Nanodiamonds are innovative nanocrystalline carbon particles able to deliver chemically conjugated miRNAs. In oncology, the use of miRNA-based therapies may represent an advantage, based on their ability to simultaneously target multiple intracellular oncogenic targets. Here, nanodiamonds were tested and optimized to deliver miR-34a, a miRNA playing a key role in inhibiting tumor development and progression in many cancers. The physical-chemical properties of nanodiamonds were investigated suggesting electrical stability and uniformity of structure and size. Moreover, we evaluated nanodiamond cytotoxicity on two breast cancer cell models and confirmed their excellent biocompatibility. Subsequently, nanodiamonds were conjugated with miR-34a, using the chemical crosslinker polyethyleneimine; real-time PCR analysis revealed a higher level of miR-34a in cancer cells treated with the different formulations of nanodiamonds than with commercial transfectant. A significant and early nanodiamond-miR-34a uptake was recorded by FACS and fluorescence microscopy analysis in MCF7 and MDA-MB-231 cells. Moreover, nanodiamond-miR-34a significantly inhibited both cell proliferation and migration. Finally, a remarkable anti-tumor effect of miR-34a-conjugated nanodiamonds was observed in both heterotopic and orthotopic murine xenograft models. In conclusion, this study provides a rationale for the development of new therapeutic strategies based on use of miR-34a delivered by nanodiamonds to improve the clinical treatment of neoplasms.

Department of Advanced Medical and Surgical Sciences DAMSS University of Campania Luigi Vanvitelli Via S M di Costantinopoli 104 80138 Naples Italy

Department of Mental and Physical Health and Preventive Medicine Pathology Unit University of Campania Luigi Vanvitelli 80138 Naples Italy

Department of Pharmacy University of Naples Federico 2 D Montesano 49 80131 Naples Italy

Department of Precision Medicine University of Campania Luigi Vanvitelli Via L De Crecchio 7 80138 Naples Italy

Institute of Biophysics 2nd Faculty of Medicine Charles University 5 Uvalu 84 15006 Prague Czech Republic

Laboratory of Precision and Molecular Oncology Biogem Scarl Institute of Genetic Research Contrada Camporeale 83031 Ariano Irpino Italy

Translational Oncology Research Unit IRCCS Regina Elena National Cancer Institute E Chianesi 53 00144 Rome Italy

See more in PubMed

Van der Meel R., Sulheim E., Shi Y., Kiessling F., Mulder W.J.M., Lammers T. Smart cancer nanomedicine. Nat. Nanotechnol. 2019;14:1007–1017. PubMed PMC

Mukherjee A., Waters A.K., Kalyan P., Achrol A.S., Kesari S., Yenugonda V.M. Lipid-polymer hybrid nanoparticles as a next-generation drug delivery platform: state of the art, emerging technologies, and perspectives. Int. J. Nanomed. 2019;14:1937–1952. PubMed PMC

Markman J.L., Rekechenetskiy A., Holler E., Ljubimova J.Y. Nanomedicine therapeutic approaches to overcome cancer drug resistance. Adv. Drug Deliv. Rev. 2013;65:1866–1879. PubMed PMC

Giarra S., Zappavigna S., Campani V., Abate M., Cossu A.M., Leonetti C., Porru M., Mayol L., Caraglia M., De Rosa G. Chitosan-Based Polyelectrolyte Complexes for Doxorubicin and Zoledronic Acid Combined Therapy to Overcome Multidrug Resistance. Pharmaceutics. 2018;10:180. PubMed PMC

Sabourian P., Yazdani G., Ashraf S.S., Frounchi M., Mashayekhan S., Kiani S., Kakkar A. Effect of Physico-Chemical Properties of Nanoparticles on Their Intracellular Uptake. Int. J. Mol. Sci. 2020;21:8019. PubMed PMC

Mitchell M.J., Billingsley M.M., Haley R.M., Wechsler M.E., Peppas N.A., Langer R. Engineering precision nanoparticles for drug delivery. Nat. Rev. Drug Discov. 2021;20:101–124. PubMed PMC

Abate M., Scotti L., Nele V., Caraglia M., Biondi M., De Rosa G., Leonetti C., Campani V., Zappavigna S., Porru M. Hybrid Self-Assembling Nanoparticles Encapsulating Zoledronic Acid: A Strategy for Fostering Their Clinical Use. Int. J. Mol. Sci. 2022;23:5138. PubMed PMC

Lee S.W.L., Paoletti C., Campisi M., Osaki T., Adriani G., Kamm R.D., Mattu C., Chiono V. MicroRNA delivery through nanoparticles. J. Contr. Release. 2019;313:80–95. PubMed PMC

Najahi-Missaoui W., Arnold R.D., Cummings B.S. Safe Nanoparticles: Are We There Yet? Int. J. Mol. Sci. 2020;22:385. PubMed PMC

Gao G., Guo Q., Zhi J. Nanodiamond-Based Theranostic Platform for Drug Delivery and Bioimaging. Small. 2019;15 PubMed

Karami P., Salkhi Khasraghi S., Hashemi M., Rabiei S., Shojaei A. Polymer/nanodiamond composites - a comprehensive review from synthesis and fabrication to properties and applications. Adv. Colloid Interface Sci. 2019;269:122–151. PubMed

Bartelmess J., Quinn S.J., Giordani S. Carbon nanomaterials: multi-functional agents for biomedical fluorescence and Raman imaging. Chem. Soc. Rev. 2015;44:4672–4698. PubMed

Moore L., Grobárová V., Shen H., Man H.B., Míčová J., Ledvina M., Štursa J., Nesladek M., Fišerová A., Ho D. Comprehensive interrogation of the cellular response to fluorescent, detonation and functionalized nanodiamonds. Nanoscale. 2014;6:11712–11721. PubMed PMC

Zhu Y., Li J., Li W., Zhang Y., Yang X., Chen N., Sun Y., Zhao Y., Fan C., Huang Q. The biocompatibility of nanodiamonds and their appiication in drug deiivery systems. Theranostics. 2012;2:302–312. PubMed PMC

Torelli M.D., Nunn N.A., Shenderova O.A. A Perspective on Fluorescent Nanodiamond Bioimaging. Small. 2019;15 PubMed PMC

Tinwala H., Wairkar S. Production, surface modification and biomedical applications of nanodiamonds: A sparkling tool for theranostics. Mater. Sci. Eng. C Mater. Biol. Appl. 2019;97:913–931. PubMed

Torelli M.D., Rickard A.G., Backer M.V., Filonov D.S., Nunn N.A., Kinev A.V., Backer J.M., Palmer G.M., Shenderova O.A. Targeting Fluorescent Nanodiamonds to Vascular Endothelial Growth Factor Receptors in Tumor. Bioconjugate Chem. 2019;30:604–613. PubMed PMC

Eldawud R., Reitzig M., Opitz J., Rojansakul Y., Jiang W., Nangia S., Dinu C.Z. Combinatorial approaches to evaluate nanodiamond uptake and induced cellular fate. Nanotechnology. 2016;27 PubMed PMC

Rupaimoole R., Slack F.J. MicroRNA therapeutics: towards a new era for the management of cancer and other diseases. Nat. Rev. Drug Discov. 2017;16:203–222. PubMed

Lu J., Getz G., Miska E.A., Alvarez-Saavedra E., Lamb J., Peck D., Sweet-Cordero A., Ebert B.L., Mak R.H., Ferrando A.A., et al. MicroRNA expression profiles classify human cancers. Nature. 2005;435:834–838. PubMed

Misso G., Di Martino M.T., De Rosa G., Farooqi A.A., Lombardi A., Campani V., Zarone M.R., Gullà A., Tagliaferri P., Tassone P., Caraglia M. Mir-34: a new weapon against cancer? Mol. Ther. Nucleic Acids. 2014;3:e194. PubMed PMC

Zhang L., Liao Y., Tang L. MicroRNA-34 family: a potential tumor suppressor and therapeutic candidate in cancer. J. Exp. Clin. Cancer Res. 2019;38:53. PubMed PMC

Diener C., Keller A., Meese E. Emerging concepts of miRNA therapeutics: from cells to clinic. Trends Genet. 2022;38:613–626. PubMed

Rossi M., Amodio N., Di Martino M.T., Caracciolo D., Tagliaferri P., Tassone P. From target therapy to miRNA therapeutics of human multiple myeloma: theoretical and technological issues in the evolving scenario. Curr. Drug Targets. 2013;14:1144–1149. PubMed

Rossi M., Amodio N., Di Martino M.T., Tagliaferri P., Tassone P., Cho W.C. MicroRNA and multiple myeloma: from laboratory findings to translational therapeutic approaches. Curr. Pharmaceut. Biotechnol. 2014;15:459–467. PubMed

Morelli E., Leone E., Cantafio M.E.G., Di Martino M.T., Amodio N., Biamonte L., Gullà A., Foresta U., Pitari M.R., Botta C., et al. Selective targeting of IRF4 by synthetic microRNA-125b-5p mimics induces anti-multiple myeloma activity in vitro and in vivo. Leukemia. 2015;29:2173–2183. PubMed PMC

Tagliaferri P., Rossi M., Di Martino M.T., Amodio N., Leone E., Gulla A., Neri A., Tassone P. Promises and challenges of MicroRNA-based treatment of multiple myeloma. Curr. Cancer Drug Targets. 2012;12:838–846. PubMed PMC

Lionetti M., Agnelli L., Lombardi L., Tassone P., Neri A. MicroRNAs in the pathobiology of multiple myeloma. Curr. Cancer Drug Targets. 2012;12:823–837. PubMed

Laube C., Oeckinghaus T., Lehnert J., Griebel J., Knolle W., Denisenko A., Kahnt A., Meijer J., Wrachtrup J., Abel B. Controlling the fluorescence properties of nitrogen vacancy centers in nanodiamonds. Nanoscale. 2019;11:1770–1783. PubMed

Huang H., Dai L., Wang D.H., Tan L.S., Osawa E. Large-scale self-assembly of dispersed nanodiamonds. J. Mater. Chem. 2008;18:1347–1352.

Krueger A. New carbon materials: Biological applications of functionalized nanodiamond materials. Chem. Eur J. 2008;14:1382–1390. PubMed

Islam N., Dihingia A., Manna P., Das T., Kalita J., Dekaboruah H.P., Saikia B.K. Environmental and toxicological assessment of nanodiamond-like materials derived from carbonaceous aerosols. Sci. Total Environ. 2019;679:209–220. PubMed

Colvin V.L. The potential environmental impact of engineered nanomaterials. Nat.biotech. 2003;21:1166–1170. PubMed

Jia G., Wang H., Yan L., Wang X., Pei R., Yan T., Zhao Y., Guo X. Cytotoxicity of carbon nanomaterials: Single wall nanotube, multi wall nanotube and fullerene. Environ. Sci. Technol. 2005;39:1378–1383. PubMed

Schrand A.M., Hens S.A.C., Shenderova O.A. Nanodiamond Particles: Properties and Perspectives for Bioapplications. Crit. Rev. Solid State Mater. Sci. 2009;34:18–74.

Burns R.C., Davies G.J. In: Properties of Natural and Synthetic Diamond». Field J.E., editor. Academic Press; 1992. pp. 395–422.

Moustakas T.D. In: Synthetic Diamond: Emerging CVD Science and Technology. Spear K.E., Dismukes J.P., editors. 1994. pp. 145–192.

Dubrovinskaia N., Dubrovinsky L., Solopova N.A., Abakumov A., Turner S., Hanfland M., Bykova E., Bykov M., Prescher C., Prakapenka V.B., et al. Terapascal static pressure generation with ultrahigh yield strength nanodiamond. Sci. Adv. 2016;2 PubMed PMC

Krueger A., Lang D. Functionality is Key: Recent Progress in the Surface Modification of Nanodiamond. Adv. Funct. Mater. 2012;22:890–906.

Patnaik S., Gupta K.C. Novel polyethylenimine-derived nanoparticles for in vivo gene delivery. Expet Opin. Drug Deliv. 2013;10:215–228. PubMed

Guo S., Huang Y., Jiang Q., Sun Y., Deng L., Liang Z., Du Q., Xing J., Zhao Y., Wang P.C., et al. Enhanced gene delivery and siRNA silencing by gold nanoparticles coated with charge-reversal polyelectrolyte. ACS Nano. 2010;4:5505–5511. PubMed PMC

Shahbazi R., Ozpolat B., Ulubayram K. Oligonucleotide-based theranostic nanoparticles in cancer therapy. Nanomedicine. 2016;11:1287–1308. PubMed PMC

Křivohlavá R., Neuhӧferová E., Jakobsen K.Q., Benson V. Knockdown of microRNA-135b in Mammary Carcinoma by Targeted Nanodiamonds: Potentials and Pitfalls of In Vivo Applications. Nanomaterials. 2019;9:866. PubMed PMC

Claveau S., Kindermann M., Papine A., Díaz-Riascos Z.V., Délen X., Georges P., López-Alemany R., Tirado Ò.M., Bertrand J.R., Abasolo I., et al. Harnessing subcellular-resolved organ distribution of cationic copolymer-functionalized fluorescent nanodiamonds for optimal delivery of active siRNA to a xenografted tumor in mice. Nanoscale. 2021;13:9280–9292. PubMed

Schrand A.M., Huang H., Carlson C., Schlager J.J., Omacr Sawa E., Hussain S.M., Dai L. Are diamond nanoparticles cytotoxic? J. Phys. Chem. B. 2007;111:2–7. PubMed

Wang L., Su W., Ahmad K.Z., Wang X., Zhang T., Yu Y., Chow E.K.-H., Ho D., Ding X. Safety Evaluation of Nanodiamond Doxorubicin Complexes in a Naïve Beagle Canine Model Using Hematologic, Histological, and Urine Analysis. Nano Res. 2022;15:3356–3366.

Perevedentseva E., Hong S.F., Huang K.J., Chiang I.T., Lee C.Y., Tseng Y.T., Cheng C.L. Nanodiamond internalization in cells and the cell uptake mechanism. J. Nanoparticle Res. 2013;15:1834.

Faklaris O., Garrot D., Joshi V., Druon F., Boudou J.P., Sauvage T., Georges P., Curmi P.A., Treussart F. Detection of single photoluminescent diamond nanoparticles in cells and study of the internalization pathway. Small. 2008;4:2236–2239. PubMed

Nie L., Zhang Y., Li L., van Rijn P., Schirhagl R. pH Sensitive Dextran Coated Fluorescent Nanodiamonds as a Biomarker for HeLa Cells Endocytic Pathway and Increased Cellular Uptake. Nanomaterials. 2021;11:1837. PubMed PMC

Wang C., Jia Q., Guo X., Li K., Chen W., Shen Q., Xu C., Fu Y. microRNA-34 family: From mechanism to potential applications. Int. J. Biochem. Cell Biol. 2022;144 PubMed

Yuan Y., Chen Y., Liu J.H., Wang H., Liu Y. Biodistribution and fate of nanodiamonds in vivo. Diam. Relat. Mater. 2009;18:95–100.

Van der Laan K., Hasani M., Zheng T., Schirhagl R. Nanodiamonds for In Vivo Applications. Small. 2018;14 PubMed

Guessous F., Zhang Y., Kofman A., Catania A., Li Y., Schiff D., Purow B., Abounader R. microRNA-34a is tumor suppressive in brain tumors and glioma stem cells. Cell Cycle. 2010;9:1031–1036. PubMed PMC

Zhao H., Chen S., Gao K., Zhou Z., Wang C., Shen Z., Guo Y., Li Z., Wan Z., Liu C., Mei X. Resveratrol protects against spinal cord injury by activating autophagy and inhibiting apoptosis mediated by the SIRT1/AMPK signaling pathway. Neuroscience. 2017;348:241–251. PubMed

Chakraborty S., Datta S., Ghosh S. Induction of autophagy under nitrosative stress: A complex regulatory interplay between SIRT1 and AMPK in MCF7 cells. Cell. Signal. 2019;64 PubMed

Lau A.W., Liu P., Inuzuka H., Gao D. SIRT1 phosphorylation by AMP-activated protein kinase regulates p53 acetylation. Am. J. Cancer Res. 2014;4:245–255. PubMed PMC

Maiuri M.C., Criollo A., Kroemer G. Crosstalk between apoptosis and autophagy within the Beclin 1 interactome. EMBO J. 2010;29:515–516. PubMed PMC

Liao H., Xiao Y., Hu Y., Xiao Y., Yin Z., Liu L., Kang X., Chen Y. Methylation-induced silencing of miR-34a enhances chemoresistance by directly upregulating ATG4B-induced autophagy through AMPK/mTOR pathway in prostate cancer. Oncol. Rep. 2016;35:64–72. PubMed

Cao Z., Kon N., Liu Y., Xu W., Wen J., Yao H., Zhang M., Wu Z., Yan X., Zhu W.G., et al. An unexpected role for p53 in regulating cancer cell-intrinsic PD-1 by acetylation. Sci. Adv. 2021;7 PubMed PMC

Wong S., Weber J.D. Deacetylation of the retinoblastoma tumour suppressor protein by SIRT1. Biochem. J. 2007;407:451–460. PubMed PMC

Sirangelo I., Borriello M., Liccardo M., Scafuro M., Russo P., Iannuzzi C. Hydroxytyrosol Selectively Affects Non-Enzymatic Glycation in Human Insulin and Protects by AGEs Cytotoxicity. Antioxidants. 2021;10:1127. PubMed PMC

Tavera- Mendoza L.E., Brown M. A less invasive method for orthotopic injection of breast cancer cells into the mouse mammary gland. Lab. Anim. 2017;51:85–88. PubMed