Antimicrobial efficacy of synthesized quaternary ammonium polyamidoamine dendrimers and dendritic polymer network

Article abstract of DOI:10.1166/jnn.2016.10656

Water treatment to mitigate microbial contaminants is a major challenge across globe paving the way to develop novel antimicrobial compounds. We aim at architecting antibacterial moiety eventually catering to vast water treatment industry. In this research study, quaternary ammonium functionalized polyamidoamine (PAMAM) dendrimer and PAMAM-ethyleneglycol dimethacrylate (EGDMA) dendritic polymer network were synthesized. These materials were characterized by various analytical techniques like ATR-FTIR, 1HNMR, DSC etc. Water soluble generation (G) 1.0 PAMAM dendrimer and water insoluble PAMAM G1.0 EGDMA dendritic polymer network were quaternized by reacting with dilute hydrochloric acid (HCl) and octyl iodide (OI) respectively. Both quaternary ammonium dendrimer products were found to exhibit potent bactericidal activity against a group of common Gram-negative and Gram-positive bacteria. 10 mg/L concentration of liquid PAMAM G1.0 QHCl was efficient to kill 100% bacteria rapidly within an incubation time of just 2 minutes. In addition, quaternary ammonium dendritic polymer network PAMAM G1.0-EGDMA Q OI demonstrated good contact killing antimicrobial property without releasing any active molecule into the surrounding medium and disinfected contaminated water within 5 minutes. Both quaternary ammonium dendrimer and dendritic polymer network showed negligible cytotoxicity in MTT assay indicating their potential as a viable antimicrobial agent.

Full text of DOI:10.1166/jnn.2016.10656

Article
Journal of
Nanoscience and Nanotechnology
Vol. 16, 998–1007, 2016
Copyright © 2016 American Scientific Publishers
All rights reserved
Printed in the United States of America
www.aspbs.com/jnn
Antimicrobial Efficacy of Synthesized
Quaternary Ammonium Polyamidoamine
Dendrimers and Dendritic Polymer Network
1
1
1
1
C. K. V. Zainul Abid , Richa Jackeray , Swati Jain , Sruti Chattopadhyay ,
2
1ꢀ∗
S. Asif , and Harpal Singh
1
Center for Biomedical Engineering, Indian Institute of Technology Delhi, New Delhi 110016, India
2
Glycoprotein Research Lab., National Institute of Immunology, New Delhi 110067, India
Water treatment to mitigate microbial contaminants is a major challenge across globe paving the
way to develop novel antimicrobial compounds. We aim at architecting antibacterial moiety eventu-
ally catering to vast water treatment industry. In this research study, quaternary ammonium function-
alized polyamidoamine (PAMAM) dendrimer and PAMAM-ethyleneglycol dimethacrylate (EGDMA)
dendritic polymer network were synthesized. These materials were characterized by various ana-
lytical techniques like ATR-FTIR, 1HNMR, DSC etc. Water soluble generation (G) 1.0 PAMAM
dendrimer and water insoluble PAMAM G1.0 EGDMA dendritic polymer network were quaternized
by reacting withDd ei l lui vt ee rhe y dd r bo yc hPl ou r bi cli sa hc ii dn g( HT Ce l c) ha nn od l oo gc tyy lt oi o: dMi d ce M( Oa sI )t er er sUp ne icv t ei v re sl yi t. yBoth quaternary
ammonium dendrim eI Pr p: r1o 7d 7u .c 1t s0 .w1 e2 r 5e . 2f o 0u 5n dO t no : eWx he i bd i ,t 0p o9 t eMn at rb a2 c0 t 1e r6 ic 0i d9a : l2 a3 c: t0i v2 ity against a group of
Copyright: American Scientific Publishers
common Gram-negative and Gram-positive bacteria. 10 mg/L concentration of liquid PAMAM G1.0
QHCl was efficient to kill 100% bacteria rapidly within an incubation time of just 2 minutes. In addi-
tion, quaternary ammonium dendritic polymer network PAMAM G1.0-EGDMA Q OI demonstrated
good contact killing antimicrobial property without releasing any active molecule into the surrounding
medium and disinfected contaminated water within 5 minutes. Both quaternary ammonium den-
drimer and dendritic polymer network showed negligible cytotoxicity in MTT assay indicating their
potential as a viable antimicrobial agent.
Keywords: Chemical Synthesis, Polymers, Nanostructures, Biomaterials.
1
. INTRODUCTION
of ordinary disinfectants like phenols, halogens, fer-
rocene, boron derivates, small quaternary ammonium com-
Microbial infection has been a serious issue through-
out the history of human existence on earth. Researchers
all over the world are working towards developing vari-
ous chemical agents which can destroy infectious micro-
6
ꢀ7
pounds (QACs) etc. Polymeric quaternary ammonium
compounds have garnered popularity due to their sta-
1
ꢀ3–5ꢀ8–10
ble and non-toxic nature.
Antimicrobial action
1
–3
of the QACs is based on their surfactant like electro-
static damaging interaction with the cytoplasmic mem-
brane of the microorganism, resulting in loss of membrane
permeability. At convenient optimum concentrations, they
organisms.
There are several types of antimicrobial
agents available in market, but the toxicity, short life
period and the fact that microbes are always evolv-
ing with formation of highly resistant strains have been
6
4
ꢀ5
cause cell leakage and eventually cell death. Literature
a serious issue of concern.
Increased hurdles have
suggests that the polymers containing QACs, not only
enhance efficacy over corresponding small QAC molecule,
but also show increased efficiency and selectivity, and pro-
prompted closure scrutiny of such disinfectants and pref-
erential development of novel non-toxic antimicrobials.
Polymeric disinfectants are a step forward in this direction
due to their lower toxicity and non-irritant properties
with prolonged period of action as compared with that
1
ꢀ4ꢀ11ꢀ12
longed lifetime.
Recently a new class of material, dendrimer, has gar-
nered attention as possible antimicrobial agent due to
their compact structure and their ability to accommodate
∗
13
Author to whom correspondence should be addressed.
large number of end functional groups. Dendrimers
998
J. Nanosci. Nanotechnol. 2016, Vol. 16, No. 1
1533-4880/2016/16/998/010
doi:10.1166/jnn.2016.10656

Abid et al.
Antimicrobial Efficacy of Synthesized Quaternary Ammonium PAMAM Dendrimers and Dendritic Polymer Network
are novel highly branched three dimensional cascad-
ing macromolecules of nanodimension that radiate from
quaternized and evaluated for antimicrobial activity to be
used as sterilizing and disinfecting agent respectively.
1
4ꢀ15
a central core.
Since molecular size and genera-
tion of dendrimers are increased stepwise via the repe-
tition of reaction sequence, their size and structure are
2
. MATERIALS AND REAGENTS
Methyl acrylate (MA), ethylene glycol dimethacrylate
EGDMA) and methylthiazolyldiphenyl-tetrazolium bro-
mide (MTT) reagent were obtained from Sigma-Aldrich
St. Louis, USA). Ethylene diamine (EDA), hydrochlo-
ric acid (HCl), octyl iodide (OI), dimethyl sulphoxide
DMSO) and azobisisobutyronitrile (AIBN) were pur-
1
4ꢀ16ꢀ17
highly controllable.
Multiple peripheral functional-
(
ity incur synergistic and combinatorial interactions with
1
8
other molecules. Polyamidoamines (PAMAM) are the
first and extensively studied dendrimer for various biomed-
ical applications due to their nanoscopic structure and
(
(
1
9–21
22
non-immunogenic nature.
Lee et al. have quaternized
chased from CDH chemicals (New Delhi, India). HPLC
grade methanol (MeOH) and toluene were obtained
from Loba Chemicals (Mumbai, India). Luria broth,
nutrient agar, bacterial strains-Escherichia coli (ATCC
G4.0 PAMAM dendrimers with methyl iodide for gene
delivery applications and observed that cationic PAMAM
dendrimers were efficient in complexing with plasmid
DNA and showed good cellular membrane penetration
efficiency with lower cytotoxicity. Modified PAMAM
dendrimer has been studied for anti-tumor drugs such
2
5922), Staphylococcus aureus (ATCC 33807), Pseu-
domonas aeruginosa (ATCC 27853) and Bacillus subtilis
(
(
ATCC 6633) were obtained from Hi-Media Laboratories
Mumbai, India). All the reagents and chemicals were used
2
3ꢀ24
as methotrexate, 5-flurouracil etc.
Amino terminated
fourth generation of PAMAM has been shown to com-
plex with folic acid using PEG4000 as a spacer, effectively
encapsulates anticancer drug and demonstrated better phar-
macokinetic, dynamic and biodistribution efficacy as com-
pared with naked drug molecules while treating mice
as received unless noted otherwise.
3. EXPERIMENTAL DETAILS
3.1. Synthesis and Quaternization of Dendrimers
3.1.1. PAMAM G1.0 Dendrimer and Quaternization
Synthesis of PAMAM dendrimers was carried out fol-
lowing a two step process, involving Michael addition of
2
5
26
induced with cancer. Recently Ghosh et al. have
reported the quaternization of G 3.0 PAMAM dendrimer
with dimethyl dodecyl amine after modification with
2
-chloroethyl isocynate to produce antimicrobial textile
ethylene diamine initiator core with methyl acrylate and
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fabrics. These treated fabrics I sPh :o 1w 7e 7d .1 e0f .f 1e c2 t 5i v. 2e 0 r5 e sOu nl t :s We de ,x h0 a9 u Ms t iav re 2a 0m 1i 6d a 0t i9o :n2 3o :f 0 r2e sulting esters with large excess
Copyright: American Scientific Publishers
16ꢀ20ꢀ29ꢀ30
against Staphylococus aureus bacteria on antimicrobial
studies. PAMAM has been known to deliver antimicro-
bial drugs27 to cells but their effect on bacteria is largely
uncharted. In one report, PAMAM derivatives have been
investigated as potential antimicrobial agents against gram
negative bacteria especially Pseudomonas aeruginosa with
of ethylene diamine as reported in literature.
In
brief, 80% w/v freshly distilled EDA was reacted with
80% w/v solution of methyl acrylate in methanol in 1:50
molar ratio for 24 h at room temperature under nitro-
gen blanket. The resultant product was purified and was
called G −0.5 dendrimer. Further, the solution of G −0.5
dendrimer in methanol (80% w/v) was carefully added to
a vigorously stirred solution of EDA in methanol (80%
2
8
0
.9–1.5 ꢁg/mL as EC .
50
In the present study, we have synthesized water
ꢀ
soluble PAMAM G1.0 dendrimer and water insoluble
poly(PAMAM G1.0 EGDMA) dendritic polymer net-
work operating on simple chemistries without employing
multistep reactions for generating secondary antibacte-
rial polycations. These liquid and solid dendrimers were
w/v) in 1:50 molar ratio at 0 C and reaction was con-
tinued for 96 hours at room temperature to obtain G 0.0
dendrimer with amine end functional groups. Above steps
were repeated with G0.0 dendrimer to obtain G1.0 den-
drimer as shown in Scheme 1. All reactions were carried
(a)
Scheme 1. Continued.
J. Nanosci. Nanotechnol. 16, 998–1007, 2016
999

Antimicrobial Efficacy of Synthesized Quaternary Ammonium PAMAM Dendrimers and Dendritic Polymer Network
(b)
Abid et al.
(c)
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Scheme 1. (a) Flowchart depicting steps for synthesis of quaternized dendrimers (b) Chemical reactions representing synthesis of different generations
PAMAM dendrimers (c) Synthesis of PAMAM G1.0-EGDMA dendrimer.
1000
J. Nanosci. Nanotechnol. 16, 998–1007, 2016

Abid et al.
Antimicrobial Efficacy of Synthesized Quaternary Ammonium PAMAM Dendrimers and Dendritic Polymer Network
out in amber colored round bottom (RB) flask and the
temperature was never allowed to increase above 30 C
300 MHz frequency was used for recording 1HNMR spec-
ꢀ
trum of synthesized compounds in CDCl solvent. Deuter-
3
to avoid undesirable side reactions due to light and heat
initiation. All addition reaction steps were followed by the
removal of excess reagents and solvents by rotary vacuum
ated dimethyl sulhpoxide (DMSO) was used as solvent
to record the nuclear magnetic resonance (1HNMR) spec-
trum of PAMAM G1.0 and PAMAM G1.0 Q HCl. Dif-
ferential scanning calorimetry (DSC) studies were carried
out using Perkin Elmer DSC-6 system. Vacuum-dried sam-
ples were loaded into calorimeteric system and thermo-
ꢀ
evaporator at temperature below 35 C.
Synthesized PAMAM G1.0 (1 mole) and 3 N HCl
(
2.5 mole) were taken in RB flask equipped with a mag-
ꢀ
netic stirrer. Temperature of the system was adjusted to
grams were obtained in the temperature range of −50 C
ꢀ
ꢀ
4
0 C and reaction was allowed to proceed for 6 h. Excess
to 200 C under nitrogen atmosphere at the heating rate
ꢀ
of HCl and water were removed by rotary vacuum evapo-
rator and the liquid product was stored in amber coloured
bottle for further characterization and use.
of 10 C/min. ZEISS EVO 50 scanning electron micro-
scope (SEM) was employed to study surface morphol-
ogy of synthesized dendritic polymeric networks. Samples
were dried under vacuum overnight and were mounted on
a metallic stub and images were taken after coating the
stub with silver to provide conduction for better resolution
of images. Solid quaternized PAMAM G1.0 dendritic net-
work of EGDMA were also thoroughly tested for its gel
content, swelling behavior and leaching parameters.
3
.1.2. PAMAM G1.0-EGDMA Dendrimer
Methanolic solution of PAMAM G1.0 (80% w/v)
Section 3.1.1) was added to a stirred solution of
ethyleneglycol dimethacrylate (EGDMA) in methanol
(
(
80% w/v) in 1:20 mole ratio, under nitrogen, over a
ꢀ
Gel content of the quaternized dendritic polymer was
determined by immersing the dry and weighed amount
period of 1 h at 0 C and continuously stirred for 24 h
at room temperature (Scheme 1). Solvent and excess of
(
1 g) of polymer in chloroform at room temperature for
EGDMA were removed by rotary vacuum evaporator at
ꢀ
ꢀ
24 h. Sample was then taken out, dried at 60 C and weight
of the sample was noted. Gel content was calculated using
the following equation.
4
0 C. Resulting pale yellow oil was further vacuum dried
ꢀ
overnight (10–1 mm Hg, 50 C) to remove any traces of
methanol and EGDMA (Yield = 98%).
ꢀ
ꢁ
ꢂꢃ
W −W
i
f
Gel Content (%) = W −
×100
3
.1.3. Dendritic Polymer Network Poly(PAMAM
i
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Copyright: American Scientific Publishers
W
i
PAMAM G1.0-EGDMA was polymerized by free-radical
polymerization technique using azo bis-isobutyronitrile
where, W
i
and W
f
are the weights of the dried polymer
before and after immersing in chloroform respectively.
Swelling studies of the dendritic polymer networks are
important because of their future use as water disinfectant
and dendritic polymer should not contaminate the water
upon degradation due to excessive swelling. Water absorp-
tion capacity of the quaternized dendritic polymer was
determined by immersing the dry polymer sample (1 g)
in distilled water. The sample was taken out from water
at various time intervals and weighed after blotting out
excess water from the surface of polymers with filter paper
for 10 seconds. They were put back in water immedi-
ately after weighing. The percent water absorption of the
synthesized polymer was calculated using the following
equation.
(
AIBN) as free-radical initiator. PAMAM G 1.0-EGDMA
and AIBN were vortexed in a glass test tube (0.06 w/w
of total dendrimer) to form a uniform solution. Glass test
ꢀ
tube was kept on a water bath at 75 C for 10 min to get
solid transparent polymer(poly(PAMAM G1.0-EGDMA).
ꢀ
Synthesized product was kept at 70 C for 1 h for com-
plete curing and was later on washed several times with
hot distilled water to remove any traces of unreacted den-
drimer/initiator from the system.
4
g of poly(PAMAM G1.0-EGDMA) was crushed into
ꢀ
small pieces and was refluxed at 40 C with 30 mL octyl
iodide for 6 h in a round bottom flask with constant stir-
ring to quaternize amino groups present in the polymer.
This quaternized product poly(PAMAM G1.0-EGDMA)
Q OI was washed several times with hot distilled water
to remove excess of octyl iodide and was dried under
vacuum.
ꢀ
ꢃ
ꢂW −W ꢃ
s
d
Percentage swelling (%) =
×100
W
d
where, W and W are the weight of the polymer in the
s
d
swollen and dry states respectively. The experiment was
repeated thrice for each specimen and average values are
reported.
To evaluate the leaching property of the prepared den-
dritic polymer network 2 g of dried quaternized dendritic
polymer network was kept in 10 mL distilled water for
a period of 30 days and UV-Visible absorption of the
water was tested regularly with a five days interval using
3
.2. Characterization
The structures of both dendrimers and dendritic poly-
meric network were extensively studied with attenuated
total reflectance-Fourier transform infra red (ATR-FTIR)
spectroscopy and spectra were recorded on Perkin-Elmer
spectrum one spectrometer attached with single bounce
diamond ATR accessory. Bruker AC 300 spectrometer of
J. Nanosci. Nanotechnol. 16, 998–1007, 2016
1001

Antimicrobial Efficacy of Synthesized Quaternary Ammonium PAMAM Dendrimers and Dendritic Polymer Network
Abid et al.
1
0 mm path length transmission micro probe connected to
a Cary 50 UV-Visible spectrophotometer (Spectral range
90–1100 nm) wavelength. After 30 days polymer was
is the lowest concentration of antimicrobial drug required
to kill an organism. Antimicrobials are usually regarded
as bactericidal if the MBC is not more than four times
1
1
1ꢀ34
taken out, dried in vacuum oven and the weight of polymer
was noted.
of the MIC.
Different concentrations (ranging from
200 mg/L to 1.25 mg/L) of PAMAM G1.0 Q OI were pre-
pared in autoclaved Luria broth solution and 1 mL of each
concentration was transferred to glass test tubes. To each
3
.3. Antimicrobial Activity of PAMAM G1.0 QHCl
and Poly(PAMAM G1.0-EGDMA) QOI
8
test tube, 100 ꢁL of 10 CFU/mL bacterial solution was
ꢀ
added and incubated for 24 h at 37 C. After this, test tubes
The antibacterial activity of liquid PAMAM G1.0 QHCl
and solid poly(PAMAM G1.0 EGDMA) QOI were evalu-
ated against E coli, Staphylococcus aureus, Pseudomonas
aeruginosa, Bacillus subtilis common Gram-negative and
Gram-positive bacteria by colony count method. 10 mL
of all bacterial cultures (10 CFU/mL) were prepared
in separate test tubes. 0.05 and 0.1 mg of PAMAM
G1.0 QHCl and poly(PAMAM G1.0 EGDMA) QOI were
added in 10 mL of bacterial contaminated water to obtain
were checked for the formation of turbidity. Turbid test
tubes indicated the growth of bacteria. 100 ꢁL was with-
drawn from each non turbid test tube and plated to find out
number of colonies formed. MIC was taken as the amount
of QAC at which no turbidity formation or increase in ini-
tial bacterial concentration was observed. MBC was taken
as the minimum concentration of quaternary ammonium
dendrimer at which there was no turbidity and a 100%
killing of test bacteria was observed.
3
1
8
5
mg/L and 10 mg/L concentration for a fixed incubation
time (2 min, 5 min and 10 min) under constant shaking.
Then 100 ꢁL aliquots were withdrawn from the above
water samples and laid over the nutrient agar plates using
3.6. In Vitro Toxicity Assessment of PAMAM G1.0
QHCl and Poly(PAMAM G1.0 EGDMA)
ꢀ
sterile glass spreader and incubated for 24 h at 37 C.
QOI by MTT Assay
All experiments were done in triplicate and negative con-
trol contained no quaternary ammonium compound. Log
reduction values of bacteria for all quaternary ammonium
compounds were calculated after two minutes of incuba-
MTT stock solution of 12 mM concentration was prepared
by dissolving 5 mg of MTT reagent in 1 mL of PBS buffer.
1.5 mg of each ammonium dendrimer and dendritic poly-
mer was added to wells of 96 well plate in triplicate and
100 ꢁL of Jurkat cell (T-lymphocyte) suspension with a
3
2
tion time using following equation.
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6
cell density of 10 cells/mL were added into each well
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Log reduction = ꢂLog initial bacterial countꢃ
ꢀ
1
0
10
Copyright: American S ca ine dn tt ihf iec pP l ua tbe li sw ha es r si ncubated at 37 C for 24 h. To each
well, 10 ꢁL of the 12 mM MTT stock solution was added
−
ꢂLog final bacterial countꢃ
10
ꢀ
and incubated at 37 C for 3 h. MTT reagent gets reduced
into purple colored formazan crystals if metabolic activ-
ities (viability) are present in the cell population. 50 ꢁL
of DMSO was added to each well to solubilize formazan
crystals formed and the contents of the wells were cen-
trifuged separately to remove all insoluble materials. After
collecting 100 ꢁl supernatant to fresh wells, absorbance
was recorded on UV spectrophotometer at 540 nm. All
experiments were done in triplicate, a negative control
where no dendritic samples added to cell suspension with
3
.4. Evaluation of Antimicrobial Efficiency of
Poly(PAMAM G1.0 EGDMA) QOI for
Repetitive Use
0
.1 mg of quaternary ammonium dendritic polymer,
poly(PAMAM G1.0 EGDMA QOI), was kept in contact
with 10 mL of bacterial solution (10 CFU/mL) for 5 min-
utes (final concentration being 10 mg/L). 100 ꢁL aliquot
was withdrawn from this solution and laid over nutrient
agar plate using sterile glass spreader. Rest of the solu-
tions was completely removed and fresh 10 mL of bacterial
solution (10 CFU/mL) was added. Same procedure was
followed for twenty five cycles. All plates were incubated
for 24 h at 37 C along with a negative control and the
8
3
5
1
0 ꢁL MTT solution and a positive control with 10 ꢁL of
the MTT stock solution added to 100 ꢁL of medium were
also included in the experiment.
8
ꢀ
growth of bacteria was examined by colony count method.
4. RESULTS AND DISCUSSION
4
.1. Synthesis and Quaternization of Dendrimers and
3
.5. Evaluation of MIC and MBC of
PAMAM G1.0 Q OI
Dendritric Network
Two amino groups of EDA react with four acrylate groups
by Michael addition generating acrylate terminated struc-
ture designated as G −0.5 dendrimer as illustrated in
Schemes 1(a) and (b). These end groups further underwent
exhaustive amidation with excess of EDA, generating G
0.0 dendrimer with amino groups at the ends. Subsequent
repetition of these two steps yielded hyperbranched yel-
low colored liquid of G1.0 dendrimer of around 100 nm
Minimum inhibitory concentration (MIC) is the lowest
concentration of an antimicrobial agent that will inhibit the
visible growth of a microorganism after overnight incu-
bation. It is a basic laboratory practice to measure the
activity of antimicrobial agent against microbe and to mon-
itor the resistance of microorganisms to an antimicrobial
agent.33 The minimum bactericidal concentration (MBC)
1002
J. Nanosci. Nanotechnol. 16, 998–1007, 2016

Abid et al.
Antimicrobial Efficacy of Synthesized Quaternary Ammonium PAMAM Dendrimers and Dendritic Polymer Network
size and having 14 quaternizable nitrogen moieties. As
seen from Scheme 1, primary and secondary amines of
PAMAM G1.0 were completely quaterized with acid HCl
yielding water soluble PAMAM QHCl dendrimer. Typi-
cally, excess of acids was used to facilitate the reaction
and to prevent intradendrimer quaternization.
The two reactive protons of primary amines of PAMAM
G1.0 converted to dimethacrylate moieties on reaction
with EGDMA having terminal double bonds which were
polymerized with AIBN to yield a highly intricate trans-
parent solid compound as represented in Scheme 1(c).
Amine groups of its core dendritic structure reacted with
long hydrocarbon salt octyl iodide resulting in quaternary
ammonium solid derivative. Octyl iodide was chosen since
it has long hydrocarbon chain which significantly improves
3
6
antimicrobial activities of QACs.
Figure 2. FTIR spectrum of (a) PAMAM G1.0 EGDMA (b)
poly(PAMAM G1.0 EGDMA) (c) poly(PAMAM G1.0 EGDMA) Q OI.
4
.2. Characterization of Synthesized
Dendrimers and Network
4
.2.1. ATR-FTIR Spectroscopy
4.2.2. NMR Spectroscopy
The IR spectrum of PAMAM G1.0 and quaternized prod-
uct, PAMAM G1.0 Q HCl are given as Figures 1(a)
1HNMR spectrum of PAMAM G 0.5 is given as
Figure 3(a). Formation of ester terminated product is
−1
and (b). A broad peak at 2952 cm and increase in
confirmed by the appearance of –O–CH peak at 3.6 ppm
3
−1
the intensity at 3244 cm is attributed to the formation
of quaternary ammonium (+NH) group. Michael addi-
tion reaction of PAMAM G1.0 with EGDMA was con-
(24 H, s). A peak corresponding to –CO–NH–CH –CH –
2
2
N– at 3.25 ppm (8 H, q) further confirmed the formation
of ester terminated product PAMAM G 0.5. Absence of
−
1
firmed by the appearance of –C C peak at 810 cm and
–O–CH peak at 3.6 ppm and presence of –NH peak at
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2
−1
ester –C O peak at 1722 cm I aP s: s1 e 7e 7n . 1f r 0o .m1 2 F5 i .g2u 0r e5 2O ( an ): .We d2 ,. 40 59 pMp ma r (2 1 06 1 H6 ,0 t9) : 2c 3o n: 0fi 2r med the formation of PAMAM
Copyright: American Scientific Publishers
Figure 2(b) shows the IR spectrum of poly(PAMAM G1.0-
G1.0 in Figure 3(b). The expanded 1HNMR spectra of (c)
PAMAM G1.0 and (d) PAMAM G1.0 QHCl proved quat-
ernization of dendrimer as new peaks at 8.50–8.64 ppm
appeared. Michael addition reaction of PAMAM G1.0
with EGDMA was confirmed through the appearance of
vinyl proton peak at 5.59 ppm and 6.13 ppm (16 H, s) as
seen from Figure 3(e). Other characteristic peaks of the
new compound are as follows: 4.40 ppm (64 H, s, –N–
CH - C(CH )–CO–O–CH –CH –O–CO–C(CH ) CH );
−1
EGDMA) in which absence of –C C peak at 810 cm
confirmed the complete utilization of double bonds in
polymerization reaction. Quaternized polymer (Fig. 2(c))
clearly showed the presence of –CH stretching of octyl
−1
−1
iodide at 2956 cm
and 2923 cm confirming the
quaternization of poly(PAMAM G1.0 EGDMA) to form
poly(PAMAM G1.0 EGDMA) Q OI.
2
3
2
2
3
2
1
.94 ppm (48 H, s, –N–CH –C(CH )–CO–O–CH –
2 3 2
CH –O–CO–C(CH ) CH ); 1.19 ppm (48 H,d, –N–
2
3
2
CH –C(CH )–CO–OCH –CH –O–CO–C(CH ) CH );
2
3
2
2
3
2
3
.29 ppm (24 H, –CO–NH–CH –CH –N–); 3.6 ppm (8 H,
2 2
t, –(CH –CH ) –N–CH –CH –N-(CH –CH ) -).
2
2 2
2
2
2
2 2
4
.2.3. Differential Scanning Calorimetry
The DSC thermograms of solid PAMAM G1.0-EGDMA
and PAMAM G1.0-EGDMA QOI are shown as Figure 4.
Shifts of the glass temperature (Tg) determined from the
thermograms are considered as a reliable criterion for
the assessment of corresponding polymeric structure after
ꢀ
modifications. Tg of PAMAM G1.0-EGDMA was 138 C
where as its quaternized version showed a slightly higher
ꢀ
Tg of 146 C due to increase in charge density with the
incorporation of octyl iodide. Lengthy inflexible chain of
octyl group also decreased the dendritic mobility which
leads to increased Tg.
Figure 1. FTIR spectrum of (a) PAMAM G1.0 (b) PAMAM G1.0
Q HCl.
J. Nanosci. Nanotechnol. 16, 998–1007, 2016
1003

Antimicrobial Efficacy of Synthesized Quaternary Ammonium PAMAM Dendrimers and Dendritic Polymer Network
Abid et al.
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Figure 3. 1HNMR spectrum of (a) PAI MP A: M1 7 G7 .0 1. 50 (. b1 ) 2P 5A .M2 A0 M5 OG 1n . 0: ; WE xep da n, d 0e d9 1 MH Na Mr R2 0s p1 e 6c t r0u 9m : 2o f3 (: c0) 2P AMAM G1.0 (d) PAMAM G1.0 Q HCl
(e) PAMAM G1.0 EGDMA.
Copyright: American Scientific Publishers
4
.2.4. Scanning Electron Microscopy
not degraded by acid an essential feature for antimicrobial
system.
The scanning electron micrographs of PAMAM dendritic
polymer network before and after quaternization are pre-
sented in Figure 5. Polymer showed more roughness in
the surface but no porosity was observed on polymer after
the quaternization. The increase in roughness and irregu-
larity of the polymer was probably due to heterogeneous
reaction of acid on polymer and the structure is intact and
4
.2.5. Gel Content
Quaternary ammonium poly(PAMAM G1.0-EGDMA Q
OI) was found to be highly insoluble in chloroform and
its gel content was evaluated as 99.94% indicating highly
cross linked structure of dendritic polymer network and its
high stability in organic solvent.
4
.2.6. Swelling Studies
The swelling behavior of the poly(PAMAM G1.0
EGDMA) and poly(PAMAM G1.0 EGDMA) Q OI in dis-
tilled water are shown in Figure 6. Quaternized dendritic
polymer showed more water swelling property (19.7%)
than its non-quaternized version (11%). Maximum per-
centage of swelling was achieved within 30 minutes after
immersing in water. Increased amount of swelling can be
attributed to the increase in charge density of the quater-
nized product. Irregular surface of quaternary polymer was
found to hold more water than the smooth non-quaternized
polymer. Interaction of water molecules increases with
quaternization suggesting that the material will hold more
water molecules as a disinfectant which is essential for
their working.
Figure 4. DSC curves of PAMAM (a) G1.0 EGDMA (b) G1.0
EGDMA Q OI.
1004
J. Nanosci. Nanotechnol. 16, 998–1007, 2016

Abid et al.
Antimicrobial Efficacy of Synthesized Quaternary Ammonium PAMAM Dendrimers and Dendritic Polymer Network
absorbance in the entire UV-Visible-Partial NIR range of
wavelengths from 190–1100 nm even though the sample
was kept in water for 30 days. This confirmed that the
synthesized solid compound was stable and no leaching of
chemical components occurred from dendritic polymer in
water. No weight loss was also observed from the poly-
mer after keeping them for one month in water which is
attributed to stable structure of the compound eventually
indicating water stability and their potential use for disin-
fection purpose.
4
.3. Evaluation of Antimicrobial Activity of
PAMAM G1.0 QHCl and Poly(PAMAM
G1.0-EGDMA) QOI
Table I compiles the antimicrobial activity of quaternary
ammonium PAMAM dendrimer and dendritic polymer net-
work against E coli, Staphylococcus aureus, Pseudomonas
aeruginosa, Bacillus subtilis, common bacterial contami-
nants present in water. The results showed that both liq-
uid quaternary ammonium dendrimer and solid dendritic
polymer network are highly effective against very high
count of common Gram-negative and Gram-positive bac-
8
teria present in 10 cfu/mL concentration. It was observed
that their antimicrobial activity increased with increase in
time of incubation and concentration. 10 mg/L PAMAM
G1.0 QHCl was efficient to kill 100% bacteria within a
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contact time of just 2 min and 10 mg/L of quaternary
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Copyright: American S ac mi e mn toi fni ci u Pm u bd l ei sn hd er i rms er showed almost double microbial
Figure 5. SEM micrographs of (a) PAMAM G1.0 EGDMA (b)
PAMAM G1.0 EGDMA Q OI.
killing property than 5 mg/L of compound. These results
highlight the potency and efficiency of synthesized den-
drimers since they are able to mitigate very high con-
8
centration of 10 CFU/mL and they would be effective
4
.2.7. Leaching Studies
against lower concentrations of bacterial usually contami-
nating residentials and hospitals. This can be attributed to
the fact that polycationic QACs work on contact mecha-
nism for killing bacteria by disrupting and permeating lipid
bilayer membrane of bacterial cell. Negatively charged
lipopolysaccharide and peptidoglycan layer of Gram nega-
tive bacteria as well as lipoteichoic acid and teichoid acid
layer in Gram-positive bacteria facilitate interaction and
eventual entry of positively charged dendrimers disrupt-
ing cellular integration. Log reduction value, denoting the
change (decrease) of microorganism population relative to
the starting inoculums, of quaternary ammonium PAMAM
dendrimer was calculated for the concentration 5 mg/L
with incubation period of 2 min (Table II). By definition,
Reactant EDA and liquid product PAMAM G1.0 were sol-
uble in water and showed strong absorption at 243 nm
and 220 nm respectively in UV-Visible spectrophotome-
ter. Synthesized dendritic network showed no significant
8
the log reduction value of 8 indicates 100% killing of 10
CFU/mL of bacterial concentration. Among all bacteria,
highest log reduction value of 7.0 was observed with the
bacteria P. aeruginosa whereas B. subtillis showed low-
est log reduction value (6.38) probably as during adverse
conditions these bacteria develop endospores consisting of
3
7
thick cell wall which can withstand harsh conditions.
Figure 6. Swelling behavior of poly(PAMAM G1.0 EGDMA) and
poly(PAMAM G1.0 EGDMA) Q OI.
Dendritic polymer network, poly(PAMAM G1.0 EGDMA)
J. Nanosci. Nanotechnol. 16, 998–1007, 2016
1005

Antimicrobial Efficacy of Synthesized Quaternary Ammonium PAMAM Dendrimers and Dendritic Polymer Network
Abid et al.
Table I. Antimicrobial evaluation of quaternary ammonium PAMAM dendrimer and dendritic polymer by colony count method.
Bacterial survival (CFlVmL)
5
mg/L dendrimers
10 mg/L dendrimers
Quaternary ammonium
dendrimers
Incubation
time (min.) E. coli P. aeruginosa S. aureus B. subtilis E. coli P. aeruginosa S. aureus B. subtilis
PAMAM G1.0 Q HCI
2
5
0
13± 4
8± 2
0
10± 2
9± 4
0
23± 6
12± 5
0
24± 7
16± 3
0
0
0
0
0
0
0
0
0
0
0
0
0
1
Poly(PAMAM G1.0 EGDMA) QOI
2
5
0
19± 5
16± 7
33± 5
41± 6
3± 1
3± 3
5± 3
6± 4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
QOI required higher contact time of 5 min for 100%
killing as antimicrobial action is through the contact of
antimicrobial polymer with the cell walls of bacteria with-
9
out releasing any active agents. Lower log reduction val-
ues (6.38–6.79) were observed for 5 mg/L poly(PAMAM
G1.0 EGDMA) QOI compared to PAMAM G1.0 Q HCl
(
Table II) indicating that due to insolubility of dendritic
polymer in water they take more time to make contact with
the microbes. These structures are new age antibacterials
offering advantage of efficient contact killing based bioci-
dal property without releasing any component like drug or
biocidal loaded polymers.
4
.4. Evaluation of MIC and MBC of
Figure 7. In-vitro toxicity assay (MTT assay) of synthesized quaternary
ammonium dendrimer and dendritic polymer network.
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PAMAM G1.0 Q HCl
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Minimum inhibitory concentration and minimum bacteri-
cidal concentration of the synthesized quaternary ammo-
nium PAMAM dendrimer was found to be 2.5 mg/L and
Copyright: American Scientific Publishers
4.6. In Vitro Toxicity Assessment of PAMAM G1.0
QHCl and Poly(PAMAM G1.0-EGDMA)
4
mg/L respectively against various tested bacteria. These
QOI by MTT Assay
values are in agreement with other reported polymeric
MTT assay results of quaternary ammonium PAMAM
dendrimer and dendritic polymer network are shown in
Figure 7. Higher the cytotoxicity of the tested material,
the lesser will be the metabolic activity of the cells lead-
ing to lower absorbance value. The results indicated that
Jurkat cells were proliferating and remained viable after
3
8ꢀ39
antimicrobial formulations
the iodine based antibacterial products.
and are better than some of
4
0ꢀ41
4
.5. Evaluation of Antimicrobial Efficiency
Poly(PAMAM G1.0 EGDMA)
QOI for Repetitive Use
2
4 h of exposure to quaternary ammonium dendrimer and
dendritic polymer network. PAMAM G1.0 QHCl showed
absorption value of 0.11 compared to 0.21 of negative con-
trol and 0.074 value of positive control, indicating more
number of surviving cells and hence its less toxic nature.
Cytotoxicity of the PAMAM showed further reduction on
polymerization as indicated by the higher value of absorp-
tion (0.19), indicating no leaching of active compounds in
water medium.
It was observed that there was no growth of bacteria in
any of the nutrient agar plats till twenty five cycles except
in the negative control. The results demonstrate its excel-
lent efficiency as antimicrobial agent even with very high
8
concentration of 10 CFU/mL of bacteria.
Table II. Comparative log reduction value observed for PAMAM G1.0
QHCl and poly(PAMAM G1.0 EGDMA) Q OI.
Contact time = 2 min
s
5. CONCLUSIONS
(
Initial bacterial concentration = 10 cfu/mL)
This study reports for first time the quaternization of
PAMAM with HCl to form water soluble antimicrobial
PAMAM dendrimer. It also illustrated direct functionaliza-
tion with EGDMA to make dendritic polymeric network
and its eventual quaternization. Both quaternary ammo-
nium dendrimer (PAMAM G1.0 QHCl) and dendritic
Dendrimer (5 mg/mL) E. Coli P. aeruginosa S. aureus B. subtilis
PAMAM G1.0
Q HCL
6.89
6.21
7.00
6.79
6.64
6.48
6.62
6.38
Poly(PAMAM G1.0
EGDMA) QHCl
1006
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Abid et al.
Antimicrobial Efficacy of Synthesized Quaternary Ammonium PAMAM Dendrimers and Dendritic Polymer Network
polymer network (poly(PAMAM G1.0-EGDMA) Q OI)
showed significant antimicrobial activity against Gram-
positive and Gram-negative bacteria. 5 mg/L water soluble
PAMAM G1.0 QHCl was found to be efficient to disin-
fect all types of tested bacteria within an incubation time
of just 2 minutes. Moreover, 10 mg/L of water insolu-
ble poly(PAMAM G1.0 EGDMA) QOI also disinfected
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ABBREVIATIONS
QAC Quaternary ammonium compounds
2
5. P. Singh, U. Gupta, A. Asthana, and N. K. Jain, Bioconjugate Chem.
G
Q
Generation
Quarternized
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Delivered by Publishing TechnologySctio. :1 1M5 ,c7 M1 6a( s2 t0 e1 0r ) U. niversity
PAMAM Polyamido amine
OI Octyl iodide
PAMAM-EGDMA Polyamido amine-ethylene glycol
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Copyright: American S 2c 8i .e nM t i. fi cK .P Cu abl al bi sr eht tea ,r sA. Kumar, A. M. McDermott, and C. Ca,
Biomacromolecules 8, 1807 (2007).
dimethacrylate.
2
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3
Acknowledgment: Financial support from Lockheed
Martin Corporation (NJ, U.S.A.) is gratefully acknowl-
edged. Authors are also thankful to CSIR, ICMR and
UGC, Government of India, for their senior research and
post-doctoral fellowships.
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Received: 23 April 2014. Accepted: 10 October 2014.
J. Nanosci. Nanotechnol. 16, 998–1007, 2016
1007