Purpose: The purpose of this study was to achieve improved cancer-specific delivery and bioavailability of radiation-sensitizing chemotherapy using radiation-guided drug delivery.

Experimental Design: Phage display technology was used to isolate a recombinant peptide (HVGGSSV) that binds to a radiation-inducible receptor in irradiated tumors. This peptide was used to target nab-paclitaxel to irradiated tumors, achieving tumor-specificity and enhanced bioavailability of paclitaxel.

Results: Optical imaging studies showed that HVGGSSV-guided nab-paclitaxel selectively targeted irradiated tumors and showed 1.48 ± 1.66 photons/s/cm2/sr greater radiance compared with SGVSGHV-nab-paclitaxel, and 1.49 ± 1.36 photons/s/cm2/sr greater than nab-paclitaxel alone (P < 0.05). Biodistribution studies showed >5-fold increase in paclitaxel levels within irradiated tumors in HVGGSSV-nab-paclitaxel–treated groups as compared with either nab-paclitaxel or SGVSGHV-nab-paclitaxel at 72 hours. Both Lewis lung carcinoma and H460 lung carcinoma murine models showed significant tumor growth delay for HVGGSSV-nab-paclitaxel as compared with nab-paclitaxel, SGVSGHV-nab-paclitaxel,and saline controls. HVGGSSV-nab-paclitaxel treatment induced a significantly greater loss in vasculature in irradiated tumors compared with unirradiated tumors, nab-paclitaxel, SGVSGHV-nab-paclitaxel, and untreated controls.

Conclusions: HVGGSSV-nab-paclitaxel was found to bind specifically to the tax-interacting protein-1 (TIP-1) receptor expressed in irradiated tumors, enhance bioavailability of paclitaxel, and significantly increase tumor growth delay as compared with controls in mouse models of lung cancer. Here we show that targeting nab-paclitaxel to radiation-inducible TIP-1 results in increased tumor-specific drug delivery and enhanced biological efficacy in the treatment of cancer. Clin Cancer Res; 16(20); 4968–77. ©2010 AACR.

Translational Relevance

Ionizing radiation can be used to induce the expression of cell surface molecules unique to cancer. Phage-displayed peptide libraries can be used to discover peptides that bind specifically to irradiated cancers. Here, we show that the amino acid sequence HVGGSSV achieved tumor-specific binding when conjugated to nanoparticles containing radiation-sensitizing paclitaxel. Radiation-guided delivery of nanoparticles improved both the bioavailability of drug delivery and the efficacy of radiotherapy in mouse models of lung cancer. Further studies are necessary to determine whether improved cancer-specific delivery of radiation-sensitizing chemotherapy translates to reduced side effects and better treatment outcomes in lung cancer patients.

Drug delivery systems have been developed to increase drug delivery to cancer and thereby enhance therapeutic response (13). Examples of drug delivery systems include liposomal doxorubicin and nanoparticle albumin-bound paclitaxel (nab-paclitaxel; ref. 4), which are not ligand-targeted systems and therefore not tumor specific (57). Nab-paclitaxel has features that make it an appropriate vehicle for drug encapsulation (810). It is a natural carrier of hydrophobic molecules such as paclitaxel, and has noncovalent binding characteristics (11). This allows paclitaxel to bind reversibly to albumin. Nab-paclitaxel binds to the albumin receptor gp60, which is ubiquitously present throughout tissues (1214), and therefore does not reduce the incidence of complications (1517).

Physical energy has been used to achieve site-specific drug delivery to cancer. For example, heat is used to release drugs from liposomes and nanocages. These technologies are complemented by use of radiation-guided peptides conjugated to nab-paclitaxel. Ligands that can specifically target receptors within tumor microvasculature have been previously investigated (3, 18). Radiation can be used to achieve site-specific expression of receptors within cancer (1924). These radiation-inducible receptors can in turn be targeted by peptides selected through phage display technology (24, 25). Nanoparticle carriers can be functionalized with these peptide ligands to enable radiation-guided delivery of chemotherapeutic drugs to tumor microvasculature (1926). This tumor-specific delivery of chemotherapy has the potential to improve treatment tolerability by reducing nonspecific delivery of cytotoxic drugs to normal tissues and improve bioavailability of chemotherapy to cancer.

We studied nab-paclitaxel as a scaffold for creating a radiation-guided drug delivery system. To increase tumor-specific delivery of paclitaxel and enhance tumor bioavailability, we functionalized nab-paclitaxel with a radiation-guided peptide (HVGGSSV) that specifically targets microvasculature within irradiated tumors. In this study, we focused on non–small cell lung cancer because concomitant chemotherapy and radiation therapy improves survival (2731). By using radiation-guided peptides conjugated to nab-paclitaxel, we retargeted nab-paclitaxel from the nonspecific albumin receptor gp60 to a radiation-inducible receptor. This approach improved tumor-specific delivery of nab-paclitaxel, enhanced bioavailability within tumors, and enhanced therapeutic efficacy in the treatment of mouse models of lung cancer.

Cell culture and reagents

The tumor cell lines used were murine Lewis lung carcinoma (LLC) and NCI-H460 human large cell lung carcinoma, obtained from the American Type Culture Collection. Nab-paclitaxel was supplied by American Bioscience, Inc. The compound was dissolved in 0.9% NaCl solution to a concentration of 5 mg/mL and administered i.v. in a concentration of 10 mg/kg (paclitaxel). In all experiments, nab-paclitaxel was given once as a single tail vein injection without premedication.

Conjugation chemistry

Nab-paclitaxel (purchased from Abraxis) was conjugated to a heterobifunctional cross-linker, succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol] ester (Pierce Biotechnology), producing a maleimide functionalized nab-paclitaxel surface. The cysteine thiol group of CGGGKKKGGGNHVGGSSV was reacted with the maleimide functionalized surface to produce HVGGSSV-labeled nab-paclitaxel. A scrambled version of HVGGSSV peptide (CGGGKKKGGGSGVSGHVN) was used as a control and conjugated to nab-paclitaxel in the same manner. For in vivo near-IR (NIR) imaging, the above conjugates were labeled with NIR fluorescent probe Alexa fluor 750. Monofunctional N-hydroxysuccinamide esters of Alexa fluor 750 were conjugated to the lysine ϵ-amino groups on the peptide for NIR fluorescence imaging. For conjugation, 1 mg Alexa fluor 750 was dissolved in 100 uL of DMSO (Sigma) and added to HVGGSSV-modified nab-paclitaxel for 1 hour in a PBS buffer (pH 7).

Animal models

Animal studies were done according to a protocol approved by Vanderbilt's Institutional Animal Care and Use Committee (IACUC). Male athymic nude mice (nu/nu) between four and six weeks old (Harlan Inc.) or C57BL6 male mice were anesthetized using a ketamine and xylazine solution before being injected s.c. in the hind legs with 1 × 106 LLC or H460 cells suspended in 100 μL sterile PBS. One week after inoculation, the tumors reached an approximate size of 0.5 to 0.8 cm in diameter, and the mice were used for studies.

Radiation and treatment protocol

Tumors were allowed to reach 0.5 to 0.8 cm3 in size before beginning treatments. All mice were anesthetized using ketamine and xylazine solution prior to irradiation to inhibit mobility during treatment. Tumors were irradiated with 300 kV X-rays using a Pantak Therapax 3 linear accelerator system (Pantak) with an adjustable collimator set to limit dosage to the tumor region only. During irradiation procedures, 1-cm-thick lead blocks were arranged above the rest of the body, leaving only the desired area on the hind limb exposed for treatment. Four hours following radiation, mice were administered drug treatments through tail vein injection.

Imaging and image analysis

In vivo NIR imaging was done with a Xenogen IVIS 200 small animal imaging system (Xenogen Inc.) with a Cy7 filter set (excitation at 680 nm and emission at 775 nm). Nude mice bearing LLC or H460 lung carcinoma tumors implanted in both hind limbs were treated with radiation. The tumor on the left side of each mouse received a radiation dose of 3 Gy, and the tumor on the right side received no radiation (sham radiation dose of 0 Gy) and served as an internal negative control. Four hours following irradiation, mice were anesthetized using an i.p. injection of ketamine and xylazine and prepared for tail vein injections of the treatment conjugates. At 72 hours postinjection, all mice were anesthetized with isoflurane and imaged with the Xenogen IVIS. All NIR images were acquired with one-second exposure time using an f/stop of 2. For quantitative comparison, regions of interest were drawn over tumors, and the total radiance (p/s/cm2/sr) for each area was measured. Results are presented as mean and SE for a group of three to six animals. Values for treated groups were compared with controls with the unpaired Student's t test.

Biodistribution analysis

Six- to eight-week-old male C57BL6 mice with s.c. LLC murine lung carcinoma tumors were injected i.v. with 10mg/kg (paclitaxel) nab-paclitaxel, targeted HVGGSSV-nab-paclitaxel, or SGVSGHV-nab-paclitaxel. Mice were sacrificed at 12 and 72 hours after administration, vital organs were excised, and blood samples were collected by cardiac puncture. Tissues were immediately homogenized and kept on ice. Each sample was spiked with an internal standard of docetaxel, followed by solid-phase extraction with acetonitrile, and centrifuged at 3,000 rpm (32). Supernatant was removed and analyzed for paclitaxel content using high performance liquid chromatography/tandem mass spectrometry. Results are presented as mean and SE for a group of three animals.

Subcutaneous tumor models

Six- to eight-week-old male athymic nude mice or C57BL6 mice were injected heterotopically with either 1 × 106 H460 human lung carcinoma cells or 106 murine LLC cells, respectively. Mice were monitored daily and tumor volume was measured manually with a caliper, using the formula: volume = length × width × height/2, derived from the formula for an ellipsoid. When tumors had reached the desired size (5-6 mm diameter), the mice were grouped (n = 5) and injected i.v. with PBS, nab-paclitaxel, targeted HVGGSSV-nab-paclitaxel, or SGVSGHV-nab-paclitaxel at a dose of 10 mg/kg of paclitaxel. All mice were grouped as follows: saline only, radiation only (3Gy × 3), nab-paclitaxel(3 Gy), nab-paclitaxel with radiation (3 Gy × 3), targeted HVGGSSV-nab-paclitaxel (3 Gy), targeted HVGGSSV-nab-paclitaxel with radiation (3 Gy × 3), SGVSGHV-nab-paclitaxel (3 Gy), and SGVSGHV-nab-paclitaxel with radiation (3 Gy × 3). Radiation treatment was administered every other day (33). Data were calculated as fold increase from the original tumor volume, with variance analyzed by the Kruskal-Wallis method (34).

Anti–tax-interacting protein 1 monoclonal antibody production and purification

Mouse.

Tax-interacting protein-1 (TIP-1) was produced in bacteria and purified using glutathione S-transferase columns. BALB/c mice were initially immunized with 50 mg of TIP-1 antigen mixed with equivalent amounts of Titermax adjuvant (CytRx Corporation) for each mouse. One month after initial immunization, mice were boosted with equivalent amounts of antigen without adjuvant two to three times at two-week intervals. The mouse anti-TIP-1 polyclonal antibody titer was evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blot methods. Mice exhibiting high immune response to TIP-1 antigen were chosen as B-cell donors. Spleen were removed and homogenized in RPMI 1640 culture medium free of serum and other additives. Spleen cells were combined with Sp2/O mouse meyloma (2 × 10 7 per spleen). Mixed cells were washed twice and centrifuged at 1,200 rpm for 8 minutes at room temperature. Supernatant was removed, and the cell pellet was lightly agitated to loosen the cells. Approximately 1 mL PEG (polyethylene glycol 1500; Roche) was added to fuse the cells. The fused cells were washed once with plain medium and finally resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gemini Bioproducts), L-glutamine, antibiotics, and hypoxanthine-aminopterin-thymidine (Sigma), plated into 24-well tissue culture plates, and incubated at 37°C in a humidified CO2 incubator. Fifteen days after fusion, hybridoma culture supernatants were removed from individual wells and transferred to separate 96-well microtiter plates for ELISA, Western blot, and antibody printing assays against TIP-1 antigen. Hybridomas that produced antibodies positive by immunoassay were chosen for subcloning by using limiting dilution. Resulting single-cell clones were retested by the aforementioned methods to detect antigen-positive monoclonal antibody–producing hybridomas. Positive hybridoma clones were transferred to individual flasks to expand cell number from one cell clone, and incubated with serum-free medium for antibody production. The antibodies produced by positive clones in serum-free medium were harvested twice a week for further monoclonal antibody purification. Filtered monoclonal antibody collected from serum-free medium was purified by protein A and protein G columns. The concentrated purified monoclonal antibody was assayed and stored at −20°C.

Rabbit and guinea pig.

TIP-1 protein was synthesized in bacteria as above. Rabbits (New Zealand white) or guinea pigs were initially immunized with 100 mg of TIP-1 antigen mixed with equivalent amounts of Titermax adjuvant (CytRx Corporation). One month after initial immunization, the animals were boosted with equivalent amounts of antigen two or three times at two-week intervals without adjuvant. The animals were sacrificed and blood was collected. Serum was separated individually when antibody titer reached desired titer, and was evaluated by ELISA and Western blot assays. Rabbit whole IgG in serum was purified through protein A and protein G columns, and guinea pig total IgG in serum was harvested through protein A columns. Anti-TIP-1 IgG was purified and sequentially passed on TIP-1 conjugated to cyanogens activated in sepharose 4B beads (Sigma). The antibody was dialyzed against PBS and then concentrated. Antibody concentration was tested by the Bradford protein assay method. The purified anti-TIP-1 IgG was stored at −20°C.

Immunohistochemistry

LLC murine lung tumors were implanted into the hind limbs of C57BL6 mice and grown to a diameter of approximately 8 to 10 mm. Tumors were irradiated with 0 Gy or 3 Gy, and administered either drug or antibody treatments approximately 5 hours after irradiation. For TIP-1 staining, formaldehyde-fixed paraffin-embedded sections were treated with TIP-1 primary antibody and antirabbit secondary antibody (Sigma), and counterstained with hematoxylin.

For colocalization study, mice received Alexa fluor 594–labeled HVGGSSV-nab-paclitaxel, SGVSGHV-nab-paclitaxel, nab-paclitaxel, or no treatment. Mice were sacrificed and tumors excised at 3 hours. Tissues were frozen in optimum cutting temperature embedding medium followed by liquid nitrogen. Cryosections of 5 μm thickness were immunostained using FITC-labeled rabbit anti–von Willebrand factor (vWF) antibody (Dako). Images were captured on a Zeiss Axiophot widefield microscope using oil immersion at ×60.

For paclitaxel staining, mice received HVGGSSV-nab-paclitaxel, SGVSGHV-nab-paclitaxel, nab-paclitaxel, or no treatment. Mice were sacrificed and tumors were excised approximately 12 to 16 hours. For detection of drug presence in tissues, a mouse anti-paclitaxel monoclonal antibody (Santa Cruz) was used (35).

For apoptosis detection, mice received HVGGSSV-nab-paclitaxel, SGVSGHV-nab-paclitaxel, nab-paclitaxel, or no treatment. Mice were sacrificed and tumors excised at 72 hours, fixed in 4% paraformaldehyde overnight, and embedded in paraffin. Sections of 8 μm thickness were immunostained for cleaved caspase-3 using appropriate primary and secondary antibodies. Vasculature was immunostained using rabbit anti-vWF antibody (Dako) and detected using 3,3′-diaminobenzidine method. Paraffin sections were counterstained with hematoxylin. Light microscopy images captured on a Zeiss Axiophot widefield microscope at ×10.

Western blotting

Tissue samples were homogenized and lysed in sample buffer, and membrane proteins were extracted using a mammalian membrane protein extraction kit (Mem-PER Eukaryotic Membrane protein extraction kit, Pierce) prior to being coprecipitated with the HVGGSSV peptide-biotin on streptavidin. Precipitated protein was then resolved on a 5% to 10% gradient SDS-PAGE. The proteins were then transferred to a polyvinylidene difluoride membrane, blocked, and probed with an anti-TIP-1 antibody and appropriate secondary antibody.

Radiation-targeted peptides

Phage-displayed peptide libraries were used to identify amino acid sequences that bind specifically to irradiated cancers. The peptide ligand HVGGSSV bound within irradiated lung cancers. Coprecipitation of the HVGGSSV peptide revealed a putative receptor, TIP-1, that binds HVGGSSV. Membrane protein Western blots showed a significant increase in the expression of TIP-1 protein at 4 and 24 hours following irradiation with 3 Gy as compared with 0 Gy untreated control tumors (Fig. 1A). Immunohistochemical staining showed significant levels of the TIP-1 membrane protein present in irradiated tumors, but not in untreated controls (Fig. 1B). NIR imaging studies showed significant targeting and binding of rabbit anti-TIP-1 IgG polyclonal antibody to irradiated tumors compared with untreated tumors and rabbit IgG controls at 72 hours (Fig. 1C). Further studies done using guinea pig and mouse anti-TIP-1 IgG antibodies validated binding within irradiated tumors (Fig. 1C). Results from NIR imaging studies showed that the radiance from anti-TIP-1 antibodies was 1.93 ± 2.04 photons/s/cm2/sr greater compared with control IgG antibodies in irradiated tumors (P ≤ 0.08; Fig. 1D).

Fig. 1.

TIP-1 receptor targeting studies. A, autoradiograph of TIP-1 protein at 1, 4, and 24 hours after irradiation with 3 Gy compared with TIP-1 protein in untreated controls (0 Gy). Proteins were coprecipitated with the HVGGSSV peptide-biotin on streptavidin. Protein was separated by PAGE and transfers were incubated with polyclonal antibody to TIP-1. B, immunohistochemical sections of untreated control and irradiated tumor. Tumor sections were stained with anti-TIP-1 antibody. C, NIR fluorescence images acquired approximately 72 hours postinjection of mice: intravenous injection of Alexa fluor 750–labeled rabbit IgG antibody (control) and rabbit anti-TIP-1 polyclonal antibody, guinea pig IgG antibody (control) and guinea pig anti-TIP-1 polyclonal antibody, and mouse IgG antibody (control) and mouse anti-TIP-1 monoclonal antibody. LLC tumors grown in both hind limbs were treated with 3 Gy (left hind limb) or 0 Gy (right hind limb). D, bar graph of radiance for anti-TIP-1 antibodies versus control IgG in irradiated tumors. Bars, mean and SE for 6 to 8 animals in each group. Unpaired Student's t test (P ≤ 0.08).

Fig. 1.

TIP-1 receptor targeting studies. A, autoradiograph of TIP-1 protein at 1, 4, and 24 hours after irradiation with 3 Gy compared with TIP-1 protein in untreated controls (0 Gy). Proteins were coprecipitated with the HVGGSSV peptide-biotin on streptavidin. Protein was separated by PAGE and transfers were incubated with polyclonal antibody to TIP-1. B, immunohistochemical sections of untreated control and irradiated tumor. Tumor sections were stained with anti-TIP-1 antibody. C, NIR fluorescence images acquired approximately 72 hours postinjection of mice: intravenous injection of Alexa fluor 750–labeled rabbit IgG antibody (control) and rabbit anti-TIP-1 polyclonal antibody, guinea pig IgG antibody (control) and guinea pig anti-TIP-1 polyclonal antibody, and mouse IgG antibody (control) and mouse anti-TIP-1 monoclonal antibody. LLC tumors grown in both hind limbs were treated with 3 Gy (left hind limb) or 0 Gy (right hind limb). D, bar graph of radiance for anti-TIP-1 antibodies versus control IgG in irradiated tumors. Bars, mean and SE for 6 to 8 animals in each group. Unpaired Student's t test (P ≤ 0.08).

Close modal

The purpose of this study was to determine whether targeting nab-paclitaxel to radiation-inducible receptors improved tumor-specific drug delivery. To evaluate the tumor targeting ability of radiation-guided nab-paclitaxel, we studied in vivo NIR fluorescence imaging of the biodistribution in mice with heterotopic lung cancer tumors. We conjugated the HVGGSSV peptide to nab-paclitaxel via a bifunctional polyethylene glycol linker. The HVGGSSV peptide was then labeled with a fluorescent probe (Alexa fluor 750) for NIR fluorescence imaging studies. A scrambled sequence peptide (SGVSGHV) was used as a negative control. Nab-paclitaxel particles with no peptide conjugation were also fluorescently labeled and used as controls. Nude mice bearing s.c. LLC tumors were treated with 3 Gy or 0 Gy radiation and administered HVGGSSV-nab-paclitaxel, SGVSGHV-nab-paclitaxel, or nab-paclitaxel. Images were acquired at 24, 48, and 72 hours postinjection. NIR images taken at 72 hours after nab administration showed binding in irradiated tumors treated with HVGGSSV-nab-paclitaxel, whereas tumors treated with SGVSGHV-nab-paclitaxel or nab-paclitaxel alone showed minimal radiance (Fig. 2A). Untreated (0 Gy) control tumors showed similar levels of radiance across all treatment groups (Fig. 2A). Irradiated tumors treated with HVGGSSV-nab-paclitaxel showed 1.48 ± 1.66 photons/s/cm2/sr greater radiance compared with SGVSGHV-nab-paclitaxel, and 1.49 ± 1.36 photons/s/cm2/sr greater than nab-paclitaxel alone (P < 0.05; Fig. 2B). We found no significant difference in radiance from tumors treated with 2 Gy compared with 3 Gy during imaging of HVGGSSV-nab-paclitaxel (data not shown). To determine whether HVGGSSV binds to TIP-1 in tumor microvasculature, antibody blocking studies were done. NIR imaging showed that preblocking the TIP-1 receptor by administration of rabbit anti-TIP-1 IgG polyclonal antibody, followed by injection of HVGGSSV-nab-paclitaxel produced a marked decrease in radiance in irradiated tumors compared with control IgG antibody (Fig. 2C). Blocking of TIP-1 resulted in a 47.6-fold drop in binding as compared with control IgG (Fig. 2D).

Fig. 2.

HVGGSSV-nab-paclitaxel targeted to irradiated tumors. A, NIR images acquired 72 hours postinjection of mice after i.v. injection of Alexa fluor 750–labeled HVGGSSV-nab-paclitaxel, SGVSGHV-nab-paclitaxel, and unconjugated nab-paclitaxel. LLC tumors grown in both hind limbs were treated with 3 Gy (left hind limb) or 0 Gy (right hind limb). All images are normalized to the same scale. Radiance was measured for both irradiated (3 Gy) and untreated (0 Gy) tumors. Color scale bar, radiance in units of photons/s/cm2/sr. B, bar graph of radiance for all treatment groups. Bars, mean and SE for five animals in each group. Unpaired Student's t test (P < 0.01). C, NIR images of TIP-1–blocked and unblocked (control) mice. To determine whether HVGGSSV specifically binds to TIP-1 in tumor microvasculature, TIP-1 blocking studies were done using the same tumor model in nude mice and NIR imaging. Tumors on the left hind limb were irradiated with 3 Gy, and mice were given 50 μg of rabbit anti-TIP-1 polyclonal antibody i.v. 4 hours later. Alexa fluor 750–labeled HVGGSSV-nab-paclitaxel was injected 2 hours after antibody administration. NIR imaging showed preblocking the TIP-1 receptor by administration of rabbit anti-TIP-1 IgG polyclonal antibody, followed by injection of HVGGSSV-nab-paclitaxel. D, bar graph of radiance for both treatment groups, with mean and SEM for three animals in each group. Radiance was measured for both TIP-1–blocked and unblocked tumors as compared with preblocking with control IgG. Unpaired Student's t test (P < 0.01).

Fig. 2.

HVGGSSV-nab-paclitaxel targeted to irradiated tumors. A, NIR images acquired 72 hours postinjection of mice after i.v. injection of Alexa fluor 750–labeled HVGGSSV-nab-paclitaxel, SGVSGHV-nab-paclitaxel, and unconjugated nab-paclitaxel. LLC tumors grown in both hind limbs were treated with 3 Gy (left hind limb) or 0 Gy (right hind limb). All images are normalized to the same scale. Radiance was measured for both irradiated (3 Gy) and untreated (0 Gy) tumors. Color scale bar, radiance in units of photons/s/cm2/sr. B, bar graph of radiance for all treatment groups. Bars, mean and SE for five animals in each group. Unpaired Student's t test (P < 0.01). C, NIR images of TIP-1–blocked and unblocked (control) mice. To determine whether HVGGSSV specifically binds to TIP-1 in tumor microvasculature, TIP-1 blocking studies were done using the same tumor model in nude mice and NIR imaging. Tumors on the left hind limb were irradiated with 3 Gy, and mice were given 50 μg of rabbit anti-TIP-1 polyclonal antibody i.v. 4 hours later. Alexa fluor 750–labeled HVGGSSV-nab-paclitaxel was injected 2 hours after antibody administration. NIR imaging showed preblocking the TIP-1 receptor by administration of rabbit anti-TIP-1 IgG polyclonal antibody, followed by injection of HVGGSSV-nab-paclitaxel. D, bar graph of radiance for both treatment groups, with mean and SEM for three animals in each group. Radiance was measured for both TIP-1–blocked and unblocked tumors as compared with preblocking with control IgG. Unpaired Student's t test (P < 0.01).

Close modal

Targeting nab-paclitaxel to irradiated cancer improves biodistribution

To determine whether HVGGSSV-nab-paclitaxel binds within tumor microvasculature, we labeled HVGGSSV-nab-paclitaxel, SGVSGHV-nab-paclitaxel, and nab-paclitaxel with fluorescent probe Alexa fluor 594 prior to injection. Approximately 3 hours after treatment, mice were sacrificed, and tumors were excised and cryopreserved for fluorescence microscopy. Tissue slices were fluorescently stained for vWF, an endothelial cell marker. Figure 3 shows strong colocalization of targeted HVGGSSV-nab-paclitaxel with vascular endothelium in irradiated tumors (Fig. 3A), but not in unirradiated tumors (Fig. 3B) or TIP-1–blocked tumors. Scrambled SGVSGHV-nab-paclitaxel did not show significant colocalization in either irradiated or unirradiated tumors. Some colocalization was observed with nab-paclitaxel treatment in both irradiated and unirradiated groups. This suggests that HVGGSSV peptide enables binding of nab-paclitaxel within irradiated tumor microvasculature as early as 3 hours after irradiation.

Fig. 3.

Colocalization HVGGSSV-nab-paclitaxel with tumor vascular endothelium. Cryosections of LLC tumors from each treatment group were stained for vascular marker vWF (green) 3 hours after treatment with 3 Gy (A) and 0 Gy (B). HVGGSSV-nab-paclitaxel, SGVSGHV-nab-paclitaxel, and nab-paclitaxel were labeled with Alexa fluor 594 (red) prior to injection.

Fig. 3.

Colocalization HVGGSSV-nab-paclitaxel with tumor vascular endothelium. Cryosections of LLC tumors from each treatment group were stained for vascular marker vWF (green) 3 hours after treatment with 3 Gy (A) and 0 Gy (B). HVGGSSV-nab-paclitaxel, SGVSGHV-nab-paclitaxel, and nab-paclitaxel were labeled with Alexa fluor 594 (red) prior to injection.

Close modal

The ability of targeted HVGGSSV-nab-paclitaxel to localize and bind specifically to irradiated tumors was examined by biodistribution analysis in mice bearing LLC murine lung carcinoma. Paclitaxel levels were quantified in tumors, blood, and organs 72 hours following i.v. injection of either HVGGSSV-nab-paclitaxel, SGVSGHV-nab-paclitaxel, or nab-paclitaxel. The results of this biodistribution analysis show that significantly higher levels of paclitaxel accumulated in irradiated tumors treated with HVGGSSV-nab-paclitaxel compared with SGVSGHV-nab-paclitaxel or nab-paclitaxel, and untreated control tumors (0 Gy) after 72 hours. Figure 4A shows >5-fold increase in paclitaxel levels within irradiated tumors in HVGGSSV-nab-paclitaxel–treated groups as compared with either nab-paclitaxel or SGVSGHV-nab-paclitaxel at 72 hours. No significant difference in paclitaxel levels were observed in any unirradiated tumors. Biodistribution in organs showed similar paclitaxel levels distributed among heart, lungs, kidneys, liver, and brain, with slightly less in the lungs and more in the brain. Figure 4B shows tumor to plasma ratios for HVGGSSV-nab-paclitaxel, SGVSGHV-nab-paclitaxel, and nab-paclitaxel at 72 hours posttreatment. HVGGSSV nab-paclitaxel had a tumor/plasma ratio approximately 4-fold higher in irradiated tumors than in unirradiated tumors, and 4-fold higher as compared with SGVSGHV-nab-paclitaxel. No significant difference was observed between irradiated and unirradiated SGVSGHV-nab-paclitaxel–treated groups. A slight increase in tumor/plasma ratio was observed in nab-paclitaxel following tumor irradiation as compared with no irradiation. HVGGSSV-nab-paclitaxel had a tumor/plasma ratio approximately 3-fold higher than nab-paclitaxel in irradiated tumors.

Fig. 4.

Biodistribution of targeted HVGGSSV-nab-paclitaxel. A, biodistribution of paclitaxel in tumor and tissues in each treatment group compared with nab-paclitaxel. Bars, mean and SE from three animals in each group. B, tumor/plasma concentration ratios at 72 hours postinjection. Bars, mean and SE from three animals in each group. C, paraffin sections immunohistochemically stained for paclitaxel presence (brown), and counterstained with hematoxylin (blue). Nuclei stained brown were scored as positive and nuclei that stained blue were scored as negative. Scale bar, 50 μm.

Fig. 4.

Biodistribution of targeted HVGGSSV-nab-paclitaxel. A, biodistribution of paclitaxel in tumor and tissues in each treatment group compared with nab-paclitaxel. Bars, mean and SE from three animals in each group. B, tumor/plasma concentration ratios at 72 hours postinjection. Bars, mean and SE from three animals in each group. C, paraffin sections immunohistochemically stained for paclitaxel presence (brown), and counterstained with hematoxylin (blue). Nuclei stained brown were scored as positive and nuclei that stained blue were scored as negative. Scale bar, 50 μm.

Close modal

Figure 4C shows immunohistochemical staining of paclitaxel in tumor tissues at 12 hours posttreatment. Greater paclitaxel staining was observed in irradiated HVGGSSV-nab-paclitaxel–treated tumors compared with unirradiated tumors, as well as SGVSGHV-nab-paclitaxel controls. Treatment with nab-paclitaxel resulted in similar paclitaxel staining between irradiated and unirradiated tumors. Irradiated tumors treated with HVGGSSV-nab-paclitaxel showed comparable levels of paclitaxel staining compared with nab-paclitaxel-treated tumors. Targeting HVGGSSV-nab-paclitaxel to irradiated tumors increased the amount of paclitaxel delivered to tumors, and simultaneously decreased the amount of paclitaxel within other organs and tissues.

Targeted nab-paclitaxel enhances therapeutic efficacy

The therapeutic efficacy of HVGGSSV-guided nab-paclitaxel was studied in two tumor models: murine LLC and NCI-H460 human large cell lung carcinoma grafted s.c. in C57 and athymic nude mice, respectively. On day 7 after tumor cell implantation, the mice were injected with 10 mg/kg i.v. of either HVGGSSV-nab-paclitaxel, SGVSGHV-nab-paclitaxel, nab-paclitaxel, or saline. Tumor volumes for each treatment group were measured manually with calipers until they reached a 4-fold increase in volume. Figure 5A and B show that radiation alone (9 Gy) achieved only a slight tumor growth delay (2 days) in LLC tumors, and 6 days in H460 tumors as compared with untreated controls. Negative SGVSGHV-nab-paclitaxel controls showed no significant growth delay in LLC tumors and only 2 days' delay in H460. After subsequent irradiation, this was improved to 2 days (LLC) and 6 days (H460). Treatment with nab-paclitaxel alone produced tumor growth delay of 2 days (LLC) and 6 days (H460), and upon additional irradiation increased to 6 days (LLC) and 11 days (H460). Both LLC and H460 lung carcinoma showed significant tumor growth delay for HVGGSSV-nab-paclitaxel as compared with nab-paclitaxel, SGVSGHV-nab-paclitaxel, and saline controls (Fig. 5A and B). HVGGSSV-nab-paclitaxel treatment achieved a growth delay of 3.4 days (LLC) and 8 days (H460). Additional irradiation increased this to 10 days (LLC) and 15 days (H460) over untreated controls. Subsequent doses of radiation improved growth delay for both HVGGSSV-nab-paclitaxel and nab-paclitaxel control. HVGGSSV-nab-paclitaxel combined with irradiation resulted in significantly greater tumor growth delay compared with tumors treated with SGVSGHV-nab-paclitaxel and irradiation or nab-paclitaxel and irradiation (P < 0.01, Kruskal-Wallis).

Fig. 5.

Therapeutic efficacy of targeted HVGGSSV-nab-paclitaxel in LLC and H460 xenografts. In tumor growth delay studies, LLC murine lung carcinoma–bearing C57 mice (A) or H460-bearing nude mice (B) were treated with either 3 Gy or 0 Gy and injected i.v. 5 hours later with either targeted HVGGSSV-nab-paclitaxel, nab-paclitaxel, SGVSGHV-nab-paclitaxel, or saline. Graphs, fold volume increase with mean and SE from five animals in each group; arrows, daily irradiation (RT) with 3 Gy every other day. HVGGSSV-nab-paclitaxel combined with irradiation resulted in significantly greater tumor growth delay compared with tumors treated with SGVSGHV-nab-paclitaxel and irradiation or nab-paclitaxel and irradiation (P < 0.01, Kruskal-Wallis). C and D, immunohistochemical stains of LLC tumor sections from each treatment group taken at 72 hours after treatment initiation. Tissue slices were probed for active caspase-3 to identify cell death (C), and endothelial cell marker vWF for vascular endothelium (D). All sections were counterstained with hematoxylin. Brown stained nuclei were scored as positive and nuclei that stained blue were scored as negative. Paraffin sections scale bar, 50 μm.

Fig. 5.

Therapeutic efficacy of targeted HVGGSSV-nab-paclitaxel in LLC and H460 xenografts. In tumor growth delay studies, LLC murine lung carcinoma–bearing C57 mice (A) or H460-bearing nude mice (B) were treated with either 3 Gy or 0 Gy and injected i.v. 5 hours later with either targeted HVGGSSV-nab-paclitaxel, nab-paclitaxel, SGVSGHV-nab-paclitaxel, or saline. Graphs, fold volume increase with mean and SE from five animals in each group; arrows, daily irradiation (RT) with 3 Gy every other day. HVGGSSV-nab-paclitaxel combined with irradiation resulted in significantly greater tumor growth delay compared with tumors treated with SGVSGHV-nab-paclitaxel and irradiation or nab-paclitaxel and irradiation (P < 0.01, Kruskal-Wallis). C and D, immunohistochemical stains of LLC tumor sections from each treatment group taken at 72 hours after treatment initiation. Tissue slices were probed for active caspase-3 to identify cell death (C), and endothelial cell marker vWF for vascular endothelium (D). All sections were counterstained with hematoxylin. Brown stained nuclei were scored as positive and nuclei that stained blue were scored as negative. Paraffin sections scale bar, 50 μm.

Close modal

To evaluate the mechanism of cell death, tumor sections were immunostained for caspase-3 expression, an apop tosis marker. As shown in Fig. 5C, HVGGSSV-nab-paclitaxel treatment induced significant apoptosis in the irradiated tumors but not in the unirradiated tumors. Treatment with nab-paclitaxel alone produced greater apoptosis in irradiated tumors compared with unirradiated tumors. Results for HVGGSSV-nab-paclitaxel and nab-paclitaxel were similar for both irradiated groups, as compared with SGVSGHV-nab-paclitaxel and negative controls. Tumor sections were stained for the endothelial cell marker vWF to evaluate tumor vascularity. As shown in Fig. 5D, HVGGSSV-nab-paclitaxel treatment induced a significantly greater loss in vasculature in irradiated tumors compared with unirradiated tumors, nab-paclitaxel, SGVSGHV-nab-paclitaxel, and untreated controls. Treatment with nab-paclitaxel decreased vascular density compared with negative controls, but did not show a significant decrease between irradiated and unirradiated tumors. There was no difference in the vessel density between the groups treated with SGVSGHV-nab-paclitaxel and untreated controls.

Cancer-selective therapy using various targeted strategies includes antibodies, peptides, aptamers, and other targeting moieties that bind to receptors or antigens that are specific to cancer (1, 3, 6, 7, 3639). Radiation can be used to induce the expression of receptors within tumor microvasculature (1922). Tumor targeting peptides were developed to bind to these radiation-inducible receptors. These peptides can then be modified with nanoparticles containing cytotoxic drugs to create radiation-guided drug delivery systems. Previously, the GIRLRG peptide was used to target the GRP78 receptors in breast and brain tumors, but not lung cancer models (22). In that study, the GRP78 ligand was conjugated to a non-Good Manufacturing Practice nanoparticle preparation, poly(valerolactoneepoxyvalerolactone-allylvalerolactone-oxepanedione), containing 11% epoxide and cross-linked with 1 equivalent of 2,2-(ethylenedioxy) bis (ethylamine) per epoxide. The current study focuses on the use of the HVGGSSV peptide for targeting the TIP-1 receptor expressed in non–small cell lung cancer models for cancer specific delivery of Abraxane (GMP-ready; ref. 24). The advantage in the present approach is that it provides a means to target Abraxane specifically to irradiated lung cancer.

These peptide ligands can be used to functionalize nanoparticle carriers for radiation-guided delivery of chemotherapeutic drugs to tumor microvasculature (1923, 26). This strategy has the potential to increase the therapeutic ratio by delivering chemotherapy specifically to tumors and reducing toxic side effects from nonspecific delivery commonly produced by systemic chemotherapy. In this study, we found that the chemotherapeutic drug nab-paclitaxel can be targeted specifically to tumors using the radiation-guided peptide HVGGSSV. Furthermore, this modified nab-paclitaxel produced a significantly improved biodistribution profile and enhanced tumor bioavailability resulting in greater therapeutic efficacy in the treatment of cancer. The biodistribution of nab-paclitaxel was significantly improved when modified with HVGGSSV. This enhanced delivery of drug to the tumor site also translated to improved tumor growth delay compared with nab-paclitaxel. Accumulation of HVGGSSV-nab-paclitaxel within blood vessels of tumors was shown at 3 hours and continued up to 72 hours.

Concomitant administration of paclitaxel enhances the efficacy of ionizing radiation and improves cure rates in lung cancer (27, 28). In the present study, this enhanced radiation sensitivity combined with diminished vascular density in tumors improved tumor growth delay compared with nab-paclitaxel alone or in combination with radiation. The primary goal of this research was to show preclinical proof of concept. We studied 2 Gy and 3 Gy, because we plan clinical trials in patients receiving 2 Gy or 3 Gy. Doses of 1 Gy and 5 Gy are clinically less meaningful. We found no difference in HVGGSSV binding in tumors treated with 2 Gy compared with 3 Gy (data not shown). Further studies are necessary to determine whether this tumor-selective delivery can translate to reduced side effects and better treatment outcomes in lung cancer patients.

D.E. Hallahan: ownership interest (including patents), GenVec Inc.; consultant/advisory board, Cumberland Pharmaceuticals, Pfizer Pharmaceuticals.

Grant Support: U.S. National Cancer Institute grants R21-CA128456-02, 5R01-CA125757-03, 5R01-CA112385-05 and P50-CA128323 (D.E. Hallahan).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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