In modern cancer therapy, conventional chemotherapy and surgery often fail [1
]. Nevertheless, with the field of gene therapy, a novel opportunity has reached clinical application [2
]. Despite all the benefits of viral gene therapy, it also comes with certain disadvantages, such as limited re-administration due to immediate inflammatory or delayed humoral or cellular immune response. In the last decades, much focus has also been put on non-viral gene therapy [5
]. Polymeric vehicles such as linear polyethylenimine (lPEI) with its repetitive diaminoethane motif can address different stages of the delivery pathway in a way similar to viruses [6
]. Polycations can stably bind and compact pDNA into “polyplexes” [9
] with virus-like nanodimensions [10
]. Size, stability, and shape of nanoparticles [11
] play a crucial role for the in vivo biodistribution and survival of nanoparticles, as well as cellular uptake processes [13
]. Following cellular uptake by endocytosis, endolysosomal escape of nanoparticles presents a significant bottleneck in the transfection process. lPEI polyplexes are quite effective at this stage due to the endosomal buffering (“proton sponge”) diaminoethane motif [14
]. Nevertheless, the toxicity [20
] and polydispersity [22
] of lPEI led us to the development of diaminoethane motif-containing building blocks suitable for solid phase synthesis (SPS) [23
]. With the help of SPS, it is possible to synthesize multifunctionalized defined oligomers for gene delivery [25
The introduction of a shielding domain into artificial gene carriers is crucial in the first phase of the delivery process to reduce unwanted interaction between blood and pDNA polyplexes. Polyethylene glycol (PEG) represents the most prominent and well-established shielding agent [31
], and has been used for shielding cationic polyplexes in numerous instances [33
]. It was recently found that PEGylation may cause an immunogenic response [39
]; thus, several alternatives have also been evaluated for this polyplex shielding, such as poly(N
-(2-hydroxypropyl)methacrylamide) (pHPMA) [45
], hydroxyethyl starch (HES) [48
], polysarcosine (pSar) [49
], oligosaccharides [46
], or proteins [52
]. Recently, recombinant PAS composed of repeats of the natural amino acid sequence proline-alanine-serine was reported as an alternative to PEG [53
Aside from having clearly positive effects such as improved blood circulation times and increased tumor accumulation, polyplex shielding may also provide negative drawbacks, such as a reduced intracellular performance and/or reduced plasmid DNA (pDNA) compaction; this dilemma can be at least partly overcome by programmed deshielding [38
]. The current paper focuses on the relation of polyplex shielding with pDNA compaction, size, shape, and stability. For this purpose, we compare oligoaminoamide co-oligomers with different PEG lengths or defined numbers of PAS repeats which were generated by SPS. We focused on the effect of the shielding block attached in T-shape topology onto a “two-arm” structure of oligoaminoamide oligomers tailored for pDNA delivery. The oligomer library for this study consisted of oligomers decorated with four or eight repetitions of PAS as well as PEG of 12, 24, or 48 ethylene oxide (EO) units in length, or a three-arm oligomer without shielding, respectively. Oligomers were compared and evaluated in different physicochemical and biological assays to elucidate the impact of shielding agent on critical polyplex characteristics.
2. Materials and Methods
2-Chlorotrityl chloride resin was purchased from Iris Biotech (Marktredwitz, Germany), 2-(1H
-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium-hexafluorophosphate (HBTU), syringe reactors (PP reactor with PE frit) from Multisyntech GmbH (Witten, Germany). All amino acids, peptide grade dimethylformamide (DMF), N
-diisopropylethylamine (DIPEA), and trifluoroacetic acid (TFA) were purchased from Iris Biotech (Marktredwitz, Germany). The cationic building block Fmoc-Stp(Boc)3
-OH (Stp: succinyl-tetraethylene pentamine; Boc: tert
-butyloxycarbonyl) was prepared as described in [57
]. 1-Hydroxy-benzotriazole (HOBt), triisopropylsilane (TIS), dimethylsulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and Triton X-100 were obtained from Sigma-Aldrich (Munich, Germany). N
-hexane and tert
-butyl methyl ether (MTBE) were obtained from Brenntag (Mühlheim an der Ruhr, Germany). Dichloromethane was obtained from Bernd Kraft (Duisburg, Germany). Fmoc-N
was purchased from Quanta Biodesign (Powell, OH, USA). 1,2-Ethanedithiol (EDT) was purchased from Sigma-Aldrich (Munich, Germany). All solvents and other reagents were acquired from Sigma-Aldrich (Munich, Germany), Iris Biotech (Marktredwitz, Germany), Merck (Darmstadt, Germany), or AppliChem (Darmstadt, Germany).
All cell culture consumables were obtained from NUNC (Langenselbold, Germany), TPP (Trasadingen, Switzerland), or Sarstedt (Nümbrecht, Germany). DMEM medium was purchased from Sigma Aldrich (Munich, Germany). RPMI medium, fetal bovine serum (FBS), cell culture media, and antibiotics were purchased from Invitrogen (Karlsruhe, Germany), glucose from Merck (Darmstadt, Germany), HEPES from Biomol GmbH (Hamburg, Germany), and sodium chloride from Prolabo (Haasrode, Belgium). Agarose NEEO Ultra-Qualität was obtained from Carl Roth GmbH (Karlsruhe, Germany) and GelRed™ from VWR (Darmstadt, Germany). Heparin sulfate was purchased from Ratiopharm (Ulm, Germany) with 5000 IU/mL. Luciferase cell culture 5× lysis buffer and d-luciferin sodium were obtained from Promega (Mannheim, Germany).
2.2. Oligomer Synthesis
Peptide synthesis was carried out using Fmoc-chemistry and l-amino acids. Prior to synthesis, 2-chlorotrityl chloride resins were loaded with the appropriate C-terminal amino acids. Coupling was carried out with 4 eq. of HBTU, 4 eq. of DIPEA, 4 eq. of HOBt, and 4 eq. of Fmoc-protected amino acid per free amine on the solid support; the solution was supplemented with 1% Triton X-100 (v/v). Amino acids and HOBt were dissolved in NMP (N-methylpyrrolidone), supplemented with Triton-X. HBTU was dissolved in DMF, and DIPEA diluted with NMP. Peptide synthesis was carried out using an automated Syro Wave (Biotage, Uppsala, Sweden) with a microwave cavity. Double couplings were performed at 50 °C for 12 min prior to five repetitive washing steps. Fmoc removal was conducted by standard deprotection methods with 40% piperidine in DMF (v/v) supplemented with 1% Triton-X for 5 × 10 min.
Resins for two-armed oligomers were loaded with Dde-l
-Lys(Fmoc)-OH, and for three-arm oligomers with Fmoc-l
-Cys(Trt)-OH, as described in the Supplementary Materials
. After loading determination and Fmoc removal, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, and Fmoc-Pro-OH (from now on called PAS) were attached sequentially, with four or eight triple sequence repeats (PAS4
) in the case of PAS-shielded oligomers, while PEG controls were synthesized by attachment of precise bifunctional Fmoc-N
-OH or Fmoc-N
-OH. At this step, an analytical cleavage of a small fraction was done and MALDI-TOF mass spectrometry of the Dde-K-(PAS)8
peptide was carried out (see Figure S6
Next, the cationic backbone of the oligomers was built, based on alternating Stp and histidine units, a lysine branching point, and terminal cysteines for cross-linkage. Therefore, Fmoc-l
-Lys(Fmoc)-OH, our cationic building block Fmoc-Stp(Boc)3
], and Boc-l
-Cys(Trt)-OH were coupled following the sequence in Table 1
. A detailed step-by-step description of the synthesis of Fmoc-Stp(Boc)3
-OH, of the cationic backbone, and of the three-arm oligomer can be found in [57
]. In the case of shielded oligomers, the resins loaded with Dde-l
were placed separately into the microwave cavity of the Syro Wave (Biotage, Uppsala, Sweden) peptide synthesizer, and synthesis was conducted under the conditions described above until the last coupling of N
In the case of non-shielded three-arm oligomer, a Cys(Trt)-OH preloaded 2-chlorotrityl resin was used and automated microwave-assisted synthesis was carried out to obtain the sequence mentioned in Table 1
. All oligomers were ended by a double coupling of Boc-Cys(Trt)-OH for 2 × 1 h at room temperature to avoid racemization.
Synthesis of cMet-targeted oligomers (containing a cMet-binding peptide, cmb) can be found in the experimental section of Supplementary Materials
After the final coupling step, the resins were dried and the products cleaved off the resin for 90 min using a mixture of TFA/EDT/H2O/TIS 94:2.5:2.5:1 (v/v) at a ratio of 10 mL per gram resin.
The cleaved oligomers were purified by size exclusion chromatography (SEC) performed with 10 mM hydrochloric acid/acetonitrile 7:3 as solvent. An ÄKTA purifier system (GE Healthcare Biosciences, Uppsala, Sweden) equipped with a Sephadex G-10 column and a P-900 solvent pump module, a UV-900 spectrophotometrical detector, a pH/C-900 conductivity module, and a Frac-950 automated fractionator was used. The product fractions were collected and combined prior to lyophilisation.
Analytical data of 1
H-NMR and reversed phase high-performance liquid chromatography (RP-HPLC) can be found in Figures S7–S30
2.3. Polyplex Formation
Indicated amounts of pCMVLuc (CMV: cytomegalovirus promotor; Luc: firefly luciferase gene) and oligomer at indicated nitrogen/phosphate (N/P) ratios were diluted in separate tubes. If not otherwise mentioned, suspension media was 20 mM HEPES buffered 5% glucose at pH 7.4 (HBG 7.4). Diluted oligomer and diluted pDNA were always mixed at similar volume.
For N/P ratio calculation, only the protonatable amino groups of the Stp units and N-terminal amines of cysteine residues were considered (N number) and related to the anionic phosphate groups (P number) in pDNA. Note that protonatable amines outside of the cationic backbone and protonatable imidazole nitrogens of histidines were not taken into account for calculation of N (for reasons of consistency with our previous work). The nucleic acid was added to the oligomer, mixed vigorously, and incubated for 30 min at room temperature under air exposure to enhance disulfide formation.
2.4. Polyplex Stability in the Presence of Salt
Polyplexes were prepared with oligomers at N/P 12 and 2 µg pCMVLuc in a total volume of 60 µL deionized water. After 30 min, 500 µL of phosphate-buffered saline (PBS) was added and a dynamic laser-light scattering (DLS) measurement with three runs (including six sub-runs each) was performed immediately. Samples were incubated at room temperature, and further measurements after 5, 30, 60, and 180 min were conducted.
2.5. Erythrocyte Adhesion in the Presence or Absence of Serum
Polyplexes were prepared at N/P 12 and 2 µg pDNA (20% Cy5 labeled) in a total volume of 60 µL HBG. After 30 min, three groups were treated differently. Either HBG, 3 × 106
erythrocytes in HBG, or serum (to a final concentration of 90%) were added. After further 30 min of incubation at 37 °C, erythrocytes containing polyplexes were sedimented by centrifugation (1500 rpm for 10 min at room temperature) and the supernatant was taken. Then, 3500 IU of heparin sulfate was added to dissociate the polyplex and determine the remaining amount of pDNA via Cy5 excitation/emission (λex
= 649 nm and emission wavelength λem
= 670 nm). Data were calculated in comparison to equally treated free pDNA. Self-quenching and efficacy of release with 3500 IU heparin can be found in Figure S1
2.6. Particle Size and Zeta Potential
Particle size and zeta potential of polyplexes were measured by dynamic laser-light scattering using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) and transmission electron microscopy (TEM) with a JEM 1011 (Jeol, Freising, Germany).
For DLS measurements, 2 µg pDNA was dissolved in 30 µL HBG and was added to an amount of oligomers corresponding to N/P 12 in 30 µL HBG. After 30 min of incubation at room temperature, 740 µL of 10 mM sodium chloride solution (pH 7.4) was added and the samples were measured. Results were plotted as Z-Average and SD out of three runs with 12 sub-runs each. Zeta potential (ZP) is displayed as average (mV) of three runs with up to 15 sub-runs each.
For TEM, samples were prepared as follows. The formvar/carbon-coated 300 mesh copper grids (Ted Pella Inc., Redding, CA, USA) were activated by mild plasma cleaning. Afterwards, the grids were incubated with 20 µL of the polyplex solution at N/P 12 for 2.5 min previously prepared in water. Excess liquid was blotted off using filter paper until the grid was almost dry. Prior to staining, the grids were washed with 5 µL of staining solution for 5 s. Then, the copper grids were incubated with 5 μL of a 2% aqueous uranylformate solution for 20 s, excess liquid was blotted off using filter paper, followed by air-drying for 30 min. Samples were then analyzed using a JEM 1011 (Jeol, Freising, Germany) operated at 80 kV.
2.7. Ethidium Bromide Compaction Assay and Heparin Stress
Polyplexes containing 2 μg pDNA were formed at N/P ratios of 6 and 12 in a total volume of 200 μL HBG. lPEI polyplexes formed at N/Ps 6 and 12 served as a positive control. HBG buffer (200 μL) served as blank, and 2 μg pDNA in 200 μL HBG buffer was considered as maximum ethidium bromide (EtBr) fluorescence intensity (100% value). These samples were prepared in parallel to the polyplexes. After 30 min of incubation at room temperature, 700 μL of EtBr solution (c = 0.5 μg/mL) were added. The fluorescence intensity of EtBr was measured after an additional 3 min incubation using a Cary Eclipse spectrophotometer (Varian, Germany) at the excitation wavelength λex = 510 nm and emission wavelength λem = 590 nm. The fluorescence intensity of EtBr was determined in relation to the 100% value. As a further experiment, 250 IU of heparin (Ratiopharm, Ulm, Germany) was added to the samples after EtBr addition to investigate polyplex stability against polyanionic stress.
2.8. Serum Stability of Polyplexes
Determination of polyplex stability against 90% FBS was determined by DLS. Polyplexes were prepared as described previously with 8 µg pDNA and oligomers at N/P 12 in a total volume of 50 µL HBG. After incubation for 30 min, 30 µL of HBG and 720 µL FBS were added to reach a final concentration of 90% FBS. Sixty microliters were placed in a DTS1070 cuvette, and t = 0 min was determined. Then, polyplexes in serum were incubated under steady shaking at 37 °C and aliquots were taken for further measurements after 30, 120, 240 min, and 24 h. Each time point represents one measurement averaged from 15 sub runs.
2.9. Cell Culture
A mouse neuroblastoma cell line (Neuro-2a) as well as the human hepatocellular carcinoma cell line Huh7 were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (1 g/L glucose), while human prostate cancer cell line (DU145) was cultured in RPMI-1640 medium. All media were supplemented with 10% fetal bovine serum, 4 mM stable glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cell lines were cultured at 37 °C and 5% CO2 in an incubator with a relative humidity of 95%.
2.10. In Vitro Gene Transfer
Cells were seeded 24 h prior to pDNA delivery in 96-well plates. For transfections of Neuro-2a and DU145, 10,000 cells/well were seeded, and 8000 cells/well were seeded in the case of Huh7. Transfection efficiency of oligomers was evaluated using 200 ng pCMVLuc per well. Polyplexes were formed at different N/P ratios in a total volume of 20 μL HBG. Before treatment, the cell culture medium was exchanged with 80 μL fresh medium containing 10% FBS. Polyplex solution was added to each well and incubated on cells at 37 °C for a determined period of time (45 min or 24 h). In the first case, medium was replaced 45 min after transfection by fresh medium. For control experiments, only fresh medium was added. In the second case, cells were incubated with polyplex solution for 24 h after initial transfection. All experiments were performed in quintuplicate. lPEI (N/P 6) was used as a positive control, and HBG buffer was used as a negative control.
For the luciferase assay, 24 h after initial transfection, medium was removed and cells were treated with 100 μL luciferase cell culture 5× lysis buffer. Luciferase activity in the cell lysate was measured by using a Centro LB 960 plate reader luminometer (Berthold Technologies, Bad Wildbad, Germany) and LAR buffer supplemented with 1 mM luciferin. Transfection efficiency was evaluated as relative light units (RLU) per well.
For the metabolic activity of transfected cells, 24 h after initial transfection, 10 µL of MTT was added to each well, reaching a final concentration of 0.5 mg/mL. Medium with unreacted dye was removed after an incubation time of 2 h at 37 °C. The 96-well plates were stored at −80 °C for at least one hour, and afterwards the purple formazan product was dissolved in 100 µL DMSO per well. The absorbance was determined by using a microplate reader (TecanSpectrafluor Plus, Tecan, Switzerland) at 530 nm with background correction at 630 nm. The relative cell viability (%) related to the buffer-treated control cells was calculated as ([A] test/[A] control) × 100%.
2.11. Cellular Association
Neuro-2a cells were seeded 24 h prior to transfection into 24-well plates at a density of 50,000 cells/well. Culture medium was replaced with 400 μL fresh growth medium 24 h after seeding the cells. pDNA polyplexes formed at N/P ratio 12 in 100 μL HBG and containing 1 μg pCMVLuc (20% of Cy5-labeled pCMVLuc) were added to each well and incubated on ice for 30 min. Subsequently, cells were washed twice with 500 µL PBS. Cells were detached with trypsin/EDTA and resuspended in PBS with 10% FBS. All experiments were performed in triplicate. Cellular association of the polyplexes was measured by excitation of Cy5 at 635 nm and detection of emission at 665 nm. DAPI (4′,6-diamidino-2-phenylindole) staining was used to discriminate between viable and dead cells. Cells were properly gated by forward/sideward scatter and pulse width for the exclusion of doublets. Data were recorded by Cyan ADP flow Cytometer (Dako, Hamburg, Germany) using Summit acquisition software (Summit, Jamesville, NY, USA) and analyzed by FlowJo 7.6.5 flow cytometric analysis software.
2.12. Cellular Internalization
Neuro-2a cells were seeded 24 h prior to transfection into 24-well plates at a density of 50,000 cells/well. Culture medium was replaced with 400 μL fresh growth medium 24 h after seeding the cells. pDNA polyplexes formed at N/P ratio 12 in 100 μL HBG and containing 1 μg pCMVLuc (20% of Cy5-labeled pCMVLuc) were added to each well and incubated at 37 °C for 45 min. Subsequently, cells were washed once with 500 µL PBS containing 1000 IU heparin for 15 min on ice to remove any polyplexes sticking to the cell surface and again washed once with 500 μL PBS only. Cells were detached with trypsin/EDTA and resuspended in PBS with 10% FBS. All experiments were performed in triplicate. Cellular internalization of the polyplexes was measured by the excitation of Cy5 at 635 nm and detection of emission at 665 nm. DAPI staining was used to discriminate between viable and dead cells. Cells were properly gated by forward/sideward scatter and pulse width for exclusion of doublets. Data were recorded by Cyan ADP flow Cytometer (Dako, Hamburg, Germany) using Summit acquisition software (Summit, Jamesville, NY, USA) and analyzed by FlowJo 7.6.5 flow cytometric analysis software.
2.13. In Vivo Gene Transfer
Animal experiments were performed in female 6-week-old nude mice, Rj: NMRI-nu (nu/nu) (Janvier, Le-Genest-St-Isle, France) which were housed in isolated ventilated cages with a 12 h day/night interval and food and water ad libitum. Huh7 (5 × 106 cells) suspended in 150 μL PBS were injected subcutaneously into the left flank. After injection, tumor size was monitored with a caliper and determined by formula a × b2/2 (a = longest side of the tumor; b = widest side vertical to a). When tumors reached a size of approximately 1200 mm3, the experiments started by intratumoral injection of 60 μL polyplex solution containing 50 μg pCMVLuc at N/P 12 in HBG. For each polymer, a group of five mice (n = 5) was treated. Mice were euthanized 48 h later, and tumors were collected to assess luciferase activity via ex vivo luciferase assay. Tumors were homogenized in 500 μL cell lysis buffer using a tissue and cell homogenizer (FastPrep®-24). To separate insoluble cell components, the samples were centrifuged at 3000× g at 4 °C for 10 min. Luciferase activity was measured in the supernatant using a Centro LB 960 luminometer. All animal experiments were performed according to the guidelines of the German law for the protection of animal life, and were approved by the local animal ethics committee.
2.14. Statistical Analysis
Statistical significance was analyzed by Student’s one-tailed t-test. Significance levels are indicated with symbols: ns p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.