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Abnormal scar identification with spherical-nucleic-acid technology

Abstract

The accurate diagnosis of scar type and severity relies on histopathology of biopsied tissue, which is invasive and time-consuming, causes discomfort and may exacerbate scarring. Here, we show that imaging nanoprobes for the live-cell detection of intracellular messenger RNA (mRNA) (also known as NanoFlares) enable measurements of the expression of connective tissue growth factor (CTGF) as a visual indicator of hypertrophic scars and keloids. During cell culture, NanoFlares enabled the distinction of hypertrophic and keloidal fibroblasts from normal fibroblasts, and the detection of changes in CTGF expression resulting from the regulatory effects of transforming growth factor-β (TGF-β) agonists and TGF-β antagonists. We also applied the NanoFlares topically to the skin of live mice and rabbits, and to ex vivo human skin models. Transepidermal penetration of the NanoFlares enabled the visual and spectroscopic quantification of underlying abnormal fibroblasts on the basis of CTGF mRNA expression. Our proof-of-concept studies of topically applied NanoFlare technology as a means of biopsy-free scar diagnosis may eventually inform therapeutic decisions on the basis of the mRNA-expression patterns of skin disorders.

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Fig. 1: In vitro assessment of NanoFlare (NF) specificity to target mRNA.
Fig. 2: Discriminating fibroblasts by CTGF expression levels using NanoFlares (NF).
Fig. 3: Monitoring TGF-β-induced CTGF expression changes with NanoFlares.
Fig. 4: NanoFlares (NFs) as a rapid screening assay to determine the mechanism of anti-hypertrophic scar drug candidates.
Fig. 5: Non-invasive detection of NanoFlare signals in live mice.
Fig. 6: Topically applied NanoFlare detection of abnormal scar cells within ex vivo skin.
Fig. 7: NanoFlare detection of abnormal scar cells in a rabbit ear wound model.
Fig. 8: Diagnosis of rabbit ear abnormal scars with CTGF NanoFlares.

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References

  1. Van de Vijver, M. J. et al. A gene-expression signature as a predictor of survival in breast cancer. N. Engl. J. Med. 347, 1999–2009 (2002).

    Article  CAS  PubMed  Google Scholar 

  2. Yao, M. et al. Gene expression analysis of renal carcinoma: adipose differentiation-related protein as a potential diagnostic and prognostic biomarker for clear-cell renal carcinoma. J. Pathol. 205, 377–387 (2005).

    Article  CAS  PubMed  Google Scholar 

  3. Zakrewsky, M., Kumar, S. & Mitragotri, S. Nucleic acid delivery into skin for the treatment of skin disease: proofs-of-concept, potential impact, and remaining challenges. J. Control. Release 219, 445–456 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Prigodich, A. E. et al. Multiplexed nanoflares: mRNA detection in live cells. Anal. Chem. 84, 2062–2066 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Halo, T. L. et al. NanoFlares for the detection, isolation, and culture of live tumor cells from human blood. Proc. Natl Acad. Sci. USA 111, 17104–17109 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Giljohann, D. A. et al. Gold nanoparticles for biology and medicine. Angew. Chem. Int. Ed. 49, 3280–3294 (2010).

    Article  CAS  Google Scholar 

  7. Chinen, A. B. et al. Nanoparticle probes for the detection of cancer biomarkers, cells, and tissues by fluorescence. Chem. Rev. 115, 10530–10574 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Yang, Y. et al. FRET nanoflares for intracellular mRNA detection: avoiding false positive signals and minimizing effects of system fluctuations. J. Am. Chem. Soc. 137, 8340–8343 (2015).

    Article  CAS  PubMed  Google Scholar 

  9. Lahm, H. et al. Live fluorescent RNA-based detection of pluripotency gene expression in embryonic and induced pluripotent stem cells of different species. Stem Cells 33, 392–402 (2015).

    Article  CAS  PubMed  Google Scholar 

  10. Choi, C. H. J., Hao, L., Narayan, S. P., Auyeung, E. & Mirkin, C. A. Mechanism for the endocytosis of spherical nucleic acid nanoparticle conjugates. Proc. Natl Acad. Sci. USA 110, 7625–7630 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wu, X. A., Choi, C. H. J., Zhang, C., Hao, L. & Mirkin, C. A. Intracellular fate of spherical nucleic acid nanoparticle conjugates. J. Am. Chem. Soc. 136, 7726–7733 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Jensen, S. A. et al. Spherical nucleic acid nanoparticle conjugates as an RNAi-based therapy for glioblastoma. Sci. Transl. Med. 5, 209ra152 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Randeria, P. S. et al. siRNA-based spherical nucleic acids reverse impaired wound healing in diabetic mice by ganglioside GM3 synthase knockdown. Proc. Natl Acad. Sci. USA 112, 5573–5578 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Sita, T. L. et al. Dual bioluminescence and near-infrared fluorescence monitoring to evaluate spherical nucleic acid nanoconjugate activity in vivo. Proc. Natl Acad. Sci. USA 114, 4129–4134 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Zheng, D. et al. Topical delivery of siRNA-based spherical nucleic acid nanoparticle conjugates for gene regulation. Proc. Natl Acad. Sci. USA 109, 11975–11980 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Alster, T. S. & Tanzi, E. L. Hypertrophic scars and keloids: etiology and management. Am. J. Clin. Dermatol. 4, 235–243 (2003).

    Article  PubMed  Google Scholar 

  17. Gold, M. H. et al. Updated international clinical recommendations on scar management: part 2—algorithms for scar prevention and treatment. Dermatol. Surg. 40, 825–831 (2014).

    CAS  PubMed  Google Scholar 

  18. Wolfram, D., Tzankov, A., Pülzl, P. & Piza-Katzer, H. Hypertrophic scars and keloids—a review of their pathophysiology, risk factors, and therapeutic management. Dermatol. Surg. 35, 171–181 (2009).

    Article  CAS  PubMed  Google Scholar 

  19. Sarrazy, V., Billet, F., Micallef, L., Coulomb, B. & Desmoulière, A. Mechanisms of pathological scarring: role of myofibroblasts and current developments. Wound Repair Regen. 19, s10–s15 (2011).

    Article  PubMed  Google Scholar 

  20. Van der Veer, W. M. et al. Potential cellular and molecular causes of hypertrophic scar formation. Burns 35, 15–29 (2009).

    Article  PubMed  Google Scholar 

  21. Grotendorst, G. R. Connective tissue growth factor: a mediator of TGF-beta action on fibroblasts. Cytokine Growth Factor Rev. 8, 171–179 (1997).

    Article  CAS  PubMed  Google Scholar 

  22. Leask, A. & Abraham, D. J. TGF-beta signaling and the fibrotic response. FASEB J. 18, 816–827 (2004).

    Article  CAS  PubMed  Google Scholar 

  23. Wang, Y. W. et al. siRNA-targeting transforming growth factor-beta type I receptor reduces wound scarring and extracellular matrix deposition of scar tissue. J. Investig. Dermatol. 134, 2016–2025 (2014).

    Article  CAS  PubMed  Google Scholar 

  24. Zhang, Z. et al. Recombinant human decorin inhibits TGF-β1-induced contraction of collagen lattice by hypertrophic scar fibroblasts. Burns 35, 527–537 (2009).

    Article  PubMed  Google Scholar 

  25. Wong, V. W., You, F., Januszyk, M., Gurtner, G. C. & Kuang, A. A. Transcriptional profiling of rapamycin-treated fibroblasts from hypertrophic and keloid scars. Ann. Plast. Surg. 72, 711–719 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Armendariz-Borunda, J. et al. A controlled clinical trial with pirfenidone in the treatment of pathological skin scarring caused by burns in pediatric patients. Ann. Plast. Surg. 68, 22–28 (2012).

    Article  CAS  PubMed  Google Scholar 

  27. Ichida, J. K. et al. A small-molecule inhibitor of TGF-beta signaling replaces sox2 in reprogramming by inducing nanog. Cell. Stem Cell. 5, 491–503 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Lin, T. et al. A chemical platform for improved induction of human iPSCs. Nat. Methods 6, 805–808 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Nestle, F. O., Di Meglio, P., Qin, J. Z. & Nickoloff, B. J. Skin immune sentinels in health and disease. Nat. Rev. Immunol. 9, 679–691 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kua, E. H. J., Goh, C. Q., Ting, Y., Chua, A. & Song, C. Comparing the use of glycerol preserved and cryopreserved allogenic skin for the treatment of severe burns: differences in clinical outcomes and in vitro tissue viability. Cell Tissue Bank. 13, 269–279 (2012).

    Article  CAS  PubMed  Google Scholar 

  31. Pasyk, K. A., Argenta, L. C. & Hassett, C. Quantitative analysis of the thickness of human skin and subcutaneous tissue following controlled expansion with a silicone implant. Plast. Reconstr. Surg. 81, 516–523 (1988).

    Article  CAS  PubMed  Google Scholar 

  32. Kloeters, O., Tandara, A. & Mustoe, T. A. Hypertrophic scar model in the rabbit ear: a reproducible model for studying scar tissue behavior with new observations on silicone gel sheeting for scar reduction. Wound Repair Regen. 15, S40–S45 (2007).

    Article  PubMed  Google Scholar 

  33. Yeo, D. C., Balmayor, E. R., Schantz, J.-T. & Xu, C. Microneedle physical contact as a therapeutic for abnormal scars. Eur. J. Med. Res. 22, 28 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Sellheyer, K. & Bergfeld, W. F. A retrospective biopsy study of the clinical diagnostic accuracy of common skin diseases by different specialties compared with dermatology. J. Am. Acad. Dermatol. 52, 823–830 (2005).

    Article  PubMed  Google Scholar 

  35. Lewandowski, K. T. et al. Topically delivered tumor necrosis factor-α-targeted gene regulation for psoriasis. J. Investig. Dermatol. 137, 2027–2030 (2017).

    Article  CAS  PubMed  Google Scholar 

  36. Avior, Y., Sagi, I. & Benvenisty, N. Pluripotent stem cells in disease modelling and drug discovery. Nat. Rev. Mol. Cell Biol. 17, 170–182 (2016).

    Article  CAS  PubMed  Google Scholar 

  37. Choi, S. M. et al. Efficient drug screening and gene correction for treating liver disease using patient-specific stem cells. Hepatology 57, 2458–2468 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Gammon, S. T., Leevy, W. M., Gross, S., Gokel, G. W. & Piwnica-Worms, D. Spectral unmixing of multicolored bioluminescence emitted from heterogeneous biological sources. Anal. Chem. 78, 1520–1527 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Beziere, N. et al. Dynamic imaging of PEGylated indocyanine green (ICG) liposomes within the tumor microenvironment using multi-spectral optoacoustic tomography (MSOT). Biomaterials 37, 415–424 (2015).

    Article  CAS  PubMed  Google Scholar 

  40. Vujačić, A. et al. Fluorescence quenching of 5,5’-disulfopropyl-3,3’-dichlorothiacyanine dye adsorbed on gold nanoparticles. J. Phys. Chem. C 117, 6567–6577 (2013).

    Article  Google Scholar 

  41. Hayes, A. J. et al. Wide versus narrow excision margins for high-risk, primary cutaneous melanomas: long-term follow-up of survival in a randomised trial. Lancet Oncol. 17, 184–192 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Anselmo, A. C. & Mitragotri, S. Nanoparticles in the clinic. Bioeng. Transl. Med. 1, 10–29 (2016).

    PubMed  PubMed Central  Google Scholar 

  43. Banga, R. J., Chernyak, N., Narayan, S. P., Nguyen, S. T. & Mirkin, C. A. Liposomal spherical nucleic acids. J. Am. Chem. Soc. 136, 9866–9869 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Banga, R. J. et al. Cross-linked micellar spherical nucleic acids from thermoresponsive templates. J. Am. Chem. Soc. 139, 4278–4281 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by the NTU-Northwestern Institute for Nanomedicine. We thank G. Yu (current location: Nanjing University of Posts and Telecommunications, China) for assistance with animal handling.

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Authors

Contributions

C.A.M., A.S.P., C.X. and D.C.Y. conceived and designed the experiments. D.C.Y. and C.W. performed the experiments. D.C.Y., C.W. and C.X. analysed and interpreted the data. D.C.Y., C.X., A.S.P. and C.A.M. wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Amy S. Paller, Chad A. Mirkin or Chenjie Xu.

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Competing interests

A patent application based on the reported data has been filed. C.A.M. is a cofounder of Aurasense (the company that co-developed and licensed the NanoFlare technology to Merck–Millipore, which produced over 1,600 commercial versions of NanoFlares sold under the trade name SmartFlares).

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Yeo, D.C., Wiraja, C., Paller, A.S. et al. Abnormal scar identification with spherical-nucleic-acid technology. Nat Biomed Eng 2, 227–238 (2018). https://doi.org/10.1038/s41551-018-0218-x

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