Role of a novel excipient poly(ethylene glycol)-b-poly(L-histidine) in retention of physical stability of insulin at aqueous/organic interface

Abstract

The aim of this study was to investigate whether a cationic polyelectrolyte, poly(ethylene glycol)-b-poly(L-histidine) diblock copolymer (PEG-polyHis), can stabilize insulin, at the aqueous/methylene chloride interface formed during the microencapsulation process. Insulin aggregation at this interface was monitored spectrophotometrically at 276 nm. The effects of protein concentration, pH of the aqueous medium, and the presence of poly(lactic-co-glycolic acid) (PLGA) in methylene chloride (MC) on insulin aggregation were observed. For the 2.0 mg/mL insulin solutions in phosphate buffer (PB), the effect of addition of Pluronic F-127 as a positive control and addition of PEG-polyHis as a novel excipient in PB was also evaluated at various insulin/polymeric excipient weight ratios. The conformation of insulin protected by PEG-polyHis and recovered after interfacial exposure was evaluated via circular dichroism (CD) spectroscopy. Greater loss in soluble insulin was observed with increasing insulin concentrations. pH 6.0 was selected for optimal ionic interactions between insulin and PEG-polyHis based on zeta potential and particle size studies. pH 4.5 and 7.4 (no ionic complexation between insulin and PEG-polyHis) were selected as controls to compare the stabilization effect of PEG-polyHis with that at pH 6.0. Incubation of PEG-polyHis with insulin at pH 6.0 drastically reduced protein aggregation, even in the presence of PLGA. PEG-polyHis and F-127 reduced insulin aggregation in noncomplexing pH conditions pointing to the role played by PEG in modulation of insulin adsorption at the interface. Far-UV (205-250 nm) CD study revealed negligible qualitative effects on soluble insulin's secondary structure after interfacial exposure. RP-HPLC and size-exclusion HPLC showed no deamidation of insulin or formation of soluble high molecular weight transformation products respectively. MALDI-TOF mass spectrometry confirmed the results from chromatographic procedures. Radioimmunoassay carried out on select samples showed that recovered soluble insulin had retained its immunoreactivity. An experimental method to simulate interfacial denaturation of proteins was designed for assessment of protein stability at the interface and screening for novel protein stabilizers. Understanding and manipulation of such polyelectrolyte-insulin complexation will likely play a role in insulin controlled delivery via microsphere formulation(s).

Figure 1

Chemical structure of the diblock copolymer PEG-poly(l-Histidine). * indicates the lone pair of electrons available for reversible protonation.

Figure 2

Ionic interactions between Insulin and PEG - polyHis are expected to prevent contacts of insulin with methylene chloride. I - II shows interfacial aggregation of insulin on methylene chloride layer. III depicts competition between PEG-polyHis and insulin for the interfacial area. IV - V shows protection of insulin against interfacial stresses an organic solvent via complexation.

Figure 3

Zeta potential of insulin/PEG-polyHis complexes at room temperature: The order of symbols left to right; PEG-polyHis, insulin, insulin: PEG-polyHis = 1:3, 1:2, 1:1, 1:2, 1:3.

Figure 4

Effect of insulin concentration (mg/ml) on the rate of insulin aggregation. The plot of % retention of soluble insulin as a function of time is shown. N=3.

Figure 5

Relative insulin aggregation as a function of pH on exposure to methylene chloride layer at 400 rpm for 4 hrs. Initial insulin concentration was set at 2.03 mg/ml. N = 3

Figure 6

Far-UV CD spectrum (205-250 nm) on exposure to methylene chloride layer at 400 rpm for 4 hrs. Initial insulin concentration was set at 0.4 mg/ml.

Figure 7

RP-HPLC chromatogram of soluble insulin recovered from systems containing polymeric excipients after interfacial exposure and comparison with an insulin standard at equivalent concentrations.

Figure 8

SE-HPLC chromatogram of soluble insulin recovered after interfacial exposure and its comparison with an insulin standard at equivalent concentrations.

Figure 9

Comparison of mass spectra of (A) Insulin Standard (B) Recovered Soluble Insulin and (C) Insulin aggregates dissolved in 6M urea and dialyzed against deionized water. Masses are mentioned in brackets below the charged species listed. Initial insulin concentrations were 60 μg/ml and were diluted with the matrix material.

Figure 10

Kinetic plot of loss of soluble insulin as a function of homogenization duration during the primary emulsion formation. Initial concentration was fixed at 2.06 mg/ml. PEG-polyHis concentration was fixed at 4.12 mg/ml (weight ratio of Insulin to PEG-polyHis was set at 1: 2). N = 3.

Figure 11

Effect of insulin to PEG-polyHistidine weight ratio on insulin aggregation. Insulin concentration was fixed at 2.07 mg/ml. N = 3

Figure 12

Comparison of insulin contents obtained from RP-HPLC and radioimmunoassay (RIA). Four different kinds of samples tested included an insulin standard (A), soluble insulin recovered following interfacial exposure without any polymeric excipient (B), soluble insulin recovered following interfacial exposure from systems with F-127 (C), and soluble insulin recovered following interfacial exposure from systems with PEG-polyHis at pH 6.0 (D). N = 2.