WO2015075747A1

WO2015075747A1 - Cell-penetrating peptide for biomolecule delivery

Info

Publication number: WO2015075747A1
Application number: PCT/IN2014/000725
Authority: WO
Inventors: Gajendra Pal Singh RAGHAVA; Ankur GAUTAM; Hemraj Santuji NANDANWAR
Original assignee: Council Of Scientific And Industrial Research
Priority date: 2013-11-19
Filing date: 2014-11-19
Publication date: 2015-05-28

Abstract

The present invention relates to cell penetrating peptide for delivery of biomolecules into an eukaryotic cell. The present invention specifically relates to a peptide based drug delivery system for delivering nucleic acids and/or proteins and/or peptides/ small molecules to cells, in vitro or in vivo.

Description

CELL-PENETRATING PEPTIDE FOR BIOMOLECULE DELIVERY

FIELD OF THE INVENTION

^' The present invention relates to cell penetrating peptides for delivery of biomolecules into a cell. The present invention specifically relates to peptide based drug delivery system.

BACKGROUND OF THE INVENTION

Most of the therapeutic molecules often do not reach the clinical trials due to their poor delivery and low bioavailability. Over the past decade, small cationic peptides known as cell penetrating peptides (CPPs) have emerged as a versatile delivery vehicle (1). Owing to their intrinsic cell internalization properties, CPPs have improved the intracellular delivery of various therapeutic molecules, including oligonucelotides (2-4), small molecules (5,6), protein (7), peptides (8), siRNA (9), nanoparticles (10) etc., which otherwise cannot cross plasma membrane barrier by their own.

The journey of CPP was started approx. 22 years ago (11) with the discovery of Tat (48-, 60) (12) and penetratin peptide (13), since then hundreds of CPPs have been discovered so far (14). Recently, a systematic cataloguing of these peptides have been carried out and their analysis has revealed that CPPs constitute a family of diverse peptides, few CPPs are derived from viral proteins such as VP22 (derived from herpes virus tegument protein) (15), Rev, some are derived from snake venom protein such as CyLOP-1 (derived from crotamin) (16), few are part of cell adhesion glycoprotein such as pVEC (derived from murine vascular endothelial-cadherin protein) and others are synthetic or designed such as oligoarginine. Though CPPs are very heterogeneous, they share some common features like CPPs are often cationic, and/or amphipathic in nature (17). Most of the CPPs are derived from natural proteins and contain high arginine content, which play crucial roles in mediating internalization into the cells (18,19). Despite of this heterogeneity in their sequences and structures, endocytosis has generally been acknowledged as major rout of internalization of CPPs (20,21), However, few CPPs have been reported to be internalize with non-endocytic process (22,23). Internalization mechanism is dependent on various factors like cell types, peptide concentration, type of conjugated cargo, temperature, etc. Although many CPPs have been identified so far, most of them have shown relatively low uptake efficiencies. Therefore, in the present study, we have used an integrated in silico and experimental approach to identify novel and efficient CPPs.

OBJECT OF THE INVENTION

The main object of the present invention is to provide cell-penetrating peptides for delivery of biomolecules into a cell.

SUMMARY OF THE INVENTION

Accordingly the present invention pertains to new cell-penetrating peptides (CPP) which exhibits high efficiency and low toxicity and the process of preparing the same and their use.

In an embodiment of the invention it provides a cell-penetrating peptide having amino acid sequence selected from the group consisting of Seq Id no. 1, 2, 3, and 4.

In an embodiment of the invention it provides use of the cell-penetrating peptide in treatment of diseases selected from the group comprising of bacterial infection; diabetes, skin disorders, cancer.

In an embodiment of the invention it provides a complex useful for delivery of a cargo inside the cell comprising a cell-penetrating peptide and a cargo wherein the cargo is selected from the group comprising of a chromophore, a protein, an antibiotic, a peptide, nucleic acid, nanoparticles, drugs.

In an embodiment of the invention it provides a method of intracellular delivery comprising:

(a) providing a cell penetrating peptide, and

(b) incubating the cell penetrating peptide obtained in step (a) with targeted cells.

In an embodiment of the invention it provides a method wherein the targeted cell is selected from the group comprising of a eukaryotic cell, prokaryotic cell. BEIEF DESCRIPTION OF THE FIGURES:

FIGURE 1. In silico approach for identification of arginine-rich protein derived peptides.

All entries from SwissProt were extracted and 15 amino acid length peptides containing nine arginine residues were fished out. Various filters (scanning of peptides for the presence of tumor homing motif (RGD), prediction using CellPPD algorithm and presence of tumor penetrating motif (RXXR, C-endRule) were applied to find out potential CPPs. Finally, seven peptides were selected for experimental validation.

FIGURE 2. Cellular uptake of FITC labeled peptides as determined by FACS analysis.

Overnight grown HeLa cells were incubated with 10 μΜ FITC-labeled peptides for 1 h, followed by washing with PBS, and trypsinization 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 FITC. Results are expressed as mean ± S.E., based on triplicates of at least two independent experiments. (*p<0.05, ***p<0.001).

FIGURE 3. Intercellular localization of FITC labeled peptides in HeLa cells.

: HeLa cells were grown on cover slips and incubated with FITC-labeled peptides (10 μΜ) for 1 h. Cells were then washed carefully twice with ice-cold PBS and immediately observed (without fixation) by confocal fluorescence microscopy. Bar = 10 μπι. Left panel the figure shows the histogram of FITC intensity.

FIGURE 4. Cytotoxicity of peptides.

HeLa cells were incubated with increasing concentrations (0, 5, 10, 20, 40 and 80 μΜ) of peptides in serum containing medium at 37°C for 24 h. Cell viability was measured by MTS assay. Viability of control (without peptide) was taken as 100 % and viabilities of cells treated with increasing concentration of peptides were plotted as percentage of control. Results are expressed as mean ± S,E, based on triplicates of at least two independent experiments.

FIGURE 5. Structural analysis of peptides. (A) Tertiary structures of all peptides were predicted using Pep-Fold. (B) Helix wheel projections were generated using PEPwheel tool of emboss.

FIGURE 6. Characterization of P8.

(A) Protein sequence of human voltage-dependent L-type calcium channel subunit alpha-ID. P8 sequence (15 amino acids, 503-517) is highlighted. (B) and (C) Time dependent uptake of P8. HeLa cells were incubated with 10 μΜ FITC-labeled P8 for increasing time periods (0, 5, 20, 30 and 60 min) followed by trypsinization (10 min) and flow cytometry quantification of peptide uptake. (D) Concentration dependent internalization of P8 peptides. HeLa cells were incubated with increasing concentration (0, 2.5, 5, 10, 20 μΜ) of FITC- labeled P8 for 30 min followed by trypsinization and flow cytometry quantification of peptide uptake. (E) Effect of serum on P8 uptake. HeLa cells were treated with 10 μΜ FITC- labeled P8 peptide with and without serum containing medium for 30 min followed by flow cytomtery analysis. Data are shown as mean ±S.E, based on triplicates of at least two independent experiments.

FIGURE 7. CD spectra of TAT and P8 peptide

CD spectra of TAT (A) and P8 peptide (B) in water and membrane mimicking environment.

FIGURE 8. Effect of low temperature and energy depletion on the uptake of P8 peptide.

(A) Distribution of P8 in HeLa cells. HeLa cells were incubated with 10 μΜ FITC-P8 for 30 min and immediately observed in CLSM. (B & C) Flow cytometry and (D) confocal microscopy analysis of P8 peptide cellular uptake at low temperature and upon depletion of cellular ATP. HeLa cells were either pre-incubated at 4°C or pre-treated with energy- depletion medium for 30 min, and then incubated with 10 μΜ peptide for 30 min under the same conditions. Uptake of P8 at 37°C was set to 100% and relative uptake of P8 at 4°C and in energy-depletion condition was plotted as percentage of control. Confocal microscopy analysis was performed in live cells. In (B), data are shown as means ± S.E, based on triplicates of at least two independent experiments. (*p<0.05; **p<0.001).

FIGURE 9. Effect of endocytosis inhibitors on uptake of P8 and TAT. Cellular uptake of P8 (A and B) and TAT (C) by HeLa cells in the presence of endocytic inhibitors were determined by flow cytometry. HeLa cells, pre-treated with CPZ, M CD, Cyt D and amiloride for 30 min at 37°C, were incubated in the presence of FITC labeled P8 and TAT (10 μΜ) at 37°C for additional 30 min, trypsinized for 10 min, washed twice with 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 fluorophore. The uptake of P8 (A) and TAT (C) in the absence of any inhibitor was set as 100% and relative uptake of P8 and TAT in the presence of inhibitors was shown as percentage of control. (B) Frequency distributions of FITC fluorescence intensity in HeLa cells incubated for 30 min with and without indicated endocytic inhibiotors.. Data are shown as mean ± S.E, based on triplicates of at least two independent experiments. (*p<0.05).

FIGURE 10. Cellular uptake of FITC labeled deletion mutants of P8 as determined by FACS analysis.

Overnight grown HeLa cells were incubated with 10 μΜ FITC-labeled peptides (P8, Tl and T2) for 1 h in serum-containing medium. After the incubation, cells were Washed twice with PBS, trypsinized at 37°C for 10 min, 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 FITC. Results are expressed as mean ± S.E., based on triplicates of at least two independent experiments.

FIGURE 11. CPP-eGFP fusion protein purification.

(A) Scheme demonstrating cloning of TAT-eGFP and P8-eGFP in pET28C expression vectors. Each expression vector construct was transformed into BL21(DE3) competent cells (Life technologies, Invitrogen), and recombinant proteins were purified obtained from the soluble fraction using affinity column chromatography. TAT-eGFP (B) and P8-eGFP (C) was confirmed by Coomassie brilliant blue staining. Lanel : protein marker, Lane2: uninduced sample, Lane3: soluble fraction after sonication, and Lane 4: purified protein.

FIGURE 12. P8 mediated delivery of GFP into HeLa cells.

Overnight grown HeLa cells were incubated with 10 μΜ TAT-eGFP, P8-eGFP and eGFP alone for 1 h in serum-containing medium. After the incubation, cells were washed twice with PBS, trypsinized at 37°C for 10 min, 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 eGFP.

FIGURE 13. Visualization of P8-eGFP in HeLa cells.

HeLa cells were grown on cover slips and incubated with P8-eGFP (10 μΜ) for 1 h. Cells were then washed carefully twice with ice-cold PBS and immediately observed (without fixation) by confocal fluorescence microscopy. Bar = 10 μηι.

FIGURE 14. Internalization of P8 in yeast and bacteria.

Yeast and bacterial cells were incubated at 37°C for 1 hr in PBS containing the FITC labeled P8 (5μΜ each). At the end of the incubation period, yeast and bacterial cells were washed twice with PBS, treated with trypsin and finally suspended in PBS. Finally, cells were mounted on slides and analyzed immediately by confocal microscope.

FIGURE 15. Internalization of CPPs in S. aureus.

Bacterial cells were incubated at 37°C for 1 hr in PBS containing the FITC labeled Penetratin, P3 and P8 (5μΜ each). At the end of the incubation period, bacterial cells were washed twice with PBS, treated with trysin and finally suspended in PBS. Finally, cells were mounted on slides and analyzed immediately by confocal microscope.

FIGURE 16. Combination of CPP-norfloxacin improves the efficacy of norfloxacin.

SA-SA-831 cells (5 x 10⁵ cfu/ml) in a microtitre plate were incubated with combination of norfloxacin and CPPs at (A) 5 μΜ and (B) 10 μΜ at 30°C for 48 hr. After the incubation, MTT dye was added to each well and kept for incubation at 30°C for 20 min. The concentration in the well at which no purple coloration observed is the MIC of antibiotic in presence of respective CPP.

DETAILED DESCRIPTION OF THE INVENTION

We have applied an in silico procedure to identify putative CPP candidates in the proteins available in SwissProt database. For searching CPP candidates, we screened peptides based on the presence of high arginine content and high positive charge. We have extracted peptides having 8/9 arginine in all the proteins available in SwissProt database using in silico approach. A window size of 15 amino acids was chosen. The extracted peptides were subjected to various filters to find out the potential CPP sequences (Figure 1). To achieve a higher success rate, the cell penetrating potential of all screened peptides were predicted using a computational algorithm, CellPPD (24), which is a support vector machine-based prediction method developed to predict highly efficient CPPs. FACS and CLSM results revealed that seven out of eight peptides have cell penetrating properties (Figure 2 and 3). Interestingly, none of the peptides showed significant membrane toxicities even at 80 μΜ concentrations (Figure 4) suggesting that these peptides can be used for future drug delivery applications.

All peptides internalized into HeLa cells except, one peptide P2 that did not internalize into the cells rather it could bind to the cell surface. Peptide P2 has RGD at N- terminus and RGD motif has been shown to selectively recognize and bind to integrins that are expressed on cell surfaces of various cancer cell types, including HeLa cells (25, 26). The lower uptake of P2 could be due to binding of P2 with integrins on HeLa cells.

A significant difference in uptake efficiencies between P8 and other peptides was observed despite the fact that these peptide h¾ve similar arginine content and charge. These results are in accordance with a earlier study by Nakase et al, where arginine-rich peptides with similar charge and arginine content internalized into HeLa cells with different efficiencies (27). In silico analysis (Figure 5) aimed at identifying features responsible for varying efficiencies of these arginine-rich peptides points to the presence of other amino acids like tryptophan and tendency to adopt helical structures. Orientation of arginine and tryptophan residues around the helix might also be responsible for differences in the uptake efficiencies. These observations are consistent with the previous findings where the presence of tryptophan and spacing of arginine residues have been found to affect the uptake efficiency of CPPs (28).

Further characterization then focused on the P8 peptide represented by SEQ ID NO 1, which showed almost 10 times higher uptake than TAT in HeLa cells. Though P8 and TAT are arginine rich peptides but being a part of human protein, P8 suppose to be less immunogenic. The superior uptake of P8 was also observed in other cell types, including CHO-K1, THP-1, MCF-7 and NCI-H522 (data not shown) suggesting that it can be used as a versatile . delivery system. P8 is a part of cytosolic domain of human voltage-dependent L- type calcium channel subunit alpha-ID protein (Figiure 6). In silico analysis revealed that P8 has a tendency to form, helical structure and helical wheel analysis demonstrates the partially amphipathic distribution of arginine and tryptophan residues. CD analysis also demonstarted that P8 adopted helicaL conformation in membrane mimicking environment (Figure 7). It has been shown that helical peptides are internalized more effectively than the peptides with random coil structures (29). Therefore, the presence of tryptophan and tendency to adopt amphipathic helical conformation might be responsible for its high uptake in live cells. Despite its higher uplilke efficiency, P8 did not show any significant membrane toxicity up to 40 μΜ concentration which is much higher than the routinely tested concentrations. However, at 80 μΜ c oncentration, it showed some toxicity as only 75% HeLa cells were viable at this concentration. High penetrating efficiency with low toxicity makes PS an ideal candidate for drug delivery vehicle. However, presence of serum retards its uptake efficiency by ~3 fold, suggesting that P8, being a highly cationic peptide, might interact non-specifically with serum proteins (Figure 6). Such effects of serum on uptake efficiency have also been reported for other CPPs (19). This inhibition of uptake efficiency by serum proteins is sometimes beneficial, particularly when the CPP is highly effective like P8; otherwise this high uptake may cause cytotoxicity.

Taking into consideration that the cellular uptake of P8 occurs through a temperature- sensitive and energy-dependent process (Figure 8), the possible involvement of endocytosis was thoroughly invesl gated by analysing P8 uptake in the presence of endocutosis inhibitors. CPZ, Μβϋϋ, or amibride did not able to block the cell entry of P8 (Figures 9). However, Cyt D inhibited P8 up take significantly. Interestingly, similar results were also obtained for •TAT uptake, suggesting that TAT and P8 enter the HeLa cells by a Cyt D sensitive pathway. These findings, particularly on TAT, are contradictory to the previous studies, which have reported significant inhibition of TAT uptake by CPZ (30, 31). These differences could be due to different experimental conditions. However, there are also few studies whose findings are in close agreement with our findings where only Cyt D blocked the entry of TAT (32). Therefore, the present study, at least, suggests that P8 may internalize into the cells by Cyt D sensitive endocytic pithway. However, involvement of mechanisms other than endocytosis cannot be ignored. Next, in order to find out the shorter peptide which has high penetration efficiency, we have synthesized two deletion mutants Tl (SEQ ID NO. 4) and T2 (SEQ ID NO. 3) of peptide P8 (with N-terminal truncation) having length 9 and 12 residues respectively. The penetration efficiency of these analogs was determined on HeLA, PC-3 and MiaPaca-2 cell lines and compared with parent P8 peptide. As seen in Figure 10, the analoge T2 (SEQ ID NO. 3) having 12 residues has shown penetration efficiency more or less equivalent to P8 peptide. Thus, analog T2 is as good as parent peptide P8.

In order to determine the drug delivery ability of P8 (SEQ ID NO. 1), we have over expressed P8-eGFP and TAT-eGFP fusion proteins (Figure 11) and determined the ability of P8 to deliver eGFP in HeLa cells and also compared with TAT. Both flow cytometry and confocal microscopy studies revealed that P8 delivered eGFP into HeLa cells (Figure 12 and 13). Internalized P8-GFP fluorescence was significantly higher in HeLa cells compared to Tat-GFP suggesting that efficiency of P8 to delivery GFP in HeLa cells is higher than TAT peptide. .These findings demonstrated that in silico screening based on simple features of known CPPs can be a promising approach to identify novel efficient CPPs. In addition, peptides identified in the present study, in particular, P8 (SEQ ID NO. 1) is a promising candidate for drug delivery applications.

Apart from internalization into mammalian cells, P8 also internalized into yeast and bacterial cells (Figure 14), which makes it suitable for drug delivery into these organisms. The increase in the antimicrobial drug resistance is one of the major international concerns and therefore, there is an urgent need to develop novel drug delivery strategy which not only should be different from the traditional ones but should have a novel mode of action also. Therefore, the present study also focuses on developing novel anti-MRSA drug formulations to reduce the burden of MRS A, which is resistant to conventional multidrugs.

Peptides have been gained a significant attention over the years as attractive therapeutic candidates. CPPs and antimicrobial peptides (AMPs) belong to class of membrane-active peptides and thus share similar characteristics (33, 34). CPPs are well exploited as drug delivery system (35) owing to their intrinsic cell penetrating abilities while AMPs are known to be used as antimicrobial agents (36). Many AMPs have been shown to be effective against various drug resistant bacterial pathogens either alone or in combinations with the existing antibiotics (37, 38). Besides their different functions, there is very thin line between CPPs and AMPs (39). Generally CPPs enter mammalian cells without causing significant membrane damage. However, in the case of bacterial cells, which differ greatly in membrane composition, CPP entry could cause cell leakage and death particularly at higher concentrations. Therefore, many CPPs have also shown some antimicrobial activity (40). But CPPs can be used as delivery vehicle at concentration which is not toxic to bacteria (low concentrations). Though CPPs have been extensively used as drug delivery vehicle in eukaryotic systems (35), particularly in cancer cells, their use as transport vehicle in bacterial system is comparatively less explored. Only a handful studies have reported the successful delivery of therapeutics into the bacterial cells using CPPs. Therefore, in the present study, we have explored whether CPPs at low concentration if used in combination with existed antibiotics, can improve the efficacy and delivery of antibiotics against MRSA. We first determined the antibacterial activity of various antibiotics and three CPPs, Penetratin, P8 (SEQ ID NO. 1) and P3 (SEQ ID NO. 2) against MRSA (SA-831 and wild type S. aureus (SA-96). A significant higher MIC of oxacillin against SA-831 confirmed the MRSA status of strain. In addition, norfloxacin is the second antibiotic which is the least effective among the rest of the antibiotics tested against SA-831 compared to wild type SA-96. All three CPPs have also shown moderate (>12.5μΜ) antibacterial activity (Table 2). Among these, Penetratin is the most effective (MIC 12.5 μΜ) against SA-831 followed by P8 (MIC 25 μΜ). P3 was^' not found to be antibacterial. This difference in antibacterial activity could be due to their different physicochemical properties. In addition, both Penetratin and P8 contain two Trp residues and the role of Trp residues in membrane penetration is well documented (28). Therefore, the relatively high antibacterial activity of Penetratin and P8 compared to P3 could be due to the presence of Trp residues in Penetratin and P8 (SEQ ID NO. 1).

Among the various causes for resistance development as reported in literature (41), trans-membrane drug permeability could be the one of the causes. The poor trans-membrane drug permeability can be improved by CPP mediated intracellular drug delivery. In the recent past, Sparr et al. (42) have achieved improved efficacy of fosmidomycin, a drug suffering from poor permeability, against Plasmodium and. Mycobacterium species by conjugating with the cell penetrating peptide octaarginine. However, in this study, authors have conjugated the fosmidomycin with octaarginine which makes the overall treatment costly and also sometime conjugation of drug to peptide alters the drug properties and thus may affect the overall efficacy of the drugs. Therefore, we have investigated the effect of CPPs on the efficacy and delivery of antibiotic without conjugating with CPPs.

In order to deliver the drug into the bacterial system, CPPs must be internalized into the MRSA without causing significant cell death. Therefore, FITC labelled CPPs were incubated with resting bacterial cells at concentration much lower than their MICs. Confocal microscopic studies revealed that all three CPPs were internalized into the MRSA at this concentration (Figure 15). The uptake of CPPs was found to be very efficient as significant intracellular FITC fluorescence was observed just after 1 hr incubation. Also at the same concentration, none of the peptides showed significant hemolysis (data not shown) suggesting that these CPPs could be used as delivery vehicle at the tested concentration in MRSA.

Considering the poor membrane permeability of norfloxacin as one of the probable reasons to be resistant against MRSA, we have treated both MRSA and wild type SA-96 with norfloxcin alone and in combination with three CPPs (5 and ΙΟμΜ). Interestingly, in all three combinations with CPPs, the MIC of norfloxcin was reduced significantly (Table 3). The P3- norfloxacin and P8-norfloxacin combination were highly effective and reduced the MiC of norfloxacin up to 128-fold while Penetratin-norfloxacin combination was moderately effective (Figure 16). In our recent study, the concept of efflux pump inhibitors as the adjuvant to reduce drug resistance has been discussed (41). But it is first time reported here the concept of CPP-antibiotic cocktail to reduce drug resistance in Gram +ve bacterium, especially MRSA strain.

EXAMPLES

The following examples are given by way of illustration therefore should not be construed to limit the scope of the Invention.

EXAMPLE: 1

In silico approach for identification of arginine-rich protein derived peptides.

In order to discover novel potential CPP candidates, we have used an in silico approach to identify all possible arginine-rich sequences in SwissProt proteins. It has been shown that eight/nine arginine residues are optimum for efficient internalization of oligo- arginines, therefore, peptides segments of window length 15 with eight/nine arginine that produce high positive charge were extracted from SwissProt proteins. As a result, total 12053 sequences were fished out and among which 7680 were found to be unique.

In order to identify cancer specific CPPs, a filter was applied to select peptides having tumor homing motif RGD (Arg, Gly, Asp) at N-terminus. This screening has resulted into 12 peptides, which have RGD motif at N-terminus. For rest 7668 peptides, we used CD-HIT software to remove peptides having similarity more than 60%, which resulted into 421 significant different peptides. In order to achieve a higher hit rate, resultant 421 peptides were submitted to CellPPD webserver to predict cell-penetrating properties of these peptides. Based upon SVM scores, we further selected 356 peptides having SVM scores more than 0.50, demonstrating high probabilities of these peptides to be CPPs. Since, it has been observed that negatively charged residues like Asp and Glu are not preferred in CPPs; we applied a filter to remove all peptides having these negatively charged residues. Remaining peptides were then scanned for the presence of RXXR motif at C-terminus. It has been previously shown that peptides with exposed RXXR motif at C-terminus (C-endRule) were internalized into tumor cells by binding to nuropilin receptor, which overexpressed on the surface of most of the cancer cells. This screening resulted into three datasets. From dataset 1, we selected one peptide having ROD at N-terminus and RXXR motif at C-terminus. From Dataset 2 and 3, we selected four and two peptides, respectively. Since it has been suggested that arginine is crucial and preferred over lysine in cell penetration, we have selected one lysine rich peptide just to compare the efficiency with arginine-rich peptides. All these peptides derived from different species including human, rat, Mycobacterium tuberculosis, mouse, viruses and Arabidopsis thaliana.

EXAMPLE: 2

Cell culture

PC3 and CHO-K1 cells (ATCC, USA) were grown in Ham's F12 medium supplemented with 10% FBS and L-glutamine (2mM). HeLa cells (ATCC, USA) were cultured in DMEM, supplemented with 10% FBS and 1% penicillin/streptomycin antibiotics. RWPE-1 cells were cultured in Keratinocyte-SFM medium supplemented with human recombinant epidermal growth factor (5ng/ml) and bovine pituitary extract (0.05 mg/ml). All cells were maintained at 37°C in humidified 5% C0₂ atmosphere.

EXAMPLE: 3

Peptide synthesis

All peptides 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. EXAMPLE: 4

Cellular uptake of FITC labeled peptides.

To quantify the FITC-labeled peptide uptake, HeLa cells (2.0 x 10⁵ per well) were seeded onto 24 well plates at 24 h before the start of experiments. Cells were washed with PBS and incubated with FITC-labeled peptides (10 μΜ) in serum containing medium for 1 h at 37°C. Following the above treatment, cells were washed with PBS and then treated with trypsin for 10 min to remove extracellular, surface bound peptides. The cells were harvested and centrifuged at 1000 rpm for 5 min. The cell pellet was washed twice and finally resuspended in PBS. FITC fluorescence was immediately measured by flow cytometry using Accuri C6 flow cytometer (BD Biosciences) by acquiring 10,000 cells. The cells were gated using forward/side scatter, to measure uptake in only live cells. Experiments were carried out twice in triplicate. Data were obtained and analyzed using C6 software (BD Biosciences). Cellular autofluorescence was subtracted and results shown are the average of three measurements in the flow cytometer. The error bars indicate the standard error. Untreated cells were used as controls.

EXAMPLE: 5

Confocal microscopy

HeLa cells (1 x 10⁵ cells) were seeded onto 12 well plates containing 16 mm glass coverslips, 24 h prior to incubation with FITC-labeled peptides. After complete adhesion, the cell culture medium was removed, and then the cells were incubated at 37 °C for 1 h in fresh medium containing the fluorescently labeled peptides (10μΜ). Cells were not fixed to avoid artifactual localization of the internalized peptides. At the end of the incubation period, culture medium was removed and coverslips were washed thoroughly with ice cold PBS and mounted on slides with antifade reagent (Invitrogen, Molecular Probes). Localization of fluorescently labeled peptides in the live cells was analyzed immediately using Nikon AIR confocal microscope.

EXAMPLE: 6

Structural analysis of peptides.

Tertiary structures of all peptides examined were predicted using Pep-Fold. The figures were generated with PyMol (W.L. DeLano, The PyMol Molecular graphics system (2002) http://www.pymol.org). Helix wheel projections were generated using PEPwheel tool of emboss.

EXAMPLE: 7

CD spectroscopy

CD spectra of TAT and P8 were recorded using a nitrogen-flushed Jasco spectropolarimeter using a 0.1 cm quartz cell. The CD spectra were recorded using a bandwidth of 2 mn, a scan speed of 50 nm/min. Spectra of TAT and P8 were recorded in the water, 90% 2,2,2-trifluoroethanol (TFE) and SDS miceller solution (CMC = 8.2 mM). Each spectrum was the average of five scans with background of the water subtracted.

EXAMPLE: 8

Cytotoxicity of peptides.

The toxicity of peptides was assessed using MTS cell proliferation assay (CellTiter 96®AQueous Non-Radioactive Cell Proliferation Assay, Promega) according to the manufacturer's protocol. In brief, HeLa and CHO-Kl cells were seeded at a density of 5 x 103 cells/well in 96-well microtiter plates in DMEM supplemented with 10% FCS at least 12 h before the start of experiment. Cells were incubated with different concentrations (5, 10, 20, 40, 80 and 100 uM, respectively) of peptides for 24 hr. Control cells did not receive any peptide treatment. At the end of incubation period, peptide solution was discarded and replaced with the MTS assay reagent. Cells were placed back into the 37°C humidified atmosphere with 5% C02 for 4 h. Samples were prepared in triplicate and the absorbance was measured at 490 nm with an Infinite F200 plate reader (Tecan Systems Inc.). The survival of cells relative to the. control (cells incubated with growth medium containing no peptide) was calculated by taking the ratio of the A₄₉₀ values.

EXAMPLE: 09

Effect of ATP depletion and low temperature on uptake of peptide

To analyze the uptake of peptide at low temperature, HeLa cells were maintained at 4°C for 30 min followed by incubation with FITC-labeled peptide (5 Μ) at 4°C for additional 30 min. Similarly, to analyze the uptake of peptide under ATP depleted conditions, HeLa cells were pretreated with 0.1% sodium azide and 50mM DOG for 30 min. in OptiMEM followed by incubation with FITC labeled peptide (5μΜ) in the presence of sodium azide and DOG for further 30 min. Thereafter, cells were washed with PBS (2x), heparinized/trypsinized and suspended in PBS. Uptake was measured by flow cytometry as described above and compared with parallel uptake at 37°C.

EXAMPLE: 10

Effect of endocytosis inhibitors

To measure the effect of various endocytosis inhibitors on the uptake of peptide, HeLa cells were pretreated for 30 min at 37 °C, in OptiMEM, with (i) 30 μΜ CPZ; (ii) 10 μΜ Cyt D; (iii) 5 μΜ arniloride; and (iv) 20 μΜ Methyl-p-cytodextrin. Cells were then incubated with the FITC-labeled peptide (10 μΜ) in the presence of each inhibitor for another 30 min in OptiMEM. After the incubation, cell were washed with PBS and treated with trypsin for 10 min at 37 °C. The uptake of peptide was measured by flow cytometry as described above.

EXAMPLE: 11

CPP-eGFP fusion protein purification.

The pTat-eGFP and pP8-eGFP expression vectors were constructed by inserting eGFP from pEGFP-Nl (Clontech) into the pET28C vector (novagen) to produce an in- frame fusion protein. Each expression vector construct was transformed into BL21(DE3) competent cells, and recombinant proteins were purified obtained from the soluble fraction using affinity column chromatography. CPP-GFP was confirmed by Coomassie brilliant blue staining.

EXAMPLE: 12

P8 mediated delivery of GFP into HeLa cells.

To quantify the P8-GFP uptake, HeLa cells (2.0 x 10⁵ per well) were seeded onto 24 well plates at 24 h before the start of experiments. Cells were washed with PBS and incubated with P8-GFP (10 μΜ) in serum containing medium for 1 h at 37°C. Following the above treatment, cells were washed with PBS and then treated with trypsin for 10 min. The cells were harvested and centrifuged at 1000 rpm for 5 min. The cell pellet was washed twice and linaliy resuspended in PBS. GFP fluorescence was immediately measured by flow cytometry using Accuri C6 flow cytometer (BD Biosciences) by acquiring 10,000 cells. The cells were gated using forward/side scatter, to measure uptake in only live cells. Experiments were carried out twice in triplicate. Cellular autofluorescence was subtracted and results shown are the average of three measurements in the flow cytometer. The error bars indicate the standard error. Untreated cells were used as controls.

EXAMPLE: 13

Visualization of P8-GFP in HeLa cells.

HeLa cells (1 x 10⁵ cells) were seeded onto 12 well plates containing 16 mm glass coverslips, 24 h prior to incubation with P8-GFP. After complete adhesion, the cell culture medium was removed, and then the cells were incubated at 37 °C for 1 h in fresh medium containing the P8-GFP (10μΜ). Cells were not fixed to avoid artifactual localization of the internalized peptides. At the end of the incubation period, culture medium was removed and coverslips were washed thoroughly with ice cold PBS and mounted on slides with antifade reagent (Invitrogen, Molecular Probes). Localization of fluorescently labeled peptides in the live cells was analyzed immediately using Nikon AIR confocal microscope.

EXAMPLE: 14

Internalization of CPPs in Saccharomyces cerevisiae and bacterial cells.

Yeast and bacrterial cell (MTCC, India) were grown independently in 5 ml YPD broth - (DIFCO) and Mueller Hinton Broth (MHB-HiMedia Laboratories) respectively at 30°C to reach OD₆₀o - 0.13. 100 μΐ of cell suspension was added in each well and then the cells were incubated at 37°C for 1 h in PBS containing the fluorescently labeled CPPs (5 μΜ each). At the end of the incubation period, cells were washed twice with PBS and finally suspended in 500 μΐ PBS. In order to remove surface bound peptides, cells were treated with trypsin for 10 min at 37°C. After trypsin treatment, cells were washed again with PBS and suspended in 250 μΐ PBS. Finally cells were mounted on slides with anti-fade reagent (Invitrogen, Molecular Probes). Localization of fluorescently labeled peptides in the bacterial cells was analyzed immediately using Nikon AIR (Japan) confocal microscope.

EXAMPLE: 15 Susceptibility of S. aureus strains

We used two type of Staphylococcal strains viz. clinical isolates MRSA-831 obtained from Government Medical College & Hospital, Sector 32, Chandigarh and Staphylococcus aureus MTCC 96 (SA-96, Non-clinical) obtained from MTCC, IMTECH, Chandigarh. The susceptibility study was carried out as per previously described method (41). Both Staphylococcal strains were inoculated in 5ml MHB independently and kept for incubation at 30°C for 6-8 hrs (to reach OD₆o₀ = 0.13). Antibiotics (100 μΐ) at a specific concentration were added in first well of 96-well microtitre plate and then exponentially diluted. Bacterial inoculum equivalent to 0.5 McFarland standards were prepared and 100 μΐ was added to give the final concentration of 5 x 10⁵ cfu/ml, and incubated at 30°C for 48 h. 20 μΐ of MTT dye (10 mg/ml wt/v in methanol) was added to each well and kept for incubation at 30°C for 20 min. Bacterial growth indicated by purple coloration adhered to cells. The antibiotic concentration in the well at which no purple coloration observed, is the MIC of antibiotic. Similarly, the susceptibility of S. aureus strains to CPPs was determined. CPPs were dissolved in water and diluted with MHB to give the starting concentration of 50 μΜ in 100 μΐ mixture of first well. Further, dilution was done till 8th well to give the final concentration in range of 50 μΜ to 0.097 μΜ. The rest of the protocol was same as used in case of MIC of antibiotics. The CPP concentration in the well at which no purple coloration observed, is the MIC of CPP.

EXAMPLE: 16

Susceptibility of S. aureus strains to CPP-Norfloxacin cocktail

Staphylococcal strains were inoculated independently in 5 ml MHB and kept for incubation at 30°C for 6-8 hrs to reach OD₆₀₀ ~ 0.13. 100 μΐ of cell suspension was added in each well to make final concentration 5 x 10⁵ cfu/ml. 100 μΐ of CPPs was added in a well to make their final concentration 1 μΜ, 5 μΜ, 10 μΜ and 15 μΜ. Then norfloxacin added in wells at various sub-inhibitory concentrations. The microtitre plate was incubated at 30°C for 48 h. 20μ1 of MTT dye was added to each well and kept for incubation at 30°C for 20 min. The concentration in the well at which no purple coloration observed is the MIC of antibiotic in presence of respective CPP. ADVANTAGES OF THE INVENTION

1. The peptides P8 (SEQ ID NO. 1) show almost 10 times higher uptake in HeLa cells than TAT peptide.

2. The peptide P8 internalized into the live cells very efficiently with an endocytic process, making it a promising candidate for further molecular and cellular investigations.

3. P8 (SEQ ID NO. 1.) has amphipathic helical orientation of amino acid residues (both tryphophans are on one side while rest of the hydrophilic residues (arginine) at the opposite face). This amphipathic distribution of residues could be responsible for its high efficiency.

4. Despite its higher uptake efficiency, the isolated peptide did not show any significant membrane toxicity up to 40 μΜ concentration, which is much higher than the routinely tested concentrations.

5. P8 is derived from human protein and due to this it suppose to be low immunogenic.

6. P8 delivered GFP protein into HeLa cells with higher efficiency than TAT peptide.

7. The combination of P8 (represented by SEQ ID NO. 1) and P3 (represented by SEQ ID NO. 2) with norfloxin improved the efficacy of norfloxacin 168 fold against MRSA. TABLE-1: PEPTIDES SLECETD FOR TfflS STUDY

S.No SEQ ID NO's Sequence Region S issProt II) Protein name Organism Organism

(aa)

PI SEQ ID NO. 5 K XKKKNKKLQQRGD 87-101 Q9SB89 DEAD-box ATP-dependent RNA helicase 27 Arabidopsis thaliana

P2 SEQ ID NO. 6 RGDGPRRRPRKRRGR 431-445 Q63003 5E5 antigen Rat

P3 SEQ ID NO. 2 RRRQKRIWRRRLIR 497-511 Q01351 Protein U4 Human herpesvirus

P4 SEQ ID NO. 7 RRVWRRYRRQRWCRR 881-895 P56877 PE-PGRS3 Mycobacterium tuberculo

P5 SEQ ID NO. 8 RRARRPRRLRPAPGR 501-515 P17471 Envelope glycoprotein B Bovine herpesvirus

P6 SEQ ID NO. 9 LLRARWRRRRSRRFR 231-245 P35375 Prostaglandin E2 receptor EP1 subtype Mouse

P7 SEQ ID NO. 10 RGPRRQPRRHRRPRR 366-370 Q9UN88 GABA receptor subunit theta Human

P8 SEQ ID NO. 1 RRWRRWNRFNRRRCR 503-517 Q01668 Voltage-dependent L-type calcium channel subunit Human

alpha- ID

TABLE 2. SUSCEPTIBILITY OF S. AUREUS STRAINS AGAINST VARIOUS

ANTIBIOTICS.

S. aureus strains Erythromycin Teicoplanin Norfloxacin Oxacillin Linezolid Vancomycin Penetratin

S. aureus 831 25 12.5 100 2000 3.9 3.12 12.5 NAA 25 (MIC, μ^πιΐ)

S. aureus MTCC

96 (MIC, μ^ιηΐ) 0.78 1.56 0.39 3.12 1.56 1.56 NAA 50 25

TABLE 3. SUSCEPTIBILITY OF CPP NORFLOXACIN COCKTAIL AGAINST S. AUREUS STRAINS.

MIC of MIC of P3 MIC P8 MIC Sub-inhib. Sub-inhib. Con. Sub-inhib. Con. of Sub-inhib. Con. S. aureus Norflox Penetratin (μΜ) (μΜ) Con. of of Penetratin P3 of P8

(Hospital 25 X Y Y X Y isolate) 1.56 ND Y Y X Y

0.78 ND ND X Y X Y

S. aureus 0.39 NAA 50 25 0.195 X X Y Y

MTCC 96

0.097 Y X X Y Y (Non-clinical)

0.012 X X X X Y

NAA: NO ANTIBACTERIAL ACTIVITY AT 50 μΜ CONCENTRATIONS OF CPPS; □: COCKTAIL EFFECT; □: NO COCKTAIL EFFECT; ND: NOT DETERMINED

REFERENCES

1. Margus, H., Padari, K., and Pooga, M. (2012) Cell-penetrating peptides as versatile vehicles for oligonucleotide delivery. Mol Ther 20, 525-533

2. Trabulo, S., Cardoso, A. L., Cardoso, A. M., Morais, C. M., Jurado, A. S., and de Lima, M. C. (2012) Cell-penetrating peptides as nucleic acid delivery systems: from biophysics to biological applications. Curr Pharm Des 3. Presente, A., and Dowdy, S. F. (2012) PTD/CPP Peptide-Mediated Delivery of siR As. Curr Pharm Des

4. Nakase, I., Tanaka, G., and Futaki, S. (2013) Cell-penetrating peptides (CPPs) as a vector for the delivery of siRNAs into cells. Mol Biosyst

5. Shi, N. Q., Gao, W., Xiang, B., and Qi, X. R. (2012) Enhancing cellular uptake of activable cell-penetrating peiptide-doxorubicin conjugate by enzymatic cleavage. Int J Nanomedicine 7, 1613-1621 6. Walker, L., Perkins, E., Kratz, F., and Raucher, D. (2012) Cell penetrating peptides fused to a thermally targeted biopolymer drug carrier improve the delivery and antitumor efficacy of an acid-sensitive doxorubicin derivative. Int J Pharm 436, 825-832

7. Nasrollahi, S. A., Fouladdel, S., Taghibiglou, C, Azizi, E., and Farboud, E. S. (2012) A peptide carrier for the delivery of elastin into fibroblast cells. Int J Dermatol 51, 923-929

8. Boisguerin, P., Giorgi, J. M., and Barrere-Lemaire, S. (2012) CPP-conjugated anti- apoptotic peptides as therapeutic tools of ischemia-reperfusion injuries. Curr Pharm Des 9. Mo, R. PL, Zaro, J. L., and Shen, W. C. (2012) Comparison of cationic and amphipathic cell penetrating peptides for siRNA delivery and efficacy. Mol Pharm 9, 299- 309 10. Xia, H., Gao, X., Gu, G., Liu, Z., Hu, Q., Tu, Y., Song, Q., Yao, L., Pang, Z, Jiang, X., Chen, J., and Chen, H. (2012) Penetratin-functionalized PEG-PLA nanoparticles for brain drug delivery. Int JPharm 436, 840-850

5 11. Heitz, F., Morris, M. C, and Divita, G. (2009) Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics. Br J Pharmacol 157, 195-206

12. Tyagi, M., Rusnati, M., Presta, M., and Giacca, M. (2001) Internalization of HIV- 1 tat requires cell surface heparan sulfate proteoglycans. J Biol Chem 276, 3254-3261

L0

13. Derossi, D., Calvet, S., Trembleau, A., Brunissen, A., Chassaing, G., and Prochiantz, A. (1996) Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. J Biol Chem 111, 18188-18193

L5 14. Gautam, A., Singh, H., Tyagi, A., Chaudhary, K., Kumar, R., Kapoor, P., and Raghava, G. P. (2012) CPPsite: a curated database of cell penetrating peptides. Database (Oxford), basOl 5

15. Elliott, G., and O'Hare, P. (1997) Intercellular trafficking and protein delivery by a 10 herpesvirus structural protein. Cell 88, 223-233

16. Jha, D., Mishra, R., Gottschalk, S., Wiesmuller, K. H., Ugurbil, K., Maier, M. E., and Engelmann, J. (201 1) CyLoP-1 : a novel cysteine-rich cell-penetrating peptide for cytosolic delivery of cargoes. Bioconjug Chem 22, 319-328

IS

17. Milletti, F. (2012) Cell-penetrating peptides: classes, origin, and current landscape. Drug Discov Today 17, 850-860

18. Futaki, S., Suzuki, T., Ohashi, W., Yagami, T., Tanaka, S., Ueda, K., and Sugiura, Y. 30 (2001) Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J Biol Chem 276, 5836-S840

19. Kosuge, M., Takeuchi, T., Nakase, I., Jones, A. T., and Futaki, S. (2008) Cellular internalization and distribution of arginine-rich peptides as a function of extracellular peptide concentration, serum, and plasma membrane associated proteoglycans. Bioconjug Chem 19, 656-664

20. Futaki, S. (2005) Membrane-permeable arginine-rich peptides and the translocation mechanisms. Adv Drug Deliv Rev 57, 547-558

21. Maiolo, J. R., Ferrer, M., and Ottinger, E. A. (2005) Effects of cargo molecules on the cellular uptake of arginine-rich cell-penetrating peptides. Biochim Biophys Acta 1712, 161- 172

22. Christiaens, B., Grooten, J., Reusens, M., Joliot, A., Goethals, M., Vandekerckhove, J., Prochiantz, A., and Rosseneu, M. (2004) Membrane interaction and cellular internalization of penetratin peptides. Eur JBiochem 271, 1187-1197

23. Kalafut, D., Anderson, T. N., and Chmielewski, J. (2012) Mitochondrial targeting of a cationic amphiphilic polyproline helix. Bioorg Med Chem Lett 22, 561-563

24. Gautam, A., Chaudhary, K., Kumar, R., Sharma, A., Kapoor, P., Tyagi, A., and Raghava, G. P. (2013) In silico approaches for designing highly effective cell penetrating peptides. J Transl Med 11, 74

25. Oba, M., Fukushima, S., Kanayama, N., Aoyagi, K., Nishiyama, N., Koyama, H., and Kataoka, K. (2007) Cyclic RGD peptide-conjugated polyplex micelles as a targetable gene delivery system directed to cells possessing alphavbeta3 and alphavbeta5 integrins. Bioconjug Chem 18, 1415-1423

26. Numata, K., Hamasaki, J., Subramanian, B., and Kaplan, D. L. (2010) Gene delivery mediated by recombinant silk proteins containing cationic and cell binding motifs. J Control Release 146, 136-143

27. Nakase, I., Hirose, H., Tanaka, G., Tadokoro, A., Kobayashi, S., Takeuchi, T., and Futaki, S. (2009) Cell-surface accumulation of flock house virus-derived peptide leads to efficient internalization via macropinocytosis. Mol Ther 17, 1868-1876 28. Rydberg, H. A„ Matson, M., Amand, H. L., Esbjorner, E. K., and Norden, B. (2012) Effects of tryptophan content and backbone spacing on the uptake efficiency of cell- penetrating peptides. Biochemistry 51, 5531-5539 29. Eiriksdottir, E., Konate, K., Langel, U., Divita, G., and Deshayes, S. (2010) Secondary structure of cell-penetrating peptides controls membrane interaction and insertion. Biochim Biophys Acta 1798, 1 119-1128

30. Duchardt, F., Fotin-Mleczek, M., Schwarz, H., Fischer, R., and Brock, R. (2007) A comprehensive model for the cellular uptake of cationic cell-penetrating peptides. Traffic 8,

848-866

31. Richard, J. P., Melikov, K., Brooks, H., Prevot, P., Lebleu, B., and Chernomordik, L. V. (2005) Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors. J Biol Chem 280, 15300-15306

32. Gomez, J. A., Chen, J., Ngo, J., Hajkova, D., Yeh, I. J., Gama, V., Miyagi, M., and Matsuyama, S. (2010) Cell-Penetrating Penta-Peptides (CPP5s): Measurement of Cell Entry and Protein-Transduction Activity. Pharmaceuticals (Basel) 3, 3594-3613.

33. Splith, K., and Neundorf, I. (2011). Antimicrobial peptides with cell-penetrating peptide properties and vice versa. Eur Biophys J 40, 387-397.

34. Henriques, S.T., Melo, M.N., and Castanho, M.A. (2006). Cell-penetrating peptides and antimicrobial peptides: how different are they? Biochem J 399, 1-7.

35. Fonseca, S. B., Pereira, M.P., and Kelley, S.O. (2009). Recent advances in the use of cell-penetrating peptides for medical^' and biological applications. Adv Drug Deliv Rev 61, 953-964.

36. Peters, B.M., Shirtliff, M.E., and Jabra-Rizk, M.A. (2010). Antimicrobial peptides: primeval molecules or future drugs? PLoS Pathog 6:el001067. 37. Choi, H., and Lee, D.G. (2012). Synergistic effect of antimicrobial peptide arenicin-1 in combination with antibiotics against pathogenic bacteria. Res Microbiol 163, 479-486.

38. Sharma, S.P., Sharma, J., Kanwar, S.S., and Chauhan, V.S. (2012). In vitro 5 antibacterial and antimalarial activity of dehydrophenylalanine-containing undecapeptides alone and in combination with drugs. Int J Antimicrob Agents 39, 146-152.

39. Bobone, S., Piazzon, A., Orioni, B., Pedersen, J.Z., Nan, Y.H., Hahm, K.S., Shin, S.Y., and Stella, L. (201 1). The thin line between cell-penetrating and antimicrobial peptides:

0 the case of Pep-1 and Pep-l-K. JPept Sci 17, 335-341.

40. Bahnsen, J.S., Franzyk, H., Sandberg-Schaal, A., and Nielsen, H.M. (2013). Antimicrobial and cell-penetrating properties of penetratin analogs: effect of sequence and secondary structure. Biochim Biophys Acta 1828, 223-232.

.5

41. Roy, S. ., Kumari, N., Pahwa, S., Agrahari, U. C, Bhutani, K.K., Jachak, S.M., and Nandanwar, H. (2013). NorA efflux pump inhibitory activity of coumarins from Mesua ferrea. Fitoterapia 90, 140-150.

'.0 42. Sparr, C, Purkayastha, N., Kolesinska, B., Gengenbacher, M., Amulic, B.,

Matuschewski, K., Seebach, D., and Kamena, F. (2013). Improved efficacy of fosmidomycin against Plasmodium and Mycobacterium species by combination with the cell-penetrating peptide octaarginine. Antimicrob Agents Chemother 57, 4689-4698.

>5

Claims

1. A cell-penetrating peptide having amino acid sequence selected from the group consisting of Seq Id no. 1, 2, 3, and 4.

2. Use of the cell-penetrating peptide as claimed in claim 1 in treatment of diseases selected from the group comprising of bacterial infection; diabetes, skin disorders, cancer.

3. A complex useful for delivery of a cargo inside the cell comprising a cell-penetrating peptide according to claim 1 and a cargo wherein the cargo is selected' from the group comprising of a chromophore, a protein, an antibiotic, a peptide, nucleic acid, nanoparticles, drugs.

4. The method of intracellular delivery comprising:

(a) providing a cell penetrating peptide of claim 1, and

(b) incubating the cell penetrating peptide obtained in step (a) with targeted cells. ^'

5. The method as claimed in claim 5, wherein the targeted cell is selected from the group comprising of a eukaryotic cell, prokaryotic cell.