Structural characterization and antibacterial activity of oligoguanidine

(polyhexamethylene guanidine hydrochloride)

Dafu Wei
a
, Qiangxiang Ma
a
, Yong Guan
a
, Fuzeng Hu
a
, Anna Zheng
a,
, Xi Zhang
b
, Zheng Teng
b
, Hua Jiang
b
a
Key Laboratory for Ultrane Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, 200237, Shanghai, China
b
Shanghai Municipal Center for Disease Control and Prevention, Shanghai, China
a b s t r a c t a r t i c l e i n f o
Article history:
Received 29 October 2008
Received in revised form 8 January 2009
Accepted 5 February 2009
Available online 20 February 2009
Keywords:
Antibacterial activity
Molecular structure
Polyguanidine oligomers
Polyguanidine oligomers, particularly polyhexamethylene biguanide (PHMB) oligomer, are used extensively
and safely as disinfectants and biocides. The antibacterial activity of PHMB is attributed to its interaction with
cellar membrane components. However, the detailed molecular structures and components of polyguanidine
oligomers are still vague and remain to be learned. In this paper, polyhexamethylene guanidine oligomer
(PHMG) was prepared by polycondensation of hexamethylenediamine and guanidine hydrochloride, and its
molecular structure and components were analyzed in detail using an electrospray ionization time-of-ight
mass spectrometry (ESI-TOF-MS). Seven types of molecular structures of PHMG were determined, including
three linear types and four cyclic or branched ones, and their relative contents varied with reaction
conditions. Furthermore, the relationship between the antibacterial activity and weight-average molecular
weight (M
w
) of PHMG was investigated. The results showed that an aqueous solution of PHMG (M
w
N640) at
a concentration as low as 1.0 ppm exhibited an antibacterial rate above 90.0%.
2009 Elsevier B.V. All rights reserved.
1. Introduction
Infectious disease remains a critically important global healthcare
issue. About 2 million people acquire bacterial infections in U.S.
hospitals eachyear, and 90,000 die as a result. One approach to control
infections is to develop materials that limit bacteria colonization on
their surfaces in an effort to prevent the rampant growth of bacteria
and the transmission of infectious organisms. Gabriel et al. introduced
three types of antimicrobial macromolecules arranged by chemical
structures, including antimicrobial peptidomimetics, facially amphi-
philic antimicrobial polymers and oligomers, and biocidal cationic
polymers [1]. The biocidal cationic polymers, like polyguanidines
(PGs) and polybiguanides (PBGs), have attracted considerable atten-
tion for their high antibacterial activity and low toxicity to humans.
They have been extensively used as topical anti-infective solutions in
ophthalmology [24], as disinfectants and biocides in water systems
[5], in topical wound [6,7], on cotton [8] and environments [9].
Recently, polyhexamethylene biguanide (PHMB) was found to have
activity against human immunodeciency virus type 1 [10].
The mode of action of PGs is supposed to be similar to that of PBGs.
The lethal action of PBGs to bacterial cell is believed that they can
disrupt the lipid cell membrane [1116]. PBGs have strongly positive
charges and can bind rapidly to the electronegative envelope of
bacteria and displace the stabilizing presence of Mg
2+
and Ca
2+
ions.
This binding is to the cytoplasmic membrane, or to lipopolysaccharide
and peptidoglycan components of the cell wall, which can change the
phospholipids environment of the membrane and cause the loss of its
bio-function, or destroy the cytoplasmic membrane and lead to the
death of the bacteria.
However, PGs and PBGs could be poorly dened chemically. On the
one hand, as polymeric materials, they consist of a mixture of molecules
with different chain lengths, different molecular structures and
comparatively low molecular weights. On the other hand, as polyelec-
trolytes, their molecular weights were difcult to characterize using
many classical characterization methods. Infrared spectra (IR) and
nuclear magnetic resonance (NMR) are common tools to characterize
the molecular structure, but their information are not sufcient [17,18],
providing no detailed information of the composition of PGs and PBGs.
Various conventional methods to determine polymeric molecular
weight, such as gel-permeation chromatography (GPC), intrinsic
viscosity, and vapor pressure osmometry (VPO) etc., are not suitable
to obtain satisfying results because of their poor compatibility with PGs
and PBGs. Therefore, the detailed information of molecular structures
and weights of PGs and PBGs have been scarcely reported, although
these are very important to control PGs' and PBGs' preparation and their
chemical reactions with other materials for further applications, as
shown in our researches [19]. Therefore, it is expected to develop a new
suitable method for the analysis of PGs and PBGs. The emergence of soft
ionization technique mass spectrometry-matrix-assisted laser desorp-
tion ionization time-of-ight mass spectrometry (MALDI-TOF-MS) can
Materials Science and Engineering C 29 (2009) 17761780
Corresponding author. Tel.: +86 21 64253343; fax: +86 21 6425 2744.
E-mail address: zan@ecust.edu.cn (A. Zheng).
0928-4931/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.msec.2009.02.005
Contents lists available at ScienceDirect
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potentially solve this problem. This technique is considered to be
suited for bio-macromolecules (such as protein) or synthetic polymers
(such as polystyrene). Feiertag et al. [20,21] rst used MALDI-TOF-MS to
analyze the structures of the oligoguandines, and found that some cyclic
structures existed in the products using 1,2-bis(2-aminoethoxy) ethane
as the starting material, while no cyclic structures in the products when
using hexamethylenediamine as the starting material. The structure
differences among the prepared polyguanidine oligomers are very
interesting and important. However, so far as we know, further reports
onthe preparationandproperties of suchproducts have not beenfound.
MALDI-TOF-MS was also used to obtain the structure information of
polyhexamethylene biguanide (PHMB) by Malley et al. [22]. It was
considered to be able to detect PHMB oligomers with polymerization
degree below 6, and six types of linear structures were identied in the
product. Therefore, an important advantage of MALDI-TOF-MS method
is that it can provide information on the repeating unit structure, end-
group, and molecular weight. It is a proper approach to analyze
polyguanides although there still remain some problems, such as the
limitation of polymerization degree of polyguanides [22]. Furthermore,
proper matrixes (such as 2, 5-dihydroxybenzoic acid aqueous solution
[21] or 6-aza-2-thiothyminemethanol solution[22]) were neededinthe
MALDI-TOF-MS analysis of PGs and PBGs. As a comparison, another soft
ionization technique mass spectrometry, i.e., electrospray ionization
time-of-ight mass spectrometry (ESI-TOF-MS), is hypothesized to be
more suitable for the structural analysis of polyguanidines with no
need of matrixes, especially for its compatibility with polycationic
compounds.
In this paper, polyhexamethylene guanidine hydrochloride
(PHMG) were prepared by polycondensation in the melt of hexam-
ethylenediamine and guanidine hydrochloride, and their structures
and molecular weights were analyzed using ESI-TOF-MS. Seven types
of structures were found in the product, three of them are linear and
other four are cyclic or branched. The interpretation of the formation
of cyclic or branched structures with respect to the mechanism of
polycondensation of PHMG was discussed. Furthermore, the relation-
ship between the antibacterial activity of PHMG and its M
w
was also
investigated.
2. Experimental
2.1. Synthesis and structural characterization of PHMG
Equimolar amounts of hexamethylenediamine (CP, Sinopharm
Chemical Reagents Co. Ltd. (Shanghai)) and guanidine hydrochloride
(99.5%, Sinopharm Chemical Reagents Co. Ltd. (Shanghai)) were
mixed in a round-bottomed three-necked ask, which is equipped
with a mechanical stirrer and vacuum system. The mixture reacted at
100 C for 60 min, and then at 170 C for a certain time. During the
reaction, by-product ammonia is neutralized by bubbling through
aqueous HCl. Thereafter the reaction continued on the condition of
removing ammonia by vacuum system. At the end of reaction, the
slightly yellow, viscous liquid solidies upon cooling giving PHMG
samples.
FT-IR (KBr) spectra of samples were recorded by a Nicolet 5700
Fourier transform infrared spectrometer.
1
H-NMR spectra of samples (D
2
O, 500 MHz) were acquired by an
AVANCE500 NMR spectrometer. Chemical shifts were reported in
(ppm) units relative to tetramethylsilane.
ESI-TOF mass spectra were acquired using a Micromass LCT time-
of-ight mass spectrometer equipped with electrospray ionization.
Sample solutions were prepared using H
2
O/CH
3
OH (1:3) mixture as
solvent. Ions were generated by electrospray ionization below 120 C.
The results were analyzed with MASSLYNXTM 3.5 system. In this work,
m/z values of the mono-isotopic peaks of any isotope distribution
were reported.
2.2. Antibacterial activity
Cultures of Staphylococcus aureus (ATCC6538) and Pseudomonas
aeruginosa (ATCC19429) incubated at 37 C over 24 h were diluted
with phosphate buffer solution (PBS, pH=7.4) to the bacterial
suspension containing 10
6
cfu/ml for later tests. 100 l bacterial
suspension was mixed with 5 ml PHMG aqueous solution or sterilized
comparative solution in tubes. After the mixture was shaking for a
certain period (2 min or 15 min), 0.5 ml of the mixture was taken out
and diluted with PBS to a desired concentration. Subsequently, 0.1 ml
diluted solutionwas spread on agar (15 ml) plates, which were further
incubated at 37 C for 48 h. The number of bacterial cells was
calculated by multiplying the number of colonies with the dilution
factors. Each test was repeated at least three times for each PHMG
concentration solution.
Antibacterial rate X was calculated by following equation:
X = A B = A 100k;
A: the number of colonies in the control solution;
B: the number of colonies in the PHMG solution.
3. Results and discussion
3.1. Structures and molecular weight analysis of PHMG by ESI-TOF mass
spectrometry
The characteristic peaks of PHMG samples FTIR spectra are: 3350
and 3170 (
NH
), 1650 (
C=N
) and 1630 cm
1
(
NH2
), which are
consistent with the data produced by other investigators [17]. In
the
1
H-NMR spectra, the peaks are: 1.34~1.57 (t, -CH
2
-), 2.612.66
(m, -NH
2
), 3.133.22 (m, -CH
2
-NH-) and 4.744.83 (t, -NH-). The
results of FTIR spectra and
1
H-NMR spectra showed the groups
structures of PHMG.
The relationship between weight-average molecular weights (M
w
)
of PHMG samples and reaction conditions was shown in Table 1. The
calculations of M
w
are based on a correlation of the measured ESI-TOF
mass peaks and their corresponding intensities. And their individual
ESI-TOF mass spectrum was listed in Fig. 1(ad). The results of M
w
of
PHMG based on the ESI-TOF mass spectra were similar to the data
produced by Albert et al. using MALDI-TOF-MS technique [21],
indicating ESI-TOF-MS is also a proper method to analyze the
molecular structure of PHMG.
From the ESI-TOF mass spectra of PHMG, an important result is
that seven series of molecular weights were observed, although
the intensities of some series were comparatively low. Their nomi-
nal masses are [1+(141)
n
+16+1] (A), [100+(141)
n
+16+1] (B),
[1+(141)
n
+58+1] (C), [100+(141)
n+m
1+100+16+1] (D),
[(141)
n
+1] (E), [(141)
n
+42+1] (F), and [(141)
n+m
1+100+1]
Da (G), respectively. The corresponding molecular structures could be
concluded as shown in Fig. 2. Among them, three types (AC) are linear,
terminated with one amine group and one guanidine group (type A),
with two amine groups (type B), and with two guanidine groups
Table 1
Relationship between M
w
of PHMG samples and reaction conditions.
Samples Reaction time (min) M
w
b
At 100 C At 170 C In vacuum
a
at 170 C
1 60 180 408
2 60 256 516
3 60 260 16 640
4 60 260 61 956
a
To remove by-product ammonia.
b
Calculated based on ESI-TOF mass spectra.
1777 D. Wei et al. / Materials Science and Engineering C 29 (2009) 17761780
(type C), respectively. The other four are branched (type D) or cyclic
(types EG).
These types of molecular structures can be explained by poly-
condensation mechanism between hexamethylenediamine and guani-
dine hydrochloride. It is understandable for the occurrence of the three
linear structures. The branched structures, such as type D or type G, are
considered to result from the branched reaction of imine group of
guanidinium. The reaction activity of the imine group was conrmed by
the fact that crosslinked products occurred when the polycondensation
was conducted at a higher temperature and extended reaction time.
Furthermore, for the cyclic structures, such as types E, F and G, it is also
reasonable that their formation results from the intramolecular
reactions between the terminal amines, which have the same reactivity
as monomers according to Flory's principle of equal reactivity of
functional groups of polymer. Thus, one can be expected that the
quantity of the cyclic structures will increase with the increase of chain
length which facilitates intramolecular reaction between the two
terminal amine groups. This was substantiated by the data given in
Table 2. Therefore, the cyclic structures exist inthe PHMGprepared from
hexamethylenediamine and guanidine hydrochloride. The derivation of
structures in the PHMGproducts perhaps as follows: the intramolecular
cyclization reactions of types A, C and D result in the formation of cyclic
structures types E, F and G, respectively. Besides, Albert et al. also
reported the existence of cyclic structures in the polyguanidine using
guanidine hydrochloride and 1, 2-bis (2-aminoethoxy) ethane as the
starting materials [21]. It is of interest whether the cyclic structures
could be obtained in the other polyguanidine using other diamines as
the starting materials, which will be investigated in our future work.
Fig. 1. ESI-TOF mass spectra of PHMG. (a) Sample 1, (b) Sample 2, (c) Sample 3, and (d) Sample 4.
1778 D. Wei et al. / Materials Science and Engineering C 29 (2009) 17761780
Except for the identication of seven types of molecular structures
in the prepared PHMG, their individual relative contents (shown in
Table 2) can be determined based on the intensities of their mass
spectra peaks. As can be seen, the contents of the cyclic components,
types E, F and G, monotonically increased with the reaction time until
their total contents reached about 20%. The contents of types A and C
also increased with the reaction time. However, the content of type B
decreased monotonically, especially there was a rapid decrease when
the polycondensation was conducted in vacuum to remove by-
product ammonia, which was probably due to an conversion of
type B to type A in the late stage of melting polycondensation.
Meanwhile, the content of type D also decreased as that of type B. The
relative content decrease of type B and type D (both terminated with
two amine groups) indicated that the reactivity of amine group of
hexamethylenediamine was higher than that of the amine group of
guanidine hydrochloride. Thus, the contents of cyclic and branched
components could be controlled by reducing the molar quantity of
hexamethylenediamine. However, the reaction rate of polycondensa-
tion also was reduced.
3.2. The relationship between antibacterial activities and molecular
weights of PHMG
In the above discussion, seven types of structures in the PHMG and
their individual relative contents were identied. Here we provided
the relationship between antibacterial activities and weight-average
molecular weight (M
w
) of PHMG (shown in Table 3). When M
w
was
above 640, 1.0 ppm PHMG aqueous solutions could inhibit over 90% of
the bacteria growth within 15 min against both S. aureus and
P. aeruginosa. On the contrast, the antibacterial rates against S. aureus
and P. aeruginosa were only 84.67% and 72.68%, respectively, when
M
w
was 516. However, all PHMG (M
w
516) aqueous solutions had
excellent antibacterial activities (N99.0%) against S. aureus and
P. aeruginosa within 15 min when the concentration increased to
5.0 ppm. This differed fromPHMB, which showed a good antibacterial
activity only when the polymerization degree was above 10 [11].
In the PHMG aqueous solution at the concentration as low as
1.0 ppm, PHMG should be molecularly distributed in the water. So, the
increase of the antibacterial activity of PHMG with its M
w
, may owe to
the increase of the total positive charges of PHMG molecule. It is
implied that PHMG would have the most excellent antibacterial
activity when the total positive charges of single PHMG molecule
match with the negative charges of bacteria. A clear correlation
between positive charges of molecule and antimicrobial activity has
been shown for the antimicrobial peptides. The optimal charge for a
maximal lytic activity is around +4. An excessive charge can have a
deleterious effect on activity, as it prevents structuring [23]. For the
oligoguanidine, the correlation between positive charges of molecule
and antibacterial activity is not determined, although our results
showed excellent antibacterial activities when the M
w
of PHMG
Fig. 2. Seven types of molecular structures (types AG) in the PHMG.
Table 2
Relative contents of each structure (types AG) in the PHMG.
Samples Reaction time
(min)
M
w
Component contents (mol%)
A B C D E F G
1 240 408 26.77 58.45 2.98 4.63 4.47 0.77 1.82
2 316 516 28.32 52.58 3.41 5.26 6.57 1.23 2.64
3 336 640
a
38.70 28.75 12.59 2.28 10.27 3.78 3.61
4 381 956
a
37.97 24.87 14.92 2.64 10.63 4.67 4.24
a
In vacuum after 320 min.
Table 3
Antibacterial activities against S. aureus and P. aeruginosa of aqueous solutions of PHMG
with different M
w
.
a
M
w
Concentration
(ppm)
Antibacterial rate (%)
S. aureus P. aeruginosa
2 min 15 min 2 min 15 min
516 10.0 100.00 100.00 100.00 100.00
5.0 99.95 100.00 99.72 99.98
1.0 50.70 84.67 68.92 72.68
640 10.0 100.00 100.00 100.00 100.00
5.0 94.6 100.00 91.35 100.00
1.0 66.59 91.03 66.76 92.92
956 10.0 100.00 100.00 100.00 100.00
5.0 99.99 100.00 87.44 100.00
1.0 56.91 95.97 69.82 98.56
a
The tests were performed in Shanghai Municipal Center for Disease Control and
Prevention.
1779 D. Wei et al. / Materials Science and Engineering C 29 (2009) 17761780
ranged from 640 to 956, corresponding 46 positive charges per
PHMG. However, further research is necessary to investigate the
antibacterial activity of PHMG with higher M
w
and nd the optimal
molecular structure. On the other hand, the increase of antibacterial
activity of PHMG also partially attributed to the relative contents
increase (as shown in the above discussion) of cyclic components
(containing more centralized positive charges compared with the
linear products) with the molecular weights. The centralized degree of
positive charges was supposed to affect the antibacterial activity.
Perhaps it is difcult to obtain the direct evidence. However, some
indirect results may favor this point. The sizes of aggregates of PHMG
increased when alkali was added in its aqueous solution. Meanwhile
the antibacterial activity was remarkably improved, which was
partially attributed to the increase of centralized degree of positive
charges. More details will be investigated in the future.
Oligoguanidine (PHMG) has excellent antibacterial activity. Its
potential application will be further evaluated. It is expected to be
used as disinfectants and biocides in water systems, or used as topical
anti-infective ingredient in ophthalmology or wound dressing.
4. Conclusion
In this paper, the structures and molecular weights of PHMG were
analyzed in detail using ESI-TOF-MS. The results indicate that ESI-TOF-
MS is a suitable approach to conveniently analyze the detailed struc-
tures of PHMG. With the help of ESI-TOF-MS, seven types of molecular
structures in the prepared PHMG were determined, in which three
structures were linear, and the other four were cyclic or branched. The
relative contents of seven structures varied with the reaction time,
and those of cyclic structures obviously increased as the reaction
prolonged due to the increase of chain length facilitating intramole-
cular reaction between the two terminal amine groups.
PHMG aqueous solution showed excellent antibacterial activities,
especially when M
w
was above 640. 1.0 ppm of such PHMG aqueous
solutions could inhibit over 90% of the bacteria growth within 15 min
against both S. aureus and P. aeruginosa. The antibacterial activity of
PHMG should be correlative with the total charges of PHMG molecule
and the contents of cyclic products in the PHMG.
Acknowledgements
The authors are grateful to the nancial support by research grants
fromthe National Natural Science Foundation of China (No 50573020)
and the Shanghai Leading Academic Discipline Project (No B502).
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