BACKGROUND: Nanomedicine approaches for cancer therapy face significant challenges, including a poor tumor accumulation, limited therapeutic efficacy, and systemic toxicity. We hypothesized that controlling the clustering of poly(acrylic acid-co-maleic acid) (PAM)-coated superparamagnetic iron oxide nanoparticles (SPIONs) would enhance their magnetic properties for improved targeting, while enabling a pH-responsive drug release in tumor microenvironments. METHODS: PAM-stabilized SPION clusters were synthesized via arrested precipitation, characterized for physicochemical and magnetic properties, and evaluated for doxorubicin loading and pH-dependent release. A dual targeting approach combining antibody conjugation with magnetic guidance was assessed in cellular models, including a novel alternating magnetic field (AMF) pre-treatment protocol. RESULTS: PAM-SPION clusters demonstrated controlled size distributions (60-100 nm), excellent colloidal stability, and enhanced magnetic properties, particularly for larger crystallites (13 nm). The formulations exhibited a pH-responsive drug release (8.5% at pH 7.4 vs. 14.3% at pH 6.5) and a significant enhancement of AMF-triggered release (17.5%). The dual targeting approach achieved an 8-fold increased cellular uptake compared to non-targeted formulations. Most notably, the novel AMF pre-treatment protocol demonstrated an 87% improved therapeutic efficacy compared to conventional post-treatment applications. CONCLUSIONS: The integration of targeting antibodies, magnetic guidance, and a pH-responsive PAM coating creates a versatile theranostic platform with significantly enhanced drug delivery capabilities. The unexpected synergistic effect of the AMF pre-treatment represents a promising new approach for improving the therapeutic efficacy of nanoparticle-based cancer treatments.

Institute of Chemical Engineering Sciences Foundation for Research and Technology Hellas 26504 Rio Patras Greece

Laboratory of Pharmaceutical Technology Department of Pharmacy School of Health Sciences University of Patras 26504 Rio Patras Greece

Regional Centre of Advanced Technologies and Materials Czech Advanced Technology and Research Institute Palacký University Olomouc 779 00 Olomouc Czech Republic

See more in PubMed

Siegel R.L., Kratzer T.B., Giaquinto A.N., Sung H., Jemal A. Cancer statistics, 2025. CA Cancer J. Clin. 2025;75:10–45. doi: 10.3322/caac.21871. PubMed DOI PMC

Zhang P., Xiao Y., Sun X., Lin X., Koo S., Yaremenko A.V., Qin D., Kong N., Farokhzad O.C., Tao W. Cancer nanomedicine toward clinical translation: Obstacles, opportunities, and future prospects. Med. 2023;4:147–167. doi: 10.1016/j.medj.2022.12.001. PubMed DOI

Anselmo A.C., Mitragotri S. Nanoparticles in the clinic: An update. Bioeng. Transl. Med. 2019;4:e10143. doi: 10.1002/btm2.10143. PubMed DOI PMC

Malik S., Muhammad K., Waheed Y. Emerging Applications of Nanotechnology in Healthcare and Medicine. Molecules. 2023;28:6624. doi: 10.3390/molecules28186624. PubMed DOI PMC

Velazquez-Albino A.C., Nozka A., Melnyk A., Good H.J., Rinaldi-Ramos C.M. Post-synthesis Oxidation of Superparamagnetic Iron Oxide Nanoparticles to Enhance Magnetic Particle Imaging Performance. ACS Appl. Nano Mater. 2024;7:279–291. doi: 10.1021/acsanm.3c04442. PubMed DOI PMC

Vangijzegem T., Lecomte V., Ternad I., Van Leuven L., Muller R.N., Stanicki D., Laurent S. Superparamagnetic Iron Oxide Nanoparticles (SPION): From Fundamentals to State-of-the-Art Innovative Applications for Cancer Therapy. Pharmaceutics. 2023;15:236. doi: 10.3390/pharmaceutics15010236. PubMed DOI PMC

Pucci C., Degl’Innocenti A., Belenli Gümüş M., Ciofani G. Superparamagnetic iron oxide nanoparticles for magnetic hyperthermia: Recent advancements, molecular effects, and future directions in the omics era. Biomater. Sci. 2022;10:2103–2121. doi: 10.1039/D1BM01963E. PubMed DOI

Wu W., Jiang C.Z., Roy V.A. Designed synthesis and surface engineering strategies of magnetic iron oxide nanoparticles for biomedical applications. Nanoscale. 2016;8:19421–19474. doi: 10.1039/C6NR07542H. PubMed DOI

Mehak Thummer R.P., Pandey L.M. Surface modified iron-oxide based engineered nanomaterials for hyperthermia therapy of cancer cells. Biotechnol. Genet. Eng. Rev. 2023;39:1187–1233. doi: 10.1080/02648725.2023.2169370. PubMed DOI

Wei H., Bruns O.T., Kaul M.G., Hansen E.C., Barch M., Wiśniowska A., Chen O., Chen Y., Li N., Okada S., et al. Exceedingly small iron oxide nanoparticles as positive MRI contrast agents. Proc. Natl. Acad. Sci. USA. 2017;114:2325–2330. doi: 10.1073/pnas.1620145114. PubMed DOI PMC

Saraiva C., Praça C., Ferreira R., Santos T., Ferreira L., Bernandino L. Nanoparticle-mediated brain drug delivery: Overcoming blood-brain barrier to treat neurodegenerative diseases. J. Control. Release. 2016;235:34–47. doi: 10.1016/j.jconrel.2016.05.044. PubMed DOI

Tietze R., Zaloga J., Unterweger H., Lyer S., Friedrich R.P., Janko C., Pöttler M., Dürr S., Alexiou C. Magnetic nanoparticle-based drug delivery for cancer therapy. Biochem. Biophys. Res. Commun. 2015;468:463–470. doi: 10.1016/j.bbrc.2015.08.022. PubMed DOI

Shiji R., Joseph M.M., Sen A., Unnikrishnan B.S., Sreelekha T.T. Galactomannan armed superparamagnetic iron oxide nanoparticles as a folate receptor targeted multi-functional theranostic agent in the management of cancer. Int. J. Biol. Macromol. 2022;219:740–753. doi: 10.1016/j.ijbiomac.2022.07.185. PubMed DOI

Danhier F. To exploit the tumor microenvironment: Since the EPR effect fails in the clinic, what is the future of nanomedicine? J. Control. Release. 2016;244 (Pt A):108–121. doi: 10.1016/j.jconrel.2016.11.015. PubMed DOI

Cheng Y.H., He C., Riviere J.E., Monteiro-Riviere N.A., Lin Z. Meta-Analysis of Nanoparticle Delivery to Tumors Using a Physiologically Based Pharmacokinetic Modeling and Simulation Approach. ACS Nano. 2020;14:3075–3095. doi: 10.1021/acsnano.9b08142. PubMed DOI PMC

Estelrich J., Escribano E., Queralt J., Busquets M.A. Iron oxide nanoparticles for magnetically-guided and magnetically-responsive drug delivery. Int. J. Mol. Sci. 2015;16:8070–8101. doi: 10.3390/ijms16048070. PubMed DOI PMC

Kolosnjaj-Tabi J., Lartigue L., Javed Y., Luciani N., Pellegrino T., Wilhelm C., Alloyeau D., Gazeau F. Biotransformations of magnetic nanoparticles in the body. Nano Today. 2017;11:280–284. doi: 10.1016/j.nantod.2015.10.001. DOI

Lapusan R., Borlan R., Focsan M. Advancing MRI with magnetic nanoparticles: A comprehensive review of translational research and clinical trials. Nanoscale Adv. 2024;6:2234–2259. doi: 10.1039/D3NA01064C. PubMed DOI PMC

Usov N.A., Rytov R.A., Bautin V.A. Properties of assembly of superparamagnetic nanoparticles in viscous liquid. Sci. Rep. 2021;11:6999. doi: 10.1038/s41598-021-86323-x. PubMed DOI PMC

Jang Y.J., Liu S., Yue H., Park J.A., Cha H., Ho S.L., Marasini S., Ghazanfari A., Ahmad M.Y., Miao X., et al. Hydrophilic Biocompatible Poly(Acrylic Acid-co-Maleic Acid) Polymer as a Surface-Coating Ligand of Ultrasmall Gd2O3 Nanoparticles to Obtain a High r1 Value and T1 MR Images. Diagnostics. 2020;11:2. doi: 10.3390/diagnostics11010002. PubMed DOI PMC

Friedrich R.P., Zaloga J., Schreiber E., Tóth I.Y., Tombácz E., Lyer S., Alexiou C. Tissue Plasminogen Activator Binding to Superparamagnetic Iron Oxide Nanoparticle-Covalent Versus Adsorptive Approach. Nanoscale Res. Lett. 2016;11:297. doi: 10.1186/s11671-016-1521-7. PubMed DOI PMC

Ulbrich K., Holá K., Šubr V., Bakandritsos A., Tuček J., Zbořil R. Targeted Drug Delivery with Polymers and Magnetic Nanoparticles: Covalent and Noncovalent Approaches, Release Control, and Clinical Studies. Chem. Rev. 2016;116:5338–5431. doi: 10.1021/acs.chemrev.5b00589. PubMed DOI

Du J.Z., Du X.J., Mao C.Q., Wang J. Tailor-made dual pH-sensitive polymer-doxorubicin nanoparticles for efficient anticancer drug delivery. J. Am. Chem. Soc. 2011;133:17560–17563. doi: 10.1021/ja207150n. PubMed DOI

Nowak-Jary J., Machnicka B. In vivo Biodistribution and Clearance of Magnetic Iron Oxide Nanoparticles for Medical Applications. Int. J. Nanomed. 2023;18:4067–4100. doi: 10.2147/IJN.S415063. PubMed DOI PMC

Reyes-Ortega F., Delgado Á.V., Iglesias G.R. Modulation of the Magnetic Hyperthermia Response Using Different Superparamagnetic Iron Oxide Nanoparticle Morphologies. Nanomaterials. 2021;11:627. doi: 10.3390/nano11030627. PubMed DOI PMC

Rajan A., Laha S.S., Sahu N.K., Thorat N.D., Shankar B. Recent advancements and clinical aspects of engineered iron oxide nanoplatforms for magnetic hyperthermia-induced cancer therapy. Mater. Today Bio. 2024;29:101348. doi: 10.1016/j.mtbio.2024.101348. PubMed DOI PMC

Couto D., Freitas M., Vilas-Boas V., Dias I., Porto G., Lopez-Quintela M.A., Rivas J., Freitas P., Carvalho F., Fernandes E. Interaction of polyacrylic acid coated and non-coated iron oxide nanoparticles with human neutrophils. Toxicol. Lett. 2014;225:57–65. doi: 10.1016/j.toxlet.2013.11.020. PubMed DOI

Cornell R.M., Schwertmann U. The Iron Oxides: Structure, Properties, Reactions, Occurrences and Uses. 2nd ed. Wiley-VCH; Hoboken, NJ, USA: 2003.

Lu Y., Huang J., Neverova N.V., Nguyen K.L. USPIOs as targeted contrast agents in cardiovascular magnetic resonance imaging. Curr. Cardiovasc. Imaging Rep. 2021;14:2. doi: 10.1007/s12410-021-09552-8. PubMed DOI PMC

Siepmann J., Peppas N.A. Modeling of drug release from delivery systems based on hydroxypropyl methylcellulose (HPMC) Adv. Drug Deliv. Rev. 2012;64:163–174. doi: 10.1016/S0169-409X(01)00112-0. PubMed DOI

Mehdaoui B., Meffre A., Carrey J., Lachaize S., Lacroix L.-M., Gougeon M., Chaudret B., Respaud M. Optimal Size of Nanoparticles for Magnetic Hyperthermia: A Combined Theoretical and Experimental Study. Adv. Funct. Mater. 2011;21:4573–4581. doi: 10.1002/adfm.201101243. DOI

Rosensweig R.E. Heating magnetic fluid with alternating magnetic field. J. Magn. Magn. Mater. 2002;252:370–374. doi: 10.1016/S0304-8853(02)00706-0. DOI

Johannsen M., Thiesen B., Wust P., Jordan A. Magnetic nanoparticle hyperthermia for prostate cancer. Int. J. Hyperth. 2010;26:790–795. doi: 10.3109/02656731003745740. PubMed DOI

Blanco-Andujar C., Walter A., Cotin G., Bordeianu C., Mertz D., Felder-Flesch D., Begin-Colin S. Design of iron oxide-based nanoparticles for MRI and magnetic hyperthermia. Nanomedicine. 2016;11:1889–1910. doi: 10.2217/nnm-2016-5001. PubMed DOI

Colombo M., Fiandra L., Alessio G., Mazzucchelli S., Nebuloni M., De Palma C., Kantner K., Pelaz B., Rotem R., Corsi F., et al. Tumour homing and therapeutic effect of colloidal nanoparticles depend on the number of attached antibodies. Nat. Commun. 2016;7:13818. doi: 10.1038/ncomms13818. PubMed DOI PMC

Markoutsa E., Papadia K., Giannou A.D., Spella M., Cagnotto A., Salmona M., Stathopoulos G.T., Antimisiaris S.G. Mono and dually decorated nanoliposomes for brain targeting, in vitro and in vivo studies. Pharm. Res. 2014;31:1275–1289. doi: 10.1007/s11095-013-1249-3. PubMed DOI

Skouras A., Papadia K., Mourtas S., Klepetsanis P., Antimisiaris S.G. Multifunctional doxorubicin-loaded magnetoliposomes with active and magnetic targeting properties. Eur. J. Pharm. Sci. 2018;123:162–172. doi: 10.1016/j.ejps.2018.07.044. PubMed DOI

Ramazi S., Salimian M., Allahverdi A., Kianamiri S., Abdolmaleki P. Synergistic cytotoxic effects of an extremely low-frequency electromagnetic field with doxorubicin on MCF-7 cell line. Sci. Rep. 2023;13:8844. doi: 10.1038/s41598-023-35767-4. PubMed DOI PMC

Xu A., Wang Q., Lv X., Lin T. Progressive Study on the Non-thermal Effects of Magnetic Field Therapy in Oncology. Front. Oncol. 2021;11:638146. doi: 10.3389/fonc.2021.638146. PubMed DOI PMC

Ashdown C.P., Johns S.C., Aminov E., Unanian M., Connacher W., Friend J., Fuster M.M. Pulsed Low-Frequency Magnetic Fields Induce Tumor Membrane Disruption and Altered Cell Viability. Biophys. J. 2020;118:1552–1563. doi: 10.1016/j.bpj.2020.02.013. PubMed DOI PMC

Omote Y., Hosokawa M., Komatsumoto M., Namieno T., Nakajima S., Kubo Y., Kobayashi H. Treatment of experimental tumors with a combination of a pulsing magnetic field and an antitumor drug. Jpn. J. Cancer Res. Gann. 1990;81:956–961. doi: 10.1111/j.1349-7006.1990.tb02673.x. PubMed DOI PMC

Ellman G.L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 1959;82:70–77. doi: 10.1016/0003-9861(59)90090-6. PubMed DOI

Markoutsa E., Pampalakis G., Niarakis A., Romero I.A., Weksler B., Couraud P.O., Antimisiaris S.G. Uptake and permeability studies of BBB-targeting immunoliposomes using the hCMEC/D3 cell line. Eur. J. Pharm. Biopharm. 2011;77:265–274. doi: 10.1016/j.ejpb.2010.11.015. PubMed DOI