NS5A and NS5B proteins of hepatitis C virus (HCV) are the main targets of compounds that directly inhibit HCV infections. However, the emergence of resistance-associated substitutions (RASs) may cause substantial reductions in susceptibility to inhibitors.Viral load and genotyping were determined in eighty-seven naïve HCV-infected patients, and the amplified NS5A and NS5B regions were sequenced by Sanger sequencing. In addition, physicochemical properties, structural features, immune epitopes, and inhibitors-protein interactions of sequences were analyzed using several bioinformatics tools.Several amino acid residue changes were found in NS5A and NS5B proteins; however, we did not find any mutations related to resistance to the treatment in NS5B. Different phosphorylation and few glycosylation sites were assessed. Disulfide bonds were identified in both proteins that had a significant effect on the function and structure of HCV proteins. Applying reliable software to predict B-cell epitopes, 3 and 5 regions were found for NS5A and NS5B, respectively, representing a considerable potential to induce the humoral immune system. Docking analysis determined amino acids involved in the interaction of inhibitors and mentioned proteins may not decrease the drug efficiency.Strong interactions between inhibitors, NS5A and NS5B proteins and the lack of efficient drug resistance mutations in the analyzed sequences may confirm the remarkable ability of NS5A and NS5B inhibitors to control HCV infection amongst Iranian patients. The results of bioinformatics analysis could unveil all features of both proteins, which can be beneficial for further investigations on HCV drug resistance and designing novel vaccines.The online version contains supplementary material available at 10.1186/s12876-021-01988-y. Hepatitis C virus (HCV) is the principal cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC), resulting in hepatic associated-disease. The HCV genome is responsible for encoding structural C, E1, and E2) and nonstructural  proteins [, and E2 Interferon (IFN) was an effective therapeutic agent for HCV infection , 6; howe, and ever since several NS5A inhibitors have been introduced [Daclatasvir (DCV) is a first-in-class HCV NS5A inhibitor with potency and broad genotype coverage in vitrotroduced . Althougtroduced . In additroduced . PreviouDuring the last decade, bioinformatics analysis has played a significant role in defining the viral and bacterial mutations, protein features and designing novel vaccines , 13. ThiA total of 87 patients with chronic hepatitis C (CHC) genotype 1a and 3a who were naïve for the treatment of HCV infection with negative antibody response against HIV and HBsAg enrolled at Shariati Hospital, Tehran University of Medical Sciences, Iran.Viral load of samples was done by qualitative RT-PCR assay  with a detection range of 12–100,000,000 IU/mL.According to the manufacture’s instruction of INNO-LiPA HCV II kit , Genotyping of HCV was performed.Complete genome sequences of HCV-1 and HCV-3a genotypes from different geographical regions were retrieved from the Los Alamos HCV sequence database. Multiple sequence alignment was performed, using CLC sequence viewer, and the consensus sequence was used to design primers for the NS5A and NS5B regions in both HCV genotypes 1a and 3a. Newly designed primers are listed in Table According to the manufacturer’s instructions commercial kit , viral RNA was extracted from 200 μL of the serum and eluted in 100 μL of elution buffer. The extracted RNA was subjected to cDNA synthesis using a random primer and M-MLV reverse transcriptase. Reverse transcription was performed at 25 °C for 10 min, followed by 42 °C for 90 min, and 72 °C for 10 min. NS5A and NS5B regions were amplified by nested-PCR in a 25 μL final volume PCR reaction contained 1 × PCR buffer, 1 mM of dNTP, and 0.2 µM of each primer.In both steps of nested-PCR, amplification consisted of 35 cycles of denaturation at 94 °C for 45 s, annealing at 54 °C for 45 s, and extension at 72 °C for 45 s, followed by a final extension at 72 °C for 8 min. The positive amplicons were purified, using a commercial gel extraction kit , followed by Sanger sequencing with the limit of detection ~ 15–20%.Substitutions with respect to the reference sequences (accession numbers for 1a reference sequence were AF009606 and for 3a it was D17763) were confirmed by CLC sequence viewer version Beta (QIAGEN).http://expasy.org/tools/protparam.html) was employed to determine the theoretical isoelectric point (pI), molecular weight, extinction coefficient, instability index, aliphatic index, and grand average hydropathy (GRAVY) ["Expasy’sProtParam” ( (GRAVY) , 15.http://www.dabi.temple.edu/disphos/pred.html) [http://www.cbs.dtu.dk/services/NetPhos/) [http://www.cbs.dtu.dk/services/NetNGlyc/) and GlycoEP (http://www.imtech.res.in/raghava/glycoep/submit.html) [http://clavius.bc.edu/~clotelab/DiANNA/) [Serine, threonine, and tyrosine phosphorylation sites prediction was carried out by DISPHOS (ed.html)  and NetPetPhos/) . N-glycoit.html) . In addiDiANNA/)  was emplwww.immuneepitope.org (http://tools.immuneepitope.org/tools/bcell/iedb_input), ABCpred software (http://www.imtech.res.in/raghava/abcpred/) [http://www.imtech.res.in/raghava/bcepred) [http://www.imtech.res.in/raghava/propred1/), propred (http://www.imtech.res.in/raghava/propred/), and Immune Epitope Database (IEDB) (http://tools.immuneepitope.org/main/) were employed to predict T-cell prediction.bcpred/) , and Bcebcepred)  were usehttp://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html) [http://www.sbg.bio.ic.ac.uk/phyre) [http://zhanglab.ccmb.med.umich.edu/I-TASSER) [http://galaxy.seoklab.org/cgi-bin/submit.cgi?type=REFINE) [http://swissmodel.expasy.org/qmean/cgi/index.cgi) [http://mordred.bioc.cam.ac.uk/~rapper/rampage.php) [https://servicesn.mbi.ucla.edu/ERRAT/), and ProSA-web (https://prosa.services.came.sbg.ac.at/prosa.php) were used.SOPMA software was applied to calculate the secondary structure at (ma.html)  and the k/phyre) . "I-TASS-TASSER) , 25 was =REFINE) . To finddex.cgi) ," Rampagage.php) , ERRAT a) was done using Hex and PatchDock servers. The parameters used for the Hex docking process were: Correation type—Shape only, FFT Mode—3D fast lite, Grid Dimension—0.6, Receptor range—180, Ligand Range—180, Twist range—360, and Distance Range—40. The parameters used for the PatchDock docking process were: Clustering RMSD: 1.5, Complex Type: Protein-small ligand.Docking with four NS5A inhibitors  and NS5A protein as well as NS5B and 2 approved NS5B inhibitors . We used the Shapiro–Wilk test to assess normal distribution, the Pearson Chi-square test for categorical data, and ANOVA for comparison of the mean values. A p-value of less than 0.05 was considered to be statistically significant.The majority of patients were male (65.7%) and 50.4% of them were under the age of 40 years old. Higher viral load defined as HCV-RNA levels ≥ 400,000 IU/mL detected in 66.1% of samples which was not statistically related to age, gender, and viral load.Comparison of the patients’ sequences and isolated reference showed several amino acid residue changes, shown in Table (100%), and for 3a genotypes, the mutations were A 21 T (47%), H 180 N (42%) and T 183 A (33.3%). The most prevalent mutations in NS5B 1a and 3a were Q 300 R, and D 444 N and N 317 G, A 366 P, I 448 V/T, respectively.For NS5A sequences, the most prevalent mutations in 1a genotype were K 107 T (100%), S 131 T (100%), and I 144 V E.coli; however, “protparam” analysis predicted that they are not stable in mammalian cells. The instability index, an estimate of the stability of a protein in a test tube, showed both proteins to be unstable. Aliphatic index, a positive factor for the increase of thermostability of proteins, indicated that both proteins are the thermostable proteins. In addition, GRAVY is a Hydropathicity index and a negative score indicates NS5A and NS5B are hydrophilic. Moreover, the results suggested insignificat differences between 1 and 3a genotypes  prediction of both proteins is summarized in Table The combination of all prediction samples showed 6, 4, 2, and 3 as the potentials B-cell epitopes in NS5A 1a, NS5A 3a, NS5B 1a, and NS5B 3a, respectively -α and ribavirin was the standard therapy for chronic hepatitis C patients as an indirect antiviral treatment without any direct target on HCV protein or RNA element. In 2016, the Clinical Management of Hepatitis C in Iran, prepared by Iran Hepatitis Network (IHN) recommended Daclatasvir, Sofosbuvir, Ledipasvir, and Ribavirin for the treatment of HCV-infected patients .In the present study, our data showed that NS5B and NS5A inhibitors may be beneficial to prescribe for Iranian HCV-infected patients. Docking analysis suggested the high energy values between NS5A and NS5B inhibitors and the 3D models of NS5 proteins. The higher docking score represented better binding ability indicated the strong attachment of inhibitors to NS5A and NS5B proteins to suppress viral functions that would be contributed to the promising treatment outcome.The highest energy values belonged to NS5A 1a with selected NS5A inhibitors; however, NS5B results did not show significant differences between 1 and 3a genotypes and NS5B inhibitors. The amino acids have located in the attachment region of NS5B and NS5B inhibitors showed different patterns in two genotypes; but, there was no mutation in the mentioned regions. The amino acids in the attachment regions of NS5A and NS5B proteins not only were highly conserved in genotypes 1a and 3a, but also they were preserved in all samples. These conserved regions provide an absolute opportunity for drug to target the viral protein easily that causes increased sensitivity to NS5 inhibitors leading to a favorable clinical treatment. There was only one substitution (E181D) in the attachment region (NS5A-1a and Elbasivir) with a frequency of 75% in NS5A 1a sequences. The substitution E181D in NS5A 1a did not affect the other NS5A inhibitors, but Elbasvir could significantly change the energy value. The presence of this mutation could result in deletion in a phosphorylation site; therefore, suggested NS5A inhibitors are functional in controlling HCV infection amongst Iranian patients.Compared with previous studies, three new mutations  were identified in NS5A sequences, which might have drug resistance effects. From 2014 to 2016, Grandal et al. , we did From 2015 to 2018, a large-scale RASs study was conducted with 878 Chinese patient’s samples from 27 provinces across the country who were infected with five HCV genotypes: 1b, 2a, 3a, 3b, and 6a. Although all patients were DAA-naïve treatment, fifty-two percent of patients had documentation of experiencing interferon-based therapy at sample collection. We did not find any substitution suggested by Lu et al. , describIn a large cohort study on Italian patients, Bertoli et al.  investigThe data of ProtParam determined the notable similarity in NS5A and NS5B amino acids in both 1a and 3a genotypes. But, the pI in NS5A showed a difference between the two genotypes which can be described by the diversity in the number of basic amino acids. Accurate prediction of the pI of viruses is beneficial for physical/chemical treatment processes and modeling virus behavior in environment .In recent years, HCV-NS5A and NS5B recombinant proteins used for different approaches including vaccine development, HCV genotyping, serological diagnostic methods, and therapeutic applications –44. TherAlthough our prediction showed that NS5A and NS5B proteins are unstable in mammalian cells for less than 2 h, Huang et al.  and PolyThe NS5A is a phosphorylated protein has been shown to be involved mostly in HCV pathogenesis, oncogenic transformation, and resistance to IFN therapy . The NS5PTMs of proteins involve the attachment of small proteins or functional groups such as glycosylation, phosphorylation, disulfide bridging, etc. to specific amino acids within the proteins that caused altering the structure and charge or hydrophobicity of proteins which are crucial for the proteins’ functioning. During viral infection, PTMs leads to increase protein solubilization, enhance protein antigenicity and virulence properties, viral replication, interferon response inhibition that play a crucial role in viral pathogenesis. In other words, host cells remove PMTs from viral proteins to inhibit viral protein synthesis, activate immune response pathways and control the virus replication to eliminate the virus by inactivating the viral proteins. Hopefully, bioinformatics and biochemaical analysis pave the way for development of new drugs to pharmacological suppress HCV- NS5A and NS5B proteins that may result in the restoration of strong immune responses, suppression of tumorigenesis that aim to eliminate the virus .In addition, previous studies showed that NS5A phosphoprotein can disrupt the host interferon-induced antiviral response. Recently, it was suggested that phosphoprotein may take part in regulating HCV RNA replication, modulate host intracellular signaling pathways, and virion assembly. The phosphorylation process is highly regulated and cellular protein kinases are responsible for this process, which are suggested as the new target for antiviral therapeutic intervention . Our anaIn line with the present data, previous studies showed alanine mutations in serine 225 led to NS5A hyper-phosphorylation reduction and replication of HCV genotype 2 , 58, 59.While our analysis predicted several phosphorylation sites for NS5B, the phosphorylation pattern showed significant differences between the two genotypes. Han et al.  reportedAccording to our result, predicted phosphorylation amino acids  in HCV NS5 amongst Iranian patients have a critical role in virus replication, pathogenesis, chronic infection, liver diseases, and antiviral therapeutic intervention . TherefoUnderstanding the process of NS5A and NS5B phosphorylation and its role in the HCV life cycle will not only reveal mechanisms of HCV-RNA replication and/or pathogenesis but may also provide new avenues for therapeutic intervention. There is some evidence showed NS5A induces liver pathogenesis, steatosis, and hepatocellular carcinoma that NS5A phosphorylation can affect these important functions directly or indirectly . FurtherThe other protein post-modification process is glycosylation that are essential for viral assembly. Glycosylation can increase protein solubility and may be critical for maintaining NS5A functional structure –66. The In our study, glycosylation prediction showed one site for 1a and three sites for 3a genotypes in which position 69 was similar in the two genotypes, which was in accordance with the report by Yamasaki et al. . In 6 ofGiven the conservation of glycosylation, several compounds have been classified as carbohydrate-binding agents (CBAs) that show promise in the inhibition of virus activity. All in all, development of broad-spectrum antivirals to target glycosylation and selective deglycosylation can impair viral replication, assembly that offer an exciting prospect for several clinical treatments . To the Prediction of disulfide bonds for NS5A sequences revealed several cysteines with significant similarities between the two genotypes. Six conserved cysteines  are common between the two genotypes, found in all patients’ sequences, which might be involved in NS5A function and structure.Previous studies showed a C-terminal disulfide bond in the NS5A structure. The structure analysis revealed the presence of a novel fold, a zinc-coordination motif, and a C-terminal disulfide bond . ConsistAs far as we know, there is a lack of published documents concerning NS5B disulfide bonds; however, our findings showed several disulfide bonds for NS5B in genotypes 1a and 3a. Since our data did not show any substitution in cysteine residues positions, these conserved sites might contribute to the fundamental activities associated with the host and HCV interactions.All things considered, disulfide post modification analyses revealed that mutations may affect the number of bonds and bond patterns. Formation of the disulfide bond locks the structure in place and mostly increases the stability and half-life of the proteins . TherefoNS5A B-cell epitope prediction showed some regions for the two genotypes, presenting the potential of this protein in inducing the humoral immune system. In comparison between the two genotypes, three regions  were similar that can be beneficial in the new HCV vaccines development. In compliance with the present study, Ikram et al.  applied Close to our prediction, Holmström et al.  indicateOther than immune-bioinformatics analyses, protein interaction data, drug resistance-related mutation, etc. were other key factors that should be analyzed to characterize protein structure. Numerous studies used the SOPMA software to predict the secondary structure of different viral and bacterial proteins –79. ThisIn our study, only epitopes located in random coil and β-turn structures were analyzed for antigenicity properties because such secondary structures are mostly located in the surfaces of the protein and are more easily to be recognized by antibodies. Different mutations did not affect the location of secondary structures and B cell epitopes . VariousBased on our findings, prescribing NS5A and NS5B inhibitors can effectively suppress HCV amongst Iranian HCV-infected patients. Despite some substitutions in NS5A and NS5B proteins, such minor mutations would not significantly affect the efficiency of the NS5 inhibitors. In addition, the suggested B cell epitopes in NS5A and NS5B proteins can remarkably trigger an immune response that can be profitable in HCV vaccine production. As phosphorylation and glycosylation are essential to form active NS5A and NS5B proteins, the yeast host not only may support PMTs but also can increase the protein expression yield. Moreover, NS5A and NS5B post-modifications including phosphorylation, glycosylation, and formation of disulfide bonds are crucial for HCV pathogenesis. PTM pathways and PTM sites are potential pharmacological targets for new therapeutic approaches. To unveil the precise PTM sites and the underlying mechanisms much work remains to be done.Additional file 1. Figs. S1-S12 illustrated the amino acids involved in docking analysis between NS5A and B with NS5 inhibitors including Daclatasvir, Elbasvir, Ledipasvir, and Ombitasvir.


The four systems differ in terms of tillage practices, N inputs, and species, which is likely to affect soil N. Field data were collected and N2O fluxes were calculated. These original new cropping systems are innovating, resulting in new combinations of agricultural practices. The data obtained could be used to improve models for parameterization and validation, and to increase the predictive accuracy of models of N losses in original conditions.With the development of agroecosystem approaches, new cropping systems have to be designed to deliver multiple ecosystem services. In this context, we assessed four innovative cropping systems, designed to reach multiple environmental and production goals, in a long-term field experiment (2009–2020) at Grignon . A wide range of measurements were made, for nutrient cycles and organic matter in particular, for an analysis of interactions occurring during the emissions of greenhouse gases. We focus here on nitrogen (N) data collected over eight years (2009–2016). The data include: nitrous oxide fluxes (N The experiment was focused on the assessment of innovative cropping systems with multiple environmental and production objectives designed to deliver an entire package of ecosystem services. Here, the data were collected for the first eight years of the field assessment.•These data have already been used to assess the environmental and production performances of the innovative cropping systems ,2.•These data can be used as a benchmark for future studies aiming to design new cropping systems to decrease nitrogen (N) losses and improve N management in northern Europe cropping systems.•These data could be used to improve parameterization and validation, to increase the predictive accuracy of models of N fluxes.•These data can be used to calculate new indicators based on the measurements of N fluxes.13−, ammonia NH4+), nitrous oxide (N2O) fluxes, crop N uptake and yield The open-access research data set is organized into five files of nitrogen (N) data . The raw, descriptive and computed data were collected over the first eight-year period for the four innovative cropping systems assessed in a long-term (2009-–2020) field trial at AgroParisTech experimental farm at Grignon  2O fluxes, (ii) soil N contents (NO3− and NH4+), (iii) aboveground plant biomass and N content at maturity, and yield, (iv) agricultural practices and (v) climate data.The following data are available: (i) N2O fluxes were calculated for the 2010–2016 period in two cropping systems: the PHEP  system and the L-GHG (less greenhouse gas emissions) system. All the other data were measured over the 2009–2016 period in all four cropping systems: the PHEP system, the L-GHG system, the No-Pest (no pesticide use) system and the L-EN (less energy consumption) system.NThe data sets involved here have already been used: (i) to assess system performances, in terms of greenhouse gas emissions, energy consumption, pesticide use and yield, over the first crop sequence  contents, measured at a depth of 0–25 cm or 0–30 cm, collected at the same time in the PHEP and L-GHG systems over the 2010–2016 period. There are thirteen columns, corresponding to: (1) year of harvest (YYYY), (2) name of cropping system (PHEP and L-GHG); (3) number of replicate (1 to 3); (4) number of plot (1 to 12); (5) species ; (6) date of measurement (DD/MM/YYYY); (7) number of the chamber (1 to 3); (8) N2O fluxes (g.m−2.s−1); (9) soil N-NH4+ content (kgN.ha−1); (10) soil N-NO3− content (kgN.ha−1); (11) soil moisture (((wet soil biomass - dry soil biomass)/ dry soil biomass)*100); (12) soil N-NH4+ content (mgN.l−1); (13) soil N-NO3− content (mgN.l−1).This file includes data for N1.23− and NH4+) contents measured at a depth of 0–150 cm, collected at three different time periods , in all four cropping systems over the 2009–2016 period. There are fifteen columns corresponding to: (1) year of harvest (YYYY), (2) name of cropping system , (3) number of replicate (1 to 3), (4) number of plot (1 to 12), (5) number of sample (1 to 2), (6) species , (7) period of measurement , (8) date of measurement (DD/MM/YYYY), (9) soil layer , (10) soil bulk density, (11) soil N-NH4+ content (kgN.ha−1), (12) soil N-NO3− content (kgN.ha−1), (13) soil moisture (((wet soil biomass - dry soil biomass)/ dry soil biomass)*100), (14) soil N-NH4+ content (mgN.l−1), (15) soil N-NO3− content (mgN.l−1).This file includes data for soil N (NO1.3−1), (11) crop aboveground N content (% of dry matter).This file contains data for the aboveground biomass and N content of the crop measured at maturity in the four cropping systems over the 2009–2016 period. There are eleven columns, corresponding to: (1) year of harvest (YYYY), (2) name of cropping system , (3) number of replicate (1 to 3), (4) number of plot (1 to 12), (5) number of sample (1 to 12), (6) species , (7) date of measurement (DD/MM/YYYY), (8) development stage (maturity or 8.0 for rapeseed 1.4−1), (12) variety, (13) amount of N applied (kg.ha−1), (14) type of N fertilizer , (15) name and N concentration of the fertilizer (%), (16) fertilizer location (surface or incorporated into the soil), (17) status of crop residue (stubble left in place or exported), (18) mean yield (tonnes of dry matter.ha−1), (19) standard deviation of yield (tonnes of dry matter.ha−1).This file contains data relating to agronomic practices and yield collected for the four cropping systems over the 2009–2016 period. There are nineteen columns, corresponding to: (1) year of harvest (YYYY), (2) name of cropping system , (3) number of replicate (1 to 3), (4) number of plot (1 to 12), (5) species , (6) crop characteristics (cover crop or catch crop species), (7) description of soil: bare soil (yes or no), (8) date of agricultural practice (DD/MM/YYYY), (9) agricultural practice , (10) depth of plowing (cm), (11) sowing density  date of measurement (DD/MM/YYYY), (2) mean daily temperature (°C), (3) daily rainfall (mm), (4) mean daily soil temperature at 10 cm below the surface (°C).22.1−1, silt content = 71.9 g.kg−1, sand content = 7.4 g.kg−1, bulk density = 1.4, CEC = 11.5 cmol+ .kg−1 and carbon content = 15.9 g.kg−1. The C/N ratio was 12.4 and the pH was 6.9  had induced changes in bulk density requiring a second measurement in 2014.The experiment took place at the AgroParisTech experimental farm at Grignon, in the Ile-de-France region . The field was characterized by a deep and homogeneous loamy clay soil (haplic luvisol according to the FAO classification 2.2e.g. date and amount of N fertilizer applications) due to environmental factors and working organization constraints The trial covered a total area of 6.2 ha, divided into three replicates . Each re2.3The PHEP system was designed to minimize environmental impact: (i) cover crops were sown before each spring species to decrease nitrate losses; (ii) pesticide uses were lessened by increasing crop diversity, lengthening the crop sequence and sowing highly resistant varieties; (iii) energy consumption was reduced by allowing plowing only once in the crop sequence, and N fertilizer amounts spread were decreased by the incorporation of legumes into the crop sequence. This cropping system was also designed to reach the maximum yield given the environmental targets, as described in 2O emissions were decreased by using appropriate decision rules to prevent N fertilizer application in climatic conditions favoring N2O emissions.In the L-GHG system, greenhouse gas emissions were limited by increasing carbon sequestration in the soil (i.e. producing large amounts of residues from both the main crop and catch crops without tillage) and NThe L-EN system was designed to reduce both direct and indirect energy consumption. Plowing was prohibited, direct sowing was implemented, the amount of N fertilizer applied was decreased by sowing many legumes and species with high N use efficiency, and target yield were decreased.e.g. hemp), the use of highly resistant varieties or species mixtures, a wide diversity of sowing dates, regular tillage and mechanical weeding to control weeds.For the No-Pest cropping system, the no-pesticide constraint was satisfied by including a wide range of species  and August. Additional measurements were carried out during specific periods: four measurements were performed over a two-week period after each N fertilizer application; two measurements were made after the faba bean harvest if a significant rainfall event occurred (>10 mm). For each plot, N2O emissions were measured manually, with three static chambers inserted into the soil at a depth of 10 cm after sowing and left in place until harvest. Each chamber covered a surface area of 0.25 m2 and the top of the chamber was 0.2 m above the soil surface. N2O emissions were measured between 10 a.m. and 3 p.m.  at each sampling date, with the same order used for monitoring on each plot. The chambers were closed for 45 min, during which the headspace air was sampled four times . Gas samples (20 mL) were collected in a syringe and immediately injected into pre-evacuated 12 mL glass vials . These vials were stored in the dark at room temperature until laboratory analyses. N2O concentrations were analyzed with a gas chromatograph equipped with an electron capture detector .Set 1: measurements performed at the same time as those for NSet 2: measurements performed at three different periods over the year (i.e. at the beginning of winter (BW) around November 15, after winter (AW) around February 15, and about eight days post harvesting (PH) of the main crop). Six samples were collected from five layers  in each plot. Three samples from each layer were pooled to generate two soil samples per layer for each plot .Two sets of soil N content data were managed and are provided in two different files:−1) with the aid of a magnetic stirrer for 30 min. After 30 min of decantation, the suspended matter has settled and the supernatant was collected. Supernatant aliquots were sent to the analytical laboratory. NO3− was determined by reaction with N-(1-naphthyl) dichloride diamine ethylene, and NH4+ was determined by reaction with sodium dichloro-isocyanurate and sodium salicylate. NO3− and NH4+ contents were analyzed in an aliquot of the extracts obtained, by colorimetry . Results were expressed both in kgN per hectare and in mgN per liter.These two groups of measurements were managed in the same way. Soil samples were collected manually with an auger and stored in a cold box (4 °C) until analysis. Water content was measured gravimetrically, according to the international standard method (NF ISO 11-465). From a soil sample (50 g), soil inorganic N was extracted in potassium chloride solution (77 g.l3.3A and B) in all years except in 2009. Seeds were separated from the vegetative parts of the plant , except for winter rapeseed, for which all aboveground parts  were pooled. All samples were oven-dried at 80 °C for 48 h. For analyses of N content, we pooled two or three samples, depending on species, which were then ground and analyzed by the Dumas combustion method Depending on species, we collected nine to twelve samples (i.e. 1 m² per sample) per plot at maturity, except for winter rapeseed, for which samples were collected at stage 8.0 3.4Yield (mean and standard deviation) were calculated on the basis of six samples  collected at maturity, with a combine harvester, from each plot. Yield unit was tonne of dry matter per hectare.3.5The management of the four cropping systems has been described in detail in 3.6Mean daily temperatures (°C) and daily rainfalls (mm) data were collected from an automated INRAE meteorological station  located 150 m from the trial.Caroline Colnenne-David: Conceptualization, Methodology, Validation, Formal analysis, Resources, Writing – original draft, Writing – review & editing, Project administration, Funding acquisition, Supervision; Gilles Grandeau: Resources; Marie-Hélène Jeuffroy: Conceptualization, Writing – original draft, Funding acquisition; Thierry Doré: Conceptualization, Writing – original draft, Writing – review & editing, Funding acquisition, Supervision.The authors declare that they have no known competing financial interests or personal relationships that have or could be perceived to have influenced the work reported in this article.


This result is confirmed by experiments conducted in real time for 60-minute exposure to blood at 100 s−1, 600 s−1, and 900 s−1 shear rates.Implantable devices fabricated from austenitic type 316L stainless steel have been employed significantly in medicine, principally because the material displays excellent mechanical characteristics and corrosion resistance. It is well known, however, that interaction of exposure of such a material to blood can initiate platelet adhesion and blood coagulation, leading to a harmful medical condition. In order to prevent undesirable surface platelet adhesion on biomaterials employed in procedures such as renal dialysis, we developed an ultrathin anti-thrombogenic covalently attached monolayer based on monoethylene glycol silane chemistry. This functions by forming an interstitial hydration layer which displays restricted mobility in the prevention of surface fouling. In the present work, the promising anti-thrombogenic properties of this film are examined with respect to platelet aggregation on 316L austenitic stainless steel exposed to whole human blood. Prior to exposure with blood, all major surface modification steps were examined by X-ray photoelectron spectroscopic analysis and surface free-angle measurement by contact angle goniometry. End-stage anti-thrombogenicity detection after 20 min of blood exposure at 100 s Coronary artery disease (CAD) is a debilitating condition where a buildup of plaque forms which causes the occurrence of arterial narrowing (stenosis) . Thus faThrombus formation occurs when medical implants or devices are in contact with whole blood such that different components of the blood such as proteins and cells adsorb onto the surface of the implant . It is vThe myriad of suggested approaches to prevent thrombus formation and re-stenosis do not necessarily display a high level of overall stability. By far the most common technology at the present time is the DES, as mentioned above. This technology, however, does not reduce the risk of developing thrombosis. Even with use of DES, anticlotting agents such as aspirin or clopidogrel are often administered, since clot formation can potentially affect how well the drug is eluted from the stent surface . In factMore recent advances in drug eluting stents include heparin-eluting stents ,23,24,25Other recent work has included phenolic-amine chemistry combined with bivalirudin, which is a thrombin inhibitor, in order to combat thrombogenesis . This laGraphene-coated stents have also been investigated for their anti-thrombogenic properties on stainless steel ,31,32. TMedical grade stainless steel is the most common material employed for stent fabrication . The reaSeveral of the surface treatments mentioned above have been used to modify the surface of 316L stainless steel in attempts to enhance its hemocompatibility properties. In the present work, we demonstrate that an ultrathin monoethylene glycol (MEG-OH), which is covalently attached to the steel surface, is capable of a dramatic reduction in platelet aggregation from blood. Such an effect has been previously shown with regard to polymeric biomaterials conventionally employed for conduits in renal dialysis and bypass surgery ,39. ThisThis monoethylene glycol coating is robust, easy to apply, low-cost, and has shown a much higher degree of anti-thrombogenicity when applied to plastic polymers, compared to many drug-eluting stents ,39. As sv/v) octadecyltrichlorosilane (OTS) in anhydrous toluene inside a glove box maintained under inert atmosphere. 2-(3-trichlorosilylpropyloxy) ethyl-trifluoroacetate (MEG-TFA) linker used for stainless steel surface silanization was synthesized as described previously [2) and anhydrous (P2O5) atmosphere.All glassware involved in the silanization process was pre-treated with 5% (eviously . All sil6) fluorescent dye were obtained from Sigma-Aldrich, Oakville, ONT, Canada. ACS grade chloroform, n-pentane, acetone, and methanol were purchased from Fisher Scientific, Ottawa, ONT, Canada. Annealed austenitic type 316L stainless steel foil, mirror-polished on both sides, with thickness of 2.0 mm, was purchased from Goodfellow Ltd., Cambridge, UK. Additionally, 1.0 mm thick, annealed, 2B mill polished on both sides, austenitic type 316L stainless steel plates  were obtained from Stainless Supply, Ltd., Monroe, NC, USA.Concentrated sulfuric acid (98%) was purchased from ACP Chemicals Inc., Saint-Leonard, QC, Canada. Anhydrous toluene, ACS grade toluene, ethanol, dimethyl sulfoxide (DMSO), octadecyltrichlorosilane (OTS), and 98% 3,3′-dihexyloxacarbocyanine iodide  finish are both achieved through mechanical polishing . Electro2 gas. All dried steel samples were then individually immersed and oxidized in piranha solution as follows: 10 mm × 10 mm × 2 mm samples  were placed within a test tube. Samples with dimensions of 50.8 mm × 25.4 mm × 1 mm (for blood flow experiments) were placed tilted against the glassware in a 120 mL beaker, which is capable of holding up to six samples. Each test tube or beaker was subsequently put in a hot water bath pre-heated to 90 °C. Piranha solution was prepared by adding three equivalents of 98% concentrated sulfuric acid to one equivalent 30% hydrogen peroxide. Approximately 2 mL of piranha solution was dispensed into each test tube, and about 100 mL of piranha solution was poured into the 120 mL beaker to fully immerse all steel samples. After 30 min, all oxidized samples were removed from the piranha solution. Each sample was then rinsed with a large amount of distilled water to wash off residual piranha solution on the surface, followed by 3 times the amount of ACS grade methanol. Next, samples were individually transferred to clean glassware and placed inside an oven maintained at 150 °C. After two hours, steel samples were taken out of the oven and left to cool down to room temperature for 15 min. Finally, each sample was plasma-cleaned using an expanded tabletop plasma cleaner with a maximum power of 30 V for 5 min. One oxidized sample was stored in a clean scintillation vial under nitrogen awaiting X-ray photoelectron spectroscopic (XPS) surface characterization.Steel samples were subjected to successive sonication in ACS grade n-pentane, ACS grade acetone, then 95% ethanol, for 20 min each. Afterwards, each sample was carefully rinsed with a large quantity of 95% ethanol, followed by a copious amount of distilled water. Cleaned stainless steel was subsequently dried under a steady stream of N3)2·6H2O) at relative humidity of 78% and 23 °C. Next, steel samples were transferred inside a glove box, along with MEG-TFA trichlorosilane linker stock solution, micropipettes, silanized glassware and Parafilm®. Next, 10 µL MEG-TFA linker was pipetted and diluted with 10 mL anhydrous toluene. Afterwards, 2.0 mL 1/1000 (v/v) MEG-TFA/anhydrous toluene solution was portioned into each silanized scintillation vial containing one 10 mm × 10 mm × 2.0 mm stainless steel sample. For 50.8 mm × 25.4 mm × 1.0 mm steel samples, a minimum of 20.0 mL 1/1000 (v/v) MEG-TFA/anhydrous toluene solution was required to fully immerse three samples of such dimension within a 150 mm diameter, 65 mm deep silanized Pyrex® petri dish. The scintillation vials containing steel samples immersed in MEG-TFA/anhydrous toluene solution were screw-capped and subsequently sealed with Parafilm®. The petri dishes were placed in a desiccator before removing from the glove box. The scintillation vials and the desiccator were then placed on a spinning plate for two hours. When silanization was completed, each stainless-steel sample was rinsed three times with ACS grade toluene, followed by five minutes of sonication in ACS grade toluene. Then, the same rinsing and sonication process was repeated with ACS grade chloroform on each steel sample, followed by drying under nitrogen stream. One MEG-TFA modified stainless steel sample was stored under nitrogen in a clean scintillation vial, as previously discussed, for surface analysis.After plasma cleaning, all oxidized steel samples intended for surface modification were placed immediately inside a humidity chamber for 24 h to facilitate water vapor physisorption onto the steel surface. The humidity chamber was maintained by an aqueous solution of magnesium nitrate hexahydrate (Mg(NOv/v) ethanol/distilled water on a spinning plate for overnight solvolysis at room temperature. Each 50.8 mm × 25.4 mm × 1.0 mm steel sample was placed in a Falcon® 50 mL conical centrifuge tube, and the volume of 1/1 (v/v) ethanol/distilled water used for solvolysis was increased to 40 mL. All steel samples were subsequently dried under gentle nitrogen stream. One MEG-OH-modified steel sample was stored under nitrogen in a clean scintillation vial, as previously discussed, for XPS surface characterization, while others were stored for later blood perfusion experiments.For solvolysis of the trifluoroacetic acid (TFA) group, 10 mm × 10 mm × 2 mm MEG-TFA surface modified steel samples, placed in 20 mL clean scintillation vials, were individually immersed in 2.0 mL 1/1 . Take-off angles of 20° and 90° relative to surface were chosen to be analyzed for each sample. Peak fitting and data analysis of all XPS spectra, including survey scans and elemental narrow scans of C1s, O1s, Si2p, F1s, Cr2p, Fe2p, Mn2p, Mo3d, Ni2p, were processed using the accompanying AvantageSurface wettability of all 316L stainless steel samples after each major step of surface modification was measured using CAM 101 optical tensiometer (contact angle meter), constructed by KSV Instruments Ltd., Monroe, CT, USA. Distilled water was consistently used as the testing liquid.® 0151 thixotropic epoxy paste adhesive . Two threaded 1/16 inch, straight, male polyethylene tubing connectors, each connected with 75.0 cm and 150.0 cm long 1/16 inch/1/8-inch (inside/outside) diameter moisture-resistant polyethylene vacuum tubing, were ultimately screwed into the glued female adaptors. All Luer connections and insertions were then sealed with Parafilm® extensively to prevent potential air or liquid leakage. The channel slide was made from clear, impact-resistant, both-sides-smooth, 0.0508 mm thick polycarbonate film, with up to 10% thickness tolerance. The polycarbonate film was cut into a size of 50.8 mm × 25.4 mm, and the flow channel was created by carving out a rectangular space with 4.0 mm width and 35.0 mm length.A custom-made, single-pass, parallel-plate blood perfusion chamber was consistently used for whole human blood surface thrombogenicity analysis . A trans® and further stabilized with small clamps.The assembly of the blood perfusion chamber proceeded as follows. Both the glass coverslip and channel slide were cleaned and sterilized with 95% ethanol. The channel slide was then gently applied onto the coverslip, making sure the entrance and exit holes were unobstructed and completely exposed within the rectangular flow channel. Afterwards, a thin layer of distilled water was sprayed onto the channel slide to serve as lubricant and to prevent the formation of air bubbles. The assembly of blood perfusion chamber was completed by sandwiching the channel slide with a 50.8 mm × 25.4 mm × 1.0 mm bare or MEG-OH-surface-modified steel plate. Each side of the chamber was extensively sealed with Parafilm−1, 300 s−1, 600 s−1, 750 s−1, and 900 s−1 were chosen for whole human blood thrombogenicity analysis on bare and MEG-OH-surface-modified 316L stainless steel plates. The corresponding volumetric flow rate and required blood volume at each chosen shear rate were calculated [In this study, shear rates of 100 slculated  and disp6, λexc/λem = 484/501 nm) fluorescent dye stock solution was prepared by dissolving 98% DiOC6 in ACS grade dimethyl sulfoxide (DMSO) at 1 mM concentration. The working solution was diluted to 10 µM concentration and stored in an aluminum-foil-wrapped vial and stored in a refrigerator. Collected whole human blood was stored in 6 mL lithium heparin Becton Dickinson plastic vacutainers. Whole human blood sample intended for 20 min surface thrombogenicity analysis was subsequently fluorescence-labeled with DiOC6 working solution at 1/100 (v/v) ratio. For 60 min surface thrombogenicity analysis, DiOC6 working solution was added to the whole human blood at 1/30 (v/v) ratio. The vacutainers containing fluorescence-labelled blood were then wrapped with aluminum foil and placed on a rotator for at least 20 min before use.3,3′-dihexyloxacarbocyanine iodide  were prepared for surface thrombogenicity analysis against whole human blood under a BX61W1 Olympus, confocal microscope , equipped with an EMC2 Q-imaging CCD camera, Rolera, QImaging,  The required exposure time was set to 300 ms without camera gain. The excitation laser source used was an arc mercury lamp, X-cite 120 PC fluorescence illumination system, EXFO, . A green fluorescent filter cube set FITC 3540B Semrock, . which combines a 35 nm wide excitation bandpass filter centered at 482 nm and a 40 nm emission bandpass filter centered at 536 nm, was used to filter fluorescent signals. The dimension of the camera chip used was 1004 × 1002 with pixel size of 8 × 8 µm, and the objective lens used was a 40× Plan N (Olympus) with a numerical aperture of 0.65. The microscope and camera setup allowed recording of live videos, from which specific frames were extracted. Extracted frames were then processed with imageJBefore starting blood perfusion, the parallel-plate flow chamber was stabilized on the testing platform and fitted under the microscope, followed by a 2.0 mL phosphate-buffered saline injection to purge and test leak tightness. One-to-two drops of distilled water were added onto the coverslip for objective lens immersion. Next, the syringe pump was turned on, and fluorescence-labeled whole human blood was pipetted into the blood reservoir. The image recording process did not start until blood reached the perfusion chamber.Angle-resolved XPS with take-off angles of 20 and 90 degrees relative to the surface was performed on mirror- then electrochemically polished 10 mm × 10 mm × 2 mm 316L stainless steel samples after each surface modification step. The principal objective of the investigation was to confirm the effectiveness of surface cleaning and activation protocol with piranha solution, as well as to establish the successful attachment of MEG-TFA (and subsequent formation of the MEG-OH adlayer) on steel samples. Elemental identification and modes characterized through deconvolution of carbon and oxygen narrow scan spectra were assigned based on the reported binding energy value obtained from the literature .2O3 and Cr(OH)3, are evident respectively at 530.1 eV and 531.7 eV  and Si (1s) relative atomic percentages at both 20 degrees and 90 degrees take-off angles . Through531.7 eV . This ob531.7 eV .The appearance of an organic fluorine peak for MEG-TFA-modified steel represents convincing evidence that at least the trifluoroacetyl group is present on the surface. An intense and broad peak due to organic silicon (2p), which is located at ~103 eV, and not observed on activated sample surface, also appears at both take-off angles. The formation of the MEG-TFA adlayer on the substrate surface is further supported by the deconvoluted C (1s) narrow scan spectra at both take-off angles, where peaks due to CF3 and C = O moieties are now apparent at ~294 eV and 289 eV, respectively. Hydrolysis of the TFA group to produce MEG-OH yields spectra at both angles that confirm the removal of the F(1s) peak. A careful inspection of the deconvoluted C (1s) and O (1s) narrow scan spectra also reveals component peak heights and areas associated with C–O and C–C/C–H increase marginally, suggesting that the MEG backbone stayed intact and undamaged when TFA solvolysis was taking place .n = 5) (n = 5), suggesting the surface has become hydrophilic and is fully saturated with hydroxyl groups. The contact angle of the MEG-TFA-modified surface returns to roughly the same level as the value for the bare sample, CA = 84.3 ± 4.6° (n = 5) due to the presence of hydrophobic TFA groups. After removal of the TFA group through solvolysis, the distal hydroxyl group of the MEG backbone is exposed, resulting in a substantial CA decrease to 19.7 ± 2.5° (n = 5). Overall, the contact angle data match the conclusions reached from the XPS surface characterization study detailed above, and agree with previously published work on MEG-OH [Untreated bare stainless steel shows a hydrophobic surface with a contact angle (CA) of 86.6 ± 2.8° (n = 5) a. Follown MEG-OH . Alongsi−1, 300 s−1, 600 s−1, 750 s−1, and 900 s−1 employed during such exposure. The dye DiOC6  chosen for this work is a commonly used cell-permeant, lipophilic, green fluorescent molecule primarily used for staining membrane-bound cellular organelles, such as mitochondria and endoplasmic reticulum [6 does not cause platelet activation, even for extended time periods, and secondly, in the presence of red blood cells only platelets and leukocytes are fluorescence-labeled since hemoglobin within red blood cells effectively quenches any fluorescence [6 is very straightforward, only requiring a minimum of 10 min of incubation at room temperature.Platelet aggregation on bare and MEG-OH surface-modified stainless steel following 20 min of interaction with whole human blood was studied for wall shear rates of 100 seticulum . Althougrescence . Further−1 and 600 s−1 shear rates are displayed in Representative frames for the bare and MEG-OH-modified surfaces as extracted after 20 min of blood perfusion at 100 sn = 5), 5.79 ± 1.39% (n = 5), 4.98 ± 0.60% (n = 5), 1.08 ± 0.08% (n = 5), and 0.94 ± 0.11% (n = 5), for the 100 s−1, 300 s−1, 600 s−1, 750 s−1, and 900 s−1 shear rates, respectively (n = 5) at 100 s−1, 0.29 ± 0.10% (n = 5) at 300 s−1, 0.37 ± 0.04% (n = 5) at 600 s−1, 0.10 ± 0.02% (n = 5) at 750 s−1, and 0.08 ± 0.01% at 900 s−1 shear rates, respectively. Student’s t-tests, assuming unequal sample variances, were performed to evaluate whether the calculated mean platelet surface percentage coverage on the bare and MEG-OH-modified steel surfaces is equal. The evaluated t-values for each blood shear rate are given in t-values at each tested shear rate are much greater than the tabulated Student’s t-critical values at degrees of freedom = 4, which is reported as 4.604. Therefore, there is a highly statistically significant difference between platelet surface coverage on bare and MEG-OH-modified steel.Briefly, in order to examine the data on a quantitative basis, the frames were subjected first to a conversion to grayscale, followed by binarization. The binarized images were then imported to image processing software. The number of black (representing the background) and white (representing the fluorescent platelet) pixels in each selected frame can be calculated. The ratio of white pixels to the total pixel number in a thresholded frame, which can be calculated by the “area fraction”, is reflective of the surface coverage area associated with platelets. The surface platelet percentage coverage on the control groups was calculated at 99% confidence to be 7.72 ± 0.38%  platelet surface coverage on bare steel correspondingly decreases for each recorded time period. The platelet surface coverage in these cases nearly triples from 20 min to 60 min, eventually reaching 20.2% and 10.7%, respectively, for each of these two shear rates. In stark contrast, MEG-OH-modified steel surfaces clearly display a remarkably high degree of resistance to surface plate adhesion, evidenced by the reduction in surface platelet coverage by over 90% under all tested shear rates after 60 min of blood perfusion, compared with the bare samples. In addition, the surface platelet micro-aggregate formation on MEG-OH-surface-modified steel is observed to maintain a minimal and steady rate, particularly under the 600 s−1 and 900 s−1 shear rates. Shear rates in arteries are typically greater than 300 s−1 [The results of these experiments for the 100 and 600 s 300 s−1 , which bThe success exhibited in this work with respect to the significant reduction in platelet adhesion to steel by surface-attached MEG-OH requires explanation. The generation of thrombi on foreign surfaces from blood is a complex process which is considered to first involve protein adsorption followed by platelet attachment . AccordiThe research described herein demonstrates that it is feasible to transfer oligoethylene silane surface chemistry, previously established for polymers, to 316L stainless steel, a widely used material for a number of biomedical implants, including stents. Surface characterization by XPS and contact angle goniometry has confirmed the successful modification of steel samples with MEG-OH coatings. Collectively, surface thrombogenicity assays involving fluorescence-labeled whole human blood has confirmed dramatic resistance to surface platelet adhesion and thrombus formation by the MEG-OH adlayer under all experimental shear rates. Statistical testing has indicated that there is a highly significant difference between platelet surface coverage on bare and MEG-OH-modified steel. The monolayer yields a reduction in surface platelet coverage by at least 90% on average, under all tested shear rates, in both end-stage and real-time blood perfusion experiments. In terms of relevance to stent technology, the oligoethylene monolayer provides a number of useful features in addition to its capability to reduce platelet aggregation. It is highly stable to standard sterilization protocols and can be stored without degradation for considerable periods of time, while also being low-cost, and simple to apply to samples. These advantages make it highly appealing for use on medical devices made of steel.Despite the attractiveness of the silane chemistry evaluated here, a number of aspects warrant further investigation. For example, high platelet concentration can cause blood to be more sensitive to coagulation triggers. In order to avoid potential systemic biases and errors, it would be beneficial to study blood samples from a number of sources in future investigations. Finally, with respect to clinical application, long-term surface stability in blood can be crucial. There is an indication that certain organosilane adlayers are susceptible to cleavage by hydrolysis when immersed in aqueous or buffer environment for extended periods of time, especially at higher or lower pH conditions . In this


Objective: To investigate the association between plasma amyloid-β (Aβ) levels and neuropsychological performance in patients with cognitive decline using a highly sensitive nano-biosensing platform.Methods: We prospectively recruited 44 patients with cognitive decline who underwent plasma Aβ analysis, amyloid positron emission tomography (PET) scanning, and detailed neuropsychological tests. Patients were classified into a normal control  or Alzheimer’s disease  group based on amyloid PET positivity. Multiple linear regression was performed to determine whether plasma Aβ  levels were associated with neuropsychological test results.Results: The plasma levels of Aβ42/40 were significantly different between the NC and AD groups and were the best predictor of amyloid PET positivity by receiver operating characteristic curve analysis [area under the curve of 0.952 ]. Although there were significant differences in the neuropsychological performance of cognitive domains  between the NC and AD groups, higher levels of plasma Aβ42/40 were negatively correlated only with verbal and visual memory performance.Conclusion: Our results demonstrated that plasma Aβ analysis using a nano-biosensing platform could be a useful tool for diagnosing AD and assessing memory performance in patients with cognitive decline. Alzheimer’s disease (AD), a neurodegenerative disease, is the most common cause of cognitive impairment and dementia . The patin vivo amyloid positron emission tomography (PET) scans has shown a high correlation with postmortem findings  analysis and findings ; howeverfindings , a more findings . With adfindings .We previously developed a sensing platform capable of analyzing biomolecules at levels in the range of fg/mL with high accuracy . Our bio40, Aβ42, and Aβ42/40) and neuropsychological performance in patients with cognitive decline, classified according to amyloid PET positivity using a highly sensitive nano-biosensing platform. Considering that neuropsychological performance in all cognitive domains, including attention, language, visuospatial, memory, and frontal/executive functions, are impaired in patients with AD ,  testing . The par testing . Patient testing  with mod testing . Patient testing . Brain M testing , or curr12 levels, folate levels, metabolite profiles, thyroid function tests, and syphilis serology. Genomic DNA was extracted from whole blood samples for apolipoprotein E (APOE) genotyping according to the manufacturer’s instructions .Laboratory tests were conducted for all participants to eliminate any other causes of cognitive impairment. These tests included complete blood counts, vitamin BWritten informed consent was obtained from all participants  prior to inclusion in the study. The study was approved by the Institutional Review Board (IRB) of Kyung Hee University Hospital (IRB No. 2018-01-023).To demonstrate an association between the level of plasma Aβ and neuropsychological performance, we collected approximately 1 mL of blood from each participant in an anticoagulant-treated heparin vacutainer tube . Human plasma was then separated from the blood using an ultra-centrifuge  and transferred into a polypropylene (PP) tube. Finally, the plasma was mixed with 1 × protease inhibitor  in 10 mM phosphate-buffered saline (PBS), and then approximately 1 mL was aliquoted into fresh 1.5-mL PP tubes. The aliquoted samples were stored frozen (−80°C) and diluted 1/100 in 1 mM PBS buffer immediately prior to plasma analysis.2) and 300/1500 Å tantalum/platinum (Ta/Pt) were deposited on a 4-inch silicon wafer. Then, the Ta/Pt layers were patterned via photolithography and reactive ion etching (RIE). The single sensing platform consisted of six pairs of interdigitated microelectrodes (IMEs) designed with 30 pairs of electrode fingers with 5-μm spacing and 300-μm length.The sensing platform was fabricated via a microelectromechanical system (MEMS) . First, 40 and Aβ42, respectively. The surface of the sensor was cleaned and chemically activated by sequential treatment with piranha cleaning solution (H2SO4:H2O2 = 4:1), 1% 3-(ethoxydimethylsilyl) propylamine (APMES), and N-(3 -dimethyl aminopropyl)-N-ethylcarbodiimide hydrochloride /N-hydroxysuccinimide (EDC/NHs) cross-linking solution. Then, 10 μL of antibody solution was dropped on the surface of the sensor to immobilize the antibody.Meanwhile, two types of antibodies, 11A50-B10 and 12F4, were immobilized on the surface sensor to analyze Aβbefore and ΔZafter, respectively) as follows:To quantify Aβ, a polydimethylsiloxane (PDMS) microfluidic channel fabricated through the MEMS process was attached to the sensor surface, and 10 μL of Aβ solution diluted into 1 mM PBS and standard human plasma was injected into the channel using a syringe . Then, uSimilar to the procedure for quantification of Aβ in PBS and standard plasma, Aβ was quantified in clinical samples to diagnose AD. A single clinical sample was quantified using three sensing platforms , and the average of 18 IME signals was used as the final output signal due to the Aβ response in human plasma.All participants underwent detailed neuropsychological tests using the standardized Seoul Neuropsychological Screening Battery . The bat2, and voxel size = 1 × 1 × 1 mm3.Structural three-dimensional (3D) T1-weighted images (T1WIs) were acquired using a 3.0-T MRI system  equipped with a 32-channel sensitivity-encoding head coil and multi-transmit technology. The T1WI sequence used was a magnetization-prepared rapid-acquisition gradient-echo sequence with the following parameters: relaxation time = 9.0 ms, echo time = 5.0 ms, flip angle = 8°, field-of-view = 256 × 256 mm3.3D static PET images were acquired 90 min after intravenous injection of 296 MBq of 18F-florbetaben using a Gemini TF 16 PET/CT scanner . CT images were acquired from the vertex to the skull base , and 10-mm PET images were immediately acquired over the same region. We used the ordered-subset expectation maximization algorithm (iteration = 3 and subset = 33) to reconstruct 3D PET images in a 128 × 128 × 20 matrix with a voxel size of 2 × 2 × 2 mmAll amyloid PET images were visually assessed and dichotomized as amyloid-positive or amyloid-negative after being reviewed by nuclear medicine physicians who were blinded to the participants’ information. Amyloid PET findings were considered positive when the brain amyloid-plaque load (BAPL) score  was 2−3,18F-florbetaben PET binding evaluations, as the comparison of the two methods resulted in a high accuracy of 100% .We also conducted a quantitative amyloid PET analysis using the cortical standardized uptake value ratio (SUVR) in all PET scans. Based on a previous study , an SUVR40 and Aβ42 in the brain. First, cortical and subcortical brain regions were automatically segmented using Freesurfer 5.3,1 following the pipeline described at https://surfer.nmr.mgh.harvard.edu/fswiki/recon-all. Second, 18F-florbetaben PET images were co-registered to the 3D T1WI-labeled data using the Statistical Parametric Mapping Version 12 program . Amyloid cortical SUVR was determined as the average of the standardized uptake value normalized by the uptake in the cerebellar gray matter, with this reference region being selected from the cerebellum external and cortex segments on MRI. Third, pattern analysis was conducted between Aβ present in the plasma and SUVR in amyloid PET using the coefficient of linear correlation. The results showed the average correlation coefficient in each brain region of interest as color-coded overlays on the representative MR image.Pattern analysis was performed with MRI and amyloid PET images to visually estimate the relative levels of plasma Aβt-test and are presented as the mean ± SD. Categorical variables were compared using the chi-squared test or Fisher’s exact test. For multiple comparisons, we performed non-parametric statistical testing as the data were not normally distributed . Multiple linear regression was performed using vascular risk factors , APOE ε4 status, and Aβ40, Aβ42, and Aβ42/40 as independent variables. Statistical significance was set at p < 0.05, in two-tailed tests. Statistical analyses were performed using SPSS  and Prism 8 . Also, to compute the area under the curve (AUC), a receiver operator characteristic (ROC) curve was created using Prism 8. We used a hybrid Wilson/Brown method to compare ROC curves and determine the 95% confidence intervals associated with the sensitivities and specificities (at Youden index thresholds).Continuous variables were compared using Student’s 42-specific antibodies were immobilized, an approximately 1.862-fold higher signal change occurred following the specific binding of Aβ42 compared with the change following the non-specific binding of Aβ40 . However, in the biosensor where Aβ42-specific antibodies were immobilized, the signal change due to the specific binding of Aβ42 was approximately 1.536-fold higher than that due to non-specific binding of Aβ40 . In addition, the average impedance variation due to buffer exchange and non-specific adsorption of plasma proteins on the surface of the platform was approximately 2.086 ± 0.585%.Our biosensing platform showed high specificity in the analysis of Aβs in 1 mM PBS, depending on the type of immobilized antibody on the surface of the platform . Because42 and Aβ40 ranged from approximately 3.241 ± 0.053%−10.684 ± 1.296% and from 1.563 ± 0.169% to 3.919 ± 0.632%, respectively, depending on the concentrations of Aβ in 1 mM PBS  or AD (n = 19) group. The average signal levels of Aβ40 (p = 0.012) and Aβ42 (p = 0.004) were significantly different between the NC and AD groups. This difference was clearly reduced when the signal level of Aβ42 was divided by that of Aβ40 (NC vs. AD: p < 0.001 for Aβ42/40) , 0.892–1.000]  and a negative percentage agreement of 0.95  with amyloid PET positivity.Aβ42/40) . In addiAβ42/40) . ROC cur2–1.000] . Particir) between plasma Aβ42/40 and amyloid PET SUVR is presented in 42/40 and amyloid PET SUVR showed moderate positive correlations in the bilateral frontal, parietal, temporal, and occipital cortices, except for the primary motor and sensory cortices, suggesting a typical pattern of regional deposition for neocortical Aβ in AD.A topographical pattern of the correlation coefficient (p = 0.002), SVLT , RCFT , COWAT phonemic total (p = 0.030), and Stroop color reading (p = 0.012) tasks between the NC and AD groups.As shown in 40, Aβ42, and Aβ42/40) and neuropsychological performance are presented in 40 and Aβ42 levels and neuropsychological performance in the study participants. However, higher levels of plasma Aβ42/40 showed a significant negative correlation with the Z-score of the SVLT , and RCFT  tasks in the study participants.The results of multivariate regression analysis between the levels of plasma Aβ  was the best predictor of amyloid PET positivity in the study participants, and our findings were consistent with previous results using plasma Aβ analysis , it was not possible to evaluate the effect of each form on the study results. Finally, our study participants were enrolled from a single memory clinic, which might limit the generalizability of the results. Despite these limitations, we suggest that our study might have clinical significance because plasma Aβ analysis using a highly sensitive nano-biosensing platform could not only be a useful non-invasive method for diagnosing AD, but also showed a significant association with memory performance in patients with cognitive decline. Further investigations with a larger sample size are necessary to clarify the possible relevance of plasma Aβ as a blood-based biomarker for precise AD diagnosis.The datasets generated for this study are available on request to the corresponding authors.The studies involving human participants were reviewed and approved by the Institutional Review Board of Kyung Hee University Hospital. The patients/participants provided their written informed consent to participate in this study.IH and JL: conception, design of the study, and final approval of the manuscript. GY, HK, KL, IH, HR, SY, K-CP, and JL: acquisition of data. GY, HK, H-GK, KL, IH, HR, G-HJ, SY, K-CP, IH, and JL: analysis and interpretation of the data. GY, HK, HR, K-CP, IH, and JL: drafting and revising the manuscript for content. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Vβ3 integrin’ on the surface of cancer cells. The second part is a new cytotoxic drug, VIP126. The third component links the two parts together and can only be sliced by the enzyme neutrophil elastase. Neutrophil elastase is found in high quantities in tumors, ensuring that VIP126 is released near the cancer cells and less around normal cells. VIP126, when delivered as a component of VIP236, kills tumor cells in mouse cancer models without the toxicity seen with standard chemotherapy. VIP236 may be a new modality for targeting cancer cells directly, while reducing the harmful effects to normal tissue.Many cancer drugs are cytotoxic, which means that they kill cancer cells effectively but are also toxic to normal cells. To overcome this problem, we designed a novel compound, VIP236, which consists of three components. The first part enables the drug to target and bind to a protein called ‘αVβ3 integrin binder linked to a modified camptothecin payload (VIP126), which is released by the enzyme neutrophil elastase (NE) in the tumor microenvironment (TME). The tumor targeting and pharmacokinetics of VIP236 were studied in tumor-bearing mice by in vivo near-infrared imaging and by analyzing tumor and plasma samples. The efficacy of VIP236 was investigated in a panel of cancer cell lines in vitro, and in MX-1, NCI-H69, and SW480 murine xenograft models. Imaging studies with the αVβ3 binder demonstrated efficient tumor targeting. Administration of VIP126 via VIP236 resulted in a 10-fold improvement in the tumor/plasma ratio of VIP126 compared with VIP126 administered alone. Unlike SN38, VIP126 is not a substrate of P-gp and BCRP drug transporters. VIP236 presented strong cytotoxic activity in the presence of NE. VIP236 treatment resulted in tumor regressions and very good tolerability in all in vivo models tested. VIP236 represents a novel approach for delivering a potent cytotoxic agent by utilizing αVβ3 as a targeting moiety and NE in the TME to release the VIP126 payload—designed for high permeability and low efflux—directly into the tumor stroma.To improve tumor selectivity of cytotoxic agents, we designed VIP236, a small molecule–drug conjugate consisting of an α Recent research interest has previously focused on antibody–drug conjugates (ADCs) that utilize antibodies to deliver potent cytotoxic agents ,2,3. In  in 2011 ,5; Kadcy in 2013 ,7; Bespo in 2017 ; Mylotar in 2017 ,10; Poli in 2019 ,13; Trod in 2020 ; and Zyn 2020 [® , Aidixi®20 [® [® , and TivVβ3 integrin-targeting moiety and a neutrophil elastase (NE)-cleavable linker with a modified camptothecin (CPT), VIP126, as the cytotoxic payload  [®) [®) [The modular composition of the SMDC enabled us to optimize the components separately while considering opportunities for synergy. We reasoned that the success of the SMDC approach would be highly dependent on the cytotoxic payload employed. The CPT class of cytotoxic agents is potent with a well-understood mechanism-of-action . Inhibit®)  and saci®) [®) . In addi®) [®) ,33. VIP1 [®) [®) . It has Vβ3 integrin-targeting moiety in VIP236 was selected based on the abundant expression of this cell surface protein on cancer cells and activated endothelial cells in a wide range of tumor types [Vβ3 integrins could suppress tumor angiogenesis without affecting quiescent endothelial cells in normal tissues [The αor types ,39,40,41 tissues . Therefo tissues . Neverth tissues .Vβ3 to arginine-glycine-aspartic acid (RGD) sequences present on many extracellular matrix proteins, has been well characterized [Vβ3 integrins presents an attractive mechanism for selective delivery of cytotoxic agents into the tumor microenvironment (TME). Indeed, αVβ3 targeting conjugates and nanoparticles have been investigated to deliver chemotherapy compounds [The binding of integrins, such as αcterized ,45,46,47ompounds ,49,50, gompounds , and siRompounds  to tumorVβ3 integrins, neutrophil infiltration and elastase expression in tumors have been shown to correlate with their metastatic potential and poor prognosis [Vβ3 integrin-targeting moiety combined with the NE-specific linker in VIP236 could be ideally suited for restricting delivery of the novel cytotoxic payload VIP126 into the TME.Extracellular enzymatic cleavage of the VIP236 conjugate is essential for the release of the active agent VIP126. The design of VIP236 with a cleavage site for NE, a highly specific protease enriched in the TME of many malignant tumors , enabledrognosis . TherefoVβ3 integrin binder and an NE-cleavable linker connected to the cytotoxic payload VIP126.Herein, we describe the pharmacokinetic characteristics and in vitro and in vivo efficacy of VIP236—a novel SMDC consisting of an αCaco-2 human colon adenocarcinoma, NCI-H1975 human non-small cell lung cancer (NSCLC), 786-O human RCC, HT-29 human CRC, LoVo human CRC, SW480 human CRC, NCI-H292 human pulmonary mucoepidermoid carcinoma, NCI-H69 human SCLC, 4T1 mouse breast cancer, and MX-1 human triple negative breast cancer cells were acquired from ATCC and cultured according to the provider’s instructions. LLC-PK1 porcine renal epithelial cells overexpressing human P-gp (L-MDR1 cells) were obtained from Prof. Schinkel at Netherlands Cancer Institute . The recombinant NCI-H1975-P-gp and NCI-H1975-BCRP NSCLC cell lines were generated by the Natural and Medical Sciences Institute  by lentiviral transduction using the plasmids pGT414_Hyg_PGP and pGT414_Hyg_BCRP. The cancer cell lines were authenticated using short tandem repeat DNA fingerprinting at DSMZ and subjected frequently to mycoplasma testing.Vβ3 binder, the modified CPT payload VIP126, and the NE-cleavable linker were synthesized at Bayer AG. The dye conjugate BAY810, consisting of the αVβ3 binder BAY812 coupled to IRDye® 800CW , and BAY813, a non-binding control conjugate of IRDye® 800CW, were also synthesized at Bayer AG. Topotecan (Hycamtin®) was purchased from GlaxoSmithKline . Irinotecan (camptothecin-11) was purchased from Sanofi-Aventis . SN38 was synthesized at Bayer AG. Doxorubicin, cisplatin, and 5-FU were purchased from Sigma-Aldrich .The SMDC VIP236, consisting of the αPapp) was calculated for both the apical to basolateral (A → B) and the basolateral to apical (B → A) direction as described by Schwab et al. [The cell permeability of the VIP126 and SN38 payloads was investigated with in vitro flux assays using Caco-2 cells  or P-gp-b et al. .3 cells/well) were plated in RPMI-1640 medium supplemented with 2% fetal calf serum (FCS) minimal media (MM) in 96-well plates and allowed to adhere at 37 °C/5% CO2 for 24 h. Then, the test compounds VIP126 and SN38 were added at concentrations of 1000–250–62.5–15–4–1–0.25 nM in MM  concentration of 0.1%) in triplicates. The cells were incubated at 37 °C/5% CO2 for 72 h. Then, Alamar Blue  was added at a 1:1 ratio, the cells were incubated at 37 °C/5% CO2 for 4 h, and fluorescence (530 nm/590 nm) was measured with a VICTOR X2 Multilabel Reader . The fluorescent signal was directly proportional to the number of viable cells. The half-maximal growth inhibition (IC50) was determined from the dose–response curves using the GraphPad Prism software.The in vitro cytotoxicity of the VIP126 and SN38 payloads was investigated in parental NCI-H1975 and in transfected NCI-H1975-P-gp and NCI-H1975-BCRP NSCLC cells. Cells (6 × 103 cells/well) were plated in their appropriate growth medium in 96-well plates, allowed to adhere at 37 °C/5% CO2 for 24 h, and then the test compounds were added at concentrations of 1 × 10−5–1 × 10−13 M in triplicates. Two identical sets of samples were prepared; the first set was treated with the test compound alone and the second set was treated with the test compound and 10 nM NE (Sigma-Aldrich). Cells were incubated at 37 °C/5% CO2 for 72 h, and the proliferation was determined using the MTT assay according to the manufacturer’s instructions. The proliferation of untreated, but otherwise identically handled, cells was defined as 100%. The half-maximal growth inhibition (IC50) was determined from the dose–response curves using the GraphPad Prism software.The antiproliferative activity of VIP236 and VIP126 was evaluated in a panel of cancer cell lines using the MTT Cell Proliferation Assay (ATCC). Briefly, cells  were inoculated subcutaneously (s.c.) with 2 × 106 786-O RCC cells suspended in phosphate-buffered saline (PBS). The mice were fed with chlorophyll-free chow  7 days prior to imaging to reduce the background fluorescence. When the 786-O tumors reached an average tumor size of 0.7 cm in diameter, the mice (n = 3 mice/group) were injected with 20 nmol of BAY810 (1.35 mg/kg), consisting of the αVβ3 binder coupled to IRDye® 800CW , according to the manufacturer’s instructions, or BAY813 (1.35 mg/kg), a non-binding control conjugate of IRDye® 800CW. As a negative control, one mouse was injected with IRDye® 800CW carboxylate (0.84 mg/kg) in a sterile 0.9% NaCl aqueous solution (Sigma-Aldrich). Tumor accumulation of the IRDye® conjugates in mice was determined 8, 24, and 48 h post administration using the LI-COR Pearl® imaging system .All animal experiments were conducted in accordance with the German Animal Welfare Law and approved by local authorities. To study the tumor homing of the αn = 2 mice/group at each time point), and the concentrations of VIP236 and VIP126 were measured by LC-MS/MS. Pharmacokinetic analysis of VIP236 and VIP126 was performed using the mean concentration–time profiles. All pharmacokinetic parameters were calculated by non-compartmental methods, as previously described [The pharmacokinetic properties of VIP236 and VIP126 in plasma and tumor were estimated in female NMRI nu/nu xenograft mice (Taconic M&B A/S) bearing 786-O human RCC tumors. The mice were treated intravenously (i.v.) via the tail vein with a single dose of VIP236 (4 mg/kg) or VIP126 (1 mg/kg). The doses of VIP236 and VIP126 were chosen so that an equimolar amount of VIP126 was administered regardless of whether it was delivered utilizing VIP236 or delivered directly. Plasma and tumor samples were collected at 2 min, 10 min, 30 min, 1 h, 2 h, 4 h, 7 h, and 24 h after treatment , MX-1 (human TNBC), NCI-H69 (human SCLC), and SW480  xenograft models.VIP236 was formulated in PBS. Irinotecan, doxorubicin, topotecan, cisplatin, and 5-FU were diluted in 0.9% NaCl. In all in vivo antitumor efficacy studies, the vehicle control group was treated with the VIP236 formulation buffer. 6 786-O cells suspended in 100% Matrigel®. The mice were randomized into control and treatment groups (n = 8) when the 786-O tumors reached an average size of 25 mm2. The 786-O tumor-bearing mice were treated with vehicle or VIP236 .In the 786-O efficacy study, immunocompromised female NMRI nu/nu mice  were inoculated s.c. with 2 × 10n = 8) when the MX-1 tumors reached an average size of 25 mm2. The MX-1 tumor-bearing mice were treated with vehicle, VIP236 at 23, 36, or 40 mg/kg (3 days on/4 days off (3 on/4 off), i.v.), irinotecan at 15 mg/kg  or 30 mg/kg , or doxorubicin at 10 mg/kg .In the MX-1 efficacy study, the in vivo-grown tumor fragments (2 mm × 2 mm × 2 mm) were implanted s.c. in immunocompromised female NMRI nu/nu mice . The mice were randomized into control and treatment groups (6 NCI-H69 cells suspended in 50% Matrigel®/50% medium. The mice were randomized into control and treatment groups (n = 8) when the NCI-H69 tumors reached an average size of 25 mm2. The NCI-H69 tumor-bearing mice were treated with vehicle, VIP236 , topotecan , or cisplatin .In the NCI-H69 efficacy study, immunocompromised female NMRI nu/nu mice  were inoculated s.c. with 3 × 106 SW480 cells suspended in 50% Matrigel®/50% medium. The mice were randomized into control and treatment groups (n = 8) when the SW480 tumors reached an average size of 25 mm2. The SW480 tumor-bearing mice were treated with vehicle, VIP236 at 23, 36, or 40 mg/kg , irinotecan at 15 mg/kg  or 30 mg/kg , or 5-FU at 100 mg/kg .In the SW480 efficacy study, immunocompromised female NMRI nu/nu mice  were inoculated subcutaneously with 3 × 102 unless ulceration or other predefined humane endpoints were met. At study termination, the animals were sacrificed by cervical dislocation under CO2 anesthesia. T/C (treatment/control) ratios were calculated using the final mean tumor areas at study or control group termination. Treatment responses were defined on the last day of the vehicle group  using the RECIST criteria [Subcutaneous tumor growth was monitored by measuring tumor area (length × width) using a caliper. Animal body weight was monitored as an indicator of treatment-related toxicity. Measurement of tumor area and body weight was performed at least twice weekly. Individual animals were sacrificed when showing >20% body weight loss or when tumors reached a maximum area of ~200 mmcriteria . Progrest-tests. p values < 0.05 were considered significant.Statistical analyses were performed using the GraphPad Prism software (version 9). Tumor area data were analyzed using one-way ANOVA followed by Dunnett’s test or paired Vβ3 integrin binder in 786-O human renal cell carcinoma (RCC) tumor-bearing mice via conjugation to a fluorescent IR800 dye using in vivo near-infrared imaging. We used an i.v. dose of 1.35 mg/kg for the IR800-coupled αVβ3 integrin binder and the non-binding IR800-coupled control conjugate or 0.84 mg/kg for the carboxylate dye negative control  per mouse, and the mice were imaged 8, 24, and 48 h post administration. The IR800-coupled αVβ3 integrin binder showed strong tumor homing in 786-O RCC tumor-bearing mice .First, we investigated the tumor homing ability of the αing mice A, whereaing mice B. Overaling mice C. Inject®) as well as the payload of the ADC sacituzumab govitecan (Trodelvy®) [50) values compared with the parental NCI-H1975 cells  efflux pump expressed on cancer cells, are known to contribute to resistance to chemotherapy treatment . The cytodelvy®) . Here, w75 cells .Vβ3 binder and the VIP126 payload, which is connected via a linker designed to be selectively cleaved by NE. Since tumor cell lines do not express NE, we evaluated the in vitro cytotoxic activity of the VIP236 conjugate in the presence or absence of NE in a panel of cancer cell lines. VIP236 showed weak antiproliferative activity in the absence of NE, with IC50 values in the two to three-digit nanomolar range. When NE was added to the culture medium to simulate the TME, the cytotoxic activity of VIP236 increased to levels comparable to the VIP126 payload alone, reaching single-digit nanomolar IC50 values  was low for VIP236 and the approximated plasma volume, suggesting limited distribution outside of plasma [ss was moderate, being 22-fold higher compared with VIP236, suggestive of higher penetration into cells. The half-life (≤1 h) was short for both compounds. Finally, the delivery of VIP126 via VIP236 resulted in reduced systemic exposure of VIP126, as demonstrated by a ~6-fold lower area under the curve value (AUCplasma) compared with direct VIP126 delivery. The reduced systemic exposure is anticipated to reduce potential adverse effects  was ~72% higher compared with direct VIP126 administration (tumor/AUCplasma) with VIP236 conjugate administration was 6 (p < 0.001) on Day 38, when the vehicle group was sacrificed , NCI-H69 small cell lung cancer (SCLC), and SW480 colorectal cancer (CRC) xenograft mouse models.p < 0.001) and with doxorubicin (p < 0.001) with less toxicity  were observed in >80% of mice across all three VIP236 doses   showed marked antitumor efficacy with a T/C of 0.06 and partial responses (PRs) in 8/8 mice  and was = 0.022) E. On Dayeatments F. In conp < 0.001) more efficacious than 5-fluorouracil (5-FU), a chemotherapy commonly used to treat CRC.In the SW480 CRC model, VIP236 treatment resulted in clear tumor growth inhibition at all three doses, similar to irinotecan . TreatmeVβ3 integrin. In the TME, the payload is cleaved by NE and enters cancer cells to induce DNA damage and cell killing [We developed a potent modified CPT payload, VIP126, to be selectively delivered to cancer cells by an SMDC targeting α killing . Many tu killing . In contVβ3 is most abundantly expressed on angiogenic endothelial cells in remodeling and pathological tissues [Vβ3 integrins play critical roles in tumor progression, resistance to cytotoxic therapy, metastasis, and the recruitment of immune and inflammatory cells . They hiewed in ,39,40,41l cancer ,67 and ml cancer . In expeal cells , raisingiewed in ). Vβ3 inhibitory antibodies, based on a humanized form of the mouse monoclonal LM609 , were tested and well tolerated [V integrin antibodies, abituzumab [Vβ3 and αVβ5 integrins, performed well in preclinical studies [Vβ3 signaling, resulting in stimulated angiogenesis [Vβ3-targeting molecules as anti-angiogenic drugs may be due to limitations intrinsic to all inhibitory approaches, in particular adaptive resistance [Although the inhibition of integrins has long been considered a viable approach to inhibit tumor angiogenesis and growth, this concept has not been successful in the clinic. Anti-αolerated ,71, as wituzumab  and inteituzumab . In addi studies ,75 and p studies . However studies ,78,79,80ogenesis . The faisistance . Functiosistance  may be tsistance . Vβ3 small molecule binder to focally concentrate a chemotherapeutic warhead to kill tumor cells and angiogenic endothelial cells. This approach does not depend on the inhibition of integrin signaling for its therapeutic effect. Using small molecules to target integrins for the delivery of cytotoxic payloads is a relatively new and untested strategy, which has not yet been advanced to clinical trials [Vβ3 integrin-targeting nanoparticles were used with limited success to deliver chemotherapeutic compounds to tumors [Our approach employs a selective integrin αl trials . Previoul trials . Howevero tumors ,49. VIP2Vβ3 ligand that demonstrated efficient tumor homing when conjugated to a fluorescent dye.VIP236 is an SMDC rather than an ADC, which affords certain advantages. SMDCs are less immunogenic and easier to synthesize . For VIPVβ3 integrin, NE expression is also correlated with poor prognosis [To further enhance the targeting specificity of the VIP236 SMDC, we engineered an NE cleavage site into the linker peptide that connects the targeting moiety to the payload and enables the release of the payload without the formation of intermediates. The cleavage site was optimized for effective and specific cleavage by NE in the TME and it is stable in blood plasma, which minimizes systemic payload release. This restricts the drug release to the extracellular regions of the tumor where high concentrations of the enzyme occur. NE is a member of a family of serine proteases that degrade elastin and other extracellular matrix proteins . It is erognosis .50 values in the mid-nanomolar range. When NE was added to the culture medium to simulate the TME, the antiproliferative activity of VIP236 increased to levels comparable to the VIP126 payload alone, with single-digit nanomolar IC50 values. This indicates that VIP236 functions as an antiproliferative compound only upon specific cleavage by a protease prevalent in the TME. The tumor-to-plasma ratios of the payload VIP126 were 10-fold enhanced when delivered as a conjugate versus direct administration. Finally, in vivo treatment with VIP236 resulted in tumor regression in all xenograft models tested , with good tolerability. VIP236 exhibited strong biologic activity in in vitro and in vivo experiments. In cultured cancer cells, the compound showed weak antiproliferative activity in the absence of NE, with ICVβ3 integrin targeting combined with payload release by NE in the TME focus the cytotoxic activity to the tumor. This TME targeting, along with the high conjugate stability in plasma and the release of VIP126 at its site of action, further supports the broadening of the therapeutic window. Moreover, the beneficial properties of VIP126  present a promising approach to overcome common tumor resistance mechanisms. These findings support further development of VIP236 as a potential treatment option for advanced or invasive cancers. The SMDC VIP236 benefits from the synergy resulting from its modular composition. Tumor homing by αVβ3 integrin on cancer cells and activated endothelial cells and to release its modified CPT payload VIP126 when selectively cleaved by NE in the TME. The αVβ3 integrin binder demonstrated efficient tumor homing in vivo. VIP126 is a novel, modified SN38 derivative with higher cellular permeability and significantly reduced efflux potential. In contrast to SN38, VIP126 also retained its cytotoxic activity in transporter-expressing mutant cell lines. A 10-fold higher tumor-to-plasma ratio of VIP126 was shown after conjugate administration compared with i.v. administration of the payload alone. Treatment with VIP236 resulted in tumor regressions in all tested xenograft models in vivo , with good tolerability.A novel SMDC VIP236 was developed and optimized to bind to α