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Bacteria-Mediated Tumor Therapy Utilizing Photothermallycontrolled TNF-# Expression via Oral Administration Jin-Xuan Fan, Zi-Hao Li, Xin-Hua Liu, Diwei Zheng, Ying Chen, and Xian-Zheng Zhang Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b05323 • Publication Date (Web): 20 Mar 2018 Downloaded from http://pubs.acs.org on March 20, 2018

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Bacteria-Mediated Tumor Therapy Utilizing Photothermally-Controlled TNF-α Expression via Oral Administration

Jin-Xuan Fan,1,§ Zi-Hao Li,1,§ Xin-Hua Liu1, Diwei Zheng1, Ying Chen1 and Xian-Zheng Zhang1,2,*

1 Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China 2 The Institute for Advanced Studies, Wuhan University, Wuhan 430072, P. R. China

* Corresponding author E-mail address: [email protected] (X.-Z. Zhang). §

J.-X. Fan and Z.-H. Li contributed equally to this work.

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Abstract Oral drug administration is widely adopted for diverse drugs, and enjoys the convenience of use and capbility to reach different parts of the body via the bloodstream. However, it is generally not feasible for biomacromolecular anti-tumor drugs such as protein and nucleic acids due to the the limited absorption through gastrointestinal tract (GIT) and the poor tumor targeting. Here, we report a non-invasive thermally-sensitive programmable therapetic system using bacteria E. coli MG1655 as an vehicle for tumor treatments via oral administration. Thermally-sensitive programmable bacteria (TPB) are transformed with plasmids expressing therapeutic protein TNF-α, and then decorated with bio-mineralized gold nanoparticles (AuNPs) to obtain TPB@Au. AuNPs and TNF-α plasmids efficaciously protected by TPB in gut can be transported into internal microcirculation via transcytosis of M cells. After that, the bacteria-based anti-tumor vehicles accumulate at tumor sites due to the anaerobic bacterial feature of homing to tumor microenvironments. In vitro and in vivo experiments verify the successful delivery of AuNPs and TNF-α plasmids by TPB. Importantly, under remote activation, the expression of TNF-α in tumor sites can be procisely controlled by the heat generated from photothermal AuNPs to exert therapeutic actions. The biological security evaluation demonstrate that this strategy would not disturb the balance of intestinal flora.

Keywords: bacterial mediation, biomineralization, synthetic biology, tumor therapy, oral administration.


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Oral administertion is the most widely used route of drug administration owing to the low risk of infection and simple procedures.1-2 Nowadays, more than 80% of drugs were taken orally for treating a variety of diseases.3 However, most of anti-tumor drugs especially protein and nucleic acids are not appropriate to be administrated orally, mainly because the gastrointestinal tract (GIT) acts as a barrier due to the strong acidic destruction, enzymatic degradation and poor drug penetration of the intestinal membrane, etc.4-6 Besides, even though some anti-tumor drugs can be absorbed via GIT, the lack of tumor-targeting capacity would lead to serious side effects.7 In that case, it is necessary to develop valueable tumor targeting drug carriers for oral delivery of anti-tumor drugs. Very recently, with the advanced development of drug delivery, specific species of bacteria are widely regarded as drug vehicles to load proteinic drugs or nanoparticles for anti-tumor oral medication.8-10 Typically, orally administered bacteria possess indomitable vitality to resist the rugged environment in stomach, which could protect cargoes from inactivating or degrading. It is reported that specific species of bacteria can be transferred by the microfold cells (M cells) of Peyer’s patches in intestinal epithelial tissues, and further invade internal microcirculation through hepatic portal vein.11-12 In addition, the majority of bacteria, particularly anaerobic bacteria such as Clostridium novyi, Salmonella typhimurium and Listeria monocytogenes, show remarkable ability to selectively home to tumor tissues by harnessing the specificity of tumor microenvironments, such as immune surveillance suppression, necrotic tumor core eutrophication and the hypoxic milieu in solid tumors.13-15 Based on these, synthetic biology and biological mineralization are utilized to realize the transportation of protein drugs and functional therapeutic nanoparticles to tumor sites via bacteria.16-18 However, utilizing bacteria-based peroral vehicles for anti-tumor drug delivery still remains to be a critical challenge, due to powerless administration in vivo and potential homeostasis disorders.19


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Here, we designed a new type of bacteria-based anti-tumor vehicle for oral delivery via non-invasive bacterium E. coli MG1655, which was transformed with plasmids expressing therapeutic proteins for tumor treatmeat and then decorated with photothermal nanoparticles.20 Briefly, as a non-invasive bacterium, E. coli MG1655 was transformed with custom-designed

plasmid

pBV220

containing

a

thermally-sensitive

promoter

(temperature-sensitive bacteriophage λ repressor cI857, TcI) and the gene of therapeutic protein (tumor necrosis factor-α, TNF-α).21-23 When the thermally-sensitive programmable bacteria (TPB) were administrated orally into gastrointestinal tract (GIT), they could survive in the rugged environment of stomach. After being transported to internal microcirculation, TPB could further target to tumor regions and colonize rapidly, accompanied by replication of the custom-designed plasmids. Few days later, a second oral dose was performed by TPB decorated with bio-mineralized photothermic gold nanoparticles (TPB@Au). After a rapid accumulation, tumor sites were irradiated by near-infrared (NIR) light for heat generation to induce expression of TNF-α, which could induce apoptotic cell death in tumor treatment. In vitro and in vivo studies confirmed that this anti-tumor system exhibited good anti-tumor efficacy. This strategy overcomes the difficulties of current oral delivery of anti-tumor drugs by bacterial vehicles to achieve therapeutic proteins synthsis via remote activation by NIR, which shows fantastic prospect for biomedicine in the future.


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Figure 1. (a) Mechanism of m-Cherry/TNF-α expression based on plasmid pBV220, (b) m-Cherry protein expression from MG1655(pBV220/m-Cherry) under different temperature, (c)

Time-dependent

m-Cherry

expression

in

MG1655

(pBV220/m-Cherry),

(d)

Temperature-dependent expression of the TNF-α protein in MG1655 (pBV220/TNF-α).

As shown in Figure 1a, in order to program the thermally-sensitive plasmid, a widely-used temperature-sensitive plasmid pBV220 containing TcI repression and tandem pR-pL operator-promoter was introduced to non-invasive bacterium E. coli MG1655.24-27 When the ambient temperature shifted from 37 °C to 45 °C, TcI repression was relieved to initiate the target gene expression. Further, we engineered two construct encodings regulated respectively by TcI, fluorescent protein m-Cherry (as a reporter protein) and human TNF-α (as a therapeutic protein).28 First, the thermal logic circuits of TPB was verified. The expression of m-Cherry was illustrated by spatial patterned bacterial variants incubated at


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37 °C and 45 °C (Figure 1b). After being incubated at 45 °C for 10 min, the level of visible red and the fluorescence intensity derived from TPB increased with time remarkably (Figure 1c and Figure S1). The phenomena above demonstrated the feasibility and effectiveness of our designing sequence. Next, the ability of temperature-stimulated human TNF-α expression was tested. As shown in Figure 1d, polyacrylamide gel electrophoresis (PAGE) revealed the presence of TNF-α protein (24 kDa) in filtered culture supernatant of TPB after incubation at 45 °C for 10 min, whereas no TNF-α was detected after incubation at 37 °C. The quantitative TNF-α expression of TPB@Au was detected by ELISA assay (Figure S2). Clearly, the expression of target therapeutic protein TNF-α, can be procisely regulated by the bacterial culture temperature. Next, photothermic gold nanoparticles were biosynthesized by TPB via spontaneous redox reaction of bacteria to form TPB@Au. As shown in Figure 2a, specific NADPH-dependent reductase (e.g. nitrate reductase) could convert Au3+ to Au0 through electron shuttle enzymatic metal reduction, leading to the deposition of AuNPs on the bacteria surface.29-30 After being incubated with HAuCl4 (1 mM) at 37 °C for 12 h, the color of bacterial suspension changed noticeably from pale yellow (HAuCl4) to purplish red (Figure 2a), indicating the presence of AuNPs. The crystal structure and phase composition of the biosynthetic AuNPs were analyzed by X-ray diffraction (XRD).31 As shown in Figure S3, three remarkable peaks were observed in XRD diffraction pattern, corresponding to the (111), (200) and (220) Bragg reflections derived from face-centered cubic gold. Meanwhile, AuNPs were detected by UV-vis spectroscopy as shown in Figure 2b. Neither the bacterial suspension nor the mixture of bacteria and HAuCl4 solution showed apparent absorption peaks, while the spectra of bacteria treated with HAuCl4 revealed a strong absorption at nearly 530 nm after 12 h of incubation at 37°C. Furthermore, it was revealed from TEM (Figure 2c-2e) and SEM (Figure 2f) micrographs that the biosynthetic AuNPs with particle sizes


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around 60 nm were randomly distributed on the surface of TPB. This is considered as beneficial for photothermal effect in previously research.32-33

Figure 2. (a) Biosynthesis mechanism of AuNPs by TPB through enzymatic reduction. (b) UV-vis spectra of TPB, TPB@Au and TPB with HAuCl4 solution. (c), (d) TEM images of TPB@Au. (e) TEM image of AuNPs on the surface of TPB@Au. (f) SEM image of TPB@Au. (g) Temperature elevation curves of TPB@Au aqueous solutions with different concentrations, as a function of irradiation time. (h) Co-culture site for tumor cells and bacteria. (i) Cytotoxicity of TPB@Au after 37 ℃ treated and TPB@Au after 45 ℃ treated against 4T1 cells. (n = 8) (j) Fluorescence live/dead cell images of 4T1 cells with blank control treatment, supernate of TPB@Au (45 ℃, 108 CFU/mL bacteria), thalluses of 108


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CFU/mL bacteria (45 ℃), thalluses of 109 CFU/mL bacteria (45 ℃). Scale bar: 200 µm. Significance between every two groups was calculated using unpaired two-tailed Student's t test unless otherwise indicated. *P < 0.05, **P < 0.01, ***P < 0.001. The values are presented as mean±SD.

Subsequently, the bacterial photothermal conversion of TPB@Au was evaluated under NIR laser irradiation utilizing an IR camera. As shown in Figure 2g and Figure S4, with the increasing concentration of TPB@Au, both the heating rate and the ultimate temperature of suspension increased accordingly, indicating that the generation of heat could be controlled accurately. Then, the heat transferred from NIR laser irradiation was used to produce TNF-α for killing tumor cells. In this study, transwell chamber was served as the co-culture site for tumor cells and bacteria. Tumor cells (mouse 4T1 breast tumor cells) were placed in the bottom chamber, and bacteria cultured in inserts was separated from tumor cells by a polycarbonate membrane with a pore size of 400 nm (Figure 2h). TPB@Au was pretreated under different intensities of NIR laser irradiations (808 nm) to maintain the temperature at 37 °C and 45 °C for 10 min. After being co-cultured with pretreated suspension of bacteria with different concentrations for 24 h, the viability of 4T1 cell was evaluated by MTT assay. As the concentration increased, TPB@Au incubated under 37 °C did not show obvious cytotoxicity to 4T1 cells, while that incubated under 45 °C killed 4T1 cells significantly (Figure 2i). Furthermore, the mechanism of cell death was explored through western blot assay, as the key signal molecules of cell necroptosis pathway caused by TNF-α, receptor interacting protein kinase-1 (RIP-1) and receptor interacting protein kinase-3 (RIP-3) up-regulated distinctly in the cells treated with TPB@Au (45 °C incubated) (Figure S5). These results indicated that the death of tumor cells was mainly resulted from TNF-α, which was expressed by light-controlled TPB@Au. Intuitively, live/dead cell strain was used to observe the cytotoxicity of TPB@Au (Figure 2j). The aforementioned observations were 8 ACS Paragon Plus Environment

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critical to verify the toxicity of the bacteria-based vehicles which entered into systemic circulation via oral administration. A lot of research has sufficiently proved that most of bacteria had the ability to be internalized by M cells with the expression of apical membrane receptors for recognizing bacteria, which distributed in interstitial spaces of intestinal epithelial cells to overlay the lymphoid follicles of intestinal Peyer’s patches.34 In this study, we speculated that TPB@Au may also enter the systemic circulation by this way (Figure 3a). The transport of bacteria in GIT is not fully explored currently and no unambiguous conclusion can be drawn from the present results. In our study, subsidiary experiments were conducted to explore the transport of TPB@Au. Glycoprotein 2 (GP-2), specially expressed in M cells, is recognized to complete antigen transcytosis by recognizing and bonding FimH (a component of type I pili on the E.coli MG1655 outer membrane). We quantified the FimH expression of different bacteria by PCR (Figure S6). The more positive zeta potential of TPB@Au compared to TPB (Table S5), is deemed to facilitate the transcytosis of TPB@Au by M cells.35 It should be noted that the exact mechanism transcytosis of bacteria has not been well established, and the detailed mechanism of transcytosis of TPB@Au prepared in this study is warranting further study. To verify the transfer process of oral administration route for the bacteria-based vehicle TPB@Au, we firstly confirmed whether it could overcome the adverse environment in the stomach, as well as the overheating generated by AuNPs. TPB@Au was cultured in acetic acid buffer (pH 3.0, 37 ℃) for 30 min as the mimic of stomach surrounding and in PBS (pH 7.4, 37 ℃) as the control. In the meantime, TPB@Au was also cultured in PBS (pH 7.4) under 45 ℃. As shown in Figure 3b and Figure S7, the fluorescence intensity and the number of colonies on solid LB agar plates demonstrate that the survival rate of TPB@Au under overheating (45 ℃) decreases to around 85%, whereas the survival rate of TPB@Au in strong acidity environment is around 20%. Also, the plate colony-counting method was used for the 9 ACS Paragon Plus Environment

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quantification of bacteria in the blood with ampicillin-resistant. As shown in Figure S8, the blood concentration of TPB at different time points after oral administration was evaluated, and at 0.5 h, the concentration of TPB in blood reached around 260 CFU/mL. These results indicate the stomach pH (pH 3.0) and photothermal conversion (overheating) could reduce bacterial viability to a certain extent, despite that numerous bacteria still survive in the harsh environment.

Figure 3. (a) Speculated scheme of oral administration route for delivery of TPB@Au. (b) Relative viability of TPB@Au at physiological pH (pH 7.4) and simulated stomach pH (pH 3.0), physiological temperature (37 ℃) and hyperthermia temperature (45 ℃). The absolute bioluminescence intensity of viable TPB@Au was defined as 100%. (n = 3) (c) Immunofluorescence images of small intestine tissues from 4T1 tumor-bearing mice oral administration of PBS and TPB@Au, respectively at 30 min and 120 min. Scale bar: 50 µm. Magnification: ×200. Bio-TEM images of small intestine section after 24 h treatment, (d1) Scale bar: 500 nm; (d2) Scale bar: 2 µm. Bio-TEM images of liver section after 24 h treatment, 10 ACS Paragon Plus Environment

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(e1) Scale bar: 500 nm; (e2) Scale bar: 2 µm. (f) Blood biochemistry indexes of mice after different treatments at 1st day and 7th day respectively. (h) Distribution of samples among the top 9 bacteria community types after different treatments. (g) Different samples comparison evaluated by ANOSIM (R Statistic: Margalef Richness Index).

Moreover, the entrance of TPB@Au into systemic circulation was detected via immunofluorescence staining (IF) and bio-TEM. After oral administration of TPB@Au, the intestinal epithelium tissues of mice were marked by Cy3-conjugated Anti-E.coli antibody at different time points. Evidenced by IF staining (Figure 3c), strong fluorescence of bacteria was observed in intestinal epithelium tissues after 30 min treatment, compared with negative control. With the time prolonging, the fluorescence of bacteria decreased significantly (120 min). As shown in Figure 3d and 3e using Bio-TEM, it was evident that TPB@Au distributed in intestine epithelial tissues and hepatic tissues after the oral administration of bacteria. These results confirmed that TPB@Au was transported into systemic circulation from gut, and could be migrated further to liver via the hepatic portal vein. In order to assess the potential impacts on intestinal microflora and homeostasis, various blood biochemistry and hematologic indexes were tested and analyzed to measure the in vivo toxicity of [email protected] As shown in Figure 3f, Table S6 and Table S7, the average value of each index was compared between the 1st day and the 7th day after oral administration. The majority of indexes was in normal range except the trace volatile of aspartate aminotransferase (AST) level after the administration, the data turned normal 7 days later, which meant TPB@Au could not bring on side effects for internal environment, and H&E staining of small intestine sections and stomach sections of mice after treated with TPB@Au by oral administration successively for 7 days and 14 days revealed the same conclusion (Figure S9). In the meantime, the system elimination of TPB@Au in major metabolic organs was detected by fluorescence of Cy3-conjugated Anti-E.coli antibody (Figure S10). Compared 11 ACS Paragon Plus Environment

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to control, numbers of bacteria were obviously observed in IF of liver, kidney and spleen at 7th day, and the amount of bacteria decreased gradually in the next few weeks, which signified that TPB@Au could be eliminated after certain period of time. Afterward, metagenome technique was used to explore the alteration of intestinal flora after oral administration of different ingredients such as AuNPs, TPB and [email protected] First, we compared the overall diversity of intestinal microbiota between all treated groups and healthy control groups, and found that the microbiota of mice showed obvious differences from those of control shortly after oral administration by analysis of similarity (ANOSIM). However, 7 days and 14 days later, the diversities in comparison to control decreased remarkably (Figure 3g). Then, nine kinds of the most abundant bacterial genus in mice intestinal environment were measured by 16S Ribosomal DNA Identification after oral administration of different ingredients (Figure 3h and Figure S11). Compared with the control group, the relative abundance of common and critical bacterial genus such as Lactobacillus, Bacteroides, Lachnospiraceae and Oscillospira showed a trend of gentle fluctuation at the 1st day. With time prolonging, the fluctuation gradually tended to be normal. At a gene level, this result demonstrated that the therapeutic method via oral administration possesses receivable biological security for intestinal microbiota.38 The ex vivo bio-distribution of TPB@Au was evaluated using 4T1 cells bearing Balb/c mice model. DiR iodide was used to mark TPB@Au and the mice were sacrificed after gavage with TPB@Au for 40 h to detect the accumulation of TPB@Au in different organs through small animal fluorescence imaging system (IVIS), and average flurescence intensities of different organs were quantitatively analyzed (Figure S12). As shown in Figure 4a (right), tumor tissue displayed high fluorescence level, while the other organs such as heart, spleen, liver and kidney displayed negligible enrichment. The result demonstrates that TPB@Au has the ability for rapid tumor-specific accumulation due to the tropism of bacteria to tumor tissue. IF staining indicated the same conclusion (Figure 4a, left). Then, we performed quantitative 12 ACS Paragon Plus Environment

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PCR (q-PCR) to study the proliferation of TPB@Au in different immune organs as well as tumors for a long period. As shown in Figure 4b and Figure S13, compared with 1st day group, after the gavage with TPB@Au for 7 days, the amount of TPB@Au remarkably increased in the tumor tissue, while no detectable change was observed in other organs. This indicated that after the rapid tumor-specific accumulation, TPB@Au could further colonize in tumor tissue and multiply gradually. However, another 7 days later, the amount of TPB@Au decreased obviously in tumor, while in kidney and spleen a rise in the amount of TPB@Au was observed, which might be caused by immune clearance.

Figure 4. (a) Fluorescence staining and images of the major organs as well as tumors after oral administration of TPB@Au for 24 h. Scale bar: 100 µm. (b) q-PCR analyze of the major organs and tumor for mice after oral administration of TPB@Au at 1st day, 7th day and 14th day, respectively. (n = 3) (c) Optical living imaging of 4T1 tumor-bearing mice after oral


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administration of TPB@Au for different time periods. (left: vertical view; right: side view) (d) In vivo photothermal images of mice after oral administration of TPB@Au and PBS.

Furthermore, the bio-distribution of TPB@Au after oral administration was evaluated in vivo as shown in Figure 4c. TPB@Au accumulated in stomach first, and with time prolonging, the fluorescence of TPB@Au increased in tumor sites, which is in accord with ex vivo experiment. Gram stain diagnosis of mice faeces verified the same process (Figure S14). To confirm that AuNPs were effectively transferred to tumor tissue, NIR-laser triggered tumor temperature change was evaluated and recorded in real time by an IR thermal imaging camera.39 As shown in thermal images (Figure 4d), the local temperature of the tumors treated with TPB@Au increased rapidly (34.4 °C to 41.8 °C) over the course of photo irradiation (1 W/cm2). For control group, hardly any temperature increase was detected. This phenomenon verified the accumulation of AuNPs in the tumor tissue through bacteria transport. 4T1 tumor bearing Balb/c mice model was used to evaluate the in vivo antitumor effect of TPB@Au following the therapeutic schedule in Figure 5a. The mice were treated with TPB@Au, TPB and PBS orally respectively. 7 days later, a second dose was performed orally. After 24 h, NIR laser was used to trigger the expression of TNF-α. The levels of TNF-α in tumors of different treated groups were detected one day after the irradiation by enzyme-linked immunosorbent assay (ELISA) and western blot assay. As shown in Figure 5b, 5c, 5g and 5j , the up-regulation of TNF-α treated with TPB@Au+NIR was much more significant than that of the other groups. Furthermore, to validate the apoptosis-promoting effect of combined treatment, the expression of the cleaved caspase-3, B-cell lymphoma 2 (Bcl-2) as well as interferon-γ (IFN-γ) in treated tumors were examined. Cleaved caspase-3 and Bcl-2 play key roles in the execution-phase of cell apoptosis, and IFN-γ represents the immune activation level in vivo.40 As shown in Figure 5c and Figure S15, compared with PBS, TPB+NIR and TPB@Au, the group treated with TPB@Au+NIR remarkably expressed the 14 ACS Paragon Plus Environment

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highest level of cleaved caspase-3 and IFN-γ, while the level of Bcl-2 was apparently the lowest.

Figure 5. (a) Therapeutic schedule of TPB@Au for inhibiting tumor growth with NIR. (b) Expression of TNF-α in tumor tissues after different treatments. (n = 3) (c) Western blotting analysis of related protein expression (caspase-3, Bcl-2, TNF-α and IFN-γ) in tumor tissues. (n = 3) (d) Tumor volumes after different treatments. (n = 6) (e) Mice weights after different treatments. (f) Representative tumor photographs of the harvested tumors after different treatments. (g) The corresponding quantitative evaluation of protein expression (TNF-α and HIF-1α) in tumors. H&E staining (h), TUNEL (i), TNF-α (j) and HIF-1α (k) of tumor tissues after different treatments. Significance between every two groups was calculated using unpaired two-tailed Student's t test unless otherwise indicated. *P < 0.05, **P < 0.01, ***P < 0.001. The values are presented as mean ± SD.


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During the treatment, TPB@Au+NIR showed excellent ability for tumor inhibition. As shown in Figure 5d, 5e and 5f, the tumor growth was greatly inhibited after 2 weeks of treatment. The average tumor volume of the mice treated with TPB@Au+NIR was less than 200 mm3, while that of PBS treated group reached 1200 mm3. The body weights demonstrated negligible side effects of this treatment. Consistent with previous results, apparent tumor cells damages were observed after the treatment of TPB@Au+NIR (Figure 5h). The maximum degree of cell apoptosis was found by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay (Figure 5i).41 In addition, compared with the other groups, TPB@Au+NIR showed much less expression of hypoxia inducible factor-1 (HIF-1α) in IF staining (Figure 5g and 5k), demonstrating significant improvement of the hypoxia environment in tumor tissues. These results showed that the oral administration route was feasible, and the programmable bacteria-mediated delivery armed with nanoparticles had efficient antitumor effect in vivo. Meanwhile, negligible toxicity of TPB@Au+NIR to normal tissues was observed from H&E staining of other organs such as heart, liver, spleen, lung and kidney (Figure S16), and H&E staining of small intestine sections and stomach sections showed limited effect for gut (Figure S17). In summary, by harnessing the protective effect and tumor-targeting capacity of E. coli MG1655, an oral-administrated bacteria-mediated anti-tumor therapetic system combining photothermal conversion nanoparticles and thermally-sensitive plasmids is developed in this study. The thermally-sensitive programmable bacteria (TPB) are decorated with bio-mineralized gold nanoparticles (AuNPs) and transformed with plasmids expressing therapeutic proteins TNF-α. The transfer process of oral administration route for the bacteria-based vehicle was studied. When the vehicles accumulate in tumor sites, the expression of TNF-α triggered by NIR irradiation leads to the death of tumor cells. Our investigation suggests that the bacteria-based anti-tumor vehicles taken orally is an effective strategy for overcoming the barrier existing in oral administration for anti-tumor treatment, 16 ACS Paragon Plus Environment

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and can achieve satisfactory tumot-targeting therapeutic effiency.

The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxxxx. Details of experimental procedures and supplementary results (PDF).

Acknowledgement J.-X. Fan and Z.-H. Li contributed equally to this work. This work was supported by the National Natural Science Foundation of China (51690152, 21721005 and 21474077).


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