Tumor-targeted delivery of silibinin and IPI-549 synergistically inhibit breast cancer by remodeling the microenvironment

Article abstract of DOI:10.1016/j.ijpharm.2020.119239

We induced changes in the tumor microenvironment (TME) through the synergistic actions of two drugs used in breast cancer therapy. The anti-fibrotic drug silibinin (SLB) targets tumor-associated fibroblasts and exerts immune-mediated anti-cancer effects.

Full text of DOI:10.1016/j.ijpharm.2020.119239

Journal Pre-proofs
Tumor-targeted delivery of Silibinin and IPI-549 synergistically inhibit breast
cancer by remodeling the microenvironment
Min Jiang, Kaiyong He, Tong Qiu, Jiahui Sun, Qi Liu, Xueqiong Zhang, Hua
Zheng
PII:
S0378-5173(20)30223-4
DOI:
https://doi.org/10.1016/j.ijpharm.2020.119239
IJP 119239
Reference:
To appear in:
International Journal of Pharmaceutics
Received Date:
Revised Date:
Accepted Date:
18 December 2019
6 March 2020
15 March 2020
Please cite this article as: M. Jiang, K. He, T. Qiu, J. Sun, Q. Liu, X. Zhang, H. Zheng, Tumor-targeted delivery
of Silibinin and IPI-549 synergistically inhibit breast cancer by remodeling the microenvironment, International
Journal of Pharmaceutics (2020), doi: https://doi.org/10.1016/j.ijpharm.2020.119239
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Tumor-targeted delivery of Silibinin and IPI-549 synergistically inhibit breast
cancer by remodeling the microenvironment
Min Jianga, Kaiyong Heb, Tong Qiua, Jiahui Suna, Qi Liuc, Xueqiong Zhanga,*,
Hua Zhenga,*
aSchool of Chemistry, Chemical Engineering and Life Sciences, Wuhan University of
Technology, Wuhan 430070, China
bHubei Institute for drug control, Wuhan 430070, China
cDepartment of Dermatology, Johns Hopkins University School of Medicine,
Baltimore, MD, 21231, USA
*Corresponding author: Xueqiong Zhang, Tel.: +86 138 8612 3736
E-mail address: zxqiong0204@163.com
Corresponding author: Hua Zheng, Tel.: +86 138 7117 4722
E-mail address: zhenghua.whut@126.com
1

ABSTRACT
We induced changes in the tumor microenvironment (TME) through the
synergistic actions of two drugs used in breast cancer therapy. The anti-fibrotic
drug silibinin (SLB) targets tumor-associated fibroblasts and exerts immune-
mediated anti-cancer effects. IPI-549, an efficient and highly selective
phosphoinositide-3-kinase-gamma (PI3Kγ) inhibitor, was applied to alter the
balance of immunosuppressive cells by inhibiting PI3Kγ molecules; it also
promotes anti-tumor immunity. We developed nanoparticle formulations to
encapsulate both drugs into the targeting carrier aminoethyl anisamide-
polyethylene glycol-polycaprolactone (AEAA-PEG-PCL) respectively. The
drugs were intravenously delivered in mice and resulted in an increase in anti-
tumor efficacy and apoptotic tumor tissue compared with either IPI-549 or SLB
alone in 4T1 breast cancer cell-derived tumors. Furthermore, a significant
reduction in regulatory T (Treg) cells and myeloid suppressor cells (MDSCs) was
observed. A normalized TME structure was also observed, including
angiogenesis suppression, antifibrotic effects and the inhibition of collagen
formation in the tumor tissue, significantly enhancing the anti-tumor effects. In
summary, this combination strategy may offer an alternative treatment for
breast cancer.
KEY WORDS: Tumor microenvironment; Phosphoinositide-3-kinase-
gamma;
Combination; Therapy; AEAA-PEG-PCL; Breast cancer;
1. Introduction
2

In recent years, the prevalence of breast cancer has shown a clear increasing trend
(Siegel et al., 2019). There is increasing evidence that breast cancer progression
depends to a large extent on the tumor microenvironment (TME (Francoa et al.,
)
2019; Houthuijzen and Jonkers, 2018), which includes a variety of immune cells,
such as tumor-associated fibroblasts (TAFs), tumor-associated macrophages
(TAMs), regulatory T (Treg) cells and myeloid suppressor cells (MDSCs). MDSCs
are a group of heterogeneous immune cell populations, including myeloid cell
precursors, immature granulocytes, monocytes, and dendritic cells and are also
known as immature immunity due to their immunosuppressive function (Lei and
Zhou, 2014; Vlachou et al., 2012). Treg cells are essential for maintaining
autoimmune homeostasis and are an important factor in inhibiting the formation of
tumor-suppressive microenvironments (Gao et al., 2014). In view of the powerful
functions of Treg cells, changes in Treg cells in tumor tissues are also an important
issue. Both Treg cells and MDSCs correlate with tumor occurrence, development
and prognosis, and their overexpression in various malignant tumor tissues has
been shown (Wallecha et al., 2013; Weed et al., 2015). A study found that breast
tumor growth in mice induces the accumulation of TAMs (Franklin et al., 2014),
which in turn increases TAM concentration in breast tumor-bearing mice. Studies
have shown that a highly selective phosphoinositide-3-kinase-gamma (PI3Kγ)
inhibitor, IPI-549, can significantly improve the response of tumors with high TAM
concentrations to immunological checkpoint blocking therapy (Janku, 2017). In
addition, silibinin (SLB) is an anti-fibrotic drug that significantly inhibits TAFs in
the tumor microenvironment (Ko et al., 2017). As the most abundant stromal cell
in TME, TAFs play a key role in immunotherapy (He et al., 2017). We targeted
TAFs with the anti-fibrotic drug, SLB, which when combined with IPI-549, may
be a novel therapeutic approach for breast cancer through adjusting these
immunosuppressive cells in the TME.
A related study (Nature, 2016) indicated that blocking PI3Kγ molecule can
effectively reduce the activity of pretumor macrophages and enhance the function
of anti-tumor macrophages to alter the balance of immunosuppressive cells and
promote anti-tumor immunity (De Henau et al., 2016; Pathria et al., 2019). In
addition, the effective inhibition and selectivity of IPI-549 is more than 100 times
that of other lipids and protein kinases and 58-282 times that of other PI3K
subtypes, with a biochemical IC50 of 16 nM (Evans et al., 2016). In a variety of
tumor models, IPI-549 increased anti-tumor immunity by reducing MDSCs and
increasing Treg cells, significantly inhibiting the growth of transplanted tumors and
improving the sensitivity of certain tumors to existing anti-cancer drugs by
synergistically enhancing the ability of existing cancer immunotherapy to eradicate
tumors (Yu et al., 2019).
SLB is an anti-tumor agent, which mechanism is through anti-oxidation, anti-lipid
peroxidation, anti-fibrosis, cell membrane stability, immunity, and regulation
(Hammam and El, 2012). Meanwhile, many reports have demonstrated that the
anti-fibrosis is associated with a reduction of alpha-smooth muscle (α-SMA)-
positive cells and procollagen mRNA (Polachi et al., 2016). In recent years, SLB
3

has been found to have anti-tumor activity and has a good inhibitory effect on breast
cancer (Hossainzadeh et al., 2019).
Unfortunately, SLB and IPI-549 have poor water solubility and low selectivity, and
few studies have targeted nanoformulations that specifically deliver SLB and IPI-
549 to tumor cells.
We report the use of the nanosedimentation method to effectively encapsulate IPI-
549 and SLB into the targeted polymer carrier aminoethyl anisamide-polyethylene
glycol-polycaprolactone (AEAA-PEG-PCL) to prepare nanoparticles, and we
evaluated the toxicity of the preparation on breast tumors in vivo and in vitro.
Ligand installed on the surface of nanocarriers can inhance nanoparticles targeting
abilities (Lin et al., 2018; Mi et al., 2019). The sigma receptor is a membrane
protein, and express on many tumor cells (van Waarde et al., 2015). Recent studies
have found that sigma receptors are highly expressed on the surface of cancer cells
and TAFs, and AEAA is a ligand for sigma receptors (Hou et al., 2018; Liu et al.,
2019). If the sigma receptor ligand AEAA is attached to the surface of the drug
delivery system, the drug can be specifically delivered to the critical immune
suppressive cells in the tumor (Huo et al., 2017; Zhao et al., 2015). Therefore, the
biodegradable amphiphilic polymer PEG-PCL was modified with AEAA, which
allows the modified nanoparticles (NPs) to release drugs in tumor tissues (Zhang
et al., 2019). We hypothesized that the two nanoparticles would actively target
breast tumors and remodel the TME synergistically to enhance anticancer efficacy
through immunotherapy.
2. Methods
2.1. Materials
IPI-549 was purchased from Chemietek (Indianapolis, IN, USA). Silibinin (SLB)
was purchased from Chengdu Mansite Biotechnology Co., Ltd. (China). Tert-
Butoxycarbonyl-Amino-Polyethylene Glycol-Amino-(Boc-NH-PEG2000-NH2)
was purchased from Shanghai Ponsure Biotechnology Co., Ltd. (China).
Polycaprolactone-carboxy (PCL5000-COOH) was purchased from Xi'an Ruixi
Biological Technology Co., Ltd. (China). p-Methoxybenzoyl chloride, 2-
bromoethylamine
trifluoroacetic
hydrobromide,
acid (TFA),
N,N-diisopropylethylamine
dichloromethane (DCM)
(DIPEA),
and N,N-
diisopropylcarbodiimide (DIC) were purchased from Aladdin Industrial Inc.
(China). Roswell Park Memorial Institute 1640 (RPMI-1640), 0.25% trypsin and
double antibody were obtained from HyClone and Thermo Fisher Scientific. Fetal
bovine serum (FBS), cell counting kit-8 (CCK-8), 4,6-diamidino-2-phenylindole
(DAPI), and 4% paraformaldehyde fixative were obtained from Beyotime Institute
of Biotechnology (Shanghai, China). Deionized water was produced by a water
purifier (Aquapro, Chongqing, China). Mouse breast cancer cells (4T1 cells) were
purchased from the China Center for Type Culture Collection; Female balb/c mice
(6-7 weeks old) were provided by Wuhan Greenson Island Biotechnology Co., Ltd.
(China) and raised at the specific pathogen-free (SPF) Animal Center of Hubei
Food and Drug Administration. The experimental unit used the license number
4

[SYXK (E) 2019-0009], the unit license number [SCKY (E) 2015-0018]. The
environment is sterilized by ultraviolet light on a regular basis, maintaining a
constant temperature of approximately 20 °C, a relative humidity of approximately
50%, a photoperiod of 12 h each day, and feeding the mice with a basic diet. All
operations are performed in an aseptic workstation in an SPF environment and in
accordance with Chinese law. All drugs and reagents were obtained from
commercial corporations without further processing.
2.2. Synthesis of AEAA-PEG-PCL
A total of 341 mg of p-methoxybenzoyl chloride (2 mmol), 410 mg of 2-
bromoethylamine hydrobromide (2 mmol), and 180 µL of DIPEA were dissolved
in 5 mL of acetonitrile and reacted at 40°C overnight; 400 mg of NH2-PEG-NH-
Boc (Mw = 2000, 0.2 mmol) was added, and the samples were stirred for 24 h.
After the reaction, the mixture was added to 30 mL of ethyl ether and centrifuged
for 10 min at 10,000 RCF; then, the tubes were stored at -20 °C for 2 h, centrifuged
at 8000 RPM and -4 °C for 5 min to compact the precipitate. Then, the waste ether
was removed, and the samples were washed with ether twice to give final purified
product (240.8 mg, yield: 60.2%).
The precipitate (240.8 mg AEAA-PEG-NH2-Boc) was resuspended in 4.5 mL of
TFA /DCM (1/2 by vol). After stirring at RT for 2 h, the solvent was removed in a
vacuum. The precipitate was redissolved in 5 mL of DCM and precipitated into the
ether. Then, the sample was centrifuged at 10,000 RCF and -4°C for 15 min to
compact the precipitate. After completion, the waste ether was removed, and the
sample was washed with ether twice. The waste ether was removed, and the
precipitate was dried under nitrogen gas for 2 - 4 h. The dry AEAA-PEG-NH2·TFA
(233.6 mg, yield: 97%) was transferred to a 20-mL vial for the next reaction.
A total of 233.6 mg of AEAA-PEG-NH2·TFA, 600 mg of PCL-COOH (Mw = 5
kDa, 0.12 mmol), 132 µL of DIPEA, and 250 µL of DIC were dissolved in 6 mL
of DCM. After stirring at RT for 48 h, the AEAA-PEG-PCL in DCM was dropwise
added into methanol. The sample was centrifuged at 10,000 RCF and -0 °C for 15
min to compact the precipitate. The waste methanol was removed, and the sample
was washed with methanol twice. Then, the waste methanol was removed, and the
precipitate was dried under nitrogen for 1 h to give AEAA-PEG-PCL (560.2 mg,
yield: 67.2%). The specific synthetic route is shown in Scheme 2. The chemical
structures of the synthesized samples above were confirmed by a Fourier infrared
spectrometer (Avator360, Nicolet, MA, USA) and Nuclear magnetic resonance
instrument (Varian, USA).
2.3. Embedding silibinin and IPI-549 into AEAA-PEG-PCL using solvent
displacement
A total of 75 mg AEAA-PEG-PCL polymer targeting vector and 5 mg of IPI-
549/SLB were accurately weighed and placed in 500 µL of anhydrous acetone.
After complete dissolution, the carrier was mixed with IPI-549 and SLB. The
mixed solution was slowly dropped continuously into distilled water with stirring
5

and stirred at 40 °C for 7 h. After completion, the reaction solution was removed
and centrifuged at 8000 RPM for 15 min, and the precipitate was removed. The
supernatant was finally passed through a 0.45-µm filter membrane and lyophilized
to obtain AEAA-PEG-PCL-IPI-549 and AEAA-PEG-PCL-SLB NPs lyophilized
powders.
2.4. Characterization of nanoparticles
The particle size distribution and polydispersity index (PDI) of the two groups of
nanoparticles were measured by dynamic light scattering (DLS) instrument
(Malvern, UK).
The morphologies of the nanoparticles were obtained using a transmission electron
microscope (TEM) (Tecnai G220 S-TWIN, FEI, USA) at an acceleration voltage
of 100 kV. Briefly, the freshly prepared nanoparticle solution (1 mg/mL) was
dropped on a copper mesh and allowed to dry naturally.
Determination of critical micelle concentration (CMC) of AEAA-PEG-PCL NPs
by pyrene fluorescence probe method with a fluorescence spectrophotometer (F-
7000, Hitachi High, Japan).
IPI-549 and SLB nanoparticles lyophilized powders were dissolved in deionized
water. The content of the IP-549 and SLB was determined by ultraviolet
spectrophotometry (UV 1101, Techcomp, China) at 267 and 288 nm. The
encapsulation rate (EE) and drug-loading capacity (DLC) of the IPI-549 and SLB
NPs were calculated according to the following formulas:
Amounts of drug in NPs
EE 
DL 
100%
100%
Total amount of drug
Amounts of drug in NPs
Total weight of NPs
2.5. In vitro SLB and IPI-549 release from NPs
The in vitro release behaviors of IPI-549 and SLB from nanoparticles were
investigated by the dynamic dialysis method in PBS (pH 7.4). First, 5 mL of freshly
prepared nanoparticle solution was taken, packaged in a dialysis bag, and placed in
a centrifuge tube containing 45 mL of PBS (pH 7.4). A constant temperature of 37
°C was maintained, and the sample oscillated for 72 h at a speed of 100 rev/min.
Subsequently, 3 mL of the drug release medium was withdrawn at the set time to
be measured by UV. The total volume of the release medium of each group was
maintained by supplementation; the cumulative release of IPI-549 and SLB was
determined using the following formula, and the release curve was generated.
45C  3
Ci
i1
n
Q% 
100%
mdrug
where Ci and Cn refer to the mass concentration of drug in the release medium at
the i and n moment, respectively.
6

2.6. Cell toxicity and in vitro synergistic effects
To verify the anticancer efficiency of the coadministered nanoparticles prepared
herein, first, in vitro cell-level simulation experiments were performed, and the
cytotoxicity of each component was determined using the CCK-8 method. First,
4T1 cells were propagated to a complete culture solution of medium/FBS/double
antibody (89/10/1 by vol) and kept in an incubator at 5% CO2 and at 37 °C. Then,
4T1 cells grown to log phase were seeded at a density of 8,000 cells/well in 96-
well plates, and the incubation time was recorded. After the cells were incubated
for 24 h and exhibited good growth, the original culture solution in each well was
discarded. Afterwards, the six groups of 100 µL of different concentrations samples
of PEG-PCL, AEAA-PEG-PCL, SLB, SLB NPs, IPI-549, IPI-549 NPs, SLB+IPI-
549, and SLB+IPI-549 NPs were plated into 96-well plates, and the cells were
further incubated for 24 and 48 h. Each sample was repeated six times, and
untreated cells acted as a control. The culture solution in each well was replaced by
100 µL of CCK-8 reagent (90%) in the dark. The cells were incubated for 2 h in
the dark, and the optical density value was measured using a microplate reader
(Multiskan FC, Thermo Scientific, Germany).
2.7. Cellular uptake behavior of nanoparticles
The cellular uptake behavior of SLB NPs+IPI-549 NPs on 4T1 cells was evaluated
using the CCK-8 assay. Because the raw material cannot spontaneously display
fluorescence, it was marked with Nile red (NR) and fluorescein isothiocyanate
(FITC green). 4T1 cells (1×105 cells/dish) were incubated in a Petri dish cell for 24
h. Afterwards, the cell culture solution was substituted by NPs containing
equivalent amounts of PEG-PCL-FITC-IPI-549, PEG-PCL-NR-SLB, AEAA-
PEG-PCL-FITC-IPI-549, and AEAA-PEG-PCL-NR-SLB, and the cells were
incubated for an additional 0.5, 2 and 4 h. The culture solution was then removed,
and the 4T1 cells were washed with PBS in duplicate. Then, 4% W/V formaldehyde
was used to fix cells for 15 min, and the cells were washed with PBS again.
Subsequently, the cell nuclei were stained with DAPI, and the cells were incubated
for 10 min. The washed culture dish was then placed under CLSM (Zeiss LSM 700)
to observe the fluorescence intensity and distribution in the cells.
2.8. Anti-tumor efficacy in mouse breast cancer cell (4T1) xenografts
After 7 days of adaptive feeding, an orthotopic tumor model was established. First,
4T1 cells were digested and resuspended in serum-free medium. Then, the cells
were resuspended thrice with PBS buffer and diluted to 2 × 106 cells/mL. The cell
suspension (200 µL) was inoculated subcutaneously into the right mammary glands
of female balb/c mice using a disposable sterile syringe. On day 8 after tumor
implantation (tumor size 50-70 mm2), the mice were randomized into 7 groups (n
= 5) as follows: untreated control (Saline); SLB (SLB in 30% PEG-400 solution,
stirred or sonicated to evenly disperse, 5 mg/kg); SLB NPs; IPI-549 (IPI-549 in
30% PEG-400 solution, stirred or sonicated to evenly disperse, 5 mg/kg); IPI-549
NPs; SLB+IPI-549; and SLB NPs + IPI-549 NPs. Each sample solution was
7

prepared according to the preset concentration and the average mouse body weight
and sterilized using a 0.45-μm sterile filter. The mice were administered the drug
(200 μL/each) via the tail vein using a disposable sterile syringe every other day.
Drug administration was performed 7 times in total. The tumor long axis and short
axis were monitored using a Vernier caliper every other day and calculated as 1/2
× (long axis) × (short axis)2. After 22 days of inoculation, the mice were sacrificed
by cervical dislocation, and the tumor tissue and major organs were collected for
further experiments. The inhibition ratio (IR) was calculated using IR (%) = [(Ws-
Wt)/Ws] × 100, where Ws and Wt refer to average tumor weights for the saline
group and each administration group, respectively.
2.9. Safety evaluation
The drug was administered 7 times in total, at an interval of once every other day.
After 22 days of administration, all mice were sacrificed, and the heart, liver,
spleen, lung and kidney of body were quickly dissected, placed in tissue bottles,
and immobilized with formalin. Then, the morphology and pathology of the
prepared tissues were studied by hematoxylin-eosin (H&E) staining, and the safety
of the nanoparticles was evaluated. Briefly, the main organs were embedded in
paraffin and sectioned, and the tissue sections were dewaxed and stained with
H&E. Subsequently, images of the prepared tissues were observed using positive
fluorescence microscopy (NIKON, Japan) to evaluate the safety of the
nanoparticles.
2.10. TUNEL assay
The TUNEL assay was used to detect apoptotic cells in tumor tissues. After the
mice were sacrificed, the intact tumors were dissected and fixed with formalin and
sectioned; then, the tumor tissue was deparaffinized and stained with DAPI
staining. Finally, the sections were washed with PBS, dried, and sealed with an
anti-fluorescence quencher. Images were captured under a fluorescence microscope
(NIKON, Japan) and quantitatively analyzed for apoptosis using Image Pro Plus
6.0.
2.11. Immunofluorescence staining
By double-labeling tumor tissue sections, protein expression in tissue cells was
determined, and the anti-tumor mechanism in vivo was explored. Tumor sections
were deparaffinized for rehydration to retrieve the antigen, naturally cooled,
washed, and dried. Then, the sections were incubated for 30 min with 1% bovine
serum albumin (BSA), and the primary antibody was incubated for 12 h at 4 °C.
The next day, the sample was treated with a secondary antibody at room
temperature for 50 min, and the nuclei were counterstained with DAPI. Finally, the
sample image was observed and acquired using a fluorescence microscope
(NIKON, Japan).
2.12.Masson trichrome staining
8

Collagen in tumor tissue was detected by Masson's trichrome assay. Tumor sections
were deparaffinized and hydrated. The sections were then stained according to the
manufacturer's instructions and finally differentiated and blocked. Collagen in
tumor tissue of images was analyzed using a vertical optical microscope and
imaged (NIKON, Japan).
2.13. Analysis of α-SMA and PARP expression using Western blot
The anti-tumor mechanism of this study was verified using Western blot assays.
First, the tumor tissue was excised and mixed with the tissue lysate. Afterwards,
the cell debris was cleared by centrifugation, and protein-loaded buffer was added
to completely denature the protein. The sample was separated by SDS-PAGE, and
the protein in the gel was transferred to a polyvinylidene fluoride (PVDF)
membrane. Then, the membrane was covered with a blocking solution with
agitation for 2 h and incubated with the indicated primary antibodies α-SMA,
PARP, and GAPDH at 4 °C overnight. The membrane was thoroughly washed with
TBST and blotted on a membrane strip, and the strip was placed in the secondary
antibody working solution, incubated at RT for 2 h in the dark, and washed
thoroughly with TBST. The PVDF membrane was placed face up between the two
films of the exposed enamel, and the mixed ECL solution was added. After 1-2
min, the residual liquid was removed, and the film was covered to start exposure.
Finally, the exposed film was developed and fixed, the archived film was scanned,
the color was removed using Adobe Photoshop, and the optical density value was
processed using grayscale analysis software (alpha EaseFC).
2.14. Statistical analysis
Quantitative data in the study are presented as the mean ± standard deviation (SD)
using GraphPad for statistical analysis, and the difference analysis was performed
using a one-way variance method. P values < 0.05 were considered significant as
follows: *, P<0.05; **, P < 0.01; ***, P < 0.001.
3. Results and Discussion
3.1. Characterization of AEAA-PEG-PCL
The chemical structures of the products at each step were characterized using FT-
IR spectra. Fig. 1 shows the infrared spectra of (A) NH2-PEG-NH-Boc, (B) AEAA-
PEG-NH-Boc, (C) AEAA-PEG-NH2·TFA, and (D) AEAA-PEG-PCL. The NH2-
PEG-NH-Boc infrared spectrum (A) shows that the absorption peak at 3380 cm-1
and 2886 cm-1 is the stretching vibration of CH in the NH and PEG segments, and
the absorption peak at 1711 cm-1 corresponds to the ester bond. The characteristic
absorption peak is C=O.
Compared with Fig. 1A, the skeletal vibration absorption peak of the benzene ring
appears in Fig. 1B; that is, the wavenumber is 1605 cm-1 and 1506 cm-1, and the
stretching vibration (1706 cm-1) of C=O is shifted to the right, which indicates the
successful synthesis of AEAA-PEG-NH-Boc.
Comparing C with B, the stretching vibration of C=O became 1681 cm-1, indicating
9

that the tert-butoxycarbonyl group was successfully substituted.
In the D diagram, the absorption peaks at 1945 cm-1 and 2866 cm-1 are the CH
stretching vibrations of the PEG and PCL segments, respectively, and the
absorption peak at 1724 cm-1 belongs to the C=O vibration of the PCL segment.
These characteristic absorption peaks indicate that PCL was successfully bonded
to AEAA-PEG-NH2·TFA. In addition to the FT-IR spectra, the 1H NMR also
clearly affirmed the successful conjugations of AEAA-PEG-NH-Boc, AEAA-
PEG-NH2·TFA, and AEAA-PEG-PCL (Fig. 2). As shown in Fig. 2, the nuclear
magnetic resonance spectrum of Fig. 2A shows that the peak is assigned to its
molecular structure, and the proton peak signal in the range of 2.89-3.62 ppm
belongs to the hydrogen on the PEG segment. The proton peak signal at 1.37 ppm
corresponds to the methyl peak of the tert-butoxycarbonyl group (Boc) at one end
of the PEG, and the proton peak signal at 6.73 ppm is attributed to the hydrogen
proton on the amino group at the other end of the PEG. In Fig. 2B, the new proton
signal peak at 8.57 ppm is the hydrogen proton signal on the amino group directly
attached to the carbonyl group, while the signal peak at 8.0 and 7.8 ppm is derived
from the benzene ring structure of AEAA. The methyl peak of the tert-
butoxycarbonyl group (Boc) in C disappeared, which confirms the successful
synthesis of AEAA-PEG-NH2.
The proton peak signals of 1.27, 1.55, 2.27, and 3.98 ppm appearing in Fig. 2D are
attributed to hydrogen on the PCL segment, indicating the successful synthesis of
the targeting vector AEAA-PEG-PCL.
3.2. Characterization of nanoparticles
A nanoparticle-based drug delivery system is an effective method to target the
delivery of hydrophobic therapeutics to tumor sites (Wang and Thanou, 2010).
Therefore, water-insoluble IPI-549 and SLB were encapsulated into NPs using the
solvent displacement method so that improve drug solubilization and enhance drug
targeting after systemic administration. As a result, the EE for IPI-549 NPs and
SLB NPs was 91.4 ± 3.8% and 76.9 ± 0.2%, respectively, and the DL for IPI-549
NPs and SLB NPs was 15.8 ± 6.4% and 12.9 ± 7.1%, respectively. The average
sizes of IPI-549 NPs (Fig. 3A) and SLB NPs (Fig. 3B) were 34.4 nm and 37.2 nm,
respectively, measured by DLS. As shown in Figs. 3 (C-D), there are spherical
configuration and relatively uniform size of these nanoparticles by TEM
measurement. Meanwhile, the CMC for the AEAA-PEG-PCL NPs were also
measured (2.41 μg/mL) (Fig. 4A).
3.3. Study on the in vitro release properties of nanoparticles
The release of IPI-549 and SLB from the NPs exhibited a different maximum
cumulative release (37.25% and 74.13%) at the same time (45 h) in pH 7.4 (Fig.
4B). The results showed that both nanoparticle formulations had a very large
sustained release effect, which significantly prolonged the time of activity of SLB
and IPI-549 and improving the bioavailability of the two drugs. In addition, the
difference in the release rates of the two nanoparticles is mainly due to the extremely
10

poor water solubility of IPI-549.
3.4. Cell toxicity and in vitro synergistic effects
We first determined the toxicity of the empty carrier PEG-PCL and the targeting
vector AEAA-PEG-PCL on murine breast cancer cells (4T1 cells). The results
showed that both vectors were biosafe. As shown in Fig. 5, after a 24-h incubation
with different concentrations of PEG-PCL solution and AEAA-PEG-PCL solution,
the toxicity of 4T1 cells was negligible. After 48 h, the targeting vector was less
toxic than PEG-PCL. The results of these studies indicate that the target carrier of
this study has good biosafety, which also plays an important role in maintaining the
low toxicity and side effects of NPs in systemic circulation.
The cell inhibition effect of SLB and IPI-549 and their NPs was evaluated (Fig. 5).
Both SLB and IPI-549 reduced the survival rate of 4T1 cells, exhibiting good
concentration-dependent and time-dependent effects. The antitumor effects of NPs
were significantly stronger than that of the free drugs. SLB NPs + IPI-549 NPs had
the highest cytotoxicity, which showed that the combined treatment was synergistic
and had a powerful inhibitory effect on tumor cells.
3.5. Cellular uptake of nanoparticles
Aminoethylanisamide enhances the cellular uptake of nanoparticles by specifically
binding to sigma receptors on the surface of tumor cells(Kim et al., 2018). Fig. 6
shows representative CLSM images of 4T1 cells incubated with AEAA-PEG-PCL-
IPI-549 NPs and AEAA-PEG-PCL-SLB NPs loaded with FITC and NR for 4 h.
With increasing incubation time, strong red and green fluorescence signals were
observed at the periphery of the cell nucleus, indicating that the nanoparticles were
effectively absorbed by the cells, thus validating the efficacy of our therapeutic
system for 4T1 cell delivery. To demonstrate the targeting ability of nanoparticles,
surface-unmodified PEG-PCL-FITC-IPI-549 and PEG-PCL-NR-SLB were also
incubated with 4T1 cells for 4 h. We can observe clearly that the red and green
fluorescence of 4T1 cells was relatively lower at each time point. The results
showed that the surface modification of NPs to aminoethyl anisamide significantly
promoted their cellular uptake in 4T1 cells.
3.6. Anti-tumor efficacy in mouse breast cancer cells (4T1) xenografts
The effects of drug-loaded nanoparticles on tumor growth were assessed. As shown
in Fig. 7A, the tumor growth rate was extremely rapid (approximately 1700 mm3
on the 16th day) in the control group. The SLB showed a less curative effect than
the IPI-549 drug at the same dose. Notably, the antitumor effects of SLB NPs and
IPI-549 NPs were significantly stronger, which is consistent with the results of the
cell toxicity experiments. The combined action of SLB NPs and IPI-549 NPs
resulted in the strongest tumor inhibition effect, in which the tumor volume was
only approximately 350 mm3 after 16 days of treatment. As shown in Fig. 7C, SLB
NPs+IPI-549 NPs significantly inhibited transplanted tumor growth in mice, and
the tumor weight (0.4 g) was lighter than that of the control group (2.6 g) (P <
11

0.001). The average inhibition rate of each group was compared, and the SLB
inhibition rate was 26.92%. The inhibition rates of SLB NPs, IPI-549, and IPI-549
NPs were 61.5%, 50% and 69.23%, respectively, which were comparable to that of
SLB+IPI-549 (57.69%). The inhibition rate of SLB NPs+IPI-549 NPs treatment
reached 84.62%, indicating that the therapeutic effect of the combined
administration of SLB NPs and IPI-549 NPs was improved. The tumor weight and
tumor volume results showed that the SLB NPs+IPI-549 NP treatment had strong
anti-tumor ability and high activity in vivo.
3.7. Safety evaluation of the different treatments
The mice treated with different drug formulations had no abnormal changes in body
weight throughout the tumor inhibition experiment (Fig. 8A). In addition, the H&E
staining observations indicated no obvious pathological changes in the hearts,
spleens, lungs, and kidneys (Fig. 8B). These findings confirmed that the NP
structures are safe to animals.
3.8. TUNEL assay
The tumor cell apoptosis index was quantitatively analyzed using a TUNEL assay.
The tumor cells in the control group exhibited very little apoptosis, while the tumor
cells in the mice treated with drugs exhibited different degrees of apoptosis.
Notably, the tumor tissue showed the highest level of cell apoptosis (46.6 ±2.7%)
in the SLB NPs + IPI-549 NPs group (Fig. 9). These results are associated with the
tumor inhibition data, indicating that the optimal therapeutic effect is produced by
the combined nanoparticles. This finding might be attributed to the synergistic
effect and the targeted release of the drug.
3.9. Suppression of cell reprogramming in the TEM
AEAA-PEG-PCL-SLB NPs and AEAA-PEG-PCL-IPI-549 NPs can efficiently
deliver SLB and IPI-549 to tumor cells. Immunoregulatory cell populations,
including MDSCs, TFA, and Treg cells, were investigated during the experiments.
These cells secrete various cytokines and chemokines to affect tumor cells (Song
et al., 2018). Treg cells are a subset of CD4 + T cells with immunosuppressive
effects that play a key role in the homeostasis of maintaining immune cells, and
their specific transcription factor Foxp3 (forkhead/winged helix transcription
factor3) is mainly expressed in the nucleus of lymphocytes (Li and Wang, 2012;
Ohta et al., 2012). Foxp3 also plays an important role in regulating the
differentiation, development and function of Tregs and is recognized as a Treg-
specific marker (Bacchetta et al., 2007; Shi and Sun, 2014). MDSCs exert
immunosuppressive functions through multiple pathways and mechanisms, and the
phenotypic marker of mouse MDSCs is CD11b + Gr-1 (Yang et al., 2015). The
results (Fig. 10) show that the percentage of MDSCs and Treg cells in the TME is
reduced after SLB treatment. The inhibitory effect of silibinin on MDSCs and Tregs
in a mouse model of breast cancer is consistent with the results of the previous
study (Castellaneta et al., 2016). Forghani et al. also reported that SLB failed to
12

inhibit tumor growth in immunodeficient mice, indicating that the anticancer
effects of SLB are immune-mediated (Forghani et al., 2014). However, when SLB
was combined with IPI-549, the MDSC and Treg cell counts were significantly
reduced even lower than those in the SLB-treated and IPI-549-treated groups. This
result is because IPI-549 can not only alter the immunosuppressive
microenvironment of tumors but can also trigger an anti-tumor immune response
(Pathria et al., 2019). Therefore, in present study, the anti-tumor immune response
is further activated by both enhancing the anti-tumor immunosuppressive effect and
its tumor-suppressive activity in the SLB NPs + IPI-549 NPs group (Fig. 10B-E).
Taken together, our data suggest that SLB and IPI-549 have synergistic effects in
anti-tumor immunosuppression.
3.10. Structural changes in the TME: TAFs, vasculature, and collagen fibers
TAFs are considered a class of cells that promote tumor cell growth and metastasis
through multiple pathways and can inhibit anti-tumor immune responses (Costa et
al., 2018; Wang et al., 2012). The effect of SLB NPs on TAFs was investigated by
staining for α-smooth muscle actin (α-SMA) (a TAF biomarker) (Lin et al., 2018).
The results indicated that both SLB and SLB NP treatments remarkably decreased
TAF expression in tumors (Fig. 10A), which is consistent with the results that SLB
demonstrated antifibrotic properties (Trappoliere et al., 2009). Furthermore, the
combination of SLB and IPI-549 NPs reduced the percentage of fibroblasts in the
tumor to 18.0 ± 3.0% (p < 0.001) (Fig. 10B). Abnormal vessel structures cause
malignant cells to migrate and prevent drug and immunocyte penetration into the
tumor mass, which further enhance the deterioration of tumor cells. We evaluated
the vascular content and distribution in tumors by immunohistochemistry of CD31
(vascular marker) in each group (Fig. 10A). Both SLB NPs and IPI-549 NPs
dramatically reduced the percentage of vascular content to 33.3 ± 7.7% and 30.5 ±
7.1% (p < 0.01), respectively. The combined drug treatment exhibited the lowest
blood vessel content of 14.7 ± 3.8% (p < 0.001). These data suggest that SLB and
IPI-549 caused structural changes in the TME, facilitating the reconstitution of
fibroblasts and the vascular network.
Similarly, SLB NPs + IPI-549 NPs also significantly reduced the matrix content of
the tumor mass. We examined collagen content and distribution as well as
morphology in the tumor by Masson trichrome staining (Fig. 11). The matrix of
collagen staining exhibited a relatively slender fibrous structure in the control
tumor. The IPI-549 NPs reduced the matrix to only 66.3 ± 7.1% (p < 0.01) of that
in the control group, while the SLB NPs reduced the matrix by approximately 33.0
± 1.9% (p < 0.001) of the untreated control. Thus, the powerful effect of SLB on
collagen in present study is consistent with the report that SLB retards collagen
accumulation fibrosis induced in rats (Boigk et al., 1997). In the SLB NPs + IPI-
549 NPs group, the collagen content was further reduced to only approximately
23.0 ± 4.8% (p < 0.001). Therefore, we conclude that SLB NPs + IPI-549 NPs exert
topgallant anti-tumor efficiency via antifibrotic, angiogenesis suppression, and
inhibition of tumor cell migration, attributing to the synergistic effect of drugs.
13

3.11. Analysis of α-SMA and PARP expression using Western blot
The results are shown in Fig. 12A. Compared with the untreated tumor, SLB NPs
+ IPI-549 NPs dramatically reduced the expression levels of the downstream
cleavage of poly ADP ribose polymerase (PARP) (apoptosis marker), which were
relatively lower than those induced by SLB/IPI-549 NPs and free SLB/IPI-549.
This finding is consistent with the TUNEL data. α-SMA expression was reduced
in tumors in the SLB NPs and SLB NPs + IPI-549 NPs groups. The results showed
that SLB has an anti-fibrotic effect and can effectively inhibit TAF production. The
combination of SLB and the IPI-549 significantly improved the TME, inhibiting
breast cancer cell aggregation.
4.Conclusion
In this study, a long-circulating nanoparticle system based on tumor targeting was
designed for breast cancer by immunotherapy. This nanoparticle system had a
“core-shell” structure containing a hydrophobic IPI-549/SLB nanocore and a
hydrophilic AEAA-PEG-PCL targeted shell and achieved both the efficient
targeting of breast tumors and the slow release of IPI-549 and SLB. In the TME,
IPI-549 and SLB decreased the amount of MDSCs and Treg cells and altered the
blood vessel and stromal structure of the tumor. IPI-549 enhanced the immune-
mediated anti-cancer effect from SLB by modulating immunosuppressive cells.
Our data provided evidence in vivo and in vitro that the codelivery of PI3Kγ
inhibitors and an anti-fibrotic drug to tumors can act synergistically by remodeling
the TME in breast cancer, which improves the oncotherapy efficiency of existing
therapies.
Conflict of interest
The authors declare that they have no conflicts of interest.
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Figure Captions
Scheme 1. Schematic illustration of establishing a mouse xenograft tumor model
and treatment by the tail vein injection of SLB NPs and IPI-549 NPs
Scheme 2. Schematic illustration of the synthesis of the AEAA-PEG-PCL
conjugate.
Fig. 1. FT-IR characterization: (A) NH2-PEG-NH-Boc; (B) AEAA-PEG-NH-Boc;
(C) AEAA-PEG-NH2·TFA; (D) AEAA-PEG-PCL.
Fig. 2. 1H NMR spectra of (A) NH2-PEG-NH-Boc; (B) AEAA-PEG-NH-Boc; (C)
AEAA-PEG-NH2·TFA; (D) AEAA-PEG-PCL.
17

Fig. 3. TEM images of (A) AEAA-PEG-PCL-IPI-549 NPs and (B) AEAA-PEG-
PCL-SLB NPs. DLS histograms of (C) AEAA-PEG-PCL-IPI-549 NPs and (D)
AEAA-PEG-PCL-SLB NPs.
Fig. 4. The critical micell concentration of AEAA-PEG-PCL NPs (A). In vitro
cumulative release profiles of IPI-549 NPs and SLB NPs in PBS (pH 7.4)
containing 0.1% (w/v) poloxamer at 37 °C (B). The results are presented as the
mean ± SD (n = 3).
Fig. 5. Viability of 4T1 cells incubated with PEG-PCL and AEAA-PEG-PCL for
24 h (A) and 48 h (B). Viability of 4T1 cells incubated with SLB, SLB NPs, IPI-
549, IPI-549 NPs, SLB+IPI-549 and SLB NPs+IPI-549 NPs for 24 h (A) and 48 h
(B). The results are presented as the mean ± SD. (n = 6, * P < 0.05, ** P < 0.01,
*** P < 0.001).
Fig. 6. CLSM images of 4T1 cells were incubated with (A) PEG-PCL NPs and (B)
AEAA-PEG-PCL NPs for 0.5 h, 2 h, and 4 h. Scale bar = 20 μm.
Fig. 7. Tumor inhibition effects of SLB, SLB NPs, IPI-549, IPI-549 NPs, SLB+IPI-
549, and SLB NPs+IPI-549 NPs on 4T1-bearing mice. 4T1-bearing mice were
injected intravenously every other day at a dose of 5 mg/kg SLB/SLB NPs or 5
mg/kg IP-549/IPI-549 NPs in all treatment groups. (A) The tumor volumes curves
of 4T1-bearing mice along the experiment duration by measured every other day.
(B) Images of the excised tumors from each group at the end of the tumor inhibition
experiment. (C) The IR and the average tumor weight of each treatment group at
the end of the point (day 22). The results are presented as the mean ± SD. (n = 5,
* P < 0.05, ** P < 0.01, *** P < 0.001).
Fig. 8. Safety evaluation of all treatment groups. (A) The average body weight
curves of 4T1-bearing mice over the course of the experiment as measured every
other day. (B) Representative H&E stained sections of major organs after treatment.
Scale bar = 100 μm.
Fig. 9. Representative TUNEL-stained sections of tumors from each group after
treatment. Tumor tissues were stained for cell apoptosis (green). Quantitative
analysis of apoptotic index (TUNEL-positive cells/total cells). Scale bar = 100 μm.
The results are presented as the mean ± SD. (n = 3, * P < 0.05, ** P < 0.01, *** P
< 0.001).
Fig. 10. Remodeling of tumor-infiltrating immune cells in the TME. Tumor tissues
were assayed for the blood vessel (CD31 in green) and tumor-associated fibroblasts
(α-SMA in red), MDSC cells (Gr-1+ in red, CD11b+ꢀin green), and Treg cells (CD4
in red, Foxp3 in green) with immunofluorescence staining (A). Flow cytometric
18

analysis was also used to quantify immune cells in the TME (B-E). Scale bar = 100
μm. The results are presented as the mean ± SD. (n = 3, * P < 0.05, ** P < 0.01,
*** P < 0.001).
Fig. 11. Tumor sections were stained with Masson trichrome. The blue color
represents collagen fibers. Scale bar = 100 μm. The results are presented as the
mean ± SD. (n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001).
Fig. 12. Western blot analysis assessment of α-SMA and PARP knockdown in
tumor tissues after different treatments. The results are presented as the mean ±
SD. (n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001).
Scheme 1
19

O
O
NH2-PEG-NH-Boc
Br
DIPEA
O
NH2
.
NH
HBr
O
Cl +
Br
O
O
O
O
H
N
H
TFA
O
O
O
N
H2N
NH
O
N
NH
DCM
H
n
n
O
O
O
H
N
C
PCL-COOH
DIPEA DIC DCM
O
N
H
N
H
n
O
O
O
Scheme 2
Fig. 1
20

Fig. 2
21

Fig. 3
Fig. 4
22

Fig. 5
23

Fig. 6
Fig. 7
24

Fig. 8
Fig. 9
25

Fig. 10
26

Fig. 11
Fig. 12
27

Graphical abstract
28

Credit Author Statement
All the authors listed have approved the manuscript that is enclosed. The
manuscript has not been previously published, nor is currently considered
for publication in any other journals.
Author contributions
Min Jiang: Conceptualization, Methodology, Software, Investigation,
Writing - Original Draft.
Kaiyong He: Validation, Formal analysis, Visualization, Software.
QiuTong: Validation, Formal analysis, Visualization.
Jiahui Sun: Resources, Writing - Review & Editing, Supervision,
Data Curation.
Qi Liu: Writing - Review & Editing, Supervision, Data Curation.
Xueqiong Zhang: Writing: Review & Editing.
Hua Zheng: Writing: Review & Editing.
29