Engineering a layer-by-layer (LbL) film for the extended release of antibiotics

The Technology:
My research involves the economic, scalable, and efficient technology of layer-by-layer (LBL) deposition. Specifically, I am interested in using electrostatics to build films with alternating polyanionic and polycationic species. The films will be functionalized by incorporating both gentamicin—a small, hydrophilic antibiotic—and a unique breed of polycations known as poly(β-amino esters). The latter are polymers that are relatively stable in the solid-state, but undergo rapid hydrolytic degradation upon contact with physiological environment (i.e. in vivo). The films are engineered to erode in top-down fashion such that the elution of contained drug can be precisely tuned. Ultimately, this work with antibiotics will be coupled to similar systems incorporating proteins and analgesics in order to achieve a complex, sequential, and hence “smart” release profile of the film contents.

The Application:
As the baby boomer generation begins to increase the demand for orthopedic implants, our technology can find excellent use in the near future. Rough numbers for infection rates in total knee arthroplasty and total hip arthroplasty are about 1 in 200 for primary surgeries and grow to 10% for first revisions. Second revisions increase even more drastically. Through my conversations with orthopedic surgeons Dr. Mitch Harris and Dr. Scott Martin (Brigham and Women’s Hospital), there would be a major market for these films on all revision arthroplasties as well as primary arthroplasties in high risk cases (e.g. patient is diabetic and obese). By applying our thin films directly to surface of orthopedic implants, we envision that a single surgery could locally provide the patient with painkillers, anti-inflammatories, antibiotics, and growth factors all in one and with optimized dosage profiles. The goal is to drastically reduce the heightened infection rates in revision arthroplasties and high risk cases. The importance of this work relates to the consequences of infection. Infected patients end up getting crushed with two more surgeries (to remove infected implant, disinfect the area, and re-implant a new prosthesis), steep hospital bills, a drawn out period of immobility, and potential for permanent limb damage and death.

The Research Problem:
Sustained delivery of gentamicin from LbL films is difficult because it is a small, hydrophilic molecule. When placed in aqueous solution, we currently find that the gentimicin will readily diffuse out of these films. Specifically, there are two domains of release: first, there is a “burst” release where a large fraction of the deposited gentamicin leaves the film within the first few hours, and this is followed by a slower release of the remaining fraction as the film erodes. Release durations beyond 24 hours are very difficult to achieve using our current method of layering the gentamicin directly into the film. Antibiotic delivery will need to reach a 2—6 week duration for any practical application.

The Proposed Solution:
Encapsulate gentamicin inside of much larger vesicles which are not as capable of diffusion within the film. The vesicles can be engineered with a charged surface so that they can be conveniently layered into the LBL superstructure. Hence, the drug is effectively “locked in place” and will need to wait until the film has sufficiently eroded before release. This method has its challenges rooted in optimizing the encapsulation efficiency, antibiotic efficacy, and delivery timeline for use in orthopedic applications. Along the way, my goal is to discover the different variables that can be adjusted to precisely tune the dosage and delivery of gentamicin.

Upper left: growth curve for (LPEI/PSS)[(CHI/HA)₅(Liposome/HA)₁]n as determined by profilometry. Notation defined below.

Lower Left: loading of liposomes by mass into (LPEI/PSS)[(CHI/HA)₅(Liposome/HA)₁]n as determined by liquid scintillation counting. This graph corresponds to liposomes with no drug loading. Recent measurements suggest that the n = 20 film would have incorporated approximately 4 µg/cm2 of gentamicin sulfate. Assuming a 1 cm diffusion length away from the substrate surface, this would be 40x greater than the minimum inhibitory concentration of gentamicin against S. aureus—the most common bacteria associated with bone infection.

Upper Right: liposomes are stabilized with poly L-lysine (PLL) to give them a robust polymer shell such that they keep their structure when being layered into the films. As fabricated, the liposomes are negatively charged. The graph shows that by increasing the mixing ratio in favor of polymer, the liposomes go from naked with a negative zeta potential to polymer-stabilized with a positive zeta potential.

Lower Right: This graph shows the activity of gentamicin-loaded liposomes against S. aureus. The liposome concentrations are an over-estimate to their true value suggesting that their activity may be even more favorable than shown here.* The minimum inhibitory concentration appears to be between 39 and 78 µg. Coupling this information with the graph in the lower left, we should be able to build films that are efficacious against this bacteria in vitro.

* The over-estimate is due to the fact that gentamicin loaded liposomes are prepared initially with an excess of gentamicin. This excess must be dialyzed out and the assumption above is that liposome loss during dialysis is negligible. It has been experimentally determined that there is about a 2% loss of liposomes due to dialysis.

Notation for films:
(X/Y)_n
X = Polymer 1
Y = Polymer 2 of opposite charge
(X/Y) = Bi-layer of X/Y
n = number of bilayers

Materials:
LPEI—Linear poly(ethyleneimine)
PSS—Poly(styrene sulfonate)
CHI—Chitosan
HA—Hyaluronan (or hyaluronic acid)
Liposomes—Prepared with a 69.5 to 30 to 0.5 mass ratio of dioleoylphosphatidylethanolamine (DOPE), n-glutaryl-DOPE, and C16-poly(ethylene glycol)-ceramide. The glutaryl component provides negative charge and the poly(ethylene glycol) component prevents aggregation of liposomes.