Relevance of biophysical interactions of nanoparticles with a model membrane in predicting cellular uptake: study with TAT peptide-conjugated nanoparticles.
Relevance of biophysical interactions of nanoparticles with a model membrane in predicting cellular uptake: study with TAT peptide-conjugated nanoparticles.
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DOI：
10.1021/mp900011h
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发表时间：
2009-09
影响因子：
4.9
通讯作者：
Labhasetwar V
中科院分区：
文献类型：
--
作者：
Peetla C;Rao KS;Labhasetwar V
该研究的目的是检验以下假设：反式激活转录因子 (TAT) 肽缀合纳米粒子 (NP) 与模型细胞膜的生物物理相互作用可以预测封装治疗剂的细胞摄取。为了检验上述假设，使用朗缪尔薄膜天平与内皮细胞模型膜（EMM）研究了负载利托那韦的聚（L-丙交酯）纳米颗粒（RNP）（与TAT肽（TAT-RNP）或乱序TAT肽（sc-TAT-RNP）缀合）的生物物理相互作用，并使用相应的人血管内皮细胞（HUVEC）来研究封装的治疗剂。生物物理相互作用是根据 EMM 表面压力 (SP) 与 NP 相互作用后随时间变化的变化以及存在 NP 时 EMM 脂质混合物的压缩等温线 (π-A) 来确定的。此外，使用 Langmuir-Schaeffer (LS) 技术与 NP 相互作用后，EMM 被转移到硅基底上。通过原子力显微镜 (AFM) 对转移的 LS 薄膜进行成像，以确定脂质形态的变化并表征 NP-膜相互作用。 TAT-RNPs 显示 EMM 的 SP 增加，这取决于与 NP 结合的肽的量和 NP 的浓度，而 sc-TAT-RNPs 和 RNPs 没有显示 SP 的任何显着变化。等温线实验显示，在 TAT-RNP 存在的情况下，平均分子面积 (mmA) 会向更高的方向转变，表明它们与 EMM 的脂质相互作用，而 sc-TAT-RNP 和 RNP 没有显示任何显着变化。 AFM 图像显示，在与 TAT-RNP 相互作用后，脂质发生凝结，表明它们渗透到 EMM 中，而 RNP 没有引起任何变化。 EMM 的表面分析和 3-D AFM 图像进一步证实了 TAT-RNP 渗透到 EMM 中，而 RNP 松散地锚定在膜上，并且数量明显少于 TAT-RNP。我们推测，形成 NP 界面的 TAT 的疏水性酪氨酸驱动了 TAT-RNP 与 EMM 的初始相互作用，然后是与膜的阴离子磷脂的静电相互作用。对于 sc-TAT-RNP，亲水性精氨酸形成不与 EMM 相互作用的 NP 界面，尽管这些 NP 上具有与 TAT-RNP 相似的阳离子电荷。单独的 TAT 肽没有显示出 SP 的任何变化，这表明当肽与载体系统缀合时会发生相互作用。与 sc-TAT-RNP 或 RNP 相比，HUVEC 对 TAT-RNP 的药物摄取更高，这表明 NP 与细胞膜脂质的生物物理相互作用在 NP 的细胞内化中发挥作用。总之，TAT 肽序列和与 NP 缀合的 TAT 量显着影响 NP 与 EMM 的生物物理相互作用，并且这些相互作用与封装药物的细胞递送相关。因此，与模型膜的生物物理相互作用可以有效地用于开发用于药物输送应用的高效功能化纳米载体系统。
The aim of the study was to test the hypothesis that the biophysical interactions of the trans-activating transcriptor (TAT) peptide-conjugated nanoparticles (NPs) with a model cell membrane could predict the cellular uptake of the encapsulated therapeutic agent. To test the above hypothesis, the biophysical interactions of ritonavir-loaded poly (L-lactide) nanoparticles (RNPs), either conjugated to a TAT peptide (TAT-RNPs) or scrambled TAT peptide (sc-TAT-RNPs), were studied with an endothelial cell model membrane (EMM) using a Langmuir film balance, and the corresponding human vascular endothelial cells (HUVECs) were used to study the uptake of the encapsulated therapeutic. Biophysical interactions were determined from the changes in surface pressure (SP) of the EMM as a function of time following interaction with NPs, and the compression isotherm (π–A) of the EMM lipid mixture in the presence of NPs. In addition, the EMMs were transferred onto a silicon substrate following interactions with NPs using the Langmuir–Schaeffer (LS) technique. The transferred LS films were imaged by atomic force microscopy (AFM) to determine the changes in lipid morphology and to characterize the NP–membrane interactions. TAT-RNPs showed an increase in SP of the EMM, which was dependent upon the amount of the peptide bound to NPs and the concentration of NPs, whereas sc-TAT-RNPs and RNPs did not show any significant change in SP. The isotherm experiment showed a shift towards higher mean molecular area (mmA) in the presence of TAT-RNPs, indicating their interactions with the lipids of the EMM, whereas sc-TAT-RNPs and RNPs did not show any significant change. The AFM images showed condensation of the lipids following interaction with TAT-RNPs, indicating their penetration into the EMM, whereas RNPs did not cause any change. Surface analysis and 3-D AFM images of the EMM further confirmed penetration of TAT-RNPs into the EMM whereas RNPs were seen anchored loosely to the membrane, and were significantly less in number than TAT-RNPs. We speculate that hydrophobic tyrosine of the TAT that forms the NP–interface drives the initial interactions of TAT-RNPs with the EMM, followed by electrostatic interactions with the anionic phospholipids of the membrane. In case of sc-TAT-RNPs, hydrophilic arginine forms the NP–interface that does not interact with the EMM, despite having the similar cationic charge on these NPs as TAT-RNPs. TAT peptide alone did not show any change in SP, suggesting that the interaction occurs when the peptide is conjugated to a carrier system. HUVECs showed higher uptake of the drug with TAT-RNPs as compared to that with sc-TAT-RNPs or RNPs, suggesting that the biophysical interactions of NPs with cell membrane lipids play a role in cellular internalization of NPs. In conclusion, TAT peptide sequence and the amount of TAT conjugated to NPs significantly affect the biophysical interactions of NPs with the EMM, and these interactions correlate with the cellular delivery of the encapsulated drug. Biophysical interactions with a model membrane thus could be effectively used in developing efficient functionalized nanocarrier systems for drug delivery applications.
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