Synthesis, photophysical characterisation and antimicrobial activity of a new anionic PAMAM dendrimer

Article abstract of DOI:10.1016/j.jphotochem.2020.112878

A new anionic dendrimer (D1) has been synthesized and characterized by modification of a poly(amidoamine) (PAMAM) dendrimer with 3-(6-nitro-1,3-dioxo-1H-benzo[de]isoquinolin-2(3 H)-yl)propanoic acid (NI1). Its photophysical characteristics have been investigated in organic solvents of different polarity and a positive solvatochromism has been observed. The effect of the pH medium on the fluorescence intensity has been investigated and it has been shown that in the highly alkaline medium the dendrimer emits intense fluorescence. The dendrimer and its monomeric structural analogue have been loaded on a cotton fabric and the release of substances from the surface of the cotton fabric has been investigated. The compounds have been screened for antimicrobial and cytotoxic activities. They have been found more active against Gram-positive bacteria. The dendrimer exhibits slightly lower activity than the monomeric analogue, but is found to be significantly less cytotoxic. The deposited on cotton fabric D1 prevents the formation of bacterial biofilm on the fabric surface.

Full text of DOI:10.1016/j.jphotochem.2020.112878

Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112878
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, A: Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Synthesis, photophysical characterisation and antimicrobial activity of a
new anionic PAMAM dendrimer
Desislava Stanevaa, Silvia Angelovab, Evgenia Vasileva-Tonkovac, Peter Grozdanovc,
,d,
Ivanka Nikolovac, Ivo Grabcheva
*
a University of Chemical Technology and Metallurgy, 1756 Sofia, Bulgaria
b Institute of Optical Materials and Technologies “Acad. Jordan Malinowski”, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
c The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
d Faculty of Medicine, Sofia University “St. Kliment Ohridski”, 1407 Sofia, Bulgaria
A R T I C L E I N F O
A B S T R A C T
Keywords:
A new anionic dendrimer (D1) has been synthesized and characterized by modification of a poly(amidoamine)
(PAMAM) dendrimer with 3-(6-nitro-1,3-dioxo-1H-benzo[de]isoquinolin-2(3 H)-yl)propanoic acid (NI1). Its
photophysical characteristics have been investigated in organic solvents of different polarity and a positive
solvatochromism has been observed. The effect of the pH medium on the fluorescence intensity has been
investigated and it has been shown that in the highly alkaline medium the dendrimer emits intense fluorescence.
The dendrimer and its monomeric structural analogue have been loaded on a cotton fabric and the release of
substances from the surface of the cotton fabric has been investigated. The compounds have been screened for
antimicrobial and cytotoxic activities. They have been found more active against Gram-positive bacteria. The
dendrimer exhibits slightly lower activity than the monomeric analogue, but is found to be significantly less
cytotoxic. The deposited on cotton fabric D1 prevents the formation of bacterial biofilm on the fabric surface.
1,8-Naphthalimide
Anionic PAMAM dendrimer
Antimicrobial activity
Antibacterial
Cotton fabric
Antimicrobial finishing
1. Introduction
Functionalization of dendrimers with photoactive groups expand
their application fields [5]. The chemical structure of the dendrimer
Dendrimers are a relatively new class of hyperbranched macromol-
ecules with unique chemical structure and functional characteristics
[1–3]. The increased attention to this class of compounds arises from the
fact that they are a new form of organization of polymeric materials, the
peculiarity of which is the combination of the properties of low molec-
ular weight and high molecular weight substances. Compared to the
small molecules and linear polymers with antimicrobial properties,
dendrimers have the potential of a single molecule to carry a large dose
of biologically active substance. In addition, dendrimer biocides are
characterized not only by increased activity but by reduced toxicity, as
well [4]. Dendrimers contain a large number of functional groups in the
branches and in the periphery, which gives great opportunities for
purposeful modification of their properties. The first one enables their
binding to the functional groups of different matrix (for example textile
materials). The second one allows multiple copies of a drug to unite in
one molecule. This may induce a multivalent effect, reminiscent of the
polyvalent interactions widely occurring in biological systems [2].
molecules makes them relevant for different application in such fields as
biomedicine, nanomedicine ecology, etc. [6–12]. Cationic or anionic
dendrimer structures can be obtained by suitable chemical methods.
They exhibit high antimicrobial activity against different pathogens
[13–17]. Some of them have more pronounced activity to Gram-positive
bacteria, while others are more active against Gram-negative bacteria.
The utilisation of antibacterial textiles in daily life, and especially in
hospital and clinical practice, may prevent various diseases and bacte-
rial infections. The production of antibacterial textiles is mainly ach-
ieved by treating textile materials with biologically active substances
[18–21]. These substances can be attached to the surface of the textile
materials through different interactions depending on their application.
In our laboratory we conduct systematic studies on the modification
of dendrimers with 1,8-naphthalimide [22–25], acridine [26], benzan-
throne [27,28], or 4-nitrobenzofurazan [29] with the aim of investi-
gating the structure - properties relationship. These dendrimers have
been probed as metal and/or pH detectors. In the last few years, our
* Corresponding author.
E-mail address: i.grabchev@chem.uni-sofia.bg (I. Grabchev).
https://doi.org/10.1016/j.jphotochem.2020.112878
Received 22 July 2020; Received in revised form 21 August 2020; Accepted 27 August 2020
Available online 29 August 2020
1010-6030/© 2020 Elsevier B.V. All rights reserved.

D. Staneva et al.
Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112878
Scheme 1. Synthesis of compounds NI1 and D1.
efforts have also been focused on the antimicrobial and anticancer ac-
tivity of such systems [30–34]. This direction was dictated by the fact
that pathogenic microorganisms are becoming more resistant to the
antibiotics used in medical practice, which requires the search and study
of new compounds with increased antimicrobial activity [35].
2.1. Synthesis of 3-(6-nitro-1,3-dioxo-1H-benzo[de]isoquinolin-2(3 H)-
yl)propanoic acid (NI1)
4-Nitro-1,8-naphthalic anhydride (2,43 g, 0.01 M), was dissolved at
50 ml ethanol and β-alanine (0.89 g, 0.01 M) was added. The mixture
was refluxed 4 hours. After cooling, the formed needle crystals were
filtered and washed with ethanol. Yield: 2.32 g, 74%.
Derivatives of 1,8-naphthalimides are cyclic imides, characterized by
hydrophobicity and large π-fused skeleton, which can easily interact
with various biological systems through non-covalent interactions, such
FT-IR (KBr) cm-1: 1741, 1708, 1654, 1590, 1524, 1346, 1300, 1231,
840, 784, 753.
as π-π stacking. This determines the wide biological and biomedical
interest to them [36–38].
1H NMR (DMSO) δ (ppm): 2.66 (t. J = 7.2 Hz, 2H, HOOCCH2-), 4.24
(t. J = 7.8H, 2H, CH2-N), 8.06 (dd, J = 12.3, 6.2 Hz 1H, Ar-H), 8.52 (d,
J = 8.2 Hz, 1H, Ar-H), 8.57 (d, J = 7.9 Hz, 1H, Ar-H), 8.60(d, J = 8.6 Hz
1H, Ar-H), 8.67 (d, J = 8.0 Hz 1H, Ar-H),12.41 (s. 1H COOH);
13C NMR (DMSO): δ (ppm) = 22.4 (HOOCCH2), 36.5 (NCH2CH2-CO),
123.2-132.2 (Ar-C), 149.5 Ar-C-NH), 162.4.2 and 163.4 (CONHCO),
172.9(COOH);
This study aims to investigate a new anionic PAMAM dendrimer,
peripherally modified with four 1,8-naphthalimide units. The basic
photophysical characteristics of the dendrimer have been investigated in
organic solvents of different polarity. The effect of pH has also been
investigated. The antimicrobial activity of the dendrimer against
different pathogens has been examined in a liquid medium and after
deposition on a cotton fabric. By gaining insight into the factors that
correlate with dendrimer structure one can design and further optimize
their properties for various applications. A computer simulation tech-
nique (DFT calculations) has been used to predict some properties of the
new PAMAM dendrimer (and to differentiate them from those of the 1,8-
naphthalimide derivative, used for its peripheral functionalization) at
the molecular level. DFT-based calculations provide valuable informa-
tion about the configuration and conformation of the molecules and
important insights into the electronic structure. To understand the
relationship between electronic structure and photophysical behaviour,
we have investigated theoretically (by means of popular and affordable
DFT method) the 1,8-naphthalimide derivative and a model system,
representing the studied new PAMAM dendrimer.
Analysis: C15H10N2O6 (314.21 g mol-1): Calc. (%): C-57.28, H 3.18, N
8.91. Found (%): C-57.30, H 3.24, N 9,02;
2.2. Synthesis of dendrimer D1
PAMAM dendrimer (0.129 g, 0.25 mmol) and (0.314 g, 1.0 mmol) of
NI1 were dissolved in 10 ml of N,N-dimethylformamide. The solution
was stirred at 25 ◦C and after 24 hours, the product has been isolated by
solvent evaporation in vacuum. The dendrimer obtained was washed
with hexane and dried at air. Yield: 0.301 g, 76 %.
FT-IR (KBr) cm-1: 1739, 1688, 1634, 1579, 1527, 1306, 1239, 1101,
823, 786, 756.
1H NMR (DMSO) δ (ppm): 2.42 (d. J = 7.1 Hz, 8H, CH2-CO), 3.10 (q.
J = 5.2 Hz, 8H, HOOCCH2-), 2.68 (d. J = 7.3 Hz, 8H, CH2-N-CH2), 3.18
(d. J = 7.2 Hz, 4H, N-CH2CH2-N), 3.32 (s. 8H, HNCH2-) 3.65 (d.
J = 6.8 Hz, 8H, N-CH2CH2-NH), 4.10 (d. J = 7.2 Hz, 8H, CON-CH2), 6.96
(bs, 4H, NH) 7.38 (t, J = 7.8 Hz, 4H, Ar-H), 7.89 (d, J = 8.2 Hz, 4H, Ar-
H), 8.20 (d, J = 8.5 Hz, 4H, Ar-H), 8.48 (t, J = 6.9 Hz, 8H, Ar-H), 8.64 (d,
J = 8.2 Hz, 4H, Ar-H), 9.42 (bs, 4H NHCO), 11.23 (s. 4H COOH); 13C-
NMR (CDCl3): δ (ppm) = 30.8 (HOOCCH2), 33.2 (NCH2CH2-CO), 34.6
2. Experimental part
Detail description of used methods for spectral characterization
microbiological activity and of compounds was done in Supplementary
material.
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D. Staneva et al.
Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112878
Table 1
Photophysical characteristics of compound D1 in organic solvents (see text).
λA
nm
ε
λF
nm
νA
-
νF
ΦF
l mol -1 cm-1
cm-1
Acetonitrile
426
421
428
430
412
414
414
422
42 500
43 900
42 700
42 800
43 900
43 200
43 300
41 800
522
518
533
538
513
511
509
520
4 317
4 448
4 603
4668
0.329
0.365
0.123
0.156
0.320
0.395
0.339
0.114
N,N-dimethylformamide
Ethanol
Methanol
Chloroform
4 778
4 587
4 508
4 466
Dichloromethane
Tetrahydrofuran
Water (pH = 7.2)
(NHCH2CH2NHCO), 42.2 ((OC)2NCH2), 46.3 (NHCH2CH2NHCO), 51.2
(NCH2CH2N), 54.9 (NCH2CH2CO), 102.2-134.0 (Ar-C), 153.1 Ar-C-
NH), 163.3 and 164.0 (CONHCO), 170.3(COOH); 173.8 (CONH).
Analysis: C82H84N14O20 (1584.56 g mol-1): Calc. (%): C-62.10, H
5.30, N 12.36. Found (%): C-62.31, H 5.19, N 12,39;
Fig. 1. Dependence of absorption (A) and fluorescence (B) maxima of den-
drimer D1 on the solvent polarity: 1) chloroform; 2) dichloromethane; 3)
tetrahydrofuran; 4) N,N-dimethylformamide; 5) acetonitrile; 6) ethanol; 7)
methanol; 8) H2O (pH = 7.2).
2.3. Treatment of cotton fabric with NI1 or D1
To the dissolved in 5 ml ethanol-water 1:4 (v/v) solution 5 mg of D1
or NI1 have been added (1 g) cotton fabric (weight 140 g/m2) at 40 ◦C
and has been immersed in this solution for 60 min. After that, the cotton
sample was washed with water and dried.
Table 2
TDDFT/B3LYP calculated λmax and f (oscillator strength) in chloroform and
methanol (solvents of different polarity).
Compound
Solvent
ε
, D
λmax, nm
f
3. Results and discussion
Chloroform
Methanol
4.7
411
422
0.33
0.32
D1(branch)
32.6
Amphyphilic dendrimers with carboxyl groups in their peripheries
show good antibacterial activity against gram-positive bacteria with low
eukaryotic toxicity [13]. In previous studies, we have shown that
cationic dendrimers [39] or neutral dendrimers and their metal com-
plexes [29–32] exhibit microbiological activity. In this regard, we per-
formed a targeted design to obtain a PAMAM dendrimer modified with
1,8-naphthalimides, in which a carboxyl group was previously intro-
duced. In the design of D1, we assumed that this dendrimer would show
good antibacterial properties with low toxicity, which is important in its
subsequent use to obtain wound dressings.
Scheme 1 shows the synthetic route for the preparation of dendrimer
D1 and its precursor HI1.3-(6-Nitro-1,3-dioxo-1H-benzo[de]iso-
quinolin-2(3 H)-yl)propanoic acid (NI1) has been obtained by reacting
of 4-nitronaphthalic anhydride with β-alanine in 2-methoxyethanol so-
lution, at 80 ◦C for 6 h according to the method described by Shaki et al.
[40]. This product has been used to modify a poly(amidoanine)
(PAMAM) dendrimer from zero generation containing four primary
amino groups, in DMF solution at room temperature for 24 h [41].
Under these conditions, only a nucleophilic replacement of the nitro
group from HI1 with an amino group from PAMAM structure is possible.
The carboxyl group has not involved in a chemical reaction to form an
imide group. The progress of the reaction was monitored by thin-layer
chromatography in a 1: 1 n-heptane-acetone elution system until NI1
was exhausted. In this case, as a high molecular weight product, D1
remained at the start as a yellow fluorescent spot. The final product D1
has been isolated by pouring the reaction mixture into water, filtering
the precipitate and washing with water.
Fig. 2. Pictorial illustration of the highest occupied and lowest unoccupied
molecular orbitals (HOMO and LUMO) ;isodensity plot, isovalue = 0.02 a.u.
dendrimer D1 absorbs in the visible spectral region at λA = 412-430 nm
(Table 1). As a result of the polarization of the 1,8-naphthalimide
structure, the dendrimer D1 emits yellow-green fluorescence with
maxima in the spectral range λF = 509-538 nm. From Fig. 1 it can be
seen that the absorption and fluorescence maxima of D1 depend on the
polarity of the organic solvents. In both cases, they are bathochromati-
cally shifted in the transition from nonpolar to polar medium, which
means that the D1 dendrimer displays a positive solvatochromism. The
3.1. Photophysical characteristics of D1
Compound NI1 contains an electron-acceptor nitro group at the C-4
position of the chromophore system, resulting in an absorption
maximum in the ultraviolet region at 344 nm in DMF solution. The
dipole moment of NI1 has been calculated to be 1.58 D. As a result of the
covalent binding of NI1 to the periphery of the PAMAM dendrimer, the
nitro group has been replaced by a secondary amino group. This altered
the polarization of the chromophore system of the 1,8-naphthalimide
Stokes shift (νA - νF), which is the difference between the absorbance and
the fluorescence maxima of the fluorescent compounds, are at the range
4317-4778 cm-1. The photophysical characteristics of the investigated
fragments (D1 (branch) has
a dipole moment of 9.00 D) and
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D. Staneva et al.
Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112878
Fig. 4. Possible protonation sites in the molecular structure of D1(branch) and
Hirshfeld charges on the nitrogen atoms.
Fig. 3. Possible solute–solvent interactions of D1(branch) system as a hydrogen
bond acceptor in polar protic solvent (methanol).
Table 3
Gibbs free energy differences (ΔG) between the protonated forms of the model
D1(branch) fragment (relative stabilities) of the protonated D1(branch) forms in
the gas phase (ΔG1) and in water (ΔG78).
dendrimer are similar to those previously obtained by us dendrimers
which have a secondary amino group as a substituent in the 1,8-naph-
thalimide structure [10,21].
Structure
ΔG1, kcal mol-1
ΔG78, kcal mol-1
TDB3LYP/6–31+G(d,p)//B3LYP/6-31+G(d,p) calculations gave the
electronic vertical excitations of the studied D1 dendrimer in chloroform
and methanol. D1 dendrimer was represented in a simplified manner by
using a model with one branch of D1 and ethylenediamine core repre-
sented by a N(CH3)2 group (Figure S1, Supporting Information). sol-
vatochromism (Table 2). The agreement between theory and UV
experimental data is excellent. A small neglectable) systematic over-
estimation of the excitation energies (, typical for TDDFT calculations is
observed for D1: 0.01 eV in chloroform and 0.05 eV in methanol
medium.
D1(branch)-H+ (N1)
D1(branch)-H+ (N2)
D1(branch)-H+ (N3)
D1(branch)-H+ (N4)
25.97
2.89
44.52
17.16
30.87
0.00
31.36
0.00
the 3-D structure of the dendrimer molecule and as a result of this
change the quantum yield decreases. DFT predicted solute–solvent in-
teractions for D1 (branch and core fragments) in a hydrogen bond donor
solvent (MeOH) are visualized in Fig. 3.
The D1(branch) first excited state is determined by HOMO–LUMO
transition with oscillator strengths f = 0.32/0.33. The frontier orbitals of
NI1 and D1(branch) systems are shown in Fig. 2. There is no difference
between the spatial distribution of the frontier orbitals - HOMOs and
LUMOs are delocalized on the 1,8-naphtalimide core for both com-
pounds (Fig. 2).
3.2. Dependence of the fluorescence intensity of D1 on the pH
pH value is a critical parameter in numerous fields, including
chemical and food industry, pharmacy, medical and biological science,
biochemical process, etc. [42]. 1,8-Naphthalimides are one of the widely
used chromophore systems for pH determination. In most of the 1,
8-naphthalimide derivatives, in which the fluorescence emission is
dependent on the pH values of the medium, the intensity of the emitted
fluorescence is higher in acidic media. This can be explained by the
Quantum yield of fluorescence (ΦF) provides information about the
quantum efficiency of the dendrimer D1 which has been measured using
a relative method. The ΦF of D1 was calculated by comparing its fluo-
rescence intensity to Rhodamine 6 G uzing as reference sample.
Su Ast n2Du
ΦF = Φst
(1)
Sst Au n2Dst
where Φst is the quantum yield of the standard (Rhodamine 6 G
Φst = 0.94);
Ast and Au are absorbance of the standard and sample;
S
st and Su are the integrated emission band areas of the standard and
sample,
nDst and nDu are the solvent refractive indexes of the standard and
sample;
It is apparent from the data in Table 1 that the polarity of the organic
solvents does not significantly affect the ΦF. The results have shown that
the two to three times lower quantum yield was obtained in polar protic
solvents (alcohols and water solutions). These solvents contain hydroxyl
groups through which hydrogen bonds can be formed with the 1,8-naph-
thalimide chromophore system. For example, effective hydrogen bonds
can be formed by the carbonyl (C=O) imide structure, resulting in a
change in the polarity of the chromophore system. Hydrogen bonds
formation is also possible with participation of the amide groups and
tertiary nitrogen atoms of the PAMAM structure, which probably alters
Fig. 5. Fluorescence spectra and dependence of the fluorescence intensity of D1
on the pH in aqueous solution.
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D. Staneva et al.
Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112878
Fig. 6. Release of the dendrimer D1 from the cotton surface at different
pH values.
Table 4
The amount at % of NI1 or D1 released from cotton fabric in aqueous solution at
different pH values.
рH =5.6
рH =7.3
рH =8.3
рH =11.2
NI1 %
D1 %
12
10
30
25
70
65
99
90
protonation of amino groups, which are most commonly linked to the
C-4 atom of the chromophore system, and the stop of the photoinduced
electron transfer and the restoration of fluorescence intensity [43]. In
some structures, such as deprotonated hydroxyl group bonded directly
to the naphthalene nucleus of 1,8-naphthalimide, the fluorescence is
enhanced in an alkaline medium [44].
To probe the dominant protonation sites on the D1 dendrimer
structure, we compared the stabilities of the different protonated forms
of the model D1(branch) fragment (Fig. 4 and Table 3). D1(branch)-H+
(N4) form appears to be the most stable one. Hirshfeld atomic charges
obtained for the optimized D1(branch) geometry also show that the
protonation preferably occurs at the ethylenediamine core (represented
by a N(CH3)2 group in our model).
The pH-dependent fluorescence intensity of dendrimer D1 was
investigated (Fig. 5). The fluorescence intensity increases with
increasing pH values of the solution. In an acidic medium, D1 emits a
low fluorescence, which is enhanced in an alkaline environment
(pH > 9). This indicates that the fluorescence emission of this dendrimer
is low in an acidic environment, which was not observed in neutral
Fig. 8. SEM micrographs of cotton fabric tested against B. cereus at 10000x
magnification: A) – initial cotton fabric before treatments; B) - bacterial biofilm
on cotton fabric before treatment with D1; C) - inhibition of biofilm formation
on cotton fabric treated with D1.
dendrimer molecules [10,23,24].
In order to obtain an antibacterial textile material, dendrimer D1 has
been deposited on cotton fabric. The amount of the deposited dendrimer
on the surface of the fabric has been examined spectrophotometrically
using absorption spectroscopy. Approximately 85% of the dendrimer
initially used (0.5 w%) has been fixed to the cotton fabric. Release of the
dendrimer from the surface of the cotton fabric has been investigated at
37 ◦C and different pH for 2 hours. The results obtained at four pH
values: pH = 5.6, 7.3, 8.3 and 11.2 are shown in Fig. 6. It is seen that in
Fig. 7. Reduction of the growth of model bacteria by cotton fabrics, treated
with NI1 and D1.
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D. Staneva et al.
Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112878
Fig. 9. Cytotoxicity values of the compounds NI1 (A) and D1 (B) towards HEp-2 cells.
acidic medium (pH = 5.6) about 10% of the deposited D1 is separated
from the surface of the fabric. At neutral pH = 7.3, 25% of dendrimer has
been released, and in a slightly alkaline medium (pH = 8.3) 65% of the
dendrimer was separated from the cotton fabric. At these pH values, the
separation follows a linear relationship. The dendrimer is most rapidly
detached in a highly alkaline medium (pH = 11.2) in which the carboxyl
groups are deprotonated and the solubility correspondingly higher. In
this case, linear dependence is observed in the first 60 min (about 70%
separation), after which the process slows down in the next hour, with
almost 90% of the dendrimer being separated at the end of the second
hour. This indicates that the pH of the medium has a significant effect on
the retention of the dendrimer on the surface of the cotton fabric. A
similar dependence has been observed in the study of the release of NI1
from the textile surface. From Table 4 it can be seen that in the case of
NI1 the amount is slightly greater and in the highly alkaline medium
almost the whole amount has released from the cotton fabric. This dif-
ference is probably due to the larger chemical structure of the dendrimer
compared to NI1 and hence to the possibility of forming more hydrogen
bonds and, therefore, to its stable fixation to the cotton fabric.
of bacterial infections is of particular importance. Antimicrobial modi-
fication of cotton surfaces is an alternative way of preventing the for-
mation of highly resistant biofilms and can be achieved by various
methods [46]. The antimicrobial effect of cotton fabrics modified by NI1
and D1 was evaluated by growth reduction of Gram-positive B. cereus
and Gram-negative P. aeruginosa.
The results showed that the cotton material treated with NI1 reduced
the growth of B. cereus by about 32% while the D1 treated sample was
less active reducing the growth by about 12% (Fig. 7). Against
P. aeruginosa, lower microbial reduction by NI1 and D1 was observed
(about 10% and 2%, respectively). It could suggest that both slow
diffusion of dendrimers from the cotton textile into the medium, and
direct contact with bacterial cells contributed to the antibacterial effect
of treated cotton fabrics. Several factors may affect considerably the
antimicrobial effect of textiles as reported by Surdu et al. [47].
It was of interest to examine the cotton surface for bacterial biofilm
formation by SEM technique. Fig. 8 shows SEM micrographs of cotton
fabric treated with D1 before and after 24 hours of contact with B. cereus.
Cotton fabric treated with D1 and untreated with bacteria has been
shown in Fig. 8A. Fig. 8B shows the formation of a stable biofilm on the
cotton fabric before treatment with D1. Applying dendrimer D1 to the
fabric significantly reduces the adhesion of bacteria and biofilm for-
mation. Only single bacteria have been detected on the fabric surface
(Fig. 8C). This means that dendrimer D1 prevents the retention of bac-
teria on the surface of the cotton tissue and from there to the formation
of a bacterial biofilm. Therefore, D1 has the potential to be used for the
preparation of antibacterial textile. Similar protective properties were
observed using 1,8-naphthalimide modified PAMAM dendrimers
[29–31].
3.3. Antimicrobial activity
In the agar diffusion test, no inhibition zones were observed by both
compounds against the model strains. In MPB, antimicrobial activity of
the compounds was tested quantitatively against the tested bacterial
strains B. cereus and P. aeruginosa. The results showed that Gram-
positive B. cereus exhibited lower resistance to the compounds
compared to Gram-negative P. aeruginosa. NI1 was found a little m ore
toxic to the tested strains than D1. NI1 inhibited completely the growth
of B. cereus at concentration of 240
95% of the growth of the strain at 450
ginosa demonstrated higher resistance towards the compounds. At the
μ
g/ml, while D1 inhibited more than
μ
g/ml. Gram-negative P. aeru-
3.5. Cytotoxicity
highest test concentration of 600 μg/ml, about 64% growth inhibition of
For the preperation of wound dressings it is important to investigatе
the citotoxicity of dendrimer D1 and 1,8-naphthalimide NI1. The eval-
uation of cytotoxicity of antimicrobials is a critical step to guarantee
their safe use. The results for the cytotoxicity of the compounds are
shown in Fig. 9. It has been found that the newly synthesized dendrimer
D1 is considerably less toxic (about 6.5 fold) to the HEp-2 cell line
compared to the monomer NI1 which can be considered as a great
advantage of the dendrimer over the corresponding monomer. This
confirms the effect observed by Grinstaff et al. that amphiphilic anionic
dendrimers exhibit a good antibacterial effect with minimal toxicity
[13]. Also dendrimer D1 was found to show lower cytotoxicity to similar
PAMAM dendrimers and hyperbranched polymers modified with 1,
8-naphthalimides or acridine [25,31,33].
P. aeruginosa was established by NI1, and no inhibition was observed by
D1. The difference in susceptibility of Gram-positive and Gram-negative
bacteria can be explained by differences in the structure of the bacterial
cell walls [45].
3.4. Antibacterial activity of modified cotton fabrics
At the contact of pathogenic microorganisms with the hydrophilic
cotton surface, especially in humid environments, may result in bacte-
rial biofilm formation. Preventing the formation of such a biofilm is
particularly important in wound dressings where they are in direct
contact with human skin or injured areas. In these cases, the prevention
6

D. Staneva et al.
Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112878
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4. Conclusion
A new zero-generation anionic PAMAM dendrimer modified with
four 1.8-naphthalimide fragments has been synthesized. Its photo-
physical characteristics were investigated in organic solvents of different
polarity and the dendrimer was found to have yellow colour and emits
yellow-green fluorescence. The position of the absorption and fluores-
cence maxima depends on the polarity of the medium. The dendrimer
and its monomeric structural analogue were deposited on a cotton
fabric. The release of both compounds from the surface of the cotton
fabric has been evaluated spectrophotometrically, and it was found that
this process is faster in a highly alkaline medium. The antimicrobial
activity of the compounds has been tested in agar, in solution, and after
their deposition on cotton cloth. Both compounds have been found to
have higher activity against the tested Gram-positive bacteria than to
Gram-negative bacteria. The dendrimer D1 has been found to be
considerably less toxic (about 6.5 fold) to the HEp-2 cell line compared
to the monomeric anionic analogue NI1. This indicates that the den-
drimer has higher potential to be applied as antimicrobial agent.
Deposited on a cotton fabric, it prevents the formation of bacterial films
and can be used for preparation of wound dressing.
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CRediT authorship contribution statement
[23] S. Yordanova, I. Grabchev, S. Stoyanov, I. Petkov, J. Photochem. Photobiol. A 283
(2014) 1–7.
Desislava Staneva: Investigation, Methodology, Writing - review &
editing. Silvia Angelova: Methodology, Investigation. Evgenia Vasi-
leva-Tonkova: Investigation. Peter Grozdanov: Investigation. Ivanka
Nikolova: Investigation. Ivo Grabchev: Supervision, Funding acquisi-
tion, Conceptualization, Methodology, Writing - original draft.
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Chim. Acta 438 (2015) 179–188.
[25] P. Bosch, D. Staneva, E. Vasileva-Tonkova, P. Grozdanov, I. Nikolova, R. Kukeva,
R. Stoyanova, I. Grabchev, Materials 12 (2019) 3020.
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[27] D. Staneva, E. Vasileva –Tonkova, I. Grabchev, J. Photochem. Photobiol. A 375
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[28] D. Staneva, S. Yordanova, E. Vasileva-Tonkova, S. Stoyanov, I. Grabchev,
J. Photochem. Photobiol. A 395 (2020), 1125062.
Declaration of Competing Interest
[29] I. Grabchev, E. Vasileva-Tonkova, D. Staneva, P. Bosch, R. Kukeva, R. Stoyanova,
Int. J. Polym. Anal. Charact. 23 (2018) 45–57.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
[30] I. Grabchev, D. Staneva, E. Vasileva-Tonkova, R. Alexandrova, M. Cangiotti,
A. Fattori, M.F. Ottaviani, J. Polym. Res. 24 (2017) 210.
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Res. 26 (2018) 332–340.
[32] I. Grabchev, E. Vasileva-Tonkova, D. Staneva, P. Bosch, R. Kukeva, R. Stoyanova,
New J. Chem 42 (2018) 7853–7862.
Acknowledgements
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I. Grabchev, Biointerface Res. Appl. Chem 8 (2018) 3053–3059.
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◦
This work was particularly supported by Grants N KOST-KП-19,
Fund “Scientific Research”, Ministry of Education and Science of
Bulgaria. S.A. acknowledges the provided access to the e-infrastructure
of the NCHDC – part of the Bulgarian National Roadmap for RIs.
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Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jphotochem.2020.
112878.
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