Zwitterionic shielded polymeric prodrug with folate-targeting and pH responsiveness for drug delivery

Article abstract of DOI:10.1039/c8tb02772b

Zwitterionic polymers are a class of polymers that acts as both Lewis base and Lewis acid in solution. These polymers not only have excellent properties of hydration, anti-bacterial adhesion, charge reversal and easy chemical modification, but also have c

Full text of DOI:10.1039/c8tb02772b

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Zwitterionic shielded polymeric prodrug with
folate-targeting and pH responsiveness for drug
Cite this:DOI: 10.1039/c8tb02772b
delivery†
a
b
a
Lei Li,a Yue Song,a Jinlin He,
Mingzu Zhang,a Jian Liu
and Peihong Ni
*
Zwitterionic polymers are a class of polymers that acts as both Lewis base and Lewis acid in solution.
These polymers not only have excellent properties of hydration, anti-bacterial adhesion, charge reversal and
easy chemical modification, but also have characteristics of long-term circulation and suppress nonspecific
protein adsorption in vivo. Here, we describe a novel folate-targeted and acid-labile polymeric prodrug
under the microenvironment of tumor cells, abbreviated as FA-P(MPC-co-PEGMA-BZ)-g-DOX, which was
synthesized via a combination of reversible addition–fragmentation chain transfer (RAFT) copolymerization,
Schiff-base reaction, Click chemistry, and a reaction between the amine group of doxorubicin (DOX) and
aldehyde functionalities of P(MPC-co-PEGMA-BZ) pendants, wherein MPC and PEGMA-BZ represent
2-(methacryloyloxy)ethyl phosphorylcholine and polyethylene glycol methacrylate ester benzaldehyde,
respectively. The polymeric prodrug could self-assemble into nanoparticles in an aqueous solution. The
average particle size and morphologies of the prodrug nanoparticles were observed by dynamic light
scattering (DLS) and transmission electron microscopy (TEM), respectively. We also investigated the in vitro
drug release behavior and observed rapid prodrug nanoparticle dissociation and drug release under a
mildly acidic microenvironment. The methyl thiazolyl tetrazolium (MTT) assay verified that the P(MPC-co-
PEGMA-BZ) copolymer possessed good biocompatibility and the FA-P(MPC-co-PEGMA-BZ)-g-DOX
prodrug nanoparticles showed higher cellular uptake than those prodrug nanoparticles without the FA
moiety. The results of cytotoxicity and the intracellular uptake of non-folate/folate targeted prodrug
nanoparticles further confirmed that FA-P(MPC-co-PEGMA-BZ)-g-DOX could be efficiently accumulated
and rapidly internalized by HeLa cells due to the strong interaction between multivalent phosphorylcholine
(PC) groups and cell membranes. This kind of multifunctional FA-P(MPC-co-PEGMA-BZ)-g-DOX prodrug
nanoparticle with combined target-ability and pH responsiveness demonstrates promising potential for
cancer chemotherapy.
Received 21st October 2018,
Accepted 17th December 2018
DOI: 10.1039/c8tb02772b
rsc.li/materials-b
a College of Chemistry, Chemical Engineering and Materials Science, State and
Local Joint Engineering Laboratory for Novel Functional Polymeric Materials,
Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application,
Suzhou Key Laboratory of Macromolecular Design and Precision Synthesis,
Soochow University, Suzhou 215123, P. R. China. E-mail: phni@suda.edu.cn;
Tel: +86-512-65882047
Introduction
During the past decades, cancer has been one of the most life-
threatening diseases in the world.1 The improvement of chemo-
therapy drugs on the aspects of their short half-time and severe
toxicity to normal tissues remains an unmet need for clinical
applications.2,3 To overcome these limitations, polymeric prodrug
nanoparticles as an emerging tool for cancer chemotherapy have
been utilized because they have characteristic properties of
increasing drug bioavailability, prolonging circulation time, and
promoting accumulation in the tumor site.4–6 In these prodrugs,
b Institute of Functional Nano and Soft Materials (FUNSOM), Soochow University,
Suzhou, 215123, P. R. China
† Electronic supplementary information (ESI) available: Experimental details and
characterization data. 1H NMR and 13C NMR spectra of PEGMA-BZ monomer.
1H NMR spectra of 4-cyano-4-ethylsulfanylthiocarbonylsulfanylentanoic acid
(CEP) and folic acid. 31P spectrum of P(MPC-co-PEGMA-BZ) copolymer. UV-vis
spectra of (A) P(MPC-co-PEGMA-BZ)-g-DOX prodrug, (B) P(MPC-co-PEGMA-BZ),
and (C) the reaction product of P(MPC-co-PEGMA-BZ) with triethylamine. UV-vis the structure of polymers plays an important role in the perfor-
spectra of free folate and FA-P(MPC-co-PEGMA-BZ)-g-DOX in ethanol. Intensity
mance and therapeutic effects.
ratios (I3/I1) as a function of Log C for (A) PMPD1 and (B) FA-PMPD1 prodrug
Zwitterionic polymers possess both cationic and anionic
nanoparticles in pH 7.4 buffer solution. TEM image of the PMPD1 prodrug
groups but still remain charge neutral as a whole,7 thus forming
nanoparticles (A) and the particle size distribution curve (B) corresponding to the
a strongly associated hydration layer through electrostatic inter-
TEM sample (scale bar 200 nm). Variation of particle size for the FA-PMPD1
prodrug nanoparticles in PBS 7.4 at different times. See DOI: 10.1039/c8tb02772b actions on the surface of drug delivery vessels and effectively
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suppressing non-specific protein adsorption and foreign body drugs onto the side chains of the polymers with acid-labile
reactions.8,9 A synthetic polymer, which was inspired by linkages, such as hydrazone,33,34 acetal,35,36 and imine,37–39
the phosphorylcholine (PC) headgroup of the bilayer cell whose cleavage enables the drug release in the tumor sites. It
membrane, has been used to prepare or modify drug delivery is well known that the typical pH of the normal cells is
carriers.10–12 Zwitterionic polymers can minimize the inter- approximately 7.4, while the pH range can be as low as 5.0 to
action between the carriers and the biological components 6.0 in the endosomes of the cancer cells and much lower in the
during the circulation process.13,14
intracellular lysosomes (pH B 4.0–5.0) at the cellular level.40,41
Based on this, a new strategy has been proposed for improving Cheng et al.42 designed and prepared acid-sensitive polylactide-
the cellular internalization efficiency and the inefficient intra- graft-doxorubicin (PLA-g-DOX) by azide–alkyne click reaction to
cellular drug release of the prodrug, that is, the cell-adhering transform acetylene functionalized PLA into PLA-graft-aldehyde
zwitterionic polymers. For example, poly(2-methacryloyloxyethyl (PLA-g-ALD), after which DOX was conjugated into the polymer
phosphorylcholine) (polyMPC),15,16 poly(sulfobetaine methacry- scaffold by the Schiff base reaction, which can release DOX from
late) (polySBMA),17 and poly(carboxybetaine methacrylate) PLA-g-DOX rapidly in an acidic medium due to the acid-labile
(polyCBMA)18,19 have been reported. Among them, polyMPC Schiff base linkage between DOX and the polymer scaffold.
especially exhibits additional promise when conjugated to
Prodrug nanoparticles can also enhance the accumulation
therapeutic proteins and chemotherapeutics. In addition, poly- of the drugs in the tumor site through the enhanced perme-
meric scaffolds with a zwitterionic 2-methacryloyloxyethyl phos- ability and retention (EPR) effect.43 For achieving good ther-
phorylcholine (MPC) group containing a phosphate anion and a apeutic efficacy, targeting ligands such as folic acid (FA),44,45
trimethylammonium cation with an inner-salt structure have biotin46,47 and RGD48,49 are introduced into the prodrug nano-
attracted more attention.20 Hence, polyMPC can be used as particles, which could be expected to reach the tumor site
a drug delivery carrier due to its zwitterionic character with effectively via active targeting. Furthermore, folate receptors a
considerable advantages such as extensive water solubility, (FRa) are most widely expressed at very low levels in normal
biocompatibility, and rapid cellular internalization.21,22 These tissues, but it is highly expressed in numerous cancers.50,51
polymers have been widely synthesized to mimic the cell Folate-based drug delivery has been widely applied in tumor
membranes and are considered as stealthy materials.23,24
targeting for its high-affinity binding to the folate receptor, which
In addition, polyMPC based polymers as drug carriers have also significantly decreases their toxic side effects compared to
also been widely investigated with respect to the prolongation of those of the non-targeted chemotherapy.52,53
the circulation time, which verified that zwitterionic copolymers’
In recent years, in the field of polymer synthesis, RAFT and
nanoparticles possess good protein resistance properties and click chemistry have been widely used. However, the folate-
accelerate blood clearance during the circulation process.25–27 targeted polymeric prodrugs prepared through Schiff base
Yusa and Ishihara prepared a type of pH-responsive polyion reaction with a combination of RAFT and click chemistry
complex (PIC) vesicles consisting of water-soluble zwitterionic methods have rarely been reported. It is important to develop
PMPC block and cationic or anionic blocks, which could be neoteric and integrative tactics to enhance the stability, cellular
collapsed under acidic conditions and the drug was efficiently internalization, intracellular drug release, and active targeting
released from the lysosomes to the cytoplasm.28 Ji et al. synthe- ability of the tumor cells simultaneously.
sized a pH-responsive PMPC-b-P(MEMA-hydrazide-DOX) prodrug,
Herein, we first synthesized a new kind of zwitterionic
which was stable in blood circulation, with efficient cellular random copolymer P(MPC-co-PEGMA-BZ) by RAFT copolymer-
uptake and rapid drug release in cancer cells.29 Yu et al.30 ization (Scheme 1). A pH-responsive polymeric prodrug was
prepared a novel pH responsive polymer–drug conjugate system prepared using the amino group of DOX to react with the
poly(2-(methacryloyloxy)ethyl choline phosphate)-b-poly(2-methoxy- aldehyde groups of the P(MPC-co-PEGMA-BZ) pendants through
2-oxoethyl methacrylate-hyd-doxorubicin) (PCP-b-PMEMA-hyd- a Schiff base reaction. Afterward, the thiol groups of P(MPC-co-
DOX), and confirmed that PCP-b-PMEMA-hyd-DOX can be easily PEGMA-BZ)-g-DOX were further reacted with the alkynyl groups
and rapidly internalized by various cancer cells. Gong et al. of the propargyl folate by click chemistry. The polymeric prodrug
reported a series of amphiphilic random-copolymers comprising could then self-assemble into nanoparticles in an aqueous
MPC, stearyl methacrylate (SMA) and trimethoxysilylpropyl solution, which could maintain the stability owing to the zwit-
methacrylate (TSMA). They found that the polymeric nano- terionic shell of the prodrug nanoparticles. Once the prodrug
particles could be stable in the presence of the cell membrane nanoparticles were internalized into the tumor site, they will
mimetic random-copolymers.31 Luo et al. developed a DOX- easily bind to the cell membrane by means of the folate-receptor-
conjugated poly(L-lysine)-block-poly(methacryloyloxyethyl phos- mediated effect and rapidly internalize into the cell. The Schiff base
phorylcholine) PLL(CB/DOX)-b-PMPC prodrug with 4-carboxy bond (benzoic-imine) could be cleaved under the endosomal/
benzaldehyde grafted, which could promote the micellar struc- lysosomal pH conditions. Then, the parent drug DOX could escape
tural disintegration in the tumor acid environment, leading to from the lysosome/endosome and diffuse into the cell nucleus,
an endosomal escape as well as a rapid drug release to inhibit which could efficiently inhibit tumor cells proliferation. We provide
the tumor proliferation.32
a convenient method for the fabrication of the folate-targeted
For enhancing the efficiency of the drug delivery and achieving zwitterionic prodrug, and the resulting FA-P(MPC-co-PEGMABZ)-g-
rapid release at tumor sites, many researchers have conjugated DOX prodrug nanoparticles have stability during circulation,
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Synthesis of P(MPC-co-PEGMA-BZ) copolymer
The P(MPC-co-PEGMA-BZ) copolymer with pendant benzalde-
hyde groups was synthesized via RAFT polymerization. All mag-
netic stirring bars and glasswares used in the experiment were
dried at 120 1C for 24 h and cooled under vacuum to eliminate
the moisture before use. Briefly, MPC (1.12 g, 3.80 mmol),
PEGMA-BZ (1.90 g, 3.04 mmol), CEP (10 mg, 0.038 mmol), and
azobis(isobutyronitrile) (AIBN, 3.20 mg, 0.019 mol) were added
into the flask together with a mixed solvent of 4 mL deionized
water and 4 mL DMSO. After being degassed through three
exhausting–refilling nitrogen cycles, the mixture was stirred
under a nitrogen atmosphere at 70 1C for 12 h. Then, the flask
was cooled to terminate the polymerization, and the raw product
was subsequently dialyzed (MWCO 7000) against Milli-Q water
for 24 h with the purpose of removing the unreacted monomers.
The final product P(MPC-co-PEGMA-BZ) copolymer was obtained
by lyophilization (2.3 g, yield: 76.0%).
Scheme 1 Illustration of zwitterionic shielded prodrug nanoparticles with
folate-targeting for efficient intracellular release of hydrophobic anti-
cancer drugs triggered by the acidic microenvironment inside the tumor
tissue.
efficient cellular internalization and good targeting ability to
tumor cells.
Synthesis of P(MPC-co-PEGMA-BZ)-g-DOX prodrug
P(MPC-co-PEGMA-BZ)-g-DOX prodrug was synthesized by Schiff
base reaction. Briefly, P(MPC-co-PEGMA-BZ) (150 mg, 0.0025 mmol),
DOXꢁHCl (80 mg, 0.147 mmol), and 0.5 mL of triethylamine (TEA)
were dissolved in a mixture of deionized water (4 mL) and DMSO
(4 mL), and subjected to ultrasound for 10 min. The mixture was
stirred at 30 1C for 48 h. The raw product was subsequently
dialyzed (MWCO 7000) against Milli-Q water with the purpose of
removing the unreacted DOX. For forming the thiol-group of
P(MPC-co-PEGMA-BZ)-g-DOX prodrug, we adjusted the dialysis
water to an alkaline environment through the addition of a
certain amount of TEA. The final product was obtained by
lyophilization (180 mg, yield: 78.3%).
Experimental section
Preparation of polyethylene glycol methacrylate ester
benzaldehyde (PEGMA-BZ) monomer
The synthesis procedure was carried out according to the
reported methods with slight modifications.54 Poly(ethylene
glycol) methacrylate (0.02 mol, 10.0 g) and 4-carboxybenzaldehyde
(0.04 mol, 6.0 g) were dissolved in anhydrous THF (120 mL) under
nitrogen atmosphere. Afterward, the THF solution (30 mL)
of N,N0-diisopropylcarbodiimide (DIC, 0.04 mol, 10.3 g) and
4-dimethylamino pyridine (DMAP, 0.01 mol, 1.22 g) was added
dropwise at ꢀ5 1C. The reaction mixture was heated to 30 1C for
48 h allowing the reaction to be completed. The solid deposited
was filtered off and most of the THF was removed by a rotary
evaporator. The crude product was further purified by silica gel
column chromatography (10.5 g, yield: 83.1%).
Preparation of FA-P(MPC-co-PEGMA-BZ)-g-DOX prodrug by
click chemistry
FA-P(MPC-co-PEGMA-BZ)-g-DOX prodrug was synthesized by
click chemistry. All magnetic stirring bars and glasswares used
in the experiment were dried at 120 1C for 24 h and cooled
under vacuum to eliminate the moisture before use. Briefly,
P(MPC-co-PEGMA-BZ)-g-DOX (100 mg, 0.0015 mmol), propargyl
folate (7.37 mg, 0.015 mmol), and AIBN (5 mg, 0.030 mmol) were
added into a flask together with 6 mL of deionized water/DMSO
(v/v, 1/1). After being degassed through three exhausting–refilling
nitrogen cycles, the mixture was stirred under a nitrogen atmo-
sphere at 70 1C for 12 h. Then, the flask was cooled to terminate
the polymerization, and the product was subsequently dialyzed
(MWCO 7000) against Milli-Q water for 48 h; the dialysis water
was adjusted to an alkaline environment. The final product was
obtained by lyophilization (95.2 mg, yield: 88.7%).
Synthesis of alkyne-functionalized folic acid (propargyl folate)
The synthesis of propargyl folate was accomplished by a method
derived from the literature reports of folate conjugation.55
Briefly, folic acid (1.0 g, 0.0022 mol) was dissolved in N,N-dimethyl-
formamide (DMF, 10 mL) and cooled in an ice-water bath.
N-Hydroxysuccinimide (260 mg, 0.0025 mol) and 1-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 440 mg,
0.0025 mol) were added, and the resulting mixture was stirred
in an ice-water bath for 30 min to give a white precipitate.
A solution of propargylamine (124 mg, 2.25 mmol) with 5 mL of
DMF was added, and the resulting mixture was stirred at 25 1C
for 24 h. The reaction mixture was poured into water (100 mL)
and stirred for 30 min to form a precipitate. The orange-yellow
Results and discussion
Synthesis and characterization of FA-P(MPC-co-PEGMA-BZ)-g-
DOX prodrug
precipitate was filtered, washed with acetone, and dried under In this study, a novel acid-cleavable FA-P(MPC-co-PEGMA-BZ)-g-
vacuum to a constant weight at 30 1C (0.8 g, yield: 71.2%).
DOX prodrug was prepared through the combination of RAFT
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Scheme 2 Synthesis routes of FA-P(MPC-co-PEGMA-BZ)-g-DOX polymeric prodrug.
copolymerization and click chemistry by the following steps, as
shown in Scheme 2. First, the P(MPC-co-PEGMA-BZ) copolymer was
synthesized by RAFT copolymerization of MPC and PEGMA-BZ
monomers using 4-cyano-4-ethylsulfanylthiocarbonyl-sulfanylenta-
noic acid (CEP) as chain transfer agent. Then, P(MPC-co-PEGMA-
BZ)-g-DOX was synthesized by the Schiff base reaction. Finally,
the targeting molecule, FA, was connected onto the terminal
group of P(MPC-co-PEGMA-BZ)-g-DOX by click chemistry to yield
the final product FA-P(MPC-co-PEGMA-BZ)-g-DOX.
The chemical structure of PEGMA-BZ monomer was verified by
1H NMR and 13C NMR analyses, which are shown in Fig. S1 and S2
in the ESI.† From these two figures, we identify three chemical shifts
Fig. 1 1H NMR spectrum of PMP1 random copolymer.
at d 10.11 ppm (peak i), d 8.23 ppm (peak g), and d 7.97 ppm (peak h)
that are all attributed to the protons of 4-carboxybenzaldehyde.
Furthermore, the characteristic signals at d 1.95 ppm, d 3.65 ppm,
d 3.86 ppm, d 4.30 ppm, d 4.52 ppm, d 5.58 ppm, and d 6.13 ppm
We also characterized the chemical structure of PMP1 random
belong to the protons of –CO–C(CH2)–CH3, –CH2–CH2–OCO–, copolymer by 31P NMR, as shown in Fig. S4 (ESI†), which could
–CH2–CH2–OCO–, and –CO–C(CH2) of PEGMA-BZ monomer in further verify the successful synthesis of PMP1 random copolymer.
Fig. S1 (ESI†), respectively. 13C NMR (100 MHz, DMSO-d6, d ppm): The GPC trace of PMP1 random copolymer in Fig. 2 exhibited a
%
191.55 (HCO–), 167.47 [CH3(CH2)–CO–O–], 165.32 (–OCO–CH2–), unimodal peak with a polydispersity index of 1.62, and the Mn
125.81 [–CO–C–(CH2)–CH3], 70.67 (–CH2–CH2–O–CH2–CH2–), 64.68 value of PMP1 random copolymer is 1.70 ꢂ 104 g molꢀ1. Then, the
(–CH2–CH2–O–CH2–CH2–), 18.54 [–C–(CH2)–CH3] (Fig. S2, ESI†). The P(MPC-co-PEGMA-BZ)-g-DOX prodrug (abbreviated as PMPD1) was
1
chemical structure of CEP was characterized by 1H NMR, as shown characterized by H NMR spectroscopy. As shown in Fig. 3, some
in Fig. S3 (ESI†). Afterward, a P(MPC-co-PEGMA-BZ) random chemical shifts at d 10.01 ppm and d 7.81–8.06 ppm of the PMPD1
copolymer was synthesized by RAFT copolymerization of MPC prodrug disappeared, indicating that the aldehyde group in the
and PEGMA-BZ monomers using CEP as chain transfer agent, side chain of the P(MPC-co-PEGMA-BZ) copolymer completely
which was P(MPC45-co-PEGMA-BZ40). For the sake of simplicity, reacted through the Schiff base reaction.
we designated the random copolymer as PMP1. As shown in
Fig. 1, all the chemical shifts can be assigned to the protons of calculated according to the H NMR analysis by the following
the PMP1 random copolymer. There are proton signals appearing at eqn (1) and (2):
The polymerization degrees of P(MPC-co-PEGMA-BZ) were
1
d 10.01 ppm (peak i), d 7.81–8.06 ppm (peak h + g), and d 3.21 ppm
(peak n) in Fig. 1, which can be attributed to the units of PEGMA-BZ
and MPC in the PMP1 random copolymer.
Ak
2
¼
(1)
An 9y
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%
Table 1 Size (Dz), size PDI, DOX content and FA content of PMPD1 and
FA-PMPD1 prodrug nanoparticles
a
%
Sample
PMPD-1
Dz (nm) Size PDIa DOX contentb (wt%) FA contentb (wt%)
134
0.258
0.233
28.2
23.2
—
0.46
FA-PMPD-1 144
a
b
Determined by DLS measurement. Determined by UV-vis
measurement.
Fig. 2 GPC trace of PMP1 random copolymer.
Fig. 4 HPLC analyses results of (A) free DOX and (B) PMPD1 prodrug.
HPLC analyses were performed with acetonitrile/water (50 : 50, v/v), as the
mobile phase at 30 1C with a flow rate of 1.0 mL minꢀ1
.
(abbreviated as FA-PMPD1). The UV-vis spectra of FA and
FA-PMPD1 prodrugs are shown in Fig. S7 (ESI†), in which we
can find that the peak at 275 nm and 500 nm could be attributed
to the specific absorption wavelengths of DOX and folate moieties
in the FA-PMPD1 prodrug, respectively, indicating that the DOX
and folate moieties have been successfully conjugated onto
the copolymer. In this study, the DOX contents of PMPD1 and
FA-PMPD1 have been calculated by eqn (S1) (ESI†), the results are
listed in Table 1. Moreover, the folate content of FA-PMPD1
prodrug was calculated to be 0.46 wt% by eqn (S2) in the ESI.†
For further verifying that the free DOX had been conjugated
to the side chains of P(MPC-co-PEGMA-BZ), HPLC was used; the
HPLC results are shown in Fig. 4. We can see that the free DOX
elutes at 5.2 min in Fig. 4(A), while the PMPD1 prodrug elutes at
3.7 min in Fig. 4(B). There are no traces at 5.2 min in Fig. 4(B),
which could confirm that P(MPC-co-PEGMA-BZ)-g-DOX has
been purified and does not contain free DOX.
Fig. 3 1H NMR spectrum of PMPD1 prodrug.
0
0
Ac þd þm 32x þ 9y
¼
(2)
Ak
2
0
0
where Ak, An, and, Ac +d +m were the integral values of the peaks
k, n, and c0 + d0 + m in Fig. 1, respectively.
%
The Mn,NMR of the P(MPC-co-PEGMA-BZ) random copolymer
was calculated using the following eqn (3):
%
Mn,NMR = 632x + 294.73y + 264.86
(3)
where 632 g molꢀ1 and 294.73 g molꢀ1 were the molecular
weights of one repeating unit of the PEGMA-BZ and MPC,
respectively, and 264.86 g molꢀ1 was the molecular weight of
the CEP. x and y represent the polymerization degrees of
PEGMA-BZ and MPC, respectively.
Self-assembly of the polymeric prodrugs
The critical aggregation concentration (CAC) represents the
The new peak at d 8.01 ppm (peak a) can be attributed to the thermodynamic stability of the nanoparticles in an aqueous
–HCQN– of the P(MPC-co-PEGMA-BZ)-g-DOX prodrug in Fig. 3, medium. When the concentration of the prodrug is higher
which demonstrates that the PMPD prodrug has been success- than the CAC value, the PMPD1 and FA-PMPD1 prodrug can
fully prepared. The chemical structure of propargyl folate has self-assemble into nanoparticles with DOX as the core and
1
been characterized by H NMR and is shown in Fig. S5 (ESI†). hydrophilic P(MPC-co-PEGMA-BZ) as the shell in an aqueous
The thiol (–SH)-functionalized P(MPC-co-PEGMA-BZ)-g-DOX solution. The CAC value of PMPD1 and FA-PMPD1 prodrug
was obtained by an aminolysis reaction. We can confirm the nanoparticles was determined by the steady-state fluorescence
successful synthesis of the thiol-functionalized prodrug due to probe method using pyrene as the probe. The CAC values of the
the discernible UV-vis peaks of the –SH group, as seen in Fig. S6 PMPD1 and FA-PMPD1 prodrug nanoparticles are 47.9 mg Lꢀ1
(B, red line) (ESI†). Subsequently, the FA-P(MPC-co-PEGMA-BZ)- and 51.8 mg Lꢀ1 as shown in Fig. S8(A and B) (ESI†), respectively.
g-DOX prodrug was prepared by click chemistry method The morphologies of PMPD1 and FA-PMPD1 nanoparticles
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Fig. 5 TEM image of FA-PMPD1 prodrug nanoparticles (A) and the particle
size distribution curve (B) corresponding to the TEM samples (scale bar
500 nm). The concentration was 0.5 mg mLꢀ1
.
were observed by TEM analysis, while the average particle
%
diameters (Dz) and size polydispersity index (size PDI) of the
prodrug nanoparticles were determined by dynamic light
scattering (DLS), and the results are listed in Table 1.
As illustrated in Fig. 5(A) and Fig. S9(A) (ESI†), the morphologies
of PMPD1 and FA-PMPD1 prodrug nanoparticles exhibited rela-
tively uniform distribution. For PMPD1 and FA-PMPD1 prodrug
nanoparticles, the corresponding particle size distribution
curves with an average diameter of 144 nm and 134 nm are
displayed in Fig. 5(B) and Fig. S9(B) (ESI†), respectively.
The stability of FA-PMPD1 prodrug nanoparticles was then
observed by DLS measurement. The prodrug nanoparticles could
maintain good stability over a period of 50 days as shown in
Fig. S10 (ESI†). The particle size distribution of FA-PMPD1
prodrug nanoparticles has almost no significant changes, which
could be ascribed to the hydrophilic property of the zwitterionic
PC moiety. These results indicate that the FA-PMPD1 prodrug
nanoparticles with stealthy PC shell could prolong circulation
time and have possibility to enhance tumor accumulation
in vivo.56,57
Fig. 6 Size change of FA-PMPD1 prodrug nanoparticles under different
conditions of pH 7.4 and pH 5.0 as determined by DLS measurements.
pH-Responsive ability and colloid stability of the prodrug
nanoparticles
To investigate the pH-responsive ability of FA-PMPD1 prodrug
nanoparticles, the particle size evolutions of FA-PMPD1 pro-
drug nanoparticles at pH 5.0 and 7.4 were determined by DLS
measurement, respectively. As shown in Fig. 6, the size of the
FA-PMPD1 prodrug nanoparticles showed no significant
change at pH 7.4 for 48 h. However, the average size of the
prodrug nanoparticles increased significantly with the increas-
ing incubation time at pH 5.0. The size of the prodrug nano-
particles further expanded to hundreds of nanometers at 48 h
intervals, or even larger. This may be caused by the aggregation
of hydrophobic segments to form larger aggregates at pH 5.0
Fig. 7 TEM images of FA-PMPD1 prodrug nanoparticles incubation at pH
7.4 (A) and pH 5.0 (B) at 48 h intervals. The concentration of the prodrug
nanoparticles was kept at 0.5 mg mLꢀ1
.
after 48 h, indicating that the FA-PMPD1 prodrug nanoparticles after 48 h, as shown in Fig. 7(B). The results could indicate that
could be remarkably destroyed in an acidic medium.
FA-PMPD1 prodrug nanoparticles could maintain the stability
In addition, we used TEM to observe the morphology under physiological conditions and the FA-PMPD1 prodrug
changes in the FA-PMPD1 prodrug nanoparticles. As shown nanoparticles could be dissociated under mildly acidic condi-
in Fig. 7(A), it should be noted that the morphology of the tions. We can see that the results obtained from the DLS
FA-PMPD1 prodrug nanoparticles could maintain the uniform measurement in Fig. 6 are consistent with those of the TEM
structure under the condition of pH 7.4 at 48 h intervals. measurement in Fig. 7, indicating that the FA-PMPD1 prodrug
However, irregular aggregates were clearly observed at pH 5.0 nanoparticles have pH-responsive properties.
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Fig. 9 Cell viabilities of L929 cells, HeLa cells, and HepG2 cells treated with
PMP1 random copolymer at different concentrations with 48 h of incubation.
drug carrier to treat some ongoing challenges in current
tumor treatment.
For evaluating their antitumor efficacy, the antiproliferation
activities of the free DOX, PMPD1, and FA-PMPD1 prodrug
nanoparticles against HeLa cells were investigated using MTT
assays; this is because the folate receptor was over-expressed in
the HeLa cells.45,58 As shown in Fig. 10, the cell viabilities of
HeLa cells decreased gradually when the DOX concentration
increased from 0.02 mg Lꢀ1 to 10 mg Lꢀ1. The half-maximal
inhibitory concentration (IC50) values of free DOX, PMPD1, and
FA-PMPD1 prodrug nanoparticles against HeLa cells were
Fig. 8 In vitro DOX release curves for PMPD1 (A) and FA-PMPD1 (B)
prodrug nanoparticles at pH 7.4 and pH 5.0. The nanoparticles concen-
tration was 1 mg mLꢀ1
.
determined to be 0.17 mg Lꢀ1, 0.76 mg Lꢀ1, and 0.62 mg Lꢀ1
,
In vitro drug release. The release profiles of PMPD1 and
FA-PMPD1 were studied at the physiological pH 7.4 and intra-
cellular acidic pH 5.0. As shown in Fig. 8, approximately 80% of
DOX was released from the PMPD1 prodrug nanoparticles after
incubation for 98 h at pH 5.0, whereas the release of DOX
was about 15% at pH 7.4. The DOX could be released from the
FA-PMPD1 prodrug nanoparticles at pH 7.4, the final prodrug
nanoparticles cumulative release rate was approximately 7.3%
after 140 h. Notably, the DOX release was evidently accelerated
at pH 5.0, and almost 70% of DOX was released from the
FA-PMPD1 prodrug nanoparticles within 140 h, as shown in
Fig. 8(B). These results indicate that the cleavage of the Schiff
base bond and the disassembly of the prodrug nanoparticles
could be accelerated at pH 5.0, which could indeed cause the
fast release of DOX from the PMPD1 and FA-PMPD1 prodrug
nanoparticles. In addition, the PMPD1 and FA-PMPD1 prodrug
nanoparticles can reduce premature drug release in the blood-
stream and promote drug burst release under endo/lysosomal
acidic condition in the tumor cells.
respectively. The in vitro antitumor efficacy of free DOX was
higher than those of the PMPD1 and FA-PMPD1 prodrug
nanoparticles because it is easily diffused through the cellular
membrane, enhancing the antitumor function of free DOX.59–61
Another reason is due to the overexpressed folate FRa receptors
in HeLa cells, which can indeed lead to enhanced cytotoxicity of
the FA-PMPD1 prodrug nanoparticles. We further monitored the
endocytosis process by a living cell-image system. The results
In vitro cytotoxicity. In this study, we used MTT assays to
investigate the cytotoxicity of the PMP1 random copolymer
against normal cells (L929 cells) and cancer cells (HeLa cells
and HepG2 cells). In Fig. 9, the cell viability assays demonstrated
that the PMP1 random copolymer has good biocompatibility
and low toxicity against L929 cells. The same tendency was
found in two other tumor cell lines, which could indicate that
the PMP1 random copolymer may be a potential prospect as a
Fig. 10 Cell viability of HeLa cells treated with free DOX, PMPD1, and
FA-PMPD1 with different DOX dosages for 48 h of incubation. The IC50
values of free DOX, PMPD1, and FA-PMPD1 were calculated by GraphPad
Prism 5 software.
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For analysing the fluorescence intensity of HeLa cells incu-
bated with free DOX, PMPD1, and FA-PMPD1 prodrug nano-
particles for fixed time intervals (1 h, 3 h, and 6 h), we recorded
the fluorescence intensity of HeLa cells using flow cytometry. As
shown in Fig. 12, the relative geometrical mean fluorescence
intensities of the HeLa cells with PMPD1 and FA-PMPD1
prodrug nanoparticles increased with increasing time. Notably,
there are stronger DOX fluorescence intensities at 6 h than at
1 h, indicating that the uptake of nanoparticles was a time-
dependent property. We could find that the fluorescence inten-
sity of the free DOX was higher than that of the PMPD1 prodrug
nanoparticles incubated for 6 h, as shown in Fig. 12(A). FA-
PMPD1 prodrug nanoparticles had significantly higher cellular
uptake than free DOX at the same incubation time, as seen in
Fig. 12(B), implying that FA could enhance the phagocytosis of
FA-PMPD1 by the over-expression of FA receptors on the HeLa
cells surface.
Fig. 11 Live cell-imaging system images of HeLa cells incubated with (A)
FA-PMPD1 nanoparticles, (B) free DOX and (C) PMPD1 nanoparticles at
different times. (The DOX dosage was 4 mg Lꢀ1.) For each panel, images
from left to right show cell nuclei stained by H 33342 (blue), DOX
fluorescence in cells (red), and overlays of the blue and red images. The
scale bars are 50 mm in all images.
Conclusions
showed that FA-PMPD1 prodrug had better endocytosis effect
(Fig. 11A) than those of free DOX (Fig. 11B) and the non-targeted
PMPD1 prodrug nanoparticles (Fig. 11C) in HeLa cells,62,63
demonstrating that the folate enhanced the targeting ability
of the FA-PMPD1 prodrug nanoparticles on internalization of
HeLa cells.
In summary, we have developed a novel pH-sensitive zwitter-
ionic prodrug FA-P(MPC-co-PEGMA-BZ)-g-DOX with a combi-
nation of RAFT and click chemistry methods. The zwitterionic
polymeric prodrug could self-assemble into nanoparticles, which
possesses good stability due to the stealthy phosphorylcholine
(PC) and PEGMA moiety under physiological conditions. The
covalently linked DOX could be released from the FA-PMPD
prodrug nanoparticles due to the cleavage of the benzoic-imine
bond at pH 5.0, and an accumulative DOX release of FA-PMPD1
prodrug nanoparticles was up to 70.0%, demonstrating a remark-
able pH-triggered drug release behavior. In addition, the in vitro
MTT assay results revealed that the PMPD and FA-PMPD prodrug
nanoparticles could exhibit superior antitumor effects against
HeLa cells with IC50 of 0.76 mg Lꢀ1 and 0.62 mg Lꢀ1 after 48 h
of incubation, respectively. Meanwhile, we utilized cellular uptake
and flow cytometry analyses to confirm that the FA-PMPD prodrug
nanoparticles possessed targeting ability. This is due to the
presence of large amount of over-expression FA receptors on HeLa
cell surface. Therefore, the cell membrane mimicking zwitterionic
FA-P(MPC-co-PEGMA-BZ)-g-DOX prodrug nanoparticles show
great potential in the application of cancer treatment.
Conflicts of interest
The authors declare no competing financial interest.
Acknowledgements
We gratefully acknowledge the financial supports from the
National Natural Science Foundation of China (21374066),
the Major Program of the Natural Science Project of Jiangsu
Higher Education Institutions (15KJA150007), the Natural
Science Foundation of Jiangsu Province (BK20171212), a Project
Funded by the Priority Academic Program Development (PAPD)
of Jiangsu Higher Education Institutions, and Soochow-Waterloo
Fig. 12 Flow cytometry curves of HeLa cells treated with (A) PMPD1 and
(B) FA-PMPD1 prodrug nanoparticles for different times (1 h, 3 h, and 6 h)
with a certain concentration of DOX (4 mg Lꢀ1).
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University Joint Project for Nanotechnology from Suzhou
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