Малоинвазивная диагностика рака легкого на основе анализа внеклеточной микроРНК крови

Введение. Одной из причин высокой смертности больных раком легкого (РЛ) является нехватка высокочувствительных диагностических маркеров этого заболевания. в качестве таковых могут быть предложены маркеры генетических и эпигенетических процессов, характерных для опухолевых клеток, например микроРНК. известно, что внеклеточная/циркулирующая  микроРНК биологических жидкостей в комплексах с белками или упакованная во внеклеточные везикулы представляет интерес для диагностики опухолевых заболеваний.

Цель исследования – выполнить сравнительный анализ экспрессии микроРНК в составе внеклеточных везикул и супернатанта плазмы крови больных РЛ и доноров и предложить на основании полученных результатов диагностическую панель для выявления пациентов с данной патологией.

Материалы и методы. из образцов крови доноров и больных РЛ методом последовательного центрифугирования была получена плазма крови. Затем из части супернатанта плазмы методом агрегации – осаждения полиэтиленгликолем/синим декстраном выделена фракция внеклеточных везикул (размером 40–150 нм). из обеих собранных фракций плазмы крови больных РЛ и доноров с использованием гуанидина изотиоцианата и октановой кислоты получены микроРНК. экспрессия 17 микроРНК, участвующих в механизмах развития РЛ, по нашим данным и данным литературы, в вышеупомянутых фракциях плазмы крови была проанализирована методом петлевой полимеразной цепной реакции с обратной транскрипцией.

Результаты. В ходе исследования во фракциях внеклеточных везикул и супернатанта плазмы крови обнаружены 29 и 10 пар микроРНК соответственно, экспрессия которых достоверно различалась между больными немелкоклеточным РЛ и донорами. Таким образом, внеклеточные везикулы плазмы обладают большим потенциалом с точки зрения диагностики РЛ на основе оценки относительной экспрессии микроРНК по сравнению с плазмой крови. Разработан диагностический алгоритм, основанный на исследовании аберрантной экспрессии 8 различных микроРНК (miRNA-30e, -1, -125b, -133, -222, -374, -425, -660) в составе 6 пар, позволяющий выявить немелкоклеточный РЛ II–IV стадии в 100 % случаев.

Заключение. внеклеточные везикулы являются более перспективными, диагностически значимыми микроРНК по сравнению с микроРНК плазмы крови. для диагностики больных немелкоклеточным РЛ предложена панель из 8 микроРНК, характеризующаяся 100 % чувствительностью и специфичностью.

1. Sung H., Ferlay J., Siegel R.L. et al. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2021;71(3):209–49. DOI: 10.3322/caac.21660

2. Eberhardt W.E., Pöttgen C., Gauler T.C. et al. Phase III study of surgery versus definitive concurrent chemoradiotherapy boost in patients with resectable stage IIIA(N2) and selected IIIB non-small-cell lung cancer after induction chemotherapy and concurrent chemoradiotherapy (ESPATUE). J Clin Oncol 2015;33(35):4194–201. DOI: 10.1200/JCO.2015.62.6812

3. Chang K., Wang H. The cancer genome atlas pan-cancer analysis project. Nat Genet 2013;45(10):1113–20. DOI: 10.3779/j.issn.1009-3419.2015.04.02

4. Sempere L.F., Azmi A.S., Moore A. microRNA-based diagnostic and therapeutic applications in cancer medicine. Wiley Interdiscip Rev RNA 2021;12(6):e1662. DOI: 10.1002/wrna.1662

5. Dhawan A., Scott J.G., Harris A.L., Buffa F.M. Pan-cancer characterisation of microRNA across cancer hallmarks reveals microRNA-mediated downregulation of tumour suppressors. Nat Commun 2018;9(1):5228. DOI: 10.1038/s41467-018-07657-1

6. He B., Zhao Z., Cai Q. et al. miRNA-based biomarkers, therapies, and resistance in cancer. Int J Biol Sci 2020;16(14):2628–47. DOI: 10.7150/ijbs.4720

7. Fredsøe J., Rasmussen A.K.I., Thomsen A.R. et al. Diagnostic and prognostic microRNA biomarkers for prostate cancer in cell-free urine. Eur Urol Focus 2018;4(6):825–33. DOI: 10.1016/j.euf.2017.02.018

8. Hu C., Meiners S., Lukas C. et al. Role of exosomal microRNAs in lung cancer biology and clinical applications. Cell Prolif 2020;53(6):e12828. DOI: 10.1111/cpr.12828

9. Yi M., Liao Z., Deng L. et al. High diagnostic value of miRNAs for NSCLC: quantitative analysis for both single and combined miRNAs in lung cancer. Ann Med 2021;53(1):2178–93. DOI: 10.1080/07853890.2021.2000634

10. Gao S., Guo W., Liu T. et al. Plasma extracellular vesicle microRNA profiling and the identification of a diagnostic signature for stage I lung adenocarcinoma. Cancer Sci 2022;113(2):648–59. DOI: 10.1111/cas.15222

11. Osipov I.D., Zaporozhchenko I.A., Bondar A.A. et al. Cell-free miRNA-141 and miRNA-205 as prostate cancer biomarkers. Adv Exp Med Biol 2016;924:9–12. DOI: 10.1007/978-3-319-42044-8_2

12. Zaporozhchenko I.A., Morozkin E.S., Ponomaryova A.A. et al. Profiling of 179 miRNA expression in blood plasma of lung cancer patients and cancer-free individuals. Sci Rep 2018;8(1):6348. DOI: 10.1038/s41598-018-24769-2

13. Li C., Yin Y., Liu X. et al. Non-small cell lung cancer associated microRNA expression signature: integrated bioinformatics analysis, validation and clinical significance. Oncotarget 2017;8(15):24564–78. DOI: 10.18632/oncotarget.15596

14. Huang W., Huang W., Hu J. et al. Two microRNA panels to discriminate three subtypes of lung carcinoma in bronchial brushing specimens. Am J Respir Crit Care Med 2012;186(11):1160–7. DOI: 10.1164/rccm.201203-0534OC

15. Wu C., Cao Y., He Z. et al. Serum levels of miR-19b and miR-146a as prognostic biomarkers for non-small cell lung cancer. Tohoku J Exp Med 2014;232(2):85–95. DOI: 10.1620/tjem.232.85

16. Boeri M., Verri C., Conte D. et al. MicroRNA signatures in tissues and plasma predict development and prognosis of computed tomography detected lung cancer. Proc Natl Acad Sci USA 2011;108(9):3713–8. DOI: 10.1073/pnas.1100048108

17. Zhou X., Wen W., Shan X. et al. A six-microRNA panel in plasma was identified as a potential biomarker for lung adenocarcinoma diagnosis. Oncotarget 2016;8(4):6513–25. DOI: 10.18632/oncotarget.14311

18. Liu M., Gao J., Huang Q. et al. Downregulating microRNA-144 mediates a metabolic shift in lung cancer cells by regulating GLUT1 expression. Oncol Lett 2016;11(6):3772–6. DOI: 10.3892/ol.2016.4468

19. Pan H.L., Wen Z.S., Huang Y.C. et al. Down-regulation of microRNA-144 in air pollution-related lung cancer. Sci Rep 2015;5:14331. DOI: 10.1038/srep14331

20. Zhang G., An H., Fang X. MicroRNA-144 regulates proliferation, invasion, and apoptosis of cells in malignant solitary pulmonary nodule via zinc finger E-box-binding homeobox 1. Int J Clin Exp Pathol 2015;8(5):5960–7.

21. Ling B., Wang G.X., Long G. et al. Tumor suppressor miR-22 suppresses lung cancer cell progression through post-transcriptional regulation of ErbB3. J Cancer Res Clin Oncol 2012;138(8):1355–61. DOI: 10.1007/s00432-012-1194-2

22. Xin M., Qiao Z., Li J. et al. miR-22 inhibits tumor growth and metastasis by targeting ATP citrate lyase: evidence in osteosarcoma, prostate cancer, cervical cancer and lung cancer. Oncotarget 2014;7(28):44252–65. DOI: 10.18632/oncotarget.10020

23. Hosseini S.A., Seidi M., Yaghoobi H. Designed miR-19a/b sponge induces apoptosis in lung cancer cells through the PI3K-PTEN-Akt pathway regulation. Mol Biol Rep 2022;49(9):8485–93. DOI: 10.1007/s11033-022-07670-0

24. Zhang X., Wang X., Chai B. et al. Downregulated miR-18a and miR-92a synergistically suppress non-small cell lung cancer via targeting Sprouty 4. Bioengineered 2022;13(4):11281–95. DOI: 10.1080/21655979.2022.2066755

25. Reis P.P., Drigo S.A., Carvalho R.F. et al. Circulating miR-16-5p, miR-92a-3p, and miR-451a in plasma from lung cancer patients: potential application in early detection and a regulatory role in tumorigenesis pathways. Cancers 2020;12(8):2071. DOI: 10.3390/cancers12082071

26. Wang Z., Pan L., Yang L. et al. Long non-coding RNA GATA6-AS1 sponges miR-324-5p to inhibit lung cancer cell proliferation and invasion. Onco Targets Ther 2020;13:9741–51. DOI: 10.2147/OTT.S256336

27. Zhang Y., Xu H. Serum exosomal miR-378 upregulation is associated with poor prognosis in non-small-cell lung cancer patients. J Clin Lab Anal 2020;34(6):e23237. DOI: 10.1002/jcla.23237

28. Ji K.X., Cui F., Qu D. et al. MiR-378 promotes the cell proliferation of non-small cell lung cancer by inhibiting FOXG1. Eur Rev Med Pharmacol Sci 2018;22(4):1011–9. DOI: 10.26355/eurrev_201802_14383

29. Cao X., Zhong W., Guo S. et al. Low expression of miR-27b in serum exosomes of non-small cell lung cancer facilitates its progression by affecting EGFR. Open Med (Wars) 2022;17(1):816–25. DOI: 10.1515/med-2022-0472

30. Zhong S., Golpon H., Zardo P. et al. miRNAs in lung cancer. A systematic review identifies predictive and prognostic miRNA candidates for precision medicine in lung cancer. Transl Res 2021;230:164–96. DOI: 10.1016/j.trsl.2020.11.012

31. Konoshenko M.Y., Lekchnov E.A., Bryzgunova O.E. et al. Isolation of extracellular vesicles from biological fluids via the aggregation-precipitation approach for downstream miRNAs detection. Diagnostics 2012;11(3):384. DOI: 10.3390/diagnostics11030384

32. Lekchnov E.A., Zaporozhchenko I.A., Morozkin E.S. et al. Protocol for miRNA isolation from biofluids. Anal Biochem 2016;499:78–84. DOI: 10.1016/j.ab.2016.01.025

33. Konoshenko M.Y., Lekchnov E.A., Bryzgunova O.E. et al. The panel of 12 cell-Free microRNAs as potential biomarkers in prostate neoplasms. Diagnostics 2020;10(1):38. DOI: 10.3390/diagnostics10010038

34. Landoni E., Miceli R., Callari M. et al. Proposal of supervised data analysis strategy of plasma miRNAs from hybridisation array data with an application to assess hemolysis-related deregulation. BMC Bioinformatics 2015;16:388. DOI: 10.1186/s12859-015-0820-9

35. Bryzgunova O.E., Zaporozhchenko I.A., Lekchnov E.A. et al. Data analysis algorithm for the development of extracellular miRNA-based diagnostic systems for prostate cancer. PLoS One 2019;14(4):e0215003. DOI: 10.1371/journal.pone.0215003

36. Bryzgunova O.E., Konoshenko M.Y., Zaporozhchenko I.A. et al. Isolation of cell-free miRNA from biological fluids: influencing factors and methods. Diagnostics (Basel) 2021;11(5):865. DOI: 10.3390/diagnostics11050865

37. Dejima H., Iinuma H., Kanaoka R. et al. Exosomal microRNA in plasma as a non-invasive biomarker for the recurrence of non-small cell lung cancer. Oncol Lett 2017;13(3):1256–63. DOI: 10.3892/ol.2017.5569

38. Kanaoka R., Iinuma H., Dejima H. et al. Usefulness of plasma exosomal microRNA-451a as a noninvasive biomarker for early prediction of recurrence and prognosis of non-small cell lung cancer. Oncology 2018;94(5):311–23. DOI: 10.1159/000487006

39. Liu Q., Yu Z., Yuan S. et al. Circulating exosomal microRNAs as prognostic biomarkers for non-small-cell lung cancer. Oncotarget 2017;8(8):13048–58. DOI: 10.18632/oncotarget.14369

40. Munagala R., Aqil F., Gupta R.C. Exosomal miRNAs as biomarkers of recurrent lung cancer. Tumor Biol 2016;37(8):10703–14. DOI: 10.1007/s13277-016-4939-8

41. Silva J., García V., Zaballos Á. et al. Vesicle-related microRNAs in plasma of nonsmall cell lung cancer patients and correlation with survival. Eur Respir J 2011;37(3):617–23. DOI: 10.1183/09031936.00029610

42. Zhuang L., Shou T., Li K. et al. MicroRNA-30e-5p promotes cell growth by targeting PTPN13 and indicates poor survival and recurrence in lung adenocarcinoma. J Cell Mol Med 2017;21(11):2852–62. DOI: 10.1111/jcmm.13198

43. Li W., Yang P., Zhong C. et al. The circ-PITX1 promotes non-small cell lung cancer development via the miR-30e-5p/ITGA6 axis. Cell Cycle 2022;21(3):304–21. DOI: 10.1080/15384101.2021.2020041

44. Dong W., Zhang H., Dai Y. et al. circRNA circFAT1(e2) Elevates the development of non-small-cell lung cancer by regulating miR-30e-5p and USP22. BioMed Res Int 2021;2021:6653387. DOI: 10.1155/2021/6653387

45. Gao W., Shen H., Liu L. et al. MiR-21 overexpression in human primary squamous cell lung carcinoma is associated with poor patient prognosis. J Cancer Res Clin Oncol 2011;137(4):557–66. DOI:10.1007/s00432-010-0918-4

46. Ning Z.Q., Lu H.L., Chen C. et al. MicroRNA-30e reduces cell growth and enhances drug sensitivity to gefitinib in lung carcinoma. Oncotarget 2017;8(3):4572–81. DOI: 10.18632/oncotarget.13944

47. Silva J., García V., Zaballos Á. et al. Vesicle-related microRNAs in plasma of nonsmall cell lung cancer patients and correlation with survival. Eur Respir J 2011;37(3):617–23. DOI: 10.1183/09031936.00029610

48. Turchinovich A., Burwinkel B. Distinct AGO1 and AGO2 associated miRNA profiles in human cells and blood plasma. RNA Biol 2012;9(8):1066–75. DOI: 10.4161/rna.21083

49. Lunavat T.R., Lesley C., Dae-Kyum K. et al. Small RNA deep sequencing discriminates subsets of extracellular vesicles released by melanoma cells – evidence of unique microRNA cargos. RNA Biol 2015;12(8):810–23. DOI: 10.1080/15476286.2015.1056975

50. Huang X., Tiezheng Y., Tschannen M. et al. Characterization of human plasma-derived exosomal RNAs by deep sequencing. BMC Genomics 2013;14:319. DOI: 10.1186/1471-2164-14-319

51. Markou A., Sourvinou I., Vorkas P.A. et al. Clinical evaluation of microRNA expression profiling in non small cell lung cancer. Lung Cancer 2013;81(3):388–96. DOI: 10.1016/j.lungcan.2013.05.007

52. Cui Y., Zhao L., Zhao S. et al. MicroRNA-30e inhibits proliferation and invasion of non-small cell lung cancer via targeting SOX9. Human Cell 2019;32(3):326–33. DOI: 10.1007/s13577-018-0223-0

53. Xu G., Cai J., Wang L. et al. MicroRNA-30e-5p suppresses non-small cell lung cancer tumorigenesis by regulating USP22-mediated Sirt1/JAK/STAT3 signaling. Exp Cell Res 2018;362(2):268–78. DOI: 10.1016/j.yexcr.2017.11.027

54. He J., Jin S., Zhang W. et al. Long non-coding RNA LOC554202 promotes acquired gefitinib resistance in non-small cell lung cancer through upregulating miR-31 expression. J Cancer 2019;10(24):6003–13. DOI: 10.7150/jca.35097

55. Liu X., Sempere L.F., Ouyang H. et al. MicroRNA-31 functions as an oncogenic microRNA in mouse and human lung cancer cells by repressing specific tumor suppressors. J Clin Invest 2010;120(4):1298–309. DOI: 10.1172/JCI39566

56. Zhu C., Wang S., Zheng M. et al. miR-31-5p modulates cell progression in lung adenocarcinoma through TNS1/p53 axis. Strahlenther Onkol 2022;198(3):304–14. DOI: 10.1007/s00066-021-01895-x

57. Hou C., Sun B., Jiang Y. et al. MicroRNA-31 inhibits lung adenocarcinoma stem-like cells via down-regulation of MET-PI3K-Akt signaling pathway. Anticancer Agents Med Chem 2016;16(4):501–18. DOI: 10.2174/1871520615666150824152353

58. Zhang J., Li D., Zhang Y. et al. Integrative analysis of mRNA and miRNA expression profiles reveals seven potential diagnostic biomarkers for non-small cell lung cancer. Oncol Rep 2020;43(1):99–112. DOI: 10.3892/or.2019.7407

59. Cheng X., Sha M., Jiang W. et al. LINC00174 suppresses non-small cell lung cancer progression by up-regulating LATS2 via sponging miR-31-5p. Cell J 2022;24(3):140–7. DOI: 10.22074/cellj.2022.7991

60. Wang Y., Shang S., Yu K. et al. miR-224, miR-147b and miR-31 associated with lymph node metastasis and prognosis for lung adenocarcinoma by regulating PRPF4B, WDR82 or NR3C2. Peer J 2020;8:e9704. DOI: 10.7717/peerj.9704

61. Meng W., Ye Z., Cui R. et al. MicroRNA-31 predicts the presence of lymph node metastases and survival in patients with lung adenocarcinoma. Clin Cancer Res 2013;19(19):5423–33. DOI: 10.1158/1078-0432.CCR-13-0320

62. Zeng X., Liu D., Peng G. et al. MiroRNA-31-3p promotes the invasion and metastasis of non-small-cell lung cancer cells by targeting forkhead Box 1 (FOXO1). Comput Math Methods Med 2022;2022:4597087. DOI: 10.1155/2022/4597087

63. Sun X., Zhang S., Ma X. Prognostic value of microRNA-125 in various human malignant neoplasms: a meta-analysis Clin Lab 2015;61(11):1667–74. DOI: 10.7754/clin.lab.2015.150408

64. Kazempour Dizaji M., Farzanegan B., Bahrami N. et al. Expression of miRNA1, miRNA133, miRNA191, and miRNA24, as good biomarkers, in non-small cell lung cancer using real-time PCR method. Asian Pac J Cancer Prev 2022;23(5):1565–70. DOI: 10.31557/APJCP.2022.23.5.1565

65. Liu S., Chen J., Zhang T. et al. MicroRNA-133 inhibits the growth and metastasis of the human lung cancer cells by targeting epidermal growth factor receptor. J BUON 2012;24(3):929–35.

66. Liu G., Li Y.I., Gao X. Overexpression of microRNA-133b sensitizes non-small cell lung cancer cells to irradiation through the inhibition of glycolysis. Oncol Lett 2016;11(4):2903–8. DOI: 10.3892/ol.2016.4316

67. Wei G., Xu Y., Peng T. et al. miR-133 involves in lung adenocarcinoma cell metastasis by targeting FLOT2. Artif Cells Nanomed Biotechnol 2018;46(2):224–30. DOI: 10.1080/21691401.2017.1324467

68. Xiao B., Liu H., Gu Z. et al. Expression of microRNA-133 inhibits epithelial-mesenchymal transition in lung cancer cells by directly targeting FOXQ1. Arch Bronconeumol 2016;52(10):505–11. DOI: 10.1016/j.arbres.2015.10.016

69. Xu M., Wang Y.Z. miR-133a suppresses cell proliferation, migration and invasion in human lung cancer by targeting MMP-14. Oncol Rep 2013;30(3):1398–404. DOI: 10.3892/or.2013.2548

70. Peinado P., Andrades A., Martorell-Marugán J. et al. The SWI/SNF complex regulates the expression of miR-222, a tumor suppressor microRNA in lung adenocarcinoma. Human Mol Gen 2021;30(23):2263–71. DOI: 10.1093/hmg/ddab187

71. Wu Q., Yu L., Lin X. et al. Combination of serum miRNAs with serum exosomal miRNAs in early diagnosis for non-small-cell lung cancer. Cancer Manag Res 2020;12:485–95. DOI: 10.2147/CMAR.S232383

72. Chen W., Li X. MiR-222-3p promotes cell proliferation and inhibits apoptosis by targeting PUMA (BBC3) in non-small cell lung cancer. Technol Cancer Res Treat 2020;19:1533033820922558. DOI: 10.1177/1533033820922558

73. Garofalo M., Romano G., Di Leva G. et al. EGFR and MET receptor tyrosine kinase-altered microRNA expression induces tumorigenesis and gefitinib resistance in lung cancers. Nat Med 2011;18:74–82. DOI: 10.1038/nm.2577

74. Mao K.P., Zhang W.N., Liang X.M. et al. MicroRNA-222 expression and its prognostic potential in non-small cell lung cancer. Sci World J 2014;908326. DOI: 10.1155/2014/908326

75. Zhong C., Ding S., Xu Y. et al. MicroRNA-222 promotes human non-small cell lung cancer H460 growth by targeting p27. Int J Clin Exp Med 2015;8(4):5534–40.

76. Sun Q., Jiang C.W., Tan Z.H. et al. MiR-222 promotes proliferation, migration and invasion of lung adenocarcinoma cells by targeting ETS1. Eur Rev Med Pharmacol Sci 2017;21(10):2385–91.

77. Hetta H.F., Zahran A.M., El-Mahdy R.I. et al. Assessment of circulating miRNA-17 and miRNA-222 expression profiles as non-invasive biomarkers in egyptian patients with non-small-cell lung cancer. Asian Pac J Cancer Prev 2019;20(6):1927–33. DOI: 10.31557/APJCP.2019.20.6.1927

78. Kim Y., Sim J., Kim H. et al. MicroRNA-374a expression as a prognostic biomarker in lung adenocarcinoma. J Pathol Transl Med 2019;53(6):354–60. DOI: 10.4132/jptm.2019.10.01

79. Wang G., Ji X., Li P. et al. Human bone marrow mesenchymal stem cell-derived exosomes containing microRNA-425 promote migration, invasion and lung metastasis by down-regulating CPEB1. Regen Ther 2022;20:107–16. DOI: 10.1016/j.reth.2022.03.007

80. Guo Z., Ye H., Zheng X. et al. Extracellular vesicle-encapsulated microRNA-425-derived from drug-resistant cells promotes non-small-cell lung cancer progression through DAPK1-medicated PI3K/AKT pathway. J Cell Physiol 2021;236(5):3808–20. DOI: 10.1002/jcp.30126

81. Zhou J.S., Yang Z.S., Cheng S.Y. et al. miRNA-425-5p enhances lung cancer growth via the PTEN/PI3K/AKT signaling axis. BMC Pulm Med 2020;20(1):223. DOI: 10.1186/s12890-020-01261-0

82. Jiang L., Ge W., Geng J. miR-425 regulates cell proliferation, migration and apoptosis by targeting AMPH-1 in non-small-cell lung cancer. Pathol Res Pract 2019;215(12):152705. DOI: 10.1016/j.prp.2019.152705.

83. Fu Y., Li Y., Wang X. et al. Overexpression of miR-425-5p is associated with poor prognosis and tumor progression in non-small cell lung cancer. Cancer Biomark 2020;27(2):147–56. DOI: 10.3233/cbm-190782

84. Yuwen D., Ma Y., Wang D. et al. Prognostic role of circulating exosomal miR-425-3p for the response of NSCLC to platinum-based chemotherapy. Cancer Epidemiol Biomark Prevent 2019;28(1):163–73. DOI: 10.1158/1055-9965.epi-18-0569

85. Fortunato O., Boeri M., Moro M. et al. Mir-660 is downregulated in lung cancer patients and its replacement inhibits lung tumorigenesis by targeting MDM2-p53 interaction. Cell Death Dis 2014;5(12):e1564. DOI: 10.1038/cddis.2014.507

86. Qi Y., Zha W., Zhang W. Exosomal miR-660-5p promotes tumor growth and metastasis in non-small cell lung cancer. J BUON 2019;24(2):599–607.

87. Moro M., Di Paolo D., Milione M. et al. Coated cationic lipid-nanoparticles entrapping miR-660 inhibit tumor growth in patient-derived xenografts lung cancer models. J Control Release 2019;308:44–56. DOI: 10.1016/j.jconrel.2019.07.006