One-Step Preparation of pH-Responsive Polymeric Nanogels as Intelligent Drug Delivery Systems for Tumor Therapy

Article abstract of DOI:10.1021/acs.biomac.8b00195

In this work, pH-responsive polypeptide-based nanogels are reported as potential drug delivery systems. By the formation of pH-sensitive benzoic imine bonds, pH-responsive nanogels are constructed using hydrophilic methoxy poly(ethylene glycol)-b-poly[N-[N-(2-aminoethyl)-2-aminoethyl]-l-glutamate] (MPEG-b-PNLG) and hydrophobic terephthalaldehyde (TPA) as a cross-linker. At pH 7.4, MPEG-b-PNLG nanogels exhibit high stabilities with hydrophobic inner cores, which allow encapsulation of hydrophobic therapeutic agents. Under tumoral acidic environments (pH ～6.4), the cleavage of benzoic imine bonds induces the destruction of MPEG-b-PNLG nanogels and leads to rapid release of their payloads. The formation and pH sensitivity of the nanogels are investigated by dynamic light scattering. These nanogels exhibit excellent stabilities in the presence of salt or against dilution. The globular morphologies of the nanogels are confirmed using transmission electron microscopy. Doxorubicin is used as a model drug to evaluate drug encapsulation and release. Finally, the anticancer activities of the drug-encapsulated nanogels are assessed in vitro.

Full text of DOI:10.1021/acs.biomac.8b00195

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One-Step Preparation of pH-Responsive Polymeric Nanogels
as Intelligent Drug Delivery Systems for Tumor Therapy
Yi Li, Quang Nam Bui, Le Thai Minh Duy, Hong Yu Yang, and Doo Sung Lee
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00195 • Publication Date (Web): 06 Apr 2018
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OneꢀStep Preparation of pHꢀResponsive Polymeric
Nanogels as Intelligent Drug Delivery Systems for
Tumor Therapy
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Yi Li1, Quang Nam Bui1, Le Thai Minh Duy, Hong Yu Yang and Doo Sung Lee*
1These authors contributed equally to this work
School of Chemical Engineering and Theranostic Macromolecules Research Center,
Sungkyunkwan University, Suwon, 16419, Republic of Korea
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ABSTRACT: In this work, pHꢀresponsive polypeptideꢀbased nanogels are reported as potential
drug delivery systems. By formation of pHꢀsensitive benzoic imine bonds, pHꢀresponsive
nanogels are constructed using hydrophilic methoxy poly(ethylene glycol)ꢀbꢀpoly[Nꢀ[Nꢀ(2ꢀ
aminoethyl)ꢀ2ꢀaminoethyl]ꢀLꢀglutamate] (MPEGꢀbꢀPNLG) and hydrophobic terephthalaldehyde
(TPA) as a crosslinker. At pH 7.4, MPEGꢀbꢀPNLG nanogels exhibit high stabilities with
hydrophobic inner cores, which allow encapsulation of hydrophobic therapeutic agents. Under
tumoral acidic environments (pH ~ 6.4), the cleavage of benzoic imine bonds induces the
destruction of MPEGꢀbꢀPNLG nanogels and leads to rapid release of their payloads. The
formation and pHꢀsensitivity of the nanogels are investigated by dynamic light scattering. These
nanogels exhibit excellent stabilities in the presence of salt or against dilution. The globular
morphologies of the nanogels are confirmed using transmission electron microscopy (TEM).
Doxorubicin is used as a model drug to evaluate drug encapsulation and release. Finally, the
anticancer activities of the drugꢀencapsulated nanogels are assessed in vitro.
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KEYWORDS: Nanogels, pHꢀsensitive, Polypeptide, Drug delivery, Anticancer
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INTRODUCTION
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In the field of nanotherapy, nanogels have been considered as potential drug delivery platforms
due to their outstanding advantages.1 In general, nanogels are prepared by crosslinking of
preformed polymeric nanoparticles or through templateꢀassisted fabrication.2 Nanogels,
especially environmentꢀresponsive nanogels, are frequently employed as drug delivery systems
(DDS), since they can fulfill many demands such as outstanding serum stabilities, high
therapeutic efficiencies, low side effects and onꢀdemand release of payloads. Intelligent nanogels
that can respond to a wide range of stimuli (e.g. pH, temperature, redox, light and glucose) have
been reported by many groups.3, 4
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The high mortality rates caused by cancer have increased the need for drug delivery systems to
improve the therapeutic efficiencies of antiꢀcancer drugs.5, 6 To treat cancers more efficiently,
smart drug delivery systems were widely developed based on the intrinsic features of various
cancers (e.g. extracellular and intracellular acidities, hypoxia and disorganized vasculatures).7, 8
Among these intrinsic features, acidic extracellular conditions, caused by anaerobic glycolysis in
the hypoxic tumor microenvironment, is considered to be a major hallmark of tumor tissues.9ꢀ11
Therefore, tumor extracellular pHꢀtargeting drug delivery systems based on different
mechanisms have been developed.12ꢀ14 Similarly, pHꢀsensitive nanogels that are prepared using
different polymers (both synthetic and natural),15, 16 or different release mechanisms (pHꢀ
triggered swollen or disassembly)17 also gained much attention for programmed drug delivery to
deliver either hydrophobic or hydrophilic therapeutic agents.18, 19 To date, pHꢀresponsive
nanogels are mainly prepared by two methods. One method is copolymerization of pHꢀ
responsive monomers and bifunctional crosslinkers. For example, Wang and coworkers
copolymerized acrylic acid and N,Nꢀbis(acryloyl)cystamine in the presence of 2,2ꢀ
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azobis(isobutyronitrile) as an initiator.18 The asꢀobtained nanogels can be further conjugated with
poly(ethylene glycol) (PEG) and loaded with drugs to generate a core/shellꢀstructured
nanomedicine.19 However, the nanogels prepared by this method is nonꢀbiodegradable and
insensitive to subtle pH decrease (drug release is accelerated only when pH is decreased to 5). In
addition, the preparation of drugꢀloaded nanogels is a complicated process and inconvenient for
real application. The other method for pHꢀresponsive nanogel preparation is crosslinking of preꢀ
formed pHꢀresponsive and selfꢀassembled micelles. For example, Chen et al. synthesized a pHꢀ
responsive methoxyl poly(ethylene glycol)ꢀbꢀpolyꢀ(N,N’ꢀdiethylaminoethyl methacrylate)
copolymer and prepared a micelle solution from this diblock copolymer.20 Next, a bifunctional
crosslinker was employed to crosslink the cores of the micelles by quaternization reaction. When
pH of the solution is decreased from 7.4 to 6.8, drugs can be released at a faster rate due to
expansion of the nanogels at this pH. In another report, Yusa et al. fabricated a pHꢀresponsive
nanogel by photoꢀcrosslinking of a preꢀformed pHꢀresponsive polymeric micelle.21 However,
these nanogels are nonꢀbiodegradable and cannot disassociate under acidic conditions.
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In this study, we propose a new approach for fabrication of pHꢀresponsive nanogels through a
facile oneꢀstep method. A waterꢀsoluble polypeptideꢀbased copolymer, methoxy poly(ethylene
glycol)ꢀbꢀpoly[Nꢀ[Nꢀ(2ꢀaminoethyl)ꢀ2ꢀaminoethyl]ꢀLꢀglutamate] (MPEGꢀbꢀPNLG), was directly
crosslinked in an aqueous solution using terepthalaldehyde (TPA) at physiological pH (pH 7.4).
The acidꢀlabile benzoic imine bonds can be cleaved under mildly acidic condition (pH 6.4). The
asꢀprepared nanogels can physically encapsulate therapeutic molecules with high stabilities
under the protection of the MPEG shell. The mechanism of pHꢀtriggered release of DOX from
this nanogel system is briefly shown in Scheme 1. Under physiological pH, the cores of the
nanogels can protect the payloads from nonspecific absorption and minimize side effects. Once
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the drugꢀloaded nanogel system accumulates at tumor sites and is exposed to the tumor acidic
conditions, the cores can be rapidly destructed due to the cleavage of benzoic imine bonds,
resulting in enhanced accumulation of drugs at tumor sites. Compared with the other reported
pHꢀresponsive nanogels, our nanogels have advantages in three aspects; (1) preparation of
nanogels as well as drug loading can be achieved in one step (direct crosslinking of solubilized
polymer through dynamic linkers), which is convenient over the other reported methods; (2) the
polymer we use is biodegradable and biocompatible, and the nanogels are responsive to subtle
pH changes; (3) a decrease in pH leads to both the release of drugs and disassociation of the
nanogels, which makes the clearance of the drug carrier possible after achieving their missions.
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Scheme 1. Formation of pHꢀresponsive MPEGꢀbꢀPNLG nanogels and their drug delivery
mechanism.
EXPERIMENTAL SECTION
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Materials. LꢀGlutamic acid γꢀbenzyl ester (BLG), 2ꢀhydroxypyridine (2ꢀHP), ethylenediamine
(EDA), terephthalaldehyde (TPA), doxorubicin hydrochloride (DOX·HCl), triethylamine (TEA),
chloroform (CHCl3) and dimethylformamide (DMF) were purchased from SigmaꢀAldrich (St.
Louis, MO, USA). Triphosgene was obtained from Tokyo Chemical Industry Co., Ltd. Methoxy
poly(ethylene glycol) amine (Mw = 5000 Da) was acquired from Sunbio.
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Synthesis of γꢀbenzylꢀLꢀglutamateꢀNꢀcarboxyanhydride (BLGꢀNCA). Daly’s method was used
to prepare the BLGꢀNCA monomer.22 Briefly, BLG (1 eq.) and triphosgene (0.6 eq.) were
dissolved in anhydrous THF at 50oC for 1 hr. The reaction was terminated by pouring the
mixture directly into cold hexane. The resultant solution was kept at ꢀ20oC overnight for
recrystallization. The next day, the filtrate was further recrystallized twice in an ethyl
acetate/hexane mixture with a volume ratio of 1:2 at ꢀ20oC for 12 hr.
Synthesis of methoxy poly(ethylene glycol)ꢀblockꢀpoly(γꢀbenzylꢀLꢀglutamate) (MPEGꢀbꢀ
PBLG). The precursor copolymer MPEGꢀbꢀPBLG was synthesized by ringꢀopening
polymerization.23ꢀ25 Briefly, MPEGꢀNH2 (1 eq.) and BLGꢀNCA (40 eq.) were dissolved in 20
mL of anhydrous CHCl3. The mixture was kept under nitrogen protection for 72 hr at room
temperature (RT). The resultant polypeptide was collected by precipitation in cold ether and
dried in vacuum oven at RT for 48 hr.
Synthesis of methoxy poly(ethylene glycol)ꢀbꢀpoly[Nꢀ[Nꢀ(2ꢀaminoethyl)ꢀ2ꢀaminoethyl]ꢀLꢀ
glutamate] (MPEGꢀbꢀPNLG). MPEGꢀbꢀPNLG was synthesized by aminolysis of MPEGꢀbꢀ
PBLG using a large excess amount of ethylene diamine (EDA).26 Briefly, a mixture of MPEGꢀbꢀ
PBLG and an excess amount of 2ꢀHP was dissolved in anhydrous DMF at 55oC and kept under
the protection of N2. Subsequently, a large excess amount of EDA was added to the mixture, and
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the mixture could stand for 72 hr. The yellowish and viscous product was collected by
precipitation in cold ether. The crude product was purified by dialyzing the yellow liquid in a
dialysis tube (3.5k MWCO) against HCl solution (0.01 M) for 1 day and deionized water (DIW)
for another 2 days. The final MPEGꢀbꢀPNLG copolymer was obtained as a white powder after 3
days of lyophilization.
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Characterization of polymers. The chemical structures of the copolymers (MPEGꢀbꢀPBLG and
MPEGꢀbꢀPNLG), the nanogels and the nanogels after disassociation were analyzed using proton
nuclear magnetic resonance spectroscopy (1HꢀNMR). 1HꢀNMR spectra were recorded on Varian
Unity Inova 500NB (500 MHz) in dimethyl sulfoxideꢀd6 or D2O at room temperature.
The molecular weights (Mn) of the polymers were measured by gel permeation
chromatography (GPC) using DMSO as an eluent and a flow rate of 1.0 mL/min. The molecular
weights were calibrated using PEG standards.
Preparation and characterization of nanogels. Firstly, 10 mg of MPEGꢀbꢀPNLG was dissolved
in 10 mL of PBS or DIW at pH 7.4. Next, a solution of TPA (10 mg/mL in THF) was added to
the aboveꢀmentioned aqueous copolymer solution, and the mixture was agitated for 1 hr at RT.
After that, THF was removed using a rotary evaporator. The obtained solution was used for
further characterizations.
FTꢀIR spectra of the MPEGꢀbꢀPNLG copolymer, nanogels and the nanogels after
disassociation at low pH were recorded using an FTꢀIR spectrometer (IFSꢀ66/S, Bruker, USA).
The spectra were recorded in the range of 650 ꢀ 4000 cmꢀ1 with a resolution of 4 cmꢀ1.
pHꢀresponsibility of the nanogels. The surface charge and particle size of the nanogels (1
mg/mL) at various pH values were characterized by dynamic light scattering (Zetasizer, Nanoꢀ
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ZS90). Different amounts of TPA were added to the MPEGꢀbꢀPNLG polymer solution to get
different crosslinking densities (the molar ratio of CHO/NH2 was adjusted from 0.2 to 1.2).
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Stability and morphology of the nanogels. To check the stability of the nanogels against salt at
37oC, the concentration of NaCl in the nanogel solutions was adjusted from 0 to 2 M. Moreover,
to check the stability of the nanogels against dilution, the concentration of the nanogels in PBS
was lowered gradually to 15.63 µg/mL. Then, the changes in particle diameter were measured
and compared with one another. On the other hand, the stability of the nanogels during long term
storage was also evaluated by size measurements. The morphologies of the MPEGꢀbꢀPNLG
nanogels were observed using transmission electron microscopy (TEM) (JEOL USA, JEMꢀ
2100F). The TEM sample was prepared by a sequential twoꢀdroplet method with the
tungstophosphoric acid solution (1% w/w in DIW) as a negative staining solution. Briefly, 10 µL
of the nanogels in DIW (pH 7.4, 1 mg/mL, CHO/NH2 = 1) was placed onto the dull side of the
grid. The suspension was dried by tapping on a filter paper. Afterward, 20 µL of the staining
solution was dropped onto the grid and removed after 30 seconds using filter paper. The stained
grid was further washed twice with DIW to remove excess amounts of the staining agent and
then allowed to be ambientꢀdried for a few hours before TEM scanning.
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Drug loading and release. An anticancer drug, DOX·HCl (1 mg), was dissolved in 500 µL of
THF that contains 5.2 mg of TEA for 24 hr. Then the DOX solution was filtered (via PTFE
syringe filters, 0.22 µm) to remove the insoluble TEA·HCl salt. Subsequently, TPA (2.6 mg) was
added to the aboveꢀmentioned solution. The DOX/TPA mixture was slowly dropped into MPEGꢀ
bꢀPNLG in PBS (7.4 mg in 10 mL) under vigorous stirring for 1 hr. THF was then evaporated
under reduced pressure to get the DOXꢀloaded nanogel solution. The untrapped DOX, trace
amount of THF and remaining TEA·HCl were further removed by dialysis (MWCO ~ 3.5K)
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against 4 L of PBS at RT. The purified DOXꢀloaded nanogels were collected after lyophilization
and then reꢀdispersed in PBS for UVꢀVis measurements. Drug loading efficiency (DLE) and
drug loading content (DLC) were calculated by the following equations:
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weight of loaded drug
ꢀ ꢁ
DLE % =
× 100%
× 100%
weight of feeding drug
weight of loaded drug
weight of nanogel
ꢀ ꢁ
DLC % =
The release of DOX from the nanogels was measured by a dialysis method as published in our
previous report with slight modifications.27 A volume of 1 mL of the DOXꢀloaded nanogel
solution (DOX concentration, 100 µg/mL) was dialyzed against 30 mL of PBS solution (pH 7.4
or 6.4) at 37oC under a shaking speed of 100 rpm. A volume of 3 mL of exterior aqueous
medium was collected at the following time intervals i.e., 1, 2, 4, 8, 12 and 24 hr, respectively.
Afterward, an equal volume of fresh media was added. The concentrations of DOX in the
collected samples were determined by UVꢀVis spectroscopy with an absorption wavelength of
490 nm.
Cytotoxicity of MPEGꢀbꢀPNLG nanogels. Human embryonic kidney cell line 293T (supplied by
ATCC, Manassas, VA, USA) was used to evaluate the biocompatibility of MPEGꢀbꢀPNLG
nanogels through a MTT viability assay. The 293T cells were cultivated and seeded in 96ꢀwell
plates at a concentration of 104 cells/well in Dulbecco’s Modified Eagle’s Medium (DMEM)
(37oC, 5% CO2). After 24 hr, MPEGꢀbꢀPNLG nanogel solutions (in PBS) were fed to wells at
various final concentrations (0 to 500 µg/mL). The 96ꢀwell plate exposed to different treatments
was further incubated at 37oC (5% CO2) for another 24 hr. The plate was filled with 20 µL of
MTT solution (5%) per well and subsequently incubated in the incubator for 2 hr (37oC, 5%
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CO2). To dissolve the formazan crystals, DMSO was added to the plate (200 µL/well), which
was darkly shielded and incubated at 60oC for 1 hr. A microplate reader was used to measure UV
absorbance of MTT at a wavelength of 570 nm.
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Cancer cellꢀkilling effect of DOXꢀloaded nanogels. The human cancer cell line, MDAꢀMBꢀ231
(ATCCHTBꢀ26), was chosen as a model to evaluate the killing effect of DOXꢀloaded nanogels
via a MTT assay (free DOX was used as the control group). A 96ꢀwell plate was prepared with
104 cells/well in Roswell Park Memorial Institute (RPMI) media. The cells were incubated with
DOXꢀloaded nanogels or free DOX samples at various DOX concentrations (0, 5, 10, 15, 20, and
25 µg/mL) for 24 hr (37oC, 5% CO2). The MTT assay was done to evaluate cell viability with a
microplate reader (570 nm).
Cell uptake. MDAꢀMBꢀ231 cells were seeded onto the 35ꢀmm cell culture dish (106 cells/dish).
DOXꢀloaded nanogels and free DOX samples were treated with the cells at the final DOX
concentration of 3 µg/mL. The internalization was characterized via fluorescenceꢀactivated cell
sorting (FACS), with PBS as a negative control. By varying treatment times (1 and 3 hr), the live
cells were trypsinized and collected through washing cycles via centrifugation, which was then
dispersed again in 200 µL of PBS. The fluorescence absorbance of cells that had taken up the
drug was monitored via a flow cytometry instrument (MACSQuant Analyzer 10, Miltenyi
Biotec.) using a population of 20,000 cells.
The cellular uptake of DOXꢀloaded nanogels and free DOX were further confirmed using
confocal laser scanning microscopy (CLSM, LSM700 Carl Zeiss) X400. Briefly, MDAꢀMBꢀ231
cells were prepared in confocal disks (1x104 cells) in RPMI. The cells were incubated with
different DOX formulations at a final concentration of 3 µg of DOX per mL for different
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treatment periods (1 and 3 hr). Nuclei were stained with 4, 6ꢀdiamidinoꢀ2ꢀphenylindole (DAPI)
for 10 min and washed twice with PBS before being fixed with 3.7% formaldehyde. Afterward,
the cells were observed using CLSM.
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RESULTS AND DISCUSSION
The route for synthesis of the copolymer MPEGꢀbꢀPNLG is illustrated in Scheme 2. The
1
chemical structures of both MPEGꢀbꢀPBLG and MPEGꢀbꢀPNLG were determined by HꢀNMR.
The number of repeating units of the PBLG block was calculated using the ratio between the
integrations of the peak corresponding to the methyl groups of MPEG (a, CH3ꢀ, 3.14 ppm) and
that of the methylene groups (f, ꢀCH2ꢀ, 5.17 ppm) (Figure 1C). The disappearance of benzyl
groups together with the appearance of the ethyl groups of EDA (peak i and peak j in Figure 1B)
confirmed the complete aminolysis of the benzyl groups. These results were summarized in
Table 1. Ringꢀopening polymerization is a living polymerization that allows the molecular
weight of the obtained polymers to be similar with the designed molecular weight. A series of
polypeptides with structures similar to PNLG have been investigated using different diamine
compounds for various applications in previous publications.23ꢀ26 Since MPEGꢀbꢀPNLG is fully
soluble in D2O, all NMR peaks can be identified in the NMR spectra. However, after
crosslinking the PNLG blocks using TPA in D2O with NaOD as a pHꢀadjusting agent, the
formation of benzoic imine bonds led to the formation of a hydrophobic PNLG/TPA core which
was protected by MPEG shells. Therefore, only proton peaks corresponding to the MPEG block
appeared in the NMR spectra. In Figure 1A, the disappearance of pendant amino groups in the
spectra confirmed formation of nanoparticles with MPEG blocks as hydrophilic shells. This
phenomenon has also been observed in several studies that are related to amphiphilic polymeric
nanoparticles.28, 29 Figure 1D shows the NMR spectrum of the MPEGꢀbꢀPNLG nanogels after
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disassociation at pH 6.4. It can be seen that the peaks that belong to the PNLG block appeared
again in the spectrum, which revealed the disassociation of MPEGꢀbꢀPNLG nanogels at low pH.
Moreover, peak k in Figure 1D corresponds to the protons on the benzene rings of the TPA
crosslinker. The molecular weight (Mn) and molecular weight distribution of MPEGꢀbꢀPNLG
copolymer were measured by GPC. A unimodal peak is observed in Figure S1, and the
copolymer has a Mn of 10,776 Da and a polydispersity index of 1.18.
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Scheme 2. Synthesis routes of MPEGꢀbꢀPBLG copolymer and MPEGꢀbꢀPNLG copolymer.
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Figure 1. 1HꢀNMR spectrums of (A) MPEGꢀbꢀPNLG nanogels, (B) MPEGꢀbꢀPNLG copolymer,
(C) MPEGꢀbꢀPBLG copolymer, and (D) MPEGꢀbꢀPNLG nanogels after disassociation.
Table 1. Characterizations of copolymers and nanogels.
Molecular Weighta) Particle Sizeb) DLEc)
DLCc)
(%)
Sample
(g/mol)
(nm)
(%)
MPEGꢀbꢀPBLG
13,769
ꢀ
ꢀ
ꢀ
MPEGꢀbꢀPNLG
Blank nanogels
11,848
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
56.83
109.1
DOXꢀloaded nanogels
64.6
6.07
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a)
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b)
c)
Calculated from HꢀNMR spectrums; Determined from DLS; Calculated from UVꢀVis
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absorbance.
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The dynamic light scattering method was used to confirm the formation of the nanogels and to
study the reversibility of the crosslinking. At pH 7.4, benzoic imine bonds between the primary
amines pendant groups of PNLG blocks and benzaldehyde groups of TPA were formed, which
subsequently induced nanoscale gelation and resulted in nanoparticles with a mean size of 56.83
± 1.99 nm. In order to investigate the reproducibility of the sizes of nanogels, statistics on sizes
of MPEGꢀbꢀPNLG nanogels from different batches is provided (Table S1). Dynamic light
scattering also confirmed that the pHꢀinduced formation of the nanogel is reversible; when the
pH is decreased from 7.4 to 6.4, the nanogels disassociated and the polymer chains were
dissolved in water. The size change in this process was shown in Figure 2A. The nanogels have
a mean size of 56.83 nm at pH 7.4; while, at pH 6.4, no particles were detected. When pH was
increased from 6.4 to 7.4, the nanoparticles with a similar mean particle size (53.26 ± 1.66 nm)
can be formed. As expected, the surface charges of the nanogels (Figure 3A) showed similar
zigzag tendencies when the pH was adjusted between 7.4 and 6.4 (the changes of zeta potential
values between pH 6.4 (18.9 and 20.1) and pH 7.4 (7.21 and 9.15) are also reversible). Figure
S2A shows the count rate of the MPEGꢀbꢀPNLG nanogel solution at different time after the
addition of TPA into the MPEGꢀbꢀPNLG polymer solution. It can be seen that the number of
nanogel particles gradually increases within 40 min after the addition of TPA but does not
further increase after 40 min, which means the balance time for formation of nanogel particles is
around 40 min. Digital images in Figure 2D show MPEGꢀbꢀPNLG solutions in PBS at pH 7.4
and pH 6.4. At pH 7.4, the solution possessed a higher turbidity due to the formation of
nanoparticles; while, at pH 6.4, the solution was highly transparent due to the cleavage of the
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imine bonds. Furthermore, by controlling the crosslinking density via controlling the ratio of
CHO/NH2, the number of particles was observed to increase remarkably when the ratio increased
from 0.2 to 1.2 (Figure 2B). The increase of the count rate indicated that a higher amount of
crosslinker led to the formation of more benzoicꢀimine bonds and thus produced more nanogel
particles. Similarly, an increase in particle size was also observed when the CHO/NH2 ratio was
increased from 0.2 to 1.2 (Figure S2B), which provided a way to control the size of nanogels.
The formation of nanogels at pH 7.4 and the disassociation of nanogels at pH 6.4 were further
confirmed by FTꢀIR analysis. It can be seen from Figure S3 that a strong absorbance peak (1650
cmꢀ1) that corresponds to the bending vibration of NꢀH bonds in the primary amines of MPEGꢀbꢀ
PNLG is clearly observed. After the formation of nanogels, the disappearance of this peak
demonstrated the disappearance of primary amine groups; while the appearance of the
absorbance peak at 1590 cmꢀ1 that corresponds to the stretching vibration of C=N bonds clearly
proved the formation of imine bonds in the nanogels. When the nanogels are disassociated at low
pH, the absorbance peak at 1590 cmꢀ1 shifted to 1650 cmꢀ1 again, demonstrating the cleavage of
imine bonds and formation of primary amine bonds.
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Figure 2. (A) Reversible size changes of MPEGꢀbꢀPNLG nanogels between pH 7.4 and 6.4. (B)
Count rates of MPEGꢀbꢀPNLG nanogel solutions with various CHO/NH2 ratios. (C) Size
distribution of MPEGꢀbꢀPNLG nanogels. (D) Digital image of nanogel solutions at pH 6.4 and
7.4. Stability of MPEGꢀbꢀPNLG nanogels at various conditions (E) addition of NaCl, and (F)
dilution of nanogel solution.
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Figure 3. (A) Reversible zeta potential changes of MPEGꢀbꢀPNLG nanogels between pH 7.4
and 6.4. (B) Size distribution comparison of blank nanogels and DOXꢀloaded nanogels. (C)
Stability of MPEGꢀbꢀPNLG nanogels with different incubation periods at 37oC. (D) Drug release
profiles of DOXꢀloaded nanogels at different pH values.
To study the stabilities of the nanogels, the size change of the nanogels against incubation
time, addition of salt and dilution were evaluated. Figure 2E showed that there were negligible
changes in the size of the nanogels when the salt concentration increased from 0.25 M to 2 M,
and Figure 2F showed the nanogels were stable when the concentration of the nanogel solutions
was diluted from 1 to 0.01 mg/mL. In human blood, sodium salts are the main osmoles that keep
various essential functions such as regulating blood volume, hydrostatic pressure, and osmotic
equilibrium.30, 31 Sodium chloride, a strong electrolyte, may critically influence the behavior of
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the polymer molecules and lead to destabilization of drug delivery systems.32 The existence of
the TPA core may prevent PNLG blocks from conformational changes under the investigated
conditions, which endows the crosslinked system with high stability. Figure 3C showed that the
nanogels remained stable after 30 days of incubation at 37oC. All these results suggest that the
MPEGꢀbꢀPNLG nanogels are suitable for drug delivery applications where high stability and
long term circulation is required.
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A model drug, DOX, was encapsulated into the nanogels with a loading efficiency of 64.6%
and a loading content of 6.07% by a solvent evaporation method. The size difference between
the empty nanogels and the DOXꢀloaded nanogels were given in Figure 3B; The DOXꢀloaded
nanogels have an average size of 109.1 nm that was higher than the size of the empty MPEGꢀbꢀ
PNLG nanogels. This observation confirmed the successful encapsulation of DOX. This volume
increase might be caused by a higher hydrophilicity of PNLG blocks that limited DOXꢀloaded
particles from forming a dense core.33 The morphologies of MPEGꢀbꢀPNLG nanogels and DOXꢀ
loaded nanogels were observed by TEM. It can be seen from Figure S4 that there is not much
morphological difference before and after loading of DOX. Since the TEM images are taken in
dried states, the size difference of MPEGꢀbꢀPNLG nanogels and DOXꢀloaded nanogels is also
not much. DOXꢀloaded nanogels were dialyzed in pH 7.4 or 6.4 media that simulate the
physiological or tumoral pH, respectively. The segregated DOX concentration in the exterior
media was measured and calculated to obtain the pHꢀdependent drug release profile (Figure 3D).
At a physiological pH, the drugꢀloaded nanoparticles with highly stable benzoic imine bonds
prevented leakage of DOX. Only 26.92% and 37.3% of DOX were released after 1 and 24 hr,
respectively. In contrast, due to the cleavage of the acidꢀlabile bonds in the core of the nanogels,
the release rate was significantly accelerated with 58.81% and 100% of drug released after 1 and
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24 hr, respectively. It is worth noting that a burst release of DOX from the nanogels is observed
within the first hour even at pH 7.4. This might be attributed to the rapid release of unremoved
free DOX or DOX that is adsorbed on the surface of the hydrophobic core of the nanogels.
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The biocompatibilities of MPEGꢀbꢀPNLG nanogels were assessed by the MTT assay using
293T cell lines. Figure 4A showed that the nanogels exhibited negligible toxicities against the
cells at both pH 7.4 and pH 6.4. The viability of the MDAꢀMBꢀ231 human carcinoma cancer cell
line was evaluated at different concentrations of DOX by a MTT assay to confirm the cancer
cellꢀkilling effect of the DOXꢀloaded nanogels. Figure 4B showed that the DOXꢀloaded
nanogels exhibited higher cytotoxicity than free DOX against the breast cancer cell line MDAꢀ
MBꢀ231. When incubating the cells with DOXꢀloaded nanogels, the cell viability decreased to
35.13 % and 18.29%, respectively, at a DOX concentration of 10 and 35 µg/mL (IC50=3.66
µg/mL). In contrast, 57.93% and 32.11% of the cells survived after treatment with free DOX at a
concentration of 10 and 35 µg/mL, respectively (IC50=15.45 µg/mL). The MDAꢀMBꢀ231 cell
line has been reported to be relatively resistant to DOX, which may be acquired during treatment
via various mechanisms such as mutation of the tumor suppressor protein p53, MDR1 overꢀ
expression, or drug efflux.34 This acquired resistance reduces the activity of DOX on killing
cancer cells; so, normally a higher dose of DOX with longer treatment time is needed.35, 36 To
overcome these resistances, various promising carriers that preserve the activity of DOX but
avoid its side effects have been studied. By conjugating DOX with cellꢀpenetrating peptides (e.g.,
TAT, Penetratin, and Maurocalcine), several reported systems could induce cell apoptosis with
higher cellꢀkilling efficiency than free DOX.37, 38 Polymerꢀbased vectors have also been
developed to enhance tumor accumulation as well as to reach an outcome inhibition to
progression of drugꢀresistant cancer cells.39, 40 In this study, the cytotoxicity of DOX against
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MDAꢀMBꢀ231 was raised up by using the pHꢀsensitive MPEGꢀbꢀPNLG nanogel as a carrier.
The malnourished cellꢀkilling effect of free DOX might be a result of the acquired drug
resistance. Conversely, nanogels with pHꢀsensitive cores can increase the intracellular retention
time of DOX or induce a mitochondriaꢀindependent apoptotic pathway.41 As a result, viability
was remarkably decreased due to the retained intracellular activities of DOX. These data
indicated that the DOXꢀloaded nanogels are promising for therapeutic applications.
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Figure 4. (A) Biocompatibility of MPEGꢀbꢀPNLG nanogels on 293T cells at various nanogel
concentrations. (B) Cancer cellꢀkilling effect of free DOX and DOXꢀloaded nanogels.
The internalization of DOXꢀloaded nanogels was further studied on MDAꢀMBꢀ231 cells (at a
DOX concentration of 3 µg/mL). The intensity of intracellular DOX was measured as a function
of treatment time in the FACS assay. The FACS histograms of free DOX and DOXꢀloaded
nanogels at 1 and 3 hr are depicted in Figure 5. This figure demonstrated that DOXꢀloaded
nanogels achieved a significant increase in intracellular concentration of the drug than free DOX.
The detected fluorescence intensity correlates proportionally with the drug taken up by the cells.
Therefore, at the same DOX concentration, the intracellular accumulation of DOX was enhanced
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by using MPEGꢀbꢀPNLG nanogels as carriers. Confocal laser scanning microscope (CLSM) was
utilized to capture the fluorescence images of intracellular DOX. It can be seen from Figure 6,
the fluorescence intensity of DOX increased in a timeꢀdependent manner. When the same DOX
concentration (3 µg/mL) is used, the amount of DOXꢀloaded nanogels taken up by cells were
notably higher than that of free DOX in all investigated time points, which was in consistent with
the FACS results. The intracellular release of DOX from nanogels could also be observed in
CLSM images (Figure 6). After incubation for 1 hr, most DOXꢀloaded nanogels are in the
cytosol; however, with the incubation time increased to 3 hr, DOX was detected to be released to
perinuclear and nuclear regions. The appearance of DOX in the nucleus could be attributed to the
release of DOX from nanogels after the cleavage of imine bonds in the intracellular acidic
environment. The improved intracellular accumulation of DOXꢀloaded nanogels might enhance
the antitumor efficiency of DOX.
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Figure 5. FACS histogram comparison of free DOX and DOXꢀloaded nanogels with various
incubation time (1 hr and 3 hr).
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Figure 6. CLSM images of cells incubated with free DOX for (A) 1 hr and (B) 3 hr and cells
incubated with DOXꢀloaded nanogels for (C) 1 hr and (D) 3 hr. (Scale bar: 50 µm)
CONCLUSIONS
In conclusion, a pHꢀsensitive and polypeptideꢀbased nanogel system was constructed from
hydrophilic PNLG. By using hydrophobic TPA as a crosslinker, the obtained nanogels could
encapsulate a hydrophobic and smallꢀmolecule drug inside its hydrophobic core. This DOXꢀ
loaded nanocarrier could rapidly release the encapsulated DOX under acidic conditions such as
an intracellular endosome. Furthermore, this system possessed a higher anticancer activity
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against MDAꢀMBꢀ231 cells than free DOX. This pHꢀsensitive drug delivery system, with its
high stability and rapid response, can be suggested as a potential platform for enhanced tumor
therapy.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information includes additional Figures S1ꢀS4 and Table S1 showing GPC of the
MPEGꢀbꢀPNLG polymer; sizes of MPEGꢀbꢀPNLG nanogels from different batches; count rates
of MPEGꢀbꢀPNLG nanogels at different gelling time; size of MPEGꢀbꢀPNLG nanogels at
different CHO/NH2 ratios; FTꢀIR spectrum of MPEGꢀbꢀPNLG copolymer, nanogels and
nanogels after disassociation; TEM images of MPEGꢀbꢀPNLG nanogels and DOXꢀloaded
nanogels. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* Eꢀmail: dslee@skku.edu (D. S. L.). Tel: +82ꢀ31ꢀ290ꢀ7282
Notes
The authors declare no competing financial interests.
ACKOWNLEDGEMENTS
This research was supported by the Basic Science Research Program through a National
Research Foundation of Korea grant funded by the Korean Government (MEST) (NRFꢀ
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2017R1A5A1070259) and the National Research Foundation of Korea (NRF) funded by The
Ministry of Science, ICT & Future Planning (NRFꢀ2017R1D1A1B03033988).
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Title: OneꢀStep Preparation of pHꢀResponsive Polymeric Nanogels as An Intelligent Drug
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Delivery System for Tumor Therapy
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Authors: Yi Li, Quang Nam Bui, Le Thai Minh Duy, Hong Yu Yang and Doo Sung Lee
Graph:
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