Introduction: Berberine (BBR), an isoquinoline alkaloid from Berberis aristata, holds a rich history in traditional medicine and is the subject of modern scientific exploration. BBR has been integral to Ayurveda and traditional Chinese medicine, addressing ailments from hypertension to inflammation. It significantly lowers lipid levels by enhancing LDL receptor expression and has undergone clinical trials for anti-hyperlipidemic effects (3-5). BBR also inhibits cell invasion, metastasis, and influences cell proliferation, demonstrating anti-inflammatory and antioxidant properties (6-8). However, its potential is constrained by poor water solubility, first-pass metabolism, and limited absorption, necessitating high doses with potential side effects (3, 4, 9-11). Therefore, a particular nano-formulation method (the hydrothermal method) has been utilized in an attempt to overcome the issue of low water solubility and increase its potential therapeutic efficacy. HSA, the predominant protein in blood plasma, is a single-chain, none-glycosylated peptide exhibiting a helical structure comprised of extended loops and turns (with concentrations of 35–50 g/L in human serum and a molecular weight of 66.5 kDa). HSA has a high affinity for a wide range of molecules including exogeneous drugs and this affinity ultimately affects the distribution and bioavailability of drugs. Thus, this study was conducted to explore the intricate details and subtle aspects of the interaction between berberine nanoparticles (nBBR) and HSA, which can further be useful in designing drug delivery systems based on plasma proteins.
Methods: The nBBR was produced through the hydrothermal method, and the obtained particles were characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS). Moreover, the formation of the nBBR-HSA complex has been investigated using biophysical methods such as fluorescence spectroscopy, isothermal titration calorimetry, and circular dichroism (CD).
Results: The hydrodynamic radius (RH) and the size distribution of the synthesized nanoparticles were measured using the dynamic light scattering. Additionally, TEM micrographs were employed to analyze the morphology of the obtained nanoparticles (Fig. 1). Furthermore, fluorescence data of HSA revealed a strong emission with a maximum peak at 340 nm, which significantly reduced in a concentration-dependent manner in the presence of nBBR (Fig. 2). The experiment was conducted at different temperatures (298, 303, and 308 K), and the data were analyzed using the Stern-Volmer equation. The 𝐾SV values (on the order of 10^5 M^(-1)) exhibited a decrease as the temperature increased. To provide a quantitative description of the energetics of the interaction, thermodynamic measurements were performed using the van’t Hoff method and ITC technique. According to the data, the formation of the nBBR-complex is associated with a positive entropy change (11.95 J.〖mol〗^(-1) K^(-1)) followed by a negative enthalpy change (-23.56 kJ.〖mol〗^(-1)). This results in a negative net ΔG^0 for the system. Moreover, negative (downward) peaks appeared in the ITC thermogram. The CD spectrum of HSA (Fig. 3) demonstrated the double minima at 208 and 222 nm, which are the characteristic of α-helical structure. Notably, the negative ellipticity increased upon the addition of nBBR, and the formation of the complex was accompanied by an enhancement in the α-helix and β sheets content percentage, along with a reduction in random coil structures.
Conclusion: In this empirical study, we synthesized berberine nanoparticles (nBBR) using the hydrothermal method and conducted a comprehensive analysis of their interaction with human serum albumin (HSA) through various biophysical techniques. Our analysis, as revealed by dynamic light scattering (DLS) and transmission electron microscopy (TEM) imaging, demonstrated that the produced particles exhibited an average size ranging from 50 to 70 nm, with a narrow size distribution and spherical morphology, aligning precisely with our anticipated outcomes. Furthermore, fluorescence data combined with Stern-Volmer analysis unequivocally confirmed the ability of nBBR to interact with HSA, forming a ground-state complex and eliciting structural alterations in the protein. Thermodynamic profile of the interaction underscored that the formation of nBBR-HSA complex is an energetically favorable, exothermic, and spontaneous process that regulates through electrostatic forces. Notably, the circular dichroism results unveiled the significant influence of nBBR in inducing conformational changes and promoting the development of a more ordered secondary structure in HSA, thereby enhancing its biological functionality. These findings collectively shed light on the binding behavior of nBBR-HSA complex, providing information that holds substantial implications in designing drug delivery systems