Mary K Lowrey, Holly Day, Kevin J Schilling, Katherine T Huynh, Cristiane M Franca, Carolyn E Schutt
{"title":"利用超声响应生物墨水在同轴三维生物打印结构中远程控制基因传递","authors":"Mary K Lowrey, Holly Day, Kevin J Schilling, Katherine T Huynh, Cristiane M Franca, Carolyn E Schutt","doi":"10.1007/s12195-024-00818-x","DOIUrl":null,"url":null,"abstract":"<p><strong>Introduction: </strong>Coaxial 3D bioprinting has advanced the formation of tissue constructs that recapitulate key architectures and biophysical parameters for in-vitro disease modeling and tissue-engineered therapies. Controlling gene expression within these structures is critical for modulating cell signaling and probing cell behavior. However, current transfection strategies are limited in spatiotemporal control because dense 3D scaffolds hinder diffusion of traditional vectors. To address this, we developed a coaxial extrusion 3D bioprinting technique using ultrasound-responsive gene delivery bioinks. These bioink materials incorporate echogenic microbubble gene delivery particles that upon ultrasound exposure can sonoporate cells within the construct, facilitating controllable transfection.</p><p><strong>Methods: </strong>Phospholipid-coated gas-core microbubbles were electrostatically coupled to reporter transgene plasmid payloads and incorporated into cell-laden alginate bioinks at varying particle concentrations. These bioinks were loaded into the coaxial nozzle core for extrusion bioprinting with CaCl<sub>2</sub> crosslinker in the outer sheath. Resulting bioprints were exposed to 2.25 MHz focused ultrasound and evaluated for microbubble activation and subsequent DNA delivery and transgene expression.</p><p><strong>Results: </strong>Coaxial printing parameters were established that preserved the stability of ultrasound-responsive gene delivery particles for at least 48 h in bioprinted alginate filaments while maintaining high cell viability. Successful sonoporation of embedded cells resulted in DNA delivery and robust ultrasound-controlled transgene expression. The number of transfected cells was modulated by varying the number of focused ultrasound pulses applied. The size region over which DNA was delivered was modulated by varying the concentration of microbubbles in the printed filaments.</p><p><strong>Conclusions: </strong>Our results present a successful coaxial 3D bioprinting technique designed to facilitate ultrasound-controlled gene delivery. This platform enables remote, spatiotemporally-defined genetic manipulation in coaxially bioprinted tissue constructs with important applications for disease modeling and regenerative medicine.</p><p><strong>Supplementary information: </strong>The online version contains supplementary material available at 10.1007/s12195-024-00818-x.</p>","PeriodicalId":9687,"journal":{"name":"Cellular and molecular bioengineering","volume":"17 5","pages":"401-421"},"PeriodicalIF":2.3000,"publicationDate":"2024-10-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11538209/pdf/","citationCount":"0","resultStr":"{\"title\":\"Remote-Controlled Gene Delivery in Coaxial 3D-Bioprinted Constructs using Ultrasound-Responsive Bioinks.\",\"authors\":\"Mary K Lowrey, Holly Day, Kevin J Schilling, Katherine T Huynh, Cristiane M Franca, Carolyn E Schutt\",\"doi\":\"10.1007/s12195-024-00818-x\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Introduction: </strong>Coaxial 3D bioprinting has advanced the formation of tissue constructs that recapitulate key architectures and biophysical parameters for in-vitro disease modeling and tissue-engineered therapies. Controlling gene expression within these structures is critical for modulating cell signaling and probing cell behavior. However, current transfection strategies are limited in spatiotemporal control because dense 3D scaffolds hinder diffusion of traditional vectors. To address this, we developed a coaxial extrusion 3D bioprinting technique using ultrasound-responsive gene delivery bioinks. These bioink materials incorporate echogenic microbubble gene delivery particles that upon ultrasound exposure can sonoporate cells within the construct, facilitating controllable transfection.</p><p><strong>Methods: </strong>Phospholipid-coated gas-core microbubbles were electrostatically coupled to reporter transgene plasmid payloads and incorporated into cell-laden alginate bioinks at varying particle concentrations. These bioinks were loaded into the coaxial nozzle core for extrusion bioprinting with CaCl<sub>2</sub> crosslinker in the outer sheath. Resulting bioprints were exposed to 2.25 MHz focused ultrasound and evaluated for microbubble activation and subsequent DNA delivery and transgene expression.</p><p><strong>Results: </strong>Coaxial printing parameters were established that preserved the stability of ultrasound-responsive gene delivery particles for at least 48 h in bioprinted alginate filaments while maintaining high cell viability. Successful sonoporation of embedded cells resulted in DNA delivery and robust ultrasound-controlled transgene expression. The number of transfected cells was modulated by varying the number of focused ultrasound pulses applied. The size region over which DNA was delivered was modulated by varying the concentration of microbubbles in the printed filaments.</p><p><strong>Conclusions: </strong>Our results present a successful coaxial 3D bioprinting technique designed to facilitate ultrasound-controlled gene delivery. This platform enables remote, spatiotemporally-defined genetic manipulation in coaxially bioprinted tissue constructs with important applications for disease modeling and regenerative medicine.</p><p><strong>Supplementary information: </strong>The online version contains supplementary material available at 10.1007/s12195-024-00818-x.</p>\",\"PeriodicalId\":9687,\"journal\":{\"name\":\"Cellular and molecular bioengineering\",\"volume\":\"17 5\",\"pages\":\"401-421\"},\"PeriodicalIF\":2.3000,\"publicationDate\":\"2024-10-27\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11538209/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Cellular and molecular bioengineering\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://doi.org/10.1007/s12195-024-00818-x\",\"RegionNum\":4,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/10/1 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"Q3\",\"JCRName\":\"BIOPHYSICS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cellular and molecular bioengineering","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1007/s12195-024-00818-x","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/10/1 0:00:00","PubModel":"eCollection","JCR":"Q3","JCRName":"BIOPHYSICS","Score":null,"Total":0}
引用次数: 0
摘要
导言:同轴三维生物打印技术推动了组织构建物的形成,这种构建物能够再现体外疾病建模和组织工程疗法所需的关键结构和生物物理参数。控制这些结构中的基因表达对于调节细胞信号传导和探测细胞行为至关重要。然而,由于致密的三维支架阻碍了传统载体的扩散,目前的转染策略在时空控制方面受到了限制。为了解决这个问题,我们利用超声响应基因递送生物墨水开发了一种同轴挤压三维生物打印技术。这些生物墨水材料中含有可产生回声的微气泡基因递送颗粒,在超声波照射下可使构建体中的细胞发生声穿透,从而促进可控转染:方法:磷脂包裹的气芯微气泡与报告转基因质粒有效载荷进行静电耦合,并以不同的颗粒浓度掺入含有细胞的藻酸盐生物墨水中。将这些生物墨水装入同轴喷嘴核心,用外鞘中的 CaCl2 交联剂进行挤压生物打印。将生成的生物打印置于 2.25 MHz 聚焦超声波下,评估微泡活化和随后的 DNA 递送及转基因表达情况:结果:建立了同轴打印参数,可使超声响应基因递送颗粒在生物打印的藻酸盐细丝中保持稳定至少 48 小时,同时保持较高的细胞活力。对嵌入细胞的成功超声穿透可实现 DNA 的递送和强大的超声控制转基因表达。转染细胞的数量可通过改变聚焦超声脉冲的数量来调节。通过改变印刷细丝中微气泡的浓度,可调节 DNA 输送的大小区域:我们的研究结果展示了一种成功的同轴三维生物打印技术,旨在促进超声控制的基因递送。这一平台可在同轴生物打印组织构建物中实现远程、时空定义的基因操作,在疾病建模和再生医学方面具有重要应用价值:在线版本包含补充材料,可查阅 10.1007/s12195-024-00818-x。
Remote-Controlled Gene Delivery in Coaxial 3D-Bioprinted Constructs using Ultrasound-Responsive Bioinks.
Introduction: Coaxial 3D bioprinting has advanced the formation of tissue constructs that recapitulate key architectures and biophysical parameters for in-vitro disease modeling and tissue-engineered therapies. Controlling gene expression within these structures is critical for modulating cell signaling and probing cell behavior. However, current transfection strategies are limited in spatiotemporal control because dense 3D scaffolds hinder diffusion of traditional vectors. To address this, we developed a coaxial extrusion 3D bioprinting technique using ultrasound-responsive gene delivery bioinks. These bioink materials incorporate echogenic microbubble gene delivery particles that upon ultrasound exposure can sonoporate cells within the construct, facilitating controllable transfection.
Methods: Phospholipid-coated gas-core microbubbles were electrostatically coupled to reporter transgene plasmid payloads and incorporated into cell-laden alginate bioinks at varying particle concentrations. These bioinks were loaded into the coaxial nozzle core for extrusion bioprinting with CaCl2 crosslinker in the outer sheath. Resulting bioprints were exposed to 2.25 MHz focused ultrasound and evaluated for microbubble activation and subsequent DNA delivery and transgene expression.
Results: Coaxial printing parameters were established that preserved the stability of ultrasound-responsive gene delivery particles for at least 48 h in bioprinted alginate filaments while maintaining high cell viability. Successful sonoporation of embedded cells resulted in DNA delivery and robust ultrasound-controlled transgene expression. The number of transfected cells was modulated by varying the number of focused ultrasound pulses applied. The size region over which DNA was delivered was modulated by varying the concentration of microbubbles in the printed filaments.
Conclusions: Our results present a successful coaxial 3D bioprinting technique designed to facilitate ultrasound-controlled gene delivery. This platform enables remote, spatiotemporally-defined genetic manipulation in coaxially bioprinted tissue constructs with important applications for disease modeling and regenerative medicine.
Supplementary information: The online version contains supplementary material available at 10.1007/s12195-024-00818-x.
期刊介绍:
The field of cellular and molecular bioengineering seeks to understand, so that we may ultimately control, the mechanical, chemical, and electrical processes of the cell. A key challenge in improving human health is to understand how cellular behavior arises from molecular-level interactions. CMBE, an official journal of the Biomedical Engineering Society, publishes original research and review papers in the following seven general areas:
Molecular: DNA-protein/RNA-protein interactions, protein folding and function, protein-protein and receptor-ligand interactions, lipids, polysaccharides, molecular motors, and the biophysics of macromolecules that function as therapeutics or engineered matrices, for example.
Cellular: Studies of how cells sense physicochemical events surrounding and within cells, and how cells transduce these events into biological responses. Specific cell processes of interest include cell growth, differentiation, migration, signal transduction, protein secretion and transport, gene expression and regulation, and cell-matrix interactions.
Mechanobiology: The mechanical properties of cells and biomolecules, cellular/molecular force generation and adhesion, the response of cells to their mechanical microenvironment, and mechanotransduction in response to various physical forces such as fluid shear stress.
Nanomedicine: The engineering of nanoparticles for advanced drug delivery and molecular imaging applications, with particular focus on the interaction of such particles with living cells. Also, the application of nanostructured materials to control the behavior of cells and biomolecules.