Abstract
The present study was designed to test the hypothesis that programmed cell death-1 (PD-1) siRNA can downregulate PD-1 expression in macrophages in culture and in tumor tissues in mice and inhibit tumor growth in a mouse model. PD-1 siRNA was encapsulated in solid lipid nanoparticles (SLNs), and the physical properties of the resultant SLNs were characterized. The ability of the PD-1 siRNA-SLNs to downregulate PD-1 expression was confirmed in J774A.1 macrophages in culture and in tumor tissues in mice. Moreover, the antitumor activity of the PD-1 siRNA-SLNs was evaluated in a mouse model. The PD-1 siRNA-SLNs were roughly spherical, and their particle size, polydispersity index, and zeta potential were 141 ± 5 nm, 0.17 ± 0.02, and 20.7 ± 4.7 mV, respectively, with an siRNA entrapment efficiency of 98.9%. The burst release of the PD-1 siRNA from the SLNs was minimal. The PD-1 siRNA-SLNs downregulated PD-1 expression on J774A.1 macrophage cell surface as well as in macrophages in B16-F10 tumors pre-established in mice. In mice with pre-established B16-F10 tumors, the PD-1 siRNA-SLNs significantly inhibited the tumor growth, as compared with siRNA-SLNs prepared with non-functional, negative control siRNA. In conclusion, the PD-1 siRNA-SLNs inhibited tumor growth, likely related to their ability to downregulate PD-1 expression by tumor-associated macrophage (TAMs).
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References
Salmaninejad A, Valilou SF, Shabgah AG, Aslani S, Alimardani M, Pasdar A, et al. PD-1/PD-L1 pathway: basic biology and role in cancer immunotherapy. J Cell Physiol. 2019;234(10):16824–37. https://doi.org/10.1002/jcp.28358.
Jiang X, Wang J, Deng X, Xiong F, Ge J, Xiang B, et al. Role of the tumor microenvironment in PD-L1/PD-1-mediated tumor immune escape. Mol Cancer. 2019;18(1):10. https://doi.org/10.1186/s12943-018-0928-4.
LaFleur MW, Muroyama Y, Drake CG, Sharpe AH. Inhibitors of the PD-1 pathway in tumor therapy. J Immunol. 2018;200(2):375–83. https://doi.org/10.4049/jimmunol.1701044.
Cui C, Yu B, Jiang Q, Li X, Shi K, Yang Z. The roles of PD-1/PD-L1 and its signalling pathway in gastrointestinal tract cancers. Clin Exp Pharmacol Physiol. 2019;46(1):3–10. https://doi.org/10.1111/1440-1681.13028.
Sun X, Zhang T, Li M, Yin L, Xue J. Immunosuppressive B cells expressing PD-1/PD-L1 in solid tumors: a mini review. Qjm. 2019. https://doi.org/10.1093/qjmed/hcz162.
Cai J, Qi Q, Qian X, Han J, Zhu X, Zhang Q, et al. The role of PD-1/PD-L1 axis and macrophage in the progression and treatment of cancer. J Cancer Res Clin Oncol. 2019;145(6):1377–85. https://doi.org/10.1007/s00432-019-02879-2.
Mariotti FR, Petrini S, Ingegnere T, Tumino N, Besi F, Scordamaglia F, et al. PD-1 in human NK cells: evidence of cytoplasmic mRNA and protein expression. Oncoimmunology. 2019;8(3):1557030. https://doi.org/10.1080/2162402x.2018.1557030.
Gong J, Chehrazi-Raffle A, Reddi S, Salgia R. Development of PD-1 and PD-L1 inhibitors as a form of cancer immunotherapy: a comprehensive review of registration trials and future considerations. J Immunother Cancer. 2018;6(1):8. https://doi.org/10.1186/s40425-018-0316-z.
Shergold AL, Millar R, Nibbs RJB. Understanding and overcoming the resistance of cancer to PD-1/PD-L1 blockade. Pharmacol Res. 2019;145:104258. https://doi.org/10.1016/j.phrs.2019.104258.
Killock D. Immunotherapy: macrophages hijack anti-PD-1 therapy. Nat Rev Clin Oncol. 2017;14(7):394. https://doi.org/10.1038/nrclinonc.2017.79.
Simon B, Harrer DC, Schuler-Thurner B, Schaft N, Schuler G, Dorrie J, et al. The siRNA-mediated downregulation of PD-1 alone or simultaneously with CTLA-4 shows enhanced in vitro CAR-T-cell functionality for further clinical development towards the potential use in immunotherapy of melanoma. Exp Dermatol. 2018;27(7):769–78. https://doi.org/10.1111/exd.13678.
Burvenich IJG, Parakh S, Parslow AC, Lee ST, Gan HK, Scott AM. Receptor occupancy imaging studies in oncology drug development. AAPS J. 2018;20(2):43. https://doi.org/10.1208/s12248-018-0203-z.
Yoo B, Jordan VC, Sheedy P, Billig AM, Ross A, Pantazopoulos P, et al. RNAi-mediated PD-L1 inhibition for pancreatic cancer immunotherapy. Sci Rep. 2019;9(1):4712. https://doi.org/10.1038/s41598-019-41251-9.
Gordon SR, Maute RL, Dulken BW, Hutter G, George BM, McCracken MN, et al. PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity. Nature. 2017;545(7655):495–9. https://doi.org/10.1038/nature22396.
Bucana CD, Fabra A, Sanchez R, Fidler IJ. Different patterns of macrophage infiltration into allogeneic-murine and xenogeneic-human neoplasms growing in nude mice. Am J Pathol. 1992;141(5):1225–36.
Cao Q, Yan X, Chen K, Huang Q, Melancon MP, Lopez G, et al. Macrophages as a potential tumor-microenvironment target for noninvasive imaging of early response to anticancer therapy. Biomaterials. 2018;152:63–76. https://doi.org/10.1016/j.biomaterials.2017.10.036.
Daldrup-Link HE, Golovko D, Ruffell B, Denardo DG, Castaneda R, Ansari C, et al. MRI of tumor-associated macrophages with clinically applicable iron oxide nanoparticles. Clinical cancer research: an official journal of the American Association for Cancer Research. 2011;17(17):5695–704. https://doi.org/10.1158/1078-0432.CCR-10-3420.
Erel-Akbaba G, Carvalho LA, Tian T, Zinter M, Akbaba H, Obeid PJ, et al. Radiation-induced targeted nanoparticle-based gene delivery for brain tumor therapy. ACS Nano. 2019;13(4):4028–40.
Wu Y, Gu W, Li L, Chen C, Xu ZP. Enhancing PD-1 gene silence in T lymphocytes by comparing the delivery performance of two inorganic nanoparticle platforms. Nanomaterials (Basel). 2019;9(2). https://doi.org/10.3390/nano9020159.
Ligtenberg MA, Pico de Coana Y, Shmushkovich T, Yoshimoto Y, Truxova I, Yang Y, et al. Self-delivering RNAi targeting PD-1 improves tumor-specific T cell functionality for adoptive cell therapy of malignant melanoma. Mol Ther 2018;26(6):1482–1493. https://doi.org/10.1016/j.ymthe.2018.04.015.
Wu Y, Gu W, Li J, Chen C, Xu ZP. Silencing PD-1 and PD-L1 with nanoparticle-delivered small interfering RNA increases cytotoxicity of tumor-infiltrating lymphocytes. Nanomedicine (Lond). 2019;14(8):955–67. https://doi.org/10.2217/nnm-2018-0237.
Kwak SY, Lee S, Han HD, Chang S, Kim KP, Ahn HJ. PLGA nanoparticles codelivering siRNAs against programmed cell death protein-1 and its ligand gene for suppression of colon tumor growth. Mol Pharm. 2019;16:4940–53. https://doi.org/10.1021/acs.molpharmaceut.9b00826.
Zhao T, Wei T, Guo J, Wang Y, Shi X, Guo S, et al. PD-1-siRNA delivered by attenuated Salmonella enhances the antimelanoma effect of pimozide. Cell Death Dis. 2019;10(3):164. https://doi.org/10.1038/s41419-019-1418-3.
Zhao T, Feng Y, Guo M, Zhang C, Wu Q, Chen J, et al. Combination of attenuated Salmonella carrying PD-1 siRNA with nifuroxazide for colon cancer therapy. J Cell Biochem. 2020;121(2):1973–85. https://doi.org/10.1002/jcb.29432.
Celec P, Gardlik R. Gene therapy using bacterial vectors. Front Biosci (Landmark Ed). 2017;22:81–95. https://doi.org/10.2741/4473.
Kulkarni JA, Witzigmann D, Chen S, Cullis PR, van der Meel R. Lipid nanoparticle technology for clinical translation of siRNA therapeutics. Acc Chem Res. 2019;52(9):2435–44. https://doi.org/10.1021/acs.accounts.9b00368.
Akinc A, Maier MA, Manoharan M, Fitzgerald K, Jayaraman M, Barros S, et al. The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs. Nat Nanotechnol. 2019;14(12):1084–7. https://doi.org/10.1038/s41565-019-0591-y.
Aldayel AM, O'Mary HL, Valdes SA, Li X, Thakkar SG, Mustafa BE, et al. Lipid nanoparticles with minimum burst release of TNF-alpha siRNA show strong activity against rheumatoid arthritis unresponsive to methotrexate. J Control Release. 2018;283:280–9. https://doi.org/10.1016/j.jconrel.2018.05.035.
Kleffel S, Posch C, Barthel SR, Mueller H, Schlapbach C, Guenova E, et al. Melanoma cell-intrinsic PD-1 receptor functions promote tumor growth. Cell. 2015;162(6):1242–56. https://doi.org/10.1016/j.cell.2015.08.052.
Hussein MR. Tumour-associated macrophages and melanoma tumourigenesis: integrating the complexity. Int J Exp Pathol. 2006;87(3):163–76. https://doi.org/10.1111/j.1365-2613.2006.00478.x.
Zhu S, Wonganan P, Lansakara PD, O'Mary HL, Li Y, Cui Z. The effect of the acid-sensitivity of 4-(N)-stearoyl gemcitabine-loaded micelles on drug resistance caused by RRM1 overexpression. Biomaterials. 2013;34(9):2327–39. https://doi.org/10.1016/j.biomaterials.2012.11.053.
Boedtkjer E, Pedersen SF. The acidic tumor microenvironment as a driver of cancer. Annu Rev Physiol. 2020;82:103–26. https://doi.org/10.1146/annurev-physiol-021119-034627.
Fennelly C, Amaravadi RK. Lysosomal biology in cancer. Methods Mol Biol. 2017;1594:293–308. https://doi.org/10.1007/978-1-4939-6934-0_19.
Dhupkar P, Gordon N, Stewart J, Kleinerman ES. Anti-PD-1 therapy redirects macrophages from an M2 to an M1 phenotype inducing regression of OS lung metastases. Cancer Med. 2018;7(6):2654–64. https://doi.org/10.1002/cam4.1518.
Pahl JH, Kwappenberg KM, Varypataki EM, Santos SJ, Kuijjer ML, Mohamed S, et al. Macrophages inhibit human osteosarcoma cell growth after activation with the bacterial cell wall derivative liposomal muramyl tripeptide in combination with interferon-gamma. J Exp Clin Cancer Res. 2014;33:27. https://doi.org/10.1186/1756-9966-33-27.
Hsu J, Hodgins JJ, Marathe M, Nicolai CJ, Bourgeois-Daigneault MC, Trevino TN, et al. Contribution of NK cells to immunotherapy mediated by PD-1/PD-L1 blockade. J Clin Invest. 2018;128(10):4654–68. https://doi.org/10.1172/jci99317.
Acknowledgments
This work was supported in part by an Egyptian Government Scholarship with bench fees (MSH), the Mannino Fellowship in Pharmacy at UT Austin (ZC), a UT Austin-Portugal CoLab project (ZC), and Via Therapeutics, LLC (through NIH R43AR074360 to JJK).
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ZC declares conflict of interest with Via Therapeutics, LLC, which has been reviewed and approved by UT Austin in accordance with its policy on objectivity in research.
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Hanafy, M.S., Hufnagel, S., Trementozzi, A.N. et al. PD-1 siRNA-Encapsulated Solid Lipid Nanoparticles Downregulate PD-1 Expression by Macrophages and Inhibit Tumor Growth. AAPS PharmSciTech 22, 60 (2021). https://doi.org/10.1208/s12249-021-01933-y
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DOI: https://doi.org/10.1208/s12249-021-01933-y