Design and Development of Natural-Product-Derived Nanoassemblies and Their Interactions with Alpha Synuclein

Abstract

Biomimetic nanoassemblies derived from natural products are considered promising nanomaterials due to their self-assembling ability and their favorable interactions with biological molecules leading to their numerous applications as therapeutic agents or as molecular probes. In this work, we have created peptide nanoconjugates of two natural products, β-Boswellic acid (BA) and β-glycyrrhetinic acid (GH). Both BA and GH are known for their medicinal value, including their role as strong antioxidants, anti-inflammatory, neuroprotective and as anti-tumor agents. To enhance the bioavailability of these molecules, they were functionalized with three short peptides (YYIVS, MPDAHL and GSGGL) to create six conjugates with amphiphilic structures capable of facile self-assembly. The peptides were also derived from natural sources and have been known to display antioxidant activity. Depending upon the conjugate, nanofibers, nanovesicles or a mixture of both were formed upon self-assembly. The binding interactions of the nanoconjugates with α-Synuclein, a protein implicated in Parkinson's disease (PD) was examined through in silico studies and FTIR, circular dichroism and imaging studies. Our results indicated that the nanoassemblies interacted with alpha-synuclein fibrils efficaciously. Furthermore, the nanoassemblies were found to demonstrate high viability in the presence of microglial cells, and were found to enhance the uptake and interactions of α-Synuclein with microglial cells. The nanoconjugates designed in this work may be potentially utilized as vectors for peptide-based drug delivery or for other therapeutic applications.

Keywords: alpha synuclein; cellular uptake; nanoassemblies; self-assembly; terpenes.

Figure 1

Chemical structures of the designed conjugates. (a) Glycyrrhetinate-GSGGL; (b) glycyrrhetinate-MPDAHL; (c) glycyrrhetinate-YYIVS; (d) Boswellate-GSGGL; (e) Boswellate-MPDAHL; (f) Boswellate-YYIVS. Blue: nitrogen; red: oxygen; grey: carbon; and white: hydrogen.

Figure 2

Binding pocket region of alpha-synuclein fibrils as determined by POCASA.

Figure 3

(a) Root mean square deviations and (b) root mean square fluctuations of BA–peptide and GH–peptide conjugates over 100 ns simulations. (Y, G and M represent the peptide components of the conjugates: YYIVS, GSGGL and MPDAHL, respectively).

Figure 4

Trajectory snapshots of GH–peptide conjugates complexed with α-Syn fibrils at 0 ns, 50 ns and at 100 ns over a 100 ns simulation. (a) GH-YYIVS, (b) GH-GSGGL and (c) GH-MPDAHL.

Figure 5

Trajectory snapshots of BA–peptide conjugates complexed with α-Syn fibrils at 0 ns, 50 ns and at 100 ns over a 100 ns simulation. (a) BA-YYIVS, (b) BA-GSGGL and (c) BA-MPDAHL.

Figure 6

Comparison of Dynamic Light Scattering Analysis for each of the nanoassemblies formed over a period of one week.

Figure 7

Scanning electron microscopy images of self-assembled BA–peptide and GH–peptide conjugates. BA = Boswellate; GH = glycyrrhetinate; M = MPDAHL; G = YYIVS; and G = GSGGL.

Figure 8

Comparison of interactions of alpha-synuclein fibrils with self-assembled nanoconjugates. (a) α-Syn fibrils (untreated); (b) BA-G nanoassemblies with α-Syn; (c) BA-Y assemblies with α-Syn; (d) BA-M nanoassemblies with α-Syn; (e) GH-G nanoassemblies with α-Syn; (f) GH-Y nanoassemblies with α-Syn; and (g) GH-M nanoassemblies with α-Syn. All samples were incubated with α-Syn for 48 h, centrifuged, washed and then imaged using confocal microscopy. Images were taken in brightfield mode. Scale bar = 4 μm.

Figure 9

Comparison of FTIR spectra of BA–peptide and GH—–peptide nanoassemblies before and after binding with α-Syn. The spectrum of neat α-Syn is also shown to the far right of the image. M = MPDAHL; G = GSGGL; Y = YYIVS.

Figure 10

Cell viability results for microglial cells after 48 h of incubation with BA–peptide and GH–peptide assemblies. Y, G and M represent the peptide components YYIVS, GSGGL and MPDAHL, respectively, while GH and BA represent the glycyrrhetinate and Boswellate components of the conjugates, respectively. Data expressed are the mean (n = 3) with error bars indicating standard deviations. * p < 0.05; ** p < 0.01.

Figure 11

Interactions of microglial cells with nanoassemblies. (a) Control untreated cells; (b) cells incubated with BA-G assemblies; (c) cells incubated with BA-G assemblies; (d) cells incubated with BA-G assemblies; (e) cells incubated with BA-G assemblies; (f) cells incubated with BA-G assemblies; (g) cells incubated with BA-G assemblies. Images are taken after 24 h of incubation. Scale bar = 50 μm.

Figure 12

(a,b) Flow cytometry analysis showing uptake of alpha-synuclein in the presence and absence of nanoassemblies.

Figure 13

Expression of interleukin 6 in the presence and absence of nanoassemblies and α-Syn fibrils. Control 1: Microglial cells with no treatment. Control 2: Microglial cells with α-Syn fibrils, no assemblies were added.