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.

Integrating osteoimmunology and nanoparticle-based drug delivery systems for enhanced fracture healing.

Fracture healing is a complex interplay of molecular and cellular mechanisms lasting from days to weeks. The inflammatory phase is the first stage of fracture healing and is critical in setting the stage for successful healing. There has been growing interest in exploring the role of the immune system and novel therapeutic strategies, such as nanoparticle drug delivery systems in enhancing fracture healing. Advancements in nanotechnology have revolutionized drug delivery systems to the extent that they can modulate immune response during fracture healing by leveraging unique physiochemical properties. Therefore, understanding the intricate interactions between nanoparticle-based drug delivery systems and the immune response, specifically macrophages, is essential for therapeutic efficacy. This review provides a comprehensive overview of the relationship between the immune system and nanoparticles during fracture healing. Specifically, we highlight the influence of nanoparticle characteristics, such as size, surface properties, and composition, on macrophage activation, polarization, and subsequent immune responses. IMPACT STATEMENT: This review provides valuable insights into the interplay between fracture healing, the immune system, and nanoparticle-based drug delivery systems. Understanding nanoparticle-macrophage interactions can advance the development of innovative therapeutic approaches to enhance fracture healing, improve patient outcomes, and pave the way for advancements in regenerative medicine.

Bone-Targeted Nanoparticle Drug Delivery System-Mediated Macrophage Modulation for Enhanced Fracture Healing.

Despite decades of progress, developing minimally invasive bone-specific drug delivery systems (DDS) to improve fracture healing remains a significant clinical challenge. To address this critical therapeutic need, nanoparticle (NP) DDS comprised of poly(styrene-alt-maleic anhydride)-b-poly(styrene) (PSMA-b-PS) functionalized with a peptide that targets tartrate-resistant acid phosphatase (TRAP) and achieves preferential fracture accumulation has been developed. The delivery of AR28, a glycogen synthase kinase-3 beta (GSK3ß) inhibitor, via the TRAP binding peptide-NP (TBP-NP) expedites fracture healing. Interestingly, however, NPs are predominantly taken up by fracture-associated macrophages rather than cells typically associated with fracture healing. Therefore, the underlying mechanism of healing via TBP-NP is comprehensively investigated herein. TBP-NPAR28 promotes M2 macrophage polarization and enhances osteogenesis in preosteoblast-macrophage co-cultures in vitro. Longitudinal analysis of TBP-NPAR28 -mediated fracture healing reveals distinct spatial distributions of M2 macrophages, an increased M2/M1 ratio, and upregulation of anti-inflammatory and downregulated pro-inflammatory genes compared to controls. This work demonstrates the underlying therapeutic mechanism of bone-targeted NP DDS, which leverages macrophages as druggable targets and modulates M2 macrophage polarization to enhance fracture healing, highlighting the therapeutic benefit of this approach for fractures and bone-associated diseases.

Identification of Periostin as a critical niche for myofibroblast dynamics and fibrosis during tendon healing.

Tendon injuries are a major clinical problem, with poor patient outcomes caused by abundant scar tissue deposition during healing. Myofibroblasts play a critical role in the initial restoration of structural integrity after injury. However, persistent myofibroblast activity drives the transition to fibrotic scar tissue formation. As such, disrupting myofibroblast persistence is a key therapeutic target. While myofibroblasts are typically defined by the presence of αSMA+ stress fibers, αSMA is expressed in other cell types including the vasculature. As such, modulation of myofibroblast dynamics via disruption of αSMA expression is not a translationally tenable approach. Recent work has demonstrated that Periostin-lineage (PostnLin) cells are a precursor for cardiac fibrosis-associated myofibroblasts. In contrast to this, here we show that PostnLin cells contribute to a transient αSMA+ myofibroblast population that is required for functional tendon healing, and that Periostin forms a supportive matrix niche that facilitates myofibroblast differentiation and persistence. Collectively, these data identify the Periostin matrix niche as a critical regulator of myofibroblast fate and persistence that could be targeted for therapeutic manipulation to facilitate regenerative tendon healing.

Development of a Nanoparticle-Based Tendon-Targeting Drug Delivery System to Pharmacologically Modulate Tendon Healing.

Tendon regeneration following acute injury is marred by a fibrotic healing response that prevents complete functional recovery. Despite the high frequency of tendon injuries and the poor outcomes, including functional deficits and elevated risk of re-injury, there are currently no pharmacological therapies in clinical use to enhance the healing process. Several promising pharmacotherapies have been identified; however, systemic treatments lack tendon specificity, resulting in poor tendon biodistribution and perhaps explaining the largely limited beneficial effects of these treatments on the tendon healing process. To address this major unmet need, we leveraged our existing spatial transcriptomics dataset of the tendon healing process to identify an area of the healing tendon that is enriched for expression of Acp5. Acp5 encodes tartrate-resistant acid phosphatase (TRAP), and we demonstrate robust TRAP activity in the healing tendon. This unexpected finding allowed us to refine and apply our existing TRAP binding peptide (TBP) functionalized nanoparticle (NP) drug delivery system (DDS) to facilitate improved delivery of systemic treatments to the healing tendon. To demonstrate the translational potential of this drug delivery system, we delivered the S100a4 inhibitor, Niclosamide to the healing tendon. We have previously shown that genetic knockdown of S100a4 enhances tendon healing. While systemic delivery of Niclosamide did not affect the healing process, relative to controls, TBP-NP delivery of Niclosamide enhanced both functional and mechanical outcome measures. Collectively, these data identify a novel tendon-targeting drug delivery system and demonstrate the translational potential of this approach to enhance the tendon healing process.

Identification of Periostin as a critical niche for myofibroblast dynamics and fibrosis during tendon healing.

Tendon injuries are a major clinical problem, with poor patient outcomes caused by abundant scar tissue deposition during healing. Myofibroblasts play a critical role in the initial restoration of structural integrity after injury. However, persistent myofibroblast activity drives the transition to fibrotic scar tissue formation. As such, disrupting myofibroblast persistence is a key therapeutic target. While myofibroblasts are typically defined by the presence of αSMA+ stress fibers, αSMA is expressed in other cell types including the vasculature. As such, modulation of myofibroblast dynamics via disruption of αSMA expression is not a translationally tenable approach. Recent work has demonstrated that Periostin-lineage (PostnLin) cells are a precursor for cardiac fibrosis-associated myofibroblasts. In contrast to this, here we show that PostnLin cells contribute to a transient αSMA+ myofibroblast population that is required for functional tendon healing, and that Periostin forms a supportive matrix niche that facilitates myofibroblast differentiation and persistence. Collectively, these data identify the Periostin matrix niche as a critical regulator of myofibroblast fate and persistence that could be targeted for therapeutic manipulation to facilitate regenerative tendon healing.

Drug Delivery Approaches to Improve Tendon Healing.

Tendon injuries disrupt the transmission of forces from muscle to bone, leading to chronic pain, disability, and a large socioeconomic burden. Tendon injuries are prevalent; there are over 300,000 tendon repair procedures a year in the United States to address acute trauma or chronic tendinopathy. Successful restoration of function after tendon injury remains challenging clinically. Despite improvements in surgical and physical therapy techniques, the high complication rate of tendon repair procedures motivates the use of therapeutic interventions to augment healing. While many biological and tissue engineering approaches have attempted to promote scarless tendon healing, there is currently no standard clinical treatment to improve tendon healing. Moreover, the limited efficacy of systemic delivery of several promising therapeutic candidates highlights the need for tendon-specific drug delivery approaches to facilitate translation. This review article will synthesize the current state-of-the-art methods that have been used for tendon-targeted delivery through both systemic and local treatments, highlight emerging technologies used for tissue-specific drug delivery in other tissue systems, and outline future challenges and opportunities to enhance tendon healing through targeted drug delivery.

Investigating the Interactions of Glioma Stem Cells in the Perivascular Niche at Single-Cell Resolution using a Microfluidic Tumor Microenvironment Model.

The perivascular niche (PVN) is a glioblastoma tumor microenvironment (TME) that serves as a safe haven for glioma stem cells (GSCs), and acts as a reservoir that inevitably leads to tumor recurrence. Understanding cellular interactions in the PVN that drive GSC treatment resistance and stemness is crucial to develop lasting therapies for glioblastoma. The limitations of in vivo models and in vitro assays have led to critical knowledge gaps regarding the influence of various cell types in the PVN on GSCs behavior. This study developed an organotypic triculture microfluidic model as a means to recapitulate the PVN and study its impact on GSCs. This triculture platform, comprised of endothelial cells (ECs), astrocytes, and GSCs, is used to investigate GSC invasion, proliferation and stemness. Both ECs and astrocytes significantly increased invasiveness of GSCs. This study futher identified 15 ligand-receptor pairs using single-cell RNAseq with putative chemotactic mechanisms of GSCs, where the receptor is up-regulated in GSCs and the diffusible ligand is expressed in either astrocytes or ECs. Notably, the ligand-receptor pair SAA1-FPR1 is demonstrated to be involved in chemotactic invasion of GSCs toward PVN. The novel triculture platform presented herein can be used for therapeutic development and discovery of molecular mechanisms driving GSC biology.

Development of Bisphosphonate-Conjugated Antibiotics to Overcome Pharmacodynamic Limitations of Local Therapy: Initial Results with Carbamate Linked Sitafloxacin and Tedizolid.

The use of local antibiotics to treat bone infections has been questioned due to a lack of clinical efficacy and emerging information about Staphylococcus aureus colonization of the osteocyte-lacuno canalicular network (OLCN). Here we propose bisphosphonate-conjugated antibiotics (BCA) using a "target and release" approach to deliver antibiotics to bone infection sites. A fluorescent bisphosphonate probe was used to demonstrate bone surface labeling adjacent to bacteria in a S. aureus infected mouse tibiae model. Bisphosphonate and hydroxybisphosphonate conjugates of sitafloxacin and tedizolid (BCA) were synthesized using hydroxyphenyl and aminophenyl carbamate linkers, respectively. The conjugates were adequately stable in serum. Their cytolytic activity versus parent drug on MSSA and MRSA static biofilms grown on hydroxyapatite discs was established by scanning electron microscopy. Sitafloxacin O-phenyl carbamate BCA was effective in eradicating static biofilm: no colony formation units (CFU) were recovered following treatment with 800 mg/L of either the bisphosphonate or α-hydroxybisphosphonate conjugated drug (p < 0.001). In contrast, the less labile tedizolid N-phenyl carbamate linked BCA had limited efficacy against MSSA, and MRSA. CFU were recovered from all tedizolid BCA treatments. These results demonstrate the feasibility of BCA eradication of S. aureus biofilm on OLCN bone surfaces and support in vivo drug development of a sitafloxacin BCA.

Microengineered 3D Tumor Models for Anti-Cancer Drug Discovery in Female-Related Cancers.

The burden of cancer continues to increase in society and negatively impacts the lives of numerous patients. Due to the high cost of current treatment strategies, there is a crucial unmet need to develop inexpensive preclinical platforms to accelerate the process of anti-cancer drug discovery to improve outcomes in cancer patients, most especially in female patients. Many current methods employ expensive animal models which not only present ethical concerns but also do not often accurately predict human physiology and the outcomes of anti-cancer drug responsiveness. Conventional treatment approaches for cancer generally include systemic therapy after a surgical procedure. Although this treatment technique is effective, the outcome is not always positive due to various complex factors such as intratumor heterogeneity and confounding factors within the tumor microenvironment (TME). Patients who develop metastatic disease still have poor prognosis. To that end, recent efforts have attempted to use 3D microengineered platforms to enhance the predictive power and efficacy of anti-cancer drug screening, ultimately to develop personalized therapies. Fascinating features of microengineered assays, such as microfluidics, have led to the advancement in the development of the tumor-on-chip technology platforms, which have shown tremendous potential for meaningful and physiologically relevant anti-cancer drug discovery and screening. Three dimensional microscale models provide unprecedented ability to unveil the biological complexities of cancer and shed light into the mechanism of anti-cancer drug resistance in a timely and resource efficient manner. In this review, we discuss recent advances in the development of microengineered tumor models for anti-cancer drug discovery and screening in female-related cancers. We specifically focus on female-related cancers to draw attention to the various approaches being taken to improve the survival rate of women diagnosed with cancers caused by sex disparities. We also briefly discuss other cancer types like colon adenocarcinomas and glioblastoma due to their high rate of occurrence in females, as well as the high likelihood of sex-biased mutations which complicate current treatment strategies for women. We highlight recent advances in the development of 3D microscale platforms including 3D tumor spheroids, microfluidic platforms as well as bioprinted models, and discuss how they have been utilized to address major challenges in the process of drug discovery, such as chemoresistance, intratumor heterogeneity, drug toxicity, etc. We also present the potential of these platform technologies for use in high-throughput drug screening approaches as a replacements of conventional assays. Within each section, we will provide our perspectives on advantages of the discussed platform technologies.