EGFR-targeted multifunctional polymersomal doxorubicin induces selective and potent suppression of orthotopic human liver cancer in vivo

Acta Biomaterialia xxx (2017) xxx–xxx

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EGFR-targeted multifunctional polymersomal doxorubicin induces selective and potent suppression of orthotopic human liver cancer in vivo Yuan Fang a, Weijing Yang a, Liang Cheng a,b,⇑, Fenghua Meng a,⇑, Jian Zhang a, Zhiyuan Zhong a,⇑ a 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, PR China b Department of Pharmaceutics, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, PR China

a r t i c l e

i n f o

Article history: Received 16 June 2017 Received in revised form 13 September 2017 Accepted 9 October 2017 Available online xxxx Keywords: Biodegradable polymersomes Liver cancer EGFR Targeted delivery

a b s t r a c t Liver cancer is a globally leading malignancy that has a poor five-year survival rate of less than 20%. The systemic chemotherapeutics are generally ineffective for liver cancers partly due to fast clearance and low tumor uptake. Here, we report that GE11 peptide functionalized polymersomal doxorubicin (GE11-PS-DOX) effectively targets and inhibits epidermal growth factor receptor (EGFR)-positive SMMC7721 orthotopic human liver tumor xenografts in mice. GE11-PS-DOX with a GE11 surface density of 10% displayed a high drug loading of 15.4 wt%, a small size of 78 nm, and glutathione-triggered release of DOX. MTT assays, flow cytometry and confocal microscopy studies revealed that GE11-PS-DOX mediated obviously more efficient DOX delivery into SMMC7721 cells than the non-targeting PS-DOX and clinically used liposomal doxorubicin (Lipo-DOX) controls. The in vivo studies showed that GE11-PSDOX had a long circulation time and an extraordinary accumulation in the tumors (13.3 %ID/g). Interestingly, GE11-PS-DOX caused much better treatment of SMMC7721 orthotopic liver tumorbearing mice as compared to PS-DOX and Lipo-DOX. The mice treated with GE11-PS-DOX (12 mg DOX equiv./kg) exhibited a significantly improved survival rate (median survival time: 130 days versus 70 and 38 days for PS-DOX at 12 mg DOX equiv./kg and Lipo-DOX at 6 mg DOX equiv./kg, respectively) and achieved 50% complete regression. Notably, GE11-PS-DOX induced obviously lower systemic toxicity than Lipo-DOX. EGFR-targeted multifunctional polymersomal doxorubicin with improved efficacy and safety has a high potential for treating human liver cancers. Statement of Significance Liver cancer is one of the top five leading causes of cancer death worldwide. The systemic chemotherapeutics and biotherapeutics generally have a low treatment efficacy for hepatocellular carcinoma partly due to fast clearance and/or low tumor uptake. Nanomedicines based on biodegradable micelle and polymersomes offer a most promising treatment for malignant liver cancers. Their clinical effectiveness remains, however, suboptimal owing to issues like inadequate systemic stability, low tumor accumulation and selectivity, and poor control over drug release. Here we report that GE11 peptidefunctionalized, disulfide-crosslinked multifunctional polymersomal doxorubicin (GE11-PS-DOX) can effectively suppress the growth of orthotopic SMMC7721 human liver tumors in nude mice. They showed significantly decreased systemic toxicity and improved mouse survival rate with 3.4-fold longer median survival time as compared to clinically used pegylated liposomal doxorubicin (Lipo-DOX) and achieving 50% complete regression. GE11-PS-DOX, based on PEG-PTMC is biodegradable, nontoxic, and easy to prepare, appears as a safe, robust, versatile and all-function-in-one nanoplatform that has a high potential in targeted chemotherapy of EGFR expressed hepatocellular carcinoma. Ó 2017 Published by Elsevier Ltd on behalf of Acta Materialia Inc.

1. Introduction ⇑ Corresponding authors at: 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, PR China. E-mail addresses: [email protected] (L. Cheng), [email protected] (F. Meng), [email protected] (Z. Zhong).

Liver cancer is one of the top 5 leading causes of cancer death in China [1] as well as in the United States [2]. Notably, about 75% of liver cancers are hepatocellular carcinoma (HCC). The clinical treatment of HCC is mainly limited to conventional surgery, liver transplantation, percutaneous ablation, transarterial

https://doi.org/10.1016/j.actbio.2017.10.013 1742-7061/Ó 2017 Published by Elsevier Ltd on behalf of Acta Materialia Inc.

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Y. Fang et al. / Acta Biomaterialia xxx (2017) xxx–xxx

chemoembolization, and more recently developed small kinase inhibitor (e.g. sorafenib) [3–6]. The systemic chemotherapeutics and biotherapeutics generally have a low treatment efficacy for HCC partly due to fast clearance and low tumor uptake [7,8]. In the past years, various nanomedicines were developed and investigated for enhanced treatment of HCC [9–13]. For example, Wang et al. reported that co-delivery of platinum and siRNA with micelles effectively improved drug accumulation and growth inhibition of SMMC7721 tumor xenografts [14]. Galactose, galactosamine or lactose-functionalized micelles and nanoparticles were extensively investigated for targeted treatment of various HCC such as HepG2 and SMMC-7721 cancers that overexpress asialoglycoprotein receptors (ASGPR) [15–18]. Glycyrrhetinic acid-functionalized nanoparticles were also reported to markedly enhance drug accumulation in the liver and treatment efficacy toward subcutaneous and orthotopic liver tumor models [19]. Interestingly, galactosamine-modified PHPMA-doxorubicin prodrug (PK2) has been translated to phase II clinical trials for targeted liver cancer therapy [20]. It should be noted, however, that ASGPR is overexpressed not only in liver cancer cells but also in healthy hepatocytes [21], rendering ASGPR-targeted nanomedicines less favorable for HCC treatment. Epidermal growth factor receptor (EGFR) is known to specifically overexpress on cancerous cells including breast, lung, pancreatic and liver cancers [22–26]. Drugs targeting to EGFR (e.g. Tarceva, Iressa, and Erbitux) have been approved by US FDA for treating advanced lung and colon cancers [27]. Anti-EGFR antibody-decorated nanomedicines have shown targetability to SMMC-7721 liver cancers [24,28]. Interestingly, Xu et al. reported that GE11, a dodecapeptide (YHWYGYTPQNVI), has a high affinity to EGFR [29]. GE11 peptide has shown to improve the targetability of liposomal drugs and polyplexes in different cancer cells in vitro and in vivo [25,30–32]. In particular, GE11-decorated RNA polyplexes [33,34] and GE11-DOX conjugates [35] have significantly attenuated growth of HepG2, Huh7 and SMMC-7721 cancer cells in vivo. Wagner et al. reported that unlike anti-EGFR antibodies and EGF, GE11 peptide does not lead to EGFR signaling activation [36]. Here, we developed and investigated GE11 peptidefunctionalized multifunctional polymersomal doxorubicin (GE11PS-DOX) for targeted chemotherapy of orthotopic SMMC7721 human liver cancer in nude mice (Scheme 1). We have recently found that PS-DOX based on poly(ethylene glycol)-b-poly (trimethylene carbonate-co-dithiolane trimethylene carbonate) (PEG-P(TMC-DTC)) diblock copolymer has superior stability, tolerability, and reduction-sensitive drug release to clinically used liposomal doxorubicin (Lipo-DOX) [37]. It was hypothesized that the functionalization of PS-DOX with GE11 peptide could achieve actively targeted treatment of EGFR-positive liver cancers. Intriguingly, our results showed that GE11-PS-DOX could treat orthotopic SMMC7721 human liver tumor xenografts significantly better than the non-targeted PS-DOX as well as clinically used Lipo-DOX.

2. Experimental section 2.1. Synthesis of PEG-P(TMC-DTC) and GE11-PEG-P(TMC-DTC) Under a nitrogen atmosphere, diphenyl phosphate (DPP, 25 mg, 100 lmol) was added to a stirred solution of NHS-PEG-OH (Mn = 7. 5 kg/mol, 75 mg, 10 lmol), trimethylene carbonate (TMC, 230 mg, 2.25 mmol) and dithiolane trimethylene carbonate (DTC, 20 mg, 0.10 mmol) in dichloromethane (DCM, 1.8 mL, total monomer concentration [M]0 of 2.0 M). The polymerization proceeded at 30 °C for 72 h. NHS-PEG-P(TMC-DTC) was recovered by twice precipitation in cold diethyl ether and vacuum drying. Yield: 86.5%. 1H

NMR (400 MHz, CDCl3, ppm): PEG: d 3.64; TMC moieties: d 2.06, 4.24; DTC moieties: d 3.02, 4.19; NHS moieties: d 2.52. Mn (1H N MR) = 32.1 kg/mol. Mn (GPC) = 38.1 kg/mol, Mw/Mn = 1.10. Subsequently, NHS-PEG-P(TMC-DTC) (100 mg, 3.1 lmol) was added to a stirred solution of GE11 peptide (YHWYGYTPQNVI, 13.8 mg, 9.0 lmol) in 1.5 mL of N,N-dimethylformamide (DMF). After 48 h reaction at r.t., GE11-PEG-b-P(TMC-DTC) was isolated via dialysis (MWCO 3500) against DMF for three times and DCM for two times followed by precipitation in cold diethyl ether and vacuum drying. Yield: 98.8%. The degree of substitution of GE11 was determined to be 92.8% by using TNBS assays performed as described previously [38]. PEG-P(TMC-DTC) was synthesized using 1,5,7-triazabicyclo[4.4. 0]dec-5-ene (TBD) as a catalyst and MeO-PEG-OH (Mn = 5 kg/mol) as a macro-initiator at a molar ratio of TBD/OH/TMC/DTC = 0.5/1/ 227/10 and a total monomer concentration of 1.0 M in DCM and at 30 °C for 12 h. Yield: 93.2%. Mn (1H NMR) = 30.8 kg/mol, Mn (G PC) = 35.3 kg/mol, Mw/Mn = 1.38. 2.2. Formation of GE11-directed polymersomal doxorubicin (GE11-PSDOX) DOX
HCl-loaded polymersomes (GE11-PS-DOX) were obtained using a pH-gradient method (pHin = 4, pHout = 7.80) [37]. Briefly, a DMF solution (10 mg/mL, 100 lL) of PEG-P(TMC-DTC) and GE11-PEG-P(TMC-DTC) at a predetermined molar ratio (GE11 molar content of 0, 5%, 10%, 19%, 29% or 39%, respectively) was dropwise added into 0.9 mL of citric acid buffer (pHin = 4.0, 10 mM) under stirring at 300 rpm to form polymersomes. After standing still for 6 h, saturated Na2HPO4 solution was added to adjust pHout to 7.80 in order to build a pH gradient across the polymersome membrane. Immediately, DOX
HCl solution in phosphate buffer (PB, 5 mg/mL) was added at a theoretical drug loading content (DLC) of 16.7 wt% or 23.1 wt% under stirring at 37 °C. The dispersions were allowed to incubate in a shaking bed (37 °C, 200 rpm) for 12 h before extensive dialysis (MWCO 3500 Da) against PB (10 mM, pH 7.4) at r.t. for 24 h. The polymersomes were selfcrosslinked during the work-up procedure, and named as GE11PS-DOX. The DOX contents in the PS-DOX and GE11-PS-DOX were determined using UV spectroscopy (ex. 480 nm) as described in supporting information. 2.3. Determination of EGFR expression on the surfaces of tumor cells Human hepatocellular carcinoma SMMC-7721 cells and human colorectal carcinoma HCT-116 cells were cultured in 6-well plates (5  105 cells/well) in 100 lL RPMI 1640 medium, and human chronic myelogenous leukemia K562 cells were in DMEM medium. The media were supplemented with 80 units/mL penicillin G, 100 lg/Ml streptomycin, and 10% FBS. After culturing for 24 h the cells were digested and fixed with 80% methanol (5 min) and permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were incubated for 1 h in PBS containing 10% normal goat serum to block non-specific protein-protein interactions followed by treatment with PE mouse anti-human EGFR antibody for 30 min at 22 °C according to the supplier’s information. Then the cells were measured by a flow cytometry as described in the supporting information. 2.4. MTT assays SMMC-7721 (high EGFR expressing) and K562 cells (low EGFR expressing) were seeded in a 96-well plate (5  103 cells/well) for 24 h to achieve 70% confluence. 20 lL of GE11-PS-DOX at GE11 molar contents of 0, 5%, 10%, 19%, 29% or 39% were added at a dosage of 10 lg DOX equiv./mL. After 4 h incubation, the cul-

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Scheme 1. (A) Chemical structure of GE11-PEG-P(TMC-DTC). (B) GE11-PS-DOX as a robust and multifunctional nanomedicine for EGFR-targeted therapy of orthotopic SMMC-7721 liver tumor-bearing mice.

ture medium was removed and replaced with fresh medium, and the cells were incubated for another 44 h. Then 10 lL of 3-(4,5-di methylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) solution in PBS (5 mg/mL) was added to incubate for 4 h. The MTTformazan generated by live cells was dissolved in 150 lL of DMSO, and the absorbance at 492 nm was measured using a microplate reader. The relative cell viability (%) was determined by comparing the absorbance at 492 nm with control SMMC-7721 cells treated only with PBS (100%). Data were given as average ± SD (n = 4). To determine the half-maximal inhibitory concentration (IC50), GE11-PS-DOX, PS-DOX and Lipo-DOX (pegylated liposomal doxorubicin, LIBODÒ) were added into SMMC-7721 cells at DOX
HCl dosages ranging from 0.5 to 40 lg/mL. The cytotoxicity of blank GE11-PS and PS to SMMC-7721 cells was determined in a similar way at polymer concentrations ranging from 0.1, 0.2, 0.3, 0.4 to 0.5 mg/mL.

were washed with phosphate buffered saline (PBS, 3) before fixation with 4% paraformaldehyde solution for 15 min and PBS wash (3). The cell nuclei were stained with 40 ,6-diamidino-2-phenylin dole (DAPI) for 10 min and washed with PBS (3). The fluorescence images were obtained using a CLSM (TCS SP5) and the DOX imaging was done with identical exposure times to allow comparison. An inhibition experiment by incubating GE11-PS-DOX with GE11 pretreated SMMC-7721 cells (100 lg/mL, 2 h) were performed. SMMC-7721 cells seeded in a 6-well plate (5  105 cells/well) were incubated with GE11-PS-DOX, PS-DOX, Lipo-DOX or free DOX
HCl in 100 lL PB (DOX
HCl dosage: 10 lg/mL) at 37 °C for 4 h. PBS was used as control. For comparison, K562 cells and GE11 pretreated SMMC-7721 cells (100 lg/mL, 2 h) were also incubated with GE11-PS-DOX. Then the cells were measured using a flow cytometry for the detection of DOX fluorescence as described in supporting information.

2.5. Intracellular DOX delivery evaluated by confocal laser scanning microscopy (CLSM) and flow cytometry

2.6. Animal models

SMMC-7721 cells were cultured on microscope coverslips in 24-well plate (5  104 cells/well). The cells were incubated with GE11-PS-DOX, PS-DOX or Lipo-DOX in 50 lL of PB (DOX
HCl dosage: 10 lg/mL) at 37 °C. After 4 h, the medium was aspirated and replaced with fresh medium, and the cells were further incubated for another 8 or 20 h. The cells on microscope coverslips

All animals were handled under protocols approved by Soochow University Laboratory Animal Center and the Animal Care and Use Committee of Soochow University. For building mouse subcutaneous liver tumor model, female Balb/c mice (5 weeks, Model Animal Research Center of Nanjing University) were inoculated subcutaneously on the right hind flank with SMMC 7721 cells (1  106 per mouse) in 50 lL PBS containing BD Matrigel. Mice

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with tumor size of ca. 200–300 mm3 were used for in vivo biodistribution and imaging studies. For building mouse orthotopic SMMC 7721 liver tumor model, the cells (1  106 per mouse) in 50 lL PBS containing BD Matrigel were slowly injected into the right upper lobe of the liver of female Balb/c mice (5 weeks) using a 29-gauge needle. The growth of the liver tumors was monitored by sacrificing one mouse at predetermined time points. About 10 days after inoculation, mouse liver tumors were ready for therapeutic studies, and after ca. 15 days liver tumors were prominent and used for in vivo imaging and biodistribution studies. 2.7. The in vivo blood circulation and in vivo biodistribution For the in vivo pharmacokinetic studies, Kunming mice were randomly grouped (n = 3) and intravenously injected with 200 lL of GE11-PS-DOX, PS-DOX and Lipo-DOX (10 mg DOX equiv./kg) in PB via tail veins. About 20 lL of blood was taken from the retro-orbital sinus of mice at different time points. Blood samples were immediately dissolved in 100 mL of Triton X-100 (1%) with brief sonification. DOX was extracted by DMF solution containing 20 mM DTT overnight at 25 °C. After centrifugation at 10 krpm for 20 min, DOX concentration in the supernatant was determined by using fluorescence measurement (ex. 480 nm, em. 560 nm). The blood circulation half-lives (t1/2,a and t1/2,b) and area under the curve (AUC) were obtained by second-order exponential decay fits [38]. To study the in vivo targeting effect of GE11-PS-DOX to SMMC 7721 liver tumors, the ex vivo fluorescent imaging studies were firstly conducted in both subcutaneous model and orthotopic model. Briefly, into Balb/c mice bearing either subcutaneous or orthotopic SMMC-7721 tumors which were randomly grouped (n = 3), 200 lL of GE11-PS-DOX or PS-DOX in PB were i.v. injected via tail veins (10 mg DOX equiv./kg). At 12 h post injection, the mice were sacrificed. The major organs or tumors were collected, washed with PBS, wiped with tissues, and weighed before taking photographs or ex vivo fluorescence imaging. For orthotopic models, Lipo-DOX was used for comparison. For the quantitative biodistribution studies, the organs were quickly homogenized and DOX was extracted by DMF solution (containing 20 mM DTT) overnight at 25 °C. After centrifugation at 10,000 rpm for 20 min, DOX concentration in the supernatant was determined by fluorescence measurement (n = 3). To evaluate maximum tolerated dose (MTD), nude mice were administered with a single dose of GE11-PS-DOX or Lipo-DOX via tail veins. The body weights were measured every two days and normalized to their initial weights. 2.8. In vivo treatment of orthotopic SMMC-7721 bearing mice For antitumor efficacy studies, Balb/c mice bearing SMMC-7721 orthotopic tumors were randomly grouped (n = 5, one for histological analysis and four for therapeutic studies) and intravenously injected via tail veins with 200 lL of GE11-PS-DOX every four days (total 10 injections) at 6 or 12 mg DOX equiv./kg. PS-DOX (12 mg DOX equiv./kg), Lipo-DOX (6 mg DOX equiv./kg) and PBS were used as controls. For Lipo-DOX, 9 injections were given due to severe toxicity. Mice were weighed every two days and the relative body weights were normalized to their initial weights. On day 44, one mouse from each group was sacrificed by cervical vertebra dislocation and the heart, liver, spleen, lung, and kidney were isolated and prepared for photographs and histological analyses. The rest mice (n = 4) were used for monitoring the survival rates. With the progress of the tumors, the mouse behavior and their symptom of liver ascites were recorded. Mice in each cohort were considered to be dead either when the mice died during treatment or when the mouse abdomen reached 100 mm due to liver ascites.

For histological analyses, the excised tumors and organs were fixed with 10% formalin, embedded in paraffin and sliced (thickness: 4 lm). The sliced tissues were mounted on glass slides, stained using hematoxylin and eosin (H&E) and observed using a digital microscope (Olympus BX41). 2.9. Statistical analyses Difference between groups was assessed using one-way ANOVA with Tukey multiple comparisons tests using Prism software, wherein ⁄p < .05 was considered significant, ⁄⁄p < .01 and ⁄⁄⁄p < 0.001 were highly significant. 3. Results and discussion 3.1. Formation and Characterization of GE11-PS and GE11-PS-DOX GE11 functionalized polymersome (GE11-PS) was obtained from PEG-P(TMC-DTC) and GE11-functionalized PEG-P(TMC-DTC) (GE11-PEG-P(TMC-DTC)) diblock copolymers. GE11-PEG-P(TMCDTC) was prepared by copolymerization of TMC and DTC using NHS-PEG-OH (Mn = 7.5 kg/mol) as an initiator followed by coupling with GE11 peptide (Scheme S1). Table 1 shows that NHS-PEG-P (TMC-DTC) had a controlled Mn of 7.5-(22.5-2.1) kg/mol with a low Mw/Mn of 1.10. 1H NMR displayed characteristic peaks of NHS moieties at d 2.6 ppm (Fig. S1A), indicating that NHS groups were intact during reaction and work-up procedure. The treatment of NHS-PEG-P(TMC-DTC) with GE11 peptide resulted in new resonances assignable to GE11 peptide at d 6.5–8.5 ppm while disappearance of NHS signals (Fig. S1B). This is in accordance with our TNBS assays, which revealed nearly quantitative GE11 functionalization. PEG-P(TMC-DTC) was synthesized with an Mn of 5.0(23.9-1.9) kg/mol and an Mw/Mn of 1.38 using MeO-PEG-OH (Mn = 5.0 kg/mol) as an initiator (Table 1, Fig. S2). The co-self-assembly of GE11-PEG-P(TMC-DTC) and PEG-P (TMC-DTC) at predetermined molar ratios yielded polymersome GE11-PS with tunable GE11 surface densities. DLS measurements showed that hydrodynamic diameters of GE11-PS increased from 59 to 78 nm with increasing GE11 contents from 0 to 39% (Fig. S3). As previous reports for micelles and polymersomes based on DTC [37,39,40], GE11-PS was automatically crosslinked during the work-up procedure owing to the ring-opening polymerization of dithiolane in the polymersomal membrane (Scheme 1). In accordance, no critical aggregation concentration (CAC) could be detected, and GE11-PS was very robust against extensive dilution or in the presence of 10% serum (Fig. S4). However, GE11-PS quickly swelled in response to 10 mM glutathione (GSH), a cytosol mimicking reductive environment, due to de-crosslinking and increased hydrophilicity of polymersomal membrane, similar to lipoic acid-crosslinked nanoparticles [41]. DOX
HCl could be efficiently loaded into GE11-PS using pH gradient method. Table 2 shows that GE11-PS-DOX at 10% GE11 surface density achieved a high drug loading content (DLC) of 15.4 wt %. GE11-PS-DOX maintained a small size and narrow distribution (Fig. 1A). TEM micrograph of GE11-PS-DOX displayed a spherical vesicular structure and an average size of ca. 60–80 nm (Fig. 1A inset). Notably, GE11-PS-DOX and PS-DOX displayed neutral surface charge (Table 2). The in vitro drug release studies revealed low DOX leakage under physiological conditions while markedly accelerated drug release in the presence of 10 mM GSH (cumulative drug release: ca. 19% versus 84% in 36 h) (Fig. 1B). It is well known that reduction sensitive nanocarriers are very attractive for fast intracellular drug release [42,43], and disulfide crosslinking of the nanocarriers could solve the conflicting requirements of the in vivo stability and fast drug release [40,41]. Hence, GE11-PS-DOX

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Y. Fang et al. / Acta Biomaterialia xxx (2017) xxx–xxx Table 1 Synthesis of PEG-P(TMC-DTC) and NHS-PEG-P(TMC-DTC). Copolymer

PEG-P(TMC-DTC) NHS-PEG-P(TMC-DTC) a b

Mw/Mnb

Mn (kg/mol) Design

1

a

5.0-(2 3 -2) 7.5-(2 3 -2)

5.0-(23.9-1.9) 7.5-(22.5-2.1)

b

H NMR

GPC

35.3 38.1

1.38 1.10

Determined by 1H NMR. Determined by GPC (standards: polystyrene, eluent: DMF, flow rate: 1.0 mL/min, 30 °C).

Table 2 Characteristics of GE11-PS-DOX (GE11 density = 10%) and PS-DOX. Polymersomes

a b c

DLEa (%)

Sizeb (nm)

PDIb

fc (mV)

10.7 15.0

60.0 59.0

76 80

0.16 0.12

-0.14 -0.82

10.2 15.4

57.0 60.7

77 78

0.11 0.15

-0.24 -0.11

DLC (wt%) theory

determineda

PS-DOX

16.7 23.1

GE11-PS-DOX

16.7 23.1

Determined by UV. The intensity-based Z-average size determined by DLS in PB (pH 7.4, 10 mM). Determined in PB (pH 7.4, 10 mM).

Fig. 1. Characterization of GE11-PS-DOX with a GE11 molar content of 10%. (A) The size distribution profile determined by DLS. Inset: TEM micrograph. (B) The in vitro DOX release in the presence or absence of 10 mM GSH in PB (pH 7.4, 10 mM) at 37 °C. Data are presented as mean ± SD (n = 3).

has the merits of small size, high drug loading, high stability and triggered drug release. 3.2. EGFR-targetability and antitumor activity of GE11-PS-DOX EGFR expression levels on the surface of several cancer cells like human hepatocellular carcinoma SMMC-7721 cells, human colorectal carcinoma HCT-116 cells and human chronic myelogenous leukemia K562 cells were studied by flow cytometry using PE antiEGFR antibody. A high EGFR expression on SMMC-7721 cells was found, which was similar to HCT-116 cells and 3.5 times higher than K562 cells (Fig. 2A). MTT assays showed that empty GE11PS was nontoxic to SMMC-7721 cells even at a concentration of 0.5 mg/mL (Fig. 2B). The effect of GE11 surface density on antitumor activity of GE11-PS-DOX was investigated in SMMC-7721 and K562 cells using MTT assays. Fig. 3A displays that GE11-PS-DOX caused the best antitumor effect to SMMC-7721 cells at a GE11 density of 10%. In contrast, GE11 density exhibited little influence on the antitumor activity of GE11-PS-DOX to K562 cells (low EGFR expression), in which cell viabilities were over 90% independent of GE11 densities. Fig. 3B shows that GE11-PS-DOX at 10% GE11

had a half-maximal inhibitory concentration (IC50) of ca. 8.4 lg DOX equiv./mL toward SMMC-7721 cells, which was about 2-fold lower than the non-targeting PS-DOX. GE11 peptide decoration was shown to enhance the antitumor efficacy of several different nanomedicines in EGFR-overexpressing cancer cells including MDA-MB-231 breast cancer cells [44], A549 lung cancer cells [31], and SMMC 7721 cells [35]. Notably, commercial liposomal doxorubicin (Lipo-DOX, LIBADOÒ) exhibited a significantly lower antitumor activity to SMMC-7721 cells than both GE11-PS-DOX and PS-DOX, possibly resulting from its slow drug release inside the cells. The EGFR-targetability and intracellular drug release of GE11PS-DOX was further studied in SMMC-7721 cells using CLSM and flow cytometry. Fig. 4A shows that the cells treated with GE11PS-DOX had much stronger DOX fluorescence than those with PS-DOX under otherwise the same conditions. Moreover, GE11PS-DOX efficiently released DOX into the cell nuclei after 24 h. The inhibition experiment results by incubating GE11-PS-DOX with GE11 pretreated SMMC-7721 cells displayed that the intracellular DOX fluorescence was drastically reduced. In comparison, weak DOX fluorescence was observed in SMMC-7721 cells treated with Lipo-DOX, corroborating their slow intracellular DOX release.

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Fig. 2. (A) EGFR expression of SMMC-7721, HCT-116 and K562 cells determined by flow cytometry using PE anti-EGFR antibody. (B) MTT assays of SMMC-7721 cells after 48 h incubation with empty GE11-PS and PS.

Fig. 3. MTT assays (n = 4). (A) The antitumor activity of GE11-PS-DOX at varying GE11 molar contents from 0 to 39% to SMMC-7721 and K562 cells (10 lg DOX equiv./mL). (B) Dependence of SMMC-7721 cell viabilities on drug concentrations. The cells were incubated for 4 h with GE11-PS-DOX, PS-DOX or Lipo-DOX and further cultured in fresh medium for 44 h. One-way ANOVA with Tukey multiple comparisons tests, *p < .05, **p < .01.

Notably, flow cytometric analyses showed that SMMC-7721 cells following 4 h treatment with GE11-PS-DOX had ca. 2 and 12- fold higher cellular DOX uptake than PS-DOX and Lipo-DOX, respectively (Fig. 4B). The pretreatment of SMMC-7721 cells with GE11 before GE11-PS-DOX incubation caused decreased fluorescence to a level comparable to that of PS-DOX. The functionalization of nanoparticles and pDNA nanocomplex with anti-EGFR antibody was reported to afford 1.5–2-fold increase in cellular uptake [24,28]. In contrast, in EGFR negative K562 cells, similar cellular uptake was observed for both GE11-PS-DOX and PS-DOX (Fig. 4C). These results confirm that GE11-PS-DOX can actively target, enter EGFR-overexpressing SMMC-7721 cells via receptormediated endocytosis, and quickly release DOX intracellularly. 3.3. In vivo pharmacokinetics and biodistribution of GE11-PS-DOX The in vivo blood circulation of GE11-PS-DOX, PS-DOX and LipoDOX was investigated in Kunming (KM) mice. The results showed that GE11-PS-DOX had a long circulation life (Fig. 5). The elimination half-life (t1/2,b) of GE11-PS-DOX was 8.27 h, similar as the nontargeted PS-DOX and commercial Lipo-DOX, but significantly

longer than free DOX
HCl (t1/2,b = 0.42 h) [45]. The area under curve (AUC) of GE11-PS-DOX was calculated to be 29.5 lg/mL
h, which was slightly higher than those of PS-DOX and Lipo-DOX (22.8 and 20.3 lg/mL
h, respectively). These results support that GE11PS-DOX possesses excellent stability in vivo and GE11 functionalization has no detrimental effect to the blood circulation time of PS-DOX. The in vivo biodistribution of GE11-PS-DOX was firstly evaluated in a subcutaneous SMMC-7721 tumor model. Interestingly, Fig. 6A reveals that tumor-bearing mice following 12 h i.v. injection of GE11-PS-DOX had the strongest DOX fluorescence in the tumor among major organs. Moreover, mice treated with non-targeting PS-DOX exhibited also strong DOX fluorescence in the tumor, though much weaker than that with GE11-PS-DOX. The quantification of DOX levels in the tumors and major organs displayed GE11PS-DOX achieved 2-fold higher tumor uptake than PS-DOX while maintaining a similar uptake in all healthy organs, illustrating enhanced selectivity of GE11-PS-DOX toward SMMC-7721 tumors. It should further be noted that a prominent tumor accumulation of ca. 13.3%ID/g (percentage injected dose per gram of tissue) was observed for GE11-PS-DOX treated tumors, which was significantly

Please cite this article in press as: Y. Fang et al., EGFR-targeted multifunctional polymersomal doxorubicin induces selective and potent suppression of orthotopic human liver cancer in vivo, Acta Biomater. (2017), https://doi.org/10.1016/j.actbio.2017.10.013

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Fig. 4. (A) CLSM of SMMC-7721 cells following 4 h treatment with GE11-PS-DOX, PS-DOX or Lipo-DOX and further 8 or 20 h culture in fresh medium. Scale bars correspond to 15 mm. (B) Flow cytometric analyses of SMMC-7721 cells following 4 h incubation with GE11-PS-DOX, PS-DOX, Lipo-DOX or free DOX. PBS treated cells and GE11 pretreated cells were used as controls. (C) Flow cytometric analyses of K562 cells following 4 h incubation with GE11-PS-DOX and PS-DOX. Drug dosage was 10 lg DOX equiv./mL.

higher than in any major organs including liver (Fig. 6B). The particularly high tumor accumulation of GE11-PS-DOX is likely a result of small size, long circulation time, and high affinity to SMMC-7721 tumor. We also studied the biodistribution of GE11-PS-DOX in orthotopic SMMC-7721 liver tumor model. The ex vivo fluorescence images of major organs at 12 h post injection demonstrated that

all three DOX nanoformulations had the strongest DOX fluorescence in the liver (Fig.6C). In the bright field pictures, the exact location of tumor in the liver was marked with a circle to guide the eyes (Fig.6C, upper row). Remarkably, GE11-PS-DOX treated mice exhibited the highest DOX fluorescence right in the circled liver tumor area. In contrast, much lower DOX fluorescence was detected in the tumor area of mice treated with Lipo-DOX or PS-

Please cite this article in press as: Y. Fang et al., EGFR-targeted multifunctional polymersomal doxorubicin induces selective and potent suppression of orthotopic human liver cancer in vivo, Acta Biomater. (2017), https://doi.org/10.1016/j.actbio.2017.10.013

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deliver and release DOX to the tumor site in the liver. Thus, it might well solve the targetability issue and achieve efficient chemotherapy of human liver cancers. The tolerability studies of GE11-PS-DOX and Lipo-DOX showed that no mice death and significant body weight loss was observed for GE11-PS-DOX group at dosages from 120 to 160 mg/kg in 10 days (Fig. S5). In contrast, Lipo-DOX exhibited significant toxicity at 20 mg DOX equiv./kg. The maximum-tolerated dose (MTD) of GE11-PS-DOX was at least 8-fold higher than Lipo-DOX. Similar increase of toleration was also reported for cNGQ-PS-DOX systems [37]. The significantly improved toleration of GE11-PS-DOX may be attributed to its excellent stability, little drug leakage in circulation and low uptake by healthy cells.

3.4. In vivo treatment of orthotopic SMMC-7721 tumor-bearing mice

Fig. 5. The in vivo pharmacokinetics of GE11-PS-DOX, PS-DOX and Lipo-DOX in KM mice (n = 5).

DOX. It should be noticed that Lipo-DOX had very high uptake by healthy liver tissues. It has been reported that galactose [9], folic acid [18] or glycyrrhetinic acid [19] modified nanosystems also showed a high uptake by normal liver tissues. GE11-PS-DOX could discriminate the cancerous cells and healthy cells, and selectively

The therapeutic performance of GE11-PS-DOX was then studied in nude mice bearing orthotopic SMMC-7721 tumors by i.v. injection at 6 or 12 mg DOX equiv./kg every four days. PS-DOX (12 mg DOX equiv./kg) and Lipo-DOX (6 mg DOX equiv./kg) were used as controls. Lipo-DOX at 12 mg DOX equiv./kg was not applied due to severe systemic toxicity. The results demonstrated that PBS treated mice showed fast tumor progression, severe liver ascites and abnormal increase of body weight (Fig.7A), and they became weaker over time and could hardly walk at the end. The mice were considered dead when their abdominal circumferences were over 100 mm. Two out of five mice treated with PBS were dead on

Fig. 6. The ex vivo fluorescence images (A) and quantitative biodistribution (n = 3) (B) of DOX in the tumors and major organs in subcutaneous SMMC-7721 tumor-bearing mice at 12 h post i.v. injection of GE11-PS-DOX or PS-DOX. One-way ANOVA with Tukey multiple comparisons tests, *p < .05. (C) The bright field (upper row) and ex vivo fluorescence (lower row) images of major organs of the orthotopic SMMC-7721 tumor-bearing mice at 12 h post i.v. injection of GE11-PS-DOX, PS-DOX or Lipo-DOX. The circles in the livers indicate the location of tumors.

Please cite this article in press as: Y. Fang et al., EGFR-targeted multifunctional polymersomal doxorubicin induces selective and potent suppression of orthotopic human liver cancer in vivo, Acta Biomater. (2017), https://doi.org/10.1016/j.actbio.2017.10.013

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Fig. 7. The in vivo antitumor efficacy of GE11-PS-DOX in orthotopic SMMC-7721 tumor-bearing nude mice (n = 6). The drug was given every 4 days (total 10 injections) at 6 or 12 mg DOX equiv./kg. PS-DOX (12 mg DOX equiv./kg), Lipo-DOX (6 mg DOX equiv./kg) and PBS were used as controls. For Lipo-DOX, 9 injections was given. (A) Body weight changes in 36 d. (B) The photograph of the mouse livers on day 44. (C) Survival curves within 180 d. The number of stars (*) represents the number of mice that died.

day 35. In contrast, the mice treated with GE11-PS-DOX and PSDOX were almost free of liver ascites in 40 days. Notably, LipoDOX treated mice showed significant body weight loss (Fig.7A) and from the second injection mice developed skin peeling and hand-foot syndrome (HFS) which was commonly occurred for Lipo-DOX, likely as a result of high systemic toxicity. One mouse of each group was sacrificed on day 44. The ex vivo photograph of excised mouse livers showed significant tumor evasion for mice treated with PBS and effective tumor inhibition by PS-DOX at 12 mg DOX equiv./kg and GE11-PS-DOX at 6 mg DOX equiv./kg. Remarkably, liver tumor was completely eradicated by the treatment with GE11-PS-DOX at 12 mg DOX equiv./kg and Lipo-DOX (Fig.7B). Moreover, the survival curves within 180 d revealed that LipoDOX had little improvement on mice survival rate (median survival time: 42 d versus 35 d for PBS group) (Fig.7C). The mice treated with PS-DOX (12 mg DOX equiv./kg) and GE11-PS-DOX (6 mg DOX equiv./kg) showed obviously longer median survival time of 70 and 72 d, respectively. Most interestingly, GE11-PS-DOX (12 mg DOX equiv./kg) group had markedly improved median survival time of 130 d without the sign of liver ascites, and achieved 50% complete regression. H&E staining of the major organs displayed that GE11-PS-DOX treatment caused no adverse effects to normal organs including liver while Lipo-DOX provoked obvious damage to the heart and liver (Fig. 8). Notably, a couple of targeted nanomedicines functionalized with anti-CD44 antibody, glycyrrhetinic acid, boronic acid, galactose or folic acid were studied for the treatment of orthotopic HepG2 [12], H22 [19,46], SK-HEP-1 [47] or Bel-7404 [48] liver

tumors. No tumor free mice were reported. Moreover, none have shown such a long survival time. This extraordinary antitumor activity of GE11-PS-DOX in orthotopic liver tumor is most probably a result of high stability and tumor accumulation, selective tumor cell internalization and fast intracellular drug release.

4. Conclusion We have demonstrated that GE11 peptide-directed polymersomal doxorubicin (GE11-PS-DOX) effectively targets and inhibits orthotopic SMMC7721 human liver tumor xenografts in mice. Notably, targetability of GE11-PS-DOX is critically dependent on GE11 molar contents, in which 10% GE11 appears the best. GE11functionalized multifunctional polymersomal doxorubicin has many interesting features: (i) it has a high drug loading of 15.4 wt%, small size of 78 nm, high stability, and glutathione-triggered release of DOX; (ii) it has a long circulation time (t1/2,b = 8.27 h) and an extraordinary accumulation in SMMC7721 tumors (13.3 % ID/g); (iii) it significantly improves mouse survival rate with 3.4fold longer median survival time as compared to Lipo-DOX control and achieves 50% complete regression; and (iv) it induces obviously lower systemic toxicity than Lipo-DOX. Hence, GE11decorated multifunctional polymersomal doxorubicin has a high potential in targeted chemotherapy of EGFR strongly expressed hepatocellular carcinoma.

Please cite this article in press as: Y. Fang et al., EGFR-targeted multifunctional polymersomal doxorubicin induces selective and potent suppression of orthotopic human liver cancer in vivo, Acta Biomater. (2017), https://doi.org/10.1016/j.actbio.2017.10.013

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Fig. 8. The microscopic images (40) of H&E stained major organs of orthotopic SMMC-7721 tumor-bearing mice treated with GE11-PS-DOX (6 or 12 mg DOX equiv./kg), PSDOX (12 mg DOX equiv./kg), Lipo-DOX (6 mg DOX equiv./kg) or PBS for 44 d. A and B indicate normal and cancerous liver tissue, respectively. Black triangles illustrate apoptotic cancer cells.

Acknowledgements This work is financially supported by research grants from the National Natural Science Foundation of China (NSFC 51473111, 51561135010, 51633005), a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, and Ph.D. Programs Foundation of Ministry of Education of China (20133201110005). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.actbio.2017.10.013. References [1] W. Chen, R. Zheng, P.D. Baade, S. Zhang, H. Zeng, F. Bray, A. Jemal, X.Q. Yu, J. He, Cancer statistics in China, CA Cancer J. Clin. 66 (2016) (2015) 115–132. [2] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, CA Cancer J. Clin. 67 (2017) (2017) 7–30. [3] P.J. Johnson, S. Qin, J.W. Park, R.T. Poon, J.L. Raoul, P.A. Philip, C.H. Hsu, T.H. Hu, J. Heo, J. Xu, L. Lu, Y. Chao, E. Boucher, K.H. Han, S.W. Paik, J. Robles-Avina, M. Kudo, L. Yan, A. Sobhonslidsuk, D. Komov, T. Decaens, W.Y. Tak, L.B. Jeng, D. Liu, R. Ezzeddine, I. Walters, A.L. Cheng, Brivanib versus sorafenib as first-line therapy in patients with unresectable, advanced hepatocellular carcinoma: results from the randomized phase III brisk-fl study, J. Clin. Oncol. 31 (2013) 3517–3524. [4] J.J. Knox, S.P. Cleary, L.A. Dawson, Localized and systemic approaches to treating hepatocellular carcinoma, J. Clin. Oncol. 33 (2015) 1835–1844. [5] H.Y. Kim, J.-W. Park, Clinical trials of combined molecular targeted therapy and locoregional therapy in hepatocellular carcinoma: past, present, and future, Liver Cancer 3 (2014) 9–17. [6] R. Qin, J.J. Knox, J.R. Strosberg, B. Tan, A. Kaubisch, A.B. El-Khoueiry, T.S. BekaiiSaab, S.R. Rousey, H.X. Chen, C. Erlichman, A phase II trial of bevacizumab plus temsirolimus in patients with advanced hepatocellular carcinoma, Invest. New Drugs 33 (2015) 241–246. [7] Y.D. Livney, Y.G. Assaraf, Rationally designed nanovehicles to overcome cancer chemoresistance, Adv. Drug Deliv. Rev. 65 (2013) 1716–1730. [8] I. Lyra-González, L.E. Flores-Fong, I. González-García, D. Medina-Preciado, J. Armendáriz-Borunda, Adenoviral gene therapy in hepatocellular carcinoma: a review, Hepatol. Int. 7 (2013) 48–58.

[9] S. Huang, S. Duan, J. Wang, S. Bao, X. Qiu, C. Li, Y. Liu, L. Yan, Z. Zhang, Y. Hu, Folic-acid-mediated functionalized gold nanocages for targeted delivery of anti-MIR-181b in combination of gene therapy and photothermal therapy against hepatocellular carcinoma, Adv. Funct. Mater. 26 (2016) 2532–2544. [10] M. Liu, Z.H. Li, F.J. Xu, L.H. Lai, Q.Q. Wang, G.P. Tang, W.T. Yang, An oligopeptide ligand-mediated therapeutic gene nanocomplex for liver cancer-targeted therapy, Biomaterials 33 (2012) 2240–2250. [11] Q. Hu, K. Wang, X. Sun, Y. Li, Q. Fu, T. Liang, G. Tang, A redox-sensitive, oligopeptide-guided, self-assembling, and efficiency-enhanced (rose) system for functional delivery of microrna therapeutics for treatment of hepatocellular carcinoma, Biomaterials 104 (2016) 192–200. [12] L. Wang, W. Su, Z. Liu, M. Zhou, S. Chen, Y. Chen, D. Lu, Y. Liu, Y. Fan, Y. Zheng, CD44 antibody-targeted liposomal nanoparticles for molecular imaging and therapy of hepatocellular carcinoma, Biomaterials 33 (2012) 5107–5114. [13] C.E. Ashley, E.C. Carnes, G.K. Phillips, D. Padilla, P.N. Durfee, P.A. Brown, T.N. Hanna, J. Liu, B. Phillips, M.B. Carter, N.J. Carroll, X. Jiang, D.R. Dunphy, C.L. Willman, D.N. Petsev, D.G. Evans, A.N. Parikh, B. Chackerian, W. Wharton, D.S. Peabody, C.J. Brinker, The targeted delivery of multicomponent cargos to cancer cells via nanoporous particle-supported lipid bilayers, Nat. Mater. 10 (2011) 389–397. [14] S. Shen, C.-Y. Sun, X.-J. Du, H.-J. Li, Y. Liu, J.-X. Xia, Y.-H. Zhu, J. Wang, Codelivery of platinum drug and sinotch1 with micelleplex for enhanced hepatocellular carcinoma therapy, Biomaterials 70 (2015) 71–83. [15] D.Q. Wu, B. Lu, C. Chang, C.S. Chen, T. Wang, Y.Y. Zhang, S.X. Cheng, X.J. Jiang, X. Z. Zhang, R.X. Zhuo, Galactosylated fluorescent labelled micelles as a liver targeting drug carrier, Biomaterials 30 (2009) 1363–1371. [16] M. Zhang, X. Zhou, B. Wang, B.C. Yung, L.J. Lee, K. Ghoshal, R.J. Lee, Lactosylated gramicidin-based lipid nanoparticles (Lac-gln) for targeted delivery of antimir-155 to hepatocellular carcinoma, J. Control. Release 168 (2013) 251–261. [17] Y. Zou, Y. Song, W. Yang, F. Meng, H. Liu, Z. Zhong, Galactose-installed photocrosslinked ph-sensitive degradable micelles for active targeting chemotherapy of hepatocellular carcinoma in mice, J. Control. Release 193 (2014) 154–161. [18] D. Zhu, W. Tao, H. Zhang, G. Liu, T. Wang, L. Zhang, X. Zeng, L. Mei, Docetaxel (DTX)-loaded polydopamine-modified TPGS-PLA nanoparticles as a targeted drug delivery system for the treatment of liver cancer, Acta Biomater. 30 (2016) 144–154. [19] C. Zhang, W. Wang, T. Liu, Y. Wu, H. Guo, P. Wang, Q. Tian, Y. Wang, Z. Yuan, Doxorubicin-loaded glycyrrhetinic acid-modified alginate nanoparticles for liver tumor chemotherapy, Biomaterials 33 (2012) 2187–2196. [20] Y.C. Shen, Z.Z. Lin, C.H. Hsu, C. Hsu, Y.Y. Shao, A.L. Cheng, Clinical trials in hepatocellular carcinoma: an update, Liver Cancer 2 (2013) 345–364. [21] J. Zhang, J.C. Garrison, L.Y. Poluektova, T.K. Bronich, N.A. Osna, Liver-targeted antiviral peptide nanocomplexes as potential anti-HCV therapeutics, Biomaterials 70 (2015) 37–47.

Please cite this article in press as: Y. Fang et al., EGFR-targeted multifunctional polymersomal doxorubicin induces selective and potent suppression of orthotopic human liver cancer in vivo, Acta Biomater. (2017), https://doi.org/10.1016/j.actbio.2017.10.013

Y. Fang et al. / Acta Biomaterialia xxx (2017) xxx–xxx [22] L. Wang, J. Yao, S. Xin, L. Hu, Z. Li, T. Song, H. Chen, MicroRNA-302b suppresses cell proliferation by targeting EGFR in human hepatocellular carcinoma SMMC-7721 cells, BMC Cancer 13 (2013) 1–9. [23] S.A. Eccles, The epidermal growth factor receptor/Erb-b/Her family in normal and malignant breast biology, Int. J. Dev. Biol. 55 (2011) 685–696. [24] J.L. Wang, G.P. Tang, J. Shen, Q.L. Hu, F.J. Xu, Q.Q. Wang, Z.H. Li, W.T. Yang, A gene nanocomplex conjugated with monoclonal antibodies for targeted therapy of hepatocellular carcinoma, Biomaterials 33 (2012) 4597–4607. [25] V. Kumar, Goutam Mondal, Surendra K. Shukla, Pankaj K. Singh, Ram I. Mahato, EGFR-targeted polymeric mixed micelles carrying gemcitabine for treating pancreatic cancer, Biomacromolecules 17 (2016) 301–313. [26] F. Ciardiello, G. Tortora, A novel approach in the treatment of cancer: targeting the epidermal growth factor receptor, Clin. Cancer Res. 7 (2001) 2958–2970. [27] G.M. Stella, M. Luisetti, S. Inghilleri, F. Cemmi, R. Scabini, M. Zorzetto, E. Pozzi, Targeting EGFR in non-small-cell lung cancer: lessons, experiences, strategies, Resp. Med. 106 (2012) 173–183. [28] P. Liu, Z. Li, M. Zhu, Y. Sun, Y. Li, H. Wang, Y. Duan, Preparation of EGFR monoclonal antibody conjugated nanoparticles and targeting to hepatocellular carcinoma, J. Mater. Sci-Mater. Med. 21 (2010) 551–556. [29] Z. Li, R. Zhao, X. Wu, Y. Sun, M. Yao, J. Li, Y. Xu, J. Gu, Identification and characterization of a novel peptide ligand of epidermal growth factor receptor for targeted delivery of therapeutics, FASEB. J. 19 (2005) 1978–1985. [30] A.M. Brinkman, G. Chen, Y. Wang, C.J. Hedman, N.M. Sherer, T.C. Havighurst, S. Gong, W. Xu, Aminoflavone-loaded EGFR-targeted unimolecular micelle nanoparticles exhibit anti-cancer effects in triple negative breast cancer, Biomaterials 101 (2016) 20–31. [31] L. Cheng, F.Z. Huang, L.F. Cheng, Y.Q. Zhu, Q. Hu, L. Li, L. Wei, D.W. Chen, GE11modified liposomes for non-small cell lung cancer targeting: preparation, ex vitro and in vivo evaluation, Int. J. Nanomed. 9 (2014) 921–935. [32] S. Song, D. Liu, J. Peng, Y. Sun, Z. Li, J.R. Gu, Y. Xu, Peptide ligand-mediated liposome distribution and targeting to EGFR expressing tumor in vivo, Int. J. Pharmaceut. 363 (2008) 155–161. [33] K. Muller, P.M. Klein, P. Heissig, A. Roidl, E. Wagner, EGF receptor targeted lipooligocation polyplexes for antitumoral siRNA and miRNA delivery, Nanotechnology 27 (2016) 464001. [34] L. Chang, G. Wang, T. Jia, L. Zhang, Y. Li, Y. Han, K. Zhang, G. Lin, R. Zhang, J. Li, Armored long non-coding RNA meg3 targeting EGFR based on recombinant ms2 bacteriophage virus-like particles against hepatocellular carcinoma, Oncotarget 7 (2016) 23988–24004. [35] M. Fan, D. Yang, X. Liang, J. Ao, Z. Li, H. Wang, B. Shi, Design and biological activity of epidermal growth factor receptor-targeted peptide doxorubicin conjugate, Biomed. Pharmacother. 70 (2015) 268–273. [36] F.M. Mickler, L. Mockl, N. Ruthardt, M. Ogris, E. Wagner, C. Brauchle, Tuning nanoparticle uptake: live-cell imaging reveals two distinct endocytosis mechanisms mediated by natural and artificial EGFR targeting ligand, Nano Lett. 12 (2012) 3417–3423.

11

[37] Y. Zou, F.H. Meng, C. Deng, Z.Y. Zhong, Robust, tumor-homing and redoxsensitive polymersomal doxorubicin: a superior alternative to Doxil and Caelyx?, J Control. Release 239 (2016) 149–158. [38] N. Zhang, Y. Xia, Y. Zou, W.J. Yang, J. Zhang, Z.Y. Zhong, F.H. Meng, ATN-161 peptide functionalized reversibly cross-linked polymersomes mediate targeted doxorubicin delivery into melanoma-bearing c57bl/6 mice, Mol. Pharm. (2017), https://doi.org/10.1021/acs.molpharmaceut.6b00800. [39] Y. Fang, Y. Jiang, Y. Zou, F.H. Meng, J. Zhang, C. Deng, H.L. Sun, Z.Y. Zhong, Targeted glioma chemotherapy by cyclic RGD peptide-functionalized reversibly core-crosslinked multifunctional poly(ethylene glycol)-b-poly(ecaprolactone) micelles, Acta Biomater. 50 (2017) 396–406. [40] Y. Zou, Y. Fang, H. Meng, F.H. Meng, C. Deng, J. Zhang, Z.Y. Zhong, Selfcrosslinkable and intracellularly decrosslinkable biodegradable micelles: a robust, simple and multifunctional nanoplatform for high-efficiency targeted cancer chemotherapy, J. Control. Release 244 (2016) 326–335. [41] W.J. Yang, Y. Zou, F.H. Meng, J. Zhang, R. Cheng, C. Deng, Z.Y. Zhong, Efficient and targeted suppression of human lung tumor xenografts in mice with methotrexate sodium encapsulated in all-function-in-one chimaeric polymersomes, Adv. Mater. 28 (2016) 8234–8239. [42] S.Y. An, S.H. Hong, C. Tang, J.K. Oh, Rosin-based block copolymer intracellular delivery nanocarriers with reduction-responsive sheddable coronas for cancer therapy, Polym. Chem. 7 (2016) 4751–4760. [43] N.R. Ko, J.K. Oh, Glutathione-triggered disassembly of dual disulfide located degradable nanocarriers of polylactide-based block copolymers for rapid drug release, Biomacromolecules 15 (2014) 3180–3189. [44] D. Hu, O. Mezghrani, L. Zhang, Y. Chen, X. Ke, T. Ci, GE11 peptide modified and reduction-responsive hyaluronic acid-based nanoparticles induced higher efficacy of doxorubicin for breast carcinoma therapy, Int. J. Nanomed. 11 (2016) 5125–5147. [45] Y.N. Zhong, J. Zhang, R. Cheng, C. Deng, F.H. Meng, F. Xie, Z.Y. Zhong, Reversibly crosslinked hyaluronic acid nanoparticles for active targeting and intelligent delivery of doxorubicin to drug resistant CD44+ human breast tumor xenografts, J. Control. Release 205 (2015) 144–154. [46] J. Wang, Z. Zhang, X. Wang, W. Wu, X. Jiang, Size- and pathotropism-driven targeting and washout-resistant effects of boronic acid-rich protein nanoparticles for liver cancer regression, J. Control. Release 168 (2013) 1–9. [47] B. Singh, Y. Jang, S. Maharjan, H.-J. Kim, A.Y. Lee, S. Kim, N. Gankhuyag, M.-S. Yang, Y.-J. Choi, M.-H. Cho, C.-S. Cho, Combination therapy with doxorubicinloaded galactosylated poly(ethyleneglycol)-lithocholic acid to suppress the tumor growth in an orthotopic mouse model of liver cancer, Biomaterials 116 (2017) 130–144. [48] D. Ling, H. Xia, W. Park, M.J. Hackett, C. Song, K. Na, K.M. Hui, T. Hyeon, pHsensitive nanoformulated triptolide as a targeted therapeutic strategy for hepatocellular carcinoma, ACS Nano 8 (2014) 8027–8039.

Please cite this article in press as: Y. Fang et al., EGFR-targeted multifunctional polymersomal doxorubicin induces selective and potent suppression of orthotopic human liver cancer in vivo, Acta Biomater. (2017), https://doi.org/10.1016/j.actbio.2017.10.013