Hypoglycemic activity and stability enhancement of human insulin–tat mixture loaded in elastic anionic niosomes

Figures & data

Figure 1. Amounts of initial insulin and insulin interacted with C-Tat-resin or Tat-C-resin or 2-mercaptoethanol-resin.

Table 1. Physical characteristics of elastic, non-elastic neutral and anionic niosomes loaded and not loaded with the peptides (insulin, Tat or the insulin–Tat mixture).

Niosomal formulations	Vesicular size (nm)	Zeta-potential (mV)	DI
Blank non-elastic neutral niosomes	166.3 ± 2.4	−2.5 ± 0.2	0.36 ± 0.08
Non-elastic neutral niosomes loaded with human insulin	194.7 ± 1.0	−2.9 ± 0.3	–
Non-elastic neutral niosomes loaded with Tat	210.8 ± 1.7	−2.8 ± 0.2	–
Non-elastic neutral niosomes loaded with human insulin-Tat mixture	183.5 ± 2.2	–2.5 ± 0.7	–
Blank elastic neutral niosomes	206.0 ± 2.5	–2.5 ± 0.3	0.90 ± 0.25
Elastic neutral niosomes loaded with human insulin	253.1 ± 2.0	–2.8 ± 0.3	–
Elastic neutral niosomes loaded with Tat	260.6 ± 2.9	–3.1 ± 0.2	–
Elastic neutral niosomes loaded with human insulin–Tat mixture	147.1 ± 0.2	–2.5 ± 0.3	–
Blank non-elastic anionic niosomes	124.4 ± 0.7	–16.6 ± 0.9	0.09 ± 0.15
Non-elastic anionic niosomes loaded with human insulin	1,102 ± 33	–4.5 ± 0.5	–
Non-elastic anionic niosomes loaded with Tat	216.8 ± 3.7	–8.1 ± 0.8	–
Non-elastic anionic niosomes loaded with human insulin–Tat mixture	162.6 ± 0.4	–8.4 ± 0.6	–
Blank elastic anionic niosomes	178.5 ± 2.6	–17.9 ± 0.6	1.01 ± 0.27
Elastic anionic niosomes loaded with human insulin	1225 ± 113	–5.5 ± 0.4	–
Elastic anionic niosomes loaded with Tat	1009 ± 57	–8.0 ± 0.5	–
Elastic anionic niosomes loaded with human insulin–Tat mixture	792.2 ± 41.9	–8.4 ± 0.5	–

The experiments were performed in triplicate and the data were presented in mean ± SD. DI, Deformability index.

Figure 2. Gel electrophoresis of niosomes loaded and not loaded with human insulin-Tat mixture (1:3 molar ratio). Lane 1 = the standard human insulin. Lane 2 = human insulin-Tat mixture (1:3 molar ratio). Lane 3 = blank non-elastic neutral niosomes. Lane 4 = non-elastic neutral niosomes loaded with the mixture. Lane 5 = blank elastic neutral niosomes. Lane 6 = elastic neutral niosomes loaded with the mixture. Lane 7 = blank non-elastic anionic niosomes. Lane 8 = non-elastic anionic niosomes loaded with the mixture. Lane 9 = blank elastic anionic niosomes. Lane 10 = elastic anionic niosomes loaded with the mixture.

Table 2. Entrapment efficiency of the peptides (insulin, Tat or the insulin–Tat mixture) in elastic, non-elastic neutral and anionic niosomes.

	Entrapment efficiency (%)
Niosomal formulations	human insulin	Tat
Blank non-elastic neutral niosomes	–	–
Non-elastic neutral niosomes loaded with human insulin	19.3 ± 1.8	–
Non-elastic neutral niosomes loaded with Tat	–	29.6 ± 3.7
Non-elastic neutral niosomes loaded with human insulin–Tat mixture	4.1 ± 2.7	2.2 ± 1.9
Blank elastic neutral niosomes	–	–
Elastic neutral niosomes loaded with human insulin	37.0 ± 3.9	–
Elastic neutral niosomes loaded with Tat	–	36.1 ± 5.1
Elastic neutral niosomes loaded with human insulin–Tat mixture	7.0 ± 5.0	2.8 ± 3.8
Blank non-elastic anionic niosomes	–	–
Non-elastic anionic niosomes loaded with human insulin	100.0 ± 0.0	–
Non-elastic anionic niosomes loaded with Tat	–	56.6 ± 0.6
Non-elastic anionic niosomes loaded with human insulin–Tat mixture	87.9 ± 2.3	16.6 ± 1.6
Blank elastic anionic niosomes	–	–
Elastic anionic niosomes loaded with human insulin	100.0 ± 0.0	–
Elastic anionic niosomes loaded with Tat	–	58.8 ± 1.4
Elastic anionic niosomes loaded with human insulin–Tat mixture	100.0 ± 0.0	27.3 ± 6.4

The experiments were performed in triplicate and the data were presented in mean ± SD.

Figure 3. The percentages of insulin released from the elastic anionic niosomes after incubated at 37 °C for 6 h.

Table 3. Physical stability of elastic anionic niosomes loaded with human insulin–Tat mixture (1:3 molar ratio) or free insulin when stored at 5 ± 3 °C, room temperature (30 ± 2 °C) and 40 ± 2 °C for 4 weeks.

		Vesicular size (nm)			Zeta-potential (mV)
Samples	Storage time (weeks)	5 ± 3 °C	30 ± 2 °C	40 ± 2 °C	5 ± 3 °C	30 ± 2 °C	40 ± 2 °C
Blank elastic anionic niosomes	Initial	172.4 ± 2.5			−17.9 ± 1.1
	1	237.4 ± 3.0	235.7 ± 2.9	606.8 ± 10.6	−17.1 ± 0.2	−17.7 ± 0.9	−17.9 ± 0.7
	2	258.4 ± 1.6	252.7 ± 2.8	392.4 ± 0.8	−17.9 ± 0.2	−17.1 ± 0.4	−18.4 ± 0.7
	3	159.7 ± 0.5	174.3 ± 0.4	409.9 ± 4.3	−17.4 ± 1.1	−18.0 ± 1.2	−18.4 ± 0.6
	4	158.3 ± 2.0	176.2 ± 1.2	383.6 ± 3.2	−17.0 ± 1.0	−17.3 ± 0.8	−18.1 ± 0.7
Insulin loaded in elastic anionic niosomes	Initial	288.1 ± 2.4			−16.7 ± 0.4
	1	470.2 ± 1.8	310.1 ± 7.3	3357 ± 90	−16.7 ± 0.6	−16.8 ± 0.8	−14.5 ± 0.6
	2	483.2 ± 6.7	573.4 ± 20.8	4385 ± 236	−15.5 ± 0.7	−15.7 ± 0.3	−13.3 ± 0.5
	3	321.3 ± 3.0	556.1 ± 15.8	4292 ± 131	−16.5 ± 0.5	−15.9 ± 0.7	−11.9 ± 0.4
	4	558.2 ± 9.7	670.6 ± 25.9	3905 ± 257	−15.7 ± 0.5	−15.3 ± 0.3	−12.3 ± 0.5
Insulin–Tat mixture of 1:3 molar ratio loaded in elastic anionic niosomes	Initial	567.6 ± 82.3			−4.7 ± 0.1
	1	5520 ± 241	2929 ± 855	8051 ± 864	−2.2 ± 0.1	−2.3 ± 0.2	−2.8 ± 0.3
	2	5939 ± 1452	6991 ± 120	(1.35 ± 0.1) × 10^4	−2.5 ± 0.1	−2.0 ± 0.1	−2.4 ± 0.2
	3	2401 ± 512	8418 ± 676	(1.18 ± 0.1) × 10^4	−2.3 ± 0.2	−2.2 ± 0.1	−1.5 ± 0.1
	4	2977 ± 740	4345 ± 178	(1.06 ± 0.2) × 10^4	−2.4 ± 0.1	−2.1 ± 0.1	−0.6 ± 0.2

The experiments were performed in triplicate and the data were presented in mean ± SD. RT, Room temperature.

Figure 4. Percentages remaining of insulin in the insulin-Tat mixture (1:3 molar ratio) loaded and not loaded in elastic anionic niosomes when stored at 5 ± 3 °C, room temperature (30 ± 2 °C) and 40 ± 2 °C for 12 weeks.

Figure 5. The blood glucose reduction percentages in alloxan-induced diabetic mice (n = 5–6) at various time intervals after treated with various insulin systems. SC, Subcutaneous injection.