In vitro release of Ad from NPs in serum

In vitro release of Ad from NPs in serum Fetal bovine serum (3 ml) was prepared with 172 μl of EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine) [Ad deaminase inhibitor (1 mg/ml), NaCl 0.9%; Sigma-Aldrich] and 4.8 μl of dipyridamole [Ad uptake inhibitor (30 mg/ml), dimethyl sulfoxide; Sigma-Aldrich]. SQAd/VitE NPs (180 μl; 2 mg/ml) were incubated at different time intervals with 180 μl of the fetal bovine serum solution in various Eppendorf vials, each for one time point. At the predetermined time intervals (i.e., 5 min, 30 min, 2 hours, 15 hours, 24 hours, and 48 hours), aliquots (100 μl) were collected and added into 500 μl of MeOH to denature and precipitate the enzymes and proteins of the serum, which were removed after centrifugation (16,000g for 10 min). To quantify the remaining SQAd bioconjugate and the released Ad, the remaining supernatants (150 μl) were evaporated to dryness at 40°C under nitrogen flow and then solubilized in 100 μl of MeOH. Quantification was performed using a reversed-phase HPLC on a Halo C18 column (4.6 mm by 250 mm, 5 μm; Interchim), a 1525 Binary LC Pump (Waters), a 2707 Autosampler (Waters), and a 2998 PDA detector (Waters). The HPLC was carried out using a gradient elution with the mobile phase composed of 80% 10 mM potassium phosphate in Milli-Q water (pH 4.0) 20% MeOH (phase A) and MeOH (phase B). Elution was carried out at a flow rate of 0.8 ml/min. The system was held at 100% of A for 8 min, followed by 1-min linear gradient from 100% A to 100% B, kept at 100% B for 12 min, and brought back to initial condition by a 1-min linear gradient from 100% B to 100% A. The sample run was maintained for 7 min with 100% A to equilibrate the column pressure. Temperature was set at 30°C, and ultraviolet detection was monitored at 260 nm for Ad and 272 nm for SQAd. The detection limit of the HPLC technique was 2 μg/ml for Ad and 10 μg/ml for SQAd. This method exhibited linearity (R2 = 0.9988) over the assayed concentration ranges (2 to 200 μg/ml).

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