CD44-Targeted and Enzyme-Responsive Photo-Cross-Linked Nanogels with Enhanced Stability for in Vivo Protein Delivery

Article abstract of DOI:10.1021/acs.biomac.1c00653

One of the biggest challenges of the protein delivery system is to realize stable and high protein encapsulation efficiency in blood circulation and rapid release of protein in the targeted tumor cells. To overcome these hurdles, we fabricated enzyme-responsive photo-cross-linked nanogels (EPNGs) through UV-triggered chemical cross-linking of cinnamyloxy groups in the side chain of PEGylation hyaluronic acid (HA) for CD44-targeted transport of cytochrome c (CC). The EPNGs showed high loading efficiency and excellent stability in different biological media. Notably, CC leakage effectively suppressed under physiological conditions but accelerated release in the presence of hyaluronidase, an overexpressed enzyme in tumor cells. Moreover, thiazolylblue tetrazolium bromide (MTT) results indicated that the vacant EPNGs showed excellent nontoxicity, while CC-loaded EPNGs exhibited higher killing efficiency to CD44-positive A549 cells than to CD44-negative HepG2 cells and free CC. Confocal images confirmed that CC-loaded EPNGs could effectively be internalized by CD44-mediated endocytosis pathway and rapidly escape from the endo/lysosomal compartment. Human lung tumor-bearing mice imaging assays further revealed that CC-loaded EPNGs actively target tumor locations. Remarkably, CC-loaded EPNGs also exhibited enhanced antitumor activity with negligible systemic toxicity. These results implied that these EPNGs have appeared as stable and promising nanocarriers for tumor-targeting protein delivery.

Full text of DOI:10.1021/acs.biomac.1c00653

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Article
CD44-Targeted and Enzyme-Responsive Photo-Cross-Linked
Nanogels with Enhanced Stability for In Vivo Protein Delivery
Hong Yu Yang,*,∥ Jia Meng Du,∥ Moon-Sun Jang,∥ Xin Wang Mo, Xin Shun Sun, Doo Sung Lee,
Jung Hee Lee,* and Yan Fu*
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ABSTRACT: One of the biggest challenges of the protein delivery system is to realize stable and high protein encapsulation
efficiency in blood circulation and rapid release of protein in the targeted tumor cells. To overcome these hurdles, we fabricated
enzyme-responsive photo-cross-linked nanogels (EPNGs) through UV-triggered chemical cross-linking of cinnamyloxy groups in the
side chain of PEGylation hyaluronic acid (HA) for CD44-targeted transport of cytochrome c (CC). The EPNGs showed high
loading efficiency and excellent stability in different biological media. Notably, CC leakage effectively suppressed under physiological
conditions but accelerated release in the presence of hyaluronidase, an overexpressed enzyme in tumor cells. Moreover, thiazolylblue
tetrazolium bromide (MTT) results indicated that the vacant EPNGs showed excellent nontoxicity, while CC-loaded EPNGs
exhibited higher killing efficiency to CD44-positive A549 cells than to CD44-negative HepG2 cells and free CC. Confocal images
confirmed that CC-loaded EPNGs could effectively be internalized by CD44-mediated endocytosis pathway and rapidly escape from
the endo/lysosomal compartment. Human lung tumor-bearing mice imaging assays further revealed that CC-loaded EPNGs actively
target tumor locations. Remarkably, CC-loaded EPNGs also exhibited enhanced antitumor activity with negligible systemic toxicity.
These results implied that these EPNGs have appeared as stable and promising nanocarriers for tumor-targeting protein delivery.
improving permeability and stability. Particularly, nanogels
possess an inner hydrated region for effective protein loading
and a hydrophilic outer shell for controlling enzymatic
degradation of proteins and inhibiting serum-triggered
aggregation of proteins that have captured considerable
attention as protein nanocarriers.16−18 Moreover, the nanogels
with suitable size can not only prolong the circulation time in
blood but also be favorably accumulated into tumor sits via the
enhanced permeability and retention (EPR) effect.19,20 These
features enable nanogels to be promising candidates for protein
1. INTRODUCTION
A growing number of protein therapeutics progressed, which
have been widely used in advanced clinical testing for treating
various severe human diseases such as malignant tumors and
diabetes mellitus.1,2 However, protein-based therapeutics
currently only perform extracellular functions for clinical
translation, such as monoclonal antibodies and cytokines,
while protein drugs have limited intracellular antitumor
activities due to their enzymatic degradation, short plasma
half-life, and inferior permeability.3−5 Therefore, design and
development of smart nanocarriers for efficient delivery of
proteins to the targeted tumor cells and triggered intracellular
liberation of cargos is vital to cancer therapy.6
Received: May 24, 2021
Revised: July 7, 2021
Published: July 21, 2021
To overcome the above limitations, various protein delivery
systems have been developed, including polymeric micelles,7−9
nanogels,10,11 hydrogels,12,13 and liposomes,14,15 which pro-
vided a promising strategy for reducing immunogenicity and
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Scheme 1. Schematic Illustration of Enzyme-Responsive Photo-Cross-Linked Nanogels (EPNGs) for High Loading Efficiency
a
and CD44-Targeted Delivery of CC in A549 Human Lung Tumor-Bearing Nude Mice
a
First, CC was loaded into the nanoparticles by multiphysical interactions with the HA-co-mPEG-co-Deta-CA polymer and was successfully cross-
linked by UV irradiation, which ensured excellent stability of the delivery nanosystems. Second, upon intravenous injection and cellular uptake, CC-
loaded EPNGs could effectively be internalized through CD44-mediated endocytosis and subsequent endo-lysosomal escape induced by the “pH-
dependent” effect of the Deta-containing polymer. Finally, Hyal-1 enzyme initiated the degradation of HA shells to release CC into cytosolic
location and caused cancer cell apoptosis.
delivery in cancer treatment. For example, Zhong et al.
constructed EGFR and CD44 dual-targeted nanogels, which
were used for the delivery of Granzyme B to breast cancers in
vitro and in vivo, leading to an enhanced curative efficacy.21
Chen and co-workers designed a hypoxia-responsive nanogel
through host−guest interactions between azobenzene and β-
cyclodextrin, which was applied to deliver Ribonuclease A and
trigger the release of proteins in 4T1 cells, achieving strong
therapeutic efficiency in vitro and in vivo.22 We recently also
fabricated pH-sensitive and CD44-targeted nanogels by
forming acid-labile cross-linked core that accomplished
intracellular-triggered release of CC to effectively induce
apoptosis of MCF-7 cancer cells.23
However, there are multiple technical issues that must be
addressed in the designing and engineering of nanogels for
systemic and cytosolic delivery of proteins. One critical hurdle
is related to the stability of nanogels. Because the poor stability
of nanogels can easily result in premature protein release in
blood circulation, leading to serious side effects and reducing
therapeutic efficiency.24,25 The second key problem is that
nanogels should be able to overcome both extracellular and
intracellular obstacles, including tumor-targeting capacity, fast
cellular internalization, effective endosomal escape, and
controlled protein release, which are critical for targeted and
cytosolic protein delivery.26,27 Furthermore, materials based on
the fabrication of the nanogels should be rapidly degraded after
completing protein delivery.28,29 By far, in response to these
challenges, many efforts have been devoted to this area, but
most can only resolve individual hurdles rather than cover all
of the issues.30,31 Thus, an effective strategy to tackle the
above-mentioned challenges in the systemic and cytosolic
delivery of proteins is still rare.
To construct an ideal nanogel, a multifunctional PEGlyated
hyaluronic acid, HA-co-mPEG-co-Deta-CA (Scheme 1), was
prepared. The HA backbone acts as an active tumor-targeting
polymer with high-binding affinity to CD44-overexpressed
cancer cells.32,33 Among its side-chain components, the mPEG
segment offers a hydrophilic shell to inhibit nonspecific
adsorption to serum proteins; the conjugated Deta compo-
nents can improve the loading efficiency with proteins and
promote endo-lysosomal escape of nanogels into the cytosol
based on the pH-dependent protonation of Deta components
in the nanogels to disrupt endo/lysosomal membrane (Scheme
1);34,35 the pendent cinnamyloxy groups can form a stable
cross-linked core by UV-induced photodimerization reaction
(Scheme 1).36 Based on this material, we fabricated enzyme-
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concentration was fixed at 50 mg/mL. The chemical cross-linking of
cinnamyloxy groups was irradiated using a Spectronics XL-1000
equipped with an 8 W lamp for 1 h. The power of light was 40 mW/
cm2 at 254 nm. To confirm the formation and stability of EPNGs,
non-cross-linked polymeric micelles (NLPMs) were prepared by the
dialysis method. HA-co-mPEG-co-Deta-CA polymer (50 mg) was
completely dissolved in 5 mL of DMSO; then, 20 mL of PBS (10
mM, pH 7.4) was added dropwise under constant stirring. The
resulting solution was dialyzed against PBS (pH 7.4) for 24 h to yield
the NLPMs. Moreover, the particle sizes of EPNGs and NLPMs in
organic solvent were further analyzed to assess the formation of
EPNGs with a Zetasizer ZS90 dynamic light scattering (DLS) device.
The biological stability of EPNGs and NLPMs was assessed using
DLS by dilution in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% FBS.
responsive photo-cross-linked nanogels (EPNGs) for targeted
delivery of cytochrome c (CC) as an apoptotic model protein,
to the cytosol of cancer cells (Scheme 1). Moreover, our
results confirmed that EPNGs displayed excellent stability and
high protein-loading efficiency. The resulting CC-loaded
EPNGs not only be uptaken via the CD44-mediated endocytic
pathway but also rapidly escaped from endo/lysosomal
compartment. Notably, CC-loaded EPNGs could also actively
target CD44-overexpressed A549 human lung tumors in nude
mice and significantly suppressed tumor growth compared to
free CC. Overall, this EPNG that incorporates multiple
features into a single nanoplatform can overcome numerous
challenges for systemic and cytosolic delivery of proteins.
2.4. Loading and Release of Protein. Briefly, CC or Cy5-CC
was dissolved in 10 mL of PBS (10 mM, pH 7.4) containing various
amounts of HA-co-mPEG-co-Deta-CA at a CC/polymer weight ratio
of 2 or 20% and stirred for 2 h. Subsequently, CC or Cy5-CC-loaded
EPNGs were prepared like those for the fabrication of empty EPNGs.
Notably, the secondary structure of CC was not affected using low-
band and high-intensity UV irradiation.37 The resulting solution was
extensively dialyzed against PBS (pH 7.4) to remove superfluous
proteins using the dialysis membrane with 300 kDa. The CC loading
content (CLC) and efficiency (CLE) were estimated by subtracting
the unloaded CC from the total amount of CC based on a calibration
UV−vis absorbance curve of the specified Cy5-CC concentrations.
CLC and CLE were estimated by the following equations
2. EXPERIMENTAL METHOD
2.1. Materials. Sodium hyaluronic acid (HA, Mn = 9100 g/mol,
degree of polymerization (DP): ∼23) was obtained from Freda
Biopharm Co., Ltd. (Shandong, China). Cinnamyl alcohol (CA),
phosphate-buffered saline (PBS), deuterium oxide (D2O), anhydrous
dimethyl sulfoxide (DMSO), cytochrome c (CC), dimethyl sulfoxide-
d6 (DMSO-d6), thiazolylblue tetrazolium bromide (MTT), fetal
bovine serum (FBS), 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic
acid) (ABTS), Dulbecco’s phosphate-buffered saline, RPMI-1640
medium, and pyridine were purchased from Sigma-Aldrich Co., Ltd.
(Missouri). N-Hydroxysuccinimide (NHS), N-(3-dimethylamino-
propyl)-N-ethylcarbodiimide hydrochloride (EDC), diethylenetri-
amine (Deta), isothiocyanate isomer (FITC), and hyaluronidase
(Hyal-1, 240 N.F.U/mg) were purchased from TCI Co., Ltd. (Tokyo,
Japan). Monomethoxy PEG amine (mPEG-NH2, Mn = 5000 Da) was
purchased from Ponsure Biological Co., Ltd. (Shanghai, China). 4-
Nitrophenyl chloroformate was purchased from Energy Chemical Co.,
Ltd. (Shanghai, China). LysoTracker Red was purchased from
Thermo Fisher Co., Ltd. (Shanghai, China). Cyanine5 (Cy5) NHS
ester was purchased from Lumiprobe Co., Ltd. Cy5-CC and FITC-
CC were prepared according to our previous research.8
CLC% = (amount of CC in EPNGs/amount of polymer and
loaded CC) × 100%
CLE% = (amount of CC in EPNGs/total amount of feeding CC)
× 100%
The release of CC from CC-loaded EPNGs was performed through
the dialysis technology. Briefly, a certain number of Cy5-CC-loaded
EPNGs were incubated in 5 mL of PBS (pH 7.4) in the presence or
absence of 150 U/mL of hyaluronidase38,39 and then poured into the
dialysis membrane (MWCO = 300 kDa). Subsequently, 3 mL of
medium was removed at designated time points and replaced by an
equal volume of a new medium. The released mass of CC was
estimated by a UV/vis spectrometer (absorption peak at 650 nm).
2.5. Confocal Imaging Assay. The cellular uptake behaviors and
endosomal escape capacity of the CC-loaded EPNGs were studied in
CD44-positive A549 cells using an LSM700 confocal laser scanning
microscope (CLSM, X400, Carl Zeiss) and 2.5 D imaging technology.
In brief, A549 cells were treated with Cy5-CC-loaded EPNGs (CC,
20 μg/mL) for 6 h. Then, the cells were washed with PBS thrice and
stained using Hoechst 33342 for 10 min. HepG2 cells were selected as
a CD44-negative cell model in this study.23,40 For the endo/lysosome
escape ability assay, A549 cells were treated with FITC-CC-loaded
EPNGs in the same control medium for designated time points (2 h
and 4 h). Then, the endo/lysosomal compartment was stained with
LysoTracker Red probe for 20 min before harvesting the cells,
followed by rinsing with PBS three times. The endo/lysosomal escape
images of CC-loaded EPNGs were obtained using a CLSM by fixing
excitation at 360 nm for Hoechst 33342 (λem = 460 nm), 580 nm for
LysoTracker (λem = 590 nm), and 650 nm for Cy5 (λem = 670 nm).
2.6. Evaluation of In Vitro Antitumor Activity. CD44-positive
A549 cells and CD44-negative HepG2 cells were seeded at a density
of 1 × 104 cells/well in a 96-well plate, which were applied to evaluate
the in vitro antitumor activity of CC-loaded EPNGs and free CC
using an MTT viability method. Two types of cells were treated with
CC-loaded EPNGs or alone CC at various protein concentrations
ranging from 5 to 100 μg/mL and incubated for 24 h. Then, 20 μL of
MTT solution in PBS (5 mg/mL) was added and further incubated
for 4 h. The supernatant was removed. DMSO (200 μL) was added to
each plate to dissolve the generated formazan. The relative cell
2.2. Preparation of HA-co-mPEG-co-Deta-CA. First, Deta-
conjugated PEGylated hyaluronic acid, HA-co-mPEG-co-Deta, was
synthesized via the amidation reaction with EDC/NHS as the
coupling agent. Briefly, HA (1 g, 0.11 mmol) was dissolved in 10 mL
of deionized (DI) water in a three-neck flask, then EDC (0.387 g, 2.02
mmol) and NHS (0.233 g, 2.02 mmol) were added to the solution for
activating 80% carboxylic groups of HA. The reaction was stirred for 2
h at room temperature (RT) under nitrogen atmosphere (N2). After
activation, mPEG-NH2 (1.10 g, 0.22 mmol) dispersed in DI water (10
mL) was injected and allowed to unceasingly react for 12 h.
Afterward, the aforementioned solution was added dropwise to the
stirred solution of DI water (20 mL) containing Deta (3.75 g, 36.4
mmol, 18 times molar to repeating unit of HA) and permitted to
further react under the same conditions for 12 h. HA-co-mPEG-co-
Deta intermediate polymer was obtained through twice dialysis
(MWCO 7000-8000) with DI water followed by freeze-drying. Yield:
93%. Furthermore, cinnamyl alcohol (CA) was conjugated to the HA-
co-mPEG-co-Deta by activating the hydroxyl group of CA with 4-
nitrophenyl chloroformate (p-NPC). In brief, the DMOS solution
containing CA (61.5 mg, 0.46 mmol) and pyridine (0.15 mL) was
added to the prepared p-NPC (92.1 mg, 0.46 mmol) solution in
DMSO (5 mL) within 30 min at 0 °C and allowed to react without
interruption at RT for 24 h in the dark under N2. Subsequently, HA-
co-mPEG-co-Deta (300 mg, 0.014 mmol) in DI water (10 mL) was
added drop by drop to the above mixture solution and allowed to
further react by employing the same reaction conditions. The final
HA-co-mPEG-co-Deta-CA was collected by dialysis against excess DI
water and lyophilization. Yield: 91%. (1H NMR, 500 MHz, D2O)
2.3. Preparation of Enzyme-Responsive Photo-Cross-Linked
Nanogels (EPNGs). EPNG nanogels were constructed by chemical
cross-linking of the cinnamyloxy groups in the HA-co-mPEG-co-Deta-
CA. In brief, HA-co-mPEG-co-Deta-CA polymer was dissolved in
phosphate-buffered saline (PBS, 10 mM, pH 7.4), where the polymer
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Scheme 2. Synthesis Route of the HA-co-mPEG-co-Deta Intermediate Polymer and the HA-co-mPEG-co-Deta-CA Polymer
viability (%) can be determined by comparing the absorbance at 540
nm with a microplate reader. Tests were done in quintuplicate, and
data are represented as mean SD. The biocompatibility of blank
EPNGs to either A549 or HepG2 cells was studied using the same
process at EPNG concentrations ranging from 50 to 500 μg/mL.
2.7. In Vivo Fluorescence Imaging and Biodistribution
Assay. Six-week-old female BALB/c mice (20 2 g) were obtained
from Seoul Oriental Bio Center, and experimental processes abided
by the guidelines of the Guide for the Care and Use of Laboratory
Animals (Institute of Samsung Biomedical of Korea). BALB/c mice
were subcutaneously injected in the right flank with A549 cells (1 ×
107 cells/mouse) to build human lung tumor model. When tumor
volumes achieved 100−200 mm3, Cy5-CC-loaded EPNGs (5 mg/kg
dose of Cy5-CC) were injected through the tail intravenous
administration. Fluorescent scans were monitored at various time
intervals (pre, 1, 4, 8, and 24 h) with a near-infrared fluorescence
imaging system (IVIS 200, Massachusetts). After 24 h scanning, the
mice were sacrificed to isolate tumor and major organs (liver, lung,
kidney, heart, and spleen) for evaluation of protein in vivo
biodistribution. Moreover, fluorescence intensities of tumor and
main organs were further quantified with Living Image 2.5 software
(Caliper Life Science).
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1
Figure 1. H NMR spectra of the HA-co-mPEG-co-Deta intermediate polymer (A) and the final HA-co-mPEG-co-Deta-CA polymer (B) in D2O.
restrain nonspecific adhesion of serum proteins;41 the Deta was
2.8. In Vivo Antitumor Efficacy and Biosafe Assessment.
A549 tumor-bearing mice were treated with CC-loaded EPNGs or
incorporated not only to improve the binding capacity of
free CC at a dosage of 5 mg of CC equivalents/kg via tail intravenous
nanogel to proteins but also to facilitate the escape of nanogels
injection every 3 days when tumor volumes increased to 150−200
from endo/lysosomal compartments. The successful synthesis
mm3. Saline was injected as a control group. The tumor volumes and
of HA-co-mPEG-co-Deta was confirmed using 1H NMR
bodyweight of mice were measured every 2 days. Tumor volumes (V)
were estimated according to the following formula: V = (L × W2)/2,
where L and W are the tumor length and width, respectively. Relative
tumor volumes were estimated as V/V0, where V0 is the initiative
tumor volume. Tumor volume, bodyweight, and survival ratios of the
mice were monitored every 3 days. The mice were gently killed when
the tumor sizes reached approximately 1000 mm3. Then, the tumors
were removed and weighed. Finally, isolated tumors and main organs
(liver, lung, kidney, heart, and spleen) were stained with hematoxylin
and eosin (H&E) and TUNEL for histological analysis and biosafety
evaluation using a digital microscope.
spectrum (Figure 1A). A distinct characteristic peak at ∼3.64
ppm associated with the mPEG protons, and the proton peaks
ranging from ∼3.1 to ∼3.5 ppm, which corresponded to the
Deta component in the HA-co-mPEG-co-Deta polymer. For
the final HA-co-mPEG-co-Deta-CA polymer, the partially
residual amine groups of HA-mPEG-Deta were used to graft
the activated CA monomers. By comparing Figure 1A,B, three
new protons peaks appeared at ∼7.9, ∼8.4, and ∼8.8 ppm,
which belonged to the cinnamyloxy groups of the HA-co-
mPEG-co-Deta-CA polymer.
The grafting degrees of mPEG, Deta, and CA of the HA-co-
mPEG-co-Deta-CA polymer were estimated by integrating the
proton signals of each component and the characteristic
methyl peaks of acetyl groups of HA, and the detailed results
are presented in Table S1.
3.2. Preparation and Properties of EPNGs. Studies
about the photodimerization of cinnamyloxy groups to form a
cyclobutane ring with low-band UV light have been previously
reported.36 Therefore, the EPNGs were easily fabricated by the
3. RESULTS AND DISCUSSION
3.1. Synthesis of the HA-co-mPEG-co-Deta-CA Poly-
mer. The HA-co-mPEG-co-Deta-CA polymer was prepared
according to a two-step reaction (Scheme 2). For PEGylated
HA-co-mPEG-co-Deta polymer, mPEG and Deta were
conjugated onto the backbone of HA by amidation reaction
using EDC/NHS as coupling agents. Its mPEG ingredient
could act as a hydrophilic shell to enhance the stability and
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Figure 2. (A) Size distribution of EPNGs and NLPMs. (B) Size change of EPNGs and NLPMs in a large quantity of DMSO solvent. (C) Stability
of EPNGs and NLPMs using 100-fold dilutions of DMEM supplement containing 10% FBS and the stability of EPNGs in PBS (pH 7.4) at 37 °C
(initial concentration of the sample was set at 1 mg/mL). (D) HR-TEM images of EPNGs.
Figure 3. In vitro release of CC from EPNGs incubated in PBS (pH
7.4) with or without Hyal-1 (150 U/mL). Data are presented as mean
SD (n = 3).
Figure 4. (A) Confocal images and 2.5D images of A549 and HepG2
photodimerization of cinnamyloxy groups on the polymer
cells incubated with Cy5-CC-loaded EPNGs for 6 h. (B) Confocal
backbone to fabricate a cross-linked core under UV irradiation.
To investigate the successful preparation and good stability of
EPNGs, non-cross-linked NLPMs as a compared group were
fabricated. As shown in Figure 2A, the particle size of EPNGs
was about 61 nm, which is smaller than that of the NLPMs. It
might be explained that the chemical cross-linking of
cinnamyloxy groups further led to the shrinking of nanogels
due to enhanced hydrophobic interactions between cinnamy-
loxy groups to weaken the electrostatic repulsion among
residual carboxy groups in the interior of nanoparticles. To
further assess the photochemical cross-linking reaction of
EPNGs, the EPNGs and NLPMs were, respectively, diluted
with the same organic solvent and their particle sizes were
evaluated by DLS measurements (Figure 2B). The diameter of
images of A549 cells incubated with FITC-CC-loaded EPNGs for 2
and 4 h, respectively. The cells were counterstained with LysoTracker
Red for endo/lysosome and Hoechst for nucleus. The yellow areas
indicate colocalization of endolysosome (red) and FITC-CC (green).
The scale bars represent 20 μm.
NLPMs distinctly decreased from the initial ∼86 to ∼10 nm by
adding a large quantity of DMSO solvent. However, the size of
EPNGs increased about 2 times from the original ∼61 to ∼120
nm after adding the same amount of solvent. This result
indicated that the EPNGs possessed integrated structure
through photo-induced cross-linking even after incubating in
highly soluble organic solvents. Besides, dilution in a lot of
DMEM containing 10% FBS was used to evaluate the stability
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Figure 5. (A) MTT assays of blank EPNGs in A549 and HepG2 cells. (B) MTT assays of CC-loaded EPNGs and free CC in A549 and HepG2
cells (n = 3).
be effectively encapsulated into EPNGs with a high CLE of
97.8% at a theoretical CLC of 2 wt % and a CLE of 80.3% at a
theoretical CLC of 20 wt %. The high loading efficiency of
protein is most possibly attributable to the strong multiphysical
interactions between CC and HA-co-mPEG-Deta-CA poly-
mers. Moreover, the negative zeta potential of CC-loaded
EPNGs slightly decreased from −10.2 to −5.6 mV with
increasing CLC from 2 to 20 wt %.
In vitro release patterns of CC from EPNGs were studied
with or without hyaluronidase 1 (Hyal-1) conditions. The
results in Figure 3 showed that the CC release was significantly
suppressed without obvious burst release (∼26% release in 24
h) in a physiological environment (pH 7.4 and 37 °C), while
more than 85% of CC was released during 24 h under Hyal-1
condition. The above results not only indicated that the
EPNGs with enhanced stability could control the unintended
burst release of CC during the blood circulation but also
confirmed that the accelerated release of CC could be triggered
by Hyal-1, which is an important enzyme in HA degradation
and abundant in the cytosol of tumor cells.44 Moreover, the
2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS)
measurement also confirmed that released CC maintained
the same bioactivity as native CC (Figure S1), indicating that
CC retained its bioactivity over the CC-loaded EPNG
Figure 6. (A) In vivo fluorescence images of A549 human lung
preparation process.
tumor-bearing nude mice at different time points following injection
3.4. Confocal Image Analysis and Antitumor Efficacy
of Cy5-CC-loaded EPNGs. Ex vivo fluorescence imaging (B) and the
of CC-Loaded EPNGs In Vitro. CD44-mediated internal-
corresponding fluorescence intensities (C) of tumor and main organs
ization and endo/lysosomal escape capacity of Cy5-CC-loaded
isolated at 36 h post-treatment.
EPNGs were evaluated by CLSM in either A549 or HepG2
of EPNGs mimicking the intravenous injection,42 which is
essential to apply these nanogels for effective delivery of
cells. Confocal images displayed strong Cy5 fluorescence in the
cytoplasm of A549 cells after 6 h treatment, indicating efficient
cellular uptake of Cy5-CC-loaded EPNGs into A549 cells
protein in vivo. As shown in Figure 2C, the NLPMs showed
(Figure 4A). In contrast, a negligible fluorescence signal was
partial dissociation against extensive dilution with DMEM
containing 10% FBS to the final concentration (∼0.01 mg/
mL) that is lower than the CMC (∼0.08 mg/mL), as the
found in CD44-negative HepG2 cells after the same incubation
time. Notably, the different internalization effects between
A549 and HepG2 cells can further be visualized with 2.5D
imaging technology without further quantitative analysis
(Figure 4A). As expected, meaningless fluorescence signals
were observed on both A549 and HepG2 cells with free Cy5-
CC after 6 h treatment, indicating that free CC could hardly be
uptaken by the tumor cells (Figure S2).45 Additionally,
exploring the endo/lysosomal escape ability of EPNGs has
become one of the key issues for intracellular protein delivery.
As shown in Figure 4B, we could clearly observe that the
FITC-CC-loaded EPNGs were mainly entrapped in the endo/
lysosomal compartments after 2 h incubation, while most CC
could successfully escape from the endo/lysosomal compart-
appearance of multiple peaks. However, the size of EPNGs was
almost constant without significant changes under the same
diluent condition and in PBS (pH 7.4) for more than 3 days,
indicating that these EPNGs containing cross-linked cores
significantly improved the stability of nanogels. Moreover, the
TEM image in Figure 2D further confirmed that EPNGs
exhibited a spherical morphology and particle size distribution
close to that the results of DLS assays.
3.3. Loading and Release of CC from EPNGs.
Cytochrome c (CC) acted as a protein drug model because
the high concentration of CC enables DNA fragmentation in
the cell nucleus to induce apoptosis of cancer cells.43 CC could
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Figure 7. In vivo antitumor efficacy of CC-loaded EPNGs in A549 tumor-bearing nude mice. Free CC and saline groups were used as controls. (A)
Changes of relative tumor volumes of A549 tumor-bearing mice after intravenous injection of saline, free CC, and CC-loaded EPNGs within 21
days. (*p < 0.05, **p < 0.01 using Student’s t-test). (B) Average weights of isolated tumor sections in three treatment groups on day 21. (C) H&E
and TUNEL staining assay of isolated tumor tissues after treatment in different groups. (D) Relative body weights of the mice within 21 days of
treatment. (E) Survival rates of the mice in different treatment groups.
ment into the cytoplasm of A549 cells in 4 h. The endo/
lysosomal escape ability of CC-loaded EPNGs might be
explained that the pH-dependent protonation of Deta
components in the EPNGs resulted in destabilization of
endo/lysosomal membrane.46 It is evident, therefore, that
EPNGs exhibited improved cellular internalization and rapid
endo/lysosomal escape in CD44-positive A549 cells.
To investigate the cytotoxicity of EPNGs, the cytotoxicity of
vacant EPNGs against both A549 and HepG2 cells was
evaluated using the standard MTT technology. The cell
viability of A549 and HepG2 cells was significantly not
changed by treatment with the same concentration of EPNGs
ranging from 5 to 500 μg/mL for 24 h (Figure 5A), indicating
that these EPNGs endowed outstanding biocompatibility. The
in vitro antitumor efficiency of CC-loaded EPNGs to A549
and HepG2 cells was further assessed by the same procedure.
In Figure 5B, free CC as a compared group showed negligible
cytotoxicity for each of the cells at various CC concentrations
ranging from 5 to 100 μg/mL, owing mainly to the unaided
cellular uptake of free CC. It is worth noting that CD44-
positive A549 cells exhibited much better cell apoptosis than
CD44-negative HepG2 cells by treatment with CC-loaded
EPNGs at an identical CC dose. Negligible cell apoptosis was
presented in HepG2 cells treated with the CC-loaded EPNGs
because the CC-loaded EPNGs have a negative charge and
HepG2 cells act as CD44-negative cancer cells, which led to
poor cellular uptake and also supported the results of confocal
imaging. Oppositely, these results also further indicated that
enhanced cell apoptosis of CC-loaded EPNGs to CD44-
overexpressed A549 cells is attributable to the combinative
contribution of CD44-mediated endocytosis and the innate
endo/lysosomal escape ability of EPNGs.
3.5. In Vivo Targeting and Biodistribution of CC-
Loaded EPNGs. In vivo near-infrared fluorescence (NIRF)
imaging was performed in A549 tumor xenograft mouse model
to evaluate CD44 targetability and biodistribution of Cy5-CC-
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Figure 8. Systemic biosafety assessment. H&E and TUNEL staining of major organs (liver, lung, kidney, spleen, and heart) from the mice bearing
A549 lung cancer xenografts treated as described in Figure 7 on day 21.
loaded EPNGs after intravenous injection using a noninvasive
imaging system. Figure 6A presents an obvious tumor
accumulation effect of Cy5-CC-loaded EPNGs at 8 h post
intravenous (i.v.) injection. The strongest fluorescence signal
in the tumor site was found at 24 h post-injection. Tumor
location fluorescence intensity still remained strong from 24 to
36 h, indicating that Cy5-CC-loaded EPNGs exhibited
superior tumor-targeting ability. The NIRF signals in tumor
and major organs withdrawn from the A549 tumor-bearing
nude mice were detected 36 h after i.v. injection. The
fluorescence signal of tumor tissues was significantly higher
than those of the liver, spleen, heart, lung, and kidney (Figure
6B), which also agreed with the quantitative results of the ex
vivo fluorescence images (Figure 6C). The corresponding high
tumor-targeting ability of Cy5-CC-loaded EPNGs is likely
attributed to EPR effect-based tumor accumulation, HA-
receptor-mediated targeting mechanism, and enhanced stabil-
ity of the nanosystem.
3.6. In Vivo Antitumor Efficiency and Biosafety
Assessment of CC-Loaded EPNGs. Lung cancer remains
the leading cause of morbidity and mortality throughout the
world.47,48 Motivated by their fast internalization, superior
endo/lysosomal escape ability, and high tumor accumulation
effect, we further assessed the in vivo antitumor efficacy of CC-
loaded EPNGs in A549 tumor-bearing mice models. When the
volume of tumors reached 150−200 mm3, saline (control
group), free CC, and CC-loaded EPNGs were intravenously
injected with identical doses of CC (5 mg/kg dose of CC).
The results in Figure 7A confirmed that tumor growth was
effectively suppressed by treatment with CC-loaded EPNGs
during the total treatment period, while rapid tumor
progression was detected for control groups receiving saline
and free CC. Moreover, these results were supported by the
tumor weight withdrawn from the mice after treatment (Figure
7B). At the end of antitumor study, the tumor tissues were
stained with the H&E and TUNEL staining to further reveal
the highest potency of CC-loaded EPNGs in eradicating A549
tumor cells (Figure 7C). Their biosafety was investigated by
monitoring the change in bodyweight and survival rate of mice
during the entire treatment period and identifying the
histopathological changes of normal organs after treatment.
Notably, CC-loaded EPNGs caused little changes in body-
weight and maintained a high survival rate of mice (Figure
7D,E), signifying that these EPNGs had excellent biosafety.
Furthermore, the H&E and TUNEL staining assays disclosed
that CC-loaded EPNGs caused negligible damage to the major
organs, including liver, lung, kidney, spleen, and heart,
indicating negligible systemic toxicity (Figure 8). These results
suggested that EPNGs are highly effective for protein
nanotherapeutics owing to its enhanced stability, which
could inhibit unintended protein release during blood
circulation and improve their tumor-targeting capacity.
4. CONCLUSIONS
We designed enzyme-responsive photo-cross-linked nanogels
(EPNGs) through UV-induced photodimerization of cinna-
myloxy groups. The EPNGs consisting of HA backbone and
cross-linked core resulted in enhanced stability and achieved
sustained release of CC. They also exhibited fast cellular
internalization via CD44-mediated cell adhesion and efficient
endo/lysosome escape ability. The CC-loaded EPNGs
endowed excellent stability to enable specific targeting of
tumor, which was confirmed by the IVIS imaging system. The
antitumor ability of CC-loaded EPNGs was better than those
of free CC and control groups in vitro and in vivo. Overall,
these results demonstrated that the excellent stability of
nanogels could markedly impact their tumor-targeting capacity
and therapeutic efficiency as protein nanocarriers for use in
cancer therapy.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.biomac.1c00653.
■
sı
Characterization method, assessment of bioactivity of
CC released from EPNGs, cell culture method, and 2.5D
images of A549 cells and HepG2 incubated with free
Cy5-CC (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Hong Yu Yang − College of Materials Science and Engineering,
Jilin Institute of Chemical Technology, Jilin City 132022, P.
R. China; orcid.org/0000-0002-0730-2779;
Email: yhyqdu@163.com
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Biomedical Research Institute, Seoul 06351, Republic of
Korea; Email: hijunghee@skku.edu
Yan Fu − College of Materials Science and Engineering, Jilin
Institute of Chemical Technology, Jilin City 132022, P. R.
China; Email: fuyanqd@163.com
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Moon-Sun Jang − Department of Radiology, Samsung Medical
Center, Sungkyunkwan University School of Medicine and
Center for Molecular and Cellular Imaging, Samsung
Biomedical Research Institute, Seoul 06351, Republic of
Korea
Xin Wang Mo − College of Materials Science and Engineering,
Jilin Institute of Chemical Technology, Jilin City 132022, P.
R. China
Xin Shun Sun − College of Materials Science and Engineering,
Jilin Institute of Chemical Technology, Jilin City 132022, P.
R. China
Doo Sung Lee − Theranostic Macromolecules Research Center
and School of Chemical Engineering, Sungkyunkwan
University, Suwon, Gyeonggi-do 16419, Republic of Korea;
orcid.org/0000-0002-7979-7459
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.biomac.1c00653
Author Contributions
∥H.Y.Y., J.M.D., and M.-S.J. contributed equally to this paper.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by National Natural Science
Foundation of China (NSFC) (no. 21805108) and Out-
standing Young Talents Program through a Science and
Technology Development Plan Program of Jilin Province
(20190103132JH). This research was also supported by the
National Research Foundation of Korea (NRF) funded by the
Korea government (no. NRF-2020R1A2C2012416).
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