Cell-penetrating peptides (CPPs), and metal-organic frameworks (MOFs) are promising as next-generation for the delivery of gene-based therapeutic agents. Oligonucleotide (ON)-mediated assembly of nanostructures composed of hierarchical porous zeolitic imidazolate framework (ZIF-8), and nanoparticles such as graphene oxide (GO), and magnetic nanoparticles (MNPs) for gene therapy are reported.
Microporous and Mesoporous Materials 300 (2020) 110173 Contents lists available at ScienceDirect Microporous and Mesoporous Materials journal homepage: http://www.elsevier.com/locate/micromeso Gene delivery using cell penetrating peptides-zeolitic imidazolate frameworks Hani Nasser Abdelhamid a, b, *, Moataz Dowaidar c, Mattias H€allbrink c, Ülo Langel c, ** a Department of Materials and Environmental Chemistry, Stockholm University, Svante Arrhenius V€ ag 16C, Stockholm, SE-106 91, Sweden Advanced Multifunctional Materials Laboratory, Department of Chemistry, Assiut University, Assiut, 71515, Egypt c Department of Biochemistry and Biophysics, Stockholm University, Svante Arrhenius V€ ag 16B, Stockholm, SE-10691, Sweden b A R T I C L E I N F O A B S T R A C T Keywords: Cell-penetrating peptides Metal-organic frameworks Hierarchical porous materials Zeolitic imidazolate frameworks Gene delivery Cell-penetrating peptides (CPPs), and metal-organic frameworks (MOFs) are promising as next-generation for the delivery of gene-based therapeutic agents Oligonucleotide (ON)-mediated assembly of nanostructures composed of hierarchical porous zeolitic imidazolate framework (ZIF-8), and nanoparticles such as graphene oxide (GO), and magnetic nanoparticles (MNPs) for gene therapy are reported Five different types of non-viral vectors (ZIF8, RhB@ZIF-8, BSA@ZIF-8, MNPs@ZIF-8, and GO@ZIF-8), and three gene therapeutic agents (plasmid, splice correction oligonucleotides (SCO), and small interfering RNA (siRNA)) were investigated The polyplexes were characterized and applied for gene transfection The materials show very low toxicity with high efficiency for luciferase transfection ZIF-8 enhances the transfection of plasmid, SCO, siRNA of CPPs by 2–8 folds The mechanism of the cell uptakes was also highlighted Data reveal cell internalization via scavenger class A (SCARA) Introduction Gene therapy, which involves the delivery of exogenous nucleic acids to target cells, has been considered a promising strategy to prevent and treat a myriad of diseases, including cancer, cystic fibrosis, inflamma tory and infectious diseases, cardiovascular diseases, Duchenne’s muscular dystrophy, AIDS, beta-thalassemia, and diabetes [1,2] How ever, the direct delivery of nucleic acid is very limited due to its insta bility in physiological conditions and its inability to penetrate the plasma membrane Thus, viral and non-viral vectors were applied as carriers Viral vectors lack security and thus are less favorable compared to non-viral vectors Features of non-viral vectors including their flexi bility in packaging nucleic acids and ease of production offer additional advantages However, many of these vectors showed low transfection efficiency Thus, peptide-based gene delivery vectors, including cell-penetrating peptide (CPPs), or protein transduction domains (PTDs) [3], are promising due to their high safety, flexibility in conjugating nucleic acids, and simple synthesis [4] They ensure-invasive delivery of therapeutic or diagnostic molecules into mammalian cells [5] They are good capping systems and may provide specific intracellular compart ments without macrophage recognition and subsequent phagocytosis, cross endothelial and epithelial barriers and enter the cytoplasm of target cells [6] Peptides are relatively small, low-cost, and are stable in a wide range of biological conditions [7] Some of these peptides have entered into Phase I, Phase II, and Phase III clinical trials [8] However, loading CPPs with gene therapeutic agents is a major challenge Metal-organic frameworks MOFs are hybrid porous materials con sisting of metal centers and organic linkers [9–11] They have been widely used for several applications such as biomedicine [12–21], biotechnology [22,23], and analytical chemistry [24,25] MOFs (e.g UiO-66, Universiteteti Oslo) was reported for co-delivery of cisplatin and siRNAs [26] UiO-66 enhances the therapeutic efficacy and over coming drug resistance in ovarian cancer cells [26] The drug release from MOFs can be stimuli via metal-ion/ligand, strand/anti-strand, or light [27] MOFs can be loaded with nucleic acids through metal –phosphate coordination interaction [26], intrinsic, multivalent coor dination between DNA backbone phosphate and unsaturated zirconium sites on MOFs [28], or encapsulation [29] MOFs offered high density (~2500 strands/particle) [28], and their surface can be easily func tionalized with terminal phosphate-modified oligonucleotides [30] They can be easily conjugated with nanoparticles that offer multi-functionalities [31] Although issues such as cost of synthesis, * Corresponding author Department of Materials and Environmental Chemistry, Stockholm University, Svante Arrhenius v€ ag 16C, Stockholm, SE-106 91, Sweden ** Corresponding author E-mail addresses: hany.abdelhamid@aun.edu.eg, chemist.hani@yahoo.com (H.N Abdelhamid), ulo.langel@dbb.su.se (Ü Langel) https://doi.org/10.1016/j.micromeso.2020.110173 Received 13 January 2020; Received in revised form March 2020; Accepted 12 March 2020 Available online 14 March 2020 1387-1811/© 2020 The Authors Published by Elsevier Inc This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) H.N Abdelhamid et al Microporous and Mesoporous Materials 300 (2020) 110173 Fig Schematic representation for the synthesis of ZIF-8 composite and their application for gene delivery Fig Characterization of ZIF-8 composite using (a) XRD, and (b-c) SEM images for GO@ZIF-8 (b) and MNPs@ZIF-8 (c) Scale bars represent 100 nm biodegradability, biocompatibility, and toxicity limit MOF applicability [32] Zeolitic imidazolate framework-8 (ZIF-8) is microporous MOFs built from zinc nodes and 2-methylimidazole (Hmim) [33–39] Zn-based MOFs have also been reported as nanocarriers due to the low toxicity of zinc ion [40] ZIF-8 was applied to deliver anticancer drugs such as curcumin (CCM) [41], doxorubicin (DOX) [42,43], camptothecin (CPT) [44], and CpG (stand for 50 —C—phosphate—G—30 , C, and G represent cytosine and guanine) oligodeoxynucleotides [45] ZIF-8 can be conju gated with other nanoparticles such as Fe3O4@PAA\AuNCs\ZIF-8 of fering tri-modal cancer imaging (magnetic resonance, computed X-ray tomography and fluorescence imaging) and chemotherapy into a single system [43] In vivo anticancer experiments indicate that CCM@ZIF-8 NPs exhibit higher antitumor efficacy compared to free CCM [41] En gineering MOFs with proteins not only improve the specificity and affinity but also increase the material biocompatibility [31] Here, we presented simple oligonucleotides (ONs) loading, and efficient release via a synergistic combination of the advantages of ZIF-8 nanoparticles as chemically tunable nanocarriers with those of CPPs, PepFects (PF), as a capping system PepFects (PF) are transportan 10 (TP10) analogs with an N-terminal fatty acid moiety [46] ZIF-8 and their composite with rhodamine B dye, bovine serum albumin (BSA), H.N Abdelhamid et al Microporous and Mesoporous Materials 300 (2020) 110173 Fig Zeta potential measurements for (a) ZIF-8, (b) RhB@ZIF-8, (c) MNPs@ZIF-8, and GO@ZIF-8 before and after modifications with CPPs, pGL3, SCO, and siRNA magnetic nanoparticles (MNPs), and graphene oxide (GO) were evalu ated Three ONs gene therapeutic agents; plasmid (pGL3), splice correction oligonucleotides (SCO), and small interfering RNA (siRNA) were investigated using HeLa cells, HeLa puLc 705 cells, and U-87 MG-luc2 cancer cells, respectively ZIF-8 and its composite improve cell transfection, and enhance cellular uptake of ONs with high biocompatibility 2.2 Synthesis of ZIF-8, RhB@ZIF-8, BSA@ZIF-8, MNPs@ZIF-8, and GO@ZIF-8 A Zn(NO3)2⋅6H2O solution (0.84 M), and Hmim (3.0 M) were pre pared by dissolving 25 g, and 62.5 g in 100 mL, and 250 mL, of deionized water, respectively MNPs, GO, and BSA solutions were prepared by dispersion of mg in mL of deionized water In a glass scintillation vial, 0.10 mL of TEA was added to 0.8 mL of the Zn(NO3)2⋅6H2O solution (0.67 mmol) [47] Then, 4.0 mL of the RhB solution (8 μmol), BSA, MNPs, or GO (1 mg mLÀ 1) was added, followed by the addition of the Hmim solution (6.9 mmol, mL) The reaction solutions were stirred for h before collection using centrifugation (13, 000 rpm, 30 min) The products were washed using water and ethanol (2 � 40 mL) and dried overnight in an oven at 85 � C The solutions of ZIF-8, RhB@ZIF-8, BSA@ZIF-8, MNPs@ZIF-8, and GO@ZIF-8 (10 mg) were prepared via dispersion in 10 mL of deionized water using ultrasonication Materials and methods Zinc nitrate hexahydrate (Zn(NO3)2⋅6H2O), 2-methylimidazole (Hmim), and triethylamine (TEA) were purchased from Sigma Aldrich (Germany) Rhodamine B (RhB), and bovine serum albumin (BSA) encapsulated ZIF-8 (RhB@ZIF-8, or BSA@ZIF-8) were prepared following literature [47] Natural graphite ( 20 ỵ 84 mesh, 99.9%) was obtained from Alfa Aesar (Great Britain) Bare magnetic nanoparticles (MNPs) [48,49], and graphene oxide (GO) [50–55] were synthesized following literature Phosphorothioate 20 -O-methyl RNA oligonucleo tides with Cy5 labeling at 50 -end were purchased from Microsynth AG, Switzerland 2.3 Cell culture HeLa cells, HeLa puLc 705 cells, U-87 MG-luc2 (7000 cells of 100 μL per well, 96 well plates) were cultured at 37 � C and 5% CO2 in 0.1 mM non-essential amino acids Dulbecco’s modified Eagles medium (DMEM) supplemented with 10% (v/v) FBS, mM L-glutamine, 100 U mLÀ penicillin, and 100 mg mLÀ streptomycin (Invitrogen, Sweden) 2.1 Synthesis of peptides CPPs; PF14 (Stearyl-AGYLLGKLLOOLAAAALOOLL-NH2), and PF221 (Stearyl-FLKLLKKFLFLKLLKKFL-amide), were synthesized on an auto mated Syro II multiple peptide synthesizer (MultiSynTech, Witten, Germany), using standard solid-phase Fmoc protocols applying Rinkamide Chem matrix resin (PCAS BioMatrix, Canada) The crude pep tides were purified using high-pressure liquid chromatography (HPLC, Germany) 2.4 Water-soluble tetrazolium salt-1 (WST-1) toxicity assay The material biocompatibility was measured for HeLa cells (7000 cells/well) using cell proliferation reagent (WST-1, Roche Diagnostics Scandinavia AB, Sweden) The cells were incubated with peptide– plasmid complexes modified ZIF-8 The wells containing the treated cells were incubated for another 24 h The cytotoxicity was measured by H.N Abdelhamid et al Microporous and Mesoporous Materials 300 (2020) 110173 Fig TEM images of ZIF-8 composite for a, c, (e) PF14, and b, d, f PF221 using a-b pGL3, c-d SCO, and e-f siRNA Scale bars represent 200 nm following the absorbance at 450 nm on Sunrise™-Tecan microplate absorbance handled using the same procedure as that for the SCO experiments described above 2.5 Transfection using pGL3 luciferase plasmid, splice correction oligonucleotides (SCO), and siRNA 2.6 Confocal laser scanning microscopy One day before the experiment 7,000 HeLa cells were seeded in serum-containing media containing PF14-Alexa 568-705ASO (MR10, 100 nM of SCO) complexes The treated with Fast-dio membrane stain according to the manufacturer’s instruction Confocal microscopy was performed using a Leica DM/IRBE epifluorescence microscope controlled using Micro-ManagerRef (Leica, Mannheim, Germany) Argon ion laser was used to excite fluorescein at 488 nm Emission was recorded between 500 and 550 nm Images were analyzed using Fiji (ImageJ) software Image stacks were filtered with the 3d mean filter using � � settings Synthesis of CPPs–ZIF-8-plasmid complexes were performed in a small vial, 80 μL of MQ water, μL of ZIF-8 (1 mg mLÀ 1) Leica DM/IRBE epifluorescence microscope controlled using Micro-ManagerRef, and μL of pGL3 (205 ng μLÀ 1) were mixed Finally, PF221 or PF14 (10.5 μL) was added The vials were incubated at room temperature for h.siRNA and PF14 were mixed in a molar ratio of 1:20 with and without ZIF-8 composite HeLa cells (7000 cells per 100 μL, 96 well plates) were used to evaluate pGL3 luciferase-expressing plasmid (Promega, USA, Qiagen Plasmid Midi kit, Qiagen, USA) pGL3-ZIF-8-CPPs (CPPs: PF221, and PF14, 10 μL) was added to HeLa cells After incubation for 24 h, the medium was decanted The cells were lysed in cell lysis buffer (Promega, USA) and were measured using GLOMAX™ luminometer (Promega, USA) The biological activity of SCO-ZIF-8-CPPs (CPPs: PF221, and PF14) was determined using HeLa puLc 705 cells (7000 cells/100 μL) The media of cells were aspirated from each well and followed by the addition of the lysis solution (10 μL, 0.2% Triton X-100 in HKR buffer) Finally, luciferase activity was measured using Promega’s luciferase assay system on GLOMAX™ 96 microplate luminometer (Promega, Sweden) U-87 MG-luc2 cells (7000 cells/100 μL) were cultured and investi gated for the transfection of siRNA After the cells have been treated with PF14-siRNA or PF14-siRNA-ZIF-8 (10 μL) for 24 h, the plates were 2.7 Characterization techniques X-ray diffraction (XRD) patterns were recorded using a PANalytical X’Pert Pro diffractometer equipped with a Pixel detector using Cu Kα1 radiation (λ ¼ 1.5406 Å, current of 40 mA, an accelerating voltage of 40 kV and a source slit of 10 mm) Transmission electron microscopy (TEM) was performed on a JEM-2100 instrument (JEOL, Japan) at an accel erating voltage of 200 kV Scanning electron microscopy (SEM) images were recorded using a JSM-7000F instrument (JEOL, Japan) at an accelerating voltage of 15.0 kV Zeta potentials were recorded using the Zetasizer Nano Z system (Malvern Panalytical Ltd, UK) H.N Abdelhamid et al Microporous and Mesoporous Materials 300 (2020) 110173 Fig TEM images of RhB@ZIF-8 composite for (a, c, e) PF14, and (b, d, f) PF221 using (a-b) pGL3, (c-d) SCO, and (e-f) siRNA Scale bars represent 200 nm 2.8 Statistical analysis hydrogen bonding Adsorption of pGL3, SCO, or siRNA was achieved via soaking ZIF-8 composite in aqueous solutions of those gene therapeutic agents The charge of the materials was determined using Zeta potential (Fig 3) All ZIF-8, except for GO@ZIF-8, have positive zeta potentials (Fig 3) The nanocomposites based on SCO for all ZIF-8 materials are positive charge materials In contrast, all materials based on siRNA have high zeta potential with negative values (Fig 3) The negative charges are due to the high phosphate group content of siRNA The morphology of the formed complexes is characterized using TEM images (Figs 4–5, Figs S2–S3), and SEM image (Fig S4) Data show the formation of protein corona of CPPs and gene therapeutic agents on the external surface of ZIFs nanocomposites The results reveal ZIF-8 nanoparticle with a size of 25–150 nm (Figs 4–5, Figs S2–S3) The images reveal a layer of CPPs and gene therapeutic agents with a size of � 3–10 � nm indication the formation of a core-shell structure (Fig S5) Data were generated from at least three independent experiments, and statistically analyzed using a two-way ANOVA test (the program of Graphpad Prism) for the statistical significance: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 Results and discussion Schematic representation for the synthesis of hierarchical porous ZIF-8 composite with RhB, BSA, MNPs, and GO is shown in Fig Adding triethylamine (TEA) to an aqueous solution of Zn2ỵ leads to the formation of ZnO (Fig 1) The molecules (RhB, and BSA), or nano particles (MNPs, and GO, Fig S1) are adsorbed into the formed ZnO that convert into hierarchical porous ZIF-8 after the addition of Hmim (Fig 1) XRD patterns confirm the formation of a pure phase of ZIF-8 composited, called RhB@ZIF-8, BSA@ZIF-8, MNPs@ZIF-8, and GO@ZIF-8 (Fig 2a) SEM images show the formation of ZIF-8 nano particles with a particle size of 25–100 nm (Fig 2b and c) The formation of polyplex consist of CPPs (PF14, and PF221), ZIF-8 composite (RhB@ZIF-8, BSA@ZIF-8, MNPs@ZIF-8, and GO@ZIF-8), and gene therapeutic agents (pGL3, SCO, and siRNA) take place via non-covalent interactions (Fig 1) PF14 and PF221 were chosen because it is an efficient delivery reagent for pGL3, SCOs, and siRNA [56] The complexation takes place via the step-by-step addition of each reagent The interactions inside the polyplex are mainly electrostatic forces and 3.1 Cell viability The WST-1 assay was used to evaluate the cytotoxicity of the formed complexes in HeLa cells (Fig 6) As shown in Fig 6, the cytotoxicity of the materials concentration-independent for the investigated molar ratio and charge ratio There is no observation for any toxicity for ZIF-8 composite with and without CPPs (Fig 6) Biocompatibility of ZIF-8 toward six different cell lines representing various body parts (kidney, H.N Abdelhamid et al Microporous and Mesoporous Materials 300 (2020) 110173 Fig WST-1 cell viability using HeLa cells for (a-b) pGL3 using charge ratio of (a) 5, and (b) 10, and (c-d) SCO using molar ratio of (c) 10, and (d) 20 skin, breast, blood, bones, and connective tissue) revealed that ZIF-8 showed insignificant cytotoxicity up to a threshold value of 30 μg mLÀ [57] It was also reported that ZIF8-CpG ODNs complexes showed no cytotoxicity compared to ZIF-8 alone [45] These results indicate that the platform of ZIF-8 and CPPs are biocompatible materials and can be applied as carriers for gene-based therapeutic agents compared to the complexes without ZIF-8 nanocomposites ZIF-8 im proves the transfection of PF14 that showed higher transfection compared to commercial transfection agent Lipofectamine™ 2000 [56] Gene silencing or knockdown using small/short interfering RNA (siRNA) for the prepared materials tested (Fig 8) siRNA is a doublestranded RNA molecule which is similar to miRNA It prevents trans lation of mRNA after transcription via interfering with the comple mentary nucleotide sequences [61] Cell transfection using siRNA (10 and 25 nM) indicates significant gene knockdown using CPPs-ZIF8 vectors (Fig 8) 3.2 Cell transfection using pGL3, SCO, and siRNA To investigate the performance of the prepared materials ZIF-8 nanocomposite, three gene therapeutic agents called pGL3, splice correction oligonucleotides, and small interfering RNA were investigated The prepared materials; ZIF-8, RhB@ZIF-8, BSA@ZIF-8, MNPs@ZIF8, and GO@ZIF-8, were applied to introduce luciferase reporter genes as a plasmid DNA (pGL3) into the HeLa cells for PF14, and PF221 (Fig 7) Two different ratios of the plasmid: ZIF-8 composite of 1:0.5 and 1:1 were investigated To account for transfection variation among wells, a control plasmid containing pGL3-PF14, or pGL3-PF221were transfected for normalization purposes Results show no transfection efficiency in the absence of CPPs (Fig 7) ZIF-8 nanocomposites increase the effi ciency of pGL3-PF14 by 2–4 folds While ZIF-8 nanocomposites improve the transfection of pGL3-PF221 by 3-8-fold (Fig 7) Results reveal that CPPs-ZIF8 is robust enough to have luciferase signals significantly higher than the background (Fig 7) Transfection of splice correction oligonucleotides (50 -CCU CUU ACC UCA GUU ACA- Cy5-labeled) using the prepared materials is also investigated (Figs S6–S9) The used SCOs are antisense oligonucleotides (ONs) with 18 bases in length [58–60] The complexes of the ternary component i.e SCO, ZIF-8 nanocomposite, and CPPs were tested for HeLa cell line with a recombinant plasmid pLuc 705 that carry the luciferase gene interrupted by a mutated human beta-globin intron IVS2-705 Results demonstrate that complexes containing ZIF-8 nano composites have higher transfection efficacy for oligonucleotides as 3.3 Mechanism of uptakes Finally, we elucidated the intracellular fate of the CPPs-ZIF8 after incubation using uptake of Scavenger class A (SCARA, Fig 9), and confocal microscopy (Fig 10) A study using curcumin loaded ZIF-8 showed intracellular distribution via the clathrin-mediated endocy tosis to the lysosomal pathway [62] In general, there are several pro posed mechanisms for gene therapeutic agents Among those proposed mechanisms, endocytosis, energy-independent translocation [3], acti vation of sphingomyelinase which converts sphingomyelin into cer amide [63], and presence of scavenger class A (SCARA) may explain the cell uptakes of the formed complexes (Fig 9) SCARA is a receptor that can bind and endocytose acetylated low-density lipoprotein [64] The experiment was performed via incubation HeLa cells with inhibitor (Dextran sulfate (Dex), polyinosinic acid (Poly I) or fucoidan (Fuc)) and the corresponding control reagents (Chondroitin sulfate (Chon), poly cytidylic acid (Poly C) or galactose (Gal)) (Fig 9) As shown in Fig 9, no transfection was observed in the presence of the inhibitors On the other side, the transfection was successfully observed in the presence of the control reagents that confirm the high impact of SCARAin the uptake of PF14–SCO-ZIF-8 The cell after transfection was also observed under a confocal mi croscope (Fig 10) There are no notable changes in cell morphology The H.N Abdelhamid et al Microporous and Mesoporous Materials 300 (2020) 110173 Fig a-b) pGL3 transfection of HeLa cells for PF14 using plasmid: ZIF-8 composite of (a) 1:0.5, and (b) 1:1 Fig Cell uptakes based on the mechanism of scavenger receptor class Fig Transfection of U-87 MG-luc2 cells with siRNA H.N Abdelhamid et al Microporous and Mesoporous Materials 300 (2020) 110173 Fig 10 Confocal microscopy images of Hela-705 cells incubated for 24 h with PF14-Alexa 568–705ASO (MR10, 100 nM ASO) complexes (in red) for (a) PF-14, (b) PF14-ZIF8, (c) PF14–RhB@ZIF8, (d) PF14-BSA@ZIF8, (e) PF14-MNPs@ZIF8, and (f) PF14-GO@ZIF8 Cell-membranes are stained with Fast-dio stain (in green) (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article) red color corresponding oligonucleotides indicate the presence of the gene therapeutic agent inside the cell revealing successful cell trans fection (Fig 10) Results indicate that most of the oligonucleotides entered the cells by endocytosis that release the gene after degradation in the cytosol (Fig 1) It is important to keep in mind that ZIF-8 nano particles can be dissolved in the acidic environment of the cancer cells (pH 5.5) [28] These results confirm that CPPs-ZIF8 is an effective gene vector for the internalization of a gene therapeutic agent into the cells hyperthermia Declaration of competing interest None CRediT authorship contribution statement Hani Nasser Abdelhamid: Conceptualization, Methodology, Writing - original draft Moataz Dowaidar: Data curation Mattias €llbrink: Visualization Ülo Langel: Supervision, Writing - review & Ha editing Conclusions Based on our presented findings, a broad gene vector based on ZIF-8 nanoparticles with tunable properties can be used to prepare effective materials for gene delivery Our protocol offers facile integration of multifunctional components via inclusion and post-synthetic modifica tions of ZIF-8 These materials may be useful for multi-targeted medical applications in both diagnosis and therapy with exceptional high cell biocompatibility The synthesized materials appear promising due to the presence of other components such as dye, protein, magnetic nano particles, and graphene oxide These reagents offered potential appli cations of the composites as a multifunctional platform for cancer theranostics involving magnetic resonance imaging, drug delivery, and Acknowledgments This work was funded by the Swedish Research Council (VR-NT), and from the Innovative Medicines Initiative Joint Undertaking under grant agreement no 115363 sources of which are composed of financial participation from the European Union’s Seventh Framework Pro gramme (FP7/2007–2013) and EFPIA companies, Swedish Cancer Foundation, Sweden, and from the European Regional Development Fund through the Center of Excellence in Molecular Cell Engineering H.N Abdelhamid et al Microporous and Mesoporous Materials 300 (2020) 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Scandinavia AB, Sweden) The cells were incubated with peptide– plasmid