Dual-ligand modification of PEGylated liposomes shows better cell selectivity and efficient gene delivery

The objective of this study was to develop an efficient dual-ligand based PEGylated liposomal delivery system that had target specificity as well as properties that would enhance cellular uptake. PEGylated liposomes (PEG-LP) were prepared by the lipid film hydration method by adding distearoyl phosphoethanolamine-polyethylene-glycol-2000 conjugate (DSPE-PEG2000) to a lipid mixture. The cyclic RGD (Arg-Gly-Asp) peptide, a specific ligand with affinity for Integrin α_vβ₃ was coupled to the distal end of the PEG on the PEG-LP (RGD-PEG-LP). Stearylated octaarginine (STR-R8) was incorporated on the surface of the RGD-PEG-LP as dual-ligand (R8/RGD-PEG-LP) that functions as a cell penetrating peptide (CPP). RGD-PEG-LP and R8/RGD-PEG-LP were preferentially taken up by caveolae-mediated and clathrin-mediated endocytosis pathways, respectively. Compared to PEG-LP, R8/RGD-PEG-LP showed an enhanced cellular uptake as well as a higher transfection efficiency in Integrin α_vβ₃ expressing cells. However, the amount of cellular uptake or gene expression by the single ligand versions was negligible, even in Integrin α_vβ₃ expressing cells. No remarkable difference in cellular uptake or gene expression was observed for cells in which the expression of targeted receptors was absent. It can be concluded that dual-ligand modified PEG-LP possesses a strong capability for the efficient internalization of PEG-LP and consequently would be an effective tool for the targeted delivery of macromolecules or chemotherapeutics through accelerated cellular uptake.     

Modification of MR molecular imaging probes with cysteine‐terminated peptides and their potential for in vivo tumour detection

One of the challenges facing superparamagnetic iron oxide (SPIO) nanoparticles is to improve their biological compatibility. While highly uniform SPIOs can be manufactured, the surfaces are hydrophobic as a result of the surfactants used in their fabrication. In this study, we developed a general strategy to fabricate an MR molecular imaging probe in one step by replacing hydrophobic surfactants with small peptides terminated with cysteine. The hydrophobic SPIO surface was transformed into a hydrophilic one by exchanging surface oleic acids with the peptides RGD–Cys or RGD–PEG–Cys. After the RGD–Cys and RGD–PEG–Cys peptide exchange, both RGD–Cys–SPIO and RGD–PEG–Cys–SPIO specifically targeted α_vβ₃‐expressing cells (A549) in vitro, with RGD–Cys–SPIO achieving this more efficiently. Furthermore, MR imaging of A549 tumors receiving RGD–Cys–SPIO or RGD–PEG–Cys–SPIO demonstrated that both the targeted particles could reach and label the α_vβ₃‐expressing tumor, much more efficiently than the non‐targeted particles (Cys–SPIO). Histology showed that the probes not only target the tumor neovasculature but also extravasate from vessels and address the tumor cells. Our study shows that directly replacing oleic acid with cysteine or cysteine‐terminated small peptides is a general strategy to transforming the hydrophobic surface of SPIO into a hydrophilic one, as well as providing targeting ligands. Such SPIOs are of interest as MR molecular imaging probes to detect for cancer in vivo. Copyright © 2010 John Wiley & Sons, Ltd.     

Near-Infrared Fluorescence Imaging of Tumor Integrin αvβ3 Expression with Cy7-Labeled RGD Multimers

Purpose Cell adhesion molecule integrin α_vβ₃ is an excellent target for tumor interventions because of its unique expression on the surface of several types of solid tumor cells and on almost all sprouting tumor vasculatures. Here, we describe the development of near-infrared (NIR) fluorochrome Cy7-labeled RGD peptides for tumor integrin targeting.Procedures Mono-, di-, and tetrameric RGD peptides were synthesized and conjugated with Cy7. The integrin specificity of these fluorescent probes was tested in vitro for receptor binding assay and fluorescence microscopy and in vivo for subcutaneous U87MG tumor targeting.Results The tetrameric RGD peptide probe with the highest integrin affinity showed the highest tumor activity accumulation and strongest tumor-to-normal tissue contrast. This uptake is integrin-specific as the signal accumulated in the tumor can be effectively blocked by unconjugated RGD peptide antagonist of integrin α_vβ₃.Conclusions Noninvasive NIR fluorescence imaging is able to detect and semiquantify tumor integrin expression based upon the highly potent tetrameric RGD peptide probe.     

Clustering and Internalization of Integrin ��v��3 With a Tetrameric RGD-synthetic Peptide

Integrin α_vβ₃ is overexpressed on neoendothelial cells and frequently on tumor cells. We have developed a peptide-like scaffold (regioselectively addressable functionalized template, RAFT), which holds four cyclo(-RGDfK-) (cRGD) motifs and proved that this molecule (called regioselectively addressable functionalized template-arginine-glycine-aspartic acid, RAFT-RGD) targets integrin α_vβ₃ in vitro and in vivo. Using fluorescence correlation spectroscopy (FCS), we measured the constant of affinity (K_D) of the RAFT-RGD for purified integrins. K_D values rose from 3.87 nmol/l for RAFT-RGD to 41.70 nmol/l for cyclo(-RGDfK-). In addition, RAFT-RGD inhibited α_vβ₃ lateral mobility in the cell membrane, probably due to the formation of integrin clusters as demonstrated by fluorescence recovery after photobleaching (FRAP). This was confirmed by electronic microscopy data, which established the formation of molecular complexes containing two integrins in the presence of RAFT-RGD but not cRGD or regioselectively addressable functionalized template-arginine-alanine- aspartic acid (RAFT-RAD). Using an enzyme-linked immunosorbent assay (ELISA), we proved that 1 µmol/l RAFT-RGD increased by 79% α_vβ₃ internalization via clathrin-coated vesicles. Conversely, cRGD was internalized without modifying α_vβ₃ internalization. Although RGD has been known for >20 years, this is the first study to formerly establish the relationships among multimeric presentation, increased affinity, and subsequent integrin-mediated cointernalization. These results strongly support the rationale for using multimeric RGD-peptides as targeting vectors for imaging, diagnosis, or therapy of cancers.     

Fast clearing RGD‐based near‐infrared fluorescent probes for in vivo tumor diagnosis

A fast clearing hydrophilic near‐infrared (NIR) dye ICG‐Der‐02 was used to constitute tumor targeting contrast agents. Cell adhesion molecule integrin α_vβ₃ served as the target receptor because of its unique expression on almost all sprouting tumor vasculatures. The purpose of this study was to synthesize and compare the properties of integrin α_vβ₃‐targeted, fast clearing NIR probes both in vitro and in vivo for tumor diagnosis. ICG‐Der‐02 was covalently conjugated to three kinds of RGD peptide including linear, monoeric cyclic and dimeric RGD to form three RGD‐based NIR probes. The integrin receptor specificities of these probes were evaluated in vitro by confocal microscopy. The dynamic bio‐distribution and elimination ratse were in vivo real‐time monitored by a near‐infrared imaging system in normal mice. Further, the in vivo tumor targeting abilities of the RGD‐based NIR probes were compared in α_vβ₃‐positive MDA‐MB‐231, U87MG and α_vβ₃‐negtive MCF‐7 xenograft mice models. Three RGD‐based NIR probes were successfully synthesized with good optical properties. In vitro cellular experiments indicated that the probes have a clear binding affinity to α_υβ₃‐positive tumor cells, with a cyclic dimeric RGD probe owing the highest integrin affinity. Dynamic bio‐distributions of these probes showed a rapid clearing rate through the renal pathway. In vivo tumor targeting ability of the RGD‐based porbes was demonstrated on MDA‐MB‐231 and U87MG tumor models. As expected, the c(RGDyK)₂‐ICG‐Der‐02 probe displayed the highest tumor‐to‐normal tissue contrast. The in vitro and in vivo block experiments confirmed the receptor binding specificity of the probes. The hydrophilic dye‐labeled NIR probes exhibited a fast clearing rate and deep tissue penetration capability. Further, the α_υβ₃ receptor affinity of the three RGD‐based NIR probes followed the order of dimer cyclic > monomer cyclic > linear. The results demonstrate potent fast clearing probes for in vivo early tumor diagnosis. Copyright © 2012 John Wiley & Sons, Ltd.     

Syndecan-1 regulates αvβ3 and αvβ5 integrin activation during angiogenesis and is blocked by synstatin,a novel peptide inhibitor

Syndecan-1 (Sdc1) is a matrix receptor shown to associate via its extracellular domain with the α_vβ₃ and α_vβ₅ integrins, potentially regulating cell adhesion, spreading, and invasion of cells expressing these integrins. Using Sdc1 deletion mutants expressed in human mammary carcinoma cells, we identified the active site within the Sdc1 core protein and derived a peptide inhibitor called synstatin (SSTN) that disrupts Sdc1''s interaction with these integrins. Because the α_vβ₃ and α_vβ₅ integrins are critical in angiogenesis, a process in which a role for Sdc1 has been uncertain, we used human vascular endothelial cells in vitro to show that the Sdc1 regulatory mechanism is also required for integrin activation on these cells. We found Sdc1 expressed in the vascular endothelium during microvessel outgrowth from aortic explants in vitro and in mouse mammary tumors in vivo. Moreover, we show that SSTN blocks angiogenesis in vitro or when delivered systemically in a mouse model of angiogenesis in vivo, and impairs mammary tumor growth in an orthotopic mouse tumor model. Thus, Sdc1 is a critical regulator of these two important integrins during angiogenesis and tumorigenesis, and is inhibited by the novel SSTN peptide.Angiogenesis, or the sprouting of new blood vessels from existing ones, occurs during development and in diseases such as diabetic retinopathy, endometriosis, psoriasis, rheumatoid arthritis, and tumor-induced angiogenesis (1). Vascular endothelial cells rely on signaling from multiple integrins during the angiogenic process (for review see reference 2), including the α_vβ₃ and α_vβ₅ integrins; signaling by the α_vβ₃ and α_vβ₅ integrin leads to endothelial cell proliferation, migration, matrix metalloprotease activation, and resistance to apoptosis (3).The α_vβ₃ and α_vβ₅ integrins are subject to regulation during angiogenesis. Fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF), two potent angiogenic factors released by tumors, induce the expression of these two integrins that collaborate with the FGF and VEGF receptors in angiogenic signaling pathways (4); disrupting angiogenic signaling by inactivation of either integrin or growth factor receptor leads to endothelial cell apoptosis (5). The integrins are often up-regulated on metastatic tumors as well, leading to enhanced invasion, proliferation, and tumor survival (6–9) by largely the same mechanisms operative in endothelial cells. For these reasons, the integrins and their regulatory mechanisms are attractive targets for the development of therapeutic drugs. Drugs that are currently being tested range from inhibitory integrin antibodies (e.g., Vitaxin [10], based on the inhibitory antibody LM609 [11]), to cyclic RGD peptides that interfere with ligand binding (e.g., cRGDfV, cilengitide, and ST1646 [12–15]), to peptidomimetics based on the RGD sequence (e.g., S247 [16]). These inhibitors have all been shown to disrupt the growth of solid tumors as well as angiogenesis.We have recently identified a regulatory mechanism by which syndecan-1 (Sdc1), a cell-surface matrix receptor, regulates the activation of the α_vβ₃ and α_vβ₅ integrins on mammary carcinoma cells and fibroblasts (17–20). The syndecans are multifunctional extracellular matrix receptors on the surface of all adherent cells (21–23). They anchor to the matrix via heparan sulfate (HS) glycosaminoglycan chains attached near the distal tips of their core proteins; these chains recognize “heparin-binding” domains present in most matrix ligands, including fibronectin (FN), laminins, vitronectin (VN), thrombospondin, and the fibrillar collagens (21). In addition, mounting evidence suggests that they assemble with and control the signaling of other cell surface receptors, including integrins. McFall et al. first described a “cell-binding domain” in the extracellular domain of Sdc4 (24, 25); this site has recently been shown to regulate β1-containing integrins on mesenchymal cells, although the exact integrin target and regulatory mechanism remain unknown (26, 27). Recombinant Sdc2 extracellular domain alters adhesion mechanisms in colon carcinoma cells, suggesting that a regulatory site also exists in its extracellular domain (28, 29). More recently, we have shown that Sdc1 is necessary for activation of the α_vβ₃ integrin on mammary carcinoma cells (17, 20). Silencing Sdc1 expression, selective deletion of amino acids in its extracellular domain, or targeted competition with domain-specific antibodies or recombinant extracellular domain protein disrupts integrin activation and matrix recognition necessary for cell spreading and invasion. Similar activation of the α_vβ₅ integrin by Sdc1 occurs on B82L fibroblasts, which rely exclusively on this integrin for attachment to VN and FN (19). These extracellular syndecan-specific regulatory sites are readily accessible to therapeutic drugs and may hold promise as targets for combating tumorigenesis and other diseases in which their regulated mechanisms play a role.Given the importance of the α_vβ₃ and α_vβ₅ integrins in angiogenesis, we examined the possibility that Sdc1 regulates these integrins on vascular endothelial cells during tumor-induced angiogenesis. We found that Sdc1 is expressed by mouse and human endothelial cells in vitro, and is expressed during angiogenesis induced by FGF or VEGF in vitro and in vivo. We found that the α_vβ₃ and α_vβ₅ integrins associate with Sdc1 and that this association can be disrupted by a peptide called synstatin (SSTN) that is derived from the active site in the Sdc1 core protein. Furthermore, SSTN is an effective inhibitor of angiogenesis in vitro and in vivo, and of mammary carcinoma formation in nude mice. These results define the Sdc1 regulatory mechanism as a critical component of the angiogenic and tumorigenic process.     

Polymeric micelle for tumor pH and folate-mediated targeting.

Novel pH-sensitive polymeric mixed micelles composed of poly(L-histidine) (polyHis; M(w) 5000)/PEG (M(n) 2000) and poly(L-lactic acid) (PLLA) (M(n) 3000)/PEG (M(n) 2000) block copolymers with or without folate conjugation were prepared by diafiltration. The micelles were investigated for pH-dependent drug release, folate receptor-mediated internalization and cytotoxicity using MCF-7 cells in vitro. The polyHis/PEG micelles showed accelerated adriamycin release as the pH decreased from 8.0. When the cumulative release for 24 h was plotted as a function of pH, the gradual transition in release rate appeared in a pH range from 8.0 to 6.8. In order to tailor the triggering pH of the polymeric micelles to the more acidic extracellular pH of tumors, while improving the micelle stability at pH 7.4, the PLLA/PEG block copolymer was blended with polyHis/PEG to form mixed micelles. Blending shifted the triggering pH to a lower value. Depending on the amount of PLLA/PEG, the mixed micelles were destabilized in the pH range of 7.2-6.6 (triggering pH for adriamycin release). When the mixed micelles were conjugated with folic acid, the in vitro results demonstrated that the micelles were more effective in tumor cell kill due to accelerated drug release and folate receptor-mediated tumor uptake. In addition, after internalization polyHis was found to be effective for cytosolic ADR delivery by virtue of fusogenic activity. This approach is expected to be useful for treatment of solid tumors in vivo.     

TAT peptide-based micelle system for potential active targeting of anti-cancer agents to acidic solid tumors.

A novel drug targeting system for acidic solid tumors has been developed based on ultra pH-sensitive polymer and cell penetrating TAT. The delivery system consisted of two components: 1) A polymeric micelle that has a hydrophobic core made of poly(l-lactic acid) (PLLA) and a hydrophilic shell consisting of polyethylene glycol (PEG) conjugated to TAT (TAT micelle), 2) an ultra pH-sensitive diblock copolymer of poly(methacryloyl sulfadimethoxine) (PSD) and PEG (PSD-b-PEG). The anionic PSD is complexed with cationic TAT of the micelles to achieve the final carrier, which could systemically shield the micelles and expose them at slightly acidic tumor pH. TAT micelles had particle sizes between 20 and 45 nm and their critical micelle concentrations were 3.5 mg/l to 5.5 mg/l. The TAT micelles, upon mixing with pH-sensitive PSD-b-PEG, showed a slight increase in particle size between pH 8.0 and 6.8 (60-90 nm), indicating complexation. As the pH was decreased (pH 6.6 to 6.0) two populations were observed, one that of normal TAT micelles (45 nm) and the other of aggregated hydrophobic PSD-b-PEG. Zeta potential measurements showed similar trend substantiating the shielding/deshielding process. Flow cytometry and confocal microscopy showed significantly higher uptake of TAT micelles at pH 6.6 compared to pH 7.4 indicating shielding at normal pH and deshielding at tumor pH. The confocal microscopy indicated that the TAT not only translocates into the cells but is also seen on the surface of the nucleus. These results strongly indicate that the above micelles would be able to target any hydrophobic drug near the nucleus.     

Spermine modified polymeric micelles with pH-sensitive drug release for targeted and enhanced antitumor therapy

Tumor targeting delivery of chemotherapeutic drugs by nanocarriers has been demonstrated to be a promising strategy for cancer therapy with improved therapeutic efficacy. In this work, we reported a novel type of active targeting micelle with pH-responsive drug release by using biodegradable poly(lactide)-poly(2-ethyl-2-oxazoline) di-block copolymers functionalized with spermine (SPM). SPM has been considered as a tumor binding ligand through its specific interaction with the polyamine transport system (PTS), a transmembrane protein overexpressed on various types of cancer cell, while its application in nano-drug delivery systems has rarely been explored. The micelles with spherical shape (∼110 nm) could load hydrophobic paclitaxel (PTX) with high capacity, and release the payload much faster at acidic pH (4.5–6.5) than at pH 7.4. This pH-responsive property assisted the rapid escape of drug from the endo/lysosome after internalization as demonstrated by confocal laser scanning microscopy images using coumarin-6 (Cou-6) as a fluorescent probe. With surface SPM modification, the micelles displayed much higher cellular uptake than SPM lacking micelles in various types of cancer cells, demonstrating tumor targeting ability. The uptake mechanism of SPM modified micelles was explored by flow cytometry, which suggested an energy-consuming sag vesicle-mediated endocytosis pathway. As expected, the micelles displayed significantly enhanced anti-cancer activity. This work demonstrates that SPM modified pH-sensitive micelles may be potential drug delivery vehicles for targeting and effective cancer therapy.

Tumor targeting delivery of SPM functionalized micelles via PTS binding and their endocytosis and pH-triggered endo/lysosome drug release for anti-cancer therapy.     

pH-responsive polymeric micelle based on PEG-poly(β-amino ester)/(amido amine) as intelligent vehicle for magnetic resonance imaging in detection of cerebral ischemic area

A series of pH-responsive polymeric micelles is developed to act as intelligent carriers to deliver iron oxide (Fe₃O₄) nanoparticles and respond rapidly to an acidic stimuli environment for magnetic resonance imaging (MRI). The polymeric micelle can be self-assembled at physiological pH by a block copolymer, consisting of a hydrophilic methoxy poly(ethylene glycol) (PEG) and a pH-responsive poly(β-amino ester)/(amido amine) block. Consequently, the Fe₃O₄ nanoparticles can be well encapsulated into polymeric micelles due to the hydrophobic interaction, shielded by a PEG coronal shell. In an acidic environment, however, the pH-responsive component, which has ionizable tert-amino groups on its backbone, can become protonated to be soluble and release the hydrophobic Fe₃O₄ nanoparticles. The Fe₃O₄-loaded polymeric micelle was measured by dynamic light scattering (DLS), superconducting quantum interference device (SQUID) and a 3.0 T MRI scanner. To assess the ability of this MRI probe as a pH-triggered agent, we utilize a disease rat model of cerebral ischemia that produces acidic tissue due to its pathologic condition. We found gradual accumulation of Fe₃O₄ nanoparticles in the brain ischemic area, indicating that the pH-triggered MRI probe may be effective for targeting the acidic environment and diagnostic imaging of pathologic tissue.     

Comparison of Ga-68-Labeled Fusarinine C-Based Multivalent RGD Conjugates and [<Superscript>68</Superscript>Ga]NODAGA-RGD—<Emphasis Type="Italic">In Vivo</Emphasis> Imaging Studies in Human Xenograft Tumors

Purpose

Multimeric arginine-glycine-aspartic acid (RGD) peptides have advantages for imaging integrin α_vβ₃ expression. Here, we compared the in vitro and in vivo behavior of three different Ga-68-labeled multimeric Fusarinine C-RGD (FSC-RGD) conjugates, whereby RGD was coupled directly, via a succinic acid or PEG linker (FSC(RGDfE)₃, FSC(succ-RGD)₃, FSC(Mal-RGD)₃). The positron emission tomography/X-ray computed tomography (PET/CT) imaging properties were further compared using [⁶⁸Ga]FSC(succ-RGD)₃ with the monomeric [⁶⁸Ga]NODAGA-RGD in a murine tumor model.

Procedure

FSC-RGD conjugates were labeled with Ga-68, and stability properties were studied. For in vitro characterization, the partition coefficient, integrin α_vβ₃ binding affinity, and cell uptake were determined. To characterize the in vivo properties, biodistribution studies and microPET/CT were carried out using mice bearing either human M21/M21-L melanoma or human U87MG glioblastoma tumor xenografts.

Results

All FSC-RGD conjugates were quantitatively labeled with Ga-68 within 10 min at RT. The [⁶⁸Ga]FSC-RGD conjugates exhibited high stability and hydrophilic character, with only minor differences between the different conjugates. In vitro and in vivo studies showed enhanced integrin α_vβ₃ binding affinity, receptor-selective tumor uptake, and rapid renal excretion resulting in good imaging properties.

Conclusions

The type of linker between FSC and RGD had no pronounced effect on targeting properties of [⁶⁸Ga]FSC-RGD trimers. In particular, [⁶⁸Ga]FSC(succ-RGD)₃ exhibited improved properties compared to [⁶⁸Ga]NODAGA-RGD, making it an alternative for imaging integrin α_vβ₃ expression.

     

In Vivo Near-Infrared Fluorescence Imaging of Integrin αvβ3 in an Orthotopic Glioblastoma Model

Purpose Expression of cell adhesion molecule integrin α_vβ₃ is significantly up-regulated during tumor growth, and sprouting of tumor vessels and correlates well with tumor aggressiveness. The purpose of this study was to visualize tumor integrin α_vβ₃ expression in vivo by using near-infrared fluorescence (NIRF) imaging of Cy5.5-linked cyclic arginine–glycine–aspartic acid (RGD) peptide in an orthotopic brain tumor model.Procedures U87MG glioma cells transfected with the firefly luciferase gene were stereotactically injected into nude mice in the right frontal lobe. Bioluminescence imaging (BLI) using d-luciferin substrate and small animal magnetic resonance imaging (MRI) using gadolinium contrast enhancement were conducted weekly after tumor cell inoculation to monitor intracranial tumor growth. Integrin α_vβ₃ expression was assessed by using a three-dimensional optical imaging system (IVIS 200) 0–24 hours after administration of 1.5 nmol monomeric Cy5.5-RGD via the tail vein. Animals were injected intravenously with both Texas Red–tomato lectin and Cy5.5-RGD prior to sacrifice to visualize peptide localization to tumor vasculature using histology.Results Fluorescence microscopy demonstrated specific Cy5.5-RGD binding to both U87MG tumor vessels and tumor cells with no normal tissue binding. NIRF imaging showed highest tumor uptake and tumor to normal brain tissue ratio two hours postinjection (2.64 ± 0.20). Tumor uptake of Cy5.5-RGD was effectively blocked by using unlabeled c(RGDyK), and injection of Cy5.5 dye alone showed nonspecific binding.Conclusions Optical imaging via BLI and NIRF offer a simple, effective, and rapid technique for noninvasive in vivo monitoring and semiquantitative analysis of intracranial tumor growth and integrin α_vβ₃ expression. This study suggests that NIRF via fluorescently labeled RGD peptides may provide enhanced surveillance of tumor angiogenesis and anti-integrin treatment efficacy in orthotopic brain tumor models.     

<Superscript>18</Superscript>F-Labeled Galacto and PEGylated RGD Dimers for PET Imaging of α<Subscript>v</Subscript>β<Subscript>3</Subscript> Integrin Expression

Purpose

In vivo imaging of α_vβ₃ has important diagnostic and therapeutic applications. ¹⁸F-Galacto-arginine–glycine–aspartic acid (RGD) has been developed for positron emission tomography (PET) imaging of integrin α_vβ₃ expression and is now being tested on humans. Dimerization and multimerization of cyclic RGD peptides have been reported to improve the integrin α_vβ₃-binding affinity due to the polyvalency effect. Here, we compared a number of new dimeric RGD peptide tracers with the clinically used ¹⁸F-galacto-RGD.

Procedures

RGD monomers and dimers were coupled with galacto or PEG₃ linkers, and labeled with ¹⁸F using 4-nitrophenyl 2-¹⁸F-fluoropropionate (¹⁸F-NFP) or N-succinimidyl 4-¹⁸F-fluorobenzoate as a prosthetic group. The newly developed tracers were evaluated by cell-based receptor-binding assay, biodistribution, and small-animal PET studies in a subcutaneous U87MG glioblastoma xenograft model.

Results

Starting with ¹⁸F-F^?, the total reaction time for ¹⁸F-FP-SRGD2 and ¹⁸F-FP-PRGD2 is about 120 min. The decay-corrected radiochemical yields for ¹⁸F-FP-SRGD2 and ¹⁸F-FP-PRGD2 are 52?±?9% and 80?±?7% calculated from ¹⁸F-NFP. Noninvasive small-animal PET and direct tissue sampling experiments demonstrated that the dimeric RGD peptides had significantly higher tumor uptake as compared to ¹⁸F-galacto-RGD.

Conclusion

Dimeric RGD peptide tracers with relatively high tumor integrin-specific accumulation and favorable in vivo kinetics may have the potential to be translated into clinic for integrin α_vβ₃ imaging.

     

Binding of α<Subscript>v</Subscript>β<Subscript>3</Subscript> Integrin-Specific Radiotracers Is Modulated by Both Integrin Expression Level and Activation Status

Purpose

Molecular imaging of α_vβ₃ integrin has exhibited real potential to guide the appropriate use of anti-angiogenic therapies. However, an incomplete understanding of the factors that influence binding of α_vβ₃ integrin-specific radiotracers currently limits their use for assessing response to therapy in cancer patients. This study identifies two fundamental factors that modulate uptake of these radiotracers.ProceduresExperiments were performed in prostate cancer (PC3) and glioblastoma (U87MG) cells, which differentially express α_vβ₃ integrin. α_vβ₃ integrin-specific radiotracers were used to investigate the effect of manipulating α_vβ₃ integrin expression or activation in cellular binding assays. β₃ integrin and α_vβ₃ integrin expression were measured by western blotting and flow cytometry, respectively. The effect of select pharmacological inhibitors on α_vβ₃ integrin activation and expression was also determined.

Results

Radiotracer binding was proportional to α_vβ₃ integrin expression when it was decreased (β₃ knock-down cells) or increased, either using pharmacological inhibitors of cell signalling or by culturing cells for different times. Studies with both small molecule and arginine–glycine–aspartic acid (RGD)-based radiotracers revealed increased radiotracer binding after activation of α_vβ₃ integrin with Mn²⁺ or talin head domain. Moreover, inhibition of fundamental signalling pathways (mitogen-activated protein kinase kinase (MEK), Src and VEGFR2) decreased radiotracer binding, reflecting reduced α_vβ₃ integrin activity.

Conclusion

Binding of small molecule ligands and radiolabelled RGD peptides is modulated by expression and activation status of α_vβ₃ integrin. α_vβ₃ integrin-specific radiotracers can provide otherwise inaccessible information of the effect of signalling pathways on α_vβ₃ integrin. This has significant implications for assessing response to anti-angiogenic therapies in clinical studies.

     

MicroPET Imaging of Integrin ��v��3 Expressing Tumors Using 89Zr-RGD Peptides

Purpose

The dimeric transmembrane integrin, ??_v??₃, is a well-investigated target by different imaging modalities through suitably labeled arginine?Cglycine?Caspartic acid (RGD) containing peptides. In this study, we labeled four cyclic RGD peptides with or without PEG functional groups: c(RGDfK) (denoted as FK), PEG₃-c(RGDfK) (denoted as FK-PEG₃), E[c(RGDfK)]₂ (denoted as [FK]₂), and PEG₄-E[PEG₄-c(RGDfK)]₂ (denoted as [FK]₂-3PEG₄), with ⁸⁹Zr (t _1/2?=?78.4 h), using the chelator desferrioxamine-p-SCN (Df) for imaging tumor integrin ??_v??₃.

Methods

The Df conjugated RGD peptides were subjected to integrin ??_v??₃ binding assay in vitro using MDA-MB-435 breast cancer cells. The ⁸⁹Zr-labeled RGD peptides were then subjected to small animal positron emission tomography (PET) and direct tissue sampling biodistribution studies in an orthotopic MDA-MB-435 breast cancer xenograft model.

Results

All four tracers, ⁸⁹Zr-Df-FK, ⁸⁹Zr-Df-FK-PEG₃, ⁸⁹Zr-Df-[FK]₂, and ⁸⁹Zr-Df-[FK]₂-3PEG₄, were labeled in high radiochemical yield (89?±?4%) and high specific activity (4.07?C6 MBq/??g). Competitive binding assay with ¹²⁵I-echistatin showed that conjugation of the RGD peptides to the Df chelator did not have significant impact on their integrin ??_v??₃ binding affinity and the dimeric peptides were shown to be more potent than the monomers. In agreement with binding results, tumor uptake of ⁸⁹Zr-Df-[FK]₂ and ⁸⁹Zr-Df-[FK]₂-3PEG₄ was significantly higher (4.32?±?1.73%ID/g and 4.72?±?0.66%ID/g, respectively, at 2 h post-injection) than the monomers ⁸⁹Zr-Df-FK and ⁸⁹Zr-Df-FK-PEG₃ (1.97?±?0.38%ID/g and 1.57?±?0.49%ID/g, respectively, at 2 h post-injection). Out of the four labeled peptides, ⁸⁹Zr-Df-[FK]₂-3PEG₄ gave the highest tumor-to-background ratio (18.21?±?2.52 at 2 h post-injection and 19.69?±?3.99 at 4 h post-injection), with the lowest uptake in metabolic organs. Analysis of late time points biodistribution data revealed that the uptake in the tumor was decreased, along with increase in the bone, which implies decomplexation of ⁸⁹Zr-Df.

Conclusion

Efficient radiolabeling of peptides with an appropriate chelator such as Df-RGD with ⁸⁹Zr was observed. The ⁸⁹Zr radiolabeled peptides provided high-quality and high-resolution microPET images in xenograft models. ⁸⁹Zr-Df-[FK]₂-3PEG₄ demonstrated the highest tumor-to-background ratio of the compounds tested. Preparation of ⁸⁹Zr peptides to take advantage of the longer half-life is unwarranted due to the relatively rapid clearance from the tumor region of peptide tracers prepared for this study and the increased uptake in the bone of transchelated ⁸⁹Zr with time (2.0?±?0.36%ID/g, 24 h post-injection).     

Engineering Clustered Ligand Binding Into Nonviral Vectors: ��v��3 Targeting as an Example

The development of techniques to efficiently deliver genes using nonviral approaches can broaden the application of gene delivery in medical applications without the safety concerns associated with viral vectors. Here, we designed a clustered integrin-binding platform to enhance the efficiency and targetability of nonviral gene transfer to HeLa cells with low and high densities of α_vβ₃ integrin receptors. Arg-Gly-Asp (RGD) nanoclusters were formed using gold nanoparticles functionalized with RGD peptides and used to modify the surface of DNA/poly(ethylene imine) (PEI) polyplexes. DNA/PEI polyplexes with attached RGD nanoclusters resulted in either 5.4- or 35-fold increase in gene transfer efficiency over unmodified polyplexes for HeLa cells with low- or high-integrin surface density, respectively. The transfection efficiency obtained with the commercially available vector jetPEI-RGD was used for comparison as a vector without clustered binding. JetPEI-RGD exhibited a 1.2-fold enhancement compared to unmodified jetPEI in cells with high densities of α_vβ₃ integrin receptors. The data presented here emphasize the importance of the RGD conformational arrangement on the surface of the polyplex to achieve efficient targeting and gene transfer, and provide an approach to introduce clustering to a wide variety of nanoparticles for gene delivery.     

Local and systemic delivery of VEGF siRNA using polyelectrolyte complex micelles for effective treatment of cancer

For efficient cancer therapy, small interfering RNA (siRNA) should be stably and efficiently delivered into the target tissue and readily taken up by cancer cells. To address these needs, a polyelectrolyte complex (PEC) micelle-based siRNA delivery system was developed for anti-angiogenic gene therapy. The interaction between poly(ethylene glycol) (PEG)-conjugated vascular endothelial growth factor siRNA (VEGF siRNA–PEG) and polyethylenimine (PEI) led to the spontaneous formation of nanoscale polyelectrolyte complex micelles (VEGF siRNA–PEG/PEI PEC micelles), having a characteristic siRNA/PEI PEC inner core with a surrounding PEG shell layer. Intravenous as well as intratumoral administration of the PEC micelles significantly inhibited VEGF expression at the tumor tissue and suppressed tumor growth in an animal tumor model without showing any detectable inflammatory responses in mice. Upon examination of the PEC micelle distribution and in vivo optical imaging following intravenously injection, enhanced accumulation of the PEC micelles was also observed in the tumor region. This study demonstrates the feasibility of using PEC micelles as a potential carrier for therapeutic siRNAs in local and systemic treatment of cancer.     

Directing integrin-linked endocytosis of recombinant AAV enhances productive FAK-dependent transduction

Recombinant adeno-associated virus (rAAV) is a widely used gene therapy vector. Although a wide range of rAAV serotypes can effectively enter most cell types, their transduction efficiencies (i.e., transgene expression) can vary widely depending on the target cell type. Integrins play important roles as coreceptors for rAAV infection, however, it remains unclear how integrin-dependent and -independent mechanisms of rAAV endocytosis influence the efficiency of intracellular virus processing and ultimately transgene expression. In this study, we examined the contribution of integrin-mediated endocytosis to transduction of fibroblasts by rAAV2. Mn(++)-induced integrin activation significantly enhanced (~17-fold) the efficiency of rAAV2 transduction, without altering viral binding or endocytosis. rAAV2 subcellular localization studies demonstrated that Mn(++) promotes increased clustering of rAAV2 on integrins and recruitment of intracellular vinculin (an integrin effector) to sites of rAAV2 binding at the cell surface. Focal adhesion kinase (FAK), a downstream effector of integrin signals, was essential for rAAV2/integrin complex internalization and transduction. These findings support a model whereby integrin activation at the cell surface can redirect rAAV2 toward a FAK-dependent entry pathway that is more productive for cellular transduction. This pathway appears to be conserved for other rAAV serotypes that contain a capsid integrin-binding domain (AAV1 and AAV6).     

精氨酸-甘氨酸-天冬氨酸肽类似物与新生血管内皮靶向治疗

背景：血管内皮是缺血、血栓、炎症、水肿、氧化应激等病理损伤中的重要部位,选择特异性的内皮细胞靶点成为药物介入治疗的关键。目的：分析和总结近年来精氨酸-甘氨酸-天冬氨酸从及精氨酸-甘氨酸-天冬氨酸多肽类似物作为特异性的内皮细胞靶点的研究。方法：分别以“RGD、整合素、靶向治疗”,“RGD、integrin、targetedtherapy”为检索词,应用计算机检索Pubmed数据库1998年1月至2011年12月有关文章。纳入有关血管新生的文献。排除与研究目的无关和内容重复者。保留42篇文献做进一步分析。结果与结论：整合素αvβ3是内皮细胞病理损伤及衄管新生时的特异靶点,参与内皮细胞的迁移、增吼、分化过程。精氨酸-甘氨酸-天冬氨酸肽及精氨酸-甘氨酸-天冬氨酸多肽类似物作为整合素和处配体相互作用的识别位点,能结合肿瘤或者病理损伤时新生血管表达增高的整合素αvβ3,介导细胞与细胞外基质及细胞之间的相互作用,可在新生血管显影、靶向药物递送、载体材料修饰中发挥重要作用。     

The alphavbeta5 integrin of hematopoietic and nonhematopoietic cells is a transduction receptor of RGD-4C fiber-modified adenoviruses

Epithelial and endothelial cells expressing the primary Coxsackie virus B adenovirus (Ad) receptor (CAR) and integrin coreceptors are natural targets of human Ad infections. The fiber knob of species A, C, D, E and F Ad serotypes binds CAR by mimicking the CAR-homodimer interface, and the penton base containing arginine-glycine-aspartate (RGD) motifs binds with low affinity to alphav integrins inducing cell activation. Here, we generated seven different genetically modified Ad vectors with RGD sequences inserted into the HI loop of fiber knob. All mutants bound and infected CAR and alphav integrin-positive epithelial cells with equal efficiencies. However, the Ads containing two additional cysteines, both N and C terminals of the RGD sequence (RGD-4C), were uniquely capable of transducing CAR-less hematopoietic and nonhematopoietic human tumor cell lines and primary melanoma cells. Both binding and transduction of RGD-4C Ad were blocked by soluble RGD peptides. Flow cytometry of cell surface integrins and virus binding to CAR-less cells in the presence of function-blocking anti-integrin antibodies indicated that the alphavbeta5 integrin, but not alphavbeta3, alphaIIbbeta3 or beta1,alpha5 or alpha6-containing integrins served as a functional transduction receptor of the RGD-4C Ads. However, in cells with low levels of alphavbeta5 integrin, the function-blocking anti-alphavbeta5 antibodies were not effective, unlike soluble RGD peptides. Collectively, our data demonstrate that the alphavbeta5 integrin is a functional transduction receptor of RGD-4C Ads in the absence of CAR, and that additional RGD receptors are targets of these viruses. The RGD-4C vectors further extend the tropism of Ads towards potential human therapies.