In this study, novel Trojan particles were engineered for direct delivery of doxorubicin (DOX) and miR-34a as model drugs to the lungs to raise local drug concentration, decrease pulmonary clearance, increase lung drug deposition, reduce systemic side effects, and overcome multi-drug resistance. For this purpose, targeted polyelectrolyte nanoparticles (tPENs) developed with layer-by-layer polymers (i.e., chitosan, dextran sulfate, and mannose-g-polyethyleneimine) were spray dried into a multiple-excipient (i.e., chitosan, leucine, and mannitol). The resulting nanoparticles were first characterized in terms of size, morphology, in vitro DOX release, cellular internalization, and in vitro cytotoxicity. tPENs showed comparable cellular uptake levels to PENs in A549 cells and no significant cytotoxicity on their metabolic activity. Co-loaded DOX/miR-34a showed a greater cytotoxicity effect than DOX-loaded tPENs and free drugs, which was confirmed by Actin staining. Thereafter, nano-in-microparticles were studied through size, morphology, aerosolization efficiency, residual moisture content, and in vitro DOX release. It was demonstrated that tPENs were successfully incorporated into microspheres with adequate emitted dose and fine particle fraction but low mass median aerodynamic diameter for deposition into the deep lung. The dry powder formulations also demonstrated a sustained DOX release at both pH values of 6.8 and 7.4.

Centre of Polymer Systems University Institute TBU Tr Tomase Bati 5678 Zlin Czech Republic

Faculty of Mechanical Engineering Brno University of Technology Technicka 2896 2 61669 Brno Czech Republic

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Ding Y, Liu W, Yu W, Lu S, Liu M, Kaplan DL, et al. Three-dimensional tissue culture model of human breast cancer for the evaluation of multidrug resistance. J Tissue Eng Regen Med. 2018;12(9):1959–1971. doi: 10.1002/term.2729. PubMed DOI

Motiei M, Aboutalebi F, Forouzanfar M, Dormiani K, Nasr-Esfahani MH, Mirahmadi-Zare SZ. Smart co-delivery of miR-34a and cytotoxic peptides (LTX-315 and melittin) by chitosan based polyelectrolyte nanocarriers for specific cancer cell death induction. Mater Sci Eng C. 2021:112258. PubMed

Gandham SK, Rao M, Shah A, Trivedi MS, Amiji MM. Combination microRNA-based cellular reprogramming with paclitaxel enhances therapeutic efficacy in a relapsed and multidrug-resistant model of epithelial ovarian cancer. Mol Ther Oncolytics. 2022;25:57–68. doi: 10.1016/j.omto.2022.03.005. PubMed DOI PMC

Lo Y-L, Lin H-C, Tseng W-H. Tumor pH-functionalized and charge-tunable nanoparticles for the nucleus/cytoplasm-directed delivery of oxaliplatin and miRNA in the treatment of head and neck cancer. Acta Biomater. 2022;153:465–480. doi: 10.1016/j.actbio.2022.09.027. PubMed DOI

Xiao J, Weng J, Wen F, Ye J. Red blood cell membrane-coated silica nanoparticles codelivering DOX and ICG for effective lung cancer therapy. ACS Omega 2020. PubMed PMC

Han W, Shi L, Ren L, Zhou L, Li T, Qiao Y, et al. A nanomedicine approach enables co-delivery of cyclosporin A and gefitinib to potentiate the therapeutic efficacy in drug-resistant lung cancer. Signal Transduct Target Ther. 2018;3(1):1–10. PubMed PMC

Shen Y, TanTai J. Co-delivery anticancer drug nanoparticles for synergistic therapy against lung cancer cells. Drug Des Dev Ther. 2020;14:4503. doi: 10.2147/DDDT.S275123. PubMed DOI PMC

Liu J, He J, Zhang M, Xu G, Ni P. A synergistic polyphosphoester-based co-delivery system of the anticancer drug doxorubicin and the tumor suppressor gene p53 for lung cancer therapy. J Mater Chem B. 2018;6(20):3262–3273. doi: 10.1039/C8TB00746B. PubMed DOI

Taratula O, Kuzmov A, Shah M, Garbuzenko OB, Minko T. Nanostructured lipid carriers as multifunctional nanomedicine platform for pulmonary co-delivery of anticancer drugs and siRNA. J Control Release. 2013;171(3):349–357. doi: 10.1016/j.jconrel.2013.04.018. PubMed DOI PMC

Xu C, Wang Y, Guo Z, Chen J, Lin L, Wu J, et al. Pulmonary delivery by exploiting doxorubicin and cisplatin co-loaded nanoparticles for metastatic lung cancer therapy. J Control Release. 2019;295:153–163. doi: 10.1016/j.jconrel.2018.12.013. PubMed DOI

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(1):1–13. doi: 10.1186/1756-9966-31-1. PubMed DOI PMC

Li J, Che L, Xu C, Lu D, Xu Y, Liu M, et al. XIST/miR-34a-5p/PDL1 axis regulated the development of lung cancer cells and the immune function of CD8+ T cells. J Recept Signal Transduct. 2022:1–10. PubMed

Abtahi NA, Naghib SM, Ghalekohneh SJ, Mohammadpour Z, Nazari H, Mosavi SM, et al. Multifunctional stimuli-responsive niosomal nanoparticles for co-delivery and co-administration of gene and bioactive compound: In vitro and in vivo studies. Chem Eng J. 2022;429:132090. doi: 10.1016/j.cej.2021.132090. DOI

Trivedi M, Singh A, Talekar M, Pawar G, Shah P, Amiji M. MicroRNA-34a encapsulated in hyaluronic acid nanoparticles induces epigenetic changes with altered mitochondrial bioenergetics and apoptosis in non-small-cell lung cancer cells. Sci Rep. 2017;7(1):1–17. doi: 10.1038/s41598-017-02816-8. PubMed DOI PMC

Sharma P, Dando I, Strippoli R, Kumar S, Somoza A, Cordani M, et al. Nanomaterials for autophagy-related miRNA-34a delivery in cancer treatment. Front Pharmacol. 2020;11. PubMed PMC

Silva AS, Sousa AM, Cabral RP, Silva MC, Costa C, Miguel SP, et al. Aerosolizable gold nano-in-micro dry powder formulations for theragnosis and lung delivery. Int J Pharm. 2017;519(1–2):240–249. doi: 10.1016/j.ijpharm.2017.01.032. PubMed DOI

Darquenne C. Deposition mechanisms. J Aerosol Med Pulm Drug Deliv. 2020;33(4):181–185. doi: 10.1089/jamp.2020.29029.cd. PubMed DOI

Frederix EMA, Kuczaj AK, Nordlund M, Belka M, Lizal F, Jedelsky J, et al. Simulation of size-dependent aerosol deposition in a realistic model of the upper human airways. J Aerosol Sci. 2018;115:29–45. doi: 10.1016/j.jaerosci.2017.10.007. DOI

Restani RB, Silva AS, Pires RF, Cabral R, Correia IJ, Casimiro T, et al. Nano-in-micro POxylated polyurea dendrimers and chitosan dry powder formulations for pulmonary delivery. Part Part Syst Charact. 2016;33(11):851–858. doi: 10.1002/ppsc.201600123. DOI

Wang Y, Kho K, Cheow WS, Hadinoto K. A comparison between spray drying and spray freeze drying for dry powder inhaler formulation of drug-loaded lipid–polymer hybrid nanoparticles. Int J Pharm. 2012;424(1–2):98–106. doi: 10.1016/j.ijpharm.2011.12.045. PubMed DOI

Ruge CA, Bohr A, Beck-Broichsitter M, Nicolas V, Tsapis N, Fattal E. Disintegration of nano-embedded microparticles after deposition on mucus: a mechanistic study. Colloids Surf B. 2016;139:219–227. doi: 10.1016/j.colsurfb.2015.12.017. PubMed DOI

Motiei M, Kashanian S. Novel amphiphilic chitosan nanocarriers for sustained oral delivery of hydrophobic drugs. Eur J Pharm Sci. 2017;99:285–291. doi: 10.1016/j.ejps.2016.12.035. PubMed DOI

Yin L, Chen Y, Zhang Z, Yin Q, Zheng N, Cheng J. Biodegradable micelles capable of mannose-mediated targeted drug delivery to cancer cells. Macromol Rapid Commun. 2015;36(5):483–489. doi: 10.1002/marc.201400650. PubMed DOI PMC

Jaynes JM, Sable R, Ronzetti M, Bautista W, Knotts Z, Abisoye-Ogunniyan A, et al. Mannose receptor (CD206) activation in tumor-associated macrophages enhances adaptive and innate antitumor immune responses. Sci Transl Med. 2020;12(530):eaax6337. PubMed PMC

Wang W, Li W, Wang J, Hu Q, Balk M, Bieback K, et al. Folate receptor mediated genetic modification of human mesenchymal stem cells via folic acid-polyethylenimine-grafted poly (N-3-hydroxypropyl) aspartamide. Clin Hemorheol Microcirc. 2017(Preprint):1–17. PubMed

De Pauw E, Vervaet C, Vanhoorne V. Formation of delta-mannitol by co-spray drying: enhancing the tabletability of paracetamol/mannitol formulations. J Drug Deliv Sci Technol. 2022;77:103907. doi: 10.1016/j.jddst.2022.103907. DOI

Liu M, Li J, Li B. Mannose-modificated polyethylenimine: a specific and effective antibacterial agent against Escherichia coli. Langmuir. 2018;34(4):1574–1580. doi: 10.1021/acs.langmuir.7b03556. PubMed DOI

Motiei M, Sedlařík V, Lucia LA, Fei H, Münster L. Stabilization of chitosan-based polyelectrolyte nanoparticle cargo delivery biomaterials by a multiple ionic cross-linking strategy. Carbohydr Polym. 2020;231:115709. doi: 10.1016/j.carbpol.2019.115709. PubMed DOI

Chavan C, Bala P, Pal K, Kale S. Cross-linked chitosan-dextran sulphate vehicle system for controlled release of ciprofloxaxin drug: an ophthalmic application. OpenNano. 2017;2:28–36. doi: 10.1016/j.onano.2017.04.002. DOI

Nikolić GS, Cakić MD, Glišić S, Cvetković DJ, Mitić ŽJ, Marković DZ. Study of green nanoparticles and biocomplexes based on exopolysaccharide by modern Fourier transform spectroscopy. Fourier Transforms-High-tech Application and Current Trends: InTech; 2017.

Gorban IE, Soldatov MA, Butova VV, Medvedev PV, Burachevskaya OA, Belanova A, et al. l-leucine loading and release in MIL-100 nanoparticles. Int J Mol Sci. 2020;21(24):9758. doi: 10.3390/ijms21249758. PubMed DOI PMC

Berghian-Grosan C, Olenic L, Katona G, Perde-Schrepler M, Vulcu A. L-Leucine for gold nanoparticles synthesis and their cytotoxic effects evaluation. Amino Acids. 2014;46(11):2545–2552. doi: 10.1007/s00726-014-1814-z. PubMed DOI

Gawali SL, Barick B, Barick K, Hassan P. Effect of sugar alcohol on colloidal stabilization of magnetic nanoparticles for hyperthermia and drug delivery applications. J Alloys Compd. 2017;725:800–806. doi: 10.1016/j.jallcom.2017.07.206. DOI

López-León T, Carvalho E, Seijo B, Ortega-Vinuesa J, Bastos-González D. Physicochemical characterization of chitosan nanoparticles: electrokinetic and stability behavior. J Colloid Interface Sci. 2005;283(2):344–351. doi: 10.1016/j.jcis.2004.08.186. PubMed DOI

Mussi SV, Parekh G, Pattekari P, Levchenko T, Lvov Y, Ferreira LA, et al. Improved pharmacokinetics and enhanced tumor growth inhibition using a nanostructured lipid carrier loaded with doxorubicin and modified with a layer-by-layer polyelectrolyte coating. Int J Pharm. 2015;495(1):186–193. doi: 10.1016/j.ijpharm.2015.08.079. PubMed DOI PMC

Gaspar DP, Serra C, Lino PR, Gonçalves L, Taboada P, Remuñán-López C, et al. Microencapsulated SLN: an innovative strategy for pulmonary protein delivery. Int J Pharm. 2017;516(1–2):231–246. doi: 10.1016/j.ijpharm.2016.11.037. PubMed DOI

Kho K, Hadinoto K. Effects of excipient formulation on the morphology and aqueous re-dispersibility of dry-powder silica nano-aggregates. Colloids Surf A. 2010;359(1–3):71–81. doi: 10.1016/j.colsurfa.2010.01.066. DOI

Yu H, Teo J, Chew JW, Hadinoto K. Dry powder inhaler formulation of high-payload antibiotic nanoparticle complex intended for bronchiectasis therapy: spray drying versus spray freeze drying preparation. Int J Pharm. 2016;499(1–2):38–46. doi: 10.1016/j.ijpharm.2015.12.072. PubMed DOI

Muhsin MD, George G, Beagley K, Ferro V, Wang H, Islam N. Effects of chemical conjugation of L-leucine to chitosan on dispersibility and controlled release of drug from a nanoparticulate dry powder inhaler formulation. Mol Pharm. 2016;13(5):1455–1466. doi: 10.1021/acs.molpharmaceut.5b00859. PubMed DOI

Lebhardt T, Roesler S, Uusitalo HP, Kissel T. Surfactant-free redispersible nanoparticles in fast-dissolving composite microcarriers for dry-powder inhalation. Eur J Pharm Biopharm. 2011;78(1):90–96. doi: 10.1016/j.ejpb.2010.12.002. PubMed DOI

Demoly P, Hagedoorn P, de Boer AH, Frijlink HW. The clinical relevance of dry powder inhaler performance for drug delivery. Respir Med. 2014;108(8):1195–1203. doi: 10.1016/j.rmed.2014.05.009. PubMed DOI

Usmani OS, Biddiscombe MF, Barnes PJ. Regional lung deposition and bronchodilator response as a function of β2-agonist particle size. Am J Respir Crit Care Med. 2005;172(12):1497–1504. doi: 10.1164/rccm.200410-1414OC. PubMed DOI

Finlay WH. The mechanics of inhaled pharmaceutical aerosols: an introduction: Academic Press; 2001.

Soong T, Nicolaides P, Yu C, Soong S. A statistical description of the human tracheobronchial tree geometry. Respir Physiol. 1979;37(2):161–172. doi: 10.1016/0034-5687(79)90068-9. PubMed DOI

Lavorini F, Pistolesi M, Usmani OS. Recent advances in capsule-based dry powder inhaler technology. Multidiscip Respir Med. 2017;12(1):1–7. PubMed PMC

Xu Y, Turan ET, Shi Z, Franzyk H, Thakur A, Foged C. Inhalable composite microparticles containing siRNA-loaded lipid-polymer hybrid nanoparticles: Saccharides and leucine preserve aerosol performance and long-term physical stability. Front Drug Deliv. 2022;2:945459. doi: 10.3389/fddev.2022.945459. DOI

Kunda NK, Alfagih IM, Dennison SR, Somavarapu S, Merchant Z, Hutcheon GA, et al. Dry powder pulmonary delivery of cationic PGA-co-PDL nanoparticles with surface adsorbed model protein. Int J Pharm. 2015;492(1–2):213–222. doi: 10.1016/j.ijpharm.2015.07.015. PubMed DOI

Chani MTS. Fabrication and characterization of chitosan-CeO2-CdO nanocomposite based impedimetric humidity sensors. Int J Biol Macromol. 2022;194:377–383. doi: 10.1016/j.ijbiomac.2021.11.079. PubMed DOI

Haddrell AE, Lewis D, Church T, Vehring R, Murnane D, Reid JP. Pulmonary aerosol delivery and the importance of growth dynamics. Ther Deliv. 2017;8(12):1051–1061. doi: 10.4155/tde-2017-0093. PubMed DOI PMC

Motiei M, Aboutalebi F, Forouzanfar M, Dormiani K, Nasr-Esfahani MH, Mirahmadi-Zare SZ. Smart co-delivery of miR-34a and cytotoxic peptides (LTX-315 and melittin) by chitosan based polyelectrolyte nanocarriers for specific cancer cell death induction. Mater Sci Eng C. 2021;128:112258. doi: 10.1016/j.msec.2021.112258. PubMed DOI

Perry JL, Tian S, Sengottuvel N, Harrison EB, Gorentla BK, Kapadia CH, et al. Pulmonary delivery of nanoparticle-bound toll-like receptor 9 agonist for the treatment of metastatic lung cancer. ACS Nano. 2020;14(6):7200–7215. doi: 10.1021/acsnano.0c02207. PubMed DOI PMC

Wei L, Surma M, Gough G, Shi S, Lambert-Cheatham N, Chang J, et al. Dissecting the mechanisms of doxorubicin and oxidative stress-induced cytotoxicity: the involvement of actin cytoskeleton and ROCK1. PLoS ONE. 2015;10(7):e0131763. doi: 10.1371/journal.pone.0131763. PubMed DOI PMC

Motiei M, Kashanian S, Lucia LA, Khazaei M. Intrinsic parameters for the synthesis and tuned properties of amphiphilic chitosan drug delivery nanocarriers. J Control Release. 2017;260:213–225. doi: 10.1016/j.jconrel.2017.06.010. PubMed DOI

Li J, Li B, Liu M. One-step synthesis of mannose-modified polyethyleneimine copolymer particles as fluorescent probes for the detection of Escherichia coli. Sens Actuators B Chem. 2019;280:171–176. doi: 10.1016/j.snb.2018.10.018. DOI

Mitchell JP, Nagel MW, Wiersema KJ, Doyle CC. Aerodynamic particle size analysis of aerosols from pressurized metered-dose inhalers: comparison of Andersen 8-stage cascade impactor, next generation pharmaceutical impactor, and model 3321 Aerodynamic Particle Sizer aerosol spectrometer. AAPS PharmSciTech. 2003;4(4):E54. doi: 10.1208/pt040454. PubMed DOI PMC

Celluzzi A, Paolini A, D’Oria V, Risoluti R, Materazzi S, Pezzullo M, et al. Biophysical and biological contributions of polyamine-coated carbon nanotubes and bidimensional buckypapers in the delivery of miRNAs to human cells. Int J Nanomed. 2018;13:1. doi: 10.2147/IJN.S144155. PubMed DOI PMC

Indorf C, Dyckmans J, Khan KS, Joergensen RG. Optimisation of amino sugar quantification by HPLC in soil and plant hydrolysates. Biol Fertil Soils. 2011;47(4):387–396. doi: 10.1007/s00374-011-0545-5. DOI