Conjugation of cell-penetrating peptides with poly(lactic-co-glycolic acid)-polyethylene glycol nanoparticles improves ocular drug delivery

Figures & data

Figure 1 Synthesis of (A) PLGA-NPs-PEG-peptide and (B) PLGA-PEG-peptide polymer followed NPs preparation.

Abbreviations: EDC, ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; FB, flurbiprofen; NHS, N-hydroxysuccinimide; NPs, nanoparticles; PEG, polyethyleneglycol; PLGA, poly(lactic-co-glycolic acid); SH, sulfhydryl group.

Figure 2 The ¹H-NMR spectra of PLGA-PEG-POD and PLGA-PEG-HIV-Tat confirm the peptide coupling to PLGA-PEG-maleimide polymer.

Notes: ¹H-NMR spectra in DMSO-d₆ at 25°C of (A) PLGA-PEG-maleimide-POD copolymer and POD peptide. We used the specified amide/amine side chain region for peptide signal integration. (B) For PLGA-PEG-maleimide-HIV-Tat and PLGA-PEG-maleimide, we clearly see a reduction of the maleimide methine protons signal intensity and the appearance of Tyr aromatic protons from HIV-Tat peptide after conjugation.

Abbreviations: ¹H-NMR, proton nuclear magnetic resonance; DMF, N,N-Dimethylformamide; DMSO, dimethyl sulfoxide-d₆; HIV-Tat, human immunodeficiency virus transactivator; Mal, maleimide; PEG, polyethyleneglycol; PLGA, poly(lactic-co-glycolic acid); POD, peptide for ocular delivery; Tyr, tyrosine.

Table 1 Physicochemical properties (with and without FB) and EE of PLGA NPs, PLGA-NPs-PEG, and PLGA-NPs-PEG-peptide

NPs composition	Size ± SD (nm)	PI ± SD	Zpot ± (mV)	EE ± SD (%)
PLGA 50:50 NPs	148.4±2.0	0.064±0.027	−29.1±1.3	–
PLGA 50:50 NPs + FB	190.5±1.5	0.065±0.020	−30.4±3.3	77.1±4.2
PLGA-NPs-PEG	164.5±1.8	0.079±0.018	−32.5±1.5	–
PLGA-NPs-PEG + FB	170.3±3.5	0.084±0.014	−30.6±0.7	75.4±2.0
PLGA-NPs-PEG-POD	203.8±1.4	0.060±0.009	33.2±1.7	–
PLGA-NPs-PEG-POD + FB	219.9±1.2	0.082±0.010	30.2±1.4	70.3±5.6
PLGA-NPs-PEG-HIV-Tat	218.4±0.9	0.075±0.045	25.2±2.6	–
PLGA-NPs-PEG-HIV-Tat + FB	210.7±0.2	0.076±0.050	20.2±1.2	76.5±3.8

Notes: Values are expressed as mean ± SD; n=3.

Abbreviations: EE, entrapment efficiency; FB, flurbiprofen; HIV-Tat, human immunodeficiency virus transactivator; NPs, nanoparticles; PEG, polyethylene glycol; PI, polydispersity index; PLGA, poly(lactic-co-glycolic acid); POD, peptide for ocular delivery; SD, standard deviation; Z_pot, zeta potential.

Table 2 Physicochemical properties (with and without FB) and EE of PLGA NPs, PLGA-PEG NPs, PLGA-PEG-peptide NPs

NPs composition	MW (kDa)	Size ± SD (nm)	PI ± SD	Zpot ± SD (mV)	EE ± SD (%)
PLGA-PEG NPs	PLGA (34) PEG (2)	105.1±0.2	0.080±0.026	−24.3±3.3	–
PLGA-PEG NPs + FB		128.9±0.9	0.079±0.017	−12.6±3.0	73.3±3.3
PLGA-PEG^® NPs	PLGA (95) PEG (5)	147.7±1.1	0.095±0.039	−24.0±0.7	–
PLGA-PEG^® NPs + FB		150.8±1.0	0.096±0.017	−20.9±1.1	78.0±2.5
PLGA-PEG-POD NPs	PLGA (34) PEG (2) POD (3.6)	85.9±0.9	0.011±0.020	15.2±0.3	–
PLGA-PEG-POD NPs + FB		103.7±0.3	0.097±0.014	18.6±1.5	82.9±0.8
PLGA-PEG-HIV-Tat NPs	PLGA (34) PEG (2) HIV-Tat (1.9)	102.8±1.1	0.012±0.010	20.0±0.4	–
PLGA-PEG-HIV-Tat NPs + FB		123.1±1.7	0.103±0.007	14.0±0.8	81.0±1.7

Notes: Values are expressed as mean ± SD; n=3.

Abbreviations: EE, entrapment efficiency; FB, flurbiprofen; HIV-Tat, human immunodeficiency virus transactivator; NPs, nanoparticles; PEG, polyethylene glycol; PEG^®, Resomer^® RGP type d5055; PI, polydispersity index; PLGA, poly(lactic-co-glycolic acid); POD, peptide for ocular delivery; SD, standard deviation; Z_pot, zeta potential.

Figure 3 In vitro release profiles of FB from (A) PLGA-NPs-PEG-peptide, (B) PLGA-PEG NPs, and PLGA-PEG-peptide NPs.

Abbreviations: FB, flurbiprofen; HIV-Tat, human immunodeficiency virus transactivator; NPs, nanoparticles; PEG, polyethyleneglycol; PEG^®, Resomer^® RGP type d5055; PLGA, poly(lactic-co-glycolic acid); POD, peptide for ocular delivery.

Figure 4 CLSM images of a cross-section of (A) corneal epithelium at depth of 10 μm, (B) pretreated with PLGA-PEG-HIV-Tat-Rho NPs, (C) pretreated with PLGA-PEG-POD-Rho NPs, (D) their corresponding phase contrast with nucleus, and (E) cell membranes.

Abbreviations: CLSM, confocal laser scanning microscopy; HIV-Tat, human immunodeficiency virus transactivator; NPs, nanoparticles; PEG, polyethyleneglycol; PLGA, poly(lactic-co-glycolic acid); POD, peptide for ocular delivery; Rho, 5(6)-carboxytetramethylrhodamine.

Figure 5 Cytotoxicity of PLGA-PEG-peptide NPs on (A) HepG2 and (B) HeLa cells lines using the MTT assay, and photographs of (C) HepG2 and HeLa cells incubated with NPs to LD₅₀ and LD₁₀₀ concentrations.

Abbreviations: HIV-Tat, human immunodeficiency virus transactivator; LD₅₀, median lethal dose; LD₁₀₀, lethal dose; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium; NPs, nanoparticles; PEG, polyethyleneglycol; PLGA, poly(lactic-co-glycolic acid); POD, peptide for ocular delivery.

Figure 6 Comparison of anti-inflammatory efficacy of PLGA-PEG NPs, PLGA-PEG-peptide NPs, and Ocufen^® in the (A) treatment and (B) prevention of ocular inflammation induced by SA in the rabbit eye.

Notes: Values are expressed as mean ± SD; *P<0.05, **P<0.01, and ***P<0.001 significantly lower than the inflammatory effect induced by SA; ^$P<0.05, ^$$P<0.01, and ^$$$P<0.001 significantly lower than anti-inflammatory efficacy of Ocufen^®.

Abbreviations: HIV-Tat, human immunodeficiency virus transactivator; NPs, nanoparticles; PEG, polyethyleneglycol; PLGA, poly(lactic-co-glycolic acid); POD, peptide for ocular delivery; SA, sodium arachidonate; SD, standard deviation.

Figure S1 MALDI-TOF mass spectra of (A) POD, (C) HIV-Tat peptides, and (B and D) the conjugation with maleimide-PEG-amine.

Abbreviations: HIV-Tat, human immunodeficiency virus transactivator; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; PEG, polyethyleneglycol; POD, peptide for ocular delivery.

Figure S2 ¹H-NMR spectra of (A) PLGA-PEG-metoxi and (B) PLGA-PEG-maleimide copolymers.

Abbreviations: ¹H-NMR, proton nuclear magnetic resonance; PEG, polyethyleneglycol; PLGA, poly(lactic-co-glycolic acid).

Figure S3 Scanning electron microscopy analysis of PLGA-PEG-POD NPs.

Abbreviations: NPs, nanoparticles; PEG, polyethyleneglycol; PLGA, poly(lactic-co-glycolic acid); POD, peptide for ocular delivery.

Figure S4 MALDI-TOF spectra of (A) Rho-POD and (B) Rho-HIV-Tat peptides.

Abbreviations: HIV-Tat, human immunodeficiency virus transactivator; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; Rho, 5(6)-carboxytetram-ethylrhodamine; POD, peptide for ocular delivery.

Figure S5 Fluorescence microscopy images of (A) PLGA-PEG-peptide-Rho NPs and (B) green emission of all scanned NPs.

Abbreviations: NPs, nanoparticles; PEG, polyethyleneglycol; PLGA, poly(lactic-co-glycolic acid); Rho, 5(6)-carboxytetramethylrhodamine.

Figure S6 Photographs of CAM 5 minutes after the addition of NPs containing FB: (A) PLGA NPs, (B) PLGA-PEG NPs, (C) PLGA-PEG-POD NPs, (D) PLGA-PEG-HIV-Tat NPs, and (E) positive control (0.1 M sodium hydroxide).

Abbreviations: CAM, chorioallantoic membrane; FB, flurbiprofen; HIV-Tat, human immunodeficiency virus transactivator; NPs, nanoparticles; PEG, polyethyleneglycol; PLGA, poly(lactic-co-glycolic acid); POD, peptide for ocular delivery.

Figure S7 Draize test after instillation of PLGA-PEG-POD NPs (A) and inflammation induced by SA (B).

Abbreviations: NPs, nanoparticles; PEG, polyethyleneglycol; PLGA, poly(lactic-co-glycolic acid); POD, peptide for ocular delivery; SA, sodium arachidonate.

Figure S8 Anti-inflammatory effect of PLGA-PEG-POD NPs in the prevention of ocular inflammation induced by SA.

Note: The images were taken 30 minutes after inducing inflammation.

Abbreviations: NPs, nanoparticles; PEG, polyethyleneglycol; PLGA, poly(lactic-co-glycolic acid); POD, peptide for ocular delivery; SA, sodium arachidonate.

Table S1 Release kinetics obtained after fitting FB release data from PLGA-NPs-PEG-peptide and PLGA-PEG-peptide NPs into Korsmeyer–Peppas model

NPs composition	Korsmeyer–Peppas model*			Transport mechanism
	r^2	k ± SD (h^−n)	n ± SD
NPs obtained following the scheme indicated in Figure 1A
PLGA-NPs-PEG-POD	0.948	7.23±1.32	0.47±0.02	Non-Fickian or anomalous diffusion
PLGA-NPs-PEG-HIV-Tat	0.974	2.67±0.07	0.63±0.01	Non-Fickian or anomalous diffusion
NPs obtained following the scheme indicated in Figure 1B
PLGA-PEG-POD NPs	0.989	2.71±1.48	0.60±0.07	Non-Fickian or anomalous diffusion
PLGA-PEG-HIV-Tat NPs	0.967	2.56±0.20	0.74±0.11	Non-Fickian or anomalous diffusion

Notes: Values are expressed as mean ± SD; n=3

*non-significant differences were observed between POD and HIV-Tat NPs obtained following schemes of Figure 1A and B.

Abbreviations: FB, flurbiprofen; HIV-Tat, human immunodeficiency virus transactivator; k, the release rate constant; n, the diffusion exponent; NPs, nanoparticles; PEG, polyethyleneglycol; PLGA, poly(lactic-co-glycolic acid); POD, peptide for ocular delivery; SD, standard deviation.

Table S2 Physicochemical properties of PLGA-PEG-peptide-Rho NPs

NPs composition	Size ± SD (nm)	PI ± SD	Zpot ± SD (mV)
PLGA-PEG-POD-Rho NPs	87.2±0.3	0.112±0.041	22.6±2.7
PLGA-PEG-HIV-Tat-Rho NPs	99.7±1.2	0.083±0.028	15.8±0.8

Abbreviations: HIV-Tat, human immunodeficiency virus transactivator; NPs, nanoparticles; PEG, polyethyleneglycol; PI, polydispersity index; PLGA, poly(lactic-co-glycolic acid); POD, peptide for ocular delivery; Rho, 5(6)-carboxytetramethylrhodamine; SD, standard deviation; Z_pot, zeta potential.