DESC:RELATED PATENT APPLICATION:

This application claims the priority to and benefit of Indian Patent Application No. 202341048749 filed on January 20, 2024; the disclosures of which are incorporated herein by reference

FIELD OF THE INVENTION:

The present invention relates to the field of synthetic peptides. Particularly the present invention relates to identification of apoptotic cells, cancer cells, extra cellular vesicles, and their application thereof. More particularly, the present invention relates to the designing of a non cell penetrating and metacyclic synthetic peptide to detect the phosphatidyl serine. Specifically, the synthetic peptides of the present invention mimic the topography of the phosphatidyl serine specific membrane binding region of Annexin V. More specifically, the present invention describes the use of synthetic peptides in atherothrombosis, in vitro diagnostics, extracellular vesicles, immunomodulation, apoptosis, and Cancer cells.

BACKGROUND OF THE INVENTION:

Cancer is the leading cause of morbidity and mortality across the world. For successful treatment of cancer, early detection is the most crucial stage. Early diagnosis involves detecting symptomatic patients as early as possible and providing them with timely and appropriate care. Early diagnosis is particularly relevant for cancers that have a long preclinical phase and a high potential for cure if detected early, such as breast, cervical, oral, laryngeal, colorectal, and skin cancers. Many researchers are studying both the diagnosis and treatment of cancer, and their main concern in cancer diagnosis is the early detection of cancer.

Synthetic peptides can be used for the early detection of cancer by selectively targeting cancer cells. Peptides can be designed to bind to specific receptors or molecules that are overexpressed on the surface of cancer cells. Synthetic peptides can mimic or inhibit the action of naturally occurring peptides or proteins in the body, making them useful as drugs for the treatment of cancer and other diseases. By incorporating a fluorescent or radioactive label, these peptides can be used as imaging agents to visualize cancer cells in the body. This approach has the potential to improve the early detection of cancer, allowing for more effective treatment and better patient outcomes.

The cancer cells have a Phosphatidylserine (PS) molecule on their surface membrane which is normally found on the inner leaflet of the normal cell membrane. This exposure of PS on cancer cells has been targeted for the development of synthetic peptides in detection and treatment of cancer. Synthetic peptides have enhanced membrane poration through binding to PS, leading to increased cell death in cancer cells.

Annexin V is known to be used to detect apoptosis in cancer cells by binding to exposed PS molecules. It is a protein that has high affinity to phosphatidylserine (PS) molecules. However, Annexin V requires calcium presence to detect cell death. Though some versions of Annexin V (pSIVA-IANBD, Novus Biologicals) have been developed to be calcium free, it still requires binding buffer. In cohesion with aforementioned observation the binding buffer (0.5mM to 2.5 mM Ca2+ buffer) does cause cell death on its own such as cell death by calcium buffer among K562 and other cell types.

The present available Annexin kit for the apoptosis market includes: BioLegend (Apotracker Green), FUJIFILM Wako Chemicals (Mag Capture Exosome Isolation kit) pSIVA-IANBD (Novus Biologicals). These are Annexin V based kits which require a binding buffer and calcium whether present or absent, in order to bind to PS and target cancer cells.

There are some synthetic peptides known to have PS specificity without the need for calcium binding. Said synthetic peptides can be linear or cyclic. WO2016089187A1 discloses the cyclic peptide (cyclo [Cys-Gln-Arg-Pro-Pro-Arg-Cys] peptide) that has a specificity to bind to the Phosphatidylserine (PS) molecule. The cyclic peptide can be used in early diagnosing a response of a drug for treating diseases associated with abnormal cell proliferation and tissues afflicted with Apoptosis associated diseases.

WO2016207626A1 discloses the fluorogenic amino acid derivatives, which can be used as optical probes. It also discloses the processes for the preparation of such compounds, the use of such compounds as probes and methods of detecting a target using such compounds as probes.

A research article by Barth et al., 2020, a fluorogenic cyclic peptide for imaging and quantification of drug-induced apoptosis, Nature Communications, 11:4027, discloses the design of a highly stable fluorogenic peptide (termed Apo-15) that selectively stains apoptotic cells in vitro and in vivo in a calcium-independent manner and under wash-free conditions. It also discloses that the phosphatidylserine is a molecular target of Apo-15. Apo-15 can be used for assessing the in vivo efficacy of anti-inflammatory and anti-cancer therapeutics.

These prior arts show phosphatidylserine specificity without the need for calcium binding and an alternative to Annexin V. But these are cell penetrating peptides acting by penetrating the cell membrane. This membrane penetration of the cyclic peptide may induce membrane rupture and potential toxicity.

Thus, there is need for new synthetic peptides that can be used for the early detection of cancer, without causing membrane rupture, cell death or inducing toxicity.

OBJECTS OF THE INVENTION:

The primary object of the present invention is to provide a novel synthetic peptides, particularly non-cell penetrating, metacyclic synthetic peptides.

Another object of the present invention is to develop a non-cell penetrating peptides to detect phosphatidylserine (PS).

Another object of the present invention is to design the metacyclic peptides mimicking the topography of the PS specific membrane binding region of Annexin V.
Yet another object of the present invention is to develop a non-cell penetrating synthetic peptides for the detection of apoptotic cells, extracellular vesicles, cancer cells and functions thereof.

SUMMARY OF THE INVENTION:
In one non-limiting embodiment of the present disclosure, synthetic non-cell permeating peptide (non-CPP) for detection of apoptotic cells, extracellular vesicles, cancer cells and related cells is disclosed. The synthetic non-cell permeating peptide (non-CPP) for detection of apoptotic cells, extracellular vesicles, cancer cells and related cells comprises amino acid sequences. Further, one or more amino acids in the sequences are selected from the group comprising of Arginine (Arg), Glycine (Gly), Aspartic Acid (Asp), Glutamic Acid (Glu), Leucine (Leu), Serine (Ser), Proline (Pro), Lysine (Lys), Cysteine (Cys), Histidine (His), Asparagine (Asn), Threonine (Thr), Phenylalanine (Phe), Tryptophan (Trp), Tyrosine (Tyr) and Alanine (Ala).

In an embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has structure of the amino acid sequences is selected from linear, metacyclic and cyclic.

In another embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has amino acid sequence disclosed in SEQ.NO: 01.

In another embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has amino acid sequence disclosed in SEQ.NO: 02.

In another embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has amino acid sequence disclosed in SEQ.NO: 03.
In another embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has amino acid sequence disclosed in SEQ.NO: 04.

In another embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has amino acid sequence disclosed in SEQ.NO: 05.

In another embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has amino acid sequence disclosed in SEQ.NO: 06.

In another embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has amino acid sequence disclosed in SEQ.NO: 07.

In another embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has amino acid sequence disclosed in SEQ.NO: 08.

In another embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has amino acid sequence disclosed in SEQ.NO: 46.

In another embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has amino acid sequence disclosed in SEQ.NO: 47.

In another embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has amino acid sequence disclosed in SEQ.NO: 48.
In another embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has amino acid sequence disclosed in SEQ.NO: 49.

In another embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has amino acid sequence disclosed in SEQ.NO: 50.

In another embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has amino acid sequence disclosed in SEQ.NO: 52.

In another embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has amino acid sequence disclosed in SEQ.NO: 54.

In another embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has amino acid sequence disclosed in SEQ.NO: 55.

In another embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has amino acid sequence disclosed in SEQ.NO: 56.

In another embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has amino acid sequence disclosed in SEQ.NO: 57.

In another embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has amino acid sequence disclosed in SEQ.NO: 58.
In another embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has amino acid sequence disclosed in SEQ.NO: 59.

In another embodiment of the present invention, the synthetic non-cell permeating peptide (non-CPP) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has the amino acid sequences are selected from non-cpp peptides, non-cpp and hairpin-loop forming peptides, or cyclic peptides mimicking annexin V functional motif and cysteine knot functional group and branches that adhere to membrane.

In one non-limiting embodiment of the present disclosure, the use of the synthetic non-cell permeating peptide (non-CPP) for detection of apoptotic cells, extracellular vesicles, cancer cells and related cells is disclosed. The synthetic non-cell permeating peptide (non-CPP) for detection of apoptotic cells, extracellular vesicles, cancer cells comprises one or more amino acids selected from the group comprising of Arginine (Arg), Glycine (Gly), Aspartic Acid (Asp), Glutamic Acid (Glu), Leucine (Leu), Serine (Ser), Proline (Pro), Lysine (Lys), Cysteine (Cys), Histidine (His), Asparagine (Asn), Threonine (Thr), Phenylalanine (Phe), Tryptophan (Trp), Tyrosine (Tyr) and Alanine (Ala).

In one non-limiting embodiment of the present disclosure, the use of synthetic non-cell permeating peptide (non-CPP) for detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has the peptide comprises amino acid sequences selected from SEQ No. 1, 2, 3, 4, 5, 6, 7, 8, 46, 47, 48, 49, 50, 52, 54, 55, 56, 57, 58, and 59.

In one non-limiting embodiment of the present disclosure, a method of detecting apoptotic cells, extracellular vesicles, cancer cells and related cells in a subject by using synthetic non-cell permeating peptide (non-CPP) is disclosed. The synthetic non-cell permeating peptide (non-CPP) for detection of apoptotic cells, extracellular vesicles, cancer cells comprises one or more amino acids selected from the group consisting of Arginine (Arg), Glycine (Gly), Aspartic Acid (Asp), Glutamic Acid (Glu), Leucine (Leu), Serine (Ser), Proline (Pro), Lysine (Lys), Cysteine (Cys), Histidine (His), Asparagine (Asn), Threonine (Thr), Phenylalanine (Phe), Tryptophan (Trp), Tyrosine (Tyr) and Alanine (Ala).

In one non-limiting embodiment of the present disclosure, the method of synthetic non-cell permeating peptide (non-CPP) for detection of apoptotic cells, extracellular vesicles, cancer cells and related cells has the peptide comprises amino acid sequences selected from SEQ No. 1, 2, 3, 4, 5, 6, 7, 8, 46, 47, 48, 49, 50, 52, 54, 55, 56, 57, 58, and 59.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Schematic representation of the metacyclic peptide mimicking the Annexin V like structure binding to PS unlike CyclicRKKWF showing a deep penetration into the membrane.
(A) Metacyclic peptide- CyGAG
(B) Annexin V
(C) Cyclic peptide- CyRKKWF

FIG. 2: Shows a comparative fluorescence images of cell line, primary tumour cell and healthy cell in presence of the DAD/DAPI drug treatment.

FIG. 3: Shows a comparison of the staining of ANNEXIN V in + Ca2 Buffer and - Ca2 Buffer. Another is for DAD2-Cy3 in + Ca2 Buffer and - Ca2 Buffer.

FIG. 4: Shows a graph of DAD2-Cy3 for comparing the Imatinib and control.

FIG. 5: Shows comparative fluorescence images of cell in presence of the DAD/DAPI drug treatment at concentration of 1 µM, 5 µM, 10 µM, 50 µM, 100 µM and control.

FIG. 6: Shows representative molecular graphics for Peptide-1.

FIG. 7: Shows representative molecular graphics for Peptide-2.

FIG. 8: Shows representative molecular graphics for Peptide-3.

FIG. 9: Shows representative molecular graphics for Peptide-4.

FIG. 10: Shows representative molecular graphics for Peptide-5.

FIG. 11: Shows representative molecular graphics for Peptide-6.

FIG. 12: Shows representative molecular graphics for Peptide-7.

FIG. 13: Shows representative molecular graphics for Peptide-8.

FIG. 14: Shows representative molecular graphics for test cyclic peptide.

FIG. 15: Shows representative molecular graphics for Peptide-cy2 (47).

FIG. 16: Shows representative molecular graphics for Peptide-cy3 (49).

FIG. 17: Shows representative molecular graphics for Peptide-cy4 (52).

FIG. 18: Shows HMC3 Cells Treated with 3µM LLOME that were checked with DAD3 with or without the presence of CEYDEY motif at the N-Terminus.

FIG. 19 shows the control Annexin V structure (PDB code: 1AXN); four domains (each with five alpha helices) of Annexin V are shown in Red (I) Yellow (II) Blue (III) and Green (IV).The Lys186 (shown at extreme right) in domain III is primarily involved in POPS interaction.

FIG. 20 shows control as the Bovine Lactadherin C2 domain (PDB code: 3BN6) anchors to the membrane through its three spikes, coloured in different shades of pink. Each spike possesses at least one aromatic amino acid (i.e. Spike1: Trp26, Spike2: Phe46 and Spike3: Phe81). The residue Arg79 (shown at cyan stick) primarily involves POPS interaction.

FIG. 21 shows representative molecular graphics for Apotracker Seq-cy1 – RKKWF

FIG. 22 shows representative moleculargraphics for
Seq1: ERGDELGGSGPKPPSKKRSCDPSHHHHHH

FIG. 23 shows representative moleculargraphics for
Seq2- ERGDELPKPPSKKRSCDPS

FIG. 24 shows representative moleculargraphics for
Seq 2 Cyclic ERGDELPKPPSKKRSCDPS

FIG. 25 shows representative moleculargraphics for
Seq 4 CEYDEYGSPKPKKRGS

FIG. 26 shows Origin of cyGAG: The distance between the centre of mass of different domains of Annexin V was calculated, as shown in Table 1. The cyGAG sequence was designed to match the topology of annexin V with half the surface area. Two Arg and two Trp AA were incorporated into the sequence (called functional AA), and each of these amino acids was separated by a tripeptide ‘Gly-Ala-Gly’ (i.e. GAG). The GAG-tripeptides provide sharp kinks in the polypeptide sequence, allowing easy cyclisation of the polypeptide (thus named cyGAG). The peptide structure was optimised through MD simulations, and it was found that cyGAG occupies a superhelical twisted structure by bringing Trp residues closer (p-p interaction). This further improves the peptide stability. The orientation of all four functional AAs of cyGAG was maintained on one side to facilitate membrane interactions.

FIG. 27 shows representative moleculargraphics for Seq 5 GAGWGAGRGAGWGAGR

FIG. 28 shows representative moleculargraphics for Seq 6 Cy2b: GAGWGAGRGAEWGCGR

FIG. 29 shows representative moleculargraphics for Seq 7 CLRNTSGFKRWKKQF

FIG. 30 shows representative moleculargraphics for Seq 7-cyc- CLRNTSGFKRWKKQF

FIG. 31 shows representative moleculargraphics for Seq 8 CRKWKKNWFDLWSD

FIG. 32 shows representative moleculargraphics for Seq 8 –Cyc CRKWKKNWFDLWSD

FIG. 33 shows representative moleculargraphics for Seq 9 PKPPSKKRSCDPS

FIG. 34 shows representative moleculargraphics for Seq 10-cy4- PKPGSKKRS

FIG. 35 shows representative moleculargraphics for Negative Control Seq 11 PEPGSEGGSCPSK.

FIG. 36 shows representative moleculargraphics for Negative Ctrl Seq 11 -Cyc PEPGSEGGSCPSK.

FIG. 37 shows representative moleculargraphics for Seq 12- WCRKWKKNWFDLWSDW.

FIG. 38 shows representative moleculargraphics for Seq 13- WSWGRKKGWSW.

FIG. 39 shows representative moleculargraphics for Seq 14- PSPGRKKGPSP.

FIG. 40 shows representative moleculargraphics for Seq 15- PSPWGKKRGWPSP.

FIG. 41 shows representative moleculargraphics for Seq 16- WFPKPSKKRSCDSFW.

FIG. 42 shows representative moleculargraphics for Seq 17- PCPWGKPKRPKGWPSP.

FIG.43 shows representative molecular graphics for Seq.17-cyc-PCPWGKPKRPKGWPSP.

DETAILED DESCRIPTION OF THE INVENTION
Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described herein. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, and components referred to or indicated in this specification, individually or collectively, and all combinations of any two or more of said steps or features.

The terminology used, in the present invention, is only for the purpose of explaining a particular embodiment and such terminology shall not be considered to limit the scope of the present invention. As used in the present disclosure, the forms "a,” "an," and "the" may be intended to include the plural forms as well, unless the context clearly suggests otherwise. The terms "comprises," "comprising," “including,” and “having,” are open ended transitional phrases and therefore specify the presence of stated features, integers, steps, methods, elements, modules, units and/or components, but do not forbid the presence or addition of one or more other features, integers, steps, methods, elements, components, and/or groups thereof.

The present invention describes the development of a non-cell penetrating metacyclic synthetic peptides to detect phosphatidyl serine (PS) for the detection of apoptotic cells, extracellular vesicles, cancer cells and related cells.

The present inventors started working on the viral protein Apoptin, since 2012. The present inventors have found that 10 amino acid peptides derived from apoptin (Seq 1: PKPPSKKRSC) is sufficient to bind to BCR-Abl protein and inhibit cancer growth in such cells even though they are resistant to chemotherapeutic drugs (Jangamreddy et al., 2015, Oncotarget, 6(1): 10134- 10145). The inventors further continued this work and during 2017-2019 remodeled the original peptide to 12 amino acid version (Seq 2: PKPPSKKRSCDPS) and tagged another cell penetrating motif (Seq 3: ERGDEL) along with His tag to make it a 29 amino acid peptide (Seq 4: ERGDELGGSGPKPPSKKRSCDPSHHHHHH) with spacer Glysine and Serine. As a control, another peptide was designed with a motif which can predicted to inhibit the cell penetration (Seq 5: EEGEELGGSGPEPGSEEGSCDPSHHHHHH).

Based on imaging data provided while testing several peptides for cell penetration and cell targeting during those experiments, it was observed that, altered peptides bind to apoptotic cells or killing the cells irrespective of the adjacent motifs attached using immunohistochemistry with anti-HIS antibodies. Based on these observations, the present inventors started to apply the reverse engineering on these sequences using in silico approaches and observed that both 29 amino acid versions (Seq 4 & 5) are non-cell penetrating and have strong preferential binding to Phosphatidylserine. Based on these observations, the present inventors have redesigned various versions of the peptides that do not show cell permeability and have strong preferential binding to apoptotic cells.

Overcoming the prior art problems, the synthetic peptides (linear and circular) of the present invention do not penetrate the cell membrane and also do not require any buffer for their function to detect phosphatidyl serine expressed on outer membrane of the cells. Thus, present inventors proposes that the present peptides can be used in the native media of the cells with FBS or without FBS or PBS without any need of special buffers to detect apoptosis and thus can give absolute cell death induced by the agent or present in the cells. Since these peptides are small and can be incorporated with few D-Amino acids or synthetic amino acids, can escape from immune detection, and can penetrate the tissues very easily. Thus, can be used under in vivo, in vitro conditions to detect apoptotic cells, Extracellular vesicles, apoptotic bodies, and cancer cells that express phosphatidyl serine on the outer membrane.

This is one of the novel approaches to design the non-cell penetrating, metacyclic synthetic peptides and in-silico studies shown that the membrane penetration of the cyclic peptide has the potential to induce membrane rupture and toxicity.

In one embodiment, the present invention provides a non-cell penetrating metacyclic synthetic peptides that detect phosphatidyl serine (PS).

In another embodiment, the present invention provides a synthetic peptide as a non-cell penetrating and binds only superficially to the PS on the membrane.

In one embodiment, the present invention provides a non-cell penetrating linear peptides, metacyclic peptides which is mimicking the Annexin V functional binding region topography.

In one embodiment, the present invention provides a tool to identify epitope interacting peptides mimicking the topography of the natural binding molecules.

Accordingly, in one aspect, the present invention provides small peptide motifs that are designed to mimic the Annexin V epitope region binding to phosphatidyl serine (PS) specifically.

While the earlier discoveries of small peptides like LIKKPF, RKKWF and CQRPPRC, are shown to bind to PS, the linear peptides are cell permeable and the latter two are cyclized and thus restricting cell penetration. The in-silico studies show the RKKWF peptide linked to BODIPY even though cyclized it shows a deep penetration into the membrane and CQRPPRC peptides shows least efficiency in binding to PS. Because of such deep cell penetration, the charged amino acids in the peptide and the fluorophore show interaction with phosphate groups along with serine, thus impacting both the cell membrane integrity and non-specific binding.

The present invention provides the peptide motifs of Apoptin and Evectin that are non-cell penetrating and/or metacyclic peptides, which modulate to imitate the structural topography of Annexin V.

In one embodiment, the present non-cell penetrating and/or metacyclic peptides provides the advantage of hovering over the membrane to bind to the PS without penetrating the membrane unlike the small peptides or their cyclic counterparts that integrate into the membrane and show interactions with phosphate group than serine specific interactions.

In another embodiment the present invention discloses the use of CEYDEY, ERGDEL, EEGEEL, CLRNTSG, FDLWSD and other non-CPP motifs.

The Non-CPP peptides at the N-terminus of the PS binding peptides are more effective in limiting the penetration and PS binding than at the C-Terminus.

These non-CPP motifs comprise presence of Arg which enhances binding to phosphatidyl serine (PS).

The Non-Cell penetrating motif of the present invention can be used with either apoptin, Evectin, Lactederin derived peptides, or other small cell penetrating peptides that bind to PS including LIKKPF, RKKWF etc.

Further the effective separation of at least 3 to 6 amino acids in between the Arg/Lys amino acids either as singlets, doublets, or triplets (homo or heteromers) in the peptide enhances the binding to PS and number of interactions. E.g., R/KXXXR/K, R/KR/KXXX(n)R/KR/K, etc.

The Glu and Arg or combination positive charged and negative charged side chains of amino acids such as Asp/Arg, Glu/Lys can be used to form a salt bridge to make a “cyclic” peptide with the PS binding peptides.

Said Cyclic peptides form either permanent Cyclization with end-to-end conjugation (COOH-NH2) or Cysteine (SH-SH) linkage mentioned including CyGAG, CyApoptin, CyEvectin, etc. with non-CPP motifs.

Said cyclic peptide is more than 9 amino acids with the mentioned sequences that hinders cell penetration.

In one embodiment of the present invention provides a metacyclic peptide (A) mimicking the Annexin V (B) like structure binding to PS unlike CyclicRKKWF (C) showing a deep penetration into the membrane as shown in FIG. 1. These metacyclic peptides (A) hover over the membrane to bind to the PS without penetrating the membrane unlike the small peptides or their cyclic counterparts (C) that integrate into the membrane and show interactions with phosphate group than serine specific interactions.

In another embodiment, the present invention provides the linear or cyclic synthetic peptides sequences as listed in Table 1.

Table 1. Non-cell penetrating metacyclic peptide sequences.
(Where, [-] means Non-CPP; FC1-Only sequence based; FC2-Only Structure based; FC4-All features)
Sr. No. Sequence CPP % (FC1) CPP % (FC2) CPP % (FC4)
1 >DAD1
ERGDELGGSGPKPPSKKRSCDPSHHHHHH [-]86.64 [-]94.08 [-]92.31
2 >DAD2
ERGDELPKPPSKKRSCDPS [-]76.57 [-]77.34 [-]87.34
3 >DAD3
PKPPSKKRSCDPS 76.06 57.27 73.46
4 >DAD4
EEGEELGGSGPEPGSEEGSCDPSHHHHHH [-]94.98 [-]89.33 [-]92.07
7 >DAD6
RGDGPKPPSKKRSCDPSHHHHHH [-]58.0 [-]79.95 [-]73.54
9 >DAD8
RGKPKPPSKKRSCDPSHHHHHH 60.89 [-]50.63 [-]60.4
10 >DAD9
RGDPKPPSKKRSCDPS 71.43 53.54 [-]53.39
11 >Test Control 1
LIKKPF- 86.64 81.84 84.28
12 >Test Control 2
RKKWF 83.94 85.87 89.05
13 >Test56
ERGDELGGSPKPKKRK [-]77.79 57.14 [-]75.72
14 >Test57
KRKKPKPSGGLEDGRE [-]76.24 [-]75.64 [-]74.84
15 >Test58
RLGGKSEGDKKPKPRE [-]81.52 56.15 [-]77.71
16 >Test59
RLGGKSEGDKKPKPRD [-]77.97 56.5 [-]73.09
17 >Test60
KLGGDSEGDKKPKPR [-]81.17 [-]61.56 [-]77.87
18 >Test61
KLGKKPKPRGDSEGD [-]84.95 [-]61.55 [-]81.02
19 >Test62
RLKGKKPKPGDSEGD [-]84.95 [-]61.55 [-]53.26
20 >Test63
KRGKKPKPGDVSEGD [-]85.41 [-]61.33 [-]68.76
21 >Test64
KPKKRKGGSERDELP [-]70.45 61.4 [-]57.93
22 >Test65
CKRGKKPKPGDVSEGD [-]86.45 [-]69.4 [-]74.93
23 >Test66
CKRGKKPKPGAVESEG [-88.52] [-54.12] [-]81.53
24 >Test67
CKRGKKPKPGAVESE [-]86.76 [-]50.16 [-]79.16
25 >Test68
KRGKKPKPGAVESEC [-87.97] [-53.34] [-]79.16
26 >Test69
CEGVESKGKKPKPR [-54.35] [- 58.46] [-]53.07
27 >Test70
CEGDVESKGKKPKPR [-62.52] [-55.31] [-]74.6
28 >Test71
CEPDPESKGKKPKPR [-69.86] [-51.5] [-]57.28
29 >Test72
CERGDELPKPPSKKRSDPS [-71.61] [-68.61] [-]87.28
30 >Test73
CERGDELPKPPSKKRSP [-59.89] [-50.11] [-]78.28
31 >Test74
CERGDELPKPPSKKRP [-65.78] [-55.76] [-]73.28
32 >Test75
CERGDELPKPPSKKPR [-57.22] [-55.76] [-]67.28
33 >Test76
CERNDEYPKPPSKKPR [-51.41] [-62.88] [-]78.28
34 >Test77
CENDEYPKPPSKKPR [-76.63] [-78.42] [-]85.38
35 >Test78
CEYDEYPKPPSKKPR [-78.2] [-80.04] [-]86.04
36 >Test79
CEYDEYPKPYSKKPR [-78.89] [-83.03] [-]86.39
37 >Test80
CEYDEYPKPYSKKYR [-78.96] [-82.56] [-]84.8
38 >Test81
CEYDEYPKPKKYR [-]82.2 [-]82.1 [-]79.8
39 >Test82
CEYDEYRKKWF [-]81.2 [-]66.85 [-]77.28
40 >Test83
CEYDEYLIKKPF [-]88.5 [-]89.17 [-]88.25
41 >Test84
CEYDEYRHRYRWRRK 75.03 78.53 68.25
42 >Test85
CEYDEYCEYRHRYRWRRK 62.83 [-53.38] [-]66.09
43 >Test86
CEYDEYCEYDEYRHRYRWRRK [-78.01] [-68.31] [-]87.32
44 >Test87
CDADEDPKPYSKKYR [-77.34] [-85.56] [-]80.31
45 >Test88
CEYDEYGSGSPKPKKYR [-]87.08 [-]93.14 [-]91.09
46 >Test89
CEYDEYGSGSPKPKKR [-]84.86 [-]91.15 [-]87.58
47 >Test90
CEYDEYGSPKPKKRGS [-] 83.83 [-] 91.15 [-]88.26
48 >DAD91
CERGDELSDPSPKPPSKKR [-]79.75 [-]78 [-]87.26
49 >DAD92
CERGDELGSPKPPSKKRGS [-]82.35 [-]72.74 [-]87.48
50 >DAD93
CEFDIWGSPKPKKRGS [-]73.68 [-]84.37 [-]78.54
51 >DAD94
CLRNTSGFKRWKKQF [-69.36] 73.35 [-]63.54

In yet another aspect of the present invention, the fluorescence images as shown in FIG. 2 depict a comparative fluorescence images of Cell line, primary tumour cell and healthy cell. These cells were treated with the drug DAD/DAPI, where cells in presence of the DAD shows a bright red fluorescence on the cell surface of the apoptotic cells in cell lines, primary cells, and primary cells without any staining of the healthy untreated cells.

FIG. 3 shows a comparison of the staining of ANNEXIN V in + Ca2 Buffer and - Ca2 Buffer. Another is for DAD2-Cy3 in + Ca2 Buffer and - Ca2 Buffer. DAD2-Cy3 does not shows any effect of presence or absence of calcium and shows a better result in both. While Annexin V shows better binding in presence of the Calcium.

FIG. 4 shows a graph of DAD2-Cy3 for comparing the Imatinib and control.

FIG. 5 shows comparative fluorescence images of cell in presence of the DAD/DAPI drug treatment at concentration of 1 µM, 5 µM, 10 µM, 50 µM, 100 µM and control.

In another aspect present invention discloses sequences of DAD versions (hereafter peptide) and control peptides. Functional residues in each peptide are highlighted.

The minimum distances between the carboxylate group of Serine residue in POPS and the functional amino acids of peptide were computed over the trajectory. The temporal changes in the minimum distances and the total number of contacts between the above-mentioned groups are shown for each peptide.

A representative molecular graphic is shown in figures 6-17 depicting salt bridges between peptide and POPS. The cyclic peptides were designed by stitching the N and C terminal of respective peptide.

>Seq1: ERGDELGGSGPKPPSKKRSCDPSHHHHHH
(Glu-Arg-Gly-Asp-Glu-Leu-Gly-Gly-Ser-Gly-Pro-Lys-Pro-Pro-Ser-Lys-Lys-Arg-Ser-Cys-Asp-Pro-Ser-His-His-His-His-His-His)

FIG. 6 shows representative molecular graphics for Peptide-1.

The early events of cell death, apoptotic, result in the expression of phosphatidyl serine (POPS) on the outer leaflet of the plasma membrane. The POPS possess a serine residue that furnishes a negative charge on the membrane at physiological pH, interacting with the positively charged AA. To form a strong salt bridge maximum allowed distance is assumed to be 4Å.

Peptide-1 shows selective POPS interaction with its positively charged AAs (i.e. Lys12, Lys16 – Arg18), and the N- terminal shows conformational changes after 500 ns time resulting in a ß hairpin arrangement, furnishing extra stability. As a consequence of such conformational changes, Arg2 becomes more available to interact with POPS leading to the increased number of contacts between the peptide and POPS.

>Seq2: ERGDELPKPPSKKRSCDPS
(Glu-Arg-Gly-Asp-Glu-Leu-Pro-Lys-Pro-Pro-Ser-Lys-Lys-Arg-Ser-Cys-Asp-Pro-Ser)

FIG. 7 shows representative molecular graphics for Peptide-2.

Peptide-2 was derived from peptide-1 by removing AA Gly7 – Gly10 (linker) and the last six Histidine (HIS-TAG). This peptide binds similarly to Peptide 1, showing more interactions after 500 ns of simulation time. During this period, the N – terminal attains a short a-helical conformation. Though peptide-2 shows more or less similar binding to that of peptide-1, the linker removal reduces the peptide disorder and enables it to build more contacts with POPS at any time point of the simulation.

>Seq3: PKPPSKKRSCDPS
(Pro-Lys-Pro-Pro-Ser-Lys-Lys-Arg-Ser-Cys-Asp-Pro-Ser)

FIG. 8 shows representative molecular graphics for Peptide-3.

Peptide-3 was obtained by removing the first six AA from peptide-2. This modification resulted in the abrogation of any regular conformation to the peptide. The positively charged, functional AA (i.e., Lys12, Lys16 – Arg18) becomes more available to interact with the POPS by making strong salt bridges with the carboxylate group of POPS.

The small size, disordered structure, and strong binding capability with POPS of peptide-3 give the advantage to use it for different stages of cell death.

>Seq4: EEGEELGGSGPEPGSEEGSCDPSHHHHHH
(Glu-Glu-Gly-Glu-Glu-Leu-Gly-Gly-Ser-Gly-Pro-Glu-Pro-Gly-Ser-Glu-Glu-Gly-Ser-Cys-Asp-Pro-Ser-His-His-His-His-His-His)

FIG. 9 shows representative molecular graphics for Peptide-4.

Peptide-4 is a negative control peptide with no functional, positively charged AA. Therefore, it cannot make any polar interaction with the POPS.

From the minimum distance graph, it is evident that the peptide is approximately 5Å separated from the POPS; however, throughout the trajectory, there are some close contacts which may arise from the sidechain hydrophobic interactions.

>Seq5: PEPGSEGGSCPSK
(Pro-Glu-Pro-Gly-Ser-Glu-Gly-Gly-Ser-Cys-Pro-Ser-Lys)

FIG. 10 shows representative molecular graphics for Peptide-5.

Peptide-5 was designed, with a single positive charge AA, as the positive control peptide. Therefore, under favorable conditions, it can make only one POPS residue. By comparing the minimum distance graphs of peptide-3, it is clear that the total contacts between peptide-5 and POPS are reduced by 50%. Further, the average minimum distance graph of peptide-5, approximately 3 – 3.5 Å, shows higher fluctuation than peptide-3, which at most places is between 2.5 – 3 Å.

>Seq6 - CLRNTSGFKRWKKQF
(Cys-Leu-Arg-Asn-Thr-Ser-Gly-Phe-Lys-Arg-Trp-Lys-Lys-Gln-Phe)

FIG. 11 shows representative molecular graphics for Peptide-6.

Peptide-6 was designed to maximize contact with the POPS membrane. The peptide attains the a-helical conformation; MD simulation suggests that the peptide is very stable on the membrane surface. After the 500ns trajectory time, it shows higher fluctuations, between 550 – 600 ns and 800 – 900 ns, though it returns to normal, maintaining POPS interactions.

>Seq-7- RKWKKNWFDLWSD
Arg-Lys-Trp-Lys-Lys-Asn-Trp-Phe-Asp-Leu-Trp-Ser-Asp
FIG. 12 shows representative molecular graphics for Peptide-7.

Peptide-7 has all the positive charged AA at N-terminal. The MD simulation trajectory suggests this peptide has stable binding on the membrane surface after and makes multiple contacts with POPS. However, the closely placed positive charged side chains of consequent AA places them in different 3D space, thus at any time point only one or two amino acids can interact with POPS.

>Seq-8- CEYDEYGSPKPKKRGS
(Cys-Glu-Tyr-Asp-Glu-Tyr-Gly-Ser-Pro-Lys-Pro-Lys-Lys-Arg-Gly-Ser)

FIG. 13 shows representative molecular graphics for Peptide-8.

Peptide-8 has a redesigned version of peptide-2; introducing aromaticity at N-terminal has led to the complete loss of any regular structure. Though there are some fluctuations in peptide–membrane distance, the average peptide–membrane separation is maintained between 2.9 – 3.4 Å, which offers several transient salt bridges, at least 7 bonds, formations with the functional AA.

Comparing results from the previous peptide-2 and peptide-3, it is evident that the negatively charged AA crowding at N-terminal might repel peptide from the negatively charged membrane surface.

>Seq-cy1- RKKWF
(Arg-Lys-Lys-Trp-Phe)

FIG. 14 shows representative molecular graphics for test cyclic peptide.

This test cyclic peptide was designed, as a positive control, to estimate the minimum number of AA residues required for cyclisation and POPS interaction. This cyclic peptide shows internalization of the overall ring, owing to the relatively higher aromaticity of the peptide. Although initially peptide shows good interaction with POPS but after about 400 ns of trajectory time interaction becomes poor thus total contact is also reduced. On average, at any time point of the trajectory, total contacts of peptide with POPS are five.

>Seq-cy2- GAGWGAGRGAGWGAGR
(Gly-Ala-Gly-Trp-Gly-Ala-Gly-Arg-Gly-Ala-Gly-Trp-Gly-Ala-Gly-Arg)

FIG. 15 shows representative molecular graphics for Peptide-cy2.

Peptide-cy2 was designed two Trp and two Arg, each separated by randomization sequence (i.e., Gly-Ala-Gly). This cyclic peptide is large enough to settle on the membrane surface without internalization. Two Arg residues are almost oppositely placed in the ring enabling the cy2 to bind with at least two POPS residues.

MD simulation clearly shows that peptide is stable on the membrane surface with an average distance of 2.5 – 3 Å, making it available for strong interaction with POPS; however, some higher fluctuations are seen between 700 – 900 ns. From the above-presented data, it can be inferred that cy2 forms more salt-bridge contacts with POPS.

>Seq-cy3- CLRNTSGFKRWKKQF
(Cys-Leu-Arg-Asn-Thr-Ser-Gly-Phe-Lys-Arg-Trp-Lys-Lys-Gln-Phe)

FIG. 16 shows representative molecular graphics for Peptide-cy3.

Peptide-cy3 is the cyclized peptide-6, is more stable on the membrane surface. In comparison to its linear form, it shows low fluctuation in the late stage of the trajectory. Overall contacts with POPS are always more than five.

>Seq-cy4- PKPGSKKRS
(Pro-Lys-Pro-Gly-Ser-Lys-Lys-Arg-Ser)

FIG. 17 shows representative molecular graphics for Peptide-cy4.

FIG. 18 shows HMC3 Cells Treated with 3µM LLOME that were checked with DAD3 with or without the presence of CEYDEY motif at the N-Terminus.

The DAD3 without the NON-CPP peptide showed intracellular localization in the control cells but showed increased staining in the treated apoptotic cells (A). The Non-CPP motif containing DAD3 sequence did not show any stating in the control healthy cells and bound to the membrane of the apoptotic cells (B).

In another embodiment, the present invention provides the linear or cyclic synthetic peptides sequences as listed in Table 2.

Table 2. Non-cell penetrating metacyclic peptide sequences for Minimum Distance Analysis.
Name Length System Version Minimum Distance Analysis
Min (Å) Max (Å)
Mean (Å)

Control 320 Annexin_V* Native 0.3 34.5 6.9
ApoGreen 5 RKKWF Lin 0.08 17.85 4.11
Cyclic 0.14 13.64 3.9
Seq 1 29 ERGDELGGSGPKPPSKK
RSCDPSHHHHHH Lin 0.05 23.3 3.95
Cyclic 0.14 21.5 4.53
Seq 2 19 ERGDELPKPPSKKR
SCDPS Lin 0.3 10.02 4.24
Cyclic 0.12 15.65 4.2
Seq 8 16 CEYDEYGSPKPKKRGS Lin 0.13 19.14 4.07
Cyclic 0.12 24.86 4.2
Seq 47 16 GAGWGAGRGAGW
GAGR Lin 0.12 14.64 4.26
Cyclic 2.7 11.8 4.1
Seq 6 15 CLRNTSGFKRWKKQF Lin 0.15 15.23 3.26
Cyclic 2.78 10 3.3
Seq 7 14 CRKWKKNWFDLWSD Lin 0.1 16.46 4.1
Cyclic 0.1 14.1 3.8
Seq 52 9 PKPGSKKRS Lin 0.09 16.45 3.67
Cyclic 0.1 9.4 3.6
Seq 5 13 PEPGSEGGSCPSK Lin 0.22 19.721 4.5
Cyclic 0.17 28.2 11.41
Seq 54 WCRKWKKNWFDLWS
DW Lin 0.11 17.9 4.59
Seq 55 WSWGRKKGWSW Lin 0.06 21.01 4.62
Seq 56 PSPGRKKGPSP Lin 0.08 22.77 4.1
Seq 57 PSPWGKKRGWPSP Lin 0.08 19.75 5.82
Seq 58 WFPKPSKKRSCDSFW Lin 0.04 15.65 3.69
Seq 59 PCPWGKPKRPKGWPSP Lin
0.14 14.15 3.3

Table 2 presents the analysis of peptide sequences for their potential to interact with target proteins. By calculating the minimum distance between a peptide sequence and a known ligand or binding site, the binding potential was predicted.
Seq 1 represents a linear, non-cell-penetrating PS-binding peptide, and its cyclic version also binds to PS. Seq 2 similarly shows a linear, non-cell-penetrating PS-binding peptide, and its cyclized version does not penetrate the cell but retains its ability to bind to PS. Seq 3 describes a PS-binding peptide that is cell-penetrating in its linear form but becomes non-cell-penetrating upon cyclization, with nearly 95% PS-binding efficiency. Seq 4 serves as a control peptide, which is non-PS-binding in both linear and cyclic forms.
Seq 6 demonstrates a linear, non-cell-penetrating peptide with more than 95% PS-binding efficiency, and cyclization does not alter its binding efficiency. Seq 7 shows a peptide that is linear, non-cell-penetrating, and cyclic in nature. The control peptide remains non-PS-binding in both linear and cyclic forms.
Seq 14 is a hairpin loop peptide with more than 80% capability to bind to PS. Lastly, Seq 17 represents a recent addition — a non-cell-penetrating hairpin loop peptide with the highest PS-binding capability, exceeding 95%. Cyclization does not further enhance its PS-binding efficiency.
The binding efficiency of peptides to PS (phosphatidylserine) varies based on their structure (linear, cyclic, or hairpin loop) and cell-penetrating ability.

In another aspect, the present invention discloses sequences of a new peptide that includes Cys amino acids to promote intra-chain cyclization, facilitating the formation of a PS-binding functional loop. The terminal amino acids are used as anchors or vice versa. These sequences are selected from SEQ. NO: 65, SEQ. NO: 66, SEQ. NO: 67, and SEQ. NO: 68.

Novel and inventive features of the present invention:
• Non-cell penetrating peptides to detect PS.
• Design of the metacyclic peptides mimicking the topography of the PS specific membrane binding region of Annexin V

The linear, metacyclic and cyclic peptides of the present invention can be used in conjugation with self-assembling peptides e.g., collagen like peptides or RGD, di, tri and tetrameric self-assembling peptides, to form multimeric peptides as delivery vehicles for drug and vaccine adjuvants.

Further the linear peptides and cyclic peptides of the present invention can be used with Pi stacking Pyrene linkage to form multimeric aggregates for drug delivery and vaccine adjuvants.

Every year, India imports at least 50,000 kits of Annexin V at an average cost of 30,000 INR, amounting to a 150 to 200 Crore market, despite the fact that India accounts for less than 1% of the global market. The global apoptosis market is valued at $5 billion, with kits accounting for up to 50% of the market and flow cytometry kits accounting for up to 33% of the market. Given that Annexin V kits account for roughly 10% of the market, the global market is estimated to be worth $500 million. The present peptide is modified for the identification and isolation of exosomes and extracellular vesicles, which has a $300 million market potential and is growing at a 25% CAGR.

In an embodiment of the present invention, a synthetic non-cell-permeating peptide (non-CPP) is provided for the detection of apoptotic cells, extracellular vesicles, cancer cells, and related cell types. The list of sequences is given below:

SEQ.NO: 01 - ERGDELGGSGPKPPSKKRSCDPSHHHHHH
SEQ.NO: 02 - ERGDELPKPPSKKRSCDPS
SEQ.NO: 03 - PKPPSKKRSCDPS
SEQ.NO: 04 - EEGEELGGSGPEPGSEEGSCDPSHHHHHH
SEQ.NO: 05 - PEPGSEGGSCPSK
SEQ.NO: 06 - CLRNTSGFKRWKKQF
SEQ.NO: 07 - CRKWKKNWFDLWSD
SEQ.NO: 08 – CEYDEYGSPKPKKRGS
SEQ.NO: 46 - ERGDELPKPPSKKRGS
SEQ.NO: 47 – GAGWGAGRGAGWGAGR
SEQ.NO: 48 - GAGWGAGRGAEWGCGR
SEQ.NO: 49 - CLRNTSGFKRWKKQF
SEQ.NO: 50 - CRKWKKNWFDLWSD
SEQ.NO: 52 - PKPGSKKRS
SEQ.NO: 54 - WCRKWKKNWFDLWSDW
SEQ.NO: 55 - WSWGRKKGWSW
SEQ.NO: 56 - PSPGRKKGPSP
SEQ.NO: 57 - PSPWGKKRGWPSP
SEQ.NO: 58 - WFPKPSKKRSCDSFW
SEQ.NO: 59 – PCPWGKPKRPKGWPSP
SEQ.NO: 65 – WSPCGKPKRPKGCWPSP
SEQ.NO: 66 – WSPCGKPKRPKGC
SEQ.NO: 67 – WFSPCGKPKRPKGCWFPSP
SEQ.NO: 68 - WFSPCGKPKRPKGC
,CLAIMS:
1. Synthetic non-cell permeating peptide (non-CPP) for detection of apoptotic cells, extracellular vesicles, cancer cells and related cells, comprising amino acid sequences wherein one or more amino acids in the sequences are selected from the group comprising of Arginine (Arg), Glycine (Gly), Aspartic Acid (Asp), Glutamic Acid (Glu), Leucine (Leu), Serine (Ser), Proline (Pro), Lysine (Lys), Cysteine (Cys), Histidine (His), Asparagine (Asn), Threonine (Thr), Phenylalanine (Phe), Tryptophan (Trp), Tyrosine (Tyr) and Alanine (Ala),

2. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein structure of the amino acid sequences is selected from linear, metacyclic and cyclic.

3. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein the peptide has amino acid sequence disclosed in SEQ.NO: 01.

4. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein the peptide has amino acid sequence disclosed in SEQ.NO: 02.

5. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein the peptide has amino acid sequence disclosed in SEQ.NO: 03.

6. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein the peptide has amino acid sequence disclosed in SEQ.NO: 04.

7. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein the peptide has amino acid sequence disclosed in SEQ.NO: 05.

8. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein the peptide has amino acid sequence disclosed in SEQ.NO:06.

9. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein the peptide has amino acid sequence disclosed in SEQ.NO:07.

10. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein the peptide has amino acid sequence disclosed in SEQ.NO:08.

11. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein the peptide has amino acid sequence disclosed in SEQ.NO:46.

12. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein the peptide has amino acid sequence disclosed in SEQ.NO:47.

13. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein the peptide has amino acid sequence disclosed in SEQ.NO:48.

14. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein the peptide has amino acid sequence disclosed in SEQ.NO:49.

15. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein the peptide has amino acid sequence disclosed in SEQ.NO:50.

16. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein the peptide has amino acid sequence disclosed in SEQ.NO:52.

17. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein the peptide has amino acid sequence disclosed in SEQ.NO:54.

18. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein the peptide has amino acid sequence disclosed in SEQ.NO:55.

19. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein the peptide has amino acid sequence disclosed in SEQ.NO:56.

20. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein the peptide has amino acid sequence disclosed in SEQ.NO:57.

21. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein the peptide has amino acid sequence disclosed in SEQ.NO:58.

22. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein the peptide has amino acid sequence disclosed in SEQ.NO:59.

23. The synthetic non-cell permeating peptide (non-CPP) as claimed in claim 1, wherein the amino acid sequences are selected from non-cpp peptides, non-cpp and hairpin-loop forming peptides, or cyclic peptides mimicking annexin V functional motif and cysteine knot functional group and branches that adhere to membrane.

24. Use of synthetic non-cell permeating peptide (non-CPP) for detection of apoptotic cells, extracellular vesicles, cancer cells and related cells, wherein the peptide comprises amino acid sequences comprising one or more amino acids selected from the group comprising of Arginine (Arg), Glycine (Gly), Aspartic Acid (Asp), Glutamic Acid (Glu), Leucine (Leu), Serine (Ser), Proline (Pro), Lysine (Lys), Cysteine (Cys), Histidine (His), Asparagine (Asn), Threonine (Thr), Phenylalanine (Phe), Tryptophan (Trp), Tyrosine (Tyr) and Alanine (Ala).

25. Use of synthetic non-cell permeating peptide (non-CPP) for detection of apoptotic cells, extracellular vesicles, cancer cells and related cells, wherein the peptide comprises amino acid sequences selected from SEQ No. 1, 2, 3, 4, 5, 6, 7, 8, 46, 47, 48, 49, 50, 52, 54, 55, 56, 57, 58, and 59.

26. A method of detecting apoptotic cells, extracellular vesicles, cancer cells and related cells in a subject by using synthetic non-cell permeating peptide (non-CPP), comprising amino acid sequences wherein one or more amino acids in the sequences are selected from the group comprising of Arginine (Arg), Glycine (Gly), Aspartic Acid (Asp), Glutamic Acid (Glu), Leucine (Leu), Serine (Ser), Proline (Pro), Lysine (Lys), Cysteine (Cys), Histidine (His), Asparagine (Asn), Threonine (Thr), Phenylalanine (Phe), Tryptophan (Trp), Tyrosine (Tyr) and Alanine (Ala).

27. A method of detecting apoptotic cells, extracellular vesicles, cancer cells and related cells in a subject by using synthetic non-cell permeating peptide (non-CPP), comprising amino acid sequences selected from SEQ No. 1, 2, 3, 4, 5, 6, 7, 8, 46, 47, 48, 49, 50, 52, 54, 55, 56, 57, 58, and 59.