cell-penetrating and antibacterial nanobioconjugates based
TRANSCRIPT
1
Cell-penetrating and antibacterial nanobioconjugates based on
immobilized BUF-II on polyetheramine (PEA)-modified magnetite
nanoparticles Perez Jessica G1., Muñoz Laura N.2, Muñoz-Camargo C.1, and Cruz Juan C.1
1Department of Biomedical Engineering, Universidad de los Andes, Bogotá, Colombia 2Department of Chemical Engineering, Universidad de los Andes, Bogotá, Colombia
Email: [email protected]
Controlled delivery of therapeutic molecules in a localized manner has become an area of interest due to its potential to reduce
drug exposure to healthy tissue and thereby to minimizing undesirable side effects. The use iron oxide nanoparticles has been
widely accepted in biomedicine due to their ability to transport a variety of biomolecules, their ease of use in the lab and most
importantly, their capability to be actively controlled by external magnetic fields. This might be potentially exploited for their
guided transport and controlled accumulation in specific organs. Buforin II (BUF-II) is an antimicrobial peptide capable of
killing bacteria by attaching to the DNA or RNA and thereby interrupting the microbe’s replication cycle. Here, we evaluated
the immobilization of BUF-II on PEA-modified magnetite nanoparticles and its potential use in a topical treatment. The
obtained nanobioconjugates were characterized via FTIR, DLS, TEM, and TGA. Biocompatibility, translocation ability, and
escape from intracellular endosomes were evaluated on mammalian cells via absorbance assays, confocal microscopy. Also,
internalization and morphological changes were evaluated upon delivery in bacteria via AFM and SEM. Finally, cellular uptake
of the nanobioconjugates was estimated via Fluorescence spectroscopy. The antibacterial assay for the nanobioconjugates
showed reduced bacterial growth of about 50% with respect to native BUF-II. More importantly, the conjugates can penetrate
the membrane of multiple human cell lines, including THP-1, HaCaT, and HFF by largely avoiding the formation of
endosomes. Moreover, they demonstrated exceptional biocompatibility due to its low cytotoxicity and hemolytic tendency.
The preparation of topical treatments was by high-speed dispersion of the BUF-II-PEA-Magnetite nanobioconjugates in
stabilized O/W and W/O emulsions of mineral oil. Hemolytic tendency and antibacterial capacities of the topical treatment
were evaluated on human erythrocytes, and strains of S. aureus and E. coli, respectively. The topical treatment maintained the
antimicrobial capacity of 50% observed for the nanobioconjugates. Altogether, these results unveil the versatility of our system
considering their separate or combined use as an antibacterial or drug delivery system. Future experiments will be directed
toward evaluating the performance of the topical treatment in an infected 3D in vitro dermo-epidermal skin model.
Keywords – Antimicrobial peptides, Buforin II, peptide immobilization, PEA-coated magnetite
I. Introduction
Drug delivery systems have become the focus of a
great variety of research in pharmacology, cancer
therapy, and even diagnostic mechanisms. These
systems address the limitations of drug release rates,
cell and tissue-specific targeting and drug stability.
They are designed using different materials and
chemical strategies, which allow the controlled
release of drugs, the delivery of small molecules, such
as systemic RNA, and the administration of localized
therapies [1]. Administration of a localized therapy is
the main goal of drug delivery in certain diseases such
as cancer, Parkinson’s and Alzheimer’s diseases.
Two main strategies have been devised to achieve
this, which are the use of drug vehicles such as
nanoparticles, and the covalent modification of the
drug with small molecules capable of temporarily
masking the drug’s activity. This method is also
known as prodrug delivery and it minimizes
premature drug activation [2].
The conjugation of peptides to the drugs is an
emerging class of prodrug delivery. This approach is
attractive as some peptides are biodegradable and
show mild to nonimmunogenic responses. At the
same time, some peptides have antimicrobial
capacity, which could potentially confer new
attributes to the conjugate. One of such peptides is
Buforin II, which exhibit both translocation and
antibacterial activity [2].
2
Buforin II (BUF-II) is a 21-amino acid
antimicrobial peptide derived from Bufo bufo
gargarizans, which can kill bacteria. This is achieved
by spontaneously translocating the cell membrane
and subsequently binding to DNA and RNA, which
ultimately leads to an interruption of the replication
cycle [3]–[5]. This amphipathic peptide is composed
by an N-terminal random coil region, an extended
helical region, a proline hinge related to the cell-
penetrating abilities, and a C-terminal regular ɑ-
helical region associated with the antimicrobial
activity [6]. Nevertheless, this peptide is highly
susceptible to proteolytic degradation by endogenous
proteases, which reduces its in vitro and in vivo
lifespan [6]–[8]. A strategy to overcome these issues
is the combination of peptide-based prodrugs with
drug delivery vehicles such as nanoparticles or
emulsions to increase the peptide stability,
permeation and retention, and reduce renal clearance
due to their nanosized dimensions [9].
Over the past few years, emulsions have been
widely used as drug carriers for different routes of
administration such as topical, ocular, intravenous,
intranasal and oral. The use of emulsions as topical
drug delivery vehicles is advantageous mainly due to
enhancement solubility of lipophilic drugs, which in
turn improves the permeation rate, their ability to
protect drugs from hydrolysis and enzymatic
degradation, and their long-term stability and ease of
manufacture at an industrial scale [10]–[12].
Emulsions whose droplets have a nanoscale size (10
to 1000 nm) are known as nanoemulsions and are
widely used as drug delivery systems in treatments
for infection of the reticuloendothelial system (RES),
enzyme replacement therapies in liver, cancer and
vaccination [13].
Another possible drug vehicle are magnetic
nanoparticles (MNPs), which can be made of iron
oxides such as magnetite. These nanoparticles allow
the remote control of their accumulation in specific
organs by means of external magnetic fields, are easy
to prepare and handle, and can be incorporated into
cell separation devices and be used, for enzyme
immobilization [14]–[16]. MNPs have also been used
as immobilization supports for biomolecules such as
epidermal growth factor [17], albumin [18],
bacitracin [19], and in previous studies, BUF-II. Due
to its antimicrobial capacity, BUF-II immobilized on
magnetite nanoparticles can also be incorporated into
O/W or W/O emulsions for the development of
topical treatments for skin wound infections. This
could be a potential route to address the increasing
resistance to treatments against different bacteria.
Our previous studies revealed that upon direct
immobilization on MNPs, BUF-II loses its
antibacterial capacity. The translocation capacity;
however, is fully preserved [20].
Here, we proposed to overcome the loss of
antibacterial activity by immobilizing BUF-II on
MNPs modified with a polymer spacer. The resulting
nanobioconjugates were characterized with the aid of
several spectroscopy and microscopy techniques.
Moreover, we evaluated the antibacterial activity and
biocompatibility. Finally, a topical treatment was
prepared by incorporating the nanobioconjuates into
O/W and W/O emulsions. The obtained formulation
was physiochemically and biologically characterized.
II. Materials and Methods
A. Materials
Iron (II) chloride tetrahydrate (98%),
dimethylformamide (DMF) (99.8%), sodium chloride
(99.9%) and sodium hydroxide (NaOH) (98%) were
obtained from PanReac AppliChem. LysoTracker Green DND-26 was obtained from Life technologies.
Glycerol (99.5%) was purchased from Fisher
Chemical. Iron (III) chloride hexahydrate (97%), tetramethylammonium hydroxide (TMAH) (25%),
(3-Aminopropyl) triethoxysilane (APTES) (98%),
LB medium, N-hydroxy succinimide (NHS) (98%), N-[3-dimethylammino)-propyl]-N’-ethyl
carbodiimide hydrochloride (EDC) (98%), dimethyl
sulfoxide (DMSO) (99%), Carbopol, Tween® 20,
Poly(ethylene glycol) 400 (PEA-400), Mineral oil, Stearic acid (98.5%), 1-Hexadecanol (Cetyl alcohol)
(95%), Span® 80, Vaseline®, Triethanolamine
(99%), Potassium hydroxide (KOH) (85%), and glutaraldehyde (25%) were purchased from Sigma-
3
Aldrich. The polyetheramine Jeffamine® M-600 was purchased from Huntsman (600 molecular weight
polypropylene glycol monoamine). Dulbecco’s
modified eagle’s medium (DMEM), fetal bovine
serum (FBS) and trypsin EDTA were obtained from Biowest. Penicillin/Streptomycin (P/S) was
purchased from Lonza. Buforin II (BUF-II-
TRSSRAGLQFPVGRVHRLLRK) and FTIC-BUF-II was synthesized in the Peptide Synthesis Facility at
Pompeu Fabra University. Purification was
performed by HPLC (>95%) and masses were confirmed via mass spectrometry as we did in a
previous work [21]. Delivery was conducted in Vero
(ATCC® CCL-81), THP-1 (ATCC® TIB-202), HFF
(ATCC® SRC-1041), and HaCaT (ATCC® CRL-2404) cells. Bacteria strains were Staphylococcus
aureus (ATCC 23235) and Escherichia coli (ATCC
25922).
B. Synthesis and functionalization of magnetite
nanoparticles
Iron oxide nanoparticles were synthesized through
co-precipitation of aqueous suspensions of ferric (500
mM) and ferrous chloride (250 mM) salts in the
presence of a 5 M sodium hydroxide solution at 90°C
and under continuous agitation. This solution was left
to cool until it reached room temperature.
The magnetite nanoparticle suspension (100 mg)
was sonicated for 10 minutes, negatively charged
with a TMAH solution (2 mL, 25% v/v), and
sonicated for 10 minutes. (3-Aminopropyl)
triethoxysilane (APTES) (200 𝜇L) was added to the
magnetite solution for silanization along with 100 𝜇𝐿
of acetic acid and sonicated for 10 minutes. Silanized
nanoparticles were washed six times with distilled
water to remove excess of APTES and suspended in
30 mL of distilled water until further use.
C. Polyetheramine oxidation and conjugation
on magnetite nanoparticles
We followed the protocol established by Feng et
al. [22] to oxidize the methoxyethyl termination of the
PEA Jeffamine® M-600. Briefly, JEFFAMINE® M-
600 and an excess amount of KMnO4 (1:2 molar
ratio) were dissolved in 150 mL of type II water. The
solution was left to react under constant magnetic
stirring for 12 h at room temperature. Upon oxidation,
the resulting MnO2 byproduct was filtered out under
vacuum obtaining a colorless homogeneous solution.
Excess KMnO4 and hydrazine byproduct were
removed by dialysis against type II water for 24 h
using a membrane cassette with a 1000 cut-off
molecular weight. A rotary evaporator was then used
to concentrate the oxidized PEA for 6 h at 70°C. PEA-
coated magnetite nanoparticles were obtained by
mixing 100 mg of silanized nanoparticles in 30 mL of
type II water along with 1 mL of 2% v/v
glutaraldehyde-type II water solution. After 30
minutes of reaction at room temperature, the oxidized
PEA solution was added on a molar ratio 1:2 and
stirred overnight. Next, PEA-coated magnetite
nanoparticles were washed 6 times with type II water.
Finally, the nanoparticles were resuspended in 30 mL
of type II water for the subsequent immobilization of
Buforin II.
D. Conjugation of BUF-II on PEA-modified
magnetite nanoparticles
To obtain the BUF-II-PEA-magnetite
nanobioconjugates (Figure 1A), 100 mg of PEA-coated magnetite were suspended in 30 mL of
distilled water and sonicated for 10 minutes. BUF-II
was conjugated to the Carboxyl groups of oxidized
PEA by its N-terminal to form amide bonds with the aid of two equivalents of EDC and NHS (with respect
to the Carboxyl groups). Briefly, 500 µL of unlabeled
or labeled BUF-II solution at 1 mg/mL in sterile NaPB were mixed with the EDC/NHS pre-dissolved
in DMF and were subsequently added to the
nanoparticle suspension. The mixture is sonicated for
5 more minutes and left to react for 24h. After conjugation, samples were thoroughly washed with
type I water and the aid of a strong permanent magnet
to remove excess reagents.
Fig. 1 Chemical structure of the nanobioconjugate magnetite-PEA-BUF-II.
4
E. O/W and W/O emulsion formulation and
preparation
Reagents for the O/W and W/O emulsions were
selected according to the protocol by Wibowo et al.
[23]. The complete formulation for both emulsions is
presented in Table 1. The global composition for the
O/W emulsion was 70% (w/w) oil phase, 4% (w/w)
surfactant mix, and 26% (w/w) aqueous phase. The
global composition of the W/O emulsion was 85%
(w/w) aqueous phase, 11% (w/w) oil phase, and 4%
(w/w) surfactant. Carbopol was selected as the
thickener and a mix 6:4 between Tween 20 and Span
80 as surfactants. This was needed to obtain a stable
emulsion with an HLB of 11.74 for the mineral oil
[24]–[26].
The emulsion was prepared by homogenizing each
phase separately at 50°C. Briefly, the aqueous phase
was stirred with a mechanical agitator equipped with
an inclined flat blades impeller, at 400 rpm for 15
minutes. Nanobioconjugates were then incorporated
in the aqueous phase during homogenization at 1
mg/mL. Subsequently, the dispersed phase was
incorporated into the continuous phase using a
peristaltic pump and a hose while maintaining
agitation of the continuous phase at 1200 rpm with the
aid of a mechanical agitator equipped with an inclined
flat blade impeller. The stirring velocity was constant
for 10 minutes, then, it was increased to 700 rpm for
5 more minutes. Finally, the emulsion was bottled and
kept at room temperature until further use.
F. Nanobioconjugate characterization
The hydrodynamic diameter was determined
through DLS (Zeta-Sizer Nano-ZS, Malvern, UK)
using a solution of magnetite in ethanol (3% w/v) at
37°C to simulate body temperature. A 200kV FEI
Tecnai F20 Super-Twin TEM was used to determine
the size of the nanobioconjugate in the solid state.
Sequential surface modifications of magnetite
nanoparticles with APTES, Oxidized PEA, and BUF-
II were evaluated via Fourier Transform Infrared
Spectroscopy (FTIR) by a Bruker Alpha II FTIR Eco-
ATR (Bruker, Germany). Spectra were collected in
the range of 4000 – 400 cm-1 with a spectral resolution
of 2 cm-1. Thermogravimetric analysis (TGA) was
carried out by ramping up the temperature of a 10 mg
sample at a rate of 10°C/min from 25 to 800°C in a
simultaneous TGA/DSC instrument (TA Instruments,
USA).
Table 1 Complete formulation for O/W and W/O emulsions
Reagent % (w/w) Concentration
O/W emulsion W/O emulsion
Distillate Water
Glycerol
Carbopol
Tween 20
PEA-400
Mineral Oil
Stearic Acid
Cetyl Alcohol
Span 80
Vaseline
25.62
1.080
0.3
2.4
0.6
62.8
1.05
1.05
1.6
2.5
57.05
2.38
0.21
0.06
0.01
7.14
0.12
0.12
2.74
0.39
G. Antibacterial Activity Assay
Antibacterial activity was assayed by the broth
microdilution assay as described by Park et al.6
Briefly, bacterial cells from S. aureus and E. coli were
cultured overnight in LB medium at 37°C. An aliquot
of this culture was then taken and incubated in fresh
medium until a McFarland standard of 0.5 was
achieved. Four concentrations (1000, 750, 500 and
100 µg/mL) of BUF-II-PEA-magnetite conjugates
were assayed, as well as two concentrations of BUF-
II (100 µM and 3.125 µM). Then, 50 µL of each
treatment were seeded by triplicate on a 96-well
microtiter plate (TPP). After bacterial cells reached
mid-logarithmic phase, cells were centrifuged,
washed three times with 10 mM sodium phosphate
buffer (NaPB, pH 7.4) and diluted 1:10.000 in the
same buffer. Then, 50 µL of the diluted cells were
added to 50 µL of each of the treatments and
incubated for 3 hours at 37°C. After incubation, 100
µL of fresh LB medium was added to each well and
incubated for 18 hours at 37°C. Inhibitory growth
5
effects were evaluated by absorbance at 620 nm [20].
Antibacterial activity for both emulsions (O/W and
W/O with nanobioconjugate) was evaluated by direct
contact using Antimicrobial Disk Susceptibility Test
as described by the Clinical and Laboratory Standards
Institute [27] and indirect contact by Broth
microdilution. E. coli and S. aureus strains were
cultured overnight in LB medium at 37°C; an aliquot
of this culture was taken and incubated in fresh LB
medium until mid-logarithmic phase was achieved
(0.5 McFarland Standard) for both tests. Bacteria
were spread over an agar petri dish using a sterile
swab and disks impregnated with the emulsion were
collocated on the center of the Petri dish. After
exposure, Petri dishes were incubated at 37°C for 18
hours. The inhibition halo size was then determined
using ImageJ. Ceftriaxone disk at 10 µg was used as
positive control. Assay by indirect contact was
evaluated by diluting the O/W emulsion at two-fold
dilutions in NaPB starting in 50% emulsion and
finishing in 6.25% emulsion, and in the W/O
emulsion without diluting it. Briefly, the evaluation
was conducted on a 96-well microtiter plate (TPP) by
adding 50 µL of the emulsions along with 50 µL of
the diluted bacteria as previously described, and
incubated for 3 hours at 37°C. After incubation 100
µL of LB medium were added to each well and finally
the microtiters were incubated at 37°C for 18 hours,
inhibitory growth was evaluated by absorbance at 620
nm.
H. Biocompatibility
Biocompatibility of the nanobioconjugates was
evaluated by assessing its cytotoxic, hemolytic and
platelet aggregation capacities. Cytotoxicity was
evaluated on Vero cells cultured at 37°C and 5% CO2
in DMEM media supplemented with 10% FBS and
1% P/S. Cells were plated at 3 x 104cells/well in a 96-
well microtiter plate and exposed to two-fold
dilutions of nanobioconjugate by triplicate starting at
100 μg/mL down to 6.25 μg/mL during 24h and 72h
of incubation at 37°C and CO2, cell toxicity was then
evaluated by the lactate dehydrogenase (LDH) assay.
Briefly, after incubation, 100 μL of supernatant was
transferred to a new 96-well plate along with 100 of
LDH reaction mixture and left to react for 30 minutes.
Absorbance from LDH release was quantified at 490
nm wavelength and 650 nm as the reference value. As
a negative control, cells without treatment were used
and as the positive control, cells were treated with
DMSO. The hemolytic activities of the
nanobioconjugate were assessed as described by
Muñoz et al.[21] Briefly, 2 x 107 erythrocytes from a
human blood healthy donor were centrifuged and
washed three times with NaCl 150 mM and then
replaced with 1 mL of PBS 1X. 50 μL of erythrocytes
were mixed by triplicate along 50 μL of the conjugate
and serially diluted concentrations in PBS 1X starting
at 100 μg/mL down to 6.25 μg/mL, in a 96-well
microtiter plate and incubated at 37°C for one hour.
Milli Q water was used as the positive control and
PBS 1X as the negative control. After incubation, the
microtiter plate was centrifuged, and the absorbance
of each supernatant was measured at 450 nm.
The effect of BUF-II-PEA-coated magnetite
nanobioconjugates, bare magnetite and PEA-coated
magnetite nanoparticles on platelet aggregation was
evaluated using platelet-rich plasma platelets (PRPs).
Positive controls of aggregation were epinephrine,
adenosine diphosphate (ADP) and collagen. PRPs
were obtained by centrifugation of a sample of human
blood in sodium citrate at 1000 rpm. Two-fold
serially diluted concentrations of the treatment
(nanobioconjugates, bare magnetite, and PEA-
coated-magnetite) were evaluated starting at 100
μg/mL down to 12.5 μg/mL after 3 minutes of
exposure to the treatments. Negative controls were
PRPs in the absence of materials and PRPs in PBS
1X. Aggregation was measured by absorbance at 620
nm.
For the emulsion, hemolytic capacity was
evaluated as biocompatibility test. Hemolysis was
assessed in human erythrocytes. Briefly, 2 x 107
erythrocytes were centrifuged and washed three times
with NaCl 150 mM and then replaced with 1 mL of
PBS 1X. O/W emulsion was diluted in two-fold serial
dilutions with PBS 1X starting at 100% of emulsion
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and ending in 6.25% emulsion, while W/O emulsion
was evaluated directly. 500 µL of emulsion were
mixed along with erythrocytes by triplicate in a 96-
well microtiter plate and incubated at 37°C for one
hour. As positive control, Triton 10X was used and as
negative control PBS 1X was used. After incubation,
the microtiter plate was centrifuged, and the
absorbance of each supernatant was read at 450 nm.
I. Cellular translocation and endosome escape
Cell translocation of the nanobioconjugate BUF-II-
PEA-coated magnetite was evaluated in monocytes
from acute monocytic leukemia (THP-1),
keratinocytes from an aneuploid immortal cell line
(HaCaT) and human foreskin fibroblasts (HFF).
Briefly, fluorescently labelled peptide (BUFII-FTIC
and BUFII-Rhodamine B) was conjugated to PEA-
coated magnetite as described above and
subsequently co-delivered with DAPI to cells at a
dilution ratio of 1:100 in 1 mL of the corresponding
medium (DMEM for HaCaT and HFF and RPMI for
THP1, 10% FBS and 1% P/S). Samples were
incubated for 1 hour at 37°C and 5% CO2 prior to
confocal observation on the Olympus FV1000
microscope.
Additionally, the ability of nanobioconjugates to
escape endosomal routes of internalization was
assessed in THP-1 cells. For this purpose, BUFII-
Rhodamine B-PEA-Magnetite conjugates were co-
delivered with the endosomal marker LysoTracker
green DND-26 and incubated for 1 hour at 37°C and
5% CO2. To confirm if the cellular internalization in
THP-1 cells was energy-independent, cells (100,000
cells/35 mm petri dish) were incubated with BUFII-
Rhodamine B-PEA-Magnetite nanobioconjugates for
1 h at 37°C and 5% CO2 prior to observation at either
37°C or 4°C. Also, a sample of the cells incubated at
37°C was incubated for 2 more hours at 4°C prior to
observation. Imaging was conducted on an Olympus
FV1000 confocal laser scanning microscope (CLSM)
with a PlanApo 60x, 1.35 NA oil-immersion
objective. Samples labelled with Rhodamine B and
DAPI were imaged by exciting with the 550 nm and
347 nm lasers, respectively. Imaging was conducted
at different positions throughout the culture depth to
ensure a complete z-stack scan. Image analysis was
performed in ImageJ® and Fiji ®
(https://imagej.net/Fiji/Downloads).
J. Bacteria translocation and Morphological
changes
Bacteria translocation and morphological changes
were evaluated in E. coli via Atomic Force
Microscopy (AFM, MFP-3D-BIO, Asylum Research,
UK) and Scanning Electron Microscope (SEM,
TESCAN Lyra 3, Czech Republic). E. coli cells were
cultivated at 37°C in LB medium until Log Phase was
achieved at a concentration equivalent to an optical
density of ~0.2 at 620 nm. Then, E. coli in the
presence nanobioconjugates were washed twice with
PBS 1X and fixed onto glass slides for 2 hours at
room temperature with 2.5% (v/v) glutaraldehyde.
After fixation, bacteria were washed twice with PBS
1X and stored at 4°C prior to the AFM measurement
[28]. AFM images were acquired by using the
Magnetic Force Microscopy module of the
instrument. Measurements were started by randomly
scanning areas of 7 x 7 µm2 with the purpose of
finding enough bacterial cells for imaging. Images
were collected for bacteria in the presence and
absence of the nanobioconjugates. Both
morphological changes, and magnetic response were
evaluated in the samples. Amplitude, height, and
voltage images were recorded simultaneously.
Topography from height images was used to calculate
the roughness of the bacterial cell surface based on
the root mean square (RMS) values, which in turn, are
based on the standard deviation of all the height
values within a given area [29]. Amplitude images
were captured to analyze size profiles of bacteria,
while voltage images helped to determine the
presence of the nanobioconjugates inside bacteria.
For SEM imaging, samples (bacteria,
nanobioconjugate in the presence of bacteria, and
only the nanobioconjugate) were cultured as
described above and subsequently washed six times
with Milli Q water, prior to fixing them on a
7
conducting tape in an aluminium base via
evaporation. SEM images were acquired at an
accelerating voltage (HV) of 10 kV to guarantee a
higher penetration into the sample and provide more
information on the layer beneath the surface. The
collected signal was acquired via secondary electrons
(SE) and backscattered electrons (BSE) signal.
Finally, image analysis was performed on ImageJ®.
K. Nanobioconjugate uptake on mammalian cells
Nanobioconjugate uptake by mammalian cells
THP-1 was evaluated by a decrease in the
fluorescence intensity upon internalization of the
labeled nanobioconjugates (BUFII-Rhodamine B-
PEA-Magnetite) in cells, using a Synergy HT Multi-
Mode Reader (BioteK) Spectrofluorometer. Briefly,
THP-1 cells were cultured in DMEM media
supplemented with 10% FBS and 1% P/S, at 37°C
and 5% CO2, until an 80% confluency was reached.
Cells were seeded at 3x104 cells/well on a 96-well
microtiter plate and exposed to the nanobioconjugates
in concentrations ranging from 6.25 µg/mL to 500
µg/mL. Cells were then incubated for 1 hour at 37°C
and 5% CO2. Fluorescence intensity was collected at
530 nm of excitation and 635 nm of emission. Data
was converted into internalization percentage with
the aid of a previously prepared calibration curve of
Intensity vs. Nanobioconjugate concentration.
III. Results and Discussion
A. Nanobioconjugate characterization
Fig 2. shows the hydrodynamic diameter
distribution of bare magnetite nanoparticles and
nanobioconjugates as determined by DLS. Bare
magnetite nanoparticles exhibited a mean
hydrodynamic diameter of 130 nm with a
polydispersity index (PI) of 14.66%. After
conjugation of BUF-II, the hydrodynamic diameter
increased to 295 nm with a PI of 25.6%. The sizes and
PIs obtained prior to the conjugation of BUF-II are
comparable to those obtained in our previous work
[20] but are larger to monodisperse MNP suspensions
obtained by others under a nitrogen-controlled
atmosphere [30] or by precisely adjusting stirring
speed during the co-precipitation process [31]. Upon
conjugation of BUF-II; however, the obtained size
and PI is larger compared with our previous work.
This can be attributed to the presence of the PEA
polymer linker on the surface that is likely to promote
aggregation. TEM images show aggregates with
individual particles exhibiting an average diameter of
7.84 nm ± 1.61 nm (Fig. 3), which agrees well with
the sizes found by Ling (about 4 nm) [32] and
Karimzadeh (14.7 nm) [33] The size of the obtained
nanobioconjugates appears attractive for applications
where a sustained accumulation of the delivery
vehicles is required for a long-lasting therapeutic
effect. This is the case of cancerous tumors where
accumulation has been reported for nanoparticles
with diameters above 20 nm. In contrast,
nanoparticles with diameters below 5.5 nm tend to be
easily cleared by the renal excretory system [32],
[33]. Sizes of aggregates from DLS and TEM
measurements are not comparable because of the
differences in the state of the samples during their
evaluation. In the first case, the sample is suspended
in an aqueous medium while in the second the
material is dried out for observation, which leads to
uncontrolled aggregation processes [34], [35].
Fig. 2 DLS histogram for the size distribution of magnetite
nanoparticles. Bare magnetite (red) and BUF-II-PEA-magnetite
(blue).
8
Fig. 3 TEM imaging of the BUF-II-PEA-magnetite
nanobioconjugates with their approximate diameters.
Surface modifications of magnetite nanoparticles
and PEA oxidation were confirmed by Fourier
transformed infrared spectroscopy (FTIR) and
thermogravimetric analysis (TGA). Fig. 4 shows the
FTIR spectra of PEA and oxidized PEA. Prior to
oxidation, FTIR spectrum of PEA shows two
absorption bands at around 1083 cm-1, which can be
attributed to a C-O-H stretching and an additional
band at 1453 cm-1, which can be correlated with the
C-H bending [36].
Fig. 4 FTIR spectra of oxidized PEA (orange), and PEA (purple).
Compared to the PEA IR spectrum, oxidized PEA
exhibits four different vibrational bands at about
1710, 1548, 1393 and 1278 cm-1, which can be
explained by the presence of C=O stretching, N-O
stretching, C-H bending and O-H stretching,
respectively. Of interest is the C=O stretching
because it can be associated with the carboxyl groups
needed for the conjugation of BUF-II. As shown in
Fig. 5, bare magnetite exhibited absorption bands at
around 632 cm-1 and 585 cm-1, which can be attributed
to the Fe-O bond of iron oxide [17], [30]. The
presence of the Si-O stretching vibration at about
1029 cm-1 and bending vibrations of C-H at 1391 cm-
1 and N-H bands at 1653 cm-1, confirmed the
silanization with APTES [31], [37]. Conjugation of
PEA on silanized magnetite was confirmed by an
absorption peak at about 1629 cm-1, which is due to
the presence of the C=O stretching in the backbone of
the polymer. Finally, conjugation of BUF-II was
verified by the presence of the amide band at 1650
cm-1, which can be related to the α-helix region in the
secondary structure of BUF-II. Further confirmation
was provided by a band at 1565 cm-1, which can be
attributed to the N-H and C-N bonds of the peptide,
as well as a band at about 1744 cm-1, which can be
explained by dehydrated carbonyl groups [38].
Fig. 5 FTIR spectra of bare magnetite (orange), PEA-coated magnetite (blue), bare BUF-II (green) and BUF-II-PEA-
magnetite nanoparticles (purple)
Conjugation efficiency of BUF-II on the surface
of magnetite nanoparticles was estimated via TGA
(Fig. 6). Magnetite and BUF-II-PEA-magnetite
(MPB) conjugates exhibited a first weight loss of
2.54% and 3.15% mainly due to dehydration of the
samples. Bare magnetite had a second weight loss of
13.04% while for the MPB was of 6.62%. These
losses are attributed to physically adsorbed organic
compound residues left by the synthesis and
9
functionalization processes. Finally, the detachment
of BUF-II from the magnetite was estimated with a
final weight loss step of 7.43%. These weight losses
agree well with those found in our previous work
[20].
Fig. 6 TGA analysis of bare magnetite (green) and BUF-II-PEA-magnetite conjugate (red)
B. Antibacterial Activity
Antibacterial activity of the nanobioconjugates
was measured by broth microdilution assay using
Gram-positive and Gram-negative bacteria: S. aureus
and E. coli. Simultaneously, we evaluated the effect
of salt in the conjugate’s activity by using low salt LB
and LB Broth medium. The values obtained are
shown in Fig. 7.
BUF-II-magnetite conjugates reduced bacterial
growth in S. aureus and E. coli up to 50% at 1 mg/mL,
at 750 µg/mL E. coli growth is reduced 35% while S.
aureus is reduced to 50%. Finally, at 500 µg/mL and
100 µg/mL, bacterial growth is also reduced by 45%
and 20% in S. aureus, 22%, and 18% in E. coli,
respectively. Nonetheless, the conjugate reduces
bacterial growth in both medium types (low salt LB
and LB Broth) at all concentrations. These data prove
that BUF-II immobilization onto PEA-coated
magnetite nanoparticles does not inhibit its
antibacterial capacity, albeit it only approaches 50%
of the native activity of BUF-II.
Fig. 7 Bacterial growth after treatments with BUF-II-PEA-
magnetite conjugates at 1 mg/mL, 750µg/mL, 500 µg/mL and 100
µg/mL (MPB 1000, MPB 750, MPB 500 and MPB 100),
gentamicin (Gen) and BUF-II at 100 µM and 3.125 µM (BUF-II
100 and BUF-II 3.125)
Antibacterial activity of emulsions was evaluated
via Antimicrobial Disk Susceptibility Tests and Broth
microdilution on E. coli and S. aureus, at 1 mg/mL of
nanobioconjugate. Table 2 shows the diameter of the
inhibition halos found for each emulsion with
dispersed nanobioconjugates in the presence of each
bacterium. According to our results, the W/O
emulsion led to a reduction in bacterial growth of
about 33% for both E. coli and S. aureus when
compared with Ceftriaxone. In contrast, the inhibition
exhibited by the O/W emulsion approached 21.2% for
S. aureus and 27% for E. coli. According to these
results, both S. aureus and E. coli can be considered
as resistant bacteria to our topical treatments.
Table 2 Diameters of inhibition zones for W/O and O/W
emulsions with dispersed nanobioconjugates in the presence of
E. coli and S. aureus
Sample Zone of inhibition (mm)
E, coli S. aureus
O/W Emulsion
W/O Emulsion
Ceftriaxone
9.47
11.5
34.86
7.29
11.34
34.85
Table 3 summarizes the results of inhibition halos
for O/W emulsions in the absence of dispersed
nanobioconjugates. The prepared emulsions inhibited
bacterial growth in about 71% for E. coli while for S.
aureus, the reduction approached 28%. The W/O
emulsion led to a reduction in bacterial growth of
10
about 64% for E. coli and of about 25% for S. aureus.
These data demonstrated a higher susceptibility for E.
coli compared with S. aureus. The reduced
antibacterial activity when compared with the
Ceftriaxone control can be attributed to the
interaction between BUF-II and some components of
the emulsions. In this regard, some studies have
demonstrated that an extremely strong reaction
between the peptides and phospholipid head group
would prevent the membrane translocation. Also, an
increase in the hydrophobicity could lead to
dimerization, which in turn leads to a decrease in the
antibacterial activity [39],[40].
In the case of emulsions prepared with the
nanobioconjugates, the reduction in antibacterial
activity was even more pronounced than in their
absence. This can be possibly explained by
considering an altered interfacial activity when
compared with the free peptide. We hypothesize that
some of the nanobioconjugates are possibly excluded
into the hydrophobic phase where accessibility to
bacterial cells is largely compromised as has been
shown for Pickering emulsions [41].
Table 3 Diameters of inhibition zones for W/O and O/W
emulsions in the absence of nanobioconjugates and in the
presence of E. coli and S. aureus
Sample Zone of inhibition (mm)
E, coli S. aureus
O/W Emulsion
W/O Emulsion
Ceftriaxone
24,88
22,38
34.86
9,61
8,87
34.85
Broth microdilution results for emulsions in the
presence of 1 mg/mL of nanobioconjugate are
presented in Fig. 8. These results showed that
emulsions with dispersed nanobioconjugates are able
to reduce bacterial growth for both E. coli and S.
aureus. In this regard, when exposed to the W/O
emulsions, the reduction approached 60% for E. Coli
and 80% for S. aureus. These results suggest that
interactions of the active antibacterial components are
stronger in the case of Gram-positive bacteria.
Fig. 8 Bacterial growth after exposure to W/O and O/W
emulsions with nanobioconjugates. O/W emulsions were diluted
in PBS (50% to 6.25% emulsion).
Additionally, antibacterial capacity was also
evaluated in emulsions prepared in the absence of the
nanobioconjugates. As observed in Fig. 9, in the
absence of the nanobioconjugates, W/O, O/W and
O/W emulsions diluted to 50% are capable of
reducing the growth of S. aureus in about 70%. In the
case of E. coli, the maximum reduction (i.e., 80%)
was observed for the O/W. Bacterial inhibition found
in the Broth microdilution agrees well with the
inhibition halos found in the Kirby-Bauer Test in
which emulsions in the absence of the
nanobioconjugates had better antibacterial activity
than those in their presence. At the same time,
antibacterial testing demonstrated higher
susceptibility for S. aureus than for E. coli.
Fig. 9 Bacterial growth after exposure to W/O and O/W
emulsions. O/W emulsions were diluted in PBS (50% to 6.25%
emulsion).
C. Biocompatibility
11
To determine the potential as a drug delivery
vehicle, the obtained BUFII-PEA-Magnetite
nanobioconjugates need to be biocompatible.
According to the ISO 10993 standard, to assure
biocompatibility of nanomaterials, a number of tests
are required including hemolysis, platelet
aggregation, and cytotoxicity [42].
Fig. 10 Assessment of the hemolytic effect of BUF-II-PEA-
magnetite conjugates (MPB) at different concentrations. In all
cases, hemolysis was below 5%. Milli Q water was used as a
positive control and PBS 1X as a negative control.
As shown in Fig. 10, hemolysis levels remained
below 5%, which complies with the ISO standard.
Fig. 11 shows that PEA-coated magnetite and BUFII-
PEA-Magnetite nanobioconjugates cause more
platelet aggregation than the positive controls
epinephrine, ADP, and collagen in all the evaluated
concentrations. A slight reduction in aggregation was
observed; however, after conjugation of BUFII on the
PEA-modified nanoparticles. Bare magnetite causes
little aggregation and consequently, it can be
categorized as a poor platelet aggregator. The
aggregation tendency of BUF-II can be in part
attributed to sharing the sequence identity of the N-
terminus of H2A histone [6], which has been reported
to induce thrombin generation and subsequently
activate the coagulation pathway. Also, extracellular
histones are capable of binding to platelets, which in
turn leads to increased calcium influx, activation, and
ultimately aggregation [43].
Fig. 11 Platelet aggregation effect caused by bare magnetite
(blue), PEA-coated magnetite nanoparticles (red) and BUF-II-
PEA-magnetite conjugates (green) compared to Adenosine
diphosphate (ADP), collagen (Col), Epinephrine (Epi) and
Buforin II (BUF-II).
Finally, Fig. 12 shows that at the highest
concentration evaluated (i.e., 100 µg/mL), cell
viability for BUF-II-PEA-magnetite
nanobioconjugates remains above 85% at 24 hours
while after 72 hours it decreases to 75%. Taken
together, these results are encouraging to proceed to
the in vivo evaluation of the developed cell-
penetrating vehicles.
Fig. 12 Cytotoxic effect of BUF-II-PEA-magnetite conjugates at
24 hours (blue) and 72 hours (red)
The hemolytic activity of O/W and W/O
emulsions in the presence and absence of the
nanobioconjugates is shown in Fig. 13 and Fig. 14,
respectively. The hemolytic tendency for the O/W
12
emulsions in both the presence and absence of the
nanobioconjugates increases linearly as a function of
the percentage of PBS added. At the highest
concentration evaluated (i.e., 50% PBS and 50%
emulsion (v/v)), the hemolysis approached 70% with
respect to the control. This could be problematic for
applications where the treatment needs to be in
contact with the blood stream. Accordingly, the
topical is best suited for superficial wounds.
Fig. 13 Assessment of hemolysis of O/W emulsions in PBS (50%
(v/v) to 6.25% (v/v) emulsion) on human erythrocytes in the
presence (blue) and in the absence of the nanobioconjugates
(green).
For the W/O emulsion prepared, compared with
the control, the hemolysis was of about 20% in
presence of the nanobioconjugates and 30% in the
absence of them. The lower hemolysis levels
observed for the W/O compared with the O/W
emulsions can be attributed to the lower stability of
the former. This, in turn, can be explained by the
sterilization process required for the anionic
surfactants Tween 20 and Span 80. In this regard, it
has been reported that when subjected to high
temperatures, these surfactants tend to degrade and
lose their surface-active properties [44]. As a result, it
is possible to have detrimental changes in the HLB
values and micelle formation tendency. This could
eventually lead to changes in membrane permeability
and unbalanced osmotic pressure [44]. At the same
time, when the hydrophobicity of the non-polar face
of the amphipathic α helix of the peptide is high, it
has a tendency to penetrate deeper into the
hydrophobic core of the cell membrane of red blood
cells thereby causing lysis [39].
Fig. 14 Assessment hemolysis of W/O emulsions without diluting on human erythrocytes in the presence (blue) and absence of the nanobioconjugates (red).
To address this issue, it has been suggested to
move to surfactants such as sodium lauryl sulfate
(SLES), sodium dodecyl sulfate (SDS) or
nonylphenol, which tend to develop electrostatic
charges that prevent coalescence of droplets and
therefore promote an increase in stability [45].
D. Cellular Delivery and Endosome Formation
To evaluate whether BUF-II maintained its
translocation capacity after immobilization, THP-1,
HaCaT, and HFF cells were exposed to the BUF-II-
FITC-PEA-Magnetite and BUF-II-Rhodamine-PEA-
Magnetite nanobioconjugates. Subsequent
internalization was assessed with the aid of confocal
microscopy. As observed in Fig. 15, we confirmed
that the nanobioconjugates effectively translocated
both cell and nuclear membranes and distributed
homogeneously at the intracellular level in all studied
cell lines. In our previous work [20], the synthesized
nanobioconjugates were only able to penetrate the
cell membrane, and we also showed that the peptide
alone remained mostly in the cell membrane.
13
The ability of the new vehicle to come across the
nuclear membrane makes it superior for a number of
applications in gene delivery compared with other
drug delivery vehicles [46].
Endosome escape upon internalization was
assessed for BUFII-Rhodamine and B-PEA-
Magnetite nanobioconjugates via colocalization in
the presence of LysoTracker Green® as an endosome
marker and changes in thermal energy. Fig. 16A and
16B show representative images for the
internalization of the nanobioconjugates and formed
endosomes in THP-1 cells. Colocalization of
Lysotracker and nanobioconjugates approached
27∓12%, which means that the remaining 73∓12%
of the conjugates can escape endosomes. Figure 16
shows that incubation at low temperatures led to a
homogeneous intracellular distribution, which
confirmed that internalization is most likely an
energy-independent and non-endocytic process [47].
The direct translocation mechanism and subsequent
internalization through a non-endocytic pathway are
advantageous for assuring that delivered drugs
remain active as they are protected from enzymatic
digestion within lysosomes [5] The ability of BUF-II
to translocate the cell membrane can be associated
with the presence of four arginine residues in its
structure. This is in line with the work by Kauffman
et al. according to which arginine-rich peptides can be
Fig. 15 (A) Confocal microscopy images of effective cellular and nuclear internalization of BUF-II-PEA-magnetite nanobioconjugates in
THP-1 cells. Scale bar corresponds to 15 µm. (B) Effective cellular and nuclear internalization of BUF-II-PEA-magnetite in HaCaT cells. Scale bar corresponds to 50 µm. (C) Effective cellular and nuclear internalization of BUF-II-PEA-magnetite conjugates in HFF cells. Scale bar corresponds to 50 µm
14
transported across the membrane without disrupting
it [48].
E. Membranal Changes and internalization in
bacteria
Fig. 17 shows AFM images of E. coli in the absence
and presence of BUF-II-PEA-magnetite
nanobioconjugates (Fig. 17A, Fig. 17B). Height
profiles (Fig. 17C) and surface roughness for a
representative bacterium cell in the absence (Fig.
17D) and presence (Fig. 17E) of nanobioconjugates
were plotted with the aid of the open-source software
Gwyddion®. The height profiles showed no
significant changes in size for the bacterium after
Fig. 16(A) Simultaneous delivery of LysoTracker Green and BUFII-Rhodamine B-PEA-Magnetite nanobioconjugates to evaluate endosome
escape in THP-1 cells. White arrows point to some of the regions where no colocalization was found between the nanobioconjugate and the Lysotracker while the yellow arrow corresponds to highly colocalized regions. Scale bar corresponds to 10 µm (B) Zoom in to an individual THP-1 cell after delivery of LysoTracker Green® and BUFII-Rhodamine B-PEA-Magnetite nanobioconjugates. White arrows point to regions of low colocalization. Scale bar corresponds to 10 µm (C) THP-1 cells incubated with the nanobioconjugates at 37°C and subsequent incubation at 4°C for 2 hours. No significant changes in fluorescence intensity upon incubation at different thermal energy support the notion that cellular internalization of BUFII-Rhodamine B-PEA-Magnetite nanobioconjugates proceeds through an energy-independent pathway. Scale bar corresponds to 10 µm.
15
exposure to the conjugates. Surface roughness also
remained unaltered in the presence of the conjugates.
Taken together, these two findings strongly suggest
that penetration of the conjugates into E. coli
proceeds without promoting significant membrane
perturbation. A mask was imposed on cells aided by
Gwyddion® to evaluate mean square roughness or
RMS of height irregularities (Sq) and mean roughness
or Sa value of height irregularities. Even though Sq
and Sa are computed from the 2nd central moment of
data values, the first is calculated from the sum of
absolute values of data differences from the mean,
while the second is from the differences from their
squares.
Table 4 summarizes the calculated values for each
parameter in the presence and absence of the
nanobioconjugates. The higher values of Sq and Sa for
the analyzed bacterium in the absence of the
conjugates suggests that upon entrance into the
microorganisms, conjugates are most likely
responsible for promoting surface perturbations that
ultimately lead to the release of weakly interacting
molecules such as buffer salts. According to a recent
AFM study on E. coli, Sq values approach 18, which
is a third of our estimation [28].
This can possibly be explained by the higher
number of bacteria considered for their studies
compared with our single-cell analysis.
Internalization of nanobioconjugates was confirmed
with the aid of the magnetic force module of the AFM
instrument. In this case, images of magnetic fields on
the sample are acquired by incorporating a magnetic
probe into the AFM. Here we explored the
electrostatic force components of the force, which are
purely attractive and generate a contrast that appears
dark for regions where a metal structure is present
[49].
Fig. 17 A AFM height images of (A) E. coli (B) E. coli in the presence of BUF-II-PEA-magnetite nanobioconjugates (C) Height profiles of E.
coli (red) and E. coli in the presence of BUF-II-PEA-magnetite nanobioconjugates (blue) (D) E. coli roughness profile for E. coli (red) and
(E) E. coli in the presence of BUF-II-PEA-magnetite nanobioconjugates (blue) (F) Electrostatic force on a single bacterium after exposure
to the BUF-II-PEA-magnetite nanobioconjugate.
16
Table 4 Differences in cell morphology for E. coli in the
presence and absence of nanobioconjugates
Parameter E. coli E. coli +
Nanobioconjugate
RMS roughness
(Sq.)
Mean
Roughness (Sa)
Surface Area
56.90 nm
47.07 nm
2.48 µm2
21.67 nm
6.99 nm
1.80 µm2
Accordingly, Fig. 17F qualitative shows that dark
regions, i.e., where the MNPs are present, located
within the bacterium cells. A similar experiment in
the absence of nanobioconjugates failed to produce
any recordable electrostatic forces (data not shown).
Further SEM imaging analysis of bacteria in the
presence of nanobioconjugates was conducted to
confirm the notion that no significant changes in the
integrity of E. coli cells occur upon internalization.
Images cells and nanobioconjugates prior to delivery
are shown in Fig. 18A and 18B, respectively.
Aggregates of the nanobioconjugates of about 200 nm
are observable in Fig. 18A with white deposits of
buffer salts surrounding them. E. coli cells in Fig. 18B
shows the typical rod-like morphology with some
superficial features that can be attributed to
electrostatically attached buffer salts. Fig. 18C shows
that after delivery of the nanobioconjugates, E. coli
cells appear with an even membrane surface and no
significant alterations in size or morphology. This
result agrees well with the absence of surface
irregularities detected by AFM.
F. Nanobioconjugate Uptake on Mammalian Cells
The efficiency of the nanobioconjugates at entering
mammalian cells was evaluated with the aid of a
spectrofluorometer. BUFII-Rhodamine B-PEA-
Magnetite nanobioconjugates were delivered and
subsequently incubated with THP-1 cells for 1 hour
at 37°C. The difference in fluorescence intensity prior
to and after delivery was correlated with the
percentage of conjugates internalized.
Fig. 19 shows the internalized percentage as a
function of the initial concentration of
nanobioconjugates to which cells were exposed. This
curve reaches saturation (i.e., 100% internalization) at
about 250 𝞵g/mL. Internalization of half of
nanobioconjugates is achieved at 50 𝞵g/mL, which
lies in the middle of the linear regime of uptake. An
internalization efficiency of about 75% was achieved
Fig. 19 SEM images of (A) nanoparticles at 79 kx. Scale bar corresponds to 500 nm, (B) E. coli at 46.2 kx. Scale bar corresponds to 2 µm and (C) Interaction between E. coli and nanoparticles at 79 kx. Scale bar corresponds to 1 µm.
Fig. 18 Nanobioconjugate uptake on THP-1 cells
17
at 100 𝞵g/mL, a concentration at which
biocompatibility is still acceptable for most delivery
applications.
IV. Conclusion
Overcoming the low-penetration capacity of
several pharmacological compounds represents a
major challenge for the pharmaceutical industry. To
potentially overcome this issue, we have recently
introduced novel cell-penetrating vehicles by
immobilizing BUF-II on polyetheramine-modified
magnetite nanoparticles. We hypothesized that by
introducing this surface spacer, the residues of BUF-
II responsible for the antimicrobial activity remain
largely accessible to block the replication machinery
of bacteria. The nanobioconjugates are highly
biocompatible as demonstrated by hemolysis levels
below 5% and acute cytotoxicity of around 85%. A
surprisingly high thrombogenicity was observed,
could be potentially useful for applications in
regeneration where a sustained platelet activation is
beneficial. Our findings show a promissory vehicle
for cell-penetration and microbial control with the
capability of escaping endocytic internalization
pathways and reaching nuclei, and a promising
approximation to new topics for the treatment of
bacterial infections.
V. Acknowledgements
This work was funded by Colciencias grant #689-
2018. Also funding from the Department of
Biomedical Engineering and by the Fondo de Apoyo
a Profesores Asistentes (Research Support Fund for
Assistant Professors [FAPA] to Carolina Munoz and
Juan C. Cruz), Universidad de los Andes, is gratefully
acknowledged. We would like to thank the
Department of Chemical Engineering for providing
access to the TGA instrument. Finally, we would to
thank the Microscopy Core Facility for access to the
instruments and technical support with the confocal
and AFM microscopes.
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