Targeted Delivery of Insoluble Cargo (Paclitaxel) by PEGylated

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Targeted Delivery of Insoluble Cargo (Paclitaxel) by PEGylated Chitosan Nanoparticles Grafted with Arg-Gly-Asp (RGD) Pi-Ping Lv,†,‡,§ Yu-Feng Ma,§,∥ Rong Yu,∥ Hua Yue,† De-Zhi Ni,†,‡ Wei Wei,*,† and Guang-Hui Ma*,† †

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, PR China ∥ Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education,West China School of Pharmacy, Sichuan University, Chengdu, 610041, China ‡ Graduate University of Chinese Academy of Sciences, Beijing, 100049, PR China S Supporting Information *

ABSTRACT: Poor delivery of insoluble anticancer drugs has so far precluded their clinical application. In this study, we developed a tumortargeting delivery system for insoluble drug (paclitaxel, PTX) by PEGylated O-carboxymethyl-chitosan (CMC) nanoparticles grafted with cyclic Arg-Gly-Asp (RGD) peptide. To improve the loading efficiency (LE), we combined O/W/O double emulsion method with temperature-programmed solidification technique and controlled PTX within the matrix network as in situ nanocrystallite form. Furthermore, these CMC nanoparticles were PEGylated, which could reduce recognition by the reticuloendothelial system (RES) and prolong the circulation time in blood. In addition, further graft of cyclic RGD peptide at the terminal of PEG chain endowed these nanoparticles with higher affinity to in vitro Lewis lung carcinoma (LLC) cells and in vivo tumor tissue. These outstanding properties enabled as-designed nanodevice to exhibit a greater tumor growth inhibition effect and much lower side effects over the commercial formulation Taxol. KEYWORDS: nanoparticles, crystallization, drug delivery, RGD peptide

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INTRODUCTION Cancer has become the leading cause of death worldwide and accounts for more than 8 million deaths every year.1 As most cases are diagnosed at advanced stage, chemotherapy is almost indispensable during the clinical treatment. Unfortunately, many chemotherapeutic anticancer agents are found to be insoluble in water because of their bulky polycyclic structure.2 Various problems derived from their hydrophobic nature make the intravenous delivery of these drugs very troublesome, and even hamper their translation from bench to bed side. For example, aggregates formed by free insoluble drug can cause embolization of blood vessels, resulting in severe safety problems. These aggregates also lead to high localized concentrations at the deposition site, which can be associated with local toxicity and lowered systemic bioavailability.3 Paclitaxel (PTX), a typical insoluble anticancer drug, has potent antineoplastic activity against various types of solid tumors such as non-small-cell lung cancer (NSCLC), ovarian cancer, and breast cancer.4 To improve its solubility and allow intravenous administration, PTX has to be formulated in a 1:1 blend of Cremophor EL/absolute ethanol as Taxol. However, Taxol can cause injury to normal tissues due to the nonspecific biodistribution of the drug in both tumors and normal tissues.5 Worse still, administration of Cremophor EL in practical © 2012 American Chemical Society

dosage form can also arouse severe side effects including hypersensitivity reaction and neurotoxicity.6 Delivery of insoluble drugs by encapsulation within nanocarriers can not only overcome the aforementioned problems of conventional free drugs but also passively transport through the new blood vessels and accumulate at tumor sites via the enhanced permeability and retention effect (EPR effect).7 Albeit promising, most of these nanocarriers cannot finally retain the solid tumor tissue due to their poor binding affinity and internalization toward tumor cells.8 To solve this problem, modification with targeting ligands onto the surface of nanocarriers becomes necessary to promote nanocarrier binding and internalization at the lesion site.9 Moreover, the rapid opsonization-mediated clearance of circulating nanocarriers from the bloodstream coupled with their high uptake by the reticuloendothelial system (RES) can also greatly compromise the drug bioavailability.10 In this aspect, surface modification such as conjunction of polyethylene glycol (PEG) chain to reduce interactions with serum proteins and mitigate uptake by phagocytic cells also becomes very crucial.11 Received: Revised: Accepted: Published: 1736

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Figure 1. Schematic illustration of RGD-PEG-CNP:PTX (A) and the conjunction of PEG chain and cyclic RGD peptide (B).

3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and glutaraldehyde were supplied by Sigma-Aldrich. Maleimide PEG2000 amine (MAL-PEG2000NH2) was from Jenkem Technology (China). All other materials used were analytical grade. Lewis lung carcinoma cells (LLC), murine fibroblast cells (NIH 3T3) and macrophage cell line (J774A.1) were supplied by American Type Culture Collection (ATCC). Dulbecco’s modified Eagle’s medium (DMEM), newborn bovine serum (NBS) and fetal bovine serum (FBS) were purchased from Gibco. PTX, MTT (3-(4,5-dimethylazolyl-2)-2,5-diphenyltetrazolium bromide), penicillin, streptomycin and Cremophor EL were purchased from Sigma-Aldrich. Taxol was supplied by Merck. DIR iodide (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide, an insoluble near-infrared (NIR) fluorescence dye), Rhodamine-phalloidin, Alexa Fluor 635 phalloidin, and DAPI (4,6-diamidino-2-phenylindole) were purchased from Invitrogen. Alexa Fluor 488 anti-mouse/rat CD61 (integrin β3 chain) antibody was bought from Biolegend (San Diego, CA, USA). Male C57BL/6 mice, 6−8 weeks of age, were purchased from Charles River Laboratories (USA). All animal experiments were performed in compliance with the institutional ethics committee regulations and guidelines on animal welfare (Animal Care and Use Program Guidelines of Peking University). Synthesis and Characterization of CMC. CMC was synthesized according to the method reported previously.12 Chitosan (10 g) was swelled and alkalized in sodium hydroxide (13.5 g) solution (water:isopropanol = 50:50 mL) at room temperature for 24 h. Monochloroacetic acid (15 g) dissolved in isopropanol (20 mL) was added into the reaction mixture dropwise for 30 min and reacted for 4 h at 50 °C. The pH of the reaction mixture was adjusted to 6.5−7.0 using hydrochloric

Therefore, the main challenge for high-performance nanocarriers of insoluble agents is to simultaneously achieve high targeting specificity in time, while avoiding nonspecific entrapment by RES. In this work, we attempted to design a targeting delivery system for the insoluble anticancer drug PTX by chitosan-based nanoparticles (Figure 1). To achieve subsequent functionalization with PEG chain and RGD ligand, the derivative Ocarboxymethyl-chitosan (CMC) was first synthesized as a nanoparticle matrix. To make full use of the EPR effect, uniform-sized PTX-loaded CMC nanoparticles (CNP:PTX) were prepared by a premix membrane emulsification process. To achieve a high loading efficiency (LE), an O/W/O double emulsion method was combined with a temperatureprogrammed solidification technique to control PTX within the matrix network as in situ nanocrystals. The obtained CNP:PTX was further modified by polyethylene glycol (PEG) chains to prolong their circulation time in the bloodstream. A synthetic cyclic peptide containing the arginine-glycineaspartate (RGD) motif was subsequently modified at the outer end of the PEG chain to enhance the binding affinity of nanoparticles toward tumor cells. Macrophage J774A.1 and Lewis lung carcinoma (LLC) cells were used to examine the stealth and tumor-cell targeting ability in vitro, respectively. The LLC-bearing mouse model was also established to in vivo test the feasibility of as-designed nanodevice for targeting delivery of PTX.

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MATERIALS AND METHODS Reagents and Materials. Chitosan (CS, Mw = 50 000) was purchased from Golden-Shell Biochemica (China). Shirasu porous glass (SPG) membrane was bought from SPG Technology (Japan). PO-500 ((hexaglycerin penta) ester) was obtained from Sakamoto Yakuhin Kogyo (Japan). 1-Ethyl1737

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1.0 mL/min. The PTX concentrations in the samples were calculated from a calibration curve. Drug LE was calculated according to the following formula:

acid to stop the reaction. The sample was extracted with water, rinsed in 70−90% ethyl alcohol to desalt, and vacuum-dried at room temperature to obtain the Na-form CMC. The H-form CMC was obtained after further acidifying the Na-form CMC with hydrochloric acid. The IR spectrum of CMC was recorded on an FT/IR-660 Fourier transform infrared spectrometer (Jasco Co., Tokyo, Japan). The substitution of carboxymethyl groups was determined from the 1H NMR spectra using the method previously reported.13 Preparation of PTX-Loaded Nanoparticles. PTX-loaded CMC nanoparticles (CNP:PTX) were first prepared by an O/ W/O double emulsion together with temperature-programmed solidification methods.14 In brief, the oil phase I (OI) was dichloromethane solution containing 50 mg/mL PTX. CMC (0.5 wt %) containing 2 wt % Tween-60 was dissolved in acetic acid buffer solution (pH 3.5), which was used as the water phase (W). The oil phase II (OII) was a mixture of liquid paraffin and petroleum ether 1:2 (v/v) containing 4 wt % PO500 as emulsifier. The volume ratio of OI, W and OII was fixed at 1:5:150. The OI and W were first mixed through ultrasonic emulsification to obtain a primary emulsion (OI/W), which was further added into OII to obtain a coarse double emulsion (OI/ W/OII). Uniform-sized nanodroplets were achieved by extruding the coarse emulsion through the SPG membrane (pore size was 0.5 μm) under high pressure (2.0 MPa). Subsequently, glutaraldehyde saturated toluene (GST) was slowly dropped into the emulsion to solidify nanodroplets into nanoparticles. Solidification of PTX-loaded nanoparticles was performed by a two-step procedure. The solidification temperature was first maintained 25 °C for 30 min and then slowly increased to (2 °C/min) 50 °C. After 10 h, the CNP:PTX was collected and washed by petroleum ether and deionized (DI) water three times.15 To prepare PTX-loaded nanoparticles functionalized by PEG chain and cyclic RGD peptide (Figure 1A), the obtained CNP:PTX (20 mg) was suspended in 5 mL of PBS (pH 6.5) followed the addition of EDC (20 mg) and NHS (7.5 mg) and further stirred at room temperature for 15 min. Afterward, MAL-PEG2000-NH2 (5 mg) was added to the mixture and stirred for 6 h at room temperature.16 The PEGylated CNP:PTX (PEG-CNP:PTX) was obtained after washing three times with DI water. To prepare RGD-grafted PEGCNP:PTX (RGD-PEG-CNP:PTX), cyclic RGD peptide (1 mg) was added to the suspension (PBS, pH 7.2) of PEGCNP:PTX and incubated for 2 h at room temperature.17 The as-obtained samples (Figure 1A) were collected and washed three times with DI water. Characterization of PTX-Loaded Nanoparticles. The coupling of PEG chains and the graft density of cyclic RGD peptide were tested by Fourier transform infrared (FTIR) spectroscopy and elemental analysis (CS-344 carbon/sulfur analyzer, LECO, St. Joseph, MI, USA), respectively. The average particle size, size polydispersity Index (PDI) and ζ potential of the samples (CNP:PTX, PEG-CNP:PTX and RGD-PEG-CNP:PTX) were determined by Zetasizer (Nanoseries, Malvern, U.K.). The surface morphology and internal structure of the RGD-PEG-CNP:PTX were observed by scanning electron microscope (SEM, JEOL, Japan) and transmission electron microscope (TEM, JEOL, Japan), respectively. The drug LE was determined in triplicate by HPLC with UV detection at 227 nm (LC-20AT, Shimadzu). The mobile phase consisted of acetonitrile−water (65:35, v/v) with a flow rate of

LE (%) =

weight amount of PTX in NP × 100% weight amount of PTX‐loaded NP (1)

To determine the in vitro drug release behavior, 10 mg of PTX-loaded nanoparticles (CNP:PTX, PEG-CNP:PTX and RGD-PEG-CNP:PTX) was dispersed in 10.0 mL of release medium (PBS and 0.1% Tween 80) and incubated at 37 °C under gentle shaking at 120 rpm. At determined time intervals, the buffer was refreshed with the same volume of release medium through centrifugation at 10000g for 10 min. 450 μL of supernatant mixed with 550 μL of acetonitrile was analyzed by HPLC as described above. The stability of PTX-loaded nanoparticles in PBS (pH 7.2) and DMEM was evaluated by its turbidity.18 The turbidity of sample was determined according to a method previously described. Briefly, the sample (100 μg/mL) was added into a quartz cell, and the corresponding absorbance was then measured at 620 nm with a spectrophotometer as a function of time. The turbidity was calculated from a standard absorbance−turbidity curve with formazin suspension as standards. Formazin was prepared by reacting hydrazine sulfate with hexamethylenetetrammonium, and standards of formazin turbidity units (FTU) were prepared by appropriate dilution. The corresponding images at 24 h were also collected. In Vitro Cellular Uptake of Nanoparticles. LLC and J774A.1 cells were both grown in DMEM supplemented with 10% (v/v) FBS, 100 U/mL of penicilin and 100 U/mL of streptomycin. The cells were incubated in a humidified incubator at 37 °C, 5% CO2. For the cellular uptake study, LLC and J774A.1 cells were seeded on two 24-well cell culture plates at the density of 1 × 105 cells per well and incubated at 37 °C for 24 h to allow cell attachment. The cells were then incubated with CNP, PEGCNP and RGD-PEG-CNP (100 μg/mL) at 37 °C for different time intervals. The cells were rinsed with PBS (pH 7.2) softly, predetached by 0.25% trypsin (for J774A.1 cell), resuspended and fixed in 3.7% paraformaldehyde. Based on the nanoparticle autofluorescence property, the cells were analyzed using a CyAn ADP 9 color flow cytometer (Beckman Coulter).19 Data were obtained from 15000 for each sample. Using the same method, LLC and J774A.1 cells were grown in a culture dish at a density of 1 × 106 cells per dish and incubated at 37 °C for 24 h. The cells were then incubated with CNP, PEG-CNP and RGD-PEG-CNP (500 μg/mL) at 37 °C for 24 h. After the cells were rinsed three times with PBS (pH 7.2) to remove the nanoparticles that were still suspended in culture medium, the total fluorescence images of nanoparticles internalized into cells were scanned using an in vivo imaging system (FX Pro, Kodak) with an excitation bandpass filter at 480 nm and an emission at 540 nm. In the confocal laser scanning microscopy (CLSM) imaging studies, LLC and J774A.1 cells were first seeded on Petri dishes for 24 h, and the medium was replaced with new medium containing 100 μg/mL of nanoparticles (CNP, PEG-CNP, RGD-PEG-CNP). After incubation for 24 h at 37 °C, the cells were washed with PBS (pH 7.2) and fixed with formalin for 30 min. The cell membrane and nuclei were stained with Alexa Fluor 635 phalloidin and DAPI, respectively. Nanoparticles were detected using their autofluorescence at 488 nm. The 1738

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fluoresence intensity was detected at 488 nm using an Infinite M200 microplate spectrophotometer (Tecan, Switzerland). The real-time fluoresence intensity of CNP, PEG-CNP and RGD-PEG-CNP in blood was calculated by subtracting the fluoresence intensity of pure blood. Tissue Distribution of PTX. The CNP:PTX, PEGCNP:PTX, RGD-PEG-CNP:PTX and Taxol (1 mg/kg of PTX in 100 μL of PBS, pH 7.2) were intravenously administered to LLC-bearing mice. After 48 h, the mouse tissues including heart, liver, spleen, lung, kidney and tumor were collected and ground by a mixer mill (RETSCH, MM400). The PTX was extracted with dichloromethane, purified with acetonitrile, and measured by HPLC. In Vivo NIR Fluorescence Imaging. To monitor the biodistribution of nanoparticles in vivo, DIR was loaded into CMC nanoparticles (CNP:DIR) according to the method described in the preparation of PTX-loaded nanoparticles (by replacing PTX with DIR). These nanoparticles were then coupled with PEG chain (PEG-CNP:DIR) and further grafted with cyclic RGD peptide (RGD-PEG-CNP:DIR). LLC-bearing mice were injected intravenously with 10 μg of samples in 100 μL of PBS (pH 7.2). At different time intervals, the mice were anesthetized and scanned using an in-vivo imaging system with an excitation bandpass filter at 750 nm and an emission filter at 790 nm. After in vivo imaging, animals were euthanized. Tumors and organs were excised and imaged. To monitor stealth effects of PEGylated nanoparticles in RES, LLC-bearing mice were injected twice a day with the CNP, PEG-CNP, RGD-PEG-CNP (10 μg in 100 μL of medium) and PBS (pH 7.2) through the tail vein, respectively. Two days later, the mice were sacrificed, and the tissues of liver and spleen were excised. Then, the tissues were fixed in formalin, embedded in paraffin, sliced, dewaxed and stained with DAPI. Finally, tissue slices were observed by CLSM. Nanoparticles and tissues were detected at 488 and 561 nm, respectively. The fluorescent images of cell nuclei, nanoparticles and tissues at 420−450 nm, 500−560 nm, and 570−600 nm were taken by CLSM. Therapeutic Study. In the subcutaneous lung cancer model, treatments were started when tumor volume was about 200 mm3 and the mice were randomly divided into five groups (n = 8). Mice of the five groups were intravenously administered PBS (100 μL, pH 7.2), Taxol, CNP:PTX, PEGCNP:PTX and RGD-PEG-CNP:PTX (1 mg/kg of PTX) once every day. Tumor sizes were measured with a digital caliper every day. Tumor volume was calculated by the formula (LW2)/2, where L is the long and W is the short tumor diameter (mm). Relative tumor volume (RTV) was calculated at each measurement time point (where RTV equals the tumor volume at a given time point divided by the tumor volume prior to initial treatment). To monitor potential toxicity, the body weight of each mouse was also measured every day. To evaluate allergic reaction of mice treated with different PTX formulations, tail vein blood of mice in different groups was collected and centrifuged after the last administration (n = 3). Serum samples were analyzed according to the procedure of mouse IgE ELISA (Immunology Consultants Laboratory, USA). After the last treatment, mouse blood was collected to test the white blood cell number (WBC) and platelets (PLT) using a hematology analyzer (MEK-7222K, Nihon Kohden Celltac E). For humane reasons, mice were sacrificed when the tumor volume was above 5000 mm3.

corresponding fluorescent images at 420−450 nm, 500−560 nm, and 650−700 nm were taken by CLSM (TCS SP5, Leica). In Vitro Cytotoxicity. To compare the cytotoxicity of blank nanoparticles on LLC cells with the commercial vehicle (Cremophor EL:ethanol) in Taxol formulation, MTT assay was employed. LLC cells were first seeded in a 96-well plate at a density of 5000 viable cells per well and cultured for 24 h at 37 °C. Then, the medium was replaced with serial dilutions of CNP, PEG-CNP, RGD-PEG-CNP (corresponding PTX concentration was 0−25 μg/mL) and commercial vehicle. After incubation for 48 h, MTT (0.5 mg/mL) solution was added to all wells, and incubated at 37 °C for 4 h, which was followed by the addition of 200 μL of isopropanol/DMSO (1:1) to dissolve the formazan crystals. Infinite M200 microplate spectrophotometer (Tecan, Männedorf, Switzerland) was used to measure the absorbance at 570 nm. Percent viability was normalized to cell viability in the absence of the samples. The cytotoxicity of free PTX, PTX-loaded nanoparticles and commercial Taxol was also evaluated on LLC cells. The cells were incubated with free PTX (PTX was dissolved in DMSO and then diluted by cells culture media, and the concentration of DMSO was less than 0.1%), CNP:PTX, PEG-CNP:PTX, RGD-PEG-CNP:PTX and Taxol (equivalent PTX concentration was 0−25 μg/mL) for 48 h at 37 °C. Cell viability was assessed by MTT assay. Expression of Cell-Suface Integrin. NIH 3T3 cells were grown in DMEM supplemented with 10% (v/v) NBS, 100 U/ mL of penicilin and 100 U/mL of streptomycin. The cells were incubated in a humidified incubator at 37 °C, 5% CO2. To determine β3 integrin expression by flow cytometry, LLC and NIH 3T3 cells were seeded on two 12-well cell culture plates at the density of 1 × 105 cells per well and incubated at 37 °C for 24 h. The cells were then harvested with PBS (pH 7.2). After centrifugation, 1.0 × 105 cells in 100 μL of PBS (pH 7.2) were mixed with monoclonal antibody (moAb, Alexa Fluor 488 anti-mouse/rat CD61 antibody, 10 μg/mL) solution (prechilled to 4 °C). The cells were incubated on ice for 30 min in the dark and subsequently washed twice with 800 μL of PBS (pH 7.2). Cell-surface fluorescence was measured using CyAn ADP 9 color flow cytometer. Data were obtained from 15000 for each sample. In the CLSM imaging studies, LLC and NIH 3T3 cells were first seeded on Petri dishes for 24 h, and the medium was replaced by new medium containing 1 μg/mL of the moAb solution. After incubation for 1 h at 4 °C, the cells were washed twice with PBS (pH 7.2) and fixed with formalin for 30 min. The cell membrane and cell nuclei were stained with Rhodamine-phalloidin and DAPI, respectively. Antibody and cell membrane were detected at 488 and 561 nm, respectively. The corresponding fluorescent images at 420−450 nm, 500− 560 nm, and 570−600 nm were taken by CLSM. Establishment of Tumor-Bearing Mouse Model. A subcutaneous syngeneic transplantable model of lung cancer was established by injecting 1 × 106 LLC cells in 100 μL of PBS (pH 7.2) subcutaneously at the right axillary fossa of male C57BL/6 mice. Blood Circulation of Different Nanoparticles after Intravenous Injection. The normal mice were injected with CNP, PEG-CNP and RGD-PEG-CNP through the tail vein. Then, 50 μL of the vein blood from mice was collected at different time intervals postinjection and immediately added into blood diluent with equal volume. Their corresponding 1739

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Figure 2. SEM (A) and TEM (B) images of RGD-PEG-CNP:PTX. PTX nanocrystals were successfully encapsulated into uniform-sized RGD-PEGCNP.

Figure 3. In vitro (A−C) and in vivo (D and E) evaluations of stealth effect of different nanoparticles. (A) Internalization kinetics (*P < 0.05) and (B) total fluorescence image of nanoparticles internalized into J774A.1 cells. (C) The corresponding CLSM images of J774A.1 cells treated with different nanoparticles. Scale bars: 10 μm. (D) Fluorescence intensity of nanoparticles in mouse blood. (E) Distribution of nanoparticles in the liver (upper panels) and spleen (lower panels) of C57BL/6 mice. Yellow dots represented fluorescent nanoparticles.

Neurotoxicity Test. Normal mice were first treated with different PTX formulations (1 mg/kg of PTX once every day). After 15 days of treatment, their motor coordinative abilities

were tested on an accelerating rotarod (TSE rotarod systems, Germany) starting at 4 rpm and accelerating to 40 rpm over a period of 300 s. The first test day was set as the 0 day. The 1740

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Figure 4. Internalization kinetics (A), total fluorescence images (B) and the corresponding CLSM images (C) of different nanoparticles internalized into LLC cells. Scale bar: 10 μm. *P < 0.05, **P < 0.01.

nanoparticles. To address this challenge, we coupled carboxymethyl with hydroxyl groups on sugar rings and synthesized CMC as nanoparticle matrix (Figures S1 and S2 in the Supporting Information). The subsequent functionalization was achieved via a bifunctional PEG chain, which was used as a spacer to connect cyclic RGD peptide and CNP (Figure 1B; Figure S3 and Table S1 in the Supporting Information). Based on the as-obtained results (Figure S4 and Table S1 in the Supporting Information), about 64% carboxyl groups were substituted by PEG chain, while 56% PEG chain was coupled to cyclic RGD peptide. The obtained RGD-PEG-CNP:PTX was systematically characterized (Figure 2; Figure S4, Figure S5 and Table S2 in the Supporting Information). By premix membrane emulsification technique, the product exhibited a small size (∼145 nm) with narrow size distribution (PDI < 0.1). The introduction of the PEG chain endowed our product with a good colloid stability. As expected, PTX nanocrystals distributed in the matrix network homogenously, resulting in a high LE (>30%). The couplings of both PEG chain and RGD peptide did not significantly influence the drug LE (fluctuation between 30% and 36%, Table S2 in the Supporting Information). Also, all these nanoparticle formulations especially PEGylated nanoparticles exhibited sustained and faster drug release behavior (Figure S6 in the Supporting Information), which would contribute to the enhancement of antitumor effects. Stealth Effects of PEGylated Nanoparticles in Vitro and in Vivo. Longer blood circulation time can offer nanocarriers more opportunities to reach the tumor site via the EPR effect.22 To ascertain whether these PEGylated nanoparticles (PEG-CNP and RGD-PEG-CNP) could possess stealth ability, murine macrophage cell line J774A.1 was selected for subsequent study. Compared with pristine

latency to fall from the rod was recorded in three trials per test day. The tests were carried out with 15 min rest between trials and repeated every 4 days. During the experiment time, the mice were still injected with different PTX formulations each day. Statistical Analysis. Statistical evaluations of data were performed by Student’s t test for two groups, and one way ANOVA for multiple groups. All results were expressed as mean ± standard error (SEM) unless otherwise noted. P < 0.05 was considered statistical significant.

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RESULTS AND DISCUSSION Physicochemical Characterization of PTX-Loaded Nanoparticles. Conventionally, hydrophobic materials are the preferred carrier matrix for water-insoluble drugs due to their similar soluble property, thus achieving a high LE.20 Unfortunately, poor drug dissolution rate usually occurred since these hydrophobic carriers are difficult to be wetted in vivo. To solve this problem, we attempted to use a hydrophilic material as the matrix. Considering the biocompatibility and biodegradability, chitosan, one of the most abundant polysaccharides in nature, was selected.21 However, multiple challenges still followed. First, it is difficult to encapsulate hydrophobic drug with hydrophilic matrix. Toward this, we combined an O/W/O double emulsion method with a temperature-programmed solidification technique. By this design, PTX might be, in principle, controlled within the matrix network as in situ nanocrystallites form, therefore facilitating the achievement of a very high LE. The second challenge we had to face was how to simultaneously functionalize this nanoparticle with PEG chain and RGD ligand, since the amino group in the chitosan chain had been already occupied during the preparation process of 1741

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Figure 5. The cytotoxicities of different PTX formulations without (A) or with (B) PTX against LLC cells. The corresponding PTX concentration in panel A refers the blank nanoparticles.

In Vitro Cytotoxicity of Different PTX Formulations. Considering the growing safety concerns about the in vivo application of nanocarriers, we first assessed the cytotoxicity of blank CNP, PEG-CNP and RGD-PEG-CNP using MTT assay. As shown in Figure 5A, our nanocarriers exhibited little cytotoxicity at the corresponding theoretical PTX concentration up to 25 μg/mL, implying their satisfactory biocompatibility. On the contrary, commercial vehicle used in Taxol showed severe cytotoxicity to LLC cells due to the introduction of Cremophor EL. The in vitro cytotoxicity of different PTX formualtions was subsequently performed on LLC cells (Figure 5B). All PTX formulations exhibited detectable cytotoxicity at the tested PTX concentrations. Notably, when the PTX concentration was above 0.5 μg/mL, viabilities of LLC cells treated with PEGCNP:PTX and RGD-PEG-CNP:PTX were rapidly decreased. However, this trend was not obvious in the group of CNP:PTX, which should be attributed to the poor internalization mentioned above. When PTX concentration was increased to 10 μg/mL, the cell viability of LLC cells treated with RGD-PEG-CNP:PTX reduced to about 10%, which was significantly lower than other groups (P < 0.05). The corresponding IC50 (concentration resulting in a 50% inhibition of cell growth) value for Taxol was 9.87 μg/mL, while that of RGD-PEG-CNP:PTX was 1.33 μg/mL. These results demonstrated the enhanced antitumor activity of RGD-PEGCNP:PTX. It should be also emphasized that, in the case of Taxol, the cytotoxic effect was mostly attributed to the excipient Cremophor EL, whereas PTX itself contributed a lot in the case of PTX-loaded nanoparticles (drug-free nanoparticles were non-cytotoxic). Biodistribution of Nanoparticles and Tissue Distribution of Drug. The above results gave us great hope and prompted us to evaluate the synergistic effect of PEG chain and cyclic RGD peptide in vivo. CNP, PEG-CNP, and RGD-PEGCNP were labeled by DIR and then injected intravenously into mice bearing LLC tumors and observed by in vivo NIR fluorescence imaging. As shown in Figure 6A, the pristine CNP (red fluorescence) mainly accumulated in the RES (liver and spleen) within 48 h due to its poor stealth property. Once the PEG chain was introduced, fluorescence signal in the RES significantly decreased, indicating that PEGylated nanoparticle could escape the entrapment of RES. Particularly, the fluorescence intensity at tumor increased markedly following the injection of RGD-PEG-CNP and was maintained up to 48

nanoparticle, a much slower cellular uptake rate was observed in the presence of PEG chain (Figure 3A). The internalization amount of these PEGylated nanoparticles was decreased by approximately 50% (P < 0.05) at 24 h, which was consistent with the subsequent fluorescence images (Figure 3B). CLSM images provide us a more vivid vision that fewer nanoparticles were internalized into J774A.1 cells after PEG conjunction (Figure 3C). The distinct results of in vitro phagocytic uptake between PEGylated and pristine nanoparticles indicated that they might exhibit great difference in in vivo pharmacokinetics. For verification, an additional animal experiment was performed by treating mice with different nanoparticles via the tail vein. As shown in Figure 3D, the half-life of CNP in blood was only 8 h, while that of PEGylated nanoparticle groups significantly increased (>30 h), indicating that PEGylation could greatly prolong the circulation time in the bloodstream. We proposed that hydrophilic PEG brushes sterically prevented the coating of opsonins onto the nanoparticles, thus reducing the nonspecific interaction of nanoparticles with RES cells. Such a pronounced fate was further demonstrated by histological sections. PEGylated CNP was sparsely observed in the liver and spleen, whereas most of CNP failed to escape from RES entrapment (Figure 3E). Improved Tumor Cell Uptake by Cyclic RGD Peptide. To achieve efficient accumulation in the lesion site, these nanoparticles should also possess high binding affinity toward tumor cells.23 We therefore evaluate the cyclic RGD effect on tumor cell uptake. As shown in Figure 4A,B, only a very small amount of CNP could be internalized into LLC cells due to the charge repulsion between CNP and the cell membrane, both negatively charged. Once PEGylated, these nanoparticles could be more extensively internalized since the PEGylation neutralized the negative charges of the CNP (P < 0.05). This behavior could be further improved by RGD modification on the PEG terminal. This is because the RGD could bind preferentially to the integrin that is highly expressed on the surface of LLC cells (Figure S7 in the Supporting Information).24 Similar results were obtained by CLSM imaging (Figure 4C). Fluorescent signal strongly presented in the group of RGD-PEG-CNP, but became weak in LLC cells after incubating with PEG-CNP, let alone the CNP. These results together reflected that the cellular uptake of RGD-PEGCNP was significantly promoted with the assistance of PEG chain and RGD peptides. 1742

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Figure 6. Biodistribution of different PTX-loaded nanoparticles and corresponding drug−tissue distribution. (A) In vivo images of real-time tumor targeting characteristics of CNP, PEG-CNP, and RGD-PEG-CNP. The images are merged images of NIRF and X-ray. (B) Representative ex vivo optical images of tumors and organs of LLC bearing mice sacrificed at 72 h: CNP (top), PEG-CNP (middle) and RGD-PEG-CNP (bottom), and the calibration bar was 600−6000 arbitrary units (a.u.). (C) Drug distribution in LLC-bearing mice after treatment with Taxol, CNP:PTX, PEGCNP:PTX and RGD-PEG-CNP:PTX for 48 h.

h, confirming the enhanced tumor targeting ability by further cyclic RGD peptide. To give more clear evidence of the stealth effect and tumor specificity, major organs (heart, liver, spleen, lung and kidney) and tumors were excised at 72 h postinjection, and similar results were observed (Figure 6B).

Only when cytotoxic anticancer drugs reach the tumor can they exert a therapeutic effect.25 We therefore evaluated the drug tissue distribution after treatment with different PTX formulations in a mouse xenograft model of LLC. As shown in Figure 6C, a high proportion of PTX accumulated into RES in the groups of Taxol or CNP:PTX. But this phenomenon did 1743

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Figure 7. Antitumor efficacies of different PTX formulations in LLC-bearing mice model. (A) Tumor volumes of mice during (I) and after (II) treatment; dashed line: stop administration. (B) Survival of mice. (C) Tumor images and the corresponding cell nuclear morphology. Scale bar: 10 μm.

detailed information about tumors and corresponding nuclear apoptosis in different groups was captured. Consistent with data of tumor growth inhibition, the RGD-PEG-CNP:PTX group induced the greatest cell apoptosis (nuclear condensation and fragmentation), and the tumor was also the smallest (Figure 7C).26 These results provide evidence that the combination of the stealth of PEG chain with the tumor celltargeting ability of cyclic RGD peptide endowed RGD-PEGCNP:PTX with significant improvement in antitumor therapeutic efficacy. Evaluation of the Side Effects of Different PTX Formulations. Although RGD-PEG-CNP:PTX had shown significant therapeutic effects compared with Taxol in vivo, the side effect level was still unclear. Considering that the treatment with Taxol can result in serious side effects including severe hypersensitivity reactions (usually type 1), hematological toxicity and neurotoxicity,27 we tested potential side effects of different PTX formulations in corresponding aspects. It has been demonstrated that IgE antibodies play an important role in mediating type-1 hypersensitivity responses. We thus selected IgE levels as the indicator for the evaluation of Taxol-associated hypersensitivity reactions (Figure 8A). The IgE level of mice treated with Taxol was significantly higher than that of the PBS group (P < 0.05), which should be ascribed to the introduction of Cremophor EL. However, little change in IgE level was found in the nanoparticle groups, suggesting that our nanoparticle formulations could reduce the

not arise when mice were treated with PEGylated nanoparticles, reconfirming the superior stealth property of PEGylated nanoparticles. As expected, no significant accumulation of PTX was observed in tumors after injection with Taxol and CNP:PTX because of their rapid metabolism. However, the presence of RGD and RGD-PEG-CNP:PTX showed 5.2 times and 2.0 times higher accumulation in tumor tissue compared with Taxol and PEG-CNP:PTX, respectively. These results again confirmed the success of our design. In Vivo Antitumor Efficacy. Having demonstrated the utility of PEG chain for prolonging circulation time in blood and the tumor targeting ability of cyclic RGD peptide, we moved on to explore the antitumor efficacy of different PTX formulations (Figure 7A). Although all the groups showed tumor inhibition compared with the PBS-treated group, the CNP:PTX exhibited only a slight antitumor effect. This efficacy was meliorated in PEG-CNP:PTX because of the prolonged circulation time. Once PTX was loaded into RGD-PEG-CNP, the tumor growth inhibition rate was the leader. Notably, a rapid tumor growth arose in the Taxol group once stopping treatment, whereas this phenomenon did not occur in the RGD-PEG-CNP:PTX group, indicating that nanoparticle-based formulation of PTX could accumulate at tumor site and achieve sustained drug release. Likewise, the median survival time of mice was significantly extended with RGD-PEG-CNP:PTX treatment in comparison with the Taxol group even though the treatment was stopped at the 15th day (Figure 7B). More 1744

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Figure 8. Side effects evaluation of different PTX formulations: (A) IgE levels in LLC-bearing mice; (B) hematological parameter change of normal mice; (C) sensorimotor coordination of normal mice was analyzed using the rotarod test; (D) body weight changes of LLC-bearing mice.

PTX could be well encapsulated into matrix network as the nanocrystallites form with a large amount. After intravenous injection, these PEGylated nanoparticles could display their superior stealth ability, therefore, resulting in a prolonged circulation time. When encountering tumor tissue, they could also extravasate via the leaky vessels by the EPR effect and achieve active tumor targeting through specific interaction between RGD and integrin receptor. Further studies on in vivo imaging and drug tissue distribution demonstrated the favorable synergistic effect of PEG chain and cyclic RGD peptide, which benefited the achievement of better antitumor efficacy, higher survival rate and less side effects compared with CNP:PTX, PEG-CNP:PTX and commercial Taxol. All these results together supported that the formulation developed in this work exhibited great potential as an effective tumor targeting delivery system for insoluble anticancer drugs. In the immunofluorescence study on histological section of tumor, we found that this system (RGD-PEG-CNP) could distribute not only in tumor parenchymal cells but also in the tumor blood vessels (Figure S10 in the Supporting Information), which might also contribute to the enhanced antitumor efficacy. In this situation, codelivery of antiangiogenesis drug (combretastatin A4 or TNP-470) and paclitaxel could, in principle, simultaneously kill the endothelial cells of newly formed blood vessels and parenchymal cells within tumor. Such a synergistic effect of this delivery system would better promote its antitumor performance.

risk of hypersensitivity reactions. The blood of mice was also collected to analyze the blood cell counts, which are often used as the indicator of hematotoxicity (Figure 8B).28 The total WBC/PLT counts of mice treated with Taxol showed an obvious decrease over the normal group (P < 0.05). Once PTX was encapsulated into nanoparticles, the WBC/PLT number fluctuated in an acceptable range, supporting that our nanoparticle formulations could protect blood cells from injury and thus avoid severe hematotoxicity. Neurotoxicity assessment was also performed by testing mouse motor performance.29 As shown in Figure 8C, little but acceptable decline of endurance time on the balance rotarod was observed in RGD-PEGCNP:PTX group. In contrast, other groups, especially Taxol, displayed significant decrease in endurance time because of their nonspecific distribution. Body weight change of mice was also recorded to comprehensively assess the side effects of PTX formulations. Evident fluctuation in body weight of mice treated with Taxol was found, whereas this phenomenon did not occur in other groups owing to their lower system toxicity (Figure 8D). This formulation with better therapeutic effect as well as lower side effects would greatly improve the patient’s quality of life.

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CONCLUSIONS To address the multiple challenges in high-performance delivery of insoluble anticancer agents, we have successfully prepared a novel nanocarrier RGD-PEG-CNP that synergistically holds many advantages. In terms of loading efficiency, 1745

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ASSOCIATED CONTENT

S Supporting Information *

Synthesis and characterization of CMC, preparation and physicochemical characterizations of CNP:PTX, PEGCNP:PTX, and RGD-PEG-CNP:PTX, and expression of cellsurface integrin. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Chinese Academy of Sciences, Institute of Process Engineering, National Key Laboratory of Biochemical Engineering, Beijing, 100190, PR China. Tel/fax: +86 10 82627072. E-mail: [email protected]; [email protected]. Author Contributions §

Both authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the 973 Program (2009CB930300), the National Nature Science Foundation of China (20820102036, 21161160555). The authors wish to thank Qiang Li and Juan Li in Institute of Process Engineering, Chinese Academy of Sciences, for their assistance in writing the manuscript.

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