Article pubs.acs.org/Biomac
Porous Quaternized Chitosan Nanoparticles Containing Paclitaxel Nanocrystals Improved Therapeutic Efficacy in Non-Small-Cell Lung Cancer after Oral Administration Pi-Ping Lv,†,‡,§ Wei Wei,†,§ Hua Yue,†,‡ Ting-Yuan Yang,† Lian-Yan Wang,† and Guang-Hui Ma*,† †
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, P. R. China. ‡ Graduate University of Chinese Academy of Sciences, Beijing, 100049, P. R. China S Supporting Information *
ABSTRACT: Clinical application of paclitaxel (PTX) is limited because of its poor solubility in aqueous media. To overcome this hurdle, we devised an oral delivery system by encapsulating PTX into N-((2-hydroxy-3-trimethylammonium) propyl) chitosan chloride (HTCC) nanoparticles. These nanoparticles were small (∼130 nm), had a narrow size distribution, and displayed high loading efficiency owing to the homogeneous distribution of PTX nanocrystals. The matrix hydrophilicity and porous structure of the obtained nanoparticles accelerated their degradation and improved drug release. In vitro and in vivo transport experiments had proved that the presence of positive charges enhanced the intestinal permeability of these nanoparticles. Further in vitro experiment of cytotoxicity showed that the PTX-loaded HTCC nanoparticle (HTCC-NP:PTX) was more effective than native PTX owing to enhanced cellular uptake. Drug distribution in tissues and in vivo imaging studies confirmed the preferred accumulation of HTCC-NP:PTX in subcutaneous tumor tissue. Subsequent tumor xenograft assays demonstrated the promising therapeutic effect of HTCC-NP:PTX on inhibition of tumor growth and induction of apoptosis in tumor cells. Additional investigation into side effects revealed that HTCC-NP:PTX caused lower Cremophor ELassociated toxicities compared with Taxol. These results strongly supported the notion that HTCC nanoparticle (HTCC-NP) is a promising candidate as an oral carrier of PTX for cancer therapy.
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Progress in the therapy of cancer has also received gifts from the development of nanotechnology and polymeric nanoparticles have been increasingly used for their potential efficacy. This is because these nanoparticles can escape from the vasculature through the leaky endothelial tissue surrounding the tumor and then accumulate in tumor tissue by the so-called enhanced permeation and retention (EPR) effect.6 Moreover, the encapsulated anticancer drugs can be released in a sustained manner at the lesion site. Additionally, the administration route can, in principle, be expanded to oral delivery with the assistant of nanoparticles, which can greatly improve patient compliance in comparison with conventional intravenous dosage forms.7 Although promising, research into the development of oral delivery system using nanoparticles for insoluble anticancer drugs is still in its infancy. The main problems that have precluded their access to practical applications are the nonuniform particle size, low capacity for drug loading, poor bioavailability, and especially slow rate of drug dissolution.8
INTRODUCTION
Lung cancer is the leading cause of cancer-related morbidity and mortality, resulting in >1.1 million deaths per year worldwide.1 About 80% lung cancer cases are non-small-cell lung cancer (NSCLC), and 65% of them have an advancedstage disease at diagnosis.2 In this situation, chemotherapy is still the mainstay treatment. Paclitaxel (PTX), a microtubule stabilizer that causes mitotic arrest, is a promising anticancer drug used for the treatment of NSCLC.3 Unfortunately, its poor solubility in water drastically limits its use in the natural form. To solve this problem, PTX has to be formulated (Taxol) at 6 mg/mL in a vehicle composed of 1:1 blend of Cremophor EL and ethanol, which can be administered by intravenous injection.4 However, this administration method can cause great distress to patients and is inconvenient for them. Because of the relatively large amount of Cremophor EL used and the nonspecific biodistribution of the drug in tumors and normal tissues, Taxol has also been associated with serious side effects, including severe hypersensitivity reactions, myelosuppression, and neurotoxicity. 5 Developing a convenient and safe delivery system for PTX to maximize the therapeutic efficacy at tumor sites while minimizing the side effects is therefore a challenging endeavor. © 2011 American Chemical Society
Received: August 2, 2011 Revised: September 25, 2011 Published: November 1, 2011 4230
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Sakamoto Yakuhin Kogyo (Japan). All other reagents used were analytical grade. SPG membrane was bought from SPG Technology (Japan). Dulbecco’s modified Eagle’s medium (DMEM), Hank’s buffered salt solution (HBSS), and fetal bovine serum (FBS) were obtained from Gibco. Oil-soluble cyanine 7 (Cy7, near-infrared fluorescence (NIRF) dye), Alexa Fluor 635 phalloidin, DAPI (4, 6diamidino-2-phenylindole), and nonessential amino acids were purchased from Invitrogen. Mouse IgE ELISA was purchased from Immunology Consultants Laboratory (USA). The human colon adenocarcinoma cell line (Caco-2) and LLC cells were obtained from the Peaking University Health Science Center (China) and cultured in DMEM containing 10% (v/v) FBS, 100 IU/ mL of penicillin G sodium, and 100 μg/mL of streptomycin sulfate. The culture medium of Caco-2 cells also contained 1% (v/v) nonessential amino acid. Both cells were maintained in an incubator supplied with 5% CO2/95% air humidified atmosphere at 37 °C. Male C57BL/6 mice, 6−8 weeks age, were purchased from the Department of Laboratory Animal Science, Peking University Health Science Center (China). All animals were caged according to principles established for the care and use of laboratory animals. Preparation of PTX-Loaded CS and HTCC Nanoparticles. SPG membrane emulsification technique and cross-linking method were used to prepare CS nanoparticle (CS-NP) and HTCC-NP, as previously described.15 O/W/O double emulsion together with temperature-programmed solidification methods were used to prepare PTX-loaded CS nanoparticle (CS-NP:PTX) and HTCC-NP:PTX. PTX was dissolved in dichloromethane to obtain oil phase I (OI). CS (0.5 wt %) or a mixture (0.5 wt %) of CS and HTCC (1:1) containing 2 wt % emulsifier were dissolved in acetic acid buffer solution (pH 3.5), which was used as the water phase (W). The oil phase II (O II) was a mixture of liquid paraffin and petroleum ether 1:2 (v/v) containing 4 wt % PO-500 as emulsifier. The volume ratio of O I, W, and OII was fixed at 1:5:150. The OI and W were first mixed through ultrasonic emulsification to obtain primary emulsion (OI/W), which was further added to OII to obtain coarse double emulsion (OI/W/ OII). Uniform-sized nanodroplets were achieved by extruding the coarse emulsion through the membrane with quite high pressure. Subsequently, glutaraldehyde saturated toluene (GST) was slowly dropped into the emulsion to solidify nanodroplets into nanoparticles. Solidification of PTX-loaded nanoparticle was performed by a twostep procedure to control PTX nanocrystals within the matrix network. The obtained emulsion droplets were first maintained 25 °C for 30 min to allow primary solidification of HTCC matrix, entrapping the O I droplets in local position. The solidification temperature was then slowly increased to (2 °C/min) and kept at 50 °C for 10 h to solidify further the HTCC nanoparticles. Finally, the PTX-loaded nanoparticles were collected and washed three times by repeated centrifugation and redispersion with petroleum ether and deionized water, respectively. At last, PTX-loaded nanoparticles were obtained. The stability of primary emulsion was indicated by its turbidity. The turbidity of primary emulsion was determined according to a method previously described.16 The effects of emulsifiers including Triton X405, Tween-60, Brij35, and Tween-20 on the stability of primary emulsion were evaluated. In brief, the primary emulsions were prepared by different emulsifiers and added to a quartz cell. The absorbance was measured at 620 nm with spectrophotometer as a function of time. The turbidity was calculated from standard absorbance−turbidity curve with formazin suspension. Formazin was prepared by reacting hydrazine sulfate with hexamethylenetetrammonium, and standards of formazin turbidity units (FTUs) were prepared by appropriate dilution. Characterization of CS-NP:PTX and HTCC-NP:PTX. The size, size distribution, and zeta potential of CS-NP:PTX and HTCCNP:PTX were analyzed by zeta potential measurement with submicrometer particle size analyzer (Brookhaven Instruments Corporation). The pore sizes of CS-NP and HTCC-NP dried by Critical Point Drying (Emitech, East Sussex, U.K.) were measured by Surface Area Analyzer (Micromeritics, Norcross, Georgia, U.S.). The surface morphology and internal structure of CS-NP:PTX and HTCCNP:PTX were observed by scanning electron microscope (SEM) and
The first challenge in the design of carriers for oral delivery of insoluble anticancer drugs is that the matrix materials should require superior properties. Hydrophobic material such as poly(lactic acid) (PLA) is widely used as the matrix of nanocarriers for water-insoluble drug to obtain high encapsulation efficiency (EE) and high loading efficiency (LE).9 However, poor drug dissolution rate and rapid clearance of these nanocarriers by reticuloendothelial system (RES) usually occurred because of their hydrophobic nature. These two hurdles can be overcome by using hydrophilic matrix, but enhancing EE and LE of hydrophobic drugs again becomes ac critical issue. The naturally biodegradable polysaccharide chitosan (CS) has attracted numerous interest in medical application because of its excellent biocompatibility, biodegradability, and the essential capacity to enhance the permeability and absorption of drugs at gastrointestinal mucosal sites.10 However, this capacity would be compromised at physiological pH owing to deprotonation effects. HTCC, a partially quaternized derivative of CS, has been characterized with good solubility and permeation-enhancing effects in neutral environments.11 The mechanism underlying permeation enhancement involves electrostatic interaction between positively charged HTCC and negatively charged sites in the tight junctions of cells, which leads to drug transport via transiently opened tight junctions. Moreover, HTCC can interact with negatively charged mucin glycoproteins, which endows it with greater mucoadhesive properties than CS.12 These superior properties have strongly promoted HTCC as an ideal candidate for oral delivery system of insoluble anticancer drugs. However, HTCC nanoparticles (HTCC-NP) prepared by traditional methods were exclusively suitable for water-soluble drugs loading,13 whereas their use as the carrier of insoluble drugs is seldom a success. In this work, we attempted to design an oral delivery system for insoluble anticancer drug PTX by HTCC nanoparticles. To solve aforementioned problems, we first added HTCC in the matrix to improve dissolution rate and bioavailability. Shirasu porous glass (SPG) membrane emulsification technique was then employed to obtain uniform-sized nanoparticles. During the subsequent preparation process, we used O/W/O double emulsion and temperature-programmed solidification methods to achieve PTX nanocrystals within the matrix network through an in situ crystallization process, which would greatly avoid the formation of large PTX crystals and escape of PTX, thus enhancing the LE and EE and release rate of PTX in the application. After characterizations of physicochemical properties and in vitro release profiles, the potential benefits of these nanoparticles for facilitating intestinal permeability were assessed on Caco-2 cells and the C57/BL6 mouse model. Lewis lung carcinoma (LLC) cells were employed for in vitro cytotoxicity and uptake assays. The tissue distribution, antitumor effects, and side effects after oral administration were also evaluated in subcutaneous lung cancer xenograft models.
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MATERIALS AND METHODS
Reagents and Materials. CS (Mw = 50 000) was purchased from Golden-Shell Biochemical, China. HTCC was synthesized according to the previously method reported.14 PTX, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), penicillin, streptomycin, Cremophor EL, and glutaraldehyde were purchased from SigmaAldrich. Taxol was obtained from a hospital pharmacy (SL Pharma, China). PO-500 ((hexaglycerin penta) ester) was supplied by 4231
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autofluorescent property at 488 nm and cell monolayer at 633 nm were observed by confocal laser scanning microscopy (CLSM) (TCS SP5, Leica). The corresponding emission wavelengths were 500−545 and 650−690 nm, respectively. Cellular Uptake and Cytotoxicity of CS-NP:PTX and HTCCNP:PTX. For the cellular uptake study, 1 × 105 LLC cells were first seeded on a 24-well cell culture plate for 24 h. The cell culture medium was then replaced with new medium containing 100 μg/mL of nanoparticles (CS-NP or HTCC-NP) and incubated at 37 °C. After incubation for different time intervals, cells were extensively washed by PBS (pH 7.4), resuspended, and fixed in 3.7% paraformaldehyde (pH 7.4). The cells were analyzed on a CyAn ADP 9 color flow cytometer (Beckman Coulter). Data were acquired from 15 000 cells per sample. Using the same method, LLC cells were treated with different PTX formulations (Taxol, CS-NP:PTX, and HTCC-NP:PTX). After incubation for 24 h, the cells were extensively washed by PBS, resuspended in cell lysing solution for 1 h. The resultant cell lysate suspensions were exposed to dichloromethane solution for 24 h to extract PTX. PTX in dichloromethane solution was obtained through centrifugation and evaporation. PTX was dissolved in 550 μL of acetonitrile mixed with 450 μL of water and determined by HPLC. In the CLSM imaging studies, cells were grown on a Petri-dish for 24 h, and the cell culture medium was replaced with new medium containing 50 μg/mL of nanoparticles. After incubation for 24 h, the cells were washed with PBS and fixed with formalin for 30 min. Cell nuclei and membrane were then stained with DAPI and Alexa Fluor 635 phalloidin, respectively. Nanoparticles were detected using their autofluorescence at 488 nm. The corresponding fluorescent images at 420−450, 500−545, and 650−690 nm were taken by CLSM. The MTT assay was used to evaluate cell viability. The cytotoxicity of blank nanoparticles was evaluated on LLC cells in comparison with the commercial vehicle (Cremophor:Ethanol) in Taxol formulation. Commercial solvent was prepared with a mixture of Cremophor EL and anhydrous ethanol (1:1, v/v). Typically, LLC cells were cultured in 96-well plates at the density of 5 × 104 per well for 24 h. Subsequently, serial dilutions (corresponding PTX concentration was 0−25 μg/mL) of CS-NP, HTCC-NP, and commercial vehicles were incubated with cells for 48 h. MTT (0.5 mg/mL) solution was added immediately to all wells. The plates were incubated with MTT for 4 h at 37 °C, developed, and measured. Each experiment was performed in triplicate and data were represented as means ± standard deviation. The in vitro cytotoxicity of CS-NP:PTX and HTCC-NP:PTX on LLC cells was subsequently investigated. The cells were incubated with CS-NP:PTX, HTCC-NP:PTX, and Taxol at equivalent PTX concentrations ranging from 1 to 25 μg/mL along with 48 h. Cell viability was also assessed with MTT assay. Intestinal Absorption of CS-NP and HTCC-NP. To evaluate intestinal absorption, two mice were orally administered with CS-NP and HTCC-NP three times (300 μL of 10 mg/mL, once every 8 h). After administration of the last dose, followed by 6 h of fasting, mice were sacrificed. The tissues of small intestine were regularly fixed, embedded, sliced, and stained with DAPI. At last, tissue slices were detected by CLSM. The excitation wavelengths of DAPI, nanoparticles, and small intestine were 405, 488, and 561 nm, respectively. The corresponding images were taken at 420−450, 500−545, and 590−630 nm, respectively. Oral Bioavailability and Tissue Distribution of PTX. A subcutaneous xenograft model of lung cancer was first established by injecting 1 × 106 LLC cells in 100 μL of mixture of PBS subcutaneously at the left axillary fossa in male C57BL/6 mice. To investigate the oral bioavailabilities of PTX-loaded nanoparticles, the CS-NP:PTX and HTCC-NP:PTX were administered orally to two groups of normal mice at the PTX dose of 10 mg/kg body weight (n = 6), respectively. The mice were sacrificed at 24 h. Organs including liver, spleen, kidney, lung, heart, intestine, and stomach were collected and then ground by a mixer mill (RETSCH, MM400). The PTX was further extracted from the tissues homogenate by incubating with dichloromethane for 24 h at room temperature. Purified PTX was obtained by combining filtration with centrifugation. After evaporation of dichloromethane, the resultant PTX was dissolved in acetonitrile
transmission electron microscope (TEM) (JEOL, Tokyo, Japan), respectively. Drug LE and EE were calculated according to the following formulas.
The samples were first immersed in acetonitrile for 24 h to extract PTX from nanoparticles. After centrifugation, PTX in supernatant was measured by reverse phase high-performance liquid chromatography (HPLC, LC-20AT, Shimadzu) using C18 column at 30 °C. The mobile phase consisted of acetonitrile−water (55:45 v/v), and the flow rate was 1.0 mL/min. The detection wavelength was 227 nm. Sample injection volume was 50 μL. The HPLC was calibrated with standard solutions of 5 to 100 μg/mL of PTX dissolved in acetonitrile (correlation coefficient of R2 = 0.99). The PTX concentrations in the samples were obtained from the calibration curve. Determination of in Vitro Drug Release. To determine the in vitro drug release behavior, 10 mg of PTX-loaded nanoparticles were dispersed in 10.0 mL of release medium (phosphate buffer solution (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 10 000g for 10 min. 450 μL of supernatant mixed with 550 μL of acetonitrile was analyzed by HPLC, as described above. Their degradation was characterized by TEM images after 10 days. In Vitro Adhesion and Permeation of Nanoparticles through Caco-2 Model. Caco-2 cells were seeded onto polycarbonate membrane filters (3 μm pore size, 1.12 cm2 growth area) inside Transwell cell culture chambers (Corning Costar) at a density of 1 × 105 cells/insert. The culture medium (0.5 mL per insert and 1.5 mL per well) was replaced every 48 h for the first 2 weeks and 24 h thereafter. After 3 weeks in culture, cell monolayers were used for the following assays. Before experiments, the integrity of the Caco-2 cell monolayer was examined by measuring the trans-epithelial electrical resistance (TEER) with a Millicell-ER system (Millipore, Billerica, Massachusetts, U.S.). Only the cell monolayers with TEER values over 800 Ω·cm2 were used. The bioadhesion of CS-NP and HTCC-NP was investigated on Caco-2 cell monolayers model in vitro. In brief, Caco-2 cells were first seeded into 16 wells of a 24-well plate and cultured for 3 weeks. Then, the cells were divided into four groups (control, CS-NP, and HTCCNP, n = 4). After incubation with the corresponding nanoparticles (100 μg/mL) at 4 °C for 2 h,17 cells were gently washed with ice-cold PBS to remove the residual adhering nanoparticles. At last, the fluorescence intensity of each sample was quantified by In Vivo Imaging Systems (FX Pro, Kodak). The excitation and emission wavelengths were 480 and 550 nm, respectively. The permeability of nanoparticles was studied across the apical to basolateral direction on the monolayer of Caco-2 cells. Nanoparticle samples (100 μg per monolayer) were suspended immediately in HBSS prior to experiments and then applied to apical buffer solution. Basolateral samples were withdrawn from the receiving chamber at different time points and immediately replaced by an equivalent volume of fresh HBSS. All of the transport experiments were conducted at least in triplicate (n = 3). The fluorescence intensities of basolateral samples could be subsequently measured at 540 nm (excitation wavelength was 480 nm) by an infinite M200 microplate spectrophotometer (Tecan, Männedorf, Switzerland). The permeation percentage of nanoparticles could be expressed as the fluorescence intensity of basolateral nanoparticles divided by its corresponding initial apical fluorescence intensity. For CLSM imaging study, Caco-2 cells monolayers were exposed to CS-NP and HTCC-NP for 6 h at 37 °C. Then, the monolayers were washed with PBS, fixed with formalin, and labeled with Alexa Fluor 635 phalloidin to define outline of cells. These two nanoparticles with 4232
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Table 1. Characteristics of HTCC-NP:PTX and CS-NP:PTX Prepared under Optimal Conditions NP
LE (%)
EE (%)
particle size (nm)
polydispersity Index
pore size (nm)
ζ potential (mV)
HTCC CS
38.91 ± 4.02 35.42 ± 3.02
83.12 ± 4.89 86.69 ± 5.52
130.27 ± 6.73 125.19 ± 5.92
0.034 0.029
13.98 ± 0.39 6.28 ± 0.48
+21.02 ± 0.27 +1.52 ± 0.53
Figure 1. SEM and TEM images of HTCC-NP:PTX. and analyzed by HPLC. The oral bioavailability of PTX was expressed as the percentage calculated by dividing the measured PTX concentration by its initial dose. On the basis of calculated oral bioavailability of PTX, we next investigated the drug tissue distribution. In this study, mice bearing LLC tumor were administered with CS-NP:PTX and HTCC-NP:PTX by oral gavage (n = 6, 10 mg/kg of PTX). As comparison group, Taxol was administrated by tail intravenous injection ((n = 6, 1 mg/kg of PTX). After treatment for 24 h, mice were euthanized, and their tissues and tumors were collected. The PTX concentrations in different tissues were examined by the same method as aforementioned. The biodistribution of PTX in normal mice was also evaluated as described above. The in vivo tumor-targeting ability of HTCC-NP:PTX was evaluated by In Vivo Imaging Systems. To monitor the distribution of nanoparticles in live animals, we replaced PTX by a fluorescence probe Cy7 to prepare Cy7-loaded HTCC-NP (HTCC-NP:Cy7) as described above. Mice bearing LLC tumors were subjected to in vivo imaging. The excitation and emission wavelengths of Cy7 were 740 and 790 nm, respectively. In Vivo Tumor Growth Inhibition Study. In the subcutaneous lung cancer model, treatments were started when tumor volume reached 100−200 mm3, and this day was designated as day 0. The mice were randomly divided into the following four groups (n = 10): PBS, Taxol, CS-NP:PTX, and HTCC-NP:PTX. Taxol (1 mg/kg of PTX) was administrated via tail intravenous injection. PBS, CSNP:PTX, and HTCC-NP:PTX (10 mg/kg of PTX) were administrated by oral administration. To detect whether blank nanoparticles own potential antitumor efficacy, an additional experiment was performed independently. PBS (500 μL per mouse), CS-NP, and HTCC-NP (500 μg per mouse) were administrated to the mice bearing LLC tumor by oral gavage (n = 10) once a day. The corresponding tumor volume data were collected by measuring tumor diameter with an electronic caliper every day. The effects of different PTX formulations on tumor growth were also assessed every day by measuring tumor diameter with an electronic caliper. Tumor volume was calculated using the formula: (L × W2)/2, where L is the longest and W is the shortest tumor diameter (millimeter). Relative tumor volume (RTV) was calculated at each measurement time point (where RTV was equal to the tumor volume at a given time point divided by the tumor volume prior to initial treatment). To monitor potential toxicity, we measured the weight of each mouse. For humane reasons, animals were killed and regarded as dead if the implanted tumor volume reached 5000 mm3. To further evaluate the hematological toxicity of different PTX formulations, we
collected 200 μL of blood of each mouse after final administration. Obtained blood was immediately evaluated by a blood cell analyzer (MEK-7222K, Japan). Detection of Allergic Reaction. Four groups of tumor bearing mice (18−22 g, n = 10) were used in allergy testing studies of PTX in three formulations (Taxol, CS-NP:PTX, and HTCC-NP:PTX). One of the four groups was used as control. The Taxol was daily administrated via tail intravenous injection at the PTX dose of 1 mg/ kg body weight. The other two formulations were administrated by intragastric administration at the PTX dose of 10 mg/kg body weight once a day. After administration with different PTX formulations for 2 weeks, tail vein blood of mice in different groups was collected and centrifuged. Serum samples were analyzed according to the procedure of Mouse IgE ELISA.
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RESULTS AND DISCUSSION Optimization of the Preparation Condition for PTXLoaded Nanoparticles. The LE and EE of drug were directly affected by the stability of double emulsion during the preparation process, so the choice of emulsifier was considered. Furthermore, the effects of different initial drug concentrations on the LE and EE of PTX were also critical issues. As shown in the Supporting Information (Figures S1−S3), Tween-60 displayed an excellent performance in terms of ensuring emulsion stability, which was consistent with the corresponding EE. The EE and LE were higher if the drug concentration was 50 mg/mL. These optimum preparation conditions were also suitable for the preparation of CS-NP:PTX, which was used as control to evaluate further the physicochemical properties of HTCC-NP:PTX in the subsequent experiments. Characterization of CS-NP:PTX and HTCC-NP:PTX. The main physicochemical characteristics of PTX-loaded nanoparticles are summarized in Table 1. High LE and EE of both nanoparticles resulting from optimized preparation conditions were observed. This would greatly lighten the burden of frequent and large dosage administration. The sizes of both nanoparticles were ∼130 nm. This was in the size range favoring intestinal uptake and subsequent accumulation in tumor tissue by the EPR effect.18 Successful preparation of nanoparticles with a narrow-size distribution (polydispersity index
