Towards Radiolabeled EGFR-Specific Peptides: Alternatives to GE11.

The human epidermal growth factor receptor (EGFR) is closely related to several cancer-promoting processes and overexpressed on a variety of tumor types, rendering it an important target structure for the imaging and therapy of several malignancies. To date, approaches to develop peptidic radioligands able to specifically address and visualize EGFR-positive tumors have been of limited success. Most of the attempts were based on the lead GE11, as this peptide was previously described to be a highly potent EGFR-specific agent. However, since it has recently been shown that GE11 exhibits an insufficient affinity to the EGFR in monomeric form to be suitable as a basis for the development of tracers based on it, in the present work we investigated which other peptides might be suitable as lead structures for the development of EGFR-specific peptidic radiotracers. For this purpose, we developed 68Ga-labeled radioligands based on the peptides D4, P1, P2, CPP, QRH, EGBP and Pep11, having been described before as EGFR-specific. In addition, we also tested three truncated versions of the endogenous EGFR ligand hEGF (human epidermal growth factor) with respect to their ability to specifically target the EGFR with high affinity. Therefore, chelator-modified labeling precursors of the mentioned peptides were synthesized, radiolabeled with 68Ga and the obtained radioligands were evaluated for their hydrophilicity/lipophilicity, stability against degradation by human serum peptidases, in vitro tumor cell uptake, and receptor affinity in competitive displacement experiments on EGFR-positive A431 cells. Although all NODA-GA-modified (NODA-GA: (1,4,7-triazacyclononane-4,7-diyl)diacetic acid-1-glutaric acid) labeling precursors could be obtained more or less efficient in yields between 5 and 74%, the 68Ga-radiolabeling proved to be unsuccessful for two of the three truncated versions of hEGF ([68Ga]Ga-8 and [68Ga]Ga-9), producing several side-products. For the other agents [68Ga]Ga-1-[68Ga]Ga-7, [68Ga]Ga-10 and [68Ga]Ga-11, high radiochemical yields and purities of ≥98% and molar activities of up to 114 GBq/µmol were obtained. In the assay investigating the radiopeptide susceptibilities against serum peptidase degradation, the EGBP-based agent demonstrated a limited stability with a half-life of only 66.4 ± 3.0 min, whereas the other tracers showed considerably higher stabilities of up to an 8000 min half-life. Finally, all radiotracer candidates were evaluated in terms of tumor cell internalization and receptor binding potential on EGFR-positive A431 cell. In these experiments, all developed agents failed to show an EGFR-specific tumor cell uptake or a relevant EGFR-affinity. By contrast, the positive controls tested under identical conditions, [125I]I-hEGF and hEGF demonstrated the expected high EGFR-specific tumor cell uptake (33.6% after 1 h, being reduced to 1.9% under blocking conditions) and affinity (IC50 value of 15.2 ± 3.3 nM). Thus, these results indicate that none of the previously described peptidic agents developed for EGFR targeting appears to be a reasonable choice as a lead structure for the development of radiopeptides for targeting of EGFR-positive tumors. Likewise, the tested truncated variants of the endogenous hEGF do not seem to be promising alternatives for this purpose.

GE11-antigen-loaded hepatitis B virus core antigen virus-like particles efficiently bind to TNBC tumor.

Purpose: This study aimed to explore the possibility of utilizing hepatitis B core protein (HBc) virus-like particles (VLPs) encapsulate doxorubicin (Dox) to reduce the adverse effect caused by its off-target and toxic side effect. Methods: Here, a triple-negative breast cancer (TNBC) tumor-targeting GE11-HBc VLP was constructed through genetic engineering. The GE11 peptide, a 12-amino-acid peptide targeting epidermal growth factor receptor (EGFR), was inserted into the surface protein loops of VLPs. The Dox was loaded into HBc VLPs by a thermal-triggered encapsulation strategy. The in vitro release, cytotoxicity, and cellular uptake of TNBC tumor-targeting GE11-HBc VLPs was then evaluated. Results: These VLPs possessed excellent stability, DOX loading efficiency, and preferentially released drug payload at high GSH levels. The insertion of GE11 targeting peptide caused improved cellular uptake and enhanced cell viability inhibitory in EGFR high-expressed TNBC cells. Conclusion: Together, these results highlight DOX-loaded, EGFR-targeted VLPs as a potentially useful therapeutic choice for EGFR-overexpressing TNBC.

Curcumin-Encapsulated Fusion Protein-Based Nanocarrier Demonstrated Highly Efficient Epidermal Growth Factor Receptor-Targeted Treatment of Colorectal Cancer.

Curcumin, a polyphenol derived from turmeric, has multiple biological functions, such as anti-inflammatory, antioxidant, antibacterial and, above all, antitumor activity. Colorectal cancer is a common malignancy of the gastrointestinal tract with an extremely high mortality rate. However, the low bioavailability and poor targeting properties of curcumin generally limit its clinical application. In the present study, we designed a fusion protein GE11-HGFI as a nanodrug delivery system. The protein was connected by flexible linkers, inheriting the self-assembly properties of hydrophobin HGFI and the targeting ability of GE11. The data show that the encapsulation of curcumin by fusion protein GE11-HGFI can form uniform and stable nanoparticles with a size of only 80 nm. In addition, the nanocarrier had high encapsulation efficiency for curcumin and made it to release sustainably. Notably, the drug-loaded nanosystem selectively targeted colorectal cancer cells with high epidermal growth factor receptor expression, resulting in high aggregated concentrations of curcumin at tumor sites, thus showing a significant anticancer effect. These results suggest that the nanocarrier fusion protein has the potential to be a novel strategy for enhancing molecular bioactivity and drug targeting in cancer therapy.

Challenges for the application of EGFR-targeting peptide GE11 in tumor diagnosis and treatment.

Abnormal regulation of cell signaling pathways on cell survival, proliferation and migration contributes to the development of malignant tumors. Among them, epidermal growth factor receptor (EGFR) is one of the most important biomarkers in many types of malignant solid tumors. Its over-expression and mutation status can be served as a biomarker to identify patients who can be benifit from EGFR tyrosine kinase inhibitors and anti-EGFR monocloncal antibody (mAb) therapy. For decades, researches on EGFR targeted ligands were actively carried out to identify potent candidates for cancer therapy. An ideal EGFR ligand can competitively inhibit the binding of endogenous growth factor, such as epidermal growth factor (EGF) and transforming growth factor-α(TGF-α) to EGFR, thus block EGFR signaling pathway and downregulate EGFR expression. Alternatively, conjugation of EGFR ligands on drug delivery systems (DDS) can facilitate targeting delivery of therapeutics or diagnostic agents to EGFR over-expression tumors via EGFR-mediated endocytosis. GE11 peptide is one of the potent EGFR ligand screened from a phage display peptide library. It is a dodecapeptide that can specifically binds to EGFR with high affinity and selectivity. GE11 has been widely used in the diagnosis and targeted delivery of drugs for radiotherapy, genetherapy and chemotherpy against EGFR positive tumors. In this review, the critical factors affecting the in vivo and in vitro targeting performance of GE11 peptide, including ligand-receptor intermolecular force, linker bond properties and physiochemical properties of carrier materials, are detailedly interpreted. This review provides a valuable vision for the rational design and optimization of GE11-based active targeting strategies for cancer treatment, and it will promote the translation studies of GE11 from lab research to clinical application.

Structure-based peptide ligand design for improved epidermal growth factor receptor targeted gene delivery.

The epidermal growth factor receptor EGFR allows targeted delivery of macromolecular drugs to tumors. Its ligand, epidermal growth factor, binds EGFR with high affinity but acts mitogenic. Non-mitogenic peptides are utilized as targeting ligands, like the dodecapeptide GE11, although its low binding affinity warrants improvement. We applied a two-step computational approach with database search and molecular docking to design GE11 variants with improved binding. Synthesized peptides underwent binding studies on immobilized EGFR using surface plasmon resonance. Conjugates of peptides coupled via heterobifunctional PEG linker to linear polyethylenimine (LPEI) were used for transfection studies on EGFR-overexpressing cells using reporter gene encoding plasmid DNA. Docking studies unraveled similarities between GE11 and the EGFR dimerization arm. By skipping non-overlapping amino acids, a less hydrophobic segment (YTPQNVI) was identified to be directly involved in EGFR binding. By replacing valine by tyrosine, a full-length version with proposed enhanced binding (GE11m3) was developed. While hydrophobic or hydrophilic segments and variations thereof exhibited low binding, GE11m3 exhibited 3-fold increase in binding compared to GE11, validating in silico predictions. In transfection studies, polyplexes with GE11m3 induced a significantly higher reporter gene expression when compared to GE11 polyplexes both on murine and human cancer cells overexpressing EGFR.

Biodistribution and Activity of EGFR Targeted Polymeric Micelles Delivering a New Inhibitor of DNA Repair to Orthotopic Colorectal Cancer Xenografts with Metastasis.

The disruption of polynucleotide kinase/phosphatase (PNKP) in colorectal cancer (CRC) cells deficient in phosphatase and tensin homolog (PTEN) is expected to lead to the loss of cell viability by a process known as synthetic lethality. In previous studies, we have reported on the encapsulation of a novel inhibitor of PNKP, namely, A83B4C63, in polymeric micelles and its activity in slowing the growth of PTEN-deficient CRC cells as well as subcutaneous xenografts. In this study, to enhance drug delivery and specificity to CRC tumors, the surface of polymeric micelles carrying A83B4C63 was modified with GE11, a peptide targeting epidermal growth factor receptor (EGFR) overexpressed in about 70% of CRC tumors. Using molecular dynamics (MD) simulations, we assessed the binding site and affinity of GE11 for EGFR. The GE11-modified micelles, tagged with a near-infrared fluorophore, showed enhanced internalization by EGFR-overexpressing CRC cells in vitro and a trend toward increased primary tumor homing in an orthotopic CRC xenograft in vivo. In line with these observations, the GE11 modification of polymeric micelles was shown to positively contribute to the improved therapeutic activity of encapsulated A83B4C63 against HCT116-PTEN-/- cells in vitro and that of orthotopic CRC xenograft in vivo. In conclusion, our results provided proof of principle evidence for the potential benefit of EGFR targeted polymeric micellar formulations of A83B4C63 as monotherapeutics for aggressive and metastatic CRC tumors but at the same time highlighted the need for the development of EGFR ligands with improved physiological stability and EGFR binding.

Inhibitory Effect of Oxaliplatin-loaded Engineered Milk Extracellular Vesicles on Tumor Progression.

BACKGROUND/AIM: Anti-cancer chemotherapy is an effective therapeutic approach. Milk extracellular vesicles (EVs) loaded with chemotherapeutics have a potential anticancer effect by acting as a drug delivery system. Thus, our study aimed to explore the effect of engineered milk extracellular vesicles. MATERIALS AND METHODS: To treat epidermal growth factor receptor (EGFR) expressing solid tumors, we established oxaliplatin-loaded milk EV conjugated with GE11 peptide (GE11Milk EVoxal), which has a high affinity to EGFR and assessed their anti-cancer effect in vitro and in vivo. RESULTS: Drug-loaded GE11Milk EVoxal showed significantly higher incorporation into EGFR expressing cancer cells compared with milk EV without GE11 conjugation (Milk EVoxal), leading to apoptosis of cancer cells. GE11Milk EVoxal also inhibited cell viability compared to milk EVoxal or oxaliplatin alone. In colorectal cancer xenograft murine model, GE11Milk EVoxal showed the maximum therapeutic effect on tumor progression. These findings indicate that GE11Milk EVoxal suppresses EGFR expressing cancer through GE11 peptide-mediated EGFR targeting and subsequently anti-cancer drug delivery. CONCLUSION: Anti-cancer drug-loaded engineered milk EVs might be a novel therapeutic approach for treating patients with EGFR expressing solid tumors.

GE11 Modified PLGA/TPGS Nanoparticles Targeting Delivery of Salinomycin to Breast Cancer Cells.

Salinomycin (Sal) is a potent inhibitor with effective anti-breast cancer properties in clinical therapy. The occurrence of various side effect of Sal greatly limits its application. The epidermal growth factor receptor (EGFR) family is a family of receptors highly expressed in most breast cancer cells. GE11 is a dodecapeptide which shows excellent EGFR affinity. A series of nanoparticles derivatives with GE11 peptide conjugated PLGA/TPGS were synthesized. Nanoprecipitation method was used to prepare the Sal loaded nanoparticles at the optimized concentration. The characterization, targeting efficacy, and antitumor activity were detected both in vitro and in vivo. Encapsulation of Sal in GE11 modified PLGA/TPGS nanoparticles shows an improved therapy efficacy and lower systemic side effect. This represents the delivery system a promising strategy to enhance the therapeutic effect against EGFR highly expressed breast cancer.

Synthesis and evaluation of [18F]FP-Lys-GE11 as a new radiolabeled peptide probe for epidermal growth factor receptor (EGFR) imaging.

INTRODUCTION: The epidermal growth factor receptor (EGFR) has emerged as an attractive target in the treatment of various cancers. Radiolabeled small molecules, antibodies, and peptides that specifically target EGFR are promising probes for tumor imaging to guide personalized treatment with EGFR-targeted drugs. This study aimed to radiolabel GE11 (an EGFR-specific targeting peptide) with 18-fluorine to develop a new EGFR-targeting positron emission tomography (PET) probe, [18F]FP-Lys-GE11, for imaging tumors overexpressing EGFR. METHODS: [18F]FP-Lys-GE11 was produced by radiolabeling a GE11 peptide with the prosthetic group 4-nitrophenyl-2-[18F]fluoropropionate ([18F]NFP). Stability in PBS and mice serum, affinity for A431 cell line, U87 and PC-3 cells uptake and blocking studies, and biodistribution of [18F]FP-Lys-GE11 were determined. 2 h dynamic and static PET scans of probe for tumor-bearing mice normal and inhibition uptake were performed. RESULTS: [18F]FP-Lys-GE11 was stable in PBS and mice serum. The Kd and Bmax values of probe for A431 were 42.43 ± 3.75 nM and 3383 ± 81.73 CPM, respectively. In cell uptake and blocking experiments, a significant reduction in radioactivity accumulation (over 4-fold) was observed by blocking U87 and PC-3 cells with unlabeled peptide. PET imaging of U87 and PC-3 tumor-bearing mice revealed clear tumor imaging (tumor radioactivity accumulation was 3.48 ± 0.44 and 3.68 ± 0.76%ID/g respectively, tumor-to-muscle ratio was 3.45 ± 0.43 and 3.64 ± 0.76 respectively). Blocking imaging revealed that the U87 tumor uptake was significantly inhibited (2.21 ± 0.41%ID/g). The biodistribution and dynamic PET imaging showed that [18F]FP-Lys-GE11 was mainly excreted by the kidneys and the rest was excreted through the bile and intestines. CONCLUSION: The current results showed that [18F]FP-Lys-GE11was a good radiolabeled peptide probe for EGFR overexpression tumor's imaging.

Synthesis and Analysis of 64Cu-Labeled GE11-Modified Polymeric Micellar Nanoparticles for EGFR-Targeted Molecular Imaging in a Colorectal Cancer Model.

Polymeric micellar nanoparticles represent versatile and biocompatible platforms for targeted drug delivery. However, tracking their biodistribution, stability, and clearance profile in vivo is challenging. The goal of this study was to prepare surface-modified micelles with peptide GE11 for targeting the epidermal growth factor receptor (EGFR). In vitro fluorescence studies demonstrated significantly higher internalization of GE11 micelles into EGFR-expressing HCT116 colon cancer cells versus EGFR-negative SW620 cells. Azo coupling chemistry of tyrosine residues in the peptide backbone with aryl diazonium salts was used to label the micelles with radionuclide 64Cu for positron emission tomography (PET) imaging. In vivo analysis of 64Cu-labeled micelles showed prolonged blood circulation and predominant hepatobiliary clearance. The biodistribution profile of EGFR-targeting GE11 micelles was compared with nontargeting HW12 micelles in HCT116 tumor-bearing mice. PET revealed increasing tumor-to-muscle ratios for both micelles over 48 h. Accumulation of GE11-containing micelles in HCT116 tumors was higher compared to HW12-decorated micelles. Our data suggest that the efficacy of image-guided therapies with micellar nanoparticles could be enhanced by active targeting, as demonstrated with cancer biomarker EGFR.

Reimaging biological barriers affecting distribution and extravasation of PEG/peptide- modified liposomes in xenograft SMMC7721 tumor.

Liposomes, as one of the most successful nanotherapeutics, have a major impact on many biomedical areas. In this study, we performed laser scanning confocal microscope (LSCM) and immunohistochemistry (IHC) assays to investigate the intra-tumor transport and antitumor mechanism of GE11 peptide-conjugated active targeting liposomes (GE11-TLs) in SMMC7721 xenograft model. According to classification of individual cell types in high resolution images, biodistribution of macrophages, tumor cells, cells with high epidermal growth factor receptor (EGFR) expression and interstitial matrix in tumor microenvironment, in addition, their impacts on intra-tumor penetration of GE11-TLs were estimated. Type I collagen fibers and macrophage flooded in the whole SMMC7721 tumor xenografts. Tumor angiogenesis was of great heterogeneity from the periphery to the center region. However, the receptor-binding site barriers were supposed to be the leading cause of poor penetration of GE11-TLs. We anticipate these images can give a deep reconsideration for rational design of target nanoparticles for overcoming biological barriers to drug delivery.

Multi-functional self-assembled nanoparticles for pVEGF-shRNA loading and anti-tumor targeted therapy.

Although RNA interference (RNAi) technology shows great potential in cancer treatment, the tumor target delivery and sufficient cytosolic transport of RNAi agents are still the main obstacles for its clinical applications. Herein, we report a functional supramolecular self-assembled nanoparticle vector for RNAi agent loading and tumor target therapy. Molecular block adamantane-grafted poly(ethylene glycol) (Ad-PEG) was modified with epidermal growth factor receptor (EGFR)-specific binding ligand GE11 or pH-sensitive fusogenic peptide GALA and then used for self-assembly with cyclodextrin-grafted branched polyethylenimine (CD-PEI), adamantane-grafted polyamidoamine dendrimer (Ad-PAMAM), and plasmid DNA containing a small hairpin RNA expression cassette against vascular endothelial growth factor (VEGF) into functional DNA-loaded supramolecular nanoparticles (GE11&GALA-pshVEGF@SNPs) based on molecular recognition and charge interaction. These functional peptides facilitated the target cell binding, internalization, and endosomal escape of GE11&GALA-pshVEGF@SNPs, resulting in increased reporter gene expression and efficient targeted gene silencing. The systemic delivery of the GE11&GALA-pshVEGF@SNPs can efficiently downregulate the intratumoral VEGF protein levels, reduce blood vessel formation, and significantly inhibit A549 xenograft tumor growth. These results reveal the potential of these multifunctional self-assembled nanoparticles as a nucleic acid drug delivery system for the treatment of lung cancer.

Modulation of Hypoxia-Induced Chemoresistance to Polymeric Micellar Cisplatin: The Effect of Ligand Modification of Micellar Carrier Versus Inhibition of the Mediators of Drug Resistance.

Hypoxia can induce chemoresistance, which is a significant clinical obstacle in cancer therapy. Here, we assessed development of hypoxia-induced chemoresistance (HICR) against free versus polymeric cisplatin micelles in a triple negative breast cancer cell line, MDA-MB-231. We then explored two strategies for the modulation of HICR against cisplatin micelles: a) the development of actively targeted micelles; and b) combination therapy with modulators of HICR in MDA-MB-231 cells. Actively targeted cisplatin micelles were prepared through surface modification of acetal-poly(ethylene oxide)-poly(α-carboxyl-ε-caprolactone) (acetal-PEO-PCCL) micelles with epidermal growth factor receptor (EGFR)-targeting peptide, GE11 (YHWYGYTPQNVI). Our results showed that hypoxia induced resistance against free and cisplatin micelles in MDA-MB-231 cells. A significant increase in micellar cisplatin uptake was observed in MDA-MB-231 cells that overexpress EGFR, following surface modification of micelles with GE11. This did not lead to increased cytotoxicity of micellar cisplatin, however. On the other hand, the addition of pharmacological inhibitors of key molecules involved in HICR in MDA-MB-231 cells, i.e., inhibitors of hypoxia inducing factor-1 (HIF-1) and signal transducer and activator of transcription 3 (STAT3), substantially enhanced the cytotoxicity of free and cisplatin micelles. The results indicated the potential benefit of combination therapy with HIF-1 and STAT3 inhibitors in overcoming HICR to free or micellar cisplatin.

Functional graphene oxide as cancer-targeted drug delivery system to selectively induce oesophageal cancer cell apoptosis.

Graphene oxides (GO) is a promising building material to fabricate desired drug delivery system due to its excellent physicochemical properties. In this study, an innovative nano-drug (Ori@GE11-GO) was constructed based on GE11 peptide functionalized GO for targeted delivery of oridonin to realize the specific recognition of tumour cells and enhance anticancer efficiency. GE11 surface modification onto GO significantly increased the cellular uptake of GO in EGFR overexpressed oesophageal cancer cells (KYSE-30 and EC109 cells) than that of normal cells, indicating the EGFR targeting effects of Ori@GE11-GO. The internalized Ori@GE11-GO could accumulate into lysosomes and significantly inhibit the viability of cancer cells. Moreover, Ori@GE11-GO could effectively induce KYSE-30 and EC109 cells cycle arrest, apoptosis, mitochondrial membrane potential (△Ψm) disruption through the activation of apoptotic signalling pathways and the inhibition of EGFR/Ras/Raf/MEK/ERK signalling pathway, showing potential use of Ori@GE11-GO for cancer treatment. Taken together, this study demonstrates a good strategy for the construction of bio-functionalized GO drug delivery nanosystem to improve the cancer targeting efficiency of anticancer medicines.

Intra-Articular Formulation of GE11-PLGA Conjugate-Based NPs for Dexamethasone Selective Targeting-In Vitro Evaluation.

Selectively targeted nanoscale drug delivery systems have recently emerged as promising intravenously therapeutic option for most chronic joint diseases. Here, a newly synthetized dodecapeptide (GE11)-polylactide-co-glycolide (PLGA)-based conjugate was used to prepare smart nanoparticles (NPs) intended for intra-articular administration and for selectively targeting Epidermal Growth Factor Receptor (EGFR). GE11-PLGA conjugate-based NPs are specifically uptaken by EGFR-overexpressed fibroblast; such as synoviocytes; which are the primarily cellular component involved in the development of destructive joint inflammation. The selective uptake could help to tune drug effectiveness in joints and to decrease local and systemic side effects. Dexamethasone (DXM) is a glucorticoid drug commonly used in joint disease treatment for both systemic and local administration route. In the present research; DXM was efficiently loaded into GE11-PLGA conjugate-based NPs through an eco-friendly nanoprecipitation method set up for this purpose. DXM loaded GE11-PLGA conjugate-based NPs revealed satisfactory ex vivo cytocompatibility; with proper size (≤150 nm) and good dimensional stability in synovial fluid. Intra-articular formulation was developed embedding DXM loaded GE11-PLGA conjugate-based NPs into thermosetting chitosan-based hydrogel; forming a biocompatible composite hydrogel able to quickly turn from liquid state into gel state at physiological temperature; within 15 min. Moreover; the use of thermosetting chitosan-based hydrogel extends the local release of active agent; DXM.

GE11 Peptide as an Active Targeting Agent in Antitumor Therapy: A Minireview.

A lot of solid tumors are characterized by uncontrolled signal transduction triggered by receptors related to cellular growth. The targeting of these cell receptors with antitumor drugs is essential to improve chemotherapy efficacy. This can be achieved by conjugation of an active targeting agent to the polymer portion of a colloidal drug delivery system loaded with an antitumor drug. The goal of this minireview is to report and discuss some recent results in epidermal growth factor receptor targeting by the GE11 peptide combined with colloidal drug delivery systems as smart carriers for antitumor drugs. The minireview chapters will focus on explaining and discussing: (i) Epidermal growth factor receptor (EGFR) structures and functions; (ii) GE11 structure and biologic activity; (iii) examples of GE11 conjugation and GE11-conjugated drug delivery systems. The rationale is to contribute in gathering information on the topic of active targeting to tumors. A case study is introduced, involving research on tumor cell targeting by the GE11 peptide combined with polymer nanoparticles.

GE11 peptide conjugated selenium nanoparticles for EGFR targeted oridonin delivery to achieve enhanced anticancer efficacy by inhibiting EGFR-mediated PI3K/AKT and Ras/Raf/MEK/ERK pathways.

Selenium nanoparticles (Se NPs) have attracted increasing interest in recent decades because of their anticancer, immunoregulation, and drug carrier functions. In this study, GE11 peptide-conjugated Se NPs (GE11-Se NPs), a nanosystem targeting EGFR over-expressed cancer cells, were synthesized for oridonin delivery to achieve enhanced anticancer efficacy. Oridonin loaded and GE11 peptide conjugated Se NPs (GE11-Ori-Se NPs) were found to show enhanced cellular uptake in cancer cells, which resulted in enhanced cancer inhibition against cancer cells and reduced toxicity against normal cells. After accumulation into the lysosomes of cancer cells and increase of oridonin release under acid condition, GE11-Ori-Se NPs were further transported into cytoplasm after the damage of lysosomal membrane integrity. GE11-Ori-Se NPs were found to induce cancer cell apoptosis by inducting reactive oxygen species (ROS) production, activating mitochondria-dependent pathway, inhibiting EGFR-mediated PI3K/AKT and inhibiting Ras/Raf/MEK/ERK pathways. GE11-Se NPs were also found to show active targeting effects against the tumor tissue in esophageal cancer bearing mice. And in nude mice xenograft model, GE11-Ori-Se NPs significantly inhibited the tumor growth via inhibition of tumor angiogenesis by reducing the angiogenesis-marker CD31 and activation of the immune system by enhancing IL-2 and TNF-α production. The selenium contents in mice were found to accumulate into liver, tumor, and kidney, but showed no significant toxicity against liver and kidney. This cancer-targeted design of Se NPs provides a new strategy for synergistic treating of cancer with higher efficacy and reduced side effects, introducing GE11-Ori-Se NPs as a candidate for further evaluation as a chemotherapeutic agent for EGFR over-expressed esophageal cancers.

GE11 peptide-conjugated nanoliposomes to enhance the combinational therapeutic efficacy of docetaxel and siRNA in laryngeal cancers.

In this study, dual therapeutic-loaded GE11 peptide-conjugated liposomes were developed and applied to enhance therapeutic efficacies of standard-of-care regimens for the treatment of laryngeal cancer. The therapeutic strategy used here was a combination treatment with the chemotherapeutic docetaxel (DTX) and siRNA against the ABCG2 gene that regulates multidrug resistance in many tumor types. Liposome-encapsulated DTX/ABCG2-siRNA molecules were targeted to recognize tumor cells of squamous morphology by conjugation to the EGFR-targeting ligand, GE11. Targeted, drug-infused liposomes were nanosized and exhibited controlled release of DTX. Presence of GE11 peptides on liposomal surfaces enhanced the quantities of liposomal constructs taken up by Hep-2 laryngeal cancer cells. GE11 peptide-conjugated liposomes also enhanced cytotoxic effects against Hep-2 laryngeal cancer cells when compared to treatment with free DTX, thereby reducing IC50 values. Additionally, GE11 peptide-conjugated liposomes had significantly increased anti-tumor and apoptotic effects. Treatments with the GDSL nanoparticle formulation inhibited tumor growth in Hep-2 xenograft-bearing nude mouse models when compared to treatments with non-targeted NP constructs. Treatment of the mouse models with GE11 peptide-conjugated liposomes mitigated toxicities observed after treatment with free DTX. Taken together, liposomal encapsulation of DTX and ABCG2-siRNA improved the anti-tumor effects of treatment with free DTX in Hep-2 cell lines, and conjugation of GE11 peptides to liposomal constructs enhanced anti-tumor efficacies and specificities in laryngeal cancer cells.

99mTc-radiolabeled GE11-modified peptide for ovarian tumor targeting.

BACKGROUND: Ovarian cancer is a serious threat for women health and the early diagnosis of this cancer might improves the survival rate of patients. The use of the targeted radiopharmaceuticals could be a non-invasive and logical method for tumor imaging. The aim of this study was to radiolabel GE11 peptide as a new specific probe for imaging of ovarian tumor. METHODS: HYNIC-SSS-GE11 peptide was labeled with 99mTc using tricine as a coligand. The 99mTc-tricine-HYNIC-SSS-GE11 peptide was evaluated for specific cellular binding in three cell lines with different levels of EGFR expression. Tumor targeting was assessed in SKOV3 tumor bearing mice. RESULTS: By using tricine as a coligand, labeling yield was more than 98% and the stability of the radiolabelled peptide in human serum up to 4 h was 96%. The in vitro cell uptake test showed that this radiolabeled peptide had a good affinity to SKOV3 cells with dissociation constant of 73 nM. The in vivo results showed a tumor/muscle ratio of 3.2 at 4 h following injection of 99mTc-tricine-HYNIC-SSS-GE11 peptide. CONCLUSIONS: Results of this study showed that 99mTc-tricine-HYNIC-SSS-GE11 peptide could be a promising tool for diagnosis and staging of ovarian cancer. 99mTc-tricine-HYNIC-SSS-GE11, a novl targeted agent for ovarian tumor imaging.

Design of smart GE11-PLGA/PEG-PLGA blend nanoparticulate platforms for parenteral administration of hydrophilic macromolecular drugs: synthesis, preparation and in vitro/ex vivo characterization.

Active drug targeting and controlled release of hydrophilic macromolecular drugs represent crucial points in designing efficient polymeric drug delivery nanoplatforms. In the present work EGFR-targeted polylactide-co-glycolide (PLGA) nanoparticles were made by a blend of two different PLGA-based polymers. The first, GE11-PLGA, in which PLGA was functionalized with GE11, a small peptide and EGFR allosteric ligand, able to give nanoparticles selective targeting features. The second polymer was a PEGylated PLGA (PEG-PLGA) aimed at improving nanoparticles hydrophilicity and stealth features. GE11 and GE11-PLGA were custom synthetized through a simple and inexpensive method. The nanoprecipitation technique was exploited for the preparation of polymeric nanoparticles composed by a 1:1weight ratio between GE11-PLGA and PEG-PLGA, obtaining smart nanoplatforms with proper size for parenteral administration (143.9±5.0nm). In vitro cellular uptake in EGFR-overexpressing cell line (A549) demonstrated an active internalization of GE11-functionalized nanoparticles. GE11-PLGA/PEG-PLGA blend nanoparticles were loaded with Myoglobin, a model hydrophilic macromolecule, reaching a good loading (2.42% respect to the theoretical 4.00% w/w) and a prolonged release over 60days. GE11-PLGA/PEG-PLGA blend nanoparticles showed good in vitro stability for 30days in physiological saline solution at 4°C and for 24h in pH 7.4 or pH 5.0 buffer at 37°C respectively, giving indications about potential storage and administration conditions. Furthermore ex vivo stability study in human plasma using fluorescence Single Particle Tracking (fSPT) assessed good GE11-PLGA/PEG-PLGA nanoparticles dimensional stability after 1 and 4h. Thanks to the versatility in polymeric composition and relative tunable nanoparticles features in terms of drug incorporation and release, GE11-PLGA/PEG-PLGA blend NPs can be considered highly promising as smart nanoparticulate platforms for the treatment of diseases characterized by EGFR overexpression by parenteral administration .