Administration of Soft Matter Lipid-DNA Nanoparticle As the

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Administration of soft matter lipid-DNA nanoparticle as the immunostimulant via multiple routes of injection in vivo Jun-O Jin, Minseok Kwak, Li Xu, Haejoo Kim, Tae Hyeong Lee, JaeOuk Kim, Qing Liu, Andreas Herrmann, and Peter Chang-Whan Lee ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00440 • Publication Date (Web): 02 Aug 2017 Downloaded from http://pubs.acs.org on August 3, 2017

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ACS Biomaterials Science & Engineering

Administration of soft matter lipid-DNA nanoparticle as the immunostimulant via multiple routes of injection in vivo Jun-O Jin,†,⊥ Minseok Kwak,‡,⊥ Li Xu,† Haejoo Kim,‡ Tae Hyeong Lee,¶ Jae-Ouk Kim,§ Qing Liu,k Andreas Herrmann,k and Peter C.W. Lee∗,¶ †Shanghai Public Health Clinical Center, Shanghai Medical College, Fudan University, 2901 Caolang Road, Shanghai 201508, China ‡Department of Chemistry, Pukyong National University, 45 Yongso-ro, Nam-gu, Busan 48513, South Korea ¶Department of Biomedical Sciences, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05535, South Korea §International Vaccine Institute, SNU Research Park, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, South Korea kUniversity of Groningen, Zernike Institute for Advanced Materials, Department of Polymer Chemistry, Nijenborgh 4, 9747 AG Groningen, The Netherlands ⊥Contributed equally to this work E-mail: [email protected] Phone: +82 (0)2 3010 2799

Abstract We elucidated the proper routes for injecting lipid-DNA micelles formulated with an oligonucleotide immune adjuvant in naïve and tumor-bearing mice. We report

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herein that after assessing six ways of administering an immunostimulatory nanoparticle (INP), several of the injection routes effectively induced dendritic cell-mediated immune-stimulation in vivo, making them amenable to cancer immunotherapies and vaccines using such DNA materials.

KEYWORDS : Adjuvants, dendritic cells, micelles, hybrid materials, nucleic acids, DNA nanotechnology

INTRODUCTION Recent advances in DNA-based materials science and nanotechnology are mainly focused on biomedicine due to the fact that DNA is an inherently nontoxic and degradable biomacromolecule common to all living organisms. 1,2 Therefore, DNA nanomaterials have been actively used as carriers of various therapeutic cargos, such as small molecule drugs for drug delivery and short oligonucleotides for RNA interference or immune-stimulation. Efficient immunotherapy seeks target cells that kill without undesired side effects, such as tissue inflammation. 3,4 Cytotoxic T lymphocytes (CTLs) are the most effective immune cells in terms of killing pathogens by antigen (Ag) recognition, 3–5 in which an Ag is transferred from Ag presenting cells (APCs), such as dendritic cells (DCs) and macrophages. 6 Differing from DCs generated in vitro, those generated in vivo have specific phenotypes and functions. The in vivo mouse myeloid DCs have two subsets: CD8α+ and CD8α− DCs. CD8α+ DCs are specialized to induce CTL activation by cross-presentation of endogenously synthesized Ags through major histocompatibility complex (MHC) class I. 7,8 Therefore, an immunologically potent material to activate and mature CD8α+ DCs, fulfilling biocompatibility and programmability at the same time, is desirable for successful immunotherapies and vaccines. Our previous study demonstrated that in vivo delivery of CpG-incorporated lipid-DNA micelle (immunostimulatory nanoparticle; INP) via intravenous (i.v.) injection promotes 2


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spleen DC activation in mice. 9 However, such an i.v. injection is undesirable for humans and animals, since the immunostimulant will spread to the peripheral tissues and induce severe inflammation in those tissues. 10–12 Such a negative effect has to be minimized in order to develop effective immunotherapies and vaccines with new formulations. Moreover, compared to popular particle formulations (e.g. liposomes, polymers and inorganics), 13–18 DNA nanomaterials, such as INP, have not yet been studied in the aspects of injection and immune response since the materials are relatively new. In this regard, quantitative studies on the INP-induced activation of CD8α+ DCs would help us find suitable in vivo administration routes.

RESULTS For these reasons, we evaluated multiple administration routes of INP in terms of the activation of CD8α+ DCs in lymphoid tissues and the production of pro-inflammatory cytokines (Scheme 1). For this study, we prepared INP composed of lipid-DNAs, the delivery vehicle, and CpG cargo (eCpG) with a small modification – the phosphorothioated immunostimulatory segment was adapted for enhanced nuclease resistance. The formation of INP is a simple matter of mixing two DNA strands, because 12-nucleotide (NT) long lipid-DNAs spontaneously aggregate into spherical micelles, and at the same time, the lipid-DNAs hybridize with a complementary segment of eCpG. The exposed space of the INP is then covered with a single-stranded, immunostimulatory segment of eCpG, known as ODN 1826 (see SI for the sequences). Introduction of randomly oriented eCpG in such a way does not change the size of nanoparticles, ca. 8 nm measured on TEM, 9 while the zeta potential of INP gets nearly double, from ζ = -10.4 mV to -20.1 mV for lipid-DNA and INP, respectively, due to the increased coverage of eCpGs. Prior to in vivo injections, we performed a cytotoxicity test of INP in a human lung epithelial cell line (BEAS-2b), because in vivo injections mostly involve contact with such epithelial tissues. We found that neither lipid-DNA, eCpG,

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harvested. In the same manner, fluorescently labeled INP was separately administered to investigate injection route-dependent biodistribution of lymphoid tissues. After harvesting the tissues, Alexa-488 positive CD8α+ DCs in the spleen, iLN and mLN were analyzed. The CD8α+ DCs in the tissues were defined by lineage− CD11c+ CD8α+ live leukocytes (Figure S2). Notably, i.v. injection resulted in markedly high percentage (from 28 to 48 %) of Alexa-488 positive DCs throughout all lymphoid tissues whereas almost no observation was found for samples injected orally (Figures 1A and S3). It is also interesting that s.c. and i.n. INP injections exhibited the highest fluorescence integrations in iLN and mLN, respectively, than those of other injections. We think that the close proximity between injection spots and lymphoid tissues may play a role in this INP uptake results (i.e. s.c. to iLN and i.n. to mLN, repsectively, see the mouse in Scheme 1). Considering these findings we then investigated effects of injection routes in immune activation. The expression levels of co-stimulatory molecules in CD8α+ DCs were measured as described in previous studies. 9,19 The s.c. and i.v. injections of INP induced substantial increases in the expression of costimulatory molecules in the spleen CD8α+ DCs (Figure 1B, grey BG). Moreover, i.m. and i.p. injection also promoted considerable increases in the co-stimulatory molecule expression in the spleen CD8α+ DCs compared to those with PBS injection. However, the oral and i.n. injections of INP did not up-regulate the expression levels of co-stimulatory molecules in the spleen CD8α+ DCs (Figure 1B, grey BG). The expression levels of co-stimulatory molecules in the iLN CD8α+ DCs were much higher by the s.c. injection than the other injection routes (Figure 1B, yellow BG). Consistent with the spleen CD8α+ DC activation, the oral and nasal administrations of INP did not promote activation of iLN CD8α+ DCs (Figure 1B, yellow BG). In the mLN CD8α+ DC activation, i.n. injection of INP promoted the highest levels compared to the other injections (Figure 1B, orange BG). Indeed, these uptake and stimulation results (Figure 1A-B) agree each other implying that immune stimulation by INP in any injection is closely related to biodistribution of the materials. To further evaluate the immune stimulatory effect of INP by different injection routes,

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the production of pro-inflammatory cytokines was measured in the serum 24 h after INP administration. Consistent with costimulatory molecule expression in CD8α+ DCs, the levels of pro-inflammatory cytokines in the serum were also dramatically elevated by s.c. and i.v. injections of INP (Figure 1C). Moreover, the i.p. and i.m. injections of INP also promoted substantial up-regulation of pro-inflammatory cytokine production in the serum compared to the PBS-treated control (Figure 1C). However, oral and nasal administrations of INP did not significantly induce elevated cytokine levels in the serum (Figure 1C). INPinduced different immune stimulatory effects by different injection routes may be due to the entrance of INP to capillary. Compared to subcutaneous, cell density is concentrated in muscle, which may interpret migration of INP to lymphoid tissue. Moreover, nucleases also contribute for stability of INP during migration to lymphoid tissues from injected area, since INP is entirely composed of deoxyribonucleotides. Consistent with poor INP uptake in oral injection (Figure 1A), the failure of immune activation by oral administration of INP may be due to the high concentration of RNases and DNases in the digestive tissues and saliva, which degraded INP. 20,21 Therefore, for the successful employment of INP to induce immune activation for treatment of diseases or vaccination, one needs to carefully consider a specific injection route corresponding to the target organs. For instance, seemingly i.n. is promising to the treatment against cancer and infection in the lung whereass.c. is more appropriate to skin cancer such as melanoma. Our finding that s.c. injection of INP efficiently promoted CD8α+ DC activation in the iLN and pro-inflammatory cytokine production prompted us to evaluate the effect of s.c. administration of INPs in the immune stimulation in melanoma tumor-bearing mice and also compared those effect to other routes of injection. The tumor micro-environment produces immune suppression molecules, which promote immune escape of cancer cells. 20,21 The immune suppression molecules produced by the tumor micro-environment interrupt the activation of DCs especially, co-stimulatory molecule expression, and Ag presentation. 22 Consequently, DCs cannot induce cancer Ag-specific immune activation, which develops an

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immune tolerance against cancer Ag, and thus the cancer cells will escape from immune responses. 20–22 For this reason, the treatment of cancer by immunotherapy requires enhancement of the activation and maturation of DCs in the tumor micro-environment. Therefore, once a tumor was well established on day 15 of B16 melanoma injection, the mice were further administered orally, i.n., s.c., i.m., i.p., and i.v. with INP for 24 h. The same flank of tumor was used for the s.c. administration of INP and the iLN of tumor flank was harvested and analyzed DC activation as a tumor-drLN. Consistent with activation of CD8α+ DCs by s.c. injection of INP in the iLN and spleen in tumor free mice, the s.c. injection of INP promoted the highest stimulatory effect in the CD8α+ in the spleen and tumor-drLN compared to other injection routes (Figure 2A). Moreover, the i.v. injection of INPs also substantially up-regulated the expression of co-stimulatory molecules in the spleen and tumor-drLN in the tumor-bearing mice. In addition, the s.c. and i.v. injections of INP in tumor-bearing mice promoted substantially increased levels of pro-inflammatory cytokines in the serum (Figure 2B). Thus, these data suggested that the s.c. and i.v. injections of INP indeed induce immune stimulation in the tumor micro-environment.

CONCLUSION In conclusion, we have demonstrated that s.c. and i.n. injections of INP promote CD8α+ DC activation in the iLN and mLN, specifically while i.v. injection so does to spleen as well. For efficient immunotherapy against cancer, DC-mediated Ag-specific immune activation is required to reduce undesired inflammation in healthy tissues. 4,6 Since s.c. injection of INP highly induced CD8α+ DC activation in the iLN and spleen, it is possible to avoid the unwanted spread of immunostimulant to health tissues through blood circulation, which potentially reduces inflammation in the tissues. Moreover, the s.c. injection of INP also promoted CD8α+ DC activation in tumor-drLN of the mouse’s flank, which indicated that the s.c. injection of INP has great potential to deliver cancerous Ag to tumor-drLN DCs.

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In addition, i.n. injection is also a promising administration route for localized delivery to diseases in upper body. According to our CpG-dose study, an optimal INP formulation was achieved with the lipid-DNAs half-filled with CpG segments through duplex formation while the other half of lipid-DNAs remain unoccuppied. 9 Thus, it is worth noting that INP can be readily equipped with Ag-DNA conjugates via duplex formation for Ag-specific immune activation. Based on the pronounced DC activations dependent on the injection routes described herein, our next task is to tackle delivery of Ag-incorporated INPs. Our finding that s.c. injection is an appropriate route of INP administration is an important step toward successful immunotherapy and vaccine applications using soft matter nanoparticle formulation.

Acknowledgement This work was supported by the Research Grant from Pukyong National University (2015).

Supporting Information Available Additional experimental results, materials and methods are available free of charge via the Internet at http://pubs.acs.org. This material is available free of charge via the Internet at http://pubs.acs.org/.

References (1) Seeman, N. C. Structural DNA nanotechnology; Cambridge University Press, 2016. (2) Linko, V.; Ora, A.; Kostiainen, M. A. DNA nanostructures as smart drug-delivery vehicles and molecular devices. Trends Biotechnol. 2015, 33, 586–594.

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(3) Dubensky, T. W.; Reed, S. G. Adjuvants for cancer vaccines. Semin. Immunol. 2010; pp 155–161. (4) Miconnet, I.; Coste, I.; Beermann, F.; Haeuw, J.-F.; Cerottini, J.-C.; Bonnefoy, J.-Y.; Romero, P.; Renno, T. Cancer vaccine design: a novel bacterial adjuvant for peptidespecific CTL induction. J. Immunol. 2001, 166, 4612–4619. (5) Cohn, L.; Delamarre, L. Dendritic cell-targeted vaccines. Front. Immunol. 2014, 5, 255. (6) Banchereau, J.; Palucka, A. K. Dendritic cells as therapeutic vaccines against cancer. Nat. Rev. Immunol. 2005, 5, 296–306. (7) Naik, S. H.; Proietto, A. I.; Wilson, N. S.; Dakic, A.; Schnorrer, P.; Fuchsberger, M.; Lahoud, M. H.; O’Keeffe, M.; Shao, Q.-x.; Chen, W.-f.; Villadangos, J. A.; Shortman, K.; Wu, L. Cutting Edge: Generation of Splenic CD8+ and CD8- Dendritic Cell Equivalents in Fms-Like Tyrosine Kinase 3 Ligand Bone Marrow Cultures. J. Immunol. 2005, 174, 6592–6597. (8) Schnorrer, P.; Behrens, G. M. N.; Wilson, N. S.; Pooley, J. L.; Smith, C. M.; ElSukkari, D.; Davey, G.; Kupresanin, F.; Li, M.; Maraskovsky, E.; Belz, G. T.; Carbone, F. R.; Shortman, K.; Heath, W. R.; Villadangos, J. A. The dominant role of CD8+ dendritic cells in cross-presentation is not dictated by antigen capture. P. Natl. Acad. Sci. U.S.A. 2006, 103, 10729–10734. (9) Jin, J.-O.; Park, H.; Zhang, W.; de Vries, J. W.; Gruszka, A.; Lee, M. W.; Ahn, D.-R.; Herrmann, A.; Kwak, M. Modular delivery of CpG-incorporated lipid-DNA nanoparticles for spleen DC activation. Biomaterials 2017, 115, 81–89. (10) Zuckerman, J. N. The importance of injecting vaccines into muscle. British Medical Journal 2000, 321, 1237–1237.

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(11) Bremer, P. T.; Schlosburg, J. E.; Lively, J. M.; Janda, K. D. Injection route and TLR9 agonist addition significantly impact heroin vaccine efficacy. Mol. Pharm. 2014, 11, 1075–1080. (12) Pittman, P. R. Aluminum-containing vaccine associated adverse events: role of route of administration and gender. Vaccine 2002, 20, S48–S50. (13) Chattaraj, S. C.; Rathinavelu, A.; Das, S. K. Biodegradable microparticles of influenza viral vaccine: comparison of the effects of routes of administration on the in vivo immune response in mice. J. Control. Release 1999, 58, 223–232. (14) Mohanan, D.; Slütter, B.; Henriksen-Lacey, M.; Jiskoot, W.; Bouwstra, J. A.; Perrie, Y.; Kündig, T. M.; Gander, B.; Johansen, P. Administration routes affect the quality of immune responses: a cross-sectional evaluation of particulate antigen-delivery systems. J. Control. Release 2010, 147, 342–349. (15) Reddy, L. H.; Sharma, R.; Chuttani, K.; Mishra, A.; Murthy, R. Influence of administration route on tumor uptake and biodistribution of etoposide loaded solid lipid nanoparticles in Dalton’s lymphoma tumor bearing mice. J. Control. Release 2005, 105, 185–198. (16) Üner, M.; Yener, G. Importance of solid lipid nanoparticles (SLN) in various administration routes and future perspectives. Int. J. Nanomed. 2007, 2, 289–300. (17) Auttachoat, W.; McLoughlin, C. E.; White Jr, K. L.; Smith, M. J. Route-dependent systemic and local immune effects following exposure to solutions prepared from titanium dioxide nanoparticles. J. Immunotoxicol. 2014, 11, 273–282. (18) Scheerlinck, J.-P. Y.; Gloster, S.; Gamvrellis, A.; Mottram, P. L.; Plebanski, M. Systemic immune responses in sheep, induced by a novel nano-bead adjuvant. Vaccine 2006, 24, 1124–1131.

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(19) Zhang, W.; Yu, X.; Kwak, M.; Xu, L.; Zhang, L.; Yu, Q.; Jin, J.-O. Maturation of dendritic cells by pullulan promotes anti-cancer effect. Oncotarget 2016, 7, 44644– 44659. (20) Finn, O. J. Cancer immunology. New Engl. J. Med. 2008, 358, 2704–2715. (21) Ma, Y.; Shurin, G. V.; Gutkin, D. W.; Shurin, M. R. Tumor associated regulatory dendritic cells. Semin. Cancer Biol. 2012; pp 298–306. (22) Kaufman, H. L.; Disis, M. L. Immune system versus tumor: shifting the balance in favor of DCs and effective immunity. J. Clin. Invest. 2004, 113, 664–667.

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Administration of Soft Matter Lipid-DNA Nanoparticle As the

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