Mixed-Generation PAMAM G3-G0 Megamer as a Drug Delivery System for Nimesulide: Antitumor Activity of the Conjugate Against Human Squamous Carcinoma and Glioblastoma Cells

Abstract

Polyhydroxylated dendrimer was synthesized from poly(amidoamine) (PAMAM) dendrimer generation 3 by addition of glycidol (G3gl). G3gl megamer was further modified by binding PAMAM G0 dendrimers by activation of G3gl with p-nitrophenylchloroformate, followed by the addition of excess PAMAM G0 and purification using dialysis. The maximum G0 binding capacity of G3gl was 12 in the case when G0 was equipped with two covalently attached nimesulide equivalents. Nimesulide (N) was converted into N-(p-nitrophenyl) carbonate derivative and fully characterized using X-ray crystallography and spectral methods. Nimesulide was then attached to G0 via a urea bond to yield G02N. The mixed generation G3gl–G02N megamer was characterized using 1H NMR spectroscopy, and its molecular weight was estimated to be 22.4 kDa. The AFM image of G3gl–G02N deposited on mica demonstrated aggregation of nimesulide-covered megamer. The height of the deposited megamer was 8.5 nm. The megameric conjugate with nimesulide was tested in vitro on three human cell lines: squamous cell carcinoma (SCC-15) and glioblastoma (U-118 MG) overexpressing cyclooxygenase-2 (COX-2), and normal skin fibroblasts (BJ). The conjugate efficiently penetrated into all cells and was more cytotoxic against SCC-15 than against BJ. Moreover, the conjugate produced a strong and selective antiproliferative effect on both cancer cell lines (IC50 < 7.5 µM).

Stepwise Glucoheptoamidation of Poly(Amidoamine) Dendrimer G3 to Tune Physicochemical Properties of the Potential Drug Carrier: In Vitro Tests for Cytisine Conjugates

“…In water, both of those conjugates as well as G3 2C26gh and G3 2C16gh showed also association with average diameter >70 nm, which dominantly contributed to a volume-averaged size (see Appendix A, Figure A1, and Table A1). Similar association was previously observed by AFM imaging for nimesulide-containing megamer G3 12G024Nim , which showed 5.1 nm size measured by DLS for dilute solution, while average height of associates deposited on mica was 9 nm [12]. The molecular association also might be responsible for reported large size (185 nm) of PAMAM G5 measured by DLS [28].…”

“…PAMAM G3 dendrimer was obtained at 5 milimolar scale according to the procedure published by Tomalia et al [1] and stored as 20.1 mM solution in methanol for further use. Then, G3 was substituted with d-glucoheptono-1,4-lactone (GHL) by the addition of solid GHL (50, 100, 150, and 200 mg) into four test tubes containing 278 mg G3 (41.2 µmol) in 3 mL DMSO each, stirred until GHL dissolved, and heated at 50 • C for 12 h. In separate synthesis, the G3 32gh compound was obtained according to the same protocol except using 20% stoichiometric excess of GHL as described before [12].…”

“…The 1 H and 13 C-NMR signal assignments for CNPC in comparison with C based on 2-D COSY ( Figure 2B) and HSQC and HMBC ( Figure 3B) NPCF is convenient acyl chloride, which was used to substitute hydrogens of both the -OH and -NH groups. Furthermore, derivatized compounds like lauryl alcohol [7] or nimesulide [12,27] were reactive towards the amine groups of PAMAM dendrimers. The CNPC was used here to covalently attach C via carbonyl linker into G3 (see Scheme 1).…”

“…The hemotoxicity of PAMAM generation 3–5 dendrimers (G3–G5) increases with generation and precludes their straightforward application as drug carriers bearing either absorbed (encapsulated) or covalently attached pro-drugs to the PAMAM core. There is a variety of PAMAM terminal amine group modifications reported till now, like PEG-ylation, acylation, and hydroxyalkylation [ 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 ], which modify not only the basicity of intrinsic nitrogen atoms but also tune surface hydrophilicity vs. hydrophobicity. On the other hand, amine groups are convenient peripheral sites to attach potential ligands like folate or biotin and are responsible for binding a conjugated carrier selectively to cancer cell membranes [ 5 , 8 , 10 , 13 , 14 , 15 , 16 , 17 , 18 ].…”

PAMAM dendrimers functionalised with an anti-TNF α antibody and chondroitin sulphate for treatment of rheumatoid arthritis

“…For this assay the ATDC5 cells were seeded at a density of accomplished. [21], [22] The PAMAM dendrimer is a branched polymer with an abundance of protons, making the identification of full structure on the 1 H-NMR and FTIR spectrum a challenge.…”