Porous degradable polyelectrolyte microspheres as vaccine vector
A polyelectrolyte, polyol technology, applied in the fields of pharmaceutical technology, molecular biology and immunology
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Embodiment 1
[0155] Embodiment 1: the synthesis of solid microsphere
[0156] Dextran sulfate (DEXS; 10kDa), poly-L-arginine (P L ARG, 100 kDa), ovalbumin (OVA; grade VII) and mannitol were obtained from Sigma-Aldrich. OVA-alexa488 and LysoTracker Red were obtained from Invitrogen. All water used in the experiments was Milli-Q grade.
[0157] Mannitol was mixed with dextran sulfate (DS) and ovalbumin (OVA; used as a model antigen in this study) in water at a ratio of 40 / 4 / 1 / 5 at 1% total solids under agitation. Concentration mix. In detail, 200 mg of mannitol, 20 mg of DS and 5 mg of OVA were dissolved in 20 ml of water. Then P L ARG was dissolved in 5 ml of water and added dropwise to the stirred mannitol / DS / OVA solution to allow L Electrostatic recombination between ARG and DS and OVA, respectively.
[0158] The mixture was then spray dried using a B290 benchtop spray dryer and collected as a dry powder. This mixture was fed into a two-fluid nozzle (diameter: 0.7 mm) on top of th...
Embodiment 2
[0159] Example 2: Particle Characterization
[0160] The morphology of the solid microspheres ( FIG. 2A ) and the porous microspheres obtained after extraction of mannitol in water ( FIG. 2B ) were visualized by scanning electron microscopy (SEM).
[0161] Most importantly, as will be suggested in more detail below by confocal microscopy, the spray-dried microspheres remain stable and do not break down into the original starting components when resuspended in an aqueous medium. As a control, we also spray dried a mixture of mannitol and OVA and observed immediate dissolution of the microparticles upon addition of water (data not shown), suggesting the need for DS / P L ARG polyelectrolyte complex framework to obtain stable microspheres. Extraction of mannitol in water leads to the formation of porous microspheres that are likely stabilized via a combination of electrostatic and hydrophobic interactions and physical entanglements between polymer and protein chains.
[0162] Con...
Embodiment 3
[0163] Embodiment 3: the mensuration of encapsulation efficiency
[0164] Quantification of encapsulation efficiency was determined by individually resuspending dried solid microspheres in phosphate buffered saline (PBS; pH 7.4). Subsequently, the microspheres were centrifuged and the OVA concentration in the supernatant was determined. When the encapsulation efficiency is defined as the amount of protein retained within the microspheres after resuspension relative to the amount of protein in the dried microspheres, an encapsulation efficiency of 100 ± 1% after resuspension in PBS was observed. This value means that no detectable amount of OVA was released from the porous microspheres following the extraction of mannitol from the microspheres after resuspension in PBS. The encapsulation efficiency of 100% into porous polyelectrolyte spheres was significantly higher than the ~50% encapsulation efficiency of the previous encapsulation of OVA into hollow polyelectrolyte multilay...
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Abstract
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