Adequately-Sized Nanocarriers Allow Sustained Targeted Drug

May 16, 2016 - We sought to evaluate this possibility using rat carotid arteries with induced neointima. Cy5-labeled polyethylene glycol-conjugated po...
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Adequately-Sized Nanocarriers Allow Sustained Targeted Drug Delivery to Neointimal Lesions in Rat Arteries Ryosuke Taniguchi,†,○ Yutaka Miura,‡ Hiroyuki Koyama,*,†,§ Tsukasa Chida,∥ Yasutaka Anraku,∥ Akihiro Kishimura,⊥,# Kunihiro Shigematsu,† Kazunori Kataoka,‡,∥,∇ and Toshiaki Watanabe† †

Division of Vascular Surgery, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan ‡ Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan § Department of Vascular Surgery, Saitama Medical Center, Saitama Medical University, 1981 Kamoda, Kawagoe, Saitama 350-8550, Japan ∥ Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan ⊥ Department of Applied Chemistry, Faculty of Engineering, Kyusyu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan # Center for Molecular Systems, Kyusyu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan ∇ Innovation Center of Nanomedicine, Kawasaki Institute of Industry Promotion, 66-20 Horikawa-cho, Saiwai-ku, Kawasaki 212-0013, Japan S Supporting Information *

ABSTRACT: In atherosclerotic lesions, the endothelial barrier against the bloodstream can become compromised, resulting in the exposure of the extracellular matrix (ECM) and intimal cells beneath. In theory, this allows adequately sized nanocarriers in circulation to infiltrate into the intimal lesion intravascularly. We sought to evaluate this possibility using rat carotid arteries with induced neointima. Cy5-labeled polyethylene glycol-conjugated polyion complex (PIC) micelles and vesicles, with diameters of 40, 100, or 200 nm (PICs-40, PICs-100, and PICs-200, respectively) were intravenously administered to rats after injury to the carotid artery using a balloon catheter. High accumulation and long retention of PICs-40 in the induced neointima was confirmed by in vivo imaging, while the accumulation of PICs-100 and PICs-200 was limited, indicating that the size of nanocarriers is a crucial factor for efficient delivery. Furthermore, epirubicin-incorporated polymeric micelles with a diameter similar to that of PICs-40 showed significant curative effects in rats with induced neointima, in terms of lesion size and cell number. Specific and effective drug delivery to pre-existing neointimal lesions was demonstrated with adequate size control of the nanocarriers. We consider that this nanocarrier-based drug delivery system could be utilized for the treatment of atherosclerosis. KEYWORDS: atherosclerosis, neointimal hyperplasia, epirubicin, nanocarrier, nanoparticle, drug delivery

1. INTRODUCTION Atherosclerosis related diseases are the leading causes of death in the United States and in most other developed countries.1 To inhibit the progression of this pathological condition, various drugs to treat patients have been developed.2 However, there are many cases for which these drugs have only limited therapeutic effects, and therefore, invasive therapies (i.e., endovascular or surgical interventions) may be required to achieve a better outcome. One of the limitations of traditional © XXXX American Chemical Society

pharmacotherapy is that therapeutic drugs with low molecular weight undergo nonspecific systemic disposition, which prevents their specific and concentrated accumulation in diseased lesions. Received: March 12, 2016 Revised: May 7, 2016 Accepted: May 16, 2016

A

DOI: 10.1021/acs.molpharmaceut.6b00219 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

River Laboratories, Yokohama, Japan) fed with alfalfa-free chow food were provided general anesthesia by inhalation of 2−2.5% isoflurane. A balloon injury of the left carotid artery was made as described in previous studies.13 In brief, after performing a midline neck incision, a 2-Fr Fogarty balloon catheter (Edwards Lifesciences, Irvine, CA) was introduced via the left external carotid artery and inserted into the left common carotid artery. The 2-Fr balloon catheter is the standard size used in the rat model, and the left side of the carotid was selected due to the easy accessibility for the surgeon. The balloon was inflated at physiological pressure and passed through the common carotid artery 3 times with constant rotation. Injection of solutions via the tail vein described below was performed under anesthesia by inhalation of 2% isoflurane. Euthanasia was performed with an overdose of anesthesia by inhalation of 5% isoflurane. Anesthesia was monitored by periodic observation of the respiration and pain response. 2.2. Preparation of Cy5-Labeled Nanocarriers. To assess the effect of particle size on the distribution of nanocarriers to arterial lesions, we prepared Cy5-labeled PIC micelles and PICsomes, which have diameters of 40 nm (PICs40), 100 nm (PICs-100), and 200 nm (PICs-200) in average, by mixing homo- or block-polycatiomer and block copolymer as previously described.14 Prior to their injection, these PIC micelles and/or PICsomes were suspended in phosphate buffered saline (PBS) so that the relative fluorescence units measured using a fluorospectrometer (NanoDrop 3300, Thermo Scientific, Wilmington, DE, USA) were equivalent in each series. Details are described in the Supporting Information. 2.3. Evaluation of the Macroscopic Distribution of Nanocarriers Using an IVIS Imaging System. Immediately after the balloon injury, 300 μL of PICs-40, PICs-100, or PICs200 was injected via the tail vein of the animal prior to anesthetic recovery (n = 12 for each size of nanocarrier). Additional sets of model rats received an injection of PICs-40, PICs-100, or PICs-200, in the same manner, at either 7 or 14 days after balloon injury, under anesthesia (n = 12 for each size of nanocarrier). The animals were euthanized with an overdose of anesthesia at 1, 8, or 24 h after injection (n = 4 for each time point). Subsequently, the left and right carotid arteries were harvested, together with the aortic arch. To premeasure the autofluorescence of the artery, carotid arteries of rat models without nanocarrier injection were prepared in the same manner (n = 4 for each postinjury day of injection). The carotid arteries and the aortic arch were gently rinsed with PBS to wash the remaining blood from the lumen. The macroscopic distribution of Cy5-labeled nanocarrier was evaluated with an IVIS imaging system (Caliper Life Sciences, Hopkinton, MA). The specimens were placed flat with the anterior side facing upward, and adequate filters were equipped in the system (excitation filter, 640 nm; emission filter, 680 nm). Emitted photons were quantified as radiant efficiency using Living Image Software (Caliper Life Sciences, Hopkinton, MA); the average radiant efficiency ([p/s/cm2/sr]/W[μ/cm2]) of the left and right carotid arteries was acquired using Region of Interest tools. The acquired values for the bilateral carotids were subtracted by the corresponding mean values of the rat carotid arteries without injection of Cy5-labeled nanocarriers. 2.4. Determination of the Microscopic Distribution of Nanocarriers by Histological Analyses. After the IVIS evaluation, arterial segments of rats harvested 1 or 24 h after the injection were dissected from the middle part of the left carotid

To overcome this problem, drug delivery systems consisting of nanoscale drug carriers (nanocarriers) can be used owing to their potentials of selective delivery to diseased lesions and increase in therapeutic efficacy with minimized side effects. Indeed, nanocarriers have been used in animal models of vascular diseases to achieve safe and targeted drug delivery.3−7 Notably, the advantages of nanocarrier systems have been observed primarily in the treatment of solid tumors, and the driving force for their targeting is better known as enhanced permeability and retention (EPR) effect.8,9 However, the pathological structure of atherosclerotic lesions is intrinsically different from that of solid tumors, and the EPR effect has not yet been elucidated in vascular diseases. In atherosclerotic lesions, the barrier provided by the endothelial layer is compromised in many ways.10 Furthermore, the intimal structure beneath the endothelial layer is formed by a network of extracellular matrix (ECM), which fills the spaces between intimal cells and deposits of other substances (i.e., calcium and lipids).11 Defects in the endothelial layer can indicate that the ECM network, along with the intimal cells and deposits, is exposed to the bloodstream. This suggests that access routes into the intimal lesion may be available for adequately sized substances in the bloodstream due to the submicron interstices in the intimal ECM network.12 Therefore, we hypothesized that nanocarriers circulating repeatedly in the bloodstream are able to specifically infiltrate and accumulate in the intimal lesions if their size is suitable for the interstices in the ECM network. The primary aim of the present study was to elucidate the potential and characteristics of nanocarrier accumulation in neointimal lesions via systemic administration of nanocarriers of different sizes to rats with neointimal lesions induced by balloon injury.13 To analyze whether nanocarriers of a particular size accumulated specifically in the induced neointimal lesions, the size of the nanocarriers must be precisely controlled within a predefined range and possess sufficient stability in vivo to maintain their structure in the bloodstream for a prolonged period. Among available nanocarriers, crosslinked polyion complex micelle (PIC micelle) and vesicle (PICsome), which are constructed by self-assembly of poly(ethylene glycol) (PEG)-based block aniomers and homocatiomers by electrostatic interaction, are expected to fulfill the aforementioned requirements.14,15 These PIC-based nanocarriers are characterized by its hydrophilic PEG components arrayed as an outer shell layer of the particles, providing high biocompatibility in vivo, and also, its chemical cross-linking, which produces persistent stability under harsh physiological conditions. More importantly, their structure and particle size are precisely controllable by the adjustment of the initial concentration of the polymers and the PEG fraction or polymer composition, providing a series of cross-linked PIC micelles and PICsomes with different diameters.16 Based on the results of our size-effect analyses, we administrated polymeric micelles of the adequate size and biomaterial similarity with PIC-based nanocarriers that incorporated epirubicin, an anthracycline drug, to a rat model of carotid injury and evaluated its therapeutic effects.17−19

2. EXPERIMENTAL SECTION 2.1. Animal Models. All animal experiments were approved by the Animal Care and Use Committee of the University of Tokyo and conducted in accordance with institutional guidelines. Male Sprague−Dawley rats (300−350 g; Charles B

DOI: 10.1021/acs.molpharmaceut.6b00219 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 1. Macroscopic accumulation of Cy5-labeled polyethylene glycol-conjugated polyion complex micelles and vesicles in arterial lesions. (A) Radiant efficiencies of the left (Lt) and right (Rt) common carotid arteries, visualized using IVIS imaging system, following the administration of 40 (left), 100 (middle), and 200 nm (right) Cy5-labeled nanocarriers (PICs-40, PICs-100, and PICs-200, respectively). Images were acquired 1, 8, and 24 h after administration, to rats immediately after (top), 7 days after (middle), and 14 days after (bottom) balloon injury (n = 4). Color scale of radiant efficiency: Min = 2.20 × 107; Max = 1.00 × 108 ([photons/sec/cm2/steradian]/[μW/cm2]). Scale bar, 5 mm. (B) Corresponding quantitation of the adjusted mean radiant efficiency (×107 [photons/sec/cm2/steradian]/[μW/cm2]) of rat carotids to time after administration (h) by nanocarrier size and day(s) after balloon injury. Data are mean ± standard deviation (SD, n = 4 per bar). *P < 0.05, †P < 0.01 vs contralateral carotid.

artery and embedded in an optimal cutting temperature (OCT) compound (Sakura Finetek, Tokyo, Japan). Transverse cryosections (10 μm thick) were cut and stained using a Hoechst 33342 solution (Invitrogen, Eugene, OR) as the nuclear stain. The distribution of Cy5-labeled nanocarriers was visualized using a confocal laser scanning microscope (Zeiss LSM 780, Jena, Germany) equipped with a Plan-Apocromat objective lens (Zeiss, Jena, Germany). In tissue sections with an evident accumulation of nanocarriers, immunostaining for CD68, α-smooth muscle actin (αSMA), or embryonic smooth muscle myosin heavy chain (SMemb) was conducted to evaluate the presence of macrophages, medial smooth muscle cells (SMCs), or neointimal SMCs, respectively. After blocking (Blocking One, Nacalai Tesque, Inc., Kyoto, Japan), monoclonal antibodies against CD68 (1:100; T-3003, BMA Biomedicals, Augst, Switzerland), αSMA (1:100; 1A4, Dako,

Glostrup, Denmark), or SMemb (1:1000; 7602, Yamasa, Tokyo, Japan) were added, and sections were incubated for 30 min at room temperature. Subsequently, the sections were incubated with Alexa Fluor 555 labeled secondary antibodies (1:300; ab150118, Abcam, Cambridge, UK) for 30 min at room temperature, stained with Hoechst 33342, and analyzed with a confocal laser scanning microscope. 2.5. Evaluation of the Distribution of Epirubicin Incorporating Polymeric Micelles (EPM) in Arterial Lesions. The effect of nanocarrier-based delivery of epirubicin to arterial lesions was performed utilizing EPM (NC-6300, NanoCarrier Co., Kashiwa, Japan).17−19 Alexa Fluor 647 labeled EPM was prepared in prior to evaluate the accumulation of the polymeric micelle (see Supporting Information). The left carotid arteries of the rats were injured using a balloon catheter in the same manner as described above. C

DOI: 10.1021/acs.molpharmaceut.6b00219 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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than those of the contralateral carotid. In contrast, samples with little Cy5 emission revealed no considerable values for the injured carotid, compared with the contralateral carotid (Figure 1B). 3.2. Microscopic Distribution of Nanocarriers in Arterial Lesions. The microdistribution of nanocarriers in the injured carotids was also assessed histologically (Figure 2). When nanocarriers were administrated immediately after balloon injury, a neointimal lesion was not observed in any of the left carotid arteries. In the carotids harvested 1 h after the injection of PICs-40, PICs-100, or PICs-200, a clear accumulation of Cy5-positive particles in the media and adventitia was observed (Figure 2). Cy5-positive particles

At 7 days after balloon injury, a 9 mg/kg dose of Alexa647labeled EPM was injected via the tail vein (n = 3). The left carotid artery was harvested 8 h after injection, and embedded in an OCT compound. Transverse cryosections were immunostained for CD68 or SMemb, treated with Hoechst 33342, and analyzed using a confocal laser scanning microscope, as described above. 2.6. Efficacy of EPM in Arterial Lesions. A 6 mg/kg dose of EPM (1.2 mL/kg; epirubicin dose, 18 mg/kg) was injected via the tail vein (EPM group, n = 4) 7, 10, and 13 days after balloon injury. In the control group, 10% sucrose/deionized water (DW, 1.2 mL/kg) or epirubicin dissolved in DW (1.2 mL/kg; epirubicin dose, 18 mg/kg) was injected into the rats 7, 10, and 13 days after the injury (vehicle and epirubicin group, respectively, n = 4 each). The body weight of the rats was measured every 2−3 days. The animals were euthanized with an overdose of anesthesia 14 days after the first injection, and the left carotid artery was excised after perfusion-fixation with 4% paraformaldehyde at 120 mmHg. Three arterial segments were dissected from the excised carotid artery 1−6 mm, 6−11 mm, and 11−16 mm from the origin of the left common carotid artery. The arterial segments were additionally fixed overnight with 4% paraformaldehyde and embedded in paraffin. Tissue cross sections (4 μm thick) were cut from the paraffin blocks and stained with hematoxylin−eosin. The areas of the neointimal and medial layer on each section were measured using a computerized digital image analysis system (ImageJ, NIH, Bethesda, MD), and the ratios of the neointimal area to the medial area were calculated (intima/media area ratio). Furthermore, the number of neointimal cells in each section was counted. The mean value of the three segments from each carotid artery was used for statistical analyses. 2.7. Statistical Analysis. All statistical analyses were performed using standard software programs (JMP Pro 10.0.2, SAS Institute, Cary, NC). Data are reported as mean ± standard deviation unless otherwise specified. The unpaired t-test was used to compare continuous variables. A P value of 20% 14 days after the first injection (Figure 5A). The animals in the EPM group also showed a steady increase in body weight after injection, although the relative weights of the EPM group were significantly lower than those of the vehicle group from 3 days after the first injection. In contrast, all rats in the epirubicin group died within 10 days of the first injection. Histological evaluation of the carotid samples was therefore performed only in the EPM and vehicle groups whose mortality rate was 0%. Formation of neointimal lesions was observed in both the EPM and vehicle groups; neointimal lesions in the EPM group were smaller, compared with those in the vehicle group (Figure 5B). The intima/media area ratio (Figure 5C) and the number of neointimal cells (Figure 5D) were significantly lower in the EPM group, compared with the vehicle group.

Figure 3. Localization of Cy5-labeled polyethylene glycol-conjugated polyion complex micelles and vesicles in arterial lesions. Representative immunofluorescence micrographs of macrophage (left) and smooth muscle cell (SMC, right) of injured carotid artery 1 h after administration of PICs-40, PICs-100, or PICs-200 to rats immediately after (top) balloon injury (n = 4). The injured carotid artery 1 or 24 h after administration of PICs-40 to rats 7 days after (middle) or 14 days after (bottom) balloon injury are also shown (n = 4). The nuclei were counterstained with Hoechst (blue). Macrophages with CD68 (red, excitation: 561 nm), SMC with α-smooth muscle actin or embryonic smooth muscle myosin heavy chain (red, excitation: 561 nm), Cy5 (yellow), and autofluorescence (green) of elastic laminae are also visualized. L, Lumen. Scale bar, 20 μm.

4. DISCUSSION In this work, we investigated the effect of the size of PIC-based nanocarriers on their distribution in rat carotid arteries that had E

DOI: 10.1021/acs.molpharmaceut.6b00219 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 4. Accumulation of Alexa 647-labeled EPM in arterial lesions. (A) Representative immunofluorescence micrographs of macrophage (left) and smooth muscle cell (SMC, right) of injured carotid artery 8 h after administration of Alexa 647-labeled EPM to rats 7 days after balloon injury (n = 3). The nuclei were counterstained with Hoechst (blue), macrophage with CD68 (red), and SMC with embryonic smooth muscle myosin heavy chain (SMemb, red). Alexa 647 (yellow, excitation: 633 nm) and autofluorescence (green) of elastic laminae are also visualized. Scale bar, 100 μm. (B) Magnification of the orange square above with SMemb stain in high power field. The entire component (top left) without nuclear stain (top right), without nuclear stain and Alexa 647 (bottom left), and without nuclear stain and SMC (bottom right) are shown. Scale bar, 10 μm.

Figure 5. Reduction of neointimal hyperplasia in arterial lesions by EPM. (A) Relative body weight change of rats administered with EPM (red), free epirubicin (blue), or vehicle (green) 3 times, with a 3 day interval, from 7 days after balloon injury (indicated by arrowheads). Data are indicated as mean ± standard deviation (SD, n = 4). (B) Photomicrographs (hematoxylin−eosin staining) of injured carotid artery of rats administered with EPM (left) or vehicle (right) at 21 days after balloon injury. N, neointima. Scale bar, 100 μm. (C,D) Intima/media area ratio (C) and total cell count of the neointima (D) of injured carotid artery in rats administered with EPM or vehicle at 21 days after balloon injury. Data are shown as mean ± SD (n = 4). *P < 0.05, †P < 0.001 vs vehicle.

been injured by a balloon catheter. There is high reproducibility in the development of neointimal lesions caused by balloon injury in rat carotid arteries.12 Since the neointima of a rat carotid is similar to an intimal lesion occurring in early stages of human atherosclerosis,20 the balloon injury model of rat carotids has been widely used as an experimental model for studying the pathophysiology of atherosclerosis.3−7 Previous investigators have elucidated many details about the process of neointimal formation using the balloon injury model in rats.12,21 Briefly, injury with a balloon catheter denudes the monolayer of endothelial cells and damages the cells of the media. In response to the damage, portions of the SMCs in the medial layer are induced to proliferate and peak 2 days after injury. Additionally, portions of the SMCs in the media migrate to the neointima beginning at 4 days after injury, and the migrated cells in the neointima start to proliferate immediately. Afterward, cell proliferation in the neointima gradually decrease until it generally ceases 14 days after injury. The intimal cells simultaneously synthesize ECM, which are accumulated in the neointima. Therefore, the proliferation of SMCs and the accumulation of ECM collectively form the neointimal lesion. Namely, nascent neointima begin to appear at 4 days after injury; however, a longer duration is required for it to be recognized as a lesion. Since our final goal was the development

of a system for atherosclerotic lesion-specific drug delivery, we needed to target the artery with clear and evident neointimal lesions. Therefore, out of our injection protocol of either 0, 7, or 14 days after balloon injury, it was more relevant to administer the nanocarriers 7 or 14 days after injury rather than immediately after injury, making our study distinct from previous studies.3−7 Our results showed that the distribution of nanocarriers in injured rat carotids was markedly different depending on whether the neointimal lesion existed at the time of administration. When nanocarriers were intravenously injected immediately after balloon injury, all 3 sizes of nanocarriers rapidly accumulated in the media and adventitia of the injured carotids. These findings may reflect microstructural damage to the carotid wall caused by the balloon injury. The passages of an inflated balloon through the carotid lumen exert excessive F

DOI: 10.1021/acs.molpharmaceut.6b00219 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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injured carotids in a manner reminiscent of PICs-40. Furthermore, most of the Alexa647-positive particles in the neointima did not colocalize with the CD68- or SMembpositive cells, which was also comparable to PICs-40. Sustained accumulation of nanocarriers in the neointima may facilitate translocation of the nanocarriers into neointimal cells via endocytosis. Following internalization, the nanocarriers would then be expected to encounter acidic endosomes and lysosomes over time. In the case of EPM, the acidic conditions within endosomes and lysosomes induce the release of free epirubicin.17 Epirubicin intercalates deoxyribonucleic acid (DNA) strands and inhibits synthesis of DNA and ribonucleic acid (RNA), suppressing cell proliferation.23 Therefore, the nanocarriers needed to be administrated when cell proliferation played a crucial role in the development of neointimal lesion in order to examine the effects of EPM on the neointimal lesion. As described above regarding the process of the neointimal formation in the rat carotid, the nascent neointima appears 4 days after balloon injury, and the intimal cells continue to proliferate mostly until 14 days after the injury. Therefore, in the rat carotid artery 4−14 days after balloon injury, the proliferation of neointimal cells is a promoter of neointimal development, indicating that EPM potentially suppresses the development of neointima by inhibiting cell proliferation. Since clear accumulation of EPM in the neointima had been confirmed in the rat 7 days after balloon injury, we considered that the administration of EPM should be scheduled 7−14 days after injury and therefore decided to inject EPM 7, 10, and 13 days after injury, which was consistent with the process of neointimal formation. To assess the effect of EPM administration on the rat neointima, we prepared two control groups: the epirubicin group and the vehicle group. Our results showed marked reduction of acute toxicity of epirubicin in the EPM group, as compared to the epirubicin group. One reason could be that EPM can circulate in vivo for a long period, with the epirubicin remaining encapsulated, due to the “stealth” effect by the PEG shell.24,25 The stealth effect of EPM leads to sustained release of free epirubicin, while the full dose of free epirubicin was delivered almost instantaneously in the epirubicin group.18 Furthermore, previous study has shown reduced accumulation of EPM into the kidney, lung, and heart, resulting in minimized side effects.19 The most important finding of this work was that EPM administration significantly suppressed the development of the rat neointima when compared to vehicle administration. The result suggests that a considerable number of EPMs were specifically accumulated in the neointimal lesion after intravenous injections, and EPM successfully delivered a sufficient amount of free epirubicin to the neointimal cells. The delivery of epirubicin inhibited the proliferation of neointimal cells, resulting in the suppression of neointimal development. According to the morphometric analyses of histological images, the number of neointimal cells was significantly lower in the EPM group than the vehicle group 14 days after the first injection, which proved the inhibitory effect of cell proliferation in the neointima. In conclusion, we were able to demonstrate that 40 nm, and not >100 nm, is an adequate nanocarrier size for drug delivery to neointimal lesions. Furthermore, we showed the pharmacological effects of epirubicin incorporated nanocarriers based on this size-effect analysis. Our findings indicate that simple modification of nanocarrier size enables the enhancement of

tensile stress on all layers of the carotid artery. This is known to cause tears in the internal elastic lamina and denudation of the endothelium.22 Such structural changes in the injured carotid may allow nanocarriers of various sizes to rapidly penetrate into the carotid media and adventitia. Indeed, the sizes of drug incorporated nanocarriers that showed therapeutic effects ranged from 40 to 160 nm in previous studies.3−6 In contrast, when nanocarriers were injected to the rats 7 or 14 days after injury, only PICs-40 had accumulated in the neointima of injured carotids to a considerable degree. Since the neointimal lesion was formed in response to the balloon injury, there were no fissures due to trauma in the microstructure of the neointima. Moreover, little regeneration of the endothelial layer was observed in rat carotid arteries 7 and 14 days after balloon injury in previous studies, indicating that the neointimal ECM, including intimal cells, was mostly exposed to the bloodstream.12,21 According to the distributional behavior, PICs-40 could infiltrate through the interstices of the ECM network of the neointima, while PICs-100 and PICs-200 could not. As observed by immunostains of the carotid samples, most PICs-40 in the neointima did not colocalize with CD68positive cells or SMemb-positive cells. Since the cell component of the neointima in the rat model consists of SMCs and a few macrophages, the findings from the immunostains indicated that PICs-40 were mostly located in the ECM network of the neointima and not in the neointimal cells. This finding supported our hypothesis of PICs-40 infiltrating into the neointima through the interstices of the ECM network. Another distinctive feature of the distribution of PICs-40 was that PICs-40 remained accumulated in the neointima for a considerable period of time. In the rat model, a significant accumulation of PICs-40 was detected 1, 8, and 24 h after injection 7 days after injury, indicating that regional accumulation was sustained for at least 23 h. Similarly, the retention period of PICs-40 was at least 16 h in the rat model 14 days after injury. There is little evidence about how long the nanocarriers have to be retained in the neointima to achieve sufficient drug delivery, which may depend upon both the properties of the specific nanocarrier and the drug being delivered. However, it is obvious that a retention period of >16 h will enable a certain level of sustained drug delivery, increasing the probability of endocytotic internalization by the neointimal cells. According to the findings of our experiments comparing the behavior of different sized nanocarriers, a 40 nm in diameter was within the adequate size range required to allow specific and sustained drug delivery to the neointimal lesions. Based on the analysis of the behavior of differently sized nanocarriers in the rat neointima, we attempted to deliver a drug to the rat neointima using drug-incorporated nanocarriers with similar biomaterial properties and then evaluated the efficacy of the drug on the neointima. To conduct this experiment, we used EPM,17−19 which consists of epirubicin covalently bound to a block copolymer consisting of PEG and polyaspartate blocks. In aqueous media, this compound spontaneously forms a micellar nanocarrier in which the PEG components are arrayed as an outer shell layer with an average diameter of approximately 49 nm (see Supporting Information). We assumed that EPM could be compared to PICs-40 due to the slight difference in particle size and half-life in the circulation of approximately 4 h.18 Alexa647-labeled EPM that were intravenously administrated to the rats 7 days after balloon injury showed clear accumulation in the neointima of G

DOI: 10.1021/acs.molpharmaceut.6b00219 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

of rapamycin-loaded nanoparticles prevented neointimal hyperplasia and reendothelialized injured artery. Circ.: Cardiovasc. Interventions 2008, 1, 209−216. (5) Uwatoku, T.; Shimokawa, H.; Abe, K.; Matsumoto, Y.; Hattori, T.; Oi, K.; Matsuda, T.; Kataoka, K.; Takeshita, A. Application of nanoparticle technology for the prevention of restenosis after balloon injury in rats. Circ. Res. 2003, 92, e62−69. (6) Banai, S.; Chorny, M.; Gertz, S. D.; Fishbein, I.; Gao, J.; Perez, L.; Lazarovichi, G.; Gazit, A.; Levitzki, A.; Golomb, G. Locally delivered nanoencapsulated tyrphostin (AGL-2043) reduces neointima formation in balloon-injured rat carotid and stented porcine coronary arteries. Biomaterials 2005, 26, 451−461. (7) Kapadia, M. R.; Chow, L. W.; Tsihlis, N. D.; Ahanchi, S. S.; Eng, J. W.; Murar, J.; Martinez, J.; Popowich, D. A.; Jiang, Q.; Hrabie, J. A.; Saavedra, J. E.; Keefer, L. K.; Hulvat, J. F.; Stupp, S. I.; Kibbe, M. R. Nitric oxide and nanotechnology: a novel approach to inhibit neointimal hyperplasia. J. Vasc Surg. 2008, 47, 173−182. (8) Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. Tumor vascular permeability and the EPR effect in macromolecular therapeutics: A review. J. Controlled Release 2000, 65, 271−284. (9) Moore, T.; Chen, H.; Morrison, R.; Wang, F.; Anker, J. N.; Alexis, F. Nanotechnologies for noninvasive measurement of drug release. Mol. Pharmaceutics 2014, 11, 24−39. (10) Tarbell, J. M. Mass transport in arteries and the localization of atherosclerosis. Annu. Rev. Biomed. Eng. 2003, 5, 79−118. (11) Libby, P. Mechanisms of acute coronary syndromes and their implications for therapy. N. Engl. J. Med. 2013, 368, 2004−2013. (12) Clowes, A. W.; Reidy, M. A.; Clowes, M. M. Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium. Lab Invest. 1983, 49, 327−333. (13) Tulis, D. A. Rat carotid artery balloon injury model. Methods Mol. Med. 2007, 139, 1−30. (14) Anraku, Y.; Kishimura, A.; Oba, M.; Yamasaki, Y.; Kataoka, K. Spontaneous formation of nanosized unilamellar polyion complex vesicles with tunable size and properties. J. Am. Chem. Soc. 2010, 132, 1631−1636. (15) Anraku, Y.; Kishimura, A.; Kobayashi, A.; Oba, M.; Kataoka, K. Size-controlled long-circulating picsome as a ruler to measure critical cut-off disposition size into normal and tumor tissues. Chem. Commun. (Cambridge, U. K.) 2011, 47, 6054−6056. (16) Kishimura, A. Development of polyion complex vesicles (PICsomes) from block copolymers for biomedical applications. Polym. J. 2013, 45, 892−897. (17) Bae, Y.; Fukushima, S.; Harada, A.; Kataoka, K. Design of environment-sensitive supramolecular assemblies for intracellular drug delivery: Polymeric micelles that are responsive to intracellular pH change. Angew. Chem., Int. Ed. 2003, 42, 4640−4643. (18) Harada, M.; Bobe, I.; Saito, H.; Shibata, N.; Tanaka, R.; Hayashi, T.; Kato, Y. Improved anti-tumor activity of stabilized anthracycline polymeric micelle formulation, NC-6300. Cancer Sci. 2011, 102, 192− 199. (19) Takahashi, A.; Yamamoto, Y.; Yasunaga, M.; Koga, Y.; Kuroda, J.; Takigahira, M.; Harada, M.; Saito, H.; Hayashi, T.; Kato, Y.; Kinoshita, T.; Ohkohchi, N.; Hyodo, I.; Matsumura, Y. NC-6300, an epirubicin-incorporating micelle, extends the antitumor effect and reduces the cardiotoxicity of epirubicin. Cancer Sci. 2013, 104, 920− 925. (20) Nakashima, Y.; Wight, T. N.; Sueishi, K. Early atherosclerosis in humans: role of diffuse intimal thickening and extracellular matrix proteoglycans. Cardiovasc. Res. 2008, 79, 14−23. (21) Clowes, A. W.; Clowes, M. M.; Fingerle, J.; Reidy, M. A. Regulation of smooth muscle cell growth in injured artery. J. Cardiovasc. Pharmacol. 1989, 14, S12−15. (22) Steele, P. M.; Chesebro, J. H.; Stanson, A. W.; Holmes, D. R.; Dewanjee, M. K.; Badimon, L.; Fuster, V. Balloon angioplasty. Natural history of the pathophysiological response to injury in a pig model. Circ. Res. 1985, 57, 105−12. (23) Minotti, G.; Menna, P.; Salvatorelli, E.; Cairo, G.; Gianni, L. Anthracyclines: molecular advances and pharmacologic developments

their accumulation and therapeutic efficacy in the rat carotid artery balloon injury model. Due to the structural similarity between rat neointima and atherosclerotic lesions in humans, we consider that this nanocarrier-based drug delivery system could be utilized for the treatment of atherosclerosis. We hope that our findings will encourage the development of safer and more effective drug incorporated nanocarriers, which can possibly lead to curing human atherosclerosis.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.6b00219. Supplementary methods and results (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-3-3815-5411. Fax: +81-3-3811-6822. E-mail: [email protected]. Present Address ○

Department of Vascular Surgery, Saitama Medical Center, Saitama Medical University, 1981 Kamoda, Kawagoe, Saitama 350-8550, Japan.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Grants-in-Aid for Young Scientists (A) (No. 24689051 to Y.M.), Grants-in-Aid for Scientific Research (B) (No. 26462100 to H.K., No. 26288082 to A.K.), and Grants-in-Aid for Challenging Exploratory Research (No. 24659584 to Y.M.) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan. This study was partially supported by the Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program) from the Japan Society for the Promotion of Science (to K.K.).

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REFERENCES

(1) Lopez, A. D.; Mathers, C. D.; Ezzati, M.; Jamison, D. T.; Murray, C. J. Global and regional burden of disease and risk factors, 2001: systematic analysis of population health data. Lancet 2006, 367, 1747− 1757. (2) Smith, S. C.; Benjamin, E. J.; Bonow, R. O.; Braun, L. T.; Creager, M. A.; Franklin, B. A.; Gibbons, R. J.; Grundy, S. M.; Hiratzka, L. F.; Jones, D. W.; Lloyd-Jones, D. M.; Minissian, M.; Mosca, L.; Peterson, E. D.; Sacco, R. L.; Spertus, J.; Stein, J. H.; Taubert, K. A. World Heart Federation and the Preventive Cardiovascular Nurses Association. AHA/ACCF secondary prevention and risk reduction therapy for patients with coronary and other atherosclerotic vascular disease: 2011 update: A guideline from the American heart association and American college of cardiology foundation. Circulation 2011, 124, 2458−2473. (3) Chan, J. M.; Rhee, J. W.; Drum, C. L.; Bronson, R. T.; Golomb, G.; Langer, R.; Farokhzad, O. C. In vivo prevention of arterial restenosis with paclitaxel-encapsulated targeted lipid-polymeric nanoparticles. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 19347−19352. (4) Reddy, M. K.; Vasir, J. K.; Sahoo, S. K.; Jain, T. K.; Yallapu, M. M.; Labhasetwar, V. Inhibition of apoptosis through localized delivery H

DOI: 10.1021/acs.molpharmaceut.6b00219 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics in antitumor activity and cardiotoxicity. Pharmacol Rev. 2004, 56, 185− 229. (24) Kataoka, K.; Kwon, G. S.; Yokoyama, M.; Okano, T.; Sakurai, Y. Block copolymer micelles as vehicles for drug delivery. J. Controlled Release 1993, 24, 119−132. (25) Gullotti, E.; Yeo, Y. Extracellularly activated nanocarriers: A new paradigm of tumor targeted drug delivery. Mol. Pharmaceutics 2009, 6, 1041−51.

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DOI: 10.1021/acs.molpharmaceut.6b00219 Mol. Pharmaceutics XXXX, XXX, XXX−XXX