Frozen serum of male SWISS mice (900 μl) was quickly thawed and then preincubated at 37°C for 30 min before the addition of 300 μl of LENK-SQ-Dig NPs or LENK-SQ-Am NPs (2 mg/ml). In the case of LENK-SQ-Diox NPs, diluted serum (30% in 5% dextrose solution) was used for the release study. At various time intervals, aliquots (80 μl) were collected and added into 320 μl of ACN to denature and precipitate the enzymes and proteins of the serum, to remove them after centrifugation (3000g for 15 min). To quantify the residual LENK-SQ bioconjugate and the released LENK, the resulting supernatants (150 μl) were evaporated to dryness at 40°C under nitrogen flow and then solubilized in 150 μl of Milli-Q water. Free peptide quantification was performed using RP-HPLC on an Uptisphere Strategy C18HQ column (4.6 mm by 100 mm, 5 μm; Interchim), a 1525 Binary LC Pump (Waters), a 2707 Auto-sampler (Waters), and a 2998 PDA detector (Waters). The HPLC was carried out using a gradient elution with the mobile phase composed of 5 mM ammonium acetate in Milli-Q water (phase A) and 5 mM ammonium acetate in ACN (phase B). Elution was carried out at a flow rate of 1 ml/min for 13 min with the linear gradient from 10 to 100% of B; then, the system was held at 100% of B with isocratic flow for 10 min. Temperature was set at 35°C, and UV detection was monitored at 257 nm. The detection limit of the HPLC technique was 0.39 μg/ml for the peptide. This method exhibited linearity (R2 = 0.99998) over the assayed concentration range (0.39 to 200 μg/ml) and demonstrated good precision with relative SD being all less than 2.01%. The accuracy corresponded to 96 ± 5%.

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