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: jg.perez10@uniandes.edu.co

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

6

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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