Nanoparticle Binding to Urokinase Receptor on Cancer Cell Surface Triggers Nanoparticle Disintegration and Cargo Release

Abstract

Cancer cell expresses abundant surface receptors. These receptors are important targets for cancer treatment and imaging applications. Our goal here is to develop nanoparticles with cargo loading and tumor targeting capability. Methods: A peptide targeting at cancer cell surface receptor (urokinase receptor, uPAR) was expressed in fusion with albumin (diameter of ~7 nm), and the fusion protein was assembled into nanoparticles with diameter of 40 nm, either in the presence or absence of cargo molecules, by a novel preparation method. An important feature of this method is that the nanoparticles were stabilized by hydrophobic interaction of the fusion protein and no covalent linking agent was used in the preparation. The stability, the cargo release, in vitro and in vivo properties of such formed nanoparticles were characterized by transmission electron microscopy, dynamic light scattering, gel shift assay, laser scanning confocal microscopy and 3D fluorescent molecular tomography. Results: The nanoparticles were stable for more than two weeks in aqueous buffer, even in the buffer containing 10% fetal bovine serum. Interestingly, in the presence of urokinase receptor, the uPAR-targeting nanoparticle disintegrated into 7.5 nm fragments and released its cargo, but not the non-targeting nanoparticles made from albumin by the same preparation method. Such nanoparticles also showed higher uptake and cytotoxicity to the receptor-expressing cancer cells in vitro and higher tumor accumulation in xenografted tumor-bearing mice in vivo compared to the non-targeting nanoparticles. Conclusion: Our results demonstrate a new function of cell surface receptor as a responsive trigger to disassemble nanoparticles, besides its common use to enrich targeting agents. Such nanoparticles were thus named receptor-responsive nanoparticles (RRNP).

Keywords: amino terminal fragment of urokinase-type plasminogen activator; cargo release; human serum albumin; receptor-triggered disintegration; urokinase receptor.

Figure 1

A) Schematic illustration of the preparation of receptor-responsive nanoparticle (RRNP). Both nanoparticles ATF-HSA:CPZ@RRNP (B) and HSA:CPZ@NP (C) showed monodispersed distribution on DLS. The TEM images and photograph of the respective nanoparticles were shown as insets. The insets at lower right panels showed that the hydrophobic cargo CPZ precipitated in aqueous buffer but was solubilized inside nanoparticles (left). D) The zeta potential of both nanoparticles (10 mM Tris-HCl, pH 8.0) was about -20 mV.

Figure 2

A) ATF-HSA:CPZ@RRNP (black curve, size ~40 nm) was disintegrated and reduced to ~10 nm in the presence of recombinant uPAR receptor at a protein molecular ratio of 1:1 (red curve) but no disintegration observed in the presence of non-uPAR membrane surface receptor HAI-1 (green curve) or BSA (blue curve). B) HSA:CPZ@NP (~40 nm, black curve) is stable in the presence of uPAR (red curve), HAI-1 (green curve) or BSA (blue curve). C) Fluorescence of ATF-HSA:CPZ@RRNP (black curve) increased in the presence of recombinant uPAR receptor at a protein molecular ratio of 1:1 (red curve), but remain unchanged in the presence of non-uPAR membrane surface receptor HAI-1 (green curve) or BSA (blue curve). D) Fluorescence of HSA:CPZ@NP (black curve) did not changed in the presence of uPAR (red curve), HAI-1 (green curve) or BSA (blue curve). E) The TEM images showed ATF-HSA:CPZ@RRNP disintegrated into small pieces after incubation with recombinant uPAR protein for 2.5 h. Left TEM image is for ATF-HSA:CPZ@RRNP whereas the right TEM is the nanoparticle containing 1:1 uPAR and was incubated for 2.5 h. F) Schematic illustration of receptor-triggered disintegration and cargo release of ATF-HSA:CPZ@RRNP.

Figure 3

A) uPAR on H1299 cell triggers the disintegration of ATF-HSA:CPZ@RRNP (red curve), however, the sizes of other three groups, H1299 incubated with HSA:CPZ@NP (green curve), HELF incubated with ATF-HSA:CPZ@RRNP (blue curve) or HELF incubated with HSA:CPZ@NP (black curve) did not change basically. B) ATF-HSA:CPZ@RRNP shows higher cellular uptakes in H1299 cell compared with HSA:CPZ@NP. The time points (8 min and 2 h) of cells uptake were enlarged and shown as insets. C) Both nanoparticles have similar cellular uptakes in HELF cells. The time points (8 min and 2 h) of cells uptake were enlarged and shown as insets. D) ATF-HSA:CPZ@RRNP shows enhanced phototoxicity on H1299 cells compared with HSA:CPZ@NP at the same drug concentration. E) Both ATF-HSA:CPZ@RRNP and HSA:CPZ@NP display no phototoxicity on HELF cells compared to H1299 cells. F) The pre-incubation of free ATF (200-fold) with H1299 cells followed by the addition of ATF-HSA:CPZ@RRNP reduced the enhanced phototoxicity of ATF-HSA:CPZ@RRNP (left column) to the level of HSA:CPZ@NP (right column). Values represent the mean of three separate experiments; bars represent standard error of the mean (SEM). The unpaired, 2-tailed Student t test was used to analyze data; * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

A) Diagram of fluorescence molecular tomography imaging instrument. B) Kinetics of cargo accumulation in the tumor sites of mice. The data were averaged from 5 mice in each group. C) Representative three-dimensional (X/Y/Z axial) profile of H22 tumor in Kunming mice post intravenous injection of nanoparticles. Slices of X/Y/Z axial profile across the center of H22 tumor in Kunming mice taken at different time points (1, 2, 4, 8, 12, 24, 48, 72, 96 h) post intravenous injection of ATF-HSA:CPZ@RRNP and HSA:CPZ@NP. D) ATF-HSA:CPZ@RRNP leads a significant reduced tumor growth rate compared with HSA:CPZ@NP-treated group and the saline-treated group. The data were averaged from 10 mice in each group. E) After 7-day photodynamic therapy, all mice were sacrificed and tumor were removed and weighed. The tumor weights of ATF-HSA:CPZ@RRNP group were significant smaller than HSA:CPZ@NP group and saline group. The data were averaged from 8 mice in each group. All bars represent standard error of the mean (SEM). The unpaired, 2-tailed Student t test was used to analyze data; * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

A) Representative organs fluorescence images of H22 tumor-bearing mice taken at different time points (6, 12, 24, 48, 72, 96 h) post intravenous injection of ATF-HSA:CPZ@RRNP (top) and HSA:CPZ@NP (bottom). B) 3D quantitative analysis showed ATF-HSA:CPZ@RRNP group showed lower drug accumulation in liver tissue than HSA:CPZ@NP group. In addition, the cargo concentration in liver decreased quickly. Other organs (kidney, spleen, lung, and heart) have significantly lower cargo concentration (in the range of 17 nM), suggesting strong safety for future application. The data were averaged with the bars representing standard error of the mean (SEM).