Opti-Biologics


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Lab Update #2: August


September 19, 2022

Informal Introduction 
The previous 30 days or so have been focused on making new peptide amphiphiles and purifying them. This is a straightforward process but can be highly time-consuming. Since I have two projects and both revolved around purification and verification, I will be looping them together. 
To purify a peptide, a crude peptide can be produced via a PS3 peptide synthesizer. Peptides with specific sequences can also be purchased from various online companies. Once a peptide is synthesized, it is usually contaminated with debris or unbound peptides, etc. A crude peptide will not be 100% pure, so the next step is to obtain a pure peptide source so that future steps are accurate in concentration. To purify a peptide, reverse-phase high-performance liquid chromatography (HPLC) with a C18 column is used. The basic idea of using HPLC is that the peptide will be separated from other molecules by running down a column so that more dissolvable smaller molecules will be separated from larger molecules. Once the peptide is dissolved in Milli-Q water and injected into the HPLC, a peak can be detected and collected. These fractions are then put under a fume hood to allow the acetonitrile (ACN) to evaporate. To verify that each fraction contains pure peptide, mass spectrometry is used. Briefly, 0.8 ul of sample is spotted onto a target plate and 0.8 ul of a matrix is added. The spots can then be analyzed via MS. Once the ACN is evaporated, the fractions can be placed into pre-weighed tubes and stored in a -80C freezer for 48 hours. Once completely frozen, the fractions can be placed into the lyophilizer, which freeze-dries the sample. 

Manuscript
The following peptides MGSHIEPGGC (AC1), KMGGTNHPEC (AC2), and CKDSPKSSKSIRFIPVST (CKD) were dissolved in Milli-Q water and purified using a reverse-phase high-performance liquid chromatography system with a C18 column. The m/z was characterized by MALDI-TOF and the expected peak is [M+H]+ =  1029, 1115, and 2021, respectively. 
Figure 1a: Peak of AC1 peptide at 1047 m/z.
Figure 1b: Peak of CKD peptide at 2022 m/z.
Figure 1c: Peak of AC2 peptide at 1137 m/z,
Informal
Once the pure peptides are fully freeze-dried, they can be conjugated to DSPE-PEG (2000)-maleimide. The purpose of this conjugation is to create a peptide amphiphile. An amphiphile is a molecule that contains a hydrophobic chain, in this case, it is DSPE, and a hydrophilic region, in this case, it is a peptide. This peptide amphiphile can form micelles if its concentration of it is higher that the critical micelle concentration. To do this conjugation, an amount of pure peptide will be mixed with DSPE-PEG (2000)-maleimide at a 10% molar excess. This solution will be purged with
nitrogen gas and set on a shaker for 72 hours. Since it is likely that not 100% of the peptide will react with DSPE-PEG (2000)-maleimide, this solution must be purified using HPLC as previously described. The same procedure will follow, including MS verification and freeze-drying the sample.
 
Manuscript
The previously described purified peptides were conjugated to DSPE-PEG (2000)-maleimide in pH 7.4 buffered Milli-Q water for 72 hours. The products were purified using a reverse-phase high-performance liquid chromatography system with a C4 column. The expected peak for the successfully conjugated peptide amphiphiles are as follows:  [M+H]+ =  3990 (AC1), 4078 (AC2), and 4963 (CKD). 
Figure 2a: Peak of CKD peptide at 2022 m/z. Peak of DSPE-PEG (2000)-CKD Peptide at 5011 m/z.
Figure 2b: Peak of AC2 peptide at 1137 m/z. Peak of DSPE-PEG (2000)-AC2 Peptide at 4151 m/z.
Informal
After conjugating the peptide to DSPE-PEG (2000)-maleimide, micelles can be synthesized via thin-film self-assembly. This procedure has been previously described. Briefly, peptide amphiphiles were dissolved in methanol at varying molar ratios. The methanol was completely evaporated using nitrogen gas and the film was left in a vacuum overnight. The following morning, the film was resuspended in triple syringe filtered Milli-Q water, vortexed at 6 speed for 30 seconds, sonicated for 15 minutes, and left at 80C for 30 minutes. The peptide amphiphile micelles (PAMs) were left at room temperature for 24 hours to allow equilibrium to be achieved. 

Manuscript 
Peptide amphiphile micelles (PAMs) were synthesized using thin-film self-assembly. Peptide amphiphiles were dissolved in methanol with DSPE-PEG(2000)-rhodamine B at a mol ratio of 90:10. The methanol was evaporated under the fume hood using nitrogen. The films were left to dry in a vacuum overnight. The films were hydrated using Milli-Q water and sonicated for 10 minutes. The resulting solution was then incubated at 80°C for 30 minutes and then left at room temperature overnight. 
Micelle size was determined by a Malvern zeta sizer ultra at 100 µM. Measurements were carried out at 25°C in replicates of three. Micelle zeta potential was determined by a Malvern zeta sizer ultra at 33 µM in replicates of three. 

Figure 3: All PAMs made in 90:10 mol ratio (DSPE-PEG(2000)-Peptide: DSPE-PEG(2000)-RhB. [n=3]
 
Informal
Previously mentioned in lab update 1, CKD peptide PAMs were unable to protect the mRNA from RNase A-mediated degradation. I hypothesized that this was due to the insufficient positive charge of the CKD PAMs since protection requires semi-intense electrostatic interaction between the PAMs and mRNA. To increase the zeta potential of CKD PAMs, I synthesized a polylysine peptide (KKKKKKKC). I purified the peptide using HPLC and verified using MALDI, as previously described. I also conjugated the polylysine peptide to DSPE-PEG (2000)-maleimide, purified, and verified using HPLC and MALDI respectively. 

Manuscript 
To increase the zeta potential of the micelles containing DSPE-PEG (2000)-CKD peptide, polylysine (KKKKKKC) peptide was synthesized using a PS3 peptide synthesizer. The crude peptide was then purified using a reverse-phase high-performance liquid chromatography system with a C18 column. The m/z was characterized by MALDI-TOF, and the expected peak is [M+H]+ = 1019 m/z. The purified peptide was conjugated to DSPE-PEG (2000)-maleimide in pH 7.4 buffered Milli-Q water and purified using a reverse-phase high-performance liquid chromatography system with a C4 column. The m/z was characterized by MALDI-TOF and the expected peak is [M+H]+ =  3961 m/z. 
Micelles were synthesized using thin-film self-assembly as previously described. Briefly, DSPE-PEG (2000)-KKKKKKKC and DSPE-PEG (2000)-CKD peptide was dissolved in methanol at varying mol ratios. Once the methanol was completely evaporated, the film was left in a vacuum overnight and resuspended in Milli-Q water. The solution was sonicated for 10 minutes and incubated at 80⁰C for 30 minutes. Following incubation, the solution was left at room temperature for 24 hours. Micelles were characterized using a Malvern zeta sizer as previously described. The results indicate that micelles composed of 50:50 and 60:40 mol ratios of DSPE-PEG (2000)-KKKKKKKC: DSPE-PEG (2000)-CKD peptide have a sufficient charge to adsorb mCherry mRNA. 
Figure 4a: Peak of polylysine peptide at 1019 m/z.
Figure 4b: Peak of DSPE-PEG (2000)-polylysine peptide at 3966 m/z.
Figure 5: Panel displaying hydrodynamic diameter, zeta potential, and polydispersity index of PAMs. All PAMs are fluorescently-labelled using rhodamine B. [n=3]
Figure 6: Panel displaying hydrodynamic diameter, zeta potential, and polydispersity index of PAMs. All PAMs do not include rhodamine B. [n=3]
Targeting the CCD Project
The first project, which briefly explained is to create a PAM that binds selectively to the cortical collecting duct (CCD). The next month is dedicated to synthesizing more AC1 and AC2 peptides as well as scrambled versions of each peptide. The scrambled peptides will serve as controls in future studies. The most immediate future study will be an in vitro cell binding assay. Briefly, PAMs will be fluorescently labeled with rhodamine B and incubated with mouse CCD cells at 50uM for 15 minutes. The cells will then be fixed, stained with DAPI, and then mounted on a microscope slide. Fluorescence microscopy will be used to visualize cell binding. 

mRNA Delivery to CCD for the Treatment of ADPKD
To ensure the PAMs can be assembled correctly, this week’s study will include triple filtering the solution using a PDVF filter prior to DLS. This will lower the polydispersity index (PDI) of my micelles. After this, an in vitro cell binding assay will be performed to visualize if the CKD peptide micelle can selectively bind to CCD cells compared to scrambled CKD peptide micelle and a methoxy micelle. Once the CKD binding is established, the micelles at either 50:50 or 60:40 (DSPE-PEG (2000)-KKKKKKKC: DSPE-PEG (2000)-CKD peptide) will be incubated with mRNA for 60 minutes at room temperature. Upon completion of the mRNA attachment, PAM/mRNA complexes will be characterized using DLS and zeta as previously described. Along with this, PAM/mRNA complexes will be loaded into a 1% agarose gel at varying weight ratios to determine if the PAM is able to retard mRNA migration down the gel thus representing successful adsorption. 

 

Meet The Author


Hello everyone, 

My name is Joshua Giblin. I am a post-bachelor researcher/research technician at USC. My interests range from nutrition to nanomedicine and also practical science to improve everyday life. Through this blog, I aim to communicate practical scientific research and present it to curious individuals so that an educated decision can be made. Thank you for reading the blog and showing your support. 

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