Cell Adhesion Molecules for Targeted Drug Delivery

NOTE Cell Adhesion Molecules for Targeted Drug Delivery ALISON L. DUNEHOO, MEAGAN ANDERSON, SUMIT MAJUMDAR, NAOKI KOBAYASHI, CORY BERKLAND, TERUNA J. SIAHAAN Department of Pharmaceutical Chemistry, The University of Kansas, Simons Research Laboratories, 2095 Constant Avenue, Lawrence, Kansas 66047

Received 17 October 2005; revised 20 April 2006; accepted 21 April 2006 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20676

ABSTRACT: Rapid advancement of the understanding of the structure and function of cell adhesion molecules (i.e., integrins, cadherins) has impacted the design and development of drugs (i.e., peptide, proteins) with the potential to treat cancer and heart and autoimmune diseases. For example, RGD peptides/peptidomimetics have been marketed as anti-thrombic agents and are being investigated for inhibiting tumor angiogenesis. Other cell adhesion peptides derived from ICAM-1 and LFA-1 sequences were found to block T-cell adhesion to vascular endothelial cells and epithelial cells; these peptides are being investigated for treating autoimmune diseases. Recent findings suggest that cell adhesion receptors such as integrins can internalize their peptide ligands into the intracellular space. Thus, many cell adhesion peptides (i.e., RGD peptide) were used to target drugs, particles, and diagnostic agents to a specific cell that has increased expression of cell adhesion receptors. This review is focused on the utilization of cell adhesion peptides and receptors in specific targeted drug delivery, diagnostics, and tissue engineering. In the future, more information on the mechanism of internalization and intracellular trafficking of cell adhesion molecules will be exploited for delivering drug molecules to a specific type of cell or for diagnosis of cancer and heart and autoimmune diseases. ß 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:1856–1872, 2006

Keywords: cell adhesion; RGD; ICAM-1; LFA-1; targeted delivery; peptide; biomaterial; diagnostic; integrin

Abbreviations used: Ad2, Adenovirus type 2; APC, antigenpresenting cells; RGE, Arg-Gly-Glu; RGD, Arg-Gly-Asp; DGE, Asp-Gly-Glu; CAMs, cell adhesion molecules; cIBR, cyclic ICAM-1 blocker right; DHFR, dihydrofolate reductase; Dox, doxorubicin; D1, domain-1; ECM, extracellular matrix, 18FBRGD, 18F-benzoyl-RGD; 18FG-RGD, 18F-galactose-RGD; FN, fibronectin; FG, fibrinogen; FMDV, foot-and-mouth disease virus; gpIIb/IIIa, glycoproteins IIb/IIIa; GPR, Gly-Pro-Arg; HCAEC, human coronary artery endothelial cells; HIV-1, human immunodeficiency virus-1; HPEV1, human parechovirus 1; HUVEC, human umbilical vascular endothelial cells; ICAM-1, intercellular adhesion molecule-1; i.v., intravenous; LDV, Leu-Asp-Val; LFA-1, leukocyte function-associated antigen-1; K, Lys; LCL, long circulating liposomes; MHC-Ag, major histocompatibility complex-antigen; MTX, methotrexate; mAb, monoclonal antibody; MadCAM-1, mucosal addressin cell

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adhesion molecule-1; HPMA, N-(2-hydroxypropyl)-methacrylamide; PTX, paclitaxel; PI3K, phosphoinositide-3-OH kinase; PECAM-1, platelet endothelial cell adhesion molecule-1; PEG, poly(ethylene glycol); PEG-PLA, poly(ethylene glycol)poly(D,L-lactic acid); PET, positron emission tomography; PRG, Pro-Arg-Gly; PKC, protein kinase C; sICAM-1, soluble ICAM-1; TCR, T-cell receptor; DOTA, 1,4,7,10-tetraazadodecane-N,N0 ,N00 ,N000 -tetraacetic acid; VCAM-1, vascular cellular adhesion molecule-1; VEGF, vascular endothelial growth factor; VN, vitronectin; VWF, von Willebrand Factor. Correspondence to: Teruna J. Siahaan (Telephone: 785-8647327; Fax: 785-864-5736) Journal of Pharmaceutical Sciences, Vol. 95, 1856–1872 (2006) ß 2006 Wiley-Liss, Inc. and the American Pharmacists Association

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INTRODUCTION In the past two decades, many cell adhesion molecules (CAMs) have been discovered, and their functions in cell morphology, locomotion, mitosis, cytokinesis, phagocytosis, and the maintenance of cell polarity have been studied.1–3 CAMs have important roles in different disease states such as cancer,3–8 thrombosis,9–11, and autoimmune diseases (e.g., rheumatoid arthritis and type-1 diabetes).12–14 Thus, researchers have been actively investigating the structure, function, and recycling mechanisms of some CAMs, as well as how to modulate them for controlling disease progression.14 In addition, CAMs have been implicated in pathogenic (i.e., virus and bacteria) infections.15–17 CAMs are glycoproteins found on the cell surface that act as receptors for cell-to-cell and cell-toextracellular matrix (ECM) adhesion.18–20 CAMs can be divided into four classes: integrins, cadherins, selectins, and the immunoglobulin superfamily. Some CAMs are internalized into the cytoplasm during the recycling process via the formation of clathrin-coated pits.16,21 Thus, the

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internalization process of CAMs can be exploited for targeting drugs into a compartment of a specific cell type (i.e., cancer and leukemic cells). In recent years, peptides, peptidomimetics, and proteins that bind to cell adhesion receptors have been investigated for targeting drugs, particles, and liposomes to specific cell-bearing cell adhesion receptors (i.e., integrin and immunoglobulin superfamily).22–24 Although structure–activity relationships of some cell adhesion peptides/ proteins have been elucidated, there are limited comprehensive reviews on their utility in targeted drug delivery, diagnostics, and biomaterial design. Thus, this review is focused on the current uses of cell adhesion peptides and proteins for drug targeting, tumor diagnostics, and biomaterial design. Specifically, these cell adhesion peptides are derived from the ECM proteins, the immunoglobulin family, and integrins. The ECM peptides (i.e., RGD) have been used to target integrins avb3 and avb5, and peptides derived from the intercellular adhesion molecule-1 (ICAM-1) have been used to target the aLb2 integrin. Finally, peptides derived from aLb2 can target ICAM-1-expressing cells.

Figure 1. The general structure of integrins adapted from Shimaoka and Springer14 showing different segments of the a- and b-subunits based on the structure of avb3. There are three possible conformations of integrins: (a) a bent conformation with a low affinity for the ligand, (b) an extended conformation with intermediate ligand affinity and closed headpiece, and (c) a high ligand affinity conformation with an extended conformation that has open headpiece when bound to RGD peptide. DOI 10.1002/jps

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INTEGRIN STRUCTURE, FUNCTION, AND INTERNALIZATION PROPERTIES General Structure Integrins are cell surface glycoprotein receptors that are formed by a heterodimer of a- and b-subunits (Fig. 1). There are about 24 known combinations of heterodimers that are assembled from 18 a-subunits (e.g., a1, aL, av) and 8 bsubunits (e.g., b1, b2, b3).14,18,25,26 A combination of a- and b-subunits provides an integrin with its own ligand binding specificity and signaling properties.26 Integrins bind to ECM proteins (e.g., fibronectin (FN), vitronectin (VN)) or cell surface immunoglubolin proteins (e.g., intercellular adhesion molecule-1 (ICAM-1), and vascular cellular adhesion molecule-1 (VCAM-1)) during cell aggregation, thrombosis, tumor migration, and angiogenesis.20 Cell surface immunoglobulin proteins such as ICAM-1 and VCAM-1 have important roles in (a) the adhesion process of leukocytes to vascular endothelium and antigenpresenting cells (APC) and (b) the signaling process during leukocyte activation in the inflammatory response.12–14,27 The leukocyte adhesion process can be partly mediated by an integrin called leukocyte function-associated antigen-1 (LFA-1, aLb2, or C11a/CD18) on leukocytes that consist of aL- and b2-subunits. One of the counterparts for LFA-1 on leukocytes is ICAM-1 on the surface of APC or vascular endothelial cells.12–14 An integrin can adopt a non-activated (Fig. 1a) or activated (Fig. 1b,c) state. Integrins bind to their ligands in the intermediate activated (Fig. 1b) or highly activated state (Fig. 1c).28–31 The activation of an integrin involves its conformational changes followed by the intracellular signaling processes, which regulates gene expression, cell growth, differentiation, and survival.32–34 During binding to ECM proteins, integrins form clusters at the cell membrane that are associated with a cytoskeletal complex to promote actin filament assembly.35,36 As a positive feedback, the reorganization of actin filaments into larger stress fibers increases integrin clustering followed by enhancement of ECM protein binding. Integrin Internalization Integrin can undergo endocytosis and exocytosis during cell locomotion and migration.37–41 In the cell locomotion process, integrins continuously form new integrin-based focal contacts at the front of the cell, which can be triggered by JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 9, SEPTEMBER 2006

vascular endothelial growth factor (VEGF) and controlled by protein kinase C (PKC).42 When the cell moves forward, the integrin interaction is released from extracellular ligands at the rear of the cells after forming new and persistent integrin connections at the front of the cell.43,44 Polarized distribution of avb3 integrins was observed in migrating neutrophils;45 this was maintained by Ca2þ-dependent release of cell adhesion followed by endocytosis of the integrin.46 Thus, integrin endocytosis has been proposed as an attractive mechanism for controlling cell signaling pathways that are stimulated by ligand binding. This signaling process allows the cell to control where and when the integrin expression is needed.16,47 Integrin internalization is regulated by a cytoplasmic domain sequence, NPXF or NPXY where X represents a non-conserved residue.48 Several bsubunits of integrin (i.e., fibronectin receptor, glycoproteins IIb/IIIa (gpIIb/IIIa), LFA-1, Mac-1, p150,95) contain two copies of a conserved NPXY or NPXF sequence. The NPXY sequence is a major recognition site for the incorporation of LDL receptors in clathrin-coated pits for internalization.48 A similar finding was observed for an integrin called Mac-1 (CD11b/CD18); mutation or truncation of the NPXF sequence on the b2subunit (CD18) suppresses the internalization of this integrin.49 Interestingly, b2- and b7-integrins selectively expressed in leukocytes have an additional tyrosine-based ‘‘endocytic signal’’ in the membrane proximal region. This endocytic signal has a YXXØ sorting motif that is found in transferrin receptors, where X is any amino acid and Ø is an amino acid with a bulky hydrophobic residue.50 Integrin-Mediated Viral and Bacterial Internalization Many viruses also utilize integrins for their budding and internalization into the host cells, and the viral–cell surface interface is mediated by interactions between viral pentons and integrin clusters.16,51 This interaction process is more complex than previously thought; several viruses can interact with unique integrin regions and activate distinct cell signaling pathways. The activation of a signaling pathway promotes viral endocytosis; for example, adenovirus internalization by av integrins requires activation of phosphoinositide-3-OH kinase (PI3K).16,52–54 Different viruses utilize different integrins for DOI 10.1002/jps

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Table 1. Binding of Virus to Integrin for Cell Internalization Virus

Integrin

Human parechovirus 1 (HPEV1) Herpes virus Foot-and-mouth disease virus (FMDV) (VP1) Human adenovirus (i.e., Ad9, Ad19p) Adenovirus type 2 (Ad2) and type 12 Rotavirus (VP4 anVP7) SA11 Rotavirus

internalization (see Tab. 1). For example, herpes virus uses a3b1 integrin55 as its receptor, but human parechovirus 1 (HPEV1) uses avb3 and avb1 integrins.16 It has been suggested that adenovirus type 2 (Ad2) utilizes aMb2 receptors for attachment followed by av integrin interactions for viral internalization.56 Ad2 virus binding to human monocytic cells via the b2-integrins can be blocked by a soluble viral penton or monoclonal antibody (mAb) to aMb2.56 Integrin recognition for the viral surface is mediated by different sequences found on the penton of viral capsid, and this recognition triggers integrin-mediated viral internalization into cell endosomes.51,54 Five Arg-Gly-Asp (RGD) sequences per viral penton of human Ad2 or Ad12 bind to five avb5 integrins on the cell surface prior to viral internalization.51 Many viruses utilize the RGD motif for integrin recognition (Tab. 1) and RGD peptides and/or mAbs to integrin have been shown to block viral cell uptake.55,57 Thus, RGDmediated gene delivery using adenovirus has been developed to reduce non-specific transfection by incorporating an RGD sequence in the HI loop of the fiber knob.58,59 Sequences other than the RGD motif have also been shown to participate during viral binding and infection, including: (a) Asp-GlyGlu (DGE) from VP4 capsid protein that binds to a2b1 integrin and (b) Gly-Pro-Arg (GPR) and LeuAsp-Val (LDV) sequences that are involved in binding between VP7 capsid protein and axb2, avb3, and a4b1 integrins.15,60 In addition, cell surface immunoglobulin proteins such as ICAM-1 are also receptors for human rhinovirus; the coat proteins of the virus recognize the domain-1 (D1) of ICAM1.61 HIV-1 that has been engineered to express ICAM-1 on its surface has enhanced viral infectivity on T cells; the uptake of this virus is mediated by LFA-1 on T cells.62 Finally, integrins have also been observed to mediate bacterial infection (i.e., Y. pseudotuberculososis) by binding to invasin on the surface of bacteria.63–65 DOI 10.1002/jps

avb3 and avb1 a3b1 avb3 avb5 aMb2, avb5 a2b1, axb2, aVb3, a4b1 a2b1, a4b1

Reference 53 55 143,144 57 51,56 15,60 145,146

DISCOVERY OF PEPTIDES THAT INHIBIT INTEGRIN-MEDIATED CELL ADHESIONS Historically, cell adhesion peptides were exploited for suppressing cell adhesion-mediated diseases such as thrombosis, inflammation, and tumor metastasis. Initially, the roles of ECM proteins were investigated in the adhesion of normal cells to tissues and the process of metastasis of cancer cells. During these investigations, Pierschbacher and Ruoslahti discovered that the RGD sequence found in FN was responsible for cell adhesion to FN and that small RGD peptides derived from FN could block cell attachment to FN (Fig. 2a).66,67 Since this discovery, many other ECM proteins such as collagens, laminin, osteonectin, VN, fibrinogen (FG), von Willebrand Factor (VWF), and thrombospondin have been found to have an RGD sequence(s); the cell surface receptors called integrins that recognized the RGD sequences were also discovered, including avb3, avb5, avb6, avb8, aIIbb3, a5b1, and a8b1 (see Fig. 2).67–69 In a similar fashion, peptides derived from ICAM-1 sequence were found to bind LFA-1 and block Tcell adhesion to epithelial and endothelial cell monolayers (Fig. 2b).12,13 Design and Selectivity of RGD Peptides Many synthetic RGD peptides and peptidomimetics that are selective to a specific integrin were designed to block cell adhesion to ECM (Fig. 2a) and developed as drugs to treat thrombosis,70 cancer,71 and wounds.72 Integrilin173,74 and Aggrastat175–77 (Fig. 3) are two antithrombic drugs that are currently on the market. These drugs were designed to selectively bind gpIIb/IIIa receptors found on the surface of platelets. During thrombosis, FG binds to gpIIb/IIIa receptors to generate platelet aggregation and, thus, Integrilin1 or Aggrastat1 blocks FG binding to gpIIb/ IIIa receptor and prevents platelet aggregation. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 9, SEPTEMBER 2006

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Figure 2. Two models for integrin-mediated cell adhesion to (a) ECM protein and (b) another cell. (a) A model for cell adhesion to fibronectin via integrin (i.e., fibronectin receptor) using the RGD sequence. The cell–ECM adhesion can be blocked by a small RGD peptide or peptidomimetics. (b) An example of leukocyte adhesion to vascular endothelial cells using ICAM-1/LFA-1 or VCAM-1/VLA-4 interactions. The cell–cell adhesion can be inhibited by peptides derived from the ICAM-1 or LFA-1 sequences. [Color figure can be seen in the online version of this article, available on the website, www.interscience.wiley.com.]

On the other hand, cyclic peptides cRGDfK, cRGDyK, and RGDC4 (Fig. 3) are selective for avb3 and avb5 integrins. Because the angiogenic endothelial cells of a solid tumor express av integrins that are not detectable in normal blood vessels, cyclic peptides cRGDfK, cRGDyK, and RGDC4 have been used to inhibit angiogenesis in solid tumors.78–81 It is interesting that these drugs were designed based on the structural understanding of the RGD sequence and its flanking residues (see below). The selectivity of an RGD peptide for a specific integrin depends on the conformation of the RGD sequence and the characteristics of its flanking residues.79,80,82 – 91 To restrict the conformation of an RGD sequence, many researchers have designed a variety of cyclic RGD peptides (Integrilin1, cRGDyK, and RGD4C in Fig. 3) that provide different secondary structures at the RGD sequence. The formation of cyclic RGD peptide can be accomplished using a peptide bond (i.e., cRGDyK) or a disulfide bond (Integrilin1). A selective cyclic RGD peptide for the gpIIb/IIIa receptor such as Integrilin1 has a turn-extendedJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 9, SEPTEMBER 2006

turn structure at the RGD sequence and an aromatic residue (i.e., Trp, Tyr, or Phe) at the Cterminal of the Asp residue.85,89,90 On the other hand, a cyclic RGD peptide (cRGDfK) that is selective for avb3 and avb5 has a g-turn at the Gly of RGD and a D-Phe amino acid (Fig. 3).81,84,87,92 The difference in backbone conformation at the RGD sequence is translated into the triad distances between (a) the positively charged guadinium group of the Arg residue and the negatively charged Asp residue (R1), (b) the carboxylic acid of the Asp residue and the aromatic group of the Phe or Tyr residue (R2), and (c) the aromatic group and the positively charged guanidium group (R3) (Fig. 4).84 – 90,92 The separation between the positive and negative charges (R1) in gpIIb/IIIa-selective peptides is longer than that in the avb3-selective peptide. In addition, the relative distances between the sidechain aromatic residue (i.e., Trp, Tyr, or Phe) and the positive charge of the Arg side chain (R3) or the negative charge of the Asp side-chain (R2) are important for RGD peptide selectivity. Using docking studies, Marinelle et al.88 proposed the DOI 10.1002/jps

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Figure 3. The structures of RGD peptides and peptidomimetics selective for gpIIb/ IIIa, avb3, and avb5 integrins. Integrilin and Aggrastat1 are antithrombic agents currently on the market that contain the RGD motif; these compounds are selective for gpIIb/IIIa integrin. RGDyK and RGD4C are cyclic peptides selective for the avb3, and avb5 integrins; these compounds have been investigated for blocking angiogenesis and tumor metastasis.

nature of interactions between the residues in the RGD peptides and in avb3 integrin. These results support the postulate that the conformation of RGD sequence and the orientation of its flanking residues determine the selectivity of the peptide for a specific integrin. The structural informations from cyclic RGD peptides have been used to design RGD peptidomimetics (i.e., Aggrastat1). One major drawback for RGD peptide and peptidomimetic drugs is that they are not orally bioavailable. Thus, both Integrilin1 and Aggrastat1 are currently delivered via intravenous (i.v.) injection. This is due to the presence of positive (guanidinium or amine group) and negative (carboxylic acid group) charges in these molecules that prevent them from partitioning into cell membranes and crossing the intestinal mucosa barrier.93–95 The presence of the charges in these molecules can be transiently masked by forming esterase-sensitive cyclic prodrugs. The cyclic prodrugs of RGD peptidomimetics were formed by linking the amino group and the DOI 10.1002/jps

carboxylic acid group via an esterase-sensitive linker such as acyloxyalkoxy,96 trimethyl lock,97,98 or coumarinic acid.99 Formation of esterase-sensitive cyclic prodrugs has been shown to improve the permeation of RGD-peptidomimetics through the intestinal mucosa.95,99,100 After the cyclic prodrug crosses the intestinal mucosa barrier, it is anticipated that it can be converted to the drug by esterase found in the blood stream. Peptides Derived from ICAM-1 Another class of important cell adhesion molecules is the immunoglobulin superfamily, including ICAM-1, VCAM-1, platelet endothelial cell adhesion molecule-1 (PECAM-1), and mucosal addressin cell adhesion molecule-1 (MadCAM-1) (Fig. 2b). ICAM-1 is involved in a co-stimulatory signal for T-cell activation during the interaction of T cells and APC.101 For antigen-specific T-cell activation, ICAM-1/LFA-1 interactions (Signal-2) along with interaction between T-cell receptor (TCR)/major histocompatibility comJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 9, SEPTEMBER 2006

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Figure 4. The general concept of recognition of RGD peptide by integrin proposed by Marinelli et al.93,96,97 The integrin recognizes the RGD peptide using a triad relationship between the guanidinium of the Arg residue, the carboxylic acid of the Asp residue, and the phenyl ring of the Phe residue. It is proposed that the distances (R1, R2, R3) between these triad recognition sites may determine the selectivity of RGD peptide to a particular integrin. Thus, the conformational restriction imposed by cyclization of the RGD sequence (i.e., b-turn, extended) may influence the selectivity of the peptide for a particular integrin (i.e., gpIIb/IIIa or avb3). [Color figure can be seen in the online version of this article, available on the website, www.interscience.wiley.com.]

plex–antigen complex (MHC-Ag) (Signal-1) form an ‘‘immunological synapse’’ at the T cell–APC interface.101 –103 Blocking the ICAM-1/LFA-1 interactions with mAbs resulted in suppression of autoimmune diseases such as diabetes,104 rheumatoid arthritis,105 and psoriasis106,107 and prevention of allograft rejection.108,109 Linear and cyclic peptides (Tab. 2) derived from the D1 of ICAM-1 have been shown to block ICAM1/LFA-1-mediated homotypic/heterotypic T-cell adhesion and mixed lymphocyte reactions.12,110– 113 Binding of the D1 of ICAM-1 to the I-domain of a-subunit of LFA-1 on leukocytes is initiated by the conformational change of LFA-1 (Fig. 2b).114,115 Interestingly, FITC-labeled cIBR peptide is internalized into T cells by LFA-1; thus, this peptide has been explored for its use in targeting drugs to leukocytes (see below).111,116 As in RGD peptide, the important motif in the cIBR peptide is the ProArg-Gly (PRG) sequence, and the conformation of this sequence is in a b-turn structure.110,117,118

Further investigation of the effect of PRG conformation on peptide selectivity for LFA-1 is currently being carried out. Peptides Derived from LFA-1 Integrin Peptides derived from the aL- and b2-subunits of LFA-1 are the first series of peptides derived from integrin sequences that were found to block cell adhesion. These peptides block mixed lymphocyte reactions and heterotypic T-cell adhesion to intestinal mucosa epithelial cell monolayers and pancreatic islet microvascular endothelium.13,112,113,119–123 Cyclic peptide cLABL derived from the I-domain of LFA-1 has been shown to bind the D1 of ICAM-1.120,121 Besides binding to ICAM-1, FITC-labeled cLABL (FITCcLABL) was found to be internalized by ICAM-1 into the cytoplasmic domain.121 Because of this internalization characteristic, cLABL peptides are being exploited to target drugs into cells.121

Table 2. ICAM-1- and LFA-1-Derived Peptides that Block ICAM-1/LFA-1Mediated Homotypic and Heterotypic T-Cell Adhesion Peptide Name cIBR cLABL LBE cLBEL cLBEC

Peptide Sequence

Origin

Cyclo(1,12)PenPRGGSVLVTGC Cyclo(1,12)PenITDGEATDSGC DLSYSLDDLRNVKKLGGDLLRALNE Cyclo(1,12)PenDLSYSLDDLRC Cyclo(1,12)PenDLRNVKKLGC

D1 of ICAM-1 aL I-domain of LFA-1 b2-subunit of LFA-1 b2-subunit of LFA-1 b2-subunit of LFA-1

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Linear and cyclic peptides from the b2-subunit of LFA-1 (i.e., LBE, cLBEL, and cLBEC, Tab. 2) were also found to inhibit homotypic T-cell adhesion and mixed lymphocyte reactions.112 These peptides were derived from a region of the b2-subunit of LFA-1 homologous to a region that binds to RGD peptide in the bIIIa-subunit of gpIIb/ IIIa integrin.113 It is interesting to find that LBE peptide can enhance the binding of anti-ICAM-1 mAb or FITC-cLABL peptide to ICAM-1 on T cells. This enhancement is presumably due to the induced conformational change in ICAM-1 as determined by induced conformational change of soluble ICAM-1 (sICAM-1) upon addition of LBE peptide monitored by fluorescence spectroscopy.119 These results indicate that LBE peptide binds to a site on ICAM-1 different from that of cLABL and induces ICAM-1 to a high affinity conformation for cLABL peptide.119

UTILIZING CELL ADHESION MOLECULES IN TISSUE DIAGNOSTICS, DRUG TARGETING, AND BIOMATERIAL DESIGN Many peptides and proteins (i.e., mAbs, transferrin, luteinizing hormone-releasing hormone (LHRH) peptide) have been used to target drugs or diagnostic agents to specific cells and organs. They also have been utilized to target particles and polymers for drug delivery. Only recently have cell adhesion molecules gained the interest of pharmaceutical scientists for use as cancer diagnostic agents by detecting the increase in integrin expression on cancer cells. These cell adhesion peptides were also being investigated to target antitumor and anti-inflammatory drugs into cells with increased expression of a specific integrin. In addition, they were used to decorate the surface of particles, liposomes, and biodegradable polymers for drug targeting and biomaterials for tissue engineering. RGD Peptides for Diagnostics Radio-labeled RGD peptides have been used to diagnose the presence of tumor cells in tissues with increased expression of avb3 and avb5 integrins compared to normal cells. 125I, 18F, 64 Cu, and 99mTc atoms were incorporated into cyclo(RGDxK) and RGDC4 peptides (Fig. 5); these labeled-peptides were used to diagnose tumor cells with higher expression of avb3 and avb5 integrins. 125I atom was incorporated at the ortho position to the hydroxyl group of the D-Tyr phenol DOI 10.1002/jps

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ring.124 18F-atom was incorporated to galactose and benzoyl groups before these groups were conjugated to the side chain of the Lys residue in cRGDyK peptide. 64Cu or 99mTc atom was incorporated to RGD peptides or RGD peptidecontaining polymers by forming organometallic complexes with N-o-bis(2-pyridylmethyl)125 or 1,4,7,10-tetraazadodecane-N,N0 ,N00 ,N000 -tetraacetic acid (DOTA).124 In animal studies using positron emission tomography (PET), 18F-galactose-RGD-peptide (18FG-RGD-peptide) quantitatively detected the pattern and enhanced expression of avb3 integrin on vascular endothelial cells of a tumor with a significant correlation to the detection with anti-av mAb.126 Unfortunately, 18FG-RGDpeptide showed a scattered characteristic of av-integrin expression in tumors in patients due to inter- and intra-individual variations in expression of avb3 integrin. Therefore, 18FG-RGDpeptide may be useful for selecting patients that are suitable for avb3 targeting therapy.126 Different methods to incorporate the radio-nuclei gave different tumor diagnostic and clearance properties of cyclo(RGDyK) peptide as diagnostic agents.124 Although 125I-RGD has the highest tumor uptake property, 64Cu-DOTA-RGD and (18F-bentoy/-RbA) have been shown to be better agents for PET imaging of av-integrin expression in breast cancer. 18FB-RGD has rapid hepatobiliary excretion and a rapid tumor washout rate while 64 Cu-DOTA-RGD has consistent and sustained blood concentration and prolonged tumor uptake. N-(2-hydroxypropyl)-methacrylamide (HPMA) polymer decorated with RGDC4 peptide and N-o-bis(2-pyridylmethyl)-L-lysine:99mTc complex (Fig. 5) has high affinity for avb3 integrin. It has been used to deliver radionuclides to tumor capillaries to kill the newly generated vessels during angiogenesis in the prostate tumor mouse model.125 In contrast, the polymer bearing a negative control Arg-Glu-Glu (RGE) peptide did not bind to tumor capillaries.125 Although this polymer is also distributed in other organs, the tumor/organ ratios increased over time, suggesting efficient retention on tumor cells compared to the rapid clearance from normal organs.125 Delivering Drugs to Integrin-Expressing Cells Using Cell Adhesion Peptides Because integrins can be internalized by cells, cell adhesion peptides have been utilized to target drugs to cancer cells and leukocytes. Cyclic RGD JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 9, SEPTEMBER 2006

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Figure 5. The structures of radiolabeled cyclic RGD peptides and HPMA polymer conjugated with cyclic RGD peptide and 99mTc complex to target avb3 and avb3 for tumor diagnostic purposes.

peptides (i.e., RGDyK and RGD4C) were conjugated to paclitaxel (PTX-RGD) and doxorubicin (Dox-RGD4C) for improving the specific delivery of these drugs to tumor cells.22 Mice bearing human breast carcinoma cells (i.e., MDA-MB-435) survived the disease when treated with DoxRGD4C, while the untreated control mice all died because of the disease.127 The conjugate showed better efficacy in suppressing tumor progression than Dox alone.127 These studies suggest that the conjugate targets avb3 and avb5 integrins on the tumor vasculature during angiogenesis. To target LFA-1 integrin on T cells, the Nterminal of cIBR peptide (Tab. 2) has been conjugated to the g-carboxylic acid of the Glu residue of methotrexate (MTX) to give MTX–cIBR conjugate to lower MTX toxicity. MTX–cIBR conjugate has activity one order of magnitude lower than MTX alone in inhibiting isolated dihydrofolate reductase (DHFR) enzyme. This lower activity is due to steric hindrance by the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 9, SEPTEMBER 2006

cIBR fragment during conjugate binding to DHFR as analyzed by molecular modeling experiments. Although the activity of the conjugate was lower than that of MTX alone, the conjugate had selective toxicity for LFA-1-expressing Molt-3 T cells compared to KB epithelial cells that are deficient in LFA-1 receptors. Because the cytotoxicity of MTX–cIBR conjugate on T cells was also blocked by anti-CD11a mAb in a concentrationdependent manner, this suggests that MTX–cIBR conjugate internalization could be partially mediated by LFA-1 receptors. The in vivo activity of MTX–cIBR conjugate after i.v. injection was compared to that of MTX alone in the rat adjuvant arthritis model. The MTX–cIBR-treated rats showed a significant suppression of arthritis (i.e., paw swelling, synovitis, periarticular inflammation, and neutrophil infiltration) compared to untreated animals. Although the MTX-treated animals have a similar suppression of arthritis at below toxic concentraDOI 10.1002/jps

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tions, these animals also have severe weight loss; on the other hand, the conjugate-treated animals continue to increase their body weights. This suggests that the conjugate has lower toxicity and a different mechanism than that of MTX alone. Further studies are being carried out to elucidate the mechanism of action of MTX–cIBR conjugate. ICAM-1 has been shown to internalize cLABL peptide derived from the I-domain of the aLsubunit of LFA-1 (i.e., cLABL peptide; Tab. 2). cLABL peptide has been conjugated with MTX to give MTX–cLABL conjugate. Because ICAM-1 is upregulated during tissue inflammation and several different cancers (i.e., lung and pancreatic cancers), this conjugate may be useful for directing drugs to inflammatory and tumor cells. The antiinflammatory activity of MTX is due to the suppression of production of cytokines such as IL-6 and IL-8. Thus, the activity of MTX–cLABL conjugate was compared to MTX in suppressing the production of these cytokines in human coronary artery endothelial cells (HCAEC) stimulated with TNF-a. Both MTX–cLABL and MTX blocked the production of IL-6 with relatively similar potency. In contrast, MTX–cLABL was 10 times less potent than MTX in blocking IL-8 production. These results suggest that MTX– cLABL is more selective in suppressing the production of IL-6 than IL-8, which is opposite to MTX. Further studies need to be carried out to elucidate the mechanism of action of the MTX–cLABL in in vitro and in vivo models of inflammation. RGD-Peptide for Particle Targeting RGD-surface-modified nanoparticles, polyplexes, and liposomes were utilized to deliver therapeutic agents to cancer cells. Inulin poly(methacrylate) nanoparticles studded with RGD peptides and loaded with doxorubicin showed drug accumulation in tumor cells compared to other tissues.128 Nanoparticles containing avb3-selective RGDpeptidomimetics were assembled by cross-linking the lipid components to entrap ATPm-Raf gene for delivery to angiogenic blood vessels in tumorbearing mice.129 These nanoparticles successfully suppressed angiogenesis of tumor vessels by blocking the Ras-Raf-MEK–ERK pathway. Similarly, RGD-PEG-containing polyethyleneimine (PEI) particles accumulated in N2A tumor tissue and selectively delivered VEGFR2 siRNA to inhibit tumor growth and angiogenesis.130 RGD DOI 10.1002/jps

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peptide was also conjugated to the phospholipids via a PEG moiety and incorporated into long circulating liposomes (LCL).24 The long half-life of these liposomes is presumably due to avoidance of uptake by reticuloendothelial systems. The presence of PEG groups forms a hydrated barrier surrounding the liposomes that shields the defects in the lipid bilayer and inhibits protein adsorption.131 LCLs loaded with a fluorescence marker adhered to the wall of tumor microvessels and formed liposome clusters. In contrast, no clustering was found for Arg-Ala-Asp (RAD)containing LCL or the parent LCL, suggesting that RGD–LCL binds to avb3 and avb5 integrins on the endothelial microvessels of tumors.24 Similarly, RGD-studded liposomes have been used to target drugs such as doxorubicin,132 VEGF,133 and DNA134 to tumor cells. RGD–Polymer Conjugate for Biomaterials Many biopolymers containing RGD peptides have been developed to aid tissue engineering, tissue regeneration, wound healing, reconstructive surgery, neural regeneration, bone grafts, and organ transplantation. Normally, the RGD peptide is covalently anchored to the polymer using a peptide, thioether, or ester bond via a spacer molecule.135 The spacer molecule is designed to avoid steric hindrance during the interaction between the RGD peptides on the polymer and the integrins on the cell surface.135 Studies have shown that the majority of the RGD binding sites on integrins can be reached when the RGD ˚ from the polymer peptide is extended 11–46 A 135,136 matrix. In general, the RGD peptides are connected to the polymer via a PEG spacer to avoid steric hindrance during peptide–integrin interactions and to increase hydrophilicity of the polymer to avoid non-specific cell adhesion.137 As a strategy to provide tissue scaffolds of biodegradable polymers, RGD peptides were conjugated to polymers to give RGD-containing poly(ethylene glycol) (RGD–PEG),138 poly[N-(2hydroxypropyl)methacrylamide] (RGD–PHP MA),139 poly(ethylene glycol)-poly(D,L,-lactic acid) (RGD-HN-PEG-PLA),137 RGD-chitosan,140 and a mixture of poly(ethylene oxide-propylene oxideethylene oxide) (PEO-PPO-PEO) and poly(DLlactic acid) (RGD-PEO-PPO-PEO/PLA).141 Polymers (i.e., PHPMA) studded with RGD peptides have been shown to promote cell attachment and growth for tissue regeneration.139,141,142 In addition to forming a peptide bond, a simple method JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 9, SEPTEMBER 2006

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was developed to modify a polymer surface by providing a reactive tip on the polymer films cast on a glass slip.137 In this case, the amino group on H2N-PEG-PLA was modified with disuccinimidyl tartarate or N-succinimidyl-3-maleimido propionate to give reactive polymers ST-NH-PEG-PLA and MP-NH-PEG-PLA (Fig. 6a and b). Thus, upon addition of peptide-containing free amine or thiol

groups, the peptide reacts with an activated group on the polymer to form a modified polymer surface such as RGD-NH-PEG-PLA (Fig. 6a) or RGE-MPNH-PEG-PLA (Fig. 6b).137 A disulfide bond has also been utilized to conjugate RGD peptide to chitosan (Fig. 6c). RGD-chitosan was designed for reconstructive surgery and allogeneic transplantation purposes.140 One potential problem with

Figure 6. Synthetic strategies to provide scaffolds that contain RGD peptides for tissue engineering. (a) Conjugation of RGD peptide to PEG-PLA polymer by forming a peptide bond between the side chain of the Lys residue in RGD peptide and the active ester on the PEG group.140 (b) Formation of a conjugate between an RGD peptide and PEG-PLA polymer via thioether bond formation.140 (c) Linear RGD peptide is conjugated to chitosan via a disulfide bond formation.141 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 9, SEPTEMBER 2006

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this conjugation method is that it is difficult to control the oxidation process to create a homogeneous material. The oxidation process may produce covalent oligomers in the chitosan as well as a dimeric form of peptide via a disulfide bond formation. Nonetheless, chondrocyte and fibroblast cells can adhere to plates coated with RGD-chitosan conjugate but not to plates coated with chitosan alone.140

CONCLUSION The use of cell adhesion peptides (i.e., RGD peptides) for tumor diagnosis and targeted drug delivery has shown some success in vitro and in vivo. The use of RGD peptides has been expanded to deliver drugs, liposomes, and nanoparticles to tumor cells that express certain integrin types. RGD peptides have also been incorporated into polymers for designing biomaterials to recruit and/or adhere to a specific type of cell. Besides RGD peptides, new adhesion peptides (i.e., ICAM1 and LFA-1 peptides) have been shown to bind to and be internalized by cell adhesion receptors. Initial data have shown that peptides mimicking specific domains of ICAM-1 and LFA-1 can deliver toxic drugs specifically to leukocytes. In the future, more information on the recycling mechanism and trafficking of cell adhesion molecules will become available, and this information may be utilized to target drugs to specific cells. Furthermore, the intracellular trafficking property of cell adhesion molecules may be used to direct drug conjugates into a specific intracellular compartment.

ACKNOWLEDGMENTS This work was supported by grants from NIH (EB-00226 and AI-063002). TJS would like to acknowledge Self Faculty Scholar and General Research funds from The University of Kansas and Pfizer Faculty Scholar funds for financial support. We also thank Nancy Harmony for proofreading this manuscript.

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