Colloidal gold-loaded, biodegradable, polymer-based stavudine nanoparticle uptake by macrophages: an in vitro study

Figures & data

Table 1 Composition of experimental nanoparticles with their drug loading and entrapment efficiency

Formulation code	Stavudine:PLGA (mg:mg)	Presence of colloidal gold	Speed of homogenization (rpm)	Amount recovereda (mg)	Drug loadingb (%)	Entrapment efficiencyc (%)
S1	20:250	No	15,000	165.4	5.69	76.83
S2	20:250	Yes	15,000	187.2	5.08	68.57
S3	25:250	No	16,000	150	7.93	87.23
S4	25:250	Yes	16,000	186.6	6.15	67.63
S5	0:250	No	15,000	152	NA	NA

Notes:

a Recovery (mg) = amount of lyophilized formulation obtained (mg) out of total amount of drug and excipients used (mg);

b Drug loading (actual) (%) = Amount of drug in nanoparticles × 100/Amount of nanoparticles obtained;

c Drug entrapment efficiency (%) = Drug loading (actual) (%) × 100/Drug loading (theoretical) (%).

Abbreviations: NA, not applicable; PLGA, poly(d,l-lactic-co-glycolic acid).

Figure 1 FTIR spectra of (A) stavudine; (B) PLGA; (C) PVA; (D) PVA and PLGA mixture; and (E) stavudine, PVA, and PLGA mixture.

Abbreviations: FTIR, Fourier transform infrared spectroscopy; PLGA, poly(d,l-lactic-co-glycolic acid); PVA, polyvinyl alcohol.

Table 2 Size distribution (Z-average [nm]), polydispersity index, zeta potential, and conductivity of experimental nanoparticulate formulations

Formulation code	Z-average (nm)	PDI	Zeta potential (mV)	Conductivity (mS/cm)
S1	1365	0.102	−12.6	0.0117
S2	1136	0.109	−8.16	0.0346
S3	211	0.424	−27.8	0.00894
S4	201	0.376	−19.6	0.00882
S5	2038	0.046	−17	0.0125

Abbreviation: PDI, polydispersity index.

Figure 2 FESEM photographs of formulations. (A) Formulation S1 prepared at 15,000 rpm shows that the particles were of micrometer sizes. (B) Formulation S2 prepared at 15,000 rpm shows that the particles were in micron range. (C) Formulation S3 prepared at 16,000 rpm shows that the particles were in nanometer range. (D) Formulation S4 prepared at 16,000 rpm shows that the particles were in nanometer range. (E) Formulation S5 prepared at 15,000 rpm shows that the particles were mostly in micrometer range. (F) SEM photograph of the gold nanoparticles shows that the particles were in submicron range with variable sizes.

Abbreviations: FESEM, field-emission scanning electron microscopy; SEM, scanning electron microscopy.

Figure 3 Size distribution of different formulations. (A) S1 (by intensity); (B) S2 (by intensity); (C) S3 (by intensity); (D) S4 (by intensity); (E) S5 (by intensity).

Figure 4 EDX data showing the presence of gold particles in gold-encapsulated polymeric nanoparticles.

Abbreviation: EDX, energy dispersive X-ray spectroscopy.

Figure 5 EDX data of colloidal gold nanoparticles alone.

Abbreviation: EDX, energy dispersive X-ray spectroscopy.

Figure 6 Release profile of didanosine from different formulations in phosphate-buffered saline (pH 7.4).

Note: Data show means (n = 3).

Table 3 Data of drug release kinetics of various formulations

Kinetic model	Formulation S1	Formulation S2	Formulation S3	Formulation S4
Zero order	R^2 = 0.890	R^2 = 0.917	R^2 = 0.886	R^2 = 0.855
	K0(μg mL^−1 h^−1) = 1.0257	K0(μg mL^−1 h^−1) = 0.9542	K0(μg mL^−1 h^−1) = 0.8198	K0(μg mL^−1 h^−1) = 0.8341
First order	R^2 = 0.966	R^2 = 0.973	R^2 = 0.931	R^2 = 0.908
	K1(h^−1) = −0.0081	K1(h^−1) = −0.0072	K1(h^−1) = −0.0063	K1(h^−1) = −0.0072
Higuchi	R^2 = 0.986	R^2 = 0.984	R^2 = 0.951	R^2 = 0.945
	KH(h^−1/2) = 9.2194	KH(h^−1/2) = 8.9496	KH(h^−1/2) = 7.8591	KH(h^−1/2) = 8.4073
Korsmeyer–Peppas	R^2 = 0.825	R^2 = 0.963	R^2 = 0.919	R^2 = 0.901
	n = 0.785	n = 0.657	n = 0.519	n = 0.453
Hixson–Crowell	R^2 = 0.945	R^2 = 0.958	R^2 = 0.918	R^2 = 0.889
	KHC(μg^1/3 t^−1) = −0.0233	KHC(μg^1/3 t^−1) = −0.0212	KHC(μg^1/3 t^−1) = −0.0189	KHC(μg^1/3 t^−1) = −0.021
Hopfenberg	R^2 = 0.9851	R^2 = 0.9679	R^2 = 0.9143	R^2 = 0.9199
	K(μg mg^−1 μm^−1)h^−1 = 0.0092	K(μg mg^−1 μm^−1)h^−1 = 0.0104	K(μg mg^−1 μm^−1)h^−1 = 0.0559	K(μg mg^−1 μm^−1)h^−1 = 0.1139

Table 4 Nanoparticle (S4) uptake by macrophages

Serial no	Concentration of nanoparticles in suspension (ng/mL medium)	Effective concentration of drug (ng/mL medium)	Incubation time (hours)	Degree of nanoparticles uptake by macrophages (number of + symbols indicates the increasing degree)
1	300	17.28	4	+
2	500	28.80	4	++

Figure 7 Confocal microscopic image of macrophages treated with (A) stavudine nanoparticles (300 ng/mL of medium) for 4 hours, and (B) stavudine nanoparticles (500 ng/mL of medium) for 4 hours.