End-functionalised polypeptide for targeted therapeutic delivery and a method for preparation thereof

Abstract

ABSTRACT END-FUNCTIONALISED POLYPEPTIDE FOR TARGETED THERAPEUTIC DELIVERY AND A METHOD FOR PREPARATION THEREOF An end-functionalized shikimic polypeptide with pendant shikimic moieties has been developed for efficient nuclear targeting. The synthetic methodology developed allows the facile and straightforward synthesis of water-soluble shikimic acid functionalized polypeptides with high molecular weights. These synthetic shikimic acid functionalized polypeptides were also found to display minimal toxicity to cells in vitro. Cellular uptake experiments reveal that shikimic acid functionalized polypeptides are selectively trafficked into the nucleus. The conjugation of these end-functionalized shikimic polypeptides to other biological entities, including, but not limited to, recombinant anticancer drugs, DNA, RNA, and CRISPR-Cas9 is a suitable alternative for delivery of these biological entities into cells affected by cancer and other genetic diseases without causing any harmful side effects or death of neighboring healthy cells, thereby opening up numerous potential therapeutic applications.

Description

FIELD OF INVENTION

[001] The present invention relates generally to end-functionalized polypeptidesand a method for preparation thereof for targeted drug delivery and morespecifically relates to a shikimic acid end-functionalized polypeptide and amethod for preparation thereof for nuclear specific targeting, wherein theshikimic acid end-functionalized polypeptide may be conjugated withvarious molecules, including, but not limited to, therapeutics, anticancerdrugs, DNA, RNA, CRISPR-Cas9 for nuclear specific applications.BACKGROUND OF INVENTION

[002] When it comes to molecular complexes, including, but not limited to, smallmolecule drugs and macromolecules, which are frequently required to enterand exit the cell during the normal cell function, faces certain level ofrestriction for access to the nucleus of the cell. Further, the nuclearmembrane consists of nuclear pore complexes (NPCs), that provide thepathways for all exchanges between the nucleoplasm and cytoplasm, hassmall pore size which confines transportation of molecules, including, butnot limited to, polymers and nanoparticles acting as drug delivery vehicles.

[003] Diseased cells like the cancer cells misfunction due to alteration in itsgenetic material inside the nucleus. For treatment of diseases like cancer,these diseased cells needs to be targeted and killed without any harmful sideeffects or death of neighboring healthy cells. The nucleus remains the targetfor several of these drugs. However, the conventional treatment techniquesshow several limitations including low or no specificity, high toxicity andconsequently a low efficacy in discriminating between target cells andhealthy cells.

[004] Many biocompatible polymers have been developed as a vehicle for drugdelivery to the target cells, wherein these drugs penetrates the cell plasmamembrane and escapes into the cytoplasm, however, it fails to overcome thenuclear membrane barrier to enter the nucleus and exert their specificfunction. More specifically, if the target cells are cancer cells, the cytosolicdrugs have to overcome many intracellular resistance mechanisms to gainaccess to the nucleus. As a result, only a small percentage of drugs deliveredinto the cytosol finally reaches the nucleus in the target cells.

[005] However, to overcome this, researchers introduced nuclear localizationsignals (NLSs) on the drug delivery systems, which can mediate its bindingto the NPCs and help internalize the resulting complex inside the nucleus,one such molecule is the TAT peptide, which consists of several arginineunits and is known to act as NLS because of its ability to permeabilize thenuclear envelope and accumulate in the nucleus. But the reported cytotoxicnature of TAT peptide excludes them from several applications. Also, theoligomeric cationic polypeptides, which can permeabilize the nuclearenvelope, do not have any specificity for cancer cells. Hence, for targeteddelivery of cytotoxic drugs into the nucleus of specific cancer cells, ligandsspecific for cancer cell receptors and NLS need to be separately incorporatedinto the delivery system design.

[006] Various developments have been made to overcome such need. One suchdevelopment was the liposome nanoparticles end-functionalized withshikimic acid, capable of binding to mannose receptors and enablingtargeted delivery. However, liposomes end-functionalized with head-groupsshikimic and quinic acid, which functionally mimic mannose, have shownvery less efficiency when transfected to dendritic cells (~3%, Srinivas, R. etal. J. Med. Chem. 2010; 53:1387-1391), thereby inhibiting the initiation ofadaptive immune responses and hence fail to function as the 'sentinels' ofthe immune system. What is truly missing in the area of drug deliveryvehicles is of biocompatible polymer that can do both the functionssimultaneously, i.e. (i) enter the cell via receptor-mediated endocytosis(RME); and (ii) exclusively trafficked to the nucleus where they interactwith the nuclear pore complex to cross the nuclear membrane.

[007] Till date no attempts have been made to develop a biocompatible polymerhaving ability to enter cell via receptor-mediated endocytosis (RME) andsimultaneously exclusively trafficked to the nucleus wherein they interactwith the nuclear pore complex to cross the nuclear membrane. In order toachieve, the present invention provides a high molecular weight shikimicacid-functionalized polypeptides that could perform the dual function oftrafficking into the cell and exclusively into the nucleus of mammalian cellsthrough specific mannose receptors and they are non-toxic to mammaliancells. In addition, the present invention provides for the method for thesynthesis of nuclear-targeting shikimic acid-functionalized polypeptides.OBJECT OF THE INVENTION

[008] It is the principle object of the invention, to provide an end-functionalizedpolypeptide having shikimic acid side chain head groups.

[009] Another object of the invention is to provide a method for synthesis of end-functionalized polypeptides containing shikimic acid as head group, therebycreating water-soluble shikimic polypeptides for efficient trafficking intothe cell nucleus mediated by the nuclear pore complex of the target cell.

[0010] Another object of the invention is to provide shikimic acid end-functionalized polypeptides with higher molecular weights.

[0011] Another object of the invention is to provide a method for synthesis of bothrandom and block copolymers containing shikimic acid functionalizedpolypeptides as pre-functionalized NCA monomer assures thepolymerization to yield a polypeptide bearing 100 % shikimic acidfunctionalization in all its side chains.

[0012] Yet another object of the invention is to show the endocytotic pathway ofthe shikimic-acid functionalized polypeptides inside the cell and thetrafficking of the shikimic-acid functionalized polypeptides into the nucleusselectively.

[0013] A further objective of the invention is to show that the water-solubleshikimic acid functionalized polypeptides synthesized exhibits minimaltoxicity to the cell in vitro.

[0014] Another object of the invention is to provide shikimic acid end-functionalized polypeptides containing sticky ends for easy conjugationand / or attachment of various therapeutics, anticancer drugs molecules,DNA, RNA, CRISPR-Cas9 for nuclear-specific treatment and diagnosticapplications in mammalian cells.SUMMARY OF INVENTION

[0015] There is a need for an end-functionalized polypeptides with shikimicmoieties which is water soluble and is capable of selectively trafficking intothe nucleus of the target cells exhibiting high efficacy, yet display minimaltoxicity to cells in vitro. Because shikimic acid is the valuable chemicalwhich has attracted worldwide attention due to its characteristicpharmaceutical application and there is a shortfall in botanically sourcedshikimic acid, it is required to synthesize water-soluble shikimicpolypeptides with high molecular weights using synthetic methodology fortargeted delivery of drugs exclusively to the nucleus of the cancerous cellswithout any harmful side effects or death of neighboring healthy cells.

[0016] Accordingly, the present invention provides an end-functionalizedpolypeptides with shikimic acid head group having the general formula Awherein R, Y, X, n & p are described herein;

[0017] According to another aspect of the invention, an end-functionalizedpolypeptides with shikimic acid head group having the general formula A isrepresented by the compounds of general formulae 1 - 5:Formula 1:wherein R, Y, X, m, n, p & q are described herein.Formula 2:wherein R, Y, X, m, n, p & q are described herein.FORMULA AFormula 3:wherein, R, Y, X, m, n, p & q are described hereinFormula 4:wherein, R, Y, m, n, p & q are described hereinFormula 5:wherein, R, Y, m, n, p & q are described herein

[0018] According to another aspect of the invention, a method for preparation ofan end-functionalized polypeptides with shikimic acid head group havingthe general formula Awherein R, Y, X, n & p are described herein; said method comprising thesteps of:(a) reacting the acetyl masked shikimic acid with 9-BBN-protected L-lysineby EDC coupling (acid-amine coupling);(b) the 9-BBN deprotection to be performed in the mixture of solventchloroform and methanol at room temperature for 24 h to afford the shikimicacid-functionalized L-lysine and also results into free amino acid precursorwhich is further reacted with tri-phosgene (C3O3Cl6) and N-methylmorpholine (NMM) in dry THF at 55 °C for 1.5 h to obtain shikimic acid functionalized L-lysine NCA monomer;(c) the resultant shikimic acid functionalized polypeptides will be purifiedby the re-precipitation method in diethyl ether, thereby resulting to bothrandom and block copolymers containing shikimic acid functionalizedpolypeptides;(d) the purified shikimic acid functionalized polypeptides having acetylprotectinggroups on the shikimic moiety is hydrolyzed using both NaOMeor hydrazine hydrate in MeOH at room temperature for 4-6 hours, and theresultant water-soluble shikimic acid end-functionalized polypeptide(shikimic polypeptide) is purified by dialysis.

[0019] According to yet another aspect of the invention, the dialysis of the waters-oluble shikimic acid end-functionalized polypeptides is carried using adialysis membrane of molecular weight cut-off 3.5 kDa to 12.5 kDa,depending upon the polymers' molecular weight, thereby resulting intoshikimic acid functionalized polypeptides with high molecular weights.

[0020] According to yet another aspect of the invention, the shikimic-acidfunctionalized polypeptides exhibit the endocytotic pathway inside the celland selective trafficking into the nucleus of the target cells.

[0021] According to yet another aspect of the invention, the cellular endocytosisis observed within 4 h at 0.2 mg / mL concentration with subsequenttrafficking to the nucleus.

[0022] According to yet another aspect of the invention, the water-solubleshikimic acid functionalized polypeptides synthesized exhibits minimaltoxicity to the cell in vitro, wherein at least 80 % of the cell remains viableup to 0.3 mg of polymers after 48 h of incubation.

[0023] According to yet another aspect of the invention, the shikimic acid end-functionalized polypeptides provides greater advantage in terms of nuclearspecific treatment and diagnostic applications as the shikimic acid end-functionalized polypeptides contains sticky ends for easy conjugation and / orattachment of various molecules / ligands / biological entities, including, butnot limited to, therapeutics, anticancer drugs molecules, DNA, RNA,CRISPR-Cas9.

[0024] These and other aspects of the present invention are set out in the followingdetailed description of the invention.BRIEF DESCRIPTION OF DRAWINGS

[0025] FIG. 1 is a schematic representation of the synthetic procedures used forthe preparation of shikimic acid end-functionalized polypeptides (Scheme1).

[0026] Reagents and conditions: (a) EDC, NHS, HOBT, DMAP, dry THF, argonatmosphere; (b) CHCl3:MeOH (10:1), 24 h, room temperature; (c) C3O3Cl6,N-methyl morpholine, dry THF, 1.5 h, 55°C; (d) Primary amine initiator(hexyl amine, propargyl amine, azido peg amine, peg amine, etc.), protonsponge, dry DMF, 36 h.; (e) NaOMe, dry MeOH or hydrazine hydrate,MeOH.

[0027] FIG. 2 depicts the selective nuclear targeting with fluorescence labeledshikimic acid end-functionalized polypeptide (FLShiPP30: 0.2 mg mL-1: d, h, and l) on RAW 264.7 antigen presenting (APC) monocyte / macrophagelike cells when cultured for 4 h (hours) then stained with DAPI (50 nM) for30 min as compared with fluorescence labeled galactose-functionalizedglycopolypeptide (FLGalGP30: 0.2 mg mL-1 : b, f, and j) and fluorescencemannose functionalized glycopolypeptide (FLMannGP30: 0.2 mg mL-1: c, g,and k). The cells were probed by confocal microscopy showing merging ofthe fluorescence signal (shown in green) and that of DAPI (shown in blue)revealing colocalization as indicated by the solid blue areas (scale bars, 50μm).

[0028] FIG. 3 depicts the selective nuclear targeting with fluorescence labeledshikimic acid end-functionalized polypeptide (FLShiPP30: 0.2 mg mL-1: b, e, and h) on MDA-MB-231 human breast adenocarcinoma cancerous cells,when cultured for 4 hours then stained with DAPI (50 nM) for 30 min ascompared with fluorescence labeled mannose functionalizedglycopolypeptide (FLMannGP30: 0.2 mg mL-1: c, f, and i). The cells wereprobed by confocal microscopy showing merging of the fluorescence signal(shown in green) and that of DAPI (shown in blue) revealing colocalizationas indicated by the solid blue areas (scale bars, 50 μm).

[0029] FIG. 4 FIG. 4 depicts the competition assay for the nuclear internalizationof fluorescence labeled shikimic acid end-functionalized polypeptide (FLShiPP30) on RAW-264.7 cells, wherein showing the confocal fluorescencemicroscopy images of RAW-264.7 cells treated with fluorescence labeledshikimic acid end-functionalized polypeptide (FLShiPP30 : a, f, and k);confocal fluorescence microscopy images of RAW-264.7 cells that werefirst pre-treated with 2.0 mM monomeric galactose followed byfluorescence labeled shikimic acid end-functionalized polypeptide addition(FLShiPP30 : b, g, and l); confocal fluorescence microscopy images ofRAW-264.7 cells that were first pre-treated with 2.0 mM monomericmannose followed by fluorescence labeled shikimic acid end-functionalizedpolypeptide addition (FLShiPP30 : c, h, and m); confocal fluorescencemicroscopy images of RAW-264.7 cells that were first pre-treated with 2.0mM monomeric shikimic acid followed by fluorescence labeled shikimicacid end-functionalized polypeptide addition (FLShiPP30 : d, i, and n); andconfocal fluorescence microscopy images of RAW-264.7 cells that werefirst pre-treated with 0.7 mM monomeric galactose, 0.7 mM monomericmannose, and 0.7 mM monomeric shikimic acid followed by fluorescencelabeled shikimic acid end-functionalized polypeptide addition (FLShiPP30 :e, j, and o); (scale bars, 50 μm).

[0030] FIG. 5 depicts cytotoxicity test of the shikimic acid end-functionalizedpolypeptides (MTT assay) of fully deprotected polymers (ShiPP30 / ShiPP40) with mammalian cancerous cells, including, but not limited to, RAW-264.7,MDA-MB-231, and PANC-1 cell lines.

[0031] FIG. 6. is a schematic representation of fluorescence labelling of Shikimicacid functionalized polypeptides (ShiPPn) (Scheme 2).

[0032] Reagent and condition: (a) FL-NHS, Na2CO3, Dry THF, r.t, 24 h, Argonatmosphere; (b) NaOMe, Dry MeOH, 15-20 min, Argon atmosphere.

[0033] FIG. 7 depicts cellular uptake study of FL-ShiPP30 and FLShiPP40 on RAW264.7 cell lines for 2 h and 4 h.

[0034] FIG 8. depicts the Colour intensity profile of cellular uptake study of FL-ShiPP30, FL-ManGP30, and FL-GalGP30 on RAW-264.7 cell lines for 4 h.

[0035] The drawings described herein above are for illustration purposes only andare not intended to limit the scope of the present disclosure in any way.DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention overcomes the limitations of the various attemptsmade by providing water soluble shikimic acid functionalized polypeptidesof high molecular weight having dual aspect, (1) ability to enter target cellvia receptor-mediated endocytosis (RME) and (2) selective trafficking intothe nucleus of the target cell, yet display minimal toxicity to cells in vitro.Further, the present invention provides a method for preparation of water-soluble shikimic acid functionalized polypeptides using syntheticmethodology. For purposes of this invention, chemical terms, chemicalelements, specific functional groups, moieties and reactivity, are disclosedthroughout and include those summarized herein.

[0037] In the following disclosure, it is to be understood that description willdescribe the invention in relation to the preferred embodiments of theinvention. Further, all components listed by generic name, if any, are hereinmeant to include or encompass all equivalents for such componentsavailable. Furthermore, the terms such as "may," "may provide for," and "itis contemplated that the present invention may" and so forth are terms usedin interchange with the terms "is," "can," "will," and like terms used assynonyms.

[0038] It is to be understood that the reference, such as "in one embodiment" or"an embodiment" in the disclosure is intended to indicate that at least anembodiment of the invention is included with a particular feature, structure,or characteristic described in connection with the embodiment. In variousplaces in the disclosure, it is not necessary that the presence of the phrase"in one embodiment" or "an embodiment" is all referring to the sameembodiment. Further, it is to be understood that the term "comprise" andvariations thereof, such as "comprising", "comprises" and "comprised" asused in this disclosure, except where the context requires otherwise, are notintended to exclude other additives, components, integers or steps.

[0039] According to an embodiment of the present invention, the presentinvention provides an end-functionalized polypeptides with shikimic acidhead group having the general formula Awherein,n = 10 to 100p = 1 to 4EquationY = O, NHOR any anti-cancer drugs;that could perform the dual function after entering into the cell, that isexhibiting the endocytotic pathway inside the cell and traffickingexclusively into the nucleus of the target cells without causing any harm tothe neighboring healthy cells.

[0040] Further in an embodiment of the present invention, an end-functionalizedpolypeptides with shikimic acid head group having the general formula Acontains sticky ends for easy conjugation and / or attachment of variousmolecules / ligands / biological entities, including, but not limited to,therapeutics, anticancer drugs molecules, DNA, RNA, CRISPR-Cas9,thereby is represented by the compounds of general formulae 1 - 5:Formula 1:wherein,m = 10 to 100; n = 10 to 100p = 1 to 4; q = 1 to 4EquationX = COOH, OH, NH2, SH Y = O, NHFormula 2:wherein,m = 10 to 100; n = 10 to 100p = 1 to 4; q = 4EquationY = O, NHOR any anti-cancer drugsFormula 3:wherein,m = 10 to 100; n = 10 to 100p = 1 to 4; q = 3, 4EquationY = O, NHFormula 4:wherein,m = 10 to 100; n = 10 to 100p = 1 to 4; q = 3EquationY = O, NHFormula 5:wherein,m = 10 to 100; n = 10 to 100p = 1 to 4; q = 2, 3EquationY = O, NH

[0041] Further in an embodiment of the present invention, a method forpreparation of an end-functionalized polypeptides with shikimic acid headgroup having the general formula Awherein,n = 10 to 100p = 1 to 4EquationY = O, NHOR any anti-cancer drugs; synthetic strategies employed for preparing thepresently described end functionalized polypeptides with shikimic acid headgroup are depicted schematically in Scheme 1 (FIG. 1), wherein said methodcomprising the steps of:(a) first, the α-amino group is protected by 9-BBN (quantitative yield),keeping the ε-amino group free and the hydroxyl groups of shikimic acid ismasked by acetyl group using acetic anhydride as a masking agent withquantitative yield;(b) reacting the acetyl masked shikimic acid with 9-BBN-protected L-lysineby EDC coupling (acid-amine coupling) to afford the desired conjugate 9-BBN-protected L-lysine-triacetyl shikimic complex (Scheme 1: complex 2);(c) further, the 9-BBN deprotection is performed in the mixture of solventchloroform and methanol at room temperature for 24 h to afford the shikimicacid-functionalized L-lysine and the resulting free amino acid precursor isfurther reacted with tri-phosgene (C3O3Cl6) and N-methyl morpholine(NMM) in dry THF at 55 °C for 1.5 h to obtain shikimic acid-functionalizedL-lysine NCA monomer, wherein NCA is purified by re-precipitation in dryhexane and anhydrous flash column chromatography (yield 80%) andformation of NCA was confirmed by FT-IR spectrum and 1H-NMR and 13CNMR determined the purity of the NCA;(d) after obtaining shikimic acid-functionalized L-lysine NCA monomer,ring-opening polymerization (ROP) of corresponding α-NCA monomer isproceeded with a primary amine initiator at room temperature, which wasconfirmed by time-dependent FT-IR spectroscopy where it indicated that aminimum of 36 h was required to consume the NCA monomer at roomtemperature entirely and the GPC analysis, FT-IR, and NMR study showedthat NCA monomer affords corresponding shikimic polypeptide (ShiPPn) inexcellent yield with no detectable side products(e) the resultant shikimic acid end-functionalized polypeptides is purified bythe re-precipitation method in diethyl ether, thereby resulting to bothrandom and block copolymers containing shikimic acid functionalizedpolypeptides;(f) the purified shikimic acid end-functionalized polypeptides having acetyl-protecting groups on the shikimic moiety is hydrolyzed using both NaOMeor hydrazine hydrate in MeOH at room temperature for 4-6 hours, and theresultant water-soluble shikimic acid functionalized-polypeptide (shikimicpolypeptide) is purified by dialysis using a dialysis membrane.

[0042] Furthermore, in an embodiment of the present invention, the synthesis ofwater-soluble shikimic acid-functionalized polypeptides is achieved viaring-opening polymerization of its corresponding NCA monomer, whereinpre-functionalized NCA monomer assures the polymerization to yield apolypeptide bearing 100 % shikimic acid moiety head groups in all its sidechains that enables the synthesis of both random and block copolymerscontaining shikimic acid functionalized polypeptides.

[0043] Further in an embodiment of the present invention, the resultant water-soluble shikimic acid end-functionalized polypeptide purified by dialysisusing a dialysis membrane yields shikimic acid end-functionalizedpolypeptide of higher molecular weight.

[0044] Furthermore, in an embodiment of the present invention, the dialysismembrane used is having a molecular weight cut-off 3.5 kDa to 12.5 kDadepending upon the polymers' molecular weight.

[0045] In an embodiment of the present invention, preliminary cellularinternalization assays are conducted to investigate the endocytotic pathwayand the trafficking of the shikimic-acid end-functionalized polypeptideinside the mammalian target cell, preferably, mammalian cancerous cells.Since cell viability study of the polymers is a prerequisite for all traffickingstudies, a cytotoxicity test (MTT assay) of fully deprotected polymers(ShiPP30 / ShiPP40) (FIG. 5 ) with mammalian cancerous cells, including, butnot limited to, RAW-264.7, MDA-MB-231, and PNPC-1 cell lines (FIG. 5)is performed, wherein the RAW 264.7 antigen presenting (APC)monocyte / macrophage like cells, which are known to be excellent hosts fortransfection studies and the other two cell lines used in the study were thehuman breast adenocarcinoma cancerous cells MDA-MB-231 andpancreatic cancer cells PNPC-1, which would allow us to evaluate itspotential for targeted delivery of anticancer drugs.

[0046] Further in an embodiment of the present invention, the cell viability test(the MTT assay) on all three cell lines demonstrates that the shikimicpolypeptides exhibit minimal toxicity to the cell in vitro.

[0047] Furthermore, in an embodiment of the present invention, to visualize thecellular entry of shikimic acid end-functionalized polypeptide upon cellularinternalization, water-soluble fluorescein-labeled shikimic acid end-functionalized polypeptide (FL-4c: FLShiPP30) were synthesized by thereaction of acetyl protected shikimic polypeptide (3c) with fluorescein-NHS(FL-NHS) followed by deprotection of acetyl group for shikimic moiety(FIG 6. Scheme 2), wherein the shikimic moiety in the poly-L-lysinebackbone of the polypeptide chain completely alters the cellularinternalization and becomes nucleus specific. We hypothesize that thenuclear membrane proteins (NPC: nuclear pore complex protein) bind to theshikimic acid moiety in polypeptides, thereby facilitating its nuclear entrypathway. It is possible that the nuclear membrane protein interacts with theshikimic polypeptide polyvalently and internalizes into the cell nucleus.

[0048] The following examples are given by way of illustration of the presentinvention and therefore should not be construed to limit the scope of thepresent invention.EXAMPLE 1: Cell Viability Test Using MTT Assay:

[0049] The cell viability test (the MTT assay) on all three cell lines demonstratesthat the shikimic acid end-functionalized exhibit minimal toxicity to the cellin vitro since at least 80 % of the cell remains viable up to 0.3 mg ofpolymers after 48 h of incubation. RAW-264.7, MDA-MB-231, andPANC1 cells were seeded in a flat bottomed 96-well plate at a density of1x10^4cells / well in DMEM containing 10% FBS. The plate was incubatedat 37°C with 5% CO2 for 24 h. GalGP30, GalGP40, ManGP30, ManGP40, ShiPP30,ShiPP40 were added to make a final concentration of 25, 75, 150, and 300μg / mL, respectively in DMEM containing 10% FBS. Cells were incubatedfor 48 hrs at 37 °C with 5% CO2. At the end of incubation media wasremoved and 110 μL solution of DMEM containing 10% FBS with filtersterilized MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) solution (0.45 mg / mL) was added into each well and furtherincubated at 37 °C with 5% CO2 for 4 h. At the end of incubation media wasaspirated from the wells and 100 μL DMSO was added to dissolve insolubleFormosan crystals formed. The absorbance was measured at 550 nm usinga microtiter plate reader. The relative % cell viability was calculated fromthe following equation: Relative % cell viability = (Atest / Acontrol) x100%(Atest is the absorbance of the sample treated cells and Acontrol is theabsorbance of the untreated cells. Each absorbance was taken to be the meanof triplicate measurements). The cell viability was represented as apercentage relative to untreated cells as a control.EXAMPLE 2: Utility of shikimic acid end-functionalized polypeptide (ShiPPn) fornuclear targeting in RAW-264.7 cells

[0050] RAW-264.7 macrophage antigen presenting cell lines were taken andcultured for 4 hours at 37°C with fluorescence-labeled (FL)glycopolypeptides and polypeptide, that is, fluorescence labeled galactose-functionalized glycopolypeptide (FLGalGP30: FIG. 2 - b, f, and j),fluorescence labeled mannose functionalized glycopolypeptide (FL-MannGP30: FIG. 2 - c, g, and k) and fluorescence labeled shikimic acid end-functionalized polypeptide (FL-ShiPP30: FIG. 2 - d, h, and l), each at aconcentration of 0.2 mg / mL. Subsequently, the cells were treated with DAPI(50 nM) for 30 min to stain the nucleus. The cultured cells were studiedusing confocal fluorescence microscopy (FIG. 2). The cells probed revealedthe distribution of fluorescence spots exhibiting the colocalization of greencolor from fluorescence-labeled shikimic acid end-functionalizedpolypeptide (FLShiPP30) with the DAPI dye, indicating that fluorescentlabeled shikimic acid end-functionalized polypeptides (FLShiPP30) werelocated inside the nucleus indicated by the solid blue areas (scale bars, 50μm) (FIG. 2).EXAMPLE 3: Cellular internalization and subsequent localization of differentlysized shikimic acid end-functionalized polypeptides:

[0051] RAW-264.7 macrophage antigen presenting cell lines were taken andcultured with differently sized shikimic acid end-functionalizedpolypeptides (FLShiPP30 and FLShiPP40) at different incubation times, thatis at 2 h and 4 h (FIG. 2 and FIG. 7) and then then stained with DAPI (50nM) for 30 min. After 2 h of incubation, very few polypeptides were presentin the nucleus and were primarily localized in the cytoplasm or theperinuclear region (FIG. 7). Notably, after 4 h of incubation, thepolypeptides were observed exclusively in the nucleus (FIG. 2).Additionally, the color intensity profile from the bright area of thecolocalized images displays an identical variation of color intensity withdistance for both the green and blue detection channels, which furtherproves that both the dyes are colocalized within the individual cellularorganelles i.e., the nucleus (FIG. 8). The above observation demonstratesthat the polypeptides can specifically internalize into the cell nucleuscompartment within 4 h.EXAMPLE 4: Utility of shikimic acid end-functionalized polypeptide (ShiPPn) fornuclear targeting in MDA-MB-231 cells

[0052] MDA-MB-231 human breast adenocarcinoma cancerous cells were takenand cultured for 4 hours at 37°C with fluorescence-labeled (FL)glycopolypeptide and polypeptide, that is, fluorescence labeled mannosefunctionalized glycopolypeptide (FLMannGP30: FIG. 3 - c, f, and i) andfluorescence labeled shikimic acid end-functionalized polypeptide (FL-ShiPP30: FIG. 3 - b, e, and h), each at a concentration of 0.2 mg / mL.Subsequently, the cells were treated with DAPI (50 nM) for 30 min to stainthe nucleus. The cultured cells were studied using confocal fluorescencemicroscopy (FIG. 3). The cells probed revealed the distribution offluorescence spots exhibiting the colocalization of green color fromfluorescence-labeled shikimic acid end-functionalized polypeptide (FL-ShiPP30) with the DAPI dye, indicating that fluorescent shikimic acid end-functionalized polypeptides (FLShiPP30) were located inside the nucleusindicated by the solid blue areas (scale bars, 50 μm) (FIG. 3).EXAMPLE 5: Competition assay for the nuclear internalization of fluorescencelabelled shikimic acid end-functionalized polypeptide (FLShiPP30) onto RAW264.7 cells:

[0053] RAW-264.7 macrophage antigen presenting cell lines were taken tounderstand the nuclear internalization and role of shikimic acid moietiespresence in the polypeptide chain in the endocytic pathway. RAW-264.7cells were divided into 5 sets where each set was pre-treated using freemonomeric galactose, mannose, and shikimic acid, respectively and then inaddition such cells were treated with florescence labeled shikimic acid end-functionalized polypeptide (FLShiPP30). The cultured cells were studiedusing confocal fluorescence microscopy (FIG. 4). Treatment of RAW-264.7cells with 2.0 mM monomeric mannose or shikimic acid for 2 h at 37°Cbefore adding the fluorescence-labeled shikimic acid end-functionalizedpolypeptide (0.2 mg / mL) and subsequent incubation for another 4 h at 37°C displays more than 65% reduction in the cellular uptake. Additionally,complete inhibition of nuclear entry of fluorescence-labelled shikimic acidend-functionalized polypeptide (FLShiPP30) is observed when the cells werepre-treated with monomeric shikimic acid (FIG. 4). However, resultsindicate that the trafficking of shikimic acid end-functionalizedpolypeptides principally follows the mannose receptor-mediatedendocytosis pathway, and the presence of shikimic acid moieties in thepolypeptide chain plays a vital role in the endocytic pathway. It has beenevident from earlier studies that mannose-6-phosphate (M6P) labeledpolypeptides (M6PGPn), the simple insertion of a phosphate group at the 6-position of mannose moiety completely alters the trafficking compared tothe mannosylated polypeptides. The M6P functionalized glycopolypeptidespecifically traffics the lysosome via M6P receptor-mediated endocytosis incancerous cells and normal cells. In contrast, the mannosylated polypeptideis distributed all over the cytoplasm. These earlier studies prompted us tostudy the characteristic differences between the trafficking behavior ofmannosylated (MannGP30), galactosylated (GalGP30), and shikimicpolypeptide (ShiPP30) in the cell line. It was observed that when RAW-264.7cells were treated with FL-galactose-GP (FLGalGP30) and FL-mannose-GP(FLMannGP30) displayed more evenly distributed fluorescence intensityinside the cells (FIG. 2) that were colocalized only sporadically with DAPI(FIG. 2). This contrasts the fluorescence labeled shikimic acid end-functionalized polypeptide (FLShiPP30) where the fluorescent-labeledpolymers inside the cell were colocalized (FIG. 2) with DAPI almostprecisely (FIG. 2). Together, these observations signify that incorporatingthe shikimic moiety in the poly-L-lysine backbone in place of mannose orgalactose completely altered its cellular internalization and became mostlynucleus specific.

[0054] The foregoing has been a description of certain non-limiting preferredembodiments of the invention. Those of ordinary skill in the art willappreciate that various changes and modifications to this description may bemade without departing from the spirit or scope of the present invention, asdefined in the following claims.

Claims

1. A compound having the general Formula A FORMULA A wherein, n = 10 to 100 p = 1 to 4 R = Equation Y = O, NH R and X = OR Curcumin OR Roscovitine OR any anti-cancer drugs.

2. A compound as claimed in Claim 1, wherein the compound is end functionalized polypeptides with shikimic acid head group.

3. A compound as claimed in Claim 1, wherein shikimic acid head group is water soluble.

4. A compound as claimed in Claim 1, wherein it is water-soluble shikimic acid functionalized-polypeptides.

5. A compound as claimed in Claim 1, wherein the shikimic acid head group contains sticky ends for easy conjugation and / or attachment of various molecules / ligands / biological entities, including, but not limited to, therapeutics, anticancer drugs molecules, DNA, RNA, CR1SPR-Cas9.

6. A compound as claimed in Claim 1, wherein the shikimic acid head group containing sticky ends conjugates and / or attaches to various molecules / ligands / biological entities, thereby is represented by the compounds of general formulae 1-5: Formula 1: wherein, m = 10 to 100; n = 10 to 100 p = 1 to 4; q = 1 to 4 Equation X = COOH, OH, NH2, SH Y = O, NH Formula 2: wherein, X m = 10 to 100; n = 10 to 100 p = 1 to 4; q = 4 Equation Y = O, NH OR any anti-cancer drugs Formula 3: wherein, m = 10 to 100; n = 10 to 100 p = 1 to 4; q = 3, 4 Equation Y = O, NH Equation Formula 4 wherein, m= 10 to 100; n= 10 to 100 p = 1 to 4; q = 3 Equation Y = O, NH Formula 5: wherein, m = 10 to 100; n = 10 to 100 p = 1 to 4; q = 2, 3 Equation Y = O, NH7. A compound as claimed in Claim 1, wherein the compound exhibits the endocytotic pathway inside the cell and selective trafficking into the nucleus of the target cells.

8. A compound as claimed in Claim 1, wherein the cellular endocytosis is observed within 4 h at 0.2 mg / mL concentration with subsequent trafficking to the nucleus.

9. A compound as claimed in Claim 1, wherein the toxicity test has been performed on RAW-264.7, MDA-MB-231, and PAN Cl cells and cell viability represented as a percentage relative to untreated cells as a control.

10. A compound as claimed in Claim 1, exhibits minimal toxicity to the cells in vitro, wherein at least 80 % of the cell remains viable up to 0.3 mg of polymers after 48 h of incubation.

11. A method for preparation of a compound having the general Formula A FORMULA A wherein, n = 10 to 100 p = 1 to 4 Equation OR Curcumin OR Roscovitine OR any anti-cancer drugs., and said method comprising the steps of: a) reacting the acetyl masked shikimic acid with 9-BBN-protected L-lysine by EDC coupling (acid-amine coupling); b) the 9-BBN deprotection to be performed in the mixture of solvent chloroform and methanol at room temperature for 24 h to afford the shikimic acid-functionalized L-lysine and also results into free amino acid precursor which is further reacted with tri-phosgene (C3O3Cl6) and N-methyl morpholine (NMM) in dry THF at 55°C for 1.5 h to obtain shikimic acid-functionalized L-lysine NCA monomer; c) the shikimic acid-functionalized L-lysine NCA monomer obtained is polymerised by ring-opening polymerization (ROP) of corresponding a-NCA monomer with a primary amine initiator at room temperature, thereby resulting to a shikimic polypeptide (ShlPPn) with no detectable side products; d) the resultant shikimic polypeptides will be purified by the re-precipitation method in diethyl ether, thereby resulting to both random and block copolymers containing shikimic acid functionalized polypeptides; e) the purified shikimic acid functionalized polypeptides having acetyl-protecting groups on the shikimic moiety is hydrolyzed using both NaOMe or hydrazine hydrate in MeOH at room temperature for 4-6 hours, and the resultant water-soluble shikimic acid end-functionalized polypeptide (shikimic polypeptide) is purified by dialysis.

12. A method as claimed in Claim 11, wherein the a-amino group is protected by 9-BBN (quantitative yield), keeping the e-amino group free and the hydroxyl groups of shikimic acid is masked by acetyl group using acetic anhydride as a masking agent with quantitative yield.

13. A method as claimed in Claim 11, wherein the reaction of acetyl masked shikimic acid with 9-BBN-protected L-lysine by EDC coupling results to a desired conjugate 9-BBN-protected L-lysine-triacetyl shikimic complex.

14. A method as claimed in Claim 11, wherein the shikimic polypeptide (ShlPP") is a polypeptide bearing 100 % shikimic acid moiety head groups in all its side chains.

15. A method as claimed in Claim 11, wherein the dialysis of the water soluble shikimic acid end-functionalized polypeptides is carried using a dialysis membrane.

16. A method as claimed in Claim 11, wherein the dialysis membrane used is having a molecular weight cut-off 3.5 kDa to 12.5 kDa, depending upon the polymers' molecular weight, thereby resulting into shikimic acid functionalized polypeptides with high molecular weights.