Combined computational and experimental studies of molecular interactions of albuterol sulfate with bovine serum albumin for pulmonary drug nanoparticles

Figures & data

Figure 1 Chemical structure of albuterol sulfate with the protonated nitrogen atom.

Note: 1, 2, and 3 are the oxygen atom of the phenolic hydroxy group, the oxygen atom of the hydroxymethlphenyl group, and the oxygen atom of the α-hydroxy group, respectively.

Figure 2 The MD process of AS–BSA complexes.

Notes: (A) RMSD of the backbone atoms of BSA and the R_g of BSA as a function of time during 5 ns MD and (B) 25–35 ns MD for the complex of BSA and AS. (C) Ligand starting positions of ten AS molecules. BSA is represented with New Cartoon colored by secondary structure and AS with VDW by name. (D) Number of hydrogen bonds (HB_n) between BSA and AS during 40 ns MD. AS1–AS10, representing different ligand starting positions, were numbered when adding AS into the cubic box randomly. (E) Positions of AS1–AS3 and AS8 on the BSA at 35 ns; the BSA is shown with QuickSurf colored by index and AS with CPK by name. (F) The hydrogen bond number (HBn) and lifetime per hydrogen bond (HB_lifetime) during the simulation of AS1, AS2, AS3 and AS8 with BSA.
Abbreviations: MD, molecular dynamics; AS, albuterol sulfate; BSA, bovine serum albumin; RMSD, root-mean-square-deviation; R_g, radius of gyration; VDW, van der Waals; CPK, Corey–Pauling–Koltun.

Table 1 Comparison of binding energy (kJ/mol) between major AS-binding sites on BSA

Binding site	Componentsa
	ΔEvdw	ΔEele	ΔGpolar	ΔGnonpolar	ΔGbind
AS1	−29.8±9.2	−1,744.3±40.1	1,510.7±29.7	−9.3±0.5	−272.7±21
AS2	−38.4±18.3	−985.2±141.2	970±162.4	−6.8±2.8	−60.4±26.5
AS3	−5±8.6	−718.5±252.8	698.8±232	−16.2±4.1	−40.9±57.6
AS8	−26.0±10.1	−939±49.3	910.8±57.9	−21.1±3.9	−75.2±14.6

Notes:

a E_vdw, van der Waals energy; E_ele, electrostatic energy; G_polar, polar solvation energy; G_nonpolar, the nonpolar solvation energy; G_bind, the binding free energy; G_bind, E_vdw + E_ele + G_polar+ G_nonpolar. The sum of G_polar and G_nonpolar represents the solvation free energy.

Abbreviations: AS, albuterol sulfate; BSA, bovine serum albumin.

Table 2 Hydrogen-bond analysis in terms of presence and average distance

Code	Donor	Acceptor	Presence (%)	Distance (Å)
HB1	Lys239 N–H	AS O2	–	4.58±1.03
HB2	Lys239 N–H	AS O3	85.71	2.96±0.61
HB3	AS O2–H	Glu243 O1	91.71	2.22±0.99
HB4	AS O2–H	Glu243 O2	90.61	2.58±1
HB5	AS N–H	Asp255 O1	67.73	3.38±0.59
HB6	AS N–H	Asp255 O2	88.71	2.22±0.8
HB7	AS O3–H	Glu251 O1	–	5.87±0.86
HB8	AS O3–H	Glu251 O2	–	5.47±1.13
HB9	AS O3–H	Asp255 O1	93.71	1.83±0.62

Note: Presence labeled “–” was <20%, which represented the hydrogen bonds between donors and acceptors being very unstable.

Abbreviations: Lys, lysine; Glu, glutamate; Asp, aspartate; AS, albuterol sulfate.

Figure 3 Critical site interactions of AS1 with BSA.

Notes: (A) Detailed interactions between AS1 and BSA; BSA is represented with NewCartoon colored by secondary structure and AS1 with line by name. (B) The DA type of water bridge between AS1 and BSA in the process of MD; BSA is represented with NewCartoon colored by secondary structure and AS1 with CPK by name. (C) Binding energy contribution of each residue of BSA; residues which decomposition energy exceed to 10 kJ/mol are labeled. The inset is the residues around the binding site.
Abbreviations: AS, albuterol sulfate (numbered 1 when adding ten albuterol sulfate molecules into the cubic box randomly); BSA, bovine serum albumin; MD, molecular dynamics; DA, donor–acceptor; CPK, Corey–Pauling–Koltun; Lys, lysine; Glu, glutamate; Asp, aspartate; W, water.

Figure 4 SPR dose–response sensorgrams of AS with immobilized BSA.

Notes: (A) Response curve of different concentrations of AS from 0.982 μM to 1.01 mM with twofold dilution. (B) Fitting of response and AS concentrations to 1:1 Langmuir binding model using Biacore T200 Evaluation software version 2.0.
Abbreviations: SPR, surface plasmon resonance; AS, albuterol sulfate; BSA, bovine serum albumin.

Table 3 Drug-loading (DL) efficiency of BSA-NPs in different AS concentrations (mean ± standard deviation; n=3)

AS concentration (mg/mL)	ABNI		ABNA
	DLa	BSA:ASb	DLa	BSA:ASb
0.5	1.2±0.3	1:2	1.9±0.03	1:4
1.0	2.5±0.3	1:4	1.9±0.7	1:4
2.0	4.1±0.9	1:8	2.3±1.2	1:4

Notes:

a Loading efficiency (mg/100 mg);

b one BSA molecule combined with various amounts of AS (mol:mol) on average.

Abbreviations: AS, albuterol sulfate; BSA, bovine serum albumin; ABNI, BSA nanoparticles carrying AS prepared by incorporation; ABNA, BSA nanoparticles carrying AS prepared by adsorption.

Figure 5 Characterization of AS-BSA-NPs.

Notes: Size distribution of ABNI (A) and ABNA (B). TEM images of ABNI (C) and ABNA (D).
Abbreviations: AS-BSA-NPs, bovine serum albumin nanoparticles carrying albuterol sulfate; ABNI, AS-BSA-NPs prepared by incorporation; ABNA, AS-BSA-NPs prepared by adsorption; TEM, transmission electron microscopy.

Table 4 Fitting model for the release kinetics of AS from AS-BSA-NPs

Model	Zero order	First order	Higuchi	Ritger–Peppas
ABNI	$\frac{M_t}{M_{\infty }}=0.19t+27.91$	$\frac{M_t}{M_{\infty }}=81.84\left(1-{\mathrm{e}}^{-0.019t}\right)$	$\frac{M_t}{M_{\infty }}=4.48t^{\frac{1}{2}}+10.42$	$\frac{M_t}{M_{\infty }}=13.38t^{0.32}$
	(R^2=0.628)	(R^2=0.99)	(R^2=0.877)	(R^2=0.869)
ABNA	$\frac{M_t}{M_{\infty }}=0.24t+20.95$	$\frac{M_t}{M_{\infty }}=91.32\left(1-{\mathrm{e}}^{-0.012t}\right)$	$\frac{M_t}{M_{\infty }}=5.18t^{\frac{1}{2}}+2.19$	$\frac{M_t}{M_{\infty }}=7.25t^{0.44}$
	(R^2=0.784)	(R^2=0.986)	(R^2=0.949)	(R^2=0.925)

Notes: M_t is the cumulative mass of AS that had been released from AS-BSA-NPs, and M_∞ is the total mass of AS in the AS-BSA-NPs; t is time (minutes).

Abbreviations: AS-BSA-NPs, BSA nanoparticles carrying albuterol sulfate; ABNI, AS-BSA-NPs prepared by incorporation; ABNA, AS-BSA-NPs prepared by adsorption.

Figure 6 In vitro release profiles of AS-BA-NPs and free AS in PBS (pH 7.2) (n=3).

Note: Error bars represent standard deviation.
Abbreviations: AS-BSA-NPs, bovine serum albumin nanoparticles carrying albuterol sulfate; ABNI, AS-BSA-NPs prepared by incorporation; ABNA, AS-BSA-NPs prepared by adsorption; PBS, phosphate-buffered saline.

Figure S1 The RMSD of the backbone atoms of the complex with respect to its initial structure during 40 ns MD simulation. The part enclosed in red (25–35 ns) indicates equilibrium.

Abbreviations: RMSD, root-mean-square-deviation; MD, molecular dynamics.

Table S1 Binding pocket analysis for initial and MD-BSA using DoGSiteScorer

Structure	nb	Pocketc	Volume (Å^3)	Surface (Å^2)	Liposurface (Å^2)	Depth (Å^2)
Initial-BSA	15	1	979.56	1,208.59	792.46	33.22
		2	972.45	1,175.37	870.87	26.29
		3	910.76	1,086.47	686.13	21.3
MD-BSAa	18	1	996.04	1,318.51	859.25	24.55
		2	905.07	1,049.02	824.14	24.67
		3	904.64	1,080.84	808.19	26.48

Notes:

a BSA after 5 ns MD simulation;

b number of BSA potential binding pockets;

c main binding pockets based on the algorithm of DoGSiteScorer. Major structural features analyzed were pocket volume, solvent-accessible surface, liposurface, and pocket depth.

Abbreviations: MD, molecular dynamics; BSA, bovine serum albumin.