Self-assembled insulin and nanogels polyelectrolyte complex (Ins/NGs-PEC) for oral insulin delivery: characterization, lyophilization and in-vivo evaluation

Figures & data

Table 1 Optimization of Ins/NGs-PEC parameters

Formula code	Parameter optimized				Parameters kept constant
	Ins:NGs ratios	pHs	Incubation time (hrs)	Stirring rate (rpm)	Ins:NGs ratios	pHs	Incubation time (hours)	Stirring rate (rpm)
InF1	1:20	-	-	-	-	4.0	6	100
InF2	1:30	-	-	-	-	4.0	6	100
InF3	1:40	-	-	-	-	4.0	6	100
InF4	1:50	-	-	-	-	4.0	6	100
	Formulations InF1–InF4 were prepared using the following parameters: different ratios of Ins:NG, pH (4.0), incubation time (6 hours) and stirring rate (100 rpm). The best formulation was selected for further studies using different pHs (InF5–InF7)
InF5	-	1.2	-	-	Optimized ratio	-	6	100
InF6	-	4.0	-	-	Optimized ratio	-	6	100
InF7	-	7.4	-	-	Optimized ratio	-	6	100
	Formulations InF5–InF7 were prepared using the following parameters: different pHs, optimized Ins:NGs ratio, incubation time (6 hours) and stirring rate (100 rpm). The best formulation was selected for further studies using different incubation time (InF8–InF10)
InF8	-	-	4	-	Optimized ratio	Optimized pH	-	100
InF9	-	-	6	-	Optimized ratio	Optimized pH	-	100
InF10	-	-	8	-	Optimized ratio	Optimized pH	-	100
	Formulations InF8–InF10 were prepared using different incubation times, optimized Ins: NGs ratio, optimized pHs and stirring rate (100 rpm). The best formulation was selected for further studies using different stirring rate (InF11–InF13)
InF11	-	-	-	0	Optimized ratio	Optimized pH	Optimized incubation time	-
InF12	-	-	-	100	Optimized ratio	Optimized pH	Optimized incubation time	-
InF13				200	Optimized ratio	Optimized pH	Optimized incubation time	-
	Formulation InF11–InF13 were prepared using different stirring rates, optimized Ins:NGs ratio, optimized pHs and optimized incubation time. The best formulation was selected for further studies

Abbreviations: In or Ins, insulin; F, formulation; NGs, nanogels.

Table 2 Treatments received by diabetic rat groups for determination of blood glucose level

Groups	Codes	Design of treatment	Treatments	No. of rats
Control-I	Wpo	Water (per oral)	Free access	6
Control-II	BFpo	Blank formulation (per oral)	Equivalent wt. to 100 IU/kg body weight	6
Control-III	Ipo	NovoRapid^® (per oral)	Insulin solution 100 IU/kg body weight	6
Control-IV	Isc	(NovoRapid^®) (subcutaneous)	5 IU/kg body weight	6
Test-V	NGI 25IU po	Ins-loaded-NGs (per oral)	25 IU/kg body weight	6
Test-VI	NGI 50IU po	Ins-loaded-NGs (per oral)	50 IU/kg body weight	6
Test-VII	NGI po 100IU	Ins-loaded-NGs (per oral)	100 IU/kg body weight	6

Abbreviations: Wpo, water per oral (po); BFpo, Blank Formulations (po); Ipo, insulin (po); Isc, insulin (sc); NGI 25 po, InF12-Tre2 containing insulin 25 IU (po); NGI 50 po, InF12-Tre2 containing insulin 50 IU (po); NGI 100 po, InF12-Tre2 containing insulin 100 IU (po).

Plate 1 Physical appearance of insulin solution at different pH values.

Figure 1 Determination of the isoelectric point in (A) Insulin, (B) Nanogels, and (C) Ins/NGs-PEC. Mean ± SD, n=3.

Table 3 Effect of Ins/NG ratios, pH, incubation times and stirring rates on Ins/NGs-PEC properties

Effect of Ins:NGs ratios on the Ins/NGs-PEC properties
Formulation code	Ins:NGs	PS (nm)	PdI	ZP (mV)	% EE
InF1	1:20	1399±55.59	1±0	−9.05±0.15	66.22±3.16
InF2	1:30	235.4±5.80	0.318±0.025	−16.00±0.65	75.02±3.31
InF3	1:40	190.4±0.90	0.186±0.023	−16.70±0.50	85.20±2.33
InF4	1:50	186.2±4.00	0.173±0.021	−15.70±0.28	82.45±3.06
Effect of pH on the Ins/NGs-PEC properties
	pH	PS (nm)	PdI	ZP (mV)	% EE
InF5	pH 1.2	8299.70±1156.20	1.000±0.00	1.38±1.06	78.50±1.50
InF6	pH 4.0	190.40±0.90	0.186±0.023	−16.70±0.50	85.20±2.33
InF7	pH 7.4	186.20±4.24	0.145±0.028	−48.60±0.50	18.42±5.06
Effect of incubation time on the Ins/NGs-PEC properties
	Incubation time (hours)	PS (nm)	PdI	ZP (mV)	% EE
InF8	4	188.13±1.95	0.133±0.01	−23.43±0.64	76.44±1.97
InF9	6	190.40±0.90	0.186±0.023	−16.70±0.50	85.20±2.33
InF10	8	191.03±1.10	0.145±0.04	−16.20±1.20	82.42±1.13
Effect of stirring rate on the Ins/NGs-PEC properties
	Stirring rate (rpm)	PS (nm)	PdI	ZP (mV)	% EE
InF11	0	249.10±4.12	0.294±0.04	−21.80±1.08	75.28±1.63
InF12	100	190.40±0.90	0.186±0.023	−16.70±0.50	85.20±2.33
InF13	200	189.33±1.74	0.151±0.035	−22.50±0.90	75.71±4.62

Abbreviations: Ins, Insulin; NGs, Nanogels; PEC, Polyelectrolyte complex; InF, Insulin loaded formulations; PS, Particle size; PdI, Polydispersity index; ZP, Zeta potential; % EE, Percentage entrapment efficiency.

Table 4 Lyophilization of InF-12 formulation using trehalose as a cryoprotectant. Mean ± SD, n=3

Trehalose (% w/v)	Formulation code	PS (nm)	PdI	ZP (mV)	% EE
0%	InF12	NM	NM	NM	NM
0.5	InF12Tre0.5	NM	NM	NM	NM
1	InF12Tre1	893.90±56.29	1.000±0.000	−27.43±1.89	74.57±4.08
2	InF12Tre2	430.50±27.61	0.588±0.094	−16.50±0.60	82.10±2.12
3	InF12Tre3	418.73±55.38	0.459±0.228	−15.80±0.67	80.71±2.89

Abbreviations: Tre, Trehalose; InF, Insulin loaded formulations; PS, Particle size; PdI, Polydispersity index; ZP, Zeta potential; % EE, Percentage entrapment efficiency; NM, Not measured.

Figure 2 In-vitro release of insulin from (A) Liquid formulation of InF-12, and (B) Lyophilized formulation of InF12-Tre2 in SGF (pH 1.2) and SIF (pH 6.8) at 37°C. Mean ± SD, n=3.

Abbreviations: SGF, Simulated gastric fluid; SIF, Simulated intestinal fluid.

Figure 3 FTIR spectra of (A) Insulin, (B) Nanogels, (C) Trehalose, (D) Lyophilized blank formulation, and (E) Lyophilized InF12-Tre2.

Figure 4 SDS-PAGE results. Lane 1: Unstained low range protein ladder; Lane 2: Insulin released from formulation; Lane 3: Standard insulin.

Figure 5 TEM of (A) InF12 before lyophilization and (B) Reconstituted lyophilized InF12-Tre2 after lyophilization.

Table 5 The stability data of InF12-Tre2 formulation stored at 5±3°C and 25±2°C/75±15%RH. Mean ± SD, n=3

Temperature: 5±3°C
Parameters	T0M	T1M	T2M	T3M
PS (nm)	430.5±27.60	425.53±30.03	411.96±22.78	421.13±24.00
PDI	0.588±0.094	0.546±0.053	0.400±0.180	0.422±0.052
ZP (mV)	−16.50±0.60	−16.73±0.64	−17.60±0.964	−16.60±0.781
Drug content (%)	100.28±3.91	97.44±3.79	98.76±4.71	98.26±4.34
Temperature: 25±2°C/75±15%RH
Parameters	T0M	T1M	T2M	T3M
Particle size (nm)	430.5±27.60	NM	NM	NM
PDI	0.588±0.094	NM	NM	NM
Zeta potential (mV)	−160.50±0.60	−22.63±1.59	−26.33±1.16	−34.70±0.50
Drug content (%)	100.28±3.91	82.49±3.68	68.17±3.15	51.39±4.59

Abbreviations: T0M, Time at zero month (immediately after preparation); T1M, Time at one month; T2M, Time at two months; T3M, Time at three months; InF, Insulin loaded formulations; PS, Particle size; PdI, Polydispersity index; ZP, Zeta potential; % EE, Percentage entrapment efficiency; RH, Relative humidity; NM, not measured.

Figure 6 In-vitro release profile of lyophilized InF12-Tre2 formulation stored at 5±3°C in (A) SGF, and (B) SIF. Mean ± SD, n=3.

Abbreviations: SGF, Simulated gastric fluid; SIF, Simulated intestinal fluid; T0M, Time at zero month (immediately after preparation); T1M, Time at one month; T2M, Time at two months; T3M, Time at three months.

Figure 7 Effect of oral administration of the three doses (25, 50 and 100 IU/kg body weight) of lnF12-Tre2 and four control groups on the percentage reductions in blood glucose level in the streptozotocin-induced diabetic rats.*Statistically significant difference to BFpo. Mean ± SD, n=6. Data points represent relative values of the initial blood glucose concentration.

Abbreviations: Wpo, water per oral; BFpo, blank formulations per oral; Ipo, insulin per oral; Isc, insulin subcutaneous; NGI 25 po, InF12-Tre2 containing insulin 25 IU per oral; NGI 50 po, InF12-Tre2 containing insulin 50 IU per oral; NGI 100 po, InF12-Tre2 containing insulin 100 IU per oral.