Shell Polymer Microspheres as a Multiply

Feb 26, 2014 - An effective strategy was developed to fabricate the novel dually thermo- and pH-responsive yolk/shell polymer microspheres as a drug d...
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Novel Smart Yolk/Shell Polymer Microspheres as a Multiply Responsive Cargo Delivery System Pengcheng Du and Peng Liu* State Key Laboratory of Applied Organic Chemistry and Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China S Supporting Information *

ABSTRACT: An effective strategy was developed to fabricate the novel dually thermoand pH-responsive yolk/shell polymer microspheres as a drug delivery system (DDS) for the controlled release of anticancer drugs via two-stage distillation precipitation polymerization and seed precipitation polymerization. Their pH-induced thermally responsive polymer shells act as a smart “valve” to adjust the diffusion of the loaded drugs in/out of the polymer containers according to the body environments, while the movable P(MAA-co-EGDMA) cores enhance the drug loading capacity for the anticancer drug doxorubicin hydrochloride (DOX). The yolk/shell polymer microspheres show a low leakage at high pH values but significantly enhanced release at lower pH values equivalent to the tumor body fluid environments at human body temperature, exhibiting the apparent tumor-environment-responsive controlled “on−off” drug release characteristics. Meanwhile, the yolk/shell microspheres expressed very low in vitro cytotoxicity on HepG2 cells. Consequently, their precise tumor-environment-responsive drug delivery performance and high drug loading capacity offer promise for tumor therapy.

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INTRODUCTION Recently, multifunctional hollow micro/nanospheres have attracted more attention owing to their potential applications in encapsulation and delivery systems for drugs, in gene delivery, and as protective shells for cells and enzymes.1−6 Among these functional hollow microspheres, stimuliresponsive polymer-based hollow microspheres have been designed as potential multifunctional drug delivery systems (DDS), enhancing specifically the accumulation of drugs in the required organ or tissues.7−10 Up to now, various approaches have been established to fabricate hollow microspheres with tailored morphology and physicochemical properties as drug carriers, such as layer-by-layer self-assembly,11,12 polymerization from templates,13 interfacial polymerization,14 and so on. On the basis of the advantage of the responsive polymer, the hollow microspheres are responsive to such external stimuli as pH, temperature, oxidation, light, and ionic strength.15−20 In the tumor-curing process, traditional anticancer drugs cannot distinguish the cancerous cells from the healthy cells, so collateral damage and adverse side effects are almost inevitable. The design of DDS for the selective release of anticancer drugs on the tumor tissues based on the difference between the tumor cells and the normal healthy cells is a challenging way to cure cancer. For example, most cancerous tissues exhibit lower extracellular pH values (pH 6.0−7.0) than the normal tissues and the bloodstream (pH 7.4), and the pH value drops further inside cells, especially inside endosomes (4.5−5.5).21 Additionally, certain malignancies show distinct local hyperthermia.22 In order to solve this formidable challenge, the thermo-responsive © 2014 American Chemical Society

polymer poly(N-isopropylacrylamide) (PNIPAM) has attracted intensive attention and has a relatively low volume phase transition temperature (VPTT) of around 32 °C in water. Thus, it is soluble below its VPTT but insoluble above its lower critical solution temperature.23,24 The VPTT could be adjusted efficiently to higher or lower temperature by incorporating functional comonomers into the microgels, depending on the hydrophilic or hydrophobic property of the comonomers.25−28 The inclusion of multiple stimuli-responsive properties into microstructures has broadened their properties and application.29−31 The design of the core−shell structured hybrid microspheres, combining the stimuli-responsive polymer as the shell and the functional inorganic nanoparticles as the core, have promising potential applications in biomedical fields.32−35 For example, Xia’s group covered gold nanocages by P(NIPAM-co-acrylamide) for controlled drug release, in which the gold nanocages have strong absorption in the nearinfrared region. The gold nanocages convert light into heat, leading to the rise in temperature and the collapse of the P(NIPAM-co-acrylamide) chains to release the drug loaded in the gold nanocages.36 Meanwhile, joining the quantum dots, magnetic nanoparticles, mesoporous material or functional hybrid microspheres, and smart polymers together has been focused to produce the core−shell structured microspheres for imaging or magnetic targeting for controlled drug release.37−41 In comparison, the responsive polymer cores can provide a new Received: February 24, 2014 Published: February 26, 2014 3060

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Scheme 1. Schematic Illustration of the Fabrication Process of the Multifunctional Yolk/Shell Microspheres for Controlled Release of Drug from the Sandwich Core/Shell Microspheres with Sacrificial Silica Interlayer

feeding volume ratio of 4:6) with AIBN as initiator in neat acetonitrile.44 A typical procedure was as follows: 3.0 mL of MAA, 2.0 mL of EGDMA, and 0.1 g of AIBN (2 wt % of monomers) were dissolved in 200 mL of acetonitrile in a 250 mL two-necked flask equipped with a fractionating column, Liebig condenser, and a receiver that was submerged in a heating mantle. The reaction mixture was heated to boiling. After boiling for 30 min, the solvent (acetonitrile) was distilled out of the reaction mixture and the reaction was ended after 100 mL of acetonitrile was distilled off. The P(MAA-co-EGDMA) microgels were purified by washing thoroughly with acetonitrile three times. Finally, the microgels were dried (vacuum, 50 °C) till a constant weight. Core−Shell MPS-Modified P(MAA-co-EGDMA)/SiO2 Microspheres. The core−shell poly(methacrylic acid-co-ethyleneglycol dimethacrylate)/silica (P(MAA-co-EGDMA)/SiO2) microspheres were prepared by encapsulating the P(MAA-co-EGDMA) microgels with an outer silica layer via a sol−gel process: 0.20 g of P(MAA-coEGDMA) microgels, 2.40 mL of ammonia, and 1.0 mL of TEOS were added into a water/ethanol mixture (40 mL/160 mL). After the mixture was vigorously stirred for 12 h, another 2.40 mL of ammonia and 1.0 mL of TEOS were added and stirred for further 12 h at room temperature. Then the P(MAA-co-EGDMA)/SiO2 microspheres were modified with MPS to introduce the reactive vinyl groups on their surface by adding 2.0 mL of MPS into the ethanol dispersion of the core−shell particles followed by being stirred for 48 h at room temperature. The core−shell MPS-modified P(MAA-co-EGDMA)/SiO2 microspheres were purified by washing with ethanol three times to remove completely the excessive MPS. The final core−shell MPS-modified P(MAA-co-EGDMA)/SiO2 microspheres were dried (vacuum, 50 °C) till a constant weight. P(MAA-co-EGDMA)/SiO2/P(NIPAM-co-MAA) Sandwich Microspheres. The P(MAA-co-EGDMA)/SiO2/P(NIPAM-co-MAA) sandwich microspheres were achieved by a facial precipitation polymerization. The different molar ratios of the comonomers NIPAM/MAA [(100 − X)/X, where X increases from 0 to 10 for the copolymer (P(NIPAM-co-MAA)-X)] were fed in the precipitation polymerization in the presence of the core−shell MPS-modified P(MAA-co-EGDMA)/SiO2 microspheres. In a typical procedure, 0.2 g of MPS-modified P(MAA-coEGDMA)/SiO2 composite microspheres was fully ultrasonicated for 2 h into 95 mL of water in a 250 mL three-neck flask to prepare the seed dispersion, and then 0.6 g of NIPAM, 15 mg of MBA, and 13.6 μL of MAA were introduced into the dispersion. Then the dispersion was ultrasonicated for 30 min at room temperature to obtain a stable dispersion. After that, the mixture was heated to 75 °C and kept at this temperature under a N2 atmosphere. After being stirred for 0.5 h, 5

role in encapsulation of molecules and drug loading for use in DDS.42,43 Furthermore, it is possible to fabricate more sophisticated and smart DDSs with improved selectivity and targeting efficiency by controlling their size distribution. In the present work, we describe a facile fabrication process for multifunctional yolk/shell microspheres, the movable PMAA cores being encapsulated with the smart pH-induced thermally responsive polymer shells, as a controlled drug release system (Scheme 1). The monodisperse poly(methacrylic acid-co-ethyleneglycol dimethacrylate) (P(MAAco-EGDMA)) microgel cores can exhibit pH responsiveness, and independently, the P(NIPAM-co-MAA) shells can respond to pH and temperature environmental stimuli respectively. The yolk/shell microspheres were used to enhance the doxorubicin hydrochloride (DOX) loading capacity and exhibited more rational release behavior, i.e., very low drug release at pH 7.4 but rapid drug release at reduced pH values (6.5 or 5.0) at 37 °C. The dually thermo- and pH-responsive characteristics of the yolk/shell microspheres could induce the controlled release due to the temperature and pH differences between tumor tissues and normal tissues, indicating their apparent tumorenvironment-responsive drug delivery performance.

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EXPERIMENTAL SECTION

Materials and Reagents. Methacrylic acid (MAA) was obtained from Tianjin Chemical Reagent II Co. and purified by vacuum distillation. [3-(Methacryloxy)propyl]trimethoxysilane (MPS) and tetraethylorthosilicate [Si(OEt)4, TEOS] was purchased from Tianjin Chemical Reagent II Co. and used as received. N-Isopropylacrylamide (NIPAM) was purchased from Aldrich. N,N′-methylenebisacrylamide (MBA) and 2,2′-azobisisobutyronitrile (AIBN) were obtained from Tianjin Chemical Co. Ltd. DOX was received from Beijing Huafeng Lianbo Technology Co., Ltd., China. Ammonia as a 25% aqueous solution was obtained from Tianjin Dongsheng Fine Chemical Reagent Factory. Hydrofluoric acid (40 wt % HF) was obtained from Tianjin Chemical Reagent Institute. Analytical grade acetonitrile was obtained from Tianjin Chemical Reagent II Co., dried over calcium hydride, and purified by distillation before use. Deionized water was used throughout. Monodisperse pH-Sensitive Poly(methacrylic acid-co-ethyleneglycol dimethacrylate) (P(MAA-co-EGDMA)) Microgels. The monodisperse poly(methacrylic acid-co-ethyleneglycol dimethacrylate) (P(MAA-co-EGDMA)) microgels were synthesized via the facile distillation precipitation copolymerization of EGDMA and MAA (with 3061

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Figure 1. TEM images of (a) P(MAA-co-EGDMA) microgels, (b) core−shell P(MAA-co-EGDMA)/SiO2 microspheres, (c) sandwich P(MAA-coEGDMA)/SiO2/P(NIPAM-co-MAA) composite microspheres, (d−h) multifunctional yolk/shell microspheres with the MAA rate of 0, 1, 3, 5, 10% for the shells with phosphotungstic acid dyeing, and (i) yolk/shell microspheres with the MAA rate of 5% for the shells with no dyeing. mL of 4 mg mL−1 KPS solution was rapidly added, and the reaction was allowed to proceed for 4 h. Finally, the sandwich microspheres were collected and washed with water by five centrifugation and redispersion cycles to remove the unreacted monomers for the further use. Multifunctional Yolk/Shell Microspheres. The resultant sandwich P(MAA-co-EGDMA)/SiO2/P(NIPAM-co-MAA) microspheres were dispersed in HF solution for 24 h with stirring. The multifunctional yolk/shell microspheres were washed with water and ethanol five times to remove the excess HF. Drug Loading. Ten milligrams of yolk/shell microspheres was added into 3 mL of 1.0 mg mL−1 DOX solution at pH 7.4. After being stirred for 48 h, the DOX-loaded yolk/shell microspheres were separated by centrifugation. The DOX concentration in the supernatant solution was analyzed with a UV−vis spectrophotometer at its maximum absorbance of 233 nm. The drug-loading capacity of the yolk/shell microspheres was calculated from the drug concentrations in solutions before and after the drug loading. Controlled Release. Ten milliliters of aqueous dispersion of the DOX-loaded yolk/shell microspheres was transferred into dialysis tubes with a molecular weight cutoff of 14 000 and immersed into 90 mL of phosphate-buffered saline (PBS) at pH 5.0, 6.5, or 7.4 at 37 °C, respectively. A 5.0 mL aliquot of the solution was taken out at certain time intervals to measure the drug concentrations in the dialysates with UV−vis spectrometry. Besides, 5.0 mL of fresh solution with the

same pH value was added after each sampling to keep the total volume of the solution constant. The cumulative release is expressed as the total percentage of the released drug over time. Cell Toxicity Assays. An MTT assay was performed to evaluate the cytocompatibility of the multifunctional yolk/shell microspheres with HepG2 cells. After the cells had been seeded in 96-well plates at densities of 1 × 104 cells per well for 24 h, the yolk/shell microspheres with pH-responsive cores were added to the cells. After a defined time, the culture medium was removed and 20 mL of 0.5 mg mL−1 MTT reagent was added, followed by incubating for another 2 h. Then 150 μL of DMSO was added into each MTT/medium well to dissolve the formazan crystals. The absorbance at 570 nm of the solutions was measured with a microplate reader (Bio-Rad, iMarkTM). The average data were presented for triplicate measurement. Characterizations. A Nicolet 8210 Fourier transform infrared spectrometer (Nicolet Instrument Inn) was used for the FT-IR spectroscopy analysis in the range of 400−4000 cm−1 with the resolution of 4 cm−1 by the KBr pellet technique. Thermogravimetric analysis (TGA) results were obtained with a TA Instrument 2050 thermogravimetric analyzer (TA Instruments, New Castle, DE) from 25 to 800 °C with a heating rate of 10 °C/min at N2 atmosphere. The morphologies of the microgels, core−shell composite microspheres, and the yolk/shell microspheres were characterized with a JEM-1200 EX/S transmission electron microscope (TEM) (JEOL, 3062

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Tokyo, Japan). The samples were ultrasonically dispersed in water for 5 min and then deposited on a copper grid covered with a perforated carbon film. The mean hydrodynamic diameter and size distribution of the P(MAA-co-EGDMA) microgels and the P(MAA-co-EGDMA)/P(NIPAM-co-MAA) yolk/shell microspheres were determined by a light-scattering system (BI-200SM, Brookhaven Instruments) equipped with a BI-200SM goniometer, BI-9000AT correlator, temperature controller, and Coherent INOVA 70C argon ion laser at 20 °C, after being dispersed in physiological saline. The dynamical light scattering (DLS) measurements were performed using 135 mW intense laser excitation at 514.5 nm and at a detection angle of 90° at 25 °C. The particle size distribution was calculated with the Brookhaven Instruments Particle sizing software. The pH values of the dispersions were adjusted directly with HCl or NaOH solution. The data are presented as the averages of three measurements.

defined silica interlayer could efficiently isolate the pHresponsive cores from the outermost polymer shells and offer extra space for yolk/shell microspheres structure. The P(MAAco-EGDMA)/SiO2/P(NIPAM-co-MAA) trilayered sandwich microspheres were prepared by the second-stage precipitation polymerization (Scheme 1).41,45 A number of experiments were initially designed to investigate the polymerizing conditions on the structure and properties of the dually thermo- and pH-responsive shell layers, in which the feeding ratios of the pH-responsive monomer MAA were controlled at 0, 1, 3, 5, and 10 mol %, and the NIPAM feeding ratio was varied 3/1 to the mass of the P(MAA-co-EGDMA)/SiO2 cores, respectively. The TEM image of the P(MAA-co-EGDMA)/SiO2/P(NIPAM-co-MAA) trilayer sandwich microspheres with uniform shape is shown in Figure 1c. The well-defined P(MAA-co-EGDMA)/SiO2/P(NIPAM-co-MAA) trilayer sandwich microspheres with narrow size distribution under the feeding ratio of MAA monomer of 3% was selected as a sample. Compared with the TGA curve of the P(MAA-co-EGDMA)/SiO2 cores, about 56% of the monomer had been polymerized to form the multifunctional polymer shells. Furthermore, both the inner P(MAA-coEGDMA) microgel cores and the outermost PNIPAM corona exhibited lower contrast in comparison with the silica layers in the TEM image. The successful formation of the PNIPAM corona could be further confirmed by the FT-IR analysis (Figure S1c, Supporting Information). The characteristic absorbance peaks at 1544, 1650, and 3300 cm−1 are associated with the vibration modes of the amide groups in the PNIPAM corona. In addition, the shoulder peak of the CO stretching of the carboxylic acid groups was still observed at 1729 cm−1 in the P(MAA-co-EGDMA)/SiO2/P(NIPAM-co-MAA) trilayer sandwich microspheres. Finally, the movable PMAA cores encapsulated with the dually thermo- and pH-responsive smart polymer shells as multifunctional yolk/shell microspheres could be achieved, after the selective removal of the inorganic silica interlayer with HF. The yolk/shell structures of the P(MAA-co-EGDMA)/ P(NIPAM-co-MAA) microspheres with PMAA cores and flexible shells are revealed by TEM analysis (Figure 1d−h), respectively. The yolk/shell microspheres could be clearly observed after being dyed with 1 wt % phosphotungstic acid solution. In comparison, there is an obvious contrast between the dyed sample and the unstained one shown in Figure 1i. Obviously, the inner P(MAA-co-EGDMA) microgel cores showed an obviously lower contrast than the outermost dyed PNIPAM layers in the TEM image. Here the cavity between the pH-responsive movable P(MAA-co-EGDMA) cores and the dually thermo- and pH-responsive smart polymer shells could provide space for the volume change of both upon environmental stimuli or drug loading.46 Environmental Stimuli Responsive Properties. The pH-responsive P(PMAA-co-EGDMA) cores were prepared as a drug cargo through a facile distillation precipitation polymerization, the tailored uniform microgels exhibiting excellent pHresponsive characteristics. The P(NIPAM-co-MAA) shells exhibit dually thermo- and pH-responsive characters. Therefore, the drug delivery can be expected to be controlled in body fluid. The DLS characterization was used to investigate the effect of the media pH on the hydrodynamic diameters of the P(MAA-co-EGDMA) microgel cores in different aqueous solution and to test the influence of the VPTT of the yolk/

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RESULTS AND DISCUSSION Preparation of Yolk/Shell P(MAA-co-EGDMA)/PNIPAM-MAA Microspheres. The fabrication process for the multifunctional yolk/shell microspheres is shown schematically in Scheme 1. Initially, the uniform pH-sensitive P(MAA-coEGDMA) cores, which were rich in carboxyl groups and can be used as the pH-responsive polymeric cores for the drug carriers with high drug-loading capacity due to the electrostatic interaction and/or hydrogen bond between the carriers and drugs, were prepared by the facile distillation precipitation polymerization.44 The TEM image (Figure 1a) shows that the P(MAA-co-EGDMA) microgels were monodisperse spheres with an average size of about 90 nm. The absorbance band at 1729 cm−1 in the FT-IR spectrum of the P(MAA-co-EGDMA) microgels (Figure S1a, Supporting Information) is associated with the stretching vibration of the CO groups of the PMAA microgels. Then a new silica shell was coated onto the P(MAA-coEGDMA) microgels by the sol−gel condensation hydrolysis of TEOS. A typical core−shell structure could be obviously observed with the P(MAA-co-EGDMA) cores (light contrast) and the silica shells (deep contrast). The TEM image of the core−shell P(MAA-co-EGDMA)/SiO2 microspheres (Figure 1b) demonstrated that the microspheres were uniform, spherical shapes in the absence of any secondary silica particles produced by the self-nucleation during the sol−gel hydrolysis. In additional, the particle size of the core−shell P(MAA-coEGDMA)/SiO2 microspheres significantly increased from 90 nm of the P(MAA-co-EGDMA) cores to 150 nm. The efficiency of encapsulation, demonstrated as the mass ratio of the outermost SiO2 shells and the polymer cores, was calculated to be 28.83% from the TGA analysis (Figure S2a, Supporting Information). The thickness of the silica shells was calculated to be about 20 nm, as half of the difference between the diameter of the P(MAA-co-EGDMA) cores and the core−shell P(MAAco-EGDMA)/SiO2 microspheres. The surfaces of the obtained core−shell P(MAA-coEGDMA)/SiO2 microspheres were modified with a commercially available silane coupling agent, MPS, to introduce the growth sites for the subsequent seed precipitation polymerization. The strong absorbance peak at 1630 cm−1 was associated with the CC groups of the immobilized MPS (Figure S1b, Supporting Information), indicating that the polymerizable groups had been successfully introduced onto the core−shell microspheres. It is favorable for the subsequent surface seed precipitation polymerization technique to form the dually thermo- and pH-responsive polymer shells. So the well3063

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Figure 2. pH dependence of the average hydrodynamic diameter of the P(MAA-co-EGDMA) microgels (a) and the P(MAA-co-EGDMA)/SiO2/ P(NIPAM-co-MAA)-3 sandwich composite microspheres (b) in the physiological saline.

Figure 3. (A) Effect of the feeding ratio of MAA on the temperature-responsive property of the hydrodynamic diameter of the yolk/shell microspheres with pH-responsive movable cores at pH 7.4 (PBS). Legend for MAA content: (a) 5 mol %, (b) 3 mol %, (c) 1 mol %, (d) 0 mol %. (B) Effect of temperature on the hydrodynamic diameter of the yolk/shell microspheres with PMAA movable cores at pH 5.0. Legend for MAA content: (a) 5 mol %, (b) 3 mol %.

Table 1. VPTT of the P(MAA-co-EGDMA)/P(NIPAM-co-MAA)-X Yolk/Shell Microspheres VPTT (°C) sample

MAA (mol %)

Dh (nm)

pH 7.4

pH 5.0

P(MAA-co-EGDMA)/PNIPAM P(MAA-co-EGDMA)/P(NIPAM-co-MAA)-1 P(MAA-co-EGDMA)/P(NIPAM-co-MAA)-3 P(MAA-co-EGDMA)/P(NIPAM-co-MAA)-5

0 1 3 5

570.8 500.7 454.2 426.9

32.0 35.0 41.0 42.5

32.0 37.0

an unstable state.48 The particle size increases because of the stretch of the PMAA chains. The P(MAA-co-EGDMA)/SiO2/P(NIPAM-co-MAA)-3 trilayered sandwich microspheres were prepared by the seed precipitation polymerization of NIPAM with MAA as the comonomer, so the polymeric shells interestingly show the pHresponsive property. The hydrodynamic diameters of the P(MAA-co-EGDMA)/SiO2/P(NIPAM-co-MAA) trilayered sandwich microspheres increased slowly from 425 to 515 nm upon increasing the media pH values (Figure 2b). Due to the same reason for the change of the PMAA chains, the electrostatic repulsion results in the higher swell ratio of the PNIPAM shells.30 On the opposite side, in low pH solution, the effect of the force is weakened, so the swelling ratio of the PNIPAM shells reduces.

shell P(MAA-co-EGDMA)/P(NIPAM-co-MAA) microspheres in physiological saline with different pH values. The hydrodynamic diameters (Dh) and size distributions of the PMAA microgels in aqueous media (constant pH from 3 to 11) were investigated with the DLS technique at 25 °C (Figure 2a). Due of the large amount of carboxyl groups, the PMAA microgels are quite sensitive to the environmental pH values. The hydrodynamic diameters of the P(MAA-co-EGDMA) microgels increased slowly from 150 to 224 nm upon increasing the media pH values, due to the electrostatic repulsion between the carboxyl anions of PMAA.47 As the pH value of the solution increases, the −COOH groups dissociate into −COO− anions, and then the PMAA chains extended substantially. Unlike in high pH media, the hydrodynamic diameter at pH 3 is an exception. It located on 3064

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Figure 4. (A) Effect of pH on the drug-loading content (■) and loading efficiency (□) for the yolk/shell P(MAA-co-EGDMA)/P(NIPAM-coMAA)-3 microspheres at 25 °C. (B) In vitro drug release from the DOX-loaded yolk/shell microspheres at different pH values at 37 °C, respectively.

comparison, after five cycles between 25 and 41 °C at pH 5.0, the diameters still return back their original state (Figure S3b, Supporting Information). One can see that the thermoresponsive recyclability can be maintained in physiological saline with different pH values. Furthermore, the scattered intensity showed the obvious reversible circulation characteristics, with the opposite response at different pH values (Figure S4, Supporting Information). The result may resulted from the shrinking of the microspheres, which made the refractive index between the yolk/shell microspheres and the solution different from their low-temperature state.49 Drug Loading. DOX was used as a model drug to investigate the potential application of the multifunctional yolk/shell P(MAA-co-EGDMA)/P(NIPAM-co-MAA)-3 microspheres. They were loaded with DOX (1:0.3 w/w) at various pH values by shaking in the dark for 48 h at 25 °C. It was evident from Figure 4 A that the drug-loading capacity was significantly affected by the media pH values. Upon increasing the pH value from 5.0 to 10.0, the drug-loading capacity increased from 0.0502 mmol g−1 (2.91 wt %) to 0.466 mmol g−1 (27.03 wt %) and the loading efficiency increased from 9.71 wt % to 90.13 wt %. Interestingly, the drug-loading capacity revealed a remarkable enhancement at pH 7.4. Obviously, in extremely low pH environment (pH 5.0), the absence of the electrostatic interaction between the carboxyl groups of the drug carriers and the positive-charged DOX resulted in the low drug-loading capacity. However, upon increasing the media pH, the drug-loading capacity shows an obvious enhancement compared with other examples in the literatures at pHs lower than 7.4,41,50 because the cavity of the yolk/shell microspheres can provide storage space that can be used for the adsorption of the DOX molecules and the PMAA cores inside the yolk/shell microspheres also can interact with the DOX molecules via hydrogen bonding, which can tremendously improve the capacity of the drug-loading. On the basis of these advantages, these yolk/shell microspheres with PMAA cores are expected to have promising application as high-loading drug carriers. The drug-loading capacity reached the maximum at pH 8.0. According to a known report, the net charge of DOX is around zero in the media with a pH value near its isoelectric point (pI = 8.25).51 The loading process of DOX was predominantly based on the physical adsorption mechanism inside of the microspheres. Thus, the electrostatic interactions between the polymer shells and drug were minimal,52 while DOX molecules would be easily adsorbed inside through the

The effects of the feeding ratios of MAA on the VPTT of the P(MAA-co-EGDMA)/P(NIPAM-co-MAA)-X (X increases from 0 to 5) at pH 7.4 in solution were evaluated as shown in Figure 3 A. With increasing temperature, the hydrodynamic diameters decreased, corresponded to a volume phase transition. As the MAA content increased in the shell of the yolk/shell P(MAA-co-EGDMA)/P(NIPAM-co-MAA) microspheres, the hydrodynamic diameters greatly increased at a certain temperature and the VPTT shifted from about 32.0 to 44.4 °C (Table 1). To study the temperature- and pH-responsive properties of the yolk/shell microspheres, the effect of environmental pH values on the VPTT of the yolk/shell P(MAA-co-EGDMA)/ P(NIPAM-co-MAA)-3 and P(MAA-co-EGDMA)/P(NIPAMco-MAA)-5 microspheres was investigated at pH 5.0 (Figure 3 B). In comparison with the test at pH 7.4, as pH decreased, the volume phase transition occurred at a lower temperature, which resulted in the VPTT of 32.0 and 37.0 °C with the MAA feeding ratios of 3% and 5%, respectively (Table 1). It can be explained by the lower charge density of the surfaces of the yolk/shell microspheres due to the deionization of the carboxylic acid groups along with the decrease in pH values.48 The results revealed that the volume phase transition of the yolk/shell P(MAA-co-EGDMA)/P(NIPAM-co-MAA)-3 microspheres could not occur in the normal physiological media (pH 7.4) with the temperature of 37.0 °C, due to their VPTT of 41.0 °C at this pH value. However, in the weakly acidic media, such as the tumor issues, their VPTT changed to 32.0 °C, so the volume phase transition occurs because the temperature at the tumor issues is higher than 37.0 °C. Thus, it could be concluded that the yolk/shell P(MAA-co-EGDMA)/P(NIPAM-co-MAA)-3 microspheres are more suitable for investigating the controlled drug release upon the temperature change induced by pH and temperature of certain tissues, especially as the tumor-environment-responsive DDS for cancer therapy. The continuous changeability of the hydrodynamic diameter and the scattered intensity of the yolk/shell P(MAA-coEGDMA)/P(NIPAM-co-MAA)-3 microspheres were also investigated by DLS at pH 7.4 or 5.0. Under the five cycles between 25 and 55 °C at pH 7.4, their diameters expressed an obviously reversible character, and their diameters still showed obvious thermo-responsiveness and can return to the original state after five cycles (Figure S3a, Supporting Information). In 3065

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parameter n of the drug release process at pH 7.4 and 37 °C has a value of 0.4237 (