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  • br The resulting dispersions of

    2020-08-30


    The resulting dispersions of cationic nanogels and anionic nanogels, respectively, were purified via dialysis (Spectra/Por dialysis membrane, MWCO: 12,000–14,000 Da) against deionized water. During dialysis, water was changed four times daily for 10 days at room temperature to remove any unreacted monomer and other impurities. The nanogels were isolated by freeze drying using a Labconco Freeze Dry System Freezone 4.5 (Kansas City, MI, USA), precooling to −52 °C, and freeze-
    Fig. 1. Synthesis scheme of core-shell nanogels. a) P2MBA anionic core nanogels (ACN); b) PDEAEMA cationic core nanogels (CCN). PEG chain lenght n = 23.
    drying at a pressure of 0.02 mbar for 72 h. After drying the nanogels, these can be used for later re-dispersion and/or characterization.
    The purity and composition of anionic and cationic core nanogels was quantified by 1H-NMR (Bruker AVANCE III HD NMR 400 MHz) using DMSOd6 (ACN) and CDCl3 (CCN). The size distribution of the nanogels was obtained by dynamic light scattering (DLS) using a Zetasizer Nano ZS (ZEN3690; Malvern Instruments, Miami, FL) equipped with a red laser of 630 nm. The angle of measurement was 90. Dialyzed and dispersed samples were analyzed. The hydrodynamic diameter (Dh) and polydispersity index (PDI) were calculated using Malvern Instruments dispersion technology software based on CONTIN analysis and the Stokes–Einstein equation for spheres; for Dh the average value of three measurements is reported [37]. For the tem-perature sensitivity determination a trend method was used from 15 °C to 55 °C in two degree steps, equilibrating 240 s once the measurement temperature was attained; the transition temperature reported is the minimum value of the first derivate of Dh with respect to temperature, representing the average temperature of the transition. In order to study the pH sensitivity, measurements were carried out at 25 °C from pH 3 to pH 10, taking three measurements every pH unit, the time for each measurement was 10 min. For all the measurements the pH of the medium was controlled using HCl (0.1 N) and NaOH (0.1 N) additions, depending on the pH of the solution. The zeta potential (ζ) was also measured using the Zetasizer Nano ZS by laser doppler microelec-trophoresis. Measurements were performed on folded capillary SB203580 at 25 °C. 
    A dispersion of anionic core nanogels with P2MBA cores and a dispersion of cationic core nanogels with PDEAEMA cores were pre-pared using 60 mg of each lyophilized nanogel, and 20 mL of deionized water under constant stirring for 48 h at pH 8.5 and 5.0, respectively, to allow the nanogel ionization and therefore their swelling. Subsequently, CDDP was added to the nanogel solution in the weight ratio of nanogel:CDDP 2:1, and the mixture was stirred for 48 h. The resulting dispersion was purified via dialysis (Spectra/Por dialysis membrane, MWCO: 12,000–140,00 Da) at pH 8.5 and 5.0 respectively, against deionized water under stirring to remove free drug and surface adsorbed drug. During dialysis at room temperature, water was changed four times every 24 h [27], with a total period of dialysis of 10 days. The concentrations of the loaded CDDP were measured with in-ductively coupled plasma spectrometry (ICP-OES) using a Perkin-Elmer equipment (model Optima 8300, USA), system power supply 6038 A, Hewlett-Packard. Afterwards, the loaded nanogels were frozen at −4 °C for 12 h and then placed into the drying chamber of a Labconco Freeze Dry System Freezone 4.5 (Kansas City, MI, USA), precooled at −52 °C. Freeze-drying was performed at a pressure of 0.02 mbar for 24 h. The drug loading capacity (DLC) and drug loading efficiency (DLE) were r> calculated by the following equations: [38]
    DLC (%, Pt) = amountofdruginparticle x 100
    (amountofpolymer + amountofdruginparticle)
    DLE(%) = amountofdruginparticle x 100
    amountofdruginfeed
    Table 1
    Physicochemical properties of nanogels CCN and ACN, such as composition, particle size, surface charge and yield.
    Sample code Feed monomer composition PEGMA:MON Cross-linker contentc Copolymer Compositiond PEGMA: MON Dh nm PDI ζ potential mV Yielde wt%
    a Monomer (MON) is DEAEMA.
    c Respect to MON.
    d Obtained by 1H-NMR. e Mass yield with respect to PEGMA + MON.
    2.4. Measurement of CDDP release profiles
    In-vitro drug release studies were performed under sink conditions in a jacketed reaction beaker placed above a magnetic stirrer and connected to a thermostated water bath at 37 °C, three different Phosphate-Buffered Saline (PBS) solutions (pH 5, 6.8 and 7.4) were used as release media. Typically, 3 mg of CDDP loaded nanogels were dispersed in 10 mL of the corresponding buffer and then placed into a dialysis bag, which was subsequently immersed in 200 mL of the release medium with moderate stirring at 37 °C. At selected time intervals, 3 mL of release medium was taken out and replenished with same vo-lume of fresh media to keep the volume of release medium unchanged. The same procedure was used to study the free drug released from the dialysis bag. Drug release experiments were performed for 7 days. In all cases, the cumulative drug release was calculated from ICP-OES mea-surements using an equipment described above.