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, comprised of a specific pattern of hydrophobic and hydrophilic amino acid repeats, driving their self-assembly^9,10 and allowing them to serve as a medium between hydrophobic and hydrophilic environments. Consequently, for the clinical delivery of hydrophobic drugs – which have extremely low bioavailability and absorption in the body due to lack of solubility in aqueous environments^11,12  – SAPs are promising as a delivery vehicle. Furthermore, their sequence pattern also implies that SAPs can be rationally designed and engineered to maximize compatibility with any given drug or compound (i.e., based on functional groups) and further assist solubility.

SAPs have been applied as effective drug delivery vehicles in many in vitro and in vivo settings^13,14,15,16. They have also shown great safety and biocompatibility. However, due to low osmolarity of SAP-drug preparations, they cannot be used for intravenous injections as in clinical settings¹³. Considering this restraint, we have recently developed a strategy which combines SAPs with amino acid solutions in order to reduce the use of toxic co-solvents and increase the formulation osmolarity, and therefore, clinical relevance. We chose to use amino acids as they are the building blocks of SAPs, are already clinically-accepted, and in combination with SAPs, they increase hydrophobic drug solubility while reducing the amount of SAP required^17,18.

We have scrutinized SAP-AA combinations as a generalized platform for hydrophobic drug solubility and subsequent delivery by creating a multi-step screening pipeline and applying it to the Src inhibitor, PP2, as a model hydrophobic compound. In this process, we examined the effect of changing components of the formulation – ultimately testing 6 different SAPs, all 20 amino acids at 2 different concentrations (low and high; low based on concentrations in existing clinical applications, and high concentrations were 2x, 3x, or 5x the clinical concentration based on the maximum solubility of each amino acid in water), and 2 different co-solvents – and selected combinations that solubilized PP2 for further analysis. This drug formulation proved to be effective as a drug delivery vehicle in cell culture, as well as in vivo models using both intratracheal and intravenous administrations. Likewise, our work touched on the versatility of SAP-AA combinations in solubilizing multiple, structurally-different hydrophobic compounds in aqueous environments; specifically, the drugs rottlerin and curcumin¹⁸. This manuscript outlines the SAP-AA formulation method and analysis of curcumin solubility as an example of the primary step in our screening pipeline. This protocol provides a simple, reproducible way to screen for the optimal SAP-AA combinations, which dissolve any given hydrophobic compound.

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 their low water solubility, and using PBS instead of water helps to maintain the low pH. Furthermore, the concentration calculations were obtained using a final volume of 40 mL for each amino acid solution. All amino acid concentrations are outlined in Table 3. Be sure to rinse the spatula in between amino acids to avoid contamination. We recommend a water rinse, followed by wiping with 70% ethanol.
4. Add 40 mL of purified water (or PBS) into each 50 mL tube using a serological pipette. Cap tubes and vortex or shake vigorously until dissolved. Water bath sonication (room temperature, 130 W, 40 kHz) can also be used to assist in the solubility process.
   NOTE: The following amino acid solutions are sensitive to light and should be covered with aluminum foil: tryptophan, phenylalanine, and tyrosine (which consist of aromatic ring-like structures) and cysteine (reactive -SH group).

2. Preparation of SAP-AA Solutions

1. Prepare 20 mL scintillation vials for the self-assembling peptides. For a given self-assembling peptide, prepare one vial per prepared amino acid solution (each combination will be made in a separate vial).
2. Using a high-performance analytical balance (with a readability down to 0.1 mg or less), weigh approximately 1 ± 0.2 mg of peptide into the bottom of each vial. Cap after weighing and record the exact weight of the peptide on the cap.
3. Pipette the appropriate volume of amino acid solution (prepared in Section 1) into each vial containing peptide, in order to reach the desired concentration of self-assembling peptide (0.1 mg/mL for long peptides with a length of 16 amino acids, or 0.2 mg/mL for shorter peptides with a length of 8 amino acids).
4. Sonicate for 10 min in a water bath sonicator (130 W, 40 kHz) at room temperature, ensuring the solutions within vials are completely immersed in the water bath.

3. Preparation of Drug-DMSO or Drug-Ethanol Stock Solutions

1. Combine 1 mg of drug (in this case, curcumin) with 100% DMSO, and another 1 mg with 100% ethanol to create two stock solutions.
   NOTE: We added 200 µL of DMSO and 400 µL ethanol to make DMSO-curcumin and ethanol-curcumin stocks that were 5 mg/mL and 2.5 mg/mL, respectively, due to varying solubility in each solvent; however, it is important to note that the concentration of stock should be adjusted depending on the hydrophobic drug of interest. Factors such as drug solubility and effective biological concentration are important in determining this value. Also, keep in mind that the stock will be diluted 100-fold and 50-fold in DMSO and ethanol formulations, respectively, when combined with SAP-AA solutions (see Section 4). It may be preferred to prepare a larger volume of stock depending on the number of formulations required – in this case, more than 1 mg of drug would be used. The stock can be stored at -20 °C; thaw on ice and vortex before use.
2. Vortex vials for 15 s to completely dissolve the drug.

4. Preparation of Drug Formulations

1. Prepare clear, 1.5 mL microcentrifuge tubes for each formulation. Be sure to label tubes with the intended self-assembling peptide, amino acid (and concentration), and co-solvent.
2. Add 10 µL of Drug-DMSO stock, or 20 µL Drug-Ethanol stock to appropriate microcentrifuge tubes.
3. Add 990 µL of SAP-AA acid solutions to the appropriate labeled microcentrifuge tubes containing Drug-DMSO stock, and 980 µL to those containing Drug-Ethanol stock. This produces 1 mL drug formulations with 1% DMSO or 2% ethanol.
   NOTE: The final concentration of all curcumin formulations was 0.5 mg/mL according to the protocol. Again, this will vary when using other hydrophobic compounds and/or beginning with a different stock concentration (see step 3.1)
4. Vortex vigorously for 30 s and allow formulations to rest for 30 min.

5. Solubility Testing

1. After rest period, vortex vigorously once again for 30 s.
2. Centrifuge the formulations at 14,220 x g for 1 min.
3. Analyze the bottom of the microcentrifuge tubes for precipitation (by visualization).

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

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.