Name: solubility of hydrophobic compounds in aqueous solution using combinations of self-assembling peptide and amino acid
Uploaded: 2017-09-20T14:00:00.000+00:00
Duration: 5 min 8 s
Description: Watch this Scientific Journal Video about Solubility of Hydrophobic Compounds in Aqueous Solution Using Combinations of Self-assembling Peptide and Amino Acid at JoVE.com

This work is supported by Canadian Institutes of Health Research, operating grants MOP-42546 and MOP-119514.

1. Preparation of Amino Acid Solutions
<ol>
	<li>Prepare and label two 50 mL conical centrifuge tubes for each amino acid (one each for both &#34;low&#34; and &#34;high&#34; concentrations).</li>
	<li>Prepare a large 2 L flask containing purified water (18.2 M&#937;&#183;cm at 25 &#176;C).</li>
	<li>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 &#34;High&#34; 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.</li>
	<li>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).</li>
</ol>
2. Preparation of SAP-AA Solutions
<ol>
	<li>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).</li>
	<li>Using a high-performance analytical balance (with a readability down to 0.1 mg or less), weigh approximately 1 &#177; 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.</li>
	<li>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).</li>
	<li>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.</li>
</ol>
3. Preparation of Drug-DMSO or Drug-Ethanol Stock Solutions
<ol>
	<li>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 &#181;L of DMSO and 400 &#181;L 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 &#8211; in this case, more than 1 mg of drug would be used. The stock can be stored at -20 &#176;C; thaw on ice and vortex before use.</li>
	<li>Vortex vials for 15 s to completely dissolve the drug.</li>
</ol>
4. Preparation of Drug Formulations
<ol>
	<li>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.</li>
	<li>Add 10 &#181;L of Drug-DMSO stock, or 20 &#181;L Drug-Ethanol stock to appropriate microcentrifuge tubes.</li>
	<li>Add 990 &#181;L of SAP-AA acid solutions to the appropriate labeled microcentrifuge tubes containing Drug-DMSO stock, and 980 &#181;L 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)</li>
	<li>Vortex vigorously for 30 s and allow formulations to rest for 30 min.</li>
</ol>
5. Solubility Testing
<ol>
	<li>After rest period, vortex vigorously once again for 30 s.</li>
	<li>Centrifuge the formulations at 14,220 x g for 1 min.</li>
	<li>Analyze the bottom of the microcentrifuge tubes for precipitation (by visualization).</li>
</ol>

<ol>
	<li>Holmes, T. C., de Lacalle, S., Su, X., Liu, G., Rich, A., Zhang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+neurite+outgrowth+and+active+synapse+formation+on+self-assembling+peptide+scaffolds.">Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds.</a> Proc. Natl. Acad. Sci. U. S. A. 97 (12), 6728-6733 (2000).</li><li>Davis, M. E., Motion, J. P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+self-assembling+peptide+nanofibers+create+intramyocardial+microenvironments+for+endothelial+cells.">Injectable self-assembling peptide nanofibers create intramyocardial microenvironments for endothelial cells.</a> Circulation. 111 (4), 442-450 (2005).</li><li>Matson, J. B., Stupp, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembling+peptide+scaffolds+for+regenerative+medicine.">Self-assembling peptide scaffolds for regenerative medicine.</a> Chem. Commun. 48 (1), 26-33 (2012).</li><li>Tatman, P. D., Muhonen, E. G., Wickers, S. T., Gee, A. O., Kim, E., Kim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembling+peptides+for+stem+cell+and+tissue+engineering.">Self-assembling peptides for stem cell and tissue engineering.</a> Biomater. Sci. 4 (4), 543-554 (2016).</li><li>Keyes-Baig, C., Duhamel, J., Fung, S. -. Y., Bezaire, J., Chen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembling+peptide+as+a+potential+carrier+of+hydrophobic+compounds.">Self-assembling peptide as a potential carrier of hydrophobic compounds.</a> J. Am. Chem. Soc. 126 (24), 7522-7532 (2004).</li><li>Kumar, P., Pillay, V., Modi, G., Choonara, Y. E., du Toit, L. C., Naidoo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembling+peptides:+implications+for+patenting+in+drug+delivery+and+tissue+engineering.">Self-assembling peptides: implications for patenting in drug delivery and tissue engineering.</a> Recent Pat. Drug Deliv. Formul. 5 (1), 24-51 (2011).</li><li>Wang, H., Yang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-peptide-based+molecular+hydrogels:+novel+gelation+strategies+and+applications+for+tissue+engineering+and+drug+delivery.">Short-peptide-based molecular hydrogels: novel gelation strategies and applications for tissue engineering and drug delivery.</a> Nanoscale. 4, 5259-5267 (2012).</li><li>French, K. M., Somasuntharam, I., Davis, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembling+peptide-based+delivery+of+therapeutics+for+myocardial+infarction.">Self-assembling peptide-based delivery of therapeutics for myocardial infarction.</a> Adv. Drug Deliv. Rev. 96, 40-53 (2016).</li><li>Zhang, S., Holmes, T., Lockshin, C., Rich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+assembly+of+a+self-complementary+oligopeptide+to+form+a+stable+macroscopic+membrane.">Spontaneous assembly of a self-complementary oligopeptide to form a stable macroscopic membrane.</a> Proc. Natl. Acad. Sci. U. S. A. 90 (8), 3334-3338 (1993).</li><li>Bowerman, C. J., Nilsson, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembly+of+amphipathic+&#946;-sheet+peptides:+insights+and+applications.">Self-assembly of amphipathic &#946;-sheet peptides: insights and applications.</a> Biopolymers. 98 (3), 169-184 (2012).</li><li>Amidon, G., Lennern&#228;s, H., Shah, V., Crison, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+theoretical+basis+for+a+biopharmaceutic+drug+classification:+the+correlation+of+in+vitro+drug+product+dissolution+and+in+vivo+bioavailability.">A theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability.</a> Pharm. Res. 12 (3), 413-420 (1995).</li><li>Shi, Y., Porter, W., Merdan, T., Li, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+intravenous+delivery+of+poorly+water-soluble+compounds.">Recent advances in intravenous delivery of poorly water-soluble compounds.</a> Expert Opin. Drug Deliv. 6 (12), 1261-1282 (2009).</li><li>Bawa, R., Fung, S. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembling+peptide-based+nanoparticles+enhance+cellular+delivery+of+the+hydrophobic+anticancer+drug+ellipticine+through+caveolae-dependent+endocytosis.">Self-assembling peptide-based nanoparticles enhance cellular delivery of the hydrophobic anticancer drug ellipticine through caveolae-dependent endocytosis.</a> Nanomedicine. 8 (5), 647-654 (2012).</li><li>Liu, J., Zhang, L., Yang, Z., Zhao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+release+of+paclitaxel+from+a+self-assembling+peptide+hydrogel+formed+in+situ+and+antitumor+study+in+vitro.">Controlled release of paclitaxel from a self-assembling peptide hydrogel formed in situ and antitumor study in vitro.</a> Int. J. Nanomed. 6, 2143-2153 (2011).</li><li>Wu, Y., Sadatmousavi, P., Wang, R., Lu, S., Yuan, Y., Chen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembling+peptide-based+nanoparticles+enhance+anticancer+effect+of+ellipticine+in+vitro+and+in+vivo.">Self-assembling peptide-based nanoparticles enhance anticancer effect of ellipticine in vitro and in vivo.</a> Int. J. Nanomed. 7, 3221-3233 (2012).</li><li>Fung, S. Y., Yang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-Assembling+Peptide+as+a+Potential+Carrier+for+Hydrophobic+Anticancer+Drug+Ellipticine:+Complexation,+Release+and+In+Vitro+Delivery.">Self-Assembling Peptide as a Potential Carrier for Hydrophobic Anticancer Drug Ellipticine: Complexation, Release and In Vitro Delivery.</a> Adv. Funct. Mater. 19 (1), 74-83 (2009).</li><li>Fung, S. -. Y., Oyaizu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+nanoscale+combinations+of+self-assembling+peptides+and+amino+acids+of+the+Src+tyrosine+kinase+inhibitor+in+acute+lung+injury+therapy.">The potential of nanoscale combinations of self-assembling peptides and amino acids of the Src tyrosine kinase inhibitor in acute lung injury therapy.</a> Biomaterials. 32 (16), 4000-4008 (2011).</li><li>Pacheco, S., Kanou, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formulation+of+hydrophobic+therapeutics+with+self-assembling+peptide+and+amino+acid:+A+new+platform+for+intravenous+drug+delivery.">Formulation of hydrophobic therapeutics with self-assembling peptide and amino acid: A new platform for intravenous drug delivery.</a> J. Control. Release. 239, 211-222 (2016).</li></ol>

The authors have nothing to disclose.

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...

SAPs are a class of biomaterials that have been studied extensively as 3D scaffolds in regenerative medicine1,2,3,4. More recently however, they have been exploited as vehicles for delivery of therapeutics due to their unique biological properties5,6,7,8. 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-assembly9,10 and allowing them to serve as a medium between hydrophobic and hydrophilic environments. Consequently, for the clinical delivery of hydrophobic drugs &#8211; which have extremely low bioavailability and absorption in the body due to lack of solubility in aqueous environments11,12&#160; &#8211; 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 settings13,14,15,16. 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 required17,18.
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 &#8211; 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 &#8211; 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.

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

solubility of hydrophobic compounds in aqueous solution using combinations of self-assembling peptide and amino acid

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 &#8211; PP2, rottlerin, and curcumin &#8211; 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.

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...

Watch this Scientific Journal Video about Solubility of Hydrophobic Compounds in Aqueous Solution Using Combinations of Self-assembling Peptide and Amino Acid at JoVE.com

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

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.

Self-assembling peptides (SAPs) are promising vehicles for the delivery of hydrophobic therapeutics for clinical applications; their amphipathic ...

solubility-hydrophobic-compounds-aqueous-solution-using-combinations

Research

Education

JoVE Journal

JoVE Core

Organic Chemistry

Medicine

Unit 1

Covalent Bonding and Structure

SCIENTISTS IN ACTION

16.7K Views.  University Health Network. Dit protocol beschrijft een klinisch-middelen uit het oplossen van de hydrofobe bestanddelen in een waterige omgeving met behulp van combinaties van het zelf monteren van peptide en aminozuur oplossingen. Onze methode is opgelost een belangrijke beperking van hydrofobe therapeutics, die veilig en efficiënt vervoermiddel oplosbaarheid en levering methoden in klinische instellingen.

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

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Chemistry

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and fold into higher order nanostructures. Structurally similar to peptides, peptoids are poly N-substituted glycines, where the side chains are attached to the nitrogen rather than the alpha-carbon. Their ease of synthesis and structural diversity allows testing of basic design principles to drive de novo design and engineering of new biologically-active and nanostructured materials.
Here, a simple manual peptoid synthesis protocol is presented that allows the synthesis of long chain polypeptoids ( up to 50mers) in excellent yields. Only basic equipment, simple techniques (e.g. liquid transfer, filtration), and commercially available reagents are required, making peptoids an accessible addition to many researchers' toolkits. The peptoid backbone is grown one monomer at a time via the submonomer method which consists of a two-step monomer addition cycle: acylation and displacement. First, bromoacetic acid activated in situ with N,N'-diisopropylcarbodiimide acylates a resin-bound secondary amine. Second, nucleophilic displacement of the bromide by a primary amine follows to introduce the side chain. The two-step cycle is iterated until the desired chain length is reached. The coupling efficiency of this two-step cycle routinely exceeds 98% and enables the synthesis of peptoids as long as 50 residues. Highly tunable, precise and chemically diverse sequences are achievable with the submonomer method as hundreds of readily available primary amines can be directly incorporated.
 
Peptoids are emerging as a versatile biomimetic material for nanobioscience research because of their synthetic flexibility, robustness, and ordering at the atomic level. The folding of a single-chain, amphiphilic, information-rich polypeptoid into a highly-ordered nanosheet was recently demonstrated. This peptoid is a 36-mer that consists of only three different commercially available monomers: hydrophobic, cationic and anionic. The hydrophobic phenylethyl side chains are buried in the nanosheet core whereas the ionic amine and carboxyl side chains align on the hydrophilic faces. The peptoid nanosheets serve as a potential platform for membrane mimetics, protein mimetics, device fabrication, and sensors. Methods for peptoid synthesis, sheet formation, and microscopy imaging are described and provide a simple method to enable future peptoid nanosheet designs.

A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and ...

Bioengineering

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles (PPAs) and Related Biomaterials

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). Traditional PAs possess charged amino acids as solubilizing groups (lysine, arginine), which are directly connected to a lipid segment or can contain a linker region made of neutral amino acids. Tuning the peptide sequence of PAs can yield diverse morphologies. Similarly, PPAs possess a hydrophobic segment and neutral amino acids, but also contain polyamine molecules as water solubilizing (hydrophilic) groups. As is the case with PAs, PPAs can also self-assemble into diverse morphologies, including small rods, twisted nano-ribbons, and fused nano-sheets, when dissolved in water. However, the presence of both primary and secondary amines on a single polyamine molecule poses a significant challenge when synthesizing PPAs. In this paper, we show a simple protocol, based on literature precedents, to achieve a facile synthesis of PPAs using solid phase peptide synthesis (SPPS). This protocol can be extended to the synthesis of PAs and other similar systems. We also illustrate the steps that are needed for cleavage from the resin, identification, and purification.

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles PPAs and Related Biomaterials

The synthesis of polyamine-based peptide amphiphiles (PPAs) is a significant challenge due to the presence of multiple amine nitrogens, which requires judicious use of protecting groups to mask these reactive functionalities. In this paper, we describe a facile method for the preparation of these new class of self-assembling molecules.

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). ...

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting self-assembled supramolecular structures. The synthetic route takes advantage of solid-phase peptide synthesis with the on-resin coupling of the dithiolane precursor molecule, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the microwave-assisted thioacetate deprotection of the peptide N-terminus before final cleavage from the resin to yield the 1,2-dithiolane modified peptide. After the high-performance liquid chromatography (HPLC) purification of the 1,2-dithiolane peptide, derived from the nucleating core of the A&#946; peptide associated with Alzheimer&#39;s disease, the peptide is shown to self-assemble into cross-&#946; amyloid fibers. Protocols to characterize the amyloid fibers by Fourier-transform infrared spectroscopy (FT-IR), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM) are presented. The methods of N-terminal modification with a 1,2-dithiolane moiety to well-characterized self-assembling peptides can now be explored as model systems to develop post-assembly modification strategies and explore dynamic covalent chemistry on supramolecular peptide nanofiber surfaces.

A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting ...

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded oligomeric strands mediated by paired dynamic covalent interactions in a manner mimicking the thermal cycling commonly employed for the self-assembly of complementary nucleic acid sequences. Primary amine monomers bearing aldehyde and amine pendant moieties are functionalized with orthogonal protecting groups for use as dynamic covalent reactant pairs. Using a modified automated peptide synthesizer, the primary amine monomers are encoded into oligo(peptoid) strands through solid-phase submonomer synthesis. Upon purification by high-performance liquid chromatography (HPLC) and characterization by electrospray ionization mass spectrometry (ESI-MS), sequence-specific oligomers are subjected to high-loading of a Lewis acidic rare-earth metal triflate which both deprotects the aldehyde moieties and affects the reactant pair equilibrium such that strands completely dissociate. Subsequently, a fraction of the Lewis acid is extracted, enabling annealing of complementary sequence-specific strands to form information-encoded molecular ladders characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The simple procedure outlined in this report circumvents kinetic traps commonly experienced in the field of dynamic covalent assembly and serves as a platform for the future design of robust, complex architectures.

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded ...

Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers can be readily functionalized and have been used in various applications such as bio-sensors and bio-devices. In this study, we introduced a planar organic molecule: copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) to dope lipid membranes. The CuPc/lipid hybrid membrane forms at the water/air interface by self-assembly. In this membrane, the hydrophobic CuPc molecules are located between the hydrophobic tails of lipid molecules, forming a lipid/CuPc/lipid sandwich structure. Interestingly, an air-stable hybrid lipid bilayer can be readily formed by transferring the hybrid membrane onto a Si substrate. We report a straightforward method for incorporating nanomaterials into a lipid bilayer system, which represents a new methodology for the fabrication of biosensors and biodevices.

We report a protocol for producing a hybrid lipid membrane at the water/air interface by doping the lipid bilayer with copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) molecules. The resulting hybrid lipid membrane has a lipid/CuPc/lipid sandwich structure. This protocol can also be applied to the formation of other functional nanomaterials.

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers ...

 Peptide Hydrogels: Methods for Environmentally Responsive Gelation of ECF-5

Preparation of Mechanically Stable Self-Assembled Peptides Hydrogels

Peptide hydrogels are highly hydrophilic, three-dimensional network gels formed by the self-assembly of nanofibers or polymers, creating water-locking networks. Their morphology closely resembles that of the extracellular matrix, allowing them to exhibit both the biological functions of peptides and responsive gelation properties. These unique characteristics have led to their extensive application in tissue engineering, three-dimensional cell culture, cancer therapy, regenerative medicine, and other biomedical fields. This article describes three methods for preparing ECF-5 peptide hydrogels using self-assembling peptides with environmentally responsive gelation processes: (1) pH-responsive gelation: varying pH levels induce the protonation or deprotonation of amino acid residues, altering electrostatic interactions between peptide molecules and promoting their self-assembly into hydrogels; (2) Metal ion addition: polyvalent metal ions chelate with negatively charged amino acid residues, acting as bridges between peptides to form a network hydrogel; (3) Solvent exchange: hydrophobic peptides are initially dissolved in non-polar organic solvents and subsequently induce self-assembly into hydrogels upon transitioning to a polar aqueous environment. These methods utilize conventional experimental procedures to facilitate peptide self-assembly into hydrogels. By designing peptide sequences to align with specific gelation-inducing conditions, it is possible to achieve finely tuned micro/nanostructures and biological functions, highlighting the significant potential of peptide hydrogels in the biomedical domain.

Author Spotlight: Improving the Production of Self-Assembling Fibers and Peptide Hydrogels for Superior Biocompatibility

This protocol presents three fast and simple preparation methods that use environmental conditions to trigger the self-assembly of peptides into hydrogels. Additionally, the characterization of peptide hydrogels is described, demonstrating that mechanically stable peptide hydrogels can be formed under these straightforward conditions.

Peptide hydrogels are highly hydrophilic, three-dimensional network gels formed by the self-assembly of nanofibers or polymers, creating water-locking ...

Biochemistry

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated media. In this article, we describe detailed protocols that enable the construction of nanofiber architectures in organic solvent mixtures through the self-assembly of uniquely addressable, single-stranded, gamma-modified peptide nucleic acid (&#947;PNA) tiles. Each single-stranded tile (SST) is a 12-base &#947;PNA oligomer composed of two concatenated modular domains of 6 bases each. Each domain can bind to a mutually complimentary domain present on neighboring strands using programmed complementarity to form nanofibers that can grow to microns in length. The SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers. In contrast with analogous DNA nanostructures, which form diameter-monodisperse structures, these &#947;PNA systems form nanofibers that bundle along their widths during self-assembly in organic solvent mixtures. Self-assembly protocols described here therefore also include a conventional surfactant, Sodium Dodecyl Sulfate (SDS), to reduce bundling effects.

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated ...

Evaluating Biofunctional Self-Assembling Peptides for Cell Adhesion and Morphology

Fabrication and Characterization of Colorectal Cancer Organoids from SW1222 Cell Line in Ultrashort Self&#45;Assembling Peptide Matrix

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of hydrogels that are biocompatible, biodegradable, and non-immunogenic. We have previously proven that SAPs, when biofunctionalized with protein-derived motifs, can mimic the extracellular matrix characteristics that support colorectal organoid formation. These biofunctional peptide hydrogels retain the original parent peptide's mechanical properties, tunability, and printability while incorporating cues that allow cell-matrix interactions to increase cell adhesion. This paper presents the protocols needed to evaluate and characterize the effects of various biofunctional peptide hydrogels on cell adhesion and lumen formation using an adenocarcinoma cancer cell line able to form colorectal cancer organoids cost-effectively. These protocols will help evaluate biofunctional peptide hydrogel effects on cell adhesion and luminal formation using immunostaining and fluorescence image analysis. The cell line used in this study has been previously utilized for generating organoids in animal-derived matrices.

Author Spotlight: Optimization of Ultrashort Peptide Matrices for Colorectal Cancer Organoids

This protocol aims to evaluate biofunctional self-assembling peptides for cell adhesion, organoid morphology, and gene expression by immunostaining. We will use a colorectal cancer cell line to provide a cost-effective way of obtaining organoids for intensive testing.

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video