Research Article Cite This: ACS Appl. Mater. Interfaces 2018, 10, 14463−14474
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Design of Mannose-Functionalized Curdlan Nanoparticles for Macrophage-Targeted siRNA Delivery Tsogzolmaa Ganbold and Huricha Baigude* School of Chemistry & Chemical Engineering, Inner Mongolia Key Laboratory of Mongolian Medicinal Chemistry, Inner Mongolia University, 235 West College Road, Hohhot, Inner Mongolia 010020, P. R. China ABSTRACT: 6-Amino-6-deoxy-curdlan is a promising nucleic acid carrier that efficiently delivers plasmid DNA as well as short interfering RNA (siRNA) to various cell lines. The highly reactive C6-NH2 groups of 6-amino-6-deoxy-curdlan prompt conjugation of various side groups including tissue-targeting ligands to enhance cell-type-specific nucleic acid delivery to specific cell lines. Herein, to test the primary-cell-targeting efficiency of the curdlan derivative, we chemically conjugated a macrophage-targeting ligand, mannose, to 6-amino-6-deoxycurdlan. The resulting curdlan derivative (denoted CMI) readily complexed with siRNA and formed nanoparticles with a diameter of 50−80 nm. The CMI nanoparticles successfully delivered a dye-labeled siRNA to mouse peritoneal macrophages. The delivery efficiency was blocked by mannan, a natural ligand for a macrophage surface mannose receptor (CD206), but not by zymosan, a ligand for the dectin-1 receptor, which is also present on the surface of macrophages. Moreover, CMI nanoparticles were internalized by macrophages only at 37 °C, suggesting that the cellular uptake of CMI nanoparticles was energy-dependent. Furthermore, CMI nanoparticle efficiently delivered siRNA against tumor necrosis factor α (TNFα) to lipopolysaccharide-stimulated primary mouse peritoneal macrophages. In vivo experiments demonstrated that CMI nanoparticles successfully delivered siTNFα to mouse peritoneal macrophages, liver, and lung and induced significant knockdown of the TNFα expression at both messenger RNA and protein levels. Therefore, our design of CMI may be a promising siRNA carrier for targeting CD206-expressing primary cells such as macrophage and dendritic cells. KEYWORDS: curdlan, short interfering RNA, macrophage, mannose receptor, TNFα
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INTRODUCTION RNA interference (RNAi) is a promising therapeutic approach that allows post-transcriptional down-regulation of gene expression by designed sequences of double-stranded short interfering RNA (siRNA).1 Endogenously generated or exogenously introduced siRNA loads to RNA-induced silencing complex and clips target messenger RNA (mRNA) at a specific site, resulting in the expedited turnover of a disease-causing problematic protein.2 SiRNA can be designed to target any mRNA at very low doses with a sustainable time period, making RNAi technology extremely valuable for curing diseases triggered by nondruggable gene products.3 However, because of the vulnerable nature of siRNA such as its high susceptibility to nuclease and other environmental elements, protection of siRNA through formulation is usually necessary prior to any type of siRNA delivery (except for highly chemically modified siRNA sequences).4,5 Naturally occurring macromolecules, with or without chemical modifications, have been extensively investigated for potential applications for in vitro and in vivo siRNA delivery.6,7 Wild-type curdlan (β-1,3-D-glucan) has shown the ability of protecting and delivering siRNA.8,9 With a series of formulations involving several types of macromolecules such as polyethylenimine (PEI) and tRNA, a siRNA delivery microparticle termed GeRP was created. When orally administered, GeRP efficiently delivered siRNA to macrophages residing in several organs © 2018 American Chemical Society
including liver, spleen, and lung and induced RNAi against tumor necrosis factor α (TNFα).10 We synthesized 6-azido-6-deoxycurdlan for the first time using triphenylphosphine, carbon tetrabromide, and sodium azide. A subsequent reduction of 6azido-6-deoxy-curdlan by NaBH4 gave 6-amino-6-deoxy-curdlan (denoted 6AC-100), which showed excellent water solubility after protonation with dilute acid, hence allowing accurate structural analysis by NMR spectroscopy.11 6AC-100, which is one of the three curdlan derivatives with full C6-amination, was selected for further chemical modifications and investigation of the gene/siRNA delivery efficiency. The 6-azide group can also directly react with alkyne through “click” chemistry.12 The alkyne-functionalized lysine residues were conjugated to 6-azidecurdlan to give a cationic polymer. When complexed with plasmid DNA, the lysine-functionalized cationic polymer formed nanoparticles, which successfully delivered a plasmid expressing green fluorescence protein in HepG2 cells. The advantages of such a chemically modified nucleic acid carrier are not only viable synthesis and access of the materials but also potentials for applications in tissue- or cell-targeted drug delivery. For example, partially aminated curdlan derivatives (such as 6AC-70, 6AC-50, etc.) may partially maintain the Received: February 3, 2018 Accepted: April 12, 2018 Published: April 12, 2018 14463
DOI: 10.1021/acsami.8b02073 ACS Appl. Mater. Interfaces 2018, 10, 14463−14474
Research Article
ACS Applied Materials & Interfaces Scheme 1. Synthesis of Mannose and Imidazole Double-Functionalized 6-Amino-6-deoxy-curdlan (CMI)
Figure 1. 13C NMR spectrum of CMI in D2O.
Macrophages are the most plastic cells in the hematopoietic system and have roles in development, homeostasis, tissue repair, and innate immunity.15 The important roles of macrophages in many diseases, such as inflammatory disease, cancer, and chronic infectious disease have prompted recent attempts to utilize macrophages as a novel therapeutic target for gene therapy.16,17 When activated, macrophages release tumor necrosis factor α (TNFα), a potent regulator of varied physiological mechanisms in multiple organ systems.18 Inhibition of TNF has proven to be remarkably effective in the treatment of rheumatoid arthritis, inflammatory bowel disease, Alzheimer’s disease, and Crohn’s disease, and this strategy has a strong scientific basis given the abundance of literature substantiating the proinflammatory role of TNFα.18,19 TNFα-based strategies are being explored in the form of “anti-TNF” therapies for inflammatory diseases that target and neutralize the TNF cytokines and thus reduce the disease activity.20 There are some approved therapeutics that block the activity of TNFα, such as etanercept, a dimeric fusion
original conformation of curdlan, which may recognize and bind to cell surface receptors such as dectin-1, the major patternrecognition receptor present on phagocytes including macrophages.13 Therefore, the macrophage-targeted delivery of therapeutics by these cationic polymers is possible. For example, we synthesized D-galactose-functionalized 6-amino-curdlan through single-step reductive amination of lactose. Resulting polymer 6AC-100Lac specifically binds to HepG2 cells through an asialoglycoprotein receptor (ASGPR). Blocking of ASGPR by N-acetyl-D-galactosamine, the ligand of ASGPR, disabled cellular penetrating ability of 6AC-100Lac but not 6AC-100Mal, a maltose-conjugated 6-amino-curdlan.14 This cell-type-specific delivery of siRNA-induced RNAi activity resulted in a significant knockdown of endogenous gene GAPDH. Such conjugations demonstrated that the 6-amino group of 6AC-100 is a highly reactive chemical bonding site and provides versatile conjugation of functional moieties. 14464
DOI: 10.1021/acsami.8b02073 ACS Appl. Mater. Interfaces 2018, 10, 14463−14474
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ACS Applied Materials & Interfaces
Figure 2. (A) GPC chromatograph of CMI. (B) Gel retardation assay of the CMI/siRNA complex. Lane 0, free siRNA and lane 1−7, CMI/siRNA nanoparticles at an amine/phosphate (N/P) ratio of 1−6:1. (C) Transmission electron microscopy (TEM) image of the CMI/siRNA complex. (D) Buffering capacity of CMI. (E) Stability of siRNA complexed with CMI in fetal bovine serum (FBS). (F) siRNA release from CMI/siRNA complexes after incubation with increasing amounts of heparin. (G) siRNA release from CMI particles at various pHs mimicking the endosomal environment.
The molecular weight distribution of CMI was analyzed by GPC analysis, which gave Mn of 12.5 kDa and Mw of 14.6 kDa (Figure 2A). The molecular weight of CMI was lower than that of 6AC100 because the conjugation of multiple moieties to the 6AC-100 backbone resulted in minor aggregation in the final product. The subsequent removal of the insoluble precipitates (which is highly substituted high molecular weight fraction) in final product resulted in a CMI with reduced molecular weight. SiRNA Binding and Nanoparticle Imaging. To evaluate the nucleic acid binding trend of CMI, a gel retardation assay was conducted to measure the requirement of the minimum charge ratio between CMI and siRNA for efficient complexing. The positive charge of CMI and the negative charge on siRNA enforce electrostatic interaction between them to form the nanoparticles. The influence of charge ratios on siRNA condensation by CMI can be estimated by the gel retardation assay (Figure 2B). The migration of siRNA in gel was retarded with the increasing amine/phosphate (N/P) ratio; at an N/P ratio of 4, the siRNA migration in gel was totally retarded, indicating that CMI and siRNA can form a complete complex through electrostatic interactions. Moreover, the dynamic light scattering measurement indicated that naked CMI nanoparticles display an average size of 16.1 nm (polydispersity index (PDI), 0.23); after siRNA was loaded to CMI, the particle size significantly increased to 105.1 nm (PDI, 0.20), indicating that CMI can assemble with siRNA in a neutral aqueous solution (pH, 7.4).27 The average hydrodynamic size (diameter) of the CMI/siRNA complex was 107.3 nm (PDI, 0.20). The ζ potential of the particle was 32.9 mV at pH 7.4, indicating the appreciable stability of the nanoparticles in the neutral solution.28 Furthermore, we observed the morphological appearance of the CMI/siRNA complex by TEM (Figure 2C). Spherical particles with diameters ranging between 50 and 80 nm encapsulating the siRNA were captured in the images taken by TEM.
protein; infliximab, a chimeric monoclonal receptor antibody; and adalimumab, a human monoclonal TNFα antibody. However, they are associated with severe infections and malignancies because of their nonselectivity.21,22 siRNAmediated knockdown of proinflammatory cytokines at the translational level offers an alternative therapeutic strategy to overcome inflammatory conditions.23 Herein, we report chemically modified curdlan for cell surface receptor-targeted siRNA delivery to macrophages.
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RESULTS AND DISCUSSION Synthesis and Characterization of CMI. To create a siRNA carrier based on naturally occurring macromolecules with active targeting capacity to primary macrophages, we chemically modified 6-amino-6-deoxy-curdlan, which exhibited a great potential for siRNA delivery to various cell lines in our previous studies.11,14,24−26 To chemically functionalize the curdlan derivative, we first conjugated mannose moieties to 6-amino-6deoxy-curdlan. This was achieved by reacting 6-amino-6-deoxycurdlan with 4-isothiocyanatophenyl α-D-mannopyranoside, which is an isothiocyanate-activated mannose that readily reacts with primary amines; then, imidazole groups were also attached to the backbone of the curdlan derivative by reacting with 4imidazoleacetic acid using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as a condensing reagent (Scheme 1). The product of the reaction was extensively purified by dialysis as well as gel permeation. Then, the structure of the resulting polymer (denoted CMI) was confirmed by 13C NMR analysis (Figure 1) and the molecular weight distribution of the polymer was measured by gel permeation chromatography (GPC). In 13C NMR, the newly attached mannose moiety showed a signal at 98.32 ppm (5) and carbon from the amide bond showed a signal at 181.21 ppm (11), confirming the successful conjugation of both mannose and imidazole side chains on the curdlan backbone. The degree of substitution, estimated from the 13C NMR spectrum, was 5.1% for mannose and 8.2% for imidazole. 14465
DOI: 10.1021/acsami.8b02073 ACS Appl. Mater. Interfaces 2018, 10, 14463−14474
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ACS Applied Materials & Interfaces
Figure 3. Cytotoxicity assay. Mouse peritoneal macrophages, HeLa cells, MDA-MB-231 cells, and A549 cells remain viable 24 h after treatment with CMI at indicated concentrations. Cell toxicity levels are expressed as percent of control (nontreated, NT).
environment.29 At pH 5.5−6.0, siRNA was effectively released from the complex (Figure 2G), indicating that CMI can release a full-length siRNA sequence from the endosome. The conjugation of imidazole may have contributed to such ability because of the “proton sponge” effect.30 Cytotoxicity Measurement. Designing a safe and effective vehicle for gene delivery is a challenge because carriers can potentially induce cellular disorders. To examine the cytotoxicity of CMI, HeLa, A549, MDA-MB-231, and mouse peritoneal macrophages were treated with various concentrations of CMI, respectively, and the cell viability was assessed by an 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Figure 3). PEI and 6AC-100 were used as comparison groups. The results showed that PEI is the most toxic for all cell types, with the cell viabilities of 50−60% at a concentration of 20 μg/mL, compared with the nontreated (NT) group. The cells treated with 6AC-100 had 70−80% cell viability at a concentration of 20 μg/mL. However, CMI was essentially nontoxic (cell viability 80%) with all of the cell types even at concentrations as high as 60 μg/mL and showed nearly the same diagram for all four types of cells. This result demonstrated that CMI nanoparticles show negligible influence on various cells compared with PEI and 6AC-100. Cellular Uptake of CMI by Macrophages. Now that CMI has a functionalized mannose group that binds to the cell surface mannose receptor (CD206) on macrophages and dendritic cells, we evaluated the cellular uptake of the CMI/siRNA complex by mouse peritoneal macrophages. To do this, we complexed CMI with a dye (fluorescein isothiocyanate (FITC))-labeled siRNA at an N/P ratio of 4 and then treated mouse peritoneal macrophages with the complex. After 6 h, the cellular uptake was captured under confocal laser scanning microscopy (Figure 4). Compared with that in the control groups (Lipofectamine 2000 and 6AC-100), significantly greater amount of the green fluorescent signal was observed located in the cytosol of macrophages. Interestingly, 6AC-100/siRNA complexes had fragile cellular uptake compared to that of lipofectamine 2000
Buffering Capacity. The efficiency of nucleic acid delivery by cationic polymers has been mainly attributed to their high buffering capacity for mediating endosomal release by the proton sponge function, which can promote the osmotic swelling of endosome, endosomal membrane rupture, and the eventual leakage of the polyplex (cationic polymer complexed with nucleic acid) into the cytosol. We measured the buffering capacity of CMI to assess its proton absorption efficiency (Figure 2D). CMI showed excellent buffering ability at a pH of 4.5−5.5, although compared to that of the starting material, the titration curve of CMI dropped slightly (data not shown). The proton-absorbing property of CMI was lower than that of 6AC-100 because of its lesser protonable amino groups, which attached with mannose or imidazole. Stability and Release Assay. The stability of the siRNA/ carrier complex in the presence of serum is one of the essential parameters for successful siRNA delivery. To investigate whether CMI protects cargo siRNA from nuclease attack in the serum, we incubated the CMI/siRNA complex with FBS (Figure 2E). Whereas the naked siRNA substantially degraded after 1 h of incubation and completely hydrolyzed after 24 h of incubation in the serum, CMI successfully protected siRNA from serum degradation, even after 24 h of incubation. These results demonstrated that CMI nanoparticles can protect siRNA against serum nuclease digestion. To investigate siRNA release from CMI/siRNA complexes, a competitive heparin displacement assay was performed in which complexes were incubated with increasing amounts of heparin as an electrostatic competition agent (Figure 2F). The result showed that, as the amount of heparin increased, the siRNA was released from the complex and visual bands were observed in the gel, whereas no visual band was observed for the complex incubated with buffer. These results suggested that the complexes will be able to dissociate to achieve intracellular release of siRNA. To assess the efficiency of endosomal release of siRNA from the CMI complex after cellular internalization, we incubated the CMI/siRNA complex in neutral pH as well as in acidic pH that mimics the endosomal 14466
DOI: 10.1021/acsami.8b02073 ACS Appl. Mater. Interfaces 2018, 10, 14463−14474
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Figure 4. Uptake of CMI/FITC-siRNA by a primary macrophage. (A) Mouse peritoneal macrophages were transfected with FITC-siRNA complexed to lipofectamine (L2K), 6AC-100, and CMI. The fluorescence signals in the cells were observed by a fluorescence microscope. (B) Quantification of the cellular uptake by flow cytometry.
quantified the cellular uptake of dye-labeled siRNA by flow cytometry analysis, which reflects the amount of sample internalized by the cells proportionally to the fluorescent intensity of siRNA. As a result, mouse peritoneal macrophages treated with CMI/siRNA complexes showed 93% cellular uptake compared with nontreated cells, whereas cells treated with L2K/ siRNA had no cellular uptake and 6AC-100 appeared to have tenuous cellular uptake (8.6%). These results demonstrated that
(L2K), which may be attributed to the receptor-mediated cellular uptake involving dectin-1, which is also present on the macrophage cell surfaces and recognizes β-glucans. In contrast, cells treated with L2K/FITC-siRNA complexes did not show any fluorescence signal. These results indicated that CMI/siRNA complexes may enter the cell through receptor-mediated endocytosis involving both the mannose receptor and the dectin-1 receptor on the surface of macrophages. Moreover, we 14467
DOI: 10.1021/acsami.8b02073 ACS Appl. Mater. Interfaces 2018, 10, 14463−14474
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ACS Applied Materials & Interfaces
Figure 5. Receptor-mediated endocytosis of CMI. (A) Uptake of CMI nanoparticles was inhibited by mannan but not by dextrin and zymosan. (B) Incubation temperature dependence of the amount of cellular uptake of CMI/FITC-siRNA complexes. (C) Cytoplasmic location of the CMI/FITCsiRNA complex at 37 or 4 °C; blue, nucleus; red, cytoplasm; green, siRNA. (D) Effect of inhibitors on cellular uptake. PC and NC represent positive control and negative control, respectively. Each value represents the mean ± standard error of the mean (SEM) (n = 4). The symbol “*” indicates significant differences (p < 0.05) compared with the PC group.
points after the transfection, was much lower at 4 °C than that at 37 °C, whereas the overall cellular uptake increased time dependently, suggesting that the energy-dependent uptake occurred at 37 °C. This result can be interpreted as follows: the fluorescence increase at 4 °C reflects the amount of CMI/ FITC-siRNA complexes bound on mannose receptor on the cellular surface and at 37 °C reflects CMI/FITC-siRNA complexes uptake by endocytosis plus the amount bound on the cellular surface (Figure 5B). In addition, to distinguish surface-bound CMI/FITC-siRNA complexes from the intracellular ones, we observed the endoplasmic location of the complex at 37 or 4 °C. Microscopic images showed large amounts of internalized CMI/FITC-siRNA complexes at 37 °C than those at 4 °C (Figure 5C). These results indicate that CMI/ FITC-siRNA complexes enter the cell through cell surface mannose-receptor-mediated endocytosis. The above results proved that CMI nanoparticles were internalized by macrophages through a mannose receptor. To further illustrate the cellular uptake mechanisms of CMI nanoparticles, we used several inhibitors of endocytic pathways to block the possible cellular uptake ways of CMI nanoparticles (Figure 5D). The inhibitors are as follows: sodium azide (NaN3), an energy-dependent endocytosis inhibitor;33 chloroquine, a clathrin-dependent endocytosis inhibitor;34 sucrose, an inhibitor of clathrin-mediated endocytosis by hyperosmotic pressure;34,35 and nystatin, an inhibitor of caveola-dependent endocytosis.36 As a result, the endocytic uptake of the CMI/FITC-siRNA complex was perceptibly inhibited by NaN3, as well as significantly inhibited by incubating at 4 °C (Figure 5B). These results suggested that CMI nanoparticles were taken up inside the macrophages through an energy-dependent endocytic process. Treatment with nystatin did not induce significant change in cellular uptake, indicating that caveola-dependent endocytosis was not involved in the endocytosis pathway of CMI
CMI/siRNA may enter macrophages through CD206 receptormediated cellular uptake; hence, CMI may be applicable for macrophage-targeted siRNA delivery. Competitive Binding and Receptor Blocking. To test whether or not CMI carries the siRNA cargo and enters the macrophage through CD206 receptor-mediated endocytosis, we conducted a competitive blocking experiment. To do this, we chose mannan (a natural ligand for the CD206 receptor) and zymosan (ligand for the dectin-1 receptor) as receptor binding competitive inhibitors to evaluate the ligand−receptor binding specificity of CMI. Mannan has a high content of mannose oligosaccharide-bearing structures, which have very high affinity for macrophage mannose receptors and could be recognized by C-type like domain 4 on the receptor in a Ca2+-dependent manner.17 We pretreated mouse peritoneal macrophages with mannan or zymosan, respectively, and then treated the cells with CMI/FITC-siRNA complexes at various concentrations, followed by quantification of the transfection efficiency using flow cytometry. Blocking the mannose receptor by mannan inhibited the cellular uptake of CMI/FITC-siRNA complexes in a dosedependent manner. At a concentration of 10 μg/mL, mannan inhibited transfection of CMI by about 50%; at 20 μg/mL, 80% of the CMI/siRNA complex was competitively inhibited for cellular uptake (Figure 5A). However, the pretreatment of macrophages with zymosan had no influence on CMI transfection, suggesting that CMI predominantly binds to CD206 instead of the dectin-1 receptor. The dectin-1 receptor recognizes β-(1,3)-glucans only in linear, unbranched chains.31,32 Because CMI has mannose side chains, it is not recognized as a ligand for the dectin-1 receptor on the macrophages. To further investigate the mechanism of CMI cellular entry, we transfected mouse primary macrophages with CMI/FITCsiRNA complexes and incubated at 4 or 37 °C, respectively. The fluorescence intensity, which was measured at different time 14468
DOI: 10.1021/acsami.8b02073 ACS Appl. Mater. Interfaces 2018, 10, 14463−14474
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Figure 6. Measurement of a TNFα expression of mouse peritoneal macrophages by LPS stimulation. (A). Measurement of LPS dose-dependent production of TNFα; (B) Measurement of a time-dependent expression of TNFα. Each value represents the mean ± SEM (n = 4). The symbol * indicates significant differences (p < 0.05) compared with the nontreatment group.
Figure 7. Macrophage-targeted siRNA delivery by CMI nanoparticles. (A) Expression of TNFα in macrophages was knocked down dose dependently. Mouse peritoneal macrophages were transfected with various concentrations of siRNA against TNFα. After 2 h, the macrophages were stimulated with LPS (10 ng/mL). Cells were harvested for the isolation of total mRNA and protein, followed by quantification by RT-qPCR and ELISA, respectively. (B) Transfection of siRNA against TNFα by lipofectamine 2000 did not induce significant knockdown even at the highest concentration of siRNA. (C) Either CMI complexed to siControl or siControl alone did not induce any silencing of TNFα in mouse peritoneal macrophages. Each value represents the mean ± SEM (n = 4). The symbol * indicates significant differences (p < 0.05) compared with the LPS-treated group.
uptake.40 Mannose receptor internalization is dependent on the interaction of dihydrophobic motifs in its cytoplasmic tail with the clathrin adapter complex AP-2, which prompts rapid internalization by recruiting to clathrin-coated pits. Mannose-Receptor-Mediated siRNA Delivery to Peritoneal Macrophages. Upon confirming the targeting efficiency as well as the cellular uptake mechanism of CMI, we tested the macrophage-targeted siRNA delivery efficiency of CMI. To do this, we treated mouse primary macrophages with
nanoparticles in macrophages. However, the cellular uptake of CMI nanoparticles was strongly abolished by sucrose and significantly inhibited by chloroquine as well. Sucrose blocks receptor recycling through the formation of clathrin microcages on the inner surface of the plasma membrane.37−39 Chloroquine inhibits clathrin-dependent endocytosis by affecting the function of clathrin and clathrin-coated vesicles.34 Chloroquine inhibits receptor-mediated endocytosis of mannose glycoconjugates by macrophages, effecting receptor recycling and thereby ligand 14469
DOI: 10.1021/acsami.8b02073 ACS Appl. Mater. Interfaces 2018, 10, 14463−14474
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Figure 8. Macrophage-targeted in vivo siRNA delivery by CMI nanoparticles. Administration (ip injection) of the CMI/siTNFα complex to mice induced significant knockdown of the LPS-stimulated expression of TNFα in peritoneal macrophages (A), liver (B), and lung (C). After ip injection of the CMI/siTNFα complex to mice, the TNFα protein level was remarkably reduced in the peritoneal fluid (D), serum (E), and bronchoalveolar lavage fluid (BALF) (F). Each value represents the mean ± SEM (n = 4). The symbol * indicates significant differences (p < 0.05) compared with the LPStreated group and siControl group.
primary macrophages, induces RNAi activity, and knocks down endogenous gene expression at both mRNA and protein levels. The preventive regulation of TNFα expression by siRNA is meaningful in clinic. LPS induces a quick immune response, for example, the expression level of TNFα in primary macrophages after LPS treatment maximizes in 1−2 h and decreases rapidly thereafter; meanwhile, RNAi activity reaches plateau 8 h after siRNA transfection. Therefore, the pretreatment of immune cells by RNAi can knock down the abruptly increased TNFα expression, potentially relieving the allergic symptoms and effectively preventing from recurrence, considering the persistence of RNAi (up to 7 days).41 Although the inhibition of TNFα by antibodies (e.g., infliximab) may increase the risk of infections such as tuberculosis, closely monitoring the circulating levels of the antibodies as well as tailoring to individual patients can potentially improve the outcome of the treatment with minimum side effects.42 In Vivo siRNA Delivery. The expression of TNFα in macrophages swiftly increases upon stimulation by immunogenic substances such as LPS. Kupffer cells in liver, alveolar macrophages in lung, and peritoneal macrophages are among the main responders during acute infection, and inhibition of TNFα in these cells can significantly alleviate the TNFα-related symptoms. To evaluate the in vivo siRNA delivery efficiency of CMI to the aforementioned organs, we administered the siTNFα-containing CMI nanoparticles by intraperitoneal (ip) injection to mice. The subsequent stimulation of mice with LPS induced prompt up-regulation of TNFα at both mRNA and protein levels (Figure 8). However, only preinjection of the CMI/siTNFα complex remarkably inhibited the expression of TNFα, whereas a control siRNA complexed to CMI did not change either mRNA or protein levels of TNFα. Such a
CMI complexed to a siRNA against TNFα. First, we stimulated mouse peritoneal macrophages with lipopolysaccharide (LPS), an important structural component of the outer membrane of Gram-negative bacteria that activates monocytes and macrophages to produce proinflammatory cytokines, such as TNFα and interleukin (Il)-1β. After various concentrations of LPS (1− 20 ng/mL) were subjected to the peritoneal macrophages, cellular mRNA and protein levels of TNFα were measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively (Figure 6A). The result showed that both mRNA and protein levels of TNFα increased with the increasing dose of LPS, with a minimum concentration of 1 ng/mL required for TNFα stimulation. Next, the cells were treated with 10 ng/mL LPS in a time-dependent manner (Figure 6B). The secretion of TNFα reached a plateau level 1 h after LPS treatment. On the basis of these results, we examined the efficacy of the TNFα silencing efficiency by CMI/siRNA complexes in peritoneal macrophages. The cells were treated with CMI/siRNA (mouse TNFα-specific siRNA) for 2 h. Then, the cells were incubated for another 2 h after adding10 ng/mL LPS, and then TNFα mRNA and protein levels were measured (Figure 7). At 10 nM concentration of siRNA, delivery by CMI induced 50% knockdown of TNFα mRNA and 35% reduction of the TNFα protein level, whereas the treatment of cells with naked siRNA did not significantly change mRNA and protein levels of TNFα. At 50 nM, siRNA delivery by CMI induced about 90% knockdown of both mRNA and protein levels of TNFα. Remarkably, siRNA transfection of primary macrophages by CMI induced RNAi in the cells within a few hours, demonstrating the quick transfection efficiency of CMI. These results demonstrated that CMI efficiently delivers siRNA to 14470
DOI: 10.1021/acsami.8b02073 ACS Appl. Mater. Interfaces 2018, 10, 14463−14474
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ACS Applied Materials & Interfaces
Figure 9. In vivo administration of CMI/siRNA complex does not induce liver toxicity. After ip injection of the CMI/siRNA complex, the serum levels of liver enzymes AST (A) and ALT (B) were analyzed by ELISA. Each value represents the mean ± SEM (n = 4).
isothiocyanate (FITC)-siRNA were synthesized by Takara Biotechnology Co., Ltd. (Dalian, Liaoning, China). Synthesis and Characterization of CMI. 6-Amino-6dexoy-curdlan (80 mg; 6AC-100)11 was dissolved in 3 mL of phosphate-buffered saline (PBS) (pH = 9.0), and 1 mL of dimethyl sulfoxide solution of 4-isothiocyanatophenyl α-Dmannopyranoside (18.7 mg) was added, followed by rotating at room temperature (r.t.) for 2 h. Then, a mixture of imidazole4(5)-acetic acid hydrochloride (31.5 mg) and 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (22.1 mg) in 0.1 M MES buffer (pH = 6.0) was added to the solution and the rotation was continued at r.t. for 6 h. After the product was purified by dialyzing (molecular weight cut-off 3500) against deionized water for 2 days, a PD-10 desalting column (GE Healthcare) was used to further purify the new product (denoted CMI), which was obtained by subsequent freeze-drying. The structure of CMI was confirmed by carbon nuclear magnetic resonance (13C NMR) analysis. Gel Permeation Chromatography Analysis of CMI. The weight-average molecular weight was determined by gel permeation chromatography (GPC) employing a refractive index detector with pullulan as a calibration standard, using a Waters HPLC instrument (Waters Co., Milford, MA). Watersoluble CMI was analyzed with a 7.8 × 300 mm2 column (Waters Co., Milford, MA) eluted with acetic acid (0.2 M)/sodium acetate (0.1 M) buffer. The flow rate was 0.8 mL/min, and the temperature was 40 °C. Electrophoretic Mobility Shift Assay. CMI/siRNA complexes were formed at various N/P ratios in a gel mobility shift assay. The mixtures were incubated at r.t. for 20 min and then subjected to electrophoresis on agarose gel (2%) for 30 min at 100 V to confirm the siRNA complexation in 1× Tris/borate/ ethylenediaminetetraacetic acid (EDTA) running buffer. After the gel was stained using 0.5 μg/mL ethidium bromide, the banding pattern was obtained using a Gel Logic 212 PRO imaging system (Carestream, Toronto, Canada). Serum Stability Assay. To evaluate the serum stability, CMI nanoparticles (8 μg) and siRNA (100 μM) were incubated with an equal volume of FBS (final concentration 50% v/v) at 37 °C. After the samples were kept at 0.5, 2, 4, 6, and 24 h, 1 μL of 0.5 M EDTA was immediately added to stop any nuclease activity. Then, 1 μL of heparin (50 μg) was added to each sample to release the siRNA from each complex and the samples were subjected to gel electrophoresis. Heparin Competition Assay. The siRNA release from CMI/siRNA complexes was assessed by co-incubation with
significant knockdown of TNFα was observed in both the mRNA level in tissue cells (liver, lung, and peritoneal cells) and the secreted protein level in body fluid (peritoneal fluid, blood, and bronchoalveolar lavage fluid (BALF)). To assess the in vivo toxicity of the CMI/siRNA complex, we collected serum from the mice injected with the complexes. The analysis of liver enzymes (AST and ALT) indicated that the CMI/siRNA complex does not increase the liver enzyme level in serum (Figure 9), demonstrating that CMI nanoparticles do not have apparent liver toxicity. Collectively, these experiments confirmed that CMI can efficiently deliver siRNA to multiple organs (liver and lung) and induce profound RNAi activity. More importantly, such RNAi activity is strictly sequence-specific and the delivery of such nucleic acid sequences by CMI nanoparticles is macrophage-specific, which involves cell surface-receptor-mediated internalization and subsequent knockdown of disease-related genes.
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CONCLUSIONS Mannose- and imidazole-functionalized 6-amino-6-deoxy-curdlan was designed for primary-macrophage-targeted siRNA delivery. When complexed with siRNA, the CMI nanoparticle has a diameter of 50−80 nm with a ζ potential of 32.9 mV. CMI delivered dye-labeled siRNA to macrophages in a significantly higher efficiency compared with other transfection reagents. The competitive blocking experiments as well as temperaturedependent experiments indicated that a CMI nanoparticle was internalized by macrophages through receptor-mediated endocytosis. The functioning receptor for CMI binding and subsequent endocytosis was the mannose receptor (CD206) not the dectin-1 receptor. CMI delivered siRNA against TNFα to LPS-stimulated primary macrophages in vitro and in vivo and induced significant silencing of TNFα at both mRNA and protein levels in a time-efficient manner. Collectively, our data suggest that the CMI nanoparticle may be a promising siRNA delivery agent.
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MATERIAL SECTION Chemicals. Curdlan was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 4-Isothiocyanatophenyl α-D-mannopyranoside was purchased from Toronto Research Chemicals (Toronto, Canada). Imidazole-4(5)-acetic acid hydrochloride and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were purchased from TCI chemicals (Tokyo, Japan) and Aladdin (Shanghai, China), respectively. TNFα gene silencing siRNA, TNFα primers, and fluorescein 14471
DOI: 10.1021/acsami.8b02073 ACS Appl. Mater. Interfaces 2018, 10, 14463−14474
Research Article
ACS Applied Materials & Interfaces
μg/mL. After incubating for 24 h, cell viability was evaluated by the MTT assay. Nanoparticle Uptake in Isolated Peritoneal Macrophages. Peritoneal macrophages were plated at a density of 1 × 106 cells in the culture medium in a 24-well plate with microscopic coverslips. After culturing for 24 h, the FITClabeled siRNA/CMI (CMI/FITC-siRNA) complex was added to the cell culture and incubated for 6 h, beginning at 20 min prior to the transfection. Final concentrations of CMI and FITCsiRNA were 20 μg/mL and 50 nM, respectively. L2K and 6AC100 were used as comparison groups. Transfected macrophages were washed with 1× PBS (pH 7.4), till complete removal of nontransfected FITC-siRNA, and fixed with 4% paraformaldehyde for 30 min. After rinsing the slides with PBS three times, the fixed cells were stained with 4′,6-diamidino-2-phenylindole for 10 min. The coverslips were detached and mounted on a glass slide by mounting media and stored at 4 °C until confocal microscopy was performed. For flow cytometer (FCM) analysis, the peritoneal macrophages were seeded at a density of 2.5 × 106 cells in a 12-well plate. The cells were transfected with CMI/ FITC-siRNA, L2K/FITC-siRNA, or 6AC-100/FITC-siRNA complexes under the same conditions as those used in confocal microscopy experiments, and then the cells were analyzed by a NovoCyte Benchtop Flow Cytometer (ACEA Biosciences, Inc., San Diego, CA). Blocking and Temperature-Dependent Transfection Efficiency. The peritoneal macrophages were seeded in a 12well plate at 2.5 × 106 cell density and cultured for 24 h. After blocking the cells with different final concentrations (10, 20, 40, and 80 μg/mL) of blocking solutions (mannan and zymosan) for 1 h, CMI/FITC-siRNA complexes were added to the cell cultures and incubated for 6 h in a cell-culturing incubator. CMI/ FITC-siRNA complexes were incubated at r.t. for 20 min before transfection. The results were assessed by FCM. For temperature-dependent cellular uptake, the cells were transfected with CMI/FITC-siRNA complexes and incubated at 4 or 37 °C. The cells were re-suspended in 0.5 mL of PBS and assessed by FCM. Then, and cytoplasmic location of the complex were assessed on an FCM and on confocal microscopy, respectively. Before capturing the cells under the confocal microscopy, the cells were prepared in the same way as described in the Nanoparticle Uptake in Isolated Peritoneal Microphages section and stained with Phalloidin-iFluor 555 (Abcam). Cellular Uptake Mechanisms of CMI Nanoparticles. To evaluate the cellular uptake mechanisms of CMI nanoparticles, we used various endocytosis inhibitors to hinder the possible cellular uptake pathway of CMI nanoparticles. The experiment was divided into three groups, namely, positive control, negative control, and treatment groups. For the treatment group, the peritoneal macrophages were treated with 0.1% (w/v) NaN3, 0.5 mM sucrose, 25 μM nystatin, and 800 μM chloroquine in serumfree Opti-MEM medium. The control groups were incubated in serum-free Opti-MEM medium. After 1 h, the cells of the treatment group and the positive control group were coincubated with the FITC-siRNA/CMI complex for another 1 h. Subsequently, flow cytometry was used to detect the cellular uptake of CMI nanoparticles. TNFα Silencing in Murine Primary Macrophages in Vitro. Mouse peritoneal macrophages were stimulated with different concentrations of LPS (1, 2.5, 5, 10, and 20 ng/mL) for 2 h or stimulated for different durations (0.5, 1, 1.5, 2, 3, 4, 6, and 12 h) with a constant concentration of LPS (10 ng/mL), and mRNA levels and protein levels of TNFα were examined by RT-
heparin as a competitive binding agent. Briefly, CMI nanoparticles (8 μg) and siRNA (100 μM) were complexed in 15 μL of 20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid buffer, pH 7.2. Complexes were then incubated with various amounts of heparin sodium salt solution (0.25, 0.5, 1, 3, 5, and 10 μg) for 15 min at r.t. or with 5 μL of water. Then, the released free siRNA was analyzed by agarose gel electrophoresis. Particle Size and ζ Potential Measurements. CMI was dissolved in water and complexed with siRNA at a 4/1 molar ratio of the nitrogen in the CMI and the phosphorus in the siRNA (abbreviated as the N/P ratio). The mixture was incubated at r.t. for 20 min, and then ζ potential values and size distributions of CMI/siRNA complexes were analyzed by a Zetasizer Nano S instrument (Malvern Instrument, U.K.) in aqueous solution (pH 7.4) in triplicates. Transmission Electron Microscopy (TEM). Shape and surface morphological examination of CMI/siRNA nanoparticles at a 4/1 N/P ratio in aqueous solution was investigated by transmission electron microscopy (TEM). After the mixed solution of CMI and siRNA at a 4/1 N/P ratio was incubated at r.t. for 20 min, two drops of the sample were placed on a copper grid, air-dried for 10 min, and then negatively stained with 2% phosphomolybdic acid solution for 2 min. The grid was allowed to air-dry for 10 min and examined under a TEM. Buffering Capacity. The buffering capacity of CMI was measured by acid−base titration. A total of 10 mg of CMI was dissolved in 10 mL of ddH2O, and the pH of the solution was adjusted to 10.0 with 1 M NaOH. In Vitro Release of siRNA from the CMI/siRNA Complex. The siRNA releasing profile from CMI/siRNA nanoparticles was examined using agarose gel electrophoresis in solutions with pH 7.4−5.0, mimicking physiological conditions (7.4), early/late endosomal conditions (6.0 and 5.5), and lysosomal conditions (5.0) according to the previous report.29 CMI/siRNA nanoparticles were complexed at an N/P ratio of 10 and incubated at 37 °C in 0.2 M citric acid sodium phosphate buffer solution (0.2 M) with pH 7.4, 6.0, 5.5, or 5.0, respectively, for 30 or 120 min. The released siRNAs were detected by loading onto 2% agarose gel and electrophoresis at 100 V for 20 min. Isolation of Peritoneal Macrophages. Animal experiments were approved by the Animal Care and Use Committee of the Inner Mongolia University. Peritoneal macrophages were harvested from Balb/c mice (Inner Mongolia University Experimental Animal Center) and the procedure can be described as follows: the mice were injected intraperitoneally with 5 mL of PBS with 5% heat-inactivated FBS and 4 μg/mL heparin and were massaged for 1−2 min. The macrophages were harvested from peritoneal lavage using a 10 mL syringe, followed by centrifugation at 450g for 10 min. The cells were suspended in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin and were plated on 12- or 24-well plates (Costar, Corning Inc.). After incubation at 37 °C in 5% CO2 for 3 h, nonadherent cells were washed off with culture medium and cells were cultivated for next experiments. Cytotoxicity Assay. HeLa cells, A549 cells, MDA-MB-231 cells, and mouse peritoneal macrophages were seeded at a density of 1.0 × 104 cells per well in 96-well plates in DMEM or RPMI 1640 medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin and cultured for 24 h at 37 °C in 5% CO2. Then, CMI and PEI and 6AC-100 as control were added to the cell at final concentrations of 10, 20, 40, and 60 14472
DOI: 10.1021/acsami.8b02073 ACS Appl. Mater. Interfaces 2018, 10, 14463−14474
ACS Applied Materials & Interfaces
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qPCR and ELISA, respectively. To investigate the transfection efficiency of CMI, the cells were transfected with TNFα siRNA (sense strand: 5′-gucucagccucuucucauuccugct-3′; antisense strand: 5′-agcaggaaugagaagaggcugagacau-3′) complexed to CMI for 4 h. As controls, cells were also treated with the same concentration of siRNA without the CMI complex or with CMI complexed to a control siRNA (siControl, Silencer negative control siRNA, Invitrogen, Carlsbad, CA). Then, 10 ng/mL LPS was added to the transfected cells and incubated for 2 more hours. Total RNA was extracted with TRIZOL (Invitrogen, Carlsbad, CA). The expression of mRNA was measured using the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit for cDNA synthesis and amplified by qPCR using iTaqTM Universal SYBR Green Supermix (Bio-Rad, Hercules, CA). The sequences of primers used for RT-qPCR were as follows: mouse β-actin primers: forward, 5′-ctaaggccaaccgtgaaaag-3′ and reverse, 5′-accagaggcatacagggaca-3′; mouse TNFα primers: forward: 5′tcttctcattcctgcttgtgg-3′ and reverse: 5′-gaggccatttgggaacttct-3′. To determine the protein level, the cell culture supernatants were analyzed using an ELISA kit for TNFα detection (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. In Vivo siRNA Delivery. Mice were reared at an animal facility under environmentally controlled conditions (24 ± 0.5 °C; humidity, 55 ± 5%) with a 12 h dark/light cycle and free access to food and water. Male Balb/c mice (7−8 weeks old) were randomly divided into four groups (four animals per group): nontreated (NT) group, LPS-treated group (LPS), control siRNA/CMI + LPS-treated group (siControl), and siTNFα/CMI + LPS-treated group (siTNFα). The doses of CMI and siRNA were 25 and 3 mg/kg, respectively. The mice were challenged with LPS (10 μg/kg) by ip injection 12 h after administration of siRNAs/CMI complexes by ip injection. The mice were sacrificed 1 h after the instillation of LPS, and the liver, lung, and peritoneal macrophages were harvested for measuring TNFα mRNA levels. Meanwhile, the peritoneal fluid, serum, and bronchoalveolar fluid (BALF) were collected for analysis of TNFα protein levels by ELISA. Additionally, to analyze the toxic effect of CMI nanoparticles on liver, the liver enzymes ALT and AST were analyzed by a biochemistry analyzer (Pronto Evolution, Italy). Statistical Analysis. Statistical significance was determined using one-way analysis of variance with a Dunnett’s multiple comparison test. p-Values of 3)-beta-D-glucans with various functional appendages. Carbohydr. Res. 2006, 341, 35−40. (13) Brown, G. D.; Herre, J.; Williams, D. L.; Willment, J. A.; Marshall, A. S.; Gordon, S. Dectin-1 mediates the biological effects of betaglucans. J. Exp. Med. 2003, 197, 1119−1124. (14) Wu, Y.; Cai, J.; Han, J.; Baigude, H. Cell Type-Specific Delivery of RNAi by Ligand-Functionalized Curdlan Nanoparticles: Balancing the Receptor Mediation and the Charge Motivation. ACS Appl. Mater. Interfaces 2015, 7, 21521−21528. (15) Wynn, T. A.; Chawla, A.; Pollard, J. W. Macrophage biology in development, homeostasis and disease. Nature 2013, 496, 445−455. (16) Pei, Y.; Yeo, Y. Drug delivery to macrophages: Challenges and opportunities. J. Controlled Release 2016, 240, 202−211. (17) Ruan, G. X.; Chen, Y. Z.; Yao, X. L.; Du, A.; Tang, G. P.; Shen, Y. Q.; Tabata, Y.; Gao, J. Q. Macrophage mannose receptor-specific gene delivery vehicle for macrophage engineering. Acta Biomater. 2014, 10, 1847−1855. (18) Tobinick, E.; Gross, H.; Weinberger, A.; Cohen, H. TNF-alpha modulation for treatment of Alzheimer’s disease: a 6-month pilot study. MedGenMed 2006, 8, 25. (19) O’Shea, J. J.; Ma, A.; Lipsky, P. Cytokines and autoimmunity. Nat. Rev. Immunol. 2002, 2, 37−45. (20) Upadhyaya, S. K. Antibodies to Tumor Necrosis Factors in the Treatment of Rheumatoid Arthritis and Spondyloarthritis: The Basic
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Huricha Baigude: 0000-0001-5479-182X Notes
The authors declare no competing financial interest.
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Research Article
ACKNOWLEDGMENTS
This research has been kindly supported by the National Natural Science Foundation of China (81560568, 21375058, and 21364006). 14473
DOI: 10.1021/acsami.8b02073 ACS Appl. Mater. Interfaces 2018, 10, 14463−14474
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