Advanced Drug Delivery Reviews 57 (2005) 1539 – 1550

www.elsevier.com/locate/addr

Biofilms and antibiotic therapy: Is there a role for combating

bacterial resistance by the use of novel drug delivery systems?
Anthony W. Smith*
Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom
Received 3 December 2004; accepted 11 April 2005
Available online 13 June 2005

Abstract

The conventional view of antibiotic resistance is one where bacteria exhibit significantly reduced susceptibility to
antimicrobials in laboratory tests by mechanisms such as altered drug uptake, altered drug target and drug inactivation. Whilst
these mechanisms undoubtedly make a major contribution to antibiotic failure in the clinic, the phenomenon of clinical failure
in spite of sensitivity in laboratory tests is also well recognised. It is in this context that attention has focussed on bacteria
growing as adherent biofilms, not only as the mode of growth of device-related infections associated for example with artificial
joints and venous catheters, but also with other chronic infections such as those occurring in the respiratory tract. Growth as a
biofilm almost always leads to a significant decrease in susceptibility to antimicrobial agents compared with cultures grown in
suspension and, whilst there is no generally agreed mechanism for the resistance of biofilm bacteria, it is largely phenotypic.
That is, when biofilm bacteria are grown in conventional laboratory suspension culture they become susceptible to anti-
microbials. A number of elements in the process of biofilm formation have been studied as targets for novel drug delivery
technologies. These include surface modification of devices to reduce bacterial attachment and biofilm development as well as
incorporation of antimicrobials—again to prevent colonisation. Electrical approaches have been used either to release
antimicrobials from device surfaces or to drive antimicrobials through the biofilm. Other technologies not specifically focussed
on biofilms include aerosolized delivery of antibiotics to the lung and formulation into liposome and polymer-based vehicles.
Liposomal systems have been widely studied, either to target antibiotics to the surface of bacterial biofilms, or by virtue of their
property of being taken up cells of the reticuloendothelial system, to target antibiotics towards intracellular bacteria. Many
polymer-based carrier systems have also been proposed, including those based on biodegradable polymers such as poly(lactide-
co-glycolide) as well as thermoreversible hydrogels. Their contribution to the prevention or resolution of infection is reviewed.
D 2005 Elsevier B.V. All rights reserved.

Keywords: Biofilms; Phenotypic resistance; Material modification; Polyurethanes; Iontophoresis; Bio-electric effect; Pulsed electromagnetic
fields; Ultrasound; Photodynamic enhancement; Liposomes; Pegylated liposomes; Biodegradable microspheres; Poly(lactide-co-glycolide);
Hydroxyapatite; Halloysite; Electrospun fibrous scaffolds; Thermoreversible gels; Infection responsive systems; Aerosols

* Tel.: +44 1225 383101; fax: +44 1225 386114.

E-mail address: a.w.smith@bath.ac.uk.

0169-409X/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2005.04.007
1540 A.W. Smith / Advanced Drug Delivery Reviews 57 (2005) 1539–1550

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1540
2. Biofilms in infection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1540
2.1. Biofilm resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1541
3. A role for drug delivery systems? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1542
3.1. Anti-biofilm approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1542
3.1.1. Material modification to prevent biofilm formation . . . . . . . . . . . . . . . . . . . . . . . 1542
3.1.2. Electrical enhancement of antimicrobial activity . . . . . . . . . . . . . . . . . . . . . . . . 1543
3.1.3. Ultrasound enhancement of antimicrobial transport . . . . . . . . . . . . . . . . . . . . . . . 1543
3.1.4. Photodynamic approaches to biofilm treatment . . . . . . . . . . . . . . . . . . . . . . . . . 1544
3.2. Liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1544
3.2.1. Liposomal delivery to biofilms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1544
3.2.2. Liposomal delivery in intracellular infection . . . . . . . . . . . . . . . . . . . . . . . . . . 1544
3.2.3. Stealth liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1545
3.3. Polymer-based antimicrobial delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1545
3.3.1. Implantable matrices, microparticles, fibrous scaffolds and thermoreversible gels.. . . . . . . 1545
3.3.2. Delivery on demand—an infection-responsive system . . . . . . . . . . . . . . . . . . . . . 1547
3.4. Aerosol delivery to the lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1547
4. Discussion and future aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1547
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1547
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1547

1. Introduction ena such as biofouling, it is now clear that microbial

In addressing the question of whether use of novel
drug delivery systems can overcome antibiotic resis-
tance, it is important to view resistance in the clinical
context. It may not be an all-or-nothing response and
decreases in susceptibility do not necessarily mean
clinical failure if sufficient antibiotic can be targeted
to the infection. It is in this scenario that novel drug
delivery systems may have some benefit. Unfortunate-
ly, a different scenario typically prevails in the clinic
where treatment fails in spite of antibiotic sensitivity
in laboratory tests. In other words, clinical failure is
often due not to infections with bacteria harbouring
mechanisms resulting in high-level antibiotic resis-
tance, but rather to bacteria that are phenotypically
resistant in vivo.
2. Biofilms in infection
There is now widespread recognition of the contri-
bution of biofilms to human infection. Previously
thought to be the concern only of industrial and
environmental microbiologists interested in phenom-
biofilms are largely responsible for the recalcitrance of
many infections to conventional antimicrobial therapy
[1,2]. A microbial biofilm is broadly defined as ad-
herent microorganisms within a polymeric matrix,
typically comprising exopolysaccharide that develops
into a complex community [3]. The composition is
often heterogeneous with water channels occurring
between glycocalyx-enclosed microorganisms in
stalk- or mushroom-like structures. The structure is
also a dynamic one and may include single or multiple
microbial species [4]. Cases of biofilm infection in-
clude the well-known examples of device-related
infections such as those associated with artificial
joints, prosthetic heart valves and catheters. Even
with the use of perioperative antimicrobial prophylax-
is and a laminar air-flow surgical environment, the
risk of intraoperative infection is still around 1% for
hip and shoulder replacement and 2% after knee
replacement [5]. With more than 200,000 hip replace-
ments and 200,000 knee replacements each year in the
United States alone, the healthcare costs are high.
Recent surveys also indicate that catheter-associated
bacteremia following catheter-related infection, is by
far the leading cause of nosocomial bloodstream in-
 A.W. Smith / Advanced Drug Delivery Reviews 57 (2005) 1539–1550
fection in intensive care units [6]. Many chronic
infections, not related to medical devices, are now
being attributed either to bacteria not growing and
being relatively dormant or growing slowly as bio-
masses or adherent biofilms on mucosal surfaces.
2.1. Biofilm resistance
Growth as a biofilm almost always leads to a large
increase in resistance to antimicrobial agents com-
pared with cultures grown in suspension (planktonic)
in conventional liquid media, with up to 1000-fold
decreases in susceptibility reported [7,8]. Currently,
there is no generally agreed mechanism to account for
the broad resistance of microbial biofilms [2,9–12].
However, the general resistance of microbial biofilms
is largely phenotypic. When biofilm organisms are
grown in conventional laboratory planktonic culture
they become susceptible to antimicrobials. Thus, the
resistance properties of biofilm bacteria mirror the
common finding of clinical failure of antibiotic ther-
apy in spite of antibiotic susceptibility in laboratory
tests. Eradication of infection by antibiotic treatment
requires elimination of all the bacteria, typically
assisted by the host defences. Specifically, biofilm
resistance can be determined by the susceptibility of
the most resistant cell. There is significant heteroge-
neity within biofilms and it is not the case that all cells
within a biofilm are always highly resistant [9,13].
But the most resistant members of a biofilm popula-
tion are typically orders of magnitude more resistant
than similar members of a planktonic population.
Subculture often fails to show the existence of resis-
tant mutants, but evidence is accumulating for the
contribution of persister cells to biofilm tolerance to
antimicrobials [14]. Recent work now offers a mech-
anistic explanation for Escherichia coli persisters that
neither grow nor die in the presence of bactericidal
agents and so are multidrug tolerant [15].
A full review of biofilm resistance is beyond the
scope of this work and it is noteworthy that to date
compelling evidence that any of the well characterised
mechanisms are uniquely responsible is lacking
[11,16]. The focus here will be a brief overview of
drug penetration into the biofilm, antibiotic inactivat-
ing enzymes, efflux and oxygen deprivation.
Many papers have investigated the possible lack
of antimicrobial penetration as an explanation of
1541
biofilm resistance [9,10,17–19] and a number of
delivery strategies have been used in an attempt to
drive agents through biofilms. However, given that
in many cases biofilms consist of stacks of cells
with aqueous channels flowing in between, impene-
trability seems highly unlikely [20], a finding con-
firmed with Klebsiella pneumoniae biofilm [21].
Commonly, the entire biofilm is coated with a com-
plex hydrophilic polymer, the glycoclayx [22] that is
typically anionic in nature. Where the antimicrobial
agent reacts chemically with exopolymer or is
adsorbed to it, then the net effect is that of having
the appearance of a penetration barrier. There will
be a similar effect if antimicrobials adsorb onto
cells, perhaps dead ones, in the outer parts of the
biofilm.
As reviewed elsewhere, resistance can be due to
production of inactivating enzymes and there is evi-
dence that the relatively large amounts of antibiotic-
inactivating enzymes such as h-lactamases which ac-
cumulate within the glycocalyx produce concentration
gradients can protect underlying cells [23,24]. In con-
trast, in other systems, enzyme inactivation appears
not to contribute to the resistance of biofilm bacteria
[21].
Efflux has also been reviewed elsewhere and the
contribution of efflux to antimicrobial resistance of
biofilms is uncertain. In one study, none of the four
multidrug efflux pump systems present in the ge-
nome of Pseudomonas aeruginosa contributed to
biofilm resistance [25]. Recent work has highlighted
the contributions of oxygen deprivation and anaer-
obic growth to antibiotic resistance. There is evi-
dence of P. aeruginosa growing anaerobically
within airway mucus of cystic fibrosis patients
[26] and microelectrode studies on colony biofilms
indicated that oxygen only penetrated to approxi-
mately 25% of the depth of the biofilm [27]. More-
over, large anoxic regions developed as the biofilm
aged and regions of active protein synthesis oc-
curred only in a narrow band at the air interface
[28]. When challenged with antimicrobials, 4-h-old
colony biofilms growing in the presence of air were
susceptible, however similar aged biofilms grown
anaerobically were much less susceptible [27]. The
authors calculated that oxygen limitation could ex-
plain 70% or more of the protection of old biofilm
cells.
1542 A.W. Smith / Advanced Drug Delivery Reviews 57 (2005) 1539–1550
3. A role for drug delivery systems?
A number of the key elements in the infectious
process and formation of biofilm have been proposed
for application of novel technologies and drug deliv-
ery systems—prevention of colonisation and biofilm
formation, accumulation at the biofilm surface and
drug penetration into the biofilm (Fig. 1). Recent
developments will be reviewed here. Given the in-
creasing use of relatively invasive medical and surgi-
cal procedures, the material properties of medical
devices have received much attention as have strate-
gies to target antimicrobials to device-related infec-
tions. Dental plaque and oral hygiene is another
common therapeutic target and there is now a consid-
erable body of work using carrier systems to target
antibiotics against intracellular infections.
3.1. Anti-biofilm approaches
3.1.1. Material modification to prevent biofilm
formation
An unadulterated surface does not exist. Any sur-
face, synthetic or otherwise, is coated with consti-
tuents of the local environment: first water and
electrolytes, then organic material. This conditioning
film often exists before the arrival of any microorgan-
isms. Firstly, there is a weak and reversible contact
between microbe and conditioning film resulting from
Brownian motion, gravitation, diffusion, or microbial
motility and involving electrostatic interactions [29].
This interaction is a function of the microbial cell
surface and the nature of the adherent surface. The
importance of electrostatic charge is highlighted by
Carrier systems to
target antimicrobials to penetration into biofilm
biofilm surface
Planktonic cells
attach to surface
Surface modification Adsorption of
to reduce adhesion/ antimicrobials
biofilm development onto surface
Fig. 1. Anti-biofilm strategies.
the failure of a mutant of Staphylococcus aureus
bearing an increased negative charge due to the lack
of d-alanine esters in its teichoic acids, to colonise
glass or polystyrene surfaces [30]. The observation
that surfaces immediately become coated with a con-
ditioning film can thwart coating-based strategies to
reduce infection resulting from haematogenous seed-
ing of medical devices such as intravascular catheters.
Put simply, the coating becomes coated. However,
given that many catheter-related infections are due
to skin organisms acquired at the time of catheter
insertion, then anti-colonisation strategies are still
worth exploring. For example, surfaces containing
immobilised long-chain N-alklyated polyvinylpyri-
dines and structurally unrelated N-alklyated polyethy-
lenimines were lethal to S. aureus, S. epidermidis, P.
aeruginosa and E. coli. The structure–activity analy-
sis revealed that for surfaces to be bactericidal, the
immobilised long polymeric chains have to be hydro-
phobic, but not excessively so, and positively charged
[31]. There are many instances where broad-spectrum
antimicrobials have been incorporated into the device
[32–36]. These are then eluted with the aim of pre-
venting biofilm formation by killing early colonising
bacteria. As noted by others, such a strategy is not
without its problems [37]. Sufficient antibiotic must
be incorporated for the duser-lifetimeT of the device
and such incorporation must not damage the proper-
ties of the material, for example, lubrication, lifetime
and host compatibility. There is also the nagging
concern that low levels of antimicrobials could favour
acquisition of antibiotic resistant organisms [38]. Wor-
ryingly, in staphylococcal species commonly associ-
ated with device-related infections, sub-inhibitory
Enhancement of drug
Development into
glycocalyx-enclosed
biofilm
Antimicrobial
impregnated matrices
 A.W. Smith / Advanced Drug Delivery Reviews 57 (2005) 1539–1550
concentrations of tetracycline and quinupristin–dalfo-
pristin enhanced biofilm development by increasing
expression of the intercellular adhesion ica locus [39].
Alternative approaches to antibiotic incorporation
have also been used. For example, rifampin and
amoxicillin have been adsorbed on polyurethanes
exhibiting acidic or basic properties, the binding af-
finity increasing with the introduction of polymer
side-chain functional groups and with matrix hydro-
philicity. The zone of inhibition from amoxicillin-
coated polymers lasted only a few hours, whereas
that from the rifampin-coated polymer lasted several
months [40]. Another approach has been to incorpo-
rate drugs into the backbone of polymers as a main
chain monomer. Biodegradable polyurethanes have
been synthesised with the fluoroquinolone antibiotic
ciprofloxacin that is released upon the action of the
macrophage-derived enzyme cholesterol esterase.
Since this enzyme recognises hydrophobic moieties,
drug release could be achieved by formulating poly-
urethanes synthesised with 1,12 diisocyanatodode-
cane with long hydrophobic monomers immediately
adjacent to the ciprofloxacin [41]. An alternative ap-
proach has been development of a liposomal hydrogel
that reduces bacterial adhesion to silicone catheter
material. Here, liposomes containing ciprofloxacin
are sequestered within a poly(ethylene glycol)-gelatin
hydrogel. Bacterial adhesion was completely inhibited
on catheter surfaces throughout a 7-day adhesion
assay [42]. Emergence of multi-drug resistant patho-
gens is always a risk when antibiotics are used both in
prophylaxis and long-term therapy as well as in con-
ventional acute therapy. Hence, there is interest in
inhibiting biofilm formation on polymer surfaces
using novel compounds that are not otherwise used.
One such compound is usnic acid, a secondary lichen
metabolite that possesses antimicrobial activity
against a number of gram-positive bacteria, including
S. aureus, Enterococcus faecalis and E. faecium.
When loaded into a modified polyurethane and placed
in infection-simulating flow cells, confocal laser scan-
ning microscopy indicated that whilst adhesion of S.
aureus occurred, usnic acid inhibited development of
biofilm [43]. P. aeruginosa biofilm did form on usnic-
acid-coated polymer, although its morphology dif-
fered from the biofilm formed on untreated polymer,
leading the authors to suggest that signalling pathways
within the biofilm may have been perturbed.
1543
3.1.2. Electrical enhancement of antimicrobial
activity
Electro-chemical approaches have been proposed
both as a means to prevent biofilm formation and also
to enhance the activity of antimicrobials against estab-
lished biofilms—the bioelectric effect. For example,
an iontophoretic approach has been taken where a
low-current power source is used to drive the release
of antimicrobial silver ions from the device [44]. The
bioelectric effect refers to the concurrent application
of antibiotics and a weak electric field to kill bacteria.
With application of direct current electric fields be-
tween 1.5 and 20 V/cm, the concentrations of anti-
biotics needed to be effective against biofilm bacteria
fell from approximately 5000 times to 4 times greater
than those necessary for planktonic bacteria in the
absence of electricity [45]. Electrolytic generation of
oxygen may be partly responsible for this bioelectric
effect, at least with the aminoglycoside antibiotic
tobramycin against P. aeruginosa biofilm [46]. En-
hanced antibiotic activity has also been shown using
pulsed electromagnetic fields (PEMF). Here, PEMF
enhanced the activity of gentamicin against 5-day
biofilms of S. epidermidis, although there was no
significant effect when gentamicin was replaced
with vancomycin [47].
3.1.3. Ultrasound enhancement of antimicrobial
transport
Ultrasound has been used to enhance antibiotic
transport through biofilms. For example, pulsed ultra-
sound over 24 h enhanced the killing by gentamicin of
E. coli biofilms on polyethylene discs implanted sub-
cutaneously into rabbits without damage to the skin
[48]. Similar results have been reported for enhanced
vancomycin activity against S. epidermidis biofilms in
vivo, although longer treatment times were required
[49]. Low-frequency ultrasound (70 kHz) of low
acoustic intensity increased the growth rate of S.
epidermidis, P. aeruginosa and E. coli grown on
polyethylene surfaces and it was hypothesized that
ultrasound increases the transport of oxygen and nutri-
ents to the cells [50]. Such increased growth would
likely enhance susceptibility to antibiotics. Using a
colony biofilm model where diffusion of antibiotic
through the biofilm could be measured, insonation
of E. coli or P. aeruginosa biofilms significantly
increased transport of gentamicin across the biofilm,
1544 A.W. Smith / Advanced Drug Delivery Reviews 57 (2005) 1539–1550
whereas in the absence of ultrasound diffusion either
did not occur or did so at a much slower rate [51].
3.1.4. Photodynamic approaches to biofilm treatment
Photodynamic approaches have been proposed as a
means to overcome resistance and to break down
biofilms [52]. Photosensitising drugs produce reactive
oxygen species that are difficult for the microorgan-
ism to defend against. Necessarily, applications are
limited to those sites where light can reach. For this
reason, the focus has been on pathogens associated
with the skin and with the oral cavity. For example,
biofilms of the oral pathogen Actinomyces viscosus
have been exposed to laser light at 666 nm in the
presence of methylene blue. Confocal microscopy
revealed that a single photomechanical wave in-
creased the penetration of methylene blue by 75%
and enhanced the photodestruction of the biofilm
[53]. Similar findings have been obtained using
multi-species biofilms of oral bacteria irradiated with
light from a helium/neon laser in the presence of
toluidine blue where greater than 95% of biofilm
bacteria were killed [54]. Comparisons of growth
phase and extracellular slime on the photodynamic
inactivation of S. epidermidis and S. aureus indicated
that slime production and stationary phase, both char-
acteristics of biofilm infections, were obstacles to this
therapy. However, use of polylysine-based cationic
photosensitisers overcame some of the growth phase
effects [55].
3.2. Liposomes
Liposomes are attractive as drug delivery/targeting
vehicles by virtue of their compatibility with biolog-
ical constituents and the range and extent of pay loads
that they can carry. In the context of antibiotics and
treatment of infection, they have been studied for their
ability to concentrate agents at biofilm interfaces
[56,57] and also to be taken up into cells harbouring
intracellular pathogens [58–62].
3.2.1. Liposomal delivery to biofilms
Jones and colleagues have reported extensive stud-
ies of the interaction between liposomes and bacterial
biofilms [56,57,63–66]. For example, using either
cationic liposomes comprising dimyristoylphosphati-
dylcholine (DMPC), cholesterol and dimethlydiocta-
decylammonium bromide (DDAB) or anionic
liposomes substituting DMPC with phosphatidylino-
sitol (PI), they noted that each bacterium in the bio-
film adsorbed independently and that the extent of
adsorption of anionic liposomes was smaller. Interest-
ingly, when targeting mixed biofilms of Streptococcus
sanguis and S. salivarius with liposomes loaded with
the bactericide triclosan, anionic liposomes were most
effective against S. sanguis, but relatively ineffective
against S. salivarius [65]. An additional approach has
been to load antibacterials into liposomes adsorbed on
the surface of zinc citrate particles, used in toothpaste
formulations, to produce solid supported vesicles
(SSVs) containing either triclosan or aqueous-soluble
penicillin-G. Anionic liposomes were prepared by
incorporation of PI into DMPC liposomes and cation-
ic liposomes were prepared by incorporation of
DDAB and cholesterol into DMPC. Whilst zinc cit-
rate is itself antibacterial, it was noted that particles
and empty liposomes had an additional or synergistic
effect, whereas particles and liposomally encapsulated
antimicrobials had an inhibitory effect on each other
against S. oralis biofilms [63]. Other oral hygiene
approaches have included liposomal encapsulation
of the enzyme glucose oxidase and horse radish per-
oxidase, that generate hydrogen peroxide and oxya-
cids in the presence of their substrates. They were
effective against S. gordonii biofilms in a manner
dependent upon liposome–biofilm and substrate–bio-
film incubation times [66].
3.2.2. Liposomal delivery in intracellular infection
The uptake of liposomes by cells of the reticulo-
endothelial system has been exploited to target anti-
biotics to those intracellular sites where parasitic
bacteria reside, and by virtue of sustained release
properties, extend the half-life of the drug in the
body. Ciprofloxacin has been incorporated with high
efficiency into DSPC/cholesterol liposomes and ex-
amined in a mouse model of Francisella tularensis
[67]. Intravenous injection of liposome-encapsulated
ciprofloxacin resulted in increased drug retention in
the lungs, liver and spleen compared with that of free
drug. Aerosolized liposomal ciprofloxacin gave com-
plete protection against a lethal pulmonary infection
of F. tularensis, whereas free ciprofloxacin was inef-
fective [67]. Caution should be exercised in extrapo-
lating data as it is clear that liposomal efficacy is
 A.W. Smith / Advanced Drug Delivery Reviews 57 (2005) 1539–1550
dependent on the infecting organism. Liposome-en-
capsulated ciprofloxacin, delivered intravenously, has
been compared with free drug in a rat model of S.
pneumoniae pneumonia and, whilst serum and lung
lavage levels were higher (peak and area under curve),
survival rates were similar [68].
An interesting development of the liposomal con-
cept has been the use of pH-sensitive liposomes in a
murine salmonellosis model [69]. Here, gentamicin
encapsulated in liposomes including a pH-sensitive
lipid fusion between unsaturated PE and N-succinyl-
dioleyl-PE gave 153- and 437-fold greater drug levels
in the liver and spleen, respectively, compared with
free drug. Overall, liposomal delivery was associated
with 10,000-fold greater activity than that of free
drug.
3.2.3. Stealth liposomes
Somewhat counter-intuitively, stealth approaches
have been adopted for delivering antibiotics. Pegy-
lated long-circulating liposomes loaded with gentami-
cin were superior to free gentamicin in a rat model of
K. pneumoniae unilateral pneumonia/septicaemic
[70]. Studies in vitro have also confirmed that pegyla-
tion of liposomes reduced their affinity for S. aureus
biofilms [64]. Here, they found that liposomes pre-
pared from the phospholipids DMPC, dipalmitoyl PC
and distearoyl PC containing DDAB (cationic) or PI
(anionic) and variable amounts of dipalmitoylpho-
sphatidylethanolamine bonded to PEG of molecular
mass 2000 (DPPE-PEG-2000), exhibited decreasing
electrophoretic mobilities and zeta potentials with
increasing DPPE-PEG-2000 incorporation. The ad-
sorption of liposomes to S. aureus biofilms followed
the Langmuir isotherm and both surface coverage and
the magnitude of the Gibbs energy of adsorption
decreased with the extent of pegylation [64].
3.3. Polymer-based antimicrobial delivery systems
Many reviews have highlighted the use of biode-
gradable polyesters as effective drug carriers includ-
ing nano- or microparticles, hydrogels, micelles and
fibrous scaffolds [71–76]. Inevitably, there are advan-
tages and disadvantages associated with each delivery
system, however these experimental approaches have
been investigated in a number of infections, including
periodontitis and osteomyelitis, as well as intracellular
1545
infections such as tuberculosis and brucellosis. Small
biodegradable microspheres are useful alternatives to
liposomes for targeting drugs to the monocyte–mac-
rophage system. They tend not to suffer from the same
difficulties of low encapsulation efficiency and stabil-
ity on storage typically exhibited by liposomal for-
mulations. Biodegradable microspheres prepared from
poly(lactide) (PLA) and its co-polymers with glyco-
lide (PLGA) can release encapsulated drugs in a
controlled way, depending on the method of microen-
capsulation and the physico-chemical properties of the
polymer and drug.
3.3.1. Implantable matrices, microparticles, fibrous
scaffolds and thermoreversible gels
Prevention and treatment of osteomyelitis, particu-
larly associated with orthopaedic implant surgery,
have been the focus of many studies. Systems
implanted at the same time as the prosthesis may be
either non-biodegradable or biodegradable. Poly-
methylmethacrylate beads impregnated with gentami-
cin have been available for about 20 years, however
since they are non-biodegradable they have to be
removed, usually about 4 weeks after insertion [77].
A number of osteoconductive/biodegradable alterna-
tives have been studied, including calcium phosphates
such as hydroxyapatite whose chemical composition
is similar to the bone mineral phase. Studies with
ciprofloxacin incorporated into hydroxyapatite and
poly(d,l-lactide) formulations implanted in the
femur of rabbits, indicated that therapeutic bone levels
were achieved over 6 weeks with release enhanced by
erosion—disintegration and bone ingrowth into the
implant [78]. Other studies using the glycopeptide
antibiotic teicoplanin, effective against S. aureus, in-
dicate that it too can be effective over several weeks
when incorporated into microspheres prepared from
PLGA (75:25) (Mr 136,000) polymer [79,80]. Other
materials studied against implant-related osteomyelitis
include poly(3-hydroxybutyrate-co-3-hydroxyvale-
rate) (PHBV). The release behaviour of sulbactam:
cefoperazone from rods comprising 7%, 14% or 22%
(mol) 3-hydroxyvalerate were representative of typi-
cal monolithic devices where a rapid early release
phase is followed by a slower and prolonged phase.
With PHBV 22 rods, this extended phase lasted for up
to 2 months, making it a promising controlled release
vehicle since treatment of device-related infections is
1546 A.W. Smith / Advanced Drug Delivery Reviews 57 (2005) 1539–1550
typically up to 6 weeks [81]. Release studies of anti-
biotics adsorbed onto the surface of hydroxyapatite
cylinders having a bimodal pore size distribution also
have a prolonged duration of release, attributed to the
small pores, combined with favourable osteoconduc-
tion properties into the large pores [82]. A number of
matrices have been formed using poly(d,l-lactide-co-
glycolide) (PLGA), including discs [83] and electro-
spun nanofibrous scaffolds [84]. In an effort to
achieve the ideal drug release pattern of no lag time
and zero-order release, lactide monomer or glycolide
monomer have been incorporated into PLGA discs
loaded with gentamicin. The idea here is that channels
will form in the disc following dissolution of the
monomer to aid the release of gentamicin. Discs con-
taining 10% monomers showed nearly zero-order re-
lease kinetics for more than 1 month [83]. Evidence
for the channel-forming properties of the monomers
came from the water uptake by the discs. After 7 days,
the amount of water absorbed by the control discs was
20%, compared with 60% in monomer-containing
discs.
Fibrous scaffolds are currently receiving attention
both as a means to prevent post-surgery adhesions and
also to release drugs in a site-specific manner. Whilst
surgical implantation is required, they could be used
where surgery is already indicated and the drug re-
lease profile can be controlled by varying the scaf-
fold’s morphology, porosity and composition. In a
recent study, cefoxitin has been incorporated into
PLGA scaffolds [84]. Here, PLGA controls the rate
of degradation whilst high molecular weight poly(lac-
tide) (PLA) confers mechanical strength to the scaf-
fold. An amphiphilic diblock co-polymer comprising
poly(ethylene glycol)-b-poly(lactide) was added to the
polymer solution to encapsulate the hydrophilic
cefoxitin sodium, which otherwise has poor solubility
in the PLGA solvent N,N-dimethyl formamide. The
drug/polymer solution was electrospun in a spinneret
at 23–27 kV. As the concentration of cefoxitin in-
creased, the scaffolds changed from a bead and string
morphology, attributed to insufficient stretching of the
polymer jet, to a fine fibrous structure of diameter
260 F 90 nm. The spinning process did not affect the
cefoxitin and following an initial burst, prolonged
release was measured for up to 1 week.
Other applications of PLGA have included incor-
poration of antibiotic-loaded microparticles into an
injectable collagen sponge, resulting in local drug
delivery combined with the tissue regeneration prop-
erties of collagen [85]. Several other matrices have
been used, including the aluminosilcate material hal-
loysite. Whilst chemically similar to kaolin, halloysite
has a tubular structure that can be loaded with drug.
Moreover, surface charge neutralisation using cationic
polymers can place an additional level of control on
drug release. In one study of tetracycline incorpora-
tion into halloysite for the treatment of periodontitis,
an initial burst of tetracycline release was followed by
a dramatic reduction in release. Coating with the
cationic polymer chitosan reduced the dburstT release
from 45% to 30% and the total release over 9 days
from 88% to 78% [86]. A key concern was delivery of
the halloysite to the gingival pocket and its subse-
quent retention. The polyoxyethylene–polyoxypropy-
lene copolymer poloxamer 407 was used for its
thermoreversible properties of being liquid below 20
8C and forming a hydrogel at higher temperatures if
sufficiently concentrated, that is also biodegradable
and relatively non-toxic. The ideal here is that the
delivery system should be a liquid at room tempera-
ture, so avoiding the need for refrigeration, and a
device-retaining gel at the temperature within the
gingival crevice. In this particular study, the transition
temperature range was narrowed by the addition of
polyethylene glycols, however this caused the storage
modulus (solidity) of the gel to drop as well [86]. This
was overcome by the addition of 1% octyl cyanoac-
rylate (OCA), a powerful tissue adhesive that poly-
merises at neutral pH. For storage, the pH of the
system was held at 4 by addition of glacial acetic
acid, and upon application to the gingival crevice
the natural buffer capacity of the tissue slowly brought
the pH of the formulation up to neutral, allowing the
OCA to polymerise. Thermoreversible polymers have
also been studied as delivery matrices in their own
right [87–89]. For example, pluronic F-127 has been
used to deliver vancomycin to treat methicillin-resis-
tant S. aureus otitis media [90]. Based on the sol–gel
phase transition measurements, a 25% w/v pluronic
solution was loaded with vancomycin and injected
through a 26 gauge needle. It changed to a gel at 36
to 37 8C that consisted of large populations of the
micelles and aqueous channels from which the van-
comycin was released. Bioabsorption studies followed
over 50 days indicated that the gel gradually disap-
 A.W. Smith / Advanced Drug Delivery Reviews 57 (2005) 1539–1550
peared leaving open spaces that initially showed some
inflammatory exudate, but by day 50 normal tissue
was observed without any inflammation, fibrosis, or
open spaces from the gel.
3.3.2. Delivery on demand—an infection-responsive
system
An interesting approach to antibiotic delivery has
been the development of systems that are responsive
to microbial infection. The rationale here is to develop
systems that release antibiotics only during infection,
recognising that prophylactic or prolonged use of
antibiotics can favour selection of resistant variants
and also lead to renal and liver toxicity with some
agents such as the aminoglycosides. One such ap-
proach has been based on the observation that
wound fluid from S. aureus infection showed high
levels of thrombin-like activity. An insoluble poly-
mer–drug conjugate was prepared in which gentami-
cin was bound to poly(vinyl alcohol) through a
thrombin-sensitive peptide linker [91]. The conjugate
released gentamicin when it was incubated with
thrombin and leucine aminopeptidase together, but
not with either component alone. Gentamicin was
also released upon incubation with S. aureus wound
fluid and the conjugate reduced the bacterial number
in an animal model of infection.
3.4. Aerosol delivery to the lung
Aerosolization offers an attractive approach to
deliver antimicrobials directly into the respiratory
tract for treatment and prophylaxis of pulmonary
infections [92,93]. Aerosolized solutions of amino-
glycosides, particularly tobramycin, are used in
patients with cystic fibrosis, where high endobron-
chial concentrations are achieved that may overcome
bacterial resistance as defined by standard suscepti-
bility testing protocols [94–96]. Other applications of
aerosol technology include aerosolized antibiotics in
mechanically ventilated patients [97]. In this in-
stance, an efficient method was achieved that deli-
vers the aerosol beyond the endotracheal tube and
drug levels in pulmonary secretions were several
orders of magnitude higher than those following
intravenous therapy. A number of questions have to
be addressed, notably whether the drug can with-
stand aerosol generation, such as the high-frequency
1547
ultrasound used in some nebulisers. The resultant
aerosol properties will depend not only on the phy-
sical process used to nebulise the drug, but also on
the intrinsic device characteristics and its perfor-
mance with a particular drug [98].
4. Discussion and future aspects
It is clear from this brief review of the current
literature that the full spectrum of modern drug deliv-
ery approaches is being applied to antimicrobial ther-
apy, but the prospects for a revolution in treatment
seem some way distant. The problem is not with the
drug delivery system; rather it is their payload. The
currently available antibiotics have a limited range of
cellular targets and were developed on the basis of
their activity against fast-growing planktonic cells in
conventional laboratory culture. It seems likely that
drug delivery systems for the treatment of infection
will only come of age when there are drugs that are
worth carrying. We are beginning to understand the
physiology of biofilm bacteria at the genomic and
proteomic levels and we must look to these studies
to yield new targets and to stimulate the design of
drugs that are worth carrying.
Acknowledgements
Work in the author’s laboratory is supported by the
BBSRC, the Lord Dowding fund for Humane Re-
search and the Department of Health.
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