Solubility of Hydrophobic Compounds in Aqueous Solution Using Combinations of Self-assembling Peptide and Amino Acid

Summary

This protocol describes a clinically-applicable means of dissolving hydrophobic compounds in an aqueous environment using combinations of self-assembling peptide and amino acid solutions. Our method resolves a major limitation of hydrophobic therapeutics, which lack safe, efficient means of solubility and delivery methods into clinical settings.

Abstract

Self-assembling peptides (SAPs) are promising vehicles for the delivery of hydrophobic therapeutics for clinical applications; their amphipathic properties allow them to dissolve hydrophobic compounds in the aqueous environment of the human body. However, self-assembling peptide solutions have poor blood compatibility (e.g., low osmolarity), hindering their clinical application through intravenous administrations. We have recently developed a generalized platform for hydrophobic drug delivery, which combines SAPs with amino acid solutions (SAP-AA) to enhance drug solubility and increase formulation osmolarity to reach the requirements for clinical uses. This formulation strategy was thoroughly tested in the context of three structurally different hydrophobic compounds – PP2, rottlerin, and curcumin – in order to demonstrate its versatility. Furthermore, we examined effects of changing formulation components by analyzing 6 different SAPs, 20 naturally existing amino acids at low and high concentrations, and two different co-solvents dimethyl sulfoxide (DMSO) and ethanol. Our strategy proved to be effective in optimizing components for a given hydrophobic drug, and therapeutic function of the formulated inhibitor, PP2, was observed both in vitro and in vivo. This manuscript outlines our generalized formulation method using SAP-AA combinations for hydrophobic compounds, and analysis of solubility as a first step towards potential use of these formulations in more functional studies. We include representative solubility results for formulation of the hydrophobic compound, curcumin, and discuss how our methodology serves as a platform for future biological studies and disease models.

Introduction

SAPs are a class of biomaterials that have been studied extensively as 3D scaffolds in regenerative medicine^1,2,3,4. More recently however, they have been exploited as vehicles for delivery of therapeutics due to their unique biological properties^5,6,7,8. SAPs naturally assemble into stable nanostructures⁹, thus providing a means of drug encapsulation and protection. SAPs are amphipathic, co....

Protocol

1. Preparation of Amino Acid Solutions

1. Prepare and label two 50 mL conical centrifuge tubes for each amino acid (one each for both "low" and "high" concentrations).
2. Prepare a large 2 L flask containing purified water (18.2 MΩ·cm at 25 °C).
3. Calculate the amount of each amino acid (in grams) to reach the desired concentrations, and weigh the appropriate amount of amino acid into their respective 50 mL centrifuge tubes using a spatula.
   NOTE: For the "High" concentration of the two negatively charged amino acids, PBS is used instead of water. We could not increase their concentrations due to the....

Results

For the hydrophobic drug, curcumin, we produced formulations using all 20 naturally existing amino acids at low concentrations, in combination with only one SAP, EAK16-II, as a proof-of-principle. We also tested formulations using both DMSO and ethanol as co-solvents. In total, this produced 40 curcumin formulations, each containing different components. It is important to note that, in our previous studies using the Src inhibitor, PP2, we included more options for SAP (total of 6) and am.......

Discussion

In the formulation procedure, there are various critical steps and points to consider in troubleshooting. First, as we are working with various components and concentrations, multiple vortex steps throughout the protocol ensure that all concentrations are uniform and correct. Some of the high-concentration, hydrophobic amino acid solutions may still not be completely dissolved after vortexing, and in this case, they can be shaken vigorously by hand to assist in the process. Likewise, it is essential that SAP-AA solutions.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
EAK16-I	CanPeptide Inc.	Custom peptide	Sequence: AEAKAEAKAEAKAEAK, N-terminus acetylation and C-terminus amidation, >95% pure by HPLC
EAK16-II	CanPeptide Inc.	Custom peptide	Sequence: AEAEAKAKAEAEAKAK, N-terminus acetylation and C-terminus amidation, >95% pure by HPLC
EAK16-IV	CanPeptide Inc.	Custom peptide	Sequence: AEAEAEAEAKAKAKAK, N-terminus acetylation and C-terminus amidation, >95% pure by HPLC
EFK8-II	CanPeptide Inc.	Custom peptide	Sequence: FEFEFKFK, N-terminus acetylation and C-terminus amidation, >95% pure by HPLC
A6KE	CanPeptide Inc.	Custom peptide	Sequence: AAAAAAKE, N-terminus acetylation and C-terminus amidation, >95% pure by HPLC
P6KE	CanPeptide Inc.	Custom peptide	Sequence: PPPPPPPKE, N-terminus acetylation and C-terminus amidation, >95% pure by HPLC
Alanine	Sigma-Aldrich	A7469-100G	L-Alanine
Isoleucine	Sigma-Aldrich	I7403-100G	L-Isoleucine
Leucine	Sigma-Aldrich	L8912-100G	L-Leucine
Methionine	Sigma-Aldrich	M5308-100G	L-Methionine
Proline	Sigma-Aldrich	P5607-100G	L-Proline
Valine	Sigma-Aldrich	V0513-100G	L-Valine
Phenylalanine	Sigma-Aldrich	P5482-100G	L-Phenylalanine
Tryptophan	Sigma-Aldrich	T8941-100G	L-Tryptophan
Tyrosine	Sigma-Aldrich	T8566-100G	L-Tyrosine
Glycine	Sigma-Aldrich	G8790-100G	L-Glycine
Asparagine	Sigma-Aldrich	A4159-100G	L-Asparagine
Glutamine	Sigma-Aldrich	G8540-100G	L-Glutamine
Serine	Sigma-Aldrich	A7219-100G	L-Serine
Threonine	Sigma-Aldrich	T8441-100G	L-Threonine
Histidine	Sigma-Aldrich	H6034-100G	L-Histidine
Lysine	Sigma-Aldrich	L5501-100G	L-Lysine
Arginine	Sigma-Aldrich	A8094-100G	L-Arginine
Aspartic Acid	Sigma-Aldrich	A7219-100G	L-Aspartic Acid
Glutamic Acid	Sigma-Aldrich	G8415-100G	L-Glutamic Acid
Cysteine	Sigma-Aldrich	C7352-100G	L-Cysteine
Dimethyl Sulfoxide	Sigma-Aldrich	D4540-500ML	DMSO
Ethanol	Sigma-Aldrich	277649-100ML	Anhydrous
Curcumin	Sigma-Aldrich	08511-10MG	Hydrophobic drug, curcumin
Rottlerin	EMD Millipore	557370-10MG	Hydrophobic drug, rottlerin
PP2	Enzo	BML-EI297-0001	Hydrophobic drug, PP2
Scintillation Vials	VWR	2650-66022-081	Borosilicate Glass, with Screw Cap, 20 mL. Vials for weighing peptide.
Falcon 50 mL Conical Centrifugation Tubes	VWR	352070	Polypropylene, Sterile, 50 mL. For amino acid solutions.