Precise Enzymatic Cleavage Sites for Improved Bioactivity of siRNA

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Precise Enzymatic Cleavage Sites for Improved Bioactivity of siRNA Lipo-Polyplexes Sören Reinhard, Yanfang Wang, Sebastian Dengler, and Ernst Wagner Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00585 • Publication Date (Web): 21 Sep 2018 Downloaded from http://pubs.acs.org on September 22, 2018

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Bioconjugate Chemistry

Precise Enzymatic Cleavage Sites for Improved Bioactivity of siRNA Lipo-Polyplexes Sören Reinhard,*,a Yanfang Wang,a Sebastian Dengler,a and Ernst Wagnera,b a

Department of Pharmacy, Pharmaceutical Biotechnology, Center of Nanoscience (CeNS), Ludwig-Maximilians-Universität, Butenandtstr. 5-13, 81377 München, Germany, b Nanosystems Initiative Munich (NIM), Schellingstr. 4, 80799 München, Germany *E-mail: [email protected] siRNA delivery • polyplexes • solid phase synthesis • biodegradable oligomers • lysosomal degradation ABSTRACT Sequence-defined cationic lipo-oligomers are potent siRNA carriers, forming stable lipo-polyplexes based on both electrostatic and hydrophobic interactions, and, after endocytosis and endosomal protonation, facilitating the delivery of siRNA into the cytosol. After completion of the nucleic acid delivery process, carriers should be readily biodegradable to ensure minimum accumulation of amphiphilic molecules that are harmful to lysosomes and other intracellular organelles. Endolysosomal enzymes may degrade a surplus of carrier molecules left over in lysosomes and thereby facilitate the generation and rapid excretion of cleavage products. By solid-phase supported synthesis, a library of sequence-defined lipo-oligomers was generated containing artificial and natural amino acids comprising precise enzymatic cleavage sites. Incorporating either short cleavable L-arginine sequences (RR), non-cleavable D-arginine linkers (rr) or varieties of both tailored the degradability of lipo-oligomers, as demonstrated upon incubation with the endolysosomal protease cathepsin B. Cleavage products were identified by MALDI-TOF mass spectrometry. The effect of improved intracellular degradation on cell tolerability was studied by transfecting Huh7-eGFPLuc and DU145-eGFPLuc cells. Positioning of enzymatic cleavage sites between a lipophilic diacyl domain and an ionizable oligocationic siRNA binding unit enabled efficient enzymatic degradation of the carrier and reduced the lytic potential under lysosomal conditions. Highly degradable carriers containing at least one L-arginine dipeptide linker significantly improved the viability of transfected cells without hampering gene silencing activity. Therefore, the precise integration of enzymatic cleavage sites in lipo-oligomers is a promising strategy towards biocompatible nucleic acid carriers. INTRODUCTION The transfer of therapeutic genes and oligonucleotides offers great opportunities for the treatment of severe diseases including genetic disorders and cancer.1-2 However, nucleic acids display limited biological stability and cannot diffuse across cellular membranes due to their size and negative charge. The delivery problem can be addressed by formulation of nucleic acids with suitable carriers. Nanoformulations like polyplexes3-13 or lipid nanoparticles (LNPs)14-24 improve the intracellular delivery of nucleic acids. Although polycations fulfill several extracellular and intracellular delivery requirements, the therapeutic window between transfer efficiency and cytotoxicity is usually narrow. Non-degradable polymers like the potent and commonly used transfection polymer polyethylenimine (PEI) show significant toxicity in a time- and concentration-dependent manner with a two-stage mechanism: Phase 1 short term toxicity results from compromised plasma membrane integrity, while phase 2 long-term (>24h) toxicity is caused by intracellular mechanisms after internalization of the polyplexes.25-26 PEI-induced damage of lysosomal and mitochondrial membranes are potential causes for late-phase cell death. The disintegration of mitochondrial membranes leads to the release of pro-apoptotic cytochrome c and energy crisis due to ATP-leakage, while perturbation of lysosomal membranes contributes to cellular stress through possible ACS Paragon Plus Environment

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release of lysosomal cathepsins.25-32 The degradation of cationic polymers in low molecular weight subunits reduces both acute toxicity and negative long-term effects, which might occur after repeated administration.13, 33-35 Biodegradable cationic polymers can be designed by introduction of ester bonds36-37, disulfides33, 38-39, ketals40, imines41, polyglutamic acid amides, and other degradable amide bonds.42-43 Solid-phase assisted synthesis enables the generation of sequence-defined cationic oligomers with various topologies and functional elements to meet specific require-ments for the delivery of nucleic acids.44-46 Such carriers can form multifunctional siRNA nanoplexes, which showed tumor cell killing in vivo after systemic application.47-49 Biodegradable sequence-defined oligomers were generated by introducing a disulfide-containing building block between a lipophilic diacyl domain and an ionizable oligocationic siRNA-binding unit. Degradable siRNA polyplexes showed higher gene silencing efficiency and lower cytotoxicity than their stable analogs.50 Pre-mature disulfide cleavage of carriers by cell surface oxidoreductases may however present a significant obstacle depending on tissue and cell type.51 Moreover, glutathione-triggered cleavage is initiated after cytosolic release and therefore effects only a small fraction of cytosolic carriers, as endosomal escape is a major bottleneck for delivery. Release of siRNAs from endosomes into the cytosol occurs at low rates (1–2 %) for lipid nanoparticles.52-53 This implies that large amounts of cationic carriers accumulate in lysosomes, which represent the terminal organelles on the endocytic pathway28, 54 unless dumped by emergency exocytosis.23 Specific degradation of nucleic acid carriers by endolysosomal enzymes therefore appears as an attractive strategy to destroy abundant carrier molecules while ensuring high extracellular stability. Lysosomes mediate the degradation of extracellular particles from endocytosis and of intracellular components from autophagy with more than 60 different types of hydrolytic enzymes.55 Endolysosomal cysteine proteases like cathepsin B are involved in protein degradation and turnover in cells.56-57 Cathepsin B, together with cathepsin L and D, is one of the most abundant lysosomal proteases with concentrations as high as 1 mM and ubiquitous expression.56 Cathepsin B has both endo- and exopeptidase activity, which was utilized for enzymetriggered, intracellular drug delivery58-60 as well as for nucleic acid delivery with degradable peptide-HPMA copolymers. Polyplexes formed with biodegradable copolymers showed similar transfection efficiency but less cytotoxicity compared to non-degradable structures.61 In this study, we evaluated the cathepsin B-triggered cleavage of oligoaminoamides based on synthetic solid-phase compatible building blocks succinoyl-tetraethylene-pentamine (Stp), which contain the pH-responsive diaminoethylene motif of the transfection polymer PEI, and different peptide linkers placed between two Stp units. A library of myristic acid- and Stpcontaining sequence-defined lipo-oligomers with tailored biodegradability was synthesized by introducing either short cleavable L-arginine dipeptides, non-cleavable D-arginine dipeptide linkers or varieties of both. Endolysosomal degradation was simulated by incubation with cathepsin B at pH 5.5 and the fragments were identified by MALDI-TOF mass spectrometry. The influence of tailored intracellular cleavability on cell tolerability and transfection efficiency was studied in Huh7-eGFPLuc and DU145-eGFPLuc cells.

RESULTS AND DISCUSSION Degradability of Test Oligomers by Cathepsin B. For solid-phase assisted synthesis of sequence-defined oligomers, natural and artificial amino acids, synthetic building blocks and fatty acids can be used. While linear peptide sequences of natural L-amino acids usually are readily cleavable by proteases, the degradability might be hampered when oligoamide structures contain artificial building blocks or are synthesized in branched configurations. Repeating units of the synthetic building block Stp provide oligoaminoamides with both nucleic acid binding and endosomal buffering capacity and therefore can be substantial parts of nucleic acid carriers. A library of test structures was synthesized to study the cathepsin Btriggered degradation of Stp-containing oligomers (Figure 1).

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Figure 1. Enzymatic degradation of test structures. A) Single L-amino acid or dipeptide linkers are placed at position X between two amide-bound units of the synthetic building block Stp. Left: Cathepsin-triggered degradation of a test structure containing a L-Arg-L-Arg linker (Acr-Stp-RR-Stpw). B) HPLC-Chromatogram of Acr-Stp-RR-Stp-w before (left) and after (right) incubation with cathepsin B

The test oligomers consist of two Stp units connected by one or two of the natural L-amino acids lysine, arginine, histidine or tyrosine, which have been used in our published nucleic acid carriers.62 Lysine and arginine bind and complex nucleic acids, histidine provides endosomal buffer capacity and tyrosines are involved in aromatic polyplex stabilization. The test structures were equipped with acridine (Acr) at the N-terminus and D-tryptophane (w) at the C-terminus to facilitate the detection of fragments by HPLC-DAD. The degradability of the test oligomers was evaluated by incubation with cathepsin B at endolysosomal pH 5.5. After 2 hours of incubation at 37 °C, the percentage of cleaved material was determined by HPLC (Table 1). Table 1. Cathepsin-triggered degradation of test structures. Test structurea N→C

Degradation b fragments

Peptide c degradation [%]

Acr-Stp-Stp-w

-

0

Acr-Stp-H-Stp-w

-

0

Acr-Stp-HH-Stp-w

-

0

-

0

Acr-Stp-Y-Stp-w Acr-Stp-YY-Stp-w Acr-Stp-K-Stp-w Acr-Stp-KK-Stp-w Acr-Stp-R-Stp-w Acr-Stp-RR-Stp-w

Acr-Stp-Y + Y-Stp-w Acr-Stp-K + Stp-w Acr-Stp-K + K-Stp-w Acr-Stp-R + Stp-w Acr-Stp-R + R-Stp-w

8.6 16 100 47 100

a

Test structures contain no linker (Acr-Stp-Stp-w) or L-His (H or HH), L-Tyr (Y or YY), L-Lys (K or KK), or L-Arg (R or RR) single amino acid or dipeptide linkers. bIdentified by MALDI-TOF mass spectrometry after HPLC. cDetermined by HPLC.

The test structure containing two amide-bound Stp units without any natural L-amino acid linkers was not degradable by cathepsin B. This enzyme resistance is most relevant information for carriers based on Stp block oligomers, and consistent with findings that length of Stp oligomers correlates with cytotoxicity.63 No cleavage was also found for test oligomers containing one or two histidines or one tyrosine. A low fraction of degraded material was ACS Paragon Plus Environment


detected when two tyrosines (8.6 %) or one lysine (16 %) were placed between the Stp units. Arginine was the preferred substrate of the enzyme with cleavage rates of 47 % with one and 100 % with two L-arginines. T-shaped Lipo-oligomers with Designed Enzymatic Degradability. Based on the results of the cathepsin-triggered degradation of test oligomers, a library of Stp- and argininecontaining t-shaped lipo-oligomers with precisely introduced cleavage sites was synthesized (Figure 2A, Table S1). Arginine was preferred over lysine as a single L-Arg linker between two amide-bound Stp units in the test oligomers showed significantly higher degradability compared to the L-Lys linker. However, the linkage via the L-Lys dipeptide showed complete cleavage in the test structure and could potentially also be integrated in lipo-oligomers as a motif for efficient enzymatic degradation.

Figure 2. A) Scheme of lipo-oligomers X1-My-X2 with cleavable single amino acid or dipeptide Larginine sequences (R or RR) or non-cleavable D-arginine linkers (r or rr) at sites X1 and X2. My is used as abbreviation for t-shape lipo-oligomers containing a di-myristic acid domain. An overview of all structures and the linkers can be found in Table S1. Y: L-tyrosine, K: L-lysine B) Enzymatic degradation only occurs when L-amino acids are incorporated. C) MALDI mass spectra of rr-My-rr (top) containing only D-Arg linkers and RR-My-RR (bottom) containing only L-Arg linkers after digestion.

Biodegradability was tailored by placing either cleavable L-arginine or non-cleavable Darginine dipeptides, or varieties of both linkers between a lipophilic di-myristic acid (MyrA) domain and a Stp-containing cationic siRNA-binding unit. The later domain contains also two tyrosine tripeptides, which are flanking the Stp-units at the C- and N-terminus and are incorporated to enhance polyplex stabilization.64 The lipo-oligomers were incubated with cathepsin B to simulate endolysosomal degradation. Fragments of cathepsin B-triggered cleavage were identified by MALDI-TOF mass spectrometry. Fragments resulting from cleavage between two L-arginines both at sites X1 and X2 were found (Figure 2B and 2C, and see Supporting Information; Analytical Data; Summarizing tables: Mass data for lipooligomers after incubation with cathepsin B). When no arginines (My), or D-arginine dipeptides were incorporated instead of L-Arg-L-Arg, no cleavage was detectable at sites X1 and X2. In all cases, cleavage of up to five of six tyrosines was detectable. Lytic Activity of Bioresponsive Lipo-Oligomers. Amphiphilic lipo-oligomers containing cationizable, bioresponsive Stp units together with hydrophobic moieties usually display a pH-responsive membranolytic activity. This was confirmed in an erythrocyte leakage assay at three pH values covering the range from early to late endosomal/lysosomal conditions. The lytic activities of all lipo-oligomers were highest at the lowest pH 5.5 (Figure 3A). Although the lytic activities at pH 7.4 were low compared to the endosomal/lysosomal conditions, an increasing erythrocyte lysis was observed for structures with more arginines (14 % for My without arginines and ~ 30 % for structures with six arginines). This indicates that the incorporation of arginines can potentially impact short term toxicity resulting from plasma membrane disruption.


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Figure 3. Erythrocyte leakage of lipo-oligomers A) at different pH conditions at 5 mM concentration and B) at endolysosomal pH 5.5 after incubation with cathepsin B-containing buffer (digested) or buffer only (native) at 2.5 mM concentration.

Bioresponsive behavior with low lytic potential at neutral pH and increasing lysis at lower pH values is highly desirable and necessary for efficient endosomal escape. However, as nucleic acid carriers accumulate in lysosomes, highly lytic structures might trigger toxicity by intracellular mechanisms. The incorporation of degradable L-arginine dipeptides as linkers between the oligocationic and lipophilic moieties should result in reduced amphiphilic character of the lipo-oligomers after cleavage, thereby reducing cytotoxicity caused by lysosome and organelle damage. Reduced lytic activity of biodegraded structures was verified in an erythrocyte leakage assay at pH 5.5 before and after incubation with cathepsin B. For this assay, the concentration of the lipo-oligomers was reduced to 2.5 mM to avoid a saturation of the assay, as the lytic activities of all lipo-oligomers at 5 mM concentration were close to 100 % at pH 5.5. All lipo-oligomers showed > 50 % lysis in native form (Figure 3B). Structures containing at least one L-Arg dipeptide sequence either at site X1 or X2 displayed significantly reduced lytic activity after degradation, while the non-degradable structures My and rr-My-rr largely retained their lytic potential after incubation with cathepsin B. Similar, but less pronounced effects were found for lipo-oligomers containing only single L-arginines as cleavage sites, in agreement with a reduced degradability of such structures (Figure S1, Table S1). Lipo-oligomers containing cleavable L-arginine linkers with reduced lytic activities at endolysosomal pH conditions after internalization and degradation could potentially cause less intracellular membrane damage and thereby display higher cell tolerability. Improved Cell Tolerability Without Hampering Gene Silencing Efficiency. The influence of improved endolysosomal degradability on cell tolerability and transfection efficiency was studied in human hepatoma Huh7-eGFPLuc and human prostate carcinoma DU145eGFPLuc cells. Lipo-polyplexes were formed by mixing lipo-oligomers with siRNA at a constant N/P value, which depicts the ratio of protonatable amines (N) of the oligomers to phosphates (P) of the siRNA. The N/P value does not present charge ratios, as only a fraction of the protonatable amines of Stp are protonated at physiological pH. Biophysical characterization showed no significant differences in size, zeta potential or siRNA binding (Figure S2, Table S2). In previous work, lipo-oligomers containing the saturated C14 short chain myristic acid were found to display efficient gene silencing, but also high lytic activity and cytotoxicity.45-46, 50 This renders MyrA-containing oligomers as good model structures to study the effect of lysosomal detoxification. However, phase 1 short term toxicity resulting from plasma membrane damage might still occur and veil any effects of improved intracellular degradability on phase 2 long-term toxicity. For this reason, incubation times on cells were kept short (4 h) and the read-out of cytotoxicity and gene silencing assays were measured after further 44 h long-term incubation in fresh media. Metabolic cell activities compared to buffer-treated cells, as an indicator for cell viability, were measured via MTT assays. Buffer-treated cells were set to 100 %. For MTT assays, only lipo-oligomers without siRNA were added to the cells. Biodegradable structures containing L-arginine dipeptides either at site X1 or X2 showed excellent cell tolerability on Huh7 cells compared to the highly toxic non-degradable My and rr-My-rr oligomers (Figure 4A). ACS Paragon Plus Environment


Figure 4. A) Metabolic activity indicating cell viability of human hepatoma Huh7-eGFPLuc (top) and human prostate carcinoma DU145-eGFPLuc cells (bottom) compared to buffer-treated cells determined by MTT assay after incubation with lipo-oligomers with tailored biodegradability. Lipooligomer solutions were used in the same concentrations as in gene silencing experiments, but without the addition of siRNA. B) Gene silencing of lipo-polyplexes (N/P 20) formed by mixing of lipooligomers and eGFP-targeted siRNA (siGFP) or control siRNA (siCtrl) compared to buffer-treated cells. C) MALDI mass spectra of cell extracts after transfection with My (left) or highly degradable RRMy-RR (right) after transfection showing both undigested lipo-oligomers (red) and fragments of endolysosomal degradation (green).

Improved cell tolerability of degradable structures was also found in DU145 cells; the nondegradable oligomers did not display as strong toxicity as in HUH7 cells. These results are in good agreement with the reduced lytic potential of enzyme-degraded carriers (Figure 3B), indicating that lysosomal cleavage enhanced cell tolerability. Gene silencing experiments were performed in cells stably expressing an eGFP-Luciferase fusion protein. Gene silencing by siGFP (light bars), resulting in decreased luciferase activity, is quantified by a standard luciferase assay. In the absence of unspecific effects, cells treated with analogous control siCtrl lipo-polyplexes (black bars) should maintain luciferase activity close to 100 % (buffertreated cells); reduced levels of luciferase activity in siCtrl treated cells are an indicator of cytotoxicity. Cells were transfected at N/P 20, as this ratio was found to enable efficient gene silencing. Improved knockdown of the eGFP-Luciferase gene in siGFP-treated cells would not be expected for biodegradable oligomers, as reduced lytic activity after enzymatic cleavage might even hamper the escape of the nucleic acid cargo into the cytosol. Notably, all siGFP lipo-polyplexes show similar levels of eGFP-Luciferase expression in both Huh7and DU145 cells, indicating that transfection efficiency is not reduced by the introduction of enzymatic cleavage sites (Figure 4B). Luciferase activities in Huh7 cells treated with My and rr-My-rr siCtrl-polyplexes are significantly reduced compared to buffer-treated cells. The unspecific reduction of gene expression for non-degradable structures is in agreement with the reduced cell viability detected in the MTT assay. Interestingly, siCtrl-polyplexes formed with structures containing at least one L-arginine dipeptide linker showed luciferase activities well above 100 %, which has previously been observed for non-toxic transfections of Huh7eGFPLuc transgenic cells (unpublished data). In this cell line, the eGFP-luciferase fusion gene is stably expressed under control of a CMV promoter. The transcription of transgenes from the CMV promoter can be up-regulated by a variety of mild stresses by activation of ACS Paragon Plus Environment

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MAP protein kinases.65 The transfection of siRNA with cationic lipo-oligomers could potentially result in stress-activation of transgene expression. However, the improved biocompatibility of degradable lipo-oligomers is supported by the results from the MTT assay, which showed metabolic cell activities similar to buffer-treated cells. The positioning of the cleavable linker did not significantly influence MTT and gene silencing assays in Huh7 cells. For DU145 cells, only lipo-oligomers containing cleavable L-arginines at site X1 (RR-My, RR-My-RR and RR-My-rr) showed significantly reduced toxicity and higher luciferase expression of siCtrl treated compared to siGFP treated cells. This indicates that not only the introduction of cleavage sites, but also their precise positioning might affect cell tolerability. Lipo-oligomers with only single L-arginines as cleavage sites at position X1 also showed improved cell viabilities in MTT and gene silencing assays in both cell lines compared to non-degradable structures or structures with degradable linkers only at site X2 (Figure S3). Integration of cleavage sites at position X1 within the more hydrophilic part of the lipo-oligomers seems preferable for these structures, possibly due to better accessibility for endolysosomal proteases and therefore more efficient degradation; cleavage site X2 is positioned at the side chain of a branching central lysine followed by a hydrophobic diacyl domain. In Huh7 cells, dipeptide linkers showed a superior effect on cell tolerability compared to single L-arginine linkers. Incorporation of cleavage sites at position X2 was only beneficial for dipeptides. At site X1, metabolic activities were ranging from 49 – 66 % for single L-Arg linkers while structures with dipeptide motifs showed similar cell viability as buffer-treated control cells. This is in accordance with a more efficient cathepsin-triggered degradation of test oligomers with dipeptide linkers. Cell viability assays in Huh7 cells were also performed with lipo-polyplexes formed by mixing of lipo-oligomers with siGFP and siCtrl, as polyplexes could potentially be less toxic than carriers in isolation. In this cell line, siRNA lipo-polyplexes showed similar results compared to MTT assays with only lipo-oligomers (Figure S4).

Oligomer Cleavage Detected in Cell Transfections. Fragments of degradation were identified from lysates of cells transfected with lipo-polyplexes in agreement with cathepsin B incubation assays, indicating that endolysosomal degradation takes place after cellular internalization (Figure 4C, and see Supporting Information; Analytical Data; Summarizing Tables: Mass data for lipo-oligomers after incubation with cathepsin B). In contrast to degradation assays with cathepsin B, significant amounts of non-degraded material were found in cell lysates, which can be attributed to non-internalized material at the cell surface, intact lipo-oligomers that were released into the cytosol or non-degraded structures in the lysosomes. Cathepsin B-triggered degradation of lipo-oligomers was studied after addition of siRNA to examine the influence of electrostatic interaction with nucleic acids on the degradability of the carrier. At N/P 1, no degradation of lipo-oligomers was detectable, indicating that the binding of nucleic acid phosphates to L-arginine side chains prevents enzymatic cleavage. When lipo-oligomers were added in excess (N/P 20), efficient cleavage similar to the degradation of lipo-oligomers without siRNA was found (see Supporting Information; Analytical Data; Summarizing Tables: Mass data for lipo-oligomers after incubation with cathepsin B).

CONCLUSION Stp-based oligoaminoamides, as used as nucleic acid transfection carriers, were found to be resistant towards enzymatic degradation by the lysosomal enzyme cathepsin B. This may present a problem for the cell metabolism, as the majority of transfection material is known to initially accumulate in the lysosomal compartment, and need to be dumped from cells23 before serious organelle damage. Biodegradability of lipo-oligomeric nucleic acid carriers can be tailored by precise integration of enzymatic cleavage sites such as a simple L-Arg ACS Paragon Plus Environment


dipeptide. Our data indicate that the cleavage sites should preferably be integrated in the hydrophilic parts of carriers. This finding opens up possibilities for further optimization by integrating and evaluating different cleavage motifs in lipo-oligomers. Similar degradation fragments were found after incubation of lipo-oligomers with the endolysosomal protease cathepsin B as in cell lysates after transfection. Introducing short cleavable L-Arg dipeptide linkers significantly improved cell tolerability after transfection without hampering gene silencing efficiency. Reduced lytic activities of degraded lipo-oligomers at endolysosomal pH conditions after internalization can be hypothesized as underlying mechanism for the decreased toxicity.

EXPERIMENTAL SECTION Materials. Protected Fmoc-α-amino acids, 2-chlorotrityl chloride resin, N,Ndimethylformamide (DMF), N,N-diisopropylethylamine (DIPEA) and trifluoroacetic acid (TFA) were purchased from Iris Biotech (Marktredewitz, Germany). Triisopropylsilane (TIS), 1hydroxybenzotriazole (HOBt), myristic acid, 1,4-dithiothreitol (DTT), super-DHB (sDHB), 9acridinecarboxylic acid hydrate and Triton X-100 were purchased from Sigma-Aldrich (Munich, Germany). Dichloromethane (DCM) was purchased from Bernd Kraft (Duisburg, Germany). 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium-hexafluorophosphate (HBTU) and microreactors were obtained from Multi-SynTech (Witten, Germany). Cell culture media, antibi-otics and fetal calf serum (FCS) were purchased from Invitrogen (Karlsruhe, Germany), HEPES from Biomol GmbH (Hamburg, Germany), MF-Millipore™ membrane filters (pore size: 0.025 µm), cathepsin B (human liver) and glucose from Merck (Darmstadt, Germany), agarose NEEO Ultra-Qualität from Carl Roth GmbH (Karlsruhe, Germany) and GelRedTM from VWR (Darmstadt, Germany). Citrate-buffered human blood was provided by the hospital of the Ludwig-Maximilians-University (Munich, Germany). Cell culture 5 × lysis buffer and D-luciferin sodium salt were obtained from Promega (Mannheim, Germany). Ready-to-use siRNA duplexes were obtained from Axolabs GmbH (Kulmbach, Germany): eGFP-targeted siRNA (siGFP) (sense: 5’-AuAucAuGGccGAcAAGcAdTsdT-3’; antisense: 5’UGCUUGUCGGCcAUGAuAUdTsdT-3’; small letters: 2’methoxy; s: phosphorothioate) for silencing of eGFPLuc; control siRNA (siCtrl) (sense: 5’-AuGuAuuGGccuGuAuuAGdTsdT-3’; antisense: 5’-CuAAuAcAGGCcAAuAcAUdTsdT-3’). All solvents and other reagents were purchased from Sigma-Aldrich (Munich, Germany), Iris Biotech (Marktredwitz, Germany), Merck (Darmstadt, Germany) or AppliChem (Darmstadt, Germany). Oligomer synthesis. Oligomers were synthesized using a 2-chlorotrityl resin preloaded with the first C-terminal amino acid as solid support (see Supporting Information). Artificial Fmocoligoamino acid Fmoc-Stp(Boc3)-OH was synthesized as described before.44 Oligomers were synthesized under standard Fmoc solid phase peptide synthesis conditions using a Syro WaveTM synthesizer (Biotage, Uppsala, Sweden). Coupling steps were carried out using 4 eq. Fmoc-amino acid, 4 eq. HOBt, 4 eq. HBTU and 8 eq. DIPEA in NMP (10 mL g−1 resin) for 2 × 90 min (double couplings). Fmoc deprotection was performed by 4 × 10 min incubation with 20 % piperidine in DMF (10 mL g−1 resin). After each coupling and deprotection step, a washing procedure with 5 × 1 min DMF incubation (10 mL g−1 resin) was performed. Symmetrical branching points were introduced using Fmoc-Lys(Fmoc)-OH, asymmetric branching in T-shape structures was introduced using Fmoc-Lys(Dde)-OH. Ddedeprotection was performed 30 × 2 min with hydrazine/DMF 1:50. Then the resin was washed with 5 × 1 min DMF, 3 × 1 min DIPEA/DMF 1:10 and 5 × 1 min DMF (10 mL g−1 resin). All oligomers were cleaved off the resin by incubation with TFA/H2O/TIS 95:2.5:2.5 (10 mL g−1 resin, 10 µmol each) for 90 min at 22 °C. The cleavage solution and the resins were cooled to 4 °C before addition. The oligomers were immediately precipitated in 40 mL of MTBE/n-hexane 1:1 at -20 °C. All oligomers were purified by HPLC and white powders were obtained. Oligomer identities were validated by mass spectrometry and 1H-NMR.


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HPLC. For lipo-oliogmer purification, samples were dissolved in H2O containing 0.1 % TFA. Column: YMC-Pack C4, 250 x 10 mm, 5 µm, 12 nm. Conditions: A: 0.1 % TFA in H2O; B: 0.1 % TFA in ACN; 5 % B for 5 min, 5-90 % B in 15 min; 2.00 mL min-1, 30 °C, λ = 280 nm. To analyze the cathepsin B-triggered cleavage of the test structures, samples were dissolved in H2O containing 0.1 % TFA either before or after digestion. Column: YMC-UltraHT Hydrosphere C18, 150 x 4.6 mm, 5 µm, 12 nm. Conditions: A: 0.1 % TFA in H2O; B: 0.1 % TFA in ACN; 5 % B for 5 min, 5-90 % B in 10 min, 90 % B for 5 min; 1.00 mL min-1, 30 °C, λ = 280 and 370 nm. Chromatograms were recorded using a Chromaster HPLC-DAD system by VWR Hitachi and analyzed using Chromaster System Manager (Ver. 1.1 by Hitachi). Oligomer digestion with cathepsin B. A cathepsin B solution (0.379 mg mL-1, 331 U mg-1) was added to an acetate-incubation buffer (sodium acetate 0,1 M, EDTA 1 mM and DTT 1 mM, pH 5.5) and activated for 5 min at 22 °C under constant shaking. The activated enzyme was added to the oligomer or polyplex sample to reach a final concentration of 0.25 µM cathepsin B. Test structures were incubated for 2 h at 37 °C at a final concentration of 0.5 mM. Lipo-oligomers were incubated for 24 h at 37 °C at a final concentration of 0.125 mM. Polyplexes formed with lipo-oligomers and siRNA at indicated N/P ratios were incubated for 24 h at 37 °C at a final concentration of 500 ng siRNA in 50 µL reaction mixture. For MALDI mass spectrometry, 1 µL of the reaction mixture was pipetted on top of a MF-Millipore membrane filter placed in 2 L deionized water and microdialyzed for 60 min. MALDI mass spectrometry. A drop of 1 µL matrix solution, consisting of 10 mg mL-1 sDHB (sum of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid) in acetonitrile/water (3:7) with 0.1 % (v/v) TFA, was spotted on a MTP AnchorChip (Bruker Daltonics, Bremen, Germany). After the sDHB matrix crystallized, 1 µL of the sample solution was added on the matrix spot. Samples were analyzed in positive ion mode using an Autoflex II mass spectrometer (Bruker Daltonics, Bremen, Germany). Erythrocyte leakage assay. Fresh, citrate-buffered human blood was washed with phosphate-buffered saline (PBS) until the supernatant was clear. The washed human erythrocyte suspension was centrifuged and the pellet was diluted to 5 × 107 erythrocytes per mL with PBS (pH 7.4, 6.5 and 5.5). To determine the pH-dependent lytic activity, a volume of 75 µL of erythrocyte suspension at the indicated pH value and 75 µL of oligomer solution (previously diluted with PBS of the respective pH) were added to each well of a V-bottom 96well plate (NUNC, Denmark), resulting in a final concentration of 5 µM oligomer per well. To determine the lyctic acitivty at the endolysosomal pH 5.5 before and after digestion, 75 µL of oligomer solution (either incubated with incubation buffer only or incubation buffer with cathespin B, diluted with PBS at pH 5.5) and a volume of 75 µL of erythrocyte suspension at pH 5.5, were added to each well of a V-bottom 96-well plate. The final concentration of lipooligomer was 2.5 µM per well. The plates were incubated at 37 °C under constant shaking for 1 h. After centrifugation, 100 µL of the supernatant was analyzed for hemoglobin release at 405 nm wavelength using a microplate reader (Spectrafluor Plus, Tecan, Männedorf, Switzerland). PBS-treated erythrocytes were set to 0 %. Triton X-100 served as positive control and was set to 100 %. Data are presented as mean value (± SD) out of quadruplicates. siRNA lipo-polyplex formation. siRNA and lipo-oligomer at indicated nitrogen/phosphate (N/P) ratios were diluted in 20 mM HEPES buffered 5 % glucose pH 7.4 (HBG) in separate tubes of equal volumes. 500 ng siRNA were dissolved in 10 µL HBG. Only protonatable nitrogens were considered in the N/P calculations. The siRNA solution was added into the lipo-oligomer solution, mixed by 5 × rapid pipetting and incubated for 40 min at RT. Cell culture. Human hepatoma Huh7-eGFPLuc cells66 and human prostate carcinoma DU145-eGFPLuc cells46, both stably transfected with an eGFPLuc gene, were used for cytotoxicity (MTT) assays and gene silencing assays. Huh7-eGFPLuc cells, stably expressing an eGFP-luciferase fusion gene under control of a CMV promoter, were cultivated in DMEM and Ham's F12 medium (50:50). DU145-eGFPLuc cells, stably ACS Paragon Plus Environment


expressing an eGFP-luciferase fusion gene under control of a PGK promoter, were grown in RPMI-1640 medium. Both culture media were supplemented with 10 % FCS, 100 U mL-1 penicillin and 100 µg mL-1 streptomycin. The cells were maintained in ventilated flasks in the incubators at 37 °C with 5 % CO2 in a humidified atmosphere. Cell lines were grown to 80 – 90 % confluency and harvested. Cytotoxicity assay (MTT assay). Cytotoxicity assays were performed using human hepatoma Huh7-eGFPLuc or human prostate carcinoma DU145-eGFPLuc cells. 5 × 103 cells per well were seeded onto 96-well plates, and medium was replaced with 80 µL fresh growth medium after 24 h. Oligomer solutions (20 µL, prepared as described in the polyplex formation section for N/P 20, but without adding siRNA) were added to each well and incubated for 4 h at 37 C. The oligomer-containing medium was replaced with 100 µL fresh growth medium and the cells were incubated for additional 44 h at 37 °C. MTT assay (Life Technologies, Darmstadt, Germany) was performed and measured using a SpectraFluor Plus microplate reader to evaluate the cytotoxicity. The experiments were performed in triplicates and the cell viability was calculated as percentage compared to untreated control cells. Reporter gene silencing assay. Gene silencing assays were performed using human hepatoma Huh7-eGFPLuc or human prostate carcinoma DU145-eGFPLuc cells. The siRNA against eGFP (siGFP) for silencing the eGFPLuc gene or its control sequence (siCtrl) was used. 5 × 103 cells per well were seeded onto 96-well plates, and medium was replaced with 80 µL fresh growth medium after 24 h. Polyplex solutions (20 µL, prepared as described above at N/P 20) were added to each well and incubated for 4 h at 37 °C. The polyplexcontaining medium was replaced with 100 µL fresh growth medium and the cells were incubated for additional 44 h at 37 °C. After transfection, medium was removed and cells were treated with 100 µL cell lysis buffer. Luciferase activity in the cell lysate was measured using a luciferase assay kit (Promega, Mannheim, Germany) and a SpectraFluor Plus microplate reader (Tecan, Männedorf, Switzerland). The experiments were performed in triplicates, and the relative light units (RLU) were presented as percentage of the luciferase gene expression obtained with HBG-treated control cells. Identification of degradation products from cell lysates. Human hepatoma Huh7eGFPLuc cells were transfected with polyplexes at N/P 20 as described in the reporter gene silencing section. A protocol for the identification of mammalian cell lines using MALDI-TOF mass spectrometry67 was adapted as follows: After transfection, medium was removed and the cells were resuspended in 100 µL of a 10 mg mL-1 solution of sDHB in acetonitrile/water (3:7) with 0.1 % (v/v) TFA. The cell suspension was stored at -80 °C for 1 h. Then the cell lysate was sonicated for 5 min at 22 °C. One µL of the sample solution was added on a dried sDHB matrix spot and MALDI mass spectrometry was performed. ASSOCIATED CONTENT Supporting Information. Experimental section: Loading of 2-chlorotrityl chloride resin with Fmoc protected amino acids, Particle size and zeta potential, Agarose gel shift siRNA binding assay, Proton 1H NMR spectroscopy Supplement figures and tables: Erythrocyte leakage of lipo-oligomers containing only single L- or D-arginines, Agarose gel shift siRNA binding assay with lipo-polyplexes, MTT assays and gene silencing of lipo-oligomers containing only single L- or D-arginines, Particle size (zaverage), polydispersity index (PDI) and zeta potential of siRNA lipo-polyplexes. Analytical data: Mass data of test structures and lipo-oligomers, 1H Proton NMR spectra of lipo-oligomers AUTHOR INFORMATION ACS Paragon Plus Environment

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Corresponding Author *Sören Reinhard, Pharmaceutical Biotechnology, Department of Pharmacy, LudwigMaximilians-Universität, Butenandtstr. 5-13, 81377 München, Germany. E-mail: [email protected] ORCID Sören Reinhard: 0000-0003-1348-5919 Ernst Wagner: 0000-0001-8413-0934 Author Contributions Experiments were performed by SR, YW and SD. The manuscript was written by SR and EW. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by German Research Foundation (DFG) Excellence Cluster Nanosystems Initiative Munich (NIM), DFG SFB1032 B4 (EW) and SFB1066 B5 (EW).

ABBREVIATIONS MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; GFP, green fluorescent protein; ATP, adenosine triphosphate; DAD, diode array detector; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

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TABLE OF CONTENTS ARTWORK


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Figure 1. Enzymatic degradation of test structures. A) Single L-amino acid or dipeptide linkers are placed at position X between two amide-bound units of the synthetic building block Stp. Left: Cathepsin-triggered degradation of a test structure containing a L-Arg-L-Arg linker (Acr-Stp-RR-Stp-w). B) HPLC-Chromatogram of Acr-Stp-RR-Stp-w before (left) and after (right) incubation with cathepsin B 55x36mm (300 x 300 DPI)



Figure 2. A) Scheme of lipo-oligomers X1-My-X2 with cleavable single amino acid or dipeptide L-arginine sequences (R or RR) or non-cleavable D-arginine linkers (r or rr) at sites X1 and X2. My is used as abbreviation for t-shape lipo-oligomers containing a di-myristic acid domain. An overview of all structures and the linkers can be found in Table S1. Y: L-tyrosine, K: L-lysine B) Enzymatic degradation only occurs when L-amino acids are incorporated. C) MALDI mass spectra of rr-My-rr (top) containing only D-Arg linkers and RR-My-RR (bottom) containing only L-Arg linkers after digestion. 55x36mm (300 x 300 DPI)


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Figure 3. Erythrocyte leakage of lipo-oligomers A) at different pH conditions at 5 mM concentration and B) at endolysosomal pH 5.5 after incubation with cathepsin B-containing buffer (digested) or buffer only (native) at 2.5 mM concentration. 40x19mm (300 x 300 DPI)



Figure 4. A) Metabolic activity indicating cell viability of human hepatoma Huh7-eGFPLuc (top) and human prostate carcinoma DU145-eGFPLuc cells (bottom) compared to buffer-treated cells determined by MTT assay after incubation with lipo-oligomers with tailored biodegradability. Lipo-oligomer solutions were used in the same concentrations as in gene silencing experiments, but without the addition of siRNA. B) Gene silencing of lipo-polyplexes (N/P 20) formed by mixing of lipo-oligomers and eGFP-targeted siRNA (siGFP) or control siRNA (siCtrl) compared to buffer-treated cells. C) MALDI mass spectra of cell extracts after transfection with My (left) or highly degradable RR-My-RR (right) after transfection showing both undigested lipo-oligomers (red) and fragments of endolysosomal degradation (green). 102x124mm (300 x 300 DPI)


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Figure S1. Erythrocyte leakage of lipo-oligomers containing only single L- or D-arginines at endo/lysosomal pH 5.5 before and after incubation with cathepsin B. 38x32mm (300 x 300 DPI)



Figure S2. Agarose gel shift siRNA binding assay with lipo-polyplexes at N/P 20. All lipo-oligomers show full siRNA binding. 38x19mm (300 x 300 DPI)


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Figure S3. A) Metabolic activity indicating cell viability of human hepatoma Huh7- (top) and human prostate carcinoma DU145-eGFPLuc cells (bottom) determined by MTT assay after incubation with lipo-oligomers containing only single L- or D-arginines. B) Gene silencing of lipo-polyplexes at N/P 20 formed by mixing of lipo-oligomers and eGFP-targeted siRNA (siGFP) or control siRNA (siCtrl). 71x60mm (300 x 300 DPI)



Figure S4. Metabolic activity indicating cell viability of human hepatoma Huh7-eGFPLuc cells determined by MTT assay after incubation with polyplexes at N/P 20 formed by mixing of lipo-oligomers and eGFP-targeted siRNA (siGFP) or control siRNA (siCtrl). 47x27mm (300 x 300 DPI)


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TOC Artwork 629x271mm (96 x 96 DPI)


Precise Enzymatic Cleavage Sites for Improved Bioactivity of siRNA

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