Targeting of albumin-embedded paclitaxel nanoparticles to tumors

Abstract

We have used tumor-homing peptides to target abraxane, a clinically approved paclitaxel-albumin nanoparticle, to tumors in mice. The targeting was accomplished with two peptides, CREKA and LyP-1 (CGNKRTRGC). Fluorescein (FAM)-labeled CREKA-abraxane, when injected intravenously into mice bearing MDA-MB-435 human cancer xenografts, accumulated in tumor blood vessels, forming aggregates that contained red blood cells and fibrin. FAM-LyP-1-abraxane co-localized with extravascular islands expressing its receptor, p32. Self-assembled mixed micelles carrying the homing peptide and the label on different subunits accumulated in the same areas of tumors as LyP-1-abraxane, showing that Lyp-1 can deliver intact nanoparticles into extravascular sites. Untargeted, FAM-abraxane was detected in the form of a faint meshwork in tumor interstitium. LyP-1-abraxane produced a statistically highly significant inhibition of tumor growth compared with untargeted abraxane. These results show that nanoparticles can be effectively targeted into extravascular tumor tissue and that targeting can enhance the activity of a therapeutic nanoparticle.

Figure 1. Localization of CREKA-abraxane in tumor tissue

Balb/c nude mice bearing MDA-MB-435 tumors (~0.5 cm³) were intravenously injected with abraxane conjugated to labeled CREKA peptide (CREKA-abraxane) or to fluorescein (FAM-abraxane), and adjusted to 20 mg/kg of paclitaxel equivalent. The mice were sacrificed by perfusion through the heart 3 hours later and tissues sections were examined for fluorescence (green). CREKA-abraxane accumulates in tumor blood vessels (A; anti-CD31, red) and co-localizes with anti-fibrin(ogen) (red) staining (B). FAM-abraxane showed some accumulation in tumor interstitium (C). Nuclei were counterstained with DAPI (blue). The results are representative of three independent experiments. Magnification: 200x.

Figure 2. Localization of LyP-1-abraxane in tumor tissue

LyP-1-abraxane was injected into nude mice bearing MDA-MB-435 tumors as described in the legend of Figure 1. LyP-1-abraxane (green) co-localizes with p32 (red), the receptor for LyP-1 (A). Cell clusters positive for LyP-1-abraxane are interspersed with podoplanin-positive structures (red), presumed to be lymphatic vessels (B), but these areas are mostly devoid of blood vessels (C). LyP-1-abraxane exhibits increased accumulation in extravascular tissue compared to FAM-abraxane (D, E, F). Blood vessels were stained with anti-CD31 and lymphatic vessels with anti-podoplanin. Nuclei were counterstained with DAPI. The results are representative of three independent experiments. Magnification: 600x (A & B), 200x (C, D & E). Quantification of fluorescence (F) in tumor micrographs (FAM-abraxane, CREKA-abraxane and LyP-1-abraxane) and liver micrographs (LyP-1-abraxane) was perfomed using Image J (NIH, USA). Five random fields were quantified per tumor and liver for LyP-1-abraxane (n=3), four random fields per tumor for CREKA-abraxane (n=3) and five random fields per tumor for FAM-abraxane (n=3). Error bars represent S.E.M.

Figure 2. Localization of LyP-1-abraxane in tumor tissue

LyP-1-abraxane was injected into nude mice bearing MDA-MB-435 tumors as described in the legend of Figure 1. LyP-1-abraxane (green) co-localizes with p32 (red), the receptor for LyP-1 (A). Cell clusters positive for LyP-1-abraxane are interspersed with podoplanin-positive structures (red), presumed to be lymphatic vessels (B), but these areas are mostly devoid of blood vessels (C). LyP-1-abraxane exhibits increased accumulation in extravascular tissue compared to FAM-abraxane (D, E, F). Blood vessels were stained with anti-CD31 and lymphatic vessels with anti-podoplanin. Nuclei were counterstained with DAPI. The results are representative of three independent experiments. Magnification: 600x (A & B), 200x (C, D & E). Quantification of fluorescence (F) in tumor micrographs (FAM-abraxane, CREKA-abraxane and LyP-1-abraxane) and liver micrographs (LyP-1-abraxane) was perfomed using Image J (NIH, USA). Five random fields were quantified per tumor and liver for LyP-1-abraxane (n=3), four random fields per tumor for CREKA-abraxane (n=3) and five random fields per tumor for FAM-abraxane (n=3). Error bars represent S.E.M.

Figure 3. Rapid extravasation of LyP-1 phage

Nude mice with MDA-MB-435 tumors were intravenously injected with 100 ml of 2×10¹⁰ plaque forming units (p.f.u.) of T7 phage displaying 415 copies of LyP-1 peptide (Cys-Gly-Gln-Lys-Arg-Thr-Arg-Gly-Cys) (A and B) or CG7C peptide (Cys-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Cys) (C) on the phage capsid protein. The mice were sacrificed 15 minutes post-injection by perfusion with PBS and tissues were collected for histology. The T7 phage was detected by immunostaining with polyclonal rabbit anti-T7. Blood vessels were stained with anti-CD31 and nuclei were counterstained with DAPI. The images are representative of 2 independent experiments. Scale bar: 50 μm

Figure 4. Peptide- targeted micelles are delivered intact to tumor tissue

Nude mice bearing MDA-MB-435 tumors (~0.5 cm³ in diameter) were intravenously injected with 100 μl of 1 mM solution of DSPE-PEG₂₀₀₀-FAM-CREKA micelles (A), DSPE-PEG₂₀₀₀-FAM-LyP-1 micelles (B), DSPE-PEG₂₀₀₀-FAM micelles (C), mixed CREKA micelles, (D) or mixed LyP-1 micelles (E). (The mixed micelles were prepared from DSPE-PEG₂₀₀₀-CREKA (unlabeled) and DSPEPEG₂₀₀₀-FAM or DSPE-PEG₂₀₀₀-LyP-1 (unlabeled) and DSPE-PEG₂₀₀₀-FAM). The mice were sacrificed 3 hours post-injection by perfusion through heart with PBS and tissues were collected for histology. Blood vessels were visualized by staining with anti-CD31 (red). Nuclei were counterstained with DAPI (blue). The images are representative of 3 experiments. Magnification: 200x (A, B, C, E). Scale bar: 50 μm (D).

Figure 5. Effect on tumor growth by LyP-1-abraxane conjugate

Nude mice bearing MDA-MB-435 xenograft tumors were treated with LyP-1-conjugated abraxane, and unmodified abraxane, free LyP-1 peptide, or saline four times a week for 3 weeks (at a paclitaxel equivalent of 3 mg/kg/day for LyP-1-abraxane and unmodified abraxane). The total cumulative dose was 30 mg/kg. LyP-1 peptide was used at a dose equivalent to what was injected on the particles. There were 10 mice per group and the treatment started when the mean tumor volume for each group was about 100 mm³. Two independent experiments were performed and gave similar results; one is shown here. LyP-1-abraxane was significantly more effective in inhibiting tumor growth than unmodified abraxane (p = 0.013), LyP-1 alone (p < 0.01) and saline (p< 0.01). Error bars represent S.E.M.