Tumor-penetrating Peptide iRGD Research Articles

Deep Tumor Penetrating Bioparticulates Inspired Burst Intracellular Drug Release for Precision Chemo-Phototherapy.

The relevance of personalized medicine has inspired research for individually concerted diagnosis and therapy. Numerous efforts are devoted to designing drug particulates with capabilities of tumor penetrating and subcellular trafficking to concurrently discharge theranostics in response to multistimulations. In this study, a bioinspired particulate, formulated with whole components of native high-density lipoproteins (HDLs) and decorated with the tumor-penetrating peptide iRGD, is proposed to promote tumor penetration of HDLs (pHDLs) together with payloads. Specifically, paclitaxel (PTX), and the NIR fluorescent probe indocyanine green (ICG) are integrated into pHDLs (pHDL/PTX-ICG) for synergetic chemo-phototherapy. Inspired by lipoproteins, pHDLs are not only restored from naturally occurring materials but also possessed artificially endowed functions, leading to an enhanced cellular uptake, higher accumulation, and deep penetration into tumors without causing appreciable adverse effects, compared to reconstituted HDLs or lipid-based nanoparticles. After intravenous administration, pHDL/PTX-ICG performs a burst of intracellular drug release and imaging-guided precision chemo-phototherapy upon NIR irradiation that completely eradicates xenograft tumors. Neither recurrence nor significant toxicity is observed due to maneuvered regional photodynamic and photothermal therapy. Taken together, pHDL/PTX-ICG is proven to be a promising platform to achieve deep tumor penetration and imaging-guided chemo-phototherapy.

Coadministration of a tumor-penetrating peptide improves the therapeutic efficacy of paclitaxel in a novel air-grown lung cancer 3D spheroid model.

Three-dimensional (3 D) cell culture platforms are increasingly being used in cancer research and drug development since they mimic avascular tumors in vitro. In this study, we focused on the development of a novel air-grown multicellular spheroid (MCS) model to mimic in vivo tumors for understanding lung cancer biology and improvement in the evaluation of aerosol anticancer therapeutics. 3 D MCS were formed using A549 lung adenocarcinoma cells, comprising cellular heterogeneity with respect to different proliferative and metabolic gradients. The growth kinetics, morphology and 3 D structure of air-grown MCS were characterized by brightfield, fluorescent and scanning electron microscopy. MCS demonstrated a significant decrease in growth when the tumor-penetrating peptide iRGD and paclitaxel (PTX) were coadministered as compared with PTX alone. It was also found that when treated with both iRGD and PTX, A549 MCS exhibited an increase in apoptosis and decrease in clonogenic survival capacity in contrast to PTX treatment alone. This study demonstrated that coadministration of iRGD resulted in the improvement of the tumor penetration ability of PTX in an in vitro A549 3 D MCS model. In addition, this is the first time a high-throughput air-grown lung cancer tumor spheroid model has been developed and evaluated.

Abstract 965: Two in one: nanotechnology based strategies for the treatment of ER+ breast cancer

Abstract Understanding endocrine resistance mechanisms is key to develop new therapeutic strategies. We focus on the role the tumor microenvironment plays as a modulator of endocrine therapy resistance in breast cancer, in particular associated to Tamoxifen. Studies show that the use of nanoparticles (NPs) as antitumor drug delivery systems is a good strategy to improve the efficacy and decrease secondary effects of conventional chemotherapies. In this context we hypothesize that a therapeutic strategy based on the use of Tamoxifen carried in NPs coated with the tumor penetrating peptide iRGD, would be more effective than conventional Tamoxifen. Neuropilin-1, that mediates iRGD induced endocytosis, has been shown to be associated to breast cancer stem cells. Thus, we postulate that our multifunctional NPs would be effective in reducing this cell population, contrary to what is observed with free Tamoxifen. Moreover, iRGD blocks the interaction between integrin β1 and fibronectin, a mechanism we have previously shown induces Tamoxifen resistance (Pontiggia et al. 2012). NPs were synthesized with polyethyleneglycol and polycaprolactone and coated with the iRGD peptide. This peptide was constructed with a FAM fluorophore in order to track the NPs. To evaluate the cell uptake, MCF-7 cells were incubated for 5 h with iRGD-NP or FAM-NP as a control. NP entry was higher when the NPs were coated with iRGD, both in 2D and in 3D cultures. Cell viability experiments revealed that Tamoxifen encapsulated in NPs was more effective than the free drug (p<0,05), and NPs coated with iRGD had an even stronger effect (p<0,01). Moreover, resistance to Tamoxifen induced by fibronectin was reversed with the iRGD-coated NPs, contrary to what is observed with the free drug. Previously, we demonstrated that treatment with Tamoxifen enhances the stem cell population in estrogen receptor-positive breast cancer cell lines (Raffo et al. 2013). In this context iRGD coated NPs lead to a decrease in breast cancer stem cells as evaluated by mammosphere assays (p<0,01). Finally, in vivo tumor homing was evaluated to test whether the iRGD peptide affected the biodistribution of the NPs. Nude mice carrying MCF-7 breast tumors were treated with iRGD-NP and control NPs. Tumors were collected at 4 and 24 hrs post inoculation. Frozen sections were analyzed by confocal microscopy revealing that only the mice treated with the iRGD-NPs had high levels of NPs in the tumor, compared to the control group. Moreover, frozen sections of kidneys, livers, lungs and spleen showed very low levels of iRGD-NPs, confirming that the targeting was effective. In vivo experiments of the impact of iRGD-NPs on tumor growth, metastasis and the stem cell population are ongoing. These results suggest that iRGD-NPs carrying Tamoxifen could be an effective strategy to avoid the development of endocrine resistance in breast cancer. Citation Format: Maria Ines Diaz Bessone, Lorena Simón-Gracia, Pablo Scodeller, Tambet Teesalu, Marina Simian. Two in one: nanotechnology based strategies for the treatment of ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 965. doi:10.1158/1538-7445.AM2017-965

Tumor-penetrating peptide iRGD conjugation effectively potentiates intratumor T cell infiltration.

e14543 Background: Poor infiltration of activated lymphocytes into solid tumors can be a fundamental factor limiting their efficacy. The purpose of this study was to determine whether a cycled tumor-penetrating peptide, iRGD, with a well-defined role in delivering drugs into extravascular tumor tissues, could facilitate lymphocytes infiltration into tumors in both the iRGD combination regimen and conjugated pattern. Methods: We used polyethylene glycol-conjugated phospholipid (PEG-lipid) derivatives, a time-efficient and versatile platform, to immobilize iRGD on T cell membrane, and examined the ability of iRGD to improve lymphocytes infiltration into tumors in multicellular spheroids and two xenograft mouse models. Furthermore, the synergy effect of iRGD modification and PD-1 gene knock out in adoptive T cell transfer immunotherapy was evaluated in a xenograft model of EBV associated gastric cancer. Results: In this study, we first showed that systematic injection of iRGD could double the number of CD3+ T cells in 4T1 tumor in BALB/C mouse and the synthetic iRGD-PEG-lipid, which could spontaneously transfer from solution to cell surfaces, had no effect to cell vitality, proliferation, phenotype and function. In adoptive transfer studies, the infiltration of T cells could be promoted by the co-administration with iRGD while T cells modified with iRGD could spread more extensively throughout both the multicellular spheroids and tumor mass. Furthermore, We demonstrated that iRGD modified T cells had superior antitumor efficiency to iRGD combination regimen due to increased T cell infiltration, and exhibited robust synergy effect with PD-1 gene knock out. Conclusions: Our study offers a simple method to target immune cells into tumors and may have the potential to enhance the clinical efficacy of adoptive T cell immunotherapy.

Tumor-penetrating peptide enhances transcytosis of silicasome-based chemotherapy for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is almost uniformly fatal; however, some improvement in overall survival has been achieved with the introduction of nanocarriers that deliver irinotecan or paclitaxel. Although it is generally assumed that nanocarriers rely principally on abnormal leaky vasculature for tumor access, a transcytosis transport pathway that is regulated by neuropilin-1 (NRP-1) has recently been reported. NRP-1-mediated transport can be triggered by the cyclic tumor-penetrating peptide iRGD. In a KRAS-induced orthotopic PDAC model, coadministration of iRGD enhanced the uptake of an irinotecan-loaded silicasome carrier that comprises lipid bilayer-coated mesoporous silica nanoparticles (MSNPs); this uptake resulted in enhanced survival and markedly reduced metastasis. Further, ultrastructural imaging of the treated tumors revealed that iRGD coadministration induced a vesicular transport pathway that carried Au-labeled silicacomes from the blood vessel lumen to a perinuclear site within cancer cells. iRGD-mediated enhancement of silicasome uptake was also observed in patient-derived xenografts, commensurate with the level of NRP-1 expression on tumor blood vessels. These results demonstrate that iRGD enhances the efficacy of irinotecan-loaded silicasome-based therapy and may be a suitable adjuvant in nanoparticle-based treatments for PDAC.

Access granted: iRGD helps silicasome-encased drugs breach the tumor barrier.

In this issue of the JCI, Liu et al. use irinotecan-loaded nanoparticles to treat pancreatic adenocarcinomas in mice. Encapsulating drugs into nanoparticles has distinct advantages: it can improve the pharmacokinetics of the drug, enhance efficacy, and reduce unwanted side effects. A drawback is that the large size of nanoparticles restricts their access to the tumor interior. Liu and colleagues show that the cyclic tumor-penetrating peptide iRGD, reported to be capable of enhancing tumor penetration by drugs, can overcome this limitation to a substantial degree when administered together with the nanoparticles. Pancreatic adenocarcinoma is a challenging malignancy to treat and in desperate need for improved treatments; therefore, advances like this are most welcome.

IRGD-targeted delivery of a pro-apoptotic peptide activated by cathepsin B inhibits tumor growth and metastasis in mice.

The use of cytolytic peptides with potential therapeutic properties is a promising approach to cancer therapy due to their convenient automated synthesis and their capacity for modifications. However, the use of cytolytic peptides is limited due to their nonspecific cytolytic activity. In this study, we designed a tumor-targeting proapoptotic system based on an amphipathic D-amino acid-modified apoptotic peptide, KLA, a variant of (KLAKLAK)2, which is fused with a linear tumor-penetrating homing peptide iRGD through specific cathepsin B (CTSB) cleavage sequences that are overexpressed in many types of tumor tissues. Our data show that the procytotoxic peptide D(KLAKLAKKLAKLA)K-GG-iRGD (m(KLA)-iRGD) is internalized into cultured tumor cells through a neuropilin-1 (NRP1)-activated pathway by iRGD delivery. Once inside the cells, the peptide triggers rapid apoptosis through both the mitochondrial-induced apoptotic pathway and the death receptor pathway in NRP1+/αvβ3/CTSB+ tumor cells. Furthermore, m(KLA)-iRGD spread extensively within the tumor tissue when it was injected into 4T1 tumor-bearing mice. The m(KLA)-iRGD peptide inhibited tumor growth to a certain degree, resulting in a significant reduction in tumor volume (P < 0.05) and the total inhibition of metastasis at the end of the treatment. These results suggest that m(KLA)-iRGD has the potential for development as a new antitumor drug.

Does ligand–receptor mediated competitive effect or penetrating effect of iRGD peptide when co-administration with iRGD-modified SSL?

Ligand-mediated targeting of anticancer therapeutic agents is a useful strategy for improving anti-tumor efficacy. It has been reported that co-administration of a tumor-penetrating peptide iRGD (CRGDK/RGPD/EC) enhances the efficacy of anticancer drugs. Here, we designed an experiment involving co-administration of iRGD-SSL-DOX with free iRGD to B16-F10 tumor bearing mice to examine the action of free iRGD. We also designed an experiment to investigate the location of iRGD-modified SSL when co-administered with free iRGD or free RGD to B16-F10 tumor bearing nude mice. Considering the sequence of iRGD, we selected the GPDC, RGD and CRGDK as targeting ligands to investigate the targeting effect of these peptides compared with iRGD on B16-F10 and MCF-7 cells, with or without enzymatic degradation. Finally, we selected free RGD, free CRGDK and free iRGD as ligand to investigate the inhibitory effect on RGD-, CRGDK- or iRGD-modified SSL on B16-F10 or MCF-7 cells. Our results indicated that iRGD targeting to tumor cells was ligand–receptor mediated involving RGD to αv-integrin receptor and CRGDK to NRP-1 receptor. Being competitive effect, the administration of free iRGD would not be able to further enhance the anti-tumor activity of iRGD-modified SSL. There is no need to co-administrate of free iRGD with the iRGD-modified nanoparticles for further therapeutic benefit.

A Novel Strategy to Improve the Therapeutic Efficacy of Gemcitabine for Non-Small Cell Lung Cancer by the Tumor-Penetrating Peptide iRGD.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, comprising approximately 75–80% of all lung cancers. Gemcitabine is an approved chemotherapy drug for NSCLC. The objective of this study was to develop a novel strategy to improve the therapeutic efficacy of Gemcitabine for NSCLC by the co-administered iRGD peptide. We showed that the rates of positive expression of αvβ3, αvβ5 and NRP-1 in the A549 cell line were 68.5%, 35.3% and 94.5%, respectively. The amount of Evans Blue accumulated in the tumor of Evans Blue+iRGD group was 2.5 times that of Evans Blue group. The rates of growth inhibition of the tumors of the iRGD group, the Gemcitabine group and the Gemcitabine+iRGD group were 8%, 59.8% and 86.9%, respectively. The results of mechanism studies showed that PCNA expression in the Gemcitabine+iRGD group decreased 71.5% compared with that in Gemcitabine group. The rate of apoptosis in the Gemcitabine+iRGD group was 2.2 time that of the Gemcitabine group. Therefore, the tumor-penetrating Peptide iRGD can enhance the tumor-penetrating ability and therapeutic efficacy of Gemcitabine in the A549 xenograft. The combined application of Gemcitabine with iRGD may be a novel strategy to enhance the clinical therapeutic efficacy of Gemcitabine in patients with NSCLC.

Nanoparticles coated with the tumor-penetrating peptide iRGD reduce experimental breast cancer metastasis in the brain.

Metastasis is the main killer in cancer; consequently, there is great interest in novel approaches to prevent and treat metastatic disease. Brain metastases are particularly deadly, as the protection of the blood-brain barrier obstructs the passage of common anticancer drugs. This study used magnetic resonance imaging (MRI) to investigate the therapeutic effects of nanoparticles coated with a tumor-penetrating peptide (iRGD) against a preclinical model of breast cancer brain metastasis. Single doses of iRGD nanoparticle were administered intravenously, and the effect on tumor growth was observed over time. iRGD nanoparticles, when applied in the early stages of metastasis development, strongly inhibited tumor progression. Overall, this study demonstrated for the first time that a single dose of iRGD nanoparticle can have a significant effect on metastatic tumor progression and nonproliferative cancer cell retention when applied early in course of tumor development. These data suggest that iRGD nanoparticles may be useful in preventatively reducing metastasis after a cancer diagnosis has been established. bSSFP MRI can be used to track nonproliferative iron-labeled cells and tumor development over time. iRGD-NW, when applied early, has a significant effect on metastatic tumor progression. Retained signal voids represent a subpopulation of nonproliferating tumor cells. Reduced cell retention and tumor burden show a role for iRGD-NW in metastasis prevention. iRGD target is universally expressed; thus, iRGD-NW should be clinically translatable.

Superior intratumoral penetration of paclitaxel nanodots strengthens tumor restriction and metastasis prevention.

Recently discovered intratumoral diffusion resistance, together with poor solubility and nontargeted distribution of chemotherapeutic drugs, has significantly impaired the performance of cancer treatments. By developing a well-designed droplet-confined/cryodesiccation-driven crystallization approach, we herein report the successful preparation of nanocrystallites of insoluble chemotherapeutic drug paclitaxel (PTX) in forms of nanodots (NDs, ≈10 nm) and nanoparticles (NPs, ≈70 nm) with considerably high drug loading capacity. Superficially coated Pluronic F127 is demonstrated to endow the both PTX nanocrystallites with excellent water solubility and prevent undesired phagocyte uptake. Further decoration with tumor-penetrating peptide iRGD, as expected, indiscriminatively facilitates tumor cell uptake in traditional monolayer cell culture model. On the contrary, distinctly enhanced performances in inward penetration and ensuing elimination of 3D multicellular tumor spheroids are achieved by iRGD-NDs rather than iRGD-NPs, revealing the significant influence of particle size variation in nanoscale. In vivo experiments verify that, although efficient tumor enrichment is achieved by all nanocrystallites, only the iRGD-grafted nanocrystallites of ultranano size realize thorough intratumoral delivery and reach cancer stem cells, which are concealed inside the tumor core. Consequently, much strengthened restriction on progress and metastasis of orthotopic 4T1 mammary adenocarcinoma is achieved in murine model, in sharp contrast to commercial PTX formulation Taxol.

Tumor-penetrating iRGD peptide inhibits metastasis.

Tumor-specific tissue-penetrating peptides deliver drugs into extravascular tumor tissue by increasing tumor vascular permeability through interaction with neuropilin (NRP). Here, we report that a prototypic tumor-penetrating peptide iRGD (amino acid sequence: CRGDKGPDC) potently inhibits spontaneous metastasis in mice. The antimetastatic effect was mediated by the NRP-binding RXXK peptide motif (CendR motif), and not by the integrin-binding RGD motif. iRGD inhibited migration of tumor cells and caused chemorepulsion in vitro in a CendR- and NRP-1-dependent manner. The peptide induced dramatic collapse of cellular processes and partial cell detachment, resulting in the repellent activity. These effects were prominently displayed when the cells were seeded on fibronectin, suggesting a role of CendR in functional regulation of integrins. The antimetastatic activity of iRGD may provide a significant additional benefit when this peptide is used for drug delivery to tumors.

Abstract 2663: Hyperthermia improves Cetuximab accumulation in pancreatic cancer mouse model

Abstract Objective: Histopathological characteristics of pancreatic carcinoma are the presence of abundant stroma that prevent mAb diffusion and consequently hampers mAb from direct attacking cancer cells. With a strong conviction that additional tool that improve mAb penetration and diffusion in dense fibroblastic solid cancer should indispensable for better therapeutic effect, we focused and employed the simple but strongest modalities, heat. Heat might increase tumor blood flow and tumor vessel permeability, leading enhanced accumulation of anticancer mAb. In this study, we aimed the increased accumulation of mAb in mouse pancreatic subcutaneous models and also boosted antitumor effects by applying moderate heat. Methods: Heat generation was completed by hot bathtub method for subcutaneous tumor in nude mice leg. Three human pancreatic cell of different stromal amount were employed (abundant: Capan-1, moderate: BxPC-3, and scant: MIAPaCa-2). At the timing of each tumors growth up to 70mm3, Cetuximab (1 mg/kg) was systematically administrated via caudal vein, subsequently applying heat to tumors for 30 min at three different temperatures of 25°C (control temperature), 37°C (intraabdominal organs), or 41°C (hyperthermia) (n = 5 each). The amount of accumulated Cetuximab were quantified by the fluorescent intensity of Alexa 488 against anti-human IgG at 24 hr, followed by figuring up the intensity of one cancer cell by dividing the total intensity value by the number of a cancer cell nuclei stained with DAPI. Enhanced antitumor effects, among irradiating various heat doses, were evaluated by the tumor volume over time. Results: In Capan-1, the fluorescent intensity per a cancer cell at 25°C, 37°C, 41°C were 675, 1130, 2332 respectively. Similarly, those in BxPC-3 were 673, 1347, 1573, and in MIAPaCa-2 were 1632, 1921, 1949, respectively. In Capan-1 and BxPC-3, the mean tumor growth on day 40 were significantly inhibited at 37°C group (934, 189 mm3), and 41°C group (491, 121 mm3) in compared with 25°C group (1385, 470 mm3) respectively. In MIAPaCa-2, however, that on day 50 was not inhibited at 37 degrees group (109 mm3), and 41 group (151 mm3) as compared with 25 group (123 mm3). Discussion: We demonstrated that hyperthermia actually contributed to measurable add-on antitumor effect for two among three pancreatic cancer models. Intending to provide more add-on effect, we are now evaluating the combined effects of hyperthermia plus applying a cyclic tumor-penetrating peptide (iRGD), which improved anticancer mAb penetration to cancer cells by binding to integrin αv/β3 or β5 and to neuropillin-1 on cancer cells. We suggested that this novel combined strategy would remarkably contribute to the enhanced accumulation of anticancer mAb in tumors and to the enhanced antitumor effects. Citation Format: Ryoichi Miyamoto, Tatsuya Oda, Shinji Hashimoto, Yoshimasa Akashi, Tomohiro Kurokawa, Yuki Inagaki, Nobuhiro Ohkohchi. Hyperthermia improves Cetuximab accumulation in pancreatic cancer mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2663. doi:10.1158/1538-7445.AM2014-2663

Doxorubicin delivery to 3D multicellular spheroids and tumors based on boronic acid-rich chitosan nanoparticles

Boronic acid-rich chitosan-poly(N-3-acrylamidophenylboronic acid) nanoparticles (CS-PAPBA NPs) with the tunable size were successfully prepared by polymerizing N-3-acrylamidophenylboronic acid in the presence of chitosan in an aqueous solution. The CS-PAPBA NPs were then functionalized by a tumor-penetrating peptide iRGD and loading doxorubicin (DOX). The interaction between boronic acid groups of hydrophobic PAPBA and the amino groups of hydrophilic chitosan inside the nanoparticles was examined by solid-state NMR measurement. The size and morphology of nanoparticles were characterized by dynamic light scattering and electron microscopy. The cellular uptake, tumor penetration, biodistribution and antitumor activity of the nanoparticles were evaluated by using three-dimensional (3-D) multicellular spheroids (MCs) as the in vitro model and H22 tumor-bearing mice as the in vivo model. It was found that the iRGD-conjugated nanoparticles significantly improved the efficiency of DOX penetration in MCs, compared with free DOX and non-conjugated nanoparticles, resulting in the efficient cell killing in the MCs. In vivo antitumor activity examination indicated that iRGD-conjugated CS-PAPBA nanoparticles promoted the accumulation of nanoparticles in tumor tissue and enhanced their penetration in tumor areas, both of which improved the efficiency of DOX-loaded nanoparticles in restraining tumor growth and prolonging the life time of H22 tumor-bearing mice.