Multifunctional Click Hyaluronic Acid Nanogels for Targeted Protein

Nov 16, 2016 - Protein therapeutics offer a most effective treatment for many human diseases, including diabetes, cardiovascular diseases, and maligna...
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Multifunctional Click Hyaluronic Acid Nanogels for Targeted Protein Delivery and Effective Cancer Treatment In Vivo Jing Chen, Yan Zou, Chao Deng, Fenghua Meng, Jian Zhang, and Zhiyuan Zhong Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.6b04404 • Publication Date (Web): 16 Nov 2016 Downloaded from http://pubs.acs.org on November 19, 2016

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Multifunctional Click Hyaluronic Acid Nanogels for Targeted Protein Delivery and Effective Cancer Treatment In Vivo Jing Chen, Yan Zou, Chao Deng*, Fenghua Meng, Jian Zhang, and Zhiyuan Zhong*

Biomedical Polymers Laboratory, and Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, People’s Republic of China.

*Corresponding authors. Tel./Fax: +86-512-65880098. E-mail: [email protected] (C. Deng), [email protected] (Z. Zhong).

ABSTRACT: Protein therapeutics offers a most effective treatment for many human diseases including

diabetes,

cardiovascular diseases,

and

malignant tumors.

Unlike

most

chemotherapeutics that often cause notorious side effects, many protein drugs possess a high specificity and reduced systemic toxicity. Notably, clinically used protein drugs are mostly limited to those taking effects extracellularly. Protein drugs having intracellular targets though represent a big family of protein biologics have not come to the clinics, due to absence of translatable intracellular protein delivery vehicles. Here we report efficient and targeted cancer protein therapy in vivo by bioresponsive fluorescent photo-click hyaluronic acid (HA) nanogels. Two intracellular protein drugs, cytochrome C (CC) and granzyme B (GrB), are loaded into the nanogels with preserved bioactivity. CC and GrB-loaded HA nanogels can effectively target and release proteins to CD44 positive MCF-7 and A549 cancer cells, yielding striking antitumor effects with a half-maximal inhibitory concentration thousands times lower than clinical chemotherapeutics. Remarkably, GrB-loaded HA nanogels at a low dose of 3.8-5.7 nmol GrB equiv./kg exhibit complete suppression of tumor growth and minimal adverse effects in nude mice bearing subcutaneous MCF-7 human breast tumor and orthotopic A549 human lung tumor xenografts. 1

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INTRODUCTION The rapidly advancing protein biotechnology has led to the discovery and development of numerous therapeutic proteins,1, 2 which have emerged as novel, more specific and effective, as well as low toxic alternatives to small-molecule drugs for various diseases like diabetes, cardiovascular diseases, and malignant tumors.3-7 Several therapeutic proteins like Cetuximab, Panitumumab, Trastuzumab, and L-asparaginase have been approved for the treatment of acute leukemia and various solid tumors.4, 8 It is surprising to note that clinically used protein drugs are limited to those taking effects extracellularly. Ying and Gu recently reported that protein nanotherapeutics can further improve the blood circulation time and therapeutic efficacy of extracellularly active proteins.5, 9 Protein drugs having intracellular targets though represent a big family of protein biologics have not advanced to the clinical stages, due to lack of a viable delivery technology.10, polymersomes,15-17 nanogels,10,

18-24

11

Various nanocarriers like liposomes,12-14

and nanoparticles25-30 have been investigated for

improved intracellular protein delivery. However, few of them have been applied in vivo because systemic delivery of proteins is much more challenging and has to deal with not only intracellular barriers but also extracellular barriers (including in vivo stability, protein degradation, tumor accumulation, etc.).31-33 In this study, we have designed and developed bioresponsive fluorescent hyaluronic acid (HA) nanogels (NGs) for facile encapsulation and active tumor-targeting intracellular delivery of apoptotic proteins including CC and GrB in vivo (Figure 1a). NGs are crosslinked hydrogel particles and have high water content, excellent biocompatibility, and abundant interior space, which makes them as attractive nanovehicles for protein loading and delivery.34-37 Kurisawa et al. reported that HA-green tea catechin NGs mediated targeted delivery of GrB to HCT-66 colon cancer cells in vitro.22 Here, HA NGs were readily prepared from

two

HA

HA-lysine-tetrazole

derivatives,

i.e.

(HA-Lys-Tet),

HA-cystamine-methacrylate by

combing

inverse

(HA-Cys-MA)

and

nanoprecipitation

and

“tetrazole-alkene” photo-click reaction (Scheme S3). Notably, photo-click reaction has shown a high specificity and efficiency and involves no any catalyst.38, 39 It has been applied to selectively label proteins in live mammalian cells.40 HA is a biodegradable and biocompatible natural material and show intrinsic targetability to CD44 positive malignant cancer cells such as human breast and lung tumor cells (MCF-7, A549), human multiple myeloma (LP1), and acute myelogenous leukemia (ALM2).41-43 Here, MA groups are conjugated to HA via a cystamine linker that endows the NGs with bioresponsivity. It is known that there exists a high redox potential in the tumor cells that can act as an internal stimulus to trigger 2

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intracellular drug release following the cleavage of disulfide bonds.44-48 It should further be noted that thus formed NGs possess intrinsic fluorescence derived from the pyrazoline cycloadducts,38 which would allow facile monitoring of NG trafficking and fate in vitro and in vivo.

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100 10 mM GSH

80 60 40

No GSH

20 0

0

10

20 30 Time (h)

40

50

Figure 1. Characterization of hyaluronic acid (HA) nanogels (NGs). a) Schematic illustration of bioresponsive fluorescent HA NGs prepared by combining inverse nanoprecipitation and catalyst-free photo-click reaction for facile encapsulation and active tumor-targeting intracellular delivery of apoptotic proteins in vivo. b) Size distribution of HA NGs obtained 3

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by DLS and TEM. c) Fluorescence spectra of HA nanodroplets before and after crosslinking at an excitation wavelength at 405 nm (the insert shows the fluorescent image of HA NGs). d) Release of CC from HA NGs at pH 7.4 and 37 °C in PB (n= 3).

RESULTS AND DISCUSSION Formation and Reduction-Sensitivity of Fluorescent HA NGs. HA NGs were fabricated by slowly adding an aqueous solution of HA-Cys-MA and HA-Lys-Tet conjugates into acetone, followed by UV irradiation. Notably, the nanodroplets dispersed in the mixed homogeneous solution (aqueous phase and organic phase) before crosslinking exhibited a small size of ca.98 nm with a low polydispersity (PDI) of 0.09 (Figure S3). HA NGs formed at a prepolymer concentration of 1.25 mg/mL following work-up displayed an average diameter of 150 nm and narrow PDI of 0.10 in water (Figure 1b). TEM image demonstrated that HA NGs possessed a spherical morphology and a size distribution close to that from DLS (Figure 1b). As expected, HA NGs had a negative surface charge of -17.6 mV (Table S1). Notably, they exhibited strong fluorescence with an emission wavelength of 450 nm (excitation 405 nm) (Figure 1c), similar to a previous report.38 As a result, they exhibited excellent stability with little size change both in PB (pH 7.4) for over one week and in 10% fetal calf serum (FBS) for 24 h at 37 oC (Figure S4a). These photo-click nanogels have better stability than physically crosslinked HA nanosystems.22, 49 The highly specific click reaction would also circumvent cross-reaction with encapsulated protein drugs, which are often reported for other chemically crosslinked HA nanocarriers.50 Significant swelling and aggregation, however, was observed in 8 h under 10 mM glutathione (GSH) condition (Figure S4b), corroborating that these HA NGs are highly sensitive to reduction. Loading and Release of CC from HA NGs. CC could readily be loaded into HA NGs with a decent loading efficiency of 89.2% at a theoretical loading content of 10 wt.%. CC-loaded NGs (CC-NGs) had a small size of 163 nm, a narrow PDI of 0.10, and a negative surface charge of -15.3 mV (Table S2). Notably, loading of CC was nearly quantitative (loading efficiency > 99 %) at a low theoretical loading content of 2 wt.%. The in vitro release studies exhibited that while protein release was inhibited (ca. 30 % release in 48 h) under a physiological condition (pH 7.4 and 37 oC), over 80% of CC was released in 10 h under 10 mM GSH condition (Figure 1d), indicating that protein release can be triggered under cytoplasmic reductive environment. The 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) assay and circular dichroism (CD) measurement revealed that released CC maintained its enzymatic bioactivity and secondary structure (Figures S5 and S6). 4

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CD44-Targetability and Apoptotic Activity. The cellular uptake behavior of HA NGs was explored using confocal laser scanning microscopy (CLSM). CLSM images show strong NG fluorescence in the cytoplasm of MCF-7 cells after 4 h incubation, supporting efficient internalization of HA NGs into MCF-7 cells (Figure 2a). In contrast, negligible NG fluorescence is observed in CD44 negative U87 cells under otherwise the same conditions. Moreover, pretreating MCF-7 cells with free HA before incubating with HA NGs significantly reduced HA NGs fluorescence. It is evident, therefore, that HA NGs can actively target to CD44-positive cancer cells. Figure S7 further shows that NGs could escape from endosomes of MCF-7 cells following 12 h incubation. MTT assays revealed that blank NGs were nontoxic towards all tested cells (i.e. L929, MCF-7, A549 and U87 cells) at a NG concentration of 800 µg/mL (Figure S8), corroborating that HA NGs have good biocompatibility.

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100 80 60 40 20 0

free GrB, MCF-7 GrB-NGs, MCF-7 GrB-NGs, A549

0.01 0.1 1 10 GrB concentration (nM)

Figure 2. Cellular uptake and MTT assays of HA NGs. a) CLSM images of cells following 8 h incubation with fluorescent HA NGs (green, 200 µg/mL). The nuclei were stained with DAPI (blue) and the scale bars correspond to 10 µm. b) Antitumor activity of CC-NGs in MCF-7 and U87 cells (4 h incubation with CC-NGs followed by 92 h culture in fresh media). c) Antitumor activity of GrB-NGs in MCF-7 and A549 cells. For the inhibitive experiments, CD44 receptors in cells were blocked by 4 h incubating with 5 mg/mL free HA. Data are presented as the average ± SD (n = 4).

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The antitumor activity of protein-loaded HA NGs was evaluated by MTT assays in CD44 receptor overexpressing MCF-7 and A549 cells.43, 51 CC, known to induce programmed cell death,52 was selected as a first model protein drug. The results showed that CC-NGs exhibited a significantly enhanced antitumor activity with a low half-maximal inhibitory concentration (IC50) of 0.52 µM, while free CC was practically nontoxic even at a high concentration of 6.2 µM due to poor cell internalization (Figure 2b). The receptor blocking experiments showed that incubating MCF-7 cells with free HA before treating with CC-NGs led to markedly reduced apoptosis of MCF-7 cells. Moreover, CC-NGs displayed also low apoptotic activity in CD44 negative U87 cancer cells. These results confirm that HA NGs are uptaken by MCF-7 cells through CD44 receptor-mediated endocytosis. In the following, we investigated the delivery of GrB that is reported to be a highly potent apoptosis mediator, and can cause the cleavage, activation, or inactivation of multiple protein substrates in the cytosol.22, 53 Strikingly, in contrast to minimal cytotoxicity of free GrB, GrB-NGs were highly potent with IC50 as low as 3.0 nM to MCF-7 cells (Figure 2c), which is over ninety thousand times lower than that of Doxil® (2.85×105 nM),54, 55 a widely used chemotherapeutics in the clinic. It should further be noted that GrB-NGs exhibited also a high cytotoxicity against A549 lung cancer cells with a remarkably low IC50 of 8.1 nM. The high potency of CC-NGs and GrB-NGs to CD44+ MCF-7 and A549 cells but not to CD44- U87 cells, demonstrating that HA-induced uptake of nanogels is directly related to the interaction between HA and CD44 receptors on the cell surface. To evaluate their pharmacokinetics and biodistribution, HA NGs were labeled with Cy5, a near-infrared (NIR) fluorescent dye. Notably, HA NGs displayed a long circulation time with the distribution and elimination half-lives of 0.45 and 4.60 h in mice, respectively (Figure S9). The in vivo biodistribution studies in MCF-7 tumor-bearing nude mice revealed a remarkable tumor accumulation of 8.12 %ID/g at 6 h post i.v. injection of Cy5-HA NGs, which was similar to liver but significantly higher than heart, spleen, lung and kidney (Figure S10), signifying that HA NGs can efficiently target to and accumulate in MCF-7 tumor. The high tumor accumulation of HA NGs is likely attributed to their active targeting property which would not only enhance tumor cell uptake but also tumor retention.31, 56 In Vivo Therapeutic Efficacy of GrB-Loaded HA NGs. MCF-7 human breast tumor-bearing nude mice with tumor volume of about 30 mm3 were used as a model. Mice were i.v. injected with GrB-NGs every three days (on day 0, 3, 6, 9) at a dosage of 0.95 or 3.8 nmol GrB equiv./kg. Interestingly, tumor growth was effectively inhibited by GrB-NGs (Figure 3a). The mice treated with 3.8 nmol GrB equiv./kg showed complete tumor 6

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suppression in 14 days, in line with the high efficacy and specificity of protein therapeutics. It should be noted that to effectively suppress tumor growth over one thousand times higher dosage (>5.8 µmol/kg) is required for chemotherapeutics like platinum, docetaxel, paclitaxel, and doxorubicin.57-61 On the contrary, fast tumor progression was discerned for control groups receiving blank NGs and PBS. The photographs of tumors excised on day 14 further corroborated efficacious treatment of MCF-7 human breast tumor by GrB-NGs (Figure 3b). Notably, mice treated with GrB-NGs, as for those with PBS and blank NGs, had little change in body weights (Figure 3c), indicating that GrB-NGs have little systemic toxicity. TUNEL assays displayed that mice treated with GrB-NGs especially at 3.8 nmol GrB equiv./kg exhibited significant tumor cell apoptosis (Figure 3d). The histological analyses revealed that GrB-NGs caused substantial tumor cell necrosis with negligible damage to the liver, heart and kidney (Figure 3d). Inefficient intratumoral penetration of nanomedicines is a limiting factor for their effective tumor therapy.62-64 Taking advantages of their intrinsic fluorescence, the penetration and distribution of HA NGs in MCF-7 tumor sections was observed using CLSM. The results showed that HA NGs while co-localized with blood vessels at 1 h post injection clearly moved away from the blood vessels and into the tumor in 6 and 24 h (Figure 3e). Intriguingly, HA NGs spread throughout the whole tumor at 24 h post injection, indicating that they possess remarkable tumor penetration ability in human MCF-7 tumor xenografts.

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Blank NGs

b

*** ***

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a Relative tumor volume (V/V0)

heart

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0.95 nmol/kg GrB-NGs Blank NGs

PBS

Figure 3. In vivo antitumor performance of GrB-NGs in subcutaneous human breast tumor xenografts. a) Tumor volume changes of mice injected with PBS, blank NGs or GrB-NGs on day 0, 3, 6 and 9 (dosage: 0.95 and 3.8 nmol GrB equiv./kg) (n= 4). b) Photographs of tumors excised on day 14. c) Body weight changes in 14 d. d) TUNEL assays and H&E staining of tumor, liver, heart, and kidney excised on day 14. The images were obtained by a Leica microscope at high magnification (400 x). e) Immunofluorescence staining of tumor at 1, 6 and 24 h post-injection. The red and green fluorescences indicate tumor blood vessels and HA NGs, respectively. The scale bar represents 50 µm. Lung cancer is a leading cause of morbidity and mortality in the world.65, 66 Here we studied the therapeutic efficacy of GrB-NGs in orthotopic human A549-luciferase (A549-Luc) lung xenografts in nude mice. When the luminescence intensity of lung tumor reaching about 2 × 104 p/s/cm2/sr, mice were treated with GrB-NGs every three days (on day 0, 3, 6, 9) at a dosage of 5.7 nmol GrB equiv./kg. The results showed that GrB-NGs group had significantly weaker tumor luminescence than blank NG and PBS groups (Figure 4a). The semi-quantitative analysis of radiance revealed that GrB-NGs caused ca. 6-fold reduction of tumor luminescence as compared to blank NGs and PBS (Figure 4b). The images of lung blocks isolated on day 15 clearly showed that GrB-NGs group had the least luminescence and 8

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tumor invasion (Figure 4c). Importantly, in contrast to blank NGs and PBS that led to gradual body weight loss, likely as a result of tumor invasion to the lung, GrB-NGs caused little change of body weights (Figure 4d), corroborating that GrB-NGs have a high treatment efficacy and little adverse effects. The histological analysis indicated that GrB-NGs group had well-organized lung tissue with single-cell-layer alveoli (Figure 4e), supporting effective suppression of tumor growth in the lung. In contrast, great lung damage including disrupted alveolar structure was observed in the blank NGs and PBS groups. Moreover, GrB-NGs group exhibited little liver, heart and kidney damage, further confirming that GrB-NGs have little nonspecific side effects. Remarkably, survival curves exhibited a 100% survival rate for GrB-NGs group within an experimental period of 40 days (Figure 4f). In comparison, control groups with PBS and blank NGs showed significantly shorter survival times (median survival times: 20 and 24 days). 9

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Figure 4. In vivo antitumor performance of GrB-NGs in orthotopic human lung tumor xenografts. a) Tumor volume change evaluated by luminescence optical imaging in mice treated with GrB-NGs, blank NGs, and PBS. b) Quantified average luminescence levels of mice treated with GrB-NGs, blank NGs, and PBS. The in vivo luminescent images were normalized and reported as photons per second per centimeter squared per steradian (p/s/cm2/sr). c) Ex vivo imaging of lung blocks excised on day 16. d) Body weight changes of mice in 15 d. e) H&E staining of lung, heart, liver and kidney sections excised from tumor-bearing mice following 15 d treatment with GrB-NGs (5.7 nmol GrB equiv./kg), blank NGs or PBS. The images were obtained by a Leica microscope at high magnification (400 x). f) Survival rates of mice in 40 d.

CONCLUSIONS In summary, our results show that reduction-sensitive fluorescent click HA NGs mediate selective and potent delivery of therapeutic proteins in vitro and in vivo. CC and GrB-loaded HA NGs can efficiently target and release proteins to CD44 overexpressing MCF-7 and A549 cancer cells, yielding striking antitumor effects with a half-maximal inhibitory concentration thousands times lower than clinical chemotherapeutics. Notably, GrB-loaded HA NGs at a low dose of 3.8-5.7 nmol GrB equiv./kg exhibit complete suppression of tumor growth and minimal adverse effects in nude mice bearing subcutaneous MCF-7 human breast tumor and orthotopic A549 human lung tumor xenografts. This represents a promising nanoplatform for targeted, safe, and efficient delivery of intracellular anticancer protein therapeutics.

EXPERIMENTAL SECTION Synthesis of HA-Lys-Tet. HA-Lys-NH2 and tetrazole (Tet) were synthesized according to previous reports.39, 43 HA-Lys-Tet was prepared by conjugating Tet to HA-Lys-NH2 using 1,3-dicyclohexyl carbodiimide (DCC, 99%, Alfa Aesar) and N-hydroxysuccinimide (NHS, 98%, J&K) as coupling agents (Scheme S1). In a typical procedure, DCC (232 mg, 1.12 mmol) was added into Tet (608 mg, 58 µmol) in DMSO (10.0 mL) under a nitrogen atmosphere. The reaction proceeded for 12 h at room temperature (r.t.) in the dark. Then, HA-Lys-NH2 (400 mg, 195 µmol amino group) in 30.0 mL anhydrous formamide and 4-dimethylamino pyridine (DMAP, 80 mg, 655 µmol) were added. The reaction continued for 48 h at 40 ºC. HA-Lys-Tet was obtained by dialysis (Spectra/Pore, MWCO 3500) in D.I. water/DMSO (1:1, v/v) and D.I. water followed by freeze-drying. Yield: 69%. 1H NMR (400 MHz, D2O/DMSO-d6, δ, Figure S1): HA: 1.82, 2.70–3.68, and 4.23–4.38; Lys: 0.92, 1.06, 10

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1.52, 2.97, 3.61 and 3.95; Tet: 7.91, 7.92 and 6.79, 6.80. The degree of substitution (DS) was determined to be 3.9 by comparing the integrals of signals at δ 6.79, 6.80 (aromatic proton in Tet) with that at δ 1.82 (methyl proton in HA). Synthesis of HA-Cys-MA. HA-Cys-MA was synthesized in two steps (Scheme S2). Firstly, MA-Cys-NH2 was synthesized by the reaction of Nα-Boc-cystamine (Boc-Cys-NH2) with methacrylic anhydride followed by the deprotection of Boc groups in TFA/ methanol (1:1, v/v) mixed solution, as previously reported.67 Under a nitrogen atmosphere, HA (50 mg, 1.43 µmol) solution in D.I. water (5 mL) activated by EDC (75.9 mg, 0.396 mmol) and NHS (22.8 mg, 0.198 mmol) was slowly added to MA-Cys-NH2 (14.5 mg, 66 µmol) solution. The reaction continued at 40 ºC for 24 h in the dark. HA-Cys-MA was obtained by dialysis (Spectra/Pore, MWCO 3500) in water/methanol (1:1, v/v) and D.I. water followed by freeze-drying. Yield: 92%. 1H NMR (400 MHz, D2O, δ, Figure S2): HA: 2.00, 2.86–3.88, and 4.44–4.52; Cys: 2.70, 3.11–3.15 and 3.56; MA: 1.92, 5.45 and 5.69. DS of MA was determined to be 3.6 by comparing the integrals of signals at δ 5.69 and 5.45 (vinyl proton in MA) with that at δ 4.44–4.52 (anomeric proton in HA). Preparation of Reduction-Sensitive Fluorescent HA NGs. Fluorescent HA NGs were prepared by combing inverse nanoprecipitation and catalyst-free “tetrazole-alkene” photo-click reaction. Typically, HA-Lys-Tet and HA-Cys-MA (1/1, mol/mol) were dissolved in phosphate buffer (pH 7.4, 10 mM) with a total polymer concentration of 1.25 mg/mL. Then, 1 mL of well mixed solution was injected into acetone solution (100 mL) via a syringe to form a homogeneous solution with a thickness of less than 0.45 cm in a big petri dish. After irradiation with UV (320-390 nm, 50 mW/cm2) for 3 min, the solvent was evaporated to obtain HA nanogel dispersion in water. NGs were obtained by dialysis (Spectra/Pore, MWCO 3500) in D.I. water and freeze-drying. Yield: 89%. For preparation of CC-loaded HA NGs (CC-NGs), 1 mL mixed solution of a certain amount of CC and HA derivatives was added into 100 mL acetone, and the following steps were performed similar to the preparation of blank NGs. The size of NGs was evaluated by dynamic light scattering (DLS, Zetasizer Nano-ZS, Malvern Instruments). Transmission electron microscopy (TEM) was conducted on a Tecnai G220 TEM (accelerating voltage of 200 kV). 10 µL of 0.2 mg/mL nanoparticle dispersion was dropped on the copper grid followed by staining with phosphotungstic acid (1 wt.%). In Vivo Antitumor Efficacy. Mice bearing subcutaneous MCF-7 human breast tumor with tumor volumes of about 30 mm3 were arbitrarily divided to four groups (n = 4), and treated with intravenous injection of GrB-NGs at concentrations of 25 µg GrB equiv./kg and 100 µg 11

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GrB equiv./kg in 200 µL of PBS via tail vein every three day. PBS saline and blank NGs were included as the negative controls. The tumor size and body weight were monitored every other day. The tumor volume was estimated according to the formula: Volume = ½*a*b*c, wherein a, b and c are the tumor dimension at the longest, widest, highest points, respectively. Relative tumor volumes were calculated as V/V0 (V0 is the tumor volume when the treatment was initiated). Mice were weighed with the relative body weights normalized to their initial weights. Orthotopic human lung tumor xenografts were established by inoculating A549 cells (1 × 7

10 cells per mouse) to the lung of nude mice. When luminescence intensity of lung grew up to about 20000 p/s/cm2/sr, A549 lung orthotopic tumor-bearing nude mice were arbitrarily divided to three groups (n = 6), and treated with intravenous injection of GrB-NGs at concentrations of 150 µg GrB equiv./kg in 200 µL of PBS via tail vein every three day. PBS saline and blank NGs were included as the negative controls. The tumor growth and body weight were monitored every three day. The tumor growth was calculated according to the luminescence intensity of lung. Mice were weighed with the relative body weights normalized to their initial weights.

ASSOCIATED CONTENT Supporting Information The supporting information is available free of charge on the ACS Publications website. Materials, in vitro release of CC, evaluation of enzymatic activity of released CC, confocal microscopy and flow cytometry measurements, in vitro cytotoxicity assays, pharmacokinetics and biodistribution, tumor penetration analysis, and histological analysis as well as additional schemes, figures, and tables as described in the text (PDF).

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (C. Deng). *E-mail: [email protected] (Z. Zhong). Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS 12

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This work was supported by the National Natural Science Foundation of China (NSFC 51273137, 51273139, 51473110 and 51225302). Z.Z. thanks the Friedrich Wilhelm Bessel Research Award from the Alexander von Humboldt Foundation.

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Multifunctional Click Hyaluronic Acid Nanogels for Targeted Protein Delivery and Effective Cancer Treatment In Vivo

Jing Chen, Yan Zou, Chao Deng*, Fenghua Meng, Jian Zhang, and Zhiyuan Zhong*

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