Stephen W. Morton

Layer-by-Layer Nanoparticles for Systemic Codelivery of an Anticancer Drug and siRNA for Potential Triple-Negative Breast Cancer Treatment

A single nanoparticle platform has been developed through the modular and controlled layer-by-layer process to codeliver siRNA that knocks down a drug-resistance pathway in tumor cells and a chemotherapy drug to challenge a highly aggressive form of triple-negative breast cancer. Layer-by-layer films were formed on nanoparticles by alternately depositing siRNA and poly-l-arginine; a single bilayer on the nanoparticle surface could effectively load up to 3500 siRNA molecules, and the resulting LbL nanoparticles exhibit an extended serum half-life of 28 h. In animal models, one dose via intravenous administration significantly reduced the target gene expression in the tumors by almost 80%. By generating the siRNA-loaded film atop a doxorubicin-loaded liposome, we identified an effective combination therapy with siRNA targeting multidrug resistance protein 1, which significantly enhanced doxorubicin efficacy by 4 fold in vitro and led to up to an 8-fold decrease in tumor volume compared to the control treatments with no observed toxicity. The results indicate that the use of layer-by-layer films to modify a simple liposomal doxorubicin delivery construct with a synergistic siRNA can lead to significant tumor reduction in the cancers that are otherwise nonresponsive to treatment with Doxil or other common chemotherapy drugs. This approach provides a potential strategy to treat aggressive and resistant cancers, and a modular platform for a broad range of controlled multidrug therapies customizable to the cancer type in a singular nanoparticle delivery system.

A Convergent Synthetic Platform for Single-Nanoparticle Combination Cancer Therapy: Ratiometric Loading and Controlled Release of Cisplatin, Doxorubicin, and Camptothecin

The synthesis of polymer therapeutics capable of controlled loading and synchronized release of multiple therapeutic agents remains a formidable challenge in drug delivery and synthetic polymer chemistry. Herein, we report the synthesis of polymer nanoparticles (NPs) that carry precise molar ratios of doxorubicin, camptothecin, and cisplatin. To our knowledge, this work provides the first example of orthogonally triggered release of three drugs from single NPs. The highly convergent synthetic approach opens the door to new NP-based combination therapies for cancer.

Controlling in Vivo Stability and Biodistribution in Electrostatically Assembled Nanoparticles for Systemic Delivery

This paper demonstrates the generation of systemically deliverable layer-by-layer (LbL) nanoparticles for cancer applications. LbL-based nanoparticles designed to navigate the body and deliver therapeutics in a programmable fashion are promising new and alternative systems for drug delivery, but there have been very few demonstrations of their systemic delivery in vivo due to a lack of knowledge in building LbL nanofilms that mimic traditional nanoparticle design to optimize delivery. The key to the successful application of these nanocarriers in vivo requires a systematic analysis of the influence of film architecture and adsorbed polyelectrolyte outer layer on their pharmacokinetics, which has thus far not been examined for this new approach to nanoparticle delivery. Herein, we have taken the first steps in stabilizing and controlling the systemic distribution of multilayer nanoparticles. Our findings highlight the unique character of LbL systems; the electrostatically assembled nanoparticles gain increased stability in vivo with larger numbers of deposited layers, and the final layer adsorbed generates a critical surface cascade, which dictates the surface chemistry and biological properties of the nanoparticle. This outer polyelectrolyte layer dramatically affects not only the degree of nonspecific particle uptake, but also the nanoparticle biodistribution. For hyaluronic acid (HA) outer layers, a long blood elimination half-life (∼9 h) and low accumulation (∼10-15% recovered fluorescence/g) in the liver were observed, illustrating that these systems can be designed to be highly appropriate for clinical translation.

Redox-responsive branched-bottlebrush polymers for in vivo MRI and fluorescence imaging

Stimuli-responsive multimodality imaging agents have broad potential in medical diagnostics. Herein, we report the development of a new class of branched-bottlebrush polymer dual-modality organic radical contrast agents--ORCAFluors--for combined magnetic resonance and near-infrared fluorescence imaging in vivo. These nitroxide radical-based nanostructures have longitudinal and transverse relaxation times that are on par with commonly used heavy-metal-based magnetic resonance imaging (MRI) contrast agents. Furthermore, these materials display a unique compensatory redox response: fluorescence is partially quenched by surrounding nitroxides in the native state; exposure to ascorbate or ascorbate/glutathione leads to nitroxide reduction and a concomitant 2- to 3.5-fold increase in fluorescence emission. This behaviour enables correlation of MRI contrast, fluorescence intensity and spin concentration with tissues known to possess high concentrations of ascorbate in mice. Our in vitro and in vivo results, along with our modular synthetic approach, make ORCAFluors a promising new platform for multimodality molecular imaging.

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.

Scalable Manufacture of Built-to-Order Nanomedicine: Spray-assisted Layer-by-Layer Functionalization of PRINT® Nanoparticles

Scalable methods, PRINT particle fabrication, and spray-assisted Layer-by-Layer deposition are combined to generate uniform and functional nanotechnologies with precise control over composition, size, shape, and surface functionality. A modular and tunable approach towards design of built-to-order nanoparticle systems, spray coating on PRINT particles is demonstrated to achieve technologies capable of targeted interactions with cancer cells for applications in drug delivery.

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.

Fluorescent Multiblock π-Conjugated Polymer Nanoparticles for In Vivo Tumor Targeting†

Highly fluorescent multiblock conjugated polymer nanoparticles with folic acid surface ligands are highly effective for bioimaging and in vivo tumor targeting. The targeted nanoparticles were preferentially localized in tumor cells in vivo, thereby illustrating their potential for diagnostic and therapeutic applications.

Layer-by-Layer Assembled Antisense DNA Microsponge Particles for Efficient Delivery of Cancer Therapeutics

Antisense oligonucleotides can be employed as a potential approach to effectively treat cancer. However, the inherent instability and inefficient systemic delivery methods for antisense therapeutics remain major challenges to their clinical application. Here, we present a polymerized oligonucleotides (ODNs) that self-assemble during their formation through an enzymatic elongation method (rolling circle replication) to generate a composite nucleic acid/magnesium pyrophosphate sponge-like microstructure, or DNA microsponge, yielding high molecular weight nucleic acid product. In addition, this densely packed ODN microsponge structure can be further condensed to generate polyelectrolyte complexes with a favorable size for cellular uptake by displacing magnesium pyrophosphate crystals from the microsponge structure. Additional layers are applied to generate a blood-stable and multifunctional nanoparticle via the layer-by-layer (LbL) assembly technique. By taking advantage of DNA nanotechnology and LbL assembly, functionalized DNA nanostructures were utilized to provide extremely high numbers of repeated ODN copies for efficient antisense therapy. Moreover, we show that this formulation significantly improves nucleic acid drug/carrier stability during in vivo biodistribution. These polymeric ODN systems can be designed to serve as a potent means of delivering stable and large quantities of ODN therapeutics systemically for cancer treatment to tumor cells at significantly lower toxicity than traditional synthetic vectors, thus enabling a therapeutic window suitable for clinical translation.

The architecture and biological performance of drug-loaded LbL nanoparticles.

Layer-by-Layer (LbL) nanoparticles are an emerging class of therapeutic carriers that afford precise control over key design parameters that facilitate improved drug and carrier pharmacokinetics, and enhanced molecular-targeting capabilities. This paper advances the development of these systems by establishing them as drug carriers, with the means to control drug release in a systemic environment and retard particle clearance from circulation, promoting improved biodistribution of the drug-containing system. Using dual-fluorescent tracking in vivo, this work establishes a robust means of screening libraries of LbL systems generated, affording simultaneous resolution over persistence and biodistribution of both the drug and carrier following systemic administration of a single particle formulation. Employing a PLGA drug-containing core as a substrate for LbL deposition, a range of coated systems were fabricated to investigate the abilities of these films to stabilize drug for delivery as well as to improve the pharmacokinetics of both the drug and carrier. Significant reductions in liver accumulation were observed for different formulations of the layered architectures within the first 30 min of systemic circulation. LbL architectures diminished liver localization of the surrogate drug, cardiogreen, by 10-25% ID/g relative to native PLGA nanoparticles and modulated carrier accumulation in the liver >50% ID/g. Further, enhanced persistence of the drug was observed with the coated systems, significantly increasing the drug half-life from 2 to 3 min for free drug and 1.87 h for the uncoated core to 4.17 h and 4.54 h for the coated systems. These systems provide an exciting, modular platform that improves the pharmacokinetic properties of the therapeutic, reduces bolus release of drug from nanoparticles, and enhances the safety and circulation half-life of the drug in vivo, proving them to be highly clinically-relevant and a promising approach for future development of molecularly-targeted and combination therapeutics.