Supramolecular self assembly of nanodrill-like structures for intracellular delivery

Abstract

Despite recent advances in the supramolecular assembly of cell-penetrating peptide (CPP) nanostructures, the tuning of size, shape, morphology and packaging of drugs in these materials still remain unexplored. Herein, through sequential ligation of peptide building blocks, we create cell-penetrating self-assembling peptide nanomaterials (CSPNs) with the capability to translocate inside cells. We devised a triblock array of Tat_48-59 [HIV-1 derived transactivator of transcription_48-59] based CPPs, conjugated to up to four Phenylalanine (Phe) residues through an amphiphilic linker, (RADA)₂. We observed that the sequential addition of Phe leads to the transition of CSPN secondary structures from a random coil, to a distorted α-helix, a β-sheet, or a pure α-helix. This transition occurs due to formation of a heptad by virtue of even number of Phe. Atomic force microscopy revealed that CSPNs form distinct shapes reminiscent of a "drill-bit". CSPNs containing two, three or four Phe, self-assemble into "nanodrill-like structures" with a coarse-twisted, non-twisted or fine-twisted morphology, respectively. These nanodrills had a high capacity to encapsulate hydrophobic guest molecules. In particular, the coarse-twisted nanodrills demonstrate higher internalization and are able to deliver rapamycin, a hydrophobic small molecule that induced autophagy and are capable of in vivo delivery. Molecular dynamics studies provide microscopic insights into the structure of the nanodrills that can contribute to its morphology and ability to interact with cellular membrane. CSPNs represent a new modular drug delivery platform that can be programmed into exquisite structures through sequence-specific fine tuning of amino acids.

Keywords: Cell penetrating peptides; Intracellular delivery; Nanodrills; Supramolecular assembly.

Figure 1. Structure and schematic representation of different CSPNs

We have highlighted the hyrophillic Tat (blue), amphiphillic (RADA)₂ (pink) and hydrophobic Fmoc and Phe (red). Within the hydrophobic region we show the aromatic regions in yellow. CSPNs that form drillbit like supramolecular assembly after sequential addition of Phe include (a). 2F-RT (coarse-twisted) (b). 3F-RT (non-twisted) and (c). 4F-RT (fine-twisted). These assemblies were visualized using tapping mode AFM (red insert shows a schematic of the nanodrills).

Figure 2. Molecular basis of secondary structure

(a). CD spectral analysis of different CSPNs was performed to elucidate secondary structure. The transition of secondary structure was observed as the number of Phe was increased from zero to four (b). The transition of secondary structure was from a highly random coil (RT) to a moderate random coil (1F-RT) followed by formation of a pure β-sheet on addition of odd number of Phe. On the other hand, even number of Phe changed the random coil to distorted α-helical (2F-RT) and formed pure α-helical (4F-RT) structure. (c) Different amino acids of 2F-RT from N-terminal was designated as abcdefg which constitutes a heptad making a helical wheel (Chain I). The condition for the formation of coiled coils (α-helical structure) is “the heptad should interact with another chain (i.e. Chain II with amino acids a’b’c’d’e’ f’g’) by the hydrophobic interactions (a-a’ and d-d’) and ionic interactions (e-g’ and g-e’).” Illustration of helical wheel diagram model of heptad interaction in two chains of 2F-RT from the N-terminal amino acid and shows hydrophobic interaction (Phe-Phe and Ala-Ala) and ionic interaction (Asp-Arg and Arg-Asp) suggesting the formation of a parallel homodimer. (d) Illustration of helical wheel diagram model of heptad interaction in two chains of 4F-RT from the N-terminal amino acid and shows hydrophobic interaction (Phe-Phe and Phe-Phe) and ionic interaction (Asp-Arg and Arg-Asp) suggesting the formation of a parallel homodimer (e) Scheme of secondary structural transition in different CSPNs based on Phe number. The regions of the CSPNs such as hydrophobic (red), hydrophilic (dark blue), cationic (light blue), anionic (green) , amphiphilic (pink) and charge per residue was also highlighted. The structural transition was dependent on the number of Phe residue per sequence. The even favours α-helical structure as depicted in the scheme.

Figure 3. Molecular basis of hydrophobicity

(a–e) Spectrofluorimetric analysis of synthetic peptides at various concentrations (2–16 µM) followed by ANS (0.2 µM ) encapsulation.. Blank and ANS alone served as controls. Hyperchromic shift in intensity ANS was observed for (c–e). (f). Shows comparative analysis of mean fluoroscence intensity of different peptides

Figure 4. Morphological analysis of self assembly

AFM images of (a, d) 2F-RT (b,e) 3F-RT and (c, f) 4F-RT were acquired in tapping mode. The primary images in panels a, b and c show the full height of the samples, whereas the color scale has been adjusted for inserts a, b and c as well as panels d, c and f in order to accentuate the unique, differentiating features of the samples. Insert (red-dotted line, a–c) shows higher magnification of CSPNs that assemble as coarse-twisted (a), non-twisted (b) and fine-twisted (c) nanodrills. Cross-sectional profile along the selected length (Top-panel) (d). A, (e) A, B, (f) A and diameter (Bottom Panel) (d) B, (e) C,D,E, (f) B, C is indicated. The cross sectional profile was used to develop schematics that show coarse twisted nanodrills with central groove (d), non-twisted nanodrill without any groove (e), fine-twisted nanodrills (f)

Figure 5. AFM analysis of ANS encapsulated 3F-RT

(a) Scans of blank 3F-RT and ANS encapsulated 3F-RT nanodrills under the similar conditions taken at different time points while continuously scanning. This demonstrates the relative mechanical instability of nanodrills loaded with hydrophobic molecules. The ANS encapsulated 3F-RT nanodrills (39 min, red insert) were subjected to cross sectional analysis given in (b) and (c). In (b–c), curves A–E represent two different orientations of the ANS encapsulated 3F-RT nanodrills. (b) Longitudinal height measurements (curves A and B) show partial dissociation of the ANS encapsulated 3F-RT nanodrills. (c) Cross-sectional measurements of 3F-RT shows nanodrills with a central groove (curve C) and without a central groove (curve D and E). This is due to different orientations of the nanodrills on the mica surface. Each nanodrill is shown with a schematic model to explain their relative orientations. The yellow circles represent ANS molecules loaded within the hydrophobic core.

Figure 6. Intracellular delivery of autophagy inducer by CSPNs

(a) Encapsulation efficiency of CSPNs to package rapamycin, an autophagy inducer (b) Kinetics of rapamycin release from CSPNs (c) The cellular uptake of Cy7 labeled 2F-RT and 3F-RT in mammalian cells (d) Mouse Embryonic fibroblasts that stably express p62-Luc were exposed to different concentrations of rapamycin loaded 2F-RT (7–250 nM) and the luciferase acitivity was measured normalized to cell viability. Rapamycin and 2F-RT serve as controls (0.05 ≥ *p > 0.01, 0.01 ≥ **p > 0.005, ***p ≤ 0.005) (e) Cy7-labeled 2F-RT (0.1 mg/kg) was injected in mice. The animals were sacrificed at 2 hours, the organs harvested and imaged using IVIS. PBS alone served as a negative control.

Figure 7. Proposed model for self assembly

Schematic representation of the self-assembly pattern observed in nanodrills (a) 2F-RT self-assembled through aromatic π-π stacking into a distorted α-helical structure which forms coarse-twisted nanodrills, (b) 3F-RT self-assembled through aromatic π-π stacking into a parallel β-sheet that forms non-twisted nanodrills (c) 4F-RT self-assembled through aromatic π-π stacking into a pure- α-helical structure which forms fine twisted nanodrills. Inserts show AFM images of the nanodrills.

Figure 8. Molecular Dynamics (MD) simulation of 2F-RT nanodrills

(a) Snapshots of the 2F-RT nanodrill starting structure (left) and after 150 ns of simulation (right). Individual peptides are colored based on their segment (top) and on their starting position in the assembly (bottom) (b). Average helix content of different segments in core and surface peptides (c). Average percentage occupancy of intermolecular salt bridges among core and surface peptides (d). Average root mean square fluctuation (RMSF) of core and surface peptides.