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Digital micelles of encoded polymeric amphiphiles for direct sequence reading and ex vivo label-free quantification

Nature Chemistry volume 15, pages 257–270 (2023)Cite this article

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Abstract

Identification and quantification of synthetic polymers in complex biological milieu are crucial for delivery, sensing and scaffolding functions, but conventional techniques based on imaging probe labellings only afford qualitative results. Here we report modular construction of precise sequence-defined amphiphilic polymers that self-assemble into digital micelles with contour lengths strictly regulated by oligourethane sequences. Direct sequence reading is accomplished with matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry, facilitated by high-affinity binding of alkali metal ions with poly(ethylene glycol) dendrons and selective cleavage of benzyl-carbamate linkages. A mixture of four types of digital micelles could be identified, sequence-decoded and quantified by MALDI and MALDI imaging at cellular, organ and tissue slice levels upon in vivo administration, enabling direct comparison of biological properties for each type of digital micelle in the same animal. The concept of digital micelles and encoded amphiphiles capable of direct sequencing and high-throughput label-free quantification could be exploited for next-generation precision nanomedicine designs (such as digital lipids) and protein corona studies.

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Fig. 1: Direct sequence reading of encoded polymeric amphiphiles and sequence-regulated self-assembly into digital micelles for label-free ex vivo quantification.
Fig. 2: Modular synthesis of sequence-defined polyurethanes with uniform chain lengths.
Fig. 3: Facile synthesis of sequence-defined polyurethanes via orthogonal isocyanate-generating chemistries.
Fig. 4: Direct reading of sequence-defined polyurethanes via MALDI tandem MS.
Fig. 5: Sequence-regulated self-assembly of encoded polymeric amphiphiles into digital micelles with tunable contour lengths.
Fig. 6: Identification and label-free quantification of digital micelles at organ and tissue slice levels.

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The authors declare that all data supporting the findings of this study are available within the article, the associated source data and its Supporting Information and can also be obtained from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

Financial support from the National Key Research and Development Program of China (2020YFA0710700), the National Natural Science Foundation of China (NNSFC) Project (51690150, 51690154, 52021002, 21925107 and U19A2094) and Fundamental Research Funds for the Central Universities is gratefully acknowledged. This work was partially carried out at the USTC Center for Micro and Nanoscale Research and Fabrication. We thank the staff from the BL19U2 beamline of the National Facility for Protein Science in Shanghai (NFPS) at the Shanghai Synchrotron Radiation Facility for assistance during SAXS data collection.

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Authors and Affiliations

  1. CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, School of Chemistry and Materials Science, Collaborative Innovation Center of Chemistry for Energy Materials (iChEM), University of Science and Technology of China, Hefei, China

    Qiangqiang Shi, Rundi Song, Jie Xu, Jiajia Tan, Xin Zhou, Jie Cen, Zhengyu Deng & Shiyong Liu

  2. Mass Spectrometry Lab, Instruments Center for Physical Science, University of Science and Technology of China, Hefei, China

    Hao Yin

  3. Center for Instrumental Analysis, Xi’an Jiaotong University, Xi’an, China

    Huimin Tong

  4. School of Chemistry, MOE Key Laboratory for Nonequilibrium Synthesis and Modulation of Condensed Matter, Xi’an Jiaotong University, Xi’an, China

    Chenhui Cui & Yanfeng Zhang

  5. College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen, China

    Xiaopeng Li

  6. State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, China

    Zhengbiao Zhang

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  1. Qiangqiang Shi
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Contributions

S.Y.L. conceived and thoroughly supervised the project. S.Y.L. and Q.Q.S. designed the experiments and analysed the data. Q.Q.S. conducted the experiments with assistance from R.D.S., J.X., J.J.T., X.Z. and J.C. MALDI–TOF MS and MALDI imaging experiments were conducted with help from H.Y. and Z.B.Z. Cryo-TEM characterization was conducted with help from H.M.T., C.H.C. and Y.F.Z. ESI-MS characterization and MS data interpretation were conducted with assistance from X.P.L. Q.Q.S., Z.Y.D., X.P.L. and S.Y.L. wrote the manuscript.

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Correspondence to Shiyong Liu.

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Extended data

Extended Data Fig. 1 Sequence reading of encoded amphiphiles.

MALDI tandem MS recorded for T-000′00000-dPEG; the benzyl-O linkage in 0′ unit (pink) remains inert and does not undergo fragmentation, indicating the bond-selective nature.

Source data

Extended Data Fig. 2 Characterization of self-assembled nanorods and quantification of encoded amphiphiles.

a, Sequence-regulated evolution of self-assembled nanorod lengths (n represents the number of digital micelles measured, data are presented as mean values ± SD). b, Standard calibration curves of sequence-defined polyurethanes relative to the control (T-010101-dPEG) for MALDI–TOF MS quantification (n = 3 independent experiments; data are presented as mean values ± SD).

Source data

Extended Data Fig. 3 Comparison of MALDI detection sensitivities for encoded amphiphiles.

a,b, S/N values in MALDI characterization of T-00000000-dPEG and T-11111111-dPEG, and MALDI samples were prepared from unimer (THF solution) and micellar state (aqueous dispersion) at varying concentrations (n = 3 independent experiments; data are presented as mean values ± SEM). c,d, MALDI–TOF mass spectra recorded for T-00000000-dPEG and T-11111111-dPEG; MALDI samples were prepared from unimer (THF solution) and micellar state (aqueous dispersion) at 0.5 μg/mL and 1.0 μg/mL, respectively. e,f, MALDI–TOF mass spectra recorded for T-000000-dPEG in the unimer state (THF solution) and (b) micellar state (aqueous dispersion) after coating onto rat spleen tissue slices and applying with DCTB/CF3COONa MALDI matrix. The negligible MS peak area difference indicated that encoded amphiphiles in the micellar NP state could be detected by MALDI with the sensitivity compared to those in the molecularly dissolved state.

Source data

Extended Data Fig. 4 Characterization of cationic lipid/siRNA/polymer hybrid NPs.

a, Schematics for the fabrication of cationic lipid/siRNA/polymer hybrid NPs assisted by the digital lipid (2C18-0101-OEG16-OH). b, TEM image and c, dynamic laser scattering (DLS) results recorded for hybrid NPs stabilized by 2C18-0101-OEG16-OH.

Source data

Extended Data Fig. 5 Comparison of cationic lipid/siRNA/polymer hybrid NPs stabilized by digital lipid versus that by DSPE-PEG.

a,b, Fluorescence emission spectra recorded for two types of FAM-siRNA-Luc hybrid NP dispersions and corresponding supernatant solutions after initially formed hybrid NPs were subjected ultrafiltration (MWCO, 100 kDa). c, Evolution of DLS sizes upon coincubation with 20% FBS. d,e, Evolution of emission intensities of two types of FAM-siRNA-Luc hybrid NPs upon co-incubation with 20% FBS. f, Uptake extents of FAM-siRNA-Luc hybrid NP stabilized by the digital lipid when co-incubated with BXPC-3 cells, as quantified by MALDI–TOF MS in a label-free manner (n = 3 biologically independent cells; data are presented as mean values ± SEM). g, CLSM images (FAM channel) of BXPC-3 cells upon co-incubation with two types of FAM-siRNA-Luc hybrid NPs, whereas naked FAM-siRNA-Luc was used as a control. Scale bar: 25 μm.

Source data

Extended Data Fig. 6 Biological behaviors of cationic lipid/siRNA/polymer hybrid NPs.

Comparison of luciferase expression levels in Pan 02-Luc cells transfected with FAM-siRAN-Luc hybrid NPs stabilized by either DSPE-PEG or the digital lipid (2C18-0101-OEG16-OH) at varying siRNA concentrations (n = 3 biologically independent cells; data are presented as mean values ± SEM).

Source data

Extended Data Fig. 7 Cell sorting protocols for the spleen organ.

Schematics of the sorting of immune cells (macrophages, T cells, B cells, and dendritic cells) and stromal cells from the spleen organ of BABL/c mice.

Extended Data Fig. 8 Quantification of cellular uptake extents.

Average absolute endocytosis mass of four types of encoded amphiphiles internalized by single stromal cell and each type immune cells (macrophage, T cell, B cell, and dendritic cell), which were sorted from the spleen organ of BABL/c mice sacrificed at a, 1 h and b, 6 h post i.v. injection of a mixture of four types of digital micelles (21 nm nanospheres and 45 nm, 78 nm, and 245 nm nanorods; 11.2 mg/mL in total, 2.8 mg/mL for each type). n.d.: not detected (n = 3 biologically independent animals; data are presented as mean values ± SEM).

Source data

Supplementary information

Supplementary Information

Supplementary Methods, Schemes 1–27, Characterization, Figs. 1–125 and Tables 1–5.

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Readable versions of tables in the Supplementary Information file.

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Shi, Q., Yin, H., Song, R. et al. Digital micelles of encoded polymeric amphiphiles for direct sequence reading and ex vivo label-free quantification. Nat. Chem. 15, 257–270 (2023). https://doi.org/10.1038/s41557-022-01076-y

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