Name: solubility of hydrophobic compounds in aqueous solution using combinations of self-assembling peptide and amino acid
Uploaded: 2017-09-20T14:00:00
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;...

<ol>
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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. 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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. 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Release. 239, 211-222 (2016).</li></ol>

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 style="border-collapse:collapse;width:1034pt" width="1379">
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">EAK16-I</td>
			<td class="xl17" style="width:106pt" width="141">CanPeptide Inc.</td>
			<td class="xl17" style="width:101pt" width="135">Custom peptide</td>
			<td class="xl20" style="width:510pt" width="680">Sequence: AEAKAEAKAEAKAEAK, N-terminus acetylation and C-terminus amidation, &#62;95% pure by HPLC</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">EAK16-II</td>
			<td class="xl17" style="width:106pt" width="141">CanPeptide Inc.</td>
			<td class="xl17" style="width:101pt" width="135">Custom peptide</td>
			<td class="xl20">Sequence: AEAEAKAKAEAEAKAK, N-terminus acetylation and C-terminus amidation, &#62;95% pure by HPLC</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">EAK16-IV</td>
			<td class="xl17" style="width:106pt" width="141">CanPeptide Inc.</td>
			<td class="xl17" style="width:101pt" width="135">Custom peptide</td>
			<td class="xl20">Sequence: AEAEAEAEAKAKAKAK, N-terminus acetylation and C-terminus amidation, &#62;95% pure by HPLC</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">EFK8-II</td>
			<td class="xl17" style="width:106pt" width="141">CanPeptide Inc.</td>
			<td class="xl17" style="width:101pt" width="135">Custom peptide</td>
			<td class="xl20">Sequence: FEFEFKFK, N-terminus acetylation and C-terminus amidation, &#62;95% pure by HPLC</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">A6KE</td>
			<td class="xl17" style="width:106pt" width="141">CanPeptide Inc.</td>
			<td class="xl17" style="width:101pt" width="135">Custom peptide</td>
			<td class="xl20">Sequence: AAAAAAKE, N-terminus acetylation and C-terminus amidation, &#62;95% pure by HPLC</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">P6KE</td>
			<td class="xl17" style="width:106pt" width="141">CanPeptide Inc.</td>
			<td class="xl17" style="width:101pt" width="135">Custom peptide</td>
			<td class="xl20">Sequence: PPPPPPPKE, N-terminus acetylation and C-terminus amidation, &#62;95% pure by HPLC</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Alanine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">A7469-100G</td>
			<td class="xl20">L-Alanine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Isoleucine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">I7403-100G</td>
			<td class="xl20">L-Isoleucine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Leucine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">L8912-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Leucine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Methionine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">M5308-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Methionine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Proline</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">P5607-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Proline</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Valine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">V0513-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Valine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Phenylalanine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">P5482-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Phenylalanine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Tryptophan</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">T8941-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Tryptophan</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Tyrosine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">T8566-100G</td>
			<td class="xl20">L-Tyrosine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Glycine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">G8790-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Glycine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Asparagine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">A4159-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Asparagine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Glutamine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">G8540-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Glutamine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Serine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">A7219-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Serine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Threonine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">T8441-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Threonine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Histidine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">H6034-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Histidine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Lysine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl17" style="width:101pt" width="135">L5501-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Lysine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Arginine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">A8094-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Arginine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Aspartic Acid</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">A7219-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Aspartic Acid</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Glutamic Acid</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">G8415-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Glutamic Acid</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Cysteine</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">C7352-100G</td>
			<td class="xl17" style="width:510pt" width="680">L-Cysteine</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Dimethyl Sulfoxide</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">D4540-500ML</td>
			<td class="xl22">DMSO</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl17" height="21" style="width:317pt;height:15.75pt" width="423">Ethanol</td>
			<td class="xl17" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl25">277649-100ML</td>
			<td class="xl22">Anhydrous</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl22" height="21" style="height:15.75pt">Curcumin</td>
			<td class="xl21" style="width:106pt" width="141">Sigma-Aldrich</td>
			<td class="xl22">08511-10MG</td>
			<td class="xl22">Hydrophobic drug, curcumin</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl22" height="21" style="height:15.75pt">Rottlerin</td>
			<td class="xl21" style="width:106pt" width="141">EMD Millipore</td>
			<td class="xl22">557370-10MG</td>
			<td class="xl22">Hydrophobic drug, rottlerin</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:317pt;height:15.75pt" width="423">PP2</td>
			<td class="xl21" style="width:106pt" width="141">Enzo&#160;</td>
			<td class="xl22">BML-EI297-0001</td>
			<td class="xl21" style="width:510pt" width="680">Hydrophobic drug, PP2</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:317pt;height:15.75pt" width="423">Scintillation Vials</td>
			<td class="xl21" style="width:106pt" width="141">VWR</td>
			<td class="xl21" style="width:101pt" width="135">2650-66022-081</td>
			<td class="xl22">Borosilicate Glass, with Screw Cap, 20 mL. Vials for weighing peptide.</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl21" height="21" style="width:317pt;height:15.75pt" width="423">Falcon 50 mL Conical Centrifugation Tubes</td>
			<td class="xl21" style="width:106pt" width="141">VWR</td>
			<td class="xl23" style="width:101pt" width="135">352070</td>
			<td class="xl22">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 ...

The overall goal of this method is to increase solubility of hydrophobic compounds in aqueous solution for potential delivery into clinical settings. This method addresses a key limitation in the medical field which is a lack of use of hydrophobic compounds in clinical settings due to their poor solubility in aqueous systems such as the blood. The main advantage of this technique is that hydrophobic compounds can be dissolved in aqueous solutions without any significant use of toxic co-solvents.First, label two 50 liter conical centrifuge tubes for each amino acid. Prepare a large two liter flask containing purified water. After calculating the amount of each amino acid for the desired concentrations, weight the appropriate amounts of amino acids into the respective centrifuge tubes using a spatula.Using a seriological pipette, ad 40 milliliters of purified water to each centrifuge tube. After capping the tubes, vortex the samples until dissolved. Using a high performance analytical balance, weigh approximately one plus or minus 0.2 milligrams of peptide into the bottom of a 20 milliliter scintillation vial.Record the exact weight of the peptide on the cap and screw the cap on the vial. Next, pipette the appropriate volume of amino acid solution into each vial in order to reach the desired concentration of self-assembling peptide Sonicate the samples for 10 minutes in a water bath sonicator at room temperature ensuring the solutions in the vial are completely immersed in the water bath. Prepare two drug stock solutions by combining one milligram of the desired of the desired drug with the appropriate amount of DMSO and another one milligram of the drug with ethanol.Then, vortex both solutions for 15 seconds to completely dissolve the drug. Following this, label 1.5 milliliter micro-centrifuge tubes for each formulation with the self assembling peptide, amino acid, and co-solvent. Add 10 microliters of the drug DMSO stock solution or 20 microliters of the drug ethanol stock solution to the appropriate micro-centrifuge tubes.Next, add 990 microliters of the self assembling peptide amino acid solutions to the micro-centrifuge tubes containing drug DMSO stock solution. Add 980 microliters of the peptide amino acid solutions to the micro-centrifuge tubes containing the drug ethanol stock solution. Vortex all drug formulations vigorously for 30 seconds.Then, cover with aluminum foil and allow formulations to sit for 30 minutes before solubility testing. After 30 minutes, vortex the formulations vigorously for 30 seconds. Then, centrifuge the formulations at 14, 220 times G for one minute.Following centrifugation, visual inspect the bottom of the micro-centrifuge tubes for precipitation. Out of the 40 formulations tested in this study, seven successfully dissolved the hydrophobic drug, curcumin. Two major trends were identified by grouping the formulations by components.Ethanol seems to be a better co-solvent for dissolving curcumin, and positively charged amino acids, lysine, and arginine seem to be optimal components for dissolving curcumin. It is interesting to note the color change for formulations containing arginine and lysine which reveal curcumin is dissolved in the alkaline condition. Once mastered, this technique can be done in two to three hours with the proper equipment and if the amino acid solutions are prepared before-hand.Following this procedure, soluble drug formulations can be further tested in vitro and in vivo in order to directly assess and compare the biological efficacy or safety of the dissolved drug. After it's development, this formulation method paved the way for in vivo delivery of a hydrophobic stock inhibitor called PP@for the prevention of acute lung injury. After watching this video, you should have a good understanding of how to apply a self-assembling peptide and amino acid combination to dissolve any given hydrophobic drug.

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