Self-assembling multifunctional peptide was designed for gene delivery systems. The multifunctional peptide (MP) consists of cellular penetrating peptide moiety (R8), matrix metalloproteinase-2 (MMP-2) specific sequence (GPLGV), pH-responsive moiety (H5), and hydrophobic moiety (palmitic acid) (CR8GPLGVH5-Pal). MP was oxidized to form multifunctional peptide dimer (MPD) by DMSO oxidation of thiols in terminal cysteine residues. MPD could condense pDNA successfully at a weight ratio of 5. MPD itself could self-assemble into submicron micelle particles via hydrophobic interaction, of which critical micelle concentration is about 0.01 mM. MPD showed concentration-dependent but low cytotoxicity in comparison with PEI25k. MPD polyplexes showed low transfection efficiency in HEK293 cells expressing low level of MMP-2 but high transfection efficiency in A549 and C2C12 cells expressing high level of MMP-2, meaning the enhanced transfection efficiency probably due to MMP-induced structural change of polyplexes. Bafilomycin A1-treated transfection results suggest that the transfection of MPD is mediated via endosomal escape by endosome buffering ability. These results show the potential of MPD for MMP-2 targeted gene delivery systems due to its multifunctionality.
Lots of peptides have been developed and used for gene delivery systems due to their advantages [
Particularly, cellular penetrating peptides such as penetratin, Tat sequence, or oligoarginine have been extensively examined in gene delivery field because they can form nanosized complexes with nucleic acids due to their cationic property and possess high cellular uptake efficiency [
In this work, we designed a self-assembling multifunctional peptide (MP, CR8GPLGVH5-Pal) in order to combine advantages of several functional peptides for gene delivery systems. It is expected that R8 moiety would condense pDNA into polyplex particles and facilitate the cellular uptake of the polyplexes [
Multifunctional peptide (CR8GPLGVH5-Pal) was purchased from GL Biochem (Shanghai, China). Thiazolyl Blue Tetrazolium Bromide (MTT reagent), agarose, and 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent) were purchased from Sigma-Aldrich (St. Louis, MO). Dimethyl sulfoxide (DMSO) was purchased from Merck (Germany). Luciferase assay system was purchased from Promega (Madison, WI). BCA protein assay kit was purchased from Pierce (Rockford, IL). Fetal bovine serum (FBS), 0.25% trypsin-EDTA, Dulbecco’s phosphate buffered saline (DPBS), and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Invitrogen (Carlsbad, CA). The luciferase reporter gene-encoding plasmid DNA, pCN-Luci, was amplified in
Multifunctional peptide dimer (MPD) was synthesized by using DMSO autooxidation reaction [
Ellman’s assay was carried out to confirm the synthesis of MPD by measuring residual thiols after dimerization of MPM. MPD was dissolved in DMSO (0.2 mM). 50
DNA condensation ability of MPD was identified by agarose gel electrophoresis assay. Agarose gel (0.7 wt%) containing ethidium bromide was prepared in Tris-Acetate-EDTA (TAE) buffer. The MPD polyplexes (0.5
In order to characterize CMC of MPD, pyrene was selected as a model drug as previously reported [
Average sizes and Zeta-potential values of MPD and MPD polyplexes were measured by Zetasizer Nano ZS (Malvern Instruments, UK) with He-Ne laser beam (633 nm) at 25°C, respectively. 1 mL of MPD (0.01 and 0.1 mM) and MPD polyplex solutions (5
The morphologies of MPD particle structures were observed by using atomic force microscopy (Park Systems, Korea). 100
Human lung adenocarcinoma epithelial cells (A549), human transformed primary embryonal kidney epithelial cells (HEK293), and mouse myoblast cells (C2C12) were grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (P/S) in humidified atmosphere containing 5% CO2 at 37°C.
Cytotoxicity of MPD was characterized by MTT assay. Cells were seeded into 96-well cell culture plates in 100
The transfection efficiency of MPD was examined by measuring luciferase transgene expression. Cells were seeded into 24-well cell culture plates in 500
In order to characterize the endosome buffering ability and transfection mechanism of MPD, transfection experiments with Bafilomycin A1 treatment were carried out. A549 cells were seeded as explained above. 200 nM of Bafilomycin A1 solutions was pretreated for 10 min before transfection. MPD polyplexes were prepared with weight ratio of 10, 20, and 30. PEI25K was used as a positive control. Polyplex solutions were treated to the cells for 4 h and the media were exchanged with fresh DMEM with 10% FBS. After 48 h of further incubation, transfection efficiency was examined as described above. Final results were presented in terms of relative transfection efficiency (transfection efficiency with Bafilomycin A1/transfection efficiency without Bafilomycin A1).
MP consists of cellular penetrating peptide moiety (R8), matrix metalloproteinase-2 (MMP-2) specific sequence (GPLGV), pH-responsive moiety (H5), and hydrophobic moiety (palmitic acid) (CR8GPLGVH5-Pal). Cellular penetrating moiety was introduced for condensation of pDNA by electrostatic interaction of cationic arginine residues and for enhancement of cellular uptake. pH-responsive moiety (H5) was introduced for facilitation of endosomal escape of polyplexes after cellular uptake by proton sponge effect. Hydrophobic palmitic acid was introduced to C-terminal of MP in order to induce the formation of micelle structure by hydrophobic interaction. R8 and other functional blocks were linked with MMP-2 specific sequence, which may lead to structural change of polyplexes by cleavage of the sequence with exposure to MMP-2.
The structure of MP was confirmed by the manufacturer. MP was dimerized to synthesize MPD by DMSO oxidation via disulfide bond formation between cysteine thiols at N-terminal. MP was insoluble in water but the synthesized MPD after oxidation was soluble in water, probably due to the facilitated formation of micelle structure by strengthened hydrophobic interaction of MPD. It is thought that disulfide bond formation between MP could cause the proximity of hydrophobic moieties (histidine moieties and palmitic acid) and ease of micelle structure formation by structural restriction.
The synthesis of MPD was examined by quantification of remaining thiols of MPD product. After DMSO oxidation, it was found that only 0.3 mole% of thiol was detected in MPD product, which means that almost MP could be dimerized to synthesize MPD.
pDNA condensing ability of MPD was investigated by agarose gel electrophoresis. As shown in Figure
Agarose gel electrophoresis result of MPD polyplexes. C: pDNA only. Numbers mean weight ratios of the polyplexes.
CMC of MPD was measured in order to confirm the formation of micelle structure by MPD. MPD solutions with various concentrations were incubated with pyrene and absorbances from specific peaks from pyrene were summed. It was reported that absorbance values sum from 4 specific peaks of pyrene would be increased under hydrophobic environment such as core part of micelle structures [
CMC measurement result of MPD micelle.
Average sizes and Zeta-potential values of MPD micelles and MPD polyplexes were measured by Zetasizer (Figure
Average sizes and Zeta-potential values measurement results of MPD micelles (a) and MPD polyplexes (b).
The morphology of MPD micelles was also observed by AFM. They showed heterogeneous structures containing small sphere or large cylinder structures at 0.01 mM, which explains large variation of average size measurement results (Figure
AFM images and height information of MPD micelles at 0.01 mM (a) and 0.1 mM (b).
In the case of MPD polyplexes, average size was 230 nm at a weight ratio of 2 and increased to about 2-3
The cytotoxicity of MPD was examined by MTT assay in various cell lines (A549, C2C12, and HEK293). As shown in Figure
MTT assay results of MPD in A549 (a), C2C12 (b), and HEK293 (c) cells.
Transfection efficiency of MPD polyplexes was investigated by measurement of luciferase transgene expression in A549, C2C12, and HEK293 cell lines (Figure
A scheme for the proposed mechanism of structural change of MPD polyplex in the presence of MMP-2.
Transfection experiment results of MPD polyplexes in A549 (a), C2C12 (b), and HEK293 (c) cells. Numbers in boxes mean weight ratios of MPD polyplexes. PEI25k polyplexes were prepared at a weight ratio of 1. (d) Average sizes measurement result of MPD polyplexes (weight ratio: 20) in the absence or presence of MMP-2.
Transfection experiments were also performed in the presence of Bafilomycin A1 in order to examine the endosome buffering ability of MPD and the transfection mechanism. Bafilomycin A1 is an inhibitor of vacuolar type ATPase, which can suppress the proton influx into endosome and disturb the proton pump effect of endosome buffering moiety, finally leading to the decrease of transfection efficiency [
Transfection experiment results with/without Bafilomycin A1 in A549 cells. WR means the weight ratios of MPD polyplexes. Relative efficiency is the ratio of transfection efficiency with Bafilomycin A1 to the transfection efficiency without Bafilomycin A1.
Self-assembling multifunctional peptide was designed and dimerized for gene delivery systems. This MPD could self-assemble to micelle structures and condense pDNA by electrostatic interaction. The cytotoxicity of MPD was concentration-dependent but low in the examined concentration range. High transfection efficiency of MPD polyplexes in cancer cells expressing high level of MMP-2 showed the potential of MPD for targeted gene delivery. In addition, it was revealed that the transfection of MPD is mediated via endosomal escape by endosome buffering ability. In the further study, encapsulation of hydrophobic drug molecules in MPD micelles would be tried for synergistic effect of drug/gene codelivery for cancer therapy and the detailed mechanism for structural change of MPD polyplex by MMP cleavage also would be revealed.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0015045) and by the Ministry of Science, ICT, and Future Planning (NRF-2014R1A1A1037692). The authors also acknowledge Professor Yan Lee for permission to take Zetasizer measurements.