Block copolymer [(l-GluA-5-BE)-: B -(l-AspA-4-BE)]-based nanoflower capsules with thermosensitive morphology and pH-responsive drug release for cancer therapy

Article abstract of DOI:10.1039/d0tb01647k

Herein, the synthesis of an amino-acid-based di-block copolymer (di-BCP) in-between an l-glutamic acid-5-benzyl ester and l-aspartic acid-4-benzyl ester [(l-GluA-5-BE)-b-(l-AspA-4-BE)] has been reported. However, the synthesis of di-BCP of [(l-GluA-5-BE)-b-(l-AspA-4-BE)] was carried out through the facile modified ring-opening polymerization (ROP) without using any surfactants and harmful chemicals. Interestingly, the synthesized [(l-GluA-5-BE)-b-(l-AspA-4-BE)] has been used to design nanoflower capsules (NFCs) with surface-functionalized nanoflakes and petals. Notably, the simple solvent propanol has been used as a dispersing medium for the di-BCP-based powder to observe morphology of NFCs. Moreover, these amino-acid-based NFCs are biocompatible, biodegradable, and bio-safe for mankind usage. Consequently, di-BCP-based NFCs show changes in morphology with different temperature conditions, i.e., at ～10 °C, ～25 °C (RT), and ～37 °C (body temperature). Furthermore, the average thickness of the surface-functionalized nanopetals has been calculated as ～324 nm (in diameter). Similarly, the average distance between petals is calculated as 3.6 μm and the pore depth is ～21 nm. Additionally, the porosity throughout the surface of capsules in-between nanopetals is an advantageous characteristic feature to improve the drug/paclitaxel (PTX) loading capacity. It is a unique and novel approach to design NFCs, which are a potential payload for nanomedicine and cancer therapy. Furthermore, NFCs were used to evaluate the loading efficacy of drugs and showed ～78% (wt/wt%) of the PTX loading. Moreover, NFCs showed ～74% drug release at physiological body temperature. Thus, NFCs showed remarkable release at acidic pH medium. However, PTX released from NFCs showed greater cell inhibition (i.e., ～79%) with an increase of the PTX concentration after 24 h incubation over HeLa (human epithelial cervical cancer) cells. Besides, PTX released from NFC showed significant (～34%) cell killing capacity. Such promising NFCs are recommended for breast, liver, and lung cancer therapeutics.

Full text of DOI:10.1039/d0tb01647k

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Materials Chemistry B
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Block copolymer [(L-GluA-5-BE)-b-(L-AspA-4-BE)]-
based nanoflower capsules with thermosensitive
morphology and pH-responsive drug release for
cancer therapy†
Cite this:DOI: 10.1039/d0tb01647k
a
Chander Amgoth, *a Shuai Chen,a Tirupathi Malavathb and Guping Tang
*
Herein, the synthesis of an amino-acid-based di-block copolymer (di-BCP) in-between an L-glutamic acid-
5-benzyl ester and L-aspartic acid-4-benzyl ester [(L-GluA-5-BE)-b-(L-AspA-4-BE)] has been reported.
However, the synthesis of di-BCP of [(L-GluA-5-BE)-b-(L-AspA-4-BE)] was carried out through the facile
modified ring-opening polymerization (ROP) without using any surfactants and harmful chemicals.
Interestingly, the synthesized [(L-GluA-5-BE)-b-(L-AspA-4-BE)] has been used to design nanoflower
capsules (NFCs) with surface-functionalized nanoflakes and petals. Notably, the simple solvent propanol has
been used as a dispersing medium for the di-BCP-based powder to observe morphology of NFCs.
Moreover, these amino-acid-based NFCs are biocompatible, biodegradable, and bio-safe for mankind
usage. Consequently, di-BCP-based NFCs show changes in morphology with different temperature
conditions, i.e., at B10 1C, B25 1C (RT), and B37 1C (body temperature). Furthermore, the average
thickness of the surface-functionalized nanopetals has been calculated as B324 nm (in diameter). Similarly,
the average distance between petals is calculated as 3.6 mm and the pore depth is B21 nm. Additionally,
the porosity throughout the surface of capsules in-between nanopetals is an advantageous characteristic
feature to improve the drug/paclitaxel (PTX) loading capacity. It is a unique and novel approach to design
NFCs, which are a potential payload for nanomedicine and cancer therapy. Furthermore, NFCs were used
to evaluate the loading efficacy of drugs and showed B78% (wt/wt%) of the PTX loading. Moreover, NFCs
showed B74% drug release at physiological body temperature. Thus, NFCs showed remarkable release at
acidic pH medium. However, PTX released from NFCs showed greater cell inhibition (i.e., B79%) with an
increase of the PTX concentration after 24 h incubation over HeLa (human epithelial cervical cancer) cells.
Besides, PTX released from NFC showed significant (B34%) cell killing capacity. Such promising NFCs are
recommended for breast, liver, and lung cancer therapeutics.
Received 3rd July 2020,
Accepted 20th August 2020
DOI: 10.1039/d0tb01647k
rsc.li/materials-b
branching capability followed by the net-work formation led to
the synthesis of the di-BCP of [(L-GluA-5-BE)-b-(L-AspA-4-BE)], as
Introduction
The design and development of amino-acid-based surface- explained by Amgoth et al.2 The self-assembly mechanism
functionalized NFCs with porosity and nanopetals for enhanced through the coordinative conjugation between two or more
drug loading and targeted release kinetics are an essential task different polymers leads to the formation of BCPs. Therefore,
for cancer therapy.1 However, material science and engineering, biocompatible, biodegradable, and bio-safe polymers with
followed by the combination of life sciences, can fulfill the active functional groups play a crucial role in synthesizing BCPs,
research area (such as drug delivery and cancer theranostics) followed by the design and development of surface-
with potential amino-acid-based carrier capsules. The charac- functionalized nanoarchitectured materials (such as capsules
teristic properties of block copolymers (BCPs) such as low and particles).3 However, BCPs have an enormous potential for
density, low molecular weight, self-assembly nature, amphiphi- use in the design and development of carrier capsules with
lic (hydrophobic and hydrophilic) nature, cross-linking and easy surface-functionalized properties such as smooth and rough
surface, porosity, fringes, and nanopetals.4 Interestingly, such
bio-inspired materials play a vital role in order to evaluate the
bio/pharmaceutical applications, especially for cancer treat-
ment. The systemic research on amino-acid-based BCPs via
a Department of Chemistry, Zhejiang University, Hangzhou-310028, China.
E-mail: tangguping@zju.edu.cn
b Department of Biochemistry and Molecular Biology, Tel Aviv University, Israel
† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0tb01647k
supra-molecular hydrophobic imprinting helps in the design
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of potential surface-functionalized nanomaterials, which can be [(L-GluA-5-BE)-b-(L-AspA-4-BE)] are a unique and novel approach
recommended for drug/cell/gene delivery systems.5 BCPs are to design NFCs with adjuvant nanoarchitectured features to
often referred to as wonder and magic polymers for drug uplift the drug delivery issues associated with cancer therapy.
delivery and cancer therapy because of their precise control The main aim of this work is to develop efficient cargo capsules
over the polymer length, size scale, chain network, branching for the targeted release of molecular therapeutic agents. NFCs
capacity, easy changeability in shape, and morphology without with thermosensitive and pH-responsive characteristics are also
using any harmful chemicals.6 Several factors certainly affect an advantageous property for cancer therapeutics.4–8 However,
the properties of the polymer nanomaterials, such as solvent, such qualitative amino-acid-based NFCs are promising drug
temperature conditions, reaction methods, purity, environment, carrier systems for various cancer treatments, such as breast,
concentration, pH of the medium, stirring and sonication lung, liver and prostate cancers.14,17
methods.7 Li et al. reported on the polysaccharides and dual-
drug response for conjugated cancer chemotherapy.8 Similarly,
different fullerene-type capsules, porous metal–organics, hydro-
gels, mesoporous metal–organic frameworks, aggregates,
Materials and methods
Materials
organic cages, and composite frameworks were also recom-
mended as promising drug delivery systems by Wu et al.9 and
As per the animal ethical committee and user permit guidelines
of Zhejiang University, China (Ethics code no: ZJU2010-1-01-
20Y), healthy and well-cultured 4–6 week-old grown rats were
purchased from the Jackson Laboratory and used for in vivo
inflammation studies. All of the experiments related to in vitro
and in vivo studies were performed according to compliance
with the relevant laws and institutional user guidelines. The
Wang et al.10,11 Ding et al. reported on the engineered nano-
medicine for enhanced tumor penetration through the transla-
tional advanced strategies for chemotherapy and the role of
materials.12 All of these carrier materials have certain limita-
tions for their use in bio/pharmaceutical applications. These
limitations include low loading capacity, burst release, side
committee has approved all of the experiments. Furthermore,
effects, cytotoxicity, biocompatibility, biodegradability, bio-
the chemicals required for the synthesis of di-BCP of [(L-GluA-5-
safety, size, shape and morphology. Such limitations can be
BE)-b-(L-AspA-4-BE)] and the development of [(L-GluA-5-BE)-b-(L-
addressed through the amino-acid-based BCPs with advanced
AspA-4-BE)]-based NFCs were purchased with high purity, and
characteristic features like porosity, larger specific surface area,
used without further purification (except tetrahydrofuran/THF
ideal particle size, shape, and time-to-time tunable properties
for cancer therapeutics.10–13 Biodegradable polymers conju-
solvent). L-Glutamic acid-5-benzyl ester (499%, Sigma Aldrich),
ammonium hydroxide (NH4OH, 498%, Sigma Aldrich),
L-aspartic acid-4-benzyl ester (499%, Sigma Aldrich), triphos-
gene (499%, Sigma Aldrich), sodium citrate, (499.8%, Sigma
Aldrich), ethanol (CH3CH2OH, 498.5%), propanol (497%,
SDFCL), THF solvent (498%, Merck), THF distillation conden-
gated with amino-acids attracted interest in the synthesis of
di-BCP of [(L-GluA-5-BE)-b-(L-AspA-4-BE)], followed by the design
and development of thermosensitive and pH-responsive NFCs.
Herein, the synthesis of amino-acid-based di-BCP of [(L-GluA-5-
BE)-b-(L-AspA-4-BE)], followed by the design and development of
ser, THF reflux mantle, three-neck round bottom (RB) flask
surface-functionalized NFCs, has been deliberated. The formula
(1 Lt), water circulation pump, Whatmann filter paper, pH meter,
and chemical structure of amino-acid-based polymers, L-GluA-5-
conical flask, beakers, MTT (methyl thiazole tetrazolium, 499.9%,
Sigma Aldrich), magnetic beads, magnetic stirrer-cum hot plate,
BE and L-AspA-4-BE, differ only by a methyl (–CH2–) group,
which is excess in L-GluA-5-BE, but their self-assembly and
formulation of NFCs are amazing for mankind usages.14–16
micropipettes, 96-well plates, sample viols, centrifuge tubes, alu-
minium foil, HeLa cells, media for cell culture (RPMI), CO2
L-GluA-5-BE and L-AspA-4-BE both have greater affinity in acidic
incubator, orbital shaker, dd-H2O, goggles, mask, gloves, apron,
environments. The acidic medium (pH = 4 and lower than 4)
liquid N2 container, Petri dish, ultra-sonic bath, FITC (Fluores-
leads to a change in the protons from an acid group to amine.
cence isothiocyanate; 498%, Sigma Aldrich), rhodamine-B
(496%, Sigma Aldrich), DAPI ((2-(4-aminophenyl)-1H-indole-6-
carboxamidine); 498%, Sigma Aldrich), paclitaxel (499%, Sigma
Aldrich).
Since L-GluA-5-BE and L-AspA-4-BE have amine and carboxyl
groups, the amine (–NH2) group can gain a proton and the
carboxyl/acid (–COOH) group can lose a proton at the same time
under acidic pH conditions. This protonation property could be
the reason for the pH-responsive nature of synthesized NFCs.
The peculiar properties of L-AspA-4-BE and L-GluA-5-BE, such as
Synthesis of [(L-GluA-5-BE)-b-(L-AspA-4-BE)]
aspartate and glutamate transamidation followed by the inter- The synthesis of di-BCP of [(L-GluA-5-BE)-b-(L-AspA-4-BE)] was
conversion of oxaloacetate, and the donation of a nitrogen atom carried out through the facile modified ring-opening polymer-
for other biosynthetic products help in the morphological ization (ROP) method. The oxidation reaction of L-glutamic acid-
changes with respect to various temperatures. However, 5-benzyl ester and L-aspartic acid-4-benzyl ester with the help of
water retention and nitrogen uptake can also be considerable triphosgene leads to the formation of N-carboxy anhydride
properties for the thermosensitive morphological changes of (NCA) or cyclic anhydride. During the oxidation reaction, the
NFCs. Nanoparticles and capsules with size ranges of 100 to acid group of L-glutamic acid-5-benzyl ester and L-aspartic acid-
200 nm are much more suitable for targeted drug delivery, as 4-benzyl ester were converted into NCA, which further helps in
reported by Zhao et al.15 and Ge et al.17 Inherently, di-BCPs of the ROP reaction. However, Fig. 1, 2 and Fig. S1 (S; ESI†) show
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the steps involved in the synthesis of di-BCP and the develop-
ment of NFCs at different temperature conditions. Fig. S2 (ESI†)
depicts the structure of L-glutamic acid-5-benzyl ester and
L-aspartic acid-4-benzyl ester, along with their NCA structures.
Initially, B2.5 g of L-glutamic acid-5-benzyl ester and L-aspartic
acid-4-benzyl ester were mixed with B1.5 g of oxidizing agent
(Triphosgene), and the reactions were performed with B50 mL
of dry THF solvent followed by stirring for 1 h at B50 1C.
The resultant NCA of both acids was used for the synthesis of
di-BCP of [(L-GluA-5-BE)-b-(L-AspA-4-BE)]. Furthermore, in a
150 mL RB flask, B2.5 g of L-glutamic acid-5-benzyl ester was
taken, and it was added to B50 mL of dry THF solvent.
Similarly, in an RB flask, B2.5 g of L-aspartic acid-4-benzyl
ester was added to B50 mL of dry THF solvent. Both were
individually allowed to stir for 30 min at room temperature (RT).
Then, both dissolved mixtures were transferred into a 250 mL
RB flask and added to B2.5 g of Pd/C (activated charcoal). The
neck of the RB was tightened with an H2 gas bladder, and
stirring was continued for 4 h at B70 1C (Fig. 1, 2 and Fig. S1,
ESI†). However, upon continuous stirring, the transparent
solution changed into a white precipitate of [(L-GluA-5-BE)-b-(L-
AspA-4-BE)]. After completion of the 4 h reaction, the remaining
solvent was decanted, and the final [(L-GluA-5-BE)-b-(L-AspA-4-BE)]
compound was freeze-dried under reduced pressure (50 bar, C-Gen
Biotech, À80 1C freeze dryer model). The lyophilized [(L-GluA-5-BE)-
b-(L-AspA-4-BE)] compound was allowed to dry at RT for 24 h.
The compound of [(L-GluA-5-BE)-b-(L-AspA-4-BE)] was obtained and
used for calculation of the final yield, which is B3.6/5 g (wt/wt),
i.e., equal to B72%, as explained by Amgoth et al.2,4,6,11,14 and
Ge et al.17
Fig. 2 Schematic (a) for NCA of L-GluA-5-BE, (b) for NCA of L-AspA-4-BE,
(c) for a mixture of di-BCP of [(L-GluA-5-BE)-b-(L-AspA-4-BE)], (d) for the
well-dried powder, (e) NFC developed at RT, (f) for NFC at body tempera-
ture, and (g) for NFC at low temperatures.
Cell culture
The human epithelia cervical carcinoma cancer cell line (HeLa)
was grown in RPMI medium supplemented with 10% heat
inactivated fetal bovine serum (FBS), 100 IU mLÀ1 penicillin,
100 mg mLÀ1 streptomycin and 2 mM of L-glutamine, and
maintained in a humidified atmosphere with 5% CO2 at
B37 1C. Cells were sub-cultured once every three days.
The anticancer drug (PTX) was used for the treatment. PTX
was dissolved in dimethyl sulfoxide (DMSO) solvent and used to
load inside the NFCs. However, treatments were started with a
bulk number of cells (B4 Â 104 cells wellÀ1) in 60 mm Petri
dishes, and continued with a stepwise increase in the concen-
trations, i.e., 1.0, 5.0, 10, 15, 20, and 25 mg mLÀ1. The inhibition
of the HeLa cells with the formulations was studied with a
multimode (plate) reader using 96-well plates.
MTT assay
The MTT assay is a colorimetric method to measure the activity
of enzymes in living cells to reduce MTT to purple formazan
crystals/dyes. Upon addition to cells, it gives purple formazan
crystals. The MTT assay was basically used to determine cell
viability, cell inhibition and cytotoxicity of potential medicines
and our developed NFCs. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) is a complex chemical dye,
which is widely used to stain cells during treatment with DPBS
buffer solution. In a 96-well plate, B4 Â 104 cells per well were
cultured and added with formulation concentrations of 1.0, 5.0,
10, 15, 20, and 25 mg mLÀ1, respectively. However, 6-wells in a
96-well plate were kept blank and considered as the control,
which means that the cells were maintained as cultured.
The developed nanoformulations [(NFCs)–(PTX)–(HeLa cells)]
were added to the rest of the wells, and incubated at B37 1C in
5% CO2 overnight. During the incubation, drug (PTX) mole-
cules came out from the NFCs and interacted with the HeLa
cells. However, the calculated amount of NFCs of ([(L-GluA-5-
BE)-b-(L-AspA-4-BE)]) was dispersed in propanol, and then
Fig. 1 Schematic illustration of the steps involved in the synthesis of
di-BCP of [(L-GluA-5-BE)-b-(L-AspA-4-BE)], followed by the design and
development of NFCs and pH-based drug release at room temperature
conditions.
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added to each well for thorough mixing with media (150 rpm 2 and Fig. S2, ESI†), as reported by Amgoth et al.2,14 and
for 4 min). The well plates were further incubated at B37 1C in Ge et al.17 Fig. S4 and S5 (ESI†) gives details about the synthesis
5% CO2 for 24 h to allow the PTX to show an effect on the of di-BCP of [(L-GluA-5-BE)-b-(L-AspA-4-BE)] and its structural
cancer cells. To those wells, 20 mL of MTT solution was added confirmation through the primary characterizations, such as
and stirred at 150 RPM for B4 min to thoroughly mix the MTT FTIR and 1H NMR. Since the NCA of both L-GluA-5-BE and
with the media. Furthermore, the plates were incubated for L-AspA-4-BE are structurally identical, they gave identical peak
24 h to allow the MTT to be metabolized with HeLa cells. positions in FTIR and 1H NMR (Fig. S4 and S5, ESI†). L-GluA-5-
The excess and unwanted residue of media was removed. The BE and L-AspA-4-BE differ via the additional methyl (–CH2–)
plates were used to read the optical density at 560 nm, and group in L-GluA-5-BE (Fig. 1, 2 and Fig. S1–S5, ESI†).
subtract the background at 670 nm. The optical density was
However, FTIR characterization for the NCA of L-GluA-5-BE
directly correlated with the cell quantity, and the obtained and L-AspA-4-BE was performed through the KBr pellet
results were used to calculate the cell viability and inhibition.
method.2,18 Fig. S4(a) (ESI†) corresponds to the reaction
between L-GluA-5-BE, followed by the conversion of NCA of
L-GluA-5-BE. Fig. S4(b) (ESI†) corresponds to the FTIR for
L-GluA-5-BE. Its peak positions at B3300 cmÀ1 (Æ10) correspond
to the free hydroxyl (–OH) group, and those at B3059 cmÀ1 (Æ10)
and B2927 cmÀ1 (Æ10) correspond to an asymmetric and sym-
metric stretch of the methyl (–CH2–) groups, respectively.
The peak positions at B1958 and B1872 cmÀ1 (Æ10) correspond
to the NCA of L-GluA-5-BE. However, the peak position at
B1744 cmÀ1 (Æ10) corresponds to an ester (R–COO–R).
The peaks at 1616, 1514, 1156, 967, and 690 cmÀ1 (Æ10)
correspond to the complex aromatic structure of L-GluA-5-BE.
Interestingly, identical FTIR peak positions and values were
observed for L-AspA-4-BE in Fig. S5(b) (ESI†).18 Furthermore,
1H NMR characterization was performed for L-AspA-4-BE.
The peak position (chemical shift, d) values (nature of proton)
were appended as follows: d value at B0.9 and 1.3 ppm, which
correspond to the methyl (–CH2–) group of benzyl, 1.9 and
2.6 ppm for the protons adjacent to the ester group, a sharp
peak at 3.8 ppm corresponds to the amide (–NH) bound proton,
another sharp peak at 7.26 ppm corresponds to the reference
Sample preparation for SEM and AFM imaging
For the freeze-dried compound of [(L-GluA-5-BE)-b-(L-AspA-4-
BE)], B0.5 g was dispersed in B1000 mL of propanol. Therefore,
the well-dispersed [(L-GluA-5-BE)-b-(L-AspA-4-BE)] in a volume of
300 mL was separated into a cryo sample vial, and further used
for ultra-sonication for 8 minutes at RT. Furthermore, the
sonicated sample was divided into three parts (samples 1, 2
and 3) at 100 mL each. A 100 mL sample was kept in an ice-bath
(temperature maintained at B10 1C), the second one was
maintained at room temperature (i.e., B25 1C), and the third
one was maintained at body temperature (i.e., B37.4 1C).
Finally, all three samples were used for the sample preparation
for various microscopic characterizations, such as SEM and
AFM imaging for morphological observations. However, the
propanol-dispersed final compound (B20 mL) was drop-casted
onto the surface of a clean and neat piece of glass, and allowed
to dry at RT for 24 h. Since the polymer samples are non-
conducting in nature, they were sputter-coated with metal (Au)
ions before the SEM imaging. Similarly, a well-dispersed
[(L-GluA-5-BE)-b-(L-AspA-4-BE)] solution of B20 mL was drop-
casted onto the surface of the mica sheet, and allowed to dry at
RT for 24 h. The samples without any sputter coating were
loaded for AFM imaging.
1
solvent CDCl3 used for H NMR characterization, and the peak
position at 8 ppm belongs to the aromatic benzene ring, as
shown in Fig. S4(c) (ESI†).
Furthermore, identical 1H NMR peak position values were
observed for L-AspA-4-BE in Fig. S5(c) (ESI†).2,18,19 The NCA-
converted L-GluA-5-BE and L-AspA-4-BE further underwent the
ring-opening polymerization with palladium activated charcoal
(Pd/C-10%) with the help of the continuous purge of hydrogen
gas in the presence of dry THF solvent. Finally, the synthesized
di-BCP of [(L-GluA-5-BE)-b-(L-AspA-4-BE)] was confirmed
Results and discussion
Morphology of [(L-GluA-5-BE)-b-(L-AspA-4-BE)]-based
nanoflower capsules
Fig. 1, 2 and Fig. S1 (ESI†) illustrate the steps involved in the through FTIR and 1H NMR characterizations, as shown in
synthesis of the AB-type di-block copolymer (di-BCP) of Fig. S3 (ESI†).
[(L-GluA-5-BE)-b-(L-AspA-4-BE)] through the facile modified
ring-opening polymerization reactions.
The final di-BCP compound was dispersed in propanol and
separated into three samples. Sample 1 was stored at low
Fig. 2(a and b) corresponds to NCA of L-GluA-5-BE and temperatures (i.e., maintained at B10 1C with the help of an
L-AspA-4-BE, and (c) shows the di-BCP of [(L-GluA-5-BE)-b-(L- ice-bath), sample 2 was stored at room temperature/RT (i.e.,
AspA-4-BE)] synthesized with the help of Pd/C (palladium B25 1C), and sample 3 was maintained at body temperature
activated charcoal) and continuous purging of hydrogen (H2) (i.e., B37.4 1C). Furthermore, under the microscopic (SEM)
gas, followed by stirring with dry THF solvent at B70 1C for imaging, sample 2 (stored at RT) gave nanoflowers with surface-
4 h.2,8,12–17 The conversion of the acid group of L-GluA-5-BE and functionalized petals and porosity.19,20 From Fig. 3 and 4, we
L-AspA-4-BE into NCA was carried out with the help of triphos- can see the morphology of the nanoflowers with evenly dis-
gene as a strong oxidizing agent (Fig. S2, ESI†). The NCA- tributed petals and flakes throughout the capsule surface.
converted L-GluA-5-BE and L-AspA-4-BE were further used for Among those, the porosity was also observed in between the
the synthesis of di-BCP of [(L-GluA-5-BE)-b-(L-AspA-4-BE)] (Fig. 1, inter-petals of the capsule surface. The gaps or inter-petal
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Fig. 3 SEM micrographs (a–c) of NFCs at lower magnifications and (d–f)
at higher magnifications. Di-BCP of [(L-GluA-5-BE)-b-(L-AspA-4-BE)] was
dispersed in propanol, and the sample was maintained at B25 1C.
Fig. 5 SEM micrographs (a–d) acquired from lower to higher magnifica-
tion for disk-type compressed capsules with nanochannels. Propanol-
dispersed di-BCP of the [(L-GluA-5-BE)-b-(L-AspA-4-BE)] sample was
maintained at B10 1C.
distances between the petals and their distribution were shown
from the SEM and FESEM micrographs. However, these NFCs
with surface-distributed petals were evidenced with various
microscopic characterizations. It is worth noting that NFCs
with petals are stable and suitable for DDSs.21 Similarly, sample
3 (maintained at body temperature, i.e., B37.4 1C) also gave
nanoflowers with surface-distributed nanopetals and porosity
(Fig. 4). These NFCs are also stable at RT and body temperature.
At considerably lower temperatures (8–12 1C), these NFCs are
sensitive and show drastic changes in morphology. From Fig. 3
and 4, it is evident that the petals are distributed throughout
the capsule surface. From the powder XRD data, the synthe-
sized di-BCP of [(L-GluA-5-BE)-b-(L-AspA-4-BE)] solid state was
confirmed as amorphous in nature. These amorphous [(L-GluA-
5-BE)-b-(L-AspA-4-BE)]-based NFCs are highly sensitive to tem-
perature conditions, as well as the concentrations, pH values,
and different solvents.2,22 From Fig. 3 and 4, the average NFCs
size was calculated as B30 mm (in diameter).
Interestingly, the NFCs (Fig. 3 and 4) are stable at RT and
body temperature, which is an advanced characteristic property
for consideration as potential cargo for selected drug delivery.
However, these NFCs are pH sensitive. Even though the tem-
perature difference is B12 1C, these NFCs show negligible
morphological changes in-between the petal arrangements.
In contrast, sample 1 dispersed at lower temperatures, such
as 8–10 1C (maintained by using ice-bath), showed a disk-type
compressed morphology of the capsules with surface-
distributed nanochannels (Fig. 5). However, the nucleation
and growth of the nanopetals depend on the dispersion solvent,
as well as the temperature of the medium.23 The surface of the
nano-scaled capsules distributed with petals and with unique
porosity is a remarkable and promising characteristic property
of NFCs for theranostic advancements.24 These advanced
features of NFCs show remarkable drug loading capacity, and
are attributed to the significant release (controlled and sus-
tained with precise doses) to malignant organs and tissues
through the pH-based release kinetics. Thus, compressed
nanochannels can also contribute to loading the drug mole-
cules throughout the surface of the disk-type spherical capsules
with certain limitations (Fig. 5). Nanoscale flowers can be a host
material to incorporate the drug molecules inside the pores and
in-between the petals.25,26 However, the interaction between
the dispersion solvent and di-BCP leads to the growth of
micron-sized capsules, along with nanoscale surface-
distributed flower-type petals. However, the powder of di-BCP
of [(L-GluA-5-BE)-b-(L-AspA-4-BE)] was dispersed in propanol
solvent, which further nucleated through the self-assembly
process between the active functional groups of the polymer
(such as ester (R–COOR), hydroxyl (–OH), amide (–NH)).
The pi-bonds with larger resonating capacity lead to the
Fig. 4 SEM micrographs (a and c) for NFCs at lower magnifications and
(b and d) at higher magnification. Di-BCP of [(L-GluA-5-BE)-b-(L-AspA-4-
BE)] was dispersed in propanol solvent, and the sample was maintained
at B37.4 1C.
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formulation of NFCs. The nanopetal growth and arrangement
on the surface of the spherical capsule is very much random,
inconsistent, and highly asymmetric. The gaps or inter-petal
distances and surface porosity show the significant character-
istic feature of the capsule to load the nanomedicines and
anticancer drugs for targeted delivery. The main driving force
to form NFCs is dependent on the dispersion solvent and the
temperature conditions of the medium.26 Moreover, the affinity
and electrostatic interaction between the active functional
groups of the synthesized di-BCP of [(L-GluA-5-BE)-b-(L-AspA-4-
BE)] lead to the formation of NFCs. In addition, the self-
assembly process coordinates with the dispersion solvent,
followed by the nucleation, which ultimately gives the
surface-distributed NFC complexes. When the propanol-
dispersed compound is maintained at different temperature
conditions, the nucleation growth of the nanopetals takes place
for the spherical nanoflower capsules. Thus, it exhibits poros-
ity, which enhances the drug loading capacity. With the change
of the dispersion solvent and temperature conditions, the
morphology, size, shape, and surface properties might have
been changed.27,28 Through the self-assembly process, the
active functional groups tend to show affinity with the disper-
sion solvent and trigger the nucleation of petals. The amphi-
philic nature of di-BCP of [(L-GluA-5-BE)-b-(L-AspA-4-BE)] with
ionic capacity formulates the spherical shaped NFCs.28 Further-
more, the amino-acid residues with nitrogen moieties or bind-
ing sites tend to contribute to the underlying process to
nucleate the petals on the capsule surface.29 With the increase
or decrease of temperature conditions, sample medium results
in morphological changes from perfect NFCs to compressed
disk-type morphology. These amino-acid-based polymer nano-
flower capsules are recommended for many applications,
such as catalysis, drug delivery, and analytical science
applications.2,17,29 The hydrophobic and hydrophilic nature of
the final compound helps in molecular imprinting to create
pores, cavities and voids on the surface of the capsule. There is
a need to understand the detailed interface science between the
synthesized block copolymer and solvent used for dispersion,
along with the temperature effect. The porosity and flake
thickness were further characterized with the help of AFM
(atomic force microscopy) imaging of non-contact mode opera-
tion. Fig. 6 and Fig. S7, S8 (ESI†) show the characteristic
features of the surface-functionalized nanocapsules.
Fig. 6 SEM image (a) depicts the hierarchy of the nanopetals on the NFC
surface. AFM image (b) for the phase(s), (c) for the 3D and (d) surface profile
of NFCs.
cost-effective and facile method to synthesize the block copoly-
mer of amino-acids, such as [(L-GluA-5-BE)-b-(L-AspA-4-BE)].
This synthesized, di-block copolymer plays a crucial role as a
potential payload for many anticancer drugs and nanomedi-
cines through the surface-active complexes.2,29–31 Furthermore,
[(L-GluA-5-BE)-b-(L-AspA-4-BE)]-based NFCs were characterized
through energy-dispersive X-ray spectroscopy (EDAX) for ele-
mental and composition analysis. Fig. S9 (ESI†) corresponds to
EDAX, and illustrates the presence of elements in the developed
NFCs of di-BCP of [(L-GluA-5-BE)-b-(L-AspA-4-BE)]. The inset
table in Fig. S9 (ESI†) shows the carbon (C) weight at
B64.55% and atomic at B72.48%; for oxygen (O), the weight
is B30.39% and atomic is B25.61%, which were calculated for
the designed NFCs.32 The remaining elements in the inset table,
such as sulphur (S) and potassium (K), appeared from a mild
amount of impurities present in NFCs. However, the thermal
stability (degradation temperature, weight loss and heat flow) of
the synthesized di-BCP of [(L-GluA-5-BE)-b-(L-AspA-4-BE)] and
developed NFCs is an essential property to perform TGA (thermo-
gravimetric analysis), as well as DSC (differential scanning calori-
metry). Fig. S10 (ESI†) corresponds to the TGA and DSC for the
synthesized di-BCP of [(L-GluA-5-BE)-b-(L-AspA-4-BE)]. The plot (a)
corresponds to TGA, and shows the spontaneous degradation of
the NFCs concerning an increase in temperature from 40 to
200 1C. Similarly, the plot (b) corresponds to DSC, and depicts the
exothermic heat flow of the NFCs for the same temperature
range. The heating and cooling rates of temperature were
adjusted to 10 1C minÀ1. Such surface-functionalized NFCs
The yellow arrows in Fig. 6(a) show the petal thickness, as
well as the distances between two particular petals.17,30 Fig. 6(b)
corresponds to the 2D phase(s) of NFCs, and shows a line scan
between two petals that depicts the gap between them. Fig. 6(c)
corresponds to the 3D image of petals with surface properties
and height profiles. Similarly, Fig. 6(d) depicts the surface
profile of NFCs with width, depth, and thickness of particular
petals on the capsule surface. The gap between those two
particular petals (petals 1 and 2) was calculated as 4.2 mm.
The average depth of the petals was measured as 21.2 nm.2,17,31
were attributed to the good surface area (B12 m2 À1), and
g
confinement of the nanomedicines and anticancer drugs for
targeted release-based on their thermal stability at body
temperature.4,11,14,33
Viability assay on [(L-GluA-5-BE)-b-(L-AspA-4-BE)] NFCs
The design and development of [(L-GluA-5-BE)-b-(L-AspA-4-BE)]- The designed NFCs of [(L-GluA-5-BE)-b-(L-AspA-4-BE)] were
based carrier capsules with characteristic surface-functionalized further used to examine the biocompatibility (cell viability)
features is a novel approach in the drug delivery fields. It is a through the MTT assay on cultured cell lines, such as HeLa
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(human epithelial cervical carcinoma cells) and HEK293
(human embryonic kidney cells). Concentrations of 1, 5, 10,
15, and 25 mg mLÀ1 [(L-GluA-5-BE)-b-(L-AspA-4-BE)] NFCs were
mixed with the cell lines. Furthermore, samples were allowed to
incubate for 24 h in a CO2 incubator. Slow agitations, followed
by stirring, were performed during the treatment hours.
Between these two cell lines, HeLa is the cancerous cell line
and HEK293 is the healthy (non-cancerous) cell line used for
the cell viability assay. Cultured cell lines of approximately
4 Â 103 cells per well were added to NFCs. After 24 h of
incubation, the 96-well plate was taken out of the incubator
and used for the cell viability study (MTT assay). Fig. 8(a) shows
the cell viability of [(L-GluA-5-BE)-b-(L-AspA-4-BE)]-based NFCs
and the results of their biocompatibility for both cell lines. The
cell viability (survival rate) for both cell lines is greater than
98% for the bare NFCs. This demonstrates the suitability and
biocompatibility of NFCs for drug delivery systems, as reported
by Amgoth et al.2,4,11,14 and Chagala et al.34
Fig. 7 Plot (a) for cumulative PTX release at acidic pH conditions, (b) for
PTX release at basic pH medium, (c) for PTX released at room temperature,
and (d) for PTX release at body temperature.
Drug release studies
The designed NFCs of [(L-GluA-5-BE)-b-(L-AspA-4-BE)] were
further used to load the anticancer drug (PTX) to evaluate the drug release of B70% was achieved.4,14,38 As the pH levels
drug-loading and release kinetics. However, PTX loading to gradually increased from 3 to 7, there was a slight decrease in
[(L-GluA-5-BE)-b-(L-AspA-4-BE)]-based NFCs was performed the release of drug molecules (i.e., pH B 4 gave 64%, pH B 5
through the incubation of capsules and PTX for 24 h in a CO2 gave 58%, pH B 6 gave 54% and pH B 7 (neutral) gave 49%).
incubator.34,35 The incubation was followed by slow stirring for Fig. 7(b) shows the percentage of cumulative drug release at
better interactions between NFCs and drug molecules. Further- basic pH (i.e., B8, 9, 10, 11 and 12). The PTX release at such
more, these drug-loaded NFCs were mixed with HeLa cells to basic pH medium showed a decreased trend (like 47, 43, 40, 36
check the cell inhibition according to the release of PTX and 31%). Similarly, Fig. 7(c) shows the drug release (68%) at
molecules. Fig. 8(b) shows the UV-Visible characterization, room temperature (RT) with acidic (pH B 3.5) medium.
which shows the absorbance of the anticancer drug PTX.35 Fig. 7(d) shows the PTX release (B74%) at body temperature
The maximum intensity of the excited UV-Visible spectra at (i.e., B37.4 1C) under acidic pH (pH B 3.5) medium. The acidic
B271 nm corresponds to the highly intense peak of PTX. release of PTX at body temperature was greater (B74%) than
The absorbance vs. wavelength plot shows the difference the release at RT and physiological conditions. The drug release
between the standard PTX plots, as well as PTX released from at different pH conditions, as well as at different temperature
NFCs. The anticancer drug PTX (B1.0 mg) was dissolved in conditions, was performed through the membrane bag dialysis
B1000 mL of DMSO (dimethyl sulfoxide), and its concentration method followed by slow stirring. During the slow agitations,
was calculated as B0.01 mmol. Interestingly, these designed the incorporated drug molecules came out and passed through
NFCs showed B780/1000 ng mgÀ1 loading of PTX (which is the dialysis membrane for the release kinetic studies.38
equal to 78%).36 Rough surfaces with surface functionalized The decanted solution of the drugs were used for UV-Visible
nanopetals and porosity are a unique property of the capsule to spectroscopic characterization with different time intervals,
significantly enhance the loading efficacy.
and those points were used to draw a cumulative drug release
These [(L-GluA-5-BE)-b-(L-AspA-4-BE)]-based NFCs loaded plot. However, these NFCs showed controlled release at differ-
with PTX and void porous space and gaps between petals were ent pH values and temperature conditions. It demonstrates the
accommodated with drug molecules in a host–guest manner. first-order release kinetics, which depends on the concen-
Furthermore, these NFCs were used for drug release kinetics tration and periods with specified dosages.38,39
through the membrane bag dialysis methods.37 However,
Cell inhibition assay
14 kDa membrane bags were used for the drug release studies
at various pH conditions, as well as two different temperatures Furthermore, these potential carrier capsules of [(L-GluA-5-BE)-
(such as RT and body temperature). Furthermore, the PTX b-(L-AspA-4-BE)] were used to evaluate the cell inhibition studies
release studies were performed in acidic (acetic acid) and on cancerous HeLa cells. Fig. 8(c) shows the cell inhibition for
neutral (PBS) conditions, and in ammonium hydroxide as the bare NFC, free PTX and nanoformulation [(NFCs)-(PTX)]. HeLa
basic medium. From Fig. 7(a), we can see the increased drug cells were used for the in vitro cell-based inhibition studies.
release trends from neutral to acidic pH levels at different time Based on the MTT assay, the data showed an increased ten-
intervals (max 24 h). More acidic conditions resulted in greater dency in cell inhibition with an increase of concentration of
drug release. When the condition was too acidic, (pH at B3), a PTX released from designed NFCs and its effect on cells. Six (6)
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different concentrations of PTX (1, 5, 10, 15, 20, and 25 mg mLÀ1
)
Apoptosis is a programmed cell-killing process, whereas our
were separately added to fixed (B4 Â 103 cells per well) numbers [(NFCs)-(PTX)-(HeLa)] showed significant cell killing within the
of HeLa cells in each case, and allowed to incubate for 24 h. 24 h treatment. Fig. S16(a–d) (ESI†) shows the apoptosis for the
The blank column shows zero cell inhibition because of the cells (HeLa) as cultured, NFCs with HeLa cells, PTX with HeLa
absence of nanoformulations. For the control, no drug-loaded cells and nanoformulation of [(NFCs)-(PTX)-(HeLa)] cells.
NFCs were added. Cells as cultured were incubated and used for
Tumor-bearing rats and anticancer efficacy studies
MTT assay analysis for zero cell inhibition. The rest of the six (6)
wells of the 96-well plate were added with nanoformulations. Healthy and well-cultured 4–6 weeks grown rats were purchased
During the incubation, the interactions between the cells and from Jackson Laboratory, and used for inflammation studies.
released drug molecules led to the cell inhibition process. The tumor growth was observed, and allowed to grow up to 150
The incubation period of 24 h with the nanoformulations and to 220 mm3 size. Rats were then randomly divided into four
cells was termed as the treatment time before the MTT assay. parts, such as PBS (pH of 7.2 with 0.02 mmol) saline, NFC
As the PTX concentration increased from B1 to 25 mg mLÀ1, the (500 mg mLÀ1) dispersed in propanol, NFC–PTX (1 : 1 equimolar
cellular inhibition also gradually increased.2,14,39
ratio of 500 mg mLÀ1), and PTX (500 mg mLÀ1 dissolved in
B1 mg mLÀ1 concentration of PTX showed B26% cell organic solvent DMSO). Furthermore, drugs were injected sub-
inhibition, and B25 mg mLÀ1 of PTX concentration showed cutaneously, and the tumor growth was illuminated and mon-
B79% of inhibition. The bare NFCs showed negligible cell itored from day 1 to day 30 (Fig. 9(a)). Similarly, Fig. 9(b) shows
inhibition. From these studies, it is evident that NFCs with the densitometric analysis and expression of COX-2, iNOS by
surface-functionalized properties showed enhanced drug load- western blot analysis for the control sample and samples with
ing efficacy, as well as the potential release to kill the cancer NFC, PTX, and its combination (such as [(NFC)–(PTX)]).
cells with preferable cell inhibition. These in vitro cell-based The expressions of COX2 and iNOS in tumor tissue were
studies showed that the characteristic properties of the novel examined by immunohistochemistry, *p o 0.05 vs. the NFC
carrier capsules of [(L-GluA-5-BE)-b-(L-AspA-4-BE)] are predomi- groups. Fig. 9(c) shows the tumor volume with respect to time in
nant for cancer treatment and other biomedical usages through days with our NFC groups and saline. Fig. 9(d) shows a gradual
the pH-responsive controlled release kinetics.37,39 Fig. 8(d) increase in the body weight of the rats post-treatment. Once the
shows the FACS (fluorescence-activated cell-sorting), and tumor growth reached the optimum size, the tumor growth
quadrant(Q) represents the cell distribution after 24 h treat- tissue was extracted, as shown in Fig. 9(a), and was used for
ment with our developed NFC-based nanoformulations. anticancer efficacy examinations. The NFC-PTX combination
Further, cells killed with various reasons such as mechanical at the same resolution shows the ruptured and fragmented
damage, medium (Q1) is measured as 4%, the necrotic cells morphology of the cells. Fig. 9(e–h) visually demonstrates the
mechanical damage (Q2) is 8%, Q3 corresponds to the viable normal tissue structure without any harm (control) and the
cells 56% and apoptotic cells (Q4) corroborates the cells killed damage on the inflammatory lesions with NFC, PTX and com-
with our developed nanoformulations (which means because of bined [(NFC)–(PTX)]. From the H and E staining in the tissue
PTX released from NFCs) has been calculated as B34%. treated with [(NFC)–(PTX)], it can be concluded that the devel-
oped nanoformulations are promising agents to kill the cancer
tumor cells. Furthermore, this was demonstrated with the
apoptosis examinations, and the detailed results have been
incorporated into the respective sections.
Cell uptake
After the cell inhibition assay, further cell-based studies were
performed to examine the morphology of the treated HeLa
cells. 0.5 mg of [(L-GluA-5-BE)-b-(L-AspA-4-BE)]-based NFCs were
dispersed in 2.5 mL of propanol. 1 mg of PTX was dissolved in
DMF, and 200 mL of the drug suspension was used to load with
NFCs, which were already dispersed in propanol. Finally, those
two were mixed through the vigorous stirring methods to load
the drug molecules inside the pores and gaps in-between the
NFC petals. The drug-loaded NFCs were then added to the wells
of the 96-well plate. Each well was filled with a fixed amount of
HeLa cells (B4 Â 103 cells per well).2,4,40 The combination of
nanoformulation and HeLa cells were allowed to incubate for
24 h under humidified conditions by using CO2 incubator. Slow
agitations were performed to release the PTX molecules from
Fig. 8 Plot (a) for cell survival rate, (b) UV-Visible for standard PTX and
NFCs and interact with HeLa cells, followed by the penetration
of the cell membrane. After 24 h of treatment, the wellplate was
released PTX from NFCs, (c) for cell inhibition, and (d) for FACS of
apoptotic cells.
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Fig. 10 LSCM images of HeLa (Human epithelial cervical carcinoma) cells.
Top to bottom images were acquired from lower to higher magnifications.
Cells were captured after 24 h treatment with PTX released from the
designed NFCs. Cells were stained with red fluorescent dye Rho-B and
green fluorescent dye FITC, followed by their bright field and merged
images (right side).
the ruptured, leaky, and fragmented morphology of the cells
after treatment with our developed nanoformulations.
The yellow arrows in Fig. 10(h) show the nucleus of the cell
with the ruptured cell morphology. The aggregate of the
ruptured cells was segregated through slow stirring methods
to separate the individual cells, as represented in Fig. 10(l), for a
better understanding of the treated cells and the cellular uptake
of nanomedicines and dye distribution.
Fig. 9 (a) Normal photograph of rat representing the dissected tumor.
(b) Densitometric analysis and expression of COX-2, iNOS by western blot
analysis for NFC, PTX and its combination. (c) Tumor volume and (d) rat
body weight as a function of time. Microscopic images (e–h) with H and E
staining of tumor tissue for the control sample and samples treated with
NFC, PTX, and NFC–PTX, respectively. Scale bars were 100 mm.
Conclusions
In conclusions, we report the simple and facile synthesis of an
amino-acid-based di-block copolymer of [(L-GluA-5-BE)-b-(L-
taken out of the incubator. Imaging was performed by laser AspA-4-BE)] between L-glutamic acid-5-benzyl ester and
scanning confocal microscopy (LSCM).38–40 Wells were added L-aspartic acid-4-benzyl ester. The synthesis was followed by
with different dyes to stains the cell organelles to understand the ring-opening polymerization (ROP) without the use of any
the ruptured morphology of the complete cells. The dyes used surfactants and precursors. Importantly, the synthesized
for the studies included FITC (fluorescence isothiocyanate), a [(L-GluA-5-BE)-b-(L-AspA-4-BE)] compound gave NFCs upon
green fluorescent dye that stains the cell membrane, and dispersion in propanol. Interestingly, [(L-GluA-5-BE)-b-(L-AspA-
DAPI, a blue fluorescent dye (2-(4-aminophenyl)-1H-indole-6- 4-BE)] showed the thermosensitive morphology of NFCs at
carboxamidine), which particularly stains the genetic material three different temperature conditions, such as low (B10 1C),
(adenine and thymine/A-T) rich regions of the cells. Similarly, RT (B25 1C) and body temperature (i.e., B37 1C). From the
rhodamine-B, a red fluorescent dye, was also added to the cells SEM micrographs, the sample maintained at lower temperature
to stain the cell membrane.
gave disk-shaped capsules with surface-distributed nanochan-
The well-stained cell samples were used for confocal micro- nels. The samples maintained at RT and body temperature gave
scopic characterization to understand the cellular uptake, NFCs with surface-functionalized nanopetals and porosity.
followed by the ruptured, swelled, and fragmented morphology Furthermore, our collective and systemic studies on drug
of the treated cells. Fig. 10 shows the HeLa cells treated with loading and release revealed the unique characteristic property,
our developed nanoformulations and the morphology of the which is the pH-responsive drug release nature of NFCs.
cells after 24 h treatment.40–42 Fig. S11 and S12 (ESI†) show the The findings in our study demonstrate the enhanced drug
confocal microscopic images of the HeLa cells that exhibit loading capacity (i.e., B78%; w/w) of NFCs because of the
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11 C. Amgoth, A. Singh, R. Santhosh, S. Yumnam, P. Mangla,
R. Karthik, T. Guping and M. Banavoth, RSC Adv., 2019,
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surface-distributed petals and porosity. Therefore, NFCs deliver
the PTX target specifically upon changing the trajectory medium.
Our in vitro and in vivo studies validate NFCs as prominent
delivery systems, which stimulate apoptosis to kill the cancer
cells. Confocal microscopic images of the cell uptake demon-
strate the inherent imaging performance of the treated (HeLa)
cells. It explores NFCs as thermosensitive and pH-responsive
potential cargo materials for cancer theranostics. Such pro-
mising NFCs with remarkable characteristic features can be
recommended for catalysis, biosynthesis, and molecular bio-
imprinting technology. In addition, it is proposed for tissue
engineering and other cancer therapeutics.
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Author contributions
C. A. performed all of the experiments, including the synthesis
of di-BCP and development of NFCs, and wrote the manuscript.
S. C. helped with the statistical data analysis. T. M. contributed
by performing the MTT assay (cell viability and inhibition)
studies. G. T. contributed through the correction of the manu-
script and valuable input.
Conflicts of interest
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23 C. Dohmen, D. Edinger, T. Frohlich, L. Schreiner, U. Lachelt,
C. Troiber, J. Radler, P. Hadwiger, H. P. Vornlocher and
E. Wagner, ACS Nano, 2012, 6, 5198.
The authors declare that there are no conflicts of interest.
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molecules, 2012, 13, 3581.
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Acknowledgements
The authors would like to acknowledge the Department of
Chemistry, Zhejiang University for financial support and research
facilities. This work has been jointly supported by the National
Natural Sciences Foundation of China (Grant No. 51873185).
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