Article 1

Broadening the spectrum of antimicrobial activity with grami- 2

cidin and cationic polymer 3

Yunys Pérez-Betancourt 1, Rachel Zaia1, Marina Franchi Evangelista1, Rodrigo Tadeu Ribeiro, Bruno Murillo 4
Roncoleta1, Beatriz Ideriha Mathiazzi and Ana Maria Carmona-Ribeiro 1,* 5

1 Biocolloids Laboratory, Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São 6
Paulo 05508-000, Brazil; y.betancourt@usp.br (Y.P-B); rachelzaia@usp.br (R.Z); 7
marina.franchi.evangelista@usp.br (M.F.E); rodrigo@iq.usp.br (R.T.R); bruno.roncoleta@usp.br (B.M.R); 8
bemathi@usp.br (B.I.M). 9
* Correspondence: amcr@usp.br; Tel.: +55-011-3091-1887. 10

Abstract: In this work, gramicidin (Gr) nanoparticles (NPs) are characterized and combined with 11
poly (diallyldimethylammonium) chloride (PDDA) antimicrobial polymer to yield antimicrobial 12
dispersions active against Gram-positive and Gram-negative bacteria, and fungus. Gr/PDDA dis- 13
persions were prepared and characterized using dynamic light-scattering for sizing, zeta-potential 14
analysis and polydispersity determinations whereas activity was obtained from colony forming uni- 15
ties counting against the microbia. Hybrid dispersions in water remained stable for at least 24 h and 16
displayed complete microbicidal effect against Staphyloccoccus aureus and Candida albicans against 17
107 and 106 viable cells per mL, respectively. Gramicidin dispersions in water examined under scan- 18
ning electron microscopy showed the Gr NPs with diameters varying from 160-200 nm that dis- 19
played a milky aspect in the assay tube, typical of particles scattering the incident light. Upon addi- 20
tion of PDDA, the slightly negative charge of Gr NPs changed its sign to positive values. Conduct- 21
ance of the Gr/PDDA dispersions was due to PDDA and was not affected by the presence of the Gr 22
NPs, showing that the interaction between Gr NPs and PDDA was relatively weak. In comparison 23
to Gr inserted in cationic bilayers that also yielded broad spectrum activity, the effect of Gr/PDDA 24
was equally broad but more complete yielding a total loss of cell viability. 25

Keywords: antimicrobial peptide; antimicrobial cationic polymer; antimicrobial cationic lipid; na- 26
Citation: Lastname, F.; Lastname, F.; noparticles; supported cationic bilayer on silica with or without gramicidin; scanning electron mi- 27
Lastname, F. Title. Biomedicines 2022, croscopy; dynamic light scattering; broad spectrum of antimicrobial activity 28
10, x. https://doi.org/10.3390/xxxxx 29

Academic Editor: Firstname Last-

name
1. Introduction 30
Received: date
Accepted: date
The antimicrobial peptide gramicidin is produced by Bacillus brevis as a mixture of 31
Published: date
six peptides where gramicidins A, B and C, represent 80, 6, and 14 of the total composi- 32
Publisher’s Note: MDPI stays neu- tion, respectively [1]. Gramicidins A, B and C are collectively called gramicidin D (Gr). Gr 33
tral with regard to jurisdictional linear pentadecapeptides can form cation-selective ion-channels spanning across lipid 34
claims in published maps and institu- membranes and adopting a β-helical dimer conformation in the bilayer [2,3]; they have 35
tional affiliations. been extensively studied as a model ion-channel [4,5]. Gramicidin contains four trypto- 36
phans per monomer, which in the channel form of gramicidin are positioned at the mem- 37
brane interface [6–8]. Gr channel function is directly related to membrane thickness [2]. 38
Copyright: © 2022 by the authors. Scheme 1 shows the chemical structure of Gr A, B and C evidencing the highly hydropho- 39
Submitted for possible open access bic nature of lateral groups in this peptide. 40
publication under the terms and
conditions of the Creative Commons
Attribution (CC BY) license
(https://creativecommons.org/license
s/by/4.0/).

Biomedicines 2022, 10, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/biomedicines

Biomedicines 2022, 10, x FOR PEER REVIEW 2 of 15

Scheme 1.Chemical structures of gramicidin D. Atypical terminations with aldehyde and 41

ethanol- amine occurs flanking an intermediate sequence of hydrophobic amino-acids residues. 42
Reproduced from https://en.wikipedia.org/wiki/File: Structure_of_Gramicidins_A_B_C.png. 43
44
Bilayers of the cationic lipid dioctadecyldimethylammonium bromide (DODAB) 45
were successfully used to reconstitute the functional Gr dimeric channel [9,10] . Whereas 46
Gr exhibits high activity against Gram-positive bacteria (and high toxicity against mam- 47
malian cells) [11–17]; DODAB shows high activity against Gram-negative bacteria [18–20]. 48
Combining DODAB bilayer with Gr broadened the spectrum of antimicrobial activity to 49
a certain extent [13,21] but the loss of bacteria viability was not complete. 50
Other interesting combination for formulating Gr would be the antimicrobial cationic 51
polymer poly (diallyl dimethyl ammonium) chloride (PDDA) also highly effective against 52
Gram-negative bacteria and fungus [22–25]. Furthermore, PDDA displayed high affinity 53
for proteins such as albumin [26,27] and ovalbumin [28] yielding nanoparticles and allow- 54
ing to foresee some affinity for peptides such as Gr. In fact, combinations of polyelectro- 55
lytes and antimicrobial peptides for coating surfaces have been reported as potent antimi- 56
crobial agents [29–31]. 57
In this work, we evaluate combinations of Gr and PDDA in water dispersions, aiming 58
at a broad and complete loss of cell viability of S. aureus, E. coli and C. albicans. Curiously, 59
Gr alone dispersed in water yielded colloidal nanoparticles of high colloid stability visu- 60
alized by scanning electron microscopy (SEM) that displayed high activity against S. au- 61
reus and C. albicans. Recalling our data for PDDA activity against Gram-negative bacteria 62
[23,32,33], and considering that in this work the Gr/PDDA combination induced a com- 63
plete loss of S. aureus and C. albicans viability we suggest that this combination achieved 64
complete and broad spectrum of activity. 65

2. Materials and Methods 66

2.1. Materials 67
D-glucose, poly (diallyldimethylammonium chloride) (PDDA) 35% w/v with very 68
low molecular weight (< 100,000) was obtained from Sigma (Steinheim, Germany), gram- 69
icidin D (a peptide mixture consisting mostly of Gr A), ethanol, chloroform, 2,2,2-trifluo- 70
roethanol (TFE) and Mueller-Hinton agar (MHA) were purchased from Sigma-Aldrich (St 71
Louis, MO, USA). Dioctadecyldimethylammonium bromide (DODAB) and KCl were pur- 72
chased from Sigma (St. Louis, MO, USA). Silica (AEROSIL OX-50) was purchased from 73
Degussa (Frankfurt, Germany). The mean particle diameter determined by the supplier 74
using transmission electron microscopy (TEM) was 50 nm. Specific surface area was 75
Biomedicines 2022, 10, x FOR PEER REVIEW 3 of 15

previously determined from Brunauer–Emmett–Teller (BET) method yielding 26.00 m2/g 76

[34]. The silica dispersion was prepared by dispersing AEROSIL OX-50 in ultrapure water 77
or in a 1mM KCl aqueous solution [35]. 78

2.2. Preparation of gramicidin and gramicidin / poly (diallyldimethylammonium chloride) 79

dispersions. 80
Gramicidin (Gr) and PDDA stock solutions at 6.4 mM Gr and 10 mg/mL PDDA, re- 81
spectively, were used to prepare 2 mL of dispersions in ultrapure water to yield the de- 82
sired final concentrations. The final TFE concentration was kept at 1% of the final volume 83
by adding 0.02 mL of appropriate Gr solutions in TFE to 2 mL pure water under stirring 84
by vortexing for 30 s. 85
Gr/PDDA dispersions were prepared by adding 0.05 mL of a stock PDDA solution 86
in water (10 mg/mL) to the previously prepared Gr dispersion (2 mL) also under stirring 87
by vortexing for 30 s. 88

2.3. Preparation of supported dioctadecyldimethylammonium bromide (DODAB) bilayers on 89

silica particles with or without gramicidin inserted in the DODAB bilayer. 90
Dioctadecyldimethylammonium bromide (DODAB) chloroform solutions yielded 91
the lipid films on the bottom of glass tubes from chloroform vaporization under a nitrogen 92
flux [36]. Any temperature above the gel-to-liquid crystalline phase transition tempera- 93
ture of DODAB bilayer would yield the interaction between the silica surface and the 94
DODAB bilayer in the liquid crystalline state. Nascimento et al. first obtained the mean 95
temperature of the main phase transition at 47–49 °C [37]; therefore, the choice of 56 °C 96
was arbitrary and defined by our water bath that kept the temperature constant at 56 °C. 97
Silica dispersion (2 mg/mL) was sonicated for further redispersing the nanoparticles; 98
titanium from the titanium macrotip was precipitated by centrifuging the sonicated dis- 99
persion at 10,000 g in a microcentrifuge. The silica dispersion in the supernatant was col- 100
lected, added to the glass tube with the DODAB film and heated at 56 °C for 1h. An aliquot 101
of the gramicidin stock solution was then added, and the tube heated for another hour 102
(final concentrations were 0.5 mM DODAB, 0.05 mM Gr and 2 mg/mL silica). Under these 103
experimental conditions all silica nanoparticles are covered by a DODAB bilayer able to 104
provide adequate microenvironment for Gr [36]. SiO2/DODAB/Gr nanoparticles had their 105
physical properties and antimicrobial activity determined as described below. 106

2.4. Determination of physical properties of Gr or Gr/PDDA or SiO2/DODAB/Gr dispersions 107

Dispersions were characterized by dynamic light scattering (DLS) for determining 108
zeta-average diameter (Dz), zeta-potential (ζ), polydispersity (P) and conductance (G) us- 109
ing a Zeta-Plus Zeta-Potential Analyzer (Brookhaven Instruments Corporation, Holts- 110
ville, NY, USA), which was equipped with a 677 nm laser and dynamic light-scattering 111
(DLS) at 90° for particle sizing. 112
From fluctuations of scattered light intensity, the decay times of the fluctuations can 113
be related to the diffusion constants and, therefore, to the sizes of the particles. Small par- 114
ticles moving rapidly cause faster decaying fluctuations than large particles moving 115
slowly. The decay times of these fluctuations are determined in the time domain using a 116
correlator. The fluctuating signal is processed by forming the autocorrelation function 117
which decays exponentially with time so that the relaxation of the fluctuations is directly 118
related to the decay constant (Γ). The decay constant is given by Equation (1): 119
Γ = Dq2 (1) 120
where q depends on the scattering angle, the wavelength of the laser light, and re- 121
fractive index of the suspending liquid and D is the translational diffusion coefficient. 122
Hydrodynamic particle diameter (Dz) is inversely related to D by the Stokes–Einstein 123
Equation (2): 124
Dz = kT/(3πηD) (2) 125
Biomedicines 2022, 10, x FOR PEER REVIEW 4 of 15

where k is the Boltzmann’s constant, T is temperature in Kelvin, η is the viscosity of 126

the suspending liquid, and D is the diffusion coefficient. Mean diameters were obtained 127
by fitting data to log-normal size distributions that do not discriminate between one, two, 128
or more different populations and always consider all scattering particles as belonging to 129
one single Gaussian population. For the size distribution data, fitting was performed by 130
the apparatus software using the non-negatively constrained least-squares (NNLS) algo- 131
rithm, which is a model-independent technique allowing multimodal distributions to be 132
achieved [38]. 133
In order to define a relative width of size distributions, the polydispersity P is given 134
by Equation (3): 135
P = µ 2Γ2 (3) 136
where μ2 is proportional to the variance of the “intensity” weighted diffusion coeffi- 137
cient distribution and carries information on the width of the size distribution. Polydis- 138
persity (P) has no units. It is close to zero (0.000 to 0.020) for monodisperse or nearly mon- 139
odisperse samples, small (0.020 to 0.080) for narrow distributions, and larger for broader 140
distributions. ζ was determined from electrophoretic mobility (μ) in 1 mM NaCl and the 141
Smoluchowski equation (Equation (4)): 142
ζ = μη/ε (4) 143
where η is the medium viscosity and ε the medium dielectric constant. 144

2.5. Determination of colloidal stability 145

The colloidal stability of Gr or Gr/PDDA dispersions was evaluated at 0, 1 and 24 h 146
after preparation from DLS and turbidimetry at 400 nm. Data were presented as zeta- 147
average diameter (Dz), zeta-potential (ζ), polydispersity (P) and absorbance at 400 nm 148
over time. 149

2.6. Determination of circular dichroism spectra for gramicidin in ethanol, trifluoroethanol, water 150
and poly (diallyldimethylammonium) chloride solutions. 151
Spectra were acquired at 25°C using a 720 Spectropolarimeter (Jasco Inc, Tokyo, Ja- 152
pan) in a 0.1 cm quartz cell with 0.5 nm wavelength increments and a 4-second response 153
in the 200–280 nm range (at a 100 nm per minute scan rate). Each spectrum is the average 154
of five scans, with a full-scale sensitivity of 10 m deg. All spectra were corrected for back- 155
ground by subtraction of appropriate blanks in the absence of Gr (PDDA solution or Gr 156
solvent). Spectra smoothing kept the overall spectral shape. The ellipticities θ (in deg 157
dmol−1 cm2) were plotted as a function of wavelength. 158

2.7. Scanning electron microscopy for Gr or Gr/PDDA dispersions 159

Dispersions were prepared at 0.05 mM Gr or 0.05 mM Gr/0.05 mg/mL PDDA in water 160
and further diluted in water by a factor of 1:2 before placing them on coverslips for drying 161
overnight at room temperature. Thereafter, samples were coated by a gold layer using a 162
Leica EM SCD 050 sputtering apparatus and examined using a Jeol JSM-6460LV scanning 163
electron microscope for obtaining the micrographs. 164

2.8. Determination of cell viability for Staphylococcus aureus and Candida albicans over a range 165
of gramicidin or gramicidin /poly (diallyldimethylammonium chloride or 166
dioctadecyldimethylammonium bromide concentrations. 167
Staphylococcus aureus American Type Culture Collection (ATCC) 29213 or Candida al- 168
bicans (ATCC 90028) were cultured from previously frozen stocks (kept at −20 °C in the 169
appropriate storage medium). Each microorganism was reactivated separately, seeded by 170
streaking technique on the plates of Mueller-Hinton agar (MHA), and incubated for 18– 171
24 h at 37 °C. Isolated colonies were suspended in an isotonic 0.264 M D-glucose solution 172
and the turbidity of either bacteria or fungus suspensions was adjusted according to tube 173
0.5 of the McFarland scale at 625 nm. The 0.264 M D-glucose solution was used instead of 174
Biomedicines 2022, 10, x FOR PEER REVIEW 5 of 15

any culture medium because cationic molecules are inactivated by the relatively high ionic 175
strength or negatively charged molecules, such as amino acids and polysaccharides. For 176
the determination of cell viability, 0.1 mL of the cell suspensions (around 10 7–108 colony- 177
forming unities per mL, CFU.mL−1) were mixed with 0.9 mL of NPs dispersions diluted in 178
the same D-glucose solution for 1 h interaction. Thereafter, aliquots of 0.1 mL were with- 179
drawn and either directly plated or diluted 10 to 10 6 times before plating on MHA plates. 180
The plates were incubated at 37 °C for 24 h. The colony forming unities (CFU) counting 181
per mL was plotted in a logarithmic scale as a function of concentration of the antimicro- 182
bial agent. When no counting was obtained, since the log function does not exist for zero, 183
the CFU counting per mL was taken as 1 so that log CFU.mL−1 was zero. 184

3. Results and Discussion 185

Table 1 shows macroscopic features and physical properties of Gr, PDDA and 186
Gr/PDDA dispersions in pure water. Surprisingly, Gr itself in water yielded turbid dis- 187
persions meaning that these dispersions were made of Gr aggregates shaped as nanostruc- 188
tures able to scatter the incident light. DLS confirmed the particulate nature of Gr disper- 189
sions in water. Nanoparticles mean hydrodynamic diameter at 0.1 mM Gr was 159±1 nm 190
displaying a slightly negative zeta-potential (-26±3 mV). The amino-acids composition of 191
Gr includes hydrophobic aminoacids such as isoleucine and valine with terminal trypto- 192
phans. The hydrophobic effect might have accounted for the intermolecular aggregation 193
of Gr and formation of nanoparticles. The low negative charge might have been due to 194
tyrosine residues of gramicidin C where the hydroxyl of the phenolic moiety might have 195
dissociated upon exposure to the water phase originating the negative charges of the oth- 196
erwise neutral nanoparticles. 197
Adding PDDA (0.25 mg/mL) to Gr nanoparticles (0.1 mM Gr) changed the zeta-po- 198
tential to positive values and increased particle size and polydispersity (Table 1). At this 199
relatively large PDDA concentration, one can expect bridging flocculation joining one or 200
more Gr nanoparticles [32,39] thereby increasing the mean particle size in the dispersion. 201
Table 1 shows also that PDDA, as a hydrophilic cationic polymer, is readily soluble in 202
water and yields transparent water solutions. 203
204
Table 1. Macroscopic appearance and physical characteristics of gramicidin (Gr) dispersions in wa- 205
ter or in poly (diallyldimethyl ammonium chloride) (PDDA) water solutions. Gr dispersions were 206
prepared from 0.031 mL of a 6.4 mM stock Gr solution in trifluoroethanol (TFE) diluted under stir- 207
ring in water (2 mL). Gr/PDDA dispersions were prepared by adding 0.05 mL of a stock PDDA 208
solution in water (10 mg/mL) to the previously prepared Gr dispersion (2 mL) also under stirring. 209

Sample Photo Dz/nm P ζ /mV G/µS Turbidity/400 nm

Pure water - - - - 5±1 0
0.25 mg/mL PDDA in water - - - 245 ± 10 0

TFE in water - - - 13 ± 2 0
Biomedicines 2022, 10, x FOR PEER REVIEW 6 of 15

0.1 mM Gr/TFE/water 159 ± 1 0.14 ± 0.01 -26 ± 3 4±2 0.43 ± 0.01

0.25 mg/mL PDDA/TFE/water - - - 246 ± 11 0

0.1 mM Gr/0.25 mg/mL PDDA/TFE/water 426 ± 31 0.24 ± 0.04 49 ± 1 236 ± 6 0.64 ± 0.01

210
Figure 1 shows the effect of Gr concentration on the physical properties of Gr disper- 211
sions. With exception of the first point at a very low Gr concentration (0.005 mM), Dz and 212
zeta-potential for the Gr nanoparticles increased with Gr concentration. Therefore, in- 213
creasing the number of Gr molecules in the nanoparticle might have increased not only 214
their sizes but also the negative charges available for colloidal stabilization. This stability 215
was favored not only by the negative charges but also by the nature of formyl and ethan- 216
olamine moieties that are covalently bound to the two ends of the Gr molecule. These 217
moieties have the possibility of forming hydrogen bonds with water and are possibly oc- 218
cupying the outer surface of the Gr nanoparticle. The origin of the negative charges in the 219
otherwise neutral Gr molecule can only be the dissociation of hydroxyl in the phenyl moi- 220
ety of tyrosine (amino-acid residue at the 11th position of the molecule). One should notice 221
that terminal or lateral carboxylic groups are absent in the Gr primary structure. The hy- 222
droxyl dissociation in the phenyl moiety of tyrosine might have derived from the close 223
association between Gr molecules required for forming the Gr nanoparticles. The Gr in- 224
termolecular aggregation would be responsible for exposure of the phenyl tyrosine moi- 225
ety to the water phase. At this point one cannot avoid to propose a molecular dynamics 226
simulation study as a possible effective tool to ascertain the driving forces for Gr aggre- 227
gation in water. 228
Polydispersity (P) for the Gr dispersions remained constant as a function of Gr con- 229
centration revealing a good colloidal stability and lack of aggregation in between nano- 230
particles (Figure 1). Taking this result in combination with the increase in particle size (Dz) 231
upon increasing Gr concentration reveals that the increase in Dz is due to increase in the 232
number of Gr molecules in each nanoparticle instead of interparticle aggregation. The 233
measurement at 0.005 mM Gr performed at unfavorable condition of too low light scat- 234
tering was the less reliable one, possibly reflecting a condition where the Gr nanoparticles 235
were not formed yet. 236
237
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238
Figure 1. Effect of gramicidin (Gr) concentration on physical properties of Gr dispersions in water. 239
Measurements were performed 30 minutes after preparation of the dispersions and contained 1% 240
of trifluoroethanol. Physical properties were the mean z-average diameter (Dz), the zeta-potential 241
(ζ), the polydispersity (P) and the conductance (G). 242
243
Figure 2 shows the effect of increasing [PDDA] over a range of [PDDA] (0 -0.1 244
mg/mL) at 0.05 mM Gr on Dz, P, zeta-potentials and conductance of the Gr/PDDA disper- 245
sions. In addition, the photos evidence the turbid but dispersed nature of the Gr/PDDA 246
dispersions in pure water. In absence of PDDA, the Gr dispersions of NPs exhibit a slightly 247
negative zeta-potential that was easily reversed at tiny amounts of PDDA. The mean zeta- 248
potential of Gr/PDDA in the dispersions was positive (Figure 2). Conductance increased 249
linearly with [PDDA]. 250
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251
Figure 2. Effect of poly (diallyldimethyl ammonium chloride) (PDDA) concentration on macro- 252
scopic aspect and physical properties of gramicidin (Gr) /PDDA dispersions at 0.05 mM Gr. Physical 253
properties were the mean z-average diameter (Dz), the zeta-potential (ζ), the polydispersity (P) and 254
the conductance (G). 255
256
The colloid stability of Gr/PDDA dispersions was assessed from dynamic light scat- 257
tering and turbidity at 400 nm measurements over 24 h showing their stable character 258
(Figure 3). 259
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260
Figure 3. Colloidal stability of gramicidin (Gr) nanoparticles in the absence or in the presence of 261
poly (diallyldimethyl ammonium chloride) (PDDA). Mean z-average diameter (Dz), zeta-potential 262
(ζ), polydispersity (P) and turbidity at 400 nm (Abs 400 nm) of gramicidin (Gr) nanoparticles in 263
water or in 0.05 mg/mL PDDA as a function of time. 264
265
In order to gain some insight into the secondary structure or conformation of the Gr 266
peptide in the Gr/PDDA dispersions, the circular dichroism spectra of Gr in different me- 267
dia were obtained. The peptide Gr assumes an intertwined conformation in ethanol and 268
a beta-helix turn in TFE. In the Gr NPs without or with PDDA, there was a significant 269
reduction in intensity of the circular dichroism of the Gr molecules, possibly due to their 270
tight packing in the Gr NPs. In addition, the Gr spectra in Gr NPs or in Gr/PDDA were 271
more similar to Gr spectrum in TFE possibly corresponding to some residual beta-helix 272
conformation for Gr individual molecules in the Gr NPs or Gr NPs/PDDA dispersions. 273
274

275
Figure 4. Circular dichroism spectra of 0.02 mM gramicidin (Gr) at 25 °C in different media. Aliquots 276
(0.02 mL) of Gr stock solutions (6.4 mM) in trifluoroethanol (TFE) or ethanol were added to 2.0 mL 277
of water, TFE or ethanol. Alternatively, aliquots of poly (diallyldimethyl ammonium chloride) 278
Biomedicines 2022, 10, x FOR PEER REVIEW 10 of 15

(PDDA) stock solution (10 mg/mL) were added to Gr dispersion under stirring by vortexing to yield 279
0.01, 0.02 and 0.05 mg/mL PDDA. 280
281
The SEM micrographs for Gr dispersions in water confirmed the occurrence of spherical NPs 282
for Gr dispersions (Figure 5a) and for Gr/PDDA dispersions (Figure 5b). 283

(a) (b) 284

Figure 5. Scanning electron micrographs of gramicidin (Gr) dispersions at 0.05 mM Gr in pure water 285
(a) or in 0.05 mg/mL poly (diallyldimethyl ammonium chloride) (PDDA) solution (b). 286
287
The apparent aggregation of Gr NPs seen in the Gr/PDDA dispersion on Figure 5b 288
might be derived from drying the Gr/PDDA dispersion before depositing the gold coating 289
for SEM visualization. This can be understood from the fact that this same dispersion in 290
water examined by DLS did not show the aggregation appearing by SEM. Dz for 0.05 mM 291
Gr/0.05 mg/ml PDDA on Figure 2 yielded a mean Dz of 240-250 nm. Thus the SEM image 292
on Figure 5b might indicate some aggregation effect on Gr/PDDA NPs derived from dry- 293
ing. This aggregation was not present in the Gr/PDDA dispersion examined by a less in- 294
vasive technique such as DLS. 295
Figure 6 shows the profile of conductance (G) as a function of [PDDA] for 0.05 mM 296
Gr dispersions in water with or without PDDA. Whereas the dispersion of Gr NP only 297
yielded a conductance similar to the one of pure water, the Gr dispersions in the presence 298
of PDDA yielded conductance due to PDDA only as depicted from the comparison be- 299
tween 0.05 mM Gr/PDDA dispersions and PDDA dispersions over the same range of 300
[PDDA]. 301
302

303
Figure 6. Conductance of a 0.05 mM gramicidin (Gr) dispersion in the absence (∆) or in the presence 304
of poly (diallyldimethyl ammonium chloride) (PDDA) (□). The control for conductance of PDDA 305
solutions in absence of Gr was performed over a range of [PDDA] (o). 306
In order to gain some insight on the relative efficacy of Gr formulations developed in 307
our group over the last decades against S. aureus, a comparison between different Gr for- 308
mulations involving or not the cationic antimicrobial lipid DODAB and its supported or 309
non-supported bilayers was presented on Figure 7. 310
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311
Figure 7. Cell viability of Staphylococcus aureus (107 –108 CFU/mL) after interacting for 1 h with gram- 312
icidin (Gr) nanoparticles or other Gr formulations with dioctadecyldimethylammonium bromide 313
(DODAB) bilayers. Cell viability in the presence of DODAB supported bilayers on silica (SiO2 314
/DODAB/Gr), on polystyrene sulfate (PSS) nanoparticles (PSS/DODAB/Gr) [21], DODAB BF [20] or 315
DODAB bilayer fragments (DODAB BF) [13], all of them incorporating Gr]. In the DODAB BF dis- 316
persions, Gr dimers in the channel conformation were previously described [13,21]. The 317
SiO2/DODAB/Gr stock dispersion was prepared at 2 mg/mL silica, 0.5 mM DODAB and 0.05 mM 318
gramicidin yielding Dz=280±5 nm, P=0.20± 0.02 and ζ=45±4. 319
320
The cationic lipid bilayer of DODAB, similarly to the cationic polymer PDDA, bears 321
quaternary ammonium moieties well known for their activity against Gram-negative bac- 322
teria but yielding a poor performance against Gram-positive ones such as S. aureus. In- 323
serting Gr dimeric channels in DODAB bilayers prepared as DODAB bilayer fragments 324
did not improve very much the activity against S. aureus (Figure 7); Gr dimeric channels 325
found a very appropriate microenvironment in the DODAB bilayer and did not leave this 326
comfortable situation to interact with the coccus [13,21]. On the other hand, using sup- 327
ported DODAB bilayers on PSS NPs [21,40] or on silica [36] with Gr in the bilayers im- 328
proved significantly the activity against S. aureus (Figure 7]. In this work, the best formu- 329
lation against S. aureus was achieved, namely, the Gr NPs (Figure 7). There was a complete 330
loss of cell viability against this Gram-negative bacterium. 331
On Figure 8, the Gr NPs combined with PDDA were evaluated against S. aureus 332
showing also a complete loss of cell viability, similarly to the one obtained with Gr NPs 333
only. PDDA by itself had previously been investigated and did not cause a complete loss 334
of viability against S. aureus [23]. 335
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336
Figure 8. Cell viability of Staphylococcus aureus (106 –108 CFU/mL) after interacting for 1 h with gram- 337
icidin (Gr) nanoparticles, PDDA/Gr nanoparticles or PDDA solutions over a range of Gr and/or 338
PDDA concentrations. Data for cell viability over a range of [PDDA] were reproduced from [23]. 339
340
On Figure 9, Gr NPs, PDDA and Gr NPs/PDDA dispersions were tested against Can- 341
dida albicans. In this case, the results showed the efficacy of Gr NPs and Gr NPs/ PDDA 342
against the fungus at very low Gr and PDDA concentrations. Testing immobilized Gr in 343
gold coatings a 90% reduction of fungus viability was reported [41]. For Gr NPs, the com- 344
plete loss of C. albicans viability on Figure 9 shows that Gr molecules are readily available 345
to interact with the fungus from the Gr NPs. The combined action of Gr NPs and PDDA 346
caused an even more lethal effect on the fungus than the effect of Gr only showing that 347
the cationic polymer is possibly paving the way of the antimicrobial peptide to the fungus 348
cell membrane across its brush cell wall [42]. 349
350

351
Figure 9. Cell viability of C. albicans (106 CFU/mL) after interacting for 1 h with gramicidin (Gr) 352
nanoparticles, PDDA/Gr nanoparticles or poly (diallyl dimethyl ammonium chloride) (PDDA) so- 353
lutions over a range of Gr and/or PDDA concentrations. Data for cell viability over a range of 354
[PDDA] were reproduced from [23]. 355

5. Conclusions 356
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The particulate nature of gramicidin D dispersions in pure water was revealed from 357
sizing, zeta-potential, polydispersity, scanning electron microscopy and colloid stability 358
analysis. Given the high activity of Gr against Gram-positive bacteria and low activity 359
against Gram-negative ones, here successful combinations of Gr NPs with the cationic 360
antimicrobial polymer PDDA were shown to broaden the spectrum of antimicrobial ac- 361
tivity effectively causing complete loss of cell viability against 106 -108 cells/mL over 1 h 362
interaction time between dispersions and microbia (S. aureus and C. albicans). In addition, 363
Gr NPs dispersions advantageously compared with other Gr formulations in supported 364
or non –supported cationic bilayers showing the higher availability of Gr to interact with 365
the microbia from the Gr NPs as compared to the Gr/ cationic bilayer formulations. 366
367

Author Contributions: Conceptualization, A.M.C-R; methodology, Y.P.-B., R.Z., M.F.E., R.T.R., 368
B.M.R., B.M.I.; formal analysis, A.M.C-R.; investigation, Y.P.-B., R.Z., M.F.E., R.T.R., B.M.R., B.M.I, 369
A.M.C-R; resources, A.M.C-R; data curation, A.M.C-R.; writing—original draft preparation, A.M.C- 370
R and Y.P-B; writing—review and editing, A.M.C-R.; supervision, A.M.C-R; project administration, 371
A.M.C-R; funding acquisition, A.M.C-R. All authors have read and agreed to the published version 372
of the manuscript. 373

Funding: This research was funded by Conselho Nacional de Desenvolvimento Científico e Tecno- 374
lógico (CNPq), grants 302758/2019-4 and 302352/2014-7, and by Fundação de Amparo à Pesquisa do 375
Estado de São Paulo (FAPESP), grant 2019/17685-2. 376

Institutional Review Board Statement: Not applicable 377

Informed Consent Statement: Not applicable. 378

Data Availability Statement: All data available are reported in the article. 379

Acknowledgments: Y.P.-B. was the recipient of a Ph.D. fellowship from CNPq (grant 140091/2019- 380
0). B.M.R. is the recipient of an undergraduate technician TT-1 fellowship from FAPESP (grant 381
2021/01245-3) and R.Z. is the recipient of the Programa Unificado de Bolsas (PUB) from Univer- 382
sidade de São Paulo (USP) granted to the project Antimicrobial Nanoparticles and their Films by 383
A.M.C-R. 384

Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the 385
design of the study; in the collection, analyses, or interpretation of data; in the writing of the manu- 386
script, or in the decision to publish the results. 387

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