Evaluation of polyamidoamine (PAMAM) dendrimers as drug carriers of anti-bacterial drugs using sulfamethoxazole (SMZ) as a model drug

Article abstract of DOI:10.1016/j.ejmech.2006.07.015

Sulfamethoxazole (SMZ), a sulfonamide with well-known anti-bacterial properties, is not freely soluble in water and causes problems in its clinical applications. In the present study we investigated the potential of ethylenediamine (EDA) core polyamidoamine (PAMAM) dendrimers as drug carriers of SMZ by aqueous solubility, in vitro release as well as anti-bacterial activity studies. Results showed that the aqueous solubility of SMZ was approximately proportional to dendrimer concentration (a 40-fold increase in solubility in 10 mg/ml G3 PAMAM dendrimer solutions compared with that in double-distilled water at 37 °C). The in vitro release of SMZ in the presence of PAMAM dendrimers was significantly slower compared to pure SMZ dissolved in ethanol. Microbiology studies showed that PAMAM dendrimers could increase the anti-bacterial activity of SMZ (a 4- or 8-fold increase in the anti-bacterial activity of SMZ in dendrimer solution compared to pure SMZ dissolved in dimethylsulfoxide (DMSO) or 0.01 M NaOH solution). The in vitro release behavior and anti-bacterial activity studies indicated that PAMAM dendrimers might be considered as potential drug carriers of sulfonamides with a sustained release behavior under suitable conditions.

Full text of DOI:10.1016/j.ejmech.2006.07.015

European Journal of Medicinal Chemistry 42 (2007) 93e98
http://www.elsevier.com/locate/ejmech
Short communication
Evaluation of polyamidoamine (PAMAM) dendrimers
as drug carriers of anti-bacterial drugs using
sulfamethoxazole (SMZ) as a model drug
a
a
a
Minglu Ma a, Yiyun Cheng a,b, , Zhenhua Xu , Peng Xu , Haiou Qu ,
*
Yujie Fang b, Tongwen Xu b, Longping Wen a,
*
a Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences,
University of Science and Technology of China, Hefei, Anhui 230027, China
b Laboratory of Functional Membranes, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China
Received 16 May 2006; received in revised form 19 July 2006; accepted 21 July 2006
Available online 7 November 2006
Abstract
Sulfamethoxazole (SMZ), a sulfonamide with well-known anti-bacterial properties, is not freely soluble in water and causes problems in its
clinical applications. In the present study we investigated the potential of ethylenediamine (EDA) core polyamidoamine (PAMAM) dendrimers
as drug carriers of SMZ by aqueous solubility, in vitro release as well as anti-bacterial activity studies. Results showed that the aqueous solubility
of SMZ was approximately proportional to dendrimer concentration (a 40-fold increase in solubility in 10 mg/ml G3 PAMAM dendrimer
solutions compared with that in double-distilled water at 37 ꢀC). The in vitro release of SMZ in the presence of PAMAM dendrimers was
significantly slower compared to pure SMZ dissolved in ethanol. Microbiology studies showed that PAMAM dendrimers could increase the
anti-bacterial activity of SMZ (a 4- or 8-fold increase in the anti-bacterial activity of SMZ in dendrimer solution compared to pure SMZ dis-
solved in dimethylsulfoxide (DMSO) or 0.01 M NaOH solution). The in vitro release behavior and anti-bacterial activity studies indicated that
PAMAM dendrimers might be considered as potential drug carriers of sulfonamides with a sustained release behavior under suitable conditions.
Ó 2006 Published by Elsevier Masson SAS.
Keywords: Polyamidoamine dendrimers; PAMAM; Drug carrier; Anti-bacterial drugs; Sulfamethoxazole
1. Introduction
including enteric and urinary tract, and respiratory tract [3].
They are preferred due to the ease of administration and wide
spectrum of anti-bacterial activity. However, the clinical use
of sulfonamides is limited mostly due to their extremely low sol-
ubility in water, rapid elimination in blood, low level of associ-
ation to plasma proteins and several side effects, which are
characterized by fever, skin rash, hepatotoxicity, lymphadenop-
athy and hematological disorders [4]. The poor solubility of sul-
fonamides restricts their use in topical and parenteral
applications. As poor solubility is generally related to a low bio-
availability, this presents a major challenge during drug formu-
lation. In order to improve the solubility of sulfonamides in
water, cyclodextrinesulfonamides complexes were prepared
to enhance dissolution and absorption rate [5,6]. However,
high costs and nephrotoxicity on parenteral administration limit
Bacterial infections remain major causes of morbidity and
mortality in hospitals around the world [1]. A new report esti-
mated that Staphylococcus aureus (S. aureus) infections alone
resulted in 9.5 billion dollars in extra hospital charges and nearly
12,000 inpatient deaths per year [2]. Sulfonamides, the develop-
ment of which is a fascinating and promising area in medicinal
chemistry, are widely used in various bacterial infections
* Corresponding authors. Hefei National Laboratory for Physical Sciences at
Microscale and School of Life Sciences, University of Science and Technol-
ogy of China, Hefei, Anhui 230027, China.
E-mail addresses: yycheng@mail.ustc.edu.cn (Y.Y. Cheng), lpwen@ustc.
edu.cn (L.P. Wen).
0223-5234/$ - see front matter Ó 2006 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2006.07.015

94
M. Ma et al. / European Journal of Medicinal Chemistry 42 (2007) 93e98
the use of cyclodextrins. Moreover, the aqueous solubility of the
commonly used cyclodextrin is insufficient to stabilize drugs at
therapeutic doses [7].
methanol, and DMSO (HPLC grade) were obtained from
Shanghai Chemical Co. (Shanghai, China). For both solubility
and in vitro release behavior studies, double-distilled water
was used.
Dendrimers are hyperbranched, monodisperse, three-
dimensional macromolecules, having defined molecular weight
and hosteguest entrapment properties. They allow the precise
control of size, shape and placement of functional groups and
combine typical characteristics of small organic molecules
and polymers that result in special physical and chemical prop-
erties [8e11]. Accordingly, dendrimers have attracted increas-
ing attention for their applications in many fields. Among
them the use of dendrimers as a drug carrier in delivery systems
has been of great interest.
2.2. Synthesis of PAMAM dendrimers
PAMAM dendrimers were synthesized according to
Ref. [20]. Ethylenediamine (10.0 g, 0.166 mol) was dissolved
in 100 ml methanol in a 1 L-round-bottomed flask. Methyl ac-
rylate (94.6 g, 0.751 mol) was added at 40 ꢀC and the system
stirred for 24 h under nitrogen. Excess methyl acrylate was re-
moved under vacuum at room temperature. A Michael addi-
tion between the amine and the acrylate yielded a product
bearing four terminal methyl ester groups, defined as the
G0.5 PAMAM. Subsequently, ethylenediamine (120 g,
2.00 mol) was dissolved in methanol and added to the G0.5
PAMAM and, after stirring for 48 h under nitrogen and remov-
ing excess reactants by vacuum distillation, a product bearing
four terminal amino groups were obtained, defined as the G1
PAMAM. By repeating the above cycle, higher generation
PAMAM dendrimers (up to G5) were synthesized. Purity of
the amine-terminated PAMAM dendrimers was characterized
via FT-IR, 1H and 13C NMR and Element analysis. The results
agreed well with that reported in the literature [11].
Polyamidoamine (PAMAM) dendrimers with an ellipsoidal
or spheroidal shape is one of the most studied starburst mac-
romolecules. Due to specific synthesis, PAMAM dendrimers
have some interesting properties, which distinguish them
from classical linear polymers, e.g. PAMAM has a much
higher amino group density comparing with conventional
macromolecules, a third generation PAMAM prepared from
ammonia core has 1.24 ꢁ 10ꢂ4 amine moieties per unit vol-
ume (cubic Angstrom units) in contrast to the 1.58 ꢁ 10ꢂ6
amine moieties per unit volume of a conventional star polymer
[11]; also, PAMAM dendrimers possess empty internal cavi-
ties and many functional end groups, which are responsible
for high solubility and reactivity. These specific properties
make dendrimers suitable for drug delivery systems [12e
15]. Drugs or other molecules can either be attached to den-
drimers’ end groups or encapsulated in the macromolecule in-
terior [16]. The high density of amino groups and special
structure in PAMAM dendrimers may be expected to have po-
tential applications in enhancing the solubility of the low
aqueous solubility drugs and as delivery systems for bioactive
materials [17]. Drugs bound to dendrimers are at early stages
of development and data on them are limited. Several authors
reported on the encapsulation of non-steroidal anti-inflamma-
tory drugs (NSAIDs) and anti-cancer drugs in dendrimers
[18,19]. However, to our knowledge there are no studies de-
voted to the solubilization of anti-bacterial drugs in the pres-
ence of dendrimers. Here, we focus on using PAMAM
dendrimers as potential drug carriers, which are emerging as
a promising group of safer and perhaps more effective alterna-
tives to traditional sulfonamides.
2.3. Solubility test
The solubility of SMZ was determined using the equilib-
rium solubility method. Excess drugs were added to 500 ml
of each test solution to ensure the drug solution reached satu-
ration. The solution was mechanically shaken for 24 h at 37 ꢀC
and then the solutions were centrifuged at 10,000 rpm for
3 min. The saturated solutions were then diluted to a proper
concentration (500ꢁ). Three repeats were conducted.
2.4. UVeVis spectroscopy
SMZ in ethanol solution or distilled water gives maximum
absorbance in UV region at its characteristic wavelength
(265 nm for SMZ). A calibration curve of SMZ was prepared
at different SMZ concentrations. PerkineElmer UVeVis
spectrometer was used to estimate the amount of drug incorpo-
rated in the dendrimer. The drugedendrimer solution was di-
luted by the same distilled water. Since the dendrimers in the
diluted solutions give no absorbance at 265 nm, the absor-
bance obtained from SMZedendrimer solution would be
solely from SMZ. This absorbance was correlated with the cal-
ibration curve and amount of SMZ was determined [21].
The aim of the present work was (1) to investigate the po-
tential of PAMAM dendrimers (G2eG4) as solubility en-
hancers of sulfonamides as exemplified by SMZ; (2) to
study the effect of concentration, generation, ion concentration
and temperature on the solubility of SMZ; and (3) to investi-
gate the in vitro release behavior and anti-bacterial activity
of SMZ in the presence of PAMAM dendrimers.
2.5. In vitro release studies
2. Experiments
In vitro release behavior of SMZ in the presence of G3
PAMAM dendrimers was investigated [22]. The SMZ was dis-
solved in dendrimer solutions and diluted by distilled water to
a final concentration of 4 mg/ml. Pure SMZ was dissolved in-
methanol (4 mg/ml) and used as control. This solution (1 ml in
2.1. Materials
SMZ was purchased from Shouguang Fukang Pharmacy
Factory (Shandong, China); ethylenediamine, methyl acrylate,

M. Ma et al. / European Journal of Medicinal Chemistry 42 (2007) 93e98
95
16
12
8
volume) was transferred to a dialysis bag (M.W. cut
off ¼ 1000) immediately. The dialysis bag was placed in
a 50 ml beaker containing 40 ml distilled water. The outer
phase was stirred continuously. After a scheduled interval of
time for 12 h, 100 ml of sample was withdrawn from the outer
phase, and the outer phase was again replenished with 100 ml
distilled water. The absorbance of the outer phase was moni-
tored at 265 nm using a spectrophotometer in order to charac-
terize the concentration of SMZ.
G2
G3
G4
4
2.6. Anti-bacterial activity test
The compounds were tested against Escherichia coli
(E. coli) for their anti-bacterial activities, using a common
Luria-Bertani liquid medium micro-dilution method as de-
scribed in Ref. [32]. Before the anti-bacterial tests, the drug
formulations in the presence/absence of dendrimer were pre-
pared as follows: SMZ was dissolved in 10 mg/ml G3 PA-
MAM dendrimer to a concentration of 2 mg/ml, while pure
SMZ was dissolved in DMSO and 0.01 M NaOH at the
same drug concentration. G3 dendrimer, DMSO and 0.01 M
NaOH solutions were also evaluated in the absence of SMZ.
When conducting the anti-bacterial activity studies, 50 ml of
Luria-Bertani liquid medium was firstly distributed from the
second to the 10th well of a 96-well plate. Then, 100 ml test
solutions prepared as above were added to the first test well
of each line, and 50 ml of scalar dilution was transferred
from the second to the 10th well. Finally, 50 ml of a microbial
suspension (w106 colony forming units, CFU/ml), obtained
from an overnight growth at 37 ꢀC, was added to each well
of the plate. The final concentration of these samples used to
evaluate the anti-bacterial activity was from 2 mg/ml (second
well) to 0.0078 mg/ml (tenth well). The plates were incubated
for 18 h at 37 ꢀC and examined by measuring the optical den-
sity in a spectrophotometer (630 nm). The concentration of
each test compound, in which the O.D. 630 nm value is lower
than 0.1, was taken as its Minimal Inhibitory Concentration
(MIC).
0
0.000
0.003
0.006
0.009
Dendrimer concentration (M)
Fig. 1. Solubility of SMZ in the presence of increasing concentration of
PAMAM dendrimers.
dendrimers can keep small guest molecules inside and make
dendrimers suitable for enhancing the solubility of drug mol-
ecules such as SMZ molecules in aqueous solutions [23]. Also,
there are tertiary amines in these internal cavities, which could
interact with the atoms of the SMZ molecules by hydrogen
bond formation [24]. Therefore, PAMAM dendrimers possess
open and internal cavities and many functional terminal
groups, which are responsible for high solubility and reactiv-
ity. These specific properties make dendrimers suitable for
drug delivery systems.
The effect of various generations of PAMAM dendrimers
(G2eG4) on the process was investigated. The results are
also shown in Fig. 1, from which it is clear that the solubility
of SMZ was affected by the generation of PAMAM dendrimer.
The solubility of SMZ in higher generation PAMAM solution
was in fact higher than those in lower ones. The solubility of
hydrophobic compounds in dendrimer solutions likely de-
pends on the dendrimer generation (size) [25]. Since the num-
ber of cavities and tertiary amines in the dendrimer increases
with generation size, at a given pH condition, higher genera-
tion dendrimer has a tendency to entrap more hydrophobic
compound inside than lower ones. In this way, we could ex-
plain why higher generation dendrimers could enhance the sol-
ubility of SMZ more efficiently than lower ones.
3. Results and discussion
3.1. The effect of dendrimer concentration and
generation on solubility of SMZ
The effect of PAMAM dendrimer concentration on solubil-
ity of sulfonamides was measured at 37 ꢀC, and the results are
shown in Fig. 1. It was observed that the extremely low water
solubility of SMZ has been significantly improved by PA-
MAM dendrimers (a 40-fold increase in solubility in 10 mg/
ml G3 PAMAM dendrimer solutions compared with that in
double-distilled water). The apparent solubility of SMZ in-
creased linearly as a function of PAMAM dendrimer solution
over the whole concentration range. The increase of solubility
of extremely low water solubility of SMZ was presumably
contributed to the internal cavities that are available to encap-
sulate SMZ molecules. Due to the specific and interesting
property of PAMAM dendrimers, the cavities in PAMAM
3.2. The effect of ion concentration on solubility of SMZ
in the presence of PAMAM dendrimers
Fig. 2 shows the effect of salt concentration (sodium chlo-
ride in this study) on solubility of SMZ in the presence of PA-
MAM dendrimers. Interestingly, high ion concentration could
inhibit the inclusion of SMZ in the cavities of PAMAM den-
drimers. Take a fourth generation PAMAM dendrimer (G4)
with a molecular weight of 6900 Da and 32 amino groups in
the outer shell for example, the approximate number of
SMZ molecules associated with each G4 PAMAM dendrimer
without any sodium chloride is calculated to be 30 according

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M. Ma et al. / European Journal of Medicinal Chemistry 42 (2007) 93e98
15
12
9
15
0 M NaCl
1 M NaCl
5 M NaCl
37°C
12
9
27°C
17°C
6
6
3
3
0
0
0.0000
0.0003
0.0006
Dendrimer concentration (M)
0.0009
0.0012
0.0015
0.0000
0.0007
0.0014
0.0021
Dendrimer concentration (M)
0.0028
Fig. 2. Solubility of SMZ in the presence of G4 PAMAM dendrimers at var-
ious salt concentrations.
Fig. 3. Solubility of SMZ in the presence of G3 PAMAM dendrimers at var-
ious temperatures.
to the slope of the line in Fig. 2; when the salt concentration is
1.0 M and 5.0 M, this number is 27 and 22, respectively. The
enhanced SMZ solubility by dendrimers was slightly greater
compared to the ones in the presence of sodium chloride
with the same concentration of PAMAM dendrimers. Welch
and Muthukumar [29] reported that varying the salt concentra-
tion in the solvent could change the shape of the intramolecu-
lar density profile of dendrimers in solution. Also they found
that a reversible between a ‘dense core’ and a ‘dense shell’
dendritic structure could be observed as the ionic strength is
cycled from high to low. Combining the simulation results
of Ref. [29] and the solubility results of this study, it is pro-
posed therefore that conformation of the PAMAM dendrimer
in solution can be tailored by varying the ion concentration
in the solvent. It was reported that the nature of the intramo-
lecular density profile and the position of the terminal groups
are critical in utilizing dendrimers as hosts in controlled re-
lease systems [26]. Ideally, the branches of dendrimers would
be highly extended at each generation of growth, with branch
termini lying at the periphery of the molecule. Several theoret-
ical studies have addressed the possibility of this occurring in
flexible dendritic systems [27,28]. Furthermore, Welch and Mu-
thukumar [29] using Monte Carlo simulations reported that the
density profiles of synthetic systems are tunable from that of the
dense core to that of the dense shell by manipulation of the salt
concentration or pH in aqueous solutions. Our data from this
study, consistent with earlier data, suggest that large changes
in molecular conformation of PAMAM dendrimers are realiz-
able. This is an important factor controlling the release of
drug molecules in a variety of environments.
Milhem et al. [30] reported that the amount of ibuprofen dis-
solved in PAMAM dendrimers was inversely proportional to
temperature. It is interesting that such result was not observed
in our experiments. In most cases, the effect of temperature
had no significant impact on the dendrimers’ host ability.
3.4. In vitro release behavior
After the anti-bacterial drug SMZ was equably dissolved in
G3 PAMAM dendrimer, the solutions were filtered through
a 0.45 mm HA filter (Millipore). UVeVis absorbance measure-
ments were carried out for the characterization of precise SMZ
concentration in the dendrimer solution. The maximum number
of SMZ numbers associated with G3 PAMAM dendrimer was
approximately 14 mol/mol of dendrimer. The in vitro release be-
havior of SMZ from the PAMAM dendrimer solution was exam-
ined in distilled water at room temperature [22]. The results are
shown in Fig. 4. After 1 h, 35.6% of the pure drug is released,
whereas only 4.8% is released from the SMZ-G3 dendrimer
70
60
50
40
30
20
pure SMZ
3.3. The effect of temperature on solubility of SMZ in the
presence of PAMAM dendrimers
10
SMZ+dendrimer
0
0
2
4
6
Time (hour)
8
10
Fig. 3 shows the effect of temperature on SMZ in the pres-
ence of G3 PAMAM dendrimer. No significant difference in
the solubility of SMZ in the presence of PAMAM dendrimers
can be observed from the curves at different temperatures.
Fig. 4. In vitro release of SMZ in G3 PAMAM dendrimer solution compared
with the pure SMZ release behavior.

M. Ma et al. / European Journal of Medicinal Chemistry 42 (2007) 93e98
97
solution. Ten hours after the system started 62.4% release was
obtained for the pure drug while 42.4% was obtained for
SMZ-G3 PAMAM solution. The release of SMZ from the
drugedendrimer solution was significantly slower compared
to pure SMZ.
drug attachment that has the ability to bind and release drugs
through several mechanisms [31]. Our work demonstrated that
encapsulation of SMZ into dendrimers led to sustained release
of the drug in vitro and an increased anti-bacterial activity. We
are in the process of conducting pre-clinical testing to evaluate
the potential of dendrimers as carrier for SMZ and other anti-
bacterial drugs. Although toxicity problems may exist, modi-
fication of the structure of dendrimers should resolve this
issue.
3.5. Anti-bacterial activities
The anti-bacterial activity of SMZ, dendrimer and SMZe
G3 dendrimer is present in Fig. 5. The results indicated that
the three compounds display anti-bacterial activity against
E. coli at proper concentrations. Interestingly, when equal
amounts of free SMZ and SMZeG3 dendrimer are considered
(the actual amount of SMZ in the dendrimer solution was
equal to the free drug used), SMZeG3 dendrimer is definitely
more potent than free SMZ dissolved in DMSO or 0.01 M
NaOH solution (a 4- or 8-fold increase in anti-bacterial activ-
ity). As pure G3 PAMAM dendrimer displayed anti-bacterial
activity against E. coli (O.D. 630 nm < 0.1) at a much higher
concentration (2.5 mg/ml, data not shown), the enhanced anti-
bacterial activity should not be contributed to dendrimer itself.
It was well-known that PAMAM dendrimers with primary
amine surface functional groups could penetrate through cell
membrane. We could presume that the enhanced anti-bacterial
activity was contributed to the dendrimers, which might favor
the interaction of the drug with its target or help SMZ with
penetration through the bacterial membrane. The precise rea-
son for this increased activity is at present unclear. Although
further investigations are necessary in this respect, the in vitro
results are very promising as they indicate that appropriate
complexation with dendrimer can increase the effectiveness
of SMZ while it was used as an anti-bacterial drug. Such a de-
velopment would increase the clinical use of SMZ.
Acknowledgements
Financial supports from the One Hundred Talent Project
and Nanomedicine Research Project (kjcx2-sw-h12-01) of
the Chinese Academy of Sciences, Anhui Talent Fund
(2004Z023), the National Natural Science Foundation of
China (30470871), and the Innovation Foundation of Graduate
Student in University of Science and Technology of China
(KD2004035) were highly appreciated.
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