Synthesis of perfluorocarbon nanoparticles.
Perfluorocarbon nanoparticles were synthesized as an oil-in-water emulsion by microfluidization as described earlier (72
). The lipid surfactant used consisted of egg lecithin (98 mole%) and dipalmitoyl-phosphatidylethanolamine (DPPE) (2 mole%) (Avanti Polar Lipids Inc.). αv
integrin–targeted nanoparticles were made by incorporating 0.1 mole% peptidomimetic vitronectin antagonist (US Patent 6,322,770) conjugated to polyethylene glycol 2000–phosphatidylethanolamine (PEG 2000–phosphatidylethanolamine) (Avanti Polar Lipids Inc.), replacing equimolar quantities of lecithin. The effective binding affinity for the αv
integrin per particle is approximately 50 pM, as reported recently by our group, due to multiple simultaneous ligand binding (23
). The specificity for αv
integrin as compared with glycoprotein IIb/IIIa (GP IIbIIIa) receptor is more than 3 orders of magnitude in binding affinity (34
Incorporation of melittin onto the nanoparticles.
Melittin-loaded nanoparticles were formulated by mixing known amounts of melittin to perfluorocarbon nanoparticles. Pure melittin peptide was produced by solid-state peptide synthesis and was obtained from Robert Mecham (Department of Cell Biology and Physiology, Washington University School of Medicine). The melittin was dissolved in 100 mM KCl (pH 7, 10 mM HEPES) at 0.1 mM, and 2 to 20 μl was added to 25 μl of nanoparticle suspension with mixing. After incubation at room temperature for 10 minutes, the nanoparticles were washed twice by centrifugation (100 g, 10 minutes) to remove the unbound melittin. The melittin in the supernatant was quantified by measuring the tryptophan fluorescence (excitation, 280 nm; emission, 350 nm). Depending on the amount of melittin added, the melittin-loaded nanoparticles yielded molar lipid/melittin ratios ranging from 3000 to 40. For animal studies and except where otherwise noted, the lipid/melittin ratio was 40.
Preparation of liposomes.
To compare the behavior of traditional liposomes to the proposed perfluorocarbon nanoparticle vehicles as peptide carriers, liposomes (98 mole% egg lecithin, 2 mole% DPPE) were synthesized as described earlier (73
The effect of melittin on bilayered liposomes and on monolayered perfluorocarbon nanoparticles was examined by TEM. The nanoparticles were sequentially stained with 1.25% osmium tetroxide, 2% tannic acid, and uranyl acetate, after which the pellet was dehydrated and embedded in Poly/Bed 812 (Polysciences Inc.). The negative stained pellet was then thin-sectioned on a Reichert-Jung Ultracut, and after placing on copper grids, was viewed on a Zeiss 902 electron microscope.
Replicating and imaging deep-etched or freeze-dried samples.
The interaction of C32 melanoma cells with either nontargeted or αv
integrin–targeted nanoparticles was studied by centrifuging a suspension of nanoemulsions on a plate of melanoma cells, followed by warming for 7 minutes at 37°C to allow nanoparticle fusion/uptake, a process referred to as spinoculation
). The samples were deep-etched and fractured at 105°C. After sequential platinum replication, 1% potassium permanganate fixation, and rotary replication, the samples were transferred to Formvar-coated grids and viewed by TEM at 100 kV (74
Human umbilical cord blood was obtained from healthy donors after informed consent. The red cells were separated, and various concentrations of melittin or melittin-loaded nanoparticles were added to a fixed number of red cells (5 × 10 cells) and incubated at 37°C for 3 hours. The release of hemoglobin was quantified by measuring the absorbance at 540 nm of the supernatant in a Microplate Reader (model 550; Bio-Rad) after centrifugation. The absorbance of the supernatant obtained by incubating the red cells in water under identical conditions was set to 100%.
Pharmacokinetics and biodistribution of nanoparticles.
For pharmacokinetic and biodistribution studies, melittin was chemically conjugated with Tc. Freshly eluted TcO (13.5 mCi) was added to the Isolink Kit (Mallinckrodt Institute of Radiology, Washington University) and heated at 100°C for 20 minutes to form the Tc tricarbonyl precursor. Melittin was added to the precursor and incubated at 55°C for 30 minutes to allow transchelation. The conjugation efficiency was determined by thin-layer chromatography and was consistently greater than 95%.
Tc-labeled melittin (free or on nanoparticles) was injected i.v. through the tail vein (1 mg/kg) in C57BL/6 mice and the blood drawn from the jugular vein at various times. At the end of 2 hours, the mice were sacrificed and the organs removed. The radioactivity was measured in a gamma counter (Wizard 1480; PerkinElmer). The pharmacokinetic data were fit by a sum of exponentials model using MATLAB to calculate the volume of distribution and the elimination half-lives.
Therapeutic efficacy of melittin-loaded nanoparticles in solid tumors.
All animal protocols were approved by the Washington University Animal Studies Committee. Mice were housed in standard 9 × 15 × 6–inch mouse cages (3–5 per cage) and were fed with rodent chow and water. Six-week-old athymic nude mice (National Cancer Institute) were implanted in the right inguinal fat pad with a cocktail of 2 million MDA-MB-435 cells (50 μl), Matrigel (50 μl), VEGF (100 ng/ml), and bFGF (100 ng/ml). At day 7 after implantation, the tumors were imaged by ultrasound. The mice were randomized into different groups and treated with either saline, nanoparticles, or melittin-loaded nanoparticles (melittin dose, 1 mg/kg) every third day starting at day 7 for a total of 5 doses. At day 22, the tumors were imaged again by ultrasound. The tumor growth rate was measured from the ratio of the starting and end tumor volumes for the 2 groups.
C57BL/6 mice (4 to 6 weeks old; National Cancer Institute) were implanted s.c. in the right flank with 1 million B16F10 melanoma cells. On day 5, the mice were randomly grouped into 3 groups and were treated with i.v. injection of either saline, nanoparticles alone, or melittin-loaded nanoparticles (melittin dose, 8 mg/kg or 1 mg/kg) every other day for a total of 4 doses. The tumor dimensions were measured using a caliper. At day 14, mice were sacrificed, blood removed for serum chemistry, and tumors or organs excised for histology. Excluding the tumor weight, there were no significant changes in the body weight among the 3 groups (P = NS for saline vs. nontargeted melittin-loaded nanoparticles vs. αvβ3 integrin–targeted melittin-loaded nanoparticles).
Paraffin-embedded formalin-fixed tumor sections were stained with H&E. For immunofluorescent staining, tumor sections were stained with anti-CD31 antibody (Blood Vessel Staining Kit, Chemicon International; Millipore) for blood vessel counts or anti-BrdU antibody (BrdU In-Situ Detection kit; BD Biosciences — Pharmingen) using the manufacturer’s instructions. The positively stained cells were detected by the DAB kit or the VIP kit (Vector Laboratories), respectively. Images were obtained using MicroSuite imaging software, version FIVE (Olympus).
Surface plasmon resonance.
Biacore X biosensor and carboxymethylated dextran chip L1 were obtained from Biacore. A uniform lipid monolayer on an L1 chip was created by injecting 35 μl of untargeted or αvβ3 integrin–targeted nanoparticles (3 μl/min). Attached nanoparticles were stabilized with 50 μM of 10 mM NaOH and exposed surface covered with 25 μl of 0.1 mg/ml BSA in PBS. C32 melanoma cells in 30 μl PBS were then injected at a flow rate of 30 μl/min and the response recorded for 60 minutes.
Ultrasound data acquisition and analysis.
A high-frequency ultrasound imaging system (Vevo 660; VisualSonics) was used to acquire backscatter data from the mouse tumors. The system was modified to output analog radio frequency (RF) data and associated trigger signals in order to permit digitization of the raw RF waveforms. Waveforms were digitized at 500 MHz with an 8-bit digitizer (CS82G; GaGe) and stored for offline analysis. The transducer probe (40 MHz wobbler, 6-mm focal length, 20 Hz frame rate) was affixed to a motorized gantry under computer control in order to enable automated scanning of the probe across the length of the tumor. Each anesthetized animal was placed on its back on a platform beneath the probe, and a small amount of ultrasound coupling gel was applied to the area proximal to the tumor. The probe was positioned so that the central area of the tumor was situated in the focal region of the transducer. RF data corresponding to cross-sectional views of the tumor was acquired at multiple sites along the length of the tumor, so that the entire tumor volume was interrogated. The probe was translated laterally (perpendicularly to the swing of the wobbler) in 100-micron steps between each scan plane acquisition.
All signal and image processing was performed with custom software plug-ins in the open-source software package ImageJ (W. S. Rasband, NIH; http://rsb.info.nih.gov/ij/). A sliding time gate (0.1 microsec Hamming window) was applied to each digitized RF waveform, and the log of the sum of the squared amplitude values within the window was calculated as the window was translated along the entire waveform. The resulting values (representative of the mean backscattered energy at each point in the A-line) for all waveforms in each scan plane was used to generate scaled, 2D cross-sectional images of the tumor. Regions were drawn by hand around the apparent tumor outline for each scan plane to obtain cross-sectional tumor area measurements. The total tumor volume was calculated based on these cross-sectional areas and the distance between scan planes.
Cell proliferation inhibition assay.
The effect of αvβ3 integrin–targeted melittin-loaded nanoparticles on mouse endothelial (2F2B) and human melanoma (C32) cancer cell proliferation was determined by the MTT assay. C32 melanoma cells express the integrin receptor αvβ3, while mouse endothelial cells were treated with 1 nM nicotine for 3 hours to overexpress the integrin receptor αvβ3.
Mode of cell death.
Annexin V–FITC (Sigma-Aldrich) and 7-AAD staining solution (BD Biosciences) were used to stain the phosphatidylserine in the outer cell membrane and nucleic acids, respectively. Cells were harvested after 1 hour of incubation at 37°C with either nontargeted or αvβ3 integrin–targeted nanoparticles and the samples analyzed by flow cytometry.
Effect of cholesterol depletion on cellular interactions.
C32 melanoma cells were treated with either 0.25 mM or 0.5 mM methyl-β-cyclodextrin (Sigma-Aldrich) for 15 minutes at 37°C to deplete cholesterol. Amplex Red Cholesterol Assay Kit (Invitrogen) was used to quantify the amount of cholesterol depleted.
Intracellular trafficking of melittin.
To track the melittin being delivered to the cells and define its localization, fluorescently labeled melittin was synthesized. FluoroTag FITC Conjugation Kit (Sigma-Aldrich) was used to conjugate FITC to the N terminus of melittin. The unconjugated FITC was separated using a G25 sephadex column. Fluorescein-melittin–loaded nanoparticles were incubated with C32 melanoma cells for 1 hour at 37°C. The cells were fixed and visualized using a Zeiss 510 confocal microscope. Confocal Z-stack images were obtained and reconstructed using the T3D package in NOEsys (Research Systems Inc.) to confirm the intracellular deposition of FITC-melittin. The experiment was repeated at 4°C and after ATP depletion that was achieved by treating the cells with 20 mM sodium azide and 50 mM 2-deoxyglucose for 15 minutes at 37°C, respectively, prior to addition of targeted FITC–melittin-loaded nanoparticles.
Cytochrome c release assay.
C32 melanoma cells were treated with varying concentrations of αvβ3 integrin–targeted melittin-loaded nanoparticles for 1 hour at 37°C, after which the mitochondria were isolated by using the Mitochondrial Isolation Kit (Pierce; Thermo Scientific). The cytochrome c present in the mitochondrial and cytosol fractions thus obtained was assayed by using the cytochrome c ELISA kit (Invitrogen). Cells treated with 10 μM camptothecin (overnight at 37°C) were taken as 100% release.
LDH release assay.
C32 melanoma cells were either treated with free melittin or αvβ3 integrin–targeted melittin-loaded nanoparticles at various concentrations for 1 hour at 37°C and the amount of LDH released quantified by using the kit from BioVision. Cells treated with 0.1% Triton X-100 were taken as 100% release.
In vivo efficacy in K14-HPV16 transgenic mice.
The in vivo efficacy of αv
integrin–targeted nanoemulsions was tested in K14-HPV16 transgenic mice. These mice express the E6 and E7 oncogenes from HPV type 16 controlled by the human keratin 14 promoter in the basal squamous epithelium and spontaneously develop epidermal cancers over a period of 12 months that closely mimic HPV-induced human cancers (45
). K14-HPV16 (4 months old) mice were obtained from Jeffrey Arbeit at Washington University. All animal protocols were approved by the Washington University Animal Studies Committee. Around 4 to 5 months, precancerous dysplastic lesions in these mice are known to undergo the angiogenic switch. A protocol was designed in which the mice (5 per group) were injected i.v. with either saline or nontargeted or αv
integrin–targeted melittin-loaded nanoparticles (melittin dose, 13 mg/kg) every third day for a total of 7 doses. On day 24 from the start of the dosing, the mice were sacrificed, blood collected for serum chemistry, and the ears along with the internal organs excised and preserved in 10% formaldehyde followed by paraffin embedding for histopathological examination (i.e., H&E staining). For each ear, randomly chosen 5-μm thick sections were imaged and the height of papillae across the entire epidermis was measured using the MicroSuite FIVE software (Olympus). The number of severe papillae (greater than 100 μm in height) were then averaged for each mouse and the 3 groups (saline, melittin-loaded nanoparticles, and targeted melittin-loaded nanoparticles; n
= 5 each group) compared by nonparametric statistics (Kruskal-Wallis test and Mann-Whitney U test).
To visualize the binding of nanoparticles, mice were injected with either nontargeted or αvβ3 integrin–targeted rhodamine nanoparticles (1 ml/kg) for 2 hours. Prior to sacrifice, mice were injected with 50 μl FITC-labeled tomato lectin (Lycopersicon esculentum; Sigma-Aldrich) for 3 minutes to delineate the vasculature. The mouse ears were excised and embedded in frozen OCT, and 50 μm sections were visualized on a fluorescent microscope (Olympus). The exposure time was set to 500 ms.
Data are represented as mean ± SD of at least 5 samples unless otherwise indicated. A 2-tailed Student’s t test was used to assess the difference between any 2 groups for the in vitro tests. Nonparametric statistics (Kruskal-Wallis test and Mann-Whitney U test) were used for in vivo studies. The CI was set at 95%, and P < 0.05 was considered significant.