The results demonstrated that the steroid-r-PDMAEMA copolymers could efficiently bind pDNA (N/P < 3.0) and then form near-spherical shape (142–449 nm) and positively-charged (+11.5 to +19.6 mV) nanoparticles. The in vitro cytotoxicity and gene transfection efficiency greatly depend on the steroid hydrophobic tail structures and steroid/PDMAEMA block ratios. Optimum transfection efficiency of the (Chol-P1/pDNA and Dios-P3/pDNA) nanoplexes could reach to 18.1–31.2% of the PEI-25K/pDNA complex. Moreover, all of the steroid-r-PDMAEMA/Cy3-pDNA nanoplexes have an obvious “lysosome localization” effect, indicating the steroid structures do not remarkably influence the intracellular localization behaviors of these nanoplexes.Using natural-based lipids to construct biocompatible, controllable and efficient nanocarriers and elucidating their structure–function relationships, was regarded as an important area for creating sustainable biomaterials. Herein, we utilized two natural steroids: cholesterol and diosgenin (bearing different hydrophobic tails) as the building blocks, to synthesize a series of natural steroid-based cationic random copolymers PMA6Chol- Using natural-based lipids to construct biocompatible, controllable and efficient nanocarriers and elucidating their structure–function relationships, was regarded as an important area for creating sustainable biomaterials. Meanwhile, researchers made great efforts to elucidate the structure–function relationships of cationic polymers so as to obtain the optimal balance between gene transfection efficiency and cytotoxicity.11–14Cationic polymers as non-viral gene carriers have been intensively studied in recent decades, due to their controllable structure and function, excellent siRNA/pDNA condensing capability, as well as low immunogenicity.15 Hydrophobic moieties, including polyesters,16–20 poly(meth)acrylate21 and natural lipids22,23 were incorporated into cationic polymers to prepare stable nanoparticles, microcapsules or liposomes. Xu et al.24 revealed that phenylalanine could enhance the gene transfection efficacy of poly(amidoamine)s (PAMAM) in SMMC-7721 and COS-7 cells. Lee et al.25,26 found that dexamethasone-conjugated polyethylenimine (PEI)/PAMAM have higher gene delivery efficiency than their unmodified counterparts in acute lung injury (ALI) animal models. Dong et al.18–20,27–31 developed a series of polyester-based cationic polymers with enhanced pDNA (or siRNA) transfection efficacy. Moreover, changing hydrophobic core in nanoparticles (NPs) could affect the delivery efficiency of NPs/gene nanoplexes by changing their endocytic pathways and intracellular siRNA release behavior. In a previous work, we disclosed that introducing proper length of hydrophobic poly(l-lactide) into poly(l-lysine) could increase pDNA binding, nanoplex stability, and improve gene transfection capability through an efficient caveolae-mediated endocytosis pathway.32,33 In some cases, introducing natural lipids, such as cholesterol, phospholipid and vitamin E, could effectively decrease cytotoxicity and improve gene transfection efficiency.34–37 Eliyahu et al.38 modified dextran-spermine conjugated cationic polymer with natural oleic acid, which showed improved transfection capability in NIH-3T cells. Likewise, Neamnark et al.39 found aliphatic lipid modification of PEI-25K would greatly enhance its transfection efficiency. Nevertheless, up to date, the relationship between hydrophobic (lipid) moiety/block ratios of cationic gene carriers and their apparent cytotoxicity and gene transfection efficiency, is still not fully addressed.Introducing hydrophobic moieties into cationic polymers could improve plasmid (pDNA) binding/loading affinity and nanoplex stability by facilitating the formation of “hydrophobic core–hydrophilic shell” structure, they could also increase membrane fusion and cellular uptake, which gave rise to enhanced gene transfection.40 Bhattacharya et al.41 disclosed that some cholesterol-modified oligoethylenimine (Cho-OEI) polymers possessed higher gene transfection efficiency than the “gold standard” bPEI-25K. Davis et al.42,43 prepared cholesterol-containing random cationic polymer gene (pDNA) carriers from cholesteryl methacrylate (CMA) and (dimethylamino)ethyl methacrylate (DMAEMA) monomers, the optimum transfection ratio was achieved on 2% cholesterol molar ratio (N/P = 20). Dufès et al.44 designed disulfide-linked cholesterol-PEGylated diaminobutyric polypropylenimine G3 dendrimers, the introduction of cholesterol could enhance the stability of nanovesicles and promote drug/gene co-delivery efficiency. Lee et al.45 synthesized cholesterol-conjugated polyamidoamine (PAM-Chol) for combined delivery of anti-inflammatory gene and drug into the lungs by inhalation, which showed higher transfection efficiency than lipofectamine 2000 and bPEI-25K in L2 lung epithelial cells. Moreover, it was revealed that introducing cholesterol to cationic gene carriers could offer them some specific biological manners e.g. Zhang et al.46 discovered that some siRNA-loaded cholesterol-grafted bioreducible poly(amidoamine)s (rPAA-Ch) nanoparticles got into cell through caveolae-dependent endocytosis pathway and localized in lysosomes. In an earlier work, we synthesized a series of cationic cholesterol-disulfide (CHOSS) lipids with low cytotoxicity and high gene transfection efficiency in COS-7 cells, their pDNA nanoplexes showed an interesting “perinucleic localization” effect.47 However, apart from cholesterol, other steroid-based cationic gene carriers were rarely prepared and studied, the structural dependence of their self-assembly properties, cytotoxicity, gene transfection capability, endocytosis and intracellular trafficking/localization, were still remain obscure.48 Noteworthy, most of the steroidal compounds have similar four-ring core skeletons but different hydrophobic tails, which made them good model molecules for further investigation of the structure–function relationships.Natural steroids  were recently utilized as renewable, biocompatible and hydrophobic lipid moieties to construct efficient gene carriers.e.g. random and block), providing an efficient molecular engineering approach for structure–function relationship study.49–51PDMAEMA has been considered as an excellent gene carrier since its high transfection efficiency with acceptable cytotoxicity and buffering capacity (known as “proton sponge effect”) for endosomal escape. PDMAEMA and its copolymers could be easily synthesized by atom transfer radical polymerization (ATRP) or reversible addition fragmentation transfer (RAFT) polymerization method with controllable molecular weights and architectures (r-PDMAEMA), including PMA6Chol-r-PDMAEMA  and PMA6Dios-r-PDMAEMA , via RAFT polymerization of cholesterol-based monomer MA6Chol (or diosgenin-based monomer MA6Dios) with the DMAEMA monomer. Their structures were characterized by nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). pDNA binding affinities of the as-synthesized steroid-r-PDMAEMA copolymers were determined by agarose gel retardation assay, the average particle sizes, zeta potentials and morphologies of steroid-r-PDMAEMA/pDNA nanoplexes were analyzed by dynamic laser scattering (DLS) and transmission electron microscope (TEM), respectively. In vitro cytotoxicity of the steroid-r-PDMAEMA copolymers was evaluated by CCK-8 assay and pDNA transfection efficiency was measured by luciferase activity assay in H1299 lung cancer cell line. Moreover, intracellular localization of the PMA6Chol4-r-PDMAEMA190 (Chol-P1)/Cy3-pDNA and PMA6Dios18-r-PDMAEMA180 (Dios-P3)/Cy3-pDNA nanoplexes was observed by fluorescence microscopy.Based on the above background, to explore the effects of steroid hydrophobic moieties on gene transfection, in this work, we prepared series of new steroid-based cationic random copolymers  were purchased from TCI, Japan. Methylacrylic acid (98.0%) was purchased from Lingfeng Chemical Co Ltd. Shanghai. Dicyclohexylcarbodiimide  and 4-dimethylaminopyridine  were purchased from GL Biochem Ltd. Azodiisobutyronitrile  was purchased from Aladdin, Shanghai and re-crystallized twice in methanol before use. Branched structural poly(ethyleneimine)  was purchased from Sigma-Aldrich. Dimethylaminoethylmethacrylate  was purchased from Aladdin, Shanghai, and purified with basic aluminum oxide to remove the inhibitor. RAFT chain transfer agent (CTA) 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT) was synthesized referred to the literature.52 The organic solvent toluene was freshly distilled before use. All other solvents and chemicals were purchased from commercial suppliers and used as-received. Luciferase assay and bicinchoninic acid (BCA) protein quantitation kits (Cat #PW0104) were supplied by Promega (USA) and Biomiga (USA), respectively. DAPI (Cat #70217321) was purchased from Roche, Switzerland. Label IT-Cy3 Nucleic acid labeling kit (Cat #MIR7010) was purchased from Mirus Bio Corporation (USA). Cell counting kit-8  and LysoTracker Green (Cat #MF8124G) were purchased from MesGen Biotech. (China). PCMV-Luc plasmid (pDNA) was kindly gifted by Prof. Yuhong Xu of school of pharmacy of Shanghai Jiaotong University. RPMI1640 medium was purchased from Hangzhou Genom Co., Ltd. Fetal bovine serum (FBS) was purchased from GIBCO . Human lung cancer cell line H1299 was generously gifted by Dr Bo Wan of Fudan University .Cholesterol (97.0%) and diosgenin (98.0%) were purchased from Acros Organics. 2.2r-PDMAEMA cationic polymers were prepared by controllable RAFT radical polymerization. Firstly, the cholesterol-based MA6Chol and diosgenin-based MA6Dios monomers were synthesized via three-step process : 5.31–5.35  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>CH of cholesterol), 4.08–4.19 (COOCH2), 3.43–3.49 (CH2OChol), 3.12–3.14 (OCHR of cholesterol), 2.53–2.63 (CH2N of DMAEMA), 2.23–2.39 ((CH3)2N of DMAEMA).1H NMR : 5.32–5.35 (CCH of diosgenin), 4.08–4.19 (COOCH2), 3.41–3.48 (CH2ODios), 3.11–3.15 (OCHR of diosgenin), 2.53–2.63 (CH2N of DMAEMA), 2.23–2.39 ((CH3)2N of DMAEMA).2.31H NMR spectra were recorded on a Bruker Avance-300 NMR spectrometer at ambient temperature, operating at 300.0 MHz for proton (1H) nuclei. Molecular weights  and their distribution (Mw/Mn) were characterized on a PerkinElmer 200 gel permeation chromatography (GPC) equipped with a refractive index detector (RI), and double TSKgel multipore HXL-M columns and a guard column  were employed with THF as the eluent at a flowing rate of 1.0 mL min−1. Polystyrene standards with narrow molecular weight distribution  were used to generate the calibration curve. The molecular weights and distribution (Mw/Mn) were calculated with attached PE GPC software.2.4r-PDMAEMA/pDNA and PMA6Dios-r-PDMAEMA/pDNA nanoplexes were prepared by mixing plasmid DNA (1.0 μg) with a predetermined amount of PMA6Chol-r-PDMAEMA or PMA6Dios-r-PDMAEMA cationic copolymers to a total volume of 30 μL. After incubation at 37 °C for 20 min, the polyplex solution was loaded onto a 1.0% agarose gel containing 0.5 μg mL−1 ethidium bromide (EB). Then gel electrophoresis was set up in 1× TAE running buffer at 100 mV and kept for 40 min. Finally, the retardation of DNA migration was observed and recorded on a UVP benchtop 2UV transilluminator instrument.Before the agarose gel assay, the PMA6Chol-2.5r-PDMAEMA/pDNA and PMA6Dios-r-PDMAEMA/pDNA nanoplexes under various N/P ratios were analyzed on a Malvern Zetasizer Nano ZS90 DLS instrument at 25 °C with incident laser beams at λ = 633 nm and a scattering angle of 90°.Average particle size and zeta potential of the as-prepared PMA6Chol-2.6r-PDMAEMA/pDNA and PMA6Dios-r-PDMAEMA/pDNA nanoplexes (N/P = 3) solution was dropped onto a 300-mesh carbon-coated copper grid, and excess fluid was removed with filter paper, then air-dried at room temperature. The morphologies of the nanoplexes were observed by TEM  with an acceleration voltage of 80 kV.The as-prepared PMA6Chol-2.7r-PDMAEMA and PMA6Dios-r-PDMAEMA cationic copolymers was examined by CCK-8 assay. First, H1299 cells were seeded in 24-well microplates with 500 μL RPMI 1640 medium containing 10% FBS at a density of 5 × 105 cells per well and incubated for 24 h. Then the predetermined amount of PMA6Chol-r-PDMAEMA (or PMA6Dios-r-PDMAEMA) cationic copolymer solution was added and incubated with human lung cancer H1299 cells (4 × 104 per well) at 37 °C for 24 h, and CCK-8 solution (10 μL per well) was added and incubated for 4 h in 96-well plates. The absorption of CCK-8 was measured at 450 nm with a background correction of 620 nm using ELISA reader (Bio-Rad). Relative cell viability (%) was calculated as (ODsample − OD0)/(ODcontrol − OD0) × 100%. All data was presented as mean value with standard deviation of three experiments done in triplicate (n = 3).Cytotoxicity of the PMA6Chol-2.85 cells per well and cultivated for 24 h. Then, the medium in microplates were replaced with serum-free RPMI 1640 medium. The as-prepared PMA6Chol-r-DMAEMA/pDNA and PMA6Dios-r-PDMAEMA/pDNA nanoplexes at various N/P ratios (1.0 μg pDNA per well) was added and kept incubation at 37 °C for 4 h. Thereafter, the medium was replaced with 500 μL of fresh RPMI 1640 medium with 10% FBS and further incubated at 37 °C for 24 h. Luciferase gene transfection assays were conducted according to the protocol of Promega luciferase assay system, and total protein contents were measured using BCA protein quantification kit. Gene transfection efficiency was calculated as relative light units per milligram of luciferase protein (RLU per mg). Commercially available bPEI-25K was conducted in the same way as the control. All transfection experiments were performed in triplicate (n = 3).H1299 cells were first inoculated in 24-well microplates with 500 μL RPMI 1640 medium containing 10% FBS at a density of 4 × 102.9r-PDMAEMA/Cy3-pDNA and PMA6Dios-r-PDMAEMA/Cy3-pDNA polyplexes was observed by fluorescence microscope. H1299 cells were seeded into 6-well plate (2 × 105 cells per well) and cultivated at 37 °C overnight in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin–streptomycin. Subsequently, the cells were separately transfected with the as-prepared Chol-P1/Cy3-pDNA or Dios-P3/Cy3-pDNA polyplexes. After 4 h incubation, the cells were washed with PBS for three times, then fixed with 4% paraformaldehyde in 0.12 M phosphate buffer, pH 7.2, at 37 °C for 30 min and washed three times in PBS buffer. Afterward, DAPI  and Lysotracker  were added and further incubated for 15 min, then washed with PBS. The fluorescence images were observed and recorded on a Nikon Ti–S invert fluorescence microscopy.Firstly, Chol-P1/Cy3-pDNA and Dios-P3/Cy3-pDNA nanoplexes were previously prepared by mixing the polymers with Cy3-pDNA (N/P = 3) and incubating at 37 °C for 30 min. Intracellular localization of the PMA6Chol-3.3.1via a three-step organic synthetic strategy. Details of the polymer preparation process and 1H NMR spectra were shown in the ESI  and (b) and Fig. S1(a) and (b)via reversible addition fragmentation transfer (RAFT) polymerization method, respectively, with the DDMAT as the chain transfer agent (CTA) and AIBN as the initiator (P ∼ 6.9) and polar carboxyl group, which makes it highly compatible to the steroid-bearing monomer and DMAEMA monomer. Moreover, compared to aromatic ring-bearing CTA,8 the electron-donating property of alkyl chain on DDMAT could bring good reactivity to thioester free radicals. By regulating the monomer feeding ratios, a series of PMA6Chol-r-PDMAEMA  and PMA6Dios-r-PDMAEMA  random copolymers were prepared, with the steroid-bearing hydrophobic weight ratios of 7–27 wt%. Notably, the PMA6Chol-r-PDMAEMA and PMA6Dios-r-PDMAEMA random copolymers showed very close average polymerization degrees of each blocks and hydrophilic/hydrophobic weight ratios, which benefits for elucidating their structure–function relationships. r-PDMAEMA and PMA6Dios-r-PDMAEMA copolymer series. 1H NMR spectrum for as-resulted Chol-P3 and Dios-P3 random copolymers, in which 1H nuclei resonance signals a (5.2 ppm) and c (3.4 ppm) could be assigned to the double bonds (CCH) and OCHR groups of steroid cores, and the proton signals b (4.1 ppm), d (2.6 ppm) and e (2.3 ppm) could be assigned to the DMAEMA moieties/units, respectively. The assigned 1H NMR resonance signals and high monomer conversions as well as low Mw/Mn values indicated the successful preparation of the steroid-r-PDMAEMA copolymers with controlled structures by one-step RAFT polymerization approach.First, the cholesterol-based monomer MA6Chol and diosgenin-based monomer MA6Dios were synthesized nitiator . DDMAT h3.2r-PDMAEMA and PMA6Dios-r-PDMAEMA random cationic copolymers, which bearing various amount of hydrophobic cholesterol and diosgenin moieties/blocks but almost equal amount of DMAEMA cationic moieties/blocks, by agarose gel retardation assay. As shown in r-PDMAEMA and PMA6Dios-r-PDMAEMA random copolymers could effectively bind and condense pDNA to completely retard its movement across the agarose gel at low N/P ratio of 2.5, indicating the strong capability for the prepared copolymers to condense pDNA into polyplexes. It is worth noting that the pDNA binding affinity could be achieved on the PMA6Chol-r-PDMAEMA and PMA6Dios-r-PDMAEMA cationic random copolymers with low hydrophobic ratios  at an N/P ratio of 1.0–1.5, while lower pDNA binding affinity (N/P ratio of 2.5) was observed on Chol-P3 and Dios-P3  copolymers. The results illustrated that the pDNA binding affinity decreased along with the increasing of steroid ratio, which may be attributed to the steric hindrance effect of the large-sized steroid moieties. Moreover, mesogen properties of the steroid moieties may lead to the formation of self-organized micro-liquid crystal aggregation statement of the cationic copolymers, which further result in the formation of rigid and large-sized nanoplex aggregates and gave rise to lower pDNA binding affinities. Additionally, the pDNA binding affinity of cholesterol-based polymers seems slightly higher than that of diosgenin-based polymers, indicating the pDNA binding affinity did not only depend on the number of hydrophobic moieties, but also on the type of steroid architectures.To investigate the effect of hydrophobic steroid moieties and hydrophilic/hydrophobic ratios on gene binding/loading efficiency, we examined the pDNA binding affinity of PMA6Chol-3.331,32 We measured the average particle size and zeta potential of the PMA6Chol-r-PDMAEMA/pDNA and PMA6Dios-r-PDMAEMA/pDNA nanoplexes by DLS with a fixed pDNA amount of 4.0 μg mL−1. As shown in r-PDMAEMA copolymers when N/P ratio was lower than 2.0, indicating the efficient pDNA condensation effect.8 Then, the size of the nanoplexes increased with the increasing addition of cationic polymers and reached a platform at the N/P ratio of 3.0–4.0. Moreover, the particle size increased along with the increasing of hydrophobic cholesterol/diosgenin weight ratio, due to the enhanced aggregation induced by the increasing of hydrophobic moieties. Notably, the size of the cholesterol-containing PMA6Chol-r-PDMAEMA/pDNA polyplexes were larger than that of their diosgenin counterparts, indicating that the particle size depended on the type of the steroid hydrophobes, which might further influence their intracellular uptake and gene transfection properties. On the other hand, zeta potentials of the PMA6Chol-r-PDMAEMA/pDNA and PMA6Dios-r-PDMAEMA/pDNA were converted from negative to positive at the N/P ratio around 1.0–1.5, and then rapidly increased and maintained at +11.5 to +19.6 mV. The results indicated that the positive surface charge of the polyplexes was suitable for their intracellular uptake.It is known that average particle size and surface charge were important factors for the cytotoxicity, cellular uptake and gene transfection efficiency of the polyplexes.3.4r-PDMAEMA/pDNA and PMA6Dios-r-PDMAEMA/pDNA nanoplexes under the N/P ratio of 3.0 by TEM. As shown in r-PDMAEMA/pDNA nanoplexes were near spherical-shaped nanoparticles with the size of 50–150 nm. Besides, PMA6Dios-r-PDMAEMA/pDNA were loose-structured nanoparticles with smaller size of 25–100 nm. At high hydrophobic ratios  cell line, the cell viability of free H1299 cells were set as 100%. As shown in −1 each polymer, while with the increasing of hydrophobic ratio , the H1299 cell viability still maintained around 80%. This may due to the formation of comparatively large-sized cationic polymer nanoparticle that diminished their electrostatic interactions with the negatively-charged plasma membrane. Similarly, Bhattacharya41et al. found that cholesterol-modified PEI have much lower cytotoxicity than unmodified PEI. Neamnark39et al. revealed that the cytotoxicity greatly relied on the structure of hydrophobic moieties, hydrophobic/hydrophilic proportion, as well as the hydrophobic modification ratio.15 Moreover, the results indicated that appropriate introducing hydrophobic steroid moieties to the cationic copolymers could further reduce the cytotoxicity, which offered the possibility for safe gene transfection.For safe gene transfection, the cytotoxicity of the cationic polymer was regarded as an essential factor.3.6in vitro gene transfection capability of the PMA6chol-r-PDMAEMA and PMA6Dios-r-PDMAEMA cationic copolymers, luciferase gene expression assay was conducted in H1299 cells. As shown in r-PDMAEMA random cationic polymers was found greatly depend on the hydrophobic/hydrophilic ratios and steroid hydrophobic tail structures. Among the copolymers, Chol-P1 and Dios-P3 showed maximum/optimum luciferase gene transfection capability at the N/P charge ratio of 3.0, and the optimum transfection efficiency could reach to 18.1–31.2% of the efficiency of commercially available “gold standard” PEI-25K.48 While higher hydrophobic cholesterol-containing Chol-P3 exhibit lower transfection capability, and the transfection efficiency enhanced with the increasing of N/P charge ratio within the range of measurement. The results were contrary to the trend of the size change of polyplexes. From this point of view, we can speculate that the low transfection efficiency of Chol-P3 may due to the large polyplex that hampered the intracellular uptake. On the contrary, Dios-P1, Dios-P2 and Dios-P3 showed increased transfection efficiency along with the increasing of diosgenin ratio, which may be attributed to their increasingly condensed micellar structures.To examine 3.7r-PDMAEMA and PMA6Dios-r-PDMAEMA copolymers in H1299 cells, the optimized Chol-P1/Cy3-pDNA and Dios-P3/Cy3-pDNA nanoplexes were accordingly prepared, which then incubated with H1299 cells for 4 h and observed under fluorescence microscope. The cell nuclei and endosome/lysosome were separately stained with DAPI (blue) and Lysotracker (green), respectively. As shown in via lipid-raft or caveolae pathway,48 then the lipoplexes were future transported into endosome–lysosome, finally localized inside cell nuclei. Thus, in this work, we can suppose that the Chol-P1/Cy3-pDNA and Dios-P3/Cy3-pDNA might be internalized into H1299 cells through the gateway of “lipid-raft/caveolae-mediated endocytosis–endosome–lysosome–cell nuclei”. Further investigation on the endocytosis pathway of the PMA6Chol-r-PDMAEMA and PMA6Dios-r-PDMAEMA nanoplexes was carried out in our lab.To further understand the intracellular fate of the pDNA nanoplexes of PMA6Chol-4.r-PDMAEMA cationic random polymers denoted as PMA6Chol-r-PDMAEMA  and PMA6Dios-r-PDMAEMA  via controllable RAFT polymerization. The steroid-r-PDMAEMA random cationic polymers could efficiently bind pDNA (N/P < 3.0) and subsequently form nanoparticles with near-spherical morphology (142–449 nm) and positive surface charge (+11.5 to +19.6 mV) in aqueous solution, and the particle size were depended on the hydrophobic/hydrophilic ratios and steroid structures. The cytotoxicity of the synthesized steroid-r-PDMAEMA random cationic polymers was relatively low, and increasing the hydrophobic/hydrophilic ratios could further diminish the cytotoxicity. The gene transfection efficiency of the steroid-r-PDMAEMA cationic random polymers was found greatly dependent on the hydrophobic/hydrophilic ratios and steroid structures, and the optimum transfection efficiency could reach to 18.1–31.2% of the efficiency of commercially available PEI-25K. The steroid-r-PDMAEMA/Cy3-pDNA polyplexes showed a well-known “lysosome localization” effect, which may facilitate their gene transfection process. Moreover, all of the steroid-r-PDMAEMA/Cy3-pDNA nanoplexes have obvious “lysosome localization” effect, indicating the steroid structures do not remarkably influence the intracellular localization behaviors of the nanoplexes. All in all, the results demonstrated the physico-chemical properties, cytotoxicity and gene transfection features could be tuned by appropriately control of steroid structures and hydrophobic/hydrophilic ratios of the steroid-based cationic polymers.In summary, by using natural steroids (cholesterol and diosgenin) as the sustainable molecular resources, we synthesized a series of steroid-There are no conflicts to declare.RA-011-D1RA00223F-s001


Z-conformation nucleic acid binding protein 1 (ZBP1), a powerful innate immune sensor, has been identified as the important signaling initiation factor in innate immune response and the multiple inflammatory cell death known as PANoptosis. The initiation of ZBP1 signaling requires recognition of left-handed double-helix Z-nucleic acid (includes Z-DNA and Z-RNA) and subsequent signaling transduction depends on the interaction between ZBP1 and its adapter proteins, such as TANK-binding kinase 1 (TBK1), interferon regulatory factor 3 (IRF3), receptor-interacting serine/threonine-protein kinase 1 (RIPK1), and RIPK3. ZBP1 activated innate immunity, including type-I interferon (IFN-I) response and NF-κB signaling, constitutes an important line of defense against pathogenic infection. In addition, ZBP1-mediated PANoptosis is a double-edged sword in anti-infection, auto-inflammatory diseases, and tumor immunity. ZBP1-mediated PANoptosis is beneficial for eliminating infected cells and tumor cells, but abnormal or excessive PANoptosis can lead to a strong inflammatory response that is harmful to the host. Thus, pathogens and host have each developed multiplex tactics targeting ZBP1 signaling to maintain strong virulence or immune homeostasis. In this paper, we reviewed the mechanisms of ZBP1 signaling, the effects of ZBP1 signaling on host immunity and pathogen infection, and various antagonistic strategies of host and pathogen against ZBP1. We also discuss existent gaps regarding ZBP1 signaling and forecast potential directions for future research. Recently, Z-conformation nucleic acid binding protein 1 (ZBP1) has become a rising star and attracts a lot of attention ,3. LaterZBP1 was originally found to be significantly upregulated in mouse peritoneal tumor stromal cells or macrophages upon stimulation with IFN-γ or LPS . Later sZBP1 is a member of both the left-handed helical Z-conformation nucleic acid (Z-NA) binding protein family containing the Zα domain and the receptor-interacting serine/threonine protein (RIP) family holding the RIP homotypic interaction motif (RHIM). ZBP1 harbors two N-terminal Z-NA binding domains , two intermediate RHIM (RHIM1 and RHIM2), and a C-terminal signal domain (SD) ,10,11,12The Zα domains of ZBP1 determine its location in the cell. The initial research on the subcellular localization of ZBP1 indicated that ZBP1 presents the punctate distribution in the cytoplasm and interacts dynamically with stress granules (SGs) and processing bodies (PBs) . SGs andIFN-I expression is mainly induced by various pattern-recognition receptors (PRRs), including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), cyclic-GMP-AMP synthase-stimulator of interferon gene (cGAS-STING) ,25. IFN-ZBP1 was initially recognized as a sensor for double-stranded DNA (dsDNA). In mouse embryo fibroblasts (MEFs) or L929 cells in response to immunostimulatory dsDNA  overexpressing ZBP1 significantly increased the IFN-I (IFN-α and IFN-β) expression . KnockdoZBP1 acts as both an ISG and the promoting IFN-I-expression gene, which will form the ZBP1-mediated IFN positive feedback loop and provoke a strong innate immune response in dsDNA stimulated cells. However, subsequent studies pointed to the limitation in the ZBP1-dependent induction of IFN-I. Deletion of the ZBP1 did not affect the B-DNA-mediated IRF3 activation and IFN-β production in A549, HepG2 cells, or mice ,31,32. ONF-κB family  play a crucial role in regulating inflammation and innate immune ,37. NF-κThe ZBP1-mediated NF-κB signaling is mechanically very similar to TLR3. TLR3 is an innate immune sensor that recognizes pathogen-associated dsRNA. TLR3 activates NF-κB signaling via toll-like receptor adapter molecule 1 (TRIF) . TRIF inAfter suffering from stress or microbial infection, the immune system of cells will initiate programmed cell death (PCD) to maintain homeostasis or obliterate infected cells . Major PZBP1 act as a master regulator of PANoptosis and plays a vital role in initiating the assembly of PANoptosome  Figure . ZBP1 PAMuch research elucidated some crosstalk between apoptosis, necroptosis, and pyroptosis, which provides a basis for studying the signaling crosstalk in ZBP1-mediated PANoptosis. CASP8 is the key connection between apoptosis and pyroptosis. FADD/CASP8 are necessary for the activation of CASP1 and cell pyroptosis in response to LPS + ATP, C. rodentium or Yersinia stimulation ,63,64. CAs a potent innate immune sensor, ZBP1 plays an essential role in immune defense against multiple pathogens infection, including viruses, bacterium, fungi, and parasites . For exaMembers of the herpesvirus family are classified as large dsDNA viruses. Based on genome sequence homologies, host or cell tropism, and replication strategies, herpesvirus is divided into alpha-, beta, and gamma-herpesviruses .WT), the M45 mutant MCMV (MCMVM45mutRHIM) infection-induced RIPK3-dependent but RIPK1/TRIF-independent necroptosis and show weaker replication in 3T3-SA or SVEC4-10 cells [M45mutRHIM virulence [WT reshapes the ZBP1-RIPK3 complex and impedes the necroptotic signaling to maintain viral normal replication [Cytomegalovirus (CMV) is β-herpesvirus and leads to extensive clinical symptoms, such as scongenital infection, retinitis, and hepatitis. Mouse cytomegalovirus (MCMV) encodes several cell death suppressors including the M38.5-encoded viral mitochondrial inhibitor of apoptosis (vMIA), the M36-encoded viral inhibitor of caspase-8 activation (vICA), and the M45-encoded viral inhibitor of RIP/RHIM activation (vIRA) . The vIR10 cells . Subsequirulence . The Zα2irulence ,83. Intrirulence ,83. The lication . In addilication ,103. Knolication .−/− BMDMs [Herpes simplex virus-1 (HSV-1) and varicella zoster virus (VZV) both belong to the α-herpesvirus. HSV-1 is known to induce cytolytic death and cause orofacial infections or encephalitis . Mice BM/− BMDMs . Consist/− BMDMs . Similar/− BMDMs . HSV-1 e/− BMDMs ,86,87  is a chronic human γ-herpesvirus that persists for life. EBV infection causes an increased risk of cancer and autoimmune diseases ,106. HowZα1ZBP1) or ADAR1 Zα (E3ZαADAR1) for E3 Zα still blocks necroptosis and maintains the normal virulence of the VACV, indicating the equivalence of Zα domain of Z-NA binding family in sensing and binding Z-NA and the significance of E3 Zα as a critical suppressor of ZBP1-induced necroptosis [VACV belongs to the poxvirus family and has a large linear dsDNA genome and replicates in the cytoplasm. VACV is used as a vaccine against smallpox and is currently being further developed as an efficient vector for novel vaccines against cancer and other infectious diseases ,111. Theroptosis ,91.IAV is a negative sense, single-stranded, lytic RNA virus and belongs to the Orthomyxoviridae family. IAV targets and lyses lung epithelial cells and fibroblasts that cause airway and lung dysfunction as well as cardiovascular diseases ,113. IAV−/−) or Zα2 deficiency of ZBP1 (ZBP1ΔZα2) substantially alleviated the inflammatory cell death and lethality of mice upon treatment with IFN and SARS-CoV-2 infection [Since 2019, the Coronavirus disease 2019 (COVID-19) pandemic has led to socioeconomic and psychological distress . COVID-1nfection . The ZBPnfection ,127. In nfection ,124. Thenfection . Hence, nfection .Genome analysis reveals the potential Z-RNA prone sequences  within the coronavirus genome including SARS-CoV, MERS, and SARS-CoV-2. SARS-CoV-2-derived flipons are mainly present in Nsp2, Nsp12, Nsp13, S, and M genes . That flFlaviviruses have a positive-sense, single-stranded RNA genome and are generally spread to vertebrate hosts by arthropods. West Nile virus (WNV) and Zika virus (ZIKV) both belong to mosquito-borne flaviviruses and cause encephalitis and severe neurological injuries ,131. ZBPYersinia pseudotuberculosis (a Gram-negative bacterium) infected or lipopoly-saccharide -treated BMDMs arises apoptosis and pyroptosis via the TRIFosome consisting of TRIF, ZBP1, RIPK1, FADD, CASP8, GSDMD [Yersinia pseudotuberculosis infection [Francisella novicida (an intracellular Gram-negative bacterium) infected BMDMs form the AIM2 PANoptosome similar to HSV-1 infection [Enteropathogenic Escherichia coli (EPEC) encoded type III secretion system (T3SS) effector EspL has a tripartite cysteine protease motif  that cleaves within the RHIM domains and degrades the RHIM-containing proteins  [8, GSDMD . After T8, GSDMD ,72. The 8, GSDMD . Lackingnfection ,136. ZBPnfection . TLR4-TRnfection . Additionfection . In addinfection . AIM2 funfection . ZBP1 acnfection . Similar3, TRIF) . The intCandida albicans and Aspergillus fumigatus infection also activate ZBP1-dependent PANoptosis and inflammation in BMDMs [−/− or ZBP1ΔZα2 BMDMs show significantly attenuated PANoptosis and the release of IL-18 after fungi infection [in BMDMs . Comparenfection . While infection ,141.Toxoplasma gondii belongs to obligate intracellular parasites and is able to invade all nucleated cells in warm-blooded animals including humans [T. gondii infection upregulates and maintains the ZBP1 expression [T. gondii in macrophages or mice and reduces host survival against oral challenge, confirming the suppression of ZBP1 on T. gondii infection [T. gondii infection [T. gondii infection [T. gondii infection could need more effort to illuminate.g humans . T. gondpression ,144. Delnfection . But thenfection ,146. Altnfection ,147. ThuSensing host-derived nucleic acid ligands causes the spontaneous activation of ZBP1, that is responsible for many auto-inflammatory diseases, such as perinatal lethality, inflammatory bowel disease (IBD), heatstroke and oxidative stress-induced injuries, and acute pancreatitis (AP). −/− mice or RIPK1mR/mR mice expressing RIPK1 with mutated RHIM develop systemic inflammation and early postnatal lethality due to ZBP1-mediated necroptosis in multiple tissues [E-KO mice (with the epidermis-specific RIPK1-knockout) suffer from skin inflammation because of ZBP1-dependent keratinocyte necroptosis [−/− mice with loss of RIPK1 kinase activity in other cells, exhibit spontaneous colonic inflammation relied on ZBP1-RIPK3 necrosome [−/− caused spontaneous necroptosis, and endogenous retroviruses (ERV)-derived complementary reads were detected in epidermal RNA [In the setting of pathogens infection, RIPK1 is an important member of the ZBP1-PANoptosome and the key to ZBP1-mediated PANoptosis ,57. Para tissues ,150,151.roptosis ,152. Theecrosome . Advancermal RNA ,154  edits of ERV-derived dsRNA and represses abnormal immune responses activated by ERVs ,158,159./mR mice ,163. ZBP/mR mice ,163. On /mR mice .−/− mice [Impaired mitochondrial DNA (mtDNA) is another endogenous ligand to activate ZBP1 . PUMA, N−/− mice ,167. Bey−/− mice . The ZBP−/− mice .Intriguingly, the latest research indicated that the ZBP1 without binding to any nucleic acid ligands can also provoke cell death when suffering from heatstroke . Heat stMoreover, the uncontrolled expression of ZBP1 and following ZBP1-dependent pyroptosis are the main reason for acute pancreatitis (AP) . The tRF+ T cells in tumor cells with radiation treatment [Although ZBP1-induced aberrant cell death and inflammation are harmful to the host, there is a positive effect in the background of tumorigenesis. Advanced solid tumors generally suffer from necroptosis . Howeverreatment . Mechanireatment . On the reatment ,179,180.reatment ,181.In addition to mtDNA, the endogenous dsRNA remains the important ligand for ZBP1-mediated tumor immunity. However, the ZBP1 sensing for endogenous dsRNA is still interdicted by ADAR1. The latest research points out that some 3’UTR of ISGs mRNA containing short interspersed nuclear elements (SINEs) or GU-type simple repeats and naturally fold into Z-type dumbbells . Such Z-+ T lymphocytes (CTLs) and CD4+ Th1 responses, compared to the empty vector plus TAAs vaccine inoculation [+ T cells and more durable stronger resistance to B16 melanoma was detected in co-vaccinated mice with pZBP1 and pTRP2 [Through genetic engineering, previous studies have applied ZBP1 to tumor therapy. Various genetic adjuvants enhance vaccine immunogenicity and strengthen adaptive immune responses ,185,186.culation . Comparend pTRP2 . In addind pTRP2 . A ZBP1-nd pTRP2 . AlthougZBP1 is another Z-NA binding protein in addition to ADAR1 in mammals. Initially, ZBP1 was shown to be a cytoplasmic DNA sensor that promotes innate immune responses, and subsequent studies confirmed the importance of ZBP1 in PANoptosis initiation. ZBP1 signaling typically relies on the combination of the Zα2 domain with active ligands, such as virus-derived Z-RNA (VACV and IAV) ,161,190.−/− mice and colitis in RIPK1−/− IECs [ZBP1-PANoptosome initiates PANoptosis and plays a double-edged sword role in anti-infection and development. ZBP1-mediated PANoptosis efficiently limits the replication of intracellular pathogens, and stimulates the recruitment of immune cells and adaptive immune responses, which is beneficial to host survival . Nonethe−/− IECs ,155, and−/− IECs . However−/− IECs ,174. Both the pathogen and the host have evolved skills to regulate ZBP1 signaling to ensure pathogen replication or immune homeostasis . RelyingIn this review, we have elucidated the ZBP1-mediated signaling mechanisms of innate immune response and PANoptosis. We also discussed the pathogens and host-developed multiple regulation strategies for ZBP1-mediated signaling; ZBP1 plays a double-effective role in infection and host health. Understanding the impact of ZBP1 signaling on host or pathogens and developing novel positive or negative regulated drugs against ZBP1 signaling will be helpful to anti-infection, alleviation of inflammatory disease, and anti-tumor immunity.


DDX41 mutations are often associated with bone marrow failure and cytopenia before overt disease manifestation. However, the mechanisms underlying these specific conditions remain elusive. Here, we demonstrate that loss of DDX41 function impairs efficient RNA splicing, resulting in DNA replication stress with excess R-loop formation. Mechanistically, DDX41 binds to the 5′ splice site (5′SS) of coding RNA and coordinates RNA splicing and transcriptional elongation; loss of DDX41 prevents splicing-coupled transient pausing of RNA polymerase II at 5ʹSS, causing aberrant R-loop formation and transcription-replication collisions. Although the degree of DNA replication stress acquired in S phase is small, cells undergo mitosis with under-replicated DNA being remained, resulting in micronuclei formation and significant DNA damage, thus leading to impaired cell proliferation and genomic instability. These processes may be responsible for disease phenotypes associated with DDX41 mutations.Myeloid malignancies with  DDX41 mutation occurs in various hematopoietic malignancies, most frequently in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) . NT. NT24]. To clarify the biological significance of DDX41, we suppressed its expression in different cell lines Fig. . DDX41 kNext, we examined cell cycle progression using U2OS cells stably expressing GFP-tagged histone H2B. DDX41-knockdown cells had a longer mitotic period, with abnormal mitosis and post-mitotic nuclear morphologies  and reduced bromodeoxyuridine (BrdU) incorporation Fig. . Slowed E)-N-hydroxy-1-phenyl-2,3-dihydroquinolin-4(1H)-imine] and [4-amino-6--ylidene)amino)-1-methyl-2-phenylquinolinium chloride hydrochloride] , which were demonstrated to inhibit ATPase activity of DDX41 [To better understand the roles of DDX41, we analyzed cell phenotypes after acute inhibition of DDX41, by using recently identified DDX41 inhibitors, [, a DNA polymerase α inhibitor  that lacks enzymatic activity but retains R-loop-binding capacity  dataset . In suppThis DepMap data may reflect our data obtained from transcriptome analyses of DDX41-knockdown cells Fig. . Gene seTo investigate the relationship between DDX41-mediated RNA splicing and transcriptional elongation, we performed IP-Western blotting. We found that DDX41 interacted with Pol II phosphorylated at S2 and S5 (pS2 and pS5) in the C-terminal domain (CTD) and that RNA did not mediate the interaction Fig. , raisingWe then performed fractionation-assisted native chromatin IP and sequencing analysis (ChIP-seq)  with an We next studied whether observed signal accumulation of Pol II at 5ʹSS was related to RNA splicing. We selected constitutively skipped exons in adequately transcribed genes in control cells for analysis  mice that express a Ddx41 mutant corresponding to human DDX41 R525H in a tamoxifen-inducible manner  immature bone marrow cells isolated from heterozygous (Ddx41R525H/WT) and WT (Ddx41WT/WT) mice were cultured in the presence of cytokines to support stem/progenitor cell growth, followed by induction of R525H mutation in Ddx41R525H/WT cells by adding (Z)-4-hydroxytamoxifen (4-OHT). Induction of the mutant was confirmed by direct sequencing  mice. Increased R-loop formation was observed in Ddx41WT/KO cells, which was further enhanced by the induction of R525H mutation (Ddx41R525H/KO)  have relatively longer cell cycles and divide at a low rate, once every 40 weeks . Therefoied mice . These iWe also showed that DNA damage caused by loss of DDX41 function was dependent on transcription-related R-loop accumulation. Although impaired RNA splicing can lead to R-loop formation , its proDDX41 mutation is clearly needed for its confirmation, given that INTS3 intron retention observed in DDX41-knockdown cells was reported to be highly exclusive to SRSF2 mutation [DDX41-related myeloid malignancies.DDX41 depletion induced mild splicing changes without any specific pattern; one possible explanation for this is that DDX41 regulates efficient splicing in the late step rather than decision of SS. However, further sequencing analysis of clinical specimens with mutation . Since omutation , regions2 incubator at 37 °C.K562 and THP-1 cells were cultured in RPMI-1640 (Sigma-Aldrich) with 10% heat-inactivated fetal bovine serum. HEK293FT, HEK293T, HeLa, U2OS and ARPE-19 cells were cultured in Dulbecco’s Modified Eagle Medium (Sigma-Aldrich) with 10% heat-inactivated fetal bovine serum. All cells were maintained in a 5% CODDX41inh-1 and DDX41inh-2 were gifts from Axcelead Drug Discovery Partners Inc.HeLa cells were synchronized to the late G2 phase or G1/S boundary by double thymidine block with or without CDK1 inhibitor, respectively see .Cell cycle distribution, incorporation of BrdU or IdU, apoptosis and γ-H2AX expression were analyzed via flow cytometry see .CLIP-seq was performed according to a previously reported method 26Sortm1(cre/Esr1)Arte (ERT2Cre) mice  to allow tamoxifen-inducible excision of floxed regions. Ddx41 heterozygous knockout mice (C57BL/6N Ddx41tm1a) were purchased from the UC Davis KOMP Repository (MMRRC Stock #047340-UCD). All mice were kept according to guidelines of the Institute of Laboratory Animal Science, Hiroshima University. The Animal Care Committee at the Japanese Foundation for Cancer Research approved all murine studies. Detailed procedures for generating Ddx41R525H cKI mice and experimental procedures with the mice are described in p values ≤0.01 being considered statistically significant unless otherwise indicated. Error bars in figures indicate standard deviation (SD).Statistical analysis was performed with R software (version 4.1.2). All statistical details of experiments are given in Additional methods are available in the Supplementary informationSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Table S1Supplementary Table S2


The impairment of the angiopoietin-1 (Ang-1)/Tie-2 signaling pathway has been thought to play a critical role in diabetic complications. However, the underlying mechanisms remain unclear. The present study aims to investigate the effects of Tie-2 glycation on Ang-1 signaling activation and Ang-1-induced angiogenesis. We identified that Tie-2 was modified by advanced glycation end products (AGEs) in aortae derived from high fat diet (HFD)-fed mice and in methylglyoxal (MGO)-treated human umbilical vein endothelial cells (HUVECs). MGO-induced Tie-2 glycation significantly inhibited Ang-1-evoked Tie-2 and Akt phosphorylation and Ang-1-regulated endothelial cell migration and tube formation, whereas the blockade of AGE formation by aminoguanidine remarkably rescued Ang-1 signaling activation and Ang-1-induced angiogenesis in vitro. Furthermore, MGO treatment markedly increased AGE cross-linking of Tie-2 in cultured aortae ex vivo and MGO-induced Tie-2 glycation also significantly decreased Ang-1-induced vessel outgrow from aortic rings. Collectively, these data suggest that Tie-2 may be modified by AGEs in diabetes mellitus and that Tie-2 glycation inhibits Ang-1 signaling activation and Ang-1-induced angiogenesis. This may provide a novel mechanism for Ang-1/Tie-2 signal dysfunction and angiogenesis failure in diabetic ischaemic diseases. Angiogenesis impairment resulting in microvascular insufficiency contributes to the pathogenesis of various cardiovascular complications in diabetes mellitus ,2 and thTie-2 is selectively expressed in endothelial cells and gene deficiency of Tie-2 leads to embryonic lethality due to abnormal vascular development, indicating its indispensable role in maintaining normal vascular function . Ang-1, Methylglyoxal (MGO), a highly reactive metabolism of glucose, significantly increases under hyperglycemic condition in diabetes ,15. The Therefore, in this study, we determined the glycation of Tie-2 in aortae derived from diabetic mice and further tested the hypothesis that MGO-induced Tie-2 glycation may inhibit Ang-1 signaling and Ang-1-induced angiogenesis.Firstly, the modification of Tie-2 was investigated in a pathological model in vivo. Specifically, 8-week-old male C57BL/6J mice were fed with a normal chow diet (ND) or a high-fat chow diet (HFD) for 16 weeks, which produced obesity and hyperglycemia . The aorMGO, which has been confirmed to significantly increase in diabetes, is known to react with arginine and lysine residues of proteins to form AGE–protein adducts, resulting in impairment of protein function. Therefore, we hypothesized that MGO may induce Tie-2 glycation. To test this hypothesis, HUVECs were exposed to MGO (100 μM) or vehicle control for 24 h and the effects of MGO on Tie-2 modification were determined. The cell lysates were immunoprecipitated with anti-Tie-2 antibody and blotted with anti-AGE antibody. The results showed that incubation with MGO significantly increased the formation of AGEs on Tie-2 immunoprecipitates a, providTo investigate the impacts of MGO-induced glycation on Tie-2 function, the activation of Ang-1 signaling pathways was determined. HUVECs were exposed to MGO (100 μM) or vehicle control for 24 h, followed by stimulation with Ang-1 (200 ng/mL) for 15 min. Cell lysates were then prepared, separated by SDS-PAGE and analyzed with immunoblotting. Ang-1 significantly increased the phosphorylation of Tie-2 in HUVECs without MGO stimulation, whereas pretreatment with MGO for 24 h inhibited Ang-1-induced Tie-2 phosphorylation a. The doTo further investigate the effects of Tie-2 glycation on Ang-1-activated endothelial cell physiological responses, HUVECs pretreated with MGO or vehicle control were seeded onto the upper chambers of a transwell, followed by exposure to Ang-1 (200 ng/mL). After 12 h, cells that had migrated to the lower chambers were stained and counted. Ang-1 significantly increased the migration of endothelial cells without MGO pre-incubation. However, pretreatment with MGO inhibited Ang-1-induced cell migration b. In agrTo clarify the relationship between Tie-2 glycation and Ang-1/Tie-2 signaling dysfunction, the effects of aminoguanidine (AG) (AGE formation inhibitor), which has been demonstrated to prevent MGO-induced protein glycation ,24, on ATo further detect the effects of Tie-2 glycation on Ang-1-induced angiogenesis ex vivo, the aortae isolated from C57BL/6J mice were pretreated with AG (100 μM) for 1 h, followed by exposure to MGO (100 μM) for 24 h. The segments of MGO-treated aortae were then cultured in rat tail collagen I gel in the presence or absence of Ang-1 (200 ng/mL) for 14 d. The histologic analysis revealed a remarkable Tie-2–AGEs colocalization in MGO-treated aortae, which were significantly inhibited by AG pretreatment a, providPrevious studies suggest that the impairment of the Ang-1/Tie-2 signaling pathway has been proposed to be implicated in the pathogenesis of diabetic microangiopathy ,9. HowevDuring diabetes mellitus, increased AGEs are well known to be implicated in diabetic microvascular complications through inducing oxidative stress, inflammation and cell apoptosis . More reTo clarify the significance of Tie-2 glycation in Ang-1/Tie-2 signaling, MGO-treated HUVECs were stimulated with Ang-1. Our results showed remarkably decreased Tie-2 phosphorylation and downstream signaling activation in MGO-treated endothelial cells compared to cells stimulated with vehicle control. Previous studies also showed Ang-1 signaling impairment in mouse heart microvascular endothelial cells cultured in high glucose , which idb/db mice compared to C57BL/6J mice [The attenuated Ang-1/Tie-2 signal transduction has been demonstrated as one of the important mechanisms underlying angiogenesis dysfunction in diabetic complications. Experiments involving cell migration and tube formation in vitro and aortic ring angiogenesis ex vivo displayed that MGO treatment significantly decreased Ang-1-induced cell migration, tube formation and vessel sprouting, whereas the inhibition of AGE formation by AG eliminated the inhibitory effects of MGO on Ang-1 angiogenic responses, further indicating the important role of Tie-2 glycation on dysfunction of Ang-1 signal transduction. Consistent with our results, previous studies also confirmed that Ang-1-induced aortic ring vessel outgrowth was greatly decreased in /6J mice . HoweverIn summary, our present study has found that Tie-2 is glycated in HFD-treated mice and in MGO-treated endothelial cells and the glycation of Tie-2 inhibits Ang-1 signaling activation and Ang-1-induced angiogenesis . The finPrimary HUVECs  and the endothelial cell medium  were obtained from ScienCell Research Laboratories . Recombinant human Ang-1  was obtained from R&D systems . MGO  and AG  were purchased from Sigma-Aldrich . Growth-factor-reduced Matrigel matrix  was purchased from BD Biosciences . Collagen I  was obtained from Corning . Antibodies against phosphorylated Tie-2  and AGEs  were purchased from Abcam . Antibodies against phosphorylated Akt , total Akt  and total Tie-2  were obtained from Cell Signaling Technology . Anti-Tie-2  and anti-AGEs  antibodies used for immunohistochemical staining were purchased from Bioss . Pierce classic immunoprecipitation kit  was obtained from Thermo Scientific . The high-fat diet (TP2330055A) and the standard chow diet (TP2330055AC) were obtained from Trophic Animal Feed High-tech Co., Ltd. .C57BL/6J mice (6–8 weeks old) were purchased from the Chongqing Medical University Animal Center (Chongqing China). These mice were maintained in a controlled environment . All procedures for animal use were approved by the Animal Care and Use Committee of Southwest Medical University .The 8-week-old male C57BL/6J mice were fed with a HFD  for 16 weeks, as described previously ,34. Age-Mouse aortae were isolated from HFD-fed and ND-treated mice, fixed, embedded in paraffin, and sectioned. The 4 μm cross-sections were then prepared and incubated with an antibody to Tie-2 (1:100) or AGEs (1:100) at 4 °C overnight, followed by exposure to horseradish peroxidase-conjugated secondary antibodies for 60 min at 37 °C. The immune complexes were determined using diaminobenzidine substrate working solution.v/v) fetal bovine serum (FBS), 1% (v/v) endothelial cell growth supplement and 1% (v/v) antibiotic solution (penicillin/streptomycin) [HUVECs were cultured in endothelial cell medium with 5% (tomycin) ,10. WhenHUVECs were seeded onto 24-well plates, allowed to grow to 90% confluence and exposed to MGO (100 μM) or vehicle control  for 24 h. The cells were then scratched with a 200 μL pipette tip, followed by stimulation with Ang-1 (200 ng/mL) or vehicle control for 24 h. Photomicrographs were taken immediately after the scratch and after Ang-1 stimulation. Image J software was used to measure the change in scratch area over time.4 cells were added to the upper chambers, followed by stimulation with Ang-1 (200 ng/mL). After 12 h, cells remaining in the upper chambers were removed and the cells that had migrated to the lower chambers were fixed with 4% paraformaldehyde, stained with 0.25% crystal violet, photographed with a microscope and counted.HUVEC migration was also performed using transwell chambers with an 8.0 μm-sized porous membrane. HUVECs cultured in 6-well plates were allowed to grow to 90% confluence and exposed to MGO (100 μM) or vehicle control for 24 h. In some experiments, the cells were pretreated with AG (100 μM) for 1 h before MGO treatment ,36. The 5/well) which were pre-coated with a growth-factor-reduced Matrigel matrix (250 μL/well), followed by stimulation with Ang-1 (200 ng/mL) for 12 h. The tubes were then photographed and the total tube length in 5 fields was quantified using Image J software [HUVECs cultured in 6-well plates were allowed to grow to 90% confluence and exposed to MGO (100 μM) or vehicle control for 24 h. In some experiments, the cells were pretreated with AG (100 μM) for 1 h before MGO treatment. The cells were then detached and seeded onto 24-well plates (1 × 10software .v/v) protease phosphatase inhibitor cocktail (Thermo Scientific). Equal samples were subjected to SDS-PAGE and transferred to polyvinylidene fluoride membranes . The membranes were blocked with rapid blocking buffer  for 15 min at room temperature and incubated with primary antibodies against phosphorylated Tie-2 (1:1000), phosphorylated Akt (1:1000), total Tie-2 (1:1000) and total Akt overnight at 4 °C. The membranes were then incubated with species-specific horseradish peroxidase-conjugated goat IgG (Beyotime) for primary antibodies for 60 min at room temperature. After stripping, the same membranes were probed with anti-GAPDH (1:1000) or anti-β-actin (1:1000) antibodies. Chemiluminescence was used to visualize the protein bands, and the band density was measured with Image J software.HUVECs cultured in 6-well plates were allowed to grow to 90% confluence and exposed to MGO (100 μM) for 24 h, followed by stimulation with Ang-1 (200 ng/mL) for 15 min. In some experiments, the cells were pretreated with AG (100 μM) for 1 h before MGO treatment. The HUVECs were then lysed in RIPA buffer  supplemented with 1%  or vehicle control for 1 h, followed by exposure to MGO (100 μM) for 24 h. The aortic rings were then embedded into collagen I gel and stimulated with Ang-1 (200 ng/mL) or vehicle control ,39. Vessp value < 0.05 was considered significant.All of the data are presented as mean ± standard deviation (SD). An unpaired t-test and one-way ANOVA followed by post hoc comparison were carried out to analyze the results. All of the statistical analyses were performed using SPSS 19.0. A


In vitro studies showed thatthe optimized fluorescent conjugate MMC(FNIR-Tag)-TOC bound primarily via somatostatin receptor subtype-2 (SSTR2), whereas itsnegatively charged counterpart with IRDye 800CW had higher off-targetbinding. NIRF imaging in cell line- and patient-derived xenograftmodels revealed markedly higher tumor contrast with MMC(FNIR-Tag)-TOC,which was attributed to increased tumor specificity. Ex vivo staining of surgical biospecimens from primary and metastatic tumors,as well as involved lymph nodes, demonstrated binding to human tumors.Finally, in an orthotopic tumor model, a simulated clinical workflowhighlighted our unique ability to use standard preoperative nuclearimaging for selecting patients likely to benefit from SSTR2-targetedFGS. Our findings demonstrate the translational potential of MMC(FNIR-Tag)-TOCfor intraoperative imaging and suggest broad utility for using FNIR-Tagin fluorescent probe development.Dye design can influence the abilityof fluorescentlylabeled imagingagents to generate tumor contrast and has become an area of significantinterest in the field of fluorescence-guided surgery (FGS). Here,we show that the charge-balanced near-infrared fluorescent (NIRF)dye FNIR-Tag enhances the imaging properties of a fluorescently labeledsomatostatin analogue.  Accurate intraoperativedetection of tumors is therefore essential and has led to the developmentof techniques that augment visual identification of cancer in theoperating room. Fluorescence-guided surgery (FGS) is a method of enhancingintraoperative visualization of tumors, which may be difficult todiscern, often through the use of exogenously administered contrastagents that target tumor tissues. This approach allows for delineationof tumors from normal tissues in real time to enable safer and moreeffective removal of cancerous lesions.5 We recently reported a chemically stable C4′-O-alkyl charge-balanced cyanine fluorophore, FNIR-Tag, thatis highly promising for biomolecule labeling and imaging.7 FNIR-Tag conjugates of antibodies or virus-likeparticles were brighter and had improved tumor targeting and reducednonspecific  uptake when compared totheir counterparts that used the highly anionic (net charge −3)fluorophore IRDye800 CW (abbreviated as IR800). FNIR-Tag could offersimilar benefits with low-molecular-weight compounds , which are inherently more sensitive to bioconjugationeffects, by reducing nonspecific interactions and altered pharmacokinetics caused by labeling with highlycharged dyes.6Cyanine dyes that emit fluorescence in the near-infrared (NIR)range (≥700 nm) provide strong depth of penetration and capitalizeon the inherently low tissue autofluorescence in this spectral range,making them a preferred class of fluorophore for FGS procedures.8 There are two key elementsthat make GEP-NETs an ideal environment for evaluating the performanceof a low-molecular-weight FNIR-Tag conjugate. First, GEP-NETs relyon surgery as a critical component of patient care but lack technologies9 that can identify small, multifocal lesions orinvolved lymph nodes, both of which are common in this disease processor tumor margins in the operating room.13 Second, these patients are administeredclinically approved radioactive somatostatin analogues for preoperativeimaging and surgical planning via a somatostatinreceptor subtype-2 (SSTR2)-targeted positron emission tomography (PET)scan,15 thereby establishing the value of the somatostatinanalogue/SSTR2 ligand-receptor axis. Since somatostatin analoguescan undergo a wide range of bioconjugation reactions without losingbinding affinity, we developed a strategy to transform the PET agent1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-Tyr3-octreotide  froma diagnostic agent into a surgical navigation tool by synthesizinga dual-labeled analogue, 68Ga-MMC(IR800)-TOC.16 The MMC  is a customizedcyclen analogue that enables facile conjugation of IR800 while alsoacting as a “radioactive linker” for noninvasive imagingand ex vivo tissue quantitation. Unlike conventionalFGS agents, inclusion of the MMC enabled quantitative comparison to 68Ga-DOTA-TOC as a benchmark and showed that dye conjugationdid not impair SSTR2 binding in cells and animal models.17 Furthermore, ex vivo staining of pancreatic NET(pNET) biospecimens showed high specificity for human SSTR2-expressingtumors and correlated with gold standard histopathology, demonstratingfor the first time that a clinical radiotracer could be adapted forFGS.Gastroenteropancreaticneuroendocrine tumors (GEP-NETs) are a heterogeneousgroup of malignancies with substantially increasing incidence andprevalence.e.g., pancreas, small intestine)prevented clear contrast at clinically desirable time points (3 hpost-injection). Thus, we hypothesized that replacing IR800 with anoptimized NIR fluorophore would reduce nonspecific interactions, restorethe favorable pharmacokinetic profile of somatostatin analogues, andprovide meaningful image contrast at time points that are compatiblewith GEP-NET surgery in a clinical setting. Here, we synthesized asecond-generation analogue, MMC(FNIR-Tag)-TOC, with the overall goalof improving tumor specificity and suitability for clinical imaging; flow rate,1 mL/min. Electrospray ionization mass spectra were acquired on aLCQ FLEET instrument (Thermo Scientific). Flash column chromatographywas performed using reversed phase .All chemicals were purchasedfrom Sigma-Aldrich  unless otherwise noted. IR800-DBCOwas purchased from LI-COR Biosciences . Reversed-phasehigh-performance liquid chromatography (HPLC) was performed on ananalytical Hitachi LaChrom system using a Kinetex C18 column  (Phenomenex) with a mobile phase of 1H NMR  δ8.10 , 7.88 , 7.64 , 7.49, 7.40–7.29 ,7.24 , 6.35 , 5.14 , 4.37, 4.14 , 3.90 , 3.75–3.70, 3.68 , 3.59 , 3.52 , 3.50–3.38 , 3.29 , 3.26 , 2.66 , 2.56–2.46, 2.23 , 2.12–2.01, 1.92 , 1.75 ,1.72  ppm. HRMS (Q-TOF) calculated for C71H93N5O15S2 (M + H)+ 1319.6099, observed 1319.6080.FNIR-Tag and HATU  were dissolved in dry DMF (1.3mL) in a 1-dram vial equipped with a magnetic stir bar. DIPEA  was added under argon, and the greensolution was stirred for 0.5 h at ambient temperature. DBCO-amine dissolved in dry DMF (350 μL)was added under argon, and the reaction was stirred for an additional0.5 h. The reaction mixture was precipitated in diethyl ether (30mL) in a 50 mL conical tube, vortexed, and centrifuged for 5 min at5000 RPM. The pellet was dissolved in 5% acetonitrile/water (10.0mL) and directly purified by automated reversed-phase flash chromatography. The green fractions werecombined and lyophilized to afford FNIR-Tag-DBCO as a fluffy green powder. 16 Briefly, a solution of azido-MMC-TOC was mixed with FNIR-Tag-DBCO  in a mixtureof water and DMSO (3:1). After stirring at 37 °C for 6 h andovernight at room temperature in the dark, the product (∼1.6mg) was purified with an ultrafiltration spin column (2000 Da molecularweight cutoff). Purified MMC(FNIR-Tag)-TOC was identified by analyticalHPLC and electrospray mass spectrometry m/z calculated for C142H193N23O34S4, 2895.66; found m/z, 1448.1 (1/2 mass) for [M + H]+. Chemical purityof 96.5% was determined by HPLC with a retention time of 6.14 minfor the product peak. Absorption curves were collected on a ShimadzuUV-2550 spectrophotometer operated by UVProbe 2.32 software. Fluorescencetraces were recorded on a Horiba PTIQuantaMaster-400 fluorometer operatedby FelixGX 4.2.2 software, with 5 nm excitation and emission slitwidths, 0.1 s integration rate, and enabled emission correction. TheHoriba PTIQuantaMaster-400 fluorometer was equipped with an integratingsphere for absolute ΦF measurements. Absolute ΦF measurements were carried out on solutions with absorbanceat λmax < 0.1. Calculated Log P (c Log P)values were calculated using ChemDraw Professional v19.1.FNIR-Tag-DBCO was conjugated to azido-MMC-TOCaccording to methods described for MMC(IR800)-TOC.17 BON–SSTR2  were cultured in DMEM medium with 10% (v/v) FBSand 250 μg/mL G418 antibiotic and maintained at 37 °C with95% humidity/5% CO2 atmosphere. HCT116-WT and HCT116-SSTR2cells were kindly provided by Dr. Buck Rogers (Washington Universityin St. Louis). BON–SSTR2 cells were courtesy of Dr. JeffreyFrost . Routine testing wasperformed to confirm the absence of mycoplasma in cell lines usingthe MycoAlert PLUS Mycoplasma Detection Kit .NCI-H69 , HCT116-WT, and HCT116-SSTR2 (transfectedSSTR2-expressing counterpart) cells were cultured as previously described.6 cells were implanted,respectively. For the NCI-H69 xenograft model, 6 × 106 cells were implanted. Experiments were conducted 3–4 weekspost-implantation when tumor size reached ∼5–10 mm maximumdiameter. To enable testing in a more clinically representative setting,an orthotopic pancreatic tumor model was also developed accordingto published methods.19 Mice were anesthetized, and asurvival surgery was performed under aseptic conditions. The pancreaswas then exposed to inject 3 × 106 BON–SSTR2cells in a Matrigel/PBS mixture  intothe head of the pancreas. Studies were conducted 8 weeks after orthotopicimplantation. To further strengthen the rigor of our in vivo studies, a bilateral PDX tumor model was prepared using proceduresapproved by The University of Iowa’s Institutional Animal Careand Use Committee. NOD scid γ mice  were injected with 1 × 106 NEC913 (SSTR2+)and 7 × 105 NEC1452 (SSTR2-) NEC cells, as previouslydescribed, and studies were performed 4 weeks after implantation.20 We used the percutaneous retro-orbital techniquefor intravenous injections and overdose of anesthesia followed bycervical dislocation as the method of euthanasia in terminal studiesunless otherwise noted.All animal studies were performed inaccordance with the ethical protocols approved by the InstitutionalAnimal Care and Use Committee (IACUC) of the University of Texas HealthScience Center at Houston. Athymic female J:Nu nude mice  were maintained on regular rodentchow and anesthetized with 1–2% isoflurane when required. Forsubcutaneous xenografts, cells were prepared in Matrigel (Corning)/PBS(1:1) and injected in the shoulders of mice. For the bilateral HCT116-SSTR2/WTxenograft model, 1.5 and 3 × 1068GaCl3 (t1/2 = 68 min)was purchased from the MD Anderson radiopharmacy  and 67Ga-citrate (t1/2 = 3.3 d) waspurchased from Cardinal Health. DOTA-TOC, MMC(IR800)-TOC, and MMC(FNIR-Tag)-TOC(20 nmol per batch) were dissolved in sodium acetate buffer and radiolabeledwith 68Ga or 67Ga using cation exchange chromatographyas previously described.17 Following purificationwith a Sep-Pak Light C18 (Waters) cartridge, radiochemical puritieswere determined by radio-HPLC using a dual scan-RAM (LabLogic) andfound to be ≥95% .Short and longer-livedGa radionuclideswere used based on experimental objectives. Generator-produced 21 Briefly,500 000 cells per cell line were incubated (triplicates) in96-well plates (Greiner Bio-One) with 100 nM of MMC(IR800)-TOC orMMC(FNIR-Tag)-TOC for 1 h at 37 °C in the presence or absenceof 100-fold excess octreotide, a potent SSTR2 agonist. After washing,cells were fixed in 4% formaldehyde and resuspended in 200 μLPBS. Untreated cells underwent the same procedure to determine backgroundautofluorescence. Flow cytometry was performed with a NIRF-equippedBD FACSAria II, and median fluorescence intensity values were determinedusing the FlowJo software (BD).Cellularuptake of the fluorescent conjugates was analyzed by flow cytometryaccording to published procedures.67Ga-DOTA-TOC, 67Ga-MMC(IR800)-TOC,or 67Ga-MMC(FNIR-Tag)-TOC in the presence (100×) orabsence of octreotide for 1 h at 37 °C. After washing to removeunbound radioligand, cell-associated radioactivity was quantifiedusing a Cobra II auto-γ counter (Packard), and the % of totalradioactivity added was calculated from a known aliquot.For radioactive uptake studies,200 000 cells (triplicates) were seeded in 96-well plates andincubated with 10 nM of 21 Briefly, HCT116-SSTR2cells were seeded in 8-well culture slides  at a density of100 000 cells/well. Following overnight attachment, cells wereincubated with 5 μM MMC(IR800)-TOC or MMC(FNIR-Tag)-TOC in thepresence or absence of 10-fold excess octreotide for 1 h at 37 °C.Cells were then washed with PBS, fixed in 4% formaldehyde, and mountedwith Vectashield containing DAPI (Vector Laboratories). Fluorescencewas detected using appropriate filter settings on a confocal microscope(Olympus FV3000) with excitation at 405 and 730 nm for the DAPI andNIRF signals, respectively.Binding and internalization ofthe fluorescent conjugates were examined by confocal microscopy aspreviously described.n = 5/group) were injected with 2 nmol (5.7 μg)of MMC(IR800)-TOCor MMC(FNIR-Tag)-TOC. In vivo imaging was performedwith the In Vivo Xtreme (Bruker) preclinical imaging system at 1 and3 h post-injection (p.i.). After euthanasia, tumors and tissues ofinterest were harvested for ex vivo imaging (3 h).Acquisition parameters for both in vivo and ex vivo imaging remained constant throughout the study:Excitation/Emission (Ex/Em) = 760 and 830 nm, respectively, exposuretime = 10 s, binning = 4×, f-stop = 1.1, field-of-view = 1.9cm. At the conclusion of macroscopic imaging, selected tissues werefixated and sectioned for immunohistopathological and mesoscopic imaging.Mice with bilateral HCT116-SSTR2/WT xenografts using the Xerra (Emit Imaging).67Ga-MMC(FNIR-Tag)-TOC and underwent in vivo (1 and 3 h) and exvivo (3 h) NIRFimaging as detailed above (n = 4/group). Resectedtissues were weighed, and γ counting was performed to quantitativelymeasure drug biodistribution as % of the injected activity per gramof tissue (%IA/g). The total injected radioactivity per mouse wasdetermined from an aliquot of injected solutions. After analyzingresults from the dose escalation study, 5 nmol was selected as theoptimal dose, and the imaging/biodistribution study was repeated at24 h p.i (n = 5). At the conclusion of γ counting,selected tissues were processed for immunohistopathological and mesoscopicimaging.NCI-H69 xenografts were injected with 2,5, and 10 nmol of n = 3) were injected with 5 nmol of MMC(FNIR-Tag)-TOC. In vivo and ex vivo NIRF imaging was conducted24 h p.i. using the benchtop IVIS Lumina S5 small animal imaging system(PerkinElmer). Acquisition parameters were Ex/Em = 740 and 790 nm,respectively, small binning, subject height = 1.5 cm, F/Stop = 2,and exposure imaging times of 2 s for in vivo and0.1 s for ex vivo. At the conclusion of macroscopicimaging, selected tissues were fixated and sectioned for immunohistopathologicaland mesoscopic imaging.Mice with bilateral NEC913/NEC1452xenografts (n = 5) were injected with 7.4 MBq  of 68Ga-DOTA-TOC and underwent PET/computed tomography (CT) imaging 1 h after injection.At 48 h after PET/CT imaging, mice were injected with 5 nmol of MMC(FNIR-Tag)-TOC,followed by in vivo NIRF imaging at 3 h p.i. as detailedabove. After euthanasia, tumors were harvested under white light,and the wound bed was re-imaged with NIRF. After a more thorough visualinspection of the wound bed under white light, suspicious lesionswere identified and harvested with the intact organ for exvivo imaging along with other tissues of interest. Tumors,suspicious lesions, and relevant non-tumor tissues were processedfor immunohistopathological and mesoscopic imaging.Mice bearingorthotopically implanted BON–SSTR2 tumors (exvivo and CFT experiments were measured with the molecularImaging (Bruker) and VivoQuant (Invicro) software, respectively. Tumor-to-backgroundratios (TBRs) were measured with respect to selected tissues of interest. TBR ratio was calculated using theformula St and Sb stand for fluorescentsignal in tumor andbackground tissue, respectively.Image analysis from NIRF For CFT two-dimensional (2D)and three-dimensional (3D) reconstruction, 2D images were obtainedfrom individual slices, and an image stack was generated to produce3D data sets. For PET/CT imaging, region-of-interest analysis wasdone with Integrated PMOD software (PMOD technologies) to standardizeuptake values and determine TBRs.21 Briefly, after peroxidase inactivation,sections were incubated with anti-SSTR2 rabbit monoclonal antibody overnight at 4 °C. After PBS washing, a secondaryantibody  was appliedfor 10 min at room temperature. For visualization, a DAB detectionkit  was used according to the manufacturer’sinstructions. Sections were then counterstained with Mayer’shematoxylin , dehydrated through two changes ofalcohol, cleared in xylene, and cover-slipped with Cytoseal 60 mountingmedium (Thermo Scientific). For FFPE sections, the slides were deparaffinizedbefore H&E and IHC staining, and antigen retrieval was performedprior to IHC staining. For mesoscopic imaging, an adjacent sectionfrom each tissue was scanned on an Odyssey (LI-COR) at 800 nm withthe highest resolution (21 μm).Tissuesof interest were embedded in paraffin or OCT to prepare formalin-fixed, paraffin-embedded(FFPE), or frozen sections, respectively. Blocks were then seriallysectioned at 5 μm thickness, and one section per block was stainedwith standard hematoxylin and eosin (H&E). Immunohistochemistry(IHC) staining was performed on adjacent sections as we previouslydescribed.n = 5) and normal pancreas (n =3) from the Institutional Tissue Bank at MD Anderson, whereas samplesof lymph nodes (n = 2) and metastatic deposits fromthe liver (n = 2) were obtained from the BiospecimensCore at the University of Iowa. The use of surgical tissues was approvedby the respective Institutional Review Boards of both institutes.Depending on availability, fresh or banked surgical biospecimens wereused to prepare frozen sections. H&E and IHC staining were performedon consecutive sections as described above, and fluorescent stainingfollowed published methods.21 In brief, frozen sectionswere thawed at room temperature and wetted with PBS. A 2 μMsolution of MMC(FNIR-Tag)-TOC was then applied to the slides for 1h at 37 °C. After incubation, the slides were washed with PBS,fixed in 4% paraformaldehyde, mounted with an antifade mounting medium, and imaged as detailed above.We obtained surgical biospecimens of pNETs(n >2)were performed with one- or two-way ANOVA along with multiple comparisonscorrection (Holm–Sidak). Family-wise significance and confidencelevels were set to P < 0.05. All data are presentedas mean ± SD. All in vitro experiments wereperformed with at least three technical and biological replicates.Statistical analysis was performedin GraphPad Prism 8.1.0. Group comparisons  using copper-freestrain-promoted alkyne-azide cycloaddition .16 Following HPLCpurification, spectral analysis revealed that both conjugates haveexcitation and emission peaks in the NIR region, with MMC(FNIR-Tag)-TOChaving slightly blue-shifted spectra (λmaxem = 788 nm) compared to MMC(IR800)-TOC(λmaxem = 795 nm) due to differing C4′ substitution . Both conjugates had thesame fluorescence quantum yield (ΦF) of 0.13 in PBS.Although both probes have similar optical properties, they have markedlydifferent physicochemical properties. Upon conjugation of the fluorophoresto the MMC, the net overall charge differs for MMC(IR800)-TOC (-5)and MMC(FNIR-Tag)-TOC (-2). cLog P values highlight that MMC(FNIR-Tag)-TOC(−6.68) is much more hydrophilic than MMC(IR800)-TOC (0.0938),suggesting that MMC(FNIR-Tag)-TOC will have improved in vivo performance. Spectral and physicochemical properties are summarizedin Supporting Figure 4B.MMC-TOC was produced accordingto prior methods and conjugated to DBCO-derivatives of IR800 and FNIR-Tag(in vitro experiments and used the IR800 counterpart as acontrol. We selected HCT116-WT (no SSTR2), HCT116-SSTR2, BON–SSTR2,and NCI-H69 cells based on their varying SSTR2 expression (HCT116-SSTR2≫ BON–SSTR2 > NCI-H69)  and established use for characterizing novel SSTR2-targetedagents.26 Flow cytometry results showedcomparable uptake between agents in HCT116-SSTR2 cells that was reducedwhen coincubated with octreotide and in the absence of SSTR2 (HCT116-WTcells) . Although theIR800 analogue had higher uptake in BON–SSTR2 and NCI-H69 celllines, it also had higher nonspecific uptake, as shown by blockingstudies, which yielded comparable specific binding ratios (1.3–2.8).We then applied the radioactive utility of the MMC to compare 67Ga-labeled FNIR-Tag and IR800 conjugates to the gold standard, 67Ga-DOTA-TOC. Both radiolabeled conjugates had uptake  comparable to the positive control, 67Ga-DOTA-TOC (8.7 ± 1.8%) in HCT116-SSTR2 cells (67Ga-MMC(FNIR-Tag)-TOC weremore representative of 67Ga-DOTA-TOC across cell linesand translated into specific binding ratios of 9.2 (HCT116-SSTR2),5.9 (BON–SSTR2), and 1.7 (NCI-H69), which compare favorablyto the clinical agent  and are higherthan those of 67Ga-MMC(IR800)-TOC . To confirm thatFNIR-Tag conjugation does not impair the agonist properties of TOC, we performed confocalmicroscopy in HCT116-SSTR2 cells and observed SSTR2-mediated internalizationthat was similar to MMC(IR800)-TOC -TOC, we performed in vivo study wasto demonstrate the feasibility of SSTR2 targeting and assess dye effectson imaging properties. Accordingly, we selected the dually implantedHCT116-SSTR2/WT xenograft model and a dose of 2 nmol per mouse, whichwe previously used to evaluate our first-generation agent.17 We injected the fluorescent conjugates in miceand performed in vivo NIRF imaging at 1 and 3 h p.i.,followed by ex vivo imaging of resected tissues.Consistent with our published results,17 clear tumor delineation was not possible with MMC(IR800)-TOC ateither time point due to high background fluorescence . Conversely, the reduction in background signalshown with other zwitterionic dye-conjugates3 was evident with MMC(FNIR-Tag)-TOC and provided excellent SSTR2-mediatedtumor localization at 3 h. Ex vivo imaging showedcomparable fluorescence in SSTR2+ tumors with both conjugates, butin the absence of SSTR2, only the FNIR-Tag conjugate produced theexpected decrease in tumor signal, an observation that extended toall normal tissues except the kidney . We measured, on average, a slight increasein tumor uptake with MMC(FNIR-Tag)-TOC and a >60% signal reductionin tumors lacking SSTR2 and in most nontarget tissues, including thoseassociated with NET surgery  .27 Thus, we examined the correlation between fluorescenceand SSTR2 distribution using mesoscopic NIRF imaging and immunohistopathology.In tumors, we found that only MMC(FNIR-Tag)-TOC had a fluorescencelocalization pattern that was consistent with IHC staining . The cumulative effects of dye optimization wereillustrated using 2D and 3D reconstructions that clearly support theuse of the second-generation agent for high-contrast in vivo imaging (Multimedia Files 1 and 2). Overall, these findings demonstrate thatMMC(FNIR-Tag)-TOC has higher tumor specificity and more favorablepharmacokinetics than its IR800 counterpart, which could translateinto increased tumor contrast along lower false-positive rates ina translational setting, thereby enhancing surgical accuracy.Fluorescence imaging at the macroscaleis subjectedto the diffusenature of photons and may obscure the true specificity of FGS agentsthat target a tumor biomarker -TOCwith the γ-emitting radionuclide 67Ga to overcomeattenuation and scattering phenomena.33 We injectedincreasing doses  of 67Ga-MMC(FNIR-Tag)-TOCinto nude mice with NCI-H69 xenografts, which endogenously expressSSTR2, and imaged at 3 and 24 h p.i. Supporting Figure 11. The qualitative correlation between fluorescenceand SSTR2 distribution (IHC) as a function of dose and time is shownin Supporting Figure 12. Semiquantitativeanalysis of fluorescence readouts (Supporting Tables 1 and 2), suggesting saturable tumor binding(1.5–2.0%IA/g) and efficient clearance . On average, fluorescentTBRs were >3.5 and >5.5 in the pancreas and small intestine,respectively,with no differences (P > 0.05) associated withdoseescalation -TOC,we first used the novel neuroendocrine carcinoma (NEC) patient-derivedxenograft (PDX) models, NEC913 (SSTR2+) and NEC1452 (SSTR2−),that more accurately recapitulate human disease34 4A. We in tissues 4D. Given tissues 4E. Overa18 Eight weeks afterimplantation, we implemented a proposed clinical workflow modeledafter the theranostic paradigm that uses 68Ga-DOTA-TOC(or 68Ga-DOTA-TATE) as a patient selection tool for patientswho may benefit from peptide-receptor radionuclide therapy -TOC. Abnormaluptake was observed in the abdomen of 4 out of 5 mice  and SSTR2-positiveregions (IHC) that correlated with fluorescence  between the intraoperative contrast agent and the clinicallyapproved PET radiotracer, 68Ga-DOTA-TOC (or 68Ga-DOTA-TATE). However, converting a radiopeptide into a fluorescentcounterpart is complicated by factors such as (i) a significant increasein molecular weight that can slow excretion, (ii) higher lipophilicitythat shifts clearance from the kidneys, which are preferred, to theliver, and (iii) a poor charge-to-hydrophobicity distribution thatis imparted by commonly used NIRF dyes.40 For our first-generationFGS agent (68Ga-MMC(IR800)-TOC), these collective effectsresulted in longer blood residence time compared with 68Ga-DOTA-TOC16 and required delayed imaging(24–48 h p.i.) to obtain meaningful contrast.17 We also observed higher clearance via thereticuloendothelial system (liver and spleen), which has significantclinical implications since it could reduce detection sensitivityfor metastases in these organs.28The somatostatin analogue/SSTR2 ligand-receptoraxis is the centralcomponent of theranostic approaches in GEP-NETs. Conceivably, adaptingSSTR2-targeted peptides for FGS could broaden the theranostic utilityby extending the visual information of preoperative PET imaging intothe operating room. This would require similar 2 Head-to-head studies showed that ZW800-1 conjugateshad lower serum binding and nonspecific tissue uptake compared withtheir anionic counterparts, resulting in higher TBRs.3 More recently, the charge-balanced, symmetrical, and PEGylatedcyanine FNIR-Tag was developed to address the problem of aggregation-inducedquenching that occurs with IR800-labeled antibodies.7 Antibodies labeled with FNIR-Tag had higher tumor uptake,reduced liver uptake, and enhanced brightness compared with IR800-labeledconjugates. Unlike IR800, FNIR-Tag reduces fluorescence signal inblood while minimizing interactions with serum proteins and cationicsurfaces. To achieve the combination of high tumor binding and lowbackground signal associated with radiolabeled somatostatin analogues,we developed the second-generation FGS agent MMC(FNIR-Tag)-TOC anddemonstrated its superior performance in cells, animal models, andhuman biospecimens. The mechanism by which FNIR-Tag mitigates nonspecificinteractions and enhances tumor contrast relative to IR800 remainsto be investigated. Previous studies showed that C4′-O-alkyl cyanines are less reactive to cellular proteins  than the C4′-O-aryl IR800.43 Here, we found that MMC(FNIR-Tag)-TOC retains tumor targeting, decreasesbackground fluorescence in liver and spleen (30–60%), and increases>2-fold kidney signal compared to the IR800 analogue -TOC in the present study. Most notably, wedemonstrated that employing a charge-balanced dye restored the lowbackground signal of the parent peptide in multiple tumor models andproduced a >60% decrease in fluorescence signal in the pancreasandsmall intestine. The lower off-target signal also improved TBRs tothe extent that it was possible to visualize tumors as early as 3h p. i. These combined effects could conceivably increase the positivepredictive value of SSTR2-targeted FGS and expand the imaging timewindow to give surgeons flexibility in administering MMC(FNIR-Tag)-TOC.While these findings suggest an improved likelihood for intraoperativetumor delineation with the second-generation conjugate, we acknowledgethat results obtained with preclinical imaging systems are not indicativeof utility with a clinical imaging device.51 However, we previously addressed this limitation by showing thefeasibility of tumor imaging with our first-generation agent in combinationwith the da Vinci Firefly imaging system and a custom-built clinicalprototype NIRF imaging system (OnLume Inc.) and anticipate similardrug-device compatibility with MMC(FNIR-Tag)-TOC.52 We also demonstrated the unique benefitof selecting DOTA-TOC as the foundation for the fluorescent analogue,as it allowed the use of standard-of-care PET imaging for “patient”selection in animal studies. This strategy is currently not possiblewith any other FGS agents since they do not have an FDA-approved nuclearimaging counterpart and may instead rely on biopsies or alternativeimaging techniques to determine target expression.Several low-molecular-weightFGS agents have advanced to clinicaltrials,55 Furthermore,adding real-time tumor imaging to minimally invasive (robotic) surgerymay compensate for its lack of tactile feedback. Given the existinguse of nontargeted NIRF dyes with commercially available surgicalrobotic systems, similar integration of MMC(FNIR-Tag)-TOC is feasibleand could facilitate quicker recovery after GEP-NET surgery.56 Finally, our FGS strategy could potentiallyextend beyond GEP-NETs by facilitating the conversion of other radiolabeledpeptides and small molecules into tumor-targeted fluorescent analogues.Thus, new imaging capabilities would become available for a varietyof tumor types that rely on surgery as their primary treatment modality.In conclusion,our data shows that the engineered dye FNIR-Tagcan produce an SSTR2-targeted FGS agent with superior tumor specificity.Clinical application of our agent may enable the detection and removalof multifocal small bowel NETs and nodal disease that may otherwisebe missed with standard surgical techniques. SSTR2-targeted FGS alsohas the potential to increase surgical accuracy in pNETs by facilitatingparenchyma-sparing R0 resections. This capability could reduce unnecessarilywide surgical margins that can cause pancreatic insufficiency in upto 40% of patients and impair quality of life.