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Precise surface functionalization of PLGA particles for human T cell modulation

Nature Protocols volume 18, pages 3289–3321 (2023)Cite this article

Subjects

Abstract

The biofunctionalization of synthetic materials has extensive utility for biomedical applications, but approaches to bioconjugation typically show insufficient efficiency and controllability. We recently developed an approach by building synthetic DNA scaffolds on biomaterial surfaces that enables the precise control of cargo density and ratio, thus improving the assembly and organization of functional cargos. We used this approach to show that the modulation and phenotypic adaptation of immune cells can be regulated using our precisely functionalized biomaterials. Here, we describe the three key procedures, including the fabrication of polymeric particles engrafted with short DNA scaffolds, the attachment of functional cargos with complementary DNA strands, and the surface assembly control and quantification. We also explain the critical checkpoints needed to ensure the overall quality and expected characteristics of the biological product. We provide additional experimental design considerations for modifying the approach by varying the material composition, size or cargo types. As an example, we cover the use of the protocol for human primary T cell activation and for the identification of parameters that affect ex vivo T cell manufacturing. The protocol requires users with diverse expertise ranging from synthetic materials to bioconjugation chemistry to immunology. The fabrication procedures and validation assays to design high-fidelity DNA-scaffolded biomaterials typically require 8 d.

Key points

  • The protocol describes the fabrication of DNA scaffolds, the bioconjugation of biomolecules with complementary DNAs, conjugate assembly onto the DNA scaffolds and their immunomodulatory effect on primary human T cells in culture.

  • Steric hindrance typically limits the use of orthogonal chemistry and covalent surface attachment strategies, whereas this DNA hybridization-based approach maintains control over the loading of each biomolecule species.

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Fig. 1: Schematic of the fabrication protocol for precision ICEps.
Fig. 2: Quality control of PLGA particles with dense DNA scaffolds.
Fig. 3: Protocol and quality checkpoints of Ab–DNA conjugation and purification.
Fig. 4: Density and ratiometric control of cargos co-loaded onto particle surfaces.
Fig. 5: ICEp activation of human T cells and their phenotypic characterization.

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Data availability

Any raw data that supports the plots within this paper are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

The authors thank Z. Gartner and B. Moser for relevant DNA synthesis, S. Douglas and K. Shen for gel imaging, W. Lim for sonication equipment, the University of California, San Francisco (UCSF) Nikon Imaging center for confocal microscopy, and V. Nguyen and the UCSF Flow Cytometry Core (RRID: SCR_018206) for their flow cytometry expertise and equipment use—funded in part by the Diabetes Research Center via the National Institutes of Health (NIH) grant P30 DK063720. P.H. was supported by the UCSF Medical Scientist Training Program training grant T32GM141323 via the National Institute of General Medical Sciences (NIGMS). X.H. acknowledges the start-up fund provided by the School of Biomedical Engineering, Science, and Health Systems at Drexel University. This work was also partially funded by the UCSF Diabetes Center Pilot and Feasibility award funded in part by NIH grant P30 DK063720 (P.H., T.D. and Q.T.), NIH grant 1U54CA244438 (T.D.), the Northern California JDRF Center of Excellence 5-COE-2019-860-S-B (Q.T.) and the JDRF grant 2-SRA-2022-1221-S-B (T.D.). We thank E. Ronin and P. Ho for helpful discussion and trainings. We also thank E. Hansen for help with protocol validation and assistance.

Author information

Authors and Affiliations

  1. Medical Scientist Training Program, University of California, San Francisco, CA, USA

    Pierce Hadley

  2. UC Berkeley–UCSF Graduate Program in Bioengineering, UCSF Mission Bay Campus, San Francisco, CA, USA

    Pierce Hadley & Tejal Desai

  3. Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA, USA

    Pierce Hadley, Yuanzhou Chen, Lariana Cline, Zhiyuan Han, Xiao Huang & Tejal Desai

  4. Diabetes Center, University of California, San Francisco, CA, USA

    Pierce Hadley, Qizhi Tang & Tejal Desai

  5. Gladstone–UCSF Institute of Genomic Immunology, San Francisco, CA, USA

    Pierce Hadley & Qizhi Tang

  6. Cell Design Institute and Center for Synthetic Immunology, University of California, San Francisco, CA, USA

    Yuanzhou Chen, Xiao Huang & Tejal Desai

  7. Department of Surgery, University of California, San Francisco, CA, USA

    Qizhi Tang

  8. School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA, USA

    Xiao Huang

  9. School of Engineering, Brown University, Providence, RI, USA

    Tejal Desai

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Contributions

P.H. and X.H. designed the experiments and drafted the manuscript. P.H. and L.C. contributed to the experiments in Figs. 2–5. Y.C. and Z.H. contributed to the experiments in Fig. 2. P.H. analyzed the data. All authors contributed to the editing of the manuscript.

Corresponding authors

Correspondence to Xiao Huang or Tejal Desai.

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Competing interests

T.D. and X.H. are inventors of a pending patent related to the technology described within this manuscript (WO2020014270A1). Q.T. is a co-founder, shareholder and scientific advisor of Sonoma Biotherapeutics. P.H., Y.C., Z.H. and L.C. declare no competing interests.

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Related links

Key reference using this protocol

Huang, X. et al. Nat. Nanotechnol. 16, 214–223 (2021): https://doi.org/10.1038/s41565-020-00813-z

Supplementary information

Reporting Summary

Supplementary Table

Supplementary Table 1. Example reaction template for DNA−PLGA conjugation.

Source data

Source Data Fig. 2

Uncropped urea–PAGE gels for Fig. 2b–d.

Source Data Fig. 3

Uncropped SDS–PAGE gel for Fig. 3b, urea–PAGE gel used for quantification in Fig. 3c and urea–PAGE gel for Fig. 3e.

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Hadley, P., Chen, Y., Cline, L. et al. Precise surface functionalization of PLGA particles for human T cell modulation. Nat Protoc 18, 3289–3321 (2023). https://doi.org/10.1038/s41596-023-00887-8

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