We show here that the clinically used nanoparticle drug, abraxane, can be modified with homing peptides, and that peptides with different tumor-targeting specificities deliver abraxane to their target sites. We describe a peptide-targeted abraxane that is more effective than untargeted abraxane in tumor treatment.
The two homing peptides used in this work both selectively target tumors, but with a very different specificity. The CREKA peptide recognizes clotted plasma proteins in tumor vessels and tumor stroma. Iron oxide nanoparticles delivered to tumors bind to the walls of tumor vessels and cause clotting in them. As this additional clotting creates new binding sites for the CREKA peptide, the homing of the nanoparticles becomes self-amplifying11
. The CREKA-coated abraxane nanoparticles and micelles studied also accumulated in tumor vessels and caused clotting in them as shown by co-localization of the particles with fibrin staining and trapping of red blood cells inside the clots.
Iron oxide particles, even when coated with PEG, tend to accumulate in the liver (although they do not cause any detectable clotting in the liver11, 24
. CREKA-abraxane or CREKA micelles showed only modest uptake into the liver. Abraxane that was modified only by FAM without a peptide was not detectable in the liver, indicating that adding the CREKA peptide causes liver binding. However, the increase obtained with CREKA coating in tumor accumulation of abraxane seemed to outweigh the slight diversion of some of the CREKA-abraxane into the liver. The LyP-1 peptide also caused some liver uptake of abraxane and micelles. The presence of basic amino acids in both the peptides might favor some liver uptake but the liver accumulation of the peptide-coated abraxane particles may not be entirely charge-based, as the net charge is +1 for CREKA and +3 for LyP-1, and the liver uptake of abraxane coated with these peptides appeared to be equal.
The distribution of LyP-1 abraxane and LyP-1 micelles within tumors was very different from that of the CREKA-coated particles. The LyP-1 particles were not detectable in blood vessels, and instead accumulated outside the blood vessels in islands of cells that were positive for the known receptor for this peptide, p3215
. The vessels in these areas are primarily lymphatics, and there are few blood vessels, which may be the reason why these areas are hypoxic19
. Tumor cells in these hypoxic (and apparently also nutrient-deficient) areas tend to be resistant to chemotherapeutic agents, which generally target dividing cells. There is also growing evidence indicating that these cells are genetically unstable, and a significant source of metastasis25–27
. Thus, the ability of LyP-1 to deliver payloads to these otherwise difficult-to-access sites opens up new possibilities in tumor therapy.
LyP-1 is a cell-penetrating peptide with activity similar to that of widely used cell-penetrating peptides such as Tat and penetratin28, 29
, except that LyP-1 is cell type-specific. The ability of LyP-1 to penetrate into tumor tissue and take payloads with it may be related to the cell type specificity of its binding and internalization. Non-selective cell-penetrating peptides are taken up by all cells in all tissues30
and will concentrate in the cells that are in or close to blood vessels, whereas LyP-1 is not consumed by these cells and can accumulate in the specific targets cells. We are also investigating the possibility that there may be an active tissue-penetrating element to the tumor homing of LyP-1.
Nanoparticles do not readily extravasate, and their penetration into tumor tissue beyond the vicinity of blood vessels is particularly problematic because of the high interstitial pressure in tumors1, 31
. Importantly, our results show that synthetic particles coated with LyP-1 extravasate and spread into tumor tissue. As the abraxane nanoparticles did not lend themselves to determining whether the particles were still intact when they reached the p32-rich extravascular sites, we studied this question by using mixed micelles that have the label on one amphiphile and the peptide on another. The presence of the label at the sites the peptide homes to indicates that LyP-1 can deliver intact micelles and other nanoparticles to extravascular sites. Synaphic targeting may make it possible to change the pharmacokinetics of a drug such that more of the drug is delivered into the tumor, rendering it more effective and thereby reducing the toxicity. We did not perform toxicity studies, but our targeted abraxane nanoparticles seem to fulfill two of these expectations: non-targeted, FAM-labeled abraxane was detectable as a fluorescent network in tumor tissue, but this network was stronger when FAM-LyP-1-abraxane was injected, and p32-rich islands were intensely positive.
Our tumor treatment results with the 3 abraxane compounds were in good agreement with the bio-distribution results. The CREKA compound was not significantly different from non-targeted abraxane. In contrast, LyP-1-abraxane improved the efficacy of abraxane in a statistically highly significant matter (p = 0.001 and 0.013 in two independent experiments; combined p = 0.007). The reason for the difference may be that, although the accumulation of CREKA-abraxane in tumor blood vessels was impressive, the diffuse spread into the tumor was similar to unmodified abraxane. The accumulation in the blood vessels alone may not be sufficient to exert an enhanced anti-tumor effect compared to untargeted abraxane. However, there was an indication that CREKA-abraxane could have been slightly more active than non-targeted abraxane. Future studies will explore the possibility LyP-1 abraxane and CREKA-abraxane might synergize, as the fact that the two peptides deliver abraxane to largely non-overlapping sites in tumor tissue suggests that combining them should result in a broader distribution of the drug than either one alone. At this time, our results indicate that LyP-1-abraxane is a promising compound for tumor treatment.