Cyclic NGR Peptide Research Articles

Synthesis and evaluation of 68Ga-labeled dimeric cNGR peptide for PET imaging of CD13 expression with ovarian cancer xenograft.

Introduction: Previous studies have shown that peptides containing the asparagine-glycine-arginine (NGR) sequence can specifically bind to CD13 (aminopeptidase N) receptor, a tumor neovascular biomarker that is over-expressed on the surface of angiogenic blood vessels and various tumor cells, and it plays an important role in angiogenesis and tumor progression. In the present study, we aimed to evaluate the efficacy of a gallium-68 (68Ga)-labeled dimeric cyclic NGR (cNGR) peptide as a new molecular probe that binds to CD13 in vitro and in vivo.Materials and Methods: A dimeric cNGR peptide conjugated with 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) was synthesized and labeled with 68Ga. In vitro uptake and binding analyses of the 68Ga- DOTA-c(NGR)2 were performed in two ovarian tumor cell lines, ES2 and SKOV3, which had different CD13 expression patterns. An in vivo biodistribution study was performed in normal mice, and micro positron emission tomography (PET) imaging was conducted in nude mice bearing ES2 and SKOV3 tumors.Results: 68Ga-DOTA-c(NGR)2 was prepared with high radiochemical purity (>95%), and it was stable both in saline at room temperature and in bovine serum at 37°C for 3 h. In vitro studies showed that the uptake of 68Ga-DOTA-c(NGR)2 in ES2 cells was higher compared with SKOV3 cells, and such uptake could be blocked by the cold DOTA-c(NGR)2. Biodistribution studies demonstrated that 68Ga-DOTA-c(NGR)2 was rapidly cleared from blood and mainly excreted from the kidney. MicroPET imaging of ES2 tumor xenografts showed the focal uptake of 68Ga-DOTA-c(NGR)2 in tumors from 1 to 1.5 h post-injection. The high-contrast tumor visualization occurred at 1 h, corresponding to the highest tumor/background ratio of 10.30±0.26. The CD13-specific tumor targeting of the 68Ga-DOTA-c(NGR)2 was further supported by the reduced uptake of the probe in ES2 tumors by co-injection of the unlabeled cold peptide. In SKOV3 tumor models, the tumor was not obviously visible under the same imaging conditions.Conclusions: 68Ga-DOTA-c(NGR)2 was easily synthesized, and it showed favorable CD13-specific targeting ability by in vitro data and microPET imaging with ovarian cancer xenografts. Collectively, 68Ga-DOTA-c(NGR)2 might be a potential PET imaging probe for non-invasive evaluation of the CD13 receptor expression in tumors.

Increasing the activity of cell adherent cyclic NGR peptides by optimizing the peptide length and amino acid character.

Cyclic peptides are an attractive modality for the development of therapeutics and the identification of functional cyclic peptides that contribute to novel drug development. The peptide array is one of the optimization methods for peptide sequences and also useful to understand sequence-function relationship of peptides. Cell adherent cyclic NGR peptide which selectively binds to the aminopeptidase N (APN or CD13) is known as an attractive tumor marker. In this study, we designed and screened a library of different length and an amino acid substitution library to identify stronger cell adhesion peptides and to reveal that the factor of higher binding between CD13 and optimized cyclic peptides. Additionally, we designed and evaluated 192 peptide libraries using eight representative amino acids to reduce the size of the library. Through these optimization steps of cyclic peptides, we identified 23 peptides that showed significantly higher cell adhesion activity than cKCNGRC, which was previously reported as a cell adhesion cyclic peptide. Among them, cCRHNGRARC showed the highest activity, that is, 1.65 times higher activity than cKCNGRC. An analysis of sequence and functional data showed that the rules which show higher cell adhesion activity for the three basic cyclic peptides (cCX1 HNGRHX2 C, cCX1 HNGRAX2 C, and cCX1 ANGRHX2 C) are related with the position of His residues and cationic amino acids.

Preparation and cellular-interaction investigation of 177Lu/FITC labeled NGR peptides

Aminopeptidase N (CD13) as the malignant cell surface marker is overexpressed on many tumor cells including lung cancer and the neovascular cell membranes. A tripeptide structure, Asn-Gly-Arg (NGR), can specifically bind to CD13. In this study, we successfully labeled various NGR peptides derivatives with FITC or 177Lu at high efficiency. The 177Lu labeling of the NGR peptides was stable in vitro. The cellular uptake of the 177Lu-labeled cyclic NGR peptides was different by varying the structure of peptides.

Development of novel cyclic NGR peptide-daunomycin conjugates with dual targeting property.

Cyclic NGR peptides as homing devices are good candidates for the development of drug conjugates for targeted tumor therapy. In our previous study we reported that the Dau=Aoa-GFLGK(c[KNGRE]-GG-)-NH2 conjugate has a significant antitumor activity against both CD13+ HT-1080 human fibrosarcoma and CD13− but integrin positive HT-29 human colon adenocarcinoma cells. However, it seems that the free ε-amino group of Lys in the cycle is not necessary for the biological activity. Therefore, we developed novel cyclic NGR peptide–daunomycin conjugates in which Lys was replaced by different amino acids (Ala, Leu, Nle, Pro, Ser). The exchange of the Lys residue in the cycle simplified the cyclization step and resulted in a higher yield. The new conjugates showed lower chemostability against deamidation of Asn than the control compound, thus they had lower selectivity to CD13+ cells. However, the cellular uptake and cytotoxic effect of Dau=Aoa-GFLGK(c[NleNGRE]-GG-)-NH2 was higher in comparison to the control especially on HT-29 cells. Therefore, this conjugate is more suitable for drug targeting with dual targeting property.

NGR-peptide-drug conjugates with dual targeting properties.

Peptides containing the asparagine-glycine-arginine (NGR) motif are recognized by CD13/aminopeptidase N (APN) receptor isoforms that are selectively overexpressed in tumor neovasculature. Spontaneous decomposition of NGR peptides can result in isoAsp derivatives, which are recognized by RGD-binding integrins that are essential for tumor metastasis. Peptides binding to CD13 and RGD-binding integrins provide tumor-homing, which can be exploited for dual targeted delivery of anticancer drugs. We synthesized small cyclic NGR peptide–daunomycin conjugates using NGR peptides of varying stability (c[KNGRE]-NH2, Ac-c[CNGRC]-NH2 and the thioether bond containing c[CH2-CO-NGRC]-NH2, c[CH2-CO-KNGRC]-NH2). The cytotoxic effect of the novel cyclic NGR peptide-Dau conjugates were examined in vitro on CD13 positive HT-1080 (human fibrosarcoma) and CD13 negative HT-29 (human colon adenocarcinoma) cell lines. Our results confirm the influence of structure on the antitumor activity and dual acting properties of the conjugates. Attachment of the drug through an enzyme-labile spacer to the C-terminus of cyclic NGR peptide resulted in higher antitumor activity on both CD13 positive and negative cells as compared to the branching versions.

Systemic and tumor-targeted delivery of siRNA by cyclic NGR and isoDGR motif-containing peptides.

The drug development of siRNA has been seriously hindered by the lack of an effective, safe and clinically applicable delivery system. The cyclic NGR motif and its isomerization product isoDGR recruit CD13 and integrin as their specific receptors, both of which are overexpressed by tumor and neovascular cells. In this study, a bi-functional peptide, named NGR-10R, was designed and tested for siRNA delivery in vitro and in vivo. Through the formation of peptide/siRNA nanoparticles, RNase resistance was greatly enhanced for the siRNAs. Both FACS and confocal assays revealed that the peptide/siRNA complexes were effectively internalized by MDA-MB-231 cells. Gene silencing assays indicated that anti-Lamin A/C siRNA delivered by NGR-10R robustly repressed gene expression in MDA-MB-231 and HUVEC (a CD13(+)/αvβ3(+) cell). Importantly, the siRNAs were efficiently delivered into tumor tissues and localized around the nuclei, as revealed by in vivo imaging and cryosection examination. In summary, NGR-10R not only efficiently delivered siRNAs into MDA-MB-231 cells in vitro but also delivered siRNAs into tumor cells in vivo, taking advantage of its specific binding to CD13 (neovascular) or αvβ3 (MDA-MB-231). Therefore, the NGR-10R peptide provides a promising siRNA delivery reagent that could be used for drug development, particularly for anti-tumor therapeutics.

Abstract A31: Cyclic NGR peptide functionalized PEG-PLGA nanoparticles as dual-targeting carrier for site specific antitumor drug delivery

Abstract CD13 receptors are abundantly overexpressed in tumor cells as well as in neovasculature. The polymeric nanoparticles (NPs) as the nanomodules for anticancer drug carriers are well known. Nowadays, targeted delivery options are becoming a fascinating approach, so CD13 receptors was selected as a targeted site and NPs as a delivery system. By combining all the issue, a cyclic NGR (cNGR) peptide as a ligand was coupled on the terminal end of polyethylene glycol-b-poly(lactic-co-glycolic acid) (PEG-b-PLGA) and prepared the dual targeted polymeric NPs (cNGR-PEG-PTX-NPs) in order to enhance the intracellular delivery of anticancer drug to the tumor cells and tumor endothelial cells via ligand-receptor interaction. In-vitro cytotoxicity studies confirmed that the presence of cNGR enhanced the cytotoxic efficiency by 2.8 folds in human umbilical vein endothelial (HUVEC) cells, while cytotoxicity was improved by 2.6 folds in human fibrosarcoma (HT-1080) cells as compared to non-specific stealth NPs. Compared with other tested NPs, cNGR-PEG-PTX-NPs revealed more cytotoxicity by inducing more apoptosis, higher cell uptake and more G2/M phase arrest. The tumor volume inhibition rate was 59.7% in case of cNGR-PEG-PTX-NPs that was comparatively more with other formulations, indicating that cNGR-PEG-PTX-NPs could more effectively inhibit tumor growth. As a consequence, the cNGR-PEG-PTX-NPs play a key role in enhancing tumor therapeutic efficiency for treatment of CD13 receptor specific solid tumor. Citation Format: Dr. Madhu Gupta, Sr., Dr. Vikas Sharma, Prof. G.P. Agrawal, Prof. S.P. Vyas. Cyclic NGR peptide functionalized PEG-PLGA nanoparticles as dual-targeting carrier for site specific antitumor drug delivery. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A31.

Development of cyclic NGR peptides with thioether linkage: structure and dynamics determining deamidation and bioactivity.

NGR peptides that recognize CD13 receptors in tumor neovasculature are of high interest, in particular due to their potential applications in drug targeting. Here we report the synthesis and structural analysis of novel thioether bond-linked cyclic NGR peptides. Our results show that their chemostability (resistance against spontaneous decomposition forming isoAsp and Asp derivatives) strongly depends on both sample handling conditions and structural properties. A significant correlation was found between chemostability and structural measures, such as NH(Gly)-CO(Asn-sc) distances. The side-chain orientation of Asn is a key determining factor; if it is turned away from HN(Gly), the chemostability increases. Structure stabilizing factors (e.g., H-bonds) lower their internal dynamics, and thus biomolecules become even more resistant against spontaneous decomposition. The effect of cyclic NGR peptides on cell adhesion was examined in A2058 melanoma cell lines. It was found that some of the investigated peptides gradually increased cell adhesion with long-term characteristics, indicating time-dependent formation of integrin binding isoAsp derivatives that are responsible for the adhesion-inducing effect.

Molecular imaging of angiogenesis after myocardial infarction by 111In-DTPA-cNGR and 99mTc-sestamibi dual-isotope myocardial SPECT

BackgroundCD13 is selectively upregulated in angiogenic active endothelium and can serve as a target for molecular imaging tracers to non-invasively visualise angiogenesis in vivo. Non-invasive determination of CD13 expression can potentially be used to monitor treatment response to pro-angiogenic drugs in ischemic heart disease. CD13 binds peptides and proteins through binding to tripeptide asparagine-glycine-arginine (NGR) amino acid residues.Previous studies using in vivo fluorescence microscopy and magnetic resonance imaging indicated that cNGR tripeptide-based tracers specifically bind to CD13 in angiogenic vasculature at the border zone of the infarcted myocardium.In this study, the CD13-binding characteristics of an 111In-labelled cyclic NGR peptide (cNGR) were determined. To increase sensitivity, we visualised 111In-DTPA-cNGR in combination with 99mTc-sestamibi using dual-isotope SPECT to localise CD13 expression in perfusion-deficient regions.MethodsMyocardial infarction (MI) was induced in Swiss mice by ligation of the left anterior descending coronary artery (LAD). 111In-DTPA-cNGR and 99mTc-sestamibi dual-isotope SPECT imaging was performed 7 days post-ligation in MI mice and in control mice. In addition, ex vivo SPECT imaging on excised hearts was performed, and biodistribution of 111In-DTPA-cNGR was determined using gamma counting. Binding specificity of 111In-DTPA-cNGR to angiogenic active endothelium was determined using the Matrigel model.ResultsLabelling yield of 111In-DTPA-cNGR was 95% to 98% and did not require further purification. In vivo, 111In-DTPA-cNGR imaging showed a rapid clearance from non-infarcted tissue and a urinary excretion of 82% of the injected dose (I.D.) 2 h after intravenous injection in the MI mice. Specific binding of 111In-DTPA-cNGR was confirmed in the Matrigel model and, moreover, binding was demonstrated in the infarcted myocardium and infarct border zone.ConclusionsOur newly designed and developed angiogenesis imaging probe 111In-DTPA-cNGR allows simultaneous imaging of CD13 expression and perfusion in the infarcted myocardium and the infarct border zone by dual-isotope micro-SPECT imaging.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-015-0081-7) contains supplementary material, which is available to authorized users.

68Ga-labeled cyclic NGR peptide for microPET imaging of CD13 receptor expression.

Peptides containing the asparagines-glycine-arginine (NGR) motif have been identified as specific ligands binding to CD13/aminopeptidase N (APN) receptor, a tumor neovascular biomarker. In this study, we synthesized a novel NGR-containing peptide (NOTA-G3-NGR), and labeled NOTA-G3-NGR with 68Ga (t1/2 = 67.7 min). The resulting 68Ga-NOTA-G3-NGR peptide was subject to in vitro and in vivo characterization. The microPET imaging results revealed that the 68Ga-NOTA-G3-NGR peptide exhibits rapid and specific tumor uptake, and high tumor-to-background contrast in a subcutaneous HT-1080 fibrosarcoma mouse model. We concluded that the 68Ga-NOTA-G3-NGR peptide has potential in the diagnosis of CD13-targeted tumor angiogenesis.

Dual Targeted Polymeric Nanoparticles Based on Tumor Endothelium and Tumor Cells for Enhanced Antitumor Drug Delivery

Some specific types of tumor cells and tumor endothelial cells represented CD13 proteins and act as receptors for Asn-Gly-Arg (NGR) motifs containing peptide. These CD13 receptors can be specifically recognized and bind through the specific sequence of cyclic NGR (cNGR) peptide and presented more affinity and specificity toward them. The cNGR peptide was conjugated to the poly(ethylene glycol) (PEG) terminal end in the poly(lactic-co-glycolic) acid PLGA-PEG block copolymer. Then, the ligand conjugated nanoparticles (cNGR-DNB-NPs) encapsulating docetaxel (DTX) were synthesized from preformed block copolymer by the emulsion/solvent evaporation method and characterized for different parameters. The various studies such as in vitro cytotoxicity, cell apoptosis, and cell cycle analysis presented the enhanced therapeutic potential of cNGR-DNB-NPs. The higher cellular uptake was also found in cNGR peptide anchored NPs into HUVEC and HT-1080 cells. However, free cNGR could inhibit receptor mediated intracellular uptake of NPs into both types of cells at 37 and 4 °C temperatures, revealing the involvement of receptor-mediated endocytosis. The in vivo biodistribution and antitumor efficacy studies indicated that targeted NPs have a higher therapeutic efficacy through targeting the tumor-specific site. Therefore, the study exhibited that cNGR-functionalized PEG-PLGA-NPs could be a promising approach for therapeutic applications to efficient antitumor drug delivery.

Synthesis and Evaluation of 64Cu-Labeled Monomeric and Dimeric NGR Peptides for MicroPET Imaging of CD13 Receptor Expression

The NGR-containing peptides have been shown to bind specifically to CD13/aminopeptidase N (APN) receptor, one of the attractive tumor vasculature biomarkers. In this study, we evaluated (64)Cu-labeled monomeric and dimeric NGR peptides for microPET imaging of CD13 receptor expression in vivo. Western blot analysis and immunofluorescence staining were performed to identify CD13-positive and CD13-negative cell lines. NGR-containing peptides were conjugated with 1,4,7,10-tetraazadodecane-N,N',N″,N‴-tetraacetic acid (DOTA) and labeled with (64)Cu (t(1/2) = 12.7 h) in ammonium acetate buffer. The resulting monomeric ((64)Cu-DOTA-NGR1) and dimeric ((64)Cu-DOTA-NGR2) peptides were then subjected to in vitro stability, cell uptake and efflux, small animal micorPET, and biodistribution studies. In vitro studies demonstrated that CD13 receptors are overexpressed in human fibrosarcoma HT-1080 cells and negative in human colon adenocarcinoma HT-29 cells. The binding affinity of (64)Cu-DOTA-NGR2 to HT-1080 cells was measured to be within low nanomolar range and about 2-fold higher than that of (64)Cu-DOTA-NGR1. For small animal microPET studies, (64)Cu-DOTA-NGR2 displayed more favorable in vivo performance in terms of higher tumor uptake and slower tumor washout in CD13-positive HT-1080 tumor xenografts as compared to (64)Cu-DOTA-NGR1. As expected, significantly lower tumor uptake and poorer tumor/normal organ contrast were observed for both (64)Cu-DOTA-NGR1 and (64)Cu-DOTA-NGR2 in CD13-negative HT-29 tumor xenografts in comparison with those in the HT-1080 tumor xenografts. The CD13-specific tumor activity accumulation of both (64)Cu-DOTA-NGR1 and (64)Cu-DOTA-NGR2 was further demonstrated by significant reduction of tumor uptake in HT-1080 tumor xenografts with a coinjected blocking dose of cyclic NGR peptide [c(CNGRC)]. The biodistribution results were consistent with the quantitative analysis of microPET imaging. We concluded that both (64)Cu-DOTA-NGR1 and (64)Cu-DOTA-NGR2 have good and specific tumor uptake in CD13-positive HT-1080 tumor xenografts. (64)Cu-DOTA-NGR2 showed higher tumor uptake and better tumor retention than (64)Cu-DOTA-NGR1, presumably due to bivalency effect and increase in apparent molecular size. (64)Cu-DOTA-NGR2 is a promising PET probe for noninvasive detection of CD13 receptor expression in vivo.

An NGR-integrated and enediyne-energized apoprotein shows CD13-targeting antitumor activity

Targeting and inhibiting angiogenesis is a promising strategy for treatment of cancer. NGR peptide motif is a tumor-homing peptide, which could bind with CD13 expressed on tumor blood vessels. Lidamycin is a highly potent antitumor antibiotic, which is composed of an apoprotein (LDP) and an active enediyne chromophore (AE). Here, an NGR-integrated and enediyne-energized apoprotein composed of cyclic NGR peptide and lidamycin was developed by a two-step procedure. Firstly, we prepared the fusion protein composed of NGR peptide and LDP by recombinant DNA technology. Then, AE was reloaded to the fusion protein to get NGR-LDP-AE. Our experiments showed that NGR-LDP could bind to CD13-expressing HT-1080 cells, whereas the recombinant LDP (rLDP) showed weak binding. NGR-LDP-AE exerted highly potent cytotoxicity to cultured tumor cells in vitro. In vivo antitumor activity was evaluated in murine hepatoma 22 (H22) model and human fibrosarcoma HT-1080 model. At the tolerable dose, NGR-LDP-AE and lidamycin inhibited H22 tumor growth by 94.8 and 66.9%, and the median survival time of the mice was 62 and 37 days, respectively. In the HT-1080 model, NGR-LDP-AE inhibited tumor growth by 88.6%, which was statistically different from that of lidamycin (74.5%). Immunohistochemical study showed that NGR-LDP could bind to tumor blood vessels. Conclusively, these results demonstrate that fusion of LDP with CNGRC peptide delivers AE to tumor blood vessels and improves its antitumor activity.

NGR Peptide Ligands for Targeting CD13/APN Identified through Peptide Array Screening Resemble Fibronectin Sequences

Peptides containing the Asn-Gly-Arg (NGR) motif are known to bind CD13 isoforms expressed in tumor vessels and have been widely used for tumor targeting. Residues flanking the NGR sequence play an important role in modulating the binding affinity and specificity of NGR for the CD13 receptor. Herein, we have used a rapid, easy, and reliable peptide array-whole cell binding assay for screening a library of NGR peptides with different flanking residues. A peptide array consisting of forty-five NGR containing peptides was synthesized on a cellulose membrane, followed by screening against CD13 positive (HUVEC and HT-1080) and CD13 negative cell lines (MDA-MB-435 and MDA-MB-231). The library screening led to the identification of five cyclic and acyclic NGR peptides that display higher binding (up to 5-fold) to CD13 positive cells with negligible binding to CD13 negative cell lines when compared to the lead sequence cyclic CVLNGRMEC. Peptides with high binding affinity for the CD13 positive cells also showed improved in vitro cellular uptake and specificity using flow cytometry and fluorescence microscopy. Interestingly, the identified peptides resemble the NGR sequences present in the human fibronectin protein. These NGR peptides are promising new ligands for developing tumor vasculature targeted drugs, delivery systems and imaging agents with reduced systemic toxicity.

Efficacy of positive contrast imaging techniques for molecular MRI of tumor angiogenesis

Superparamagnetic iron oxide particles (SPIOs) are promising contrast agents for molecular MRI. To improve the in vivo detection of iron-based contrast media, positive contrast imaging techniques have been developed. Here, the efficacy of two positive contrast techniques, white marker and susceptibility gradient mapping (SGM), were evaluated for molecular MRI of tumor angiogenesis and compared with conventional negative contrast gradient echo (GE) imaging. In vitro, cylindrical phantoms containing varying iron oxide concentrations were used to measure the response of positive contrast techniques. In vivo, tumor bearing mice were used as a model for tumor angiogenesis. Mice were injected with unlabeled SPIOs (n = 5) or SPIOs labeled with cyclic NGR peptide (cNGR) (n = 5), which homes specifically to angiogenic microvessels. Pre- and post-contrast GE and white marker images were acquired. Subsequently, SGM images and R(2)(*) maps were calculated. For image analysis, the contrast-to-noise ratio (CNR) and the percentage of enhanced voxels (EVs) in the tumor rim and core were calculated. In vitro, the linear increases in MRI signal response for increasing iron oxide concentration were much stronger for SGM than white marker. In vivo, the CNR of GE, white marker and SGM imaging was 5.7, 1.2 and 6.2, respectively, with equal acquisition times. Significant differences in the percentage of EVs between the tumor rim and core were found using R(2)(*) mapping, GE and SGM (p < 0.05). The two contrast agents had significantly different percentages of EVs by R(2)(*) mapping and SGM in the rim (p < 0.001). The in vivo efficacy of white marker and SGM was evaluated for molecular MRI relative to GE imaging and R(2)(*) mapping. Only SGM, and not white marker, can be used to transfer the negative contrast from targeted SPIOs in a positive contrast signal without loss of CNR.

Synthesis of novel cyclic NGR/RGD peptide analogs via on resin click chemistry

Targeted drug deliveries as well as high resolution imaging of cancerous tissues and organs via specific cancer cell markers have become important in chemotherapeutic interventions of cancer treatment. Short peptides such as RGD and NGR are showing promising results for targeted drug delivery and in vivo imaging. We have applied on resin Huisgen's 1,3-dipolar cycloaddition to synthesize new cyclic RGD and NGR peptide analogs. Preliminary binding assays of these new analogs by fluorescence polarization indicates specific binding to purified CD13 (Aminopeptidase N) and cell lysates from MCF-7 and SKOV-3 cancer cell lines.

Bioreducible BPEI-SS-PEG-cNGR polymer as a tumor targeted nonviral gene carrier

The work demonstrated development of multifunctional gene carrier which has incorporated reducible moiety, tumor targeting ligands as well as PEG to achieve efficient release of pDNA, enhanced tumor-specificity and long circulation, respectively. In our successful one-pot synthesis of multifunctional polymer, low molecular weight branched polyethylenimine (BPEI) was thiolated with propylene sulfide, and mixed with α-Maleimide-ω-N-hydroxysuccinimide ester polyethylene glycol (MAL–PEG–NHS, MW: 5000), and cyclic NGR peptide. The structural elucidation of the cNGR conjugated reducible BPEI containing disulfide bond (BPEI-SS-PEG-cNGR), was done by NMR and GPC study. Complex formation as well as reducible property of the polymer was confirmed by gel retardation assay. In order to achieve efficient tumor targeting, we have used cNGR peptide which is known to bind to CD13 overexpressed in neovasculature endothelial cells. Tumor target-specificity of polymer was established by carrying out competitive inhibition assay with free cNGR peptide. Cellular uptake of polymers was evaluated by confocal laser scanning microscope (CLSM). Finally, addition of free cNGR and buthionine sulfoximine (BSO) reduced transfection efficiency synergistically, which implied that multifunctional polymer-mediated gene transfection took place tumor-specifically and via GSH-dependent pathway.

Synthesis and in vitro evaluation of cyclic NGR peptide targeted thermally sensitive liposome

The Asn–Gly–Arg (NGR) motif in both cyclic and linear form has previously been shown to specifically bind to CD13/aminopeptidase N that is selectively overexpressed in tumor vasculature and some tumor cells. However, previous versions of cyclic NGR used a liable disulfide bridge between cysteine residues that may be problematic for liposome targeting due to disulfide bond formation between adjacent peptides on the liposomal surface. In this study, we report the design, synthesis, and characterization of a novel cyclic NGR-containing peptide, cKNGRE, which does not contain a disulfide bridge. cKNGRE was synthesized in good yield and purity and attached to the fluorescent reporter Oregon Green (cKNGRE-OG) and lysolipid-containing temperature sensitive liposomes (LTSLs). The identity of cKNGRE was verified with NMR and mass spectral techniques. In vitro fluorescence microscopy evaluation of cKNGRE-OG demonstrated binding and active uptake by CD13 + cancer cells and minimal binding to CD13 − cancer cells. The cKNGRE-OG ligand displayed 3.6-fold greater affinity for CD13 + cancer cells than a linear NGR-containing peptide. Affinity for CD13 + cancer cells was similarly improved 10-fold for both the cyclic and linear NGR when presented in a multivalent fashion on the surface of an LTSL. cKNGRE-targeted LTSLs rapidly released (> 75% in < 4 s) doxorubicin at 41.3 °C with minimal release at 37 °C. These results demonstrate the ability to synthesize a cKNGRE-targeted temperature sensitive liposome that lacks a disulfide bridge and has sufficient binding affinity for biological applications.

High-yield expression, purification and characterization of tumor-targeted IFN-α2a

IFN-alpha has been shown to be effective against hematologic malignancies. However, it is ineffective against most solid tumors and has not been satisfactory because of its toxicity. The NGR (Asn-Gly-Arg) peptide is a tumor-homing peptide. In order to increase the anti-tumor activity of IFN-alpha2a and lower the dose, we coupled a cyclic NGR peptide with the C terminus of IFN-alpha2a (named IFN-alpha2a-NGR). The fusion protein was expressed in E. coli and purified by ion-exchange chromatography. The purity of IFN-alpha2a-NGR was >98% and the final purification yield of IFN-alpha2a-NGR was approximately 18 mg/L. The anti-tumor efficacy and the binding ability of IFN-alpha2a-NGR with tumor vasculature were investigated in vitro and in vivo. Our study has demonstrated that the anti-tumor efficacy of IFN-alpha2a-NGR is significantly increased in comparison with IFN-alpha2a, and IFN-alpha2a-NGR could selectively target tumor vessels. These data indicate that the tumor-homing peptide (NGR) can enhance the therapeutic efficacy of IFN-alpha2a against tumors.