Article pubs.acs.org/molecularpharmaceutics

Methotrexate-Loaded PEGylated Chitosan Nanoparticles: Synthesis, Characterization, and in Vitro and in Vivo Antitumoral Activity Juan Chen,† Liuqing Huang,† Huixian Lai,† Chenghao Lu,† Ming Fang,† Qiqing Zhang,‡ and Xuetao Luo*,† †

Department of Materials Science and Engineering, College of Materials, Xiamen University, Xiamen 361005, P. R. China Research Center of Biomedical Engineering, College of Materials, Xiamen University, Xiamen 361005, P. R. China

‡

ABSTRACT: Cancer nanotherapeutics are rapidly progressing and being implemented to solve several limitations of conventional drug delivery systems. In this paper, we report a novel strategy of preparing methotrexate (MTX) nanoparticles based on chitosan (CS) and methoxypoly(ethylene glycol) (mPEG) used as nanocarriers to enhance their targeting and prolong blood circulation. MTX and mPEG-conjugated CS nanoparticles (NPs) were prepared and evaluated for their targeting efficiency and toxicity in vitro and in vivo. The MTX−mPEG−CS NP size determined by dynamic light scattering was 213 ± 2.0 nm with a narrow particle size distribution, and its loading content (LC %) and encapsulation efficiency (EE) were 44.19 ± 0.64% and 87.65 ± 0.79%, respectively. In vitro release behavior of MTX was investigated. In vivo optical imaging in mice proved that MTX was released from particles subsequently and targeted to tumor tissue, showing significantly prolonged retention and specific selectivity. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay obviously indicated that the higher inhibition efficiency of MTX−mPEG−CS NPs meant that much more MTX was transferred into the tumor cells. A significant right-shift in the flow cytometry (FCM) assay demonstrated that MTX-loaded nanoparticles were far superior to a pure drug in the inhibition of growth and proliferation of Hela cells. These results suggest that MTX−mPEG−CS NPs could be a promising targeting anticancer chemotherapeutic agent, especially for cervical carcinoma. KEYWORDS: methotrexate, chitosan, mPEG, cellular uptake, targeted delivery

1. INTRODUCTION Methotrexate (MTX), a stoichiometric inhibitor of dihydrofolate reductase, is clinically used for the treatment of cancer, autoimmune diseases, and induction of abortion with misoprostol.1 However, its clinical efficacy is often compromised by undesirable side effects, due to its possessing low permeability (C logP = 0.53), poor aqueous solubility (0.01 mg/mL),2 short plasma t1/2 (2−10 h), and nonspecific drug delivery.3 Therefore, the main aim is to reduce its side effects and toxicity, promote cellular uptake, and improve tumor targeting. From a literature survey, considerable research efforts have been directed toward novel MTX delivery systems, such as nanoparticles,4−6 microspheres,7 prodrug and drug conjugates,8 liposomes,9 and miscellaneous multiparticulate systems.10 Among these, nanoparticles seem a promising tool for site specific delivery,3 based on the fact that they will release the active pharmacological moiety on tumor cells owing to the enhanced permeability and retention (EPR) effect11,12 and passive drug targeting. Whereas clinical applications of such formulations of MTX are still in infancy, the problems of sizedependent property, stability, and evaluation of tumor tissue distribution are the challenges remained to be resolved.3 The biodistribution of a given drug is a major factor for the success of chemotherapy.13 Much research3 was focused on the preparation and in vitro release behavior of MTX-loaded © XXXX American Chemical Society

nanoparticles, while few focused on performances in cellular uptake and in vivo antitumoral activity. In this paper, we focus on intracellular distributions and vivo antitumoral activity of the MTX-loaded nanosystem. Furthermore, recently polymeric micelles (PMs) have been proven effective for site-specific delivery of anticancer drugs to tumor and reduce its side effects. The inherent properties of PMs, such as size in the nanorange, stability in plasma, longevity in vivo, and pathological characteristics of tumor, allow them to target the tumor site by a passive mechanism. PMs formed from an amphiphilic block copolymer are suitable for encapsulation of poor watersoluble, hydrophobic anticancer drugs.14 In recent years, there is significant interest in the development of nanoparticles drug delivery approach,4,6,7,9 which has been studied and proved a challenging advantageous tool to minimize the adverse effects and maximize its therapeutic outcome with the specificity and selectivity. Polymer-based nanoparticles have received increasing attention13,15,16 for their Special Issue: Emerging Technology in Evaluation of Nanomedicine Received: May 5, 2013 Revised: October 1, 2013 Accepted: October 28, 2013

A

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respectively. All care and handling of animals were performed with the approval of Institutional Authority for Laboratory Animal Care. 2.3. Synthesis of CS NPs, mPEG−CS NPs, FA−CS NPs, and FA−mPEG−CS NPs. These NPs were synthesized using a method of ion-induced combined with chemical cross-linking. First, CS NPs were synthesized. Briefly, CS (125 mg) was dissolved in 100 mL of acetic acid (AC, 0.2 M, pH 4.9). Under intensive stirring, 18 mL of sodium triphosphate (STPP) solution (2 mg/mL) was slowly added to the CS solution. To this mixture, 10 mL of aqueous glutaraldehyde (GA, 25%, v/v) was added dropwise. After 12 h of reaction under stirring, they were isolated by centrifugation at 15 000 rpm for 20 min. The deposits were resuspended in 10 mL of water at room temperature, then 60 mg of sodium borohydride (NaBH4) was added to turn the CN bond of CS into C−N one. Subsequently, the NPs were dispersed in hydrochloric acid (HCl, 1 M) for 12 h and dialyzed against water overnight at room temperature to eliminate the nonreacted reactants. The solution was freeze-dried to obtain the resultant CS NPs. Last, the NPs were redispersed in phosphate buffer solution (PBS) before use. Methoxypolyethylene glycol propionic acid (mPEG−SPA, 50 mg) was added to CS NPs (5 mL, 10 mg/mL), and the reaction mixture was stirred for 4 h at room temperature to obtain mPEG−CS NPs. FA (1 mL, 5 mg/mL) was added to CS NPs and mPEG−CS NPs (5 mL, 10 mg/mL), respectively, in the presence of catalyst, 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC, 25 mg), and the stirring was continued for 1 h at room temperature to get yellow FA−CS NPs and FA− mPEG−CS NPs. The deposits were washed with water and centrifuged at 15 000 rpm for 20 min three times until there was no FA determined in the supernatant by UV spectroscopy at 303 nm using a UV-2500 spectrometer (Beckman, USA). 2.4. Synthesis of MTX−CS NPs and MTX−mPEG−CS NPs. A sample of 10 mg of MTX was dissolved in 2 mL of pH 7.4 PBS (0.067 M). Then, 0.5 mL of the solution (5 mg/mL) was added to CS NPs or mPEG−CS NPs (1 mL, 10 mg/mL), in the presence of catalyst, 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC, 25 mg), and the stirring was continued for 1 h at room temperature to get yellow MTX−CS NPs and MTX−mPEG−CS NPs. Subsequently, the suspension was isolated by centrifugation at 15 000 rpm for 20 min. The loading content (LC, weight percentage of drug in NPs) and encapsulation efficiency (EE) of MTX-loaded NPs were determined by the supernatant fluid. The absorbance of MTX was measured at 303 nm by UV−vis spectrophotometry (Beckman, USA). The mean values from replicates ± SD were obtained. LC of MTX−mPEG −CS NPs was determined using the following equation:

excellent biocompatibility. Furthermore, nanosized polymer therapeutic agents can circulate in the bloodstream for long periods of time, allowing them to reach the target site. In addition, chemical modification of polymer therapeutic agents with ligands capable of specifically binding receptors that are overexpressed in cancer cells can markedly augment therapeutic efficiency.13 Chitosan (CS) which is derived from natural sources has been considered as a safer anticancer nanocarrier since it is known as a biocompatible, biodegradable, and low toxic material with high cationic potential.17−19 The pKa of chitosan is 6.3−7 and only partly will be ionized in physiological pH. It is composed from glucosamine units with a free amino group on the second carbon. In physiological pH, it becomes a polyelectrolyte owing to the protonation of the −NH2 groups, modified with a secondary amine can enhance ionization. The equilibrium reaction described the state of ionization as follows:19 Chit‐NH 2 + H3O+ ↔ Chit‐NH3+ + H 2O

CS nanoparticles (NPs) have been investigated as a promising drug carrier for targeted delivery to specific sites and cancer therapy.16,17,20−22 The ionotropic gelation combined with chemical cross-linking technique is an effective way to obtain stability and size-controlled NPs.23 Poly(ethylene glycol), PEG, is a synthetic polymer. Thanks to several advantages of PEG−drug conjugates, such as prolonged residence in the body, decreased degradation by metabolic enzymes, or reduction or elimination of protein immunogenicity,24,25 PEG modifying polymer now plays a significant role in drug delivery.26−28 Furthermore, as the hydrophilic segment of polymeric nanoparticles, they are rarely recognized by the reticuloendothelial system (RES),29 allowing prolonged circulation in the bloodstream.13 Upon this sense, we report a novel strategy of preparation MTX nanoparticles based on CS and PEG used as nanocarries to enhance their targeting and prolong blood circulation. Moreover, the tumor distribution was investigated, and the targeting efficiency and toxicity were evaluated in vitro and in vivo. Then the results were compared with free drug and FA− mPEG NPs, given the circumstance that various literature reports30−36 that the folate receptor (FR) was universally considered as the receptor for mediating selective targeting of drugs to cancer cells. The folate receptor (FR), one of the most exploited targets, is a membrane glycoprotein of 38 kDa, which is present as three different isoforms: two GPI-anchored membrane proteins, FR-α, FR-β, and a soluble form called FRγ.37

2. EXPERIMENTAL DETAILS 2.1. Materials. Chitosan with molecular weight 150 000 (deacetylation degree of 95%) was purchased from Zhejiang Aoxing (China). Folic acid (FA) and MTX were obtained from BBI (USA). The 2000 Da succinimidyl ester of methoxypolyethylene glycol propionic acid (SPA-mPEG) was supplied by Jiaxing Biomatrik (China). DCl (D-form for NMR study) and deuterium oxide (D2O) were purchased from Aladdin (China). Other reagents were analytical grade and used without further purification. 2.2. Animals. Mice weighing 20 ± 2 g were obtained from Experimental Animal Center of Xiamen University, provided with standard food and water ad libitum. The temperature and relative humidity were maintained at 23 ± 2 °C and 55 ± 5%,

LC(wt %) =

weight of the drug in nanoparticles × 100 weight of the nanoparticles (1)

EE(wt %) =

weight of the drug in nanoparticles × 100 weight of the feeding drug (2)

2.5. Synthesis of Rhodamine B and Fluorescein Isothiocyanate (FITC) Labeled NPs. A sample of 5 mg of rhodamine B or FITC was dissolved in 1 mL of dimethyl sulfoxide (DMSO). Then, 0.2 mL of the solution was added to B

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NPs; FA−mPEG−CS NPs and MTX−mPEG−CS NPs) was added to the medium, respectively, followed by incubating for further 24 h. After that, the excess NPs were removed, and the wells were washed with ice-cold PBS. Subsequently, the cells were harvested by trypsinization. Finally, the cells were imaged with a laser scanning confocal microscope (LSCM, Leica, Germany). NPs and cell nuclei were expressed in green and blue, respectively. 2.9. In Vivo Optical Imaging of NPs in Animals. Animal procedures were in agreement with the guidelines of Institutional Animal Care Use Committee. Mouse hepatoma-22 cells were implanted subcutaneously into the right leg of four weeks old male mice. Then 0.2 mL (5 mg/mL) of rhodamine Blabeled of NPs (including CS NPs, FA−CS NPs, MTX−CS NPs, mPEG−CS NPs; FA−mPEG−CS NPs and MTX− mPEG−CS NPs) was injected into different mice through the tail vein, when tumors reached 0.4−0.6 cm in average diameter (7 days after implant). After 12 h, the animals were sacrificed, and then various organs and tumors were taken out. A fluorescence imaging study of various organs and tumors was performed immediately using Maestro EX (CRI, Maestro, USA). The intracellular distribution analysis of tumor sections was assessed by LSCM. 2.10. Cell Viability Assays. The cell viability of MTX encapsulated in NPs was tested using the MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in Hela cells. Cells were seeded into a 96-well cell culture plate at 5 × 104/well and then incubated for 24 h at 37 °C with 5% CO2. After that, they were exposed to free MTX solution and MTX−mPEG−CS NPs suspension at concentrations of 5, 10, and 20 μM. Moreover, blank NPs (NPs without drug) of comparable concentrations were tested in parallel. The following equation was used to calculate the viability of cell growth:

1 mL of 10 mg/mL of all NPs we had made above, respectively. Afterward, 1 mL of 2 M sodium carbonate/sodium bicarbonate (Na2CO3/NaHCO3) buffer was added to the suspension. After 12 h of reaction at 4 °C, the suspension was dialyzed against water overnight at room temperature to get rid of nonreacted reactants. 2.6. Nanoparticle Characterization. The size and zeta potential of NPs were determined using a Marvent Nanozs (Marvent, U.K.). For this end, NPs were diluted in distilled water to make a 1 mg/mL suspension. The morphologies of NPs were analyzed by scanning electron microscopy (SEM) with LEO-1530 (LEO, Germany) and transmission electron microscope (TEM) with JEM-2100 (JEOL, Japan). The TEM sample was prepared by placing a drop of the nanoparticle suspension onto a copper grid coated with carbon. After having been dried, the sample was observed directly. Fourier transform infrared spectroscopy (FTIR) spectra were measured using Nicolet AVATR 360 spectrometer (Nicolet, USA) from samples in KBr pellets. For preparing the samples, 1 mL (5 mg/mL) of different kinds of suspensions were centrifuged in 2 mL centrifuge tube. After that, the depositions were freeze-dried and mixed with 200 mg spectroscopy grade KBr. Then, the mill was used for powdering the mixture to give the KBr discs. 1 H nuclear magnetic resonance spectroscopy (NMR) spectra were carried out in 2% DCl/D2O (v/v) using AvanceIII 500 MHz NMR spectrometer (Bruker, Switzerland). The absolute molecular weight (MW) of the NPs were measured by a gel permeation chromatograph (GPC, Waters, USA) equipped with a multiangle laser light scattering detector (MALLS, Wyatt, USA). The samples were dissolved in 0.5 M sodium acetate buffer (pH 6.0). The mobile phase was 0.5 M sodium acetate buffer (pH 6.0), and the flow rate was 0.5 mL/ min. The injection volume was 0.2 mL (10 mg/mL). 2.7. In Vitro Release Study. In vitro release assessments from MTX-loaded mPEG−CS NPs were carried out for 144 h in PBS at pH 6.4, 7.4, and 8.4, respectively. An aliquot of NPs (5 mg/mL) was placed in dialysis bag (cutoff 3 kDa) and suspended in 100 mL of PBS at 37 °C, followed by gentle magnetic stirring (100 rpm). The suspension was put into the dialysis membranes with 3000 molecular weight cutoff. Periodically, 2 mL of medium was withdrawn and replaced with an equal volume of fresh medium. Subsequently, the sample was analyzed by UV-2550 spectrophotometry (Beckman, USA). The experimental procedure was performed in triplicate. The cumulative release percentage (CR %) of MTX at each time point was calculated according to the equation listed below: CR% =

Mt × 100 Mi

cell viability(%) =

mean of a value of treatment group mean of a value of control × 100

(4)

2.11. Cell Cycle Analysis by Flow Cytometry. Hela cells were cultured in 60 mm Petri dishes for 24 h at a density of 2 × 105 cells/mL at 37 °C with 5% CO2. Then they were treated with MTX−mPEG−CS NPs on cell cycle distribution in comparison with CS NPs, mPEG−CS NPs, and FA−mPEG− CS NPs. After 24 h, the cells were harvested by trypsinization, washed in cold PBS twice, fixed with ice-cold 70% ethanol, resuspended in 200 μL of DNA extraction buffer, and then incubated for 30 min at 25 °C. Finally, the samples were kept in dark conditions for 0.5 h and measured with the Beckman Coulter ADC Epics XL flow cytometer (Beckman, USA). 2.12. Statistical Analysis. All obtained data were expressed as th emean ± standard deviation. Statistics were performed with a one-way analysis of variance (ANOVA) followed by a Tukey test to determine the differences between the data using SPSS software (Chicago, USA). A value of p < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01).

(3)

where Mt is the amount of drug released at time t and Mi is the initial amount of drug loaded in the NPs. 2.8. In Vitro Confocal Imaging of Cellular Uptake. Human cervical carcinoma (Hela) cell lines, grown in modified Eagle’s medium (MEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C and 5% CO2, were supplied by Shanghai Institutes for Biological Sciences (SIBS). Cells (5 × 107/L) were plated on 14 mm glass coverslips and allowed to adhere for 12 h. Then 0.2 mL (1 mg/mL) of FITC-labeled of NPs (including CS NPs, FA−CS NPs, MTX−CS NPs, mPEG−CS

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterizations of NPs. Chitosan (CS) is widely used as a drug nanocarrier due to its structure characteristics of having reactive amino groups in the backbone, and in consequence it can chemically conjugate various molecules.17,19,21,38,39 CS NPs was achieved through a series C

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Scheme 1. Synthesis of CS NPs

Scheme 2. Synthesis of MTX−mPEG−CS NPs

of reactions (Scheme 1). According to a wealth of literature,40,41 FA and mPEG could be successfully conjugated to the surface of CS. The chemical scheme for conjugating MTX onto the surface of the mPEG−CS NPs through an amide bond is shown in Scheme 2. FTIR spectroscopy was used to confirm that MTX was successfully conjugated to the surface of NPs. FTIR spectra of CS NPs, mPEG, mPEG−CS NPs, MTX, and MTX−mPEG−CS NPs are shown in Figure 1. The CS spectrum (Figure 1A) presents characteristic IR absorption peaks at 3461 cm−1 assigned to stretching vibration of −NH2 and −OH groups, at 1601 cm−1 associated with N−H bending mode in the primary amine, at 1113 cm−1 related to the C−O stretching vibration. The characteristic peaks of the mPEG unit are shown in peaks at 2886 and 1739 cm−1 (Figure 1B). Moreover, for the free MTX (Figure 1D), the peaks at 1640 and 1601 cm−1 relate to carboxylate and amide CO.42

There are new peaks that appear in the MTX−mPEG−CS NPs (Figure 1E): 2886 cm−1 (typical signals of mPEG), 1606 cm−1 (typical signals of MTX), 3469 cm−1 (amide N−H stretch), 1658 cm−1 (amide I band), and 1630 cm−1 (amide II band). In addition, the 3469 cm−1 of the amide N−H stretch becomes wider. All of these indicated that mPEG and MTX were conjugated to CS NPs. The chemical structures of NPs were also confirmed by 1H NMR. Figure 2 exhibits the 1H NMR spectra of CS NPs, mPEG, mPEG−CS NPs, MTX, and MTX−mPEG−CS NPs, respectively. The 1H NMR spectrum of CS NPs is shown in Figure 2A. The signals were observed around 3.44−3.63 ppm due to the hydrogen atoms (H3−H6) in the chitosan ring.43 The peak of solvent (D2O) is at 4.6−4.7 ppm. In Figure 2C, a new peak at 3.6 ppm is attributed to mPEG (s, −CH−CH),44 suggesting that mPEG chains were successful introduced to the D

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negatively charged character of the cell plasma membrane.31 Also, the PEGylation reduced the zeta potential values, owing to the presence of PEG chains shielding the positive charges. In addition, the loading content (LC, weight percentage of drug in NPs) and encapsulation efficiency (EE) of MTX-loaded NPs were 44.19 ± 0.64% and 87.65 ± 0.79%, respectively. Rhodamine B and FITC were chemically linked to the NPs. Their LCs were 8.82 ± 0.34% and 8.16 ± 0.51%, respectively. The molecular weight increase of mPEG−CS NPs and MTX−mPEG−CS NPs was measured by GPC-MALLS, shown in Table 2. The results indicated that 99 mPEG and 1562 MTX were attached to the CS NPs, confirming that mPEG and MTX were successfully conjugated to CS NPs. SEM and TEM observations were performed to assess MTX−mPEG−CS NPs morphology. Figure 4A illustrates that the shape of NPs is spherical. The minority is homodispersed, while the majority aggregate together due to sticky and viscous of CS NPs suspension. The morphology of MTX−mPEG−CS NPs was shown in Figure 4B, which illustrates a spherical shape. 3.2. In Vitro Release Study. The cumulative amount of MTX released from the MTX−mPEG−CS NPs is illustrated in Figure 5, performed at pH 6.4, 7.4, and 8.4 PBS at 37 °C, respectively. The release of MTX from NPs displayed biphasic with an initial burst release of 1% after 1 h due to MTX absorbed on the surface of NPs, which might be attributed to that drug molecules were loosely incorporated into them, such as by electrostatic interaction between the ionized carboxyl groups of MTX and the positively charged amino groups of CS in the particle surface, while possessing a rather sustained release of 7% within 144 h as a result of amido linkage between TMX and CS. Moreover, a high value of pH may promote amide hydrolysis. The release of it demonstrates pH dependence. In addition, PEG plays a crucial role in drug release. Thanks to it possessing high chain mobility in an aqueous environment and having a large excluded volume, the PEG molecules in the outer layer can inhibit MTX release from of NPs.13 3.3. In Vitro Confocal Imaging of Cellular Uptake. To demonstrate the ability of MTX-loaded NPs to release endocytosed materials into the cell cytoplasm compared with FA-loaded NPs, the uptake of FITC-labeled NPs into Hela cells was examined by a laser scanning confocal microscope (Figure 6). MTX−mPEG−CS NPs (Figure 6F) show the highest intracellular FITC concentration than others NPs, visualized by a bright green punctuated distribution of FITC. According to the physicochemical characteristics of the nanocarrier and the nature of the target cells, two main internalization pathways may occur with phagocytosis and endocytic pathway. Physicochemical characteristics, such as surface charge, particle size, shape, and surface properties, played a crucial role in the cellular of NPs.31 Furthermore, in the absence of mPEG, the FITC concentration of MTX−CS NPs (Figure 6C) is also higher than that of FA−CS NPs (Figure 6B), while lower than that of MTX−mPEG−CS NPs (Figure 6F), suggesting that MTX−mPEG−CS NPs are more targeting and sustained. This is partly due to the reason that the PEG possesses long chains and a large volume that tended to incline drug degradation in vivo.31,46 More importantly, the chemical structure of MTX is similar to that of FA and FA has been widely used for the targeted delivery to folate receptor (RF) overexpressing cancer cell lines such as Hela. Folate-based targeting systems show great potential for future clinical diagnostic and therapeutic

Figure 1. FTIR spectra of CS NPs (A), mPEG (B), mPEG−CS NPs (C), MTX (D), and MTX−mPEG−CS NPs (E).

Figure 2. 1H NMR spectra of CS NPs (A); mPEG (B), mPEG−CS NPs (C), MTX (D), and MTX−mPEG−CS NPs (E) recorded in D2O/DCl at 500 MHz.

CS NPs. Figure 2D shows the specific peaks of MTX, 6.6−6.8 ppm owing to the benzoyl group and 7.5−8.0 ppm due to the 2,4-diamino-6-pteridinyl group, in agreement with the literature.45 Nevertheless, the new peaks appeared at 7.4, 7.8, and 8.5 ppm in Figure 2E, which presented the right shift of the intrinsic proton peak. Specific peaks of glucosamine ring (C1− C6) of CS were confirmed at 3.5−4.0 ppm, and their peaks overlapped with that of ethylene group of mPEG. Above results demonstrated that MTX was chemically combined with mPEG−CS NPs. As apparent from Figure 3 and Table 1, the mean size and size distribution of the different kinds of NPs revealed that it had a narrow size distribution with an average diameter about 210 nm, indicating that the size of NPs was considered to be determined by the backbone of chitosan. Furthermore, the particle size distribution was quite homogeneous, showing low polydispersity index (PDI) values. The surface charge of the different kinds of NPs was about 40 mV (Figure 3A), which displayed better association and internalization rates due to the E

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Figure 3. Zeta potential (A) and size distribution (B) of NPs.

Table 1. Average Size, PDI, and Zeta Potential of NPs (n = 3) sample name

size (nm)

PDI

zeta potential (mV)

CS NPs mPEG−CS NPs MTX−mPEG−CS NPs

208.8 ± 0.9 211.1 ± 1.5 213.4 ± 2.0

0.112 ± 0.003 0.109 ± 0.023 0.113 ± 0.035

43.3 ± 0.5 41.4 ± 0.8 43.6 ± 0.6

human tumors causes a wide range of tumor targets.37 Drug loaded NPs are anticipated to be hydrolyzed to MTX by appropriate aminopeptidases localized in the vicinity of the tumor.47 MTX was bound to the nanocarrier via an amide bond. The half-life of MTX-loaded NPs with an amide bond is stable in the presence of plasma aminopeptidases.48 Theoretically, cellular uptake is an important step for potentially improving drug delivery to tumor cells. The more drugs enter tumor tissue, the more tumor cells are killed. Therefore, the MTX−mPEG−CS NP is superior to the FA−mPEG−CS NP, a targeted carrier, in terms of cellular uptake. 3.4. In Vivo Optical Imaging of NPs in Animals. To further investigate the particle distribution in various organs and tumor tissue, rhodamine B fluorescence imaging study of various organs and tumor tissue was performed immediately with hepatoma-22 bearing mice using Maestro EX (CRI), after

Table 2. Relationship of the Molecular Weight of NPs sample name

Mn (× 106)

Mw (× 106)

DS

CS NPs mPEG−CS NPs MTX−mPEG−CS NPs

0.98 1.18 1.89

1.10 1.30 2.27

99a 1562b

Degree of substitution (DS) of mPEG = [(Mn of mPEG−CS NPs − Mn of CS NPs)]/2000, where 2000 refers to the molecular weight of mPEG. bDegree of substitution (DS) of MTX = [(Mn of MTX− mPEG−CS NPs − Mn of mPEG−CS NPs)]/454.44, where 454.44 refers to the molecular weight of MTX. a

applications. The receptor/ligand complex can be induced to internalize via endocytosis, which may facilitate the cytosolic delivery of therapeutic agents. The high receptor affinity and lack of normal tissue receptor expression create high tumoral specificity. Also, high frequency of overexpression among

Figure 4. Scanning electron microscopy (A) and transmission electron microscopy images (B) of MTX−mPEG−CS NPs. F

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(Figure 7). As reported, FA has been widely used as a targeting ligand for anticancer strategies.49 FA modified NPs can readily enter FR overexpressed tumor cells by endocytic mechanisms. Chitosan is promising as a novel macromolecule for specific liver targeting drug delivery system and the liver accumulation of drug polymer conjugated. The surface modification of nanoparticles with PEG, hydrophilic polymers, reduces the interfacial energy in an aqueous environment and prevents unwanted aggregation due to secondary interactions between nanoparticles. Furthermore, the surface decoration of nanoparticles with mPEG may minimize recognition by proteins and cells in the body, allowing the NPs to circulate in the blood for a longer period of time and increasing the possibility that it will reach the target site. After intravenous injection, NPs can encounter tumor tissue, extravasate via the leaky vessels by the EPR effect, and achieve active tumor targeting through specific interaction.13 PEG-based NPs are rarely recognized by the RES system.13,28,50 Therefore, the results suggest that PEGylated NPs can dramatically prolong the circulation half-life. What is more, MTX−mPEG−CS NPs possess superior targeted selectivity for tumor tissue. 3.5. Cell Viability Assays. The viability of Hela cells was estimated after being exposed to free MTX and MTX−mPEG− CS NPs suspensions at different concentrations for 24 h by an MTT assay in Figure 9. As apparent from the figure, within the investigated range of concentrations, the viability of the cells was significantly affected by MTX−mPEG−CS NPs accompanied with a concentration-dependent manner. The higher inhibition efficiency of MTX−mPEG−CS NPs obviously demonstrates that much more MTX was transferred into the tumor cells, which clearly proving that the activity of MTX− mPEG−CS NPs against the tumor cells was greater than that of the free drug. Free MTX is found to be pumped off the cell cytosol by P-glycoprotein, and the availability of free MTX at its intracellular site of action depends on a passive diffusion mechanism.5 In contrast, the markedly enhanced cytotoxicity of MTX−mPEG−CS NPs via the nanosized particles means that there was a significant reverse effect of drug resistance. NPs could reduce the multiple drug resistance that characterizes

Figure 5. In vitro cumulative release of MTX from NPs in PBS buffer at pH 6.4 (■), 7.4 (red ●), and 8.4 (blue ▲) (37 °C, n = 3).

that biodistribution analysis of tumor sections was assessed by LSCM. The results are shown in Figure 7 and Figure 8, respectively. As described in Figure 7, the strongest red fluorescence intensity of the NPs was mainly deposited in tumor tissue and next in the liver after blood circulated for 7 days. However, there is almost no red fluorescence intensity displayed in the lungs, heart, spleen, or kidney. These indicate that NPs were accumulated mainly in tumor, partly in liver, and little or nothing in other organs. Furthermore, in the presence of PEG, MTX-loaded NPs have a good selectivity in tumors. Figure 8 also demonstrates that all five kinds of NPs could enter tumor tissues. Moreover, a predominant amount of NPs distributes in euangiotic moieties. In addition, there is no significant different fluorescence intensity between FA−CS NPs and MTX−CS NPs. However, the fluorescence intensity of MTX−mPEG−CS NPs is notably stronger than that of FA− mPEG−CS NPs. This is consistent with the results of in vitro cellular uptake (Figure 6) and distribution in various organs

Figure 6. Laser scanning confocal images of Hela cells after incubated with fluorescently labeled NPs for 24 h, CS NPs (A), FA−CS NPs (B), MTX−CS NPs (C), mPEG−CS NPs (D), FA−mPEG−CS NPs (E), and MTX−mPEG−CS NPs (F). Nuclei stained with DAPI signal in blue; green fluorescence signal from NPs. G

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Figure 7. In vivo fluorescence images of dissected organs of mouse-bearing heptoma-22 sacrificed 12 h after intravenous injection of CS NPs (a), FA−CS NPs (b), MTX−CS NPs (c), mPEG−CS NPs (d), FA−mPEG−CS NPs (e), and MTX−mPEG−CS NPs (f); 1, tumor; 2, lung; 3, heart; 4, spleen; 5, kidney; 6, liver. All images were acquired under the same conditions (0.2 mL NPs per mouse).

Figure 8. LSCM images of intracellular distributions of dissected tumor tissue of mouse-bearing heptoma-22 sacrificed 12 h after intravenous injection of CS NPs (A), FA−CS NPs (B), MTX−CS NPs (C), mPEG−CS NPs (D), FA−mPEG−CS NPs (E), and MTX−mPEG−CS NPs (F). All images were acquired under the same conditions (0.2 mL NPs per mouse). Red fluorescence signal from NPs.

many anticancer drugs by a mechanism of cellular internalization of the drug by endocytosis.51 The result of MTT assay further confirms that MTX−mPEG−CS NPs possess a specific drug delivery. 3.6. Cell Cycle Analysis by Flow Cytometry. As shown in Figure 10, exposure of Hela cells to the same concentration of mPEG−CS NPs, FA−mPEG−CS NPs, and MTX−mPEG− CS NPs suspensions for 48 h produced a significant right-shift in the flow cytometry (FCM) analysis (Figure 10A), compared with CS NPs, the negative control. The values of geometrical mean (GMean) of intracellular fluorescence intensity of CS NPs, mPEG−CS NPs, FA−mPEG−CS NPs, and MTX− mPEG−CS NPs are 1.0 ± 0.02, 3.53 ± 0.02, 9.81 ± 0.04 and 16.68 ± 0.03, respectively (Figure 10B). It is evident that PEG plays a significant role in cellular uptake. We have observed that the designed formulation could increase the drug uptake into the target cells. With the introduction of hydrophilic polymers, PEG, the in vivo fate would be modulated owing to the stealth effect.13,26,28,35 PEGylation induced passive targeting by the EPR effect11,12 and PEG chains serve as a hydration shell on the surface of the nanoparticles to retard the release of hydrophobic

Figure 9. Viability of Hela cells treated with MTX (black ■) and MTX−mPEG−CS NPs (red ■) at various concentrations for 24 h (n = 3, **p < 0.01).

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Figure 10. Flow cytometry results of Hela cells incubated with CS NPs (a), mPEG−CS NPs (b), FA−mPEG−CS NPs (c), and MTX−mPEG−CS NPs (d) for 48 h. A: Profiles of fluorescence intensities. The red range was a negative control group. B: Relationship between NPs concentration and GMean of fluorescence intensities (n = 3, **p < 0.01).

drugs. As expected, fluorescence intensity in FA−mPEG−CS NPs treated cells is stronger than that in mPEG−CS NPs and CS NPs treated cells because there are overexpressed FR in Hela cells,52 which promotes the FA-mediated NPs selective targeting the cancer cells. Furthermore, the intracellular fluorescence intensity in the cells treated with MTX− mPEG−CS NPs is approximately twice as that of FA− mPEG−CS NPs, indicating that more amount of MTX loaded NPs was transferred into Hela cells. Being similar to the chemical structure of FA, MTX can also act as the receptor’s natural ligand. The result of FCM is in agreement with that of in vitro confocal imaging of cellular uptake, in vivo optical imaging of NPs in animals, and MTT assay.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the key jointed Foundation of the National Science Foundation of China-Yunnan (no. U1137601). We thank Prof. Hu Chen (Medical College of Xiamen University) for his skillful technical help in the animal experiments.

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ABBREVATIONS MTX, methotrexate; CS, chitosan; PEG, poly(ethylene glycol); STPP, sodium triphosphate; mPEG, methoxypoly(ethylene glycol); SPA-mPEG, methoxypoly(ethylene glycol) propionic acid; PBS, phosphate buffer solution; HCl, hydrochloric acid; GA, glutaraldehyde; NaBH4, sodium borohydride; FA, folic acid; RF, folate receptor; EDC, 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride; NPs, chitosan nanoparticles; LSCM, laser scanning confocal microscope; FITC, fluorescein isothiocyanate; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; FCM, flow cytometry

4. CONCLUSION In summary, we have demonstrated a new efficient preparation procedure for MTX-loaded mPEG−CS NPs with a suitable nanosize as an intracellular delivery system for improving tumor cells selectivity, especially for the FR-overexpressing tumor. Furthermore, the MTX−mPEG−CS NPs showed high entrapment efficiency and enhanced cytotoxicity on HeLa in comparison to free MTX. In vitro and in vivo tumor cellular uptake and biodistribution studies illustrated that MTX− mPEG−CS NPs could availably enter into tumor tissues, which were superior to FA, a targeting ligand for nanocarriers. Based on the overall results, a conclusion may be reached is that MTX−mPEG−CS NPs could be a promising targeting anticancer chemotherapeutic agent.

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REFERENCES

(1) Yoon, S.; Choi, J. R.; Kim, J.-O.; Shin, J.-Y.; Zhang, X.; Kang, J.-H. Influence of reduced folate carrier and dihydrofolate reductase genes on methotrexate-induced cytotoxicity. Cancer Res. Treat. 2010, 42, 163−171. (2) Kasim, N. A.; Whitehouse, M.; Ramachandran, C.; Bermejo, M.; Lennernäs, H.; Hussain, A. S.; Junginger, H. E.; Stavchansky, S. A.; Midha, K. K.; Shah, V. P. Molecular properties of WHO essential drugs and provisional biopharmaceutical classification. Mol. Pharmaceutics 2004, 1, 85−96. (3) Khan, Z. A.; Tripathi, R.; Mishra, B. Methotrexate: a detailed review on drug delivery and clinical aspects. Expert Opin. Drug Delivery 2012, 9, 151−169.

AUTHOR INFORMATION

Corresponding Author

*Department of Materials Science and Engineering, College of Materials, Xiamen University, No. 422, South-Siming Road, Fujian, Xiamen 361005, P. R. China. Tel.: +86-592-2184881. Fax: +86-592-2188503. E-mail: [email protected]. I

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(4) Chen, Y. H.; Tsai, C. Y.; Huang, P. Y.; Chang, M. Y.; Cheng, P. C.; Chou, C. H.; Chen, D. H.; Wang, C. R.; Shiau, A. L.; Wu, C. L. Methotrexate conjugated to gold nanoparticles inhibits tumor growth in a syngeneic lung tumor model. Mol. Pharmaceutics 2007, 4, 713− 722. (5) Nogueira, D. R.; Tavano, L.; Mitjans, M.; Perez, L.; Infante, M. R.; Vinardell, M. P. In vitro antitumor activity of methotrexate via pHsensitive chitosan nanoparticles. Biomaterials 2013, 34, 2758−2772. (6) Thomas, T. P.; Huang, B. H.; Choi, S. K.; Silpe, J. E.; Kotlyar, A.; Desai, A. M.; Zong, H.; Gam, J.; Joice, M.; Baker, J. R. Polyvalent Dendrimer-Methotrexate as a Folate Receptor-Targeted Cancer Therapeutic. Mol. Pharmaceutics 2012, 9, 2669−2676. (7) Liang, L. S.; Jackson, J.; Min, W. X.; Risovic, V.; Wasan, K. M.; Burt, H. M. Methotrexate loaded poly(L-lactic acid) microspheres for intra-articular delivery of methotrexate to the joint. J. Pharm. Sci. 2004, 93, 943−956. (8) Smal, M. A.; Dong, Z.; Cheung, H.; Asano, Y.; Escoffier, L.; Costello, M.; Tattersall, M. H. Activation and cytotoxicity of 2-αaminoacyl prodrugs of methotrexate. Biochem. Pharmacol. 1995, 49, 567−574. (9) Kuznetsova, N. R.; Sevrin, C.; Lespineux, D.; Bovin, N. V.; Vodovozova, E. L.; Meszaros, T.; Szebeni, J.; Grandfils, C. Hemocompatibility of liposomes loaded with lipophilic prodrugs of methotrexate and melphalan in the lipid bilayer. J. Controlled Release 2012, 160 (2), 394−400. (10) Kang, H.; Kim, J.-D.; Han, S.-H.; Chang, I.-S. Self-aggregates of poly (2-hydroxyethyl aspartamide) copolymers loaded with methotrexate by physical and chemical entrapments. J. Controlled Release 2002, 81, 135−144. (11) Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J. Controlled Release 2000, 65, 271−284. (12) Matsumura, Y.; Maeda, H. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res. 1986, 46, 6387−6392. (13) Park, J. H.; Lee, S.; Kim, J.-H.; Park, K.; Kim, K.; Kwon, I. C. Polymeric nanomedicine for cancer therapy. Prog. Polym. Sci. 2008, 33, 113−137. (14) Kedar, U.; Phutane, P.; Shidhaye, S.; Kadam, V. Advances in polymeric micelles for drug delivery and tumor targeting. Nanomed.: Nanotechnol. Biol. Med. 2010, 6 (6), 714−729. (15) Canal, F.; Sanchis, J.; Vicent, M. J. Polymer-drug conjugates as nano-sized medicines. Curr. Opin. Biotechnol. 2011, 22, 894−900. (16) Dash, T. K.; Konkimalla, V. B. Polymeric Modification and Its Implication in Drug Delivery: Poly-epsilon-caprolactone (PCL) as a Model Polymer. Mol. Pharmaceutics 2012, 9, 2365−2379. (17) Agnihotri, S. A.; Mallikarjuna, N. N.; Aminabhavi, T. M. Recent advances on chitosan-based micro- and nanoparticles in drug delivery. J. Controlled Release 2004, 100, 5−28. (18) Chua, B. Y.; Al Kobaisi, M.; Zeng, W. G.; Mainwaring, D.; Jackson, D. C. Chitosan Microparticles and Nanoparticles as Biocompatible Delivery Vehicles for Peptide and Protein-Based Immunocontraceptive Vaccines. Mol. Pharmaceutics 2012, 9, 81−90. (19) Lu, B.; Wang, C. F.; Wu, D. Q.; Li, C.; Zhang, X. Z.; Zhuo, R. X. Chitosan based oligoamine polymers: synthesis, characterization, and gene delivery. J. Controlled Release 2009, 137, 54−62. (20) Rinaudo, M.; Pavlov, G.; Desbrieres, J. Influence of acetic acid concentration on the solubilization of chitosan. Polymer 1999, 40 (25), 7029−7032. (21) Ravi Kumar, M. N. V. A review of chitin and chitosan applications. React. Funct. Polym. 2000, 46, 1−27. (22) Vinsova, J.; Vavrikova, E. Recent advances in drugs and prodrugs design of chitosan. Curr. Pharm. Des. 2008, 14, 1311−1326. (23) Gan, Q.; Wang, T.; Cochrane, C.; McCarron, P. Modulation of surface charge, particle size and morphological properties of chitosan− TPP nanoparticles intended for gene delivery. Colloids Surf., B 2005, 44, 65−73.

(24) Pasut, G.; Veronese, F. M. Polymer-drug conjugation, recent achievements and general strategies. Prog. Polym. Sci. 2007, 32, 933− 961. (25) Li, C.; Wallace, S. Polymer-drug conjugates: Recent development in clinical oncology. Adv. Drug Delivery Rev. 2008, 60, 886−898. (26) Hong, M.; Zhu, S.; Jiang, Y.; Tang, G.; Sun, C.; Fang, C.; Shi, B.; Pei, Y. Novel anti-tumor strategy: PEG-hydroxycamptothecin conjugate loaded transferrin-PEG-nanoparticles. J. Controlled Release 2010, 141, 22−9. (27) Li, W. J.; Zhan, P.; De Clercq, E.; Lou, H. X.; Liu, X. Y. Current drug research on PEGylation with small molecular agents. Prog. Polym. Sci. 2013, 38, 421−444. (28) Otsuka, H.; Nagasaki, Y.; Kataoka, K. PEGylated nanoparticles for biological and pharmaceutical applications. Adv. Drug Delivery Rev. 2003, 55, 403−419. (29) Frank, M. M.; Hamburger, M. I.; Lawley, T. J.; Kimberly, R. P.; Plotz, P. H. Defective reticuloendothelial system Fc-receptor function in systemic lupus erythematosus. New J. Med. 1979, 300, 518. (30) Elnakat, H.; Ratnam, M. Distribution, functionality and gene regulation of folate receptor isoforms: implications in targeted therapy. Adv. Drug Delivery Rev. 2004, 56, 1067−1084. (31) Hillaireau, H.; Couvreur, P. Nanocarriers’ entry into the cell: relevance to drug delivery. Cell. Mol. Life Sci. 2009, 66, 2873−96. (32) Low, P. S.; Henne, W. A.; Doorneweerd, D. D. Discovery and development of folic-acid-based receptor targeting for imaging and therapy of cancer and inflammatory diseases. Acc. Chem. Res. 2008, 41, 120−129. (33) Roger, E.; Kalscheuer, S.; Kirtane, A.; Guru, B. R.; Grill, A. E.; Whittum-Hudson, J.; Panyam, J. Folic Acid Functionalized Nanoparticles for Enhanced Oral Drug Delivery. Mol. Pharmaceutics 2012, 9, 2103−2110. (34) Spiegelstein, O.; Eudy, J. D.; Finnell, R. H. Identification of two putative novel folate receptor genes in humans and mouse. Gene 2000, 258, 117−125. (35) Canal, F.; Vicent, M. J.; Pasut, G.; Schiavon, O. Relevance of folic acid/polymer ratio in targeted PEG-epirubicin conjugates. J. Controlled Release 2010, 146, 388−399. (36) Vandana, M.; Sahoo, S. K. Reduced Folate Carrier Independent Internalization of PEGylated Pemetrexed: A Potential Nanomedicinal Approach for Breast Cancer Therapy. Mol. Pharmaceutics 2012, 9, 2828−2843. (37) Sudimack, J.; Lee, R. J. Targeted drug delivery via the folate receptor. Adv. Drug Delivery Rev. 2000, 41 (2), 147−162. (38) Amoozgar, Z.; Park, J. Y.; Lin, Q. N.; Yeo, Y. Low MolecularWeight Chitosan as a pH-Sensitive Stealth Coating for Tumor-Specific Drug Delivery. Mol. Pharmaceutics. 2012, 9, 1262−1270. (39) Lu, B.; Wang, C. F.; Wu, D. Q.; Li, C.; Zhang, X. Z.; Zhuo, R. X. Chitosan based oligoamine polymers: Synthesis, characterization, and gene delivery. J. Controlled Release 2009, 137, 54−62. (40) Braumuller, H.; Wieder, T.; Brenner, E.; Assmann, S.; Hahn, M.; Alkhaled, M.; Schilbach, K.; Essmann, F.; Kneilling, M.; Griessinger, C.; Ranta, F.; Ullrich, S.; Mocikat, R.; Braungart, K.; Mehra, T.; Fehrenbacher, B.; Berdel, J.; Niessner, H.; Meier, F.; van den Broek, M.; Haring, H. U.; Handgretinger, R.; Quintanilla-Martinez, L.; Fend, F.; Pesic, M.; Bauer, J.; Zender, L.; Schaller, M.; Schulze-Osthoff, K.; Rocken, M. T-helper-1-cell cytokines drive cancer into senescence. Nature 2013, 494, 361−365. (41) E-Sherbiny, I. M.; Smyth, H. D. C. Controlled release pulmonary administration of curcumin using swellable biocompatible microparticles. Mol. Pharmaceutics 2012, 9, 269−280. (42) Dhanikula, R. S.; Hildgen, P. Influence of molecular architecture of polyether-co-polyester dendrimers on the encapsulation and release of methotrexate. Biomaterials 2007, 28, 3140−3152. (43) Fernandez-Megia, E.; Novoa-Carballal, R.; Quinoa, E.; Riguera, R. Optimal routine conditions for the determination of the degree of acetylation of chitosan by H-1-NMR. Carbohydr. Polym. 2005, 61 (2), 155−161. J

dx.doi.org/10.1021/mp400269z | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

Article

(44) Sagara, K.; Kim, S. W. A new synthesis of galactosepoly(ethylene glycol)-polyethylenimine for gene delivery to hepatocytes. J. Controlled Release 2002, 79 (1−3), 271−281. (45) Li, Y.; Kwon, G. S. Micelle-like structures of poly(ethylene oxide)-block-poly(2-hydroxyetbyl aspartamide)-methotrexate conjugates. Colloids Surf., B 1999, 16 (1−4), 217−226. (46) Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.; Torchilin, V.; Langer, R. Biodegradable long-circulating polymeric nanospheres. Science 1994, 263 (5153), 1600−1603. (47) Šubr, V.; Strohalm, J.; Hirano, T.; Ito, Y.; Ulbrich, K. Poly[N-(2hydroxypropyl)methacrylamide] conjugates of methotrexate: Synthesis and in vitro drug release. J. Controlled Release 1997, 49 (2−3), 123−132. (48) Smal, M. A.; Dong, Z.; Cheung, H. T. A.; Asano, Y.; Escoffier, L.; Costello, M.; Tattersall, M. H. N. Activation and cytotoxicity of 2α-aminoacyl prodrugs of methotrexate. Biochem. Pharmacol. 1995, 49 (4), 567−574. (49) Shoji-Kawata, S.; Sumpter, R.; Leveno, M.; Campbell, G. R.; Zou, Z. J.; Kinch, L.; Wilkins, A. D.; Sun, Q. H.; Pallauf, K.; MacDuff, D.; Huerta, C.; Virgin, H. W.; Helms, J. B.; Eerland, R.; Tooze, S. A.; Xavier, R.; Lenschow, D. J.; Yamamoto, A.; King, D.; Lichtarge, O.; Grishin, N. V.; Spector, S. A.; Kaloyanova, D. V.; Levine, B. Identification of a candidate therapeutic autophagy-inducing peptide. Nature 2013, 494, 201−206. (50) Hu, F. Q.; Zhang, Y. Y.; You, J.; Yuan, H.; Du, Y. Z. pH triggered doxorubicin delivery of PEGylated glycolipid conjugate micelles for tumor targeting therapy. Mol. Pharmaceutics 2012, 9, 2469−2478. (51) Moysan, E.; Bastiat, G.; Benoit, J. P. Gemcitabine versus Modified Gemcitabine: A Review of Several Promising Chemical Modifications. Mol. Pharmacol. 2013, 10, 430−444. (52) Lu, Y.; Low, P. S. Folate-mediated delivery of macromolecular anticancer therapeutic agents. Adv. Drug Delivery Rev. 2002, 54, 675− 693.

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