Anti-Angiogenic Activity Of Bevacizumab-Bearing Dexamethasone-Loaded PLGA Nanoparticles For Potential Intravitreal Applications

Figures & data

Table 1 Optimization Of PEI Ratio In W Phase On eBev-DPPNs

Batch	Ratio Of PEI In W Phase (w/v, %)	DPPNs			eBev-DPPNs			BE (%)
		Size (d.nm)	PDI	Zeta Potential (mV)	Size (d.nm)	PDI	Zeta Potential (mV)
1	0	146.0 ± 1.3	0.093 ± 0.036	−3.61 ± 0.90	-	-	-	-
2	0.3	154.4 ± 2.5	0.146 ± 0.090	13.42 ± 1.62	189.9 ± 5.4	0.256 ± 0.057	0.36 ± 0.89	44.21 ± 0.46
3	0.4	155.7 ± 3.3	0.131 ± 0.077	19.47 ± 2.89	190.4 ± 3.9	0.290 ± 0.066	−0.16 ± 0.55	66.30 ± 2.01
4	0.5	162.8 ± 3.0	0.162 ± 0.089	23.25 ± 1.87	207.4 ± 6.2	0.236 ± 0.038	1.06 ± 0.25	78.95 ± 2.57
5	0.6	175.0 ± 2.8	0.121 ± 0.033	28.20 ± 2.03	217.7 ± 5.3	0.279 ± 0.049	0.85 ± 0.37	85.64 ± 0.28
6	0.7	184.2 ± 1.9	0.032 ± 0.014	26.76 ± 3.27	221.0 ± 8.4	0.257 ± 0.033	−0.15 ± 0.89	82.98 ± 1.28
7	0.8	190.3 ± 5.7	0.168 ± 0.021	26.21 ± 3.45	222.7 ± 9.2	0.293 ± 0.067	0.33 ± 0.61	86.46 ± 1.38

Notes: The eBev-DPPNs are fabricated at DPPNs to bevacizumab at a ratio of 1:1. Binding efficiency is defined as percentage of the amount of reacted bevacizumab on the amount of bevacizumab used during the preparation process. Data are expressed as mean ± SD, n = 3.

Abbreviations: PEI, Polyethylenimine; DPPNs, dexamethasone-loading PLGA/PEI nanoparticles; eBev-DPPNs, electrostatically-conjugated bevacizumab-bearing DPPNs; PDI, polydispersity index; BE, binding efficiency.

Table 2 Optimization Of The Ratio Of DPPNs And Bevacizumab On eBev-DPPNs

Batch	Bevcizumab-to-DPPNs (w/w)	eBev-DPPNs			BE (%)
		Size (d.nm)	PDI	Zeta Potential (mV)
8	1:2	193.8 ± 8.2	0.341 ± 0.081	0.86 ± 1.13	90.70 ± 1.59
9	3:4	215.6 ± 5.7	0.249 ± 0.037	0.89 ± 0.72	89.35 ± 1.81
10	1:1	217.7 ± 5.3	0.279 ± 0.049	0.85 ± 0.37	85.64 ± 0.28
11	5:4	248.2 ± 2.1	0.032 ± 0.045	0.44 ± 0.63	63.28 ± 0.73
12	3:2	253.3 ± 3.7	0.327 ± 0.019	0.51 ± 0.28	50.08 ± 0.55

Notes: The eBev-DPPNs are fabricated at 0.6% PEI ratio in W phase. Binding efficiency is defined as percentage of the amount of reacted bevacizumab on the amount of bevacizumab used during the preparation process. Data are expressed as mean ± SD, n = 3.

Abbreviations: DPPNs, dexamethasone-loading PLGA/PEI nanoparticles; eBev-DPPNs, electrostatically-conjugated bevacizumab-bearing DPPNs; PDI, polydispersity index; BE, binding efficiency.

Table 3 Characteristics Of The Optimized Formulations

Formulation	Size (d.nm)	PDI	Zeta Potential (mV)	DL (%)	EE (%)	BE (%)
DPNs	146.0 ± 1.3	0.093 ± 0.036	−3.61 ± 0.90	10.87 ± 0.96	65.21 ± 5.81	–
DPPNs	175.0 ± 2.8	0.121 ± 0.033	28.20 ± 2.03	11.14 ± 0.62	66.88 ± 3.62	–
eBev-DPPNs	217.7 ± 5.3	0.279 ± 0.049	0.85 ± 0.37	9.50 ± 0.30	56.97 ± 1.93	85.64 ± 0.28
cBev-DPNs	201.3 ±7.2	0.318 ± 0.084	0.31 ± 1.15	9.34 ± 0.07	56.01 ± 0.33	81.75 ± 2.12

Note: Data are expressed as mean ± SD, n = 3.

Abbreviations: DPNs, dexamethasone-loading PLGA nanoparticles; DPPNs, dexamethasone-loading PLGA/PEI nanoparticles; eBev-DPPNs, electrostatically-conjugated bevacizumab-bearing DPPNs; cBev-DPNs, chemically-conjugated bevacizumab-bearing DPNs; PDI, polydispersity index; PDI, polydispersity index; DL, drug loading; EE, encapsulation efficiency; BE, binding efficiency.

Figure 1 Characteristics of the eBev-DPPNs and cBev-DPNs. (A) Size distribution of eBev-DPPNs based on DLS; (B) Size distribution of cBev-DPNs based on DLS; (C) SEM images of eBev-DPPNs; (D) SEM images of cBev-DPNs. Data are expressed as mean ± SD, n = 3.

Abbreviations: eBev-DPPNs, electrostatically-conjugated bevacizumab-bearing DPPNs; cBev-DPNs, chemically-conjugated bevacizumab-bearing DPNs; DLS, dynamic light scattering; SEM, scanning electron microscopy.

Figure 2 Stability of the eBev-DPPNs and cBev-DPNs. (A) Particle size; (B) PDI; (C) Binding efficiency of bevacizumab in PBS; (D) Particle size; (E) PDI; (F) Binding efficiency of bevacizumab in the vitreous humor of rabbit eyes. Data are expressed as mean ± SD, n = 3.

Abbreviations: eBev-DPPNs, electrostatically-conjugated bevacizumab-bearing DPPNs; cBev-DPNs, chemically-conjugated bevacizumab-bearing DPNs.

Figure 3 Structural stability of the bevacizumab on the surface of the eBev-DPPNs and cBev-DPNs.

Notes: (A) SEC-HPLC chromatograms; (B) CD spectra; (C) Fluorescence spectra.

Abbreviations: eBev-DPPNs, electrostatically-conjugated bevacizumab-bearing DPPNs; cBev-DPNs, chemically-conjugated bevacizumab-bearing DPNs.

Figure 4 (A) In vitro release of dexamethasone from the eBev-DPPNs and cBev-DPNs; (B) In vitro release of bevacizumab from the eBev-DPPNs and cBev-DPNs. Data are expressed as mean ± SD, n = 3.

Abbreviations: eBev-DPPNs, electrostatically-conjugated bevacizumab-bearing DPPNs; cBev-DPNs, chemically-conjugated bevacizumab-bearing DPNs.

Figure 5 Induction of apoptosis by nanoparticles on HUVECs. Data are expressed as mean ± SD, n = 6.

Abbreviations: eBev-DPPNs, electrostatically-conjugated bevacizumab-bearing DPPNs; cBev-DPNs, chemically-conjugated bevacizumab-bearing DPNs; PI, propidium iodide.

Figure 6 Migration inhibition by nanoparticles on HUVECs in wound healing assays. (A) Representative images of HUVECs migration; (B) Migration number of HUVECs. Data are expressed as mean ± SD, n = 6. **p < 0.01, ***p < 0.001 vs control; #p < 0.05, ##p < 0.01, ###p < 0.001 vs eBev-DPPNs.

Abbreviations: eBev-DPPNs, electrostatically-conjugated bevacizumab-bearing DPPNs; cBev-DPNs, chemically-conjugated bevacizumab-bearing DPNs.

Figure 7 Invasion inhibition by nanoparticles on HUVECs in Transwell assays. (A) Representative images of HUVECs invasion; (B) Invasion number of HUVECs. Data are expressed as mean ± SD, n = 6. **p < 0.01, ***p < 0.001 vs control; #p < 0.05, ###p < 0.001 vs eBev-DPPNs.

Abbreviations: eBev-DPPNs, electrostatically-conjugated bevacizumab-bearing DPPNs; cBev-DPNs, chemically-conjugated bevacizumab-bearing DPNs.

Figure 8 Tube formation inhibition by nanoparticles on HUVECs. (A) Representative images of HUVECs tube formation; (B) Tube formation capacity of HUVECs. Data are expressed as mean ± SD, n = 6. **p < 0.01, ***p < 0.001 vs control; ##p < 0.01, ###p < 0.001 vs eBev-DPPNs.

Abbreviations: eBev-DPPNs, electrostatically-conjugated bevacizumab-bearing DPPNs; cBev-DPNs, chemically-conjugated bevacizumab-bearing DPNs.

Figure 9 VEGF secretion inhibition by nanoparticles on HUVECs. (A) VEGF secretion at 12 h; (B) VEGF secretion at 24 h; (C) VEGF secretion at 48 h; (D) VEGF secretion at 72 h. Data are expressed as mean ± SD, n = 6. *p < 0.05, **p < 0.01 vs control without CoCl₂; #p < 0.05, ##p < 0.01, ###p < 0.001 vs control with CoCl₂; ^p < 0.05, ^^p < 0.01 vs eBev-DPPNs with CoCl_2.

Abbreviations: VEGF, vascular endothelial growth factor; eBev-DPPNs, electrostatically-conjugated bevacizumab-bearing DPPNs; cBev-DPNs, chemically-conjugated bevacizumab-bearing DPNs.

Figure 10 Anti-angiogenesis effects in vivo on chorioallantoic membranes. (A) Representative images of angiogenesis; (B) The amount of blood vessel on chorioallantoic membranes. Data are expressed as mean ± SD, n = 10. **p < 0.01, ***p < 0.001 vs control; #p < 0.05, ##p < 0.01, ###p < 0.001 vs eBev-DPPNs.

Abbreviations: eBev-DPPNs, electrostatically-conjugated bevacizumab-bearing DPPNs; cBev-DPNs, chemically-conjugated bevacizumab-bearing DPNs.

Figure 11 The inhibition of CNV area on laser-induced CNV rabbits model. (A) Representative images of the leakage area of CNV; (B) The intensity of CNV area on rabbits fundus. Data are expressed as mean ± SD, n = 3. *p < 0.05, ***p < 0.001 vs control; #p < 0.05, ##p < 0.01, ###p < 0.001 vs eBev-DPPNs.

Abbreviations: eBev-DPPNs, electrostatically-conjugated bevacizumab-bearing DPPNs; cBev-DPNs, chemically-conjugated bevacizumab-bearing DPN; CNV, Choroidal neovascularization.