Co-delivery of bufalin and nintedanib via albumin sub-microspheres for synergistic cancer therapy

Article abstract of DOI:10.1016/j.jconrel.2021.08.049

Albumin nanoparticles represent an approved anti-tumor drug delivery system. However, there is only one albumin nanoparticle product (paclitaxel-albumin nanoparticle) on the market. The application of albumin carriers is limited by the lack of universal preparation technology and insufficient targeting effect. Herein, we developed multifunctional albumin sub-microspheres prepared by coaxial-electrospray technology to co-delivery bufalin and nintedanib for tumor-targeted combination therapy. The biguanide and ursodeoxycholic acid dual-modified multifunctional albumin was synthesized to enhance the anti-tumor effect and tumor target efficiency. Coaxial-electrospray technology was utilized in preparing albumin sub-microspheres with a core-shell structure that enables payload efficiency and stability. More importantly, the in vitro and in vivo experiments demonstrated that the multifunctional albumin sub-microspheres possessed superior tumor target efficiency. Furthermore, nintedanib and bufalin combined therapy relieved the tumor microenvironment and exerted a synergistic therapeutic effect. Therefore, this work provides a novel method for fabricating an albumin-based drug delivery system and a potential efficient combination therapeutic strategy for tumor treatment.

Full text of DOI:10.1016/j.jconrel.2021.08.049

Journal of Controlled Release 338 (2021) 705–718
Contents lists available at ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
Co-delivery of bufalin and nintedanib via albumin sub-microspheres for
synergistic cancer therapy
,
,
,*
Ying Xua *, Yulong Liua, Qi Liub, Shengzhe Lua c, Xiaolin Chena, Wenrong Xud, Feng Shia
a College of Pharmacy, Jiangsu University, Zhenjiang 212013, China
b Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
c Chia Tai Tianqing Pharmaceutical Group Co., Ltd, Lianyungang, Jiangsu 222062, China
d Jiangsu Key Laboratory of Medical Science and Laboratory Medicine, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Albumin nanoparticles represent an approved anti-tumor drug delivery system. However, there is only one al-
bumin nanoparticle product (paclitaxel-albumin nanoparticle) on the market. The application of albumin carriers
is limited by the lack of universal preparation technology and insufficient targeting effect. Herein, we developed
multifunctional albumin sub-microspheres prepared by coaxial-electrospray technology to co-delivery bufalin
and nintedanib for tumor-targeted combination therapy. The biguanide and ursodeoxycholic acid dual-modified
multifunctional albumin was synthesized to enhance the anti-tumor effect and tumor target efficiency. Coaxial-
electrospray technology was utilized in preparing albumin sub-microspheres with a core-shell structure that
enables payload efficiency and stability. More importantly, the in vitro and in vivo experiments demonstrated that
the multifunctional albumin sub-microspheres possessed superior tumor target efficiency. Furthermore, ninte-
danib and bufalin combined therapy relieved the tumor microenvironment and exerted a synergistic therapeutic
effect. Therefore, this work provides a novel method for fabricating an albumin-based drug delivery system and a
potential efficient combination therapeutic strategy for tumor treatment.
Multifunctional albumin sub-microspheres
Coaxial-electrospray technology
Bufalin
Nintedanib
Synergistic anti-tumor effect
Tumor microenvironment remodeling
1. Introduction
medicine, as well as induce the changes of the protein structure [4,5]. In
addition, the drug loading efficiency of most present preparations, such
Albumin nanoparticles have attracted wide interest as an anti-cancer
drug carrier system. As a kind of biomaterial, albumin has excellent
biocompatibility, non-immunogenicity, and in vivo stability [1,2].
What’s more, it is reported that albumin has a targeting effect, which is
mainly dependent on the affinity to gp60 (albondin) and secreted pro-
tein acidic and rich in cysteine (SPARC) on tumor vascular epithelium
and tumor cells, respectively [3]. The existing methods for preparing
albumin-based macro/nanocarriers, such as glutaraldehyde crosslinking
or high-pressure homogenization, have to use organic solvent or pro-
duce heat, which may affect the stability and activity of the incorporated
as nab-technology, is primarily affected by the drug properties and af-
finity with albumin. Until now, only one albumin-based nanoparticle
product (Abraxane®) has been approved [6]. What’s more, albumin
nanoparticles prepared by nab-technology indicated low stability and
poor drug blood retention because of their weak colloidal stability [7,8].
It is an urgent need to develop new technology for preparing albumin
macro/nanoparticles.
Electrostatic spray technology is a novel technology that could
fabricate nanofibers or micro/nanoparticles by conducting high voltage
from a metal capillary tip to induce a charge on the droplet’s surface. It
Abbreviations: BF, bufalin; BGA, p-biguanylbenzoic acid; C6, coumarin 6; ND, nintedanib; UA, ursodeoxycholic acid; BG-BSA, BGA modified albumin; UA-BSA, UA
modified albumin; BG-UA-BSA, UA and BGA modified albumin; C6-P-sMPs, C6 loaded native albumin sub-microspheres; C6-BP-sMPs, C6 loaded BGA modified
albumin sub-microspheres; C6-UP-sMPs, C6 loaded UA modified albumin sub-microspheres; C6-BUP-sMPs, C6 loaded dual modified albumin sub-microspheres; DiR-
P-sMPs, DiR loaded native albumin sub-microspheres; DiR-BUP-sMPs, DiR loaded dual modified albumin sub-microspheres; BF-BUP-sMPs, BF loaded dual modified
albumin sub-microspheres; ND-BUP-sMPs, ND loaded dual modified albumin sub-microspheres; BF-ND-P-sMPs, BF and ND co-loaded native albumin sub-micro-
spheres; BF-ND-BP-sMPs, BF and ND co-loaded BGA modified albumin sub-microspheres; BF-ND-UP-sMPs, BF and ND co-loaded UA modified albumin sub-micro-
spheres; BF-ND-BUP-sMPs, BF and ND co-loaded dual modified albumin sub-microspheres; BF-ND-FITC-BUP-sMPs, FITC labeled BF-ND-BUP-sMPs.
* Corresponding authors.
E-mail addresses: xying@ujs.edu.cn (Y. Xu), shifeng@ujs.edu.cn (F. Shi).
https://doi.org/10.1016/j.jconrel.2021.08.049
Received 12 March 2021; Received in revised form 20 August 2021; Accepted 30 August 2021
Available online 1 September 2021
0168-3659/© 2021 Published by Elsevier B.V.

Y. Xu et al.
Journal of Controlled Release 338 (2021) 705–718
is worth noting that the liquid solvent was vaporized as the particles
formed. In particular, the preparation process is a simple and quick one-
step technique of combining encapsulation and solidification and
avoiding heating [9,10]. Moreover, for the coaxial technology, the
organic solvent and the water solution could spray from the inner and
outer axial, respectively. For example, the albumin is dissolved in water
as the outer solution, while the hydrophobicity drug is dissolved in an
organic solvent as the inner solution. Then the outer and inner solutions
are then injected through a coaxial tube into a high voltage electric field
and ejected simultaneously. The contact time and area of the two phases
are very limited, which benefits the stability and activity of biological
medicine and materials. Besides, the coaxial technology can form micro/
nanoparticles with shell-core structure, therefore decreasing the drug’s
burst effect [11,12]. Currently, several researchers have focused on
preparing micro/nanoparticles for drug delivery by using electrostatic
spray technology. The commonly used materials included poly (lactic-
co-glycolic acid) (PLGA), polylactic acid (PLA), chitosan, and other
polymeric or polysaccharide materials. As a spherical protein form, al-
bumin is lacking viscoelastic properties, leading to poor spin-ability. In
that case, albumin is rarely used in electrostatic spray technology. Only
a few studies are focusing on electrospinning albumin fibers. To our best
knowledge, there are very few reports about applying coaxial-
electrostatic spray technology to prepare albumin micro/nanoparticles.
Cancer therapy has always been the focus of pharmaceutical research
[13–16]. Although some research progress has been made, the severe
side effects of chemotherapeutic agents cannot be ignored. Targeted
therapy is an excellent method to solve the above problems [17–19]. The
tumor target effect of nanoparticles solely depending on the passive
effect is insufficient [20]. Some researches focus on decorating active
targeting moiety on the nanoparticles to increase the targeting effect of
the vehicle. For example, bile acid can be taken up by hepatocytes and
has a targeting effect on the liver [21]. Moreover, the modification of
ursodeoxycholic acid (UA) can increase the targeting effect of the car-
riers to hepatoma cells [21,22]. In that case, UA decorated albumin
vehicle can be used for targeted hepatocellular carcinoma (HCC) ther-
apy. Metformin is an antihyperglycemic biguanides drug widely used in
the clinical treatment of type 2 diabetes. Recent researches have shown
that metformin exhibited potential anti-tumor activity [23–25] via
activation of adenosine 5‘monophosphate-activated protein kinase
pathway (AMPK, which plays a key role as a master regulator of cellular
energy homeostasis) [26], and inhibition of the mammalian target of
rapamycin (mTOR, which is a highly conserved serine/threonine kinase
that controls cell growth and metabolism) [27]. Moreover, biguanide-
rich transporters are proved to have the ability to mediate cell uptake
[28]. In this case, the biguanidinyl group (p-biguanylbenzoic acid, BGA)
was synthesized and decorated on the albumin as a functional group to
enhance the anti-tumor effect and cell uptake.
that ND is a promising candidate to remodel the TME.
Herein, we reported a UA and BGA dual-decorated albumin sub-
microsphere by using coaxial electrostatic spray technology, co-
delivery of BF and ND. The UA and BGA dual-decorated albumin was
synthesized to improve the target efficiency, as well as the anti-tumor
effect. Subsequently, the BF and ND co-loaded dual-decorated albumin
sub-microspheres were prepared by coaxial electrostatic spray technol-
ogy, and the physicochemical properties were characterized. The co-
axial electrostatic spray technology was applied to prepare drug-loading
albumin microspheres. The fabricated multifunction albumin carriers
exhibited excellent biocompatibility and promoted transcytosis of the
drug in tumor cells. Furthermore, the BF and ND combination therapy
with the multifunctional albumin microspheres facilitated targeted de-
livery, elicited the synergistic anti-tumor efficacy, and exhibited TME
regulation effect.
2. Materials and methods
2.1. Materials
Nintedanib (ND) was provided by Nanjing Aosaikang Pharmaceu-
tical Co., Ltd. Bufalin (BF) was purchased from Chengdu Desite Bio.
Dicyandiamide (DCD), p-aminobenzoic acid (PABA) and coumarin 6
were purchased from Aladdin Industrial Corporation. 1-(3-Dimethyla-
minopropyl)-3-ethyl carbodiimide hydrochloride (EDC, 98%), and N-
Hydroxysulfosuccinimide sodium salt (sulfo-NHS, 98%) were purchased
from Sigma-Aldrich. Polyvinylpyrrolidone (PVP) was purchased from
Huzhou Zhanwang Pharmaceutical Co., Ltd. DiIC18(7)1,1′-dio-
ctadecyltetramethylindotricarbocyanine Iodide (DiR) was purchased
from Bailingwei Technology Co., Ltd. All other chemicals, reagents, and
solvents were obtained from Sinopharm Group Chemical Reagent Co.,
Ltd. (Shanghai, China).
2.2. Synthesis and characterization of modified albumin
2.2.1. Synthesis and characterization of p-biguanylbenzoic acid
p-Biguanylbenzoic acid (BGA) was synthesized as references re-
ported with some modification [38,39]. The general synthesis was
shown in Scheme 1(A). Briefly, 0.80 g of dicyandiamide (DCD) was
added in a hydrochloric acid solution which contained 0.67 g of p-
aminobenzoic acid (PABA). The reaction has proceeded for 6 h at 80 ◦C
under stirring. After that, the reacted mixture was processed by
decompression-evaporation and recrystallization by acetone. Next, the
recrystallization product was dissolved in water, followed by adjusting
pH to 4.4 for precipitation. After vacuum drying, the white product of
BGA was obtained. The structure of BGA was characterized by Fourier
transform infrared spectrum (FTIR, Nicolet Nexus 470, Thermo Scien-
tific, USA), Nuclear magnetic resonance (1H NMR, AVANCEII 400 MHz,
BRUKER, Switzerland), and Mass spectrometer (MS, Thermo LXQ,
Thermo Scientific, USA).
Bufalin (BF), an anti-tumor monomer from Chinese medicine
Chansu, exhibits significant anti-tumor activities in many tumor cell
lines, which has an excellent ability to inhibit proliferation and invasion
of HCC [29–31]. However, its clinical application is limited by severe
side effects, especially the extremely cardiotoxicity [32]. Available
strategies for improving tumor targeting distribution and decrease the
cardiotoxicity of bufalin are strongly desired. On the other side, some
studies reveal that angiogenesis and tumor microenvironment (TME),
which impede the drug from entering deep into the tumor, play a crucial
role in HCC progress and lead to insufficient therapeutic efficacy. Nin-
tedanib (ND) is a small molecule inhibitor of multiple tyrosine kinases
that targets bind to the ATP-binding sites within the kinase domains not
2.2.2. Synthesis and characterization of modified albumin
Ursodeoxycholic acid (UA) modified albumin (UA-BSA) was pre-
pared by the carbodiimide method [40], as shown in Scheme 1(B). 13
mg of EDC and 10 mg of sulfo-NHS were added into 0.7 mL of anhydrous
DMF. 0.3 mL of tetrahydrofurans (contains 16 mg of UA) was then added
to it. And the reaction was proceeded at 0 ◦C for 4 h and then at 20 ◦C
overnight. After then, sulfo-NHS active ester of UA was added drop-wise
to 20 mL of 0.2 M NaHCO3 solution (pH 8.5) containing 1 mM albumin,
under stirring and continued for 8 h at room temperature. The reacted
mixture was transferred into dialysis and dialyzed against double
distilled water (DD water) for 48 h at room temperature, following by
centrifuging at 12,000 for 10 min. Finally, the white product of UA-BSA
was obtained after lyophilization. BGA modified albumin (BG-BSA) was
prepared as the same procedure except that UA was substituted by BGA
(9 mg).
only of VEGFR 1–3 and PDGFR α/β, but also FGFR 1–4 and c-Src [33,34].
ND can reverse epithelial-mesenchymal transition (EMT) in carcinoma
and exhibit vital antiangiogenic functions by directly affecting cell types
involved in angiogenesis, including endothelial cells, pericytes, and
smooth muscle cells [35,36]. The anti-cancer efficiency of ND on tar-
geting multiple signaling ways within the TME is presented via sup-
pressing tumor growth and metastasis [37]. These achievements suggest
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Y. Xu et al.
Journal of Controlled Release 338 (2021) 705–718
Scheme 1. Synthetic schematic of modified albumin. (A) Synthetic route of p-biguanylbenzoic acid (BGA). (B) Synthetic schematic of modified albumin.
BG-UA-BSA was prepared as follows: 70 mg of UA-BSA was dissolved
in NaHCO3 solution (pH 8.5), a certain amount of sulfo-NHS active ester
of BGA (9 mg) in DMF was added drop by drop under stirring and
continued for 8 h at room temperature. BG-UA-BSA was obtained after
centrifugation and dialysis to remove the unreacted reagents. Fluores-
cein isothiocyanate (FITC)-labeled BG-UA-BSA was also prepared for
core-shell structure observation.
2.2.3. Substitution degree of modified albumin
The substitution degrees of UA and BGA on UA-BSA were determined
by MALDI-TOF-MS (AB 5800, AB SCIEX, USA). Briefly, the molecular
weights of native and modified albumin (UA-BSA, BG-BSA, and BG-UA-
BSA) were measured by MALDI-TOF-MS, and the substitution degree
was calculated as the following equations,
Scheme 2. Diagrammatic sketch of coaxial electrostatic spray technology.
0.4 mm of inner axial and 1.2 mm of outer axial), then sprayed out
simultaneously under the voltage of 20 kV with the flow rate of 0.05
mm/min and 0.07 mm/min, respectively. The deposition distance was
23 cm, the temperature was 25 ◦C, with the humidity of 30%. The
powder of BF and ND co-loaded dual-modified albumin sub-
microspheres (BF-ND-BUP-sMPs) were obtained. The single drug-
loaded preparations (BF-BUP-sMPs and ND-BUP-sMPs) were prepared
as the same method except that only BF or ND was added into the inner
solution. Non-modified and single-modified albumin were also used to
prepare the BF-ND-P-sMPs, BF-ND-BP-sMPs, and BF-ND-UP-sMPs,
respectively. Coumarin 6 (C6) or DiR was used as the fluorescent
probe loading in the sub-microspheres for cell experiments or in vivo-
imaging, respectively.
ХBG1 = (MBGꢀ BSA—MBSA)/MBG
ХUA = (MUAꢀ BSA—MBSA)/MUA
ХBG2 = (MBGꢀ UAꢀ BSA—MUAꢀ BSA)/MBG
(1)
(2)
(3)
X
BG1 and XBG2 are the substitution degrees of BGA in BG-BSA and BG-
UA-BSA, respectively; XUA is the substitution degree of UA in UA-BSA;
M
BSA, MBG-BSA, MUA-BSA, and MBG-UA-BSA are the molecular weight of
native BSA, BG-BSA, UA-BSA, and BG-UA-BSA detected by MALDI-TOF-
MS, respectively; MBG and MUA are the molecular weight of BGA, and
UA, which are 203 and 374, respectively.
2.4. Characterization of modified albumin sub-microspheres
2.3. Preparation of bufalin and nintedanib co-loaded dual modified
albumin sub-microspheres by coaxial electrostatic spray technology
2.4.1. Morphology, core-shell structure, particle size, zeta potential, and
encapsulation efficiency
The morphology of BF-ND-BUP-sMPs was observed by scanning
electron microscope (SEM, S-4800 II FESEM, Hitachi High-
Technologies, Japan) and transmission electron microscopy (TEM,
Tecnai 12, Philips company, Holland). BF-ND-FITC-BUP-sMPs were
prepared by FITC-labeled BG-UA-BSA. The core-shell structure of BF-
ND-FITC-BUP-sMPs was detected by a laser scanning confocal micro-
scope (LSCM, TCS SP5 II, Leica, Germany). The particle size and zeta
potential of BF-ND-BUP-sMPs were analyzed using a dynamic light
BF and ND co-loaded dual modified albumin sub-microspheres were
prepared by coaxial electrostatic spray technology, as shown in Scheme
2. Briefly, an equal weight of ND and BF were dissolved in 10 mL PVP
ethanol solution (2 mg/mL) as the inner solution. BG-UA-BSA and
polyethylene oxide (PEO, 0.7% w/v) were dissolved in 10 mL pH 7.4 PBS
solution as the outer solution. The inner drug solution and outer carrier
solution were injected into coaxial spray system (with the diameter of
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Y. Xu et al.
Journal of Controlled Release 338 (2021) 705–718
scattering method (DLS, Mastersizer 3000, Malvern). The encapsulation
efficiencies of BF and ND in sub-microspheres were detected by ultra-
filtration centrifugation and high-performance liquid chromatography
(HPLC) method. Briefly, 0.5 mL of BF-ND-BUP-sMPs were placed in an
ultrafiltration centrifuge tube (molecular weight cutoff, MWCO, 100
kDa). After centrifugation at 10,000 rpm for 5 min under 4 ◦C, the free
drug was separated and then detected by HPLC method. The encapsu-
lation efficiency (EE) of BF or ND loaded in sub-microspheres was
calculated as the following equation:
BUP-sMPs.
The inhibitory effects of different formulations (BF-ND-P-sMPs, BF-
ND-BP-sMPs, BF-ND-UP-sMPs, and BF-ND-BUP-sMPs) on HepG2 cells
were also determined by MTT assay to evaluate the enhanced anti-tumor
effect of the target preparations.
2.6. In vitro cellular uptake
HepG2 cells were seeded in 6 well plate at a density of 1 × 106 cells/
well and preincubated for 24 h. After that, the culture medium was
replaced by different concentrations of C6 labeled preparations and
incubated for 2 h, included C6-P-sMPs, C6-BP-sMPs, C6-UP-sMPs, and
C6-BUP-sMPs (with 40 ng/mL of C6 concentration), respectively. Sub-
sequently, the cells were stained with 4′,6-diamidino-2-phenylindole
(DAPI) (KeyGen Biotech, China) and observed by fluorescence micro-
scopy (ECLIPSE Ti, Japan, Nikon eclipseTi-E).
ꢀ
)
/
EE% = Wt ꢀ Wf
× 100%
(4)
Wt
Where the Wt is the total dose of BF or ND (
μ
g); Wf is the free drug in
the filtrate (
μg).
2.4.2. X-ray diffraction analysis
The state of BF and ND entrapped in BF-ND-BUP-sMPs was charac-
terized using X-Ray Diffraction Analysis (XRD, D8 ADVANCE, BRUKER,
Germany). The analysis samples, including BF, ND, blank BUP-sMPs, the
mixture of BF with blank BUP-sMPs, the mixture of ND with blank BUP-
sMPs, and BF-ND-BUP-sMPs, were scanned at a range of 5◦ ~ 80◦.
2.7. In vivo biodistribution and targeting efficiency study
All the experimental protocols have followed the principles of lab-
oratory and animal care of the university. Jiangsu University Animal
Center (Zhenjiang, China) provided male ICR mice (20–25 g, 5 weeks).
H22 cells were purchased from Jiangsu KeyGEN BioTECH Co., Ltd.,
Nanjing, China). The tumor model was built via subcutaneous injection
of H22 cells (5 × 106 cells) into each mouse’s forelimb. When the tumor
volumes reached about 200 mm3, mice were divided randomly into
three groups and intravenously injected with DiR solution, DiR-P-sMPs,
2.4.3. In vitro drug release
In vitro release experiments of BF and ND from BF-ND-BUP-sMPs
were performed via the dialysis method. BF-ND mixed solution (solu-
bilized by 0.1% Tween 80) and BF-ND-BUP-sMPs (corresponding to 0.4
mg of BF and 0.55 mg of ND) were transferred into dialysis bags
(MWCO, 14000 Da) with or without serum and placed in 200 mL of pH
7.4 PBS medium (0.1% Tween 80) at 37 ± 0.5 ◦C and oscillating at 100
rpm. Samples (0.5 mL) were withdrawn at predetermined time points,
while the same volume of fresh medium was added. After centrifugation
at 10,000 rpm for 10 min, the drug content in the supernatant was
determined by the HPLC method.
and DiR-BUP-sMPs (at a dose of 200 μg DiR/kg body weight) via tail
vein, respectively. The in vivo DiR fluorescence images of mice were
taken under anesthesia at 2, 4, 8, and 24 h post-injection by an in vivo
imaging system (In vivo xtremII, Germany, Burker) to evaluate the bio-
distribution and target effect. After then, the mice were sacrificed, and
the major organs and tumors were harvested for ex vivo imaging and
quantitating.
2.5. In vitro cell cytotoxicity
2.8. In vivo synergy therapeutic effect
2.5.1. Cell cultures
HepG2 cell line was purchased from Jiangsu KeyGEN BioTECH Co.,
Ltd. (Nanjing, China), and was cultured in Dulbecco’s Modified Eagle’s
Medium-HG (DMEM-HG, HyClone) supplemented with fetal bovine
serum (FBS, 10% v/v; Gibco), penicillin (100 U/mL) and streptomycin
When average tumor volume reached to 100 mm3, H22 tumor-
bearing mice were randomly divided into eight groups (n = 5) and
intravenously injected with PBS (pH 7.4), BF-ND Solution, BF-BUP-
sMPs, ND-BUP-sMPs, BF-ND-P-sMPs, BF-ND-BP-sMPs, BF-ND-UP-sMPs,
and BF-ND-BUP-sMPs at a dose of 0.8 mg/kg of BF and/or 1.05 mg/kg
of ND, respectively. The administrations were started at day 0 and
repeated every day for 5 times, and the mice were weighed, and tumors
were measured with a vernier caliper every day. The tumor volumes
were calculated as V = a × b2/2, where a and b were the longest and
shortest diameter, respectively. Mice were sacrificed 24 h after the last
administration, and the tumors were harvested. Tumor tissues were
histologically evaluated with hematoxylin and eosin (H&E) staining and
terminal deoxynucleotidyltransferase-mediated nick end labeling
(TUNEL) assay, using a commercial apoptosis detection kit (Promega,
Madison, WI). The images were observed by fluorescence microscopy
(ECLIPSE Ti, Japan, Nikon eclipseTi-E), and the apoptotic index was
calculated.
(100 μg/mL). The cells were cultured in an incubator (Thermo Electron
Corporation) operating at 37 ◦C under 5% CO2.
2.5.2. Cytotoxicity evaluations
In vitro cytotoxicity evaluations were divided into three parts to
determine the biocompatibility of modified albumin, the synergistic
anti-tumor effect of BF and ND in preparations, and the enhanced anti-
tumor effect of the formulations. The cytotoxicities of modified albumin
and blank BUP-sMPs on HepG2 cells were briefly evaluated by the MTT
method. HepG2 cells were inoculated on 96 well plate at a density of 3 ×
104 cells/well and preincubated for 24 h. Then the culture medium was
replaced with different concentrations of native BSA, BG-BSA, UA-BSA,
and blank BUP-sMPs diluted in serum-free DMEM-HG medium, respec-
tively. After 24 h of incubation, the cell viability was detected by MTT
assay and measured with a microplate reader (800TS, Bio-Tek, USA) at
570 nm. Each point was performed in triplicate.
2.9. Immunohistochemical and immunofluorescence study
The cytotoxicity of BF-BUP-sMPs, ND-BUP-sMPs, and BF-ND-BUP-
sMPs on HepG2 cells was determined as the same method, and the
synergistic anti-tumor effect was evaluated by the combination index
(CI), which was calculated by the following equation:
Vessels and tumor-associated fibroblasts (TAF) were characterized
by platelet endothelial cell adhesion molecule-1 (CD31) and α-smooth
muscle Actin (α-SMA, Mouse-anti-alpha smooth muscle actin, Abcam,
England). Nuclei were counterstained with DAPI. The Masson Tri-
chrome assay was carried out to detect collagen in tumor tissues. To
quantify the fields, three randomly selected immunofluorescent or
immunohistochemical staining microscopic fields were quantitatively
analyzed using Image J.
CI = B/BX + N/NX
(5)
Where B and N represent the half inhibitory concentration (IC50) of
BF or ND in BF-ND-BUP-sMPs groups, respectively; BX represents the
IC50 of BF in BF-BUP-sMPs groups and Nx represent the IC50 of ND in ND-
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Y. Xu et al.
Journal of Controlled Release 338 (2021) 705–718
2.10. In vivo safety evaluation
cmꢀ 1) in DCD disappeared in the spectrum of BGA, while the absorption
peaks of C N (1647 cmꢀ 1) and aromatic ring δC-H (1584 and 1536
–
–
Major organs, including hearts, livers, spleens, lungs, and kidneys of
tumor-bearing mice treated with PBS, BF-ND solution, and BF-ND-BUP-
sMPs, respectively, were harvested and stained with H&E for histo-
pathological evaluation.
cmꢀ 1) and benzene (852 cmꢀ 1) were found in the spectrum of BGA. It
can be seen from 1H NMR spectra in Fig. 1(B), δ = 10.22 was the
–
characteristic peak of C N bond, the characteristic peak of a carboxyl
group (COOH) at δ = 12.52, while, δ = 2.50 and δ = 3.56 were the
characteristic peaks of deuterium DMSO and water solvent, respectively.
Mass spectrometry of Fig. 1(C) showed that the molecular weight of BGA
(containing H+) was 222, consistent with the molecular weight of the
compound (221). These results indicated that BGA was successfully
synthesized.
2.11. Statistical analysis
All the experiments were performed at least three times, and the data
were expressed as the mean ± standard deviation (SD). The ANOVA
followed by a post hoc Student–Newman–Keuls test (SPSS 13; SPSS Int.,
Chicago, IL, USA) was applied to compare the experimental groups and
the corresponding controls. Significant differences between or among
groups were acceptable at *P˂0.05, **P˂0.01, and ***P˂0.001.
3.1.2. Substitution degree of BGA and UA on modified albumin
According to Fig. 2, the molecular weight of native and modified
albumin detected by MALDI-TOF-MS were 66,486 of native albumin,
69,695 of UA-BSA, 74231of BG-BSA, and 75,613 of BG-UA-BSA,
respectively. The increase of molecular weight of modified albumin
indicated that UA or BGA was covalently coupled on the albumin suc-
cessfully. For the single modified albumin, the substitution degree of UA
on UA-BSA was 14.3%, while, that of BGA on BG-BSA was 63.6%. BG-
UA-BSA was the dual-modified albumin, in which BGA was covalently
bound on UA-BSA to obtain the final product, with the substitution
3. Result and discussion
3.1. Characterization of BG-BSA, UA-BSA, and BG-UA-BSA
3.1.1. FTIR, 1H NMR, and MS of BGA
–
–
As shown in Fig. 1(A), the absorption peak of C N (2207– 2162
–
Fig. 1. (A) FTIR spectra of DCD, PABA, and BGA (a. DCD; b. PABA; c. BGA). (B) 1H NMR spectra of BGA. (C) Mass spectra of BGA.
709

Y. Xu et al.
Journal of Controlled Release 338 (2021) 705–718
Fig. 2. Determination of molecular weight of (A) BSA, (B) UA-BSA, (C) BG-BSA, and (D) BG-UA-BSA by MALDI-TOF-MS.
degree of BGA was 48.6%. Protein has abundant amino acid residues,
such as hydroxyl, sulfhydryl, amino, and imidazole. There are about 60
free lysine amino groups in a BSA molecule [41]. In the previous ex-
periments, we found that too high a degree of modification would affect
the solubility of the albumin; the substitution degree should be no more
than 70%.
technology has universal applicability and advantage in albumin nano/
microsphere preparation.
3.2.2. Characterization of BF-ND-BUP-sMPs
As shown in Fig. 3(A), BF-ND-BUP-sMPs exhibited a spherical
morphology with good dispersion and smooth appearance, no obvious
adhesion was observed. TEM was also used to visualize the morphology
of BF-ND-BUP-sMPs. As shown in Fig. 3(B), small particles were wrap-
ped in microparticles. The particle size of BF-ND-BUP-sMPs detected by
DLS was 879 ± 56 nm, and the polydispersity index (PDI) was 0.16. The
zeta potential was ꢀ 5.86 ± 1.12 mV. The DLS results are consistent with
that of SEM and TEM.
3.2. Preparation and characterization of BF-ND-BUP-sMPs
3.2.1. Preparation of BF-ND-BUP-sMPs
The modified albumin water solution was sprayed by outer coaxial,
while the drug/PVP organic solution was sprayed by inner coaxial
simultaneously. Therefore, albumin-based sub-MPs with core-shell
structure were prepared by coaxial electrostatic spray technology.
Compared with conventional preparation methods, electrostatic spray
technology was simpler, convenient, moderate, processing in one step
without heating. However, electrospun technology was only used in
albumin nanofibers production before [42]. There was limited research
on albumin micro/nanoparticles fabrication basing electrostatic spray
technology due to the poor spin-ability of the spherical form of the
protein. Furthermore, the water-soluble albumin is not stable in the
organic solvent; however, the most antineoplastic drugs are lip-
ophilicity. In that case, searching for a solvent to co-dissolve the drug
and albumin is a big challenge in traditional preparation methods, as
well as by single-axis electrostatic spray technology. Our team innova-
tively- apply coaxial electrostatic spray technology on albumin micro-
particle preparation, in which the spin-ability is improved by adding a
specific ratio of PEO. The albumin and the drug are dissolved in the
water or organic solvent, respectively, without needing to be concerned
about the difficulty of solvent selection. Coaxial electrostatic spray
The green fluorescent ring of FITC labeled albumin and the black
core could be observed clearly from the LSCM photo (Fig. 3(C)), which
indicated that the drug-albumin sub-microspheres with core-shell
structure were prepared successfully through coaxial electrostatic
spray technology.
The BF-ND-BUP-sMPs held a similar high EE with 78.0% of BF and
76.2% of ND, respectively. Hence, the properties of the drug have no
significant effect on loading efficiency in the vehicles prepared by co-
axial electrostatic spray technology. This preparation method exhibited
general applicability to drugs, indicating that the fabricated albumin
vehicle could be used as a multi-drug platform.
As shown in Fig. 3(D), the XRD spectrums of BF and ND had a
characteristic diffraction peak around 10◦ - 30◦, while there was no
obvious crystal peak in the spectrum of blank albumin nanoparticles.
The characteristic diffraction peak of the BF and ND could still be
observed in the XRD spectrum of the physical mixture of BF and ND with
albumin sMPs. However, the characteristic diffraction peak of BF or ND
disappeared in BF-ND-BUP-sMPs, which indicated that the drug was
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Journal of Controlled Release 338 (2021) 705–718
Fig. 3. (A) SEM of BF-ND-BUP-sMPs; (B) TEM of BF-ND-BUP-sMPs; (C) LSCM of BF-ND-FITC-BUP-sMPs for core-shell structure observation. (D) X-ray diffraction
pattern of BF, ND, blank BUP-sMPs, the physic mixture of BF and blank BUP-sMPs, the physic mixture of ND and blank BUP-sMPs, and BF-ND-BUP-sMPs. (E) In vitro
drug release curve of BF from solution or BF-ND-BUP-sMPs with or without serum. (F) ND from solution and BF-ND-BUP-sMPs with or without serum.
encapsulated in albumin nanoparticles in an amorphous state.
and delivery ability to enhance the drug action. The cell cytotoxicity of
modified albumin was detected using MTT assay, and the results were
shown in Fig. 4(A). Native BSA and UA-BSA revealed negligible cyto-
3.2.3. In vitro drug release
The in vitro release profiles of BF and ND from the preparations in pH
7.4 PBS medium (0.1% Tween 80) with or without serum were exam-
ined and shown in Fig. 3 (E) and Fig. 3(F). Compared with the BF-ND
solution, the release of BF and ND from BF-ND-BUP-sMPs was much
slower. Besides, no obvious burst effect was observed in the release
curve of BF-ND-BUP-sMPs. The results demonstrated that BUP-sMPs had
the sustained release effect and avoided burst release of BF and ND from
the microspheres, which presumably attributed to the core-shell struc-
ture. In addition, the release of BF or ND from BUP-sMPs was rather slow
with the existence of serum which indicated the excellent stability of
albumin sub-microparticles.
toxicity on HepG2 cells at the concentration of 500 μg/mL, which
exhibited excellent biocompatibility. BG-BSA and blank BUP-MPs
showed a little effct on the cell growth at high concentraion. The re-
sults demonstrated that the modified albumin is safe, and BGA modifi-
cation may enable the anti-tumor potential.
3.4. In vitro evaluation of the synergistic anti-tumor effect
As shown in Fig. 4(B), BF, ND, BF-BUP-sMPs, and ND-BUP-sMPs
exhibited an obvious inhibition effect on HepG2 cells, which was posi-
tively proportional to the drug concentrations. Compared to free BF and
ND, BF-BUP-sMPs and ND-BUP-sMPs exerted stronger inhibition effects.
The IC50 of BF-BUP-sMPs and ND-BUP-sMPs were 4.54 ± 0.67 and 7.53
± 1.13 µmol/L, respectively, which were much lower than that of free BF
(14.03 ± 1.89 µmol/L) and free ND (22.70 ± 2.21 µmol/L), indicating
3.3. In vitro biocompatibility evaluation
Ideal vehicles need to have good biocompatibility to prove the safety
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Journal of Controlled Release 338 (2021) 705–718
Fig. 4. In vitro cytotoxicity evaluation. (A) In vitro cytotoxicity of native BSA, UA-BSA, BG-BSA, and blank BUP-sMPs on HepG2 cells at various concentrations.
(Points, mean ± SD, n = 3); (B) In vitro cytotoxicity and IC50 of BF, ND, BF-BUP-sMPs and ND-BUP-sMPs on HepG2 cells; (C) In vitro cytotoxicity of BF-ND-P-sMPs, BF-
ND-UP-sMPs, BF-ND-BP-sMPs, BF-ND-BUP-sMPs groups on HepG2 cells; (D) IC50 of BF and ND in different formulations on HepG2 cells. (Points, mean ± SD, n = 3).
(E) Representative fluorescence microscopy images of HepG2 cells after incubation with C6-P-sMPs, C6-BP-sMPs, C6-UP-sMPs, and C6-BUP-sMPs (40 ng/mL of C6
concentration) for 2 h.
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Journal of Controlled Release 338 (2021) 705–718
that the vehicles could enhance the drug action powerfully. While, as
shown in Fig. 4(D), the IC50 of BF and ND in the co-delivery formulation
(BF-ND-BUP-sMPs) were 0.51 and 0.72 µmol/L, respectively, which
were significantly lower than that of the single drug formulation (4.54
µmol/L of BF in BF-BUP-sMPs and 7.53 µmol/L of ND in ND-BUP-sMPs).
The CI of BF and ND in the compound formulation was 0.208. CI was a
commonly used index to evaluate the synergistic effect of the compound
preparation. As literature reported [43], CI < 1 suggested the two drugs
had a synergistic effect, CI > 1 suggested the two drugs had an antag-
onism effect, while CI = 1 suggested there was no interaction between
the two drugs. The CI of BF and ND in the compound formulation was far
less than 1, which indicated the synergistic effect of the two drugs in the
co-delivery vehicle.
effect of BGA, which was consistent with the literature reports [44,45].
The results indicated that BGA could synergistically enhance the anti-
tumor effect. Moreover, BUP-sMPs fabricated by dual-modified BSA
showed the strongest inhibitory effect on the growth of tumor cells,
demonstrating that the dual-modified albumin carrier could enhance the
anti-tumor effect through the targeted mediation of UA and the anti-
tumor effect of BGA.
3.5. Cellular uptake
The transcellular transportability and targeting efficiency of the
fabricated vehicles were evaluated on the HepG2 cells, as shown in
Fig. 4(E). Under fluorescence microscopy, only a small amount of C6-P-
sMPs is taken up by HepG2 cells. While the C6-BP-sMPs demonstrated
higher cellular uptake efficiency than C6-P-sMPs. It indicated that BGA
modification could enhance the uptake of the vehicle, presumably
attributed to the cell member affinity of the guanidine, which was
consistent with the literature report [28]. Moreover, the uptake of C6-
UP-sMPs was significantly stronger than that of C6-P-sMPs and C6-BP-
sMPs, which indicated that UA modification enhanced the tumor
cellular uptake through the bile acid-mediated transport effectively.
The cell inhibition effect of the BF and ND delivered by different
modification albumin was also evaluated by MTT assay (Fig. 4(C)). As
shown in Fig. 4 (D), the IC50 of BF and ND in different formulations
exerted a similar tendency, that was, BF-ND-P-sMPs > BF-ND-UP-sMPs
> BF-ND-BP-sMPs > BF-ND-BUP-sMPs. The results indicated that UP-
sMPs could enhance the cytotoxicity probably by UA mediated target
effect compared with P-sMPs. While, the BP-sMPs fabricated by BG-BSA
exhibited stronger cytotoxicity, mainly attributed to the antineoplastic
Fig. 5. (A)In vivo time-lapse NIR fluorescence images of the tumor-bearing mice treated with free DiR, DiR-P-sMPs, or DiR-BUP-sMPs; (B) Representative ex vivo
fluorescence images; (C) Quantitative analysis of fluorescence intensity in the tumor after i.v. injection with free DiR, DiR-P-sMPs, and DiR-BUP-sMPs, respectively,
***P < 0.001; (D) Quantitative analysis of fluorescence intensity in excised organs from tumor-bearing mice at 24 h post-injection. The data were presented as the
mean ± SD, ***P < 0.001.
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Notably, the C6-BUP-sMPs group showed the strongest fluorescence
intensities. The results illustrated that the dual-modified albumin sub-
microspheres efficiently promoted the tumor celluar uptake through
the guanidine-based membrane affinity and UA-mediated active
transport.
through gp60 and SPARC [46]. As shown in Fig. 5(C), the maximum
fluorescence intensities of DiR-P-sMPs and DiR-BUP-sMPs in tumor tis-
sue were observed at 4 h and 8 h post-injection, respectively. In
particular, DiR-BUP-sMPs exhibited the strongest fluorescence intensity
in tumors among the three groups. The fluorescence intensity of DiR-
BUP-sMPs was 1.46 and 1.71 times higher than that of DiR-P-sMPs at
4 h and 8 h post-injection in the tumor, respectively. These results
indicated that the decorating targeting ligand of UA facilitated the
accumulation of the vehicles in the tumor and possessed the excellent
tumor targeting effect.
3.6. In vivo biodistribution and targeting efficiency
Fig. 5 (A) showed the real-time fluorescence images of H22 tumor-
bearing mice after injection of DiR solution or DiR loaded sub-
microspheres. Comparing with DiR solution, despite decorating target-
ing ligands or not, DiR-loaded albumin sub-microspheres exhibited
much stronger and more durable fluorescence at tumor tissue. This
might be due to the enhanced albumin accumulation in the tumor
Consistent results were received from ex vivo fluorescence images
showed in Fig. 5(B) and (D). In excised tumor tissues, the fluorescence of
the DiR solution was extremely faint. Meanwhile, the DiR-BUP-sMPs
group possessed the strongest fluorescence intensity, indicating the
Fig. 6. In vivo antitumor studies. (A) Tumor volumes of mice treated with different preparations. (B) The tumor inhibition ratio of mice treated with different
preparations. (C) Ex vivo tumor images of mice treated with different preparations. (D) Micrographs of H&E stained tumor sections from different groups (×40). (E)
Apoptosis of tumor cells detected by TUNEL staining (×40). (F) The quantitative analysis of the TUNEL staining section by image J. The results are displayed as mean
± SD, *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group.
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Journal of Controlled Release 338 (2021) 705–718
tumor target effect of the dual-modified albumin sub-microspheres. It
was noted that even the free DiR exhibited relatively higher fluorescence
in the liver. We speculated that the liver was the major metabolic organ
of DiR, which lead to the high fluorescence [47]. Moreover, the BUP-
sMPs increased the fluorescence intensity not only in the tumor but
also in the liver. The following reasons may interpret this. First, the liver
is the main distribution and metabolism organ for nano-formulations
[48]. Second, the bile acid receptor is not only expressed in the tumor
but also the liver [21]. In that case, the UA increased the distribution of
the sub-MPs both in the liver and in the tumor through the bile acid
receptor-mediated active transport.
were: BF-ND-BUP-sMPs > BF-ND-BP-sMPs > BF-ND-UP-sMPs > BF-ND-
P-sMPs. Compared with native albumin carriers, single modified albu-
min particles (BF-ND-BP-sMPs and BF-ND-UP-sMPs) enhanced the anti-
tumor effect significantly. The enhancement of tumor inhibition effect of
BP-sMPs may be attributed to the synergistic anti-tumor effect of
biguanide, which consisted of the literature report [49] and in vitro cell
experiments results. While the UP-sMPs improved the tumor suppres-
sion rate through the UA-mediated tumor target effect and accumula-
tion. The dual-modified BUP-sMPs exhibited the strongest inhibition
effect by taking advantages of biguanide generated synergistic anti-
tumor effect and UA-mediated targeted effect.
On the other side, compared with the control group, all drug-treated
groups elicited a tumor suppression effect. The mice receiving mono-
therapy of BF albumin preparations elicited a significant anti-tumor
effect. While ND monotherapy also showed a certain degree of anti-
cancer effects. Furthermore, the combination of BF and ND albumin
sub-MPs exhibited the maximum tumor growth inhibition effect. The
tumor inhibitory rate of BF-BUP-sMPs, ND-BUP-sMPs, and BF-ND- BUP-
sMPs were 66.5%, 58.7%, and 84.2%, respectively. The results
demonstrated that the synergy achieved by BF-ND-BUP-sMPs leads to
the superior anti-tumor effect in the combined group. The in vivo anti-
tumor study confirmed that the combination therapeutic strategies
together with the dual-modified BUP-sMPs could make the most of the
3.7. In vivo inhibition of tumor growth
In vivo tumor inhibition studies were performed to further confirm
the targeting and therapy effect of the dual-modified albumin sub-
microspheres and combination therapeutic strategies. As shown in
Fig. 6(A), (B), and (C), despite decorating ligands or not, BF and ND co-
loaded albumin sub-microspheres exhibited a much stronger inhibition
effect on tumor growth compared to the BF and ND solution, which
indicated that the albumin sub-microspheres could deliver the drug to
tumor and enhance the anti-tumor effect. Furthermore, the orders of the
anti-tumor effect of the native and modified albumin sub-microspheres
Fig. 7. (A) Left and middle: immunofluorescent staining microscopic images of tumor sections stained with CD31-antibody or
stained with DAPI (blue), blood vessels were marked by CD31-antibody (green), and the TAFs in tumors were marked by -SMA antibody (green). Right: immu-
nohistochemical staining microscopic images of tumor sections stained with Masson’s Trichrome. The blue color represents collagen fibers. The scale bar was 20 m.
α-SMA antibody. The nuclei were
α
μ
Quantitative analysis was performed on (B) CD31, (C) α-SMA, and (D) collagen fibers by Image J. The results are displayed as mean ± S.D., *P < 0.05, **P < 0.01,
***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Journal of Controlled Release 338 (2021) 705–718
anti-tumor effect.
multifunction albumin carrier. The maximum antiangiogenic effect was
achieved from the combination of BF and ND in BUP-sMPs (Fig. 7(B)).
The results implied that the angiogenesis inhibitory effect was mainly
attributed to ND, which was one of the potential anti-tumor mechanisms
of the synergistic therapy strategy.
As shown in Fig. 6(E) and (F), the TUNEL staining results also indi-
cated that the combination treatment of BF and ND increased the
apoptosis of the tumor cells. What’s more, the tumor cell apoptosis of the
combination treatment was further enhanced by delivering the BUP-
sMPs, which indicated that this novel-designed vehicle could enlarge
the therapeutic effect efficiently.
The α-SMA, which was stained with green fluorescence in the second
column in Fig. 7(A), was the marker of TAFs. Similar to the CD31 ten-
dency in the tumor of mice treated with different formulations, ND-BUP-
sMPs significantly attenuated the α-SMA levels, which indicated that ND
3.8. Vasculature, tumor-associated fibroblasts (TAF), and collagen fibers
changes in the tumor microenvironment (TME)
had a strong inhibition effect on TAFs. Besides, the albumin sub-
microspheres significantly enhanced the anti-TAFs effect of ND.
Furthermore, BF-ND-BUP-sMPs exhibited the strongest inhibition effect
on TAFs (Fig. 7(C)).
Angiogenesis, TAF, and stroma, which are the important components
of the TME, play an oncogenic role in tumorigenesis and support tumor
cell survival, enhance cell migration and chemotherapy resistance. The
angiogenesis inhibition abilities of the monotherapy or combination
therapy groups were investigated by blood vessel marker (CD31). As
shown in Fig. 7(A), vessels stained with green fluorescence were abun-
dant and randomly distributed in the PBS group. This phenomenon is
consistent with the literature report [50]. Abnormal blood vessel dis-
tribution was mainly caused by the hypoxic tumor environment, which
would be the obstacle of the chemotherapeutic drugs to infiltrate into
the tumor mass. Both the combined drug solution group and the BF-BUP-
sMPs group exhibited weak antiangiogenic effects. While ND-BUP-sMPs
presented a much stronger angiogenesis inhibitory effect than the so-
lution group, which benefited from the targeted delivery effect of the
The collagen level, representing the stroma distribution in tumor
sections, is detected by Masson’s Trichrome staining [50]. As shown in
Fig. 7(A), collagen fibers were stained with blue color. The over-
expressed stroma structure revealed by collagen staining in the tumor
section was observed in the control group. In contrast, the collagen
levels were decreased in all of the drug-treated groups (Fig. 7(D)). ND-
BUP-sMPs group showed a stronger collagen inhibition effect than the
control group and BF-BUP-sMPs group, which indicated that ND could
decrease the stroma in the tumor effectively. It is worth noting that the
mice treated with BF-ND-BUP-sMPs resulted in the lowest collagen
content in the tumor section compared with other groups, illustrating
that BF-ND-BUP-sMPs have the strongest inhibitory effect on the
Fig. 8. Safety evaluations. (A) Body weight curves of tumor-bearing mice treated with different formulations. The results are displayed as (mean ± SD), n = 5. (B)
Representative histological images of the H&E-stained heart, liver, spleen, lung, and kidney sections (×40) from the tumor-bearing mice treated by PBS, BF-ND
solution, and BF-ND-BUP-sMPs, respectively.
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Taken together, the results indicated although the tumor growth
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This research was supported by China Postdoctoral Science Foun-
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Declaration of Competing Interest
The authors declare no conflicts of interest.
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