Target-Amplified Drug Delivery of Polymer Micelles Bearing

6 days ago - Bioorthogonal chemistry together with biomarker-installing techniques are very promising in the amplification of the tumor targeting effi...
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Target-Amplified Drug Delivery of Polymer Micelles Bearing Staudinger Ligation Peng Zhang, Xiaoke Zhang, Cheng Li, Sensen Zhou, Wei Wu, and Xiqun Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b10295 • Publication Date (Web): 14 Aug 2019 Downloaded from pubs.acs.org on August 16, 2019

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Target-Amplified Drug Delivery of Polymer Micelles Bearing Staudinger Ligation Peng Zhang‡, Xiaoke Zhang‡, Cheng Li, Sensen Zhou, Wei Wu*, Xiqun Jiang* Department of Polymer Science & Engineering, College of Chemistry & Chemical Engineering, Nanjing University, Nanjing, 210093, China.

‡ Peng Zhang and Xiaoke Zhang contributed equally to this work * Corresponding to [email protected], [email protected]

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ABSTRACT:

Bioorthogonal chemistry together with biomarker-installing techniques is very promising in the amplification of the tumor targeting efficiency of nanomedicine. In this work, we newly synthesized an amphiphilic block copolymer polyoxazoline-block-polycaprolactone (POX-PCL), in which a certain number of amino groups were dangled in side chain of the POX block and then functionalized into triarylphosphine groups for active tumor targeting via Staudinger ligation. By using the block copolymer self-assembly, the Staudinger ligation reagents contained and drugloaded reactive micelles were prepared with the hydrodynamic diameter of ~74 nm. Such drugloaded reactive POX-PCL micelles exhibited significant tumor target ability through the Staudinger ligation between the micelles and the tumors metabolically labeled with azide group, as demonstrated by a series of in vitro and in vivo evaluations. In this work, a novel method was proposed for the application of Staudinger ligation in the nanomedicine for amplifying the tumor targeting ability and antitumor activity of nanodrugs.

KEYWORDS: Micelles; Drug delivery; Bioorthogonal reaction; Staudinger ligation reaction; Polyoxazoline-b-polycaprolactone block copolymer

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1. Instruction Bioorthogonal chemistry which could occur in living cells without effect on native biochemical processes has been widespread used for researching and regulating biological processes.1-4 DNA, peptide, and protein could be decorated on cells robustly and specifically through bioorthogonal chemistry.5 In living bodies, the combination of metabolic labeling techniques and bioorthogonal chemistry is very promising and useful in the enhancement of disease diagnosis and treatment because artificial targets can be created in objective cells by metabolic labeling, and highly specific bioorthogonal reactions can be performed in vivo.6,7 So far, a lot of bioorthogonal chemical groups have been used as targeting groups, and they can be incorporated into cells with the biofunctional stuff in a more facile and low-cost way than proteins, antibodies, aptamers, etc.8 Furthermore, the bioorthogonal chemical targeting strategy can overcome the receptor saturation or receptor-free issues occurring in disease sites.9-11 As an endogenous metabolic product, sialic acid residues often reside at the ends of glycan chains which decorate around the cells.12 Through a biologically metabolic process of sialic acid precursors, some chemically functional groups, like azides, could be modified on the cell surfaces.13 These functional groups could then covalently coupled with exogenous reagents containing complementary groups. It’s extremely attractive to modify the cell surfaces with azides by metabolic labeling, whereby a biosynthetic precursor containing azide functional group, Nazidoacetylmannosamine (Ac4ManNAz) is ingested by cell-surface glycoproteins and transformed into N-azidoacetyl-sialic acid by the biosynthetic machinery of cells. This method has been used to modify proteins, glycans, and lipids in living bodies with various reactive probes.14-17 Bertozzi and co-workers have developed Staudinger-Bertozzi ligation reaction between azides and triarylphosphines. This ligation reaction has exquisite chemoselectivity and can be carried on in

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living systems, allowing the chemical modification to be carried out without any effect on the natural state of cells.18 The ability to modify glycans around the cells in living system allows us to create new imaging and therapeutic targets for disease detection and treatment, for example, cancer diagnosis and therapy.4,19 Although the bioorthogonal reaction of Staudinger ligation and azide has successfully been demonstrated in living mice, the two coupling partners in StaudingerBertozzi ligation reaction are still limited in the small molecular systems.18,31,32 The combination of nano drug carriers and Staudinger ligation for targeting drug delivery has not been studied much. Nano drug delivery systems have provided a new avenue to enhance drug accumulation in disease sites and minimize undesired side effects in health tissues.20 In order to further enhance the specificity of nanocarriers to lesions, active targeting strategy has been proposed by decorating nanocarriers with targeting groups, like antibodies, aptamers and peptides, which could enhance the targeting efficiency by binding with the receptors surrounded the target cells.21-25However, this strategy is not effective when the target cells of interest are short of proper receptors or the difference of endogenous receptor between diseased and normal cells is little. For example, the common receptors in breast cancer patients, such as estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 do not overexpressed in triple negative breast cancer patients.26 Furthermore, only 20% of breast cancer patients have high expression levels of HER2.27 In this case, manually manipulating cell-surface to create artificial targets in disease sites is highly desirable and falls within the scope of bioorthogonal chemistry.28,29 On the other hand, bioorthogonal chemistry can also be used to add the new receptors in cells of interest to further amplify the difference between diseased and normal cells.30 We herein reported the first example of target-amplified polymer nanomedicine via metabolic cell-labeling and Staudinger-Bertozzi bioorthogonal reaction in vitro and in vivo as shown in

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Scheme 1. The polymer nanomedicine is a class of polymer micelles comprised of an amphiphilic block copolymer, polyoxazoline-block-polycaprolactone (POX-PCL) bearing triarylphosphine side groups in POX block and doxorubicin (DOX) enriched in the micelle cores. The azide groups acting as receptors were decorated around the tumor cell surfaces and installed by metabolic glycoengineering. The validity of Staudinger-Bertozzi reaction was demonstrated in targeting drug delivery of polymer micelles in vitro and in vivo. Polyoxazolines (POXs) are a kind of hydrophilic pseudo polypeptides, which have good biocompatibility, low immunogenicity, desirable protein repellency.33-37 Moreover, unlike poly(ethylene glycol) (PEG) which has shown an accelerated blood clearance in vivo when repeated injection occurs, POXs have a stable pharmacokinetic behavior over multiple injections.34-38 The detailed experiment and route for synthesis of amphiphilic POX-PCL block copolymer is shown in Scheme 1b. POX terminated with propargyl group was initially constructed through

copolymerization

of

2-methyl-2-oxazoline

and

2-[N-tert-butyloxycarbonyl-5-

aminopentyl]-2-oxazoline using propargyl p-toluenesulfonate as an initiator. 3-azido-1-propanol was used to initiate ε-caprolactone to synthesize PCL terminated with an azide group by ringopening polymerization. The molecular weight of PCL was determined to be 10400 Da based on 1H-NMR

measurement. After removing the protective groups of tert-butyloxycarbonyl in POX by

trifluoroacetic acid, two blocks of POX and PCL were covalently connected by alkyne-azide click chemistry to generate the amphiphilic block copolymer POX-PCL bearing amylamine side groups in POX block. Also, through 1H-NMR measurement, the molecular weight of POX was determined to be 7800 Da. Thereafter, 2-(diphenylphosphino)terephthalic acid 1-methyl 4-Nsuccinimidyl diester reacted with the amino groups in POX block, forming the Staudinger ligationbearing POX-PCL block copolymer. Each POX block contains about 4.1 Staudinger reagents. The

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detailed structure and characterization of the involved compounds are presented in Supporting Information (Scheme S1 and S2, Figure S1-S14 in the SI).

Scheme 1. a) Schematic illustration of specifically modifying tumor cells with azide groups in vivo and subsequent drug targeting through Staudinger ligation; b) Synthesis route of the Staudinger ligation reagent-labeled POX-PCL amphiphilic block copolymer.

2. Experimental Section 2.1. Materials. D-Mannosamine hydrochloride, calcium hydride (CaH2), 6-aminohexanoic acid, di-tert-butyl dicarbonate, CuSO4·5H2O, dry methanol, doxorubicin (DOX), ascorbic acid, propargyl alcohol, bis(2-dimethylaminoethyl)methylamine (PMDETA), dimethylaminopyridine (DMAP), triethylamine, N,N-diisopropylethylamine, 2-chloroethyl aminehydrochloride, Nhydroxysuccinimide (NHS), 4-toluene sulfonyl chloride, 3-chloro-1-propanol, sodium azide, NEthyl-N'-(3-dimethylaminopropyl)carbodiimide

hydrochloride

(EDC·HCl),

1-methyl-2-

aminoterephthalate, adiphenylphosphane and 4’,6-diamidino-2-phenylindole (DAPI) were obtained from Adamas-beta and directly used as received. THF was dried by sodium and distilled

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under nitrogen atmosphere. 2-Methyl-2-oxazoline (MOx), N,N-dimethylformamide (DMF), acetonitrile, ε-caprolactone were dried by CaH2 and distilled under vacuum. 2.2. Synthesis of Staudinger ligation reagent-labeled POX-PCL (12). 10 mg of StaudingerNHS and 100 mg of amino-containing POX-PCL were mixed with 2 mL of dry DMF. Then a drop of triethylamine was dropped into the mixture and stirred for one night away from light. The mixture was precipitated in methanol and washed three times with diethyl ether. The molecular weight of this polymer was about 18200 ( calculated by Figure S14, 1H NMR)and each polymer chain contains about 4.1 Staudinger reagents. 2.3. Synthesis of FITC-labeled POX-PCL and NIR-797-labeled POX-PCL. 1 mg fluorescein isothiocyanate, 10 mg of Staudinger-NHS and 100 mg of amino-containing POX-PCL were mixed with 2 ml of DMF. Then a small amount of triethylamine was dropped into the mixture and stirred for one night. The result mixture was precipitated in methanol and washed three times with diethyl ether. NIR-797-labeled POX-PCL was also prepared as above. 2.4. Fabrication of DOX-loaded POX-PCL micelles. 2 mg of DOX and 20 mg of Staudinger ligation reagent-labeled POX-PCL were mixed with 3 ml of DMF and the resulting solution was then dialysed against PBS buffer (pH 7.4) for 2 days to give the DOX-loaded POX-PCL micelles. (MWCO = 14 KDa). Preparations of FITC-labeled DOX-loaded POX-PCL micelles and NIR-797labeled DOX-loaded POX-PCL micelles were similar to the above method by replacing POX-PCL copolymers with corresponding fluorescent labeled copolymers. Hydrodynamic diameters and size distributions of micelles were evaluated by dynamic light scattering (DLS, Brookhaven BI9000AT system. Transmission electron microscopy (TEM) (JEOL TEM-100, Japan) was used to characterize the morphology and size of micelles in condition of lacking water. Micelles were dripped onto the copper grid and dried at room temperature before examination.

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2.5. Drug-loading content and entrapment efficiency. Concentration of DOX in micelles was determined with ultraviolet spectroscopy at 480 nm. Standard DOX was used to prepare the calibration curve. A solution of lyophilized DOX loaded micelles containing DMF was prepared and evaluated. Drug-loading content (DL) and the drug entrapment efficiency (DE) were calculated as follows. DL (%) = (mass of DOX in micelles)/(mass of micelles in solution) × 100% DE (%) = (mass of DOX in micelles)/(total mass of DOX added in solution) × 100% 2.6. Drug release characteristics in vitro. DOX-loaded POX-PCL micelles were re-dissolved with deionized water. The resulting solution was then poured into a dialysis bag and submerged in pH 5.4 and pH 7.4 PBS buffer (5 mL, 0.01M) respectively. This release system was placed in the shaker at 37 °C and stirred in the dark at a rate of 100 r/min. The dialysate was replaced regularly with fresh medium and fluorescence spectrometer (RF-5301PC, SHIMADZU, Japan) was used to evaluate the concentration of DOX in the dialysate at 480 nm. 2.7. In vitro cellular uptake and flow cytometry. Murine colon tumor cells, CT26 tumor cells were cultured on glass bottomed dishes containing 2 ml of DMEM medium dissolved with Ac4ManNAz (50 μM) or without Ac4ManNAz. The cell density was controlled at a density of 3 ×104 cells per well. After three days of incubation at 37 °C with 5% CO2, cells were carefully washed with fresh PBS (pH 7.4) and FITC-labeled DOX-loaded POX-PCL micelles (200 μg/ml) was added and cultured for another 4 h. Then, the medium was replaced with fresh PBS and formaldehyde glutaraldehyde was added to fix the cells before observation with a confocal laser scanning microscope (CLSM; LSM 710, Zeiss, Germany). CT26 cells treated and non-treated with Ac4ManNAz as above were cultured with FITC-labeled DOX-loaded POX-PCL micelles (200 μg/ml) for 4 h and analysed by Flow Cytometer (BD Accuri C6).

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2.8. Cytotoxicity assay of the DOX-loaded micelles in vitro. To evaluate the vitro cytotoxicity of the DOX-loaded POX-PCL micelles and free DOX, CT26 cells treated or non-treated with Ac4ManNAz (50 μM) as above were incubated with a density of 5 ×103 cells per well for 24 hours. Fresh culture medium containing free DOX, DOX-loaded POX-PCL micelles at various concentrations was used to replace the culture medium, respectively, and incubated for another 48 h. Then 20 μL of MTT solution (5 mg/ml) was dropped into each well, and continued to culture for 4 h before 150 μl of DMSO was added. Absorbance of each well were observed at 490 nm with a microplate reader (Huadong, DG-5031, NJ). 2.9. Near-infrared fluorescence (NIRF) imaging in vivo. All the animal tests were approved by Animal Care and Use Committee, Nanjing University. About 5×105 H22 tumor cells were translated into both the upper limb armpits of ICR mice by subcutaneous injection. As the tumor grew up to about 50 mm3, Ac4ManNAz (50 μl in 50 μM of Ac4ManNAz, 70% aqueous DMSO) (right tumor) or saline (left tumor) were peritumoral injected into the mice once a day for 3 days, respectively. [29] After intravenous injection of NIR-797-labeled DOX-loaded POX-PCL micelles (6 mg/kg of DOX), the MaestroTM in vivo imaging system (CRi, USA) was used to observe the biodistribution and tumor accumulation of micelles marked by the intrinsic fluorescence of NIR797 on predetermined times. Then, the tumor-bearing mice were sacrificed and organs, such as heart, liver, spleen, lungs, kidneys, brain, intestine, stomach, and tumor were collected and imaged to study the tissue distribution of micelles. CT26 tumors were also induced into male BALB/c mice on both sides of flank and the tumor accumulation and distribution of NIR-797-labeled DOXloaded POX-PCL micelles were also observed as above. 2.10. Penetration of micelles. In order to follow the tracks of micelles in tumor tissues, micelles were labeled with FITC and intravenous injected into Ac4ManNAz pretreated mice bearing H22

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and CT26 double tumors, respectively. After 30 hours, tumors were bisected and finely sliced for histological analysis. A solution of DAPI was dropped to stain cell nucleus. After multiple rinses steps, observation of these sections was carried out with a confocal microscope (Zeiss LSM 710). 2.11. DOX accumulation and biodistribution in vivo. To characterize the distribution of micelles in vivo, two tumor-bearing mice models were prepared by subcutaneous injection of H22 tumor cells in both sides of the flank. Ac4ManNAz (50 μl in 50 μM of Ac4ManNAz, 70% aqueous DMSO) and saline as control were intratumoral injected into the right and left tumors for three days, respectively. Free DOX and POX-PCL micelles containing DOX were mixed with 0.01 M PBS and intravenously injected into mice bearing H22 tumor according to 6 mg/kg equivalent DOX doses, respectively. The collected blood samples at the scheduled time were separated by centrifugation at 13000 r/min for 12 min to obtain plasma. Then, the mice were sacrificed, and major organs like heart, liver, spleen, lung, kidney and tumor were collected and weighed respectively. These samples were homogenized with a medium containing 70% ethanol and 0.3 N HCl, After purification by centrifugation, the concentrations of DOX were measured by a fluorescence spectrometer with an excitation wavelength of 480 nm and emission wavelength of 590 nm. 2.12. In vivo antitumor activity of micelles loaded with DOX. Male mice (25~30 g) bearing with H22 tumor were randomized into 5 groups (each group with 10 mice). As the tumor grew to about 50 mm3, Ac4ManNAz (50 μl in 50 μM of Ac4ManNAz, 70% aqueous DMSO) and saline as control were intratumoral injected into the right and left tumors for three days respectively. Then, all groups received only one intravenous injection. DOX-loaded POX-PCL micelles (6 mg/kg eq. Ac4ManNAz+), DOX-loaded POX-PCL micelles (6 mg/kg eq. PBS (Ac4ManNAz-)), PBS (Ac4ManNAz+) and PBS (Ac4ManNAz-), free DOX (6 mg/kg) were administrated into the mice

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by intravenous injection, respectively. The volume of tumor (V) was calculated by formula as followed. V = d2 × D/2, d and D in this formula represent the short and the long diameter of the tumor, respectively. Weight of the mice was measured every two days, and monitoring the survival state of mice in the whole experiment. 2.13. Statistical analysis. Differences in data were compared with Student’s t-test and were not statistically significant if the P values were greater than 0.05.

3. Results and Discussions 3.1. Construction of DOX-loaded reactive POX-PCL micelles. Based on Staudinger ligationbearing POX-PCL amphiphilic block copolymer, DOX-loaded reactive POX-PCL micelles were constructed by molecular self-assembly in water solution. The drug-loading content was measured to be about 7.3% and the drug entrapment efficiency was about 75%. The size and morphology of the DOX-loaded reactive POX-PCL micelles were examined by transmission electron microscopy (TEM) and dynamic light scattering (DLS), as shown in Figure 1A and Figure 1B, respectively. TEM observation shows that the DOX-loaded reactive POX-PCL micelles are spherical with a narrowly distributed diameter of about 35 nm. Meanwhile, the hydrodynamic diameter of the micelles obtained from DLS is about 74 nm and Size-distribution by intensity was also presented in Figure S15. The size measured by DLS is larger than that measured through TEM because of the hydration of the micelles in aqueous solution. Surface charge of the particles in aqueous medium has been measured by Zetaplus. Before and after DOX loading, the surface charge of the micelles was -14.9 mV and -10.5 mV, respectively, indicating that DOX is predominantly located into the particle interior. The release characterization in vitro of DOX from micelles was investigated at 37 °C in 0.01M PBS with pH 5.5 and 7.4, respectively (Figure 1C). As presented

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in Figure 1C, release of the DOX is sustained, pH-dependent and no initial burst. The DOX release at pH 7.4 is significantly slower than that at pH 5.5. Within the monitoring duration, when the pH is 7.4, only 20% of the loaded DOX is released from the nanocarriers. However, with the same period the release of DOX from the micelles increases up to 42% as the pH is 5.5. The pHdependence of release rate should be owing to the enhanced protonation of the amino groups in DOX molecules at pH 5.5 comparing to that at pH 7.4, which increases the solubility of DOX in aqueous medium.39,40,41

Figure 1. A) Typical TEM image of POX-PCL micelles indicates that the average micelles diameter is 35 nm; B) The average hydration diameter of POX-PCL micelles measured by DLS is 74 nm; C) In vitro release profiles of DOX-loaded POX-PCL micelles in the media of 0.01 M PBS with pH 5.5 and 7.4, respectively.

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Figure 2. CLSM images of CT26 cells after incubating with the FITC-labeled DOX-loaded POXPCL micelles at 37 ºC for 4 h, “+” represents the cells treated with Ac4ManNAz; “-” represents the cells untreated with Ac4ManNAz. All the scale bars are 20 μm.

3.2. In vitro targeting effect and cytotoxicity. In order to verify the targeting effect of the DOX-loaded reactive POX-PCL micelles via Staudinger ligation, the Ac4ManNAz-treated colon CT26 cancer cells and untreated CT26 cells were cultured with the FITC-labeled reactive POXPCL micelles containing DOX, respectively, for the same period at 37 ºC. The cell uptake of the micelles in these two types of cells was observed and compared by confocal laser scanning microscopy (CLSM). The green and red signals in the CLSM images are from the fluorescence of FITC and DOX, respectively. As shown in the CLSM images (Figure 2), the cellular uptake of the micelles in Ac4ManNAz-treated CT26 cells is much more than that in the tumor cells without

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Ac4ManNAz treatment. The mean intensity of FITC in Ac4ManNAz-treated CT26 cells is 4.67 folds higher than that in the cells without Ac4ManNAz treatment (Figure S16), suggesting that the azide groups can be successfully decorated on the surfaces of Ac4ManNAz-treated cells and the Staudinger-Bertozzi ligation reaction between the micelles and the cells occurs in vitro. More importantly, this result indicates that the Staudinger ligation between the micelles and the cells facilitates significantly the cell uptake of the functionalized nanocarriers. Flow cytometry was used to further demonstrate the targeting effect of this micelles and the result is presented in Figure 3. As shown in Figure 3, the cell accumulation based on tumor targeting in vitro of the micelles via Staudinger ligation is almost 3 times more compared to that without Staudinger ligation, indicating again enhanced targeting effect of micelles based on Staudinger ligation.

Figure 3. A) Flow cytometry analysis of FITC-labeled DOX-loaded POX-PCL micelles; B) Quantitative data of flow cytometry analysis.

The targeting effect of the DOX-loaded reactive POX-PCL micelles to the Ac4ManNAz-treated cells can also be demonstrated by the cytotoxicity test. First, the viability of CT26 cells incubated

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Figure 4. A) Cytotoxicity of the DOX-loaded POX-PCL micelles against the Ac4ManNAz-treated (blue) and untreated (red) CT26 cells, and DOX against untreated CT26 cells (black) in vitro; B) The half maximal inhibitory concentration (IC50) of the DOX-loaded reactive POX-PCL micelles against the Ac4ManNAz-treated (blue) and untreated (red) CT26 cells, and DOX against untreated CT26 cells (black).

with Ac4ManNAz was tested, and the result shows no cytotoxicity at high concentration after 48 h (Figure S17). The in vitro cytotoxicity of the DOX-loaded reactive POX-PCL micelles against the Ac4ManNAz-treated and untreated CT26 cells was subsequently evaluated with a control of DOX against untreated CT26 cells. The inhibitory rates of cell growth were measured after 48 h culture with different concentrations of DOX and DOX-loaded reactive POX-PCL micelles by MTT method. As presented in Figure 4A, the DOX-loaded POX-PCL micelles exhibits higher cytotoxicity in the Ac4ManNAz-treated tumor cells than that in the untreated cells, but it is still lower than the cytotoxicity of free DOX due to the sustained release of drug from micelles. The half maximal inhibitory concentration (IC50) of the reactive micelles containing DOX for the Ac4ManNAz-treated CT26 cells is about 35.05 μg/mL. In contrast, the IC50 of the micelles for the

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untreated cells is 46.23 μg/mL (Figure 4B). Obviously, the cytotoxicity difference of the micelles in the Ac4ManNAz-treated and untreated CT26 cells is caused by the Staudinger ligation that endows the micelles with targeting ability to the Ac4ManNAz-treated CT26 cell and enhances the cell uptake of micelles.

Figure 5. NIR fluorescence images of the mice bearing CT26 (top) and H22 tumors (bottom), respectively, after intravenous injection of NIR-797-labeled DOX-loaded POX-PCL micelles, and images of Near Infrared fluorescence in vitro from tumors and organs dissected at 60 hour p.i.. The tumors are in both the armpits of the mice with the right tumor treated by Ac4ManNAz.

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3.3. In vivo active tumor targeting efficiency of the DOX-loaded reactive POX-PCL micelles via Staudinger ligation. To evaluate the tumor targeting efficiency of the DOX-loaded reactive POX-PCL micelles via Staudinger ligation in vivo, we established CT26 subcutaneous tumor model in both armpits of mice and injected directly Ac4ManNAz solution into the tumor on the right side to metabolically label the tumor cells with azide groups. We prepared near-infrared dye, NIR-797-labeled DOX-loaded POX-PCL micelles bearing Staudinger ligation reagents and injected the labeled reactive micelles into the mice bearing tumor through tail vein. The administrated mice were imaged at 30 hours and 60 hours post injection (p.i.). As presented in the images (Figure 5), the fluorescence signals from the micelles can be found in the tumor and liver regions. The fluorescence intensity of the signals in the right tumor at 60 h p.i. is much stronger than that at 30 h p.i.. Importantly, at both the time points, the signals in the right tumor are much stronger than that in the left one, confirming that the Staudinger ligation plays a significant role in this tumor targeting therapy, since the right tumor is metabolically labeled with azide groups. At 60 h p.i., the main organs were dissected immediately from the mice and examined by NIR imaging. In the ex vivo images, the strong fluorescence intensity could be found from tumors, liver and spleen, and the fluorescence intensity from the right tumor is very higher than that from the left tumor, which is consistent with the in vivo imaging. We further studied the targeting effect of the micelles by using the mice bearing murine hepatic H22 tumors in both armpits with the right tumor labeled metabolically with azide groups. NIR fluorescence imaging shows the similar result to the case of CT26 tumor models. From the quantitative analysis based on fluorescence intensity of major organs and tumors, it is found that the micelles concentration in metabolically labeled tumor is 1.72 and 1.48 folds higher than unlabeled tumor in CT26 and H22 tumor-bearing mice, respectively (Figure S18). This target-enhanced magnitude based on Staudinger ligation-bearing

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POX-PCL micelles is in well agreement with other bioorthogonal reactions occurred in small or biomacromolecular systems in vivo.11,42,43

Figure 6. A) CLSM images of the CT26 tumor slices dissected at 30 h after intravenous injection of the FITC-labeled DOX-loaded POX-PCL micelles; B) CLSM images of the H22 tumor slices dissected at 30 h after intravenous injection of the FITC-labeled DOX-loaded POX-PCL micelles. “+” represents the tumor treated by Ac4ManNAz and “-” represents untreated tumor. The nucleus of cells was stained by DAPI.

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By the use of CT26 or H22 tumors bearing mice, we further assessed the targeting effect of the reactive micelles by imaging the slices of the tumors dissected at 30 h after intravenous injection of the FITC-labeled micelles through CLSM. As presented in Figure 6, the red signals are from the DOX transported by the micelles, the green signals are from the FITC-labeled micelles and the blue signals are from the nucleus stained by DAPI. It can be seen that both green and red signals in the images of the Ac4ManNAz-treated tumor slices are stronger than those of the untreated tumors for both CT26 (Figure 6A) and H22 (Figure 6B) tumor models. The quantitative analysis of the images shows that for the CT26 tumor models, the intensity of the green signals in the Ac4ManNAz-treated tumor slices is 3.15 times of that in the untreated tumor slices. Whereas, the value is 1.82 for the H22 tumor models (Figure S20). This result is consistent with the in vivo NIR imaging. It can also be seen that some of the signals from DOX do not co-localize with the signals from the micelles, suggesting that part of DOX has been released from the micelles and penetrated deep in tumor tissues. 3.4. Tumor targeting and antitumor effect of DOX-loaded micelles in vivo. The drug delivery efficiency of the tumor targeting reactive POX-PCL micelles in vivo was further investigated by quantitatively measuring the DOX concentrations in major organs. The DOXloaded reactive POX-PCL micelles and free DOX were intravenously injected into the H22 bearing mice in both armpits with the right one labeled metabolically with azide groups. At pre-set time point after injection, the tumors and organs were collected. The concentrations of DOX in different organs were evaluated by fluorescence spectrometry after extraction of DOX from organs homogenates (Figure 7A and B). In the free DOX group, much more drug accumulated in lung and kidney comparing to other tissues, and no significant difference in DOX concentration is found

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Figure 7. A) Biodistributions of DOX in major organs of mice bearing H22 tumor at several time points after intravenous injection of free DOX; B) Biodistributions of DOX in major organs of mice bearing H22 tumor at several time points after intravenous injection of DOX-loaded reactive POX-PCL micelles; C) Concentrations of DOX in plasma at different time points after treatments with free DOX; D) Concentrations of DOX in plasma at different time points after treatments with DOX-loaded POX-PCL micelles. The tumors are in both the armpits of the mice with the right tumor treated by Ac4ManNAz. (0.01 < *P ≤ 0.05; **P ≤ 0.01; P > 0.05 is statistically considered as non-significant and denoted as “n.s.”)

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for azide-labeled and azide-free tumors. In contrast, for the case of the DOX-loaded reactive POXPCL micelles, high DOX concentration is found in the tumor treated. The DOX concentration in the azide-labeled tumors is 1.23, 1.21, 1.22 folders higher than that in the azide-free tumors at 12 h, 24 h, 48 h, respectively, and this comparison has a statistical significance, verifying the targeting effect of the micelles via Staudinger ligation. In addition, high DOX concentration is found in liver and spleen for reactive POX-PCL micelles. The half-life (t1/2) of DOX in DOX-loaded POX-PCL micelles is 3.3 hours (Figure 7D), which is significantly longer than free DOX (t1/2 = 18 min).44 To evaluate the effect of the Staudinger ligation in tumor treatment, we investigated the antitumor efficiency of the DOX-loaded reactive POX-PCL micelles by using the model mice bearing the Ac4ManNAz-treated H22 tumors and untreated H22 tumors, respectively. In this work, 5 groups of the test animals were involved: Group A bearing azide-labeled tumors treated by Staudinger ligation reagent-contained and DOX-loaded POX-PCL micelles; Group B bearing azide-free tumors treated by DOX-loaded POX-PCL micelles without Staudinger ligation reagent; Group C bearing azide-free tumors treated by free DOX; Group D bearing azide-free tumors treated by PBS and group E bearing azide-labeled tumors treated by PBS. Based on the results presented in Figure 8A and B, the Staudinger ligation reagent-labeled DOX-loaded POX-PCL micelles show the best antitumor efficiency and make the mice survival time longest among the three DOX formulations. The rate of tumor growth from all the groups increases in the order of group A < group B < group C < groups D ≈ group E. The tumor growth inhibitions (TGIs) of the group A, B and C on day 15 p.i. are 79.9%, 69.9%, 46.4%, respectively. Survival curve for each groups of mice are presented in Figure 8B. On day 50 after the treatments, the number of the survival mice in the group A, B, C, E and D are 4, 3, 2, 1, 0, respectively. The median survival time for the group A, B, C, E and D is 44, 39, 33, 21 and 19

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Figure 8. A) In vivo tumor growth curves of H22 tumor bearing mice which treated with different methods; B) Kaplan-Meier curves showing survival of tumor-bearing mice in various groups; C) Evolution of body weight of mice receiving different treatments. “+” represents the groups with the tumors treated by Ac4ManNAz and “-” represents the groups with untreated tumors. Data are expressed as mean ± S.D. (n = 10).

days, respectively. Within the 15 days p.i., all test groups were monitored, especially for the weight (Figure 8C) and clinical situation, showing there were no significant differences among different treatments, include the PBS-treated groups, demonstrating the good tolerance of drug imposed on the experimental mice. The good antitumor performance of the Staudinger ligation reagent-

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contained and DOX-loaded POX-PCL micelles is attributable to the Staudinger ligation between the micelles and the azide-labeled tumors, which enhances effectively the antitumor efficiency and tumor accumulation of the DOX-loaded micelles.

4. Conclusions In conclusion, we synthesized a novel amphiphilic POX-PCL block copolymer which had a certain number of amino side groups in the POX block and further functionalized into Staudinger ligation reagent, triarylphosphine groups for active tumor-targeting via Staudinger ligation. By using the block copolymer self-assembly, the Staudinger ligation contained and DOX-loaded reactive micelles were prepared with hydrodynamic diameter of ~74 nm. The investigation on the cytotoxicity, cell uptake, biodistribution of DOX and antitumor properties demonstrated that the Staudinger ligation could indeed significantly enhance the efficiency of tumor targeted therapy of the micelles in vitro and in vivo. As shown in images from NIR observation and slice optical analysis, the tumor accumulation of the micelles in vivo via Staudinger ligation increased 1.5 to 3.2 folds compared to that without Staudinger ligation, leading to enhanced antitumor activity and prolong the lifetime of tumor-bearing mice.

Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxxxxxxxxx. The detailed synthesis and characterization of the involved compounds, and complementary results including cytotoxicity and biodistribution.

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AUTHOR INFORMATION Corresponding Author *Email: [email protected], [email protected]. Author Contributions ‡Peng

Zhang and Xiaoke Zhang contributed equally to this work

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by National Key R&D Program of China (2017YFA0701301 and 2017YFA0205400) and the Natural Science Foundation of China (No. 51690153 and 21720102005), and the Program for Changjiang Scholars and Innovative Research Team in University.

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REFERENCES (1) Qin, L-H.; Hu, W.; Long, Y-Q. Bioorthogonal Chemistry: Optimization and Application Updates During 2013–2017. Tetrahedron Lett. 2018, 59, 2214-2228. (2) Thirumurugan, P.; Matosiuk, D.; Jozwiak, K. Click Chemistry for Drug Development and Diverse Chemical-Biology Applications. Chem. Rev. 2013, 113, 4905-4979. (3) Lang, K.; Chin, J. W. Bioorthogonal Reactions for Labeling Proteins. ACS Chem. Biol. 2014, 9, 16-20. (4) Yoon, H. Y.; Shin, M. L.; Shim, M. K.; Lee, S.; Na, J. H.; Koo, H. Artificial Chemical Reporter Targeting Strategy Using Bioorthogonal Click Reaction for Improving Active-Targeting Efficiency of Tumor. Mol. Pharmaceutics. 2017, 14, 1558-1570. (5) Seitchik, J. L.; Peeler, J. C.; Taylor, M. T.; Blackman, M. L.; Rhoads, T. W.; Cooley, R. B.; Refakis, C.; Fox, J. M.; Mehl, R. A. Genetically Encoded Tetrazine Amino Acid Directs Rapid Site-Specific In Vivo Bioorthogonal Ligation with trans-Cyclooctenes. J. Am. Chem. Soc. 2012, 134, 2898–2901. (6) Wang, H.; Tang, L.; Liu, Y.; Dobrucki, I. T.; Dobrucki, L. W.; Yin, L. C.; Cheng, J. J. In Vivo Targeting of Metabolically Labeled Cancers with Ultra-Small Silica Nanoconjugates. Theranostics. 2016, 6, 1467–1476. (7) Chang, P. V.; Prescher, J. A.; Sletten, E. M., Baskin, J. M.; Miller, I. A.; Agard, N. J.; Lo A.; Bertozzi, C. R. Copper-free Click Chemistry in Living Animals. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 1821–1826. (8) Wang, H.; Gauthier, M.; Kelly, J. R.; Miller, R. J.; Xu, M.; Jr, W. D. O.; Cheng, J. J. Targeted Ultrasound-Assisted Cancer-Selective Chemical Labeling and Subsequent Cancer Imaging using Click Chemistry. Angew. Chem., Int. Ed. 2016, 55, 5452–5456.

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Page 26 of 31

(9) Amin, M. L.; Joo, J. Y.; Yi, D. K.; An, S. S. A. Surface Modification and Local Orientations of Surface Molecules in Nanotherapeutics. J. Controlled Release. 2015, 207, 131–142. (10) Yoon, H. Y.; Koo, H.; Kim, K.; Kwon, I. C. Molecular Imaging Based on Metabolic Glycoengineering and Bioorthogonal Click Chemistry. Biomaterials. 2017, 132, 28–36. (11) Lee, S.; Koo, H.; Na, J. H.; Han, S. J.; Min, H. S.; Lee, S. J.; Kim, S. H.; Yun, S. H.; Jeong, S. Y; Kwon, I. C.; Choi, K.; Kim, K. Chemical Tumor-targeting of Nanoparticles Based on Metabolic Glycoengineering and Click Chemistry. ACS Nano. 2014, 8, 2048–2063. (12) Chinoy, Z. S.; Bodineau, C.; Favre, C.; Moremen, K. W.; Duran, R.V.; Friscourt, F. Selective Engineering of Linkage-Specific α2,6-N-Linked Sialoproteins Using Sydnone-Modified Sialic Acid Bioorthogonal Reporters. Angew. Chem., Int. Ed. 2019, 58, 4281-4285. (13) Hu, F.; Mao, D.; Kenry; Cai, X.; Wu, W.; Kong, D.; Liu, B. A Light-Up Probe with Aggregation-Induced Emission for Real-Time Bio-orthogonal Tumor Labeling and Image-Guided Photodynamic Therapy. Angew. Chem., Int. Ed. 2018, 57, 10182-10186. (14) Chang, P. V.; Prescher, J. A.; Hangauer, M. J.; Bertozzi, C. R. Imaging Cell Surface Glycans with Bioorthogonal Chemical Reporters. J. Am. Chem. Soc. 2007, 129, 8400–8401. (15) Cohen, A. S.; Dubikovskaya, E. A.; Rush, J. S.; Bertozzi, C. R. Real-time Bioluminescence Imaging of Glycans on Living Cells. J. Am. Chem. Soc. 2010, 132, 8563–8565. (16) Schoffelen, S.; Eldijk, M. B.; Rooijakkers, B.; Raijmakers, R.; Heck, A. J. R.; Hest, J. C. M. Metal-free and PH-controlled Introduction of Azides in Proteins. Chem. Sci. 2011, 2, 701–705. (17) Graaf, A. J.; Kooijman, M.; Hennink, W. E.; Mastrobattista, E. Nonnatural Amino Acids for Site-Specific Protein Conjugation. Bioconjugate Chem. 2009, 20, 1281–1295. (18) Prescher, J. A.; Dube, D. H.; Bertozzi, C. R. Chemical Remodelling of Cell Surfaces in Living Animals. Nature. 2004, 430, 873–877.

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(19) Du, L. H.; Qin, H.; Ma, T.; Zhang, T.; Xing, D. In Vivo Imaging-Guided Photothermal/ Photoacoustic Synergistic Therapy with Bioorthogonal Metabolic Glycoengineering Activated Tumor Targeting Nanoparticles. ACS Nano. 2017, 11, 8930–8943. (20) Devadasu, V. R.; Bhardwaj, V.; Ravi, K. M. N. V. Can Controversial Nanotechnology Promise Drug Delivery? Chem. Rev. 2013, 113, 1686–1735. (21) Zhao, M.; Liu, Y.; Hsieh, R. S.; Wang, N.; Tai, W.; Joo, K-I.; Wang, P.; Gu, Z.; Tang, Y. Clickable Protein Nanocapsules for Targeted Delivery of Recombinant p53 Protein. J. Am. Chem. Soc. 2014, 136, 15319–15325. (22) Dhar, S.; Gu, F. X.; Langer, R.; Farokhzad, O. C.; Lippard, S. J. Targeted Delivery of Cisplatin to Prostate Cancer Cells by Aptamer Functionalized Pt(IV) Prodrug-PLGA-PEG Nanoparticles. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 17356–17361. (23) Zhong, Y.; Meng, F.; Deng, C.; Zhong, Z. Ligand-Directed Active Tumor-Targeting Polymeric Nanoparticles for Cancer Chemotherapy. Biomacromolecules. 2014, 15, 1955-1969. (24) Gu, X.; Wei, Y.; Fan, Q.; Sun, H.; Cheng, R.; Zhong, Z.; Deng, C. cRGD-Decorated Biodegradable Polytyrosine Nanoparticles for Robust Encapsulation and Targeted Delivery of Doxorubicin to Colorectal Cancer In Vivo. J. Controlled. Release. 2019, 301, 110-118. (25) Huang, K.; He, Y.; Zhu, Z.; Guo, J.; Wang, G.; Deng, C.; Zhong, Z. Small, Traceable, Endosome-Disrupting, and Bioresponsive Click Nanogels Fabricated via Microfluidics for CD44Targeted Cytoplasmic Delivery of Therapeutic Proteins. ACS Appl. Mater. Interfaces. 2019, 11, 22171-80. (26) Foulkes, W. D.; Smith, I. E.; Reis-Filho, J. S. Triple-Negative Breast Cancer. N. Engl. J. Med. 2010, 363, 1938-1948.

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Page 28 of 31

(27) Cronin, K. A.; Harlan, L. C.; Dodd, K. W.; Abrams, J. S.; Ballard-Barbash, R. Populationbased Estimate of the Prevalence of HER-2 Positive Breast Cancer Tumors for Early Stage Patients in the US. Cancer Invest. 2010, 28, 963-968. (28) Xie, R.; Dong, L.; Huang, R.; Hong, S.; Lei, R.; Chen, X. Targeted Imaging and Proteomic Analysis of Tumor-Associated Glycans in Living Animals. Angew. Chem., Int. Ed. 2014, 53, 14082–14086. (29) Koo, H.; Lee, S.; Na, J. H.; Kim, S. H.; Hahn, S. K.; Choi, K.; Kwon, I. C.; Jeong, S. Y.; Kim, K. Bioorthogonal Copper-Free Click Chemistry In Vivo for Tumor-Targeted Delivery of Nanoparticles. Angew. Chem., Int. Ed. 2012, 51, 11836–11840. (30) Yao, Q.; Lin, F.; Fan, X.; Wang, Y.; Liu, Y.; Liu. Z.; Jiang, X; Chen, P. R.; Gao, Y. Synergistic Enzymatic and Bioorthogonal Reactions for Selective Prodrug Activation in Living Systems. Nat. Commun. 2018, 9, 5032. (31) Vocadlo, D. J.; Hang, H. C.; Kim, E-J.; Hanover, J. A.; Bertozzi, C. R. A Chemical Approach for Identifying O-GlcNAc-modified Proteins in Cells. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 9116-9121. (32) Hangauer, M. J.; Bertozzi, C. R. A FRET-Based Fluorogenic Phosphine for Live-Cell Imaging with the Staudinger Ligation. Angew. Chem., Int. Ed. 2008, 47, 2394-2397. (33) Lorson, T.; Lübtow, M. M., Wegener, E.; Haider, M. S.; Borova, S.; Nahm, D.; Jordan, R.; Sokolski-Papkov, M.; Kabanov, A. V.; Luxenhofer, R. Poly(2-oxazoline)s Based Biomaterials: A Comprehensive and Critical Update, Biomaterials. 2018, 178, 204–280. (34) Grube, M; Leiske, M. N.; Schubert, U. S.; and Nischang, I. POx as an Alternative to PEG? A Hydrodynamic and Light Scattering Study. Macromolecules. 2018, 51, 1905–1916.

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(35) Wyffels, L.; Verbrugghen, T.; Monnery, B. D.; Glassner, M.; Stroobants, S.; Hoogenboom, R.; Staelens, S. μPET Imaging of the Pharmacokinetic Behavior of Medium and High Molar Mass 89Zr-Labeled Poly(2-ethyl-2-oxazoline) in Comparison to Poly(ethylene glycol). J. Controlled. Release. 2016, 235, 63–71. (36) Kronek, J.; Kroneková, Z.; Lustoň, J.; Paulovičová, E.; Paulovičová, L.; Mendrek, B. In Vitro Bio-immunological and Cytotoxicity Studies of Poly(2-oxazolines). J. Mater. Sci.: Mater. Med. 2011, 22, 1725–1734. (37) Luxenhofer, R.; Sahay, G.; Schulz, A.; Alakhova, D.; Bronich, T. K.; Jordan, R.; Kabanov, A. V. Structure-property Relationship in Cytotoxicity and Cell Uptake of Poly(2-oxazoline) Amphiphiles. J. Controlled. Release. 2011, 153, 73–82. (38) He, Z.; Wan, X.; Schulz, A.; Bludau, H.; Dobrovolskaia, M. A.; Stern, S. T.; Montgomery, S. A.; Yuan, H.; Li, Z.; Alakhova, D.; Sokolsky, M.; Darr, D. B.; Perou, C. M.; Jordan, R.; Luxenhofer, R.; Kabanov, A. V. A High Capacity Polymeric Micelle of Paclitaxel: Implication of High Dose Drug Therapy to Safety and In Vivo Anti-cancer Activity. Biomaterials. 2016, 101, 296–309. (39) Guo, Y.; Chu, M.; Tan, S.; Zhao, S.; Liu, H.; Otieno, B. O.; Yang, X.; Xu, C.; Zhang, Z. Chitosan-g-TPGS Nanoparticles for Anticancer Drug Delivery and Overcoming Multidrug Resistance. Mol. Pharmaceutics. 2014, 11, 59-70. (40) Guo, X.; Shi, C.; Yang, G.; Wang, J.; Cai, Z.; Zhou, S. Dual-Responsive Polymer Micelles for Target-Cell-Specific Anticancer Drug Delivery. Chem. Mater. 2014, 26, 4405-4418. (41) Wang, L.; Zhang, J.; Song, M.; Tian, B.; Li, K.; Liang, Y.; Han, J.; Wu, Z. A ShellCrosslinked Polymeric Micelle System for PH/Redox Dual Stimuli-Triggered DOX on-demand Release and Enhanced Antitumor Activity. Colloids Surf., B. 2017, 152, 1-11.

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(42) Wang, H.; Wang, R.; Cai, K.; He, H.; Liu, Y.; Yen, J.; Cheng, J. J. Selective In Vivo Metabolic Cell-Labeling-Mediated Cancer Targeting. Nat. Chem. Biol. 2017, 13, 415-424. (43) Lee, S.; Jung, S.; Koo, H.; Na, J. H.; Yoon, H. Y.; Shim; M. K. Nano-Sized Metabolic Precursors for Heterogeneous Tumor-targeting Strategy Using Bioorthogonal Click Chemistry In Vivo. Biomaterials. 2017, 148, 1-15. (44) Yang, C. C.; Wang, X.; Yao, X. K.; Zhang, Y.; Wu, W.; Jiang, X. Q. Hyaluronic Acid Nanogels with Enzyme-Sensitive Cross-Linking Group for Drug Delivery. J. Controlled. Release. 2016, 235, 63–71.

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