Dendrimer-Based Biocompatible Zwitterionic Micelles for Efficient

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Dendrimer-Based Biocompatible Zwitterionic Micelles for Efficient Cellular Internalization and Enhanced Anti-Tumor Effects Longgang Wang, Linlin Zhu, Matthew T. Bernards, Shengfu Chen, Haotian Sun, Xiaolei Guo, Weili Xue, Yanshuai Cui, and Dawei Gao ACS Appl. Polym. Mater., Just Accepted Manuscript • DOI: 10.1021/acsapm.9b00026 • Publication Date (Web): 17 Apr 2019 Downloaded from http://pubs.acs.org on April 17, 2019

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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.

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ACS Applied Polymer Materials

Dendrimer-Based

Biocompatible

Zwitterionic

Micelles for Efficient Cellular Internalization and Enhanced Anti-Tumor Effects Longgang Wang,a Linlin Zhu,a Matthew T. Bernards,b Shengfu Chen,c Haotian Sun,c Xiaolei Guo,a Weili Xue,c Yanshuai Cuia,d* and Dawei Gaoa*

a. Key

Laboratory of Applied Chemistry, College of Environmental and Chemical

Engineering, Yanshan University, Qinhuangdao, 066004, P.R. China.

b.Department

of Chemical and Materials Engineering, University of Idaho, Moscow,

83844, U.S.A.

c. Key

Laboratory of Biomass Chemical Engineering of Ministry of Education, College of

Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, P.R. China.

ACS Paragon Plus Environment

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ACS Applied Polymer Materials 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

d. State

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Key Laboratory of Metastable Materials Science and Technology, Yanshan

University, Qinhuangdao, 066004, P.R. China

* Corresponding author: [email protected]; [email protected].

KEYWORDS: biocompatibility, nonspecific protein adsorption, dendrimers, enhanced internalization efficiency, zwitterionic micelles

ABSTRACT: Zwitterionic materials have been employed to achieve excellent biocompatibility and well-suppressed nonspecific protein adsorption of nanoparticles. However, a thick and compact zwitterionic layer may prevent the modified nanoparticles from entering tumor cells, resulting in low cellular internalization efficiency. To address this problem, new biocompatible micelles (PPIMYC) with a thin zwitterionic layer were designed and prepared. Zwitterionic generation 2 polypropylene imine dendrimers (G2 PPI) serve as the hydrophilic external shell, and N-(2-mercaptoethyl)oleamide serve as the hydrophobic internal core. Drug-loaded dendritic micelles (PPIMYC-DOX-Ce6) were


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also prepared by self-assembly of PPIMYC with doxorubicin (DOX) and chlorin e6 (Ce6) to demonstrate chemo-photodynamic dual therapy. As potential drug delivery systems for anti-tumor therapy, PPIMYC-DOX-Ce6 exhibited sustained drug release under acidic conditions, and high stability in fibrinogen solution. In addition, cytotoxicity studies showed enhanced efficiency of PPIMYC-DOX-Ce6 in killing HeLa cells as compared to free DOX with or without irradiation (660 nm laser). More importantly, both flow cytometry and fluorescence microscopy results indicated that the cellular uptake efficiency of DOX was significantly enhanced in PPIMYC-DOX-Ce6 treated cells relative to free DOX treated cells. The intracellular internalization of PPIMYC-DOX-Ce6 was more efficient under acidic pH, representing the tumor environment, as compared to normal pH. This results from the pH sensitivity of the zwitterionic layer. Temperature was the only environmental factor affecting the cellular internalization process. It is believed that the enhanced intracellular internalization efficiency is due to the thin zwitterionic layer of PPIMYC-DOX-Ce6. The preparation scheme of zwitterionic micelles would offer a new strategy to design novel anti-tumor drug delivery systems with enhanced cellular internalization efficiency and high stability in complex medium.


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1. INTRODUCTION Cancer is considered to be pernicious disease that causes many human deaths. Chemotherapy still remains the primary method of treatment for cancer.1-4 Most of conventional chemotherapeutic drugs such as doxorubicin (DOX), methotrexate and camptothecin have shortcomings such as low selectivity, poor solubility in water, large side effects and multi-drug resistance.5,6 Nanomaterial based drug delivery systems (DDS), such as liposomes, dendrimers, and polymeric micelles,7,8 have advantages because they can be passively transported and accumulate into tumor tissues via the enhanced permeability and retention (EPR) effect.9-13 However, the stability of intravenously injected DDS is degraded by proteins in blood. They can also be recognized and subsequently eliminated by reticuloendothelial system (RES).14-18 In addition, the selective accumulation of DDS in tumor cells is crucial to maximize their anti-tumor effect. The entry of DDS is controlled by the tumor cell membrane, which is composed of amphiphilic phospholipids. Thus, the interactions of DDS with blood proteins and cancer cells membrane are two of the major problems which need to be addressed to improve the drug accumulation inside cancer cells.


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Self-assembled polymeric micelles are one class of promising DDS, which have been used for targeted delivery of many anticancer drugs.19,20 Surface modification of polymeric micelles with hydrophilic materials is one of the most effective strategies to reduce the interaction between polymeric micelles and blood components.21 Non-ionic poly(ethylene glycol) (PEG) is widely used hydrophilic substance to suppress nonspecific protein adsorption on nanoparticle surfaces.22,23 This suppression is attributed to the formation of a hydration layer via hydrogen bonding at the interface.24-27 However, PEG has many drawbacks such as oxidative damage and generation of anti-PEG antibodies.28-30 As substitutes, zwitterionic materials are more stable and compatible in

vivo.31 The enhanced property of zwitterionic materials is ascribed to their stronger water binding ability relative to PEG and their biomimetic structure.32-36 However, thick and compact zwitterionic layers on polymer micelles reduce the interaction between polymer micelles and tumor cells, leading to lower cellular uptake efficiency of nanoparticles.37,38 Zwitterionic materials with pH-responsive properties were studied to increase the cellular uptake efficiency of zwitterionic polymer micelles in tumor tissue. The pH in


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normal tissue and tumor tissue is about 7.4 and 6.5, respectively. Some zwitterionic groups undergo protonation in an acidic environment, changing their zeta potential from slightly negative in the normal tissue environment to positive in the tumor tissue environment. Thus, the affinity between zwitterionic polymer micelles and negatively charged tumor cell membrane is enhanced under acidic conditions. For example, Lu et

al.

prepared

pH-responsive

zwitterionic

polymer

micelles

using

poly(benzyl

methacrylate)-block-poly[N-(2-hydroxypropyl) acrylamide-co-N-(3-(4-morpholino)propyl) acrylamide-co-acrylic acid].37 Wu et al. synthesized a surface charge convertible zwitterionic

nanoparticles

using

taurine,

N,N-bis

(acryloyl)

cystamine

and

dodecylamine.28 Ou et al. reported surface-adaptive mixed-shell micelles with biodegradable poly(ε-caprolactone) and poly(2-methacryloyloxyethyl phosphorylcholine) or poly(β-amino ester).38 However, the uptake efficiency of drugs encapsulated in the zwitterionic polymer micelles in all of these examples was still lower than that of the free drug alone. One of the reasons could be the very thick zwitterionic shell in these systems. The thick layer reduces the interactions between the polymer micelles and the cell membrane. In contrast, liposomes have better fusion ability with the cell membrane


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due to their structural similarities.39 The main component of liposomes are phospholipids, which have a zwitterionic head group and a hydrophobic tail. Mimicking the structure of phospholipid may be a new method to prepare zwitterionic polymer micelles with higher cellular uptake efficiency. The mechanism of interaction between nanoparticles and cell membranes is also important for tuning the cellular uptake efficiency of nanoparticles by tumor cells. However, the mechanism is still unclear as described in many reports.39 In this work, we synthesized a novel biocompatible zwitterionic polymer PPIMYC to mimic the structure of phospholipids to some extent (Scheme 1). PPIMYC is composed of hydrophilic and zwitterionic G2 PPI and a hydrophobic N-(2-mercaptoethyl)oleamide. The zwitterionic G2 PPI also offers many functional groups for further modifications. PPIMYC self-assembled micelles were used to encapsulate the hydrophobic chemotherapeutic drug DOX and the photosensitizer Ce6 for combinatorial chemophotodynamic dual therapy applications. The morphology, size, and zeta potential of drug-loaded micelles were carefully measured. The enhanced cellular internalization of the drug-loaded micelles was studied using fluorescence microscopy and flow


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cytometry, and the corresponding mechanism was investigated. Meanwhile, the enhanced cytotoxicity of drug-loaded micelles was studied by cell culture using an MTT assay.


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Scheme 1. Scheme of the Intracellular Drug Release from Zwitterionic Micelles PPIMYC-DOX-Ce6

2. EXPERIMENTAL SECTION 2.1.Materials Doxorubicin hydrochloride (DOX·HCl), dithiothreitol (DTT), and maleic anhydride were obtained from Aladdin Chemical Co., Ltd (Shanghai, China). Chlorpromazine hydrochloride, amiloride hydrochloride, genistein, G2 PPI, and fibrinogen were obtained from Sigma-Aldrich Co., Ltd (USA). Cystamine dihydrochloride was purchased from


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Energy Chemical Technology Co., Ltd (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Sijiqing Biological Engineering Materials Co., Ltd (China). Hoechst 33342 was acquired from Beyotime Biotechnology Co., Ltd (Shanghai, China). Cysteamine was purchased from J&K Chemical (Beijing, China). Dichlorofluorescin diacetate (DCFH-DA) was obtained from Beijing Jinming Biotechnology Co., Ltd (Beijing, China). Chlorin e6 (Ce6) was purchased from Ji’nan Daen Pharma Tech Co., Ltd (China). Dialysis bags were obtained from Spectrum Laboratories Inc., (USA). 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) and 1-hydroxybenzotriazole (HOBt) were purchased from GL Biochem Co., Ltd (Shanghai, China). Sodium hydroxide (NaOH), dichloromethane (CH2Cl2), anhydrous magnesium sulfate (MgSO4), oleic acid, ethyl acetate (EtOAc), petroleum ether, triethylamine (TEA), hydrochloric acid (HCl), anhydrous sodium sulfate (Na2SO4), methanol, ether, and acetone were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). All of the chemicals were used without further purification. 2.2.Characterization


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The 1H NMR spectra were measured in deuterated reagents using a Bruker AVANCE III 400 M spectrometer. The hydrodynamic size and zeta potential of nanoparticles were detected using DLS on a Zetasizer Nano-ZS90 system (Malvern, UK) at a scattering angle of 90 °C. Each sample had triplicate measurements. The UV-Vis spectra of nanoparticles were recorded on a spectrometer (UV 2550, Japan) at room temperature. A fluorescence spectrometer (F-7000, Hitachi, Japan) was used to measure the concentration of DOX and Ce6 in drug-loaded micelles. In addition, the size and morphology of nanoparticles in a dried state were measured by TEM (HT 7700, Japan). For TEM measurements, 10 μL samples were dropped onto carbon film and dried in the air. After 3 h, phosphotungstic acid (10 μL) was dropped onto carbon film and dried. The fluorescent intensity of DOX internalized by cells was analyzed using a fluorescen microscope and flow cytometry (BD FACSCalibur, USA). Meanwhile, SpectraMax M2 was used to record the cell viability following the MTT assay. 2.3.Synthesis of N,N’-(disulfanediylbis(ethane-2,1-diyl))dioleamide The cystamine was synthesized by previously reported procedures.40 Colorless oily liquid was obtained. Yield: 82 %. 1H NMR (400 MHz, DMSO-d6, δ): 2.79 (t, 4H, 2CH2N)


11


2.71

(t,

4H,

2CH2S),

1.98

(s,

4H,

2NH2).

Page 12 of 63

N,N’-(disulfanediylbis(ethane-2,1-

diyl))dioleamide was synthesized according to previous methods.41 In brief, oleic acid (20.3 g, 0.072 mol), HOBt (19.5 g, 0.144 mol), and EDC·HCl (27.6 g, 0.144 mol) were dissolved in CH2Cl2. Cystamine (3.7 g, 0.024 mol) was dissolved in CH2Cl2 and added into the above solution and reacted for 24 h. The products were purified by column chromatography (EtOAc/petroleum ether, v/v=1:1) to give final product. Yield: 52 %. 1H NMR (400 MHz, CDCl3, δ): 6.32 (s, 2H, 2NHCO), 5.34 (m, 4H, 2CH2NH), 3.57 (q, 4H, 2CH2S), 2.83 (t, 4H, 4CHCH), 2.22 (t, 4H, 2CH2CO), 2.01 (m, 8H, 4CH2CH), 1.63 (m, 4H, 2CH2CH2CO), 1.30 (m, 40H, 2CHCH2(CH2)6 and 2CHCH2(CH2)4), 0.88 (m, 6H, 2CH3). 2.4.Synthesis of N-(2-mercaptoethyl)oleamide The synthesis of N-(2-mercaptoethyl)oleamide was according to previously reported methods.42,43 N,N’-(disulfanediylbis(ethane-2,1-diyl))dioleamide (120.0 mg, 0.176 mmol) was dissolved in CH2Cl2. DTT (54.3 mg, 0.352 mmol) and TEA (35.6 mg, 0.352 mmol) were dissolved in CH2Cl2 and added. The mixture was deoxygenated for 30 min. The organic phase was extracted with 1 M HCl and dried with anhydrous Na2SO4 overnight.


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The obtained product is a colorless liquid. Yield: 62 %. 1H NMR (400 MHz, CDCl3, δ): 6.32 (s, H, NHCO), 5.34 (m, 2H, CH2NH), 3.57 (q, 2H, CH2S), 2.83 (t, 2H, 2CHCH), 2.22 (t, 2H, CH2CO), 2.01 (m, 4H, 2CH2CH), 1.63 (m, 2H, CH2CH2CO), 1.30 (m, 20H, CHCH2(CH2)6 and CHCH2(CH2)4), 0.88 (m, 3H, CH3). 2.5.Synthesis of maleic anhydride modified G2 PPI (PPIM) PPIM was prepared according to previously reported steps.44 G2 PPI (20.0 mg, 2.59×10-2 mmol) and maleic anhydride (40.6 mg, 0.414 mmol) were dissolved in DMSO and reacted for 24 h. The product was dialyzed against deionized water with a dialysis bag (MWCO=500). The final solution was lyophilized to give a solid. Yield: 91 %. 1H NMR (400 MHz, D2O, δ): 6.18 (d, 8H, 8CHCONH), 5.79 (d, 8H, 8CHCOOH), 3.17 (s, 16H, 8CH2NH2), 2.70 (d, 36H, 18CH2N), 1.74 (s, 24H, 12CH2CH2NH2), 1.46 (s, 4H, 2CH2CH2N). 2.6.Synthesis of N-(2-mercaptoethyl)oleamide modified PPIM (PPIMY) PPIM

(20.0

mg,

1.284×10-2

mmol)

was

dissolved

in

CH3OH.

N-(2-

mercaptoethyl)oleamide (35.1 mg, 0.103 mmol) was dissolved in CH2Cl2 and added into the above solution. The mixture was reacted for 24 h. The product was dialyzed against


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CH3OH with dialysis bags (MWCO=1000). Yield: 85 %. 1H NMR (400 MHz, CD3OD, δ): 6.18 (d, 5H, 5CHCONH), 5.88 (d, 5H, 5CHCOOH), 5.28 (m, 6H, 3CH2NH), 3.55 (q, 6H, 3CH2S),

2.99

(s,

8CH2NH2

from

PPI),

2.42-2.73

(m,

6CHCH

from

N-(2-

mercaptoethyl)oleamide and 18CH2N from PPI), 1.81-2.08 (m, 3CH2CO and 6CH2CH from N-(2-mercaptoethyl)oleamide and 12CH2CH2CH2 from PPI), 1.68 (s, 6H, 3CH2CH2CO), 1.50 (s, 4H, 2CH2CH2N), 1.19 (m, 60H, 3CHCH2(CH2)6 and 3CHCH2(CH2)4), 0.88 (m, 9H, 3CH3). 2.7.Synthesis of cysteamine modified PPIMY (PPIMYC) Cysteamine (79.3 mg, 1.027 mmol) was dissolved in CH3OH. The solution was added into the 1.284×10-2 mmol PPIMY solution. PPIMYC was precipitated with anhydrous diethyl ether and centrifuged. Yield: 57 %. 1H NMR (400 MHz, CD3OD, δ): 5.39 (m, 6H, 3CH2NH), 3.64 (d, 8H, 8COCH), 3.44 (q, 6H, 3CH2S), 3.20 (s, 8CH2NH2 from PPI), 2.96 (d, 8H, 8CHCOOH), 2.79 (t, 6H, 6CHCH), 2.58 (s, CH2 from PPI), 2.21(t, 6H, 3CH2CO), 1.99 (m, 12H, 6CH2CH), 1.72-1.85 (d, CH2 from PPI), 1.62 (s, 6H, 3CH2CH2CO), 1.31 (m, 60H, 3CHCH2(CH2)6 and 3CHCH2(CH2)4), 0.92 (m, 9H, 3CH3). 2.8.Preparation of micelles


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Micelles were prepared according to previously reported dialysis methods.30 PPIMYC (9.5 mg, 3.21×10-3 mmol) dissolved in CH3OH was dropped into deionized water, stirred for 3 h and dialyzed against deionized water to obtain blank micelles. PPIMYC (9.5 mg, 3.21×10-3 mmol) was dissolved in CH3OH. DOX·HCl (186 μg, 3.21×10-4 mmol) and 5 μL TEA were mixed in CH3OH to obtain hydrophobic DOX. Ce6 (192 μg, 3.21×10-4 mmol) was dissolved in DMSO. The above three solutions were mixed and reacted for 3h. The resulting solution was dialyzed against deionized water to obtain DOX and Ce6 loaded micelles (PPIMYC-DOX-Ce6) micelles. PPIMYC-DOX micelles were prepared using similar procedure. Drug-loaded micelles were lyophilized, and dissolved in solution of DMSO and methanol. The fluorescence intensity (λex=480 nm, λem=592 nm) was determined to calculate the loading capacity of DOX. The fluorescence intensity (λex=405 nm, λem=660 nm) was determined to calculate the loading capacity of Ce6. Drug loading content (DLC) and drug loading efficiency (DLE) were calculated by the following equations 1 and 2:


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weight of encapsulated drug in micelles  100 % total of encapsulated drug and micelles weight of encapsulated drug in micelles DLE（%）=  100 % weight of drug in feed

DLC（%）=

Page 16 of 63

(1) (2)

2.9.Critical micelle concentration (CMC) of blank micelles Pyrene was used to measure the CMC.37 Pyrene (6×10-2 mM) was dissolved in acetone, and the acetone was evaporated spontaneously. Blank micelles were added with the concentrations from 4.39×10-5 to 0.72 mg/mL in water. The excitation wavelength used for the test was 334 nm. The fluorescence intensity at wavelengths of 374 nm and 384 nm was recorded. The ratios of fluorescence intensity at 374 nm (I374) and 384 nm (I384) were plotted as a function of the logarithm of the blank micelles concentrations. 2.10. Drug release assays in vitro The release curve of DOX from PPIMYC-DOX-Ce6 micelles was studied using a dialysis method. A solution of PPIMYC-DOX-Ce6 micelles (1 mL) was put into a dialysis bag (MWCO=8000-14000). The dialysis bag was immersed into 50 mL phosphate buffered saline (PBS) solution at pH 6.5 or 7.4. At selected time


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intervals, 1 mL of released solution was taken out and replaced with 1 mL PBS solution. The concentration of DOX was measured using F-7000 spectrometer at an excitation wavelength of 480 nm. 2.11. Stability of micelles The stability of blank micelles and drug-loaded micelles against fibrinogen was studied through the evaluation of size changes of mixed solutions. The size was detected by using DLS at 37 °C. Fibrinogen (0.5 mg/mL), blank micelles (1 mg/mL), and drug-loaded micelles (1 mg/mL) were prepared in PBS solution. The detailed steps of the cell experiment are shown in the supporting information. 3. RESULTS AND DISCUSSION 3.1.Synthesis and characterization of PPIMYC The detailed synthesis steps of the dendrimer-based biocompatible zwitterionic polymer (PPIMYC) are illustrated in Scheme 2. Cystamine (1, Figure S1) was prepared with an 82 % yield according to previous methods. N,N’-(disulfanediylbis(ethane-2,1diyl))dioleamide (2, Figure S2) was synthesized with a 52 % yield via an amidation


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reaction between cystamine and oleic acid, where EDC·HCl was used as the carbonyl activating

reagent

and

HOBt

was

used

as

the

organocatalyst.

N-(2-

mercaptoethyl)oleamide (3, Figure S3) was synthesized with a 62 % yield through the reduction

reaction

of

N,N’-(disulfanediylbis(ethane-2,1-diyl))dioleamide

using

dithiothreitol (DTT). PPIM (4) was obtained with an acylation reaction of the G2 PPI with maleic

anhydride.

The

product

of

the

reaction

of

PPIM

with

N-(2-

mercaptoethyl)oleamide was named PPIMY (5). PPIMY was synthesized via a thiol-ene reaction of the PPIM with sulphydryls of N-(2-mercaptoethyl)oleamide. The product of the reaction of PPIMY with cysteamine was named PPIMYC (6). PPIMYC was obtained

via a thiol-ene reaction of the PPIMY with sulphydryls of cysteamine.

Scheme 2. Synthesis of the Dendrimer-Based Biocompatible Zwitterionic Polymer PPIMYC. (A) The Synthesis of Cystamine; (B) The Detailed Synthesis Steps of N-(2mercaptoethyl)oleamide; (C) The Detailed Synthesis Steps of PPIMYC


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(A) H2N

S

NaOH

NH2

S

H 2N

• 2HCl

S

S

NH2

1 EDC•HCl, HOBt

(B)

H 2N

S

NH2

S

+

COOH

O

H N

S

S

O

DTT

N H

2 H N

HS

O

3


19


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(C ) NH2 O

O

O

O

G2 PPI

G2 PPI

N H

O

3 OH 8

thiol-ene reaction

4 (G2 PPI)

(PPIM) O

O

HN

G2 PPI

OH O S

N H

H N

7 H 2N

SH

thiol-ene reaction

O COOH

1

5 (PPIMY) O HN

G2 PPI N H

S

NH2

COOH H O N S

7

O COOH

1

6 (PPIMYC)

1H

NMR spectra (Figure 1) were used to confirm the successful preparation of the

biocompatible zwitterionic polymer. The 1H NMR spectrum of G2 PPI (Figure 1A) shows four characteristic peaks, which correspond to the four kinds of protons in G2 PPI. The 1H

NMR spectrum of PPIM (Figure 1B) shows new characteristic peaks at 5.79 ppm and

6.18 ppm, which were assigned to the double bond -CH=CH- (indicated by bold H), and


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the other peaks are from the G2 PPI. G2 PPI molecule was modified by about eight maleic anhydride molecules through the comparison of the area of double bond to the other areas. PPIM was reacted with N-(2-mercaptoethyl)oleamide and cysteamine. PPIMY (Figure 1C) shows two new characteristic peaks which are derived from groups of N-(2-mercaptoethyl)oleamide. One peak was at 0.81 ppm and is assigned to -CH3, and the other peak was at 1.19 ppm and is assigned to CHCH2(CH2)4 and CHCH2(CH2)6. This result indicates sulphydryls of N-(2-mercaptoethyl)oleamide were reacted with double bond groups of PPIM via thiol-ene additions. It was calculated that about 1 hydrophobic N-(2-mercaptoethyl)oleamide molecule was successfully reacted with 1 G2 PPI molecule. The 1H NMR spectrum of PPIMYC (Figure 1D) shows the peaks at 5.79 and 6.18 ppm are fully disappeared, which suggests that the double bonds of PPIMY were fully reacted with cysteamine. There were about 7 cysteamine molecules were conjugated to 1 G2 PPI molecule. The molecular weights of G2 PPI, PPIM, PPIMY, and PPIMYC were measured by MALDI-TOF mass spectrometry to be 773.12 g/mol (Figure S4), 1557.54 g/mol (Figure S5), 1897.37 g/mol (Figure S6) and 2438.29 g/mol (Figure S7).


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Figure 1. 1H NMR spectra of (A) G2 PPI, (B) PPIM, (C) PPIMY and (D) PPIMYC. The results indicate that one hydrophobic N-(2-mercaptoethyl)oleamide molecules and seven cysteamine molecules were successfully conjugated to one G2 PPI molecule. 3.2.Characterization of blank micelles and drug-loaded micelles. The

zwitterionic

polymer

of

PPIMYC

consists

of

hydrophobic

N-(2-

mercaptoethyl)oleamide segments and superhydrophilic zwitterionic dendritic segments. PPIMYC self-assembled into micelles in aqueous solutions, where hydrophobic N-(2-mercaptoethyl)oleamide was the core and the hydrophilic zwitterionic segment was the shell. Instead of using a high molecular weight linear polymer as the hydrophilic portion as done in previous work,1,30,45,46 we used low molecular weight zwitterionic dendritic G2 PPI as the hydrophilic portion. It should be noted that each PPIMYC molecule contains seven pairs of zwitterionic groups which provide a thin zwitterionic layer. In contrast, linear zwitterionic polymers often result in a thick layer. The thin zwitterionic layer may impart stealth properties to the carrier, while increasing the cellular uptake efficiency. In addition, the prepared PPIMYC molecule contains both super-


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hydrophilic zwitterionic head groups and hydrophobic fatty tails. PPIMYC molecules have a similar structure to phospholipids to some extent. In order to investigate whether free drugs were successfully encapsulated in the micelles of PPIMYC-DOX-Ce6, the UV-Vis spectra and fluorescence spectra of samples were measured. Figure 2A shows the characteristic absorption peak of free DOX and free Ce6 at 480 nm and 641 nm, respectively. The UV-Vis spectrum of PPIMYC-DOX-Ce6 micelles also displays a broad absorption band at 508 nm and 670 nm, which were derived from encapsulated DOX and Ce6, respectively. Thus, these results confirmed that the two drugs were successfully encapsulated within the micelles. It should be noted that a noticeably broader and red shifted absorption band were observed, which may be due to the hydrophobic and π-π interactions between the porphyrin core of Ce6 and the aromatic ring of DOX. In addition, PPIMYC-DOX-Ce6 had considerably strong absorbance around 670 nm, which indicates that the system could absorb near-infrared light, which has great tissue penetration ability in vivo.47 Fluorescence spectrum of PPIMYC-DOX-Ce6 (Figure 2B) also shows similar results. At excitation wavelengths of 480 nm and 405 nm, PPIMYCDOX-Ce6 exhibited emission peaks at 575 nm and 660 nm, which are derived from free DOX and free Ce6, respectively. The results suggest that DOX and Ce6 are encapsulated inside the micelles. Moreover, the color of blank micelles, free drugs, and drug-loaded micelles also confirm this result as shown in Figure 2C. It has been reported that hydrophobic drugs are encapsulated into nanoparticles because of the hydrophobic interactions between hydrophobic sections of the nanoparticles and drug molecules.48,49


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In PPIMYC-DOX micelles, the DLC and DLE of DOX were 5.98 % and 53.05 %, respectively. Moreover, in PPIMYC-DOX-Ce6 micelles, the DLC and DLE of DOX were 2.66 % and 35.95 %, respectively, while the DLC and DLE of Ce6 were 2.86 % and 38.75 %, respectively. Figure 2D shows the critical micelle concentration (CMC) value of the blank micelles is 11.25 μg/mL. The value of the CMC is affected by the ratio of the hydrophilic segment to the hydrophobic segment. CMC values decrease with increasing hydrophobic sections.17

Figure 2. (A) The UV-Vis spectra and (B) the fluorescence spectra of DOX, Ce6 and PPIMYCDOX-Ce6 micelles. (C) Images of blank micelles, free drugs, and drug-loaded micelles. (D) Plot of the intensity ratio (I374/I384) as a function of the logarithm of blank micelles concentrations. The results indicate that DOX and Ce6 are successfully encapsulated in the zwitterionic micelles.


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The size and morphology of the nanoparticles affect the interaction between the nanoparticles and cells.38 TEM samples were prepared by dissolving micelles in deionized water before dropping onto TEM grids. The TEM results show that the sizes of blank micelles (Figure S8), PPIMYC-DOX micelles (Figure S9), and PPIMYC-DOXCe6 micelles (Figure 3B) were 47.17 ± 22.15 nm, 77.58 ± 23.24 nm and 8.91 ± 5.03 nm, respectively. TEM results also demonstrate that all micelles were spherical. The PPIMYC-DOX-Ce6 micelles (Figure 3A) had the most uniform spherical shape and the smallest particle size across all samples. The size of the PPIMYC-DOX micelles was larger than that of the blank micelles after loading DOX. This may be due to repulsive forces such as van der Waals interactions, between hydrophobic DOX and the hydrophobic core of micelles, leading to expansion of the core of the nanoparticles and increased size.50,51 The size of the PPIMYC-Ce6 micelle in water was 9.5 nm. The size of PPIMYC-Ce6 micelles was much smaller than PPIMYC-DOX micelles, which indicated the interaction of PPIMYC-Ce6 micelles was significantly different from PPIMYC-DOX micelles. This may be due to the presence of Ce6 enhancing the hydrophobic interaction during selfassembly52. The interaction between PPIMYC and Ce6 possessed a dominant force in the selfassembly process, which made the size of the PPIMYC-DOX-Ce6 micelles close to PPIMYC-


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Ce6 micelles. Reassembly may occur during the formation of the PPIMYC-DOX-Ce6 micelles, resulting in fewer PPIMYC molecules than the blank micelles and PPIMYCDOX micelles. Figure S10 shows that the volume-based hydrodynamic sizes of blank micelles, PPIMYC-DOX micelles, and PPIMYC-DOX-Ce6 micelles are 50.9 nm, 334.9 nm and 9.2 nm in deionized water, respectively. The sizes measured by TEM are considerably smaller than that measured by DLS because the samples tested by TEM were in a dry state on grids while the samples measured by DLS were in a solvated state in solution.53


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Figure 3. (A) TEM image of PPIMYC-DOX-Ce6 from an aqueous solution and corresponding (B) histogram of the size distribution. The TEM sample was stained with phosphotungstic acid. (C) The hydrodynamic size of PPIMYC-DOX and PPIMYC-DOXCe6 micelles measured by DLS in PBS solutions at pH 5.5, 6.5, 7.4 and corresponding (D) zeta potential. Meanwhile, the sizes and zeta potentials of drug-loaded micelles in phosphate buffered saline (PBS) solutions with different pH values were evaluated. As shown in


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Figure 3C, the sizes of PPIMYC-DOX micelles and PPIMYC-DOX-Ce6 micelles were 26.18 nm and 11.32 nm at pH 7.4, 28.86 nm and 9.70 nm at pH 6.5, and 58.19 nm and 17.83 nm at pH 5.5, respectively. The size of both systems increased with the decreasing pH, indicating the reduced stability of the micelles. As shown in Figure 3D, the zeta potentials of PPIMYC-DOX micelles and PPIMYC-DOX-Ce6 micelles were -1.1 mV and -3.3 mV at pH 7.4, 0.7 mV and 1.0 mV at pH 6.5, and 2.7 mV and 1.8 mV at pH 5.5, respectively. The results demonstrate that the zeta potentials of PPIMYC-DOX micelles and PPIMYC-DOX-Ce6 micelles are reversed at pH 7.4 in the normal physiological microenvironment as compared to an acidic pH in the tumor microenvironment. At pH 7.4, the exterior carboxyl groups have a slightly negative charge. At acidic pH, these primary amine groups protonate and the resulting charge on the micelles leads to a stronger attraction with the negatively charged tumor cell membrane surface. This could improve their cellular uptake efficiency.38 In short, the TEM and DLS results confirm that PPIMYC-DOX-Ce6 micelles have small size, narrow size distribution, spherical shape, and responsive zeta potential, which will impart the nanoparticles with a high penetration ability in tumor tissues.54


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3.3.Stability of blank micelles and drug-loaded micelles in fibrinogen. Intravenously, these DDS will encounter many blood proteins. The strong interaction between DDS and proteins will lead to the quick clearance of the DDS by the RES. Fibrinogen can be easily activated by DDS due to charge or hydrophobic interactions in blood. Thus, the stability in fibrinogen solution is commonly evaluated using DLS before the use of DDS in vivo.55 As shown in Figure S11A, the volume-based diameter of fibrinogen in pH 7.4 PBS was about 23.0 nm, and the volume-based diameter of PPIMYC micelles was 10.4 nm. When PPIMYC micelles were mixed with fibrinogen, no observable aggregation was seen. The mixed solution was also clear. The results indicate that interaction between PPIMYC micelles and fibrinogen are weak enough to maintain a stable mixed solution. Figure S11B and Figure 4 show that PPIMYC-DOX micelles and PPIMYC-DOX-Ce6 micelles have similar results to PPIMYC micelles. Thus, PPIMYC micelles and drug-loaded micelles are stable in fibrinogen solution. This is beneficial for avoiding the capture of the micelles by the RES. The high stability in fibrinogen solution is due to the hydration of the zwitterionic shell of dendritic G2 PPI


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through electrostatic hydration at physiological pH. In short, the prepared micelles have high stability in proteins.

Figure 4. The volume-based diameters of 1 mg/mL PPIMYC-DOX-Ce6 micelles and 0.5 mg/mL fibrinogen in pH 7.4 PBS solution. The results demonstrate that the zwitterionic micelles are stable in the fibrinogen solution. 3.4. In vitro release of PPIMYC-DOX-Ce6. For DDS, the controlled release behavior of the drugs instead of burst release is particularly important after they are injected into blood.51 Thus, many methods have been developed to prevent the burst release of drugs in normal tissue and to achieve enhanced release in tumor tissue. To achieve this goal, ionizable groups have been introduced because they undergo conformational changes and solubility changes in


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different pH.50 The pH of tumor tissue is slightly acidic, which is lower than that of normal healthy tissue. The effect of pH on the release behavior of DOX was investigated by using fluorescence spectrometry. The PPIMYC-DOX-Ce6 micelles were placed in PBS solutions with a pH of 6.5 or 7.4 to mimic the acidic tumor tissue environment or normal tissue environment, respectively. As shown in Figure 5, sustained release of DOX from PPIMYC-DOX-Ce6 micelles was observed both at pH 6.5 and pH 7.4. Burst release of the DOX was not observed. The cumulative DOX release at pH 6.5 was faster and to a greater extent than that at pH 7.4. After 35 h, the cumulative release of DOX from PPIMYC-DOX-Ce6 micelles was around 40.88 % at pH 6.5, as compared to about 25.06 % at pH 7.4. The enhanced cumulative release of DOX at pH 6.5 can be attributed to an improved solubility of DOX, due to the protonation of the -NH2 group of DOX at lower pH.30,37 This acid-sensitive DDS could enhance the therapeutic effect against the tumor cells and reduce toxic side effects on normal cells. In short, the prepared PPIMYC-DOX-Ce6 micelles release more DOX under slightly acidic pH than at normal physiological pH.


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Figure 5. The cumulative DOX release curve of PPIMYC-DOX-Ce6 micelles in pH 6.5 and pH 7.4 PBS solutions. The results indicate that PPIMYC-DOX-Ce6 micelles have sustained and enhanced release of DOX at pH 6.5. 3.5.Cytotoxicity study. The relative cytotoxicity of G2 PPI and PPIMYC micelles against HeLa cells was evaluated with an MTT assay and visualizing the cell morphologies.56 Figure 6A shows the relative cell viabilities following exposure to G2 PPI or PPIMYC micelle as a function of their exposure concentrations at pH 7.4. The cell viability following exposure to G2 PPI and PPIMYC micelles was 81.3 % and 92.4 %, respectively, when the maximum exposure concentration of 440 μg/mL was reached. The cell viability after exposure to


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PPIMYC micelles with a 660 nm laser at a power of 300 mW cm-2 for 3 min was 94.1 %, which indicates that cell viability is not affected by this irradiation condition. Figure 6C shows the less spread morphology of HeLa cells after incubation with G2 PPI. Figure 6D shows the morphology of HeLa cells after incubation with PPIMYC, which was similar to the control groups (Figure 6B). Thus, the zwitterionic micelle PPIMYC possessed better biocompatibility than G2 PPI in vitro.

Figure 6. (A) The cell viability following exposure to G2 PPI and PPIMYC. Cell morphologies of HeLa cells after incubation with (B) medium, (C) G2 PPI, and (D)


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PPIMYC at a concentration of 440 μg/mL at pH 7.4. * indicates significant differences (P < 0.05) between G2 PPI and PPIMYC group. The results indicate that the zwitterionic micelles PPIMYC possess better biocompatibility than G2 PPI in vitro. The cytotoxicity of drug-loaded micelles was also measured using an MTT assay.56 Figure 7 shows the relationship between HeLa cell viability and the concentration of DOX and Ce6 in free Ce6, PPIMYC-Ce6, PPIMYC-Ce6 + Laser, free DOX, PPIMYCDOX, PPIMYC-DOX-Ce6, and PPIMYC-DOX-Ce6 + Laser at pH 7.4. All of the samples showed increasing cytotoxicity with increasing concentration of DOX and Ce6. When the DOX and Ce6 concentration of each sample reached 1.8 µg/mL, the cell viability in wells treated with free Ce6, PPIMYC-Ce6, PPIMYC-Ce6 + Laser, free DOX, PPIMYCDOX, PPIMYC-DOX-Ce6, and PPIMYC-DOX-Ce6 + Laser were 85.14 %, 67.53 %, 50.24 %, 68.27 %, 56.93 %, 45.24 %, and 15.08 %, respectively. In addition, the cell viability for control cells irradiated with a 660 nm laser at a power of 300 mW cm-2 for 3 min was 98.15 %, which indicated that cell viability was not affected by this irradiation condition. The cytotoxicity of PPIMYC-DOX-Ce6 micelles was higher than that of PPIMYC-DOX and PPIMYC-Ce6 + Laser micelles, which confirms the synergistic effect


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of DOX and Ce6. It should be noted that both PPIMYC-DOX and PPIMYC-DOX-Ce6 micelles show a better inhibition rate against HeLa cells as compared to free DOX, which is different from most zwitterionic micelles. The mechanism of enhanced cytotoxicity should be investigated. Among the six systems investigated here, the PPIMYC-DOX-Ce6 + Laser system has the highest cytotoxicity. The cytotoxicity of PPIMYC-DOX-Ce6 micelles was enhanced upon irradiation from a 660 nm laser at a power of 300 mW cm-2 for 3 min compared to the system without irradiation. The Ce6 encapsulated in PPIMYC-DOXCe6 micelles was photosensitizer, which could generate reactive oxygen species (ROS) under a 660 nm laser irradiation to induce cell death.47 Therefore, the cytotoxicity of the PPIMYC-DOX-Ce6 + Laser was enhanced by the laser irradiation for 3 min.


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Figure 7. Cell viability of HeLa cells after treatment with varying concentrations of free Ce6, PPIMYC-Ce6 micelles (with or without laser irradiation), free DOX, PPIMYC-DOX micelles, and PPIMYC-DOX-Ce6 micelles (with or without laser irradiation) at pH 7.4. The results indicate that irradiated PPIMYC-DOX-Ce6 micelles have the highest cytotoxicity. 3.6.Generation of intracellular ROS. The cytotoxicity of PPIMYC-DOX-Ce6 micelles under a 660 nm laser irradiation was significant enhanced compared with that of PIMMYC-DOX-Ce6 micelles alone. Thus, DCFH-DA was used to investigate whether enhanced cytotoxicity can be attributed to intracellularly generated ROS from the PPIMYC-DOX-Ce6 micelles upon laser


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irradiation. DCFH-DA can freely pass through the cell membrane and has no fluorescence. After DCFH-DA entered the cell, intracellular ROS oxidized DCFH which was generated by DCFH-DA to produce the green fluorescent substance DCF.47 Thus, the relative level of ROS in the cells can be evaluated by detecting the fluorescence of DCF. HeLa cells were treated with serum-free medium containing free DOX, PPIMYCDOX, PPIMYC-Ce6 + Laser, PPIMYC-DOX-Ce6, and PPIMYC-DOX-Ce6 + Laser at pH 7.4 at the concentration of 1.8 µg/mL DOX and 1.8 µg/mL Ce6. Figure 8 shows that the PPIMYC-DOX-Ce6 + Laser system had the highest intensity of fluorescent signal in the cells, indicating the PPIMYC-DOX-Ce6 was inside the cells and produced the most ROS. In short, the Ce6 in the PPIMYC-DOX-Ce6 was excited by irradiation to generate ROS, which enhances the cytotoxicity, inducing cell apoptosis.57,58


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Figure 8. Intracellular ROS level evaluated by DCFH-DA Fluorescence images of HeLa cells after incubation with different samples at pH 7.4 for 24 h. BF means bright field. The results indicate the enhanced ROS level in the PPIMYC-DOX-Ce6 + Laser system resulted in increasing cytotoxicity against HeLa cells. 3.7.Cellular uptake. To investigate the mechanism of enhanced cytotoxicity of prepared micelles as compared with free DOX, the cellular uptake of free DOX, PPIMYC-DOX micelles, and PPIMYC-DOX-Ce6 micelles at pH 6.5 and 7.4 was studied using both flow cytometry and fluorescence microscopy. Figure 9 shows that the cellular uptake of PPIMYC-DOX


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micelles and PPIMYC-DOX-Ce6 micelles were higher than that of free DOX. Detailed flow cytometry results are shown in Figure S12 and Figure S13.

Figure 9. Quantitative analysis of the mean fluorescence intensity of DOX in HeLa cells treated with free DOX, PPIMYC-DOX micelles, and PPIMYC-DOX-Ce6 micelles for 20 h (DOX 1.8 μg/mL) at two pH values (7.4 and 6.5). Non-exposed cells were used as controls. * indicates significant differences (P < 0.05) between pH 7.4 group and pH 6.5 group. The results indicate that drug-loaded micelles have enhanced cellular uptake compared with free DOX.


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As a representative example, the average fluorescence intensities of free DOX, PPIMYC-DOX micelles, and PPIMYC-DOX-Ce6 micelles at pH 6.5 were 59.79, 116.59, and 182.83, respectively. Thus, the relative magnitudes of the mean fluorescence intensities were: PPIMYC-DOX-Ce6 micelles > PPIMYC-DOX micelles > free DOX. Similar trends were also found at pH 7.4. Compared with free DOX, much more DOX was taken up and then released inside HeLa cells from drug-loaded micelles. The average fluorescence intensity of PPIMYC-DOX micelles and PPIMYC-DOX-Ce6 micelles increased with decreasing pH, which is due to the enhanced affinity between drug-loaded micelles and HeLa cells. The zwitterionic drug-loaded micelles have the highest cellular uptake efficiency relative to the reported zwitterionic micelle literature.37,38 The enhanced cellular uptake could be attributed to the special structure of the drug-loaded micelles, which have small particle size, thinner zwitterionic layer and the amphiphilic structure. Meanwhile, the cellular uptake of drug-loaded micelles was also studied by fluorescence microscopy. Figure 10 shows representative fluorescent microscopy images of HeLa cells after incubation with free DOX, PPIMYC-DOX micelles, and


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PPIMYC-DOX-Ce6 micelles at pH 7.4 for 20 h, respectively. It is worth noting that the fluorescence signal of PPIMYC-DOX-Ce6 micelles was higher than free DOX and PPIMYC-DOX micelles at identical exposure times, indicating the highest cellular uptake efficiency of PPIMYC-DOX-Ce6. The result was consistent with the results of the flow cytometry. In addition, the free DOX is widely distributed in HeLa cells, suggesting that free DOX can enter cells by passive diffusion.45 The DOX in the drug-loaded micelles was concentrated within the HeLa cells due to the presence of the DOX carrier. Therefore, the prepared micelle carriers enhance the cellular uptake of DOX, leading to the higher cytotoxicity of PPIMYC-DOX-Ce6 micelles compared to free DOX.


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Figure 10. Representative fluorescent microscopy images of HeLa cells incubated with free DOX, PPIMYC-DOX micelles, and PPIMYC-DOX-Ce6 micelles (DOX 1.8 μg/mL) at pH 7.4 after 20 h. The red fluorescence signal of DOX indicates that drug-loaded micelles have enhanced cellular uptake compared with free DOX. 3.8.Determination of the endocytic pathways of micelles. When cells are incubated with nanoparticles, there are two known internalization pathways for nanoparticles according to previous literature. One is the passive, physical penetration of nanoparticles, and the other is active endocytosis. Both experimental and theoretical results show that the size, shape, surface chemistry, and ligand arrangement


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of nanoparticles affect their passive penetration or active endocytosis.39 In order to better explain the cellular uptake phenomenon, the endocytic pathways of PPIMYCDOX micelles and PPIMYC-DOX-Ce6 micelles were studied. Temperatures of 4 °C and 37 °C were used to study whether the cellular uptake pathway is energy dependent. Three different endocytosis inhibitors, chlorpromazine, amiloride, and genistein, were used to inhibit clathrin-mediated endocytosis, micropinocytosis, and caveolin-mediated endocytosis, respectively.59,60 Figure 11A and Figure 11B show the results of the endocytic pathway analysis for PPIMYC-DOX micelles and PPIMYC-DOX-Ce6 micelles at pH 7.4, respectively. As shown in Figure 11A, the cellular uptake of PPIMYC-DOX micelles was affected by temperature and chlorpromazine hydrochloride, indicating that clathrin-mediated endocytosis was the primary endocytosis pathway for PPIMYC-DOX micelles, and this process is dependent on energy. Figure 11B shows that the cellular uptake of PPIMYC-DOX-Ce6 micelles was only affected by temperature, which could be due to small particle size, thinner zwitterionic layer and the amphiphilic structurer of PPIMYCDOX-Ce6 micelles. Thus, PPIMYC-DOX-Ce6 micelles and PPIMYC-DOX micelles enter the cell through different pathways.


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Figure 11. Relative uptake efficiency of (A) PPIMYC-DOX micelles and (B) PPIMYCDOX-Ce6 micelles by HeLa cells treated with various endocytosis inhibitors and different temperatures at pH 7.4 for 6 h. * indicates significant differences (P < 0.05) compared with 37 °C group, & indicates without significant differences (P > 0.05) compared with 37 °C group. The results indicate that PPIMYC-DOX-Ce6 micelles and PPIMYC-DOX micelles enter the cell through different pathways. 4. CONCLUSION We prepared new dendrimer-based biocompatible zwitterionic micelles, which were composed

of

hydrophilic

zwitterionic

G2

PPI

and

hydrophobic

N-(2-

mercaptoethyl)oleamide. The drug-loaded micelles exhibited undetectable interactions


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with fibrinogen in an aqueous solution, and acid-sensitive sustained drug release. In addition, cytotoxicity assays demonstrated that the blank micelles had good biocompatibility, while drug-loaded micelles exhibited enhanced cytotoxicity compared with free DOX. This was attributed to the enhanced cellular uptake of DOX in drugloaded micelles. Temperature was only the factor that affected the cellular uptake of PPIMYC-DOX-Ce6 micelles. Therefore, the utilization of PPIMYC-DOX-Ce6 micelles overcame the difficulties of delivering free DOX into cells, providing a novel drug delivery system. ASSOCIATED CONTENT

Supporting Information.

Electronic Supporting Information (ESI) is available: 1H NMR spectrum of cystamine, N,N’-(disulfanediylbis(ethane-2,1-diyl))dioleamide

and

N-(2-mercaptoethyl)oleamide;

MALDI-TOF mass spectrum of G2 PPI, PPIM, PPIMY, PPIMYC; TEM images of blank micelles and PPIMYC-DOX micelles; The size distribution of blank micelles, PPIMYCDOX, and PPIMYC-DOX-Ce6 micelles in an aqueous solution by DLS; The volume-


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based diameters of PPIMYC micelles and PPIMYC-DOX micelles in fibrinogen solution; Flow cytometry analysis of cellular uptake in Hela cells at pH 7.4 and pH 6.5 after treatment by free DOX, PPIMYC-DOX, and PPIMYC-DOX-Ce6 micelles.

AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected].

*E-mail: [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

The authors appreciate financial support from the National Natural Science Foundation of China (21474085, 21674092, and 51750110495 (MTB)), the National Development Project on Key Basic Research (973 Project, 2015CB655303), Natural Science Foundation of Hebei Province (B2017203229), Youth Foundation Project


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supported by the Hebei Education Department of China (QN2015034), China Postdoctoral Science Foundation (2016M601284), Postdoctoral Science Foundation of Hebei Province (B2016003017), the Doctor Fund of Yanshan University (B915), and the Young Teacher Research Program of Yanshan University (15LGB018).

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TOC：


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The dendrimer-based zwitterionic polymer micelles were fabricated to increase their cellular uptake by cancer cells.


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Dendrimer-Based Biocompatible Zwitterionic Micelles for Efficient

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