Intranasal delivery of Huperzine A to the brain using lactoferrin-conjugated N-trimethylated chitosan surface-modified PLGA nanoparticles for treatment of Alzheimer’s disease

Figures & data

Figure 1 The synthesis routes of TMC and Mal-TMC.

Note: R represents the maleimide group.

Abbreviations: TMC, N-trimethylated chitosan; Mal-TMC, maleimide–TMC; SMP, N-Succinimidyl 3-maleimidopropionate.

Table 1 Different levels of variables in the Box–Behnken design

Variables	Level
	Low	Medium	High
Independent variables
A = Polymer concentration (mg/mL)	2.5	6.25	10
B = Theoretical drug loading (%)	5	15	25
C = PVA concentration (mg/100 mL)	0.5	1	1.5
Dependent variables	Desired constraints
Y1= Entrapment efficiency (%)	Maximize
Y2= Diameter of particles (nm)	Minimize

Abbreviation: PVA, polyvinyl alcohol.

Figure 2 ¹H-NMR spectra of (A) chitosan, (B) TMC, and (C) Mal-TMC.

Abbreviations: NMR, nuclear magnetic resonance; TMC, N-trimethylated chitosan; Mal-TMC, maleimide–TMC.

Table 2 Effect of independent variables on dependent variables

Run	Independent variables			Dependent variables
	Polymer concentration (mg/mL) (A)	Theoretical drug loading (%) (B)	PVA concentration (mg/100 mL) (C)	Entrapment efficiency (%) (Y1)	Diameter of particles (nm) (Y2)
1	2.5	15	1.5	4.25	140
2	10	15	1.5	28.57	175
3	6.25	15	1	77.90	124
4	2.5	15	0.5	18.58	161.7
5	6.25	5	1.5	19.00	138.5
6	10	5	1	46.78	165
7	6.25	5	0.5	60.92	153.7
8	6.25	15	1	79.31	123
9	10	15	0.5	36.34	223.9
10	10	25	1	23.12	193.6
11	6.25	25	0.5	23.45	185
12	2.5	5	1	18.34	146.9
13	6.25	15	1	76.02	119
14	6.25	15	1	80.01	121
15	6.25	25	1.5	29.34	142.6
16	6.25	15	1	80.22	126
17	2.5	25	1	13.63	134

Abbreviation: PVA, polyvinyl alcohol.

Figure 3 3D response surface plots showing the effect of independent variables on dependent variables. The y-axis represents the desirability of dependent variables. The sign represents the site of the optimal formulation.

Abbreviation: PVA, polyvinyl alcohol.

Table 3 Characterization of Huperzine A-loaded nanoparticles

Formulation	Diameter of particles (nm)	Polydispersity index	Zeta potential (mV)	Entrapment efficiency (%)
PLGA NPs	78.1±3.7	0.182±0.027	−21.2±0.8	83.2±9.2
TMC NPs	125.4±9.1	0.197±0.025	+36.3±4.0	77.0±3.9
Lf-TMC NPs	153.2±13.7	0.229±0.078	+35.6±5.2	73.8±5.7

Note: Values represent the mean ± SD (n=3).

Abbreviations: PLGA, polylactide-co-glycoside; NPs, nanoparticles; TMC, N-trimethylated chitosan; Lf, lactoferrin.

Figure 4 In vitro release study of HupA from free Huperzine A, HupA PLGA NPs, HupA TMC NPs and HupA Lf-TMC NPs in 0.1 M pH 7.4 PBS.

Abbreviations: HupA, Huperzine A; PLGA, polylactide-co-glycoside; Lf, lactoferrin; TMC, N-trimethylated chitosan; NPs, nanoparticles; PBS, phosphate buffered saline.

Figure 5 Binding efficiency of mucin to NPs. Values represent the mean ± SD (n=3). Statistically significant differences with PLGA NPs are marked with (**) for p<0.01.

Abbreviations: PLGA, polylactide-co-glycoside; Lf, lactoferrin; TMC, N-trimethylated chitosan; NPs, nanoparticles.

Figure 6 Cytotoxicity of free HupA, HupA loaded (A) and unloaded NPs (B) incubated for 24 h in 16HBE cells. Values represent the mean ± SD (n=3). Statistically significant differences with PLGA NPs are marked with * for p<0.05 and ** for p<0.01.

Abbreviations: HupA, Huperzine A; PLGA, polylactide-co-glycoside; Lf, lactoferrin; TMC, N-trimethylated chitosan; NPs, nanoparticles.

Figure 7 Cellular uptake of NPs. Fluorescence microscopy images of cellular uptake of Nile red NPs in 16HBE (A) and SH-SY5Y cells (B); original magnification ×200. Mean fluorescence intensity on flow cytometry of coumarin-6 NPs in (C) 16HBE and (D) SH-SY5Y cells. Values represent the mean ± SD (n=3). Statistically significant differences with PLGA NPs are marked with ** for p<0.01. Statistically significant differences with TMC NPs are marked with # for p<0.05 and ## for p<0.01.

Abbreviations: PLGA, polylactide-co-glycoside; Lf, lactoferrin; TMC, N-trimethylated chitosan; NPs, nanoparticles.

Figure 8 In vivo and ex vivo fluorescence images of organs of mice. (A) In vivo imaging of mice at 0.5, 1, 4, and 8 h after intranasal administration of DiR-loaded PLGA NPs, TMC NPs, and Lf-TMC NPs at a dose of 0.5 mg DiR/kg of body weight. (B) Ex vivo imaging of organs excised from mice at 8 h after intranasal administration.

Abbreviations: DiR, 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanineiodid; PLGA, polylactide-co-glycoside; Lf, lactoferrin; TMC, N-trimethylated chitosan; NPs, nanoparticles.

Table 4 Pharmacokinetic parameters following intranasal administration of Huperzine A-loaded nanoparticles

Formulation	Tissue	Kel (h^−1)	Tmax (h)	Cmax (ng/mL or ng/g)	AUC0–12h (ng h/mL or ng h/g)	DTI
PLGA NPs	Plasma	0.2414	1	180.0±14.4	593.4±63.0
	OB	0.2883	1	393.0±31.9	1,632.2±151.7	1
	CR	0.3156	1	245.1±18.6	1,120.5±133.5	1
	CL	0.2096	1	187.2±30.8	719.8±82.6	1
	HI	0.2402	2	195.6±19.9	885.0±96.4	1
TMC NPs	Plasma	0.2114	0.25	189.9±19.8	739.2±72.8
	OB	0.1709	1	552.0±38.2	2,970.8±206.3***	1.6±0.1***
	CR	0.1957	2	312.0±32.3	1,796.2±182.9**	1.4±0.1***
	CL	0.1681	2	287.7±19.9	1,791.9±211.1**	1.8±0.1***
	HI	0.1752	2	223.1±22.5	1,491.4±130.2**	1.6±0.1***
Lf-TMC NPs	Plasma	0.1948	0.25	160.0±23.7	809.7±81.0*
	OB	0.1884	2	681.0±39.3	4,219.6±316.7***,##	2.0±0.1***,##
	CR	0.2005	1	423.0±38.4	2,283.8±113.6***,#	1.6±0.1***
	CL	0.1902	1	314.6±27.0	2,138.9±207.4***,#	1.9±0.1***
	HI	0.1669	1	269.4±26.9	1,857.5±155.8***,##	1.9±0.1***,##

Notes: Values represent the mean ± SD (n=3). Statistically significant differences from PLGA NPs:

* p<0.05,

** p<0.01,

*** p<0.001. Statistically significant differences from TMC NPs:

# p<0.05,

## p<0.01.

Abbreviations: PLGA, polylactide-co-glycoside; NPs, nanoparticles; TMC, N-trimethylated chitosan; Kel, elimination rate constant; T_max, peak-reaching time; C_max, the maximum concentration; AUC, area under concentration-time curve; DTI, drug targeting index; Lf, lactoferrin; OB, olfactory bulb; CR, cerebrum with hippocampus removal; CL, cerebellum; HI, hippocampus.

Figure 9 Brain distribution of Hup ANPs. (A) Blood concentration–time profiles of HupA following intranasal administration of HupA PLGA NPs, TMC NPs, and Lf-TMC NPs. Data represented the mean ± SD (n=3). Brain biodistribution of HupA following intranasal administration of HupA loaded NPs in the (B) olfactory bulb, (C) cerebrum with hippocampus removed, (D) cerebellum, and (E) hippocampus.

Abbreviations: HupA, Huperzine-A; PLGA, polylactide-co-glycoside; Lf, lactoferrin; TMC, N-trimethylated chitosan; NPs, nanoparticles.