Protein-drug conjugate programmed by pH-reversible linker for tumor hypoxia relief and enhanced cancer combination therapy

Article abstract of DOI:10.1016/j.ijpharm.2020.119321

Combining functional proteins with small molecular drugs into one entity may endow distinct synergistic advantages. However, on account of completely different physicochemical properties of such payloads, co-delivery through systemic administration for therapeutic purpose is challenging. Herein, we designed the protein-drug conjugate HSAP-DC-CAT (human serum albumin/Pt (IV)-dibenzocyclooctyne/chlorin e6-catalase) by modification of CAT and cisplatin pro-drug loaded HSA with pH-sensitive azide linker 3-(azidomethyl)-4-methyl-2,5-furandione (AzMMMan) followed by click chemistry assembly with DC. The dynamic covalent bonds between linker and proteins, on the one hand, can bridge proteins and small molecular drugs in the intermediate state for systemic delivery in the harsh in vivo environment; on the other hand, it can trigger traceless cleavage and release of drugs and proteins with full bioactivity in acidic microenvironment of tumor. The multifunctional HSAP-DC-CAT provides efficient cytosolic transduction in vitro, excellent blood half-lives after systemic administration, and significant antitumor outcome via integrated cisplatin-based chemotherapy and Ce6-based photodynamic therapy enhanced by catalase-induced manipulation of tumor hypoxia microenvironment. This study describes a universal formulation strategy for protein and small molecular drug by a bifunctional linker through amide reaction and click chemistry, with traceless in vivo release of therapeutic units.

Full text of DOI:10.1016/j.ijpharm.2020.119321

InternationalJournalofPharmaceutics582(2020)119321
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Protein-drug conjugate programmed by pH-reversible linker for tumor
hypoxia relief and enhanced cancer combination therapy
Xican Zhanga, Liangzhu Fengb,⁎⁎, Ziliang Dongb, Xiaoqian Xina, Zhijuan Yangb, Dashi Dengb,
Ernst Wagnerc, Zhuang Liub, Xiaowen Liua,⁎
a Clinical Translational Center for Targeted Drug, Department of Pharmacology, School of Medicine, Jinan University, Guangzhou 510632, Guangdong Province, China
b Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Soochow University, Suzhou
215123, China
c Pharmaceutical Biotechnology, Center for System-based Drug Research, Ludwig-Maximilians-Universität München, Butenandtstrasse 5-13, D-81377 Munich, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
Protein-drug
Cancer
Combinational therapy
pH-reversible
Combining functional proteins with small molecular drugs into one entity may endow distinct synergistic ad-
vantages. However, on account of completely different physicochemical properties of such payloads, co-delivery
through systemic administration for therapeutic purpose is challenging. Herein, we designed the protein-drug
conjugate HSAP-DC-CAT (human serum albumin/Pt (IV)-dibenzocyclooctyne/chlorin e6-catalase) by mod-
ification of CAT and cisplatin pro-drug loaded HSA with pH-sensitive azide linker 3-(azidomethyl)-4-methyl-2,5-
furandione (AzMMMan) followed by click chemistry assembly with DC. The dynamic covalent bonds between
linker and proteins, on the one hand, can bridge proteins and small molecular drugs in the intermediate state for
systemic delivery in the harsh in vivo environment; on the other hand, it can trigger traceless cleavage and
release of drugs and proteins with full bioactivity in acidic microenvironment of tumor. The multifunctional
HSAP-DC-CAT provides efficient cytosolic transduction in vitro, excellent blood half-lives after systemic ad-
ministration, and significant antitumor outcome via integrated cisplatin-based chemotherapy and Ce6-based
photodynamic therapy enhanced by catalase-induced manipulation of tumor hypoxia microenvironment. This
study describes a universal formulation strategy for protein and small molecular drug by a bifunctional linker
through amide reaction and click chemistry, with traceless in vivo release of therapeutic units.
Tumor microenvironment
Dynamic bond
1. Introduction
the acidic TME, though negatively impairs most cancer treatments,
provides a unique chemical environment for the design of tumor-
Solid tumor microenvironments (TME) impose diverse biological
barriers including tumor hypoxia, reduced pH and other hostile features
on the effective cancer treatments (Estrella et al., 2013; Maas et al.,
2012; Trédan et al., 2007). Several pioneering strategies, enabling ef-
ficient TME modulation by smart nanodrug delivery systems (NDDSs)
(Jain, 2013; Mura et al., 2013; Torchilin, 2014), have been proposed to
benefit these tumor characteristic parameters for better therapeutic
specificity and efficiency. Given that the negative role of tumor hypoxia
on cancer treatment, it has been shown that catalase and its mimics
integrated NDDSs are able to efficiently normalize the TME for hypoxia
relief by promoting the decomposition of endogenous H2O2 to generate
O2, thereby providing a favorable condition for photodynamic therapy
(PDT) (Shi et al., 2019) and other oxygen-consuming cancer therapy
(Prasad et al., 2014; Song et al., 2016b; Zhang et al., 2017). Meanwhile,
acidity-responsive NDDS, enabling significantly enhanced therapeutic
specificity and efficacy (Danhier et al., 2010; Du et al., 2018; Liu et al.,
2014).
Ascribing to their robust targeting or therapeutic potencies of those
proteins and peptides (Carter, 2011; Ibraheem et al., 2014; Lagassé
et al., 2017; Leader et al., 2008; Han et al., 2010), protein conjugates
have found to be effective biopharmaceuticals with improved se-
lectivity and prolonged blood circulation time, particularly for these
PEGylated biotherapeutics (Dozier and Distefano, 2015; Mishra et al.,
2016; Turecek et al., 2016). However, considering these PEGylated
biotherapeutics are commonly obtained via the irreversible covalent
conjugation, their therapeutic effects are often remarkably diminished
as the PEG corona would restrict the interaction between these bio-
therapeutics and their targets (Bailon et al., 2001; Foser et al., 2003; Li
⁎ Corresponding author.
⁎⁎ Corresponding author.
E-mail addresses: lzfeng@suda.edu.cn (L. Feng), xwliu231@jnu.edu.cn (X. Liu).
https://doi.org/10.1016/j.ijpharm.2020.119321
Received 17 February 2020; Received in revised form 4 April 2020; Accepted 7 April 2020
Availableonline11April2020
0378-5173/©2020ElsevierB.V.Allrightsreserved.

X. Zhang, et al.
InternationalJournalofPharmaceutics582(2020)119321
et al., 2016). In addition, combinational therapies of proteins and small
molecular drugs would greatly expand the therapeutic potency through
realization of synergistic effect between the heterogeneous ther-
apeutics. However, one key dilemma for the optimal therapeutic out-
come is the rational formulation of therapeutic protein-drug(Holland
et al., 2019), which should protect the proteins from in vivo degrada-
tion and denaturation, as well as prolong the circulation time and en-
hance biocompatibility of small molecular drugs. So far, very few
samples have been investigated in this field, and to the best of our
knowledge, the reversible covalent binding of therapeutic proteins and
small molecular drugs has not been well studied.
Lambda 750 UV-VIS-NIR spectrophotometer. Transmission electron
microscopy (TEM) was used to characterize the morphology of HSAP-
DC-CAT nanoparticles. The hydrodynamic diameters of conjugates
(HSAP-DC-CAT, HSA-DC-CAT, HSAP-DC-HSA and mixture CAT/Ce6/
HSAP) with or without acidic buffer incubation were determined by a
Zetasizer Nano-ZS (Malvern Instruments, UK). The conjugation assess-
ment of click chemistry (HSAP-DC-CAT, HSA-DC-CAT and HSAP-DC-
HSA) and traceless release behavior of conjugates (HSAP-DC-CAT)
treated with acidic buffer was determined by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) or Zetasizer Nano-ZS.
The portable dissolved oxygen meter was used to detect the O2
production treated by free catalase and HSAP-DC-CAT with different
concentrations of H2O2, by recording the dissolved oxygen concentra-
tion continuously.
The method of detecting singlet oxygen was reported previously
(Chen et al., 2017; Li et al., 2013). In simply, singlet oxygen sensor
green reagent (SOSG, molecular probes) was dissolved in methanol
To this end, we previously developed azidomethyl-methylmaleic
anhydride (AzMMMan) based pH-responsive linker for the reversible
PEGylation or conjugation of several different proteins, which showed
efficient intracellular delivery and controlled release of proteins with
their bioactivity unimpaired (Liu et al., 2016; Liu et al., 2017; Maier
and Wagner, 2012). In the present study, we developed the dynamic
covalent bond-assisted programmed approach to assemble two small
therapeutic drugs with proteins, which could improve the in vivo
pharmacokinetics and pharmacodynamics of small molecule drugs and
construct a favorable therapeutic microenvironment by proteins for
synergistic cancer therapy. For the purpose of decreasing cisplatin as-
sociated side effects (Johnstone et al., 2016; Tolan et al., 2016; Wang
et al., 2019), the cisplatin(IV)-succinic acid pro-drug (Pt(IV)SA) was
conjugated with human serum albumin (HSAP) (Feng et al., 2016).
Then AzMMMan was applied as pH-sensitive bifunctional linker to
couple with remaining amino groups of HSAP and catalase (CAT) via
the pH reversible acyl substitution. In the following, the DBCO coupled
photosensitizer Ce6 (Chin et al., 2008; Tan et al., 2016; Tian et al.,
2011) was utilized as the cross-linker to initiate the conjugation of these
AzMMMan modified proteins (HSAP, CAT) by strain-promoted azide
alkyne cycloaddition (SPAAC). The obtained protein-drug of HSAP-DC-
CAT showed remarkably enhanced pharmacokinetics and pharmaco-
dynamics of small molecule drugs and well-protected catalytic perfor-
mance towards H2O2 decomposition. Collectively, HSAP-DC-CAT con-
sisted of four functional units: (i) cisplatin pro-drug (IV) conjugated
with HSA to decrease cisplatin associated side effects, for site-directed
chemotherapy by conversion into cytotoxic Pt (II) within the in-
tracellular reducing tumor target microenvironment; (ii) small ther-
apeutic drug photosensitizer Ce6 for photodynamic therapy; (iii) CAT,
an antioxidant enzyme, to overcome tumor hypoxia by specific catalytic
behavior in decomposing H2O2 to produce O2, thus providing a favor-
able TME for the PDT by Ce6; and (iv) the dynamic covalent protein-
drug conjugation by an acidic reversible and click reactive bifunctional
linker AzMMMan, thus mediating traceless release of unmodified pro-
teins and small molecule drugs at the acidic tumor target micro-
environment.
firstly and added to the prepared samples ([SOSG]
= 2.5 µM,
[Ce6] = 1 µM). Then samples were irradiated by 660 nm light at
5 mW/cm2 in nitrogen atmosphere. The measurement of SO generation
was determined by SOSG fluorescence signals at 530 nm with excitation
at 494 nm.
The catalase activity of HSAP-DC-CAT was determined by the Góth
method (Góth, 1991). Briefly, 0.5 mL hydrogen peroxide (50 mM) was
added to free catalase or HSAP-DC-CAT solution and incubated for
1 min at 37 °C. Then the reaction was terminated by adding 0.5 mL
ammonium molybdate (32.4 mM) by forming stable yellow complexes
with the residual H2O2. After cooling down to 25 °C, the absorbance of
each sample was measured at 400 nm by the UV–VIS-NIR spectrometer
to determine catalase activity. The relative activity of catalase was
calculated by the following equation: relative catalytic activity =
(absorbance of H2O2 with PBS – absorbance of H2O2 with HSAP-DC-
CAT) / (absorbance of H2O2 with PBS – absorbance of H2O2 with free
CAT). To measure the stability of catalase against protease digestion,
both free catalase and HSAP-DC-CAT were incubated with protease K
(0.5 mg/ml) at 37 °C. At different time points, the catalase activity of
samples was measured by aforementioned Góth method.
2.3. Synthesis of 3-(azidomethyl)-4-methyl-2,5-furandione (AzMMMan)
The BrMMMan intermediate was synthesized by using a previous
protocol (Liu et al., 2016; Maier and Wagner, 2012). In brief, di-
methylmaleic anhydride (5.04 g, 40 mmol), N-bromosuccinimide
(14.24 g, 80 mmol), and benzoyl peroxide (0.2 g, 0.83 mmol) were
added into an adaptive round-bottom flask and dissolved in carbon
tetrachloride (CCl4, 250 mL). The reaction was stirred for 5 h under
refluxing. Then the mixture was cooled down to room temperature,
benzoyl peroxide (0.2 g, 0.83 mmol) was added as catalyst to the
mixture again, the reaction was stirred for another 5 h under refluxing.
Then the mixture was agaun cooled down to room temperature, and
solids were removed by filtration. The obtained solution was washed
two times with DI water (100 mL) and one time with saturated sodium
chloride solution by separating funnel, then the organic solution dried
over anhydrous sodium sulfite for 3 h and concentrated to a thick
yellow oil by rotary evaporator. The crude intermediate was purified by
chromatography on a silica gel column (elution with petroleum ether/
ethyl acetate 4:1). Finally, BrMMMan intermediate (purity 96%, 1.1 g)
was obtained (Fig. S2a). BrMMMan: 1H NMR (500 MHz, CDCl3) δ
(ppm) = 2.18 (s, 3H, eCH3), 4.18 (s, 2H, eCH2-Br), 7.26 (CHCl3).
The AzMMMan was similarly synthesized as previously reported
(Liu et al., 2016; Maier and Wagner, 2012). Briefly, BrMMMan
(1.5 mmol), sodium azide (1.6 mmol) and dry acetone (15 mL) were
added into a 50 mL round-bottom flask. The reaction was stirred for
12 h under room temperature. The crude product was obtained by fil-
tering and evaporating. After that, the final product was purified by
silica gel column (eluent, hexane/ethyl acetate = 7:3). AzMMMan
2. Materials and methods
2.1. Materials
Catalase solution (≥35,000 units/mg) was purchased from Aladdin.
Chlorin e6 was purchased from J&K Scientific Ltd. Human serum al-
bumin, dimethyl sulfoxide, N-(3-dimethylaminopropyl)-N-ethylcarbo-
diimide hydrochloride crystalline, N-hydroxysuccinimide, N-hydro-
xysulfosuccinimide sodium salt and 3-(4,5-dimethylthiazol-2-yl)-2.5-
diphenyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich.
RPMI-1640 medium and fetal bovine serum were obtained from
Thermo Fisher Scientific Inc. All other chemicals were purchased from
China National Pharmaceutical Group Corporation and used directly
without any further purification.
2.2. Characterization and methods
UV-VIS-NIR absorbance spectra were recorded by the Perkin Elmer
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X. Zhang, et al.
InternationalJournalofPharmaceutics582(2020)119321
(95.5 mg) was obtained after evaporating the eluent (Fig. S2b).
AzMMMan: 1H NMR (500 MHz, CDCl3), δ (ppm) = 2.23 (s, 3H, eCH3),
4.29 (s, 2H, eCH2-N3), 7.26 (CHCl3).
Culture Collection (ATCC) and cultured in RPMI-1640 medium in-
cluding 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin
at 37 °C, 5% CO2.
To study the cellular internalization efficiency of HSAP-DC-CAT and
free Ce6, 4T1 cells were seeded into 12 well plates and then incubated
with HSAP-DC-CAT and free Ce6 (Ce6, 10 µM) for 4 h. Confocal ima-
ging and flow cytometry were carried out to study the internalization of
HSAP-DC-CAT and control group.
To detect the reactive oxygen species (ROS) in vitro, 4T1 cells were
incubated with free Ce6, HSAP-DC-HSA and HSAP-DC-CAT separately.
DCFH-DA was used as fluorescence probe. The ROS level in these
groups were confirmed by confocal imaging.
2.4. Synthesis of DBCO-Ce6
DBCO-PEG4-CH2CH2NH2 in this experiment was used to introduce
DBCO groups onto Ce6 by the reaction of amino groups with carboxyl
groups of Ce6. In brief, Ce6 (1 eq) was pre-dispersed in DMSO and
activated by EDC (5 eq) and NHS (5 eq) for 1 h. Then DBCO-PEG4-
CH2CH2-NH2 (2.5 eq) and TEA (5 eq) were added into the solution and
the mixture was reacted for 12 h, resulting in DBCO-Ce6.
For studying the chemotherapy effect when treated with HSAP-DC-
CAT or free cisplatin, 4T1 cells in 96-well plates were incubated with
different concentrations of free cisplatin or HSAP-DC-CAT for 24 h. The
standard MTT assay was used for measuring the cell relative viabilities.
For combinational therapy of PDT and chemotherapy, 4T1 cells
were seeded into 96-well plates and then incubated with various con-
centrations of free Ce6 and HSAP-DC-CAT. After 4 h incubation, sam-
ples were incubated for 30 min with or without irradiation by 660 nm
light at 5 mW/cm2. After 24 h of additional incubation, MTT assay was
used for determining the relative viabilities of cells.
For enhanced combinational therapy of PDT and chemotherapy,
4T1 cells were firstly seeded into 96-well plates and cultured in a hy-
poxic cell incubator (5% CO2, 94% N2, 1% O2) for 24 h, and subse-
quently incubated with different concentrations of HSAP-DC-CAT and
free Ce6 for 4 h. Then these samples were irradiated by 660 nm light at
5 mW/cm2 for 30 min with or without adding 100 µM H2O2 in advance.
After that, samples were transferred into normal condition and in-
cubated for 24 h with fresh media. MTT assay was carried out to
measure the cell viabilities after various treatments.
In order to find out whether HSAP-DC-CAT in the acidic environ-
ment would enhance their deep penetration, multicellular tumor
spheroids (MCTS) were used as model system, to evaluate migration
ability of HSAP-DC-CAT in different pH conditions. In briefly, MCTS
were firstly incubated with HSAP-DC-CAT at pH 6.5 or 7.4 for 24 h and
then washed with normal PBS. Then, MCTS were fixed by 4% for-
maldehyde and dehydrated by sugar solutions for frozen section. The
MCTS slices were stained with DAPI for confocal imaging to determine
the migration of HSAP-DC-CAT (Fig. S7).
2.5. Modification of proteins with AzMMMan
AzMMMan was used to reversibly introduce azide groups into cat-
alase, or serum albumin (HSA) by the reaction of excessive maleic an-
hydride group from AzMMMan with amino groups of the proteins.
Briefly, AzMMMan (10 mg) dissolved in 50 µL acetonitrile was slowly
added into catalase solution (10 mg) in Hepes buffer pH 9.0 and reacted
for 4 h. CAT-AZ was obtained after ultrafiltration by a centrifugal filter
device (molecular weight cut-off, MWCO = 10 kDa) to remove ex-
cessive free AzMMMan.
Succinic acid-derivatized cisplatin prodrug (Pt (IV)SA) was pre-
pared by oxidation and acidification of cisplatin according to a previous
protocol (Feng et al., 2016). HSA (10 mg, 1 eq) reacted with Pt (IV)SA
(0.653 mg, 10 eq) which was first activated by EDC and NHS in DMSO
for 0.5 h. The mixture was stirred for 12 h in PBS (pH 8.0) in the dark.
HSA-Pt (IV) was obtained after centrifugation at 4500 rpm for 5 min to
remove possible precipitate and ultrafiltration by a centrifugal filter
device (molecular weight cut-off, MWCO = 10 KDa) to remove free
small molecules. Pt (IV)-HSA-AZ was then prepared analogously as
described for CAT-AZ.
2.6. Synthesis of protein-drug conjugates by click chemistry
HSAP-DC-CAT conjugates were prepared by the click chemistry
reaction of DBCO-Ce6 with azide groups of AzMMMan-modified pro-
teins (Fig. S3). In brief, DBCO-Ce6 (1 eq to 1 eq AzMMMan from pro-
teins-AZ) was used to bridge CAT-AZ (5 mg) and Pt (IV)-HSA-AZ (5 mg)
in PBS (pH 8.0), the mixture reacted for 6 h, then the resulting protein
conjugates were PEGylated.
2.9. Animal experiment
As a control, HSAP-DC-HSA were prepared by the similar procedure
by replacement of CAT with HSA as described in the synthesis of HSAP-
DC-CAT. HSA-DC-CAT without cisplatin was also prepared analogously
except that no Pt (IV)SA was previously introduced onto HSA (Fig. S3,
Fig. S4). Furthermore, the CAT/Ce6/HSAP mixture as another control
was obtained by simply physical mixing of CAT, Ce6 and HSAP.
The drug loading rate of these conjugates were calculated by using
the following equations: Ce6 loading rate = determined Ce6 content in
conjugate/initial Ce6 content for DBCO-Ce6 conjugation; Pt loading
rate = determined Pt content in conjugate/initial Pt content for con-
jugation with HSA (Table S1).
To establish the animal model, female nude mice were purchased
from Nanjing Peng Sheng Biological Technology Co Ltd. All animal
experiments were performed according to the guidelines for the pro-
tection of animal life and were approved by Laboratory Animal Ethics
Committee in Soochow University. For tumor inoculation, 4T1 cells
(about 2 × 106) were suspended in 50 µL PBS and finally injected
subcutaneously into the back of mice.
For in vivo imaging and distribution studies, HSAP-DC-CAT or CAT/
Ce6/HSAP (Ce6, 0.5 mg/ml, 200 µL) was intravenously injected into
mice (Ce6 dose: 5 mg/kg). Blood was drawn from tail vein of mice at
different time points after intravenous injection. The mice were then
sacrificed, organs including liver, spleen, kidneys, heart, lung and
tumor were obtained and used for ex vivo imaging by a Maestro in vivo
optical imaging system.
For in vivo combinational therapy, nude mice with 4T1 tumors
(~100 mm3) were divided into five groups: (1) Untreated; (2) i.v. in-
jected with HSAP-DC-CAT and irradiated with 660 nm light at 4 h p.i.;
(3) i.v. injected with HSAP-DC-CAT without irradiation; (4) i.v. injected
with HSAP-DC-HSA and irradiated by 660 nm light at 4 h p.i. ; (5) i.v.
injected with HSA-DC-CAT and irradiated by 660 nm light at 4 h p.i.
The dose of Ce6 and cisplatin were kept at 5 mg/kg and 85 µg/kg,
respectively. The weight of mice of different groups were recorded (Fig.
S8), lengths and widths of tumor measured with a digital caliper were
2.7. pH-responsive release of proteins
HSAP-DC-CAT, HSA-DC-CAT, HSAP-DC-HSA and mixture CAT/
Ce6/HSAP were incubated in PBS buffer (pH 7.4, pH 6.0) for 8 h, and
the hydrodynamic diameters of these treated conjugates were de-
termined by dynamic light scattering (DLS) (Fig. S5). Additionally, the
protein release from various conjugates in different acidic conditions
was evaluated by SDS-PAGE (Fig. S6).
2.8. Cell experiment
Murine breast cancer cells (4T1) were obtained from American Type
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X. Zhang, et al.
InternationalJournalofPharmaceutics582(2020)119321
recorded every two days for 2 weeks. The tumor volume was calculated
by the formula: width2 xlength /2.
The histological properties of tumors with various treatment were
analyzed by hematoxylin and eosin (H&E) staining assay. The H&E
staining tumor slices were collected. The damaged stated of various
tumors were then assessed by confocal imaging.
(~652 nm), the red shift in HSAP-DC-CAT was observed, likely a result
from the covalent bonding between Ce6 and HSAP or CAT (Chen et al.,
2017). Meanwhile, HSAP-DC-CAT exhibited two new absorption bands
at 402 nm and 504 nm compared to HSA, indicated the successful
conjugation of CAT (Fig. 2a). The fabricated HSAP-DC-CAT showed
uniform size with average diameter around ~120–200 nm as revealed
by transmission electron microscopy (TEM) imaging (Fig. 2b), colla-
boratively demonstrated by the hydrodynamic diameters of 164 nm
measured with DLS (Fig. 2c). In addition, HSA-DC-CAT and HSAP-DC-
HSA showed again the increased size compared to CAT/Ce6/HSA
mixture (Fig. S4), in corresponding to the SDS-PAGE results in Fig. S3,
as result of the click conjugation. All these complementary analyses
verified the successful conjugation of CAT-AZ, Pt (IV)-HSA-AZ with
DBCO modified Ce6 by click chemistry.
3. Results and discussion
In the few recent decades, protein-based therapeutics including
their drug conjugates presented a great opportunity for treatment of a
variety of human diseases, owning to high specificity and high potency
derived from their amino-acid-based secondary and tertiary structure
(Chen et al., 2017; Song et al., 2016a; Song et al., 2016b). However, the
interaction force between proteins and formulation counterpart should
be rational balanced. The interaction should be strong enough to
withstand the in vivo hostile environment. In additional, the interaction
should not influence the bioactivity of protein therapeutics and drugs,
result in reducing the therapeutic outcome. For example, PEGylated
interferon-α2a, as FDA approved in 2001 for treating hepatitis, retains
only 10% activity of unmodified interferon-α2a (Bailon et al., 2001;
Foser et al., 2003).
3.2. Acidic pH reversibility
The AzMMMan linker offers the opportunity for acidic pH-reversible
modifications of proteins (Liu et al., 2016; Liu et al., 2017; Maier and
Wagner, 2012). To evaluate the acidic sensitivity of HSAP-DC-CAT,
HSAP-DC-CAT was incubated at different pH conditions (pH 7.4, 6.5).
The DLS measurement together with TEM observation showed a re-
duction of size of HSAP-DC-CAT from 164 nm to 21 nm when incubated
in acidic pH 6.5 buffer (Fig. 2d), collaboratively demonstrated by the
size shrinking of HSA-DC-CAT, HSAP-DC-HSA and HSAP-DC-HSA (Fig.
S5). Meanwhile, HSAP-DC-CAT treated with normal buffer (pH 7.4) did
not show obvious alteration of size. This indicates that the HSAP-DC-
CAT conjugate formulation could be dissociated under acidic environ-
ment. The conjugates without or with treatment in acidic buffer were
also analyzed by SDS-PAGE to confirm successful conjugation and dy-
namic release under acidic conditions (Fig. S3, Fig. S6). The SDS-PAGE
gel of HSAP-DC-CAT showed a lower shift distance compared to the
unmodified HSA and CAT, which demonstrates successful assembly of
proteins with DC, as also revealed by aforementioned TEM and DLS
measurement. Meanwhile, the HSAP-DC-CAT treated with acidic buf-
fers displayed a similar migration compared to that of HSA and CAT,
verifying again that the covalent binding of HSA-AZ, CAT-AZ with DC
could be cleavage triggered in acidic environment (Fig. 2d, Fig. S5, Fig.
S6).
Inspired by the pH-dissociation of HSAP-DC-CAT conjugates, which
might be beneficial for cancer therapy along with the shrink size of the
conjugates in the acidic TME, their penetration into the solid tumor was
investigated. The multicellular tumor spheroids (MCTS) model of solid
tumor was incubated with HSAP-DC-CAT conjugates at different pH
conditions (pH 7.4, pH 6.5). The fluorescence of MCTS treated with
HSAP-DC-CAT at pH 6.5 was stronger than treatment at pH 7.4, as
revealed by confocal imaging (Fig. S7). This indicates a better in-
tratumoral penetration in the acidic microenvironment as a result of the
dynamic formulation strategy.
3.1. Dynamic covalent bond-assisted protein-drug formulation HSAP-DC-
CAT
Protein-drug conjugates present promising biopharmaceuticals for
clinical usage. Nonetheless, the conjugation of proteins to their drug
counterparts should be rationally designed to avoid inactivation of their
functional activities (Chen et al., 2017; Leader et al., 2008; Song et al.,
2016a). To circumvent this possible pitfall, we explored our previous
developed tumor acidity responsive bifunctional AzMMMan (Liu et al.,
2016; Liu et al., 2017; Maier and Wagner, 2012) as the dynamic
covalent linker for the fabrication of a multifunctional protein-drug
conjugate for effective cancer treatment. For this purpose, we chose
CAT as a therapeutic protein model, pro-drug Pt (IV) and Ce6 as small
molecule drugs to study this novel type of protein-drug conjugate
(Fig. 1, Fig. S1). In briefly, the pro-drug Pt (IV)-COOH was firstly
conjugated with parts of amino groups of HSA and free pro-drug Pt (IV)
was removed by ultrafiltration, the anchored Pt (IV) was quantified by
inductively coupled plasma mass spectrometry (ICP-MS). In the fol-
lowing, CAT and Pt (IV) loaded HSA were then coupled with excess
acidic reversible bifunctional linker AzMMMan (Fig. S2) by the reaction
of maleic anhydride groups with amino groups of the proteins. After
that, the HSAP-DC-CAT were obtained through a crosslinking process
by click chemistry of DBCO coupled Ce6 (DC) with AzMMMan modified
proteins by strain-promoted azide alkyne cycloaddition. The resulting
protein conjugates were PEGylated. Besides of this, HSAP-DC-HSA
conjugates without CAT, HSA-DC-CAT conjugates without pro-drug Pt
(IV) and CAT/Ce6/HSAP mixture without AzMMMan were also pre-
pared by similar covalent reaction or simple physical mixing for the
following control experiments.
3.3. Enzyme activity assessment
The conjugates were then analyzed by SDS-PAGE to confirm the
successful assembly (Fig. S3). Compared to the unmodified CAT, HSA
and CAT/Ce6/HSA mixture, the SDS-PAGE gel revealed the shortest
migration for HSAP-DC-CAT, HSAP-DC-HSA and HSA-DC-CAT con-
jugates, which could be explained by the successful click conjugation of
DC with AzMMMan modified HSAP, HSA and CAT. The loading con-
tents of Pt (IV) and Ce6 of HSAP-DC-CAT conjugates were 74.2% and
73.2% respectively determined by ICP-MS and UV–VIS-NIR spectra
(Table S1).
As revealed by the UV–VIS–NIR absorption spectra of CAT, CAT-AZ,
HSA, Pt(IV)-HSA-AZ, Ce6 and HSAP-DC-CAT, HSAP-DC-CAT showed a
new absorption band around 664 nm compared to CAT and CAT-AZ
(Fig. 2a), which derived from the interaction of photosensitizer Ce6
with HSA. Interestingly, compared with the absorption peak of free Ce6
Catalase, one of the highest catalytic efficiency enzyme in organs to
decompose H2O2 to H2O and O2, has been investigated to adjust the
hypoxia condition in the TME (Song et al., 2016a). To demonstrate that
the HSAP-DC-CAT would also catalyze the decomposition of H2O2, an
abundant metabolite in the tumor tissue, to H2O and O2, the catalytic
performance of HSAP-DC-CAT and control groups were rationally stu-
died by a portable dissolved oxygen probe. Compared to the un-
modified CAT, the cross-linked HSAP-DC-CAT also performed high
catalytic activity of decomposition of H2O2 to generate O2 in 2 min.
(Fig. 3a), though with a reduced efficiency which could be explained by
the partial inhibition of the enzyme after conjugation, but this expected
to be restored when the enzyme traceless cleavage release in acidic
environment. As expected, the catalytic ability of H2O2 to O2 by HSAP-
DC-CAT is a concentration dependent process (Fig. 3a). However, in the
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InternationalJournalofPharmaceutics582(2020)119321
Fig. 1. A scheme illustrating the synthesis process of HSAP-DC-CAT and traceless pH-responsive release of payloads. (a) HSAP-DC-CAT were fabricated by click
chemistry of DBCO-Ce6 with AzMMMan modified HSAP and CAT. (b) Schematic description of diminishing solid tumor by accumulated HSAP-DC-CAT and de-
gradation of HSAP-DC-CAT triggered by acidic tumor microenvironment.
absence of catalase-containing group, a negligible O2 was detected
which indicated H2O2 solution was rather stable at the experimental
condition (Fig. 3a).
Motived by the highly efficient generation of O2 catalysis by HSAP-
DC-CAT, the generation of singlet oxygen (SO) was then studied, which
would be greatly beneficial for PDT in a tumor hypoxia micro-
environment. The concentration of SO was measured by the singlet
oxygen sensor green (SOSG) probe (Chen et al., 2017), when HSAP-DC-
CAT treated with or without 0.5 mM H2O2, which was to mimic the
metabolic TME. It is noteworthy that high SO generation was detected
when treated HSAP-DC-CAT together with H2O2, owing to the efficient
production of O2 by catalytic decomposition of H2O2. Meanwhile, the
generation of SO treated only with HSAP-DC-CAT or free H2O2 was
much lower than that of treated with both reagents (Fig. 3b). This si-
mulation experiment of TME could indirectly prove that the HSAP-DC-
CAT would reconstruct a beneficial therapeutic parameter of abundant
SO for the following PDT.
Though the activity of CAT from HSAP-DC-CAT to decompose H2O2
would be partially inhibited compared to unmodified CAT, but this
could be beneficial in vivo physiological environment, as unmodified
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InternationalJournalofPharmaceutics582(2020)119321
Fig. 2. Characterization of HSAP-DC-CAT. (a)
UV-via-NIR spectra of free CAT, HSA, Ce6, CAT-
AZ, Pt(IV)-HSA-AZ and HSAP-DC-CAT. (b) TEM
imaging of HSAP-DC-CAT in buffer (pH 7.4). (c)
DLS measurement of free CAT, HSA and HSAP-
DC-CAT in buffer (pH 7.4). (d) DLS measurement
of HSAP-DC-CAT in different pH conditions (pH
7.4, 6.5) and TEM imaging of HSAP-DC-CAT
treated in pH 6.5 buffer.
CAT was exposed to a hostile environment in the presence of proteases,
which would result in the frequent deactivation of unmodified CAT. To
determine whether the conjugation would be beneficial for the pro-
tection of CAT bioactivity, HSAP-DC-CAT and free CAT were treated
with proteases, and activities of CAT from both were investigated by the
Góth method (Góth, 1991). Compared to the unmodified CAT, most of
which lost its bioactivity in 24 h during treatment with protease K
(0.5 mg/ml), CAT from HSAP-DC-CAT conjugates could maintain most
enzyme bioactivity (69.5%) ever after 24 h when incubated with the
same concentration of protease K applied for unmodified CAT (Fig. 3c).
This might offer an optimal protection strategy of the CAT for its tar-
geting delivery to TME, as well as for other therapeutic proteins which
perform their functions in vivo.
scanning microscopy (CLSM) (Fig. 4a) and flow cytometry (Fig. 4b),
thus providing the internalized Pt (IV) as anchored in HSAP-DC-CAT
the proper location to perform its therapeutic cancer cell killing effect.
Consequently, cell viability assays (MTT) were performed with various
concentration (Pt: 0.625 μM, 1.25 μM, 2.5 μM, 5 μM) of HSAP-DC-CAT
and free cisplatin quantified by ICP-MS to investigate the biological
activity and effect on 4T1 cell viability after 24 h incubation. Both
HSAP-DC-CAT and free cisplatin showed a cytotoxicity towards to 4T1
cancer cells which was concentration dependent, with HSAP-DC-CAT at
5 µM presenting most efficient cell killing ability (Fig. 4c).
In the following we wanted to evaluate whether the conjugated CAT
from HSAP-DC-CAT could improve the intracellular concentration of
reactive oxygen species (ROS), which would be expected to enhance the
therapeutic effect for PDT. The intracellular generation of ROS by
adding ROS detector DCFH-DA was imaged after incubation with Ce6,
HSAP-DC-HSA and HSAP-DC-CAT conjugates. It was found that gen-
eration of ROS was much higher when treated with HSAP-DC-CAT
conjugates than that of Ce6 and HSAP-DC-HSA treatment without CAT
(Fig. 4d), as revealed by the green signal only observed from HSAP-DC-
3.4. Intracellular delivery and cytotoxicity
After the characterization of HSAP-DC-CAT, their cellular inter-
nalization and in vitro cytotoxicity was investigated. HSAP-DC-CAT was
efficiently internalized into 4T1 cells, as indicated by the confocal laser
Fig. 3. Enzyme activity assessment. (a) Oxygen generation treated with free CAT or HSAP-DC-CAT in the presence of various H2O2 concentrations. (b) The generation
of singlet oxygen determined by the increased SOSG fluorescence for free H2O2 and HSAP-DC-CAT with or without adding H2O2. (c) Relative bioactivity of free CAT
and HSAP-DC-CAT after treatment with proteinase K assay.
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InternationalJournalofPharmaceutics582(2020)119321
Fig. 4. Intracellular delivery and cytotoxicity. (a) Confocal fluorescence images of 4T1 cells after incubation with free Ce6 and HSAP-DC-CAT. Blue and red colors
represented DAPI-stained cell nuclei and Ce6 fluorescence respectively. (b) Flow cytometry of 4T1 cells after incubation with free Ce6 and HSAP-DC-CAT. (c) Relative
cell viability of 4T1 treated with cisplatin and HSAP-DC-CAT at different Pt concentrations. (d) Reactive oxygen species (ROS) in 4T1 cells were imaged by the
confocal laser scanning microscope (CLSM) by adding DCFH-DA, after incubation with free Ce6, HSAP-DC-HSA and HSAP-DC-CAT. (e) Relative cell viability of 4T1
treated with free Ce6 and HSAP-DC-CAT with or without irradiation at a various concentrations of Ce6. (f) Relative cell viability of 4T1 treated with HSAP-DC-CAT
with or without adding H2O2 in the presence of different Ce6 concentrations under irradiation. P values were calculated by Student’s t-test (***p < 0.001). (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
CAT group.
Having demonstrated that HSAP-DC-CAT assisted ROS generation,
mentioned paragraph.
Furthermore, considering that CAT could help to decompose en-
dogenous H2O2 within the TME to produce oxygen, we also checked
whether the therapeutic outcome would be further improved with
exogenous H2O2, to mimic TME under nitrogen atmosphere. The cell
cytotoxicity of HSAP-DC-CAT treatment in the presence of H2O2 (for
enhanced ROS generation) under NIR irradiation showed encouraging
therapeutic effects; cancer cells viability treated with 2.5 μM (quanti-
fied by Ce6) HSAP-DC-CAT in the presence of H2O2 under NIR irra-
diation was only 1.3%, far lower than that treatment without H2O2
incubation (Fig. 4f), as a result of enhanced PDT improved by the
generation of ROS by catalytic degradation of H2O2 with CAT. To the
best of our knowledge, this is the lowest concentration of Ce6 for potent
the 4T1 cancer cell viability was evaluated by MTT after treated with
HSAP-DC-CAT conjugates at various concentrations without or with
light irradiation. Compared to the treatment with Ce6 or HSAP-DC-CAT
without light irradiation, the combinational treatment by HSAP-DC-
CAT under NIR irradiation showed the highest cell killing ability to-
wards to 4T1 cancer cells, as revealed only 3.1% cells viability when
treated at the 2.5 μM conjugated Ce6 (Fig. 4e), which is much more
effective than previously reported(Chen et al., 2017). It has to be
mentioned that HSAP-DC-CAT presumably display a higher cytotoxicity
than cells treated with Ce6 under NIR irradiation, due to the extra sy-
nergistic therapeutic effect from Pt (IV), as also explained in the above-
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InternationalJournalofPharmaceutics582(2020)119321
Fig. 5. In vivo pharmacokinetic behaviors of HSAP-DC-CAT. (a) The blood circulation of CAT/Ce6/HSAP mixture and HSAP-DC-CAT conjugates after tail intravenous
injection (3 mice each group). (b) In vivo fluorescence imaging of 4T1 tumor-bearing mice with intravenous injection of CAT/Ce6/HSAP and HSAP-DC-CAT at
different time points, and ex vivo fluorescence images of major organs at 24 h post injection. Li, Sp, Ki, He, Lu and Tu stand for liver, spleen, kidney, heart, lung and
tumor, respectively. (c) Quantification of ex vivo fluorescence images for those organs (24 h p.i) from b). P values were calculated by Student’s t-test (***p < 0.001).
cancer cell killing. In sum, HSAP-DC-CAT showed an excellent cancer
cell killing ability in a synergistic setting of a programmed drug for-
mulation strategy.
resulting in inferior blood circulation.
After confirming suitable in vivo circulation of HSAP-DC-CAT, mice
bearing subcutaneous 4T1 tumors were imaged by a Maestro EX in vivo
imaging system at different time points post injection of HSAP-DC-CAT
or the CAT/Ce6/HSAP mixture. The fluorescence of Ce6 from both
HSAP-DC-CAT and mixture CAT/Ce6/HSAP were diffuse within the
whole body of mouse at the early time points (Fig. 5b). Subsequently,
the mouse showed excellent tumor accumulation with injected HSAP-
DC-CAT, as visible by the strong fluorescence of Ce6 at tumor tissue
even at 24 h post-injection, whereas much weaker fluorescent signals
were observed at the tumor of mice injected with CAT/Ce6/HSAP
(Fig. 5b). The semi-quantitative analysis of fluorescence distribution
through various tissues and organs confirmed that mice treated with
HSAP-DC-CAT performed much higher tumor accumulation
(40.6–16.8%) for potential therapeutics (Fig. 5c), while mice treated
with CAT/Ce6/HSAP mixture showed higher accumulation in kidney.
The superior tumor accumulation of HSAP-DC-CAT might be con-
tributed by the covalent binding strategies for the protein-drug for-
mulation, as also discussed in the previous section.
3.5. In vivo pharmacokinetics and pharmacodynamics
Considering that the remarkable in vitro cancer cells killing ability
by treatment with HSAP-DC-CAT, the in vivo pharmacokinetics and
pharmacodynamics of HSAP-DC-CAT were then investigated. Herein,
the simple physical mixture of CAT, Ce6 and HSAP (CAT/Ce6/HSAP)
was carried out as the control experiment, to verify whether the dy-
namic covalent bondage of fabricated HSAP-DC-CAT would enhance
their in vivo behavior and therapeutic efficacy. Mice were in-
travenously injected with the dynamic covalent bond conjugate HSAP-
DC-CAT and the control mixture CAT/Ce6/HSAP at the same dose of
Ce6 (5 mg/kg). The in vivo behavior of conjugates and control mixture
was tracked at different time points post injection based on the fluor-
escence of Ce6. As detected by the fluorescence of Ce6 in the extracted
blood at various time, the blood circulation half-lives of HSAP-DC-CAT
was calculated and found to be much longer than that of CAT/Ce6/
HSAP mixture (8.05 h compared to 2.74 h, Fig. 5a). HSAP-DC-CAT
apparently were sufficiently stable in the blood circulation environ-
ment, with the covalent binding between components to resist the
hostile environment in the blood. On the contrary, the simple physical
mixture of CAT/Ce6/HSAP by intermolecular forces, which are much
weaker than covalent bonding, might be easily dissociated in the blood,
Motivated by the excellent blood circulation half-lives and superior
tumor accumulation of the HSAP-DC-CAT, their combination therapy at
different conditions was performed in vivo with the 4T1 tumor model
(Fig. 6a). To investigate the therapeutic outcome by treatment with
HSA-DC-CAT or other control groups, the mice were divided into five
groups (five mice for one group): untreated, HSAP-DC-CAT without
irradiation, HSA-DC-CAT (Ce6, 5 mg/kg) with 660 nm light for 1 h,
8

X. Zhang, et al.
InternationalJournalofPharmaceutics582(2020)119321
Fig. 6. In vivo combination therapy. (a) Scheme of HSAP-DC-CAT behavior in TME. (b) Tumor growth curves of mice (n = 5 per group) in different treatment groups
(i.v. injection). Untreated; HSAP-DC-CAT irradiated at 660 nm light at 4 h post-injection (p.i.); HSAP-DC-CAT without irradiation; HSAP-DC-HSA irradiated at
660 nm light at 4 h p.i.; HSA-DC-CAT irradiated at 660 nm light at 4 h p.i.; (c) Average tumor weight after various treatments. P values were calculated by Student’s t-
test: *p < 0.1, *p < 0.01, ***p < 0.001 (n = 5). d) H&E stained tumor slices from different groups collected 24 h p.i (scale bars: 100 μm).
HSAP-DC-HSA (Ce6, 5 mg/kg; Pt, 85 µg/kg) with 660 nm light for 1 h,
HSAP-DC-CAT (Ce6, 5 mg/kg; Pt, 85 µg/kg) with 660 nm light for 1 h.
After various treatments, the tumor size was monitored every two days
for two weeks with a caliper (Fig. 6b). The therapeutic outcome was
most encouraging; the mice treated with HSAP-DC-CAT under NIR ir-
radiation showed best antitumor effects among the various treatment
groups, presumably due to the combination effects of PDT with Ce6, Pt
for chemotherapeutic activity, CAT to improve the ROS generation for
enhanced PDT (Fig. 6a), acidic cleavage of DC for deeper intratumoral
penetration of therapeutics (Fig. S7). The HSA-DC-CAT group without
Pt chemotherapeutic activity, the HSAP-DC-CAT group without NIR
irradiation for PDT, the HSAP-DC-HSA group without CAT to improve
ROS generation, all showed lesser therapeutic effects compared to the
optimal protein-drug conjugate formulation HSAP-DC-CAT (Fig. 6b).
The therapeutic outcome was also evaluated by the tumor weight of
various treatments. It again revealed that the HSAP-DC-CAT group with
660 nm light had the most effective therapeutic effect with smallest
tumors (225 mg weight) compared to other control groups (heavier
than 600 mg) (Fig. 6c). Meanwhile, none of the treatments with the
different protein-drug formulation showed obvious cytotoxicity, as the
weight of mice maintained normal physical state during treatments
(Fig. S8). Furthermore, the histology analysis of tumor slices collected
after various treatments was performed by hematoxylin and eosin (H&
E) staining (Fig. 6d). The most efficient tumor cell destruction was
observed for the treatment by HSAP-DC-CAT with 660 nm light, while
other control groups, for example HSAP-DC-HSA (pink line) without
CAT to improve ROS generation, had reduced or negligible damaged
cancer cells. Overall, our study demonstrated the high efficiency of
tumor inhibition by dynamic covalent bond conjugate formulation of
the combination protein-drug conjugate HSAP-DC-CAT.
4. Conclusion
In this study, a balanced protein-drug conjugate HSAP-DC-CAT with
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X. Zhang, et al.
InternationalJournalofPharmaceutics582(2020)119321
an acidic-reversible bifunctional linker (AzMMMan) by amidation and
click chemistry has been first developed. The conjugated HSAP-DC-CAT
integrated small molecular drugs for chemotherapy (Pt pro-drug) and
enhanced PDT (Ce6) with an enzyme (CAT) for hypoxia normalization
of TME. The covalent binding of proteins and drugs would benefit both
for favorable in vivo circulation and control release of payloads for
better therapeutic outcome. Pharmacokinetic studies showed the pro-
longed circulation half-lives of dynamic covalent conjugated HSAP-DC-
CAT (8.05 h), as compared to the physical mixture of CAT/Ce6/HSAP
(2.74 h), demonstrating the advantages of conjugation. Meanwhile, the
HSAP-DC-CAT conjugates accumulated in the tumor tissue could bring
about synergistic effect by small molecular drugs Ce6, pro-drug Pt (IV)
with therapeutic protein to improve the hypoxia microenvironment of
tumor. Given that abundant amino groups on proteins, we expect the
pH-labile trace-less conjugation method should be applicable for many
therapeutic proteins with reactive small molecular drugs.
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Methodology, Data curation. Ernst Wagner: Writing - review & editing.
Zhuang Liu: Project administration, Supervision. Xiaowen Liu: Project
administration, Funding acquisition, Conceptualization, Methodology,
Supervision, Writing - original draft, Writing - review & editing.
Declaration of Competing Interest
Liu, X., Zhang, P., Rödl, W., Maier, K., Lächelt, U., Wagner, E., 2017. Toward artificial im-
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The authors declare that they have no known competing financial
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This work was supported by the startup funding from Jinan
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Supplementary data to this article can be found online at https://
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