Chemo-Enzymatic Synthesis of Linear and Branched Cationic Peptides: Evaluation as Gene Carriers.

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Chemo-Enzymatic Synthesis of Linear and Branched Cationic Peptides: Evaluation as Gene Carriersa Jose Manuel Ageitos, Jo-Ann Chuah, Keiji Numata* Cationic peptides such as poly(L-lysine) and poly(L-arginine) are important tools for gene delivery since they can efficiently condense DNA. It is difficult to produce cationic peptides by recombinant bacterial expression, and its chemical synthesis requires several steps of protection/deprotection and toxic agents. Chemo-enzymatic synthesis of peptides is a clean chemistry technique that allows fast production under mild conditions. With the aim to simplify the production of cationic peptides, the present work develops an enzymatic reaction which enables the synthesis of linear cationic peptides and, through terminal functionalization with tris(2aminoethyl)amine, of branched cationic peptide conjugates, which show improved DNA complex formation. Cytotoxicity and transfection efficiency of all the chemo-enzymatically synthesized cationic peptides are evaluated for their novel use as gene delivery agents. Synthesized peptides exhibit transfection efficiencies comparable to previously reported monodisperse peptides. Chemo-enzymatic synthesis opens the door for efficient production of cationic peptides for their use as gene delivery carriers.

The study of non-viral gene delivery systems is an emerging area since these vectors enable internalization of genetic material into cells[1,2] with reduced immunogenicity compared to viral vectors.[3] Most non-viral gene vectors contain cationic sequences that form electrostatic Dr. J. M. Ageitos, Dr. J.-A. Chuah, Dr. K. Numata Enzyme Research Team, Biomass Engineering Program Cooperation Division, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako-shi Saitama 351-0198, Japan E-mail: [email protected] a Supporting Information is available online from the Wiley Online Library or from the author.

ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

complexes with the negatively charged DNA.[2] For example, cationic peptides composed of L-lysine or Larginine can condense DNA, thereby mimicking the function of histones in eukaryotic cells.[4] Poly(L-lysine) [poly(K)] is one of the most highly studied peptides with the ability to condense DNA.[5–7] Nevertheless, the use of poly(K) as gene carrier is limited by its cytotoxicity, which increases with its molecular weight.[3,8] Although poly(K) is able to bind to the negatively charged cellular membrane, its transfection efficiency is usually low due to its strong interaction with DNA, which leads to inefficient intracellular release,[9] and it tends to damage the cellular membrane.[8] The role of poly(L-arginine) [poly(R)] in DNA packaging has been widely studied and employed for gene delivery[4,10,11] because L-arginine is the main component of

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1. Introduction

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J. M. Ageitos, J.-A. Chuah, K. Numata www.mbs-journal.de

protamine, a nuclear protein that replaces histones during spermatogenesis.[12] It is generally recognized that nonaarginine is one of the most efficacious protein transduction domains composed of natural L-amino acids,[13] while different repetition units have also been studied.[9,10] Peptides containing arginine have shown lower cellular toxicity than other cationic peptides[14,15] since oligo(Larginine) [oligo(R)] promotes cellular internalization through energy-dependent endocytosis, thereby reducing membrane disruption.[13,16] On the other hand, branched and dendrimeric cationic peptides have shown increased transfection efficiency compared to linear peptides owing to their superior capacity for DNA condensation.[17] Although their high charge density allows easy insertion into membranes and can facilitate endosomal escape, dendrimers have shown low water solubility and exhibit elevated cytotoxicity.[18,19] One of the drawbacks on the production of cationic peptides is their multistep synthesis using toxic chemicals. Even it is possible to produce polypeptides by recombinant expression in microbial systems[20]; this technique is challenging to apply for expression of cationic peptides given that they are toxic to micro-organisms and it is limited to linear peptides.[21] In turn, chemical synthesis of peptides allows precise control of the amino acid sequence at the expense of multiple-step reactions involving protection/deprotection of functional groups that increase the cost of production.[22] Moreover, N-carboxylanhydride ring-opening polymerization allows the synthesis of high molecular weight peptides, but it requires the use of phosgene derivatives under anhydrous conditions with pure monomers.[23,24] Another alternative for peptide synthesis is based on chemo-enzymatic reaction using enzymes such as proteases[25,26] or lipases.[27] Enzymes enable selective reaction due to their substrate specificity. Under appropriate conditions, hydrolases can act as transferases, allowing peptide synthesis.[26,28] The kinetically controlled synthesis (KCS) reaction is initiated by the formation of an acyl-enzyme intermediate between the serine or cysteine residue in the active site and the ester group of an activated amino acid (usually ethyl or methyl ester). [26] Control of the kinetics of enzymatic equilibrium is conducted using high concentration of monomer, their amine group competes with water as nucleophile for acyl-enzyme complex promoting the aminolysis and peptide bond formation[29] (Scheme 1). KCS can be considered as clean chemistry technique[30] that allows a rapid and efficient synthesis of peptides[28,31,32] without the requirement of specialized equipment. In our previous work, we reported that proteinase K is a highly active protease that allows the terminal functionalization with tris(2-aminoethyl) amine, producing flexible star-shape conjugates.[28] Even though length (5–15 residues)[9,14] of

short cationic peptides studied in gene delivery is in a similar range than peptides produced by KCS (4–14), their synthesis is challenging since their solubility promotes hydrolysis of the product.[33] Despite escalating interest in poly(R) and oligo(R), there are no complete studies of KCS using L-arginine.[34] While several attempts for KCS of lysine containing peptides can be found in literature,[23,31,33–36] there are no studies of any KCS cationic peptides as gene delivery carriers. With the aim of developing a technique that enables production of linear and branched cationic peptides in a simple and precise way, KCS of cationic peptides mediated by proteinase K has been studied. In the present work, we have synthesized oligo(L-lysine) [oligo(K)], oligo(R), and oligo(Llysine-co-L-arginine) [oligo(KR)] in a one-pot reaction without using organic solvents or deprotection steps. In order to improve DNA complex formation, we have chemo-synthesized cationic peptide conjugates by terminal functionalization with Tris(2-aminoethyl) amine (Scheme 1). In this way, the present report is the first study of the enzymatic synthesis of branched cationic peptide conjugates. We evaluated the cytotoxicity, DNA condensation, and transfection efficiency of the linear and star-shaped cationic peptides for their use as gene delivery agents. Our results show that it is possible to synthesize peptide-based gene carriers by enzymatic synthesis with similar transfection efficiencies than classical organic synthesis ones.

2. Experimental Section 2.1. Materials L-Arginine methyl ester hydrochloride (Arg-Mt) was purchased from Wako Pure Chemical Industries (Osaka, Japan). L-Lysine ethyl ester hydrochloride (Lys-Et), tris(2-aminoethyl) amine (TREN), poly(L-arginine) hydrochloride [poly(R), molecular weight 5–15 kDa], and trifluoroacetic acid (TFA) were purchased from Sigma–Aldrich (St. Louis, MO). Poly(L-lysine) hydrochloride [poly(K), molecular weight cut-off (MWCO): 8 kDa] was purchased from Peptide Institute, Inc. (Osaka, Japan). Standard chemicals were purchased from Wako Chemical Co. (Kanagawa, Japan). Before use, commercial poly(K) and poly(R) were dialyzed against Milli-Q water using dialysis membranes (Spectra/Por1 Biotech cellulose ester (CE); MWCO: 100–500 Da; Spectrum Laboratories, Inc., Rancho Dominguez, CA), and peptide solutions were frozen at –80 8C and lyophilized. Proteinase K (21 U mg1) was purchased from Wako Pure Chemical Industries (Osaka, Japan) and used without further purification. Commercial enzymatic unit was defined as the amount of proteinase K that generates peptides equivalent to 1 mmol of tyrosine using 20 mg mL1 of hemoglobin as substrate in phosphate buffer pH 7.5 containing 6 M urea as the coloring substance reacted with Folin–Ciocalteu reagent at 37 8C for 1 min under the above conditions.[37] Activity of proteinase K was measured using Protease Colorimetric Detection Kit (Sigma– Aldrich) following manufacturer instructions. One enzymatic

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Scheme 1. Kinetically controlled synthesis of linear and star-shaped cationic peptides mediated by proteinase K.

unit (U) was defined as the quantity of enzyme that liberates 1 mmol of tyrosine equivalents per min per mL in pH 7.5 phosphate buffer at 37 8C using 0.65% (w/v) casein as substrate.[37,38] 21 U mL1 of commercial enzyme activity is equivalent to 6.6 0.3 U mL1.

Conditions for synthesis were based on previous reports[23,31] and optimized conditions for oligo(L-phenylalanine) synthesis with proteinase K.[28] Reactions were performed with stirring (600 rpm)


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in a EYELA ChemiStation (Tokyo, Japan) at 40 8C for 3 h. Reactions were carried out in triplicate in 25 mL glass reaction tubes at a final volume of 5 mL, employing various concentrations of monomer (0– 0.8 M) and 6.6 U mL1 proteinase K in 1 M phosphate buffer pH 8. Reaction mixtures were heated to 100 8C for 5 min for inactivation of the enzyme and cooled to 25 8C. Proteinase K was removed by centrifugal filters (Amicon Ultra-4, MWCO: 10 kDa, Merck Millipore, Germany). The eluate was dialyzed twice against 5 L Milli-Q water using cellulose dialysis membranes (Spectra/Por1 Biotech cellulose ester (CE); MWCO: 100–500 Da) for 24 h to remove unreacted monomers, low molecular weight products and buffer. The peptide solution was frozen at –80 8C and lyophilized. Reaction yield was calculated by gravimetric analysis[28,36] as the percent of theoretical product obtained after the purification procedure described above. Purified reaction products were characterized by 1H NMR[28,31] and confirmed by matrix-assisted laser desorption/ionization time-of-flight mass analysis (MALDI-TOF). [31]

2.3. pH and Time Course Study The evolution of pH during synthesis reactions was monitored using a pH meter LAQUA act D-71 (Horiba Scientifics, Kyoto, Japan). Reactions were stopped at different time points (0–180 min) and treated as described in general synthesis. Reactions performed at pH 10.6 were carried out in 1 M sodium carbonate buffer. Amino acids protonation states were calculated using MarvinSketch 5.10.4 (ChemAxon Ltd., Budapest, Hungary).

2.4. Synthesis of Oligo(KR) and TREN Based Peptides For synthesis of oligo(KR), an equimolar ratio (0.2 M) of Lys-Et and Arg-Mt was employed in 1 M phosphate buffer pH 8 containing 6.6 U mL1 proteinase K. The reactions were processed as described above in general synthesis. Functional termination of peptides was performed using TREN as a nucleophile during the aminolysis extension step in the synthesis reaction (Scheme 1) as described in Ageitos et al.[28] For the synthesis reaction, a 6:1 ratio of monomer (Lys-Et, Arg-Mt, or both):TREN in 1 M phosphate buffer pH 8 with 6.6 U mL1 proteinase K was employed. The reactions were processed as described in general synthesis.

2.5. Synthesis of Oligo(KR) and TREN Based Peptides For synthesis of oligo(KR), an equimolar ratio (0.2 M) of Lys-Et and Arg-Mt was employed in 1 M phosphate buffer pH 8 containing 6.6 U mL1 proteinase K. The reactions were processed as described above in general synthesis. Functional termination of peptides was performed using TREN as a nucleophile during the aminolysis extension step in the synthesis reaction (Scheme 1) as described in Ageitos et al.[28] For the synthesis reaction, a 6:1 ratio of monomer (Lys-Et, Arg-Mt, or both):TREN in 1 M phosphate buffer pH 8 with 6.6 U mL1 proteinase K was employed. The reactions were processed as described in general synthesis.

NMR System 500 (500 MHz) spectrometer (Varian Medical Systems, Palo Alto, CA) at 25 8C controlled with VnmrJ. The lyophilized samples (10 mg mL1) were suspended in deuterated dimethyl sulfoxide (DMSO-d6) containing 1% trifluoroacetic acid (TFA). One hundred twenty-eight scans were recorded. Tetramethylsilane (TMS) was used as an internal reference at 0.00 ppm. Data were processed and analyzed by ACD/NMR Processor Academic Edition, version 12.01 (Advanced Chemistry Development, Inc., Toronto, On, Canada, www.acdlabs.com, 2010). The average degree of polymerization (DPavg) of peptides was calculated using the relative integration values of amine a carbon to internal chain a carbon.[28,31] 1H NMR annotated spectra available in Supporting information.

2.7. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Aspectrometry MALDI-TOF data were acquired using Autoflex speed L MALDI-TOFMS system (Bruker, Bremen, Germany) following the protocol of Baker and Numata.[32] a-Cyano-4-hydroxycinnamic acid (Sigma) was dissolved in a mixture containing 0.1% TFA, 50% acetonitrile, and 50% water and used as the matrix. Lyophilized peptide was dissolved in Milli-Q water and mixed with the matrix at a 1:1 ratio. The prepared sample (2 mL) was spotted on the target plate and allowed to air-dry at room temperature. The acquired data were processed by Flex analysis 3.4 and Polytools 1.18 (Bruker Daltonik GmbH, Bremen, Germany) to analyze the molecular weight and degree of oligomerization of samples.[31] MALDI-TOF analysis was used to determine the maximum degree of polymerization (DPmax) and to confirm the production of peptides.

2.8. Cell Cytotoxicity Assays Human embryonic kidney (HEK) 293 cells were cultured in 96-well micro plates (8 103 cells/well) and incubated overnight at 37 8C in Dulbecco’s modified Eagle’s medium (DMEM) (Wako Chemical Co.), supplemented with 10% fetal bovine serum.[28] Stock solutions of oligo(K), TREN(K), oligo(R), TREN(R), oligo(KR), TREN(KR), poly(K), and poly(R) were dissolved in Dulbecco’s phosphate buffered saline [D-PBS()] (pH 7.4) and added to each well containing DMEM medium with a final concentration of 10–5000 mg/mL. Negative control reactions were performed with addition of D-PBS(). Positive cytotoxicity controls were performed by incubation with cell lysis buffer (Promega Corporation, Madison, WI). Following incubation for 24 h at 37 8C, cell viability was evaluated using CellTiter 961 AQueous One Solution Cell Proliferation Assay kit (Promega Corporation). Cell viability was calculated as follows: [absorbance at 490 nm of the cell culture incubated with sample]/ [absorbance at 490 nm of the negative control] 100. Each experiment was repeated three times.

2.9. Preparation of DNA Complexes 2.6. NMR Characterization 1

H NMR, gCOSY (two-dimensional J-correlation spectroscopy with gradient coherence selection) spectra were recorded on a Varian

Plasmid DNA (pDNA) encoding Green Fluorescence Protein (GFP), (phMGFP, 4707 bp, Promega, WI) or Luciferase (pGL3, 4818 bp, Promega) were amplified in competent DH5a cells and purified


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using Plasmid Giga Kits (Qiagen, Hilden, Germany). [39] The cationic peptide pDNA complexes (pDNA complexes) were prepared at different N/P ratios, the molar ratio between peptide NH2 radical groups and DNA phosphate groups. pDNA stock solution was diluted in Milli-Q water to 40 ng mL1, added to an equal volume of the appropriate concentration of cationic peptides, and mixed by 20 s of fast pipetting. Complexes were incubated at 25 8C for 30 min. In order to study the effect of calcium on the stability of complexes,[14] calcium chloride was added to complexes after 30 min of incubation to a final concentration of 50 mM and incubated at 25 8C for 30 min before use. The experimental details of peptide-pDNA complexes characterization are available in the Supporting information.

2.10. Qualitative Evaluation of Gene Transfection pDNA complexes containing 1.2 mg of phMGFP were added to each glass-bottom dish plate culturing HEK cells (approximately 7 000 cells cm2) and incubated for 6 h at 37 8C. Cells were rinsed with PBS, and media was exchanged to media without pDNA complexes. Cells were visualized after 12, 24, and 36 h. Qualitative evaluation of GFP expression in HEK293 cells was performed by fluorescence confocal laser scanning microscopy (LSM 700, Carl Zeiss Jena, Germany). Lipofectamine 2000 (Invitrogen, Carlsbad, CA) was employed as the positive control vector.

2.11. Quantitative Evaluation of Gene Transfection HEK293 cells were seeded in 24-well plates with a density of 8 103 cells/well. After 24 h of incubation at 37 8C, pDNA complexes containing 1.2 mg of pGL3 were added to each plate (n ¼ 4) and incubated and processed as described above. After 72 h of incubation, quantitative evaluation of Luciferase expression was carried out by the Luciferase assay system (Promega) following the manufacturer’s instructions. The amount of protein in each sample was determined using Microplate BCA protein assay kit (Thermo Scientific, Waltham, MA). Lipofectamine 2000 was employed as positive control vector.

3. Results and Discussion 3.1. Synthesis of Oligo(L-lysine) Given that monomer concentration is the driving force of KCS (Scheme 1), we studied the synthesis reaction at different concentrations of L-lysine ethyl ester hydrochloride (Lys-Et) (Figure 1(A)) and achieved the optimal yield at 0.2 M. The DPavg of 0.2 M reaction previous to dialysis was lower than the observed in the dialyzed product (536 21 g mol1 and 719 27 g mol1, respectively). Based on the analysis of the eluate of dialysis, the presence of peptides with an average molecular weight of 458 18 g mol was confirmed (Figure S1). The dialyzed product showed a similar molecular weight to the cationic peptides employed in previous gene delivery studies.[9,14] Since synthesis conditions were

optimized to maximize the yield of those cationic peptides, dialysis procedure was selected in order to remove low molecular-weight products, which are not capable of condensing DNA. DPavg of the purified peptides was calculated by 1H NMR. The DPavg of the purified peptides after the dialysis was slightly higher than that before the dialysis, because the lower molecular fraction of the peptides was removed by the dialysis (Figure S1). The MALDI-TOF spectrum of oligo(K) (Figure S2) exhibited fragmentation of the last lysine residue, as previously reported.[40] The maximum DP (DPmax) of oligo(K) (16.3 0.3) was obtained at 0.8 M Lys-Et, but the DPavg increased as the monomer concentration was reduced (Figure 1(A)). A time course study of KCS using 0.2 M Lys-Et showed that the yield of oligo(K) followed a cumulative curve as a function of time with a maximum yield at 60 min, after which the yield decreases (Figure 1(B)). In our previous studies of KCS, it was observed that monomer concentration has the highest influence on reaction yield.[28] Other parameters such as enzyme concentration and temperature affected the reaction kinetics, while the alkaline pH of the reaction promoted synthesis, as aminolysis requires a neutral amine.[25,33] In


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Figure 1. Synthesis of oligo(L-lysine) from L-lysine ethyl ester. A) Effect of monomer concentration on yield (bar) and degree of polymerization (~ maximum, & average) in the synthesis of oligo(L-lysine) after 3 h. B) Time course study of yield (&) and pH (*) in reaction of 0.2 M L-lysine ethyl ester. Reactions were carried out in 1 M phosphate buffer pH 8 containing proteinase K (6.6 0.3 U mL1). Error bars represent the standard deviation of replicates.



oligo(L-phenylalanine) KCS, the maximum yield was obtained at 0.6 M monomer after 2 h[28]; however, in the case of Lys-Et, the maximum yield was obtained at 0.2 M after 1 h (Figure 1(B)). This behavior may be due to different affinities between proteinase K and the monomers: the Michaelis constant (KM) of esterase activity for lysine is 1.75 times higher than that for phenylalanine, and the catalytic efficiency is 6 times smaller.[41] The yield of KCS is higher for hydrophobic peptides than for hydrophilic peptides since hydrophobic peptides tend to precipitate, which reduces hydrolysis[36,42] at expenses to reduce their DPmax. In spite of the different reaction properties, it was possible to obtain a 56% yield of oligo(K) in 1 h (Figure 1(B)). KCS of peptides usually follows an exponential cumulative curve as a function of time, which generally plateaus at maximum yield.[28,42] In order to characterize the reaction, conversion of Lys-Et to oligo(K) was studied by 1H NMR spectroscopy.[31] Monomer conversion increased as a function of time (Figure S3), proportional to the variation in pH observed during the reaction (R2 ¼ 0.9959). This indicates that pH variation is a good indicator of monomer conversion, as previously reported.[31,43] After 3 h, 79 6% of conversion was achieved, and this value was similar to that reported for trypsin (65%) and bromelain (76%). [26,36] Maximum monomer conversion did not correspond with maximum yield accumulation during the time course study (Figure 1 (B)). Since the eluate from the dialysis of reactions showed the presence of peptides under the MWCO (Figure S1); the reduction in yield observed in the second half of the KCS of oligo(K) (Figure 1(B)) is due to the removal, via dialysis, of low molecular weight products mainly generated by hydrolysis, according to previous reports.[23,33,36] Previous results[31,36] have shown that monomer conversion and yield accumulation in KCS is not always related, since conversion into products that are eliminated during purification (such as low molecular weight ones) do not increase yield. A similar overall reaction pattern was reported by Aso and Kodaka with trypsin,[33] wherein the hydrolysis of oligo(K) was promoted over time, resulting in the formation of dimers and trimers. KCS reactions were carried out in 1 M phosphate buffer pH 8; after adding monomer (L-lysine ethyl ester dihydrochloride), neutralization of HCl groups and a subsequent decrease in the initial pH was observed (time 0, Figure 1(B)). The reaction pH was stabilized for at least 10 min at 40 8C before initiating synthesis. After adding the enzyme, a decrease of pH due to enzymatic activity was observed. When the pH of the KCS reaction fell below 7.4, the overall reaction yield decreased (Figure 1(B)). As proteinase K has a broad pH range (4.0–12.5) and is active for several hours at pH 6.5–10,[44] its activity should not be drastically influenced by the pH range produced in the reactions. Thus, the reduction in yield of oligo(K) below pH 7.4 (Figure 1(B)) can be explained by

promotion of the hydrolysis pathway (Scheme 1). This pattern may be due to a decrease in the concentration of uncharged monomers at lower pH such that peptides under the MWCO are produced even in the presence of excess monomer.[33] Lys-Et has two pKa values[35]; below pH 7.4, more than 50% of monomers have a- and e-amino groups protonated, whereas above pH 7.4, more than 50% of monomers have a neutral a-amino group, while e-amino group remains protonated until pH 10.2. Since only deprotonated amino groups are suitable for aminolysis,[33] oligo(K) production is favored above pH 7.4.[24,31] A similar pH range was described by Qin et al. for bromelain-mediated synthesis of oligo(K) (pH 7–8, optimal 7.6), [23] and Aso and Kodaka[33] reported an optimal pH of 10 for trypsin-catalyzed synthesis of oligo(K) (pH 7–11). The difference in optimal pH can be attributed to bromelain instability above pH 8.[42,45] When the pH of 0.2 M Lys-Et reactions was maintained above 7.4, the yield increased to 60.6 6.8% in 2 h. The yield of oligo(K) was relatively higher than the reported yields using papain[31,36,46] (23–40%) and trypsin[33] (50%), whereas yield of 80% was reported by using papain and e-protected L-lysine as calalyst and monomer.[36] Low yield was observed when substrate concentration was 0.6 and 0.8 M (Figure 1(A)), which indicates that the buffer cannot neutralize the released proton during the reaction. The similar behavior was described in trypsin-catalyzed synthesis of oligo(K). [33] An increase in buffer concentration can enhance production of peptides, but the buffer concentrations over 1 M provided precipitation of buffer and monomer, suggesting that reduction of monomer concentration helps to control the reaction. When KCS of oligo(K) was performed at pH 10.6 using proteinase K, the formation of a- and e-links (up to 6%) between peptides was observed. In two-dimentional 1H NMR two-dimensional J-correlation spectroscopy with gradient coherence selection (gCOSY) analysis (Figure S4), the correlation of e-CH2 (3.10 ppm) with e-NH (8.18 ppm), and d-CH2 (1.44 ppm) was observed as reported by Lai et al. for e-poly(K). [47] Although it is possible to perform the KCS of e-oligo(L-lysine) at pH 10.6, the reaction yield decreases to approximately 10%, with a DPavg of 10 and DPmax of 15. The reactivity of the terminal e-amino group (Figure S4) is dependent on its deprotonation above pH 10.2. Proteinase K has shown the ability to form bonds between such reactive terminal groups.[28] Reduction of yield at pH 10.6 can be attributed to the loss of the ethyl ester group by alkaline saponification [33] and the steric effect of e-amino links in the extension of KCS. 3.2. Synthesis of Oligo(L-Arginine) In a similar manner to oligo(K), the maximum yield of oligo(R) was observed at 0.2 M monomer (Figure 2(A)). Reactions were carried out with L-arginine ethyl ester and


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Figure 2. Synthesis of oligo(L-arginine) from L-arginine methyl ester. A) Effect of monomer concentration on yield (bar) and degree of polymerization (~ maximum, & average) in the synthesis of oligo(L-arginine) after 3 h. B) Time course study of yield (&) and pH (*) in reaction of 0.2 M L-arginine methyl ester. Reactions were carried out in 1 M phosphate buffer pH 8 containing proteinase K (6.6 0.3 U mL1). Error bars represent the standard deviation of replicates.

methyl ester (Arg-Mt), which showed no differences in yield (26.0 and 24.6%, respectively) or DPavg. Arg-Mt was selected due to its thermal stability, and the different carboxyl end allowed identification of the terminal group in the co-

synthesis with Lys-Et (Figure 3(A)). The maximum peptide length was achieved at 0.6 M Arg-Mt. The DPavg and DPmax were similar between 0.6 and 0.2 M, but the highest yield and lowest dispersity were observed for 0.2 M monomers (Figure 2(A)). In the time course study (Figure 2(B)), yield accumulation followed an exponential cumulative curve as a function of time. The reaction pH decreased proportionally with the yield accumulation (R2 ¼ 0.9771) (Figure S5A). Similar to Lys-Et, the conversion of 0.2 M Arg-Mt increased proportionally with pH (R2 ¼ 0.9675) as the reaction progresses. Arg-Mt conversion was higher than the one observed in the synthesis of oligo(K), reaching 93 3% after 3 h of reaction (Figure S3). Different conversion rate can be attributed to the different affinity of proteinase K toward the monomers, since KM of arginine is six times lower than lysine.[41] However, the lower yield of oligo(R) can be attributed to the higher turnover rate of lysine (1.3 s1 mM1) as compared with arginine (0.3 s1 mM1), and the affinity of proteinase K for peptides with terminal lysine (689 s1 mM1)[41] that facilitates elongation of products. In the case of KCS with 0.2 M Arg-Mt, the pH did not decrease below the pKa of the monomer (pH 7.0) during the time course studied (180 min), indicating that the decrease in pH did not affect the yield. However, the pH decreased below 7 for 0.4 M Arg-Mt (Figure S5B), and the yield decreased correspondingly because the monomers must be deprotonated for reaction to occur. A direct correlation between yield and pH variation was observed [oligo(R): 0.2 M, R2 ¼ 0.9771 and 0.4 M, R2 ¼ 0.9394; oligo(K): 0.2 M, R2 ¼ 0.9894]. It is therefore possible to observe the strong influence of pH on the KCS. Although the importance of pH in these reactions is well-known, most studies carried out using KCS tend to explain the differences in terms of the optimal pH for the protease[23,28,38,42] without considering the protonation state of the monomer or the relative abundance of the reactive amine form. Performing KCS

Figure 3. Characterization of oligo(L-lysine-co-L-arginine) [oligo(KR)]. A) 1H NMR study of oligo(KR) depicting proton assignments. B) MALDITOF-MS spectrum of oligo(KR).


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Full Paper

Chemo-Enzymatic Synthesis of Linear and Branched Cationic Peptides: Evaluation as Gene Carriersa Jose Manuel Ageitos, Jo-Ann Chuah, Keiji Numata* Cationic peptides such as poly(L-lysine) and poly(L-arginine) are important tools for gene delivery since they can efficiently condense DNA. It is difficult to produce cationic peptides by recombinant bacterial expression, and its chemical synthesis requires several steps of protection/deprotection and toxic agents. Chemo-enzymatic synthesis of peptides is a clean chemistry technique that allows fast production under mild conditions. With the aim to simplify the production of cationic peptides, the present work develops an enzymatic reaction which enables the synthesis of linear cationic peptides and, through terminal functionalization with tris(2aminoethyl)amine, of branched cationic peptide conjugates, which show improved DNA complex formation. Cytotoxicity and transfection efficiency of all the chemo-enzymatically synthesized cationic peptides are evaluated for their novel use as gene delivery agents. Synthesized peptides exhibit transfection efficiencies comparable to previously reported monodisperse peptides. Chemo-enzymatic synthesis opens the door for efficient production of cationic peptides for their use as gene delivery carriers.

The study of non-viral gene delivery systems is an emerging area since these vectors enable internalization of genetic material into cells[1,2] with reduced immunogenicity compared to viral vectors.[3] Most non-viral gene vectors contain cationic sequences that form electrostatic Dr. J. M. Ageitos, Dr. J.-A. Chuah, Dr. K. Numata Enzyme Research Team, Biomass Engineering Program Cooperation Division, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako-shi Saitama 351-0198, Japan E-mail: [email protected] a Supporting Information is available online from the Wiley Online Library or from the author.

ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

complexes with the negatively charged DNA.[2] For example, cationic peptides composed of L-lysine or Larginine can condense DNA, thereby mimicking the function of histones in eukaryotic cells.[4] Poly(L-lysine) [poly(K)] is one of the most highly studied peptides with the ability to condense DNA.[5–7] Nevertheless, the use of poly(K) as gene carrier is limited by its cytotoxicity, which increases with its molecular weight.[3,8] Although poly(K) is able to bind to the negatively charged cellular membrane, its transfection efficiency is usually low due to its strong interaction with DNA, which leads to inefficient intracellular release,[9] and it tends to damage the cellular membrane.[8] The role of poly(L-arginine) [poly(R)] in DNA packaging has been widely studied and employed for gene delivery[4,10,11] because L-arginine is the main component of

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Figure 4. Two-dimensional J-correlation spectroscopy with gradient coherence selection (gCOSY) 1H NMR study of tris(2-aminoethyl) amideoligo(L-lysine-co-L-arginine) [TREN(KR)] peptides depicting proton assignments.

Table 1. Molecular weight and degree of polymerization of cationic peptides employed in cytotoxicity and gene transfection experiments.

Mwa) [Da]

Oligo(K) TREN(K) Oligo(R) TREN(R) Oligo(KR) TREN(KR)

704.0 165.1 399.2 93.6 1023.9 89.8 455.6 40.0 1045.9 85.3 711.6 58.0

DPavga) [n]

Highest Mass Peakb) [Da]

5.5 2.0 6.6 2.3 7.4 4.4

1024.0 122.2 1103.2 64.0 1405.8 127.0 1002.3 78.1 1278.9 53.3 1342.4 63.5

Calculated by 1H-NMR. b) Average of highest mass peak detected by MALDI-TOF.

peptides. For instance, commercial poly(R) with molecular weight 5–15 kDa migrated at 2.4 kDa, suggesting that the apparent molecular weight in SDS–PAGE was shifted due to the cationic charges. Even apparent mass obtained by this technique was not precise, SDS–PAGE allowed to observe the distribution of peptides. Linear peptides have a narrow


distribution, while TREN-derived peptides showed two bands with apparent molecular masses that differed from that of the linear peptides. These results are coherent with the NMR and MALDI-TOF analyses that showed presence of linear and branched peptides and the low molecular weight of TREN-derived peptides (Table 1).


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molecular weight (Table 1) and/or branched structures. Although the cationic peptides synthesized in this study showed reduced cytotoxicity in vitro as compared with poly(K) and poly(R) (Figure 5), in vivo cytotoxicity and immune responses of these low molecular-weight cationic peptides need to be further evaluated in future work.

Figure 5. Human embryonic kidney (HEK) 293 cell viability after 24 h in the presence of several concentrations of cationic peptides (10–5000 mg/mL). Oligo(K): oligo(L-lysine); TREN(K): tris(2-aminoethyl) amide-oligo(L-lysine); Poly(K): poly(L-lysine); Oligo(R): oligo(Larginine); TREN(R): tris(2-aminoethyl) amide-oligo(L-arginine); Poly(R): poly(L-arginine); Oligo(KR): oligo(L-lysine-co-L-arginine); TREN(KR): tris(2-aminoethyl) amide-oligo(Llysine-co-L-arginine). Values are normalized to the viability of the negative control. Error bars represent the standard deviation of replicates.

3.6. Cytotoxicity of Cationic Peptides Cytotoxicity of the synthesized peptides was evaluated by incubating HEK 293 cells for 24 h with several concentrations of the cationic peptides. Commercial poly(K) and poly(R) were employed as control samples (Figure 5). Cytotoxicity of cationic peptides is assumed to be caused by attachment of the cationic peptides to negatively charged cell membranes,[17] and loss of membrane integrity increases with increased molecular weight of cationic peptides.[49] Herein, cytotoxicity increased with concentration for all the cationic peptides. The chemoenzymatically synthesized peptides are composed of a polydisperse molecular weight; being the peptides employed in the present study different to previous reports of monodisperse cationic peptides. Poly(K) and poly(R) showed higher toxicity than the cationic peptides synthesized by KCS, and TREN(K) and TREN(KR) presented toxicity similar to the linear peptides. The reduction in cytotoxicity of peptides synthesized in the present study can be attributed to their low degree of polymerization (Table 1) compared to commercial samples.[39,49–51] Oligo(K) and TREN(K) showed less than 20% cytotoxicity even at 2500 mg mL1. Oligo(R) demonstrated similar cytotoxicity to both oligo(K) (Figure 5) and chemically synthesized oligo(R). [14] TREN(R) showed an increase in cytotoxicity compared with oligo(R) for concentrations over 100 mg mL1, which is in agreement with a recent study that showed higher toxicity for dendritic or hyperbranched cationic peptides than for linear peptides due to their resistance to proteases.[18] TREN(KR) and TREN(K) showed less cytotoxicity than linear oligo(KR) and oligo(K), potentially due to their lower

3.7. pDNA Condensation and Gene Transfection

Cationic peptide complexes with pDNA were prepared at different N/P ratios (the molar ratio between peptide NH2 groups and DNA phosphate groups). The electronic stability of pDNA complexes was evaluated by a gel retardation assay, which showed that the presence of calcium ions enhanced the stability of complexation (Figure S7A) to a similar extent as cationic peptides.[14,52] Previous studies showed that calcium ions condense DNA by neutralizing the phosphate groups, thereby producing smaller and more stable complexes.[14,52] The cationic peptides synthesized in this study exhibited formation of ionic complexes with pDNA at a presence of 50 mM calcium ion (Figure S7 and S8). Higher N/ P ratios were required for observable retardation with peptides synthesized by KCS, which have polydisperse molecular weights, compared with those of non-disperse peptides.[9,14] Even though it was reported that cationic oligomers can form DNA complexes,[4] the efficiency of complex formation strongly depended on the lengths and types of cationic peptides employed.[9] Additionally, the TREN-branched peptides, especially TREN(KR) and TREN(R), formed more stable complexes than linear peptides (Figure S6). In order to study the function of the peptide in pDNA condensation, the hydrodynamic diameters of the pDNA-peptide complexes were measured by dynamic light scattering (DLS). Calcium ions showed an ability to induce efficient pDNA condensation, whereas increasing the N/P ratio caused a corresponding linear increase in complex sizes (Table S1) and a more positive z potential (Figure S7). The surface charge of the complexes at low N/P ratio was more positive than the reported cellular membrane charge of HEK cells (–20 mV), [53] allowing membrane association and reducing interaction with serum proteins.[54] At N/P ratios from 0.1 to 10, the linear cationic peptides formed slightly smaller complexes with more positive charge than the branched peptides (Figure S8). The mechanism of cationic peptide-induced DNA condensation is not completely elucidated. Several mathematical models suggest


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Figure 6. Confocal laser scanning micrographs of HEK293 cells after 36 h transfection with phMGFP peptide complexes at an N/P ratio of 1. Each image is an overlay of GFP and DIC images. A) Oligo(L-lysine). B) Oligo(L-arginine). C) Oligo(L-lysine-co-L-arginine). D) Tris(2-aminoethyl) amide-oligo(L-lysine). E) Tris(2-aminoethyl) amide-oligo(L-arginine). F) Tris(2-aminoethyl) amide-oligo(L-lysine-co-L-arginine). White bars represent 50 mm.


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that cationic peptides bind in the major groove of DNA, allowing hydrogen-bonding interactions between the cationic charges and the DNA phosphate backbone.[10,55] The specific binding interactions of lysine with DNA differ from those of arginine peptides.[9,10] During DNA condensation, oligo(K) was reported to yield weaker interhelical attraction forces and stronger short-range repulsion than oligo(R). [10] Therefore, DNA complexation with oligo(K) requires longer peptides to stabilize specific major groove binding over other binding locations.[9,10] In fact, pDNA complexes with oligo(R) showed more positive charge than those with oligo(K) at low N/P ratios (Figure S8), indicating that oligo(R) more efficiently forms complexes with pDNA. In vitro transfection experiments with two reporter genes were performed with HEK293 cells to evaluate the suitability of the cationic peptides synthesized in this study as vectors for gene delivery. HEK293 cells were incubated with complexes of pDNA encoding green fluorescent protein (GFP) reporter gene. All complexes at N/P ¼ 1 demonstrated successful transfection of the pDNA based on the expression of GFP (Figure 6). For quantitative analysis of transfection efficiency, HEK293 cells were transfected with pDNA encoding the luciferase gene (Figure 7). Luciferase assays allow the study of the expression in reference to the amount of protein, being possible to determine the transfection efficiency in a quantitative and comparable manner in all the cells exposed to the complexes.[39] Transfection efficiency of the KCS peptides was lower than that reported using fusion peptide of silk and oligo(K)[39,56] or higher molecular-weight cationic polymers such as chitosan and dendritic poly(lysine) [57] but similar to that

reported using oligo(K) and oligo(R). [9,14] Oligo(K) and oligo(R) showed higher transfection efficiencies at lower N/ P ratios (0.1–10), whereas the optimum ratio for oligo(KR), TREN(K), and TREN(R) was N/P ¼ 50. Oligo(R) and oligo(K), which were prepared by KCS, showed transfection efficiencies comparable to their chemically synthesized counterparts.[9,14] Generally, longer cationic peptides show better condensation of DNA than shorter sequences,[6,50] the results obtained are coherent with the average molecular weight of the synthesized cationic peptides (Table 1). As observed in Figure 1 and 2, the average molecular weight of peptides can be tuned up by using different monomer concentrations or through further purification steps, at the expense of reducing the yield of reaction. As recently described by Qin et al.,[36] by using eprotected L-lysine as monomer, it is possible to reduce polydispersity even though this improvement required the use of organic solvents and additional deprotection steps. Higher molecular-weight cationic peptides will not only increase complexation efficiency but also cytotoxicity of peptides.[39,49–51,56,57] Functionalization with TREN did not increase the transfection efficiency at low N/P ratios, while pDNA complexation, surface charge, and transfection efficiency were increased at high N/P ratios (Figure S7 and S8, Figure 7). Differences in transfection efficiency, size, and surface charge between linear and branched peptides can be due to the DPavg;[9,14] specifically, the branched peptides with TREN showed lower molecular weights (Table 1). The low transfection efficiency for TREN(K) indicates stronger length dependence for lysine peptides in pDNA condensation and release;[9] thus, the negatively charged surface of TREN(K) complexes and the lack of

Figure 7. Luciferase transfection assay after 72 h incubation of HEK293 cells with pDNA ion complexes at N/P ratios of 0.1 to 100. Oligo(K): oligo(L-lysine); Oligo(R): oligo(L-arginine); TREN(K): tris(2-aminoethyl) amide-oligo(L-lysine); TREN(R): tris(2-aminoethyl) amide-oligo(Larginine); Oligo(KR): oligo(L-lysine-co-L-arginine); TREN(KR): tris(2-aminoethyl) amide-oligo(L-lysine-co-L-arginine); Poly(K): poly(lysine); Poly(R): poly(arginine); C LIPO: Lipofectamine 2000. DNA: Transfection with pDNA encoding luciferase with 50 mM CaCl2 (N/P ¼ 0). Error bars represent the standard deviation of replicates (n ¼ 4).


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correlation between N/P ratio and size (Table S1) suggest deficient complex formation due to the low molecular weight of the polymer (Table 1). pDNA complexes prepared with 50 mM calcium (N/P ¼ 0) showed smaller hydrodynamic diameters (Table S1) and similar surface charges compared to pDNA complexes with cationic peptides at low N/P ratios (Figure S7). Furthermore, pDNA complexed with calcium ions demonstrated a lower transfection efficiency than complexes with cationic peptides (Figure 7), suggesting that cationic peptides function as gene carriers and are required for efficient gene transfection.

4. Conclusion This is the first report on the synthesis of oligo(L-arginine), and branched cationic peptides synthesized by chemoenzymatic polymerization as well as the suitability of those cationic peptides for DNA condensation and gene delivery. Chemo-enzymatic synthesis of peptides mediated by proteases is considered green chemistry as it allows peptide production in aqueous solution without complicated synthesis steps. The lengths of the peptides produced in this synthetic approach vary from dimers to octadecamers depending on the protease and synthesis conditions employed. In the present work, we have studied the conditions for synthesis of cationic oligopeptides for gene delivery.[14] In our previous work, we studied the suitability of proteinase K for KCS of aromatic peptides, but its use for production of cationic peptides has never been studied. The KCS of cationic peptides is shown to be strongly dependent on monomer concentration and pH. Based on our results, the hydrolysis pathway is considered to predominate at pH’s below the pKa of monomer amines, even at high concentrations of monomer (Scheme 1). This is the first report of cationic peptides produced by chemo-enzymatic synthesis to be assayed for gene delivery and is also one of the first detailed reports of oligo(L-arginine) synthesis. Additionally, through terminal functionalization, we synthesized branched or star-shaped cationic peptides that improved DNA complexation. Branched cationic peptides produce more stable DNA complexes than corresponding linear peptides; however, this modification did not directly improve gene delivery efficiency due to the limited molecular weight achieved by the branched peptides. The chemo-enzymatically synthesized cationic peptides showed similar transfection efficiencies to chemically synthesized cationic peptides with similar degree of polymerization, indicating that cationic peptides do not need to be monodisperse to produce complexes and effect efficient gene transfection. Chemo-enzymatic synthesis of cationic peptides mediated by proteinase K simplifies the production of functional peptides with a green chemistry.

Received: November 6, 2014; Revised: January 30, 2015; Published online:DOI: 10.1002/mabi.201400487 Keywords: chemo-enzymatic synthesis; gene delivery; oligo(Larginine); oligo(L-lysine); proteinase K

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Acknowledgements: This study was financially supported by the RIKEN Biomass Engineering Program.



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Cationic nanogels as Trojan carriers for disruption of endosomes.

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