1. Introduction
Nucleic acid-based therapeutics may offer treatment opportunities for various diseases at the transcriptional and translational levels [
1]. However, intracellular delivery of nucleic acids is limited due to their large molecular weight and polarity. Additional challenges such as low in vivo stability and rapid enzymatic degradation limit the use of nucleic acids for therapeutic purposes [
2]. In order to overcome these issues, an efficient delivery system is required to deliver the therapeutic nucleic acids into the target cells, tissues, and organs.
The research of the past 30 years has led to the discovery of safe and efficient delivery vectors for nucleic acids including viral vectors, lipid nanoparticles, cationic polymers, and inorganic nanoparticles [
1,
2]. Despite the numerous delivery vectors available for different applications, only a few of them have managed to reach the clinical stage. For example, siRNA formulations in LNP or with GalNAc are in clinical use [
3]. However, these methods are specific to hepatocytes, whereas delivering into extrahepatic tissues remains an unresolved challenge in the field [
4]. Hence, there is an urgent need for efficient delivery systems especially for in vivo applications. One of the potential delivery vector types for in vivo applications is the cell-penetrating peptides (CPPs), which are relatively short peptides, consisting of approximately 5–30 amino acid residues. CPPs have been used as nonviral delivery vectors for the intracellular delivery of various bioactive cargos, including nucleic acids [
5,
6,
7,
8,
9]. Several CPP-based molecules with therapeutic potential have reached different stages of clinical trials [
10], and the CPP-based neuromodulator Daxxify has been approved by the FDA. A major advantage of CPPs is their ability to enter the cells in a noninvasive way. CPPs are therefore considered a safe and efficient delivery platform for therapeutic molecules [
11].
In our previous efforts, we have focused on designing nucleic acid delivery methods for in vivo applications and we have introduced the NickFect (NF) series of CPPs which are proven to be efficient for gene delivery in vivo. During the sequential development of CPP iterations, we have shown that altering the amino acid in the seventh position of Stearyl-TP10 (PepFect3) linking together neuropeptide galanin motif and mastoparan residues led to an improved nucleic acid delivery vector [
6]. One of the key findings was that replacing the “linker” amino acid Lys
7 with Orn
7, and continuing synthesis via the ε-NH
2 group instead of α-NH
2 of the linker amino acid Ornithine resulted in a CPP with a significant rise of the pDNA transfection efficiency for in vitro applications [
7,
8]. In further works, we increased the in vivo delivery efficacy of the CPP by optimizing the charge distribution in the C-terminal part of the peptide sequence. We introduced the CPP NF55 [
8], which achieved potent in vivo delivery of pDNA and siRNA [
9]. This enabled us to create a platform for the delivery of nucleic acids which could contribute to the clinical development of nucleic acid-based therapeutics.
The aim of our current work was to further explore the importance of the linker region in the CPP NF55 in the delivery of a pDNA type nucleic acid in vivo. We compared alternative linker regions in the parent NF55 sequence with the aim of obtaining optimal pDNA condensation into nanoparticles and enhanced nucleic acid delivery in vivo. For this, we (a) used amino acids with different side chain lengths as linker amino acids and (b) compared the effect of the synthesis from the α-amino group (“linear” peptides) versus the side chain amino group (“kinked” peptides) of the linker amino acid.
2. Materials and Methods
2.1. Peptide Synthesis
Peptides were synthesized on an automated peptide synthesizer Biotage Initiator+ Alstra (Biotage, Uppsala, Sweden) using the fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis strategy. For the synthesis of peptides, where the chain is continued via the ε-NH2 group of the linker amino acid, Boc-L-Dap(Fmoc)-OH, Boc-L-Dab(Fmoc)-OH, Boc-L-Orn(Fmoc)-OH, and Boc-L-Lys(Fmoc)-OH (Iris Biotech GmbH, Marktredwitz, Germany) were used. The reaction was carried out in DMF by coupling the amino acid (5 eq) using HOBT and HBTU (5 eq) as coupling reagents and DIEA (10 eq) as an activator base to Rink amide ChemMatrix resin (Biotage, Uppsala, Sweden) with a loading of 0.41 mmol/g (0.125 mmol, 1 eq) which was used as a solid phase to obtain C-terminally amidated peptides. Stearic acid (5 eq) was coupled to the N-terminus of peptidyl resin manually in DCM using the same strategy as when coupling amino acids.
Cleavage was performed following the standard protocol with trifluoroacetic acid (TFA), 2.5% triisopropylsilane, and 2.5% ultrapure water for 2 h at room temperature. Peptides were purified by reversed phase high-performance liquid chromatography on a C3 column (Agilent Zorbax 300SB-C3, 5 μm, 250 × 9.4 mm) using a gradient of acetonitrile–water containing 0.1% TFA. The purity of peptides was confirmed by reverse-phase ultraperformance liquid chromatography using a C18 column (ACQUITY UPLC BEH130 C18, 1.7 μm, 100 × 2.1 mm) and a solvent system of acetonitrile (B)–water (A) containing 0.1% TFA with a gradient of B = 5–80%. The molecular weight of the peptides was analyzed by matrix-assisted laser desorption–ionization and time-of-flight mass spectrometry (Bruker Daltonics GmbH & Co. KG, Bremen, Germany). The specific concentration of the peptides was achieved by diluting accurately weighed substances and the absorption of tyrosine.
2.2. The Formation of CPP–pDNA Complexes
The CPP–pDNA complexes were prepared by mixing plasmid DNA (0.5 μg of EGFP encoding pEGFP (size 4731 bp; Clontech Laboratories Inc., Montain View, CA, USA ) per well for 24-well plate transfection or 0.1 μg firefly luciferase encoding pLuc (size 10,060 bp) per well for a 96-well plate) with CPPs at a CPP–pDNA charge ratio (CR) of 1:1 to 4:1 (if not indicated otherwise) in MilliQ water, followed by incubation for 30 min at room temperature. CR was calculated theoretically considering the net positive charges of the peptide and the negative charges in the pDNA backbone.
PEI MAX (MW = 40,000; Polysciences, Inc., Warrington, PA, USA) was mixed with plasmid DNA in MilliQ water at the indicated N/P ratio. Thereafter, a complex solution was incubated for 20 min at room temperature.
For in vivo studies, the pLuc was mixed with the CPP at CR2 in MilliQ water, wherein the dose per animal was 2.5 mg/kg. After 30 min of incubation at room temperature, glucose was added to the complexes to obtain an isotonic solution with a final injection volume of 336 µL, which was immediately injected intravenously via the tail vein.
2.3. Characterisation of the CPPs
The secondary structure of the peptides was determined by CD spectroscopy using a Chirascan CD spectrometer (Applied Photophysics Ltd., Leatherhead, UK). For measurement, peptides in ultrapure water with a concentration of 100 μM were transferred to a quartz cuvette (Hellma GmbH & Co. KG, Müllheim, Germany) with an optical path length of 1 mm. The signal was recorded for wavelength intervals between 185 nm and 260 nm, using a 1 nm bandwidth. Based on the obtained CD spectra, the secondary structures of the peptides in water were calculated using BeStSel secondary structure prediction server [
12].
2.4. The Assessment of ξ-Potential of the CPP–pDNA Complexes
For the measurements of ξ-potential, dynamic light scattering studies were performed using a Zetasizer Nano ZS apparatus (Malvern Panalytical, Malvern, UK). Complexes between the pLuc and the CPP were formed as described previously and diluted 10-fold prior to the measurements.
2.5. The Assessment of the CPP–pDNA Complex Formation by Quantitation of Nucleic Acid Intercalating Dye, the Stability of the Complexes towards Enzymatic Degradation by Proteinase K, and pDNA Displacement from the Complexes by Heparin Sodium Salt
To evaluate the complexation of pDNA by the CPP and the stability of formed complexes to enzymatic degradation by Proteinase K, we quantified the accessible pDNA with Quant-iT PicoGreen (PG) (Thermo Fisher Scientific, Vantaa, Finland). Briefly, the preformed CPP–pLuc complexes were transferred to a black opaque 96-well plate and diluted with 115 µL of MilliQ water. Thereafter, 10 µL of diluted fluorescent DNA intercalating dye (Quant-iT PicoGreen) was added to the complexes and after 5 min of incubation, the initial accessibility of pDNA was quantified by measuring the fluorescence (λex = 492, λem = 535). For assessing the stability of the complexes towards enzymatic degradation, 10 µL of Proteinase K (>21 U) was added after ensuring the formation of complexes. The fluorescence was measured over an indicated period at 25 °C and the values were normalized against the fluorescence of free (uncomplexed) pDNA at the same concentration as used for the complexes. For assessing the stability of the complexes against the displacement of pDNA by heparin, the preformed CPP–pDNA nanoparticles were coincubated with different concentrations of heparin sodium salt, and measured fluorescence was normalized against the fluorescence of free pDNA.
2.6. The Assessment of the CPP–pDNA Complex Formation by Gel Migration Assay
To investigate the ability of the NF55 analogues to interact with the pDNA and limit its migration in agarose gel, complexes were formed between the NF55 analogues and pLuc (size 4731 bp), as indicated in
Section 2.2 at CR2. After incubation, samples were diluted with MilliQ water 2 times, and the loading dye was added. The samples were transferred to an agarose gel tooth. Gel electrophoresis was performed in agarose gel (1% agarose gel in 1× TAE buffer) in 1× TAE buffer at 80 mA for 1 h. The accessible pDNA was visualised under UV light using ethidium bromide. As a ladder, ZipRuler Express DNA ladder 2 was used (Thermo Fisher Scientific, Vantaa, Finland).
2.7. The Analysis of the Nanoscopic Structure of the CPP–pDNA Complexes by Atomic Force Microscopy Combined with Infrared Spectroscopy (AFM-IR)
Spatially-resolved infrared measurements were carried out using a combination of atomic force microscopy and nanoscale infrared spectroscopy on an Anasys NanoIR2-s instrument. Complexes between CPP and pLuc were formed at CR2 as described in
Section 2.2. Droplets from the complex mixtures were then placed onto Au-coated substrates, which were dried under a mild vacuum in a desiccator. Data collection was carried out in contact mode by scanning surface areas of 2 × 2 μm
2 or 3 × 3 μm
2 at a resolution of 256 × 256 pixels. The cantilever was equipped with ContGB-G probes, which had a spring constant of 0.2 N/m and a tip radius of 25 nm. Infrared spectra were obtained by positioning the probe on the top of a specific nanoparticle and illuminating the substrate with laser light in the infrared range of 1570–1800 cm
−1. Initial image processing was performed using Analysis Studio software version 3.14, which eliminated noise from the spectra using Fast Fourier transform filters (FFT, 10 points). Further visualization enhancements were carried out using Gwyddion software version 2.51 [
13].
2.8. Cell Culture Maintenance
CHO-K1 and A549 cells were cultured in a humidified environment at 37 °C, 5% CO2, and cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) for the CHO-K1 cell line and in RPMI-1640 for the A549 cell line. Both media were supplemented with GlutaMax, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin.
2.9. The Transfection of pDNA in Cell Culture
For the plasmid delivery assay, 10,000 CHO-K1 or A549 cells per well were seeded in a serum-containing media 24 h prior to each experiment into a 96-well plate. The luciferase encoding plasmid pLuc was mixed with CPPs at the indicated CR, as described in
Section 2.2 and the cells were incubated with complexes for 24 h in a media with serum or without serum. After incubation, the cells were washed with 1× PBS and lysed by treating the cells with 30 µL of 0.1% Triton X-100 in PBS at +4 °C for 30 min. Thereafter, 20 µL of cell lysate was transferred to a black frame and a white well 96-well plate and luciferase activity was measured using a GloMax 96 Microplate Luminometer (Promega Biotech AB, Stockholm, Sweden) after treatment with a luciferin-containing solution. Acquired results were normalized against total protein content, which was obtained by treating the remaining cell lysate with a Pierce BCA Protein Assay kit (Thermo Scientific, Vantaa, Finland) and determined by measuring the absorbance at 562 nm.
For flow cytometry analysis used to determine the transfected cell population, 50,000 CHO-K1 cells per well in serum-containing media were seeded 24 h prior to each experiment into a 24-well plate. The eGFP encoding plasmid was mixed with the CPPs at CR3 as described in
Section 2.2 and cells were incubated with the complexes for 24 h in a media with serum or without serum. Following incubation, cells were washed with 1× PBS, detached from the plate using trypsin-EDTA (0.25%), resuspended in 1× PBS containing 1% FBS, and transferred to 1.5 mL tubes. Flow cytometry was performed using Attune NxT Flow Cytometer equipped with a 488 nm argon laser, where the population of viable cells was determined from a plot of forward scattered vs. side scattered light. Attune NxT Software 3.2.1 software was used to analyze a minimum of 10,000 events per sample from the viable cell population. Results were shown as a percentage of GFP-positive cells from the viable cell population.
2.10. The Quantification of Reporter Levels In Vivo
All animal experiments and procedures were approved by the Estonian Laboratory Animal Ethics Committee (approvals no. 110 12 June 2017 and no. 203 22 September 2021). For in vivo experiments, male and female, 8-week-old BALB/c mice were used.
Complexes were formed as described in
Section 2.2. The expressed reporter gene levels were assessed by using bioluminescence live animal imaging IVIS (details under
Section 2.11) 16 h after injecting the complexes. After imaging the mice were sacrificed, tissues were harvested, and bioluminescence was detected from the tissue homogenates (details under
Section 2.12).
2.11. Bioluminescence Live Animal Imaging
To assess the expression levels of luciferase-encoding pDNA, in vivo bioluminescence imaging was performed using the IVIS Lumina II (PerkinElmer, Inc., Waltham, MA, USA). For this, a solution of D-luciferin (PerkinElmer, Inc., Waltham, MA, USA) in DPBS free of Mg2+ and Ca2+, which was sterile-filtered with 0.2 µm syringe filters prior to in vivo use, was administered by intraperitoneal injection, at the dose of 150 mg/kg. Throughout the imaging process, the mice were anaesthetized by using isoflurane (3% for induction, 1% for maintenance). Photon emissions from the live animals were quantified 10 min later with an exposure time of 1 min. Regions of interest (ROI) were quantified as average radiance (photons s−1 cm−2 sr−1), represented by colour scale (IVIS Living Image 4.0).
2.12. The Quantification of Luciferase Activity from the Tissues Ex Vivo
To evaluate the amount of luminescence from the tissue homogenates, the whole tissues were first lysed by adding 700 µL of T-PER Tissue Protein Extraction Reagent (Thermo Scientific, Vantaa, Finland) followed by homogenization using Precellys24 Tissue Homogenizer (Bertin Corp., Rockville, MD, USA). Thereafter, the samples were shaken for 15 min at 3000 rpm and then centrifuged (3 min at 10,000× g and at +4 °C). The supernatant was transferred to a clean tube and 500 µL of the lysis buffer was added to the pellet, followed by shaking and centrifugation. The supernatant was collected and mixed with the previous one, followed by gentle mixing. From this, 20 µL were transferred to a black frame white well 96-well plate and the amount of luciferase was measured by using the Promega luciferase assay system according to the manufacturer’s protocol in combination with GloMax 96 Microplate Luminometer (Promega Biotech AB, Stockholm, Sweden). An average RLU (relative light unit) of two technical replicates was normalized against the total protein content, which was obtained by treating 10 µL of 20× diluted tissue lysate with a Pierce BCA Protein Assay Kit (Thermo Scientific, Vantaa, Finland) and determined by measuring the absorbance at 562 nm.
2.13. The Assessment of Safety of the NF55 Analogues
Cell viability was assessed by using a CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) (Promega Biotech AB, Stockholm, Sweden) according to the manufacturer’s instructions. For this, 10,000 CHO-K1 cells per well were seeded 24 h prior to the experiment in a 96-well plate in a serum-containing media. Before transfecting the cells with the complexes (CPP and pLuc complexes formed at CR3), the media on the cells was changed for a phenol red-free, serum-free DMEM media. Per well, 20 µL of the MTS reagent was added 20 h post-transfection, and the absorbance of the formazan product was measured at 490 nm after 2–4 h incubation with the reagent. The results are shown as the percentage of viable cells normalized to untreated cells (the latter are defined as 100%).
In vivo safety was evaluated by measuring the alanine aminotransferase and the aspartate aminotransferase levels from the blood of mice injected with nanoparticles formed between NF55 analogues and pDNA. Additionally, the mice were weighed preinjection with the CPP-pDNA complexes and prior to sacrifice.
2.14. Statistical Analysis
The statistical analyses were done using GraphPad Prism version 9.3.1 (Graphpad Software, San Diego, CA, USA). The values in all experiments are shown as the mean ± SEM of 2–3 independent experiments including at least 3 replicates per experiment, if not indicated otherwise.