Nanoparticle-induced vascular blockade in human prostate cancer

Abstract

The tumor-homing pentapeptide CREKA (Cys-Arg-Glu-Lys-Ala) specifically homes to tumors by binding to fibrin and fibrin-associated clotted plasma proteins in tumor vessels. Previous results show that CREKA-coated superparamagnetic iron oxide particles can cause additional clotting in tumor vessels, which creates more binding sites for the peptide. We have used this self-amplifying homing system to develop theranostic nanoparticles that simultaneously serve as an imaging agent and inhibit tumor growth by obstructing tumor circulation through blood clotting. The CREKA nanoparticles were combined with nanoparticles coated with another tumor-homing peptide, CRKDKC, and nanoparticles with an elongated shape (nanoworms) were used for improved binding efficacy. The efficacy of the CREKA peptide was then increased by replacing some residues with nonproteinogenic counterparts, which increased the stability of the peptide in the circulation. Treatment of mice bearing orthotopic human prostate cancer tumors with the targeted nanoworms caused extensive clotting in tumor vessels, whereas no clotting was observed in the vessels of normal tissues. Optical and magnetic resonance imaging confirmed tumor-specific targeting of the nanoworms, and ultrasound imaging showed reduced blood flow in tumor vessels. Treatment of mice with prostate cancer with multiple doses of the nanoworms induced tumor necrosis and a highly significant reduction in tumor growth.

Figure 1

Combining CREKA-NWs with nanoworms coated with another tumor-homing peptide enhances homing efficiency. (A) Iron oxide nanoworms coated with FAM-labeled CREKA peptide were injected intravenously (5 mg of iron per kilogram of body weight) into nude mice bearing orthotropic 22Rv1 human prostate tumors. The mice had been preinjected with Ni-liposomes to reduce uptake by the reticuloendothelial system. Tumors were harvested 5 hours later, and tumor sections were stained with antibodies and examined by confocal microscopy (the 5-hour time point was found to be optimal for nanoworm homing with regard to accumulation of the nanoworms in the tumor and clearance of nanoworms from the blood). The CREKA-coated particles are green; blood vessels and clotting were visualized separately with anti-CD31 (magenta) or antifibrin(ogen) staining (red); nuclei were stained with DAPI (blue). Bars represent 200 μm. (B) Nanoworms coated with FAM-labeled CRKDKC or CGKRK were injected intravenously, and the tissues were collected and processed as in panel A. CRKDKC- or CGKRK-coated particles are green; blood vessels visualized with anti-CD31 are magenta (white indicates colocalization of magenta and green) and those visualized with anti-fibrin(ogen) staining are red (yellow indicates colocalization of red and green); nuclei were stained with DAPI (blue). Large vessels were selected for the panels on the right because intravascular clotting (which is not promoted by CRKDKC-NWs or CGKRK-NWs) is most apparent in larger vessels. Bars represent 200 μm (left and middle panels) and 100 μm (right panels). (C) A mixture of nanoworms coated with rhodamine-labeled CREKA (red) and FAM-labeled CRKDKC (green) was injected intravenously (2.5 mg of iron per kilogram of each nanoworm preparation), and the tissues were collected and processed as in A and stained for fibrin(ogen) (magenta); nuclei were stained with DAPI (blue). Bars represent 200 μm. (D) Mice were injected with the indicated materials as in panels A, B, or C. The sections stained with anti-fibrin(ogen) antibody were subjected to image analysis with Scanscope to quantify fibrin(ogen)-positive areas. The insets show examples of anti-fibrin(ogen) immunostaining in the tumor rim (left) and interior (right) from mice injected with the nanoworm mixture. Bars represent 50 μm. Statistical analyses were performed with analysis of variance. Error bars represent SEM (n = 5-6); **P < .01.

Figure 2

CEUS imaging of blood circulation in tumors of mice treated with peptide-coated nanoworms. (A) Mice preinjected with Ni-liposomes were subsequently injected with a mixture of CREKA-NWs and CRKDKC-NWs and, after the indicated periods of time, injected with an ultrasound contrast agent. CEUS and conventional ultrasound (US) images obtained at the different time points are shown. The images are representative of 3 tumors imaged. (B) Enhancement-analysis curves of blood flow in different tumor regions and the surrounding tissue from experiments described in panel A. The orientation of the tumors is slightly different between the time points because the mice were anesthetized for each scan and reintroduced to the ultrasound instrument; n = 3.

Figure 3

Tumor accumulation of the CREKA peptide and its N/Cα-methylated variants. Mice bearing orthotropic 22Rv1 xenograft tumors were injected intravenously with 200 μg of FAM-labeled CREKA or N/Cα-methylated CREKA peptides, which were allowed to circulate for 3 hours. This time point highlights the differences between nonmodified CREKA and some of the methylated variants (D). The mice were perfused through the heart with PBS, and the organs shown were collected and viewed under ultraviolet light. (A) Quantification of fluorescence with ImageJ software. Several N/Cα-methylated CREKA analogs produced stronger fluorescence than unmodified CREKA. Statistical analyses were performed with analysis of variance. Error bars show SEM (n = 3-4); **P < .01; ***P < .001. (B-C) Representative images from mice injected with the CREKA or CR(NMe)EKA peptides (B, 22Rv1 xenografts; C, LAPC9 xenografts). In the top panels, white dotted lines show where the organs were placed in a macroscopic examination, and the yellow lines outline the tumor. The bottom panels show confocal images of tumor sections from mice injected with the peptides (green) indicated above. Blood vessels were visualized with anti-CD31 (red); nuclei were stained with DAPI (blue). Bars represent 200 μm. (B right panels) Representative confocal image fields illustrate the localization of the CR(NMe)EKA peptide (green) in relation to anti-fibrin(ogen) (red) and anti-fibronectin (magenta) staining used as markers of tumor stroma; nuclei were stained with DAPI (blue). Bar represents 50 μm. (C right panels) Quantification of fluorescence with ImageJ software. Statistical analysis was performed with Student t test. Error bars show SEM (n = 3); **P < .01. (D) Quantification of fluorescence with ImageJ software 15 minutes or 3 hours after peptide injection into 22Rv1 tumor–bearing mice. CR(NMe)EKA produced stronger fluorescence over time than unmodified CREKA. Statistical analysis was performed with Student t test. Error bars show SEM (n = 3-4); ***P < .001. (NMe) and (CMe) indicate an N- or Cα-methylated residue, respectively.

Figure 4

CRE(CMe)KA-NW homing to 22Rv1 tumors. Mice bearing orthotropic 22Rv1 xenograft tumors were injected intravenously with 200 μg of FAM-labeled CRE(CMe)KA or 5 mg of iron per kilogram of nanoworms coated with FAM-CRE(CMe)KA. The peptide was allowed to circulate for 3 hours, and nanoworms were allowed to circulate for 5 hours. The mice were then perfused through the heart with PBS, and the tumors were collected. Tumor sections were stained with CD31 or anti-fibrin(ogen) (red) and examined by confocal microscopy. Nanoworms are green; nuclei were stained with DAPI (blue). Bars represent 200 μm. n = 3.

Figure 5

Improved tumor homing of nanoworms coated with an N-methylated CREKA peptide analog. Nanoworms coated with FAM-labeled CREKA peptide or its N-methylated variant, CR(NMe)EKA, were injected intravenously into mice bearing 22Rv1 tumors (total dose 5 mg of iron per 1 kg). (A) Tumors were harvested 5 hours later, and tumor sections were stained with antibodies and examined by confocal microscopy. CR(NMe)EKA-NWs are green; blood vessels and clotting were visualized separately with anti-CD31 or anti-fibrin(ogen) staining (red). Nuclei were stained with DAPI (blue). Bars represent 100 μm (50 μm in the inset). (B) T2-weighted magnetic resonance images (fast spin echo, repetition time = 6.4 seconds, echo time = 69 ms). CREKA-NWs or CR(NMe)EKA-NWs were injected intravenously into tumor-bearing mice. The particles were allowed to circulate for 7-8 hours (the time determined in preliminary experiments to be optimal for differential homing). Gray-scale images of axial planes through the tumors are shown. Gadolinium (Gd) and Feridex (iron) were used as reference standards. n = 3-4.

Figure 6

Nanoworm distribution and effects on intravascular clotting, tumor apoptosis, and tumor therapy. Mice bearing 2-week-old orthotopic xenografts of 22Rv1 human prostate cancer were injected intravenously with nanoworms coated with peptides through a 5-kDa PEG spacer. The nanoworms were administered every other day for 14 days (5 mg of iron per kilogram per day, total cumulative dose 35 mg/kg). (A) Tumor sections were stained with anti-CD31 (red); CR(NMe)EKA-NW/CRKDKC-NW combination is shown as green; nuclei were stained with DAPI (blue). Bars represent 200 μm. The necrotic area at the center of the tumor is autofluorescent. (B) CEUS imaging and analysis showed reduction in tumor blood flow at the end of treatment. The images are representative of n = 3. (C) Staining with hematoxylin and eosin showed a large necrotic area (arrow) in the middle of a typical tumor treated with the CR(NMe)EKA-NW/CRKDKC-NW combination and occluded vessels in the viable rim of these tumors (broken arrows). A tumor of a similar size from a mouse treated with CRKDKC-NWs alone is shown for comparison. (D) Apoptosis analysis by TUNEL staining is shown as red; nanoworm combination is shown as green; nuclei were stained with DAPI. Bars represent 200 μm.

Figure 7

Tumor treatment with targeted nanoworms. Mice bearing orthotopic xenografts of 22Rv1 or LAPC9 human prostate cancer (2 weeks or 10 days after inoculation, respectively) were injected intravenously with nanoworms coated with peptides through a 5-kDa PEG spacer. The particles were administered every other day for 14 days (5 mg of iron per kilogram per day, total cumulative dose 35 mg/kg). (A) Tumor volume 1 day after the last injection in the 22Rv1 model is shown. Statistical analyses were performed with analysis of variance. Error bars show SEM (n = 10-12); **P < .01; ***P < .001. Similar results were obtained in 2 independent experiments. (B) Mice bearing LAPC9 tumors were treated as described in panel A, and survival was monitored over time (n = 8 per group). The arrow indicates the day the nanoworm treatment was stopped.