Redox and pH Dual-Responsive Polymeric Micelles with Aggregation

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Biological and Medical Applications of Materials and Interfaces

Redox and pH Dual-Responsive Polymeric Micelle with AggregationInduced Emission Feature for Cellular Imaging and Chemotherapy Weihua Zhuang, Yangyang Xu, Gaocan Li, Jun Hu, Boxuan Ma, Tao Yu, Xin Su, and Yunbing Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02890 • Publication Date (Web): 08 May 2018 Downloaded from http://pubs.acs.org on May 8, 2018

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ACS Applied Materials & Interfaces

Redox and pH Dual-Responsive Polymeric Micelle with Aggregation-Induced Emission Feature for Cellular Imaging and Chemotherapy Weihua Zhuang, Yangyang Xu, Gaocan Li*, Jun Hu, Boxuan Ma, Tao Yu, Xin Su, and Yunbing Wang*

National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu 610064, China Corresponding Author * E-mail: [email protected],

* E-mail: [email protected]

ABSTRACT: Intelligent polymeric micelles for antitumor drug delivery and tumor bioimaging have drawn a broad attention due to the reduced systemic toxicity, enhanced efficacy of drugs and potential application of tumor diagnosis. Herein, we developed a multifunctional polymeric micelle system based on pH and redox dual responsive mPEG-P (TPE-co-AEMA) copolymer for stimuli triggered drug release and

aggregation-induced

emission

(AIE)

active

imaging.

These

mPEG-P

(TPE-co-AEMA) based micelles showed excellent biocompatibility and emission property, exhibiting great potential application for cellular imaging. Furthermore, antitumor drug doxorubicin (DOX) could be encapsulated during self-assembly process with high loading efficiency, and a DOX-loaded micelle system with the size of 68.2 nm and narrow size distribution could be obtained. DOX-loaded micelles

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demonstrated great tumor suppression ability in vitro and the dual-responsive triggered intracellular drug release could be further traced. Moreover, DOX-loaded micelles could efficiently accumulate in tumor site because of EPR effect and long-circulation of micelles. Comparing with free DOX, DOX-loaded micelles exhibited better anti-tumor effect and significantly reduced adverse effects. Given the efficient accumulation targeting to tumor tissue, dual-responsive drug release and excellent aggregation-induced emission property, this polymeric micelle would be a potential candidate for cancer therapy and diagnosis.

KEYWORDS: polymeric micelles, dual responsive, aggregation-induced emission, drug delivery, bioimaging.

1 INTRODUCTION Chemotherapy is still one of the most common strategies for cancer treatment. However, conventional chemotherapeutics are restricted by the serious adverse effects and poor solubility.1-3 An innovative strategy has been provided utilizing the enhanced permeability and retention (EPR) effect for tumor targeted antitumor drug delivery.4 Since then, a large number of long-circulating nanoparticles have been developed for antitumor drugs delivering in a controlled way, which significantly reduce the adverse effects and improve antitumor efficacy.5-6 Among these reported nanoparticles, polymeric micelle has showed a great prospect in clinical application and several polymeric micelle drug delivery systems have reached clinical trials.7-8 However, the


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low bioavailability and limited improvement in therapeutic effect due to the slow drug release of conventional micelles are still the urgent problems to improve anticancer efficacy. Recently,

intelligent

polymeric

micelles

responding

to

the

special

microenvironment of tumor tissue have been developed and the stimuli-triggered drug release has been confirmed to significantly improve antitumor efficacy.9-13 One of the most common strategies is to introduce pH response to design smart nanoparticles due to the great pH variation among cellular compartments such as endosome and lysosome (pH ~ 5.0)，extracellular tumor tissue (pH ~ 6.5) and normal tissue (pH 7.4).9,14 Conventional method is to introduce pH sensitive chemical bonds to nanocarriers, such as acetal, imine and orthoester, which can be cleaved by acid, yet suffers from storage stability.10, 15-16 Therefore, the development of new kinds of more intelligent pH-responsive polymers, such as 2-azepane ethyl methacrylate (PAEMA), which is hydrophobic when pH is higher than 6.8 while can convert rapidly to be hydrophilic when pH is lower than 6.8,17 is in high demand. In addition, redox response based on disulfide bond is widely utilized to design intelligent nanocarriers because of the much higher intracellular concentration of glutathione (GSH) (approximately 2 to 10 mM) in tumor cells and much lower concentration in extracellular space (approximately 2 to 20 µM).10 Nanocarriers equipped with redox-responsive feature have been proven to improve the therapeutic effect.18-19 Furthermore, in order to make the best of the special microenvironment of tumor, dual-responsive or multi-responsive nanoparticles exhibiting improved antitumor



efficacy compared to the single stimuli-responsive nanoparticles due to the rapid release of drugs have been developed.20-22 Recently, nanocarriers with simultaneous bioimaging and intelligent drug delivery have received more and more attention.10, 23 To achieve this goal, development of “visible” nanocarriers is in urgent need. Conventional method is to encapsulate chromophores as fluorescent probes. However, traditional chromophores are usually restricted by aggregation-caused quenching (ACQ) effect with dramatic decrease in their fluorescence efficiency in the high concentration.24-25 In 2001, a new kind of fluorescent molecules emitting strong fluorescence in the aggregated state was firstly reported by Tang’s group known as aggregation-induced emission (AIE) effect.26 In view of this unique characteristic, AIE-based fluorescent molecules have been widely employed for bioimaging and monitoring the in vivo and intracellular drug delivery especially for drugs without fluorescence.27-30 In addition, AIE-based fluorogen, such as tetraphenylethene (TPE), can also be introduced as hydrophobic segment of the nanocarriers to monitor the distribution of carriers. In this work, AIE labeled polymeric micelles have been prepared based on mPEG-P (TPE-co-AEMA) copolymer for bioimaging and pH/redox dual responsive drug release at targeting sites (Scheme 1). DOX, a kind of antitumor drug, is encapsulated in the core of micelles with uniform particle size of about 68.2 nm. Furthermore, accelerated drug release of drug-loaded micelles with dual response to acidic medium and high level of GSH has been achieved and these DOX-loaded micelles exhibit great antitumor efficacy in vitro and great cellular imaging ability. The enhancement


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of antitumor efficacy in vivo response to pH/redox stimuli and AIE active bioimaging make this functional polymeric nanocarrier a prominent candidate for cancer theranostics.

Scheme 1. Illustration of DOX-loaded polymeric micelles based on mPEG-P (TPE-co-AEMA) for ultrasensitive pH and redox triggered drug release and bioimaging.

2 EXPERIMENTS 2.1 Materials and Methods



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4-Cyanopentanoic acid dithiobenzoate was obtained from Sigma-Aldrich. Poly (ethylene glycol) methyl ether amine (mPEG-NH2) was obtained from Shanghai Fansheng Biotechnology Co., Ltd. (Shanghai, China) and used without further purification. Doxorubicin hydrochloride (DOX·HCl) was obtained from Adamas Reagent, Ltd. (Shanghai, China). TPE-OH was prepared according to previous work.29 2.2 Synthesis of Poly (TPE-co-AEMA) TPE-SS-MA (0.40 g, 0.66 mmol) and 4-cyanopentanoic acid dithiobenzoate (96.0 mg, 0.34 mmol) were added to the solution of AEMA (0.58, 2.76 mmol) in THF (10 mL). The tube was sealed and degassed with three cycles of freeze-pump-thaw procedure. After reaction at 70 oC for 24 h, the solution was allowed to dialyze against deionized water (MCWO = 1000) for 24 h. Poly (TPE-co-AEMA) was obtained through freeze-drying. 2.3 Synthesis of mPEG-P (TPE-co-AEMA) Under an atmosphere argon, the solution of poly (TPE-co-AEMA) (0.30 g, 0.12 mmol), EDC·HCl (24.52 mg, 0.13 mmol) and NHS (14.72 mg, 0.13 mmol) in THF/DCM (v/v = 1/1, 20 mL) was stirred at room temperature for 24 h. The solution of PEG5k-NH2 (0.47 g, 0.093 mmol) and TEA (10.34 mg, 0.10 mmol) in DCM (10 mL)

was

added

dropwise

into

the

stirring

solution

of

poly

(TPE-co-AEMA)-COO-NHS. The resulting solution was allowed to stir at room temperature for another 24 h. The solvent was removed under vacuum and the residue was redissolved in THF (20 mL). Afterwards, the solution was dialyzed against


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deionized water (MWCO = 5000) for 48 h and filtrated. mPEG-P (TPE-co-AEMA) was obtained by freeze drying. 2.4 Preparation and Characterization of mPEG-P (TPE-co-AEMA) Micelles DOX-loaded mPEG-P (TPE-co-AEMA) micelles were prepared via dialysis. Briefly, 20 mg of mPEG-P (TPE-co-AEMA) copolymer, DOX • HCl (4 mg) and TEA (10 µL) were totally dissolved in the mixed solvent (THF/DMF, 1/1, v/v, 4 mL). Afterwards, the resulting solution was added dropwise into stirring deionized water (10 mL, pH = 7.4) and the mixture was allowed to stir at room temperature for 4 h. The resulting solution was dialyzed (MWCO = 3500) against deionized water (pH = 7.4) for 24 h to remove the unencapsulated drug. Drug loading content and drug loading efficacy were measured using the reported method.31 Blank micelles were prepared similarly without the addition of drug. DOX-loaded micelles were lyophilized for further application. The AIE behavior of mPEG-P (TPE-co-AEMA) micelles was investigated by fluorescence spectra (excited at 330 nm) with the same concentration of copolymer in pure water or THF. The redox-sensitive behavior of mPEG-P (TPE-co-AEMA) micelles was investigated by incubating the micelles with 10 mM GSH and the particle size changes were monitored by DLS at selected time interval. The influence of pH and 10 mM GSH on particle size was also studied by TEM after incubating micelles in medium (pH 6.5) containing 10 mM GSH for 4 h. 2.5 In Vitro Drug Release The dual stimuli-responsive of polymeric micelles was further evaluated by in vitro



drug release of DOX-loaded mPEG-P (TPE-co-AEMA) micelles at different mediums. Briefly, 2 mL drug-loaded micelles solution (1 mg/mL) was transferred to a dialysis tube (MWCO = 3500) and the tube was immersed into 40 mL release medium with different pH or different concentrations of GSH. At selected time interval, 2 mL release medium was removed to measure the release amount of drug and 2 mL fresh release medium was added. The whole processer was kept in the dark with continuous shaking. 2.6 Cellular Imaging of mPEG-P (TPE-co-AEMA) Micelles 4T1 cells were planted in glass dishes (diameter = 35 mm, 1×104 per dish) and incubated for 24 h. Then, mPEG-P (TPE-co-AEMA) micelles were added with a total TPE concentration of 50 µM. The culture medium was removed and the cells were washed with PBS for three times after incubation for 1 h and 3 h. The cultured cells were imaged by confocal laser scanning microscopy (CLSM) excited at 405 nm. As for DOX-loaded micelles, after incubation for 1 h, 3 h and 5 h after the addition of DOX-loaded micelles (10 µg DOX/mL), the cells were washed by PBS for 3 times and imaged by CLSM. 2.7 In Vivo Antitumor Effect Studies All animal experiments were carried out according to the institutional and NIH guidelines for the care and use of research animals. Subcutaneous 4T1 models were built in the right back area of BALB/c mice (20 ± 2 g) at a density of 1 × 106 per mouse. After the volume reached approximately 100 mm3 (tumor volume V (mm3) = 1/2 × L × W2, where L and W stood for the length and the width of the tumor), the


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mice were randomized to three groups (n = 7) and the treatment were started. Free DOX and DOX-loaded micelles (5 mg DOX/Kg body weight) were separately injected via the tail vein of mice once every 4 days for 4 times in total. And physiological saline with the same volume was injected into the control group. The adverse effects and real-time antitumor efficacy were evaluated by monitoring the changes of body weight and tumor volume every two days. 2.8 Ex Vivo Fluorescence Imaging Study DOX-loaded mPEG-P (TPE-co-AEMA) micelles were injected into 4T1 tumor bearing female BALB/c mice (5 mg DOX/Kg body weight). At pre-selected time interval, the major organs included hearts, livers, spleens, lungs and kidneys as well as tumors were excised after the mice were sacrificed. Ex vivo imaging was carried out on a Maestro Imaging System. 2.9 Histological Examination and Immunohistochemical Analysis On the 21st day, all mice were sacrificed and the major organs as well as tumor were excised, which were washed with PBS before being fixed with 4% formaldehyde. After dehydrating with gradient ethanol, Organs and tumor were embedded in paraffin blocks to prepare the tissue sections of 5 µm. After dewaxing and staining, histopathological evaluation of these tissue sections was carried out with haematoxylin and eosin (H&E). CD31, Ki-67 and TUNEL were analyzed according to literatures.32-34

3 RESULTS and DISCUSSION 3.1 Synthesis of mPEG-P (TPE-co-AEMA) Copolymer



mPEG-P (TPE-co-AEMA) copolymer was obtained via several reactions as shown in Scheme 2. The 1H NMR results confirmed that the synthesis of HO-SS-MA (Figure S1), TPE-tB (Figure S2), TPE-COOH (Figure S3), TPE-SS-MA (Figure S4) and AEMA (Figure S5) was successful. After the monomers were prepared, P (TPE-co-AEMA) was obtained by reversible addition-fragmentation chain transfer polymerization (RAFT), which was confirmed by the 1H NMR (Figure S6). The degree of polymerization (DP) of TPE-SS-MA and AEMA were calculated as 1 and 8, respectively, and the molecular weight distribution (MWD) of P (TPE-co-AEMA) was determined to be 1.16 in THF (Figure S7). Finally, mPEG-P (TPE-co-AEMA) copolymer was obtained through amidization between the terminal amino of PEG5K-NH2 and the terminal carboxyl group of P (TPE-co-AEMA). The characteristic peaks of mPEG5k-NH2 and P (TPE-co-AEMA) and the full extent of amidation was confirmed by 1H NMR analysis shown in Figure 1, which was also in according with the GPC result (MWD = 1.26) in Figure S7. Therefore, the mPEG-P (TPE-co-AEMA) copolymer studied in this work could be defined as mPEG5k-p (TPE1-co-AEMA8), with a molecular weight of 7.6 kDa.


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Scheme 2. Synthesis of mPEG-P (TPE-co-AEMA) copolymer.



Figure 1. 1H NMR spectrum of mPEG-p (TPE-co-AEMA) copolymer in CDCl3. 3.2 Characterization and In Vitro Drug Release of mPEG-P (TPE-co-AEMA) Micelles Hydrophobic antitumor drug DOX was encapsulated in the core of mPEG-P (TPE-co-AEMA) micelles due to the π-π staking and hydrophobic interaction between DOX and TPE. As shown in Figure 2A, the particle size of blank micelles was determined to be 50.7±2.4 nm with a small PdI of 0.092, while an increased size of DOX-loaded micelles to 68.2±2.0 nm with a PdI of 0.142. The small particle size of DOX-loaded mPEG-P (TPE-co-AEMA) micelles would be suitable for nanoparticles accumulating in tumor tissue via EPR effect. Moreover, TEM was further conducted to study the morphology of DOX-loaded mPEG-P (TPE-co-AEMA) micelles (Figure 2B), which was fairly uniform sphere in shape but smaller than the result analyzed by DLS owing to the dehydration of hydrophilic shell during the sample preparation for TEM. The micellar morphology was further evaluated by AFM (Figure S8A), showing well-defined spheroidal in shape, which was in accordance with the result of TEM. PAEMA was supposed to endow micelles with ultrahigh pH


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sensitive ability,35-36 which would trigger drug release and the potential increase of zeta potential would be beneficial for cellular uptake of micelles. As expected, the size of DOX-loaded micelles quickly increased form ~50 nm to ~75 nm (Figure 2C) at pH 6.5, and the zeta potential changed to 6.81±0.59 from -0.17±0.52 (Figure 2H), which would promote cellular uptake of these DOX-loaded mPEG-P (TPE-co-AEMA) micelles. In addition, the redox-responsive feature of micelles could be triggered by 10 mM GSH and the size changes was shown in Figure 2D, which changed from unimodal peak to multimodal peaks after incubation for 6 h. Furthermore, when incubated at pH 6.5 with 10 mM GSH, the disassembly of micelles would be more quickly and the size distribution was detected to be changed to multimodal peaks after incubation for 2 h (Figure 2E), which was much quicker than that at pH 6.5 or with 10 mM GSH. TEM image in Figure 2F and AFM image in Figure S8B further confirmed the disassembly of micelles triggered by acid and GSH, in which obvious aggregates could be observed. Moreover, the changes of particle size triggered with or without GSH (10 mM) at pH 7.4 and pH 6.5 were shown in Figure 2G, which were in accordance with the result in Figure 2C, Figure 2D and Figure 2E. The disassembly of drug-loaded micelle promoted synergistically by acid and GSH, was expected to accelerate drug release in target site, thereby enhancing therapeutic effect. Figure 2I showed that drug release of DOX-loaded mPEG-P (TPE-co-AEMA) micelles was triggered by GSH or individual acid, which about 55% or 80% of drug release after 48 h, respectively. However, drug release was more quickly in acidic medium containing 10 mM GSH, with more than 95% of drug



release after 48 h, showing the efficient response of DOX-loaded micelles to the microenvironment of tumor and expected enhanced antitumor efficacy.

Figure 2. Particle size distribution of blank and DOX-loaded mPEG-P (TPE-co-AEMA) micelles determined by DLS (A); TEM image of DOX-loaded mPEG-P (TPE-co-AEMA) micelles (B); Changes of size distribution of DOX-loaded micelles incubated at pH 6.5 (C); Size changes of DOX-loaded micelles incubated with GSH (10 mM) (D); Size changes of DOX-loaded micelles incubated at pH 6.5 with GSH (10 mM) (E); TEM image of DOX-loaded mPEG-P (TPE-co-AEMA) micelles after incubation at pH 6.5 with 10 mM GSH for 4 h (F); Size changes of DOX-loaded micelles triggered with or without GSH (10 mM) at pH 7.4 and pH 6.5 (G); Zeta potential of DOX-loaded micelles at pH 7.4 and pH 6.5 (H); In vitro drug


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release of DOX-loaded mPEG-P (TPE-co-AEMA) micelles triggered with or without GSH (10 mM) at pH 7.4 and pH 6.5 (I). 3.3 AIE Behavior and Cellular Imaging Unique AIE feature was shown for the TPE group labeled mPEG-P (TPE-co-AEMA) micelles. A very weak fluorescent emission could be observed when the copolymer was dissolved in THF. However, a strong fluorescence emerged for mPEG-P (TPE-co-AEMA) in aqueous solution due to the aggregation state of TPE (Figure 3A). Moreover, dynamic changes of AIE characteristics along the incubation in medium with or without GSH (10 mM) at pH 7.4 and pH 6.5 were investigated. As shown as Figure S9, Figure S10 and Figure S11, when the micelles were in an acidic medium, the fluorescence intensity would slightly decrease due to the relatively looser aggregation state. And in medium (pH = 7.4) containing 10 mM GSH, the fluorescence intensity would slightly increase, which might be attributed to the release of TPE and the formation of new aggregation state of TPE in water. In addition, when the micelles were in an acidic medium with 10 mM GSH, the fluorescence intensity would quickly decrease due to the looser aggregation state of TPE. However, the fluorescence intensity would increase due to the new aggregation state of TPE in water with time. This great AIE feature made these micelles to be a potential fluorescence probe for cell imaging.



Figure 3. FL spectra of mPEG-P (TPE-co-AEMA) micelles in water/THF (A); In vitro cytotoxicity of mPEG-P (TPE-co-AEMA) micelles (B); CLSM imaging of 4T1 cells cultured with mPEG-P (TPE-co-AEMA) micelles for 2 and 4 h (C). Scale bar: 10 µm. It was believed that biocompatibility was quite important for a good fluorescent probe in biological application. 4T1 cells and HeLa cells were utilized to evaluate the potential cytotoxicity of mPEG-P (TPE-co-AEMA) micelles by MTT assay. As shown in Figure 3B, the relative cell viability of both two cells was around 100% even with the concentration of blank micelles up to 200 µg/mL, indicating the great


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biocompatibility of mPEG-P (TPE-co-AEMA) micelles. The cellular imaging ability of mPEG-P (TPE-co-AEMA) micelles was further investigated by CLSM. The CLSM images of 4T1 cells in Figure 3C confirmed the powerful bioimaging ability of these mPEG-P (TPE-co-AEMA) micelles. In addition, blue fluorescence was mainly found in cytoplasm and became stronger with time went on, indicating more micelles were endocytosed by cells. 3.4 In Vitro Antitumor Efficacy 4T1 cells and HeLa cells were used to study the antitumor efficacy of DOX-loaded mPEG-P (TPE-co-AEMA) micelles. As shown in Figure 4, DOX-loaded mPEG-P (TPE-co-AEMA) micelles exhibited great antitumor efficacy as compared with free DOX. The cellular growth inhibition ability of DOX-loaded micelles was evaluated by IC50, and the IC50 value of DOX-loaded micelles was 1.94 µg/mL and 2.63 µg/mL for 4T1 cells and HeLa cells. The IC50 of free DOX was 0.78 µg/mL and 0.99 µg/mL for 4T1 cells and HeLa cells, respectively. In spite of the great anticancer ability of DOX-loaded mPEG-P (TPE-co-AEMA) micelles, free DOX was more toxic, which was due to that DOX-loaded micelles were uptaken by tumor cells via endocytic pathways and drug should have escaped from endosome so as to play the role of tumor killers.



Figure 4. In virto cytotoxicity of DOX-loaded mPEG-P (TPE-co-AEMA) micelles against 4T1 cells and HeLa cells after 24 h and 48 h. 3.5 Cellular Imaging of DOX-loaded mPEG-P (TPE-co-AEMA) Micelles Nanocarriers equipped with AIE feature could be used for monitoring intracellular drug delivery and made these carriers visible. In this study, the fluorescence of mPEG-P (TPE-co-AEMA) micelles could be used to trace intracellular drug delivery and the drug could be released rapidly from DOX-loaded mPEG-P (TPE-co-AEMA) micelles at target site to enhance antitumor efficacy. As shown in Figure 5, blue fluorescence of TPE and red fluorescence of DOX could be easily observed in cytoplasm after co-cultured with DOX-loaded micelles for 1 h and the red fluorescence overlapped almost completely with blue fluorescence, indicating DOX-loaded mPEG-P (TPE-co-AEMA) micelles could be quickly uptaken by 4T1


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cells. In addition, both red fluorescence and blue fluorescence became stronger over time, indicating more micelles were uptaken by cells. After incubation for 5 h, while blue fluorescence still distributed in the cytoplasm, red fluorescence could be found in cell nucleus, which suggested that DOX-loaded mPEG-P (TPE-co-AEMA) micelles could rapidly release the cargo and the released drug could escape from the lysosome and reach the target site.

Figure 5. CLSM images of 4T1 cells cultured with DOX-loaded mPEG-P (TPE-co-AEMA) micelles for 1, 3 and 5 h. Scale bars: 10 µm. 3.6 Ex Vivo Optical Imaging



The biodistribution of DOX-loaded micelles was important to evaluate the side effects and antitumor effect of the drug delivery system (Figure 6). As shown in Figure 6A, no fluorescent emission could be observed in tumors and major organs of mice treated with saline. In contrast, DOX-loaded mPEG-P (TPE-co-AEMA) micelles treated group exhibited obvious fluorescence of DOX in livers, kidneys and tumors, indicating that DOX-loaded mPEG-P (TPE-co-AEMA) micelles could efficiently accumulate and retain in tumor tissue to inhibit tumor growth. After injection for 12 h, the tumor signal was very evident, suggesting the efficient accumulation of DOX-loaded micelles. In addition, the intensity of fluorescence was enhanced over time, and the fluorescence signal reached the peak after injection for 48 h, indicating more drug-loaded micelles were accumulated in target site due to EPR effect, which could be further confirmed by the fluorescence percentage of DOX in Figure 6B. Fluorescent emission could also be observed in other organs, which was because of the body circulation of DOX-loaded micelles.

Figure 6. Ex vivo fluorescent images of tumors and organs from the tumor-bearing


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mice administrated with DOX-loaded mPEG-P (TPE-co-AEMA) micelles (A); Fluorescence percentage of DOX in major organs, data were calculated based on fluorescence signals of DOX counted by a Maestro imaging system (B, n = 3). 3.7 In Vivo Antitumor Efficacy A model of 4T1 breast cancer-bearing female BALB/c mice was used to evaluate the tumor growth inhibition ability of DOX-loaded mPEG-P (TPE-co-AEMA) micelles. As excepted, saline treated mice did not exhibit any tumor growth inhibition effect and the tumor volume reached ~1000 mm3 at the end of a therapy period. However, free DOX and DOX-loaded mPEG-P (TPE-co-AEMA) micelles exhibited effective tumor growth inhibition with tumor volumes of ~500 and ~370, respectively (Figure 7A, *p < 0.05; **p < 0.01). The better antitumor efficacy of DOX-loaded mPEG-P (TPE-co-AEMA) micelles could be attributed to its long-circulation in vivo, targeted accumulation, longer retention in tumor tissue, stimuli triggered drug release and potential lysosome escape of drug. The DOX-induced toxicity was evaluated by body weight loss of mice. There was no body weight loss for saline treated mice. However, obvious side effects could be observed for these free DOX treated mice with severe body weight loss. On the contrary, mice treated with DOX-loaded micelles after each administration only showed a slight body weight loss, indicating a significant lower toxicity than free DOX. Therefore, it could be confirmed that DOX-loaded mPEG-P (TPE-co-AEMA) micelles could be a potential choice for the treatment of cancer with an improved antitumor efficacy and decreased side effects.



Figure 7. (A) Tumor volume of mice treated with saline, free DOX and DOX-loaded micelles (*p < 0.05, **p < 0.01 vs control group); Changes of mice body weight after different treatment (B). Mice were injected with saline, DOX-loaded micelles and free DOX at different time of day 0, 4, 8 and 12. 3.8 Histological Studies To further study the toxicity and anticancer efficacy of DOX-loaded mPEG-P (TPE-co-AEMA) micelles, histological tissues slides of major organs and tumors after 21 days’ treatment were performed (Figure 8). As we know, the serious toxicity would limit the long-term application of DOX. Figure 8 showed that obvious cardiotoxicity could be observed in mice treated with free DOX, while no obvious abnormity was observed for the cardiac tissue of DOX-loaded micelles treated mice. Meanwhile, compared with DOX-loaded micelles, free DOX exhibited more focal necrosis and inflammation in lung, spleen and liver. Moreover, DOX-loaded micelles treated mice exhibited more tumor necrosis lesions than free DOX treated mice, indicating the higher tumor inhibition ability of DOX-loaded mPEG-P (TPE-co-AEMA) micelles. It was suggested that the DOX-loaded mPEG-P (TPE-co-AEMA) micelles might


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enhance antitumor efficacy with the slight side effects.

Figure 8. Images of H&E assays for tumors and major organs after different treatment after 21 d (all tissue: 200×). 3.9 Immunohistochemical Analysis The tumor inhibition ability of DOX-loaded mPEG-P (TPE-co-AEMA) micelles was



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further evaluated by immunohistochemical analysis. Tumor angiogenesis was the leading cause of invasion and metastasis of tumors, which could be marked by using CD31 to reflect the antitumor efficacy by tumor microvessel density (MVD). The CD31 images in Figure 9 demonstrated that DOX-loaded micelles treated mice exhibited the least angiogenesis compared with saline and free DOX treated mice, suggesting DOX-loaded micelles could significant reduce angiogenesis thereby inhibit tumor growth in line with in vivo tumor growth inhibition in Figure 7. Besides, the Ki-67 protein expression, an important tumor proliferation marker, was significantly reduced in DOX-loaded micelles treated mice, indicating the less cell proliferations activation. In addition, the tumor cell apoptosis was also studied via TUNEL assay. Figure 9 showed that ~10% and ~52% of apoptotic tumor cells were observed for saline treated mice and free DOX treated mice. In contrast, DOX-loaded micelles treated mice exhibited the highest apoptotic tumor cells with an apoptotic proportion up to ~63%, indicating efficient inhibition of tumor growth for DOX-loaded

micelles.

Overall,

this

multifunctional

DOX-loaded

mPEG-P

(TPE-co-AEMA) micelle system could inhibit tumor angiogenesis and tumor proliferation as well as trigger apoptosis of tumor cells, resulting into a better antitumor efficacy.


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Figure 9. Immunohistochemical (IHC) analysis of 4T1 tumor (CD31, Ki-67 and TUNEL) (n=6, all tissue: 400×). Brown area of CD31 images indicated CD31-positive, and the capillary number was analyzed in each section (MVD) (**p < 0.01 vs saline, *p < 0.05 vs free DOX); Ki-67 density was calculated by Ki-67-positive area to total area (**p < 0.01 vs saline, **p < 0.01 vs free DOX); TUNEL-positive staining and the apoptotic indices were evaluated by counting the number of the apoptotic cells to the total cells in each microscopic field of view (**p < 0.001 vs saline, *p < 0.05 vs free DOX). The scale bars were 200 µm.



4 Conclusion In this work, AIE active fluorescent polymeric micelles with pH and redox dual-response have been developed for intelligent DOX delivery and bioimaging. DOX-loaded mPEG-P (TPE-co-AEMA) micelles with pH ultrasensitive feature can rapidly respond to acid environment resulting in the accelerated drug release as well as the enhancement of particle and zeta potentialwith the potential promotion of the endocytosis of micelles. In addition, high level of GSH can also accelerate drug release with the further destruction of micellular structure, showing great antitumor efficacy in vitro. Furthermore, the highly effective tumor tissue accumulation and rapid drug release triggered by acid and redox environment of tumor endow these DOX-loaded micelles with higher tumor growth inhibition efficacy and lower side effect compared with free DOX. This multifunctional polymeric micelle would provide new insights for designing high-efficiency visible nanocarriers.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected],

* E-mail: [email protected]

Author Contributions


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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This research was financially supported by the National 111 Project of Introducing Talents of Discipline to Universities (No. B16033), the National Natural Science Foundation of China (Projects 21502129), and China Postdoctoral Science Foundation Funded Project (2017M612956). We would be grateful to the help of Mr. Chenghui Li (Analytical & Testing Center, Sichuan University) of taking laser scanning confocal images.

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Table of Content

DOX-loaded mPEG-P (TPE-co-AEMA) polymeric micelles have been developed for combining AIE active bioimaging and cancer therapy with ultrasensitive to acidic condition and high concentration level of GSH. mPEG-P (TPE-co-AEMA) micelles exhibited great cellular imaging ability and excellent environment triggered drug release, showing great antitumor efficacy with significantly reduced side effects compared to free DOX.


Redox and pH Dual-Responsive Polymeric Micelles with Aggregation

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