A Novel Micelle-Forming Material Used for Preparing a Theranostic

Article pubs.acs.org/jmc

A Novel Micelle-Forming Material Used for Preparing a Theranostic Vehicle Exhibiting Enhanced in Vivo Therapeutic Efficacy Hsiao-Ping Chen,† Ming-Hong Chen,†,‡,§,∥ Fu-I Tung,⊥ and Tse-Ying Liu*,†,# †

Institute of Biomedical Engineering, National Yang-Ming University, Taipei, Taiwan, R.O.C. Division of Neurosurgery, Department of Surgery, Taipei Tzu Chi Hospital, Taipei, Taiwan, R.O.C. § Department of Surgery, School of Medicine, Tzu Chi University, Hualien City, Taiwan, R.O.C. ∥ Department of Biomedical Engineering, Ming Chuang University, Taipei, Taiwan, R.O.C. ⊥ Department of Orthopaedic Surgery, Taipei City Hospital, Taipei, Taiwan, R.O.C. # Biophotonics & Molecular Imaging Research Center (BMIRC), National Yang-Ming University, Taipei, Taiwan, R.O.C. ‡

S Supporting Information *

ABSTRACT: A new micelle-forming material, folic acid-conjugated carboxymethyl lauryl chitosan (FA-CLC), and superparamagnetic iron oxide (SPIO) nanoparticles were used for preparing an imaging-guided drug vehicle (the FA-CLC/SPIO hybrid micelle) that demonstrates targeted delivery, imaging, and controlled release of hydrophobic agents. We found that the ratio of viable normal cells to tumor cells was increased prominently after delivery of camptothecin (CPT)-loaded FA-CLC/SPIO micelles and therapeutic sonication. In addition, a magnetic field could enhance the tumor-targeting effect of FA-CLC/ SPIO micelles. Therefore, after sequential administration of magnetic attraction to CPT-loaded FA-CLC/SPIO micelles, and therapeutic sonication, the in vivo therapeutic efficacy of CPT was markedly enhanced. However, a nonfocused magnetic field could enhance the undesirable accumulation of iron-containing vehicles in the liver if the tumor (i.e., magnetic attraction site) is near the liver. We propose that magnetic attraction must be carefully applied, far from the liver.

■

INTRODUCTION Chemotherapy is one of the main treatments used in cancer therapy. Unfortunately, antitumor agents also inevitably produce side effects. Therefore, it is critical to understand how to lower the administration dose of an antitumor agent without reducing its effectiveness in killing tumor cells. Recently, an image-guided DDS (i.e., a drug-delivery system featuring drug-encapsulation and imaging functions) combined with targeted-delivery and triggered-release functions was developed in order to help lower the dose and systemic toxicity of highly toxic pharmaceutical agents administered in the human body.1−4 This enabled the realization of the concept of integrating diagnosis and therapy within a single system, which has been widely accepted and applied in trials for several diseases.5−7 One of the most important strategies to lower the systemic toxicity of an antitumor agent is to develop drug vehicles that feature passive targeting (i.e., the enhanced permeation and retention (EPR) effect) and/or active targeting functionalities.6,8−10 In addition, vehicles that exhibit pH-triggered release behaviors have been developed to ensure that the encapsulated drug can be released in intracellular acidic compartments.11−13 However, it remains challenging to prepare a vehicle that can encapsulate hydrophobic agents, demonstrate targeted delivery, © XXXX American Chemical Society

exhibit magnetic resonance (MR) imaging functionality, and display pH-triggered release behavior. In this study, we developed a new micelle-forming material, folic acid (FA)conjugated carboxymethyl lauryl chitosan (FA-CLC), and used it together with superparamagnetic iron oxide (SPIO) nanoparticles to prepare a hybrid micelle, the FA-CLC/SPIO micelle. CLC is a pH-sensitive, cationic, and amphiphilic chitosan derivative that was developed by our group and used as a biocompatible material to encapsulate hydrophobic drugs.14 In this study, we expected the cationic micelle-forming material, the FA-CLC molecules, to target angiogenic vessels that overexpress negatively charged phosphatidylserine residues, and to target FA receptor-overexpressing tumor cells within tumor tissues.15−17 SPIO nanoparticles have been employed to exhibit MR imaging functionality and demonstrate magnetic targeted delivery.18−20 Therefore, targeted delivery and controlled release of hydrophobic agents could be achieved using FA-CLC/SPIO micelles by means of targeting to angiogenic vessels (i.e., through cationic CLC and magnetic attraction) and tumor cells (i.e., through the EPR effect and FA-mediated targeting) in tumor tissues, followed by pHReceived: September 26, 2014

A

DOI: 10.1021/jm501996y J. Med. Chem. XXXX, XXX, XXX−XXX

Article

Journal of Medicinal Chemistry

Figure 1. Cationic FA-CLC/SPIO micelle designed for employing combined strategies (targeted delivery, US-enhanced internalization, and pHtriggered release) to overcome the barriers against the delivery of drugs.

micelles that were designed for magnetic guidance and sonication-enhanced delivery. The effects of US and the CPT-loaded FA-CLC/SPIO micelles on the ratio of viable normal cells to viable tumor cells in vitro were investigated. The possible negative effects of US bombardment and magnetic guidance were also addressed. Furthermore, we studied the in vivo therapeutic efficacy of sequentially administering a magnetic field to the CPT-loaded FA-CLC/SPIO micelles, and therapeutic sonication. Our results provide valuable fundamental information that can support future design and fabrication of theranostic agents featuring imaging and thermochemotherapy functionalities.

triggered drug release inside tumor cells. This report presents the first study in which CLC was used for preparing cationic hybrid micelles designed for combining targeted delivery, MR imaging functionality, and pH-triggered release of hydrophobic agents, all of which warrant systemic investigation. Ultrasound (US) is a noninvasive, radio-free, easily focused, and cost-effective form of energy that is used to overcome transvascular and/or transcellular barriers against drug delivery.21−24 However, this usage poses a major challenge because US bombardment might trigger early leakage of drugs from vehicles before the completion of extravasation and/or endocytosis (i.e., it might trigger drug release in the intravascular and/or extracellular space rather than in intracellular acidic compartments). Therefore, we developed organic/inorganic hybrid micelles to demonstrate limited USinduced drug leakage and adequate pH sensitivity for targeted delivery, as shown in Figure 1: Because the camptothecin (CPT)-loaded FA-CLC/SPIO micelles feature targeted functionalities, they should become highly concentrated in the intravascular space of tumor tissues and/or the extracellular space of tumor cells under a static magnetic field. Subsequently, the tumor tissue is bombarded using therapeutic sonication in order to enhance the site-specific internalization of the micelles. After internalization (i.e., after translocation from the extracellular to the intracellular space), the release of the encapsulated drugs should be triggered from the pH-sensitive vehicles that localize in intracellular acidic compartments. This hypothesis was tested in this study. We did not focus on the positive effect of US on transvascular and transcellular delivery, which has been widely described;21−24 instead, we compared the US-induced increment of FA-conjugated vehicles internalized into FA-positive tumor cells with the vesicles that were internalized into normal cells. This increment was highly correlated with the effect of the CPT-loaded FA-CLC/SPIO micelles on the ratio of viable normal cells to viable tumor cells when the therapeutic sonication was applied, a finding that has rarely been reported and warrants further exploration. Our main objective in this study was to use the new micelleforming material, CLC, to prepare novel FA-CLC/SPIO

■

RESULTS AND DISCUSSION Characterization of FA-Conjugated CLC and FA-CLC/ SPIO Micelles. Our main objective in this study was to prepare multifunctional drug vehicles by using FA-conjugated CLC as a micelle-forming material. FA-CLC conjugates were synthesized by chemically linking FA with CLC by using EDC/NHS as a carboxyl-activating agent (Scheme 1). The 1H NMR spectrum of CLC is shown in Figure 2A), in which ring protons (C1 to C6) corresponding to the chemical shifts between δ 3.6 and 4.1 ppm were observed. The chemical shifts at δ 4.03 and δ 4.10 ppm were designated to the protons of NCH2CO and OCH2CO of CLC, respectively, and the chemical shifts at δ 0.88 ppm (CH3−), δ 1.2−1.8 ppm (−CH2−), and δ 2.11 ppm (−C−CH2−CO) were assigned to the protons from the lauryl chain. These chemical shits assigned to CLC were also present in the 1H NMR spectrum of FA-conjugated CLC (Figure 2B), with a slight upfield shift of 0.2 ppm. Furthermore, an intensified chemical shift at δ 2.58 ppm was observed because of the conjugation of FA molecules.25,26 The characteristic peak from δ 6.71−8.29 ppm assigned to the aromatic protons of the FA molecules was observed in FA-conjugated CLC.27 These results indicated that FA was successfully conjugated with CLC, and this was further supported by the UV−vis spectrum shown in Figure 2C: a characteristic peak was observed at 285 nm in the spectra of FA and FA-conjugated CLC, which confirmed the successful synthesis of FA-conjugated CLC. B


Article

Journal of Medicinal Chemistry Scheme 1. Structures of FA, CLC, and FA-Conjugated CLC

43.26°, and 62.87° were present in the diffraction pattern of FA-CLC/SPIO micelles and these were assigned to the (311), (400), and (440) planes of Fe 3 O 4 (JCPD 02-1035), respectively. These results suggest that the obtained micelles were composed of Fe3O4 nanoparticles. The loading efficiency of SPIO was characterized using TGA and was determined to be 74.1%. Moreover, monosized SPIO nanoparticles featuring a mean size of 4 nm were assembled in the FA-CLC/SPIO micelles, which was attributed to the hydrophobic interaction between lipophilic ligands on the SPIO surface and the hydrophobic moieties on the CLC molecules. This was expected to enable efficient encapsulation of hydrophobic agents, which was demonstrated by the results presented in Table 1: the EE and LE of CPT in the FA-CLC/SPIO micelles were 63.6% and 4.9%, respectively. The EE of CPT in the FACLC/SPIO micelles was not very high because CPT is not extremely hydrophobic. Figure 3D presents images of FACLC/SPIO micelles that had and had not been exposed to

The morphology of the FA-CLC/SPIO micelles was characterized using TEM. As shown in Figure 3A, an inorganic/organic hybrid-micelle structure featuring an average particle size of 122 nm was successfully prepared through the incorporation of the amphiphilic FA-conjugated CLC with hydrophobic SPIO nanoparticles. The TEM sample was dried and vacuumed, and thus it is reasonable that the particle size measured using TEM was smaller than that measured using the DLS method (138 nm, inset in Figure 3A). This result suggested that CLC exhibited an extremely high micelleforming ability that can be used for preparing nanovehicles by means of a facile sonication method. The phase of the nanoparticles incorporated in the core of the FA-CLC/SPIO micelles was characterized by performing HR-TEM (Figure 3B). The HR-TEM photographs of the inorganic nanoparticles incorporated in the micelles revealed lattice fringes of 0.2581 nm that were assigned to the (311) planes of crystalline Fe3O4. The XRD patterns (Figure 3C) showed that peaks at 35.65°, C


Article


Figure 2. 1H NMR spectra of (A) CLC and (B) FA-conjugated CLC. (C) UV−vis spectra of CLC, FA, and FA-conjugated CLC.

magnetic attraction; the FA-CLC/SPIO micelles attracted by the magnet are brown in color. These results suggest that the

micelles developed in this study can be readily attracted using magnetism. The magnetic properties of the FA-CLC/SPIO D


Article


Figure 3. (A) TEM images and particle-size distribution of CPT-loaded FA-CLC/SPIO micelles. (B) HR-TEM image of inorganic nanoparticles incorporated within FA-CLC/SPIO micelles. (C) XRD patterns of CLC, SPIO, and FA-CLC/SPIO micelles. (D) Magnetization−magnetic field strength (M−H) curve of CPT-loaded FA-CLC/SPIO micelles, which were suspended in solution (inset) without (left) and with (right) the application of a magnetic field.

Table 1. Characteristics of CPT-Loaded FA-CLC/SPIO Micelles vehicle CPT-loaded FA-CLC/SPIO micelle

particle size (nm)

zeta potential (mV)

EE of CPT (%)

LE of CPT (%)

137.9

11.6 (pH 7.4) 48.5 (pH 5.2)

63.6

4.9

micelles were characterized using a SQUID system. Figure 3D shows the magnetization−magnetic field strength (M−H) curve of the micelles; no hysteresis loop was observed, indicating that the drug vehicle exhibited superparamagnetic behavior, which is expected to demonstrate MR T2 image contrast. Effect of Ultrasound on FA-Mediated Internalization. We characterized the biocompatibility of the CLC/SPIO micelles (Figure 4) and determined that irrespective of FA conjugation, these micelles did not induce marked cytotoxicity in normal L929 cells, FA-negative A549 tumor cells, and FApositive MDA-MB-231 tumor cells. Moreover, confocal imaging of the FA-negative A549 and FA-positive MDA-MB231 cells that had taken up Nile red-loaded FA-CLC/SPIO micelles (Figure 5A) showed that the red fluorescence emitted from MDA-MB-231 cells was stronger than that emitted from A549 cells. This result indicates that the hydrophobic model agent (Nile red) loaded in FA-CLC/SPIO micelles could be

Figure 4. Viability of L929 fibroblasts (normal cells), A549 lungcancer cells (FA-negative tumor cells), and MDA-MB-231 breastcancer cells (FA-positive tumor cells) incubated with various concentrations (micelle weight/culture medium volume = 2.5, 5, and 10 μg/mL) of CLC/SPIO micelles and FA-CLC/SPIO micelles for 24 h.

specifically taken up by FA-positive tumor cells. To confirm that the vehicles were specifically internalized by the FApositive MDA-MB-231 cells, we used ICP to determine the uptake amount of the SPIO-containing drug vehicles (Figure 5B), which showed that the amount of SPIO nanoparticles internalized by MDA-MB-231 cells at 6 h was higher than that E


Article


Figure 5. (A) Confocal images of FA-negative A549 and FA-positive MDA-MB-231 cells that had taken up Nile red-loaded FA-CLC/SPIO micelles added for 24 h. Scale bar, 30 μm. (B) The iron content in A549 and MDA-MB-231 cells measured using ICP-AES after incubating the cells with FACLC/SPIO micelles for 6 and 24 h. (C) Flow cytometric histogram profiles of normal (L929) and FA-positive tumor (MDA-MB-231) cells cultured with Nile red-loaded FA-CLC/SPIO micelles and exposed to various sonication conditions. Sonication parameters: frequency 1 MHz, duty ratio 20%, power density 0.4 W/cm2, sonication time 20 min.

tumor cells than in normal cells, indicating that sonication affected the amount of FA-CLC/SPIO micelles taken up to a greater extent in FA-positive tumor cells than in normal cells. This could be explained based on the following mechanisms. First, US enhanced the convection and diffusion of the extracellular fluid and also the fluidity of cell membrane, and thus the possibility of FA ligand−receptor coupling and subsequent FA-specific internalization could be increased.28 Second, Hauser et al. reported that both fluid-phase and receptor-mediated endocytosis can be enhanced by lowintensity pulsed therapeutic US.29 This might enhance both nonspecific and specific internalization. Lastly, another nonspecific pathway of US-enhanced internalization of the micelles could be the US-dependent formation of a porous plasma membrane, with the pore size being 100 nm.30 The first 2 mechanisms support our flow

internalized by A549 cells. Furthermore, the increase in the uptake amount between 6 and 24 h was greater in FA-positive MDA-MB-231 cells than in FA-negative A549. This quantitative analysis supported our qualitative confocal imaging results (Figure 5A) and suggested that the targeting capability of FA was retained after the conjugation process; thus, FA-CLC/ SPIO micelles together with the encapsulated hydrophobic agent can be specifically delivered into FA-positive breasttumor cells. Moreover, the US-induced increment of the FAconjugated vehicles internalized into FA-positive tumor cells and normal cells was investigated by using flow cytometry (Figure 5C): The amount of the model agent internalized into normal cells was lower than that internalized into FA-positive tumor cells in the absence of sonication, and the amounts of the agent internalized into both normal and FA-positive tumor cells were enhanced by sonication. Most importantly, the USinduced increase in internalization was higher in FA-positive F


Article


Figure 6. (A) Cumulative-release profiles of CPT-loaded FA-CLC/SPIO micelles in PBS buffers of various pH and under distinct sonication treatments. (B) TEM images of CPT-loaded FA-CLC/SPIO micelles immersed in PBS buffers of various pH for 4 h. In vitro MR (C) T2-weighted spin−echo images and (D) R2-concentration plot of FA-CLC/SPIO micelle suspensions obtained at various micelle concentrations (0−30 μg/mL); the micelle suspensions had been incubated for 4 h in the medium (pH 7.4 and 5.2) after receiving 20 min sonication. Sonication parameters: frequency 1 MHz, duty ratio 20%, power density 0.4 W/cm2, sonication period between 20 and 40 min (sonication time 20 min).

long lauric chain) of CLC and the lipophilic SPIO nanoparticles, and thus this organic/inorganic hybrid micelle is more rigid than normal polymeric micelles are. Therefore, the vehicle developed in this study was not affected by the short-term therapeutic sonication that we applied. By contrast, the release of drugs from the FA-CLC/SPIO micelles was considerably active under acidic conditions (Figure 6A), which can be attributed to CLC being pH sensitive because CLC molecules bear COOH and NH2 moieties. In a neutral environment, the COOH moiety tends to be ionized (to COO−), which lowers the net charge of FA-CLC/SPIO micelles, whereas in an acidic environment, the net charge of the micelle is determined by the protonated NH2 (NH2 → NH3+) and the deionized COO− (COO− → COOH). This was supported by the measurement of the zeta potential of FA-CLC/SPIO micelles at distinct pH (Table 1), which showed that the zeta potential of FA-CLC/ SPIO micelles at pH 7.4 and 5.2 was 11.6 and 48.5, respectively. The alternation of the molecular charge (i.e., interaction) of CLC might induce a change in vehicle structure and a release of the drug. This possibility was confirmed by means of TEM observation (Figure 6B), which revealed that FA-CLC/SPIO micelles retained a solid core structure and did not exhibit substantial changes in morphology after receiving sonication for 20 min and then being incubated at pH 7.4 for 4 h. However, under the same sonication conditions but after incubating the

cytometry result showing that sonication induced greater internalization of FA-CLC/SPIO micelles into FA-positive tumor cells than into normal cells under the tested conditions (i.e., under the vehicle concentration and US parameters used). Effect of Ultrasound on Drug-Release Behavior. We determined that the advantage of using therapeutic US in vitro was that sonication-induced increase in internalization of FACLC/SPIO micelles was higher in tumor cells than in normal cells (Figure 5C). However, US bombardment could potentially destroy vehicles and induce the early release of drugs in intravascular and extracellular spaces in vivo.31 Therefore, we examined the impact of therapeutic sonication on the in vitro drug-release behavior of FA-CLC/SPIO micelles. The results in Figure 6A show that the influence of short-term sonication (20 min) on drug release could almost be ignored. The effect of sonication on the release of drugs from micelle-based vehicles is debated: whereas certain researchers have reported developing US-sensitive micelles,32 other reports have suggested that micelle-based vehicles are not sensitive to US because the micelles are considerably smaller than the US wavelength. Furthermore, the lack of a bubble (air-core) structure and its corresponding resonance effect also make micelles unresponsive to sonication.33 In this study, the FACLC/SPIO micelles were assembled through the hydrophobic interaction that occurs between the hydrophobic moiety (the G


Article

Journal of Medicinal Chemistry micelles at pH 5.2 for 4 h, the solid structure transformed into a structure that appeared hollow. The TEM micro-characterization of single vehicles was supported by the macrocharacterization preformed using MR imaging. The T2 imageconcentration map and its R2-concentration plot corresponding to Figure 6B are shown in Figure 6C,D: the slope of the R2concentration plot acquired from the CPT-loaded FA-CLC/ SPIO micelles that had been sonicated from 20 min and then incubated for 4 h in the pH 5.2 medium was markedly different from the slope obtained in the case in which the micelles were incubated in the pH 7.4 medium after the same sonication. This result can be ascribed to the alteration of the distance between SPIO nanoparticles.22 In summary, the pH-sensitive nature of CLC molecules might provide the force required to alter the structure of micelles and induce drug release in acidic compartments. Furthermore, the MR T2 signal of the FACLC/SPIO micelles was pH dependent, which implied that the vehicle developed in this study could be considered a pHsensitive MR imaging-contrast agent. Effect of Ultrasound on Endolysomal Escape. We also investigated the time course of internalization of the FA-CLC/ SPIO micelles into MDA-MB-231 tumor cells in vitro by using LysoSensor Green and confocal microscopy. The LysoSensor reagent is a pH indicator that partitions into acidic compartments, within which the reagent exhibits a pH-dependent increase in fluorescence intensity upon acidification. By using this methodology, we obtained separate images in which red fluorescence represented the model drug (Nile red) and green fluorescence represented the acidic organelles (i.e., endolysomal compartments). In merged images, spots featuring a clear contour and bright yellow fluorescence represented the model drug within intact acidic compartments (i.e., the colocalization of the model drug and the acidic compartment); moreover, some of the bright yellow spots transformed and showed orange fluorescence, and these represented the colocalization of highly intense red fluorescence (i.e., a high concentration of released Nile red) with green fluorescence. Areas without a clear contour that exhibited orange color in the merged images might represent the drug escaping form disrupted endolysomal compartments. The images presented in Figure 7A show at 2 h after administration, internalized vehicles were observed (red fluorescence in the separated image); at 4 h, the release of the model drug from the vehicles was observed in acidic compartments (bright yellow in merged images); and at 6 h, large amounts of the drug had been released from the vehicles and had escaped from the acidic compartments (orange in the merged images). This might be ascribed to the cationic CLC molecules because cationic polymers such PEI and chitosan in endosome were reported to increase the influx of Cl− and water, which resulted in osmotic swelling of the endosome, rupture of the endosome membrane, and intracellular release from endosome (i.e., the proton sponge effect was triggered).34,35 In this study, we used therapeutic sonication (frequency 1 MHz, power density 0.4 W/cm2, duty ratio 20%, sonication time 20 min) to enhance the internalization of drug-loaded FACLC/SPIO micelles that were administrated and delivered to the extracellular space of tumor cells. As shown in Figure 7B, at 2 h after sonication, an increased amount of vehicle (red fluorescence) was observed in the separated images and the release of the model drug from the vehicles was observed in acidic compartments (bright yellow in the merged images). At 4 h, we observed several spots and orange areas (i.e., the drug

Figure 7. Confocal images of MDA-MB-231 breast-cancer cells incubated with LysoSensor Green and Nile red-loaded FA-CLC/SPIO micelles (A) without and (B) with sonication; the images show the intercellular trafficking of acidic compartments and vehicles. Scale bar, 20 μm. (C) Magnified images of MDA-MB-231 breast-cancer cells incubated with LysoSensor Green and Nile red-loaded FA-CLC/SPIO micelles without and with sonication for 4 h. Sonication parameters: frequency 1 MHz, duty ratio 20%, power density 0.4 W/cm2, sonication time 20 min. Scale bar, 10 μm.

was highly concentrated in acidic organelles and/or had escaped from endolysomal compartments into the cytoplasm) in the tumor cells. The results shown in Figure 7A,B suggested that therapeutic sonication can enhance the internalization and accelerate the subsequent endolysomal escape of the model drug carried by FA-CLC/SPIO micelles. The detailed mechanism that can explain the influence of US on the kinetics of intracellular trafficking remains unknown; these aspects were beyond the main focus of this study and they require further investigation. However, regardless of the pathway involved, our results shown in the magnified confocal images of the samples (obtained at 4 h after administration) that had and had not received sonication (Figure 7C) revealed that (1) the internalized vehicles remained mostly colocalized with the acidic compartments after receiving sonication; and (2) the orange fluorescence observed in the sample that received sonication was stronger than that observed in the sample that had not received sonication. These results indicated that the internalized vehicles were delivered to endolysomal compartments, that this was followed by the release of the drug from the vehicles and the subsequent escape of the drug from the acidic compartments, and that this process was potently accelerated by therapeutic sonication. Effect of Ultrasound on in Vitro Cytotoxicity. The results shown in Figures 5−7 suggested that therapeutic sonication can enhance tumor-specific internalization of FACLC/SPIO micelles into breast-cancer cells coupled with limited US-induced intravascular and/or extracellular drug H


Article


In Vivo Tumor Targeting. The results shown in Figure 8 imply that the ratio of viable normal cells to tumor cells was increased while tumor cells colocalized with sonication region. Therefore, we used a therapeutic US system equipped with power density/frequency/duty ratio control, and we achieved tumor targeting through the cationic CLC, the EPR effect and FA-mediated endocytosis. It is known the optimal size range of particulate drug carriers that is required to extravasate from most tumor blood vessels into the tumor interstitium (i.e., EPR effect) is 50−150 nm.8 In pilot experiments, we determined that the FA-CLC/SPIO micelles featuring an average particle size of around 140 nm (a size that can be tuned by adjusting the amount of CLC and solvent) exhibited the most satisfactory passive accumulation. The ability of FA-CLC/SPIO micelles to enable tumor-targeted delivery of hydrophobic agents was examined by using Cy5.5 as the vehicle-tracking agent (i.e., Cy5.5 was covalently conjugated with FA-CLC/SPIO micelle) and a fluorescence-imaging technique. Fluorescence images captured of the tumor-bearing mice before and after injecting them with the Cy5.5-conjugated FA-CLC/SPIO micelle suspension are shown in Figure 9A−C. Comparing the leftside mouse in Figure 9A with the corresponding mice in Figure 9B showed that at 1.5 h after intravenous injection, substantial tumor-targeted accumulation of micelle was observed in the tumor-bearing mouse. The distribution of micelles in tumor and various organs is shown in Figure 9C, in which the signal of Cy5.5-conjugated FA-CLC/SPIO micelles observed in tumor was the strongest compared to other organs. In addition, tumor-targeted accumulation of micelle was confirmed by comparing the sliced MR images of the tumor before (Figure 9D) and after (Figure 9E) injection, which showed that the dark contrast assigned to the superparamagnetic micelles was clearly detected at the tumor site (Figure 9E). The optically integrated images and the sliced MR images revealed that both the optical signal derived from the Cy5.5-conjugated micelles and the MR T2 signal from SPIO nanoparticles were detected at the tumor site. This result indicated that the FA-CLC/SPIO micelles exhibited satisfactory tumor-specific targeted delivery of hydrophobic agents, which was ascribed primarily to the effect of the particle size (i.e., passive targeting) and FA conjugation (i.e., active targeting). However, as shown in Figure 9C, a light signal of Cy5.5-conjugated FA-CLC/SPIO micelles was found in the liver. It was ascribed to the fact that vehicles containing Fe are recognized to be readily captured by Kupffer cells attached to liver sinusoids.36,37 Effect of Magnetic Attraction on in Vivo Tumor Targeting. As shown in Figure 9C, Fe-containing vehicles were inevitably accumulated in the liver. In order to enhance the efficacy of targeted delivery, we used a magnetic attraction to guide CPT-loaded FA-CLC/SPIO micelles because most previous studies have reported positive effects of magnetic attraction on targeted delivery. We added a static magnetic field to the subcutaneous tumor model and then injected the Cy5.5conjugated FA-CLC/SPIO micelles and performed fluorescence imaging (Figure 9A−C). When the magnetic field was applied to further enhance the targeting effect, we found that the fluorescent signal in tumor was increased. This was supported by the sliced MR images of the tumors acquired before and after injection under magnetic attraction; an enhanced T2 image contrast at the tumor site was observed in the mice that had been exposed to the magnetic field (Figure 9F,G). However, as shown in Figure 9C, the fluorescent signal observed in the liver was also increased when magnetic

release, followed by a pH-sensitive release of drugs and endolysomal escape to produce cytotoxicity. To confirm that this occurs, we investigated the in vitro cytotoxic effect of CPTloaded FA-CLC/SPIO micelles on normal and breast-cancer cells that did or did not receive 20 min sonication (Figure 8).

Figure 8. Effects on cell viability of CPT-loaded FA-CLC/SPIO micelles evaluated using the MTT assay. L929 and MDA-MB-231 cells were cultured with drug-loaded vehicles and were exposed or not exposed to sonication. Sonication parameters: frequency 1 MHz, duty ratio 20%, power density 0.4 W/cm2, sonication time 20 min. CPT concentration in the culture medium was 0.49 μg/mL.

Before the test, we confirmed 2 parameters. First, the US used in this study was only slightly cytotoxic in vitro in normal and breast-cancer cells that were incubated without drugs or drugloaded vehicles. Second, the CPT concentration (0.49 μg CPT/ mL of culture medium) used in the cell-viability test was not noticeably harmful to normal cells when sonication was not applied (the in vitro cell viability was around 80%). Therefore, as the threshold, this CPT concentration was applied in all in vitro cell-viability tests shown in Figure 8. At this dosage, the viability of the tumor cells incubated with the CPT-loaded FACLC/SPIO micelle suspension in the absence of sonication was 47% (i.e., it was slightly lower than 50%). Under the condition in which sonication is applied, as mentioned earlier in this section (Figure 5C), both nonspecific and FA-mediated internalization could be facilitated. Thus, the in vitro viabilities of normal cells and tumor cells incubated with CPT-loaded FACLC/SPIO micelles were respectively lowered to 65% and 20% when the 20 min therapeutic sonication was applied (Figure 8). Nevertheless, the US-induced increase in internalization was higher in FA-positive tumor cells than in normal cells (Figure 5C). The ratio of viable normal cells to tumor cells was increased considerably, from 1.70 (without sonication; 80/47 = 1.70) to 3.25 (with a therapeutic sonication; 65/20 = 3.25), after they had received vehicles and sonication (Figure 8). This could be used to predict the normal and tumor cells located in the tumor tissue (in sonication region). On the other hand, the results shown in Figure 8 also could be used to predict the normal cells that were located far from the tumor tissue (i.e., the cells that did not receive sonication). The ratio of viable normal cells in these healthy organs (i.e., organs that did not receive sonication) to tumor cells (i.e., cells that received sonication) was increased markedly, from 1.7 to 4.0 (80/20 = 4.0), after the administration of drug-loaded vehicles and sonication. I


Article


Figure 9. In vivo fluorescence images of tumor-bearing nude mice acquired (A) before and (B,C) after injecting Cy5.5-conjugated FA-CLC/SPIO micelle suspensions and not exposing (MF−) or exposing (MF+) the tumors to magnetic fields. T2-weighted MR images of tumor sites acquired from tumor-bearing nude mice (D) before and (E) after injection of the FA-CLC/SPIO micelle suspension (3.87 mg iron/kg body weight), in the absence of magnetic attraction (MF−). T2-weighted MR images of tumor sites acquired from tumor-bearing nude mice (F) before and (G) after injection of the FA-CLC/SPIO micelle suspension (3.87 mg iron/kg body weight), coupled with exposure to magnetic attraction (MF+). Iron content in cells was revealed by performing Prussian blue staining on tissue sections obtained from tumors (H) before injection, and after injection of the FA-CLC/SPIO micelle suspension (I) without (MF−) and (J) with (MF+) exposure to magnetic attraction. Iron content in cells was revealed by performing Prussian-blue staining on tissue sections obtained from the liver (K) before injection, and after injection of the FA-CLC/SPIO micelle suspension (L) without (MF−) and (M) with (MF+) exposure to magnetic attraction. MF strength was 5800 G.

observed in the tumor (Figure 9J) and the liver (Figure 9M) were slightly and markedly increased, respectively. This finding indicated that the undesirable accumulation of FA-CLC/SPIO micelles in the liver was enhanced by the magnetic flux emitted from the attraction site (i.e., tumor site). In Vivo Therapeutic Efficacy. The blood concentration of CPT-time profiles of the tumor-bearing nude mice administrated with CPT solution and CPT-loaded FA-CLC/SPIO micelle suspension were recorded and shown in Figure 10A. The pharmacokinetic parameters were calculated via using the noncompartmental model fitted by a pharmacokinetic software (WinNonlin, Version 6.3). As shown in Table 2, values of halflife and MRT of CPT loaded in FA-CLC/SPIO micelle were higher than those of free CPT, implying that CPT molecules loaded in micelles were more stable in blood circulation than free CPT. It might be attributed to the effect that the lactone form of CPT was protected and maintained by FA-CLC/SPIO micelles with prolonged circulation, sustained release and EPR behaviors. Hence, total clearance was decreased. Subsequently,

attraction was applied. Nevertheless, the signal increment observed in the tumor was higher than that observed in the liver. The results shown in Figure 9A−C suggested that magnetic attraction can concurrently enhance the accumulation of FA-CLC/SPIO micelles in tumors and in the liver, if the tumor site (i.e., magnetic attraction site) is near the liver. This was further supported by the results of the potassium ferrocyanide trihydrate staining test, a histochemical analysis that is used for probing the iron content in tissues (Figure 9H− M): the blue spots assigned to iron were not observed in tissue sections of the tumor (Figure 9H) and the liver (Figure 9K) obtained from the animal that had not been injected with FACLC/SPIO micelles; however, at 1.5 h after injection, blue spots were observed in the sections of the tumor (Figure 9I) and the liver (Figure 9L) obtained from the animal that had not been exposed to magnetic attraction. The density of the blue spots observed in the tumor was substantially higher than that in the liver. When the magnetic field was applied to further enhance the targeting effect, we found that the blue spots J


Article


Figure 10. (A) Blood concentration of CPT-time curves of tumor-bearing nude mice after intravenous administration of CPT or CPT-loaded FACLC/SPIO micelles at a dose of 1.5 mg CPT/kg bodyweight. (B) The influence of sequentially administering magnetic attraction and therapeutic sonication on the in vivo therapeutic efficacy of CPT-loaded FA-CLC/SPIO micelles in tumor-bearing nude mice was evaluated by measuring the tumor size in mice exposed to distinct therapeutic conditions for 30 days. Group-1 mice were treated with PBS and received sonication (US+). Group-2 mice were treated with a CPT/DMSO/saline solution and received sonication (US+). Group-3 mice were treated with the CPT-loaded FACLC/SPIO micelle/saline suspension but did not receive magnetic attraction (MF−) and sonication (US−). Group-4 mice were sequentially administered magnetic attraction (MF+, 1.5 h), CPT-loaded FA-CLC/SPIO micelle/saline suspension, and sonication (US+) for 3 cycles. MF strength was 5800 G. The CPT dosage administrated to mice in Groups 2, 3, and 4 was 0.35 mg/kg body weight per cycle. Sonication parameters: frequency 1 MHz, duty ratio 20%, power density 0.4 W/cm2, sonication time 20 min. The data are shown as means ± SD, *P < 0.05 and **P < 0.01. In vivo acute toxicity studies of mice administered differing treatments. (C) Hepatic function (aspartate aminotransferase, AST; alanine aminotransferase, ALT) and (D) renal function (blood urea nitrogen, BUN; creatinine, CREA). The normal range of AST is 54 to 298 U/L, ALT is 17 to 132 U/L, BUN is 12 to 33 mg/dL, and CREA is 0.2 to 0.9 mg/dL.

To investigate the in vivo therapeutic efficacy of sequentially administering magnetic attraction, CPT-loaded FA-CLC/SPIO micelles, and therapeutic sonication in tumor-bearing mice, we measured the size of the tumors in the mice. Because the effect of magnetic attraction on in vivo tumor-targeted drug delivery and the influence of therapeutic sonication on in vitro cytotoxicity of the CPT-loaded FA-CLC/SPIO micelles had been confirmed, we did not separately investigate the contributions of magnetic attraction and sonication; based on considering animal welfare, we sought to reduce the number of animals sacrificed. The results in Figure 10B show that the size of the tumors increased considerably in the mice that were periodically administered PBS (Group 1, control group) and CPT/DMSO/PBS (Group 2, CPT group). Furthermore, the tumor-bearing mice that were periodically administered CPTloaded FA-CLC/SPIO micelle suspensions (Group 3) exhibited a larger effect of the drug than did the mice in the control and CPT groups. Interestingly, the tumor-bearing mice that sequentially received 3 cycles of magnetic attraction, CPTloaded FA-CLC/SPIO micelle suspensions, and sonication

Table 2. Pharmacokinetic Analysis for Tumor-Bearing Nude Mice after Intravenous Administration of CPT and CPTLoaded FA-CLC/SPIO Micelles at a Dose of 1.5 mg CPT/kg Body Weight (n = 6) samples CPT-loaded FA-CLC/ SPIO micelles CPT

AUC0→∞ (h·μg/mL)

t1/2 (h)

CL (mL/h)

MRT (h)

Vss (mL)

7.73

6.14

4.85

8.40

40.73

1.15

0.79

32.53

0.77

25.13

The pharmacokinetic parameters were calculated by WinNonlin noncompartmental analysis (Version 6.3). Data were expressed as means. AUC = area under the curve, t1/2 = half-life in plasma, CL = total clearance, MRT = mean residence time, Vss = distribution volume at steady state.

AUC0→∞ value of CPT loaded in the FA-CLC/SPIO micelles was higher than that of free CPT, suggesting that FA-CLC/ SPIO micelle is a suitable vehicle to encapsulate and deliver CPT. K


Article


to prepare novel FA-CLC/SPIO micelles that exhibit encapsulation capability for hydrophobic antitumor agents, targeted delivery, MR imaging functionality, limited USinduced leakage of drug, and substantial pH-triggered release. In our in vitro tests, the US-induced increment of FA-CLC/ SPIO micelles internalized into FA-positive tumor (MDA-MB231) cells was higher than that internalized into normal cells under the tested conditions. Therefore, after incubation with the CPT-loaded FA-CLC/SPIO micelles, the ratio of viable normal cells to tumor cells was increased markedly, from 1.70 (without sonication) to 3.25 (with therapeutic sonication). The cationic FA-CLC/SPIO micelles demonstrated satisfactory tumor-targeted delivery in vivo, which was enhanced by applying magnetic attraction. After three cycles of magnetic attraction were administered sequentially to CPT-loaded FACLC/SPIO micelles, and therapeutic sonication, tumor growth was potently inhibited. Our results suggest that magnetic guidance applied together with sonication helped enhance the therapeutic efficacy of the CPT loaded in the FA-CLC/SPIO micelles, but that magnetic attraction must be carefully applied and controlled, especially when the tumor site is near organs that can trap iron-containing nanoparticles.

(Group 4) demonstrated the highest therapeutic efficacy. These results clearly demonstrated that sequential administration of magnetic attraction and sonication enhanced the therapeutic efficacy of CPT loaded in the FA-CLC/SPIO micelles. The in vivo results shown in Figure 10B together with the in vitro results shown in Figures 5−9 support the hypothesis tested in this study that sequentially administering magnetic attraction, CPT-loaded FA-CLC/SPIO micelles, and therapeutic sonication enables efficient delivery of CPT molecules into the intracellular target site in tumor cells, which substantially increases the in vivo therapeutic efficacy of CPT used at a given dosage. To verify the side effects of the proposed treatment (i.e., Group 4 in Figure 10B), we performed biochemical blood analyses on days 1 and 7. As shown in Figure 10C,D, no abnormalities in the liver and kidney functions occurred after receiving the proposed treatment. This study marks the first phase of an investigation into the newly developed FA-CLC/SPIO micelles. In this phase, our main aim was to use a new micelle-forming material to prepare hydrophobic drug-loaded micelles, which were designed for employing combined strategies (i.e., FA- and MF-targeted delivery, US-enhanced delivery, and pH-triggered release) to overcome the barriers against the delivery of drugs. We sequentially administered magnetic guidance and therapeutic sonication in order to enhance targeted delivery and FAmediated internalization, respectively. Positive effects of US and magnetic attraction on therapeutic efficacy have been reported previously. Conversely, in this study, we also investigated the possible negative effects of US and magnetic attraction; these effects have not been extensively discussed in the literature related to US-enhanced delivery and magnetic guidance. Therefore, we tested whether the application of US is accompanied by negative effects such as US-induced early drug release and US-induced internalization of drug-loaded vehicles by normal cells. This concern might be addressed to a certain extent by using the normal therapeutic US facility with controlled orientation, depth, and power density, which is highly available and feasible in hospital. On the other hand, periodically administering low concentrations of vehicles that feature enhanced targeted-delivery and limited US-induced release functionalities might offer a feasible strategy for lowering the risk of US-enhanced delivery of drugs to healthy tissues. This is the reason that we developed the proposed vehicles and used magnetic attraction to further remedy the risk of US-enhanced delivery of drugs to healthy tissues. Although magnetic guidance coupled with sonication exerted a positive effect on in vivo therapeutic efficacy, using a nonfocused magnetic field could enhance the undesirable accumulation of Fe-containing vehicles in the liver if the tumor (i.e., magnetic attraction site) is near the liver (Figure 9). This is the reason why we suggest that magnetic attraction must be carefully applied far from the liver. Moreover, the US parameters (i.e., the frequency, sonication time, power density, and duty ratio), drug loading, and the administration amounts of the vehicle applied in the in vitro study must be finely tuned in order to achieve maximal efficacy in vivo and minimal side effects of the CPT-loaded FA-CLC/SPIO micelles. These aspects were not the immediate focus of this study, and they will be investigated further in the next phase of our work.

■

EXPERIMENT SECTION

Materials. Chitosan (MW 200 kDa), oleic acid, 1,2-hexadecanediol, oleylamine, benzyl ether, phenyl ether, lauric anhydride, FA, 2(N-morpholino)ethanesulfonic acid (MES), dimethyl sulfoxide (DMSO), glutaraldehyde, Fe(acac)3, CPT, sodium bicarbonate, 1ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), thiazolyl blue tetrazolium bromide (MTT), and Nile red were purchased from Sigma-Aldrich. RPMI-1640 medium, Dulbecco’s phosphate-buffered saline (PBS), 0.25% trypsin, DAPI (4′,6-diamidino-2-phenylindole), and LysoSensor Green DND-189 were purchased from Invitrogen. Human breast adenocarcinoma (MDA-MB-231) cells, human lung epithelial carcinoma (A549) cells, and normal fibroblasts (L929) were obtained from Bioresource Collection and Research Center (Taiwan). Preparation of Drug-Loaded FA-CLC/SPIO Micelles. Hydrophobic SPIO nanoparticles were synthesized using the thermal decomposition method.38 Briefly, Fe(acac)3 (2 mmol) was mixed with 1,2-hexadecanediol (10 mmol), oleic acid (6 mmol), and oleylamine (6 mmol). The mixture was dissolved in phenyl ether (20 mL) and refluxed at 100 °C for 30 min under a nitrogen flow to remove water and oxygen, and then heated at 200 °C for 1 h and subsequently at 265 °C for 30 min. After cooling to room temperature, the black-brown mixture was collected by centrifugation at 6000 rpm for 10 min, and then washed three times with excess ethanol to remove the solvent. The SPIO nanoparticles were collected and redispersed in ethanol. Previously, our group synthesized CLC by using N,O-carboxymethyl chitosan (NOCC) as a precursor.14 The NOCC compositions (2 g) were dissolved in distilled water (50 mL) and stirred for 24 h. The resulting solutions were mixed with methanol (50 mL), following which lauric anhydride was added at a concentration of 30 mmol. When the reaction finished, the reaction mixture was dialyzed (MW cutoff: 14 000 Da) into distilled water (1 L), which was changed every 3−6 h for 2 day. After the sample was dried, CLC was obtained. FA conjugation was performed using a previously described procedure39 with modification. Briefly, FA (10 mmol) was mixed with EDC (20 mmol) and MES buffer (pH 5.5, 5 mL) for 30 min. The resulting solution was mixed with NHS solution (50 mmol) at room temperature. After reaction for 5 h, the solution obtained was mixed with 10 mL of the CLC aqueous solution (1% v/v) overnight in the dark to synthesize FA-conjugated CLC. Finally, the reaction mixture was sequentially dialyzed (MW cutoff: 14 000 Da) with phosphate buffer (pH 7.4), DI water/ethanol solution and ethanol for 4 days. The clearance of free FA residue after dialysis was confirmed by gel

■

CONCLUSIONS A new micelle-forming material, CLC, was successfully conjugated with FA and incorporated with SPIO nanoparticles L


Article


positive) that were incubated with various vehicle concentrations (micelle weight/culture-medium volume = 2.5, 5, and 10 μg/mL) of CLC/SPIO micelles and FA-CLC/SPIO micelles for 24 h.25,26 To confirm the in vitro active-targeting behavior of the vehicles developed in this study, FA-CLC/SPIO micelles loaded with Nile red, a hydrophobic fluorescent dye with a similar molecular weight to CPT, were incubated with FA-negative cells (A549) and FA-positive cells (MDA-MB-231) for 6 and 24 h, after which confocal imaging was performed. For these intracellular-uptake studies, Nile red (0.5 mg/mL in DMSO) was encapsulated in the FA-conjugated CLC/SPIO micelles. Typically, cells were seeded onto glass coverslips in 12-well tissue-culture plates (5 × 104 cells/well) and incubated for 24 h. Next, the medium was removed and the cells were washed twice with PBS, and then 2 mL of RPMI-1640 medium containing Nile red-loaded FACLC/SPIO micelles (micelle weight/culture-medium volume = 10 μg/mL) was added into each well. Following incubation for another 24 h, the cells on the coverslips were washed several times with PBS and then fixed with 4% formaldehyde for 10 min. After being washed three times with PBS, the cells were treated for 10 min with DAPI (Invitrogen) to stain the nuclei. Finally, the cells were examined using a laser confocal microscope (Olympus FV1000). To determine the iron content in cells, samples were dissolved using nitric acid (2%) and then characterized using ICP-AES. The influence of US on the in vitro internalization of CPT-loaded FA-CLC/SPIO micelles into tumor cells (MDA-MB-231) was evaluated. Cells were cultured in medium (4 mL) containing Nile red-loaded FA-CLC/SPIO micelles (micelle weight/culture-medium volume = 10 μg/mL) and treated with and without sonication during the initial 20 min. After incubation for 2, 4, and 6 h, the cells were washed with PBS, and then LysoSensor Green DND-189 (pH 5.2 endosome/lysosome probe; Invitrogen) was added in a culture medium that did not contain fetal bovine serum. After incubation for 30 min, the cells were washed with PBS and then examined using a laser confocal microscope. LysoSensor Green and Nile red were excited at 405 and 543 nm, respectively. The influence of US on the in vitro cytotoxic effect of CPT-loaded FA-CLC/SPIO micelles on tumor cells (MDA-MB-231) and normal cells (L929) was evaluated using the MTT assay. Cells were seeded in 6 cm cell-culture dishes at a density of 6 × 104 cells (in 4 mL of medium) per dish and incubated overnight to allow the cells to attach to the dishes. The tumor cells and normal cells were cultured with sample-containing media (culture medium for control groups; CPTloaded FA-CLC/SPIO micelles for experimental groups) and treated or not treated with sonication. The concentration of CPT-loaded FACLC/SPIO micelles in the culture medium was 10 μg/mL (i.e., 10 μg/ mL × 4.9% = 0.49 μg CPT/mL). The results of our pilot tests showed that the viability of normal cells cultured with the free-CPT-containing medium at the same CPT concentration (0.49 μg/mL) was >80%. Therefore, the concentration of the CPT-loaded FA-CLC/SPIO micelles in the culture medium used for the experimental groups was acceptable. US treatment involved applying therapeutic US (Metron Accusonic Plus therapeutic US machine, Australia) at a frequency of 1 MHz, a duty ratio of 20%, and a power density of 0.4 W/cm2 during the initial 20 min. After incubation for 24 h, the MTT reagent was added and the cytotoxicity was measured. Cellular uptake of the model dye was then analyzed using a BD FACSCalibur flow cytometer. In Vitro Drug-Release Test. The influence of therapeutic sonication and pH on the drug-release behavior of the CPT-loaded FA-CLC/SPIO micelles was investigated by measuring the amount of CPT released from the CPT-loaded vehicles into PBS (5 mL, pH 7.4 and 5.2), without and with the application of therapeutic sonication (frequency 1 MHz, power density 0.4 W/cm2, duty ratio 20%) during the period from 20 to 40 min (sonication time 20 min). The cumulative amount of CPT released was measured based on the UV− vis absorbance at various time points.

permeation chromatography and size exclusion chromatography (GPC/SEC) test shown in Figure S1 (Supporting Information). As shown, the signal assigned to FA molecule was not observed in the GPC/SEC result of FA-conjugated CLC sample after dialysis. It implies that our dialysis process designed for FA-conjugated CLC, sequential dialysis by ethanol/DI solution and DI water, is proper for subsequent tests. Finally, the products was dried for further use.40 The lipophilic SPIO nanoparticles and the model agents (Nile red or DiR or CPT) were loaded in the FA-CLC/SPIO micelles by using the self-assembly route. Typically, the SPIO nanoparticle suspension (10 μg/μL) was mixed with hexane, the model drug/DMSO solution (1.0 μg/μL), and the FA-conjugated CLC aqueous solution (1% w/v), and then the samples were sonicated in an ice bath. The free model agents that were not encapsulated in the superparamagnetic micelles were removed using the magnetic separation method. After the centrifugal process and redispersion for the precipitation were repeated, the free SPIO nanoparticles could be separated from SPIO-loaded micelles. Characterization of FA-CLC/SPIO Micelles. The conjugation of FA with CLC was confirmed by obtaining the 1H NMR spectrum (Bruker Avance III 400 MHz NMR Spectrometer; Germany) and by using a UV−visible (UV−vis) spectrophotometer (JASCO Corp., V630). The morphology of the CPT-loaded FA-CLC/SPIO micelles was examined by performing transmission electron microscopy (TEM) and high-resolution TEM (Hitachi H-7100) at 200 keV. The crystallographic phase of SPIO was identified by performing X-ray diffraction (XRD; Rigaku TTRAX III) at a scanning rate of 4° (in units of 2θ min−1) over a 2θ range of 10° to 70°. The magnetic properties of the FA-CLC/SPIO micelles were characterized by using a superconducting quantum interference device (SQUID; MPMS7) at 300 K. The mean diameter and the zeta potential of the drug vehicles were measured using dynamic light scattering (DLS; Malvern, ZS90). The SPIO and iron content was confirmed using thermogravimetric analysis (TGA; TA Instruments Q500) and inductively coupled plasma atomic emission spectroscopy (ICP-AES; Kontron S-35), respectively. The drug encapsulation efficiency (EE) and loading efficiency (LE) of the CPT-loaded FA-CLC/SPIO micelles were quantified (n = 3) by first destroying the micelles in PBS (pH 5.2) by using low-frequency sonication (37 kHz for 30 min) and then measuring the absorption intensity of the UV−vis spectra at 367 nm under the condition in which the concentration was lower than the solubility limit of CPT in PBS. EE and LE were calculated using the following equations: EE (%) =

mass of CPT encapsulated in vehicles × 100% mass of CPT in feed

LE (%) =

mass of CPT encapsulated in vehicles × 100% mass of vehicles

In Vitro Cytotoxicity and Intracellular-Uptake Studies: Cell Culture and MTT Assay. Cells were seeded in 96-well tissue-culture plates at a density of 1 × 104 cells per well in RPMI-1640 medium, which was supplemented with 10% fetal calf serum and contained 100 U/mL penicillin and 100 μg/mL streptomycin; cells were grown at 37 °C in a 5% CO2 atmosphere. After overnight incubation, the culture medium was replaced with sample-containing medium and the cultures were incubated for 24 h. The number of viable cells was determined using the MTT assay: 20 μL of the MTT solution (5 mg/ mL in PBS) was added into each well and after incubation for 4 h, 200 μL of DMSO was used to dissolve the formazan crystals. Finally, the optical density at 570 nm was measured using a Tecan Infinite 200 plate reader. Untreated cells were used as the control. The results of the cytotoxicity measurements (n = 4) are expressed as cell viability (%), which was calculated using the following equation:

cell viability (%) =

absorance(sample) × 100% absorance(control)

We measured the viability of L929 fibroblasts (FA negative), A549 lung-cancer cells (FA negative), and MDA-MB-231 cancer cells (FA M


Article


Group 3 received three cycles of CPT-loaded FA-CLC/SPIO micelle/ PBS suspension (3.87 mg Fe/kg body weight per cycle). The mice in Group 4 sequentially received three cycles of magnetic attraction (1.5 h), CPT-loaded FA-CLC/SPIO micelle/PBS suspension (3.87 mg Fe/ kg body weight per cycle), and sonication. The CPT dosage administrated to mice of Groups 2, 3, and 4 was 0.35 mg/kg body weight per cycle. The sonication conditions used for Groups 2 and 4 were the same as those used for Group 1. The tumor sizes were measured every 5 days by using vernier calipers and the tumor volume was calculated using this equation: tumor volume = (tumor length) × (tumor width)2/2. The growth curves of the MDA-MB-231 tumor in the four groups of mice were recorded. Pharmacokinetic Analysis in Mice. The tumor-bearing nude mice were administered intravenously with CPT solution or CPTloaded FA-CLC/SPIO micelle suspension at a dose of 1.5 mg CPT/kg bodyweight. The blood samples were obtained at 0.5, 1, 3, 6, 12, and 24 h after administration. CPT was extracted using dichloromethane and methanol (4:1). The organic phase was isolated and dried. To determine the concentration of CPT, high-performance liquid chromatography (HPLC) was used. The mobile phase was composed of 45:55 (v/v) acetonitrile trimethylamine acetate and water at a flow rate of 1 min/mL. The excitation and emission wavelengths of fluorescence detector were set at 367 and 430 nm, respectively. The concentrations of CPT in plasma were recorded at each time point. Statistical Analysis. All results are presented as means ± standard deviation (SD), and the statistical significance of the difference between various samples was determined using Student’s t test; * indicates P < 0.05, which was considered statistically significant, and ** indicates P < 0.01, which was considered highly significant.

cumulative CPT release (%) total amount of CPT released from vesicles at each time point = amount of CPT initially loaded into the vehicles × 100% In vitro MR imaging experiments were performed to probe the T2 imaging ability and the structural changes in FA-CLC/SPIO micelles in media under distinct pH and sonication conditions by using a 7T MR imaging instrument (Bruker S300 BIOSPEC/MEDSPEC). The transverse-relaxation rate (R2 = 1/T2) was determined by probing transverse images by using a two-dimensional (2D) spin−echo MR sequence featuring 30 echoes and an echo time of 22.5 ms. The imaging parameters were a repetition time of 2000 ms, a field of view (FOV) of 60 × 60 mm, slice thickness of 1 mm, a 256 × 256 imaging matrix, and a flip angle of 180°. The FA-CLC/SPIO micelle suspensions of various micelle concentrations were used in the in vitro MR imaging tests. The corresponding SPIO concentration of each sample was confirmed by performing TGA. In Vivo Tumor Targeting by Means of MR/IVIS Dual-Model Imaging. Female nude mice (6−8 weeks old, 20−25 g) were purchased from the Laboratory Animal Center of National Yang-Ming University. All animals used in our experiments were treated and housed following a protocol approved by the Institutional Animal Care and Use Committee of National Yang-Ming University (NYMIACUC). To grow the model tumor, MDA-MB-231 cells (2 × 106) suspended in 100 μL of PBS were injected into the left shoulder of each mouse. The tumor-bearing mice were used when the tumor volume reached approximately 100 mm3. We used fluorescence and MR imaging to investigate the effect of magnetic attraction on the in vivo tumor targeting of FA-CLC/SPIO micelles in the nude mice that bore MDA-MB-231 breast-cancer tumors. The FA-CLC/SPIO micelle suspension (150 μL) was injected intravenously through the tail vein in two groups of mice at a dose of 3.87 mg Fe/kg body weight. One group was not exposed to magnetic attraction, whereas the other group was exposed to magnetic attraction by placing a magnet (surface magnetic field (MF) strength = 5800 G) on top of the tumor for 1.5 h. Optical imaging was performed using an IVIS spectrum imaging system (IVIS Xenogen, Alameda, CA) to probe Cy5.5-conjugated FA-CLC/SPIO micelles (i.e., dye was covalently bound with micelle) and DiR-loaded FA-CLC/SPIO micelles (dye was physically loaded in micelle). MR images of the abdomen were acquired before and after the intravenous injection was administered together with respiratory gating control; imaging was performed using an MR imaging system (Bruker S300 BIOSPEC/ MEDSPEC) equipped with a 3.5 cm circular surface coil (G060 Micro-imaging gradient insert) and a T2 2-D fast low-angle shot sequence. The parameters used were the following: TR/TE = 100 ms/ 8 ms; flip angle = 40°; FOV = 30 × 30 mm; slice thickness = 1 mm; and imaging-matrix size = 256 × 256. After characterization by means of optical imaging, the livers and tumor tissues of the sacrificed mice were collected and immediately fixed in 4% formalin for 30 min and then embedded in paraffin. The iron content in the liver and tumor tissues was determined semiquantitatively by staining with Prussian blue. The sections were incubated in Prussian blue solution (2 mL) containing hydrochloride (1%) and potassium ferrocyanide(II) trihydrate (1%) for 30 min, rinsed, and then counterstained with neutral red. The iron staining was examined using an optical microscope. In Vivo Antitumor Therapeutic Efficacy. The in vivo therapeutic efficacy of sequentially administering three cycles of magnetic attraction, the CPT-loaded FA-CLC/SPIO micelle suspension, and therapeutic sonication was investigated by measuring the size of tumors in the tumor-bearing mice. We separated 12 tumor-bearing mice into four groups (n = 3/group) and treated them with distinct sample suspensions (150 μL) by means of tail-vein injection every 5 days for three cycles (at 0, 5, and 10 days). The mice in Group 1 received saline solution and sonication (frequency 1 MHz, duty ratio 20%, power density 0.4 W/cm2) for 20 min. The mice in Group 2 received CPT/DMSO/saline solution and sonication. The mice in

■

ASSOCIATED CONTENT

S Supporting Information *

Figures showing GPC/SEC results of free FA, FA-conjugated CLC before dialysis, and FA-conjugated CLC after dialysis, and of free Cy5.5, Cy5.5-conjugated CLC before dialysis, and Cy5.5-conjugated CLC after dialysis. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jm501996y.

■

AUTHOR INFORMATION

Corresponding Author

*Tel.: +886-2-28267923. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We are grateful to the National Science Council of the Republic of China (NSC 101-2628-E-010-002-MY3 and NSC 102-2627E-010-001), the Department of Health, Taipei City Government (10301-62-044), and Yen Tjing-Ling Foundation (CI103-13) for their financial support. We thank the 7T Animal MRI Core Lab of the Neurobiology and Cognitive Science Center at National Taiwan University and the Brain Research Center at National Yang-Ming University (3T MRI) for technical and facility support. The authors thank Prof. Hsin-Ell Wang (National Yang-Ming University) for the pharmacokinetic software support.

■

ABBREVIATIONS USED DDS, drug-delivery system; EPR, enhanced permeation and retention; FA, folic acid; CLC, carboxymethyl lauryl chitosan; SPIO, superparamagnetic iron oxide; Cy5.5, cyanine 5.5 NHS ester; CPT, camptothecin; PEI, polyethylenimine; EDC, 1ethyl-3-(3-(dimethylamino)propyl)carbodiimide; NHS, Nhydroxysuccinimide; DAPI, 4′,6-diamidino-2-phenylindole; N


Article


(16) Dicheva, B. M.; Hagen, T. L. Mt; Li, L.; Schipper, D.; Seynhaeve, A. L. B.; Rhoon, G. Cv.; Eggermont, A. M. M.; Lindner, L. H.; Koning, G. A. Cationic thermosensitive liposomes: a novel dual targeted heat-triggered drug delivery approach for endothelial and tumor cells. Nano Lett. 2013, 13, 2324−2331. (17) Strieth, S.; Dunau, C.; Kolbow, K.; Knuechel, R.; Michaelis, U.; Ledderose, H. Phase I clinical study of vascular targeting fluorescent cationic liposomes in head and neck cancer. Eur. Arch. Oto-RhinoLaryngol. 2013, 270, 1481−1487. (18) Tanaka, K.; Narita, A.; Kitamura, N.; Uchiyama, W.; Morita, M.; Inubushi, T.; Chujo, Y. Preparation for highly sensitive MRI contrast agents using core/shell type nanoparticles consisting of multiple SPIO cores with thin silica coating. Langmuir 2010, 14, 11759−11762. (19) Wu, S. C.; Chen, Y. J.; Lin, Y. J.; Wu, T. H.; Wang, Y. M. Development of a mucin4-targeting SPIO contrast agent for effective detection of pancreatic tumor cells in vitro and in vivo. J. Med. Chem. 2013, 22, 9100−9109. (20) Kim, D. K.; Mikhaylova, M.; Wang, F. H.; Kehr, J.; Bjelke, B.; Zhang, Y.; Muhammed, M. Starch-coated superparamagnetic nanoparticles as MR contrast agents. Chem. Mater. 2003, 23, 4343−4351. (21) Bohmer, M. R.; Klibanov, A. L.; Tiemann, K.; Hall, C. S.; Gruell, H.; Steinbach, O. C. Ultrasound triggered image-guided drug delivery. Eur. J. Radiol. 2009, 70, 242−253. (22) Liu, T. Y.; Huang, T. C. A novel drug vehicle capable of ultrasound-triggered release with MRI functions. Acta Biomater. 2011, 7, 3927−3934. (23) Grull, H.; Langereis, S. Hyperthermia-triggered drug delivery from temperature-sensitive liposomes using MRI-guided high intensity focused ultrasound. J. Controlled Release 2012, 161, 317−327. (24) Husseini, G. A.; Christensen, D. A.; Rapoport, N. Y.; Pitt, W. G. Ultrasonic release of doxorubicin from pluronic p105 micelles stabilized with an interpenetrating network of N,N-diethylacrylamide. J. Controlled Release 2002, 83, 303−305. (25) Yang, S. J.; Lin, F. H.; Tsai, K. C.; Wei, M. F.; Tsai, H. M.; Wong, J. M. Folic acid-conjugated chitosan nanoparticles enhanced protoporphyrin IX accumulation in colorectal cancer cells. Bioconjugate Chem. 2010, 21, 679−689. (26) Dubé, D.; Francis, M.; Leroux, J. C.; Winnik, F. M. Preparation and tumor cell uptake of poly(n-isopropylacrylamide) folate conjugates. Bioconjugate Chem. 2002, 13, 685−692. (27) Yoo, M. K.; Park, I. K.; Lim, H. T.; Lee, S. J.; Jiang, H. L.; Kim, Y. K. Folate−PEG−superparamagnetic iron oxide nanoparticles for lung cancer imaging. Acta Biomater. 2012, 8, 3005−3013. (28) Gao, Z. G.; Fain, H. D.; Rapoport, N. Controlled and targeted tumor chemotherapy by micellar-encapsulated drug and ultrasound. J. Controlled Release 2005, 102, 203−222. (29) Hauser, J.; Ellisman, M.; Steinau, H. U.; Stefan, E.; Dudda, M. L.; Hauser, M. Ultrasound enhanced endocytotic activity of human fibroblasts. Ultrasound Med. Biol. 2009, 35, 2084−2092. (30) Mehier-Humbert, S.; Bettinger, T.; Yan, F.; Guy, R. H. Plasma membrane poration induced by ultrasound exposure: implication for drug delivery. J. Controlled Release 2005, 104, 213−222. (31) Rapoport, N. Combined cancer therapy by micellar-encapsulated drug and ultrasound. Int. J. Pharm. 2004, 277, 155−162. (32) Wang, J.; Pelletier, M.; Zhang, H.; Xia, H.; Zhao, Y. Highfrequency ultrasound-responsive block copolymer micelle. Langmuir 2009, 25, 13201−13205. (33) Husseini, G. A.; Pitt, W. G. Micelles and nanoparticles for ultrasonic drug and gene delivery. Adv. Drug Delivery Rev. 2008, 60, 1137−1152. (34) Behr, J. P. The proton sponge: a trick to enter cells the viruses did not exploit. Chimia Int. J. Chem. 1997, 51, 1−2. (35) Huang, M.; Fong, C. W.; Khor, E.; Lim, L. Y. Transfection efficiency of chitosan vectors: effect of polymer molecular weight and degree of deacetylation. J. Controlled Release 2005, 106, 391−406. (36) Geraldes, C.; Laurent, S. Classification and basic properties of contrast agents for magnetic resonance imaging. Contrast Media Mol. Imaging 2009, 4, 1−23.

RPMI-1640 medium, Roswell Park Memorial Institute medium; AST, aspartate aminotransferase; ALT, alanine aminotransferase; BUN, blood urea nitrogen; CREA, creatinine; HR-TEM, high-resolution transmission electron microscopy; XRD, X-ray diffraction; DLS, dynamic light scattering; TGA, thermogravimetric analysis; SQUID, superconducting quantum interference device; ICP-AES, inductively coupled plasma atomic emission spectroscopy; DI water, deionized water; MF, magnetic field; US, ultrasound; IVIS spectrum, in vivo imaging system spectrum; FOV, field of view; TR, repetition time; TE, echo time; AUC, area under the curve; t1/2, half-life in plasma; CL, total clearance; MRT, mean residence time; Vss, distribution volume at steady state; GPC/SEC, gel permeation chromatography and size exclusion chromatography

■

REFERENCES

(1) Arias, J. L. Advanced methodologies to formulate nanotheragnostic agents for combined drug delivery and imaging. Exp. Opin. Drug Delivery 2011, 8, 1589−1608. (2) Iyer, A. K.; He, J.; Amiji, M. M. Image-guided nanosystems for targeted delivery in cancer therapy. Curr. Med. Chem. 2012, 19, 3230− 3240. (3) Terreno, E.; Uggeri, F.; Aime, S. Image guided therapy: the advent of theranostic agents. J. Controlled Release 2012, 161, 328−337. (4) Fokong, S.; Theek, B.; Wu, Z.; Koczera, P.; Appold, L.; Jorge, S. Image-guided, targeted and triggered drug delivery to tumors using polymer-based microbubbles. J. Controlled Release 2012, 163, 75−81. (5) Menon, J. U.; Jadeja, P.; Tambe, P.; Yuan, K. Vu. B.; Nguyen, K. T. Nanomaterials for photo-based diagnostic and therapeutic applications. Theranostics 2013, 3, 152−166. (6) Kelkar, S. S.; Reineke, T. M. Theranostics: combining imaging and therapy. Bioconjugate Chem. 2011, 22, 1879−1903. (7) Brambilla, D.; Droumaguet, B. L.; Nicolas, J.; Hashemi, S. H.; Wu, L. P.; Moghimi, S. M.; Moghimi, P.; Couvreur, K. Nanotechnologies for Alzheimer’s disease: diagnosis, therapy, and safety issues. Nanomedicine 2011, 7, 521−540. (8) Petros, R. A.; DeSimone, J. M. Strategies in the design of nanoparticles for therapeutic applications. Nat. Rev. Drug Discovery 2010, 9, 615−627. (9) Sudimack, J.; Lee, R. J. Targeted drug delivery via the folate receptor. Adv. Drug Delivery Rev. 2000, 41, 147−162. (10) Ye, F.; Barrefelt, A.; Asem, H.; Abedi-Valugerdi, M.; El-Serafi, I.; Saghafian, M. Biodegradable polymeric vesicles containing magnetic nanoparticles, quantum dots and anticancer drugs for drug delivery and imaging. Biomaterials 2014, 35, 3885−3894. (11) Liang, K.; Such, G. K.; Johnston, A. P.; Zhu, Z.; Ejima, H.; Richardson, J. J.; Cui, J.; Caruso, F. Endocytic pH-triggered degradation of nanoengineered multilayer capsules. Adv. Mater. 2014, 26, 1901−1905. (12) Zhao, S.; Tan, S.; Guo, Y.; Huang, J.; Chu, M.; Liu, H. pHsensitive docetaxel-loaded D-α-tocopheryl polyethylene glycol succinate-poly (β-amino ester) copolymer nanoparticles for overcoming multidrug resistance. Biomacromolecules 2013, 14, 2636−2646. (13) Yang, Y. Q.; Zhao, B.; Li, Z. D.; Lin, W. J.; Zhang, C. Y.; Guo, X. D. pH-sensitive micelles self-assembled from multi-arm star triblock co-polymers poly (ε-caprolactone)-b-poly (2-(diethylamino) ethyl methacrylate)-b-poly (poly (ethylene glycol) methyl ether methacrylate) for controlled anticancer drug delivery. Acta Biomater. 2013, 9, 7679−7690. (14) Liu, T. Y.; Lin, Y. L. Novel pH-sensitive chitosan-based hydrogel for encapsulating poorly water-soluble drugs. Acta Biomater. 2010, 6, 1423−1429. (15) Krasnici, S.; Werner, A.; Eichhorn, M. E.; Schmitt-Sody, M.; Pahernik, S. A.; Sauer, B. Effect of the surface charge of liposomes on their uptake by angiogenic tumor vessels. Int. J. Cancer 2003, 105, 561−567. O


Article

Journal of Medicinal Chemistry (37) Berry, C. C. Progress in functionalization of magnetic nanoparticles for applications in biomedicine. J. Phys. D: Appl. Phys. 2009, 42, 224003. (38) Zeng, H.; PMR; Wang, S. X.; Sun, S. Shape-controlled synthesis and shape-induced texture of MnFe2O4 Nanoparticles. J. Am. Chem. Soc. 2004, 126, 11458−11459. (39) Setua, S.; Menon, D.; Asok, A.; Nair, S.; Koyakutty, M. Folate receptor targeted, rare-earth oxide nanocrystals for bi-modal fluorescence and magnetic imaging of cancer cells. Biomaterials 2010, 31, 714−729. (40) Mansouri, S.; Cuie, Y.; Winnik, F.; Shi, Q.; Lavigne, P.; Benderdour, M.; Beaumont, E.; Fernandes, J. C. Characterization of folate-chitosan-DNA nanoparticles for gene therapy. Biomaterials 2006, 27, 2060−2065.

P


A Novel Micelle-Forming Material Used for Preparing a Theranostic

Recommend Documents