This study recognized biologically produced gold nanoparticles (AuNPs) as multiple cargo carriers with a perspective of drug delivery into specialized tumor cells in vivo. Paclitaxel (PTX), transferrin, and antimiR-135b were conjugated with AuNPs and their uptake by mouse tumor cells in an induced breast cancer model was investigated. Each of the above-mentioned molecules was conjugated to the AuNPs separately as well as simultaneously, loading efficiency of each cargo was assessed, and performance of the final product (FP) was judged. After tumor induction in BALB/c mice, sub-IC50 doses of FP as well as control AuNPs, PTX, and phosphate buffered saline were administered in vivo. Round AuNPs were prepared using Fusarium oxysporum and exhibited a size of 13 ± 1.3 nm and a zeta potential of -35.8 ± 1.3 mV. The cytotoxicity of individual conjugates and FP were tested by MTT assay in breast tumor cells 4T1 and nontumor fibroblasts NIH/3T3 cells. The conjugation of individual molecules with AuNPs was confirmed, and FP (size of 54 ± 14 nm and zeta potential of -31.9 ± 2.08 mV) showed higher 4T1-specific toxicity in vitro when compared to control conjugates. After in vivo application of the FP, transmission electron microscopy analyses proved the presence of AuNPs in the tumor cells. Hematoxylin and eosin staining of the tumor tissue revealed that the FP group exhibited the highest amounts of inflammatory, necrotic, and apoptotic cells in contrast to the control groups. Finally, qPCR results showed that FP could transfect and suppress miR-135b expression in vivo, confirming the tumor-targeting properties of FP. The capacity of biologically produced gold nanoparticles to conjugate with multiple decorative molecules while retaining their stability and effective intracellular uptake makes them a promising alternative strategy superior to current drug carriers.

Center of Molecular Structure Institute of Biotechnology Czech Academy of Sciences Vestec 252 20 Czech Republic

Department of Medical Sciences Biological Nanoparticles in Medicine Research Center Shahrood Branch Islamic Azad University Shahrood 9WVM 5HC Iran

Department of Medical Sciences Shahrood Branch Islamic Azad University Shahrood 9WVM 5HC Iran

Faculty of Health Studies Technical University of Liberec Liberec 46001 Czech Republic

Institute of Analytical Chemistry Czech Academy of Sciences Brno 602 00 Czech Republic

Institute of Microbiology Czech Academy of Sciences Praha 142 20 Czech Republic

Zobrazit více v PubMed

Waks A. G.; Winer E. P. Breast cancer treatment: a review. JAMA 2019, 321 (3), 288–300. 10.1001/jama.2018.19323. PubMed DOI

Lei S.; Zheng R.; Zhang S.; Wang S.; Chen R.; Sun K.; Zeng H.; Zhou J.; Wei W. Global patterns of breast cancer incidence and mortality: A population-based cancer registry data analysis from 2000 to 2020. Cancer Commun. 2021, 41 (11), 1183–1194. 10.1002/cac2.12207. PubMed DOI PMC

Wu H.-C.; Chang D.-K.; Huang C.-T. Targeted therapy for cancer. J. Cancer Mol. 2006, 2 (2), 57–66.

Li Y.; Gao Y.; Zhang X.; Guo H.; Gao H. Nanoparticles in precision medicine for ovarian cancer: From chemotherapy to immunotherapy. Int. J. Pharm. 2020, 591, 119986.10.1016/j.ijpharm.2020.119986. PubMed DOI

Wang J.; Xu B. Targeted therapeutic options and future perspectives for HER2-positive breast cancer. Signal Transduction Targeted Ther. 2019, 4 (1), 34.10.1038/s41392-019-0069-2. PubMed DOI PMC

Perez H. L.; Cardarelli P. M.; Deshpande S.; Gangwar S.; Schroeder G. M.; Vite G. D.; Borzilleri R. M. Antibody-drug conjugates: current status and future directions. Drug Discovery Today 2014, 19 (7), 869–881. 10.1016/j.drudis.2013.11.004. PubMed DOI

Zardavas D.; Irrthum A.; Swanton C.; Piccart M. Clinical management of breast cancer heterogeneity. Nat. Rev. Clin. Oncol. 2015, 12 (7), 381–394. 10.1038/nrclinonc.2015.73. PubMed DOI

Wood K. C. Mapping the pathways of resistance to targeted therapies. Cancer Res. 2015, 75 (20), 4247–4251. 10.1158/0008-5472.CAN-15-1248. PubMed DOI PMC

Huang P. S.; Oliff A. Drug-targeting strategies in cancer therapy. Curr. Opin. Genet. Dev. 2001, 11 (1), 104–110. 10.1016/S0959-437X(00)00164-7. PubMed DOI

Vine K. L.; Lobov S.; Chandran V. I.; Harris N. L. E.; Ranson M. Improved pharmacokinetic and biodistribution properties of the selective urokinase inhibitor PAI-2 (SerpinB2) by site-specific PEGylation: implications for drug delivery. Pharm. Res. 2015, 32 (3), 1045–1054. 10.1007/s11095-014-1517-x. PubMed DOI

Yahyaei B.; Nouri M.; Bakherad S.; Hassani M.; Pourali P. Effects of biologically produced gold nanoparticles: toxicity assessment in different rat organs after intraperitoneal injection. AMB Express 2019, 9 (1), 38.10.1186/s13568-019-0762-0. PubMed DOI PMC

Yahyaei B.; Arabzadeh S.; Pourali P. An alternative method for biological production of silver and gold nanoparticles. JPAM 2014, 8, 4495–4501.

Pourali P.; Badiee S. H.; Manafi S.; Noorani T.; Rezaei A.; Yahyaei B. Biosynthesis of gold nanoparticles by two bacterial and fungal strains, Bacillus cereus and Fusarium oxysporum, and assessment and comparison of their nanotoxicity in vitro by direct and indirect assays. Electron. J. Biotechnol. 2017, 29, 86–93. 10.1016/j.ejbt.2017.07.005. DOI

Pourali P.; Yahyaei B.; Afsharnezhad S. Bio-Synthesis of Gold Nanoparticles by Fusarium oxysporum and Assessment of Their Conjugation Possibility with Two Types of β-Lactam Antibiotics without Any Additional Linkers. Microbiology 2018, 87 (2), 229–237. 10.1134/s0026261718020108. DOI

Pourali P.; Dzmitruk V.; Pátek M.; Neuhöferová E.; Svoboda M.; Benson V. Fate of the capping agent of biologically produced gold nanoparticles and adsorption of enzymes onto their surface. Sci. Rep. 2023, 13 (1), 4916.10.1038/s41598-023-31792-5. PubMed DOI PMC

Pourali P.; Neuhöferová E.; Dzmitruk V.; Benson V. Investigation of Protein Corona Formed around Biologically Produced Gold Nanoparticles. Materials 2022, 15 (13), 4615.10.3390/ma15134615. PubMed DOI PMC

Pourali P.; Benada O.; Pátek M.; Neuhöferová E.; Dzmitruk V.; Benson V. Response of Biological Gold Nanoparticles to Different pH Values: Is It Possible to Prepare Both Negatively and Positively Charged Nanoparticles?. Appl. Sci. 2021, 11 (23), 11559.10.3390/app112311559. DOI

Pourali P.; Baserisalehi M.; Afsharnezhad S.; Behravan J.; Ganjali R.; Bahador N.; Arabzadeh S. The effect of temperature on antibacterial activity of biosynthesized silver nanoparticles. BioMetals 2013, 26 (1), 189–196. 10.1007/s10534-012-9606-y. PubMed DOI

Yahyaei B.; Pourali P. One step conjugation of some chemotherapeutic drugs to the biologically produced gold nanoparticles and assessment of their anticancer effects. Sci. Rep. 2019, 9 (1), 10242.10.1038/s41598-019-46602-0. PubMed DOI PMC

Naimi-Shamel N.; Pourali P.; Dolatabadi S. Green synthesis of gold nanoparticles using Fusarium oxysporum and antibacterial activity of its tetracycline conjugant. J. Mycol. Med. 2019, 29 (1), 7–13. 10.1016/j.mycmed.2019.01.005. PubMed DOI

Roy N.; Gaur A.; Jain A.; Bhattacharya S.; Rani V. Green synthesis of silver nanoparticles: an approach to overcome toxicity. Environ. Toxicol. Pharmacol. 2013, 36 (3), 807–812. 10.1016/j.etap.2013.07.005. PubMed DOI

Składanowski M.; Golinska P.; Rudnicka K.; Dahm H.; Rai M. Evaluation of cytotoxicity, immune compatibility and antibacterial activity of biogenic silver nanoparticles. Med. Microbiol. Immunol. 2016, 205 (6), 603–613. 10.1007/s00430-016-0477-7. PubMed DOI PMC

Křivohlavá R.; Neuhüferová 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 (6), 866.10.3390/nano9060866. PubMed DOI PMC

Uva P.; Cossu-Rocca P.; Loi F.; Pira G.; Murgia L.; Orrù S.; Floris M.; Muroni M. R.; Sanges F.; Carru C.; et al. miRNA-135b contributes to triple negative breast cancer molecular heterogeneity: Different expression profile in Basal-like versus non-Basal-like phenotypes. Int. J. Med. Sci. 2018, 15 (6), 536–548. 10.7150/ijms.23402. PubMed DOI PMC

Hua K.; Jin J.; Zhao J.; Song J.; Song H.; Li D.; Maskey N.; Zhao B.; Wu C.; Xu H.; et al. miR-135b, upregulated in breast cancer, promotes cell growth and disrupts the cell cycle by regulating LATS2. Int. J. Oncol. 2016, 48 (5), 1997–2006. 10.3892/ijo.2016.3405. PubMed DOI

Kesarwani P.; Tekade R. K.; Jain N. Spectrophotometric estimation of paclitaxel. Int. J. Adv. Pharm. Sci. 2011, 2 (1), 29–32.

Pavelek Z.; Vyšata O.; Tambor V.; Pimková K.; Vu D. L.; Kuča K.; Št’ourač P.; Vališ M. Proteomic analysis of cerebrospinal fluid for relapsing-remitting multiple sclerosis and clinically isolated syndrome. Biomed. Rep. 2016, 5 (1), 35–40. 10.3892/br.2016.668. PubMed DOI PMC

Procházková E. k.; Kucherak O.; Stodůlková E.; Tošner Z. k.; Císařová I.; Flieger M.; Kolarik M.; Baszczyňski O. NMR Structure elucidation of naphthoquinones from Quambalaria cyanescens. J. Nat. Prod. 2021, 84 (1), 46–55. 10.1021/acs.jnatprod.0c00930. PubMed DOI

Ackerson C. J.; Sykes M. T.; Kornberg R. D. Defined DNA/nanoparticle conjugates. Proc. Natl. Acad. Sci. U.S.A. 2005, 102 (38), 13383–13385. 10.1073/pnas.0506290102. PubMed DOI PMC

Wang H.-Q.; Deng Z.-X. Gel electrophoresis as a nanoseparation tool serving DNA nanotechnology. Chin. Chem. Lett. 2015, 26 (12), 1435–1438. 10.1016/j.cclet.2015.10.019. DOI

Vajedi F. S.; Dehghani H.; Zarrabi A. Design and characterization of a novel pH-sensitive biocompatible and multifunctional nanocarrier for in vitro paclitaxel release. Mater. Sci. Eng.: C 2021, 119, 111627.10.1016/j.msec.2020.111627. PubMed DOI

Hazekawa M.; Nishinakagawa T.; Kawakubo-Yasukochi T.; Nakashima M. Evaluation of IC50 levels immediately after treatment with anticancer reagents using a real-time cell monitoring device. Exp. Ther. Med. 2019, 18 (4), 3197–3205. 10.3892/etm.2019.7876. PubMed DOI PMC

Li C.; Price J. E.; Milas L.; Hunter N. R.; Ke S.; Yu D.-F.; Charnsangavej C.; Wallace S. Antitumor activity of poly (L-glutamic acid)-paclitaxel on syngeneic and xenografted tumors. Clin. Cancer Res. 1999, 5 (4), 891–897. PubMed

Pourali P.; Nouri M.; Ameri F.; Heidari T.; Kheirkhahan N.; Arabzadeh S.; Yahyaei B. Histopathological study of the maternal exposure to the biologically produced silver nanoparticles on different organs of the offspring. Naunyn-Schmiedeberg’s Arch. Pharmacol. 2020, 393 (5), 867–878. 10.1007/s00210-019-01796-y. PubMed DOI

Pourali P.; Dzmitruk V.; Benada O.; Svoboda M.; Benson V. Conjugation of microbial-derived gold nanoparticles to different types of nucleic acids: evaluation of transfection efficiency. Sci. Rep. 2023, 13 (1), 14669.10.1038/s41598-023-41567-7. PubMed DOI PMC

England C. G.; Miller M. C.; Kuttan A.; Trent J. O.; Frieboes H. B. Release kinetics of paclitaxel and cisplatin from two and three layered gold nanoparticles. Eur. J. Pharm. Biopharm. 2015, 92, 120–129. 10.1016/j.ejpb.2015.02.017. PubMed DOI PMC

Manivasagan P.; Bharathiraja S.; Bui N. Q.; Lim I. G.; Oh J. Paclitaxel-loaded chitosan oligosaccharide-stabilized gold nanoparticles as novel agents for drug delivery and photoacoustic imaging of cancer cells. Int. J. Pharm. 2016, 511 (1), 367–379. 10.1016/j.ijpharm.2016.07.025. PubMed DOI

Ng C. T.; Tang F. M. A.; Li J. J. e.; Ong C.; Yung L. L. Y.; Bay B. H. Clathrin-mediated endocytosis of gold nanoparticles in vitro. Anat. Rec. 2015, 298 (2), 418–427. 10.1002/ar.23051. PubMed DOI

De Jong J.; van Diest P. J.; Baak J. Number of apoptotic cells as a prognostic marker in invasive breast cancer. Br. J. Cancer 2000, 82 (2), 368–373. 10.1054/bjoc.1999.0928. PubMed DOI PMC

Koren E.; Fuchs Y. Modes of regulated cell death in cancer. Cancer Discovery 2021, 11 (2), 245–265. 10.1158/2159-8290.CD-20-0789. PubMed DOI

Liu Z.-g.; Jiao D. Necroptosis, tumor necrosis and tumorigenesis. Cell Stress 2020, 4 (1), 1–8. 10.15698/cst2020.01.208. PubMed DOI PMC

Calaf G. M.; Ponce-Cusi R.; Carrión F. Curcumin and paclitaxel induce cell death in breast cancer cell lines. Oncol. Rep. 2018, 40 (4), 2381–2388. PubMed

Wang T. H.; Wang H. S.; Soong Y. K. Paclitaxel-induced cell death: where the cell cycle and apoptosis come together. Cancer 2000, 88 (11), 2619–2628. 10.1002/1097-0142(20000601)88:11<2619::AID-CNCR26>3.0.CO;2-J. PubMed DOI

Yahyaei B.; Pourali P.; Hassani M. Morphological Change of Kidney after Injection of the Biological Gold Nanoparticles in Wistar Rats. J. Anim. Biol. 2020, 13 (1), 109–119.

Zhou Y.; Zhou J.; Wang F.; Yang H. Polydopamine-based functional composite particles for tumor cell targeting and dual-mode cellular imaging. Talanta 2018, 181, 248–257. 10.1016/j.talanta.2018.01.003. PubMed DOI

Pourali P.; Svoboda M.; Neuhöferová E.; Dzmitruk V.; Benson V.. Accumulation and toxicity of biologically produced gold nanoparticles in different types of specialized mammalian cells. Biotechnol. Appl. Biochem. 202410.1002/bab.2575, Online publication ahead of print. PubMed DOI

Chaumet A.; Wright G. D.; Seet S. H.; Tham K. M.; Gounko N. V.; Bard F. Nuclear envelope-associated endosomes deliver surface proteins to the nucleus. Nat. Commun. 2015, 6 (1), 8218.10.1038/ncomms9218. PubMed DOI PMC

Degors I. M.; Wang C.; Rehman Z. U.; Zuhorn I. S. Carriers break barriers in drug delivery: endocytosis and endosomal escape of gene delivery vectors. Acc. Chem. Res. 2019, 52 (7), 1750–1760. 10.1021/acs.accounts.9b00177. PubMed DOI PMC

Egorova E. A.; Lamers G. E.; Monikh F. A.; Boyle A. L.; Slütter B.; Kros A. Gold nanoparticles decorated with ovalbumin-derived epitopes: effect of shape and size on T-cell immune responses. RSC Adv. 2022, 12 (31), 19703–19716. 10.1039/D2RA03027F. PubMed DOI PMC

Daniele R.; Brazzale C.; Arpac B.; Tognetti F.; Pesce C.; Malfanti A.; Sayers E.; Mastrotto F.; Jones A. T.; Salmaso S.; et al. Influence of folate-targeted gold nanoparticles on subcellular localization and distribution into lysosomes. Pharmaceutics 2023, 15 (3), 864.10.3390/pharmaceutics15030864. PubMed DOI PMC

Yoo J.-W.; Irvine D. J.; Discher D. E.; Mitragotri S. Bio-inspired, bioengineered and biomimetic drug delivery carriers. Nat. Rev. Drug Discovery 2011, 10 (7), 521–535. 10.1038/nrd3499. PubMed DOI

Bechara C.; Sagan S. Cell-penetrating peptides: 20 years later, where do we stand?. FEBS Lett. 2013, 587 (12), 1693–1702. 10.1016/j.febslet.2013.04.031. PubMed DOI

Cesbron Y.; Shaheen U.; Free P.; Levy R. TAT and HA2 facilitate cellular uptake of gold nanoparticles but do not lead to cytosolic localisation. PLoS One 2015, 10 (4), e012168310.1371/journal.pone.0121683. PubMed DOI PMC

Davis M. E.; Zuckerman J. E.; Choi C. H. J.; Seligson D.; Tolcher A.; Alabi C. A.; Yen Y.; Heidel J. D.; Ribas A. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 2010, 464 (7291), 1067–1070. 10.1038/nature08956. PubMed DOI PMC

Tu Y.; Zhao L.; Billadeau D. D.; Jia D. Endosome-to-TGN trafficking: organelle-vesicle and organelle-organelle interactions. Front. Cell Dev. Biol. 2020, 8, 163.10.3389/fcell.2020.00163. PubMed DOI PMC

Le Blanc I.; Luyet P.-P.; Pons V.; Ferguson C.; Emans N.; Petiot A.; Mayran N.; Demaurex N.; Fauré J.; Sadoul R.; et al. Endosome-to-cytosol transport of viral nucleocapsids. Nat. Cell Biol. 2005, 7 (7), 653–664. 10.1038/ncb1269. PubMed DOI PMC

Behzadi S.; Serpooshan V.; Tao W.; Hamaly M. A.; Alkawareek M. Y.; Dreaden E. C.; Brown D.; Alkilany A. M.; Farokhzad O. C.; Mahmoudi M. Cellular uptake of nanoparticles: journey inside the cell. Chem. Soc. Rev. 2017, 46 (14), 4218–4244. 10.1039/C6CS00636A. PubMed DOI PMC

Pourali P.; Svoboda M.; Benada O.; Dzmitruk V.; Benson V. Biological Production of Gold Nanoparticles at Different Temperatures: Efficiency Assessment. Part. Part. Syst. Charact. 2023, 40 (12), 2200182.10.1002/ppsc.202200182. DOI

Clarance P.; Luvankar B.; Sales J.; Khusro A.; Agastian P.; Tack J.-C.; Al Khulaifi M. M.; Al-Shwaiman H. A.; Elgorban A. M.; Syed A.; et al. Green synthesis and characterization of gold nanoparticles using endophytic fungi Fusarium solani and its in-vitro anticancer and biomedical applications. Saudi J. Biol. Sci. 2020, 27 (2), 706–712. 10.1016/j.sjbs.2019.12.026. PubMed DOI PMC

Hosseinzadeh N.; Shomali T.; Hosseinzadeh S.; Raouf Fard F.; Pourmontaseri M.; Fazeli M. Green synthesis of gold nanoparticles by using Ferula persica Willd. gum essential oil: production, characterization and in vitro anti-cancer effects. J. Pharm. Pharmacol. 2020, 72 (8), 1013–1025. 10.1111/jphp.13274. PubMed DOI

Khanzada B.; Akthar N.; Bhatti M. Z.; Ismail H.; Alqarni M.; Mirza B.; Mostafa-Hedeab G.; Batiha G. E.-S. Green synthesis of gold and iron nanoparticles for targeted delivery: an in vitro and in vivo study. J. Chem. 2021, 2021 (1), 1–16. 10.1155/2021/1581444. DOI

Mukherjee S.; Sau S.; Madhuri D.; Bollu V. S.; Madhusudana K.; Sreedhar B.; Banerjee R.; Patra C. R. Green synthesis and characterization of monodispersed gold nanoparticles: toxicity study, delivery of doxorubicin and its bio-distribution in mouse model. J. Biomed. Nanotechnol. 2016, 12 (1), 165–181. 10.1166/jbn.2016.2141. PubMed DOI

Paciotti G. F.; Zhao J.; Cao S.; Brodie P. J.; Tamarkin L.; Huhta M.; Myer L. D.; Friedman J.; Kingston D. G. Synthesis and evaluation of paclitaxel-loaded gold nanoparticles for tumor-targeted drug delivery. Bioconjugate Chem. 2016, 27 (11), 2646–2657. 10.1021/acs.bioconjchem.6b00405. PubMed DOI PMC

Hale S. J.; Perrins R. D.; Garcıa C. E.; Pace A.; Peral U.; Patel K. R.; Robinson A.; Williams P.; Ding Y.; Saito G.; et al. DM1 loaded ultrasmall gold nanoparticles display significant efficacy and improved tolerability in murine models of hepatocellular carcinoma. Bioconjugate Chem. 2018, 30 (3), 703–713. 10.1021/acs.bioconjchem.8b00873. PubMed DOI

Govindaraju S.; Roshini A.; Lee M.-H.; Yun K. Kaempferol conjugated gold nanoclusters enabled efficient for anticancer therapeutics to A549 lung cancer cells. Int. J. Nanomed. 2019, 14, 5147–5157. 10.2147/ijn.s209773. PubMed DOI PMC

Paris J. L.; Villaverde G.; Gómez-Graña S.; Vallet-Regí M. Nanoparticles for multimodal antivascular therapeutics: Dual drug release, photothermal and photodynamic therapy. Acta Biomater. 2020, 101, 459–468. 10.1016/j.actbio.2019.11.004. PubMed DOI PMC