The 20-mer PO oligonucleotides bearing a 5-aminohexyl tether were purchased from Beijing Tsing Biotech Co., Ltd. (Beijing, China). The antisense and sense sequences used here were 5’-GGGAGTTACTTGCCAACTTG-3’ and 5’-CAAGTTGGCAAGTAACTCCC-3’, respectively. (HEPES), HCl (30%), acetonitrile (ACN), sodium chloride, sodium acetate, methanol, ammonium acetate, and ethanol (96%) were obtained from Merck (Darmstadt, Germany). p-SCN-Bn-NOTA) was purchased from Macrocyclics (Dallas, TX, USA). Foetal bovine serum (FBS) was obtained from Biosharp. Glen-Pak DNA purification cartridges were purchased from Glen Research. Pure water (18.2MΩ cm) was used in all reactions. Unless stated otherwise, all other chemicals used for tracer synthesis were obtained either from Acros (Geel, Belgium) or Sigma-Aldrich.
Aqueous triethylammonium acetate (TEAA) (1 M, pH 7.0) was prepared with 1 M acetic acid and 1 M TE (Sigma-Aldrich). A Thermo LCQ Deca XP plus Mass Spectrometer was used to record ESI-MS spectra. Products were isolated and purified using a HPLC C18 column (1200 series, Zorbax ODS 4.6 × 250 mm). HPLC conditions were as follows: A/B gradient; A: 2% acetonitrile in 0.1 M TEAA (pH 7.0); B: 50% acetonitrile in 0.1 M TEAA (pH 7.0); elution: 1 mL/min. A 68Ge-68Ga generator (ITG Isotope technologies) was used to produce [68Ga] Cl3.
Oligonucleotides were functionalized at the 5′ end with hexylamine to optimize the bioconjugation and radiolabelling conditions. Figure 1 showed the conjugation of p-SCN-Bn-NOTA and MALAT-1 ASO and subsequent 68Ga complexation. MALAT-1 (100 nmol) possessing 5’-aminoalkyl linkers was dissolved in NaHCO3 (15 μL of 0.1 M). Equivalents of p-SCN-Bn-NOTA (20 mg/mL) were added for 12 h at room temperature. Modified MALAT-1 ASO was then separated via the DNA purification cartridge (GlenPak) and products were purified via. HPLC. MALAT-1-ASO was ethanol-sodium acetate precipitated, HPLC purified and vacuum concentrated. NOTA-ASO samples were aliquoted, lyophilized, and frozen until use.
68Ga-labelling of MALAT-1 antisense oligonucleotides (68Ga–MALAT-1 ASO)
The synthetic route is shown in Figure 1. We produced 68Ga as [68Ga]Cl3 in the 68Ge/68Ga generator via elution with 0.05 N aq. HCl. The 68Ga3+ eluate was mixed with the solubilized bioconjugate (5-12 nmol) that was dissolved in 1M HEPES (final pH 4.0–4.2). Reactions proceeded for 10 min at room temperature. 68Ga–MALAT-1 ASO was separated on purification cartridges and sense oligodeoxynucleotides (SO) were treated as described for ASO. Radio-HPLC was used for the confirmation of yields.
Quality control and stability
The synthesized 68Ga-MALAT-1 ASO was assayed for purity via HPLC, pH using indicator paper and the absence of suspended precipitates by analytical HPLC.
In Vitro Stability Analysis
In vitro stability studies of 68Ga-MALAT-1 ASO were performed in foetal bovine serum as well as in phosphate-buffered saline (PBS) solution. Briefly, 50 µL of 68Ga-MALAT-1 ASO (1 MBq) was mixed with FBS at 37°C for 30 min, 60 min, and 2h. Acetonitrile (0.5 mL) was then added to precipitate serum proteins and radio-HPLC was performed to determine serum stability. Fifty microliters of 68Ga-MALAT-1 ASO (1 MBq) was mixed with PBS (10 mM, 450 µL) for 2 h and radio-HPLC was used to confirm stability.
Partition coefficient studies
Partition coefficients (Log P) of 68Ga-MALAT-1 ASO were measured through the assessment of the distribution of radioactivity in 1-octanol and phosphate buffer in a 2 mL centrifuge tube. A 10 µL of 68Ga-MALAT-1 ASO solution was added to PBS + 1-octanol (total volume: 1 mL) and centrifuged (5 min at 5000 rpm). A total of 2 samples (50 μL) taken from each layer were assayed in a γ counter. Partition coefficients (log Po/w) are shown as the log-counts in 1-octanol vs PBS layers (n=3).
ICR female mice (body weight 18–20 g), and BALB/c nude mice (6 to 8 weeks of age) were purchased from Jiangsu Gempharmatech. The mice were raised and managed on a standard diet with free water access at our institute. All animal protocols were approved by our internal review board and followed the standard care procedures for animal use (National Research Council of USA, 1996).
For the pharmacokinetic studies, six female ICR mice weighting 18–20 g was administrated with 68Ga-MALAT-1 ASO (7.4 MBq, 0.2 mL) by IV injection into the caudal vein. Blood samples (10 μL) were obtained from the tail at various time points from 3 min to 120 min following 68Ga-MALAT-1 ASO injection. Radioactivity was calculated as the % of dose per g of tissue per body weight (%ID/g) expressed over time. Pharmacokinetics were assayed using DAS, version 2.1.1.
Evaluation of cellular uptake
MHCC-LM3 cells (a gift from the Key Lab, State Health Commission) were cultured in complete DMEM plus 10% FBS and 100 mg/mL pen/strep. 5’(Cy3.0)-MALAT-1 ASO was transfected into cells using Lipofectamine 2000 and confirmed by immunofluorescence .
MHCC-LM3 cells seeded into 6-well plates (2.5× 106 cells per well) to 80% confluency were treated with 68Ga-MALAT-1 ASO (final concentration of 100 nM, 0.55 MBq 68Ga-MALAT-1 ASO) for different times (30, 60, 90,120, and 240 min). Mixtures were then washed in ice-cold PBS and centrifuged to discard the supernatants. The radioactivity in the precipitate was quantitatively measured on a γ-counter. Blocking cellular uptake by the addition of excess PO-ASO (1 µM) was studied. These experiments were repeated four times under the same conditions.
Normal ICR female mice (18–20 g) were randomly divided into four groups (n=5), and the radiolabelled preparation (0.1 mL, 0.37 MBq) was IV administered into the tail vein. After 15, 30, 60, and 120 min, individual mice were sedated with CO2-O2 and blood, brain, heart, liver, spleen, lung, kidney, stomach, intestine, muscle, pancreas, thyroid, fat, bone, thymus, adrenal gland, and urinary bladder samples were taken and weighed. Radioactivity in each organ was assessed on a γ-counter. Values are shown as the percentage of the injected dose per gram (% ID/g) which corrected for background and decay to maintain consistency.
Biodistribution in MHCC-LM3 xenograft-bearing nude mice
BALB/c nude mice (average weight 20±3 g, age 4-to 6-wk-old) were inoculated subcutaneously with 2.5×106 tumour cells into the right axilla. Tumor growth was monitored for 3-4 weeks until the tumors reached 0.5-1.0 cm3 in volume.
Model mice were divided into four groups (n=5), which were individually injected with 3.7 MBq of 68Ga-MALAT-1 ASO via the tail vein under anaesthesia. For the non-blocked studies, mice were sacrificed and their blood, tumor and organ samples were harvested and weighed at 30, 60, and 120 min post-injection. For the blocked group of mice, we injected unlabelled MALAT-1 P-O ASO (100 nmol) followed by 0.1 Ml of 68Ga-MALAT-1 ASO (370kBq) into the tail vein after 30 min. Organ radioactivity was measured as described and expressed as % ID/g.
In vivo micro-PET/CT imaging
Mice with MHCC-LM3 tumours were divided into group 1 (antisense group), 2 (sense group) and 3 (blocked group), (n=4 for each group). Mice were injected with 0.1 mL of 68Ga-MALAT-1 ASO (3.7 MBq) via tail vein. The images were performed at 30, 60 and 90 min after injection using a micro PET/CT scanner (Siemens Inveon MultiModality system) while the mice were maintained under isoflurane anaesthesia (1.5% isoflurane, 3% oxygen). A blocking study was performed in which 100 nmol of unlabelled MALAT-1 ASO was intravenously administered 30 min before the intravenous injection of 68Ga-MALAT-1 ASO (3.7MBq). Three-dimensional volumes of interest (VOIs) were used for the assessment of %ID/g and standard uptake value (SUV) in selected organs.
Quantitative data were showed as the mean ± SD, and means were compared via one-way ANOVA with SPSS V.22.0 software. P-values ≤ 0.05 were considered significant differences.