Targeting multiple mediators of sepsis using multifunctional tannic acid-Zn2+-gentamicin nanoparticles

Abstract

⋅Design of multifunctional nanoparticles that can target multiple mediators of sepsis⋅A new strategy of treating inflammatory disease with anionic therapeutic nanomaterials Recent research points to the pathogenic role of cell-free DNA (cfDNA) as damage-associated molecular pattern molecules in activating the toll-like receptor (TLR) pathways and inducing inflammation. We, and others, have been using cationic nanostructures to scavenge cfDNA molecules as a new strategy to modulate sepsis. To reduce toxicity, we hypothesize that the incorporation of antibacterial and antioxidation components into an anion nanostructure can offer the multifunctionality of cfDNA scavenging, antibacterial effect, and antioxidation to improve therapeutic efficacy while reducing side effects. We screened a number of nanoparticle (NP) compositions and showed that the tannic acid-Zn2+-gentamicin NPs (TA-Zn-Gen NPs) with multiple anti-sepsis activity could reduce multiple organ damage and achieve a 60% survival rate in a severe sepsis animal model. Our study suggests a new strategy of treating inflammatory diseases with anionic nanomaterials. Treatments that target single mediators of sepsis have failed to reduce its high mortality rate. Here we developed multifunctional tannic acid-Zn2+-gentamicin nanoparticles (TA-Zn-Gen NPs) that target multiple mediators of sepsis to improve sepsis treatment. TA-Zn-Gen NPs with lower gentamicin content possessed net negative surface charge but still bound cell-free DNA (cfDNA) with high affinity. The TA-Zn-Gen NPs exhibited five modes of anti-sepsis activity: (1) scavenged cfDNA and inhibited cfDNA-initiated activation of toll-like receptors and NF-κB signaling; (2) inhibited activated macrophage-induced macrophage recruitment; (3) scavenged reactive oxygen species (ROS) and reduced ROS-induced DNA damage and cell death; (4) inhibited nitric oxide production induced by lipopolysaccharides; and (5) potent antibacterial activity. The NPs reduced multiple organ damage and increased the survival rate of mice with severe sepsis. Together, the results demonstrate the potency of targeting multiple mediators for sepsis treatment, and support the development of multifunctional NPs for treating other intractable inflammation-related diseases. Treatments that target single mediators of sepsis have failed to reduce its high mortality rate. Here we developed multifunctional tannic acid-Zn2+-gentamicin nanoparticles (TA-Zn-Gen NPs) that target multiple mediators of sepsis to improve sepsis treatment. TA-Zn-Gen NPs with lower gentamicin content possessed net negative surface charge but still bound cell-free DNA (cfDNA) with high affinity. The TA-Zn-Gen NPs exhibited five modes of anti-sepsis activity: (1) scavenged cfDNA and inhibited cfDNA-initiated activation of toll-like receptors and NF-κB signaling; (2) inhibited activated macrophage-induced macrophage recruitment; (3) scavenged reactive oxygen species (ROS) and reduced ROS-induced DNA damage and cell death; (4) inhibited nitric oxide production induced by lipopolysaccharides; and (5) potent antibacterial activity. The NPs reduced multiple organ damage and increased the survival rate of mice with severe sepsis. Together, the results demonstrate the potency of targeting multiple mediators for sepsis treatment, and support the development of multifunctional NPs for treating other intractable inflammation-related diseases. IntroductionSepsis is a life-threatening systemic inflammatory response to fungal, bacterial, or viral infection.1Hotchkiss R.S. Moldawer L.L. Opal S.M. Reinhart K. Turnbull I.R. Vincent J.L. Sepsis and septic shock.Nat. Rev. Dis. Primers. 2016; 2: 16045Crossref PubMed Scopus (607) Google Scholar,2Angus D.C. van der Poll T. Severe sepsis and septic shock.N. Engl. J. Med. 2013; 369: 840-851Crossref PubMed Scopus (1769) Google Scholar Despite advances in sepsis treatment, the mortality rate of sepsis remains high, and over eight million people die annually due to sepsis.3Arnold C. News Feature: the quest to solve sepsis.Proc. Natl. Acad. Sci. 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A nanotrap improves survival in severe sepsis by attenuating hyperinflammation.Nat. Commun. 2020; 11: 3384Crossref PubMed Scopus (20) Google Scholar we hypothesized that a multifunctional NP that targeted multiple factors simultaneously could achieve a greater anti-sepsis therapeutic effect. Here, we developed multifunctional TA-Zn-Gen NPs that consist of tannic acid, Zn2+, and gentamicin for improved sepsis treatment. We synthesized a series of TA-Zn-Gen NPs with different gentamicin contents in a simple, low-cost, scalable, one-pot process at ambient temperature (Figure 1A), and characterized the NPs in terms of their physicochemical properties and anti-sepsis therapeutic activity in vitro and in vivo, with a focus on five mechanisms of anti-sepsis therapeutic activity (Figures 1B and 1C): (1) binding and scavenging inflammatory cfDNA and inhibition of cfDNA-induced TLR activation and NF-κB signaling; (2) inhibition of activated macrophage-induced macrophage recruitment; (3) scavenging of ROS and inhibition of ROS-induced DNA damage and cell death; (4) inhibition of bacterial LPS-induced NO production; and (5) antibacterial activity and inhibition of bacteria-induced inflammation.Results and discussionCharacterization of TA-Zn-Gen NPsWe fabricated a series of TA-Zn-Gen NPs with increasing amounts of gentamicin (Table S1). The chemical structures of tannic acid and gentamicin are shown in Figure S1. As-constructed TA-Zn-Gen NPs displayed spherical morphology (Figure S2). The size, polydispersity index (PDI), and zeta potential of the NPs increased with increasing gentamicin content (Figures 2A–2C ). Intriguingly, when the tannic acid/gentamicin (TA:Gen) weight ratio was increased from 1:0.5 to 1:1, the NP size increased sharply from ∼200 nm to over 2 μm in diameter, and the surface charge flipped from negative to positive. Since anionic NPs are favorable for prolonging blood circulation time and enhancing accumulation in inflamed sites,33Jin Q. Deng Y. Chen X. Ji J. Rational design of cancer nanomedicine for simultaneous stealth surface and enhanced cellular uptake.ACS Nano. 2019; 13: 954-977PubMed Google Scholar we selected the three smaller, anionic NPs for further investigation. Element analysis confirmed the presence of nitrogen (due to gentamicin) in the NPs (Figure 2D), and inductively coupled plasma mass spectroscopy (ICP-MS) confirmed the presence of Zn (Figure 2D). C=O and C=C characteristic peaks of tannic acid34Abouelmagd S.A. Abd Ellah N.H. Amen O. Abdelmoez A. Mohamed N.G. Self-assembled tannic acid complexes for pH-responsive delivery of antibiotics: role of drug-carrier interactions.Int. J. Pharm. 2019; 562: 76-85Crossref PubMed Scopus (26) Google Scholar were observed in the Fourier transform infrared spectroscopy (FTIR) spectra of the NPs (Figure 2E), confirming the presence of tannic acid. No crystalline peak was observed in X-ray diffraction (XRD) spectra (Figure 2F), indicating an amorphous structure. Stability analysis of the TA-Zn-Gen NPs showed no statistically significant change in size after 2 days of incubation in phosphate buffered solution (PBS), Fetal bovine serum (FBS), or Dulbecco's modified Eagle’s medium (DMEM) containing 10% FBS, indicating their stability under physiological condition (Figure S3).Figure 2Characterization of TA-Zn-Gen NPsShow full caption(A–C) (A) Size, (B) PDI, and (C) zeta potential of TA-Zn-Gen NPs with increasing gentamicin content. TA:Gen weight ratio = (1) 1:0.125, (2) 1:0.25, (3) 1:0.5, (4) 1:1, (5) 1:2. A sharp increase in particle size and PDI and a change from net negative to positive surface charge occurred between a TA:Gen weight ratio of 1:0.5 and 1:1.(D–F) (D) ICP-MS and element analysis, (E) FTIR spectra, and (F) XRD spectra of TA-Zn-Gen NPs 1–3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DNA binding affinity of TA-Zn-Gen NPsIn preliminary experiments, we were surprised to observe that TA-Zn-Gen NPs with a net negative surface charge (NPs 1–3) exhibited concentration-dependent absorption of Cy5-labeled CpG (a short single-stranded DNA [ssDNA] oligonucleotide that activates TLR9) (Figure S4). We further evaluated the calf thymus DNA binding affinity of the NPs in Tris-EDTA (TE) buffer with and without 10% FBS by measuring unbound PicoGreen-labeled DNA (Figures 3A and 3B). Despite their net negative surface charge, TA-Zn-Gen NPs 1–3 exhibited high DNA binding affinity. TA-Zn-Gen NPs with increasing gentamicin content displayed increasing DNA binding affinity, possibly due to electrostatic interactions between DNA and gentamicin. Interestingly, free tannic acid also exhibited DNA binding (Figure 3A), possibly due to hydrogen bonding between tannic acid and the phosphate backbone of DNA. The addition of 10% FBS reduced DNA binding in all groups, but the competitive interactions due to serum proteins were overcome by increasing the amount of NPs (Figure 3B). To investigate whether charged molecules in the cytoplasm of cells cause dissociation of TA-Zn-Gen/DNA complexes, we performed GFP plasmid transfection experiments using TA-Zn-Gen 3 NPs as a plasmid DNA carrier. A CCK-8 assay was used to determine the cytotoxicity of the NPs to RAW 264.7 murine macrophage cells (Figure 3C), and a nontoxic NPs concentration of 100 μg/mL was chosen for transfection experiments. Following transfection of HEK 293 cells, bright fluorescence was observed in the positive control group (using Lipofectamine 3000 as DNA carrier), but negligible fluorescence was observed when TA-Zn-Gen 3 NPs were used as the DNA carrier (Figure S5). These results suggest that robust binding between the NPs and plasmid prevented release of the GFP-encoding plasmid for transcription and translation.Figure 3DNA binding affinity and cytotoxicityShow full caption(A and B) DNA binding affinity of NPs at different NP:DNA mass ratios (see legend) in TE buffer without FBS (A) and with 10% FBS (B). [Calf thymus DNA] = 0.5 μg/mL.(C) Cytotoxicity of NPs to RAW 264.7 cells.(D) Colocalization of FITC-labeled TA-Zn-Gen NPs and Cy5-labeled CpG in lysosomes (CLSM images). Scale bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Anti-inflammatory effect of TA-Zn-Gen NPs in vitroInflammatory circulating nucleic acids such as CpG DNA activate TLR9 signaling following endocytosis, leading to recruitment of MyD88 and NF-κB signaling.35Takeshita F. Takeshita F. Gursel I. Ishii K.J. Suzuki K. Gursel M. Klinman D.M. Signal transduction pathways mediated by the interaction of CpG DNA with toll-like receptor 9.Semin. Immunol. 2004; 16: 17-22Crossref PubMed Scopus (157) Google Scholar Therefore, we investigated the internalization of the TA-Zn-Gen NPs and their ability to block CpG-induced TLR9 activation in vitro. FITC-labeled TA-Zn-Gen NPs were internalized with high efficiency by RAW 264.7 cells, as observed by confocal laser scanning microscopy (CLSM) (Figures 3D and S6). Colocalization of FITC-labeled NPs and Cy5-CpG fluorescence was observed in endolysosomal compartments (Figure 3D), confirming the internalization of TA-Zn-Gen NPs via endocytosis, which might be beneficial for blocking recognition between CpG and TLR9.We then measured NP inhibition of nucleic acid-induced TLR activation by using HEK-Blue human TLR (hTLR) cells and monitoring downstream NF-κB signaling (Figure S7). HEK-Blue hTLR3, hTLR4, and hTLR9 cells were constructed by co-transfecting the hTLR gene and an optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene into HEK 293 cells. An IFN-β minimal promoter fused to five NF-κB and AP-1 binding sites was designed to control the expression of SEAP reporter gene. TLR agonist treatments initiate the expression of NF-κB and AP-1, which induce production of SEAP, which is detected by using Quanti-Blue reagent and measuring the optical density (OD) at 620 nm. We tested three TLR agonists: (1) CpG Bw006 ssDNA oligonucleotide, a TLR9 ligand; (2) Poly (I:C), a synthetic double-stranded RNA analog that activates TLR3 signaling; and (3) LPS, a TLR4 ligand. The NPs alone did not activate the TLRs (Figure S8). Consistent with the previous nucleic acid binding results, the NPs inhibited CpG-induced activation of HEK-Blue hTLR9 cells and inhibited Poly (I:C)-induced activation of HEK-Blue hTLR3 cells in a NP dose-dependent manner, regardless of the presence or absence of FBS (Figures 4A, 4B, 4D, 4E, and S9). However, the NPs did not inhibit LPS-induced HEK-Blue hTLR4 activation (Figures 4C and 4F ). Together, these results demonstrate that the TA-Zn-Gen NPs specifically inhibit nucleic acid (DNA or RNA but not LPS)-induced TLR activation and downstream NF-κB signaling.Figure 4Inhibition of nucleic acid-induced TLR activation and activated macrophage-induced macrophage migrationShow full caption(A–F) Activation of HEK-Blue (A) hTLR9, (B) hTLR3, and (C) hTLR4 cells in the absence of FBS after different treatments for 24 h. Activation of HEK-Blue (D) hTLR9, (E) hTLR3, and (F) hTLR4 cells in the presence of FBS after different treatments for 24 h. The legend indicates NPs:agonist mass ratios.(G) Schematic of inhibition of nucleic acid-induced TLR activation by TA-Zn-Gen NPs.(H and I) (H) TNF-α mRNA and (I) TNF-α cytokine generated by macrophages after different treatments for 24 h.(J) Inhibition of activated macrophage-induced macrophage migration, measured using a transwell assay. Data are expressed as mean ± SD. Statistical comparisons of groups were performed using a Student's t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To our surprise, tannic acid alone also inhibited nucleic acid-induced activation of TLR9 and TLR3. We then performed a further experiment to prove that the anti-inflammation activity of TA-Zn-Gen NPs was derived from the nucleic acid scavenging capability instead of the function of tannic acid. In this experiment, we mixed each agonist (CpG, Poly (I:C), or LPS) with TA-Zn-Gen NPs at different NPs concentrations for 1 h, then centrifuged the mixture to precipitate TA-Zn-Gen/agonist complexes, and used the supernatant to treat HEK-Blue hTLR cells. When a high NP concentration was used, the supernatant treatment resulted in lower levels of HEK-Blue hTLR9 and hTLR3 cell activation (Figure S10). This result indicated that NP inhibition of TLR activation was derived from nucleic acid scavenging (Figure 4G).Activation of TLR9 by inflammatory circulating cfDNA has been suggested to play a critical role in the progression of sepsis.36Tsujimoto H. Ono S. Efron P.A. Scumpia P.O. Moldawer L.L. Mochizuki H. Role of toll-like receptors in the development of sepsis.Shock. 2008; 29: 315-321Crossref PubMed Scopus (186) Google Scholar, 37Zha Z. Yue X. Ren Q. Dai Z. Uniform polypyrrole nanoparticles with high photothermal conversion efficiency for photothermal ablation of cancer cells.Adv. Mater. 2013; 25: 777-782Crossref PubMed Scopus (617) Google Scholar, 38Atalan N. Acar L. Yapic N. Kudsioglu T. Ergen A. Yilmaz S.G. Isbir T. The relationship between sepsis-induced immunosuppression and serum toll-like receptor 9 level.In Vivo. 2018; 32: 1653-1658Crossref PubMed Scopus (6) Google Scholar We next used CpG Bw006 to initiate TLR9 activation in RAW 264.7 cells and evaluated the anti-inflammation effect of the TA-Zn-Gen NPs in terms of tumor necrosis factor alpha (TNF-α) transcription and translation. TA-Zn-Gen NP treatment reduced transcription and translation of TNF-α (Figures 4H and 4I), indicating NPs inhibition of nucleic acid-induced inflammation in vitro.Inhibition of activated macrophages-induced macrophages recruitmentMacrophage accumulation in inflamed sites aggravates inflammation, and reducing activated macrophage-induced macrophage recruitment is a promising strategy for alleviating inflammation in sepsis.39Guo J. Li D. Tao H. Li G. Liu R. Dou Y. Jin T. Li L. Huang J. Hu H. Zhang J. Cyclodextrin-derived intrinsically bioactive nanoparticles for treatment of acute and chronic inflammatory diseases.Adv. Mater. 2019; 31: 1904607Crossref Scopus (54) Google Scholar CpG Bw006-activated RAW 264.7 macrophages recruited a large number of macrophages from the upper side of a transwell chamber to the lower side (Figures 4J and S11) due to chemotaxis induced by release of attractants by activated macrophages. When TA-Zn-Gen NPs were added, macrophage migration was sharply reduced. Thus, the TA-Zn-Gen NPs not only inhibit nucleic acid-initiated TLR activation but also inhibit activated macrophage-induced macrophage migration once macrophages are activated.ROS scavenging and protection from ROS-induced cell damageROS have emerged as an important factor in the pathophysiology of sepsis.40Mantzarlis K. Tsolaki V. Zakynthinos E. Role of oxidative stress and mitochondrial dysfunction in sepsis and potential therapies.Oxid. Med. Cell Longev. 2017; 2017: 5985209Crossref PubMed Scopus (132) Google Scholar Since tannic acid is a natural antioxidant that has been explored for use as an ROS scavenger, we examined the ROS scavenging ability of TA-Zn-Gen NPs. The ⋅OH scavenging capability of the TA-Zn-Gen NPs was assessed by monitoring the absorption kinetics at 650 nm of oxidized 3,3′,5,5′-tetramethylbenzidine (TMB) generated from the oxidation of TMB by ⋅OH. A Fenton reaction between Cu2+ and H2O2 was used to produce ⋅OH. The TA-Zn-Gen NPs markedly reduced the generation of oxidized TMB by ⋅OH in a NPs dose-dependent manner (Figures 5A and S12), indicating that the NPs scavenge hydroxyl radicals. Next, the oxidation of xanthine by xanthine oxidase was used to produce O2⋅⁃, and the O2⋅⁃ scavenging ability of the TA-Zn-Gen NPs was characterized by measuring the fluorescence of ethidium, the product of hydroethidine oxidation by O2⋅⁃ at 610 nm.41Yao J. Cheng Y. Zhou M. Zhao S. Lin S. Wang X. Wu J. Li S. Wei H. ROS scavenging Mn3O4 nanozymes for in vivo anti-inflammation.Chem. Sci. 2018; 9: 2927-2933Crossref PubMed Google Scholar The fluorescence intensity decreased with increasing NP concentration (Figure 5B), indicating the elimination of O2⋅⁃ by the NPs. The intracellular ROS level was then evaluated by using cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) as an indicator. Bright green fluorescence was observed in tert-butyl hydroperoxide (TBHP)-treated cells upon stimulation (Figures 5C and 5D), demonstrating the successful induction of oxidation pressure. With the introduction of the TA-Zn-Gen NPs, the fluorescence intensity in cells decreased significantly, indicating ROS scavenging by the NPs in vitro, which was further confirmed by detecting the 2',7'-dichlorofluorescein (DCF) fluorescence at 520 nm with multiwell plate reader via excitation at 490 nm (Figure S13).Figure 5ROS scavenging capability and protection on ROS-induced DNA damage and cell deathShow full caption(A and B) (A) ⋅OH and (B) O2⋅⁃ scavenging capability of the TA-Zn-Gen NPs. (A) 1, HT; 2, HTC; 3, HTC/gentamicin; 4, HTC/tannic acid; 5, HTC/TA-Zn-Gen 1; 6, HTC/TA-Zn-Gen 2; 7, HTC/TA-Zn-Gen 3. T, TMB; H, H2O2; C, Cu2+.(C) Intracellular ROS imaging of macrophages after treatments. BF, bright field. Scale bar, 100 μm.(D) Semi-quantitative fluorescence intensity of images in Figure 5C evaluated by Image J.(E) NPs protection from ROS-induced DNA damage assessed using a plasmid nicking assay and agarose gel electrophoresis. The positions of lanes from gels were rearranged to make them easy to compare.(F) Phosphorylated histone variant H2AX imaging of macrophages following different treatments. Scale bar, 100 μm.(G) Protective effect of NPs on ROS-induced cell death (CCK-8 assay). Data are expressed as mean ± SD. Statistical comparisons of groups were performed using Student's t test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Next, the effect of TA-Zn-Gen NPs on ROS-induced cell DNA damage was evaluated through a plasmid nicking assay.42Li Y. Li Z. Hou H. Zhuang Y. Sun L. Metal chelating, inhibitory DNA damage, and anti-inflammatory activities of phenolics from rambutan (Nephelium lappaceum) peel and the quantifications of geraniin and corilagin.Molecules. 2018; 23: 2263Crossref Scopus (26) Google Scholar In this experiment, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) was used as an ROS source to convert supercoiled DNA into the nicked form. Supercoiled DNA and nicked DNA possess different electrophoretic mobilities and can be separated by agarose gel electrophoresis. The supercoiled plasmid DNA strand was damaged by AAPH treatment, and a high fraction of the nicked form was observed in a gel electrophoretogram (Figures 5E and S14). In contrast, the formation of the nicked form decreased dramatically when TA-Zn-Gen NPs were added, demonstrating protection from ROS-induced DNA damage by the NPs. Next, NP protection against cellular DNA damage was determined by measuring the amount of phosphoryl