吳嬌，楊唐斌，柳長(zhǎng)柏

(1.三峽大學(xué)腫瘤微環(huán)境與免疫治療湖北省重點(diǎn)實(shí)驗(yàn)室，湖北宜昌443002；2.三峽大學(xué)醫(yī)學(xué)院，湖北宜昌443002)

腫瘤歸巢肽及其在腫瘤靶向治療中的應(yīng)用

吳嬌1，2，楊唐斌2，柳長(zhǎng)柏1，2

(1.三峽大學(xué)腫瘤微環(huán)境與免疫治療湖北省重點(diǎn)實(shí)驗(yàn)室，湖北宜昌443002；2.三峽大學(xué)醫(yī)學(xué)院，湖北宜昌443002)

腫瘤歸巢肽(THPs)是一類對(duì)腫瘤組織或血管具有歸巢效應(yīng)的多肽，它能識(shí)別和結(jié)合腫瘤組織或血管表面的特異性受體或標(biāo)志物。除了原位腫瘤，THPs還能識(shí)別并結(jié)合血管源性或轉(zhuǎn)移性腫瘤。因此，THPs可將抗腫瘤藥物直接靶向遞送至腫瘤組織、細(xì)胞中，能夠減少或消除藥物耐受及毒副作用。本文將綜述迄今人們對(duì)THPs的認(rèn)識(shí)、開(kāi)發(fā)及其在抗腫瘤診斷及治療應(yīng)用中所取得的進(jìn)展。

腫瘤歸巢肽；靶向遞送；抗腫瘤；靶向治療

惡性腫瘤的早期診斷對(duì)其治療至關(guān)重要。尋找、鑒定具有腫瘤靶向特性的分子不僅可以作為探針靶向腫瘤組織顯像，從細(xì)胞和分子水平檢測(cè)腫瘤，更能實(shí)現(xiàn)藥物的靶向遞送而備受關(guān)注。無(wú)論是腫瘤的分子顯像還是靶向治療，都需要腫瘤特異性配體作為橋梁。迄今常用的分子靶向遞送載是抗體或其片段，但因其分子量大，細(xì)胞膜穿透能力差，且易被吞噬系統(tǒng)攝取，其應(yīng)用受到極大限制。近年腫瘤歸巢肽研究拓寬了靶向配體開(kāi)發(fā)的范圍。

1 腫瘤歸巢肽的概述及分類

每個(gè)組織細(xì)胞表面分子都有其特異性的表達(dá)模式，腫瘤細(xì)胞表面分子表達(dá)模式與正常細(xì)胞不同，且在其發(fā)生、發(fā)展的不同階段會(huì)呈現(xiàn)“階段特異性”改變[1-2]。研究發(fā)現(xiàn)，有一類能與腫瘤組織、細(xì)胞表面分子相結(jié)合的多肽被稱為腫瘤歸巢肽(Tumor homing peptides，THPs)，不僅可以識(shí)別腫瘤細(xì)胞，還可以識(shí)別、結(jié)合實(shí)體腫瘤生長(zhǎng)中非常重要的腫瘤新生血管[3]。因此，近年THPs已成為一種有效的腫瘤診斷與治療的靶向工具，在抗腫瘤應(yīng)用中備受關(guān)注[4]。THPs被分為兩類：選擇性和非選擇性。

1.1 非選擇性THPs某些THPs能結(jié)合多種類型的腫瘤細(xì)胞，它們能識(shí)別多種腫瘤細(xì)胞共表達(dá)的分子標(biāo)志物，這些標(biāo)志物大多在腫瘤血管生成中扮演重要角色。RGD[5]和NGR[6]是最早發(fā)現(xiàn)的兩個(gè)THPs。這兩種肽都是通過(guò)識(shí)別腫瘤血管內(nèi)皮細(xì)胞受體而實(shí)現(xiàn)其靶向作用的，這些靶受體在新生血管中的表達(dá)呈上調(diào)趨勢(shì)[7]。其他非選擇性THPs還包括F3和Lyp-1，其中F3靶向白血病和乳腺癌血管，Lyp-1則能結(jié)合乳腺癌轉(zhuǎn)移瘤淋巴管上的受體或標(biāo)志物[8-9]。

1.2 選擇性THPs通過(guò)噬菌體展示篩選技術(shù)，先后發(fā)現(xiàn)了一些能識(shí)別特定腫瘤類型的THPs。例如：識(shí)別胃癌BCG823細(xì)胞的AADNAKTKSFPV肽序列[10]；識(shí)別胰腺癌轉(zhuǎn)基因小鼠模型的CRGRRST肽序列[11]；識(shí)別食管癌細(xì)胞的YSXNXW肽序列[12]等。靶向各種腫瘤類型的代表性THPs總結(jié)如表1所示。

2 THPs的篩選方法

THPs的篩選方法主要有噬菌體展示肽技術(shù)和珠-化組合技術(shù)。下面對(duì)這兩種技術(shù)進(jìn)行簡(jiǎn)單介紹：

2.1 噬菌體展示肽庫(kù)(Phage Display Peptide Library，PDPL)噬菌體展示技術(shù)是利用基因工程方法將外源基因定點(diǎn)插入到噬菌體基因中，融合的外源蛋白或多肽表達(dá)在噬菌體表面，形成噬菌體蛋白(肽)庫(kù)。該融合蛋白或多肽具有獨(dú)立的空間構(gòu)象，易于被特異性受體識(shí)別、結(jié)合。噬菌體肽庫(kù)經(jīng)過(guò)與靶組織或靶細(xì)胞孵育、洗脫、收集，得到靶向噬菌體，對(duì)靶向噬菌體進(jìn)行DNA的提取及序列測(cè)定，進(jìn)而得到靶向性THPs。噬菌體展示技術(shù)包括體內(nèi)篩選、體外篩選和附加體內(nèi)篩選三種方式[13]。

2.2 珠-化組合庫(kù)(One Bead One Compound，OBOC)珠-化組合庫(kù)于1991年由Lam等[14]首次提出。該方法是將肽顯示在數(shù)以萬(wàn)計(jì)的珠子表面，每個(gè)珠子只結(jié)合一種肽，運(yùn)用拆分和組合方法形成組合庫(kù)[15]。運(yùn)用數(shù)以萬(wàn)計(jì)的肽庫(kù)可以篩選出靶向同一目標(biāo)的聯(lián)合肽。該技術(shù)篩選的肽性質(zhì)多樣，可以包含D-氨基酸、非天然氨基酸、有機(jī)基團(tuán)、翻轉(zhuǎn)、環(huán)形或分支結(jié)構(gòu)甚至非肽基團(tuán)。含有非天然氨基酸和非肽基團(tuán)的肽更能耐受蛋白水解酶。通過(guò)珠化組合庫(kù)的方法已經(jīng)分離出多種腫瘤細(xì)胞系的肽配體[16-17]。

表1 靶向各腫瘤類型的代表性腫瘤歸巢肽

3 THPs的應(yīng)用

由于能特異性結(jié)合腫瘤細(xì)胞和腫瘤新生血管系統(tǒng)，THPs已經(jīng)作為顯像劑和抗癌藥物的選擇性靶向遞送載體，廣泛應(yīng)用于癌癥的診斷和治療研究中。

3.1 腫瘤的診斷THPs作為顯像劑的靶向遞送載體在腫瘤的診斷中可發(fā)揮重要功能，因此，THPs發(fā)現(xiàn)不久便迅速被開(kāi)展應(yīng)用于腫瘤的早期診斷研究。將THPs與放射性核素通過(guò)螯合劑形成復(fù)合物，這種復(fù)合物能夠通過(guò)THPs靶向腫瘤組織或血管，進(jìn)而通過(guò)放射性核素經(jīng)電子發(fā)射斷層掃描成像術(shù)顯示腫瘤的位置、大小和形態(tài)等。Liang等[18]最近開(kāi)發(fā)了一種涂布鐵鉑的半胱氨酸納米顆粒作為MRI/CT成像對(duì)比劑對(duì)惡性神經(jīng)膠質(zhì)瘤進(jìn)行診斷。結(jié)果顯示該復(fù)合物具有優(yōu)良的生物相容性和良好的MRI/CT成像能力，顯示了其在腦惡性神經(jīng)膠質(zhì)瘤診斷中的良好潛能。另外一些THPs，如RGD/NGR等被分別螯合放射性核素68Ga[19]、64Cu[20]，并已成功應(yīng)用于臨床診斷中[21]。

3.2 腫瘤的治療THPs能靶向遞送多種抗癌劑進(jìn)入腫瘤組織，包括細(xì)胞毒性藥物、抗癌蛋白、促凋亡肽及納米顆粒等進(jìn)而實(shí)現(xiàn)腫瘤的靶向治療。

3.2.1 細(xì)胞毒性藥物1998年，Arap等[22]首次運(yùn)用THPs遞送抗癌劑進(jìn)行荷瘤小鼠的靶向治療，發(fā)現(xiàn)細(xì)胞毒性藥物阿霉素作為抗癌劑與RGD或NGR進(jìn)行組裝，顯示了抑制腫瘤生長(zhǎng)的效果比單用阿霉素明顯提高。隨后，有研究人員用脂質(zhì)體將阿霉素先進(jìn)行封裝，再耦聯(lián)上THPs，顯示更佳的抗腫瘤效果[23]。迄今，已有整合素結(jié)靶向THPs修飾的脂質(zhì)體攜帶阿霉素(ATN-161，Ac-PHSCNK-NH2)進(jìn)入抗癌治療的二期臨床試驗(yàn)[24]。

3.2.2 藥用蛋白質(zhì)抗癌藥用蛋白質(zhì)如TNF-α，通常具有全身毒性而在應(yīng)用中受到極大限制，運(yùn)用靶向遞送策略能降低這種毒性。有研究將hPK5(纖溶酶原的一個(gè)蛋白水解片段，也是內(nèi)源性血管生長(zhǎng)抑制因子)與CNGRC融合形成重組肽，作用于人類肺癌和結(jié)腸癌的移植瘤小鼠，顯示與野生型hPK5相比，重組肽抗血管新生作用更強(qiáng)，能更好抑制腫瘤生長(zhǎng)[25]。

3.2.3 促凋亡肽促凋亡肽如KLA能引起哺乳動(dòng)物細(xì)胞線粒體腫脹進(jìn)而導(dǎo)致線粒體依賴性細(xì)胞凋亡。THPs修飾的KLA已經(jīng)作為一種促凋亡肽用以選擇性殺傷腫瘤血管和腫瘤細(xì)胞。在荷瘤小鼠研究中，THPs與KLA形成的共軛物已經(jīng)成功地將促凋亡肽送入腫瘤組織，并誘導(dǎo)了腫瘤血管內(nèi)皮細(xì)胞的凋亡、抑制了腫瘤的生長(zhǎng)[26]。

3.2.4 THPs修飾的納米顆粒在腫瘤診斷和治療中的應(yīng)用納米顆粒廣泛用于藥物的遞送研究中，它增強(qiáng)藥物附著和吸收[27]。THPs修飾的納米顆粒，既可實(shí)現(xiàn)納米顆粒的靶向性，也可增強(qiáng)THPs的細(xì)胞穿透能力，實(shí)現(xiàn)互補(bǔ)相乘效果。研究證實(shí)能夠?qū)HPs與納米顆粒進(jìn)行組裝，用于靶向腫瘤的診斷與治療應(yīng)用[28]。

隨著納米顆粒的應(yīng)用，腫瘤的診斷治療學(xué)也有了發(fā)展與突破[29]。近年來(lái)流行將診斷試劑和治療藥物同時(shí)與THPs連接組裝，靶向腫瘤組織以實(shí)現(xiàn)更加特異性的診斷和個(gè)體化治療。Wu等[30]開(kāi)發(fā)了一種聚乙烯乙二醇-聚乳酸與THPs修飾的殼聚糖納米顆粒，包裹熒光成像劑和抗癌藥物紫杉醇，用于膠質(zhì)母細(xì)胞瘤荷瘤小鼠體內(nèi)，實(shí)現(xiàn)了良好的腫瘤靶向殺傷作用。類似的研究也顯示了有效的抗多形性惡性膠質(zhì)瘤作用[31]。

近些年，研究人員將THPs(如環(huán)形RGD)與放射性核素和阿霉素等結(jié)合制備了多種具診斷和靶向治療功效的納米顆粒，如SPIO納米顆粒[32]、黃金納米顆粒[33]及單分子膠束[34]，同時(shí)實(shí)現(xiàn)腫瘤靶向歸巢、顯像診斷和抗癌治療。盡管納米顆粒介導(dǎo)的診斷治療法在抗腫瘤應(yīng)用中仍然處于初級(jí)階段，但它們?cè)谂R床前期研究中的成功，為個(gè)體用藥和靶向治療提供了良好基礎(chǔ)，在抗腫瘤治療應(yīng)用中具有良好前景。

4 展望

本文介紹了THPs的類型、鑒定方法及其在靶向抗腫瘤研究中的應(yīng)用。THPs-納米系統(tǒng)的開(kāi)發(fā)促進(jìn)了腫瘤診斷治療學(xué)的發(fā)展，為抗腫瘤治療提供了一種新的、有效的手段。迄今，已經(jīng)有部分研究運(yùn)用THPs對(duì)腫瘤進(jìn)行診斷和治療已經(jīng)進(jìn)入了臨床試驗(yàn)[35-36]。然而，THPs仍然存在不足，如其穩(wěn)定性和半衰期等是主要被關(guān)注的問(wèn)題[37]。多肽在體內(nèi)容易被多種酶降解，但這種缺陷可以通過(guò)多種方法克服，如包括插入D-氨基酸或非天然氨基酸、環(huán)化肽以及碳、氮端修飾等。此外，其穿透細(xì)胞膜的能力仍然有限，尋找同時(shí)具有靶向識(shí)別和細(xì)胞膜穿透能力的THPs可能成為研究熱點(diǎn)。

[1]Joyce JA,Laakkonen P,Bernasconi M,et al.Stage-specific vascular markers revealed by phage display in a mouse model of pancreatic islet tumorigenesis[J].Cancer Cell,2003,4(5):393-403.

[2]Hoffman JA,Giraudo E,Singh M,et al.Progressive vascular changes in a transgenic mouse model of squamous cell carcinoma[J].Cancer Cell,2003,4(5):383-391.

[3]Weis SM,Cheresh DA.Tumor angiogenesis:molecular pathways and therapeutic targets[J].Nature Medicine,2011,17(11):1359-1370.

[4]Di Matteo P,Mangia P,Tiziano E,et al.Anti-metastatic activity of the tumor vascular targeting agent NGR-TNF[J].Clinical&Experimental Metastasis,2015,32(3):289-300.

[5]Pasqualini R,Koivunen E,Ruoslahti E.Alpha v integrins as receptors for tumor targeting by circulating ligands[J].Nat Biotechnol, 1997,15(6):542-546.

[6]Arap W,Pasqualini R,Ruoslahti E.Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model[J].Science,1998, 279(5349):377-380.

[7]Desgrosellier JS,Cheresh DA.Integrins in cancer:biological implications and therapeutic opportunities[J].Nature Reviews Cancer, 2010,10(1):9-22.

[8]Porkka K,Laakkonen P,Hoffman J A,et al.A fragment of the HMGN2 protein homes to the nuclei of tumor cells and tumor endothelial cells in vivo[J].Proc Natl Acad Sci USA,2002,99(11): 7444-7449.

[9]Laakkonen P,Porkka K,Hoffman J A,et al.A tumor-homing peptide with a targeting specificity related to lymphatic vessels[J].Nat Med, 2002,8(7):751-755.

[10]Zhang WJ,Sui YX,Budha A,et al.Affinity peptide developed by phage display selection for targeting gastric cancer[J].World J Gastroenterol,2012,18(17):2053-2060.

[11]Li ZJ,Cho CH.Peptides as targeting probes against tumor vasculature for diagnosis and drug delivery[J].J Transl Med,2012,10(Suppl 1):S1.

[12]Zhi M,Wu K,Hao Z,et al.Screening of specificbinding peptide targeting blood vessel of human esophageal cancer in vivo in mice[J]. Chinese Med J,2011,124(4):581-585.

[13]McCafferty J,Schofield D.Identification of optimal protein binders through the use of large genetically encoded display libraries[J]. Curr Opin Chem Biol,2015,26C:16-24.

[14]Lam KS,Salmon SE,Hersh EM,et al.A new type of synthetic peptide library for identifying ligand-binding activity[J].Nature,1991, 354(6348):82-84.

[15]Bononi FC,Luyt LG.Synthesis and cell-based screening of One-Bead-One-Compound Peptide Libraries[J].Peptide Libraries: Methods and Protocols,2015:223-237.

[16]Gao Y,Amar S,Pahwa S,et al.Rapid lead discovery through iterative screening of One Bead One Compound Libraries[J].ACS Combinatorial Science,2014,17(1):49-59.

[17]Garcia-Martin F,Matsushita T,Hinou H,et al.Fast epitope mapping for the anti-MUC1 monoclonal antibody by combining a One-Bead-One-Glycopeptide Library and a Microarray Platform[J]. Chemistry-AEuropean Journal,2014,20(48):15891-15902.

[18]Liang S,Zhou Q,Wang M,et al.Water-soluble L-cysteine-coated FePt nanoparticles as dual MRI/CT imaging contrast agent for glioma[J].Int J Nanomedicine,2015,10:2325-2333.

[19]Kim JH,Lee JS,Kang KW,et al.Whole-body distribution and radiation dosimetry of68Ga-NOTA-RGD,a positron emission tomography agent for angiogenesis imaging[J].Cancer Biotherapy and Radiopharmaceuticals,2012,27(1):65-71.

[20]Chen K,Ma W,Li G,et al.Synthesis and evaluation of64Cu-labeled monomeric and dimeric NGR peptides for MicroPET imaging of CD13 receptor expression[J].Molecular Pharmaceutics,2012,10 (1):417-427.

[21]Lee S,Kang SW,Ryu JH,et al.Tumor-homing glycol chitosan-based optical/PET dual imaging nanoprobe for cancer diagnosis[J].Bioconjugate Chemistry,2014,25(3):601-610.

[22]Arap W,Pasqualini R,Ruoslahti E.Chemotherapy targeted to tumor vasculature[J].Current Opinion in Oncology,1998,10(6):560-565.

[23]Yang Y,Yan Z,Wei D,et al.Tumor-penetrating peptide functionalization enhances the anti-glioblastoma effect of doxorubicin liposomes [J].Nanotechnology,2013,24(40):405101.

[24]Dai W,Yang T,Wang Y,et al.Peptide PHSCNK as an integrin α 5 β 1 antagonist targets stealth liposomes to integrin-overexpressing melanoma.Nanomedicine:Nanotechnology[J].Biology and Medicine, 2012,8(7):1152-1161.

[25]Jiang W,Jin G,Ma D,et al.Modification of cyclic NGR tumor neovasculature-homing motif sequence to human plasminogen kringle 5 improves inhibition of tumor growth[J].PLoS One,2012,7(5): e37132.

[26]Ma JL,Wang H,Wang YL,et al.Enhanced peptide delivery into cells by using the synergistic effects of a cell-penetrating peptide and a chemical drug to alter cell permeability[J].Mol Pharmaceutics, 2015,12(6):2040-2048.

[27]Nehoff H,Parayath NN1,Domanovitch L,et al.Nanomedicine for drug targeting:strategies beyond the enhanced permeability and retention effect[J].International Journal of Nanomedicine,2014,9: 2539-2555.

[28]Gao H,Zhang Q,Yang Y,et al.Tumor homing cell penetrating peptide decorated nanoparticles used for enhancing tumor targeting delivery and therapy[J].International Journal of Pharmaceutics,2015,478 (1):240-250.

[29]Gautam A,Kapoor P,Chaudhary K,et al.Tumor homing peptides as molecular probes for cancer therapeutics,diagnostics and theranostics[J].Current Medicinal Chemistry,2014,21(21):2367-2391.

[30]Wu J,Zhao J,Zhang B,et al.Polyethylene glycol-polylactic acid nanoparticlesmodifiedwithcysteine-arginine-glutamicacid-lysine-alanine fibrin-homing peptide for glioblastoma therapy by enhanced retention effect[J].International Journal of Nanomedicine, 2014,9:5261-527.

[31]Zhao J,Zhang B,Shen S,et al.CREKA peptide-conjugated dendrimer nanoparticles for glioblastoma multiforme delivery[J].J Colloid Interface Sci,2015,450:396-403.

[32]Huang RY,Chiang PH,Hsiao WC,et al.Redox-sensitive polymer/ SPIO nanocomplexes for efficient magnetofection and MR imaging of human cancer cells[J].Langmuir,2015[Epub ahead of print].

[33]Alkilany AM,Boulos SP,Lohse SE,et al.Homing peptide-conjugated gold nanorods:the effect of amino acid sequence display on nanorod uptake and cellular proliferation[J].Bioconjugate Chemistry, 2014,25(6):1162-1171.

[34]Chung EJ,Cheng Y,Morshed R,et al.Fibrin-binding,peptide amphiphile micelles for targeting glioblastoma[J].Biomaterials,2014,35 (4):1249-1256.

[35]Pastorino F,Brignole C,Loi M,et al.Nanocarrier-mediated targeting of tumor and tumor vascular cells improves uptake and penetration of drugs into neuroblastoma[J].Front Oncol,2013,3:190.

[36]Sha H,Zou Z,Xin K.Tumor-penetrating peptide fused EGFR single-domain antibody enhances cancer drug penetration into 3D multicellular spheroids and facilitates effective gastric cancer therapy[J].J Control Release,2015,200:188-200.

[37]Craik DJ,Fairlie DP,Liras S.The future of peptide-based drugs[J]. Chem Biol Drug Des,2013,81:136-144.

Tumor homing peptides and their applications in tumor targeting therapy.

WU Jiao1,2,YANG Tang-bin2,LIU Chang-bai1,2.1.Hubei Province Key Laboratory of Tumor Microenvironment and Immunotherapy,China Three Gorges University,Yichang 443002,Hubei,CHINA;2.Medical School of China Three Gorges University,Yichang 443002,Hubei,CHINA

Tumor homing peptides(THPs)are a kind of peptide which specifically bind to the tumor cells and tumor vessels.THPs have ability to recognize and bind to primary tumor as well as metastatic tumors through the specific receptors or bio-markers on the surface of the tumor cells or tumor vessels.In consequence,THPs can be used as delivery tools for anti-tumor drugs targeted to tumor tissues and cells directly,which will be able to reduce or eliminate the drug resistance and side effects.This review will summarize the development and applications of THPs in the tumor diagnosis and the anti-tumor therapies.

Tumor homing peptides;Delivery vector;Anti-tumor;Targeted therapy

R730.5

A

1003—6350（2016）01—0082—03

10.3969/j.issn.1003-6350.2016.01.029

2015-06-24)

國(guó)家自然科學(xué)基金(編號(hào)：81201766)；湖北省自然科學(xué)基金(編號(hào)：2010CDB10705)；湖北省高等學(xué)校優(yōu)秀中青年創(chuàng)新團(tuán)隊(duì)計(jì)劃項(xiàng)目(編號(hào)：T201203)

柳長(zhǎng)柏。E-mail：cbliu@ctgu.edu.cn