Advancing Bone-Targeted Drug Delivery: Leveraging Biological Factors and Nanoparticle Designs to Improve Therapeutic Efficacy.

Designing targeted drug delivery systems to effectively treat bone diseases ranging from osteoporosis to nonunion bone defects remains a significant challenge. Previously, nanoparticles (NPs) self-assembled from diblock copolymers of poly(styrene-alt-maleic anhydride)-b-poly(styrene) (PSMA-b-PS) delivering a Wnt agonist were shown to effectively target bone and improve healing via the introduction of a peptide with high affinity to tartrate-resistant acid phosphatase (TRAP), an enzyme deposited by the osteoclasts during bone remodeling. Despite these promising results, the underlying biological factors governing targeting and subsequent drug delivery system (DDS) design parameters have not been examined to enable the rational design to improve bone selectivity. Therefore, this work investigated the effect of target ligand density, the treatment window after injury, specificity of TRAP binding peptide (TBP), the extent of TRAP deposition, and underlying genetic factors (e.g., mouse strain differences) on TBP-NP targeting. Data based on in vitro binding studies and in vivo biodistribution analyses using a murine femoral fracture model suggest that TBP-NP-TRAP interactions and TBP-NP bone accumulation were ligand-density-dependent; in vitro, TRAP affinity was correlated with ligand density up to the maximum of 200,000 TBP ligands/NP, while NPs with 80,000 TBP ligands showed 2-fold increase in fracture accumulation at day 21 post injury compared with that of untargeted or scrambled controls. While fracture accumulation exhibited similar trends when injected at day 3 compared to that at day 21 postfracture, there were no significant differences observed between TBP-functionalized and control NPs, possibly due to saturation of TRAP by NPs at day 3. Leveraging a calcium-depletion diet, TRAP deposition and TBP-NP bone accumulation were positively correlated, confirming that TRAP-TBP binding leads to TBP-NP bone accumulation in vivo. Furthermore, TBP-NP exhibited similar bone accumulation in both C57BL/6 and BALB/c mouse strains versus control NPs, suggesting the broad applicability of TBP-NP regardless of the underlying genetic differences. These studies provide insight into TBP-NP design, mechanism, and therapeutic windows, which inform NP design and treatment strategies for fractures and other bone-associated diseases that leverage TRAP, such as marrow-related hematologic diseases.

Delivery route considerations for designing antigen-specific biomaterial strategies to combat autoimmunity.

Disease modifying drugs and biologics used to treat autoimmune diseases, although promising, are non-curative. As the field moves towards development of new approaches to treat autoimmune disease, antigen-specific therapies immunotherapies (ASITs) have emerged. Despite clinical approval of ASITs for allergies, clinical trials using soluble ASITs for autoimmunity have been largely unsuccessful. A major effort to address this shortcoming is the use of biomaterials to harness the features unique to specific delivery routes. This review focuses on biomaterials being developed for delivery route-specific strategies to induce antigen-specific responses in autoimmune diseases such as multiple sclerosis, type 1 diabetes, rheumatoid arthritis, and celiac disease. We first discuss the delivery strategies used in ongoing and completed clinical trials in autoimmune ASITs. Next, we highlight pre-clinical biomaterial approaches from the most recent 3 years in the context of these same delivery route considerations. Lastly, we provide discussion on the gaps remaining in biomaterials development and comment on the need to consider delivery routes in the process of designing biomaterials for ASITs.

Enhancing the functionality of self-assembled immune signals using chemical crosslinks.

Multiple sclerosis (MS) is an autoimmune disease that develops when dysfunctional autoreactive lymphocytes attack the myelin sheath in the central nervous system. There are no cures for MS, and existing treatments are associated with unwanted side effects. One approach for treating MS is presenting distinct immune signals (i.e., self-antigen and immunomodulatory cues) to innate and adaptive immune cells to engage multiple signaling pathways involved in MS. We previously developed immune polyelectrolyte multilayer (iPEM) complexes built through layer-by-layer deposition of self-antigen - myelin oligodendrocyte glycoprotein (MOG) - and toll-like receptor antagonist, GpG to treat MS. Here, glutaraldehyde-mediated stable cross-links were integrated into iPEMs to load multiple classes of therapeutics. These cross-linked iPEMs maintain their immunological features, including the ability of GpG to blunt toll-like-receptor 9 signaling and MOG to expand T cells expressing myelin-specific T cell receptors. Lastly, we show that these functional assemblies can be loaded with a critical class of drug - mTOR inhibitors - associated with inducing regulatory T cells. These studies demonstrate the ability to incorporate small molecule drugs in reinforced self-assembled immune signals juxtaposed at high densities. This precision technology contributes new technologies that could drive antigen-specific immune response by simultaneously modulating innate and adaptive immunity.

Engineering the lymph node environment promotes antigen-specific efficacy in type 1 diabetes and islet transplantation.

Antigen-specific tolerance is a key goal of experimental immunotherapies for autoimmune disease and allograft rejection. This outcome could selectively inhibit detrimental inflammatory immune responses without compromising functional protective immunity. A major challenge facing antigen-specific immunotherapies is ineffective control over immune signal targeting and integration, limiting efficacy and causing systemic non-specific suppression. Here we use intra-lymph node injection of diffusion-limited degradable microparticles that encapsulate self-antigens with the immunomodulatory small molecule, rapamycin. We show this strategy potently inhibits disease during pre-clinical type 1 diabetes and allogenic islet transplantation. Antigen and rapamycin are required for maximal efficacy, and tolerance is accompanied by expansion of antigen-specific regulatory T cells in treated and untreated lymph nodes. The antigen-specific tolerance in type 1 diabetes is systemic but avoids non-specific immune suppression. Further, microparticle treatment results in the development of tolerogenic structural microdomains in lymph nodes. Finally, these local structural and functional changes in lymph nodes promote memory markers among antigen-specific regulatory T cells, and tolerance that is durable. This work supports intra-lymph node injection of tolerogenic microparticles as a powerful platform to promote antigen-dependent efficacy in type 1 diabetes and allogenic islet transplantation.

Nanoparticle-Mediated Delivery of Micheliolide Analogs to Eliminate Leukemic Stem Cells in the Bone Marrow.

Micheliolide (MCL) is a naturally occurring sesquiterpene lactone that selectively targets leukemic stem cells (LSCs), which persist after conventional chemotherapy for myeloid leukemias, leading to disease relapse. To overcome modest MCL cytotoxicity, analogs with ≈two-threefold greater cytotoxicity against LSCs are synthesized via late-stage chemoenzymatic C-H functionalization. To enhance bone marrow delivery, MCL analogs are entrapped within bone-targeted polymeric nanoparticles (NPs). Robust drug loading capacities of up to 20% (mg drug mg-1 NP) are obtained, with release dominated by analog hydrophobicity. NPs loaded with a hydrolytically stable analog are tested in a leukemic mouse model. Median survival improved by 13% and bone marrow LSCs are decreased 34-fold following NPMCL treatments versus controls. Additionally, selective leukemic cell and LSC cytotoxicity of the treatment versus normal hematopoietic cells is observed. Overall, these studies demonstrate that MCL-based antileukemic agents combined with bone-targeted NPs offer a promising strategy for eradicating LSCs.

Macrophage depletion increases target specificity of bone-targeted nanoparticles.

Despite efforts to achieve tissue selectivity, the majority of systemically administered drug delivery systems (DDSs) are cleared by the mononuclear phagocyte system (MPS) before reaching target tissues regardless of disease or injury pathology. Previously, we showed that while tartrate-resistant acid phosphatase (TRAP) binding peptide (TBP)-targeted polymeric nanoparticles (TBP-NP) delivering a bone regenerative Wnt agonist improved NP fracture accumulation and expedited healing compared with controls, there was also significant MPS accumulation. Here we show that TBP-NPs are taken up by liver, spleen, lung, and bone marrow macrophages (Mϕ), with 76 ± 4%, 49 ± 11%, 27 ± 9%, and 92 ± 5% of tissue-specific Mϕ positive for NP, respectively. Clodronate liposomes (CLO) significantly depleted liver and spleen Mϕ, resulting in 1.8-fold and 3-fold lower liver and spleen and 1.3-fold and 1.6-fold greater fracture and naïve femur accumulation of TBP-NP. Interestingly, depletion and saturation of MPS using 10-fold greater TBP-NP doses also resulted in significantly higher TBP-NP accumulation at lungs and kidneys, potentially through compensatory clearance mechanisms. The higher NP dose resulted in greater TBP-NP accumulation at naïve bone tissue; however, other MPS tissues (i.e., heart and lungs) exhibited greater TBP-NP accumulation, suggesting uptake by other cell types. Most importantly, neither Mϕ depletion nor saturation strategies improved fracture site selectivity of TBP-NPs, possibly due to a reduction of Mϕ-derived osteoclasts, which deposit the TRAP epitope. Altogether, these data support that MPS-mediated clearance is a key obstacle in robust and selective fracture accumulation for systemically administered bone-targeted DDS and motivates the development of more sophisticated approaches to further improve fracture selectivity of DDS.

Biomaterials for Orthopaedic Diagnostics and Theranostics.

Despite widespread use of conventional diagnostic methods in orthopaedic applications, limitations still exist in detection and diagnosing many pathologies especially at early stages when intervention is most critical. The use of biomaterials to develop diagnostics and theranostics, including nanoparticles and scaffolds for systemic or local applications, has significant promise to address these shortcomings and enable successful clinical translation. These developments in both modular and holistic design of diagnostic and theranostic biomaterials may improve patient treatments for myriad orthopaedic applications ranging from cancer to fractures to infection.

Reduction of leukemic burden via bone-targeted nanoparticle delivery of an inhibitor of C-chemokine (C-C motif) ligand 3 (CCL3) signaling.

Leukemias are challenging diseases to treat due, in part, to interactions between leukemia cells and the bone marrow microenvironment (BMME) that contribute significantly to disease progression. Studies have shown that leukemic cells secrete C-chemokine (C-C motif) ligand 3 (CCL3), to disrupt the BMME resulting in loss of hematopoiesis and support of leukemic cell survival and proliferation. In this study, a murine model of blast crisis chronic myelogenous leukemia (bcCML) that expresses the translocation products BCR/ABL and Nup98/HoxA9 was used to determine the role of CCL3 in BMME regulation. Leukemic cells derived from CCL3-/- mice were shown to minimally engraft in a normal BMME, thereby demonstrating that CCL3 signaling was necessary to recapitulate bcCML disease. Further analysis showed disruption in hematopoiesis within the BMME in the bcCML model. To rescue the altered BMME, therapeutic inhibition of CCL3 signaling was investigated using bone-targeted nanoparticles (NP) to deliver Maraviroc, an inhibitor of C-C chemokine receptor type 5 (CCR5), a CCL3 receptor. NP-mediated Maraviroc delivery partially restored the BMME, significantly reduced leukemic burden, and improved survival. Overall, our results demonstrate that inhibiting CCL3 via CCR5 antagonism is a potential therapeutic approach to restore normal hematopoiesis as well as reduce leukemic burden within the BMME.

Ligand Density Controls C-Type Lectin-Like Molecule-1 Receptor-Specific Uptake of Polymer Nanoparticles.

The newest generation of drug delivery systems (DDSs) exploits ligands to mediate specific targeting of cells and/or tissues. However, studies investigating the link between ligand density and nanoparticle (NP) uptake are limited to a small number of ligand-receptor systems. C-type lectin-like molecule-1 (CLL1) is uniquely expressed on myeloid cells, which enables the development of receptors specifically targeting treat various diseases. This study aims to investigate how NPs with different CLL1 targeting peptide density impact cellular uptake. To this end, poly(styrene-alt-maleic anhydride)-b-poly(styrene) NPs are functionalized with cyclized CLL1 binding peptides (cCBP) ranging from 240 ± 12 to 31 000 ± 940 peptides per NP. Unexpectedly, the percentage of cells with internalized NPs is decreased for all cCBP-NP designs regardless of ligand density compared to unmodified NPs. Internalization through CLL1 receptor-mediated processes is further investigated without confounding the effects of NP size and surface charge. Interestingly, high density cCBP-NPs (>7000 cCBP per NP) uptake is dominated by CLL1 receptor-mediated processes while low density cCBP-NPs (≈200 cCBP per NP) and untargeted NP occurred through non-specific clathrin and caveolin-mediated endocytosis. Altogether, these studies show that ligand density and uptake mechanism should be carefully investigated for specific ligand-receptor systems for the design of targeted DDSs to achieve effective drug delivery.

Fracture-Targeted Delivery of ß-Catenin Agonists via Peptide-Functionalized Nanoparticles Augments Fracture Healing.

Despite several decades of progress, bone-specific drug delivery is still a major challenge. Current bone-acting drugs require high-dose systemic administration which decreases therapeutic efficacy and increases off-target tissue effects. Here, a bone-targeted nanoparticle (NP) delivery system for a ß-catenin agonist, 3-amino-6-(4-((4-methylpiperazin-1-yl)sulfonyl)phenyl)-N-(pyridin-3-yl)pyrazine-2-carboxamide, a glycogen synthase kinase 3 beta (GSK-3ß) inhibitor, was developed to enhance fracture healing. The GSK-3ß inhibitor loading capacity was found to be 15 wt % within highly stable poly(styrene-alt-maleic anhydride)-b-poly(styrene) NPs, resulting in ∼50 nm particles with ∼ -30 mV surface charge. A peptide with high affinity for tartrate-resistant acid phosphatase (TRAP), a protein deposited by osteoclasts on bone resorptive surfaces, was introduced to the NP corona to achieve preferential delivery to fractured bone. Targeted NPs showed improved pharmacokinetic profiles with greater accumulation at fractured bone, accompanied by significant uptake in regenerative cell types (mesenchymal stem cells (MSCs) and osteoblasts). MSCs treated with drug-loaded NPs in vitro exhibited 2-fold greater ß-catenin signaling than free drug that was sustained for 5 days. To verify similar activity in vivo, TOPGAL reporter mice bearing fractures were treated with targeted GSK-3ß inhibitor-loaded NPs. Robust ß-galactosidase activity was observed in fracture callus and periosteum treated with targeted carriers versus controls, indicating potent ß-catenin activation during the healing process. Enhanced bone formation and microarchitecture were observed in mice treated with GSK-3ß inhibitor delivered via TRAP-binding peptide-targeted NPs. Specifically, increased bone bridging, ∼4-fold greater torsional rigidity, and greater volumes of newly deposited bone were observed 28 days after treatment, indicating expedited fracture healing.

The effects of particle shape and size on T2 relaxation in magnetic resonance imaging.

Superparamagnetic iron oxide nanoparticles have recently been developed as T2 contrast agents for magnetic resonance imaging. Here we report the dependence of the phase relaxivity, r2, on the particle shape. We show that the size dependence of the relaxivity for spherical particles can be generalized to spheroidal particles. In addition, we show that the saturation of relaxivity above a certain size observed in spherical particles does not occur in the spheroidal particles investigated.