Lung cancer combination treatment: Evaluation of the synergistic effect of cisplatin prodrug, vinorelbine and retinoic acid when co-encapsulated in a multi-layered nano-platform

Article abstract of DOI:10.2147/DDDT.S251749

Purpose: Lung cancer remains the leading cancer-associated deaths worldwide. Cisplatin (CIS) was often used in combination with other drugs for the treatment of non-small cell lung cancer (NSCLC). Prodrug is an effective strategy to improve the efficiency of drugs and reduce the toxicity. The aim of this study was to prepare and characterize CIS prodrug, vinorelbine (VNR), and all-trans retinoic acid (ATRA) co-delivered multi-layered nano-platform, evaluating their antitumor activity in vitro and in vivo. Methods: Cisplatin prodrug (CISP) was synthesized. A multi-layered nano-platform contained CISP, VNR and ATRA were prepared and named CISP/VNR/ATRA MLNP. The physicochemical properties of CISP/VNR/ATRA MLNP were investigated. In vitro cytotoxicity against CIS-resistant NSCLC cells (A549/CIS cells) and Human normal lung epithelial cells (BEAS-2B cells) was investigated, and in vivo anti-tumor efficiency was evaluated on mice bearing A549/CIS cells xenografts. Results: CISP/VNR/ATRA MLNP were spherical particles with particle size and zeta potential of 158 nm and 12.3 mV. CISP/VNR/ATRA MLNP (81.36%) was uptake by cancer cells in vitro. CISP/VNR/ATRA MLNP could significantly inhibit the in vivo antitumor growth and suspended the tumor volume from 1440 mm3 to 220 mm3. Conclusion: It could be concluded that the CISP/VNR/ATRA MLNP may be used as a promising system for lung cancer combination treatment.

Full text of DOI:10.2147/DDDT.S251749

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Open Access Full Text Article
O R I G I N A L R E S E A R C H
Lung Cancer Combination Treatment: Evaluation of
the Synergistic Effect of Cisplatin Prodrug,
Vinorelbine and Retinoic Acid When
Co-Encapsulated in a Multi-Layered Nano-Platform
This article was published in the following Dove Press journal:
Drug Design, Development and Therapy
Zhen Liang1
Purpose: Lung cancer remains the leading cancer-associated deaths worldwide. Cisplatin
Juan Li2
(CIS) was often used in combination with other drugs for the treatment of non-small cell
Budong Zhu2
1Department of Thoracic Surgery I, Key
lung cancer (NSCLC). Prodrug is an effective strategy to improve the efficiency of drugs and
reduce the toxicity. The aim of this study was to prepare and characterize CIS prodrug,
vinorelbine (VNR), and all-trans retinoic acid (ATRA) co-delivered multi-layered nano-
Laboratory of Carcinogenesis and
platform, evaluating their antitumor activity in vitro and in vivo.
Translational Research (Ministry of
Education), Peking University Cancer
Hospital and Institute, Beijing, 100142,
People’s Republic of China; 2Key labora-
tory of Carcinogenesis and Translational
Research (Ministry of Education/Beijing),
Day Oncology Unit, Peking University
Cancer Hospital & Institute, Beijing
100142, People’s Republic of China
Methods: Cisplatin prodrug (CISP) was synthesized. A multi-layered nano-platform con-
tained CISP, VNR and ATRA were prepared and named CISP/VNR/ATRA MLNP. The
physicochemical properties of CISP/VNR/ATRA MLNP were investigated. In vitro cyto-
toxicity against CIS-resistant NSCLC cells (A549/CIS cells) and Human normal lung
epithelial cells (BEAS-2B cells) was investigated, and in vivo anti-tumor efficiency was
evaluated on mice bearing A549/CIS cells xenografts.
Results: CISP/VNR/ATRA MLNP were spherical particles with particle size and zeta
potential of 158 nm and 12.3 mV. CISP/VNR/ATRA MLNP (81.36%) was uptake by cancer
cells in vitro. CISP/VNR/ATRA MLNP could significantly inhibit the in vivo antitumor
growth and suspended the tumor volume from 1440 mm3 to 220 mm3.
Conclusion: It could be concluded that the CISP/VNR/ATRA MLNP may be used as
a promising system for lung cancer combination treatment.
Keywords: lung cancer, combination treatment, synergistic effect, cisplatin prodrug, multi-
layered nano-platform
Introduction
Lung cancer is the leading cause of cancer death worldwide.1 In 2019, one-quarter
of all cancer deaths are due to lung cancer in the USA.2 There are only 18% of all
patients with lung cancer live 5 years or more after diagnosis.3 Non-Small Cell
Lung Cancer (NSCLC), the most common type of lung cancer, accounts for more
than 84%.2,4 Based on the NCCN Guidelines, surgery, radiotherapy, and systemic
therapy are the three modalities most commonly used therapeutic regimens for
NSCLC patients. Among them, chemotherapy is recommended for patients with
stage IV (advanced) NSCLC and preoperative or postoperative chemotherapy for
patients with NSCLC at stage IB-IIIA, and all aforementioned patients account for
about 80% of NSCLC cases.4–7
Correspondence: Budong Zhu Key
laboratory of Carcinogenesis and
Translational Research (Ministry of
Education/Beijing), Day Oncology Unit,
Peking University Cancer Hospital &
Institute, No. 52 Fu-Cheng Road, Hai-
Dian District, Beijing 100142, People’s
Republic of China
Email zhubdbju@163.com
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http://doi.org/10.2147/DDDT.S251749

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Platinum-based chemotherapy (cisplatin, carboplatin, Factory (Beijing, China). Injectable soya lecithin (ISL)
and their combination regimens) is recommended and ben- was obtained from Nanjing Well Pharmaceutical Co., Ltd
(Beijing, China). Hyaluronic acid (HA, 300 kDa) was
kindly provided by Bloomage Biotechnology Co., Ltd
(Jinan, China). CIS, VNR, ATRA, EDC·HCl, NHS,
dimethyldioctadecylammoniumbromide (DDAB) were
obtained from Sigma-Aldrich China (Shanghai, China).
Dulbecco’s modified eagle’s medium (DMEM), fetal
bovine serum (FBS), and 3-(4,5-dimethyl-2-thiazolyl)-
2,5-diphenyl-2-H-tetrazolium bromide (MTT) were pur-
chased from Invitrogen Corporation (Carlsbad, CA).
eficial for patients with stage IV NSCLC and preoperative
and postoperative therapy in the NCCN guidelines.8–10 The
main mechanism of antitumor effect for cisplatin (CIS) is to
form CIS-genomic DNA interstrand and intrastrand cross-
links, and hinder transcription and/or DNA replication, then
lead to cell death. However, it is severe for the adverse
reactions of CIS including nephrotoxicity, myelosuppres-
sion, and some patients are not able to tolerate it.11,12
Therefore, various researchers have devoted to new strate-
gies to overcome these obstacles, including producing CIS
prodrug, using nanotechnology, applying combination ther-
apy, etc.13–15 The current standard combination chemother-
apy of most patients with NSCLC remains platinum-based
strategy containing vinorelbine (VNR), gemcitabine, pacli-
taxel, docetaxel, and pemetrexed.16
Synthesis of Cisplatin Prodrug
Cisplatin prodrug (CISP) was synthesized by conjugating
the amino group of tryptophan (TRP) with the carboxy-
lated CIS (Figure 1).29 Briefly, H2O2 was added into CIS
(300 mg, dissolved with DMSO) to form CIS-(OH)2, in
which succinic anhydride (SA, 100 mg) was added and
stirred for 10 h to obtain CIS-(COOH)2 (CIS-SA).
EDC·HCl (50 mg) and NHS (30 mg) were dissolved in
CIS-SA with stirring, in which TRP (50 mg, dissolved in
water) was added and stirred for 24 h. Finally, the solution
was dialyzed against water for another 24 h and lyophi-
lized to obtain CISP (CIS-SA-TRP). ESI-MS m/z calcu-
lated for [C15H22Cl2N4O6Pt]+: 619.06 [M + H]+; found:
Combinations of two or more therapeutic agents or
strategies have been explored for its unique advantages,
such as improved drug efficacy, reduced adverse reactions,
and multidrug resistance.17,18 All-trans retinoic acid
(ATRA), the biological active metabolites of vitamin A,
has been proven to have anticancer activity including lung
cancer, leukemias, head/neck cancer, etc.19–22 Recent stu-
dies have investigated combination therapy of ATRA and
CIS for lung cancer; results have shown that the combina-
tion brings enhancing therapeutic efficacy, and decreasing
nephrotoxicity and drug resistance.23–25 However, the low
water solubility of ATRA hinders its clinical application.
Nanocarriers have emerged as platforms for cancer ther-
apy, because they can offer advantages over free drugs
including improved intracellular penetration, enhanced dis-
tribution of drugs into a selected tissue, extended circulating
half-life and long shelf life.26 Multi-layered nanoparticle
platforms have been applied for combination drugs delivery
to tumors.27,28 In this study, a novel CIS prodrug (CISP)
was synthesized and a multi-layered nano-platform was
constructed for the delivery of CISP, VNR, and ATRA. In
vitro and in vivo performances were evaluated, summar-
ized, and discussed.
1
619.37. H-NMR analysis (DMSO-d6, 300 mHz, ppm):
peaks at 2.03 belong to CIS, the signals attributed to TRP
are at 3.06, 7.18, and 10.08, the formation of carboxy and
amino groups was confirmed by the peaks at 2.65, 2.41,
and 4.85, respectively.
Preparation of Multi-Layered
Nano-Platform
A multi-layered nano-platform contained CISP, VNR, and
ATRAwere prepared and named CISP/VNR/ATRA MLNP
(Figure 2). Firstly, CISP and VNR loaded inner core was
constructed by a solvent displacement technique.30 Briefly,
CISP (20 mg), VNR (20 mg), and PLGA (100 mg) was
dissolved in acetone (5 mL) and added drop-wise into
20 mL of water (contained 1% CBP, w/v) which was stirred
(300 rpm) at room temperature for 6 h to form solution-1.
Secondly, lipid mixed with ATRA as the middle layer was
produced by a nanoprecipitation technique.31 Briefly,
ATRA (20 mg), ISL (100 mg), and DDAB (1%, w/v)
were dissolved in acetone (5 mL), then added dropwise to
the solution-1 which was stirred (400 rpm) at room tem-
Materials and Methods
Materials
Poly (D, L-lactic-co-glycolic) (PLGA, 50:50, MW 10,000)
was provided by Beijing TopScience Bio-technology Co.,
Ltd (Beijing, China). Carbopol 940 (CBP, MW 1000,000)
was obtained from Shanghai Renmin Pharmaceutical perature for 6 h to get solution-2. Finally, positively charged
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Liang et al
Figure 1 Synthesis of cisplatin prodrug. Cisplatin prodrug (CISP) was synthesized by conjugating the amino group of tryptophan (TRP) with the carboxylated CIS.
Figure 2 A multi-layered nano-platform contained CISP, VNR and ATRA were prepared and named CISP/VNR/ATRA MLNP. The TEM image showed that the CISP/VNR/
ATRA MLNP was spherical particles with inner cores and coats on the outer surface.
HA (the surface layer) was coated onto the negatively nano-platform contained CISP, VNR and ATRA but do not
charged middle layer.32 HA (100 mg) was dissolved in
contain HA was prepared by the similar method without
water and added to solution-2 which was stirred (400 rpm)
at room temperature for 4 h to produce CISP/VNR/ATRA
MLNP.
using HA as the outer layer, named CISP/VNR/ATRA NP.
HA CISP, VNR and ATRA mixed solution were prepared
by dissolving the three drugs in water (contained 1%
Pluronic F68, w/v), named Free CISP/VNR/ATRA.
A multi-layered nano-platform contained CIS (not pro-
drug), VNR and ATRA were prepared by the similar
method using CIS instead of CISP, named CIS/VNR/
ATRA MLNP. A multi-layered nano-platform contained
CISP and VNR was prepared and by the similar method
without using ATRA, named CISP/VNR MLNP. Multi-
layered nano-platforms contained single CISP, VNR or
ATRA were prepared and by the similar method using
CISP, VNR or ATRA only (without other drugs), named
CISP MLNP, VNR MLNP, or ATRA MLNP. Blank multi-
layered nano-platform was prepared and by the similar
method without using any drugs, named Blank MLNP. A
Characterization of Multi-Layered
Nano-Platform
The morphology of the CISP/VNR/ATRA MLNP was
observed by transmission electron microscopy (TEM)
using an H-7650 transmission electron microscope
(Hitachi, Tokyo, Japan).33 Average particle size, polydisper-
sity index (PDI) and zeta potential of all the MLNPs were
determined by dynamic light scattering using a Zetasizer
Nano ZS90 (Malvern Instruments, Malvern, U.K.).34
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VNR/ATRA NP were added to A549/CIS cells which were
previously incubated in a 24-well plate (1 × 105 cells/well,
24 h). The medium containing the formulations was dis-
carded and the cells were washed three times with
D-Hank’s solution and photographed by fluorescence
microscopy. To quantify the uptake efficiency, cells were
analyzed by a flow cytometer (BD Biosciences, San
Jose, CA).
Drug Encapsulation and Loading Efficiency
Drugs were isolated from the MLNPs were measured by
redispersed the formulations in 0.1% Tween 80 and cen-
trifuged (20,000 rpm, 15 min) and the supernatant was
collected.35 The CIS content in MLNPs was determined
by atomic absorption spectroscopy.13 The VNR and ATRA
content were determined using HPLC at 269 nm and 340
nm.35,36 The drug encapsulation efficiency (EE) and load-
ing efficiency (LE) of drugs loaded MLNPs were calcu-
lated by equations: EE (%) = The amount of drugs
encapsulated in MLNPs/Total amount of drugs
added×100%; LE (%) = The amount of drugs encapsulated
in MLNPs/The amount of MLNPs×100%.
Cytotoxicity
The cytotoxicity of MLNP was evaluated on A549/CIS
and BEAS-2B cells by MTT assay.40 Briefly, all kinds of
MLNPs, CISP/VNR/ATRA NP and Free CISP/VNR/
ATRA at different concentrations were added to A549/
CIS or BEAS-2B cells which were previously incubated
in 96-well plates (3 × 104 cells/well, 24 h). After removal
of the medium, MTT solution (20 µL, 5 mg/mL in PBS)
was added to each well. DMSO (200 µL) was added 4
h later and the absorbance at 570 nm was recorded.
Serum Stability
The serum stability of MLNPs was evaluated in PBS
mixed with FBS (1:1, v/v).37 MLNP formulations were
incubated with 50% FBS (v/v) solution for 72 h. At 1, 2, 4,
8, 24, 48, and 72 h, 1 mL of formulation was taken out and
the particle size and PDI were measured by the same
methods described in the “Characterization of multi-
layered nano-platform” section.
Synergistic Effect
The synergistic effect of CISP/VNR/ATRA MLNP was eval-
uated by Chou-Talalay method.41 Combination index (CI) is
applied for the evaluation of synergy or antagonism, which is
n
In Vitro Drug Release
calculated by the equation: CI = ∑ðCÞi=ðCxÞi.42 (C)i: the
The release study was conducted using a dialysis
method.38 Briefly, MLNP formulations (2 mL each) were
sealed into separate dialysis bags, which were immersed
into PBS mixed with FBS (1:1, v/v) solutions (48 mL
each, 37°C) under shaking (100 rpm). Aliquots (2 mL)
were taken out from the solution periodically, and the
amount of drugs released from MLNP was analyzed by
the same methods described in the “Drug encapsulation
and loading efficiency” section.
i¼1
concentrations of drugs in the combination; (Cx)i: the con-
centrations of drugs used alone. CI < 1 indicated synergy,
closer to zero means higher synergy efficiency; CI > 1
represented antagonism.
Animals
BALB/c nude mice (6–8 weeks) were purchased from
Beijing Vital River Laboratory Animal Technology Co.,
Ltd (Beijing, China) and the mice are maintained and
treated in compliance with the guidance of the National
Institutes of Health for the care and use of laboratory
animals (NIH Publications No. 8023, revised 1978) and
approved by the Animal Care and Use Committee of
Peking University Cancer Hospital & Institute. A549/CIS
cells (1×107) were injected into the abdominal cavity of
mice to produce NSCLC xenograft mice.
Cells
The CIS-resistant NSCLC cells (A549/CIS cells) and
Human normal lung epithelial cells (BEAS-2B cells)
were obtained from American Type Culture Collection
(ATCC, Manassas, VA, USA) and cultured (37°C, 5%
CO2) in DMEM medium (10% FBS, 100 U/mL penicillin,
0.1 mg/mL streptomycin).
In Vivo Tumor Inhibition
NSCLC xenograft mice were randomized into 10 groups
Cellular Uptake
The uptake MLNP by A549/CIS cells was tested using (6 mice/group). CISP/VNR/ATRA MLNP, CIS/VNR/
coumarin 6 (C6) as an indicator.39 Briefly, C6 (40 mg) was ATRA MLNP, CISP/VNR MLNP, CISP MLNP, VNR
dissolved along with PLGA (in acetone) in the preparation MLNP, ATRA MLNP, CISP/VNR/ATRA NP, Free CISP/
section. C6 contained CISP/VNR/ATRA MLNP and CISP/ VNR/ATRA, Blank MLNP, and 0.9% normal saline (NS)
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was intravenously injected every 3 days at an equivalent BUN levels were assayed as indicators of renal and
dose of CISP, VNR, and/or ATRA (2 mg/kg).43 The tumor
hepatic function.
volumes (V) of mice were measured every 3 days and
calculated by the equation: V = length × width2/2. The
Statistical Analysis
tumor inhibition efficiency (TRE) was evaluated by the
The non-parametric data were analyzed using Friedman test
equation: TIE (%) = (Tumor weight of the mice treated
with NS group - Tumor weight of the mice treated with
formulations/Tumor weight of the mice treated with NS
group × 100. At the end of study (day 18), the macro-
scopic images of tumor tissues were presented.
(n=6). *P < 0.05 was considered statistically significant.
Results
Characterization of Multi-Layered
Nano-Platform
The TEM image showed that the CISP/VNR/ATRA
MLNP was spherical particles with inner cores and coats
on the outer surface (Figure 2). Particle sizes, PDIs, and
zeta potentials were summarized (Figure 3). The size, PDI,
and zeta potential of CISP/VNR/ATRA MLNP were about
158 nm, 0.11, and 12.3 mV. Similar results were found in
other MLNPs, which illustrated the loading of drugs did
not change the size and surface charge of the particles. The
size of CISP/VNR/ATRA NP (127 nm) was smaller than
CISP/VNR/ATRA MLNP, with a reversed surface charge
(−13.5 mV). The EE and LE of drugs loaded MLNP were
around 90% and 5%, respectively (Figure 4). The sizes
and PDIs of MLNPs remained unchanged during the
serum stability test, indicating the MLNPs may be stable
in the plasma when administrated in vivo.
In Vivo Tissue Distribution
After intravenously injected with CISP/VNR/ATRA
MLNP, CIS/VNR/ATRA MLNP, CISP/VNR/ATRA NP,
and Free CISP/VNR/ATRA as the “In vivo tumor inhibi-
tion” section, mice were sacrificed at 1 h and 48 h.44 Major
tissues (tumor, lung, heart, kidney, liver, and spleen) were
harvested, weighed, and dissolved in the concentrated
nitric acid by heating up and evaporated to dryness, and
then re-dissolved in 0.1 N HCl for tissue distribution
analysis by the same methods described in the “Drug
encapsulation and loading efficiency” section.
Systemic Toxicity
The systemic toxicity of the formulations were evaluated
by the changes of body weights and the variations of
alanine aminotransferase (ALT), lactate dehydrogenase
(LDH), and blood urea nitrogen (BUN).45 The body
weights of mice were measured every 3 days and
recorded. At the end of the study, blood was collected
In Vitro Drug Release
All the three drugs showed sustained release from MLNPs.
CISP and VNR exhibited similar release profiles, showing
complete release at 48 h (Figure 5). However, ATRA were
and centrifuged to obtain the serum. ALT, LDH, and released from MLNPs in different manners compared with
Figure 3 Particle sizes (A), PDIs (B), and zeta potentials (C) of the particles.
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Figure 4 The EE (A) and LE (B) of drugs loaded particles.
Figure 5 In vitro drug release profiles of CISP (A), VNR (B) and ATRA (C). All the three drugs showed sustained release from MLNPs. CISP and VNR exhibited similar
release profiles, but ATRA were released from MLNPs in different manners compared with CISP and VNR.
CISP and VNR. Faster release was observed by ATRA, it MLNP and CISP/VNR/ATRA NP (P < 0.05), while no
took 36 h to totally release from MLNPs. CISP/VNR/
ATRA MLNP showed slower drug release than that of
non-HA coating CISP/VNR/ATRA NP.
significant difference was found on BEAS-2B cells. The
cell viability was more remarkably inhibited by drugs loaded
MLNPs and CISP/VNR/ATRA NP formulations than Free
CISP/VNR/ATRA on A549/CIS cells (P < 0.01). However,
BEAS-2B cells viability differences among the formulas
were not that obvious. The synergistic cytotoxicity was
evaluated by CI values on the bases of IC50 values of
different formulations. The effect of three-drug combination
treatment was compared with the effect of treatment with
each drug alone. Figure 8 shows the CI values were <1 when
the fraction of affected cells at the intervals of 20%-80%. CI
values closer to 0 means higher synergy effects. More
remarkable combination effect was exhibited by CISP/
VNR/ATRA MLNP than that of CISP/VNR MLNP, indicat-
ing the better efficiency brought by ATRA.
Cellular Uptake
The cellular uptake efficiency of CISP/VNR/ATRA MLNP
and non-HA coating CISP/VNR/ATRA NP was compared
by the images and quantitative results (Figure 6). The
result clearly showed that more CISP/VNR/ATRA
MLNP (81.36%) were uptake by cancer cells in compar-
ison with CISP/VNR/ATRA NP (43.33%) (P < 0.01).
Remarkably higher uptake efficiency may be explained
by the HA coating that improved the uptake of the parti-
cles, which was going to be discussed later.
Synergistic Cytotoxicity
Concentration deepened manners were found in all drugs
contained formulations on both A549/CIS and BEAS-2B
cells (Figure 7). On A549/CIS cells, CISP/VNR/ATRA
In Vivo Tumor Inhibition
In vivo tumor inhibition ability of CISP/VNR/ATRA MLNP
was better than CISP/VNR/ATRA NP (P < 0.01), which
MLNP exhibited higher cytotoxicity than CIS/VNR/ATRA could be the evidence of the tumor-targeted ability of the
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Figure 6 The cellular uptake efficiency of CISP/VNR/ATRA MLNP and non HA coating CISP/VNR/ATRA NP were compared by the images and quantitative results.
multi-layered nano-platform with an HA coat (Figure 9). CISP/VNR/ATRA (Figure 10) (P < 0.05). In the meantime,
CISP/VNR/ATRA MLNP also exhibited higher tumor inhi-
bition efficiency than double drugs and single drug-loaded
MLNPs (P < 0.01), this may attribute to the synergy effects
of the three agents. Compare CISP/VNR/ATRA MLNP
with CIS/VNR/ATRA MLNP, more significant antitumor
efficacy was found by prodrug-based CISP/VNR/ATRA
MLNP (P < 0.05). Drugs loaded nanoparticles illustrated
stronger in vivo tumor inhibition effects than Free CISP/
VNR/ATRA (P < 0.05).
drugs distribution in kidney and heart was lower for MLNP
and CISP/VNR/ATRA NP formulations than the Free CISP/
VNR/ATRA (P < 0.05). These results may influence the
systemic toxicity of these systems when delivered in vivo.
Systemic Toxicity
No significant change of body weight was found on drugs
loaded nanoparticles groups (Figure 11A). However, free
CISP/VNR/ATRA exhibited obvious body weight lost,
which may indicate the systemic toxicity. ALT, LDH,
and BUN levels illustrated that drug-loaded nanoparticle
In Vivo Tissue Distribution
Higher tumor accumulation was found on MLNP formula-
tions in comparison with CISP/VNR/ATRA NP and Free groups did not change the parameters compared with
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Figure 7 In vitro cytotoxicity. Concentration deepened manners were found in all drugs contained formulations on both A549/CIS (A) and BEAS-2B cells (B). *P < 0.05.
et al, produced a cisplatin conjugated 1.2-distearoyl-sngly-
cero-3-phosphoethanolamine-N-mPEG3000 to reverse cispla-
tin resistance in ovarian cancer.47 As the start of the research,
a CIS prodrug: CIS-SA-TRP was synthesized using an amino
acid: tryptophan. Tryptophan is the precursor of serotonin, an
important neurotransmitter, and one of the essential amino
acids in human body.48 It could be used as nutrition supple-
ments and anticonvulsant. As a tranquilizer, it can regulate
mental rhythm and improve sleep.49 Tryptophan was used to
produce CIS prodrug here and the in vivo effects will be
discussed later.
A
multi-layered nano-platform CISP/VNR/ATRA
MLNP was constructed by CISP and VNR loaded poly-
meric core, ATRA contained middle layer, and HA-coated
outer layer. TEM image of CISP/VNR/ATRA MLNP was
spherical particles with inner cores and coats on the outer
surface. The size and zeta potential of CISP/VNR/ATRA
MLNP and other MLNPs are similar, which illustrated the
loading of drugs did not change the size and surface
charge of the particles.50 CISP/VNR/ATRA NP showed
a smaller size and reversed negative surface charge than
CISP/VNR/ATRA MLNP, indicating the HA coating
enlarged the size and bring negative surface charge.34
HA is reported to prevent unnecessary protein adsorption
and opsonization in the blood stream and also has targeting
ability to the CD44 overexpressing cancer cell line.51,52
The sizes and PDIs of MLNPs remained unchanged during
the serum stability test, indicating the MLNPs may have
good stability when administrated in vivo. This was in
accordance with the founding of Li et al, who concluded
Figure 8 The synergistic cytotoxicity was evaluated by CI values on the bases of
IC50 values of different formulations. The effect of three-drug combination treat-
ment was compared with the effect of treatment with each drug alone.
control groups, while free drugs caused significant
increase of these data (Figure 11B–D) (P < 0.05).
Discussion
In this study, CIS prodrug (CISP) was synthesized and used as
an antitumor agent, which was loaded in a multi-layered nano-
platform. CIS prodrugs were widely used to overcome the
main problems that limit the clinical use of cisplatin include
acquired resistance and serious side effects such as neurotoxi-
city and nephrotoxicity, what is closely related to the lack of
tumor selectivity of cisplatin.46 Examples included that Ling that no significant size changes means the proteins in the
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Figure 9 In vivo tumor inhibition ability of CISP/VNR/ATRA MLNP, CISP/VNR/ATRA NP and Free CISP/VNR/ATRA. *P < 0.05.
Figure 10 In vivo tissue distribution of CISP/VNR/ATRA MLNP, CISP/VNR/ATRA NP and Free CISP/VNR/ATRA. *P < 0.05.
serum did not adsorb on the nanoparticles and did not drugs in the presence of serum suggests that this multi-
cause aggregation, thus leading to increase in particle layered nano-platform may be more effective as an anti-
size.37
tumor agent with sustained release and excellent stability
The sustained release of drugs from nanocarriers may in the blood.55
be due to homogenous entrapment of the drugs, and the
The cellular uptake efficiency of CISP/VNR/ATRA
anticancer drugs could maintain continuous efficiency.53 In MLNP and non-HA coating CISP/VNR/ATRA NP was
the section, drugs showed sustained release from MLNPs. evaluated on A549/CIS cells, which have been reported
Faster release was observed by ATRA when compared to overexpress CD44 receptors on cell surface.56
with CISP and VNR, this may be explained by the differ- Remarkably higher uptake efficiency of CISP/VNR/
ent position of drugs in the multi-layered carriers. CISP ATRA than CISP/VNR/ATRA NP. This phenomenon
and VNR are encapsulated in the inner core, while ATRA clearly demonstrates the role of HA-CD44 interaction in
are loaded in the middle layer. The depth of drugs loaded terms of enhancing cellular uptake.57 HA-coated multi-
in the system can affect the release behavior.54 Drugs in layered nano-platform may penetrate high overexpressed
the outer layers may release more quickly than the inner CD44 cells (A549 cells) through a receptor-mediated
layers. So the effective and specific release manner of endocytosis.57 The penetration of drugs can be improved
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Figure 11 Drugs loaded nanoparticles groups presented no significant change of body weight (A). ALT (B), LDH (C), and BUN (D) levels illustrated that drugs loaded
nanoparticles groups did not change the parameters compared with control groups, while free drugs caused significant increase of these data. *P < 0.05.
by HA surface layer thus could reduce the drug dose, that of CISP/VNR MLNP, indicating the better efficiency
attenuate side effect, and overcome drug resistance.58
Combination therapy using nanocarriers containing
brought by ATRA.62
In vivo tumor inhibition ability of CISP/VNR/ATRA
CIS has received much interest in cancer therapy to over- MLNP was better tumor than CISP/VNR/ATRA NP,
come CIS-resistant.59 For example, Cheng et al developed which suggests the multi-layered nano-platform with an
a CIS and Curcumin co-loaded liposomal delivery system HA coat may increase the stability and the concentration
to deliver into hepatocellular carcinoma cells.60 CIS and of drugs in the blood and tumor.63 The results were sum-
Curcumin co-encapsulated lipid-polymer hybrid nanopar- marized by Liu L et al that multi-layered nano-platform
ticles were prepared by Li et al which showed and higher can also promote the apoptosis of lung cancer cells
cytotoxicity than free drugs.61 Our study evaluated three in vivo, thereby promoting subsequent apoptotic signaling
agents encapsulated in one multi-layered nano-platform. and cell apoptosis, which is consistent with that in vitro.64
The cell viability was more remarkably inhibited by CIS/ The in vivo distribution of drugs loaded in MLNPs fol-
VNR/ATRA MLNP than CISP/VNR/ATRA NP on A549/ lowed the order: tumor > liver > lung > spleen > kidney >
CIS cells but not BEAS-2B cells. These were in line with heart, which is in accordance with the conclusion drawn
the cellular uptake results that HA-coated multi-layered by Liu J et al.65 They argued that these results were
nano-platform may penetrate high overexpressed CD44 attributed to the efficient accumulation of MLNPs in the
cells (A549 cells) and exhibited more prominent cytotoxi- tumors through HA targeting to the CD44 receptor. Body
city. To further determine the synergistic cytotoxicity, CI weight, ALT, LDH, and BUN parameters of MLNPs
values were evaluated by on the bases of IC50 values of remained stable, which could be the evidence of the side
different formulations. More remarkable combination effects were successfully alleviated.45 Our future study
effect was exhibited by CISP/VNR/ATRA MLNP than will devote to the application of this nano-platform from
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Liang et al
11. Campbell AB, Kalman SM, Jacobs C. Plasma platinum levels: rela-
tionship to cisplatin dose and nephrotoxicity. Cancer Treat Rep.
1983;67(2):169–172.
12. Matsui K, Masuda N, Uchida Y, et al. Determinants of myelosuppres-
sion in the treatment of non-small cell lung cancer with
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bench to bedside. Our group has devoted to the design and
evaluation of these nano-platforms, which will be even-
tually used to treat cancer patients.
Conclusion
A multi-layered nano-platform CISP/VNR/ATRA MLNP was
prepared. It has layered morphology with particle size and zeta
potential of 158 nm, and 12.3 mV. As high as 81.36% of CISP/
VNR/ATRA MLNP was uptake by cancer cells in vitro. CISP/
VNR/ATRA MLNP could significantly inhibit the in vivo
antitumor growth and suspended the tumor volume from
1440 mm3 to 220 mm3. CISP/VNR/ATRA MLNP may be
used as a promising system for lung cancer combination
treatment.
16. Wang G, Wang Z, Li C, et al. RGD peptide-modified, paclitaxel
prodrug-based, dual-drugs loaded, and redox-sensitive lipid-polymer
nanoparticles for the enhanced lung cancer therapy. Biomed
Pharmacother. 2018;106:275–284. doi:10.1016/j.biopha.2018.06.137
17. Al-Attar T, Madihally SV. Recent advances in the combination deliv-
ery of drug for leukemia and other cancers. Expert Opin Drug Deliv.
2020;22:1–11.
Disclosure
The authors report no conflicts of interest for this work.
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