Design and Validation of PEG-Derivatized Vitamin E Copolymer for

Subscriber access provided by United Arab Emirates University | Libraries Deanship

Article

Design and Validation of PEG-derivatized Vitamin E Copolymer for Drug Delivery into Breast Cancer Yanping Li, Qinhui Liu, Wenyao Li, Ting Zhang, Hanmei Li, Rui Li, Lei Chen, Shiyun Pu, Jiangying Kuang, Zhiguang Su, Zhirong Zhang, and Jinhan He Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00292 • Publication Date (Web): 14 Jul 2016 Downloaded from http://pubs.acs.org on July 19, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 44

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

Bioconjugate Chemistry

Design and Validation of PEG-derivatized Vitamin E Copolymer for Drug Delivery into Breast Cancer

Yanping Li1, 2, Qinhui Liu2, Wenyao Li1, Ting Zhang1, Hanmei Li4, Rui Li1, Lei Chen1, Shiyun Pu1, Jiangying Kuang3, Zhiguang Su3, Zhirong Zhang4, Jinhan He1, 2*

1

Department of Pharmacy, 2Laboratory of Clinical Pharmacy and Adverse Drug

Reaction, 3Molecular Medicine Research Center, Collaborative Innovation Center of Biotherapy, West China Hospital of Sichuan University, Chengdu, China, 610041. 4

Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of

Education, Sichuan University, Chengdu, China, 610041.

Corresponding author: Jinhan He, Department of Pharmacy, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital of Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu, Sichuan, China. Tel. 86-28-85426416, Email: [email protected].

1

ACS Paragon Plus Environment


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

Table of Contents

2


Page 2 of 44

Page 3 of 44

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


ABSTRACT

This study examined the ability of amphiphilic poly(ethylene glycol) (PEG) derivatives to assemble into micelles for drug delivery. Linear PEG chains were modified on one end with hydrophobic vitamin E succinate (VES), and PEG and VES were mixed in different molar ratios to make amphiphiles, which were characterized in terms of critical micelle concentration (CMC), drug loading capacity (DLC), serum stability, tumor spheroid penetration and tumor targeting in vitro and in vivo. The amphiphile PEG5K-VES6 (PAMV6), which has a wheat-like structure, showed a CMC of 3.03×10-6 M, good serum stability and tumor accumulation. The model drug, pirarubicin (THP) could be efficiently loaded into PAMV6 micelles at a DLC of 24.81%. PAMV6/THP micelles were more effective than THP solution at inducing cell apoptosis and G2/M arrest in 4T1 cells. THP-loaded PAMV6 micelles also inhibited tumor growth much more than free THP in a syngeneic mouse model of breast cancer. PAMV6-based micellar systems show promise as nanocarriers for improved anticancer chemotherapy.

Key Words: Vitamin E, polymeric micelles, breast cancer, nanomedicine, sustained

drug delivery

3



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

Page 4 of 44

INTRODUCTION

Chemotherapy, one of the most important tools in clinical cancer treatment, works by inhibiting growth of primary tumors and suppressing proliferation of metastatic tumor cells.1 Although many anticancer drugs have been approved, their clinical application is often compromised by poor water solubility, short persistence in circulation and systemic toxicity. Nanotechnology to encapsulate anticancer drugs in nanoparticles, liposomes, dendrimers and micelles has opened up new possibilities for sustained, controlled, targeted delivery, thereby leading to higher effective concentrations at the tumor site, with fewer off-target side effects.2-5 Polymeric micelles bearing a shell of polyethylene glycol (PEG) have attracted considerable attention as drug delivery systems because of their ease of preparation, good biocompatibility and high efficiency.6 Such micelles have advantages over other drug delivery systems because their amphiphilic nature means that they can encapsulate

water-insoluble

agents

in

their

hydrophobic

core,

improving

bioavailability, while the hydrophilic PEG shell can significantly prolong persistence in the circulation.7, 8 The small size of micelles (10-100 nm) helps them pass through the leaky vasculature via the enhanced permeability and retention effect (EPR), making them highly effective at passive tumor targeting.9 One of the disadvantages of traditional polymeric micelles is that they include a large amount of inert carrier materials that add to the cost and toxicity risk of drug delivery systems.10 This has led researchers to explore using micelle components that themselves have therapeutic effects.11 Vitamin E acts via various mechanisms to inhibit growth of a 4


Page 5 of 44

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


range of tumors,12, 13 and PEGylated vitamin E has been used in copolymers and micelles of several multifunctional drug delivery systems.14-16 In these systems, vitamin E succinate is esterified with PEG 1000 to form

D-α-tocopheryl

PEG

succinate 1000 (TPGS), a highly water-soluble amphiphilic molecule.17 TPGS shows several disadvantages as a micelle component: it has a high critical micelle concentration (CMC) of 0.2 mg/mL, and its relatively short PEG chains do not prolong persistence in circulation long enough to avoid accumulation of micelles in liver and spleen. Therefore, TPGS is usually mixed with other lipids or copolymers to form micelles. Research groups have taken different approaches to improving the properties of TPGS as a micelle carrier. Conjugating tocopheryl succinate with PEG 2000 leads to TPGS2K, which has a much lower CMC than TPGS, allowing formation of stable drug-loaded micelles without addition of other lipids or polymers.18 A study screening different PEG lengths (PEG 2000 or PEG 5000) and different molar ratios of PEG to vitamin E (1:1 to 1:2) found that conjugating one PEG 5000 to two vitamin E molecules gave the lowest CMC and highest loading capacity and stability.19 Another study confirmed that conjugating PEG to two vitamin E molecules reduced the CMC.20 What remains unclear is how the molar ratio of PEG to vitamin E affects the properties of TPGS-based micelles in vitro or in vivo. The stability and loading capacity of micelle carriers relies to a large extent on hydrophobic interactions between the encapsulated drug and the hydrophobic segment of amphipathic polymers.21 Vitamin E-based micelle systems likely involve other 5



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

carrier-drug interactions as well, since the benzene ring and long alkyl chain in vitamin E raise the possibility of hydrophobic interactions, hydrogen bonding and π-π stacking.19,

22

This suggests that increasing VES density should increase binding

affinity between the hydrophobic chains and the encapsulated drug. Unfortunately, linear PEG molecules feature the same number of hydroxyl groups for VES coupling, regardless of length.23 This highlights the need to develop PEG derivatives with a large number of available hydroxyl groups to maximize drug loading capacity and formulation stability. To address these issues in the design of amphiphilic micelles for sustained anticancer drug delivery, we developed an amphiphilic multi-hydroxyl PEG derivative coupled to vitamin E that has a wheat-like structure. These derivative-based micelles show good stability and high drug loading capacity. We also prepared the PEG-vitamin E amphiphiles in different molar ratios of PEG and vitamin E and systematically examined the structure, stability and drug delivery efficacy of the resulting micelles in vitro and in vivo. Micelles prepared with the optimal molar ratio were then loaded with the drug pirarubicin (THP) (Scheme 1) and examined for efficacy against breast cancer cells in vitro and in vivo.

6


Page 6 of 44

Page 7 of 44

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


Scheme 1. Schematic diagram of PAMV6 micelles for the tumor cell-specific delivery of THP. (A) Graphical elucidation of the PAMV6/THP micelles; (B) Graphical elucidation of tumor cell targeting delivery of PAMV6/THP micelles. PAMV6/THP micelles could penetrate trough the leaky vasculature due to the EPR effect, and target to tumor cells where THP would be released and enter into nuclei to induce the tumor cell apoptosis.

RESULTS AND DISCUSSIONS Synthesis and identification of PAMVn amphiphiles The polyether PEG macromolecule is widely used in drug delivery system for its biocompatibility and bioavailability.28-30 Linear PEG has limited positions for conjugating hydrophobic chains, so derivatizing PEG to introduce multiple hydroxyl groups can improve the density of hydrophobic chains. To synthesize these derivatives, we started with mPEG with a molecular weight of 5000 Da. Ally glycidyl 7



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

ether (AGE) was coupled to the terminal hydroxyl to form the intermediate, mPEG-(AGE-ME)n, which has multiple double bonds on the side chains. The number of double bonds was determined by the molar mass of AGE added to the reaction. The intermediate then reacted with 2-mercaptoethanol in an addition reaction on the double bonds and thiol group to afford various PEG derivatives with different numbers of terminal hydroxyl groups. Conjugating these PEG derivatives covalently to VES in the presence of condensing agent catalyst led to PEG-derivatized VES amphiphiles carrying different VES densities (Figure 1). The synthesis of PAMVn was confirmed by 1H-NMR spectra (Figure S1). The peaks at 3.468 ppm and 3.509-4.000 were assigned, respectively, to CH3O and OCH2-CH2OH of mPEG5K. AGE polymerization was confirmed by peaks at 5.320 and 5.883 ppm, belonging to the carbon-carbon double bond. The molar ratio of added AGE to mPEG5K was calculated from the ratio of the integrated peak area for the double bond (C=C) in AGE to the integrated peak area for the methoxyl group (CH3O) in mPEG5K. Addition of 2-mercaptoethanol was reflected in the disappearance of peaks for C=C and the appearance of peaks at 2.664-2.878 ppm. Addition of VES was reflected in the appearance of peaks at 0.836-2.634 ppm in CDCl3. Disappearance of peaks at 0.836-2.634 ppm in D2O coincided with the self-assembly of PAMVn amphiphiles. MALDI-TOF mass spectrometry indicated the following molecular weights for amphiphiles (in Da): PMAV1, 5554.43; PAMV3, 7077.18; PAMV6, 8887.54; and PAMV10, 11514.41 Da (Figure S2). The respective VES density on these amphiphiles 8


Page 8 of 44

Page 9 of 44

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


is calculated to be about 1, 3, 6 and 10 per molecule.

Figure 1. Synthesis route of PAMVn

Preparation and characterization of PAMVn micelles To investigate how VES density affects micelle properties, we synthesized four PEG-derivatized vitamin E amphiphiles: PAMV1, PAMV3, PAMV6 and PAMV10. The first three of these copolymers readily self-assembled into nanomicelles in aqueous solution, with particle size ranging from 50 to 80 nm and polydispersity index < 0.20 based on dynamic light scattering (Figure 2A). Transmission electron microscopy of PAMV6 micelles revealed spherical particles of a uniform size consistent with the light scattering results (Figure 2B). Similar results were obtained for PAMV1 and PAMV3 micelles. Amphiphilic polymer materials with a suitable hydrophile lipophile balance (HLB) value should be able to form stable micelles.31, 32 PAMV10 did not form uniform nanomicelles, presumably because the high VES density destroyed the 9



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

balance between hydrophilic and hydrophobic chains. Thus, PAMV10 amphiphiles were excluded from subsequent experiments. CMC is an important parameter of micelle stability, so CMC was determined for the PEG-VES amphiphiles using pyrene as a fluorescent probe. The ratio of fluorescence intensity I338/I333 was plotted as a function of logarithmic concentration of the amphiphiles, and the inflection point was taken as the CMC (Figure S3): PAMV1, 17.50 nM; PAMV3, 8.78 nM; and PAMV6, 3.03 nM (Figure 2D). All three CMCs were much lower than that of commercial TPGS (133.33 nM), perhaps reflecting the higher VES density and therefore stronger hydrophobic tails. Lower CMC should translate to greater stability in suspension and greater resistance to dissociation even upon dilution into bloodstream. The micelles should remain stable in blood until reaching the target tissue; if they aggregate or dissociate in the bloodstream, they will tend to be trapped by the reticuloendothelial system (RES), significantly affecting their biodistribution. Thus micelle stability in 50% FBS was examined. In addition, we measured the efficiency of THP loading into PAMV1, PAMV3 and PAMV6 at different carrier-to-drug mass ratios using ultrafiltration. At a minimal mass ratio of 5.0:1, PAMV1 and PAMV3 micelles were stable for less than 1 h in serum, and drug loading efficiency (DLE) was only 54.58% for PAMV1 micelles and 64.80% for PAMV3 micelles (Table 1). In contrast, a carrier-to-drug mass ratio of 2.5:1 was sufficient for PAMV6 to form stable micelles with THP, giving a DLC of 24.81% and DLE of 82.37%. Higher carrier-to-drug input ratios were associated with higher DLE and greater colloidal 10


Page 10 of 44

Page 11 of 44

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


stability of PEG-VES micelles. Increasing VES density at constant carrier-to-drug ratio led to increases in DLE and DLC as well as greater serum stability. PAMV6 micelles showed the greatest stability, remaining intact for over 72 h in 50% FBS. Based on DLC and serum stability, we ranked the three amphiphiles as follows: PAMV6 > PAMV3 > PAMV1.

Table 1. Physicochemical characterization (including size, PDI, drug loading capacity, drug loading efficiency and serum stability) of THP-loaded PAMVn micelles Micelles

PAMV1

PAMV3

PAMV6

Carrier/THP ratio 1:5.0 1:7.5 1:10 1:5.0 1:7.5 1:10 1:2.5 1:5.0 1:7.5 1:10

Size (nm) 60.43±1.22 55.78±2.60 50.34±0.60 75.32±0.59 62.07±0.38 56.05±1.72 98.42±1.38 89.30±2.54 78.23±0.78 61.63±1.38

PDI 0.38 0.23 0.20 0.32 0.28 0.19 0.21 0.24 0.20 0.19

DLC (%) 9.10 8.53 8.98 10.80 10.17 8.96 24.81 16.50 11.68 9.05

DLE (%) 54.58 72.49 98.75 64.80 86.45 98.54 82.37 98.20 98.89 99.50

Stability in FBS (h) Milky 2.5 4.0 Milky 4.0 8.0 48 72 120 >168

The kinetics of THP release were measured from different formulations using a dialysis method in which the release medium was PBS (pH 7.4) containing 0.2% (w/v) Tween-80. During the first 8 h of dialysis, 80% of total THP was released from THP solution and from PAMV1 and PAMV3 micelles; PAMV6 micelles, in contrast, showed slow, sustained release with no burst phase (Figure 2E). The resulting ranking in terms of sustained release, PAMV6 > PAMV3 > PAMV1, mirrors that of in vitro stability. Hydrophobic interactions, hydrogen bonding and π-π stacking are likely to contribute 11



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

to affinity between THP and vitamin E, which contains a benzene ring and long alkyl chain.19,

22

Increasing VES density should further increase interactions among

hydrophobic chains, further stabilizing the micelles. Indeed, the wheat-like structure of the hydrophobic chains of PAMV6 likely creates a binding pocket that enhances the interaction between carrier and drug, which may help explain why these micelles showed the greatest stability of the variations we tested. Since micelles are injected intravenously, it is important to determine whether the surfactants on the micelle surface affect the integrity of host cell membranes. We found that while polyethylenimine (PEI) caused significant hemolysis in a dose-dependent manner (Figure 2F), PAMV1, PAMV3 and PAMV6 micelles showed negligible hemolytic activity ( PAMV3 > PAMV1. These results suggest that increasing VES density improved penetration efficiency through the solid tumor. These results confirm that PAMVn micelles can penetrate deep into tumor spheroids. 14


Page 14 of 44

Page 15 of 44

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


To further examine whether this penetration would translate into efficient intracellular release of drug cargo, and in particular would allow THP to enter nuclei to exert antitumor effects, we examined the intracellular distribution of THP-loaded PAMV6 micelles using confocal laser scanning microscopy. In the case of both free THP and PAMV6/THP micelles, red THP fluorescence localized mainly to the DAPI-stained cell nucleus (Figure 3C). This suggests that PAMV6/THP micelles are internalized into cells, where they release THP that enters the nucleus.

Figure 3. Tumor spheroids penetration and intracellular distribution of PAMVn micelles. (A) In vitro cytotoxicity of PAMVn in L929 cells and HUVEC cells by MTT assay. Data represent mean ± SD (n = 6); (B) Fluorescence distribution of 4T1 tumor spheroids after incubation with different rhodamine 123 (RHO) loaded PAMVn micelles (10 µM) for 12 h. Z-stack images were obtained from the top toward 15



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

spheriod equatorial plane in 30 µm thickness. Scale bars represent 200 µm; (C) Confocal laser scanning microscopy images of 4T1 cells after treatment with PAMV6/THP micelles for 4 h. Scale bars represent 40 µm.

In vivo and ex vivo imaging Nanocarriers should circulate in the blood long enough to localize and perfuse into the target organ. If they do not persist in systemic circulation, nanoparticles will be cleared rapidly without reaching the target site. Therefore we analyzed the pharmacokinetics of PAMVn micelles in rats (Figure S4A). Injecting animals with DiD solution led to a spike in plasma concentration of DiD that rapidly decreased and was gone completely within 2 h of administration. This suggests that free DiD is rapidly eliminated from the blood. Encapsulating DiD into PAMVn micelles substantially increased the area under the curve (AUC), mean retention time (MRT) and elimination half-life (t1/2), indicating much longer persistence in the circulation and much higher bioavailability of PAMVn micelles (Figure S4B). Based on persistence in circulation, we ranked the amphiphiles in the order: PAMV6> PAMV3> PAMV1, which likely reflects greater micelle stability with increasing VES density. To assess tumor targeting efficiency of PAMVn micelles in vivo, the biodistribution of DiD-loaded PMAVn micelles in mice bearing 4T1 xenografts was measured using near-infrared fluorescence imaging. After injection with DiD-loaded PAMVn micelles, fluorescence intensity in the tumor increased between 1 h and 24 h, indicating that PAMVn micelles prolonged the persistence of DiD in the circulation (Figure 4A). 16


Page 16 of 44

Page 17 of 44

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


Increasing VES density increased micelle accumulation in the tumor, suggesting that dense VES density stabilized the micelles in the blood (Figure 4B-C). These results were confirmed by semi-quantitative analysis at 24 h after administration (Figure 4D): PAMVn micelles accumulation at 24 h was greater at the tumor site than among all major organs, indicating that the micelles accumulated at the tumor site, presumably due to the EPR effect. PAMVn micelles did not accumulate preferentially in liver or spleen, implying that they suffered minimal non-specific scavenging by the RES. Based on tumor targeting efficiency, we ranked the three amphiphiles in the order PAMV6 > PAMV3 > PAMV1. The relatively high targeting efficiency of all three amphiphiles may reflect that their sizes were within the range of 50-100 nm considered ideal for the EPR effect.36-38 It likely also reflects the longer persistence circulation with higher VES density, since longer persistence is associated with more efficient EPR effect.39

Figure 4. In vivo and ex vivo imaging. Mice were randomly divided into 4 groups: 1, 17



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

DiD solution; 2, PAMV1/DiD micelles; 3, PAMV3/DiD micelles; 4, PAMV6/DiD micelles. (A) In vivo imaging of 4T1 bearing Balb/c mice 1, 6 and 24 h after treated with different formulations; (B) Ex vivo imaging of 4T1 tumors stripped from mice 1, 6 and 24 h after treated with different formulations; (C) Ex vivo imaging of major organs 24 h after treated with different formulations; (D) Semi-quantitative fluorescence intensity of major organs, Data represent mean ± SD (n = 5).

These various experiments demonstrate that the molar ratio of PEG to VES significantly affects in vitro and in vivo properties of PEG-derivatized vitamin E amphiphiles, including CMC, DLC, serum stability, tumor penetration, in vivo pharmacokinetics and tumor targeting. The ratio in PAMV6 micelles appeared to be the best, since they showed the lowest CMC, highest DLC and longest serum stability. Moreover, PAMV6 micelles delivered loaded cargo into tumor spheroids most efficiently. PAMV6 micelles showed long persistence in circulation and good tumor targeting ability via the EPR effect, indicating that they are well-suited to overcoming systemic biological barriers. Therefore we conducted subsequent experiments only with PAMV6.

Antitumor efficacy in vitro The doxorubicin derivative THP was selected as the model drug to validate the efficacy of PAMV6 micelles. THP is a cytotoxic agent that inhibits the activities of type II topoisomerase and DNA polymerase.40 It is widely used in the clinic because it 18


Page 18 of 44

Page 19 of 44

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


has proven effective against several kinds of solid tumors, acute leukemia, and malignant lymphoma, and because it has a reasonable therapeutic index.41, 42 The antitumor activity of PAMV6/THP micelles against 4T1 breast cancer cells in culture was evaluated in terms of cell viability, cell cycle distribution and apoptosis. Treating cells with blank PAMV6 micelles led to viabilities above 90%, indicating low cytotoxicity of the micelle carrier (Figure 5A). While both THP in solution and PAMV6/THP micelles inhibited cell proliferation in a dose-dependent manner, PAMV6/THP micelles led to lower cell viabilities than free THP at various concentrations. This may reflect that free THP and encapsulated THP enter cells by different pathways, with free drug entering mainly by passive diffusion and encapsulated drug entering mainly by endocytosis. Since THP can delay the cell cycle at G2/M, drug-induced apoptosis often occurs after cells have stopped dividing.43 Therefore we treated cells with different THP formulations, then stained them with PI and analyzed their distribution in the cell cycle. Treatment with PAMV6/THP micelles led to a significantly higher proportion of cells in G2/M (52.50%) than THP in solution (27.85%) (Figure 5B), and it led to significantly greater proportions of cells in apoptosis based on annexin V-FITC/PI staining (Figure 5C). DAPI staining confirmed that encapsulated THP induced apoptosis more efficiently than free drug: the nuclei of untreated 4T1 cells showed no evidence of segmentation or fragmentation, while cells treated with PAMV6/THP micelles showed severe shrinkage and their nuclei showed condensation, fragmentation and apoptotic bodies (Figure 5D). In addition, treating cells with 19



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

Page 20 of 44

PAMV6/THP micelles activated the apoptotic maker caspase-3 to a greater extent than free THP (Figure 5E). These results indicate that PAMV6/THP micelles enhanced THP-induced cell apoptosis in vitro, suggesting an ability to potentiate the drug’s chemotherapeutic efficacy in vivo.

Figure 5. In vitro antitumor effects of PAMV6/THP micelles on 4T1 cells. (A) Cell viability assay of 4T1 cells treated with different formulations. Data represent mean ± SD (n = 5); (B) Flow cytometry analysis of the cell cycle for 4T1 cells after incubation with free THP and PAMV6/THP micelles (100 ng/mL) for 24 h. Data represent mean ± SD (n = 3). **p < 0.01 with respect to the control,

##

p < 0.01 with

respect to free THP; (C) Apoptosis of 4T1 cells induced by free THP and PAMV6/THP micelles (100 ng/mL) after 48 h incubation. Data represent mean ± SD 20


Page 21 of 44

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


(n = 3). **p < 0.01 with respect to the control, ##p < 0.01 with respect to free THP. (D) DAPI staining of fragmented chromatin or apoptotic bodies in 4T1 cells after 48 h incubation with PAMV6/THP micelles; (E) Western blotting of caspase-3 and cleaved caspase-3.

Antitumor efficacy in vivo To test in vivo antitumor efficacy of PAMV6/THP micelles, mice bearing 4T1 xenografts were treated with saline, free THP or PAMV6/THP micelles. All treatments led to slower growth in tumor volume than the saline control (Figure 6A and C). Tumor inhibition was greater with PAMV6/THP micelles than with free THP at THP doses of 2.5 mg/kg (87.88% vs 55.29%) and 5.0 mg/kg (98.60% vs 62.40%). In fact, average tumor volume barely changed in animals treated with PAMV6/THP micelles at 5.0 mg/kg THP during the 25-day treatment period. The fact that all treatment groups gained weight suggests that THP was associated with minimal toxicity despite the antitumor effects (Figure 6B). The ability of THP formulations to suppress proliferation in xenograft tumors was analyzed by immunostaining tumor sections for Ki-67, while their ability to induce necrosis was assessed by staining sections with hematoxylin-eosin. The rate of Ki-67-positive cells was higher in sections from saline- or free THP-treated animals than in sections from PAMV6/THP-treated mice, indicating that the encapsulated drug reduced active cell proliferation to the greatest extent, helping to explain the strong antitumor effects (Figure 6F). The encapsulated drug also led to greater necrosis in 21



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

tumor sections than the free drug did, and this histopathology was dose-dependent, while little or no such pathology was observed in sections from saline-treated animals (Figure 6E). These in vivo experiments indicate that PAMV6/THP micelles showed greater antitumor efficacy than free THP at an equivalent dosage, consistent with the in vitro results.

Figure 6. In vivo antitumor efficacy of THP formulations in 4T1 bearing Balb/C mice. 22


Page 22 of 44

Page 23 of 44

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


Mice were randomly divided into 5 groups: 1, Saline; 2, THP 2.5 mg/kg; 3, PAMV6/THP 2.5 mg/kg; 4, THP 5 mg/kg; 5, PAMV6/THP 5 mg/kg (n = 8 for each group). (A) Body weight of mice receiving different treatments; (B) Tumor growth curves of mice receiving different treatments; (C) Image of tumors stripped from mice on the day 24 after six consecutive treatment; (D) Weight of tumors on the day 24 after treatment with different formulations. The IRT of different groups were also calculated and listed; (E) H&E and Ki-67 staining analysis of 4T1 tumor sections. The cytoplasm was dyed light red by eosin, while the cell nucleus was stained blue with hematoxylin in H&E staining (scale bar represents 200 µm). The brown and blue stains indicate Ki-67-positive staining areas and nuclei respectively in the Ki-67 assays (scale bar represents 50 µm).

CONCLUSIONS We have designed and synthesized a series of PEG-derivatized VES amphiphiles with varying molar ratios of PEG to VES. Systematic studies showed that VES density significantly affected the in vitro and in vivo properties of PEG-VES based micelles, such as stability, drug loading capacity and tumor targeting efficiency. PAMV6, with its wheat-like structure, showed the lowest CMC, greatest stability in serum, and the highest tumor distribution and retention. Pirarubicin (THP) was loaded into PAMV6 micelles with a DLC of 24.81%, which compares favorably to previously reported THP nanoformulations. PAMV6/THP micelles led to significantly higher antitumor efficacy both in vitro and in vivo. Thus, PAMV6-based micelle systems are a 23



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

promising strategy for potentiating the efficacy and mitigating the toxicity of cytotoxic chemotherapeutics.

EXPERIMENTAL SECTION Materials mPEG5K, ally glycidyl ether (AGE) and β-mercaptoethanol were obtained from Sigma-Aldrich (St. Louis, USA). D-α-Tocopheryl succinate was purchased from J&K scientific (Beijing, China). Pirarubicin (THP) was provided by Zhejiang Hisum Pharmaceutical Co., Ltd. (China). 1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindodi carbocyanine perchlorate (DiD) was purchased from Biotium (California, USA). Pyrene, 4',6-diamidino-2-phenylindole (DAPI), propidium iodide (PI), RNase A and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetra-zolium bromide (MTT) were also purchased from Sigma Aldrich (St. Louis, USA). Annexin V-FITC/PI apoptosis detection kit was purchased from BD biosciences (CA, USA). Fetal bovine serum (FBS) was obtained from Biological Industries Israel Beit Haemek LTD. (Kibbutz Beit-Haemek, Israel). Caspase-3 antibody was purchased from Boster Biotech (Wuhan, China). β-actin antibody and horseradish peroxidase (HRP)-labeled secondary antibodies were purchased from Abcam (California, USA). All other chemicals and reagents were analytical grade and were obtained commercially. Male Sprague-Dawley rats (200 ± 20 g) and female 6-10 week-old BALB/C (18-22 g) mice were purchased from Dashuo biotechnology (Jianyang, China). All animal experiments were performed in accordance with the principles of care and use of 24


Page 24 of 44

Page 25 of 44

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


laboratory animals and were approved by the Experiment Animal Administrative Committee of Sichuan University.

Synthesis of the copolymer PAMVn Synthesis of mPEG5K/(AGE)n 24. The methoxyl-PEG 5000 (mPEG5K, 10 g) was suspended in nonaqueous dioxane (60 mL) and dried for 3 h at 100 °C through oil pump-reduced pressure. Sodium hydride (60%, w/w; 0.2 g) was added and stirred at 60 °C for 1 h. Then AGE was added to the reaction mixture and stirred another 48 h at 60 °C. Water was added to stop the reaction, and the pH was adjusted to neutral using hydrochloric acid. Solvent was removed by evaporation, the residue was dissolved by dichloromethane, then it was dried using waterless sodium sulfate and filtered. The supernatant was condensed by evaporation. After three precipitations with diethyl ether, mPEG5K/(AGE)n was obtained as the product , where n indicates the number of monomers. Synthesis of mPEG5K/(AGE-ME)n

25

. 2-mercaptoethanol was added to the C=C

side chain of mPEG5k/(AGE)m. Briefly, mPEG5k/(AGE)n (1.0 g) was dissolved in 6 mL methanol. 2-mercaptoethanol was added dropwise to the solution and stirred for 48 h. Next, the methanol was removed by evaporation. Then the residue was dialyzed against distilled water for 3 days through a cellulose membrane (1000 Da). The solution was lyophilized and the final product was obtained. Synthesis D-α-tocopheryl

of

mPEG5K/(AGE-ME-VES)n.

mPEG5K/(AGE-ME)n

and

succinate were weighed and dissolved in DCM together with EDC 25



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

and HoBt in a stoichiometric ratio of 1:1:2:2. To the mixture was added 100 µL of DIPEA, and the mixtire was left in a nitrogen environment with stirring for 24 h at room temperature. The solvent was concentrated by evaporation, and the residue was dialyzed against distilled water for 3 days through a cellulose membrane (8000-14000 Da). The solution was filtered and lyophilized to yield a white powder.

Preparation and characterization of micelles Micelles were prepared by the thin-film hydration method. PAMVn was dissolved in chloroform, which was removed by rotary evaporation, and the thin film was hydrated in PBS (pH 7.4). Pirarubicin (THP)-loaded micelles were prepared by adding THP to the organic solution prior to solvent evaporation. DiD and rhoadamine 123 (RHO) were also added to the solution before solvent evaporation to prepare DiD-labeled or RHO-labeled micelles. Particle size distribution and zeta potential of PAMVn micelles were measured using dynamic light scattering on a Zetasizer Nano ZS90 instrument (Malvern, UK). Micelle morphology was examined by transmission electron microscopy (H-600, Hitachi, Japan) after staining samples with 2% phosphotungstic acid. To determine drug loading efficiency (DLE), residual free THP was removed by ultrafiltration, and the concentration of THP was determined by LC-MS as described previously 26. Drug loading capacity (DLC) and DLE were calculated using the following equations: DLC (%) = weight of drug loaded / total weight of micelles × 100%, DLE (%) = weight of drug loaded / weight of drug input × 100% 26


Page 26 of 44

Page 27 of 44

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


In vitro stability of micelles in serum In order to investigate the serum stability of micelles, variations in the particle size of micelles were assayed in the presence of fetal bovine serum (FBS). Micelles were mixed with an equal volume of 50% FBS diluted in PBS and incubated at 37 °C. At predetermined time points, 0.2 mL of the sample was diluted in 1 mL PBS, and micelle size was measured using dynamic light scattering (Zetasizer, Nano-ZS90, Malvern, UK).

In vitro release In vitro release of THP from loaded micelles was investigated using the dialysis method. THP-loaded micelles or free THP in solution (1.0 mL) was placed into dialysis bags with a molecular weight cut-off of 8000-14000 Da. Then the dialysis bags were immersed in 200 mL PBS (pH 7.4) containing 0.2% (w/v) Tween-80 and incubated in an incubator at 37 °C with constant shaking at 130 rpm. At predetermined time points, 1.0 mL of release medium was sampled and replaced with the same volume of fresh medium. Then samples were diluted with methanol and THP concentrations were determined using LC-MS.

Determination of critical micelle concentration (CMC) CMCs of different PAMVn micelles were determined using pyrene as a fluorescence probe. Briefly, pyrene in THF (50 µL, 6.0×10−5 M) was added to 5-mL volumetric flasks, and the solvent was left to evaporate for 24 h in order to form a 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

Page 28 of 44

pyrene film in the flasks. Then various concentrations of PAMVn solution were added to each volumetric flask and kept on a shaker at 37 °C for 24 h to reach equilibrium. Steady-state fluorescence spectra were measured at room temperature using a fluorescence

spectrophotometer

(RF-5301

PC,

Shimadzu,

Kyoto,

Japan).

Fluorescence emission spectra were obtained at excitation wavelengths of 333 and 338 nm. The fluorescence intensity ratio I338/I333 was analyzed as a function of micelle concentration.

Hemolytic effect of micelles Blood samples were collected from the femoral artery of rats, and heparin was immediately added to 10-mL samples to prevent coagulation. Red blood cells were separated from plasma by centrifugation at 200 g for 10 min at 4 °C, washed three times with 30 mL ice-cold saline, then diluted to 2% (v/v) with saline and utilized immediately in the hemolysis assay. Suspensions of red blood cells (1 mL) were treated with blank PAMV1, PAMV3 or PAMV6 amphiphiles or with PEI at various concentrations (0.0001, 0.001, 0.01, 0.1, and 1.0 mg/mL). The suspensions were incubated at 37 °C in an incubator shaker for 4 h, then centrifuged at 200 g for 10 min at 4 °C. Supernatant (100 µL) from each sample was transferred into a 96-well plate. Hemoglobin release was determined by absorbance at 540 nm using a microplate reader. In parallel, red blood cells were treated with 2% Triton X-100 as a positive control or with saline as a negative control. Hemoglobin release was calculated as (ODsample−ODnegative control)/(ODpositive control−ODnegative control) × 100%. 28


Page 29 of 44

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


Micelle uptake by tumor spheroids Tumor spheroids of 4T1 cells were formed using the hanging drop method as described previously 27. The wells of 24-well plates were coated with a thin layer of agarose solution (2%, w/v; 0.3 mL) and filled with 0.9 mL of culture medium. Cells (20 µL, 500 cells) were suspended on the lid of the 24-well plates. The spheroids were transferred to the bottom of the well after 3 days, and allowed to grow for another 2 days to reach a size of 200 µm. The tumor spheroids were treated with free RHO, PAMV1/RHO micelles, PAMV3/RHO micelles or PAMV6/RHO micelles at the equivalent of 5 µM RHO. After 12-h incubation, spheroids were rinsed three times with ice-cold PBS and fixed with 4% paraformaldehyde at room temperature for 15 min. Then the spheroids were transferred to glass-bottom petri dishes and covered by glycerophosphate. Fluorescence intensity was observed using a confocal microscope (LSM710, Carl Zeiss, Germany).

Intracellular distribution of drug-loaded micelles The intracellular distribution of THP-loaded PAMV6 micelles was observed using confocal laser scanning microscopy. 4T1 cells were seeded into the confocal dish at a density of 1×105 per well for 24 h. Cells were incubated for 4 h at 37 °C with THP in solution or PAMV6/THP micelles (5 µg/mL THP), washed with cold PBS and fixed. Cells were stained with DAPI to label nuclei and visualized using confocal laser scanning microscopy.

29



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

In vivo and ex vivo imaging study To investigate how efficiently PAMVn micelles targeted tumors, the biodistribution of DiD-labeled micelles was analyzed in BALB/C mice bearing 4T1 tumors. To establish the murine breast cancer model, female BALB/C mice aged 6-10 weeks were injected subcutaneously in the right flank with 4T1 cells (5×105 cells in 100 µL PBS). When tumors reached 500 mm3 after 21 days, mice were randomly assigned to receive an intravenous tail vein injection of DiD in solution or DiD-labeled micelles of PAMV1, PAMV3 or PAMV6 (0.2 mg/kg DiD each) (n = 5 animals per condition). Mice were imaged using an in vivo imaging system (Quick View 3000, Bio-Real, Austria) at 1, 6 and 24 h. Then mice were executed by cervical dislocation, and intact major organs were excised and imaged using in vivo imaging system. Ex vivo fluorescence images were semi-quantitated to give estimates of fluorescence intensity in different organs.

MTT assay 4T1 cells were seeded in 96-well plates at a density of 1×104 per well and cultured at 37 °C for 24 h in the presence of 5% CO2. Serial dilutions of free THP or THP-loaded PAMV6 micelles in FBS-free medium were added to the wells, which were incubated another 48 h. Cytotoxicity of the different treatments against 4T1 cells was evaluated using the MTT assay.

Cell cycle assay 30


Page 30 of 44

Page 31 of 44

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


4T1 cells were seeded in 6-well plates at a density of 1×105 per well and incubated for 24 h. Then THP in solution or PAMV6/THP micelles (100 ng/mL) were added and the cells were incubated another 24 h; cells were collected, washed and fixed with 75% ethanol (prechilled to 0 °C) at 4 °C for 0.5 h. Then cells were resuspended in PBS and treated successively with 0.1% Triton X-100, RNase (0.1 mg/ml) and propidium iodide (0.1 mg/ml). The cell cycle distribution was determined using flow cytometry (Cytomics FC500; Beckman Coulter, Brea, CA, USA) and analyzed with Multicycle software (Phoenix Flow Systems, San Diego, CA, USA).

Cell apoptosis assay 4T1 cells were seeded into 6-well plates as for the cell cycle assay (section 2.7.2). After incubation for 24 h, THP solution or PAMV6/THP micelles (100 ng/mL) were added and the cells were incubated another 48 h. Cells were collected and washed twice with cold PBS, then the degree of apoptosis was quantified using the Annexin V-FITC/ propidium iodide Apoptosis Detection Kit (BD Biosciences, CA, USA). To observe nuclear morphology, cells were seeded into the confocal dish, stained with DAPI and observed using confocal laser scanning microscopy. To assay levels of caspase-3 using Western blotting, cells were lysed with RIPA lysis buffer and total protein quality was determined using a BCA Kit (Pierce; Rockford IL, USA). Total protein samples were resolved on 10% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and incubated with rabbit primary antibodies against mouse β-actin and caspase-3. Then membranes were 31



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

incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibodies and amount of binding was quantitated using the Immobilon Western HRP Substrate (Millipore, USA) and imaged by a Bio-Rad ChemiDoc MP System (Bio-Rad Laboratories, USA).

In vivo antitumor efficacy The murine breast cancer model was established as described for the biodistribution study (section 2.6). On day 6, when tumors had reached a size of 50-100 mm3, mice were randomly assigned to receive multiple injections of one of the following (n = 10 animals per condition): saline, free THP (2.5 mg/kg), PAMV6/THP micelles (2.5 mg/kg), free THP (5.0 mg/kg) and PAMV6/THP micelles (5.0 mg/kg). Mice were injected once daily through the tail veins every three days for a total of 6 times, and tumor volume and body weight were measured. On day 31 after tumor inoculation, the mice were executed and the tumors were weighed and sectioned. Sections were paraffin-embedded, stained with hematoxylin and eosin (H&E) and immunostained against Ki67 according to the manufacturer’s instructions.

Statistical analysis Differences between two groups were assessed for significance using Student’s t test, while differences among more than two groups were assessed using one-way ANOVA. Thresholds for statistical differences and statistically significant differences were, respectively, P

Design and Validation of PEG-Derivatized Vitamin E Copolymer for

Recommend Documents