Amphiphilic diblock terpolymer PMAgala-b-P(MAA-co-MAChol)s with attached galactose and cholesterol grafts and their intracellular pH-responsive doxorubicin delivery

Article abstract of DOI:10.1021/acs.biomac.5b01227

In this work, a series of diblock terpolymer poly(6-O-methacryloyl-d-galactopyranose)-b-poly(methacrylic acid-co-6-cholesteryloxy hexyl methacrylate) amphiphiles bearing attached galactose and cholesterol grafts denoted as the PMAgala-b-P(MAA-co-MAChol)s were designed and prepared, and these terpolymer amphiphiles were further exploited as a platform for intracellular doxorubicin (DOX) delivery. First, employing a sequential RAFT strategy with preliminarily synthesized poly(6-O-methacryloyl-1,2:3,4-di-O-isopropylidene-d-galactopyranose) (PMAIpGP) macro-RAFT initiator and a successive trifluoroacetic acid (TFA)-mediated deprotection, a series of amphiphilic diblock terpolymer PMAgala-b-P(MAA-co-MAChol)s were prepared, and were further characterized by NMR, Fourier transform infrared spectrometer (FTIR), gel permeation chromatography (GPC), differential scanning calorimetry (DSC), and a dynamic contact angle testing instrument (DCAT). In aqueous media, spontaneous micellization of the synthesized diblock terpolymer amphiphiles were continuously examined by critical micellization concentration assay, dynamic light scattering (DLS), and transmission electron microscopy (TEM), and the efficacies of DOX loading by these copolymer micelles were investigated along with the complexed nanoparticle stability. Furthermore, in vitro DOX release of the drug-loaded terpolymer micelles were studied at 37 °C in buffer under various pH conditions, and cell toxicities of as-synthesized diblock amphiphiles were examined by MTT assay. Finally, with H1299 cells, intracellular DOX delivery and localization by the block amphiphile vectors were investigated by invert fluorescence microscopy. As a result, it was revealed that the random copolymerization of MAA and MAChol comonomers in the second block limited the formation of cholesterol liquid-crystal phase and enhanced DOX loading efficiency and complex nanoparticle stability, that ionic interactions between the DOX and MAA comonomer could be exploited to trigger efficient DOX release under acidic condition, and that the diblock terpolymer micellular vector could alter the DOX trafficking in cells. Hence, these suggest the pH-sensitive PMAgala-b-P(MAA-co-MAChol)s might be further exploited as a smart nanoplatform toward efficient antitumor drug delivery.

Full text of DOI:10.1021/acs.biomac.5b01227

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Article
Amphiphilic Diblock Terpolymer PMAgala-b-P(MAA-co-
MAChol)s With Attached Galactose and Cholesterol Grafts
and Their Intracellular pH-responsive Doxorubicin Delivery
Zhao Wang, Ting Luo, Ruilong Sheng, Hui Li, Jingjing Sun, and Amin Cao
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01227 • Publication Date (Web): 18 Dec 2015
Downloaded from http://pubs.acs.org on December 25, 2015
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Amphiphilic Diblock Terpolymer
PMAgalaꢀbꢀP(MAAꢀcoꢀMAChol)s With Attached
Galactose and Cholesterol Grafts and Their
Intracellular pHꢀresponsive Doxorubicin Delivery
Zhao Wang, Ting Luo, Ruilong Sheng, Hui Li, Jingjing Sun and Amin Cao*
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CAS Key Laboratory of Synthetic and Selfꢀassembly Chemistry for Organic Functional
Molecules, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345
Lingling Road, Shanghai 200032, China
*Author for correspondence:
Phone: +86-21-5492-5303, fax: +86-21-5492-5537
E-mail: acao@ sioc.ac.cn
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ABSTRACT
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In this work, a series of diblock terpolymer poly(6ꢀOꢀmethacryloylꢀDꢀgalactopyranose)ꢀbꢀ
poly(methacrylic acidꢀcoꢀ6ꢀcholesteryloxy hexylmethacrylate) amphiphiles bearing attached
galactose and cholesterol grafts denoted as the PMAgalaꢀbꢀP(MAAꢀcoꢀMAChol)s were designed
and prepared, and these terpolymer amphiphiles were further exploited as a platform for
intracellular doxorubicin (DOX) delivery. First, employing a sequential RAFT strategy with
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preliminarily
synthesized
poly(6ꢀOꢀmethacryloylꢀ1,2:3,4ꢀdiꢀOꢀisopropylideneꢀDꢀ
galactopyranose) (PMAIpGP) macroꢀRAFT initiator and a successive TFAꢀmediated
deprotection, a series of amphiphilic diblock terpolymer PMAgalaꢀbꢀP(MAAꢀcoꢀMAChol)s
were prepared, and were further characterized by NMR, FTIR, GPC, DSC and dynamic contact
angle testing instrument (DCAT). In aqueous media, spontaneous micellization of the
synthesized diblock terpolymer amphiphiles were continuously examined by critical
micellization concentration assay, DLS and TEM, and the efficacies of DOX loading by these
copolymer micelles were investigated along with the complexed nanoparticle stability.
Furthermore, in vitro DOX release of the drugꢀloaded terpolymer micelles were studied at 37 oC
in buffer under various pH conditions, and cell toxicities of asꢀsynthesized diblock amphiphiles
were examined by MTT assay. At last, with H1299 cells, intracellular DOX delivery and
localization by the block amphiphile vectors were investigated by invert fluorescence
microscopy. As a result, it was revealed that the random copolymerization of MAA and MAChol
comonomers in the second block limited the formation of cholesterol liquidꢀcrystal phase, and
enhanced DOX loading efficiency and complex nanoparticle stability, and that ionic interactions
between the DOX and MAA comonomer could be exploited to trigger efficient DOX release
under acidic condition, and the diblock terpolymer micellular vector could alter the DOX
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trafficking in cells. Hence, these suggest the pHꢀsensitive PMAgalaꢀbꢀP(MAAꢀcoꢀMAChol)s
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might be further exploited as a smart nanoꢀplatform toward efficient antiꢀtumor drug delivery.
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KEYWORDS
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Terpolymer Amphiphiles; Galactose; Cholesterol; pHꢀresponsive; Doxorubicin; Delivery
INTRODUCTION
In the recent decades, synthetic glycopolymers with saccharide grafts such as galactose (GAL),
mannose (MAN), lactobionic acid (LBA) and so forth have attracted broad interests. It provides
a great potential toward advanced drug/gene delivery, tissue engineering, organ regeneration as
well as clinical diagnostics and biomedical imaging.1–4 Up to date, it has already been uncovered
that the bioactive GAL and LBA grafts could give rise to favorable waterꢀsolubility instead of
polyethylene glycol (PEG). In addition, they have strong and specific binding to the
asialoglycoprotein receptors (ASGPR), which is overꢀexpressed on human hepatocyte surface.5
Therefore, the GALs and/or LBAs have been extensively exploited as potent blocks in designing
translocating vectors for drug delivery systems targeting human liver cancer cells.6–8 For instance,
two series of diblock P(MEO2MAꢀcoꢀOEGMA)ꢀbꢀPMAGP and P(MAGPꢀcoꢀDMAEMA)ꢀbꢀ
PPDSMA copolymer amphiphiles with GAL grafts were prepared by Pan et al.9,10, and their
selfꢀassembled supramolecular nanoparticles could be uptaken by HepG2 cells in a more
efficient way than HeLa cells. Amphiphilic sugarꢀfunctional polycarbonate micelles also exhibit
strong ability targeting HepG2 cells in vitro11. Alternatively, Ahmed et al.12 explored the
hemocompatibility of hyperbranched glycopolymers, and thereby convinced the presence of
these glycopolymers would not lead to clot formation, red blood cell aggregation and any
immune response. Taking the advantages of LBAs and reductionꢀresponsive disulfide linkages
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into account, glycoꢀnanoparticles have been reported to bear reductionꢀsensitive sheddable LBA
shells as potent hepatomaꢀtargeting anticancer drug delivery vectors13. Likewise, polymeric gene
vectors bearing mannitols and some other saccharides have also been revealed to be able to
stimulate glycoꢀnanoparticle cellular uptake and trafficking via selectively activated endocytic
and intracellular trafficking gateways.14,15 Moreover, Yang et al.16 recently uncovered that two
factors of enhanced permeabilityꢀandꢀretention effect (EPR) and lectinꢀmediated
hepatomaꢀtargeting ability simultaneously influenced the doxorubicin (DOX) delivery in vivo by
biodegradable polycarbonate micellar nanoparticles with GAL surface grafts. Since a PEG with
its molecular weight higher than 40 KDa may encounter the metabolic issue of accumulation in
tissues and organs17, alternative hydrophilic polymers bearing the saccharides like GAL and LBA
have been expected with stealth effect and biological functions.
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On the other hand, cholesterol as an essential component of the plasma membrane, has been
known to be involved in many cellular processes such as membrane property regulation,
steroidogenesis, bile acid synthesis and cellular signal transduction.18,19 Meanwhile, a
NiemannꢀPick C1ꢀlike transmembrane protein (NPC1) mediates its intracellular uptake and
transportation in living cells,20 and the cholesterols translocate through lysosomeꢀperoxisome
membrane contacts19. As for its potential application in drug delivery, cholesterols attached onto
the Nꢀ(2ꢀhydroxypropyl) methacrylamide (HPMA)ꢀbased copolymer drug conjugates could
result in lowered blood clearance and enhanced accumulation in tumors than their dodecyl or
oleic acid conjugated counterparts21, and the strong hydrophobic interactions between the
cholesterols and anticancer drugs played crucial roles in enhancing paclitaxel (PTX) drug
loading levels22. Notably, the cholesterol conjugates of amphiphilic copolymer vectors were
further found to be capable of altering the DOXꢀloaded complex nanoparticle cellular
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internalization mechanism23. In addition, cholesterol as an interesting natural rodꢀlike mesogen
has recently been explored for the structural design of new liquid crystalline (LC) organics,
functional synthetic lipids and linearꢀdendritic block LC copolymers.24–27 In particular, tailored
block structural LC copolymer amphiphiles bearing grafted cholesterols like poly(ethylene
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glycol)ꢀbꢀpoly(cholesteryl
acryloyoxy
ethyl
carbonate)s,
poly(6ꢀcholesteryloxyhexyl
and poly(glyceryl
methacrylate)ꢀbꢀpoly(2ꢀhydroxyethyl
methacrylate)s
methacrylate)ꢀbꢀpoly(6ꢀcholesteryloxyhexyl methacrylate)s were found to selfꢀassemble in
aqueous media into nanoꢀobjects with finely ordered hierarchical structures due to the cholesterol
hydrophobic interactions and mesogen stacking, and a number of unique morphologies as
periodically layered rodꢀlike micelles, ellipsoidal vesicles, solid spheres and nanoꢀtubes have
been disclosed.28–33 Moreover, Lu et al.34 and Li et al.35 separately prepared amphiphilic graft
copolymers and reductionꢀsensitive diblock copolymers bearing pendant cholesterols, and their
selfꢀassembled nanoparticles and vesicles were further explored for DOX and calcein release,
respectively. However, challenges of slow and less responsive release in cells still remained,
which were due to the strong cholesterol hydrophobic interaction and highly ordered solid LC
phase structure, and these impede their practical application as efficient drug delivery vectors.
As for a polymeric micellar drug vector, utilizing cholesterol grafts seems to be very helpful
for decreasing drug leakage and initial burst release, and enhancing micelle stability under
infinite dilution via physical crossꢀlinking by cholesterol interactions27,35. To further realize the
controlled drug release by the cholesterolꢀbased copolymer amphiphiles, additional responsive
constituents seem to be important for triggering the efficient drug release. It has been known that
human tumors exhibit feature acidicity of pH=6.5~7.2, and intracellular organelles like
earlyꢀendosome and lateꢀlysosome tend to be more acidic with pH=5~6 and 4~5, respectively.36
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Therefore, these pH differences have thus been extensively exploited for rational design of
pHꢀresponsive drug release carriers.37, 38 The methacrylic acid (MAA) monomer has a pKa of
5~6,39 and the presence of biocompatible MAA comonomers in amphiphilic copolymers could
enhance the interactions between the copolymer carriers and doxorubicins (DOX) through strong
ionic interactions under physiological condition, ultimately leading to efficient drug loading.40–42
Taking the advantages of MAA, galactose and cholesterol mesogen into account, in this work,
we first designed and prepared a new series of amphiphilic diblock terpolymer
poly(6ꢀOꢀmethacryloylꢀDꢀgalactopyranose)ꢀbꢀpoly(methacrylic acidꢀcoꢀ6ꢀcholesteryloxyhexyl
methacrylate)s hereby denoted as PMAgalaꢀbꢀP(MAAꢀcoꢀMAChol) via RAFT copolymerization,
and molecular structures of asꢀsynthesized diblock terpolymers were further characterized by
means of NMR, FTIR and GPC. Then, the PMAgalaꢀbꢀP(MAAꢀcoꢀMAChol) supramolecular
nanoparticles with/without DOX drug payloads were examined in terms of drug loading
efficiency, micellar particle morphologies and stability. Cytotoxicity of these prepared diblock
terpolymer amphiphiles was evaluated by MTT assay with H1299 cells. Finally, in vitro DOX
drug release and their intracellular delivery by the PMAgalaꢀbꢀP(MAAꢀcoꢀMAChol) vectors
were examined and discussed.
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EXPERIMENTAL SECTION
Materials
Cholesterol (97%), Dꢀgalactose (99%) and methacryloyl chloride (98%) were purchased from
Shanghai Sinopharm Chemical Reagent Co. Ltd (China) and utilized asꢀreceived. N,N’ꢀazobis
(isobutyronitrile) (AIBN, 98%, Shanghai Sinopharm Chemical Reagent Co. Ltd) was
recrystallized twice from methanol prior to use. Tertꢀbutyl methacrylate (tBMA, 99%, Sigma &
Aldrich) was purified via basic alumina gel column chromatography. RAFT agent of
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4ꢀcyanoꢀ4ꢀ(dodecylsulfanyl thiocarbonyl) sulfany pentanoic acid (CDP) was prepared as referred
to the literature43. 6ꢀCholesteryloxyhexyl methacrylate (MAChol) was synthesized and further
purified as previously reported29. Toluene was refluxed over metallic sodium and distilled before
use. Fluorescent probe of pyrene (95%) for CMC measurement was bought from Aladdin
Chemical Reagent Co. Ltd (China). All other organics were commercially supplied and used
asꢀreceived. In addition, cellulose dialysis membrane (MWCO 3500) was purchased from
Shanghai Green Bird Science & Technology Development Co. Ltd (China). Doxorubicin (DOX,
98%) was purchased from Zhejiang Hisun Pharmaceutical Co. Ltd (China). Sodium dodecyl
sulfate (SDS, 99%), fetal bovine serum (FBS, Cat#10099ꢀ141) and bovine serum albumin (BSA,
Cat#0332) were purchased from Genebase Gene Tech Co. Ltd (China), Invitrogen (USA) and
Amresco (USA), respectively. Thiazoyl blue tetrazolium bromide (MTT, Cat#M5655) was
supplied by Sigma & Aldrich (USA). PARP antibody (Cat#9542) was received from Cell
Signaling Technology, Inc (USA). RPMI1640 (Cat#11875ꢀ093) and DMEM (Cat#11995ꢀ065)
mediums were purchased from GIBCO (USA). βꢀtubulin antibody (Cat#MAN1003), sodium
pyruvate solution (100X, Cat#MG6096), antibiotics penicillin (100 IU/mL) and streptomycin
(100 ꢁg/mL) (Cat#MG7989), Lꢀglutamine (Cat#MG8042), horseradish peroxidaseꢀlinked rabbit
antiꢀmouse immunoglobulin (Cat#MAN4001) and horseradish peroxidaseꢀconjugated goat
antiꢀmouse immunoglobulin G (Cat#MAN4002) were supplied by MesGen (China).
Chemiluminescence kit (Cat#34077) was purchased from Pierce (USA). H1299 and HepG2 cell
lines were kindly gifted by Dr. Bo Wan of Key Laboratory of Genetic Engineering of Fudan
University (Shanghai, China).
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Instrumental Characterization
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NMR spectra Routine 1H NMR spectra were recorded on a Varianꢀ300 FTꢀNMR spectrometer
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operated at 300.0 MHz for proton nuclei, and C NMR characterization was implemented on a
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Bruker Avanceꢀ400 FTꢀNMR spectrometer (100.0 MHz for 13C nuclei) with CDCl3 or DMSOꢀd6
as the solvents. Measurements of NMR spectra were conducted at ambient temperature with
tetramethylsilane (TMS) as internal chemical shift reference. In addition, temperatureꢀresolved
1H NMR spectra were recorded on a Bruker Avanceꢀ400 FTꢀNMR spectrometer (400.0 MHz for
proton nuclei) with a BVT2000 temperature controller and pyridineꢀd5 as the solvent.
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Mass spectra ESIꢀMS were routinely measured on a Varian SATURN 2000 instrument.
FTIR spectra FTIR spectra were recorded on a BioꢀRad FTSꢀ185 spectrometer at room
temperature with 64 scans spanning a spectral range of 4000～500 cmꢀ1 with a resolution of 4.0
cmꢀ1. Samples were first wellꢀdissolved in an organic solvent (chloroform or pyridine), and then
dropꢀcasted on potassium bromide (KBr) pellets and dried prior to the measurement.
Elemental analysis Oxygen elemental analysis was routinely conducted on an Elementar vario
EL Ⅲ system (German).
Gel permeation chromatography (GPC) Molecular weights (Mn, Mw) and polydispersity
(Mw/Mn) of the synthesized polymers were routinely measured at 35°C on a PerkinElmer 200
GPC equipped with a refractive index detector (RI). THF was utilized as the eluent at a flowing
rate of 1.0 mL/min, and polystyrene standards (Polymer laboratories, UK) were applied to
calibrate the GPC traces.
Dynamic light scattering (DLS) Particle sizes and zeta potentials of the amphiphilic micelles
were analyzed at 25°C or 37°C on a Malvern Zetasizer Nano ZS90 dynamic light scattering
instrument with laser incident beam at λ=633 nm and a fixed scattering angle of 90°.
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Transmission electronic microscopy (TEM) Micelle morphologies were investigated by TEM
(JEOLꢀ1230, JEOL Co. Ltd, Japan) with an acceleration voltage of 80 KV. Typically, micelle
solution with a concentration of 1.0 mg/mL was dropped onto a 300ꢀmesh carbonꢀcoated copper
grid, and excess fluid was removed with filter paper and finally airꢀdried at room temperature.
The morphologies of the micelles were immediately observed without further stain.
Preparation of new amphiphilic diblock terpolymers
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Synthesis of 6-O-Methacryloyl-1,2:3,4-di-O-isopropylidene-D-galactopyranose (MAIpGP)
monomer MAIpGP was synthesized in a way as partially referred to the literatures9,44. In brief,
Dꢀgalactose was protected via reacting with acetone in the presence of concentrated H2SO4 (98%)
and anhydrous CuSO4, and then esterified with methacryloyl chloride in ice bath. After
purification via column chromatography, the MAIpGP products were collected as white solids
(yield of two steps: 64%).
1H NMR (CDCl3, δ in ppm): 6.13 (m, 1H, =CHH), 5.58 (m, 1H, =CHH), 5.53 (d, 1H,
Galactopyranose (Gal)−H at 1 position), 4.62 (m, 1H, Gal−H at 3 position), 4.30 (m, 4H, Gal−H
at 2, 4 and 6 position), 4.05 (m, 1H, Gal−H at 5 position), 1.93 (s, 3H, CH3CR=CH2), 1.53ꢀ1.33
(m, 12H, (CH3)2COO).
13C NMR (CDCl3, δ in ppm): 167.2, 136.0, 125.8, 109.6, 108.8, 96.3, 71.1, 70.7, 70.5, 66.1,
63.6, 25.9, 25.0, 24.4, 18.3.
FTIR (in cmꢀ1): 2978, 2926, 1716, 1384, 1258, 1208, 1176, 1164, 1113, 1066, 1006, 936, 900,
865.
ESIꢀMS [M+H+] (in m/z): 329.1 (Calc.: 329.2).
RAFT polymerization of MAIpGP Polymerization of MAIpGP monomer was conducted by
controlled RAFT polymerization. Typically, MAIpGP (656.0 mg, 2.0 mmol), CDP (40.4 mg, 0.1
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mmol) and AIBN (3.3 mg, 0.02 mmol) were dissolved in freshly distilled toluene (4.4 mL), and
placed into a Schlenk tube equipped with magnetic stirrer. The mixture was deoxygenated by
freezeꢀpumpꢀthawing for three times, and then immersed into an oil bath thermostated at 80°C
for 8 h. At predetermined time intervals, the reaction mixture was sampled by a syringe, and the
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monomer conversion and molecular weight were analyzed by H NMR and GPC. The reaction
was stopped by cooling in ice bath, and precipitated in cooled dry hexane, and the collected
precipitates were dried under vacuum to finally obtain yellowish PMAIpGP powders.
1H NMR (CDCl3, δ in ppm): 5.53 (d, Gal−H at 1 position), 4.62 (m, Gal−H at 3 position),
4.35ꢀ3.90 (m, Gal−H at 2, 4, 5 and 6 position), 2.35 (s, HOOCCH2R).
FTIR (in cmꢀ1): 2988, 2932, 1732, 1382, 1256, 1212, 1166, 1115, 1070, 1004, 892.
Synthesis of a series of diblock terpolymer PMAIpGP-b-P(tBMA-co-MAChol) precursors and
final PMAgala-b-P(MAA-co-MAChol) amphiphiles PMAIpGPꢀbꢀP(tBMAꢀcoꢀMAChol)s diblock
terpolymers were prepared in a way similar to that for the PMAIpGP. In brief, predetermined
t
amounts of PMAIpGP, AIBN, BMA and MAChol dissolved in freshly distilled toluene were in
turn added into a Schlenk tube with magnetic stirrer. After three cycles of freezeꢀpumpꢀthawing,
the Schlenk tube was immersed into an oil bath preheated at 80°C, and kept reaction for 16 h.
After the precipitation for three times in anhydrous methanol, final yellowish powder terpolymer
products were obtained after the drying under vacuum for 12 h. Furthermore, the asꢀprepared
terpolymer precursors were deprotected at room temperature in mixed trifluoroacetic acid (TFA)
and dichloromethane (DCM) (1/2, v/v) for 32 h, and PMAgalaꢀbꢀP(MAAꢀcoꢀMAChol)
amphiphiles were prepared by repeated precipitation in anhydrous cold methanol and drying
under vacuum .
PMAIpGP-b-P(tBMA-co-MAChol):
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1H NMR (CDCl3, δ in ppm): 5.53 (d, Gal−H at 1 position), 5.34 (d, =CHR of cholesterol), 4.65
(m, Gal−H at 3 position), 4.35ꢀ3.75 (m, Gal−H at 2, 4, 5 position and ꢀCH2COOR), 3.45 (t,
CH2OR of cholesterol), 3.12 (m, OCHR of cholesterol).
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FTIR (in cmꢀ1): 2933, 2866, 1728, 1458, 1382, 1368, 1255, 1212, 1142, 1072, 1005.
PMAgala-b-P(MAA-co-MAChol):
1H NMR (pyridineꢀd5, 80°C, δ in ppm): 6.80ꢀ5.40 (Gal−H at 1 position and–OH), 5.54 (=CHR
of cholesterol), 5.20ꢀ4.00 ppm (GlaꢀH at 2, 3, 4, 5 position and ꢀCH2COOR), 3.63 (CH2OR of
cholesterol), 3.35 (OCHR of cholesterol).
FTIR (in cmꢀ1): 3363, 2933, 2866, 1726, 1466, 1366, 1254, 1147, 1103.
Preparation of diblock terpolymer amphiphiles and their DOX-loaded micelles
The terpolymer amphiphiles and their DOXꢀloaded micelles were prepared in a way as
following: In brief, 10.0 mg of each synthesized terpolymer amphiphile was first dissolved in 1
mL of THF/DMSO (1/1, v/v), and stirred at room temperature for 8 h. Then, 5 mL phosphate
buffer (pH 7.4, 10 mM) was added dropwise into the mixture under vigorous stirring. The
resultant solution was further dialyzed against deionized water for 48 h using a preꢀswollen
cellulose dialysis membrane (MWCO 3500) to remove residual solvent. In a similar way, DOX
(10.0 mg) and triethylamine (TEA, 2 mol equiv. to DOX) were dissolved in 1 mL of DMSO, and
stirred for 4 h, then 40.0 mg of terpolymer amphiphilie preliminarily dissolved in 4 mL of
THF/DMSO (1/1, v/v) was again added. Afterwards, 20 mL of phosphate buffer (pH=7.4, 10
mM) was gradually placed into above mixture, and the DOXꢀloaded terpolymer amphiphilie
micelle solution was dialyzed in deionized water for 48 h to give drugꢀloaded amphiphilie
micelle aqueous solution. The DOX loading levels were accordingly measured by UVꢀVis
spectrophotometer (UVꢀ2800, Hitachi, Japan). Lyophilized DOXꢀloaded terpolymer micelles
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were dissolved in DMSO, and the DOX concentration was evaluated according to a standard
curve, and DOX loading content (DLC) and loading efficiency (DLE) were further calculated
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according to the following formulas:45
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Weight of loaded drug
ꢀ
ꢃ
DLC ꢁꢂ% =
× 100%
Weight of loaded drug and polymers
Weight of loaded drug
DLEꢀ%ꢃ =
× 100%
Weight of drug in feed
Critical micelle concentration (CMC)
Pyrene was utilized as fluorescence probe to determine the CMC of asꢀsynthesized terpolymer
amphiphiles. Briefly, 10 ꢁL of pyrene/acetone solution (1.2×10ꢀ4 M) was placed into the
terpolymer micelle aqueous solution under various mass concentration of 1×10ꢀ5～5×10ꢀ1
mg/mL to adjust final 6.0×10ꢀ7 M pyrene in micelle solution, then kept at room temperature in
dark for 24 h before the measurement. Fluorescence spectra were recorded on a fluorescence
spectrophotometer (Fꢀ7000, Hitachi, Japan) with an excitation wavelength of 334 nm and
scanning wavelength of 350 ～450 nm, and fluorescence intensity ratios (I394/I374) were
calculated, and plotted as a function of terpolymer concentration. The CMC for each terpolymer
amphiphilie was routinely determined as ever reported11.
Stability of DOX-loaded terpolymer micelles
Stability of the DOXꢀloaded terpolymer amphiphile micelles (1.0 mg/mL) was examined by
DLS, and the SDS (100 ꢁg/mL) was employed as a destabilizing agent22, and light scattering
intensity was continuously monitored by DLS at 37°C over a period of 48 h. Similarly, average
particle sizes of the DOXꢀloaded micelles in the presence of 10% FBS, BSA (0.3%, m/v) and 2
M NaCl were separately analyzed after 24 h incubation.
DOX release in vitro
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DOX release profiles were examined at 37°C under different pH for the drug loaded
terpolymer amphiphile micelles in three kinds of media: (a) acetate buffer, pH=4.5, 10 mM; (b)
phosphate buffer, pH=6.5, 10 mM; (c) phosphate buffer, pH=7.4, 10 mM. In brief, each
DOXꢀloaded micelle solution (2.0 mg/mL, 3 mL) was placed into a cellulose dialysis membrane
(MWCO 3500), and immersed into the aboveꢀmentioned media at 37°C under gentle shaking. At
predetermined time intervals, 2 mL release media was sampled for UVꢀVis analysis, and an
equal volume of fresh media was added, and in vitro DOX release was routinely analyzed using
a UVꢀVis spectrophotometer10.
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MTT assays
Cytotoxicities of asꢀprepared terpolymer micelles were assayed with H1299 cells by MTT
assay kit. Cells were first seeded into 96ꢀwell microplate (6×103 cells/well) with RPMIꢀ1640
medium supplemented with 10% FBS, 1% Lꢀglutamine, antibiotics penicillin (100 IU/mL) and
streptomycin (100 ꢁg/mL), and incubated for 24 h. Then, the medium was aspirated and replaced
by 100 ꢁL fresh medium with 10% FBS supplemented with micelle solution under various
concentration of 0, 20, 50, 80 and 100 ꢁg/mL, and kept cultivation at 37 °C under 5% CO2 for
another 30 h. Afterwards, 20 ꢁL of MTT solution (5.0 mg/mL) was placed into the microplate
replaced with 100 ꢁL FBSꢀfree fresh medium, and further incubated for another 2 h. 100 ꢁL of
DMSO was added into each well to dissolve the MTTꢀformazan, and light absorbances at λ=490
were measured on microplate reader (BioTek, ELX800) with λ=630 nm as the reference. As a
result, relative cell viability was evaluated with n=5 as following:
Cell viability (%)=(OD490(sample)ꢀOD630(sample))/(OD490(control)ꢀ OD630(control))×100%
In a similar way, cell viabilities were also examined for the DOXꢀloaded diblock terpolymer
amphiphile micelles and free DOX with H1299 and HepG2 cell lines. As for the MTT assay with
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HepG2 cell line, the HepG2 cells were first cultivated with DMEM medium supplemented with
10% FBS, 1% Lꢀglutamine, antibiotics penicillin (100 IU/mL), streptomycin (100 ꢁg/mL) and
sodium pyruvate (1mM). After 24 h incubation, the medium was aspirated, and replaced by 100
ꢁL fresh medium (with 10% FBS) supplemented with different amounts of DOXꢀloaded
terpolymer micelles or free DOX to adjust final DOX concentration of 0.3, 1, 3, 5, 10 and 15
ꢁg/mL, and kept incubation at 37°C under 5% CO2 for 30 h. Thereafter, cell viability was thus
assayed by the MTT assay kit.
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Intracellular DOX release
Cellular uptake and intracellular DOX release of the DOXꢀloaded micelles and free DOX
control were examined with H1299 cells on fluorescence microscopy. First, the cells were
cultured on microscope slides in 6ꢀwell plate (5×105 cells/well) using RPMIꢀ1640 medium with
10% FBS, 1% Lꢀglutamine, antibiotics penicillin (100 IU/mL) and streptomycin (100 ꢁg/mL),
then the cells were incubated at 37°C with either DOXꢀloaded micelles or free DOX for 1 and 6
h. After the removal of culture medium, the cells were rinsed three times with 1×PBS, and
stained cell nuclei with Hoechst 33342, then fluorescence images were recorded on Nikon TiꢀS
invert fluorescence microscopy.
Western-blot assay
H1299 cells preliminarily treated with either free DOX or DOXꢀloaded micelles for 30 h were
harvested and extracted with RIPA lysis buffer, and then incubated at 95°C for 15 min. Total
protein was separated by 8% SDSꢀPAGE, and blotted to nitrocellulose transfer membranes. The
membranes were further blocked with 3% BAS TBST (20 mM Tris–HCl, pH 7.6, 137 mM NaCl,
and 0.01% Tweenꢀ20) for 1 h at the room temperature, followed by incubation with PARP or
βꢀtubulin primary antibodies at 4°C over night. After extensive washing with TBST, the
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membranes were allowed to be reprobed with horseradish peroxidaseꢀlinked antiꢀmouse
immunoglobulin in 5% BSA TBST for 40 min, and unreacted antibodies were separated with
TBST for three times. Then, the membranes were subjected to westernꢀblot analysis with the
signals from the primary antibody amplified by horseradish peroxidaseꢀconjugated goat
antiꢀmouse immunoglobulin G. Finally, the protein bands were detected and recorded by a
chemiluminescence kit.
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RESULTS AND DISCUSSION
Synthesis and Characterization of Diblock Terpolymer PMAgala-b-P(MAA-co-MAChol)s
As for the preparation of functional glycopolymers, controlled radical polymerization has been
widely employed as an effective synthetic approach.46 Lowe et al47 reported the first RAFT
polymerization of 6ꢀOꢀmethacryloylꢀ1,2:3,4ꢀdiꢀOꢀisopropylideneꢀDꢀgalactopyranose (MAIpGP)
monomer, and several dithiobenzoateꢀtype chain transfer agents (CTAs) have been explored in
the MAIpGP RAFT polymerization.9,10,44 In this work, as shown in Scheme 1, the monomers of
MAIpGP and MAChol were accordingly synthesized with good yields. Figures 1A and 1B depict
1H NMR spectra for the MAIpGP and MAChol monomers, and the resonance signals were
accordingly assigned9,29. Furthermore, the PMAIpGP was prepared under an optimized reaction
temperature of 80ºC in toluene through RAFT polymerization with AIBN and the synthesized
4ꢀcyanoꢀ4ꢀ(dodecylsulfanylthiocarbonyl) sulfany pentanoic acid (CDP) as the initiator and CTA,
respectively, and the initial CTA/initiator molar ratios were hereby optimized to be 1/0.2.10
Figure 2A presents 1H NMR spectrum of the PMAIpGP product, and the 1H resonance signals (a,
1
h) could be assigned to the H nuclei neighboring anomeric carbon in sugar ring and methylene
group of the CTA moiety, demonstrating successful RAFT polymerization. To make more clear
the MAIpGP RAFT polymerization kinetics, Figure 3 shows the results of polymerization
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kinetics study at 80°C in toluene under a MAIpGP/CDP/AIBN molar ratio of 20/1/0.2, and the
Ln([M]0/[M]t) vs reaction time (t) plots as seen in Figure 3A tend to exhibit a good linear
relationship, inferring a pseudoꢀfirst order polymerization kinetics for the MAIpGP monomer.
Moreover, Figure 3B shows the MAIpGP monomer conversion dependence of number average
molecular weight (Mn) by GPC, indicating a linear relationship with polydispersity indices
(Mw/Mn) lower than 1.2, demonstrating controlled polymerization of the PMAIpGP.
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To achieve amphiphilic diblock terpolymer PMAgalaꢀbꢀP(MAAꢀcoꢀMAChol)s, the purified
PMAIpGP were continuously employed as the macroꢀRAFT agents for successive random
t
copolymerization of MAChol and BMA comonomers at 80°C in toluene to give
PMAIpGPꢀbꢀP(tBMAꢀcoꢀMAChol) precursors as shown in Scheme 1C, and Figure S1 depicts a
t
copolymerization time dependence of the MAChol and BMA comonomer conversion under an
initial MAChol/tBMA/macroꢀRAFT/AIBN feeding molar ratio of 15/25/1/0.2, and the observed
similar consumption rates for these two comonomers may indicate the occurrence of random
RAFT copolymerization of the second block. The resulted random comonomer sequence may be
helpful for tuning the interactions and ordering of cholesterol mesogens and successive physical
1
crossꢀlinking of PMAgalaꢀbꢀP(MAAꢀcoꢀMAChol) micelles34,35. Figure 2B shows typical H
NMR spectrum for asꢀresulted PMAIpGPꢀbꢀP(tBMAꢀcoꢀMAChol), and GPC traces of the
PMAIpGP macroꢀRAFT agent and asꢀsynthesized series of diblock terpolymers are presented in
Figure 4. In general, Table 1 summarizes synthetic results for the PMAIpGP macroꢀRAFT agent
1
and PMAIpGPꢀbꢀP(tBMAꢀcoꢀMAChol)s, and the assigned H NMR resonance signals and high
monomer conversions as well as low Mw/Mn values substantiated successful preparation of the
PMAIpGP18ꢀbꢀP(tBMAꢀcoꢀMAChol) precursors with narrow molecular weight distribution.
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So far, trifluoroacetic acid (TFA) has already been known as a standard reagent to remove the
tertꢀbutyl and isopropylidene groups of protected galactoses, and has been reported to have less
influence on the other type of ester structures and cholesterol skeleton.47ꢀ49 In this work, a
reaction conditions of TFA/DCM (1/2, v/v) for 32 h was employed to deprotect the terpolymer
PMAIpGP18ꢀbꢀP(tBMAꢀcoꢀMAChol) precursors. Figure S2C depicts FTIR spectra for the
PMAIpGP18ꢀbꢀP(tBMA16ꢀcoꢀMAChol12) and achieved diblock terpolymer amphiphile product,
and remarkable new emergence of broad IR absorption around 3300 cmꢀ1 assignable to –OH
stretch vibration of the resulted terpolymer amphiphile could be observed, indicating efficient
tertꢀbutyl and isopropylidene removals. Since the PMAgalaꢀbꢀP(MAAꢀcoꢀMAChol) terpolymer
amphiphiles could not be well dissolved at the room temperature in the most of commonly used
deuterated solvent, temperatureꢀresolved 1H NMR spectra with pyridineꢀd5 solvent were recorded
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as shown in Figure S3, and the conditions of pyridineꢀd5 solvent at 80°C was used to record H
NMR spectrum with better resolution for the PMAgala18ꢀbꢀP(MAA16ꢀcoꢀMAChol12) as shown in
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Figure 2C. The theoretical and experimental resonance intensity ratios of the H signals at
δ=3.55~3.75 ppm (ꢀCH2ꢀOꢀCHR2 of cholesterol grafts) to those at δ=4.00~5.20 ppm (ꢀCH2COOꢀ
and the GlaꢀH at 2, 3, 4, 5 position) were estimated to be 1/5.50 and 1/5.13, respectively. As a
1
result, the galactose graft preservation could further be estimated to reach 91.7%. Since the H
resonance signals of tertꢀbutyl and isopropylidene groups around δ=1.38~1.48 ppm overlapped
with the signals of cholesterol skeleton, to give a deep insight into the deprotection of diblock
terpolymer precursors, additional diblock copolymer analogues of PMAIpGP18ꢀbꢀPtBMA18 and
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their deprotected PMAgala18ꢀbꢀPMAA18 were synthesized, and H NMR spectra of the
PMAIpGP18ꢀbꢀPtBMA18 in CDCl3 and PMAgala18ꢀbꢀPMAA18 in DMSOꢀd6 are depicted in
1
Figures S2A and S2B. It could be clearly seen that after the deprotection in TFA/DCM, H
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resonance signals at δ=1.40~1.47 ppm attributable to the tertꢀbutyl and isopropylidene
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thoroughly disappeared, demonstrating efficient TFAꢀcatalyzed removal of protecting groups.
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Furthermore,
oxygen
elemental
analysis
for
the
diblock
terpolymer
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PMAIpGP18ꢀbꢀP(tBMA16ꢀcoꢀMAChol12) precursor and PMAgala18ꢀbꢀP(MAA16ꢀcoꢀMAChol12)
amphiphile were conducted with the resulted oxygen elemental percentages of 19.88% and
23.41%, respectively, which agrees well with their corresponding theoretical values of 20.40%
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and 24.53% within the instrumental limit. Hence, the experimental evidences of H NMR and
oxygen elemental analysis substantiate efficient deprotection with negligible cleavage of
cholesterol/galactose grafts.
Furthermore, Figure S4 presents the recorded pictures of waterꢀdroplets on the thin film
surfaces of asꢀsynthesized series of diblock terpolymer amphiphiles, and the calculated contact
angles tend to decrease from 106.4±1.3°, 104.3±0.9°, 100.2±2.1° to 93.0±2.5° with increasing
MAA comonomer content, demonstrating the more hydrophilic physical properties. As for their
thermal properties, thermostated polarized optical microscopic (POM) pictures and temperature
modulated DSC traces are shown in Figures S5 and S6A, respectively. PMAChol18
homopolymer prepared by similar RAFT polymerization was observed to show feature fanꢀtype
texture of smectic A (SmAd) LC phase as previously reported33,50, and glass transition
temperatures (Tg) of the prepared diblock terpolymer amphiphiles were evaluated to increase
from 27.7°C, 42.0°C, 46.0°C to 54.9°C along with increasing MAA comonomer content. Since
the MAA repeating units are ionic comonomers, the phenomena of Tg increase in DSC traces for
the terpolymer amphiphiles with more MAA units could be interpreted for their strong hydrogen
bonding and ionic interactions in between51. Moreover, no obvious liquid crystal phase transition
was observed for the aboveꢀsynthesized copolymers during the DSC cooling and heating scans
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(Figure S6B and S6C). These results may be accounted for the disturbed cholesterol stacking due
to the presence of MAA comonomers in vicinity, and this would be beneficial for tuning micellar
physical crossꢀlinking, DOX loading and intracellular drug release as discussed below.
Preparation of DOX-loaded Supramolecular Micelles by the PMAgala-b-P(MAA-
co-MAChol)s
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To further exploit aboveꢀsynthesized PMAgalaꢀbꢀP(MAAꢀcoꢀMAChol)s as potential DOX
delivery vectors, their CMC in aqueous media were measured with pyrene as the fluorescent
probe as shown in Figure S7, and very low CMC were calculated to be 1.41~2.82 mg/L for the
PMAgala18ꢀbꢀPMAChol14 and the series of PMAgala18ꢀbꢀP(MAAꢀcoꢀMAChol)s as summarized
in Table 2, and these results suggest strong hydrophobic association of cholesterol grafts, which
seems favorable for high micelle stability against extreme dilution during blood circulation in
vivo.52 Figure 5 depicts particle sizes and morphologies of the PMAgala18ꢀbꢀP(MAA26ꢀcoꢀ
MAChol9) micelles characterized by DLS and TEM, respectively, and monodisperse spherical
micelles could be observed by TEM with an average particle size of 40.3 nm, which is smaller
than 94.9 nm in wet state by DLS due to the micelle shrinkage upon dehydration.10 In general,
Table 2 summarized the micelle sizes for the PMAgala18ꢀbꢀPMAChol14 and the series of
PMAgala18ꢀbꢀP(MAAꢀcoꢀMAChol)s, and the micelles with particle sizes of 48.9~140.2 nm
formed, strongly depending on their copolymer comonomer composition. Herein, it is
noteworthy that under physiological environment, larger size terpolymer micelles could be
observed for the PMAgala18ꢀbꢀP(MAAꢀcoꢀMAChol)s with higher MAA comonomer contents,
and this could be interpreted for the increased hydrophilicity and negative charge repulsion of the
carboxyl grafts.
In contrast, the DOXꢀloaded PMAgala18ꢀbꢀPMAChol14 and PMAgala18ꢀbꢀP(MAAꢀcoꢀ
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MAChol)s complex micelles showed particle sizes, decreasing from 243.9 nm to 95.8 nm with
increasing the MAA comonomer contents, and the drugꢀloaded diblock terpolymer micelles with
particle sizes smaller than 200 nm seem favorable for being accumulated in tumor vasculature
via EPR.53 Concerning the decrease in micelle particle size for the amphiphilic terpolymers with
more MAA comonomer, it could be accounted for the reasons that after drug loading under
neutral condition, positively charged DOX (pKa=8.2)54 would interact with negatively charged
MAA carboxyl groups (pKa=5~6), and the charge neutralization would further decrease their
waterꢀsolubility and give rise to more compacted solid micelles with decreased particle sizes and
enhanced complexed micelle stability. Moreover, as seen in Table 2, under neutral condition, all
DOXꢀloaded micelles showed zetaꢀpotentials of ꢀ12.2 to ꢀ26.9 mV, and the negative surface
charge may prevent their interactions with serum proteins, and thus be favorable for long
circulation in vivo.55 Moreover, πꢀπꢀstacking, electrostatic interaction and hydrogen bonding
between the polymer and drug have been well harnessed to increase drug loading efficiency and
micelle stability.56,57 In a view of drug loading efficacy (DLE) for the PMAgala18ꢀbꢀPMAChol14
and PMAgala18ꢀbꢀP(MAAꢀcoꢀMAChol)s micelles, the incorporation of a third MAA comonomer
led to remarkably increased DLE from 47.1% to 91.2%, demonstrating that the cholesterol and
MAA could synergistically improve the DOX encapsulation. As for the copolymer amphiphiles
bearing cholesterols, it has been known that the cholesterols in micellar core could result in high
drug loading due to hydrophobic interactions between the cholesterols and drugs22,58, however,
the cholesterols could also spontaneously form cholesterol LC phase, and thus decrease their
interactions with drug molecules22. On the evidences of DOX loading by the synthesized diblock
terpolymer PMAgala18ꢀbꢀP(MAAꢀcoꢀMAChol)s, it could be demonstrated that hydrophobic
association and ionic interaction between the MAA comonomer carboxyl and DOX significantly
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enhanced DOX loading for the diblock terpolymers, and random comonomer sequences in the
second block limited cholesterol LC phase formation as evidenced in Figure S5B and S5C.
Figure 6 presents stability assay results of the DOXꢀloaded complex micelles in aqueous
media by DLS with SDS destabilizing agent (100 ꢁg/mL) as referred59. It could be seen that the
light intensity of diblock PMAgala18ꢀbꢀPMAChol14/DOX micelle solution dropped down to 75%
within 48 h after adding SDS, while the DOXꢀloaded complex micelles by three
PMAgala18ꢀbꢀP(MAAꢀcoꢀMAChol)s showed excellent stability against SDS. Meanwhile, no
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significant
particle
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PMAgala18ꢀbꢀP(MAA26ꢀcoꢀMAChol9)/DOX micelles under 2 M NaCl or 0.3% (m/v) BSA, and
only slight swelling could be detected in 24 h with 10% FBS. These results inferred that the
copolymerization of MAChol and MAA comonomers as the second block endowed excellent
stability of DOXꢀloaded micelles.
Responsive DOX Release of Drug Loaded Diblock Terpolymer Micelles
Figure 7 depicts pH dependence of particle sizes for the DOXꢀloaded diblock terpolymer
PMAgala18ꢀbꢀP(MAA26ꢀcoꢀMAChol9) micelles at 37°C in buffer solution by DLS. It could be
seen that in acetate buffer (pH=4.5), the initial terpolymer micelle size was around 90~100 nm,
and the micelle sizes gradually increased along with the incubation time (0.5~16 h), and finally
dual dispersive peaks respectively centering around 120 nm and 1000 nm were detected due to
the destruction and aggregation of the DOXꢀloaded complex micelles.60 In contrast, after 24 h
incubation in PBS buffer (pH=7.4), the DOXꢀloaded PMAgala18ꢀbꢀP(MAA26ꢀcoꢀMAChol9)
micelles were just slightly swelled. Therefore, these results demonstrate strong pH dependence
of the drugꢀloaded terpolymer micelle sizes. Figure 8 shows in vitro DOX release profiles by the
diblock PMAgala18ꢀbꢀPMAChol14 and diblock terpolymer PMAgala18ꢀbꢀP(MAAꢀcoꢀMAChol)s
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in various media. At pH=7.4, the drug slowly released, and only 7% DOX were released from the
drugꢀloaded PMAgala18ꢀbꢀP(MAA26ꢀcoꢀMAChol9) micelles, in comparison no more than 30%
DOX were released from the diblock PMAgala18ꢀbꢀPMAChol14 micelles, this may infer that the
copolymerization of MAA comonomer could not only stabilize the DOXꢀloaded terpolymer
micelles, but also limit the DOX release at pH=7.4. At pH=6.5, the DOX release obviously
accelerated, and cumulative drug release could reach 30~47% within 50 h. When pH was further
decreased to 4.5, very fast and high cumulative DOX release could be observed for the
amphiphilic drug carriers of PMAgala18ꢀbꢀPMAChol14 (57.7%), PMAgala18ꢀbꢀP(MMA5ꢀ
coꢀMAChol14) (78.0%), PMAgala18ꢀbꢀP(MAA16ꢀcoꢀMAChol12) (75.0%) and PMAgala18ꢀbꢀ
P(MAA26ꢀcoꢀMAChol9) (90.3%), respectively. These results might be interpreted for the facts
that under pH condition below the pKa (5~6) of MAA carboxyl groups, ionic interactions
between the encapsulated DOX and MAA comonomers in amphiphile micelle cores were
significantly weakened while waterꢀsolubility of the DOX molecules simultaneously increased
due to the protonation of amino groups, and these finally led to responsive terpolymer micelle
disassembly and fast DOX releases under the acidic environment.
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Cell Viability Assay and Intracellular DOX Delivery by PMAgala-b-P(MAA-co-MAChol)
Micelles
Continuously, cell toxicities of asꢀprepared diblock PMAgala18ꢀbꢀPMAChol14 and the series of
terpolymer PMAgala18ꢀbꢀP(MAAꢀcoꢀMAChol)s were assayed with human lung cancer H1299
cells, and the MTT assay results are shown in Figure S8. After 30 h incubation, negligible slight
MTT toxicity could be observed under the amphiphile mass concentration up to 100 ꢁg/mL,
indicating excellent biocompatibility of the asꢀprepared terpolymer amphiphiles. Since the
terpolymer PMAgala18ꢀbꢀP(MAA26ꢀcoꢀMAChol9) has shown highly efficient DOX loading and
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pH responsive release, its DOXꢀloaded micelles were further employed to examine their cancer
cell proliferation inhibition and intracellular DOX delivery. Figure 9 depicts H1299 and HepG2
cell viabilities after 30 h incubation in the presence of the PMAgala18ꢀbꢀP(MAA26ꢀcoꢀ
MAChol9)/DOX micelles and free DOX under a series of DOX dosages. It could be seen that the
presence of either DOXꢀloaded terpolymer micelles or free DOX unambiguously resulted in
DOXꢀdosage dependent cell viability, and relatively lower tumor cell inhibition exhibited for the
DOXꢀloaded terpolymer micelles under the same DOX dosage in both cell lines. The IC50 values
(half maximal inhibitory concentration) of DOXꢀloaded micelles were 13.34 ꢁg DOX euqiv/mL
and 8.58 ꢁg DOX euqiv/mL toward H1299 cell line and HepG2 cell line, respectively, which are
both higher than that of free DOX (6.74 ꢁg/mL toward H1299 cell line and 6.67 ꢁg/mL toward
HepG2 cell line, respectively). This results indicated that the DOXꢀloaded complex micelles
could be uptaken efficiently and release DOX in both H1299 and HepG2 cells, and the relatively
lower tumor cell inhibition properties might be due to their different endocytic mechanisms as
compared with free DOX and prolonged DOX release from the complex micelles as indicated in
the in vitro DOX release profiles (Figure 8).61,62 Furthermore, since H1299 cells generally have
the merits of easy morphology observation with good reproducibility during cell biological study,
thus H1299 cells were chosen to give further insights into the DOX endocytosis and intracellular
trafficking. As shown in Figure 10, for the free DOX, after 1 h incubation, strong red DOX
fluorescence could be observed predominantly in cell nuclei, and the fluorescence intensity
localized in the cell nuclei was found to become stronger after 6 h incubation. This may be due
to fact that the DOX small molecules could freely penetrate through plasma membrane via fast
diffusion and rapid intracellular traffic to cell nuclei. As for the DOXꢀloaded diblock terpolymer
micelles, red DOX fluorescence was predominantly observed in cytoplasm after 1 h incubation,
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and significant DOX accumulation in cell nuclei could be seen after 6 h incubation, inferring that
the DOXꢀloaded terpolymer micelles may be internalized into cells via endocytosis, and further
release DOX in cytoplasm, and then deliver DOX into cell nuclei. These evidences are well
consistent with the results of cell viability assay shown in Figure 9A and similar results as
reported in literature56.
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Alternatively, it has already been known that the DOX is a topoisomerase inhibitor which
could cause DNA damage and cancer cell inhibition mainly by apoptosis. PARP, a 116 kDa
nuclear poly(ADPꢀribose) polymerase, has been known to be involved in DNA repair in response
to environmental stress63, and it is one of the main cleavage targets of caspaseꢀ3 in vivo64. The
PARP cleavage occurs between Asp214 and Gly215, which could separate the PARP Nꢀterminal
DNA binding domain (24 kDa) from the Cꢀterminal catalytic domain (89 kDa)64, and the
cleavage of PARP would facilitate cellular disassembly and serve as a marker of cells undergoing
apoptosis65. Figure 11 shows H1299 cell morphological images (A~C) recorded by brightꢀfield
microscopy with free DOX as the control, and obvious cell growth inhibition could be observed
after the incubation for 30 h under a fixed DOX dosage of 10 ꢁg/mL for the free DOX and
DOXꢀloaded diblock terpolymer micelles. Moreover, Figure 11D presents the western blot
analytic results for the cells treated with either free DOX or DOXꢀloaded terpolymer micelles,
and notably upꢀregulated PARP cleavages could be clearly observed with βꢀtubulin as the
internal reference, demonstrating the apoptosis of H1299 cells in the presence of either free DOX
or DOXꢀloaded terpolymer micelles. The westernꢀblot experimental evidences indicate that the
DOX delivered by the diblock terpolymer complex micelles could effectively result in cell
apoptosis despite possible different trafficking routes of free DOX and DOXꢀloaded terpolymer
complex micelles. Therefore, these infer that new diblock terpolymer micelles with efficient
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DOX loading and pHꢀresponsive drug release may be further developed as future drug delivery
carriers as shown in Scheme 2, and continuous studies concerning liver cancer cell targeting and
DOXꢀloaded complex particle autophagy in cells are now ongoing in this lab.
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CONCLUSION
In this study, we designed and successfully synthesized a series of amphiphilic diblock
terpolymer PMAgalaꢀbꢀP(MAAꢀcoꢀMAChol)s with sideꢀattached galactose and cholesterol
grafts. Structural and physical characterization demonstrated controlled RAFT copolymerization
of the diblock terpolymers, and random copolymerization of the MAA and MAChol in the
second block limited the formation of cholesterol LC phase, and the diblock terpolymers
exhibited increased waterꢀsolubility when increasing their MAA comonomer composition. In
aqueous solution, the PMAgala18ꢀbꢀPMAChol14 and PMAgala18ꢀbꢀP(MAAꢀcoꢀMAChol)s could
selfꢀassemble into micelles, and the diblock terpolymers could more efficiently encapsulate DOX
with a DLE up to 91.2% with better complex micelle stability. In vitro DOX release profiles
demonstrated high stability of the DOXꢀloaded terpolymer micelles under neutral condition, and
significantly fast responsive DOX release with cumulative release up to 90.3% could be
observed, and the higher MAA content of a diblock terpolymer tended to result in higher
cumulative DOX release, strongly depending on the pH of buffer solution. MTT assay with
H1299 cells indicated slight cytotoxicity of the PMAgala18ꢀbꢀPMAChol14 and PMAgala18ꢀbꢀ
P(MAAꢀcoꢀMAChol)s. The results of fluorescence microscopy revealed that the DOX
encapsulated in the synthesized diblock terpolymer PMAgala18ꢀbꢀP(MAA26ꢀcoꢀMAChol9)/DOX
micelles could be uptaken and delivered into cell nuclei in an efficient way, and their
intracellular trafficking pathway may be altered as compared with the free DOX control. In
addition, westernꢀblot assay of the PARP biomarker indicated efficient apoptosis of H1299 cells
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by the PMAgala18ꢀbꢀP(MAA26ꢀcoꢀMAChol9)/DOX micelles similarly as DOX control. Taking
advantages of the DOXꢀloaded PMAgalaꢀbꢀP(MAAꢀcoꢀMAChol) micelles, these diblock
terpolymers may be anticipated as potent vectors for controlled drug delivery in vivo.
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ASSOCIATED CONTENT
Supporting Information Synthesis and 1H NMR spectra of diblock PMAIpGP18ꢀbꢀPtBMA18 and
PMAgala18ꢀbꢀPMAA18, FTIR spectra of diblock terpolymer PMAIpGP18ꢀbꢀP(tBMA16ꢀcoꢀ
1
MAChol12) and PMAgala18ꢀbꢀP(MAA16ꢀcoꢀMAChol12), temperatureꢀresolved H NMR spectra
of the PMAgala18ꢀbꢀP(MAA16ꢀcoꢀMAChol12) amphiphile in pyridineꢀd5, kinetic plots of RAFT
t
polymerization of MAChol and BMA comonomers, POM images of the PMAChol18
homopolymer, images of water contact angle, temperatureꢀmodulated DSC and routine DSC
curves and CMC plots of asꢀprepared copolymer amphiphiles, cell viability assay of copolymer
micelles. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: acao@ sioc.ac.cn
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
ACKNOWLEDGMENT
The authors are grateful to the financial supports partially from National Natural Science
Foundation of China (21174160, 21002116 and 21372251).
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