Specifically Increased Paclitaxel Release in Tumor and Synergetic

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Specifically increased paclitaxel release in tumor and synergetic therapy by a hyaluronic acid-tocopherol nanomicelle Hanbo Zhang, Wei Li, Xiaomeng Guo, Fenfen Kong, Zuhua Wang, Chunqi Zhu, Lihua Luo, Qingpo Li, Jie Yang, Yongzhong Du, and Jian You ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 25 May 2017 Downloaded from http://pubs.acs.org on May 27, 2017

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ACS Applied Materials & Interfaces

Specifically increased paclitaxel release in tumor and synergetic therapy by a hyaluronic acid-tocopherol nanomicelle Hanbo Zhang, Wei Li, Xiaomeng Guo, Fenfen Kong, Zuhua Wang, Chunqi Zhu, Lihua Luo, Qingpo Li, Jie Yang, Yongzhong Du, and Jian You*

College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, Zhejiang 310058, P. R. China

* Corresponding Author: Jian You, College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, Zhejiang, China. Office: 086-571-88208443 Fax: 086-571-88208439 Email: [email protected].

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Abstract Recently, interest in tumor-targeting and site-specific drug release from nanoparticles as a means of drug delivery has increased. In this study, we reported on a smart nanosized micelle formed by hyaluronic acid (HA) conjugated with D-α-Tocopherol Succinate (TOS) using a disulfide bond as the linker (HA-SS-TOS, HSST). HSST micelles can specifically bind to the CD44 receptors that are overexpressed on cancer cells. The high levels of glutathione (GSH) in tumor cells selectively break the disulfide bond linker. This effect results in the synchronous release of the payload and a TOS fragment. These two components subsequently demonstrate synergetic anticancer activity. First, we demonstrated that drug release from HSST occurred rapidly in physiological high redox conditions and inside cancer cells. Significant GSH-triggered drug release was also observed in vivo. Furthermore, an in vivo biodistribution study indicated that the HSST micelles efficiently accumulated into tumor sites, primarily due to an enhanced permeability and retention (EPR) effect and the efficient binding to cancer cells that overexpressed the CD44 receptor. Interestingly, the synchronous release of paclitaxel (PTX) and the TOS fragment from PTX-loaded HSST caused synergetic tumor cell killing and tumor growth inhibition. Our work presented a useful candidate for a drug delivery system that could specifically accumulate into tumor tissue, selectively release its payload and a TOS fragment, and thus display a synergetic anticancer effect. Keywords: redox-responsive, CD44-targeting, hyaluronic acid, D-α-tocopherol succinate, synergetic tumor therapy 2 ACS Paragon Plus Environment

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1. Introduction In the past decade, nanomedicine has received considerable attention in the area of cancer therapy for its notable ability to improve anticancer activity and substantially reduce the side effects of drugs.1-3 In this context, self-assembled nanosized polymeric micelles offer many advantages such as prolonged blood circulation, continuous accumulation in tumor site and increased solubility of hydrophobic drugs.3-5 However, as a drug delivery system, the micelles are plagued with some urgent problems including the lack of controlled drug release at the target site and nonspecific biodistribution in vivo.6, 7 Furthermore, it should be noted that most micelles are formed from physiologically inert materials that lack biocompatibility and biodegradability,4,

11 and 12

and thus play the roles only of

encapsulating the drugs and delivering them to the targets. Redox-responsive nanoparticles that are sensitive to the differences in the redox potential between normal and tumor tissues and between the intracellular and extracellular environment are one of the most studied stimulus-responsive drug delivery systems.9 Such nanoparticles selectively release their payloads in cancer cells due to the 100- to 1 000-fold higher glutathione level in cancer cells compared with normal cells. This process meets the requirement of ‘switch on/off’ processes with spatial, temporal and dosage control of drug release.8-10 In this work, a smart micelle was designed using hyaluronic acid (HA) conjugated with D-α-tocopherol succinate (TOS) through the redox-sensitive cystamine linkage of a disulfide bond (HA-SS-TOS, HSST). CD44, which serves as a hyaluronic acid 3 ACS Paragon Plus Environment


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receptor and is overexpressed on the cell surfaces of many malignant tumors, was expected to mediate uptake of the HSST micelles into cancer cells to achieve tumor-targeting and enhancing treatment efficacy.13-15 TOS, a vitamin E derivative, has frequently been investigated and has been shown to suppress some types of cancer by inhibiting DNA biosynthesis in the cancer cells,19, 20 which induces cancer cell apoptosis,21 inhibits tumor angiogenesis and suppresses tumor growth and metastasis.22 These observations suggested that the simultaneous administration of paclitaxel (PTX) and TOS might result in a synergetic effect with an enhanced anticancer efficacy. Therefore, in our designed system, TOS serves not only as the hydrophobic core of the micelle for the encapsulation of the hydrophobic PTX but also as an antitumor agent itself that provides a synergetic therapy with the payload. Many HA derivatives have been designed so far and were employed to shuttle PTX molecules for CD44 mediated tumor-targeting therapy

14, 24 and 25

. However, most of

them lacked effective control of drug release. We hypothesized that the disulfide bond linkage between HA and TOS would induce a rapid release of the PTX payload and the TOS in redox environment of tumors and thus achieve an enhanced anticancer therapy. The stability of HSST in various redox environments was investigated. The redox-responsive release behavior was further confirmed in vitro and in vivo. The CD44-mediated tumor-targeting delivery and the anticancer activity of the PTX-loaded HSST were studied. For comparison, analogous micelles that were insensitive to the strongly reducing physiological environment (HA-CC-TOS, HCCT) were also synthesized using an analogous aliphatic chain bond as linker. 4 ACS Paragon Plus Environment

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2. Results 2.1. Synthesis and characterization of HSST and HCCT conjugates Cystamine-conjugated HA (HA-CYS) and 1, 6-diaminohexane-conjugated HA (HA-HMDA) were first synthesized by amine-reactive coupling. Then, the carboxyl group on TOS was activated by EDC and NHS, followed by coupling with amino group on HA-CYS or HA-HMDA. Therefore, the only chemical structure difference between

HA-SS-TOS

(HSST,

redox-responsive)

and

HA-CC-TOS

(HCCT,

non-redox-responsive) was the linkage between the HA backbone and the TOS fragment (Scheme 1 and Figure S1). Figure 1 shows the 1H NMR of HA, HA-HMDA, HA-CYS, HCCT and HSST. The peaks at ~0.95 ppm, ~1.92 ppm, ~2.87 ppm and ~2.89 ppm belonged to protons of the methyl hydrogen in TOS (12H, -CH-(CH3)-CH3 and -CH2-CH-(CH3)-CH2-), N-acetyl in HA (3H, -CO-CH3), methylene in cystamine (2H, -CH2-CH2-NH2) and methylene in 1, 6-diaminohexane (2H, -CH2-CH2-NH2), respectively

(Figure

S2-4).

Successful

conjugation

of

cystamine

and

1,

6-diaminohexane onto the backbone of HA was confirmed by the appearance of peaks at ~2.80 and ~2.82 ppm in the HA-CYS and HA-HMDA as shown in Figure 1, and the DS of CYS and HMDA were 9.9% (CYS/HA, mol/mol) and 10.2% (HMDA/HA, mol/mol), respectively. Successful conjugation of TOS onto the backbones of HA-CYS and HA-HMDA was confirmed by the appearance of a peak at ~0.90 ppm in HSST and HCCT as shown in Figure 1, and the DS of TOS were 2.9% and 3.0% (molar amount of TOS against HA), respectively. It was quite important that the redox-responsive HSST and non-redox-responsive HCCT shared a similar DS, which 5 ACS Paragon Plus Environment


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ensured a similar formation of HSST and HCCT micelles when they were dispersed in aqueous medium.

Scheme 1. Illustration of CD44 receptor targeting and intracellular GSH-triggered drug release of HSST.

2.2 Preparation and characterization PTX-loaded micelles When the amphiphilic HSST micelles were dispersed in distilled water containing pyrene, the concentration of HSST at the crosspoint of the I1/I3 ratio which indicates the CMC of micelles was 48.0 µg/mL, which was similar to that for HCCT (64.9 µg/mL) (Figure 2A). The particle sizes of the HSST and HCCT micelles were 114 ± 11.6 nm and 108 ± 16.2 nm, respectively, as determined by DLS (Table 1). The spherical morphology of these micelles was confirmed by TEM (Figure 2B). The surface zeta potentials were -20.9 ± 4.3 mV and -18.2 ± 4.2 mV for the HSST and HCCT micelles, respectively. The loading of the PTX into the HSST and HCCT 6 ACS Paragon Plus Environment

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micelles did not cause significant changes in the particle sizes or zeta potentials of these micelles (Table 1). The results indicated that HSST and HCCT demonstrated similar physicochemical properties on the basis that they shared the same DS and similar chemical structures except for the linker bridge between HA backbone and TOS fragment. The encapsulation efficiency of HSST and HCCT, which was calculated by comparing the amount in micelles to the feeding amount of PTX, was always over 70% (w/w) and was similar for HSST and HCCT over a range of PTX feeding ratios from 5% to 40% (w/w) (Figure 2D). With a high PTX feeding ratio, the drug loading could be up to 27.2% and 28.5% (w/w) for HSST and HCCT, respectively. Those results suggested that both HSST and HCCT could efficiently encapsulate PTX.

Figure 1.

1

H NMR spectra of HA, HA-HMDA, HA-CYS, HA-CC-TOS and

HA-SS-TOS, from the bottom to the top. 7 ACS Paragon Plus Environment


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Figure 2. Characteristics of HSST and HCCT. (A) Cross point of intensity ratio (I1/I3) of pyrene, which indicates the critical micelle concentration of HSST (48.0 µg/mL) and HCCT (64.9 µg/ml), from the top to the bottom. (B) TEM images of HSST (left) and HCCT (right). (C) The encapsulation efficiency (EE) and (D) drug-loading (DL) at various PTX feeding ratio (5% - 40%, w/w).

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Table 1. Size, polydispersity index (PDI), and zeta potential of the blank and PTX-loaded HA-SS-TOS and HA-CC-TOS micelles. HA-SS-TOS

Feeding

HA-CC-TOS

ratio (%)

dn (nm)

PDI

ζ (mV)

dn (nm)

PDI

ζ (mV)

0

114 ± 11.6

0.195

-20.9 ± 4.3

108 ± 16.2

0.175

-18.2 ± 4.2

5

116 ± 21.3

0.133

-25.8 ± 5.4

119 ± 23.3

0.186

-25.7 ± 10.4

10

95.5 ± 13.7

0.164

-25.9 ± 5.2

96.2 ± 16.7

0.139

-24.5 ± 5.1

20

95.1 ± 15.1

0.157

-23.6 ± 14.6

98.8 ± 18.9

0.189

-24.3 ± 16.1

30

111 ± 16.6

0.100

-23.6 ± 12.6

117 ± 17.6

0.093

-23.2 ± 6.4

40

120 ± 19.4

0.105

-24.3 ± 5.7

128 ± 20.2

0.153

-24.7 ± 15.6

The dn, PDI, and ζ values represent the hydrodynamic diameter, polydispersity index, and micelle zeta potential, respectively.

2.3 GSH-triggered drug release To investigate the GSH-triggered drug release of HSST micelles under various redox conditions in vitro, drug-loaded HSST micelles were incubated in a reducing solution consisting of a PBS solution containing GSH or tumor lysates. As shown in Figure 3A, PTX release from the PTX@HSST in the medium without GSH (PTX@HSST + 0 mM GSH) reached approximately 30% (w/w) after 6 hours, which was similar to that released from the PTX@HCCT micelles with GSH in medium 9 ACS Paragon Plus Environment


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(PTX@HCCT + 10 mM GSH) and PTX@HCCT without GSH in medium (PTX@HCCT + 0 mM GSH) after the same incubation time. Interestingly, the PTX release from PTX@HSST micelles was obviously accelerated in the medium containing 10 mM GSH (PTX@HSST + 10 mM GSH), which induced a significantly higher cumulative release of 55.3% (w/w) at 6 hours (Figure 3A) and almost 100% (w/w) PTX release after 48 hours (Figure S5). In contrast, the PTX release from the PTX@HCCT micelles (PTX@HCCT + 10 mM GSH and PTX@HCCT + 0 mM GSH) or from PTX@HSST micelles (PTX@HSST + 0 mM GSH) was only approximately 60% (w/w) at 48 hours (Figure S5). The results indicated that PTX could achieve an clear GSH-responsive release from PTX@HSST micelles, which could be attributed to the redox-responsive linkage of disulfide bond between HA and TOS. The GSH-triggered release behavior was further confirmed by incubating NR@HSST or NR@HSST in PBS containing GSH. The fluorescence intensity change that indicated the NR release from the NR@HSST micelles showed an obvious time- and GSH-dependent profile, while the NR@HCCT presented an almost unaltered fluorescence signal, which demonstrated that few NR molecules were released from the NR@HCCT micelles under the same conditions (Figure 3B and C). To investigate the GSH-triggered release behavior in a simulated tumor environment, NR@HSST or NR@HCCT were dispersed into solutions containing S180 tumor lysate, followed by the measurements of the NR fluorescence. NR@HSST produced a moderately greater NR signal than NR@HCCT at 0.001, 0.01, and 0.1 X concentrations of the S180 tumor lysate (Figure 3D and E). It was found 10 ACS Paragon Plus Environment

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that NR@HCCT also demonstrated an enhanced NR release with increasing concentrations of tumor lysate. A possible explanation was that complex components of the tumor lysate could induce partial release of NR from the NR@HCCT micelles. For example, many types of metabolic enzymes such as hyaluronidase in the tumor lysate could also cause some breakdown of the micelles even in the absence of a redox-responsive linkage in the micelle matrix.23

Figure 3. Selective redox sensitivity of drug-loaded HSST and HCCT. (A) GSH triggered PTX accumulative release from PTX@HSST and PTX@HCCT micelles within 12 hours (n = 3) on incubation with PBS containing 0 and 10 mM GSH. NR fluorescent images (B) and quantification (C) of NR@HSST and NR@HCCT 11 ACS Paragon Plus Environment


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micelles after incubation with different concentrations of GSH in PBS buffer for 10, 30 and 60 min (n = 4). NR fluorescent images (D) and quantification (E) of NR@HSST and NR@HCCT micelles after incubation with different concentrations of S180 tumor lysate for 15 min. Initial S180 supernatant lysate was considered as 1 X concentration, then diluted by PBS

into 0.01 and 0.001 X concentration, (n = 4).

*p

Specifically Increased Paclitaxel Release in Tumor and Synergetic

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