A microphysiological system for screening lipid nanoparticle−mRNA complexes predicts in vivo heart transfection efficacy

Data availability

The main data supporting the results in this study are available within the Article and its Supplementary Information. All raw and analysed datasets generated during the study are available from the corresponding authors on request. Source data are provided with this paper.

Code availability

Contractile activity and action potential waveforms of the cardiac micromuscles generated in the cardiac MPS were analysed with in-house code. The code for the motion analysis is available via GitHub at https://computationalphysiology.github.io/mps_motion and for fluorescence analysis via GitHub at https://computationalphysiology.github.io/mps.

References

GBD 2017 Disease and Injury Incidence and Prevalence Collaborators Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet 392, 1789–1858 (2018).
Tsao, C. W. et al. Heart disease and stroke statistics—2022 update: a report from the American Heart Association. Circulation 145, e153–e639 (2022).

Article PubMed Google Scholar
Gabriel-Costa, D. The pathophysiology of myocardial infarction-induced heart failure. Pathophysiology 25, 277–284 (2018).

Article CAS PubMed Google Scholar
Savarese, G. et al. Global burden of heart failure: a comprehensive and updated review of epidemiology. Cardiovasc. Res. 118, 3272–3287 (2023).

Article PubMed Google Scholar
Yang, Q., Fang, J., Lei, Z., Sluijter, J. P. G. & Schiffelers, R. Repairing the heart: State-of the art delivery strategies for biological therapeutics. Adv. Drug Deliv. Rev. 160, 1–18 (2020).

Article CAS PubMed Google Scholar
Ingber, D. E. Human organs-on-chips for disease modelling, drug development and personalized medicine. Nat. Rev. Genet. 23, 467–491 (2022).

Article CAS PubMed PubMed Central Google Scholar
Cho, S., Discher, D. E., Leong, K. W., Vunjak-Novakovic, G. & Wu, J. C. Challenges and opportunities for the next generation of cardiovascular tissue engineering. Nat. Methods 19, 1064–1071 (2022).

Article CAS PubMed PubMed Central Google Scholar
Wadman, M. FDA no longer has to require animal testing for new drugs. Science 379, 127–128 (2023).

Article CAS PubMed Google Scholar
Loskill, P., Marcus, S. G., Mathur, A., Reese, W. M. & Healy, K. E. muOrgano: a Lego-like plug & play system for modular multi-organ-chips. PLoS ONE 10, e0139587 (2015).

Article PubMed PubMed Central Google Scholar
van Haasteren, J., Li, J., Scheideler, O. J., Murthy, N. & Schaffer, D. V. The delivery challenge: fulfilling the promise of therapeutic genome editing. Nat. Biotechnol. 38, 845–855 (2020).

Article PubMed Google Scholar
Park, H. J., Yang, F. & Cho, S. W. Nonviral delivery of genetic medicine for therapeutic angiogenesis. Adv. Drug Deliv. Rev. 64, 40–52 (2012).

Article CAS PubMed Google Scholar
Paunovska, K. et al. A direct comparison of in vitro and in vivo nucleic acid delivery mediated by hundreds of nanoparticles reveals a weak correlation. Nano Lett. 18, 2148–2157 (2018).

Article CAS PubMed PubMed Central Google Scholar
Hatakeyama, H. et al. Development of a novel systemic gene delivery system for cancer therapy with a tumor-specific cleavable PEG-lipid. Gene Ther. 14, 68–77 (2007).

Article CAS PubMed Google Scholar
Fang, Y. et al. Cleavable PEGylation: a strategy for overcoming the “PEG dilemma” in efficient drug delivery. Drug Deliv. 24, 22–32 (2017).

Article CAS PubMed PubMed Central Google Scholar
Deirram, N., Zhang, C., Kermaniyan, S. S., Johnston, A. P. R. & Such, G. K. pH-Responsive polymer nanoparticles for drug delivery. Macromol. Rapid Commun. 40, e1800917 (2019).

Article PubMed Google Scholar
Gao, W., Chan, J. M. & Farokhzad, O. C. pH-Responsive nanoparticles for drug delivery. Mol. Pharm. 7, 1913–1920 (2010).

Article CAS PubMed PubMed Central Google Scholar
Palanikumar, L. et al. pH-Responsive high stability polymeric nanoparticles for targeted delivery of anticancer therapeutics. Commun. Biol. 3, 95 (2020).

Article CAS PubMed PubMed Central Google Scholar
Zhao, S. et al. Acid-degradable lipid nanoparticles enhance the delivery of mRNA. Nat. Nanotechnol. 19, 1702–1711 (2024).

Article CAS PubMed PubMed Central Google Scholar
Mathur, A. et al. Human iPSC-based cardiac microphysiological system for drug screening applications. Sci. Rep. 5, 8883 (2015).

Article CAS PubMed PubMed Central Google Scholar
Tveito, A. et al. Inversion and computational maturation of drug response using human stem cell derived cardiomyocytes in microphysiological systems. Sci. Rep. 8, 17626 (2018).

Article PubMed PubMed Central Google Scholar
Huebsch, N. et al. Metabolically driven maturation of human-induced-pluripotent-stem-cell-derived cardiac microtissues on microfluidic chips. Nat. Biomed. Eng. 6, 372–388 (2022).

Article CAS PubMed PubMed Central Google Scholar
Charwat, V. et al. Validating the arrhythmogenic potential of high-, intermediate-, and low-risk drugs in a human-induced pluripotent stem cell-derived cardiac microphysiological system. ACS Pharm. Transl. Sci. 5, 652–667 (2022).

Article CAS Google Scholar
Charrez, B. et al. In vitro safety ‘clinical trial’ of the cardiac liability of drug polytherapy. Clin. Transl. Sci. 14, 1155–1165 (2021).

Article CAS PubMed PubMed Central Google Scholar
Charrez, B. et al. Heart muscle microphysiological system for cardiac liability prediction of repurposed COVID-19 therapeutics. Front. Pharm. 12, 684252 (2021).

Article CAS Google Scholar
Nance, E. A. et al. A dense poly(ethylene glycol) coating improves penetration of large polymeric nanoparticles within brain tissue. Sci. Transl. Med. 4, 149ra119 (2012).

Article PubMed PubMed Central Google Scholar
Armstrong, J. K., Wenby, R. B., Meiselman, H. J. & Fisher, T. C. The hydrodynamic radii of macromolecules and their effect on red blood cell aggregation. Biophys. J. 87, 4259–4270 (2004).

Article CAS PubMed PubMed Central Google Scholar
Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

Article CAS PubMed Google Scholar
Giacomelli, E. et al. Human-iPSC-derived cardiac stromal cells enhance maturation in 3D cardiac microtissues and reveal non-cardiomyocyte contributions to heart disease. Cell Stem Cell 26, 862–879.e11 (2020).

Article CAS PubMed PubMed Central Google Scholar
Kedmi, R., Ben-Arie, N. & Peer, D. The systemic toxicity of positively charged lipid nanoparticles and the role of Toll-like receptor 4 in immune activation. Biomaterials 31, 6867–6875 (2010).

Article CAS PubMed Google Scholar
Li, Y. et al. Cationic liposomes induce cytotoxicity in HepG2 via regulation of lipid metabolism based on whole-transcriptome sequencing analysis. BMC Pharm. Toxicol. 19, 43 (2018).

Article Google Scholar
Hubrecht, R.C. & Carter, E. The 3Rs and humane experimental technique: implementing change. Animals 9, 754 (2019).

Article PubMed PubMed Central Google Scholar
Huebsch, N. et al. Miniaturized iPS-cell-derived cardiac muscles for physiologically relevant drug response analyses. Sci. Rep. 6, 24726 (2016).

Article CAS PubMed PubMed Central Google Scholar
Evers, M. J. W. et al. Delivery of modified mRNA to damaged myocardium by systemic administration of lipid nanoparticles. J. Control. Release 343, 207–216 (2022).

Article CAS PubMed Google Scholar
Mohamed, T. M. A. et al. Regulation of cell cycle to stimulate adult cardiomyocyte proliferation and cardiac regeneration. Cell 173, 104–116.e112 (2018).

Article CAS PubMed PubMed Central Google Scholar
Sun, J. et al. CCND2 modified mRNA activates cell cycle of cardiomyocytes in hearts with myocardial infarction in mice and Pigs. Circ. Res. 133, 484–504 (2023).

Article CAS PubMed PubMed Central Google Scholar
Qian, L. & Srivastava, D. Direct cardiac reprogramming: from developmental biology to cardiac regeneration. Circ. Res. 113, 915–921 (2013).

Article CAS PubMed Google Scholar
Zangi, L. et al. Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction. Nat. Biotechnol. 31, 898–907 (2013).

Article CAS PubMed PubMed Central Google Scholar
Qin, W., Chen, X. & Liu, P. Inhibition of TGF-β1 by eNOS gene transfer provides cardiac protection after myocardial infarction. J. Biomed. Res. 24, 145–152 (2010).

Article CAS PubMed PubMed Central Google Scholar
Jiao, Y. et al. Research progress of nucleic acid delivery vectors for gene therapy. Biomed. Microdevices 22, 16 (2020).

Article CAS PubMed Google Scholar
Fernandopulle, M. S. et al. Transcription factor-mediated differentiation of human iPSCs into neurons. Curr. Protoc. Cell Biol. 79, e51 (2018).

Article PubMed PubMed Central Google Scholar
Shi, Y. et al. Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons. Nat. Med. 24, 313–325 (2018).

Article CAS PubMed PubMed Central Google Scholar
Tian, R. et al. CRISPR interference-based platform for multimodal genetic screens in human iPSC-derived neurons. Neuron 104, 239–255.e212 (2019).

Article CAS PubMed PubMed Central Google Scholar
Trevor, C. R. et al. Transcription activator-like effector nuclease (TALEN)-mediated CLYBL targeting enables enhanced transgene expression and one-step generation of dual reporter human induced pluripotent stem cell (iPSC) and neural stem cell (NSC) lines. PLoS ONE 10, e0116032 (2015).

Article Google Scholar
Lian, X. et al. Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. Proc. Natl Acad. Sci. USA 109, E1848–E1857 (2012).

Article CAS PubMed PubMed Central Google Scholar
Zhang, H., Shen, M. & Wu, J. C. Generation of quiescent cardiac fibroblasts derived from human induced pluripotent stem cells. Methods Mol. Biol. 2454, 109–115 (2022).

Article CAS PubMed PubMed Central Google Scholar
Wang, X. et al. Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery. Nat. Protoc. 18, 265–291 (2023).

Article CAS PubMed Google Scholar
Huang, Y. L., Walker, A. S. & Miller, E. W. A photostable silicon rhodamine platform for optical voltage sensing. J. Am. Chem. Soc. 137, 10767–10776 (2015).

Article CAS PubMed PubMed Central Google Scholar
Tamer, M. A. et al. Regulation of cell cycle to stimulate adult cardiomyocyte proliferation and cardiac regeneration. Cell 173, 104–116.e12 (2018).

Article Google Scholar

Download references

Acknowledgements

This work was funded in part by the California Institute for Regenerative Medicine DISC2-14045 (to K.E.H., N.M. and D.S.), the Rider Award from Innovative Genomics Institute−UC Berkeley (to K.E.H., N.M. and G.N.), the Siebel Stem Cell Institute seed grant award (to K.E.H. and G.N.) and Agilent Technologies Inc. (to K.E.H. and N.M.). N.M. was supported by funding from the Innovative Genomics Institute and the CRISPR Cures for Cancer Initiative. B.R.C. was supported by the National Institutes of Health (R01-AG072052, R01-HL130533, R01-HL13535801, P01-HL146366), the California Institute for Regenerative Medicine (INFR6.2-15527) and the Charcot-Marie-Tooth Association. B.R.C. acknowledges support through a gift from the Roddenberry Foundation and Pauline and Thomas Tusher. We thank M. West (QB3 Cell and Tissue Analysis Facility, UC Berkeley) and the Molecular Imaging Center at UC Berkeley for assistance with image acquisition, analysis and flow cytometry; We thank H. Finsberg and S. Wall from Simula Research Laboratory (Oslo, Norway) for their collaboration in the physiological analysis of the cardiac microtissues.

Author information

Authors and Affiliations

Department of Bioengineering, California Institute for Quantitative Biosciences (QB3), University of California at Berkeley, Berkeley, CA, USA

Gabriel Neiman, Hesong Han, Sheng Zhao, Brian Siemons, Niren Murthy & Kevin E. Healy
Gladstone Institutes, San Francisco, CA, USA

Mauro W. Costa, Tomohiro Nishino, Yu Huang, Shyam Lal & Deepak Srivastava
Roddenberry Center for Stem Cell Biology and Medicine at Gladstone, San Francisco, CA, USA

Mauro W. Costa, Tomohiro Nishino, Yu Huang, Shyam Lal & Deepak Srivastava
Innovative Genomics Institute, University of California at Berkeley, Berkeley, CA, USA

Hesong Han, Sheng Zhao & Niren Murthy
Department of Materials Science and Engineering, University of California at Berkeley, Berkeley, CA, USA

Tammy K. Ng & Kevin E. Healy
Gladstone Institute of Cardiovascular Disease, San Francisco, CA, USA

Kenneth Wu, Luke M. Judge & Bruce R. Conklin
Department of Pediatrics, University of California San Francisco, San Francisco, CA, USA

Luke M. Judge
Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA, USA

Bruce R. Conklin
California Institute for Quantitative Biosciences (QB3), University of California San Francisco, San Francisco, CA, USA

Bruce R. Conklin
Department of Medicine, University of California San Francisco, San Francisco, CA, USA

Bruce R. Conklin
Department of Pediatrics, UCSF School of Medicine, San Francisco, CA, USA

Deepak Srivastava
Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA, USA

Deepak Srivastava

Authors

Gabriel Neiman
Mauro W. Costa
Hesong Han
Sheng Zhao
Tammy K. Ng
Brian Siemons
Tomohiro Nishino
Yu Huang
Shyam Lal
Kenneth Wu
Luke M. Judge
Bruce R. Conklin
Deepak Srivastava
Niren Murthy
Kevin E. Healy

Contributions

G.N., M.W.C., H.H., D.S., N.M. and K.E.H. designed the experimental studies, and performed data analysis. G.N., T.K.N. and B.S. performed the in vitro experimental studies and iPS cell cultures. M.W.C., T.N., Y.H. and S.L. performed in vivo experimental studies. H.H. and S.Z. generated the in-house acid-degradable LNP components and performed LNP characterization studies. K.W., L.M.J. and B.R.C. designed and engineered the genetically modified XR1 WTC-11 hiPS cell-Cre reporter line used to generate the cardiac micromuscles.

Corresponding authors

Correspondence to Deepak Srivastava, Niren Murthy or Kevin E. Healy.

Ethics declarations

Competing interests

K.E.H. and B.S. have a financial relationship with Organos Inc., and hence may benefit from the commercialization of the results of this research. N.M. and H.H. have a financial relationship in Opus Biosciences, and may benefit from the commercialization of the results of this research. D.S. is a scientific co-founder, shareholder and director of Tenaya Therapeutics. The other authors declare no competing interests.

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Peer review information

Nature Biomedical Engineering thanks Milica Radisic and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Cardiac MPS features and function.

A. Photograph of a cardiac MPS under fluorescent lighting with fluidic tubing. B. Layout of a multiplexed cardiac MPS device comprising four parallel tissue chambers with individual cell loading ports and a common media inlet (in) and outlet (out)²⁴. The media channels run parallel to each tissue chamber, and the fenestration barrier provides protection from fluid shear stress while allowing for the exchange of media via diffusion. Anchor pillars located at both extremities of the tissue chamber provide attachment points to keep the cardiac muscle elongated and prevent collapsing. C. Brightfield images of the MPS with cardiac tissues formed using XR1 WTC iCMs. D. SEM micrograph of the MPS showing the 2μm endothelial-like barriers connecting the nutrient channel and the tissue chamber which allows for diffusive transport of drugs and molecules. Simulated velocity profile of flow in the nutrient channel and barrier interface, demonstrating mass transport to the tissue is exclusively diffusive.

Extended Data Fig. 2 Enhanced diffusivity of PEG polymers in the Cardiac MPS.

A. Time-course experiment showing the diffusion of 2 kDa PEG conjugated to FITC over time. Images were captured every 15 mins. Scale bars, 100 μm. B. FRAP experiment was performed after the PEG and Dextran polymers reached a steady state. Finitial is the time regime that corresponds to the initial fluorescence before bleaching; F0 is the fluorescence measurement immediately after photobleaching; Ft>0 corresponds to the recovery of fluorescence after photobleaching; Ft»0 corresponds to the maximal recovery of fluorescence at the end of the experiment. C. Half-time of recovery (sec) and diffusion coefficient (μm²/s) were estimated for the different polymers in the FRAP assay. Global one-way ANOVA was performed followed by post-hoc Tukey analysis.

Extended Data Fig. 3 XR1 WTC hiPSC Cre-reporter line development.

A. Schematic illustration of the development of a genetically modified hiPSC Cre-reporter line by TALEN-Mediated homologous recombination. B. eGFP expression in the XR1 hiPSC upon Cre recombinase plasmid transfection (n = 5).

Extended Data Fig. 4 ADP-LNPs can transfect human iCMs in 2D.

A. Flow cytometry analysis of 2D iCMs after RNA iMAX and LNP-1/eGFP mRNA transfection (controls). B. Flow cytometry analysis of 2D iCMs after RNA iMAX, LNP-1/Cre rec mRNA and Tat-Cre rec protein transfection (controls).

Extended Data Fig. 5 ADP-LNPs can transfect human iCMs in the cardiac MPS.

A. Fluorescent microscopy images showing the eGFP+ cells within the XR1 cardiac micromuscles after exposure to naked Cre rec mRNA, Tat-Cre rec protein, and LNP-1/Cre rec mRNA. Each condition included 3 to 5 biological replicates (n = 3–5). Scale bars, 20 μm. B. Representative distribution of eGFP+ cells within the center of the cardiac MPS (dotted vertical lines). Each point represents an individual cell. Green line is the cut off for eGFP+ cells. Every condition was normalized to 1100 cells.

Extended Data Fig. 6 Isogenic XR1 cardiomyocyte and cardiac fibroblast differentiation.

A. A schematic diagram showing the protocol for small molecule-directed differentiation to hiPSC-derived cardiomyocytes and cardiac fibroblasts. Created with BioRender. B. Representative flow cytometry analysis showing protein levels of hcFb markers such as CD90, DDR2, and Vimentin. The protein expression for the endothelial marker CD31 was also assessed. C. Representative brightfield image of the hcFbs in culture and immunofluorescence images showing the positive expression for hcFB markers such as Collagen Type I, CD90, DDR2, and TE-7 (n = 3). Scale bars, 50 μm. D. mRNA expression analysis of genes that are significantly expressed in cardiac fibroblasts such as COL1A1, GATA4, POSTN, and TBX20 relative to their pluripotent state. Statistical significance was assessed using a two-sided unpaired Student’s t-test.

Extended Data Fig. 7 ADP-LNP/Luc accumulates in hearts with reduced liver targeting.

A. Schematic illustration describing transfection of Ai6 mouse with either Luc mRNA or Cre mRNA delivered by 2 kDa 10% ADP-LNPs and LNP-1. Created with BioRender. B. Characterization of the region used in quantification presented in Fig. 6b. upon 15 seconds of exposure. C. Normalized images of Liver (top, exposure 30 seconds) and hearts (bottom, exposure 300 seconds) show clear increase in heart luciferase activity in 2 kDa 10% ADP-LNP when compared to LNP-1 with a small decrease in overall off-target liver expression.

Extended Data Fig. 8 ADP-LNP formulations showed lower accumulation in both the liver and spleen compared to LNP-1.

Quantification of luciferase activity in isolated liver (A), and spleen (B) following intracardiac delivery of 2 kDa ADP-LNP formulations (1% and 10% PEGylation) and the standard LNP-1 formulation (n = 3). ADP-LNP formulations show reduced off-target activity in the liver and spleen compared to LNP-1. Global one-way ANOVA was performed followed by post-hoc Dunnet analysis.

Extended Data Fig. 9 Immunofluorescence control for sections shown on Fig. 7c.

Absence of signal when no primary antibody for Tnni or aSMA and no conjugated IB4 were used (neg control, left), while genetic ZsGreen1 labeling upon Cre-mediated deletion shows CM with distinct sarcomeres highlighted with yellow lines (middle) and DAPI marking cell nucleus (right). Scale bar, 10mM.

Supplementary information

Supplementary Information

Supplementary Tables 1−4 and Fig. 1.

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Supplementary Video 1

Time-course experiment showing the diffusion of 2 kDa PEG conjugated to FITC into the cardiac MPS over 15 h. Images were captured every 15 mins in brightfield. Related to Figure S2.

Supplementary Video 2

Time-course experiment showing the diffusion of 2 kDa PEG conjugated to FITC into the cardiac MPS over 15 h. Images were captured every 15 mins in FITC channel. Related to Figure S2.

Supplementary Video 3

Volumetric nuclear segmentation performed in the whole cardiac micromuscle. Related to Fig. 3.

Supplementary Video 4

3D cardiac micromuscle showing an eGFP+ cardiomyocyte population upon 2 kDa 10% ADP-LNP/Cre mRNA delivery. Related to Fig. 3.

Supplementary Video 5

3D cardiac micromuscle upon exposure of 2 kDa 10% ADP-LNPs/CRE mRNA. cardiac micromuscle composition is 80% iCMs-20%hcFb. iCMs are identified based on the Cardiac Troponin T positive expression (red) and hcFb based on the Vimentin positive expression (yellow). Related to Fig. 5.

Supplementary Video 6

Brightfield videos of the cardiac MPS showing the displacement vectors used to estimate the peak twitch amplitude before LNP delivery. Related to Fig. 5.

Supplementary Video 7

Brightfield videos of the cardiac MPS showing the displacement vectors used to estimate the peak twitch amplitude 4 days after LNP delivery. Related to Fig. 5.

Supplementary Video 8

Infarcted mouse heart following intramyocardial injection of 2 kDa 10% ADP-LNP/CRE mRNA. Related to Fig. 7.

Supplementary Video 9

Non-infarcted mouse heart following intramyocardial injection of 2 kDa 10% ADP-LNP/CRE mRNA. Related to Fig. 7.

Supplementary Video 10

Luciferase mRNA was used as a control in infarcted mouse hearts. Related to Fig. 7.

Supplementary Video 11

Luciferase mRNA was used as a control in non-infarcted mouse hearts. Related to Fig. 7.

Supplementary Video 12

Representative slice view showing ZsGreen1 fluorescence signal throughout the non-infarcted mouse heart. Related to Fig. 7.

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Neiman, G., Costa, M.W., Han, H. et al. A microphysiological system for screening lipid nanoparticle−mRNA complexes predicts in vivo heart transfection efficacy. Nat. Biomed. Eng (2025). https://doi.org/10.1038/s41551-025-01523-4

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Received: 28 February 2024
Accepted: 29 August 2025
Published: 03 November 2025
Version of record: 03 November 2025
DOI: https://doi.org/10.1038/s41551-025-01523-4