Molecular design, assembled behavior and conformation
In order to study the molecular design and their assembly behavior, we modular designed and synthesized seven molecules (Table 1), which respectively were BIVA probe: M1 (mPEG-GPAKLVFFGC(IR783)GRGD); the non- FAP-α tailoring molecule: M2 (mPEG-AGGKLVFFGC(IR783)GRGD) scrambled response tailoring motif of Gly-Pro-Ala with Ala-Gly-Gly; the non-labeling molecule of M1: M3 (mPEG-GPAKLVFFGCGRGD) removed IR783 labeling; FAP-α tailoring residue of M1: R-M1 (AKLVFFGC(IR783)GRGD); the non-targeting molecule: M4 (mPEG-GPAKLVFFGC(IR783)GDTG) scrambled targeting tailoring motif of Arg-Gly-Asp with Asp-Thr-Gly; the non-labeling molecule of M2: M5 (mPEG-AGGKLVFFGCGRGD) removed IR783 labeling; FAP-α tailoring residue of M3: R-M3 (AKLVFFGCGRGD). All the synthesized procedure (Supplementary Fig. 1) and characterizations of these seven molecules can be found in SI (Supplementary Figs. 2–8). As seen in Table 1, although scrambled the tailoring motif of M2 and targeting motif of M4, the critical assembly concentration (CAC) of these two molecules in aqueous solution was like that of M1, both of which were above 500 μM. Without IR783 labeling, molecules of M3 and M5 decreased the CAC value in solution relative to M1 and M2, which could be attributed to the proximity of the steric hindrance of hydrophilic IR783 to the self-assembly motif (Supplementary Fig. 9). When we removed the long-term circulation motif of mPEG2000 tail of M1 and M3, the CAC of the truncated residues of R-M1 and R-M3 dramatically decreased by more than two orders of magnitude (Supplementary Fig. 10). The CAC was quantitatively calculated by fluorescence probe of pyrene. All the results indicated that both hydrophilic mPEG tail and IR783 labeling contributed to the solubility of molecules in aqueous solution.
From the molecular dynamics (MD) simulation calculations, we observed that both the backbones of M1 and the residual R-M1 of M1 were β-hairpin conformations (Fig. 2 and Supplementary Fig. 11). The mPEG tail was close to the self-assembly motif through multiple hydrophobic interactions to stabilize its conformation, including hydrogen bonds VAL4:CYS8, ARG10:ALA1, ARG10: IR783, ARG10:ASP12, ARG10:ASP12, and salt-bridge ARG10:ASP12 on both sides of the hairpin. Interestingly, the labeling of IR783 was perpendicular to the β-hairpin backbone and mPEG tail, which formed a significant steric hindrance preventing the further intermolecular assembly. When the mPEG motif was tailored, the backbone of R-M1 remained its β-hairpin structures by hydrophobic interactions, hydrogen bonds GLY9:PHE6, LYS2:CASP12, ARG10:CASP12, LYS2:CASP12, ARG10:CASP12 and salt-bridges LYS2:CASP12, ARG10:CASP12 on both sides of hairpin, while the IR783 showed an obvious intramolecular rearrangement, resulting in its alignment parallel to the backbone. When the mPEG motif was tailored28, the IR783 was arranged parallel to the backbone, the hydrophilicity of molecule decreased and the hydrogen bonds on the self-assembled surface were exposed. The decrease of hydrophilicity of molecule and the exposure of hydrogen bonding surface of self-assembled motif were conducive for the occurrence of intermolecular dynamic assembly. So that the tailoring of the mPEG motif promotes the occurrence of intermolecular dynamic assembly.
The molecular interaction details including hydrogen bond (blue dotted-line), salt-bridge (red dotted-line) and hydrophobic interaction. Red part: IR783; Gray part: mPEG2000; Green part: peptide.
To further evaluate the assembled structures, the corresponding circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy, and wide-angle X-ray scattering (WAXS) spectroscopy were applied. As shown in Fig. 3a and Supplementary Table 1, CD spectrum of M3 assemblies had a positive band at λ = 193 nm and two negative bands at λ = 208 nm, and λ = 225 nm respectively, which implied a β-sheet and α-helix hybrid structure. In contrast, under the same concentration, M1 molecules had a random coil secondary structure in CD spectrum as monomers which the concentration is lower than CAC. The FITR spectra of M1 and M3 in Fig. 3b observed the intermolecular interactions. The peaks at 1629 cm−1, 1675 cm−1, and 1698 cm–1 of M3 indicated anti-parallel β-sheet structure (represent by green arrow heads), peaks at 1648 cm−1 and 1663 cm−1 indicated parallel beta sheet structure (represent by blue arrow heads), the existence of 1654 cm−1 indicate α-Helix structure in M1 which verified the results deduced by CD in Fig. 3a29,30. The evidence indicated that without IR783 labeling, the M3 molecule was easier to assemble than the M1 in the absence of IR783, and the driving forces of assembly depend on the multiple hydrogen bonds and other weak interactions of the self-assembly motif. After tailoring the hydrophilic balance of mPEG, the R-M1 exhibited a well-ordered β-sheet assembled secondary structures with a typical strong positive band at 196 nm and a wide negative band at 216 nm (Fig. 3c). The R-M1 molecules had a rapid dynamic assembly (within few minutes), and the assemblies in aqueous solution had an obvious Tyndall phenomenon. As a homologous sequence with amyloid β-protein (Aβ), the self-assembly motif with peptide sequence of KLVFFGCG had similar aggregation kinetics to (Aβ)42 peptide, which occurred via dynamic growth from oligomers to amyloid fibrils31. The aggregation started from the freshly isolated monomers of R-M1, and precipitates were separated in 1 min and 1 h, respectively. The FTIR spectra of these two samples (Fig. 3d) exhibited completely different spectral features. The one separated rapidly showed a broad peak at 1634 cm−1, which was identified as oligomer; while the one with extended aggregated time had three peaks at 1698 cm−1, 1688 cm−1, and 1629 cm−1, which were ascribed to the as anti-parallel β-sheet fibrils32,33. After analyzed the CD spectra of M1, M3, and R-M1 (Fig. 3e), it can be clearly seen that the main secondary conformation of M1 was Random, the M3 was hybrid of Helix and Beta, and the R-M1 was Beta (Supplementary Table 1). All the results confirmed the conclusion from FTIR spectra. Characteristic of nucleated growth procedure (Fig. 3f), the aggregation curves with ThT trace had a growth phase for primary process from the initial 17 min, an elongation phase for surface-catalyzed secondary process between 17 and 30 min, and a final plain phase after 30 min. The dynamic growth procedure was like the 8-anilino-1-naphthalenesulfonic acid (ANS) stained curve (Supplementary Fig. 12). As known, ANS was sensitive to hydrophobic interaction34. When R-M1 were in the initial oligomer, the fluorescence intensity of ANS increased due to the enhanced hydrophobicity. When the molecules were elongated and stacked in higher ordered nanofibrils, the blue shift of ANS in the β-sheet structures reduced the fluorescence. The molecular packing mode of well-ordered fibrils of R-M1 in Fig. 3g observed a weak reflection at 4.9 Å as laminates space and a strong broad reflection at 10.3 Å as sheet space, which was illustrated in the inserted figure. The fibril morphology was characterized by transmission electron microscopy (TEM) imaging (Fig. 3f). The statistical calculation of the fiber diameter in TEM images was 5.0 ± 0.4 nm (Supplementary Fig. 13), which was corresponded to the theoretical calculated two molecules length of R-M1. We assumed that the nanofibers were assembled by twisted R-M1 molecules centered on self-assembly motif.
a CD spectra of M1 and M3 in DI water under a concentration of 200 μM. b The FTIR spectra of M1 and M3, powder samples collected from freeze dried sample solutions. c The typical β-sheet CD spectra of R-M1 in DI water (insert figure: the Tyndall phenomenon) under a concentration of 100 μM. d The FTIR spectra of dynamic growth of R-M1, powder samples collected from freeze dried sample solutions in different period. e Analysis of secondary structure composition of M1, M3, and R-M1 based on CD spectra. f, Elongation-nucleation growth procedure with ThT staining. The mean of data of three samples with the same conditions is shown and data are presented as mean values ± SD (n = 3) g, The WAXS spectrum and illustration of the R-M1 fibrils, powder sample collected from freeze dried sample solution. h, The TEM images of nanofibers morphology of R-M1.
Specific enzyme tailoring induced nanofibril assembly
To further investigate the FAP-α specific tailoring and BIVA probe assembly in situ simultaneously (Fig. 4a), the high-performance liquid chromatography (HPLC), TEM, and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were applied for the characterizations purpose. To specify the cutting of the response tailoring motif (Gly-Pro-Ala), pre-synthesized molecules of R-M3, M3 and M5 were set as controls. After incubation with FAP-α for 12 h, M3 molecules were totally cleaved by the enzyme (Fig. 4b), resulting in truncated residues with different retention times compared to M3 (29.5 min). Comparing the residues peaks with R-M3, the primary sharp peak at 27.4 min can be identified by the R-M3 control (27.6 min). In the meantime, the wide peak at 14.6 min might be the remaining PEG residue. In sharp contrast, after incubation of FAP-α with M5, there was no change of the retention peak, which double confirmed that the GPA was the FAP-α specific recognized sequence and the molecule was cut between the amino acid of Pro and Ala. The retention time of M1 after co-incubation with inactivated FAP-α was similar to that of M3 (Supplementary Fig. 14). After hydrolyzed by FAP-α, the residues of M1 in situ assembled into nanofibers structures under the complex buffer (Fig. 4c and Supplementary Fig. 15). M1 could not be assembled after co-incubation with the inactivated FAP-α, which further indicated that the assembly of M1 could be ascribed to the FAP-α tailoring (Supplementary Fig. 16). The tailored residues were identified by MALDI-TOF (Fig. 4d), which revealed that M1 was cut into two parts of R-M1 and PEG residue. Additionally, when the molecules of M1 were incubated with MIA PaCa-2 cells for 2 h, the cell lysis was observed to split into layers. MALDI-TOF confirmed that R-M1 in the precipitate might be the assembly induced precipitate, and the supernatant obviously contained the PEG residues (Supplementary Fig. 17). In order to further evaluate the specificity of M1 for different enzymes, including FAP-α, pepsin, pancreatin, lipase, and BSA, we used ThT (Thioflavin T) as a detection probe (Fig. 4e). After co-incubation with M1 for 12 h, only FAP-α group strikingly enhanced the fluorescence intensity, which indicated that M1 molecule had specificity for FAP-α induced in situ assembly.
a The illustration the working mechanism of BIVA probe based on FAP-α catalytic hydrolysis. b HPLC curves of M3 and M5 after incubation with FAP-α in buffer. R-M3, M3 and M5 were synthesized controls. The TEM image (c), and the MALDI-TOF (d), results of M1 after tailoring by FAP-α in buffer, the red arrows represent the assembled fibrils of M1 after incubation with FAP-α. e The enzyme specificity of M1 in buffer. Buffer: 50 mM Tris, 1 M NaCl, 1 mg/mL, BSA, pH 7.5; Concentration of M1 and M3: 100 μM; Concentration of FAP-α: 50 μM; incubation time: 12 h. The mean of data of four samples with the same conditions is shown and data are presented as mean values ± SD (n = 4). p = 2.04E-22 < 0.001, p values were performed with one-way ANOVA by post hoc Tukey’s test for the indicated comparison.
Enhanced targeting and in situ high efficiency nanofiber formation located cell outline
As designed, the bioactivated in vivo assembly (BIVA) was a triggered and synchronous dynamic assembly system with active targeting cooperative aggregation/assembly induced retention (AIR) effect. Compared with active targeting mechanism dependent on binding constant Kd, BIVA effect showed an amplified mechanism based on primary binding constant Kd and secondary assembly rate constant Ka (Fig. 5a). To further confirm this hypothesis, the M1 and M2 were equally labeled by FITC for cell imaging (Fig. 5b). To simulate the dynamic physiological condition, the cell culture medium was replaced every 15 min. After 1 h of incubation with M1 and M2 at the same molecule molar concentration, there occurred significant differences between the two molecules (Fig. 5c). The M1 with BIVA effect had a higher fluorescence retention rate on the cell membrane, while the M2 with active targeting mechanism significantly reduced the fluorescence signal during the dynamic incubation. The huge difference can be explained by the fact that the secondary assembly rate broke the balance between the targeted ligand and receptor, and thus tended to a stable assembly interaction. The retention efficiency depended on the assembly rate constant Ka. Meanwhile, the rapid dynamic assembly of BIVA probe in situ around the cell membrane, and contributed to the efficient formation and retention of nanofibers on the cell profile. When the incubation time was delayed up to 2 h, some components could be endocytosed into the cells, but most of them were found on the membranes (Supplementary Fig. 18). As shown in Fig. 5d, most molecules of M1 were assembled and located on the cell membrane within 1 h of incubation. Then, the isolated cells were collected and lysed, the extracted cell membrane fragments were stained with ThT. Interestingly, the nanofibers on the membrane were all stained by ThT, and the correlation coefficient between ThT and FITC fluorescence was high up to 0.83 (Fig. 5e). In order to verify the influence of the addition of the targeting module of the probe molecules, we explored the imaging of R-M1 and M4 on cells. After co-incubation with R-M1, which was an insoluble suspended mixture, the fluorescence signal could be observed both in medium and on the cell membrane (Supplementary Fig. 19d). While the cells were treated by M4, only a small amount of fluorescence signal could be observed on cell membrane (Supplementary Fig. 19e). The phenomenon observed by R-M1 indicated that with RGD motif, the assembled R-M1 still remain the specific bonding capability to cell membrane. Without RGD bonding, the M4 molecules could also be tailored by FAP-α and the assembled nanofibers exhibited non-specific interaction on the membranes. Moreover, once pre-treated the cells with RGD peptides (Supplementary Fig. 19b) or FAP-α inhibitors (Supplementary Fig. 19e), the cell membrane bonding and retention of the M1 signals were significantly reduced, which validated that the RGD recognition and FAP-α induced nanofibers formation both contributed to the attach on the membranes.
a Illustration of BIVA effect: active targeting and assembly/aggregate induced retention (AIR) effect. b Chemical structure of FITC labeling. c 2D and 3D confocal images of M2 with active targeting property and M1 with BIVA effect on MIA PaCa-2 cells after incubation for 1 h. d The distribution of M1 on MIA PaCa-2 cell. e ThT staining of the lysed cell membrane of MIA PaCa-2. f, Illustration of migration inhibition after treated by BIVA probe. g The image of the migrated cells after treatment of PBS (blank), M2, M4, and M1. Scale bar: 100 μm. h The pie diagram of quantitative statistical calculation of the migrated cells (blue) in different groups. The blank control was set as 100% migrated cells.
In order to further understand the contribution of the active targeting and AIR effect during locating of the cells, the cell transwell experiment was used to quantitively evaluate interference of the cell migration based on M1, M2, and M4 (Fig. 5f). As expected, untreated cells were easy to migrate to the lower chamber, while the cells treated with active targeting molecule M2 and assembled molecule M4, the migration of cell were reduced. Under the same molar concentration, the M1 treatment group had the most inference on cell migration (Fig. 5g). According to the quantitative statistical calculation of the number of cells (Fig. 5h and Supplementary Fig. 20), the results clearly verified that the BIVA effect indeed had a high trapping and localization efficiency around cells. Based on those results, we speculate M1 can be more enriched in the tumor site and form fibers through assembly to inhibit tumor cell migration compared to M2 and M4 which have no ability to target and adhere on the cell membrane. Although the targeting group of M2 and BIVA group of M1 had significant interaction with cells, there was no obvious cytotoxicity at high concentration of 300 μM (Supplementary Fig. 21).
Metabolic difference and optimized biodistribution enhanced imaging
In order to reduce systemic error and individual difference, we constructed the subcutaneous pancreatic cancer model in mice for quantitatively calculating of the metabolic data. First, the subcutaneous tumor model of the right hind leg had no overlap with other organs, and it can reduce the organs depth difference caused system error. Secondly, compared with the orthotopic tumor, the size of the subcutaneous tumor was more controllable, reducing the individual difference between the experimental groups, and making the experimental data more reliable. According to the time-dependent in vivo NIR images, there were significant differences in the fluorescence distribution among ICG, M2, and M1 mice (Fig. 6a). The representative small molecule probe was ICG, which showed rapid distribution and elimination all over the mice body with no obviously specific targeting effect on tumor tissue. The results of near infrared (NIR) imaging showed that ICG completed its metabolic clearance within 8 h. Moreover, the M2 probe with active targeting capability was distributed and accumulated in the tumor area within 0.1–24 h. The metabolized rate from tumor and other tissues seemed no obvious difference. However, the M1 probe based on the BIVA effect optimized the biodistribution, which accumulated more signal in the tumor area and enhanced retention in tumor long lasted up to 96 h. Meantime, the non-tumor tissues showed lower signal distribution resulting a short elimination time. Based on NIR imaging, the M1 with higher imaging contrast enable to have a stable detection window within 8 h-96 h attributing to the enhanced targeting and metabolic difference of the BIVA probe. Meanwhile, the photoacoustic imaging after i.v. injection of M1 and M2 for 12 h clearly show the big difference on the tumor signal and the surrounding tissues, the M1 had more accumulation in tumor than that of M2 (Supplementary Fig. 22).
a The time-dependent NIR fluorescence image of mice bearing MIA PaCa-2 cells after intravenous administration of ICG, M2 and M1 with a dose of 16 mg/kg. The images acquired at time intervals from 0.1 h to 120 h are managed with the same procedure (The circles represent the locations of the tumors). b The blood circulation curve of ICG, M2 and M1 based on exponential curve fitting. The t1/2 value was the blood circulation half-life. c The time-dependent quantitative calculation of the average fluorescence intensity in tumor area and the area under the curve (AUC) of ICG, M2 and M1. The mean of three biological replicates is shown and data are presented as mean values ± SD (n = 3).
Otherwise, the molecules of M1 and M2 with a mPEG tail, both had long-time circulation half-life (t1/2), which were 110 ± 3 min and 102 ± 4 min, respectively (Fig. 6b). The blood circulation half-life (t1/2) of ICG was 2.5 ± 0.5 min. The short t1/2 was related to the rapid distribution and elimination behavior in vivo. In order to understand the contribution of these elements to effective availability of imaging probe. The significant parameter of pharmacokinetic: area under the curve (AUC) in tumor tissue was obtained according to the quantitative calculation from fluorescence signal. After quantitative calculation the concentration of probe in tumor area without background signal subtracted, the time-dependent curve of M1 and M2 were carried out (Fig. 6c). The area under the curve ranges from 0 h to 120 h (AUC 0-120 h) of M1 was 3.6 times more than that of M2, which mean that with a single dose administration, the average fluorescence intensity distribution per unit area of M1 in tumor tissue was 3.6-fold higher than that of M2. The time to peak of M1 was 4 h later than M2, about at 12 h. The highest signal on tumor of M1 was 1.8 times higher than M2. In addition, the signal elimination of M1 from tumor was quite slow, only 27.6% was reduced between 12 h and 120 h, while the fluorescence signal of M2 was disappeared completely in the same time interval. Finally, we obtained a stable intraoperative navigation window between 8 h and 96 h for our BIVA probe (M1). In conclusion, both the long-term blood circulation and the dynamic enzyme tailoring helped the continuous accumulation in tumor area. The in-situ assembly in tumor tissues slowed down the dynamic elimination and prolong the elimination time, which contributed to maintaining the imaging signal during surgery operation. The FAP-α specific tailoring and assembling of M1 differed the tumor from the other tissues, which offered better contrast and biodistribution. To evaluate the imaging property of BIVA probe, the orthotopic pancreatic tumor mice model was built. After intravenous injected M1 and M2 molecules with a dose of 16 mg/kg for 12 h, the mice were sacrificed. When dissected the spleen, the high contrast signal was clearly observed on the orthotopic tumor area (Fig. 7a, Supplementary Fig. 23). Then, all the important organs were dissected for ex vivo imaging. The significant difference between M1 and M2 on the tissue biodistribution. For BIVA probe M1, the distribution on tumor had obviously selectivity, and the molecules had part retention in the metabolic organs (e.g., liver and kidney). Whereas, the M2 exhibited no significant difference in the biodistribution of lung, kidney, and tumor, but most of the molecules were stuck in liver. The huge difference between the two molecules can be explained by the high specific recognition of FAP-α to M1, which was conducive to efficient molecular tailoring and assembly in tumor, while the M2 was non-specific cleavage and accumulation in liver during metabolism. The quantitative analysis results also confirmed the conclusion (Fig. 7b). The signal accumulation of FAP-α specific BIVA probe M1 was twice as much as that of M2. Under the same blood circulation time, organ selectivity depended on the specificity of substrate to target enzyme. Its accumulation amount relied on the cleavage rate of enzyme and aggregation efficiency of molecular residues. The primary nucleation of assembly can induce long lasting growth of the fibril in tumor, reduce the metabolic rate, and achieve the retention and accumulation of tumor. When pre-treated the mice with M1 and M2 with the same dose for 48 h (Supplementary Fig. 24), the signal in liver were reduced, which could be explained as the dynamic metabolism by the liver. The signal on orthotopic tumor or small tumor still clearly observed after 48 h M1 treatment. Upon the individual differences, we validated 6 mice under surgery to induce orthotopic tumor in pancreatic head. All the positive results were obtained including the small sized tumor around 2 mm in the diameter (Fig. 7c). The ex vivo dissection in Fig. 7d provided a fantastic imaging contrast on tumor and the around the spleen tissue, which visualized and identified the small tumor (~ 2 mm) both on 2D and 3D images (Supplementary Fig. 25). The statistical results of fluorescence signal on tumor were over 9 folds higher than the surrounding spleen tissue (Fig. 7e). The whole tumor histologic section in Supplementary Fig. 26 stained by Congo Red fully viewed the fibril distribution the tumor after 12 h M1 treatment. As known, the FAP-α was a membrane located protein, overexpressed on tumor associate fibroblast cell and pancreatic cell surface. The FAP-α specific BIVA probe M1 were well depicted the tumor margin and interstitial space, which concentrated the signal in tumor for better bioimaging, but the M2 has no obvious Congo Red, which means there was no assembly inside tumor. In order to validate the M1 deep penetration in tumor after 48 h M1 administration, we merged the tumor histochemical staining with Hochest3342 (blue), CD31 (green) and M1 (red). As seen in Fig. 7f, most of the M1 were far away from the green colored blood vessels and the signals were uniform distributed in tumor. The whole tumor section both confirmed the conclusion above (Supplementary Fig. 27). Once stained by Congo Red (Supplementary Fig. 28), the tumor slices were easily observed the red distribution, which mean that the deep penetrated M1 were transformed to nanofibers.
a The in vivo NIR images of small orthotopic pancreatic tumor by M1 and M2, and the ex vivo of organ biodistribution including heart, liver, spleen, lung, kidney, and tumor. The mean of three biological replicates is shown (n = 3). b The quantitative analysis of average fluorescence intensity per organ area p = 0.000844 < 0.001, (n = 3). c The in vivo NIR images of orthotopic pancreatic tumor with individual difference with the same i.v. dose administration for 12 h. The mean of five biological replicates is shown (n = 5). d The small size (~2 mm diameter) orthotopic pancreatic tumor images and its ex vivo signal distribution. e The quantitative calculation of the signal in tumor area and healthy spleen area. The mean of eight biological replicates is shown. (n = 10) f The tumor histochemical staining with Hochest3342 (blue), CD31 (green) and M1 (red) post i.v. injection of M1 for 48 h. The yellow arrows pointed at the blood vessels. Bars of the up layer: 200 μm; Bars of the bottom layer: 50 μm. Data: mean ± standard deviation. Injection dose (i.v. administration): 16 mg/kg. Statistical analysis: one-way t test followed by post hoc Tukey’s test, ***p < 0.001.
Acute toxicity evaluation to organs
The acute toxicity evaluation of M1 to mice were verified by blood biochemistry, hemograms, and histological analysis. The representative biomarkers of liver function included alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), total protein (TP), and albumin concentration (ALB). Compared to the healthy group (PBS), there was no obvious hepatic toxicity after i.v. injection of 16 mg/kg of M1 for 24 h (Fig. 8a). In addition, the hematological assessment results including creatinine (CREA), white blood cells (WBC), red blood cells (RBC), hematocrit (HCT), mean corpuscular volume (WCV), mean corpuscular hemoglobin concentration (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell volume distribution width-coefficient of variation (RDW-CV), platelet (PLT)and mean platelet volume (MPV) were carried out (Fig. 8b). All the above indicators in PBS group and M1 group appeared normal, which was basically consistent with the normal range reported in the literature. Then the mice from PBS group and M1 group were sacrificed for the further histological section analysis of the significant organs. After Hematoxylin and Eosin (H&E) staining, the slices of heart, liver, spleen, lung, kidney, and pancreas were compared and evaluated (Fig. 8c). There was no noticeable organ damage and tissue injury of the two groups. All the evidence revealed that under the imaging dose, the BIVA probe had no acute toxicity performance.
a Liver function indicators: alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), total protein (TP) and albumin concentration (ALB). The mean of four biological replicates is shown and data are presented as mean values ± SD (n = 4). b Blood biochemical indicators: creatinine (CREA), white blood cells (WBC), red blood cells (RBC), hematocrit (HCT), mean corpuscular volume (WCV), mean corpuscular haemoglobin concentration (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell volume distribution width-coefficient of variation (RDW-CV), platelet (PLT)and mean platelet volume (MPV). The mean of four biological replicates is shown and data are presented as mean values ± SD (n = 4). c Histologic sections of different organs: heart, liver, spleen, lung, kidney and pancreas compared with healthy group (PBS). Staining: H&E; Injection dose of M1. (i.v. administration): 16 mg/kg; Administration time: 24 h. Scale bar 200 μm.
