Anionic amphiphilic dendrimers as antibacterial agents

Article abstract of DOI:10.1021/ja806912a

An anionic amphiphilic dendrimer is reported that possesses increased cytotoxicological potency against prokaryotic cells compared to eukaryotic cells. The half-maximal effective concentration (EC₅₀) for the dendrimer against Bacillus subtilis, a Gram-positive bacterial strain, was measured to be 4.1 × 10^-5 M, while that against human umbilical vein endothelial cells (HUVEC) was more than 36× greater at a value of 1.5 × 10^-3 M. EC₅₀ ratios for two commercial amphiphiles, sodium dodecyl sulfate (SDS) and Triton X-100, in addition to a similar synthesized dendritic structure were at most only 3.8× greater. Furthermore, the observed EC₅₀ values appear to be correlated to the critical aggregation constant (CAC) in solution suggesting a mechanism of action for these anionic amphiphilic dendrimers related to their supramolecular structures. Copyright

Full text of DOI:10.1021/ja806912a

Published on Web 10/09/2008
Anionic Amphiphilic Dendrimers as Antibacterial Agents
Steven R. Meyers, Frank S. Juhn, Aaron P. Griset, Nathanael R. Luman, and Mark W. Grinstaff*
Departments of Biomedical Engineering and Chemistry, Metcalf Center for Science and Engineering,
Boston UniVersity, Boston, Massachusetts
Received September 1, 2008; E-mail: mgrin@bu.edu
Dendritic macromolecules, due to their structure, unique proper-
ties, and precise compositions, are of significant interest1 and are
finding uses in an ever-increasing number of medical applications.2
This is especially evident in the drug delivery area where the
dendritic structure enables the attachment of a multitude of drugs
or targeting moieties as well as the opportunity to control phar-
macokinetics through alterations in generation number.3 Our interest
lies in the synthesis and evaluation of dendritic macromolecules
composed of building blocks that are natural metabolites or known
to be biocompatible for ocular tissue repair,4 cartilage tissue
engineering,5 and drug delivery.6 To expand the biomedical
applications of dendrimers and our understandings of the resulting
structure-activity relationships, we are investigating anionic den-
dritic macromolecules as antibacterial agents. Herein, we report
the antibacterial activity of an anionic amphiphilic dendrimer and
the striking selectivity in its cytotoxicity toward a prokaryotic Gram-
positive bacterium compared to a eukaryotic human cell.
There is a significant global need for new antibacterials and
alternative mechanisms of action given the rise in resistance among
bacteria.7 Of the various known antibacterial agent classes, am-
phiphilic compounds act through perturbation and disruption of the
Figure 1. Structures of the two dendritic anionic amphiphiles, 1 and 2,
SDS, 3, and Triton X-100, 4.
prokaryotic membrane.8 We hypothesized that amphiphilic anionic
dendrimers may exhibit antibacterial activity with minimal eukary-
otic cell cytotoxicity, since dendrimers with terminal anionic charges
are generally noncytotoxic and have low toxicity in zebrafish whole
animal development studies.9 On the other hand, cationic dendrim-
ers, some of which have antibacterial properties if the positive
charge is properly shielded,10 have repeatedly shown cytotoxicity
against a variety of eukaryotic cell lines.3e,11 In addition, there are
many reports of linear polycationic agents but only a few descrip-
tions of linear polyanionic antibacterial agents (e.g., sulfonated
polystyrene).12 Consequently, we synthesized a series of surface-
block anionic amphiphilic dendrimers composed of succinic acid,
glycerol, and myristic acid possessing various numbers of acid and
alkyl functionalities.13 Based on the physicochemical properties of
these amphiphilic anionic dendrimers, we identified two potential
candidates, dendrimers 1 and 2 (Figure 1). Both dendrimers were
synthesized in 9 steps with an overall yield of 30 and 28%,
respectively, for evaluation of antibacterial activity (see Supporting
Information). Additionally, linear anionic amphiphile sodium do-
decyl sulfate (SDS), 3, and neutral-charge amphiphile Triton X-100,
4, were added to the evaluation as positive controls with known
antibacterial activity (i.e., disruption of the cytoplamic membrane
and protein solublization).8
Cytotoxicological experiments were conducted against a wild-
type Gram-positive bacterial strain (Bacillus subtilis AG174).
Bacteria were cultured until logarithmic growth was achieved, and
then dilutions were added to LB broth with various concentrations
of compounds 1-4 and the constituents of the dendrimers: glycerol,
succinic acid, and myristic acid along with an untreated negative
control. The turbidity of the wells was monitored for 9 h, and the
resulting cytotoxicities are shown in Figure 2. As expected,
Figure 2. Cytotoxicity of the compounds against Gram-positive B. subtilis.
Absorbances determined by measuring the turbidity of the cell-containing
medium and reported as a fraction of untreated bacteria turbidity over the
same 9 h period (n ) 3; mean ( SD).
commercial amphiphiles 3 and 4 proved to be toxic while myristic
acid, succinic acid, and glycerol were not toxic to the B. subtilis
strain over the concentration range tested. Significantly, we observed
antibacterial activity for the synthesized anionic amphiphilic
dendrimers 1 and 2, though the amplitude of the sigmoidal curve
was comparatively compressed. The half-maximal effective con-
centration (EC50) for 1 and 2 are 6.0 × 10-5 and 4.1 × 10-5 M,
respectively. A partial explanation for this effect was obtained from
further kinetic studies which suggested a bacteriostatic mechanism
of action that required ∼1.5 h to slow the growth significantly
compared to an untreated control.
We next examined the eukaryotic cytotoxicity by evaluating all
the compounds against a primary cell line of human umbilical vein
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10.1021/ja806912a CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
biological, and clinical interest, as antibacterials such as these would
be maximally effective against microbial infections without harming
the host. Moreover, 2 can be prepared easily in good yield and in
the future may provide a cost-effective route for preparation.
Continued efforts in the synthesis of new dendritic macromolecules,
characterization of their unique properties, and evaluation in
clinically important indications will lead to new solutions for a
variety of health care needs.
Acknowledgment. The authors would like to thank the NIH
and BU for partial support. We also thank Dr. Tim Gardner.
Supporting Information Available: Experimental materials and
methods for all procedures. This material is available free of charge
via the Internet at http://pubs.acs.org.
Figure 3. Cytotoxicity of the compounds against HUVEC. Absorbances
are calculated as a percentage of the untreated cells over a 24 h time period
(n ) 3; mean ( SD).
References
Table 1. Experimental Properties of the Dendritic Amphiphiles 1
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1
2
SDS
Triton
CAC (M)
HUVEC EC50(M)
2.0 × 10-4 1.1 × 10-5 1.0 × 10-3 2.4 × 10-4
1.3 × 10-4 1.5 × 10-3 5.4 × 10-4 1.1 × 10-4
B. Subtilis EC50(M) 6.0 × 10-5 4.1 × 10-5 1.4 × 10-4 8.8 × 10-5
ratio EC50
2.2
g36
3.8
1.3
a CMCs for SDS and Triton are from ref 15.
endothelial cells (HUVECs). Low-passage number HUVECs were
equilibrated in a subconfluent monolayer and challenged with varied
concentrations of the compounds for 24 h. The resultant cell
viabilities were determined using a tetrazolium assay (Figure 3).
As seen before with the Gram-positive bacteria, glycerol, myristic
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