Self-assembled nanoparticles based on modified cationic dipeptides and DNA: novel systems for gene delivery
© Panda et al.; licensee BioMed Central Ltd. 2013
Received: 27 February 2013
Accepted: 14 June 2013
Published: 21 June 2013
Gene therapy is most effective when delivery is both efficient and safe. However, it has often proven difficult to find a balance between efficiency and safety in case of viral or polymeric vectors for gene therapy. Peptide based delivery systems may be attractive alternatives but their relative instability to proteolysis is a major concern in realizing their potential application in biomedical sciences. In this work we report gene delivery potential of nanoparticles (Nps) synthesized from cationic dipeptides containing a non-protein amino acid α, β-dehydrophenylalanine (∆Phe) residue.
Dipeptides were synthesized using solution phase peptide synthesis method. Nps were formed using self-assembly. Nps were characterized using light scattering, electron microscopy. Transfection efficiency was tested in hepatocellular carcinoma (HuH 7) cells.
The cationic dipeptides condensed plasmid DNA into discrete vesicular nanostructures. Dipeptide Nps are non-cytotoxic, protected the condensed DNAs from enzymatic degradation and ferried them successfully inside different types of cells. GFP encoding plasmid DNA loaded dipeptide Nps showed positive transfection and gene expression in HuH 7 cells.
The cationic dipeptide Nps can successfully deliver DNA without exerting any cytotoxic effect. Owing to their simple dipeptide origin, ease of synthesis, enhanced enzymatic stability as well unmatched biocompatibility, these could be successfully developed as vehicles for effective gene therapy.
KeywordsCationic Dipeptide Plasmid Delivery Nanoparticle
Gene therapy continues to hold great promise in the field of medicine. Replacing defective genes with functional genes offers many exciting possibilities for treating dreaded diseases such as cancers, autoimmune disorders and neurodegenerative diseases . Efficient gene delivery, fundamental to the success of gene therapy remains a complex process with many possible rate-limiting steps. The most serious intracellular barriers are lysosomal degradation, nucleolytic degradation in the cytosol and inefficient delivery to the nucleus [2–4], whereas, extracellular barriers include nucleolytic degradation in the serum, recognition by the reticulo-endothelial system and nonspecific delivery . Gene delivery in humans requires carriers that will transfer DNA into the nuclei of target cells. These carriers must be efficient in transfection, safe for human use, protect DNA from degradation before arriving at the target cell and possibly hold targeting qualities for the specific delivery of gene to required cells or tissue.
Most early applications of gene therapy were concentrated around viral vectors for efficient gene delivery. However, many of these vectors raised undesirable immune responses, hindering their therapeutic effect . Synthetic gene delivery methods surpassed viral vectors in many ways, such as low cytotoxicity, reduced ability to provoke an immune response, ease of manufacturing and scale up and high adaptability. Over the years many chemical synthetic approaches using polymers with ability to accommodate larger DNA molecules compared to viral vectors, have been developed. Nanoparticles (Nps) loaded with plasmid DNA may serve as efficient sustained release gene delivery systems due to their rapid escape from the degradative endo-lysosomal compartment to the cytoplasmic compartment . Successful oligonucleotide delivery to HeLa cells using anionic liposomes , lipid-derived Nps that carry oligonucleotides either in their core or via covalent attachment have been reported to have significant efficacy in vivo and in vitro. Many research groups have used quantum dots , magnetic  and gold Nps [12, 13] as well as carbon nanotubes  and enabled successful siRNA/antisense delivery [10, 14]. Much attention was given to the biodegradable and biocompatible poly PLGA polymers . Polycationic polymer based Nps as non-viral gene delivery vectors, have also been developed and polyethylenimines is currently the most popular polymer used to deliver genes into various cell types, including neurons. Polyethylenimines is able to condense genes into small Nps and protect the DNA from degradation by nucleases. Polymeric complexes such as PLGA– polyethylenimines Nps have been demonstrated as new delivery systems to carry genes to the lung epithelium . Cationic bovine serum albumin conjugated with poly(ethyleneglycol)–poly(lactide) nanoparticle, was also developed as a promising brain drug delivery carrier with low toxicity .
We have been working with very short peptides that self-assemble to form different kind of Nps. Here we report DNA condensation and cell delivery potential of cationic dipeptide Nps (CdNps) such as Arg-∆Phe and Lys-∆Phe. The cationic dipeptides were non-cytotoxic and could condense different plasmid DNAs into discrete Nps enhancing their cellular uptake and delivery, and may represent a novel platform that can be further developed for DNA delivery into cells of different kinds.
Materials used in this study are: THF, NMM, DMF, piperidine, DIPCDI, IBCF, TIPS, TFA, HFIP, phenol, DL-threo-phenylserine, MTT (Sigma-Aldrich, Munich, Germany) Boc-Arg(Mtr)-OH, Boc-Lys(Boc)-OH (Novabiochem, Merck, Darmstadt, Germany) sodium acetate, ethyl acetate, acetonitrile (Spectrochem Pvt Ltd, Mumbai, India), anhydrous sodium sulfate, citric acid (Merck, Munich, Germany), human ovarian cancer (HeLa), hepatocellular carcinoma (HuH-7) and human dermal fibroblast (L929) cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). RPMI-1640, DMEM, NHS-modified Alexa-488 (Invitrogen, Life Technologies Corp. NY, USA). Plasmids used in the study were generous gifts from Dr Reddy’s laboratory, ICGEB, India. Except for method 2.8 in which plasmids of different lengths [in base pairs (bp)] were used and the transfection experiment where EGFP plasmid was used, all other studies were carried out with pgem plasmid DNA.
Assembly of dipeptides
Assembly of dipeptides into Nps in water was done from their HFIP stock solutions at a concentration of 2 mg/ml. Briefly, 2 mg of cationic peptides were first dissolved in 20 μl of HFIP and then diluted with 1 ml of filtered MilliQ water to form self-assembled Nps.
Dynamic Light Scattering (DLS) studies
Light scattering studies were performed in Photocor complex (Photocor, Moscow, Russia) using multiple tau digital correlator. For DLS measurement samples were prepared in dust free environment. Experiments were carried out at an angle of 90° using 632 nm laser at room temperature. Data represent mean of three different sets.
Loading DNA in preformed peptide Nps
Arg-∆Phe and Lys-∆Phe, Nps (2 mg/ml) were prepared as described above and were titrated with increasing concentrations of plasmid DNA (25, 37.5, 50, 62.5, 75, 100 μg) and analyzed using DLS to determine the optimum DNA concentration needed to form compact nanostructures.
Determination of entrapment efficiency of DNA in Arg-∆Phe and Lys-∆Phe Nps
Formation of DNA-peptide Nps by Arg-∆Phe and Lys-∆Phe
For making DNA-peptide Nps, 100 μg of plasmid DNA was titrated with increasing amounts of peptides (100, 500, 1000, 2000 μg) and analyzed using DLS.
Effect of plasmid DNA size on their interaction with Arg-∆Phe and Lys-∆Phe
Peptides (2 mg/ml) were incubated with 100 μg of plasmids with varying sizes for 30 min. The DNA-peptide Nps so obtained were analyzed using DLS and electron microscopy.
Transmission Electron Microscopy (TEM)
DNA-peptide Nps were adsorbed on a 300 mesh copper grid with carbon coated formvar support and stained with 1% uranyl acetate and viewed under transmission electron microscope operating at an accelerated voltage of 120 kV (Tecnai 12 BioTWIN, FEI Netherlands). Photomicrographs were digitally recorded using a Megaview II (SIS, Germany) digital camera. Image analysis was carried out using Analysis II (Megaview, SIS, Germany) and ImageJ (http://rsb.info.nih.gov/ij/) software packages.
Localization of DNA in DNA-peptide Nps
In order to determine the exact location of plasmid DNA in DNA-peptide Nps. plasmid DNA was first complexed with platinum using ULYSIS DNA labeling kit and then incubated with Arg-∆Phe (as better Nps were obtained with Arg-∆Phe) for 30 min. Nps so formed were analyzed under TEM.
Cellular uptake of DNA-peptide Nps
For estimating the cellular uptake of DNA-peptide Nps, plasmid DNA and peptide Nps were labeled with Alexa-488 using ULYSIS DNA labeling kit (Sigma-Aldrich, Munich, Germany) and NHS-modified Alexa-588 dyes (Invitrogen, Life Technologies Corp. NY, USA) respectively. DNA-peptide Nps were prepared as described above. HeLa cells were first cultured in tissue culture dishes in RPMI-1640 media, trypsinized and seeded in 6 well plates (1 x 105 cells/well). After 12 hrs of culture, cells were incubated with plasmid DNA, peptide Nps, DNA-peptide Nps for 18 hrs, washed with serum and phenol red free media and imaged under fluorescence microscope.
Nuclear localization of DNA-peptide Nps
HeLa cells were seeded on cover slips placed in 6-well plates, allowed to grow for 12 hrs for cell adherence and spreading and subsequently incubated with DNA peptide Nps carrying Alexa-488 labeled DNA for 12 hrs. After this, media was removed, cells were treated with DAPI for nucleus staining, washed and visualized under fluorescent and bright-field microscopes.
Agarose gel electrophoresis of DNA peptide Nps
Complex formation between Nps and plasmid DNA was analyzed by 1% agarose gel electrophoresis. Free plasmid DNA (4 μg) and DNA peptide Nps complexes with peptide/DNA ratio of 20/1 (4 μg DNA/80 μg of peptide in 40 μl nuclease free water) were run on a 1% (w/v) agarose gel with TBE buffer (89 mmol/l Tris, 89 mmol/l boric acid, and 2 mmol/l EDTA pH 8.0) at 75 V for 90 minutes. DNA was visualized by staining gels with ethidium bromide (0.5 g/l) and images were acquired using an UV trans-illuminator (Vilber, Lourmat, France). Stability of DNA peptide Nps towards enzymatic degradation was determined by incubating plasmid DNA (4 μg) and DNA peptide Nps complexes with peptide/DNA ratio of 20/1 (4 μg DNA/80 μg of peptide in 40 μl nuclease free water) with 5 μl of DNase I solution (10 μg/ml in DNase/Mg2+ digestion buffer, which consisted of 50 mM Tris–HCl, pH 7.6, and 10 mM MgCl2) each at 37°C for 30 min, and degradation of plasmid DNA was analyzed by 1% agarose gel electrophoresis.
Cytotoxicity of DNA-peptide Nps
HeLa and L929 cells were cultured in RPMI-1640 and DMEM supplemented with 10% FBS, and maintained on TCTP plates at 37°C in a humified atmosphere containing 5% CO2, till 70-80% confluency. Cells were then harvested by trypsinization, washed and resuspended in RPMI-1640/DMEM medium (supplemented with 10% FBS). Live cells were counted by trypan blue dye exclusion test using a hemocytometer, diluted in completed media, seeded in 96 well plates (2 x 104 cells/well). After 12 hrs, cells were incubated with 20 μl each of dipeptide and DNA-dipeptide Nps (2 mg/ml) for a period of 24 hrs. As a control, cells were incubated with 20 μl of PBS. MTT assay was used to determine the cell viability of treated cells. The percentage viability was calculated using the equation given below:
Percentage viability of cells = [(Absorbance of MTT formazan produced by cells grown in presence of nps /Absorbance of MTT formazan produced by cells grown on TCTP control plate)] × 100.
In vitro transfection efficiency of DNA peptide Nps
HuH-7 cells were cultured in DMEM supplemented with 10% FBS and 1% pencillin-streptomycin and maintained on TCTP plates at 37°C in a humified atmosphere containing 5% CO2, till 70-80% confluency. 24 h prior to transfection cells were seeded in 6 well plates (2 x 105). DNA-Nps were prepared by incubating 4 μg of EGFP encoding plasmid DNA with 80 μg each of dipeptides Lys-∆Phe and Arg-∆Phe in 40 μl of nuclease free water. Nps so formed were visualized under TEM to determine particle size and morphology as described above. On the day of transfection, the culture medium in each well was replaced with 1 ml of complete medium containing 1 μg of DNA loaded in DNA-peptide Nps at (1: 200 DNA to peptide ratio) and 1 μg of DNA in lipofectamine. The cells were then incubated for 48 hrs, washed to remove excess Nps and DNA and analysed using fluorescence microscopy for GFP expression.
The results are presented as the mean ± SD calculated over at least three data points.
Results and discussion
Cationic polymer based systems have been widely used to condense DNA inside their core to protect it from intra-cellular nucleases and achieve efficient delivery [16, 20]. Arg-∆Phe and Lys-∆Phe, due to the presence of positive charges on them, were used to condense plasmid DNA of various sizes into discrete Nps. Plasmid DNAs were loaded on to dipeptide Nps either by incubating them with preformed Nps or by the formation of DNA-nanoparticle complexes by slow mixing of the aqueous solution of cationic dipeptides and plasmids.
From the above study, the optimum peptide to DNA ratio needed to form suitably compacted DNA-peptide Nps was found to be 20:1 (w/w). Entrapment efficiency determined from centrifugation experiments of the fluorescence labeled DNA showed almost 95% entrapment in both Arg-∆Phe and Lys-∆Phe Nps at this peptide to DNA ratio.
Cationic polymers condense DNA to yield compact Nps with well defined sizes [16, 24]. Nanoparticle formation was also observed when the cationic dipeptides were added to plasmid DNA. While plasmid DNA alone in water (100 μg/ml) formed irregular structures (no regular structure was found under TEM) with a mean Rh of 450 nm, it condensed into smaller and monodispersed Nps with the addition of increasing concentration of the cationic peptides. Nps with an average Rh of 120 nm were formed by the addition of 2 mg of Lys-∆Phe (Figure 2b) to the plasmid DNA. Thus, in this case nanoparticle formation occurred by the condensation of plasmid DNA in presence of cationic dipeptides and size of the resultant Nps depended on the amount of peptide added. Similar observation was seen in case of BSA-plasmid DNA polyplexes, where an increase in BSA/plasmid DNA mass ratio led to decrease in overall particle size of the resultant polyplexes . Likewise, addition of increasing concentration Arg-∆Phe also led to DNA condensation and decrease in overall particle size (Figure 2d). It was also observed that Arg-∆Phe led to DNA condensation at lower concentration as compared to Lys-∆Phe (250 μg of Arg-∆Phe vs 2000 μg Lys-∆Phe for same amount of DNA). Similar observation has been shown earlier where better DNA condensation was obtained with ariginine homopeptides than lysine ones . This could be explained by the difference in the nature of electrostatic interactions and DNA binding capacity between lysine and arginine residues . Where there is only one amine group in the lysine side chain guanidinium side chain of arginine has three amine groups. This promotes zwitterion hydrogen bonding of arginine with the phosphate group as well as guanine base of DNA, providing high binding strength and assembly than lysine .
Effect of DNA size on the formation of DNA-peptide Nps was determined by incubating cationic dipeptides with plasmids of various sizes. Lys-∆Phe formed particles with mean Rh of 70 nm with pl, 117 nm with pgem, 165 nm with dh, 171 with md, and 112 with sod (Additional file 2: Figure S1a). Similarly, Arg-∆Phe formed particles with Rh of 103 nm with pl, 134 nm with pgem, 280 nm with dh, 479 nm with md and 280 nm with sod (Additional file 2: Figure S1b). Thus it was observed that there was no correlation between the size of DNA-peptide Nps with plasmid length (in base pairs) (Additional file 2: Figure S1).
It was also observed that in most cases Nps formed by the combination of Arg-∆Phe with plasmid DNAs were more regular as compared to those formed by the Lys-∆Phe and plasmid DNAs (Figure 4). This correlates with our DLS results that showed better DNA condensation and assembly with Arg-∆Phe as compared to Lys-∆Phe.
A major hurdle in gene or siRNA delivery is the degradation of DNAs by intra and inter-cellular nucleases. Hence an ideal gene delivery vehicle should not only facilitate intracellular delivery of loaded DNAs but also protect them from degradation by inter or intra-cellular nucleases [32–34]. In order to test the stability of DNA-peptide Nps, Nps were digested with DNase I and analyzed using agarose gel electrophoresis. Free plasmid DNA was degraded into smaller fragments. However, DNA-peptide Nps digested by DNase I were retained around sample pore (Figure 6b). These results showed that DNA was largely protected from enzymatic degradation in the DNA-dipeptide Nps.
The work presented here showed that cationic dipeptides containing a chemically modified amino acid, ∆Phe, easily formed discrete nanoparticles with plasmid DNA of different lengths. Dipeptide nanoparticles protected plasmid DNA from enzymatic degradation and easily ferried them inside mammalian cells, both in nucleus and cytoplasm. GFP encoding plasmid DNA loaded Nps showed positive transfection and gene expression in HuH 7 cells. The dipeptides described here are easy to synthesize and also demonstrated no visible cytotoxic effect in tested mammalian cells. To conclude the study brings to light the potency of cationic dipeptide based systems as gene delivery vehicles and certainly adds new dimensions to non-viral based gene delivery.
Dynamic light scattering
Plasmid DNA of 5.2 kb [Topo 2.1 vector + mondehydroascorbate reductase gene]
Plasmid DNA of 6.1 kb [pET14b vector + dehydroascorbate reductase gene]
Plasmid DNA of 5.8 kb [pET14b + superoxide dismutase gene]
Plasmid DNA of 5.26 kb [pGreen + Rd2qA gene]
Plasmid DNA of 3.69 kb [PL12R34H]
Human cervical cancer
Human hepatocellular carcinoma
Bovine serum albumin
Fetal calf serum
tissue culture treated polystyrene
phosphate buffered saline
transmission electron microscopy
enhanced green fluorescent protein
Arginine-α, β-dehydrophenylalanine and Lys-∆Phe
This work is supported from a Nanoinitiative grant from Department of Science and Technology, India, Nanotechnology grant from Department of Biotechnology, India and core funding at ICGEB, New Delhi, India. JJP also thanks Unesco-loreal for women in science for fellowship.
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