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Cellular Effects and Delivery Propensity of Penetratin Is Influenced by Conjugation to Parathyroid Hormone Fragment 1-34 in Synergy with pH
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2018-01-09 00:00:00 , DOI: 10.1021/acs.bioconjchem.7b00687 Mie Kristensen , Line Hagner Nielsen 1 , Kinga Zor 1 , Anja Boisen 1 , Malene Vinther Christensen , Jens Berthelsen 2 , Hanne Mørck Nielsen
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2018-01-09 00:00:00 , DOI: 10.1021/acs.bioconjchem.7b00687 Mie Kristensen , Line Hagner Nielsen 1 , Kinga Zor 1 , Anja Boisen 1 , Malene Vinther Christensen , Jens Berthelsen 2 , Hanne Mørck Nielsen
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The cell-penetrating peptide (CPP) penetratin has demonstrated potential as a carrier for transepithelial delivery of cargo peptides, such as the therapeutically relevant part of parathyroid hormone, i.e., PTH(1-34). The purpose of the present study was to elucidate the relevance of pH for PTH(1-34)–penetratin conjugates and coadministered penetratin with PTH(1-34) regarding transepithelial permeation of PTH(1-34) and cellular effects. Transepithelial permeation was assessed using monolayers of the Caco-2 cell culture model, and effects on Caco-2 cellular viability kinetics were evaluated by using the Real-Time-GLO assay as well as by microscopy following Tryphan blue staining. Morphological Caco-2 cell changes were studied exploiting the impedance-based xCELLigence system as well as optically using the oCelloscope setup. Finally, the effect of pH on the folding propensity of the PTH(1-34)–penetratin conjugate and its ability to disrupt lipid membranes were assessed by circular dichroism (CD) spectroscopy and the calcein release assay, respectively. The transepithelial PTH(1-34) permeation was not pH-dependent when applying the coadministration approach. However, by applying the conjugation approach, the PTH(1-34) permeation was significantly enhanced by lowering the pH from 7.4 to 5 but also associated with a compromised barrier and a lowering of the cellular viability. The negative effects on the cellular viability following cellular incubation with the PTH(1-34)–penetratin conjugate were moreover confirmed during real-time monitoring of the Caco-2 cell viability as well as by enhanced Tryphan blue uptake. In addition, morphological changes were primarily observed for cells incubated with the PTH(1-34)–penetratin conjugate at pH 5, which was moreover demonstrated to have an enhanced membrane permeating effect following lowering of the pH from 7.4 to 5. The latter observation was, however, not a result of better secondary folding propensity at pH 5 when compared to pH 7.4.
中文翻译：
Penetratin的细胞效应和传递倾向受pH协同与甲状旁腺激素片段1-34结合的影响
细胞穿透肽（CPP）渗透素已显示出作为跨运输上皮运输货物肽（例如甲状旁腺激素的治疗相关部分，即PTH（1-34））的载体的潜力。本研究的目的是阐明pH值对PTH（1-34）-penetratin共轭物的影响，以及将Penetratin与PTH（1-34）共同施用对PTH（1-34）的经上皮渗透和细胞作用的影响。使用Caco-2细胞培养模型的单层膜评估上皮渗透性，并使用Real-Time-GLO测定以及色氨酸蓝染色后的显微镜观察对Caco-2细胞活力的影响。利用基于阻抗的xCELLigence系统以及使用oCelloscope装置进行光学分析，研究了形态学Caco-2细胞的变化。最后，分别通过圆二色性（CD）光谱和钙黄绿素释放测定法评估了pH对PTH（1-34）-渗透素缀合物折叠倾向的影响及其破坏脂质膜的能力。当应用共同给药方法时，跨上皮PTH（1-34）的渗透不受pH的影响。但是，通过应用缀合方法，通过将pH从7.4降低到5，PTH（1-34）的渗透显着增强，但同时也与屏障的破坏和细胞活力的降低有关。此外，在实时监测Caco-2细胞生存力以及增强的色氨酸蓝摄取过程中，证实了与PTH（1-34）-渗透素缀合物孵育后对细胞生存力的负面影响。此外，
更新日期：2018-01-09
中文翻译：
Penetratin的细胞效应和传递倾向受pH协同与甲状旁腺激素片段1-34结合的影响
细胞穿透肽（CPP）渗透素已显示出作为跨运输上皮运输货物肽（例如甲状旁腺激素的治疗相关部分，即PTH（1-34））的载体的潜力。本研究的目的是阐明pH值对PTH（1-34）-penetratin共轭物的影响，以及将Penetratin与PTH（1-34）共同施用对PTH（1-34）的经上皮渗透和细胞作用的影响。使用Caco-2细胞培养模型的单层膜评估上皮渗透性，并使用Real-Time-GLO测定以及色氨酸蓝染色后的显微镜观察对Caco-2细胞活力的影响。利用基于阻抗的xCELLigence系统以及使用oCelloscope装置进行光学分析，研究了形态学Caco-2细胞的变化。最后，分别通过圆二色性（CD）光谱和钙黄绿素释放测定法评估了pH对PTH（1-34）-渗透素缀合物折叠倾向的影响及其破坏脂质膜的能力。当应用共同给药方法时，跨上皮PTH（1-34）的渗透不受pH的影响。但是，通过应用缀合方法，通过将pH从7.4降低到5，PTH（1-34）的渗透显着增强，但同时也与屏障的破坏和细胞活力的降低有关。此外，在实时监测Caco-2细胞生存力以及增强的色氨酸蓝摄取过程中，证实了与PTH（1-34）-渗透素缀合物孵育后对细胞生存力的负面影响。此外，
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