Polymer and Peptide Conjugation
The synthesis, detailed methodology, and complete characterization of this nanocarrier system is described in our previous publication. 14
For the synthesis of the PLGA-PEG-peptide construct, an established EGFR specific peptide was used to achieve active targeting with the nanoparticle formulation: YHWYGYTPQNVIGGGGC; the carboxyl terminal cysteine of the peptide reacts with the maleimide of the PLGA-PEG construct. The peptide was synthesized by Tufts University Core Facility, Boston, MA. For the conjugation, 50:50 poly(DL-lactide-co
-glycolide) (PLGA) with an inherent viscosity of 0.15–0.25 (Durect Lactel®
Adsorbable Polymers; Pelham, AL) was used. To synthesize the PLGA-PEG-peptide targeting construct, amine-poly(ethylene glycol) PEG-maleimide (MW 2000; JenKem Technology; Allen, TX) was used, while m-PEG-amine (MW 2000; LaysanBio; Arab, AL) was used to create a (non-targeted) PLGA-PEG construct.
Nanoparticle and Drug Solution Preparations
EGFR targeted and non-targeted polymer blend nanoparticles were synthesized using a solvent displacement method. Poly(ε-caprolactone) (average MW 14.8 kDa; Polysciences, Inc., Warrington, PA) was used as the primary nanoparticle constituent. Briefly, the PLGA-PEG-peptide conjugate (or the PLGA-PEG conjugate for non-targeted particles), PCL, and therapeutic agents were dissolved in 2 mL 50/50 acetonitrile/DMF, and placed in a 37°C water bath for 10 minutes to facilitate dissolution. This polymer/drug solution was added dropwise to 20 mL distilled, deionized water while stirring. The preparation was covered with aerated parafilm, allowed to stir overnight, centrifuged at 10,000g for 30 minutes, and then resuspended in deionized water. To synthesize targeted nanoparticles the PLGA-PEG-peptide conjugate was added to the PCL nanoparticle formulation at 20% w/w total polymer, with an additional 10% w/w of PLGA-PEG conjugate. For the non-targeted nanoparticles, the PLGA-PEG conjugate was added at a concentration of 20% w/w total polymer. Dual agent loaded nanoparticles were synthesized with a 10:1 molar ratio of lonidamine to paclitaxel. Scanning electron microscopy (SEM) images of the nanoparticles were obtained using a Hitachi S-4800 microscope.
Drug solutions for animal treatments were prepared as Cremophor® EL (polyoxyethylated castor oil) stocks. Each mL contained 3 mg paclitaxel, 12 mg lonidamine, 527 mg of Cremophor® EL (BASF, Mount Olive, NJ, USA), and 49.7% (v/v) dehydrated alcohol, USP (Thermo Fisher Scientific, Waltham, MA). Drug solutions for HPLC calibration were prepared in 50:25:25 (v/v) methanol, acetonitrile, water mix. Docetaxel was used as an internal standard at a concentration of 85 µg/ml. Lonidamine and paclitaxel standard curves were prepared at the following concentrations; 200 µg/ml, 40 µg/ml, 8 µg/ml, 1.6 µg/ml, 0.32 µg/ml, 0.2 µg/ml, 0.16 µg/ml, 0.08 µg/ml, and 0.064 µg/ml. To determine the extraction efficiency, organs and plasma from control mice (untreated) were spiked with each concentration of lonidamine and paclitaxel used in the solution standard curve at µg/ml or ug/g of tissue. The y-axis of each calibration curve (data not shown) was plotted as the peak area of drug (lonidamine or paclitaxel) divided by the peak area of docetaxel, while the x-axis was the concentration. The percent recovery was then determined by first dividing the slope of calibration curve in spiked tissue by the slope of the standard curve of drug solution and then multiplying this value by 100.
Cell Culture and Hypoxia
The MDA-MB-231 cells were obtained from ATCC (Manassas, VA). Cells were incubated at 37°C and maintained in RPMI-1640 media (Mediatech, Inc; Manassas, VA) supplemented with 10% fetal bovine serum (Gemini Bio-products; West Sacramento, CA) and 1% penicillin/streptomycin/amphotericin B mixture (Lonza; Walkersville, MD). To create hypoxic conditions using low-oxygen gas; cell culture flasks were placed in a modular incubation chamber (Billups-Rothenberg, Inc.; Del Mar, CA), flushed with a 0.5% O2, 5% CO2, nitrogen balanced gas for five minutes, and incubated at 37°C for five days.
Orthotopic Tumor Model Development
The protocol for animal experiments described in this article was approved by Northeastern University’s Institutional Animal Care and Use Committee. Female nu/nu mice were obtained from Charles River Laboratories (Wilmington, MA) and were housed in sterile cages on a 12:12 light/dark cycle with ad libitum acess to food and water. To establish the xenografts, mice were anesthetized with isoflurane and approximately 2 million human breast cancer cells suspended in a 100 µl of a 50:50 mix of matrigel and serum free medium was injected into the mammary fat pad of the mice using pre-chilled, sterile syringes with 27 gauge, ½” needles. Tumor size was measured every other day using Vernier calipers in two dimensions. Individual tumor volumes (V) were calculated using the formula V = [length × (width)2]/2 where length is the longest diameter and width is the shortest diameter perpendicular to length.
Treatment and Tissue Preparation
Once tumors reached 100 mm3, the animals were selected for experimental treatment. A total of 48 mice were used to assess treatment in the pharmacokinetic study (not including control mice). Mice were randomly allotted to the three treatment groups (paclitaxel and lonidamine loaded EGFR-targeted nanoparticles, paclitaxel and lonidamine loaded non-targeted nanoparticles, and paclitaxel and lonidamine solution). Each group was further divided into four subgroups based on post-administration time points for analysis/animal sacrifice (1 hour, 3 hours, 6 hours, and 24 hours). Data from all four subgroups (1 hour, 3 hours, 6 hours, and 24 hours) was used for plasma and tumor tissue biodistribution analysis and to determine pharmacokinetic parameters; biodistribution in the vital organs was conducted at 1 hour and 6 hours post-administration. Treatment was administered via tail vein injection as a single 125 µL dose of 80 mg/kg lonidamine and 20 mg/kg paclitaxel.
At the established post-administration time points, 4 animals from each of the three treatment groups were euthanized by carbon dioxide inhalation. Pre-sacrifice, blood samples were collected from the mice via retro-orbital bleeding; approximately 200 µL of blood was collected. Before bleeding, the mice were anesthetized by isoflurane inhalation. StatSpin®
Microtubes (StatSpin, Inc., Norwood, MA) and capillaries were used for blood collection, and were immediately centrifuged for plasma collection. After euthanasia, the tumor mass, liver, lungs, kidneys, spleen, and heart were harvested and weighed. Tissue and plasma samples were then prepared with adaptations according to established methods for the extraction of lonidamine in preparation for HPLC analysis 19
. Organs were first spiked with a solution of the docetaxel internal standard (85 µg/ml or per gram of tissue), then the organs were homogenized in a 10:1 (v/w) ratio of buffer (10 mM Tris, 1mM EDTA, and 10% (v/v) glycerol (pH 7.4)). Samples were then centrifuged for 15 minutes at 16,000g
, extracted with ethyl acetate at a 1:2 (v/v) ratio, evaporated under nitrogen, and resuspended in 100µl of 50:25:25 (v/v) methanol, acetonitrile, water. Plasma samples were collected from the StatSpin®
Microtubes and acidified with 1M HCL at a ratio of 1.5:1 (v/v). The sample was then extracted with ethyl acetate at a (v/v) ratio of 1:1.3, evaporated under nitrogen, and re-suspended in the initial plasma volume of 50:25:25 (v/v) methanol, acetonitrile, water.
HPLC Method Development and Analysis
The HPLC method was developed as an adaptation of established methods for lonidamine and paclitaxel quantification. An isocratic, reversed-phase HPLC method was developed using a SunFire C18 Column (5 µm particle size, 4.6×250 mm) (Waters; Milford, MA). The system used was a Waters system (Separations Module, 2695; Waters Corporation, Milford, MA) consisting of two pumps, an autosampler, and UV-detector (Model 2487). The user interface consisted of Waters Empower chromatography data software, which was used for instrument management, data acquisition, and processing. The mobile phase consisted of acetonitrile and water (51:49, v/v) containing 0.1% trifluoracetic acid. The column was equilibrated at a flow rate of 1 mL/min. The elution was monitored at 230 nm. The sample injection volume was 80 µl. HPLC glass vial inserts (Waters; Milford, MA) were used for samples with low volume yields.
Pharmacokinetic Parameter Data Analysis
Pharmacokinetic parameters were determined by using a recently published, free Microsoft Excel add-in, “PKSolver”. The specifications of this program as well as validation was published in the January 2010 edition of Computer Methods and Programs in Biomedicine
Non-compartmental analysis was used to determine the pharmacokinetic parameters.
Preparation of Dye-Loaded Nanoparticles and Animal Imaging
Nanoparticles were loaded at 1% w/w with the near IR fluorescent dye DiR (Invitrogen; Carlsbad, CA). The loading efficiency of both non-targeted and targeted nanoparticles was between 65–70% as determined by measuring the supernatant intensity of six nanoparticle batches relative to a standard curve of DiR near infra-red fluorescence. There was also minimal release of the dye from the nanoparticles in PBS for up to 24 hours. The nanoparticles were administered at the same w/v ratio as the paclitaxel/lonidamine combination nanocarriers (20 mg nanoparticle polymer/ 125 µl).
This study used 14 female athymic mice with 100 mm3 tumors established in the mammary fat pad. Seven mice were injected with each formulation; non-targeted nanoparticles loaded with DiR or EGFR-targeted nanoparticles loaded with DiR. Imaging was done at the following post-administration time points; 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, and 6 hours. At the established time points, mice were euthanized via carbon dioxide inhalation and rapidly imaged using a Kodak FX Imaging Station (Rochester, NY). Fluorescent fields were directly overlaid on the x-ray images using the Kodak software.
Statistical Analysis and Graphing
All graphs were made using GraphPad Prism® Software. The plasma concentration and tumor concentration graphs were plotted as mean and coefficient of variation (%) values of data obtained from HPLC analysis; the biodistribution data was plotted as mean and standard deviation values. For each data point, n=4. GraphPad Prism® Software was also used to analyze data and determine significance by using one-way ANOVA and Tukey's Multiple Comparison Test.