Modelling protein therapeutic co-formulation and co-delivery with PLGA nanoparticles continuously manufactured by microfluidics
Modelling protein therapeutic co-formulation and co-delivery with PLGA nanoparticles continuously manufactured by microfluidics
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DOI：
10.1039/c9re00395a
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发表时间：
2020-02-01
影响因子：
3.9
通讯作者：
Sarmento, Bruno
中科院分区：
文献类型：
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作者：
Martins, Claudia;Chauhan, Veeren M.;Sarmento, Bruno
将蛋白质治疗剂配制成聚（乳酸-共-乙醇酸）（PLGA）的纳米颗粒（NP）提供了关键特征，例如通过可能的靶向配体连接而防止清除、持续释放和减少副作用。这些纳米颗粒还提供了蛋白质组合治疗的潜力，预计将利用协同生物反应，避免多次给药方案和随后的错误给药。由于负载蛋白质的PLGA NP的常规制造仍然与低通量相关，因此已经建立了新的连续制造方法，例如微流体。本文中，提出了通过微流体连续制造的PLGA NP用于由缀合至荧光素（FITC）或四甲基罗丹明（TRITC）异硫氰酸酯的牛血清白蛋白（BSA）组成的两种模型蛋白的共制剂和递送。获得了100 nm的蛋白质共配制的NP，单分散的并且具有70%的缔合效率。微流体设置允许每天约7 g颗粒的生产速率，并表现出放大能力。模型蛋白以受控的方式释放，并且其二级结构没有显著变化。在巨噬细胞样细胞中的研究证明，蛋白质共配制的PLGA NP不损害代谢活性（>70%）。与游离蛋白质对照相比，当共配制到PLGA NP中时，蛋白质的细胞缔合高约2倍。此外，共配制的蛋白质的细胞缔合比单独加载有每种蛋白质类型的NP的物理混合物高4倍。这项工作已经证明了连续制造的PLGA NP用于共同配制和增强共同递送的模型蛋白的细胞缔合的有效性，为未来的蛋白质组合纳米疗法提供了概念验证基础。
Formulating protein therapeutics into nanoparticles (NPs) of poly(lactic-co-glycolic acid) (PLGA) provides key features such as protection against clearance, sustained release and less side effects by possible attachment of targeting ligands. These NPs also offer the potential for protein combination therapy, which is expected to exploit synergetic bioresponses, avoid multiple dosage regimens and consequent mis-dosing. Since the conventional manufacture of protein-loaded PLGA NPs is still associated with low-throughput, new continuous manufacturing methods such as microfluidics have been established. Herein, PLGA NPs continuously manufactured through microfluidics are proposed for co-formulation and -delivery of two model proteins consisting of bovine serum albumin (BSA) conjugated to either fluorescein (FITC) or tetramethylrhodamine (TRITC) isothiocyanates. Protein co-formulated NPs of 100 nm, monodispersed and with 70% of association efficiency were obtained. The microfluidic setup allowed a production rate of around 7 g of particles per day and demonstrated scale-up capacity. Model proteins were released in a controlled manner and without significant changes in their secondary structure. Studies in macrophage-like cells proved that protein co-formulated PLGA NPs did not impair metabolic activity (>70%). The cellular association of the proteins was around 2-times higher when co-formulated into PLGA NPs, compared to the free protein controls. Moreover, the cellular association of the co-formulated proteins was 4-times higher than the physical mixture of NPs individually loaded with each protein type. This work has demonstrated the effectiveness of continuously manufactured PLGA NPs for co-formulating and enhancing the cellular association of co-delivered model proteins, providing a proof-of-concept foundation for future protein combination nanotherapies.