Chitosan-based rechargeable long-term antimicrobial
and biofilm-controlling systems
Zhengbing Cao, Yuyu Sun
Biomedical Engineering Program, University of South Dakota, Sioux Falls, South Dakota 57107
Received 24 October 2007; revised 18 February 2008; accepted 25 February 2008
Published online 9 May 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32040
Abstract: Chitosan, a natural cationic polymer, strongly
binds anionic antibiotics (e.g., rifampin) through the for-
mation of ionic complexes. The new system shows sus-
tained rifampin release, leading to potent antimicrobial
and biofilm-controlling functions against gram-positive
bacteria including Staphylococcus epidermidis and Staphylo-
coccus aureus, which are responsible for a wide range of
medical device-related infections, for longer than 30 days.
INTRODUCTION
Microbial colonization and biofilm formation on
medical devices significantly contribute to the
increasing problem of foreign body-related infections
(FBRIs).1–16
A biofilm is formed when free-floating microor-
ganisms attached to a surface. First, colonies of bac-
teria facilitate the arrival of other cells to adhere by
beginning to build a matrix that holds the biofilm to-
gether. Once colonization started, the biofilm grows
through a combination of cell division and recruit-
ment. The development of biofilm provides to cells
more antibiotic resistance. In fact, it has been
reported that bacteria living in a biofilm are up to
1000 times more resistant to biocides than free-float-
ing bacteria.17–24 As a result, FBRIs are difficult to
treat, often leading to serious morbidity and mortal-
ity, excess length of hospital stay, and extra costs.
Although numerous antimicrobial medical devices
have been reported to control biofilms and reduce
infection rates for short-term uses,1,5,25 most of these
systems cannot provide long-term protections
because of the high release rate of antibiotics from
device materials. After a short period of time (e.g.,
days), most of the drugs are released, and inhibitory
effects are lost.
Correspondence to: Y. Sun; e-mail: yuyu.sun@usd.edu
Ó 2008 Wiley Periodicals, Inc.
Moreover, the released drugs can be recharged to further
extend antimicrobial durations. Drug release mechanisms
and potential applications of the new system are also dis-
cussed. Ó 2008 Wiley Periodicals, Inc. J Biomed Mater Res
89A: 960–967, 2009
Key words: antimicrobial; rechargeable; chitosan; biofilm-
controlling
To extend antimicrobial duration, different bind-
ing systems were used to create localized concentra-
tions of antibiotics on material surfaces. For instance,
cationic surfactants including tridodecylmethyl am-
monium chloride and benzalkonium chloride were
adsorbed on device surfaces, permitting adhesion of
anionic antibiotics to the surfaces through ionic
interactions.26 The bound antibiotics are then slowly
released during applications, extending the antimi-
crobial duration up to several days. The effectiveness
of this process has been demonstrated in short-term
applications,27 but because drugs can only be
adsorbed onto device surfaces, these systems have
limited capability for adsorbing antibiotics, and they
cannot provide long-term antimicrobial functions.
Also, release of the cationic surfactants into the
blood system could be a concern.28 To solve these
problems, new polymer materials (mainly polyur-
ethanes) bearing cationic functional groups in the
side chains have been recently synthesized, and
these can bind and then slowly release anionic anti-
microbial agents.29,30 This approach eliminates the
cationic surfactant concerns, and the antimicrobial
durations can be extended to weeks. Although these
are encouraging results, since new functional groups
were introduced into polymer structures, the bio-
compatibility and safety of the new polymers should
be vigorously tested before they can be used in real
applications.
Here, we report a simple approach in which chito-
san is used as rechargeable carriers of anionic antibi-
otics so as to prolong antimicrobial actions. Chitosan
LONG-TERM ANTIMICROBIAL AND BIOFILM-CONTROLLING SYSTEMS
is a natural cationic polymer produced by deacetyla-
tion of chitin, an abundant natural polysaccharide
that exists in the exoskeleton of crustaceans and the
cell walls of fungi. Chitosan is hypoallergenic,31 bio-
compatible,32 antimicrobial,33,34 biodegradable,35 and
has excellent blood-clotting functions,35,36 making it
an attractive candidate for a wide range of biomedi-
cal applications. We found that chitosan could bind
anionic antibiotics (e.g., rifampin) strongly through
complex formation. The new system provided anti-
microbial and biofilm-controlling functions for lon-
ger than 1 month. Moreover, the released antibiotics
could be repeatedly recharged to regenerate the anti-
microbial functions to further extend the antimicro-
bial durations, providing an innovative approach to
controlling biofilm-formations on a number of highly
needed long-term medical devices.
MATERIALS AND METHODS
Materials
Chitosan flakes were purchased from Fisher Scientific
(Fair Lawn, NJ) and used as received. Rifampin (95%) was
provided by Sigma-Aldrich (Milwaukee, WI). Other chemi-
cals were analytical grade and used without further purifi-
cation.
Preparation of chitosan films
Chitosan films were prepared according to a method
reported by Zeng and Ruckenstein.37 Briefly, around 1 g of
chitosan flake was dissolved in 50 mL of 2 vol % of acetic
acid. After filtration, the chitosan solution was poured into
a Petri dish (100 3 15 mm) and allowed to dry in a fume
hood overnight at 258C and 50% RH. The resultant films
(thickness: 0.1–0.2 mm) were immersed in 1 mol/L aque-
ous NaOH solution at room temperature under constant
stirring for 1 h to neutralize the acetic acid. The films were
then thoroughly washed with deionized water, air-dried,
and stored in a desiccator for further use.
Binding rifampin onto chitosan films
Chitosan films were immersed in saturated rifampin
aqueous or methanol solutions for a certain period of time
at room temperature under constant shaking (solid to liq-
uor ratio: 1:100). Afterwards, the films were taken out,
washed thoroughly with deionized water, air-dried, and
stored in a desiccator. To determine the rifampin content
in the films, a small piece of the film (ca. 1 3 1 cm) was
dissolved in 50 mL of 2 vol % of acetic acid. The solution
was serially diluted, and rifampin content in the solutions
was determined with a Beckman DU1 520 UV–vis spectro-
photometer at k 5 476 nm by comparing the working
961
curve generated from solutions with known rifampin con-
centrations.
Releasing rifampin from chitosan films
A series of rifampin-containing chitosan films (ca. 1 3 1
cm) were immersed in 10 mL of sterile phosphate buffered
saline (PBS) individually under constant shaking (30 rpm)
at 378C. The PBS was changed daily. Rifampin concentra-
tion in the PBS releasing solution was determined at days
1, 3, 6, 10, 20, and 30 with UV–vis, as described earlier.
Each test was repeated five times.
Antimicrobial activity
Parallel to the releasing studies, the antimicrobial activ-
ity of the rifampin-containing chitosan films after different
releasing periods was assessed with a Kirby-Bauer (KB)
technique by measuring the zone of inhibition created by
the chitosan film or chitosan-rifampin film on agar plate
coated with bacteria. Two gram-positive bacteria, Staphylo-
coccus epidermidis (S. epidermidis, ATCC # 35984) and Staph-
ylococcus aureus (S. aureus, ATCC # 6538), were used as
model microorganisms. These species were selected
because they are among the most isolated microorganisms
that are responsible for a wide range of FBRIs, particularly
central venous catheter-related bloodstream infections.1–16
Both species were purchased from American Type Culture
Collections (ATCC).
In the microbial studies, S. epidermidis and S. aureus
were grown in tryptic soy broth solution at 378C for 24 h.
The bacteria solution was diluted to desired densities with
the same broth solution. The surface of a tryptic soy agar
plate was overlaid with 1 mL of 108 to 109 colony forming
units per milliliter (CFU/mL) of S. epidermidis or S. aureus
broth solution, respectively.38,39 The plates were then
allowed to stand at 378C for 4 h. A rifampin-containing
chitosan film (ca. 1 3 1 cm) was placed onto the surface of
each of the bacteria-containing agar plate. The film was
gently pressed with a sterile forceps to ensure full contact
between the film and the agar. The same procedure was
also applied to pure chitosan films to serve as controls.
After incubation at 378C for 24 h, the size of the inhibition
zone around the films (if any) was measured. Each test
was repeated three times.
Rechargeability of the released antibiotics
During the antimicrobial duration studies, after 30 days
of antibiotic release, the resulting films were recharged
with the corresponding rifampin solutions under the same
experimental conditions used in the preparation of the first
generation of rifampin-containing chitosan films. After-
wards, the recharged films were retested in the antimicro-
bial duration and antimicrobial activity studies, as
described earlier.
Journal of Biomedical Materials Research Part A
962 CAO AND SUN

Figure 2. Chitosan films before (Left) and after (Right)

Figure 1. Chemical structure of rifampin. rifampin treatment (Rifampin treatment condition: rifam-
pin concentration was 2 wt % in methanol; time 5 24 h;
temperature 5 258C).
Biofilm-controlling function

S. epidermidis and S. aureus were grown in tryptic soy

broth solution at 378C for 24 h, as described earlier. The
bacteria were harvested by centrifuge, washed with sterile
PBS, and then resuspended in PBS to densities of 107 to
108 CFU/mL. A rifampin-containing chitosan film (ca. 1 3
1 cm) was immersed in 10 mL of each of the bacterial PBS
suspension. The mixture was gently shaken at 378C for 60
min. The films were taken out of the bacteria solution,
gently washed with sterile PBS (3 3 10 mL) under none-
flowing condition to remove loosely attached cells. The
resulting films were immersed into tryptic soy broth solu-
tions, which were incubated at 378C for 72 h. At the end
of incubation, the films were rinsed gently with 0.1M so-
dium cacodylate buffer (SCB), and fixed with 3% glutaral-
dehyde in SCB at 48C for 24 h. After being gently washed
with SCB, the samples were dehydrated through an alco-
hol gradient,40 dried in a critical point drier, mounted onto
sample holders, sputter coated with gold-palladium, and
observed under a LEO 1530 scanning electron microscope
(Leo, Germany). The same procedure was also applied to
pure chitosan films to serve as controls.
mg/cm3 of adsorbed rifampin. From 2 wt % of
rifampin methanol solution, however, the binding
amount in the film is as high as 54.8 mg/cm3. This
difference is most likely caused by the different solu-
bility of the drug. The solubility of rifampin in water
is less than 0.1 wt % at 258C. Therefore, during drug
binding, only less than 0.1 wt % of rifampin was
available to the amino groups of chitosan; the major-
ity of the drugs were just suspended as small par-
ticles in the solution, leading to relatively low drug
binding amount. Methanol, however, could dissolve
as high as 2.0 wt % of rifampin, which could signifi-
cantly promote the drug binding process.
The effects of drug binding time are shown in Fig-
ure 4. With the increase of binding time, drug con-
tent in the resulting films increases rapidly until a
relatively constant value is reached after 24 h. In
such a system, drug binding is affected by three
stages: (1) the diffusion of rifampin through bulk

RESULTS AND DISCUSSION

Binding rifampin onto chitosan films

As shown in Figure 1, one rifampin molecule con-

tains three anionic phenolic groups, which after dis-
association could form ionic complexes with the
amino groups of chitosan. Therefore, chitosan films
strongly bind rifampin. As a direct observation, pure
chitosan films are clear, but after treatment with
rifampin, the films become dark red (rifampin has a
crimson color), which cannot be removed upon
repeated washing with distilled water (see Fig. 2),
suggesting strong binding force between chitosan
and the drug.
The influences of drug binding conditions on
rifampin content in chitosan films were investigated.
Figure 3. Influence of rifampin solvents in the binding
Figure 3 shows the effects of rifampin solvents. solution on binding amount on chitosan films (the original
From 2 wt % of rifampin aqueous suspensions, after rifampin content in the solution was 2 wt %; the binding
24 h of drug binding, the resulting film contains 37.7 time was 24 h, and the binding temperature was 258C).

Journal of Biomedical Materials Research Part A

LONG-TERM ANTIMICROBIAL AND BIOFILM-CONTROLLING SYSTEMS
Figure 4. Influence of drug binding time on rifampin
content in chitosan films in methanol (rifampin concentra-
tion: 2% wt; temperature: 258C).
solution to the surface of chitosan films, (2) the
adsorption of rifampin molecules on this surface,
and (3) the diffusion of rifampin from the surface to
the interior of the chitosan sample. The second stage
and third stage is accompanied by ionic complex for-
mation between chitosan and the drug. Therefore, it
is reasonable to assume that on immersion of a chi-
tosan film in rifampin methanol solution, with suffi-
cient agitation, rapid equilibrium between rifampin
in the solution and film surface can be reached
because the amino groups of chitosan strongly
attract the phenolic groups of rifampin. Diffusion of
rifampin into the film then proceeds. The interior of
chitosan film may be assumed to contain rifampin
solutions in its free volume. It is through these free
volumes that rifampin diffuses, being bound to the
amino groups of chitosan molecules. The fact that it
takes about 24 h to reach drug binding equilibrium
indicates that the majority of adsorbed rifampin may
be bound to the interior of chitosan films, which can
lead to relatively long antimicrobial durations, as
described in the following sections.
Releasing rifampin from chitosan films
Figure 5 summarizes the release rate of rifampin
from chitosan films into PBS. In the first week of
release a typical burst effect is observed: the release
rate decreases rapidly from (158.3 6 1.16) lg/cm2
per day in day 1 to (50.9 6 5.06) lg/cm2 per day in
day 3, and then to (3.17 6 0.13) lg/cm2 per day in
day 6. After that, the decreasing trend of release rate
becomes much less obvious: on day 10, the rate is
(0.58 6 0.013) lg/cm2 per day, and even after 30
days, the film still releases (0.16 6 0.015) lg/cm2 of
rifampin per day.
963
This phenomenon can be explained by the com-
bined action of complex disassociation and drug dif-
fusion.41 In the chitosan-rifampin system, disassocia-
tion of the ionic bonds will release rifampin from
the surfaces of the films into the surrounding envi-
ronment. This may partly account for the initial
burst effect of drug release. As surface rifampin mol-
ecules leave the system, drugs in the bulk of the
films will migrate to the surfaces to provide antimi-
crobial functions. In such a system, there is equilib-
rium between rifampin disassociation and rifampin
binding. Only the disassociated rifampin (free drug)
is available for diffusion. The bound drugs cannot
migrate. The bonding may be provided mainly by
ionic interactions between the amino groups of chito-
san and the phenolic groups of the drug, although
other interactions such as hydrogen bonding, van
der Waals forces, hydrophobic interactions, and so
forth, may also play a role. All these interactions
tend to attract and hold the drugs on chitosan mole-
cules. Although diffusing within the films, the free
drugs can become bound drug again through the
establishment of ‘‘new’’ interactions with other bind-
ing sites in the film matrix. Therefore, the amount of
bound drug will be much higher than the amount of
free drug. This coupled disassociation and diffusion
release mechanism41 may be the major reason for the
sustained drug release of the new system, which can
lead to relatively long antimicrobial durations.
Antimicrobial activity
The antibacterial activity of the rifampin-contain-
ing chitosan films was assessed in a Kirby-Bauer
test. As shown in Figure 6, pure chitosan does not
show any inhibition zones against S. epidermidis or S.
aureus. On the other hand, chitosan films containing
Figure 5. Rifampin release rate from chitosan into sterile
PBS (the original rifampin content was 54.8 mg/cm3).
Journal of Biomedical Materials Research Part A
964 CAO AND SUN

Figure 6. Zone of inhibition study of (A) pure chitosan film against S. aureus, (B) rifampin-containing chitosan film
against S. aureus, (C) pure chitosan film against S. epidermidis, and (D) rifampin-containing chitosan film against S. epider-
midis. The rifampin content in the film was 54.8 mg/cm3.

54.8 mg/cm3 of bound rifampin provide a clear zone
against both species (ca. 15.2 mm for S. aureus and
17.6 mm for S. epidermidis), indicating potent antimi-
crobial activities. As expected, with the increase of
releasing time in PBS, the zone sizes gradually
decrease. However, even after 30 days of release, the
samples can still generate an inhibition zone of
about 1.0 mm against S. aureus and 1.7 mm against
S. epidermidis [see Figs. 7(A) and 8(A) for details].
This excellent antimicrobial durability is most likely
attributable to the disassociation coupled with diffu-

Figure 7. Inhibition zones of (A) chitosan films contain- Figure 8. Inhibition zones of (A) freshly prepared rifam-
ing 54.8 mg/cm3 of bound rifampin, and (B) recharged pin-containing chitosan films, and (B) recharged chitosan
chitosan films against S. aureus after different periods of films, against S. epidermidis after different periods of drug
drug releasing time. releasing time.

Journal of Biomedical Materials Research Part A

LONG-TERM ANTIMICROBIAL AND BIOFILM-CONTROLLING SYSTEMS 965

Figure 9. Biofilm-controlling function of (A) pure chitosan film against S. aureus, (B) chitosan film containing 54.8 mg/
cm3 of rifampin against S. aureus, (C) pure chitosan film against S. epidermidis, (D) chitosan film containing 54.8 mg/cm3 of
rifampin against S. epidermidis, (E) sample D, after 15 days of drug release in PBS, against S. epidermidis, and (F) sample D,
after 30 days of drug release in PBS, against S. epidermidis.

sion release mechanism41 of the new system, as
described earlier.
It is also interesting to note that the released drugs
are rechargeable. After 30 days of releasing, the
resulting films were treated with 2 wt % of rifampin
methanol solution again. UV–vis study indicated
that more than 95% of the released drugs were
recharged, and zone of inhibition studies showed
that the antimicrobial functions of the films were
regenerated after recharging, as shown in Figures
7(B) and 8(B).
The rechargeability of the released drugs in the
new system can be a very attractive feature for a
number of medical applications to further extend
antimicrobial durations even after the devices are
fabricated/inserted based on patients’ needs to
achieve long-term protection. For instance, if the
new system is used as antimicrobial coatings of
long-term central venous catheters, the recharging
treatments can be performed by using a conven-
tional ‘‘antibiotic lock’’ technique42–44 in situ when
the devices are not in use. Normally, ‘‘antibiotic
lock’’ treatment may take weeks to clear infec-
tions;36–39 in the new system for recharging, how-
ever, one or two days may be enough to charge
most of the released drugs (see Fig. 4 for effects of
drug binding time on drug content in the films),
making it a potentially simple and practical
approach to control long-term medical device related
infections.

Journal of Biomedical Materials Research Part A

966
Biofilm-controlling function
As noted earlier, the formation and development
of biofilms can cause serious medical device-related
infections.1–18 Because the rifampin-containing chito-
san films are able to effectively kill microbes, it is
highly possible that they could prevent the forma-
tion of biofilms. To evaluate this effect, Figure 9 dis-
plays the SEM results of the samples in biofilm-con-
trolling studies. After 72 h of growth in bacterial
broth solutions, a large quantity of S. aureus [Fig.
9(A)] or S. epidermidis [Fig. 9(C)] attach to and aggre-
gate on pure chitosan surfaces, indicating formation
and development of biofilms. On the surfaces of chi-
tosan films with 54.8 mg/cm3 of bound rifampin,
however, no adherent bacteria can be observed,
pointing to potent biofilm-controlling functions.
Similar to antimicrobial activities, the biofilm-con-
trolling function of the new system is both durable
and rechargeable. As an example, Figure 9(D,E) show
the SEM results of the biofilm-controlling functions of
rifampin-containing chitosan films against S. epidermi-
dis after different periods of releasing (the original
rifampin content was 54.8 mg/cm3). After 15 days or
30 days of release, although scattered bacteria can be
observed on the film surfaces, no biofilms are formed.
Moreover, after 30 days of release, the resulting films
were recharged, and the biofilm-inhibiting activity of
the samples was regenerated, further confirming that
the biofilm-controlling functions of the new system
are rechargeable, pointing to a potentially simple and
practical approach to achieve long-term antimicrobial
effects on a number of biomedical applications.
CONCLUSIONS
Chitosan strongly binds anionic antibiotics such as
rifampin through ionic complex formation. Drug bind-
ing capacity is significantly affected by drug solvents
and drug binding time. The rifampin-containing chito-
san samples demonstrate sustained drug releasing
behaviors. The new system provides potent, durable,
and rechargeable antimicrobial and biofilm-controlling
functions against gram-positive bacteria including S.
epidermis and S. aureus, which are among the most iso-
lated species that are responsible for a wide range of
medical device-related infections, particularly central
venous catheter-related bloodstream infections. These
attractive features make the new system potential can-
didates in the antimicrobial treatment for a broad
range of highly need long-term medical devices.
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Journal of Biomedical Materials Research Part A