Co-delivery of Doxorubicin and Interferon-γ by Thermosensitive Nanoparticles for Cancer Immunochemotherapy

Article abstract of DOI:10.1021/acs.molpharmaceut.8b00564

A dual-sensitive nanoparticle delivery system was constructed by incorporating an acid sensitive hydrazone linker into thermosensitive nanoparticles (TSNs) for co-encapsulating doxorubicin (DOX) and interferon γ (IFNγ) and to realize the co-delivery of chemotherapy and immunotherapy agents against melanoma. DOX, a chemotherapeutic drug, was conjugated to TSNs by a pH-sensitive chemical bond, and IFNγ, a potent immune-modulator, was absorbed into TSNs through the thermosensitivity and electrostatics of nanoparticles. Consequently, the dual sensitive drug-loaded TSN delivery systems were successfully built and showed an obvious core-shell structure, good encapsulation efficiency of drugs, sustained and sensitive drug release, prolonged circulation time, as well as excellent synergistic antitumor efficiency against B16F10 tumor bearing mice. Moreover, the combinational antitumor immune responses of hydrazone bearing DOX/IFNγ-TSN (hyd) were strengthened by activating Th1-type CD4⁺ T cells, cytotoxic T lymphocytes, and natural killer cells, downregulating the expression levels of immunosuppressive cytokines, such as IL10 and TGFβ, and upregulating the secretion of IL2 and TNFα. Taken together, the multifunctional TSNs system provides a promising strategy for multiple drugs co-delivery with distinct properties.

Full text of DOI:10.1021/acs.molpharmaceut.8b00564

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Article
Co-delivery of Doxorubicin and Interferon-gamma by
Thermosensitive Nanoparticles for Cancer Immunochemotherapy
Yijia Yin, Qian Hu, Chenfeng Xu, Qi Qiao, Xianya Qin,
Qingle Song, Yang peng, Yongdan Zhao, and Zhiping Zhang
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00564 • Publication Date (Web): 16 Jul 2018
Downloaded from http://pubs.acs.org on July 17, 2018
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Molecular Pharmaceutics
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Coꢀdelivery
of
Doxorubicin
and
Thermosensitive
Cancer
Interferonꢀgamma
Nanoparticles
by
for
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Immunochemotherapy
a
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Yijia Yin , Qian Hu , Chenfeng Xu , Qi Qiao a, Xianya Qin , Qingle Song , Yang
Peng b *, Yongdan Zhao a and Zhiping Zhang a,c,d *
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a Tongji School of Pharmacy
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b Tongji Hospital, Tongji Medical College
c National Engineering Research Centre for Nanomedicine
d Hubei Engineering Research Centre for Novel Drug Delivery System, Huazhong
University of Science and Technology, Wuhan 430030, P.R. China
Corresponding Authors
*Prof. Dr. Zhiping Zhang
Fax and phone: +86ꢀ27ꢀ83601832
Eꢀmail: zhipingzhang@mail.hust.edu.cn
*Prof. Dr. Yang Peng
20
21
Eꢀmail: peterpengyang@126.com
22 Abstract
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A dualꢀsensitive nanoparticle delivery system was constructed by incorporating
acid sensitive hydrazone linker into thermosensitive nanoparticles (TSNs) for
coꢀencapsulating doxorubicin (DOX) and interferon γ (IFNγ), to realize coꢀdelivery of
chemotherapy and immunotherapy agents against melanoma. DOX,
a
chemotherapeutic drug, was conjugated to TSNs by a pHꢀsensitive chemical bond and
IFNγ, a potent immuneꢀmodulator, was absorbed into TSNs through the
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thermosensitivity and electrostatics of nanoparticles. Consequently, the dual sensitive
drugꢀloaded TSNs delivery system were successfully built and showed an obvious
coreꢀshell structure, good encapsulation efficiency of drugs, sustained and sensitive
drug release, prolonged circulation time as well as excellent synergistic antitumor
efficiency against B16F10 tumor bearing mice. Moreover, the combinational
antitumor immune responses of hydrazone bearing DOX/IFNγꢀTSN (hyd) were
strengthened by activating Th1ꢀtype CD4+ T cells, cytotoxic T lymphocytes and
natural killer cells, downregulating the expression levels of immunosuppressive
cytokines, such as IL10 and TGFβ, and upregulating the secretion of IL2 and TNFα.
Taken together, the multifunctional TSNs system provides a promising strategy for
multiple drugs coꢀdelivery with distinct properties.
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Keywords: thermosensitive nanoparticle, doxorubicin, IFNγ, chemotherapy,
immunotherapy
1 Introduction
Nowadays, cancer has been recognized as one of main cause of death due to its
high heterogeneity. It is predicted that the high cancer mortality will continue to
increase up to approximately 22 million deaths by 2030 1, 2. As a powerful therapy
strategy, immunotherapy has recently received more extensive attention with the
function of specifically activating selfꢀdefense system to combat tumors and prevent
recurrence due to the effect of immune memory 3ꢀ5. One of the promising
immunotherapy approaches against tumors is cytokines therapy, which can regulate
the immune system to trigger antitumor immune responses and has shown partially
effective activity against melanoma both in preclinical and clinical cancer therapy 6, 7
.
Unfortunately, cytokine can be rapidly eliminated in vivo, making difficult to achieve
good inhibition against tumor. Recent breakthroughs have revealed that the antitumor
effect of cytokines could be further enhanced when combined with chemotherapeutic
drugs 8ꢀ10. In turn, immunotherapy has compensated the deficiency of chemotherapy,
such as poor specificity and strong drug resistance, which can not only kill tumors
specifically, but also increase the sensitivity of tumors to chemotherapeutic drugs 11, 12
Base on this, the combination of chemotherapy and immunotherapy, typically
.
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chemotherapeutics and cytokines, has become an attractive strategy in synergistic
treatment of tumors, providing a promising strategy for immunochemotherapy against
melanoma 13, 14
.
Interferon γ (IFNγ) is a multifunctional cytokine mainly produced by activated T
cells and NK cells, playing a vital role in coordinating innate and adaptive immune
system to recognize and kill tumors. Briefly, IFNγ can not only act directly on tumor
cells by increasing the immunogenicity of tumor cells, but also can indirectly activate
macrophages or NK cells to inhibit tumor angiogenesis and kill tumors 15. Meanwhile,
it can also promote the proliferation and differentiation of tumorꢀspecific Th1 CD4+ T
cells and cytotoxic T lymphocytes (CTLs) to enhance antitumor immune responses
16ꢀ18. Doxorubicin (DOX), a widely used cytotoxic chemotherapeutic drug, can induce
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apoptosis of tumors as
a
nonꢀspecific anticancer agent and promote
immunomodulatory effects with the dosage decreased 19. It has been reported that low
dosage of DOX can induce immunogenic cell death (ICD) in tumor cells, resulting in
a flourishing immune response against tumors with reduction of the doseꢀrelevant
cytotoxicity 20ꢀ22
.
To explore the synergetic effect of immunotherapy and chemotherapy, here we
employed thermosensitive nanoparticles (TSNs), which were consisted of
poly(lactideꢀcoꢀglycolide) conjugated DOX (PLGAꢀDOX) as the core and
PEO–PPO–PEO copolymer (pluronic F127) as the shell 23, 24. Briefly, the hydrophobic
end PPO of F127 would be surrounded by the copolymer PLGAꢀDOX, and the PEO
layer exposed to the outside to form a hydrophilic shell structure by the hydrophobic
force to make up the coreꢀshell structure of TSNs 25, 26. In our preꢀexperiment, DOX
cannot be efficiently loaded into TSNs by the classical nanoprecipitation method. In
addition, free DOX loaded TSNs showed rapid drug release and low stability. In order
to improve the loading and delivery efficiency of drugs into TSNs, DOX was partly
attached to the backbone of PLGA polymer via an acid sensitive hydrazone linker to
synthesize polymeric drug conjugate PLGAꢀDOX (hyd) 27, 28. Next, PLGAꢀDOX (hyd)
aggregated as the core, subsequently absorbed IFNγ in opposite charge into the shell
via electrostatic interactions and the thermosensitive swelling/deswelling property of
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F127 to form DOX/IFNγꢀTSN (hyd) nanoparticles, for realizing combinational
chemotherapy and immunotherapy. To prolong the circulation time of both drugs,
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mPEGꢀPLGA was also incorporated into the TSNs formulation . It was shown that
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DOX/IFNγꢀTSN (hyd) would encounter hydrolysis in the acidic environment of
lysosome (pH 5ꢀ6) to bring about an intracellular pHꢀtriggered continuous release of
DOX. Meanwhile, DOX generated an inhibition effect on nucleus of tumor cells,
inducing tumor cells apoptosis. In addition, with the shield of F127, IFNγ showed a
thermoꢀsensitive loading into TSNs, and could sustainably deliver cytokine to tumor
site without losing bioactivity. Taken together, coꢀloading DOX and IFNγ by TSNs
displayed an effective drug delivery strategy and accumulation at tumor site as well as
significantly enhanced the tumoricidal activity through combinational
immunochemotheray.
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2 Materials and methods
2.1 Materials and reagents
PLGA (RG502H, 50:50) and mPEG5kꢀPLGA45k were bought from Shandong
Institute of Medical Instruments, China. Doxorubicin hydrochloride (DOX·HCl) was
obtained from Beijing Huafeng United Technology Co., China. Pluronic F127 was
purchased from Sigma Aldrich, St. Louis, MO, USA. Recombinant murine IFNγ was
obtained from Peprotech, USA. 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl) carbodiimide
hydrochloride (EDC·HCl), Nꢀhydroxysuccinimide (NHS), 4ꢀcarboxybenzaldehyde
(TPA), 4ꢀdimethylaminopyridine (DMAP) were all purchased from Aladdin, Shanghai,
China. Dulbecco's modified eagle medium (DMEM), penicillinꢀstreptomycin, fetal
bovine serum (FBS) and trypsin without EDTA were purchased from Hyclone, USA.
alanine aminotransferase (AST), aspartate aminotransferase (ALT), urea nitrogen
(BUN) and creatinine (Cre) assay kits were purchased from Nanjing Jiancheng
Bioengineering Institute, China. All the solvents used were of analytical grade and
produced by Sinopharm Chemical Reagent Co. Ltd., China. ELISA kits for IFNγ, IL2,
TNFα, TGFβ and IL10 were obtained from Dakewe biotech, China. The antiꢀmouse
monoclonal antibodies against the following murine antigens were purchased from
BD Pharmingen™, including FITCꢀconjugated antiꢀCD11c, PEꢀconjugated antiꢀCD86,
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PEꢀCy5ꢀconjugated antiꢀCD3e, PEꢀconjugated antiꢀCD4, FITCꢀconjugated antiꢀCD8a,
APCꢀCy7ꢀconjugated antiꢀNK1.1 and PEꢀCy7ꢀconjugated antiꢀCD69. Mouse
lymphocyte isolating solution was purchased from Dakewe biotech, China.
2.2 Cells and animals
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Murine melanoma cell line B16F10 (syngeneic with C57BL/6) was cultured in
DMEM with 10% FBS, 100 IU/mL of penicillin, and 100 ꢁg/mL of streptomycin in a
humidified atmosphere incubator with 5% CO2 at 37 ºC.
Female 5ꢀ6 weeks C57BL/6 mice were purchased from Hunan SJA Laboratory
Animal Co., China. SpragueꢀDawley (SD) rats (200 ± 20 g) were bought from the
Laboratory Animal Resources of Huazhong University of Science and Technology
(HUST). All animals were maintained under specific pathogenꢀfree condition in the
animal center of HUST. All the animal experiments were performed in compliance
with the guidelines approved by the regulations of Chinese law and the local Ethical
Committee Quantita.
2.3 Synthesis and characterization of PLGAꢀDOX (hyd)
PLGAꢀDOX (hyd) was synthesized by a threeꢀstep procedure. Firstly, TPA was
activated by NHS. Briefly, 1.5 g TPA, 4.0 g EDC·HCl and 2.3 g NHS were dissolved
in acetonitrile, and then stirred at room temperature (RT) for 24 h under nitrogen
protection. After completion of reaction, the mixture was rinsed with water for three
times. The solvent was dried by anhydrous sodium sulfate and then removed under
vacuum to obtain TPAꢀNHS. Next, 2.8 g PLGA, 0.148 g TPAꢀNHS and 0.122 g
DMAP were dissolved in dichloromethane, and the mixture was then stirred at RT for
16 h. The product was precipitated by diethyl ether and then washed to remove the
unreacted TPAꢀNHS. The residual ether was removed by vacuum drying to obtain
PLGAꢀTPA. Finally, doxorubicin hydrochloride (69.6 mg, 0.12 mmol), PLGAꢀNHS
(580 mg, 0.1 mmol) and triethylamine (TEA, 50 ꢁL, 0.36 mmol) were dissolved in 3
mL N, Nꢀdimethylformamide (DMF) and reacted at RT in the dark for 24 h. The
mixture was dialyzed with a dialysis bag (MWCO 3500) against deꢀionized water for
24 h, followed by lyophilization to obtain PLGAꢀDOX (hyd) as a red powder.
1HNMR spectra (Bruker AMꢀ400 spectrometer, Switzerland) of TPAꢀNHS and
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PLGAꢀDOX (hyd) were obtained by dissolving in d6ꢀDMSO and CDCl3 for future
analysis, respectively.
2.4 Synthesis and characterization of PLGAꢀDOX (ami)
PLGAꢀDOX (ami) was synthesized by a twoꢀstep procedure. Firstly, 500 mg
PLGA, 34 mg EDC·HCl and 21 mg NHS were dissolved in 4 mL DMF at RT for 24 h.
Next, doxorubicin hydrochloride (104 mg, 0.2 mmol) and TEA (50 ꢁL, 0.4 mmol)
were dissolved in 2 mL DMF, and then added dropwise to the PLGA mixture with
stirring for 24 h at RT in the dark under N2 protection. After the reaction was
completed, the product was precipitated with cold methanol and washed for three
times. The precipitate was then dissolved with 5mL DMF and dialyzed with a dialysis
bag (MWCO 3,500) against DMF for 24 h. Then the product was lyophilized for 48 h
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to obtain a red powder. HꢀNMR spectrum of PLGAꢀDOX (ami) was achieved by
using CDCl3 as solvent for analysis.
2.5 Preparation and Characterization of DOX/IFNγꢀTSN (hyd) and
DOX/IFNγꢀTSN (ami)
DOX loaded TSNs were fabricated by a simple nanoprecipitation method
according to our previous work 25. Briefly, 10 mg PLGAꢀDOX (hyd) and 10 mg
mPEGꢀPLGA were dissolved in 3 mL acetonitrile, followed by addition of 300 mg
F127. The mixture was gently stirred at RT for 4 h and then slowly added to 20 mL
deionized water with stirring overnight at RT to evaporate the solvent. After
centrifugation at 12000 rpm for 15 min, the precipitant was resuspended in pH7.4
phosphateꢀbuffered saline (PBS) for further use.
To obtain the DOX/IFNγꢀTSN (hyd), IFNγ was incubated with DOXꢀTSN (hyd)
at 4 ºC for 2 h with continuous stirring. After centrifugation at 12000 rpm for 20 min,
free IFNγ was removed, and the precipitation was reꢀdispersed in PBS for another use.
DOXꢀTSN (ami) and DOX/IFNγꢀTSN (ami) were prepared in the similar way as
DOXꢀTSN (hyd) and DOX/IFNγꢀTSN (hyd), respectively.
Transmission electron microscope (TEM JEMꢀ1230, Japan) was utilized to
confirm the structure and morphology of TSNs, DOXꢀTSN (hyd) and DOXꢀTSN (ami)
via negative staining method with 0.5% phosphotungstic acid. The particles size
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distribution and zeta potential of different TSNs were determined by dynamic light
scattering (DLS, Zeta Plus, Brookhaven Instruments, USA). To monitor the
thermosensitivity of drug loaded TSNs, the variation of particle size at 4 ºC, 25 ºC and
37 ºC was measured. Each performance was repeated in triplicate independently.
The encapsulation efficiency (EE) of DOX and IFNγ were determined by
microplate reader with excitation/emission wavelength at 485/590 nm and
enzymeꢀlinked immunosorbent assay (ELISA) kits, respectively. Briefly,
DOX/IFNγꢀTSN (hyd) and DOX/IFNγꢀTSN (ami) were dissolved in DMSO to
measure the concentration of DOX with a UV detector of microplate reader. The EE
was calculated according to the following formula:
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EE（%）= ꢀꢁꢂꢃꢄꢅ ꢂꢆ ꢇꢂꢀꢈꢉꢈ ꢈꢊꢃꢋ×100%
ꢀꢁꢂꢃꢄꢅ ꢂꢆ ꢅꢂꢅꢀꢇ ꢈꢊꢃꢋ
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2.6 In vitro drug release kinetics
The in vitro release profile of DOX was performed by a dialysis method in pH5.0
or pH7.4 PBS containing 0.2% Tween 80 as release media. Briefly, 5 mL freshly
collected DOX/IFNγꢀTSNs (hyd) or DOX/IFNγꢀTSNs (ami) were performed in
dialysis bag (MWCO 3500) against 50 mL release media at 37 ºC with shaken for 100
rpm. At preꢀdetermined time point, 50 mL of dialysis solution was removed and
replaced with equal volume fresh release media for continuous release. The released
DOX was analyzed by HPLC with a reverse phase Restek C18 column (150 mm × 4.6
mm, pore size 5 ꢁm) at a flow rate of 1.0 mL/min. The mobile phase was consisted of
0.1% trifluoroacetic acid and acetonitrile (75:25, v/v). The fluorescence detector was
set at 470/585 nm (excitation/emission).
Different from DOX, the release behavior of IFNγ was investigated in vitro by
centrifugation. In brief, freshly prepared IFNγ loaded TSNs (100 ng/mL) were
resuspended in 1 mL pH5.0 or pH7.4 PBS containing 0.2% BSA at 37 ºC with shaken
for 100 rpm. At designed interval, the samples were centrifugated at 12000 rpm for 10
min. 1 mL of supernatant was removed and the precipitation was redissolved with
equal volume fresh PBS. The released IFNγ was then measured by ELISA kit.
2.7 In vivo pharmacokinetics
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In vivo pharmacokinetic study was performed in healthy female SD rats (200 ±
20 g), which were randomly divided into two groups（n=3）by intravenously (i.v.)
injection of free DOX/IFNγ or DOX/IFNγꢀTSN (hyd) with the doses of 2.5 mg
DOX/kg and 2.5 ꢁg IFNγ/kg. At the preꢀdetermined time point, the blood sample was
taken from the eye socket of SD rats, followed by centrifugation at 3000 rpm for 10
min. 100 ꢁL of plasma was collected and used to detect the remaining amount of IFNγ
at each time point by ELISA kit. The other 100 ꢁL plasma was precipitated with equal
volume methanol to remove excess protein, and then mixed with 1 mL chloroform to
extract DOX by vigorous vortex. After centrifugation at 12000 rpm for 10 min, the
organic phase was collected for HPLC analysis. Pharmacokinetic parameters were
calculated using the drug and statistics (DAS) software (version 2.1.1, Mathematical
Pharmacology Professional Committee, China).
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2.8 In vivo antitumor efficacy against melanoma
0.2 mL B16F10 cells were subcutaneously inoculated in the right axillary region
of 5ꢀ6 weeks female C57BL/6 mice (5 × 104 cells per mouse). When tumor volume
reached approximately 50 mm3, mice were randomly divided into five groups (n=7),
which were treated with saline or DOX/IFNγꢀTSN (hyd) with different dosage of
IFNγ. The dosage of DOX in DOX/IFNγꢀTSN (hyd) was selected as 2.5 mg/kg
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according to our previous research . However, to further explore whether different
dosage of IFNγ has various effect on melanoma, IFNγ was selected with the
administration dosage of 10 ꢁg/kg, 5 ꢁg/kg, 2.5 ꢁg/kg and 1 ꢁg/kg. In addition, to
investigate the combinational efficiency of immunochemotherapy and test the
difference of antitumor effect between DOX/IFNγꢀTSN (hyd) and DOX/IFNγꢀTSN
(ami), the experiments were performed as follows: saline, free DOX (2.5 mg/kg), free
IFNγ (2.5 ꢁg/kg), free DOX/IFNγ (2.5 mg/kg, 2.5 ꢁg/kg), DOXꢀTSN (2.5 mg/kg
DOX equivalent), IFNγꢀTSN (2.5 ꢁg/kg), DOX/IFNγꢀTSN (hyd) (2.5 mg/kg DOX
equivalent, 2.5 ꢁg/kg IFNγ) and DOX/IFNγꢀTSN (ami) (2.5 mg/kg DOX equivalent,
2.5 ꢁg/kg IFNγ). The formulations were injected every three days for four times by
tail vein. The tumor growth was monitored by measuring the longest (L) and widest
(W) diameters of tumor and calculated through the formula (L × W2)/2, and the body
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weight was also observed during the treatment procedure every two days. Mice were
sacrificed at 15 days after first administration. Meantime, the tumor sections were
weighted and the main organs were resected for hematoxylin and eosin (H&E)
staining.
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To evaluate the systematic toxicity, the whole blood was collected from mice and
then centrifugated at 3000 rpm for 10 min. After that, the supernatant was sampled for
detecting the levels of AST, ALT, BUN and Cre to evaluate systematic toxicity
according to the manufacturer’s instruction. All samples were repeated three times.
2.9 Immune cells infiltration in tumor microenvironment
After the combinational treatment, the tumors were obtained, weighed and
digested by collagenase (0.32 mg/mL, Sigma) and DNAse I (100 ꢁg/mL, Sigma) in
hanks solution at 37 ºC to incubate for 70ꢀ80 min. Then the tumor lysate was filtered
through a 100 µm cell strainer (BD Biosciences). After centrifugation at 2000 rpm for
5 min, the cell clumps were collected and purified by 3 mL lymphocyte separation
solution. The collected cells were counted and incubated with Fc blocking antibody
(antiꢀCD16/32) at 4 ºC for 10 min to prevent nonspecific binding. The tumor
infiltrating immune cells were stained with follows antibodies, DC (CD11c+), mature
DC (CD11c+CD86+), NK cells (CD3−NK1.1+), CTLs (CD3+CD8+) and T helper cells
(CD3+CD4+), respectively. Besides, the expression of CD69 in tumor tissues was
further detected as the activated marker.
2.10 Immune analysis in peripheral lymphoid organs
The maturation of DCs in tumor draining lymph nodes (DLN) and inguinal
lymph nodes (ILN) were measured at the same time as the expression of immune cells
in tumor microenvironment. When the animal experiment was completed, DLN and
ILN were stripped from axillary and inguinal region in mice, respectively. Then the
collected corresponding lymph nodes were grindered, washed and filtered through a
cell strainer. After centrifugation at 2000 rpm for 5 min, the resulting cell pellet was
reꢀdispersed in 100 ꢁL PBS. After that, the cells were incubated with 1 ꢁL
CD16/CD32 antibody at 4 ºC in the dark for 10 min, then added with 1 ꢁL
FITCꢀCD11c antibody and 1 ꢁL PEꢀCD86 antibody for incubation in the dark for 30
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min at 4 ºC, washed with 1 mL PBS and centrifuged at 2000 rpm for 5 min. The cells
were collected and tested.
2.11 Cytokine secretion and infiltration of CTLs in tumor microenvironment
When the tumor bearing mice were sacrificed after treatment, the tumors were
weighed in each group, homogenized for 30 s using a highꢀspeed homogenizer and
then centrifuged at 8000 rpm for 20 min. The expression levels of IL2, IL10, TNFα
and TGFβ in the supernatant were measured by ELISA kits. In addition, to study the
infiltration of CTLs in tumor environment, another tumor sections in each group were
collected and determined by immunofluorescence staining of PEꢀCy5.5ꢀCD3e,
FITCꢀCD8a and DAPI. Vessels were characterized by CD31 and neutrophils were
stained with Ly6G by immuneꢀhistochemical analysis in tumor resection of different
treatment groups.
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2.12 Statistical analysis
Data of all measurements including in vitro and in vivo presented as mean ±
standard deviation (SD). Oneꢀway analysis of variance (ANOVA) was used to
determine significance. A value of *p < 0.05 indicates statistical difference.
3
Results.
3.1 Synthesis of PLGAꢀDOX (hyd) and PLGAꢀDOX (ami) conjugates
The PLGAꢀDOX conjugates were synthesized in two different ways, including
an acidꢀsensitive hydrazone linkage (PLGAꢀDOX (hyd)) and a relatively stable amide
linkage (PLGAꢀDOX (ami)) as a comparison. The synthesis route and chemical
structures of these two conjugates were performed in Scheme 1. The pHꢀsensitive
PLGAꢀDOX (hyd) conjugate was constructed via hydrazone linkage between a
primary amine group of DOX and a hydroxylꢀterminal end group of PLGA. The
synthesis spectrum of TPAꢀNHS (400 MHz, CDCl3, δ): 10.1 (s, 1H), 8.31 (m, 1H),
8.17 (m, 1H), 2.91 (s, 1H) was shown in Figure S1, yield: 90.7%. Next, the
characterizations of PLGAꢀTPA and PLGAꢀDOX (hyd) were exhibited in Figure S2
showing that PLGAꢀDOX (hyd) was successfully synthesized as evidenced by the
disappeared aldehyde group on benzene ring of PLGAꢀTPA about 10 ppm, and new
appeared peaks of phenolic hydroxyl on the structure of DOX at 13 ppm and 14 ppm.
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The conjugation extent of DOX in PLGAꢀDOX (hyd) was 10.1% (w/w). On the
contrary, the PLGAꢀDOX (ami) conjugate was constructed by an amide linkage
between the amine group of DOX and the carboxyl group of PLGA with conjugation
extent of 9.7% (w/w). The corresponding spectrum was displayed in Figure S3. It was
obvious that the area ratio of a, b and c in copolymer on the 1HꢀNMR chart was 1:2:3,
which represented the peaks of ꢀCH, ꢀCH2 and ꢀCH3 on PLGA, respectively.
Meanwhile, the phenolic hydroxyls of DOX were appeared on the backbone of
polymerꢀDOX conjugate at about 13 ppm and 14 ppm, respectively. The results
indicated that DOX was successfully attached into PLGA polymer to form
PLGAꢀDOX (ami) conjugate.
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Scheme 1. The synthesis procedure of (a) PLGAꢀDOX (hyd) and (b) PLGAꢀDOX
(ami).
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3.2 Characterization of drugs loaded thermosensitive nanoparticle
To test the properties of these two kinds of nanoparticles, here we investigated
the morphology and size of TSNs by TEM and DLS, respectively. As shown in Figure
1a, both blank TSNs and DOX loaded TSNs exhibited obviously spherical coreꢀshell
structure. As shown in Figure 1b, compared to blank TSNs with a size around 100 nm,
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DOXꢀconjugated TSNs resulted in a slight increase in particle size. However, the size
exhibited no obvious distinction on DOX loaded TSNs with or without IFNγ 30. In
addition, the zeta potentials of blank TSNs, DOXꢀTSN (hyd) and DOXꢀTSN (ami)
were about ꢀ19.30 ± 1.24, ꢀ34.62 ± 3.21 and ꢀ14.77 ± 3.2 mV, respectively, implying
that the hydrazone bearing TSNs (hyd) had a strong negative charge. It showed more
potential for encapsulate IFNγ by electrostatic adherence. In Figure 1c, attributing to
the thermosensitive property of TSNs, the hydrodynamic particle size of blank TSNs
was gradually decreased as temperature increased, with diameter of 208.2 ± 4.9 nm,
100.5 ± 6.3 nm and 89.5 ± 4.0 nm at 4 ºC, 25 ºC and 37 ºC, respectively 25. Moreover,
DOXꢀTSN (hyd) and DOXꢀTSN (ami) still maintained the temperature responsive
tendency after drug loading. As shown in Figure S5, both DOXꢀTSN (hyd) and
DOXꢀTSN (ami) showed good stability in pH7.4 PBS for 7 days. Furthermore, as
depicted in Table S1, the EE of DOX and IFNγ into DOX/IFNγꢀTSN (hyd) were
improved to 40.5 ± 0.6% and 96.7 ± 1.2% through a dualꢀsensitive loading
approaches. By contrast, DOX/IFNγꢀTSN (ami) showing a relative lower EE of drugs,
with 36.9 ± 0.5% of DOX and 84.8 ± 3.2% of IFNγ coꢀloaded at the same condition.
In vitro cumulative release profile of DOX from DOX/IFNγꢀTSN (hyd) and
DOX/IFNγꢀTSN (ami) were conducted to compare the release behavior of hydrazone
bondꢀconjugated DOX and amide bondꢀconjugated DOX in pH5.0 and pH7.4 PBS at
37 ºC (Figure 1d). DOXꢀTSN (ami) exhibited a pretty weak release of DOX with 17.4
± 1.9% and 13.7 ± 1.8% within 7 days in pH 5.0 and pH 7.4 respectively. It suggested
that DOXꢀTSN (ami) was quite stable in bloodstream and lysosome environment. In
contrast, the cumulative release profile of DOXꢀTSN (hyd) showed a pHꢀdependent
hydrolysis in pH5.0 with almost 88% of DOX released within 4 days, while a
relatively slow release in physiological environment at pH7.4. The results showed that
DOXꢀTSN (hyd) could release DOX more rapidly than DOXꢀTSN (ami). Moreover,
the release behavior of IFNγ from TSNs was also performed in Figure 1e. There was
no obvious difference of release kinetics between pH5.0 and pH7.4, indicated that
changes in pH value had no significant influence on the cumulative release of IFNγ.
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In general, the DOX/IFNγꢀTSN (hyd) demonstrated to act as an effective and
sustained drug delivery vehicle for immunochemotherapy.
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Figure 1. Characterizations of different TSNs formulations. (a) TEM images of blank
TSN, DOXꢀTSN (hyd) and DOXꢀTSN (ami). The scale bar represented 200 nm. (b)
Hydrodynamic diameter distribution and zeta potential in PBS (pH7.4). (c)
Thermoꢀsensitivity of nanoparticles at 4 ºC, 25 ºC and 37 ºC, respectively. (d) In vitro
cumulative release profiles of DOX from DOXꢀTSN (hyd) and DOXꢀTSN (ami). (e)
The release behavior of IFNγ from TSNs in pH5.0 and 7.4 PBS. (*p < 0.05, n = 3).
3.3 In vivo pharmacokinetics study
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Due to the outstanding release kinetics of DOX in vitro, the following studies
were performed with hydrazone bearing DOX/IFNγꢀTSN (hyd) rather than
DOX/IFNγꢀTSN (ami). The pharmacokinetics behavior of drugs in vivo were
performed by i.v. injection of free DOX/IFNγ and DOX/IFNγꢀTSN (hyd) into healthy
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SD rats. The concentrations of DOX in blood over time were shown in Figure 2a,
suggesting that incorporation of PLGAꢀDOX to DOX/IFNγꢀTSN (hyd) could prolong
the circulation time of DOX compared to free DOX, which was quickly eliminated in
blood. Moreover, as shown in Figure 2b, free IFNγ was unstable in vivo and more
easily to be cleared within 8 h, while DOX/IFNγꢀTSN (hyd) exhibited a significantly
increased AUC, a higher t1/2 and a reduced CL over free drug under the protection of
nanoparticles. The corresponding pharmacokinetic parameters were shown in Table
S2. Taken together, the prolonged circulation of DOX and IFNγ in DOX/IFNγꢀTSN
(hyd) might be attributed to the structure and good biocompatibility of TSNs, which
was in favor of the drug accumulation in tumor for an improved antitumor efficacy.
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Figure 2. In vivo pharmacokinetics study. The concentration of (a) plasma DOX,
PLGAꢀDOX and (b) IFNγ in SD rats after i.v. administration of free DOX/IFNγ and
DOX/IFNγꢀTSN (hyd), respectively (n = 3).
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3.4 In vivo antitumor activity of drugsꢀloaded TSNs
B16F10 cells were injected subcutaneously into the right flank of C57BL/6 mice and
treatments were performed when tumors reached approximately 50 mm3, 31. To
investigate the optimal dosage of IFNγ, we chose a range of IFNγ with dosages at 10
ꢁg/kg, 5 ꢁg/kg, 2.5 ꢁg/kg and 1 ꢁg/kg. It has been discovered that lowꢀdose DOX, as
a broadꢀspectrum antitumor drug, can exert superior therapeutic effects of
immunochemotherapy against tumors, so the dosage of DOX was set as 2.5 mg/kg 21,
22. The in vivo tumor suppression effect of DOX/IFNγꢀTSN (hyd) at various dosages
of IFNγ were exhibited in Figure 3a and 3b. Tumor dissection picture and tumor
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volume revealed that IFNγ at 10 ꢁg/kg and 1 ꢁg/kg showed an modest effect on
inhibiting the progression of tumors when compared with saline. However, both 5
ꢁg/kg and 2.5 ꢁg/kg dosages of IFNγ were more effective than above dosages and
showed enhanced antitumor ability. It may be attributed to that the overall antitumor
effect of IFNγ depends on the relative balance between active and inhibitory effect
against tumor. IFNγ at high dosage can upꢀregulate the expression of PDꢀL1 on the
surface of tumor cells which promotes the progress of tumor, and produce
pathological effects. Thus, by avoidance of immunology resistance mediated by
upꢀregulated PDꢀL1 and the doseꢀinduced toxicity, IFNγ of 2.5 ꢁg/kg was chosen as
the optimal dosage for further study. In addition, the tumor weight of different
dosages of IFNγ and DOX of 2.5 mg/kg coꢀloaded DOX/IFNγꢀTSNs (hyd) were
shown in Figure 3c, which exhibited a corresponding tendency. In comparison to the
saline group, the tumor inhibitory rate of DOX/IFNγꢀTSN (hyd) significantly
increased with inhibitory rate of 80.5 ± 1.54%, 85.3 ± 4.03% and 70.2 ± 0.19% at
IFNγ dosage of 5 ꢁg/kg, 2.5 ꢁg/kg and 1 ꢁg/kg, respectively. As shown in Figure 3d,
the relative body weight ratio of different TSNs groups was slightly increased as time
extended after treatment, implying that TSNs formulations had no obvious toxicity
against tumor bearing mice.
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Figure 3. In vivo antitumor activity of TSNs with different dosage of IFNγ in B16F10
tumor bearing mice. (a) Pictures of tumors dissection. (b) Tumor growth curves. (c)
Tumor weight. (d) Body weight change of tumorꢀbearing mice. Data was represented
as mean ± SD. (*p < 0.05, n=7).
To further explore the combinational antitumor efficacy of drugs loaded TSNs in
B16F10 tumorꢀbearing mice, different formulations were injected with saline, DOX,
IFNγ, DOX/IFNγ, DOXꢀTSN, IFNγꢀTSN or DOX/IFNγꢀTSN (hyd) every 3 day for 4
continuous times. We wondered whether the delivery of lowꢀdose DOX with IFNγ
based on TSNs by hydrazone bond or amide bond could boost different antitumor
efficiency in B16F10 bearing mice. The group of DOX/IFNγꢀTSN (ami) was also
added to compare the antitumor effects between these two different kinds of drugs
loaded nanoparticles. The tumor excision picture and tumor weight were shown in
Figure 4a and 4b. Compared to saline group, free DOX and IFNγ showed a significant
antitumor ability with tumor inhibition rate of 62.6 ± 3.7% and 60.1 ± 3.7%,
respectively. Moreover, DOX/IFNγ showed a higher tumor inhibition efficacy than
free DOX and IFNγ or their corresponding drugꢀloaded nanoparticles due to the
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combinational antitumor effect of immunochemotherapy. Notably, DOX/IFNγꢀTSN
(hyd) was more effective than DOX/IFNγꢀTSN (ami) with tumor inhibitory rate was
of 88.4 ± 1.7%. It was suggested that the sustained release of DOX and IFNγ was
beneficial for a chronic suppression of tumors. In addition, DOX/IFNγꢀTSN (hyd)
provided an opportunity to prolong the circulation time, realize the sustained release
of DOX and IFNγ, leading to a better antitumor effect compared to free DOX/IFNγ
with the assistance of nanoparticles. The same tendency of growth curves of different
TSNs were shown in Figure 4c. There is no distinct difference in body weight in all
treatment groups in Figure 4d. The images of H&Eꢀstained tumor sections were
displayed in Figure 4e. It was obvious that the necrosis area of DOX/IFNγꢀTSN (hyd)
was relatively much more than the control mice, implying a synergistic antitumor
effect against melanoma. From the above results, we found that TSNs
coꢀencapsulating DOX and IFNγ via pH sensitive linker and electrostatic interaction
could achieve a better combinational therapeutic effect on melanoma than the other
groups, especially the amide linkage bearing DOX/IFNγꢀTSN (ami).
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Figure 4. In vivo combinational antitumor activity of TSNs in B16F10 tumor bearing
mice after treatment with various formulations every 3 days for 4 times via tail vein
injection. (a) Pictures of the extracted tumors. (b) Tumor weight. (c) Tumor growth
curves. (d) Body weight change rate of tumorꢀbearing mice. (e) Representative H&E
sections of tumors after treatments. The scale bar is 250 µm. Data are presented as the
mean ± SD (*p < 0.05, n=6).
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3.5 In vivo safety evaluation of TSNs after treatment
To evaluate the systemic safety induced by TSNs, we harvested the whole blood
and vital organs from B16F10 bearing mice after receiving four times treatment. The
H&Eꢀstained tissues of different formulations were shown in Figure 5a, showing no
obvious pathological lesions in heart, liver, spleen, lung and kidney. After the mice
were sacrificed, the serum was separated and analyzed by blood biochemistry kit. As
shown in Figure 5bꢀe, the levels of AST, ALT, BUN and Cre were all in no abnormal
injury signs, suggesting no significant hepatotoxicity and renal toxicity from TSNs
formulations. The results suggested that coꢀdelivery of lowꢀdose DOX and IFNγ
mediated by TSNs had a good biocompatibility in vivo and could serve as a safe
delivery system for future application.
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Figure 5. Representative H&E sections of heart, liver, spleen, lung and kidney after
four times treatments. The scale bar is 50 µm. Serum chemistry levels of (b) AST, (c)
ALT, (d) BUN and (e) Cre (n=3).
3.6 Synergistic therapeutic mechanism of drugs loaded TSNs
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DOX/IFNγꢀTSN (hyd) has shown effective tumor regression on B16F10
melanoma models. To describe the role of immune response mediated by TSNs
formulations, we investigated the immune cell populations in lymph nodes and tumor
tissues by flow cytometry after four times treatment. Notably, DCs are the most
effective APCs and act as a bridge between innate and adaptive immunity 32. ICD
effect caused by low dose of DOX could promote the maturation of DCs. Briefly,
apoptotic tumor antigens could send “eat me” signals to DCs, which promotes
immature DCs to capture and process antigens, and then presents to CTLs in lymph
nodes to activate CTLsꢀmediated antitumor immune responses 33, 34. In addition, IFNγ
plays a central role in coordinating antitumor immune response, such as promoting
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the maturation of DCs . The percentages of mature DCs (CD11c+CD86+) in DLN
and ILN showed in Figure 6a and 6b. Obviously, DOX/IFNγꢀTSN (hyd) exhibited the
strongest ability in maturing DCs compared with other treatment groups. The mature
DCs in DOX/IFNγꢀTSN (hyd) were 2.2ꢀfold and 2.3ꢀfold than DOXꢀTSN in DLN and
ILN, respectively. In addition, the maturation of DCs in the tumor microenvironment
is crucial for immunochemotherapy mediated antitumor immune responses 36. In
Figure 6c, the number of activated DCs in DOX/IFNγꢀTSN (hyd) was enhanced up to
7.9ꢀfold of saline, 3.4ꢀfold of DOX/IFNγ and 2.7ꢀfold of IFNγꢀTSN. Taken together,
by synergistically promoting the maturation of DCs, DOX and IFNγ coꢀloaded TSNs
could promote DCsꢀmediated antitumor immune responses.
IFNγ can promote the differentiation and expansion of tumorꢀspecific Th1 CD4+
T cells and CTLs 37. To further explore T cellꢀspecific antitumor immune response
induced by TSNs, we analyzed the numbers of activated CTLs (CD3+CD8+CD69+)
and Th1 CD4+ T cells (CD3+CD4+CD69+) in tumor tissues as shown Figure 6d and 6e.
The results showed that the amount of activated CTLs in DOX/IFNγꢀTSN (hyd) was
sharply enhanced with 11.1ꢀfold of saline, 2.3ꢀfold of DOX/IFNγ and 1.84ꢀfold of
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IFNγꢀTSN, respectively. The same tendency was shown in the number of activated
CD4+ T cells with 2.2ꢀfold of DOX/IFNγ and 2.03ꢀfold of IFNγꢀTSN. In addition,
IFNγ can promote the activation of NK cells and mediate innate antitumor immune
responses 38. As shown in Figure 6f, the number of NK cells (CD3ꢀNK1.1+) was
exhibited, implying that coꢀdelivery of DOX and IFNγ by TSNs has shown a strong
activation ability of NK cells compared with other treatments. The number of NK
cells was increased with 2.4ꢀfold in the tumor environment. Taken together,
DOX/IFNγꢀTSN (hyd) could mediate innate and specific antitumor immune responses
by regulating the immune system, including promoting DCs maturation, infiltration
and proliferation of T cells and NK cells in tumor microenvironment.
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Figure 6. Infiltration of immune effector cells in tumor. Percentage of mature DCs
(CD11c+CD86+) (a) in DLN. Percentage of CD11c+CD86+ (b) in ILN and the amount
of mature DCs (c) in tumor. Number of (d) activated CD4+ cells (CD3+CD4+CD69+),
(e) activated CTLs (CD3+CD8+CD69+) and the number of (f) NK cells (CD3ꢀNK1.1+)
in tumor. Data are presented as mean ± SD (*p < 0.05, n=3).
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3.7 Cytokines levels in tumor microenvironment
To further understand the antitumor immune mechanism in tumor
microenvironment, we investigated the expression level of cytokines, including TGFβ,
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IL10, TNFα, and IL2. Cytokines play an important role in the activation of immune
system. Immuneꢀmodulators such as IL10 and TGFβ are immunosuppressive
cytokines, which can inhibit the activity of T and NK cells, and thus negatively affect
the immune response against tumors. It has been reported that IFNγ can inhibit the
Th2ꢀtype immune response and TGFβ secretion so that modulate their
immunosuppressive effects. As shown in Figure 7a and 7b, compared with saline,
DOX/IFNγꢀTSN (hyd) significantly inhibited the production of TGFβ and IL10 with
10ꢀfold and 4.5ꢀfold lower than saline, respectively. The immune response mainly
depends on the balance between Th1 and Th2 cell subsets. IFNγ can regulate Th1
activity against tumors, so the expression level of IL2 in DOX/IFNγꢀTSN (hyd) group
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was increased up to 1.8 times compared with saline (Figure 7c) . In addition, IFNγ
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can promote the expression of TNFα by indirectly activating macrophages
.
Therefore, the concentration of TNFꢀα in DOX/IFNγꢀTSN (hyd) was higher than
other groups and with a 2.0ꢀfold increase compared with saline in Figure 7d. Overall,
DOX/IFNγꢀTSN (hyd) showed an outstanding antitumor immune response that could
promote T helper type 1 (Th1) lymphocyteꢀmediated response, which was much more
effective than Th2 lymphocytes.
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Figure 7. The expression of cytokines in tumor microenvironment in vivo. Levels of
(a) TGFβ, (b) IL10, (c) IL2, and (d) TNFα in tumor (n = 3). Data are presented as the
mean ± SD (*p < 0.05).
3.8 Immunofluorescence assay and immunoꢀhistochemical analysis of immune
cells
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The antitumor immune response can be activated when a large number of
immune cells infiltrated into the tumor site. CTLꢀmediated tumor inhibiting effect
induced by DOX and IFNγ was evaluated by immunofluorescence staining of CD3
and CD8 cells. As shown in Figure 8a, the strongest fluorescence intensity was
exhibited in DOX/IFNγꢀTSN (hyd) compared with saline and other treatment groups,
indicating that there were more CTLs infiltrated at the tumor site. It was conducive to
induce CD8+ T cells mediated immune response. The results were consistent with the
activated CTLs in tumor environment by flow cytometry.
The immune cell populations at the tumor site were examined by
immunohistochemistry after the last injection. CD31 is a platelet/endothelial cell
adhesion molecule that is expressed on vascular endothelial cells, and often used as an
index to assess tumor angiogenesis 41. By observing the degree of blood vessel
enrichment in tumor sections of tumor bearing mice, the inhibitory effect of IFNγ on
tumor vessels was investigated. As shown in Figure 8b, the brown site in tumor
represented the blood vessels. The saline group had rich intratumoral blood vessels.
However, the number of vessels in DOX/IFNγꢀTSN (hyd) was significantly decreased
than that of other groups, which consisted of large vascular lumen and microꢀvessels,
indicating that combination therapy is beneficial to reduce neovascularization of
tumors. To test neutrophils infiltration, we stained tumor sections with Ly6G, was also
shown in Figure S4, DOX/IFNγꢀTSN (hyd) exhibited strong positive expression than
other groups, showing the tumor inhibitory effects against melanoma.
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Figure 8. Analysis of CTLs (CD3+CD8+) infiltration in tumor section and
antiꢀangiogenic effects. (a) Confocal microscopy images of CTL infiltration in 24 h
after the last administration, the scale bar is 200 ꢁm. (b) Immunoꢀhistochemical
analysis of CD31 tumor vascularization. The scale bar is 50 µm.
4 Discussion
In this study, we successfully constructed the thermosensitive nanoparticles to
coꢀdeliver chemotherapeutic drug DOX and cytokine IFNγ by chemical coupling and
electrostatic adsorption. In order to verify the superior pHꢀsensitive and tumor
inhibition efficacy of DOX/IFNγꢀTSN (hyd), we chose the amide bondꢀconjugate
DOX/IFNγꢀTSN (ami) as a comparison. DOX was attached to the copolymer
backbone of PLGA via an acid sensitive hydrazone linker or nonꢀsensitive amide
linker. Obviously, TSNs exhibited an obvious coreꢀshell structure with PLGA carrying
DOX as the solid core and an amphipathic block copolymer F127 as the shell to form
pHꢀsensitive DOX/IFNγꢀTSN (hyd) and nonꢀsensitive DOX/IFNγꢀTSN (ami)
nanoparticles, both of which could have good loading efficiency of DOX and IFNγ. In
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contrast to DOX/IFNγꢀTSN (ami), the pHꢀsensitive DOX/IFNγꢀTSN (hyd) showed a
sustained release ability of DOX in vitro and exerted an excellent synergetic antitumor
effect on B16F10 tumor bearing mice. In addition, it could not only induce apoptosis
of tumors by prolonged circulation time of DOX and IFNγ, but also stimulate immune
system to activate innate and adaptive immune responses to kill tumors by synergetic
delivery of chemotherapeutic and immunotherapeutic agents.
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A major advantage of our delivery system is the acid sensitive property of
DOX/IFNγꢀTSN (hyd), which could bring about an intracellular pHꢀtriggered
sustained release of DOX for as long as 7 days in pH 5.0. The sustained release may
be derived from the stable covalent bond between PLGA and DOX, the prolonged
circulation time of mPEGꢀPLGA and the shied of F127. Moreover, due to the
chemical coupling bond and negative charge of TSNs, the loading efficiency of DOX
and IFNγ into TSNs (hyd) was significantly enhanced when compared with
DOX/IFNγꢀTSN (ami). In addition, DOX/IFNγꢀTSN (hyd) has exhibited more
effective regression of melanoma in combinational mode. We hypothesized that it
may be contributed to the relatively quick and continuous release of DOX from TSNs
(hyd) at pH 5.0, resulting in an excellent antitumor response by regulating immune
response to achieve synergistic antitumor effect. Another advantage is the
thermosensitivity of encapsulating IFNγ into TSNs via thermosensitive
swelling/deswelling properties of pluronic F127. Briefly, IFNγ entered the PEO layer
of F127 through electrostatic force and diffusion at 4 ºC. As the temperature rose from
25 ºC to 37 ºC, the shell layer further shrank and wrapped the cytokines tightly to
avoid leakage of IFNγ. Therefore, drug loaded TSNs could accumulate at tumor sites
by passive target in a smaller size under physiological conditions at 37 ºC. It could not
only enhance the loading efficiency, prolong the drug release kinetics and improve the
blood circulation time of IFNγ, but also have the ability to stimulate immune response
against melanoma. Taken together, pH and temperature dualꢀsensitive features of
TSNs facilitated the coꢀdelivery of smallꢀmolecule chemotherapeutic drug and
biomacromolecule cytokine with great difference in physicochemical properties, and
exerted a flourishing antitumor immune response on melanoma.
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Low dosage of DOX can cause ICD effects and mediate antitumor immune
response. Briefly, apoptotic tumor cells could release tumor associated antigens,
which could be captured by immature DCs, then processed and presented upon
maturation to stimulate CTLs mediated specific antitumor immune responses. IFNγ,
as an important immunomodulatory molecule, could also promote DCs maturation
and release corresponding cytokines to activate CTLs, CD4+ T cells, NK cells and
modulate immune response against melanoma. Therefore, from the results of
immunoassay mechanism analysis, compared to other treatment groups, DOX and
IFNγ coꢀloaded TSN (hyd) exhibited a remarkable increase in the mature DCs in DLN,
ILN and tumor as well as the amount of activated CTLs, CD4+T cells and NK cells at
tumor sites, for realizing a synergistic antitumor immune responses against melanoma.
Moreover, IFNγ also displayed an inhibition action of immunosuppressive cytokines
TGFβ and IL10, and weakened their suppressive effects on immune system. In
summary, the combination of DOX and IFNγ exhibited an enhanced
immunochemotherapy activity against tumor, based on the synergetic immune
mechanism.
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5 Conclusion
We successfully prepared a dualꢀsensitive nanoparticles delivery system for cancer
treatment that combined chemotherapeutic drug DOX and cytokine IFNγ. Different
from nonꢀsensitive encapsulation of DOX into TSNs, DOX/IFNγꢀTSN (hyd) via
hydrazone bond showed good loading efficiency and sustained release behavior of
DOX and IFNγ in vitro, extended circulation time in blood and an excellent inhibition
efficiency in subcutaneous B16F10 tumor model. Meanwhile, DOX/IFNγꢀTSN (hyd)
can activate the immune system to generate a profound synergistic therapeutic
efficacy. TSNs could be further developed to simultaneously deliver hydrophobic
drug with genes, fluorescent dyes or negativeꢀcharged cytokines and so on. It may
provide a broad prospect for the development of multiꢀdrug coꢀdelivery vectors.
Supporting Information
Figure S1.1HꢀNMR spectrum of TPAꢀNHS in d6ꢀDMSO.
Figure S2. 1HꢀNMR spectra of PLGAꢀTPA and PLGAꢀDOX (hyd) in CDCl3.
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Figure S3. 1HꢀNMR spectrum of the copolymer PLGAꢀDOX (ami) in CDCl3.
Figure S4. Immunoꢀhistochemical analysis of Ly6G in tumor sites. The scale bar is
50 µm.
Figure S5. The stability of DOX loaded TSNs in PBS for 7 days.
Table S1. The EE (%) of DOX and IFNγ in DOX/IFNγꢀTSN (hyd) and
DOX/IFNγꢀTSN (ami), respectively.
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Table S2. Pharmacokinetic parameters of free DOX/IFNγ and DOX/IFNγꢀTSN (hyd)
in rats after i.v. injection at equivalent dose of all agents.
Corresponding Author
*Prof. Dr. Zhiping Zhang
Fax and phone: +86ꢀ27ꢀ83601832
Eꢀmail: zhipingzhang@mail.hust.edu.cn
*Prof. Dr. Yang Peng
Eꢀmail: peterpengyang@126.com
Notes
The authors have declared that no competing financial interest.
Author Contributions
The manuscript was written through contributions of all authors. All authors have
given approval to the final version of the manuscript.
Acknowledgements
This work was supported by the National Natural Science Foundation of China
(81673374 and 81373360), the Fundamental Research Funds for the Central
Universities (2015ZDTD048 and 2018KFYYXJJ019). The authors thank the
Analytical and Testing Center of Huazhong University of Science and Technology for
TEM.
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