Investigating Alginate Loading and Release For Controlled Payload Delivery Applications

Abstract

One of the main challenges faced when trying to treat metastatic cancer is that disease can persist in specific locations in the body that show poor drug penetration, or pharmacological sanctuary sites. One such example is the lymph node, as many small-molecule drugs display poor uptake in the lymph node lobule. This phenomenon is important in the context of metastatic cancer, as it allows cancer cells to “hide” in the lymph node lobules, unaffected by chemotherapies or other cancer-targeting drugs.

One approach to targeting cancer cells in the lymph node is the use of Trojan horse delivery vehicles, where drug payloads are hidden in a cell-mimetic vehicle. Polymers, such as alginate, are often used to manufacture these delivery vehicles because of their biocompatibility and biodegradability, which enable the creation of a tailored drug delivery platform.

It was hypothesized that microfluidic techniques could be leveraged to optimize microparticle core fabrication, drug and molecule loading, and drug release onto 4T1 cancer cells. Alginate was used to create microparticles by incorporating synthetic strategies to improve stability and enable tuned release parameters. To investigate this hypothesis, two aims were established: (1) Develop an alginate droplet generating protocol for monodispersed droplets and efficient solvent transfer and (2) Determine payload functionality and release from the MPs along with how a material change will impact release.

To develop a reliable protocol for generating monodispersed alginate droplets with efficient solvent transfer, two fabrication approaches were evaluated: dropwise emulsion and flow-focusing microfluidics. The flow-focusing device demonstrated the narrowest size distribution; however, the dropwise emulsion method offers size tunability and has a much higher throughput. Following several washing steps with octanol, PBS, and isopropanol, the microparticles were left completely in the aqueous phase. Additionally, storing the particles at 4 °C and in a buffer that contains calcium and magnesium helped maintain particle stability and prevent premature degradation by maintaining alginate crosslinks. This aim helped establish a robust droplet generation protocol, along with optimal washing and storage conditions.

To evaluate loading across molecules of different sizes, particles were encapsulated with 500 kDa FITC-dextran and Doxorubicin (DOX). Both molecules were successfully encapsulated with comparable loading efficiencies, indicating that molecules of significantly different sizes can be loaded. When particles made via the dropwise emulsion method were loaded with DOX in concentrations greater than 1 mg/mL, significant clumping and aggregation occurred; however, particles were still loaded with drug. When particles were crosslinked with 2% and 6% acetic acid, similar levels of DOX encapsulation were achieved under identical loading conditions.

To test the efficacy of DOX post-encapsulation and release, the efficacy of free DOX on 4T1 cells was first confirmed. After 48 hours, free DOX demonstrated a clear dose-dependent reduction in cell viability; however, the lack of statistical significance between the higher concentration groups (0.35 mg/mL, 0.5 mg/mL, and 1 mg/mL), suggests that maximal cytotoxicity is reached at relatively low DOX concentrations. To then confirm that DOX remains effective after encapsulation, microparticles were incubated in saline to allow DOX release, and the resulting DOX-containing saline was applied to 4T1 cells. It was found that cells treated with free DOX had a viability comparable to that of the free DOX treatment group at the same concentration. Lastly, particles crosslinked with both 2% and 6% acetic acid were loaded with DOX and placed with 4T1 cells. After 48 hours, the cell viability in all DOX treatment groups showed no significant differences from each other but were different from that of unloaded particles. This aim helped demonstrate that DOX remains functional post encapsulation and that unloaded particles have no negative impacts on cell viability, while showcasing that a small (4%) change in acetic acid concentration does not impact loading or release.

This study showcased that this alginate microparticle platform is a robust and adaptable system for drug delivery, capable of encapsulating diverse molecules while preserving therapeutic efficacy. This work highlights the potential of emulsion and microfluidic fabrication techniques for designing tunable and effective drug delivery systems for cancer therapy and potential lymph node targeting downstream.