Platinum (II)-coordinated Portulaca oleracea polysaccharides as metal-drug based polymers for anticancer study

Article abstract of DOI:10.1016/j.colsurfb.2021.111628

Novel polysaccharide-platinum conjugated polymers bearing alendronate on Portulaca oleracea polysaccharides (PPS) were designed and synthesized. Their chemical structures and properties were characterized by Fourier transform infrared spectroscopy (FT-IR), ¹H NMR and ³¹P NMR spectroscopy, Thermogravimetric analysis (TGA), X-ray powder diffraction (XRD), UV–vis spectrophotometer (UV–vis) and other analysis methods. The results demonstrated that alendronate can be used as the linker of Portulaca oleracea polysaccharides and platinum compounds. Portulaca oleracea polysaccharides-alendronate (PPS-ALN) conjugates exhibited stronger antioxidant ability than PPS. The cytotoxicity assay to cancer cells was tested in vitro, and the Portulaca oleracea polysaccharides-alendronate-platinum (PPS-ALN-Pt) conjugates strongly inhibited the proliferation of cancer cells than PPS and PPS-ALN. The evaluation of complexes affinity toward supercoiled plasmid DNA, displayed a high DNA interaction. Interestingly, the platinum conjugates displayed immunological competence in HeLa cells by cellular immunofluorescence assay. Besides, the cellular platinum accumulation of PPS-ALN-Pt conjugates was higher than that of cisplatin in HeLa cells, implying that the polysaccharide-platinum conjugated polymers might have a synergistically therapeutic application in metal anticancer drug delivery.

Full text of DOI:10.1016/j.colsurfb.2021.111628

Colloids and Surfaces B: Biointerfaces 201 (2021) 111628
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces
journal homepage: www.elsevier.com/locate/colsurfb
Platinum (II)-coordinated Portulaca oleracea polysaccharides as metal-drug
based polymers for anticancer study
,
,
,
Qianqian Hana b, Lirong Huangc, Ying Wanga b, Shixin Suna *, Hao Huanga, Fei Lia,
Fangtian Wanga, Ligen Chend, Hongmei Zhanga *, Yanqing Wanga *
,
,
a Institute of Environmental Toxicology and Environmental Ecology, Yancheng Teachers University, Yancheng City, Jiangsu Province, 224051, People’s Republic of China
b Chemistry and Chemical Engineering, Nanjing University of Technology, Nanjing City, Jiangsu Province, 210009, People’s Republic of China
c Cardio-Thoracic Surgery, Yancheng First People’s Hospital, Yancheng, 224006, People’s Republic of China
d Department of Bioengineering School of Marine and Bioengineering, Yancheng Institute of Technology, Yancheng, 224054, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Novel polysaccharide-platinum conjugated polymers bearing alendronate on Portulaca oleracea polysaccharides
(PPS) were designed and synthesized. Their chemical structures and properties were characterized by Fourier
transform infrared spectroscopy (FT-IR), 1H NMR and 31P NMR spectroscopy, Thermogravimetric analysis (TGA),
X-ray powder diffraction (XRD), UV–vis spectrophotometer (UV–vis) and other analysis methods. The results
demonstrated that alendronate can be used as the linker of Portulaca oleracea polysaccharides and platinum
compounds. Portulaca oleracea polysaccharides-alendronate (PPS-ALN) conjugates exhibited stronger antioxidant
ability than PPS. The cytotoxicity assay to cancer cells was tested in vitro, and the Portulaca oleracea
polysaccharides-alendronate-platinum (PPS-ALN-Pt) conjugates strongly inhibited the proliferation of cancer
cells than PPS and PPS-ALN. The evaluation of complexes affinity toward supercoiled plasmid DNA, displayed a
high DNA interaction. Interestingly, the platinum conjugates displayed immunological competence in HeLa cells
by cellular immunofluorescence assay. Besides, the cellular platinum accumulation of PPS-ALN-Pt conjugates
was higher than that of cisplatin in HeLa cells, implying that the polysaccharide-platinum conjugated polymers
might have a synergistically therapeutic application in metal anticancer drug delivery.
Portulaca oleracea polysaccharides
Alendronate
Platinum-based conjugates
Antitumor activity
Immunocompetence
1. Introduction
method in the literature [11–13]. Polymer drug sustained-release sys-
tems have always attracted the attention of researchers and are at the
Cancer is one of the diseases that seriously threaten human health,
and has been intimately and considerably concerned by researchers.
Cisplatin is a kind of critical anticancer drugs, which has potent effects
on a variety of solid tumors [1,2]. However, the severe side effects of
cisplatin have limited its clinical application, which led to the devel-
opment of platinum-based alternative strategies [3,4]. Polymer-drug
delivery has been widely used and applied (e.g. hydrophobic drugs,
amphiphilic copolymers) [5,6]. Polymer-drug delivery systems can
enhance the transport efficiency of loaded drugs to solid tumors [7], as
the enhanced permeability and retention effect can prolong the blood
circulation and selectively distribute the delivered drugs in the organ-
ism, thereby reducing the side effects [8–10].
forefront of scientific and technological progress [14–16]. Poly-
saccharide biodegradable polymer composites are widely used to con-
trollably transmit and release of medicinal polysaccharides at specific
locations via spontaneous reactions according to the signals generated
under external conditions [17–19]. It possesses several advantages (e.g.
pH-responsive release, biocompatibility, targeting) compared with other
drug delivery methods, especially in the aspect of targeted drug de-
livery, and can release drugs according to the physiological environ-
ments [20–22].
Portulaca oleracea is an annual plant with outstanding medicinal and
nutritional values widely distributed in tropical-temperate regions [23].
There are multiple biological and active substances in Portulaca oleracea
after purification, including Portulaca oleracea polysaccharides (PPS)
which is one of the major bioactive components of the traditional Chi-
nese herb Portulaca oleracea. A kind of plant medicine of PPS apply for
Moreover, Pt-based anticancer drugs constitute polymer-drug de-
livery which can lessen the platinum-based antitumor drugs toxicity in
the process of delivery to healthy organs and prove the feasibility of this
* Corresponding authors.
E-mail addresses: asunshixin@163.com (S. Sun), hxzhm@126.com (H. Zhang), wyqing76@126.com (Y. Wang).
https://doi.org/10.1016/j.colsurfb.2021.111628
Received 30 August 2020; Received in revised form 15 January 2021; Accepted 13 February 2021
Available online 18 February 2021
0927-7765/© 2021 Elsevier B.V. All rights reserved.

Q. Han et al.
Colloids and Surfaces B: Biointerfaces 201 (2021) 111628
the diagnosis and treatment of dysentery, eczema, erysipelas or used as
antipyretic or germicidal agent. PPS also exhibits a wide range of bio-
logical effects, including strong antioxidant effect [24], antiviral effect
[25], immunomodulatory effect [26], anti-microbial activity [27], and
anticancer effect [28,29].
reduction, loaded into a dialysis membrane (MWCO: 3500 Da), dialyzed
in 2 L deionized water for 3 days, changed the water three times per day
to remove small molecules of flavonoids and acids and so forth, and
removed solid impurities by centrifuging at 8000 rpm for 15 min and
then filtrating under reduced pressure. The dialysate was stored in a
freeze-dried bottle in a refrigerator at ꢀ 20 ◦C for at least 4 h and then be
lyophilized at ꢀ 55 ◦C to obtain the pretreated Portulaca oleracea poly-
saccharides, storing the dried sample at 4 ◦C.
Nowadays, more and more attentions have been paid to the appli-
cation of natural products. PPS is natural plant polysaccharides with
extensive pharmacological effects. However, only few reports of the
preparation of new chemotherapy drugs and the formation of drug de-
livery systems using Portulaca oleracea polysaccharides as the carbon
skeleton. In addition, PPS modified with sulphate is more cytotoxic to
tumor cells (such as HeLa and HepG2 cells) in vitro than PPS. In general,
PPS has potential application in cancer treatment [30,31].
Oxidized Portulaca oleracea polysaccharides (PPSꢀ CHO) were pre-
pared according to the reported method with minor modifications [32,
33]. An aqueous solution of sodium periodate (1.8 g, 160 mL) was
dropped into the purified PPS (1.5 g, 140 mL) solution dropwise. The
◦
reaction was mixed in the dark at 40 C for about 48 h, quenched by
In this work, hydroxyl groups in PPS were oxidized to form alde-
hydes, and then reacted with the amino group in alendronate (ALN) to
form Schiff base. As shown in Scheme 1, the bisphosphonate acid group
of ALN was coordinated with the platinum precursor [Pt (NH3)2(OH2)2]
in aqueous solution to prepare new platinum-based polysaccharide
drugs (PPS-ALN-Pt conjugates). Multiple analysis means were used to
characterize the above samples. The antitumor activity and antioxidant
activity of the conjugates were respectively studied using MTT assay,
DPPH and ABTS assays. The interaction between PPS-ALN-Pt conjugates
and DNA was confirmed by gel electrophoresis. This may provide a new
system for cisplatin delivery.
ethanol and then stirred for another 1 h. The reaction solution was
transferred into a dialysis bag (MWCO: 3500 Da) and dialyzed in
deionized water for 72 h to remove the unreacted substance, wadding
product was obtained by freeze-drying. Hydroxylamine hydro-
chloride/sodium hydroxide (NH2OH⋅HCl/NaOH) titration method was
determined for the quantitative determination aldehyde group content
of PPSꢀ CHO which was 1.9 mmol/g according to the reported method
[34].
2.3. Synthesis of PPS-ALN conjugates
PPS-ALN (Portulaca oleracea polysaccharides-alendronate) conju-
gates were prepared via the Schiff ; base reaction. PPSꢀ CHO (0.5 g) and
alendronate sodium trihydrate (0.623 g) were completely dissolved in
45 mL and 30 mL HAc/NaAc (0.1 M) buffers with the pH value at
approximately 5.0, respectively. Alendronate sodium trihydrate solution
was slowly dripped into a round bottom flask containing PPSꢀ CHO
solution and stirred in the dark under nitrogen protection at 50 ◦C for 48
h. After the reaction, the solution was used in distilled water with
dialysis bag (MWCO: 3500 Da) for 3 days [35,36].
2. Materials and methods
2.1. Materials
Alendronate sodium trihydrate (ALN⋅3H2O, > 97 %), sodium peri-
odate (NaIO4), hydroxylammonium chloride (NH2OH⋅HCl), cisplatin,
triethylamine (TEA), and 3-(4, 5-dimethyl-thiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT, ≥ 97.0 %) were obtained from Aladdin
(Shanghai, China). Silver nitrate (AgNO3) were obtained from Sino-
pharm Chemical Reagent Co., Ltd. Dulbecco’s Modified Eagle’s Medium
(DMEM), RPMI-1640 medium (RPMI), penicillin/streptomycin were
procured from Biyuntian Biotechnology Co., Ltd. Supercoiled pUC19
DNA/MspI (HpaII) Marker were obtained from Thermo Fisher Scientific
(USA). Portulaca oleracea polysaccharides (PPS) (purity, 50 %) was
provided from Ciyuan Biotechnology, Shanxi. Anti-Calreticulin anti-
body and Alexa Fluor® 488 were supplied from Abcam (Shanghai)
Trading Co., Ltd. Other chemical reagents were used within the allow-
able dose range.
2.4. Preparation of aquatic cisplatin complexes [Pt (NH3)2(OH2)2]2+
The aquatic cisplatin complexes were prepared according to previous
reports with slight modifications [37]. Cisplatin (50 mg, 0.168 mmol)
solution and silver nitrate (57 mg, 0.333 mmol) were stirred by over-
night in the dark at room temperature, obtained an aquatic cisplatin
complexes solution. After the reaction, the solution was filtered out by
centrifugation (10,000 rpm, 1 h) and then was precipitated with 0.45 μm
water phase filter to remove the fine silver nitrate precipitate. The ac-
quired solution was stored at 4 ◦C and protected from light with tin foil
for short-term using.
2.2. Preparation of oxidation of PPS (PPSꢀ CHO) and phosphorylated
2.5. Synthesis of PPS-ALN-Pt conjugates
Portulaca oleracea polysaccharides (5 g, purity 50 %) was weighed
accurately, dissolved in 110 mL deionized water, filtrated with pressure
PPS-ALN conjugates were soluble in 20 mL deionized water, to which
TEA was added, to give a weak alkaline solution. The above aquatic
cisplatin complexes was added dropwise to the prepared conjugates
solution, stirred it by nitrogen protection in the dark at 37 ◦C for 48 h.
The conjugated solution was loaded into a dialysis bag (MWCO: 3500
Da) and then was dialyzed with periodically changed deionized water
for 3 days. The solution was lyophilized at ꢀ 55 ◦C to obtain dry powder
which was stored into a brown bottle at 4 ◦C and the above synthesis
steps can be repeated. The Pt contents of PPS-ALN-Pt conjugates were
determined by ICP-OES (Perkin Elmer, Optima 4300 DV, USA), which
value was 15.94 % in average value of three times determinations. Af-
terwards, the synthesized samples were used for the later experiments.
2.6. Characterization
The FT-IR spectra were measured on a spectrometer (Bruker, VER-
TEX 80/Raman II, Switzerland) at selected wavenumber in a range from
4000 to 500 cmꢀ 1 at room temperature and 40 % RH. 1H NMR spectra
Scheme 1. The synthesis process of PPS-ALN-Pt conjugates.
2

Q. Han et al.
Colloids and Surfaces B: Biointerfaces 201 (2021) 111628
were recorded using a Bruker AVANCE III HD 400 MHz (Germany) with
D2O as the solvents. 31P NMR spectra were measured using a Bruker
AVANCE III HD 500 MHz (Germany). UV–vis absorption standard curve
experiment of a phosphate group and free radical scavenging assays
were conducted on a LAMBDA 35 spectrophotometer (Perkin Elmer Ltd.,
USA) at 25 ◦C. The TGA curves of samples were performed by a thermal
gravimetric instrument (STA449F5 Jupiter, Germany) which set tem-
perature in a range from 25 to 850 ◦C under dynamic argon with a
heating rate of 10 ◦C/min. The XRD patterns was measured on a
diffractometer (Malvern Panalytical X’Pert3 Powder X-ray Diffractom-
eter, Netherland), on which samples were observed at a speed of 5◦/min
with the scanning range of 2θ (5 to 80◦) and operated in continuous
mode.
2.9. Radical scavenging activity
2.9.1. ABTS radical scavenging assessment
•
ABTS free radical cation (ABTS+ ) was produced by a chemical
oxidation reaction with potassium persulfate, followed by the method of
•
previous literature [40]. ABTS+ was generated by mingling an equal
volume of 0.662 mg/mL potassium persulfate solution and 3.84 mg/mL
ABTS aqueous solution, allowing the reaction laid aside in darkness for
12~16 h at room temperature. Afterwards, 2.0 mL of the reaction
mixture solution was fixed in a volumetric flask (50 mL) with deionized
water to obtain the working solution. Aqueous solutions of PPS, ALN,
PPS-ALN and PPS-ALN-Pt conjugates with different concentrations from
0 to 0.10 mg/mL were dissolved in deionized water (2 mL). They were
•
mixed with freshly prepared ABTS+ solution (2 mL), which was incu-
2.7. Determination of phosphate radical content in PPS-ALN-Pt
conjugates
bated in the dark at room temperature for 5 min. The absorbance of the
resulting solution was determined by a UV–vis spectrophotometer
(PerkinElmer Lambda 365, USA) at 734 nm. Three sets of parallel ex-
periments were conducted, and the data were averaged three times. The
percentage of ABTS radical scavenging effect is counted using Eq. (2).
Firstly, the measurement procedure of the phosphate radical stan-
dard curve was based on the report with some modifications [38]. The
concentration of phosphate standard radical liquid was purchased from
Aladdin (Shanghai, China) and diluted from 1 mg/L to 0.1 mg/L. The
scheme firstly obtained a Tris buffer solution which MgCl2⋅6H2O (120
mg) and trimethyl aminomethane (Tris, 3.6 g) were completely soluble
in 300 mL double-distilled water (DDIW), and then the value of pH was
adjusted to 7.0 with HCl (1 M). The formulation of quantitative phos-
phorus reagent was obtained by mixing an equal volume of 3 M H2SO4
solution, 20 % ascorbic acid solution and 3% ammonium molybdate
solution evenly. 0, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 3.50, 4.00, 4.50
and 5.00 mL of the double-distilled water was accurately supplemented
into the polyethene tube, and standard phosphate solution (0.1 mg/mL)
was then injected until the total volume to 5 mL. Tris buffer solution (3
mL) was added into each tube. After the solution of tube was slowly
shaken up, it was immediately added quantitative phosphorus reagent
(3 mL) and treated with a water bath at 45 ◦C for 0.5 h. The absorbance
of the solution at 580 nm (A580) was measured after cooling to room
temperature [39]. The standard curve of phosphate radical was estab-
lished between A580 and phosphate radical concentration.
Scavenging effect (%) = (1 - Asam ple / Ablank) × 100 %
(2)
Where A
is the measured absorbance value of ABTS free radical
solution abnladnkA
is the measured absorbance value of the sample
sample
mixed with ABTS free radical solution.
2.9.2. DPPH radical scavenging assessment
The antioxidant capacity of PPS, ALN, PPS-ALN and PPS-ALN-Pt
•
conjugates was monitored using DPPH assay [41]. The sample solu-
tions of PPS, ALN, PPS-ALN and PPS-ALN-Pt conjugates from 0 to 0.20
mg/mL were dissolved in deionized water (2 mL) and then were mixed
with freshly prepared DPPH (2 mL, 0.05 mg/mL) ethanol solution. The
solutions were sufficiently shaken up and reacted for 30 min in the dark
at room temperature. The absorbance of the resulting mixed solution
was determined by a UV–vis spectrophotometer (PerkinElmer Lambda
365, USA) at 517 nm. We conducted three sets of parallel experiments,
and the data were averaged three times. The scavenging effect of DPPH
free radical scavenging effect is counted by Eq. (2), where A blank is the
measured absorbance value of DPPH free radical solution and A sample is
the measured absorbance value of the sample mingled with DPPH
solution.
The molybdenum blue colorimetry was applied to determine the
radical phosphate content of the sample [38]. PPS-ALN-Pt conjugates
(0.1 g) was dissolved in a tube with concentrated sulfuric acid (1 mL)
and dense nitric acid (1 mL), and then the solution was heated until
smokeless. The solution was cooled and then was added into 30 % H2O2
aqua solution (1 mL) for one more slow heating. Above steps were
duplicated until no smoke appeared in the conical flask, and the solution
was currently pale yellow or colorless. After adding hydrochloric acid (6
M, 1 mL) in the conical flask, the powder sample was heated to complete
decomposition. Afterwards, the above solution was shifted in a volu-
metric flask (50 mL) and fixed the volume with DDIW. The absorbance
of the solution at A580 was detected by a UV–vis spectrophotometer
(PerkinElmer Lambda 365, USA). With the standard curve obtained by
the above means, the content of phosphate radical was figured out.
2.10. Cell culture and cytotoxicity assay
The relative cytotoxicity of PPS, ALN, cisplatin, PPS-ALN and PPS-
ALN-Pt conjugates was tested on the HeLa, MCF-7, LO-2, and A549
cells expressed by the half-maximal inhibitory concentration (IC50). The
cell viability of four cells to cytotoxicity was determined by MTT assay
[42]. HeLa, MCF-7, LO-2, and A549 cells were seeded in RPMI 1640 or
DMEM medium containing with 10 % fetal bovine serum (FBS), 100
U/mL penicillin/streptomycin, and incubated in a constant temperature
containing 5% CO2 incubator at 37 ◦C. The cells were seeded in the
96-well plates contained 3500 cells of per well, which was cultured for
24 h at 37 ◦C. Then medium in the 96-well plates switched with fresh
medium. Cells were treated with PPS, ALN, cisplatin, PPS-ALN and
PPS-ALN-Pt conjugates medium solution in gradient concentration,
respectively, incubated at 37 ◦C.
2.8. Determination of degree of substitution (DS)
The degree of substitution (DS) that is assumed as the average
number of a phosphate group (-PO3H2) on each sugar residue can be
calculated by the formula Eq. (1):
After incubation for 48 h, the cells were cultured by adding MTT
DS = (162 + M) × C / (M + C)
(1)
solution (20 μL, 5 mg/mL in PBS) in each well and subsequently incu-
bated for 4 h at 37 ◦C. It was necessary to carefully suck out the culture
solution in each hole owing to terminate the culture. The cells precipi-
Where 162 is the relative molecular mass of one monosaccharide res-
idue, M stands for the molar mass of substituent (-PO3H2), and C is the
content of PO34ꢀ
.
tate in the 96-well plates were dissolved by dimethyl sulfoxide (200 μL),
which was shaken well and immediately measured with the absorbance
of 490 nm (OD490). The absorbance of samples at 490 nm (OD490) was
detected by a microplate reader (Spectra Max M5, Molecular Devices
Company, USA). The evaluation of cell cytotoxicity is calculated
3

Q. Han et al.
Colloids and Surfaces B: Biointerfaces 201 (2021) 111628
according to the formula Eq. (3):
2.14. Cellular Pt uptake and DNA plastination
Cell viability (%)= (ODsam ple – ODblank)/(ODcontrol – ODblank)×100 %
(3)
HeLa cells were selected for uptake studies and cultured in a 6-well
plate (Corning) at a density of 105 cells/mL, and incubated for 24 h
under standard growth conditions. The culture medium in 6-well plate
switched with fresh growth media. Meanwhile, the cells were treated
Where the sample is the presence of the drug sample, and the blank is the
absence of the drug sample.
with cisplatin (45 μg/mL) and PPS-ALN-Pt conjugates (284 μg/mL) in
2.11. Agarose gel electrophoresis assay
fresh growth media for 24 h [46]. HeLa cells were harvested and washed
twice with PBS (4 ◦C), and then were digested with concentrated nitric
acid (100
%, 50
μ
L) for 2 h at 95 ◦C, followed by adding hydrogen peroxide (30
DNA is deemed as the major target of platinum-based compounds,
and therefore the interaction between PPS-ALN-Pt conjugates and
supercoiled pUC19 DNA was studied by gel electrophoresis with
ethidium bromide as the probe [43]. The molecular weight of super-
coiled pUC19 plasmid DNA is 1.74 × 106 Da, containing 2686 base pairs.
The concentration of the corresponding nucleotide is calculated as 696
μ
L) and hydrochloric acid (50 μ
L) at 95 ◦C to give a thoroughly
homogenized solution [47]. Water was then added to dilute the solu-
tions until to 1 mL, and the Pt content was determined by ICP-MS using a
standard Plasma-Quad II instrument (VG Elemental, Thermo Optek-
Corp). The average of three groups of repeated experimental data was
regarded as the final result.
μ
M according to the conversion formula, and the formal drug to nucle-
otide molar ratios (ri) varied from 0.2 to 1.4. The supercoiled pUC19
DNA (200 ng) and different concentrations of PPS-ALN-Pt conjugates
dissolving in Trisꢀ HCl/NaCl buffers (50 mM, pH = 7.4) were formed as
3. Results and discussion
the sample solution until total volume reached 10 μL, which was incu-
3.1. FT-IR analysis
bated at 37 ◦C for 1 day in the dark. After incubation, all reactions were
quenched by adding 5 × loading buffer (bromophenol blue, 2 μL). The
Fourier transform infrared spectroscopy (FT-IR) was applied to
recognize the types of functional groups in the material. The FT-IR
spectra of PPS, PPSꢀ CHO, ALN, PPS-ALN and PPS-ALN-Pt conjugates
were schematized in Fig. S1. As shown in Fig. S1(A), the band observed
at 3500~3200 cmꢀ 1 and 2995~2839 cm-1 was belonged to the ꢀ OH
group stretching vibration, as well as the ꢀ CH3 and ꢀ CH2 groups
stretching vibration, respectively. The emerging characteristic absorp-
tion peak of PPSꢀ CHO at 1725, 1455, 1322, 1275, 1112 cmꢀ 1 were
found in FT-IR spectra. NaIO4 was utilized for oxidizing two adjacent
hydroxyl groups in sugar ring to the aldehyde group [48]. The new peak
samples were loaded on a 1% agarose gel-forming electrophoresis and
conducted at 90 mV for 2 h in TAE buffers (40 mM Tris-acetate, 1 mM
EDTA). The agarose gel was stained with ethidium bromide solution (0.5
μ
g/mL) for 5 min and visualized by UV Transillumination (American,
BIO-RAD ChemiDoc™ XRS + gel imaging system).
2.12. Nuclear staining assay
In the tested cells of MTT assay, the values of cisplatin and ALN for
HeLa cells were the smallest, and the IC50 values of the four substances
decreased significantly. Therefore, in the subsequent bioassay experi-
ments, we chose HeLa cells as studying cells.
of 1725 cmꢀ 1 (CO ) was assigned to the aldehyde group in PPSꢀ CHO,
–
–
which demonstrated that PPS were successfully oxidated as shown in
Fig. S1(A). The -C = N- stretching vibration peak of PPS-ALN conjugates
at 1641 cmꢀ 1 overlapped with other functional groups in PPSꢀ CHO that
HeLa cells were grown in a 6-well cell culture plate containing
chemotherapy drugs, and cultured for 24 h. Then, the medium was
removed from the 6-well plate, and HeLa cells were gently rinsed with
cold PBS. After trypsin treatment, the cells were harvested. HeLa cells
were washed twice with cold PBS, fixed with 4% paraformaldehyde for
cannot be discerned shown in Fig. S1(B). However, the characteristic
ꢀ 1
–
– –
peaks of P O or P
O
C from 1152 and 924 cm indicated that
–
PPS-ALN conjugates was successfully synthesized [49]. The vibrational
peak of functional groups about 1322 and 537 cmꢀ 1 of PPS-ALN-Pt
conjugates spectra was broadened, which belonged to -N-H- stretching
vibration. It was preliminarily proved that the platinum complex grafted
onto PPS-ALN-Pt conjugates by coordination.
10 min at room temperature, and dyed with 20 μL of Hoechst 33,342 for
15 min. Ultimately, HeLa cells were washed with PBS for twice to
remove the probe residue. The cell nucleus images were immediately
observed and captured through an inverted fluorescence microscope
(LEICA M165 FC, Germany).
3.2. 1H NMR analysis
1
The H NMR spectra of PPS, PPS-ALN and PPS-ALN-Pt conjugates
dissolving in D2O were indicated in Fig. 1(A). It can be seen from the 1H
2.13. Cellular immunofluorescence assay
NMR spectra of PPS that the signals at 4.0~5.0 ppm and 5.0~5.6 ppm
belonged to β-configuration and α-configuration glucose, respectively.
HeLa cells were selected and treated with chemotherapy drugs. The
distribution of calreticulin in HeLa cells were detected by immunoflu-
orescence assay [46]. HeLa cells were cultured in DMEM medium. When
the cell density was about 50~60 %, the PPS, ALN, cisplatin, PPS-ALN
and PPS-ALN-Pt conjugates was added and cultured continuously for
24 h. The IC50 value of each sample was taken as the final concentration
of each sample according to the MTT experiment. Then the culture so-
lution was taken out from a 20 mm laser scanning confocal culture dish,
washed with PBS, and covered with 4% polyformaldehyde for 20 min,
rinsed three times in PBS for 5 min. Samples were permeabilized with
0.1 % Triton X-100 for 10 min, washed third in PBS for 5 min. Then, 5%
BSA was blocked for 1 h, and 1/500 anti-Calreticulin antibody was
incubated. After the first anti incubation, samples were rinsed three
times in PBS for 5 min. Subsequently, goat antibody to Rabbit IgG (Alexa
Fluor® 488) was cultured for 1 h carrying fluorescent markers, and DAPI
was applied to label the nucleus [45]. Images were acquired on a Zeiss
confocal microscope (LMS710, Germany), using the Zen™ 178 software
manipulated pictures.
The signal peak at approximately 1.88 ppm and 1.75 ppm in the 1H NMR
spectrum was the characteristic peak of the methylene group protons of
ꢀ CH2CH2C(OH)(PO3H2)2 in ALN of PPS-ALN and PPS-ALN-Pt conju-
gates [50]. As depicted in 1H NMR spectra of PPS-ALN-Pt conjugates, the
appearance of proton peaks at approximately 1.83 ppm and 1.11 ppm
was characteristic peak of the methylene group protons and amino
hydrogen, indicated that PPS-ALN-Pt conjugates were successfully syn-
thesized by coordination of platinum complex with biphosphate.
3.3. 31P NMR analysis
The formation of the complex conjugate was confirmed by 31P NMR
(500 MHz, D2O or DMSO-d6) [51,52]. 31P NMR spectra of ALN, PPS-ALN
and PPS-ALN-Pt conjugates were characterized and shown in Fig. 1(B).
The water solubility of ALN is 5 mg/mL. In order to make the phos-
phorus spectrum better presented, 10 mg of ALN was dissolved with
DMSO-d6 (600
μ
L). The 31P NMR spectra of ALN was the signal peaks at
4

Q. Han et al.
Colloids and Surfaces B: Biointerfaces 201 (2021) 111628
Fig. 1. (A) 1H NMR spectra of PPS, PPS-ALN and PPS-ALN-Pt in D2O; (B) 31P NMR spectra of ALN (DMSO-d6), PPS-ALN and PPS-ALN-Pt (D2O).
diffractometer with the 2θ ranges of 5~80◦ as showed in Fig. 2. PPS had
only a broad diffraction peak at 2θ ≈ 19.72◦, suggesting that PPS was
powder diffraction patterns with typical amorphous materials amor-
phous in nature. Previous studies had indicated that polysaccharides
with amorphous state properties were more stable [55]. The diffraction
pattern of the PPS-ALN and PPS-ALN-Pt conjugates had no distinct peaks
and emerged a dispersion state. The results demonstrated that the
modified polysaccharide molecule not only underwent a chemical
structure change, but the cross-linking of the structure led to dispersion
state.
– –
approximately 18.02 ppm. The P CP
signal peak of ALN also
appeared in the 31P NMR spectra of PPS-ALN conjugates at approxi-
mately 18.02 ppm. As shown in Fig. 1(B), the signal of 31P NMR spectra
was about 39.10 ppm, which belonged to the two phosphorus atoms in
the bisphosphonate group of PPS-ALN-Pt conjugates. They were
magnetically equivalent and transferred to lower field. The low-field
displacement of the phosphorus nuclei at approximately 39.10 ppm
was caused by the coordination of phosphonate oxygen with the metal
platinum. In general, the measurements of FT-IR combined with NMR
spectra confirmed that PPS-ALN and PPS-ALN-Pt conjugates were suc-
cessfully synthesized.
3.7. Antioxidant activity analysis
3.4. DS of the phosphate radical of PPS-ALN-Pt conjugates
Portulaca oleracea polysaccharides were studied for the potential
antioxidant or scavenging activities [56]. Therefore, the antioxidant test
was used to detect whether the modified products still possessing the
ability to scavenge free radicals. The antioxidant capacities of PPS, ALN,
PPS-ALN and PPS-ALN-Pt conjugates at measurement concentrations
from 0 to 0.10 or 0 to 0.20 mg/mL were estimated by most frequently
used free radical scavenging assays (ABTS and DPPH methods) pre-
sented in Fig. 3. The ABTS and DPPH scavenging free radical effect of
PPS-ALN conjugates increased with the increase of concentration up to
0.1 mg/mL (> 89.5 %) and 0.19 mg/mL (> 77.1 %), respectively.
Moreover, the scavenging activities of PPS-ALN and PPS-ALN-Pt
conjugates increased significantly with the increase of concentration
in DPPH radical scavenging assay. As shown in Fig. 3, PPS-ALN-Pt
conjugates showed the ability of scavenging free radicals in different
rules, which still had certain antioxidant capacity. The scavenging free
According to standard curve regression equation of phosphate anion
in Fig. S2 (Y = 0.0732 X + 0.0445, R2 = 0.9997), the content of PO34ꢀ
from PPS-ALN-Pt conjugates was calculated as 10.95 %. Owing to two
phosphate radicals are contained in ALN, the content of PO34ꢀ should be
adjusted to 5.47 %. Therefore, the contents of the phosphate group
-PO3H2 was accordingly 4.66 %. The DS of -PO3H2 in PPS-ALN-Pt con-
jugates was calculated as 0.139 under Eq. (1). On the other hand, it was
exactly indicated that phosphorylation had been successfully carried
out.
3.5. TGA analysis
Thermal stability of these samples was investigated by a thermal
gravimetric instrument as shown in Fig. S3. The modification improved
the thermal stability of PPS-ALN-Pt copolymer. Compared with the pure
Portulaca oleracea polysaccharides, the first mass loss around 52~110 ◦C
may be the loss of water. Followed by the mass loss of the phosphate
group and coordination bond was the region of near 170~400 ◦C, and its
decomposition speed was accelerated. Finally, the carbon structure of
the polysaccharide ring [53] could be decomposed at 400~850 ◦C.
Fig. S3(b) showed TGA curves of ALN, in which the first step mass loss of
ALN around 110~140 ◦C was due to loss crystal water. The second stage
at approximately 245~500 ◦C, corresponding to 30 % mass loss, was the
primary thermal decomposition owing to decomposition of phosphate.
The final remaining mass of ALN was about 49 % of the initial mass,
which had the best thermal stability among these samples. After grafting
with ALN and Pt, the skeleton structure of PPS became more stable
which made it have better stability at high temperature.
3.6. XRD analysis
X-ray powder diffraction (XRD) spectra was used to reflect the
crystalline degree of powder samples [54]. The diffraction patterns of
PPS, PPS-ALN and PPS-ALN-Pt conjugates were measured using X-ray
Fig. 2. XRD patterns of PPS (a), PPS-ALN (b) and PPS-ALN-Pt (c).
5

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Colloids and Surfaces B: Biointerfaces 201 (2021) 111628
Fig. 3. ABTS (A) and DPPH (B) radical scavenging activities of PPS, ALN, PPS-ALN and PPS-ALN-Pt.
radical assays results of PPS had the hydrogen-donating capacity of
antioxidants. The hydroxyl groups of ALN provided a hydrogen supply
capacity, which made PPS-ALN conjugates stronger antioxidant. Under
the combined coordination of Pt and PPS-ALN conjugates made the
decrease of hydroxyl groups of PPS-ALN-Pt conjugates which kept
relatively stable antioxidant activity.
On the other hand, IC50 values of cisplatin to HeLa, MCF-7, LO-2, and
A549 cells were 0.087, 3.15, 1.23, 5.88 μg/mL (0.29, 10.49, 4.10, 19.57
μ
M), confirmed that cisplatin had a strong inhibitory effect on cell
growth. The IC50 values of ALN to HeLa and LO-2 cells were smaller than
MCF-7 and A549 cells, moreover, IC50 values of HeLa cells were the
smallest that is ALN had the most toxic for HeLa cells. The precursor of
cisplatin coordinated with PPS-ALN formed PPS-ALN-Pt conjugates,
which had similar IC50 values in HeLa and LO-2 cells. PPS-ALN-Pt
conjugates had a similar effect to inhibit cells proliferation for HeLa,
MCF-7, LO-2, and A549 cells. It was impersonally indicated that the
antitumor activity of cisplatin remained when the precursor was coor-
dinated with PPS-ALN conjugates containing carrier. However, the IC50
values of PPS-ALN-Pt conjugates were higher than cisplatin among four
cell lines, which was related to the amount of grafted cisplatin hydrate in
the conjugates.
3.8. Cytotoxicity evaluation
The effect of PPS, ALN, cisplatin, PPS-ALN and PPS-ALN-Pt conju-
gates on cell viability was detected by MTT experiment. HeLa, MCF-7,
LO-2, and A549 cells were respectively incubated with PPS, ALN,
cisplatin, PPS-ALN and PPS-ALN-Pt conjugates, the cells viabilities of
them were then detected. The IC50 values of these sample against HeLa,
MCF-7, LO-2, and A549 cells were depicted in Fig. 4. PPS had a lower
antiproliferative effect on HeLa and LO-2 cells compared with MCF-7,
and A549 cells, giving IC50 values of PPS 1113.3, 1113.4, 867.1 and
3.9. Plasmid DNA interaction with PPS-ALN-Pt conjugates
481.4
μg/mL for HeLa, LO-2, MCF-7, and A549 cells, respectively.
Nevertheless, PPS-ALN-Pt conjugates had approximately the same
The unwrapping of the closed circular and supercoiled pUC19
plasmid DNA by interacting with PPS-ALN-Pt conjugates were detected
using an electrophoretic mobility shift experiment. The plasmid pUC19
had a double-stranded shape and existed as a supercoiled structure.
Since strand break was introduced, the superhelix form of DNA was
broken into the nicked circular or the linear form. When one of the
strands was severed, the super curl form would loosen, forming a
notched circular structure. When both strands were split, a line shape
would emerge. DNA molecules with the same molecular weight needed
IC50 values, which were 284.29, 262.1 and 233.8 μg/mL, respectively,
for HeLa, MCF-7 and A549 cells. The results of PPS-ALN-Pt conjugates
confirmed that they had a particular effect on cancer cells. PPS-ALN-Pt
conjugates was more cytotoxic than PPS, ALN, and PPS-ALN conjugates
–
–
toward the tested tumor cells. The P O of phosphate radicals of PPS-
ALN conjugates can be coordinated with the amino group on cisplatin
but not be exposed to the outside, so the PPS-ALN-Pt conjugates did not
exhibit selectivity.
Fig. 4. The IC50 value of A549, MCF-7, HeLa and LO-2 cells treated with PPS, ALN, PPS-ALN and PPS-ALN-Pt (A) and A549, MCF-7, HeLa and LO-2 cells treated with
cisplatin (B).
6

Q. Han et al.
Colloids and Surfaces B: Biointerfaces 201 (2021) 111628
to migrate differently according to their spatial structure of the DNA
molecules during electrophoresis [47]. The electrophoretic migration
speed of the closed-loop helix structure was the fastest, followed by the
linear structure and the open-loop structure. In the wake of PPS-ALN-Pt
conjugates concentration, the migration rate of DNA after treating with
the drug was slightly delayed compared with that of free supercoiled
DNA as seen in Fig. S4. It brought to light conclusion that the helicity of
DNA could be interfered by PPS-ALN-Pt conjugates, which led to the
change of DNA replication mechanisms and the processing disruption of
this DNA eventually triggered cell apoptosis.
cells. The expression of calreticulin in HeLa cells was studied by
immunofluorescence assay. When drugs stimulated cells to undergo the
process of apoptosis, CRT transferred from intracellular to the cell
membrane. As shown in Fig. 6, DAPI staining (blue fluorescence)
assisted in locating the nucleus and the antibody labelled CRT (green
fluorescence) of HeLa cells in the control group also expressed. The
expression CRT of HeLa cells after treated with PPS, PPS-ALN and
PPS-ALN-Pt conjugates was in turn increased. CRT induced by cisplatin
was slightly higher than control cells. Moreover, the induction of CRT by
PPS-ALN-Pt conjugates also moderately increased when compared to
that of control cells. Therefore, PPS-ALN-Pt conjugates played a partic-
ular role in regulating the immunogenicity of HeLa tumor cells and also
induced apoptosis correspondingly.
3.10. Nuclear staining
Fluorescence microscopy was used to analyze the nuclear morpho-
logical changes of HeLa cells staining with Hoechst 33,342, which
confirmed the apoptosis of cells [57]. The ability of PPS, PPS-ALN and
PPS-ALN-Pt conjugates to induce apoptosis was studied. The nuclear
morphology of HeLa cells, after incubated with PPS, PPS-ALN and
PPS-ALN-Pt conjugates at different concentrations for 24 h, was
observed by a fluorescence microscope. As depicted in Fig. 5, The nu-
cleus of HeLa cells in the control group was in weak blue fluorescence
with a regular round outline. The nucleus morphology of HeLa cells
treated with PPS at IC50 value resembled that of the control group,
illustrating that PPS had no effect on HeLa cells nucleation. However,
the nucleus morphology of HeLa cells incubated with cisplatin, PPS-ALN
and PPS-ALN-Pt conjugates at different concentrations displayed
condensation, nucleus shrinkage, and even nuclear fragmentation. On
the other hand, the nucleus morphology changed of HeLa cells treated
with PPS-ALN-Pt conjugates was more visible. In a nutshell, PPS-ALN-Pt
conjugates had a significant induction effect on apoptosis of HeLa cells
having a concentration-dependent manner.
3.12. Cellular Pt uptake and DNA platinization
The cellular accumulation of Pt in HeLa cells was detected by ICP-
MS. As shown in Table 1, PPS-ALN-Pt conjugates have a higher plat-
inum binding protein effect on HeLa cells than cisplatin, due to the
excellent biocompatibility of polysaccharides. Generally, the reported
DNA platinum was approximately 1%. Due to the different factors such
as the action time, cell type, drug concentration, etc., the ability of
cisplatin and PPS-ALN-Pt conjugates to platinate nuclear DNA in this
experiment was high. By formula conversion calculation, the ability of
PPS-ALN-Pt to platinate nuclear DNA in cells was higher than cisplatin
from the cellular uptake data of Table 1. On the basis of above studies,
PPS-ALN-Pt conjugates displayed slightly higher platinization ability
than cisplatin, which was attributed to the higher cellular uptake.
4. Conclusion
In summary, a sugar ring carrier containing biphosphate group
bridging ligand was rationally designed and synthesized. The coupling
3.11. The distribution of CRT in HeLa cells
1
Pt complexes of PPS-ALN conjugates were characterized by FT-IR, H
Calcium reticulin (CRT), a highly conserved multifunctional protein,
is widely distributed in the cell nucleus, endoplasmic reticulum and cell
membrane [58]. Experimental data displayed that CRT was firmly
related to the occurrence and development of tumors. CRT was trans-
ferred to the surface of the cell membrane, aiming to mediate the
immunogenicity death of tumor cells [59]. So, it played an essential role
in regulating the immunogenicity of tumor cells. CRT can be synthesized
in normal cells, apoptotic cells, tumor cells and partial drug treatment
NMR, 31P NMR, XRD, TGA analysis. The results of PPS-ALN-Pt conju-
gates had good thermal stability. In terms of scavenging active radicals,
PPS-ALN conjugates were significantly more potent than PPS, while the
antioxidant activity of PPS-ALN-Pt conjugates was kept relatively stable,
indicating that it had a certain extent antioxidant capacity to reduce the
occurrence of some inflammation. MTT assay results illustrated that
PPS-ALN-Pt conjugates had lower cytotoxicity in comparison with
cisplatin and ALN against HeLa cells. PPS-ALN-Pt conjugates interfered
Fig. 5. Morphological alterations of the nuclei of HeLa cells after incubation with PPS (1114 μg/mL), PPS-ALN (265 and 530 μg/mL), PPS-ALN-Pt (142 and 284 μg/
mL) and cisplatin (0.225 and 0.445
μ
g/mL) for 24 h and staining with Hoechst 33342.
7

Q. Han et al.
Colloids and Surfaces B: Biointerfaces 201 (2021) 111628
Fig. 6. The distribution of calreticulin in HeLa cells after incubation with control, cisplatin (0.0871 μg/mL), PPS (1114 μg/mL), PPS-ALN (530 μg/mL) and PPS-ALN-
Pt (284 μg/mL) for 24 h. Scale bars: 20 μm.
Overall, the easily prepared PPS-ALN-Pt conjugates could be a prom-
ising strategy for anti-tumor pharmacotherapy.
Table 1
Cellular uptake of Pt and platinization of cellular DNA in HeLa cells after
exposure to cisplatin (45
μ
g/mL) and PPS-ALN-Pt (284
PPS-ALN-Pt
μ
g/mL) for 24 h a.
cisplatin
CRediT authorship contribution statement
ng Pt/mg Protein
191.76 ± 2.96
16.00 ± 0.50
8.83 ± 0.21
7.82 ± 0.11
Qianqian Han: Conceptualization, Investigation, Methodology,
Writing - original draft. Lirong Huang: Resources, Conceptualization,
Investigation. Ying Wang: Visualization, Investigation. Shixin Sun:
Visualization, Investigation. Hao Huang: Visualization, Investigation.
Fei Li: Visualization, Investigation. Fangtian Wang: . Ligen Chen:
Visualization, Investigation. Hongmei Zhang: Writing - review & edit-
ng Pt/
a
μg DNA
Average of three measurements.
with the helicity of DNA by gel electrophoresis assay and possess binding
ability with DNA by cellular uptake assay. We explored that PPS-ALN-Pt
conjugates played a significant role in regulating immunogenicity and
inducing apoptosis of tumor cells by immunofluorescence assay.
ing, Supervision. Yanqing Wang: Writing
-
review
&
editing,
Supervision.
8

Q. Han et al.
Colloids and Surfaces B: Biointerfaces 201 (2021) 111628
cytotoxicity in drug-resistant lung cancer cells, Colloids Surf. B 195 (2020),
111256.
Declaration of Competing Interest
[21] X. Wang, C.C. Yang, Y.J. Zhang, X. Zhen, W. Wu, X.Q. Jiang, Delivery of platinum
(IV) drug to subcutaneous tumor and lung metastasis using bradykinin-
potentiating peptide-decorated chitosan nanoparticles, Biomaterials 35 (2014)
6439–6453.
The authors declare no conflict of interest.
Acknowledgement
[22] T. Moreira, R. Francisco, E. Comsa, S. Duban-Deweer, V. Labas, A. Teixeira-Gomes,
L. Combes-Soia, F. Marques, A. Matos, A. Favrelle, C. Rousseau, P. Zinck, P. Falson,
H. Garcia, A. Preto, A. Valente, Polymer “ruthenium-cyclopentadienyl” conjugates-
New emerging anti-cancer drugs, Eur. J. Med. Chem. 168 (2019) 373–384.
[23] E.S. Elkhayat, S.R. Ibrahim, M.A. Aziz, Portulene, a new diterpene from Portulaca
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This work was supported by the National Natural Science Foundation
of China (Project No. 21571154), the "Six Talent Peaks Project" in
Jiangsu Province, the Jiangsu Fundament of “Qinglan Project” and “333
Project”.
[24] Y.-P. Li, L.-H. Yao, G.-J. Wu, X.-F. Pi, Y.-C. Gong, R.-S. Ye, C.-X. Wang, Antioxidant
activities of novel small-molecule polysaccharide fractions purified from Portulaca
oleracea L, Food Sci. Biotechnol. 23 (2014) 2045–2052.
Appendix A. Supplementary data
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