Multivesicular liposomes for sustained release of bevacizumab in treating laser-induced choroidal neovascularization

Figures & data

Figure 1. (A) The biological activity of bevacizumab: (a) the effect of various stabilizer on bevacizumab activity; (b) the bevacizumab activity of Bev-MVLs containing 10% HAS was statistically significantly different from without HAS (mean ± SD, n = 3, **p < .01). (B) The morphological examination of Bev-MVLs: (a) light micrograph of Bev-MVLs particles at 400× magnification; (b) SEM image of Bev-MVL particles. (C) Particle size distribution of Bev-MVLs. (D) The morphology of FB-MVLs was observed at 400× magnification (a) bright-field image; (b) fluorescent image.

Figure 2. (A) In vitro release of Bev-MVLs (a) in three different mediums: normal saline, PBS (pH 7.4), and vitreous fluids; (b) coupled with different phospholipids (DOPC, EPC, and SPC) in vitreous fluids (mean ± SD, n = 3). (B) The morphological change of MVLs-DOPC at days 1, 3, 5, 7, 9, and 11 in vitreous fluids. (C) The results of SDS-PAGE for bevacizumab released from MVLs at scheduled intervals. Line 1: Marker; Line 2: free bevacizumab as a reference; Lines 3–9: bevacizumab released from MVLs at days 13, 11, 9, 7, 5, 3, and 1.

Table 1. The model parameters for the release of MVLs.

	Model	Equation	R^2	n
MVLs-DOPC	Zero order	Q = 6.917t + 9.008	0.947
	First order	Q = 105.729 (1-e^−0.137^t)	0.997
	Higuchi	Q = 26.517t^1/2 − 7.474	0.985
	Ritger–Peppas	lnQ =0.741lnt +2.696	0.992	0.741
	Weibull	lnln1/(1 − Q/100)) = 0.920lnt −1.741	0.989
MVLs-EPC	Zero order	Q = 6.907t + 30.514	0.687
	First order	Q = 96.036 (1-e^−0.492^t)	0.991
	Higuchi	Q = 27.872t^1/2 + 12.558	0.874
	Ritger–Peppas	lnQ =0.559lnt +3.450	0.884
	Weibull	lnln1/(1 − Q/100) = 0.868lnt −0.759	0.976	0.559
MVLs-SPC	Zero order	Q = 7.17t + 23.921	0.788
	First order	Q = 96.963 (1-e^−0.340^t)	0.990
	Higuchi	Q = 29.443t^1/2 + 3.281	0.954
	Ritger–Peppas	lnQ = 0.556lnt +3.355	0.961	0.556
	Weibull	lnln1/(1 − Q/100) = 0.835lnt −0.928	0.989

Figure 3. (A) In vivo imaging of SD rats after intravitreal injection of CB and CB-MVLs at 0.5, 1, 3, 7, and 14 days, respectively. (B) The concentrations of Bev-MVLs and Bev-S at 3, 7, 14, 21, 28, 42, and 56 days in vitreous humor, and (C) in aqueous humor. (D) The comparison of bevacizumab concentrations between the vitreous and aqueous humors after intravitreal injection of Bev-MVLs, and (E) of Bev-S (mean ± SD, n = 3).

Table 2. Pharmacokinetic parameters of Bev-MVLs and Bev-S in the vitreous and aqueous humors following intravitreal injection (mean ± SD, n = 3).

	Pharmacokinetic parameters	Bev-MVLs	Bev-S
Vitreous humor	t1/2 (days)	8.84 ± 1.01*	4.45 ± 0.20
	Cmax (μg/mL)	207.28 ± 6.35*	163.11 ± 6.54
	AUC0–t (μg/mL × days)	3474.33 ± 255.95*	1587.09 ± 184.26
	MRT0–t (days)	15.03 ± 0.95*	10.19 ± 0.77
	t1/2 (days)	8.26 ± 2.0*	4.23 ± 0.13
Aqueous humor	Cmax (μg/mL)	46.61 ± 5.16	32.22 ± 2.84
	AUC0–t (μg/mL × days)	582.38 ± 109.08*	318.44 ± 60.21
	MRT0–t (days)	11.33 ± 0.89	10.05 ± 0.95

*p < .05.

Figure 4. (A) OCT images of intraretinal layers cross-section (a) before photocoagulation; (b) 7 days after photocoagulation; (c) 14 days after photocoagulation; and (d) 21 days after photocoagulation. (B) FFA images: (a) before photocoagulation; (b) 7 days after photocoagulation; (c) 14 days after photocoagulation; and (d) 21 days after photocoagulation. (C) Histological images: (a) normal group without laser induction; (b) control group with 35 days after laser induction; (c) Bev-S group with 28 days after administration; and (d) Bev-MVLs group with 28 days after administration (white arrows indicate CNV lesion margins, scale bar, 50 μm). (D) BN rats treated with either Bev-S or Bev-MVLs had significantly thinner neovascular membranes than those in the control group (*p < .05, **p < .01, mean ± SD, n = 5).