Login Register Cart 0
Generic placeholder image

Current Drug Delivery

Editor-in-Chief

ISSN (Print): 1567-2018
ISSN (Online): 1875-5704

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe
Research Article

Macrophage-Mediated Delivery of Fe3O4-Nanoparticles: A Generalized Strategy to Deliver Iron to Tumor Microenvironment

Author(s): Cong Wu, Guozhong Zhang, Zhihao Wang and Hongcan Shi*

Volume 19, Issue 9, 2022

Published on: 25 May, 2022

Page: [928 - 939] Pages: 12

DOI: 10.2174/1567201819666220426085450

Price: $65

Abstract

Background: Iron is used to alter macrophage phenotypes and induce tumor cell death. Iron oxide nanoparticles can induce macrophage polarization into the M1 phenotype, which inhibits tumor growth and can dissociate into iron ions in macrophages.

Objective: In this study, we proposed to construct high expression of Ferroportin1 macrophages as carriers to deliver Fe3O4-nanoparticles and iron directly to tumor sites.

Methods: Three sizes of Fe3O4-nanoparticles with gradient concentrations were used. The migration ability of iron-carrying macrophages was confirmed by an in vitro migration experiment and monocyte chemoattractant protein-1 detection. The release of iron from macrophages was confirmed by determining their levels in the cell culture supernatant, and we constructed a high expression of ferroportin strain of macrophage lines to increase intracellular iron efflux by increasing membrane transferrin expression. Fe3O4-NPs in Ana-1 cells were degraded in lysosomes, and the amount of iron released was correlated with the expression of ferroportin1.

Results: After Fe3O4-nanoparticles uptake by macrophages, not only polarized macrophages into M1 phenotype, but the nanoparticles also dissolved in the lysosome and iron were released out of the cell. FPN1 is the only known Fe transporter; we use a Lentiviral vector carrying the FPN1 gene transfected into macrophages, has successfully constructed Ana-1-FPN1 cells, and maintains high expression of FPN1. Ana-1-FPN1 cells increase intracellular iron release. Fe3O4-nanoparticles loaded with engineered Ana-1 macrophages can act as a “reservoir” of iron.

Conclusion: Our study provides proof of strategy for Fe3O4-NPs target delivery to the tumor microenvironment. Moreover, increase of intracellular iron efflux by overexpression of FPN1, cell carriers can act as a reservoir for iron, providing the basis for targeted delivery of Fe3O4-NPs and iron ions in vivo.

Keywords: Macrophage, polarization, Fe3O4-nanoparticles, cell carrier, ferroportin1, lentiviral transfection.

« Previous Next »
Graphical Abstract

[1]
de Melo-Diogo, D.; Pais-Silva, C.; Dias, D.R.; Moreira, A.F.; Correia, I.J. Strategies to improve cancer photothermal therapy mediated by nanomaterials. Adv. Healthc. Mater., 2017, 6(10), 1700073.
[http://dx.doi.org/10.1002/adhm.201700073] [PMID: 28322514]
[2]
Souho, T.; Lamboni, L.; Xiao, L.; Yang, G. Cancer hallmarks and malignancy features: Gateway for improved targeted drug delivery. Biotechnol. Adv., 2018, 36(7), 1928-1945.
[http://dx.doi.org/10.1016/j.biotechadv.2018.08.001] [PMID: 30077715]
[3]
El Sayed, S.M. Enhancing anticancer effects, decreasing risks and solving practical problems facing 3-bromopyruvate in clinical oncology: 10 years of research experience. Int. J. Nanomedicine, 2018, 13, 4699-4709.
[http://dx.doi.org/10.2147/IJN.S170564] [PMID: 30154655]
[4]
Bobo, D.; Robinson, K.J.; Islam, J.; Thurecht, K.J.; Corrie, S.R. Nanoparticle-based medicines: A review of FDA-approved materials and clinical trials to date. Pharm. Res., 2016, 33(10), 2373-2387.
[http://dx.doi.org/10.1007/s11095-016-1958-5] [PMID: 27299311]
[5]
Pang, L.; Zhang, C.; Qin, J.; Han, L.; Li, R.; Hong, C.; He, H.; Wang, J. A novel strategy to achieve effective drug delivery: Exploit cells as carrier combined with nanoparticles. Drug Deliv., 2017, 24(1), 83-91.
[http://dx.doi.org/10.1080/10717544.2016.1230903] [PMID: 28155538]
[6]
Wang, P.; Wu, W.; Gao, R.; Zhu, H.; Wang, J.; Du, R.; Li, X.; Zhang, C.; Cao, S.; Xiang, R. Engineered cell-assisted photoactive nanoparticle delivery for image-guided synergistic photodynamic/photothermal therapy of cancer. ACS Appl. Mater. Interfaces, 2019, 11(15), 13935-13944.
[http://dx.doi.org/10.1021/acsami.9b00022] [PMID: 30915833]
[7]
Anselmo, A.C.; Gilbert, J.B.; Kumar, S.; Gupta, V.; Cohen, R.E.; Rubner, M.F.; Mitragotri, S. Monocyte-mediated delivery of polymeric backpacks to inflamed tissues: A generalized strategy to deliver drugs to treat inflammation. J. Control. Release, 2015, 199, 29-36.
[http://dx.doi.org/10.1016/j.jconrel.2014.11.027] [PMID: 25481443]
[8]
Tsukamoto, H.; Fujieda, K.; Miyashita, A.; Fukushima, S.; Ikeda, T.; Kubo, Y.; Senju, S.; Ihn, H.; Nishimura, Y.; Oshiumi, H. Combined blockade of IL6 and PD-1/PD-L1 signaling abrogates mutual regulation of their immunosuppressive effects in the tumor microenvironment. Cancer Res., 2018, 78(17), 5011-5022.
[http://dx.doi.org/10.1158/0008-5472.CAN-18-0118] [PMID: 29967259]
[9]
Binnewies, M.; Roberts, E.W.; Kersten, K.; Chan, V.; Fearon, D.F.; Merad, M.; Coussens, L.M.; Gabrilovich, D.I.; Ostrand-Rosenberg, S.; Hedrick, C.C.; Vonderheide, R.H.; Pittet, M.J.; Jain, R.K.; Zou, W.; Howcroft, T.K.; Woodhouse, E.C.; Weinberg, R.A.; Krummel, M.F. Un-derstanding the tumor immune microenvironment (TIME) for effective therapy. Nat. Med., 2018, 24(5), 541-550.
[http://dx.doi.org/10.1038/s41591-018-0014-x] [PMID: 29686425]
[10]
Taniguchi, K.; Hikiji, H.; Okinaga, T.; Hashidate-Yoshida, T.; Shindou, H.; Ariyoshi, W.; Shimizu, T.; Tominaga, K.; Nishihara, T. Essential role of lysophosphatidylcholine acyltransferase 3 in the induction of macrophage polarization in PMA-treated U937 cells. J. Cell. Biochem., 2015, 116(12), 2840-2848.
[http://dx.doi.org/10.1002/jcb.25230] [PMID: 25994902]
[11]
DeNardo, D.G.; Ruffell, B. Macrophages as regulators of tumour immunity and immunotherapy. Nat. Rev. Immunol., 2019, 19(6), 369-382.
[http://dx.doi.org/10.1038/s41577-019-0127-6] [PMID: 30718830]
[12]
Ostuni, R.; Kratochvill, F.; Murray, P.J.; Natoli, G. Macrophages and cancer: From mechanisms to therapeutic implications. Trends Immunol., 2015, 36(4), 229-239.
[http://dx.doi.org/10.1016/j.it.2015.02.004] [PMID: 25770924]
[13]
Daldrup-Link, H.E.; Golovko, D.; Ruffell, B.; Denardo, D.G.; Castaneda, R.; Ansari, C.; Rao, J.; Tikhomirov, G.A.; Wendland, M.F.; Corot, C.; Coussens, L.M. MRI of tumor-associated macrophages with clinically applicable iron oxide nanoparticles. Clin. Cancer Res., 2011, 17(17), 5695-5704.
[http://dx.doi.org/10.1158/1078-0432.CCR-10-3420] [PMID: 21791632]
[14]
Ansari, C.; Tikhomirov, G.A.; Hong, S.H.; Falconer, R.A.; Loadman, P.M.; Gill, J.H. Development of novel tumor-targeted theranostic nanoparti-cles activated by membrane-type matrix metalloproteinases for combined cancer magnetic resonance imaging and therapy. Small, 2014, 10, 566-575.
[http://dx.doi.org/10.1002/smll.201301456]
[15]
Zanganeh, S.; Hutter, G.; Spitler, R.; Lenkov, O.; Mahmoudi, M.; Shaw, A.; Pajarinen, J.S.; Nejadnik, H.; Goodman, S.; Moseley, M.; Coussens, L.M.; Daldrup-Link, H.E. Iron oxide nanoparticles inhibit tumour growth by inducing pro-inflammatory macrophage polarization in tumour tissues. Nat. Nanotechnol., 2016, 11(11), 986-994.
[http://dx.doi.org/10.1038/nnano.2016.168] [PMID: 27668795]
[16]
Alphandéry, E. Biodistribution and targeting properties of iron oxide nanoparticles for treatments of cancer and iron anemia disease. Nanotoxicology, 2019, 13(5), 573-596.
[http://dx.doi.org/10.1080/17435390.2019.1572809] [PMID: 30938215]
[17]
Peng, F.; Setyawati, M.I.; Tee, J.K.; Ding, X.; Wang, J.; Nga, M.E.; Ho, H.K.; Leong, D.T. Nanoparticles promote in vivo breast cancer cell intravasation and extravasation by inducing endothelial leakiness. Nat. Nanotechnol., 2019, 14(3), 279-286.
[http://dx.doi.org/10.1038/s41565-018-0356-z] [PMID: 30692675]
[18]
Cairo, G.; Recalcati, S.; Mantovani, A.; Locati, M. Iron trafficking and metabolism in macrophages: Contribution to the polarized phenotype. Trends Immunol., 2011, 32(6), 241-247.
[http://dx.doi.org/10.1016/j.it.2011.03.007] [PMID: 21514223]
[19]
Sindrilaru, A.; Peters, T.; Wieschalka, S.; Baican, C.; Baican, A.; Peter, H.; Hainzl, A.; Schatz, S.; Qi, Y.; Schlecht, A.; Weiss, J.M.; Wlaschek, M.; Sunderkötter, C.; Scharffetter-Kochanek, K. An unrestrained proinflammatory M1 macrophage population induced by iron impairs wound healing in humans and mice. J. Clin. Invest., 2011, 121(3), 985-997.
[http://dx.doi.org/10.1172/JCI44490] [PMID: 21317534]
[20]
Laskar, A.; Eilertsen, J.; Li, W.; Yuan, X.M. SPION primes THP1 derived M2 macrophages towards M1-like macrophages. Biochem. Biophys. Res. Commun., 2013, 441(4), 737-742.
[http://dx.doi.org/10.1016/j.bbrc.2013.10.115] [PMID: 24184477]
[21]
Hu, G.; Guo, M.; Xu, J.; Wu, F.; Fan, J.; Huang, Q.; Yang, G.; Lv, Z.; Wang, X.; Jin, Y. Nanoparticles targeting macrophages as potential clini-cal therapeutic agents against cancer and inflammation. Front. Immunol., 2019, 10, 1998.
[http://dx.doi.org/10.3389/fimmu.2019.01998] [PMID: 31497026]
[22]
Zhu, L.; Yang, T.; Li, L.; Sun, L.; Hou, Y.; Hu, X.; Zhang, L.; Tian, H.; Zhao, Q.; Peng, J.; Zhang, H.; Wang, R.; Yang, Z.; Zhang, L.; Zhao, Y. TSC1 controls macrophage polarization to prevent inflammatory disease. Nat. Commun., 2014, 5, 4696.
[http://dx.doi.org/10.1038/ncomms5696] [PMID: 25175012]
[23]
Chanmee, T.; Ontong, P.; Konno, K.; Itano, N. Tumor-associated macrophages as major players in the tumor microenvironment. Cancers (Basel), 2014, 6(3), 1670-1690.
[http://dx.doi.org/10.3390/cancers6031670] [PMID: 25125485]
[24]
Yang, C.Y.; Hsiao, J.K.; Tai, M.F.; Chen, S.T.; Cheng, H.Y.; Wang, J.L.; Liu, H.M. Direct labeling of hMSC with SPIO: The long-term influ-ence on toxicity, chondrogenic differentiation capacity, and intracellular distribution. Mol. Imaging Biol., 2011, 13(3), 443-451.
[http://dx.doi.org/10.1007/s11307-010-0360-7] [PMID: 20567925]
[25]
Jin, R.; Liu, L.; Zhu, W.; Li, D.; Yang, L.; Duan, J.; Cai, Z.; Nie, Y.; Zhang, Y.; Gong, Q.; Song, B.; Wen, L.; Anderson, J.M.; Ai, H. Iron ox-ide nanoparticles promote macrophage autophagy and inflammatory response through activation of toll-like Receptor-4 signaling. Biomaterials, 2019, 203, 23-30.
[http://dx.doi.org/10.1016/j.biomaterials.2019.02.026] [PMID: 30851490]
[26]
Arbab, A.S.; Wilson, L.B.; Ashari, P.; Jordan, E.K.; Lewis, B.K.; Frank, J.A. A model of lysosomal metabolism of dextran coated superpara-magnetic iron oxide (SPIO) nanoparticles: implications for cellular magnetic resonance imaging. NMR Biomed., 2005, 18(6), 383-389.
[http://dx.doi.org/10.1002/nbm.970] [PMID: 16013087]
[27]
Soenen, S.J.H.; Himmelreich, U.; Nuytten, N.; Pisanic, T.R., II; Ferrari, A.; De Cuyper, M. Intracellular nanoparticle coating stability deter-mines nanoparticle diagnostics efficacy and cell functionality. Small, 2010, 6(19), 2136-2145.
[http://dx.doi.org/10.1002/smll.201000763] [PMID: 20818621]
[28]
Zhang, Z.; Zhang, F.; An, P.; Guo, X.; Shen, Y.; Tao, Y.; Wu, Q.; Zhang, Y.; Yu, Y.; Ning, B.; Nie, G.; Knutson, M.D.; Anderson, G.J.; Wang, F. Ferroportin1 deficiency in mouse macrophages impairs iron homeostasis and inflammatory responses. Blood, 2011, 118(7), 1912-1922.
[http://dx.doi.org/10.1182/blood-2011-01-330324] [PMID: 21705499]
[29]
Donovan, A.; Brownlie, A.; Zhou, Y.; Shepard, J.; Pratt, S.J.; Moynihan, J.; Paw, B.H.; Drejer, A.; Barut, B.; Zapata, A.; Law, T.C.; Brugnara, C.; Lux, S.E.; Pinkus, G.S.; Pinkus, J.L.; Kingsley, P.D.; Palis, J.; Fleming, M.D.; Andrews, N.C.; Zon, L.I. Positional cloning of zebrafish fer-roportin1 identifies a conserved vertebrate iron exporter. Nature, 2000, 403(6771), 776-781.
[http://dx.doi.org/10.1038/35001596] [PMID: 10693807]
[30]
Knutson, M.D.; Oukka, M.; Koss, L.M.; Aydemir, F.; Wessling-Resnick, M. Iron release from macrophages after erythrophagocytosis is up-regulated by ferroportin 1 overexpression and down-regulated by hepcidin. Proc. Natl. Acad. Sci. USA, 2005, 102(5), 1324-1328.
[http://dx.doi.org/10.1073/pnas.0409409102] [PMID: 15665091]
[31]
Ravasi, G.; Pelucchi, S.; Russo, A.; Mariani, R.; Piperno, A. Ferroportin disease: A novel SLC40A1 mutation. Dig. Liver Dis., 2020, 52(6), 688-690.
[http://dx.doi.org/10.1016/j.dld.2020.03.013] [PMID: 32360131]
[32]
Liang, H.; Guo, J.; Shi, Y.; Zhao, G.; Sun, S.; Sun, X. Porous yolk-shell Fe/Fe3O4 nanoparticles with controlled exposure of highly active Fe(0) for cancer therapy. Biomaterials, 2021, 268, 120530.
[http://dx.doi.org/10.1016/j.biomaterials.2020.120530] [PMID: 33296795]
[33]
Mahmoudi, M.; Sant, S.; Wang, B.; Laurent, S.; Sen, T. Superparamagnetic iron oxide nanoparticles (SPIONs): development, surface modifi-cation and applications in chemotherapy. Adv. Drug Deliv. Rev., 2011, 63(1-2), 24-46.
[http://dx.doi.org/10.1016/j.addr.2010.05.006] [PMID: 20685224]
[34]
Tian, F.; Chen, G.; Yi, P.; Zhang, J.; Li, A.; Zhang, J.; Zheng, L.; Deng, Z.; Shi, Q.; Peng, R.; Wang, Q. Fates of Fe3O4 and Fe3O4@SiO2 nano-particles in human mesenchymal stem cells assessed by synchrotron radiation-based techniques. Biomaterials, 2014, 35(24), 6412-6421.
[http://dx.doi.org/10.1016/j.biomaterials.2014.04.052] [PMID: 24814428]
[35]
Wu, C.; Xu, Y.; Yang, L.; Wu, J.; Zhu, W.; Li, D. Negatively charged magnetite nanoparticle clusters as efficient MRI probes for dendritic cell labeling and in vivo tracking. Adv. Funct. Mater., 2015, 25, 3581-3591.
[http://dx.doi.org/10.1002/adfm.201501031]
[36]
Zhang, S.; Shu, W.V.; Yiru, S.; Yunqi, X. Shi LGAS. Cytotoxicity studies of Fe3O4 nanoparticles in chicken macrophage cells. R. Soc. Open Sci., 2020, 7(4), 191561.
[http://dx.doi.org/10.1098/rsos.191561] [PMID: 32431865]
[37]
Cairo, G.; Bernuzzi, F.; Recalcati, S. A precious metal: Iron, an essential nutrient for all cells. Genes Nutr., 2006, 1(1), 25-39.
[http://dx.doi.org/10.1007/BF02829934] [PMID: 18850218]
[38]
Hentze, M.W.; Muckenthaler, M.U.; Galy, B.; Camaschella, C. Two to tango: Regulation of Mammalian iron metabolism. Cell, 2010, 142(1), 24-38.
[http://dx.doi.org/10.1016/j.cell.2010.06.028] [PMID: 20603012]
[39]
Soenen, S.J.H.; Nuytten, N.; De Meyer, S.F.; De Smedt, S.C.; De Cuyper, M. High intracellular iron oxide nanoparticle concentrations affect cellular cytoskeleton and focal adhesion kinase-mediated signaling. Small, 2010, 6(7), 832-842.
[http://dx.doi.org/10.1002/smll.200902084] [PMID: 20213651]
[40]
Biswas, S.K.; Mantovani, A. Orchestration of metabolism by macrophages. Cell Metab., 2012, 15(4), 432-437.
[http://dx.doi.org/10.1016/j.cmet.2011.11.013] [PMID: 22482726]
[41]
Recalcati, S.; Locati, M.; Gammella, E.; Invernizzi, P.; Cairo, G. Iron levels in polarized macrophages: Regulation of immunity and autoim-munity. Autoimmun. Rev., 2012, 11(12), 883-889.
[http://dx.doi.org/10.1016/j.autrev.2012.03.003] [PMID: 22449938]
[42]
Zhou, Y.; Que, K.T.; Zhang, Z.; Yi, Z.J.; Zhao, P.X.; You, Y.; Gong, J.P.; Liu, Z.J. Iron overloaded polarizes macrophage to proinflammation phenotype through ROS/acetyl-p53 pathway. Cancer Med., 2018, 7(8), 4012-4022.
[http://dx.doi.org/10.1002/cam4.1670] [PMID: 29989329]
[43]
Ovais, M.; Guo, M.; Chen, C. Tailoring nanomaterials for targeting tumor-associated macrophages. Adv. Mater., 2019, 31(19), e1808303.
[http://dx.doi.org/10.1002/adma.201808303] [PMID: 30883982]
[44]
Ahamed, M.; Akhtar, M.J.; Khan, M.A.M.; Alhadlaq, H.A.; Alshamsan, A. Cobalt iron oxide nanoparticles induce cytotoxicity and regulate the apoptotic genes through ROS in human liver cells (HepG2). Colloids Surf. B Biointerfaces, 2016, 148, 665-673.
[http://dx.doi.org/10.1016/j.colsurfb.2016.09.047] [PMID: 27701048]
[45]
Huang, H.; Chen, J.; Lu, H.; Zhou, M.; Chai, Z.; Hu, Y. Iron-induced generation of mitochondrial ROS depends on AMPK activity. Biometals, 2017, 30(4), 623-628.
[http://dx.doi.org/10.1007/s10534-017-0023-0] [PMID: 28608291]
[46]
Gaharwar, U.S.; Meena, R.; Rajamani, P. Iron oxide nanoparticles induced cytotoxicity, oxidative stress and DNA damage in lymphocytes. J. Appl. Toxicol., 2017, 37(10), 1232-1244.
[http://dx.doi.org/10.1002/jat.3485] [PMID: 28585739]
[47]
Zhang, Y.; Hai, Y.; Miao, Y.; Qi, X.; Xue, W.; Luo, Y.; Fan, H.; Yue, T. The toxicity mechanism of different sized iron nanoparticles on hu-man breast cancer (MCF7) cells. Food Chem., 2021, 341(Pt 2), 128263.
[http://dx.doi.org/10.1016/j.foodchem.2020.128263] [PMID: 33038805]
[48]
Kruger, M.J.; Smith, C. Postcontusion polyphenol treatment alters inflammation and muscle regeneration. Med. Sci. Sports Exerc., 2012, 44(5), 872-880.
[http://dx.doi.org/10.1249/MSS.0b013e31823dbff3] [PMID: 22033514]
[49]
Visser, J.G.; Smith, C. Development of a transendothelial shuttle by macrophage modification. J. Tissue Eng. Regen. Med., 2018, 12(4), e1889-e1898.
[http://dx.doi.org/10.1002/term.2620] [PMID: 29193878]
[50]
Chang, Y.N.; Guo, H.; Li, J.; Song, Y.; Zhang, M.; Jin, J.; Xing, G.; Zhao, Y. Adjusting the balance between effective loading and vector mi-gration of macrophage vehicles to deliver nanoparticles. PLoS One, 2013, 8(10), e76024.
[http://dx.doi.org/10.1371/journal.pone.0076024] [PMID: 24116086]
[51]
Visser, J.G.; Van Staden, A.D.P.; Smith, C. Harnessing macrophages for controlled-release drug delivery: Lessons from microbes. Front. Pharmacol., 2019, 10, 22.
[http://dx.doi.org/10.3389/fphar.2019.00022] [PMID: 30740053]
[52]
Zhang, W.; Wang, M.; Tang, W.; Wen, R.; Zhou, S.; Lee, C.; Wang, H.; Jiang, W.; Delahunty, I.M.; Zhen, Z.; Chen, H.; Chapman, M.; Wu, Z.; Howerth, E.W.; Cai, H.; Li, Z.; Xie, J. Nanoparticle-laden macrophages for tumor-tropic drug delivery. Adv. Mater., 2018, 30(50), e1805557.
[http://dx.doi.org/10.1002/adma.201805557] [PMID: 30368972]
[53]
Wang, C.; Li, K.; Li, T.; Chen, Z.; Wen, Y.; Liu, X.; Jia, X.; Zhang, Y.; Xu, Y.; Han, M.; Komatsu, N.; Zhao, L.; Chen, X. Monocyte-mediated chemotherapy drug delivery in glioblastoma. Nanomedicine (Lond.), 2018, 13(2), 157-178.
[http://dx.doi.org/10.2217/nnm-2017-0266] [PMID: 29173008]
[54]
Zheng, H.; Li, J.; Luo, X.; Li, C.; Hu, L.; Qiu, Q.; Ding, J.; Song, Y.; Deng, Y. Murine RAW264.7 cells as cellular drug delivery carriers for tumor therapy: A good idea? Cancer Chemother. Pharmacol., 2019, 83(2), 361-374.
[http://dx.doi.org/10.1007/s00280-018-3735-0] [PMID: 30506269]
[55]
Qiyi, F.; Yanping, L.; Jian, H.; Ke, C.; Jinxing, H. Xiao, Kai Uptake, distribution, clearance, and toxicity of iron oxide nanoparticles with different sizes and coatings. Sci. Rep., 2018, 8(1), 2082.
[http://dx.doi.org/10.1038/s41598-018-19628-z]
[56]
Sun, W. Study on biotransportation, biotransformation and metabonomic of magnetic Fe3O4- Nanoparticles in vivo; Jilin National University: Changchun, 2013.

Rights & Permissions Print Cite
Article Metrics
48
1
© 2024 Bentham Science Publishers | Privacy Policy