Materials Science and Engineering C 49 (2015) 51–57

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Porous hydrophilic core/hydrophobic shell nanoparticles for particle size and drug release control Shilei Hao, Bochu Wang ⁎, Yazhou Wang Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400030, China

a r t i c l e

i n f o

Article history: Received 28 July 2014 Received in revised form 17 October 2014 Accepted 5 December 2014 Available online 9 December 2014 Keywords: Core/shell nanoparticles Drug delivery systems Porous hydrophilic coating Drug release Particle size distribution

a b s t r a c t Polymeric nanoparticle has been developed for drug delivery during the past decades. However, the size of hydrophilic nanoparticles would increase in the aqueous environment due to water absorption, and then influence the in vivo biodistribution and drug release behavior. In the present study, the metronidazole-loaded porous Eudragit® RS (ERS)/poly(methyl methacrylate) (PMMA) core/shell nanoparticles were prepared by coaxial electrospray. Compared to the hydrophilic ERS nanoparticles, the porous hydrophilic core/hydrophobic shell nanoparticles displayed a slower drug release, and the release rate can be adjusted to change the surface area and particle size. In addition, the porous core/shell nanoparticles could maintain a stable particle size distribution in simulated body fluid for 8 h, which can be attributed to the bioinert nature of PMMA coating. And porous core/ shell nanoparticles showed slight in vitro cytotoxicity and good cellular internalization property. The results demonstrated that the prepared porous hydrophilic core/hydrophobic shell nanoparticles is a potential candidate for delivering drugs, which can also be used as a platform and further modified into targeted drug delivery systems for clinical application. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Polymeric nanoparticles have been extensively applied for delivering drugs during the past decades due to the high surface to volume ratio, modifiable platform, controlled drug release property, tunable size and shape, and good biodegradability [1,2]. And various therapeutic agents such as anti-cancer drugs [3], vaccines [4], proteins, nucleic acid [5] and other drugs have been incorporated into polymeric nanoparticles to improve their bioavailability [6]. In addition, a number of polymers have been explored to synthesize the polymeric nanoparticles, including poly(lactic acid) [7], poly(lactide-co-glycolide) [8], poly(methylmethacrylate) [9], chitosan [10,11], cyclodextrin [12], gelatin [13], and other polymers [14]. The size of polymeric nanoparticles significantly affects their in vivo absorption and biodistribution after oral administration, and small nanoparticle showed increased GI uptake [15]. Meanwhile, polymeric nanoparticles have been widely used for intravenous application, and the particle size also influences their circulation time, biodistribution and the extent of cellular uptake by phagocytosis and endocytosis [16, 17]. In addition, the size of nanoparticles has a pronounced influence ⁎ Corresponding author. E-mail address: [email protected] (B. Wang).

http://dx.doi.org/10.1016/j.msec.2014.12.029 0928-4931/© 2014 Elsevier B.V. All rights reserved.

on the release kinetics due to the different surface area/volume ratios [18]. However, the size of swellable polymeric nanoparticles would change in aqueous environment due to the water absorption [19], which would affect the in vivo biodistribution of polymeric nanoparticles and drug release profiles. The aim of present study was to prepare a porous hydrophilic core/ hydrophobic shell nanoparticles by coaxial electrospray to control the particle size distribution and drug release behavior. The size of polymeric nanoparticles can remain stable because of the hydrophobic shell, and the drug release rate can be controlled by changing the surface area and the particles size. Eudragit® RS (ERS), a pH-independent swellable polymer, was chosen as the core material [20], and the hydrophilic ERS nanoparticles were entrapped into the porous hydrophobic poly(methyl methacrylate) (PMMA) shell [21]. Furthermore, coaxial electrospray has been applied to prepare the core/shell polymeric micro/nanoparticles [22,23], and the most outstanding features of electrospray are rapid and efficient, i.e. preparation of the solid nanoparticle by one step and entrapment of the drug into nanoparticles without loss (100% entrapment efficiency) [24]. We have prepared the high drug loaded pH-sensitive nanoparticles and core/shell nanoparticles using single-axial electrospray and coaxial electrospray, respectively [24,25]. In addition, the structure of porous hydrophilic core/hydrophobic shell nanoparticles was observed by the scanning electron microscope

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(SEM) and transmission electron microscopy (TEM), and the cytotoxicity assay, cellular uptake and the in vitro release profile were also investigated. 2. Materials and methods 2.1. Materials Eudragit ® RS (MW = 320 kDa, ERS) was a kind gift from Evonik Industries (Essen, Germany). Poly(methyl methacrylate) (MW = 98 kDa, PMMA) was supplied by the LG Chem. Ltd. (Taejon, Korea). Metronidazole (MTZ) was kindly gifted by the Southwest Pharmaceutics Co. Ltd. (Chongqing, China). Caco-2 cells were supplied by the Institute of Pathology, Southwest Hospital (Chongqing, China). MTT was supplied by the Amresco (Solon, USA). Fluorescein isothiocyanate (FITC) was purchased from the Sigma (Missouri, USA). Rhodamine B (RhB) was purchased from the Sangon Biotech Co. Ltd. (Shanghai, China). All other materials and reagents used in the study were analytical grade. 2.2. Preparation of porous core/shell nanoparticle The porous ERS/PMMA nanoparticles with core/shell structure were prepared using coaxial electrospray method. ERS (100 mg) and MTZ (35 mg) were dissolved in 10 mL of dichloromethane as the inner fluid, and PMMA was dissolved in dichloromethane at different concentrations (from 1% to 4%, w/v) as the outer fluid. The inner and outer diameters of inner nozzle are 0.3 mm and 0.5 mm respectively, and the inner diameter of outer nozzle is 1.0 mm. The polymer solutions were loaded in plastic syringes and supplied by separate syringe pump (Langer, China). The flow rates of inner fluid and outer fluid were 200 μL/h and 400 μL/h, respectively. While, the nozzle was connected to the positive electrode (+ 15 kV) of a high-voltage power supply (Dongwen, China), aluminum foil was placed vertically to the coaxial nozzle as a collector, and the needle-to-collector distance was kept at 90 mm. The core/shell structure of porous ERS/PMMA nanoparticles was observed by fluorescence microscopy. The green (FITC) and red (RhB) dye were added into the inner fluid and outer fluid, respectively, and the FITC-loaded porous core/shell nanoparticles were also prepared for the cellular uptake study. In addition, the ERS nanoparticles were also prepared using single-axial electrospray. The inner diameter of nozzle is 1.0 mm, and the flow rate of fluid was 400 μL/h. Other fabrication parameters were the same as those in the preparation of core/shell nanoparticles. 2.3. Measurement of the particle size and zeta potential Particle size distribution, polydispersity index (PDI) and zeta potential of porous ERS/PMMA core/shell nanoparticles were measured by photon correlation spectroscopy and electrophoretic laser Doppler anemometry respectively using a Zetasizer (Nano ZS90, Malvern, UK). 2.4. Observation of the morphology The surface morphology of porous ERS/PMMA core/shell nanoparticles was observed by SEM and TEM. A piece aluminum foil loaded with nanoparticle was coated with gold metal under vacuum for SEM observation (EVOLS25, Zeiss, Germany). The nanoparticle solution were dropped on copper grids, natively stained by phosphotungstic acid and dried at room temperature for TEM observation (Tecnai G2 20, FEI, USA). And the core/shell structure of ERS/PMMA nanoparticles was also characterized by fluorescence microscopy (DMI 4000B, Leica, Germany). The green dye (FITC) and red dye (RhB) were excited at 488 and 543 nm, respectively. In addition, the morphology of drugloaded porous ERS/PMMA core/shell nanoparticles (2% of PMMA) after incubation in simulated body fluid (SBF, pH 7.4) for different times (2, 4, 6 and 8 h) was also observed by SEM.

2.5. Determination of the entrapment efficiency and loading capacity The encapsulation efficiency (EE) and loading capacity (LC) of porous ERS/PMMA core/shell nanoparticles were determined as follows: MTZ-loaded nanoparticles were incubated in dichloromethane to dissolve the nanoparticles with stirring for 1 min at 100 rpm, and then PBS solution (pH 7.4) was added into the solution. The mixture solution was stirring at 300 rpm for 4 h to evaporate the organic solvent completely. Finally, the solution was separated by a centrifuge (5417R, Eppendorf, Germany) at 12,000 rpm for 30 min and analyzed using a spectrophotometer at 300 nm (Lambda 900UV, PerkinElmer, USA). The EE and LC were calculated by the following equations:

LC ¼

Amount of Drug in Nanoparticles  100% Amount of Nanoparticles

ð1Þ

EE ¼

LC of Nanoparticles  100%: LC of Nanoparticles in Theory

ð2Þ

2.6. Fourier transform infrared spectroscopy analysis The chemical structure and complex formation of PMMA, ERS, MTZ, and MTZ-loaded porous ERS/PMMA nanoparticles were analyzed by a FT-IR spectroscopy (5DX/550II, Nicolet, USA), the samples used for the FT-IR spectroscopic characterization were prepared by grinding the dry specimens with KBr and pressing them to form disks. 2.7. X-ray diffraction analysis The XRD experiments were carried out using an X-ray diffractometer (6000X, Shimadzu, Japan). PMMA, ERS, MTZ, and MTZ-loaded porous ERS/PMMA nanoparticles, and physical mixture of drug and polymers were analyzed in the 2θ ranging from 5° to 45° with a step width of 0.04° and a count time of 2 s. 2.8. Differential scanning calorimetry The thermal properties of PMMA, ERS, MTZ, and drug-loaded porous ERS/PMMA nanoparticles, and physical mixture of drug and polymers were measured by differential scanning calorimetry (DSC, 204F1, Netzsch). The temperature scanning rate was 10 °C/min and scanned up to 200 °C. 2.9. Specific surface area analysis BET specific surface area of nanoparticles with different PMMA concentrations were calculated from nitrogen adsorption–desorption isotherms determined at 77 K using a surface area analyzer (Micromeritics ASAP2020M, USA), and the sample was outgassed under a vacuum at 298 K for at least 8 h. 2.10. In vitro release studies The release studies of MTZ from porous ERS/PMMA core/shell nanoparticles prepared with different PMMA concentrations and ERS nanoparticles were investigated and conducted as follows: 20 mg of MTZloaded nanoparticles and 3 mL of SBF were put into a dialysis tube (MWCO: 12,000) and then the dialysis tube was placed in 30 mL of SBF at 37 °C and kept under shaking at 100 rpm. At specific time intervals, the medium (1 mL) was taken and replaced with fresh SBF. The concentration of the released drug was determined by UV spectrophotometry.

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2.11. In vitro cytotoxicity assays The in vitro cytotoxicity of porous ERS/PMMA core/shell nanoparticles was detected using a methyl thiazolyl tetrazolium (MTT) assay. Caco-2 cells were seeded in 96-well plates at a density of 1 × 104 cells per well. For the concentration-dependent cytotoxicity experiments, the cells were incubated with the MTZ-loaded porous ERS/PMMA core/shell nanoparticle suspension (1% PMMA) at concentrations from 25 to 800 mg/L for 24 h, and for the time-dependent cytotoxicity experiments, the cells were further incubated with drug-loaded porous ERS/ PMMA core/shell nanoparticles with different PMMA concentrations (400 mg/L) and blank porous core/shell nanoparticles (1% PMMA, without drug) for 12, 24, 36 and 48 h. After the addition of 20 μL MTT solution (5 g/L), absorption at 490 nm was measured with a microplate reader (Model 3550, Bio-Rad, USA). The results were expressed as the percentage of reduction in cell growth/viability compared to untreated control wells, whereas, cells that were not incubated with MTT were used as a blank to calibrate the spectrophotometer. 2.12. Particle cellular uptake studies To study cellular uptake of nanoparticles by fluorescence microscopy, Caco-2 cells were seeded in the 24-well plate (5 × 104 per well) and incubated with FITC-loaded porous ERS/PMMA core/shell nanoparticle suspension (200 mg/L). At the designated time intervals, the cells were washed three times with PBS, and images were obtained under an inverted fluorescence microscopy. 2.13. Statistical analysis All measurements were performed in triplicate and data were presented as mean ± standard deviation (SD). Three batches of nanoparticles were prepared for each formulation. For selected evaluation tests, the means of all tested formulations were compared with each other by means of a one-way ANOVA with the one paired Student's t-test. The statistical significance level (P) was set at ≤0.05. 3. Results and discussion 3.1. Preparation of porous core/shell nanoparticles The porous hydrophilic core/hydrophobic shell nanoparticles were first prepared by coaxial electrospray method in the present study, and the most outstanding advantages of electrospray are rapid and efficient with the features of preparation of solid nanoparticles in one step and high EE (nearly 100%). And the ultrahigh EE of porous core/shell nanoparticles prepared with different PMMA concentrations was observed (Table 1). While, the LC of porous core/shell nanoparticles decreased from to 10.43% to 3.73% with the increased PMMA concentrations. Electrospray has the potential to prepare the high drug loaded particles because of the ultrahigh entrapment efficiency [25]. However, the maximum loading capacity of MTZ was 10.43% in the present study, which was due to the limited solubility of MTZ in dichloromethane. In addition, the size of core/shell nanoparticles increased from 58.3 to 389.7 nm with the PMMA concentrations that increased from 1% to 4%, and the trends could be explained by the fact that the increase of

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polymer concentration is thought to increase the resistance of the solution to be separated into droplets, leading to an increase of particle size. Meanwhile, the values of PDI were smaller than 0.300, indicating a narrow particle size distribution, and the porous ERS/PMMA core/shell nanoparticles displayed negative zeta potential ranging from −9.39 to − 23.09 mV. Furthermore, the BET surface area of nanoparticles decreased from 30.06 to 5.89 m2/g with the increased PMMA concentrations from 1% to 4%, which may be due to the increase of particle size. And the maximum surface area of produced porous ERS/PMMA nanoparticles was approximately 30 m2/g, doesn't display a large surface area. The reason probably resulted from the core–shell structure of porous nanoparticles.

3.2. Characterizations of porous core/shell nanoparticles The morphology of ERS nanoparticles and porous ERS/PMMA core/ shell nanoparticles prepared with different PMMA concentrations was observed using SEM, TEM and fluorescence microscopy. Fig. 1A shows the SEM image of produced ERS nanoparticles using single-axial electrospray. The mean particle diameter of ERS nanoparticles was approximately 60 nm, and a few of pores were observed on the nanoparticle surface. In addition, the produced nanoparticles were not perfectly spherical in shape, which was probably due to the inhomogeneous distribution of polymer molecules on the surface region during the drying process. Fig. 1B shows the TEM image of ERS nanoparticles, and the porous structure can't be found from the TEM observation. Furthermore, the fluorescence characteristics of the dual-fluorescence ERS/PMMA nanoparticles with core/shell structure were examined using fluorescence microscopy. The green and red emissions originated from the FITC and RhB dye in the core and shell layers, respectively. Fig. 1C, D and E shows the fluorescent performance of ERS/PMMA core/shell nanoparticles (4% PMMA) excited by different wavelength lasers. Fig. 1C shows the ERS/FITC green fluorescence signal, whereas the red fluorescence signal in Fig. 1D corresponded to the PMMA/RhB. And Fig. 1E is a composite image showing co-localization of ERS/FITC and PMMA/RhB fluorescence, which further reveals the core/shell structure of ERS/PMMA nanoparticles. The porous structure of ERS/PMMA nanoparticles prepared with different PMMA concentrations (from 1% to 4%) were also observed by SEM and TEM (Fig. 2). The size of nanoparticles increased as the increase of PMMA concentration, and some cavities were observed on the nanoparticle surface. Volatility of solvent would contribute to the formation of interior and surface pores of nanoparticles, but the shrinkage can be found on nanoparticle surface because the fast solvent evaporation would drive the polymer concentration to rise rapidly during the electrospray [26]. In addition, the porous ERS/PMMA nanoparticle was also observed by TEM. Compared to the TEM image of ERS nanoparticles (Fig. 1B), the obvious porous structure of core/shell nanoparticles can be found by the TEM observation, but the boundary of outer layer and inner layer was ambiguous, which may result from the diffusion of inner solution into the shell layer. However, the outer layer of porous PMMA could form a protective coating around the core of nanoparticles, and the gray level was different in the region of core and shell of nanoparticles. The produced ERS/PMMA core/shell nanoparticles are also not perfectly spherical in shape.

Table 1 Effect of PMMA concentrations on characteristics of the MTZ-loaded porous ERS/PMMA core/shell nanoparticles (mean ± S. D., n = 3). PMMA concentrations

1% 2% 3% 4%

MTZ

Size

Zeta potential

BET surface area

LC/(%)

EE/(%)

nm

PDI

(mV)

(m2/g)

10.43 ± 0.91 6.55 ± 0.10 4.78 ± 0.32 3.73 ± 0.19

99.86 ± 3.42 100.09 ± 0.92 100.37 ± 1.80 99.58 ± 1.37

58.3 ± 2.1 103.4 ± 3.0 211.2 ± 2.1 389.7 ± 2.8

0.201 ± 0.012 0.229 ± 0.103 0.274 ± 0.082 0.209 ± 0.014

−9.39 ± 3.01 −14.18 ± 2.97 −18.11 ± 0.05 −23.09 ± 2.74

30.06 ± 2.18 24.22 ± 1.15 10.15 ± 0.33 5.89 ± 1.53

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Fig. 1. SEM image (A) and TEM image (B) of ERS nanoparticles. (C) The core of FITC-loaded porous ERS/PMMA core/shell nanoparticles obtained by screening out the green light (4% PMMA). (D) The shell of FITC-loaded porous ERS/PMMA core/shell nanoparticles obtained by screening out the red light. (E) Composite images from D to C.

Fig. 3A shows the FT-IR spectra of PMMA, ERS, MTZ and drug-loaded porous ERS/PMMA nanoparticles. Several characteristic bands of PMMA can be found contributing to the a-methyl group vibrations (at 751 cm−1), acrylate carboxyl group (at 988 cm− 1, 1065 cm−1, 841 cm−1 and 1732 cm−1), and the C–H bond stretching vibrations of the –CH3 and –CH2– groups (at 2953 cm−1 and 2997 cm−1) [27]. And some characteristic bands can be observed in the FT-IR spectra of ERS, including –COOR stretching (1150 cm−1, 1240 cm−1), C_O ester vibration (1732 cm−1), and CHX vibrations (2950 cm−1, 1450 cm−1). In addition, several characteristic bands assigned to O–H stretching (3219 cm−1 and 3101 cm−1), N–O stretching (1535 cm−1), C_C aromatic stretching (1487 cm−1) and C–OH, C–O stretching (1074 cm−1) can be found in the FT-IR spectra of MTZ. The characteristic bands of MTZ, ERS, and PMMA can be found in the spectra of physical mixture, while, no new bands except the characteristic bands of drug and polymers can be observed in the spectra of drug-loaded porous ERS/

PMMA nanoparticles, indicating that there were no chemical reaction in the preparation of drug-loaded nanoparticles [28]. Fig. 3B displays the XRD patterns of PMMA, ERS, MTZ, drug-loaded porous ERS/PMMA nanoparticles and physical mixture of drug and polymers. The diffractogram of PMMA and ERS indicated the amorphous structures, and MTZ displayed several strong peaks in the range of 5° to 45°, indicating a high degree of crystallinity. While, the specific peaks of MTZ at 12°, 13.5°, 17°, 18°, 24.5°, 25.5°, 27° and 28° appeared in the diffractogram of physical mixture, and the crystallinity of drug decreased after being encapsulated into nanoparticles, indicate an amorphous structure of drug-loaded nanoparticles, but several weak peaks at 12°, 13.5°, 24.5°, 25.5°, 27° and 28° were observed in the diffractogram of nanoparticles. The similar results can be found in the DSC analysis of ERS, PMMA, MTZ, and drug-loaded nanoparticles and physical mixture of drug and polymers (Fig. 4). MTZ showed a single endothermic peak at 160 °C corresponding to the melting of the drug, and

Fig. 2. SEM images and TEM images of produced porous ERS/PMMA core/shell nanoparticles prepared with different PMMA concentrations.

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Fig. 3. (A) The FT-IR spectra of PMMA, ERS, MTZ, drug-loaded porous ERS/PMMA nanoparticles, and physical mixture of drug and polymers. (B) The XRD patterns of PMMA, ERS, MTZ, drug-loaded porous ERS/PMMA nanoparticles, and physical mixture of drug and polymers.

the characteristic peak of MTZ can be observed in the thermograms of physical mixture. The fact that there was no endothermic peak at 160 °C in the thermograms of drug-loaded nanoparticles suggest that the nanoencapsulation process produces a marked decrease in crystallinity of drug and/or confers to this drug a nearly amorphous state [29]. 3.3. In vitro drug release The in vitro cumulative release profiles of MTZ from ERS nanoparticles and porous ERS/PMMA core/shell nanoparticles in SBF were studied (Fig. 5A). ERS nanoparticles displayed a fast drug release in SBF compared to the porous ERS/PMMA core/shell nanoparticles, which was due to the hydrophility of ERS nanoparticles. In addition, the release rate of MTZ from porous ERS/PMMA core/shell nanoparticles was lower in the case of the porous core/shell nanoparticles prepared with higher PMMA concentration. The reason could be that the increase of

PMMA concentration could lead to the decrease of surface area of nanoparticle, and the porous hydrophobic shell controls the drug diffusion speed. Furthermore, the nanoparticle size decreased and the drug loading capacity increased as the increase of PMMA concentration. Small nanoparticles could supply high surface area/volume ratio, and the drug release rate was higher in the case of formulations containing higher amount of drug. So the drug release rate from porous ERS/ PMMA core/shell nanoparticles can be controlled by changing the surface area and particle size. The size of hydrophilic particles would increase in aqueous environment due to water absorption, and then influence the release profile of drugs. Porous hydrophilic core/hydrophobic shell nanoparticles could maintain a stable particle size distribution because of the hydrophobic coating in the outer layer. As shown in Fig. 5B, ERS nanoparticles began to swell after incubation in SBF for 2 h, and the shape of nanoparticles turned from irregular to spherical. In addition, some fused ERS nanoparticles can be observed at 8 h, and the particle size increased from approximately 60 nm to approximately 200 nm. The size of porous ERS/PMMA core/shell nanoparticles (2% PMMA) was kept at approximately 100 nm within 8 h, and the pores on the porous nanoparticle surfaces were covered due to the swelling of core ERS nanoparticles. However, the porous ERS/PMMA core/shell nanoparticles were still not perfectly spherical in shape, and some cavities can be observed on the nanoparticle surface. Furthermore, the pore size on the nanoparticle would also influence the drug release rate, and the release medium comes into the core through the channel and the dissolved drug can diffuse via the channel. However, the pore size formed by solvent volatilization is difficult to control using the electrospray method. 3.4. In vitro cytotoxicity assays

Fig. 4. The DSC curves of PMMA, ERS, MTZ, drug-loaded porous ERS/PMMA nanoparticles, and physical mixture of drug and polymers.

The cytotoxicity of MTZ-loaded porous ERS/PMMA core/shell nanoparticles at different concentrations (from 25 to 800 mg/L) was investigated by MTT assay using Caco-2 cells (Fig. 6A). The cell viability increased as the increase of nanoparticle concentration, and the significant decrease of cell viability was not observed until the concentration of nanoparticles increased to 400 mg/L. In subsequent studies, the cytotoxicity of MTZ-loaded porous ERS/PMMA core/shell nanoparticles

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Fig. 5. (A) In vitro release profiles of MTZ from ERS nanoparticles and porous ERS/PMMA core/shell nanoparticles prepared with different PMMA concentrations. (B) SEM images of MTZloaded ERS nanoparticles and MTZ-loaded porous ERS/PMMA core/shell nanoparticles after incubation with SBF for different times.

prepared with different PMMA concentrations (from 1% to 4%) and blank nanoparticles (1% PMMA, without drug) after incubation for different times was determined (Fig. 6B). The cell viability decreased linearly as the decrease of PMMA concentration, and the cytotoxicity of blank nanoparticles showed no significant difference with the control group, which indicated that the cytotoxicity of drug-loaded porous core/shell nanoparticles resulted from the toxicity of drug. In addition, the cytotoxicity of nanoparticles also increased with the increase of incubation time. But the cell viabilities were higher than 70% after treatment with MTZ-loaded porous ERS/PMMA core/shell nanoparticles, indicating that the cytotoxicity of produced porous core/shell nanoparticles was slight in vitro [9].

3.5. Particle cellular uptake studies The FITC-loaded porous ERS/PMMA core/shell nanoparticles were incubated with Caco-2 cells for different times to investigate the cellular internalization property (Fig. 6C). Some nanoparticles survived onto the surface of cells after washing with PBS, and the fluorescence intensity was weak, indicating that only a few nanoparticles had been internalized by the cells after incubation for 1 h. The fluorescence intensity became strong after incubation for 2 h, and a stronger fluorescent intensity was observed after incubation for 4 h, which suggested that the uptake

of porous ERS/PMMA core/shell nanoparticles by the Caco-2 cells increased over time. 4. Conclusions In summary, the porous ERS/PMMA core/shell nanoparticles were successfully prepared by a coaxial electrospray method. The porous and core/shell structures of produced ERS/PMMA nanoparticles can be found by fluorescence and electron microscopy. Compared with hydrophilic ERS nanoparticles, porous hydrophilic core/hydrophobic shell nanoparticles could provide a slower drug release due to the porous hydrophobic shell, and the release rate from porous hydrophilic core/ hydrophobic shell nanoparticles can be adjusted by changing the surface area and particle size. Moreover, the particle size distribution of porous core/shell nanoparticles was stable after incubation in SBF for 8 h because of the bioinert nature of PMMA coating. The results of MTT assay and cellular internalization study further confirmed its safeness and effectiveness as a drug carrier. Due to the good stability and drugcontrolled release property, the porous hydrophilic core/hydrophobic shell nanoparticles have the potential to be used for clinical application loading with different therapeutic agents. However, the porous hydrophobic shell may influence the in vivo distribution of nanoparticles, and the porous core/shell nanoparticles can be also used as a platform and further modified into a targeted drug delivery system.

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Fig. 6. (A) Cell viability after treatment with different concentrations of MTZ-loaded porous ERS/PMMA core/shell nanoparticles (1% PMMA) after 24 h. (B) cell viability after treatment with blank porous core/shell nanoparticles and MTZ-loaded porous ERS/PMMA core/shell nanoparticles (400 mg/L) prepared with different PMMA concentrations after incubation for different times (**P ≤ 0.01, *P ≤ 0.05 as compared with control group). (C) Fluorescence microscopic images of Caco-2 cells after incubation with FITC-loaded porous ERS/PMMA core/ shell nanoparticles for different times, magnification = 200×.

Acknowledgments The authors acknowledge the financial assistance provided by the National Basic Research Program of China (973 Program, Grant No. 2014CB541603) and National Natural Science Foundation of China (Grant No. 31200713). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]

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hydrophobic shell nanoparticles for particle size and drug release control.

Polymeric nanoparticle has been developed for drug delivery during the past decades. However, the size of hydrophilic nanoparticles would increase in ...
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