Fabrication of Acidic pH-Cleavable Polymer for Anticancer Drug Delivery Using a Dual Functional Monomer

Article abstract of DOI:10.1021/acs.biomac.8b01001

The preparation of tumor acidic pH-cleavable polymers generally requires tedious postpolymerization modifications, leading to batch-to-batch variation and scale-up complexity. To develop a facile and universal strategy, we reported in this study design and successful synthesis of a dual functional monomer, a-OEGMA that bridges a methacrylate structure and oligo(ethylene glycol) (OEG) units via an acidic pH-cleavable acetal link. Therefore, a-OEGMA integrates (i) the merits of commercially available oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA) monomer, i.e., hydrophilicity for extracellular stabilization of particulates and a polymerizable methacrylate for adopting controlled living radical polymerization (CLRP), and (ii) an acidic pH-cleavable acetal link for efficiently intracellular destabilization of polymeric carriers. To demonstrate the advantages of a-OEGMA (M_n = 500 g/mol) relative to the commercially available OEGMA (M_n = 300 g/mol) for drug delivery applications, we prepared both acidic pH-cleavable poly(?-caprolactone)₂₁-b-poly(a-OEGMA)₁₁ (PCL₂₁-b-P(a-OEGMA)₁₁) and pH-insensitive analogues of PCL₂₁-b-P(OEGMA)₁₈ with an almost identical molecular weight (MW) of approximately 5.0 kDa for the hydrophilic blocks by a combination of ring-opening polymerization (ROP) of ?-CL and subsequent atom transfer radical polymerization (ATRP) of a-OEGMA or OEGMA. The pH-responsive micelles self-assembled from PCL₂₁-b-P(a-OEGMA)₁₁ showed sufficient salt stability, but efficient acidic pH-triggered aggregation that was confirmed by the DLS and TEM measurements as well as further characterizations of the products after degradation. In vitro drug release study revealed significantly promoted drug release at pH 5.0 relative to the release profile recorded at pH 7.4 due to the loss of colloidal stability and formation of micelle aggregates. The delivery efficacy evaluated by flow cytometry analyses and an in vitro cytotoxicity study in A549 cells further corroborated greater cellular uptake and cytotoxicity of Dox-loaded pH-sensitive micelles of PCL₂₁-b-P(a-OEGMA)₁₁ relative to the pH-insensitive analogues of PCL₂₁-b-P(OEGMA)₁₈. This study therefore presents a facile and robust means toward tumor acidic pH-responsive polymers as well as provides one solution to the trade-off between extracellular stability and intracellular high therapeutic efficacy of drug delivery systems using a novel monomer of a-OEGMA with dual functionalities.

Full text of DOI:10.1021/acs.biomac.8b01001

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Article
Fabrication of Acidic pH-Cleavable Polymer for Anti-
Cancer Drug Delivery Using a Dual Functional Monomer
Luping Zheng, Xiaolong Zhang, Yunfei Wang, Fangjun Liu, Jinlei Peng, xuezhi
zhao, Huiru Yang, Liwei Ma, Baoyang Wang, Cong Chang, and Hua Wei
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01001 • Publication Date (Web): 14 Aug 2018
Downloaded from http://pubs.acs.org on August 14, 2018
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Fabrication of Acidic pH-Cleavable Polymer for Anticancer
Drug Delivery Using a Dual Functional Monomer
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Luping Zheng,†,# Xiaolong Zhang,†,# Yunfei Wang,† Fangjun Liu,† Jinlei Peng,† Xuezhi Zhao,†
Huiru Yang,† Liwei Ma,† Baoyan Wang,† Cong Chang*,‡, and Hua Wei*,†
† State Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous Metal
Chemistry and Resources Utilization of Gansu Province, and College of Chemistry and
Chemical Engineering, Lanzhou University, Lanzhou, Gansu 730000, China
‡ Department of Pharmaceutics, School of Pharmacy, Hubei University of Chinese Medicine,
Wuhan, Hubei 430065, China
# These authors contributed equally to this paper.
*Corresponding authors
E-mail addresses: weih@lzu.edu.cn (H. Wei) or 1126@hbtcm.edu.cn (C. Chang)
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Abstract: The preparation of tumor acidic pHꢀcleavable polymers generally requires tedious
postꢀpolymerization modifications, leading to batchꢀtoꢀbatch variation and scaleꢀup complexity.
To develop a facile and universal strategy, we reported in this study design and successful
synthesis of a dual functional monomer, aꢀOEGMA that bridges a methacrylate structure and
oligo(ethylene glycol) (OEG) units via an acidic pHꢀcleavable acetal link. Therefore aꢀOEGMA
integrates (i) the merits of commercially available oligo(ethylene glycol) monomethyl ether
methacrylate (OEGMA) monomer, i.e., hydrophilicity for extracellular stabilization of
particulates and a polymerizable methacrylate for adopting controlled living radical
polymerization (CLRP), and (ii) an acidic pHꢀcleavable acetal link for efficiently intracellular
destabilization of polymeric carries. To demonstrate the advantages of aꢀOEGMA (Mn = 500
g/mol) relative to the commercially available OEGMA (Mn = 300 g/mol) for drug delivery
applications, we prepared both acidic pHꢀcleavable poly(εꢀcaprolactone)21ꢀbꢀpoly(aꢀOEGMA)11
(PCL21ꢀbꢀP(aꢀOEGMA)11) and pHꢀinsensitive analogues of PCL21ꢀbꢀP(OEGMA)18 with an
almost identical molecular weight (MW) of approximately 5.0 kDa for the hydrophilic blocks by
a combination of ringꢀopening polymerization (ROP) of εꢀCL and subsequent atom transfer
radical polymerization (ATRP) of aꢀOEGMA or OEGMA. The pHꢀresponsive micelles selfꢀ
assembled from PCL21ꢀbꢀP(aꢀOEGMA)11 showed sufficient salt stability, but efficient acidic pHꢀ
triggered aggregation that was confirmed by the DLS and TEM measurements as well as further
characterizations of the products after degradation. In vitro drug release study revealed
significantly promoted drug release at pH 5.0 relative to the release profile recorded at pH 7.4
due to the loss of colloidal stability and formation of micelle aggregates. The delivery efficacy
evaluated by flow cytometry analyses and an in vitro cytotoxicity study in A549 cells further
corroborated greater cellular uptake and cytotoxicity of Doxꢀloaded pHꢀsensitive micelles of
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PCL21ꢀbꢀP(aꢀOEGMA)11 relative to the pHꢀinsensitive analogues of PCL21ꢀbꢀP(OEGMA)18. This
study therefore presents a facile and robust means toward tumor acidic pHꢀresponsive polymers
as well as provides a one solution to the tradeoff between extracellular stability and intracellular
high therapeutic efficacy of drug delivery systems using a novel monomer of aꢀOEGMA with
dual functionalities.
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Introduction
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In the past several decades, stimuliꢀresponsive polymers capable of responding to various bioꢀ
relevant triggers including pH, temperature, and redox potential for structural change or
dissociation toward enhanced intracellular therapeutic efficacy have drawn great attention.1ꢀ10
More recently, the development of lightꢀsensitive polymers11ꢀ13 and nanodiamond
(ND)@polymer hybrid systems14ꢀ17 have emerged as innovative platforms toward improved
efficacy and safety of cancer nanomedicine applications.
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Due to the notable pH gradients between the normal and tumor tissues as well as in different
cellular compartments, that is, pH 5.5–6.0 for endosomes and pH 4.5–5.0 for lysosomes, while
the extracellular pH of tumor microenvironment is pH 6.8, which is slightly lower than the
physiological pH 7.4,18ꢀ20 various pHꢀsensitive polymeric carriers containing acidic pHꢀcleavable
links, such as acetal,21ꢀ23 hydrazone,24ꢀ26 orthoester,27,28 and carbamate29 have been successfully
designed and synthesized to realize efficiently intracellular deformation of carriers and
subsequently promoted release of loaded cargoes.
Although tremendous progresses have been made in this field of research, the preparation of
tumor acidic pHꢀcleavable polymers generally requires tedious postꢀpolymerization
modifications, leading to batchꢀtoꢀbatch variation and scaleꢀup complexity.30,31 Specifically, it
remains a synthetic challenge to develop polymers containing multiple acidꢀcleavable links with
readily and precisely modulated polymer composition and functionalities. For this purpose, we
reported in this study a facile and universal strategy by designing and successfully synthesizing a
dual functional monomer, aꢀOEGMA that bridges a methacrylate structure and oligo(ethylene
glycol) (OEG) units via an acidic pHꢀcleavable acetal link (Scheme 1). Therefore the dual
functions of aꢀOEGMA lies in (i) the merits of commercially available oligo(ethylene glycol)
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monomethyl ether methacrylate (OEGMA) monomer, i.e., hydrophilicity for extracellular
stabilization of particulates and a polymerizable methacrylate for adopting controlled living
radical polymerization (CLRP), and (ii) an acidic pHꢀcleavable acetal link30,31 for efficiently
intracellular destabilization of polymeric carries.
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To demonstrate the advantages of aꢀOEGMA (Mn = 500 g/mol) relative to the commercially
available OEGMA (Mn = 300 g/mol) for drug delivery applications, we prepared both acidic pHꢀ
cleavable poly(εꢀcaprolactone)21ꢀbꢀpoly(aꢀOEGMA)11 (PCL21ꢀbꢀP(aꢀOEGMA)11) and pHꢀ
insensitive analogues of PCL21ꢀbꢀP(OEGMA)18 with an almost identical molecular weight (MW)
of approximately 5.0 kDa for the hydrophilic blocks, which was shown in our previous study32 to
provide sufficient stability for the selfꢀassembled micelles, by a combination of ringꢀopening
polymerization (ROP) of εꢀCL and subsequent atom transfer radical polymerization (ATRP) of
aꢀOEGMA or OEGMA. The delivery efficacy of acidic pHꢀcleavable micelles was evaluated by
an in vitro drug release study, confocal microscopy measurements, flow cytometry analyses as
well as an in vitro cytotoxicity study, and compared with the pHꢀinsensitive analogues.
Scheme 1. Schematic illustration of structure and merits of aꢀOEGMA monomer developed in
this study.
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Experimental Section
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Materials, polymer synthesis and characterizations, in vitro drug loading and drug release study,
and in vitro biological assays are provided in the supporting information (SI).
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Synthesis of 2-(vinyloxy)ethyl methacrylate (VEMA)
VEMA was prepared from 2ꢀ(vinyloxy)ethanol and methacryloyl chloride in the presence of
triethylamine (TEA).33 Briefly, 2ꢀ(vinyloxy)ethanol (6.281 mL, 70 mmol), TEA (9.730 mL, 70
mmol) was dissolved in 100 mL of anhydrous DCM. A solution of methacryloyl chloride (6.159
mL, 63.64 mmol) in anhydrous DCM was added dropwise to the above mixture at 0oC under N2
flow with stirring for a period of 30 min. Then the reaction mixture was further stirred at room
temperature for 4 h. After reaction, a white byproduct TEA.HCl was filtrated out. The crude
product was concentrated and purified by column chromatography with eluents of ethyl
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acetate/hexane (1/30, v/v) to obtain 8.30 g of colorless liquid (yield, 84%). H NMR (400 MHz,
CDCl3, ppm, Figure S1): δ 6.51 (dd, J = 14.4, 6.8 Hz, 1H), 6.15 (s, 1H), 5.59 (s, 1H), 4.40 –
4.37 (m, 2H), 4.24 (dd, J = 14.0, 2.0 Hz, 1H), 4.06 (dd, J = 6.8, 2.4 Hz, 1H), 3.95 – 3.93 (m, 2H),
1.95 (s, 3H).
Synthesis of a-OEGMA (Scheme 2)
aꢀOEGMA was prepared from VEMA and mPEG (Mn= 350 g/mol) in the presence of a small
catalytic amount of PTS according to the reported procedures.34 Briefly, VEMA (4.685 g, 30
mmol), mPEG (10.50 g, 30 mmol), PTS (114.13 mg, 0.6 mmol) was dissolved in 60 mL of
anhydrous DCM at room temperature under N2 flow with stirring for a period of 30 min. The
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reaction mixture was later stirred at 35oC for 3 days. After reaction, the reaction mixture was
concentrated, and further purified by column chromatography with eluents of ethyl
acetate/hexane (1/2, v/v) to obtain 11.08 g of colorless liquid (yield, 73%).
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Scheme 2. Synthesis of a dual functional monomer, aꢀOEGMA.
Results and Discussion
Synthesis of PCL-b-P(a-OEGMA)
The vinyloxy group of VEMA, which acts as an “anchor” site, could be functionalized by
alcohols, 35 polyols,36 and triazoles37 to generate various functional methacryloyloxy acetals as
promising monomers, comonomers, crossꢀlinking agents, building blocks, and starting materials
for the synthesis of biologically active substances, therefore VEMA was used as a precursor for
aꢀOEGMA monomer in this study.
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Successful synthesis of aꢀOEGMA monomer is confirmed by H NMR analysis (Figure 1),
which reveals the presence of all the characteristic signals of each proton including peak a and b
at 6.12 and 5.57 ppm attributed to the vinyl protons of VEMA, peak d at 3.35 ppm assigned to
PEG units, and peak c and f at 4.84ꢀ4.79, and 1.34ꢀ1.31 ppm attributable to the protons of acetal
methine and adjacent methyl. More importantly, the ratio of the integrated intensity of peak g to f
was calculated to be 2/3, strongly supporting equivalent coupling of VEMA and mPEG. Further
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characterizations of aꢀOEGMA also confirm its successful synthesis including the appearance of
a characteristic signal at 99.8 ppm attributed to the acetal link in 13C NMR spectrum (Figure S2),
the presence of all the characteristic absorbance bands in the FTꢀIR spectrum (Figure S3), and
the distribution of molecular ion peaks recorded in the high resolution mass spectrum (HRMS)
(Figure S4).
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Figure 1. 1H NMR spectrum of aꢀOEGMA in CDCl3.
To investigate the utility of this new monomer for polymer construction, aꢀOEGMA was next
used to prepare target acidic pHꢀcleavable amphiphilic diblock copolymer, PCLꢀbꢀP(aꢀOEGMA)
by ATRP using ROPꢀgenerated PCLꢀiBuBr as a macroinitiator (Scheme 3). The degree of
polymerization (DP) of PCL was first determined to be 21 by 1H NMR analysis (Figure S5).
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Scheme 3. Synthesis of acidic pHꢀcleavable diblock copolymer, PCLꢀbꢀP(aꢀOEGMA) by
integrated ROP and ATRP.
To evaluate the polymerization properties of aꢀOEGMA by ATRP, a series of polymers was
synthesized by varying polymerization time. The molecular parameters of the resulting PCLꢀbꢀ
P(aꢀOEGMA)s were summarized in Table 1. Taking PCL21ꢀbꢀP(aꢀOEGMA)11 (Run 3 in Table
1) as an example, the DP of aꢀOEGMA was determined to be 11 by comparing the ratio of
integrated intensity of peak f attributed to acetal methine to that of peak d assigned to PCL units
(Figure 2a). All the three polymers show unimodal and symmetrical SEC elution peaks as well
as a clear shift of the SEC elution traces toward higher molecular weight with increasing
polymerization time (Figure 2b), which demonstrates wellꢀcontrolled chain extension of aꢀ
OEGMA by ATRP. The precisely controlled polymerizations of aꢀOEGMA by CLRP technique
are reflected by the pseudoꢀfirstꢀorder kinetics (Figure 2c) and narrow PDI (< 1.3) (Table 1)
during the polymerization process, which offers a robust route toward acidic pHꢀcleavable
hydrophilic building block.
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Table 1. Summary of a series of PCLꢀbꢀP(aꢀOEGMA) polymers prepared at different
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polymerization time using PCL21 as a macroꢀinitiator.
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Mn (kDa)
PDIc
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4.31
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a Polymerization conditions, [aꢀOEGMA]0: [PCL]0: [bpy]0: [CuBr]0= 20: 1: 2: 1, [OEGMA]0=
0.5 mol/L in anisole for run 1, 2 and 3. [OEGMA]0: [PCL]0: [bpy]0: [CuBr]0= 30: 1: 2: 1,
[OEGMA]0= 0.5 mol/L in anisole for run 4
b Determined by 1H NMR analysis
c Determined by SECꢀMALLS
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Figure 2. (a) 1H NMR spectrum of PCL21ꢀbꢀP(aꢀOEGMA)11 in CDCl3. (b) SEC elution traces of
PCL21ꢀiBuBr and PCLꢀbꢀP(aꢀOEGMA) prepared at different polymerization time (eluent: DMF)
(c) ATRP pseudoꢀfirstꢀorder kinetics plot of PCLꢀbꢀP(aꢀOEGMA).
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To demonstrate the advantages of aꢀOEGMA relative to the commercially available OEGMA
for drug delivery applications, PCL21ꢀbꢀP(OEGMA)18 diblock copolymer (Figure S6 and 3, Run
4 in Table 1) with an almost identical MW for the hydrophilic block was also prepared as a pHꢀ
insensitive analogue using the commercially available OEGMA monomer (Mn= 300 g/mol and
4~5 pendent EO units).
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PCL21
PCL21-b-P(a-OEGMA)11
PCL21-b-P(OEGMA)18
Figure 3. SEC elution traces of PCL, and PCL21ꢀbꢀP(aꢀOEGMA)11 and PCL21ꢀbꢀP(OEGMA)18
using DMF as an eluent.
Characterization of PCL21-b-P(a-OEGMA)11 micelles
The CMC of PCL21ꢀbꢀP(aꢀOEGMA)11 was investigated using pyrene as a fluorescence probe.
The fluorescence intensity keeps relatively constant at low concentrations, but increases
dramatically at a certain concentration, indicating the formation of micelles. Such concentration
was determined to be 9.33 mg/L for PCL21ꢀbꢀP(aꢀOEGMA)11 (Figure S7) and 7.98 mg/L for
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PCL21ꢀbꢀP(OEGMA)18 (Figure S8). The slightly lower CMC value of PCLꢀbꢀP(OEGMA)18
relative to that of PCLꢀbꢀP(aꢀOEGMA)11 implies the greater thermodynamic stability38 of pHꢀ
insensitive micelles over pHꢀsensitive ones, which results likely from the different packing
behaviors38ꢀ40 caused by the hydrophilic POEGMA moiety with different DPs and brushꢀlike
structures, i.e., P(aꢀOEGMA)11 with long OEG brush and short DP vs P(OEGMA)18 with short
OEG brush and long DP, albeit their almost identical MW.
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To evaluate the stability of selfꢀassembled micelles, DLS was next used to determine the
average hydrodynamic size of micelles selfꢀassembled by PCL21ꢀbꢀP(aꢀOEGMA)11 and PCL21ꢀbꢀ
P(OEGMA)18 in both ddH2O and PBS (pH 7.4, 150 mM) at various polymer concentrations. The
mean size recorded at different polymer concentrations ranges from approximately 110 to 150
nm (Figure S9 and S10). Such change is most likely relevant to the different aggregation number
of polymer chains for micelle formation at various polymer concentrations above the CMC.
Therefore the largest size at 0.5 mg/mL of all the three concentrations probably results from the
greatest aggregation number at this concentration. However, the statistical insignificance
between each value implies the stability of both micelle formulations in salt medium and in
diluted conditions.
The morphology of micelles selfꢀassembled by PCL21ꢀbꢀP(aꢀOEGMA)11 and PCL21ꢀbꢀ
P(OEGMA)18 was visualized by TEM. Both polymers selfꢀassembled into uniform nanoparticles
with regularly spherical shape (Figure 4a and S11) and similar dimension in a dried state. The
difference of the particle size between TEM and DLS measurements could be ascribed to the
hydration effect, that is, the hydrophilic PEG segments are solvated in an aqueous phase and can
be determined by DLS measurements but difficult to be observed under TEM observation.
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Figure 4. TEM image (a) and size distribution (b) of PCL21ꢀbꢀP(aꢀOEGMA)11 micelles at a
polymer concentration of 0.5 mg/mL.
Acidic pH-triggered aggregation of PCL21-b-P(a-OEGMA)11 micelles
To verify the acidic pHꢀtriggered cleavage of acetal links, the size change of PCL21ꢀbꢀP(aꢀ
OEGMA)11 micelles incubated at different pH values of 7.4, 6.8 and 5.0 for various periods was
monitored by DLS (Figure 5a).
The micelle size increased slightly from 118.5 to 143.9 nm after incubation at pH 7.4 for 48 h,
however, such change is statistical insignificance, which implies the colloidal stability of the
selfꢀassembled micelles at the physiological pH.
In addition to the stability required at pH 7.4 for realizing long circulation, polymeric micelles
must maintain certain structural integrity in the extracellular tumor microenvironment with a
weakly acidic pH of 6.8 to facilitate efficiently cellular uptake. The micelles were thus incubated
at pH 6.8 to evaluate the stability under this circumstance. The mean size kept almost stable
around approximately 140 nm after incubation for 12 h, and reached a plateau of 220 nm with
further incubation of 24 and 72 h. Such increase of micelle size is likely relevant to the minority
cleavage of the acetal links, leading to a slight aggregation of the micelles.
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However, incubation at pH 5.0 significantly promotes disassembly of micelles, which is
reflected by the dramatically increased size and a broader size distribution. Notably, a bimodal
distribution was clearly recorded for 48 h. The main population centered at approximately 504
nm probably implies the majority of the selfꢀassembled micelles have been subjected to acidic
pHꢀtriggered hydrolysis of acetal links and significant aggregation due to the deshielidng of
stabilizing corona composed of pendant OEG brushes. A few micelles remain intact, as
evidenced by the appearance of a small population centered at 122 nm, which is close to the
mean size of the stable micelles (Figure 4b). The disappearance of this small population as well
as similarly recorded mean sizes for prolonged incubation time of 72 and 96 h probably indicate
the complete hydrolysis within 72 h.
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To confirm the size variation monitored by DLS, TEM observation of PCL21ꢀbꢀP(aꢀ
OEGMA)11 micelles after incubation at pH 6.8 and 5.0 for 24 h was performed (Figure S12a and
b). Wellꢀdispersed micelles with regularly spherical shape and uniform size were maintained
even after incubation at pH 6.8 for 24 h (Figure S12a), demonstrating the stability of pHꢀ
sensitive micelles at the pH of tumor microenvironment. The slightly increased size compared to
that observed in water (Figure 4a) is most likely due to the minority cleavage of the acetal links
in the micelle structure, which exerts negligible effect on the stability of the selfꢀassembled
micelles. However, incubation at pH 5.0 for 24 h led to formation of densely arranged micelle
aggregates with irregularly spherical shape and nonuniform dimension (Figure S12b). More
importantly, the formation of aggregates is strongly evidenced by a significantly increased mean
size and broader size distribution at pH 5.0 relative to those observed in water and pH 6.8 as well
as the clearly visualized coalescences of individual small micelles. Taken together, the TEM
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results are in good agreement with the DLS data, confirming the stability of pHꢀsensitive
micelles at pH 6.8, and occurrence of aggregation at pH 5.0.
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1H NMR analysis was also utilized to verify the acidic pHꢀtriggered hydrolysis of acetal links.
10 mg of PCL21ꢀbꢀP (aꢀOEGMA)11 was first dissolved in 4.5 mL of H2O followed by addition of
hydrochloric acid aqueous solution (36 wt%, 1 mL) and stirred at 25 °C for 24 h. Note that the
transparent polymer solution became cloudy gradually, likely due to the detachment of
stabilizing pendant OEG brushes and significantly reduced solubility of the degraded products.
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The mixture was finally freezeꢀdried and subjected to H NMR analysis (Figure 5b), which
reveals presence of characteristic signals of PEG and PCL moieties, but complete loss of
characteristic peaks of acetal links at δ 4.79–4.84 ppm (highlighted using green color and arrow
in the structural formula and 1H NMR spectrum, respectively). The results support acidꢀcleavage
of acetal links in PCL21ꢀbꢀP (aꢀOEGMA)11.
In addition, SECꢀMALLS analyses were used to determine the MW of the degraded products.
The waterꢀinsoluble precipitates were collected by filtration and subjected to vacuumꢀdrying
prior to SECꢀMALLS measurements. As expected, the degraded products without pendant OEG
brushes show significantly decreased MW relative to the parent PCL21ꢀbꢀP(aꢀOEGMA)11, and
slightly increased MW compared to PCL21ꢀiBuBr (Figure 5c), which confirms the scission of
acetal links rather than the hydroplysis of polyester under acidic condition.
To further provide an insight into the acidicꢀpH triggered degradation of PCL21ꢀbꢀP(aꢀ
OEGMA)11, the fully degraded product, PCL21ꢀbꢀP(2ꢀhydroxyethyl methacrylate)11 (PCL21ꢀbꢀ
P(HEMA)11) was also synthesized by ATRP of HEMA using PCL21ꢀiBuBr macroinitiator given
that the full degradation of the hydrophilic P(aꢀOEGMA) block generates the P(HEMA) moiety
with somewhat hydrophilicity. The successful synthesis of PCL21ꢀbꢀP(HEMA)11 with desired
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polymer composition was confirmed by 1H NMR (Figure S13) and SECꢀMALLS (Figure S14)
analyses. The average size of the selfꢀassemblies formed by PCL21ꢀbꢀP(HEMA)11 was
determined by DLS to be 422.6 and 467.7 nm in water and PBS (pH 7.4), respectively (Figure
S15), which probably indicates the formation of large micelle aggregates due to the significantly
decreased hydrophilicity of P(HEMA) block relative to P(aꢀOEGMA) segment. TEM
observation of PCL21ꢀbꢀP(HEMA)11 reveals dense arrangement of nanoparticles with irregularly
spherical shape and nonuniform size (Figure S12c), further confirming selfꢀassembly of PCL21ꢀ
bꢀP(HEMA)11 into unstable aggregates. The results agree well with the acidic pHꢀtriggered
aggregation of PCL21ꢀbꢀP(aꢀOEGMA)11 micelles revealed by DLS (Figure 5a) and TEM
(Figure S12b) measurements.
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PCL21
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Water-insoluble
precipitates
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(c)
(b)
Chemicalshift (ppm)
Figure 5. (a) DLSꢀmonitored size change of PCL21ꢀbꢀP(aꢀOEGMA)11 micelles after incubation
at different pH values of 7.4, 6.8 and 5.0 for various periods at a polymer concentration of 0.25
mg/mL. (b) 1H NMR spectrum of the degraded products of PCL21ꢀbꢀP(aꢀOEGMA)11treated with
hydrochloric acid in CDCl3. (c) SEC elution traces of PCL21ꢀiBuBr, PCL21ꢀbꢀP(aꢀOEGMA)11,
and waterꢀinsoluble precipitates after degradation of PCL21ꢀbꢀP(aꢀOEGMA)11 by hydrochloric
acid.
In vitro drug loading and drug release study
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Dox was chosen as a modal drug and encapsulated in PCL21ꢀbꢀP(aꢀOEGMA)11 and PCL21ꢀbꢀ
P(OEGMA)18 micelles by dialysis method. The slightly lower CMC value and greater stability of
PCLꢀbꢀP(OEGMA)18 micelles relative to PCLꢀbꢀP(aꢀOEGMA)11 formulations contribute to the
smaller size and greater drugꢀloading capacity of PCLꢀbꢀP(OEGMA)18 micelles (Table 2).
To validate the acidic pHꢀtriggered drug release, in vitro drug release profiles were investigated
in buffer solutions with three different pHs of 7.4, 6.8, and 5.0, respectively (Figure 6). The
Doxꢀloaded PCL21ꢀbꢀP(aꢀOEGMA)11 micelles showed almost identical drug release behaviors at
pH 7.4 and 6.8, demonstrating their apparent stability at the physiological pH and the pH of
tumor microenvironment. Shift of release medium from pH 7.4 or 6.8 to 5.0 promoted Dox
release from PCL21ꢀbꢀP(OEGMA)18 micelles due to the increased solubility of protonated Dox
under the acidic conditions.41, 42 More importantly, nearly 90% of Dox was released at pH 5.0 for
Doxꢀloaded PCL21ꢀbꢀP(aꢀOEGMA)11 micelles in 72 h, whereas approximately 50% Dox release
was recorded at pH 5.0 for Doxꢀloaded PCL21ꢀbꢀP(OEGMA)18 micelles in the same period. The
significantly increased cumulative drug release is attributed to the acidic pHꢀtriggered cleavage
of acetal links for efficient destabilization of PCL21ꢀbꢀP(aꢀOEGMA)11 micelles and formation of
micelle aggregates toward promoted drug release.
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Table 2. Summarized properties of blank and drugꢀloaded PCL21ꢀbꢀP(aꢀOEGMA)11 and PCL21ꢀ
bꢀP(OEGMA)18 micelles.
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CMC
DLC
EE
Micelle constructs
Size (nm)
PDI
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(mg/L)
(%)
(%)
PCL21ꢀbꢀP(aꢀOEGMA)11
PCL21ꢀbꢀP(OEGMA)18
109.6
105.3
147.6
124.7
0.283
0.216
0.250
0.271
9.33
ꢀ
ꢀ
7.98
ꢀ
ꢀ
Doxꢀloaded PCL21ꢀbꢀP(aꢀOEGMA)11
Doxꢀloaded PCL21ꢀbꢀP(OEGMA)18
ꢀ
ꢀ
5.76
6.16
5.88
6.78
Figure 6. In vitro drug release profiles of Doxꢀloaded PCL21ꢀbꢀP(aꢀOEGMA)11 and PCL21ꢀbꢀ
P(OEGMA)18 micelles at different conditions.
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In vitro cellular uptake and cytotoxicity studies
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DAPI
Dox
Merged
Dox
Dox-loaded
PCL21-b-P(OEGMA)18
Dox-loaded
PCL21-b-P(a-OEGMA)11
Figure 7. Confocal imaging of free DOX (red), Doxꢀloaded PCL21ꢀbꢀP(OEGMA)18 and PCL21ꢀbꢀ
P(aꢀOEGMA)11 micelles uptake in A549 cells (nuclei stained blue with DAPI). Cells were
treated with free DOX or polymer constructs at 25% of its IC50 value to minimize cell death.
The cellular uptake efficiency was first evaluated using confocal microscopy. Dox fluorescence
was clearly observed in the cytoplasm and/or nuclei of cells incubated with free Dox or both
Doxꢀloaded micelles for 4 h (Figure 7), indicating that both micelle constructs can efficiently
transport the anticancer drug to the cells and subsequently undergo intracellular trafficking. Then
the cellular uptake was further quantified using flow cytometry (FCM) analyses (Figure 8a). The
mean fluorescence intensity of A549 cells treated with both Doxꢀloaded micelles was clearly
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observed in 4 h. The cells treated with free Dox presented much higher fluorescence intensity
than the cells incubated with micelle formulations most likely due to the faster diffusion rate of
Dox in cells with a mechanism of direct membrane permeation. Interestingly, the Doxꢀloaded
PCL21ꢀbꢀP(aꢀOEGMA)11 micelles mediated slightly higher fluorescence intensity with statistical
significance relative to the pHꢀinsensitive analogues, which probably implies the stronger ability
for the pHꢀsensitive micelles to deliver the anticancer drug into the cancer cells.
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Finally, the cytotoxicity of Doxꢀloaded PCL21ꢀbꢀP(aꢀOEGMA)11 and PCL21ꢀbꢀP(OEGMA)18
micelles
to
A549
cells
was
assessed
by
3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ5ꢀ(3ꢀ
carboxymethoxyphenyl)ꢀ2ꢀ(4ꢀsulfophenyl)ꢀ2Hꢀtetrazolium (MTS) cell viability assay. Both
blank micelles were nonꢀtoxic to A549 cells up to a concentration of 3.8 mg/mL (Figure. 8b).
The half maximal inhibitory concentration (IC50) of free Dox, Doxꢀloaded PCL21ꢀbꢀP(aꢀ
OEGMA)11 and PCL21ꢀbꢀP(OEGMA)18 micelles is 3.64 (3.13, 4.24), 128.5 (122.2, 135.1), and
194.7 (180.3, 210.3) ꢁg/mL, respectively (Figure 8c). The increased IC50 of Doxꢀloaded
micelles relative to free Dox is likely attributed to the slower internalization mechanism and the
release kinetics of free drug from the micelles. Most importantly, the Doxꢀloaded PCL21ꢀbꢀP(aꢀ
OEGMA)11 micelles showed significantly greater therapeutic efficacy, i.e., lower IC50 value than
the pHꢀinsensitive counterparts due to the efficient destabilization and aggregation of micelles
and subsequently promoted drug release in the intracellular acidic pH environment.
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*
(b)
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Figure 8. (a) Quantitive measurements of the mean fluorescence intensity after incubation with
free Dox, Doxꢀloaded micelles of PCL21ꢀbꢀP(aꢀOEGMA)11 and PCL21ꢀbꢀP(OEGMA)18 in A549
cells via flow cytometry (4 h incubation, Dox concentration = 20 ꢁg/mL, and 10000 cells were
counted). The data were expressed as mean ± SD, n = 3 (*p < 0.01). Viability of A549 cells
exposed to (b) blank micelles of PCL21ꢀbꢀP(OEGMA)18, PCL21ꢀbꢀP(a-OEGMA)11 and (c) free
Dox, Doxꢀloaded micelles of PCL21ꢀbꢀP(aꢀOEGMA)11 and PCL21ꢀbꢀP(OEGMA)18.
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Conclusions
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In summary, to develop a facile and universal alternative toward tumor acidic pHꢀresponsive
polymers, we reported in this study successful synthesis of a dual functional monomer, aꢀ
OEGMA that integrates the merits of commercially available OEGMA monomer for both
extracellular stabilization of particulates and CLRPꢀmediated polymerization properties, and an
acidic pHꢀcleavage acetal link for efficient intracellular destabilization of polymeric carriers.
Amphiphilic block copolymers, PCL21ꢀbꢀP(aꢀOEGMA)11 with pendant acidic pHꢀcleavable OEG
brushes were further prepared using this monomer. The delivery efficacy evaluated by an in vitro
drug release study, flow cytometry analyses as well as an in vitro cytotoxicity study further
confirmed promoted drug release at pH 5.0, greater cellular uptake and cytotoxicity of Doxꢀ
loaded pHꢀsensitive micelles of PCL21ꢀbꢀP(aꢀOEGMA)11 relative to the pHꢀinsensitive
analogues of PCL21ꢀbꢀP(OEGMA)18. The aꢀOEGMA monomer developed herein therefore
presents a facile and robust means toward tumor acidic pHꢀresponsive polymers as well as
provides a one solution to the tradeoff between extracellular stability and intracellular high
therapeutic efficacy of drug delivery systems.
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ASSOCIATED CONTENT
1
13
1
Experimental details, H, C NMR, FTꢀIR, HRMS data of VEMA, H NMR specta of PCLꢀ
iBuBr, and PCL21ꢀ ꢀP(OEGMA)18, CMC determinations of PCL21ꢀ ꢀP( ꢀOEGMA)11 and
PCL21ꢀ ꢀP(OEGMA)18, TEM images and size distributions of PCL21ꢀbꢀP(OEGMA)18 micelles,
b
b
a
b
average size of PCL21ꢀbꢀP(aꢀOEGMA)11 and PCL21ꢀbꢀP(OEGMA)18 micelles at various
polymer concentrations in water and PBS (pH 7.4, 150 mM), TEM images of PCL21ꢀbꢀP(aꢀ
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OEGMA)11 micelles after incubation at pH 6.8 and 5.0 for 24 h, and micelle aggregates formed
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by PCL21ꢀbꢀP(HEMA)11, 1H NMR spectrum and SEC elution trace of PCL21ꢀ
bꢀP(HEMA)11,
7
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size distributions of PCL21ꢀbꢀP(HEMA)11 in water and PBS (pH 7.4, 150 mM), standard curve of
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Dox in PBS (pH 7.4) are available in Figure S1ꢀS16.
AUTHOR INFORMATION
L. Zheng and X. Zhang contributed equally to this paper.
Corresponding Authors
weih@lzu.edu.cn (H. Wei)
1126@hbtcm.edu.cn (C. Chang)
Notes
The authors declare no competing financial interest.
ORCID
Hua Wei: 0000ꢀ0002ꢀ5139ꢀ9387
ACKNOWLEDGMENT
The authors acknowledge the financial support from the National Natural Science Foundation of
China (51473072 and 21504035), the Thousand Young Talent Program, and the Open Research
Fund of State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of
Applied Chemistry, Chinese Academy of Sciences.
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References
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