N8 acetylspermidine

N¹-acetylspermine (ASP) and N⁸-acetylspermidine (ASD) standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). (N¹,N¹²-diacetylspermine) DAS was synthesized in-house, and confirmed via liquid chromatography retention time matching and tandem MS/MS spectral-matching. Deuterium-labeled DAS (DAS-d6) was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Ammonium formate, LC-MS grade acetonitrile, water, formic acid, and methanol were purchased from Fisher Scientific (Hampton, NH, USA). Chromatographic separation was attained using an ACQUITY UPLC HSS PFP column (1.8 μm particle size, 2.1 mm, 100 mm) purchased from Waters Corporation (Milford, MA, USA). One milliliter, 96-well plates were purchased from Eppendorf (Hamburg, Germany). 96-well 0.2-micron PVDF filter plates were obtained from Agilent (Santa Clara, CA, USA). 12-[[(cyclo-hexylamino) carbonyl]amino]-dodecanoic acid (CUDA) was purchased from Cayman Chemicals (Ann Arbor, MI, USA). Urine and saliva were provided by Dr. Peter Belafsky with appropriate IRB approval (#708419).

Polyamine binding was measured as described earlier 24 (link)42 (link). The [³H]-spermidine competition assay was performed using 10 µM of [³H]-spermidine and the indicated concentrations (Fig. 6B) of the following acetyl polyamines; N1-acetylspermidine (Wako), N8-acetylspermidine (Sigma-Aldrich) and N1, N8-diacetylspermidine (Wako).

Gas chromatography-mass spectrometry (GC-MS) was used to determine the levels of various polyamines in MNPs@SiO₂(RITC)-treated cells. The polyamines putrescine, spermidine, spermine, N¹-acetylputrescine, N¹-acetylcadaverine, N¹-acetylspermidine, N⁸-acetylspermidine, N¹-acetylspermine, and cadaverine as well as the internal standard (IS) 1,6-diaminohexane were from Sigma-Aldrich (USA). GC-MS analyses were performed using an Agilent 6890N gas chromatograph interfaced with an Agilent 5975B mass-selective detector and an ultra cross-linked capillary column (Agilent Technologies, USA)12 (link). Analyses were performed in both scan and selected-ion monitoring (SIM) modes. The mass range scanned was 50 to 600 u at a rate of 0.99 scans/s for analyses in scan mode. For SIM mode, three characteristic ions for each polyamine were used for peak identification and quantification as described elsewhere25 (link).