Dynamic light scattering
Hydrodynamic diameter was measured on a Malvern Zetasizer Nano ZS instrument (Southborough, MA) with back scattering detector (173°, 633 nm laser wavelength) in batch mode at 25° C using a low volume quartz cuvette (pathlength 10 mm). Samples were prepared at a concentration of 2 mg/mL in water, 10 mM NaCl, PBS, or saline (154 mM NaCl), and filtered through a 0.2 μm filter (Anotop 10 Plus, Whatman) before a minimum of twelve measurements per sample were made. Hydrodynamic size is reported as the intensity-weighted average over all size populations (Z-avg), and as the intensity weighted and volume-weighted average over a particular range of size populations corresponding to the most prominent peak in the % intensity and % volume distributions (Int-Peak and Vol-Peak), respectively.
A Malvern Zetasizer Nano ZS instrument was used to measure zeta potential at 25° C. Samples were prepared at a concentration of 2 mg/mL in 10 mM NaCl and loaded into pre-rinsed folded capillary cells for the zeta potential measurements. Sample pH was measured before and after the zeta potential measurements. An applied voltage of 120 V was used. The trace shown in the Supporting Information
represents the average of at least three measurements.
HPLC-based assessments of purity
The chromatographic system used to measure sample purity consisted of a degasser (Agilent G1379A, Palo Alto, CA), capillary pump (Agilent G1378A), micro well-plate autosampler (Agilent G1377A), Zorbax 300SB-C8 column (1.0 mm ID × 150 mm, 3.5 μm, Agilent), and a diode array detector (Agilent G1315B). The mobile phase consisted of water/acetonitrile (A/B, HPLC Grade, 0.14% (w/v) trifluoroacetic acid) at a flow rate of 50 μL/min. The elution gradient was 40% MeCN for 5 minutes, ramp to 80% MeCN in 80 minutes, hold at 80% MeCN for 5 minutes, and ramp down to 40% MeCN in 10 minutes. The sample volume injected was 5 μL at a concentration of 2 mg/mL in HPLC-grade water, and the eluted sample was detected at 227 nm, the absorbance maximum. Samples were run in triplicate.
HPLC-based assessment of paclitaxel release
Samples of 1 were prepared at 1 mg/mL in water, water at pH 2, acetonitrile, and acetonitrile at pH 2. Over the course of the 3 day experiment, samples were mixed on a rotary shaker placed in an incubator set at 37° C. An additional sample of 1 (prepared in water) was spiked with 100 μg/mL paclitaxel. The amount of free paclitaxel was determined by RP-HPLC after 3 days. The chromatographic system consisted of a degasser (Agilent G1379A, Palo Alto, CA), capillary pump (Agilent G1378A), micro well-plate autosampler (Agilent G1377A), Zorbax 300SB-C8 column (1.0 mm ID × 150 mm, 3.5 μm, Agilent), and a diode array detector (Agilent G1315B). The mobile phase consisted of water/acetonitrile (A/B, HPLC Grade, 0.14% (w/v) trifluoroacetic acid) at a flow rate of 50 μL/min. The elution gradient was 40% MeCN for 5 minutes, ramp to 80% MeCN in 40 minutes, hold at 80% MeCN for 5 minutes, and ramp down to 40% MeCN in 10 minutes. The sample volume injected was 5μL and the eluted sample was detected at 210 nm. Samples were run in triplicate.
Molecular weight determination by size-exclusion chromatography (SEC) and asymmetric flow field-flow fractionation (AFFF) with multi-angle laser light scattering (MALLS) and refractive index (RI) detection
To calculate the value of dn/dc (the change in the refractive index with a change in concentration) needed for the determination of the molecular weight, samples were prepared in PBS at concentrations of 0.1, 0.5, 1, 2, and 3 mg/mL. Briefly, dry sample (typically 30 mg) was added to a pre-weighed 1.8 mL cryo-vial, and 1.5 mL Milli-Q water was added to give a concentration of 20 mg/mL. The solution was then frozen using dry ice and lyophilized overnight. The lyophilized sample was then weighed and the actual sample weight determined. A control (1.8 mL cryo-vial with 1.5 mL water) was run in parallel with the sample to correct for any water present in the cryo-vial. To the known lyophilized sample, an appropriate volume of Milli-Q water was added to give a concentration of 20 mg/mL. Three hundred (300) μL aliquots (corresponding to 6 mg of sample) were prepared from this stock sample solution, frozen and lyophilized overnight. To the 6 mg sample, 1 mL PBS was added to give a stock concentration of 6 mg/mL. Samples were prepared at the desired concentrations by diluting the stock solution with PBS. The determination of dn/dc was first performed manually by injecting 1-mL pure solvent (PBS) into the RI detector (Optilab rEX, Wyatt Technology, Santa Barbara, CA) using a 1 mL disposable syringe. This produces the pure solvent baseline. Next, each sample, starting with the lowest concentration, was manually injected (700 μL) with a new 1 mL disposable syringe. After all the samples were injected, pure PBS was injected again for baseline determination. ASTRA (v18.104.22.168, Wyatt Technology) was used to calculate dn/dc.
The SEC-MALLS apparatus comprised an isocratic pump (Agilent G1310A, Palo Alto, CA), well-plate autosampler (Agilent G1329A), and TosoHaas TSK gel Guard PW 06762 (7.5 mm ID × 7.5 cm, 12 μm) and TSKgel G4000PW 05763 (7.5 mm ID × 30 cm, 17 μm, 500 Å) columns (TosoHaas, Montgomeryville, PA). The size exclusion column was connected in-line to a light scattering (MALLS) detector (DAWN EOS, 690 nm laser, Wyatt Technology, Santa Barbara, CA) and a Refractive Index (RI) detector (Optilab rEX, Wyatt Technology, Santa Barbara, CA). The isocratic mobile phase was PBS (1x, pH 7.5, Sigma D1408, St. Louis, MO) at a flow rate of 1 mL/min. Sample concentration was 3 mg/mL in PBS and filtered through a 0.2 μm filter before 100 μL was injected into the chromatographic system. Dendrimer 1 was analyzed via asymmetric flow field-flow fractionation (AFFF) with multi-angle laser light scattering (MALLS) and refractive index (RI) for molecular weight determination.
The AFFF-MALLS measurements were performed using instrumentation from Wyatt Technology Corp. (Santa Barbara, CA, USA), which consists of Eclipse2 AFFF controller and DAWN EOS scattering detector and Optilab rEX refractive index detector.. The thickness of the AFFF separation channel was defined by a 490 μm PEEK spacer, and a nominal 10 kDa regenerated cellulose membrane served as the flow partition membrane. One hundred (100) μL of sample was injected into the separation channel and focused against the partitioning membrane for 5 min at a focusing flow rate of 1.0 mL/min. The samples were then separated by a constant cross flow of 1.0 mL/min during a period of 40 min. The same dn/dc value was used to determine the molecular weight. Two un-filtered samples with concentrations of 3.0 mg/mL and one 0.2 μm-filtered sample with a concentration of 3.0 mg/mL were injected.
Paclitaxel release in plasma
In order to determine the concentration of free PTX in 1, stock solutions of 1 (with concentration of 50, 100, 200, 300, and 400 μg/mL) were prepared in PBS. Aliquots of these stock solutions were analyzed for free PTX using an HPLC assay. In order to determine the rate of release of bound PTX from 1 in plasma, 1 was incubated in plasma (human, rat and mouse) at 37° C for 48 hours. At selected time intervals (0.5, 1, 2, 4, 6, 8, 24, and 48 hours), 100 μL aliquots of the plasma samples were collected and prepared for analysis by acetonitrile precipitation. Standard calibration samples were prepared by spiking 100 μL of blank plasma with known paclitaxel concentrations ranging from 0.125 to 25 μg/mL. Plasma samples, 100 μL, were mixed with 0.5 mL ice-cold acetonitrile that contained 5 μL of 2.5 μg/mL docetaxel as an internal standard. The samples were then centrifuged at 14000 rpm for 20 minutes to remove precipitated protein. The organic layer was transferred to a clean borosilicate glass tube and evaporated to dryness under a pressured nitrogen gas blowing concentrator (TurboVap® LV) at 40° C. The extraction residue was reconstituted in 1 mL of 20% acetonitrile in water, and 50 μL aliquots were injected into an HPLC system. PTX extraction efficiency in plasma was determined to be between 85–90%. HPLC analysis was performed using a Shimadzu system (LC-20AT pump, SPD-20A UV, a SIL-20AC auto injector, a C-R3A integrator), and C-18 Zorbax column (5 μm, 4.6 × 150 mm). Note: this is a different HPLC system than was employed earlier.
Chromatographic separations were achieved using a water and acetonitrile gradient elution method (25% acetonitrile from 0 to 5 min, increased linearly to 80% acetonitrile from 5 to 15 min, decreased linearly to 25% acetonitrile from 15 to 17 min), injection volume of 50 μL, UV detection at λmax 227 nm, and a flow rate of 1.0 mL/min. The column temperature was 25° C, and the column regeneration time between injections was 8 min. The internal standard, docetaxel, and analyte, paclitaxel, elution times were 14.73 and 15.03 min, respectively. Peak area ratio was used to calculate paclitaxel concentrations from the standard calibration curves obtained using tissue matrix standards. A separate calibration in matrix was conducted for each set of tissues analyzed. Data was acquired and processed with LC solution® chromatography software from Shimadzu.
Cytotoxicity: Cell lines HepG2, LLC-PK-1, and LS174T
Briefly, 1 and paclitaxel were diluted to 0.000003–300 μM paclitaxel equivalents of 1, Taxol and Abraxane (for LS174T). Cells were plated in 96-well, microtiter plate format at a density of 50,000 cells per well for HepG2, 25,000 cells per well for LLC-PK1, and 20,000 cells per well for LS174T. Cells were pre-incubated for 24 hours prior to test material addition, reaching an approximate confluence of 80%. Cells were then exposed to test material or media control for 4, 24, 48 and 72 hours in the dark, and cytotoxicity was determined using the MTT cell viability and LDH membrane integrity assays. IC50 values were approximated from the 48 and 72 hour MTT dose-response curves.
LS174T MTT cytotoxicity assay-short exposure
Using a cell plating density of 20,000 cells per well, cells were plated in 96-well, microtiter plate format. Cells were preincubated for 24 h prior to test material addition, reaching an approximate confluence of 80%. Both 1 and Abraxane were diluted to 0.000005–500 μM paclitaxel equivalent in cell culture media. Cells were then exposed to test material or control media for 1 h, washed with media, and fresh media added. Cytotoxicity was evaluated 6 days later by the MTT cell viability. IC50 values were approximated from the MTT dose-response curves.
Caspase 3 activity was measured by the Apo-One Homogenous Caspase-3/7 Assay kit (Promega Cat. #TB293, Promega Corp., Madison, WI). Briefly, the test materials were diluted in cell culture media to 0.000003–300 μM paclitaxel equivalents. LS174T cells were plated in 96-well microtiter plate format at 20,000 cells per well in RPMI 1640 cell culture media (2 mM L-glutamine, 10% FBS). The negative control was cell culture media. The positive control was 10 mM acetaminophen. Cells were preincubated for 24 hours prior to addition of test sample, reaching an approximate confluence of 80%. Cells were treated with test samples in the dark for 24 and 48 hours. Following the treatment period, the test materials were removed. Cells were washed with media, and then incubated for 1 hour with kit reagents. Caspase 3 activity was measured using a microtiter plate spectrophotometer at excitation 485 nm and emission 530 nm.
In vitro immunological characterization of sterility
NCL protocols STE-1.3 (turbidity Limulus Amoebocyte
Lysate (LAL)) and STE-2 were followed; complete experimental details can be found on the NCL website (http://ncl.cancer.gov/assay_cascade.asp
). Compound 1
was tested at 1 mg/mL concentration.
Hemolysis and platelet aggregation
NCL protocols ITA-1 and ITA-2 were followed for this assay; complete experimental details can be found on the NCL website (http://ncl.cancer.gov/assay_cascade.asp
). Hemolysis was assessed at four concentrations of 1
(1.0, 0.2., 0.04 and 0.008 mg/mL) using Triton-X as the positive control and PBS as the negative control. Three independent samples were prepared for each concentration and analyzed in duplicate (%CV<25).
NCL protocol ITA-5 was followed for this assay; complete experimental details can be found on the NCL website (http://ncl.cancer.gov/assay_cascade.asp
). Dendrimer 1
was tested at a concentration of 1 mg/mL. Two independent samples were prepared and each sample was analyzed in duplicate. PBS and cobra venom factor were used as the negative and positive control, respectively.
In vivo toxicity
Immediately prior to dosing, both 1 and Abraxane were suspended in PBS. The animal model utilized was 7-week-old female athymic nu/nu mice, three animals per treatment group. The study included four treatment groups (10, 25, 50 and 100 mg paclitaxel/kg) for both 1 and Abraxane, with PBS vehicle control. Treatments were administered by tail vein at 5 mL/kg body weight, once per week for three consecutive weeks. Animals were monitored daily for mortality, and signs of pharmacologic or toxicologic effects. Body weights were measured twice weekly and at study termination. Moribund animals (>20% loss in body weight) and animals surviving to planned study termination on day 21 were euthanized by CO2 asphyxiation, and blood was collected by cardiac puncture for hematological and clinical chemistry analysis. Animals were acclimated to the study environment for two weeks prior to study initiation. Animal rooms were kept at 50% relative humidity, 68–72º F with 12 h light/dark cycles. Mice were housed by treatment group, with 3 animals/cage (Thoren), with ¼″ corncob bedding. Animals were allowed ad libitum access to Purina 5L79 rodent chow and RO water. Statistical analyses were conducted using the software program Statistica version 7.1 (StatSoft, Inc., Tulsa, OK). Statistical differences for parametric data were determined by ANOVA, with post-hoc comparisons by Dunnet’s T test or Neuman-Keuls test. Nonparametric data was analyzed by the Kruskal-Wallis ANOVA with multiple comparisons test.
NCI-Frederick is accredited by AAALAC International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the “Guide for Care and Use of Laboratory Animals” (National Research Council; 1996; National Academy Press; Washington, D.C.)
Tissue Culture and Animal Model
The PC-3 cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA). PC-3 cells or PC-3-h-luc (permanently transfected with luciferase gene) were cultured in T-media at 37º C in an atmosphere of 5% CO2 and were passaged at 75% confluence in P150 plates. T-media was supplemented with 5% Fetal Bovine Serum (FBS) and 1 × Penicillin/Streptomycin. Cultured cells were harvested from monolayer using PBS and trypsin/EDTA, and suspended in T-media with 5% FBS. The cell suspension was then mixed 1:1 with Matrigel™ and injected subcutaneously (2.5 × 106 cells per injection, injection volume 100 μL) into the front flanks (for biodistribution) or the nape of the neck (for treatment) of male SCID (Severe combined immunodeficiency) mice with 6 – 8 weeks of age. After the cell injection, the animals were monitored three times a week by general observations. The tumor was noticed to grow in the first week and allowed to grow three weeks to reach a palpable size for biodistribution or therapeutic efficacy evaluation studies.
Biodistribution studies in SCID mice bearing PC-3 xenografts
For biodistribution study, 12 mice bearing two tumors (50 – 500 mg) in the front flanks were randomized into 3 groups (n = 4; 4 h, 24 h, and 48 h). Each mouse was intravenously injected with 100 μL of 125I-radiolabeled 1 (~5 μCi/mouse). Mice were sacrificed at 4 h, 24 h, and 48 h post-injection (i.p.). Organs of interest (blood, heart, lung, fat, liver, spleen, kidney, stomach, intestines, muscle, femur, thyroid, brain, and tumors) were harvested and weighed, and radioactivity was quantified by γ-counter. Standards were prepared, weighed, and counted along with the samples for %ID/g and %ID/organ calculation.
Therapeutic Efficacy Evaluation
PC-3-h-luc tumors were allowed to grow in the nape of the neck for three weeks prior to the first administration of 1, which was dissolved in Dulbecco’s Phosphate Buffered Saline (DPBS) at a concentration of 50 mg PTX/mL. The tumor-bearing mice were randomized into 5 groups for treatment (n = 6): 100 mg/kg PTX equivalents (single dose; 100s); 100 mg/kg PTX equivalents (double doses administered with a 7-day interval; 100d); 200 mg/kg PTX equivalents (single dose; 200s); 200 mg/kg PTX equivalents (double doses administered with a 7-day interval; 200d); and a PBS control. The administration was through the tail vein. Tumor size in mm3 was estimated using the formula (π/6)W2 × L, where “L” is the longest diameter of the tumor and “W” is the width perpendicular to the longest diameter. The tumor size was measured every other day and the tumor cell viability was monitored by a Bioluminescence Imaging system (BLI) weekly. To minimize the individual animal difference, the tumor volume ratio (tumor size change in a mouse) at a given day was referenced to the tumor size of the same mouse at day 4, which was normalized to 1 for all animals. Statistical analysis was performed by using Prism statistic program (GraphPad, San Diego, CA). Body weights were monitored throughout.
Bioluminescent imaging (BLI)
In vivo BLI was used to noninvasively monitor the tumor cell viability in mice bearing PC-3-h-luc tumor. Prior to imaging, mice were anesthetized by inhaling an isoflurane-oxygen mixture (3% isoflurane, 3% oxygen) and then injected intradermally with 80 μL of 40 mg/mL of D-Luciferin (Gold Biotechnology Inc., St. Louis, MO) in PBS. An IVIS Cooled Charge Coupled Device (CCD) camera apparatus (Xenogen Corp., Alameda, CA) was used for capturing photon emissions from the tumors with an acquisition time between 5 sec to 2 min depending on the photon intensity. Living Image software package from Xenogen was used for imaging data analysis in the regions of interest (ROI) and the maximum values was obtained in photon/second/cm2/steradian. Prism statistic program (GraphPad, San Diego, CA) was used for statistic data analysis.