In Vivo Biodistribution of Mixed Shell Micelles with Tunable

Jan 2, 2013 - 2Tianjin Key Laboratory of Molecular Nuclear Medicine, Institute of Radiation Medicine, Chinese Academy of Medical Science and...
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In Vivo Biodistribution of Mixed Shell Micelles with Tunable Hydrophilic/Hydrophobic Surface Hongjun Gao,1 Jie Xiong,1 Tangjian Cheng,1 Jinjian Liu,2 Liping Chu,2 Jianfeng Liu,*,2 Rujiang Ma,1 and Linqi Shi*,1 1

Key Laboratory of Functional Polymer Materials, Ministry of Education, and Institute of Polymer Chemistry, Nankai University, Tianjin, China 2 Tianjin Key Laboratory of Molecular Nuclear Medicine, Institute of Radiation Medicine, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China S Supporting Information *

ABSTRACT: The miserable targeting performance of nanocarriers for cancer therapy arises largely from the rapid clearance from blood circulation and the major accumulation in the organs of the reticuloendothelial system (RES), leading to inefficient enhanced permeability and retention (EPR) effect after intravenous injection (i.v.). Herein, we reported an efficient method to prolong the blood circulation of nanoparticles and decrease their deposition in liver and spleen. In this work, we fabricated a series of mixed shell micelles (MSMs) with approximately the same size, charge and core composition but with varied hydrophilic/hydrophobic ratios in the shell through spontaneously self-assembly of block copolymers poly(ethylene glycol)-block-poly(L-lysine) (PEG-b-PLys) and poly(N-isopropylacrylamide)-block-poly(aspartic acid) (PNIPAM-b-PAsp) in aqueous medium. The effect of the surface heterogeneity on the in vivo biodistribution was systematically investigated through in vivo tracking of the 125I-labeled MSMs determined by Gamma counter. Compared with single PEGylated micelles, some MSMs were proved to be significantly efficient with more than 3 times lower accumulation in liver and spleen and about 6 times higher concentration in blood at 1 h after i.v.. The results provide us a novel strategy for future development of long-circulating nanocarriers for efficient cancer therapy.

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INTRODUCTION

In the absence of specific design features, nanoparticles are generally removed from circulation within 10 min by the effective capture of the macrophages through phagocytosis mediated by opsonization.11,12 After i.v., a variety of serum proteins called opsonins (including immunoglobins, blood clotting factors, such as fibronectin and thrombospondin, and components of the complement system13) bind to the particle surface, which are recognized by the scavenger receptors on the macrophage cell surface and removed from the blood circulation.11,12 As reviewed by Alexis and co-workers,14 many physiochemical properties including composition, size, core properties, surface modifications, and targeting ligand functionalization can substantially affect the in vivo biodistribution of nanoparticles. Various strategies have been studied for increasing blood residence time and organ specific accumulation by reducing the level of nonspecific uptake, delaying opsonization, and increasing the extent of tissue specific accumulation. Kataoka et al.15 found that micelles possessing slight anionic charge on the surface exhibited less uptake into the liver and spleen because of the reduced nonspecific uptake due to the electrostatic repulsion between the cellular surface and the negatively micelles. Wooley et al.16,17 reported that, in

Nanoparticles have attracted intensive interest in biomedical applications as tumor diagnostics agents and drug delivery nanocarriers in cancer therapy because of the unique and tailorable properties.1−5 Tumor targeting strategies utilizing multifunctional nanoparticles rely largely on passive targeting achieved by the enhanced permeability and retention (EPR) effect due to leaky tumor blood vessels and the defective lymphatic drainage in solid tumor.6,7 Although benefited from the EPR effect, efficient cancer therapy is greatly challenged by the fact that the nanoparticles are rapidly removed from the blood and the majority of nanoparticles are accumulated in the organs of the reticuloendothelial system (RES) after intravenous injection (i.v.), subsequently leading to low particle concentration in blood and short contact time with the target tumor site.8 Park et al.9 pointed out that less than 5% of the total administered nanoparticles were actually delivered to the intended target site due to the mainly deposited in liver and spleen. Longevity in the bloodstream, that is, giving the nanoparticles sufficient time to reach the intended tumor targets, is highly demanded for an ideal drug delivery system.10 To increase the therapeutic efficacy in cancer therapy, the abilities with reduced capture by the RES and prolonged circulation in blood should be paid more attention for ideal nanoparticles. © 2013 American Chemical Society

Received: November 2, 2012 Revised: January 2, 2013 Published: January 2, 2013 460

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dride (BLA-NCA) and ε-(benzyloxycarbonyl)-L-lysine N-carboxyanhydride (Lys(Z)-NCA) were synthesized by the Fuchs-Farthing method using bis(trichloromethyl) carbonate (triphosgene) according to ref 26. N-Isopropylacrylamide (NIPAM) from TCI was purified by recrystallization from hexane and dried in a vacuum. 1-Ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl; ≥98%, Fluka), N-hydroxysuccinimide (NHS; ≥97%, Fluka), trifluoroacetic acid, hydrogen bromide (HBr; 45% in acetic acid), L-tyrosine (99%, Alfa), Na125I (PerkinElmer, Inc., U.S.A.), and other chemicals and solvents were used as received. NCI-H460 cell line was kindly provided by Dr Yong Wang (Institute of Radiation Medicine, Chinese Academy of Medical Sciences, Tianjin, China). The 293T cell line was purchased from Keygen Co. (Nanjing, China). The BALB/c mice (average 22 g) were purchased from the Laboratory Animal Center of The Academy of Military Medical Sciences (Beijing, China). Synthesis of Polymers. All polymers, including PEG-b-PLys, PNIPAM-b-PAsp, PAsp, and PLys, used in this work were synthesized by ring-opening polymerization (ROP) and atom transfer radical polymerization (ATRP). Detailed polymerization procedures and the characterization of these polymers are described in Supporting Information. Preparation of MSMs. Micellization was carried out by mixing specific amounts of the solution of PEG113-b-PLys40 and PNIPAM80-bPAsp43 with four different weight ratio of PEG segment to PNIPAM segment (WPEG/WPNIPAM) as 10/0, 7/3, 5/5, and 3/7, called as MSMs0, MSMs-30, MSMs-50, and MSMs-70, as shown in Table S1. The N/ C (amine/carboxylate) ratio was kept at 1 for each solution through adjustment of adding calculated amounts of homopolymer PAsp42 or PLys18. For the preparation of MSMs-0, only block copolymer PEG-bPLys and homopolymer PAsp were used. After stirring at room temperature for 1 h, 20× amounts of EDC·HCl was added to each solution for cross-linking between the -NH2 of PLys part and the −COOH of PAsp part. The final micelle solutions were stirring steadily at room temperature overnight. Subsequently, each solution was centrifuged in an Amicon Ultra-4 device (Millipore) with an ultrafiltration membrane (molecular weight cutoff = 100 k) at a speed of 4000 rpm for 5 min each time before lyophilization. The centrifugation was performed five times using Milli-Q water as washing agent. The MSMs in 10 mM PBS buffer (pH 7.4 with 150 mM NaCl) were incubated in 45 °C water bath for several hours before characterization. For the preparation of tyrosine-conjugated polymeric micelles, the PEG113-b-P(Lys39-co-LysTyr1) block copolymer was added into the PEG113-b-PLys40 solution before micellization. Characterization. 1H NMR spectra were recorded on a Varian UNITY-plus 400 M NMR spectrometer at room temperature with tetramethylsilane (TMS) as an internal standard. The number-average molecular weight (Mn) and weight-average molecular weight (Mw) were measured by gel permeation chromatography (GPC) at 25 °C with a Waters 1525 chromatograph equipped with a Waters 2414 refractive index detector. GPC measurements were carried out using THF or DMF as eluents with a flow rate of 1.0 mL/min, respectively. Polystyrene standards were used for calibration. Dynamic light scattering (DLS) experiments at a 90° scatter angle were performed on a laser light scattering spectrometer (BI-200SM) equipped with a digital correlator (BI-9000AT) at 636 nm at required temperature. All samples were obtained by filtering through a 0.45 μm Millipore filter into a clean scintillation vial. Transmission electron microscopy (TEM) measurements were performed using a Philips T20ST electron microscope at an acceleration voltage of 100 kV. To prepare the TEM samples, the sample solution was dropped onto a carbon-coated copper grid and dried slowly at required temperature. The zeta potential values were measured on a Brookheaven ZetaPALS (Brookheaven Instrument, USA). The instrument utilizes phase analysis light scattering at 37 °C to provide an average over multiple particles. Evaluation of In Vitro Stability. The stability of the MSMs was evaluated in 10 mM PBS (pH 7.4, 150 mM NaCl) in the presence of physiologically relevant concentrations of bovine serum albumin (BSA, 45 g/L). The micelle solutions were incubated at 45 °C for several hours and then the temperature was kept at 37 °C. At various time

comparison to non-mPEGylated analogs, mPEGylated shell cross-linked (SCK) nanoparticles exhibited longer blood circulation time. And the blood circulation time of the mPEGylated SCKs was well correlated with the densities of PEG on the surface of particles. Li et al.18 reported that, compared with 80 nm AuNPs, most of which were accumulated in RES, the 20 nm AuNPs showed prolonged circulation in blood and reduced captured in liver and spleen. Nanoparticles with small size, for example, 20 nm, presented significant prolongation in blood and reduced sequestration by liver. It seemed that smaller nanoparticles possessed better biological behaviors with longer circulating time and less deposition in RES tissues compared with larger nanoparticles. The multifunctional intravascular nanoparticles, ranging from 70 to 200 nm,19−21 would be much more easily captured in RES tissues, especially in the liver. Although investigators have proposed various strategies to obtain better nanocarriers, limited progress has been achieved and the major bottleneck still lies in the great challenge of RES. In this case, further endeavors in search of more efficient strategies are still needed in designing the ideal drug delivery system. As we know, the inevitable opsonic protein adsorption on the surface of nanoparticles during blood circulation leads to specific recognition by macrophages, resulting in rapid elimination from the bloodstream. Thus, to some extent, the unfavorable interactions with plasma proteins could be logically associated with prolonged retention in blood. According to the literature, compositional heterogeneity of the nanoparticle has a synergistic effect in discouraging thermodynamically favorable interactions between the surface and the proteins.22−25 The monolayer-protected metal nanoparticles with controlled microphase-separated domains composed of hydrophilic and hydrophobic regions on the particles’ shell were proved to be effective in avoiding nonspecific adsorption of a variety of proteins.25 Later investigations22,24 suggested that the surface heterogeneity with hydrophilic/hydrophobic regions presented resistance of protein adsorption and high level hydrophobicity could enhanced the antifouling ability for the film consisting of a heterogeneous surface made up of hydrophilic and hydrophobic segments. Because the interactions between nanoparticles and opsonic proteins in plasma play a vital role for the in vivo fate of nanoparticles, we tend to investigate the effect of surface heterogeneity on the longevity and in vivo biodistribution of nanoparticles. In this work, we fabricated a series of mixed shell micelles (MSMs) with varied hydrophilic and hydrophobic domains on the surface. The core of the MSMs was labeled with iodine-125 through the chloramine-T method. The in vivo biodistribution of MSMs were systematically investigated at various time points after i.v. in BALB/c mice determined by Gamma counter and Gamma-camera imaging. Interestingly, the MSMs with proper microphase separated surface exhibited significant improvements in prolonging blood retention and reducing accumulation in liver and spleen. Thus, this work provides a novel strategy toward prolonged circulation of nanoparticles for efficient drug delivery system.

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EXPERIMENTAL SECTION

Materials. DMF was dehydrated using activated molecular sieves (4 Å) and distilled over CaH2 under reduced pressure. N-Butylamine, dichloromethane (CH2Cl2), and methanol (MeOH) were redistilled before use. α-Methoxy-ω-aminopoly(ethylene glycol) (CH3O-PEG113NH2; Mw = 5000; Mw/Mn = 1.05) was purchased from Aladdin and used after dried under vacuum. β-benzyl L-aspartate-N-carboxyanhy461

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Figure 1. General schematic illustrating the formation of the MSMs with microphase separated surface (left) and the illustration of MSMs radiolabeling for biodistribution study (right). radioactivity in the blood and tissue samples were measured using a γcounter, respectively. Statistical Analysis. One-way analysis of variance (ANOVA) were using for the statistical analysis. The quantitative data of organ accumulation were expressed as a percentage of injected doses per gram of tissue (%ID/g). For the blood clearance curve, the total activity was calculated assuming that the blood constitutes 8% of the total weight of mice. The results were presented as mean ± standard deviation.

points, the required volume of the solutions were taken and analyzed by DLS for size measurement. MTT Assay for Cell Viability. NCI-H460 and 293T cells were separately seeded in 96-well plate at an initial density of 104 cells/well in 100 μL RPMI1640 complete media containing 10% FBS at 37 °C in humidified 5% CO2 atmosphere. After an incubation of 24 h, the culture medium of each well was replaced with 100 μL of fresh medium containing various concentrations of MSMs-0, MSMs-30, MSMs-50, or MSMs-70. A total of 24 h later, the culture media were replaced with 25 μL of MTT solution (1 mg/mL final concentration), and the cells were further incubated for another 4 h. Then, the solution was replaced with 150 μL of DMSO and the plates were slightly shaken for 10 min. The optical absorbance was measured at 570 nm using a microplate reader (Labsystem, Multiskan, Ascent, Finland). Cells without micelles were used as the control. Radiolabeling of MSMs. The micelles were 125I-labeled on the phenol group of the tyrosine residues of the side chains of PLys units using chloramine-T method. Briefly, a solution of Na125I in 10 mM phosphate-buffered saline (PBS; 18.5 MBq, PerkinElmer, Inc., U.S.A.) was added to a solution of tyrosine-conjugated MSMs in water (900 μL, 1.0 mg/mL). The reaction mixture was incubated at room temperature for 30 min. Then, the reaction was quenched by the addition of a solution of sodium peroxodisulfate in 10 mM PBS (100 μL, 40 mM). After shaking for a few minutes, the unreacted 125I and other chemicals were removed by centrifugation in an Amicon Ultra-4 device with an ultrafiltration membrane (molecular weight cutoff = 100 k) at a speed of 4000 rpm for three times (each 5 min). The centrifugation was performed for 5 times using 10 mM PBS as washing agent to remove unreacted, free 125I ions from the 125I-labeled micelles. The labeling rates and radiochemical purities of the MSMs were detected by radioactive thin-layer chromatography (TLC) scanner (90% ethanol and 10% deionized water as mobile phase). The solution of 125I-labeled micelles mixed with the unlabeled micelles was incubated in 45 °C water bath prior to the biodistribution study. In Vivo Biodistribution Experiments. The animal studies were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (Tianjin, revised in June 2004) and adhered to the Guiding Principles in the Care and Use of Animals of the American Physiological Society. In detail, after incubation in a 45 °C water bath overnight, the micelles solutions were kept in 37 °C for 0.5 h. The BALB/c mice (n = 5) were injected intravenously via the tail vein with 125I-labeled MSMs at a dose of 5 mg/kg, respectively. At different time points (1, 4, 8, 24, and 48 h), blood samples were collected before sacrifice. Gamma-camera imaging was performed at specific time points after i.v. using a small-animal in vivo imaging instruments (KODAK IS in vivo FX, KODAK, New Haven, CT). Mice were sacrificed and tissues including heart, liver, spleen, lung, renal, stomach, large intestine, small intestine, genital, thyroid, skeletal muscle, and brain were harvested for each group at different time points. The tissues were weighted and the amounts of

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RESULTS AND DISCUSSION Formation of MSMs with Microphase Separated Surface. To evaluate the effect of the microphase separation on the in vivo biodistribution of MSMs, first of all, we should obtain well-designed nanoparticles with approximately the same core composition, size, and charge but with a varied hydrophilic/hydrophobic ratio on the surface of the micelle. Combined with electrostatic self-assembly and the phase transition of thermosensitive polymers, the MSMs were easily fabricated and tuned for desire properties. As shown in Figure 1, we prepared a series of MSMs with a polyion complex (PIC) core of PLys/PAsp and a mixed shell of PEG/PNIPAM through coassembly of PEG113-b-PLys40 and PNIPAM80-bPAsp43 based on the spontaneous electrostatic interaction between the PLys and PAsp parts in water at room temperature. Four kinds of surface microphase-separated MSMs with gradient hydrophilicity/hydrophobicity, of which the weight ratios of PEG segment to PNIPAM segment (WPEG/ WPNIPAM) were 10/0, 7/3, 5/5, and 3/7, respectively (MSMs-0, MSMs-30, MSMs-50, and MSMs-70 for short), were obtained by varying the relative amounts of the polymers. Only PEG-bPLys and homopolymer PAsp were used for the fabrication of MSMs-0, which was single PEGylated without the PNIPAM segment on the surface. The MSMs were chemical cross-linked with the addition of 20 times extra amounts of EDC·HCl, resulted in compact PIC core containing cross-linked PLys and PAsp parts. Without core-cross-linking, the micelles will gradually disassociate into single polymers under physiological conditions. Thermo-stimuli-induced collapse of PNIPAM chains could create microphase separation on the surface of the micelle.27−33 At room temperature, for example, 25 °C, both PEG and PNIPAM segments were hydrophilic, resulting in a mixed shell around the PIC core. Above 32 °C, the lower critical solution temperature (LCST) of PNIPAM,34 PNIPAM collapsed onto the cross-linked compact PIC core, forming as hydrophobic domains. Jiang et al.29 reported similar core-cross462

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linked MSMs, of which separated microdomains formed with mixed hydrophilic PEG and hydrophobic P2VP segments in the shell under stimuli of pH change. Without core-cross-linking, phase reverse might happen for the MSMs containing the PIC core and the mixed PEG/PNIPAM shell at high temperature. As reported in Stuart’s work,30 at 25 °C, the PEO and PNIPAM chains appeared to be randomly mixed on the surface of a noncross-linked PIC core. At higher temperature, 60 °C, onionlike complexes were formed, consisting of a hydrophobic PNIPAM inner core, a mixed PIC shell and a PEO corona. The PNIPAM segments transited into the inner core as a hydrophobic core under temperature increasing above the LCST. Moreover, the PIC micelles were sensitive to the ionic strength. In our work, without core-cross-linking, the addition of salt will disrupt the PIC core of PAsp and PLys parts. The MSMs disassociated into polymers which could be seen from the DLS measurement that the intensity and size of the MSMs decreased over time (data not shown). After core-cross-linking, the possibility of phase reverse after temperature increasing can be prevented and the PIC cross-linked core kept stable in 10 mM PBS (pH 7.4, 150 mM NaCl). In our system, the phase transition of the mixed shell could be easily realized without pH adjustment or addition of organic solvents which is a significant property for potential use of protein or gene delivery. Combined with the advantage of smart phase transition under temperature increase, PNIPAM could be considered as a good model as hydrophobic polymer at physiological environment to study the effect of tunable hydrophilic/ hydrophobic surface on the in vivo biodistribution of nanoparticles. With selective formulation, the MSMs possessed similar uniformity and nanosize around 100 nm under pH 7.4 in PBS buffer with 150 mM NaCl at 37 °C determined by DLS and TEM (Figure 2, Table 1, and Figure S6). As mentioned above,

Table 1. Physicochemical Characteristics of Different MSMs with Varied PEG/PNIPAM Ratios sample codes

PEG/PNIPAM (w/w)

MSMs-0 MSMs-30 MSMs-50 MSMs-70

10:0 7:3 5:5 3:7

a b

zeta potentiala (mv) −7.8 −8.7 −8.9 −7.1

± ± ± ±

0.6 0.3 0.8 0.4

Dhb (nm) 94.0 ± 3.8 90.7 ± 2.6 94.4 ± 4.2 101.3 ± 3.9

Measured in 10 mM PBS (pH 7.4, 150 mM NaNO3) at 37 °C. Measured in 10 mM PBS (pH 7.4, 150 mM NaCl) at 37 °C.

or PLys18 was selectively used to adjust the composition of the PIC core with a zero-total charge. The N/C (amine/ carboxylate) ratio was kept at approximately 1 to obtain consistent and slightly negative charge under physiological conditions as reported that slightly negatively charged micelles reduced the nonspecific uptake by liver and spleen to some extent attributed to the electrostatic repulsion between negatively charged micelles and cellular surface.15 For detecting the MSMs in BALB/c mice, iodine-125 was employed as an effective tracking tracer after i.v.. A tyrosine residue was easily conjugated onto the side chain of block copolymer PEG-b-PLys, resulted in the copolymer poly(ethylene glycol)-block-poly( L -lysine-co- L-lysine-Tyrosine) (PEG-b-P(Lys39-co-LysTyr1)) containing 2.5% tyrosine group at the side chain of PLys (Supporting Information). A small portion of PEG-b-P(Lys-co-LysTyr) was added into the PEG-bPLys solutions before micellization. As showed in Figure 1, after core-cross-linking, the MSMs were 125I-labeled according to the chloramine-T method by utilizing phenol groups of tyrosine incorporated into the core of MSMs. The resultant labeled MSMs were purified by centrifugation with an ultrafiltration membrane (molecular weight cutoff = 100 k) at a speed of 4000 rpm for 5 min each time. After washing with PBS buffer for three times during the purification, free 125I ions could be efficiently removed from the micelle solution, while the 125I-labeled MSMs remained at the original position shown in Figure 3B (the full data of the four MSMs were shown in Figure S7). The labeling rates for all the MSMs were around 33−39% and the radiochemical purities for the 125I-labeled MSMs after purification were higher than 96%, as summarized in Table 2. In Vitro Stability and Cell Viability. In drug delivery system, the stability of nanocarriers is one of the most important factors determining the efficacy in controlled drug delivery. In this study, the stability of MSMs was assessed by DLS measurements in 10 mM PBS buffer (pH 7.4 with 150 mM NaCl) in the presence of physiologically relevant concentration of bovine serum albumin (BSA, 45 g/L). As shown in Figure S8A−C, the size distribution of the MSMs-0, MSMs-30, and MSMs-50 changed little after incubation at 37 °C for 48 h. The size around 7 nm could be attributed to the high concentration of BSA which contributing main intensity in DLS measurements. The larger size could be attributed to the MSMs. During 48 h, the size distribution of the MSMs remained at around 100 nm and no apparent size variation was observed after incubation at 37 °C for MSMs-0, MSMs-30, and MSMs-50. This indicated these MSMs could keep stable in PBS buffer (pH 7.4 with 150 mM NaCl) in the presence of physiologically relevant concentration of BSA. However, size variation appeared for MSMs-70 in the presence of BSA at 48 h as shown in Figure S8D. The intensity of the MSMs-70 around 100 nm decreased and the size distribution broadened at 48 h.

Figure 2. (A) Hydrodynamic diameter distribution of different MSMs (MSMs-0, MSMs-30, MSMs-50, and MSMs-70) in 10 mM PBS buffer (pH 7.4 with 150 mM NaCl) measured by DLS at 37 °C. (B) TEM image of MSMs-50 at 37 °C. Scale bar for MSMs, 100 nm; magnification, 72000×; scale bar (inset), 50 nm. Images of other three MSMs with similar morphology were shown in Supporting Information, Figure S6.

the desired size of multifunctional nanocarriers ranges from 70 to 200 nm. The hydrodynamic diameter of MSMs in this work is about 100 nm, as shown in Table 1 (94.0, 90.7, 94.4, and 101.3 nm for MSMs-0, MSMs-30, MSMs-50, and MSMs-70, respectively), which is a good model to study the biodistribution of nanoparticles. The zeta potential of the MSMs was demonstrated to be about −8 mv, as shown in Table 1. For the preparation of MSMs, homopolymer PAsp42 463

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Figure 4. Cell viability of different MSMs (MSMs-0, MSMs-30, MSMs-50, and MSMs-70) against (A) NCI-H460 and (B) 293T cells measured by MTT assay.

125

Figure 3. Representative TLC chromatograms of I-labeled MSMs (MSMs-0) before (A) and after purification (B). The 125I-labeled MSMs remained at the original position while the free 125I ions were removed after purification.

Table 2. Labeling Rates and Radiochemical Purities of Labeled MSMs

125

sample code

labeling rate (%)

radiochemical purity (%)

MSMs-0 MSMs-30 MSMs-50 MSMs-70

37.69 33.86 38.54 37.29

100 100 96.73 100

higher than 0.5 mg/mL. At the concentration of 1 mg/mL or 2 mg/mL, the cell viability tended to be lower than 50%. Similar results were presented in Figure 4B. The cell viability tested with both cancer and normal cell lines indicated that all MSMs had good biocompatibility. The PNIPAM segments, which were considered as nonbiodegradable polymers, were employed as a good model of hydrophobic domain at physiological environment. In the practical application of drug delivery system, other biodegradable polymers could be utilized as hydrophobic parts with much better biocompatible properties. In Vivo Biodistribution of MSMs. To investigate the effect of surface microphase separation, the MSMs were chemical labeled with iodine-125 using chloramine-T method. The in vivo fate of the MSMs was monitored by radioactive count using a Gamma counter and Gamma-camera imaging using a small-animal in vivo imaging instrument (KODAK IS in vivo FX, KODAK, New Haven, CT). The accumulation of micelles in various tissues (including heart, liver, spleen, lung, renal, stomach, large intestine, small intestine, genital, thyroid, skeletal muscle, brain, and blood) after i.v. at a dose of 5 mg MSMs/kg (weight of mice) in BALB/c mice is represented in Figure 5. For the MSMs with different WPEG/WPNIPAM on the particle surface, the liver and spleen were the major hosts, while other organs had relatively low uptake of particles. It was suggested that the foreign nanoparticles could be rapidly cleared out of blood circulation and accumulated in RES organs, such as liver, spleen, and so on. For different MSMs, the accumulation amounts in liver and spleen differed from each other. It can be seen from Figure 5A that most of the MSMs-0 were deposited in the liver and spleen and less than 5%ID/g accumulated in any other tissues and only 4.5%ID/g remained in blood circulation at the initial time point of 1 h. This was in good agreement of the points of Park that the majority (>95%) of administered nanoparticles are accumulated in RES organs.9

I-

Furthermore, large particles up to several hundred nanometers which contributed non-negligible intensity for the scattering of particles were observed. But, as for calculation by number average in DLS, the few number of these large particles indicated trace aggregation in the presence of BSA at 48 h. The slight aggregation of MSMs-70 might be attributed to the large hydrophobic area exposed on the micelle interacted hydrophobically with the BSA. Biocompatibility is also quite important in practical application. To assess the cytotoxicity of different micelles (MSMs-0, MSMs-30, MSMs-50, and MSMs-70), both NCIH460 and 293T cells were used in the MTT assays. Four MSMs exhibited negligible toxicity evaluated by cell viability assays carried out at different doses of micelles (from 1 μg/mL to 2 mg/mL) as shown in Figure 4. Because they mainly consisted of PEG and poly(amino acid), all the MSMs studied were found to be nontoxic at all concentrations evaluated. In Figure 4A, compared with the control group (CTL), in which no MSMs but PBS buffer was added in the NCI-H460 cell solutions, all MSMs showed good biocompatibility. The cell viability was as high as 100% at lower concentration for all MSMs. The toxicity of the MSMs was dose dependent. The MSMs showed some toxicity when the concentration was 464

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Figure 5. In vivo biodistribution of four different MSMs labeled with 125I. Tissues were harvested and weighted at five various time points (1, 4, 8, 24, and 48 h) after initial inject via the tail vein of BALB/c mice with MSMs of (A) MSMs-0, (B) MSMs-30, (C) MSMs-50, and (D) MSMs-70, respectively (5 mg MSMs/kg mice body weight; data are expressed as percent injected dose per gram (%ID/g) ± standard deviation, n = 5).

The deposition of the MSMs-0 in liver at 1 h after i.v. (53.4% ID/g) was 1.2-, 3.6-, and 2.2-fold higher than that of the MSMs-30 (45.9%ID/g), MSMs-50 (14.9%ID/g), and MSMs70 (24.2%ID/g), respectively. At each time point, the uptake of the MSMs-0 in the liver was several times higher than that of MSMs-50 and MSMs-70. According to Figure 5, there was a significant contrast at 1 h after i.v. that nanoparticles accumulated in spleen for MSMs-0 (248.0%ID/g) was 2.6-, 4.4-, and 12.2-fold higher than that of the MSMs-30 (94.5%ID/ g), MSMs-50 (56.2%ID/g), and MSMs-70 (20.4%ID/g), respectively. Up to 8 h, invisible amounts of MSMs-0 deposited in tissues except liver and spleen can be seen in Figure 5A and less than 1%ID/g remained in blood. These results indicate that the MSMs-0 with single PEGylation without hydrophobic domains tended to be much easier cleared out of bloodstream and accumulated higher in RES organs than other MSMs with microphase separated surface. Generally, the MSMs with hydrophobic domains (MSMs-30, MSMs-50, and MSMs-70) exhibited reduced accumulation in liver and spleen compared with MSMs-0 and the particles accumulated in them declined with the increase of the PNIPAM content in the mixed shell (Figure 5). The MSMs30 with fewer PNIPAM collapsed domains exhibited similar blood clearance feature with MSMs-0, while with less accumulation in liver and spleen and higher uptake in other tissues (especially heart, renal, lung and stomach) during the whole testing period. It is worth pointing out that the accumulation of the MSMs-50 in liver and spleen was 15% ID/g and 60%ID/g, respectively, at 1 h after i.v., much more less than those of MSMs-0. As for the MSMs-70, the deposition level in liver and spleen was similar to MSMs-50, but with relatively less concentration in blood over test period. A remarkable difference represented in Figure 6 that MSMs-50

Figure 6. Blood clearance curves of MSMs in mice after i.v. (data are expressed as percent injected dose (%ID) ± standard deviation, n = 5).

showed considerably several times higher amounts in blood than other MSMs during the circulation time. At the initial time of 1 h after i.v., the nanoparticles circulated in blood for MSMs50 (46.7%ID) was 5.9-, 6.1-, and 4.2-fold higher than that of the MSMs-0 (7.9%ID), MSMs-30 (7.7%ID), and MSMs-70 (11.1% ID), respectively. The MSMs-50 had the notably highest level of blood retention in comparison to the other MSMs at each time point up to 24 h when few particles remained in blood for all different MSMs. The relative higher amounts of nanoparticles retained in blood seemed to be logical correlated with the lower sequestration in liver and spleen. The notable difference of blood retention suggested MSMs-50 was a good candidate in efficient contacting with the tumor tissue for improved cancer therapy. The Gamma-camera imaging with in vivo imaging system was utilized to monitor the in vivo biodistribution of MSMs at various time points. The Gamma-camera images of MSMs at various time point shows the radioactive intensity of 125I465

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relatively unfavorable interactions with opsonic proteins. Therefore, we believe that with the synergistic effect of the hydrophilic and hydrophobic domains, there will be less opsonic proteins binding to the surface of the MSMs after i.v., thus, leading to prolonged retention and higher nanoparticle concentration in blood compared with single PEGylated micelles. Without the microphase separation, the MSMs-0 performed the most undesirable behaviors of major accumulation in liver and spleen and minor retention in blood as shown in Figure 5. The relative higher blood concentration of MSMs-50 may be ascribed to the specific microphase separation with well-balanced hydrophilic and hydrophobic domains contributing less interaction with opsonic proteins in plasma. As for the MSMs-70, fewer particles remained in blood might be due to the slight aggregation of particles because of the rich domain of hydrophobic PNIPAM which would much more easier hydrophobically interacted with plasma proteins, resulted in further aggregation tended for elimination from plasma as shown in Figure S8D. Compared with MSMs-50, MSMs-30 and MSMs-70 exhibited less contribution to prolonged circulation in blood indicating that a substantial and proper hydrophobicity is needed to impart the surface of micelles to achieve optimal biological behaviors.

labeled MSMs in mice over the test period up to 48 h. For comparison of different MSMs, clearly shown in Figure S9, the Gamma-camera images of MSMs after i.v. directly indicated that particles were mainly accumulated in liver (mainly in the yellow area) for all MSMs and the concentration of MSMs distributed in body decreased over time. The MSMs were mainly captured by liver and excreted over time under the degradation of liver cells. While at the same time point, for example, the time point of 4 h, the MSMs-50 and MSMs-70 presented less intensive dispersal in liver compared with MSMs-0 and MSMs-30, which indicated fewer MSMs accumulated in liver. The images further confirm the varied biodistribution of the MSMs in mice after i.v. Nanoparticles can be rapidly removed from blood circulation by RES through phagocytosis mediated by opsonization and cleared by mainly Kupffer cells of the liver and to a lesser extent the macrophages of the spleen and the bone marrow.35,36 Although the PEGylation appeared to be an efficient candidate for reducing the RES uptake, the interactions between PEG and proteins in various systems were demonstrated.37−39 Single PEGylation strategy is not enough for the sake of significant prolonging the blood retention, especially for the larger nanocarriers. Although increasing PEG density on the surface of nanoparticles could realize better circulating behaviors, the highly PEGylated nanoparticles are difficult to obtain. As mentioned, the compositional heterogeneity of nanoparticles with hydrophilic and hydrophobic domains on the surface has a synergistic effect in preventing unspecific protein adsorption.22−25 It is worthwhile to note that combined with proper hydrophobic domains on the surface, the increased amounts of circulating nanoparticles might be due to the unfavorable interactions between MSMs and opsonins in plasma. As illustrated in Figure 7, the PIC core of the MSMs was covered

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CONCLUSION We found that the MSMs with proper surface microphase separation showed highly improved biodistribution behaviors with reduced accumulation in liver and spleen and prolonged blood circulation. In addition, it is feasible to manipulate the in vivo biodistribution by simply varying the ratio of hydrophobic and hydrophilic segments on the surface of nanoparticles. Such a particle design approach is a powerful technique in engineering smart nanoparticles toward efficient cancer therapy. This strategy can be appropriately combined with available strategies in designing long-circulating nanoparticles. More dramatic advances could be achieved utilizing the combinations of different strategies for improvements in reduced accumulation in RES organs and prolonged circulation in blood. The results in this paper give us a guideline for future development of MSMs as promising carriers in drug delivery system.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed synthesis procedures, 1H NMR, formulation, TEM, TLC chromatograms of 125I-labeled MSMs, size distribution of MSMs, and Gamma-camera images are available. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

Figure 7. Schematic representation of generic proteins in plasma (top) and the MSMs (bottom). The yellow and green contour line on top of the MSMs shows the hydrophobic PNIPAM and the hydrophilic PEG domains of the particle, respectively.

*E-mail: [email protected] (L.S.); [email protected] (J.L.). Notes

The authors declare no competing financial interest.

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with molecular to nanoscale surface heterogeneities created by the interpenetrated segments of hydrophilic PEG and thermoresponsively collapsed hydrophobic segment of PNIPAM. The structure of microphase separated surface and the conformation of the proteins are not quite matched. There will always be regions of attraction and adjacent regions of repulsion on the rough surface when interacting with proteins, leading to

ACKNOWLEDGMENTS

We thank the Science Foundation of China (No. 91127045, 50830103, 20904025, and 81171371) and the National Basic Research Program of China (973 Program, No. 2011CB932503) for financial support. 466

dx.doi.org/10.1021/bm301694t | Biomacromolecules 2013, 14, 460−467

Biomacromolecules

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(34) Akiyoshi, K.; Kang, E. C.; Kurumada, S.; Sunamoto, J.; Principi, T.; Winnik, F. M. Macromolecules 2000, 33, 3244−3249. (35) Brain, J. D.; Molina, R. M.; DeCamp, M. M.; Warner, A. E. Am. J. Physiol. 1999, 276, L146−L154. (36) Ogawara, K.; Yoshida, M.; Higaki, K.; Kimura, T.; Shiraishi, K.; Nishikawa, M.; Takakura, Y.; Hashida, M. J. Controlled Release 1999, 59, 15−22. (37) Liu, Z.; Gu, J.; Wu, M.; Jiang, S.; Wu, D.; Wang, Q.; Niu, Z.; Huang, Y. Langmuir 2012, 28, 11957−61. (38) Shah, N. B.; Vercellotti, G. M.; White, J. G.; Fegan, A.; Wagner, C. R.; Bischof, J. C. Mol. Pharm. 2012, 9, 2146−2155. (39) Gref, R.; Luck, M.; Quellec, P.; Marchand, M.; Dellacherie, E.; Harnisch, S.; Blunk, T.; Muller, R. H. Colloids Surf., B 2000, 18, 301− 313.

REFERENCES

(1) Riehemann, K.; Schneider, S. W.; Luger, T. A.; Godin, B.; Ferrari, M.; Fuchs, H. Angew. Chem., Int. Ed. 2009, 48, 872−897. (2) Wolinsky, J. B.; Grinstaff, M. W. Adv. Drug Delivery Rev. 2008, 60, 1037−1055. (3) Zhang, L.; Gu, F. X.; Chan, J. M.; Wang, A. Z.; Langer, R. S.; Farokhzad, O. C. Clin. Pharmacol. Ther. 2008, 83, 761−769. (4) Sanvicens, N.; Marco, M. P. Trends Biotechnol. 2008, 26, 425− 433. (5) Simberg, D.; Duza, T.; Park, J. H.; Essler, M.; Pilch, J.; Zhang, L. L.; Derfus, A. M.; Yang, M.; Hoffman, R. M.; Bhatia, S.; Sailor, M. J.; Ruoslahti, E. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 932−936. (6) Maeda, H.; Bharate, G. Y.; Daruwalla, J. Eur. J. Pharm. Biopharm. 2009, 71, 409−419. (7) Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. J. Controlled Release 2000, 65, 271−284. (8) Owens, D. E.; Peppas, N. A. Int. J. Pharm. 2006, 307, 93−102. (9) Bae, Y. H.; Park, K. J. Controlled Release 2011, 153, 198−205. (10) Gref., R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.; Torchilin, V. P. Science 1994, 263, 1600−1603. (11) Ogawara, K.; Yoshida, M.; Takakura, Y.; Hashida, M.; Higaki, K.; Kimura, T. Biochem. Biophys. Acta, Gen. Subj. 1999, 1472, 165−172. (12) Ogawara, K.; Yoshida, M.; Kubo, J.; Nishikawa, M.; Takakura, Y.; Hashida, M.; Higaki, K.; Kimura, T. J. Controlled Release 1999, 61, 241−250. (13) Moghimi, S. M.; Hunter, A. C.; Murray, J. C. Pharmacol. Rev. 2001, 53, 283−318. (14) Alexis, F.; Pridgen, E.; Molnar, L. K.; Farokhzad, O. C. Mol. Pharm. 2008, 5, 505−515. (15) Yamamoto, Y.; Nagasaki, Y.; Kato, Y.; Sugiyama, Y.; Kataoka, K. J. Controlled Release 2001, 77, 27−38. (16) Sun, G.; Hagooly, A.; Xu, J.; Nystrom, A. M.; Li, Z. C.; Rossin, R.; Moore, D. A.; Wooley, K. L.; Welch, M. J. Biomacromolecules 2008, 9, 1997−2006. (17) Sun, X. K.; Rossin, R.; Turner, J. L.; Becker, M. L.; Joralemon, M. J.; Welch, M. J.; Wooley, K. L. Biomacromolecules 2005, 6, 2541− 2554. (18) Zhang, G.; Yang, Z.; Lu, W.; Zhang, R.; Huang, Q.; Tian, M.; Li, L.; Liang, D.; Li, C. Biomaterials 2009, 30, 1928−1936. (19) Zhao, J.; Liu, C.; Yuan, Y.; Tao, X.; Shan, X.; Sheng, Y.; Wu, F. Biomaterials 2007, 28, 1414−1422. (20) Fang, C.; Shi, B.; Pei, Y. Y.; Hong, M. H.; Wu, J.; Chen, H. Z. Eur. J. Pharm. Sci. 2006, 27, 27−36. (21) Chang, T.; Powanda, D. Artif. Cells, Blood Substitutes, Biotechnol 2003, 31, 231−247. (22) Wong, S. Y.; Han, L.; Timachova, K.; Veselinovic, J.; Hyder, M. N.; Ortiz, C.; Klibanov, A. M.; Hammond, P. T. Biomacromolecules 2012, 13, 719−726. (23) Hung, A.; Mwenifumbo, S.; Mager, M.; Kuna, J. J.; Stellacci, F.; Yarovsky, I.; Stevens, M. M. J. Am. Chem. Soc. 2011, 133, 1438−1450. (24) Baxamusa, S. H.; Gleason, K. K. Adv. Funct. Mater. 2009, 19, 3489−3496. (25) Jackson, A. M.; Myerson, J. W.; Stellacci, F. Nat. Mater. 2004, 3, 330−336. (26) DALY, W. H.; POCHE, D. Tetrahedron Lett. 1988, 29, 5859− 5862. (27) Walther, A.; Barner-Kowollik, C.; Muller, A. Langmuir 2010, 26, 12237−12246. (28) Ma, R. J.; Shi, L. Q. Macromol. Biosci. 2010, 10, 1397−1405. (29) Cheng, L.; Zhang, G. Z.; Zhu, L.; Chen, D. Y.; Jiang, M. Angew. Chem., Int. Ed. 2008, 47, 10171−10174. (30) Voets, I. K.; Moll, P. M.; Aqil, A.; Jerome, C.; Detrembleur, C.; de Waard, P.; de Keizer, A.; Stuart, M. J. Phys. Chem. B 2008, 112, 10833−10840. (31) Srinivas, G.; Pitera, J. W. Nano Lett. 2008, 8, 611−618. (32) Ma, R. J.; Wang, B. L.; Xu, Y. L.; An, Y. L.; Zhang, W. Q.; Li, G. Y.; Shi, L. Q. Macromol. Rapid Commun. 2007, 28, 1062−1069. (33) Hui, T. R.; Chen, D. Y.; Jiang, M. Macromolecules 2005, 38, 5834−5837. 467

dx.doi.org/10.1021/bm301694t | Biomacromolecules 2013, 14, 460−467