Lipid liquid-crystalline nanoparticles as a suitable platform for accommodating sensitive membrane proteins: monitoring the activity of HMG-CoA reductase

Abstract

Biological molecules such as integral membrane proteins, peptides, and nucleic acids that are not soluble or sufficiently stable in aqueous solutions can be stabilized through encapsulation in lipid nanoparticles. Discovering the potential of lipid liquid-crystalline nanoparticles opens up exciting possibilities for housing sensitive membrane proteins. Lipid mesophases provide an environment that protects the cargo, usually a drug, from rapid clearance or degradation. This study employed the mentioned platform to stabilize a different cargo-an essential transmembrane enzyme, HMG-CoA reductase (HMGR). The nanostructured lipid liquid-crystalline (LLC) nanoparticles known as hexosomes are selected as a convenient nanocontainer for the redox-active protein for real-time monitoring of its functions in the bulk of the solution and point to the applicability of the proposed platform in the evaluation of therapeutic functions of the protein by standard physicochemical methods. Instead of using detergents, which usually affect the functions and stability of sensitive membrane proteins, we provide a suitable environment, protecting them in the bulk of the solution against other present species, e.g., toxic compounds or degrading proteins. The objective was to optimize the composition and structure of the lipid nanoparticles to meet the needs of such sensitive and flexible membrane proteins as HMGR and compare the functioning of the encapsulated enzyme with that of the same protein free in the aqueous solution. The catalytic reaction of HMGR involves the 4-electron reduction of HMG-CoA to mevalonate and CoA while simultaneously oxidizing NADPH to NADP⁺. Subsequently, mevalonate is transformed into cholesterol. The hexosomes we selected as lipid nano-containers were composed of monoolein, 1-oleoyl-rac-glycerol (GMO), Pluronic^® F127, and poly(ethylene glycol) (PEG). These specific structural characteristics of the lipid nanoparticles were found optimal for enhancing the stability of HMGR. We characterized these hexosomes using dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), and cryogenic electron microscopy (Cryo-TEM) methods, both with and without the encapsulated protein. In our innovative approach, the enzyme activity was assessed by monitoring changes in NADPH concentration outside the nanocarrier. We tracked fluctuations in NADPH levels during the catalytic reaction using two independent methods: UV-Vis spectrophotometry and cyclic voltammetry. Significantly, we could demonstrate the inhibition of the nano-encapsulated enzyme by fluvastatin, an enzyme inhibitor and cholesterol-lowering drug. This paves the way for the discovery of new enzymatic inhibitors and activators as therapeutic agents controlling the activity of membrane proteins, thereby inspiring future cholesterol-lowering therapies in our case and, in general, further research and potential new treatments.

Keywords: HMG-CoA reductase; NADPH; Bioelectrochemistry; Cyclic voltammetry; Hexosome; Lipid nanoparticles; Transmembrane enzyme.

Scheme 1

Rate-limiting reaction in cholesterol biosynthesis catalyzed by HMG-CoA reductase

Scheme 2

Chemical structures of a) monoolein 1-oleoyl-rac-glycerol (GMO) and b) polymer Pluronic®F127 (x = 95, y = 62, z = 95), and c) poly(ethylene glycol) (PEG) (n = 200)

Fig. 1

Representative small-angle X-ray scattering diffraction patterns were obtained for hexosomes (1, black line) and proteohexosomes (2, orange line)

Fig. 2

Calculated Gaussian distributions of the hexosomes (1, black) and proteohexosomes (2, orange) sizes determined using the DLS method and the correlation coefficient in time for DLS measurements (dashed lines—1 and 2)

Fig. 3

Cryo-TEM images visualizing examples of hexosomes (A) and proteohexosomes (B). The scale bar is 100 nm. Inset: fast Fourier transform (FFT) analysis of the dispersions

Fig. 4

Monitoring HMGR (in the form of solution) activity in the solution: A The relative change in the absorbance of NADPH measured at 340 nm during 10 min of catalytic reaction (1, yellow line). The initial concentration of NADPH in the cell was 0.4 mmol/L with 0.2 mmol/L HMG-CoA. Absorbance in the presence of fluvastatin (C = 10^–5 mol/L) in the solution (2, purple line). The absorbance of NADPH over 10 min but without HMGR in the solution (3, blank sample, red line). B The normalized spectra recorded after 10 min of the catalytic reaction (1, yellow line) and absorbance showing inhibition in the presence of 10^–5 mol/L fluvastatin (2, purple line). The absorbance of NADPH during 10 min but without HMGR in the solution (3, blank sample, red line)

Fig. 5

HMGR activity in the solution form (yellow) and incorporated into hexosomes (orange) measured using the UV–Vis method and calculated using Eq. 2

Fig. 6

Cyclic voltammograms obtained for 40 μmol/L ABTS (1, blue line) and in the presence of 0.4 mmol/L NADPH (2, yellow line) in PBS pH 7.4. Scan rate: 10 mV/s. Inset: redox catalysis of NADPH and ABTS (adapted from) [64].

Fig. 7

Cyclic voltammograms obtained for HMGR solution (A) and hexosomes with HMGR (B) in PBS pH 7.4 in the presence of 0.2 mmol/L HMG-CoA, 0.4 mmol/L NADPH, and 40 μmol/L ABTS. Scan rate: 10 mV/s

Fig. 8

Conversion percentage of NADPH to NADP⁺ in the HMGR-catalyzed reaction monitored spectrophotometrically (A) and voltammetrically (B) during reduction of HMG-CoA to mevalonate in PBS pH 7.4 in the presence of 0.2 mmol/L HMG-CoA (1, yellow for HMGR solution and 2, orange for HMGR entrapped in hexosomes) and 0.4 mmol/L NADPH (and 40 μmol/L ABTS in the case of CV)

Fig. 9

Time dependence of the ABTS-related oxidation peak current in the presence (purple and red lines) and absence (yellow and orange lines) of 10^–5 mol/L fluvastatin recorded for HMGR solution (purple and yellow), and hexosomes containing HMGR (red and orange) in the presence 0.2 mmol/L HMG-CoA, 0.4 mmol/L NADPH and 40 μmol/L ABTS. I_c is the peak current in the presence of NADPH, and I_d is the diffusion current of ABTS in the absence of NADPH