PPD  is developed to mediate plasmid-encoding shPD-L1 delivery by introducing multiple interactions  and polyproline II (PPII)-helix conformation, which downregulates PD-L1 expression on tumour cells to relieve the immunosuppression of T cells. Zebularine (abbreviated as Zeb), a DNA methyltransferase inhibitor (DNMTi), is used for the epigenetic regulation of the tumour immune microenvironment, thus inducing DC maturation and MHC I molecule expression to enhance antigen presentation. PPD plus Zeb combination therapy initiates a systemic anti-tumour immune response and effectively prevents tumour relapse and metastasis by generating durable immune memory. This strategy provides a scheme for tumour treatment and the inhibition of relapse and metastasis.Immunotherapy has become a powerful cancer treatment, but only a small fraction of patients have achieved durable benefits due to the immune escape mechanism. In this study, epigenetic regulation is combined with gene therapy-mediated immune checkpoint blockade to relieve this immune escape mechanism.  While immunotherapy is a promising cancer treatment option, durable benefits are often rare due to immune escape. Here, the authors combine epigenetic regulation with gene therapy-mediated immune checkpoint blockade and show efficient anti-tumour effects and immune response in vivo. However, clinical trials have shown that only a small fraction of patients acquired durable benefits4 mainly due to the immune escape mechanism in cancer7. One of the reasons for this is the low levels of tumour antigens9 and insufficient ability of antigen-presenting cells (APCs) to present antigens to T cells11, all of which cause poor cytotoxic T-lymphocyte responses. Equally important is that T cells have immune checkpoint-dependent immunosuppression, e.g., programmed cell death 1 (PD-1) receptor expressed on T cells binds to its two ligands, PD-1 ligand 1 (PD-L1) and ligand 2 (PD-L2), which are upregulated on tumour cells. This results in T cell failing to exert its effector functions in tumour immune surveillance14.Immunotherapy has become a powerful treatment strategy for cancer in the past few years and checkpoint inhibitors have revolutionized the field of oncology by activating the anti-tumour immune response16, photodynamic therapy18, and chemotherapy-mediated immunogenic cell death20. However, photothermal and photodynamic therapies are susceptible to photothermal agents, photosensitizers, and light. In addition, chemotherapeutic agents usually have severe toxic side effects on normal tissues22. Therefore, an efficient and safe strategy is urgently needed to generate an ideal condition to boost antigen-specific immune response. Recently, it has been reported that epigenetic therapy such as DNA methyltransferase inhibitors (DNMTi) could not only upregulate the expression of tumour-associated antigens but also induce the enhancement of major histocompatibility complex (MHC) class I molecules on tumour cells via cancer testis antigen (CTA), thereby ultimately mounting the visibility of tumour cells to the adaptive immune system26.Generally speaking, tumour cell fragments could be used as tumour antigens, which were achieved by photothermal therapy27. The PD-1 receptor is a crucial immune checkpoint molecule and is mainly expressed on mature cytotoxic T lymphocytes (CTLs) in peripheral tissues and the tumour microenvironment28. PD-1 signaling is mediated via the engagements of PD-L1 and PD-L2, resulting in T-cell exhaustion. A number of monoclonal antibodies targeting PD-1 or PD-L1 have demonstrated beneficial treatments against several tumour types, including melanoma30, hepatic carcinoma31, non-small cell lung cancer32, and Hodgkin’s lymphoma33. Nevertheless, effectiveness in only a minority of patients and resistance after initial response are usually observed34. Moreover, PD-1 or PD-L1 targeted antibody therapies may cause severe side effects, such as immune-related adverse events (irAEs) associated with inflammation and toxicity36. Gene therapy-guided immune checkpoint blockade could be used as an alternative strategy of reversing T-cell exhaustion while avoiding the side effects caused by PD-1/PD-L1-targeted antibody therapies.Moreover, immune checkpoints are crucial regulatory pathways that invalidate T-cell-mediated tumour killingHerein, an efficient strategy of utilizing epigenetic regulation combined with gene therapy-guided immune checkpoint blockade is described, which simultaneously increases the levels of tumour antigens, enhances the ability of APCs to present antigens and reverses T-cell exhaustion Fig. . More sp38. Among them, polyethylenimine with a molecular weight of 25 kDa (denoted as PEI25k) was recognized as the gold standard40; however, its severe cytotoxicity limited its clinical application41. Although low-molecular-weight polyethylenimine such as PEI1.8k (polyethyleneimine with a molecular weight of 1800 Da) had low cytotoxicity, its poor transfection could not live up to expectations. Herein, ‘molecular string’ p-toluene sulfonyl arginine (abbreviated as RT) was introduced onto PEI1.8k and its effect on the transfection capability and biocompatibility of PEI1.8k was studied.Polycationic gene carriers have drawn increasing attention because of their low immunogenicity, high loading efficiency, and abundant sources1H nuclear magnetic resonance (NMR) spectra indicated the successful synthesis of PEI-RTs , and PEI-Arg(NO2) were 9.8, 10.2, 10.6, 10.4, 10.7, and 10.8, respectively , and Orn(Tos), the optimal transfection efficiencies of the corresponding polycations were considerably higher than that of PEI-Arg or PEI-Orn. The reason should be that introducing hydrophobic groups increased the hydrophobic interactions between carrier and cell membrane (or DNA), thereby boosting the cellular uptake. (5) For cationic ‘molecular string’ including hydrophobic groups, the only differences existed in the hydrophobic groups, such as PEI-RT3 and PEI-Arg(NO2), and their transfection efficiencies were dramatically different, the optimal transfection efficiency of PEI-RT3 was 2.8-fold higher than that of PEI-Arg(NO2). The hydrophobicity of p-toluene sulfonyl was stronger than that of nitro group, thus accelerating the cellular uptake of PD. (6) When the hydrophobic groups of ‘molecular string’ were the same and the difference simply lay in the category of amino acids, such as PEI-RT3 and PEI-Orn(Tos), the optimal transfection efficiency of PEI-RT3 was 4.92-fold higher than that of PEI-Orn(Tos), which was owning to the strong hydrogen-bonding interactions between guanidine group of PEI-RT3 and cell membrane (or DNA). (7) For cationic ‘molecular string’ including hydrophobic groups, such as PEI-RT3, PEI-Arg(NO2), and PEI-Orn(Tos), their optimal transfection efficiencies were considerably higher than that of the ‘golden standard’ PEI25k. (8) Moreover, DNA transfection was also determined in 4T1, HeLa, and MCF-7 cells, and similar results were obtained  and PEI-RT3, their structural units had larger molecular weight than PLL-Arg. Therefore, the charge density of PEI-Arg(NO2)/DNA or PD was lower than that of PEI-Arg/DNA. For PEI-Orn(Tos), its ‘molecular string’ merely contained one positive charge and the zeta potential of PEI-Orn(Tos)/DNA was lower than that of PEI-Arg(NO2)/DNA or PD at the same mass ratio. According to gene transfection and zeta potential data, an appropriate positive surface charge is essential for efficient gene transfer.As various ‘molecular strings’ were introduced onto PEI1.8k, these polycations exhibited remarkable differences in gene transfection efficiency. This phenomenon is closely related to the physicochemical properties of the polycation/DNA complexes. The zeta potentials of polycations/DNA complexes were first measured at a mass ratio of 5/1. These polycations/DNA complexes exhibited significantly different zeta potentials at the same mass ratio  and PEI-RT3, the efficient DNA condensation was due to the strong hydrogen bonding and hydrophobic interactions between polycations and DNA. Based on gene transfection and particle size results, the particle size of polycation/DNA was an important factor in gene transfer.Moreover, the particle sizes of carrier/DNA were determined  between PEI-RT3 and cell membrane (or DNA), which contributed to DNA loading, condensation, and endocytosis, thereby enhancing gene transfer. Therefore, PEI-RT3 was selected for further study.42. After the transfection efficiency was characterized, the cytotoxicity of carrier/DNA complexes was measured at various mass ratios. Unsurprisingly, at high mass ratios of 20/1 and 10/1, PEI25k/DNA exhibited serious cytotoxicity in B16F10 cells -NCA and mPEG-b-PLG with 17 structural units  spectra showed that mPEG-b-PLG adopted a PPII-helix conformation  in four cell lines . Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Endo/lysosome and DNA were labelled with Lysotracker Green (green) and Cy5 (red), respectively. Yellow fluorescent dots indicated the overlap of endo/lysosome (green) and carrier/DNA complexes (red), and reflected the colocation of endo/lysosome and complexes. Images were taken at different time points . The yellow fluorescent dots were clearly observed at 1 h for PD and PPD groups in B16F10 cells, implying that PD or PPD entered the endo/lysosome  of PPD was 8.97 μg mL−1 h, which was also much higher than that of other groups. These results suggested that the shielding function of mPEG-b-PLG could significantly enhance the circulatory half-life of PD in vivo.These results indicated that PPD possessed an efficient gene transfer in vitro. Nevertheless, the blood circulation time needed to be determined for the in vivo application of a polymeric gene delivery system. Herein, a pharmacokinetic study was performed to evaluate the blood circulation time of PPD in vivo. Carrier/Cy5-DNA nanoparticles were injected intravenously and the concentrations of Cy5-DNA were detected at different time points . The Cy5-DNA concentration of PPD group was considerably higher than that of the other groups  effect51. The distribution of PPD complexes within tumours was further characterized. PPD complexes were found to effectively accumulate in tumour tissue and penetrate within tumour  was selected as the therapeutic gene, and PPD was intravenously administered into B16F10 tumour-bearing mice. Meanwhile, Zeb was injected paratumourally  molecules after administration  was designed. As shown in Supplementary Fig. b mRNA expression in mPEG-b-PLG/PEI-RT3/siH2-Kb (denoted as PP/siH2-Kb) group was significantly downregulated in B16F10 cells. Moreover, the anti-tumour effect of PPD plus Zeb accompany with MHC I depletion was studied in B16F10 tumour-bearing mice. PP/siH2-Kb complexes were injected intratumourally along with PPD plus Zeb treatment. The anti-tumour efficacy of PPD plus Zeb was weakened after MHC I depletion in B16F10 tumour model  staining images indicated that PPD plus Zeb exhibited the most significant tumour tissue destruction Fig. . Moreove36. Among them, Th17 cells were upregulated in inflammatory organ tissues of autoimmune diseases, suggesting the percentage of Th17 cells could serve as a parameter to evaluate the irAEs of immune checkpoint inhibitor-mediated immunotherapy56. Herein, the anti-tumour efficacy and irAEs of PPD plus Zeb versus aPD-L1 plus Zeb were evaluated. Compared to PPD group, aPD-L1 hardly inhibited B16F10 tumour growth. Moreover, PPD plus Zeb group exhibited a more efficient anti-tumour effect than aPD-L1 plus Zeb group  cells in the spleens of tumour-bearing mice was detected at the end of treatment. It was found that aPD-L1 and aPD-L1 plus Zeb group had considerably higher percentage of Th17 cells than the PBS group, which suggested the possibility of irAEs when the systematic administration of aPD-L1 monoclonal antibody in cancer therapy  and deoxyazacytidine (DAC), Zeb showed better biocompatibility in B16F10 and 4T1 cells  was ~15%. Zeb group showed a higher percentage of DC maturation than PBS and shPD-L1 groups at 30%, which was twice that of PBS or shPD-L1 group. The reason should be that the tumour-associated antigens  generated by Zeb induced DC maturation. In addition, the percentage of mature DCs increased in the lymph nodes in PPD group compared with that in the PBS or shPD-L1 group. This was because PD-L1 downregulation induced tumour cell death to a certain extent and encouraged the production of tumour-associated antigen for DC maturation. Moreover, the highest percentage of mature DCs in the spleen was observed in PPD plus Zeb group at almost 40%. These results indicated that PPD plus Zeb could effectively induce DC maturation.To verify this conjecture, the immune cells in lymph nodes and tumour tissues were analyzed by flow cytometry after treatment. Mice receiving various formulations exhibited significantly different percentages of mature DCs Fig. . For PBS+ T and CD4+ T cells in the tumour tissues of B16F10 tumour-bearing mice were analyzed after treatment. The higher percentage of CD3+ CD8+ T cells in tumour tissues of Zeb and PPD groups was obtained compared with that in PBS or shPD-L1 groups  in PPD + Zeb group was considerably higher than that of PBS group  and M2 cells (CD206+F4/80+ in CD11b+) were significantly decreased after PPD plus Zeb combination therapy  and immunoactivated cytokines including IFN-γ, TNF-α, GzmB, IL-6, and IL-1β. Moreover, PD-L1 and immunosuppressive cytokines including IL-10 and TGF-β were significantly downregulated after PPD plus Zeb treatment. These results suggested that PPD plus Zeb combination therapy could upregulate the levels of some CTA genes  accompanied by enhancement of MHC I (H2-Db and H2-Kb) and downregulate the PD-L1 expression in tumour tissue, resulting in an increase in immunoactivated cytokines and a decrease in immunosuppressive cytokines, all of which were beneficial to inhibit tumour growth. Based on the analysis of immunocytes, CTAs, and cytokines, PPD plus Zeb combination therapy could transform B16F10 tumours from an immunosuppressive phenotype into an immunoactivated phenotype.To further verify that PPD plus Zeb can elicit in vivo anti-tumour immune response, CD8ups Fig. . This fiion Fig. . Similaron Figs. . Furtheroup Fig.  and the apy Fig.  and 84. apy Fig. . Moreove58. Encouraged by the strongly stimulating in vivo anti-tumour immune response of PPD plus Zeb combination therapy, we wonder whether PPD plus Zeb combination therapy can prevent tumour relapse. Tumour re-challenge experiment was conducted to simulate the relapse  in PPD plus Zeb group was considerably higher than that in the control group  and central memory T cells (denoted as TCMs) of CD8oup Fig. . In addioup Fig. . TEMs selts Fig. . On the lts Fig. . Moreovelts Fig.  and 89. Furthermore, when 4T1 tumour-bearing mice receiving PPD plus Zeb treatment were re-implanted with 4T1 tumour, PPD plus Zeb-treated animals could significantly inhibit tumour relapse by inducing an efficient immune memory response  is characterized by estrogen receptor-, progesterone receptor-, and HER2-negative, which is extremely aggressive with a high risk of metastasisapy Fig. . Compareapy Fig. . Moreoveapy Fig.  and the apy Fig. . Moreoveapy Fig.  and 95. apy Fig.  and 97. apy Fig. . This fi+T and CD4+T cells  were remarkably upregulated after PPD plus Zeb treatment. These results suggested that PPD plus Zeb combination therapy not only downregulated PD-L1 expression on tumour cells to relieve the immunosuppression of T cells, but also induced the enhancement of MHC I molecules on tumour cells via CTA, thereby ultimately mounting the visibility of tumour cells to the adaptive immune system in a humanized mouse model. According to the anti-tumour effect in humanized mice, PPD plus Zeb combination therapy has broad prospects for clinical application.Furthermore, to verify PPD plus Zeb-mediated anti-tumour effect in humanize mouse model, a humanized plasmid-encoding shPD-L1 (abbreviated as hu-shPD-L1) was designed. According to the RT-qPCR assay, mPEG-b-PLG/PEI-RT3/hu-shPD-L1 (denoted as PPD) significantly downregulated PD-L1 expression on MDA-MB-231 cells, whereas mPEG-b-PLG/PEI-RT3/humanized control shRNA (denoted as PPD-Control) did not significantly lower the level of PD-L1 on MDA-MB-231 cells  by introducing multiple interactions  and PPII-helix conformation. The anti-tumour effects of this polymeric gene delivery system in vivo were explored. With the plasmid-encoding shPD-L1 as a therapeutic gene, PPD mediating shPD-L1 delivery combined with epigenetic regulation  synergistically relieved the immune escape mechanism of tumour cells, thus promoting an in vivo anti-tumour immune response and enhancing the efficacy of tumour treatment. This treatment could also initiate a systemic anti-tumour immune response and a durable immune memory effect that effectively inhibited tumour relapse and metastasis, respectively. Moreover, PPD plus Zeb combination therapy could also significantly inhibit tumour growth in humanized mice. Epigenetic regulation combined with gene therapy-mediated immune checkpoint blockade was carried out to relieve the immune escape mechanism of tumour cells. This strategy provides a therapeutic scheme for cancer immunotherapy and opens up a platform for cancer treatment.2)-OH was purchased from Enlai Biological Technology Co., Ltd . Trifluoroacetic acid (TFA) and triphosgene were obtained from Xiya Reagent Co., Ltd. N, N-dimethyl formamide (DMF) and anhydrous ether were obtained from Beijing Chemical Factory. P-toluene formyl chloride, p-toluene sulfonyl chloride, diisopropylethylamine (DIPEA), and 1-hydroxybenzotriazoleanhydrous (HOBT) were obtained from Dibai Chemical Technology Co., Ltd . EDC hydrochloride (EDCI) was obtained from J&K Scientific Ltd . N-hydroxysuccinimide was obtained from TCI Co., Ltd . Then, 33% HBr in CH3COOH was obtained from ACROS. mPEG-NH2 (Mn = 2000) was obtained from Beijing Jenkem Technology Co., Ltd . Dialysis bag was obtained from Yuanye Biological Technology Co., Ltd . Calf thymus DNA was obtained from Sigma (USA). The plasmid DNA (pDNA) expressing mouse shPD-L1 (named as shPD-L1) was obtained by Sangon . The sense strand sequence for shPD-L1 was 5′-CCCACAUAAAAAACAGUUGdTdT-3′. The pDNA expressing control shRNA was named as Control shRNA. The sense strand sequence for Control shRNA was 5′-GTTCTCCGAACGTGTCACGTT-3′. The pDNA expressing human shPD-L1 (named as hu-shPD-L1) was obtained by Genepharama , the sense sequence for hu-shPD-L1 was 5′-GCCGACTACAAGCGAATTACTT-3′. Zeb was obtained from TCI Co., Ltd . AC and DAC were obtained from J&K Scientific Ltd . DAPI was obtained from Sigma. CCK-8 was obtained from Beyotime Biotechnology Co., Ltd . Plasmid-encoding luciferase (pGL3), luciferin substrate, and cell lysate were obtained from Promega . GL3 luciferase-siRNA double strands  and nonspecific control siRNA double strands (sense: 5′-AGCUUCAUGAGUCGCAUUCdTdT-3′ and antisense: 5′-GAAUGCGACUCAUGAAGCUdTdT-3′) were obtained from FASMAC (Japan). H2-Kb siRNA double stands (sense: 5′-CCAGACCUCUGAUCUGUCUdTdT-3′ and antisense: 5′-AGACAGAUCAGAGGUCUGGdTdT-3′) and control siRNA double strands (sense: 5′-UUCUCCGAACGUGUCACGUdTdT-3′ and 5′-ACGUGACACGUUCGGAGAAdTdT-3′) were obtained Genepharama . RT-qPCR primers were obtained from Sangon , and the primer sequences were shown in Supplementary Table Branched PEI25k and PEI1.8k was purchased from Sigma (USA). Boc-Orn(Boc)-OH, Boc-Arg(Pbf)-OH, Boc-Orn(Tos)-OH, Boc-Arg(Tos)-OH, and H-Glu(Bzl)-OH were purchased from GL Biochem Ltd . Boc-Arg): 7.65 , 7.28 , 3.88 , 2.52–3.62 , 2.33 , 1.76 , 1.50 . The synthesis procedure of other PEI-RTs containing different numbers of RT string was similar.PEI1.8k  was dissolved in 10 mL of deionized water. The mixture of Boc-Arg (Tos)-OH , HOBT , and EDCI  were dissolved with 50 mL of DMF and stirred at room temperature for 1 h. Then PEI1.8k solution and DIPEA  were added and stirred at room temperature for 72 h. After that, the mixture solution was dialyzed , and lyophilized, then the white solid was obtained. The white solid was added into 30 mL of TFA and reacted at room temperature for 4 h. Then the mixture solution was precipitated with cold anhydrous ether, filtered, and dried in vacuum, dialyzed , and lyophilized, and the white flocculent solid was obtained . P-toluoyl chloride  was dissolved in 8 mL of THF. The p-toluoyl chloride solution was added into the ice bath of PEI1.8k solution drop by drop. Then DIPEA  was added and reacted in ice bath for 12 h. After that, the mixture solution was dialyzed  for 72 h, lyophilized, and the white solid was obtained. . 1H NMR ): 7.62 , 7.21 , 2.42–3.60 .PEI1.8k  was dissolved in 10 mL of deionized water. P-toluene sulfonyl chloride  was dissolved in 12 mL of THF. P-toluene sulfonyl chloride solution was added into the ice bath of PEI1.8k solution drop by drop. Then DIPEA  was added and reacted in ice bath for 12 h. After that, the mixture solution was dialyzed  for 72 h, lyophilized, and the white solid was obtained. . 1H NMR ): 7.65 , 7.32 , 2.18–3.25 .PEI1.8k  was dissolved in 10 mL of deionized water. 1H NMR ): 3.57 , 2.52–3.40 , 1.55–1.80 .PEI1.8k  was dissolved in 10 mL of deionized water. The mixture of Boc-Orn(Boc)-OH , HOBT , and EDCI  were dissolved with 50 mL of DMF and stirred at room temperature for 1 h. Then PEI1.8k solution and DIPEA  were added and stirred at room temperature for 72 h. After that, the mixture solution was dialyzed , lyophilized, and the white solid was obtained. The white solid was added into 30 mL of TFA and reacted at room temperature for 4 h. Then the mixture solution was precipitated with cold anhydrous ether, filtered and dried in vacuum, dialyzed  and lyophilized, the white flocculent solid was obtained . 1H NMR ): 3.97 , 2.62–3.80 , 1.85 , 1.58 .PEI1.8k  was dissolved in 10 mL of deionized water. The mixture of Boc-Arg(Pbf)-OH , HOBT , and EDCI  were dissolved with 60 mL of DMF and stirred at room temperature for 1 h. Then PEI1.8k solution and DIPEA  were added and stirred at room temperature for 72 h. After that, the mixture solution was dialyzed , lyophilized, and the white solid was obtained. The white solid was added into 40 mL of TFA and reacted at room temperature for 4 h. Then the mixture solution was precipitated with cold anhydrous ether, filtered and dried in vacuum, dialyzed  and lyophilized, the white flocculent solid was obtained . 1H NMR ): 7.65 , 7.36 , 3.62 , 2.24-3.46 , 1.21–1.62 .PEI1.8k  was dissolved in 10 mL of deionized water. The mixture of Boc-Orn(Tos)-OH , HOBT , and EDCI  were dissolved with 50 mL of DMF and stirred at room temperature for 1 h. Then PEI1.8k solution and DIPEA  were added and stirred at room temperature for 72 h. After that, the mixture solution was dialyzed , lyophilized, and the white solid was obtained. The white solid was added into 40 mL of TFA and reacted at room temperature for 4 h. Then the mixture solution was precipitated with cold anhydrous ether, filtered and dried in vacuum, dialyzed  and lyophilized, the white solid was obtained . 2)-OH , HOBT , and EDCI  were dissolved with 60 mL of DMF and stirred at room temperature for 1 h. Then PEI1.8k solution and DIPEA  were added and stirred at room temperature for 72 h. After that, the mixture solution was dialyzed , lyophilized, and the white solid was obtained. The white solid was added into 40 mL of TFA and reacted at room temperature for 4 h. Then the mixture solution was precipitated with cold anhydrous ether, filtered and dried in vacuum, dialyzed  and lyophilized, the white solid was obtained . 1H NMR ): 3.76 , 2.42–3.56 , 1.75 , 1.56 .PEI1.8k  was dissolved in 10 mL of deionized water. The mixture of Boc-Arg(NOL-Glu (OBzl)-OH  and triphosgene  were added into a dry three-mouth flask, then 500 mL of THF was added and reacted for 2 h at 50 °C until the solution was clear. Then the mixture solution is naturally cooled to room temperature and precipitated with 2 L of cold hexane, filtered, the white powder was dissolved with 250 mL of ethyl acetate, washed with cold deionized water for three times, dried with anhydrous magnesium sulfate, and stored overnight at −20 °C. Then the mixture solution was filtered and the filtrate was dried under vacuum condition, recrystallized with THF and hexane, the white solid was obtained . 1H NMR : 7.33-7.45 , 6.44 , 5.16 , 4.28 , 2.25–2.38 , 2.08–2.20 .To obtain mPEG-b-PLG, Glu (OBzl)-NCA needed to be synthesized. Briefly, H-2000-NH2  was added into the above solution, and reacted for 72 h at room temperature. Then the mixture solution was dialyzed against deionized water for 72 h , lyophilized, and the white solid was obtained. The white solid was dissolved in 20 mL of TFA, 33% HBr in CH3COOH (4 mL solution/1 g solid) was added slowly and reacted for 4 h at room temperature, the mixture solution was precipitated with anhydrous ether, filtered, dried under vacuum condition, dialyzed for 72 h (cut-off 2000 Da), lyophilized, and the white solid was obtained . 1H NMR : 4.58 , 3.67 , 3.33 , 2.42 , 1.80–2.22 .Glu(OBzl)-NCA  was dissolved in 20 mL of DMF, mPEG3 was used as the eluent for mPEG-b-PLG determination, respectively. Molecular weights were calculated by a calibration curve of linear mPEG2k standards.To obtain the molecular weight and molecular weight distribution of PEI-RT3 or mPEG-b-PLG, gel permeation chromatography measurements were carried with a Waters 515 chromatography column. Then, 0.1 M of NaNO−1) was mixed with 250 μL of PEI-RT3 solution (1 mg mL−1). After that, 1700 μL of deionized water was added. After 20 s vortex, the above mixture solution was incubated for 20 min at room temperature to obtain PD complexes. The other carrier/DNA complexes were prepared in a similar manner. For the preparation of PPD complexes, 50 μL of DNA (1 mg mL−1) was mixed with 250 μL of PEI-RT3 solution (1 mg mL−1), then 1650 μL of deionized water was added. After 20 s vortex, the above mixture solution was incubated for 20 min at room temperature, then 50 μL of mPEG-b-PLG was added. After 20 s vortex, the above mixture solution was incubated for 20 min at room temperature to obtain PPD complexes.The preparation of carrier/DNA complexes was described as the following steps. Then, 50 μL of DNA . Individual data were shown based on quintuplicate independent experiments. In addition, DLS technique was also used to determinate hydrodynamic particle size of nanoparticles with Zetasizer Pro . The morphologies of PPD and PD were characterized with scanning electron microscopy (SEM) containing XL-30ESEM-FEG SEM system .−1 in deionized water. The pH value of mPEG-b-PLG solution was adjusted to the desired value (pH 6.8 or 7.4). The mean residue molar ellipticity was calculated wth the following formulas: Ellipticity ([θ] in deg cm2 dmol−1) = (millidegrees × mean residue weight)/(path length in millimetres × concentrations of polypeptide in mg mL−1). The CD spectra were measured at room temperature.The CD spectra of mPEG-b-PLG was measured with J-815 CD spectrometer . The sample of mPEG-b-PLG was prepared at concentration of 0.2 mg mL2) B16F10 (TCM36), 4T1, HeLa (TCHu187), MCF-7 (TCHu74), and Huh-7 (TCHu182) cells were purchased from Shanghai Cell Bank (Chinese Academy of Sciences). The cells were cultured in DMEM medium containing 10% (v/v) fetal bovine serum at 37 °C in 5% (v/v) carbon dioxide  were added into cells containing plates (0.2 μg pDNA per well) and incubated for 48 h. the cells was lysed with 50 μL of cell lysate per well and frozen at −80 °C for 30 h. After melting at room temperature, 25 μL of cell lysate per well was mixed with 25 μL of luciferase substrate. Relative light units (RLUs) were obtained by a luminometer and normalized to total protein content . The luciferase activity was expressed as RLU/mg protein) and the data were shown as mean ± SD based on triplicate independent experiments.DNA transfection assay was conducted in B16F10, 4T1, HeLa, and MCF-7 cells. Luciferase plasmid DNA (pGL3) was used as reporter gene. Cells were seeded in 96-well plate at a density of 104 cells per well and incubated for 24 h. The culture medium was replaced with fresh culture medium and carrier/siRNA complexes was added (0.2 μg siRNA per well) and incubated for 48 h. Then the cells were lysed with 50 μL of cell lysate per well and frozen at −80 °C for 30 min. After melting at room temperature, 25 μL of cell lysate per well was mixed with 25 μL of luciferase substrate. RLUs were obtained by luminometer and normalized to total protein content . The luciferase activity was expressed as RLU/mg protein) and the relative luciferase activity was calculated by comparing untreated control cells. The gene silencing data were shown as mean ± SD based on triplicate independent experiments.Huh-7 Luc cells were seeded in a 96-well plate at a density of 104 per well and incubated with 24 h. After that, the culture medium was replaced with fresh culture medium at a volume of 180 μL per well. Carrier/pDNA complexes at different mass ratios  were added into cells containing plates (0.2 μg pDNA per well) and incubated for 24 h. Then the culture medium was replaced with 10% (v/v) CCK-8 containing culture medium and incubated for 1 h. The cells were detected at 450 nm and the absorption value was obtained by Bio-Rad 680 microplate reader. Cell viability (%) was calculated based on this equation: cell viability (%) = (Asample/Acontrol) × 100, where Asample and Acontrol were the absorbance values of sample well and control well, respectively. The data were shown as mean ± SD based on triplicate independent experiments.The cytotoxicity of carrier/pDNA was characterized by CCK-8 assay. B16F10 cells (or other kinds of cells) were seeded in a 96-well plate at a density of 105 cells per well and incubated for 24 h. After that, the culture medium was replaced with fresh culture medium and carrier/Cy5-DNA complexes were added and incubated for 3 h. Then the cells were digested, centrifuged (0.1 g), and washed with PBS for three times. The cells were detected with Guava EasyCyte low cytometer. In addition, CLSM was carried out to observe the cellualr uptake of carrier/DNA directly. Cells were seeded on a coverslip containing six-well plates at a density of 105 cells per well and incubated for 24 h. The culture medium was replaced with fresh medium. Carrier/Cy5-DNA complexes were added and incubated for 3 h. Then cells were washed with PBS for three times and fixed for 10 min by 4% paraformaldehyde. Nucleus was stained with DAPI for 10 min. The coverslips were taken out, placed on glass slides and enclosed with glycerol. The prepared samples were observed with CLSM (ZEISS LSM780).The cellular uptake efficiency of carrier/DNA complexes was detected by flow cytometry. Cells were seeded in 24-well plates at a density of 105 cells per well and incubated for 24 h. Then the culture medium was replaced with fresh culture medium and carrier/Cy5-DNA complexes (1 mg Cy5-DNA per well) were added and incubated for 1, 4, and 7 h. After that, the cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min. Nucleus were stained with 1 mg mL−1 of DAPI (1.5 μL per well) for 10 min, and endo/lysosome were stained with 75 nM of Lysotracker Green for 1 h. Finally, the coverslips were taken out and placed on the glass slides, enclosed with glycerol. The samples were observed by CLSM  and the correlation coefficient R-values were obtained from CLSM images (ZEN software).B16F10, 4T1, HeLa, or MCF-7 cells were seeded in six-well plates containing coverslips at a density of 1 × 107 cells/mL) were transplanted into the left flank of mice and tumour growth was monitored. For primary B16F10 tumour therapy, the solid tumour model was formed after 4 days, and the tumour volumes were about 50 mm3. The second tumour as distant tumour model  was inoculated into the right flank of mice after 10 days. For distribution and gene transfection in B16F10 tumour-bearing mice, solid B16F10 tumour model was formed after 8 days and tumour volumes were about 500 mm3. For anti-tumour efficacy study, solid B16F10 tumour model was formed after 4 days and the tumour volumes were about 50 mm3. In addition, for the construction of 4T1 tumour model, 4T1 cells were large scale expanded in vitro and cell suspensions in PBS  were transplanted into the left flank of mice and tumour growth was monitored. For primary 4T1 tumour therapy, the solid tumour model was formed after 7 days and the tumour volumes were about 100 mm3, and the second tumour as distant tumour model  was inoculated into the right flank of mice after 14 days. For distribution and gene transfection in 4T1 tumour-bearing mice, solid 4T1 tumour model was formed after 12 days and tumour volumes were about 500 mm3. For anti-tumour efficacy study, solid 4T1 tumour model was formed after 7 days and the tumour volumes were about 100 mm3.Six-week-old female C57BL/6 and BALB/c mice were obtained from Vital River Company . All animal procedures were strictly perfromed based on the Guidelines for the Care and Use of Laboratory Animals of Jilin University and approved by the Animal Ethics Committee of Jilin University. B16F10 cells were large scale expanded in vitro, and cells suspensions in PBS , PEI-RT3/Cy5-DNA (5/1), and mPEG-b-PLG/PEI-RT3/Cy5-DNA (1/5/1), respectively. The blood was collected by tain vein and transferred to heparinized centrifuge tubes. Then the blood was diluted with delionied water and then transferred into a black 96-well plate. The sample was detected at The distribution of carrier/DNA complexes in vivo was detected and analyzed by ex vivo bioluminescence imaging assay. Then, 200 μL of carrier/Cy5-DNA (1 mg Cy5-DNA/1 kg body weight) was administered intravenously into B16F10 (or 4T1) tumour-bearing mice. After 24 h, mice were killed, and tumour and major organs  were collected and imaged with Maestro In Vivo Imaging System .B16F10 or 4T1 tumour-bearing mice were intravenously administered with mPEG-b-PLG/PEI-RT/Cy5-DNA (PPD) complexes. After 24 h, mice were killed and tumour tissue was collected. The frozen sections of tumour were stained with DAPI and observed under fluorescence microscope for the distribution analysis of carrier/DNA complexes.Transfection activity of carrier/pDNA in vivo was measured by ex vivo bioluminescence imaging assay. pDNA encoding red fluorescence protein was served as a reporter gene. Then, 200 μL of pDNA, PEI1.8k/pDNA, PEI-RT3/pDNA, and mPEG-b-PLG/PEI-RT3/pDNA were intravenously injected into B16F10 (or 4T1) tumour-bearing mice, respectively (1 mg pDNA/1 kg body weight). After 48 h, the mice were killed, tumour and main organs  were collected and analyzed with Maestro In Vivo Imaging System .pDNA complexes. After 48 h, mice were killed and tumour tissue was collected. The frozen sections of tumour were stained with DAPI and observed under fluorescence microscope for the distribution analysis of expressed protein.To characterize the distribution of the expressed protein within tumours, the plasmid-encoding RFP was used as reporter gene, and B16F10 or 4T1 tumour were intravenously administered with mPEG-b-PLG/PEI-RT/3, mPEG-b-PLG/PEI-RT/shPD-L1 (PPD) ternary complexes were administered intravenously to the tumour-bearing mice. Then the mice were killed and tumours were collected and the levels of PD-L1 in DCs, macrophages, and tumour cells in tumour tissue were analyzed by flow cytometry.To characterize gene transfection of carrier/shPD-L1 in different cell types in the tumour microenvironment, the levels of PD-L1 expressed on various cell lines in tumours were studied. GFP-B16F10 cells were selected to construct the tumour model. When the tumour volume arrived at 200 mm3) were randomly divided into 6 groups (10 mice per group), namely PBS, Zeb, shPD-L1, PD, PPD, and PPD plus Zeb. Tumour-bearing mice were intravenously administered with 200 μL of PBS, shPD-L1, PD, and PPD every other day for a total of five times, respectively. Dose of shPD-L1 was 1 mg kg−1. For Zeb and PPD plus Zeb group, Zeb was paratumourally administered to tumour-bearing mice every other day for a total of five times. Dose of Zeb was 10 mg kg−1. Body weight was recorded every day for 12 days. Tumour size was measured every day for 12 days and calculated according to the equation: V = L × W2 × 0.5, where ‘V’ represents tumour volume, ‘L’ represents the longer diameter, and ‘W’ represents the shorter diameter. In addition, during various DNMTi treatments, for PPD plus AC or PPD plus DAC, AC or DAC was paratumourally administered to tumour-bearing mice every other day for a total of five times, and dose of AC or DAC was 5 mg kg−1. Furthermore, during aPD-L1 involved tumour therapy, for aPD-L1 or aPD-L1 plus PPD group, aPD-L1 was injected intraperitoneally into tumour-bearing mice every other day for a total of five times, dose of aPD-L1 was 7.5 mg kg−1. After treatment, mice were killed and spleens were collected for the flow cytometry analysis of Th17 cells. During exploring the role of MHC I molecules in Zeb plus PPD treatment, for PPD plus Zeb plus PP/siH2-Kb group, the dose of siH2-Kb was 1 mg kg−1. For 4T1 tumour treatment, 60 female BABL/c  were randomly divided into 6 groups (10 mice per group), namely PBS, Zeb, shPD-L1, PD, PPD, and PPD plus Zeb. Tumour-bearing mice were intravenously administered with 200 μL of PBS, shPD-L1, PD, and PPD every other day for a total of seven times respectively. Dose of shPD-L1 was at 1 mg kg−1. For Zeb and PPD plus Zeb group, Zeb was paratumourally administered to tumour-bearing mice every other day for a total of seven times. Dose of Zeb was 10 mg kg−1. Body weight was recorded every day for 24 days. Tumour size was measured every day for 24 days and calculated according to the equation: V = L × W2 × 0.5.pDNA encoding shPD-L1 (named as shPD-L1) was a therapeutic gene. For B16F10 tumour treatment, 60 female C57BL/6 mice  and renal function markers creatinine, uric acid, blood urea nitrogen  in serum were measured by ELISA assay .To evaluate in vivo safety of PPD complexes, the blood of tumour-bearing mice was collected after treatment. Age- and weight-matched healthy mice were used as control. The blood was centrifuged at 0.9 × 3) were randomly divided into 6 groups (10 mice per group), namely, PBS, Zeb, shPD-L1, PD, PPD, and PPD plus Zeb. Tumour-bearing mice were intravenously administered with 200 μL of PBS, Zeb, shPD-L1, PD, PPD, or PPD every other day for a total of five times, respectively. Dose of shPD-L1 was 1 mg kg−1. For Zeb and PPD plus Zeb group, Zeb was paratumourally administered to tumour-bearing mice every other day for a total of five times. Tumour size was measured every day. When the tumour volume of mice arrived at 1000 m3, the mice was defined as dead. For 4T1 tumour treatment, 60 female BABL/c  were randomly divided into 6 groups (10 mice per group), namely PBS, Zeb, shPD-L1, PD, PPD, and PPD plus Zeb. Tumour-bearing mice were intravenously administered with 200 μL of PBS, shPD-L1, PD, and PPD every other day for a total of seven times, respectively. Dose of shPD-L1 was 1 mg kg−1. For Zeb and PPD plus Zeb group, Zeb was paratumourally administered to tumour-bearing mice every other day for a total of seven times. Dose of Zeb was 10 mg kg−1. Tumour size was measured every day. When the tumour volume of mice arrived at 1000 m3, the mice was defined as dead.The survival rate of tumour-bearing mice was carried out both in B16F10 and 4T1 tumour models. For B16F10 tumour treatment, 60 female C57BL/6 mice  and tumour tissues, the RT-qPCR assay was carried out. Total RNA of cancer cells or tumour tissues were extracted from cancer cells by RNA extraction kit and RNA content was measured. Then RNA reverse-transcription assay was conducted by RNA reverse-transcription kit and acquired cDNA content was measured. Finally, qPCR assay of acquired cDNA was conducted with SYBR green technology by qPCR Kit and analyzed with Roche Light Cycler.7 cells/mL) was transplanted into the left flank of mice. The solid B16f10 tumour model was formed after 4 days and mice were treated as we designed. After treatment, B16F10 or MC38 or LLC cells  were transplanted into the right flank of mice as designed to study the tumour relapse. Right flank tumour was measured every other day and calculated according to the equation: V = L × W2 × 0.5. Moreover, 6-week-old female BABL/c mice (eight mice per group) was injected with 4T1 cells  was transplanted into the left flank of mice. The solid 4T1 tumour model was formed after 7 days and mice were treated as we designed. After treatment, 4T1 or CT26 cells  cells were transplanted into the right flank of mice as designed to study the tumour relapse. Right flank tumour was measured every other day and calculated according to the equation: V = L × W2 × 0.5.The tumour relapse in B16F10 (or 4T1) tumour-bearing mice was studied. Six-week-old female C57BL/6 mice (six mice per group) was injected with B16F10 cells  was transplanted into the left flank of mice. The solid tumour model was formed after 7 days and mice were treated as we designed. After treatment, half of mice in PPD group and all mice in other groups were killed, the liver, spleen and lung were collected and further analyzed. In addition, the remaining mice in PPD were intravenously administered with 4T1 cells . After 7 days, the liver and lung were collected and analyzed.Six-week-old female BABL/c mice (12 mice for PPD and other groups were based on 6 mice per group) was injected with 4T1 cells  for 4 H, then CD34+ cells were intravenously administered at dose of 1 × 105 cells per mice. After 20 weeks, the contents of human and mouse derived CD45+ cells in mouse peripheral blood were detected by flow cytometry to test the qualified rate of Hu-HSC-NPG reconstruction . The relevant data were presented in Supplementary Table 6 cells per mice, and Hu-HSC-NPG mice were randomly divided into two groups (three mice per group): PBS, PPD plus Zeb. When the average tumour volumes reached 50 mm3, Hu-HSC-NPG mice were administered with PPD plus Zeb or PBS every two days for total five times.Hu-HSC-NPG model was obtained from Beijing Vitalstar Biotechnology Co., Ltd, and the construction process of this model was as follows: CD34diated 1. Gy for 4Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Nanoparticle internalisation is crucial for the precise delivery of drug/genes to its intracellular targets. Conventional quantification strategies can provide the overall profiling of nanoparticle biodistribution, but fail to unambiguously differentiate the intracellularly bioavailable particles from those in tumour intravascular and extracellular microenvironment. Herein, we develop a binary ratiometric nanoreporter (BiRN) that can specifically convert subtle pH variations involved in the endocytic events into digitised signal output, enabling the accurately quantifying of cellular internalisation without introducing extracellular contributions. Using BiRN technology, we find only 10.7–28.2% of accumulated nanoparticles are internalised into intracellular compartments with high heterogeneity within and between different tumour types. We demonstrate the therapeutic responses of nanomedicines are successfully predicted based on intracellular nanoparticle exposure rather than the overall accumulation in tumour mass. This nonlinear optical nanotechnology offers a valuable imaging tool to evaluate the tumour targeting of new nanomedicines and stratify patients for personalised cancer therapy. Quantification of intratumoral nanoparticles internalisation in vivo is crucial but challenging. Here, the authors develop a binary ratiometric nanoreporter that can quantify internalisation and predict nanotherapeutic responses based on intracellular nanoparticle exposure. Over ten nanomedicines have been approved for cancer treatments, including PEGylated liposomal doxorubicin (Doxil/Caelyx), paclitaxel-albumin nanoparticle (Abraxane), etc3. However, cancer patients presented heterogeneous therapeutic responses to nanomedicines in clinical trials due to the extreme variability of enhanced permeability and retention (EPR) effect of solid tumours characterised by the complex tumour microenvironment5. Efforts have been made to identify cancer patients with high EPR effect (i.e. accumulation) to achieve remarkable therapeutic response8. Nevertheless, an important truth that cannot be ignored is nanomedicines with high tumour accumulation sometimes fail to achieve enhanced therapeutic response due to the inadequate target exposure (e.g. Doxil versus free doxorubicin)9. It is increasingly clear that drug exposure at the intracellular targets rather than the overall level in the tumour tissues acts as one of the most important factors to achieve the optimal therapeutic response11. Thus, quantifying the intracellular bioavailability of nanomedicines, including internalised amount and percentage in solid tumours would be a suitable indicator for the prediction of drug access to intracellular targets and therapeutic efficacy.Theranostic nanomedicines have made significant contributions in oncology through precise delivery of drugs into specific tissues with mitigated side-effects12, mass spectrometric14 and radioactive15 detections have been extensively used to quantify the accumulation of nanomedicines in solid tumours. However, these methodologies fail to offer the information on the intracellular delivery efficiency of accumulated nanoparticles in tumour mass due to the incapability of differentiating the internalised nanoparticles from those in tumour intravascular and extracellular spaces. Several strategies such as chemical etching17, fluorescence quenching16 and bioluminescence reaction18 have been extensively leveraged to exclude the extracellularly distributed nanoparticles from endocytosis analysis, and provide two-dimensional (2D) information at cellular or ex vivo level. Despite the successful quantification of the nanoparticle internalisation in vitro, achieving real-time quantification of nanomedicines internalisation kinetics in living subjects remains a significant challenge19.Conventional methods based on fluorescence spectroscopicee ~6.0) to report nanoparticle internalisation events due to the fast formation of early endosome after endocytosis (~5 min)22. Our BiRN is a two-modular nanoreporter system with a transition pH (pHt) at 6.326, and each module is fluorescently encoded with a unique near-infra-red fluorophore. The ‘OFF-ON’ module keeps ‘OFF’ in tumour bloodstream and extracellular space (pHe ~6.6–6.9)27, and immediately switches ‘ON’ upon a sudden pH drop in early endosome after endocytosis. The ‘always-ON’ module acts as an internal standard for ratiometric quantitation of nanoparticle internalisation. After binary ratiometric processing, the nanoparticles in tumour microvasculature and extracellular space were removed, allowing the real-time measurement of the cellular internalisation in living mice. Enabled by BiRN, we demonstrate the successful quantitation of nanoparticle internalisation in various animal tumour models. Furthermore, the prediction of therapeutic outcomes of cancer nanomedicines, including paclitaxel (PTX)-conjugated nanoparticle, which has identical physicochemical properties to BiRN, as well as Doxil was accomplished by patient stratification based on intracellular nanoparticle exposure in tumour mass  that can unambiguously differentiate intracellularly and extracellularly located nanoparticles, permitting quantitative imaging of nanoparticle internalisation in vivo Fig. . We chosass Fig. . These cb-poly(2-(diisopropyl amino)ethyl methacrylate) (PEG-b-PDPA) polymer with a pHt at 6.3 via the atom transfer radical polymerisation method  and ‘OFF-ON’ modular polymer (Fmax/Fmin > 100-fold) (Supplementary Table Fmax/Fmin = 0.9–1.1) at pH ranging from 5.0 to 7.4; the ‘OFF-ON’ module renders two-order magnitude signal amplification (Fmax/Fmin > 100-fold) when pH drops below the threshold of 6.3 (Supplementary Table We synthesised the poly(ethylene glycol)-ON/OFF = 0.21), and high fluorescence activation ratio of 111-fold; ‘always-ON’ module presented a constant fluorescence signal (Fmax/Fmin = 1.03) in a broad pH range  and dynamic light scattering (DLS) analyses showed that the BiRN was well-dispersed spherical structure with a diameter of 25.6 ± 2.2 nm at pH 7.4 and dissociated into unimer with a diameter of 7.6 ± 1.1 nm at pH 5.4 Fig. , b. Fluonge Fig. , demonstnge Fig. .We directly monitor the internalisation of BiRN in A549 tumour cells without medium removal. As expected, the Cy3.5 signal homogenously distributed in the medium in ‘always-ON’ manner, whereas the BDP signal kept ‘OFF’ in the extracellular space  were administrated into mice bearing 4T1 breast tumours by intravenous injection. As early as 10 min post-injection, the BiRN nanoparticles rapidly accumulated into the tumour sites as indicated by the observable Cy7.5 signal, while no significant nanoparticles were endocytosed by cells within tumours due to the undetectable Cy5 and O/A signals, revealing the negligible internalisation of accumulated nanoparticle at early time-points. The Cy7.5 signals increased continuously over 24 h due to the passive accumulation of BiRN into tumour tissues through EPR effect  for main organs, such as the heart, lung, spleen, liver, kidney, tumour and muscle  percentage presented a linear standard curve in a concentration-independent manner Fig. , which a25 . The tumour internalisation of BiRN was further investigated in the dose escalation study. We found the efficiency of nanoparticle internalisation into tumour tissues decreased at a high dose of 40 mg kg−1 probably due to the saturation of nanoparticle internalisation  on the stability of BiRN, the pHe of interstitial fluid in all tumour models was determined by needle microelectrode protocol29. We found that the average pHe value is 6.78 ± 0.20, none of the tumour interstitial pHe below the pHt of BiRN that guaranteed the exclusive activation inside cells, permitting the accurate measurement of nanoparticle internalisation in solid tumours  and patient-derived xenograft (PDX) oesophageal carcinoma models. In all six tumour models, we successfully quantified the BiRN internalisation in solid tumours and other organs/tissues Fig. –22. To eurs Fig. . Heat maurs Fig. . Only 10To further investigate the microscopic internalisation of nanoparticle in living tumour-bearing mice, 4T1 tumours grown in dorsal window chambers were established to assess the function of BiRN in vivo by dual-photon intravital fluorescence microscopy. At 3 h post-injection of BiRN, the ‘always-ON’ signal distributed throughout the tumour microcirculation homogenously and extravasated in the tumour extravascular spaces, while the ‘OFF-ON’ signal was only colocalized with dot-shaped ‘always-ON’ signal in the extravascular region Fig. . We deteAfter the animal imaging, the excised tumours were collected for tissue sectioning. The fluorescence imaging of the whole-mount tumour sections Fig.  displaye30 and toxicity31 has shown great potential for the evaluation of treatment response and biosafety. To investigate whether quantification of BiRN internalisation can help to predict nanotherapeutic efficacy, we introduced a pH/cathepsin B hierarchical-responsive nanomedicine recently reported by our group32. The nanomedicine was prepared through self-assembly of PEG-b-PDPA polymer-paclitaxel conjugate with enzyme cleavable Gly–Phe–Leu–Gly (GFLG) tetrapeptide linker (PEG-b-P(DPA-r-PTX), also abbreviated as PDPA-PTX. In our hypothesis, PDPA-PTX keeps micelle state in tumour microcirculation and extracellular space, but rapidly dissociated in early endosome once being internalised. After being translocated into lysosomes, the tetrapeptide linker can be cleaved by cathepsin B and the released PTX was transported into the cytoplasm, leading to effective tumour suppression  was comparable to that of free drug  on a per PTX basis. In contrast, dynasore treatment led to a ~90% decrease in anti-tumour efficacy of PDPA-PTX  due to the marginal uptake of PDPA-PTX . Results demonstrated the feasibility of using intracellular nanoparticle exposure  as surrogate indicator for intracellular drug bioavailability  underwent in vivo imaging at 24 h post-administration of BiRN  in percentage of tumour volume change among the low- and high-endocytosis groups 2 days after treatment with PDPA-PTX, and the differences were even more significant (P = 0.002 at day 10) with time lapse  between the level of intracellular nanoparticle and tumour inhibition percentage was observed on day 16 . Furthermore, a good correlation was observed between fluorescence intensity of OFF-ON module and the released PTX content  was observed between Cy5 and doxorubicin signals at single-cell level  with endocytosis amount rather than accumulation  better growth inhibition than low group  was chosen as model nanomedicine for further experiments. We first performed the correlation analysis of intracellular BiRN nanoprobes and Doxil. After sequential injection of BiRN and Doxil to 4T1 tumour-bearing mice, flow cytometry analyses of cell suspensions from tumour tissues provided conclusive evidence that good correlation , which is essential for chemically differentiating the extracellular and endocytic pH. Secondly, the ultra-fast disassembly (<5 ms) of the nanosystem enables the real-time recording of the endocytic events without any lag time. Thirdly, our design exploits pH differences between acidic tumour microenvironment and early endosome, which is a recognised hallmark of cell internalisation regardless of tumour types. Moreover, fluorescence imaging provides a real-time, noninvasive, convenient and lower-cost paradigm for quantitative measurement of the intracellular bioavailability of nanomedicines33.We have described a two-modular nanoreporter technology for quantitative imaging of cellular internalisation in living animals. This BiRN reporter successfully converts the subtle pH change involved in the endocytic events (ΔpH < 0.6) into digitised ratiometric signal output, permitting the precision quantification of cell internalisation in vivo. While, conventional pH sensors can chemically resolve continuous variations during the endosome maturation process, but fail to accurately report the endocytic events. The present nanoreporter was successfully engineered with several critical functions that enable the directly gated counting of nanoparticle internalisation in living subjects without any sample processing. Firstly, positive cooperativity in the self-assembly of pH-responsive block copolymer drives the sharp pH response  of the nanoparticles resided in solid tumours were internalised into the cell components, while ~80% of the nanoparticles were sequestered in the acellular regions of the tumours at 24 h. More importantly, our BiRN technology demonstrated that intracellular nanoparticle exposure exhibited a strong correlation with anti-tumour efficacy of nanomedicines, whereas nanoparticle accumulation in tumour mass presented a poor correlation with therapeutic nanomedicine efficacy due to the high heterogeneity of internalisation efficiency between individual subjects. Hence, quantifying the intracellular bioavailability of nanomedicines rather than the overall accumulation in solid tumours is a suitable indicator for the prediction of anti-tumour efficacy and patient stratification for personalised treatment.In summary, we successfully quantified the internalisation of nanoparticles that accumulated in the solid tumours at whole animal, intravital, excise tissue and tissue section levels by the BiRN technology. This internalisation nanoreporter offers a powerful tool to predict anti-tumour efficacy of different types of nanomedicines by patient stratification based on intracellular exposure of nanoparticle in tumour mass. This nanotechnology-enabled paradigm also advances a valuable tool to evaluate the tumour-targeting efficacy of new targeted nanomedicines, to uncover the dynamic Nano–Bio interaction in solid tumours.N-hydroxysuccinimidyl (NHS) esters of different fluorescent dyes were purchased as follows: BDP, Cy3.5, Cy5 and Cy7.5 from Lumiprobe Corporation; ICG-Sulfo-OSu from AAT Bioquest Inc; BDP650 NHS ester from Life Technologies. Ultracentrifugal filter units (MWCO = 100 kDa) were obtained from Merck Millipore. Foetal bovine serum (FBS) was purchased from Gemini Bio-products Inc. Cell culture media, penicillin-streptomycin and 0.25% trypsin-EDTA were obtained from M&C gene technology Ltd. Paclitaxel (PTX) was purchased from Dalian Meilun Biotechnology Co. Ltd. Monomers, such as 2-(diisopropylamino) ethyl methacrylate (DPA-MA) and 2-aminoethyl methacrylate (AMA) were purchased from Polysciences Company. Initiator V65 was purchased from Yuanye Biotechnology Co. Ltd. Other reagents and solvents were from Thermo Fisher Scientific or Sigma-Aldrich Inc.The b-PDPA-AMA3 copolymer was synthesised by atom transfer radical polymerisation (ATRP) method23. Briefly, PEG5k-Br (0.1 mmol), DPA-MA (8 mmol), AMA (0.3 mmol) and PMDETA (0.1 mmol) were charged into a reaction flask. Then, the above substances were dissolved by the addition of the mixed solvents of 2-propanol (2 mL) and N′N-dimethylformamide  into the flask. Next, CuBr (0.1 mmol) was added into the reaction flask under nitrogen atmosphere after three cycles of freeze-pump-thaw to remove oxygen, and the reaction was maintained under vacuum for 12 h at 40 °C. After the reaction, the reaction mixture was diluted with 10 mL tetrahydrofuran (THF), and the catalyst was removed by neutral aluminium oxide column. Most of the eluent was removed by rotovap. The residue was dialysed in distilled water for 24 h and then lyophilised to obtain white powder. The purified PEG-b-PDPA-AMA3 copolymer was characterised by 1H 400 MHz NMR and gel permeation chromatography (GPC). Molecular weight (Mn and Mw) and PDI of PEG-b-PDPA-AMA3 were 22.27, 27.39 and 1.23KDa , respectively.PEG-−1) was added into a solution of PEG-b-PDPA-AMA3 copolymer in DMF (50 mg mL−1). Then, the solution was stirred overnight in the dark. The purified PEG-b-PDPA-Dyen copolymers were obtained by preparative GPC to remove the unconjugated dye molecules. The resultant dye-conjugated polymers were lyophilised and kept at −20 °C until further use. The conjugation efficiency of the dye to each polymer was determined using UV-Vis spectrophotometry. The molar feed ratios of different dyes to AMA and corresponding conjugation ratios were listed in Supplementary Table For fluorescent labelling of the copolymer, the dye-containing DMF solution 10 mg mL0 mg mL−12 atmosphere, then the solution was cooled to 0 °C. DCC (1.2 mM) in DCM (5 mL) was added into the reaction flask with continuous stirring. After stirring for 36 h at room temperature, the reaction mixture was filtrated, and then precipitated with diethyl ether twice. The product were further vacuum-dried overnight.Firstly, macro chain transfer agent, PEG-CTA, was synthesised by esterification reaction. In brief, monomethoxy PEG (1.0 mM), DMAP (0.1 mM) and DCT (1.2 mM) were dissolved by the addition of anhydrous dichloromethane  under Nb-P(DPA-r-PTX) was synthesised by reversible addition-fragmentation chain transfer (RAFT) polymerisation method32. PEG-DCT (0.03 mmol), DPA-MA (3.7 mM), MA-GFLG-PTX (0.2 mM) and V65 (0.01 mM) were charged into a reaction flask. Then, the above substances were dissolved by the addition of anhydrous dioxane into the flask. After three cycles of freeze-pump-thaw to remove oxygen, and the polymerisation was maintained under 52 °C for 36 h. The product was purified by precipitating in large excess of cold ether, and washed three times. According to the 1H 400 MHz NMR, the degree of polymerisation of PDPA block was estimated to be ~76.6. The conjugation capacity of PTX was ~11.9%, which calculated by 1H NMR peak area relative proportion of the benzene ring in PTX and the methyl end group of the polymer.Next, the PEG-b-PDPA-Dyen (n = 0–3) polymer (5 mg) was dissolved in methanol (1 mL). Subsequently, the solution was dropped into 10 mL distilled water under continuous sonication. The organic solvent was removed through the micro-ultrafiltration system (100 kDa) for five times. Finally, the nanoparticle solution was concentrated to 4 mg mL−1 (based on the polymer mass) by ultrafiltration as a stock solution for further research.In a typical procedure, PEG-−1 with PBS buffer of different pH. The samples were excited at 630 nm with a slit width of 5 nm, and emission spectra were collected from 650 to 720 nm. The peak intensity of emission spectra was used for the quantitative assessment of the pH-sensitive properties. The value of RF (Fmax/Fmin), pHt and ΔpH10–90% are shown in Supplementary Table ex/λem) of dyes used to quantify pH-sensitivity are as follows: BDP (488/500–600 nm), Cy3.5 (575/590–700 nm), BDP650 (630/650–750 nm), Cy5 (630/650–750 nm), Cy7.5 (780/795–900 nm) and ICG (780/795–900 nm).The fluorescence emission spectra of nanoprobes in PBS buffers with different pH were measured by a fluorescence spectrometer . Taking Cy5-labelled nanoprobe for example, the nanoparticle stock solution was diluted to 0.1 mg mLRF ≥ 100). Accordingly, four dyes  with low activation ratio (RF ≤ 10) were chosen as potential ‘always-ON' fluorophores. Subsequently, a series of hybrid nanoparticles comprised of ‘OFF-ON' modules and potential ‘always-ON' modules with different molar ratios were prepared as mentioned above. Followed by fluorescence characterisation, the hybrid nanoparticles were diluted to 0.1 mg mL−1 in phosphate-buffered saline  with different pH from 5.4 to 7.2 . The samples were successively excited with two excitation wavelengths to obtain the fluorescence emission spectra of ‘OFF-ON' and ‘always-ON' modules on a fluorescence spectrometer .After fluorophores screening of ‘OFF-ON' and ‘always-ON' modules among monochrome nanoprobes. Two dyes  were selected as fluorophores of ‘OFF-ON' module due to their high activation ratios (−1 or hydrochloric acid solution (pH = 5.4), and then negatively stained by 1% phosphotungstic acid. The TEM images were captured on a transmission electron microscope .Transmission electron microscope  was used to visualise the morphology of nanoparticles at different states. The samples were diluted with Milli-Q water to 1 mg mL−1) at various pH values were acquired on an IVIS in vivo imaging system  using auto-exposure with excitation/emission band-pass filter (620 ± 10 nm/670 ± 20 nm). The appropriate filter pairs for different dyes were selected according to respective excitation and emission maximum.Fluorescent images of nanoprobe solution  at 30 min after adding K+ (140 mM), Ca2+ (5 mM), Cl− (114 mM), SO42− (20 mM) ions, GSH (10 mM), papain (5 μM), H2O2 (1 μM) and •OH (1 μM). K+, Ca2+, Cl−, SO42− ions, GSH, papain, H2O2 stock solutions were prepared by directly diluting commercially available KCl, CaCl2, NaCl, Na2SO4, GSH and papain respectively. •OH was produced from Fenton reaction between H2O2 and FeCl2.The fluorescence stability of BiRN in the presence of ions, GSH, protease or ROS were determined. The fluorescence intensities of BiRN solution (100 µg/ml) in phosphate buffers  were determined by an IVIS in vivo imaging system  using auto-exposure with band-pass filters  or in vivo imaging station . The fluorescence of nanoprobes was determined by diluting nanoprobes to a final concentration of 0.1 mg mLb-P(DPA-r-PTX) was prepared as mentioned above. PTX release from PDPA-PTX micelle was performed by incubation of the micelle in Mcilvaine’s buffer (pH 6.0) at 37 °C in the presence of papain (5.0 µM) for 48 h. The stability of PDPA-PTX micelle in PBS buffer (pH 7.4) was monitored for 24 h at 37 °C. At pre-determined time-points, the released PTX amount in samples was analysed by HPLC  at UV wavelength of 230 nm. An Agilent SB-C18  was maintained at 40 °C and a flow rate of 1 mL min−1 was used, with Milli-Q water (30%) and acetonitrile (70%) used as mobile phases34.Firstly, PDPA-PTX micelle comprised of PEG-2000 (molar ratio = 10:5:1) were dissolved in trichloromethane and subsequently the solvent was removed by rotovap at 37 °C to form a thin lipid film. The resulting film was hydrated with ammonium sulfate solution (123 mM), followed by sonication for 30 min. The prepared liposomes were passed through a Sephadex G-50 column equilibrated with PBS buffer  to replace the extra-liposomal solution. Next, DOX·HCl was added into the eluted liposome suspension and incubated at 70 °C for 30 min. The separation of liposome from unloaded DOX was performed by chromatography using a Sephadex G-50 column. The concentration of DOX was determined at 495 nm utilising ultraviolet–visible (UV–Vis) spectrophotometer  after lysis of liposomes with Triton X-100.Liposomes were prepared by the traditional thin film-hydration method and DOX was loaded into the liposomes via the remote loading with an ammonium sulfate gradient. Briefly, HSPC, Chol and DSPE-mPEG2-containing humidified atmosphere at 37 °C.4T1 and MCF-7 breast carcinoma, A549 lung cancer cells, Panc02 and BxPC-3 pancreatic cancer cells, RAW264.7 macrophage and NIH/3T3 fibroblast were obtained from National Infrastructure of Cell Line Resource. HN5 head and neck cancer cell line was established from a head and neck cancer patient and provided by Dr. Jinming Gao Lab at University of Texas Southwestern Medical Centre. Above cell lines were maintained in complete medium (RPMI 1640 or DMEM with 10% FBS and antibiotics) in 5% CO2 atmosphere to allow cell adherence and growth. For real-time imaging, cell nuclei were counterstained with Hoechst 33342 (5 μg mL−1) firstly and washed with PBS. Subsequently, confocal images were captured by a confocal microscope  under 60× oil objective lens using 405 nm , 488 nm  and 561 nm  lasers. The DAPI (450/35), FITC (515/30 nm), TRITC (605/75 nm) emission filters were used for Hoechst 33342, BDP and Cy3.5 imaging, respectively. Image acquisition was performed as soon as the addition of the culture medium containing BiRN (100 μg mL−1), followed by real-time imaging for 30 min with a time interval of 1 min between frames. The raw images were processed by NIS-Elements viewer software (Nikon) and the ratiometric images were generated by ImageJ software (NIH).The cells were seeded in glass-bottom dishes and incubated for 24 h at 37 °C, 5% COTo investigate the kinetics of BiRN internalisation by living cells, time-resolved qualitative and quantitative experiments were separately measured in a Nikon confocal microscope  and a flow cytometry .−1), the cells were precooled on ice for 10 min, pulsed with BiRN solution (100 μg mL−1) at 4 °C for 10 min, washed thrice with ice-cold PBS, and chased by imaging at 37 °C for 30 min with a time interval of 3 min. Three channels, time-lapse images were collected using Nikon confocal microscope equipped with a 60× objective lens. Acquisition settings were optimised for each channel to prevent photobleaching of fluorophores. Frames were extracted using the NIS-Elements viewer software (Nikon) and the ratiometric images were generated by ImageJ software (NIH).For confocal imaging, after counterstained with Hoechst 33342 (5 μg mL−1) at 4 °C. After pulsed for 10 min (cell binding) and washed three times, the samples were chased at 37 °C  for 2 h by measuring the fluorescent intensity of each channel for single cell on flow cytometer (Beckman Coulter) with 488 and 561 nm laser. The 488 and 561 nm lasers were used to excite BDP and Cy3.5 respectively, and appropriate compensation settings prevented crosstalk between channels. CytExpert 2.3 software was used for quantitative data collection.For quantitative analysis, the cell suspension obtained by trypsinization were put on ice for 10 min, followed by centrifugation and re-suspension in BiRN solution (100 μg mL5 cells/well) were seeded into 6-well plates and cultured until adherence. To explore the cellular uptake mechanism, the cells were pre-treated with various endocytosis inhibitors, including 25 mM EIPA, 5 mM chlorpromazine, 15 mM nystatin or 40 mM dynasore for 0.5 h. Then, 100 μg mL−1 BiRN was added into each well and incubated for 2 h. The cells were collected by trypsinization, washed thrice and analysed on a flow cytometry.4T1 cells (5 × 103 cells/well) were seeded into 96-well plates and incubated overnight. Then, the cells were pre-incubated with 40 mM dynasore for 0.5 h. Free PTX or PDPA-PTX micelle were added at final PTX concentrations of 0.001–30 μM in media and incubated for 1 h at 37 °C, followed by the replacement of fresh medium. Finally, cell viability was quantitatively evaluated by MTT assays after another 70 h incubation.The cytotoxicity of free PTX and PDPA-PTX micelle against 4T1 cells in the absence and presence of dynasore was measured by MTT assay. 4T1 cells  were purchased from Vital River Laboratory Animal Centre (China). Animals were housed in groups of 4–5 per cage, maintained at a temperature of ~25 °C in a humidity-controlled environment with a 12 h light/dark cycle. The BALB/c mice were subcutaneously inoculated with 4T1 tumour cells (1 × 106 cells per mouse) on their right flanks. Two to three weeks after implantation (once tumour reached about 200 mm3 in volume), the tumour-bearing mice were subjected to imaging studies. To demonstrate the broad application of BiRNs for real-time monitoring of tumour accumulation and cellular endocytosis, subcutaneous tumour models, including MCF-7 breast carcinoma, Panc02 and BxPC-3 pancreatic cancers, A549 lung carcinoma, were developed. Orthotopic tumour models, including HN5 head-neck cancer, and 4T1 mammary carcinoma were established. PDX oesophageal tumour model in NOD SICD mice was also developed. Human oesophageal tumours were obtained from Peking University Cancer Hospital. The study was approved by the ethics committee of Peking University Cancer Hospital and with the patients’ informed consent. The PDX tumours were cut into ~2 × 2 × 2 mm small fragments and surgically engrafted into right flanks of NOD SCID mice. One month after implantation (tumour volume reached ~100–150 mm3), the tumour-bearing mice were used for imaging studies. For the evaluation of nanotherapeutic efficacy, 1 week later (the tumours reached a volume of 50 mm3), 4T1 tumour-bearing mice were treated with Doxil after ratiometric imaging.All animal care and procedures were carried out in compliance with ethical compliance and approval by the Animal Ethics Committee of Peking University. Female ex/λem: 620 ± 10 nm/670 ± 20 nm for Cy5 and 780 ± 10 nm/845 ± 20 nm for Cy7.5). The mean fluorescence intensities of two channel images with varying endocytosis percentages were measured using Living Image software (PerkinElmer). The ratio value as a function of endocytosis percentage is plotted according to the formula:For the establishment of the calibration curve, BiRN diluted in the medium with pH 7.4 and pH 5.4 were used to simulate nanoprobes with micelle state in extracellular matrix and with unimer state in endocytic organelles, respectively. Taking PBS buffer as medium, the nanoprobes with constant polymer weight were diluted separately in two kinds of pH buffers (50 μL each) to pre-calculated concentrations, and the samples with varying ‘turn on' (endocytosis) percentages (proportion of nanoparticles in pH 5.4) were obtained. The samples with 0, 5, 10, 20, 30, 40, 50 and 100% endocytosis percentages were prepared, and then placed in a black 384-well plate, followed by fluorescence imaging using an IVIS in vivo imaging system . Fluorescence images were captured using auto-exposure with band-pass filters (λ−1) to evaluate the effect of nanoprobes concentrations on the slopes of calibration curves.To establish a calibration curve under mimetic physiological condition, a series of tissues homogenates with two pH were prepared to dilute BiRN. Fresh tissues including the heart, lung, spleen, liver, kidney, tumour and muscle were collected immediately after tumour-bearing mice were transcardially perfused with 20 mL PBS buffer. The excised tissues were cut into small pieces, weeded out connective tissues, weighed and homogenised in PBS with pH 7.4 or pH 5.4 using a homogeniser . The samples of tissue homogenates with varying endocytosis percentages were prepared following the aforementioned dilution method and imaged on an in vivo imaging system. The calibration curve of each tissue homogenate was used to calculated corresponding endocytosis percentages. BiRN was diluted in PBS and tumour homogenates with varying overall concentrations  were obtained by diluting nanoparticles with tumour homogenates. The supernatant liquid obtained by centrifugation (9.6 × 103xg) were subjected to fluorescence intensity measurement on a fluorescence spectrometer . Then, a linear relationship between fluorescence intensity and concentrations were established using the linear regression method. For intratumoural nanoparticle accumulation quantification, tumours (n = 4) were dissected at pre-determined time-points post-injection of nanoparticle. The homogenates and centrifugal supernatant of tumours were prepared by the above-mentioned methods. The amount of intratumoural nanoparticles were calculated from the linear standard curves.To establish a calibration curve for quantification, fresh tumours were collected from A549 tumour-bearing mice. The excised tumours were cut into small fragments, weeded out connective tissues, weighed and homogenised in acid methanol (0.5 mL/100 mg tumour) using a homogeniser . A series of standard samples with different nanoparticle concentrations  received BiRN nanoprobes intravenously for ratiometric fluorescence imaging to monitor tumour accumulation and internalisation of nanoparticles. The images were captured on an IVIS in vivo imaging system  with time lapse using auto-exposure with a band-pass filter . To demonstrate the broad application of BiRN and compare the difference between various tumours in accumulation and endocytosis, BiRN was administrated intravenously into other six tumour models mentioned above. Then, the mice were monitored by sequential acquisition at 670 and 845 nm at designated time-points. Mice were euthanized after the last imaging session, followed by resection of the tumour and organs. The dissected tumours and organs were fluorescently imaged immediately. The images of Cy5 and Cy7.5 channels were extracted using Living Image software (PerkinElmer) and the ratiometric images were generated by ImageJ software (NIH).The 4T1 tumour-bearing mice (35. 4T1 tumour cells (1 × 106) were harvested in up to 20 μL of phenol red–free culture medium and injected between the dermis and the exposed fascia. Over weeks, tumours reached several millimetres in size and became vascularised. Intravital imaging was performed on a dual-photon fluorescence microscope . Mice were anaesthetised with avertin  on a heated microscope stage and 100 μL of the nanoprobes (40 mg kg−1) were administered intravenously, followed by imaging at pre-determined time points. A 25× water immersion objective (NA 1.0) was used for data collection. The nanoprobe was excited at 1000 nm and the fluorescent signals of BDP and Cy3.5 were detected at 510 and 610 nm, respectively. Image analysis was performed in Leica LAS X software (Germany).For visualisation of nanoprobe distribution in living animal, female nude mice (20–22 g) were implanted with a dorsal skin window chamber following the protocol previously published−1 was injected intravenously into 4T1 tumour-bearing mice. At 24 h post-injection, the mice were euthanized and the tumour tissues were immediately excised, embedded in O.C.T. tissue freezing medium (Leica), and snap frozen. Cryostat sections of 10 μm thickness were thawed briefly, counterstained with Hoechst 33342 (5 μg mL−1) and mounted. Whole slides were scanned on an automated quantitative pathology imaging system  using DAPI, FITC and Texas red excitation filters at 20× magnification.For the micro-distribution studies of nanoprobes in tissues, BiRN at a dose of 40 mg kg2. Animals were placed on a heated blanket under anaesthesia by avertin  during the experiment. After calibration of pH metre by two pH buffers, the reference electrode (2 mm OD) was placed under the skin adjacent to tumour by a shallow incision, firstly. The needle electrode (0.75 mm OD) for pH was inserted into the centre of tumour, and was held still for a moment until pH readings stabilised. Electrodes were calibrated again at the end of each measurement. Measurements with two directions were taken at each position and three positions were selected at each tumour.pH measurements were carried out on a pH metre  with needle electrode for pH and reference electrode  when tumour volume reached to 200 mmn = 32) with an average tumour volume of 50 mm3 were fluorescently imaged at 24 h post-administration of BiRN (20 mg kg−1). The heterogeneity of EPR effect and internalisation amount of nanoparticles among tumours were quantified by the Cy7.5 and Cy5 signals over the entire tumour area, respectively. After imaging, three-quarters of the mice received the PDPA-PTX micelle with a single dose of 20 mg kg−1 PTX by intravenous administration. For tumour progression monitoring, caliper measurements were performed every two days to monitor the tumour volume (V = Length × width2/2). Pulmonary metastasis was detected on 17th day by bioluminescence imaging with an IVIS in vivo imaging system . At the end of the experiment, the GraphPad Prism 8 was used for correlation analysis between tumour progression and tumour accumulation as well as the internalisation of nanoparticles.Orthotopic 4T1-Luc tumour-bearing mice (n = 13) with average volume of 200 mm3 were collected at 48 h after co-injection of BiRN (20 mg kg−1) and PDPA-PTX . The excised tumours were cut into small fragments, weeded out connective tissues, weighed and homogenised in pH 6.0 Mcilvaine buffer (0.3 mL/100 mg tumour) using homogeniser . The resulted tumour homogenates were lysed by three times freeze-thaw. The lysate (20 µL) was divided into two parts for intratumoural total and released PTX content determination, respectively. The PTX in tumour homogenates was extracted with methanol . Docetaxel (DTX) was spiked as the internal standard before the addition of the extraction solvent. For PTX contents determination, LC-MS analysis was carried out on a Triple TOF 4600 mass spectrometer  equipped with a TurboV® ionisation source and LC-20AD liquid chromatography system . A C18 column  was maintained with a flow rate of 1 mL min−1 at 40 °C. Milli-Q water with 0.1% formic acid and acetonitrile with 0.1% formic acid were used as mobile phases A and B, respectively. Masses detected in ESI+ mode were 876.5/308.1 for free PTX and 830.6/549.4 for DTX.4T1 tumours (n = 35) with average tumour volume of 50 mm3 were fluorescently imaged at 24 h post-injection of BiRN (20 mg kg−1). The heterogeneity of EPR effect and internalisation of nanoparticle among tumours were quantified by the Cy7.5 intensity and O/A value over the entire tumour area, respectively. After imaging, the mice received the doxorubicin liposome (5 mg kg−1) every other day for three times by intravenously administration. For the correlation study, 20 of mice were euthanized and tumours were immediately excised. Then, tumours were cut into small fragments and subsequently incubated in 2 mL of PBS containing 0.5 mg mL−1 collagenase IV and 0.2 mg mL−1 DNase I for 30 min at 37 °C while shaking. Tumour lysate was filtered using a 40 μm cell strainer  to remove large aggregates. The filtrate was washed three times with PBS, followed by lysis of red blood cells by lysis buffer (BioLegend). Finally, the tumour cell samples were subjected to flow cytometry  to quantify fluorescent signals of Cy5 and doxorubicin. The rest of mice were used for tumour progression monitoring. Caliper measurements were performed every two days to monitor the tumour progression by the volume (V = Length × width2/2). At the end of the experiment, the Graphpad Prism 8 software was used for correlation analysis between tumour progression and tumour accumulation as well as internalisation of nanoparticles.Orthotopic 4T1 tumour-bearing mice  depending on the number of treatment groups and distribution.Data were expressed as mean ± s.d. Statistical analyses were performed using GraphPad Prism and Origin. Significant differences of quantitative data were analysed by conducting an independent-samples Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Reporting Summary


Our goal was to investigate the frequency of specific signs and symptoms following sexual assault-related non-fatal strangulation (NFS) and to explore the interaction between assault characteristics and physical exam findings.This retrospective observational study included all adults (>18 years) reporting strangulation during sexual assault who presented for a forensic sexual assault exam at one of six urban community hospitals contracted with a single forensic nurse agency. Demographic information, narrative elements, and physical exam findings were abstracted from standardized sexual assault reporting forms. We analyzed data with descriptive statistics and compared specific variables using chi-square testing.P = 0.02).Of the 580 subjects 99% were female, with a median age of 27 (interquartile range 22–35 years). The most common injury location was the neck (57.2%), followed by the mouth (29.1%). We found that 19.1% of the victims had no injuries evident on physical exam and 29.8% reported a loss of consciousness. Eye/eyelid and neck findings did not significantly differ between subjects who reported blows to the head in addition to strangulation and those who did not. The time that elapsed between assault and exam did not significantly correlate with the presence of most head and torso physical exam findings, except for nose injury (Slightly more than half of the victims who reported strangulation during sexual assault had visible neck injuries. Other non-anogenital findings were present even less frequently, with a substantial portion of victims having no injuries documented on physical exam. The perpetrators’ use of blows to the head may account for many of the non-anogenital injuries observed, but not for the neck and eye/eyelid injuries, which may be more specific to non-fatal strangulation. More research is needed to definitively establish strangulation as the causal mechanism for these findings, and to determine whether any long-term neurologic or vascular sequelae resulted from the observed injuries. While most emergency physicians (EP) rarely perform forensic sexual assault (SA) exams, the emergency department (ED) remains a critical access point for SA referral and resources, particularly in rural areas or in departments where a sexual assault nurse examiner (SANE) is not available on-call.Strangulation is not uncommonly reported in the course of SA. Zilkens et al 2016) estimated the frequency of non-fatal strangulation in SA victims to be about 7%,016 estimDespite the consistency with which non-fatal strangulation is reported in SA survivors and the potential severity of risk that a history of strangulation confers, little is known about the specific injury patterns resulting from SA-related strangulation. Plattner et al (2003) attempted to generate a classification system for strangulation-related injury severity based on a retrospective chart review of 134 cases but went on to note that 95 71%) of these cases did not fit criteria for just one category of classification.1% of the18Complicating matters further, medical documentation has been shown to significantly impact outcomes in the minority of SA cases that do go to trial,We performed a retrospective observational study describing the demographics, assault characteristics, and signs/symptoms present among adult SA victims presenting for forensic evaluation between January 2006– May 2019. An exemption to informed consent was granted by our institutional review board due to the absence of any personally identifying information recorded in our dataset.The study population consisted of all adult (>18 years) SA victims reporting strangulation who were examined by a single forensic nurse examination agency contracted with six urban, community hospitals in the Southern California area between January 1, 2006–May 30, 2019. We excluded those victims who were unsure whether they had been strangled due to loss of consciousness, amnesia related to drug/alcohol use, or blunt head injury. We included those victims who reported strangulation even if a strangulation addendum form (as described in the study protocol) was not attached to their record. We excluded those victims whose recollection of the assault was so unclear, or so sparsely documented, that it was uncertain whether they had been strangled.What do we already know about this issue?Strangulation is a common injury sustained during sexual assault that can cause injury ranging from temporary pain or dyspnea to carotid dissection, coma, or death.What was the research question?What is the frequency, nature, and severity of the injuries associated with non-fatal strangulation in survivors of sexual assault?What was the major finding of the study?About 57% of subjects reporting strangulation during their assault had an associated positive finding on physical exam of the neck. Almost 20% had no positive physical exam findings.How does this improve population health?Understanding the injury patterns in sexual assault is essential to providing sensitive, trauma-informed care to this highly vulnerable population.The patient exams were performed by 38 SANE professionals who had been trained in accordance with the educational standards for the Office of Emergency Services in California and the International Association of Forensic Nurses. All exams were documented using the State of California Emergency Management Agency  Form 2–923. The median number of exams performed by each nurse examiner was six, with an interquartile range of 2–12.75. During the forensic examination, all study subjects underwent a standardized interview, external physical exam with photo documentation, pelvic exam with photo documentation , and external anogenital exam with photo documentation . The Cal EMA 2–923 form contents were entered into a database management software TACT , which was then used to retrospectively identify all eligible subjects flagged as reporting strangulation during the assault. We then used the original Cal EMA 2–923 form as the source document for study data abstraction. Study variables were entered into a spreadsheet in Microsoft Excel .We collected demographic data including the victims’ gender, age, and race. The method of strangulation  the perpetrator’s relationship to the victim, and the victim’s pregnancy status were also reported. The presence of weapons , alcohol or drug use in the 24 hours preceding the assault, loss of consciousness, and a history of blows to the head during the assault were recorded, along with the presence or absence of specific symptoms: nausea/vomiting; breathing difficulty; urinary incontinence; difficulty swallowing; and voice changes. Each of these items was specifically included as a discrete question on the Cal EMA 2–923 form. The presence of any physical exam finding was reported for the following categories: face; head (excluding face); eyes/eyelids; ear; nose; mouth; under chin; neck; chest; and shoulders. We recorded the time that elapsed between the assault and the victim’s presentation as a quantitative variable. In cases where the assault occurred over several hours, the midpoint of the given time range between assault and exam was recorded. All time intervals were rounded to the nearest half hour.Spreadsheet data were imported and analyzed with descriptive statistics using R version 3.6.3 . In addition to calculating percentage of positive physical exam findings in the entire study population, we performed several exploratory analyses to evaluate whether injury patterns differed based on specific assault characteristics. First, we calculated the difference in percentage of positive physical exam findings between victims who reported blows to the head vs those who did not, along with a 95% confidence interval (CI), for each physical exam category. Next, we calculated a difference in percentage between IPV vs a collapsed non-IPV cohort  along with a 95% CI for each physical exam category. Finally, a chi-square test with Yates’ continuity correction was performed to evaluate for interaction between selected study variables for hypothesis-generating purposes. For continuous variables, data was first logarithmically transformed to generate a more normal distribution and then broken up into tertiles. The tertiles were treated as discrete categories and subjected to chi-square testing as previously described.≥18 years of age in the TACT database search, which returned all SA victims who were flagged as reporting strangulation. Ultimately, we included 580 of these subjects in the study database  were present in 57.2% of our study population, which is higher than previous estimates of physical exam findings in non-fatal strangulationnot show a significant percent difference among the battering vs non-battering populations, namely, for eyes/eyelids and neck. The similarity across these two populations suggests that neck findings and eye/eyelid findings might be specific to strangulation, although further research involving a control population would be required to definitively demonstrate this.We calculated the difference in percentage between physical exam findings in victims who reported blows to the head vs those who did not to ascertain the extent to which our findings might be accounted for by physical blows rather than strangulation. Because our study was not designed to isolate the exam findings among strangled vs non-strangled SA victims, we used this comparison instead as an indicator of whether our results might be systematically biased by the presence of injuries from an alternative mechanism. Unsurprisingly, we found that subjects who reported battering had higher rates of positive findings in most categories. More interesting were those categories that did P <0.001). Petechiae and subconjunctival hemorrhage (SCH) have both been reported as potential candidates for predictors of strangulation severity.Another variable that might be expected to have an impact upon injury patterns is the use of ligature vs manual strangulation. Few comparisons or conclusions can be drawn about the impact of ligature vs manual strangulation on injury patterns from our data, as only a small number of assailants 2.6%) used a ligature. This low prevalence of ligature use has been observed in other populations of SA-related non-fatal strangulation, as well..6% used Our sample demonstrates a high prevalence of IPV-related assaults, which is consistent with prior epidemiologic studies of assailant types in non-fatal strangulation,Much of the literature related to non-fatal strangulation has grown out of the pioneering work by Strack et al (2001) in their landmark, 300-subject case series of IPV-related, non-fatal strangulation, which showed even higher rates of reported strangulation without physical exam findings, on the order of 50% with no visible injury.We also used our data to explore a hypothesized differential prevalence of physical exam findings in people of color. Previous studies have shown that Black women experience higher rates of SA than their White counterparts within specific age and socioeconomic subcategories.Our study had several significant limitations in addition to those already addressed. First and foremost, we only studied individuals over the age of 18; thus, our data does not necessarily generalize to a child/teenage population. Additionally, there is substantial inherent sampling bias in any study of individuals voluntarily presenting for a forensic SA. Estimates of the proportion of SA victims who present for forensic exam range from around 14–21%Finally, between 9–12% of subjects were missing data for each physical exam category. The similarity across all exam categories likely reflects the portion of exam reports wherein a physical exam section was erroneously omitted or left blank. Although this likely introduced a degree of bias into our results (as described above), we felt it was more important to include the subjects with missing exam data to gain a more accurate picture of assault characteristics and victim demographics.In sexual assault survivors who have been strangled, the presence of non-anogenital physical exam findings is inconsistent, and the absence thereof does not undermine the veracity of a reported history of strangulation. Neck findings were seen much more commonly than any other facial, head, or torso injuries. The time elapsed between exam and assault did not significantly interact with most of the exam finding categories, nor did the victim’s age. Further research is necessary to determine a causal relationship between the variables we examined and specific physical exam findings.