Elkhashab Marwa, Barreto Goncalo, Fauconnier Maxime, Le Bourlout Yohann, Creemers Laura B, Nieminen Heikki J, Howard Kenneth A
Interdisciplinary Nanoscience Center (iNANO) and Department of Molecular Biology, Aarhus University, Aarhus C, Denmark.
Medical Ultrasonics Laboratory (MEDUSA), Department of Neuroscience and Biomedical Engineering, Aalto University, Espoo, Finland.
Drug Deliv. 2025 Dec;32(1):2464921. doi: 10.1080/10717544.2025.2464921. Epub 2025 Feb 18.
Antisense oligonucleotides (ASOs) have reached the clinic; however, they lack tissue specificity. Albumin is a plasma-abundant macromolecule that has been shown to accumulate in inflamed tissues. In this work, we have designed a recombinant human albumin (rHA)-based biomolecular assembly incorporating a DNase-resistant phosphorothioate-based complementary oligonucleotide (cODN) and an anti-ADAMTS5 ASO for potential delivery to inflamed sites. Ultrasound (US) was used to trigger ASO release from the assembly and enhance internalization into articular cartilage.
A phosphorothioate cODN was conjugated to rHA through a maleimide cross-linker after which, a therapeutic ADAMTS5-specific gapmer ASO was annealed to the cODN. ASO release was assessed after exposing the biomolecular assembly to different US conditions using an US-actuated medical needle operating at 32.2 kHz. Gene silencing efficiency of US-treated anti-ADAMTS5 ASO was assessed in human primary chondrocytes isolated from osteoarthritic patients. US-mediated ASO penetration into articular cartilage was assessed on bovine articular cartilage.
ASO release was observed after exposure to US waves in continuous mode conditions that did not compromise ASO gene silencing efficiency in human chondrocytes. Furthermore, US increased ASO internalization into bovine articular cartilage after 30 min of application without detrimental effects on chondrocyte viability.
A medical needle driven by continuous US waves at 32.2 kHz has the capability of disassembling a duplex oligonucleotide and enhancing released ASOs internalization into articular cartilage. This work offers the potential delivery and the local triggered release of ASOs at the surface of articular cartilage providing potential benefits for the treatment of diverse cartilage pathologies.
反义寡核苷酸（ASO）已进入临床应用；然而，它们缺乏组织特异性。白蛋白是血浆中丰富的大分子，已被证明可在炎症组织中蓄积。在本研究中，我们设计了一种基于重组人白蛋白（rHA）的生物分子组装体，其包含一种抗核酸酶的硫代磷酸酯基互补寡核苷酸（cODN）和一种抗ADAMTS5的ASO，用于潜在地递送至炎症部位。使用超声（US）触发ASO从组装体中释放，并增强其进入关节软骨的内化。
通过马来酰亚胺交联剂将硫代磷酸酯cODN与rHA偶联，之后将治疗性ADAMTS5特异性缺口mer ASO与cODN退火。使用工作在32.2kHz的超声驱动医用针头，将生物分子组装体暴露于不同的超声条件后，评估ASO的释放情况。在从骨关节炎患者分离的人原代软骨细胞中评估经超声处理的抗ADAMTS5 ASO的基因沉默效率。在牛关节软骨上评估超声介导的ASO渗透进入关节软骨的情况。
在连续模式条件下暴露于超声波后观察到ASO释放，且这种条件不会损害人软骨细胞中ASO的基因沉默效率。此外，施加超声30分钟后，超声增加了ASO进入牛关节软骨的内化，且对软骨细胞活力无有害影响。
由32.2kHz连续超声波驱动的医用针头能够拆解双链寡核苷酸，并增强释放的ASO进入关节软骨的内化。这项工作为ASO在关节软骨表面的潜在递送和局部触发释放提供了可能，为治疗多种软骨疾病带来潜在益处。
