Role of hydroxypropyl-β-cyclodextrin on freeze-dried and gamma-irradiated PLGA and PLGA–PEG diblock copolymer nanospheres for ophthalmic flurbiprofen delivery

Figures & data

Table 1 Physicochemical properties and entrapment efficiency of PLGA and PLGA–PEG NSs with and without FB

NS composition	MW (KDa)	Size ± SD (nm)	PI ± SD	ζ-potential ± SD (mV)	EE ± SD (%)
PLGA 75:25	98	202.0 ± 1.8	0.076 ± 0.009	−25.7 ± 1.3	–
PLGA 75:25 + FB		232.8 ± 1.9	0.048 ± 0.045	−25.8 ± 1.2	94.6 ± 0.4
PLGA 50:50	34	205.3 ± 1.4	0.051 ± 0.003	−34.0 ± 0,5	–
PLGA 50:50 + FB		197.5 ± 1.4	0.062 ± 0.022	−33.5 ± 0.4	90.3 ± 3.2
PLGA 50:50–PEG 5%	95 PLGA + 5 PEG	146.8 ± 2.1	0.081 ± 0.014	−22.5 ± 0.3	–
PLGA 50:50–PEG 5% + FB		147.7 ± 1.8	0.052 ± 0.043	−22.2 ± 0.2	87.7 ± 3.7
PLGA 50:50–PEG 10%	45 PLGA + 5 PEG	121.3 ± 0.9	0.090 ± 0.002	−17.3 ± 0.4	–
PLGA 50:50–PEG 10% + FB		123.3 ± 1.6	0.094 ± 0.063	−17.0 ± 0.6	80.5 ± 5.0
PLGA 50:50–PEG 15%	28 PLGA + 5 PEG	90.2 ± 0.9	0.094 ± 0.021	−12.8 ± 0.2	–
PLGA 50:50–PEG 15% + FB		96.8 ± 0.8	0.085 ± 0.019	−12.3 ± 1.7	75.0 ± 2.4

Note: Mean ± SD, n = 3.

Abbreviations: EE, entrapment efficiency; FB, flurbiprofen; MW, molecular weight of PLGA and PLGA–PEG copolymers used for NS preparations; NSs, nanospheres; PI, polydispersity index; PLGA, Poly(D,L-lactide-co-glycolide); PLGA–PEG, poly(D,L-lactide-co-glycolide) with poly(ethylene glycol); SD, standard deviation; ζ-potential, zeta potential.

Figure 1 Particle size and PI of reconstituted FB-loaded NSs in the presence of different cryoprotectants.

Note: Error bars represent ± SD.

Abbreviations: FB, flurbiprofen; HPβCD, hydroxypropyl-β-cyclodextrin; NS, nanospheres; PI, polydispersity index; PLGA, Poly(D,L-lactide-co-glycolide); PLGA–PEG, poly(D,L-lactide-co-glycolide) with poly(ethylene glycol); SD, standard deviation.

Table 2 Physicochemical properties and entrapment efficiency of freeze-dried PLGA and PLGA–PEG NSs with and without FB before and after γ-sterilization with 5% (w/v) HPβCD

NS composition	Size ± SD (nm)		PI ± SD		ζ-potential ± SD (mV)		EE ± SD (%)
	Before*	After	Before*	After	Before*	After	Before*	After
PLGA 75:25	213.3 ± 1.3	213.9 ± 2.5	0.052 ± 0.034	0.082 ± 0.020	−23.3 ± 2.1	−22.9 ± 1.3	–	–
PLGA 75:25 + FB	234.3 ± 2.1	232.9 ± 4.0	0.080 ± 0.007	0.061 ± 0.013	−23.1 ± 0.5	−22.5 ± 0.8	97.1 ± 1.2	97.0 ± 1.4
PLGA 50:50	207.9 ± 0.3	209.4 ± 5.0	0.036 ± 0.015	0.081 ± 0.020	−31.6 ± 2.1	−32.1 ± 1.4	–	–
PLGA 50:50 + FB	206.1 ± 2.1	204.9 ± 4.0	0.083 ± 0.027	0.101 ± 0.034	−31.7 ± 1.1	−31.6 ± 1.4	91.7 ± 3.7	92.5 ± 2.1
PLGA 50:50–PEG 5%	153.2 ± 1.6	151.5 ± 2.8	0.124 ± 0.008	0.110 ± 0.015	−20.5 ± 0.4	−19.9 ± 1.2	–	–
PLGA 50:50–PEG 5% + FB	150.4 ± 1.6	150.2 ± 1.4	0.105 ± 0.009	0.118 ± 0.009	−20.4 ± 0.1	−20.1 ± 2.0	90.7 ± 1.8	90.4 ± 0.8
PLGA 50:50–PEG 10%	126.6 ± 0.8	124.5 ± 0.7	0.073 ± 0.011	0.084 ± 0.007	−15.2 ± 0.5	−15.8 ± 3.2	–	–
PLGA 50:50–PEG 10% + FB	128.8 ± 1.2	124.9 ± 0.6	0.084 ± 0.014	0.089 ± 0.020	−14.9 ± 0.1	−13.9 ± 0.3	83.3 ± 2.0	83.5 ± 3.4
PLGA 50:50–PEG 15%	94.5 ± 0.4	94.5 ± 0.4	0.115 ± 0.010	0.130 ± 0.012	−10.5 ± 2.3	−10.7 ± 1.3	–	–
PLGA 50:50–PEG 15% + FB	96.2 ± 1.1	95.7 ± 0.3	0.115 ± 0.014	0.131 ± 0.004	−10.4 ± 1.0	−9.9 ± 1.4	79.6 ± 0.9	80.3 ± 1.6

Notes: Mean ± SD, n = 3.

* Freeze-dried NSs containing 5% (w/v) HPβCD (P > 0.05 in size, ζ-potential and EE against nonirradiated samples).

Abbreviations: EE, entrapment efficiency; FB, flurbiprofen; HPβCD, hydroxypropyl-β-cyclodextrin; NSs, nanospheres; PI, polydispersity index; PLGA, Poly(D,L-lactide-co-glycolide); PLGA–PEG, poly(D,L-lactide-co-glycolide) with poly(ethylene glycol); SD, standard deviation; ζ-potential, zeta potential.

Table 3 Values of glass transition temperature for raw polymer, empty NSs, and FB-loaded NSs before and after γ-irradiation

	Tg (°C)
	Nonirradiated	γ-irradiated
PLGA 75:25 raw polymer	59.44	52.84*
PLGA 75:25 NS	57.62	53.51*
PLGA 75:25 + FB NS	47.25	47.44
PLGA 50:50 raw polymer	54.18	49.78*
PLGA 50:50 NS	49.05	46.43*
PLGA 50:50 + FB NS	44.48	45.87
PLGA 50:50–PEG 5% raw polymer	49.09	34.58*
PLGA 50:50–PEG 5% NS	48.45	43.76*
PLGA 50:50–PEG 5% + FB NS	41.41	42.54
PLGA 50:50–PEG 10% raw polymer	46.09	35.99*
PLGA 50:50–PEG 10% NS	43.33	37.95*
PLGA 50:50–PEG 10% + FB NS	42.45	40.51
PLGA 50:50–PEG 15% raw polymer	43.82	33.85*
PLGA 50:50–PEG 15% NS	42.53	38.56*
PLGA 50:50–PEG 15% + FB NS	39.55	38.78

Note:

* P < 0.05 against nonirradiated samples.

Abbreviations: FB, flurbiprofen; NSs, nanospheres; PLGA, Poly(D,L-lactide-co-glycolide); PLGA–PEG, poly(D,L-lactide-co-glycolide) with poly(ethylene glycol); T_g, glass transition temperature.

Figure 2 In vitro release profiles of FB from: (A) PLGA and PLGA–PEG NSs, (B) PLGA and PLGA-NSs in the presence of 5% (w/v) HPβCD, (C) PLGA and PLGA-NSs in the presence of 5% (w/v) HPβCD after sterilization.

Note: Error bars represent ± SD.

Abbreviations: FB, flurbiprofen; HPβCD, hydroxypropyl-β-cyclodextrin; NS, nanospheres; PLGA, Poly(D,L-lactide-co-glycolide); PLGA–PEG, poly(D,L-lactide-co-glycolide) with poly(ethylene glycol); SD, standard deviation.

Table 4 Release kinetics obtained after fitting FB release data from NSs into Korsmeyer–Peppas model

NS composition	Korsmeyer–Peppas model			Transport mechanism
	r^2	k ± SD (h^−n)	n ± SD
PLGA 75:25	0.9637	1.64 ± 0.38	0.28 ± 0.04	Fickian diffusion
PLGA 50:50	0.9451	4.59 ± 0.45	0.16 ± 0.02	Fickian diffusion
PLGA 50:50–PEG 5%	0.9404	5.02 ± 0.51	0.16 ± 0.02	Fickian diffusion
PLGA 50:50–PEG 10%	0.9563	5.65 ± 0.44	0.15 ± 0.01	Fickian diffusion
PLGA 50:50–PEG 15%	0.9511	5.49 ± 0.49	0.16 ± 0.02	Fickian diffusion
PLGA 75:25/HPβCD	0.9922	0.21 ± 0.03	0.56 ± 0.03	Non-Fickian or anomalous diffusion
PLGA 50:50/HPβCD	0.9945	0.15 ± 0.02	0.68 ± 0.03	Non-Fickian or anomalous diffusion
PLGA 50:50–PEG 5%/HPβCD	0.9950	0.15 ± 0.02	0.68 ± 0.03	Non-Fickian or anomalous diffusion
PLGA 50:50–PEG 10%/HPβCD	0.9949	0.26 ± 0.03	0.60 ± 0.02	Non-Fickian or anomalous diffusion
PLGA 50:50–PEG 15%/HPβCD	0.9989	0.23 ± 0.01	0.62 ± 0.01	Non-Fickian or anomalous diffusion

Note: Mean ± SD, n = 3.

Abbreviations: FB, flurbiprofen; HPβCD, hydroxypropyl-β-cyclodextrin; k, release rate constant; n, diffusional exponent; NSs, nanospheres; PLGA, Poly(D,L-lactide-co-glycolide); PLGA–PEG, poly(D,L-lactide-co-glycolide) with poly(ethylene glycol); SD, standard deviation.

Figure 3 Confocal images of a cross-section of the corneal epithelium at depth of 10 μm after 2 hours of incubation with: (A) Rd-PLGA NS; (B) Rd-PEG-PLGA NSs; (C) Rd-NSs containing 5% (w/v) HPβCD.

Abbreviations: HPβCD, hydroxypropyl-β-cyclodextrin; NS, nanospheres; PLGA, Poly(D,L-lactide-co-glycolide); PLGA–PEG, poly(D,L-lactide-co-glycolide) with poly(ethylene glycol); Rd, rhodamine-6G.

Table 5 Corneal hydration levels and permeation coefficients of different NS formulations containing FB

NS composition	HL (%)	Papp (×10^−6 cm · s^−1)
PLGA 50:50	77.95 ± 1.06	17.72 ± 2.54
PLGA 50:50–PEG 5%	79.01 ± 3.25	17.15 ± 5.12
PLGA 50:50–PEG 10%	78.43 ± 1.99	17.60 ± 4.65
PLGA 50:50–PEG 15%	78.64 ± 2.67	17.07 ± 3.14
PLGA 75:25/HPβCD	79.15 ± 2.15	8.73 ± 3.97
PLGA 50:50/HPβCD	78.16 ± 2.65	8.80 ± 3.62
PLGA 50:50–PEG 5%/HPβCD	79.46 ± 1.68	9.67 ± 1.95
PLGA 50:50–PEG 10%/HPβCD	77.56 ± 1.04	9.19 ± 1.39
PLGA 50:50–PEG 15%/HPβCD	78.26 ± 0.95	8.45 ± 3.97

Note: Mean ± SD, n = 6.

Abbreviations: FB, flurbiprofen; HL, corneal hydration level; HPβCD, hydroxypropyl-β-cyclodextrin; NS, nanospheres; P_app, permeation coefficient; PLGA, Poly(D,L-lactide-co-glycolide); PLGA–PEG, poly(D,L-lactide-co-glycolide) with poly(ethylene glycol); SD, standard deviation.

Figure 4 Permeation of FB through isolated rabbit cornea from PLGA and PLGA–PEG NSs with and without HPβCD.

Note: Error bars represent ± SD.

Abbreviations: FB, flurbiprofen; HPβCD, hydroxypropyl-β-cyclodextrin; NS, nanospheres; PLGA, Poly(D,L-lactide-co-glycolide); PLGA–PEG, poly(D,L-lactide-co-glycolide) with poly(ethylene glycol); SD, standard deviation.

Figure 5 (A and B) Particle size, PI, and ζ-pot of the sterilized NS powders stored at 4°C, 25°C, and 40°C after 6 months of storage; (C and D) Particle size, PI, and ζ-pot of the rehydrated samples after 1 and 3 months of storage at 4°C, 25°C, and 40°C.

Notes: Error bars represent ± SD; *P < 0.05.

Abbreviations: HPβCD, hydroxypropyl-β-cyclodextrin; NS, nanospheres; PI, polydispersity index; PLGA, Poly(D,L-lactide-co-glycolide); PLGA–PEG, poly(D,L-lactide-co-glycolide) with poly(ethylene glycol); SD, standard deviation; ζ-pot, zeta potential.

Figure 6 DSC thermograms of γ-irradiated PLGA and PLGA–PEG NSs containing 5% (w/v) HPβCD stored 6 months at 4°C and after rehydrated and stored for 3 months in the aqueous form at the same temperature.

Abbreviations: DSC, differential scanning calorimetry; HPβCD, hydroxypropyl- β-cyclodextrin; NS, nanospheres; PLGA, Poly(D,L-lactide-co-glycolide); PLGA–PEG, poly(D,L-lactide-co-glycolide) with poly(ethylene glycol).