(274b) Award Submission: Silver Nanoparticle-Embedded Polymersome Nanocarriers for the Treatment of Antibiotic-Resistant Infections

Introduction

Antibiotics have been extensively used since their
commercialization in the 1930s to treat patients suffering from a wide variety
of infectious diseases. Unfortunately, however, these drugs have been used so
prevalently over the last 80 years that the bacteria they were designed to kill
have begun to evolve and adapt, rendering them ineffective. According
to the Center for Disease Control, at least 2 million people acquire serious
infections from antibiotic resistant bacteria each year, and over 23,000 die as
a direct result. Even when alternative treatments exist, patients with
antibiotic-resistant infections have significantly higher mortality rates, and
survivors often have increased hospital stays and long-term complications.
These infections cost an estimated $20 billion in excess direct healthcare
expenses.1 Infections caused by Gram-negative
bacteria are particularly difficult to treat because these bacteria have a
robust and hydrophobic outer lipopolysaccharide membrane that can impede the
influx of drugs into the cell.2  Unfortunately,
the number of new antibiotic drugs in the pipeline has also been rapidly
decreasing, largely due to the fact that new drugs are extremely expensive to
bring to market, and antibiotics are less financially lucrative to develop when
compared to treatments for chronic conditions. Thus, the need to develop
alternative strategies to treat such antibiotic-resistant bacteria, while still
utilizing existing drugs, has never been more urgent than today.

Over the past decade, interest in using nanomedicine-based
approaches to combat difficult infections has rapidly grown due to the many
advantages offered over conventional treatment with free antibiotics. This
study explored encapsulating the drug inside nano-sized
structures called polymersomes (that is, artificial
vesicles made from biodegradable, high molecular weight, amphiphilic
block co-polymers). These vesicles typically display a spherical morphology and
are composed of hydrated hydrophilic coronas both at the inside and outside of
a hydrophobic polymer membrane. This allows for hydrophilic bioactive
materials to be loaded into the particle's aqueous core, and hydrophobic
bioactive materials to be loaded into the particle's membrane bilayer. Along
these lines, metallic nanoparticles have long been investigated as potential
antibacterial agents due to their many unique physiochemical properties
which are not present at the macro scale. Among these metals,
silver is perhaps the most well-known for its
antimicrobial effects. Recent studies have shown that there may even be a
synergistic effect when silver nanoparticles and antibiotics are used
simultaneously to treat a Gram-negative infection.3 However,
there is little information regarding whether a combined treatment is
sufficient to overcome bacteria which display genetic
antibiotic resistance. Additionally, there has been almost no investigation
into the effect of dually encapsulated antibiotics and nanoparticles.

Thus, for all of the above reasons, the objective of the
present in vitro study was to design, characterize, and optimize a polymersome nanocarrier to
co-localize and deliver both antibiotics and hydrophobic silver nanoparticles,
while protecting the drug from hydrolysis by β-lactamase enzymes, into a
single "nanoformulation" in order to kill antibiotic-
resistant bacteria. Specifically, these particles were then tested for efficacy
against a strain of Escherichia coli (E. coli) which had been genetically
modified to be antibiotic- resistant.

Results

Silver nanoparticle-embedded polymersomes
(AgPs) were synthesized using a modified
stirred-injection technique. Monodispersed
hydrophobic silver nanoparticles 5 nm in diameter were
suspended in an organic solvent containing dissolved mPEG-PDLLA.
This mixture was injected through a syringe atomizer at high speed into
actively stirring phosphate buffered saline (PBS, pH 7.4) containing the
antibiotic ampicillin. The resulting suspension was allowed to dialyze against
PBS to remove the organic solvent and non-encapsulated drug.

Physicochemical characterization was performed to assess AgPs size, surface charge, and loading. Transmission
electron microscopy (TEM) revealed polymersomes of
highly uniform size and shape with clusters of silver nanoparticles embedded
inside (Figure 1A). These silver clusters frequently appeared as a single layer
of nanoparticles that was off-center from the nanoparticle core, suggesting
that they may be intercalated into the membrane bilayer. Dynamic light
scattering (DLS) indicated that the average hydrodynamic diameter was 104.3 nm
± 15.6 nm (Figure 1B). The AgPs surface was found to
have a near neutral zeta potential of 0.315 mV ± 1.13 mV at pH 7.4. The number
of silver nanoparticles embedded per polymersome was
quantified from TEM images. The nanoparticles were shown to load in a normal
tailed distribution with an average of 9.29 ± 6.07 silver nanoparticles per polymersome (Figure 1C) . The mass
of silver loaded was estimated using the density of silver and the volume of a
5 nm sphere.

Figure
1: Physiochemical
characterization of the AgPs

The growth and proliferation of a 106 colony
forming units mL-1 (CFU mL-1) suspension
of ampicillin-resistant E. coli was
examined by measuring the optical density at 600 nm (OD600)
for 24 hours following treatment with volumes of AgPs
containing a silver : ampicillin (Ag : Amp) ratio of
1:0.28 (Figure 2A), 1:0.44 (Figure 2B), or 1:0.64 (Figure 2C). Ampicillin-
loaded AgPs displayed significant bacteriostatic
action against the E. coli,
manifesting as a delay in the time taken to reach exponential growth phase.
This response was dose-dependent, with higher concentrations of ampicillin
producing a longer delay in bacterial growth. Bacteria treated with ampicillin
concentrations above 55 μg mL-1 failed
to proliferate within 48 hours. In the absence of silver nanoparticles, no
bacteriostatic effect was observed for all ampicillin concentrations tested.
This suggests that the presence of silver potentiates the therapeutic efficacy
of ampicillin. AgPs without ampicillin like- wise
produced no therapeutic benefit. Additionally, no significant differences were
observed between bacteria treated with free ampicillin (200 μg mL-1),
PBS, AgPs without ampicillin, and ampicillin loaded polymersomes (200 μg mL-1) without
silver nanoparticles. When bacteria were treated with sub-optimal
concentrations of AgPs, bacterial growth was always
observed within 17 hours. The time to exponential phase was found to vary with
both silver concentration and ampicillin loading.

Figure
2: Bacterial growth inhibition from
the AgPs

A Bliss Model was utilized to determine the degree of
synergy for different silver and ampicillin combinations.4 Drug
interactions were found be synergistic (S > 0) in all cases where ampicillin
was supplied at concentrations of 24 μg mL-1 and
above. At lower concentrations, no synergism was observed (S = 0). The degree
of synergy was dose-dependent and increased with both silver and ampicillin
concentrations. The therapeutic benefit of ampicillin reached a plateau at 50 μg mL-1 over a range of silver concentrations due
to complete inhibition of bacterial growth. When the silver concentration was
held constant, the degree of synergism was directly determined by the amount of
ampicillin loaded.

Interactions between E.
coli and AgPs were visualized using TEM (Figure 3).
Indentation of the bacterial cell membrane was observed in regions of AgPs contact. Silver nanoparticles inside AgPs appeared to be polarized in an orientation
perpendicular to the bacterial cell membrane, suggestive of hydrophobic
interactions with the outer cell membrane. In order to assess physical
intracellular changes caused by AgPs, cells treated
with an intermediate particle concentration (44 μg mL-1 Amp,
1 Ag:0.44 Amp) for 24 hours were sectioned. Bacteria
in contact with AgPs displayed significant protein
aggregation and diffuse widening of the cell envelope. This phenomenon has been
observed by other researchers following silver ion treatment, and has been
shown to correlate with increased membrane permeability and protein misfolding due to disulfide bond disruption.4 Regions
of the cell envelope with little to no AgPs contact
appeared morphologically normal.

Figure
3: Transmission electron
micrograph displaying the AgPs aggregating around an E. coli bacterium.

Discussion

This work has many important implications. A long-lasting, single-particle treatment capable of
overcoming antibiotic resistance would be extremely beneficial in the clinic.
This potential is compounded by the fact that the initial bacteria density
found in vivo is rarely as high as the CFU studied here. It has been shown that
silver nanoparticles are also effective at treating bacterial persister cells, and therefore AgPs
may show promise for biofilm-forming infections.5 One of the most
commonly occurring bacterial infections, urinary tract infection, is frequently
caused and perpetuated by biofilm-forming (and often antibiotic-resistant)
strains of E. coli.6 Additionally,
the absence of a significant cytotoxic effect from AgPs
towards human fibroblasts is a promising sign for toleration by mammalian
cells. Reports of toxicity associated with nanoscale
silver in vivo have been varied, and toxicity is generally considered to depend
on nanoparticle size, concentration, and surface coating. As designed, the AgPs particle is easily loaded with a variety of aqueous
drugs, and combinations thereof, opening avenues for the creation of a library
of therapeutic particles.

References

[1] Antibiotic Resistance Threats in
the United States, Centers 
for Disease Control and Prevention, 2013. 


[2] J. M.
Pages et al, The porin and the permeating antibiotic:
a selective diffusion barrier in Gram-negative bacteria, Nat. Rev. Microbiol.,
2008, 6, 893.

[3] P. Li
et al, Synergistic antibacterial effects of β-lactam antibiotic combined
with silver nano- particles, Nano.,
2005, 16(9), 1912.

[4] J. R. Morones-Ramirez
et al, Silver Enhances Antibiotic Activity Against Gram-Negative Bacteria, Sci. Transl. Med., 2013, 5(190),
190ra81. 


[5] D.
Roe et al, Antimicrobial Surface Functionalization of Plastic Catheters by
Silver Nanoparticles, J. Antimicrob. Chemother., 2008, 61(4), 869–876.

[6]
T. Mah, Biofilm-Specific Antibiotic Resistance, Future Microbiology, 2012, 7(9), 1061.