摘要
与传统的微粒制备方法(如机械粉碎与研磨、溶液结晶、化学反应等)相比,超临界流体微粒化技术具有产品纯度高、几何形状均一、粒径分布窄、制造工艺简单、操作适中等许多优点,尤其对热敏感、结构不稳定和具有生物活性的物系的处理具有明显优势。超临界流体抗溶剂-雾化SAS-A(Supercritical Anti-Solvent Atomization, SAS-A)技术以PGSS和SEDS技术为基础,综合了PGSS和SEDS的优点,适合于处理含水体系。本文介绍一套自行设计及搭建的SAS-A实验装置,以该技术研究制备高分子、蛋白质微粒,以及它们的复合微粒;探讨压力、溶液流量和溶液浓度等工艺参数对形成的微粒粒径、粒径分布及蛋白质活性的影响;计算实验涉及体系的高压相平衡,为该技术的进一步开发提供理论指导。
本文首先研究了实验体系所涉及的二元和三元系统的高压相行为。用PR状态方程对CO2+丙酮体系的高压汽液平衡进行计算,取得较好的效果。对CO2+乙醇,CO2+水,乙醇+水三个二元体系的高压汽液平衡计算也取得了满意的效果。对CO2+乙醇+水三元体系高压汽液平衡数据进行了预测,所得结果基本上与实验值相符。因此,可以运用这些计算方法确定SAS-A技术过程中体系所处的状态,从而和所制备的微粒联系起来。
用SAS-A工艺分别以N2和CO2从PEG6000的丙酮溶液中成功制备了PEG微粒。结果表明,所制备的微粒形态基本为球形,并且粒径分布可以方便地控制在1-5μm之间。在N2系统中,PEG微粒随预膨胀压力增大而减少,粒径分布变窄;低PEG/丙酮溶液流量下制备的微粒粒径分布较窄;高PEG溶液浓度所制备的微粒粒径要比低溶液浓度下明显大,粒径分布也宽;在研究范围内,喷嘴尺寸对微粒粒径影响不大。在CO2系统中,增大预膨胀压力会造成不规则微粒增多,低流量和低浓度更有利于制备更小的微粒。N2系统制备的微粒的形态和大小对操作条件不敏感,因此,更适合制备PEG微粒。以CO2作为超临界流体,探讨了SAS-A技术从PEG6000的乙醇水溶液中制备PEG微粒,并研究控制喷嘴前后压力不同(即控制喷嘴前压力P1和喷嘴后压力P2)对实验结果的影响。喷嘴前后压力差降低会造成雾化效果不好;固定P1,升高P2造成抗溶剂效果增大;当P1接近乙醇/CO2混合物的临界点压力,P2为大气压时具有最好的雾化效果。以SAS-A工艺用CO2成功地从BSA/乙醇/水溶液中制备了BSA微粒。研究各种操作参数对微粒形态、平均粒径和蛋白质活性的影响。结果表明,在各种操作条件下制备的BSA产品均为球形微粒,且粒径均在3μm以下。通过改变操作条件可以有效地控制微粒的形态和大小:增大压力使得粒径变小,粒径分布变窄;降低溶液流量会使微粒变小,粒径分布变窄;增高BSA浓度会明显增大微粒粒径,粒径分布也随之变宽;乙醇浓度对微粒的影响不大。在所有操作条件下获得的BSA微粒的活性损失在0-25%之间。溶液流量和乙醇浓度是影响BSA活性的主要因素,流量越小或乙醇浓度越高,BSA活性损失越大。
用SAS-A技术研究制备不同PEG/BSA进料配比下的高分子和蛋白质复合微粒。结果表明,不同进料配比下的微粒大部分为球形;但从产品的溶出度来看,低BSA含量情况下的控释效果较好,说明采用PEG可以对BSA进行包裹并获得控释效果。
Microparticles with narrow particle size distributions are widely used in many fields, such as biochemistry, materials, pharmaceuticals, cosmetics, etc., due to their excellent chemical properties and physical effects. As a result, the generation of microparticles has been the focus of numerous research projects in the past decade. Supercritical fluid assisted technologies allow the generation of particles that are difficult or even impossible to be achieved by classical methods such as milling, crystallization, spray drying or chemical reaction, particularly for substances with thermal sensitivity, structure instability or bioactivity. In this thesis, a supercritical antisolvent-atomization (SAS-A) apparatus was established for micronization of polymer, protein, and their microcomposites from organic or aqueous solutions. The SAS-A process combined the advantages of PGSSTM and the SEDSTM process, could vary two operating pressures to control particle size and morphology.
Prior to the micronization of the studied systems, the vapor-liquid equilibrium (VLE) of the binary CO2/acetone, CO2/ethanol, CO2/water, and water/ethanol systems, and the ternary CO2/ethanol/water system were investigated by the Peng-Robinson equations of state (PR-EoS) under various temperature and pressure in order to provide theoretical points for directing the micronzation process. The studies showed that the PR-EoS was suitable for describing the VLE of the mentioned systems, and therefore will be used for discussing the states of the processing fluids in the SAS-A process.
The SAS-A process was applied to the generation of polyethylene glycol 6000 (PEG) microparticles by using high pressure CO2 or N2 from PEG/acetone solutions under the downstream pressure of 0.1MPa and other operating conditions. The effects of the nozzle size, pre-expansion pressure, PEG/acetone solution flow rate and PEG concentration in acetone on the particle morphology and particle size were investigated at 50°C. Results showed that the N2-assisted process could produce spherical particles with mean sizes of 1-5μm, while the CO2-assisted process produced spherical, irregular, or agglomerated particles. No obvious difference could be found in acetone residue, crystallinity and melting point of the PEG particles obtained from the two processes. What more, using the same apparatus, PEG microparticles were successfully generated by using ethanol/water instead of acetone. Through changing the operating pressure, the process was possible to switch between atomization or anti-solvent precipitation, and spheres, crystals or both were obtained. A low value of the pressure before the nozzle (such as of 8 MPa that is close to the critical pressure of the binary ethanol/CO2 mixture), and a low value of the pressure after the nozzle (such as atmospheric pressure) were suggested to obtain desired particles.
The SAS-A process was employed to produce bovine serum albumin (BSA) protein particles from ethanol/water solutions at 50°C. The effect of the operating pressure, the flow-rate of the protein/ethanol/water feed, the protein concentration, and the ethanol content in the ethanol/water, on the morphology, size and bioactivity of the produced BSA particles was evaluated. The formed primary particles were spherical and discrete with sizes of 0.1-3μm; the statistical number- average particle size was 0.4-1.1μm. The loss of the activity of the BSA powders varied between 0 to 25% of the original BSA materials depending on the processing conditions, in particular, on the flow-rate of the protein/ethanol/water solution and the ethanol content in the solution. At mild operating pressure of 8MPa, relatively low temperature of 50°C, relatively high solution flow rate about 3-5mL/min, protein concentration about 20-30mg/mL, and the ethanol fraction less than 25% in mass, it was possible to produce BSA microparticles with almost no loss of its bioactivity by the SAS-A process.
As a continuation, BSA/PEG composite microparticles were investigated by using the SAS-A process. The effects of the BSA to PEG mass ratio on the particle morphology and the release profiles of BSA of the produced composites were examined. Results showed that BSA was encapsulated by PEG successfully, but the encapsulation efficiency should be improved further.
引文
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