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Keywords:

  • indium;
  • angiogenesis;
  • integrin;
  • nanoparticles;
  • Vx2;
  • cancer

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Earlier tumor detection can improve 5-year survival of patients, particularly among those presenting with cancers less than 1 cm in diameter. ανβ3-Targeted 111In nanoparticles (NP) were developed and studied for detection of tumor angiogenesis. Studies were conducted in New Zealand white rabbits implanted 12 days earlier with Vx-2 tumor. ανβ3-Targeted 111In/NP bearing ∼10 111In/NP vs. ∼1 111In/NP nuclide payloads were compared to nontargeted radiolabeled control particles. In vivo competitive binding studies were used to assess ligand-targeting specificity. ανβ3-Integrin-targeted NP with ∼10 111In/NP provided better (p < 0.05) tumor-to-muscle ratio contrast (6.3 ± 0.2) than ∼1 111In/NP (5.1 ± 0.1) or nontargeted particles with ∼10 111In/NP (3.7 ± 0.1) over the initial 2-hr postinjection. At 18 hr, mean tumor activity in rabbits receiving ανβ3-integrin-targeted NP was 4-fold higher than the nontargeted control. Specificity of the NP for the tumor neovasculature was supported by in vivo competition studies and by fluorescence microscopy of ανβ3-targeted fluorescent-labeled NP. Biodistribution studies revealed that the primary clearance organ in rabbits as a %ID/g tissue was the spleen. Circulatory half-life (t1/2β) was estimated to be ∼5 hr using a 2-compartment model. ανβ3-Targeted 111In perfluorocarbon NP may provide a clinically useful tool for sensitively detecting angiogenesis in nascent tumors, particularly in combination with secondary high-resolution imaging modalities, such as MRI. © 2007 Wiley-Liss, Inc.

Data accumulated over the last 25 years in the surveillance, epidemiology and end results cancer registry support the principle that earlier tumor detection improves 5-year survival of patients with either localized or regional invasive breast carcinoma.1 Improvements in survival were correlated with an overall downward shift in tumor size distribution, with particular advantage noted among patients presenting with cancers less than 1 cm. A widespread desire to detect and treat cancer earlier has spawned interest in molecular imaging and genomic–proteomic technologies, which in combination with new strategies to treat cancer may further improve cancer survival.

One approach to identifying small solid tumors has involved early detection of angiogenesis by targeting unique biosignatures of neovascular endothelium, such as αvβ3-integrin. We have previously demonstrated that paramagnetic perfluorocarbon emulsions targeted to the αvβ3-integrin can be used to detect the neovasculature of tumors ∼30 mm3 at clinical field strengths (1.5 T). Because perfluorocarbon nanoparticles (NP) have a nominal particle size of 250 nm and are constrained within the vasculature, access to αvβ3-integrin expressed on extravascular macrophages, smooth muscle and other cells is sterically precluded. MRI provides outstanding high-resolution images of even minute tumors enhanced by the bound paramagnetic NP; however, the procedure requires a priori knowledge of the tumor location to position coils, establish a field-of-view and acquire images. Identification of minute tumors in unknown locations may require the higher sensitivity of a radionuclide signal such as 111In or 99mTc, which can be detected robustly over a larger region-of-interest.

Numerous radiolabeled αvβ3-integrin or vitronectin antagonists, including antibodies, peptides, peptidomimetics and disintegrins, have been explored as tumor vasculature targeting agents.2, 3, 4, 5, 6, 7, 8, 9, 10, 11 Although these agents can be exquisitely specific for αvβ3-integrin, their penetration beyond the circulation allows binding to a cadre of nonendothelial sources. The biodistribution of perfluorocarbon NP to reticuloendothelial (RES) organs is well-known and previously reported,12 but the potential for higher radionuclide payloads and their intravascular distribution make them attractive agents for rapid identification of nascent tumors in non-RES regions, including the brain, head and neck, lung, breast and prostate.

In this study, we developed 111In-labeled ανβ3-targeted NP and explored their potential for sensitive detection of cancer-induced angiogenesis. Specifically, we (i) estimated the pharmacokinetics of ανβ3-targeted 111In NP in rabbits, (ii) studied the biodistribution of perfluorocarbon NP into the major particulate clearance organs, (iii) prepared and compared the effectiveness of high (10 111In/nanoparticle) vs. lower (1 111In/nanoparticle) nuclide payloads and (iv) microscopically characterized the spatial localization and vascular constraint of ανβ3-targeted fluorescent NP within the tumor.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Preparation of ανβ3-targeted perfluorocarbon nanoparticles

ανβ3-Targeted 111In perfluorocarbon nanoparticles (NP) were prepared by emulsification of 20% (v/v) perfluorooctylbromide, 1.5% (w/v) of a surfactant comixture, 1.7% (w/v) glycerin and water for the balance.13, 14, 15 The surfactant comixture generally included 97.9 mol % lecithin (Avanti Polar Lipids), 0.1 mol % vitronectin antagonist,16 conjugated to PEG2000-phosphatidylethanolamine15 (Avanti Polar Lipids), and 1 mol % of a lipophilic chelate,17 methoxy-DOTA-caproyl-phosphatidylethanolamine (MeO-DOTA-PE, Dow Chemical Company). The surfactant components were prepared as published elsewhere,13 combined with PFOB and distilled deionized water and emulsified (Model S110, Microfluidics) at 20,000 PSI for 4 min. Particle sizes were nominally 242 nm (polydispersity index of 0.231), determined at 37°C with a laser light scattering submicron particle analyzer (Zetasizer 4, Malvern Instruments). Bioactivity of the ανβ3-integrin targeted NP was confirmed using an in vitro vitronectin cell adhesion assay previously reported.18

Citrate shuttle radiolabeling of nanoparticles

In contradistinction to the free chelate, efficient solid-phase coupling of multiple 111In nuclides to NP proved difficult and poorly reproducible with direct coupling methods because of the variable hydrolysis and precipitation of the metal. This problem was resolved utilizing citrate, a weak chelator, as a shuttle that weakly complexed with the 111In and minimized hydrolysis. Subsequent addition of ανβ3-integrin-targeted NP rich in surface methoxy-benzyl DOTA, a strong chelator, favorably competed the 111In from the citrate, yielding reproducible labeling. Specifically, 250 μl of 0.5 M sodium citrate (pH 5.7) was combined with 40 MBq of 111InCl3 in 0.04 M HCl (250 μl). The indium-citrate buffer was mixed with ανβ3-integrin-NP in ratios to produce particles with ∼1 or ∼10 nuclides each. Following overnight incubation in a ∼40°C shaker bath (50 rpm), free DTPA was added to the reaction mixture for 5 min to scavenge the free radionuclide. Coupling was assessed by thin layer chromatography (TLC) at ambient temperature.

An aliquot of the above mixture was applied to silica gel-coated paper and developed in 0.1 M ammonium acetate (pH 5.5): methanol:water (20:100:200, v/v). One centimeter strips were counted with an automatic gamma counter (Wizard 3, model 1480, PerkinElmer). Radioactive nanoparticle payload was calculated as the ratio of radioactivity per microliter assessed by TLC associated with the NP to the number of particles per microliter of emulsion based on their nominal size and perfluorocarbon concentration. Coupling efficiency of 111In to the NP ranged from 50 to 70% for the high (10 nuclides/particle) and 85 to 90+% for 1 nuclide/particle formulations. Equivalent total dosages of NP among treatments were maintained by addition of unlabeled, nontargeted emulsion to the high specific activity injectate.

Preparation of fluorescent ανβ3-targeted nanoparticles

Microscopic assessment of ανβ3-targeted NP distribution within the tumor was facilitated by incorporation of AlexaFluor 488 coupled to caproyl-phosphatidylethanolamine or rhodamine-phosphatidylethanolamine (Avanti Polar Lipids) into the surfactant at 0.5 mol %. AlexaFluor 488-caproyl-phosphatidylethanolamine was synthesized by dissolving 7.8 μmol AlexaFluor 488 carboxylic succinimidyl ester (Molecular Probes) in 1.4 ml dimethylformamide and mixing it with 10 μmol caproylamine phosphatidylethanolamine (Avanti Polar Lipids) in 200 μl chloroform at 37°C for 1 hr. Following addition of 200 μl of chloroform, reaction temperature was increased to 50°C and continued overnight. TLC using a reverse phase hydrocarbon (C18) impregnated silica gel and a mobile phase consisting of 0.1 M sodium acetate buffer (pH 5.6):methanol:water at a ratio of 20:100:200 was performed to monitor and purify the conjugated product from the uncoupled AlexaFluor dye. The fluorescent lipid was recovered at the origin, extracted with chloroform:methanol (3:1), and evaporated to dryness until use.

Vx-2 rabbit tumor model

Animals were maintained and physiologically monitored throughout these studies in accordance with protocol and procedures approved by the Animal Studies Committee at Washington University Medical School.

Male New Zealand white rabbits (∼2.5 kg) were anesthetized with intramuscular ketamine and xylazine (65 and 13 mg/kg, respectively). The left hind leg of each animal was shaved, sterile prepped and infiltrated locally with MarcaineTM prior to placement of a small incision above the popliteal fossa. A 2 × 2-mm2 Vx-2 carcinoma tumor fragment (NCI tumor depository) was freshly obtained from a donor animal and implanted at a depth of ∼0.5 cm within the fossa. Anatomical planes were approximated and secured with a single absorbable suture, and the skin incision was sealed with Dermabond™ skin glue. Following the tumor-implantation procedure, the effects of xylazine were reversed with yohimbine, and animals were allowed to recover.

Twelve to 16 days after Vx-2 implantation, rabbits were anesthetized with 1–2% IsofluraneTM, intubated, ventilated and positioned 3 cm below the high energy pinhole collimator equipped with a single 3-mm aperture and mounted to the clinical Genesys gamma camera (Philips Medical Systems) operating in planar mode. Intravenous and intraarterial catheters were placed in opposite ears of each rabbit, and used for systemic injection of NP and arterial blood sampling/physiologic monitoring. Dosages of labeled NP were calibrated for activity immediately prior to use with a Capintec CRC-15R well counter.

Following intravenous injection, dynamic nuclear images (matrix:128 × 128) were acquired using two 20% windows centered at 170 and 244 keV at baseline and serially, every 15 min for 2 hr. DICOM images were exported to a Unix workstation and later analyzed with ImageJ software (NIH.gov). Anatomical landmarks were identified on each frame and regions-of-interest (ROI) of comparable size were manually placed around the tumor signal, muscle and background regions to determine average pixel activity.

After imaging, animals were euthanized and tumors resected, weighed and quickly frozen in OCT for routine histopathology and selective immunohistochemistry. In 2 animals, testicles were excised as a positive control to confirm neovascularity, which develops continuously in the spermatic cords. Acetone-fixed, frozen tissues were sectioned (5 μm) and routinely stained with hematoxylin and eosin or immunostained for ανβ3-integrin (LM-609, Chemicon International). Immunohistochemistry was performed using the Vectastain® Elite ABC kit (Vector Laboratories), and developed with the Vector® VIP kit. Microscopic images were obtained using a Nikon E800 research microscope and digitized with a Nikon DXM1200 camera.

Experimental design

Pharmacokinetics.

Basic pharmacokinetic parameters of radiolabeled NP were estimated in 6 New Zealand white rabbits administered ανβ3-targeted 111In NP (11 MBq/kg) bearing 10 111In/particle via ear vein bolus injection. Blood was sampled via a separate venous access at baseline and 2, 5, 10, 20, 30, 45, 60, 90 and 120 min following injection, weighed, counted in an automatic gamma well counter (Wizard 1480, Perkin Elmer), and the results normalized for slight volume differences. For each animal, a simple biexponential model, y = A0eat + B0ebt, was fit to the data, from which estimates of distribution volume, elimination rates and clearance were derived using standard kinetic modeling equations for an open 2 compartment model.19

Biodistribution of perfluorocarbon nanoparticles.

The biodistribution of ανβ3-targeted perfluorocarbon NP was determined 3-hr postinjection in New Zealand white rabbits randomly administered intravenous dosages of 0.25 ml/kg (n = 3), 0.5 ml/kg (n = 3) and 1.0 ml/kg (n = 3). Rabbits were euthanized and the primary particulate clearance organs (i.e., lung, spleen, liver, lymph node, bone marrow and kidney) were excised, weighed and prepared for perfluorocarbon analysis.

Perfluorocarbon concentration was determined with gas chromatography using flame ionization detection (Model 6890, Agilent Technologies, Wilmington, DE). Weighed tissue aliquots were extracted in 20% potassium hydroxide in ethanol. Two milliliters of internal standard (0.1% octane in freon) was added, and the mixture was sealed in a serum vial. The sealed vial contents were vigorously vortexed and then continuously agitated on a shaker for 30 min. The lower extracted layer was filtered through a silica gel column and stored at 4–6°C for analysis. Initial GC column temperature was 30°C and ramped upward at 10°C/min to 145°C. All samples were assayed in duplicate and the results were expressed as % ID/g ± SD of tissue.

Molecular imaging.

In vivo detection of angiogenesis in ∼12d Vx-2 tumors was studied in 15 New Zealand rabbits, which were randomized to receive 22 MBq/kg of either:

  • 1
    ανβ3-integrin-targeted NP with ∼10 111In/NP (n = 3).
  • 2
    ανβ3-integrin-targeted NP with ∼1111In/NP (n = 4).
  • 3
    ανβ3-integrin-targeted nonradioactive NP given (3:1) with ανβ3-integrin targeted NP with ∼10 111In/NP (i.e., competition group, n = 4).
  • 4
    Nontargeted NP with ∼10 111In/NP (n = 2).
  • 5
    Nontargeted NP with ∼1111In/NP (n = 2).

Dynamic planar 111In images were acquired every 15 min following injection for 2 hr as described earlier. Tumors with a cuff of surrounding tissues were excised after imaging and fixed in formalin or OCT for histology.

An additional 8 rabbits with Vx-2 tumors were administered either ανβ3-integrin-targeted (n = 4) or nontargeted (n = 4) NP with ∼10 111In/NP and imaged at 18 hr (n = 4) or 48 hr (n = 4). At 18 hr, rabbits were scanned dynamically every 15 min for 2 hr. At 48 hr, one 15-min image acquisition was performed.

Fluorescence imaging

Microscopic localization of NP within and around the tumor was studied in a separate cohort of Vx-2 implanted rabbits (n = 2), which received ανβ3-targeted NP (0.1 ml/kg) with AlexaFluor 488 cyan dye incorporated into the surfactant. The fluorescent NP were administered with a 10-fold excess of nontargeted, nonlabeled NP to minimize passive accumulation within the neovasculature and allowed 1 hr to circulate. Animals were killed, and the tumor was removed, rinsed repeatedly in phosphate buffered saline and frozen in OCT medium. Frozen tumor sections (4 μm) were counterstained with DAPI to identify nuclei. Photomicrographs of green AlexaFluor NP and DAPI-labeled nuclei were superimposed to assess the distribution of the contrast agent with respect to other cellular elements. Adjacent sections were stained with RAM-11 (Dako) to delineate macrophage distribution within the tumor.

In a separate cohort of animals (n = 2), ανβ3-targeted NP (0.1 ml/kg) labeled with rhodamine and FITC-lectin (Vector Laboratories), a general stain for vascular endothelium, were administered intravenously. The ανβ3-targeted rhodamine NP (0.1 ml/kg) were given 2 hr before the FITC-lectin, in concert with nuclear imaging protocol, and the fluorescent lectin was given about 15 min before euthanasia. Rabbits were extensively perfused with saline before tissue extraction to remove unbound fluorescent labels, before embedding the tumors in OCT for frozen sectioning and microscopy.

Statistical analysis

All variables are presented as mean ± standard deviation (SD). General linear models including Student's t tests and ANOVA using SAS (SAS Institute) were employed for the analysis of continuous variables. Least significant difference method was used for mean separations at an alpha level of 0.05.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Coupling of indium to ανβ3-nanoparticles

In the present study, we sought to achieve high surface payloads of 111In per nanoparticle. Initially, several direct couple labeling methods were studied using 0.1 M ammonium acetate buffer (pH 5.5), 0.2 M sodium carbonate, 0.2 M sodium hydroxide or 10% v/v triethylamine buffer in combination with heating to 65°C for 30 min; all approaches yielded generally poor results secondary to hydrolysis of the free metal. However, with the addition of 0.5 M sodium citrate, 111In hydroxide formation was reduced to <2%. Although coupling of 111In to free DOTA chelate in solution was accomplished with stoichiometric precision, the efficiency of coupling of 111In to methoxybenzyl-DOTA onto the NP was poorer.

Pharmacokinetics of nanoparticles

The pharmacokinetics of 111In ανβ3-NP (∼10 111In/NP) was defined in 6 rabbits. Figure 1 illustrates a 2-compartment modeling of the data from 1 rabbit over the initial 2 hr. Based upon the coefficients and rate estimates derived from these data, the β elimination half-life (t1/2β) of the NP was estimated to be 309 ± 136 min. The volume of distribution (VD) and clearance (Cl) were calculated to be 380 ± 66 and 0.68 ± 0.12 ml/min, respectively. The data suggest that perfluorocarbon NP exhibit a circulatory half-life that is more than adequate to reach and saturate any vascular receptor. The volume of distribution was approximately twice as large as estimates of the circulatory volume, reflecting uptake and clearance by the reticuloendothelial system.

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Figure 1. The pharmacokinetic profile depicting the distribution and clearance from circulation of ανβ3-targeted 111In nanoparticles (NP) with ∼10 111In/NP. Percent injected dose (ID) in blood vs. time postinjection is presented for 1 animal over the initial 2 hr. A 2-compartment biexponential model was applied to the data from each animal. Estimates of β elimination half-life, volume of distribution and clearance were calculated and are presented as a mean ± SD, n = 6.

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Biodistribution of perfluorocarbon NP was measured directly in the lung, spleen, liver, bone marrow, kidney and lymph node (Fig. 2). Perfluorocarbon content was greatest in the spleen as % ID/g tissue, with concentrations increasing from (1.9 ± 1.1), (3.0 ± 2.8) and (3.7 ± 0.8)% ID/g for the 0.25-, 0.5- and 1.0-ml/kg emulsion dosages, respectively. At the 1.0-ml/kg emulsion dosage level, liver perfluorocarbon content was 15% (0.6% ± 0.1% ID/g) of that measured in the spleen. In general, the perfluorocarbon concentrations of the remaining tissues were less.

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Figure 2. The biodistribution of perfluorocarbon nanoparticles in rabbits injected with nanoparticle emulsion at dosages of 0.25, 0.5 and 1.0 ml/kg (n = 3/dose). Tissue perfluorocarbon content was measured directly by gas chromatography and results are presented as % ID/g ± SD tissue.

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111In imaging of the Vx-2 neovasculature

Dynamic imaging was conducted for 2-hr postintravenous injection and the tumor-to-muscle ratio (TMR) of mean pixel intensity in rabbits given 111In ανβ3-NP bearing ∼10 111In/NP was compared to animals receiving 111In ανβ3-NP with a 3-fold competitive dosage of nonlabeled ανβ3-NP (Fig. 3, top). 111In ανβ3-NP produced high TMR contrast (6.5 ± 1.3) within 15 min of injection, which persisted throughout the 2-hr period and averaged 6.3 ± 0.2. Blockade of integrin receptors with nonlabeled ανβ3-NP lowered the TMR contrast at 15 min to 4.5 ± 1.3, and this difference persisted over the 2 hr of serial imaging, averaging 4.1 ± 0.2 (p < 0.05). Nontargeted 111In NP (Fig. 3, middle) demonstrated lower TMR contrast at 15 min (3.8 ± 0.5) and over 2 hr (3.7 ± 0.1) than did the integrin-targeted formulation (p < 0.05). The tumor contrast response of the nontargeted and competition treatments did not differ (p > 0.05). At 2 hr, the percent injected dose (% ID) at the tumor site of rabbits administered 111In ανβ3-NP was (1.20 ± 0.3)% ID, which was higher than the dosage retained in animals receiving the equivalent nontargeted NP, (0.60 ± 0.1)% ID (p < 0.05). Collectively, these results support the superior contrast enhancement obtained with ανβ3-integrin targeting vs. radiolabeled NP alone, and suggests that the overall signal from the neovasculature in the integrin-targeted rabbits reflects both contrast due to specific binding and passive entrapment.

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Figure 3. The ratio of tumor-to-muscle signal was determined immediately after contrast injection and serially every 15 min in rabbits implanted with Vx-2 after receiving 22 MBq/kg (i.v.) of: top ανβ3-targeted 111In nanoparticles (NP) with ∼10 111In/NP vs. ανβ3-targeted nonlabeled (competition); middle ανβ3-targeted or nontargeted 111In nanoparticles with ∼10 111In/NP and, bottom ανβ3-targeted 111In nanoparticles with ∼10 111In/NP vs. ∼1 111In/NP. Values presented represent the mean ± SD; *p < 0.05 over 2-hr study.

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In Figure 3 (bottom), signal enhancement relative to muscle of 111In ανβ3-NP with ∼10 111In/NP was superior (p < 0.05) over 2 hr to particles formulated with ∼1 111In/NP (5.1 ± 0.1). However, the average contrast achieved with the lower activity agent was not different (p > 0.05) from the signal obtained with a nontargeted formulation bearing ∼1 111In/NP (data not shown). These data suggest that increasing the payload of radioisotope per nanoparticle is important for maximizing the tumor angiogenic contrast.

Another cohort of 8 rabbits was examined after 18 hr (∼3 circulating half-lives) and 48 hr (∼8 circulating half-lives) to assess the persistence of the targeted nuclear signal. Figure 4 illustrates 18-hr image of 2 rabbits (1 targeted and 1 control), which received equivalent radioactive dosages of 111In NP and exhibited similar muscle background counts. The contrast of the integrin-targeted formulation was greater than that of the nontargeted agent, reflecting longer persistence of the integrin-bound agent in comparison to the passively entrapped NP. For animals receiving 111In ανβ3-NP, the average percent injected dose at the tumor site was 4 times greater (p < 0.05; 0.48% ± 0.05% ID) than that left in animals receiving the nontargeted control (0.10% ± 0.06% ID). At 48-hr postinjection, the signal from tumor and muscle were substantially lower and indistinguishable between groups (p > 0.05).

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Figure 4. The 18-hr 111In planar image (15 min scan, 128 × 128 matrix) of rabbits implanted ∼12 days previously with Vx-2 tumor following 22 MBq/kg (i.v.) of nontargeted (a) or ανβ3-targeted (b) 111In nanoparticles (NP) bearing ∼10 111In/NP. Both images were windowed to have identical dynamic ranges as shown in (b). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Localization of ανβ3-nanoparticles

Histological analysis of ανβ3-integrin expression revealed that the expression of ανβ3-integrin occurred asymmetrically along tissue interfaces between tumor and adjacent vascular structures within connective tissue fascia and vessel adventia, which is consistent with the location of the tumor implant within the popliteal fossa. The upregulated expression of ανβ3-integrin extended beyond the tumor capsule and was recognized in nearby vascular structures associated with muscle fascia (Fig. 5). ανβ3-Integrin expression was also detected with the targeted 111In NP in maturing testicular epididymis, which was confirmed by histology (not shown). Macrophages, another abundant source of ανβ3-integrin, were identified with RAM-11 staining and found densely distributed within the tumor core, and rarely in connective tissue surrounding the tumor (not shown).

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Figure 5. (a) Microscopic image (×4) of Vx-2 adenocarcinoma adjacent to muscle and stained for ανβ3-integrin, which appear as dark brown (purple) streaks (white arrows) within the intervening connective tissue. Higher magnification regions of relatively sparse (b) (×40) and dense regions (c) (×20) of ανβ3-integrin positive neovessels identified on primary image. White arrows identify neovascular expansion within connective tissue (black arrow) between tumor and muscle.

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Fluorescence microscopy of frozen tumor tissues showed that the ανβ3-targeted AlexaFluor particles were within the capsular interface with adjacent muscle (Fig. 6), corresponding to the distribution noted for ανβ3-integrin positive vessels (Fig. 5). Immunohistological costaining of ανβ3-integrin positive vessels with LM609 in rabbits was competitively inhibited by bound ανβ3-targeted AlexaFluor 488 NP. The fluorescent contrast of ανβ3-targeted AlexaFluor 488 NP did not overlap with the distribution of macrophages revealed by RAM 11 staining (not shown).

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Figure 6. (a) Light microscopic image (×4) of Vx-2 adenocarcinoma and capsule. Note necrosis towards the center and cellular proliferation occurring around the periphery of the tumor. (b) Fluorescent microscopy image (×20) of tumor capsule region depicted in (a). The green signature of vessels retaining ανβ3-integrin targeted AlexaFluor 488 nanoparticles within the capsule (yellow arrows). Blue DAPI staining represents cellular nuclei within the connective tissue. Only rare ανβ3-integrin targeted AlexaFluor 488 nanoparticles were detected within the tumor proper.

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Intravenous coadministration of ανβ3-targeted rhodamine NP and FITC-lectin, a vascular endothelial marker, revealed a close spatial correlation between the 2 markers. FITC-lectin was found throughout the vasculature including the neovessels as shown in Figure 7. Rhodamine NP were predominantly located in the smaller vessels and colocalized with the FITC-lectin.

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Figure 7. Fluorescent microscopy image (×40) of ανβ3-integrin targeted rhodamine nanoparticles (middle) and FITC-lectin (top) and the merged images obtained from the tumor capsule region. Note the ανβ3-integrin targeted rhodamine nanoparticles and the FITC-lectin are spatially colocalized (bottom). Rhodamine nanoparticles were not found in the extravascular spaces of the tumor or capsule. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The present study reports initial experiments designed to develop and investigate the utility of 111In ανβ3-NP for the detection of angiogenesis in the rabbit Vx2 tumor model. A simple citrate-shuttle method was established to limit the hydrolysis of 111In, which yielded reproducible labeling of the NP at payloads of ∼10 nuclides per particle. Preliminary pharmacokinetic estimates of 111In ανβ3-NP in rabbits revealed an average β-elimination half-life of ∼300 min. Biodistribution of the NP in primary clearance organs was greatest in the spleen when expressed on a %ID/g tissue basis. 111In ανβ3-NP provided a high TMR signal, which was better for the formulations with 10 nuclides per particle vs. 1. In vivo competitive blockade of the vascular ανβ3-integrin receptors significantly decreased the targeted signal to the nontargeted control level. Fluorescence microscopy studies demonstrated that the ανβ3-NP were concentrated within the tumor capsule in regions rich in neovasculature. ανβ3-Targeted rhodamine NP colocalized with FITC-lectin, a vascular endothelial marker, and further demonstrated the constrained biodistribution of the NP to the vasculature.

111In ανβ3-NP provided a high sensitivity, low-resolution signal from the tumor neovasculature that was well recognized within 15–30 min after injection and which persisted for hours. The level of nuclear contrast was markedly greater and conspicuous much sooner than our previous experience with the paramagnetic version of the agent used in cancer studies with rabbits15 and mice.18 The nuclear signal noted for the 111In ανβ3-NP was attributable to both specific binding and passive entrapment of particles within the dismorphic, “net-like” tumor angiogenic vasculature.20 No accumulation of particles occurred in muscle, where vessels are plentiful, normal and mature. Therefore muscle, a nonclearance organ for this agent, provided an excellent estimate of the background contrast because of the persistent circulation of radioactive particles within the blood pool.

In the present study, we demonstrated that fluorescence labeled NP colocalize with FITC-lectin in the vasculature, which strongly suggests that the agent is confined to the vasculature. The criticality of size to extravasation of particles has been reported by others,21, 22 who collectively suggest that diagnostic or therapeutic agents greater in size than 120 KDa or 100 nm, have virtually no potential for extravasation unless vascular integrity is disrupted, such as by hyperthermia.

Traditional nuclear probes, such as small molecules and peptides, rapidly extravasate from the vasculature into extracellular spaces, which permits these agents to reach biochemical epitopes presented on or in tumor cells. However, the broader biodistribution of small probes allows identification of many biomarkers, like ανβ3-integrin, to be recognized on nonendothelial cells, for example, activated macrophages within a tumor. Moreover, since the small probe labels permeate the entire extravascular space of an organ, rapid clearance is required to reduce the background level to recognize targeted uptake. In the perfluorocarbon nanoparticle situation, the nuclear contrast signal can only be derived from the vasculature because of its size. Therefore, the rapid enhancement of tumor neovasculature because of the ligand-specific binding of NP to ανβ3-integrin and nonspecific entrapment in the angiogenic “net” is not attributable to extravascular tissue background levels and does not require clearance for target recognition. In addition, many small molecule probes have excellent receptor binding features, but exhibit rapid off-rates as the radioligands are cleared from the body. In contradistinction, the neovascular tumor contrast produced by the 111In ανβ3-NP was both rapid and persistent, which could offer high flexibility for imaging patients with SPECT in a clinical setting.

Because the nuclear signal attributable to NP represents both specifically bound and passively entrapped particles, direct characterization of the expression of the integrin cannot be easily deconvolved with this technique. However, the recognition of a potential tumor lesion based on the overall neovascular signal could rapidly identify potential ROI that would guide high-resolution 1H magnetic resonance imaging (MRI). This could be applied to cancers evolving in many important regions of the body including brain, head and neck, breast and prostate. MRI depends on proper coil positioning and field-of-view placement, which requires a priori knowledge of tumor location. The 111In ανβ3-NP neovascular signal could be used to identify occult tumors or metastases in high-risk patients, and when appropriate, further study with a highly focused, high resolution MRI examination could follow. Moreover, the perfluorocarbon core of the NP can provide a 19F MR signal, which indicates the spatial location of the 111In ανβ3-NP at the tumor site.23, 24

In summary, ανβ3-targeted 111In NP were developed and studied for use as sensitive beacons of angiogenesis in nascent tumors. 111In ανβ3-NP provided a high tumor signal, which was better for the formulations with 10 nuclides per particle than 1. In vivo competitive blockade of the vascular ανβ3-integrin receptors significantly decreased the targeted signal to the nontargeted control level. 111In ανβ3-NP were constrained to the circulation by size and accumulated in nascent tumors as a function of both specific binding to and passive entrapment within the neovasculature. In an individual animal, this early, strong signal may be clinically used to identify occult tumors or metastases and guide follow-on high resolution MRI.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We extend sincere appreciation to Mr. Ralph Fuhrhop for formulation chemistry and to Mr. John Allen for his preparation and maintenance of the Vx-2 tumor model. In addition, we want to extend appreciation to Drs. Keith Frank and Jim Simon for their significant contributions to the lipophilic chelate chemistry employed in these studies.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Elkin EB,Hudis C,Begg CB,Schrag D. The effect of changes in tumor size on breast carcinoma survival in the U.S. 1975–1999. Cancer 2005; 104: 114957.
  • 2
    Haubner R,Wester HJ,Burkhart F,Senekowitsch-Schmidtke R,Weber W,Goodman SL,Kessler H,Schwaiger M. Glycosylated RGD-containing peptides: tracer for tumor targeting and angiogenesis imaging with improved biokinetics. J Nucl Med 2001; 42: 32636.
  • 3
    Haubner R,Wester HJ,Reuning U,Senekowitsch-Schmidtke R,Diefenbach B,Kessler H,Stocklin G,Schwaiger M. Radiolabeled α(v) β3 integrin antagonists: a new class of tracers for tumor targeting. J Nucl Med 1999; 40: 106171.
  • 4
    Janssen ML,Oyen WJ,Dijkgraaf I,Massuger LF,Frielink C,Edwards DS,Rajopadhye M,Boonstra H,Corstens FH,Boerman OC. Tumor targeting with radiolabeled α (v) β(3) integrin binding peptides in a nude mouse model. Cancer Res 2002; 62: 614651.
  • 5
    McQuade P,Knight LC,Welch MJ. Evaluation of 64Cu- and 125I-radiolabeled bitistatin as potential agents for targeting α v β 3 integrins in tumor angiogenesis. Bioconjug Chem 2004; 15: 98896.
  • 6
    Chen X,Park R,Hou Y,Khankaldyyan V,Gonzales-Gomez I,Tohme M,Bading JR,Laug WE,Conti PS. MicroPET imaging of brain tumor angiogenesis with 18F-labeled PEGylated RGD peptide. Eur J Nucl Med Mol Imaging 2004; 31: 10819.
  • 7
    Chen X,Park R,Shahinian AH,Bading JR,Conti PS. Pharmacokinetics and tumor retention of 125I-labeled RGD peptide are improved by PEGylation. Nucl Med Biol 2004; 31: 119.
  • 8
    Chen X,Park R,Shahinian AH,Tohme M,Khankaldyyan V,Bozorgzadeh MH,Bading JR,Moats R,Laug WE,Conti PS. 18F-labeled RGD peptide: initial evaluation for imaging brain tumor angiogenesis. Nucl Med Biol 2004; 31: 17989.
  • 9
    Chen X,Park R,Tohme M,Shahinian AH,Bading JR,Conti PS. MicroPET and autoradiographic imaging of breast cancer α v-integrin expression using 18F- and 64Cu-labeled RGD peptide. Bioconjug Chem 2004; 15: 419.
  • 10
    Onthank DC,Liu S,Silva PJ,Barrett JA,Harris TD,Robinson SP,Edwards DS. 90Y and 111In complexes of a DOTA-conjugated integrin αvβ3 receptor antagonist: different but biologically equivalent. Bioconjug Chem 2004; 15: 23541.
  • 11
    Sadeghi MM,Krassilnikova S,Zhang J,Gharaei AA,Fassaei HR,Esmailzadeh L,Kooshkabadi A,Edwards S,Yalamanchili P,Harris TD,Sinusas AJ,Zaret BL et al. Detection of injury-induced vascular remodeling by targeting activated αvβ3 integrin in vivo. Circulation 2004; 110: 8490.
  • 12
    McGoron AJ,Pratt R,Zhang J,Shiferaw Y,Thomas S,Millard R. Perfluorocarbon distribution to liver, lung and spleen of emulsions of perfluorotributylamine (FTBA) in pigs and rats and perfluorooctylbromide (PFOB) in rats and dogs by 19F NMR spectroscopy. Artif Cells Blood Substit Immobil Biotechnol 1994; 22: 124350.
  • 13
    Lanza GM,Wallace KD,Scott MJ,Cacheris WP,Abendschein DR,Christy DH,Sharkey AM,Miller JG,Gaffney PJ,Wickline SA. A novel site-targeted ultrasonic contrast agent with broad biomedical application. Circulation 1996; 94: 333440.
  • 14
    Flacke S,Fischer S,Scott MJ,Fuhrhop RJ,Allen JS,McLean M,Winter P,Sicard GA,Gaffney PJ,Wickline SA,Lanza GM. Novel MRI contrast agent for molecular imaging of fibrin: implications for detecting vulnerable plaques. Circulation 2001; 104: 12805.
  • 15
    Winter PM,Caruthers SD,Kassner A,Harris TD,Chinen LK,Allen JS,Lacy EK,Zhang H,Robertson JD,Wickline SA,Lanza GM. Molecular imaging of angiogenesis in nascent Vx-2 rabbit tumors using a novel αvβ3-targeted nanoparticle and 1.5 tesla magnetic resonance imaging. Cancer Res 2003; 63: 583843.
  • 16
    Harris TD,Kalogeropoulos S,Nguyen T,Liu S,Bartis J,Ellars C,Edwards S,Onthank D,Silva P,Yalamanchili P,Robinson S,Lazewatsky J, et al. Design, synthesis, and evaluation of radiolabeled integrin αvβ3 receptor antagonists for tumor imaging and radiotherapy. Cancer Biother Radiopharm 2003; 18: 62741.
  • 17
    Winter P,Athey P,Kiefer G,Gulyas G,Frank K,Fuhrhop R,Robertson D,Wickline S,Lanza G. Improved paramagnetic chelate for molecular imaging with mri. J Magn Magn Mater 2005; 293: 5405.
  • 18
    Schmieder AH,Winter PM,Caruthers SD,Harris TD,Williams TA,Allen JS,Lacy EK,Zhang H,Scott MJ,Hu G,Robertson JD,Wickline SA,Lanza GM. Molecular MR imaging of melanoma angiogenesis with αvβ3-targeted paramagnetic nanoparticles. Magn Reson Med 2005; 53: 6217.
  • 19
    O'Flaherty EJ. Toxicants and drugs: kinetics and dynamics. New York: Wiley, 1981.
  • 20
    Baluk P,Morikawa S,Haskell A,Mancuso M,McDonald DM. Abnormalities of basement membrane on blood vessels and endothelial sprouts in tumors. Am J Pathol 2003; 163: 180115.
  • 21
    Kong G,Braun RD,Dewhirst MW. Hyperthermia enables tumor-specific nanoparticle delivery: effect of particle size. Cancer Res 2000; 60: 44405.
  • 22
    Weissleder R,Bogdanov A,Jr,Tung CH,Weinmann HJ. Size optimization of synthetic graft copolymers for in vivo angiogenesis imaging. Bioconjug Chem 2001; 12: 2139.
  • 23
    Morawski AM,Winter PM,Yu X,Fuhrhop RW,Scott MJ,Hockett F,Robertson JD,Gaffney PJ,Lanza GM,Wickline SA. Quantitative “magnetic resonance immunohistochemistry” with ligand-targeted (19)F nanoparticles. Magn Reson Med 2004; 52: 125562.
  • 24
    Caruthers SD,Neubauer AM,Hockett FD,Lamerichs R,Winter PM,Scott MJ,Gaffney PJ,Wickline SA,Lanza GM. In vitro demonstration using (19)F magnetic resonance to augment molecular imaging with paramagnetic perfluorocarbon nanoparticles at 1.5 tesla. Invest Radiol 2006; 41: 30512.