Center for Chemistry and Biomedicine, Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innrain 80/82 6020, Innsbruck, Austria; Department of Pharmacy, Universitas Mandala Waluya, A.H.Nasution, Kendari 93231, Southeast Sulawesi, Indonesia.
Center for Chemistry and Biomedicine, Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbruck, Innrain 80/82 6020, Innsbruck, Austria.
Int J Pharm. 2024 Dec 25;667(Pt A):124844. doi: 10.1016/j.ijpharm.2024.124844. Epub 2024 Oct 24.
This study aimed to evaluate the cellular uptake of nanostructured lipid carriers (NLCs) decorated with polyphosphate coated linear and loop cell-penetrating peptides (CPPs).
Linear-CPPs and loop-CPPs were synthesized via ring-opening polymerization and anchored on the surface NLCs, followed by coating with polyphosphate (PP). These nanocarriers (NCs) were characterized in terms of particle size, polydispersity index (PDI), and zeta potential. Cell viability and hemolysis, as well as enzyme-induced charge conversion via phosphate cleavage by free and membrane-bound intestinal alkaline phosphatase (IAP) were investigated. Cellular uptake studies by Caco-2 and HEK cells were quantitatively analyzed by flow cytometry and visualized by confocal microscopy.
A shift in charge from positive to negative was obtained for both linear- and loop-CPPs-NLCs by coating with PP. PP-linear-CPPs-NLCs and PP-loop-CPPs-NLCs exhibited a particle size < 270 nm and a PDI of approximately 0.3. They had a minor effect on cell viability and caused in a concentration of 0.1 % (m/v) around 10 % hemolysis within 24 h. IAP triggered the cleavage and release of monophosphate from the surface of NLCs causing charge conversion from -22.2 mV to + 5.3 mV (Δ27.5 mV) for PP-linear-CPPs-NLCs and from -19.2 mV to + 11.9 mV (Δ31.1 mV) for PP-loop-CPPs-NLCs. Inhibition of alkaline phosphatase activity on Caco-2 and HEK cells confirmed the involvement of this enzyme in charge conversion. PP-linear-CPPs-NLCs showed on Caco-2 cells a higher uptake than PP-loop-CPPs-NLCs, whereas on HEK cells uptake of both types of NLCs was on the same level. The results of cellular uptake were confirmed visually by confocal microscopy.
CPPs-NLCs coated with polyphosphate are a promising approach to overcome the polycationic dilemma and to enhance cellular uptake.
本研究旨在评估用聚磷酸盐修饰的线性和环穿透肽（CPP）修饰的纳米结构脂质载体（NLC）的细胞摄取。
通过开环聚合合成线性 CPP 和环 CPP，并将其锚定在 NLC 表面，然后用聚磷酸盐（PP）进行涂层。这些纳米载体（NCs）在粒径、多分散指数（PDI）和 Zeta 电位方面进行了表征。研究了细胞活力和溶血，以及通过游离和膜结合的肠道碱性磷酸酶（IAP）诱导的磷酸盐裂解引起的酶诱导电荷转换。通过流式细胞术对 Caco-2 和 HEK 细胞的摄取进行了定量分析，并通过共聚焦显微镜进行了可视化。
用 PP 涂层后，线性和环 CPP-NLCs 的电荷从正变为负。PP-线性 CPP-NLCs 和 PP-环 CPP-NLCs 的粒径均<270nm，PDI 约为 0.3。它们对细胞活力影响较小，在 0.1%（m/v）浓度下，24 小时内引起约 10%的溶血。IAP 触发 NLC 表面单磷酸盐的裂解和释放，导致 PP-线性 CPP-NLCs 的电荷从-22.2mV 转换为+5.3mV（Δ27.5mV），PP-环 CPP-NLCs 的电荷从-19.2mV 转换为+11.9mV（Δ31.1mV）。在 Caco-2 和 HEK 细胞中碱性磷酸酶活性的抑制证实了该酶在电荷转换中的参与。PP-线性 CPP-NLCs 在 Caco-2 细胞中的摄取量高于 PP-环 CPP-NLCs，而在 HEK 细胞中，两种类型的 NLCs 的摄取量相同。共聚焦显微镜的摄取结果证实了这一点。
用聚磷酸盐修饰的 CPP-NLC 是一种很有前途的方法，可以克服多阳离子困境并增强细胞摄取。
