Assessment of the Pharmacokinetics, Disposition, and Duration of Action of the Tumour-Targeting Peptide CEND-1.

CEND-1 (iRGD) is a bifunctional cyclic peptide that can modulate the solid tumour microenvironment, enhancing the delivery and therapeutic index of co-administered anti-cancer agents. This study explored CEND-1's pharmacokinetic (PK) properties pre-clinically and clinically, and assessed CEND-1 distribution, tumour selectivity and duration of action in pre-clinical tumour models. Its PK properties were assessed after intravenous infusion of CEND-1 at various doses in animals (mice, rats, dogs and monkeys) and patients with metastatic pancreatic cancer. To assess tissue disposition, [3H]-CEND-1 radioligand was administered intravenously to mice bearing orthotopic 4T1 mammary carcinoma, followed by tissue measurement using quantitative whole-body autoradiography or quantitative radioactivity analysis. The duration of the tumour-penetrating effect of CEND-1 was evaluated by assessing tumour accumulation of Evans blue and gadolinium-based contrast agents in hepatocellular carcinoma (HCC) mouse models. The plasma half-life was approximately 25 min in mice and 2 h in patients following intravenous administration of CEND-1. [3H]-CEND-1 localised to the tumour and several healthy tissues shortly after administration but was cleared from most healthy tissues by 3 h. Despite the rapid systemic clearance, tumours retained significant [3H]-CEND-1 several hours post-administration. In mice with HCC, the tumour penetration activity remained elevated for at least 24 h after the injection of a single dose of CEND-1. These results indicate a favourable in vivo PK profile of CEND-1 and a specific and sustained tumour homing and tumour penetrability. Taken together, these data suggest that even single injections of CEND-1 may elicit long-lasting tumour PK improvements for co-administered anti-cancer agents.

The Antitumour Activity of a Curcumin and Piperine Loaded iRGD-Modified Liposome: In Vitro and In Vivo Evaluation.

Lung cancer is one of the most common cancers around the world, with a high mortality rate. Despite substantial advancements in diagnoses and therapies, the outlook and survival of patients with lung cancer remains dismal due to drug tolerance and malignant reactions. New interventional treatments urgently need to be explored if natural compounds are to be used to reduce toxicity and adverse effects to meet the needs of lung cancer clinical treatment. An internalizing arginine-glycine-aspartic acid (iRGD) modified by a tumour-piercing peptide liposome (iRGD-LP-CUR-PIP) was developed via co-delivery of curcumin (CUR) and piperine (PIP). Its antitumour efficacy was evaluated and validated via in vivo and in vitro experiments. iRGD-LP-CUR-PIP enhanced tumour targeting and cellular internalisation effectively. In vitro, iRGD-LP-CUR-PIP exhibited enhanced cellular uptake, suppression of tumour cell multiplication and invasion and energy-independent cellular uptake. In vivo, iRGD-LP-CUR-PIP showed high antitumour efficacy, mainly in terms of significant tumour volume reduction and increased weight and spleen index. Data showed that iRGD peptide has active tumour targeting and it significantly improves the penetration and cellular internalisation of tumours in the liposomal system. The use of CUR in combination with PIP can exert synergistic antitumour activity. This study provides a targeted therapeutic system based on natural components to improve antitumour efficacy in lung cancer.

Engineering a HEK-293T exosome-based delivery platform for efficient tumor-targeting chemotherapy/internal irradiation combination therapy.

Exosomes are nanoscale monolayer membrane vesicles that are actively endogenously secreted by mammalian cells. Currently, multifunctional exosomes with tumor-targeted imaging and therapeutic potential have aroused widespread interest in cancer research. Herein, we developed a multifunctional HEK-293T exosome-based targeted delivery platform by engineering HEK-293T cells to express a well-characterized exosomal membrane protein (Lamp2b) fused to the αv integrin-specific iRGD peptide and tyrosine fragments. This platform was loaded with doxorubicin (Dox) and labeled with radioiodine-131 (131I) using the chloramine-T method. iRGD exosomes showed highly efficient targeting and Dox delivery to integrin αvß3-positive anaplastic thyroid carcinoma (ATC) cells as demonstrated by confocal imaging and flow cytometry in vitro and an excellent tumor-targeting capacity confirmed by single-photon emission computed tomography-computed tomography after labeling with 131I in vivo. In addition, intravenous injection of this vehicle delivered Dox and 131I specifically to tumor tissues, leading to significant tumor growth inhibition in an 8505C xenograft mouse model, while showing biosafety and no side effects. These as-developed multifunctional exosomes (denoted as Dox@iRGD-Exos-131I) provide novel insight into the current treatment of ATC and hold great potential for improving therapeutic efficacy against a wide range of integrin αvß3-overexpressing tumors.

68 Ga-labeling of internalizing RGD (iRGD) peptide functionalized with DOTAGA and NODAGA chelators.

The dual interaction with integrins and neuropilin-1 receptor is the peculiar feature of iRGD peptide. Hence, in the present study, two iRGD peptide analogs were synthesized with DOTAGA and NODAGA as bifunctional chelator and aminohexanoic acid as a spacer for radiometalation with 68 GaCl3 . Negatively charged 68 Ga-DOTAGA-iRGD and neutral 68 Ga-NODAGA-iRGD radiotracers were investigated through in vitro cell uptake studies and in vivo biodistribution studies. Significant internalization of radiotracers in murine melanoma B16F10 cells was observed during in vitro studies. During in vivo studies, tumor uptake was higher for neutral 68 Ga-NODAGA-iRGD, but 68 Ga-DOTAGA-iRGD exhibited better tumor-to-blood ratio due to faster blood clearance. High kidney uptake of the two radiotracers was the limitation, which needs to be resolved through modification either in the peptide backbone or spacer/chelator.

Zerumbone Induces Apoptosis in Breast Cancer Cells by Targeting αvß3 Integrin upon Co-Administration with TP5-iRGD Peptide.

Cell-penetrating peptides (CPPs) are highly promising tools to deliver therapeutic molecules into tumours. αVß3 integrins are cell-matrix adhesion receptors, and are considered as an attractive target for anticancer therapies owing to their roles in the process of metastasis and angiogenesis. Therefore, this study aims to assess the effect of co-administration of zerumbone (ZER) and ZERencapsulated in hydroxypropyl-ß-cyclodextrin with TP5-iRGD peptide towards cell cytotoxicity, apoptosis induction, and proliferation of normal and cancerous breast cells utilizing in vitro assays, as well as to study the molecular docking of ZER in complex with TP5-iRGD peptide. Cell viability assay findings indicated that ZER and ZERencapsulated in hydroxypropyl-ß-cyclodextrin (ZER-HPßCD) inhibited the growth of estrogen receptor positivebreast cancer cells (ER+ MCF-7) at 72 h treatment with an inhibitory concentration (IC)50 of 7.51 ± 0.2 and 5.08 ± 0.2 µg/mL, respectively, and inhibited the growth of triple negative breast cancer cells (MDA-MB-231) with an IC50 of 14.96 ± 1.52 µg/mL and 12.18 ± 0.7 µg/mL, respectively. On the other hand, TP5-iRGD peptide showed no significant cytotoxicity on both cancer and normal cells. Interestingly, co-administration of TP5-iRGD peptide in MCF-7 cells reduced the IC50 of ZER from 7.51 ± 0.2 µg/mL to 3.13 ± 0.7 µg/mL and reduced the IC50 of ZER-HPßCD from 5.08 ± 0.2 µg/mL to 0.49 ± 0.004 µg/mL, indicating that the co-administration enhances the potency and increases the efficacy of ZER and ZER-HPßCD compounds. Acridine orange (AO)/propidium iodide (PI) staining under fluorescence microscopy showed evidence of early apoptosis after 72 h from the co-administration of ZER or ZER-HPßCD with TP5-iRGD peptide in MCF-7 breast cancer cells. The findings of the computational modelling experiment provide novel insights into the ZER interaction with integrin αvß3 in the presence of TP5-iRGD, and this could explain why ZER has better antitumor activities when co-administered with TP5-iRGD peptide.

iRGD decorated lipid-polymer hybrid nanoparticles for targeted co-delivery of doxorubicin and sorafenib to enhance anti-hepatocellular carcinoma efficacy.

The combination of doxorubicin (DOX) with sorafenib (SOR) has proven an effective strategy to enhance anti-hepatocellular carcinoma (HCC) efficacy. However, respective in vivo pharmacokinetic profiles and different endocytosis capacities of these two drugs greatly hinder their current application. Herein, the tumor-targeting peptide iRGD decorated lipid-polymer hybrid nanoparticles (NPs) with a shell-core structure were developed for co-delivery of DOX and SOR (DOX+SOR/iRGD NPs). After the drug ratio was optimized, the stabilized DOX+SOR/iRGD NPs were prepared. Through the iRGD-integrin recognition, DOX+SOR/iRGD NPs showed synergistic cytotoxicity, pro-apoptotic ability and enhanced internalization rate in human liver cancer HepG2 cells. In vivo pharmacokinetic result demonstrated that an extended circulation and bioavailability of DOX+SOR/iRGD NPs than free drugs. More importantly, DOX+SOR/iRGD NPs significantly enhanced antitumor efficiency in HCC xenograft mouse models. Overall, this study describes a promising nanoparticulate drug co-delivery strategy to combine clinical anticancer drugs and enhance anti-HCC efficacy.

iRGD Tumor-Penetrating Peptide-Modified Nano-Delivery System Based on a Marine Sulfated Polysaccharide for Enhanced Anti-Tumor Efficiency Against Breast Cancer.

BACKGROUND: Breast cancer is a common malignancy in women. Conventional clinical therapies for breast cancer all display moderate clinical efficacies and limitations. It is urgent to explore the novel and combined therapeutic strategies for breast cancer to meet clinical demand. METHODS: An iRGD tumor-penetrating peptide-modified nano-delivery system (denoted as iRGD-PSS@PBAE@JQ1/ORI nanoparticles) based on a marine sulfated polysaccharide was developed by codelivery of JQ1 (BET inhibitor) and oridonin (ORI, bioactive diterpenoid derived from traditional Chinese medicine herb). The iRGD-PSS@PBAE@JQ1/ORI NPs, surface modified with iRGD peptide conjugated propylene glycol alginate sodium sulfate (iRGD-PSS). The antitumor efficacy was evaluated both in vitro and in vivo. RESULTS: The prepared iRGD-PSS@PBAE@JQ1/ORI NPs effectively enhanced the tumor targeting and cellular internalization of JQ1 and ORI. Thus, JQ1 exerted the reversal effect on immune tolerance by decreasing the expression of PD-L1, while ORI displayed multiple antitumor effects, such as antiproliferation, inhibition of intracellular ROS production and inhibition of lactic acid secretion. CONCLUSION: Our data revealed that iRGD peptide could significantly improve the cellular internalization and tumor penetration of the nano-delivery system. The combination of JQ1 and ORI could exert synergistic antitumor activities. Taken together, this study provides a multifunctional nanotherapeutic system to enhance the anti-tumor efficiency against breast cancer.

Microfluidic preparation and in vitro evaluation of iRGD-functionalized solid lipid nanoparticles for targeted delivery of paclitaxel to tumor cells.

Solid lipid nanoparticles (SLNs) can combine the advantages of different colloidal carriers and prevent some of their disadvantages. The production of nanoparticles by means of microfluidics represents a successful platform for industrial scale-up of nanoparticle manufacture in a reproducible way. The realisation of a microfluidic technique to obtain SLNs in a continuous and reproducible manner encouraged us to create surface functionalised SLNs for targeted drug release using the same procedure. A tumor homing peptide, iRGD, owning a cryptic C-end Rule (CendR) motif is responsible for neuropilin-1 (NRP-1) binding and for triggering extravasation and tumor penetration of the peptide. In this study, the Paclitaxel loaded-SLNs produced by microfluidics were functionalized with the iRGD peptide. The SLNs proved to be stable in aqueous medium andwere characterized by a Z-average under 150 nm, a polydispersity index below 0.2, a zeta-potential between -20 and -35 mV and a drug encapsulation efficiency around 40%. Moreover, in vitro cytotoxic effects and cellular uptake have been assessed using 2D and 3D tumour models of U87 glioblastoma cell lines. Overall, these results demonstrate that the surface functionalization of SLNs with iRGD allow better cellular uptake and cytotoxicity ability.

Effective Triple-Negative Breast Cancer Targeted Treatment Using iRGD-Modified RBC Membrane-Camouflaged Nanoparticles.

INTRODUCTION: Triple-negative breast cancer (TNBC) has the high degree of malignancy and aggressiveness. There is no targeted therapy drug. Many studies have shown that RBC membrane-coated nanoparticles achieve biological camouflage. In addition, the RGD module in the iRGD mediates the penetration of the vector across the tumor blood vessels to the tumor tissue space. Therefore, we developed iRGD-RM-(DOX/MSNs) by preparing MSNs loaded with doxorubicin as the core, and coating the surface of the MSNs with iRGD-modified RBC membranes. METHODS: iRGD-RM-(DOX/MSNs) were fabricated using physical extrusion. In addition, their physical and chemical characterization, hemolytic properties, in vivo acute toxicity and inflammatory response, in vitro and in vivo safety, and qualitative and quantitative cellular uptake by RAW 264.7 cells and MDA-MB-231 cells were evaluated and compared. Furthermore, we examined the antitumor efficacy of iRGD-RM-(DOX/MSN) nanoparticles in vitro and in vivo. RESULTS: iRGD-RM-(DOX/MSNs) have reasonable physical and chemical properties. iRGD-RM-(DOX/MSNs) escaped the phagocytosis of immune cells and achieved efficient targeting of nanoparticles at the tumor site. The nanoparticles showed excellent antitumor effects in vivo and in vitro. CONCLUSION: In this study, we successfully developed biomimetic iRGD-RM-(DOX/MSNs) that could effectively target tumors and provide a promising strategy for the effective treatment of TNBC.

Transcatheter arterial chemoembolization (TACE) with iRGD peptide in rabbit VX2 liver tumor.

PURPOSE: Transcatheter arterial chemoembolization (TACE) is the first-line therapy for unresectable hepatocellular carcinoma (HCC). However, its therapeutic effects are hampered by the poor distribution of anticancer drugs in tumors. iRGD, a novel tumor-penetrating peptide, enhances the penetration distance and therapeutic efficacy of anticancer drugs. Herein, we evaluated the therapeutic effects of iRGD coupled with TACE in the rabbit VX2 liver tumor model. SUBJECTS AND METHODS: This study had two stages: tumor permeability assay and anticancer efficacy evaluation. In the tumor permeability assay, we coadministered TACE with either iRGD + lipiodol-doxorubicin emulsion (LDE) or LDE in the rabbit VX2 liver tumor model. We evaluated the doxorubicin (DOX) distribution at predetermined times by immunofluorescence microscopy. To evaluate anticancer efficacy, we administered saline, LDE, or iRGD + LDE to tumor-grafted rabbits. We measured tumor volume using magnetic resonance scanning. We quantified the expression levels of Bax, Bcl-2, and cleaved caspase-3 using Western blot (WB) analysis and determined the apoptosis rate in tumor cells using transferase-mediated dUTP nick-end labeling assay. RESULTS: The iRGD + LDE infusion significantly increased the DOX concentration and DOX penetration in tumors compared with the LDE infusion (P < 0.05). The antitumor efficacy of the iRGD + LDE in tumor inhibition was higher than that of the other treatments (P < 0.05). Besides, iRGD + LDE induced more apoptosis (P < 0.05). CONCLUSIONS: We demonstrated that iRGD coadministered with TACE is effective against HCC.

The construction of in vitro nasal cavity-mimic M-cell model, design of M cell-targeting nanoparticles and evaluation of mucosal vaccination by nasal administration.

In order to better evaluate the transport effect of nanoparticles through the nasal mucosa, an in vitro nasal cavity-mimic model was designed based on M cells. The differentiation of M cells was induced by co-culture of Calu-3 and Raji cells in invert model. The ZO-1 protein staining and the transport of fluorescein sodium and dexamethasone showed that the inverted co-culture model formed a dense monolayer and possessed the transport ability. The differentiation of M cells was observed by up-regulated expression of Sialyl Lewis A antigen (SLAA) and integrin ß1, and down-regulated activity of alkaline phosphatase. After targeting M cells with iRGD peptide (cRGDKGPDC), the transport of nanoparticles increased. In vivo, the co-administration of iRGD could result in the increase of nanoparticles transported to the brain through the nasal cavity after intranasal administration. In the evaluation of immune effect in vivo, the nasal administration of OVA-PLGA/iRGD led to more release of IgG, IFN-γ, IL-2 and secretory IgA (sIgA) compared with OVA@PLGA group. Collectively, the study constructed in vitro M cell model, and proved the enhanced effect of targeting towards M cell with iRGD on improving nasal immunity.

Bacteriophage MS2 As a Tool for Targeted Delivery in Solid Tumor Chemotherapy.

Bacteriophage MS2 was employed for targeted delivery of an apoptosis-inducing agent, Tl+, into a tumor tissue. The targeted delivery was ensured by iRGD peptide, a ligand of integrins presumably located on the surface of endotheliocytes of the tumor tissue neovasculature and certain tumor cells. The synthesized peptide was conjugated to MS2 capsid proteins. Tl+ ions from TlNO3 penetrated the phage particles and tightly bound to phage RNA. Peptide-modified MS2 preparations filled with Tl+ caused cell death in two types of cultivated human breast cancer cells and effected necrosis of these tumor xenografts in mice. Neither peptide-conjugated bacteriophage MS2 without Tl+ nor the phage filled with Tl+ but without the peptide or the same phage with the non-conjugated peptide in solution produced such effects. The preparation exhibited no acute toxicity at a therapeutic dose.

Tumor-Penetrating Peptide-Functionalized Redox-Responsive Hyperbranched Poly(amido amine) Delivering siRNA for Lung Cancer Therapy.

Biosafety and the targeting ability of gene delivery systems are critical aspects for gene therapy of cancer. In this study, we report the synthesis and use of redox-responsive poly(amido amine) (PAA) with good biocompatibility and biodegradation as a gene carrier material. A tumor-specific tissue penetration peptide, internalizing-RGD (iRGD) was then conjugated to PAA with an amidation reaction. In experiments using H1299 cells, PAA-iRGD was found to have a lower cytotoxicity and higher cellular uptake efficiency compared to PAA. An siRNA, specific to epidermal growth factor receptor (EGFR) that is overexpressed on the lung cancer cell surface and often targeted in lung cancer treatment, was designed to silence EGFR (i.e., siEGFR) for delivery by the gene carrier PAA-iRGD. EGFR gene silencing, apoptosis, antiproliferation, and antitumor effects of PAA-iRGD/siEGFR were evaluated in vitro and in vivo. PAA-iRGD/siEGFR displayed a much higher gene silencing ability compared to PAA and polyethylenimine (25 kDa), significantly inhibited the proliferation and migration of H1299 cells, and elicited significant cell apoptosis. Moreover, intravenously injected PAA-iRGD/siEGFR inhibited lung tumor growth in vivo. These results suggest that PAA-iRGD with good biocompatibility, biodegradation, and targeting ability could be a promising gene delivery system for gene therapy of cancers.

Peptide-targeted, stimuli-responsive polymersomes for delivering a cancer stemness inhibitor to cancer stem cell microtumors.

Often cancer relapses after an initial response to chemotherapy because of the tumor's heterogeneity and the presence of progenitor stem cells, which can renew. To overcome drug resistance, metastasis, and relapse in cancer, a promising approach is the inhibition of cancer stemness. In this study, the expression of the neuropilin-1 receptor in both pancreatic and prostate cancer stem cells was identified and targeted with a stimuli-responsive, polymeric nanocarrier to deliver a stemness inhibitor (napabucasin) to cancer stem cells. Reduction-sensitive amphiphilic block copolymers PEG1900-S-S-PLA6000 and the N3-PEG1900-PLA6000 were synthesized. The tumor penetrating iRGD peptide-hexynoic acid conjugate was linked to the N3-PEG1900-PLA6000 polymer via a Cu2+ catalyzed "Click" reaction. Subsequently, this peptide-polymer conjugate was incorporated into polymersomes for tumor targeting and tissue penetration. We prepared polymersomes containing 85% PEG1900-S-S-PLA6000, 10% iRGD-polymer conjugate, and 5% DPPE-lissamine rhodamine dye. The iRGD targeted polymersomes encapsulating the cancer stemness inhibitor napabucasin were internalized in both prostate and pancreatic cancer stem cells. The napabucasin encapsulated polymersomes significantly (p < .05) reduced the viability of both prostate and pancreatic cancer stem cells and decreased the stemness protein expression notch-1 and nanog compared to the control and vesicles without any drug. The napabucasin encapsulated polymersome formulations have the potential to lead to a new direction in prostate and pancreatic cancer therapy by penetrating deeply into the tumors, releasing the encapsulated stemness inhibitor, and killing cancer stem cells.

iRGD-functionalized PEGylated nanoparticles for enhanced colon tumor accumulation and targeted drug delivery.

AIM: To enhance the tumor accumulation and targeted drug delivery for colon cancer therapy, iRGD peptide was introduced to the surface of PEGylated camptothecin-loaded nanoparticles (NPs). METHODS: Cellular uptake, targeting specificity, biodistribution and antitumor capacity were evaluated. RESULTS: The functionalization of iRGD facilitated tumor accumulation and cellular uptake of NPs by Colon-26 cells. Furthermore, the resultant iRGD-PEG-NPs remarkably improved the therapeutic efficacy of camptothecin in vitro and in vivo by inducing a higher degree of tumor cell apoptosis compared with PEG-NPs. CONCLUSION: iRGD-PEG-NP is a desired drug delivery system to facilitate the drug accumulation in orthotopic colon tumor tissues and further drug internalization by colon cancer cells.

iRGD peptide as effective transporter of CuInZnxS2+x quantum dots into human cancer cells.

In this paper, iRGD peptide-mediated quantum dots (QDs) delivery was studied. In the first step, dodecanethiol-capped CuInZnxS2+x (ZCIS) QDs were prepared and subsequently transferred into water using a standard and facile ligand exchange approach involving 3-mercaptopropionic acid (MPA). ZCIS@MPA nanocrystals possess a photoluminescence quantum yield (PL QY) of 25%, a PL emission centered at ca. 640nm and low distributions in size and shape. Next, the iRGD peptide was electrostatically associated to ZCIS@MPA QDs. After cytotoxicity evaluation, the tumor-targeting and penetrating activities of the iRGD/QD assembly were investigated by confocal microscopy. The experiments performed on various cancer cell lines revealed a high penetration ability of the assembly, while the bare QDs were not internalized. Additionally, imaging experiments were conducted on three-dimensional multicellular tumor spheroids in order to mimic the tumor microenvironment in vivo. iRGD/QD assemblies were found to be evenly distributed throughout the whole HeLa spheroid contrary to normal cells where they were not present. Therefore, iRGD/QD assemblies have a great potential to be used as targeted imaging agents and/or nanocarriers specific to cancer cells.