Development and characterization of glutathione-conjugated albumin nanoparticles for improved brain delivery of hydrophilic fluorescent marker

Figures & data

Figure 1. Particle size distribution of (a) fluorescein sodium-loaded BSA nanoparticles; (b) glutathione-conjugated fluorescein sodium-loaded BSA nanoparticles.

Figure 2. SEM image of (a) fluorescein sodium-loaded BSA nanoparticles; (b) glutathione-conjugated fluorescein sodium-loaded BSA nanoparticles.

Table 1. Optimization of formulation variables to improve entrapment of fluorescein sodium inside the BSA nanoparticles.

Modified variable	Variable value	% Process yield	% Drug loading	% Entrapment efficiency	Particle diameter (nm)	Zeta potential (mV)
Polymer concentration	5 mg/mL	72.27 ± 6.77	15.16 ± 0.30	36.52 ± 4.81	221.5 ± 10.8	−37.1 ± 1.2
	10 mg/mL	75.54 ± 5.78	15.70 ± 0.24	37.10 ± 3.56	285.8 ± 5.9	−37.0 ± 1.0
	15 mg/mL	80.83 ± 7.15	16.20 ± 0.28	39.03 ± 5.10	330.7 ± 16.3	−34.5 ± 1.5
Fluorescein sodium to polymer ratio	1:1	70.75 ± 7.12	18.24 ± 0.58	25.81 ± 4.10	230.9 ± 11.2	−37.2 ± 1.1
	1:2	72.27 ± 8.25	15.16 ± 0.41	36.52 ± 3.73	221.5 ± 15.2	−35.4 ± 1.3
	1:5	84.1 ± 8.10	7.39 ± 0.35	37.26 ± 4.81	213.6 ± 10.6	−35.8 ± 1.3
Electrolyte concentration (NaCl)	0.05 M	84.3 ± 6.55	10.48 ± 0.43	52.80 ± 5.71	228.1 ± 7.3	−34.3 ± 1.7
	0.1 M	87.4 ± 7.05	11.25 ± 0.54	60.69 ± 6.03	235.7 ± 8.6	−34.1 ± 1.6
NaCl (0.1 M) þ 1 drop of concentrated HCl*	pH (2–3)	78.95 ± 6.72	12.41 ± 0.48	67.73 ± 5.12	285.8 ± 16.4	−38.5 ± 2.1

Mean ± SD, where n = 3.

*Addition of concentrate HCl modulate entrapment efficiency to the little extent but on the contrary caused aggregation of particles and hence this approach was restricted for further development.

Table 2. Effect of amount of EDAC on conjugation of glutathione with BSA nanoparticle.

Amount of EDAC (per 60 mg BSA nanoparticles)	Thiol groups (glutathione) µmol per gram of BSA	Thiol groups (glutathione) per BSA Nanoparticle	Particle diameter (nm)	Zeta potential (mV)
40 mg	30.11 ± 5.68	641 ± 121	252.5 ± 5.40	−45.5 ± 1.2
60 mg	35.11 ± 8.69	755 ± 187	270.9 ± 3.78	−47.3 ± 1.0
80 mg	32.12 ± 7.79	710 ± 173	268.1 ± 5.65	−46.6 ± 1.3

Table 3. Effect of EDAC reaction time on conjugation of glutathione with BSA nanoparticles.

EDAC incubation time (min) (per 60 mg BSA nanoparticles)	Thiol groups (glutathione) µmol per gram of BSA	Thiol groups (glutathione) per BSA nanoparticle	Particle diameter (nm)	Zeta potential (mV)
30	31.37 ± 6.05	667 ± 139	254.3 ± 5.84	−45.8 ± 1.0
45	35.11 ± 8.69	755 ± 187	270.9 ± 3.78	−47.3 ± 1.0
90	32.48 ± 7.15	713 ± 159	268.5 ± 5.85	−46.7 ± 1.2

Figure 3. In vitro release profile of fluorescein sodium in phosphate buffer pH 7.4 from different formulations.

Figure 4. In vitro release profile of fluorescein sodium in phosphate buffer pH 4 from different formulations.

Figure 5. Effect of fluorescein sodium-loaded formulations on (a) MDCK-MDR1 cell viability; (b) primary neuro-glial cell viability.

Figure 6. Apparent permeability of fluorescein sodium-loaded formulations through MDCK-MDR1 cell monolayer at different time interval.

Table 4. The apparent permeability (P_app) of fluorescein sodium solution and glutathione-conjugated and unconjugated BSA nanoparticles loaded with fluorescein sodium across in vitro MDCK-MDR1 cell monolayer (n = 3).

Apparent permeability coefficient	Fluorescein sodium solution	Fluorescein sodium-loaded BSA nanoparticles	Glutathione-conjugated fluorescein sodium-loaded BSA nanoparticles
Papp (cm/s) × 10^−5	0.253 ± 0.01	0.784 ± 0.05^a	1.89 ± 0.08^b,c

Mean ± SD (n = 3).

Nanoparticles equivalent to fluorescein sodium concentration 100 μg/mL were used for the study.

Level of significance according to the Tukey test:

^ap < 0.001 fluorescein sodium solution versus fluorescein sodium BSA NPs.

^bp < 0.001 fluorescein sodium solution versus glutathione-conjugated fluorescein sodium BSA NPs.

^cp < 0.001 fluorescein sodium BSA NPs versus glutathione-conjugated fluorescein sodium BSA NPs.

Figure 7. Uptake of fluorescein sodium-loaded formulations by primary neuro-glial cells under fluorescent (left) and phase contrast (right) microscopy. (a) Blank control, (b) Fluorescein sodium solution, (c) Fluorescein sodium-loaded BSA nanoparticles, (d) Glutathione-conjugated fluorescein sodium-loaded BSA nanoparticles.

Figure 8. Fluorescein sodium concentrations (ng/mL) in different organs after intravenous injection of nanoparticles formulations.

Table 5. Fluorescein sodium concentrations (ng/g organ weight) in different organs after intravenous injection of BSA nanoparticles.

		Formulation
Sr. No.	Organ	Fluorescein sodium solution	Fluorescein sodium BSA NPs	Glutathione-conjugated fluorescein sodium BSA NPs
1	Brain	159.90 ± 24.40	476.41 ± 30.88^a	1439.20 ± 35.25^c,d
2	Lungs	1209.77 ± 69.63	1530.36 ± 105.53^a	1842.61 ± 68.91^c,d
3	Liver	1742.90 ± 52.69	2446.70 ± 110.67^a	1986.05 ± 70.94^c,d
4	Spleen	1080.51 ± 61.50	1250.81 ± 45.32^b	1024.47 ± 50.74^d
5	Kidney	2589.72 ± 204.27	1584.22 ± 110.76^a	1887.04 ± 86.51^c,d

Mean ± SD (n = 6).

Level of significance according to the Tukey test:

^ap < 0.001 fluorescein sodium solution versus fluorescein sodium BSA NPs.

^bp < 0.01 fluorescein sodium solution versus fluorescein sodium BSA NPs.

^cp < 0.001 fluorescein sodium solution versus glutathione-conjugated fluorescein sodium BSA NPs.

^dp < 0.001 fluorescein sodium BSA NPs versus glutathione-conjugated fluorescein sodium BSA NPs.