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  • br On the other hand only

    2020-08-30


    On the other hand, only few reports about CDDP loading in CN have been found in the literature [30–32]. It is well-known that nano-particles with a positive charge are earlier removed from the blood-stream than those with a negative charge by the immune system. However, recent studies with macrophages show that the uptake is surface charge intensity-dependent [21]. Furthermore, cellular uptake is facilitated when cationic vectors [33] are designed as nanocarriers, probably due to the negatively charged cell membrane, which can at-tract the CNs. Recently, it was demonstrated that CNs with a network of poly(2-dimethyl amino)ethyl methacrylate (PDMAEMA) were capable of endosome disruption via proton sponge mechanism [31].
    Both types of nanogels (AN and CN) can present pros and cons for its synthesis, loading and release of drugs, and cell viability assays. In the present contribution, a direct comparison between two types of well-defined PEGylated nanogels with positive and negative surface charges, was carried out: i) cationic core nanogels (CCN) based on poly(N,N-diethylaminoethyl methacrylate) and b) anionic core nanogels (ACN) based on poly(2-methacryloyloxy benzoic acid). In addition, these systems show pH-sensitivity, which may allow the loading and pH-sensitive release of CDDP. Subsequently, the systems were tested for in-vitro studies on lung cancer cell lines NCI-H1437. The main aim of this work is to compare the capacity of anionic and cationic polymeric na-nogels as carriers of CDDP in terms of efficiency, drug-loading and 
    delivery, and its translation to cytotoxicity in lung cancer tumor cells, to offer the reader the pros and cons of both systems.
    2. Materials and methods
    N, N-(diethylamino) ethyl methacrylate (DEAEMA, Sigma-Aldrich 99%) and methacrylic anhydride (Sigma-Aldrich 94%) were purified by distillation under reduced pressure prior to use. Poly (ethylene glycol) methyl ether methacrylate (PEGMA, Mn =950 g/mol Sigma-Aldrich) and ethylene glycol dimethacrylate (EGDMA, Sigma-Aldrich 98%), were purified by passing through an inhibitor remover column for hy-droquinones (Sigma-Aldrich). Fluorescein O,O′-diacrylate (FDA, Sigma-Aldrich 98%), 2-hydroxybenzoic SB-203580 (99%), Ammonium persulfate (APS, 98%), N,N’-dimethylaminopyridine (DMAP, 99%), cis-diammi-nedichloridoplatinum (II) (CDDP, 98%) were supplied by Sigma-Aldrich, hydrochloric acid (HCl, 36.9%), dichloromethane (DCM, 99.5%) were supplied by Fermont, deuterated chloroform (CDCl3,
    99.8%), deuterated dimethyl sulfoxide (DMSO-d6, 99.8%) were sup-plied by Acros organics and triethylamine (TEA, Acros 99%), all were used as received. Phosphate buffer solutions were prepared at 0.05 M total concentration, NaOH (1 N) and HCl (2 N) solutions were prepared by using deionized water purified by a BARNSTEAD apparatus.
    2.2. Synthesis and characterization of anionic and cationic PEGylated nanogels
    For the synthesis of anionic nanogels, (2-methacryloyloxi benzoic acid) monomer (2MBA) was synthesized following a previously re-ported methodology [34]. Anionic nanogels containing P2MBA cores (denoted as ACN) were prepared following a previous procedure [35] with several modifications via surfactant-free emulsion polymerization (SFEP) method with different ratios of PEGMA:P2MBA monomers, using EGDMA or FDA crosslinker, and the APS initiator (Fig. 1a). As example of a detailed protocol, the synthesis of one ACN is described: 2MBA (2.75 g, 12.1 mmol) was mixed with the proper amounts of PEGMA with Mn =950 g/mol (2.25 g, 2.3 mmol) and were dispersed in 350 mL of deionized water at room temperature for 30 min, in constant agitation (300 rpm). Then the pH was adjusted to 5.0 using a NaOH solution (1 N), afterwards 70 μL of EGDMA (0.072 g, 0.363 mmol) in 130 mL of deionized water were added, completing a volume of 480 mL and the mixture was placed into a 1 L jacketed glass reactor (Syrris, model Atlas Potassium, Royston UK). The monomer mixture was bub-bled with nitrogen for 60 min for the removal of any dissolved oxygen, then it was heated to 70 °C and vigorously stirred using the mechanical device of the reactor at 350 rpm. The initiator APS (0.111 g, 0.484 mmol) was dissolved in 20 mL of deionized water and it was added to the mixture. The polymerization was allowed to continue for 90 min and was stopped by cooling. Anionic nanogels using FDA crosslinker were denoted as ACNF.
    Cationic nanogels with PDEAEMA cores (denoted as CCN) were prepared, by scaling-up and modification of a previously reported methodology [36]. The nanogels have been prepared by surfactant free emulsion polymerization (SFEP), using PEGMA (Mn =950 g/mol), EGDMA or FDA, and APS as polymerizable stabilizer, crosslinker and thermal initiator, respectively (Fig. 1b). Details of the synthesis are described in the Supplementary material. Cationic nanogels using FDA crosslinker were denoted as CCNF.