Nisarg J. Shah

Tunable dual growth factor delivery from polyelectrolyte multilayer films

A promising strategy to accelerate joint implant integration and reduce recovery time and failure rates is to deliver a combination of certain growth factors to the integration site. There is a need to control the quantity of growth factors delivered at different times during the healing process to maximize efficacy. Polyelectrolyte multilayer (PEM) films, built using the layer-by-layer (LbL) technique, are attractive for releasing controlled amounts of potent growth factors over a sustained period. Here, we present PEM films that sequester physiological amounts of osteogenic rhBMP-2 (recombinant human bone morphogenetic protein-2) and angiogenic rhVEGF₁₆₅ (recombinant human vascular endothelial growth factor) in different ratios in a degradable [poly(β-amino ester)/polyanion/growth factor/polyanion] LbL tetralayer repeat architecture where the biologic load scaled linearly with the number of tetralayers. No burst release of either growth factor was observed as the films degraded. The release of rhBMP-2 was sustained over a period of 2 weeks, while rhVEGF₁₆₅ eluted from the film over the first 8 days. Both growth factors retained their efficacy, as quantified with relevant in vitro assays. rhBMP-2 initiated a dose dependent differentiation cascade in MC3T3-E1S4 pre-osteoblasts while rhVEGF₁₆₅ upregulated HUVEC proliferation, and accelerated closure of a scratch in HUVEC cell cultures in a dose dependent manner. In vivo, the mineral density of ectopic bone formed de novo by rhBMP-2/rhVEGF₁₆₅ PEM films was approximately 33% higher than when only rhBMP-2 was introduced, with a higher trabecular thickness, which would indicate a decrease in the risk of osteoporotic fracture. Bone formed throughout the scaffold when both growth factors were released, which suggests more complete remodeling due to an increased local vascular network. This study demonstrates a promising approach to delivering precise doses of multiple growth factors for a variety of implant applications where control over spatial and temporal release profile of the biologic is desired.

Graphene Multilayers as Gates for Multi-Week Sequential Release of Proteins from Surfaces

The ability to control the timing and order of release of different therapeutic drugs will play a pivotal role in improving patient care and simplifying treatment regimes in the clinic. The controlled sequential release of a broad range of small and macromolecules from thin film coatings offers a simple way to provide complex localized dosing in vivo. Here we show that it is possible to take advantage of the structure of certain nanomaterials to control release regimes from a scale of hours to months. Graphene oxide (GO) is a two-dimensional charged nanomaterial that can be used to create barrier layers in multilayer thin films, trapping molecules of interest for controlled release. Protein-loaded polyelectrolyte multilayer films were fabricated using layer-by-layer assembly incorporating a hydrolytically degradable cationic poly(β-amino ester) (Poly1) with a model protein antigen, ovalbumin (ova), in a bilayer architecture along with positively and negatively functionalized GO capping layers for the degradable protein films. Ova release without the GO layers takes place in less than 1 h but can be tuned to release from 30 to 90 days by varying the number of bilayers of functionalized GO in the multilayer architecture. We demonstrate that proteins can be released in sequence with multi-day gaps between the release of each species by incorporating GO layers between protein loaded layers. In vitro toxicity assays of the individual materials on proliferating hematopoietic stem cells (HSCs) indicated limited cytotoxic effects with HSCs able to survive for the full 10 days of normal culture in the presence of Poly1 and the GO sheets. This approach provides a new route for storage of therapeutics in a solid-state thin film for subsequent delivery in a time-controlled and sequential fashion.

Characterization of tunable FGF-2 releasing polyelectrolyte multilayers.

Fibroblast growth factor 2 (FGF-2) is a potent mediator of stem cell differentiation and proliferation. Although FGF-2 has a well-established role in promoting bone tissue formation, flaws in its delivery have limited its clinical utility. Polyelectrolyte multilayer films represent a novel system for FGF-2 delivery that has promise for local, precisely controlled, and sustained release of FGF-2 from surfaces of interest, including medical implants and tissue engineering scaffolds. In this work, the loading and release of FGF-2 from synthetic hydrolytically degradable multilayer thin films of various architectures is explored; drug loading was tunable using at least three parameters (number of nanolayers, counterpolyanion, and type of degradable polycation) and yielded values of 7-45 ng/cm(2) of FGF-2. Release time varied between 24 h and approximately five days. FGF-2 released from these films retained in vitro activity, promoting the proliferation of MC3T3 preosteoblast cells. The use of biologically derived counterpolyanions heparin sulfate and chondroitin sulfate in the multilayer structures enhanced FGF-2 activity. The control over drug loading and release kinetics inform future in vivo bone and tissue regeneration models for the exploration of clinical relevance of LbL growth factor delivery films.

A nanoparticle-based combination chemotherapy delivery system for enhanced tumor killing by dynamic rewiring of signaling pathways.

Nanoparticles containing two drugs released at different times effectively kill tumor cells. Timing the One-Two Punch Morton et al. developed a dual-drug, time-delayed nanoparticle delivery system for treating cancer. The nanoparticles contained two drugs (one in the membrane and one in the center) and were coated to target the nanoparticles to cancer cells. Cancer cells took up the nanoparticles. The first drug quickly escaped the nanoparticle, sensitizing the cells to the second drug, which escaped more slowly. In mice, tumors from cells that respond to the first drug were reduced when the mice were treated with the dual-drug nanoparticles, but the tumors continued to grow in mice receiving only single-drug therapy. This time-delayed, nanoparticle-mediated drug delivery may avoid the resistance that cancer cells develop to chemotherapy. Exposure to the EGFR (epidermal growth factor receptor) inhibitor erlotinib promotes the dynamic rewiring of apoptotic pathways, which sensitizes cells within a specific period to subsequent exposure to the DNA-damaging agent doxorubicin. A critical challenge for translating this therapeutic network rewiring into clinical practice is the design of optimal drug delivery systems. We report the generation of a nanoparticle delivery vehicle that contained more than one therapeutic agent and produced a controlled sequence of drug release. Liposomes, representing the first clinically approved nanomedicine systems, are well-characterized, simple, and versatile platforms for the manufacture of functional and tunable drug carriers. Using the hydrophobic and hydrophilic compartments of liposomes, we effectively incorporated both hydrophobic (erlotinib) and hydrophilic (doxorubicin) small molecules, through which we achieved the desired time sequence of drug release. We also coated the liposomes with folate to facilitate targeting to cancer cells. When compared to the time-staggered application of individual drugs, staggered release from tumor-targeted single liposomal particles enhanced dynamic rewiring of apoptotic signaling pathways, resulting in improved tumor cell killing in culture and tumor shrinkage in animal models.

Osteophilic Multilayer Coatings for Accelerated Bone Tissue Growth

Osteophilic modular nanostructured multilayers containing hydroxyapatite nanoparticles complexed with a natural polymer chitosan create an osteoconductive surface for mesenchymal stem cells (MSCs). Coupled with the sustained release of physiological amounts of osteoinductive bone morphogenetic protein over several days from degradable poly(β-amino ester) based multilayers, this single coating results in a synergistic accelerated and upregulated differentiation of MSCs into osteoblasts laying down new bone tissue on orthopedic implants.

Adaptive growth factor delivery from a polyelectrolyte coating promotes synergistic bone tissue repair and reconstruction

Significance A critical challenge in the field of tissue repair is effective bone repair and reconstruction. The clinical standard of extracting bone from another area in the body or from donors is severely hampered by short supply, pain, and concerns about disease transmission. In this study, we developed a polymer-based nanolayered coating that carries active biological drugs in physiologically relevant amounts for tissue repair, with tunable release properties to induce bone repair. Using a rodent model, we observed that these coatings yield mature, mechanically stable bone that bridges large defects and restores the native form. This system is a potent strategy for safe and precise tissue repair and has the potential to significantly boost successful outcomes for bone repair. Traumatic wounds and congenital defects that require large-scale bone tissue repair have few successful clinical therapies, particularly for craniomaxillofacial defects. Although bioactive materials have demonstrated alternative approaches to tissue repair, an optimized materials system for reproducible, safe, and targeted repair remains elusive. We hypothesized that controlled, rapid bone formation in large, critical-size defects could be induced by simultaneously delivering multiple biological growth factors to the site of the wound. Here, we report an approach for bone repair using a polyelectrolye multilayer coating carrying as little as 200 ng of bone morphogenetic protein-2 and platelet-derived growth factor-BB that were eluted over readily adapted time scales to induce rapid bone repair. Based on electrostatic interactions between the polymer multilayers and growth factors alone, we sustained mitogenic and osteogenic signals with these growth factors in an easily tunable and controlled manner to direct endogenous cell function. To prove the role of this adaptive release system, we applied the polyelectrolyte coating on a well-studied biodegradable poly(lactic-co-glycolic acid) support membrane. The released growth factors directed cellular processes to induce bone repair in a critical-size rat calvaria model. The released growth factors promoted local bone formation that bridged a critical-size defect in the calvaria as early as 2 wk after implantation. Mature, mechanically competent bone regenerated the native calvaria form. Such an approach could be clinically useful and has significant benefits as a synthetic, off-the-shelf, cell-free option for bone tissue repair and restoration.

Surface-mediated bone tissue morphogenesis from tunable nanolayered implant coatings.

A multilayered implant coating promotes bone formation and prevents implant loosening and failure. Implant Coating Builds Bone With an aging population comes more and more surgical implants to stabilize broken hips and replace worn-down joints. Despite their widespread application, these biomedical implants can loosen by not integrating fully with the host tissue; this requires revision surgery and increases patient morbidity. In response, Shah and colleagues designed a series of biochemical coatings that can be applied to both polymer (PEEK) and metal (titanium) surfaces to help implants develop a strong interface with existing bone. The so-called layer-by-layer assembly consisted of two parts: a base coating of several “osteoconductive” layers beneath degradable layers containing human BMP-2—a protein that promotes bone growth. These degradable layers were designed to control the release of BMP-2 over time, rather than delivering the protein all at once. In vivo in rat tibiae, Shah et al. found that the implants with the two-part coating were more difficult to pull out compared with single-coating or uncoated implants. This indicated better bonding between the coating materials and the host bone, and was confirmed on a cellular level by observing bone tissue on the surface of removed implants. In a rodent model, integration of implants with the multilayered coating was calculated to be stronger than the standard bioactive bone cement and other coatings tested in an animal model that are currently used in the clinic. Through a successful proof-of-concept demonstration in rodents, Shah et al. show that this new layered approach could prevent implant loosening and associated morbidity in patients. Nevertheless, before moving into people, further testing will be needed in a larger animal model to confirm that the implants integrate with existing bone in a load-bearing environment. The functional success of a biomedical implant critically depends on its stable bonding with the host tissue. Aseptic implant loosening accounts for more than half of all joint replacement failures. Various materials, including metals and plastic, confer mechanical integrity to the device, but often these materials are not suitable for direct integration with the host tissue, which leads to implant loosening and patient morbidity. We describe a self-assembled, osteogenic, polymer-based conformal coating that promotes stable mechanical fixation of an implant in a surrogate rodent model. A single modular, polymer-based multilayered coating was deposited using a water-based layer-by-layer approach, by which each element was introduced on the surface in nanoscale layers. Osteoconductive hydroxyapatite (HAP) and osteoinductive bone morphogenetic protein–2 (BMP-2) contained within the nanostructured coating acted synergistically to induce osteoblastic differentiation of endogenous progenitor cells within the bone marrow, without indications of a foreign body response. The tuned release of BMP-2, controlled by a hydrolytically degradable poly(β-amino ester), was essential for tissue regeneration, and in the presence of HAP, the modular coating encouraged the direct deposition of highly cohesive trabecular bone on the implant surface. In vivo, the bone-implant interfacial tensile strength was significantly higher than standard bioactive bone cement, did not fracture at the interface, and had long-term stability. Collectively, these results suggest that the multilayered coating system promotes biological fixation of orthopedic and dental implants to improve surgical outcomes by preventing loosening and premature failure.

Synthesis of Highly Stable Sub-8 nm TiO2 Nanoparticles and Their Multilayer Electrodes of TiO2/MWNT for Electrochemical Applications

Next-generation electrochemical energy storage for integrated microsystems and consumer electronic devices requires novel electrode materials with engineered architectures to meet the requirements of high performance, low cost, and robustness. However, conventional electrode fabrication processes such as doctor blading afford limited control over the electrode thickness and structure at the nanoscale and require the incorporation of insulating binder and other additives, which can promote agglomeration and reduce active surface area, limiting the inherent advantages attainable from nanoscale materials. We have engineered a route for the synthesis of highly stable, sub-8 nm TiO2 nanoparticles and their subsequent incorporation with acid-functionalized multiwalled carbon nanotubes (MWNTs) into nanostructured electrodes using aqueous-based layer-by-layer electrostatic self-assembly. Using this approach, binder-free thin film electrodes with highly controllable thicknesses up to the micrometer scale were developed with well-dispersed, nonagglomerated TiO2 nanoparticles on MWNTs. Upon testing in an Li electrochemical half-cell, these electrodes demonstrate high capacity (>150 mAh/gel(ectrode) at 0.1 A/gel(ectrode)), good rate capability (>100 mAh/gel(ectrode) up to 1 A/g(electrode)) and nearly no capacity loss up to 200 cycles for electrodes with thicknesses up to 1480 nm, indicating their promise as thin-film negative electrodes for future Li storage applications.

Multilayer Thin Film Coatings Capable of Extended Programmable Drug Release: Application to Human Mesenchymal Stem Cell Differentiation

The promise of cellular therapy lies in healing damaged tissues and organs in vivo as well as generating tissue constructs in vitro for subsequent transplantation. Postnatal stem cells are ideally suited for cellular therapies due to their pluripotency and the ease with which they can be cultured on functionalized substrates. Creating environments to control and successfully drive their differentiation toward a lineage of choice is one of the most important challenges of current cell-based engineering strategies. In recent years, a variety of biomaterials platforms have been prepared for stem cell cultures, primarily to provide efficient delivery of growth or survival factors to cells and a conductive microenvironment for their growth. Here, we demonstrate that repeating tetralayer structures composed of biocompatible poly(methacrylic acid), poly(acrylamide), and poly(ethylene oxide)-block-poly(ε-caprolactone) micelles arrayed in layer-by-layer films can serve as a payload region for dexamethasone delivery to human mesenchymal stem cells (MSCs). This architecture can induce MSC differentiation into osteoblasts in a dose-dependent manner. The amount of Dex loaded in the films is controlled by varying the deposition conditions and the film thickness. Release of Dex is tuned by changing the amount of covalent cross-linking of multilayers via thermal treatments. The multilayer architecture including payload and cell-adhesion region introduced here are well suited for extended cell culture thus affording the important and protective effect of both Dex release and immobilization. These films may find applications in the local delivery of immobilized therapeutics for biomedical applications, as they can be deposited on a wide range of substrates with different shapes, sizes, and composition.

Synthetic nanoscale electrostatic particles as growth factor carriers for cartilage repair

Abstract The efficient transport of biological therapeutic materials to target tissues within the body is critical to their efficacy. In cartilage tissue, the lack of blood vessels prevents the entry of systemically administered drugs at therapeutic levels. Within the articulating joint complex, the dense and highly charged extracellular matrix (ECM) hinders the transport of locally administered therapeutic molecules. Consequently, cartilage injury is difficult to treat and frequently results in debilitating osteoarthritis. Here we show a generalizable approach in which the electrostatic assembly of synthetic polypeptides and a protein, insulin‐like growth factor‐1 (IGF‐1), can be used as an early interventional therapy to treat injury to the cartilage. We demonstrated that poly(glutamic acid) and poly(arginine) associated with the IGF‐1 via electrostatic interactions, forming a net charged nanoscale polyelectrolyte complex (nanoplex). We observed that the nanoplex diffused into cartilage plugs in vitro and stimulated ECM production. In vivo, we monitored the transport, retention and therapeutic efficacy of the nanoplex in an established rat model of cartilage injury. A single therapeutic dose, when administered within 48 hr of the injury, conferred protection against cartilage degradation and controlled interleukin‐1 mediated inflammation. IGF‐1 contained in the nanoplex was detected in the joint space for up to 4 weeks following administration and retained bioactivity. The results indicate the potential of this approach as an early intervention therapy following joint injury to delay or even entirely prevent the onset of osteoarthritis.