Description:FIELD OF INVENTION
The present invention relates to a cell-penetrating peptide sequence derived from spider toxin, Latarcin 1 and nuclear localization sequence from SV40 Simian virus T-antigen (LDP-NLS). More particularly the present invention relates to chimeric cell-penetrating peptide sequence derived from spider toxin, Latarcin 1 and nuclear localization sequence from SV40 Simian virus T-antigen used for cellular cargo delivery and possessing antimicrobial property.

BACKGROUND OF THE INVENTION
Spider venoms comprise a group of cytolytic peptide components such as lycotoxins, cupiennins, oxypinins and latarcins. These toxins range in length from 25-48 amino acid residues with a pI greater than 10.2. The toxins are linear, amphipathic peptides that possess antimicrobial and hemolytic property (Rash and Hodgson 2002; Vassilevski, Kozlov, and Grishin 2009).
Cell-penetrating peptide (CPPs) are a class of membrane-active peptides that possess inherent ability to cross the plasma membrane barrier and deliver conjugated cargo molecules inside the cells without affecting the viability of the cells. They are generally 5-30 amino acids in length with abundance of cationic residues. CPPs have been employed as vectors for delivery of therapeutic molecules such as oligonucleotides, proteins, nanoparticles and small molecular weight compounds inside the mammalian cells (Farkhani et al. 2014; Lehto et al. 2016; Lin et al. 2016; Zhang et al. 2016). A few of them have also been reported to be efficient delivery vectors for plant cells as well (Chugh and Eudes 2008a).
CPPs can be derived from natural sources such as Tat derived from transactivator of transcription of human immunodeficiency virus and penetratin derived from antennapedia homeodomain or synthetic such as oligoarginines and MAP (Derossi et al. 1996; Frankel and Pabo 1988; Mitchell et al. 2000; Oehlke et al. 1998). CPPs have been also identified from animal toxins such as crotamine from snake venom, maurocalcine and imperatoxin from spider venoms (Estève et al. 2005; Gurrola et al. 2010; Kerkis et al. 2004). However, spider venom peptides, despite being rich in membrane-active peptides, have not been yet characterized as potential CPPs with no cytotoxic effect to cells.
Membrane-active property of the Latarcins is taught in Kuzlov et al (Kozlov et al. 2006) disclose the isolation, purification and antimicrobial activity of seven classes of Latarcin peptides, Ltc 1, Ltc 2a, Ltc 3a, Ltc 3b, Ltc 4a, Ltc 4b, Ltc 5 from venom of spider, Lachesana tarabaevi. Latarcins show antimicrobial activity against gram-negative and gram-positive bacteria and yeasts. Ltc 2a and Ltc 1 show hemolytic activity.
RU2302466 discloses Latarcin peptides with antibacterial activity. The peptides, Lamartine LtAMP-3a and LtAMP-3b show antimicrobial activity against gram-positive and gram-negative bacteria.
Samsonova O V et al (Samsonova, Kudryashova, and Feofanov 2011) disclose that excision of three N-terminal residues from Ltc1-K peptide considerably reduces the cytotoxicity of the peptide for eukaryotic cells. The peptide simultaneously increases the selectivity of the antibacterial activity against certain bacterial species.
Rothan et al (Rothan et al. 2014) disclose the inhibitory activity of the Ltc-1 peptide against dengue virus in the infected cells.
Although, these peptides possess membrane-active property, none of them have been characterized as a cell-penetrating peptide (CPP). Accordingly, there is a need in the art for a cell penetrating biological carrier with minimum cytotoxic activity to the host cell.

OBJECTIVES OF THE INVENTION
The main objective of this invention is to overcome the conventional problems in the prior art.
An objective of the present invention is to provide a cell-penetrating peptide sequence derived from spider toxin, Latarcin 1 and nuclear localization sequence from SV40 Simian virus T-antigen.
An objective of present invention is in silico screening of potential cell-penetrating peptides from spider toxins.
Another objective of the present invention is the in vitro analysis of the cell-penetrating activity of the screened peptides in mammalian and plant cells and plant tissues.
Yet another objective of the present invention is to provide a method of delivery of cargo macromolecules inside mammalian and plant cells.
Another objective of the present invention is to assess the antimicrobial activity of the peptides against various pathogens.

SUMMARY OF THE INVENTION
According to an embodiment the present invention relates to a cell penetrating peptide sequence:
XWRRKLKXLXPXKKXKV wherein X is selected from amino acid R,K or A. In another embodiment the present invention relates to method for cellular delivery, comprising the steps of complexation of a cell-penetrating peptide sequence comprising latarcin-derived peptide and nuclear localization sequence with the peptide sequence: KWRRKLKKLRPKKKRKV having SEQ ID NO 1 or AWRRKLKALAPAKKAKV having SEQ ID NO: 4 with a cargo molecule to obtain a complex and thereafter administering the complex to a targeted mammalian or plant cell or tissue.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
Some of the objects of the invention have been set forth above. These and other objects, features, aspects and advantages of the present invention will become better understood with regard to the following description, appended claims and accompanying drawings where:
FIGURE 1 illustrates the method of in silico screening of the peptide sequence and formation of LDP-NLS.

FIGURE 2 illustrates the comparison of cellular uptake of chimeric peptide, LDP-NLS and variants, LDP, Mut-LDP-NLS and NLS in HeLa cells and nuclear localization of chimeric peptide. (A) illustrates uptake of LDP-NLS peptide as observed from green fluorescence by cells and no uptake in cells with peptides, LDP, Mut-LDP-NLS and NLS. (B) illustrates the cytoplasmic and nuclear localization of (fluorescein isothiocyanate) FITC-LDP-NLS peptide in HeLa cells as assessed from green fluorescence from the peptide and blue fluorescence from the nuclear dye, Hoechst 33342. (C) illustrated quantification of uptake of FITC-tagged peptides inside the cells as carried out by flow cytometry analysis.

FIGURE 3 demonstrates the quantitative uptake of LDP-NLS peptide in HeLa cells as assessed by confocal microscopy (A) and flow cytometry (B) and its affect on viability on HeLa cells (C), assessed from MTT assay.

FIGURE 4 illustrates the cytotoxicity of LDP-NLS and LDP in HeLa cells as assessed from membrane integrity assay.
FIGURE 5 illustrates the uptake of LDP-NLS-?-galactosidase complex in HeLa cells (A). Untreated cells. (B). Cells treated with ?-galactosidase enzyme but without any peptide. (C) Cells treated with X-gal buffer alone without any peptide or enzyme. (D), Cells treated with non-covalent complex of LDP-NLS peptide and ?-galactosidase enzyme. (E), Cells treated with non-covalent complex of Mut-LDP-NLS peptide and ?-galactosidase enzyme

FIGURE 6 illustrates the uptake of LDP-NLS, LDP, Mut-LDP-NLS and NLS peptides in wheat mesophyll protoplasts (A) and root tissues (B) Uptake of FITC-tagged peptides inside the cells is illustrated by confocal microscopy (A) and epifluorescence microscopy (B). Nuclear localization of LDP-NLS peptide is illustrated in A(c).

FIGURE 7 demonstrates the quantitative uptake of LDP-NLS peptide (A) in wheat protoplasts. Its affect on their viability is illustrated in (B) as percentage viability of protoplasts against different concentration of peptide.

FIGURE 8 illustrates the uptake of LDP-NLS-?-glucuronidase complex in wheat root tissues. A. Untreated root tissues. B, Tissues treated with ?- glucuronidase enzyme only without any peptide. C, Tissues treated with staining buffer only. D- E, Tissues treated with LDP-NLS and NLS peptides only. F, Tissues treated with non-covalent complex of NLS peptide and ?-glucuronidase enzyme. G, Tissues treated with non-covalent complex of LDP-NLS peptide and ?- glucuronidase enzyme.

FIGURE 9 illustrates the gel-retardation analysis (A) and DNase protection (B) of the pBT10GUS plasmid with LDP-NLS peptide. Gel retardation analysis (A) of various ratios of plasmid to LDP-NLS peptide indicates that the movement of plasmid in gel is completely inhibited at plasmid: peptide ratio of 1:2 and higher ratios 1:3, 1:4 and 1:5 (Lanes 7 to 10). Lane 1 represents 100 bp DNA ladder. Lane 2 and 3 represents plasmid and peptide, respectively. Lane 4 to 6 represents movement of plasmid complexed with peptide at ratio of 1:0.25, 1:0.5 and 1:1, respectively. Protection of plasmid from DNase was assessed by DNase protection assay (B). Intact plasmid bands are observed in the gel corresponding to plasmid: peptide ratios of 1:2 to 1:5 (Lanes 6-9). No band is observed in lanes corresponding to Lane 2 and 3 that represents movement of DNase- treated plasmid and peptide, respectively. Faint bands observed in Lanes 4 and 5 corresponding to plasmid: peptide ratio of 1:0.5 and 1:1, respectively.

DETAILED DESCRIPTION OF INVENTION
The present invention will be described with respect to preferred embodiments. The invention is not limited thereto but only by the claims.

Identification of Sequences

SEQ ID NO: 1 KWRRKLKKLRPKKKRKV
SEQ ID NO: 2 KWRRKLKKLR
SEQ ID NO: 3 PKKKRKV
SEQ ID NO: 4. AWRRKLKALAPAKKAKV

In one embodiment the present invention relates to a cell-penetrating peptide sequence characterized by in-silico analysis comprising peptide derived from Latarcin-1 and nuclear localization sequence from Simian Virus T40 antigen. The Latarcin derived peptide nuclear localization sequence (LDP-NLS) being:
XWRRKLKXLXPXKKXKV; wherein X is selected from amino acid R,K or A i.e.
Cell-penetrating peptide sequence is derived from the fusion of spider toxin-derived sequence, Latarcin-1 and nuclear localization sequence from Simian Virus T40 antigen.
In an embodiment the present invention relates to a peptide sequence being:
KWRRKLKKLRPKKKRKV ; having SEQ ID NO: 1.
Schematic of the in-silico screening of the peptide with potential cell-penetrating ability from spider venom toxins, Latarcins, is illustrated in Figure 1.
The cell penetrating peptide sequence of the present invention is formed from 6th -15th latarcin-derived peptide sequence: KWRRKLKKLR having SEQ ID NO: 2 and nuclear localization sequence: PKKKRKV having SEQ ID NO: 3 The peptide sequence with SEQ ID NO: 2 is modified at 6th amino acid of Latarcin 1 toxin from methionine to lysine with sequence KWRRKLKKLRPKKKRKV.
In one embodiment the present invention relates to a mutant version of peptide with SEQ ID 1 having SEQ ID NO: 4 obtained by replacing lysine and arginine residues at 1st, 8th, 10th, 12th and 15th positions in LDP-NLS peptide with alanine residues with sequence AWRRKLKALAPAKKAKV.

The present peptide sequence exhibit cell-penetrating ability in mammalian and plant cells and tissues, the peptide shows nuclear and cytoplasmic localization inside the mammalian and plant cells along. FIGURE 2 illustrates the comparison of cellular uptake of peptides (A). LDP-NLS shows localization in both cytoplasm and nucleus of HeLa cells (B). Variants, LDP, Mut-LDP-NLS and NLS show negligible uptake inside HeLa cells (A). FIGURE 3, illustrates that uptake of peptide increases with increase in concentration from 2.5 to 40 ?M (A & B) of the sequence.
Further, the peptide of the present invention does not cause cytotoxicity to HeLa cells even at high concentration as assessed by MTT assay (B).
In one embodiment the present invention illustrates the complexation of the cell-penetrating peptide sequence with a cargo molecule as illustrated in Figure 9 i.e. binding of cargo molecule, pBT10GUS plasmid to LDP-NLS peptide. Cargo molecule comprises of a peptide, a polypeptide, or a protein, a polysaccharide, a lipid, a lipoprotein, a glyco lipid, a nucleic acid, a small molecule drug or a toxin, a nanoparticle and an imaging or contrast agent. The invention also embodies the delivery of cargo by the cell-penetrating peptide in mammalian cells and plant tissues as illustrated in Figure 5 and Figure 8.
Figure 5 illustrates delivery of ?-galactosidase enzyme by LDP-NLS peptide in HeLa cells that turn blue as a result of enzymatic action of ?-galactosidase inside cells after histochemical staining (D). Figure 8 illustrates wheat root tissues treated with LDP-NLS non-covalently complexed with ?-glucuronidase enzyme in mass ratio of 3:1 turns dark blue after histochemical staining (G) confirming delivery of cargo.
Another aspect of the invention is the antimicrobial activity exhibited by the toxin-derived peptide and chimeric peptide against various gram-positive, gram-negative bacteria and filamentous fungi. LDP-NLS shows antimicrobial effect at around a concentration range of 2.5 -10 µM against several microorganisms.

In another embodiment the present invention relates to a method for cellular cargo delivery for diagnostic and therapeutic applications as well as for transfection, comprising the steps of:
(a) complexation of a cell-penetrating peptide sequence comprising latarcin-
derived peptide and nuclear localization sequence with the peptide sequence: KWRRKLKKLRPKKKRKV having SEQ ID NO 1 or AWRRKLKALAPAKKAKV having SEQ ID NO: 4 with a cargo molecule to obtain a complex;
(b) administering the complex to a targeted mammalian or plant cell or tissue.

The above mentioned cargo molecule can be selected from a group comprising of a peptide, a polypeptide, or a protein, a polysaccharide, a lipid, a lipoprotein, a glyco lipid, a nucleic acid, a small molecule drug or a toxin, a nanoparticle and an imaging or contrast agent. The delivery of cargo by the cell-penetrating peptide in mammalian cells and plant tissues as illustrated in Figure 5 and Figure 8.
In a preferred embodiment the complexation of a cell-penetrating peptide sequence comprising latarcin-derived peptide and nuclear localization sequence with the peptide sequence: KWRRKLKKLRPKKKRKV having SEQ ID NO 1 is done with a cargo molecule such as fluorescein isothiocyanate (FITC). FITC is conjugated to the LDP-NLS peptide via ?-amino group of first amino acid of the SEQ ID NO 1, i.e. lysine K using pyridine and 4-dimethylaminopyridine in solution phase at ambient temperature and mildly basic pH. Thereafter purification and lyophilization of FITC conjugated lysine is done. Further purified and lyophilized FITC conjugated lysine is conjugated to the peptide WRRKLKKLRPKKKRKV on wang resin by solid phase peptide synthesis to complete the synthesis of FITC-labelled peptide. Thereafter cleaving, purification and lyophilization of FITC-LDP-NLS peptide is done and FITC-LDP-NLS peptide complex is administered to a targeted mammalian or plant cell or tissue.

The invention is now being further explained by way of non-limiting examples:
Example 1 Peptide synthesis, Cell culture and Preparation of Plant Material
Peptides were custom synthesized by F-moc solid-phase peptide synthesis from GenPro Biotech, New Delhi, India with more than 95% purity. For assessing the uptake of peptides inside the cells by confocal microscopy and flow cytometry, the peptides were labelled with FITC at N-terminus.
For cell culture HeLa cells (from National Centre for Cell Science, Pune, India) were employed for assessment of the cell-penetrating ability of the peptides. The cells were cultured at 37?C in 5% CO2 in Dulbecco’s modified Eagle’s medium (Cell Clone, Genetix Biotech Asia Pvt. Ltd., India) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution (Gibco, Invitrogen, India).
For Preparation of Plant Material- Wheat mesophyll protoplasts were isolated from 6-8 day old seedlings of wheat (Triticum aestivum var HD2967) as described by Mahalakshmi et al, 1993 (Mahalakshmi, Maheshwari, and Khurana 1993).
1 mm root tips were excised from 6-8-day old wheat seedlings to assess the uptake of FITC-labelled peptides as well as for cargo delivery mediated by peptides.

Example 2. Confocal Microscopy, Flow cytometry and MTT assay
HeLa cells were seeded at a density of 300,000 onto glass coverslides in 6-well plates and cultured for 24 h. The cells were then washed with phosphate buffered saline (pH 7.4) and incubated with 2.5-20 ?M of FITC-labelled LDP-NLS and 20 ?M of LDP, NLS and Mut-LDP-NLS peptides in serum-free media for 1 h at 37?C in 5% CO2. For assessing the localization of LDP-NLS peptide with nucleus and lysosomes in HeLa cells, co-localization of the peptide with Hoechst 33342 was assessed. Briefly, after incubation of HeLa cells with the peptide for 1 h, cells were washed and treated with Hoechst 33342 dye (5 ?g/ml) for 20 mins at 37?C in 5% CO2. Further, cells were washed thoroughly with PBS. To quench the extracellular fluorescence, cells were treated with trypan blue (0.05% in PBS) for 5 minutes. Cells were subsequently washed thrice with PBS and analyzed using confocal laser scanning microscope (Olympus fluoview FV1000, Japan) at 60X magnification. 405 nm and 473 nm lasers were used to sequentially excite the Hoechst 33342 and FITC dyes.
Uptake of FITC-labelled peptides in wheat protoplasts and root tips were assessed by treating the cells and tissues with 20 ?M of each of the peptides followed by washing, trypsinization, washing and analysis by confocal microscopy and epifluorescence microscopy, respectively. Quantitative uptake of peptides in wheat protoplasts was carried out by fluorimetry.
Flow cytometry: Comparative uptake of peptides in HeLa cells was assessed by incubating the cells with 2.5-20 ?M of FITC-labelled LDP-NLS and 20 ?M of LDP, NLS and Mut-LDP-NLS peptides in serum-free media for 1 h at 37?C in 5% CO2. Dose-dependent cellular uptake of LDP-NLS peptide was assessed by incubating HeLa cells with increasing concentration of peptide for 1 h. The cells were then washed with PBS and were further treated with trypsin (1mg/ml) for 10 min to remove extracellular membrane-bound peptides. The cells were resuspended in PBS and washed twice by centrifugation and finally suspended in PBS for quantification by flow cytometry (BD FACSAria III, Becton Dickinson, USA). A total of 10,000 events were recorded and live cells were gated by forward/side scattering. Data was obtained and analyzed using FACS Diva ver 6.0 software.
MTT assay- Effect of LDP-NLS and LDP on the viability of cells was assessed by MTT assay. HeLa cells were seeded into 96-well plates at a density of 10,000 cells/well and cultured for 24 h. The cells were then incubated with different concentrations of LDP-NLS and LDP in serum-free media for 24 h at 37?C in 5% CO2. Cells incubated only in media without any peptide were used as the control and those treated with 0.1% triton X-100 were used for toxicity comparison. The cells were then incubated with MTT (1mg/ml) in PBS for for 4 h. The formazan crystals so formed were dissolved in dimethyl sulfoxide and the optical density was measured at 540 nm with reference wavelength of 620 nm on a microplate reader (Multiskan GO microplate spectrophotometer, Thermo Scientific, USA). The experiments were conducted in triplicate.
Example 3: Membrane integrity assay and cell viability
To assess the membrane integrity of HeLa cells treated with LDP-NLS and LDP, LDH-cytotoxicity assay kit was used according to manufacturer’s instructions (Biovision, USA). Briefly, the cells seeded in a 96-well plate at a density of 10,000 cells/well and cultured for 24 h were treated with increasing concentrations of LDP-NLS and LDP in serum-free medium for 1 h. Further, 100 ?l of supernatant from reaction media was mixed with 100 ?l of assay reagent for specified period of time. Further, absorbance was measured at 495 nm using a microplate reader (Multiskan GO microplate spectrophotometer, Thermo Scientific, USA). Membrane integrity of the cells was assessed by lactate dehydrogenase assay. This was studied by calculating percentage cytotoxicity as a measure of lactate dehydrogenase release from the cells when membrane integrity is compromised. LDP caused increased release of LDH from the cells as compared to LDP-NLS peptide as demonstrated in FIGURE 3. LDP-NLS was observed to be significantly less cytotoxic to the cells (P-value > 0.05) as compared to LDP (P-value <0.01) when compared to the cells that were not treated with peptides. The maximum cytotoxicity of LDP as measured from LDH release was ascertained to be approximately 60% at the highest concentration tested.

FDA viability assay: In order to assess the effect of LDP-NLS peptide on the viability of plant cells, wheat protoplasts were treated with increasing concentration of non-fluoresceinated peptide for 1 h at room temperature. Viability of protoplasts was then assessed by treating them with 0.01% fluorescein diacetate (FDA) for 5 min and then counting the viable protoplasts exhibiting green fluorescence under microscope. Protoplasts that were not treated with any peptides served as control.
Example 4. Cargo delivery ability
Cargo delivery ability of LDP-NLS peptide in HeLa cells was assessed by using ?-galactosidase enzyme (Sigma Aldrich) as the cargo molecule. LDP-NLS peptide at concentration of 20 ?M was non-covalently complexed with ?-galactosidase (1 ?g) enzyme at room temperature. The mixture was incubated at room temperature for 30 min for complex formation to take place. HeLa cells cultured in 24 well plate were then treated with peptide-enzyme complex at molar ratio of 2:1 for 1 h at 37?C in 5% CO2. The cells were washed thoroughly to remove extracellular peptide-enzyme complex and fixed with solution containing 2% formaldehyde and 0.2% glutaraldehyde. The cells were then assessed for cargo delivery by histochemical staining using X-gal staining buffer (4 mM potassium ferrocyanide, 4 mM potassium ferricyanide, 2 mM magnesium chloride, 1 mg/ml X-gal in PBS) for overnight at 37?C as demonstrated in FIGURE 5 (D). No staining was observed in the cells treated with enzyme alone without any peptide or histochemical buffer containing the substrate alone (A-C). Cells treated with Mut-LDP-NLS-cargo complex were stained light blue indicating slight uptake of peptide-cargo molecule inside the cells (E).
FIGURE 8 illustrates the uptake of LDP-NLS-?-glucuronidase complex in wheat root tissues. ?--glucuronidase was non-covalently complexed with peptide at room temperature for 30 min and then added to root tissues. Incubation of tissues with peptide-enzyme complex was carried out for 1 h at room temperature. Tissues were subsequently washed with PBS, fixed and then treated with GUS buffer at 37°C. Development of blue color is a result of enzymatic hydrolysis of substrate by ?-glucuronidase enzyme. A, Untreated root tissues. B, Tissues treated with ?- glucuronidase enzyme only without any peptide. C, Tissues treated with staining buffer only. D- E, Tissues treated with LDP-NLS and NLS peptides only. F, Tissues treated with non-covalent complex of NLS peptide and ?-glucuronidase enzyme. G, Tissues treated with non-covalent complex of LDP-NLS peptide and ?- glucuronidase enzyme.
FIGURE 9 illustrates the binding of cargo molecule, pBT10GUS plasmid to LDP-NLS peptide. Gel retardation analysis (A) of various ratios of plasmid to LDP-NLS peptide indicates that the movement of plasmid in gel is completely inhibited at plasmid: peptide ratio of 1:2 and higher ratios 1:3, 1:4 and 1:5 (Lanes 7 to 10). Lane 1 represents 100 bp DNA ladder. Lane 2 and 3 represents plasmid and peptide, respectively. Lane 4 to 6 represents movement of plasmid complexed with peptide at ratio of 1:0.25, 1:0.5 and 1:1, respectively. Protection of plasmid from DNase was assessed by DNase protection assay (B). Intact plasmid bands are observed in the gel corresponding to plasmid: peptide ratios of 1:2 to 1:5 (Lanes 6-9). No band is observed in lanes corresponding to Lane 2 and 3 that represents movement of DNase- treated plasmid and peptide, respectively. Faint bands could be observed in Lanes 4 and 5 corresponding to plasmid: peptide ratio of 1:0.5 and 1:1, respectively.
Example 5. Translocation in plant cells
FITC-labelled peptides are assessed for their translocation in plant cells. Wheat mesophyll protoplasts and root tissues were used to study the CPP activity of the peptides. FIGURE 6 represents the confocal microscopy and epifluorescence microscopy images of wheat protoplasts (A) and root tissues (B), respectively. Wheat mesophyll protoplasts and root tips isolated from 6-8-day old wheat seedlings were treated with FITC-tagged peptides for 1 h at room temperature. The cells and tissues were further washed with CPW solution, trypsinized for 10 minutes and further washed with CPW solution. Uptake of FITC-tagged peptides inside the cells was analyzed by confocal microscopy (A) and epifluorescence microscopy (B). Nuclear localization of LDP-NLS peptide is evident in Panel A(c).

FIGURE 7 represents the graphical representation for uptake of increasing concentration of LDP-NLS peptide in wheat protoplasts (A) and its affect on the viability of the cells as assessed by FDA viability assay (B). However, viability of protoplasts was maintained up to 60% at concentration up to 20 ?M. At higher concentrations, viability of protoplasts declined to less than 50%. Quantification of protoplasts treated with increasing concentration of FITC-tagged peptide for 1 h at room temperature was carried out by fluorimetry (A). Viability of protoplasts treated with increasing concentration of non-fluoresceinated LDP-NLS peptide was assessed by staining with fluorescein diacetate for 5 min. Viability is expressed as percentage viability of protoplasts against different concentration of peptide (B).

Example 6. ?-glucuronidase delivery in wheat root tips
LDP-NLS peptide was non-covalently complexed with ?-glucuronidase in the mass ratio of 3:1. After 1 h, the mixture was added to root tips of wheat for 1 h at room temperature. The root tips were then thoroughly washed and stained overnight with histochemical buffer containing X-gluc as described by Howard et al, 1992 (Howard et al. 1992).
Example 7. Gel retardation assay and DNase protection assay. LDP-NLS peptide was complexed with pBT10GUS plasmid at different mass ratio of plasmid: peptide ranging from 1:0.25 to 1:5. The assays were carried as described by Chugh et al, 2008 (Chugh and Eudes 2008b).
Example 8. Determination of antimicrobial activity of peptides
Two fold microdilution technique was followed in determining the minimum inhibitory concentrations of the peptide against microbes. Two-fold dilution of the peptides were done and treated against inoculum sizes of 5×105 cfu/ml for bacteria, 2×104 spores/ml for fungal spores and 2×105 spores/ml for 24 hours to germinate into hyphae when tested against fungal hyphae. The minimum concentration at which the peptide completely inhibits the growth of microbes is recorded as MICs against the particular microorganism.

Example 9: Criticality of cell-penetrating and antimicrobial ability data
A mutant version of LDP-NLS peptide was designed by replacing lysine and arginine residues at 1st, 8th, 10th, 12th and 15th positions in LDP-NLS peptide with alanine residues. Table 1 indicates the peptide sequences investigated for cell-penetrating and antimicrobial ability.
TABLE 1
Peptide name Sequence
LDP KWRRKLKKLR
LDP-NLS KWRRKLKKLRPKKKRKV
Mut-LDP-NLS AWRRKLKALAPAKKAKV
NLS PKKKRKV

The peptides were investigated for their cell-penetrating ability in HeLa cells and wheat protoplasts and root tissues. Uptake studies in cells with FITC-labelled peptides revealed that the chimeric peptide, LDP-NLS was most effective as a CPP in mammalian and plant cells. The peptide exhibited nuclear localization along with cytoplasmic localization.
Results of the various experiments conducted to study cell-penetrating and cargo-delivery ability of peptides in HeLa cells as well as wheat protoplasts and tissues are described below. Further, antimicrobial activity of the peptide was also tested against bacteria and fungi.
FIGURE 2 demonstrates the confocal images for the uptake of various peptides in HeLa cells. Translocation of peptides into HeLa cells were assessed by confocal laser scanning microscopy. The results demonstrate that HeLa cells treated with LDP-NLS peptides exhibited significant fluorescence. LDP exhibited significantly lesser uptake at the same concentration as LDP-NLS peptide and fluorescence was localized to the vesicles in the cytoplasm. To examine whether enhanced uptake of the LDP-NLS peptide as compared to LDP is due to the cell-penetrating ability of NLS sequence, the uptake of NLS sequence in HeLa cells was also examined. NLS showed negligible uptake in the cells as observed from lack of fluorescence in the cells treated with NLS. Since, positively charged amino acids are reported to be significant in the uptake of peptide in the cells, Mut-LDP-NLS with substitutions of lysine and arginine with alanine in both LDP and NLS regions was used as the negative control. The peptide did not exhibit significant cellular fluorescence.
HeLa cells were grown on cover slips in 6-well plate and treated with 20 µM of FITC-tagged peptides for 1 h at 37°C. Cells were further washed with PBS and then analyzed by confocal microscopy. Uptake of LDP-NLS peptide was significant as observed from green fluorescence exhibited by cells (A). HeLa cells treated with LDP, Mut-LDP-NLS and NLS did not show significant fluorescence inside the cells (A). LDP-NLS showed localization in both cytoplasm and nucleus of HeLa cells (B). Partial localization of the peptide in the nucleus was confirmed when cells were stained with nuclear dye, Hoechst 33342 (Pearson’s coefficient of 0.58 ? 0.04). This indicates that the NLS present in the peptide plays an important role in the transport of peptide inside the nucleus. To assess the nuclear localization of LDP-NLS peptide, HeLa cells were treated with Hoechst 33342 for 20 mins after peptide treatment and then washed thrice with PBS and analyzed by confocal microscopy. Partial localization of the peptide in the nucleus was observed from co-localization analysis of Hoechst 33342 (blue fluorescence) with FITC-LDP-NLS (green fluorescence) (Pearson’s coefficient of 0.58 ? 0.04). Quantification of uptake of peptide inside the cells by flow cytometry revealed that the peptide uptake followed a pattern similar to the confocal microscopy with highest uptake of LDP-NLS peptide inside the cells and comparatively, lesser uptake of LDP (C). Hence, from confocal microscopy and flow cytometry data, LDP-NLS was adjudged to be the best CPP candidate.
Example 10: Antimicrobial activity of the peptides, LDP-NLS and LDP was assessed against various bacteria and fungi by two-fold microdilution technique. Table 2 illustrates the antimicrobial property of LDP-NLS and LDP alone against representative microorganisms.
TABLE 2
S. No. Microbes MIC’s of Peptides (?M)
LDP-NLS LDP
1. MRSA 2.5 2.5
2. Bacillus subtilis 5 5
3. E.coli >10 5
4 Salmonella typhimurium >10 5
5. Xanthomonas oryzae 10 5
6. Xanthomonas campestris 10 5
7. Fusarium solani 2.5(spores)
30(hyphae) 5(spores)
40(hyphae)
8. Candida albicans 10 >10

Gram-positive, gram-negative bacteria, filamentous fungi and yeast are tested for antimicrobial activity of the peptide. LDP-NLS shows antimicrobial effect at around a concentration range of 2.5 -10 µM against the microorganisms tested except Salmonella typhimurium and Escherichia coli. LDP also inhibits microorganisms at concentration equal to LDP-NLS peptide. However, LDP-NLS enters microbial cells and targets the intracellular molecules to exert its antimicrobial activity and possess less cytotoxicity as compared to LDP peptide against mammalian cells. Hence, LDP-NLS is an excellent peptide antibiotic with low cytotoxic effect in host cells.
FIGURE 4 illustrates the cytotoxicity of LDP-NLS and LDP in HeLa cells. Percentage cytotoxicity of LDP and LDP-NLS on HeLa cells was assessed by LDH release assay. HeLa cells were treated with different concentrations of peptides. Percentage cytotoxicity was calculated as a measure of LDH release. LDP-NLS was observed to be significantly less cytotoxic to the cells (P-value > 0.05) as compared to LDP (P-value <0.01) when compared to the cells that were not treated with peptides.

Claims:1. A cell penetrating peptide sequence: wherein X is selected from amino acid R,K or A.
2.The cell penetrating peptide sequence as claimed in claim 1, wherein said sequence is formed from 6th-15th latarcin-derived peptide sequence:
KWRRKLKKLR having SEQ ID NO: 2
and nuclear localization sequence: PKKKRKV having SEQ ID NO: 3
3.The cell penetrating peptide sequence as claimed in claim 2, wherein said 6th amino acid is modified to lysine with sequence KWRRKKKKLRPKKKRKV and having SEQ ID NO: 1.
4.The cell penetrating peptide sequence as claimed in claim 1, wherein said sequence is AWRRKLKALAPAKKAKV having SEQ ID NO: 4.

5.The cell penetrating peptide sequence as claimed in claim 1, wherein said sequence forming a complex with a cargo molecule.
6.The cell penetrating peptide sequence as claimed in claim 4, wherein said cargo selected from a group consisting of a peptide, a polypeptide, or a protein, a polysaccharide, a lipid, a lipoprotein, a glyco lipid, a nucleic acid, a small molecule drug or a toxin, a nanoparticle and an imaging or contrast agent.
7.The cell penetrating peptide sequence as claimed in claim 1, wherein said sequences showing nuclear cytoplasmic localization inside a mammalian or a plant cell.
8.A method for cellular delivery, comprising the steps of:
(a)complexation of a cell-penetrating peptide sequence comprising latarcin-
derived peptide and nuclear localization sequence with the peptide sequence: KWRRKLKKLRPKKKRKV having SEQ ID NO 1 or AWRRKLKALAPAKKAKV having SEQ ID NO: 4 with a cargo molecule to obtain a complex;
(b)administering the complex to a targeted mammalian or plant cell or tissue.
9.The method as claimed in claim 8, wherein said cargo selected from a group consisting of a peptide, a polypeptide, or a protein, a polysaccharide, a lipid, a lipoprotein, a glyco lipid, a nucleic acid, a small molecule drug or a toxin, a nanoparticle and an imaging or contrast agent.