摘要

第一部分 基于液态金属的柔性电极介导原位电穿孔转染用于直接心肌重编程

目的：成年心肌细胞基本没有再生能力，缺血性心脏病如心肌梗死后，会不可避免导致心肌细胞损伤坏死和数量减少，只能通过心脏成纤维细胞活化增殖，合成分泌各种炎性介质和细胞外基质形成纤维化，对心脏进行瘢痕修复，严重可导致心功能不全和心力衰竭。目前各种药物和非药物治疗手段只能延缓疾病进展，无法从根源上解决心肌细胞坏死减少的问题。本研究期望通过开发基于液态金属的柔性电子器件，能在体内外进行电穿孔转染，向细胞内递送各种核酸物质，介导直接心肌重编程（Direct cardiac reprogramming, DCR），将成纤维细胞转分化为诱导生成的心肌细胞，最终能在皮肤或心脏进行原位电穿孔，实现心肌组织的异位或原位再生。

方法：我们基于液态金属开发两种柔性电极，通过单次丝网印刷办法得到了能用于皮肤和心脏电穿孔的两用电极；通过激光刻蚀结合双次丝网印刷法得到能进行多通道标测和定向电穿孔的心外膜电极，分别在体外、离体、在体水平测试电穿孔转染基因递送的能力，同时在体内水平进行原位心外膜电生理标测。构建了两种标记策略的报告基因鼠，分别为Myh6-Cre R26R-tdTomato小鼠和PDGFRα-DreER R26R-rox-tdTomato小鼠，能分别在体内外水平进行谱系示踪，追踪细胞发生命运变化。分离培养了原代皮肤和心脏成纤维细胞，用于体外电穿孔转染进行DCR；诱导基因修饰小鼠同源重组，启动永久性荧光蛋白标记，用于在体电穿孔转染。最后围绕DCR进行一系列效果评估，包括实时荧光定量PCR评估相关基因表达水平、报告基因荧光蛋白的表达和定量分析、诱导生成的心肌细胞的电生理特性、免疫荧光染色判断心肌特异性亚细胞结构的生成与谱系示踪共定位和超声心动图检查心梗后小鼠心功能变化等。

结果：制备了具有300%以上拉伸能力的液态金属柔性电极，可以产生大于250V/cm的电场强度用于电穿孔转染，同时心外膜电极可形成可定制化多通道电极阵列用于心外膜电生理标测，并发放定向电穿孔。体外电穿孔转染可以实现EGFP DNA、EGFP mRNA和荧光肽修饰的miRNA，并且进一步递送重编程组合可以将原代成纤维细胞转分化为产生α辅肌动蛋白和肌钙蛋白T等亚细胞结构的肌细胞或心肌样细胞。谱系示踪结果表明，体内外DCR早期（最早在第4天）可以启动心肌特异性启动子Myh6的表达，并且上调一系列心肌相关基因的表达，最终下调成纤维细胞相关的基因表达。PDGFRα启动子终身追踪了成纤维细胞的命运，DCR后流式细胞分析定量确认了有15%~20%的体内转化效率。钙成像分析了体内外DCR诱导生成的心肌细胞的电生理特性，体内表现为150~200次/分的频率，均有自发搏动。

结论：液态金属介导的直接心肌重编程在体外可以将原代成纤维细胞转分化为诱导生成的心肌细胞，在皮肤可以异位生成肌组织，在心脏可以实现原位心肌再生修复，进而改善乃至逆转如缺血性心脏病等导致的心功能受损。体内外方法单用或连用有望成为治疗心肌梗死等缺血性心脏病和或少肌症的新技术。

第二部分 基于液态金属的电生理标测和消融一体化心外膜共形贴附电极

目的：包括房颤在内的各种快速性心律失常，当药物作用无效或难以治疗时，需要进一步通过心脏电生理标测指导根治性消融手术来挽救病人生命。然而，心外科缺乏相匹配的电生理标测工具，现有的基于导管的电生理标测电极缺乏同时具有柔性、可拉伸性（无法与搏动的心脏形成稳定无缝的电极-组织界面）和高效性（需要逐点、逐区域移动导管进行全心标测），并且难以稳定贴靠和完全准确重建解剖结构，因此无法精确定电异质性区域，这会导致指导医生进行偏差的消融治疗，从而引发副损伤。因此有必要在心脏标测中部署更高密度、更高分辨率的独立多通道柔性电极，能与心外膜共形贴附，能早期识别潜在的疾病基质和电异质性，从而为心脏疾病干预和管理提供更多的潜在机会。

方法：开发了一种快速可定制化的双次丝网印刷液态金属的方法来制造柔性心外膜电极贴片，通过工程制图软件快速分别绘制电路导线图案和触点电极点阵设计，通过激光切割打孔预先制备暴露电极触点的封装层薄膜，在同样的热塑性聚氨酯薄膜上利用液态金属油墨丝网印刷底层导线电路后，同封装层热压结合即可得到选择性触点暴露的封装电极；再次在此基础上按照触点电极点阵丝网印刷液态金属油墨，将垂直连接通道进一步导通和填满电极点阵凹槽。电极通过连接柔性印刷电路板可以适配多通道电生理信号采集系统，并且通过尾线焊接和标准接头插板可以连接临床级三维标测系统。通过原代成年小鼠心肌细胞测试了心外膜电极贴片的生物相容性、记录细胞场电位和体外脉冲电场消融的能力。通过逐步过渡的大鼠、兔和犬在体实验，建立具有心外膜心律失常基质的急性心肌梗死和心室颤动等疾病模型来评估心外膜电极贴片评估电生理异常基质和传导的能力，验证该电极贴片在不同级别动物中的心脏电生理标测能力，在大鼠体内验证了脉冲电场消融的能力。

结果：该电极贴片具有完全的柔性和可拉伸性，可以卷曲、折叠后仍然迅速恢复其原始形状时保持正常功能，被保护的独立多通道电极可以进行高质量的心外膜电生理记录。此外，该电极贴片进行了高密度电生理标测，以准确识别大鼠、兔和犬模型的电压和传导特性。对于急性心肌梗死诱发的心室颤动模型，可以分析出体表心电图难以识别的异常传导。还连接了Ensite Velocity三维标测系统，展示了电极贴片重建三维电解剖模型的能力，并且具有正交双向的标测能力，在双极电位的识别上也有较高的信噪比。在体外心肌细胞脉冲电场消融中明确了2中基线阈值参数，可以完全消融心肌细胞，但是最大限度保留了非心肌细胞的组分；在体内心外膜脉冲电场消融中，产生肉眼可见的消融损伤灶，并且即刻消灭局部心肌组织的近场电位。

结论：该电极为心外科提供了前所未有的电生理标测工具，并且具有原位消融能力，与传统电极相比具有明确的优势和补足，为危及生命的心律失常提出了诊疗一体化和同步化的新策略。

Abstract

Liquid Metal Based Flexible Electrodes Mediated In Situ Electroporation Transfection For Direct Cardiac Reprogramming

Purpose: Adult myocardial cells have little regenerative capacity. Ischemic heart disease, such as myocardial infarction (MI), inevitably leads to myocardial cell injury, necrosis, and reduced cardiomyocytes. Activation and proliferation of cardiac fibroblasts synthesize various inflammatory mediators and extracellular matrix to form fibrosis, resulting in scar repair of the heart, which can ultimately lead to heart failure. Currently, various drugs and surgical treatments can only delay the progression of the disease and cannot solve the problem of reducing myocardial loss fundamentally. This study aims to develop flexible electronic devices based on liquid metal to achieve in vitro and in vivo electroporation transfection, delivering various nucleic acid reagents into cells, mediating direct cardiac reprogramming (DCR), and converting fibroblasts into induced cardiomyocytes. Ultimately, in situ or ectopic regeneration of myocardial tissue can be achieved on the skin or heart.

Methods: We developed two types of flexible electrodes based on liquid metal, which were obtained using a single silk screen printing method for both skin and cardiac electroporation. By combining laser etching with double silk screen printing, we obtained an epicardial electrode patch capable of multi-channel recording and directional electroporation. We tested the ability of electroporation mediated gene delivery in vitro, ex vivo, and in vivo, and conducted in vivo epicardial electrophysiological recordings. Two types of reporter gene mice were constructed, namely Myh6-Cre R26R-tdTomato mice and PDGFRα-DreER R26R-rox-tdTomato mice, which could be used for lineage tracing at both in vitro and in vivo levels, lineage tracing the cell fate. Primary skin and cardiac fibroblasts were isolated and cultured for in vitro electroporation mediated transfection for DCR. After induced homologous recombination in gene-modified mice, permanent fluorescent protein labeling was initiated for in vivo electroporation transfection. Finally, a series of evaluations were performed around DCR, including real-time fluorescence quantitative PCR to evaluate the relative expression levels of cardiac genes, expression and quantitative analysis of reporter gene fluorescent proteins, electrophysiological characteristics of induced cardiomyocytes, immunofluorescence staining to determine the generation of myocardium-specific subcellular structures and co-localization with lineage tracing, and echocardiography to check changes in cardiac function in mice after myocardial infarction and DCR.

Results: Flexible electrodes based on liquid metal with over 300% stretching ability were prepared, capable of generating electric field strengths greater than 250 V/cm for electroporation transfection. Meanwhile, the epicardial electrode could form customizable multi-channel electrode arrays for epicardial electrophysiological recording and perform directional electroporation. In vitro electroporation transfection could achieve transfection of EGFP DNA, EGFP mRNA, and fluorescent peptide-modified miRNA. Furthermore, further delivery of reprogramming combinations could transdifferentiate primary fibroblasts into myocytes or myocyte-like cells that produce subcellular structures such as α-actinin and cardiac troponin T. Lineage tracing results showed that early in vitro and in vivo DCR (as early as day 4) could initiate the expression of the cardiac-specific promoter Myh6 and upregulate the expression of a series of cardiac-related genes, ultimately downregulating the expression of fibroblast-related genes. PDGFRα promoter permanently tracked the fate of fibroblasts, and flow cytometry analysis after DCR confirmed a conversion efficiency of 15% to 20% in vivo. Calcium imaging analyzed the electrophysiological characteristics of induced cardiomyocytes induced by in vitro and in vivo DCR, with spontaneous beating frequencies of 150 to 200 times per minute from in vivo DCR.

Conclusion: Liquid metal-mediated direct cardiac reprogramming can transdifferentiate primary fibroblasts into induced cardiomyocytes in vitro, generate muscle tissue ectopically on the skin, and achieve in situ myocardial regeneration and repair in the heart, thereby improving or even reversing cardiac dysfunction caused by ischemic heart disease and other conditions. In vitro and in vivo methods alone or in combination are expected to become new technologies for treating ischemic heart disease and sarcopenia.

An Epicardial Conformal Electrode for Electrophysiological Mapping and Ablation Based on Liquid Metal

Purpose: Including various rapid cardiac arrhythmias such as atrial fibrillation, when pharmacological interventions prove ineffective or challenging, further guidance through cardiac electrophysiological mapping is necessary to direct life-saving curative ablation procedures. However, cardiac surgery lacks appropriately matched electrophysiological mapping tools. Existing catheter-based electrophysiological mapping electrodes lack the simultaneous features of flexibility and stretchability (incapable of forming a stable seamless electrode-tissue interface with the pulsating heart) and efficiency (requiring point-by-point, regional catheter movements for comprehensive cardiac mapping). Moreover, they struggle with stable contact and thus complete and accurate reconstruction of anatomical structures, failing to precisely identify electro-heterogeneous regions. This deficiency can lead to deviations in ablation therapy, resulting in collateral damage. Hence, there is a need to deploy higher density, higher resolution independent multichannel flexible electrodes in cardiac mapping, which can conformally adhere to the epicardium, enabling early identification of potential disease substrates and electro-heterogeneity, thereby providing more opportunities for intervention and management of cardiac diseases.

Methods: A rapid and customizable dual-step screen printing method for liquid metal was developed to manufacture flexible epicardial electrode patches. Circuit wiring patterns and electrode grid designs were swiftly drawn using engineering drawing software. Laser cutting was utilized to pre-prepare thin film encapsulation layers exposing electrode contact points. On the same thermoplastic polyurethane film, a base layer of circuit wiring was screen printed using liquid metal ink. Upon bonding with the encapsulation layer through thermal press, selectively exposed electrode contacts were obtained. Subsequently, liquid metal ink was screen printed based on the electrode grid, facilitating vertical interconnect access to further conduct and fill electrode grid recesses. The electrodes, via connection to flexible printed circuit boards, were compatible with multi-channel electrophysiological signal acquisition systems. Moreover, through tail wire soldering and standard connector plug boards, connectivity to clinical-grade three-dimensional mapping systems was established. The biocompatibility of epicardial electrode patches, their ability to record cellular field potentials, and their capability for in vitro pulse field ablation were tested using primary adult mouse cardiomyocytes. Progressively transitioning to in vivo experiments in rats, rabbits, and dogs, acute myocardial infarction and ventricular fibrillation disease models with epicardial arrhythmogenic substrates were established to evaluate the electrodes' capacity for assessing electrophysiological abnormal substrates and conduction. Validation of the electrode patches' cardiac electrophysiological mapping capability across different animal tiers and pulse field ablation capability in rats were conducted in vivo.

Results: The electrode patches exhibited complete flexibility and stretchability, swiftly recovering their original shape and retaining normal functionality even after being curled or folded. The encapsulated independent multichannel electrodes, when protected, facilitated high-quality epicardial electrophysiological recordings. Additionally, the electrode patches underwent high-density electrophysiological mapping, accurately identifying voltage and conduction characteristics in rat, rabbit, and dog models. For the ventricular fibrillation model induced by acute myocardial infarction, abnormal conduction patterns, undetectable by surface electrocardiography, were analyzed. Connection to the Ensite Velocity three-dimensional mapping system demonstrated the electrode patches' capability to reconstruct three-dimensional anatomical models and exhibited orthogonal bidirectional mapping capabilities, with a high signal-to-noise ratio in bipolar potential identification. In vitro pulse field ablation of cardiomyocytes elucidated two baseline threshold parameters, enabling complete myocardial cell ablation while maximizing retention of non-myocardial components. In vivo epicardial pulse field ablation generated visible ablation lesions and immediately extinguished near-field potentials in local myocardial tissue.

Conclusion: This electrode provides cardiac surgery with an unprecedented electrophysiological mapping tool, coupled with in situ ablation capability, offering distinct advantages and supplementation compared to traditional electrodes. It presents a novel strategy for integrated and synchronized diagnosis and treatment of life-threatening arrhythmias.