Relevance of biophysical interactions of nanoparticles with a model membrane in predicting cellular uptake: study with TAT peptide-conjugated nanoparticles

Abstract

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.

Figure 1

The change in surface pressure (SP) of the endothelial cell model membrane (EMM) following interaction with NPs. (a) Effect of TAT peptide amount and its sequence conjugated to NPs on the change in surface pressure of the EMM. A 500 μL aliquot of NP suspension (5 mg/mL) was injected into the subphase consisting of 50 mL buffer, and the change in SP of the EMM was recorded immediately with time. Arrow in the figure indicates the point of addition of NPs into subphase, (b) Effect of concentration of unconjugated and conjugated RNPs on the change in SP of the EMM following interaction after 20 min. TAT₂₀₀-RNPs were tested at different concentrations whereas RNPs and sc-TAT₂₀₀-RNPs were tested at the highest concentration used in this experiment. NP concentration in the buffer = 50 μg/mL Key for (a): 1, EMM without NPs; 2, sc-TAT₂₀₀-RNPs; 3, RNPs; 4, TAT₂₀-RNPs; 5, TAT₂₀₀-RNPs; 6, TAT₅₀₀-RNPs.

Figure 2

Effect of TAT peptide sequence conjugated to RNPs on the compression isotherm of the endothelial cell model membrane (EMM) lipid mixture. The EMM lipid mixture was spread on the buffer surface at 0 mN/m surface pressure (SP); a suspension of NPs was injected into the subphase prior to compression. NP concentration in the buffer = 50 μg/mL. Key: 1, EMM without NPs; 2, sc-TAT₂₀₀-RNPs; 3, RNPs; 4, TAT₂₀₀-RNPs.

Figure 3

Surface morphology of the endothelial cell model membrane (EMM) following interaction with NPs. Langmuir–Schaeffer films were transferred onto a silicon substrate following interaction with NPs for 20 min, and the imaging was carried out using AFM in tapping mode in air. (a) EMM alone, (b) EMM following interaction with RNPs, and (c) EMM following interaction with TAT₂₀₀-RNPs. The EMM was transferred at the SP 29 mN/m for (a), whereas (b) and (c) were transferred at the SP 31, 36 mN/m, respectively. The corresponding zoom images for (a), (b), (c) are (d), (e), (f). The phase angle scale was 50° for all images. The height scales for the images were- a, = 3 nm; b, c, = 150 nm. The section analysis was carried out on the AFM height images across the white lines. The scan size for (a–c) = 10 μm and (d–f) = 2 μm.

Figure 4

Effect of amount of TAT peptide bound to NP surface and the peptide sequence on uptake of ritonavir in HUVEC cells. Cells (3 × 10⁴ cells) were incubated with either ritonavir in solution, or unconjugated and conjugated RNPs (drug concentration = 5 μM) for different time periods. Data are expressed as mean ± s.e.m (n = 3).

Figure 5

Viability of HUVEC cells incubated with RNPs and TAT-RNPs conjugated to different amount of peptide. HUVEC cells were grown in 96-well plates at a seeding density of 5,000 cells per well until confluency was attained. Cells were then incubated with RNPs or TAT-RNPs and cell viability was assessed using a MTS assay at different time points following incubation. Ritonavir solution or TAT peptide alone did not show any toxicity (data not included in figure). The values represent percentage of viable cells relative to untreated cells. Data are expressed as mean ± s.e.m. (n = 3).

Figure 6

Energy minimized confirmation of TAT peptide and sc-TAT peptide. Minimum energy conformations were generated using a MM2 force field. In TAT peptide, tyrosine is at the beginning of the peptide sequence (top), whereas in sc-TAT peptide, it occupies the central portion of the peptide. Tyr-Tyrosine; Arg-Arginine.

Figure 7

Schematic representation of the interaction of unconjugated, TAT peptide, and sc-TAT peptide conjugated RNPs with the EMM. (a) EMM alone, (b) EMM interacting with RNPs, (c) EMM interacting with sc-TAT-RNPs, and (d) EMM interacting with TAT-RNPs.