Efficient synthesis of polymer prodrug by thiol-acrylate michael addition reaction and fabrication of Ph-responsive prodrug nanoparticles

Article abstract of DOI:10.1021/acs.bioconjchem.8b00531

In this study, an efficient method is proposed for the synthesis of polymer prodrug with acid-liable linkage via thiol-acrylate Michael addition reaction of the camptothecin with tethering acrylate group and polymer scaffold containing multiple thiol grou

Full text of DOI:10.1021/acs.bioconjchem.8b00531

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Article
Efficient Synthesis of Polymer Prodrug by Thiol-Acrylate Michael Addition
Reaction and Fabrication of pH-Responsive Prodrug Nanoparticles
Chao-Ran Xu, Liang Qiu, Cai-Yuan Pan, Chun-Yan Hong, and Zong-Yao Hao
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00531 • Publication Date (Web): 03 Aug 2018
Downloaded from http://pubs.acs.org on August 5, 2018
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Bioconjugate Chemistry
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Efficient Synthesis of Polymer Prodrug by Thiolꢀ
Acrylate Michael Addition Reaction and Fabrication
of pHꢀResponsive Prodrug Nanoparticles
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†
‡
†
*,†
*,§
ChaoꢀRan Xu , Liang Qiu , CaiꢀYuan Pan , ChunꢀYan Hong , ZongꢀYao Hao
†
CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and
Engineering, University of Science and Technology of China, Hefei, Anhui, 230026, China.
‡
Institute of Biophysics, Hebei University of Technology, Tianjin, 300401, China.
§
Institute of Urology, Anhui Medical University, Hefei, Anhui, 230026, China.
Eꢀmail: hongcy@ustc.edu.cn, haozongyao@163.com.
ABSTRACT: In this study, an efficient method is proposed for the synthesis of polymer prodrug
with acidꢀliable linkage via thiolꢀacrylate Michael addition reaction of the camptothecin with
tethering acrylate group and polymer scaffold containing multiple thiol groups. The polymer
scaffold P(HEO MA)ꢀbꢀP(HEMAꢀDHLA) is prepared by reversible additionꢀfragmentation
2
chain transfer (RAFT) polymerization of the methacrylate of lipoic acid (HEMAꢀLA) using
poly(2ꢀ(2ꢀhydroethoxy) ethyl methacrylate) (PHEO MA) as macroꢀRAFT agent, followed by
2
reduction of the disulfides in lipoic acid (LA) groups to give polymer scaffold with dihydrolipoic
acid (DHLA) pendent groups. Acrylateꢀtethering camptothecin (ACPT) is connected to
P(HEO MA)ꢀbꢀP(HEMAꢀDHLA) via Michael addition reaction between thiol and acrylate with
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a high coupling efficiency (95%). Amphiphilic polymer prodrug P(HEO MA)ꢀbꢀP(HEMAꢀ
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DHLAꢀCPT) spontaneously selfꢀassembles into nanoparticles in an aqueous solution, and
exhibits a CPT loading content as high as 40.1%. The prodrug nanoparticles with the acidꢀliable
βꢀthiopropionate linkages can release CPT under acidic condition, and the prodrug nanoparticles
show similar cytotoxicity to HeLa cells as free CPT. Overall, the prodrug nanoparticles with high
drug loading contents and acidꢀliable linkages are promising for pHꢀresponsive anticancer
therapy.
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Introduction
Chemotherapy is often used for cancer treatment, however, most anticancer drugs have some
drawbacks, such as poor water solubility, rapid blood clearance, low tumor selectivity, and side
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4,5
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effects for healthy tissues. Nanosized vehicles including liposomes, vesicles, polymer
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ꢀ11
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nanoparticles
and some inorganic materials
are used as carriers of anticancer drugs to
greatly enhance their performances in clinical. Among these nanocarriers, polymer nanoparticles
have attracted considerable attention to perform drug delivery in cancer therapy. Significant
advances on polymer nanoparticles encapsulating anticancer drugs in their cores have been
reported, but some obstacles including burst release, poor drug loading content and low
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4,15
miscibility of drugs with polymer matrix need to be overcome.
A promising approach to
addressing these problems is to conjugate free drug with polymer scaffold to form polymer
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prodrug, which has been widely investigated in recent years.
Polymer prodrugs can improve
the therapeutic efficacy of conventional small molecule drugs by increasing their loading
capacity and stability in the blood circulation, decreasing their exposure to normal tissues, and
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controlling their release in response to extracellular or intracellular stimuli.
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The conjugation of drugs to preꢀsynthesized polymers is one of the main strategies for the
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synthesis of polymer prodrugs. The “conjugation to” strategy is to graft drugs on the side
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chains of polymers via organic reactions.
Organic and platinum anticancer drugs have been
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linked to the side chains of preformed polymers via coupling strategies mainly including imine
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formation, carbodiimide reaction,
amidation reaction,
DielsꢀAlder coupling reaction
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and photochemical [2 + 2] addition reaction. However, these reactions have lower reaction
efficiency compared with copperꢀcatalyzed azideꢀalkyne cycloaddition (CuAAC) reaction. Drugs
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can be coupled to polymer scaffold efficiently via CuAAC reaction, but the residual Cu species
in the reaction products are hard to be fully removed, which is not suitable for biomedical
applications. The Michael addition reaction between thiol and acrylate doesn’t need heavy
metallic salts, which is known as a highly efficient and “green” reaction in bioconjugation
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chemistry. Additionally, the thiolꢀacrylate Michael addition reaction can generate pHꢀ
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responsive βꢀthiopropionate linkers. It is reported that the βꢀthiopropionate linkage can
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8,39
hydrolyze in acidic solution at a sustained rate,
thus, it can be used for controlled drug
release. So far literatures on drug release via the hydrolysis of βꢀthiopropionate linkage are few.
Zou et al. synthesized paclitaxelꢀconjugated linear block copolymer with βꢀthiopropionate
linkages by the postꢀfunctionalization of paclitaxel molecule onto the polymer backbone, and the
conjugation efficiency was 50% when the feed molar ratio of the paclitaxel to polymer scaffold
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was 20 to 1. Recently, our group fabricated camptothecinꢀbonded hyperbranched star
copolymer by atom transfer radical polymerization (ATRP) of prodrug monomer with βꢀ
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thiopropionate linkage in the presence of copper salt.
In this paper, Michael addition reaction between thiol and acrylate is used for synthesizing
polymer prodrug with βꢀthiopropionate linkages via the “conjugation to” approach. This
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synthetic method can control the drug loading capacity, improve the drugꢀloading efficiency and
enable the conjugated drugs to be controllably released in response to acidic milieu. As shown in
Scheme 1A, the polymer scaffold P(HEO MA)ꢀbꢀP(HEMAꢀDHLA) was synthesized by RAFT
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polymerization of HEMAꢀLA using PHEO MA as macroꢀRAFT agent, and successive reduction
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reaction of lipoic acid groups in P(HEO MA)ꢀbꢀP(HEMAꢀLA). ACPT was then conjugated to
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the polymer scaffold via Michael addition reaction with a high efficiency, forming polymer
prodrug P(HEO MA)ꢀbꢀP(HEMAꢀDHLAꢀCPT) with βꢀthiopropionate linkages. Moreover, the
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drug loading capacity of polymer prodrug could be controlled via adjusting the feed molar ratio
of ACPT and polymer scaffold. The polymer prodrug spontaneously selfꢀassembled into prodrug
nanoparticles in water (Scheme 1B). The prodrug nanoparticles were easily internalized by
cancer cells via cell endocytosis, and CPT was released in a controlled manner owing to the
hydrolysis of βꢀthiopropionate linkage in the subacid condition. Such prodrug nanoparticles have
potential applications in anticancer chemotherapy.
Scheme 1. Schematic illustration of A) the synthesis of polymer prodrug P(HEO MA)ꢀbꢀ
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P(HEMAꢀDHLAꢀCPT) with acidꢀliable linkage, and B) fabrication of pHꢀresponsive prodrug
nanoparticles for drug delivery.
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Results and Discussion
Synthesis and Characterization of Polymer Prodrug with Acidꢀliable βꢀThiopropionate
Linkages
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In this study, the polymer prodrug was synthesized by conjugating acrylateꢀtethering
camptothecin (ACPT) to polymer scaffold containing multiple thiol groups, and the detailed
synthetic route was shown in Scheme 1. PHEO MA was synthesized via the RAFT
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polymerization of HEO MA, which was further used as macroꢀRAFT agent in the RAFT
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polymerization of HEMAꢀLA. The block copolymer P(HEO MA)ꢀbꢀP(HEMAꢀLA) was
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successively reduced into polymer scaffold P(HEO MA)ꢀbꢀP(HEMAꢀDHLA) with thiol groups.
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ACPT was then connected to the polymer scaffold via Michael addition reaction between
acrylate and thiol, forming polymer prodrug P(HEO MA)ꢀbꢀP(HEMAꢀDHLAꢀCPT). The
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P(HEO MA) component in the polymer prodrug is hydrophilic, which is used for protecting the
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covalently conjugated drugs from undesirable external influence.
o
The RAFT polymerization of HEO MA was carried out in tetrahydrofuran at 70 C for 6 h,
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using 4ꢀcyanopentanoic acid dithiobenzoate (CPADB) as chain transfer agent and
azobisisobutyronitrile (AIBN) as initiator. The feed molar ratio of [HEO MA]/[CPADB]/[AIBN]
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was fixed at 50/1/0.1. The GPC curve of PHEO MA (Figure S1A in the supporting information)
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was unimodal, and the numberꢀaverage molecular weight (Mn,GPC) and the molecular weight
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distribution (M /M ) were 13500 g/mol and 1.09, respectively. In the H NMR spectrum of
w
n
PHEO MA (Figure 1A), the characteristic peaks of PHEO MA emerged at δ 4.15 ppm (b), 3.75
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ppm (c) and 3.58 ppm (d), corresponding to the CH groups on the side chain of PHEO MA.
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Meanwhile, the representative peaks at δ 7.88 ppm, 7.54 ppm and 7.38 ppm appeared in the
spectrum, which were attributed to the aromatic protons from CPADB end group. The Mn,NMR of
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PHEO MA was calculated based on the integral values of the signals at 4.15 ppm (b) and
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.2~8.0 ppm (a), and PHEO MA with degree of polymerization (DP) = 44 and M
= 7700
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n,NMR
g/mol was used in the following study.
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The block copolymer P(HEO MA)ꢀbꢀP(HEMAꢀLA) was synthesized by RAFT
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polymerization of HEMAꢀLA using PHEO MA as macroꢀRAFT agent with a feed molar ratio of
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HEMAꢀLA]/[PHEO MA]/[AIBN] = 40/1/0.1 in THF at 70 C for 6 h. Compared with Figure
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A, new peaks at δ 4.27 ppm (a), 2.37 ppm (i), 1.2~1.8 ppm (j, k, l), 3.53 ppm (m), 3.08 ppm (n),
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.47 ppm (o) and 1.92 ppm (p) emerged in the H NMR spectrum of P(HEO MA)ꢀbꢀP(HEMAꢀ
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LA) (Figure 1B), corresponding to the CH and CH groups on the side chain of P(HEMAꢀLA)
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block. The DP of P(HEMAꢀLA) block was calculated based on integral values of the signals at δ
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.47 ppm (o) and 4.0~4.5 ppm (a, b). The polydispersity index (PDI) value of the copolymer
measured by GPC (Figure S1A) was 1.21, and block copolymer P(HEO MA) ꢀbꢀP(HEMAꢀ
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LA) was used in the following experiments.
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Figure 1. H NMR spectra of PHEO MA (A) and P(HEO MA)ꢀbꢀP(HEMAꢀLA) (B) recorded in
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CDCl3.
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Under mild condition, the LA groups in the P(HEO MA)ꢀbꢀP(HEMAꢀLA) could be reduced to
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DHLA pendent groups by NaBH , giving P(HEO MA)ꢀbꢀP(HEMAꢀDHLA). After the reduction
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reaction, the signals of CH and CH protons (n and m) of P(HEO MA)ꢀbꢀP(HEMAꢀDHLA) near
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both sulfur atoms at δ 3.08 ppm and 3.53 ppm in Figure 1B shifted to δ 2.71 ppm and 2.95 ppm,
respectively; and the CH resonances (o, p) of P(HEO MA)ꢀbꢀP(HEMAꢀLA) at δ 2.47 and 1.96
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ppm in Figure 1A merged together at δ 1.90 ppm (Figure 2A), suggesting the disappearance of
cisꢀtrans isomerism in the DHLA pendent groups. GPC curves (Figure S1B) showed almost no
change in the molecular weight or PDI of polymer after reduction, and Fourier transform infrared
spectrum (Figure S2) confirmed the successful synthesis of P(HEO MA)ꢀbꢀP(HEMAꢀDHLA)
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ꢀ
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from P(HEO MA)ꢀbꢀP(HEMAꢀLA), as the thiol peak emerged at 2560 cm . The above analysis
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results showed that the disulfides in lipoic acid groups have almost converted to thiol groups,
which were available for the following Michael addition reaction. Then ACPT was coupled to
P(HEO MA)ꢀbꢀP(HEMAꢀDHLA) by Michael addition reaction between thiol and acrylate,
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forming polymer prodrug P(HEO MA)ꢀbꢀP(HEMAꢀDHLAꢀCPT). In the H NMR spectrum of
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polymer prodrug (Figure 2B), the CPT resonances (s, t, u, v, w, x, y) at δ 7.4~8.5 ppm and
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.1~5.8 ppm appeared. Meanwhile, representative new peaks emerged at δ 2.10 and 2.70 ppm (i
and z), which were attributed to CH protons on the newly formed βꢀthiopropionate linkages.
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Such results suggested that the polymer prodrug P(HEO MA)ꢀbꢀP(HEMAꢀDHLAꢀCPT) was
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synthesized successfully via the “conjugation to” approach.
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Figure 2. H NMR spectra of P(HEO MA)ꢀbꢀP(HEMAꢀDHLA) (A) and P(HEO MA)ꢀbꢀ
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P(HEMAꢀDHLAꢀCPT) (B) recorded in CDCl3.
As is well known, the Michael addition reaction of thiol and acrylate usually has almost
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quantitative conversion under mild reaction condition. In this manuscript, the conjugation of
ACPT to the polymer scaffold was carried out using triethylamine (TEA) as catalyst, and the
reaction was performed at room temperature in THF for 24 h. When the feed molar ratio of
[ACPT]/[DHLA] was 2:1, the drug loading content (DLC) of the polymer prodrug (Prodrugꢀ1)
was 40.1% and the drug loading efficiency (DLE) was as high as 88%. Upon decreasing of
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ACPT]/[DHLA] ratios to 1:1 and 1:2, the DLCs of the polymer prodrugs (Prodrugꢀ2 and
Prodrugꢀ3) decreased to 26.6% and 16.6%, and the DLEs increased to 90% and 95%,
respectively. The DLCs of polymer prodrugs could be controlled via adjusting the feed ratio of
ACPTꢀtoꢀDHLA in the Michael addition reaction, and detailed results were listed in Table 1.
GPC traces of the polymer prodrugs shown in Figure S3 revealed that the molecular weight of
polymer prodrug increased with the increase of the feed molar ratio of [ACPT]/[DHLA]. All the
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above results verified that this synthetic method endowed the polymer prodrug controllable drug
loading contents, high drugꢀloading efficiency and acidꢀliable βꢀthiopropionate linkages.
Table 1. Characterizations of prodrug nanoparticles with different drug loading content
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Sample
Feed molar ratio of
Diameter(TEM)
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Diameter(DLS)
(nm)
PDI(DLS)
DLC(UV)
(%)
DLC(Theory)
(%)
DLE
(%)
a
[
ACPT]/[DHLA]
Prodrugꢀ1
Prodrugꢀ2
Prodrugꢀ3
2:1
1:1
1:2
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0.168
0.152
0.110
40.1
26.6
16.6
45.7
29.6
17.4
88
90
95
a
[
ACPT] and [DHLA] refer to the acrylate of CPT and dihydrolipoic acid pendent group in the block copolymer P(HEO MA) ꢀ
2 44
bꢀP(HEMAꢀDHLA) , respectively;
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b
The average diameter of prodrug nanoparticles determined by TEM;
c
The numberꢀaverage diameter of prodrug nanoparticles measured by DLS;
d
The polydispersity index (PDI) of prodrug nanoparticles measured by DLS.
e
f
DLC refers to drug loading content, which was determined by ultraviolet quantitative analysis;
The theoretical DLC is calculated according to the feed weight ratio of CPT/(CPT + polymer scaffold) in the Michael addition
reaction;
g
The drug loading efficiency was calculated according to the equation: Drug loading efficiency (DLE) (%) = DLC
measured/theoretical DLC × 100%.
Fabrication of Prodrug Nanoparticles
Polymeric nanoparticles have been proven to be promising vehicles for targeted delivery of
drugs, because their nanoscopic sizes can protect them from being taken up by the phagocytic
cells of the reticuloendothelial system (RES), and they can passively accumulate in the solid
4
3,44
tumor through the enhanced permeability and retention (EPR) mechanism.
Herein, the
polymer scaffold P(HEO MA)ꢀbꢀP(HEMAꢀDHLA) is hydrophilic, but the covalently coupled
2
CPT in polymer prodrug is almost waterꢀinsoluble. The polymer prodrug P(HEO MA)ꢀbꢀ
2
P(HEMAꢀDHLAꢀCPT) could spontaneously selfꢀassemble into nanoparticles in water. Polymer
prodrug (12 mg) was dissolved in 1.0 mL of dimethyl sulfoxide (DMSO), and deionized water
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(
7.0 mL) was then added into the solution slowly through a syringe pump. The mixture was
dialyzed against deionized water, forming prodrug nanoparticles with a concentration of 1.5
mg/mL.
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Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to
characterize the formed assemblies. As shown in Figure 3A, prodrug nanoparticles fabricated
from prodrugꢀ1, prodrugꢀ2 and prodrugꢀ3 had similar diameters, which were characterized by
TEM. The DLS curves were shown in Figure S4, and the sizes of prodrug nanoparticles detected
by DLS were listed in Table 1. The sizes obtained from DLS were slightly larger than the sizes
from TEM, because prodrug nanoparticles were swollen in DLS measurement and collapsed in
TEM measurement. The critical micelle concentration (CMC) of prodrug nanoparticles was
determined using pyrene as a fluorescence probe. As shown in Figure 3B, the CMCs of prodrug
nanoparticles increased from 5.75 to 7.33 ꢁg/mL with the decrease of CPT contents in polymer
prodrugs, because camptothecin occupied the hydrophobic region of prodrug nanoparticles.
These low CMC values indicated that prodrug nanoparticles were stable in water. Overall, the
CPTꢀincorporated nanoparticles were successfully prepared, and prodrug nanoparticles
fabricated from prodrugꢀ1 were used in the following measurements.
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Figure 3. (A) TEM images of prodrug nanoparticles (a: prodrugꢀ1, b: prodrugꢀ2, c: prodrugꢀ
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); (B) Plot of the I /I ratio from pyrene excitation spectra versus the concentration of
339 332
polymer prodrugs on a log scale.
Release of CPT from Prodrug Nanoparticles
As has been reported previously, the hydrolysis of βꢀthiopropionate in acidic solution at a
relatively slow rate is driven by the generation of a partial positive charge on the ester carbonyl
45
carbon because of the inductive effect of sulfur atom. The prodrug nanoparticles with βꢀ
thiopropionate linkages were thus expected to be pHꢀsensitive. In vitro drug release experiments
of the prodrug nanoparticles were performed in the phosphate buttered saline (PBS) solution at
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pH 5.0, 6.0 and 7.4, respectively, and typical release profiles were shown in Figure 4. When
prodrug nanoparticles were incubated at pH 7.4, almost no CPT was released, indicating that the
prodrug nanoparticles were stable in the neutral solution, because βꢀthiopropionate linkage
3
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between CPT molecule and the polymer scaffold was unable to hydrolyze at pH 7.4.
Upon
treatment of prodrug nanoparticles at pH 6.0, 23.4% (for 24 h) and 50.4% (for 96 h) of CPT in
the nanoparticles were released. When the drug release was performed at pH 5.0, the release rate
of CPT from prodrug nanoparticles increased, and 38.4% (for 24 h) and 79.7% (for 96 h) of CPT
were released, this is due to that the hydrolysis rate of βꢀthiopropionate linkages increased with
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1
the decrease of pH values. It was clearly shown that the release of CPT from prodrug
nanoparticles in a subacid condition needed a couple of days to accomplish, which was
appropriate for controlled drug release in a sustained manner. It is speculated that, in the acidic
condition, such as in the cancer cells, CPT can be controllably released from the prodrug
nanoparticles.
Figure 4. In vitro CPT release profiles of prodrug nanoparticles fabricated from prodrugꢀ1 after
incubation in PBS buffer at pH values of 5.0, 6.0 and 7.4, respectively.
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In Vitro Cytotoxicity Assay
The cytotoxicity of CPT, polymer scaffold P(HEO MA)ꢀbꢀP(HEMAꢀDHLA) and prodrug
2
nanoparticles with acidꢀliable linkages against HeLa cells was investigated. Before evaluating
the cytotoxicity of prodrug nanoparticles, it was necessary to study the cytotoxicity of the
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nanocarriers, because biocompatible nanocarriers could reduce the side effect in drug delivery.
The cells were incubated with P(HEO MA)ꢀbꢀP(HEMAꢀDHLA) at various concentrations in the
2
o
Dulbecco’s modified Eagle’s medium (DMEM) at 37 C and 5% CO for 24 hours, and the cells
2
cultured with free DMEM were used as control. As shown in Figure 5A, P(HEO MA)ꢀbꢀ
2
P(HEMAꢀDHLA) was almost nontoxic to HeLa cells even at a concentration of 500 ꢁg/mL,
4
7
which was in accordance with the wellꢀrecognized biocompatibility of PHEO MA. The
2
cytotoxicity of prodrug nanoparticles was compared with that of CPT, and the result was shown
in Figure 5B. Prodrug nanoparticles showed similar cytotoxicity to free CPT, and the IC50
(inhibitory concentration for 50% cell viability) of prodrug nanoparticles was 3.05 ꢁg/mL, which
was slightly higher than that of free CPT (IC = 2.01 ꢁg/mL). Moreover, in vitro experiment of
5
0
cell apoptosis was conducted to quantitatively detect and differentiate live, apoptotic and
necrotic cells. Figure 5C showed that cells treated with prodrug nanoparticles at CPT
concentration of 5 ꢁg/mL demonstrated 16.0% early apoptotic (AV+/PIꢀ) populations and 83.0%
necrotic/late apoptotic cells (AV+/PI+), which was comparable to free CPT (10.1% early
apoptotic populations and 89.6% necrotic/late apoptotic cells). The above results revealed that
prodrug nanoparticles exhibited anticancer activity via releasing covalently conjugated CPT
molecules in response to the acidic microenvironment of cancer cells.
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Figure 5. Relative cell viability values of HeLa cells evaluated by MTT assay after incubation
with different concentrations of samples: (A) the polymer scaffold P(HEO MA)ꢀbꢀP(HEMAꢀ
2
DHLA), (B) the prodrug nanoparticles (black line) compared with free CPT (red line); (C)
Annexin V/PI assay of free CPT and prodrug nanoparticles at the CPT concentration of 5 ꢁg/mL
after incubation for 48 h.
Cellular Uptake of the Prodrug Nanoparticles
To further investigate the pHꢀresponsive intracellular release of CPT molecules from prodrug
nanoparticles, confocal laser scanning microscope (CLSM) was used to study the internalization
of the prodrug nanoparticles by cancer cells. HeLa cells were incubated with free CPT or
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prodrug nanoparticles at CPT equivalent doses of 3.0 ꢁg/mL for 4 h, and then the cells were
stained with calcein, which could label survived cells with green fluorescence. CLSM images in
Figure 6A revealed that blue fluorescence of CPT mainly appeared at the perinuclear region of
HeLa cells, and green fluorescence of calcein appeared in the overall cells. This observation
indicated that prodrug nanoparticles were internalized by HeLa Cells. Moreover, HeLa cells
cultured with free CPT displayed relatively weaker blue fluorescence compared with HeLa cells
cultured with prodrug nanoparticles. The quantitative results by flow cytometry analysis (Figure
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B) further demonstrated that prodrug nanoparticles exhibited approximately 1.5ꢀfold cellular
internalization amount compared with free CPT. The probable reason is that the poor water
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solubility of free CPT makes it difficult for CPT to be endocytosed by cells.
Prodrug
nanoparticles overcame this obstacle successfully, and it was easier for prodrug nanoparticles to
realize cell endocytosis compared with free CPT, because small size of nanoparticle was
available for cellular uptake and PHEO MA in prodrug nanoparticles could improve the cells’
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absorption of nanopartilces.
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Figure 6. (A) Confocal laser microscopic images of HeLa cells incubated with fresh DMEM
medium containing free CPT (a) or prodrug nanoparticles (b) for 4 h at a CPT (blue) equivalent
dose of 3.0 ꢁg/mL. The cells were then counterstained with calcein (green); (B) Cellular uptake
by flow cytometry for free CPT and prodrug nanoparticles at a CPT equivalent dose of 3.0
ꢁ
g/mL.
Antitumor Efficacy
Therapeutic potentials of the prodrug nanoparticles were further evaluated using subcutaneous
tumor model of Male 7ꢀweekꢀold CDꢀ1 (ICR) mice. ICR mice bearing subcutaneous tumors (~
3
5
0 mm ) were treated with PBS, free CPT and prodrug nanoparticles at a dose of 5 mg
equivalent CPT per kilogram mouse, respectively. As shown in Figure 7, the tumor of ICR mice
3
treated with PBS continued to grow for 12 days, and the tumor volume increased from 50 mm to
3
3
26 mm . Prodrug nanoparticles showed better tumor growth suppression than CPT, and the
3
tumor volume of mice treated with prodrug nanoparticles was only 73 mm after 12 d. The high
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anticancer efficacy was presumably attributed to the synergistic effect of tumors’ (the enhanced
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permeability and retention) effect
and promoted cellular internalization of prodrug
nanoparticles. In contrast, CPT is difficult for iv administration because its water solubility is
poor. Such prodrug nanoparticles have potential applications in anticancer chemotherapy.
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Figure 7. Tumor growth curves of Male 7ꢀweekꢀold CDꢀ1 (ICR) mice after intravenous
injection of PBS, free CPT and prodrug nanoparticles. Data are expressed as means ± SEM, n =
3
. **P < 0.005, ***P < 0.0001.
Conclusions
In summary, a method for the synthesis of polymer prodrugs with high drugꢀloading
efficiency, acidꢀliable linkages and controllable drug loading capacity was proposed. ACPT was
efficiently coupled to the polymer scaffold P(HEO MA)ꢀbꢀP(HEMAꢀDHLA) via acrylateꢀthiol
2
Michael addition reaction, forming polymer prodrug P(HEO MA)ꢀbꢀP(HEMAꢀDHLAꢀCPT)
2
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with pHꢀresponsive βꢀthiopropionate linkage. The drug loading contents of polymer prodrugs
could be controlled via adjusting the feed molar ratio of [ACPT]/[DHLA] in the Michael
addition reaction. Then the polymer prodrug selfꢀassembled into nanoparticles in water with a
CPT loading content as high as 40.1%. Owing to the hydrolysis of βꢀthiopropionate linkage in
acidic solution, most of CPT was released from the prodrug nanoparticles at pH 5.0 in 4 days.
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Cell viability studies revealed that the polymeric carrier P(HEO MA)ꢀbꢀP(HEMAꢀDHLA) was
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of low cytotoxicity, whereas prodrug nanoparticles displayed similar cytotoxicity to free CPT,
showing high anticancer activity. Moreover, prodrug nanoparticles were more easily internalized
by HeLa cells compared with free CPT. In vivo anticancer experiments revealed that the prodrug
nanoparticles showed better tumor growth suppression than free CPT. Therefore, this study
provides an approach for fabrication of prodrug nanoparticles in anticancer chemotherapy.
Experimental Section
Materials.
Camptothecin (CPT, TCI, >97%), triethylamine (TEA, Aladdin, 99.5%), diethylene glycol
(
TCI, 99.5%), 2ꢀhydroxyethyl methacrylate (HEMA, TCI, >95%), lipoic acid (LA, Energy
Chemical, 98%), methacryloyl chloride (Energy Chemical, 97%), acryloyl chloride (Energy
Chemical, 98%), N,N’ꢀdicyclohexylcarbodiimide (DCC, Aladdin, 99.0%), 4ꢀ
dimethylaminopyridine (DMAP, Aladdin, 99%) and calcein (TCI) were used as received.
Azobisisobutyronitrile (sigma, 98%) was recrystallized from ethanol. Tetrahydrofuran (THF)
was refluxed over sodium for 24 hours and distilled prior to use. 4ꢀCyanopentanoic acid
42
dithiobenzoate (CPADB) was synthesized according to the previous report. All other reagents
with analytical grade were purchased from Shanghai Chemical Reagent Co and used as received.
Instrumentations.
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All NMR spectra were recorded on a Bruker AV300 NMR spectrometer using CDCl as
3
solvent. Mass spectrum analysis was performed by using a LCꢀMS instrument (Thermo
Scientific, LTQ Orbitrap XL), and the system was equipped with an ESI source. Molecular
weight and molecular weight distribution (M /M ) were determined on a Waters 150C gel
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w
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permeation chromatography (GPC) instrument equipped with microstyragel columns (500, 10
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and 10 Å) and a RI 2414 detector set at 30 C; monodispersed polystyrene standards were used
in the calibration of the numberꢀaverage molecular weight M and M /M , and DMF was used as
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an eluent at a flow rate of 1.0 mL/min. Ultraviolet (UV)/visible (Vis) light measurement was
performed on a Unico (United Products & Instruments, Inc., NJ, USA) UV/Vis 2802PCS
spectrophotometer. Transmission electron microscopy (TEM) observations were conducted on a
JEMꢀ100SX electron microscope at an acceleration voltage of 100 kV. Dynamic light scattering
(
DLS) measurements were carried out on a Dynapro light scattering instrument (model Dynaproꢀ
o
99E) at 25 C with an 824.3 nm laser, and the data were analyzed with DYNAMICS V6
software. Confocal laser scanning microscopy (CLSM) observations were conducted on a Leica
TCS SP5 microscope. Fourier transform infrared spectra were acquired on Vectorꢀ22 fourier
infrared spectrometer.
Synthesis of 2ꢀ(2ꢀhydroethoxy) ethyl methacrylate (HEO MA).
2
Diethylene glycol (1.06 g, 10 mmol) and TEA (1.51 g, 15 mmol) were dissolved in
o
chloroform (250 mL). The mixture was stirred at 0 C for 20 minutes, and then methacryloyl
chloride (1.04 g, 10 mmol) in 50 mL of chloroform was added dropwise. The reaction was kept
at room temperature for 12 hours. The reaction mixture was concentrated and the crude product
was purified by column chromatography using a mixed eluent of nꢀhexane/ethyl acetate (2:1,
1
v/v) to give the product (1.13 g, yield: 65.2%). H NMR (300 MHz, CDCl , δ, ppm): 6.10 (d, J =
3
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.43 Hz, 1H), 5.60 (d, J = 1.67 Hz, 1H), 4.14 (t, J = 3.57 Hz, 2H), 3.74 (t, J = 6.51 Hz, 4H), 3.57
13
(t, J = 5.54 Hz, 2H), 1.98 (s, 3H). C NMR (75 MHz, CDCl , ppm): δ 167.2, 136.1, 125.2, 70.0,
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69.4, 65.1, 61.3, 17.9.
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Synthesis of acrylateꢀtethering camptothecin (ACPT).
Camptothecin (2.0 g, 6.0 mmol) and TEA (1.01 g, 10.0 mmol) were dissolved in chloroform
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(350 mL). The mixture was stirred at 0 C for 20 minutes, and then acryloyl chloride (0.594 g,
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.6 mmol) in 20 mL of chloroform was added dropwise. The reaction was kept at room
temperature for 24 hours. The reaction mixture was concentrated and the crude product was
purified by column chromatography using a mixed eluent of nꢀhexane/ethyl acetate (1:1, v/v) to
1
give the product (1.6 g, yield: 80%). H NMR (300 MHz, CDCl , δ, ppm): 8.40 (m, 1H), 8.21
3
(m, 1H), 7.94 (m, 1H), 7.82 (m, 1H), 7.67 (m, 1H), 7.23 (m, 1H), 6.52 (d, J = 1.78 Hz, 1H) and
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.98 (d, J = 1.64 Hz, 1H), 6.27 (dd, 1H), 5.71 and 5.43 (m, 2H), 5.29 (m, 2H), 2.4~2.1 (m, 2H),
13
1.01(t, J = 2.96 Hz, 3H). C NMR (75 MHz, CDCl , ppm): δ 167.5, 166.5, 156.9, 152.7, 148.9,
3
1
6
45.8, 145.3, 131.9, 131.3, 130.4, 129.0, 128.4, 128.2, 127.9, 127.3, 126.6, 119.9, 97.5, 76.3,
+
5.4, 53.7, 31.9, 7.9. MS: [MꢀH ] = 403.12, found at 403.1286.
Synthesis of methacrylate of lipoic acid (HEMAꢀLA).
Hydroxyethyl methacrylate (1.31 g, 0.01 mol), DMAP (0.6 g, 0.005 mmol) and DCC (3.84 g,
0
.02 mol) were dissolved in dichloromethane (70 mL). Lipoic acid (2.06 g, 0.01 mol) in 10 mL
of dichloromethane was added dropwise into the mixture at room temperature, and the solution
was stirred for 12 hours. The reaction mixture was then concentrated and the crude product was
purified by column chromatography using a mixed eluent of nꢀhexane/ethyl acetate (4:1, v/v) to
give the product (2.23 g, yield: 70.1%). The monomer is not stable at room temperature, and it
o
1
should be stored at low temperature (ꢀ20 C). H NMR (300 MHz, CDCl , δ, ppm): 6.14 (d, J =
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.56 Hz, 1H), 5.58 (t, J = 1.72 Hz, 1H), 4.32 (s, 4H), 3.54 (m, 1H), 3.12 (m, 2H), 2.53 (m, 1H),
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3
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.33 (t, J = 7.40 Hz, 2H), 1.9~1.8 (m, 4H), 1.7~1.2 (m, 6H). C NMR (75 MHz, CDCl , ppm): δ
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173.1, 167.2, 136.0, 125.8, 62.2, 62.0, 40.2, 38.5, 34.6, 33.9, 29.0, 28.0, 17.9.
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Synthesis of Poly(2ꢀ(2ꢀhydroethoxy) ethyl methacrylate) (PHEO MA).
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HEO MA (2.61 g, 0.015 mol), AIBN (5 mg, 0.03 mmol), CPADB (84 mg, 0.3 mmol) and
2
THF (6 mL) were added into a 10 mL tube. After three freezeꢀvacuumꢀthaw cycles, the
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polymerization tube was sealed under vacuum. The polymerization was carried out at 70 C for 6
h. The polymerization tube was cooled to room temperature, and then the reaction mixture was
added dropwise into excess diethyl ether to obtain a precipitate. The precipitation process was
repeated for three times with an isolated yield of 87.1%. The polymer was dried in a vacuum
oven for 24 hours.
Synthesis of P(HEO MA)ꢀbꢀP(HEMAꢀLA).
2
PHEO MA (M = 7700 g/mol, 770 mg, 0.1 mmol), HEMAꢀLA (1.272 g, 4.0 mmol), AIBN
2
n
(1.64 mg, 0.01 mmol) and THF (8 mL) was added into a 10 mL tube. After three freezeꢀvacuumꢀ
thaw cycles, the polymerization tube was sealed under vacuum. The polymerization was carried
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out at 70 C for 6 h. The polymerization tube was cooled to room temperature, and then the
polymer was obtained by precipitation into excess diethyl ether. The precipitation process was
repeated for three times with an isolated yield of 30.9%. The obtained solid was dried in a
vacuum oven at room temperature for 24 hours.
Synthesis of P(HEO MA)ꢀbꢀP(HEMAꢀDHLA).
2
P(HEO MA) ꢀbꢀP(HEMAꢀLA) (M = 11500 g/mol, 115 mg, 0.01 mmol) was added into
2
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n
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THF (10 mL). To this solution, NaBH (12 mg, 0.32 mmol) was added at 0 C, then the reaction
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mixture was kept for 2 hours at room temperature. The final solution was dialyzed against
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deionized water for 24 hours, and it should be kept in the dark place. The product was obtained
as a white solid by lyophilization with an isolated yield of 87.0%.
Synthesis of Polymer Prodrug P(HEO MA)ꢀbꢀP(HEMAꢀDHLAꢀCPT).
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n
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8 ꢁmol), THF (20 mL) and a drop of TEA were added into a 50 mL flask. The reaction was
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kept at 50 C for 24 hours. After the reaction mixture was cooled to room temperature, the
solution was added dropwise into an excess of diethyl ether to obtain the polymer. The
precipitation process was repeated for three times. The precipitate was dried at room temperature
in a vacuum oven for 24 hours with an isolated yield of 87.6%.
Fabrication of Prodrug Nanoparticles.
Polymer prodrug P(HEO MA)ꢀbꢀP(HEMAꢀDHLAꢀCPT) (12 mg) was dissolved in DMSO
2
(1.0 mL) and stirred vigorously at room temperature for 1 hour. Deionized water (7.0 mL) was
added into the solution through the syringe pump at a rate of 3.0 mL/h while stirring. After
stirring for 5 h, the solution was loaded into a dialysis bag (MWCO 3500) and dialyzed against
deionized water for 12 h, forming prodrug nanoparticles at a concentration of 1.5 mg/mL. The
drug loading content (DLC) of the nanoparticles was calculated by UV quantitative analysis. In
ꢀ1
UV analysis, the UV absorbance at 365 cm of a series of CPT solutions (0.005, 0.1, 0.05, 0.1,
0
.25 mg/mL) in DMSO was measured. Figure S6 showed the plot of UV absorbance versus
concentration of CPT in DMSO. Polymer prodrug (1.0 mg) was dissolved in DMSO (1.0 mL)
and the concentration of CPT was obtained according to the standard curve. The drug loading
content was obtained according to the followed formula:
DLC(UV) = (weight of loaded CPT/weight of polymer prodrug) × 100%
Critical micelle concentration (CMC) measurement.
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Pyrene was used as a fluorescence probe to determine the critical micelle concentration of
prodrug nanoparticles. The aqueous solution of prodrug nanoparticles at a concentration of 1.5
ꢀ5
mg/mL was diluted to various concentrations from 0.75 mg/mL to 8.125 × 10 mg/mL. Then 50
ꢀ5
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ꢁ
L acetone solution of pyrene at a concentration of 6 × 10 M was added into the solution of
prodrug nanoparticles at various concentrations. The excitation spectra of all samples were
recorded. The emission wavelength was fixed at 374 nm while the excitation wavelength was
fixed in the range of 300ꢀ370 nm. The I /I values of all solutions were recorded, and CMC
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39 332
values were shown in Figure 3B.
Release of CPT from Prodrug Nanoparticles.
The drug release experiments were performed in PBS buffer at pH 5.0, 6.0 and 7.4,
respectively. The specific experimental procedure was as follows: an aqueous dispersion of CPTꢀ
loaded prodrug nanoparticles (2.0 mg/mL, 2.0 mL) was transferred to a dialysis bag with a
membrane molecular weight cutꢀoff of 3.5 KDa, and then the dialysis bag was immersed in 60
mL of PBS buffer solution at pH 5.0 or 6.0 or 7.4. At predetermined times, 2 mL of sample from
the solution out of the dialysis bag was taken for estimating the amount of drug released, which
was replaced with 2 mL of PBS buffer with identical pH value. UV absorbance of the dialysis
ꢀ1
solution at 365 cm was monitored to determine the CPT releasing profile. This process could
last for 4 days. Each measurement was done in triplicate, and the data was shown as the mean
value plus a standard deviation (± SD).
Cytotoxicity Evaluation.
The cytotoxicity of CPT, P(HEO MA)ꢀbꢀP(HEMAꢀDHLA) and prodrug nanoparticles was
2
evaluated by MTT assays. HeLa cells were cultured in a 96ꢀwell plate at a density of 5000 cells
per well overnight using DMEM supplemented with 10% fetal bovine serum (FBS). The
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solutions of free CPT, P(HEO MA)ꢀbꢀP(HEMAꢀDHLA) and prodrug nanoparticles in PBS
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buffer (50 mM, pH 7.4) were diluted into samples with various concentrations. The cells were
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then incubated with samples at various concentrations for 24 hours at 37 C and 5% CO . Cells
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without treatment were used as control. The medium was removed and replaced with fresh
DMEM media containing 0.5 mg/mL MTT reagent. After 4 hours, the cultured medium was
removed. DMSO (150 ꢁL) was then added, and absorbance of the solution in each well was
detected at 490 nm by a microplate reader.
For further analysis of cell apoptosis, Annexin V/PI assay was carried out. HeLa cells were
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cultured in a 24ꢀwell plate at a density of 10 cells per well overnight using DMEM with 10%
FBS. Then the DMEM was removed, followed by addition of fresh DMEM containing CPT or
prodrug nanoparticles at a CPT concentration of 5 ꢁg/mL. After the cells were cultured for 4 h,
the medium was replaced with DMEM with 10% FBS. The incubation was continued for 48 h.
Then, Annexin V/PI staining for apoptotic cells was performed according to manufacturer’s
protocols, and quantitative apoptotic measurements were performed by flow cytometric analysis.
Cellular Uptake of the Prodrug Nanoparticles.
HeLa cells were cultured in glassꢀbottom petri dishes at a density of 8000 cells per well. They
o
were incubated for 24 h at 37 C and 5% CO before treatment. Fresh DMEM medium (100 ꢁL)
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containing CPT or prodrug nanoparticles at a CPT equivalent dose of 3.0 ꢁg/mL was added into
each well. After cells were incubated for 4 h, the cultured medium was removed. The cells were
rinsed two times with PBS solution, and the cells were stained with calcein (green stain) for 15
minutes. Then the cells were rinsed with PBS solution to remove redundant calcein. After the
above treatment, the cells were observed by confocal laser scanning microscope (CLSM)
immediately. The CPT was excited at 405 nm and the emitted light was detected between 400
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and 500 nm. The calcein was excited at 495 nm and the emitted light was detected between 500
and 600 nm. For flow cytometric analysis, the cells were washed for three times with PBS
solution, detached by trypsin treatment, and suspended in PBS for flow cytometry
measurements.
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In Vivo Antitumor Efficacy of Free CPT and Prodrug Nanoparticles.
The tumor models were established at the right flank of Male 7ꢀweekꢀold CDꢀ1 (ICR) mice by
7
inoculation with 10 4T1 cells suspended in 100 ꢁL PBS. To exhibit the antitumor efficacy of
3
prodrug nanoparticles, ICR mice bearing subcutaneous tumors (~ 50 mm ) were treated with
PBS, free CPT and prodrug nanoparticles, respectively, at a dose of 5 mg equivalent CPT per
kilogram mouse. Tumor size (V) was measured every two days, and tumor size was calculated
2
from the equation V = a × b /2 (a and b are the longest and shortest diameters of tumor,
respectively.). After day 12, mice were sacrificed and tumors were excised according to standard
protocol.
Supporting Information.
GPC data for the obtained polymers, FTIR spectrum of P(HEO MA)ꢀbꢀP(HEMAꢀDHLA), DLS
2
curves of prodrug nanoparticles, photographs of the representative tumors and standard curves of
CPT in DMSO are available in Figure S1ꢀ6.
*
Corresponding Author
Eꢀmail: hongcy@ustc.edu.cn, haozongyao@163.com.
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Acknowledgments
The authors gratefully acknowledge the financial support from the National Natural Science
Foundation of China under contrast Nos. 21525420 and 21774113.
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Notes
The authors declare no competing financial interest.
References
(1) Park, J. H., Lee, S., Kim, J. H., Park, K., Kim, K., Kwon, I. C. (2008) Polymeric
nanomedicine for cancer therapy. Prog. Polym. Sci. 33, 113ꢀ137.
(2) Fox, M. E., Szoka, F. C., Frechet, J. M. J. (2009) Soluble polymer carriers for the treatment
of cancer: The importance of molecular architecture. Acc. Chem. Res. 42, 1141ꢀ1151.
(3) Riehemann, K., Schneider, S. W., Luger, T. A., Godin, B., Ferrari, M., Fuchs, H. (2009)
Nanomedicineꢀchallenge and perspectives. Angew. Chem. Int. Ed. 48, 872ꢀ897.
(4) Linderoth, L., Fristrup, P., Hansen, M., Melander, F., Madsen, R., Andresen, T. L., Peters, G.
H. (2009) Mechanistic study of the sPLA ꢀmediated hydrolysis of a thioꢀester pro anticancer
2
ether lipid. J. Am. Chem. Soc. 131, 12193ꢀ12200.
(5) Fouladi, F., Steffen, K. J., Mallik, S. Enzymeꢀresponsive liposomes for the delivery of
anticancer drugs. (2017) Bioconjug. Chem. 28, 857ꢀ868.
(6) Geng, Y., Discher, D. E. (2005) Hydrolytic degradation of poly(ethylene oxide)ꢀblockꢀ
polycaprolactone worm micelles. J. Am. Chem. Soc. 127, 12780ꢀ12781.
26
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1
2
3
4
5
6
7
8
9
(
7) Soussan, E., Cassel, S., Blanzat, M., RicoꢀLattes, I. (2009) Drug delivery by soft matter:
matrix and vesicular carriers. Angew. Chem. Int. Ed. 48, 274ꢀ288.
(8) Wang, W., Ma, X., Yu, X. (2017) pHꢀresponsive polymersome based on PMCPꢀbꢀPDPA as a
1
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1
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0
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0
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7
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9
0
1
2
3
4
5
6
7
8
9
0
drug delivery system to enhance cellular internalization and intracellular drug release. Chin. J.
Polym. Sci. 35, 1352ꢀ1362.
(9) Sun, Y., Du, X. Q., He, J. L., Hu, J., Zhang, M. Z., Ni, P. H. (2017) Dualꢀresponsive coreꢀ
crosslinked polyphosphoesterꢀbased nanoparticles for pH/redoxꢀtriggered anticancer drug
delivery. J. Mater. Chem. B 5, 3771ꢀ3782.
(10) Lu, L., Zou, Y., Yang, W. J., Meng, F. H., Deng, C., Cheng, R., Zhong, Z. Y. (2015)
Anisamideꢀdecorated pHꢀsensitive degradable chimaeric polymersomes mediate potent and
targeted protein delivery to lung cancer cells. Biomacromolecules 16, 1726ꢀ1735.
(11) Wang, L. H., Hong, C. Y. (2017) Synthesis and selfꢀassembly of responsive branched
polymers and their biomedical applications. Acta. Polym. Sin. 2, 200ꢀ213.
(12) Prencipe, G., Tabakman, S. M., Welsher, K., Liu, Z., Goodwin, A. P., Zhang, L., Henry, J.,
Dai, H. (2009) PEG branched polymer for functionalization of nanomaterials with ultralong
blood circulation. J. Am. Chem. Soc. 131, 4783ꢀ4787.
(13) Ali, M. R. K., Wu, Y., Han, T., Zang, X., Xiao, H., Tang, Y., Wu, R., Fernandez, F. M., Elꢀ
Sayed, M. A. (2016) Simultaneous timeꢀdependent surfaceꢀenhanced raman spectroscopy,
metabolomics, and proteomics reveal cancer cell death mechanisms associated with gold
nanorod photothermal therapy. J. Am. Chem. Soc. 138, 15434ꢀ15442.
27
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1
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9
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1
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5
6
(
14) Kamaly, N., Xiao, Z., Valencia, P. M., RadovicꢀMoreno, A. F., Farokhzad, O. C. (2012)
Targeted polymeric therapeutic nanoparticles: design, development and clinical translation.
Chem. Soc. Rev. 41, 2971ꢀ3010.
0
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2
3
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9
0
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(15) Nicolas, J., Mura, S., Brambilla, D., Mackiewicz, N., Couvreur, P. (2013) Design,
functionalization strategies and biomedical applications of targeted biodegradable/biocompatible
polymerꢀbased nanocarriers for drug delivery. Chem. Soc. Rev. 42, 1147ꢀ1235.
(16) Albert, A. (1958) Chemical aspects of selective toxicity. Nature 182, 421ꢀ423.
(17) Lee, S., Saito, K., Lee, H.ꢀR., Lee, M. J., Shibasaki, Y., Oishi, Y., Kim, B.ꢀS. (2012)
Hyperbranched double hydrophilic block copolymer micelles of poly(ethylene oxide) and
polyglycerol for pHꢀresponsive drug delivery. Biomacromolecules 13, 1190ꢀ1196.
(18) Wang, H., Tang, L., Tu, C., Song, Z., Yin, Q., Yin, L., Zhang, Z., Cheng, J. (2013) Redoxꢀ
responsive, coreꢀcrossꢀlinked micelles capable of onꢀdemand, concurrent drug release and
structure disassembly. Biomacromolecules 14, 3706ꢀ3712.
(
19) Bao, Y., Boissenot, T., Guegain, E., Desmaele, D., Mura, S., Couvreur, P., Nicolas, J.
2016) Simple synthesis of cladribineꢀbased anticancer polymer prodrug nanoparticles with
(
tunable drug delivery properties. Chem. Mater. 28, 6266ꢀ6275.
(20) Wang, Y., Huang, P., Hu, M., Huang, W., Zhu, X., Yan, D. (2016) Selfꢀdelivery
nanoparticles of amphiphilic methotrexateꢀgemcitabine prodrug for synergistic combination
chemotherapy via effect of deoxyribonucleotide pools. Bioconjug. Chem. 27, 2722ꢀ2733.
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7
8
9
(
21) Maksimenko, A., Bui, D. T., Desmaele, D., Couvreur, P., Nicolas, J. (2014) Significant
tumor growth inhibition from naturally occurring lipidꢀcontaining polymer prodrug nanoparticles
obtained by the drugꢀinitiated method. Chem. Mater. 26, 3606ꢀ3609.
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0
(22) Sun, C. Y., Dou, S., Du, J. Z., Yang, X. Z., Li, Y. P., Wang, J. (2014) Doxorubicin
conjugate of poly(ethylene glycol)ꢀblockꢀpolyphosphoester for cancer therapy. Adv. Healthc.
Mater. 3, 261ꢀ272.
(23) Delplace, V., Couvreur, P., Nicolas, J. (2014) Recent trends in the design of anticancer
polymer prodrug nanocarriers. Polym. Chem. 5, 1529ꢀ1544.
(24) Scarano, W., Duong, H. T., Lu, H., De Souza, P. L., Stenzel, M. H. (2013) Folate
conjugation to polymeric micelles via boronic acid ester to deliver platinum drugs to ovarian
cancer cell lines. Biomacromolecules 14, 962ꢀ975.
(25) Shen, Y. Q., Jin, E. L., Zhang, B., Murphy, C. J., Sui, M. H., Zhao, J., Wang, J. Q., Tang, J.
B., Fan, M. H., Van Kirk, E., et al. (2010) Prodrugs forming high drug loading multifunctional
nanocapsules for intracellular cancer drug delivery. J. Am. Chem. Soc. 132, 4259ꢀ4265.
(26) Louage, B., Nuhn, L., Risseeuw, M. D., Vanparijs, N., De Coen, R., Karalic, I., Van
Calenbergh, S., De Geest, B. G. (2016) Wellꢀdefined polymerꢀpaclitaxel prodrugs by a graftingꢀ
fromꢀdrug approach. Angew. Chem. Int. Ed. 55, 11791ꢀ11796.
(27) Dasari, M., Acharya, A. P., Kim, D., Lee, S., Lee, S., Rhea, J., Molinaro, R., Murthy, N.
(2013) HꢀGemcitabine: A new gemcitabine prodrug for treating cancer. Bioconjug. Chem. 24, 4ꢀ
8.
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