Nanolayer multi-agent scaled delivery from implant surface

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2016.Cataloged from PDF version of thesis.Includes bibliographical references.One of the important problems in the field of orthopedic medicine is the ability to create a stable bone-materials interface with an implant, particularly when faced with the difficult condition of bone infection. Only recently have we come to understand the significance of addressing infection during the bone wound healing process; however, to apply this understanding toward an effective treatment requires the ability to deliver exacting amounts of therapeutics of different types over the appropriate timeframes in and around the implant. This task must be accomplished while maintaining the mechanical integrity of the implant materials and allowing for bone integration on their surfaces. Here we present a novel, particularly enabling next-generation implant solution for both eradication of an established biofilm within the bone cavity and accelerated bone repair via the controlled delivery of antibiotic and growth factor in sequence from stable nanometer scale coatings on the implant surface. Infection is by far the most common reason for complications, which often lead to complete removal of implants (74.3%). Infection significantly increases morbidity, and places huge financial burdens on the patient and the healthcare system-projected to exceed $1.62 billion/year by 2020. Because infection is much more common in implant replacement surgeries, these issues greatly impact long-term patient care for a continually growing part of the population. For revision arthroplasty of an infected prosthesis, a prolonged and expensive twostage procedure requiring two surgical steps and a 6-8 week period of joint immobilization exists as today's gold standard. A single-stage revision is preferred as an alternative; however, traditional bulk polymer systems such as bone cement cannot load sufficient amounts of therapeutic to eradicate existing infection, are insufficient or infeasible for the release of sensitive biologic drugs that considerably aid in bone regeneration, and lead to substandard mechanical properties and retarded bone repair. To address these issues, we created conformal, programmable, and degradable dual therapy coatings (~500 nm thick) in a layer-by-layer fashion using the enabling nanofabrication tool of electrostatic multilayer assembly. The nanolayered construct allows large loadings of each drug, thus enabling ultrathin film coatings to carry sufficient treatment and precise independent control of release kinetics and loading for each therapeutic agent in an infected implant environment. The coating architecture was adapted to allow early release of antibiotics contained in top layers sufficient to eliminate infection, followed by sustained release above the MIC over several weeks; whereas, the underlying BMP-2 growth factor layers enabled a long-term sustained release of BMP-2, which induced more significant and mechanically competent bone formation than a short-term burst release. In rats, the successful growth factor-mediated osteointegration of the multilayered implants with the host tissue improved bone-implant interfacial strength by impressive amounts (15-fold) when compared with the bare implant control, and yields a mechanical bond 17-fold higher than that created with the use of clinically available bioactive bone cement. Here we focused on dual delivery of an antibiotic and a growth factor owing to the urgent need for enhanced infection-reducing and tissue-integrating strategies in orthopedic applications, but the excellent modularity of multilayers for incorporation and release of diverse therapeutics suggests this approach should be also applicable to different implant applications such as vascular graft and artificial heart implants for which the risks of infection are often ignored. Our findings demonstrate the potential of this layered release strategy to introduce a durable implant solution, ultimately an important step forward in the design of biomedical implant release coatings for multiple medical applications. In addition to focusing on multi-therapeutic multilayer coatings for macroscale implants and scaffolds, I have also extended the work to understand release properties of the therapeutic agents, guided by predictive mathematical modeling of the release mechanisms involved in polyelectrolyte multilayer films and cell uptakes based on the principles of polymer physics and molecular and cellular biology. The potential impact of this work is substantial: introduce the next-generation biomaterials and implantable devices, save billions of dollars in the healthcare cost, and directly benefit the rapidly growing current and future generations of patients relying on medical device.by Jouha Min.Ph. D

Tunable staged release of therapeutics from layer-by-layer coatings with clay interlayer barrier

In developing new generations of coatings for medical devices and tissue engineering scaffolds, there is a need for thin coatings that provide controlled sequential release of multiple therapeutics while providing a tunable approach to time dependence and the potential for sequential or staged release. Herein, we demonstrate the ability to develop a self-assembled, polymer-based conformal coating, built by using a water-based layer-by-layer (LbL) approach, as a dual-purpose biomimetic implant surface that provides staggered and/or sustained release of an antibiotic followed by active growth factor for orthopedic implant applications. This multilayered coating consists of two parts: a base osteoinductive component containing bone morphogenetic protein-2 (rhBMP-2) beneath an antibacterial component containing gentamicin (GS). For the fabrication of truly stratified composite films with the customized release behavior, we present a new strategy—implementation of laponite clay barriers—that allows for a physical separation of the two components by controlling interlayer diffusion. The clay barriers in a single-component GS system effectively block diffusion-based release, leading to approximately 50% reduction in bolus doses and 10-fold increase in the release timescale. In a dual-therapeutic composite coating, the top GS component itself was found to be an effective physical barrier for the underlying rhBMP-2, leading to an order of magnitude increase in the release timescale compared to the single-component rhBMP-2 system. The introduction of a laponite interlayer barrier further enhanced the temporal separation between release of the two drugs, resulting in a more physiologically appropriate dosing of rhBMP-2. Both therapeutics released from the composite coating retained their efficacy over their established release timeframes. This new platform for multi-drug localized delivery can be easily fabricated, tuned, and translated to a variety of implant applications where control over spatial and temporal release profiles of multiple drugs is desired.National Institutes of Health (U.S.) (National Institute on Aging 5R01AG029601-03)National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051

In-situ monitoring of drug release from therapeutic eluting polyelectrolyte multilayers under static and dynamic conditions

The release profiles of gentamicin sulfate (GS) from [chitosan (CHI)/poly(acrylic acid) (PAA)/GS/PAA]n polyelectrolyte multilayers were investigated in situ using an innovative lab-on-fiber (LOF) optofluidic platform that mimics physiologically relevant fluid flow in a microenvironment. The LOF was constructed by enclosing in a flow-enabled and optically coupled glass capillary a long-period fiber grating both as a substrate for LbL growth of [CHI/PAA/GS/PAA]n and a measurement probe for GS release. We show that the LOF is very robust in monitoring the construction of the [CHI/PAA/GS/PAA]n multilayers at monolayer resolution as well as evaluating the rate of GS release with high sensitivity. The release processes in the LOF under static and a range of dynamic conditions are evaluated, showing a faster release under dynamic condition than that under static condition due to the varying circumstance of GS concentration gradient and the effect of flow-induced shear at the medium-multilayer interface. The LOF platform has the potential to be a powerful test bed to facilitate the design and evaluation of drug-eluting polyelectrolyte thin films for their clinical insertion as part of patient care strategy

Stretchable, Nano‐Crumpled MXene Multilayers Impart Long‐Term Antibacterial Surface Properties

Abstract Infections are a significant risk to patients who receive medical implants, and can often lead to implant failure, tissue necrosis, and even amputation. So far, although various surface modification approaches are proposed for prevention and treatment of microbial biofilms on indwelling medical devices, most are too expensive/complicated to fabricate, unscalable, or limited in durability for clinical use. Here, this work presents a new bottom‐up design for fabricating scalable and durable nano‐patterned coatings with dynamic topography for long‐term antibacterial effects. This work shows that MXene layer‐by‐layer (LbL) self‐assembled coatings—with finely tunable crumpled structures with nanometer resolution and excellent mechanical durability—can be successfully fabricated on stretchable poly(dimethylsiloxane) (PDMS). The crumpled MXene coating with sharp‐edged peaks shows potent antibacterial effects against Staphylococcus aureus and Escherichia coli. In addition, this work finds that on‐demand dynamic deformation of the crumpled coating can remove ≥99% of adhered bacterial cells for both species, resulting in a clean surface with restored functionality. This approach offers improved practicality, scalability, and antibacterial durability over previous methods, and its flexibility may lend itself to many types of biomaterials and implantable devices

Lab-on-fiber optofluidic platform for in situ monitoring of drug release from therapeutic eluting polyelectrolyte multilayers

A lab-on-fiber (LOF) optofluidic platform that provides physiologically relevant microenvironment was developed by integrating a long period grating (LPG) coupled with high order cladding mode to achieve high index sensitivity and a liquid-tight capillary tube assembly as a microfluidic chamber for LPG to mimic physiologically relevant microenvironment. We demonstrate the utility of LOF for in situ monitoring the construction of the [chitosan (CHI)/poly (acrylic acid) (PAA)/gentamicin sulfate (GS)/PAA]n multilayers at monolayer resolution as well as evaluating the rate of GS release at a flow rate of 0.127 mL/min at 37 °C in real time. We reveal that GS is released at a faster rate under the dynamic flow condition than in a static medium. Our findings underscore the importance of conducting drug release studies in physiologically relevant conditions.National Science Foundation (U.S.) (Grant DMR-1206669)Czech Republic. Ministry of Education, Youth, and Sports (Grant LH 11038

Role of silica nanoparticles in monitoring and prolonging release of drug-eluting polyelectrolyte coatings using long-period fiber grating platform

Silica nanoparticles (SNPs) were synergistically integrated with long-period grating (LPG) platform and the process of layer-by-layer (LbL) assembly to enable monitoring controlled release of drug-eluting polyelectrolyte coatings. The SNPs afforded a high surface area for increased drug loading as well as enhanced evanescent field overlap. In addition, the SNPs positioned within the LbL coatings acted as diffusion barrier layer, leading to prolonged release profile. SNPs with different sizes were respectively immobilized on the LPG using poly allylamine hydrochloride (PAH). In-situ monitoring of drug-eluting LbL coating [chitosan (CHI)/Poly arylic acid (PAA)/Gentamicin sulfate (GS)/PAA]n was carried out on LPG with a sensitivity of 8.1 nm shift/tetralayer for LPG with 1 layer of SNPs with 50 nm in diameter. LPG without the SNPs for the monitoring of [CHI/PAA/GS/PAA]n shows a sensitivity of 2.4 nm shift/tetralayer, indicating the significant ability of SNPs in enhancing the LPG sensitivity. Drug release measurement carried out on the LPG platform revealed an increased release time for LbL-SNP drug delivery system compared with that of LbL alone.National Science Foundation (Grant ECCS-1611155

Integrated microHall magnetometer to measure the magnetic properties of nanoparticles

© 2017 The Royal Society of Chemistry. Magnetic nanoparticles (MNPs) are widely used in biomedical and clinical applications, including medical imaging, therapeutics, and biological sample processing. Rapid characterization of MNPs, notably their magnetic moments, should facilitate optimization of particle synthesis and accelerate assay development. Here, we report a compact and low-cost magnetometer for fast, on-site MNP characterization. Termed integrated microHall magnetometer (iHM), our device was fabricated using standard semiconductor processes: an array of Hall sensors, transistor switches, and amplifiers were integrated into a single chip, thus improving the detection sensitivity and facilitating chip operation. By applying the iHM, we demonstrate versatile magnetic assays. We measured the magnetic susceptibility and moments of MNPs using small sample amounts (∼10 pL), identified different MNP compositions in mixtures, and detected MNP-labeled single cells

Designer Dual Therapy Nanolayered Implant Coatings Eradicate Biofilms and Accelerate Bone Tissue Repair

Infections associated with orthopedic implants cause increased morbidity and significant healthcare cost. A prolonged and expensive two-stage procedure requiring two surgical steps and a 6–8 week period of joint immobilization exists as today’s gold standard for the revision arthroplasty of an infected prosthesis. Because infection is much more common in implant replacement surgeries, these issues greatly impact long-term patient care for a continually growing part of the population. Here, we demonstrate that a single-stage revision using prostheses coated with self-assembled, hydrolytically degradable multilayers that sequentially deliver the antibiotic (gentamicin) and the osteoinductive growth factor (BMP-2) in a time-staggered manner enables both eradication of established biofilms and complete and rapid bone tissue repair around the implant in rats with induced osteomyelitis. The nanolayered construct allows precise independent control of release kinetics and loading for each therapeutic agent in an infected implant environment. Antibiotics contained in top layers can be tuned to provide a rapid release at early times sufficient to eliminate infection, followed by sustained release for several weeks, and the underlying BMP-2 component enables a long-term sustained release of BMP-2, which induced more significant and mechanically competent bone formation than a short-term burst release. The successful growth factor-mediated osteointegration of the multilayered implants with the host tissue improved bone-implant interfacial strength 15-fold when compared with the uncoated one. These findings demonstrate the potential of this layered release strategy to introduce a durable next-generation implant solution, ultimately an important step forward to future large animal models toward the clinic

Binary Targeting of siRNA to Hematologic Cancer Cells In Vivo Using Layer‐by‐Layer Nanoparticles

Using siRNA therapeutics to treat hematologic malignancies has been unsuccessful because blood cancer cells exhibit remarkable resistance to standard transfection methods. Herein, the successful delivery of siRNA therapeutics with a dual-targeted, layer-by-layer nanoparticle (LbL-NP) is reported. The LbL-NP protects siRNA from nucleases in the bloodstream by embedding it within polyelectrolyte layers that coat a polymeric core. The outermost layer consists of hyaluronic acid (a CD44-ligand) covalently conjugated to CD20 antibodies. The CD20/CD44 dual-targeting outer layer provides precise binding to blood cancer cells, followed by receptor-mediated endocytosis of the LbL-NP. This siRNA delivery platform is used to silence B-cell lymphoma 2 (BCL-2), a pro-survival protein, in vitro and in vivo. The dual-targeting approach significantly enhances internalization of BCL-2 siRNA in lymphoma and leukemia cells, which leads to significant downregulation of BCL-2 expression. Systemic administration of the dual-targeted, siRNA-loaded nanoparticle induces apoptosis and hampers proliferation of blood cancer cells, both in cell culture and in orthotopic non-Hodgkin's lymphoma animal models. These results provide the basis for approaches to targeting blood-borne cancers and other diseases and suggest that LbL nanoassemblies are a promising approach for delivering therapeutic siRNA to hematopoetic cell types that are known to evade transfection by other means