Pullulan-Based Nanoparticle-HSA Complex Formation and Drug Release Influenced by Surface Charge

Abstract

The nanomaterial composition of nanoparticles and their protein adsorption in the blood is of great significance in the design of drug-loaded nanoparticles. To explore the interaction between the different surface components of nanoparticles (NPs) and protein, we synthesized three kinds of pullulan NP polymers: cholesteric hydrophobically (CH) modified pullulan (CHP), CH-modified animated pullulan (CHAP), and CH-modified carboxylated pullulan (CHSP). Pullulan NPs were prepared by the dialysis method. Dynamic light scattering was used to determine the charge and size of the three NPs. The size of NPs was altered by the number of charge groups when polymers contain the same degree of cholesterol substitution. The zeta potentials were + 12.9, - 15.4, and - 0.698 mV for CHAP, CHSP, and CHP, respectively, and the dimensions were 116.9, 156.9, and 73.1 nm, respectively. Isothermal titration calorimetry was used to determine the thermodynamic changes of NPs with different surface charge, and the effect of human serum albumin (HSA) on the titration was investigated. The changes of enthalpy and entropy demonstrated an interaction between NPs and HSA; the binding constant (K_b) for CHSP, CHP, and CHAP was 1.41, 27.7, and 412 × 10⁴ M^-1, respectively, with the positive charge for CHAP-HSA, uncharged for CHP-HSA, and negative charge for CHSP-HSA complex. Fluorescence and circular dichroism spectroscopy were used to determine the protein structure change after the complexation between NPs and HSA. The NP and HSA complexation is a complicated process composed of protein α-helical content reduction and the peptide chain extension; CHP NPs had the largest reduction in HSA α-helical content. The drug release rates of all compounds of NP and HSA were significantly lower than those of free drug and drug-loaded NPs after 48 h. The highest and lowest rates were observed in CHSP-HSA and CHP-HSA, respectively. The drug release was significantly influenced by the adsorption of HSA on NPs, and the size and surface charge of NPs played an important role in this process.

Keywords: Binding constant; Drug release; Human serum albumin; Nanoparticles; α-Helix.

Fig. 1

Synthesis of CHP, CHSP, and CHAP polymers

Fig. 2

FTIR spectra of CHP (a), CHAP (b), and CHSP (c)

Fig. 3

¹HNMR spectra for CHP (a), CHSP (b), and CHAP (c) NPs

Fig. 4

Zeta potential (a) and size distribution (b) of CHP NPs (), CHAP NPs (), and CHSP NPs ()

Fig. 5

Isothermal titration calorimetry data for HSA titration into a CHP, b CHSP, and c CHAP NPs at 25 °C. NP concentration in the cell (250 μL) was 12 μM and the protein concentration in the syringe was 230 μM. Upper graphs show raw data, and lower graphs show integrated heats

Fig. 6

A Fluorescence spectra for HSA (1.5 × 10^− 5 mol/L) without (a) and with (b) CHSP, (c) CHP and (d) CHAP NPs with the same concentration (4.2 × 10^− 6 mol/L). B HSA emission intensity with CHSP, CHP, and CHAP NPs at 343 nm over time

Fig. 7

Fluorescence spectra of HSA (1.5 × 10^− 5 mol/L) with CHSP (A), CHP (B), and CHAP (C) at different concentrations (a) 0, (b) 2.07 × 10⁻⁷, (c) 3.31 × 10⁻⁷, (d) 4.14 × 10⁻⁷, (e) 8.28 × 10⁻⁷, (f) 20.7 × 10⁻⁷, (g) 33.1 × 10⁻⁷, and (h) 41.4 × 10⁻⁷ mol/L. D Plots (n = 7) for F₀/(F₀ − F) vs 1/[Q], Q is the concentration of CHSP (–◆–), CHP (–■–) and CHAP (–▲–), respectively

Fig. 8

A CD spectra for (a) free HSA, (b) CHSP–HSA, (c) CHAP–HSA, and (d) CHP–HSA in solution at 25 °C. The samples were complex I. B CD spectra for (a) free HSA, (b) CHSP–HSA, (c) CHAP–HSA, and (d) CHP–HSA in solution at 25 °C. The samples were complex II. C Ellipticity at 208 nm for HSA interacting with CHSP (–▲–), CHAP (–●–) and CHP (–■–) over time

Fig. 9

Mitoxantrone (MTO) release of pullulan NPs in phosphate-buffered saline (PBS) at 37 °C in vitro (□: free mitoxantrone, ○: CHP, △: CHAP, ▽: CHSP, ◁: CHAP–HSA,◇: CHP–HSA, ▷: CHSP–HSA)

Fig. 10

Adsorption of HSA to NPs