PEG-Detachable Polymeric Micelles Self-Assembled from Amphiphilic

Subscriber access provided by UNIV OF LOUISIANA

Biological and Medical Applications of Materials and Interfaces

PEG-Detachable Polymeric Micelles Self-Assembled from Amphiphilic Copolymers for Tumor-AcidityTriggered Drug Delivery and Controlled Release Mengzhen Xu, Canyang Zhang, Junguang Wu, Huige Zhou, Ru Bai, Ziyi Shen, Fangling Deng, Ying Liu, and Jing Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b13059 • Publication Date (Web): 15 Jan 2019 Downloaded from http://pubs.acs.org on January 15, 2019

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 40 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

ACS Applied Materials & Interfaces

PEG-Detachable

Polymeric

Micelles

Self-Assembled

from

Amphiphilic

Copolymers for Tumor-Acidity-Triggered Drug Delivery and Controlled Release Mengzhen Xu 1,3,#, Can Yang Zhang 1,#, Junguang Wu 1 , Huige Zhou 1 , Ru Bai 1 , Ziyi Shen 2, Fangling Deng4 , Ying Liu 1,*, and Jing Liu 1,2,*

1CAS

Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety & CAS

Center for Excellent in Nanoscience, National Center for Nanoscience and Technology of China, University of Chinese Academy of Sciences, Beijing 100190, China 2The

College of Life Sciences, Northwest University (NWU), Xi’an 710069, China

3Center

for Nanoscale Science and Technology, Academy for Advanced

Interdisciplinary Studies, Peking University, Beijing 100871, China 4Wuhan

University, Hubei 430072, China

* Corresponding authors: [email protected], [email protected] #These

authors contributed equally.

The authors declare no competing financial interests.

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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

Page 2 of 40

Abstract: The development of intelligent biomaterials system that can efficiently accumulate at the tumor site and release drug in a controlled way is very important for cancer chemotherapy. PEG is widely selected as hydrophilic shell to acquire prolonged circulation time and enhanced accumulation at tumor site, but it also restrains the cellular transport and uptake and leads to insufficient therapeutic efficacy. In this work, a PEG-detachable pH-responsive polymer form micelles from copolymer cholesterol grafted

poly(ethylene

glycol)

methyl

poly(ethylene glycol) methyl ether

ether-Dlabile-poly(β-amino

ester)-Dlabile-

(MPEG-Dlabile-PAE-g-Chol) is developed to

overcome aforementioned challenges based on pH value changes among normal physiological, extracellular (pHe) and intracellular (pHi) environment. PEGylated doxorubicin (DOX)-loaded polymeric micelles (DOX-PMs) can accumulate at the tumor site via enhanced permeability and retention (EPR) effect, and PEG shell is detachable induced by cleavage of pHe-labile linker between PEG segment and the main chain. Meanwhile, the pHi-sensitive poly(β-amino ester) (PAE) segment is protonated and has high positive charge. The detachment of PEG and protonation of PAE facilitate cellular uptake of DOX- polymeric micelles (PMs) by negatively charged tumor cells, along with the escape from endo/lysosome due to “proton-sponge” effect. The DOX molecules are controlled release from the carries at specific pH values. The results demonstrate that DOX-PMs have capability of show high therapeutic efficacy and negligible cytotoxicity compared with free DOX in vitro and in vivo. Overall, we anticipate that this PEG-detachable and tumor-acidity-responsive polymeric micelle can mediate effective and biocompatible drug delivery “on-demand”


Page 3 of 40 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


with clinical application potential. Keywords: PEG-detached, micelles, pH-sensitive, drug delivery, controlled release

Introduction Over the past decades, plenty efforts has been made to treat cancers threatening human health seriously, while persistent efforts have been made in the development of efficient methods to prevent and treat tumors around the world, especially chemotherapy.17Several

barriers have been strode and some significant progress has been made, as an

example, improved bioavailability through the increased water solubility of drugs and increased accumulation attributing to the enhanced permeability and retention (EPR) effect.8-9However, traditional chemotherapy still exhibits many weak points such as low therapeutic efficacy, uncontrolled drug release behavior and a series of side effects caused by indistinguishable and nonspecific drug delivery.10-12Inspired by the features of cancer, it is promising to develop smart bioresponsive drug delivery systems for overcoming these obstacles.13-16The different physiological stimuli between tumor microenvironment (pH,17-20 temperature,21-22 redox environment,23 enzyme,24 and so forth) and normal tissue undoubtedly provides a notion for the design of therapeutic agents delivery systems. Among these differences, pH gradient has been used widely because of its obvious difference and easy-implementation.25 It is well known that the endosomal and lysosomal pH (5.0~6.5) and extracellular pH around tumor cells (6.5~7.0) are much lower than normal physiological pH (7.2~7.4), attributing to low



oxygen perfusion and increased levels of lactic acid caused by tumor excessive proliferation.26-28 Based on the understanding of these mechanisms and variations, an army of pH-responsive drug delivery systems with high expectations have been designed for targeted drug delivery and controlled release, including polymeric micelles, liposomes, gels, liquid emulsion and nanoparticles, etc.29-33 In these drug delivery system, polymeric micelles formed from amphiphilic copolymers have special core-shell structure, showing a host of advantages of high biocompatibility, low cytotoxicity, high drug loading capacity as well as high delivery efficiency.34-38 Furthermore, the copolymers could be easily modified by various ligands to prepare more multi-functional systems.39 For instance, PEGylation has been extensively utilized to fabricate stealth surface of the nanoparticels, in order to stabilize the drug loading system, weaken interaction with protein and reduce reticuloendothelial systems (RES) clearance during the long-time biological circulation.40-41 Whereas, quite a few works have shown that PEGylation minimized the cellular uptake of therapeutic nanoparticles by tumor cells, resulting in decreased therapeutic efficacy.42-45 Nowadays, a slice of approaches have been used to enhance cellular internalization and uptake. It is well known that drug delivery systems could be conjugated with targeting ligands (folate, RDG, antibody, etc., which could selectively bound with overexpressed receptors on tumor cells) and/or cell penetrating peptides (e.g., arginine, showing high positive zeta potential and special guanidino).4, 45-51 However, normal cells also have the same/like receptors with high binding efficacy, interacting with high positively charged nanoparticles at the same time, leading to inspiring results in vitro but


Page 4 of 40

Page 5 of 40 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


formidable dilemma in vivo studies.52-53 Therefore, nanotherapeutics should be protected well in the systemic circulation, and the PEG-shell should be removed around the tumor tissues, probably resulting in increased accumulation at site of tumor and enhanced internalization by tumor cells. Yang and his coworkers54 developed a multifunctional drug carrier based on polymer methoxy poly(ethylene glycol)-labile linker-octadecane amine (PEG-b-C18). This linker is stable at normal physiological conditions (ca. pH 7.4) but cleaves in extracellular tumor environment (pH < 6.8). Hence, the drug-loaded PMs could be shielded by the PEG corona in the circulation and shed the shell in a slight acidic environment, resulting in drug targeted delivery and controlled release. Wang et al

43

designed a novel tumor acidity-sensitive polymeric

vector based on copolymer methoxy poly(ethylene glycol)-Dlinkm-arginine-poly(εcaprolactone) (PEG-Dlinkm-R9-PCL) for active targeted siRNA delivery. The Dlinkm is a degradable bridged bond that would cleave in the extracellular tumor tissues (pH 6.2~6.9), leading to cell penetrating peptides nona-arginines exposure. The experiments showed that this nanoparticle could enhance cellular uptake in tumor and improve the therapeutic efficacy. In this work, our purpose is to develop self-assembly polymeric micelles for effective and efficient drug delivery and controlled drug release based on pH gradient. The polymeric micelle is self-assembled from a cholesterol grafted amphiphilic triblock copolymer poly(ethylene glycol) methyl ether-Dlabile-poly(β-amino ester)-Dlabilepoly(ethylene glycol) methyl ether (MPEG-Dlabile-PAE-g-Chol) prepared by Michaeltype polymerization. The hydrophilic MPEG segment could form the protective shells



distributed on the outside of the nanoparticels, providing stable drug delivery system and prolonged circulation time in vivo. The pH-sensitive PAE segment is like middle loop in the sandwich, showing hydrophobicity at pH > pKa and hydrophilicity at pH < pKa because of nonprotonation/protonation of amino residues in PAE. The hydrophobic PAE segment would be helpful to increase the stability of the micelles, and the hydrophilic PAE segment which exhibits high positive zeta potential could increase the cell internalization and cellular uptake.55 The key component of mammalian cell membranes, hydrophobic cholesterol, is selected to construct the core in order to improve biocompatibility and drug loading capacity of this system.56 The polymeric shell is conjugated with PAE layer using Dlabile, benzoyl imine bond, which is labile to cleave in the slight acidic environment (pH < 6.8). In the extracellular matrix of solid tumor, the benzoyl imine bond would cleave gradually and PEG is removed increasingly.57 Meanwhile, the PAE residues begin to ionize and zeta potential increases rapidly.58 After internalization, MPEG or PAE have been detached or protonated mostly in the much more acidic endo/lysosomes, respectively. Based on the above understanding, effective and efficient drug targeted delivery and controlled release system could be achieved (Scheme 1).


Page 6 of 40

Page 7 of 40 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


O

Self-assembly

DOX

O

O n-1

Hydrophilic

CO

C H

N

H N

O

O O

O

N

pH-labile

pH-sensitive

H

H H

H N C

O C

O

O

O n-1

O

O

O

Hydrophobic

H N

O

O

X

O

O CH3

CH3 CH3

MPEG-Dliable-PAE-g-Chol

DOX-PMs

Endosome escape

Scheme 1. Schematic illustration of self-assembled dox-loaded polymeric micelles and drug targeted delivery and controlled dox release depending on pH values.

Experimental section Materials. Methyl ether poly(ethylene glycol) (MPEG, Mn = 4000, Sigma Aldrich). Triethylamine (TEA, > 99 %, Sigma Aldrich). Doxorubicin hydrochloride (DOX-HCl) was purchased from Beijing Hua Feng Co. LTD (Beijing, China). Hexane-1,6dioldiacrylate (HDD, 99 %), 1,3-diaminopropane (DAP, 99 %), p-Formylbenzoic acid (FA, 99 %), N,N-diisopropylethylamine (DIEA), 3-aminopropanol (AP, 97 %), cholesteryl chloroformate (Chol, 99 %), 4-dimethylaminopyridine (DMAP), and N,N’dicyclohexylcarbodiimide (DCC) were obtained from Alfa Aesar. N, Ndimethylformamide (DMF), dichloromethane (DCM), pyrene (> 99 %), n-hexane, dimethyl sulfoxide (DMSO), chloroform and other reagents were used without purification unless specified. Cell Vounting Kit-8 (CCK-8) was got from Dojindo Laboratoties (Japan). Breast carcinoma cell (MDA-MB-231) was bought from the



American Type Clulture Collection (ATCC). FITC was got from Beijing Lab Lead Biotechnology Co., Ltd. Male BALB/c nude mice were purchased from Beijing Vitalriver Experimental Animal Technology Co. Ltd. Synthesis of MPEG-Dlabile-PAE-g-Chol block copolymer Synthesis of MPEG-Dlabile. Firstly, MPEG (80 mg, 0.02 mmol), DCC (41 mg, 0.2 mmol) and DMAP (6.1 mg, 0.05 mmol) were dissolved in DCM (50 mL) with stirring, resulting in a clear solution. FA (45 mg, 0.3 mmol) was added and the mixture solution was mixed for 24 h at room temperature. The resulting solution was filtered and the resulting solution was concentrated by rotary evaporators. The resulting solution was precipitated with cooled isopropanol (ten times, v/v) for 1 h. After filtration, the solid was washed with isopropanol and diethyl ether (1:1, v/v) three times to obtain MPEGCHO. Secondly, MPEG-CHO (413.2 mg, 0.1 mmol) and DAP (10.4 mL, 0.125 mmol) were mixed and dissolved in DMSO (100 mL), resulting in a clear solution. The solution was stirred at 40 °C for 4 h. After concentration, the resulting solution was precipitated with hexane and dried under vacuum for 48 h to obtain MPEG-Dlabile. Synthesis of PAE. This pH-sensitive part was prepared via Michael-type step polymerization as described in previous work.59 Topically, HDD (1.2 mol) was placed in round-bottom flask under the atomosphere of N2, then cooled in an ice bath, and then the AP (1 mol) was dropwise added. The heterogeneous mixture was stirred at 90 °C for 6 h. The product was solved in DCM and then precipitated in hexane for three times to obtain PAE.


Page 8 of 40

Page 9 of 40 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


Synthesis of MPEG-Dlabile-PAE-Dlabile-MPEG. The triblock polymers were synthesized using MPEG-Dlabile as the diamine and PAE as the diacrylate, respectively. MPEG-Dlabile (419.8 mg, 0.1 mmol) was dissolved in anhydrous chloroform (50 mL) under nitrogen, and then the chloroform solution of PAE was added dropwise. The reaction was went on at 60 °C for 96 h under nitrogen. The resulting solution was precipitated with cooled hexane three times and dried to obtain the triblock polymers. Synthesis of MPEG-Dlabile-PAE-g-Chol. MPEG-Dlabile-PAE-Dlabile-MPEG (1 mmol) was dissolved with anhydrous DCM, then 15 mmol TEA was added. The DCM solution of Chol (15 mmol) was added into the flask dropwise. The solution was stirred at room temperature for 24 h. After purification, the resulting solid was dried under vacuum for 2 days to get designed copolymers. Synthesis of FITC labeled MPEG-Dlabile-PAE-g-Chol. FITC was labeled on the synthetic copolymer according to the reference and our previous work.60-61 In brief, MPEG-Dlabile-PAE-g-Chol (200 mg) was dissolved in DMSO (5 mL), and the resulted solution was mixed with FITC (2 mg) with stirring moderately. The reaction was conducted at 90~100 °C in darkness. After dialysis and lyophilization, FITC labeled MPEG-Dlabile-PAE-g-Chol was received for further study. Characterization Proton nuclear magnetic resonance (1H-NMR). 1H-NMR analysis was executed on copolymer solutions in CDCl3-d or DMSO-d6 with tetramethylsilane (TMS) as an internal standard at 25 °C using a Bruker AVANCE III 400 (Switzerland) spectrometer



operating at 400 MHz. To confirm the removal of PEG segments, copolymers were incubated in solutions of different pH (7.4 and 6.5) for 2 h, then the absence of benzoic imine and presence of formyl group were monitored by 1H-NMR. Fourier transform infrared spectroscopy (FT-IR). FT-IR spectrophotometer (Nicolet Nexus for Euro, USA) was used to verify the structure of the polymer. The transparent tablets were formed by mixture of polymer and potassium bromide (KBr), and were detected with FT-IR. The spectra were taken with a resolution of 2 cm-1 from 400 to 4,000 cm-1. Gel permeation chromatography (GPC). We confirmed the average molecular weight (Mw) and polydispersity index (PDI, Mw/Mn) by GPC adopting an Agilent1200 series GPC system and RI detector. THF or DMF was selected as the mobile phase and we use a flow rate of 1 mL/min. Transmission electron microscopy (TEM). The morphology of blank and drugloaded nanoparticels at different pH solutions was detected by TEM (Tecnai G2 20 STWIN, Japan) operation at 200 kV. To monitor the changes of pH-sensitive polymeric micelles, the received polymeric micelles were incubated with different pH of 7.4, 6.5, 6.0 and 5.0, then the solutions were lyophilized to obtain samples for TEM detection. Dynamic light scattering (DLS) analysis. The particle size, distribution and zeta potential of drug-loaded and blank polymeric micelles at different pH values were confirmed by DLS (Malvern Zetasizer Nano S, Malvern, UK). In brief, after dialysis, the solution was filtered through 0.45 μm pore size filter, and the resulting sample was


Page 10 of 40

Page 11 of 40 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


tested in a 1.0 mL quartz cuvette, using scattering angle 90°and a diode laser of 800 nm at 25 °C. Potentiometric titration. Acid-base titrations were used to ensure the copolymer base dissociation constant (pKa). In brief, the copolymer (50 mg) was dispersed in 50 mL ultrapure water. The pH value of the solution was regulated to 3~4 using HCl solution. Then, the solution was adjusted by NaOH (0.1 mol/mL) at increments with 100 μL. The pH values were recorded by an automatic titration titrator (METTLER TOLEDO FE20, CHE) at 25 °C to obtain the titration profile. Critical micelle concentration (CMC) detection. The CMCs of copolymers at different solutions with pH of 7.4, 6.8, 6.5, 6.0 and 5.0 were confirmed by the fluorescence probe technique, using pyrene as a fluorescence probe according to our previous protocol.37 Briefly, a series of samples with different concentrations (0.0001– 0.1 mg/mL) containing certain concentration of pyrene (6×10-7 M) were prepared firstly. After being equilibrated in the dark for 24 h at room temperature, the samples were detected using a fluorescence spectrophotometer (F-4500, Hitachi, Japan) and the excitation spectra were scanned at wavelengths 300~350 nm. Preparation of DOX-loaded and blank polymeric micelles The DOX-PMs and blank micelles were prepared by the dialysis method as reported.62 In brief, 20 mg DOX-HCl was dissolved in DMF (40 mL), followed by the dropwise addition of TEA (0.01 mL for per 10 mg of drug). After being stirring for 2 h, 80 mg polymer was added. The mixture was transferred into dialysis bag (molecular weight



cutoff of 3500Da), and dialyzed against 2 L of ultrapure water for 48 hours at 25 °C. The ultrapure water was changed every 2 h for the first 12 h and then every 6 h. Subsequently, the solution was filtered by 0.45 μm pore size filter, and the polymeric micelles were obtained after lyophilization. The blank micelles were prepared without drug using the same protocal above. DOX loading content (LC) and encapsulation efficiency (EE) were determined by UVvis spectrophotometer.37In brief, 1 mg DOX-PMs powder dissolved totally in 10 mL DMSO, the concentration of DOX was calculated depengding on a standard curve of free DOX/DMSO solution. LC and EE were calculated using Equations (1) and (2):

LC (%) 

Wt 100 % Wm

(1)

EE (%) 

Wt 100 % Wi

(2)

where, Wt was the weight of drug loaded in the polymeric micelles, Wm was the weight of drug-loaded polymeric micelles, Wi was the weight of initial drug in feed. Each sample analyzed in triplicates. DOX release from PMs in vitro The DOX controlled release behavior from micelles in PBS with pH 7.4, 6.5 6.0 and 5.0 which were used to simulate the tumor extra- and intra-cellular microenvironment were studied using dialysis method at 37 °C. Quite low drug concentration was performed to acquire sink conditions.37 Briefly, we dispersed 5 mg DOX-PMs in 5 mL (Ve) respective PBS buffer solution in dialysis bag (MWCO 3500Da) and allowed to


Page 12 of 40

Page 13 of 40 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


stabilize for 0.5 h. The dialysis bag was placed in the corresponding PBS solution (45 mL) in a beaker which was placed in a water bath of 37 °C and stirred at rate of 110 rpm. 1 mL solution was taken for the drug concentration detection using UV-vis spectrophotometry at 480 nm and 1 mL fresh respective PBS was added. The cumulative drug release percent (Er) was calculated by the following Equation (3). At each pH value, the experiments were performed in triplicate. 𝑛―1

𝐸𝑟 =

5 × ∑1

𝐶𝑖 + 50 × 𝐶𝑛

𝑚𝑑𝑜𝑥

× 100%

(3)

where, mdox means the amount of DOX in the micelles, and Ci means the concentration of DOX in the ith sample. Cell culture MDA-MB-231 (Human breast cancer cell line) was kept in our laboratory. The cell culture was similar as our previous work.61 The cells were kept in complete DMEM medium (WISENT Inc.), at the condition of 37 °C, 5 % CO2 and 10 % humidity, and with 10 % (v/v) fetal bovine serum (FBS). Confocal laser scanning imaging of cellular uptake The cellular uptake was determined by confocal laser scanning microscopy (CLSM, Perkin Elmer Ultra View Vox system, USA). Cells were seeded into 35 mm dishes for 24 h at 37 °C, 5 % CO2 and 10 % humidity, then one part was incubated with DOXPMs (equal to 2.5 µg/mL free DOX) for 4 h, and another part was incuabated with free DOX (1.5 µg/mL) and DOX-PMs (equal to 1.5 µg/mL free DOX) for different time



intervals (24, 36 and 48 h). After this, MDA-MB-231 cells were collected and washed with PBS for three times and stained 15 min with Hoechst 33342. Finally, cells were observed by CLSM, and the emission of samples was detected at 520 nm (green for FITC), 580 nm (red for DOX) and 460 nm (blue for nucleus) with 488 nm and 350 nm excitation wavelength, respectively. Fluorescence measurement by flow cytometry for cellular uptake MDA-MB-231 cells were incubated in plates of 6-well and cultured for 24 h at 37 °C, 5 % CO2 and 10 % humidity, and then incubated with the 1.5 µg/mL free DOX or DOXPMs (equal to 1.5 µg/mL free DOX) for 4 h at different pH, respectively. Then cells were washed with ice-cold PBS for three times and digested with TE for 3 min. The cell suspension was centrifuged for 5 min at 800 g at 4 °C , then resuspended in 100 µL ice-cold PBS for quantification of cellular DOX by flow cytometry (BD Biosciences Accuri C6), n=3. Cytotoxicity test A cell counting kit-8 (CCK-8) (Kumamoto Techno Research Park, Japan) was used to determine the cell viability. Firstly, cells were plated into 96-well microplates (Costar, Corning, NY) with the concentration of 3000 cells/well. After incubating (37 °C, 5 % CO2 and 10 % humidity), culture medium was removed and replaced with the complete medium with various concentrations of free DOX, blank and DOX-PMs for 24 h and 48 h (n = 5), respectively. Meanwhile, wells unexposed to materials were regarded as control. Finally, a mixture of the tetrazolium reagentand the complete medium (1: 10)


Page 14 of 40

Page 15 of 40 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


was added into 96-well microplates with the volume of 150 μL/well. Cell viability was calculated at 450 nm (reference wavelength 650 nm) by an Infinite M200 microplate reader (Tecan, Durham, USA). Animal studies Female BALB/c nude mice (six weeks old) were obtained from Beijing Vitalriver Experimental Animal Technology Co. Ltd, housed in a disinfected animal room with a 12 h light/dark cycle, with body weights in the range of 17.0 g to 20.0 g, were used in this work.

Mice were allowed to acclimatize for 1 week before the start of

experiments. A 100 µL mixture of 1 × 107 murine MDA-MB-231 cells and growth factor (1: 1, v/v) was subcutaneously inoculated in the right hind leg of each mouse. Tumor volumes were measured by a vernier caliper and calculated as tumor volume (V) = ab2/2 (a and b were the length and width of the tumor, respectively). About ten days later, when the tumor volume reached about 100 mm3, twenty mice were divided into four groups randomly. Tumor-bearing mice were i.v. injected with PBS, 4 mg kg-1 free DOX, 36 mg kg-1 polymer and DOX-PMs (equal to 4 mg kg-1 free DOX), respectively. Tumor growth and body weight were measured every two days in the following time. At 30 days post-injection, all the mice were sacrificed, their organs and blood were harvested for subsequent detection. Blood biochemistry and pathology Blood biochemical analysis was conducted through blood collection, by removing the



eyeball of mice, and then the major organs were harvested carefully, fixed in 10 % neutral buffered formalin, processed routinely into paraffin, stained with hematoxylin and eosin (H&E), and examined by a microscope at last. For blood analysis, about 1 mL blood was collected from mice with different treatment, and after 3 h maintained in room temperature, were separated by centrifugation with 800 g into cellular and plasma fractions. Statistical analysis The experimental data were presented as the mean ± standard deviation (SD). Student’s t-test (Excel, 2007) was used to analyze the data. Statistical significance was considered to be significant when P < 0.05.

Results and discussion Synthesis and characterization of the MPEG-Dlabile-PAE-g-Chol block copolymer Scheme 2 shows the synthetic route of MPEG-Dlabile-PAE-g-Chol copolymer. Firstly, the terminal hydroxyl groups of MPEG were activated by DCC/DMAP system and conjugated with aldehyde groups of p-formylbenzoic acid, followed by reaction with 1,3-diaminopropane, resulting in amino-capped MPEG monomer containing pHsensitive Schiff base bonds (MPEG-Dlabile). Then, diacrylate ester-ended PAE monomer was prepared using Michael-type polymerization as shown in our previous work.59 Afterwards, the MPEG-Dlabile was conjugated with PAE on the two terminals, resulting in PEG-modified PAE block polymer (MPEG-Dlabile-PAE-Dlabile-MPEG).


Page 16 of 40

Page 17 of 40 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


Finally, the cholesterol used as hydrophobic segment to increase the stability and the drug loading capacity of the system was grafted on the tail of PAE via esterification reaction, leading to the designed copolymers MPEG-Dlabile-PAE-g-Chol. The final polymer was received after purification and lyophilization for subsequent studies. The chemical structures of synthesized copolymers were carefully ensured by

1H

NMR and FT-IR. As described in Fig. S1, the signals at 10.13 (a), 8.15 (b) and 7.97 (c) ppm were ascribed, respectively, to the introduced aldehyde group (-CHO) and the benzene ring (β, γ -CH-). After conjugation of 1,3-diaminopropane on the terminal, the peak (a) disappeared and peaks 1.61 (h), 2.97 (i) and 3.12 (j) ppm appeared as well as peak 8.45 (m) ppm (-CH=N-), indicating the synthesis of amino-capped PEG monomer. As shown in Fig. S2, the peaks at 1704 cm-1, 1650 cm-1 and 762 cm-1 were ascribed to C=O stretching vibration of aldehyde, C=N bending vibration of the amino group and C-H out-of-plane vibration of para-substituted benzene ring, respectively. The results showed that the amino-ended PEG monomer with Schiff base was prepared successfully. As shown in Fig. S3, the PAE monomer was prepared successfully using Michael addition as reported.59 Subsequently, the block polymer MPEG-Dlabile-PAEDlabile-MPEG was synthesized and the chemical structure was verified by 1H NMR and FT-IR (Fig. S4 and S5). The presence of peaks at signals 8.40 (m), 8.15 (b) and 7.97 (c) ppm and the absence of peaks at signals 5.89 (c), 6.21 (b) and 6.40 (a) (Fig. S3) and 10.13 ppm (data not shown) indicated that the synthesis of this block polymer. Furthermore, Fig. S5 demonstrated that the PEG and benzene ring were present. After cholesterol grafted, the characteristic peaks at signal 5.40 (i) ppm and 0.50-2.0 ppm



Page 18 of 40

were present in the 1H NMR spectrum of MPEG-Dlabile-PAE-g-Chol (Fig. 1). Combining with the FT-IR spectrum (Fig. S6), the designed copolymer was synthesized successfully. The grafting ratio of cholesterol was 55 % verified by integral the characteristic peaks area of (i) and (d, e) according to result of 1H NMR. O O

O

DCM, DCC/DMAP

OH

HOOC

n-1

CHO

MPEG

O

O

RT, 24 h

O

CHO

C

n-1

MPEG-CHO

FA

O

O O

O

O

CHO

C

H2N

NH2

n-1

DMSO 40 ℃ , 4 h

90 ℃ , 6 h

O

CO

n-1

N

HO

HDD O

NH2

O

O

n-1

x

Chloroform

N

O

O

x

O O

O

N

H N

O

O X

O

O

PAE

HO

60 ℃ , 96 h

O

H N

O

PAE

O

N

C H

O

O

O

O

H C N

C

MPEG-Dlabile

O

O

O

O

O

NH2

O

AP

O

N

O

O

O

O

H C

C

n-1

MPEG-Dlabile

O O

O

O

O

DAP

MPEG-CHO H2N

HO

O

H N C

O C

O

O

O n-1

O

HO

MPEG-Dlabile-PAE-Dlabile-MPEG O H

H H

Cl O

RT, 24 h

CH3

TEA

CH3 CH3

O

O

O n-1

CO

C H

N

H N

O

O O

O

N

X

O

H

H N C

O

O

O H

H

H N

O

O

O CH3

CH3 CH3

MPEG-Dliable-PAE-g-Chol

Scheme 2. Synthetic route of MPEG-Dlabile-PAE-g-Chol copolymer.

Fig. 1 1H NMR spectrum of MPEG-Dlabile-PAE-g-Chol in d-CDCl3.


O C

O

O

O n-1

Page 19 of 40 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-responsibility of the copolymer We then investigated the pH-responsiveness of copolymer in vitro. Firstly, the pKa of copolymer MPEG-Dlabile-PAE-g-Chol was evaluated by an acid-base titration (Fig. 2A). The pH value raised rapidly at the beginning of base addition, and reached a plateau, attributing to the ionized amine groups in PAE moieties. Then it increased sharply again as with the addition of NaOH solution, indicating the amine groups of PAE units have protonated completely from pH 6.37-6.78. The pKa was determined about 6.55 according to the defined reference.63 After that, GPC spectrum (Fig. 2B) exhibited that the copolymer (pH 6.5, 2 h, 37 °C) displayed divisive dual peaks compared with the single peak of copolymer (pH 7.4, 2 h, 37 °C), demonstrating the presence of detached PEG segment because of the cleavage of Schiff base bonds. Furthermore, the aldehyde proton peak was not observed after incubation of copolymer in PBS at 7.4 (2 h, 37 °C), showing the stable attachment of PEG segment which could protect system well, achieving extended circulation time (Fig. 2C). However, after incubation of copolymer in PBS at pH 6.5 for 2 h, the characteristic peak of aldehyde (10.13 ppm) was emerged, indicating the presence of MPEG-CHO due to detachment of PEG segment from the main chain (Fig. 2D). Meanwhile, the intensity of the aldehyde proton peaks at different pH were investigated by 1H NMR, it almost broke completely after incubation at pH 5.0 for 2 h (Fig. S7). In a word, the results revealed that PEG segment could be stable in normal physiological conditions (pH 7.4) and detached in the weakly acidic condition (e.g. pH 6.5), suggesting the pH-responsibility of the copolymer and PEG detachment



ability.

A

11 10 9 8 7 6 5 4 3

pH

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

Page 20 of 40

0.0

B

MPEG-Dliable-PAE-g-Chol pH 6.5-2 h

300

MPEG-Dliable-PAE-g-Chol pH 7.4-2 h

280

MPEG pH 6.5-0 h

260

30

240 25

220 200

pKa 6.55

180

20

160 140

15

120 100

10

80 60 40

5

20

0.5

1.0

1.5

2.0

2.5

3.0

16

18

20

V0.1 M NaOH（mL（

C

22

24

26

28

30

Time (min)

D

Fig. 2 Confirmation of the pH-responsibility of the copolymer. (A) Acid-base titration profile of the pH-responsive copolymer MPEG-Dlabile-PAE-g-Chol. (B) GPC spectra of copolymer MPEG-Dlabile-PAE-g-Chol incubated in PBS at pH 7.4 and 6.5 for 2 h. 1H NMR spectra of copolymer MPEG-Dlabile-PAE-g-Chol incubated in PBS at pH 7.4 (C) and 6.5 (D) for 2 h.

To further evaluate the self-assembly and pH-sensitivity of the copolymer, CMCs of the copolymer MPEG-Dlabile-PAE-g-Chol were measured as a function of pH value, as shown in Fig. 3A. The CMCs of copolymer in PBS at pH 7.4, 6.0 and 5.0 were 7.3 mg/L, 14.7 mg/L and 20.5 mg/L, respectively (Fig. S8). The CMCs increased obviously with decreasing of pH because of the protonation of pH-stimuli PAE. The Schiff base bond was sensitive at pH 6.0 leads to PEG detachment. Meanwhile, partial PAE


0

0

-20 5

10

15

20

5

2

Page 21 of 40 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


segments were protonated and converted from hydrophobic to hydrophilic, resulting in the increase of CMCs compared with pH of 7.4 (from 7.3 mg/L to 14.7 mg/L). With the decrease of pH values (pH 5.0), the CMCs were increased sharply (20.5 mg/L) attributing to more ionized amine moieties of PAE and raised polymer molecular polarity, which required a stronger driving force for micellar formation to overcome the greater electrostatic repulsive force.64 These findings indicated the pH-stimuli and selfassembly of amphiphilic copolymer. Next, we monitored the zeta potential and hydrodynamic diameter of blank polymeric micelles at different pH values to further study the pH-sensitivity of micelles. As expected in Fig. 3B, when the pH was higher than 7.4, the nanoparticle size of polymeric micelles slightly increased with the pH increasing because of aggregation of micelles. As with the pH values declined, the particle size increased moderately (7.4~6.5) and dramatically (6.5~5.0) with the pH decreasing. The reason could be that the hydrophilic PEG segment detached in the weak acidic conditions, and PAE segment started to become hydrophilic because of protonation of amino groups. As pH reduced, amine segment of PAE were completely ionized, indicating that pH-sensitive PAE segment transformed from hydrophobic to hydrophilic, leading to greater hydrophobic interaction for micelle formation and swollen polymeric micellar structure. As seen from the profiles of zeta-potential as a function of pH values (Fig. 3C), it increased with the pH decreasing (from + 5 mV to + 45 mV) because of detachment of PEG and protonation amine residues in PAE moieties, resulting in high positive charge on the surface of the nanoparticles which could enhance the tumor cellular uptake because of



electrostatic effect. As seen in Fig. 3D, the peak shifted to right with decreasing of pH, and the polydispersity index (PDI) was low, displaying the similar change trends of hydrodynamic particle size. To observe the detachment of PEG and changes in particle size dependent on pH values visually, after incubation in PBS at different pH values TEM was used to image the morphology of polymeric micelles (Fig. 3D and E). The micelles took a spherical morphology in various PBS buffer solutions with different pH values. At pH 7.4, the particle size was almost 60 nm, and increased to 97 nm (pH 6.5), 116 nm (pH 6.0) and 185 nm (pH 5.0) as pH decreased. The change trend was similar to the result of DLS above. At pH 5.0, incompact corona could be observed distinctly, indicating swollen core/shell polymeric micelle. DOX-PMs size has no obvious change in PBS within 48 h, indicating that DOX-PMs had an excellent stability. (Fig. S9)

B

C

160 Size (nm)

15 10

120 80

5 0

5.5

6.0

6.5

7.0

5

7.5

6

7 pH

Mean size: 59 nm PDI: 0.205

25

30

pH=7.4 Intensity (%)

20 15 10 5 0

1

10

E

100 1000 Size (d.nm)


25

10000

20 15 10

20 10

5


25 20 15 10 5

1

10

100 1000 Size (d.nm)

10000

0

6 30

pH=6.0

50 nm

5 0

30

9

50 nm

7 pH

8

9


25 20

pH=5.0

15 10

50 nm

5 1

10

100 1000 Size (d.nm)

pH=6.5

pH=7.4

200 nm

8

30

pH=6.5 Intensity (%)

30

40

0

40 5.0

pH

D

50

Intensity (%)

CMC (mg/L)

20

200

Zeta potential (mV)

25

A

Intensity (%)

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

Page 22 of 40

200 nm

10000

0

1

pH=6.0

10

100 1000 Size (d.nm)

10000

pH=5.0

200 nm

Fig. 3 Preparation and characterization of self-assembled polymeric micelles. (A)


Page 23 of 40 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


The CMCs of copolymer MPEG-Dlabile-PAE-g-Chol as a function of pH. Particle size (B) and zeta-potential (C) of polymeric micelles dependent on the pH value. (D) Particle size, distribution , TEM images (Scale bars: 50 nm) and (E) TEM images (Scale bars: 200 nm) of polymeric micelles after incubation in PBS at pH 7.4, 6.5, 6.0 and 5.0 for 2 h.

Release of DOX from micelles in vitro DOX was encapsuled in the polymeric micelles by dialysis method, confirmed by the UV-vis scanning spectra, there is no absorption of blank PMs at wavelengths ranging from 600 to 400 nm, whereas DOX-PMs exhibited a characteristic peak of DOX at 520−425 nm. (Fig. S10) The actual drug loading content and encapsulation efficiency were about 11.2 % and 53.5 %, respectively. To confirm the drug release performances dependent on pH values, in vitro release profiles of DOX from DOX-PMs were performed in PBS with pH 7.4 (physiological conditions), 6.5 (extracellular environment), 6.0 and 5.0 (intracellular environment), as shown in Fig. 4A. It was obvious that the DOX release rate was accelerated significantly with pH value decreasing, especially from pH 7.4 to pH 5.0. In the case of pH 7.4, the DOX-PMs were compact with PEG-shell and PAE/cholesterol-core, leading to a slow DOX release rate and low accumulative release, only about 15 %, 25 % and 30 % in 2 h, 24 h and 120 h, respectively, indicating that the overwhelming majority of DOX was entrapped in the core, protected well and with negligible drug burst release. At pH 6.5, DOX release rate was accelerated slightly, and drug cumulative release were 18 %, 44 % and 60 % for 2



h, 24 h and 120 h, respectively. This could be due to the detachment of PEG and partial ionized amine groups of PAE segments, resulting in few DOX molecules released from the polymeric micelles. In contrast, when the pH decreased to 6.0 or less, the DOX release rate and cumulative release increased markedly, about 50 % or 65 % in 24 h and about 70 % and 85 % after 120 h for DOX-PMs at pH 6.0 or 5.0, respectively, attributing to the swollen and loose micellar structure by more and more protonated amine groups which caused PAE segment became hydrophilic sharply after detachment of PEG segment. The in vitro drug release performances showed that DOX molecules were able to be controlled release from the polymeric micelles based on the changes of pH value, indicating the DOX release rate and cumulative release were slow and slight in normal physiological and extracellular conditions while accelerated sharply in intracellular environment. In addition, the increased positive charge density of the micelle surface (Fig. 3C) forced enhanced electrostatic repulsion effect between hydrophilic chains which was conducive to swelling and loose of drug-loaded polymeric micelles. In summary, the drug release rates were slow in the physiological and extracellular environment, while accelerated sharply in intracellular environment. In order to further investigate the drug release behavior from polymeric matrix, the release mechanism at different conditions was roughly analyzed using following comprehensive semi-empirical Equations (4) established by Peppas.65

log(

where,

Mt

and

M

Mt )  n log t  log k M

(4)

were drug accumulative release amount at time t. k and n were

figured out to ensure the drug release mechanism and rate. The release mechanism was


Page 24 of 40

Page 25 of 40 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


not understood well and simply classified as diffusion, swelling-, erosion- controlled or a combination of these mechanisms until now.66 When n = 0.43 or 0.85, the release mechanism was determined as Fickian diffusion or swelling-controlled, respectively. When n < 0.43 or 0.43 < n < 0.85, it was defined as the combination of diffusion and erosion-controlled or anomalous transport mechanism, respectively.67 The theoretical fitted curve and calculated n and k based on experimental data were shown in Fig. 4B and Table 1. The DOX release profiles from drug-loaded micelles were divided into two stages, 0~10 h and 10~152 h. In the case of pH 7.4, 6.8 and 6.5 at the first stage, the n values were close to 0.43, indicating that the drug release behaviors corresponded to the Fickian diffusion because of drug molecular released from the core through the nanopores. With increase in time, the n values obviously decreased, much lower than 0.43, demonstrating that the diffusion and erosion control was the key factor because of swelling of the micelles. At pH 6.0 and 5.0, the n values were lower than 0.43 for both of stages, suggesting that drug release behaviors fitted the combination of diffusion and erosion-controlled at the conditon. As seen from the Table 1, the k values increased from 0.097 to 0.395 for the first stage and from 0.105 to 0.545 for the second stage with pH decreased from 7.4 to 5.0, displaying that the drug release rates from the polymeric micelles increased with decreasing pH values. The reason could be that the DOX molecules were protected well in the PEGylated polymeric micellar core at pH 7.4, resulting in slow drug release rate compared with weak acidic environment. With pH decreased, PAE segment transformed from hydrophobic to hydrophilic block because the protonation of amine groups was one of the main cause for accelerating drug release



rate. As an overall result, the detachment of PEG segment and more dissociative hydrogen ions were able to facilitate the drug release rate from the polymeric matrix.

80

0.0

B

pH 6.5 pH 5.0

-0.2

0.0

pH 7.4

-0.2

-0.6 -0.8 -1.0

60

-1.4 -0.5 0.0 -0.2

0

0.0

0.5

1.0 1.5 log t (h)

2.0

20 40 60 80 100 120 140 160 Time (h)

-1.0

-0.5 0.0

0.0

pH 6.0

-0.2

-0.4 -0.6 -0.8 -1.0

0

-0.8

-1.4

2.5

log (Mt/M)

20

-0.6

-1.2

-1.2

40

pH 6.5

-0.4

-0.4 log (Mt/M¥)

pH 7.4 pH 6.0

log (Mt/M¥)

100

log (Mt/M)

A

Accumulative Release (%)

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

Page 26 of 40

-1.2 -1.4 -0.5

0.5

1.0

log t (h)

1.5

2.0

2.5

pH 5.0

-0.4 -0.6 -0.8 -1.0 -1.2

0.0

0.5

1.0 1.5 log t (h)

2.0

2.5

-1.4 -0.5

0.0

0.5

1.0 1.5 log t (h)

2.0

2.5

Fig. 4 DOX release profiles in vitro (A) and plots of log (Mt/ M) against log t for DOX release (B) from drug-loaded micelles at pH 7.4, 6.5, 6.0 and 5.0 solutions.

Table 1. Release exponent (n), rate constant (k) for drug-loaded polymeric micelles at different pH values, T = 37 °C. pH

n (0-10 h)

k (0-10 h)

Controlled

n (10-152

k (10-152

Controlled

Mechanism

h)

h)

Mechanism

7.4

0.430

0.097

Fickian

0.143

0.105

6.8

0.431

0.125

diffusion

0.139

0.133

Diffusion

6.5

0.427

0.131

0.189

0.252

and erosion

6.0

0.383

0.277

Diffusion and

0.194

0.317

control

5.0

0.363

0.395

erosion control

0.155

0.545

Cytotoxicity and cellular uptake in vitro We compared cytotoxicity of DOX-PMs with free DOX and blank copolymers with


Page 27 of 40 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


human breast cancer cell lines MDA-MB-231. We found that even a the concentration of 200 µg/mL copolymer, the cell viability was still more than 95 % for 24 h and 48 h, indicating that the synthetic copolymer showed a relative low cytotoxicity for MDAMB-231 cells (Fig. S11). Fig. 5A and B exhibited the cytotoxicity of free DOX and DOX-PMs for 24 h and 48 h, respectively. It could be seen that the cell viability reduced obviously with the DOX concentration increased for both of free DOX and DOX-PMs. Interestingly, at 24 h, free DOX showed much higher cytotoxicity compared with DOXPMs, and induced the cell viability sharply declined with the increasing of concentration. Relatively, the toxicity of DOX-loaded system began to emerge with the DOX concentration increased from 0.1 to 20 μg/mL for 48 h, because a large amount of DOX molecule has been released in the matrix. The results demonstrated that DOXPMs showed time- and concentration-dependent cytotoxicity for MDA-MB-231 cells, meanwhile, obtained sustained drug release effect, which is beneficial for subsequent applications. Since the internalization is critical for toxicity and further therapeutic effect, we investigated the cellular uptake of DOX-PMs using the confocal microscopy (red fluorescence for DOX and the green fluorescence for carrier) and flowcytometry. As shown in Fig. 5C, after 24 h, lots of DOX-PMs were found in the cytoplasm, resulting from the enhanced cellular uptake caused by the detachment of PEG segment and high positive charge on the surface as mentioned above. Furthermore, most DOX had not been released due to the sustained controlled released effect, which is consistent with the results before. Comparable, red fluorescence had separated from green at 36 h,



indicating that DOX molecules had released and a small amount entered into cell nucleus. At last in 48 h, red fluorescence was overlapped with blue, which proved almost all DOX molecules had in the nucleus. The reason could be that the DOX-PMs escaped from the endo/lysosomes because of “Proton-Sponge” effect attributed to the protonation of amine groups in PAE moieties. In order to investigate the influence of pH-triggered charge change on cellular uptake, free DOX and DOX-PMs were incubated with MDA-MB-231 cells at different pH values (pH = 7.4, 6.5 and 5.0) for 4 h (Fig. 5D, E and F). As shown in Fig. 5F, for free dox, the cellular uptake is pHindependent, and DOX distributed in the nucleus most after 4 h incubation. For DOXPMs, however, when under the physiological pH about 7.4, MDA-MB-231 cells with extremely weak fluorescence intensity can be detected, indicating that less DOX-PMs enter cells under this condition. However, the fluorescence intensity significantly enhanced at pH 6.5 and 5.0, which mimicking the tumor microenvironment and lysosomal environment, respectively. Furthermore, most of the DOX-PMs localized in cytoplasm, different from free DOX in nucleus, which is consistent with Fig. 5C, and is likely to the reason of no significant cytotoxicity of DOX-PMs within 24 h in Fig. 5A. We also made quantitative measurements at different pH values by flow cytometry analysis (Fig. 5D). Coincident with fluorescence images, MDA-MB-231 cells showed a faster fluorescence intensity increase at pH 6.5 and 5.0 than that of pH 7.4, after treated with DOX-PMs, which further confirmed the pH-dependent cellular uptake and drug release of DOX-PMs. In addition, we tested the fluorescent emission of DOXPMs at different pH values (Fig. S12), indicating the emission intensity of DOX-PMs


Page 28 of 40

Page 29 of 40

obviously enhanced in acidic environment. According to Fig. 5D-F, cellular uptake of free DOX is more than DOX-PMs within 4 h incubation, which is probable due to the quick free diffusion of DOX rather than the ATP-required endocytosis of DOX-PMs.68

Cell viability (%)

A

130 120 110 100 90 80 70 60 50 40 30 20 10 0

B

24 h

Free Dox

0.1

130 120 110 100 90 80 70 60 50 40 30 20 10 0

1

48 h

C

Dox-PMs

5

24 h

36 h

48 h

20 g/mL

10

Free Dox

Control

Dox-PMs

Cell viability (%)

D

Mean fluorescence intensity (a.u.)

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


0.1

1

14000

5

10

20 g/mL

E

DOX

DOX-PMs

12000 10000

6.5

7.4

8000

***

6000

***

6.5

7.4

5.5

4000 2000

DOX-PMs

DOX-PMs 0

7.4

F

7.4

6.5

5.5

7.4

6.5

6.5

5.5 5.5

7.4

7.4

6.5

5.5

5.5

DOX DOX-PMs

DOX-PMs

DOX

DOX-PMs DOX

6.5

DOX

Fig. 5 The cytotoxicity for MDA-MB-231 cells induced by the treatment with free DOX or DOX-PMs for 24 h (A)DOX and 48 h (B) in concentration gradients. (C) Confocal microscopy image of cells incubated with DOX-PMs for different time intervals. (D and E) Fluorescence measurements of MDA-MB-231 cells incubated with DOX-PMs and free DOX at pH 7.4, pH 6.5 and pH 5.0 for 4 h. (F) Confocal laser scanning microscopy images of cellular uptake with DOX-PMs and free DOX at pH 7.4, pH 6.5 and pH 5.0 for 4 h incubation. The concentration of free DOX and DOX in PMs were


5.5


equivalent (Scale bars: 50 μm). ***p 0.05, are calculated by t-test. (E) Representative histological images of major organs (scale bar, 20 μm). All data expressed as mean ± s.d. (5 mice per group).

Above all, since biocompatibility and biosafety of nanomedicine is an essential concern for further biomedical applications in clinic, the prepared DOX-PMs exhibit great advantage in safety. As shown in Fig. 6D, in consideration of the toxicity of DOX, the body weight of free DOX group sharply declined at day 14 post injection, while there was no obvious decrease in DOX-PMs group compared to other control groups, which is consistent with the results of major organ weight in Fig. S13. Moreover, as shown in H&E staining images of major organs, no observable toxicity was emerged in DOXPMs group, while obvious cardiotoxicity was found in free DOX group. In addition, our results about blood biochemistry analysis (Fig. S14) indicated that CK, ALT, AST, CREA and BUN, which represent the function of heart, liver and kidney respectively, in free DOX group exhibited significant difference compared to normal control by ttest, while DOX-PMs group showed no distinction. This might be attributed to the sustained drug release of DOX-PMs. In short, our results indicated that no obvious loss of body weight (major organs weight) and no observed changes of blood biochemistry were noticed in DOX-PMs group. whereas, free DOX group exhibited remarkably toxicity. The findings demonstrated the relative biosafety of prepared DOX-loaded


Page 32 of 40

Page 33 of 40 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


PMs.

Conclusions With rapid development of nanotechnology, various multi-functional NPs have been thoroughly studied and extensively used as drug delivery carriers for diagnosis and treatment of cancer. These NPs with proper size are able to accumulate in the site of cancer due to the enhanced permeability and retention (EPR) effect.69-70 However, there are still a series of challenges for further application in clinic, such as low cellular uptake. As reported42-43, hydrophilic PEG shell could decrease the internalization of NPs after accumulation at site of tumor due to the negative charge of PEG-shielded NPs. Therefore, in this study, we have developed a PEG-detachable pH-responsive polymeric micelle self-assembled from amphiphilic copolymer MPEG-Dlabile-PAE-gChol consisting of pH-labile bonds and pH-sensitive blocks. Here, pH-sensitive PAE blocks and hydrophobic cholesterol segments formed the core of nanocarriers to load DOX via hydrophobic interaction, on which surrounded with PEG shell conjugating with pH-labile bonds. Our nanocarriers show detachment of PEG shell properties correlated to slight acidity in extracellular tumor microenvironment, and pH-triggered drug release properties due to the protonation of PAE segments in intracellular tumor microenvironment, which locally deliver and release the loaded drugs on-demand for improved therapeutic efficacy in cancer chemotherapy.

5-7

We anticipate this study

shows the development of an efficient and controlled release anticancer drug delivery



system based on pH-responsive copolymer, and provide a potential strategy for design of efficient functional stimuli-responsive nanocarriers.

Associated content Supporting Information The Supporting Information is available free of charge on the ACS Publications website. 1H

NMR spectra of MPEG-CHO, MPEG-Dliable in d-CDCl3; Fourier transform

infrared spectra of MPEG, MPEG-CHO and MPEG-Dliable; 1H NMR spectra of PAE in d-CDCl3; 1H NMR spectra of MPEG-Dliable-PAE-Dliable-MPEG in d-CDCl3; Fourier transform infrared spectra of PAE and MPEG-Dliable-PAE-Dliable-MPEG; Fourier transform infrared spectra of MPEG-Dliable-PAE-g-Chol; 1H-NMR spectra of MPEG-Dlabile-PAE-g-Chol incubated in PBS at pH 7.4, 6.5 and 5.0 for 2 h; Plot of intensity ratios (i.e. I338/I336) as function of logarithm of MPEG-Dliable-PAE-g-Chol concentrations (mg/mL); In vitro particle size stability of the DOX-PMs in PBS; UVvis scanning spectra of Blank PMs, DOX-PMs and DOX; The cytotoxicity for MDAMB-231 cells induced by the treatment with polymer for 24 and 48 h; The PL spectra of same amount DOX-PMs incubation at differet pH and free DOX and blank PMs; The weight of major organs; Blood biochemistry analysis of the mice treated with PBS, DOX, polymer and polymer-DOX.


Page 34 of 40

Page 35 of 40 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


Author Information Corresponding Author *E-mail: [email protected] *E-mail: [email protected] ORCID Mengzhen Xu: 0000-0003-4798-3529 Can Yang Zhang: 0000-0002-6975-5781 Junguang Wu: 0000-0002-9666-6549 Jing Liu: 0000-0002-8740-4600 Author Contributions All authors have given approval to the ﬁnal version of the manuscript. #These

authors contributed equally.

Notes The authors declare no competing ﬁnancial interest.

Acknowledgments This work was financially supported by the National Science Fund for Excellent Young Scholars (31622026), the National Natural Science Foundation of China (U1532122), the

National

Key

Research

and

Development

Plan

(2017YFC1600204,

2016YFA0203204) and the National Basic Research Program of China from the Ministry of Science and Technology (2016YFA0201600 and 2016YFE0133100).



References (1) Cheng, R.; Feng, F.; Meng, F.; Deng, C.; Feijen, J.; Zhong, Z. Glutathione-Responsive Nano-Vehicles as a Promising Platform for Targeted Intracellular Drug and Gene Delivery. J. Control. Release 2011, 152, 2-12. (2) Hu, Y.; Du, Y.; Liu, N.; Liu, X.; Meng, T.; Cheng, B.; He, J.; You, J.; Yuan, H.; Hu, F. Selective RedoxResponsive Drug Release in Tumor Cells Mediated by Chitosan Based Glycolipid-Like Nanocarrier. J. Control. Release 2015, 206, 91-100. (3) Chiang, Y. T.; Yen, Y. W.; Lo, C. L. Reactive Oxygen Species and Glutathione Dual Redox-Responsive Micelles for Selective Cytotoxicity of Cancer. Biomaterials. 2015, 61, 150-161. (4) Pearce, T. R.; Shroff, K.; Kokkoli, E. Peptide Targeted Lipid Nanoparticles for Anticancer Drug Delivery. Adv. Mater. 2012, 24, 3803-3822. (5) Jiang, S.; Yang, Z.; Di, S.; Hu, W.; Ma, Z.; Chen, F.; Yang, Y. Novel Role of Forkhead Box O 4 Transcription Factor in Cancer: Bringing Out the Good or the Bad. Semin. Cancer Biol. 2018, 50, 1-12. (6) Li, T.; Jiang, S.; Yang, Y. Database Selection and Heterogeneity-More Details, More Credibility. JAMA Oncol. 2018, 4, 1295-1295. (7) Yang, Z.; Jiang, S.; Cheng, Y.; Li, T.; Hu, W.; Ma, Z.; Chen, F.; Yang, Y. FOXC1 in Cancer Development and Therapy: Deciphering Its Emerging and Divergent Roles. Ther. Adv. Med. Oncol. 2017, 9, 797-816. (8) Wang, J.; Mao, W.; Lock, L. L.; Tang, J.; Sui, M.; Sun, W.; Cui, H.; Xu, D.; Shen, Y. The Role of Micelle Size in Tumor Accumulation, Penetration, and Treatment. ACS Nano 2015, 9, 7195-7206. (9) Kataoka, K.; Harada, A.; Nagasaki, Y. Block Copolymer Micelles for Drug Delivery: Design, Characterization and Biological Significance. Adv. Drug Deliv. Rev. 2001, 47, 113-131. (10) Blum, R. H.; Carter, S. K. Adriamycin: A New Anticancer Drug with Significant Clinical Activity. Ann. Intern. Med. 1974, 80, 249-259. (11) Shan, K.; Lincoff, A. M.; Young, J. B. Anthracycline-Induced Cardiotoxicity. Ann. Intern. Med. 1996, 125, 47-58. (12) Bao, Y.; Yin, M.; Hu, X.; Zhuang, X.; Sun, Y.; Guo, Y.; Tan, S.; Zhang, Z. A Safe, Simple and Efficient Doxorubicin Prodrug Hybrid Micelle for Overcoming Tumor Multidrug Resistance and Targeting Delivery. J. Control. Release 2016, 235, 182-194. (13) Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-Responsive Nanocarriers for Drug Delivery. Nat. Mater. 2013, 12, 991-1003 (14) Ikeda, M.; Tanida, T.; Yoshii, T.; Hamachi, I. Rational Molecular Design of Stimulus-Responsive Supramolecular Hydrogels Based on Dipeptides. Adv. Mater. 2011, 23, 2819-2822. (15) Huang, X.; Sun, Y.; Soh, S. Stimuli-Responsive Surfaces for Tunable and Reversible Control of Wettability. Adv. Mater. 2015, 27, 4062-4068. (16) Tokarev, I.; Minko, S. Stimuli-Responsive Porous Hydrogels at Interfaces for Molecular Filtration, Separation, Controlled Release, and Gating in Capsules and Membranes. Adv. Mater. 2010, 22, 34463462. (17) Johnson, R. P.; Jeong, Y.-I.; Choi, E.; Chung, C.-W.; Kang, D. H.; Oh, S.-O.; Suh, H.; Kim, I. Biocompatible Poly(2-hydroxyethyl methacrylate)-b-poly(L-histidine) Hybrid Materials for pH-Sensitive Intracellular Anticancer Drug Delivery. Adv. Funct. Mater. 2012, 22, 1058-1068.


Page 36 of 40

Page 37 of 40 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


(18) Guo, X.; Wang, L.; Duval, K.; Fan, J.; Zhou, S.; Chen, Z. Dimeric Drug Polymeric Micelles with AcidActive Tumor Targeting and FRET-Traceable Drug Release. Adv. Mater. 2018, 30, 1705436. (19) Wang, Y.; Wei, G.; Zhang, X.; Xu, F.; Xiong, X.; Zhou, S. A Step-by-Step Multiple Stimuli-Responsive Nanoplatform for Enhancing Combined Chemo-Photodynamic Therapy. Adv. Mater. 2017, 29, 1605357. (20) Guo, X.; Shi, C.; Wang, J.; Di, S.; Zhou, S. pH-Triggered Intracellular Release from Actively Targeting Polymer Micelles. Biomaterials. 2013, 34, 4544-4554. (21) Wan, J.; Geng, S.; Zhao, H.; Peng, X.; Zhou, Q.; Li, H.; He, M.; Zhao, Y.; Yang, X.; Xu, H. DoxorubicinInduced Co-Assembling Nanomedicines with Temperature-Sensitive Acidic Polymer and Their In-SituForming Hydrogels for Intratumoral Administration. J. Control. Release 2016, 235, 328-336. (22) Schmaljohann, D. Thermo- and pH-Responsive Polymers in Drug Delivery. Adv. Drug Deliv. Rev. 2006, 58, 1655-1670. (23) Zhang, H.; Wang, K.; Zhang, P.; He, W.; Song, A.; Luan, Y. Redox-Sensitive Micelles Assembled from Amphiphilic mPEG-PCL-SS-DTX Conjugates for the Delivery of Docetaxel. Colloids Surf., B 2016, 142, 8997. (24) Wei, X.; Luo, Q.; Sun, L.; Li, X.; Zhu, H.; Guan, P.; Wu, M.; Luo, K.; Gong, Q. Enzyme- and pHSensitive Branched Polymer–Doxorubicin Conjugate-Based Nanoscale Drug Delivery System for Cancer Therapy. ACS Appl. Mater. Inter. 2016, 8, 11765-11778. (25) Wang, W.; Cheng, D.; Gong, F.; Miao, X.; Shuai, X. Design of Multifunctional Micelle for TumorTargeted Intracellular Drug Release and Fluorescent Imaging. Adv. Mater. 2012, 24, 115-120. (26) Lim, E.-K.; Huh, Y.-M.; Yang, J.; Lee, K.; Suh, J.-S.; Haam, S. pH-Triggered Drug-Releasing Magnetic Nanoparticles for Cancer Therapy Guided by Molecular Imaging by MRI. Adv. Mater. 2011, 23, 24362442. (27) Hubbell, J. A. Enhancing Drug Function. Science 2003, 300, 595. (28) Fleige, E.; Quadir, M. A.; Haag, R. Stimuli-Responsive Polymeric Nanocarriers for the Controlled Transport of Active Compounds: Concepts And Applications. Adv. Drug Deliv. Rev. 2012, 64, 866-884. (29) Jain, A.; Agarwal, A.; Majumder, S.; Lariya, N.; Khaya, A.; Agrawal, H.; Majumdar, S.; Agrawal, G. P. Mannosylated Solid Lipid Nanoparticles as Vectors for Site-Specific Delivery of an Anti-Cancer Drug. J. Control. Release 2010, 148, 359-367. (30) Gao, Z. G.; Lee, D. H.; Kim, D. I.; Bae, Y. H. Doxorubicin Loaded pH-Sensitive Micelle Targeting Acidic Extracellular pH of Human Ovarian A2780 Tumor in Mice. J. Drug Target. 2005, 13, 391-397. (31) Gabizon, A.; Catane, R.; Uziely, B.; Kaufman, B.; Safra, T.; Cohen, R.; Martin, F.; Huang, A.; Barenholz, Y. Prolonged Circulation Time and Enhanced Accumulation in Malignant Exudates of Doxorubicin Encapsulated in Polyethylene-glycol Coated Liposomes. Cancer Res. 1994, 54, 987. (32) Wu, W.; Zhang, Q.; Wang, J.; Chen, M.; Li, S.; Lin, Z.; Li, J. Tumor-Targeted Aggregation of pHSensitive Nanocarriers for Enhanced Retention and Rapid Intracellular Drug Release. Polym. Chem. 2014, 5, 5668-5679. (33) Sarker, D. K. Engineering of Nanoemulsions for Drug Delivery. Curr. Drug Deliv. 2005, 2, 297-310. (34) Kedar, U.; Phutane, P.; Shidhaye, S.; Kadam, V. Advances in Polymeric Micelles for Drug Delivery and Tumor Targeting. Nanomed-Nanotechnol 2010, 6, 714-729. (35) Wilhelm, S.; Tavares, A. J.; Dai, Q.; Ohta, S.; Audet, J.; Dvorak, H. F.; Chan, W. C. W. Analysis of Nanoparticle Delivery to Tumours. Nat. Rev. Mater. 2016,1. (36) Shin, D. H.; Tam, Y. T.; Kwon, G. S. Polymeric Micelle Nanocarriers in Cancer Research. Front. Chem. Sci. Eng. 2016, 10, 348-359. (37) Zhang, C. Y.; Yang, Y. Q.; Huang, T. X.; Zhao, B.; Guo, X. D.; Wang, J. F.; Zhang, L. J. Self-Assembled



pH-Responsive MPEG-b-(PLA-co-PAE) Block Copolymer Micelles for Anticancer Drug Delivery. Biomaterials. 2012, 33, 6273-6283. (38) Ramezani, M.; Shamsara, J. Application of DPD in the Design of Polymeric Nano-Micelles as Drug Carriers. J. Mol. Graphics Modell. 2016, 66, 1-8. (39) Bui, Q. N.; Li, Y.; Jang, M.-S.; Huynh, D. P.; Lee, J. H.; Lee, D. S. Redox- and pH-Sensitive Polymeric Micelles Based on Poly(β-amino ester)-Grafted Disulfide Methylene Oxide Poly(ethylene glycol) for Anticancer Drug Delivery. Macromolecules. 2015, 48, 4046-4054. (40) Qin, Z.; Chen, Y.; Zhou, W.; He, X.; Bai, F.; Wan, M. Synthesis and Properties of Polymer Brushes Composed of Poly(diphenylacetylene) Main Chain and Poly(ethylene glycol) Side Chains. Eur. Polym. J. 2008, 44, 3732-3740. (41) Gref, R.; Domb, A.; Quellec, P.; Blunk, T.; Müller, R. H.; Verbavatz, J. M.; Langer, R. The Controlled Intravenous Delivery of Drugs Using PEG-Coated Sterically Stabilized Nanospheres. Adv. Drug Deliv. Rev. 2012, 64, 316-326. (42) Sun, C.-Y.; Liu, Y.; Du, J.-Z.; Cao, Z.-T.; Xu, C.-F.; Wang, J. Facile Generation of Tumor-pH-Labile Linkage-Bridged Block Copolymers for Chemotherapeutic Delivery. Angew. Chem. Int. Edit. 2016, 128, 1022-1026. (43) Sun, C.-Y.; Shen, S.; Xu, C.-F.; Li, H.-J.; Liu, Y.; Cao, Z.-T.; Yang, X.-Z.; Xia, J.-X.; Wang, J. Tumor Acidity-Sensitive Polymeric Vector for Active Targeted siRNA Delivery. J. Am. Chem. Soc. 2015, 137, 15217-15224. (44) Knop, K.; Hoogenboom, R.; Fischer, D.; Schubert, U. S. Poly(ethylene glycol) in Drug Delivery: Pros and Cons as Well as Potential Alternatives. Angew. Chem. Int. Edit. 2010, 49, 6288-6308. (45) Graf, N.; Bielenberg, D. R.; Kolishetti, N.; Muus, C.; Banyard, J.; Farokhzad, O. C.; Lippard, S. J. αVβ3 Integrin-Targeted PLGA-PEG Nanoparticles for Enhanced Anti-tumor Efficacy of a Pt(IV) Prodrug. ACS Nano 2012, 6, 4530-4539. (46) Zhang, C. Y.; Kos, P.; Müller, K.; Schrimpf, W.; Troiber, C.; Lächelt, U.; Scholz, C.; Lamb, D. C.; Wagner, E. Native Chemical Ligation for Conversion of Sequence-Defined Oligomers into Targeted pDNA and siRNA Carriers. J. Control. Release 2014, 180, 42-50. (47) Garate, A.; Santos, E.; Pedraz, J. L.; Hernández, R. M.; Orive, G. Evaluation of Different RGD Ligand Densities in the Development of Cell-Based Drug Delivery Systems. J. Drug Target. 2015, 23, 806-812. (48) Mekuria, S. L.; Debele, T. A.; Chou, H.-Y.; Tsai, H.-C. IL-6 Antibody and RGD Peptide Conjugated Poly(amidoamine) Dendrimer for Targeted Drug Delivery of HeLa Cells. J. Phys. Chem. B 2016, 120, 123130. (49) Brezden, A.; Mohamed, M. F.; Nepal, M.; Harwood, J. S.; Kuriakose, J.; Seleem, M. N.; Chmielewski, J. Dual Targeting of Intracellular Pathogenic Bacteria with a Cleavable Conjugate of Kanamycin and an Antibacterial Cell-Penetrating Peptide. J. Am. Chem. Soc. 2016, 138, 10945-10949. (50) Morshed, R. A.; Muroski, M. E.; Dai, Q.; Wegscheid, M. L.; Auffinger, B.; Yu, D.; Han, Y.; Zhang, L.; Wu, M.; Cheng, Y.; Lesniak, M. S. Cell-Penetrating Peptide-Modified Gold Nanoparticles for the Delivery of Doxorubicin to Brain Metastatic Breast Cancer. Mol. Pharm. 2016, 13, 1843-1854. (51) Shrestha, N.; Araújo, F.; Shahbazi, M.-A.; Mäkilä, E.; Gomes, M. J.; Herranz-Blanco, B.; Lindgren, R.; Granroth, S.; Kukk, E.; Salonen, J.; Hirvonen, J.; Sarmento, B.; Santos, H. A. Thiolation and CellPenetrating Peptide Surface Functionalization of Porous Silicon Nanoparticles for Oral Delivery of Insulin. Adv. Funct. Mater. 2016, 26, 3405-3416. (52) Parker, N.; Turk, M. J.; Westrick, E.; Lewis, J. D.; Low, P. S.; Leamon, C. P. Folate Receptor Expression in Carcinomas and Normal Tissues Determined by a Quantitative Radioligand Binding Assay.


Page 38 of 40

Page 39 of 40 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


Anal. Biochem. 2005, 338, 284-293. (53) Han, S.-S.; Li, Z.-Y.; Zhu, J.-Y.; Han, K.; Zeng, Z.-Y.; Hong, W.; Li, W.-X.; Jia, H.-Z.; Liu, Y.; Zhuo, R.-X.; Zhang, X.-Z. Dual-pH Sensitive Charge-Reversal Polypeptide Micelles for Tumor-Triggered Targeting Uptake and Nuclear Drug Delivery. Small 2015, 11, 2543-2554. (54) Gu, J.; Cheng, W.-P.; Liu, J.; Lo, S.-Y.; Smith, D.; Qu, X.; Yang, Z. pH-Triggered Reversible “Stealth” Polycationic Micelles. Biomacromolecules 2008, 9, 255-262. (55) Lynn, D. M.; Langer, R. Degradable Poly(β-amino esters): Synthesis, Characterization, and SelfAssembly with Plasmid DNA. J. Am. Chem. Soc. 2000, 122, 10761-10768. (56) Zhang, C. Y.; Chen, Q.; Wu, W. S.; Guo, X. D.; Cai, C. Z.; Zhang, L. J. Synthesis and Evaluation of Cholesterol-Grafted Pegylated Peptides with pH-Triggered Property as Novel Drug Carriers for Cancer Chemotherapy. Colloids Surf., B 2016, 142, 55-64. (57) Ding, C.; Gu, J.; Qu, X.; Yang, Z. Preparation of Multifunctional Drug Carrier for Tumor-Specific Uptake and Enhanced Intracellular Delivery through the Conjugation of Weak Acid Labile Linker. Bioconjugate. Chem. 2009, 20, 1163-1170. (58) Lynn, D. M.; Amiji, M. M.; Langer, R. pH-Responsive Polymer Microspheres: Rapid Release of Encapsulated Material within the Range of Intracellular pH. Angew. Chem. Int. Edit. 2001, 40, 17071710. (59) Zhang, C. Y.; Xiong, D.; Sun, Y.; Zhao, B.; Lin, W. J.; Zhang, L. J. Self-Assembled Micelles Based on pH-Sensitive PAE-g-MPEG-Cholesterol Block Copolymer for Anticancer Drug Delivery. Int. J. Nanomed. 2014, 9, 4923-4933. (60) Wu, D.-Q.; Lu, B.; Chang, C.; Chen, C.-S.; Wang, T.; Zhang, Y.-Y.; Cheng, S.-X.; Jiang, X.-J.; Zhang, X.Z.; Zhuo, R.-X. Galactosylated Fluorescent Labeled Micelles as a Liver Targeting Drug Carrier. Biomaterials. 2009, 30, 1363-1371. (61) Hu, K.; Zhou, H.; Liu, Y.; Liu, Z.; Liu, J.; Tang, J.; Li, J.; Zhang, J.; Sheng, W.; Zhao, Y.; Wu, Y.; Chen, C. Hyaluronic Acid Functional Amphipathic and Redox-Responsive Polymer Particles for the Co-Delivery of Doxorubicin and Cyclopamine to Eradicate Breast Cancer Cells and Cancer Stem Cells. Nanoscale 2015, 7, 8607-8618. (62) Zhang, C. Y.; Wu, W. S.; Yao, N.; Zhao, B.; Zhang, L. J. pH-Sensitive Amphiphilic Copolymer Brush Chol-g-P(HEMA-co-DEAEMA)-b-PPEGMA: Synthesis and Self-Assembled Micelles for Controlled AntiCancer Drug Release. RSC Adv. 2014, 4, 40232-40240. (63) Shen, Y.; Tang, H.; Zhan, Y.; Van Kirk, E. A.; Murdoch, W. J. Degradable Poly(β-amino ester) Nanoparticles for Cancer Cytoplasmic Drug Delivery. Nanomedi-Nanotechnol 2009, 5, 192-201. (64) Guo, X. D.; Zhang, L. J.; Chen, Y.; Qian, Y. Core/Shell pH-Sensitive Micelles Self-Assembled from Cholesterol Conjugated Oligopeptides for Anticancer Drug Deliv. AIChE J. 2010, 56, 1922-1931. (65) Siepmann, J.; Peppas, N. A. Modeling of Drug Release from Delivery Systems Based on Hydroxypropyl Methylcellulose (HPMC). Adv. Drug Deliv. Rev. 2001, 48, 139-157. (66) Young, C. R.; Dietzsch, C.; Cerea, M.; Farrell, T.; Fegely, K. A.; Rajabi-Siahboomi, A.; McGinity, J. W. Physicochemical Characterization and Mechanisms of Release of Theophylline from Melt-Extruded Dosage Forms Based on a Methacrylic Acid Copolymer. Int. J. Pharm. 2005, 301, 112-120. (67) Li, Y.; Li, H.; Wei, M.; Lu, J.; Jin, L. pH-Responsive Composite Based on Prednisone-Block Copolymer Micelle Intercalated Inorganic Layered Matrix: Structure and in Vitro Drug Release. Chem. Eng. J. 2009, 151, 359-366. (68) Mao, J.; Li, Y.; Wu, T.; Yuan, C.; Zeng, B.; Xu, Y.; Dai, L. A Simple Dual-pH Responsive Prodrug-Based Polymeric Micelles for Drug Delivery. ACS Appl. Mater. Inter. 2016, 8, 17109-17117.



Page 40 of 40

(69) Maeda, H.; Nakamura, H.; Fang, J. The EPR Effect for Macromolecular Drug Delivery to Solid Tumors: Improvement of Tumor Uptake, Lowering of Systemic Toxicity, and Distinct Tumor Imaging in Vivo. Adv. Drug Deliv. Rev. 2013, 65, 71-79. (70) Nichols, J. W.; Bae, Y. H. EPR: Evidence and Fallacy. J. Control. Release 2014, 190, 451-464.

TOC Tumor microenvironment

PEG

pH-labile bond

Self-assembly PAE

DOX

Cholesterol

Endocytosis Drug delivery “on-demand”


Intracellular

PEG-Detachable Polymeric Micelles Self-Assembled from Amphiphilic

Recommend Documents