A pH-responsive poly(ether amine) micelle with hollow structure for controllable drug release

Abstract

An effective strategy was developed to fabricate pH-responsive poly(ether amine) hollow nanoparticles for the controllable loading and release of anticancer drugs. The amphiphilic poly(ether amine) containing coumarin groups (gPEAC) was first synthesized, which was further modified with myristic acid to form comb-like poly(ether amine) (acPEAC). The nanoscale micelles formed by acPEAC in aqueous solution were cross-linked through the photo-dimerization of coumarin groups. Then hollow micelles were obtained by removing their hydrophobic cores of myristic ester. The anticancer drug doxorubicin (DOX) was directly loaded into the micelles without any organic solvent. The DOX-loaded micelles displayed desirable pH-sensitive release behaviours. Moreover, the hollow micelles exhibit great potential for loading drugs and controllable release, and drug-loaded micelles indicate comparable anticancer efficacy as free DOX. This work highlights the potential for the rational design of functional polymers as smart drug carriers.

---

Introduction

Recently, the design and synthesis of nanoscale hollow nanoparticles have attracted more and more attention owing to their potential applications in encapsulation and delivery systems for chemotherapeutic drugs, gene, and proteins.^1–10 Especially, stimuli-responsive polymer based hollow nanoparticles have been synthesized with enhanced accumulation and controllable release of drugs in the desired position.^8,11–15 The hollow nanoparticles are usually responsive to such external stimuli as pH,^{4,9,11,16–19} temperature,^20,21 oxidation,^22,23 light,^24,25 etc.^26–30 Generally, the strategies for fabricating the hollow nanoparticles mainly include colloid-templated layer-by-layer (LbL) self-assembly,^31–34 polymerization from template,³⁵ interfacial polymerization³⁶ and so on.

Smart drug delivery systems for selective release of drugs on the tumor tissues are usually based on the subtle difference between the normal cells and tumor cells. For example, most cancerous tissues possess lower extracellular pH values (pH 6.0–7.4) than the normal ones and the bloodstream (pH 7.4), and the pH value drops further in tumor cells, especially in endosomes (4.5–5.5).^4,11,16–19 So pH-responsive polymers have attracted intensive attention, which could enable effective drug delivery when a nanocarrier was endocytosed and distributed in endosomal/lysosomal compartments of cells at a tumor site.^4,11,16–19 Some functional polymers including peptide amphiphile,³⁷ hydrazine,³⁸ carboxyphenylboronic acid,³⁹ PNIPAAm,⁴⁰ and PAH/PSS⁴¹ have been utilized to construct pH-responsive drug delivery systems.

Poly(ether amine) (PEA) is a multiple stimuli-responsive polymer with good hydrophilicity and biocompatibility.^25,42–46 Such material attracts our interest for its simple and “green” synthesis process. And they display sensitive response by the subtle changes of pH, temperature or ionic strength in the environment. The variable monomer structure and presence of pendant hydroxyl groups could further facilitate the synthesis of multifarious polymers with the desired function.^44,47 For example, the introduction of light-sensitive coumarin units were used to obtain photosensitive poly(ether amine) (PEAC).^25,45,48

Herein, as an extension of our previous work of multi-stimuli responsive PEAC,²⁵ we synthesized a well-defined amphiphilic comb poly(ether amine) (acPEAC) containing coumarin units and poly(ethylene glycol) (PEG). Then the micelles formed by acPEAC, with a hydrophobic core of myristyl chains and a hydrophilic corona of poly(ethylene glycol), was cross-linked with the photo-dimerization of coumarin units. Finally we obtained hPEAC with a hollow structure by removed the hydrophobic core of myristyl chains through hydrolysis. The hollow nanoparticles were used to encapsulate doxorubicin hydrochloride (DOX) and exhibited controllable release behaviour and potent cytotoxicity.

Experimental details

Materials

Myristic acid, N,N-dicyclohexylcarbodiimide (DCC), trifluoroacetic acid (TFA) and 4-dimethylaminopyridine (DMAP) were purchased from Aladdin Industrial Inc., Shanghai, China. Doxorubicin (DOX) in the form of a hydrochloride salt was purchased from Hisun Pharmaceutical Co., Ltd Zhejiang, China. Methoxy polyethylene glycol amine (mPEG-NH₂, M_n = 2000 g mol⁻¹) was purchased from YareBio Co, Shanghai, China. 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Co, Shanghai, China.

Measurements

¹H NMR spectra were characterized on a Bruker AVANCE 400 M spectrometer in CDCl₃ at 25 °C. Chemical shifts were given in parts per million with respect to tetramethylsilane (TMS) as an internal reference. Gel permeation chromatography (GPC) measurements were conducted with a Waters 410 GPC equipped with Waters Styragel column (HT4 + HT3) using CDCl₃ as the eluent, the molecular weights were calibrated with polystyrene standards, and the flow rate was set at 1.0 mL min⁻¹ at 35 °C. FT-IR spectra were recorded by the US Nicolet company's AVATAR360 type collector, collection range: 4000–400 cm⁻¹. DLS measurements were performed by Malvern Zetasizer Nano ZS90, and the scattering angle was fixed at 90°. TEM studies were performed on a JEM-1011 electron microscope operating at an acceleration voltage of 100 kV. Samples were prepared by drop-casting onto Formvar Support Films and then air-dried at room temperature before measurement. The amount of the released DOX was measured using UV-vis spectroscopy (UV-2450PC, Shimadzu) at 480 nm wavelength and calculated on the basis of following calibration curve 1 (pH 7.4) calibration curve 2 (pH 5.0) and calibration curve 3 (in DI water) using different concentrations of free DOX (2.5–100 μg mL⁻¹) in the same buffer solution or DI water:

y = 0.032x − 0.001, R2 = 0.9995 (1)

y = 0.0178x + 6 × 10⁻¹⁷, R2 = 0.9999 (2)

y = 0.0187x + 0.0118, R2 = 0.9992 (3)

here y is the absorption intensity of DOX, x is the concentration of DOX, and R is the correlation coefficient.

Synthesis of gPEAC

The mixture of mPEG-NH₂ (0.001 mol) and 4-methyl-5,7-bi(2,3-epoxypropoxy) coumarin (DEMC) (0.001 mol) were refluxed in anhydrous ethanol (10 mL) for 24 h with nitrogen protection. The synthesis of DEMC was conducted as a previous publication we have reported.²⁵ After the reaction, the mixture was poured into n-hexane and the precipitation was dried in vacuum.

Synthesis of acPEAC

The mixture of gPEAC (0.001 mol), myristic acid (0.002 mol), and DCC (0.002 mol) were added to a flask equipped with a nitrogen inlet tube. Then 15 mg DMAP and 20 mL CH₂Cl₂ were added. After 24 h reaction at room temperature, CH₂Cl₂ were removed through rotary evaporation. Then 10 mL ethyl acetate was added to dissolve the crude product, followed by centrifugation to remove precipitation. At last, the supernatant was precipitated in n-hexane and dried in vacuum.

Synthesis of hPEAC

The acPEAC aqueous solution (2 mg mL⁻¹) was subjected to UV-irradiation using a UV light at an intensity of 75 mW cm⁻² at 365 nm for 20 minutes to get the photo-dimerized crosslinked micelles (di-acPEAC). Then, trace TFA were added and reacted at room temperature for 12 h. Most solvent and TFA were removed through rotary evaporation. Then pour the solution into n-hexane and the precipitation was dried in vacuum to get hPEAC.

Preparation of gPEAC/acPEAC/hPEAC micelles

The gPEAC/acPEAC/hPEAC is an amphiphilic polymer, and can be dispersed directly in water to form micelles. 100 mg gPEAC/acPEAC/hPEAC was added into 20 mL DI water and stirred for 30 minutes.

Di-acPEAC was prepared by the following method. acPEAC (50 mg) was dissolved in DI water (50 mL) at room temperature. The solution was subjected to UV-irradiation using a UV light (UV LED Glue Curing Machine, UPUL008, Japan) at an intensity of 75 mW cm⁻² at 365 nm. To study the cross-linking kinetics of the micells, a certain amount of sample solution was made from the irradiated solution at predetermined intervals and subjected to UV-vis observation. UV-vis absorption spectra were recorded on a Shimadzu UV-2401PC UV-vis spectrophotometer with a 1.0 cm path length quartz cell.

The photo-dimerization degree (PD) was calculated from the UV-vis spectra by comparing the peak absorption at 320 nm assigned to the coumarin by the following equation:²⁵

PD (%) = [A₀ − A_t]/A₀

here, A₀ and A_t are the peak absorptions centered at 320 nm. 0 and t represent before irradiation and after t time of irradiation with UV light of 365 nm, respectively.

Critical micelle concentration (CMC) measurements

Steady state fluorescence spectra were obtained by a Perkin-Elmer LS50B luminescence spectrometer. The gPEAC/acPEAC/hPEAC solutions with various concentrations from 0.0006 to 1 g L⁻¹ were added to a series of volumetric flasks. The emission wavelength was set at 320 nm for fluorescence excitation spectra. The spectra were recorded at a scan rate of 500 nm min⁻¹. Excitation and emission slit widths are 0.75 nm, emission spectra recorded between 340–600 nm generated.

Evaluation of gPEAC/acPEAC/hPEAC stability in the fetal bovine serum

The gPEAC/acPEAC/hPEAC solutions were mixed with equal volumes of PBS solution (pH 7.4, 0.01 M) containing 10% fetal bovine serum (FBS, GIBCO) and incubated at 37 °C. At various time points, 5 mL aliquots of the solutions were removed and analyzed by DLS.

Preparation of DOX-loaded micelles (DOX@gPEAC/DOX@acPEAC/DOX@hPEAC)

DOX-loaded micelles was prepared by the following method. 20 mg DOX·HCl and 20 μL TEA were dissolved in DI water (5 mL), stir for 30 minutes, then 200 mg gPEAC/acPEAC/hPEAC was added to the solution. After 2 h, 15 mL water was added slowly to the mixture, dropwise. After stirred for another 24 h, the mixture was transferred to a 3500 Da molecular weight cut off dialysis bag and dialyzed for 48 h. The water was replaced every 8 h, and finally, the mixture in the dialysis bag was freeze-dried to give red sponge-like micellar powder. To determine the drug loading content, the UV absorbance of the aqueous solutions of the powder at 480 nm was measured based on the standard calibration curve obtained from free DOX in DI water.

Drug loading content (DLC) and drug loading efficiency (DLE) were calculated according to the following formula:^25,49

DLC (wt%) = (weight of loaded drug/weight of drug loaded micelles) × 100%

DLE (wt%) = (weight of loaded drug/weight of drug in feed) × 100%

In vitro DOX release

The freeze-dried DOX-loaded micellar powders were dissolved in phosphate saline buffer (pH 7.4) and acetate buffer (pH 5.0) at a concentration of 1 mg mL⁻¹. The above solutions were transferred into a dialysis bag. The bag was then immersed into a container with 20 mL of buffer solution at the same pH value as that in the bag. The outer phase of the buffer solution was oscillated at 37 °C (50 rpm). At selected time intervals, 3 mL of the external buffer was withdrawn for UV-vis analysis and replaced with the same amount of fresh buffer solution. The released number of DOX was determined from the absorbance at 480 nm with the help of the calibration curve of DOX in the same buffer. Then the accumulative weight and relative percentage of the released DOX were calculated as a function of incubation time.

Cell lines

Three cell lines, HeLa (cervical cancer cells, human), HepG2 (hepatocellular carcinoma cells, human) and L929 (skin fibroblast cells, mouse), supplied by the Medical Department of Jilin University, China, were chosen for cell tests. HeLa, HepG2 and L929 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO) supplied with 10% heat-inactivated FBS 2 mM L-glutamine, 100 U mL⁻¹ penicillin, and 100 μg mL⁻¹ streptomycin (Sigma), at 37 °C in a humidified atmosphere containing 5% CO₂.

Cytotoxicity

The cytotoxicity of micelles was examined by MTT assay. All sample solutions were diluted with DMEM to obtain preset concentrations. HeLa and HepG2 cells were seeded into 96-well plates with a density of 10⁴ cells per well and incubated in DMEM (100 mL) for 24 h. Then six concentrations (1.0–0.03 μg mL⁻¹) of gPEAC, acPEAC and hPEAC micelles were added to the wells, and three parallel wells for each sample were used at a specific concentration. After co-incubation with cells for 48 h, 20 μL of MTT solution in PBS (5 mg mL⁻¹) was added to each well and the plate was incubated for another 4 h at 37 °C. After that, the medium containing MTT was removed, and 150 μL of DMSO was added to each well to dissolve the MTT formazan crystals. Finally, the plates were shaken for 5 min, and the absorbance of formazan product was measured at 490 nm by a Bio-Rad 680 microplate reader.

For anticancer activity analyses, we also use MTT assay to evaluate the cytotoxicity of DOX@gPEAC, DOX@acPEAC and DOX@hPEAC micelles and free DOX against HeLa cells with different DOX dosages from 0.001 to 100 μg mL⁻¹.

Cellular uptake studies

Cellular uptakes by HeLa cells were examined using confocal laser scanning microscope (CLSM). HeLa cells were seeded in 6-well culture plates (a sterile coverslip was put in each well) at a density of 1 × 10⁵ cells per well and allowed to adhere for 24 h. Then the cells were treated with free DOX (5 μg mL⁻¹) or DOX-loaded PEAC/diPEAC (5 μg mL⁻¹, equivalent DOX concentration). After incubation for 0.5 h, 4 h and 24 h at 37 °C, the supernatant was carefully removed and the cells were washed three times with ice-cold PBS and fixed with 4% formaldehyde. After the nucleus was stained with DAPI, the slides were mounted. CLSM images were captured via confocal microscope (Carl Zeiss LSM 710) under the same conditions.

Results and discussion

Synthesis of poly(ether amine)

Poly(ether amine) was synthesized through ring-open polymerization of DEMC in the presence of mPEG-NH₂. DEMC containing coumarin was made in our previous work.²⁵ The product of gPEAC was modified with myristic acid to synthesize acPEAC as shown in Scheme 1. Sequentially, the cross-linked nanoparticles were obtained by the photo-crosslinking of coumarin units of acPEAC micelles; finally, their hydrophobic cores were removed through hydrolysis, and we got hPEAC which has a hollow structure. The preparation process was shown in Scheme 1.

Scheme 1 Schematic illustration of the fabrication process of the hollow micelles.

The structures of the above polymers were validated by ¹H NMR spectra (Fig. 1). The typical peaks of PEG and coumarin could be observed clearly in Fig. 1. The methylene and methine protons were overlapped and gave the signals at 3.75–3.30 ppm. The chemical shifts at 6.0, 6.4 and 6.5 ppm in CDCl₃ were pointed to the protons of coumarin groups, which exist in ¹H NMR spectra of both gPEAC and acPEAC. The intensity is attenuated in hPEAC owing to the photo-dimerization of coumarin. Three signals at 0.9, 1.2 and 1.6 ppm were the characteristic peaks of the myristic acid in the acPEAC. For hPEAC, the signals at 0.9 and 1.6 ppm were disappeared, indicating the successful remove of myristyl chains. GPC was utilized to determine the molecular weights M_n and (M_w/M_n). M_n and polydispersity for gPEAC is 0.87 × 10⁴ and 2.72, respectively, while the number is 1.18 × 10⁴ and 1.72 for acPEAC.

Fig. 1 ¹H NMR spectra of mPEG-NH₂, gPEAC, acPEAC and hPEAC in CDCl₃.

FTIR spectra of gPEAC, acPEAC and hPEAC were shown in Fig. 2. A strong stretching vibration of hydroxyl peaks at 3400 cm⁻¹ were obsered in gPEAC, acPEAC and hPEAC, but no corresponding peaks in DEMC and mPEG-NH₂. In addition, compared with mPEG-NH₂, new peaks at 1722 and 1616 cm⁻¹ were assigned to stretching vibration of C=O, and the aromatic ring of the coumarin moieties appeared in the FT-IR spectra of gPEAC, acPEAC and hPEAC, showing the successful introduction of coumarin groups into them. Coumarin has a strongest ultraviolet absorption peak around 320 nm, as shown in Fig. 3. Thus, DEMC, gPEAC and acPEAC have a maximum absorption peak around 320 nm, but the peak for hPEAC almost disappeared due to the photo-dimerization reaction.

Fig. 2 FTIR spectra of mPEG-NH₂, gPEAC, acPEAC, hPEAC and DEMC.

Fig. 3 UV-vis spectra of DEMC, gPEAC and acPEAC.

Poly(ether amine) nanoparticles

All the synthesized poly(ether amine) containing coumarin units (PEAC) can form micelles in aqueous solution by directly dispersing in water without any organic solvent. Coumarin itself is a very good fluorescent dye, so the PEACs do not require additional fluorescence probe, such as pyrene, to detect the critical micelle concentration (CMC). The CMCs calculated directly by using the fluorescence of coumarin according to the ref. 25, 50 and 51. As shown in Fig. 4, the CMCs were calculated to be 7.8 × 10⁻² g L⁻¹, 0.9 × 10⁻² g L⁻¹ and 3.5 × 10⁻² g L⁻¹ for gPEAC, acPEAC and hPEAC, respectively.

Fig. 4 Intensity from the fluorescence spectra with PEAC in different concentration. Inset: fluorescence emission spectra with different concentration, (A) gPEAC, (B) acPEAC, (C) hPEAC. Data were presented as mean ± standard deviation (n = 3).

Photo-dimerization of coumarin were operated upon 365 nm irradiation as illustrated in Scheme 1. acPEAC was dispersed in DI water with the concentration of 1 mg mL⁻¹. Under illumination at 365 nm for different time, nanoparticles with different degree of cross-linking were obtained. The process of cross-linking was monitored by the UV-vis spectrophotometer. As shown in Fig. 5A, maximum absorption intensity at 320 nm decreased gradually with time, indicating the occurrence of dimerization. In Fig. 5B, the photo-dimerization degree of acPEAC increased to 50% rapidly in the first 5 minutes and increased to 70% in the next 15 minutes.

Fig. 5 UV-vis spectra of acPEAC micelle upon 365 nm irradiation with different time (A), photo-dimerization degree of acPEAC as a function of irradiation time based on the absorbance change at 320 nm (B). Data were presented as mean ± standard deviation (n = 3).

Characterization of the micelles

DLS was utilized to study the size distribution of the micelles. The average diameter of gPEAC micelles was 160 nm (Fig. 6A). After esterification and hydrolysis reactions, the size of acPEAC and hPEAC changed to 220 nm and 170 nm, respectively (Fig. 6B and C). The diameter of DOX@gPEAC micelle was 190 nm (Fig. 6D) and the size of DOX@acPEAC and DOX@hPEAC changed to 250 nm and 200 nm (Fig. 6E and F). The results were listed in Table 1. The micelles of gPEAC had the hydrophobic core of coumarin units and the hydrophilic corona of PEG. When hydrophobic myristyl chains were introduced into the polymer, the inner core of micelles were enriched. With the support of hydrophobic myristyl chains, the size of acPEAC micelles was larger than that of gPEAC micelles. After removing myristyl chains, the micelles lost the internal support and shrank. However, the chain movements were limited due to the dimerization of coumarin units. Thus, the size of hPEAC micelles is little larger than that of gPEAC micelles.

Fig. 6 DLS results of gPEAC micelles (A), acPEAC (B), hPEAC (C), DOX@gPEAC (D), DOX@acPEAC (E) and DOX@hPEAC (F); TEM images of PEAC micelles (G), acPEAC (H), hPEAC (I), DOX@gPEAC (J), DOX@acPEAC (K) and DOX@hPEAC (L). (Bar = 500 nm.)

Table 1 The average diameter of micelles test by DLS and TEM

Micelle	DLS (nm)	TEM (nm)
gPEAC	160	110
acPEAC	220	120
hPEAC	170	100
DOX@gPEAC	190	130
DOX@acPEAC	250	150
DOX@hPEAC	200	135

---

The average diameter of gPEAC, acPEAC and hPEAC micelles tested by TEM was about 110 nm (Fig. 6G), 120 nm (Fig. 6H) and 100 nm (Fig. 6I), respectively. For their DOX-loaded micelles, the diameter was about 130 nm (Fig. 6J), 150 nm (Fig. 6K) and 135 nm (Fig. 6L), respectively. The average diameters were summarized in Table 1. Micellar size measured by TEM was smaller than DLS due to the volume shrinkage in the drying process of the preparation of the TEM sample.^52,53 All particles presented good dispersibility, even the cross-linked particles, implying that there was no micellar aggregation during the process of cross-linking.

Stability of micelles

The stability of the gPEAC, acPEAC and hPEAC micelles was studied in PBS (pH 7.4) containing 10% FBS at 37 °C by monitoring the particle size change as a function of time. As it was shown in Fig. 7, there was no significant change in size during 24 h, suggesting that neither aggregation nor destabilization was induced by serum proteins.

Fig. 7 Particle size and PDI of micelles in PBS (pH 7.4) containing 10% FBS at 37 °C for different time periods determined by DLS. Data were presented as mean ± standard deviation (n = 3).

Drug loading and release in vitro

DOX was used as a model anticancer drug to investigate the drug loading and release of different micelles. Drug-loading was carried out by directly dispersing DOX and PEACs together into water without any organic solvents or auxiliaries. DOX content in feed is 10 wt% for different polymeric micelles. The drug loading contents (DLC) and the drug loading efficiency (DLE) of DOX@gPEAC were only 4.12 wt% and 41.2%. The data were increased to 6.26 and 62.6%m for DOX@acPEAC. DOX@hPEAC had the highest DLC and DLE (7.43 and 74.3%) which was much high than those of DOX@gPEAC. The results indicates that the hollow structure has the advantage of drug loading.

The all PEACs exhibited pH-response behaviors. Fig. 8 show the effect of pH value on Z-average diameters of gPEAC, acPEAC, hPEAC micelles. These three micelles presented similar pH-responsive behavior. Their diameters decreased with increasing pH value until pH 7.4. There was an apparent transition point around pH 7.4. Tertiary amines uniformly distributed along the polymer backbones, which is protonated at low pH. The degree of protonation goes down with increasing pH. Thus, strong electrostatic repulsion of the protonated tertiary amines at low pH made the PEACs micelles loose, which lead to the large diameters. The unique property of the micelles is considerable for controlling the release of the drug.

Fig. 8 Z-average diameters of gPEAC, acPEAC, hPEAC micelles at room temperature and different pH. Data were presented as mean ± standard deviation (n = 3).

The in vitro DOX release from DOX@gPEAC, DOX@acPEAC and DOX@hPEAC was investigated in two different buffered solutions (pH 7.4 and 5.0) at 37 °C. As shown in Fig. 9, burst release of DOX was not observed for these micelles at pH 7.4 and pH 5.0, demonstrating DOX was well entrapped in them. At pH 7.4 (Fig. 9A), only approximately 20% of DOX was released from DOX@gPEAC micelles during 60 h, while about 66% of DOX was released under pH 5.0 (Fig. 9B). The similar behavior was observed in DOX@acPEAC micelles, and the release amount was about 17% from DOX@acPEAC at pH 7.4 for 60 h, compared with a 53% release at pH 5.0. The release for DOX@hPEAC was much lower than the above micelles which was 11% at pH 7.4 and 28% at pH 5.0. The cross-linking showed a certain influence on the release rate, because the cross-linking structure made the micellar more compact, and suppressed the release of DOX from the micelles. The sustained release of DOX from the micelles was conducive to the extension of drug circulation time and protects the drug from enzyme degradation, rapid renal clearance and interactions with serum proteins and thus would significantly enhance drug delivery to tumors. These data also indicated that all PEAC micelles possess pH-sensitivity, which is useful for drug delivery to obtain a long-term circulation in blood and an effective drug release at the tumor site.

Fig. 9 In vitro release profiles of DOX from DOX@gPEAC, DOX@acPEAC, and DOX@hPEAC micelles at pH 7.4 (A) and pH 5.0 (B) at 37 °C. Data were presented as mean ± standard deviation (n = 3).

Cell uptake

The cellular uptake and intracellular release of DOX@hPEAC were investigated by CLSM. Free DOX and DOX@hPEAC were incubated with HeLa cells at 37 °C for 0.5 h, 4 h and 24 h. Red fluorescence located mainly in the cellular nuclei after 0.5 h of incubation with free DOX (Fig. 10A). However, when the cells were incubated with DOX@hPEAC micelles, the fluorescence was observed mainly in the cytoplasm rather than the cell nuclei (Fig. 10D). After 4 h incubation, the fluorescence in the nuclei of the cells became stronger for free DOX (Fig. 10B). The cells incubated with DOX@hPEAC micelles for 4 h emitted enhanced fluorescence in the cytoplasm as well as weak fluorescence in nuclei (Fig. 10E). When the incubation period increased to 24 h, the fluorescence in the nuclei of the cells became weaker for free DOX (Fig. 10C). For DOX@hPEAC micelles, the red fluorescence in the cytoplasm increased with time and slightly increased in the nuclei (Fig. 10F). These data suggest that the internalization mechanism of DOX@hPEAC micelles is different from that of free DOX.

Fig. 10 CLSM-images of HeLa cells incubated with free DOX and DOX@hPEAC micelles for 0.5 h, 4 h, and 24 h. For each row, images from left to right were the cells with nucleus stained with DAPI (blue), with DOX (red) fluorescence, and overlapped images. (Bar = 20 μm.)

Cytotoxicity

MTT assay was applied to evaluate the cytotoxicity of polymer micelles by using HeLa and HepG2 cells. After incubation with gPEAC, acPEAC and hPEAC micelles at gradient concentrations from 1.0 to 0.03 μg mL⁻¹ for 24 h, cells showed high viability (80% and above) even the concentrations of micelle up to 1 mg mL⁻¹, displaying that these micelles have good cytocompatibility and could be used as drug carriers (Fig. 11).

Fig. 11 Cell viability of HeLa and HepG2 cells incubated with PEAC and diPEAC micelles. Data were presented as mean ± standard deviation (n = 3).

The in vitro cytotoxicities of DOX-loaded micelles and the free DOX were evaluated by MTT assay against HeLa and L929 cells with different DOX dosages from 0.001 to 100 μg mL⁻¹. As shown in Fig. 12, the half maximal inhibitory concentration (IC50) values for free DOX, DOX@gPEAC, DOX@acPEAC and DOX@hPEAC against HeLa and L929 cell lines were 0.48, 2.34, 4.22, 5.88 μg mL⁻¹ and 1.98, 4.48, 7.02, 10.18 μg mL⁻¹, respectively. These results demonstrated that DOX-loaded micelles were able to enter the cells and exhibited a suitable pharmacological effect on cancer cells and normal cells. Free DOX showed higher inhibition for cancer cell proliferation than that of the DOX-loaded micelles, which had been seen in the previous literature.^54,55 The lower cytotoxicity of DOX-loaded micelles can be attributed to the slow release of DOX from micelles as evidenced by the in vitro release in Fig. 9 and delayed nuclear uptake in HeLa cells as validated by internalization studies by CLSM (Fig. 10).

Fig. 12 Cell viability of HeLa (A) and L929 (B) cells against DOX, DOX@gPEAC, DOX@acPEAC and DOX@hPEAC micelles after cultured for 48 h with different DOX dosages.

Conclusions

In summary, a coumarin-containing pH-responsive polymeric hollow nanoparticle has been successful designed and prepared. The hollow nanoparticles display excellent stability, and load drugs easily in the absence of organic solvents. The hollow nanoparticles have been exploited as carriers for loading of anticancer drug DOX, exhibiting a pH-responsive controllable release. DOX-loaded micelles indicate effective cellular uptake and comparable cytotoxicity with free DOX. This hollow nanoparticle is expected to be used for environment-responsive release of drugs in biological systems.

Acknowledgements

The authors would like to thank the financial support from National Natural Science Foundation of China (No. 51203043, No. 51403051, No. 21502042) and Developing Program of Natural Science of Henan province (No. 142300410122).

Notes and references

1. Y. J. Wang, V. Bansal, A. N. Zelikin and F. Caruso, Nano Lett., 2008, 8, 1741–1745.
2. X. W. Lou, L. A. Archer and Z. Yang, Adv. Mater., 2008, 20, 3987–4019.
3. T. Y. Liu, K. H. Liu, D. M. Liu, S. Y. Chen and I. W. Chen, Adv. Funct. Mater., 2009, 19, 616–623.
4. C. Fan, T. Bian, L. Shang, R. Shi, L. Z. Wu, C. H. Tung and T. Zhang, Nanoscale, 2016, 8, 3923–3925.
5. J. X. Huang, W. B. Li, Y. Li, C. D. Luo, Y. C. Zeng, Y. H. Xu and J. H. Zhou, J. Mater. Chem. B, 2014, 2, 6848–6854.
6. P. Xu, S. Y. Li, Q. Li, E. A. Van Kirk, J. Ren, W. J. Murdoch, Z. J. Zhao, M. Radosz and Y. Q. Shen, Angew. Chem., Int. Ed., 2008, 47, 1260–1264.
7. L. F. Tan, T. L. Liu, X. L. Ren, C. H. Fu, B. Liu, J. Ren and X. W. Meng, ACS Appl. Mater. Interfaces, 2016, 8, 11237–11245.
8. P. C. Du and P. Liu, Langmuir, 2014, 30, 3060–3068.
9. M. Y. Wu, Y. Chen, L. X. Zhang, X. Y. Li, X. J. Cai, Y. Y. Du, L. L. Zhang and J. L. Shi, J. Mater. Chem. B, 2015, 3, 766–775.
10. L. Praveen, S. Saha, S. K. Jewrajka and A. Das, J. Mater. Chem. B, 2013, 1, 1150–1155.
11. F. J. Xu, E. T. Kang and K. G. Neoh, Biomaterials, 2006, 27, 2787–2797.
12. S. R. Deka, A. Quarta, R. D. Corato, A. Riedinger, R. Cingolani and T. Pellegrino, Nanoscale, 2011, 3, 619–629.
13. Y. Chen, H. R. Chen, D. P. Zeng, Y. B. Tian, F. Chen, J. Feng and J. L. Shi, ACS Nano, 2010, 4, 6001–6013.
14. J. Z. Du and S. P. Armes, J. Am. Chem. Soc., 2005, 127, 12800–12801.
15. K. Pafiti, Z. X. Cui, D. Adlam, J. Hoyland, A. J. Freemont and B. R. Saunders, Biomacromolecules, 2016, 17, 2448–2458.
16. R. Mo, Q. Sun, J. W. Xue, N. Li, W. Y. Li, C. Zhang and Q. N. Ping, Adv. Mater., 2012, 24, 3659–3665.
17. H. M. He, S. Chen, J. Z. Zhou, Y. Dou, L. Song, L. Che, X. Zhou, X. Chen, Y. Jia and J. X. Zhang, Biomaterials, 2013, 34, 5344–5358.
18. X. W. Ding, Y. Liu, J. H. Li, Z. Luo, Y. Hu, B. L. Zhang, J. J. Liu, J. Zhou and K. Y. Cai, ACS Appl. Mater. Interfaces, 2014, 6, 7395–7407.
19. D. Schmaljohann, Adv. Drug Delivery Rev., 2006, 58, 1655–1670.
20. K. J. Mackenzie and M. B. Francis, J. Am. Chem. Soc., 2013, 135, 293–300.
21. U. Rocha, C. J. D. Silva, W. F. Silva, I. Guedes, A. Benayas, L. M. Maestro, M. A. Elias, E. Bovero, F. C. J. M. V. Veggel and J. A. G. Solé, ACS Nano, 2013, 7, 1188–1199.
22. L. S. Yan, L. X. Yang, H. Y. He, X. L. Hu, Z. G. Xie, Y. B. Huang and X. B. Jing, Polym. Chem., 2013, 3, 2403–2412.
23. W. Zhang, W. H. Lin, Q. Pei, X. L. Hu, Z. G. Xie and X. B. Jing, Chem. Mater., 2016, 28, 4440–4446.
24. A. A. Aimetti, A. J. Machen and K. S. Anseth, Biomaterials, 2009, 30, 6048–6054.
25. H. Z. He, Y. R. Ren, Y. G. Dou, T. Ding, X. M. Fang, Y. Q. Xu, H. Xu, W. K. Zhang and Z. G. Xie, RSC Adv., 2015, 5, 105880–105888.
26. W. A. Morgan, Y. Dingg and P. H. Bach, Renal Failure, 2009, 20, 441–450.
27. M. Motornov, Y. Roiter, I. Tokarev and S. Minko, Prog. Polym. Sci., 2010, 35, 174–211.
28. L. Zhou, Z. W. Chen, K. Dong, M. L. Yin, J. S. Ren and X. J. Qu, Biomaterials, 2014, 35, 8694–8702.
29. Y. Qi, J. N. Shen, Z. W. Zhang, H. J. Yu and Y. Li, Adv. Drug Delivery Rev., 2013, 65, 1699–1715.
30. N. Li, C. H. Guo, Z. Y. Duan, L. Z. Yu, K. Luo, J. Lu and Z. W. Gu, J. Mater. Chem. B, 2016, 4, 3760–3769.
31. A. Elbakry, E. C. Wurster, A. Zaky, R. Liebl, E. Schindler, P. B. Kreisel, T. Blunk, R. Rachel, A. Goepferich and M. Breunig, Small, 2012, 8, 3847–3856.
32. S. Y. Zhang, J. T. Wu, Y. L. Zhang, J. X. Leng, W. P. Yang, Z. G. Zhang and J. Y. Zhao, Sci. Rep., 2015, 5, 15114.
33. D. I. Gittins and F. Caruso, Adv. Mater., 2000, 12, 1947–1949.
34. M. Keeney, X. Y. Jiang, M. Yamane, M. Lee, S. Goodman and F. Yang, J. Mater. Chem. B, 2015, 3, 8757–8770.
35. G. D. Fu, Z. Shang, L. Hong, E. T. Kang and K. G. Neoh, Macromolecules, 2005, 38, 7867–7871.
36. S. Yang and H. Liu, J. Mater. Chem., 2006, 16, 4480–4487.
37. A. Ghosh, M. Haverick, K. Stump, X. Yang, M. F. Tweedle and J. E. Goldberger, J. Am. Chem. Soc., 2012, 134, 3647–3650.
38. F. Wang, G. M. Pauletti, J. Wang, J. Zhang, R. C. Ewing, Y. Wang and D. Shi, Adv. Mater., 2013, 25, 3485–3489.
39. Z. Luo, K. Y. Cai, Y. Hu, B. L. Zhang and D. W. Xu, Adv. Healthcare Mater., 2012, 1, 321–325.
40. K. S. Soppimath, D. C. Tan and Y. Y. Yang, Adv. Mater., 2005, 17, 318–323.
41. H. Chen, T. Moore, B. Qi, D. C. Colvin, E. K. Jelen, D. A. Hitchcock, J. He, O. T. Mefford, J. C. Gore and F. Alexis, ACS Nano, 2013, 7, 1178–1187.
42. Y. R. Ren, X. S. Jiang, G. L. Yin and J. Yin, J. Polym. Sci., Part A: Polym. Chem., 2010, 48, 327–335.
43. Y. R. Ren, X. S. Jiang, R. Liu and J. Yin, J. Polym. Sci., Part A: Polym. Chem., 2009, 47, 6353–6361.
44. R. Wang, X. S. Jiang, G. L. Yin and J. Yin, Polymer, 2011, 52, 368–375.
45. W. Rui, Y. Bing, X. S. Jiang and Y. Jie, Adv. Funct. Mater., 2012, 22, 2606–2616.
46. W. T. Lu, X. X. Wang, R. Cheng, C. Deng, F. H. Meng and Z. Y. Zhong, Polym. Chem., 2015, 6, 6001–6010.
47. X. S. Jiang, C. Di, Y. Bing and Y. Jie, ACS Appl. Mater. Interfaces, 2011, 3, 1749–1756.
48. X. S. Jiang, R. Wang, Y. R. Ren and J. Yin, Langmuir, 2009, 25, 9629–9632.
49. H. H. Kuang, H. Y. He, J. Hou, Z. G. Xie, X. B. Jing and Y. B. Huang, Macromol. Biosci., 2013, 13, 1593–1600.
50. T. J. V. Prazeres, M. Beija, F. V. Fernandes, P. G. A. Marcelino, J. P. S. Farinha and J. M. G. Martinho, Inorg. Chim. Acta, 2012, 381, 181–187.
51. J. Q. Jiang, Q. Z. Shu, X. Chen, Y. Q. Yang, C. L. Yi, X. Q. Song, X. Y. Liu and M. Q. Chen, Langmuir, 2010, 26, 14247–14254.
52. K. Hayakawa, T. Yoshimura and K. Esumi, Langmuir, 2003, 19, 5517–5521.
53. C. Feng, Z. Shen, Y. G. Li, L. N. Gu, Y. Q. Zhang, G. L. Lu and X. Y. Huang, J. Polym. Sci., Part A: Polym. Chem., 2009, 47, 1811–1824.
54. X. T. Shuai, A. Hua, N. Nasongkla, S. Kim and J. M. Gao, J. Controlled Release, 2004, 98, 415–426.
55. B. Younsoo, F. Shigeto, H. Atsushi and K. Kazunori, Angew. Chem., Int. Ed., 2003, 42, 4640–4643.

---