Transport of proteins dissolved in organic solvents across biomimetic membranes.

Abstract

Using lipid-impregnated porous cellulose membranes as biomimetic barriers, we tested the hypothesis that to afford effective transmembrane transfer of proteins and nucleic acids, the vehicle solvent should be able to dissolve both the biopolymers and the lipids. While the majority of solvents dissolve one or the other, ethanol and methanol were found to dissolve both, especially if the protein had been lyophilized from an aqueous solution of a pH remote from the protein's isoelectric point. A number of proteins, as well as RNA and DNA, dissolved in these alcohols readily crossed the lipidized membranes, whereas the same biopolymers placed in nondissolving solvents (e.g., hexane and ethyl acetate) or in those unable to dissolve lipids (e.g., water and dimethyl sulfoxide) exhibited little transmembrane transport. The solubility of biopolymers in ethanol and methanol was further enhanced by complexation with detergents and poly(ethylene glycol); significant protein and nucleic acid transport through the lipidized membranes was observed from these solvents but not from water.

Protein refolding in predominantly organic media markedly enhanced by common salts

“…Although enzymes do not have to be dissolved in organic solvents to work as catalysts (and indeed they usually are not) (Koskinen and Klibanov, 1996), in order to explore protein folding in nonaqueous media the latter should dissolve both folded and unfolded proteins. Fortunately, over the last several years it has been found that, under optimal conditions, proteins are indeed highly soluble in more than a dozen neat organic solvents (Bromberg and Klibanov, 1995;Chin et al, 1994).…”

Non-Aqueous Systems for Formulation Development—Proteins

“…The insulin remained in its native conformation with increased thermal stability (T m ), when compared with water Manning et al, 1995). Insulin (pH 3.0) solubility in EtOH increased from 14 mg/ml to 830 mg/ml with the addition of SDS (Bromberg and Klibanov, 1995). Similar results were obtained with α-chymotrypsin in sodium bis(2-ethylhexyl) sulfosuccinate (Manning et al, 1995).…”

“…MeOH >10 (Chin et al, 1994) TFE >10 (Chin et al, 1994) Ethylene glycol >10 (Chin et al, 1994) DMSO >100 (Houen, 1996) Glycerol >100 (Houen, 1996) Myoglobin Ethanol 0.59 (Bromberg and Klibanov, 1995) Methanol >5 (Bromberg and Klibanov, 1995) Salmon Calcitonin 70% EtOH 50 (Stevenson and Tan, 2000) 70% DMSO 50 (Stevenson and Tan, 2000) Water 100 (Stevenson and Tan, 2000) Ribonuclease Ethanol <0.005 (Bromberg and Klibanov, 1995) Methanol 0.61 (Bromberg and Klibanov, 1995) Table 3. Solubility of proteins in co-solvent and non-aqueous conditions.…”

“…For example, lysozyme (pI 11.0) demonstrated a 1000-fold decrease in solubility in 56% acetonitrile/44% DMSO when reconstituted from pH 10.0, as compared with pH 2.0 (Chin et al, 1994). The solubility of several proteins was characterized as a function of pH of the lyophilized material (Bromberg and Klibanov, 1995). For example, lysozyme lyophilized at pH 3.0 and 6.0 yielded solubilities in EtOH of 250 μg/ml and 200 μg/ml, respectively.…”

“…Bovine Serum Albumin Ethanol 0.01 (Houen, 1996) DMF 0.01 (Houen, 1996) Acetonitrile 0.02 (Houen, 1996) 1-Propanol 0.03 (Houen, 1996) Methanol 0.8 (Bromberg and Klibanov, 1995) DMSO 5.12 (Houen, 1996) Glycerol 20 (Houen, 1996) Cyclosporine A Water 0.02 (Seeback et al, 1989) 40% Glycofurol 0.8 (Seeback et al, 1989) 35% PEG 400 0.8 (Seeback et al, 1989) 30% PG 0.2 (Seeback et al, 1989) 40% EtOH 3.6 (Seeback et al, 1989) Cytochrome C Ethanol <0.005 (Bromberg and Klibanov, 1995) Methanol 1.4 (Bromberg and Klibanov, 1995) Chapter 12: Non-Aqueous Systems for Formulation Development-Proteins 365 Glucagon Ethanol 1.1 (Bromberg and Klibanov, 1995) Glucagon Like Peptide DMSO 80 (Stevenson, 1999) Water 150 (Stevenson, 1999) Growth Hormone Methanol ND (Bromberg and Klibanov, 1995) Ethanol 2.4 (Bromberg and Klibanov, 1995) Insulin 1-Octanol 0.005 Ethanol 0.32 (Bromberg and Klibanov, 1995) Methanol >5 (Bromberg and Klibanov, 1995) Insulin:SDS (1:6) 1-Octanol 3 Leuprolide DMSO 370 Lysozyme Acetonitrile 0.03 (Houen, 1996) Isoamyl alcohol 0.08 (Houen, 1996) 1-Octanol <0.05 …”