Browsing by Subject "Ph-responsive"
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Item Multi-responsive microencapsulated nanogels for the oral delivery of small interfering RNA(2014-12) Knipe, Jennifer Marie; Peppas, Nicholas A., 1948-; Paul, Donald; Ellison, Christopher; Contreras, Lydia; Suggs, LauraMulti-responsive, anionic poly(methacrylic acid-co-N-vinyl-2-pyrrolidone) microscale hydrogels (microgels) encapsulating polycationic nanoscale hydrogels (nanogels) were synthesized with either degradable or nondegradable crosslinks. The pH-responsive volume phase transition of these formulations was consistent with the pH transition experienced during intestinal delivery, as the hydrogels swelled at pH values greater than pH 5. The physicochemical characteristics of the nondegradable formulations were evaluated by microscopy, potentiometric titration, Fourier transform infrared spectroscopy, and thermal gravimetric analysis. The nondegradable formulations successfully loaded and released a model protein in physiological buffers, but the ability of the microgels to release the nanogels upon exposure to intestinal conditions was inadequate. Therefore, microgels containing enzyme-degradable oligopeptide crosslinks were synthesized then characterized using Fourier transform infrared spectroscopy, electron microscopy, confocal microscopy, and ImageStream flow cytometry. Degradation of the microgels upon incubation in trypsin solutions, simulated gastric fluid, or simulated intestinal fluid was evaluated by measuring the change in relative turbidity over time. Microgels were degraded specifically by the enzyme trypsin, and the rate of degradation was dependent upon the microgel to trypsin concentration ratio; for all ratios tested, degradation was complete within 4 hours. The cytocompatibility of the enzyme-degraded microgels encapsulating nanogels was evaluated in both a human and a murine cell line; at microgel concentrations less than 0.4 mg/ml the cell viability was greater than 90%. Confocal microscopy was used to obtain Z-stack images of the cells following incubation with the microgels, confirming that nanogels were released from the degraded microgels and subsequently inteRNAlized by RAW 264.7 murine macrophage cells. The microencapsulated nanogels were able to load siRNA via electrostatic complexation with loading efficiencies ranging from 60-80%. Incubation of loaded microgels in simulated intestinal fluid with reduced trypsin concentrations or in rat intestinal fluid resulted in successful degradation of the microgel matrix and release of a detectable amount of viable siRNA. The degraded microgels with nanogels transfected the two different cell lines with up to 20% silencing efficiency. Though the knockdown efficiency is not as high as that of nanogels alone, the microgel results are consistent and reproducible across two cell lines.