Hyaluronic acid-shelled disulfide-crosslinked nano-polymersomes for

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Hyaluronic acid-shelled disulfide-crosslinked nanopolymersomes for ultrahigh-efficiency reactive encapsulation and CD44-targeted delivery of mertansine toxin Yue Zhang, Kaiqi Wu, Huanli Sun, Jian Zhang, Jiandong Yuan, and Zhiyuan Zhong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17718 • Publication Date (Web): 22 Dec 2017 Downloaded from http://pubs.acs.org on December 23, 2017

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ACS Applied Materials & Interfaces

Hyaluronic acid-shelled disulfide-crosslinked nano-polymersomes for ultrahigh-efficiency reactive encapsulation and CD44-targeted delivery of mertansine toxin Yue Zhang1, Kaiqi Wu1, Huanli Sun1,*, Jian Zhang1, Jiandong Yuan2 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, P. R. China. 2

BrightGene Bio-Medical Technology Co., Ltd., Suzhou, 215123, P. R. China.

* Corresponding authors. Tel/Fax: +86-512-65880098, Email: [email protected] (H. Sun); [email protected] (Z. Zhong)

ABSTRACT It was and remains a big challenge for cancer nanomedicines to achieve high and stable drug loading while fast drug release in the target cells. Here, we report on novel hyaluronic acid-shelled disulfide-crosslinked biodegradable polymersomes (HA-XPS) self-assembled from hyaluronic acid-b-poly(trimethylene carbonate-co-dithiolane trimethylene carbonate) diblock copolymer for ultrahigh-efficiency reactive encapsulation and CD44-targeted delivery of mertansine (DM1) toxin, a highly potent warhead for clinically used antibody-drug 1 ACS Paragon Plus Environment

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conjugates. Remarkably, HA-XPS showed quantitative encapsulation of DM1 even with a high drug loading content of 16.7 wt.%. DM1-loaded HA-XPS (HA-XPS-DM1) presented a small size of ~80 nm, low drug leakage under physiological condition and fast glutathione-triggered drug release. MTT assays revealed that HA-XPS was non-cytotoxic while HA-XPS-DM1 was highly potent to MDA-MB-231 cells with an IC50 comparable to free DM1. The in vitro and in vivo inhibition experiments indicated that HA-XPS could actively target to MDA-MB-231 cells. Notably, HA-XPS-DM1 while causing little adverse effects could effectively inhibit tumor growth and significantly prolong survival time in MDA-MB-231 human breast tumor-bearing mice. HA-XPS-DM1 provides a novel and unique treatment for CD44-positive cancers. KEYWORDS: Hyaluronic acid; reduction-sensitive; polymersomes; breast tumor; targeted chemotherapy

1. INTRODUCTION Antibody-drug conjugates (ADCs) that are currently used in the clinics or clinical trials for targeted cancer treatment represent the most advanced nanomedicines.1-4 However, ADCs including trastuzumab mertansine (T-DM1)5,6 have typically a low drug conjugation content (less than 2.0 wt.%).7 Moreover, ADCs also suffer drawbacks of high cost and possible immune responses following repeated administration. Interestingly, in spite of its clinical significance, targeted delivery of DM1 other than ADCs has received significantly less attention than for other clinically used anticancer drugs like paclitaxel and doxorubicin.8,9 One probable reason is that highly toxic DM1 drug demands nanosystems with far more stringent requirements, which include efficient and stable drug encapsulation preferably via covalent conjugation as well as cell-specific drug delivery. To this end, we have designed 2 ACS Paragon Plus Environment

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reduction-sensitive hyaluronic acid-SS-DM1 prodrug10 and micellar DM1 prodrug11 that showed targeted treatment of breast tumor and melanoma-bearing mice, respectively. It takes, nevertheless, multiple steps to synthesize these DM1 prodrugs. Nano-polymersomes that structurally mimic liposomes are a particularly versatile class of delivery vehicles.12-17 Notably, nano-polymersomes are capable of encapsulating not only water soluble drugs (e.g. doxorubicin hydrochloride,18-22 proteins23-25 and nucleic acids26-28) but also lipophilic drugs (e.g. paclitaxel29,30 and docetaxel31,32). In the past years, aiming at enhancing drug therapeutic efficacy and reducing off-target toxicity, various functional polymersomes have been developed to achieve tumor-targeted33-35 and/or stimuli-sensitive drug release.36-41 We recently found that cNGQ and ATN-161 peptide-functionalized disulfide-crosslinked

biodegradable

glycol)-b-poly(trimethylene

polymersomes

carbonate-co-dithiolane

based

on

trimethylene

poly(ethylene carbonate)

(PEG-b-P(TMC-co-DTC)) copolymer could efficiently load and deliver doxorubicin hydrochloride to lung tumor and melanoma bearing mice, respectively.42,43 More interestingly, these polymersomes showed reactive loading of DM1 onto the vesicular membrane.44 Here, we report on novel hyaluronic acid-shelled disulfide-crosslinked biodegradable polymersomes (HA-XPS) self-assembled from HA-b-P(TMC-co-DTC) diblock copolymer for high-efficiency reactive encapsulation and CD44-targeted delivery of DM1 (Scheme 1). Notably, HA, a biotic and natural polysaccharide, acts as not only biocompatible hydrophilic shell but also active targeting ligand to CD44. Different groups reported that nanomedicines containing HA ligand or shell can actively target to CD44 overexpressed tumor cells in vitro and in vivo.45-50 Biodegradable P(TMC-co-DTC) block forms robust and reduction-sensitive vesicular membrane that allows not only reactive loading of DM1 via thiol-disulfide exchange 3 ACS Paragon Plus Environment


reaction but also triggered fast release of DM1 inside the cancer cells. Notably, our results revealed that HA-XPS achieved stable and quantitative DM1 loading even at a high theoretical drug loading content (DLC) of 16.7 wt.%. DM1-loaded HA-XPS (HA-XPS-DM1) demonstrated good targetability to CD44-positive MDA-MB-231 human breast tumor in nude mice and significantly better tumor suppression than free DM1. HA-XPS has appeared as an ideal platform for targeted delivery of DM1 to CD44-positive tumors.

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Scheme 1. Illustration of hyaluronic acid-shelled disulfide-crosslinked biodegradable polymersomes (HA-XPS) for high-efficiency reactive encapsulation and CD44-targeted delivery of mertansine (DM1) in MDA-MB-231 human breast tumor-bearing nude mice.

2. EXPERIMENTAL SECTION 2.1. Synthesis of HA-b-P(TMC-co-DTC) block copolymer. 5 ACS Paragon Plus Environment


HA-b-P(TMC-co-DTC) block copolymer was acquired via click reaction of HA-propargylamine with N3-P(TMC-co-DTC). HA-propargylamine was obtained as we previously reported (Figure S1, 1H NMR spectrum).51 Under a nitrogen atmosphere, HA-propargylamine (278 mg, 0.035 mmol) in 5 mL of dimethylsulfoxide (DMSO), copper(II) sulfate pentahydrate (CuSO4·5H2O, 1.45 mg, 6.0 µmol) in 0.1 mL of water, and sodium ascorbate (NaAsc, 2.38 mg, 12 µmol) in 0.1 mL of water were sequentially added to a stirred DMSO (5.0 mL) solution of N3-P(TMC-co-DTC) (500 mg, 29 µmol) followed by reacting at 50 °C in the dark. After 24 h, the reaction solution was extensively dialyzed against DMSO for 24 h, saturated aqueous EDTA solution for 24 h and deionized water for several times before lyophilization. Yield: 84%. 1H NMR (600 MHz, D2O/DMSO-d6 = 1/10) δ (ppm): 1.79, 3.03-3.67, and 4.41-4.49 (HA); 1.91 and 4.10 (TMC); 3.03 and 4.11 (DTC).

2.2. Fabrication of HA-XPS-DM1 and HA-XPS. Typically, 100 µL of HA-b-P(TMC-co-DTC) (10 mg/mL) and 10 µL of DM1 (10 mg/mL) in DMSO were mixed well and injected into 900 µL of phosphate buffer (PB, 10 mM, pH 7.4) under vigorous stirring. The resulted dispersion following 24 h shaking at 200 rpm and 37 °C was dialyzed (Spectra/Pore, MWCO 14000) against PB containing 0.2% Tween 80 for 48 h to remove DMSO and unreacted DM1. HA-XPS was prepared similarly to HA-XPS-DM1 but without adding DM1. The colloidal stability of HA-XPS against 100-fold dilution or 10% fetal bovine serum (FBS) was investigated using dynamic light scattering (DLS). To verify that DM1 was conjugated to polymersomes via disulfide bonds, HA-XPS-DM1 with or without 10 mM dithiothreitol (DTT) treatment, was subjected to HPLC measurements. 6 ACS Paragon Plus Environment

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To determine DLC and the drug loading efficiency (DLE), HA-XPS-DM1 was lyophilized and dissolved in acetonitrile containing 10 mM DTT for HPLC measurements. DLC and DLE were calculated using the following equations: DLC (wt.%) = (weight of loaded drug/total weight of loaded drug and polymer) × 100 DLE (%) = (weight of loaded drug/weight of drug in feed) × 100

2.3. In vitro cell experiments. The experimental details regarding the in vitro cell culture, MTT assay, flow cytometry and confocal microscopy measurements are presented in the Supporting Information.

2.4. In vivo pharmacokinetics and imaging. The mice were handled under protocols approved by Soochow University Laboratory Animal Center and the Animal Care and Use Committee of Soochow University. The healthy BALB/c mice were weighed around 20 g (n = 3). Cyanine5-labeled HA-XPS (Cy5-HA-XPS) (1.5 µg Cy5 equiv./mouse) was intravenously injected via tail vein. At determined time points, blood samples (~20 µL) were collected from the orbit. 100 µL of Triton X-100 (1%) and 600 µL of DMSO were added to extract Cy5 for 48 h at 25 ºC. Cy5 level in the supernatant was measured via ﬂuorescence spectrophotometer (ex. 640 nm, em. 668 nm). The blood circulation half-lives (t1/2,α and t1/2,β) were obtained according to the following second-order exponential decay ﬁts: C = A1 × exp (-αt1) + A2 × exp (-βt2) + C0 For in vivo imaging studies, BALB/c mice were injected in the hind flank with 1 × 107 MDA-MB-231 cells containing matrigel in 50 µL of PBS to establish subcutaneous human breast tumor model. When tumor sizes reached 100-200 mm3, Cy5-HA-XPS was 7 ACS Paragon Plus Environment


administrated via tail vein injection. The fluorescent scans were performed at 3, 6, 9, 12 and 24 h post i.v. injection using a near-infrared fluorescence imaging system (IVIS Lumina II). The mouse injected with free HA (50 mg/kg) 0.5 h before the administration of Cy5-HA-XPS was used as a control.

2.5. In vivo antitumor efficacy. MDA-MB-231 tumor-bearing mice were treated with HA-XPS-DM1 or free DM1 at a dosage of 0.4 or 1.0 mg DM1 equiv./kg when tumor sizes reached 80 mm3. The drug was intravenously injected every three days for 4 times in total. PBS was injected as a control. The tumor length (L) and width (W) were measured every other day using calipers and the weight of mice were recorded at the same time. Tumor volumes (V) were calculated based on the equation of V = 0.5 × L × W2. Relative tumor volumes (V/V0) and body weights were calculated with respect to their initial data just before the treatment. Mice were considered to be dead when the tumor volume increased to 1000 mm3.

3. RESULTS AND DISCUSSION 3.1. Synthesis of HA-b-P(TMC-co-DTC) block copolymer. HA-b-P(TMC-co-DTC) copolymer was synthesized via click reaction between HA-propargylamine and N3-P(TMC-co-DTC) (Scheme 2). N3-P(TMC-co-DTC) was readily prepared by ring opening copolymerization of TMC with DTC using 2-azidoethanol as an initiator and diphenyl phosphate (DPP) as a catalyst ([TMC]0/[DTC]0/[I]0 = 147/10.4/1) in dichloromethane at 40 °C. 1H NMR spectrum of N3-P(TMC-co-DTC) clearly showed signals at δ 3.73 attributable to 2-azidoethylene end group (Figure S2). The Mn of N3-P(TMC-co-DTC) was determined to be 16.9 kg/mol, close to the designed value of 17 kg/mol, by comparing the integrals of signals at δ 2.05 (methylene protons of TMC moieties) 8 ACS Paragon Plus Environment

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and 3.03 (dithiolane methylene protons of DTC moieties) to 3.73 (methylene protons of 2-azidoethylene), respectively. Gel permeation chromatography (GPC) showed that N3-P(TMC-co-DTC) had a unimodal distribution with a low Mw/Mn of 1.13. These results are consistent with previous reports that DPP induces controlled ring-opening (co)polymerization of TMC.52,53

Scheme 2. Synthesis of HA-b-P(TMC-co-DTC) block copolymer. Conditions: (i) DPP, 40 °C, 72 h; (ii) CuSO4, NaAsc, N2, DMSO/H2O, 50 °C, 24 h.

The click reaction between HA-propargylamine and N3-P(TMC-co-DTC) proceeded at 50 °C in DMSO/H2O for 24 h.

1

H NMR spectrum showed besides peaks of

N3-P(TMC-co-DTC) also signals originated from HA at δ 1.79, 3.03-3.67, and 4.41-4.49 (Figure 1). FTIR spectra displayed that the absorptions at 2108 and 2171 cm-1 owing to the azide in N3-P(TMC-co-DTC) and alkynyl group in HA-propargylamine, respectively, completely disappeared in HA-b-P(TMC-co-DTC) (Figure 2), similar to a previous report for HA-b-poly(ε-caprolactone) copolymer obtained via click reaction.54 Besides, characteristic absorption peaks related to both HA (3390 and 1650 cm-1) and P(TMC-co-DTC) (1740 cm-1) were detected, supporting formation of HA-b-P(TMC-co-DTC) block copolymer. 9 ACS Paragon Plus Environment


~

~

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~ D 2O

DMSO-d6 d

c,e b HA f a 5.0

4.5

4.0 3.5 3.0 2.5 Chemical Shift (ppm)

2.0

1.5

Figure 1. 1H NMR spectrum (600 MHz, D2O/DMSO-d6 = 1/10) of HA-b-P(TMC-co-DTC).

HA-propargylamine

N3-P(TMC-co-DTC)

HA-b-P(TMC-co-DTC)

Figure

2.

4000

3500

3000

FTIR

spectra

of

2500 2000 1500 -1 Wavenumber (cm )

HA-propargylamine,

HA-b-P(TMC-co-DTC) polymers. 10 ACS Paragon Plus Environment

1000

500

N3-P(TMC-co-DTC)

and

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3.2 Preparation of hyaluronic acid-shelled disulfide-crosslinked nano-polymersomes. HA-b-P(TMC-co-DTC) formed monodisperse polymersomes (HA-XPS) with an average size of 103 nm and a low polydispersity (PDI) of 0.09, as revealed by DLS (Figure 3A). Atomic force microscopy (AFM) image of HA-XPS showed a spherical morphology with a size consistent with DLS (Figure 3B). Furthermore, the average height of the air-dried HA-XPS was ~8.5 nm, which was more than 10-fold lower than their diameter (Figure 3C). The observed high diameter-to-height ratio is in line with the deformation of polymersomes upon drying, confirming the vesicular structure of HA-XPS.55,56 HA-XPS showed a high colloidal stability against extensive dilution or 10% FBS (Figure 3D), supporting that vesicular

membrane

is

self-crosslinked,

as

for

polymersomes

formed

from

PEG-b-P(TMC-co-DTC) block copolymers.42-44

Figure 3. Characterization of HA-XPS. (A) Size distribution determined by DLS; (B) AFM image in the air-dried state; (C) The cross-sectional height profile along the line in B; (D) Colloidal stability against 100-fold dilution to a final concentration of 10 µg/mL and in 10% 11 ACS Paragon Plus Environment


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FBS.

Figure 4. (A) HPLC curves of HA-XPS-DM1 before and after DTT treatment. Free DM1 was used as a control; (B) In vitro release of DM1 from HA-XPS-DM1 in the presence or absence of 10 mM GSH (n = 3).

Table 1. Characterization of HA-XPS-DM1 in PB. DLC (wt.%) Entry

b

Size (nm)a

PDIa

DLEb (%)

Theory

Determined

1

9.1

9.1

82

0.06

> 99

2

16.7

16.7

83

0.03

> 99

a

Determined by DLS at 25 °C with a polymer concentration of 1.0 mg/mL.

b

Determined by HPLC.

Interestingly, addition of DM1 during self-assembly of HA-b-P(TMC-co-DTC) yielded polymersomal prodrug with a smaller size of ca. 82 nm and a low PDI of 0.06. Moreover, HPLC analyses indicated quantitative loading of DM1 at a theoretical DLC of 9.1 or 16.7 wt.% (Table 1). HPLC curves showed absence of free DM1 in HA-XPS-DM1 (Figure 4A), supporting that DM1 is chemically conjugated to HA-b-P(TMC-co-DTC). However, after adding 10 mM DTT, DM1 peak was discerned exactly the same for free drug, corroborating reduction-triggered release of DM1 in its native form. This clean, stable and quantitative 12 ACS Paragon Plus Environment

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conjugation of drug to nanocarriers during fabrication is unique and has many advantages over prodrugs and prodrug nanoparticles that require multi-step synthesis and purification.10,11 Notably, HA-XPS-DM1 has about 10-fold higher drug loading levels than clinically used T-DM1.5,6 The in vitro drug release studies showed that less than 10% of DM1 was released in 24 h under pH 7.4 at 37 °C, while over 80% DM1 release was observed under a reducing environment (Figure 4B). These results suggest that HA-XPS-DM1 has good stability and fast responsive drug release. The high stability will markedly reduce drug leakage in circulation, thus abating systemic toxicities. It should further be noted that HA-XPS-DM1 following lyophilization could be nicely re-dispersed, which renders it easy for storage and use.

3.3. In vitro antitumor efficacy of HA-XPS-DM1. MTT assays showed that HA-XPS-DM1 was highly potent against CD44 overexpressing MDA-MB-231 cells with a low IC50 of ca. 0.11 µg DM1 equiv./mL, which was comparable to free DM1 (Figure 5A), supporting effective delivery and release of DM1 into MDA-MB-231 cells. Importantly, blank HA-XPS was nontoxic towards MDA-MB-231 cells with concentrations ranging from 0.01 to 1.0 mg/mL (Figure 5B).



A

B 80

120

Free DM1 HA-XPS-DM1

Cell Viability (%)

100 Cell Viability (%)

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 40 20 0

100 80 60 40 20 0

0.01 0.05 0.1 0.5 1.0 HA-XPS Concentration (mg/mL)

1E-3 0.01 0.1 1 10 DM1 Concentration (µg/mL)

Figure 5. (A) MTT assays of HA-XPS-DM1 and free DM1 in MDA-MB-231 cells. The cells were incubated with drug for 4 h and then cultured in fresh medium for another 68 h (n = 4). (B) MTT assays of blank HA-XPS in MDA-MB-231 cells after 48 h incubation (n = 4).

To study its cellular uptake, HA-XPS was labeled with Cy5. Flow cytometry data showed that Cy5-HA-XPS was efficiently taken up by CD44-positive MDA-MB-231 cells following 4 h incubation. The cellular uptake was markedly reduced when MDA-MB-231 cells were pretreated using free HA before adding Cy5-HA-XPS (Figure 6A), corroborating receptor-mediated uptake of HA-XPS by MDA-MB-231 cells. However, for CD44-negative L929 cells, the cellular uptake of Cy5-HA-XPS was ~2.2 fold lower than that for MDA-MB-231 cells and no obvious change was observed after pretreating cells with free HA (Figure 6B). Polymersomes based on hyaluronan-b-poly(γ-benzyl glutamate) block copolymer were reported to mediate targeted delivery of doxorubicin to CD44 overexpressed breast tumor cells in vitro and in vivo.57,58 CLSM images displayed strong Cy5 fluorescence in MDA-MB-231 cells following 4 h incubation with Cy5-HA-XPS (Figure 6C). On the contrary, little Cy5 fluorescence was discerned in MDA-MB-231 cells pretreated with free HA, confirming active targeting ability of HA-XPS to MDA-MB-231 cells.


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Figure 6. Flow cytometry studies of (A) MDA-MB-231 and (B) L929 cells following 4 h incubation with Cy5-HA-XPS. PBS was used as a control. (C) CLSM images of MDA-MB-231 cells after 4 h incubation with Cy5-HA-XPS (scale bar: 20 µm). Cells pretreated with 5 mg/mL of HA for 4 h (+ Free HA) were used in the competitive inhibition experiments.

3.4. In vivo pharmacokinetics and therapeutic efficacy of HA-XPS-DM1.

Figure 7A showed that HA-XPS had a long elimination half-life (t1/2,β = 3.14 h) in the BALB/c mice. Near-infrared fluorescence imaging revealed that Cy5-HA-XPS quickly and strongly accumulated in MDA-MB-231 tumor xenografts in nude mice (Figure 7B). Cy5 fluorescence in the tumor site reached the maximum at 9 h after injection. Notably, comparably lower tumor accumulation of Cy5-HA-XPS was observed in free HA pretreated mice. It is evident that HA-XPS has a long circulation time and good targeting ability to 15 ACS Paragon Plus Environment


MDA-MB-231 human breast tumor.

Figure 7. (A) In vivo pharmacokinetics of Cy5-HA-XPS in BALB/c mice (n=3). (B) In vivo fluorescence images of MDA-MB-231 human breast tumor-bearing nude mice at different time points following injection of Cy5-HA-XPS. Free HA (50 mg/kg) pre-treated group was used as a control.

The toleration studies in BALB/c mice showed a maximum-tolerated dose of 2.0 mg DM1 equiv./kg for HA-XPS-DM1 (Figure S3), which was over 2 times better than free 16 ACS Paragon Plus Environment

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DM1.10,44,59 The therapeutic studies were then performed in MDA-MB-231 human breast tumor-bearing nude mice with HA-XPS-DM1 at a dosage of 0.4 or 1.0 mg DM1 equiv./kg or with free DM1 at 0.4 mg/kg. The treatment was repeated every three days for 4 times. As shown in Figure 8A, HA-XPS-DM1 with a dose of 0.4 mg DM1 equiv./kg caused significantly more effective tumor inhibition than free DM1 (*p < 0.05), supporting that HA-XPS-DM1 has good targeting ability and DM1 is released into MDA-MB-231 cells. Further improved treatment was achieved for HA-XPS-DM1 at an increased dosage of 1.0 mg DM1 equiv./kg, in which tumor growth was largely suppressed within 16 d. The photos of tumor lumps collected on day 16 confirmed that tumor volume followed an order of HA-XPS-DM1 (1.0 mg DM1 equiv./kg) < HA-XPS-DM1 (0.4 mg DM1 equiv./kg) < free DM1 (0.4 mg/kg) < PBS (Figure 8B). Notably, no body weight changes were observed for all treatment groups (Figure 8C), indicating that HA-XPS-DM1 even with a dose of 1.0 mg DM1 equiv./kg didn’t cause obvious adverse effects. Additionally, mice treated with HA-XPS-DM1 (1.0 mg DM1 equiv./kg) had a median survival time of 48 d, which was significantly improved over free DM1 and HA-XPS-DM1 at 0.4 mg DM1 equiv./kg (Figure

8D). Interestingly, one mouse in the HA-XPS-DM1 (1.0 mg DM1 equiv./kg) group became tumor-free and survived over an experimental period of 60 d. The pronounced adverse effects of highly toxic maytansinoids are a major barrier for their clinical translation.60-62 The improved targetability and treatment effect as well as reduced systemic toxicity observed for HA-XPS-DM1 over free DM1 renders them interesting for further investigation.



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Figure 8. In vivo antitumor efficacy of HA-XPS-DM1 in MDA-MB-231 tumor bearing nude mice with 4-doses treatment. Free DM1 and PBS groups were used as controls. (A) Change of relative tumor volumes following treatments (n = 6). * p

Hyaluronic acid-shelled disulfide-crosslinked nano-polymersomes for

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