Histone H2A-peptide-hybrided upconversion mesoporous silica nanoparticles for bortezomib/p53 delivery and apoptosis induction

Article abstract of DOI:10.1016/j.colsurfb.2019.110674

The design and development of advanced gene/drug codelivery nanocarrier with good biocompatibility for cancer gene therapy is desirable. Herein, we reported a gene delivery nanoplatform to synergized bortezomib (BTZ) for cancer treatment with histone H2A-hybrided, upconversion luminescence (UCL)-guided mesoporous silica nanoparticles [UCNPs(BTZ)?mSiO₂-H2A]. The functionalization of H2A on the surface of UCNPs(BTZ)?mSiO₂ nanoparticles realized the improvement of biocompatibility and enhancement of gene encapsulation and transfection efficiency. More importantly, then UCNPs(BTZ)?mSiO₂-H2A/p53 induced specific and efficient apoptotic cell death in p53-null cancer cells and restored the functional activity of tumor suppressor p53 by the success of co-delivery of BTZ/p53. Moreover, the transfection with UCNPs(BTZ)?mSiO₂-H2A/p53 in p53-deficient non-small cell lung cancer cells changed the status of p53 and substantially enhanced the p53-mediated sensitivity of encapsulated BTZ inside the UCNPs(BTZ)?mSiO₂/p53. Meanwhile, core-shell structured mesoporous silica nanoparticles UCNPs?mSiO₂ as an UCL agent can detect the real-time interaction of nanoparticles with cells and uptake/penetration processes. The results here suggested that the as-developed UCNPs(BTZ)?mSiO₂-H2A/p53 nanoplatform with coordinating biocompatibility, UCL image, and sustained release manner might be desirable gene/drug codelivery nanocarrier for clinical cancer therapy.

Full text of DOI:10.1016/j.colsurfb.2019.110674

Colloids and Surfaces B: Biointerfaces 186 (2020) 110674
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces
journal homepage: www.elsevier.com/locate/colsurfb
Histone H2A-peptide-hybrided upconversion mesoporous silica
nanoparticles for bortezomib/p53 delivery and apoptosis induction
a
a
a,
a
a
a
a
Jiamin Rong , Pengcheng Li , Yakun Ge *, Hongling Chen , Jie Wu , Renwen Zhang , Jun Lao ,
b,
a,
Dawei Lou *, Yuanxin Zhang *
College of Biology & Food Engineering, Jilin Institute of Chemical Technology, Jilin, Jilin, 132022, PR China
a
b
Department of Analytical Chemistry, Jilin Institute of Chemical Technology, Jilin, Jilin 132022, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Histone H A
2
Gene delivery
p53
Apoptosis
Upconversion
The design and development of advanced gene/drug codelivery nanocarrier with good biocompatibility for
cancer gene therapy is desirable. Herein, we reported a gene delivery nanoplatform to synergized bortezomib
(
BTZ) for cancer treatment with histone H2A-hybrided, upconversion luminescence (UCL)-guided mesoporous
silica nanoparticles [UCNPs(BTZ)@mSiO -H2A]. The functionalization of H2A on the surface of UCNPs(BTZ)@
mSiO nanoparticles realized the improvement of biocompatibility and enhancement of gene encapsulation and
transfection efficiency. More importantly, then UCNPs(BTZ)@mSiO -H2A/p53 induced specific and efficient
apoptotic cell death in p53-null cancer cells and restored the functional activity of tumor suppressor p53 by the
success of co-delivery of BTZ/p53. Moreover, the transfection with UCNPs(BTZ)@mSiO -H2A/p53 in p53-de-
ficient non-small cell lung cancer cells changed the status of p53 and substantially enhanced the p53-mediated
sensitivity of encapsulated BTZ inside the UCNPs(BTZ)@mSiO /p53. Meanwhile, core-shell structured meso-
porous silica nanoparticles UCNPs@mSiO as an UCL agent can detect the real-time interaction of nanoparticles
with cells and uptake/penetration processes. The results here suggested that the as-developed UCNPs(BTZ)@
mSiO -H2A/p53 nanoplatform with coordinating biocompatibility, UCL image, and sustained release manner
might be desirable gene/drug codelivery nanocarrier for clinical cancer therapy.
2
2
2
2
2
2
2
1. Introduction
promotion of autophagy, or enhancement of DNA repair can effectively
recover its tumor suppressor ability in p53-mutation or p53-null cell
lines and even trigger a variety of anti-proliferative effects by enhan-
cing the activity of wt-p53 in p53-defficent cells [6–10]. Thus, plasmid
DNA encoding wt-p53 gene-mediated protein expression in human
cancer cells provides a promising strategy for tumor gene therapy.
Nevertheless, the prerequisite of successful gene therapy is a safe
and effective gene vehicle that can deliver the therapeutic genes and/or
chemotherapeutic drugs to a specific targeted tissue or organ. Several
inorganic non-viral vehicles including silica, iron oxide, and gold na-
noparticles have been widely investigated in the past few years owing
to ease preparation, specific binding, low cytotoxicity and side effects,
facile surface functionalization [11]. Among them, owing to their un-
ique nanochannels and mesopores, mesoporous silica nanoparticles
(MSNs) exhibited excellent capabilities, such as high drug payload,
stimuli-controlled release, co-delivery of drug and gene [12,13].
Moreover, due to their stable and rigid framework, MSNs were more
resistant to heating, pH variations, mechanical stress, and hydrolysis-
induced degradation compared to other polymer-based carriers [14].
It's now widely accepted that the human tumorigenesis and pro-
gression arise from mutations involving constitutive activation of on-
cogenes and inactivation of tumor suppressor genes [1]. In particular,
the status of p53 is closely correlated with tumor progression, even
more than 50% of human tumors carry mutations of p53 gene [2]. The
mutations of p53 gene have been implicated in the regulation of cycle
arrest, apoptosis and response to cell stress including oncogene acti-
vation, DNA damage, hypoxia [3,4]. Notably, p53 mutations often
caused the loss of wild-type (wt) activity and the formation of protein
with suppressor ability against wt-protein [3]. Also, some mutations
resulted in the acquisition of novel oncogenic functions, commonly
referred as to mutant gain-of-function (GOF) p53 containing induction
of the angiogenic factors, metastasis, resistance to specific anti-cancer
drugs and inactivation of other members of the p53 family [5].
Therefore, exogenous p53 gene therapy that replaces p53 or introduces
p53 mediating tumor regression through induction of apoptosis, cell
arrest, alternation of metabolism, prevention of ROS accumulation,
⁎
Corresponding authors.
E-mail addresses: yakunge@126.com (Y. Ge), 1054100603@qq.com (D. Lou), yxzhang@jlict.edu.cn (Y. Zhang).
https://doi.org/10.1016/j.colsurfb.2019.110674
Received 26 August 2019; Received in revised form 15 November 2019; Accepted 24 November 2019
Available online 28 November 2019
0927-7765/ © 2019 Elsevier B.V. All rights reserved.

J. Rong, et al.
Colloids and Surfaces B: Biointerfaces 186 (2020) 110674
However, one of the main drawbacks for these nanoparticles is that
there is no sufficient surface positive charge to absorb the therapeutic
negative DNA to a specific tissue or organ and a lack of aqueous solu-
bility within the size window of bacteria and could potentially trigger
acute immune response in vivo [15,16]. To overcome these short-
comings, the linkage of polymer or positively charged protein on MSN
surface has been actively studied to improve its biocompatibility and/or
enhance its transfection efficiency [17]. For example, recombinant
biomimetic chimeric peptides, novel tissue penetrating peptideticle
with charge-structure switching in tumor microenvironment, nano-
biomimetic carriers composing of either HWYG (HNH) or Gp41 (GNH)
fusogenic peptides were designed and genetically engineered to mimic
viral properties to overcome these obstacles in gene transfection pro-
cess [18]. What's more, the conjunction of cationic peptides, such as
H2A, blood-brain barrier (BBB) penetrating peptide K16ApoE, HIV
TAT-derived peptide, has been shown to be favorable for improving the
surface positive charge and facilitating the adsorption of gene, resulting
in reduced cytotoxicity, immunogenicity profiles, and improving
transfection efficiency [19–23]. In addition, this strategy can enhance
the DNA-condensing capacity and nuclear localization ability of or-
ganic/inorganic hybrid polyplexes, improving the DNA-delivery cap-
ability, and the biocompatibility. Altogether, these unique advantages
of natural positively charged protein modification have endowed these
organic/inorganic hybrid polyplexes with superior transfection effi-
ciency than other polymers.
Plasmids p3XFLAG-CMV-p53 and pEGFP-N3 were amplified in
Escherichia coli DH5α and purified using an Axygen Plasmid Maxi kit.
The Annexin-V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection
kit I was purchased from BioTeke Corporation (Beijing, China).
Mitochondrial membrane potential assay kit with JC-1 probe, caspase
activity assay kits were purchased from Bestbio Company (Shanghai,
China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), propidium iodide (PI), 4,6-diamidino-2-phenylindile (DAPI),
and all other chemicals used in the present study were obtained from
Sigma-Aldrich (St. Louis, MO, USA). Trizol, ExTaq reverse transcriptase,
PrimeScript™RT Master Mix were obtained from TaKaRa (Dalian,
China).
2.2. Cell culture
Human cervical cancer cells HeLa and non-small lung cancer cell
lines NCI-H1299 were purchased from the Type Culture Collection of
the Chinese Academy of Sciences (Shanghai, China) and cultured in
Dulbecco’s modified Eagle’s medium or 1640 medium supplemented
with 10% (v/v) fetal bovine serum, 100 unites/mL penicillin, and 100
μg/mL streptomycin at 37 °C in a humidified atmosphere of 5% CO
2
.
2.3. Synthesis of NaYF
4
:Yb3+Er3+ (UCNPs)
The β−NaYF
to previous report with slight modifications [25]. In a typical proce-
dure, the YCl , YbCl , and ErCl solutions were prepared by dissolving
the corresponding Y , Yb and Er in excess hydrochloric acid
aqueous solution to form the rare-earth chloride compounds. And then,
the resultant YCl (0.8 mmol), YbCl (0.18 mmol), and ErCl (0.02
:Yb3+Er3+ core (UCNPs) was synthesized according
4
Herein, gene vehicles β−NaYF
mSiO -H2A) were synthesized through the conjunction of natural his-
tones (calf thymus) to UCNPs@mSiO surface via the EDC/NHS-medi-
:Yb3+Er3+@mSiO
-H2A (UCNPs@
4 2
2
3
3
3
2
2
O
3
2
O
3
2 3
O
ated coupling reaction. The polyplexes were characterized with their
high payload of gene, preferably efficiency of in vitro gene transfection,
and good biocompatibility. Additionally, BTZ were loaded into the
3
3
3
mmol) were poured into a three-necked bottle and homogenized for 30
min at 80 °C to evaporate hydrochloric acid, and then 6 mL of OA and
15 mL of ODE were added at room temperature. The mixture was
stirred vigorous at 150 °C for 30 min to yield a clear solution. After that,
it was cooled down to 30 °C and 10 mL methanol solution containing
mesopore of the UCNPs@mSiO
2
-H2A to forming the complexes UCNPs
(
BTZ)@mSiO -H2A, which is a potential and specific inhibitor of 26S
2
proteasome and can induce the apoptosis of prostate cancer cell lines
via stimulating p53 expression [24]. Then, those were applied as a p53
gene/drug (bortezomib) co-delivery vector and its up-conversion ima-
ging, therapeutic efficacy, and possible molecular mechanisms in HeLa
4
NaOH (2.5 mmol) and NH F (4 mmol) were quickly added dropwise
into the above-mentioned solution. The reaction temperature was
raised to 50 °C and the solution was kept at this temperature for 2 h. To
remove methanol, the resulting solution was heated for 2 h at 70 °C.
Subsequently, the prepared solution was further vacuumized for 10 min
at 100 °C and kept at 300 °C under a nitrogen flow with vigorous
stirring for 1 h. After cooled down to room temperature, the resulting
nanoparticles were precipitated with excess ethanol, collected by cen-
trifugation (10,000 × g for 10 min), and then rinsed three times with
ethanol/cyclohexane (v/v = 1:1, 10 mL) solution. The obtained na-
noparticles were dispersed in 10 mL of cyclohexane, which was applied
directly in the next experiment.
(
p53-wt) cells and NCI-H1299 (p53-null) cells were systematically
evaluated.
2. Materials and methods
2.1. Materials
All the chemical reagents were directly utilized without further
purification. Rare earth oxides (Y
Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC,
8%), and N-Hydroxysulfosuccinimide sodium salt (sulfo-NHS, 98%)
were obtained from Alfa Aesar (Ward Hill, USA). Ammonium nitrate
NH NO3, 98.5%), cetyltrimethylammonium bromide (CTAB, 99%),
2 3 2 3 2
O , Yb O and Er O3, 99.9.9%), 1-
9
2 2
2.4. Synthesis of UCNPs(BTZ)@mSiO -NH
(
4
sodium hydroxide (NaOH, 99.9%), and cyclohexane (99.5%) were all
purchased from Beijing Chemical Reagent Company (Beijing, China).
Oleic acid (OA, 90%), Tetraethyl orthosilicate (TEOS, 99.9.9%), octa-
decene (ODE, 90%), 3-aminopropyltriethoxysilane (APTES, 98%), am-
The synthesis of core-shell structured UCNPs@mSiO
2
was con-
ducted as previously protocol with slight modifications [26]. Briefly, 2
−1
mL of cyclohexane solution containing UCNPs (5 mg mL ) was mixed
with 20 mL of CTAB (100 mg) aqueous solution. The mixture was
stirred vigorously to remove the cyclohexane at room temperature and
monium fluoride (NH
from Sigma-Aldrich Chemical Co. Ltd (St. Louis, MO, USA). The rare
earth chlorides of YCl , YbCl , and ErCl were prepared by dissolving
4
F, 99.99%), bortezomib (98%) were purchased
−1
generate a transparent solution (0.5 mg mL ). And then a mixture of
20 mL of distilled water, 3 mL of ethanol, and 150 μL of NaOH (2 M)
were added into 10 mL of above-mentioned mixture. The solution was
heated to 70 ℃ and then150 μL of TEOS was added dropwise to the
obtained mixture and the reaction was continued for 3 h. The as-syn-
3
3
3
the corresponding rare earth oxides in a hydrochloric acid solution
followed by evaporating the hydrochloric acid solution.
The antibodies against procaspase 3, procaspase 8, procaspase 9,
Bcl-2, Bax, Bak, cytochrome c, p53, PARP, β-actin and horseradish
peroxidase (HRP)-labeled goat anti-mouse IgG were purchased from
Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Histone H2A (calf
thymus) was purchased from Sigma-Aldrich Chemical Co. Ltd (St. Louis,
MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal
bovine serum (FBS) were obtained from Gibco (Grand Island, USA).
2
thesized UCNPs@mSiO was collected by the centrifugation at 12,000
× g for 15 min, washed with ethanol 3 time and then dried overnight in
the vaccum oven at 60 ℃. And then, 100 μL of APTES was added into
250 mL of ethanol solution containing 100 mg of as-prepared UCNPs@
mSiO
2
nanoparticles and refluxed at 78 ℃ for 36 h. Finally, NH
2
-
modified UCNPs@mSiO
2
(UCNPs@mSiO -NH was obtained by
2
2
)
2

J. Rong, et al.
Colloids and Surfaces B: Biointerfaces 186 (2020) 110674
centrifugation, washed with distilled water and ethanol. To remove
without FBS (pH 7.4) and replenished with transfection composites.
CTAB surfactant, further purification of UCNPs@mSiO
formed for cleaning the mesopores of nanoparticles. The as-synthesized
UCNPs@mSiO -NH were re-dispersed in the solution that contained 40
mL of ethanol and 10 mL of ethanol solution including NH NO (10
mg). The mixture was refluxed at 60 ℃ for 5 h, collected by cen-
trifugation (10,000 × g for 10 min), and then washed with ethanol 3
times. Afterward, the product was re-dispersed in the distilled water.
This process was repeated three times to eliminate all the residue CTAB
as excessively as possible. Finally, the product was dried by lyophili-
zation for overnight to obtain the solid power. Next, The UCNPs@
2
-NH
2
was per-
2
After 5 h incubation at 37 °C under 5% CO , the cells were rinsed twice
with PBS and continued to culture in 1 mL of DMEM with 10% FBS for
another 24 h. Afterwards, the transfected cells were washed with PBS
three times and visualized using a Nikon DS-Ri2 fluorescence micro-
scopy (Osaka, Japan). The transfection efficiency was determined by
evaluating the level of GFP expression using the Beckman Flow
Cytometry Analyzer (Beckman CytoFLEX, Suzhou, China).
2
2
4
3
2.9. MTT assay
mSiO
BTZ)@mSiO
procedure, 20 mg of as-prepared UCNPs@mSiO
00 μL bortezomib solution and then the resultant mixture was stirred
at room temperature for 48 h to form UCNPs(BTZ)@mSiO -NH
2
was used as reservoirs to load bortezomib to prepared UCNPs
-NH as described in a previously report [27]. In a typical
-NH was mixed with
The cytotoxicity of nanocarriers were evaluated using MTT assay.
(
2
2
Briefly, NCI-H1299 and HeLa cells were plated in a 96-well plate at a
2
2
density of 5 × 103 cells per well and cultured at 37 °C under 5% CO
2
2
atmosphere overnight. Equal amount of fresh medium containing car-
riers or carrier/p53 nanocomposites replaced the corresponding
medium. After 24 h of incubation, 20 μL of MTT solution in PBS (5 mg
2
2
.
2
2.5. Synthesis of UCNPs(BTZ)@mSiO -H2A
−
1
mL ) was added and plates were incubated for another 4 h at 37 °C.
Afterward, the medium was discarded and 150 μL of DMSO was added
into each well for dissolving formazan crystals. Finally, the plates were
shaken for 10 min, and the absorbances at 495 nm were detected using
at a BioTek CYTATION 5 imaging reader (Vermont, USA).
The UCNPs(BTZ)@mSiO
ated grafting reaction of carboxyl group of H2A on the primary amino
group of UCNPs(BTZ)@mSiO -NH . Firstly, H2A (0.8 g, 0.057 mM) and
2
-H2A were prepared by EDC/NHS-medi-
2
2
EDC (0.11 g, 0.57 mM) were added into 10 mL of phosphate-buffered
saline (PBS) buffer (pH 7.2), then 0.6 mg of NHS was added dropwise to
the mixture, and gently stirred for 30 min at room temperature.
2.10. Hemolysis assay
Afterward, 10 mg of UCNPs(BTZ)@mSiO
mixed with the resulting mixture and the reaction proceeded for 3 h at
7 °C with reciprocating oscillation (130 rpm). The H2A-functionalized
UCNPs(BTZ)@mSiO was centrifugated at 12,000 × g for 10 min at 4
C and redispersed in 0.5 mL PBS (pH 7.2) buffer.
2 4
-NH nanoparticles was
The endosome escape potential of nanocomposites was evaluated
using hemolysis assay. In a typical procedure, healthy human whole
blood was collected into heparinized vacutainers under aseptic condi-
tions. The erythrocytes (108 cells/200 μL) obtained from whole blood
by centrifugation (5000 × g for 5 min) were mixed with various mass
ratios of carrier/p53 nanocomposites in phosphate buffer saline (PBS)
pH 7.4 or pH 5.4 and incubated at 37 °C with slight shaking for 1 h.
Afterward, the mixture was centrifuged at 12,000 × g for 5 min and
absorbance of the supernatant was detected at 450 nm using a BioTek
CYTATION 5 imaging reader (Vermont, USA). The Triton X-100 (1%)
was used a positive control, and PBS (pH 7.4 and 5.4) was use a ne-
gative control.
3
2
°
2.6. Characterization
Transmission electron microscopy (TEM) was conducted using a FEI
G2 S-Twin TEM (FEI Co., USA) with a field emission gun at 200 kV. The
X-ray diffraction (XRD) patterns were performed on a D8 advance dif-
fractometer (Bruker Co., Germany) with graphite monochromatized Cu
Kα (0.15406 nm) radiation. Fourier transform infrared (FTIR) spectra
were recorded on a Bruker Vertex 70 spectrometer using the KBr pellet
technique. Zeta potential was carried out using Malvern Zetasizer Nano
ZS (Malvern Instruments Ltd., UK). The upconversion luminescence
2.11. Drug loading and release
(
UCL) imaging of cells was conducted using a Nikon Ti-S fluorescent
2
The as-synthesized UCNPs(BTZ)@mSiO -H2A was collected and
washed three times with PBS to remove unbinding BTZ by centrifuga-
tion (12,000 g for 10 min). The absorbance at 270 nm of UCNPs(BTZ)@
microscopy (Nikon, Tokyo, Japan) equipment with a CW NIR laser at
8
08 nm.
2
mSiO -H2A and supernatant solution were examined using a BioTek
CYTATION 5 imaging reader (Vermont, USA). The loading efficiency
was calculated with the following equation: loading efficiency =
2
.7. Gel retardation assay
The pDNA package potential of UCNPs@mSiO
2
-H2A was evaluated
[
(initial weight - supernatant weight)/initial weight] × 100%. Then,
using gel retardation assay, which indicated the free migration of
pDNA. In a typical procedure, UCNPs@mSiO -H2A/ p3XFLAG-CMV-
UCNPs(BTZ)@mSiO -H2A was dispersed in 2 mL PBS (pH = 7.4) and
2
2
was stirred at room temperature. At indicated time intervals, samples
were subjected to centrifugation (12,000 × g for 10 min) and then the
supernatant was replaced with 2 mL of fresh PBS. The amount of re-
leased BTZ was determined with a UV–vis spectrophotometer at a wa-
velength of 270 nm.
p53 composites with different N:P ratios were prepared in loading
buffer and incubated for 30 min at room temperature before the ex-
periment. The binding ability to pDNA was then performed on a 1%
agarose gel at 100 v for 1 h. The bands were visualized using ethidium
bromide (EB) through BioDoc-It® 220 Imaging System.
2.8. In vitro gene transfection assay
2.12. DAPI staining
NCI-H1299 and HeLa cells were seeded in a 24-well tissue culture
NCI-H1299 and HeLa cells were incubated with UCNPs(BTZ)@
mSiO -H2A/p53 nanocomposites with 1 μg of plasmid for 24 h. The
2
4
plate at a density of 5 × 10 cells per well and cultured as monolayer
cells overnight (70–80% confluence). Transfection composites were
prepared at indicated mass ratios by mixing the plasmid DNA (pEGFP-
N3 or p3XFLAG-CMV-p53) with serum-free medium containing H2A,
UCNPs@mSiO -NH , PEI25 K, or UCNPs@mSiO -H2A and incubated
2 2 2
for 30 min at room temperature before in vitro experiments.
cells were then washed twice with ice-cold PBS and incubated with
DAPI stock solution (100 ng/mL in PBS) for 5 min in the dark.
Afterward, the cells were rinsed with PBS twice and fixed with ice-cold
ethanol (75%) for 5 min at 4 °C in the dark. The immobilized cells were
washed with ice-cold PBS twice and observed using a Nikon DS-Ri2
fluorescence microscopy (Osaka, Japan).
Subsequently, medium was discarded, cells were washed with DMEM
3

J. Rong, et al.
Colloids and Surfaces B: Biointerfaces 186 (2020) 110674
2
.13. Cell apoptosis analysis by FACS
were centrifuged at 10,000 × g for 10 min, pellets were discarded and
supernatants were incubated with the corresponding caspase-specific
fluorogenic substrate Ac-DEVD-AFC (caspase-3), Ac-LETD-AFC (cas-
pase-8), and Ac- LEHD-AFC (caspase 9) for 30 min. The samples were
recorded at 405 nm using a BioTek CYTATION 5 imaging reader
(Vermont, USA)
Apoptosis in NCI-H1299 and HeLa cells were determined using the
Annexin V/Propidium Iodide (PI) Apoptosis Detection Kit. Briefly, 3 ×
1
5
0 cells per well were seeded in 6-well plates overnight. After trans-
fected with carrier/p53 nanocomposites for 24 h, the cells were col-
lected, washed with ice-cold PBS twice, and then incubated with 100 μL
of binding buffer containing Annexin V-FITC (5 μL) and PI (5 μL) for 10
min in the dark at room temperature. Subsequently, the samples were
added to 400 μL binding buffer and then the apoptosis were determined
by a flow cytometry using the Beckman Flow Cytometry Analyzer
2.17. The detection of mitochondrial membrane potential measurement
using JC-1 probe
The changes of mitochondrial membrane potential (MMP) were
measured by detecting the switch of probe JC-1 from red to green
fluorescent. Briefly, NCI-H1299 and HeLa cells were plated in 6-well
(
Beckman CytoFLEX, Suzhou, China). The acquisition and analysis of
data were performed using FlowJo software version 7.6 (Tree Star, Inc.,
Ashland, OR, USA).
5
plates with a density of 3 × 10 cells overnight. After transfecting with
carrier/p53 nanocomposites (2:1, w/w) for 24 h, the cells were washed
with PBS. Then the cells were trypsinized and collected through cen-
trifugation at 2000 × g for 2 min. Subsequently, the resulted cells were
washed with cold JC-1 buffer and were resuspended in 500 μL of JC-1
buffer solution. Finally, the cells were determined by a flow cytometry
using the Beckman Flow Cytometry Analyzer (Beckman CytoFLEX,
Suzhou, China). The acquisition and analysis of data were performed
using FlowJo software version 7.6 (Tree Star, Inc., Ashland, OR, USA).
2
.14. RT-PCR analysis
RT-PCR was utilized for evaluating the mRNA expression level of
5
p53 gene. The cells were seeded in a 6-well plate at 2 × 10 of cells per
well and cultured overnight. After transfection, the culture medium was
removed and the cells were rinsed with PBS twice. Total RNA was ex-
tracted using TRIzol reagents and 1 μg of total RNA was use as tem-
plates for preparing the cDNA. The synthesized cDNA was employed as
a template for PCR amplification of p53 gene as mentioned below:
denaturation (94 °C for 30 s), annealing (55 °C for 30 s), and extention
2.18. Statistical analysis
(
72 °C for 1 min) for 30 cycles. Specific primers for β−actin (5′-TCT
The Statistical Program for Social Sciences software (SPSS 19.0,
SPSS, Inc., IL, USA) was used for statistical analysis. All quantitative
data are expressed as the mean ± standard, and statistical differences
were evaluated using analysis of variance and the Least Significant
Difference post hoc test and considered to be presented at p < 0.05.
Except mentioned, all experiments were repeated in triplicate in three
independent experiments.
GGCACCACACCTTCTACAATG-3′ for the forward primer and 5′-GGAT
AGCACAGCCTGGATAGCAA-3′ for the reverse primer) and p53 (
5
5
used for the RT-PCR assay. The products of PCR were detected by 1%
agarose gel electrophoresis and visualized with a BioDoc-It® 220
Imaging System.
′-GGCTCTGACTGTACCACCATCCA-3′ for the forward primer and
′-GGCACAAACACGCACCTCAAAG-3′ for the reverse primer) were
3
. Results and discussion
.1. Synthesis and characterization of UCNPs@mSiO
The stepwise preparation process of the precursors and final UCNPs
2
.15. Western blotting analysis
3
2
-H2A
NCI-H1299 and HeLa cells were plated in 100 mm dishes at a
6
density of 2 × 10 cells overnight. The cells were then transfected with
free BTZ, UCNPs(BTZ)@mSiO
2
-NH
2
/p53, or UCNPs(BTZ)@mSiO
2
-
(BTZ)@mSiO -H2A/p53 nanocomplex is shown in Scheme 1. First of
2
H2A/p53 nanocomposites at 37 °C for 24 with un-transfected cells as
negative cells. After transfection, the cells were collected, washed with
PBS three times, and total proteins were extracted by the Total Protein
Extraction Kits (Bestbio Company, China). The concentration of protein
was quantified using the BCA Protein Aaasy Kit (Beyotime®
Biotechnology, China). Subsequently, 30−50 μg of proteins was sub-
jected to the 12% SDS-PAGE and transferred to the polyvinylidene
fluoride (PVDF) membranes (0.22 μm) for 2 h at 60 V by using a Mini
Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA). The
membranes were blocked with PBS with 5% nonfat milk power and
all, uniform size and monodisperse UCNPs were synthesized through a
conventional thermal decomposition method according to previously
report [25]. As shown in Fig. 1A, the TEM image shows that oleic acid
stabilized UCNPs are monodispersed with about 25 nm particle size.
From the high-resolution TEM (HRTEM) image (Figure S1), lattice
spacing value of a single NaYF
which is consist with the D-spacing (220) plane of hexagonal NaYF
confirmed good crystallinity of NaYF UCNPs. After coating a meso-
porous silica shell (mSiO ) on upconversion nanocrystals and APTES-
functionalized, the particle diameter of core-shell UCNPs@mSiO -NH
nanocrystals increased from 25 nm to 61 nm (Fig. 1B). By using (1-
ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride/N-hy-
droxysuccin-imide (EDC/NHS)-mediated couple reaction between NH
modified UCNPs@SiO and histone H2A, UCNPs@SiO -H2A nano-
particles were synthesized [28]. The conjugation of H2A at the
UCNPs@mSiO nanocrystals surface didn't change the mean diameter
4
UCNPs was measured to be about 2.6 Å,
4
and
4
2
2
2
0.1% Tween-20 for 1 h at room temperature. After washed with ice-cold
PBS three times, the membranes were incubated with specific primary
antibodies (dilution ratio: 1:500) overnight at 4 °C. The membranes
were rinsed with ice-cold PBS three times and were incubated with
peroxidase-conjugated secondary antibody (dilution ratio: 1:2000) for 2
h at room temperature. The blots were visualized using enhanced de-
veloper with PCA and Luminol followed by scotography in a darkroom.
2
-
2
2
2
of nanoparticles and still maintained monodispersity in water (Fig. 1C).
As shown in Figure S 2a, the characteristic vibration peaks of Si-O-Si
−
1
−1
2.16. Caspase activity
(1081 cm ), Si-OH (961 cm ) in the FTIR spectrum confirmed the
success of silica-coating [29]. Additionally, the vibration peak at 1640
−
1
−1
Caspase activity was detected using the corresponding Caspase
cm-1 and 2935 cm or 2962 cm was ascribe to the vibration of N-H
in the NH group and CH stretching vibrations, respectively (Figure S
2b) [30]. The presence of H2A is demonstrated by the appearance of
Activity Assay Kit. After transfected with carrier/p53 nanocomposites
2:1, w/w) for 24 h, the cells were collected by trypsinization, washed
with ice-cold PBS twice, and harvested via centrifugation for 5 min at
000 × g. The resulted cells were lysed with 100 μL of lysis buffer [50
mM HEPES (pH 7.4), 0.1% Triton X-100, 10% sucrose, 5 mM EGTA, and
mM EDTA] and incubated 30 min in ice. The resulted suspensions
2
2
(
−
1
−1
−1
unique peaks at 1456 cm
(C–N), 1526 cm
(CO), 1663 cm]
−1
4
(NH), 3088 cme
(NHe) from internal vibration of the amid bonds
(Figure S 2c) [30]. The zeta potential change from -12.1 ± 2.71 mV to
23.5 ± 2.83 mV further confirmed the successful coupling of H2A
5
4

J. Rong, et al.
Colloids and Surfaces B: Biointerfaces 186 (2020) 110674
2
Scheme 1. Synthetic procedure of UCNPs(BTZ)@mSiO -H2A/p53.
(
Figure S3). The sizes of UCNPs, UCNPs@mSO
2
, and UCNPs@mSiO
2
-
2
blood cells was examined for UCNPs@mSiO -H2A complexes over the
−1
H2A were also determined through dynamic light scattering (DLS)
measurements, and the effective hydrodynamic diameters were shown
in Figure S4, which matches the TEM results. In the X-ray diffraction
concentration range 6.25−400 μg mL , similar to the results of the
PBS negative control, suggesting the excellent blood compatibility
(Fig. 2C). Meanwhile, Triton X100 destroyed a great of number of red
blood cells (RBCs) compared with that of different concentration of
3
+
3+
pattern of oleic acid stabilized NaYF
diffraction peaks can be indexed to hexagonal phase NaYF
crystals, which is well matched with the JCPDS standard card
28–1192), indicating the core is composed of hexagonal phase NaYF
4
: Yb Er
nanocrystals, all the
: Yb3 Er
+
3+
UCNPs@mSiO
4
2
-H2A nanoparticles (Fig. 2D). Taken together, UCNPs@
mSiO -H2A exhibited excellent biocompatibility with high tolerance to
2
(
4
no-specific protein adsorption and low hemolytic activity after H2A
modification, making them optimal candidate as gene delivery vehicle
crystals (Fig. 1D). Moreover, under 980 laser irradiation, the trans-
parent colloidal solutions of nanocrystals before and after the mod-
ification exhibited similar fluorescence emission spectra with three
2
centered peaks at 522, 543, and 653 nm corresponding to the
H
11/
3.3. In vitro transfection efficiency
4
4
4
4
3+
2
- I15/2,
S3/2- I15/2, and
F
9/2
-
4
I
15/2 transition of Er
-H2A stained NCI-
H1299 cells showed brighter UCL (Figure S5). In the final procedure of
the assemble process, the UCNPs@mSiO -H2A was mixed with p53 as a
model gene, which was absorbed on the surface H2A to assemble a final
UCNPs@mSiO -H2A/p53. The condensation capability of UCNPs@
mSiO -NH and UCNPs@mSiO -H2A with pDNA was determined via
gel retardation experiment (Fig. 1F). Entire pDNA retardation was ob-
served to be induced by UCNPs@mSiO -H2A/pDNA (0.6:1). Further-
more, the UCNPs@mSiO -H2A showed relatively high binding affinity
to pDNA and could form nanoscale complexes with pDNA.
ions, respec-
tively (Fig. 1E) [31]. Meanwhile, UCNPs@SiO
2
2
The In vitro transfection efficiency of the UCNPs@mSiO -H2A na-
nocomplexes was firstly quantified using pEGFP-N3 as the reporter
gene in NCI-H1299 and HeLa cells by flow cytometry. As shown in
2
Fig. 3A, UCNPs@mSiO
highly gene transfection efficiency compared to UCNPs@mSiO
2
-H2A/pDNA (N:P = 0.8:1) complexes exhibited
2
-NH /
2
2
2
2
2
pDNA (N:P = 1:1), H2A/pDNA (N:P = 1:15) [33], and PEI25 K/pDNA
(N:P = 1:10) [34] complexes in NCI-H1299 and HeLa cells. In HeLa
2
cells, transfection efficiency of UCNPs@mSiO
H2A/pDNA (N:P = 1:15), and PEI25 K/pDNA (N:P = 1:10) were
9.62%, 10.64%, and 57.19%, respectively, whereas UCNPs@mSiO
H2A/pEGFP-N3-mediated transfection realized higher transfection
ratio of 80.96%. For NCI-H1299 cells, UCNPs@mSiO -H2A/pEGFP-N3
resulted in even higher transfection ratios (86.94%) compared to
UCNPs@mSiO -NH /pEGFP-N3 (11.58%), H2A/pEGFP-N3 (12.09%),
2 2
-NH /pDNA (N:P = 1:1),
2
2
-
3.2. In vitro cytotoxicity analysis and hemolysis assay
2
Since the in vitro cytotoxicity of nanoparticles is a critical pre-
2
2
PEI25 K/pEGFP-N3 (59.77%), respectively. The improved transfection
efficiency was probably ascribed to the introduction of histone H2A
moieties increasing the penetrating capabilities and uptake efficiency.
Moreover, the lower cytotoxicity of calf histone H2A increases pro-
cessing and presentation of antigenic peptide-major histocompatibility
complexes to T cells in macrophages decreasing immunological rejec-
tion response [35,36]. These changes of the gene expression of en-
hanced green fluorescent protein pEGFP-N3 was imaged using a
fluorescence microscope, which showed that there were much more
GFP-positive cells in the HeLa or PC-3 cells incubated with UCNPs@
requisite and important factor for clinical applications, the potential
cytotoxicity of UCNPs@mSiO -H2A was determined using an MTT
assay on HeLa and NCI-H1299 cells. As demonstrated in Fig. 2A and B,
no obviously cytotoxicity is observed with various investigated con-
2
centrations. Moreover, comparing to UCNPs, UCNPs@mSiO
histone H2A modification improves the biocompatibility of UCNPs@
mSiO -H2A because the conjugation of H2A on UCNPs@mSiO surface
2 2
-NH ,
2
2
substantially optimizes cationic charge density and thereby relieves
damage of cell membranes.
Additionally, the presence of serum is a major factor influencing the
gene transfection efficiency in cationic polymer-mediated transfection
process due to nonspecific binding between carriers and serum proteins.
More importantly, the adsorption of serum proteins on the gene vehicle
surfaces can result in thrombosis, blood coagulation functional failure,
and eventually clearance of nanoparticles by the reticuloendothelial
system (RES) [32]. As shown in the present study, no hemolysis of red
mSiO
mediated transfection (Fig. 3B). Indeed, the results suggested that the
H2A-modified UCNPs@mSiO -H2A complexes possessed significantly
2 2
-H2A/DNA compared to UCNPs@mSiO2-NH , PEI25K- or H2A-
2
advantages over many other cationic polymer-based transfection re-
gents.
5

J. Rong, et al.
Colloids and Surfaces B: Biointerfaces 186 (2020) 110674
Fig. 1. The characterization of UCNPs@SiO
2
-H2A. (A) TEM micrographs of NaYF
4
:20 %Yb3+,2%Er3+ upconversion nanoparticles (UCNPs), (B) mesoporous silica-
coated UCNPs@mSiO2, and (C) UCNPs@mSiO
The UCL spectra of as-synthesized UCNPs or UCNPs@mSiO
mSiO -H2A in complex with 1 μg of pEGFP-N3 plasmid at various mass ratio.
2
-H2A. (D) XRD patterns and the corresponding standard card (JCPDS No,28–1192) of the as-synthesized UCNPs. (E)
2
-H2A under 980 nm NIR laser excitation. (F) Gel retardation assay of UCNPs@mSiO -NH or UCNPs@
2
2
2
3
.4. Drug loading and release
measuring the UV–vis absorption band of bortezomib at 270 nm. Sub-
sequently, UCNPs(BTZ)@mSiO -NH and UCNPs(BTZ)@mSiO -H2A
was incubated with p3XFLAG-CMV-p53 plasmids at room temperature
for 2 h to form UCNPs(BTZ)@mSiO /p53 and UCNPs(BTZ)@mSiO
H2A/p53 complexes. In vitro drug release profile over period of 96 h in
phosphate-buffer saline (PBS, pH 7.4) at room temperature are shown
in Fig. 4A. Cumulative drug release profiles indicated that more than
2
2
2
Previous investigations have demonstrated that bortezomib induced
p53-mediated apoptosis in human glioma cells [37]. Furthermore,
UCNPs@mSiO -H2A exhibited excellent biocompatibility, hemolysis,
and high p53 gene transfection efficiency. Thus, we anticipated that
synergistic effects could be realized by co-delivery of bortezomib and
2
2
-
2
the p53 gene using UCNPs@mSiO
UCNPs@mSiO -H2A was used as reservoirs for delivering bortezomib.
The encapsulation ratio of bortezomib was calculated to be 41.2% by
2
-H2A as the nanoplatform. Herein,
78.7% of drug discharged from UCNPs(BTZ)@mSiO
8 h, followed by a sustained and slow release (11.5% of drug released
within 96 h). Compared to UCNPs(BTZ)@mSiO /p53, UCNPs(BTZ)@
2
complexes within
2
2
6

J. Rong, et al.
Colloids and Surfaces B: Biointerfaces 186 (2020) 110674
cancer, prostate cancer, breast cancer, colon cancer, and gastric [39].
To further confirm this opinion, the cytotoxicity comparison of free
bortezomib and UCNPs(BTZ)@mSiO
HeLa and NCI-H1299 cells. As shown in Fig. 5A-D, compared to free
BTZ, UCNPs(BTZ)@mSiO -H2A/p53 transfection group significantly
2
-H2A/p53 assay was performed in
2
reduced the viability in HeLa and NCI-H1299 cells. In particular, NCI-
H1299 became more sensitive to equivalent concentration of BTZ,
which suggested that the nanohybrids composed of silica nanoparticles
and histone H2A benefited efficient p53 gene transfection. To further
determine the effect of p53 status on BTZ-induced apoptosis, the per-
centage of carrier/p53 complexes-induced apoptotic cells was ex-
amined using flow cytometry. As shown in Fig. 5E, obviously apoptosis
was observed in HeLa and NCI-H1299 cells for the UCNPs(BTZ)@
mSiO
mSiO
2
-H2A/p53 transfection groups. For HeLa cells, the UCNPs(BTZ)@
-H2A/p53 transfection resulted in 20.46% of cell apoptosis for 48
2
h, whereas H2A, UCNPs(B TZ)@mSiO2-NH
2
2
, or free BTZ mediated
.42%, 2.35%, and 15.4% of cell apoptosis, respectively. For NCI-
H1299 cells, UCNPs(BTZ)@mSiO
2
-H2A/p53 (42.86%) resulted in even
-NH /p53
(0.97), free
BTZ (19.04%), respectively. These data indicated that NCI-H1299 be-
came more sensitive to UCNPs(BTZ)@mSiO -H2A-meidated p53
higher apoptosis ratios compared to UCNPs(BTZ)@mSiO
42.86%), H2A/p53 (1.87%), UCNPs(B TZ)@mSiO2-NH
2
2
(
2
2
transfection than HeLa cells due to different status of p53 gene in these
two tumor cell lines. Meanwhile, apoptosis-induced effects of UCNPs
(
2
BTZ)@mSiO -H2A/p53 in both HeLa and NCI-H1299 have obviously
differences, suggesting the introduction of wt-p53 gene into p53-null
NCI-H1299 cells enhance gene expression and tumor suppressor cap-
ability. However, HeLa cells with wt-p53 were less affected by the
exogenous p53 gene expression and the inhibitory effect was lower than
that of NCI-H1299. Also, the chromatin condensation and apoptotic
bodies of typical characteristics of apoptosis were seen in HeLa and
NCI-H1299 cells after p53 transfection, whereas these morphologic
changes were more evident in UCNPs(BTZ)@mSiO
than others group (Fig. 5F).
2
-H2A/p53 group
To elucidate the relationship between early apoptosis and p53 gene
transfection, we evaluated the expression levels of p53 mRNA by real-
time q-RT-PCR. As shown in Fig. 6A and B, compared to negative
control, free BTZ (1.09-fold), UCNPs(BTZ)@mSiO
transfection group (1.76-fold), UCNPs(BTZ)@mSiO -H2A induced 4.17-
fold increase in relative transcriptional level of p53 mRNA in NCI-
H1299 cells. In HeLa cells, though UCNPs(BTZ)@mSiO -H2A trans-
2 2
-NH -mediated
2
2
fection enhanced p53 mRNA expression level (2.06-fold), there were no
obviously differences between others groups. The results demonstrated
that UCNPs(BTZ)@mSiO -H2A successively triggered the exogenous
2
p53 mRNA expression in NCI-H1299 and HeLa cells. The obviously
changes of p53 protein expression further confirmed the introduction of
exogenous p53 gene, which were consist with the results as above-
mentioned in qRT-PCR assay. As shown in Fig. 6C and B, after UCNPs
Fig. 2. The inhibitory effect of NCI-H1299 (A) and HeLa (B) induced by UCNPs,
(BTZ)@mSiO
p53 protein was seen in NCI-H1299 and HeLa cells, whereas in UCNPs
(BTZ)@mSiO -NH -transfected group or free BTZ, no obviously change
is found. These data demonstrate that the UCNPs(BTZ)@mSiO -H2A
can efficiently transfer p53 gene into the cells and restore the function
of p53.
2
-H2A/p53 transfection for 24 h, high level expression of
UCNPs@mSiO
UCNPs@mSiO
2
-NH
2 2
, or UCNPs@mSiO -H2A. (C) Relative hemolytic activity of
2
-H2A at pH 7.4 and 5.4 and (D) visual observation of hemolysis
2
2
at pH5.4. The hemolysis of Triton X-100 was defined as 100%. The data are
indicated as mean values ± SD of three experiments.
2
mSiO
2
-H2A/p53 exhibited a continuous and slow release profile. The
The p53-mediated apoptosis was involved in the cleavage of pro-
caspase 3 to form the active caspase 3 and then elicited the caspase
cascade for apoptotic cell death [40]. As shown in Fig. 6 C and D, the
expression levels of procaspase 3 is dramatically decreased in HeLa and
sustained release properties facilitated the drug accumulation in the
tumor cells following endocytosis and reduced drug release in normal
tissue, providing a potential to enhance the long-term chemotherapy
effect and reduce toxic side effect [38].
PC-3 cells transfected with UCNPs(BTZ)@mSiO
much lower than in the UCNPs(BTZ)@mSiO -H2A /p53 transfection
group in NCI-H1299 cells. Also, the UCNPs(BTZ)@mSiO -H2A/p53
2
-H2A/p53 complexes,
2
2
3
.5. Induction of apoptosis by p53 transfection
transfection decreased the expression levels of procaspase 9 and did not
obviously affect the expression levels of procaspase 8 in NCI-H1299
cells. Besides, remarkable increase of relative activities of caspase 3 and
caspase 9 and no improvement of procaspase 8 were consist with the
aforementioned results (Fig. 6E and F). Due to the caspase 9 involved in
The status of tumor suppressor p53 has profound effects on the
cancer cell sensitivity to chemotherapeutic agent, bortezomib, which
have been applied to treat various types of solid tumors including lung
7

J. Rong, et al.
Colloids and Surfaces B: Biointerfaces 186 (2020) 110674
Fig. 3. Transfection efficiency assay of H2A, UCNPs@mSiO
cytometry and (B) fluorescence microscopy.
2
-NH
2
, PEI25 K, and UCNPs@mSiO
2
-H2A in HeLa and NCI-H1299 cells was detected using (A) flow-
the mitochondria-mediated apoptotic signal pathway, we then detected
the expression levels of Bax (pro-apoptotic protein) and Bcl-2 (anti-
apoptotic protein), which regulated the formation of mitochondrial
outer membrane permeabilization pore (MOMPP) and could promote or
inhibit apoptosis [41–43]. As illustrated in Fig. 6 D, the expression le-
vels of Bax and Bak sharply increased in NCI-H1299 after UCNPs(BTZ)
increase of permeability of mitochondria membrane, and the leak-out
of apoptotic effectors (Figure S6). These results further demonstrate
that UCNPs(BTZ)@mSiO -H2A/p53 transfection can trigger a mi-
2
tochondria-mediated apoptosis through p53-restoration mode.
@mSiO
2
-H2A/p53 transfection, whereas the expression level of Bcl-2
4. Conclusion
decreased and the expression ratio of Bcl-2/Bax obviously declined
suggesting the formation of MOMPP and the trigger of apoptosis.
Meanwhile, UCNPs(BTZ)@mSiO -H2A/p53 transfection upregulated
2
the expression levels of cytochrome c. Finally, the change of mi-
tochondrial membrane potential (MMP) was detected using JC-1 by
flow cytometry after p53 gene transfection. As we have seen, the NCI-
In summary, we successfully synthesized H2A-hybrid silica-coating
upconversion nanoparticles and efficiently applied it to codelivery p53
gene and bortezomib. This carrier is characterized with low cytotoxi-
city, low hemolysis activity, high payload of gene and drug, high gene
transfection efficiency, and upconversion image. Furthermore, UCNPs
H1299 cell population after UCNPs(BTZ)@mSiO
2
-H2A/p53 transfec-
2
(BTZ)@mSiO -H2A- mediated p53 transfection could restored the
tion obviously shifted downward suggesting a decline in MMP, the
function of p53 in NCI-H1299 cells (p53 null), enhanced the sensitivity
of NCI-H1299 to BTZ, and induced the apoptosis of mitochondria-
2 2 2
Fig. 4. Cumulative profiles of BTZ release from UCNPs@mSiO -NH and UCNPs@mSiO -H2A nanocomplexes in pH 7.4 PBS buffer.
8

J. Rong, et al.
Colloids and Surfaces B: Biointerfaces 186 (2020) 110674
Fig. 5. In vitro cytotoxic activity of free BTZ treatment for 24 h on human cervical cancer HeLa(A), and non-small lung cancer cells NCI-H1299 (B). The inhibitory
effect of UCNPs(BTZ)@mSiO -H2A nanocomplexes with equivalent concentration of BTZ on HeLa (C) and NCI-H1299 cells (D) for 24 h. (E) The apoptosis-induced
effects of H2A, UCNPs(BTZ)@mSiO -NH , free BTZ, and UCNPs(BTZ)@mSiO -H2A on HeLa and NCI-H1299 for 48 h. (F) Morphologic observation of HeLa and NCI-
2
2
2
2
H1299 cells treated with H2A, UCNPs(BTZ)@mSiO
2
-NH
2
2
, free BTZ, and UCNPs(BTZ)@mSiO -H2A on HeLa and NCI-1299 for 48 h. Scale bars for all images are 50
μm.
9

J. Rong, et al.
Colloids and Surfaces B: Biointerfaces 186 (2020) 110674
2 2 2
Fig. 6. Relative expression level of p53 mRNA induced by free BTZ, UCNPs(BTZ)@mSiO -NH /p53, and UCNPs(BTZ)@mSiO -H2A/p3 transfection in (A) HeLa or
(B) NCI-H1299 cells. Western blotting analysis of apoptosis-related protein expression (C and D) and relative activity of caspase-3, -8, -9 (E and F) in HeLa and NCI-
H1299 cells treated with free BTZ, UCNPs(BTZ)@mSiO -NH , or UCNPs(BTZ)@mSiO -H2A, respectively.
2
2
2
10

J. Rong, et al.
Colloids and Surfaces B: Biointerfaces 186 (2020) 110674
mediated signal pathway. This investigation provides an efficient
strategy for delivering gene or gene/drug to treat human malignancies.
1334–1346.
[
10] D. Martinez-Zapien, F.X. Ruiz, J. Poirson, A. Mitschler, J. Ramirez, A. Forster,
A. Cousido-Siah, M. Masson, P.S. Vande, A. Podjarny, G. Travé, K. Zanier, Nature
5
29 (2016) 541–545.
[11] N.F. Sun, Z.A. Liu, W.B. Huang, A.L. Tian, S.Y. Hu, Crit. Rev. Oncol. Hematol. 89
2014) 352–357.
12] Y. Gao, Y. Chen, X. Ji, X. He, Q. Yin, Z.W. Zhang, J.L. Shi, Y.P. Li, ACS Nano 5
2011) 9788–9798.
[13] N. Singh, A. Karambelkar, L. Gu, K. Lin, J.S. Miller, C.S. Chen, M.J. Sailor,
S.N. Bhatia, Bioresponsive mesoporous silica nanoparticles for triggered drug re-
lease, J. Am. Chem. Soc. 133 (2011) 19582–19585.
Associated Content
(
[
Supporting Information. Electronic Supplementary Information
ESI) available: HRTEM, FTIR, Zeta potential, UCL image, MMP.
Author Contributions
Y.K.G., D.W.L., and Y.X.Z. conceived and designed the experiments.
J.M.R., P.C.L., H.L.C., J.W., performed the experiments. R.W.Z. and J.L.
conducted the data analysis, and Y.X.Z. produced the manuscript.
(
(
[
14] R. Vago, V. Collico, S. Zuppone, D. Prosperi, M. Colombo, Pharmacol. Res. 111
2016) 619–641.
[15] X.J. Liang, C. Chen, Y. Zhao, P.C. Wang, Methods Mol. Biol. 596 (2010) 467–488.
(
[
[
[
[
16] I.I. Slowing, J.L. Vivero-Escoto, C.W. Wu, S.Y. Lin Victor, Advanced Drug Deliver.
Rev. 60 (2018) 1278–1288.
17] D. Tarn, C.E. Ashley, M. Xue, E.C. Carnes, J.I. Zink, C.J. Brinker, Acc. Chem. Res. 46
Declaration of competing Interest
(
2013) 792–801.
18] A. Majidi, M. Nikkhah, F. Sadeghian, S. Hosseinkhani, Eur. J. Pharm. Biopharm.
07 (2016) 191–204.
19] M. Alipour, A. Majidi, F. Molaabasi, R. Sheikhnejad, S. Hosseinkhani, Int. J. Cancer
43 (2018) 2017–2028.
The authors declare that there was no conflict of interest in the
preparation of this manuscript.
1
1
[
[
[
20] M. Alipour, S. Hosseinkhani, R. Sheikhnejad, R. Cheraghi, Sci. Rep. 7 (2017) 41507.
21] D. Balicki, C.D. Putnam, P.V. Scaria, E. Beutler, PNAS 99 (2002) (2002) 7467–7471.
22] K.M. Ahlschwede, G.L. Curran, J.T. Rosenberg, S.C. Grant, G. Sarkar, R.B. Jenkins,
S. Ramakrishnan, J.F. Poduslo, K.K. Kandimalla, Nanomedicine 16 (2019) 258–266.
23] Z.Y. Kang, Q.B. Meng, K.L. Liu, J. Mater. Chem. B Mater. Biol. Med. 7 (2019)
Acknowledgment
This project was financially supported by the Special Foundation for
Industry Innovation of Development and Reform Commission of Jilin
[
1
824–1841.
(
grant no. 2018C049-4), the Foundation of Education Department of
[
24] S.A. Williams, D.J. McConkey, Cancer Res. 63 (2003) 7338–7344.
Jilin Province (grant no. 2016133), the Youth Foundation of Jilin
Science and Technology Bureau (grant no. 20166026, 201750259), the
Major Programs of the Jilin Institute of Chemical Technology (grant no.
[25] B.B. Ding, H.Y. Peng, H.S. Qian, L. Zheng, S.H. Yu, Adv. Mater. Interfaces 3 (2016)
1500649.
[
[
[
[
[
[
26] C.X. Li, Z.Y. Hou, Y.L. Dai, D.M. Yang, Z.Y. Cheng, P.A. Ma, J. Lin. Biomater. Sci. 1
2013) 213–223.
27] J. Shen, G.S. Song, M. An, X.Q. Li, N. Wu, K.C. Ruan, J.Q. Hu, R.G. Hu, Biomaterials
5 (2014) 316–326.
28] N.C. Fan, F.Y. Cheng, J.A. Ho, C.S. Yeh, Angew. Chem. Int. Ed. 51 (2012)
806–8810.
29] X.J. Kang, Z.Y. Cheng, C.X. Li, D.M. Yang, M.M. Shang, P.A. Ma, G.G. Li, N. Liu,
J. Lin, J. Phys. Chem. C. 115 (2011) 15801–15811.
30] F. Wang, D.K. Chatterjee, Z.Q. Li, Y. Zhang, X.P. Fan, M.Q. Wang, Nanotechnology
(
20180101).
3
Appendix A. Supplementary data
8
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.110674.
1
7 (2006) 5786–5791.
31] G.F. Wang, Q. Peng, Y.D. Li, Acc. Chem. Res. 44 (2011) 322–332.
References
[32] J. Yang, X.F. Hao, Q. Li, M. Akpanyung, A. Nejjari, A.L. Neve, X.K. Ren, J.T. Guo,
Y.K. Feng, C.C. Shi, W.C. Zhang, ACS Appl. Mater. Inter. 9 (2017) 4485–4497.
[
[
[
33] H.P. Liu, I. Söderhäll, Dev. Comp. Immunol. 31 (2007) 340–346.
34] H. Hu, K.M. Xiu, S.L. Xu, W.T. Yang, F.J. Xu, Bioconjug. Chem. 24 (2013) 968–978.
35] J.G. Dohlman, D.J. Pillion, L.A. Rokeach, M.P. Ramprasad, Biochem. Biophys. Res.
Commun. 181 (1991) 787–796.
[
1] D. Hanahan, R.A. Weinberg, Cell 144 (2011) 646–674.
[2] V.J.N. Bykov, S.E. Eriksson, J. Bianchi, K.G. Wiman, Nat. Rev. Cancer 18 (2018)
8
9–102.
[3] M.J. Duffy, N.C. Synnott, P.M. McGowan, J. Crown, D. O’Connor, W.M. Gallagher,
Cancer Treat. Rev. 40 (2014) 1153–1160.
[36] S. Audouy, G. Molema, L. de Leij, D. Hoekstra, J. Gene Med. 2 (2000) 465–476.
[37] C. Li, J.Q. Hu, W.Y. Li, G.S. Song, J. Shen, Biomater. Sci. 5 (2017) 77–88.
[38] A. Prokop, J.M. Davidson, J. Pharm. Sci. 97 (2008) 3518–3590.
[39] X. Ling, D. Calinski, A.A. Chanan-Khan, M.X. Zhou, F.Z. Li, J. Exp. Clin. Cancer Res.
[
4] C. Kandoth, M.D. McLellan, F. Vandin, K. Ye, B.F. Niu, C. Lu, M.C. Xie, Q.Y. Zhang,
J.F. McMichael, M.A. Wyczalkowski, et al., Nature 502 (2013) 333–339.
5] P.A.J. Muller, K.H. Vousden, Nat. Cell Biol. 15 (2013) 2–8.
[
[
[
2
9 (2010) 8–17.
6] K.T. Bieging, S.S. Mello, L.D. Attardi, Nat. Rev. Cancer 14 (2014) 359–370.
7] X. Zeng, Y.X. Sun, W. Qu, X.Z. Zhang, R.X. Zhuo, Biomaterials 31 (2010)
[
[
[
[
40] Y.G. Shi, Mol. Cell 9 (2002) 459–470.
41] G. Fiskum, J. Korean Neurotraumatol. Soc. 17 (2000) 843–855.
42] P. Bernardi, P. Bonaldo, Ann. NY Acad. Sci 1147 (2008) 303–311.
43] C.P. Baines, Annu. Rev. Physiol. 72 (2010) 61–80.
4
771–4780.
[8] Y.Y. He, Y. Nie, L. Xie, H.M. Song, Z.W. Gu, Biomaterials 35 (2014) 1657–1666.
[9] S.K. Misraa, S. Naz, P. Kondaiah, S.A. Bhattacharya, Biomaterials 35 (2014)
11