Design of Mannose-Functionalized Curdlan Nanoparticles for

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Biological and Medical Applications of Materials and Interfaces

Design of mannose functionalized curdlan nanoparticles for macrophage targeted siRNA delivery Tsogzolmaa Ganbold, and Huricha Baigude ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b02073 • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 12, 2018

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ACS Applied Materials & Interfaces

Design of mannose functionalized curdlan nanoparticles for macrophage targeted siRNA delivery Tsogzolmaa Ganbold, 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, PR China

*Correspondence to: Dr. Huricha Baigude, School of Chemistry & Chemical Engineering, Inner Mongolia University, 235 West College Road, Hohhot, Inner Mongolia Autonomous Region, 010020 P.R.China, Fax: +86-471-4993165, e-mail: [email protected]

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Abstract 6-Amino-6-deoxy-curdlan is a promising nucleic acids 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 primary cell targeting efficiency of curdlan derivative, we chemically conjugated a macrophage targeting ligand, mannose, to 6-amino-6-deoxy-curdlan. The resulting curdlan derivative (denote CMI) readily complexed with siRNA and formed nanoparticles with a diameter of 50 nm to 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 macrophage surface mannose receptor (CD206), but not by zymosan, a ligand for dectin-1 receptor, which also presents on the surface of macrophages. Moreover, CMI nanoparticles were internalized by macrophage only at 37 oC, suggesting that the cellular uptake of CMI nanoparticles was energy dependent. Furthermore, CMI nanoparticle efficiently delivered siRNA against TNFα to LPS 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 TNFα expression at both mRNA and protein level. 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 posttranscriptional down-regulation of gene expression by designed sequences of double strand short interfering RNA (siRNA).1 Endogenously generated or exogenously introduced siRNA loads to RNA induced silencing complex (RISC) and clips target mRNA at specific site, resulting in the expedited turnover of disease coursing 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 non-druggable gene products.3 However, because of the vulnerable nature of siRNA such as highly susceptible 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, has 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 to protect and deliver siRNA.8-9 With a serious of formulations involving several types of macromolecules such as PEI and tRNA, a siRNA delivery microparticle termed GeRP was created. When orally administered, the GeRP efficiently delivered siRNA to macrophages reside in several organs including liver, spleen and lung, and induced RNAi against TNFα.10 We synthesized 6-azido-6-deoxy-curdlan for the first time using triphenylphosphine, carbon tetrabromide and sodium azide. A subsequent reduction of 6-azido-6-deoxy-curdlan by NaBH4 gave 6-amino-6-deoxy-curdlan (denoted as 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 3



derivatives with full C6-amination, was selected for further chemical modifications and investigation of 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-azide-curdlan to give a cationic polymer. When complex with plasmid DNA, the lysine functionalized cationic polymer formed nanoparticles, which successfully delivered a plasmid expression green fluorescence protein (GFP) 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 original conformation of curdlan, which may recognize and bind to cell surface receptors such as Dectin-1, the major pattern-recognition receptor present on phagocytes including macrophages.13 Therefore, macrophage targeted delivery of therapeutics by these cationic polymers is possible. For example, we synthesized D-galactose functionalized 6amino-curdlan through single step reductive amination of lactose. The resulting polymer 6AC100Lac specifically binds to HepG2 cells through 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-aminocurdlan.14 This cell type specific delivery of siRNA induced RNAi activity, resulting in a significant knockdown of endogenous gene GAPDH. Such conjugations demonstrated that 6amino group of 6AC-100 is highly reactive chemical bonding site and provides versatile conjugation of functional moieties. 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 4


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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 (TNFα), a potent regulator of varied physiological mechanisms in multiple organ systems.18 Inhibition of TNF has proven to be remarkably effective in treatment of rheumatoid arthritis (RA), inflammatory bowel disease (IBD), Alzheimer’s disease and Crohn’s disease, and this strategy has a strong scientific basis given the abundance of literature substantiating the pro-inflammatory role of TNFα.18,19 TNFα-based strategies are being explored in the form of “anti-TNF” therapies for inflammatory diseases which 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 protein; infliximab, a chimeric monoclonal receptor antibody; and adalimumab, a human monoclonal TNFα antibody. However, they are associated with severe infections and malignancies due to their non-selectivity. 21, 22 siRNA-mediated knockdown of pro-inflammatory cytokines at the translational level offers an alternative therapeutic strategy to overcome inflammatory condition.23 Herein we report chemically modified curdlan for cell surface receptor targeted siRNA delivery to macrophage. RESULTS AND DISCUSSION Synthesis and characterization of CMI. In order to create a siRNA carrier based on naturally occurring macromolecule with active targeting capacity to primary macrophages, we chemically modified 6-amino-6-deoxy-curdlan, which exhibited 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-6-deoxy-curdlan. This was 5



achieved by reacting 6-amino-6-deoxy-curdlan with 4-isothiocyanatophenyl α-Dmannopyranoside, which is an isothiocyanate activated mannose that readily react with primary amines; then, imidazole groups were also attached to the backbone of the curdlan derivative by reacting with 4-imidazoleacetic acid using EDC as a condensing reagent (Scheme 1). The product of the reaction was extensive purified by dialysis as well as gel permeation. Then, the structure of resulting polymer (denoted CMI) was confirmed by 13C NMR analysis (Figure 1), and the molecular weight distribution of the polymer was measured by GPC. In 13C NMR, the newly attached mannose moiety showed a signal at 98.32 ppm (5), and carbon from the amide bond showed 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 (DS), estimated from 13C NMR spectrum, was 5.1% for mannose and 8.2% for imidazole. 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 6AC-100 because the conjugation of multiple moieties to 6AC-100 backbone resulted in minor aggregation in the final product. The subsequent removing of the insoluble precipitates (which is highly substituted high molecular weight fraction) in final product resulted in a CMI with reduced molecular weight.

Scheme 1. Synthesis of mannose- and imidazole double functionalized 6-amino-6-deoxy-curdlan (CMI). 6


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Figure 1. 13C NMR spectrum of CMI in D2O. SiRNA binding and nanoparticle imaging. In order 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 on gel retardation assay (Figure 2B). The migration of siRNA in gel was retarded with increasing ratio of amine: phosphate (N/P); at N/P ratio of 4, siRNA migration in gel was totally retarded, indicating that CMI and siRNA can form a complete complexation through electrostatic interaction. Moreover, DLS measurement indicated that naked CMI nanoparticles display an average size of 16.1 nm (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 assembled with siRNA in neutral aqueous solution (pH, 7.4)27. The average hydrodynamic size (diameter) of CMI/siRNA complex was 107.3 nm (PDI, 0.20). The zeta potential of the particle was 32.9 mV at pH 7.4, indicating the appreciable stability of the nanoparticles in the neutral solution28. Furthermore, we observed the morphological appearance of CMI/siRNA complex by TEM 7



(Figure 2C). Spherical particles with diameters ranging between 50 nm to 80 nm encapsulating the siRNA were captured on the images taken on TEM. 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 proton sponge function, which can promote 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 buffering capacity of CMI to assess its proton absorption efficiency (Figure 2D). CMI showed excellent buffering ability at pH 4.5 to 5.5, although compared with starting material, the titration curve of CMI dropped slightly (data not shown). The proton-absorbing property of CMI was lower than 6AC-100, due to their lesser protonable amino groups, which attached with mannose or imidazole.

Figure 2. (A) GPC chromatographe of CMI; (B) Gel retardation assay of CMI/siRNA complex. Lane 0; free siRNA and lane 1-7; CMI/siRNA nanoparticles at N/P ratio of 1-6:1; (C) TEM image of 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 pH mimicking endosomal environment. 8


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Stability and release assay. The stability of 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 CMI/siRNA complex with FBS (Figure 2E). While 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 was observed in the gel, while 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 CMI/siRNA complex in neutral pH, as well as acidic pH that mimic endosome environment29. At pH 5.5 ~ 6.0, siRNA was effectively released the complex (Figure 2G), indicating that CMI can release full length siRNA sequence from the endosome. The conjugation of imidazole may have contributed to such ability due to the “proton sponge” effect30. 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-231as well as mouse peritoneal macrophages were treated with various concentration of CMI, respectively, and the cell viability was assessed by MTT assay (Figure 3). PEI and 6AC-100 were used as comparison groups. The results showed that PEI is 9



most toxic for all cell types, with the cell viabilities of 50-60% at a concentration of 20 µg/mL compared with the non-treated group. The cells treated with 6AC-100 had 70-80% cell viability at concentration of 20 µg/mL. However, CMI was essentially nontoxic (cell viability 80%) with all of the cell types even at concentration as high as 60 µg/mL and showed nearly same diagram for all 4 types of cell. This result demonstrated that CMI nanoparticles show negligible influence on various cells compared with PEI and 6AC-100.

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 concentration. Cell toxicity levels are expressed as percent of control (no transfection, NT). Cellular uptake of CMI by macrophages. Now that CMI has functionalized mannose group that binds to cell surface mannose receptor (CD206) on macrophages and dendritic cells, we evaluated cellular uptake of CMI/siRNA complex by mouse peritoneal macrophages. To do this, we complexed CMI with a dye (FITC)-labeled siRNA at N/P ratio of 4, and then treated mouse peritoneal macrophages with the complex. After 6 h, the cellular uptake was captured under the confocal laser scanning microscopy (Figure 4). Compared with the control groups (Lipofectamine 2000 and 6AC-100), significantly greater amount of green fluorescent signal was observed locating in the cytosol of macrophages. Interestingly, 6AC-100/siRNA complexes had 10


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fragile cellular uptake compared to lipofectamine 2000 (L2K), which may attribute 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 didn’t show any fluorescence signal. These results indicated that CMI/siRNA complexes may enter the cell through receptor-mediated endocytosis involving both mannose receptor and dectin-1 receptor on the macrophages surface. Moreover, we 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 non-treated cells, while cells treated with L2K/siRNA had no cellular uptake and 6AC-100 appeared tenuous cellular uptake (8.6%). These results demonstrated that CMI/siRNA may enter macrophages through CD206 receptor mediated cellular uptake, hence CMI may be applicable for macrophage targeted siRNA delivery. Competitive bind and receptor blocking. To test whether or not CMI carries siRNA cargo and enters macrophage through CD206 receptor mediated endocytosis, we conducted a competitive blocking experiment. To do this, we chose mannan (a natural ligand for CD206 receptor) as well as zymosan (ligand for 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 receptor and could be recognized by the C-type like domain 4 (CTLD4) 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 transfection efficiency using flow cytometry. 11



Blocking the mannose receptor by mannan inhibited the cellular uptake of CMI/FITC-siRNA complexes in a dose dependent manner. At a concentration of 10 µg/mL, mannan inhibited transfection of CMI by about 50%; at 20 µg/mL 80% of CMI/siRNA complex was competitively inhibited for cellular uptake (Figure 5A). However, pretreatment of macrophages with zymosan had no influence on CMI transfection, suggesting that CMI predominantly binds to CD206 instead of dectin-1 receptor. 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 dectin-1 receptor on the macrophages.

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Figure 4. Uptake of CMI/FITC-siRNA by primary macrophage. (A) Mouse peritoneal macrophages were transfected with FITC-siRNA complexed to lipofectamine (L2K), 6AC-100 and CMI, respectively. The fluorescence signal in the cells were observed by fluorescence microscope. (B) Quantification of the cellular uptake by flow cytometry.

To further investigate the mechanism of CMI cellular entry, we transfected mouse primary macrophages with CMI/FITC-siRNA complexes and incubated at 4 oC or 37 oC, respectively. The fluorescence intensity, which was measured at different time point after the transfection, was much lower at 4 oC than that at 37 oC, while overall cellular uptake increased time dependently, suggesting that energy-dependent uptake occurred at 37 oC. This result can be interpreted that the fluorescence increase at 4 oC reflects the amount of CMI/FITC-siRNA complexes bound on mannose receptor on the cellular surface, and at 37 oC reflects CMI/FITC-siRNA complexes uptake by endocytosis plus bound on the cellular surface (Figure 5B). In addition, to distinguish surface-bound CMI/FITC-siRNA complexes from intracellular ones, we observed the endoplasmic location of the complex at 37 oC or 4 oC. Microscope images showed large amount of internalized CMI/FITC-siRNA complexes at 37 oC than at 4 oC (Figure 5C). These results indicate that CMI/FITC-siRNA complexes enter the cell through cell surface mannose receptor mediated endocytosis.

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Figure 5. Receptor mediated endocytosis of CMI. (A) Uptake of CMI nanoparticles was inhibited by mannan, but not dextrin and zymosan; (B) Incubation temperature dependence of the amount of cellular taken-up of CMI/FITC-siRNA complexes. (C) The cytoplasmic location of CMI/FITC-siRNA complex at 37 oC or at 4 oC; blue, nucleus; red, cytoplasm; green, siRNA. (D) The effect of inhibitors on cellular uptake. PC and NC represent positive control and negative control, respectively. Each value represents the mean ± SEM (n=4). The symbol “*” indicates significant difference (p

Design of Mannose-Functionalized Curdlan Nanoparticles for

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