Dual-responsive doxorubicin-conjugated polymeric micelles with

Subscriber access provided by Kaohsiung Medical University

Article

Dual-responsive doxorubicin-conjugated polymeric micelles with aggregation-induced emission active bioimaging and charge conversion for cancer therapy Xin Su, Boxuan Ma, Jun Hu, Tao Yu, Weihua Zhuang, Li Yang, Gaocan Li, and Yunbing Wang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.bioconjchem.8b00671 • Publication Date (Web): 08 Nov 2018 Downloaded from http://pubs.acs.org on November 9, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

Dual-Responsive Doxorubicin-Conjugated Polymeric Micelles with AggregationInduced Emission Active Bioimaging and Charge Conversion for Cancer Therapy

Xin Su1, Boxuan Ma1, Jun Hu, Tao Yu, Weihua Zhuang*, Li Yang, Gaocan Li and Yunbing Wang*

National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu 610064, China;

Xin Su and Boxuan Ma contributed equally to this work.

Corresponding authors. E-mail: [email protected] (Weihua Zhuang) E-mail: [email protected] (Yunbing Wang)

ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT In recent years, intelligent polymeric micelles with multi-functions are in urgent demand for cancer diagnosis and therapy. Herein, pH and redox dual-responsive prodrug micelles with aggregation-induced emission (AIE) active cellular imaging and charge conversion have been prepared for combined chemotherapy and bioimaging based on a novel doxorubicin-conjugated amphiphilic PMPC-PAEMA-P (TPE-coHD)-ss-P (TPE-co-HD)-PAEMA-PMPC copolymer. The doxorubicin is conjugated via a pH cleavable imine linkage and can be packed in the hydrophobic core along with the glutathione (GSH)-sensitive disulfide bond. The DOX-conjugated inner core is sealed with a pH-responsive PAEMA as the “gate”, which would rapidly open in the acidic condition, following the drug release and charge conversion-mediated acceleration of endocytosis. After an efficient internalization, the disulfide bond can be cleaved by the high concentration of GSH causing the further accelerated drug release. Meanwhile, intracellular drug delivery can be traced due to the AIE behavior of micelles. Moreover, great tumor inhibition in vitro and in vivo has been demonstrated for these DOXconjugated micelles. This smart prodrug micelle system would be a desirable drug carrier for cancer therapy and bioimaging. INTRODUCTION In the past decades, cancer has been one of the world’s most devastating diseases with about 14.1 million new cases every year1. Currently, radiotherapy2 ， chemotherapy3 and surgical intervention4 are still the main strategies for cancer therapy in clinic. Anticancer drugs for chemotherapy can inhibit tumor growth but also accompany with


Page 2 of 34

Page 3 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


multiple side effects to main organs5. Compared to normal tissues, more structural damage was found in tumor blood vessels6, where the micelles with a size under 200 nm can aggregate via enhanced permeability and retention (EPR) effect7. Thus, a number of polymeric micelles have been developed to improve anti-cancer efficiency and reduce the side effects by loading hydrophobic drugs in the core of the micelles via EPR effect and releasing the cargos in target sites8. However, numerous problems occur during the process of circulation, accumulation, internalization and drug release, which would limit target-site drug delivery9. For example, hydrophobic drugs are often encapsulated into the core of micelles via hydrophobic effect, which can easily leak out during blood circulation, leading to reduced drug accumulation at the target-site10. Meanwhile, on account of providing a good hemocompatibility and great stability during circulation, the shells of micelles are usually composed of strong hydrophilic polymer and the nanoparticles are often electronegative, which can also reduce micellar endocytosis11. What’s more, making drugs and nanoparticles visible in vivo are also in urgent demand for cancer therapy and diagnosis12. In order to reduce the leakage of drugs during the transportation in blood stream, prodrug offers an effective strategy to develop nano-carriers with neglectable drug leakage13-15. Environment-responsive prodrugs have been widely developed, which are stable in physiological environment but rapidly release the cargos at tumor targets due to the specific tumor microenvironment, such as redox-environment, lower pH and specific enzymes. Disulfide bond is often utilized for engineering nanocarriers



responsive to high level of glutathione (GSH)

Page 4 of 34

16,17

with accelerated drug release and

enhanced anti-tumor effect due to the extremely higher intracellular concentration of GSH (2 - 10 mM) in tumor cells, which is much higher than that in the extracellular environment (2-20 μM)18. Acidic pH values are also found in different sites of tumor tissue19, such as extracellular matrix (pH 6.5-7.2), endosomes (5.0-6.5) and lysosomes (4.5-5.0)20-22. Hence, pH-sensitive structures, for instance, imine, acetal and orthoester have been developed for drug delivery, which can be broken under acid condition. In addition, polymers with pH-triggered hydrophilic-hydrophobic conversion such as poly (2-(tetramethyleneimino) ethyl methacrylate) (PTEMA)

23

, poly (2-(dibutylamino)

ethyl methacrylate) (PDEMA) 24 and poly (2-azepane ethyl methacrylate) (PAEMA)25 have been introduced into pH-responsive drug delivery systems, which also show charge conversion feature via protonation effect and can consequently turn into electropositive in acid condition, resulting in enhanced cellular internalization by combining with electronegative cytomembrane26. Besides intelligent drug release, ideal nanoparticles should also be visible so as to monitor the biodistribution of nanoparticles and trace the intracellular drug delivery. Fluorescent dyes are widely used to trace nanoparticles. However, conventional dyes are often limited by the aggregation-caused quenching (ACQ) effect27. Since 2001, a series of fluorescent molecules with aggregation-induced emission (AIE) effect have been developed, which emit strong fluorescence in the aggregated state28. Tetraphenylethene (TPE), a typical molecule with AIE effect29, exhibits significant AIE phenomenon in virtue of the restriction of their intramolecular rotations in the


Page 5 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


aggregation state and has been diffusely used as a fluorescent bioprobe in biosensing and imaging30. Therefore, TPE can be introduced into polymeric micelles as the fluorescent bioprobe to trace the biodistribution of micelles. In this work, we have successfully synthesized a novel DOX-conjugated multiblock copolymer PMPC-PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-PAEMA- PMPC with pH and redox dual responsiveness and AIE active bioimaging. Poly (2methacryloyloxyethyl phosphorylcholine) (PMPC), known as an excellent hydrophilic polymer with zwitterionic phosphorylcholine30, was selected as the hydrophilic segment owing to its high hydration by ionic solvation, which could avoid rapid clearance by the reticuloendothelial system (RES)

31

. It was expected that PMPC-

PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-PAEMA-PMPC prodrug copolymer could self-assemble into core-shell structural micelles with TPE inside the core, which endowed these micelles with great bioimaging ability (Scheme 1). In addition, under tumor acidic conditions, the pH-controlled “gate” PAEMA would open due to the hydrophilic transformation, allowing accelerated drug release and charge convention with enhanced endocytosis of micelles. Furthermore, the high level of GSH in tumor cells would promote micellar structure transformation with further accelerated drug release. Therefore, these multifunctional micelles were anticipated to improve antitumor efficacy with reduced side effects. The cellular imaging, intracellular drug delivery, ex vivo drug biodistribution, in vitro and in vivo antitumor assay were further carried out in detail.



Page 6 of 34

Scheme 1. Illustration of DOX-conjugated PMPC-PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-PAEMA-PMPC polymeric micelles with pH and redox responsive drug release, charge conversion and AIE bioimaging. RESULTS AND DISCUSSION Synthesis of PMPC-PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-PAEMA-PMPC PMPC-PAEMA-P

(TPE-co-HD)-ss-P

(TPE-co-HD)-PAEMA-PMPC

multiblock

copolymer was synthesized step-by-step via reversible addition-fragmentation chain transfer polymerization (RAFT) with CTA-ss-CTA as the chain transfer agent. The exhaustive synthetic route was shown in Figure 1.


Page 7 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Synthetic route of PMPC-PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)PAEMA-PMPC.



The chemical structures of the copolymer in each step were characterized using 1H NMR spectroscopy. The 1H NMR spectra in Figure S1, Figure S2 and Figure S3 confirmed the successful preparations of CTA-ss-CTA, HD-MA and TPE-MA, respectively. Afterwards, P (TPE-co-HD)-ss-P (TPE-co-HD) was obtained via RAFT polymerization of HD-MA and TPE-MA using CTA-ss-CTA as the chain transfer agent. The degree of polymerization (DP) of PTPE was calculated to be 6 based on the integral ratio of the peaks at δ 7.35-6.65 ppm (PTPE, b, 19H) and δ 8.0-7.34 ppm (CTA-ss-CTA, a, 5H) (Figure S4), while the DP of PHD-Boc was calculated to be 2 (c, 4H; d, 18H). The molecular weight distribution of P (TPE-co-HD)-ss-P (TPE-co-HD) was determined to be 1.31 by GPC (Figure S5). As shown in Figure S6 for the 1H NMR result of PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-PAEMA, the characteristic peaks of PAEMA (b, 4H; c, 8H) were found at δ 4.03 ppm and δ 2.70 ppm, and the DP of PAEMA was calculated to be 20. Furthermore, the molecular weight distribution of PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-PAEMA was determined to be 1.29 by GPC (Figure S7). The 1H NMR spectrum of PMPC-PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-PAEMA-PMPC was shown in Figure S8. The characteristic peak of TPE (a, δ 7.2-6.45 ppm) and the characteristic peak of PMPC (b, δ 4.24 ppm; c, δ 4.15 ppm; d, δ 4.00 ppm; e, δ 3.71 ppm) could be easily found, suggesting the successful synthesis of the copolymer. The DP of PMPC was calculated to be 40 based on the integral ratio of the peak a (PTPE, 19H) and peak c (-CH2-CH2-PO4-, 2H). Therefore, the PMPCPAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-PAEMA-PMPC could be defined as PMPC20-PAEMA10-P (TPE3-co-HD1)-ss-P (TPE3-co-HD1)-PAEMA10-PMPC20 with a


Page 8 of 34

Page 9 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


molecular weight of 19000 g/mol. After that, the copolymer was treated with TFA, and the t-Butyloxy carbonyl (Boc) group (a, δ 1.40 ppm) was disappeared in Figure S9, revealing the successful remove of the Boc group. Subsequently, the exposed amino groups could be used as the functional groups to conjugate the DOX via a pH-sensitive imine linkage. As shown in Figure 2, the characteristic peak of DOX could be easily found (h, δ 8.01-7.59 ppm; j, δ 2.32-2.08 ppm). What’s more, the 13C NMR and FT-IR spectrums of final product were also provided in Figure S10 and S11.

Figure 2. 1H NMR spectrum of DOX-conjugated PMPC-PAEMA-P (TPE-co-HD)-ssP (TPE-co-HD)-PAEMA-PMPC in CD3OD and DMSO-d6 (1:1, V/V). Preparation and the Dual-Response of DOX-Conjugated Polymeric Micelles The dual-responsive micelles with AIE imaging and charge conversion could be formed by self-assembling from the PMPC-PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)PAEMA-PMPC copolymer with the PMPC as hydrophilic shells and the other segments



as the hydrophobic cores. The particle size of blank micelles was 141.2 nm with a narrow size distribution of 0.153 (Figure 3A). After DOX was conjugated to the HD segments, the particle size of drug-loaded micelles was 121.0 nm with a distribution of 0.227. Moreover, the morphology of DOX-conjugated micelles was further observed by TEM (Figure 3G), which was well-defined spherical particles and the particle size measure by TEM was about 90 nm. The size measured by TEM was smaller than the DLS result owing to the shrinkage of the micellar shell during the sample preparation. Then, drug loading content (DLC) and drug loading efficacy (DLE) were calculated to be 4.0 % and 20.9 % measured by UV, which was in keeping with the result based on 1

H NMR result. The CMC for PMPC-PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-

PAEMA-PMPC prodrug micelles was determined to be 7.56 μg/mL, which provided a powerful supply for prodrug micellar stability after the high concentration of micelles diluted by blood in human body (Figure S12). When the micelles accumulated in the tumor tissue by EPR effect, the drug-loaded micelles would first response to the acidic extracellular environment. PAEMA, serving as a pH-sensitive “gate”, was hydrophobic at pH higher than 6.8 and in a state of closed, which could protect the cargos from leakage during blood circulation. However, when pH was lower than 6.8, this PAEMA “gate” would rapidly open and changed from hydrophobic to hydrophilic, allowing drug release and charge convention. What’s more, the imine linkage between DOX and HD segments would be cleaved resulting in DOX release. As shown in Figure 3B, the particle size significantly decreased to about 74 nm after incubated at pH 6.0 for 6 h with larger size distribution attributed to the stretch of


Page 10 of 34

Page 11 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


PAEMA and HD block after becoming hydrophilic and the hydrophobic core would be shrunk, which was further proved by TEM image in Figure 3H. In addition, the protonation of PAEMA together with the exposed amino groups in HD segments would lead to the charge conversion. When incubated at pH 7.4, the micelles maintained a stable negative zeta potential at a range of -0.5 mV to -2 mV (Figure 3C). However, when at pH 6.0, the zeta potential of these micelles rapidly increased, which reached to +5, +8 and +10 mV after 1, 4 and 6 h, respectively. This ingenious charge conversion feature of the micelles could result in a strong binding force between cytomembrane and the micelles, which enhanced the endocytosis of these prodrug micelles.



Figure 3. (A) The particle size of blank and drug-loaded micelles measured by DLS. (B) Size variation of prodrug micelles at pH 6.0. (C) Zeta potential variation of prodrug micelles at different pH over time measured by DLS. (D) Size variation of prodrug micelles in 10 mM GSH. (E) Size variation of prodrug micelles at pH 6.0 and 10 mM GSH. (F) Variation trend of particle size at different pH and different concentrations of GSH. TEM image of prodrug micelles in pH 7.4 (G), pH 6.0 (H), 10 mM GSH (I) and 10mM GSH at pH 6.0 (J). On the other hand, the disulfide bond was introduced to these micelles, which could be broken by the intracellular high concentration of GSH. As shown in Figure 3D, when incubated in medium contained 10 mM GSH, the size distribution quickly changed to multimodal, suggesting the breaking of disulfide bond. Meanwhile, TEM image in Figure 3I showed obviously reduced particle size, which confirmed the cleavage of disulfide bond and reconstruction of micellar structure. Interestingly, when micelles were incubated in medium contained 10 mM GSH at pH 6.0, the micellar distribution changed to unimodal after 6 h (Figure 3E), and the particle size decreased to about 63 nm (Figure 3F) due to the total cleavage of disulfide bond and formation of new micellar structure, which was further confirmed by TEM image in Figure 3J. Therefore, the environment of acid and redox would synergistically accelerate micellar structural reorganization, which was hopeful to release drug intelligently.


Page 12 of 34

Page 13 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. In vitro drug release of produrg micelles in different pH and different concentrations of GSH. Scale bars were 50 nm. In Vitro Drug Release Behavior The drug release behavior of DOX-conjugated prodrug micelles was investigated at 37 o

C in different pH and different concentrations of GSH. As shown in Figure 4E, less

than 35% of the drug was released from the micelles after 48 h at physiological pH (7.4), which indicated a great stability of these prodrug micelles when transported in blood stream. In addition, only about 50% of the cargos were released in the medium containing 10 mM GSH. However, when at pH 6.0, the PAEMA transformed to hydrophilic, which provided a channel for drug ejectment and the conjugated DOX was set free due to the cleavage of imine linkage. As a result, about 80% of the cargos were released after 48 h at pH 6.0. Furthermore, following the pH-sensitivity, the destruction



of the disulfide bond triggered by intracellular redox environment of tumor cells would further accelerate drug release due to the transformation of micellar structure and more than 90% of the drug was released after incubation at pH 6.0 and 10 mM GSH for 12 h. Therefore, this step-by-step dual-response to the extracellular pH and intracellular GSH would lead to a smart drug release, resulting in better antitumor effect. AIE Behavior and Cellular Imaging Aside from the drug release behavior, the ability of tracing the intracellular drug distribution of these micelles was equally important. As shown in Figure 5A, these micelles exhibited strong fluorescence in pure water, while the FL intensity reduced with the increase of THF fraction owing to the good solubility of the TPE groups in THF, which changed from aggregation state to dissolved state and led to the fluorescence quenching. Furthermore, the cellular bioimaging ability of these micelles was investigated by confocal laser scanning microscopy (CLSM). Blue fluorescence of TPE was clearly observed in cytoplasm after incubation with the micelles for 2 h (Figure 5 B). With the extension of time, the blue fluorescence intensity of TPE was significantly enhanced, indicating more micelles were internalized by cells, which might be ascribed to the charge conversion of PAEMA and PHD segments. Thus, these fluorescence micelles would be practical for bioimaging.


Page 14 of 34

Page 15 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. (A) FL spectrum of the micelles in different solvent systems. (B) CLSM images of 4T1 cells after co-culture with blank micelles for 2 h, 4 h and 6 h. Scale bars were 10 μm. Intracellular Drug Tracking and In Vitro Tumor Inhibition Owing to the AIE feature of the TPE groups, the intracellular drug release of these prodrug micelles could be traced by fluorescence imaging. DOX-conjugated micelles were incubated with 4T1 cells for 2 h, 4 h, and 6 h, and then observed by CLSM. As shown in Figure 6A, the blue fluorescence of TPE was clearly visible in the cytoplasm after 2 h, where the red fluorescence of DOX was observed overlapping, indicating that DOX had not escaped from the micelles yet. With prolonged incubation time, more red fluorescence was separated from the blue fluorescence. When the incubation time was increased to 6 h, the red fluorescence of DOX could be observed in the cell nucleus, which was distinguished with the blue fluorescence of TPE. The statistic of Pearson’s correlation coefficient (Rr) was further calculated from CLSM result (Figure S13), which demonstrated an increasingly obvious separation between DOX and TPEconjugated micelles, suggesting the released drug could efficiently escape from



micelles and result in an efficient tumor inhibition.

Figure 6. (A) CLSM image of 4T1 cells co-cultured with DOX-conjugated micelles for 2 h, 4 h and 6 h. Scale bars were 10 μm; (B) In vitro cytotoxicity of DOX-conjugated micelles and free DOX against 4T1; (C) HeLa cells after incubation of 48 h. Before conjugating DOX, the blank micelles exhibited excellent biocompatibility against 4T1 cells and HeLa cells investigated by MTT assays (Figure S14), where the relative cell viability of both two cells was around 100% even the concentration of blank micelles up to 200 μg/mL. On the other hand, the DOX-conjugated micelles performed strong power in killing both 4T1 and HeLa cells (Figure 6B, Figure 6C and Figure S15). As shown in Figure 6B and 6C, 75% of 4T1 cells and 80% of HeLa cells were exterminated after 48 h at a DOX concentration of 10 μg/mL. The cellular growth inhibition ability of DOX-loaded micelles was evaluated by IC50 (half maximal inhibitory concentration), and the IC50 values of DOX-conjugated micelles were 1.98 and 0.57 μg/mL for 4T1 cells and HeLa cells, respectively. The IC50 values of free DOX


Page 16 of 34

Page 17 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


were 0.68 and 0.27 μg/mL for 4T1 cells and HeLa cells, respectively. The antitumor effect of the prodrug micelles was similar with that of free DOX, indicating the outstanding in vitro tumor inhibition efficiency of these DOX-conjugated micelles. In Vivo Anti-Tumor Activity and Ex Vivo Optical Imaging To evaluate the anti-cancer efficiency of DOX-conjugated micelles, a xenograft tumor model was established on mice followed by intravenous injection with free DOX, DOX-loaded conjugated micelles and saline as the control. As shown in Figure 7A, on the 21st day after first injection, the tumor volume of saline treated group reached about 1400 mm3, while the group treated with free DOX and DOX-conjugated micelles exhibited effective tumor growth inhibition with tumor volumes of about 510 and 350 mm3 (*p < 0.05), respectively. The great antitumor efficacy of these DOX-conjugated micelles could be attributed to their charge conversion and dual-responsive drug release. In addition, body weight was measured to evaluate the toxicity of drug to mice. As shown in Figure 7B, severe body weight loss was detected in mice treated with free DOX. By comparison, the body weight of the DOX-conjugated micelles treated group showed a slight rise as well as the control group, suggesting a significant lower toxicity of the DOX-conjugated micelles. Owing to the improved antitumor efficacy and decreased side effects, these micelles could be considered as a potential choice for cancer therapy. The biodistribution and accumulation of DOX-conjugated micelles in different organs directly influenced the micellar antitumor effect along with the side effects. The ex vivo optical imaging of major organs and tumors at different time was shown in



Figure 7C, where the control group showed scarcely any fluorescent emission in major organs or tumor. However, the group treated with DOX-conjugated micelles showed clear fluorescence of DOX in livers, kidneys and tumors. The fluorescent intensity at tumors was increased gradually over time, suggesting that DOX-conjugated micelles could efficiently accumulate in tumor issues, which could be further proved by the fluorescence intensity of DOX average signal in Figure 7D. The efficient accumulation of DOX-conjugated micelles would help to enhance antitumor efficacy and reduce the side effects. Besides, the fluorescent emission could also be detected in other organs, which was on account of the body circulation of the DOX-conjugated micelles.


Page 18 of 34

Page 19 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. The variation of tumor volume (A) and body weight (B) after injection with

saline solution, free DOX and DOX-conjugated micelles over 21 days (*p < 0.05); (C) Ex vivo fluorescent images of organs and tumors after tumor-bearing mice were injected with DOX-conjugated micelles at predetermined time; (D) Fluorescence intensity of heart, liver, spleen, lung, kidney and tumor were calculated based on fluorescence signals of DOX (n = 6). Histological and Immunohistochemical Analysis In order to further investigate the organ toxicity and antitumor efficacy of DOXconjugated micelles, the histological tissue slides of tumor, heart, liver, spleen, lung and kidney of the mice were performed as shown in Figure S16. Significantly visible metastases could be observed in major organs of control group but rarely seen in the organs treated with DOX-conjugated micelles. Moreover, free DOX treated group showed more focal necrosis and inflammation in major organs (liver, spleen and kidney) compared with that of DOX-conjugated micelles treated group, indicating that the prodrug micelles could actually reduce the side effect of DOX and showed outstanding organ compatibility. In addition, a certain degree of tissue necrosis was observed in tumor tissues treated with both free DOX and prodrug micelles, where the prodrug micelles exhibited much higher tumor inhibition effect. To accurately characterize the tumor inhibition, the growth, invasion and metastasis of solid tumors were estimated by immunohistochemical analysis (Figure 8A). The Ki67 protein was a cellular marker for proliferation and the expression of Ki-67 was significantly reduced in DOX-conjugated micelles treated group in contrast with free



DOX and control group (Figure 8A and Figure 8B), indicating a lower cancer cell propagation of micelles treated mice. What’s more, CD31 was used primarily to evaluate the degree of tumor angiogenesis and the number of angiogenesis was rarely observed in the tumor tissue treated with DOX-conjugated micelles compared with free DOX and control group as shown in Figure 8C. It was illustrated that the DOXconjugated micelles could significantly inhibit the tumor growth by reducing tumor vasculogenesis. Last but not least, TUNEL was a method for detecting apoptotic DNA fragmentation, which could be used to reflect antitumor effect, which was used to identify and quantify apoptotic cells (Figure 8D). Compared with the saline group (9.9%), 57.5% of apoptotic cells were observed for DOX-conjugated micelles and 47.7% for free DOX, suggesting DOX-conjugated micelles possessed the best antitumor effect according to above-mentioned systematic evaluation. The DOX-conjugated micelles could effectively inhibit angiogenesis, restrain tumor cell proliferation and encourage apoptosis, resulting in high-efficiency in tumor inhibition.


Page 20 of 34

Page 21 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8. (A) Immunohistochemical (IHC) analysis of the tumor (Ki-67, CD31 and TUNEL); The scale bars were 500 μm; (B) The Ki-67 density in each image was calculated by Ki-67-positive area/total area (**p < 0.01); (C) Brown area of CD31 images indicated CD31-positive (**p < 0.01); (D) TUNEL-positive staining and the apoptotic indices were measured as the ratio of apoptotic cells to the total tumor cells in each microscopic field view (**p < 0.01). CONCLUSION In this work, intelligent multifunctional DOX-conjugated micelles with dual-response, charge conversion and bioimaging feature have been prepared for efficient cancer therapy and bioimaging. These prodrug micelles can maintain stable at physiological environment, but immediately respond to the specific tumor microenvironment. The acidic environment of tumor tissue would trigger PAEMA changing from hydrophobic to hydrophilic and open a channel for drug release, along with the charge convention to enhance endocytosis. Moreover, high concentration of GSH in tumor cells would accelerate micellar structure transformation and further accelerate drug release so as to obtain desirable antitumor efficacy. Furthermore, the intracellular drug delivery can be traced by the AIE fluorophore TPE. The efficient accumulation in tumor targets, excellent antitumor in vivo and obvious reduction of side effects make these DOXconjugated micelle system highly attractive as a drug carrier for cancer therapy and bioimaging. This kind of smart multifunctional polymeric micelle would promote the development of nanocarriers for theranostic and offer new strategy for designing novel nanoparticles for nanomedicine.



Page 22 of 34

MATERIALS AND METHODS Materials Doxorubicin hydrochloride (DOX•HCl) and 3-(4, 5-Dimethyl-thiazol-2-yl)-2, 5diphenyl tetrazolium bromide (MTT) were purchased from Chengdu Best Reagent Co., LTD (Chengdu, China). 2-methacryloyloxyethyl phosphorylcholine (MPC) was purchased from Nanjing Natural Science and Technology Institute. 4-(1, 2, 2triphenylvinyl) phenol (TPE-OH) was synthesized according to previously literature32. 4-Cyanopentanoic acid dithiobenzoate (CTA) was obtained from Sigma-Aldrich. Methacryloyl

chloride,

N,

N’-dicyclohexylcarbodiimide

(DCC),

4-

(dimethylamino)pyridine (DMAP), 2,2’-azobis(isobutyronitrile) (AIBN), di-tert-butyl dicarbonate, trifluoroacetic acid (TFA) and all other reagents and solvents were purchased from Chengdu KeLong Chemical Reagent Company (Chengdu, China) and used as received. Synthesis of CTA-ss-CTA CTA-ss-CTA was synthesized through esterification between CTA and bis (2hydroxyethyl) disulfide. Under argon (Ar) atmosphere, CTA (0.60 g, 2.15 mmol) was dissolved in dry dichloromethane, and then bis (2-hydroxyethyl) disulfide (0.17g, 1.07 mmol), DCC (0.45 g, 2.15 mmol) and DMAP (52.53 mg, 0.43 mmol) were added. The mixed solution was stirred for 24 h at room temperature. The product CTA-ss-CTA was purified by chromatography on silica gel (dichloromethane : ethyl acetate = 5 : 1). The yield of CTA-ss-CTA was 65.3 %. 1H NMR (400 MHz, CDCl3): 7.36-7.94 (m, 10H, C6H5), 4.41-4.35 (t, 4H, -CH2CH2OCOCH2), 2.97-2.89 (t, 4H, -SCH2CH2), 2.78-2.59


Page 23 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(m, 4H, -CH2CH2CNSCH3) ， 2.49-2.40 (m, 4H, -COCH2CH2), 1.94 (s, 6H, CH2CNSCH3). Synthesis of HD-MA 1, 6-diamino-hexan (HD) (4.00 g, 34.42 mmol) and triethylamine (TEA) (5 mL, 35.97 mmol) were dissolved in methanol, and the methanol solution of di-tert-butyl dicarbonate (7.51 g, 26.70 mmol) was dropwise added to the HD solution. After 12 h, the reaction mixture was concentrated and re-dissolved in DCM, followed by extraction with 1 M HCl aq, and the obtained aqueous phase was alkalinized by NaHCO3 aq (satd.) and extracted with DCM. The product Boc-HD-NH2 was purified by removing the solvent under reduced pressure (yield 70.2 %). Under Ar atmosphere, the Boc-HD-NH2 (1.90 g, 8.78 mmol) and TEA (1.80 mL, 12.95 mmol) were dissolved in 100 mL tetrahydrofuran (THF), then methacryloyl chloride in 10 mL THF (850 μL, 8.78 mmol) was added dropwise. The mixed solution was stirred overnight at room temperature. After filtration and concentration, the product was dissolved with DCM and purified by column chromatography (dichloromethane : methanol = 20 : 1). The yield of HD-MA was 82.6 %. 1H NMR (400 MHz, CDCl3): 5.67 (s, H, -COCH2CH3), 5.31 (s, H, -COCH2CH3), 3.11 (m, 4H, NHCH2), 1.44 (s, 9H, -COOC(CH3)3). Synthesis of TPE-MA TPE-OH (2.70 g, 7.75 mmol) and TEA (1.35 mL, 9.71 mmol) were dissolved in 100 mL THF, and methacryloyl chloride (750 μL, 7.75 mmol) in 10 mL THF was added dropwise. The mixed solution was stirred overnight at room temperature. After



filtration and concentration, the product was purified by column chromatography (dichloromethane : n-hexane = 1 : 1). The yield of TPE-MA was 67.2 %. 1H NMR (400 MHz, CDCl3): 7.17-6.78 (m, 19H, -COOC6H4CC6H5C(C6H5)2), 6.29 (s, H, COCH2CH3), 5.71 (s, H, -COCH2CH3). Synthesis of P (TPE-co-HD)-ss-P (TPE-co-HD) CTA-ss-CTA (162 mg, 0.24 mmol), AIBN (32 mg, 0.192 mmol), TPE-MA (0.60 g, 1.44 mmol) and HD-MA (0.28 g, 0.98 mmol) were dissolved in a schlenk flask with 10 mL THF. The flask was sealed and degassed with three cycles of freeze-pump-thaw procedure. The reaction was carried out at 70 oC with stirring overnight, followed by dialysis (MWCO = 1000) against deionized water for 24 h. The product was obtained by freeze drying and the yield was 90.7 %. Synthesis of PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-PAEMA AEMA was synthesized according to previously literature33. Briefly, N-(2hydroxyethyl) hexamethyleneimine (4.00 g, 27.9 mmol) and TEA (7.60 mL, 55.8 mmol) were dissolved in 150 mL THF, and methacryloyl chloride (4.60 mL, 44.6 mmol) in 20 mL THF was added dropwise under the protection of Ar. The mixed solution was stirred for 24 h at room temperature. After filtration and concentration, AEMA was purified by column chromatography (ethyl acetate : n-hexane = 1 : 4). P (TPE-co-HD)-ss-P (TPE-co-HD) (0.30 g, 0.1 mmol), AIBN (8.21 mg, 0.05 mmol) and AEMA (0.36 g, 1.69 mmol) were dissolved in a schlenk flask with 5 mL THF. The flask was sealed and degassed with three cycles of freeze-pump-thaw procedure, and the resulting solution was stirred at 70 oC for 24 h. PAEMA-P (TPE-co-HD)-ss-P


Page 24 of 34

Page 25 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(TPE-co-HD)-PAEMA was purified by dialyzing (MWCO = 3500) against deionized water for 2 days. Then the pink product was gained after freeze drying and the yield was 93.5%. Synthesis of PMPC-PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-PAEMA-PMPC Briefly, AIBN (4.92 mg, 0.03 mmol), PAEMA-P(TPE-co-HD)-ss-P(TPE-co-HD)PAEMA (0.5 g, 0.1 mmol) and MPC (1 g, 3.39 mmol) were dissolved in 10 mL of solvent (THF/MeOH, 1:1, v/v) in a schlenk flask. After three cycles of freeze pumpthaw procedure, the mixture was allowed to stir at 70 oC overnight. Then, the reaction solution was dialyzed against deionized water (MWCO = 3500) for 2 days, and the product was obtained by freeze drying. Afterwards, the resulting copolymer (1.40 g, 0.074 mmol) was dissolved in solvent (THF/MeOH, 1:1, v/v), and 3 mL TFA was added to abandon Boc group at room temperature for 24 h, followed by neutralization with NaHCO3 aq (satd.) and dialysis (MWCO = 3500) against deionized water for 24 h. The final product was obtained by freeze drying and the yield was 91.9%. Preparation of DOX-Conjugated Prodrug Polymeric Micelles 4 mg DOX, 5 μL TEA and 20 mg PMPC-PAEMA-P(TPE-co-HD)-ss-P(TPE-co-HD)PAEMA-PMPC copolymer were dissolved in 2 mL dissolvent (THF/MeOH, 1/1, v/v). The mixture was stirred at room temperature for 24 h. Then the solution was added dropwise to 10 mL of deionized water (pH = 7.4) under vigorous stirring. After 1 h, the solution was transferred into a dialysis bag (MWCO = 3500) and dialyzed against deionized water (pH = 7.4) for 2 days. Then the solution was filtered with a 220 nm filter membrane. The blank micelles were prepared in the same way without the



addition of DOX. The drug loading content (DLC) and drug loading efficacy (DLE) were measured with UV spectroscopy at 480 nm, which were calculated as the following equations. DLC(%)=

Weight of DOX in prodrug micelles × 100% Weight of prodrug micelles

DLE(%)=

Weight of DOX in prodrug micelles × 100% Weight of the feeding DOX

pH and Redox Sensitive Behavior Dynamic light scattering (DLS) measurements was used to investigate the pH and redox sensitive behavior of the polymeric micelles by monitoring the size and zeta potential variation of micelles in PBS solutions with different pH and different concentrations of GSH. The particle size and zeta potential of the micelles was measured at preselected time intervals (0 h, 0.5 h, 1 h, 2 h, 4 h and 6 h). What’s more, the morphology of micelles was further investigated by TEM after incubation in different pH and concentration of GSH for 6 h. The AIE Behavior of TPE Groups In order to cognize the fluorescence behavior of TPE groups, THF with different volume fraction was added to polymeric micelles solution with the same concentration of copolymer and the AIE behavior of the samples were investigated by fluorescence spectra with exciting of 330 nm. In Vitro Drug Release The DOX released from drug-conjugated micelles was measured under different mediums at 37 °C. Typically, 1 mL solution of DOX-conjugated micelles (1 mg/mL) was added into a dialysis tube (MWCO = 3500) at 37 oC in PBS (20 mL) with different


Page 26 of 34

Page 27 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


pH or different concentrations of GSH, which was performed with incessantly shaking for 48h in the dark. At selected time interval, 2 mL solution of release medium was taken out and another 2 mL fresh solution was added. The amount of released DOX was measured by UV−vis spectrometer at 480 nm. Characterization The 1H NMR spectra was recorded on a spectrometer operating at 400 MHz (Bruker AMX-400). The particle size and the zeta potential of the micelles were analyzed with a Malvern Zetasizer Nano ZS at room temperature. Transmission electron microscopy (TEM) measurements were operated on a Hitachi H-600 transmission electron microscope with an accelerating voltage of 75 KV and the samples were dyed with 2% phosphotungstic acid before measurement. The molecular weight distribution of synthetic products was determined by gel permeation chromatography (GPC) (Agilent 1260) using THF as the eluent at a flow rate of 1 mL/min at 40 oC. In Vitro Cytotoxicity Assays The cytotoxicity of blank and DOX-conjugated PMPC-PAEMA-P (TPE-co-HD)-ss-P (TPE-co-HD)-PAEMA-PMPC polymeric micelles was evaluated by MTT assays. Human cervical cancer (HeLa) cells and mouse breast cancer (4T1) cells were seeded in 96-well plates (5000 cells per well) and incubated with DMEM and RPMI-1640 medium containing 10% (v/v) fetal bovine serum and 1% (w/v) penicillin-streptomycin at 37 oC in 5% CO2, 95% humidified atmosphere for 24 h. The culture medium was removed and new medium contained different concentrations of the blank micelles, free DOX and DOX-conjugated micelles were added and the cells were incubated for



another 24 or 48 h. Then 20 µL MTT solution (5 mg/mL) was added. After 4 h, the medium was removed and 200 µL DMSO was added to dissolve the formazan. The absorbance of solutions was detected at 490 nm on a Bio-Rad microplate reader. Cellular Uptake and Intracellular Drug Release The cellular uptake of DOX-conjugated polymeric micelles was evaluated by confocal laser scanning microscopy (CLSM). 4T1 cells were seeded in glass dishes (1×10 4 per dish) and incubated for 24 h. Then DOX-conjugated micelles were added with the ultimate DOX concentration of 10 μg/mL. The cells were allowed to incubated for 2 h, 4 h and 6 h, then the culture medium was removed and washed with PBS for 3 times before observing with CLSM. The DOX was excitated at 488 nm and the TPE was excited at 405 nm to trace the intracellular drug delivery. Ex Vivo Imaging Study Animal studies were approved by the Sichuan Provincial Committee for Experimental Animal Management and scrupulously abide by the Guiding Principles in the Care and Use of Animals of the American Physiological Society. BALB/c mice (female, 18-20 g) were purchased from West China Experimental Animal Center of Sichuan University (China) and housed in SPF class animal facility at a controlled temperature of 20-22 oC, relative humidity of 50-60 % and 12 h light-dark cycles with access to commercial rat pellet diet and deionized water ad libitum. Animal xenograft tumor model was established by subcutaneous injecting 4T1 suspension cells with the amount of 1×106 per mouse. The site of injection was at the right back of mice closed to the hind limbs. When the tumor volume reached about 300


Page 28 of 34

Page 29 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mm3, the mice were injected with DOX-conjugated micelles at a dose of 5 mg DOX/kg body weight via the tail vein. After 6 h, 12 h, 24 h and 48 h, the mice were sacrificed and the major organs (heart, liver, lung, spleen and kidney) were harvested as well as the tumors. After washed with PBS for three times, the tumors and organs were performed with ex vivo fluorescence imaging using a CRI Maestro Imaging System (Cambridge Research & Instrumentation, Inc., USA). In Vivo Antitumor Efficacy 4T1 tumor-bearing mice (BALB/c) were used for investigating the antitumor efficacy of DOX-conjugated micelles. When the tumor volume approximately reached to 100 mm3, the tumor-bearing mice were randomly divided into 3 groups and mice were injected with saline solution, free DOX and DOX-conjugated micelles at selected time points (day 0, 4, 8 and 12). The free DOX and DOX-conjugated micelles were injected at a dose of 5 mg DOX/kg body weight. The body weight and tumor volume of the mice were measured every two days. The tumor volume was calculated by the equation: tumor volume (mm3) = length × width 2/2. At the 21st day, all the mice were sacrificed. The specimens of mean organs (heart, liver, spleen, lung and kidney) were excised from each group, washed by PBS, fixed with 4% formaldehyde and after dehydrating with gradient ethanol, organs and tumor were embedded in paraffin blocks to prepare the tissue sections of 5 μm. Sections were stained with hematoxylin and eosin (H&E) for histopathological evaluation. Moreover, for tumor tissue, CD3134, Ki-6735 and TUNEL36 were analyzed according to literatures. We selected one representative paraffin wax block from each group. Consecutive 4 mm thick sections were cut from



each study block and used for the immunohistochemical study. Statistical Analysis Comparisons between groups were analyzed by a Student’s t test to assess statistical significance, and p values of

Dual-responsive doxorubicin-conjugated polymeric micelles with

Recommend Documents