Liposomes containing glycocholate as potential oral insulin delivery systems: preparation, in vitro characterization, and improved protection against enzymatic degradation

Figures & data

Figure 1 Effects of homogenization pressure (A) and number of cycles (B) on recombinant human insulin entrapment efficiency and particle size of sodium glycocholate liposomes, when cycles fixed at four and pressure 300 bar for each.

Note: Data expressed as means ± standard deviations (n = 3).

Abbreviation: EE, entrapment efficiency.

Figure 2 Effects of soybean phosphotidylcholine:sodium glycocholate ratio (A), recombinant human insulin:soybean phosphotidylcholine ratio (B), water:ether volume ratio (C), pH of the inner water phase (D), ionic strength of the hydration buffer (E), and hydration time (F) on recombinant human insulin entrapment efficiency.

Note: Data expressed as means ± standard deviations (n = 3).

Abbreviations: SPC, soybean phosphotidylcholine; SGC, sodium glycocholate; rhINS, recombinant human insulin.

Figure 3 Particle size and distribution of recombinant human insulin-loaded sodium glycocholate liposomes prepared under optimal conditions.

Figure 4 Circular dichroism spectra of free insulin solution and insulin released from sodium glycocholate liposomes prepared under various extreme stress conditions at a recombinant human insulin concentration of 20 μg/mL. The insert indicates circular dichroism spectrum of liposomal recombinant human insulin for the optimal formulation.

Abbreviations: SPC, soybean phosphotidylcholine; SGC, sodium glycocholate; rhINS, recombinant human insulin; I.S., ionic strength.

Table 1 Blood glucose level after subcutaneous administration of 0.5 IU/kg recombinant human insulin released from different sodium glycocholate liposomal formulations in mice. Data expressed as means ± standard deviations (n = 6)

SG liposome formulation	Blood glucose level
	0 hours (mmol/L)	1 hour (mmol/L)	Reduction (%)
Free rhINS solution	10.70 ± 2.71	4.96 ± 1.44	53.1 ± 12.6
Blank SGC liposomes	9.93 ± 1.59	9.76 ± 1.16	1.2 ± 6.3*
At maximum SGC amount (SPC:SGC ratio 2:1)	10.98 ± 2.74	4.61 ± 1.06	57.7 ± 3.9
At maximum rhINS:SPC ratio of 0.0025:1	10.21 ± 1.59	4.35 ± 0.50	56.9 ± 6.9
At maximum ether:water ratio of 7:1	10.09 ± 1.84	5.16 ± 1.39	49.1 ± 9.7
At the strongest ionic strength (1.2)	8.48 ± 1.17	3.83 ± 1.28	52.9 ± 20.0
At extreme homogenization conditions (500 bar, 6 cycles)	7.94 ± 1.35	3.40 ± 0.85	57.3 ± 6.4
At combination of extreme stress conditions	8.83 ± 2.2	3.78 ± 0.69	55.4 ± 11.0

Note:

* P < 0.05, compared with free rhINS solution.

Abbreviations: rhINS, recombinant human insulin; SGC, sodium glycocholate; SPC, soybean phosphotidylcholine.

Figure 5 Transmission electron micrographs of conventional liposomes (A), blank (B), and recombinant human insulin-loaded sodium glycocholate liposomes (C).

Figure 6 Leakage of recombinant human insulin from liposomes as measured by entrapment efficiency in buffers with different pH values.

Note: Data expressed as means ± standard deviations (n = 3).

Figure 7 Protection of recombinant human insulin from pepsin degradation by liposomes with different types of bile salts, ie, sodium glycocholate, sodium taurocholate, sodium deoxycholate, (A) and different soybean phospholipid, ie, soybean phosphotidylcholine:sodium glycocholate ratios (B) for 4 hours at 37°C.

Note: Data expressed as means ± standard deviations (n = 3).

Abbreviations: SPC, soybean phosphotidylcholine; SGC, sodium glycocholate; rhINS, recombinant human insulin; CH, cholesterol.

Figure 8 Protection of recombinant human insulin from trypsin degradation by liposomes with different type of bile salts, ie, sodium glycocholate, sodium taurocholate, sodium deoxycholate, (A) and different soybean phospholipid, ie, soybean phosphotidylcholine:sodium glycocholate ratios (B) for 4 hours at 37°C.

Note: Data expressed as means ± standard deviations (n = 3).

Abbreviations: SPC, soybean phosphotidylcholine; SGC, sodium glycocholate; rhINS, recombinant human insulin; CH, cholesterol.

Figure 9 Protection of recombinant human insulin from α-chymotrypsin degradation by liposomes with different type of bile salts, ie, sodium glycocholate, sodium taurocholate, sodium deoxycholate (A) and different soybean phospholipids, ie, soybean phosphotidylcholine:sodium glycocholate ratios (B) for 4 hours at 37°C.

Note: Data expressed as means ± standard deviations (n = 3).

Abbreviations: SPC, soybean phosphotidylcholine; SGC, sodium glycocholate; rhINS, recombinant human insulin; CH, cholesterol.