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

Abstract 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.


Introduction
Ligand-mediated targeting of anticancer therapeutic agents is a useful strategy for improving anti-tumor efficacy [1]. This targeting effect relies on ligand binding to receptors which are either uniquely expressed or overexpressed on the target cells. Now, many tumor-homing peptides and tumor-penetrating peptides have been used as ligands to deliver anticancer drug delivery systems specifically to the tumor site [2,3].
iRGD (CRGDK/RGPD/EC), a tumor-homing and tumorpenetrating peptide, has been reported [4]. The mechanism by which iRGD homes on the tumor involves iRGD being able to target tumors by initially binding to av-integrin and then undergoing proteolytic cleavage in the tumor to produce CRGDK/R which has an affinity for neuropilin-1 (NRP-1) resulting in tissue penetration [4]. iRGD-modified nanocarriers [5][6][7][8][9][10] and iRGD-conjugated drug or factor [11][12][13] have been reported to improve the anti-tumor activity. More interestingly, it has been reported that co-administration of free iRGD can enhance the efficacy of anti-cancer drugs including those with a low-molecular weight (doxorubicin), nanoparticles (nab-paclitaxel and doxorubicin liposomes) and monoclonal antibodies (trastuzumab) [14]. This effect has been also observed in gemcitabine co-administered with free iRGD peptide [15], in paclitaxel-loaded MT1-AF7pconjugated nanoparticles co-administered with free iRGD [16] and in methoxypoly(ethylene glycol)-block-poly-(L-glutamic acid)-loaded cisplatin combined with free iRGD [17]. These exciting results suggest that iRGD can be used as a penetrating agent for improving anti-tumor activity when co-administered with anti-cancer drugs or anti-cancer drug delivery systems.

Cells and animals
Murine B16-F10 cells (Chinese Academy of Sciences Cells Bank, Shanghai, China) and human breast adenocarcinoma MCF-7cells (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China) were cultivated according to the recommended instructions.
Female C57BL/6 mice weighing 20-25 g (6-8 weeks) were obtained from the Experimental Animal Center of Peking University Health Science Center (Beijing, China). Female BALB/c nude mice (initially weighing 20-25 g) were purchased from the Academy of Military Medical Sciences (Beijing, China). All care and handling of the animals were performed according to the requirements of the Institutional Authority for Laboratory Animal Care of Peking University. For preparation of the tumor-bearing model, C57BL/6 mice or BALB/c nude mice received an s.c. inoculation of 100 ml B16-F10 cell suspension (1 Â 10 6 ) in the right armpit.
In vivo anti-tumor activity of iRGD-SSL-DOX When the tumor volume reached about 150-200 mm 3 , the tumor-bearing C57BL/6 mice were treated with physiological saline as control, free iRGD (4 mmol/kg), SSL-DOX, SSL-DOX co-administered with free iRGD (4 mmol/kg), iRGD-SSL-DOX, and iRGD-SSL-DOX co-administered with free iRGD (4 mmol/kg). The DOX formulations were all given via the tail vein on days 12, 16 and 20, at a dose of 3 mg/kg. Throughout the study, mice were weighed and tumors were measured with calipers twice a week. Tumor volumes were calculated from the formula: On day 22 after tumor inoculation, one or two mice in each group were executed, and the tumors were collected for the preparation of histological sections. TUNEL staining of the paraffin-embedded tissue sections was performed according to the standard protocols provided by the manufacturers.
The survival time was calculated from the day of B16-F10 cell inoculation (0 day) to the day of death. Kaplan-Meier survival curves were plotted for each group.

Confocal immunofluorescence microscopy study
In order to investigate the microcosmic characteristics of distribution differences of the SSL, we used DiI as a tracer and examined the location of iRGD-SSL-DiI, RGD-SSL-DiI and SSL-DiI, with or without co-administration of iRGD or RGD. When the tumor volume reached about 200 mm 3 , the tumor-bearing BALB/c nude mice were treated with SSL-DiI, SSL-DiI + RGD, SSL-DiI + iRGD, RGD-SSL-DiI, RGD-SSL-DiI + RGD, RGD-SSL-DiI + iRGD, iRGD-SSL-DiI, iRGD-SSL-DiI + RGD and iRGD-SSL-DiI + iRGD. The dose of all DiI formulations was 1600 ng per mouse. Free iRGD or free RGD co-administered with DiI formulations was injected at a dose of 4 mmol/kg. At 3, 6 or 12 h after administration, the mice were sacrificed, and the tumors were harvested and frozen in OCT embedding medium. Tumor sections (8 mm) were incubated with 10% BSA for 3 h at room temperature, and nuclei were counterstained with Hoechst 33258 (5 mg/ml). The sections were placed in gel/mount medium (Biomeda, Foster City, CA) and observed under a confocal microscope (Leica, Germany). Finally, sections were investigated by a confocal laser scanning microscope (CLSM, Leica SP5, Germany) and were quantified with a Leica Qwin image analysis software (TUNEL, immunofluorescence).

Flow cytometry
B16-F10 cells were seeded at a density of 3 Â 10 5 cells/well in 6-well plates and incubated at 37 C for 24 h. Then, the medium was replaced with SSL-coumarin-6, GPDC-SSL-coumarin-6, RGD-SSL-coumarin-6, CRGDK-SSLcoumarin-6 or iRGD-SSL-coumarin-6 (the final concentration of coumarin-6 was 150 ng/ml). The plates were divided in two groups (with or without trypsin treatment). For group I, without trypsin treatment, after a 2-h incubation at 37 C, the cells were washed three times with PBS solution. For group II, with trypsin treatment, SSL-coumarin-6, GPDC-SSL-coumarin-6, RGD-SSL-coumarin-6, CRGDK-SSL-coumarin-6 or iRGD-SSL-coumarin-6 (the final concentration of coumarin-6 was 150 ng/ml) was added in 50 ml trypsin solution (250 mg/ml) and incubated for 5 min at 37 C, and then the soybean inhibitor (50 ml, 30 mg/ml) was added to stop the reaction. The above solutions were then incubated with the cells for 2 h at 37 C. Finally, the cells were washed three times with PBS, and the cells in groups I and II were harvested by trypsinization, centrifuged at 1000 rpm for 5 min, resuspended in 500 ml PBS medium and examined using an FACScan (Becton Dickinson, San Jose, CA). The coumarin-6 in the cells was excited with an argon laser (467 nm), and fluorescence was detected at 502 nm.
In another flow cytometer experiment, the MCF-7 cells were seeded at a density of 2 Â 10 5 cells/well in 6-well plates and processed as described above using a B16-F10 cell line.

Competition experiment
For the competition experiment, B16-F10 cells were seeded at a density of 3 Â 10 5 cells/well in 6-well plates and incubated at 37 C for 24 h. After that, cells were pre-incubated with 1 mM free RGD, free CRGDK or free iRGD peptides for 30 min to saturate the receptor. Then, the medium was replaced with SSL-coumarin-6, RGD-SSL-coumarin-6, CRGDK-SSL-coumarin-6 or iRGD-SSL-coumarin-6 (the final concentration of coumarin-6 was 150 ng/ml) for a 2-h incubation at 37 C. Then, the cells were washed three times with PBS. All cells were harvested by trypsinization and centrifuged at 1000 rpm for 5 min and resuspended in 500 ml PBS medium and tested using an FACScan (Becton Dickinson, San Jose, CA). The coumarin-6 in the cells was excited with an argon laser (467 nm), and fluorescence was detected at 502 nm.
In another competition experiment, MCF-7 cells were seeded at a density of 2 Â 10 5 cells/well in 6-well plates and treated as described above using a B16-F10 cell line.

Statistical analysis
All data are shown as the mean ± SD. One-way analysis of variance (ANOVA) was used to determine significance among groups, after which post-hoc tests with the Bonferroni correction were used to compare differences between individual groups. Statistical significance was set at p50.05.

Results and discussion
In the present study, we designed a co-administration experiment involving B16-F10 tumor bearing C57BL/6 mice to examine the anti-tumor activity of iRGD-SSL-DOX when co-administered with free iRGD. As shown in Figure 1(A), the tumor growth was significantly inhibited in the iRGD-SSL-DOX and SSL-DOX treatment groups compared with the physiological saline-treated group used as a control (p50.01). iRGD-SSL-DOX significantly inhibited the growth of B16-F10 tumors compared with that in the SSL-DOX treatment group (p50.01). In addition, we observed that the anti-tumor activity of free iRGD was not significantly different from that seen in the control group (p40.05, Figure 1A).
As shown in Figure 1(A), our results showed that the antitumor activity of iRGD-SSL-DOX co-administered with free iRGD was similar to that of iRGD-SSL-DOX (p40.05). The median survival time of mice treated with iRGD-SSL-DOX co-administered with free iRGD (44 days) was also similar to that of mice treated with iRGD-SSL-DOX (46 days, p40.05), as shown in Figure 1(B). We also evaluated the effect of tumor cell apoptosis by TUNEL analysis staining of tumor tissue sections. As shown in Figure 2(E-G), tumors from the iRGD-SSL-DOX co-administered free iRGD-treated group exhibited similar cell apoptosis compared with the iRGD-SSL-DOX-treated group (p40.05). The data supporting these findings are shown in Figure 2(G).
In addition, similar results were also observed between SSL-DOX co-administered free iRGD group and SSL-DOX group, as shown in Figures 1 and 2.
In the light of these results, we believe that free iRGD has no effect when co-administered with iRGD-SSL-DOX or SSL-DOX. It has been reported that the plasma half-life of free iRGD is only about 8 min [19], so, if free iRGD is rapidly eliminated after intravenous administration, it will have no effect. Therefore, we designed an experiment to investigate the location of iRGD-modified SSL and RGD-modified SSL when co-administered with free iRGD or free RGD. We selected DiI as a fluorescent agent to prepare iRGD-SSL-DiI, RGD-SSL-DiI and SSL-DiI. B16-F10 tumor-bearing BALB/c nude mice were given intravenous iRGD-SSL-DiI, RGD-SSL-DiI and SSL-DiI, with or without co-administered free iRGD or free RGD. Then, 3, 6 or 12 h after administration, the mice were euthanized, and the tumor tissues were collected and cut into sections. These tumor sections were stained and observed under a confocal microscope. The obtained results indicated that the red fluorescence intensity of DiI in the tumor sections in the iRGD-SSL-DiI treatment group was stronger than that in the RGD-SSL-DiI and SSL-DiI treatment groups, confirming the targeting effect of iRGD-modified SSL ( Figure 3). We observed that the fluorescence intensity of iRGD-SSL-DiI co-administered with free iRGD was significantly reduced compared with that of iRGD-SSL-DiI (p50.01). Similar results were also observed for iRGD-SSL-DiI co-administered with free RGD compared with that of iRGD-SSL-DiI (p50.01). In addition, the inhibitory effect of free iRGD on iRGD-SSL-DiI was higher than that of free RGD on iRGD-SSL-DiI. We also found that the fluorescence intensity was reduced when RGD-SSL-DiI was co-administered with free iRGD or free RGD compared with that of RGD-SSL-DiI (p50.01). Interestingly, the fluorescence intensity of SSL-DiI was not increased following co-administration of free iRGD or free RGD (p40.05). Considering these findings, we believe that co-administration of free iRGD or free RGD would not increase the cumulative effect of SSL-DiI and might even reduce the accumulation of iRGD-SSL-DiI or RGD-SSL-DiI. In the case of ligandmodified delivery systems, the targeting effect relies on ligand-receptor specific binding. If the receptor sites are occupied by free ligand, the ligand-modified delivery system will not be able to bind to these receptor sites. Therefore, the targeting effect of this ligand-modified delivery system will be reduced [20]. We believe that if free iRGD or free RGD ligand occupied the receptor sites, this would result in a reduced ligand-receptor binding effect of iRGDmodified SSL.
In the case of MCF-7 cells, free CRGDK or free iRGD could bind to NRP-1 receptors. The targeting effect of CRGDK-SSL-coumarin-6 and iRGD-SSL-coumarin-6 was significantly reduced [ Figure 7(C1, C2, D1 and D2); Table  S4]. We also found that the inhibitory effect of free CRGDK was higher than that of free iRGD on CRGDK-SSL-coumarin-6 and iRGD-SSL-coumarin-6 [ Figure 7(C1, C2, D1 and D2); Table S4], showing that free CRGDK is more tightly bound to NRP-1 receptor than free iRGD. Since av-integrin is negatively expressed in MCF-7 cells, free RGD and free iRGD have no significant effects on the targeting of RGD-SSL-coumarin-6 [ Figure 7(B1 and B2); Table S4]. However, it was also found that free RGD, free CRGDK and free iRGD have no significant effects on SSL-coumarin-6 in both B16-F10 cells and MCF-7 cells [Figures 6(A1 and A2) and 7(A1 and A2); Tables S3 and S4]. These in vitro inhibitory results were similar to those obtained in our in vivo experiments.
It is well known that RGD is more commonly used as a targeting ligand for drug delivery systems [22][23][24][25]. Also, CRGDK has been used as a targeting ligand for modified nanocarriers [26][27][28]. Many iRGD-modified nanocarriers have also been reported [5][6][7][8][9][10]. Compared with the ligands of RGD and CRGDK, we suggest that iRGD could bind to both the av-integrin and NRP-1 receptors which are overexpressed in tumor endothelial cells or tumor cells, highlighting the potentially wide targeting applications for the iRGD ligand.
Considering our present results, we suggest that the mechanism of iRGD targeting to tumor cells takes place via a ligand-receptor-mediated targeting effect, which is by both RGD to av-integrin receptors and CRGDK to NRP-1 receptors, as shown in Figure 8. The triggering penetration effect of iRGD peptide when co-administrated with drug or drug delivery systems should be investigated in detail.
It has also been reported that some targeting ligands, such as hyaluronic acid, can be directly absorbed on the surface of nanocarrier systems to achieve a targeting effect [29]. In the case of reports of co-administration of free iRGD increasing the efficacy of anticancer drugs or nanoparticles, we suggest that if free iRGD is combined with an anti-cancer drug or absorbed on the surface of nanoparticles, the targeting of these complexes would be iRGD mediated. This hypothesis should be investigated in greater detail.
In fact, the characteristic of SSL, such as size, surface properties as well as the ratio of iRGD on the SSL, is also a factor which might affect this conclusion. We would consider these factors in future research. In addition, for other nanocarrier system (such as micelle or nanoparticle), this conclusion should be also further investigated.

Conclusions
Considering our present results, we suggest that iRGD targeting to tumor cells is ligand-receptor mediated, involving RGD to av-integrin receptor or/and CRGDK to NRP-1 receptor. The administration of free iRGD would not be able to further enhance the anti-tumor activity of iRGDmodified SSL being competitive effect.