(L-457) Creation of pH Sensitive Liposomes for Drug Delivery

Saturday, October 14, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row L - Poster # 457

Presenting Author(s)

AS

Ananya Shivakumar (she/her/hers)

Undergraduate Researcher at University of Pittsburgh
University of Pittsburgh
Pittsburgh, Pennsylvania, United States

Co-Author(s)

AM

Alireza Mohammadzadeh

Mentor and PhD student
University of Pittsburgh, United States

Primary Investigator(s)

SL

Steven Little, PhD

William Kepler Whiteford Endowed Professor of Chemical and Petroleum Engineering, Bioengineering
University of Pittsburgh, United States

Introduction:: Liposomes are spherical nanoparticle vesicles that are mainly composed of lipids with an aqueous interior surrounded by a phospholipid bilayer and which typically range from 25 nm to 2.5 μm in diameter¹. Due to liposomes’ amphiphilic nature, they have been widely used for carrying both hydrophilic and hydrophobic cargos². They are often utilized to deliver medicine or biopharmaceuticals to the site of disease. Many liposomes are synthesized to be photoresponsive, thermoresponsive, or pH sensitive such that the liposomes degrade when exposed to light, heat, or acidic environments. Liposomes whose lipid layer consist of dioleoylphosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) are often utilized to create pH sensitive liposomes; however, this is an expensive formulation, especially in the realm of agricultural purposes in which a cheaper formulation is preferred. As a result, a less expensive alternative has been suggested to provide pH sensitive liposome release using soybean lecithin, cholesterol, and CHEMS.

Materials and Methods::

Liposome Preparation Liposomes were created using a microfluidic hydrodynamic flow focusing chip then purified using a dialysis bag (12-14 kDa MWCO). The lipid phase consisted of soybean lecithin, cholesterol, and CHEMS which were mixed in a 3.4:1:10.6 molar ratio in 20 mL of IPA and filtered twice using a 0.22 um filter. The aqueous phase consisted of 0.116 mg/mL of Rhodamine B Dye which was also filtered twice using a 0.22 um filter. The dye acts as a drug excipient in order to more easily record the cargo release. The total flow rate (TFR) was 300 μL/min and the flow rate ratio (FRR) was 1:2 (lipid:aqueous) such that liposomes consisting of an aqueous, cargo containing, core were produced in the outlet channel. Refer to Figure 1. The resulting liposomal solution was then purified in a dialysis bag for at least three days in phosphate buffered saline (PBS) to remove any excess lipid or dye molecules.

Quantifying pH Sensitivity After purification, the dialysis method was used to determine the liposome dye release profile. Liposomes were kept in dialysis bags at either pH 7.4 PBS or pH 4 PBS buffer and the sink solution was sampled at different time points over the course of one week (1:1, donor volume:sink volume). The fluorescence was then quantified using a spectrophotometer plate reader at an excitation of 560 nm and emission of 590 nm. Total fluorescence was quantified by heating the sample to 90°C for 45 minutes, then vortexing, and then measuring the fluorescence.

Results, Conclusions, and Discussions:: Total fluorescence after heating the original liposome solution to 90°C was taken and the concentration of dye was 5079 ng/mL. Fluorescence measurements of the sink solution taken at various time points were quantified and converted to concentrations using a predetermined standard curve for Rhodamine B at pH 4 and pH 7.4. Three replicates (n=3) per time point were taken and the average and standard deviation were calculated and are represented in the graph. The concentration of Rhodamine B dye at each time point of the pH 4 sink solution exceeds that of pH 7.4 which indicates pH-sensitive release as shown in Figure 2. The same formulation with different molar ratios may be used in an attempt to obtain greater cargo release.

Acknowledgements (Optional): : I would like to thank Alireza Mohammadzadeh for his continued support and guidance on this project and I would also like to thank Dr. Steven Little, PhD for supporting this project. In addition, this research was funded partly by the Swanson School of Engineering at University of Pittsburgh through the Summer Undergraduate Research Internship (SURI) program and by the National Institute of Health.

References (Optional): :

1. Immordino, M. L., Dosio, F., & Cattel, L. (2006). Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential. International journal of nanomedicine, 1(3), 297.

2. Revathy, T., Jayasri, M. A., & Suthindhiran, K. (2016). Antimicrobial magnetosomes for topical antimicrobial therapy. In Nanobiomaterials in Antimicrobial Therapy (pp. 67-101). William Andrew Publishing.