 , 8.16–8.12 , 8.11–8.06 , 7.99 , 7.94–7.87 , 7.75 , 7.66 , 7.62–7.46 , 7.39 , 6.95 , 6.67 , 6.41  ESI–MS (MeCN): m/z = 903.10 ([M + H]+). UV-Vis  (CH3CH2OH): 287(21275), 585(6125). Anal. calc. for C55H36N4O3Ru: C, 73.24; H, 4.02; N, 6.21; found: C, 73.51; H, 4.03; N, 6.21. FTIR : 1597, 1462, 1350, 702.Complex s report , with ciRu-2 was synthesized similarly to the procedure of complex Ru-1, with cis-[Ru(dmp)2Cl2] in place of cis-[Ru(dip)2Cl2]. Yield: 76.5%. 1H NMR  δ 8.55 , 8.00 , 7.97–7.89 , 7.85–7.74 , 7.76–7.67 , 7.28 , 7.03 , 6.76 , 6.40 , 6.14 , 3.02 , 2.97 , 1.95 , 1.94 . ESI-MS (MeCN): m/z = 655.09 ([M + H]+). UV-Vis  (CH3CH2OH): 273(38250), 554(6500). Anal. calc. for C35H28N4O3Ru: C, 64.31; H, 4.32; N,8.57; found: C, 64.29; H, 4.31; N, 8.60. FTIR : 1597, 1462, 1350, 702.Complex 2 incubator. The IC50 values in All cell lines were cultured in Roswell Park Memorial Institute 1640 culture media supplemented with 10% fetal bovine serum and incubated at 37°C in a 5% CO5 cells per well) and grown overnight at 37°C in a 5% CO2 incubator. The cells were incubated with the different concentrations  of Ru(II) complexes for different time intervals . After the incubation, the cells were harvested and washed twice with PBS. Cell Mitochondria Isolation Kit was used to extract the nuclear, mitochondrial, and cytoplasmic fractions of the A549 cells. The pellets were digested with 3 mL concentrated nitric acid and 1 mL perhydrol for 24 h and then diluted to 5 mL with ultrapure water. Finally, ICP-MS was used to determine the amount of Ru(II) complexes uptaken by A549 cells.A549 cells were seeded into six-well plates  complex in aqueous NaCl was added to an equal volume of octanol, and the mixture was shaken for 48 h at 200 rpm at 25°C to allow partitioning. The aqueous layer was separated from the octanol layer after the sample was centrifuged at 3000 rpm for 10 min. The Ru(II) content in the aqueous layer was measured by ICP-MS. Finally, Log Po/w values were calculated according to the equation of Log Po/w = Log ([Ru]o/[Ru]w).Log μg/mL) for 10 min, washed twice with PBS, and then photographed using an inverted fluorescence microscope. Second, after incubation with different concentrations  of Ru-1 and Ru-2 for 24 h, respectively, cells were harvested and washed twice with PBS and then resuspended in 500 μL binding buffer. The suspension was stained with 5 μL Annexin V-FITC and 10 μL PI at room temperature for 15 min and then analyzed using the flow cytometer.First, A549 cells were incubated in six-well plates for 12 h and then exposed to Ru(II) complexes for 24 h. After incubation, cell nuclei were stained with Hoechst 33342 (5 μM) of Ru-1 and Ru-2 for 12 h, the A549 cells were trypsinized and washed twice with PBS. For microscope observation, the collected cells were incubated in complete medium containing JC-1 (10 μg/mL) for 30 min and washed with PBS twice and then imaged by an inverted fluorescence microscope . For flow cytometry analysis, the cells were trypsinized and washed twice with PBS and then incubated in 500 μL PBS containing JC-1 (10 μg/mL) for 30 min at 37°C. The cells were analyzed by flow cytometer immediately. The intracellular ROS level of A549 cells was detected after DCFH-DA stain, as illustrated in our new publication [The mitochondrial membrane potential (MMP) of A549 cells was analyzed by using an inverted fluorescence microscope and flow cytometry. After pretreatment with different concentrations  in water. An inverted microscope was used to observe the hatching and growth of the zebrafish embryos with and without Ru-1 every 24 h. The ethical protocols used for the in vivo zebrafish embryo study were performed in line with the ethics regulations of Guangdong Medical University.The in Ru-1 and Ru-2 were synthesized by the reaction of cis-[Ru(N-N)2Cl2]  and salicylic acid in the presence of sodium hydroxide. The crude product was purified by chromatography over alumina. The obtained compounds were analyzed by elemental analysis, ESI-MS, 1H NMR, FTIR, and UV-Vis absorption spectroscopy, and the results were shown in Figures Ru-1 and Ru-2 in water and CH3CN solution at 298 K was analyzed by UV-Vis absorption spectroscopy -dip or dmp (π∗) and Ru(dπ)-SA (π∗). The intense band occurring between 250 nm and 300 nm is attributed to an intra-ligand (IL) π-π∗ transition by comparing with the spectra of other Ru(II)-polypyridyl complexes  and cis-[Ru(dmp)2Cl2] were incubated with four cancer cell lines, A549, HepG2, MCF-7, HeLa, and one normal cell line BEAS-2B, at various concentrations for 48 h. The cell viability was obtained by the MTT assay. The measured IC50 values were listed in cis-[Ru(dip)2Cl2] and cis-[Ru(dmp)2Cl2] displayed no/low cytotoxicity, Ru-1 and Ru-2 recorded anticancer effect in all of the tested cell lines, suggesting that the coordination of polypyridine-Ru(II) center with SA is a crucial factor for the observed cytotoxic activities. It should be noted that although Ru-1 (IC50 = 54.3 ± 3.4 μM) and Ru-2 (IC50 = 48.1 ± 3.7 μM) show comparable low cytotoxicity towards normal BEAS-2B cells, Ru-1 displays higher anticancer activity than Ru-2, especially for A549 cell line. Hence, Ru-1 displays better selectivity between cancer cells and normal cells, indicating that it has a preferable therapeutic profile against cancer, especially lung cancer cells. This is further supported by the selectivity index (SI) assay (Ru-2 (1.6) and cisplatin (0.8), Ru-1 (4.9) demonstrates significantly higher safety profile.To evaluate the I) assay . ComparePo/w values were used to estimate the lipophilicity quantitatively. As illustrated in Ru-1 (log Po/w = 1.4 ± 0.1) shows higher log Po/w value than that of Ru-2 (log Po/w = 0.9 ± 0.02), suggesting Ru-1 has higher lipophilicity. The above result is in line with the report that there is a positive correlation between hydrophobicity and cytotoxicity for metal-based anticancer agents [The lipophilicity of a drug plays a vital role in its cytotoxicity , 40–42. r agents .μM Ru(II) complexes  and Ru-2 (85.4 ± 9.3 ng) at low drug concentration (5 μM). With the extension of time and increase of concentration, the uptake levels for both Ru-1 and Ru-2 increased. The maximum cellular uptake was reached after 6 h of exposure to Ru-1 (432.5 ± 12.4 ng) and Ru-2 (235.2 ± 16.6 ng) at the concentration of 20 μM, and Ru-1 has a 1.8-fold better cell entry than Ru-2. The subcellular distribution of Ru-1 in A549 cells was further studied. As displayed in Ru-1 was mostly accumulated in the cell nucleus, followed by mitochondria. These results imply that Ru-1 may lead to mitochondria and nucleus damage and subsequent cancer cell apoptosis.It is generally acknowledged that the cellular uptake feature of transition metal-based compounds is an essential factor for their biological activities –47. As romplexes , respecttrations . In a shRu-1 and Ru-2 on A549 cells, their impact on apoptosis induction was further studied via Hoechst 33342 staining technique. As presented in Ru-1 and Ru-2, apoptotic features including chromatin condensation, nuclear fragmentation, and plasma membrane blebbing were observed. Also, at the same concentration, complex Ru-1 induced more severe apoptosis than Ru-2 in the tested cells. To further ascertain the apoptosis induced by these Ru(II) complexes, flow cytometric analysis was carried out. As shown in Ru-1 and Ru-2 significantly induced apoptosis in A549 cells in a concentration-dependent manner. After treatment with Ru-1 and Ru-2 at 5, 10, and 20 μM for 24 h, respectively, the percentages of late apoptotic/necrotic cells increased from 7.13% (5 μM) to 20.78% (20 μM) for Ru-1 and from 0.00% (5 μM) to 10.19% (20 μM) for Ru-2, and the percentages of early apoptotic A549 cells were 14.71% (5 μM), 16.27% (10 μM), and 21.57% (20 μM) for Ru-1 and 2.79% (5 μM), 15.83% (10 μM), and 15.47% (20 μM) for Ru-2, respectively.Apoptosis, an active form of cell death, plays a vital role in cell development and survival by clearing away damaged or unwanted cells . ApoptosRu-1 on key apoptosis protein caspase-3/8/9 and poly (ADP-ribose) polymerase (PARP) were analyzed by western blotting , with about 3.0- and 1.9-fold higher than the control group for Ru-1 and Ru-2, respectively.Increasing intracellular ROS levels can trigger cell apoptosis . Various2−) and hydrogen peroxide (H2O2). Based on the above information, these Ru(II) complexes may stimulate the mitochondria-based ROS generation. This speculation correlates with our previous publication [As reported, mitochondria are both a source and target of ROS . The comlication , and theRu-1 (10 μM) and Ru-2 (10 μM) for 12 h, the cells presented well-formed comet tails, demonstrating the existence of severe DNA fragmentation. Increasing the concentration to 20 μM recorded markedly higher levels of comet tails, showing that more DNA fragmentation has occurred. For quantitative comparison, the length of DNA tails in microscopy images was quantified by ImageJ, and the quantification of DNA tails in the comet assay was displayed in Ru-1 induced more DNA damage than that induced by Ru-2 based on the same external concentrations  [The comet tail observed does not mean that the ruthenium complexes directly damage DNA. As reported, the excess intracellular ROS could attack DNA, resulting in DNA damage , 59. So,duction) . In addiduction) , 63, 64,The apoptosis-mediated cell death has been commonly measured using the comet assay to detect DNA damage of cells after treatment with complexes. Comets with almost all DNA in the tail are often referred to as “hedgehog” comets and are widely assumed to represent apoptotic cells. In summary, the title complexes could induce mitochondrial dysfunction and the generation of intracellular ROS, which may indirectly lead to DNA damage.Ru-1 with the concentration close to IC50 value (12.5 μM), the cumulative hatch rate and lethality rate display no difference compared with those of the control group. When the concentration of Ru-1 was lower than 50 μM, all zebrafish embryos can develop into juvenile zebrafish after 96 h. Even if the concentration of Ru-1 was up to 100 μM, the cumulative hatch rate was acceptable, and the lethality rate was lower than 35% after 96 h. Furthermore, when the concentration of Ru-1 was lower than 100 μM, no malformation was observed at 96 h  complexes have the potential to develop into lung cancer therapeutic agents with safety profiles.In summary, our study extensively evaluated the anticancer effect of two novel Ru(II) complexes containing O, O-chelated ligands in A549 human lung cancer cells, which was mediated through inducing cell apoptosis. In addition, our results provided evidence that these complexes enhanced the level of intracellular ROS, induced a decrease of MMP, and indirectly led to DNA damage. Also, further studies showed that


The aim to develop a highly stable near-infrared (NIR) photoinduced tumor therapy agent stems from its considerable potential for biological application. Due to its long wavelength, biological imaging exhibits a high signal-to-background ratio, deep tissue penetration and maximum permissible light power, which can minimize damage to an organism during photoinduced tumor therapy.NIR998, NIR1028, NIR980, NIR1030, and NIR1028-S) based on aza–boron–dipyrromethene (aza-BODIPY) dyes with donor–acceptor-donor structures have been rationally designed and synthesized by harnessing the steric relaxation effect and intramolecular photoinduced electron transfer (IPET). These fluorophores exhibit an intense range of NIR-II emission, large Stokes shift (≥ 100 nm), excellent photothermal conversion performance, and superior stability against photobleaching. Among the NIR-II fluorophores, NIR998 possesses better NIR-II emission and photothermal conversion performance. NIR998 nanoparticles (NIR998 NPs) can be encapsulated by liposomes. NIR998 NPs show superior stability in the presence of light, heat, and reactive oxygen nitrogen species than that of indocyanine green NPs, as well as a higher photothermal conversion ability (η = 50.5%) compared to other photothermal agents. Finally, under the guidance of photothermal imaging, NIR998 NPs have been proven to effectively eliminate tumors via their excellent photothermal conversion performance while presenting negligible cytotoxicity.A class of stable NIR-II fluorophores (NIR998 NPs have excellent photothermal conversion performance and negligible dark cytotoxicity, so they have the potential to act as photothermal agents in biological applications.Utilizing IPET and the steric relaxation effect can effectively induce NIR-II emission of aza-BODIPY dyes. Stable  The development of stable near-infrared (NIR) fluorophores, especially NIR-II fluorophores, has captured considerable interest due to their high-quality imaging with their higher performance and lower health risks. NIR fluorophores emit longer wavelengths to product biological imaging that achieves a high signal-to-background ratio, deep tissue penetration, and maximum permissible light power –4. Thus,Currently, there are two main methods of constructing NIR-II organic small molecular fluorophores. The first involves changing the heterocyclic substitutions and conjugation length to construct polymethines derivates . The secAza-boron-dipyrromethene (aza-BODIPY) dyes provide many advantages over cyanine derivatives, such as better stability, strong absorbance, and tunable photophysical properties . They haNIR998, NIR1028, NIR980, NIR1030, and NIR1028-S) were rationally developed via the steric relaxation effect and intramolecular photoinduced electron transfer (IPET)  possessed excellent water solubility and biocompatibility and achieved better resistance to photobleaching, heat and reactive oxygen nitrogen species stability than that of indocyanine green (ICG) NPs. The concentration-dependent temperature change and excellent enhanced permeability and retention (EPR) effects of NIR998 NPs made them attractive for photothermal imaging of mice  staining and immunohistochemical analyses have revealed the negligible dark cytotoxicity of NIR998 NPs. Therefore, IPET and steric relaxation effect are effectively strategies for exerting NIR-II emission of aza-BODIPY dyes and NIR998 NPs with superior stability and high photothermal conversion capabilities have exhibited promising clinical applications as diagnostic reagents.In this work, a series of aza-BODIPY dye-based NIR-II fluorophores  were rationally developed by introducing other alkyl aniline derivatives or aromatic ring skeletons with low steric bulk at positions 2 and 7 of the aza-BODIPYs skeleton.The D–A–D system with intramolecular photoinduced electron transfer properties is an effective strategy for developing low-band gap NIR dyes for biological application in vivo . Aza-BODNIR998, NIR1028, NIR980, NIR1030, and NIR1028-S) were obtained by treating X-3 with BF3·OEt2 in the presence of diisopropylethylamine. They were fully demonstrated by mass spectrometry (MS) and nuclear magnetic resonance (NMR).As shown in Additional file NIR998, NIR1028, NIR980, NIR1030, or NIR1028-S corresponded to MAP, there was an obvious red shift as 123 nm, 124 nm, 89 nm, 104 nm, or 40 nm, respectively, with the increase in solvent polarity  by recording the changes in their maximal absorption. Commercial dyes (ICG and S1451) were used as the control. The absorption spectra of all NIR-II dyes showed slight changes after irradiation for 10 min. In marked contrast, the maximal absorption of commercial dyes such as ICG and S1451 reduced distinctively. For example, after 10 min irradiation, the maximal absorption of ICG and S1451 decreased to 75% and 71%, respectively, of the original value while the remaining NIR-II dyes were great than 97%  with excellent biocompatibility were acquired via encapsulation. The concentration of NIR998 NPs was calculated as 400 μM  (pH = 7.4) was 115 nm, which was larger than the transmission electron microscopy (TEM) results as 75 ± 5 nm due to swelling of the core and liposome in PBS  showed a similar absorption shape, which corresponded to that of NIR998 in the DMSO solution , photothermal imaging, and tumor photothermal therapy capabilities. The photoacoustic (PA) signal was produced by heat-induced molecular vibration and possessed intense NIR absorption with negligible luminescence, which has been adapted as a nonradiative conversion process for improved photothermal conversion capability [NIR998 NPs solutions was explored in various concentrations. NIR998 NPs showed an obvious temperature increase with as the rising concentration or light power  solution showed a high temperature increase up to 26 °C under irradiation , which helped to effectively suppress tumor growth, indicating that NIR998 NPs could serve as promising photothermal agent for tumor photothermal therapy. The NIR998 NPs solutions also showed a concentration-dependent PA signal intensity with excellent linearity (R2 = 0.9893)  resistance were investigated in comparison with ICG NPs, which has been verified by the Food and Drug Administration (FDA) for clinical application and demonstrates similar absorption to that of NIR998 NPs [NIR998 NPs and ICG NPs in PBS (pH = 7.4) was explored after heating and cooling cycles. The temperature increase (35.9 °C) of NIR998 NPs exhibited negligible changes during seven cycles, whereas the temperature changes of ICG NPs (27.4 °C) distinctly reduced to 54% (14.8 °C) and 28% (7.6 °C) of the original number after the first and second cycles, respectively  was further explored under irradiation. As shown in Fig. NIR998 NPs solution displayed negligible changes in colors and absorption spectra, even after 15 min of irradiation, whereas the ICG NPs solution exhibited an obviously changed color and decreased maximal absorption. For example, the visible green color disappeared, and only 22% of the ICG NPs solution remained after 15 min of irradiation. These results further confirm the superior antiphotobleaching ability of NIR998 NPs.The destroyed agents usually induced misguidance, poor therapeutic efficacy, and/or even serious side effects –46. The R998 NPs , 48. Thenitrite HO2) resisd to 54% 4.8 °C and to 54% 4.8 °C an− and H2O2 are considerable important signal molecules and they are over expressed in many diseases [NIR998 NPs were investigated by recording the absorption spectra with H2O2, ONOO− or without either. The absorption spectra of NIR998 NPs solutions showed negligible change in the H2O2, and the maximal absorption of NIR998 NPs solutions with ONOO− was still greater than 94% of the original number  compared with that of blood or normal tissue [NIR998 NPs to be responsive under acidic conditions. The absorption spectra of NIR998 NPs in different pH (6.4–7.4) solutions were then explored. The characteristic absorption spectra of NIR998 NPs displayed negligible change in the pH range -2,5-diphenyltetrazolium bromide (MTT) assay was used to assess the dark cytotoxicity of NIR998 NPs to SKOV3 cells. NIR998 NPs with various concentrations (0–200 μM) were incubated with SKOV3 cells for 24 h. SKOV3 cells treated with various concentrations of NIR998 NPs showed high viability, which exhibited a slight decrease as the concentration of NIR998 NPs increased. For example, the viabilities were approximately 84% and 78% even at the high concentrations of 100 μM and 200 μM, respectively, , cell viabilities were below 25% and above 97% of the original value after incubation in NIR998 NPs and PBS, respectively. These results indicate the negligible phototoxicity under 808 nm light and excellent photothermal therapeutic performance of NIR998 NPs. Finally, 0.5 W cm−2 was selected for subsequent tests.To select a reasonable light power for the following tests, SKOV3 cells were incubated with NIR998 NPs. SKOV3 cells were treated with NIR998 NPs (20 μM) plus light irradiation, NIR998 NPs, light irradiation, or PBS. Dead cells and apoptotic cells were distinguished by propidium iodide (PI) and Annexin V-FITC, respectively. SKOV3 cells that were treated with NIR998 NPs, light irradiation, or PBS displayed high viability    was investigated, due to the accessibility of infrared thermal imaging instruments and easy manipulation. A poor temperature increase (3.6 °C) was observed before injection of NIR998 NPs  NIR998 NPs plus light irradiation; II) NIR998 NPs ; III) PBS + light irradiation ; and IV) PBS, respectively. Group I served as the experimental group. Group II, III, and IV were used as the control. Mice weight, health condition, and tumor volume were important indexes of tumor therapy results. The mice were monitored at the same time every other day before the next therapy. All mice showed good overall condition, while the weight (~ 20 g) exhibited a similar change trend  with D–A–D’ structures have been rationally developed using the steric relaxation effect and IPET. These fluorophores exhibit an intense range of NIR-II emissions, large Stokes shift (≥ 100 nm) and excellent photothermal conversion performance, and superior stability against photobleaching. Among them, NIR998 with better NIR-II emission and photothermal conversion capability was used to prepare NIR998 NPs by liposome encapsulation. NIR998 NPs show better stability than ICG NPs in the presence of light, heat and reactive oxygen nitrogen species, as well as high photothermal conversion performance (η = 50.5%). The excellent photothermal conversion ability and concentration-dependent photoacoustic signal make NIR998 NPs excellent photothermal imaging contrast agents and photothermal agents for effectively guiding tumor elimination under mild conditions. The highly efficient tumor photothermal therapeutic performance demonstrated by NIR998 NPs through H&E staining and immunohistochemical analyses have revealed the negligible cytotoxicity of NIR998 NPs and phototoxicity of light irradiation, which reinforce the potential use of NIR998 NPs as diagnostic reagents for clinical applications. Unfortunately, NIR998 NPs showed weaker NIR-II emission, which presents a disadvantage to NIR-II fluorescence imaging. Therefore, in future research, we will explore more reasonable strategies for the methods and designs used to develop NIR-II-fluorophore-based NPs with stronger NIR-II emissions.In summary, a class of aza-BODIPY-dyes-based NIR-II fluorophores  and mass spectra (Bruker) were used to reveal the chemical structures of compounds. TEM and dynamic light scattering (Nano ZS90) were used for confirming morphology and particle size of nanoparticles, respectively. An ultraviolet–visible light (UV–Vis) spectrophotometer (Cintra 2020) and spectrofluorometer (Horiba Fluoromax-4) were used to record the absorption emission spectra of the samples. Confocal luminescence imaging was conducted using an Olympus IX81 laser scanning confocal microscope. Photothermal images were measured with an NIR thermal imager (FLIR E40). The power density was measured with a VLP-2000 laser power meter.−2) and without irradiation were obtained. Finally, the photothermal conversion efficiency of the samples was obtained following the methods of a previous study [Temperature changes in the sample solutions were monitored with an infrared thermal imaging system. Temperature changes of the sample solution (300 μL) in different concentrations using different levels of light power were obtained. The processes of temperature increase and decrease in the sample solution (20 μM) with irradiation  was added for further 4 h incubation. Then, 150 μL DMSO was added, and OD570 was measured with an enzyme-linked immunosorbent assay reader. Next, the cell viability was obtained following the method of a previous report.23 In addition, SKOV3 cells were treated with PBS or NIR998 NPs (20 μM). They were irradiated (808 nm) in various light power conditions for 6 min. Finally, they were treated following the steps used for the above operation.The dark toxicity assay of −2, 6 min); NIR998 NPs (20 μM); or NIR998 NPs plus irradiation. The redundant NIR998 NPs were washed with fresh complete medium. Propidium iodide (PI) and annexin V-FITC were used to differentiate between dead cells and apoptotic cells for the subsequent confocal microscopy and flow cytometry analyses.SKOV3 cells were treated with PBS (pH = 7.4); light irradiation . All in vivo experiments were performed with approval from the Medical Department of Shenzhen University.3 were used for photothermal imaging of mice under irradiation  before and after intravenous injection of NIR998 NPs .All imaging and therapy exploration of mice were launched in line with standard principles and guidelines. SKOV3-tumor-bearing nude mice with tumor volume of approximately 100 mmNIR998 NPs  in serum with time after their intravenous injection in mice. The absorption of free NIR998 obtained from major organs and tumors was used to assess the biodistribution and accumulation of NIR998 NPs after intravenous injection  for 6 h. The major organs and tumors were collected, broken, digested, ultrasonic destroyed after nanoparticles injection in different time. Then, the free molecule was extracted to test their near-infrared absorption.The pharmacokinetics study was conducted by monitoring the NIR absorption of 3 were assigned into four groups. They were treated with (I) NIR998 NPs  + light irradiation ; (II) NIR998 NPs; (III) light irradiation; or (IV) PBS. The first group served as experimental groups, and the rest groups worked as control groups. The tumors were irradiated after intravenous injection of NIR998 NPs for 6 h. The size of SKOV3 was obtained by V = LW2/2, where L and W are the tumor length and width, respectively. The body weight and tumor size of mice were collected every two days for 30 days. At day 30, the major organs and blood of mice were collected for H&E staining and immunohistochemical analyses, respectively.SKOV3 tumor-bearing mice with a tumor volume about 100 mmNIR998. 1–3  dissolved into the mixture of diisopropylethylamine (4 mL) and dry CH2Cl2 (20 mL) at 0 °C. Then, BF3·OEt2 (6.40 mmol) was dropped. They reacted for 8 h at 0 °C. The final reaction solution was quenched by methanol. The precipitate was obtained by vacuum filter. The final blue solid NIR998  was acquired by column chromatography. 1H NMR  δ(ppm) = 8.08 , 7.96 , 7.80 , 6.85 , 6.72 , 6.65 , 3.50–3.31 , 2.85 , 2.01 , 1.23–1.18 . 13C NMR  δ(ppm) = 154.34, 147.83, 144.40, 139.94, 130.34, 128.90, 127.87, 121.05, 120.12, 118.23, 113.02, 110.03, 109.23, 47.67, 44.38, 43.40, 27.36, 21.21, 11.75, 10.08. MALDI-TOF–MS m/z: 804.992.Synthesis and characterization of NIR1028. 2–3  dissolved into the mixture of diisopropylethylamine (4 mL) and dry CH2Cl2 (20 mL) at 0 °C. Then BF3·OEt2 (6.40 mmol) was dropped. They reacted for 8 h at 0 °C. The final reaction solution was quenched by methanol. The precipitate was obtained by vacuum filter. The final blue solid NIR1028  was acquired by column chromatography. 1H NMR  δ(ppm) = 8.05 , 7.59 , 6.81 , 6.71 , 3.43 , 3.24 , 2.78 , 2.02–1.97 , 1.21 . 13C NMR  δ(ppm) = 155.15, 148.81, 144.87, 143.31, 141.31, 131.36, 128.18, 121.23, 121.02, 119.35, 114.37, 111.03, 50.12, 44.42, 28.01, 21.96, 12.78. MALDI-TOF–MS m/z: 829.173.Synthesis and characterization of NIR980. 3–3  dissolved into the mixture of diisopropylethylamine (4 mL) and dry CH2Cl2 (20 mL) at 0 °C. Then BF3·OEt2 (6.40 mmol) was dropped. They reacted for 8 h at 0 °C. The final reaction solution was quenched by methanol. The precipitate was obtained by vacuum filter. The final blue solid NIR980  was acquired by column chromatography. 1H NMR  δ(ppm) = 8.19–8.09 , 7.68–7.60 , 7.44–7.33 , 7.18–7.13 , 7.00–6.96 , 6.83–6.73 , 4.44 , 3.48 , 1.43 , 1.26 . 13C NMR  δ(ppm) = 155.08, 154.11, 150.05, 148.64, 131.08, 128.47, 122.19, 120.154, 115.21, 116.23, 115.21, 110.13, 107.36, 47.61, 44.60, 15.60, 12.81. MALDI-TOF–MS m/z: 773.271.Synthesis and characterization of NIR1030. 4–3  dissolved into the mixture of diisopropylethylamine (4 mL) and dry CH2Cl2 (20 mL) at 0 °C. Then BF3·OEt2 (6.40 mmol) was dropped. They reacted for 8 h at 0 °C. The final reaction solution was quenched by methanol. The precipitate was obtained by vacuum filter. The final blue solid NIR1030  was acquired by column chromatography. 1H NMR  δ(ppm) = 8.18 , 7.77–7.66 , 7.57 , 7.38–7.28 , 6.76 , 3.48 , 1.26 . 13C NMR  δ(ppm) = 156.08, 155.11, 150.65, 149.64, 132.08, 129.47, 125.24, 123.19, 121.54, 118.39, 116.23, 111.43, 111.13, 108.36, 44.60, 12.81. MALDI-TOF–MS m/z: 719.242.Synthesis and characterization of NIR1028-S. 5–3  dissolved into the mixture of diisopropylethylamine (4 mL) and dry CH2Cl2 (20 mL) at 0 °C. Then BF3·OEt2 (6.40 mmol) was dropped. They reacted for 8 h at 0 °C. The final reaction solution was quenched by methanol. The precipitate was obtained by vacuum filter. The final blue solid NIR1028-S  was acquired by column chromatography. 1H NMR  δ(ppm) = 8.32 , 8.16–8.12 , 7.89–7.85 , 7.43–7.35 , 7.10 , 6.77–6.73 , 3.46 , 1.24 . 13C NMR  δ(ppm) = 156.00, 149.68, 145.01, 141.01, 140.48, 135.49, 134.43, 132.02, 125.45, 125.05, 124.64, 124.08, 122.21, 118.28, 117.07, 111.43, 44.61, 12.80. MALDI-TOF–MS m/z: 750.264.Synthesis and characterization of NIR998 NPs (ICG NPs). 20 mg DSPE-mPEG5000 dissolved into 8 mL deionized water under sonication . 1.5 mg NIR998 (ICG) in 4 mL THF was dropped in above mixture quickly under sonication . The prepared solution was stirred by blowing its surface with argon at 50 ºC overnight. The blue solution was washed with PBS (pH = 7.4) by a centrifugal-filter for three times. The final concentrated blue solution was used for next experiments.The preparation of NIR998 in DMSO was acquired. The linear relationship between concentration and the absorption of NIR998 at 859 nm  in DMSO was then obtained. NIR998 NPs solution (150 μL) was dried. Then, their absorption in DMSO was got. Finally, according to above linear relationship, the concentration of NIR998 NPs solution was obtained as about 400 μM.A concentration-dependent absorption of Additional file 1: Scheme S1. Synthetic routs and chemical structures of NIR-II dyes, respectively. Fig. S1. UV−vis absorption spectra of (a) NIR998, (b) NIR1028, (c) NIR980, (d) NIR1030 and (e) NIR1028-S in different solvents, respectively. The absorption intensity has been normalized. Fig. S2. Absorption spectra of NIR-II dyes and commercial dyes (ICG and S1451) in DMSO before and after irradiation of 808 nm at 0.2 W cm−2 for 10 min, respectively. Fig. S3. (a) UV–vis absorption spectra and (b) the absorption at 859 nm of NIR998 with various concentrations in DMSO, respectively. Fig. S4. (a) The transmission electron microscopy photos and (b) dynamic light scattering results of NIR998 (10-5 M) in PBS (pH = 7.4). Fig. S5. (a), (b), (c), (d) are dynamic light scattering results of NIR998 (10-5 M) in PBS (pH = 7.4) during half a month, respectively. Fig. S6. (a) Concentrations  and (b) light power dependent (20 μM) temperature rise of NIR998 NPs in PBS (pH = 7.4) in different time, respectively. (c) and (e) are temperature change of NIR998 NPs (20 μM) solutions and PBS (pH = 7.4) under irradiation , respectively. After the temperature reached to plateau, light irradiation was stopped. (d) and (f) are time constants of NIR998 NPs (20 μM) solutions and PBS (pH = 7.4) for acquiring photothermal conversion of NIR998 NPs, respectively. Fig. S7. Photoacoustic intensity of NIR998 NPs with various concentrations in PBS (pH = 7.4). Inset: photoacoustic intensity images of NIR998 NPs with various concentrations in PBS (pH = 7.4) (λEx = 808 nm).  Fig. S8. (a) and (b) are photothermal circulation stability of NIR998 NPs (20 μM) and ICG NPs (20 μM) in PBS (pH = 7.4) under irradiation , respectively. The solutions of samples was irradiated until its temperature reached to plateau. The irradition then stopped. When its temperature decrease to ambient temperature, we then repeated above process for seven times. (c) and (d) are absorption spectra of NIR998 NPs (20 μM) and ICG NPs (20 μM) in PBS (pH = 7.4) with the absence or presence of H2O2 (200 μM) or ONOO− (200 μM), respectively. Fig. S9. Absorption spectra of NIR998 NPs (20 μM) in PBS (pH = 7.4) with various pH (6.4 – 7.4), respectively. Fig. S10. Confocal fluorescence images of SKOV3 cells treated with NIR998 NPs (20 μM) plus light irradiation , NIR998 NPs only, light irradiation only or PBS only, respectively. Dead cells and apoptotic cells were distinguished by propidium iodide (PI) and Annexin V-FITC, respectively. Fig. S11. (a), (b), (c) are dynamic light scattering results of NIR998 NPs in presence of serum proteins in vitro with time, respectively. Fig. S12. (a) The absorption spectra of NIR998 NPs in serum after intravenous injection of NIR998 NPs  with time. (b) Time-dependent-concentrations curves of NIR998 NPs after their intravenous injection in mice. Fig. S13. (a) and (b) are the absorption spectra of free NIR998 obtained from major organs and tumors after intravenous injection of NIR998 NPs  for 6 h and 48 h, respectively. Fig. S14. The absorption spectrum of NIR998 NPs (10-5 M) and excrement of mice in PBS (pH = 7.4) after intravenous injection of NIR998 NPs  for 48 h. Fig. S15. Representative images of mice with SKOV3 tumor under different treatments during tumors therapy, respectively. Fig. S16. SKOV3 tumors of mice with different treatments after 30 days tumor therapy, respectively. Fig. S17. Blood test parameters in terms of basophils (BASO), eosnophilshaem (EO), monocyte (MONO), lymphocyte (LYMPH), neutrophile (NEUT) and mean corpuscular hemoglobinregulation (MCH) of SKOV3 tumor mice treated with NIR998 NPs plus light irradiation, NIR998 NPs, light irradiation after 30 days treatments, respectively. Healthy mice act as control. Figs. S18-S32 are NMR and MALDI-TOF-MS of NIRII dyes.