Asian Journal of Pharmaceutical Sciences 18 (2023) 100796

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journal homepage: www.elsevier.com/locate/AJPS

Original Research Paper

Relaxin-encapsulated polymeric metformin

nanoparticles remodel tumor immune
microenvironment by reducing CAFs for efficient
triple-negative breast cancer immunotherapy

Hongyan Zhang a,b,1, Liying Chen a,1, Yue Zhao a,1, Ningchao Luo a, Jingbin Shi a, Shujun Xu a,
Lisha Ma a, Menglin Wang c, Mancang Gu a, Chaofeng Mu a, Yang Xiong a,b,∗
a School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China
b Academy of Chinese Medical Science, Zhejiang Chinese Medical University, Hangzhou 310053, China
c Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill,

NC 27599, United States

a r t i c l e i n f o a b s t r a c t

Article history:
Received 25 November 2022
Revised 14 January 2023
Accepted 9 February 2023
Available online 25 February 2023
Keywords:
Cancer-associated fibroblasts
Plasmid encoding relaxin
Lipid nanoparticles
Polymeric metformin
PD-L1 antibody
Cancer-associated fibroblasts (CAFs) are one of the most abundant stromal cells in the
tumor microenvironment which mediate desmoplastic response and are the primary driver
for an immunosuppressive microenvironment, leading to the failure of triple-negative
breast cancer (TNBC) immunotherapy. Therefore, depleting CAFs may enhance the effect
of immunotherapy (such as PD-L1 antibody). Relaxin (RLN) has been demonstrated to
significantly improve transforming growth factor-β (TGF-β) induced CAFs activation and
tumor immunosuppressive microenvironment. However, the short half-life and systemic
vasodilation of RLN limit its in vivo efficacy. Here, plasmid encoding relaxin (pRLN) to
locally express RLN was delivered with a new positively charged polymer named polymeric
metformin (PolyMet), which could increase gene transfer efficiency significantly and have
low toxicity that have been certified by our lab before. In order to improve the stability of
pRLN in vivo, this complex was further formed lipid poly-γ -glutamic acid (PGA)/PolyMet-
pRLN nanoparticle (LPPR). The particle size of LPPR was 205.5 ± 2.9 nm, and the zeta
potential was +55.4 ± 1.6 mV. LPPR displayed excellent tumor penetrating efficacy and
weaken proliferation of CAFs in 4T1luc /CAFs tumor spheres in vitro. In vivo, it could reverse
aberrantly activated CAFs by decreasing the expression of profibrogenic cytokine and
remove the physical barrier to reshape the tumor stromal microenvironment, which enabled
a 2.2-fold increase in cytotoxic T cell infiltration within the tumor and a decrease in
immunosuppressive cells infiltration. Thus, LPPR was observed retarded tumor growth
by itself in the 4T1 tumor bearing-mouse, and the reshaped immune microenvironment
further led to facilitate antitumor effect when it combined with PD-L1 antibody (aPD-L1).

∗
Corresponding author.
E-mail address: xyxnb@126.com (Y. Xiong).
1
These authors contributed equally to this work.
Peer review under responsibility of Shenyang Pharmaceutical University.

https://doi.org/10.1016/j.ajps.2023.100796
1818-0876/© 2023 Shenyang Pharmaceutical University. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
2 Asian Journal of Pharmaceutical Sciences 18 (2023) 100796
Altogether, this study presented a novel therapeutic approach against tumor stroma using
LPPR to achieve a combination regimen with immune checkpoint blockade therapy against
the desmoplastic TNBC model.
1. Introduction
Breast cancer is the 2nd most prevalent cancer worldwide and
the main reason for death in women [1]. Furthermore, triple
negative breast cancer (TNBC) represents one of the most
malignant tumors than other breast cancer subtypes with
a poorer prognosis, relapse proneness, and drug resistance
[2,3]. Conventional therapeutic approaches for TNBC are
traditional surgery, chemotherapy, and hormone therapy.
However, these therapies display limited therapeutic benefits
due to molecular and morphological heterogeneity [4,5].
Immunotherapy (such as PD-L1 antibody) has shown promise
in recent years for cancer treatment with a high mutational
burden, which include melanoma [6], lung [7], and ovarian
cancers [8]. Of note, PD-L1 antibody (aPD-L1) provides a long-
lasting response rate of up to 40% in melanoma [9]. In
comparison, TNBC possesses elevated PD-L1 expression, but
patients exhibited only a 20% sustained response rate treated
with aPD-L1 immunotherapy [10]. One of the reasons for the
treatment failure is the abundant geographic or central tumor
fibrosis in TNBC tumor microenvironment (TME) [11,12],
which acts as a barrier to immune checkpoint blockade (ICB)
therapy [13].
TME is a complex arrangement of tumor cells, reactive
matrix cells, blood vessels, immune cells, and extracellular
matrix (ECM), all of which constitute a disorganized and
aggressive ecological niche compared to well-organized,
homeostatic healthy tissue [14,15]. As critical effector cells
that mediate desmoplasia, hyper-proliferative cancer-
associated fibroblasts (CAFs) are essential for tumor
progression [16]. Notably, CAFs are also responsible for
recruiting immunosuppressive cells, confining cytotoxic T
lymphocytes (CTLs), and guarding cancer cells from immune
surveillance [17]. Altogether, this relatively ineffectiveness of
aPD-L1 therapy might be attributed by the abundance of CAFs
in TNBC [18–20]. In recent years, nanoparticles have provided
several smart delivery systems based on active targeting of
CAFs and immunotherapy, such as liposomes [21–23] and
polymeric nanoparticles [24,25]. Many efforts have been
made both to eradicate CAFs and to reshape their function.
Nano-strategies focused on exhausting CAFs are emerging to
augment immunotherapy of breast cancer [26].
Among the most prominent characteristics of the dense
stroma is the excessive expression of transforming growth
factor β (TGF-β) [27,28]. Firstly, under normal conditions,
normal fibroblasts are typically present in a resting state.
In TME, they can be stimulated by growth factors of TGF-
β to differentiate into CAFs [29]. This activation induces
the strengthen of collagen deposition, reshaping ECM, and
formation dense stromal TME. Moreover, TGF-β holds a
© 2023 Shenyang Pharmaceutical University.
This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
key role in the recruitment, activation and infiltration of
regulatory T cells(Treg), macrophages, and myeloid-derived
suppressor cells (MDSC) in TME [30]. Significantly, TGF-β
activation in CAFs is thought to be a deciding factor of CD8+
T cell exclusion and low response to aPD-1/PD-L1 [31,32].
Therefore, damaging CAFs by TGF-β secretion inhibition may
be promising to facilitate immunotherapy [33–35].
Relaxin (RLN) is a potent antifibrogen that suppresses
fibroblast activation upon binding to its principal receptor
RXFP1 (Relaxin family peptide receptor type 1) [33]. Relaxin
displays a potent anti-fibrotic based on its specific capacity to
suppress pro-fibrotic cytokine-mediated abnormal fibroblast
proliferation, differentiation and matrix production [36]. It
is well-documented that RLN has a powerful anti-fibrotic
activity to down-regulate TGF-β-induced collagen production
and contraction of lung, cardiac, renal, and hepatic fibrosis
[37]. Important to its function, RLN also have maintained
anti-tumor efficacy attributed to its stromal modulation
function by significantly inhibited TGF-β/pSmad2 signaling
pathway [33,38]. Nevertheless, the direct applicability of RLN
was constrained by its relatively small size (6 kDa), potential
off-target effects, short circulating half-life, continuous
administration, and adverse effects such as systemic
vasodilation [37,39]. Therefore, a feasible approach to delivery
of RLN plasmids (pRLN) into tumor tissues by generating local
expression of RLN may be a prospective strategy.
In view of this concern, we introduced the polymeric
metformin (PolyMet) to form PolyMet-pRLN complex. PolyMet,
which our lab formulated in previous studies [40–42], was a
highly positively charged polymer chain for effective delivery
of genes to beat the bottleneck of “stabilization dilemma” of
pDNA delivery [43]. PolyMet satisfies a few standards of an
effective gene carrier based on small molecular, excellent
tolerability and low toxicity [44,45]. Several experimental
studies have provided that pDNA develops a loose and
non-stable complex when mixed with only polycationic
polymers owing to its low molecular weight [44] and charge
density [46,47]. Hence, in this work, liposomes were utilized
to develop complexes to develop a novel lipid poly-γ -
glutamic acid (PGA)/PolyMet-pRLN nanoparticle (LPPR) to
guard pDNA from dissociation or removal prior to reaching
the tumor. To the end, LPPR, as an in-situ reservoir, could
deliver pRLN to achieve local RLN expression in tumor. LPPR
with a positive charge in physiological conditions displayed
increased penetrating efficacy and efficient intracellular
drug release in 4T1luc /CAFs tumor spheres due to pRLN.
We further assessed the antitumor effect of LPPR in 4T1
tumor bearing mouse. LPPR diminished CAFs to change
the immunosuppressive environment into an immune
stimulating state with increased cytotoxic T-cell infiltration.
Furthermore, LPPR eventually led to an improvement of aPD-
 Asian Journal of Pharmaceutical Sciences 18 (2023) 100796 3

Scheme 1 – The proposed mechanism of LPPR in combination with aPD-L1 mediates removal of CAFs and immune cell
infiltration. (A) LPPR formulation. (B) Effective delivery of RLN gene through LPPR with aPD-L1 disrupts the
immunosuppressive and desmoplastic TME by targeting CAFs (a. RLN gene in LPPR could silence abnormally active CAFs; b.
aPD-L1 blocks the binding of PD-1 and PD-L1 and promotes tumor cell killing by CD8+ T cells). (C) LPPR triggers the
eradication or continuous remission of aggressive TNBC through synergy with aPD-L1.

L1 therapeutic efficacy. Our findings suggested the effective
delivery of the RLN gene through LPPR, as the ideal candidate,
had the dual roles of addressing abundant tumor fibrosis and
immunosuppressive microenvironment, finally facilitated
checkpoint blockade in TNBC (Scheme 1).
2. Material and methods
2.1. Material
RLN plasmid was obtained from Sino Biological Inc. Linear
PEI hydrochloride (MW = 8000), HOBt, EDC·HCl, and Coumarin
6 were purchased from Aladdin (Shanghai, China). DOTAP
were bought from Advanced Vehicle Technology (Shanghai,
China). BCA protein assay kits and DAPI were obtained
from Beyotime (Shanghai, China). CellTracker Green 5-
chloromethylfluorescein diacetate (CMFDA) was acquired
from Yeasen Biotechnology Co., Ltd (Shanghai, China). D-
K+ was purchased from PerkinElmer (USA). All the other
chemicals were sourced from Sigma-Aldrich, except where
otherwise mentioned.
Antibodies: Anti-mouse PD-L1 (clone 10F.9G2) were
obtained from BioXcell (West Lebanon, NH). Antibodies for
flow cytometry, Western blotting and immunofluorescence
staining are listed in Tables S1 and S2 in Supplementary
Material.
Cell line: The 4T1luc cells were generously supplied by
Sichuan University (Sichuan, China). 4T1luc cell line stably
expresses the firefly luciferase gene, which was introduced via
lentiviral transduction.
Experimental animals: Mice (5–7 weeks old, female,
BALB/c) were acquired from Shanghai SLAC Laboratory
Animal Co., Ltd and bred at the Animal Experimental Research
Center of Zhejiang Chinese Medical University. All animal
studies were conducted in compliance with NIH guidelines for
laboratory research and were authorized by the Animal Care
Committee of Zhejiang Chinese Medical University. 6 × 105
4T1luc cells were injected orthotopically into the mammary
fat pad of BALB/c female mice. Treatment was initiated when
the 4T1luc tumor volume reached 100 mm3 in 8–10 d after
inoculation, the mice were randomly divided into 4 groups
(n = 5).
2.2. Synthesis of PolyMet
PolyMet (MW = 4,300 Da) was synthesized as previously
described [40,41]. Briefly, PEI (0.4 g) and dicyandiamide (4 g)
were initially mixed in 20 ml deionized water. Next, HCl (4 ml)
was added and oil bath (100 °C) for 4 h. Then the product was
4 Asian Journal of Pharmaceutical Sciences 18 (2023) 100796

Fig. 1 – Physicochemical characterization of LPPR. (A) Agarose gel electrophoresis of PolyMet-pRLN complex. Lane M, DNA
ladder; other lanes, PolyMet-pRLN complex prepared at different N/P ratios. (B) Size and surface charge of
PGA/PolyMet-pRLN at different ratios (1:1, 2:3, 3:2). (C) Size and surface charge of PGA/Lipid at various ratios (1:4, 1:8, 1:16,
1:24). (D) TEM image and size distribution of LPPR (scale bar, 50 nm). (E) Zeta potentials of LPPR. (F) Release profile of pRLN
from LPPR at various pH. Means ± SD (n = 3).

purified through ultrafiltration centrifuge tube (MW = 3500),
washed with deionized water for 2 times, and freeze-dried to
obtain PolyMet.
2.3. Preparation of LPPR
The pDNA (pRLN) complexation efficiency with PolyMet was
investigated by gel retardation assay with different N/P ratios.
PolyMet-pRLN complex were induced at various N/P from
2 to 8 by adding PolyMet with a particular concentration
into pRLN solution. The resulting PolyMet-pRLN complex
was incubated at 25 °C for 15 min. Agarose gels (1%, w/v)
were prepared containing ethidium bromide (1 mg/ml) in
TAE buffer. PolyMet-pRLN complex were softly blended with
loading dye bromophenol blue and xylenol (9:1, v/v) and added
to various wells of the agarose gel. The gel was resolved in TAE
buffer at 75 V for 20 min, imaged on a UV detector.
Next, PGA was added in distilled water with PolyMet-
pRLN complex (pRLN = 50 μg) in different molar ratios
(2:3, 1:1 and 3:2) to form the core of lipid nanoparticles.
Meanwhile, the lipid membrane of LPPR was prepared by
thin-layer dispersion. Briefly, DOTAP (20 mM, 1 ml) and
cholesterol (20 mM, 1 ml) were dissolved in chloroform,
and the solvent was eliminated by evaporation to produce
a homogeneous lipid membrane. The lipid membrane
was then hydrated and extruded sequentially through the
polycarbonate membrane to develop cationic liposomes
(10 mM) (Millipore, Billerica, MA). Finally, to identify the
optimum amount of liposomes coating on the core, various
molar ratios (1:4, 1:8, 1:16, 1:24) of PGA to DOTAP were
investigated.
2.4. Characterization of LPPR
The size and surface charge of LPPR were estimated by
ZetaSizer (Malvern ZetaSizer, Malvern, UK). The surface
morphology images of LPPR were acquired by transmission
electron microscopy (TEM, JEM-2100, JEOL, Japan). The Lipid
poly-γ -glutamic acid (PGA)/PolyMet nanoparticle (LPP), which
was LPPR without loading relaxin plasmids, and coumarin
6-labeled LPPR nanoparticles were prepared with the same
procedures as LPPR. The amount of pRLN released from LPPR
was confirmed with Hoechst 33,258 nucleic acid staining
(EX = 360 nm, Em = 460 nm) [48].
2.5. In vitro release of pRLN from LPPR
The in vitro release profile of pRLN from LPPR was carried out
by dialysis membrane method. In short, 2 ml LPPR solution
(0.3 mg pRLN) was put in a dialysis bag (MWCO = 3500) and
placed in 30 m release medium (pH 7.4 and pH 5.6), then
shaken at 75 rpm with the temperature of 37 ± 0.2 °C. One
milliliter of release medium at predetermined time points (0.5,
1, 2, 4, 8, 12, 24, 48, 72 h) were removed and supplemented with
an equal amount of fresh medium.
2.6. LPPR penetration by confocal laser scanning
microscopy in 4T1luc/CAFs tumor spheres
The CAFs and 4T1luc cell were mixed evenly at a ratio of 1:1
and placed in ultra-low adsorption 96 well cell plates (6.0 × 104
cells/well) to form an in vitro co-culture mixed tumor model.
The plates were cultured and photographed daily under an
 Asian Journal of Pharmaceutical Sciences 18 (2023) 100796 5

Fig. 2 – The effect of LPPR in penetration effect and proliferation of CAFs in 4T1luc /CAFs tumor spheres. (A) Intracellular
distribution and tracing of Coumarin 6-labled LPPR or LPP in 4T1luc /CAFs tumor spheres after 48 h of incubation. (B) Living
cell imaging of CAFs in 4T1luc /CAFs tumor spheres after LPP or LPPR treatment by CLSM (Scale bar=50 nm).

inverted fluorescent microscope to observe morphological
changes in the tumor spheres.After incubation for 48 h, the
4T1luc /CAFs tumor spheres were treated with coumarin 6-
labeled LPP and LPPR with the coumarin-6 concentration at
0.2 μg/ml. After incubation with LPP and LPPR for 48 h, the
tumor spheroids were washed by PBS and then immobilized
them with 4% paraformaldehyde (PFA). The cell nucleus was
dyed with DAPI (5 μg/ml). The tumor spheres were scanned
layer-by-layer in “Z-stack” mode by a CLSM (Zeiss, Germany)
to observe the penetration of Coumarin 6-labeled LPP and
LPPR in 4T1luc /CAFs tumor spheres (DAPI: Ex = 358 nm,
Em = 461 nm; Coumarin 6: Ex = 466 nm, Em = 504 nm).
2.7. Effect of LPPR on the proliferation of CAFs in
4T1luc/CAFs tumor spheres
CAFs were incubated with CMFDA (10 μm) for 25 min, and
replaced with fresh medium. The CAFs treated by CMFDA were
rinsed with PBS and imaged. Green fluorescence indicated
successful CMFDA staining. 4T1luc and CMFDA stained CAFs
were then used to prepare 4T1luc /CAFs tumor spheres. After
incubated with LPP and LPPR for 48 h, the tumor spheres were
stained with DAPI. The intracellular fluorescent pictures were
captured by a CLSM for three-dimensional imaging (CMFDA:
Ex = 492 nm, Em = 517 nm). Finally, the proliferation of CAFs in
6 Asian Journal of Pharmaceutical Sciences 18 (2023) 100796

Fig. 3 – LPPR in combination with aPD-L1 therapy on 4T1luc tumor model. (A) Combination regimen of LPPR in combination
with aPD-L1 for antitumor therapy. (B) Tumor growth curves of 4T1luc tumors. (C) The tumor weight. (D) The changes in
tumor luminescence intensity of tumor-bearing mice. (E) Relative BLIrel ratio curve after treatment with different drugs for
different times in vivo. (F) Changes in relative body weight of 4T1luc -bearing mice. (G) The tumor image of various
treatments after 14 d (H) H&E and TUNEL staining of treated 4T1luc tumors. (n = 5, ∗ P < 0.05, ∗∗ P < 0.01).

4T1luc /CAFs tumor spheres was evaluated by the fluorescence
intensity of CMFDA.
2.8. In vivo antitumor efficacy
Mice bearing 4T1luc allografts were randomly grouped and the
treatments were started on Day 7. Saline, aPD-L1 (5 mg/kg,
ip), LPPR (1.5 mg/kg pRLN, iv) and LPPR + aPD-L1(1.5 mg/kg
pRLN, iv + 5 mg/kg aPD-L1, ip) were administered. d-luciferin
(150 mg/kg) was intraperitoneally injected into anesthetized
mice, and the BLI was documented to evaluate the 4T1luc
growth by an IVIS imaging system every 3 d The body weight
and the tumor volume (Vt ) of mice were calculated every 2 d
using Eq. 1.
Vt =(A × B2 )/2 (1)
Where A was the longest and B was the shortest diameter
of the tumor.
The animals were sacrificed 18 d after tumor transplant,
the tumor, and major organs (heart, liver, spleen, lung, and
kidney) were excised for further H&E and TUNEL staining.
2.9. Immune status investigation
Infiltrating immune cells including CTLs (CD3+ CD4− CD8+ ), T
effector cell (Teff, CD3+ CD4+ CD8− ), Treg (CD3+ CD4+ Foxp3+ ),
MDSC (CD11b+ Gr1+ ), dendritic cell (DC, CD11c+ MHC class
Ⅱ+ ) and M2 macrophage (CD11b+ F4/80+ CD206+ ) in the TME
were profiled by flow cytometry (FCM). Single cell suspensions
acquired first by adding dissociation buffer (200 U/ml
collagenase I, 200 U/ml collagenase IV, and 100 μg/ml DNase
I) to tumors and incubating at 37 °C for 60 min in the shaker.
The single cell suspensions were stained with fluorescently
labeled antibodies for analyzing the expression of surface
markers in immune cells (BD FACS LyricTM Flow Cytometer).
The results were analyzed via CytExpert software.
 Asian Journal of Pharmaceutical Sciences 18 (2023) 100796 7

Fig. 4 – LPPR in combination with aPD-L1 treatment attenuates 4T1luc desmoplastic reaction. (A) Western blot analysis of
FAP-α, TGF-β, α-SMA and GAPDH expression in the 4T1luc tumor model after various therapy. (B) Protein expression levels
are normalized to GAPDH. RT-PCR analysis of (C) TGF-β, FGF2, PDGF-b and PDGF-c expression in the 4T1luc tumor model. (D)
Confocal microscopy is identified α-SMA and CD3+ in tumor tissues after different drug treatments. The quantitative results
of (E) α-SMA and CD3+ are shown as percent (%) of the total cell number. Scale bar represents 100 μm (n = 3, ∗ P < 0.05, ∗∗ P <
0.01 ∗∗∗ P < 0.001).

RT-PCR was carried out for measuring the amount of
interferon gamma (IFN-γ ), tumor necrosis factor-α (TNF-α),
Interleukin-4 (IL-4), Interleukin-10 (IL-10), TGF-β, fibroblast
growth factor 2 (FGF 2), platelet growth factor b and c
(PDGF-b and PDGF-c). The RNeasy R Microarray Tissue Mini
Kit was utilized to extract RNA from tumor tissue. cDNA
was synthesized and amplified via PCR. Glycerol-3-phosphate
dehydrogenase (GAPDH) was served as the housekeeping
control.
Tumor tissues collected from tumor-bearing mice were
immediately placed in 4% PFA for overnight fixation followed
by paraffin embedding. Afterwards, the slides were soaked
in 5% BSA for 15 min after dewaxing, antigen recovery and
permeabilization. The sections were incubated with anti-
CD3 and anti-alpha smooth muscle actin (α-SMA) primary
antibody overnight at 4 °C, followed by secondary antibody
for 1 h to label CD3 and α-SMA, which were the markers of
T cell and fibrosis in tumor respectively. The cell nuclei were
then stained with DAPI. Images were obtained using a VS120
S6 Slide scanner (Olympus, Japan).
2.10. Western blot
To assess the expression of protein in 4T1luc tumors, tumor
tissues collected from tumor-bearing mice were lysed using
radioimmunoprecipitation assay (RIPA) buffer (containing 1%
protease inhibitor and 1% phosphatase inhibitor), and protein
concentration was determined using a BCA assay. Samples
were diluted and heated at 95 °C for 6 min. Proteins were
separated using 10% SDS-polyacrylamide gel electrophoresis
and then transferred to polyvinylidene difluoride membranes.
Membranes were blocked with 5% nonfat dry milk in TBST
8 Asian Journal of Pharmaceutical Sciences 18 (2023) 100796

and then incubated with primary antibody overnight (4 °C).

Subsequently, the PVDF membranes were incubated with
secondary antibodies and the bands were detected using
the Pierce ECL Western Blotting substrate (Bio-Rad) for
chemiluminescent detection. ImageJ was utilized to quantify
the relative levels of protein expression with GAPDH as an
internal control.

2.11. In vivo safety evaluation

On Day 18 after tumor transplant, the serum was separated.

Creatinine (CREA), blood urea nitrogen (BUN), serum alanine
aminotransferase (ALT), and aspartate transaminase (AST)
were detected the hepatorenal functions. Major organs
consisting of heart, liver, lung, kidney, and spleen were
gathered, and immobilized for H&E staining to assess organ-
specific toxicity.

2.12. Statistical analysis

Statistical results were presented as mean ± SD. The statistical

significance was estimated by one-way ANOVA or two-tailed
Fig. 5 – LPPR in combination with aPD-L1 changed
t-test, where a P value < 0.05 was deemed statistically
cytokines in the TME using quantitative RT-PCR.
significant.
Intra-tumoral changes of (A) Th1 and (B) Th2 cytokines.
n = 3, ∗ P < 0.05, ∗∗ P < 0.01 ∗∗∗ P < 0.001.

Fig. 6 – LPPR in combination with aPD-L1 therapy remodeled the TIME on 4T1luc tumor model. (A-B) FCM analysis of CD8+
(gated on CD3+ ), (C) CD4+ T cells (gated on CD3+ ), (D) Treg, (E) Activated DCs, (F) MDSC and (G-H) M2-Macrophage in the
4T1luc tumors (n = 3). ∗ P < 0.05, ∗∗ P< 0.01, ∗∗∗ P < 0.001.
 Asian Journal of Pharmaceutical Sciences 18 (2023) 100796 9

of nanoparticles by damaging the CAFs in the tumor

3. Results and discussion sphere.

3.1. Preparation and of characterization LPPR

3.4. Antitumor efficacy of LPPR in combination with
aPD-L1 therapy in 4T1 bearing mice
PolyMet (MW = 4,300 Da) was synthesized via a one-step
reaction (Fig. S1A) [40,41]. Since the fully protonated state of
Having demonstrated that LPPR was able to damaging the
guanidine at physiological pH, the highly positively charged
CAFs in vitro, we next examined whether this tactic enhanced
PolyMet could be utilized to condensate pRLN. Electrophoresis
the tumor inhibitory effect of aPD-L1 in the TNBC. Firstly,
assays (Fig. 1A) suggested PolyMet thoroughly condense pRLN
orthotopic 4T1luc bearing mice were treated in accordance
at a mass ratio above 4:1. PGA with PolyMet-pRLN in a ratio
with the timeline illustrated in Fig. 3A. As compared with PBS
of 3:2 resulted in the smallest core (173.6 ± 0.3 nm) with
group, monotherapies of LPPR exhibited a restricted efficacy
a zeta potential of −45.5 ± 2.0 mV (Fig. 1B). The molar
in inhibiting the growth of 4T1luc tumors. Unsurprisingly, 4T1
ratio of PGA to DOTAP was selected at 1ࢼ16 to provide an
tumors with low levels of immunogenicity and strong levels
appropriate positive charge and a favorable size to maintain
of immunosuppression did not react to αPD-L1. Notably, the
its stability in vivo (Fig. 1C). The average particle size of
combination of LPPR with aPD-L1 showed improved antitumor
LPPR was 205.5 ± 2.9 nm. TEM images showed the spherical
properties, suggesting sufficient capacity to sensitize TNBC to
morphology of LPPR (Fig. 1D). An increase in zeta potential
aPD-L1 in combination with LPPR (Fig. 3B and 3C). No body
from −45.5 ± 2.0 mV for the PGA/(PolyMet-pRLN) complex to
weight loss was detected in the treatment period, supporting
+55.4 ± 1.6 mV for LPPR indicated successful coating of the
the security of the combination therapy (Fig. 3D). In vivo BLI
cationic lipid bilayer on the core (Fig. 1E). The stabilization of
imaging and quantification analysis of 4T1luc -bearing mouse
LPPR in a serum-containing medium was studied. The particle
receiving therapies also demonstrated that LPPR + aPD-L1
size of LPPR was maintained at 215 ± 10 nm and the PDI was
group could prevent tumor progression during the period of
always between 0.1–0.2 over 48 h (Fig. S2). From these data, it
treatment in accordance with the tumor images (Fig. 3E–3G).
could be concluded that LPPR has excellent physical stability
A further assessment of the antitumor efficacy of LPPR was
in serum-containing media.
performed by using H&E and TUNEL staining to study the
apoptosis of 4T1luc cells. All groups displayed various levels
3.2. In vitro release of pRLN from LPPR
of tumor inhibition, with LPPR + aPD-L1 generated maximal
tumor apoptosis (Fig. 3H).
To estimate the DNA plasmid release profile of LPPR in vitro,
pRLN in LPPR was released in PBS at pH 7.4 and pH 5.6. The
3.5. LPPR reshaped the stromal microenvironment
pRLN released from LPPR showed an initial slow release at
first, followed by a continuous fast release step, and finally
Accumulating experimental evidence demonstrated
reached a stable release. The initial slow release was lasted for
the importance of intra-tumoral T-cell infiltration for
8 h, with approximately 10% and 15% of pRLN released from
the antitumor efficacy of aPD-L1 immunotherapy [52].
LPPR at pH 7.4 and 5.6. The continuous fast release profile of
Unfortunately, CAFs engage in tumor-tumor biologic
LPPR until 48 h reached a steady state, in which nearly 35%
interactions by secreting various cell factors and ECM
and 60% of pRLN release at pH 7.4 and 5.6. At last, a negligible
proteins, such as α-SMA and FAP-α to facilitate tumor
pRLN release was observed after 48 h. These results showed
proliferation and limit penetration of T cells [53,54]. Moreover,
that pRLN release from LPPR in vitro demonstrated a sustained
TGF-β was the most potent and ubiquitous profibrogenic
release profile.
cytokine promoting CAFs activation and ECM deposition [11].
Therefore, after three consecutive dosing regimens, tumors
3.3. LPPR decreased the proliferation of CAFs and thus
were analyzed by western blot to assess α-SMA, FAP-α, and
facilitated its penetration into 4T1luc/CAFs tumor spheres
TGF-β (Fig. 4A). All pRLN-containing treatments (LPPR group
and LPPR + aPD-L1 group) clearly reduced TGF-β, FAP-α
The in vitro 4T1luc /CAFs tumor spheres preserved a matrix and
and α-SMA, signaling the damage of CAFs. LPPR + aPD-L1
three-dimensional (3D) structure resembling the TME in vivo
achieved a reduction in α-SMA (>50%), FAP (>40%) and TGF-β
[49,50]. CAFs, as the predominant stromal cells in TME, play
(>30%) compared with PBS (Fig. 4B). RT-PCR demonstrated
a vital role in the restricted penetration of drug in the tumor
that TGF-β was reduced 2.7-fold by LPPR + aPD-L1 treatment
tissue [26,51]. Here, the in vitro CAFs proliferation caused by
(P<0.001). Other pro-fibroblastic growth cytokines which
LPPR was evaluated by the fluorescence intensity of CMFDA. It
participate in the facilitation of collagen synthesis and
was found that LPPR could damage CAFs more efficiency than
suppression of extracellular matrix degradation such as FGF
LPP (Fig. 2B).
2, PDGF-b and PDGF-c [55], also displayed markedly reduced
To observe the penetration ability of LPPR after acting
levels in comparison to the PBS group (Fig. 4C). Likewise,
on CAFs, Coumarin 6 was used as a fluorescent probe in
the LPPR group markedly alleviated the tumor inhibition by
the encapsulated liposomes. The results demonstrated that
significantly diminishing fibrosis (α-SMA expression) and
Coumarin 6-labeled LPP was located on the border of the
recruiting CD3+ T cells in IHC (Fig. 4D–4E). These results
tumor, while coumarin 6-labeled LPPR could penetrate deeper
suggested that the combination of LPPR with αPD-L1 could
regions of the tumor (Fig. 2A). All these results demonstrated
diminish the stromal barrier within TNBC due to the presence
that pRLN could effectively increase the penetration
of LPPR.
10 Asian Journal of Pharmaceutical Sciences 18 (2023) 100796
Fig. 7 – Toxicity evaluation for LPPR in combination with aPD-L1 therapy. (A) Serum ALT, (B) AST, (C) BUN, and (D) CREA
levels. (E) H&E staining of major organs after different treatments. Scale bar = 200 μm, n = 3.
3.6. LPPR remodels the immune status to improve
aPD-L1 therapy in 4T1 bearing mice
As we demonstrated LPPR have impact on damaging CAFs,
the relationship between therapy and immune response
was evaluated further. Firstly, we detected the variations
in immune cytokines in the TME. We discovered elevated
relative mRNA expression of TNF-α and IFN-γ after treatment
with LPPR + aPD-L1 (Fig. 5A) [56]. In contrast, the RLN gene
therapy substantially downregulated T helper cell 2 (Th2)
cytokine signal including IL-4 and IL-10 (Fig. 5B). Notably, IL-
4 has a profibrotic function by promoting the differentiation
of quiescent resident fibroblast to myofibroblast [57]. Upon
stimulation with IL-4 and IL-10, the recruited myeloid cells
will differentiate toward immunosuppressive phenotypes
(DC, M2 macrophage, and MDSC, etc.) [58]. Together, these
immunosuppressive factors block T cell proliferation and
differentiation, thereby provoke the death of functional CTLs
and facilitate tumor cell evasion from immune surveillance
[59].
T-infiltrating lymphocytes, especially CD8+ cytotoxic T
cell-mediated cellular immunity is the primary mechanism
of antitumor immunity [60]. As expected, deactivation of
CAFs by LPPR significantly reduced intra-tumoral IL-4 and IL-
10 (Fig. 5B), which resulted in the increased infiltration of
CD3+ (1.3-fold increase), CD8+ (2.2-fold increase) and CD4+ T
cells (1.5-fold increase) into TME compared to PBS (Figs. S3
and 6A–6C). Apart from directly impairing T cell function,
activated CAFs should be responsible for recruiting circulating
myeloid cells and Treg. A significantly reduced number of
immunosuppressive Treg was noted in the LPPR-treated group
(Fig. 6D). With the combination of LPPR and aPD-L1, the
number of Treg was reduced from 7.96% in PBS group to 4.03%.
We further investigated the DCs population in TME by FCM.
Activated DCs were significantly higher in each treatment
group compared to the PBS, and the combination group
displayed the largest population of DC cells (Fig. 6E). Upon the
stimulation by the suppressive Th2 cytokines (e.g., IL-4 and IL-
10), these infiltrated myeloid cells differentiated into MDSC
and M2 macrophages [61]. It has been shown that recruited
MDSC and M2 macrophages were negatively correlated with
the amount of CTLs in the TME, leading to poor prognosis
in TNBC (Fig. 6F–6H). These observations demonstrated the
capability of combined infiltration enhancement of CTLs by
LPPR and aPD-L1 in TNBC immunotherapy. Therefore, we
suggested LPPR, which apparently reversed the resistance
mechanism of aPD-L1 treatment by damaging the CAFs,
stimulating the anti-tumor immunity and unleashing the
ability of aPD-L1 to destroy tumors.
3.7. Biosafety evaluation
The safety of LPPR + aPD-L1 therapy was further investigated
in vivo. BALB/c mice orthotopically bearing 4T1luc were
administered with PBS, aPD-L1, LPPR, and LPPR + aPD-L1. No
obvious weight reductions were observed during the various
 Asian Journal of Pharmaceutical Sciences 18 (2023) 100796
treatments (Fig. 4E). Additionally, we gathered peripheral
blood for serum biochemical analysis. No abnormal variation
in serum ALT, AST, BUN, or CREA were detected in each
treatment group, indicating high liver and kidney safety of
the regimen (Fig. 7A-7D). Finally, H&E staining showed that no
histological abnormalities in the heart, liver, lungs, spleen, and
kidneys in any treatments (Fig. 7E). The above results indicated
through the whole treatment, no adverse hematological or
histological toxicity were observed from LPPR combined with
aPD-L1.
3.8. Discussion
Accumulating evidence shows that CAFs are key factors
in the restricted penetration of drug in the tumor tissue.
CAFs contribute to the immunosuppressive cells in TME by
secreting proinflammatory cytokines and chemokines [62,63].
Owing to the importance of intra-tumoral T-cell infiltration,
αPD-L1 could achieve the optimum antitumor efficacy.
Unfortunately, the stromal barrier and immunosuppressive
milieu limited penetration of T cells into the TME, which
led to unresponsiveness to αPD-L1 immunotherapy. Not
surprisingly, the TME containing CAFs drastically limits the
promises of effective immunotherapeutic and checkpoint
inhibitors for the treatment of TNBC. Accordingly, it is
appealing to design a multifunctional therapy that is capable
of exhausting CAFs and reshaping TME.
RLN has been shown to be efficacious in inhibiting or
reversing fibrosis in cardiac and renal fibrosis models by TGF-
β/pSmad2 signaling pathway [33]. But, concerns regarding
enzymatic degradation and rapid elimination are often raised
in its application. To overcome the drawbacks of RLN, the
plasmid DNA encoding RLN was used to locally express RLN
in the TME. But, how to effectively deliver pRLN to tumor
sites to remove CAFs has become a critical issue. In the
present study, we novelty introduced PolyMet, which our lab
formulated in previous studies, to deliver pRLN effectively
to form LPPR nanoparticles. The advantage of PolyMet was
multifold including the highly positively charged potential
and synthesize easily. Of note, there have been no studies
on developing carrier by PolyMet to delivery pDNA and
combination with aPD-L1 on TNBC.
In this study, we provided a therapeutic idea by LPPR that
loaded with antifibrotic pRLN was tested in murine TNBC.
The pRLN could damage CAFs and decrease the stromal
deposition to recruit CTLs. In an alternative perspective, the
anti-fibrosis therapy could broad the engagement between
αPD-L1 and tumor cells. Through the whole treatment, no
adverse hematological or histological toxicity were observed
from LPPR combined with αPD-L1. As a result, the combination
of checkpoint inhibitors with LPPR should enhance its efficacy
in TNBC.
4. Conclusion
The abnormally dense fibrosis network in TNBC formed a
therapeutic barrier that hinders penetration and antitumor
immunotherapy. In this study, we for the first time
11
demonstrated that delivery the plasmid encoding RLN
by PolyMet damaged CAFs and tumor growth as well as
potentiated the effect of aPD-L1 in TNBC. LPPR with a positive
charge in physiological conditions displayed increased
tumor penetrating efficacy and weaken proliferation of
CAFs in 4T1luc /CAFs tumor spheres to overcome the stromal
microenvironment in vitro. Additionally, LPPR markedly
enhanced the tumor immune microenvironment (TIME)
and therapeutic efficiency of aPD-L1 by promoting fibrosis
regression and CD8+ T cell infiltration in the mouse model of
TNBC, allowing the immunosuppressive environment shifted
to an immunostimulatory state. Furthermore, this LPPR-
mediated combined therapy with aPD-L1 displayed significant
anti-tumor potentials in vivo and excellent biosafety. Taken
together, we present LPPR as a combination strategy based
on antifibrotic and ICB therapy which can be used as a
powerful TME regulator to achieve enhanced antitumor
potential.
Conflicts of interest
The authors have declared that no competing interest exists.
Acknowledgments
We gratefully acknowledge the efforts Professor Leaf Huang
and his group in synthesizing PolyMet. We appreciate
the great experimental support from the Public Platform
of Medical Research Center, Academy of Chinese Medical
Science, Zhejiang Chinese Medical University. This work was
funded by the Medical and Health Science and Technology
Program of Zhejiang Province (2021KY813), the National
Natural Science Foundation of China (82174095), and the
National Natural Science Foundation of Zhejiang Province
(LZ22H290001).
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ajps.2023.100796.
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