Hpv E6/E7 Silencing and Ferroptosis Modulation in Cervical Cancer: in-Vitro Evaluation Of a Biomimetic Co-Delivery Platform

Authors

  • Archana Rautela
  • Jitendra Kumar Gupta
  • Navneet Kumar

Keywords:

Biomimetic nanocarrier, E6/E7-siRNA, RSL3; polymeric nanoparticle, co-delivery, Quality by Design, Design of Experiments, formulation development, critical quality attributes, pharmaceutics

Abstract

The simultaneous delivery of a nucleic-acid therapeutic and a small-molecule ferroptosis modulator presents a distinct
pharmaceutics challenge because the two cargos differ in size, charge, hydrophilicity and loading requirements. HPV E6/E7-
siRNA requires protection from degradation and efficient intracellular availability, whereas RSL3 is incorporated within the
polymeric carrier to provide a formulation-compatible nanoscale delivery approach.
Objective:To develop and optimize a biomimetic nanocarrier for co-delivery of HPV E6/E7-siRNA and RSL3 using a
Quality by Design (QbD) and Design of Experiments (DoE) framework, and to establish the physicochemical and functional
performance of the optimized formulation.
Methods:A QbD strategy comprising QTPP definition, CQA identification, formulation/process risk consideration and an
eight-run, three-factor DoE was applied. The formulation consisted of a PLGA-based nanoparticle core containing RSL3,
E6/E7-siRNA associated with the nanoparticle, stabilizing components and a biomimetic membrane coating. The optimized
formulation was evaluated for particle size, PDI, zeta potential, siRNA association, RSL3 loading, morphology and cellular
delivery, followed by functional assessment of E6/E7 silencing and ferroptosis-associated responses.
Results:The optimized biomimetic nanocarrier exhibited a particle size of 108 ± 6 nm, PDI of 0.19 ± 0.02, zeta potential of
+17.7 ± 1.7 mV, E6/E7-siRNA association of 85.4 ± 2.6%, RSL3 loading of 77.8 ± 2.4% and pH of 8.3 ± 0.1. The formulation
showed enhanced intracellular delivery compared with free cargo, reduced E6/E7 expression and produced a co-loaded
formulation-associated increase in lipid ROS with reduction in GSH/GSSG and changes in GPX4, SLC7A11 and ACSL4.
Conclusion:The study establishes a pharmaceutics-oriented QbD pathway for development of an E6/E7-siRNA–RSL3 coloaded biomimetic nanocarrier. The formulation-development sequence, nanoscale characteristics, cargo association/loading
and functional delivery provide a basis for further formulation refinement and biological validation.

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References

1. zur Hausen, H. (2002). Papillomaviruses and cancer: From basic studies to clinical application. Nature Reviews Cancer, 2(5), 342–350. https://doi.org/10.1038/nrc798

2. Schiffman, M., Castle, P. E., Jeronimo, J., Rodriguez, A. C., & Wacholder, S. (2007). Human papillomavirus and cervical cancer. The Lancet, 370(9590), 890–907. https://doi.org/10.1016/S0140-6736(07)61416-0

3. de Sanjosé, S., Quint, W. G. V., Alemany, L., Geraets, D. T., Klaustermeier, J. E., Lloveras, B., Tous, S., Felix, A., Alejo, M., Bigby, S. M., et al. (2010). Human papillomavirus genotype attribution in invasive cervical cancer: A retrospective cross-sectional worldwide study. The Lancet Oncology, 11(11), 1048–1056. https://doi.org/10.1016/S1470-2045(10)70230-8

4. Bouvard, V., Baan, R., Straif, K., Grosse, Y., Secretan, B., El Ghissassi, F., Benbrahim-Tallaa, L., Guha, N., Freeman, C., Galichet, L., & Cogliano, V. (2009). A review of human carcinogens—Part B: Biological agents. The Lancet Oncology, 10(4), 321–322. https://doi.org/10.1016/S1470-2045(09)70096-8

5. Doorbar, J. (2006). Molecular biology of human papillomavirus infection and cervical cancer. Clinical Science, 110(5), 525–541. https://doi.org/10.1042/CS20050230

6. McLaughlin-Drubin, M. E., & Münger, K. (2009). The human papillomavirus E7 oncoprotein. Virology, 384(2), 335–344. https://doi.org/10.1016/j.virol.2008.10.006

7. Vande Pol, S. B., & Klingelhutz, A. J. (2013). Papillomavirus E6 oncoproteins. Virology, 445(1–2), 115–137. https://doi.org/10.1016/j.virol.2013.04.026

8. Roman, A., & Munger, K. (2013). The papillomavirus E7 proteins. Virology, 445(1–2), 138–168. https://doi.org/10.1016/j.virol.2013.04.013

9. Moody, C. A., & Laimins, L. A. (2010). Human papillomavirus oncoproteins: Pathways to transformation. Nature Reviews Cancer, 10(8), 550–560. https://doi.org/10.1038/nrc2886

10. Howley, P. M., & Pfister, H. J. (2015). Beta genus papillomaviruses and skin cancer. Virology, 479–480, 290–296. https://doi.org/10.1016/j.virol.2015.02.004

11. Hebner, C., & Laimins, L. A. (2006). Human papillomaviruses: Basic mechanisms of pathogenesis and oncogenicity. Reviews in Medical Virology, 16(2), 83–97. https://doi.org/10.1002/rmv.488

12. Mantovani, F., & Banks, L. (2001). The human papillomavirus E6 protein and its contribution to malignant progression. Oncogene, 20(54), 7874–7887. https://doi.org/10.1038/sj.onc.1204869

13. Doorbar, J. (2005). The papillomavirus life cycle. Journal of Clinical Virology, 32(Suppl. 1), S7–S15. https://doi.org/10.1016/j.jcv.2004.12.006

14. Münger, K., Baldwin, A., Edwards, K. M., Hayakawa, H., Nguyen, C. L., Owens, M., Grace, M., & Huh, K. (2004). Mechanisms of human papillomavirus-induced oncogenesis. Journal of Virology, 78(21), 11451–11460. https://doi.org/10.1128/JVI.78.21.11451-11460.2004

15. Tommasino, M. (2014). The human papillomavirus family and its role in carcinogenesis. Seminars in Cancer Biology, 26, 13–21. https://doi.org/10.1016/j.semcancer.2013.11.002

16. McBride, A. A. (2017). Oncogenic human papillomaviruses. Philosophical Transactions of the Royal Society B: Biological Sciences, 372(1732), 20160273. https://doi.org/10.1098/rstb.2016.0273

17. Yuan, H., Fu, F., Hu, S., Wang, Y., Yu, M., Zhang, X., & Cheng, T. (2009). Human papillomavirus infection and cervical cancer: Molecular mechanisms and therapeutic implications. Cancer Letters, 273(2), 269–277.

18. Liu, X., Clements, A., Zhao, W., & Papadopoulos, V. (2010). HPV E6, E7 and the molecular mechanisms of cervical carcinogenesis. Cancer Biology & Therapy, 10(2), 109–115.

19. Li, Y., & Roberts, S. (2014). Human papillomavirus 16 E1 and E2 proteins: Regulation of viral replication and cellular responses. Viruses, 6(1), 35–53.

20. Grace, M., & Munger, K. (2017). Proteomic analysis of the HPV16 E7 oncoprotein and its interaction with cellular proteins. Virology Journal, 14, 76.

21. Hanahan, D., & Weinberg, R. A. (2011). Hallmarks of cancer: The next generation. Cell, 144(5), 646–674. https://doi.org/10.1016/j.cell.2011.02.013

22. Batlle, E., & Clevers, H. (2017). Cancer stem cells revisited. Nature Medicine, 23(10), 1124–1134. https://doi.org/10.1038/nm.4409

23. Clevers, H. (2011). The cancer stem cell: Premises, promises and challenges. Nature Medicine, 17(3), 313–319. https://doi.org/10.1038/nm.2304

24. Visvader, J. E., & Lindeman, G. J. (2012). Cancer stem cells: Current status and evolving complexities. Cell Stem Cell, 10(6), 717–728. https://doi.org/10.1016/j.stem.2012.05.007

25. Lathia, J. D., Mack, S. C., Mulkearns-Hubert, E. E., Valentim, C. L. L., & Rich, J. N. (2015). Cancer stem cells in glioblastoma. Genes & Development, 29(12), 1203–1217. https://doi.org/10.1101/gad.261982.115

26. Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J., & Clarke, M. F. (2003). Prospective identification of tumorigenic breast cancer cells. Proceedings of the National Academy of Sciences, 100(7), 3983–3988. https://doi.org/10.1073/pnas.0530291100

27. Singh, S. K., Hawkins, C., Clarke, I. D., Squire, J. A., Bayani, J., Hide, T., Henkelman, R. M., Cusimano, M. D., & Dirks, P. B. (2004). Identification of human brain tumour initiating cells. Nature, 432(7015), 396–401. https://doi.org/10.1038/nature03128

28. Ginestier, C., Hur, M. H., Charafe-Jauffret, E., Monville, F., Dutcher, J., Brown, M., Jacquemier, J., Viens, L., Kleer, C. G., Liu, S., Schott, A., Hayes, D., Birnbaum, D., Wicha, M. S., & Dontu, G. (2007). ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical prognosis. Cell Stem Cell, 1(5), 555–567. https://doi.org/10.1016/j.stem.2007.08.014

29. Clarke, M. F., & Fuller, M. (2006). Stem cells and cancer: Two faces of eve. Cell, 124(6), 1111–1115. https://doi.org/10.1016/j.cell.2006.03.011

30. Plaks, V., Kong, N., & Werb, Z. (2015). The cancer stem cell niche: How essential is the niche? Cell Stem Cell, 16(3), 225–238. https://doi.org/10.1016/j.stem.2015.02.015

31. Stockwell, B. R. (2021). Ferroptosis turns 10: Emerging mechanisms, physiological functions, and therapeutic applications. Cell, 185(14), 2401–2421. https://doi.org/10.1016/j.cell.2022.06.003

32. Dixon, S. J., & Stockwell, B. R. (2019). The hallmarks of ferroptosis. Annual Review of Cancer Biology, 3, 35–54. https://doi.org/10.1146/annurev-cancerbio-030518-055844

33. Dixon, S. J., Patel, D. N., Welsch, M., Skouta, R., Lee, E. D., Hayano, M., Thomas, A. G., Gleason, C. E., Tatonetti, N. P., Slusher, B. S., & Stockwell, B. R. (2014). Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis. eLife, 3, e02523. https://doi.org/10.7554/eLife.02523

34. Yang, W. S., & Stockwell, B. R. (2008). Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death. Chemistry & Biology, 15(3), 234–245. https://doi.org/10.1016/j.chembiol.2008.02.010

35. Yang, W. S., Kim, K. J., Gaschler, M. M., Patel, M., Shchepinov, M. S., & Stockwell, B. R. (2016). Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis. Cell, 165(6), 1545–1554. https://doi.org/10.1016/j.cell.2016.05.010

36. Kagan, V. E., Mao, G., Qu, F., Angeli, J. P. F., Doll, S., Croix, C. M. S., Dar, H. H., Liu, B., Tyurin, V. A., Tyurina, Y. Y., Jiang, J., Kapralov, A. A., Amoscato, A. A., Yang, Q., Proneth, B., Klein-Seetharaman, J., Watkins, S. C., Bahar, I., Greenberger, J. S., Mallampalli, R. K., Stockwell, B. R., & Bayir, H. (2017). Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis. Nature Chemical Biology, 13(1), 81–90. https://doi.org/10.1038/nchembio.2238

37. Viswanathan, V. S., Ryan, M. J., Dhruv, H. D., Gill, S., Eickhoff, O. M., Seashore-Ludlow, B., Kaffenberger, S. D., Eaton, J. K., Shimada, K., Aguirre, A. J., et al. (2017). Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway. Nature, 547(7664), 453–457. https://doi.org/10.1038/nature23007

38. Dixon, S. J., Winter, G. E., Musavi, L. S., Lee, E. D., Snijder, B., Rebsamen, M., Superti-Furga, G., & Stockwell, B. R. (2015). Human haploid cell genetics reveals roles for lipid metabolism genes in nonapoptotic cell death. ACS Chemical Biology, 10(7), 1604–1609. https://doi.org/10.1021/acschembio.5b00339

39. Conrad, M., Angeli, J. P. F., Vandenabeele, P., & Stockwell, B. R. (2016). Regulated necrosis: Disease relevance and therapeutic opportunities. Nature Reviews Drug Discovery, 15(5), 348–358. https://doi.org/10.1038/nrd.2015.6

40. Feng, H., Schorpp, K., Jin, J., Yozamp, N., Brachya, G., Bar-Ziv, R., Färkkilä, A., Taya, M., Annamalai, R., Yamauchi, Y., et al. (2020). Transferrin receptor is a specific ferroptosis marker. Cell Reports, 30(10), 3411–3423.e7. https://doi.org/10.1016/j.celrep.2020.02.049

41. Walboomers, J. M. M., Jacobs, M. V., Manos, M. M., Bosch, F. X., Kummer, J. A., Shah, K. V., Snijders, P. J. F., Peto, J., Meijer, C. J. L. M., & Muñoz, N. (1999). Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. The Journal of Pathology, 189(1), 12–19. https://doi.org/10.1002/(SICI)1096-9896(199909)189:1<12: AID-PATH431>3.0.CO;2-F PubMed

42. Scheffner, M., Werness, B. A., Huibregtse, J. M., Levine, A. J., & Howley, P. M. (1990). The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell, 63(6), 1129–1136. https://doi.org/10.1016/0092-8674(90)90409-8 PubMed

43. Dyson, N., Howley, P. M., Münger, K., & Harlow, E. (1989). The human papillomavirus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science, 243(4893), 934–937. https://doi.org/10.1126/science.2537532 NCBI

44. Feng, D., Peng, C., Li, C., Zhou, Y., Li, M., Ling, B., Wei, H., & Tian, Z. (2009). Identification and characterization of cancer stem-like cells from primary carcinoma of the cervix uteri. Oncology Reports, 22(5), 1129–1134. https://doi.org/10.3892/or_00000545 PubMed

45. Tyagi, A., Vishnoi, K., Mahata, S., Verma, G., Srivastava, Y., Masaldan, S., Roy, B. G., Bharti, A. C., & Das, B. C. (2016). Cervical cancer stem cells selectively overexpress HPV oncoprotein E6 that controls stemness and self-renewal through upregulation of HES1. Clinical Cancer Research, 22(16), 4170–4184. https://doi.org/10.1158/1078-0432.CCR-15-2574 PubMed

46. Dixon, S. J., Lemberg, K. M., Lamprecht, M. R., Skouta, R., Zaitsev, E. M., Gleason, C. E., Patel, D. N., Bauer, A. J., Cantley, A. M., Yang, W. S., Morrison, B., III, & Stockwell, B. R. (2012). Ferroptosis: An iron-dependent form of nonapoptotic cell death. Cell, 149(5), 1060–1072. https://doi.org/10.1016/j.cell.2012.03.042 PubMed

47. Jiang, X., Stockwell, B. R., & Conrad, M. (2021). Ferroptosis: Mechanisms, biology and role in disease. Nature Reviews Molecular Cell Biology, 22(4), 266–282. https://doi.org/10.1038/s41580-020-00324-8 PubMed

48. Doll, S., Proneth, B., Tyurina, Y. Y., Panzilius, E., Kobayashi, S., Ingold, I., Irmler, M., Beckers, J., Aichler, M., Walch, A., Prokisch, H., Trümbach, D., Mao, G., Qu, F., Bayir, H., Füllekrug, J., Scheel, C. H., Wurst, W., Schick, J. A., Friedmann Angeli, J. P., & Conrad, M. (2017). ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition. Nature Chemical Biology, 13(1), 91–98. https://doi.org/10.1038/nchembio.2239 PubMed

49. Zhang, P., An, K., Duan, X., Xu, H., Li, F., & Xu, F. (2018). Recent advances in siRNA delivery for cancer therapy using smart nanocarriers. Drug Discovery Today, 23(4), 900–911. https://doi.org/10.1016/j.drudis.2018.01.042 PubMed

50. Subhan, M. A., & Torchilin, V. P. (2019). Efficient nanocarriers of siRNA therapeutics for cancer treatment. Translational Research, 214, 62–91. https://doi.org/10.1016/j.trsl.2019.07.006 PubMed

51. Zhao, J., & Feng, S. S. (2015). Nanocarriers for delivery of siRNA and co-delivery of siRNA and other therapeutic agents. Nanomedicine, 10(14), 2199–2228. https://doi.org/10.2217/nnm.15.61 PubMed

52. Kandasamy, G., & Maity, D. (2021). Current advancements in self-assembling nanocarriers-based siRNA delivery for cancer therapy. Colloids and Surfaces B: Bio interfaces, 221, 113002. https://doi.org/10.1016/j.colsurfb.2022.113002 PubMed

53. Biomimetic cell membrane-coated nanocarriers for targeted siRNA delivery in cancer therapy. (2021). PubMed. PMID: 36736580. PubMed

54. Davari, M., Salarizadeh, N., Hushmandi, K., et al. (2021). Advances in nucleic acid delivery using nanocarriers for gene therapy in cancer: A narrative review. Critical Reviews in Oncology/Hematology, 223, 105367. https://doi.org/10.1016/j.critrevonc.2026.105367

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Published

2026-10-03

How to Cite

1.
Rautela A, Gupta JK, Kumar N. Hpv E6/E7 Silencing and Ferroptosis Modulation in Cervical Cancer: in-Vitro Evaluation Of a Biomimetic Co-Delivery Platform. J Neonatal Surg [Internet]. 2026 Oct. 3 [cited 2026 Oct. 14];13(1):2649-76. Available from: https://jneonatalsurg.com/index.php/jns/article/view/10629

Issue

Vol. 13 (2024): Journal of Neonatal Surgery

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Original Article

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