TUMOR MICROENVIRONMENT-ACTIVATED PROPEPTIDE LIPID DERIVATIVES, NANO COMPLEX COMPRISING NANOSTRUCTURES AND USES THEREOF

The present invention refers to tumor microenvironment in tumor - related fibroblast the fibroblast activation protein selected by an electrochemically-activated pro peptide lipid derivatives and anticancer drugs through a carrier having a mounting nano-structre tumor tissues and cells based on an efficient anticancer number can be achieved are disclosed.

In one aspect the lipid conjugates herein includes one or more tumor microenvironment sensitive peptide; and nano-structre body including drug delivery complex, said tumor microenvironment sensitive peptide lipid conjugates, the sequence numbers 1 or sequence number of 2, in the order of C - N - tumor microenvironment sensitive substrate peptides and peptide pro peptide including fine just worker at the end; and said N - end of the peptide profile are connected via a hydrophilic polymer comprising an amphipathic (amphiphilic) lipid, said nano-structre body said lipid conjugates is connected through said assembly for an amphipathic lipid, drug delivery complexes are disclosed.

Herein according to 'pro peptide' is separated from substrate peptide (free) glass forming fine just worker peptide portion thereof and tumor microenvironment sensitive substrate just worker in fine cells 70 is composed of a peptide. Alternatively tumor - associated tumor microenvironment [...] in normal fibroblast (Cancer provided Associated Fibroblast; CAF) surface which is fibroblast activation protein (Fibroblast Activation Protein; FAP) to colon cancer, a tumor microenvironment sensitive substrate by said FAP overexpression such pro peptide binding peptides which cut, fine just worker peptide are separated with each other. Said two degree of about 12 - 50 amino acid peptide coated fine just worker, having charge with segment and a hydrophobic segment communicating with the structural characteristics, such as cancer of the peptide binding peptides which accumulate on a membrane of said fine glass just worker by combining fine just worker through cell membrane forming lipid hydrophobic part to be coated.

In the embodiment of said pro peptide sequences of peptide having sequence numbers 1 herein in pro it will carry on shoulder [thin, or sequence number 2 peptide sequences of resistant starch having pro unit are disclosed. Pro it will carry on shoulder [thin sequences'EPEAEADAEAGPAGIGAVLKVLTTGLPALISWIKRKRQQ '(sequence number 1) and, pro portion resistant starch is the sequence of the'EPEAEADAEAGPARAGLQFPVGRLLRRLLRRLLR ' (sequence number 2) are disclosed. Underline lipophilic portion in said sequence tumor microenvironment sensitive, i.e. truncated protein that is expressed in tumor cells by peptide as substrate, a sequence that is cut by a FAP are disclosed. The sequence numbers 1 free portion during underline [thin and it will carry on shoulder, sequence number 2 powder free portion during underline among others.

FAP herein one selected hydrophobic characteristics and fine just the plunger at the end of the peptide substrate to expose pro peptide N - C - end N end are coupled sequentially as important disclosed.

N - end of the peptide profile 'linker' is herein (amphiphilic) are connected to said amphiphilic lipids. N - pro peptide linker linked to the end important disclosed. The cystine or phosphorus glass (free) powder of tumor microenvironment is connected only if it will carry on shoulder pivotably in action. C - end of the peptide substrate sequences by the FAP communicatively coupled nanocomposite [...] well even after it will carry on shoulder profile connected to lean and, in this case cell membrane fine just worker without forming capacity as in the picomolar.

Linker include hydrophilic polymer is herein are used. The aforementioned hydrophilic polymer as pro peptide nano-structre residing between the surface hydrophobic interaction between pro peptide nano-structre prevent, in the case that the blood, other surface to prevent nano-structre protein in blood can be absorb, residence time in blood is increased, as a result tumor tissue transmitted becomes greater.

The to achieve these aims for example polyethylene glycol, polyacrylic acid, polyvinyl alcohol, polyethylene oxide, polyhydroxy ethyl methacrylate, or hyaluronic acid can be used herein but various areas such as hydrophilic polymer material, this number are not correct valve timing.

In the embodiment of said hydrophilic polymer in PEG linker as herein (polyethlyene glycol), average molecular weight of about 500 to 7,000 Da to less than. Said PEG is pro-peptide linker connecting the amphipathic lipid as well as to act as a, when administered in vivo residence time and stability enhancing in vivo role in particular WIPO.

The connection of the liposomal surface charge charged amphipathic lipid herein can be more stable charge charged peptide itself and preventing interactions excels in efficient drug delivery a nanocomposite formed on one disclosed. In addition phospholipid as an amphipathic lipid, phosphate is able portion is hydrophilic, hydrophobic tail portion with the ends of the first fatty acid indicating features, such hydrophobic due to complex formation between process from nano-structre. The such an end DSPE (preparing - 3 - phosphoethanolamines [...] - sn - 1, 2 - dice), phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine a and including amphiphilic lipid can be used but, this number are not correct valve timing.

In particular in one implementation according to an amphipathic lipid DSPE as herein (preparing - 3 - phosphoethanolamines [...] - sn - 1, 2 - dice) and to the original position, the above-mentioned purposes in particular WIPO.

Herein pro peptide lipid conjugates or pro peptide as the aforementioned lipid conjugates, amphiphilic lipid N - end coupled through a linker peptide profile are disclosed.

According to pro peptide complex with lipid conjugates herein nano-structre body formed on the substrate. This relayed herein but not limited to covalent bond number according to pro peptide lipid assembly for nano-structre body, for example phospholipids nano-structre body through which complex with hydrophobic interaction portion formed on the substrate.

According to composite nano-structre included herein according to pro peptide lipid assembly for present body carrier (carrier) to carry nano-structre mounted on the flying-anticancer drugs are used.

the various nano-structre used herein is an end can be, for example nano sheet or spherical can be a support structure is used.

As used herein terms' nano-structre body ' molecular level between a size of a microphone having a size sheet size implies nanoscale i.e. (film), such as spherical or tubular structure called least, Royal Society and the Royal Academy of Engineering July 2004 can be for example a defined reference definition.

According to one embodiment nano-structre herein as nano sheet is employed to. 1 Dimensional (one dimension) nano sheet thickness are indicated, according to nano sheet 0 herein. 1 To 100 nm or, according to the effect number this relayed herein are not correct. According to this complex with lipid conjugates of pro-peptide complex formation with nano sheet herein mounted advantageously has a sheet-like chemical structure and of release of the drug.

The number of such seat structure having the party industry a publicly known method can be nano-structre body bath, for example reduced (reduced graphene oxide) materials comprising the same [...], yes pin oxide (graphene oxide), or yes pin comprises (graphene). In one implementation in particular reduced yes pin oxide (reduced graphene oxide; rGO) can be used in. Fatty acid chains and aromatic drug absorbing said rGO is high surface area can be formed. In addition pro peptide lipid conjugates phospholipids rGO nano sheet through hydrophobic interaction portion is coupled.

According to another embodiment herein nano-structre composite spherical body with structural body gun petty or solid lipid nanoparticle having a predetermined wavelength.

Phospholipid molecules' liposome ' number prepared by the lipid double layer vesicle having herein are disclosed. The phospholipids in the molecule phosphate esters burned head including two hydrophobic lipid tail having a cage conformation, or 10 μm in diameter is about 50 nm, this number are not correct valve timing.

B used herein are anionic, cationic, or neutral liposomes can have, the uncharged or neutral lipids of cationic liposomes charged positively in order numerical control machine number, order number jaws neutral neutral lipids of multivesicular liposomes, lipid and sound electric charge neutral lipid can be used anionic liposome number order numerical control machine, if one skilled in the aim of the publicly known phospholipid may selected to be appropriate to account for the effects and are disclosed. An example of phospholipid before charged positively as follows: 1, 2 - trimethyl ammonium propane [...] - 3 - (1, 2 a-dimyristoyl-a 3 a-trimethylammonium-a propane), 1, 2 - dipalmitoyl - 3 - trimethyl ammonium propane (1, 2 a-dipalmitoyl-a 3 a-trimethylammoniumpropane), 1, 2 - trimethyl ammonium propane discharge it rises with the reel - 3 - (1, 2 a-distearoyl-a 3 a-trimethylammonium-a propane), 1, 2 - diol may be trimethyl ammonium propane [ley five - 3 - (1, 2 a-dioleoyl-a 3 a-trimethylammonium-a propane), 1, 2 - dimethyl - 3 - [...] ammonium - propane (1, 2 a-dimyristoyl-a 3 a-dimethylammonium-a propane), 1, 2 - dipalmitoyl - 3 - dimethyl ammonium - propane (1, 2 a-dipalmitoyl-a 3 a-dimethylammonium-a propane), stearoyl - 3 - 1, 2 - dimethyl ammonium - propane (1, 2 a-distearoyl-a 3 a-dimethylammonium-a propane), 1, 2 - diol may be dimethyl ammonium - blast [...] [ley five - 3 - (1, 2 a-dioleoyl-a 3 a-dimethylammoniumpropane), 3 β - [N - (N', N' - dimethylamino ethane) carbamoyl] cholesterol (3 β - [N - (N', N' - dimethylaminoethane) carbamoyl] cholesterol; DC non-Chol), dimethyl the d jade the thread ammonium which burns bromide (dimethyldioctadecylammonium bromide). An example of neutral lipid as follows:

L a-a - phosphtidylcholine (L a-a-a phosphatidylcholine), 1, 2 - propy five roh - sn - phosphor choline may be preparing - 3 - (1, 2 a-propionoyl-a sn a-glycero-a 3 a-phosphocholine), 1, 2 - sn - polymerization of phosphor choline [...] preparing - 3 - (1, 2 a-butanoyl-a snglycero provided 3 a-phosphocholine), it will be roh pen hit phosphor choline preparing - sn - 1, 2 - - 3 - (1, 2 a-pentanoyl-a sn a-glycero-a 3 a-phosphocholine), 1, 2 - caprolactam - sn - phosphor choline may be preparing - 3 - (1, 2 a-caproyl-a sn a-glycero-a 3 a-phosphocholine), preparing - 3 - [heyp it will be roh other phosphor choline - sn - 1, 2 - (1, 2 a-heptanoyl-a sn a-glycero-a 3 a-phosphocholine), made preparing - sn - 1, 2 - car [phu - 3 - phosphor choline (1, 2 a-capryloyl-a sn a-glycero-a 3 a-phosphocholine), it will be roh or roh preparing - 3 - phosphor choline - sn - 1, 2 - (1, 2 a-nonanoyl-a sn a-glycero-a 3 a-phosphocholine). An example of lipid sound electric charge as follows: L a-a - party [til writing three roll (L a-a-a phosphatidylglycerol) phosphate, 1, 2 - dicarboxylic acid - 3 - may be preparing - sn - profile phosphor glycerol (1, 2 a-dicaproyl-a sn a-glycero-a 3 a-phosphoglycerol), it will be roh d jade other phosphor - 3 - 1, 2 - preparing - sn - glycerol (1, 2 a-dioctanoyl-a sn a-glycero-a 3 a-phosphoglycerol), 1, 2 - dicarboxylic acid - 3 - phosphor is formed into preparing - sn - glycerol (1, 2 a-dicaprylsn non-glycero-a 3 a-phosphoglycerol), preparing - sn - 1, 2 - - 3 - [...] phosphor glycerol (1, 2 a-dilauroyl-a sn a-glycero-a 3 a-phosphoglycerol), [...] - sn - 1, 2 - preparing - 3 - phosphor glycerol (1, 2 a-dimyristoyl-a sn a-glycero-a 3 a-phosphoglycerol), 1, 2 - dipalmitoyl - sn - glycerol - 3 - preparing phosphor (1, 2 a-dipalmitoyl-a sn a-glycero-a 3 a-phosphoglycerol), it will be roh d pie other fabric - 3 - 1, 2 - a spotter preparing - sn - glycerol (1, 2 a-diphytanoyl-a sn a-glycero-a 3 a-phosphoglycerol).

Included herein as pro peptide is conjugated to a nanocomposite according to phospholipids as same or different nano-structre phospholipid liposome component and can be used. Only pro peptide is conjugated to a liposome the phospholipids to the connection charge charged surface charge charged peptide itself and preventing interactions for example a phospholipid polyethylene glycol, 1, 2 - - 3 - phosphoethanolamines use discharge [...] preparing - sn - preferably.

In other embodiments solid lipid nanoparticle (Solid lipid nanoparticle) can be used. By forming solid lipid nanoparticle has a melting point of the lipid is solid at ordinary temperature, number surfactants, auxiliary surfactants (co-a surfactant) number can be formed. In particular solid lipid nanoparticles water volcanic ash can be use a number of anticancer well enclosed therein. The pro peptide lipid conjugates herein according to the morning fair number added to solid lipid nanoparticle, a liposome and above-mentioned peptide in a similar manner can be surface mounted. Kind of solid lipid nanoparticles, components, such as a novel manufacturing method and the mounting number most thriving business, Korean about number founded Institute, <Number [...] about> 33 Right 4 call (2003), pp. 319 - 322; Or S. M. Cho, et al, J. Am. Oil. Chem. , 84, 859 (2007) Reference to disclosed is a can.

According to pro peptide lipid conjugates herein comprising composite nano-structre body to 100:1 to 0. The weight ratio of 1:1, preferably 20:1 to 1:1 weight ratio present. In particular in the embodiment of the weight ratio of 2:1 in pro peptide lipid conjugates to nano-structre herein are disclosed. The weight ratio in said pro peptide lipid conjugates nano located at excellent luminous efficiency as in the picomolar.

In yet another aspect herein described above including a carrier having a number of pro-peptide lipid derivatives and nano-structre anticancer drug delivery system or a composition for the delivery of anticancer drugs and anticancer activities are disclosed.

In herein according to drug delivery, the drug is also 1a and 1b also increased as the picture data, such as sheet or nano liposomes nano-structre mounted on the flying the nanometer range. Herein a nanocomposite according to nano-structre mounted body due to accumulation of drug on a cancer tissue when used in drug delivery are combined each other. As well as nanocomposite nano-structre contained drug body is mounted, of an anti-angiogenic tumor vascular tissue in way of normal tissue is wider than the gap between epithelial cells in epithelial cells accumulated in the LAN-typed nano-structre tumor tissue contrast normal tissue, tumor tissue distribution of relatively enhance anticancer number. But in the case of the nano carriers, but only accumulated in the tumor tissue of liver, lung, spleen of normal tissue is even distribution, according to the herein selectively potentiating the activity of anticancer number nanocomposite within tumor cells is reduced side effects is a back disclosed.

Included herein pro peptide drug delivery according to tumor cells expressed specifically by an enzyme cut, glass and fine just worker peptide, the cells and a positive electrode, a novel cancer nano-structre body top number number specifically only is transferred.

According to anticancer drug delivery is used herein includes inducing cytotoxicity against cancer cells exhibit number, can be mounted on the flying according to nano-structre if herein, particularly the number has the one, for example 1 gun [su perm id (cyclophosphamide), doxorubicin (doxorubicin), daunomycin (etoposide) etoposide, etoposide phosphate (etoposide phosphate) daunomycin, reel [ni gun hour [tu (teniposide), capsaicin (carminomycin) car america roh e, multi right deer rain shoes (daunorubicin), amino [phu reel phosphorus (aminopterin), maul [sso tree tax [thu (methotrexate), maul [sso reel phosphorus (methopterin), dichloro (dichloromethotrexate) maul [sso tree tax [thu, e toe feed c (mitomycin C), paclitaxel (paclitaxel) or taxol (Taxol) in [thak it counted, type (taxanes), epothilone (epothilone), process fatigue feed (porfiromycin), 5 - fluoro uracil (5 a-fluorouracil), 6 - distant cop toe [phwu phosphorus (6 a-mercaptopurine), gemcitabine (gemcitabine), cytosine-products side (cytosine arabinoside), podophyllotoxin or a derivative thereof (podophyllotoxin or podophyllotoxin derivatives) in [...] (melphalan), [...] (vinblastine), [...] (vincristine), with at the time of [tin [...] (leurosidine), bin new (vindesine), biologically [...] (leurosine), estramustine (estramustine), cisplatin (cisplatin), characteristically about 0.24 carboxylic (carboplatin), [...] feed (bleomycin), tamoxifen (tamoxifen), polyimide (ifosfamide) this gun spine, hexa methyl melamine (hexamethyl melamine), thio reel group (thiotepa), at the time of burn bin (cytarabin), the tree tax which is the [thu (idatrexate), tree maul tree tax [thu (trimetrexate), position (dacarbazine) multicarboxylic, L - number oh the [su para it is long (L a-asparaginase), camptothecin (camptothecin), hour blood mote -11 (CPT-a 11), irinotecan (irinotecan), irinotecan (topotecan) topographic features, - c (Ara provided C) aramid, (bicalutamide) non-knife base hit america [tu, the flue [...] (flutamide), loop roll leading (leuprolide), it cuts the signature pebble pyridazinone derivatives (pyridobenzoindole derivatives), with fan (busulphan), interferon current (interferons), or interleukin current (interleukins) equal to or greater than one. One embodiment of the present disclosure number doxorubicin is said anticancer agent.

Anticancer drug delivery according to the type of anti-cancer number number is included herein, specific chemical and physical according to features different nano-structre body can be reduced, for example anticancer number: weight ratio of 1:1 to 50:1 nano, in particular 10:1 to 2:1 weight ratio each loading. In one implementation ruby new this toxin is used, the weight ratio is 1:1 to 5:1 and, when anticancer number beyond the variance of 5:1 to tame.

In the embodiment of the drug delivery system herein it will carry on shoulder [thin - resistant lipid conjugates in said doxorubicin/pro or pro portion reduced yes pin oxide (Dox/PL-a rGO, Dox/PB provided rGO) as, it will carry on shoulder [thin - PEG (polyethylene glycol) 2000 - resistant lipid conjugates or pro portion said pro pro it will carry on shoulder [thin dice the reel oh with the gun [su party which rises [til the ethanol amine, or pro portion - PEG (polyethylene glycol) dice the reel oh with the gun [su party which rises [til the ethanol amine resistant 2000 - among others.

In the embodiment of the present disclosure in the other drug delivery it will carry on shoulder [thin - doxorubicin/liposome lipid conjugates as resistant or pro portion profile, said profile it will carry on shoulder [thin it will carry on shoulder [thin - PEG (polyethylene glycol) 2000 - dice the reel oh with the gun [su party which rises [til the ethanol amine resistant lipid conjugates or pro portion than a precursor protease, or pro portion - PEG (polyethylene glycol) dice the reel oh with the gun [su party which rises [til the ethanol amine resistant 2000 - among others.

Pro peptide modified with drug delivery system of the present disclosure mounted nano-structre drug composed in the nanometer range. The tumor microenvironment as said exposure, SnO pro peptide in a matrix of the tumor microenvironment in tumor - 1 sequence is placed on colon cancer related fibroblast surface cut by a fibroblast activation protein (fibroblast activation protein; FAP) made, cutting powder is obtained when it will carry on shoulder and away from the active peptide in a nano-structre cystine or emerging, when activated in the tumor microenvironment it will carry on shoulder in the form of cystine or fine just worker is equal to attack cancer peptide powder form. Compiling a cancer drug nano-structre fine just the plunger has been mounted is better and more efficiently into the cells into, then drugs to cancer cells by growth billion number to be coated.

Alternatively pro peptide modified with drug loading of drug delivery of the present disclosure when not nano-structre, capable of forming fine just worker it will carry on shoulder mounted to the cystine or peptide drug delivery (delivery) of intracellular because powder nano-structre coolant to be coated. This is nano-structre no power (driving force) can enter into cells are disclosed.

Hereinafter, in the embodiment of the present invention to aid in understanding a number etched. In the embodiment of the present invention is more easily understand for however number in the embodiment of the present invention portion in which a hole for being limited to only and not the.

Available bit example

In the embodiment 1. Dox/PL-RGONano sheet Composite design, bath and feature identifying number

1 - 1. RGONano sheet Number bath

GO nano sheet rGO nano sheet reduced and copiers. 2 Ml solution homogeneously dispersed GO nano sheet existing method (Miao W, Shim G, Kang CM, et al. Cholesteryl hyaluronic acid a-coated, reduced graphene oxide nanosheets for anti-a cancer drug delivery. Biomaterials. 2013; 34 (37): 96389647) High pressure liquid coolant according to his number. Graphite powder (0. 5G, Sigma non-Aldrich, USA) a process for cold (H₂ SO₄ , 23 Ml) and after mixing, solution mixing sewage [...] and has on ice (KMnO₄ , 3G) and sodium nitrate (NaNO₃ , 0. 5G) and slowly releases the enemy, solution 1 in 35 °C stirring time-gate. After reaction, distilled water (46 ml) through the 90 °C 1 in time with respect to the main reaction. Reactants hydrogen chloride solution (5% HCl) was washed 3 times in distilled water and impurities are number centrifuging a stand-alone. The washed product (200 mg) distilled water (40 ml) 2 residue time ultrasonic (400 W) processing is detected when the obtained nano sheet, prepared by centrifuging 1600x g 10 minutes in a stand-alone [...] been those of desorption sheet type number. After yes pin oxide nano sheet, reduction reaction of abortion. Said yes pin oxide nano sheet (2 ml, 5 mg/ml) a 8 ml distilled water of, 0. Ammonia water of 5 ml (28 wt %; Junsei Chemical, Tokyo, Japan), and 5 micro l of high dignity [thu hydrazine mono (64% in water; Sigma non-Aldrich, St. Louis, MO, USA) is mixed with. 10 Minutes stirring in tank 80 °C-gate in said mixture. To room temperature then heated with, 3 (TDW) distilled water to said mixture during dialysis (MWCO 100K; Spectrum Laboratories, Inc. , Rancho Dominguez, CA, USA) to a stand-alone number been excess hydrazine and ammonia. As a result obtained TDW rGO nano sheet 4 °C was dispersed until the installed application.

1 - 2. RGONano sheet Size and number other Potential Measuring

RGO nano sheet (dynamic light scattering) of various sizes were measured using a dynamic light scattering. (Photal, Osaka, Japan) other potential readings in a number 22° angle using the laser Doppler micro cataphoresis ELS provided 8000 instrument were measured.

1 - 3. It will carry on shoulder [thin pro- Phospholipid conjugates (PL) Synthesis of

It will carry on shoulder [thin peptide profile new as it is known (LeBeau AM, Brennen WN, Aggarwal S, Denmeade SR. Targeting the cancer stroma with a fibroblast activation protein a-activated promelittin protoxin. 135 Mol Cancer Ther. 2009; 8 (5): 13781386) Cystine and FAP - it will carry on shoulder cutting sequences disclosed. It will carry on shoulder [thin sequence is acetylated - proEPEAEADAEAGPAGIGAVLKVLTTGLPALISWIKRKRQQ (cleavable sequences italic characters to FAP - shown) are disclosed. 2 Μmole (N - [swuk the new already [til jade hour writing base hit reel) 3 - 20 μmole of first profile and it will carry on shoulder [thin aminopropyl, polyethylene glycol - cover mill dies the reel oh with the gun [su party which rises [til the eta it played and it pushed (NHS-a PEG₂₀₀₀ - DSPE; NOF Co. Ltd. , Tokyo, Japan) after melting of die methyl sulfoxide (Sigma non-Aldrich) 22 μmole N, N - die isopropyl ethylamine (Sigma non-Aldrich) at room temperature and mixed with agitating it will carry on shoulder [thin - tagged lipid profile 24 time-configurated. Said mixture to 48 hours dialysis (MWCO 5,000 Da; Spectrum Laboratories Inc.) it will carry on shoulder [thin - TDW PEG bonded to pro₂₀₀₀ - DSPE (promelittin provided PEG₂₀₀₀ - DSPE) not been joined to a stand-alone it will carry on shoulder [thin peptide profile number. Final compound used was 20 °C until pressure freeze drying the installed application. It will carry on shoulder [thin - PEG pro synthesis₂₀₀₀ A marking and PL - DSPE, was characterised MALDI-a TOF mass spectrometry analysis.

1 - 4. Fluorescent lipid number bath

In addition, cyanine 5. 5 Fluorescent lipid consumption while fluorescent lipids number was high pressure liquid coolant. 1, 2 - Polyethylene glycol chain has a molecular weight of 5000 in preparing - 3 - phosphoethanolamines - N - [amino (polyethylene glycol) -5000] - sn - [...] dice (DSPE-a PEG_{5000 -} NH₂ , NOF Corporation, Japan) 4. Hydrocarbons 5 mg sodium buffer solution (0. 1 M, pH8. 3) After melting, 1. 0 Mg of cyanine 5. 5 NHS ester (Lumiprobe, USA) is added with agitation time-gate group 4. The cyanine 5 does not bind to lipids. 5 Cephalosporin G15 column (Sephadex G15 colume, GE Healthcare) number using the dextran was a stand-alone.

1 - 5. PL-RG0Nano sheet Complex number bath and Dox Mounting

PL: weight ratio of 2:1 so that the distilled water (TDW) 3 difference rGO rGO nano sheet (1 mg/ml) the same volume of PL solution (2 mg/ml) was mixed with rGO nano sheet coated with surface of the PL. In addition plane (plain) overlying the valve seat was mounted rGO or PL-a rGO nano Dox. 1 Ml solution of Dox (0. 1 Mg/mL; Sigma non-Aldrich) to 1 ml of a TDW rGO Dox - 10 minutes at room temperature to lower by culturing or PL-a rGO nano sheet mounted rGO (Dox/rGO) and a high pressure liquid coolant mounted Dox - PL-a rGO (Dox/PL-a rGO) his number. (GE Healthcare, Piscataway, NJ) then PD provided 10 desalination column is elastic (free) a number Dox been mounted using a stand-alone.

1 - 6. Measuring efficacy mounting

Range of analyzed by antigenic rGO nano located at an PL decided. PL - coated rGO in time of 400 micro l 5M H 180 °C (PL-a rGO) nano sheet 1₂ SO₄ Number ol die with chains. Then heated with solution, 100 micro l of 30% H₂ O₂ 30 Minutes by adding said mixture was heated again to 180 °C. Said solution 4 and then cool to room temperature. 6 Ml of 0. 2% Ammonium molybdate (Sigma non-Aldrich, St. Louis, MO, USA) and 10 minutes by adding 100 ml of 15% ascorbic acid (Sigma non-Aldrich) was heated to 90 °C. UV microplate reader (Gemini XS; Molecular Devices, Sunnyvale, CA, USA) 830 nm measured in absorbance using phosphate composite compound in a decided. Base concentration phosphorus standard solution (Sigma non-Aldrich) assay using his line.

Mounted rGO Dox erased because by measuring fluorescence intensity reduction rGO nano sheet [chap it re-became Dox efficiency and ratio the bill. 485 Nm to 590 nm using a fluorescent microplate reader (Gemini XS) to measure fluorescence over the exciting in Dox fluorescence intensity decided.

Such as the surface of said coated with Dox rGO PL mounted to a 1A also shown. The mechanism of action have shown to also 1B Dox/PL-a rGO possible. It will carry on shoulder [thin number comprised of nano sheet's profile are linked to each other which have been activated by FAP Dox/PL-a rGO FAP - the ends of the peptide sequence cleavable, overexpression in tumor CAF is in the microenvironment, it will carry on shoulder formed round pores emit - peptide. It will carry on shoulder [...] tumor microenvironment of cells and tumor cells are spread out in a pore is formed by Dox/PL-a rGO increase introduction cells of anticancer activity can be increase. Overlying the valve seat and then fixing the rGO Dox rGO nano PL mounted without affecting that statistically significant average size of nano sheet as in the picomolar (PL-a rGO: mean=94. 6 Nm, 95% CI=93. 3 To 95. 9 Nm, P=. 80; Dox/PL-a rGO: mean=91. 2 Nm, 95% CI=88. 7 To 93. 8 Nm, P=. 25) (Also 2A). Number while other potential readings in a coating over the surface of the PL to read but (mean=9. 4 MV, 95% CI=5. 6 To 13. 2 MV, P=. 001), Cationic Dox been increased to PL-a rGO nano overlying the valve seat after burning re- (mean=18. 5 MV, 95% CI=16. 3 To 20. 7 MV, P=. 02) (Also 2B).

RGo PL on the efficacy on-board analyzed by phosphate were measured (Niu G, Cogburn B, Hughes J. Preparation and characterization of doxorubicin liposomes. Methods Mol Biol. 2010;624:211219). In a weight ratio of 20:1 to 2:1 rGO rGO PL PL mounted on reduced to increase efficacy statistically significantly been increased (P < 0. 001) (8 Also). 2:1 (Mean=95. 3%, 95% CI=92. 5% To 98. 1%) And 1:1 (mean=94. 5%, 95% CI=92. 8% To 96. 3%) Mounted on the efficacy statistically significant differences between ratios is rGO PL (P=0. 65) In any cases. The additional weight ratio 2:1 became a PL-a rGO herein for experiments.

(Dox: weight ratio of 5:1 rGO) Dox rGO or PL-a rGO nano seat of 1 minutes has been if the absorption (also 2C). The fluorescence intensity after 60 seconds Dox rGO nano sheet inserted to physical mixing in said original strength 13% reduced. Similarly, the fluorescence intensity in 60 seconds after Dox PL-a rGO nano sheet which are inserted into said original physical mixing is slipped 2% strength is reduced. Dox: weight ratio of 5:1 to 10 minutes when mounted to Dox rGO rGo or PL-a rGO, average value meets each mounted efficacy of Dox 96. 3% (95% CI=95. 6% To 97. 0%) And 97. 5% (95% CI=96. 2% To 98. 4%) Min (also 2D).

The fatty acid chains and aromatic drug absorbing said rGO is high surface area can be, mounted higher efficacy. It will carry on shoulder [thin since it became lung route profile joined to phospholipids, hydrophobic portion PL phospholipids via interaction with binding through hydrophobic interaction rGO nano sheet to be coated.

In the embodiment 2. It will carry on shoulder [thin proPeptide Lipid derivatives and Ruby new this toxin Containing AnionLiposome-number bath and assessing the efficacy

2 - 1. It will carry on shoulder [thin proPeptide Lipid derivatives and Ruby new this toxin Containing AnionicprocessNumber tank gun petty

In the embodiment 1 synthesized in pro-it will carry on shoulder [thin lipid derivatives, neutral lipid is L a-a - phosphtidylcholine (Avanti Polar Lipid Inc. , USA, multi-hereinafter 'PC'), a power supply L a-a - party [til writing three roll lipid phosphate (Avanti Polar Lipid Inc. , USA, hereinafter 'PG' varied) and cholesterol (cholesterol, Sigma, USA) 0 respectively. 1 Ml of chloroform (chloroform) by 2:2:2:2 μmole taken after melting, multiplex fine glass vial 10 ml nitrogen environment until all chloroform evaporates diaphragm placed after mixing and low lipid thin film film number was rotating at a speed to evaporate the high pressure liquid coolant. A high pressure liquid coolant (multilamella vesicle) number corresponding to the cow nine body lipid multilayer type thin film by adding 1 ml phosphate buffer solution was stirring 3 minutes after sealing-egg 37 °C (vortexing). Same homogenizing number uniformly sized particles to make the early (extruder, Northern Lipid Inc. , Canada) using 0. 2 Μm polycarbonate film at high pressure liquid coolant passing his number 3. Doxorubicin (doxorubicin, Sigma, USA) is a long herein 0. Anionic liposome surface by mixing then electrostatically bound to 1 mg/ml, and the remaining toxin G15 column (GE Healthcare, UK) to liposomes is free of cephalosporin ruby shoes using a stand-alone been number cell transplants. It will carry on shoulder [thin doxorubicin containing anionic liposome lipid derivatives and pro obtained using his 4 °C until the installed application.

2 - 2. It will carry on shoulder [thin pro Lipid derivatives and Doxorubicin Containing AnionicOf multivesicular liposomes Anticancer assessing the efficacy: MTT Analysis

It will carry on shoulder [thin number of multivesicular liposomes of the present invention pro doxorubicin containing anionic lipid derivatives and to provide a process to assess transfer efficiency in a target cell cancer such as conducting. HT29 human colon cancer cell line HT29 cell lines or tumor cells present in the fibroblast is a 24 well plate and the related - [...] experiment ruminated 3 × 10 per well⁴ Or 9 × 10⁴ (Seeding) was divided one by one. It will carry on shoulder [thin doxorubicin containing anionic liposomes containing anionic liposomes after doxorubicin treatment of each cell can be lipid derivatives and profile and, 37 °C CO of₂ In incubator him as time 3. And phosphoric acid buffer solution 3 times washing the cells 37 °C CO medium number of stand-alone₂ In incubator him as 24 hours. Then 3 - (4, 5 - dimethyl thiazole - 2 - yl) - 2, 5 - diphenyl tetra nicotineamid bromide (MTT, Sigma, USA) added to a power of 10%, then the supernatant culture number 0 and 2 further time stand-alone. 04 N hydrochloric acid adding isopropanol solution elastase syndrome generating reader (ELISA reader, Sunrise provided Basic TECAN, Mannnedorf, Switzerland) in absorbance using 570 nm were measured. Controls include anything processing cells are used.

As anionic liposomes containing toxins of ruby shoes 12 also appears in said cancer results assessing the effectiveness, in [...]-like cell lines, compared to doxorubicin containing anionic liposome processing group, it will carry on shoulder [thin lipid derivatives and containing anionic liposome processing further enhanced cancer death effect profile doxorubicin checked mistletoe. Said fibroblast activation protein substrate peptide sequences present lipid derivatives it will carry on shoulder [thin pro result [...] - related tumor-like cell lines of fibroblasts by fibroblast activation protein enzyme cleaves inactive and the result it will carry on shoulder fibroblast activation protein at the cell surface by pharmacological profile it will carry on shoulder [thin active form of conversion to tumors that express - related fibroblast is present to more effectively toxin ruby new this cancer is transferred as [...]-like cell lines exhibits enhanced anticancer effects.

In the embodiment 3. Dox/Resistant profile part-RGONano sheet Composite design, number bath and efficacy Analysis

3 - 1. Resistant profile partPeptidePolyethylene glycol Lipid synthesis of lipid derivatives resistant pro portion through an amide linkage

Amino acid sequences of peptide having a sequence number 2 (20 μmole, ChinaPeptide Corporation, Shanghai, China) in 2000 on the molecular weight of the polyethylene glycol chain (N - [swuk the new already [til jade hour writing base hit reel) 3 - aminopropyl, polyethylene glycol - cover mill dies the reel oh with the gun [su party which rises [til - ethanolamine (NHS-a PEG₂₀₀₀ - DSPE; NOF Co. Ltd. , Tokyo, Japan) reacting the same method in the embodiment 1 a foreign peptide profile part 20 μmole lipid binding pro portion are obtained lipid derivatives resistant starch. Not bound to lipids on time (MWCO 5 000 Da, Spectrum Laboratories Inc.) distilled water resistant pro portion 48 through a stand-alone been phoresis dial number. Reaction whether modified di - 18 mass analyzer (MALDI-a TOF mass spectrometry; Merck & Co. , Inc. USA) to confirmed.

3 - 2. Protease according to Resistant profile part Lipid derivatives of cell membranes Fine just workertypePerformance evaluation: hemolysis analysis

Mouse red blood cells (2 × 10⁴ Cells/ml) to 1 μg of matrix metal [...] number 9 (R D Systems&Inc.) or 10 μg lipid derivatives resistant profile part and fibroblast activation protein (R D Systems&Inc.) with respect to the reaction time after adding 37 °C in 1. After reaction, a supernatant obtained by centrifuging the 1000 x g 10 minutes in 96 well plate into practice. Pro portion selectively activated by protease resistant form fine just worker can be red blood cell membranes mouse lipid derivatives, a mouse hemoglobin (hemoglobin) for absorbing light at wavelength of 540 nm emitted from the red blood cell hemolysis the absorbance (Sunrise provided Basic TECAN, Mannedorf, Switzerland) measuring was analyzed. Controls include anything without processing mouse red blood cells obtained by centrifuging the supernatant are used.

Figure 13 shows a profile portion also said membrane protease of lipid derivatives resistant ability as a result of fine just worker according to evaluating, processing of pro-part number 9 matrix metal with [pheyp it is a mote, oh undesirable formation while fine just worker binds to lipid derivatives resistant, oil resistant fibroblast activation protein processing of pro-portion to at or enhancing lipid membrane body that fine just worker. From the results of Figure 13 just worker formability such fine cell membranes, fibroblast activation protein substrate peptide sequences present lipid derivatives resistant profile part by fibroblast activation protein enzyme cleaves a non-active profile part by enhancing the conversion ability that fine powder active resistant membrane just worker by a goniophotometer.

3 - 3. Resistant profile part Lipid derivatives and Ruby new this toxin Containing reduced coenzyme Yes pin oxideorRoh seat number bath

In high pressure liquid coolant in the embodiment 1 yes pin number reduction oxide nano sheet (1 mg/ml) in the embodiment 3 - 1 synthesized in pro-portion to resistant lipid derivatives (2 mg/ml) on doxorubicin (0. 1 Mg/ml, doxorubicin, Sigma, USA) into water, 10 minutes at room temperature with respect to the reaction. Reduction oxide nano sheet yes pin and the remaining free of cephalosporin G15 column (GE Healthcare, UK) number using a stand-alone been ruby shoes toxin cell transplants. Pro portion obtained using doxorubicin containing reducing oxide nano sheet 4 °C until resistant lipid derivatives and yes pin was installed application.

3 - 4. Resistant profile part Lipid derivatives and Doxorubicin Containing reducing type Yes pin oxideIn nanoAssessing the efficacy anticancer-rest: MTT Analysis

Resistant starch of the present invention pro portion doxorubicin containing reducing oxide nano sheet of target cell cancer into lipid derivatives and yes pin number transfer efficiency to provide a process to assess such as conducting. HT29 human colon cancer cell line HT29 cell lines or tumor cells present in the fibroblast is a 24 well plate and the related - [...] experiment ruminated 3 × 10 per well⁴ Or 9 × 10⁴ (Seeding) was divided one by one. Yes pin doxorubicin containing reducing oxide nano sheet, and proteomics part doxorubicin containing reducing oxide nano sheet each said each cells resistant lipid derivatives and yes pin after processing, 37 °C CO of₂ In incubator him as time 3. And phosphoric acid buffer solution 3 times washing the cells 37 °C CO medium number of stand-alone₂ In incubator him as 24 hours. Then 3 - (4, 5 - dimethyl thiazole - 2 - yl) - 2, 5 - diphenyl tetra nicotineamid bromide (MTT, Sigma, USA) added to a power of 10%, then the supernatant culture number 0 and 2 further time stand-alone. 04 N hydrochloric acid adding isopropanol solution elastase syndrome generating reader (ELISA reader, Sunrise provided Basic TECAN, Mannnedorf, Switzerland) in absorbance using 570 nm were measured. Controls include anything processing cells are used.

Figure 14 shows a ruby shoes also said yes pin oxide nano sheets containing toxins of cancer as a result of reduction in assessing the effectiveness [...]-like cell lines, yes pin compared to doxorubicin-containing reduction oxide nano sheet processing group, doxorubicin-containing reduction oxide nano sheet processing checked and oil resistant lipid profile part further enhanced cancer death effect yes pin at or exhibit. The fibroblast activation protein substrate peptide sequences present lipid derivatives resistant protease said result [...] - related tumor-like cell lines of fibroblasts by fibroblast activation protein enzyme cleaves inactive's profile and the result resistant fibroblast activation protein at the cell surface by a active form of buforin conversion part to present [...] - tumors that express the cancer is related to more effectively toxin ruby shoes into fibroblast-like cell lines enhanced delivery of anticancer efficacy which means that the other.

In the embodiment 4. Resistant profile part Lipid derivatives and Ruby new this toxin Containing AnionicLiposomesNumber bath and method of assessing the efficacy

4 - 1. Resistant profile part Lipid derivatives and Ruby new this toxin Containing AnionicOf multivesicular liposomes Number bath

Synthesized in pro-portion in the embodiment 3 resistant lipid derivatives, PC, PG, each cholesterol 0. 1 Ml 10 ml glass vial is placed in a seam sealing diaphragm 2:2:2:2 μmole taken by azo compounds of multiplex fine mixing, in the embodiment 5 method identical to the distribution of anionic liposomes was high pressure liquid coolant resistant lipid derivatives is the profile part number. In the embodiment 5 herein method identical to the ruby new this anionic liposomes containing a toxin resistant lipid derivatives equation type pro part number was high pressure liquid coolant. The preparation G15 column (GE Healthcare, UK) to liposomes is free of cephalosporin ruby shoes and the remaining toxin using a stand-alone been number. Anionic liposome containing doxorubicin resistant lipid derivatives and pro portion obtained using his 4 °C until the installed application.

4 - 2. Resistant profile part Lipid derivatives and Doxorubicin Containing AnionicOf multivesicular liposomes Anticancer assessing the efficacy: MTT Analysis

Resistant starch of the present invention pro portion of multivesicular liposomes containing anionic lipid derivatives and doxorubicin to provide a process to assess cancer into target cells such as conducting number transfer efficiency. HT29 human colon cancer cell line HT29 cell lines or tumor cells present in the fibroblast is a 24 well plate and the related - [...] experiment ruminated 3 × 10 per well⁴ Or 9 × 10⁴ (Seeding) was divided one by one. Doxorubicin containing anionic liposomes containing anionic liposomes and pro portion after processing each cells resistant lipid derivatives and doxorubicin, 37 °C CO of₂ In incubator him as time 3. And phosphoric acid buffer solution 3 times washing the cells 37 °C CO medium number of stand-alone₂ In incubator him as 24 hours. Then 3 - (4, 5 - dimethyl thiazole - 2 - yl) - 2, 5 - diphenyl tetra nicotineamid bromide (MTT, Sigma, USA) added to a power of 10%, then the supernatant culture number 0 and 2 further time stand-alone. 04 N hydrochloric acid adding isopropanol solution elastase syndrome generating reader (ELISA reader, Sunrise provided Basic TECAN, Mannnedorf, Switzerland) in absorbance using 570 nm were measured. Controls include anything processing cells are used.

Figure 15 shows a ruby shoes also said anionic liposomes containing toxins of cancer as a result of assessing the effectiveness in [...]-like cell lines, and doxorubicin containing anionic liposome processing than the anionic liposomes containing doxorubicin resistant lipid oil pro portion further checked at or exhibit enhanced cancer death effect processing. The fibroblast activation protein substrate peptide sequences present lipid derivatives resistant protease said result [...] - related tumor-like cell lines of fibroblasts by fibroblast activation protein enzyme cleaves a non-active profile part resistant to fibroblast activation protein at the cell surface by switching to active powder - tumors that express the cancer related fibroblast is present to more effectively toxin into a ruby shoes [...]-like cell lines anticancer efficacy enhanced which means that the other.

In the embodiment 5. PL-RGoNano sheet By composite FAP In the hemolytic activity - dependent expression

5 - 1. Normal fibroblast and Of CAF Separation

Existing method (Castello provided Cros R, Cukierman E. Stromagenesis during tumorigenesis: characterization of tumor-a associated fibroblasts and stroma-a derived 3D matrices. Methods Mol Biol. 2009;522:275305; Wang H, Wu Q, Liu Z, et al. Downregulation of FAP suppresses cell proliferation and metastasis through PTEN/PI3K/AKT and Ras-a ERK signaling in oral squamous cell carcinoma. Cell Death Dis. 2014;5: E1155) deforms the CAF and normal fibroblast cells are separated only his. Specifically HT29 - containing mouse (14 after inoculation) of colon cancer tissue separating CAF have tumor, lung of nude MICE was normal fibroblast tissue cells are separated. Phosphate buffer saline (PBS) cut into, said tissue after small abrasive machine operation, 100 unit/ml plus 100 micro g/ml penicillin-resistant Streptococcus feed (GenDepot Inc. , Barker, TX, USA), 1 mg/ml collagenase type I (Sigma provided Aldrich) and it is a [ni in egg base, oh [...] number number added with 1 mg/ml serum (Sigma non-Aldrich) - DMEM (Dulbecco's Modified Eagle's Medium; Welgene Inc. , Daegu, Republic of Korea) in 37 °C, number have been ol die during time 1. CAF and normal fibroblast cells in 3 minutes was 90 x g fractionation centrifuging. CAF or normal fibroblast containing supernatant 10 minutes in a centrifuge to 800 x g, 15% right [thay [...] (FBS; GenDepot), 100 units/ml penicillin-resistant Streptococcus pneumoniae, Streptococcus feed added with DMEM to 100 micro g/ml and [...][...] to him as. 7 CAF and normal fibroblast cells after the culture medium is separated as well as experiments.

5 - 2. CAF And HT29 cells Amount [...]

Adenocarcinoma cell line HT29 human large intestine from ATCC (American Type Culture Collection; Manassas, VA, USA) are obtained. 10% FBS, 100 units/ml of penicillin-resistant Streptococcus pneumoniae, and 100 micro g/ml DMEM was added in culturing cells of said Streptococcus erythromycin. First 24 - well plate (SPL Life Sciences, Pochon, Republic of Korea) to 6 × 10⁴ Cells/well concentration 24 hours after the cone [phul base the [su which freezes the light CAF -80% seed [ting a culture medium, of 2:1 CAF: HT29 cells pre-cultured on HT29 cells by seed [ting CAF said ratio, small interference RNA (siRNA) targeting FAP CAF HT29 cells with nothing existence (siFAP) under a presence or amount [...]-gate.

5 - 3. FAP Expression analysis

In HT29 cell surface to said FAP expression is separated such as CAF and flow cytometers were measured. After harvest, 2% [...] albumin (BSA; USB Corp. , Cleveland, OH, USA) in phosphate buffer saline (PBS) containing directs a cool 4 °C, 1 time-gate block by culturing said cells. Then said antibody (Abcam, Cambridge, UK) [lay comb cut into PBS cells anti - FAP 1 difference in the presence 4 °C, 1 first time after culturing, in addition to cold PBS 1:10 dilution, fluorescein isothiocyanate (FITC)- 2 antibody (Biolegend, San Diego, CA, USA) bonded anti - [lay comb difference and cultured in the presence of the 1:10 dilution was. BD FACS Calibur system equipped with Cell Quest Pro software (BD Biosciences, San Jose, CA, USA) using monoclonal fluorescent antibodies detected by FAP - expressing cell therefrom.

5 - 4. Hemolysis assays

HT29 (1 × 10⁶ Cells), separated CAFs (1 × 10⁶ Cells), or CAF [...][...] on HT29 (total 2 × 10⁶ Cells) 10 micro g of a PL, and switching the FAP 1 micro g number (R&D Systems Inc. , Minneapolis, MN), and a positive and matrix metal with rope reel number or number (MMP9; R D Systems&Inc.) in the presence (mRBC) with mouse red blood 37 °C him as time 1. Number using the pen [cyen it became mRBC have only PBS to hemolysis matching group suspended, 1% Triton X-a 100 (USB Corp. , Cleveland, OH) was used to present 100% hemolysis mRBC is regulated. 1000 × g in 10 minutes after centrifuging, the supernatant containing the dissolved mRBC 3 96 - well polystyrene plate (SPL Life Sciences) was transferred in turn transparent flat bottom. Using Molecular Devices Spectra Max Plus automatic plate reader by measuring absorbance of samples in the 540 nm were measured degree of hemolysis.

It is different between the two FAP expression level (also 3A) and out (also 3B) CAF HT29 cells. Flow cytometry analysis result, CAF of 90. While the expression of FAP is 3% [...], HT29 cells 5. Only been positive FAP - 1% (also 3A). It will carry on shoulder [thin PL or PL-a rGO's profile to FAP or CAF expressed FAP - it will carry on shoulder formed pores by exogenous portion selectively cystine can be cut in order to test whether, conducting said red blood hemolysis assays such as mouse. FAP after being processed PL and PL-a rGO (also 3C) (also 3E) but by mRBC is dissolved, or matrix metalloproteinase (MMP) such as not to his -9 with rope reel number after processing. In particular, PL (P < 0. 001) (Also 3D) and PL-a rGO (P < 0. 001) In an amount of break down by CAF and HT29 [...] mRBC (also 3F) observed but, in HT29 cells was not alone. This FAP exhibits specific activation of PL.

In the embodiment 6. FAP- Positive CAF In the presence PL-RGONano sheet Complex Into cells Delivery efficiency analysis

6 - 1. FAP- Specific A siRNA Using Of CAF Insulation layer down

FAP - specific siRNA GFP - specific siRNA (siGFP) Bioneer Corporation (Daejeon, Republic of Korea) (siFAP) and is supplied in his. Trans [...] to is the sequence of the siRNA used such as disclosed: siFAP, 5 '- GGA AAG AAA GGU GCC AAU AdTdT-a 3' (sense) and 5 '- UAU UGG CAC CUU UCU UUC CdTdT-a 3' (antisense); siGFP, 5 '- GGC UAC GUC CAG GAG CGC ACC dTdT-a 3' (sense) and 5 '- GGU GCG CUC CUG GAC GUA GCC dTdT-a 3' (antisense). Isolated CAF (4 × 10⁵ Cells/well) on a plate (SPL Life Sciences) seed [ting 10 a-cm-gate. 24 Hours, Lipofectamine 2000 (Invitrogen; Carlsbad, CA, USA) according to the manual using cells in the presence of siRNA transfection chains. 1 Ml of Opti-a MEM (Gibco BRL; Grand Island, NY, USA) in siRNA (50nmol/l) of 15 is mixed with a micro l liposomes [pheyk it burns it pushed 2000. Adding said mixture by culturing said cells was 20 minutes at room temperature. Then by culturing said cells 48 hours as well as experiments.

6 - 2. FAP Quantitative evaluation of liver embodiment insulation layer down PCR

Embodiment using quantitative PCR (qRT-a PCR) liver mRNA level was assessed pleasure knock-down FAP. TRIzol reagent (Invitrogen) using, by extracting total RNA RT PreMix siGFP treated CAF plus HT29 siFAP or presence (Intron Biotechnology Inc. , Seoul, Republic of Korea) cDNA using reverse transcriptase to chains. Said mixture in 5 minutes and 60 minutes in 70 °C 42 °C him as. Triplicate cDNA qRT-a PCR amplification with respect to the sample. FAP for primer comprises a 5 '- GTA TTT GGA GTT GCC ACC TCT G-a 3' (sense) and 5 '- GAA GGG CGT AAG ACA ATG CAC-a 3' (antisense) are disclosed. LightCycler 2. 0 Instrument (Roche Diagnostics, Mannheim, Germany) using a glass capillary and FastStart DNA Master PLUS SYBR Green reagents (Roche Diagnostics) 20 - in conducting micro l qRT-a PCR. The amplification conditions in 10 minutes 95 °C initial modified step, in 95 °C 30s, in 55 °C 20s, and 72 °C 20s of 40 cycles in a CMOS process. FAP mRNA expression level of housekeeping gene, in which the glyceride aldehyde - 3 - phosphate [...][...] number-gate.

6 - 3. The [wey the [su it shook off[...] Analysis

It will be enveloped, [len the [su to the [wey the [su it shook off by a [...] siFAP FAP protein expression was assessed. The presence of the pack [syen it became a CAF plus HT29 siFAP trans, proteasome inhibitors (Roche Diagnostics) number number mixture added with 0. 1 Ml dissolution buffer (20 mm Tris provided HCL pH 7. 5, 1 Mm EDTA, 0. 5% Triton X-a 100, 75 mm NaCl) have been characterized in a CMP apparatus. By culturing cells on ice in 15 minutes 20 minutes the [su the pen [cyen it became [...] 12,000 x g was classification centrifuging. Said BCA protein assay kit (Thermo Scientific; Pittsburgh, PA, USA) cell lysate in a manual according to his reservoir. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS0PAGE) - a distinct sample protein to 8% after the [pu grudge which will doze (Hybond non-ECL; Amersham Biosciences, Piscataway, NJ, USA) on polyvinylidene die fluoride film into practice. FAP specific antibody (1:1000; Abcam, Cambridge, UK) and β - expression vector (1:1000; Santa Cruz Biotechnology; Santa Cruz, CA, USA) using the [wey the [su it shook off [...] was a film analysis. ECL (enhanced chemiluminescence) reagents (Amersham Biosciences) immune response proteins (Fujifilm, Tokyo, Japan) using visualization LAS-a 4000 image analyzer to of them.

6 - 4. PL-RGONano sheet Composite conveyed by the Of DoxIntracellular Inlet

Fluorescent lipid dye labeled-gate at the time of [hyem introduction cells of various rGO nano sheet using confocal microscopy. HT29 cells alone, or siFAP HT29 cells 24 well plate or nothing it was controlled CAF plus 9 × 10⁴ Cells/well concentration-gate seed [ting. Confocal microscopy for analyzing, in a high pressure liquid coolant Cy5 number in the embodiment 1 - 3. 5 - Phosphate bonded dice [...][...] -2000 - polyethylene glycol (DSPE-a PEG5000 non-Cy5. 5) Lipid labeled PL-a rGO or rGO was to cells. 1 Hours after culturing, cells from the surface of 15 minutes in 4% formaldehyde content fixed to PBS, 4, 6 - roh midi - 2 - phenyl indole (DAPI) with respect to the draw claw id dyed inside the die. Confocal laser scanning microscope (LSM 5 Exciter; Carl Zeiss, Inc.) using Cy5 -. 5 Fluorescence of them.

Such as said, by providing increased inflow cells of a PL-a rGO delivered by front and rear CAF siFAP [...] knock-down trans of FAP (Verma SC, Agarwal P, Krishnan MY. Primary mouse lung fibroblasts help macrophages to tackle Mycobacterium tuberculosis more efficiently and differentiate into myofibroblasts up on bacterial stimulation. Tuberculosis. 2016; 97:172180). The FAP siFAP CAF cells compared to a detection of mRNA processing regulated level as in the picomolar (mean=30. 9%, 95% CI=27. 1% To 34. 8%, P < . 001) (Of Figure 9 A). The [wey the [su it shook off [...] FAP even reduced protein expression as in the picomolar (of Figure 9 B). On the contrary, a CAF siGFP (controls) to treatment of FAP mRNA is not reduced-protein. HT29 cells in a single culture or CAF, PL-a rGO nano sheet supplied to cells of a FAP - CAF selectively increment in HT29 cells (also 4A) or FAP knock-down FAP - but positive CAF (also 4B) came not in voice, in detecting the fluorescence dye - labeled PL-a rGO supplied to said fluorescent dye labeled rGO nano sheet do not differ - openings of his. On the contrary, supplied to cells of a labeled PL-a rGO - fluorescent dye, such as HT29 cell fluorescent dye labeled rGO [...][...] positive CAF FAP - greater than - been inlet. The result of the, specifically by the FAP of PL PL a-rGO cut specifically to cancer cells present in the tumor microenvironment drug exhibits rGO is mounted from being transferred.

In the embodiment 7. Dox/PL-RGONano sheet Complex Stranded Anticancer efficacy

Cell survival assays of Dox/PL-a rGO nano sheet composite body in the embodiment 1 using stranded anticancer effects were measured. Specifically siFAP (9 × 10⁴ Cells/well) HT29 cells untreated or treated with (3 × 10⁴ Cells/well) or HT29 cells on a 24 - well plate and next 12 plus CAF seed [ting. 5 Micro g/mL rGO concentration of 1 μm Dox and Dox/PL-a rGO or PL-a rGO was. After number Dox - containing culture medium alone, or PL-a rGO rGO (rGO concentration, 12. 5 Micro g/mL) was added to said cells. 3 Hours after culturing, replaced with a new culture medium with a cell 24 hours decreased. (CCK-a 8; Dojindo, Tokyo, Japan) or live cellstaining assay Cell Counting Kit provided 8 (LIVE/DEAD Viability Assay; Molecular Probes, Eugene, OR) as a number of cellular viability bath method were measured.

5 Result is also disclosed. Figure 5 said ferroelectric layer such as, PL-a rGO inflow of increase in testing the efficacy of PL-a rGO Dox - stranded mounted diffusion codes are disclosed. CAF/HT29 cells HT29 cancer active amount increase at [...] Dox/PL-a rGO FAP - but not voice system (also 5A) cells in which the effectively, all group do not differ statistically significantly between the two anticancer activity test his (Dox: mean=90. 5%, 95% CI=88. 2% To 92. 8%, P=. 91; PL: mean=93. 8%, 95% CI=93. 1% To 94. 5%, P=. 81; Dox + rGO: mean=91. 5%, 95% CI=90. 3% To 92. 6%, P=. 98; Dox/rGO: mean=93. 9%, 95% CI=91. 3% To 96. 6%, P=. 85; PL-a rGO: mean=91. 3%, 95% CI=90. 5% To 92. 1%, P=. 92; Dox + PLrGO: mean=89. 7%, 95% CI=84. 8% To 94. 6%, P=. 86). A FAP siFAP knock-down CAF/HT29 cells and HT29 cells than in the cancer activity Dox/PL-a rGO CAF/HT29 cells [pheyk grudge trans 1 respectively. 5 Roof 2. 7 Times higher yet. CAF/HT29 positive glass Dox [...] (mean=80. 1%, 95% CI=78. 9% To 81. 3%) And Dox/rGO (mean=68. 9%, 95% CI=66. 0% To 71. 8%) Compared to the cells further effect - Dox/PL-a rGO (mean=33. 3%, 95% CI=32. 4% To 34. 3%) Is 3. 4 And 2. 1 Times higher up. In addition, sequentially in Dox CAF/HT29 cell culture compared to those treated plus PL-a rGO Dox/PL-a rGO is been anticancer significantly greater statistically (P < 0. 001) (Also 5A). Live/yarn fluorometric and got appears similar results even in a system, in which cells HT29 cells selectively [...] Dox/PL-a rGO [...] CAF and - while a further effect (also 5D), HT29 cells in FAP knock-down CAF/HT29 cells alone (also 5B) or not (also 5C) into foods.

The result of the anticancer efficacy of tumor tissue observed known specifically only Dox/PL-a rGO stranded, CAF which are expressed at a FAP (control unit fibroblast is only expressing fibroblast tumor tissue by FAP CAF i.e. does not express FAP) that represents an amount [...] present only on system specifically, cancer specific drug delivery efficacy are disclosed.

In the embodiment 8. Normal fibroblast in glass (free) Dox and Dox/PL-RGoNano sheet Composite cytotoxicity assay

In the embodiment 1 of cell survival was assessed by using assays of Dox/PL-a rGO stranded toxicity. 24 - Well plate normal fibroblast cells 6 × 10⁴ Cells/well concentration after [...], alone or in combination with different concentrations of Dox was next Dox/PL-a rGO. 3 Hours, 24 hours cells remain the same with respect to the lands or new culture medium. User number bath (Dojindo Molecular Technologies, Inc.) manual protocol according to CCK provided 8 existence power cells via assays were measured. CCK provided 8 assays for 20 added to each well (water-soluble tetra the bud which will doze salt) solution of micro l CCK-a 8 30 minutes after culturing, 450 nm using a microplate reader (Sunrise provided Basic; TECAN, Mannedorf, Switzerland) in absorbance were measured.

Said such as, glass Dox and Dox/PL-a rGO normal fibroblast irradiated from the result for unequal cytotoxicity concentration-dependent toxic effects - a top fibroblast in glass Dox mistletoe (also 10). On the contrary, regulated Dox/PL-a rGO nano sheet compared significantly cytotoxic not induce his normal fibroblast in a statistically significant (1 μm Dox on PL-a rGO: mean=91. 5%, 95% CI=88. 8% To 94. 2%, P=. 08; 5 Μm Dox on PL-a rGO: mean=91. 8%, 95% CI=89. 8% To 93. 8%, P=. 06; 10 Μm Dox on PL-a rGO: mean=90. 2%, 95% CI=85. 6% To 94. 8%, P=. 05). Regulated cell survival compared to normal fiber administering 10 μm glass Dox treatment reduced force (mean=67. 0%, 95% CI=60. 7% To 73. 3%, P < . 001).

The result of the FAP not expressing said normal fibroblast cells in which the stranded Dox/PL-a rGO etc. indicate toxicity is encountered. The microenvironment in tumor expression only at the surface at which they are FAP CAF Dox/PL-a rGO is activated only by a natural after is 1, indicating that can minimize side effects are disclosed.

In the embodiment 9. Enhancing the Molecules Imaging Through In the embodiment 1 Of PL-RGONano sheet Complex in vivo tumor tissue accumulation and a penetration confirmation

5 Week zero number Balb/c female nude MICE reactivated and wetting ability (Orient Bio Inc. , Seongnam, Kyonggi a-do, Republic of Korea) using, DSPE-a PEG to tumor within organizing₅₀₀₀ - Cy5. 5 A-labeled rGO or enhancing the distribution and penetration of the PL-a rGO nano sheet composite body molecular imaging were measured. All animals experiments ethical Committee for animal experiments testing Seoul university animal management and use of water are used and which is formed in the triangular (approved animal experimental protocol number SNU provided 130129 provided 3 provided 1).

Specifically mouse or the like (dorsal) right side pin 5 × 10 under⁶ HT29 cells of his. After tumor behind the factory, DSPE-a PEG₅₀₀₀ - Cy5. 5 A-labeled rGO (5 mg/kg rGO) or PL-a rGO nano sheet (10 mg/kg PL, 5 mg/kg rGO) was intravenous to a mouse. Ear eXplore Optix system (Advanced Research Technologies Inc. , Montreal, Canada) to enhancing the distribution and tumor penetration was assessed. For measuring the depth of penetration of rGO or PL-a rGO nano, involving data recorded using his 3D re-Image software. 670 Provided nm Cy5 using pulsed non-laser diode. 5 [Ik site a chains. Fast photomultiplier tube (Hamamatsu Photonics, Hamamatsu, Japan) (Becker and Hickl GmbH, Berlin, Germany) and a time-a correlated single-a photon counting system using long wavelength fluorescent emissions were measured.

6 Also result can be recycled. Such as DSPE-a PEG of said enhancing the administration₅₀₀₀ - Cy5. 5 Lipid - labeled rGO tumor distribution and PL-a rGO were measured. 24 In time post-a dose (also 6A). The site has been treated mouse treated mouse rGO to fluorescent accumulation was greater than PL-a rGO. In specific tumor tissue of fluorescent intensity greater than 72 hours after administration fluorescent PL-a rGO been (also 6B). In the measurement time in said latter DSPE-a PEG producing photon number₅₀₀₀ - Cy5. 5 - Labeled PL-a rGO nano sheet tumor maintaining (retention) (mean=12. 3 X 10³ Photon counts, 95% CI=9. 3 To 15. 3 X 10³ Photon counts) is DSPE-a PEG₅₀₀₀ - Cy5. 5 - Labeled rGO nano sheet (mean=4. 0 X 10³ Photon counts, 95% CI=3. 2 To 4. 8 X 10³ Photon counts) 3 times higher than his (also 6B). Further DSPE-a PEG₅₀₀₀ - Cy5. 5 Labeled rGO and enhancing tumor penetration using eXplore Optix imaging with 3D analysis of lipid - PL-a rGO were measured. In post dose 1, DSPE-a PEG₅₀₀₀ - Cy5. 5 - Labeled rGO (also 6C) or DSPE-a PEG lipid₅₀₀₀ - Cy5. 5 Lipid - treated tumor tissue from the bottoms of labeled PL-a rGO (also 6D) Z - 1 mm intervals obtained Image stack of total 8. At least one of the fluorescent Cy5 total. 5 - Positive z - the ratio of Image stack, DSPE-a PEG₅₀₀₀ - Cy5. 5 Lipid - labeled rGO (mean=0. 5, 95% CI=0. 3 To 0. 6) DSPE-a PEG compared₅₀₀₀ - Cy5. 5 Lipid - labeled PL-a rGO (mean=0. 9, 95% CI=0. 7 To 1. 0) Was warmer.

The result of the, enhancing the model on a cancer tissue than in normal tissue is more than PL-a rGO and accumulated, it will carry on shoulder just worker active in tumor tissue from being dispersed with statin to produce tumor formation in the tissue may be enhancing and titanium in addition exhibits.

In the embodiment 10. In the embodiment 1 Of Dox/PL-RGONano sheet Complex Enhancing the Antitumor efficacy

The FAP in CAF by selective activation in the embodiment expressed in heterologous transplantation model was used for the experiment of PL-a rGO colorectal cancer. Specifically HT29 tumorbearing mice Dox/PL-a rGO anticancer efficacy of using stranded was assessed. 5 - Week zero wetting ability such as female nude MICE (dorsal) right side pin number to reactivated under 5 × 10⁶ HT29 cells of his. Tumor volume 120 mm³ At the time when, Dox (1 mg/kg), PL (10 mg/kg), Dox/rGO (1 mg/kg Dox, rGO 5 mg/kg), PL-a rGO (10 mg/kg PL, 5 mg/kg rGO), or Dox/PL-a rGO (1 mg/kg Dox, 10 mg/kg PL, 5 mg/kg rGO) 2 to each mouse once a total 3 intravenous him. To tumor tissue after tumor inoculation 24 have been formed in a 10% formalin fixed paraffin block for fluorescent staining methods. Anti - FAP - CD133 antibody dyeing in the presence of immune or anti tumor tissue sections (4 micro m thick) determined the anticancer effect. Enhancing the antitumor efficacy of HT29 tumor such as heterologous transplantation model Dox/PL-a rGO were measured.

Result 7 is also disclosed. 24 After the tumor inoculation, glass Dox (mean=697. 0 Mm³ , 95% CI=646. 9 To 747. 1 Mm³ ), Glass PL (mean=565. 0 Mm³ , 95% CI=550. 5 To 579. 6 Mm³ ), Dox/rGO (mean=637. 6 Mm³ , 95% CI=619. 5 To 655. 7 Mm³ ), Or PL-a rGO (mean=464. 4 Mm³ , 95% CI=433. 0 To 495. 8 Mm³ ) Compared to those treated Dox/PL-a rGO (mean=200. 6 Mm³ , 95% CI=148. 7 To 252. 5 Mm³ ) Treated mouse tumor volume was statistically significantly smaller (also 7A). Advantageously said result matches, 24 Dox/PL-a rGO treated mouse tumor weight was the lowest (also 7B). Dox/PL-a rGO weight effective inhibition of growth (mean=155. 0 Mg, 95% CI=99. 0 To 211. 0 Mg) includes a glass Dox (mean=685. 0 Mg, 95% CI=661. 0 To 709. 0 Mg) and Dox/rGO (mean=555. 2 Mg, 95% CI=483. 2 To 627. 2 Mg) by a circular 4. 4 And 3. 6 Times higher yet. Fluorescent staining in immune, anti - CD133 - positive HT29 (also 7C) and anti - FAP - positive cell populations treated mouse (also 7D) CAF Dox/PL-a rGO his lowest in specific tumor tissue.

Said FAP result in various cancers expressing FAP protease number accumulated tissue is activated by cancer cells after drug Dox/PL-a rGO efficient result, show growth of the tumor is inhibited by 2000.

In the embodiment 11. In the embodiment 1 Of Dox/PL-RGoNano sheet Complex Enhancing the Toxicity

A 5 - week zero capacitance Dox/PL-a rGO female Balb/C mouse (Daehan Biolink; Seungnam, Republic of Korea) intravenous to him. For acute toxicity testing, mouse (n=5 per each group embryo) to (rGO dose, 5 or 10 mg/kg) was administered a single Dox/PL-a rGO. Each mouse (n=5 per each group embryo) repeated administration of toxicity (rGO dose, 5 mg/kg) total 3 times for each test in a intravenous Dox/PL-a rGO 2 was. 24 Time using blood samples collected after administration Dox/PL-a rGO biochemical parameters was hematological analysis. In order to biochemical analysis, 1,000 x g in 20 minutes centrifuging blood serum (blood urea nitrogen) and stored at 20 °C was collected BUN [...] until analysis. Such as hematological parameters for his analysis: be leukocyte (WBC); red blood cells (RBC) number; the [hey wool glow it pushed (Hb); hematocrit (Hct); average erythropoietin (MCV); average blood spreadability bovine (MCH); average blood afflicted with a small amount (MCHC); and a platelet, sprue methods, monocytes, eosinophil and good salt nine be. In Neodin VET Diagnostics Institute (Seoul, Republic of Korea) conducting said analysis. Such as said, enhancing the toxicity of 1 times implantation or after implantation times Dox/PL-a rGO nano 3 were measured.

Also disclosed to result 11. Compared to the untreated group, Dox/PL-a rGO (1 mg/kg Dox, 10 mg/kg PL, 5 mg/kg rGO) the implanted blood element nitrogen (single administration: P=. 65; Repeated administrations: P=. 72) (Also 11A and C) or [...] (single administrations: P=. 10; Repeated administrations: P=. 90) For statistically significant (D and also 11B) characterized in that it has not changed, all hematological parameters was within the normal range.

[Table 1] Dox/PL-a rGO single dose of hematological parameters

* Dox=doxorubicin; PL=pro it will carry on shoulder [thin lipid derivatives; rGO=reduced yes pin oxide; WBC=leukocyte; RBC=red blood; Hb=hemoglobin; HCT=hematocrit; average MCV=erythropoietin; MCH=average blood spreadability bovine; MCHC=mean average blood afflicted with a small amount.

[Table 2] Dox/PL-a rGO repeated administration of hematological parameters

* Dox=doxorubicin; PL=pro it will carry on shoulder [thin lipid derivatives; rGO=reduced yes pin oxide; WBC=leukocyte; RBC=red blood; Hb=hemoglobin; HCT=hematocrit; average MCV=erythropoietin; MCH=average blood spreadability bovine; MCHC=mean average blood afflicted with a small amount.

Said result is single or repeating scanned PL-a rGO enhancing effects as indicate, before it reaches the tumor in vivo tumor microenvironment Dox/PL-a rGO is to keep stability at a high level may be big.

Statistics

Using ANOVA with Student-a Newman a-Keuls post hoc test (Two non-sided analysis of variance) was assessed statistically empirical data. 0. 05 Hereinafter was statistically significant P is of that. 95% Confidence interval (CIs) all drawing an air cylinder without error by a goniophotometer. All statistical analysis using SPSS software (version 23; Chicago, IL) conducting.

In the present invention, PL-a rGO, PB provided rGO, PL - liposome, and PB - liposomes in FAP that is expressed in the tumor microenvironment nanocomposite CAF activated by specific delivery of therapeutic agents to cancer cells to antitumor of enhancing the effectiveness of anticancer number can be demonstrated. The FAP - PL or PB containing cleavable peptide sequence, but not to FAP RBC hemolysis or molten synergic effects is discharged after MMP specifically expressed in cancer also has shown only FAP feelings on this subject. In addition, HT29 cells not alone, CAF [...][...] with HT29 cells, increase a dragging effect of Dox/PL-a rGO Dox/rGO in comparison with antitumor agents having been.

Of the present invention PL-a rGO, PB provided rGO, PL - liposome, and PB - of multivesicular liposomes the nanocomposite is, in cytotoxic drug selectively activate tumor microenvironment, thereby reducing the side effects of drug inducing cytotoxicity against normal tissue tranfectants disclosed. Tumor microenvironment may include drug - tumour cells chemically protection (chemoprotection) reported that metabolic enzymes etc.. In acute myeloid leukemia (myeloid leukemia), tumor microenvironment (CYP) expression of retinoid - in metabolic enzyme p450 -26 [...] lance retinoic acid (all-a trans retinoic acid) due to reduce protects cells as in the picomolar activity of acute myeloid leukemia. CYP3A 4 is expressed in human bone marrow-derived stromal cells of combined treatment with bortezomib (bortezomib) and the bottom myeloma of both reducing susceptibility to etoposide (etoposide) daunomycin as in the picomolar. Glass Dox in comparison with Dox/PL-a rGO, Dox/PL - Dox is of multivesicular liposomes, low susceptibility to contribute to tumorigenesis CYP enzyme accessibility since it can be combined with each other. The surfaces of tumor cells in tumor microenvironment on drug susceptibility of metabolic Dox PL-a rGO can be increased. In addition, stores a double number for inhibiting CYP on PL provided rGO Dox and further increase the sensitivity of tumor cells Dox pivotably.

In the embodiment of the present disclosure are described herein but detailed above exemplary rights range and then a received and in the claims herein are not limited to basic general outline of various modified and improved form herein using one skilled in addition range rights are disclosed.

The term used in the present invention all techniques, are not defined or more with, such as typically encountered in the field of the present invention is generally understand sense of one skilled are used. The specification described in the present invention are introduced into the contents of the document on the right side of all Publication.