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Examining the Cellular Transport Pathway of Fusogenic Quantum Dots Conjugated With Tat Peptide.

ABSTRACT: Understanding the underlying transport mechanism of biological delivery is important for developing delivery technologies for pharmaceuticals, imaging agents, and nanomaterials. Recently reported by our group, SDots are a novel class of nanoparticle delivery systems with distinct biointerface features and excellent fusogenic capabilities (i.e., strong ability to interact with the hydrophobic portions of biomembranes). In this study, we investigate the cellular transport mechanism of SDots conjugated with Tat peptide (SDots-Tat) by live-cell spinning-disk confocal microscopy combined with molecular biology methods. Mechanistic studies were conducted on the following stages of cellular transport of SDots-Tat in HeLa cells: cellular entry, endosomal escape, nucleus entry, and intranuclear transport. A key finding is that, after escaping endosomes, SDots-Tat enter the cell nucleus via an importin β-independent pathway, bypassing the usual nucleus entry mechanism used by Tat. This finding implies a new approach to overcome the nucleus membrane barrier for designing biological delivery technologies.

SUBMITTER: Dai J

PROVIDER: S-EPMC9184739 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Examining the Cellular Transport Pathway of Fusogenic Quantum Dots Conjugated With Tat Peptide.

Dai Jie J Wang Jun J Yang Xuan X Xu Zixing Z Ruan Gang G

Frontiers in bioengineering and biotechnology 20220527

Understanding the underlying transport mechanism of biological delivery is important for developing delivery technologies for pharmaceuticals, imaging agents, and nanomaterials. Recently reported by our group, SDots are a novel class of nanoparticle delivery systems with distinct biointerface features and excellent fusogenic capabilities (i.e., strong ability to interact with the hydrophobic portions of biomembranes). In this study, we investigate the cellular transport mechanism of SDots conjug ...[more]

PMID: 35694230

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Project description: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), conjugated to either a TAT peptide (TAT-RNPs) or a 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 (pi-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 toward 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 the 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.

Dataset Information

Examining the Cellular Transport Pathway of Fusogenic Quantum Dots Conjugated With Tat Peptide.

Publications

Examining the Cellular Transport Pathway of Fusogenic Quantum Dots Conjugated With Tat Peptide.

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