Dimeric human Î²-defensin 3 as a universal platform for intracellular

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Dimeric human #-defensin 3 as a universal platform for intracellular delivery of nucleic acid cargos Hyo Young Kim, Jeong-Eun Jang, and Dae-Ro Ahn ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00024 • Publication Date (Web): 14 Jun 2018 Downloaded from http://pubs.acs.org on June 18, 2018

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ACS Applied Bio Materials

Dimeric Human β-Defensin 3 As a Universal Platform for Intracellular Delivery of Nucleic Acid Cargos

Hyo Young Kim1, Jeong-Eun Jang1, and Dae-Ro Ahn1,2*

1

Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science

and Technology (KIST), Hwarangno 14-gil 5, Seongbuk-gu, Seoul 02792, Republic of Korea 2

Division of Biomedical Science and Technology, Korea University of Science and

Technology (UST), Hwarangno 14-gil 5, Seongbuk-gu, Seoul 02792, Korea

1

ACS Paragon Plus Environment

ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT Functional nucleic acids including siRNA, mRNA, and plasmid DNA are promising bioactive molecules to regulate cellular functions uncontrollable by conventional small molecule regulators. To realize successful cellular applications of these nucleic acids, an intracellular gene delivery vehicle with high efficiency and low cytotoxicity is required. Here, we report the dimerization of human β-defensin 3 (DhBD3) promoted by the interaction between β-strands and the application of DhBD3 for efficient delivery of various nucleic acid cargos. DhBD3 with multiple cationic residues could be complexed with various types of polyanionic DNA and RNA. DhBD3 could intracellularly deliver both small and large nucleic acid cargos loaded by complexation to regulate the expression level of target proteins, showing its potential as a universal platform for nucleic acid delivery. In addition, as DhBD3 is a human-derived material with high biocompatibility and can be robustly prepared by an inexpensive method, it is a promising gene delivery system that can be employed for biomedical purposes.

KEYWORDS β-defensin, dimeric β-defensin3, self-aggregation, gene delivery vehicles, nucleic acid cargos

2


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INTRODUCTION Intracellular delivery of functional nucleic acids that regulate the expression of specific proteins associated with various cellular functions provides an efficient regulatory approach distinct from traditional small molecule regulators.1-2 However, as these nucleic acids are hardly cell-impermeable due to their polyanionic character, an efficient vehicle for the intracellular delivery of the nucleic acid cargos is required to realize gene-based manipulation of cellular functions.3 Natural vehicles for the delivery of nucleic acid cargos include viral vectors, which are particularly optimized for the delivery of plasmid DNA for transfection. While some viral vectors, such as adeno-associated virus (AAV), have shown a practical utility in clinical area,4 the transgene size in viral vehicles is limited. Alternative to viral vehicles, non-viral gene delivery vehicles have also been investigated for decades.57

Among the various non-viral vectors,8-11 one of the most popular systems is

cationic lipid-based liposomes including Lipofectamine (LF). However, they often show considerable cytotoxicity particularly when complexed with nucleic acid cargos, which may not be acceptable in many intracellular applications.12 For improved biocompatibility, vehicles made of materials intrinsically derived from the human body have been considered. Previously, DNA and RNA nanoparticles have been employed as vehicles for the delivery of siRNA.13-14 While DNA and RNA materials are highly biocompatible, they are limited for the delivery of small nucleic acid cargos. Recently, extracellular vesicles (EVs) released from cells have been suggested as a gene delivery vehicle.15 While EVs can be regarded as a versatile vehicle for the delivery of a wide range of nucleic acid cargos, the 3



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preparation of EVs is very costly and laborious, as there is no method for the robust, large-scale production of EVs.16

Figure 1. Schematic representation of DhBD3 which can be self-aggregated or complexed with nucleic acid cargos. The hBD structure induced by three disulfide bonds (red lines) provides three β-strands (green arrows). One of the βstrands in hBD interacts with the β-strand in another hBD to form the dimer DhBD3. DhBD3 can self-aggregate to form nanoparticles. In the presence of nucleic acids, DhBD3 can be complexed with the nucleic acids due to the multiple cationic residues of the peptide.

Human β-defensins are cationic peptides naturally expressed in the human body that work as a defensive agent in the innate immune system by exerting 4


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antimicrobial activity against various invading pathogenic bacteria.17-18 The antimicrobial activity of β-defensin is based on the strong interaction between the positively charged residues of dimeric β-defensin and the negatively charged lipopolysaccharides in the bacterial cell, which leads to cell lysis through the formation of a membrane pore complex.19-22 Inspired by this fact, we hypothesized that dimeric human β-defensins (DhBDs) can also interact with the negatively charged mammalian cell membrane and penetrate into the intracellular region. At the same time, the highly cationic DhBDs can be complexed with polyanionic functional DNA and RNA to intracellularly deliver nucleic acid cargos. To prove the potential of DhBDs as a gene delivery system, we here employ DhBD3 as the vehicle for the delivery of nucleic acid cargos, as hBD3 has an affordable size for chemical synthesis and is relatively abundant in cationic residues compared to other hBDs.23 After preparing and characterizing DhBD3, we find that DhBD3 has high cellular uptake and low cytotoxicity. We also demonstrate that DhBD3 can be complexed with various small and large nucleic acids to successfully deliver nucleic acid cargos into cells for the regulation of the downstream protein expression.

RESULTS The hBD3 peptide was prepared by solid-phase peptide synthesis. It was dimerized via the interaction between β-strands of the monomers organized by three intramolecular disulfide linkages formed after spontaneous oxidation in solutions as previously reported.24-25 DhBD3 showed a mobility relevant to the expected molecular weight of the dimer (10.3 kDa) in tricine polyacrylamide gel electrophoresis. DhBD3 was transformed back into hBD3 (5.15 kDa) by breaking 5



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the disulfide bonds using a reducing agent such as dithiothreitol (DTT) (Fig. 2A). The

dimerization

was

also

characterized

by

matrix-assisted

laser

desorption/ionization time of flight (MALDI-TOF) mass spectrometry (Fig. S1). The hydrodynamic size of DhBD3 at 50 µM, as measured by dynamic light scattering (DLS), was 615 – 712 nm, much larger than that of the monomer hBD3, which indicates that DhBD3 formed self-aggregated particles (Fig. 2B) at high concentrations. The size of the aggregate in the dried state was estimated to be approximately 100 - 200 nm by transmission emission microscopy (TEM) (Fig. 2B, inset). Although the β-strands with hydrophobic residues in the amphiphilic monomer hBD3 have the potential to facilitate self-aggregation in a similar manner to other β-strand-based peptide aggregation processes,26 the intermolecular hydrophobic interaction in the monomer appears to be relatively weaker than that in the dimer. The relatively higher aggregation potential of the dimer compared with the monomer was indeed evidenced by the fact that the critical aggregation concentration (CAC) of DhBD3 (20 µM) is much lower than that of hBD3 (>120 µM) (Fig. 2C).

6


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Figure

2.

Characterization

of

DhBD3.

(A)

Tricine-polyacrylamide

gel

electrophoresis of DhBD3 in the presence and absence of DTT. (B) Hydrodynamic diameters of hBD3 and DhBD3 (50 µM) measured by DLS. The inset shows a TEM image of self-aggregated DhBD3. (C) Critical aggregation concentration (CAC) of DhBD3 determined using DLS.

Figure 3. Cellular properties of DhBD3. (A) Uptake efficiency of hBD3 and DhBD3 (10 µM) into HeLa cells. (B) Confocal fluorescence microscopy images of HeLa cells treated with Cy5-labeled DhBD3 (10 µM, red). The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). The scale bar: 20 µm. (C) Viability of HeLa cells treated with DhBD3 at various concentrations. (D) The level of interleukin-6 (IL-6) released from RAW264.7 cells after treatment with DhBD3 (10 7



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µM). The IL-6 level released from the cells treated with lipopolysaccharides (LPS, 10 µg/mL) is displayed as a positive control.

To examine the cellular uptake property of DhBD3, we treated HeLa cells with Cy5-labeled DhBD3 and analyzed the uptake efficiency using flow cytometry. Dimerization greatly enhanced the cellular uptake efficiency of hBD3, possibly due to the increased numbers of cationic residues. Compared with hBD3, DhBD3 showed 25-fold higher cellular uptake efficiency (Fig. 3A). The enhanced uptake level was also revealed by fluorescence microscopy images of the cells treated with DhBD3 which clearly illustrates cytoplasmic distribution of the dimeric peptide after uptake (Fig. 3B).The cellular uptake of DhBD3 was further increased at the concentrations higher than CAC (Fig. S2). To investigate the mechanisms involved in the cellular uptake of DhBD3 at lower and higher concentrations than CAC, we performed endocytosis inhibitor assays below CAC and above CAC. As the result, the cellular uptake of DhBD3 at the concentration below CAC substantially decreased in the presence of methyl-β-cyclodextrin (MβCD), an inhibitor of the caveolae-dependent endocytosis while the presence of the macropinocytosis inhibitor (5-(N-ethyl-N-isopropyl) amiloride, EIPA) and the inhibitor of the clathrindependent endocytosis (chlorpromazine, CPZ) only slightly decreased the uptake level (Fig. S3A). This indicates that caveolae-dependent endocytosis is the main mechanism for the cellular uptake of DhBD3 at the concentration below CAC (10 µM). However, at the concentration above CAC (50 µM), the uptake of DhBD3 was more affected by the presence of CPZ and EIPA, suggesting that clathrindependent endocytosis and macropinocytosis mechanisms are involved in the 8


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intracellular delivery of the self-aggregated DhBD3. (Fig. S3A). The cell viability was not significantly affected by high concentrations of DhBD3, showing the low cytotoxicity of the peptide in both non-aggregated and self-aggregated forms.(Fig. 3C). A previous study have shown that hBD3 induce no significant hemolysis at up to 500 µg/mL.27 To examine whether DhBD3 is also a non-hemolytic material, we performed hemolysis assay of DhBD3. As the result, DhBD3 did not induce considerable hemolysis (Fig. S4). In addition, when in vitro immunostimulation was estimated by the IL-6 level upon treatment of macrophage cells (RAW264.7) with DhBD3, the cytokine level induced by DhBD3 was found to be negligible, indicating the low immunogenicity of DhBD3 (Fig. 3D). These results show that DhBD3 derived from humans is a highly biocompatible material that can be potentially employed as a delivery vehicle for biomedical purposes. Having discovered the great potential of DhBD3 as a delivery system, we investigated whether DhBD3 can be used as a transfection agent for the delivery of plasmid DNA into cells. We attempted to load plasmid DNA expressing enhanced green fluorescence protein (pEGFP) on DhBD3 by taking advantage of the multiple cationic residues in DhBD3 that can complex with polyanionic DNA. The complex of pEGFP (10 ng) with DhBD3 (pEGFP@DhBD3) was verified by the substantially decreased mobility of the DNA in agarose gel electrophoresis (Fig. S5A). The complexation was completed at 10 µM DhBD3, which is lower than the CAC. Thus, pEGFP@DhBD3 was prepared at 10 µM DhBD3 for further experiments (Fig. 4A). The hydrodynamic size of the pEGFP@DhBD3 complex measured by DLS was 1.99 - 2.69 µm in solution (Fig. 4B), while the size in the dried state estimated under TEM was approximately 600 nm (Fig. 4B, inset). The zeta-potential value of DhBD3 9



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(+25 mV) was greatly decreased after complexation with polyanionic pEGFP which could mask the positively charged residues of DhBD3 (Fig. S6, red). The cellular uptake of pEGFP@DhBD3 was based on the caveolae-dependent endocytosis and micropinocytosis (Fig. S3B).

Figure 4. Cell transfection with a DNA plasmid using DhBD3 as a transfection agent. (A) An EGFP-expressing plasmid (pEGFP, 10 ng) was complexed with DhBD3 at various concentrations. The complexation level was analyzed by agarose 10


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gel electrophoresis (Fig. S5A). (B) The hydrodynamic size of the pEGFP@DhBD3 complex (10 ng pEGFP complexed with 2 µM DhBD3) measured by DLS. The inset shows a TEM image of the pEGFP@DhBD3 complex. (C) Flow cytometric analysis of HeLa cells expressing EGFP after transfection with pEGFP. The significance (***) indicates P

Dimeric human Î²-defensin 3 as a universal platform for intracellular

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