Influence of supramolecular layer-crosslinked structure on stability of dual pH-Responsive polymer nanoparticles for doxorubicin delivery

Article abstract of DOI:10.1016/j.jddst.2018.03.008

Undesired physiological instability remains a major limitation for nanoparticle-based drug delivery. To overcome this issue, a dual pH-responsive supramolecular layer-crosslinked nanoparticles (PCB-b-PCD/PBM-b-PDPA NPs, PDM NPs), which consisted of pH-responsive hydrophobic poly(diisopropylethyl methacrylate) (pKa ≈6.3) as the core, hydrophilic poly((methacrylic acid betaine) methyl methacrylate) as the shell and pH-responsive supramolecular crosslinked layer based on β-cyclodextrin and benzimidazole (pKa <6.0), was prepared. Effects of this supramolecular layer-crosslinked structure on dilution and stored stability, protein adsorption, and pH-responsibility were investigated. PDM NPs exhibited lower critical aggregation concentrations, good unimodal distribution and better dilution stability in comparison with non-crosslinked PCB-PDPA NPs. Moreover this pH-responsive supramolecular layer-crosslinked structure did not only influence the anti-protein adsorption ability, but also reduced the disintegrated pH (from 6.3 to below 6.0) of PDM NPs, which leads to the DOX was released from PDM NPs at the mildly acid condition effectively and sustainably in vitro. Therefore, this pH-responsive layer-crosslinked NPs held promising potentials as a smart nanocarriers for drug delivery.

Full text of DOI:10.1016/j.jddst.2018.03.008

Accepted Manuscript
Influence of supramolecular layer-crosslinked structure on stability of dual pH-
Responsive polymer nanoparticles for doxorubicin delivery
Hailiang Feng, Yu Sun, Jianhua Zhang, Liandong Deng, Anjie Dong
PII:
S1773-2247(17)31035-3
10.1016/j.jddst.2018.03.008
JDDST 601
DOI:
Reference:
To appear in: Journal of Drug Delivery Science and Technology
Received Date: 3 December 2017
Revised Date: 19 February 2018
Accepted Date: 5 March 2018
Please cite this article as: H. Feng, Y. Sun, J. Zhang, L. Deng, A. Dong, Influence of supramolecular
layer-crosslinked structure on stability of dual pH-Responsive polymer nanoparticles for doxorubicin
delivery, Journal of Drug Delivery Science and Technology (2018), doi: 10.1016/j.jddst.2018.03.008.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Influence of Supramolecular Layer-Crosslinked Structure on
Stability of Dual pH-Responsive Polymer Nanoparticles for
Doxorubicin Delivery
a
a
a
a, b *
a, b
Hailiang Feng, Yu Sun, Jianhua Zhang, Liandong Deng,
Anjie Dong
a
Department of Polymer Science and Technology, Key Laboratory of Systems Bioengineering
of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin
University, Tianjin 300072, China
b
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin
3
00072, China
*
Corresponding author:
Prof. Deng Liandong
Department of Polymer Science and Technology, Key Laboratory of Systems Bioengineering
of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin
University, Tianjin 300072, China
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin
3
00072, China
E-mail: dengliandong@tju.edu.cn
Mobile: +86 13116145498
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1 -

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Abstract
Undesired physiological instability remains a major limitation for nanoparticle-based
drug delivery. To overcome this issue, a dual pH-responsive supramolecular
layer-crosslinked nanoparticles (PCB-b-PCD/PBM-b-PDPA NPs, PDM NPs), which
consisted of pH-responsive hydrophobic poly(diisopropylethyl methacrylate) (pKa ≈
6
.3) as the core, hydrophilic poly((methacrylic acid betaine) methyl methacrylate) as
the shell and pH-responsive supramolecular crosslinked layer based on β-cyclodextrin
and benzimidazole (pKa < 6.0), was prepared. Effects of this supramolecular
layer-crosslinked structure on dilution and stored stability, protein adsorption, and
pH-responsibility were investigated. PDM NPs exhibited lower critical aggregation
concentrations, good unimodal distribution and better dilution stability in comparison
with non-crosslinked PCB-PDPA NPs. Moreover this pH-responsive supramolecular
layer-crosslinked structure did not only influence the anti-protein adsorption ability,
but also reduced the disintegrated pH (from 6.3 to below 6.0) of PDM NPs, which
leads to the DOX was released from PDM NPs at the mildly acid condition effectively
and sustainably in vitro. Therefore, this pH-responsive layer-crosslinked NPs held
promising potentials as a smart nanocarriers for drug delivery.
Keywords: drug delivery, pH-responsive, nanoparticles, supramolecular
layer-crosslinked structure, host-guest recognition
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1
. Introduction
In the past decades, polymer nanocarriers have received widespread attention for
anticancer drug delivery [1, 2]. The amphiphilic copolymer could self-assemble into
nanoparticles (NPs) with core-shell structure [3], with several advantages, such as
gathered in tumor tissue via the enhanced permeability and retention (EPR) effect [4],
enhancing the solubility of various hydrophobic anticancer drugs by the hydrophobic
core [5], prolonging the cycle time and half-life period in blood circulation by the
hydrophilic shell [6], improving the bioavailability and therapeutic effect of
anticancer drug [7], and decreasing the side effects toward to normal tissue [8].
However the conventional NPs could face several barriers when applied in vivo [9].
Once NPs is injected into the body through intravenous injection, it will be diluted in
the bloodstream immediately and combine with blood components, which harm the
NP equilibrium [10-12]. As a result, the vast clinical application of conventional NPs
are limited.
To focus on this problem, physical stabilization of NPs often involves the
manipulation of amphiphilicity, architecture and crystallinity, which are related to the
polymer composition, length of hydrophobic segment and the block length ratio.
[
13]Besides these, the method of constructing core-crosslinked and shell-crosslinked
structure is popular and crucial strategy to overcome the disintegration of NPs.[14, 15]
In these methods, the assembly of the copolymer is not only based on the hydrophobic
interaction, but also stabilized by the formation of crosslinks either in the core or in
the shell of the NPs [16-18]. These crosslinks based on covalent bonds contain
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addition of bifunctional reagents and direct reaction of pendent functionalities, such as
thiol groups and diselenide groups [19-23]. Wu and coworkers reported a
core-crosslinked pH-sensitive degradable NPs based on poly(ethylene
glycol)-b-poly(mono-2,4,6-trimethoxy
benzylidene-pentaerythritol
carbonate-co-acrylogy carbonate) (PEG-b-P(TMBPEC-co-AC)) diblock copolymer
includes acid-sensitive acetal and photo-crosslinkable acryloy groups in the
hydrophobic polycarbonate core [24]. This NPs based on photo-crosslinked displayed
high stability at pH 7.4 and hydrolyzed at pH of 4.0 and 5.0 improving the release
status of paclitaxel (PTX). Wang and coworkers prepared a pH/ redox dual triggered
shell-crosslinked
glucosamine-poly(styrene-alt-maleic
NAG-P(St-alt-MA) -b-PSt ) via post-crosslinked strategy using cystamine as
polymer
nanoparticles
based
anhydride) -b-polystyrene
130
on
N-acetyl
5
8
(
5
8
130
crosslinks between carboxyl groups of hydrophilic shell [16]. This shell-crosslinked
structure enhanced the stored stability of the NPs and avoided the premature drug
leakage to some exact. These nano-carriers based on covalent bonds could maintain
cargo with long-term stabilization to some exact, but the formation of crosslinked
structure based on covalent bonds always need light or chemical reaction, which
increase the complexity of experiments [16, 17, 19, 20].
Compared with crosslinked structure based on covalent bonds, the
supramolecular crosslinked structure based on non-covalent bonds attracts copious
research attentions [25-27]. Amount various non-covalent bonds, the host-guest
interaction based on β-cyclodextrin (β-CD) and bebzimidazole (BM), one of the most
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popular non-covalent bonds, has been applied versatile in biotechnology, particular in
improving intracellular drug release, due to its unique pH-responsiveness [28, 29].
The host-guest structure based on β-CD and BM can maintain stable at pH 7.4 and it
will be disintegrated at pH below 6.0, due to the protonation of BM (pKa < 6.0) [30].
Chen and coworkers reported a pH-sensitive supramolecular amphiphilic NPs based
on the host-guest interaction between β-CD of β-cyclodextrin-terminated
poly( -lactide) (CD-PLLA) and BM of benzimidazole-modified poly(ethylene glycol)
L
(PEG-BM), which could improve the doxorubicin release due to the host-guest
structure dissociation at pH 5.5 [31]. To our knowledge, several literatures almost
reported the effect of the supramolecular structures based on β-CD and BM on the
formation of NPs and promoting the drug release from NPs, there is little literature
reported about the effect of the upramolecualr layer-crosslinked structure on the
stability of NPs.
Here, as shown in Scheme 1, a novel dual pH-sensitive NPs with supramolecular
layer-crosslinked structure (PDM NPs) based on poly((methacrylic acid betaine)
methyl
methacrylate)-b-poly(single
6-methyl
acryloyl
ehylenediamine
β-cyclodextrin)-b-poly(diisopropylethyl methacrylate) (PCB-b-PCD-b-PDPA) and
poly((methacrylic acid betaine) methyl methacrylate)-b-poly(benzimidazole ethyl
methacrylate)-b-poly(diisopropylethyl methacrylate) (PCB-b-PBM-b-PDPA) was
designed and prepared. The effects of supramolecular layer-crosslinked structure on
the dilution stability, protein adsorption, stored stability, pH-responsibility, drug
release and safety of PDM NPs were mainly investigated and the cellular uptake
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against C6 cells was also investigated.
2
2
. Experimental
.1 Materials
Benzimidazole (BM; Energy Chemical, 98.0%), β-cyclodextrin (β-CD; Aladdin,
9.5%), Methacryloyl chloride (Aladdin, 95.0%), P-toluenesulfonic anhydride (Ts O;
9
2
Sigma-Aldrich, 99.0%), 2-(diisopropylamino) ethyl methacrylate (DPA;
Sigma-Aldrich, 97.0%) and trifluoracetic acid (TFA; Aladdin, 99.0%) were used
without any purification. Ethylenediamine (Aladdin) was dried over calcium hydride
(
CaH ; Aladdin, reagent grade, 95.0%) for 48 h and distilled before used.
2
2
,2’-azobis(2-methylpropionitrile) (AIBN; J&K Scientific, 98%) was recrystallized in
ethanol before used. N,N-dimethylformamide (DMF, reagent grade, 95.0%) was
purchased from Li’an Long Bohua Pharmaceutical Chemical Co. Ltd. (Tianjin,
China) and refluxed over CaH and distilled under reduced pressure before used.
2
Doxorubicin (DOX, 99.0%) was purchased from Beijing Huafeng United Technology
Co. Ltd (Beijing, China).
2
.2 Synthesis of PCB-b-PCD-b-PDPA and PCB-b-PBM-b-PDPA
The synthesis routes of PCB-b-PCD-b-PDPA and PCB-b-PBM-b-PDPA was shown
in Figure 1. Firstly, single 6-methyl acryloyl ehylenediamine β- cyclodextrin
(
β-CD-EMA) was synthesized via three-step method described by the literature [32,
3]. And benzimidazole ethyl methacrylate (BMMA) was synthesized as reported
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3
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previously [34]. (Detailed experimental produces were reported in the Supporting
Information)
Subsequently, P(CB-tBu)-b-PCD-b-PDPA was synthetized via continuous
reversible addition fragmentation chain transfer (RAFT) polymerization, using AIBN
as the initiator and CTAm as the chain transfer agent in DMF under argon atmosphere
according to our previously reported method [35]. Firstly, CB-tBu (4.3 mmol), CTAm
(0.1 mmol) and AIBN (0.01 mmol) were dissolved in anhydrous DMF (5 mL) in 25
mL Schlenk flask. After degassing via three freeze-pump-thaw cycles, the flask was
placed in thermostatic oil bath at 70 °C for 24 h in Ar atmosphere. The reaction
solution was dialyzed against pure water for 48 h, then lyophilized to obtain the
powder of P(CB-tBu)-CTAm. Secondly, P(CB-tBu)-b-PCD was synthesized using
P(CB-tBu)-CTAm as the chain transfer agent. The polymerization reaction was
carried out at 70 °C in Ar atmosphere using β-CD-EMA (0.8 mmol) and AIBN with a
1
0:80:1 of [P(CB-tBu)-CTAm]:[monomer]:[initiator] molar ratio. The resulting
P(CB-tBu)-b-PCD-CTAm solution was dialyzed against pure water for 48 h, then was
lyophilized to obtain the powder of P(CB-tBu)-b-PCD-CTAm. Thirdly,
P(CB-tBu)-b-PCD-b-PDPA was synthesized via method of the first step, using
P(CB-tBu)-b-PCD-CTAm as the chain transfer agent, DPA as the monomer and
AIBN
P(CB-tBu)-b-PCD-CTAm]:[monomer]:[initiator] molar ratio. The powder of
P(CB-tBu)-b-PCD-b-PDPA was obtained.
Finally, PCB-b-PCD-b-PDPA was performed via hydrolysis of
7 -
as
the
inititar
with
a
10:400:1
of
[
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P(CB-tBu)-b-PCD-b-PDPA. 0.1 mmol the copolymer was charged and dissolved into
1
mL TFA at room temperature for 6 h. Subsequently, the TFA was removed by
rotary evaporation and the residue was dissolved in DMF and dialyzed against
deionized water for 48 h, and lyophilized to obtain power products of
PCB-b-PCD-b-PDPA. The PCB-b-PBM-b-PDPA and PCB-b-PDPA were synthesized
via the same method of RAFT polymerization and hydrolysis reaction.
2
.3 Preparation of pH-Responsive Supramolecular Layer-Crosslinked PDM NPs
PDM NPs was prepared via solvent evaporation method as reported [36]. 5 mg
PCB-b-PCD-b-PDPA and 5 mg PCB-b-PBM-b-PDPA block polymer were dissolved
in 2 mL 2,2,2-trifluoroethanol (TFE) then added into 10 mL phosphate buffer saline
(PBS, pH 7.4, 10mM, 0.14 M NaCl) dropwise under magnetic stirring. After TFE was
allowed to evaporate for 24 h at 25 °C by magnetic stirring, PDM NPs was formed via
the host-guest recognition between β-CD and BM. The non-crosslinked nanoparticles
(PCB-PDPA NPs) were prepared via the same method.
2
.4 Critical Aggregation Concentration (CAC) measurement of PDM NPs
CAC of PDM NPs were measured at room temperature via steady state fluorescence
probe method utilizing pyrene as the fluorescence probe [37]. Firstly 1 mL pyrene
-6
THF solution (3×10 mol/L) was put into a centrifuge tube and THF was evaporated
at room temperature. Then the NPs stock solution (1 mg/mL) was diluted to different
0
-1
-1
-2
-3
-4
-5
-6
concentrations (1×10 , 5×10 , 1×10 , 1×10 , 1×10 , 1×10 , 1×10 , 1×10 mg/mL)
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using the same buffer. Subsequently, 5 mL NPs dispersion were added into the
-7
centrifuge tube with pyrene, the final pyrene concentration was 6×10 mol/L.
The fluorescence spectra of each sample was measured on Varian fluorescence
spectrophotometer (Varian, USA) with the excitation wavelength of 390 nm and the
detection range was from 250 nm to 360 nm. The I333 and I337 values were recorded as
the maximum emission intensity at ca. 333 and 337 nm, respectively. The I /I
3
37 333
ratio was plotted as a function of polymer concentration. The CAC value of PDM NPs
was the intersection point of fitting curves. The CAC of PCB-PDPA NPs was
investigated via the same method.
2
.5 Influence Study of pH-responsive supramolecular layer-crosslinked structure
on stabilities of NPs
.5.1 Dilution stability Study of NPs
PDM NPs dispersions were diluted with concentration from 1 mg/mL to 1×10
2
-3
mg/mL instantaneously. By using the same method, PCB-PDPA NPs dispersions with
different concentration were prepared. Then the size and PDI of each sample was
detected via DLS to obtain hydrodynamic diameter (D ) distribution.
h
2
.5.2 Protein Adsorption Study of NPs
Fetal bovine serum (FBS) was used as a model protein to examine protein adsorption
capacity of PDM NPs. Briefly, 2mL PDM NPs dispersion (1 mg/mL) was incubated
with 2 mL of a solution of 10% FBS in PBS (pH 7.4, 10 mM, 0.14 M NaCl) at 37 °C
for 2, 4, 6, 24, 36 and 48 h respectively. To illustrate the protein adsorption capacity
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indirectly, the size and PDI of each sample was measured via DLS. The protein
adsorption of PCB-PDPA NPs was investigated via the same method.
2
.5.3 Stored stability Study of NPs
To find the stored stability of PDM NPs, PDM NPs dispersion (1 mg/mL) was put in
a shaking air-bathing constant temperature vibrator at 37 °C. At predetermined time
intervals (0, 1, 2 and 5 d), 2 mL PDM NPs dispersion was withdrawn and measured
via DLS to record the PDI change of PDM NPs and PCB-PDPA NPs respectively. As
the control group, stored stability of PCB-PDPA NPs dispersion was carried out via
the same method.
2
.5.4 pH-Responsive Study of NPs
Compared to PCB-PDPA NPs as control group, to discuss the effect of
supramolecular layer-crosslinked structure based on β-CD and BM on the
pH-responsiveness of PDM NPs, the pH of PDM NPs or PCB-PDPA NPs dispersion
(1 mg/mL) were regulated from 7.4 to 6.8, 6.5, 6.0, 5.5, 5.0 and 4.5 respectively. Then
each sample was measured via DLS to record the size and PDI of PDM NPs or
PCB-PDPA NPs respectively.
2
.6 Characterization
2
.6.1 1H Nuclear Magnetic Resonance (1H NMR) Spectroscopy
1
H
NMR spectra of β-CD-EMA, BMMA, PCB-b-PCD-b-PDPA and
PCB-b-PBM-b-PDPA were recorded on Varian INOVA 500 spectrometer operating at
00 MHz (Varian Inc., Palo Alto, USA) in CDCl or DMSO-d and tetramethylsilane
5
3
6
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(TMS) as the internal standard.
2
.6.2 Dynamic Light Scattering (DLS)
Size and size distribution index (PDI) of each NPs dispersion were determined by
dynamic light scattering (DLS, zetasizer, Nano, Malvern, UK) at an operating
wavelength of 633 nm with a scattering constant angle of 173°. The diameter and PDI
of NPs were obtained from the average of three measurement.
2
.6.3 Transmission Electron Microscope (TEM)
Morphologies of each NPs sample was observed and recorded by using a Hitachi
H600 TEM system at operated voltage of 75 kV. For TEM test, each sample was
prepared by adding a drop of NPs dispersion onto the copper grid, and then the
sample was air-dried and measured at room temperature.
2
.7 Preparation of DOX-Loaded PDM NPs
Doxorubicin (DOX) was chosen as a model drug to examine the loading and release
capacity of PDM NPs. Typically, 5 mg PCB-b-PCD-b-PDPA and 5 mg
PCB-b-PBM-b-PDPA and 1.5 mg DOX were dissolved in 2 mL TFE, then the mixture
solution was added dropwise into 10 mL PBS buffer (pH 7.4, 10 mM, 0.14 M NaCl)
under magnetic stirring at a theoretical drug loading content of 15 wt%. After the
mixture was stirred at 25 °C for 24 h to remove TFE, each sample was centrifuged to
precipitate the unloaded DOX and then the dispersion of DOX-loaded PDM NPs was
obtained. The dispersion of DOX-loaded PCB-PDPA NPs was obtained via the same
method. The amount of DOX was determined via UV-visible spectrophotometer at
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detecting wavelength of 480 nm. Drug loading content (DLC) and drug loading
efficiency (DLE) were calculated by the following equations:
weight of loaded drug
DLC ꢀ%ꢁ=
×100%
weight of drug -loaded micelles
weight of loaded drug
DLE ꢀ%ꢁ=
×100%
weight of drug in feed
2
.8 In Vitro Drug Release Study
5
mL DOX-loaded PDM NPs or 5 mL DOX-loaded PCB-PDPA NPs was transferred
into a dialysis bag (MWCO = 8000), respectively. It was immersed in 15 mL different
pH phosphate buffer saline (PBS, pH 7.4 and pH 6.4, 10 mM, 0.14 M NaCl) and
acetate buffer saline (pH 5.0, 10 mM, 0.14 M NaCl) solutions in a shaking air-bathing
constant temperature vibrator at 37 °C. At predetermined time intervals, 10 mL
release medium was withdrawn and replenished with an equal volume of fresh buffer.
The amount of released DOX was measured and recorded based on the absorbance
intensity at detecting wavelength of 488 nm with UV-visible spectroscopy. The
cumulative drug release amount (E ) was calculated by the following equation:
r
n-1
V ∑ C +V C
n
e
1
i
0
Erꢀ%ꢁ=
×100%
mDOX
Wherein V is the volume of the replaced media (V = 10 mL), V is the whole
ꢂ
ꢂ
ꢃ
volume of the release media (V = 15 mL), C is the concentration of DOX in the
ꢃ
ꢄ
sample. And mꢅꢆꢇ is the amount of DOX in the nanoparticles.
2
.9 Cell Uptake Studies
To investigate the effect of supramolecular layer-crosslinked structure based on β-CD
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and BM on the cellular uptake experiments, DOX (positive control group),
DOX-loaded PCB-PDPA NPs (negative control group) or DOX-loaded PDM NPs
(experimental group) was performed on flow cytometry and fluorescence microscope
against C6 cells respectively. For the flow cytometry, C6 cells were seeded in six-well
4
plates at 1×10 cells per well in 1 mL complete DMEM and cultured for 24 h. Then
the cells were treated with free DOX, DOX-loaded PCB-PDPA NPs and DOX-loaded
PDM NPs respectively at an equivalent DOX concentration in fresh culture medium
and were incubated at 37 °C for 2 and 4 h. Thereafter, each sample was prepared for
flow cytometry analysis by removing the cell growth media, washing with PBS (pH
4
7
.4, 10 mM, 0.14 M NaCl) and treating with trypsin [38]. Data for 1.0×10 gated
events were collected and analysis was means of a BD FACSCalibur flow cytometer
and CELLQuest software.
For the fluorescence microscope studies, C6 cells were seeded in six-well plates at
4
1
×10 cells per well. After 24 h of culture, the cells were treated with free DOX,
DOX-loaded PCB-PDPA NPs and DOX-loaded PDM NPs respectively at an
equivalent DOX concentration in fresh culture medium at pH 7.4 and were incubated
at 37 °C for 2 and 4 h. Next the above incubated cells were thoroughly washed twice
with PBS (pH 7.4, 10 mM, 0.14 M NaCl) to remove excess free DOX or DOX-loaded
NPs dispersion. Then the cells were fixed with 4% formaldehyde for 30 min at room
temperature and then the cell nuclei were stained with 4,6-diamidino-2-phemylindole
(
DAPI). After replacement with PBS (pH 7.4, 10 mM, 0.14 M NaCl), the
fluorescence images were obtained by using fluorescence microscope [38].
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2
.10 Cell Viability Assays
The biocompatibility and relative cytotoxicity of NPs against C6 cells were estimated
by MTT assay. In the MTT assay, C6 cells were seeded into 96-well plates with a
3
density of 8×10 cells per well in 100 ꢀL medium. After 24 h of incubation, removed
the culture medium and added PCB-PDPA NPs or PDM NPs (or DOX-loaded PDM
NPs or DOX-loaded PCB-PDPA NPs) dispersion with pH 7.4 PBS (10 mM, 0.14 M
NaCl) at different concentrations respectively. After another 24 h of incubation, the
medium was replaced by 100 ꢀL fresh medium. Then 20 ꢀL MTT solution (5 mg/mL)
was added. After another 4 h of incubation, the medium was replaced completely, and
2
00 ꢀL was added into each well to extract the formazan product formed by viable
cells. The absorbance of the solution was measured on a Bio-Rad 680 microplate
reader at 570 nm. And the relative cell viability (%) was studied via comparing the
absorbance at 570 nm with control, which contained only culture medium. Dada were
presented as average ± SD (n = 4).
2
.11 Statistical Analysis
The ANOVA analysis and Tukey-Kramer test were utilized for the statistical analysis.
Values of *p < 0.05, **p < 0.01 and ***p < 0.001 were applied to denote various
statistical significance.
3
. Results and Discussion
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3
.1 Synthesis and of PCB-b-PCD-b-PDPA and PCB-b-PBM-b-PDPA
As shown in Figure 1, using β-CD-6-EMA or BMMA as comonomer,
PCB-b-PCD-b-PDPA and PCB-b-PBM-b-PDPA were synthesized through RAFT
polymerization. Firstly, β-CD-6-EMA and BMMA were synthesized successfully by
the method reported before [32-34]. Subsequently, using AIBN as the initiator and
CTAm as the chain transfer agent, P(CB-tBu)-b-PCD-b-PDPA or
P(CB-tBu)-b-PBM-b-PDPA was synthesized via continuous RAFT polymerization of
CB-tBu, β-CD-6-EMA or BMMA, and DPA, in DMF under argon atmosphere.
Finally, the tert-butyl groups on CB-tBu units were selectively hydrolysized in TFA
for 6 h to obtain the PCB-b-PCD-b-PDPA and PCB-b-PBM-b-PDPA.
As shown in Figure 2A, 2B and Figure S1, S2, all characteristic signals of
β-CD-6-EMA and BMMA could be found clearly in accordance with the
characteristic peaks reported by literature [32-34]. β-CD-6-EMA was characterized by
1
H NMR (500 MHz, DMSO-d , δ ppm): 1.79 (o, 3H, s), 4.81 (a, 7H, dd), 5.35 (q, 1H,
6
1
s), 5.79 (p, 1H, s), 7.94 (n, 1H, s). BMMA was also characterized by H NMR (500
MHz, CDCl , δ ppm): 2.05 (h, 3H, s), 5.58 (i, 1H, s), 6.05 (j, 1H, s), 7.42 (b + c, 2H,
3
t), 7.82 (a + d, 2H, t). These results indicated that β-CD-EMA and BMMA monomers
had been successfully synthesized, respectively.
Subsequently, it can be seen from Figure 2C the P(CB-tBu)-b-PCD-b-PDPA
1
was characterized by H NMR (500 MHz, DMSO-d , δ ppm): 1.22 (b, 20H, br), 1.39
6
(i, 12H, br), 1.51 (n, 9H, br), 3.45 (l, 6H, br), 7.45 (p, 1H, d). As shown in Figure 2D,
1
the P(CB-tBu)-b-PBM-b-PDPA was characterized by H NMR (500 MHz, DMSO-d ,
6
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d ppm): 1.22 (b, 20H, br), 1.33 (i, 12H, br), 1.51 (n, 9H, br), 3.45 (l, 6H, br), 7.42 and
7
.82 (p, 4H, d). Moreover, the disappearance of the shape peak at 1.51 ppm in Figure
2
C and 2D, represented that the tert-butyl group on CB-tBu unit in
P(CB-tBu)-b-PCD-b-PDPA and P(CB-tBu)-b-PBM-b-PDPA were hydrolyzed
respectively. These results proved that the PCB-b-PCD-b-PDPA and
PCB-b-PBM-b-PDPA were synthesized successfully. The degree of polymerization
and molecular weight could be obtained by the results integral areas of characteristic
peaks with the known CTAm unit (Table 1).
3
.2 Preparation of PDM NPs
As amphiphilic copolymers, PCB-b-PCD-b-PDPA and PCB-b-PBM-b-PDPA were
liable to self-assemble into NPs in aqueous solution with hydrophobic PDPA core,
zwitterionic hydrophilic PCB shell and supramolecular layer-crosslinked structure
based on host-guest recognition between β-CD and BM. The size, size distribution
and morphology of PDM NPs detected via DLS and TEM were shown in Figure 3
and summarized in Table 2. Comparing to PCB-PDPA NPs with larger size (189 nm)
and wider PDI (0.152), PDM NPs had an average size at 176 nm and a quit low
particle distribution index (PDI) at 0.075. This could be ascribed to the fact that the
supramolecular layer-crosslinked structure based on β-CD and BM promoted the
copolymer chain aggregating and NPs shrinking. The morphologies of NPs visualized
by TEM were also shown in Figure 3C and 3D. This result reflected that stable PDM
NPs and PCB-PDPA NPs with an approximate spherical shape and average size
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around at 150 and 160 nm respectively, could be formed in aqueous solution. All these
results revealed that PDM NPs and PCB-PDPA NPs were prepared successfully.
CAC is one of the most physicochemical parameters of the aggregation behavior
of amphiphilic copolymer in dilute aqueous solution [37]. CAC of
PCB-b-PCD/BM-b-PDPA and PCB-b-PDPA in water was determined by the
fluorescence technique with pyrene as a probe, which is a hydrophobic probe used in
aqueous media commonly. The fluorescence intensity of pyrene is closely related to
the environment where pyrene exposed, and NPs has an ability to increase the
solubility of pyrene obviously. The CAC of amphiphilic copolymer was showed in
Figure 4 and summarized in Table 2. The maximum emission wavelength of pyrene
was moved from 337 nm to 333 nm gradually when the copolymer concentration was
-6
reduced from 1 mg/mL to 1×10 mg/mL. CAC values of PCB-b-PCD/BM-b-PDPA
and PCB-b-PDPA were determined to be 0.89 and 3.2 ꢀg/mL, respectively. Because
the main driving force to form NPs was hydrophobic interaction of hydrophobic
segments. The supramolecular layer-crosslinked structure based on the host-guest
recognition between β-CD and BM enhanced the hydrophobic interaction [31, 39].
Therefore, the introduction of the supramolecular layer-crosslinked structure between
hydrophobic segment and hydrophilic segment of PCB-b-PDPA could reduce the
CAC obviously and increase the stability of PDM NPs.
3
.3 Effect of supramolecular layer-crosslinked structure on stabilities of NPs
It is well-known that the physical stability of amphiphilic copolymer NPs is of
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extremely important for their biological applications, as the stability not only
determine the half-life period, but also directly affect the absorption and physiological
distribution of drug [40]. Due to the dilution of the blood, it is easy for traditional
amphiphilic polymer NPs to disintegrate and release encapsulated therapeutic agents.
Thus the dilution stability is an important parameter for polymer NPs, which
guarantee the delivery of anticancer drug to tumor site without pre-releasing. The
effect of supramolecular layer-crosslinked structure based on host-guest recognition
between β-CD and BM on the dilution stability of NPs was investigated by detecting
the size of NPs before and after dilution via DLS. As shown in Figure 5A, the size
and PDI of PDM NPs was increased slightly and the unimodal distribution was still
maintained after diluted 1000 times. However, compared with PDM NPs, the
distribution of PCB-PDPA NPs changed from unimodal distribution to bimodal
distribution when the PCB-PDPA NPs dispersion was diluted at the same times.
Additionally, PCB-PDPA NPs with micro-size particle was also appeared after
dilution 1000 times. These results indicated that the introduction of supramolecular
layer-crosslinked structure based on β-CD and BM enhanced the dilution stability of
PDM NPs, providing a way to reduce the leakage of drug during the blood circulation.
It is well accepted that when NPs enters the blood circulation, the opsonic
proteins can be adsorbed to the surface of NPs, and then it could be recognized by the
scavenger receptors of macrophage cells [34]. This will cause the quick clearance of
NPs from blood circulation by reticuloendothelial system (RES) [6]. To assess the
protein adsorption, NPs were incubated in pH 7.4 PBS (10 mM, 0.14 M NaCl) with
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1
0% FBS for predetermined intervals, then the size was detected via DLS. It could be
seen from Figure 5B that the size of PDM NPs and PCB-PDPA NPs was increased
slightly (about 20 nm increasing at 48 h) and remained stable with the prolongation of
incubation time. This results was attributed to that the hydrophilic zwitterionic PCB
corona could prevent a large nmber of protein adsorption of NPs [41-43], and it also
indicated that the supramolecular layer-crosslinked structure between hydrophobic
segment and hydrophilic segment did not hinder the anti-protein adsorption of NPs.
The stored stability is also an important parameter of polymer NPs. If the storage
stability of NPs is poor, the NPs will aggregate resulting a large PDI during storage
period. To assess the effect of supramolecular layer-crosslinked structure on the stored
stability, PDM NPs and PCB-PDPA NPs dispersion were stationary at 37 °C for
predetermined intervals and the PDI was detected by DLS. As shown in Figure 5C,
PDI of PDM NPs was not changed significantly and remained stable as time
prolonged. Contrarily, the PDI of PCB-PD NPs was risen gradually from about 0.18
to 0.22 with time extending. This result indicated that supramolecular
layer-crosslinked structure effectively increased the stored stability of PDM NPs and
avoided the aggregation of NPs.
To confirm the effect of supramolecular layer-crosslinked structure based on
host-guest recognition between β-CD and BM on the pH-responsiveness of NPs, the
size of NPs in a series of different water buffers (pH 7.4, 6.8, 6.5, 6.0, 5.6, 5.0 and 4.5)
were determined via DLS. It could be found from Figure 5D and 5E that the size of
PDM NPs and PCB-PDPA NPs were not alterative from pH 7.4 to 6.8. However, PDI
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of PCB-PDPA NPs was becoming wider with pH decline from pH 7.4 to 6.8. When
the pH decreased to 6.5, the size and PDI of PDM NPs remain stable. On the contrary,
there was noticeable change for PCB-PDPA NPs, whose PDI began to change from
unimodal distribution to bimodal distribution. Such difference between PDM NPs and
PCB-PDPA NPs was due to tow reasons. On one hand, the PDPA segment (pKa ≈ 6.3)
transformed from hydrophobic chain to hydrophilic chain when pH was below the
transition pH, causing PCB-PDPA NPs instability and disassembling, on the other
hand, the supramolecular layer-crosslinked structure based on β-CD and BM (pKa <
6
.0) promoted the stability of PDM NPs when pH above 6.0 [44-46]. When pH was
below 5.6, the particle sizes of both PDM NPs and PCB-PDPA NPs were changed
obviously, and multi-peak distribution was appeared. These results indicated that both
PDM NPs and PCB-PDPA NPs were disassembled due to the disintegration of
supramolecular layer-crosslinked structure and protonation of PDPA. These results
were corresponding to the fact reported that BM could form host-guest recognition
complexes in aqueous solution at pH 7.4, and when the pH was below 6.0, the amino
of BM protonated, leading to a disassembly of the host-guest complexes [39, 47],
Therefore, the introduction of pH-sensitive supramolecular layer-crosslinked structure
not only increased the stability of nanoparticles, but also reduce the disintegrated pH
of PDM NPs, which might endow PDM NPs with abilities to reduce the leakage of
therapeutic agents and enhance the safety of carriers in blood circulation.
3
.4 In Vitro Release
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Doxorubicin (DOX), as one of the most effective anthracycline antitumor drugs to
treat a wide range of malignancies [38, 48], was chosen as a model drug to evaluate
the effects of supramolecular layer-crosslinked structure based on host-guest
recognition between β-CD and BM on drug loading and in vitro release behaviors of
PDM NPs. DOX-loaded PDM NPs and DOX-loaded PCB-PDPA NPs were prepared
at a copolymer concentration of 1 mg/mL and theoretical drug loading content of 15
wt% via nano-precipitation method. The characteristic of DOX-loaded NPs which
included size, particle size distribution, DLC and DLE were detected via UV-vis
spectrophotometer and summarized in Table 2.
The in vitro drug release of DOX-loaded PDM NPs and DOX-loaded
PCB-PDPA NPs were carried out under the physiological environment (pH 7.4) and
endosomal compartments (pH=6.4 and 5.0) using a dialysis method at a nanoparticle
concentration of 1.0 mg/mL at 37 °C. As shown in Figure 6, the release rate of DOX
from DOX-loaded PDM NPs or DOX-loaded PCB-PDPA NPs was obviously faster at
acidic environment (pH 5.0) than that at pH 7.4 and 6.4. Moreover, the cumulative
release amount of DOX-loaded PDM NPs was lower than that of DOX-loaded
PCB-PDPA NPs at pH 5.0. Briefly, the cumulative release amount of DOX release
from DOX-loaded PDM NPs was about 60% compared to 90% of DOX release from
DOX-loaded PCB-PDPA NPs at pH 5.0 within 12 h. And the cumulative release
amount of DOX-loaded PDM NPs was equal with that of DOX-loaded PCB-PDPA
NPs pH 6.4 and pH 7.4. These results were mainly attributed to the pH-responsive
property of hydrophobic PDPA core and supramolecular layer-crosslinked structure
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based on host-guest recognition between β-CD and BM [40]. Although the
hydrophilicity of the PDPA core of PDM NPs and PCB-PDPA NPs was enhanced
with the change of pH from 7.4 to 6.4, the hydrophobicity still played a main role in
the PDPA core of PDM NPs and PCB-PDPA NPs. Thus, the cumulative release
amount of DOX-loaded PDM NPs was equal with that of DOX-loaded PCB-PDPA
NPs at this condition. When the pH was below 6.3, the hydrophobic PDPA core of
NPs protonated completely [44-46], resulting in the disintegration of PCB-PDPA NPs.
This led to the rapid release of DOX from DOX-loaded PCB-PDPA NPs at pH 5.0.
Differently, due to the supramolecular layer-crosslinked structure based on β-CD and
BM (pKa < 6.0) lagged the disassembly of PDM NPs at pH 5.0, the DOX released
from DOX-loaded PDM NPs was slower than that from DOX-loaded PCB-PDPA
NPs.
3
.5 In Vitro Cell Uptake
To investigate the effect of the supramolecular layer-crosslinked structure of NPs on
cellular uptake behavior, the fluorescence microscope was used against C6 cells. As
shown in Figure 7 and S6 free DOX exhibited an obvious fluorescence signal at 2 h
and the fluorescence intensity was decreased quickly with prolongation of incubation
time to 4 h. In contrast, the fluorescence intensity of DOX-loaded PDM NPs or
DOX-loaded PCB-PDPA NPs was increased from 2 h to 4 h. The fluorescence
intensity of DOX-loaded PDM NPs was increased obviously, compared to a slight
increasing of DOX-loaded PCB-PDPA NPs. Because the cellular uptake mechanism
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of free DOX was through passive diffusion basically [38], leading to the high
fluorescence intensity of free DOX. Because the supramolecular layer-crosslinked
structure reduced the sudden release of DOX and promoted the DOX release from
DOX-loaded PDM NPs more sustainably, the fluorescence intensity increased faster
than that of DOX-loaded PCB-PDPA NPs. This results were accordance with the
results of the in vitro drug release assay and indicated that PDM NPs could achieve
intracellular sustained release of DOX preliminarily.
3
.6 In Vitro Cytotoxicity Assay
The good biocompatibility and low cytotoxicity of drug carrier are two crucial
characters for their clinic applications. In this experiment, the in vitro biocompatibility
and cytotoxicity of blank PDM NPs and blank PCB-PDPA NPs toward C6 cells were
evaluated utilizing MTT assay. As shown in Figure 8A, the PDM NPs and
PCB-PDPA NPs showed very low cytotoxicity against C6 cells after 24 h incubation,
and the cell viability rate higher than 85% even the NPs concentrations up to 1.0
mg/mL. This result reflected that the PDM NPs and PCB-PDPA NPs could be used
safely as biocompatible carriers for an efficient intracellular drug delivery.
Figure 8B showed the cell viabilities of C6 cells after incubation with free DOX,
DOX-loaded PCB-PDPA NPs and DOX-loaded PDM NPs for 24 h. The in vitro half
maximal inhibitory concentration (IC ) values were 0.5, 5, 1 ꢀg/mL for free DOX,
5
0
DOX-loaded PCB-PDPA NPs and DOX-loaded PDM NPs, respectively. Because free
DOX enter the cells quickly by passive diffusion and distribute into the cell nucleus
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easily [34], free DOX showed the highest inhibition effect against C6 cell
proliferation. Additionally, most NPs enter cells by endocytosis [49], which leads to
the DOX was released slowly from DOX-loaded PCB-PDPA NPs and DOX-loaded
PDM NPs. Thus, there were less DOX in C6 cells for PDM NPs and PCB-PDPA NPs,
leading to low cytotoxicity of DOX-loaded PCB-PDPA NPs and DOX-loaded PDM
NPs. Compared to DOX-loaded PCB-PDPA NPs, the higher cytotoxicity of
DOX-loaded PDM NPs was attributed that the supramolecular layer-crosslinked
structure enhanced the DOX release more sustainably and effectively, leading to kill
tumor cells more effectively. This result was accordance with the results of the in vitro
drug release assay and in vitro cell uptake assay.
4
. Conclusion
In this study, a novel dual pH-sensitive supramolecular layer-crosslinked PDM NPs
was designed and prepared based on host-guest recognition between β-CD and BM.
This supramolecular layer-crosslinked structure successfully improved the stability of
PDM NPs at pH 7.4, including dilution stability, anti-protein adsorption stability and
stored stability. Compared to the rapid disassembly to release DOX of DOX-loaded
PCB-PDPA NPs, this supramolecular layer-crosslinked structure reduced the
disassembly pH from 6.3 to below 6.0, realizing the intracellylar sustained DOX
release preliminary. Further, DOX-loaded PDM NPs displayed a higher cytotoxicity
than DOX-loaded PCB-PDPA NPs against C6 cells. Therefore, this dual
pH-responsive supramolecular layer-crosslinked PDM NPs might hold great
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potentials as intelligent nanocarriers for hydrophobic anticancer drug delivery.
Conflict of Interests
The authors declare that there is no conflict of interests.
Funding Status
This work was financially supported by the National Natural Science Foundation of
China [grant number 81371667], [grant number 31470963], [grant number 31470925]
and [grant number 31670121].
Supporting Information
Supporting Information is available online.
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