Electronic Supplementary Information
Low-transition-temperature mixtures (LTTMs) for dissolving proteins and for drug formulation
Erzheng Su a, b and Alexander M. Klibanova
a Departments of Chemistryand Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, U.S.A.
b Enzyme and Fermentation Technology Laboratory, College of Light Industry Science and Engineering, Nanjing Forestry University, Nanjing 210037, China.
Table of Contents:
1.Experimental Section 2
1.1 Materials 2
1.2 Preparation of LTTMs for dissolving proteins and containing aspirin 2
1.3 Dissolution of proteins in LTTMs 2
1.4 Determination of lysozyme activity 2
1.5 LTTM characterization 3
1.6 Irreversible lysozyme thermoinactivation studies at extreme pH conditions 3
1.7 Thermal stability of aspirin in a LTTM and in an aqueous buffer 4
2. Other Supporting Tables and Figures 4
Table S1. LTTMs prepared for dissolving proteins in this work 4
Table S2. Preparation of aspirin-containing LTTMs 5
Figure S1. Correlation of the lysozyme solubility with LTTMs’ characteristics 7
References 8
1. Experimental Section
1.1 Materials
Chemicals used for preparing LTTMs were from Alfa Aesar (USA) and were of the highest purity available. Bovine serum albumin(MW 66 kDa),bovine pancreatic α-chymotrypsinogen A(MW 26 kDa), subtilisin Carlsberg(MW 27 kDa),hen egg-white lysozyme(MW 14 kDa), and dried Micrococcus lysodeikticuscecells (ATCC No. 4698) were from Sigma-Aldrich (USA) and used without further purification. Nile red, 4-nitroaniline,N,N-diethyl-4-nitroaniline, N,N-diethyl-4-nitroaniline, and acetaminophen were also from Sigma-Aldrich. Aspirin was from Spectrum Chemical Mfg. (USA). All other chemicals or solvents were obtained from Alfa Aesar or TCI (USA).
1.2 Preparation of LTTMs for dissolving proteins and containing aspirin
Prior to LTTM preparation, all chemicals used were dried for 120 h by vacuum freeze-drying method and the solvents by incubation with 3Å molecular sieves for a week. The LTTMs were prepared by heating various hydrogen-bond acceptors (HBA)and donors (HBD) at different molar ratios to 80℃(when levulinic acid,glycerol, ethylene glycol, 1,2-propanediol, and 2-(2-aminoethoxy)ethanol were used as HBDs) or 100℃withconstantstirring until a homogeneous liquid formed. Table S1 lists the HBAs, HBDs, and their molar ratiosused for the preparation of LTTMs for dissolving proteins.The LTTMs formed were further dehydrated by incubating with 3Å molecular sieves for several days before protein solubility experiments and LTTM characterization. Table S2 lists the HBAs, HBDs, and theirmolar ratiosused to make aspirin-containing LTTMs.
1.3 Dissolution of proteins in LTTMs
The solubility of proteins in neat LTTMs was measured by placing a solid protein (typically 30 mg) into a 2-mL screw-cap scintillation vial, followed by addition of 1.0 g of a LTTM. The resultant mixture was shaken at 37°C and 600 rpm for 16 h and then centrifuged at 16,000 g for 2 h at 37°C. An aliquot of the supernatant was withdrawn and diluted 10-fold with double-distilled water. The protein content was determined using the Bradford assay.1
1.4. Determination of lysozyme activity
The catalytic activity of lysozyme was determined bymeasuring the rate of lysis of M. lysodeikticus dried cells as described by Avanti et al.2 with some modifications. Briefly, 2.5 mL of a 200 μg/ml of M. lysodeikticussuspensionin a PBS buffer (66 mM phosphate, pH 6.2) was mixed with 25μL of lysozyme solutions in plastic disposable cuvettes.Immediately after mixing, the cuvette was placed in a UV/Vis spectrophotometer, and the absorbance was measured at 450 nm as a function of time at 37℃.
1.5. LTTM characterization
The viscosities of the LTTMs were measured using a Brookfieldviscometer at 37℃.3 The pH of LTTMs were measured using a Mettler-Toledo pH meter pre-calibrated using standard pH buffers.4 The ENR and the Kamlet-Taft polarity parameters β and π* were determinedusing the solvatochromic dyes.5-7 Stock solutions of all dyes were prepared inabsolute ethanol in pre-cleaned glass vials.The required amount of dyes was weighed with a precision of ±0.1 mg. An appropriate amount of the dye solution from the stock was transferred to a 1-mL quartz cuvette. Ethanol was evaporatedusing a gentle stream of high-purity N2 gas. A pre-calculated amount of a LTTM was directly added to the cuvette to a final dye concentration of 0.01 mM,and the solution was thoroughly mixed.A UV/Vis spectrophotometer was used for acquisition of the molecular absorbancedata. All measurements were performed in triplicatestarting from sample preparation, and the results were averaged. The ENR data were calculated from eqn. (1): ENR/kcal mol-1 = 28,600/λmax;NR, whereλmax;NR isthe wavelength corresponding to the maximum of absorbance ofNile Red. The Kamlet-Taft parameter π* was obtained by measuring the wavelength of the maximum absorbance, νmax in kK (kiloKeyser, 10-3 cm-1), of the dye N,N-diethyl-4-nitroaniline, calculated from eqn. (2):π* = (νmax-ν0)/S, where ν0 = 27.5 kK and S = -3.18. The Kamlet-Taft parameter β was obtained by measuring the relative difference of solvatochromism between 4-nitroaniline (1) and N,N-diethyl-4-nitroaniline (2), calculated from eqn. (3): β = [1.035ν(2)max –ν(1)max + 2.64]/2.8, where ν(2)max andν(1)max are the wavelengths corresponding to maximum of absorbance ofN,N-diethyl-4-nitroaniline and 4-nitroaniline, respectively.
1.6. Irreversible lysozyme thermoinactivation studies at extreme pH conditions
Stopperedtest tubes containing 0.5 mg/mL solutions oflysozyme in an aqueous buffer or a LTTM were placed in a 100°C glycerol bath. Periodically, aliquots were withdrawn, diluted 10-fold with an aqueous buffer (66 mM phosphate, pH 6.2), and assayed for thelysozyme activity toward M. lysodeikticusdried cells at pH 6.2. HCl-KCl buffer (pH 1.47), NaCO3-NaHCO3 buffer (pH 9.93), TBAB:LA (pH 1.47), and ChCl:urea (pH 9.93) were used to prepare lysozyme solutions.
1.7 Thermal stability of aspirin in a LTTM and in an aqueous buffer solution
A LTTM [choline chloride:aspirin (1:2)]was transferred to a clean vial containing a stir bar. The vial was immersed in a glycerol bathheated to 100°C, and the LTTM was stirred at 300 rpm until its temperature reached 100°C. Following incubation for various time periods, the LTTM samples were diluted appropriately, and their absorbances at 300 nm (the maximum of absorbance of salicylic acid) were measured spectrophotometrically. The concentration of salicylic acid in the LTTM was calculated using a previously obtained standard curve and the residual concentration of aspirin using the mass balance. Similar procedures were used to investigate the thermal stability of aspirin in an aqueous buffer solution. A 0.57 M acetate buffer, pH of 2.5, was used to prepare the aspirin aqueous solution.
2. Supporting Tables and Figures
Table S1. LTTMs usedfor dissolving proteins in this work
Entry / Abbreviation / Hydrogen-bond acceptor / Hydrogen-bond donor / Molar ratio / Appearance at room temperature1 / ChCl:LA / Choline chloride / Levulinic acid / 1:1 / Solid precipitate after formation
2 / 1:2 / Transparent liquid
3 / ChCl:OA / Choline chloride / Oxalic acid / 1:1 / Transparent liquid
4 / ChCl:MA / Choline chloride / Malonic acid / 1:1 / Faint yellow liquid
5 / ChCl:PAA / Choline chloride / Phenylacetic acid / 1:1 / Solid precipitate after formation
6 / 1:2 / Transparent liquid
7 / ChCl:urea / Choline chloride / Urea / 1:2 / Transparent liquid
8 / ChCl:Gly / Choline chloride / Glycerol / 1:2 / Transparent liquid
9 / ChCl:EG / Choline chloride / Ethylene glycol / 1:2 / Transparent liquid
10 / ChCl:PPD / Choline chloride / 1,2-Propanediol / 1:2 / Transparent liquid
11 / ChCl:Fru / Choline chloride / D-(−)-Fructose / 1:2 / Dark-brown viscous liquid
12 / ChCl:Glu / Choline chloride / D-(+)-Glucose / 1:2 / Brown viscous liquid
13 / ChCl:Xyl / Choline chloride / Xylitol / 1:1 / Transparent viscous liquid
14 / ChCl:Sor / Choline chloride / D-Sorbitol / 1:1 / Faint-yellow viscous liquid
15 / ChCl:Gla / Choline chloride / L-Glutamic acid / 1:2 / Yellow viscous liquid
16 / ChBt:LA / Choline bitartrate / Levulinic acid / 1:2 / Faint-yellow viscous liquid
17 / ChBt:Gla / Choline bitartrate / L-Glutamic acid / 1:2 / Dark-brown viscous liquid
18 / ChBt:urea / Choline bitartrate / Urea / 1:2 / Dark-brown viscous liquid
19 / ChBt:PPD / Choline bitartrate / 1,2-Propanediol / 1:2 / Solidification after formation
20 / ChBt:EG / Choline bitartrate / Ethylene glycol / 1:2 / Solid precipitate after formation
21 / BE:LA / Betaine / Levulinic acid / 1:2 / Transparent liquid
22 / BE:Glu / Betaine / L-Glutamic acid / 1:2 / Faint-yellow viscous liquid
23 / BE:urea / Betaine / Urea / 1:2 / Transparent viscous liquid
24 / BE:PPD / Betaine / 1,2-Propanediol / 1:2 / Solid precipitate after formation
25 / BE:EG / Betaine / Ethylene glycol / 1:2 / Solid precipitate after formation
26 / ZnCl2:PPD / Zinc chloride / 1,2-Propanediol / 1:4 / Transparent liquid
27 / ZnCl2:EG / Zinc chloride / Ethylene glycol / 1:4 / Transparent liquid
28 / TPAB:LA / Trimethylphenylammonium bromide / Levulinic acid / 1:2 / Gray paste
29 / TPAB:EG / Trimethylphenylammonium bromide / Ethylene glycol / 1:2 / Dark-blue paste
30 / TBAB:LA / Tetrabutylammonium bromide / Levulinic acid / 1:2 / Faint-yellow liquid
31 / TBAB:EG / Tetrabutylammonium bromide / Ethylene glycol / 1:2 / Transparent liquid
32 / TBAI:LA / Tetrabutylammonium iodide / Levulinic acid / 1:2 / Brown paste
33 / TBAI:EG / Tetrabutylammonium iodide / Ethylene glycol / 1:2 / Solidification after formation
34 / TEAC:LA / Tetraethylammonium chloride / Levulinic acid / 1:2 / Transparent liquid
35 / TEAC:EG / Tetraethylammonium chloride / Ethylene glycol / 1:2 / Transparent liquid
36 / ChCl:AOE / Choline chloride / 2-(2-Aminoethoxy)ethanol / 1:2 / Solidification after formation
37 / ChCl:CSA / Choline chloride / (−)-Camphor-10-sulfonic acid / 1:2 / Solid precipitate after formation
38 / BE:AOE / Betaine / 2-(2-Aminoethoxy)ethanol / 1:2 / Unable to form
39 / BE:CSA / Betaine / (−)-Camphor-10-sulfonic acid / 1:2 / Solidification after formation
40 / Zn Cl2:AOE / Zinc chloride / 2-(2-Aminoethoxy)ethanol / 1:2 / Faint-yellow cloudy liquid
41 / Zn Cl2:CSA / Zinc chloride / (−)-Camphor-10-sulfonic acid / 1:2 / Black paste
42 / TPAB:AOE / Trimethylphenylammonium bromide / 2-(2-Aminoethoxy)ethanol / 1:2 / Solid precipitate after formation
43 / TPAB:CSA / Trimethylphenylammonium bromide / (−)-Camphor-10-sulfonic acid / 1:2 / Solid precipitate after formation
44 / TBAB:AOE / Tetrabutylammonium bromide / 2-(2-Aminoethoxy)ethanol / 1:2 / Faint-yellow liquid
45 / TBAB:CSA / Tetrabutylammonium bromide / (−)-Camphor-10-sulfonic acid / 1:2 / Dark-brown viscous liquid
46 / TEAC:AOE / Tetraethylammonium chloride / 2-(2-Aminoethoxy)ethanol / 1:2 / Yellow liquid
47 / TEAC:CSA / Tetraethylammonium chloride / (−)-Camphor-10-sulfonic acid / 1:2 / Solid precipitate after formation
Table S2. Preparation of aspirin-containing LTTMs
Entry / Hydrogen-bond acceptor / Hydrogen-bonddonor 1 / Hydrogen-bond
donor 2 / Molar ratio / Appearance at room temperature
1 / Choline chloride / Aspirin / None / 1:1 / Viscous transparent liquid
2 / 1:2 / Viscous transparent liquid
3 / 1:3 / Viscous transparent liquid
4 / 2:1 / Unable to form a liquid
5 / Choline bitartrate / Aspirin / None / 1:2 / Unable to form a liquid
6 / Betaine / Aspirin / None / 1:2 / Viscous transparent liquid
7 / L-Histidine / Aspirin / None / 1:2 / Unable to form a liquid
8 / Glycine / Aspirin / None / 1:2 / Unable to form a liquid
9 / L-Proline / Aspirin / None / 1:2 / Unable to form a liquid
10 / L-Glutamic acid / Aspirin / None / 1:2 / Unable to form a liquid
11 / D-Glucose / Aspirin / None / 1:1 / Unable to form a liquid
12 / Choline chloride / Aspirin / L-Proline / 1:1:1 / Viscous transparent liquid
13 / Betaine / Aspirin / L-Proline / 1:1:1 / Viscous transparent liquid
14 / Choline chloride / Aspirin / D-Glucose / 1:1:1 / Unable to form a liquid
15 / Choline chloride / Aspirin / Tartaric acid / 1:1:1 / Unable to form a liquid
3:1:1 / Viscous transparent liquid
16 / Choline chloride / Aspirin / Malic acid / 1:1:1 / Unable to form a liquid
2:1:1 / Viscous transparent liquid
17 / Choline chloride / Aspirin / Citric acid / 1:1:1 / Viscous transparent liquid
2:1:1 / Unable to form a liquid
18 / Choline chloride / Aspirin / Urea / 1:1:1 / Unable to form a liquid
2:1:1 / Viscous transparent liquid
19 / Choline chloride / Aspirin / Glycine / 1:1:1 / Unable to form a liquid
2:1:1 / Unable to form a liquid
20 / Choline chloride / Aspirin / Xylitol / 1:1:1 / Transparent liquid
21 / 2:1:0.5 / Unable to form a liquid
22 / 2:1:0.65 / Initially formed a liquid, 48 h later cloudy solidified
23 / 2:1:0.75 / Initially formed a liquid, 5 days later mostly solidified
24 / 2:1:1 / Transparent liquid
25 / 2:1:1.5 / Initially formed a liquid, 5 days later solidified
26 / 2:1:1.75 / Initially formed a liquid, 4 days later solidified
27 / 2:1:2 / Initially formed a liquid, 5 h later started to turn solidified
28 / 2:1:3 / Initially formed a liquid, 24 h later solidified
29 / 2:1:4 / Initially formed a liquid, 24 h later solidified
30 / 2:1:5 / Initially formed a liquid, 24 h later solidified
31 / 3:1:1 / Unable to form a liquid
32 / Choline chloride / Aspirin / D-Mannitol / 2:1:0.25 / Unable to form a liquid
33 / 2:1:0.5 / Unable to form a liquid
34 / 2:1: 0.75 / Initially formed a liquid, which 5 days later became very viscous
35 / 2:1:1 / Initially formed a liquid, 11 days later white crystals forming
36 / 2:1:1.25 / Unable to form a liquid
37 / 2:1:1.5 / Initially formed a liquid, completely solid 5 days later
38 / 2:1:1.75 / Initially formed a liquid, solid 5 days later
39 / 2:1:2 / Initially formed a liquid, solidified 24 h later
40 / 2:1:3 / Unable to form a liquid
41 / Choline chloride / Aspirin / D-Sorbitol / 2:1:0.5 / Unable to form a liquid
42 / 2:1:0.65 / Initially formed a liquid, started solidifying 3 days later
43 / 2:1:0.75 / Initially formed a very viscous liquid, solid started forming on one side 5 days later
44 / 2:1:1 / Initially formed a liquid, but turned into a white solid in 12 days
45 / 2:1:1.25 / Formed an extremely viscous liquid, white crystals began forming on the surface 3 days later
46 / 2:1:1.5 / Initially formed a liquid, 48 h later completely solid
47 / 2:1:2 / Initially formed a liquid, after 10 days completely solid
48 / 2:1:2.25 / Initially formed a viscous liquid, began solidifying 3 h later
49 / 2:1:2.5 / Initially formed a liquid, within 18 h formed numerous white crystals
50 / 2:1:3 / Initially formed a liquid, completely solidified in 4 days
Figure S1. Attempts to correlate the lysozyme solubility in LTTMs with the latter’s characteristics. (a) ENR, (b) β, (c) π*, (d) viscosity, and (e) pH. The dependences obtained for BSA, CTgen, and subtilisin were qualitatively similar to those for lysozyme and equally devoid of any discernible correlations.
References
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