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Cationic Polyphosphazene Vesicles for Cancer Immunotherapy by Efficient in Vivo Cytokine IL-12 Plasmid Delivery Menghua Gao, Xiumei Zhu, Liping Wu, and Liyan Qiu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00433 • Publication Date (Web): 18 May 2016 Downloaded from http://pubs.acs.org on May 24, 2016
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Biomacromolecules
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Cationic Polyphosphazene Vesicles for Cancer Immunotherapy
2
by Efficient in Vivo Cytokine IL-12 Plasmid Delivery
3
Menghua Gao,‡ Xiumei Zhu,‡ Liping Wu, ‡ Liyan Qiu*†
4 5 6
†
Ministry of Education (MOE) Key Laboratory of Synthesis and Functionalization,
7
Department of Polymer Science and Engineering, Zhejiang University, 38 Zheda Road,
8
Hangzhou 310027, China
9 10 11 12
‡ College of Pharmaceutical Sciences, Zhejiang University, 866 Yu-Hang-Tang Road, Hangzhou 310058, China * Corresponding author. Tel.: +86 571 87952306; Fax: +86 571 87952306 E-mail address:
[email protected] 13 14 15 16 17 18 19 20 21 22 23 1
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ABSTRACT: To circumvent the severe toxicity of the systemic delivery of IL-12 protein
2
and the limits of local administration of IL-12 gene, we constructed a polymersome system
3
for systemic delivery of recombinant murine IL-12 plasmid (pmIL-12) based on amphiphilic
4
polyphosphazenes containing weakly cationic N,N-diisopropylethylenediamine (DPA) as
5
hydrophobic groups and monomethoxy poly(ethylene glycol) (mPEG) as hydrophilic tails.
6
By simple dialysis method, pmIL-12 was successfully loaded into polymersomes due to the
7
combination effect of physical encapsulation and electrostatic interaction. This pmIL-12
8
polymersome delivery system was validated with good biocompatibility and stability despite
9
of serum protein and DNase challenging. The results of in vivo anti-tumor experiments
10
showed that intravenous injection of pmIL-12 polymersomes achieved significant
11
suppression of tumor growth in BALB/c mice bearing CT-26 colon carcinoma. The analysis
12
revealed that the mechanism was related to the anti-tumor immune response induced by
13
efficient transfection of pmIL-12 polymersomes, which maybe involved lymphocytes
14
infiltration and angiogenic inhibition at the tumor site.
15 16
Keywords: Interleukin-12; Polyphosphazene; Polymersome; Gene delivery; Cancer
17
immunotherapy
18 19 20 21 22 2
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INTRODUCTION
2
Over the last few decades, several types of cytokines have been involved in effective
3
antitumor immunetherapies.1 Among them, interleukin 12 (IL-12), produced by
4
antigen-presenting
5
immune-modulatory functions.2 It can promote the activity of cytotoxic T lymphocytes and
6
natural killer (NK) cells, and induce the production of interferon-γ (IFN-γ). Its antitumor
7
activity has been investigated in a number of clinical trials for the treatment of advanced solid
8
tumors and hematologic malignancies as either a monotherapy or in combination with other
9
therapies.1 However, traditional intravenous administration of the recombinant IL-12 protein
10
often results in severe adverse effects, including fever, vomiting, headache, and sometimes
11
life-threatening experiences.3 Therefore, IL-12 gene delivery is developed to be an alternative
12
with reduced toxicity compared with the protein.
cells,
is
considered
the
most
potent
cytokine
with
various
13
Gene therapy is a rapidly evolving area in medicine with great therapeutic potential.
14
Besides cationic lipids,4 polycations are the most widely used non-viral gene vectors, such as
15
poly (L-lysine),5 polyethylenimine,6 chitosan and polyamidoamine,7 which can complex with
16
negatively-charged gene through electrostatic adsorption. Since excess cationic polymers are
17
often used to achieve cellular uptake, lysosomal escape, and higher transfection efficiency,
18
their aggregation with serum proteins can hardly be avoided, which leads to quick elimination
19
of gene by the reticuloendothelial system and a low accumulation level in the target tissues.
20
Therefore, a large number of researchers have tried local delivery of IL-12 gene by means of
21
intratumoral, intramuscular or intraperitoneal injection.8-10 The physical method of
22
electroporation was also used to improve the in vivo transfection efficiency of IL-12 gene.11 3
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Although local delivery may effectively suppress tumor growth in animal tumor models, its
2
application can be hardly extended to hematologic malignancies or the solid tumors within
3
internal organs.
4
In this study, we specially designed a novel polymersome system self-assembled by
5
amphiphilic polyphosphazenes for murine IL-12 plasmid (pmIL-12) delivery. Recently,
6
polymersomes have been recognized as a novel class of nanocarriers for water-soluble
7
chemotherapeutics due to their high stability as compared to liposomes.12 Though in theory,
8
polymersomes contain an aqueous lumen for the loading of genetic substances, only a few
9
research groups have reported on the application of polymersomes in gene delivery.13,14 In
10
addition, their research was focused on siRNA delivery, with little involving the in vivo study
11
of gene transfection efficiency. This highlights the considerable difficulty of developing DNA
12
delivery systems based on polymeric vesicles. On one hand, the formation of polymersomes
13
is a complicated process determined by the molecular weight of the polymer, the fraction of
14
each block and the effective interaction energy between monomers.15 On the other hand,
15
DNA are biomacromolecules with remarkably high molecular weight and particular spatial
16
structures, so their loading is restricted by the volume of the aqueous lumen of the
17
polymersomes, resulting in poor loading efficiency via physical encapsulation alone.16
18
Therefore, we chose the weakly cationic N,N-diisopropylethylenediamine (DPA) as
19
hydrophobic side groups with hydrophilic monomethoxy poly(ethylene glycol) chains for the
20
construction of polyphosphazene vesicles. DPA is able to efficiently condense pmIL-12 and
21
further improve the loading capacity of polymersomes through both physical encapsulation
22
and electrostatic interaction. The polymersome-DNA delivery system was also expected to 4
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display a slightly positive charge in the physiological milieu, which could significantly
2
reduce the cytotoxicity. Besides, polyphosphazene with 2-dimethylaminoethylamine
3
(DMAEA) as side groups was proved to be degradable under slightly acidic and neutral
4
conditions.17 Since the chemical structure of DPA is similar to that of DMAEA, the
5
polyphosphazene containing DPA in this study has great potential for biodegradability. After
6
loading pmIL-12 into the polyphosphazene vesicles, a series of in vitro and in vivo
7
experiments were performed to evaluate the stability, hemocompatibility, cellular uptake,
8
transfection efficiency and anti-tumor effect. In addition, immunostaining and enzyme-linked
9
immune sorbent assay (ELISA) were conducted to elucidate the tumor suppression
10
mechanism.
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EXPERIMENTAL SECTION
12
Materials, Cell Lines and Animals. Hexachlorocyclotriphosphazene and monomethoxy
13
poly(ethylene glycol) (mPEG2k) were purchased from Acros Organics (Belgium). Aminated
14
mPEG2k (mPEG2k-NH2) was prepared via esterification and amidation reaction.18
15
N,N-diisopropylethylenediamine (DPA) was purchased from Alfa Aesar. The plasmid of
16
recombinant murine IL-12 (pmIL-12) was a gift from Dr. Binfeng Lu at Pittsburgh University
17
of Medicine (USA). Blank vector plasmid (pcDNA3.1 (+)) was purchased from TaKaRa
18
(TaKaRa Bio., Dalian, China). 4S Green EB (Ethidium Bromide) was bought from Sangon
19
Biotech (Shanghai, China). The ELISA kits of mouse IL-12p70 and IFN-γ were purchased
20
from R&D Systems (Minneapolis, MN, USA). DNase I was purchased from Takara bio
21
company (Dalian, China). Bovine Serum Albumin (BSA) and the fluorescent dye DiI were
22
purchased from Beyotime Biotechnology (Shanghai, China). Plasmid was labelled with Cy3 5
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using Label IT® TrackerTM CyTM3 kit (Mirus, Madison, WI, USA). The collagenase Ⅳ
2
solution and dextran-FITC were bought from sigma (Sigma chemical, St. Louis, MO, USA).
3
Fluorescence-labeled antibodies for flow cytometry including: anti-CD3ε (145-2C11),
4
anti-CD4 (RM4-5), anti-CD8α (53-6.7), anti-NK1.1 (PK136), anti-Hamster IgG1 (A19-3),
5
anti-Rat IgG2a (R35-95), anti-Rat IgG2a (R35-95), anti-Mouse IgG2a (G155-178) were
6
purchased
7
immunohistochemical staining (anti-CD4, anti-CD8 and anti-CD31) were bought from
8
Abcam (Abcam Inc., Cambridge, MA, USA). All other chemicals were commercially
9
available.
from
BD
Biosciences
(San
Diego,
CA,
USA).
Antibodies
for
10
B16 murine malignant melanoma cell line and CT-26 murine colon adenocarcinoma cell
11
line were purchased from the cell bank of Chinese Academy of Sciences (Shanghai, China).
12
Cells were incubated in RPMI 1640 medium supplemented with 10% fetal bovine serum
13
(FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in a humidified 5%
14
CO2-containing atmosphere. BALB/c female mice were obtained from Shanghai SLAC
15
Laboratory Animal Co. Ltd, China and used at 6-8 weeks of age. Animal experiments were
16
performed in accordance with the guidelines for the welfare of animals approved by Zhejiang
17
University.
18
Synthesis and Characterization of Amphiphilic Poly[(PEG)x(DPA)yphosphazene]
19
(PEDP). PEDP polymer was synthesized by sequentially nucleophilic substitution reaction of
20
mPEG2k-NH2 and DPA with chlorine atoms on poly(dichlorophosphazene) (PDCP)
21
backbone as previously described19 except that the ratio of hydrophilic/hydrophobic groups
22
was adjusted so as to form polymersomes. Firstly, PDCP was obtained by a ring-open 6
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polymerization of hexachlorocyclotriphosphazene at 250 °C. Then mPEG2k-NH2 (2.5 g) was
2
dissolved in dry toluene containing dry triethylamine (TEA) (1.22 mmol), and this solution
3
was slowly added into PDCP (0.5 g) in dry toluene under stirring at 30 °C for 24 h.
4
Subsequently, the temperature was set to 50 °C and excess amount of DPA (1.25 g) in dry
5
toluene was added with dry TEA (8.5 mmol) slowly into the reaction mixture and the mixture
6
was stirred for another 24 h. All procedures were carried out under the protection of nitrogen.
7
The final product was collected and purified by ether precipitation twice and dialysis against
8
water for two days.
9
The chemical structure of PEDP was confirmed by 1H nuclear magnetic resonance (1H
10
NMR) and Fourier transform infrared spectroscopy (FT-IR). 1H NMR spectrum was
11
performed using a NMR spectrometer (DMX-500, Brulcer Co., Zurich, Switzerland) with 10
12
mg/mL of PEDP in CDCl3 solution. Samples for FT-IR spectra were prepared with KBr
13
pressed method and detected with FT-IR spectrometer (Nicolet 6700, Themo Fisher scientific
14
LLC, NY, USA). The molecular weight of PEDP was measured by gel permeation
15
chromatography (GPC, D40, TSK CO., Kyoto, Japan) and dimethylformamide (DMF) was
16
used as solvent.
17
Self-Assemble Behavior of PEDP. Considering PEDP is amphiphilic, we tried to prepare
18
its self-assembled nano-system by both solvent-free method and solvent-displacement
19
method20. For solvent-free method, PEDP was directly dispersed into pH 7.4 phosphate
20
buffer saline (PBS) medium. As for solvent-displacement method, PEDP was first dissolved
21
in DMF. Then pH 7.4 PBS buffer with the equal volume was added dropwise into PEDP
22
solution under vigorous stirring to yield a homogenous dispersion. The organic solvent was 7
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then excluded by dialysis against the same PBS buffer. The resulted samples from these two
2
methods were observed by transmission electron microscopy (TEM, JEM-1230, JEOL
3
Company, Japan).
4
Besides, to further identify the nano-structure of PEDP self-assemblies via dialysis, PEDP
5
vesicles were simultaneously loading two kinds of fluorescent dyes as follows. The
6
hydrophobic fluoresent dye (DiI) was dissolved in DMF along with PEDP copolymer while
7
hydrophilic fluoresent agent (Dextran-FITC) was dissolved in pH 7.4 PBS. Then the aqueous
8
phase was added dropwise into the organic phase, and the mixture was dialyzed against PBS
9
buffer to remove DMF. The resultant nanoparticles were observed under two-photon
10
microscope (Olympus IX81-FV1000, Olympus, Tokyo, Japan).
11
Preparation and Characterization pmIL-12-Loaded Polymersomes and Complexes.
12
The pmIL-12 loaded PEDP polymersomes (pmIL-12 PMs) were prepared by dialysis method.
13
Briefly, PEDP were dissolved in DMF at the concentration of 20 mg/mL and the pmIL-12 (1
14
mg/mL) solution was added under vortex at various weight ratios. After a few minutes, the
15
solution was transferred into a dialysis bag (molecular-weight cutoff=14,000 Da) and
16
dialyzed against PBS buffer (pH 7.4) for 5 h. The complexes of pmIL-12 and PEDP
17
(pmIL-12 CPs) were simply prepared by incubating them in PBS buffer (pH 7.4) at room
18
temperature for 20 min. The particle size and zeta potential of pmIL-12 PMs and pmIL-12
19
CPs were evaluated by dynamic laser scanning (DLS) on a Malvern Zetasizer (Nano ZS90,
20
Herrenberg, Germany). Their morphology was also observed by TEM.
21
DNA Retardation. Gel retardation assay was applied to evaluate the DNA condensation
22
efficiency of pmIL-12 PMs and pmIL-12 CPs. The solution was subjected on 1% agarose gel 8
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stained with 4S Green EB and the electrophoresis was performed at 100 mV for 40 min. The
2
gel was illuminated on a Gel Image System (Tanon 1600, Shanghai, China) to show the
3
location of pmIL-12.
4
DNase I Protection. For the DNase I protection assay, pmIL-12 PMs containing 0.5 µg
5
pmIL-12 were prepared at various weight ratios, followed by incubated with 1 U DNase I for
6
0, 10, 20, 40 and 60 min. The DNase I reaction was stopped by the addition of 50 mM EDTA
7
solution and heated in 70 °C for 10 min. The alteration in absorbance at 260 nm was
8
measured at different time intervals using UV-Vis spectrophotometer (PuXi General
9
Instrument Co., Beijing, China). The naked pmIL-12 treated with DNase I served as control.
10
Bovine Serum Albumin (BSA) Adsorbing. To assess the stability of pmIL-12 PMs to
11
serum, pmIL-12 PMs containing 0.5 µg pmIL-12 at various weight ratios were incubated
12
with BSA standard solution at concentrations of 0.2, 0.4, 0.6, 0.8 and 1 mg/mL for 1 h at
13
37 °C. Then the alteration in turbidity at 350 nm was measured with UV-Vis
14
spectrophotometer. The naked pmIL-12 treated with BSA solution served as control.
15
Hemolysis Test. Hemolysis test was performed with pmIL-12 PMs using whole blood
16
from BALB/c mice. Briefly, blood was collected in anticoagulative tubes and washed with
17
normal saline for 3 times. The erythrocyte in precipitate was resuspended with saline at a
18
dilution of 1:10 (v/v). PmIL-12 PMs with three different weight ratios (PEDP/pmIL-12 = 25,
19
125 and 500) were prepared and the final concentrations of PEDP at 1 mg/mL or 2 mg/mL
20
were evaluated. The 100 µL diluted erythrocyte dispersion was added into 900 µL
21
polymersomes suspension and the tubes were incubated at 37°C for 1 h. After the incubation,
22
the tubes were centrifuged at 800 rpm for 5 min and the absorbance of the supernatant was 9
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detected at 545 nm with ultraviolet spectrophotometer (TU-1900, Pgenenal, Beijing, China).
2
Saline solution and distilled water were used negative and positive control, respectively. The
3
hemolysis rate (HR %) was calculated with (Dsample - Dnc) / (Dpc - Dnc) × 100%, where
4
Dsample, Dnc, Dpc represent the absorbance of the samples, the negative control and the
5
positive control.
6
In Vitro Cytotoxicity. To measure the cytotoxicity of PEDP and pmIL-12 PMs, MTT
7
assay was performed at various concentrations of polymers. B16 and CT-26 cells were seeded
8
on 96-well plates at 2 × 103 cells/well and cultured for 24 h. PEDP and pmIL-12 PMs were
9
added and incubated for additional 48 h. Then the viability of cells was evaluated with the
10
MTT assay. Cells without treatment served as control and results were represented as
11
percentage viability of control cells.
12
Cellular Uptake and Distribution. PmIL-12 was labelled with Cy3 according to the
13
Label IT® Tracker kit for 0.5 µL dye per µg pmIL-12 before use. The cellular uptake of
14
Cy3-pmIL-12 PMs at various weight ratios was quantitatively evaluated by fluorescence
15
intensity. B16 and CT-26 cells were seeded on 12-well plates at 1 × 105 cells/well and
16
incubated for 24 h. Cy3-pmIL-12 PMs were added at 0.5 µg Cy3-pmIL-12/well. After
17
incubation for 1 h or 4 h, cells were washed with cold PBS twice before lysis with 300 µL of
18
the RIPA lysis buffer. Fluorescence intensity of Cy3-pmIL-12 in 200 µL cell lysate was
19
determined by a fluorescence microplate reader (Flonstation II, ND industries, USA) at
20
λex=550 nm, λem=570 nm and protein content was measured with the BCA kit.
21
The cellular distribution of Cy3-pmIL-12 PMs was also observed with Confocal Laser
22
Scanning Microscope (CLSM, Leica TCS SP Spectral Confocal Microscope, Germany). 10
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Cells were incubated with polymersomes for 4 h with 1 µg Cy3-pmIL-12/well. Then cells
2
were washed with cold PBS for three times, stained with Hoechst 33258 and LysoTracker
3
Green, fixed with 4% paraformaldehyde and visualized under CLSM.
4
Endocytosis Pathways. To explore the mechanism of cell internalization with the
5
polymersomes, the cellular uptake assay was performed at 4 °C or in the presence of various
6
endocytic inhibitors including chlorpromazine (CPZ, 10 µg/mL), genistein (200 µg/mL),
7
methyl-β-cyclodextrin (mβCD, 6.5 mg/mL), dynasore (25.78 µg/mL) and wortmannin (10
8
µg/mL) for 30 min. Cy3-pmIL-12 PMs were added and the cells went through a further 2-h
9
uptake at 37 °C. The cells were collected and fluorescence intensity was measured as above.
10
Results were presented with the uptake percentage compared to cells without endocytic
11
inhibitor at 37 °C for 2 h.
12
In Vitro Transfection. B16 cells and CT-26 cells were seeded at 3 × 105 cells/well on
13
six-well plates and incubated for 24 h before transfection. PmIL-12 PMs were added with 2
14
µg of pmIL-12 in each well after the cells were replaced with fresh medium containing 10%
15
FBS. Cells were further incubated for 48 h before assessment of the IL-12 expression. The
16
secreted levels of mIL-12 in culture supernatants were measured with mouse IL-12p70 kits.
17
In Vivo Tumor Growth Inhibition. BALB/c mice were subcutaneously inoculated in the
18
right flank with CT-26 cells (1.5 × 106 cells/mouse). About a week later, diameter of the
19
established tumors reached 5-8 mm and mice were divided into 3 groups for treatment (7
20
mice in each group). PEDP polymersomes loading pcDNA3.1(+) vector plasmid (pVec PMs)
21
at PEDP/pcDNA3.1(+) weight ratio of 125 were prepared as pmIL-12 PMs. Then pmIL-12
22
PMs and pVec PMs were separately injected into mice via the tail vein at a dose of 15 µg 11
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pmIL-12 or pcDNA3.1(+) per mouse. Mice treated with normal saline were used as control.
2
Treatment was performed at every other day for a total five times (on days 0, 2, 4, 6 and 8).
3
During the gene therapy, tumor sizes were measured and the volumes were calculated with
4
the formula v = 0.5 × a × b2, where a represents the maximum diameter and b is the minimum
5
diameter.
6
ELISA for IL-12 and IFN-γ in Serum and Tumors. During and after the treatments
7
tumors and blood samples were taken from three mice out of each group to determine the
8
level of IL-12 and IFN-γ. Blood samples were collected from orbit and serum was separated
9
by centrifuging at 10,000 rpm for 10 min at 4 °C. After the tumors were removed and
10
weighted, the tumor samples were cut into pieces and homogenized with certain volume of
11
saline containing 10% protease inhibitor cocktail. The whole process was performed on ice.
12
The homogenized solution was subsequently centrifuged for 10 min at 10,000 rpm at 4 °C.
13
The cytokine levels in serum and tumor samples were then measured with murine IL-12 and
14
IFN-γ ELISA kits.
15
Tumor-Infiltrating Lymphocytes (TILs). For the analysis of lymphocytes infiltrating in
16
the tumor site, about 1 mg tumor tissues were removed from three mice in each group on day
17
12, which were minced into about 1 mm3 pieces and digested with collagenase Ⅳ solution
18
for 1 h at 37 °C. The digested tissues were dispersed with pipette and passed through a cell
19
strainer with the diameter of 74 µm. Then the cells were collected and counted under a
20
microscopy. For flow cytometry analysis, 2 × 105 viable cells from each sample were washed
21
with cold PBS buffer and stained with fluorescence-labeled antibodies against murine CD4+,
22
CD8+ and NK cells for 30 min on ice. After washed with cold PBS twice, the cells were 12
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analyzed by a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Immunohistochemical Analysis. For the determination of lymphocytes (CD4+ and CD8+)
2 3
infiltration
and
vessel
density,
tumors
from
each
group
were
removed
for
4
immunohistochemical analysis on day 12. The resected tumors were fixed in 4% buffered
5
formalin and embedded in paraffin. The embedded tumor sections were treated with 3% H2O2
6
for 10 min, immersed in boiling 0.01 M citrate buffer (pH 6.0) for a few minutes and blocked
7
with normal goat serum for 20 min. Then the sections were stained with anti-mouse CD4,
8
CD8 and CD31 mAbs containing 0.1% BSA at 4 °C overnight. After washed with PBS, the
9
slices were incubated with the secondary antibody for 20 min at 37 °C and stained with
10
diaminobenzidine (DAB) system. Finally the section were counterstained with hematoxylin
11
solutions and observed under a microscope (Olympus, Tokyo, Japan).
12
Statistical Analysis. Data are generally presented as the mean ± standard deviation (SD).
13
The statistical significance was determined using the analysis of variance (one way ANOVA)
14
and p < 0.05 was considered significant.
15
RESULTS AND DISCUSSION
16
Synthesis of PEDP Polymer. PEDP polymer containing mPEG (hydrophiphilic chain) and
17
DPA (hydrophobic group) was synthesized via heat polymerization of backbone, followed by
18
substitution reaction of side groups (Figure 1A). The resultant polymer was characterized by
19
1
20
corresponded to -OCH2CH2- and -OCH3 groups in mPEG; the peaks at 2.39-3.01 and 1.07
21
ppm were assigned to -CH2-, -CH- and -CH3 groups in DPA, respectively. According to the
22
chemical structure and
H NMR and FT-IR. As shown in Figure 1B, the peaks at 3.54 and 3.30 ppm were separately
1
H NMR spectrum of PEDP, it can be obtained that 13
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MmPEG*3/MDPA*12=A3.30/A1.07 (MmPEG and MDPA: the mole weight of mPEG and DPA; A3.30
2
and A1.07: the peak areas of chemical shifts at 3.30 and 1.07 ppm corresponding to the protons
3
of methyl ether units in mPEG and the protons of methyl group in DPA, respectively). Thus
4
the mole ratio of mPEG/DPA was calculated as 0.3:1.7. The detailed FT-IR peak analysis is
5
as follows (Figure 1C): mPEG at 2884 cm-1 (-CH2- stretching vibration), 1467 cm-1 (-CH2-
6
deformation vibration), and 1108 cm-1 (-C-O- stretching vibration); polyphosphazene
7
backbones at 1351 and 1021 cm-1 (-N=P- stretching absorption band), 950 cm-1 (-N-P-
8
stretching vibration); DPA at 1525 and 3322 cm-1 (-NH- stretching vibration), 2969 cm-1
9
(-CH3 stretching vibration), 2884 cm-1 (-CH- stretching vibration). The number-averaged
10
molecular weight of PEDP was 33694 with the polydispersity index of 1.25 as determined by
11
GPC.
12 13 14
Figure 1. Synthesis and characterization of amphiphilic polyphosphazene PEDP. (A) Synthesis route, (B) 1H NMR spectrum in CDCl3, and (C) FT-IR spectrum.
15 16 14
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Biomacromolecules
Self-assemble
Behavior
of
Amphiphilic
PEDP.
polymers
with
a
dual
2
hydrophobic-hydrophilic character generally tend to self-assemble in an aqueous solution.
3
However, the formation of polymersomes is a quite complicated process and the structural
4
features of polymersomes including intermembrane dynamics are greatly influenced by the
5
characteristics of the polymers and preparation methods.21 In our previous research, we
6
synthesized a series amphiphilic graft polyphosphazenes containing hydrophobic
7
ethyl-p-aminobenzoate (EAB) and hydrophilic mPEG at various weight ratios.22 It was
8
confirmed that increasing the content of hydrophobic side would facilitate the transformation
9
from micelles to polymersomes. Based on these results, we designed PEDP with relatively
10
high DPA content and investigated its self-assemble behavior in water. As shown in Figure
11
2A, the samples aggregated into small clusters with anomalous morphologies by solvent-free
12
method. However, as a distinct contrast, PEDP nanoparticles prepared by dialysis method
13
displayed spherical and symmetrical structure containing dark hydrophobic membrane
14
around central aqueous reservoir, which is typical characteristic of polymersomes (Figure
15
2B). This phenomenon suggested that vesicle formation by polymer unimers may be a
16
dynamic equilibrium process. In the case of directly dispersing PEDP in the aqueous media,
17
the polymer aggregation occurred before the polymer molecules accomplished ordered
18
arrangement via self-assembly. During the dialysis process, however, the concentration of
19
organic solvent DMF was gradually decreased, thus vesicles formed under the conditions
20
where their internal contents were continuously hyperosmotic.23 The mean diameter of PEDP
21
polymersomes was measured as 206.28 ± 3.99 nm with polydipersity index of 0.307 ± 0.022
22
by DLS analysis. In order to verify the vesicular formation, two-photon microscope images 15
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1
of PEDP polymersomes simultaneously loading dextran-FITC (hydrophilic) and DiI
2
(hydrophobic) were taken and showed in Figure 2C. The merge image showed yellow
3
fluorescence (overlay of green and red) was surrounded by red one, confirming water-soluble
4
dextran-FITC was in the lumen and lipophilic DiI in the membrane. This feature was
5
compliant with the vesicular structure as previously reported.19
6
According to the mole ratio of mPEG/DPA, the mPEG weight fraction fPEG(w) was
7
calculated as 0.67. The hydrophilic fraction fPEG(w) was reported to be a predominant issue to
8
the morphology transition of self-assemblies. Until now, polymeric vesicles are mostly
9
prepared by diblock or multiblock copolymers.24-26 For amphiphilic diblock copolymers,
10
bilayer vesicles are generally formed while fPEG(w) falls in the range of 0.20-0.42.27 However,
11
the molecular structure of amphiphilic graft polyphosphazenes is much more complex
12
compared to diblock copolymer. Indeed, self-assembled vesicular formation of other kinds of
13
polyphosphazenes with fPEG(w) higher than 0.5 was observed in our previous studies.18,28 For
14
example, amphiphilic graft polyphosphazenes containing hydrophobic EAB and hydrophilic
15
mPEG self-assembled into polymersomes while fPEG(w) was in the range of 0.67 to 0.79.
16
Besides, according to the study of Kim’s group, the preparation method also plays an
17
important role in the formation of polymersomes.29 Also different from the self-assemble
18
behavior in water, polymersomes might be prepared in water/solvent mixtures with fPEG(w) of
19
3~60%.30,31 Therefore, it can be seen that the dialysis method is an important factor for the
20
formation of PEDP polymersomes while the mole ratio of mPEG/DPA was 0.3:1.7.
16
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Biomacromolecules
1 2 3
Figure 2. Schematic formation and TEM images (30,000×magnification) of PEDP nanoparticles prepared by (A) solvent-free method and (B) solvent-displacement method. (C)
5
Confocal images (100×magnification) of PEDP polymersomes simultaneously loading hydrophilic dye dextran-FITC (green), hydrophobic dye DiI (red). White arrows in the merge
6
pictures pointed the typical polymersome structure.
4
7 8
Preparation and Characterization of pmIL-12 PMs. PmIL-12 was encapsulated into
9
PEDP polymersomes using dialysis method. The morphology of pmIL-12 PMs was observed
10
by TEM (Figure 3A). Compared to the blank polymersomes (Figure 2B), pmIL-12 PMs
11
became solid particles with increased density inside, suggesting that pmIL-12 was loaded in
12
the aqueous core of PEDP polymersomes. The size of pmIL-12 PMs was reduced from 233.4 17
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1
± 25.9 nm to 179.1 ± 18.8 nm and the zeta potential was decreased from 7.06 ± 0.14 mV to
2
3.49 ± 0.37 mV with decreasing the PEDP/pmIL-12 weight ratio from 500 to 10 (Figure 3C).
3
However, when PEDP and pmIL-12 were simply mixed in PBS buffer (pH 7.4), the resultant
4
pmIL-12 CPs had irregular shapes (Figure 3B) and larger sizes (247.1 nm at PEDP/pmIL-12
5
weight ratio of 10:1 and 295.2 nm at PEDP/pmIL-12 weight ratio of 20:1). In addition,
6
particle size distribution graphs indicated that pmIL-12 PMs (Figure 3A) have narrower
7
distribution compared to pmIL-12 CPs (Figure 3B).
8 9 10 11 12
Figure 3. TEM images (30,000×magnification) and size measurement of (A) pmIL-12 PMs and (B) pmIL-12 CPs at PEDP/pmIL-12 weight ratio of 125. (C) Size and zeta potential of pmIL-12 PMs at various PEDP/pmIL-12 weight ratios. (D) Agarose gel electrophoresis of naked pmIL-12, pmIL-12 PMs and pmIL-12 CPs at different weight ratios.
13 14
DNA Encapsulation and Complexes. As a family of carriers for drug delivery,
15
polyphosphazenes
display
unique
characteristics
with
regard
16
biocompatibility and high versatility that they can be modified to possess various properties 18
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biodegradability,
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Biomacromolecules
1
for
2
polyphosphazenes to condense plasmid DNA by electrostatic interaction.32-34 Considering the
3
protonated side group DPA in PEDP polymer, pmIL-12 may complex with PEDP in aqueous
4
solution by electrostatic interaction. Thus, a gel retardation assay was used to confirm the
5
pmIL-12 loading capacity of PEDP polymersomes. As shown in Figure 3D, pmIL-12
6
migration was almost completely retarded at the PEDP/pmIL-12 weight ratio of 1:1 in the
7
pmIL-12 PMs. However, a large amount of free pmIL-12 was observed in pmIL-12 CPs at
8
this ratio and DNA was not fully adsorbed until the weight ratio of PEDP/pmIL-12 increased
9
to 10:1. This difference was due to the different interaction modes between pmIL-12 and
10
PEDP in these two groups. PmIL-12 was adsorbed by PEDP polymers via electrostatic
11
interaction when mixed with PEDP in PBS buffer (pH 7.4). However, using the dialysis
12
method, pmIL-12 was not only condensed with PEDP like pmIL-12 CPs, but also physically
13
encapsulated into the aqueous lumen of PEDP polymersomes. Therefore, by taking advantage
14
of both the physical space allocated by the aqueous lumen of PEDP polymersomes as well as
15
electrostatic adsorption, more pmIL-12 was loaded by PEDP polymersomes than PEDP
16
unimer solution. In addition, the loading of pmIL-12 was simultaneously completed with the
17
formation of PEDP polymersomes during dialysis against PBS.35 Based on the above results
18
and discussion, pmIL-12 PMs prepared by dialysis method were chosen to undergo the
19
following investigation.
20
different
needs.
The
previous
investigations
generally
synthesized
cationic
DNase I Protection. The physiological barriers to DNA delivery include enzymatic
21
degradation,
instability
in
biological
fluids,
inefficient
22
lysosome-trapping.36 The degradation of DNA by endonucleases, such as DNase I, has been 19
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cellular
uptake
and
Biomacromolecules
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1
one of the major obstacles to successful gene therapy.37 Thus, a perfect vector should be
2
capable of protecting the gene from the abundant amounts of DNase I in vivo. The
3
absorbance at 260 nm (OD260 nm) was used as a measure of DNase I protection since intact
4
DNA possess hypochromicity and the value of OD260 nm would rapidly increase upon the
5
stimulation of enzyme and heat.38 As shown in Figure 4A, naked pmIL-12 was rapidly
6
degraded by DNase I within 60 min, according to the significant increase in OD260
7
However, OD260 nm of pmIL-12 PMs at various weight ratios of PEDP to pmIL-12 barely
8
changed within 60 min after treatment with DNase I, indicating no significant pmIL-12
9
degradation. The results demonstrated that PEDP polymer could effectively condense
10
nm.
pmIL-12 into polymersomes and protect them from enzymatic digestion.
11
BSA Adsorbing. Generally, polycation/DNA complexes may bind negative charged serum
12
albumin and form aggregates at a very low protein concentration (less than 0.5 mg/mL),
13
which results in rapid plasma clearance and low transfection efficiency.39-42 Therefore, the
14
stability of pmIL-12 PMs in the presence of BSA (0~1 mg/mL) was evaluated by the
15
turbidity change in OD350 nm. As shown in Figure 4B, pmIL-12 PMs at the PEDP/pmIL-12
16
weight ratio of 10 exhibited a significant increase in OD350 nm, especially at 1 mg/mL of BSA.
17
As PEDP/pmIL-12 weight ratios increased, pmIL-12 PMs tended to be more stable. The
18
result suggested that pmIL-12 PMs displayed low protein adsorption and aggregation
19
tendency. On one hand, pmIL-12 PMs carried weak positive-charged surface at about + 5 mV.
20
On the other hand, as the polymer ratio increased, the ratio of PEG residues around the
21
polymersomes was also increased. It was reported that the modification of hydrophilic stealth
22
PEG residues on the surface was able to increase the serum-stability of nanocarriers.43 20
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Biomacromolecules
1 2 3 4 5 6 7 8 9 10
Figure 4. (A) DNase I protection was determined by alteration in OD260 nm after pmIL-12 PMs at various weight ratios incubating with DNase I for 10, 20, 40 and 60 min. **p < 0.01 vs. Naked pmIL-12. (B) BSA challenging was evaluated by pmIL-12 PMs at various weight ratios incubating with different concentrations of BSA. OD350 nm was measured for the variation in turbidity. *p < 0.05 and **p < 0.01 vs. PEDP/pmIL-12 = 10. (C) Hemolysis test of pmIL-12 PMs. a and h were positive control (PC) and negative control (NC). The polymer concentration in b, d, f was 1 mg/mL and in c, e, g was 2 mg/mL, while the corresponding PEDP/pmIL-12 ratio in b and c was 25, in d and e was 125, and in f and g was 500. The statistical conclusions of HR % in different groups were provided below.
11 12
Determination of Hemolysis Activity. Nanoparticles with certain surface properties
13
(especially surface charge) may damage erythrocyte membranes and cause hemocytolysis.44
14
Thus the in vitro hemolysis test is an internationally recognized standard for the examination
15
of the hemocompatibility of medical devices and materials.45 Normally, polymers are
16
considered safe for medical use when the HR% is less than 5%.46 As shown in Figure 4C,
17
good hematocompatibility was observed at the PEDP/pmIL-12 ratio of 25 and 125, regardless 21
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1
of whether the polymer concentration was 1 mg/mL or 2 mg/mL, though the increasing
2
polymer concentration resulted in a slightly higher percentage of HR%. When the
3
PEDP/pmIL-12 ratio was increased to 500, HR% increased significantly and was much
4
higher than 5%, indicating that erythrocytes membranes were disrupted. This phenomenon
5
may be caused by the fact that more positive charge of PEDP were neutralized by the
6
negative charged pmIL-12 at a lower ratio of PEDP/pmIL-12 compared with high ratios.
7
Therefore, an appropriate PEDP/pmIL-12 ratio should be chosen for in vivo application via
8
intravenous injection.
9
Evaluation of Cytotoxicity in Vitro. As depicted in Figure 5A and 5B, the cytotoxicity of
10
PEDP increased as the polymer concentration increased probably due to its cationic character.
11
However, the toxicity of pmIL-12 PMs was reduced especially in CT-26 cells compared with
12
the polymer because negatively charged plasmids were speculated to partly neutralize the
13
cationic polymer. Furthermore, the well-organized hydrophilic PEG shield around the
14
polymersomes may also help alleviate the cytotoxicity. The percentage of cell survival was
15
both over 70% in B16 and CT-26 cells at the PEDP/pmIL-12 weight ratios lower than 125.
16 17 18
Figure 5. In vitro cytotoxicity of PEDP polymer and pmIL-12 PMs in (D) B16 and (E) CT-26 cells was determined by MTT assay at various weight ratios of PEDP/pmIL-12, the related
19
polymer concentration was 10, 25, 50, 125, 250 and 500 µg/mL. *p < 0.05 and **p < 0.01. 22
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Biomacromolecules
1
Cellular Uptake. Cellular uptake plays an important role in gene transfection as it is the
2
first step in anti-tumor action. Therefore, the 1 h and 4 h-uptake level of Cy3-pmIL-12 PMs
3
in B16 and CT-26 cells was evaluated. Figure 6A and 6B showed the increase of
4
fluorescence intensity in both cells as time within 4-h incubation. The internalization of
5
pmIL-12 was significantly promoted by polymer vehicles compared with the naked
6
Cy3-pmIL-12. The Cy3-pmIL-12 uptake percentage in CT-26 cells was positively related to
7
the ratio of the polymer. In contrast, the fluorescence intensity in B16 cells remained strong at
8
different ratios of PEDP/pmIL-12 after 4-h incubation. This may be caused by the different
9
membrane properties in different cells. The internalization of Cy3-pmIL-12 was further
10
confirmed with confocal microscopy. CLSM images in Figure S1 revealed that both B16 and
11
CT-26 cells treated with naked Cy3-pmIL-12 only showed red fluorescence around the cell
12
membrane. However, Cy3-pmIL-12 PMs were taken up by both cells, and fluorescence was
13
widely distributed in both the cytoplasm and cell nucleus. This indicated that Cy3-pmIL-12
14
PMs were not trapped in the lysosome, which is another key step in determining the gene
15
transfection efficiency.
16 17 18 19 20
Figure 6. Cellular uptake of Cy3-labelled pmIL-12 PMs at various PEDP/pmIL-12 weight ratios was detected in (A) B16 and (B) CT-26 cells after incubating with naked Cy3-pmIL-12 and Cy3-pmIL-12 PMs for 1 h and 4 h. Results were expressed as the fluorescence intensity of per µg protein. *p < 0.05. 23
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1
Endocytosis Pathway. The internalization mechanism and pathway of pmIL-12 PMs was
2
then explored using various endocytosis inhibitors. CPZ inhibits clathrin-mediated
3
endocytosis, while genistein and mβCD suppress the caveolae pathway in different ways;
4
dynasore prevents both clathrin- and caveolae- mediated endocytosis and wortmannin
5
suppresses micropinocytosis. As shown in Figure S2, cellular uptake was decreased to 40%
6
at 4 °C in both B16 and CT-26 cells, indicating that the internalization of pmIL-12 PMs was
7
mostly completed by means of energy-dependent endocytosis. CPZ and genistein
8
significantly lowered the uptake percentage to below 70% in both cells while mβCD and
9
dynasore decreased the uptake in B16 cells to below 80%. These results suggested that
10
clathrin- and caveolae-mediated energy-dependent endocytosis is the main internalization
11
pathway of pmIL-12 PMs.47
12
Transfection Efficiency in Vitro. The in vitro IL-12 expression in both cells was measured
13
by ELISA kits to evaluate the transfection efficiency of pmIL-12 PMs (Figure 7). The
14
expression of IL-12 was much higher in the pmIL-12 PMs compared to the naked pmIL-12
15
group, which suggested that PEDP polymersome facilitated the expression of pmIL-12 as a
16
potential transfection reagent. The transfection efficiency was increased with the
17
PEDP/pmIL-12 weight ratio increasing from 10 to 250 in CT-26 cells. However, once the
18
PEDP/pmIL-12 weight ratio increased to 500, IL-12 expression levels slightly decreased
19
probably due to the noticeable cytotoxicity of polymeric carriers at this ratio. These
20
phenomena were also observed in B16 cells, though IL-12 expression in B16 cells was much
21
lower than in CT-26 cells (about 2.5-fold lower at PEDP/pmIL-12 ratio of 125). To be noticed,
22
such effective pmIL-12 transfection was achieved in the presence of serum mainly due to the 24
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1
Biomacromolecules
stability of PEDP polymersomes against BSA adsorption (Figure 4B).
2 3 4
Figure 7. In vitro transfection efficiency of pmIL-12 PMs at various PEDP/pmIL-12 weight ratios in B16 and CT-26 cells determined by ELISA assay. *p < 0.05 and **p < 0.01.
5 6
Tumor Growth Inhibition. Tumor growth and the body weight of mice with different
7
treatments are graphically presented in Figure 8A and 8B. During the first 10 days, tumor
8
growth was remarkably inhibited after mice were successively received five times of
9
pmIL-12 PMs injection when compared with the mice receiving pVec PMs or saline injection.
10
Though the tumor sizes in three groups were all increased after the administration (Day 10),
11
the tumor suppression in pmIL-12 PMs group was significantly stronger than pVec PMs or
12
saline groups. The positive result in vivo demonstrated that pmIL-12 PMs was stable enough
13
in serum to accumulate and function in the tumor tissue, which was consistent with the in
14
vitro BSA challenging and hemolysis result. Moreover, our previous research further
15
confirmed this point that the high concentration of DPA-grafted polyphosphazenes labeled by
16
FITC can be detected at 12 h after intravenous injection.48 The body weight of mice in
17
pmIL-12 and pVec PM groups gradually increased as time, similar to the saline group,
25
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1
demonstrating that all mice were in generally good condition without serious adverse effect
2
throughout the experiment. Our prior result also proved that the polyphosphazene containing
3
DPA was mild to liver cells unless free -NH2 was introduced into the side groups.48
4
6
Figure 8. Anti-tumor effect in BALB/c mice bearing CT-26 murine colon adenocarcinoma after intravenous administration of saline, pVec PMs and pmIL-12 PMs. Arrows showed the
7
five times of injection. n = 7 mice for each group. (A) Tumor volumes were measured for 16
8
days. **p < 0.01 compared to the saline and pVec PMs groups. (B) The body weight changes
9
of all animals throughout the experiment days.
5
10 11
IL-12 Levels in Serum and Tumor. To determine the in vivo transfection efficiency of
12
pmIL-12 PMs, the expression of IL-12 in serum and tumor was analyzed by ELISA. As
13
shown in Figure 9A, the level of IL-12 in tumor remarkably increased after the injection of
14
pmIL-12 PMs, but not after the injection of pVec PMs. During the five injections of pmIL-12
15
PMs, the expression of IL-12 gradually increased (on day 5 and day 9); after that, the
16
expression remained stable at a high level (on day 12 and 16), probably due to the longer
17
half-time of IL-12 compared with other cytokines49 and also because, the transfected cells
18
may express the protein continuously. The level of IL-12 in serum also increased and was
19
maintained at about 40 pg/mL (the maximum tolerated dose is 500 ng/kg3) after injections of
20
pmIL-12 PMs (Figure 9B), which was much lower than in tumor tissue. The higher level of 26
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Biomacromolecules
1
IL-12 in tumor than in serum suggested that the immune response was mainly induced in
2
tumor microenvironment, and the abundant cytokine production in the lesions cell or tissue
3
was on the critical need for immunotherapy.9
4 5 6
Figure 9. Analysis of IL-12 and IFN-γ expression level in serum and tumor tissue on day 0, 5, 9, 12 and 16 were provided: (A) IL-12 levels in tumor; (B) IL-12 levels in serum; (C) IFN-γ
7
levels in tumor; (D) IFN-γ levels in serum. *p < 0.05 and **p < 0.01.
8 9
IFN-γ Levels in Serum and Tumor. In the tumor immunological process, the IL-12
10
protein generally induces the production of IFN-γ, and in turn, IFN-γ has the powerful ability
11
to stimulate phagocytes and dendritic cells to produce IL-12 and augment the immune
12
response. However, the half-life of IFN-γ is very short.50 The level of IFN-γ was analyzed by
13
ELISA in this study to indirectly examine the biologically activity of IL-12. As shown in
14
Figure 9C, the IFN-γ expression in tumor dramatically increased during injection days (on 27
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1
day 5 and day 9) and peaked at about 340 pg/mL, indicating that a strong immune response
2
was aroused on the early days. This was maintained at around 100 pg/g tumor after the
3
injections (on day 12 and 16), implying sustained expression of IL-12. On the other hand, it
4
revealed that the induced immune response was much srtong at the initial days while it kept
5
stable at the follow-up days. As for the IFN-γ level in serum (Figure 9D), it was as the same
6
low as it in pVec PMs group. This result was consistent with the low expression level of
7
IL-12 in serum.
8
Lymphocytes Infiltration in Tumor. The mechanism of action for pmIL-12 PMs was
9
mostly based on the action of immunologic effector cells. Besides administration route and
10
dose, the specific tumor type affects the subset of immunologic effector cells stimulated by
11
IL-12 gene therapy. For mouse hepatocellular carcinoma, IL-12 gene therapy was entirely
12
dependent on NK cells and partially on T cells.8 In murine B16F10 melanoma and metastatic
13
mammary cancer, CD4+ and CD8+ T cells both mediated tumor suppression action.9,50 As for
14
murine squamous cell carcinoma, tumor eradication was associated with CD8+ T cell
15
infiltration.51 Therefore, it is necessary to investigate individually about the mechanism of
16
pmIL-12 PMs in gene immunotherapy.
17
Lymphocytic infiltration in tumors was quantified by flow cytometry analysis of
18
phenotypic characterization. As shown in Figure 10, four types of potential effector
19
populations were stained with fluorescence-labeled antibodies: CD3+CD4+ (CD4+ T cell),
20
CD3+CD8+ (CD8+ T cells), CD3-NK1.1+ (NK cells) and CD3+NK1.1+ (natural killer T cells,
21
NKT cells). The percentage of the last three types of cells was significantly increased in the
22
pmIL-12 PMs group, compared with the pVec PMs and the saline group. The percentage of 28
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1
CD3+CD8+ cells in tumors increased from 11.90 ± 1.50% in the pVec PMs group to 22.33 ±
2
1.87% in the pmIL-12 PMs group, while CD3-NK1.1+ cells increased from 0.30 ± 0.10% to
3
0.81 ± 0.24%, and CD3+NK1.1+ cells increased from 16.99 ± 1.99% to 26.47 ± 2.07%. These
4
data indicated that the treatment of pmIL-12 PMs recruited immunologic effector cells,
5
including CD8+ T cells, NK cells and NKT cells, to the disease site. Specially, NKT cell
6
subsets were particularly clarified in recent years and considered to mediate the cytotoxicity
7
of IL-12 by an NK-like effector mechanism after activation by IL-12.52,53 In addition, the
8
infiltration of CD8+ T cells was also confirmed by immunostaining assay (Figure 11E-H,
9
arrows in panels G and H indicate the positive cells stained by brown), while CD4+ T cell
10
migration was not observed at the tumor tissue (Figure 11A-D). Therefore, pmIL-12 PMs
11
were presumed to induce a CD8+ T cell, NK cell and NKT cell-dependent anti-tumor effect in
12
the murine CT-26 colon adenocarcinoma model, but the individual contribution of these
13
infiltrated lymphocytes needs further exploration.54
14
Antiangiogenic Effect in Tumor. The anti-angiogenesis activity of IL-12 also plays a
15
critical role in the anti-tumor therapy.55 We stained the tumor vessels with an endothelial cell
16
maker, CD31. As shown in Figure 11I-L, the representative areas with the highest vascularity
17
from each group were observed under microscope (Olympus CKX41, Tokyo, Japan) at
18
magnification of × 400. It was obvious that the blood vessels in image I (saline group) and J
19
(pVec PMs group) had large lumens. After treatment with pmIL-12 PMs, however, the
20
formation of new tumoral vessels was remarkably inhibited and only immature neovessels
21
were observed (indicated with the arrows in Figure 11K and L). These results proved that
22
the anti-angiogenic effect also played a vital role in the immunologic function of pmIL-12 29
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PMs therapy, the mechanism of which was reported to be related to the decreasing expression
2
of the growth factor (VEGF) on vascular endothelial cells.10,50
3
5
Figure 10. Analysis of lymphocytes infiltration in tumor tissue by flow cytometry. BALB/c mice were treated with saline, pVec PMs and pmIL-12 PMs. Changes in leukocyte subset of
6
(A) CD3+CD4+, (B) CD3+CD8+, (C) CD3-NK1.1+ and (D) CD3+NK1.1+ were measured. **p
7
< 0.01 vs. saline group and pVec PMs group.
4
8 9 10
Figure 11. Immunostaining of CD4+ (A, B, C, D), CD8+ (E, F, G, H) and CD31+ (I, J, K, L) cells in CT-26 tumors on day 12 after intravenous administration of saline, pVec PMs and
12
pmIL-12 PMs. For pmIL-12 PMs groups, two representative pictures were provided from two samples. All images were obtained under microscopy at 400×magnification. Arrows showed
13
the representative cells and microvessels.
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1
CONCLUSION
2
In our study, the hydrophobic group DPA and hydrophilic group mPEG were chosen to
3
synthesize the amphiphilic grafted polyphosphazene. Using dialysis method, polymersome
4
formation and pmIL-12 loading were simultaneouly completed in a dynamic process by
5
gradual removal of solvent. Due to the combination effect of physical encapsulation and
6
electrostatic interaction, pmIL-12 was efficiently loaded into PEDP polymersomes and
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pmIL-12 PMs were subsequently demonstrated to be serum-stable and hemocompatible. The
8
pmIL-12 PMs-mediated tumor suppression mechanism in the murine CT-26 colon
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adenocarcinoma model is closely related to the high expression level of cytokines in the
10
tumor microenvironment. Besides, anti-angiogenic effect and tumor lymphocytic infiltration
11
were probably involved, especially for CD8+ T cells, NK cells and NKT cells infiltration. On
12
account of the enhanced permeation and retention (EPR) effect, in vivo protection and
13
delivery ability, such polymersomes based on amphiphilic graft polyphosphazenes display
14
great potential as non-viral carriers for systemic gene delivery.
15 16
ASSOCIATED CONTENT
17
Supporting information
18
Results of confocal images of cellular uptake and the endocytosis pathways (Figure S1 and
19
Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org.
20 21
AUTHOR INFORMATION
22
Corresponding author 31
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*Tel.: +86 571 87952306. Fax: +86 571 87952306. E-mail address:
[email protected].
2
Notes
3
The authors declare no competing financial interest.
4 5
ACKNOWLEDGMENTS
6
This work was financially supported by National Natural Science Funds for Excellent Young
7
Scholar (81222047). The authors would like to thank Dr. Binfeng Lu for his presenting us
8
pmIL-12.
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