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.
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.
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.
 Antibiotic Resistance Threats in the United States, Centers for Disease Control and Prevention, 2013.
 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.
 P. Li et al, Synergistic antibacterial effects of β-lactam antibiotic combined with silver nano- particles, Nano., 2005, 16(9), 1912.
 J. R. Morones-Ramirez et al, Silver Enhances Antibiotic Activity Against Gram-Negative Bacteria, Sci. Transl. Med., 2013, 5(190), 190ra81.
 D. Roe et al, Antimicrobial Surface Functionalization of Plastic Catheters by Silver Nanoparticles, J. Antimicrob. Chemother., 2008, 61(4), 869–876.
 T. Mah, Biofilm-Specific Antibiotic Resistance, Future Microbiology, 2012, 7(9), 1061.