All animal procedures were performed in compliance with Boston Children’s Hospital guidelines, and protocols were approved by the Institutional Animal Care and Use committee.
Log-D measurement for TNP-470
Aqueous solubility is one of the important chemical properties affecting oral absorption of a drug. In order to predict the intestinal absorption of TNP-470, we measured its log-D value which is a parameter of hydrophobicity determined by the ratio of drug concentration in octanol to that in water at 25 °C (Analiza). High log-D values (>2) indicate low water solubility and hence a poor oral availability of a drug. For this study log-D values of TNP-470 (30 mM) were measured at plasma and stomach pHs: pH = 7.4 and pH = 2, respectively.
Preparation of Lodamin: mPEG-PLA-TNP-470 polymeric micelles
TNP-470 (D. Figg) was conjugated to a diblock co-polymer using a two-step reaction (). In the first step succinated mPEG2000-PLA1000 with free carboxic acid end-groups (Advanced Polymers Materials) was reacted with ethylenediamine (Sigma-Aldrich). Succinated mPEG-b-PLA-OOCCH2CH2COOH (500 mg) was dissolved in DMSO and reacted with ethyl(diethylaminopropyl) carbodiimide (EDC) and a catalyst N-hydroxysuccinimide (NHS) in a molar ratio of 1:10:20 (Polymer:EDC:NHS, respectively). A fivefold molar excess of ethylendiamine was added and reacted for 4 h at 25 °C. The polymer solution was then dialyzed (MWCO 1000, Spectra/Por Biotech Regenerated Cellulose, VWR) against DMSO leading to a 65% reaction efficiency. In the second step, the amine-containing polymer was mixed with TNP-470 (350 g), dissolved in DMSO and the solution was stirred for 4 h at 25 °C. The polymeric micelles were formed by dialyzing the DMSO solution of the conjugate against double distilled water (d.d.w.) using a regenerated cellulose dialysis bag (MWCO 1000, Spectra/Por Biotech Regenerated Cellulose, VWR) to obtain micelles with high incorporation efficiency (>90%) and 0.8–1% free drug (wt/wt). The micelles were then lyophilized and stored at −20 °C in a dry environment until use.
Prepartion of fluorescently labeled mPEG-PLA micelle
Rhodamine-labeled polymeric-micelles were formed using a similar protocol. The mPEG-PLA was conjugated via the N-terminal amine group with lissamine rhodamine B sulfonyl chloride (Molecular Probes) in DMSO. For green fluorescent polymer micelles, a commonly used hydrophobic marker 6-coumarin (Sigma-Aldrich) at 0.1% wt/wt was added to the polymeric solution before the final dialysis step.
Characterization of conjugation
NMR spectrometer analysis (NERCE/BEID, Harvard Medical School) was conducted for each reaction step and mass spectrometry (Proteomic core, Harvard Medical School) was performed on the conjugate. To evaluate the efficiency of ethylendiamine binding to mPEG-PLA (first reaction step, ) and TNP-470 binding to the polymer by the amine (second step, ), we used the colorimetric amine detection reagent: 2,4, 6-trinitrobenozene sulfonic acid (TNBSA) (Pierce). TNBSA reacts with primary amines to produce a yellow product whose intensity was measured at 450 nm. To calculate amine concentration in the polymer a linear calibration curve of amino acid was used. To measure TNP-470 loading into polymeric micelles, we incubated 10 mg/ml Lodamin in 500 μl NaOH (0.1 N) to accelerate PLA degradation. After an overnight incubation with shaking (100 r.p.m) at 37 °C, we added acetonitrile to the samples (1:1 NaOH:acetonitrile) and analyzed for TNP-470 concentration. TNP-470 concentration was measured using HPLC (System Gold Microbore, Beckman Coulter). A 20-μl portion of each sample was injected into a Nova-pak C18 column (3.9 mm × 150 mm i.d.; Waters) and analyzed using a calibration curve of TNP-470. TNP-470 binding to amine was also measured using TNBSA reagent and was confirmed by subtracting the nonbound drug from the total drug added to the reaction.
Lodamin size, morphology and in vitro TNP-470 release
The particle size distribution of Lodamin was measured by Dynamic Light Scattering (DLS, DynaPro, Wyatt Technology). The measurements were done at 25 °C using Dynamic V6 software. Lodamin (1.5 mg/ml) dispersed in d.d.w. was measured in 20 successive readings of the DLS.
To study the morphology of Lodamin, TEM images were taken on the day of preparation and 1 week after preparation. Polymer micelles dispersed in d.d.w. were imaged with cryo-TEM (JEOL 2100 TEM, Harvard University—CNS). To study the kinetic release of TNP-470 (without its chlorine), Lodamin (20 mg) was incubated with either 1 ml PBS pH = 7.4 or simulated gastric fluid (HCL:d.d.w. pH = 1.2). Every few days, supernatant was taken and analyzed for TNP-470 concentration and a cumulative release graph of TNP-470 was determined. TNP-470 concentration was measured using HPLC. TNP-470 was detected as a peak at 6 min with 50% acetonitrile in water at the mobile phase. The flow rate was 1 ml/min, and the detection was monitored at 210-nm wavelength.
Murine LLC and B16/F10 melanoma cells were obtained from American Type Culture Collection. HUVECs were purchased from Cambrex. The cells were grown and maintained in medium as recommended by the manufacturers. Dulbecco’s Modified Eagle’s Medium with 10% FBS was used for tumor cells and EMB-2 (Cambrex Bio Science) containing 2% FBS and EGM-2 supplements was used for HUVECs.
Uptake of polymeric micelles by HUVECs and their localization in cells
To evaluate the uptake of polymeric micelles by HUVEC, we used 6-coumarin-labeled mPEG-PLA micelles or rhodamine conjugated to mPEG-PLA. HUVECs were seeded in a 24-well plate (2 × 104 per well) in EGM-2 medium (Cambrex) and were allowed to attach overnight. Fluorescent-labeled micelles (10 mg/ml) were suspended over a bath sonicator for 5 min, and 20 μl of the suspension was added to the cultured cells. After the designated time points (20 min, 2, 4, 7 and 24 h), the cells were washed three times with PBS and analyzed by FACS or alternatively fixed with 4% parformaldehyde. For confocal microscopy, cells were mounted using DAPI containing Vectashild (Vector Laboratories). Optical sections were scanned using Leica TCS SP2 AOBS, a ×40 objective equipped with 488-nm argon, 543-nm HeNe and 405-nm diode lasers. To study Lodamin internalization into endothelial cells, we used confocal microscopy to co-localize 6-coumarin–labeled polymeric micelles with endolysozome. Live HUVECs were imaged in different time points after addition of labeled micelles to cell medium (15 μg/ml) up to 1 h. At this point, LysoTracker Red (Molecular Probes) was added to the medium for the detection of acidic intracellular vesicles: endosomes and lysosomes. After 20 min of incubation, cells were imaged by confocal microscopy using optical sections with 488-nm argon, 543-nm HeNe and 405-nm diode lasers.
To further verify that Lodamin internalization occurs through endocytosis, we measured cell uptake in cold conditions (4 °C) in comparison to cell uptake at 37 °C. HUVECs were plated in a concentration of 15,000 cells/ml in two 24-well plates for 24 h. Fluorescence-labeled polymeric micelles (15 μg/ml) were added and incubated at different time points: 20, 30, 40 and 60 min (n = 3) in 4 °C and in 37 °C. After the designated time points, the cells were washed three times with PBS and lysed with 100 μl lysis buffer (BD Biosciences). Cell extracts were measured for fluorescent signal in a Wallac 1420 VICTOR plate-reader (Perkin-Elmer Life Sciences) with excitation/emission at 488 nm/530 nm.
HUVEC growth and proliferation
HUVECs were exposed to different concentrations of Lodamin equivalent to 50–1,000 nM free TNP-470 (0.12–2.4 mg/ml micelles) and incubated in a low serum medium for 48 h at 37 °C. To rule out a possible cytotoxic effect of the carrier, empty micelles were added to HUVECs at the same concentration as the control (4.8 mg/ml). A WST-1 proliferation assay (Roche Diagnostics) was used. Cell viability was calculated as the percentage of formazan absorbance at 450 nm of treated versus untreated cells. Data were derived from quadruplicate samples in two separate experiments. The effect of Lodamin (60 nM TNP-470 equivalent every other day) on HUVEC growth rate was evaluated by daily counting of HUVECs up to 5 d and comparing this to the number of untreated cells or cells treated with vehicle (same concentration as Lodamin).
Corneal micropocket assay
To evaluate the antiangiogenic properties of Lodamin, the corneal micropocket angiogenesis assay was performed as previously detailed29
. Pellets containing 80 ng carrier-free recombinant human bFGF or 160 ng VEGF (R&D Systems) were implanted into micropockets created in the cornea of anesthetized mice. Mice were treated daily with 15 mg/kg TNP-470 equivalent of Lodamin for 6 d, and then the vascular growth area was measured using a slit lamp. The area of neovascularization was calculated as vessel area by the product of vessel length measured from the limbus and clock hours around the cornea, using the following equation: vessel area (mm2
) = (π × clock hours × vessel length (mm) × 0.2 mm).
Body distribution, intestinal absorption and toxicity of Lodamin
For all biodistribution studies we used a fluorescent marker for tracking Lodamin. Mice were administered 6-coumarin–labeled mPEG-PLA by oral gavage for 3 d (100 μl of 1.5 mg/ml). On the third day of treatment, after 8 h of fasting, animals were killed and spleen, kidney, brain, lungs, liver, intestine, stomach and bladder were collected. The fluorescent 6-coumarin was extracted from the tissues by incubation with formamide for 48 h at 25 °C. Samples were centrifuged and signal intensity of fluorescence of supernatants was detected with a Wallac 1420 VICTOR plate-reader (Perkin-Elmer Life Sciences) with excitation/emission at 488 nm/530 nm. The results were normalized to protein levels in the corresponding tissues. Tissue autofluoresence was corrected by subtracting the fluorescent signal of nontreated mouse organs from the respective readings in treated mice. Similarly, levels of fluorescent signal in mouse sera were measured at different time points (1, 2, 4, 8, 24, 48 and 72 h) using excitation/emission readings at 488 nm/530 nm.
To analyze cell uptake in the different tissues in tumor-bearing mice, we administered orally mPEG-PLA-rhodamine micelles (100 μl of 1.5 mg/ml) or water to C57Bl/6J mice bearing LLC tumors (200 mm3) for 3 d. Organs were removed, incubated for 50 min in collagenase (Liberase Blendzyme 3; Roche Diagnostics) in 37 °C to obtain a single-cell suspension. These suspensions were analyzed by FACS to quantify the uptake of micelles into different tissue cells when compared to those in the untreated mouse.
To evaluate intestinal absorption, mPEG-PLA-rhodamine micelles were orally administered to C57Bl/6J mice after 8 h of fasting. After 2 h mice were killed and 2.5-cm segments of the small intestine were removed, washed and analyzed by histology and confocal microscopy. The rhodamine-labeled polymeric micelles were detected by confocal microscopy (Leica TCS SP2 AOBS) with a 488-nm argon laser line. Actin filaments were stained with phalloidin-FITC (Sigma) and nuclei were stained by DAPI (Sigma). To further study the uptake of Lodamin in the intestine, high-resolution images were made with cryo-TEM. Intestines from treated (as above) and untreated mice were excised and immersed immediately in a freshly prepared 4% paraformaldehyde in PBS pH 7.4 for 2 h at 25 °C. The samples were washed in PBS, transferred to a 30% sucrose solution overnight at 4 °C and embedded in OCT and kept at −80 °C until processing. Fifteen sections, each 10 μm thick, were prepared and processed for confocal microscopy or TEM. Four TEM samples were fixed for 30 min in freshly prepared 2% paraformaldehyde, 2.5% glutaraldehyde, 0.025% CaCl2 in 0.1 M sodium cacodylate buffer, pH 7.4 and subsequently postfixed for 30 min in 1% osmium tetroxide in 0.1 M sym collidine buffer, pH 7.4 at 25 °C, stained en bloc in 2% uranyl acetate, dehydrated and embedded under inverted plastic capsules. Samples were snapped free of the glass cover-slips by a cycle of rapid freezing and thawing. Thin sections were cut en face with diamond knives using a LEICA UCT Ultramicrotome. Specimens were examined using a JEOL 2100 TEM.
To exclude tissue toxicity, histological analysis (H&E) of liver, intestine, lung and kidneys was conducted (Beth-Israel pathology department). To further exclude liver toxicity, we analyzed serum levels of the liver enzymes AST and ALT (done at Shriners Burns Hospital). These studies were performed on mice treated with Lodamin for 20 d (15 mg/kg TNP-470 equivalent per day) and compared to untreated mice (n = 3–4).
Oral administration of Lodamin in vivo and primary tumor experiments
Animal procedures were performed in the animal facility at Children’s Hospital Boston using 8-week-old C57Bl/6J male mice (Jackson Laboratories).
For tumor experiments: LLC cells (1 × 106) or B16/F10 melanoma cells (1 × 106) were implanted subcutaneously in 8-week-old C57Bl/6J male mice. Oral availability of free TNP-470 was compared to that of Lodamin. A dose of 30 mg/kg every other day of free TNP-470 and an equivalent dose of Lodamin (Lodamin, 6 mg in 100 μl/d per mouse) were administered to LLC tumor-bearing mice (~100 mm3) and tumor growth was followed for 18 d. Free drug was given as a suspension in d.d.w. and freshly prepared before each dose. Additionally, we compared different doses and frequencies of Lodamin treatment: 15 mg/kg every day, 15 mg/kg every other day and 30 mg/kg every other day. To eliminate any possible effect of the vehicle (polymer without drug), we gave one group of mice micelles without drug and tumor progression was compared to water-treated mice.
For the melanoma tumor experiment, a daily dose of 15 mg/kg Lodamin was administered to B16/F10 melanoma-bearing mice. In all experiments, tumor size and animal weight were monitored every 2 d. Tumor volume was measured with calipers in two diameters as follows: (width)2
× (length) × 0.52. Note that all the above Lodamin doses are presented as TNP-470 equivalent.
Oral administration of Lodamin and liver metastasis experiments
To examine the effect of oral Lodamin treatment on metastasis development and prevention, liver metastases were generated by spleen injection. C57Bl/6J mice (n = 14) were anaesthetized with isoflurane and prepared for surgery. A small abdominal incision was made in the left flank and the spleen was isolated. B16/F10 tumor cells in suspension (50 μl, 5 × 105 in DMEM medium without serum) were injected into the spleen with a 30-gauge needle, and the spleen was returned to the abdominal cavity. The wound was closed with stitches and metal clips. After 2 d mice were divided into two groups: one was treated daily with oral Lodamin (15 mg/kg) using gavage and the second group was administered water by gavage. After 20 d, we terminated the experiment. Mice were killed and autopsied, livers and spleens were removed by surgical dissection, imaged and histology was carried out. Liver and spleen tissues were stained with H&E to evaluate tissue morphology and detect metastasis. Immunohistochemistry was carried out to specifically detect B16/F10 cells in the liver using anti-mouse melanoma antibody (HMB45, abCAM) and using DAB staining.
Evaluation of neurotoxicity with balance beam motor coordination test
A slightly modified balance beam motor coordination test30
was performed on three groups of mice: oral Lodamin–treated mice (30 mg/kg eq. every other day), free TNP-470 (30 mg/kg every other day) subcutaneously injected mice and water-treated mice (administered by gavage). The mice were pretreated for 14 d (n
= 4–5 per group) and then the mice were allowed to acclimate to the procedure room for 1 h, after which they were trained in three trials to cross a wide (20 mm width × 1 m length) balance beam. All the mice crossed the wide beam without making foot-slip errors. The mice were then trained on a narrow (4 mm width × 1 m length) beam for three trials. At the end of the training trials, no freezing behavior was observed, and the mice would start to walk within 4 s of being placed on the beam. The mice were then videotaped as they performed in three test trials of three beam crossings each—a total of nine crossings per mouse. The three trials were separated by at least 1 h to avoid fatigue of the mice. Videotaped crossings were scored for number of foot-slip errors and time to cross. All experiments and scoring of the different groups were performed by a blinded investigator.
Lodamin effect on angiogenesis, proliferation and apoptosis in tumor tissues
Histologic evaluation of tissue was performed on 8-μm thick frozen sections of LLC tumors that were removed from two random Lodamin-treated or untreated mice 14 d after treatment (15 mg/kg every day. TNP-470 equivalent). Tumors were sectioned and analyzed for cell markers, 20 microscope fields (×400) were imaged.
Tissues were stained with H&E to detect tissue morphology. Immunohistochemistry was carried out using Vectastain Elite ABC kit (Vector Laboratories). Primary antibodies included CD31 (BD Biosciences) for microvessel staining and anti-Ki-67 (DAKO) for proliferating cell. Detection was carried out using a 3,3′-diaminobenzidine chromogen, which results in a positive brown staining. Apoptotic cells were detected by reacting the tissues with TUNEL using a kit (Roche) following the company’s protocol. Vessels were detected in the same tissues by anti-CD31 and secondary FITC anti-mouse antibody (Jackson ImmunoResearch) conjugated antibody (green) and nuclei were detected by DAPI (blue).
In-vitro data are presented as mean ± s.d., whereas in vivo data are presented as mean ± s.e.m. Differences between groups were assessed using unpaired two-tailed Student’s t-test, and P < 0.05 was considered statistically significant.