Inorganic theranostic nanoparticles for Tuberculous Meningitis

结核性脑膜炎(TBM)是最严重的结核病感染形式，死亡率非常高，占儿童和HIV阳性成人结核病(TB)病例的2%-5%，导致永久性的神经学后果和残疾。临床上迫切需要开发直接在大脑内输送抗菌剂/抗炎药的载体来治疗和诊断早期脑损伤。PHD研究项目将集中在4个任务上：(1)合成尺寸可控的治疗纳米颗粒(&lt；100 nm)，以实现最佳的血脑屏障穿透。(2)用pH响应型聚合物修饰NPs，使NPs被吸收并进入血流。(3)NPs的物理化学表征。(4)NPs跨细胞分裂制剂的体外试验。任务1：NPs的合成。博士生将设计和开发球形(MSN)和花形(MSNFS)形状的多孔二氧化硅纳米颗粒，其尺寸可控(&lt；100 nm)，其中包含一线抗生素，以实现最佳的血脑屏障交叉。硅网将通过改进的STöber方法获得。不同浓度的治疗性离子(Ce和用于磁共振成像的Fe-)将通过后接枝的方法被结合到MSN中。任务2：用pH响应型聚合物修饰NPs。为了使新的治疗性纳米粒能够口服，将合成一个双聚合壳。MSNS/MSNFS将用pH响应性聚合物(Eudradit E100)装饰，这种聚合物是可溶的，在胃的pH值(高达5)时会膨胀，允许NPs被吸收并进入血液。此外，一种与pH无关的渗透性聚合物(Eudradit RL 100)将用于装饰MSN和MSNF。Eudradit RL 100可避免药物在胃和肠道中释放。COOH-MSNS和COOH-MSNFS将被功能化，使用碳二亚胺作为偶联剂，与L-多巴分子被证明强烈地改善了它们的血脑屏障交叉。任务3：MSNS和MSNFS的物理化学表征。利用电感耦合等离子体质谱研究了MSNS/MSNFS@CeO2-Fe3O4在不同生物介质中的离子释放动力学。这些纳米粒子将使用一系列技术进行表征，包括TEM-EDX、核磁共振、DLS、SAXS/WAXS、BET和高效液相。我们将使用SQUID磁强计来研究纳米粒子的磁性。任务4：MSNS/MSNFS@CeO2-Fe3O4转胞制剂的体外测试。最有希望的MSNS/MSNFS@CeO2-Fe3O4体系将被选作体外测试。选择MSN/MSNFS@CeO2-Fe3O4培养细胞系和原代星形胶质细胞、小胶质细胞和神经元。新系统对健康神经元(星形胶质细胞、小胶质细胞、神经元)和感染THP-1小胶质细胞的毒性将通过四甲基偶氮唑蓝和乳酸脱氢酶检测。ROS的产生也将被测量，并与纳米陶瓷的氧化状态相关联。炎症将通过测量细胞因子的释放来分析。为了研究MSNS/MSNFS@CeO2-Fe3O4跨越血脑屏障的能力，并在必要时调整纳米药物配方，将使用单一培养和由Robert Wilkinson教授在克里克开发的TBM、小胶质细胞和脑内皮细胞单层的专门、独特的共培养模型，融合多学科的体外研究，使用由Robert Wilkinson教授开发的TBM感染BBB的Transwell模型。在屏障和病变细胞中，将监测一系列细胞和分子生物学过程，包括纳米载体的药物疗效、生物反应性、颗粒摄取、转化、颗粒降解/降解产物、移位、功能和BBB通透性/破坏。

Tuberculous meningitis (TBM) is the most severe form of tuberculosis infection with very high mortality and accountsfor 2-5% of all tuberculosis (TB) cases among children and HIV-positive adults, causing permanent neurologicalconsequences and disability. There is an urgent clinical need to develop vehicles to deliver antimicrobials/antiinflammatoriesdirectly inside the brain to treat and diagnose early stage TBM.The PhD research project will focus on 4 tasks: (1) synthesis of theranostics nanoparticles (NPs) with controlled size(<100 nm) for optimal BBB crossing. (2) Decoration of NPs with pH responsive polymer which allows NPs to be absorbedand pass into the blood stream. (3) NPs physicochemical characterisation. (4) In vitro testing of NPs transcytosisformulation.Task 1: Synthesis of NPs. The PhD student will design and develop spherical (MSNs) and flower (MSNFs) shapemesoporous silica NPs with controlled size (<100 nm) containing first line antibiotics for optimal BBB crossing. The silicanetwork will be obtained by a modified Stöber method. Different concentrations of therapeutic ions (Ce, and Fe - forMRI imaging) will be incorporated into MSNs using a post grafting method.Task 2: Decoration of NPs with pH responsive polymer. A double polymeric shell will be synthesised in order toenable the oral administration of the new therapeutic NPs. MSNs/MSNFs will be decorated with pH responsive polymer(Eudragit E100) which is soluble and swells at gastric pH (up to 5), allowing NPs to be absorbed and pass into the bloodstream. Moreover, a permeable pH independent polymer (Eudragit RL 100) will be used to decorate MSNs and MSNFs.Eudragit RL 100 avoids drug release in the stomach and in the gut. COOH-MSNs and COOH-MSNFs will befunctionalised, using carbodiimide as coupling reagents, with the L-Dopa molecule that is shown to strongly improvetheir BBB crossing.Task 3: MSNs and MSNFs physicochemical characterisation. Ion release kinetics of the MSNs/MSNFs@CeO2-Fe3O4 in different biological media will be performed by ICP-MS. The NPs will be characterised using a range oftechniques including TEM-EDX, NMR, DLS, SAXS/WAXS and BET and HPLC. The magnetic properties of the NPs willbe studied by using a SQUID magnetometer. XPS and EELS will be used to measure the oxidation state of the ceria indifferent media.Task 4: In vitro testing of MSNs/MSNFs@CeO2-Fe3O4 transcytosis formulations.The most promising MSNs/MSNFs@CeO2-Fe3O4 systems will be selected for testing in vitro. Cell lines and primaryastrocytes, microglia, and neurons will be cultured with selected MSNs/MSNFs@CeO2-Fe3O4. The toxicity of the newsystem to healthy neuronal (astrocyte, microglia, neurons) and infected THP-1 microglial cells will be tested by usingMTT and LDH assays. ROS production will also be measured and correlated to the oxidation state of the nanoceria.Inflammation will be assayed by measuring cytokine release. The antibacterial properties of the NPs system will bemeasured using a colony forming assay.To investigate the ability of the MSNs/MSNFs@CeO2-Fe3O4 to cross the BBB and, if necessary, adjust the nano-drugformulation, a convergence of multi-disciplinary in vitro studies, using monocultures and specialised, unique co-culturemodels, of TBM, microglia and brain endothelial cell monolayers being developed at the Crick by Prof. Robert Wilkinsontranswell model of the TBM infected BBB will be used. In both the barrier and diseased cells, a range of cell andmolecular biological processes will be monitored, including nanocarrier drug efficacy, bioreactivity, particle uptake,transformation, particle degradation/products of degradation, translocation, and function and BBBpermeability/disruption.