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Optimizing Exosomes for Precision Drug Delivery

 

Group 41: Louie Zhao

Mentor: Dr. Pranav Sharma, Xosomix

 

Background

 

• Company Focus: Xosomix develops exosome-based therapeutics, using innate as well as drug loaded exosomes for treatment of neurological diseases such as Autism and Alzheimer’s Disease.
• Background: Exosomes are nanoscale, vesicles from cells that naturally carry proteins, RNAs, and other molecules between cells, enabling targeted communication and cargo delivery.
• Potential: Their ability to cross biological barriers (like the blood-brain barrier) and deliver complex therapeutic cargo makes exosomes a powerful and target for precision medicine.

Project Objective

 

• Increase Targeting Specificity: Engineer exosomes with neuron-targeting capability to enhance delivery precision and reduce off-target effects in neurological disorders.
• Develop Scalable Production Methods: Optimize exosome production method scalable to large-scale commercial manufacturing under Current Good Manufacturing Practice (CGMP) regulations.
• Optimize Cargo Loading: Optimize efficient loading of therapeutic cargo into lumen as well as surface of exosomes.

 

Project goals

 

1. Develop Scalable Production Methods: Optimize exosome isolation and cargo-loading techniques to enable large-scale, reproducible manufacturing suitable for clinical use. ​
2. Optimize Exosome Loading.

 

Methods

 

Optimizing Exosome Harvesting and Purification

 

Comparison Groups

 

• Hollow Fiber TFF

• Flat Sheet TFF

 

Purification steps

 

A human fibroblast cell line (CRL-4061) was cultured and expanded from one 10 mL cell flask into eight 100 mL cell flasks. 25 mL of cell media (DMEM) with 10% exosome-free Bovine serum albumin (BSA) and FGF (Fibroblast Growth Factor) was added to each flask and grown over the course of a week. After the cells have reached a desired concentration, the conditioned media (CM) is collected, and the cells are split into a concentration of about 100,000 cells per flask for the next growth cycle. 

 

Tangential Flow Filtration

 

With the CM, tangential flow filtration (TFF), which is a method that separates and purifies biomolecules using ultrafiltration membranes is performed on the 200 mL of CM collected. This is to concentrate the exosomes within the media into a 1 mL which will later be concentrated further. 6 total exosome samples from TFF were collected.

 

Size Exclusion Chromatography

 

Size exclusion chromatography (SEC), which separates molecules based on their size, with larger molecules filtering faster than smaller ones. This was then performed on the 1mL sample from the TFF earlier. This allows us to concentrate the exosomes into about 150 uL which is the final product that will be sent to the external company. We will also aliquot 5 uL of exosome stock to assess the protein concentration to determine the number of exosomes in ug. 

 

Protein Estimation

 

Using the Pierce Micro BCA Protein Assay Kit, a three-component version of BCA reagents, optimized to measure total protein concentration of dilute protein solution, we will assess the protein concentration of each exosome sample collected. After 8 weeks, 6 exosome samples from the mouse stem cell line were collected and a microBCA test was performed. The aliquot samples were plated on a microBCA 96 well plate with standards and blanks. After a 2 hour incubation, the samples were read on a cell imaging reader to measure the absorbance. The protein numbers were then calculated on an excel spreadsheet from the data given by the imaging reader.

 

Western Blot

 

            To assess the concentration of certain proteins (ALIX and BSA) To assess ALIX and BSA levels in exosome preparations via Western blot, exosome samples are lysed in RIPA buffer with protease inhibitors. Sample are then loaded onto a gel to go under electrophoresis. After electrophoresis, proteins are transferred to a PVDF or nitrocellulose membrane. The membrane is blocked in 5% milk or BSA in TBST for one hour, then incubated overnight at 4°C with primary antibodies against ALIX (~95 kDa, an exosome marker) and BSA (~66 kDa, a common serum contaminant). After washing, secondary antibodies are applied for one hour. A strong ALIX signal indicates successful exosome isolation, while BSA presence suggests serum contamination.

 

Quantification

 

            To quantify the results from the Western blot, MetaMorph imaging software is used. First, a high-resolution grayscale TIFF image of the developed blot is generated from the membrane. If necessary, the image is converted to grayscale to ensure accurate intensity measurements. Using the region tool, a rectangular region of interest (ROI) is drawn around each protein band, with consistent size and shape applied to all bands for reliable comparison. The integrated intensity of each ROI—representing the product of the area and the mean gray value—is then measured.

To account for background noise, an identical ROI is placed in a region of the membrane without any bands, and its integrated intensity is measured. This background value is subtracted from each band’s signal to yield background-corrected intensities. These corrected values can be normalized to a reference sample or internal loading control across blots. To assess exosome purity, the ratio of ALIX to BSA intensity is calculated, where a high ALIX and low BSA signal indicates a cleaner exosome preparation with minimal serum contamination.

 

Exosome Cargo Delivery Assay

 

Comparison Groups

 

• Hypotonic Loading

• Isotonic Loading

• Control Group

 

Sample Collection

 

A human fibroblast cell line (CRL-4061) was subcultured and plated into a 96-well plate at a density of 2,500 cells per well and incubated overnight. Three solution conditions were prepared: control buffer, hypotonic, and isotonic each with a final volume of 100 µL. All solutions contained 10 mM phosphate buffer, made by adding 10 µL of a 100 mM phosphate buffer stock. For the control buffer, 3 µL of 5 M NaCl was added to reach a final NaCl concentration of 150 mM (isotonic), followed by 87 µL of ddH₂O. No exosomes or avidin were included in the control buffer. The hypotonic solution was prepared by mixing 10 µL phosphate buffer, 10 µL of exosomes, and 10 µL of avidin, then adding 70 µL of ddH₂O to reach a total of 100 µL (no NaCl was added). The isotonic solution contained 10 µL phosphate buffer, 3 µL of 5 M NaCl (to achieve 150 mM NaCl), 10 µL exosomes, and 10 µL avidin, with 67 µL ddH₂O added to reach a final volume of 100 µL.

After initial incubation, the hypotonic solution was converted to isotonic by removing 3 µL from the tube and replacing it with 3 µL of 5 M NaCl, adjusting the salt concentration to 150 mM while maintaining the total volume. To remove unbound avidin, both hypotonic and isotonic samples were processed through spin columns by centrifugation at 12,000 g, allowing free avidin to pass through while retaining exosome-bound avidin. The resulting solutions were then loaded into five identical wells per condition (20 µL per well) and incubated for four hours.

After incubation, the media was removed, and cells were fixed with 4% paraformaldehyde. Cell nuclei were stained with DAPI. Imaging was performed using the Cytation 5 cell imaging reader. For each well, 15 images were acquired—five fields of view for each of three imaging channels: brightfield (to define cell boundaries), DAPI (to visualize nuclei), and GFP (to detect avidin fluorescence and quantify exosome uptake).

 

Quantification

 

            To quantify exosome uptake, MetaMorph imaging software was used to analyze exosome internalization within cells. Brightfield images were first used to manually define cell boundaries using the region tool. A statistical background correction was then applied to the corresponding GFP images to reduce background noise. The defined cell boundary regions were transferred onto the background-corrected GFP images, and both the average and integrated fluorescence intensity were measured for each cell. This process was repeated across all image sets for the entire assay. Once data collection was complete, the average intensity per well was calculated and plotted as a bar graph. Higher average fluorescence intensity corresponded to more efficient exosome loading and uptake.

 

Figures

 

 

 

 

 

 

 

 

 

Acknowledgements

 

I would like to express my gratitude and many thanks to my mentor Dr. Pranav Sharma, CEO of Xosomix for the aid and guidance of this research project with great mentorship and Dr. Alyssa Taylor Amos of UCSD Bioengineering for steering the Capstone Program.