{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,8]],"date-time":"2026-01-08T01:51:41Z","timestamp":1767837101462,"version":"3.49.0"},"reference-count":80,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2022,12,1]],"date-time":"2022-12-01T00:00:00Z","timestamp":1669852800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Italian Ministry of Enterprises and Made in Italy","award":["CN-RNA"],"award-info":[{"award-number":["CN-RNA"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>In this article, we used monolayer two dimensional (2D) and 3D multicellular spheroid models to improve our understanding of the gene delivery process of a new modified cationic hyper-branched cyclodextrin-based polymer (Ppoly)-loaded plasmid encoding Enhanced Green Fluorescent Protein (EGFP). A comparison between the cytotoxicity effect and transfection efficiency of the plasmid DNA (pDNA)-loaded Ppoly system in 2D and 3D spheroid cells determined that the transfection efficiency and cytotoxicity of Ppoly\u2013pDNA nanocomplexes were lower in 3D spheroids than in 2D monolayer cells. Furthermore, histopathology visualization of Ppoly\u2013pDNA complex cellular uptake in 3D spheroids demonstrated that Ppoly penetrated into the inner layers. This study indicated that the Ppoly, as a non-viral gene delivery system in complex with pDNA, is hemocompatible, non-toxic, high in encapsulation efficiency, and has good transfection efficiency in both 2D and 3D cell cultures compared to free pDNA and lipofectamine (as the control).<\/jats:p>","DOI":"10.3390\/pharmaceutics14122690","type":"journal-article","created":{"date-parts":[[2022,12,2]],"date-time":"2022-12-02T02:18:06Z","timestamp":1669947486000},"page":"2690","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Hyper-Branched Cationic Cyclodextrin Polymers for Improving Plasmid Transfection in 2D and 3D Spheroid Cells"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1171-8670","authenticated-orcid":false,"given":"Yousef Khazaei","family":"Monfared","sequence":"first","affiliation":[{"name":"Dipartimento Di Chimica and NIS, Universit\u00e0 di Torino, Via P. Giuria 7, 10125 Torino, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mohammad","family":"Mahmoudian","sequence":"additional","affiliation":[{"name":"Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz 5166-15731, Iran"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2660-9522","authenticated-orcid":false,"given":"Claudio","family":"Cecone","sequence":"additional","affiliation":[{"name":"Dipartimento Di Chimica and NIS, Universit\u00e0 di Torino, Via P. Giuria 7, 10125 Torino, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2581-0555","authenticated-orcid":false,"given":"Fabrizio","family":"Caldera","sequence":"additional","affiliation":[{"name":"Dipartimento Di Chimica and NIS, Universit\u00e0 di Torino, Via P. Giuria 7, 10125 Torino, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1784-4926","authenticated-orcid":false,"given":"Sanya","family":"Haiaty","sequence":"additional","affiliation":[{"name":"Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz 5166-15731, Iran"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hamid Reza","family":"Heidari","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz 5166-15731, Iran"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Reza","family":"Rahbarghazi","sequence":"additional","affiliation":[{"name":"Department of Applied Cell Sciences, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz 5166-15731, Iran"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8203-0673","authenticated-orcid":false,"given":"Adri\u00e1n","family":"Matencio","sequence":"additional","affiliation":[{"name":"Dipartimento Di Chimica and NIS, Universit\u00e0 di Torino, Via P. Giuria 7, 10125 Torino, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Parvin","family":"Zakeri-Milani","sequence":"additional","affiliation":[{"name":"Liver and Gastrointestinal Diseases Research Centre, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz 5166-15731, Iran"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Francesco","family":"Trotta","sequence":"additional","affiliation":[{"name":"Dipartimento Di Chimica and NIS, Universit\u00e0 di Torino, Via P. Giuria 7, 10125 Torino, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,12,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1136\/archdischild-2012-302158","article-title":"Gene therapy in cystic fibrosis","volume":"99","author":"Armstrong","year":"2014","journal-title":"Arch. Dis. Child."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"743","DOI":"10.1002\/cpt.2543","article-title":"Gene Therapy for Rare Neurological Disorders","volume":"111","author":"Flotte","year":"2022","journal-title":"Clin. Pharmacol. Ther."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"3","DOI":"10.4103\/gjtm.gjtm_87_21","article-title":"Gene therapy\u2013Principles and applications","volume":"7","author":"Pathak","year":"2022","journal-title":"Glob. J. Transfus. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"186","DOI":"10.1089\/nat.2013.0464","article-title":"Target gene knockdown by 2\u2032, 4\u2032-BNA\/LNA antisense oligonucleotides in zebrafish","volume":"24","author":"Itoh","year":"2014","journal-title":"Nucleic Acid Ther."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"e3015","DOI":"10.1002\/jgm.3015","article-title":"Gene therapy clinical trials worldwide to 2017: An update","volume":"20","author":"Ginn","year":"2018","journal-title":"J. Gene Med."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1016\/j.ijpharm.2013.11.041","article-title":"Gene therapy and DNA delivery systems","volume":"459","author":"Ibraheem","year":"2014","journal-title":"Int. J. Pharm."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"310","DOI":"10.1039\/D0MA00353K","article-title":"Capsid-like biodegradable poly-glycolic acid nanoparticles for a long-time release of nucleic acid molecules","volume":"2","author":"Guido","year":"2021","journal-title":"Mater. Adv."},{"key":"ref_8","first-page":"GE01","article-title":"Non viral vectors in gene therapy-an overview","volume":"9","author":"Ramamoorth","year":"2015","journal-title":"J. Clin. Diagn. Res. JCDR"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Hardee, C.L., Ar\u00e9valo-Soliz, L.M., Hornstein, B.D., and Zechiedrich, L. (2017). Advances in non-viral DNA vectors for gene therapy. Genes, 8.","DOI":"10.3390\/genes8020065"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"525","DOI":"10.1007\/s00253-020-11028-6","article-title":"Progress of cationic gene delivery reagents for non-viral vector","volume":"105","author":"Ma","year":"2021","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"120291","DOI":"10.1016\/j.ijpharm.2021.120291","article-title":"Strategies for cancer gene-delivery improvement by non-viral vectors","volume":"596","year":"2021","journal-title":"Int. J. Pharm."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Wahane, A., Waghmode, A., Kapphahn, A., Dhuri, K., Gupta, A., and Bahal, R. (2020). Role of lipid-based and polymer-based non-viral vectors in nucleic acid delivery for next-generation gene therapy. Molecules, 25.","DOI":"10.3390\/molecules25122866"},{"key":"ref_13","first-page":"17","article-title":"Recent trends in Nanotech Delivery for Gene Therapy","volume":"2","author":"Pathak","year":"2022","journal-title":"Med. Pharm. Sci."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1146\/annurev-bioeng-071813-104938","article-title":"Viral vectors for gene therapy: Translational and clinical outlook","volume":"17","author":"Kotterman","year":"2015","journal-title":"Annu. Rev. Biomed. Eng."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"eaan4672","DOI":"10.1126\/science.aan4672","article-title":"Gene therapy comes of age","volume":"359","author":"Dunbar","year":"2018","journal-title":"Science"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1358","DOI":"10.1021\/acs.molpharmaceut.8b01307","article-title":"Self-assembled cationic \u03b2-cyclodextrin nanostructures for siRNA delivery","volume":"16","author":"Singh","year":"2019","journal-title":"Mol. Pharm."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1007\/s13346-020-00724-5","article-title":"Cyclodextrins in drug delivery: Applications in gene and combination therapy","volume":"10","author":"Haley","year":"2020","journal-title":"Drug Deliv. Transl. Res."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Matencio, A., Hoti, G., Monfared, Y.K., Rezayat, A., Pedrazzo, A.R., Caldera, F., and Trotta, F. (2021). Cyclodextrin Monomers and Polymers for Drug Activity Enhancement. Polymers, 13.","DOI":"10.3390\/polym13111684"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"115763","DOI":"10.1016\/j.carbpol.2019.115763","article-title":"Study of oxyresveratrol complexes with insoluble cyclodextrin based nanosponges: Developing a novel way to obtain their complexation constants and application in an anticancer study","volume":"231","author":"Matencio","year":"2020","journal-title":"Carbohydr. Polym."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Matencio, A., Rubin Pedrazzo, A., Difalco, A., Navarro-Orcajada, S., Khazeai Monfared, Y., Conesa, I., Rezayat, A., L\u00f3pez-Nicol\u00e1s, J.M., and Trotta, F. (2021). Advances and Classification of Cyclodextrin-Based Polymers for Food-Related Issues. Polymers, 13.","DOI":"10.3390\/polym13234226"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Lucia Appleton, S., Navarro-Orcajada, S., Mart\u00ednez-Navarro, F.J., Caldera, F., L\u00f3pez-Nicol\u00e1s, J.M., Trotta, F., and Matencio, A. (2021). Cyclodextrins as Anti-inflammatory Agents: Basis, Drugs and Perspectives. Biomolecules, 11.","DOI":"10.3390\/biom11091384"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"132","DOI":"10.1016\/j.tifs.2020.08.009","article-title":"Applications of cyclodextrins in food science. A review","volume":"104","author":"Matencio","year":"2020","journal-title":"Trends Food Sci. Technol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1068","DOI":"10.1021\/bc990072j","article-title":"New class of polymers for the delivery of macromolecular therapeutics","volume":"10","author":"Gonzalez","year":"1999","journal-title":"Bioconjugate Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"445","DOI":"10.1046\/j.1525-1500.1998.00051.x","article-title":"Viral and nonviral gene delivery vectors for cancer gene therapy","volume":"22","author":"Cristiano","year":"1998","journal-title":"Cancer Detect. Prev."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"6346","DOI":"10.3390\/s90806346","article-title":"Recent findings concerning PAMAM dendrimer conjugates with cyclodextrins as carriers of DNA and RNA","volume":"9","author":"Arima","year":"2009","journal-title":"Sensors"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1046","DOI":"10.1016\/j.jconrel.2020.11.011","article-title":"Cyclodextrin based natural nanostructured carbohydrate polymers as effective non-viral siRNA delivery systems for cancer gene therapy","volume":"330","author":"Mousazadeh","year":"2021","journal-title":"J. Control. Release"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2149","DOI":"10.1016\/j.biomaterials.2010.11.052","article-title":"Engineered silk fibroin protein 3D matrices for in vitro tumor model","volume":"32","author":"Talukdar","year":"2011","journal-title":"Biomaterials"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.addr.2014.02.008","article-title":"Critical analysis of 3-D organoid in vitro cell culture models for high-throughput drug candidate toxicity assessments","volume":"69","author":"Astashkina","year":"2014","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"vii","DOI":"10.1016\/j.addr.2014.04.001","article-title":"Cell-based drug testing; this world is not flat","volume":"69","author":"Grainger","year":"2014","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"6468","DOI":"10.1038\/srep06468","article-title":"Three-dimensional in vitro co-culture model of breast tumor using magnetic levitation","volume":"4","author":"Jaganathan","year":"2014","journal-title":"Sci. Rep."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"601","DOI":"10.1016\/j.cell.2007.08.006","article-title":"Modeling tissue morphogenesis and cancer in 3D","volume":"130","author":"Yamada","year":"2007","journal-title":"Cell"},{"key":"ref_32","first-page":"3","article-title":"The new frontier of three-dimensional culture models to scale-up cancer research","volume":"2343","author":"Jensen","year":"2022","journal-title":"Phys. Exerc. Nat. Synth. Prod. Health Dis."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1186\/s12935-022-02662-3","article-title":"Three-dimensional (3D) cell culture: A valuable step in advancing treatments for human hepatocellular carcinoma","volume":"22","author":"Khafaga","year":"2022","journal-title":"Cancer Cell Int."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2048","DOI":"10.1007\/s13346-022-01147-0","article-title":"Three-dimensional (3D) liver cell models-a tool for bridging the gap between animal studies and clinical trials when screening liver accumulation and toxicity of nanobiomaterials","volume":"12","author":"Tutty","year":"2022","journal-title":"Drug Deliv. Transl. Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"2103","DOI":"10.1021\/mp1000795","article-title":"A three-dimensional coculture of enterocytes, monocytes and dendritic cells to model inflamed intestinal mucosa in vitro","volume":"7","author":"Leonard","year":"2010","journal-title":"Mol. Pharm."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1016\/j.drudis.2012.10.003","article-title":"Three-dimensional cell culture: The missing link in drug discovery","volume":"18","author":"Breslin","year":"2013","journal-title":"Drug Discov. Today"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Poornima, K., Francis, A.P., Hoda, M., Eladl, M.A., Subramanian, S., Veeraraghavan, V.P., El-Sherbiny, M., Asseri, S.M., Hussamuldin, A.B.A., and Surapaneni, K.M. (2022). Implications of three-dimensional cell culture in cancer therapeutic research. Front. Oncol.","DOI":"10.3389\/fonc.2022.891673"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"427","DOI":"10.1023\/A:1011037807261","article-title":"Water-soluble and low molecular weight chitosan-based plasmid DNA delivery","volume":"18","author":"Lee","year":"2001","journal-title":"Pharm. Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"116545","DOI":"10.1016\/j.lfs.2019.116545","article-title":"Enhanced penetration and cytotoxicity of metformin and collagenase conjugated gold nanoparticles in breast cancer spheroids","volume":"231","author":"Abdolahinia","year":"2019","journal-title":"Life Sci."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"118747","DOI":"10.1016\/j.carbpol.2021.118747","article-title":"Stimulus-responsive drug\/gene delivery system based on polyethylenimine cyclodextrin nanoparticles for potential cancer therapy","volume":"276","author":"Mousazadeh","year":"2022","journal-title":"Carbohydr. Polym."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"142","DOI":"10.1016\/j.ijpharm.2018.03.021","article-title":"\u03b5-Poly-l-Lysine\/plasmid DNA nanoplexes for efficient gene delivery in vivo","volume":"542","author":"Mandal","year":"2018","journal-title":"Int. J. Pharm."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1023","DOI":"10.1038\/nrd1576","article-title":"Cyclodextrin-based pharmaceutics: Past, present and future","volume":"3","author":"Davis","year":"2004","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"5806","DOI":"10.1039\/D0GC02247K","article-title":"Sustainable synthesis of cyclodextrin-based polymers by exploiting natural deep eutectic solvents","volume":"22","author":"Cecone","year":"2020","journal-title":"Green Chem."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"2586","DOI":"10.3762\/bjoc.10.271","article-title":"Synthesis and characterization of a hyper-branched water-soluble \u03b2-cyclodextrin polymer","volume":"10","author":"Trotta","year":"2014","journal-title":"Beilstein J. Org. Chem."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1642","DOI":"10.1021\/acscentsci.8b00601","article-title":"Critical assessment of the interaction between DNA and choline amino acid ionic liquids: Evidences of multimodal binding and stability enhancement","volume":"4","author":"Sahoo","year":"2018","journal-title":"ACS Cent. Sci."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1039\/b210714g","article-title":"Novel solvent properties of choline chloride\/urea mixtures","volume":"39","author":"Abbott","year":"2003","journal-title":"Chem. Commun."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1016\/j.ecoenv.2017.05.034","article-title":"Ecotoxicological evaluation of magnetic ionic liquids","volume":"143","author":"Sintra","year":"2017","journal-title":"Ecotoxicol. Environ. Saf."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"9142","DOI":"10.1021\/ja048266j","article-title":"Deep eutectic solvents formed between choline chloride and carboxylic acids: Versatile alternatives to ionic liquids","volume":"126","author":"Abbott","year":"2004","journal-title":"J. Am. Chem. Soc."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"523","DOI":"10.1002\/app.27661","article-title":"Synthesis and characterization of starch piperinic ester and its self-assembly of nanospheres","volume":"108","author":"Han","year":"2008","journal-title":"J. Appl. Polym. Sci."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"1671","DOI":"10.1080\/17425247.2017.1248398","article-title":"Molecularly imprinted cyclodextrin nanosponges for the controlled delivery of L-DOPA: Perspectives for the treatment of Parkinson\u2019s disease","volume":"13","author":"Trotta","year":"2016","journal-title":"Expert Opin. Drug Deliv."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1016\/j.matpr.2021.12.486","article-title":"Concentration dependent electrochemical performance of aqueous choline chloride electrolyte","volume":"53","author":"Singh","year":"2022","journal-title":"Mater. Today Proc."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"450","DOI":"10.1039\/C7PY01847A","article-title":"Acid-sensitive poly (\u03b2-cyclodextrin)-based multifunctional supramolecular gene vector","volume":"9","author":"Zhang","year":"2018","journal-title":"Polym. Chem."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"194","DOI":"10.1038\/sj.gt.3302159","article-title":"Dextran\u2013spermine polycation: An efficient nonviral vector for in vitro and in vivo gene transfection","volume":"11","author":"Hosseinkhani","year":"2004","journal-title":"Gene Ther."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1124\/pr.58.1.8","article-title":"Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery","volume":"58","author":"Khalil","year":"2006","journal-title":"Pharmacol. Rev."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"2512","DOI":"10.1007\/s11095-012-0958-3","article-title":"Effects of particle size and surface modification on cellular uptake and biodistribution of polymeric nanoparticles for drug delivery","volume":"30","author":"Kulkarni","year":"2013","journal-title":"Pharm. Res."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"2436","DOI":"10.1039\/C8AY00386F","article-title":"Key factors in FTIR spectroscopic analysis of DNA: The sampling technique, pretreatment temperature and sample concentration","volume":"10","author":"Han","year":"2018","journal-title":"Anal. Methods"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"64","DOI":"10.4236\/jbnb.2016.72008","article-title":"Hemocompatibility of albumin nanoparticles as a drug delivery system\u2014An in vitro study","volume":"7","author":"Elblbesy","year":"2016","journal-title":"J. Biomater. Nanobiotechnology"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1016\/j.diamond.2013.06.011","article-title":"Preparation and blood compatibility of carbon\/TiO2 nanocomposite","volume":"38","author":"Chen","year":"2013","journal-title":"Diam. Relat. Mater."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1016\/S0144-8617(97)00006-4","article-title":"Synthesis of anionic poly (ethylene glycol) derivative for chitosan surface modification in blood-contacting applications","volume":"32","author":"Amiji","year":"1997","journal-title":"Carbohydr. Polym."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"2244","DOI":"10.1016\/j.eurpolymj.2007.03.042","article-title":"Synthesis and characterization of PEG modified N-trimethylaminoethylmethacrylate chitosan nanoparticles","volume":"43","author":"Zhu","year":"2007","journal-title":"Eur. Polym. J."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1016\/S0168-3659(99)00090-5","article-title":"Poly (ethylenimine) and its role in gene delivery","volume":"60","author":"Godbey","year":"1999","journal-title":"J. Control. Release"},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1023\/B:PHAM.0000016251.42392.1e","article-title":"Partial acetylation of polyethylenimine enhances in vitro gene delivery","volume":"21","author":"Forrest","year":"2004","journal-title":"Pharm. Res."},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1016\/j.ijpharm.2006.12.032","article-title":"Influence of acyl chain length on transfection mediated by acylated PEI nanoparticles","volume":"337","author":"Nimesh","year":"2007","journal-title":"Int. J. Pharm."},{"key":"ref_64","first-page":"921","article-title":"Alkylcarboxylate grafting to polyethylenimine: A simple approach to producing a DNA nanocarrier with low toxicity","volume":"11","author":"Oskuee","year":"2009","journal-title":"J. Gene Med. A Cross-Discip. J. Res. Sci. Gene Transf. Its Clin. Appl."},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"290","DOI":"10.1016\/j.msec.2015.01.016","article-title":"A simple approach for producing highly efficient DNA carriers with reduced toxicity based on modified polyallylamine","volume":"49","author":"Oskuee","year":"2015","journal-title":"Mater. Sci. Eng. C"},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1016\/j.actbio.2018.11.051","article-title":"Calcium phosphate nanoparticle-mediated transfection in 2D and 3D mono-and co-culture cell models","volume":"84","author":"Sokolova","year":"2019","journal-title":"Acta Biomater."},{"key":"ref_67","doi-asserted-by":"crossref","first-page":"4648","DOI":"10.1021\/acsbiomaterials.2c00277","article-title":"Three-Dimensional in Vitro Models: A Promising Tool To Scale-Up Breast Cancer Research","volume":"8","author":"Rafiee","year":"2022","journal-title":"ACS Biomater. Sci. Eng."},{"key":"ref_68","doi-asserted-by":"crossref","unstructured":"Mikhail, A.S., Eetezadi, S., and Allen, C. (2013). Multicellular tumor spheroids for evaluation of cytotoxicity and tumor growth inhibitory effects of nanomedicines in vitro: A comparison of docetaxel-loaded block copolymer micelles and Taxotere\u00ae. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0062630"},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1186\/s12645-020-00074-4","article-title":"Curcumin-enriched Gemini surfactant nanoparticles exhibited tumoricidal effects on human 3D spheroid HT-29 cells in vitro","volume":"12","author":"Zibaei","year":"2021","journal-title":"Cancer Nanotechnol."},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1016\/j.taap.2016.01.016","article-title":"Development of complex-shaped liver multicellular spheroids as a human-based model for nanoparticle toxicity assessment in vitro","volume":"294","author":"Karczmarczyk","year":"2016","journal-title":"Toxicol. Appl. Pharmacol."},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1016\/j.mrgentox.2017.12.005","article-title":"A three-dimensional in vitro HepG2 cells liver spheroid model for genotoxicity studies","volume":"825","author":"Shah","year":"2018","journal-title":"Mutat. Res. \/Genet. Toxicol. Environ. Mutagen."},{"key":"ref_72","doi-asserted-by":"crossref","first-page":"403033","DOI":"10.1016\/j.mrgentox.2019.03.006","article-title":"The comet assay applied to HepG2 liver spheroids","volume":"845","author":"Elje","year":"2019","journal-title":"Mutat. Res.\/Genet. Toxicol. Environ. Mutagen."},{"key":"ref_73","doi-asserted-by":"crossref","first-page":"1411","DOI":"10.2147\/IJN.S189888","article-title":"Distribution of SiO2 nanoparticles in 3D liver microtissues","volume":"14","author":"Fleddermann","year":"2019","journal-title":"Int. J. Nanomed."},{"key":"ref_74","doi-asserted-by":"crossref","first-page":"10548","DOI":"10.1038\/s41598-019-47114-7","article-title":"Three-dimensional HepaRG spheroids as a liver model to study human genotoxicity in vitro with the single cell gel electrophoresis assay","volume":"9","author":"Mandon","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_75","doi-asserted-by":"crossref","first-page":"3260","DOI":"10.1016\/S0006-3495(04)74374-X","article-title":"Tissue electroporation: Quantification and analysis of heterogeneous transport in multicellular environments","volume":"86","author":"Canatella","year":"2004","journal-title":"Biophys. J."},{"key":"ref_76","doi-asserted-by":"crossref","first-page":"3036","DOI":"10.1016\/j.biomaterials.2011.12.049","article-title":"The use of an in vitro 3D melanoma model to predict in vivo plasmid transfection using electroporation","volume":"33","author":"Marrero","year":"2012","journal-title":"Biomaterials"},{"key":"ref_77","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1038\/cgt.2015.22","article-title":"A transfection method of PS-asODNs targeting ANGPTL4 in multicellular structures of hepatocarcinoma cell line","volume":"22","author":"Kong","year":"2015","journal-title":"Cancer Gene Ther."},{"key":"ref_78","doi-asserted-by":"crossref","first-page":"1160","DOI":"10.1002\/jgm.947","article-title":"Optimising non-viral gene delivery in a tumour spheroid model","volume":"8","author":"Mellor","year":"2006","journal-title":"J. Gene Med.: A Cross-Discip. J. Res. Sci. Gene Transf. Its Clin. Appl."},{"key":"ref_79","doi-asserted-by":"crossref","first-page":"e153","DOI":"10.1038\/mtna.2014.5","article-title":"Multicellular tumor spheroids as a model for assessing delivery of oligonucleotides in three dimensions","volume":"3","author":"Carver","year":"2014","journal-title":"Mol. Ther.-Nucleic Acids"},{"key":"ref_80","doi-asserted-by":"crossref","first-page":"49713","DOI":"10.1021\/acsami.1c14761","article-title":"Designed antitumor peptide for targeted siRNA delivery into cancer spheroids","volume":"13","author":"Cirillo","year":"2021","journal-title":"ACS Appl. Mater. 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