EGFR and CD44 Dual-Targeted Multifunctional Hyaluronic Acid

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EGFR and CD44 Dual-Targeted Multifunctional Hyaluronic Acid Nanogels Boost Protein Delivery to Ovarian and Breast Cancers in Vitro and in Vivo Jing Chen, Jia Ouyang, Qijun Chen, Chao Deng, Fenghua Meng, Jian Zhang, Ru Cheng, Qing Lan, and Zhiyuan Zhong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06879 • Publication Date (Web): 04 Jul 2017 Downloaded from http://pubs.acs.org on July 6, 2017

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ACS Applied Materials & Interfaces

EGFR and CD44 Dual-Targeted Multifunctional Hyaluronic Acid Nanogels Boost Protein Delivery to Ovarian and Breast Cancers in Vitro and in Vivo Jing Chen1, Jia Ouyang2, Qijun Chen1, Chao Deng*,1, Fenghua Meng1, Jian Zhang1, Ru Cheng1, Qing Lan2, and Zhiyuan Zhong1,* 1

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. 2

Department of Neurosurgery, The Second Affiliated Hospital of Soochow University,

Suzhou, 215004, People’s Republic of China.

ABSTRACT Protein drugs with intracellular targets like Granzyme B (GrB) have demonstrated great proliferative inhibition activity in cancer cells. Their clinical translation, however, relies on development of safe, efficient and selective protein delivery vehicles. Here, we report that EGFR and CD44 dual-targeted multifunctional hyaluronic acid nanogels (EGFR/CD44-NGs) boost protein delivery to ovarian and breast cancers in vitro and in vivo. EGFR/CD44-NGs obtained via nanoprecipitation and photoclick chemistry from hyaluronic acid derivatives with tetrazole, GE11 peptide/tetrazole and cystamine-methacrylate groups had nearly quantitative loading of therapeutic proteins like cytochrome C (CC) and GrB, a small size of ca. 165 nm, excellent stability in serum, and fast protein release under a reductive condition. Flow cytometry assays showed that EGFR/CD44-NGs exhibited over 6-fold better uptake in CD44 and EGFR-positive SKOV-3 ovarian cancer cells than CD44-NGs. In accordance, GrB-loaded EGFR/CD44-NGs (GrB-EGFR/CD44-NGs) displayed enhanced caspase activity and growth inhibition in SKOV-3 cells as compared to GrB-loaded CD44-NGs 1

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(GrB-CD44-NGs) control. Intriguingly, the therapeutic studies in SKOV-3 human ovarian carcinoma and MDA-MB-231 human breast tumor xenografted in nude mice revealed that GrB-EGFR/CD44-NGs at a low dose of 3.85 nmol GrB equiv./kg induced nearly complete growth suppression of both tumors, which was obviously more effective than GrB-CD44-NGs, without causing any adverse effects. EGFR and CD44 dual-targeted multifunctional hyaluronic acid nanogels have appeared as a safe and efficacious platform for cancer protein therapy. KEYWORDS: dual-targeting, nanogels, redox-sensitive, protein delivery, cancer therapy

1. INTRODUCTION Protein drugs with intracellular targets like Granzyme B (GrB) represent an interesting class of biologics that have demonstrated great proliferative inhibition activity in various cancer cells.1-3 The clinical translation of protein therapeutics is, however, limited by their low plasma stability, scarce cellular uptake and fast degradation in the endo/lysosomes of cells.2,

4

In the past years, various nanocarriers like liposomes, nanoparticles, and

polymersomes have been developed to transport and release proteins into target cells.5-12 Nanogels (NGs) with a watery environment, high protein loading capacity, and excellent protein compatibility are particularly appealing for protein delivery.13-17 For example, pH-sensitive NGs have been designed to release proteins like cytochrome C (CC), asparaginase, antigen, and α-glucosidase in the endo/lysosomal compartments of target cells.18-22 Enzyme-responsive NGs crosslinked via furin-degradable peptide demonstrated intracellular triggered release of proteins (caspase-3, bovine serum albumin, and transcription factor Klf4), in which caspase-3-loaded NGs caused obvious apoptosis of HeLa cells.23 Taking advantage of high reducing potential in tumor cells, reduction-sensitive NGs have been explored for intracellular protein delivery. For example, Van Nostrum and coworkers

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found that tether of antigen (ovalbumin) in dextran NGs via disulfide bonds efficiently released ovalbumin into dendritic cells, achieving a strong curative efficacy toward melanoma in vivo.24, 25 We recently reported that redox-sensitive hyaluronic acid (HA) NGs obtained by combining nanoprecipitation and photoclick reaction accomplished triggered intracellular release of therapeutic proteins like CC and GrB.26, 27 In recent years, dual-targeted nanotherapeutics that selectively target to two different cancer biomarkers presented in the tumor angiogenic blood vessels, tumor microenvironment, and/or tumor cells have been explored to achieve synergistic targeting effect. For example, Chen et al. reported that angiogenic blood vessel and heparan sulfate proteoglycan dual-targeted nanoparticulate paclitaxel afforded enhanced uptake in HUVEC cells as well as improved glioma penetration and anti-glioma efficacy in vivo.28 Neovasculature and tumor cell dual-targeted nanotherapeutics was reported to achieve enhanced therapeutic effect in both U87 glioblastoma and highly invasive breast cancer compared to mono-targeted counterparts.29, 30 Several dual-targeted nanosystems addressing two different receptors on tumor cells such as CD44/αvβ3, α5β1/α6β4, and CD44/folate receptors, have been developed to boost specificity and uptake in B16F10 and SKOV-3 cells, respectively.31-33 CD44/αvβ3 dual-targeted nanoparticles following the loading of docetaxel exhibited enhanced therapeutic efficacy in both subcutaneous B16F10 melanoma and metastatic lung tumors.31 It should be noted that no such dual-targeted nanosystem has been reported for delivery of protein therapeutics. Here, we report on EGFR and CD44 dual-targeted multifunctional hyaluronic acid nanogels (EGFR/CD44-NGs) for enhanced delivery of therapeutic proteins to SKOV-3 human ovarian carcinoma and MDA-MB-231 human breast tumor xenografts in mice (Figure 1). EGFR/CD44-NGs were readily prepared from hyaluronic acids grafted with tetrazole (HA-g-Tet), GE11 peptide and tetrazole (HA-g-GE11/Tet), and cystamine-methacrylamide

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(HA-g-Cys-MA), by combing inverse nanoprecipitation and “tetrazole-alkene” photo-click reaction. HA is a natural material that shows intrinsic targetability to CD44 receptors overexpressed in many malignant cancer cells and cancer stem cells.34-37 GE11 (YHWYGYTPQNVI) is a short peptide that has shown a high affinity to epidermal growth factor receptor (EGFR).38, 39 Notably, many cancer cells including SKOV-3 human ovarian and MDA-MB-231 human breast cancer cells are known to overexpress both CD44 and EGFR.36,

40-42

We hypothesized that EGFR/CD44-NGs would achieve not only better

selectivity but also more efficient target cell uptake in SKOV-3 human ovarian and MDA-MB-231 human breast cancer models, leading to enhanced protein therapy.

Figure 1. Schematic illustration of EGFR and CD44 dual-targeted multifunctional hyaluronic acid nanogels (EGFR/CD44-NGs) for enhanced delivery of therapeutic proteins to SKOV-3 human ovarian carcinoma and MDA-MB-231 human breast tumor xenografts in mice.

2. EXPERIMENTAL SECTION 4


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2.1. Synthesis of HA-g-GE11/Tet. HA-g-GE11/Tet was prepared by coupling amino groups of GE11 with carboxyl groups of HA-g-Tet in the presence of 4-(4,6-dimethoxy1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM). In a typical example, GE11 (7.7 mg, 5 mmol) was added into a stirred solution of HA-g-Tet (44 mg, 1 mmol, degree of substitution (DS) of Tet is 3.9) in water (2.2 mL) followed by adjusting the pH to about 6.4~6.7 using 2 M NaOH. Then, 2 mg of DMTMM (7.5 mmol) was added, and the reaction proceeded for 24 h at 35 ºC. The product was isolated by extensive dialysis (Spectra/Pore, MWCO 3500) against D.I. water followed by lyophilization. Yield: 64%.

1

H NMR

(D2O/DMSO-d6, 600 MHz, δ): HA: 1.82, 2.70–3.68, and 4.23–4.38; Lys: 0.92, 1.06, 1.52, 2.97, 3.61 and 3.95; Tet: 7.91, 7.92 6.79 and 6.80; GE11: 7.64, 8.05, 8.20. The DS of GE11 was determined to be 2.2 by the 2, 4, 6-trinitrobenzene-1-sulfonic acid (TNBS) method. In brief, the GE11 in dialysis solution was collected and concentrated by lyophilization. After re-dissolving in a sodium chloride solution (0.5 wt.%, 500 µL) containing 25% dioxane, a TNBS aqueous solution (5 wt.%, 500 µL) with 8 wt.% sodium bicarbonate was added. The mixed solution was incubated at 37 °C for 90 min, and the GE11 amount was determined by measuring the absorbance at λ = 450 nm with UV spectroscopy. Free GE11 with concentrations of 5-400 µg/mL were employed to obtain the standard curves. GE11 conjugated on HA-g-Tet was calculated by subtracting the GE11 in dialysis medium from GE11 in feed.

2.2.

Preparation

of

Dual-Targeting

Redox-Sensitive

and

Fluorescent

EGFR/CD44-NGs. Dual-targeting redox-sensitive EGFR/CD44-NGs were prepared by combining inverse nanoprecipitation method and catalyst-free “tetrazole-alkene” photoclick reaction. Typically, HA grafted with tetrazole (HA-g-Tet), GE11 peptide and tetrazole (HA-g-GE11/Tet), and cystamine-methacrylamide (HA-g-Cys-MA) were dissolved in

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phosphate buffer (PB, pH 7.4, 10 mM) at Tet/MA molar ratio of 1/1 to obtain a polymer solution (1.25 mg/mL). 1 mL of the polymer solution was injected into acetone (100 mL) via a syringe to form a homogeneous solution, followed by UV irradiation (320-390 nm, 50 mW/cm2) for 3 min. The nanogels were isolated by evaporation of acetone solvent, extensive dialysis (Spectra/Pore, MWCO 3500) against D.I. water, and freeze-drying. By changing the amount of HA-g-GE11/Tet, EGFR/CD44-NGs with varying GE11/HA molar ratios of 0, 0.48, 0.96, 1.44, and 1.92 were obtained.

2.3. Loading and in Vitro Release of Proteins. Protein-loaded nanogels were prepared by injecting 1 mL solution of HA derivatives and proteins (CC and GrB) mixture at protein feed ratios of 2 and 20 wt.% to 100 mL acetone, followed by photoclick cross-linking as described above. The CC loading content was calculated by subtracting the amount of unloaded protein from CC in feed. The in vitro release of CC from EGFR/CD44-NGs and CD44-NGs was investigated at 37 °C under two different conditions: (i) PB (10 mM, pH 7.4), and (ii) PB (10 mM, pH 7.4) containing 10 mM glutathione (GSH). CC-loaded nanogel suspension was transferred to a dialysis tube with an MWCO of 12,000–14,000. The dialysis tube was immersed into 25 mL of appropriate buffer and shaken at 37 °C. At set time intervals, 5.0 mL of the release medium was taken out and replenished with an equal volume of fresh medium. To avoid oxidation of GSH, the release media were bubbled with argon. The concentration of CC was determined by UV measurement (absorbed peak at 408 nm). Release experiments were conducted in triplicate. The results are presented as the average ± standard deviation. The circular dichroism (CD) spectrum of the released proteins from EGFR/CD44-NGs was analyzed by CD spectrometer (Jasco).

2.4. In Vivo Antitumor Efficacy. The mice were handled under protocols approved by the

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Animal Care and Use Committee of Soochow University. SKOV-3 human ovarian and MDA-MB-231 human breast tumors were established by subcutaneously inoculating SKOV-3 and MDA-MB-231 cells (1 × 107 cells per mouse) to nude mice at the left flank. When tumor volume reached about 100 mm3, tumor-bearing mice were randomly divided into 4 groups (6 mice each group), and

intravenously injected with GrB-EGFR/CD44-NGs or

GrB-CD44-NGs at a dosage of 100 µg GrB equiv./kg via tail vein every three day (on day 0, 3, 6, 9). PBS and blank HA nanogels were included as the negative controls. The tumor volume and body weight were measured on alternate days. The tumor volume was calculated according to the formula Volume = ½×L×W2, wherein L and W are the tumor dimension at the longest and widest point, respectively. Relative tumor volumes were calculated as V/V0 (V0 is the tumor volume on day 0). Tumor inhibition rate (TIR, %) was calculated using the formula TIR = 1-Wt/Wc, wherein Wt is the tumor weight of the treatment group, and Wc is the tumor weight of the PBS control group. The relative body weights were normalized to their initial weights.

2.5. Histological Analysis and Western Blot Assay. At the end of the treatment, mice of all groups were sacrificed, and the tumor, liver, kidney and heart were excised. The tissues were fixed with 4% paraformaldehyde solution and embedded in paraffin. The sliced organ tissues mounted on the glass slides were stained by hematoxylin and eosin (H&E) and observed by a digital microscope (Leica Q Win). For terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, the dewaxed and rehydrated tumor tissue sections were incubated with proteinase K for 15 min at 37 °C, rinsed twice with PBS. Positive TUNEL staining was visualized by optical microscopy (Olympus Bx41). For Western blot assay, tumor tissues from mice were homogenized in cold radio-immunoprecipitation assay (RIPA) buffer (Pierce, Rockford, IL, USA) with protease

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inhibitors (Roche Diagnostics, Mannheim, Germany) on ice. The protein concentration of tumor samples was measured by BCA kit (Pierce, Rockford, IL, USA). Equal amount of total proteins from tumors was separated on 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes following the incubation overnight at 4 oC with the corresponding primary antibody were treated with secondary antibody at room temperature for 1 h. After washing with Tris-buffered saline with Tween-20 (TBST) for three times, the fluorescence signals were detected using Super Signal ECL (Pierce, Rockford, IL, USA).

3. RESULTS AND DISCUSSION 3.1. Preparation of EGFR and CD44 Dual-Targeted Nanogels (EGFR/CD44-NGs) and Protein Release. EGFR/CD44-NGs were prepared via nanoprecipitation and photo-click reaction from hyaluronic acid grafted with tetrazole (HA-g-Tet), GE11 peptide and tetrazole (HA-g-GE11/Tet), and cystamine-methacrylamide (HA-g-Cys-MA). HA-g-GE11/Tet was readily obtained by conjugating GE11 onto HA-g-Tet via amidation reaction in the presence of DMTMM (Figure 2). 1H NMR data of HA-g-GE11/Tet showed besides the characteristic signals of HA-g-Tet clear peaks at δ 7.64, 8.05 and 8.20 that corresponded to GE11 peptide (Figure S1), indicating GE11 was successfully conjugated to HA-g-Tet. The DS of GE11 determined by TNBS method was 2.2.

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N N N N

O O

NH

O HN O O HO

OH

OH

HOOC

O HO O OH

O HO NH

x

HO O OH

NH

y

n

O

O

HA-g-Tet

O O

N N N N

NH

O NH

HN O O HO

OH O HO O OH

OH

HOOC HO O OH

O HO NH x

NH

O HO y-z

O

O

OH

O HO O OH

NH z O

n

HA-g-GE11/Tet

Figure 2. Synthesis of hyaluronic acid grafted with GE11 peptide and tetrazole (HA-g-GE11/Tet). Condition: GE11 (YHWYGYTPQNVI), DMTMM, pH 6.4~6.7, H2O, 35 oC, 48 h.

Dual-targeting fluorescent EGFR/CD44-NGs were prepared by dropwise adding the mixed aqueous solution of HA-g-(Cys-MA), HA-g-Tet and HA-g-GE11/Tet derivatives into acetone, followed by crosslinking via catalyst-free “tetrazole-alkene” photoclick chemistry. Figure 3A shows a typical distribution of EGFR/CD44-NGs. EGFR/CD44-NGs with a GE11/HA molar ratio of 0.96 had an average diameter of ca. 155 nm with a low polydispersity (PDI) of 0.15. TEM images revealed that EGFR/CD44-NGs had a spherical morphology and a size distribution close to that determined by DLS (Figure 3A). Remarkably, EGFR/CD44-NGs 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 3B). Significant swelling, however, was observed for EGFR/CD44-NGs in 8 h under 10 mM GSH, corroborating that the nanogels are highly sensitive to reduction conditions (Figure 3C). Interestingly, EGFR/CD44-NGs displayed strong fluorescence with an emission wavelength

9



of 450 nm (Figure S2). NGs following 8 h treatment with 10 mM GSH exhibited negligible change of fluorescence intensity, signifying that fluorescence quenching does not occur in NGs. A

B

20

15 10

Intensity (%)

Intensity (%)

20

100 nm

5 0

EGFR/CD44-NGs 10% FBS, 24 h PB pH 7.4, 7 d

15 10 5 0

100 1000 Size (nm)

C

100

1000 Size (nm)

10000

D

100

30

Cumulative release (%)

40 Intensity (%)

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No GSH, 0 h No GSH, 24 h GSH, 4 h

20 10 0 10

100

1000

10 mM GSH

80 60

EGFR/CD44-NGs

CD44-NGs

40

No GSH

20 0

0

10 20 30 40 50 60 Time (h)

Size (nm)

Figure 3. Characterization of EGFR/CD44-NGs with GE11/HA molar ratio of 0.96. (A) Size and size

distribution determined by DLS and TEM. (B) Colloidal stability at a concentration of 1 mg/mL in PB (pH 7.4, 10 mM) and 10% FBS. (C) Size change in response to GSH. (D) Release of CC at pH 7.4 and 37 °C in PB (n = 3).

Proteins were loaded into nanogels at a theoretical drug loading content (DLC) of 2 or 20 wt.%. Remarkably, quantitative loading of CC (loading efficiency > 99%) was accomplished at a theoretical DLC of 2 wt.%. Even at a theoretical DLC of 20 wt.%, a high loading efficiency of 73.8-78.1%, which corresponded to a DLC of 15.6-16.3% was obtained (Table 1). Notably, CC-loaded CD44-NGs and EGFR/CD44-NGs maintained a small size (164-168 nm), independent of GE11 contents (Table 1). Moreover, zeta potential of NGs increased

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from -14.2 mV to -15.6 mV with increasing GE11/HA molar ratios from 0/1 to 1.92/1, signifying that the amount of GE11 on the surface of NGs can be controlled by adjusting the amount of HA-g-GE11/Tet. The in vitro release studies showed that while CC release was inhibited (∼30% release in 48 h) under a physiological condition (pH 7.4 and 37 °C), over 80% of protein was released from EGFR/CD44-NGs in 10 h under 10 mM GSH condition (Figure 3D), indicating that protein release can be triggered under a reductive environment. Given the fact that GrB has a similar isoelectric point to CC (11 versus 10.7), we assumed that GrB would also be quantitatively loaded into EGFR/CD44-NGs at a theoretical DLC of 2 wt.%. Circular dichroism (CD) measurement revealed that released GrB had the same secondary structure as native GrB (Figure S3), indicating that proteins maintain their secondary structure over the nanogel preparation process.

Table 1. Characteristics of CC-loaded CD44-NGs and EGFR/CD44-NGs (theoretical DLC = 20 wt.%). Size (nm)a

PDIa

1

GE11/HA (mol/mol) 0

DLC (%)

DLE (%)

0.17

Zeta (mV)b -14.2

164

16.3

78.1

2

0.48

165

0.16

-14.4

16.2

77.5

3

0.96

167

0.14

-14.6

16.2

77.2

4

1.44

167

0.15

-14.9

15.8

75.3

5

1.92

168

0.16

-15.4

15.6

73.8

Entry

a

Determined by DLS at 25 °C in water.

b

Measured using a Zetasizer Nano-ZS equipped with a standard capillary electrophoresis cell.

3.2. Cellular Uptake and in Vitro Antitumor Activity of EGFR/CD44-NGs. Cellular uptake behavior of intrinsically fluorescent NGs was investigated in SKOV cells using flow cytometry (FCM). The results showed that cells treated with dual-targeting EGFR/CD44-NGs had significantly higher NG fluorescence intensity than that treated with mono-targeting 11



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CD44-NGs (Figure 4A), indicating dual-targeting ligands (GE11+HA) confer improved cellular uptake. Unexpectedly, EGFR/CD44-NGs with a GE11/HA molar ratio of 0.96 displayed the best cellular uptake. Further increasing GE11/HA molar ratios to 1.44 and 1.92 resulted in obviously decreased cellular uptake. It has been reported that ligand density plays a significant role, in which deficient ligand density would lead to insufficient affinity to cells, while excessive ligand on nanoparticles would either cause steric hindrances or consume too many receptors, and thus result in decreasing binding of nanoparticles.43-45 EGFR/CD44-NGs with GE11/HA molar ratio of 0.96 were employed for the following in vitro and in vivo studies. The inhibition experiments showed that cells pretreated with free HA, GE11 or HA+GE11 exhibited significantly reduced cellular uptake (Figure 4B), in which cells pretreated with HA or GE11 alone displayed 1.6-fold higher fluorescence than that pretreated with HA+GE11. These results support that the internalization of EGFR/CD44-NGs in SKOV cells is mediated by both EGFR and CD44 receptors. The confocal microscopy showed strong fluorescence of EGFR/CD44-NGs inside the SKOV-3 cells following 4 h incubation, supporting efficient cellular internalization (Figure S4). Figure 4C displayed that CC was trapped in endosome in 4 h while most CC was delivered into cytoplasm of SKOV-3 cells in 12 h. Endosomal escape is a critical bottleneck for intracellular protein delivery.46-48 The endosomal escape of protein-loaded NGs might be further promoted by introducing cell-penetrating peptides like H5WYG, GALA, and TAT.49, 50 It is evident, therefore, that EGFR and CD44 dual targeted nanogels show enhanced cellular uptake and effective endosomal escape in SKOV-3 cells.

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A

B 150

150 PBS 0.96

120

0 1.44

0.48 1.92

120

90

Counts

Counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


60

PBS EGFR/CD44-NGs plus HA plus GE11 plus GE11 + HA

90 60 30

30 0 10

0 10

100 1000 10000 100000 Fluorescence intensity

C

Cy5-CC

Lysosomes

100 1000 10000 100000 Fluorescence intensity

DAPI

Merge

4h

12 h

Figure 4. (A) Flow cytometry of SKOV cells following 4 h incubation with intrinsically fluorescent

CD44-NGs and EGFR/CD44-NGs at a concentration of 200 µg/mL (0, 0.48, 0.96, 1.44, and 1.92 denoted the molar ratios of GE11/HA in NGs). (B) Flow cytometry of SKOV cells pre-treated with free HA, GE11 or HA+GE11 for 4 h prior to incubation with EGFR/CD44-NGs (GE11/HA molar ratio of 0.96). (C) CLSM images of Cy5-CC-loaded EGFR/CD44-NGs (Table 1, Entry 3) in SKOV cells. The scale bars correspond to 10 µm.

GrB was used as a model therapeutic protein to study the protein delivery performance of EGFR/CD44-NGs in SKOV-3 cells. Interestingly, MTT assays revealed that GrB-EGFR/CD44-NGs with EGFR/HA molar ratio of 0.96 had the best antitumor activity (Figure 5A), which is in consistent with the cellular uptake results. The cells treated with GrB-loaded EGFR/CD44-NGs at EGFR/HA molar ratio of 0.96 exhibited a cell viability of 19.8% at GrB concentration of 30.8 nM, which was significantly lower than that for

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GrB-loaded CD44-NGs under otherwise the same conditions. In contrast, free GrB displayed minimal cytotoxicity at a concentration of 30.8 nM likely due to inefficient cellular uptake (Figure S5). To select negative controls, we studied the CD44 and EGFR expressions in different cells. The results showed that unlike SKOV-3 and MDA-MB-231cells, MCF-7 and U87 cells were CD44+/EGFR- and CD44-/EGFR-, respectively (Figure S6). Notably, GrB-EGFR/CD44-NGs exhibited a similar antitumor activity to GrB-CD44-NGs in CD44+/EGFR- MCF-7 cells (Figure S7A). Moreover, both GrB-CD44-NGs and GrB-EGFR/CD44-NGs induced a considerably lower cytotoxicity to CD44-/EGFR- U87 cells than to MCF-7 and SKOV-3 cells (Figure S7B). These results confirm dual targetability of EGFR/CD44-NGs. Moreover, pretreatment of SKOV-3 cells by free HA, GE11 or HA + GE11 prior to adding EGFR/CD44-NGs resulted in markedly increased cell viability (Figure 5B). The cell viability increased from 19.8% to 68.4% by pretreatment with HA + GE11 at GrB concentration of 30.8 nM. The effective growth inhibition of SKOV-3 cells indicates that EGFR/CD44-NGs can efficiently deliver and release GrB into the cytosol. One feature of the apoptosis is the activation of caspase enzymes, especially caspases-3/7.51 Apo-ONE™ Homogeneous Caspase3/7 Assay exhibited that cells treated with GrB-EGFR/CD44-NGs had over 1.5-fold higher caspase activity than that treated with GrB-CD44-NGs (Figure 5C), corroborating more efficient delivery of GrB by EGFR/CD44-NGs. Importantly, SKOV-3 cells following 48 h treatment with blank CD44-NGs and EGFR/CD44-NGs at concentrations of 0.5 and 1 mg/mL exhibited close to 100% cell viability (Figure S8), indicating that these nanogels are non-cytotoxic.

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B

A 0 1.44

0.48 1.92

0.96

**

40

**

20

Cell viability (%)

60 Cell viability (%)

0

EGFR/CD44-NGs plus HA plus GE11 plus HA + GE11

120 100 80 60 40 20 0

***

*

***

**

* *

15.4

30.8 GrB concentration (nM)

15.4

30.8

GrB concentration (nM)

C Caspase 3/7 activity (fold)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


12 10 8 6 4 2 0

**

s Gs Gs NG -N -N k 4 4 n 4 4 D D Bla R/C B-C F Gr G B-E Gr

Figure 5. MTT assays. (A) Viability of SKOV-3 cells following treatment with GrB-loaded

CD44-NGs and EGFR/CD44-NGs with varying GE11/HA molar ratios of 0, 0.48, 0.96, 1.44, and 1.92. (B) Viability of SKOV-3 cells pretreated with free HA, GE11, and HA+GE11 for 4 h, respectively, prior to adding GrB-loaded EGFR/CD44-NGs (GE11/HA molar ratio of 0.96). Data are presented as the average ± SD (n=4). (C) Caspase-3/7 activity of SKOV-3 cells treated with GrB-loaded CD44-NGs and EGFR/CD44-NGs (GE11/HA molar ratio of 0.96). The medium was aspirated following 4 h incubation and the cells were further cultured in fresh medium for another 92 h at 37 °C. *p < 0.05, **p < 0.01, ***p < 0.005.

3.3. In Vivo Therapeutic Efficacy of GrB-Loaded EGFR/CD44-NGs in SKOV-3 Ovarian Tumor. Ovarian cancer is one of the most deadly cancers in women.52,

53

The

therapeutic performance of GrB-EGFR/CD44-NGs was firstly evaluated in SKOV-3 ovarian tumor-bearing nude mice. The results demonstrated that tumor growth was effectively 15



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Page 16 of 27

inhibited by GrB-loaded NGs at a low dose of 3.85 nmol GrB equiv./kg, in which GrB-EGFR/CD44-NGs displayed 2-fold smaller tumor burden than GrB-CD44-NGs on day 15 (Figure 6A). Similar results were also observed for docetaxel or doxorubicin-loaded dual-targeted nanoparticles toward CD44/αvβ3 and folate/nuclear localization signal receptors in KB and B16F10 tumor-bearing mice.31, 54 In contrast, as for PBS group, mice treated with blank EGFR/CD44-NGs had fast tumor growth. The photographs of tumor blocks isolated on day 15 further corroborated that GrB-EGFR/CD44-NGs afforded most effective suppression of

tumor

growth

(Figure

GrB-EGFR/CD44-NGs

had

6B).

Figure

significantly

shows

6C

lower

tumor

that weights

mice than

treated

with

those

with

GrB-CD44-NGs (mono-targeted control). The calculation of tumor inhibition rate (TIR) revealed that mice treated with GrB-EGFR/CD44-NGs and GrB-CD44-NGs had a TIR of 87% and 71%, respectively (Figure 6C), signifying the important role of dual targeting on improving the therapeutic efficacy of protein nanotherapeutics. Importantly, GrB-CD44-NGs and GrB-EGFR/CD44-NGs caused little body weight changes (Figure 6D), indicating that these nanogel systems have negligible systemic toxicity.

16


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GrB-EGFR/CD44-NGs Blank EGFR/CD44-NGs

B

** **

12 10 8 6 4 2 0

GrB-CD44-NGs PBS

**

Relative tumor volume (V/V0)

A

0 2 4 6 8 10 12 14 16 Time (d)

1.2

** **

0.8 0.4

**

0.0

s s s S PB 4-NG 4-NG 4-NG 4 4 4 /CD B-CD R/CD FR F Gr G -EG kE B n r a G Bl

100 80 60 40 20 0

D 120

Relative body weight (%)

Tumor weight (g)

C Tumor inhibition rate (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


100 80 60 40

0 2 4 6 8 10 12 14 16 Time (d)

Figure 6. In vivo antitumor performance of GrB-loaded NGs in SKOV-3 ovarian tumor-bearing nude

mice. (A) Tumor volume changes of mice treated with PBS, blank EGFR/CD44-NGs, GrB-CD44-NGs and GrB-EGFR/CD44-NGs, respectively. The drug was given on day 0, 3, 6, and 9 at a dosage of 3.85 nmol GrB equiv./kg. (B) Photographs of tumor blocks collected from different treatment groups on day 15. (C) Tumor weight and tumor inhibition rate (TIR) of SKOV-3 tumor-bearing mice received different treatments on day 15. (D) Body weight changes of mice in different treatment groups within 15 d. Data are presented as mean ± SD (n= 6). **p < 0.01.

TUNEL assays displayed significant apoptosis in the tumor tissue of mice that treated with GrB-CD44-NGs or GrB-EGFR/CD44-NGs (Figure 7A). GrB-EGFR/CD44-NGs caused more apoptosis than GrB-CD44-NGs. The histological analysis using H&E staining revealed that GrB-EGFR/CD44-NGs caused widespread necrosis in the tumor tissue (Figure 7A) while little damage to the healthy organs like liver, heart and kidney (Figure S9). GrB has been reported to be a potent antitumor protein, and can induce both cell apoptosis and

17



necrosis by boosting the expression of active caspase-3, caspase 9, PARP, Bax and truncated protein gtBid.55 Western blot assays demonstrated that GrB-loaded NGs had remarkably high expression of active caspase-3, caspase-9, and PARP (Figure 7B), signifying that GrB released from NGs generated active caspases that resulted in cell apoptosis. In comparison to GrB-CD44-NGs, GrB-EGFR/CD44-NGs exhibited much higher expression of active caspase-3, caspase-9, and PARP, confirming that dual-targeted GrB-EGFR/CD44-NGs cause enhanced cell apoptosis. Meanwhile, GrB-loaded NGs group displayed also high expression of gtBid (Figure 7B), which combined with Bax could generate therapeutic protein CC, further boosting antitumor efficacy.

TUNEL

A

H&E

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

18


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Page 19 of 27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


B

active caspase-3 active caspase-9 active PARP Bax gtBid actin Figure 7. (A) H&E staining and TUNEL assays. (B) Western blot analysis of tumor blocks collected

from different treatment groups on day 15.

3.4.

In

Vivo

Therapeutic

Efficacy

of

GrB-Loaded

EGFR/CD44-NGs

in

Triple-Negative Breast Tumor. Encouraged by their high antitumor efficacy in ovarian cancer, we further studied the therapeutic performance of GrB-EGFR/CD44-NGs in EGFR and CD44-overexpressing MDA-MB-231 triple-negative breast cancer xenografts in mice. Triple-negative breast cancer is another aggressive and refractory malignancy in women.56 Figure

8A shows that GrB-EGFR/CD44-NGs

effectively

suppressed

growth

of

MDA-MB-231 tumor, which was significantly more efficient than GrB-CD44-NGs. The photographs of tumor blocks isolated on day 18 further corroborated that treatment with GrB-EGFR/CD44-NGs GrB-EGFR/CD44-NGs

resulted group

in

had

the a

TIR

smallest of

82%

tumor

size

(Figure

(Figure

S10A).

8A).

Therefore,

GrB-EGFR/CD44-NGs could efficiently suppress the tumor growth in both SKOV-3 and MDA-MB-231 tumor xenografts at a remarkably low dose of 3.85 nmol GrB equiv./kg, which 19



is over 1000 times lower than doxorubicin and platinum-loaded nanotherapeutics.57-59 The histological analysis revealed that GrB-EGFR/CD44-NGs caused widespread necrosis in the tumor tissue with little damage to the liver, heart and kidney (Figure 8B). Moreover, little body weight change was observed in mice treated with either GrB-EGFR/CD44-NGs or GrB-CD44-NGs (Figure S10B). These results further confirm that GrB-EGFR/CD44-NGs afford enhanced treatment of EGFR and CD44-overexpressing cancers without causing obvious systemic side effects.

PBS ** **

14 12 10 8 6 4 2 0

Blank EGFR/CD44-NGs GrB-CD44-NGs

**

A Relative tumor volume (V/V0)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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GrB-EGFR/CD44-NGs

0

3

6

9

12 15 18 21

Time (d)

20


Page 21 of 27

Liver

Heart

Tumor

B

Kidney

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8. In vivo antitumor performance of GrB-CD44-NGs and GrB-EGFR/CD44-NGs in MDA-MB-231 breast tumor-bearing nude mice. (A) Tumor volume changes of mice treated with PBS, blank EGFR/CD44-NGs, GrB-CD44-NGs and GrB-EGFR/CD44-NGs, respectively. The drug was given on day 0, 3, 6, and 9 at a dosage of 3.85 nmol GrB equiv./kg. The right images displayed typical tumor blocks collected from different treatment groups on day 18. (B) H&E staining assays of tumor and healthy organs in different treatment groups. **p < 0.01

4. CONCLUSIONS We have demonstrated that EGFR and CD44 dual-targeted multifunctional hyaluronic acid nanogels (EGFR/CD44-NGs) boost protein delivery to EGFR and CD44-overexpressing SKOV-3 human ovarian cancer and MDA-MB-231 human breast cancer in vitro and in vivo. EGFR/CD44-NGs have many advantages such as tunable EGFR densities, small size, high loading of protein, effective endosomal escape, and rapidly reduction-triggered release of protein. The installation of GE11 peptide on hyaluronic acid nanogels has shown to greatly enhance the cellular uptake and cytoplasmic release of GrB-loaded EGFR/CD44-NGs in

21



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SKOV-3 cancer cells, which in turn leads to elevated caspase expression in cytoplasm, and antitumor activity. It is of particular interest to note that GrB-loaded EGFR/CD44-NGs effectively suppress growth of SKOV-3 ovarian carcinoma and MDA-MB-231 human breast tumors without causing any adverse effects at a low dose of 3.85 nmol GrB equiv./kg. This represents a first proof-of-concept study on dual-targeted nanogels to achieve enhanced delivery of therapeutic proteins. The dual-targeting approach appears to be capable of simultaneously enhancing cancer cell selectivity and receptor-mediated internalization. EGFR and CD44 dual-targeted hyaluronic acid nanogels have emerged as a multifunctional platform for cancer protein therapy.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental on materials, characterization, cellular uptake of NGs, in vitro cystotoxicity assay; 1H NMR spectrum for HA-g-GE11/Tet; CD spectra of protein, MTT assay of blank NGs; histological analysis of health organs like heart, kidney, and liver of SKOV-3 tumor bearing nude mice in different treatment groups; body weight change, tumor weight, and tumor inhibition rate of MDA-MB-231-bearing mice in different treatment groups.

AUTHOR INFORMATION Corresponding Authors E-mail: [email protected]. Tel: +86-512-65884933. E-mail: [email protected]. Tel: +86-512-65880098.

Notes The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (NSFC 51473110, 51403147 and 51633005), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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EGFR and CD44 Dual-Targeted Multifunctional Hyaluronic Acid

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