Enhanced oral bioavailability of insulin using PLGA nanoparticles co-modified with cell-penetrating peptides and Engrailed secretion peptide (Sec)

Figures & data

Table 1. Sequences of N-terminally stearylated CPPs and Sec used in this study.

Name	Sequence
STR-R8	Stearyl-RRRRRRRR
STR-Tat	Stearyl-GRKKRRQRRRP
STR-Pen	Stearyl-RQIKIWFQNRRMKWKK
STR-Sec	Stearyl-QSLAQELGLNERQIKI

Figure 1. Schematic illustration of STR-CPPs and STR-Sec co-modified nanoparticles loaded with Ins-SPC complex. SE represents the low molecular weight emulsifier.

Figure 2. Transmission electron microscopy (TEM) images of Pen-NPs (A) and Sec-Pen-NPs (B). The scale bar represents 200 nm.

Table 2. Characteristics of coumarin-6 (C6)-loaded nanoparticles (mean ± SD, n = 3).

Formulations	Size (nm)	PDI	Zeta potential (mV)	EE% of C6	DL% of C6
Unmodified NPs	134.2 ± 7.5	0.054 ± 0.011	−7.7 ± 1.0	80.65 ± 1.33	0.019 ± 0.003
R8-NPs	116.1 ± 4.3	0.208 ± 0.031	39.5 ± 2.1	74.49 ± 5.36	0.017 ± 0.001
Tat-NPs	116.8 ± 4.7	0.190 ± 0.024	30.9 ± 0.7	75.51 ± 3.84	0.018 ± 0.002
Pen-NPs	135.1 ± 6.1	0.219 ± 0.050	37.6 ± 1.5	78.25 ± 4.17	0.019 ± 0.002
Sec-R8-NPs	153.7 ± 8.5	0.213 ± 0.037	31.1 ± 0.8	80.42 ± 2.08	0.019 ± 0.003
Sec-Tat-NPs	141.4 ± 7.8	0.166 ± 0.012	28.0 ± 0.5	78.80 ± 1.69	0.019 ± 0.003
Sec-Pen-NPs	158.6 ± 8.2	0.217 ± 0.048	34.3 ± 1.3	79.83 ± 1.71	0.019 ± 0.002

PDI, polydispersity index; EE, encapsulation efficiency; DL, drug loading.

Table 3. Characteristics of insulin-loaded nanoparticles (mean ± SD, n = 3).

Formulations	Size (nm)	PDI	Zeta potential (mV)	EE% of insulin	DL% of insulin
Unmodified NPs	159.8 ± 9.0	0.078 ± 0.015	−15.6 ± 2.3	59.60 ± 2.32	2.42 ± 0.01
Pen-NPs	147.5 ± 10.4	0.193 ± 0.030	28.3 ± 3.1	62.71 ± 3.14	2.51 ± 0.02
Sec-Pen-NPs	163.1 ± 7.2	0.205 ± 0.041	24.7 ± 4.5	65.19 ± 3.36	2.67 ± 0.04

Figure 3. In vitro release of insulin from insulin-loaded nanoparticles (unmodified NPs, Pen-NPs, Sec-Pen-NPs) in simulated intestinal medium (PBS, pH 6.8) without trypsin at defined intervals. Each data point was expressed as the mean ± SD (n = 3).

Figure 4. Remained insulin of insulin solution and insulin-loaded nanoparticles (unmodified NPs, Pen-NPs, Sec-Pen-NPs) after incubation in simulated intestinal medium (PBS, pH 6.8) with trypsin at defined intervals. Each data point was expressed as the mean ± SD (n = 3).

Figure 5. Transcellular transport of coumarin-6-loaded nanoparticles through Caco-2 cell monolayers at defined intervals. Each data point was expressed as the mean ± SD (n = 3).

Table 4. P_app of coumarin-6-loaded nanoparticles across the Caco-2 cell monolayers (mean ± SD, n = 3).

Formulations	Papp (×10^−6cm/s)
Unmodified NPs	2.54 ± 0.33
R8-NPs	4.45 ± 0.16
Tat-NPs	4.18 ± 0.13
Pen-NPs	5.45 ± 0.53
Sec-R8-NPs	4.88 ± 0.49
Sec-Tat-NPs	4.67 ± 0.55
Sec-Pen-NPs	11.09 ± 0.54

Figure 6. Fluorescence microscopy images of Caco-2 cell monolayer incubated with coumarin-6-loaded nanoparticles: unmodified NPs (A), R8-NPs (B), Tat-NPs (C), Pen-NPs (D), Sec-R8-NPs (E), Sec-Tat-NPs (F), Sec-Pen-NPs (G) for 2 h at 37 °C.

Figure 7. Cellular uptake of coumarin-6-loaded nanoparticles by Caco-2 cells at defined intervals. Each data point was expressed as the mean ± SD (n = 3).

Figure 8. CCK-8 assays of cell viability after incubation of coumarin-6-loaded nanoparticles with Caco-2 cells for 2 h. Each data point was expressed as the mean ± SD (n = 3).

Figure 9. Plasma insulin concentration in rats after ileal segments administration of saline, insulin-loaded nanoparticles (unmodified NPs, Pen-NPs, Sec-Pen-NPs) at a dose of 10 IU/kg and s.c. administration of insulin solution (1 IU/kg). Each data point was expressed as the mean ± SD (n = 7).

Table 5. Pharmacokinetic and pharmacodynamic parameters in rats after ileal segments administration of insulin loaded nanoparticles (Unmodified NPs, Pen-NPs, Sec-Pen-NPs) and s.c. administration of insulin solution (mean ± SD, n = 7).

	Ins (s.c.)	Unmodified NPs	Pen-NPs	Sec-Pen-NPs
Dose (IU/kg)	1	10	10	10
AUC (ng × h/mL)	268.45 ± 9.24	161.37 ± 55.59	299.97 ± 52.21	513.98 ± 69.24
Cmax (ng/mL)	135.57 ± 20.01	30.10 ± 10.67	53.92 ± 9.46	90.23 ± 33.31
Tmax (h)	0.5	4.0	4.0	4.0
BA (%)	100	6.01 ± 2.07	11.17 ± 2.05	19.10 ± 2.58
AAC (%glu. reduc. × h)	238.25 ± 62.24	133.76 ± 37.66	218.71 ± 31.11	350.55 ± 64.42
PA (%)	–	5.61 ± 1.59	9.18 ± 1.32	14.72 ± 2.73

AUC: the area under the plasma insulin concentration-time curve; C_max: maximum plasma concentration; T_max: time at which C_max is attained; BA: relative bioavailability; AAC: the area above the blood glucose levels-time curve; PA: pharmacological bioavailability.

Figure 10. Blood glucose levels in rats after ileal segments administration of saline, insulin-loaded nanoparticles (unmodified NPs, Pen-NPs, Sec-Pen-NPs) and s.c. administration of insulin solution. Each data point was expressed as the mean ± SD (n = 7).