WO2018173077A1 - Chemically modified cell-penetrating peptide for intracellular delivery of nucleic acids - Google Patents

Chemically modified cell-penetrating peptide for intracellular delivery of nucleic acids Download PDF

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WO2018173077A1
WO2018173077A1 PCT/IN2018/050172 IN2018050172W WO2018173077A1 WO 2018173077 A1 WO2018173077 A1 WO 2018173077A1 IN 2018050172 W IN2018050172 W IN 2018050172W WO 2018173077 A1 WO2018173077 A1 WO 2018173077A1
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imt
cell
delivery
cells
pdna
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PCT/IN2018/050172
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French (fr)
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Gajendra Pal Singh RAGHAVA
Ankur GAUTAM
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Council Of Scientific & Industrial Research
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • the present invention relates to a chemically modified cell-penetrating peptide for intracellular delivery of nucleic acid.
  • the present invention relates to a peptide- based system, which is suitable for intracellular delivery of nucleic acid.
  • the present system comprises of at least one component X, which is attached covalently to a cell-penetrating peptide Y.
  • a cell-penetrating peptide based vehicle is described which has the ability to deliver plasmid DNA into the cells without causing significant cell death.
  • the present invention will be useful to develop novel transfection reagent for the delivery of nucleic acid and could be useful for the development of peptide based nucleic acid delivery systems.
  • nucleic acid-based therapeutics e.g. plasmids, small interfering RNAs, short oligonucleotides, and their analogues
  • plasmids small interfering RNAs
  • short oligonucleotides short oligonucleotides
  • analogues e.g. a nucleic acid-based therapeutics
  • CPPs cell-penetrating peptides
  • CPPs appear to be very promising for the delivery of various nucleic acids based therapeutics; however, the entrapment of nucleic acid-CPP complexes into the vesicular compartments post endocytosis leading to the degradation in lysosome remains the major obstacle in their further progress into the clinics (17).
  • IMT-P8 (SEQ ID NO: l) that has already been reported (18).
  • IMT-P8 is arginine-rich peptide and derived from voltage-dependent L-type calcium channel subunit alpha- ID (18).
  • IMT-P8 is capable of internalizing into a variety of cells very efficiently through a rapid, dose-dependent process. Since IMT-P8 enters into the cells through endocytosis, complexes of IMT-P8 and nucleic acid supposed to be entrapped into the endosomes and this is the reason that despite its efficient cell-penetrating ability, DNA delivery using IMT-P8 is poor.
  • the main objective of the present invention is therefore to provide chemically modified cell- penetrating peptides for efficient delivery of nucleic acid into a cell which obviate the drawbacks of the hitherto reported prior art.
  • the present invention provides peptide-based system for efficient intracellular nucleic acid delivery which exhibits low toxicity, process of preparing the same and use thereof.
  • Conjugation of fatty acid/ lipid moieties to the cell penetrating peptide IMT-P8 represented by SEQ ID NO: l significantly improved its DNA delivery efficiency, which was further, enhanced in the presence of chloroquine.
  • DNA delivery efficiency of IMT-P8 was relatively poor. But DNA delivery efficiency significantly enhanced when stearic acid was conjugated to N-terminus of IMT-P8 (STR-IMT-P8).
  • GFP fluorescence protein (GFP) fluorescence was enhanced when HeLa cells were treated with chloroquine along with IMT-P8/ DNA and S TR-EVIT-P 8/DN A complexes.
  • the transfection efficiency was not higher than the commercially available transfection reagent Lipofectamine 2000 (LF 2000).
  • the present invention provides chemically modified cell-penetrating peptide represented by the general formula 1;
  • X represents a fatty acid moiety of length between 2 to 16 carbon atoms or more, covalently attached to the linker Z consisting of length between 2 to 6 carbon atoms or more, covalently attached to a 15 mer peptide A which comprises RRWRRW motif at N-terminus, and/or R/KXXR/K motif at C-terminus, and/or contains nine arginine/lysine residues and/or having 9 positively charged amino acids.
  • the present invention provides chemically modified cell-penetrating peptides represented by general formula 1;
  • X represents a fatty acid moiety of length between 2 to 16 carbon atoms, covalently attached to the linker Z consisting of length between 2 to 6 carbon atoms, covalently attached to the cell penetrating peptide comprising 15 amino acids represented by SEQ ID NO: l .
  • the SEQ ID NO: l represents a 15 mer peptide comprising the amino acids "RRWRRW RF RRRCR" denoted by IMT-P8.
  • the present invention provides a chemically modified cell- penetrating peptide characterized in that it comprises an amino acid sequence comprising XZRRWRRW RE RRRCR, wherein X represents a lipid moiety and Z represents a linker.
  • the present invention provides chemically modified cell-penetrating peptide represented by the general formula 1, useful for intracellular delivery of nucleic acids.
  • the present invention provides the 15 mer peptide represented by SEQ ID NO: 1, which contains "RRWRRW" motif at N-terminus and/or R/KXXR/K motif at C-terminus and/or contains nine arginine/ lysine residues and/or having 9 positively charged amino acids.
  • the present invention provides a molecule represented by general formula 1, wherein Z is a linker consisting of length between 2 to 6 carbon atoms or more, covalently attached to the cell penetrating peptide [CPP] A preferably represented by SEQ ID NO: l .
  • the present invention provides a molecule represented by general formula 1, wherein X is a fatty acid moiety of length between 2 to 16 carbon atoms, covalently attached to the linker Z.
  • the present invention provides a complex comprising a cell- penetrating peptide, wherein the cargo (plasmid DNA) is a nucleic acid non-covalently linked to the cell penetrating peptide represented by the general formula 1.
  • the present invention provides a nucleic acid non-covalently linked to the peptide A for intracellular delivery.
  • the present invention provides a process for the intracellular delivery of nucleic acids comprising the steps of:
  • step (b) incubating the cell penetrating peptide described in step (a) with target cells that is the cells which are to be transfected.
  • the present invention provides a process wherein the target cell is selected from the group comprising of a eukaryotic cell or prokaryotic cell.
  • the present invention provides a chemically modified cell- penetrating peptide stearyl-IMT-P8 (STR-IMT-P8), represented by general formula 1, which can deliver plasmid DNA into the cells without much toxicity and thus can serve as a potential lead candidate for further optimization in order to develop effective gene delivery vectors.
  • STR-IMT-P8 chemically modified cell- penetrating peptide stearyl-IMT-P8
  • FIGURE 1 Characterization of IMT-P8/ pDNA complexes.
  • A Agarose gel images of IMT-P8/ pDNA complexes made at different N/P ratio.
  • B Hydrodynamic diameter and
  • C zeta potential of IMT-P8/ pDNA complexes at different N/P ratio.
  • D TEM images of IMT- P8/ pDNA complexes (N/P ratio 9).
  • FIGURE 2 Cellular uptake of IMT-P8/ pDNA complexes as determined by FACS analysis. Overnight grown HeLa cells were treated with IMT-P8/ pDNA complexes at different N/P ratio (5-12) for 4 h, followed by incubation in complete medium for further 20 h. Subsequently, cells were washed with PBS, and trypsinized at 37°C for 10 min. Finally, cells were suspended in PBS, and subjected to flow cytometry. The uptake is measured as mean cellular fluorescence from the flow cytometric analysis of all live cells positive for GFP.
  • FIGURE 3 Characterization of STR-IMT-P8/ pDNA complexes.
  • A Agarose gel images of STR-IMT-P8/ pDNA complexes made at different N/P ratio.
  • B Hydrodynamic diameter and
  • C zeta potential of STR-IMT-P8/ pDNA complexes at different N/P ratio.
  • D TEM images of STR-IMT-P8/ pDNA complexes (N/P ratio 9).
  • FIGURE 4 Cellular uptake of STR-IMT-P8/ pDNA complexes as determined by FACS analysis. Overnight grown HeLa cells were treated with STR-FMT-P8/ pDNA complexes at different N/P ratio (0-12) for 4 h, followed by incubation in complete medium for further 20 h. Subsequently, cells were washed with PBS, and trypsinized at 37°C for 10 min. Finally, cells were suspended in PBS, and subjected to flow cytometry. The uptake is measured as mean cellular fluorescence from the flow cytometric analysis of all live cells positive for GFP. FIGURE 5. Intercellular fluorescence of GFP in HeLa cells.
  • HeLa cells were grown on cover slips and transfected with STR-IMT-P8/ pDNA complexes at different N/P ratio (0-12) for 4 h, followed by incubation in complete medium for further 20 h. Cells were then washed carefully twice with PBS and immediately observed (without fixation) by confocal fluorescence microscopy.
  • FIGURE 6 Effect of chloroquine on uptake of IMT-P8/ pDNA and STR-IMT-P8/pDNA complexes.
  • FIGURE 7 Cytotoxicity of peptides.
  • HeLa cells were incubated with IMT-P8/ pDNA and STR-FMT-P8/ pDNA complexes at N/P ratio 9. Also, cells were transfected with Lipofectamine 2000 (equal amount of pDNA i.e. 500 ng) as per the manufacturer's protocol. Cell viability was measured by MTT assay. Viability of control (without transfection) was taken as 100 % and viabilities of transfected cells were plotted as percentage of control.
  • Lipofectamine 2000 equal amount of pDNA i.e. 500 ng
  • HeLa cells are immortal human cell line used in scientific research.
  • molecule represented by general formula 1 refers to same type of molecule[s] and are used interchangeably throughout the specification.
  • a cell-penetrating peptide represented by SEQ ID NO: 1 comprising 15 amino acids and denoted as IMT-P8 which has already been reported in the inventors' own prior publication 3380DEL2013 is used for the purposes of the present invention.
  • the said peptide is capable of internalizing into a variety of human cancer cells very efficiently without causing significant membrane damage.
  • the cargo delivery capability of IMT-P8 was studied (19).
  • IMT-P8 delivered the Green fluorescent protein (GFP) and pro-apoptotic peptide, KLA into human cancer cells.
  • GFP Green fluorescent protein
  • KLA pro-apoptotic peptide
  • the nucleic acid delivery capability of IMT-P8 is not yet studied. Therefore, the main aim of the present invention was to examine the nucleic acid delivery capability of IMT-P8.
  • IMT-P8 in order to determine the suitability of IMT-P8 as nucleic acid carrier, first of all it was assessed whether IMT-P8 can condense plasmid DNA and can form nanocomplexes. To address this, IMT-P8 and pDNA was mixed at different ratios (increasing N/P ratios) and agarose gel retardation assay was performed with IMT-P8/ pDNA mixture and results of this assay confirmed that IMT-P8 could condense plasmid DNA and formed nano complexes at higher charge ratios (CRs) as shift in the mobility of the pDNA was observed at higher CRs. The results of dynamic light scattering and transmission electron microscopy also supported this and confirm that IMT-P8 form nanocomplexes with pDNA.
  • CRs charge ratios
  • IMT-P8 can deliver the plasmid DNA into the cytoplasm.
  • IMT-P8/pDNA complexes were made and incubated with the HeLa cells.
  • EVIT-P8/ pDNA complexes no GFP fluorescence was observed inside the HeLa cells.
  • the GFP intensity was equal to untreated control cells suggesting that IMT-P8 could not deliver plasmid DNA successfully into the cytoplasm. This could be due to the possibility that IMT-P8/pDNA internalized into the cells by endocytosis and then entrapped into the endosomes and could not escape from the endosomes.
  • EVIT-P8 mediated plasmid DNA delivery in the presence of chloroquinine was performed, which is known to facilitate destabilization of endosomes and subsequent release of entrapped biomolecules (20).
  • IMT-P8 efficiently condensed the plasmid DNA, but when transfection was performed with EVIT-P8/pDNA complexes in HeLa cells in the presence of chloroquine, only slight increase in GFP fluorescence intensity was observed compared to cells treated without chloroqunine suggesting that only small amounts of EVIT-P8/pDNA complexes were internalizedinto the cells.
  • the effect of chloroquine was more pronounced with STR-IMT- P8, demonstrating significant increase in GFP fluorescence intensity after treatment with chloroquine.
  • Transfection using cationic lipid formulations is the most common and efficient way to introduce plasmid DNA into the cells.
  • Many transfection reagents are commercially available for the delivery of nucleic acids into the mammalian cells.
  • Lipofectamine 2000 (LF 2000) is one of the most widely used transfection reagent, which consists of is lipid-based formulations. Therefore, the transfection efficiency of STR-IMT-P8/chloroquine co treatment was compared with the commercially available transfection reagent LF 2000.
  • LF2000 The efficiency of LF2000 was superior to that of plasmid delivery by STR-IMT-P8 and chloroquine treatment
  • cytotoxicity caused by LF2000 was significantly high compared to STR-FMT-P8
  • the viability was reduced to 65% in case of LF2000 whereas more than 85% cells were viable when treated with STR-IMT-P8 with chloroquine.
  • STR-IMT-P8 can deliver plasmid DNA in the eukaryotic cells with less cytotoxicity and it could be a potential lead molecule for future nucleic acid delivery investigations.
  • Both CPPs, IMT-P8 and STR-IMT-P8 were synthesized by solid phase peptide synthesis strategy using Fmoc (N-(9-fluronyl)-methoxycarbonyl) chemistry in O.Olmmole scale on a Protein Technologies Inc, USA, PS-3 peptide synthesizer as described in reference 3380DEL2013.
  • Stearic acid was conjugated to N-terminus of IMT-P8 with an amino- hexanoic acid (Ahx) linker.
  • HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10%) fetal bovine serum (FBS) and 1%> penicillin/streptomycin antibiotics and were maintained at 37°C in humidified 5% C0 2 atmosphere.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • penicillin/streptomycin antibiotics were maintained at 37°C in humidified 5% C0 2 atmosphere.
  • Plasmid DNA and CPPs (IMT-P8 and STR-IMT-P8) binary complexes were prepared as follows: 500 ng pEGFP-Nl plasmid DNA expressing GFP, expressing eGFP, was mixed with IMT-P8 and STR-IMT-P8 in order to obtained desired N:P ratios (1 to 12) in Milli-Q water in 50 ⁇ (1/lOth of the final treatment volume). N:P ratio that is the the ratios of moles of the amine groups of peptide to those of the phosphate ones of DNA were calculated theoretically, taking into account the positive charges of the peptide and negative charges of the plasmid. The mixture was kept undisturbed for 1 h at room temperature to allow formation of stable complex.
  • the stabilities of CPPs (IMT-P8 and STR-IMT-P8)/plasmid DNA binary complexes with different N:P ratio were evaluated by agarose gel electrophoresis assay.
  • Binary complexes of CPPs (IMT-P8 and STR-IMT-P8) and pDNA were prepared at N/P ratios ranging from 0 (DNA only) to 12 as described above.
  • Complexes were electrophoresed on a 1% agarose gel in 1 x Tris-acetate-EDTA (TAE) buffer at 90 V for 40 min. Images were captured using the alphalmager HP System.
  • Physiochemical properties like hydrodynamic mean diameter (size) and ⁇ (zeta)-Potential (surface charge) of the CPP (IMT-P8 and STR-IMT-P8)/pDNA nanocomplexes was determined by DLS studies using a Zetasizer Nano ZS apparatus (Malvern Instruments, Worcester, UK). CPPs/pDNA complexes resulting from the addition of IMT-P8 and STR- IMT-P8 were formulated according to the protocol, as described above, and assessed in disposable low volume cuvettes. Data were represented as mean ⁇ S.D.
  • IMT-P8/pDNA and STR-FMT-P8/ pDNA complexes were formed at N/P ratio 9 as described above. Complexes were air-dried on copper grids coated with carbon film. Transmission electron microscopy (TEM) images of complexes were obtained using a JEOL JEM 2100 transmission electron microscope.
  • TEM Transmission electron microscopy
  • HeLa cells Q x lO 5 were seeded 24 h prior to experiments onto 24-well plates.
  • Cells were treated with IMT-P8/pDNA and STR-IMT-P8/pDNA complexes at different charge ratios (CRs) and incubated for 4 h in serum-free Opti-MEM® media (Gibco, Invitrogen). After 4h, medium was replaced with fresh complete medium and cells were allowed to grow further 20 h.
  • Opti-MEM® media Gibco, Invitrogen
  • LF2000 Lipofectamine 2000
  • the complexes were formed and transfection was performed according to the manufacturer's protocol.
  • HeLa cells (1 x 10 5 cells) were seeded onto 12- well plates containing 16 mm glass coverslips, 24 h prior to the start of the experiment. After complete adhesion, cells were treated with IMT-P8/pDNA and STR-IMT-P8/pDNA complexes at different N/P ratios for 4 h in Opti-MEM. Thereafter, medium was replaced with fresh complete medium and incubated for another 20 h. Subsequently, culture medium was removed and coverslips were washed thoroughly with PBS (3x, 2 minutes) and mounted on glass slides with antifade reagent (Gibco, Invitrogen). GFP fluorescence in the live cells was analyzed immediately using Nikon AIR confocal microscope.
  • chloroquine In order to determine the effect of chloroquine on plasmid DNA delivery, flow cytometry analysis was performed with chloroquine. For this, after complex formation and before treatment of cells, chloroquine (final concentration 100 ⁇ ) was added to the peptide/ pDNAcomplex solution. Four hours after addition of the complexes and chloroquine to cells, cell medium was replaced with fresh medium in order to avoid toxicity effects. Thereafter, cells were washed and FACS was performed as described above.
  • solublization reagent 2% SDS in 50% DMF
  • solublization reagent 2% SDS in 50% DMF
  • absorbance was taken at 570 nm with a microplate reader (Tecane).
  • the survival of cells relative to the control was calculated by taking the ratio of the absorbance at 570 nm values. All the experiments were performed in triplicates.
  • IMT-P8 In order to determine whether or not IMT-P8 is capable of forming nano complexes with plasmid DNA, agarose gel retardation assay was performed with binary complexes (IMT-P8/ pDNA). It was found that EVIT-P8/pDNA complexes were progressively stabilized by increasing N/P ratios as mobility of plasmid DNA was reduced with increasing N/P ratios ( Figure 1 A). These results suggested that IMT-P8 was able to condense and form complexes with plasmid DNA. The hydrodynamic diameter and surface charge (zeta potential) of these nanoparticles was determined by using dynamic light scattering (DLS). The results are shown in Figure IB.
  • DLS dynamic light scattering
  • Figure 3A demonstrates that similar to IMT-P8, STR-IMT-P8 also condense and form complexes with plasmid DNA.
  • STR-EVIT-P8/ pDNA complexes were found to be stable as significant retardation in pDNA mobility was observed on 1% agarose gel ( Figure 3A). These complexes were almost immobilized and no bands were detected corresponding to position of naked pDNA.
  • DLS experiments suggested that the size of STR-EVIT-P8/ pDNA complexes was in the range of 60-120 nm ( Figure 3B), which was smaller than the size of IMT-P8/pDNA complexes.
  • MTT assay was performed with HeLa cells after co-incubation of peptide /pDNA complexes and chloroquine. Cytotoxicity caused by LF2000 was also compared. As shown in Figure 7, in case of EVIT-P8/ pDNA complexes, around 90% cells are viable while in case of STR-EVIT-P8/ pDNA, around 85% cells are viable. In case of LF2000, significant cytotoxicity was observed compared to EVIT-P8 and STR-IMT-P8 and around only 65% cells were viable. ADVANTAGES OF THE INVENTION
  • the chemically modified cell penetrating peptide STR-IMT-P8 (molecule represented by general formula 1) shows higher plasmid DNA delivery capability than the wild type IMT-P8 (SEQ ID NO: 1) in HeLa cells.

Abstract

The present invention relates to chemically modified cell-penetrating peptides for efficient intracellular nucleic acid delivery which exhibits low toxicity, process of preparing the same and use thereof. The present invention specifically relates to a peptide-based nucleic acid delivery into the cell. Conjugation of fatty acid/ lipid moieties to the cell penetrating peptide IMT-P8 represented by SEQ ID NO: 1 significantly improved its DNA delivery efficiency, which was further, enhanced in the presence of chloroquine.

Description

CHEMICALLY MODIFIED CELL-PENETRATING PEPTIDE FOR INTRACELLULAR DELIVERY OF NUCLEIC ACIDS FIELD OF THE INVENTION
The present invention relates to a chemically modified cell-penetrating peptide for intracellular delivery of nucleic acid. In particular, the present invention relates to a peptide- based system, which is suitable for intracellular delivery of nucleic acid. The present system comprises of at least one component X, which is attached covalently to a cell-penetrating peptide Y. Being negatively charged, nucleic acid based therapeutics cannot be internalized into the cell by their own. Therefore, intracellular delivery of nucleic acid based therapeutics remains a big challenge. In the present invention, a cell-penetrating peptide based vehicle is described which has the ability to deliver plasmid DNA into the cells without causing significant cell death. The present invention will be useful to develop novel transfection reagent for the delivery of nucleic acid and could be useful for the development of peptide based nucleic acid delivery systems.
BACKGROUND OF THE INVENTION
Past few decades have witnessed a considerable progress in gene therapy field. Plethora of nucleic acid-based therapeutics (e.g. plasmids, small interfering RNAs, short oligonucleotides, and their analogues) have been developed over the decades that have the potential to be applied in numerous pathological conditions, including cancer (1-2). However, despite the immense pharmacological potential, only a handful of such molecules have made it to the clinic so far (3). The major obstacles in their in vivo clinical applications are the low bioavailability and poor intracellular delivery (4).
Successful gene therapy requires efficient cellular uptake and subsequently release of nucleic acid to their target site where they can exert their effect. Being negatively charged macromolecules, nucleic acid based therapeutics cannot cross the plasma membrane barrier by their own. Thus, their cellular uptake and subsequent intracellular trafficking of these macromolecules to their target sites remains a big challenge (5). Therefore, development of efficient delivery systems, which are capable of delivering these molecules to the right place into the cell where they are most effective and can exert their therapeutic effect, is the need of the hour. Considerable efforts have been made in this direction to achieve this goal (6-8). In order to improve the delivery of oligonucleotides, many viral (9) and non-viral delivery (10) systems have been developed over the years, but most of these delivery systems have some limitations like high toxicity, undesirable side effects and low delivery yield. Among the non-viral delivery systems, classes of small peptides known as cell-penetrating peptides (CPPs) have recently gained increasing attention as efficient delivery vectors to bypass the problem of poor membrane permeability of these charged macromolecules (11).
To date, plethora of studies have reported the successful intracellular delivery of variety of therapeutic molecules e.g., proteins (12), peptides (13), small molecules (14), plasmid DNA (15), nanoparticles (16), etc. using CPPs both in vitro and in vivo, which makes CPP a versatile delivery system. CPPs appear to be very promising for the delivery of various nucleic acids based therapeutics; however, the entrapment of nucleic acid-CPP complexes into the vesicular compartments post endocytosis leading to the degradation in lysosome remains the major obstacle in their further progress into the clinics (17).
Based on these findings, the present invention explores the plasmid DNA delivery capability of the cell-penetrating peptide, IMT-P8 (SEQ ID NO: l) that has already been reported (18). IMT-P8 is arginine-rich peptide and derived from voltage-dependent L-type calcium channel subunit alpha- ID (18). IMT-P8 is capable of internalizing into a variety of cells very efficiently through a rapid, dose-dependent process. Since IMT-P8 enters into the cells through endocytosis, complexes of IMT-P8 and nucleic acid supposed to be entrapped into the endosomes and this is the reason that despite its efficient cell-penetrating ability, DNA delivery using IMT-P8 is poor.
Therefore, keeping in view the drawbacks of the hitherto reported prior art, the inventors of the present invention realized that there exists a dire need to chemically modify the CPPs such that their endosomal escape property and thus intracellular DNA delivery efficiency is improved. OBJECTIVES OF THE INVENTION
The main objective of the present invention is therefore to provide chemically modified cell- penetrating peptides for efficient delivery of nucleic acid into a cell which obviate the drawbacks of the hitherto reported prior art.
Another objective of the present invention is to provide chemically modified cell-penetrating peptides which exhibit low toxicity thereby delivering the nucleic acids intracellularly without causing significant cell death. Yet another objective of the present invention is to provide novel transfection reagents for the intracellular delivery of nucleic acids which could be useful for the development of peptidebased delivery systems.
SUMMARY OF THE INVENTION
The present invention provides peptide-based system for efficient intracellular nucleic acid delivery which exhibits low toxicity, process of preparing the same and use thereof. Conjugation of fatty acid/ lipid moieties to the cell penetrating peptide IMT-P8 represented by SEQ ID NO: l significantly improved its DNA delivery efficiency, which was further, enhanced in the presence of chloroquine. In the present invention, we have shown that DNA delivery efficiency of IMT-P8 was relatively poor. But DNA delivery efficiency significantly enhanced when stearic acid was conjugated to N-terminus of IMT-P8 (STR-IMT-P8). Flow cytometry and confocal laser scanning microscopy results demonstrated an enhanced green fluorescence protein (GFP) fluorescence inside the HeLa cells transfected with STR-IMT-P8/ pDNA (pEGFP-Nl) complexes compared to cells transfected with IMT-P8/pDNA complexes. In addition, GFP fluorescence was further enhanced when HeLa cells were treated with chloroquine along with IMT-P8/ DNA and S TR-EVIT-P 8/DN A complexes. However, the transfection efficiency was not higher than the commercially available transfection reagent Lipofectamine 2000 (LF 2000). Interestingly, the cytotoxicity of EVIT-P8/ DNA and STR-IMT-P8/ DNA complexes were significantly low compared to Lipofectamine 2000. In conclusion, it can be suggested that stearylation of IMT-P8 improves the endosomal escape of IMT-P8/ pDNA complexes and released the plasmid DNA into the cytoplasm without causing significant toxicity. Thus, STR-IMT-P8 could serve as an effective lead molecule for future gene delivery investigations.
In an embodiment, the present invention provides chemically modified cell-penetrating peptide represented by the general formula 1;
X-Z-A
general formula 1 wherein, X represents a fatty acid moiety of length between 2 to 16 carbon atoms or more, covalently attached to the linker Z consisting of length between 2 to 6 carbon atoms or more, covalently attached to a 15 mer peptide A which comprises RRWRRW motif at N-terminus, and/or R/KXXR/K motif at C-terminus, and/or contains nine arginine/lysine residues and/or having 9 positively charged amino acids. In another embodiment, the present invention provides chemically modified cell-penetrating peptides represented by general formula 1;
X-Z-SEQ ID NO:l
general formula 1
wherein, X represents a fatty acid moiety of length between 2 to 16 carbon atoms, covalently attached to the linker Z consisting of length between 2 to 6 carbon atoms, covalently attached to the cell penetrating peptide comprising 15 amino acids represented by SEQ ID NO: l .
In yet another embodiment of the present invention, the SEQ ID NO: l represents a 15 mer peptide comprising the amino acids "RRWRRW RF RRRCR" denoted by IMT-P8.
In still another embodiment, the present invention provides a chemically modified cell- penetrating peptide characterized in that it comprises an amino acid sequence comprising XZRRWRRW RE RRRCR, wherein X represents a lipid moiety and Z represents a linker. In another embodiment, the present invention provides chemically modified cell-penetrating peptide represented by the general formula 1, useful for intracellular delivery of nucleic acids. In yet another embodiment, the present invention provides the 15 mer peptide represented by SEQ ID NO: 1, which contains "RRWRRW" motif at N-terminus and/or R/KXXR/K motif at C-terminus and/or contains nine arginine/ lysine residues and/or having 9 positively charged amino acids.
In yet another embodiment, the present invention provides a molecule represented by general formula 1, wherein Z is a linker consisting of length between 2 to 6 carbon atoms or more, covalently attached to the cell penetrating peptide [CPP] A preferably represented by SEQ ID NO: l .
In still another embodiment, the present invention provides a molecule represented by general formula 1, wherein X is a fatty acid moiety of length between 2 to 16 carbon atoms, covalently attached to the linker Z. In yet another embodiment, the present invention provides a complex comprising a cell- penetrating peptide, wherein the cargo (plasmid DNA) is a nucleic acid non-covalently linked to the cell penetrating peptide represented by the general formula 1.
In another embodiment, the present invention provides a nucleic acid non-covalently linked to the peptide A for intracellular delivery.
In still another embodiment, the present invention provides a process for the intracellular delivery of nucleic acids comprising the steps of:
(a) providing a chemically modified cell penetrating peptide represented by the general formula 1, and
(b) incubating the cell penetrating peptide described in step (a) with target cells that is the cells which are to be transfected.
In yet another embodiment, the present invention provides a process wherein the target cell is selected from the group comprising of a eukaryotic cell or prokaryotic cell.
In yet another embodiment, the present invention provides a chemically modified cell- penetrating peptide stearyl-IMT-P8 (STR-IMT-P8), represented by general formula 1, which can deliver plasmid DNA into the cells without much toxicity and thus can serve as a potential lead candidate for further optimization in order to develop effective gene delivery vectors.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
FIGURE 1. Characterization of IMT-P8/ pDNA complexes. (A) Agarose gel images of IMT-P8/ pDNA complexes made at different N/P ratio. (B) Hydrodynamic diameter and (C) zeta potential of IMT-P8/ pDNA complexes at different N/P ratio. (D) TEM images of IMT- P8/ pDNA complexes (N/P ratio 9).
FIGURE 2. Cellular uptake of IMT-P8/ pDNA complexes as determined by FACS analysis. Overnight grown HeLa cells were treated with IMT-P8/ pDNA complexes at different N/P ratio (5-12) for 4 h, followed by incubation in complete medium for further 20 h. Subsequently, cells were washed with PBS, and trypsinized at 37°C for 10 min. Finally, cells were suspended in PBS, and subjected to flow cytometry. The uptake is measured as mean cellular fluorescence from the flow cytometric analysis of all live cells positive for GFP.
FIGURE 3. Characterization of STR-IMT-P8/ pDNA complexes. (A) Agarose gel images of STR-IMT-P8/ pDNA complexes made at different N/P ratio. (B) Hydrodynamic diameter and (C) zeta potential of STR-IMT-P8/ pDNA complexes at different N/P ratio. (D) TEM images of STR-IMT-P8/ pDNA complexes (N/P ratio 9).
FIGURE 4. Cellular uptake of STR-IMT-P8/ pDNA complexes as determined by FACS analysis. Overnight grown HeLa cells were treated with STR-FMT-P8/ pDNA complexes at different N/P ratio (0-12) for 4 h, followed by incubation in complete medium for further 20 h. Subsequently, cells were washed with PBS, and trypsinized at 37°C for 10 min. Finally, cells were suspended in PBS, and subjected to flow cytometry. The uptake is measured as mean cellular fluorescence from the flow cytometric analysis of all live cells positive for GFP. FIGURE 5. Intercellular fluorescence of GFP in HeLa cells.
HeLa cells were grown on cover slips and transfected with STR-IMT-P8/ pDNA complexes at different N/P ratio (0-12) for 4 h, followed by incubation in complete medium for further 20 h. Cells were then washed carefully twice with PBS and immediately observed (without fixation) by confocal fluorescence microscopy.
FIGURE 6. Effect of chloroquine on uptake of IMT-P8/ pDNA and STR-IMT-P8/pDNA complexes.
Overnight grown HeLa cells were co-treated with (A) IMT-P8/ pDNA complexes, (B) STR- IMT-P8/ pDNA complexes and chloroquine (100 μΜ) at N/P ratio 9 for 4 h, followed by incubation in complete medium for further 20 h. Also, cells were transfected with Lipofectamine 2000 as per the manufacturer's protocol. Thereafter, medium was removed, cells were washed with PBS, and trypsinized at 37°C for 10 min. Finally cells were suspended in PBS, and subjected to flow cytometry. The uptake is measured as mean cellular fluorescence from the flow cytometric analysis of all live cells positive for GFP. (C) The transfection efficiency of STR-IMT-P8 (at N/P ratio 9) was compared with Lipofectamin 2000.
FIGURE 7. Cytotoxicity of peptides.
HeLa cells were incubated with IMT-P8/ pDNA and STR-FMT-P8/ pDNA complexes at N/P ratio 9. Also, cells were transfected with Lipofectamine 2000 (equal amount of pDNA i.e. 500 ng) as per the manufacturer's protocol. Cell viability was measured by MTT assay. Viability of control (without transfection) was taken as 100 % and viabilities of transfected cells were plotted as percentage of control.
DETAILED DESCRIPTION OF THE INVENTION LIST OF ABBREVIATIONS USED IN THE INVENTION
Figure imgf000009_0001
LF2000 Lipofectamin 2000
TEM Transmission electron microscopy
DLS Dynamic light scattering
CRs Charge ratios
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide pDNA Plasmid DNA
FBS fetal bovine serum
PBS Phosphate buffered saline
SDS Sodium dodecyl sulfate
DMF Dimethylformamide
DETAILS OF BIOMATERIALS USED IN THE INVENTION
Reagent Source
IMT-P8 Chemically Synthesized at CSIR-IMTECH
STR-FMT-P8 Chemically Synthesized at CSIR-IMTECH
LF2000 Invitrogen
Opti-MEM® media Gib co, Invitrogen
DMEM media Gib co, Invitrogen
FBS Gib co, Invitrogen
Penicillin, Gib co, Invitrogen
streptomycin
Phosphate buffered Gib co, Invitrogen
saline
Antifade reagent Gib co, Invitrogen
HeLa cells They are immortal human cell line used in scientific research.
Kind gift from Dr. Amit Tuli's laboratory, Sr. scientist, CSIR- IMTECH.
Agarose Invitrogen
MTT Sigma Aldrich
Tris Sigma Aldrich
EDTA Sigma Aldrich
Stearic acid Sigma Aldrich
DMF Sigma Aldrich Chloroquine Sigma Aldrich
The terms "molecule represented by general formula 1", "chemically modified cell- penetrating peptides represented by general formula 1", "stearyl-IMT-P8", "stearyl-SEQ ID No. l", "stearyl-linker-SEQ ID NO: l" refer to same type of molecule[s] and are used interchangeably throughout the specification.
A cell-penetrating peptide represented by SEQ ID NO: 1 comprising 15 amino acids and denoted as IMT-P8 which has already been reported in the inventors' own prior publication 3380DEL2013 is used for the purposes of the present invention. The said peptide is capable of internalizing into a variety of human cancer cells very efficiently without causing significant membrane damage. In a recent study, the cargo delivery capability of IMT-P8 was studied (19). In that study, it was shown that IMT-P8 delivered the Green fluorescent protein (GFP) and pro-apoptotic peptide, KLA into human cancer cells. However, the nucleic acid delivery capability of IMT-P8 is not yet studied. Therefore, the main aim of the present invention was to examine the nucleic acid delivery capability of IMT-P8.
In order to determine the suitability of IMT-P8 as nucleic acid carrier, first of all it was assessed whether IMT-P8 can condense plasmid DNA and can form nanocomplexes. To address this, IMT-P8 and pDNA was mixed at different ratios (increasing N/P ratios) and agarose gel retardation assay was performed with IMT-P8/ pDNA mixture and results of this assay confirmed that IMT-P8 could condense plasmid DNA and formed nano complexes at higher charge ratios (CRs) as shift in the mobility of the pDNA was observed at higher CRs. The results of dynamic light scattering and transmission electron microscopy also supported this and confirm that IMT-P8 form nanocomplexes with pDNA.
Next, it was examined whether IMT-P8 can deliver the plasmid DNA into the cytoplasm. For this, IMT-P8/pDNA complexes were made and incubated with the HeLa cells. Unfortunately, after addition of EVIT-P8/ pDNA complexes, no GFP fluorescence was observed inside the HeLa cells. The GFP intensity was equal to untreated control cells suggesting that IMT-P8 could not deliver plasmid DNA successfully into the cytoplasm. This could be due to the possibility that IMT-P8/pDNA internalized into the cells by endocytosis and then entrapped into the endosomes and could not escape from the endosomes. Similar results were also reported earlier for plasmid delivery by several unmodified CPPs like oligoarginine, TP- 10 where poor delivery of plasmid DNA was reported due to the endosomal entrapment of CPP/pDNA complexes (20). Therefore, to overcome the limitations of endosomal entrapment, the inventors of the present invention sought to conjugate the hydrophobic stearic acid moiety to the N-terminus of IMT- P8 and its effect on plasmid delivery was evaluated.
Agarose gel retardation assays with STR-IMT-P8/pDNA mixtures demonstrated that similar to IMT-P8, STR-IMT-P8 also condensed the plasmid DNA and formed nanocomplexes, which are stabilized by increasing CRs. These results were further confirmed by DLS measurements and TEM analysis. Additional experiments pertaining to plasmid delivery using STR-IMT-P8 revealed a significant increase in GFP fluorescence intensity in HeLa cells suggesting that STR-IMT-P8 could deliver the plasmid DNA into the HeLa cells. This increase in GFP fluorescence intensity in case of plasmid delivery using STR-IMT-P8 as compared to unmodified IMT-P8 could be due to the increased escape of STR-IMT- P8/pDNA complexes from endosomes due the stearic acid moiety. Similar effect of stearic acid was reported for stearylated TP- 10 and many other CPPs like (RxR)4 (21). Next, in order to further confirm that poor delivery of plasmid DNA by unmodified IMT-P8 was due to the entrapment of EVIT-P8/pDNA complexes into endosomes, EVIT-P8 mediated plasmid DNA delivery in the presence of chloroquinine was performed, which is known to facilitate destabilization of endosomes and subsequent release of entrapped biomolecules (20). Interestingly, IMT-P8 efficiently condensed the plasmid DNA, but when transfection was performed with EVIT-P8/pDNA complexes in HeLa cells in the presence of chloroquine, only slight increase in GFP fluorescence intensity was observed compared to cells treated without chloroqunine suggesting that only small amounts of EVIT-P8/pDNA complexes were internalizedinto the cells. The effect of chloroquine was more pronounced with STR-IMT- P8, demonstrating significant increase in GFP fluorescence intensity after treatment with chloroquine. These results suggested thatsimilar to EVIT-P8, IMT-P8/pDNA (a small fraction) and STR-IMT-P8/pDNA complexes were also internalized into the HeLa cells by endocytosis and after internalization, these complexes were entrapped into the endosomes resulting in poor delivery of plasmid DNA particularly in case of EVLT-P8. In case of STR-IMT-P8/pDNA complexes, the substantial increase in GFP fluorescence intensity (compared to IMT-P8/pDNA) with chloroquine treatment demonstrates that addition of stearic acid to N-terminus of IMT-P8 promotes the endosomal escape and thus eventually delivery of pDNA into the cytoplasm. However, it could be possible that a fraction of entrapped STR-IMT-P8/pDNA still remains in endosomal compartments.
Transfection using cationic lipid formulations is the most common and efficient way to introduce plasmid DNA into the cells. Many transfection reagents are commercially available for the delivery of nucleic acids into the mammalian cells. Lipofectamine 2000 (LF 2000) is one of the most widely used transfection reagent, which consists of is lipid-based formulations. Therefore, the transfection efficiency of STR-IMT-P8/chloroquine co treatment was compared with the commercially available transfection reagent LF 2000. The efficiency of LF2000 was superior to that of plasmid delivery by STR-IMT-P8 and chloroquine treatment However, at the same time, the cytotoxicity caused by LF2000 was significantly high compared to STR-FMT-P8, The viability was reduced to 65% in case of LF2000 whereas more than 85% cells were viable when treated with STR-IMT-P8 with chloroquine. Taken all results together, it can be suggested that STR-IMT-P8 can deliver plasmid DNA in the eukaryotic cells with less cytotoxicity and it could be a potential lead molecule for future nucleic acid delivery investigations.
Peptide synthesis
Both CPPs, IMT-P8 and STR-IMT-P8 were synthesized by solid phase peptide synthesis strategy using Fmoc (N-(9-fluronyl)-methoxycarbonyl) chemistry in O.Olmmole scale on a Protein Technologies Inc, USA, PS-3 peptide synthesizer as described in reference 3380DEL2013. Stearic acid was conjugated to N-terminus of IMT-P8 with an amino- hexanoic acid (Ahx) linker.
Cell culture
HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10%) fetal bovine serum (FBS) and 1%> penicillin/streptomycin antibiotics and were maintained at 37°C in humidified 5% C02 atmosphere. Peptide/ DNA complex formation
Plasmid DNA and CPPs (IMT-P8 and STR-IMT-P8) binary complexes were prepared as follows: 500 ng pEGFP-Nl plasmid DNA expressing GFP, expressing eGFP, was mixed with IMT-P8 and STR-IMT-P8 in order to obtained desired N:P ratios (1 to 12) in Milli-Q water in 50 μΐ (1/lOth of the final treatment volume). N:P ratio that is the the ratios of moles of the amine groups of peptide to those of the phosphate ones of DNA were calculated theoretically, taking into account the positive charges of the peptide and negative charges of the plasmid. The mixture was kept undisturbed for 1 h at room temperature to allow formation of stable complex.
Gel retardation assay
The stabilities of CPPs (IMT-P8 and STR-IMT-P8)/plasmid DNA binary complexes with different N:P ratio were evaluated by agarose gel electrophoresis assay. Binary complexes of CPPs (IMT-P8 and STR-IMT-P8) and pDNA were prepared at N/P ratios ranging from 0 (DNA only) to 12 as described above. Complexes were electrophoresed on a 1% agarose gel in 1 x Tris-acetate-EDTA (TAE) buffer at 90 V for 40 min. Images were captured using the alphalmager HP System.
Dynamic light scattering (DLS) measurements
Physiochemical properties like hydrodynamic mean diameter (size) and ζ (zeta)-Potential (surface charge) of the CPP (IMT-P8 and STR-IMT-P8)/pDNA nanocomplexes was determined by DLS studies using a Zetasizer Nano ZS apparatus (Malvern Instruments, Worcester, UK). CPPs/pDNA complexes resulting from the addition of IMT-P8 and STR- IMT-P8 were formulated according to the protocol, as described above, and assessed in disposable low volume cuvettes. Data were represented as mean ± S.D.
Electron Microscopy
IMT-P8/pDNA and STR-FMT-P8/ pDNA complexes were formed at N/P ratio 9 as described above. Complexes were air-dried on copper grids coated with carbon film. Transmission electron microscopy (TEM) images of complexes were obtained using a JEOL JEM 2100 transmission electron microscope.
In vitro DNA transfection
HeLa cells Q x lO5) were seeded 24 h prior to experiments onto 24-well plates. Cells were treated with IMT-P8/pDNA and STR-IMT-P8/pDNA complexes at different charge ratios (CRs) and incubated for 4 h in serum-free Opti-MEM® media (Gibco, Invitrogen). After 4h, medium was replaced with fresh complete medium and cells were allowed to grow further 20 h. In case of Lipofectamine 2000 (LF2000) (Invitrogen, Carlsbad, CA), the complexes were formed and transfection was performed according to the manufacturer's protocol. After this treatment, medium was removed and HeLa cells were washed twice with phosphate buffered saline (PBS) and detached from the plate by trypsinization for 10 min at 37 °C. Cells were suspended in ice-cold PBS containing 5% FBS and centrifuged (900 xg for 5 min). Supernatant was removed and the resulting cell pellet was resuspended in ice-cold PBS. Green fluorescence protein (GFP) intensity in live cells was measured by flow cytometry using Accuri C6 flow cytometer (BD Biosciences) by acquiring 10,000 live cells. Data were obtained and analyzed using CFlow Sampler (BD Biosciences).
Confocal Laser Scanning Microscopy
In order to visualize the GFP fluorescence, HeLa cells (1 x 105 cells) were seeded onto 12- well plates containing 16 mm glass coverslips, 24 h prior to the start of the experiment. After complete adhesion, cells were treated with IMT-P8/pDNA and STR-IMT-P8/pDNA complexes at different N/P ratios for 4 h in Opti-MEM. Thereafter, medium was replaced with fresh complete medium and incubated for another 20 h. Subsequently, culture medium was removed and coverslips were washed thoroughly with PBS (3x, 2 minutes) and mounted on glass slides with antifade reagent (Gibco, Invitrogen). GFP fluorescence in the live cells was analyzed immediately using Nikon AIR confocal microscope.
Effect of chloroquine on plasmid DNA delivery using peptides
In order to determine the effect of chloroquine on plasmid DNA delivery, flow cytometry analysis was performed with chloroquine. For this, after complex formation and before treatment of cells, chloroquine (final concentration 100 μΜ) was added to the peptide/ pDNAcomplex solution. Four hours after addition of the complexes and chloroquine to cells, cell medium was replaced with fresh medium in order to avoid toxicity effects. Thereafter, cells were washed and FACS was performed as described above.
Cytotoxicity assay
To assess the cytotoxicity of CPP -plasmid DNA complexes, HeLa cells were seeded on 24- well plate, 24 h before the start of the experiment. Next day, medium was replaced with Opti-MEM and peptide/ pDNA complexes at N/P ratio 9 (IMT-P8/pDNA, and STR-FMT- P8/pDNA) were formed as described above and added into the cells. In addition, equal amount of CPPs alone (IMT-P8 and STR-IMT-P8) were also incubated to the cells. Cells were further incubated for 4 h at 37° C in C02 incubator in the presence of complexes. Thereafter, medium containing complexes were replaced with fresh medium containing 10% FBS and cells were allowed to grow further for 20 h. Transfection with LF2000 was carried out according to the manufacturer's protocol. Subsequently, medium was removed from each well and cells were trypsinized and resuspended into the complete medium. 100 μΐ of these cells were added into each well of 96-well plate. Cell viability of these cells were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. 10 μΐ of MTT reagent (5mg/ml) was added in each well and incubated the cells for 4 h. Thereafter, 100 μΐ of solublization reagent (2% SDS in 50% DMF) was added to each well and plate was kept at gentle shaking to dissolve the formazen crystal. Absorbance was taken at 570 nm with a microplate reader (Tecane). The survival of cells relative to the control (cells incubated with growth medium containing no peptide) was calculated by taking the ratio of the absorbance at 570 nm values. All the experiments were performed in triplicates.
EXAMPLES
The following examples are given by way of illustration only and therefore should not be construed to limit the scope of the present invention in any manner.
1. EXAMPLE 1
IMT-P8/ pDNA nanocomplex formation
In order to determine whether or not IMT-P8 is capable of forming nano complexes with plasmid DNA, agarose gel retardation assay was performed with binary complexes (IMT-P8/ pDNA). It was found that EVIT-P8/pDNA complexes were progressively stabilized by increasing N/P ratios as mobility of plasmid DNA was reduced with increasing N/P ratios (Figure 1 A). These results suggested that IMT-P8 was able to condense and form complexes with plasmid DNA. The hydrodynamic diameter and surface charge (zeta potential) of these nanoparticles was determined by using dynamic light scattering (DLS). The results are shown in Figure IB. As shown in Figure IB, the hydrodynamic diameter of complexes are in the range of 150-300 nm. Under these conditions, the surface charge (ζ-Potential) was negative at N/P ratio 1 but with increasing CR, ζ-Potential was also progressively increased (Figure 1C). At CR2 and above, ζ-Potential was positive and was almost in the range of +15 to +20 mv. These results suggested that EVIT-P8 could form complexes with pDNA at different CRs in the range of 150-300 nm. Similar nano-complexes (at N/P ration 9) were also seen in TEM (Figure ID).
2. EXAMPLE 2
Poor delivery of pEGFP-Nl plasmid DNA into HeLa cells using IMT-P8
In order to examine if IMT-P8 can deliver pEGFP-Nl DNA into the HeLa cells, HeLa cells were transfected with pEGFP-Nl plasmid complexed with IMT-P8 at different CRs (5-12) followed by analysis of GFP fluorescence intensity in HeLa cells by flow cytometry. The results is shown in Figure 2A. As shown, no significant GFP fluorescence was observed at any CRs suggesting that EVIT-P8 could not able to deliver pEGFP-Nl successfully into the cells.
3. EXAMPLE 3
STR-IMT-P8/ pDNA nanocomplex formation
Figure 3A demonstrates that similar to IMT-P8, STR-IMT-P8 also condense and form complexes with plasmid DNA. STR-EVIT-P8/ pDNA complexes were found to be stable as significant retardation in pDNA mobility was observed on 1% agarose gel (Figure 3A). These complexes were almost immobilized and no bands were detected corresponding to position of naked pDNA. DLS experiments suggested that the size of STR-EVIT-P8/ pDNA complexes was in the range of 60-120 nm (Figure 3B), which was smaller than the size of IMT-P8/pDNA complexes. Similar to EVIT-P8/ pDNA complexes, ζ-Potential was negative at N/P ratio 1 and was positive at successive N/P ratios (Figure 3C). TEM analysis of STR- IMT-P8/ pDNA complexes at N/P ratio 9 confirmed the formation of nanoparticles similar to IMT-P8/ pDNA.
4. EXAMPLE 4
Increased delivery of pEGFP-Nl plasmid DNA into HeLa cells using STR-IMT-P8
The effect of stearylation of IMT-P8 on delivery of pEGFP-Nl plasmid DNA into HeLa cells was determined. Overnight grown HeLa cells were incubated with preformed STR- IMT-P8/ pDNA complexes at different CRs from 1-12 and subsequently the fluorescence intensity of GFP was analyzed by flow cytometry. As shown in Figure 4A, with increasing CR 3 - 12, significant increase in GFP fluorescence intensity was observed in HeLa cells. These results suggested that STR-IMT-P8 could able to deliver pEGFP-Nl plasmid DNA successfully into the HeLa cells. Similar observation was observed when these cells were examined under the confocal microscope (Figure 5). As shown in Figure 5, a few cells expressing GFP was seen at CR 1-3 and slightly more number of cells could be seen at higher CRs at CR5-CR12.
5. EXAMPLE 5
Effect of chloroquine on plasmid DNA delivery using IMT-P8 and STR-IMT-P8
Since it has been reported earlier that IMT-P8 internalized into HeLa cells through endocytosis, it is very likely that IMT-P8/ pDNA and STR-EVIT-P8/ pDNA complexes were also internalized by endocytosis and it is possible that these complexes might be entrapped into the endosomes and could not release into the cytoplasm. Therefore, to check this possibility, HeLa cells were incubated with EVIT-P8/ pDNA and STR-IMT-P8/ pDNA complexes in the presence of chloroquine (ΙΟΟμΜ) followed by analysis by flow cytometry. As shown in Figure 6, a 2-fold increase in GFP fluorescence intensity was observed after the chloroquine treatment. Similar results were obtained when effect of chloroquine was examined for uptake of STR-IMT-P8/ pDNA complexes. However, in both the cases, the GFP fluorescence intensity was much lower than the LF2000 treated cells. These results suggested that like IMT-P8, IMT-P8/ pDNA and STR-FMT-P8/ pDNA complexes were internalized by endocytosis and major fraction of these complexes entrapped into the endosomes.
6. EXAMPLE 6
Cytotoxicity associated with plasmid DNA delivery by STR-IMT-P8
In order to know whether plasmid DNA delivery by EVIT-P8 and STR-IMT-P8 can cause cytotoxicity, MTT assay was performed with HeLa cells after co-incubation of peptide /pDNA complexes and chloroquine. Cytotoxicity caused by LF2000 was also compared. As shown in Figure 7, in case of EVIT-P8/ pDNA complexes, around 90% cells are viable while in case of STR-EVIT-P8/ pDNA, around 85% cells are viable. In case of LF2000, significant cytotoxicity was observed compared to EVIT-P8 and STR-IMT-P8 and around only 65% cells were viable. ADVANTAGES OF THE INVENTION
• The chemically modified cell penetrating peptide STR-IMT-P8 (molecule represented by general formula 1) shows higher plasmid DNA delivery capability than the wild type IMT-P8 (SEQ ID NO: 1) in HeLa cells.
• It is simple in production, upscalable and cost effective.
• The plasmid delivery of STR-IMT-P8 was low as compared to commercially available transfection reagent lipofectiamin, nevertheless on the upper side it was less toxic as compared to Lipofectamin 2000; which causes significant toxicity in HeLa cells upon transfection.
SEQUENCE LISTING
<110> CSIR, IN
<120> CHEMICALLY MODIFIED CELL-PENETRATING PEPTIDE FOR
INTRACELLULAR DELIVERY OF NUCLEIC ACIDS
<130> 201711010408
<160> 1
<170> Patentln version 3.5
<210> 1
<2\ \> 15
<212> PRT
<213> artificial sequence
<220>
<221> cell penetrating peptide
<223> novel and chemically synthesized in the lab
<400> 1
Arg Arg Tip Arg Arg Tip Asn Arg Phe Asn Arg Arg Arg Cys Arg
1 5 10 15
REFERENCES
(1.) Sharma V K et al. (2014) Nucleic acid therapeutics: basic concepts and recent developments. RSC Adv., 4, 16618-16631
(2.) Burnett JC and Rossi JJ. (2012) RNA-Based Therapeutics: Current Progress and Future Prospects. Chemistry and Biologyl9 (1), 60-71
(3.) Margus H, Padari K and Pooga M (2012) Cell-penetrating Peptides as Versatile Vehicles for Oligonucleotide Delivery. Molecular Therapy; 20 3, 525-533.
(4) Boisguerin P, et al. (2015) Delivery of therapeutic oligonucleotides with cell penetrating peptides. Adv Drug Deliv Rev.; 87:52-67. (5.) Were LL Munyendo et al. (2012) Cell Penetrating Peptides in the Delivery of Biopharmaceuticals. Biomolecules. 2(2): 187-202.
(6.) Lehto T, et al. (2016) Andaloussi S. Peptides for nucleic acid delivery. Adv Drug Deliv Rev.
(7.) Lehto T, et al. (2012) Cell-penetrating peptides for the delivery of nucleic acids. Expert Opin Drug Deliv. ;9(7):823-36.
(8.) Svensen N, et al. (2012). Peptides for cell-selective drug delivery. Trends Pharmacol Sci. 2012 Apr;33(4): 186-92.
(9.) Walther W, and Stein U. (2000) Viral vectors for gene transfer: a review of their use in the treatment of human diseases. Drugs, 60:249-271. (10.) Gao X et al. (2007) Nonviral gene delivery: what we know and what is next. AAPS J, 9:E92-104.
(11.) Hoyer J and Neundorf I. (2012) Peptide vectors for the nonviral delivery of nucleic acids. Acc Chem Res.;45(7): 1048-56.
(12.) Nasrollahi SA, et al. (2012) A peptide carrier for the delivery of elastin into fibroblast cells. Int. J. Dermatol. 51 :923-929.
(13.) Boisguerin P (2013) CPP-conjugated anti-apoptotic peptides as therapeutic tools of ischemia-reperfusion injuries. Curr. Pharm. Des.19:2970-2978. (14.) Shi NQ et al. (2012) Enhancing cellular uptake of activable cell-penetrating peptide- doxorubicin conjugate by enzymatic cleavage. Int. J. Nanomedicine;7: 1613-1621.
(15.) Liu BR et al. (2016) Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells. PLoS One; l l(3):e0150439.
(16.) Xia H et al. (2012) Penetratin-functionalized PEG-PLA nanoparticles for brain drug delivery. Int. J. Pharm. 436:840-850.
(17.) Shete HK et al. (2014) Endosomal escape: a bottleneck in intracellular delivery. J Nanosci Nanotechnol. 14(l):460-74. Review. (18.) Gautam A et al (2015) Identification and characterization of novel protein derived arginine-rich cell penetrating peptides. European Journal of Pharmaceuticals and Biopharmaceuticals. 89:93-10
(19.) Gautam A et al. (2016) Topical Delivery of Protein and Peptides Using Novel Cell Penetrating Peptide IMT-P8. Scientific Reports. 6:26278. (20.) Lehto T et al. (2011) A peptide-based vector for efficient gene transfer in vitro and in vivo. Mol Ther. 2011 Aug; 19(8): 1457-67.
(21.) Lehto T et al. (2010). Delivery of nucleic acids with a stearylated (RxR)4 peptide using a non-covalent co-incubation strategy. J Control Release. 141(1):42-51.

Claims

We claim:
1. A chemically modified cell-penetrating peptide represented by the general formula 1;
X-Z-SEQ ID NO:l
(1)
wherein, X represents a fatty acid moiety of length between 2 to 16 carbon atoms, covalently attached to linker Z of length between 2 to 6 carbon atoms.
2. The chemically modified cell-penetrating peptide as claimed in claim 1, wherein Z is, covalently attached to the peptide represented by SEQ ID NO: 1.
3. The chemically modified cell-penetrating peptide as claimed in claim 1, wherein X covalently attached to the linker Z.
4. The chemically modified cell-penetrating peptide as claimed in claim 1, wherein X represents stearic acid.
5. The chemically modified cell-penetrating peptide as claimed in claim 1, useful for intracellular delivery of nucleic acids in to a cell.
6. The chemically modified cell-penetrating peptide as claimed in claim 1, wherein the cell is either a prokaryotic cell or eukaryotic cell.
7. The chemically modified cell-penetrating peptide as claimed in claim 1, wherein a cargo is a nucleic acid non-covalently linked to the peptide represented by SEQ ID NO: l.
8. A process for the intracellular delivery of nucleic acids comprising:
(a) providing a chemically modified cell penetrating peptide represented by the general formula 1, and (b) incubating the cell penetrating peptide described in step (a) with targeted cells.
9. The process as claimed in claim 8, wherein the targeted cell is selected from the group comprising of a eukaryotic cell or prokaryotic cell.
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* Cited by examiner, † Cited by third party
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JP7438376B2 (en) 2020-01-30 2024-02-26 メディトックス インク． Peptides that inhibit the formation of SNARE complexes and their uses

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