Location of a single histidine within peptide carriers increases mRNA delivery

Abstract Background Previously, we determined that four‐branched histidine‐lysine (HK) peptides were effective carriers of plasmids and small interfering RNA. In the present study, we compared several branched HK carriers and, in particular, two closely‐related H3K4b and H3K(+H)4b peptides for their ability as carriers of mRNA. The H3K(+H)4b peptide differed from its parent analogue, H3K4b, by only a single histidine in each branch. Methods A series of four‐branched HK peptides with varied sequences was synthesized on a solid‐phase peptide synthesizer. The ability of these peptides to carry mRNA expressing luciferase to MDA‐MB‐231 cells was investigated. With gel retardation and heparin displacement assays, the stability of HK polyplexes was examined. We determined the intracellular uptake of HK polyplexes by flow cytometry and fluorescence microscopy. The size and polydispersity index of the polyplexes in several media were measured by dynamic light scattering. Results MDA‐MB‐231 cells transfected by H3K(+H)4b‐mRNA polyplexes expressed 10‐fold greater levels of luciferase than H3K4b polyplexes. With gel retardation and heparin displacement assays, the H3K(+H)4b polyplexes showed greater stability than H3K4b. Intracellular uptake and co‐localization of H3K(+H)4b polyplexes within acidic endosomes were also significantly increased compared to H3K4b. Similar to H3K(+H)4b, several HK analogues with an additional histidine in the second domain of their branches were effective carriers of mRNA. When combined with DOTAP liposomes, H3K(+H)4b was synergistic in delivery of mRNA. Conclusions H3K(+H)4b was a more effective carrier of mRNA than H3K4b. Mechanistic studies suggest that H3K(+H)4b polyplexes were more stable than H3K4b polyplexes. Lipopolyplexes formed with H3K(+H)4b markedly increased mRNA transfection.


| INTRODUCTION
Delivery of mRNA has been considered as an excellent alternative to plasmids because it can be translated to a protein in the cytosol without entering the nucleus to become functional. Therefore, mRNA can successfully express proteins in non-dividing cells. 1 Although degradability of mRNA may in some ways be advantageous to reduce toxicity, 2,3 the susceptibility of mRNA to enzymatic degradation with reduced translation accounts for significant problems. Consequently, the development of carriers that can protect mRNAs from degradation, facilitate cellular uptake and enhance buffering capacity to improve endosomal escape has a high priority. Among them, non-viral carriers, including polymers and lipid-based agents such as lipopolymers and liposomes, have been used for mRNA delivery. [4][5][6][7][8] Of these, liposomes are the most studied and are effective carriers of mRNA. 1,[9][10][11] For example, Zohra et al. 1,11 found that DOTAP liposomes coated with carbonate apatite showed high luciferase mRNA transfection efficiency in both mitotic and non-mitotic cells.
There have been only a limited number of studies demonstrating the utility of polymers as mRNA carriers. [6][7][8][12][13][14][15][16][17][18][19][20][21][22][23] Qiu et al. 21 synthesized an RNA delivery vector, PEG12KL4, in which the synthetic cationic KL4 peptide was attached to a linear 12-mer of PEG. With intratracheal administration, these carriers mediated significantly more effective mRNA transfection in the lungs of mice than naked mRNA. Moreover, based on the studies of Kataoka and colleagues, 12 Chan et al. 22 compared several repeating units of aminoethylene groups (2, 3, or 4) conjugated as side chains to a PEGylated polyaspartamide backbone. The carrier with the side branch of four-repeating units, tetraethylenepentamine, had the best luciferase mRNA delivery efficiency in vitro and effectively delivered luciferase mRNA injected intracerebroventricularly with no significant immune response. Interestingly, by altering the alkyl length between amines, Jarzebinska et al. 6 found an oligoalkylamine that significantly enhanced mRNA delivery.
This oligoalkylamine had a high buffering capacity between pH 6.2 and 6.5, a pH range that has been associated with endosomal lysis and escape of nucleic acids. Several investigators have also utilized either peptide-liposomes or lipopolymers to stabilize the vector to deliver mRNA in vivo. With few exceptions, 13,15 lipid-polymer hybrids or liposome-polymer combinations are required or at least greatly enhance the systemic delivery of mRNA. [6][7][8]19,20,24 Our laboratory has focused on developing histidine-rich peptides to deliver nucleic acids, including mRNA. Although several groups have used histidine-or imidazole-containing polymers to enhance the delivery of relatively small molecular weight small interfering RNA (siRNA) and large molecular weight plasmid DNA, [25][26][27][28][29][30][31][32] few of these modified polymers have been used to deliver mRNA. These studies have primarily focused on lipopolyplexes delivering mRNA for vaccines targeting tumors. [33][34][35][36] For example, Mockey et al. 35 added histidylated liposomes to a PEGylated histidylated polylysine-mRNA polyplex to form a lipopolyplex for melanoma mRNA delivery, which significantly enhanced the immune response to the translation product. Recently, this group decorated lipopolyplexes with a "tri-antenna α-D-mannopyranoside" ligand, which increased their specificity toward dendritic cells and improved the antitumor immune response. 37,38 In the present study, we compared histidine-rich peptide carriers and, in particular two close analogues, H3K4b and H3K(+H)4b peptides, for their ability as carriers of mRNA in vitro. H3K4b has four repeating motifs of -KHHH-in each branch, whereas the H3K(+H)4b has a similar repeating pattern but has an additional histidine in the second -HHHK motif of its branches (Table 1). We determined that the H3K(+H)4b peptide was a significantly better carrier of mRNA than H3K4b.

| Peptides
HK peptides were synthesized on a Ranin Voyager synthesizer (Tucson, AZ, USA) by the biopolymer core facility at the University of Maryland or by Genscript (Piscataway, NJ, USA) as described previously. 39,40 To ensure a purity of 90% or greater, 39 Table 1.
Nomenclature of four-branched HK polymers: (i) for H3K4b, the dominant repeating sequence in its terminal branch is -HHHK-, thus "H3K" is part of the name; the "4b" refers to the number of terminal branches; (ii) there are four -HHHK-motifs in each branch of H3K4b and analogues. The first motif is closest to the lysine core; (iii) H3K (+H)4b is a four-branched analogue of H3K4b in which one extra histidine is inserted in the second -HHHK-motif of the terminal branch of H3K4b; (iv) H3K(1+H)4b and H3K(3+H)4b have an extra histidine in the first and third motifs, respectively; (v) for H3K (1,3+H) 4b, there is an extra histidine in both the first and third motifs of the branches; (vi) for H-H3K(+H)4b and HH-H3K(+H)4b, these peptides are analogous to H3K(+H)4b, except they have one or two additional histidines at the N-terminal ends of the branches; (vii) for H4K4b, the predominant pattern in the branches is -HHHHK-; (viii) for H2K4b, the predominant pattern in the branches is -HHK-. Transfection with HK lipopolyplexes was carried out in a manner similar to that described above, with a few exceptions. In brief, the HK peptide (4 μg) in Opti-MEM was mixed initially with mRNA (1 μg) for 30 minutes. This was followed by adding the DOTAP cationic liposome (1,2-dioleoyl-3-trimethyl ammonium-propane; 1 μg; Roche, Basel, Switzerland) for an additional 30 minutes. The Opti-MEM mixture (50 μl) was then added dropwise to the cells.

| Heparin displacement assays
H and K represent L-histidines and L-lysines, respectively. The lower case "k" in the sequence of H3k(+H)4b represents D-lysines. Extra histidines are underlined within the terminal branch sequences with H3K4b used as a reference. The numbers above the H3K4b peptide and analogues represent the four repeating motifs of the branch. Branched peptides emanate from a 3-lysine core.
control sample was prepared with the same amount of mRNA, water and Sybr Gold dye. For the heparin displacement, instead of water, heparin salt (Sigma-Aldrich) solutions at different concentrations (0.5, 1, 1.5, 2 and 3 μg/μl) were used, and the polyplexes were incubated at 37 C for 30 minutes before the addition of Sybr Gold.
Displacement of mRNA from polyplexes with heparin was also performed with gel electrophoresis. After polyplexes were formed, different concentrations of heparin (0.5, 1.0, 1.5, 2.0 and 3.0 μg/μl; volume 20 μl) were incubated with these at 37 C for 30 min. The polyplexes were then loaded on the agarose gel (1% gel; 20 μl loading volume; 10X BlueJuice Gel loading buffer) and electrophoresis was carried out followed by staining with SG as described above. Images were acquired via an ultraviolet imager (ChemiDoc Touch; Bio-Rad).

| In vitro uptake of HK polyplexes by fluorescence microscopy
With the mRNA labeled with Cy5, HK polyplexes at a ratio of 4:1 (HK: mRNA) were prepared as described for in vitro transfection. The  2.10 | Particle size, polydispersity index (PDI) and zeta potential The size, PDI and zeta potential were determined with the Zetasizer to the disposable zeta cell.

| Statistical analysis
The results, reported as the mean ± SD, represent three separate data measurements unless otherwise indicated. Except where stated, results were analyzed using a two-tailed t-test. p < 0.05 was considered statistically significant (SigmaPlot, San Jose, CA, USA).

| H3K(+H)4b is a significantly better carrier than H3K4b
Both H3K4b and H3K(+H)4b have shown promise as carriers of nucleic acids in vitro. 40,43 Despite these previous findings, H3K(+H)4b was markedly better as a carrier of mRNA compared to its close  Table S1). Moreover, the buffering capacity does not appear to be an essential factor in their transfection differences because the percentage of histidines (by weight) in H3K4b and H3K(+H)4b is 68.9% and 70.6%, respectively. Furthermore, the pH titration curves of H3K4b and H3K(+H)4b corroborated minimal differences in their buffering profile (Figure 2).

| Gel retardation and heparin displacement assays indicate differences in their stability
The retardation assay showed the effect of different weight ratios of mRNA and HK polypeptides ( Figure 3). The retardation effect increased with higher HK peptide to mRNA weight ratios. Free mRNA and partially retarded mRNA was markedly less at the ratios of 1:2 and 1: 1 (w:w; peptide:mRNA) of H3K(+H)4b compared to the same ratios of H3K4b. With ratios of 2:1 and 4:1, the mRNA was completely entrapped by the H3K(+H)4b polyplex, whereas with the ratio of 4:1, the mRNA was completely retarded by the H3K4b polyplex. These results suggest that the H3K(+H)4b forms a more stable polyplex, and this may play a role with respect to why H3K(+H)4b is more effective as a carrier compared to H3K4b.
Further confirmation that the H3K(+H)4b peptide binds more tightly to the mRNA was demonstrated with the heparin-binding assay ( Figure 4A, 4B). Particularly at the lower concentrations of heparin, mRNA was released by the H3K4b more rapidly than by the H3K(+H)4b peptide. These data, together with the size of polyplexes in different media ( Table 2), suggest that H3K(+H)4b polyplexes may be more stable than the H3K4b polyplexes. Nevertheless, if a peptide such as H2K4b forms a polyplex that is too stable, this may also reduce mRNA transfection ( Figure 4B). Release of the HK polymers, including H2K4b, from the polyplex is likely critical for the interaction of the HK polymer with endosomal membrane with subsequent lysis of the endosomes. 36 Interestingly, other HK polyplexes that showed effective mRNA transfection had stabilities similar to the H3K(+H)4b polyplexes when exposed to heparin (see Supporting information, Figure S1).

| Increased intracellular localization of H3K (+H)4b polyplexes compared to H3K4b
With the mRNA labeled with cyanine-5, we compared the uptake of polyplexes were imported into the cells more than H3K4b polyplexes (see Supporting information, Figure S2). Similar to these results, fluorescence microscopy indicated that H3K(+H)4b polyplexes localized within the acidic endosomal vesicles significantly more than H3K4b polyplexes [H3K4b versus H3K(+H)4b; p < 0.001] ( Figure 5). Interestingly, irregularly-shaped H3K4b polyplexes, which did not overlap endocytic vesicles, were likely extracellular aggregates and were not observed with H3K(+H)4b polyplexes ( Figure 5A). As evidenced by the heparin displacement assay (Figure 4; see also Supporting information, Figure S1), the reduced stability of H3K4b-mRNA polyplexes provides a rationale for their irregular-shapes compared to the H3K(+H)4b polyplexes.
F I G U R E 1 Comparison of H3K(+H)4b and H3K4b peptides as carriers of mRNA. To form polyplexes, the mRNA (1 μg) was mixed with three different ratios of the HK (4, 8 and 12 μg) polymer for 30 minutes. The mRNA polyplexes were then added to the cells as described in the Materials and methods, and luciferase activity was measured 24 hours later. ***p < 0.001; ****p < 0.0001, H3K(+H)4b versus H3K4b F I G U R E 2 Titration of different HK peptide solutions. Solutions of polymers (5 mg/ml) [H2K4b, H3K4b, H3K(+H)4b and H4K4b] were adjusted to pH 3 (initial volume 1 ml) and then 5-μl aliquots of 0.05 N NaOH were stepwise added and the pH was measured

| Transfection of mRNA with HK carriers with extra histidine in the second motif is essential for mRNA transfection
All the HK peptides with an extra histidine in the second -HHHK motif of the branches were effective carriers of mRNA ( Figure 6; see also Supporting information, Table S2). Of these peptides, H3k(+H)4b was determined to be the optimal carrier of mRNA [H3k(+H)4b versus H3K(+H)4b; p < 0.05]. With this peptide, the L-lysines were replaced with D-lysines, and the enhanced stability of this polyplex may be the reason why this peptide was a better carrier than H3K(+H)4b. This was based on prior antimicrobial studies in which replacement of L-lysines with D-lysines suggested that H3k4b [a close analog of H3k (+H)4b] was more stable to enzymatic degradation, had greater antimicrobial activity, and had no observed cytotoxicity to human cells. 44 Exposure to trypsin provided further support that the H3k (+H)4b-mRNA polyplexes had enhanced stability to enzymatic degradations compared to the H3K(+H)4b polyplexes (see Supporting information, Figure S3). Interestingly, additional histidines in locations other than the second motif do not appear to be a critical factor in enhancing mRNA transfection ( Figure 6; see also Supporting information, Table S2) Figure 7 and Tables 1; see also Supporting information, Table S2).
H3K(1+H)4b and H3K(3+H )4b have an extra histidine in first and third domains, respectively. Thus, the location of the histidines in the branches appears to be important.
Although the data for Figures 6 and 7 were obtained with the ratio of 4:1 (w/w, HK:mRNA), analogous results were generally found at 8:1 and 12: 1 ratios during the initial screening (see Suppoting information, Table S2)  Table S2). Compared to other branched HK polymers, H2K4b had the highest percentage of lysines.
Previously, we determined that HK peptides and cationic liposomes (i.e. DOTAP) significantly increased transfection with plasmids 45 and, consequently, we investigated whether these liposomes together with HK peptides enhanced mRNA transfection.
Notably, the H3K(+H)4b and H3k(+H)4b carriers were significantly better carriers of mRNA than the DOTAP liposomes (p < 0.001) ( Figure 8; see also Supporting information, Figure S4). We determined that the combination of H3K(+H)4b and DOTAP liposomes was synergistic in the ability to carry mRNA into MDA-MB-231 cells ( Figure 8; see also Suppoting information, Figure S4). The combination was approximately 3-fold and 8-fold more effective as carriers of mRNA than the polymer alone and the liposome carrier, respectively [H3K(+H)4b/liposomes versus liposomes or H3K(+H)4b; p < 0.01].
Notably, not all HK peptides demonstrated improved activity with DOTAP liposomes. The combination of H3K4b and DOTAP carriers was less effective than the DOTAP liposomes as carriers of luciferase mRNA (see Supporting information, Figure S4).
As stated previously, the D-isomer, H3k(+H)4b, was the most effective polymeric carrier ( Figure 6). The D-isomer/liposome carrier of mRNA was almost 4-fold and 10-fold more effective than the H3k (+H)4b alone and liposome carrier, respectively ( Figure 8). Although the D-H3k(+H)4b/liposome combination was modestly more effective than the L-H3K(+H)4b/liposome combination, this comparison was not statistically different. In prior studies, the four-branched HK peptide, H2K4b, was a good carrier of large molecular weight plasmids, 28 although it was a poor carrier of the relatively low molecular weight siRNA. 40 Previous data from our laboratory showed that the two histidine-rich peptides, H3K4b and H3K(+H)4b, were effective carriers of siRNA, 40,52 although H3K(+H)4b appeared to be modestly more effective. 53 Moreover, the H3K4b carrier of siRNA induced cytokines to a significantly greater degree in vitro and in vivo than the H3K(+H)4b-siRNA polyplexes. 53 In the present study, we determined that the H3K(+H) polymers based on prior studies. 45 Investigators have determined that incorporating helper lipids such as DOPE with DOTAP into liposomes may significantly enhance the cytotoxic T-cell response. 54 Furthermore, substituting an imidazole/histamine liposome for DOTAP liposomes, together with an histidylated polymer, markedly augmented the silencing activity of siRNA. 33 Although this later study utilized siRNA, these differences between lipid preparations may extend to mRNA. Alternatively, we are also investigating whether HK lipopolymers will be effective carriers of mRNA. Conjugating fatty acyl chains or cholesterol to polymeric carriers of mRNA have markedly enhanced their ability to transfect cells and tissues in vivo. 6,24,55 In summary, we have investigated a series of four-branched HK peptides for mRNA delivery. Among them, we found that H3K(+H)4b was a significantly more effective carrier of mRNA than H3K4b. With [or H3k(+H)4b] than with either carrier alone. Based on these initial promising studies, more investigations are warranted for these mRNA transfection agents.