retroductal-nanoparticle-injection-to-the-murine-submandibular-gland

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally ...

production-stable-monodisperse-phospholipid-coated-microbubbles-at

Production of Stable Monodisperse Phospholipid-coated Microbubbles at Room Temperature Using a Microfluidic Flow-focusing Device

Monodisperse microbubbles have demonstrated significant potential in enhancing the efficacy of ultrasound contrast agents for both imaging and therapeutic ...

University of Rochester

Erasmus MC

Despite great potential, delivery remains as the most significant barrier to the widespread use of siRNA therapeutics. siRNA has delivery limitations due to susceptibility to RNase degradation, low cellular uptake, and poor tissue-specific localization. Here, we report the development of a hybrid nanoparticle (NP)/hydrogel system that overcomes these challenges. Hydrogels provide localized and sustained delivery via controlled release of entrapped siRNA/NP complexes while NPs protect and enable efficient cytosolic accumulation of siRNA. To demonstrate therapeutic efficacy, regenerative siRNA against WW domain-containing E3 ubiquitin protein ligase 1 (Wwp1) complexed with NP were entrapped within poly(ethylene glycol) (PEG)-based hydrogels and implanted at sites of murine mid-diaphyseal femur fractures. Results showed localization of hydrogels and controlled release of siRNA/NPs at fractures for 28 days, a timeframe over which fracture healing occurs. siRNA/NP sustained delivery from hydrogels resulted in significant Wwp1 silencing at fracture callus compared to untreated controls. Fractures treated with siRNA/NP hydrogels exhibited accelerated bone formation and significantly increased biomechanical strength. This NP/hydrogel siRNA delivery system has outstanding therapeutic promise to augment fracture healing. Owing to the structural similarities of siRNA, the development of the hydrogel platform for in vivo siRNA delivery has myriad therapeutic possibilities in orthopaedics and beyond.

Controlled and sustained delivery of siRNA/NPs from hydrogels expedites bone 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 β-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-3β) inhibitor, was developed to enhance fracture healing. The GSK-3β 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 β-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-3β inhibitor-loaded NPs. Robust β-galactosidase activity was observed in fracture callus and periosteum treated with targeted carriers versus controls, indicating potent β-catenin activation during the healing process. Enhanced bone formation and microarchitecture were observed in mice treated with GSK-3β 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.

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

pH-responsive diblock copolymers provide tailorable nanoparticle (NP) architecture and chemistry critical for siRNA delivery. Here, diblock polymers varying in first (corona) and second (core) block molecular weight (M), corona/core ratio, and core hydrophobicity (%BMA) were synthesized to determine their effect on siRNA delivery in murine tenocytes (mTenocyte) and murine and human mesenchymal stem cells (mMSC and hMSCs, respectively). NP-mediated siRNA uptake, gene silencing, and cytocompatibility were quantified. Uptake is positively correlated with first block M in mTenocytes and hMSCs (p ≤ 0.0005). All NP resulted in significant gene silencing that was positively correlated with %BMA (p < 0.05) in all cell types. Cytocompatibility was reduced in mTenocytes compared to MSCs (p < 0.0001). %BMA was positively correlated with cytocompatibility in MSCs (p < 0.05), suggesting stable NP are more cytocompatible. Overall, this study shows that NP-siRNA cytocompatibility is cell type dependent, and hydrophobicity (%BMA) is the critical diblock copolymer property for efficient gene silencing in musculoskeletal cell types.

Diblock Copolymer Hydrophobicity Facilitates Efficient Gene Silencing and Cytocompatible Nanoparticle-Mediated siRNA Delivery to Musculoskeletal Cell Types.

Impaired fracture healing is a major clinical problem that can lead to patient disability, prolonged hospitalization, and significant financial burden. Although the majority of fractures heal using standard clinical practices, approximately 10% suffer from delayed unions or non-unions. A wide range of factors contribute to the risk for nonunions including internal factors, such as patient age, gender, and comorbidities, and external factors, such as the location and extent of injury. Current clinical approaches to treat nonunions include bone grafts and low-intensity pulsed ultrasound (LIPUS), which realizes clinical success only to select patients due to limitations including donor morbidities (grafts) and necessity of fracture reduction (LIPUS), respectively. To date, therapeutic approaches for bone regeneration rely heavily on protein-based growth factors such as INFUSE, an FDA-approved scaffold for delivery of bone morphogenetic protein 2 (BMP-2). Small molecule modulators and RNAi therapeutics are under development to circumvent challenges associated with traditional growth factors. While preclinical success has shown promise experimentally, drug delivery has become a major hurdle stalling clinical translation. Therefore, this review overviews current therapies employed to stimulate fracture healing pre-clinically and clinically, including a focus on drug delivery systems for growth factors, parathyroid hormone (PTH), small molecules, and RNAi therapeutics, as well as recent advances and future promise of fracture-targeted drug delivery.

Development of Controlled Drug Delivery Systems for Bone Fracture-Targeted Therapeutic Delivery: A Review.

Locally opening up the endothelial barrier in a safe and controlled way is beneficial for drug delivery into the extravascular tissue. Although ultrasound-induced microbubble oscillations can affect the endothelial barrier integrity, the mechanism remains unknown. Here we uncover a new role for F-actin in microbubble-mediated endothelial gap formation. Unique simultaneous high-resolution confocal microscopy and ultra-high-speed camera imaging (10 million frames per second) reveal that single oscillating microbubbles (radius 1.3-3.8 μm; n = 48) induce sonoporation in all cells in which F-actin remodeling occurred. F-actin disruption only mainly resulted in tunnel formation (75 %), while F-actin stress fiber severing and recoil mainly resulted in cell-cell contact opening within 15 s upon treatment (54 %) and tunnel formation (15 %). F-actin stress fiber severing occurred when the fibers were within reach of the microbubble's maximum radius during oscillation, requiring normal forces of ≥230 nN. In the absence of F-actin stress fibers, oscillating microbubbles induced F-actin remodeling but no cell-cell contact opening. Together, these findings reveal a novel mechanism of microbubble-mediated transendothelial drug delivery, which associates with the underlying cytoskeletal F-actin organization.

Ultrasound-activated microbubbles mediate F-actin disruptions and endothelial gap formation during sonoporation.

Ultrasound contrast agents, comprised of phospholipid-coated microbubbles, can be produced as monodisperse populations using a microfluidic flow-focusing device. However, microbubble coalescence remains a significant challenge. High production temperatures (e.g., 55 °C) can be used to suppress coalescence, but it complicates the microfluidic device design and is incompatible with targeting agents and drug conjugates. This study investigates the production of monodisperse microbubbles at room temperature with the addition of the amphiphilic surfactant Pluronic F68. Two 1,2-distearoyl--glycero-3-phosphocholine (DSPC)-based phospholipid formulations were investigated: F1, containing 1,2-dipalmitoyl--glycero-3-phosphoethanolamine--[carbonyl-methoxypolyethylene glycol] (DPPE-PEG5000), and F2, which included both DPPE-PEG5000 and polyoxyethylene(40) stearate (PEG40-stearate). We characterized the size stability and acoustic behavior of monodisperse microbubbles produced with various Pluronic F68 concentrations. Adding 5-10 mol % Pluronic F68 was found to effectively suppress coalescence and facilitated the production of monodisperse microbubbles that remained shelf stable for at least 7 days. Acoustic attenuation measurements revealed a shell stiffness ranging from 0.78 to 0.93 N/m for these microbubbles. The 10 mol % Pluronic F68 addition (10PF) demonstrated superior monodispersity and was selected for further experiments. Upon dilution, the size and resonance frequencies of both F1-10PF and F2-10PF decreased over time, though F2-10PF showed better stability compared to F1-10PF for both metrics. Both F1-10PF and F2-10PF exhibited a stronger subharmonic scattering intensity than SonoVue (clinical approved microbubbles), which offers potential for blood pressure sensing. Our study shows that incorporating Pluronic F68 facilitates the production of monodisperse microbubbles at room temperature that are stable long-term and have excellent acoustical properties, with the F2-10PF formulation demonstrating better stability than the F1-10PF.

Influence of Pluronic F68 on Size Stability and Acoustic Behavior of Monodisperse Phospholipid-Coated Microbubbles Produced at Room Temperature.

Ultrasound insonification of microbubbles has been shown to increase vascular permeability to locally enhance drug delivery. For more effective and controllable therapeutic outcomes, uniform acoustic responses from microbubbles and a deeper understanding of the biophysical mechanisms of drug delivery are critical. In this study, we investigated the impact of monodisperse microbubble size and their dynamics on cellular responses and drug delivery outcomes in endothelial cells. Monodisperse microbubbles with radii of 1.5, 2.2, 2.7, and 2.9 μm were produced using a microfluidic flow-focusing device. Upon insonification (2 MHz, 220 kPa peak negative pressure, 10 cycles), the microbubble oscillation was captured in real-time at 10 million frames per second using ultra-high-speed imaging, while confocal microscopy was employed to observe cellular responses in both 2D and 3D. For the 65 microbubbles studied, the 2.2 μm microbubbles, i.e., corresponding to the resonant radius at 2 MHz ultrasound, exhibited the highest sonoporation rate (75 %), induced the largest membrane perforations (a median value of 78 μm) and highest intracellular drug uptake. The 1.5 μm microbubbles achieved a comparable sonoporation rate (73 %), yet with significantly smaller membrane perforations (a median value of 20 μm), lower intracellular drug uptake, and highest occurrence of transendothelial drug delivery pathways (64 %). Mechanistically, microbubble-generated shear stress was identified as the significant factor driving sonoporation, while normal stress did not show significance. In conclusion, our study highlights the importance of carefully selecting the microbubbles size to maximize microbubble-mediated drug delivery outcomes and facilitate safe translation of monodisperse microbubbles into clinical practice.

ArticleTotal : 2	Year
Publication title Cited by 10	2018
Publication title	2025

Article	Year
Controlled and sustained delivery of siRNA/NPs from hydrogels expedites bone fracture healing. Biomaterials\| PubMed ID: 28601703	2017
Fracture-Targeted Delivery of β-Catenin Agonists via Peptide-Functionalized Nanoparticles Augments Fracture Healing. ACS nano\| PubMed ID: 28881139	2017
Diblock Copolymer Hydrophobicity Facilitates Efficient Gene Silencing and Cytocompatible Nanoparticle-Mediated siRNA Delivery to Musculoskeletal Cell Types. Biomacromolecules\| PubMed ID: 28960967	2017
Development of Controlled Drug Delivery Systems for Bone Fracture-Targeted Therapeutic Delivery: A Review. European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V\| PubMed ID: 29471078	2018
Ultrasound-activated microbubbles mediate F-actin disruptions and endothelial gap formation during sonoporation. Journal of controlled release : official journal of the Controlled Release Society\| PubMed ID: 39500409	2024
Influence of Pluronic F68 on Size Stability and Acoustic Behavior of Monodisperse Phospholipid-Coated Microbubbles Produced at Room Temperature. ACS applied materials & interfaces\| PubMed ID: 39898998	2025
Dependence of endothelial drug delivery on monodisperse microbubble dynamics. Journal of controlled release : official journal of the Controlled Release Society\| PubMed ID: 40409374	2025

Yuchen Wang

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