Evaluating methods to create protein functionalized catanionic vesicles

Abstract

Catanionic surfactant vesicles (SVs) composed of sodium dodecylbenzenesulfonate (SDBS) and cetyltrimethylammonium tosylate (CTAT) have potential applications as targeted drug delivery systems, vaccine platforms, and diagnostic tools. To facilitate these applications, we evaluated various methods to attach proteins to the surface of SDBS/CTAT vesicles. Acid phosphatase from wheat germ was used as a model protein. Acid phosphatase was successfully conjugated to vesicles enriched with a Triton-X 100 derivative containing an unsaturated ester. Enzymatic activity of acid phosphatase attached to vesicles was assessed using an acid phosphatase assay. Results from the acid phosphatase assay indicated that 15 ± 3% of the attached protein remained functional but the presence of vesicles interferes with the assay. DLS and zeta potential results correlated with the protein functionalization studies. Acid phosphatase functionalized vesicles had an average diameter of 175 ± 85 nm and an average zeta potential of -61 ± 5 mV in PBS. As a control, vesicles enriched with Triton-X 100 were prepared and analyzed by DLS and zeta potential measurements. Triton X-100 enriched vesicles had an average diameter of 140 ± 67 nm and an average zeta potential of -49 ± 2 mV in PBS. Functionalizing the surface of SVs with proteins may be a key step in developing vesicle-based technologies. For drug delivery, antibodies could be used as targeting molecules; for vaccine formulation, functionalizing the surface with spike proteins may produce novel vaccine platforms.

Figure 1:. Schematic representation of surface functionalization strategies

In method A, a bifunctional linker containing a hydrophobic anchor is incorporated into vesicles and then the functionalized vesicles react with protein. In method B, an electrostatic anchor is conjugated to a protein and the protein conjugate is mixed with pre-formed vesicles.

Figure 2:

Schematic representation of Triton X-100 esterification.

Figure 3:. Acid phosphatase assay results of acid phosphatase functionalized vesicles and other acid phosphatase samples.

Sample key: AP control = acid phosphatase control sample; BV with AP = bare vesicles mixed with acid phosphatase; TXV with AP: Triton X-100 enriched vesicles mixed with acid phosphatase; AP SVs: acid phosphatase functionalized vesicles. Results from a single trial of different acid phosphatase samples. Acid phosphatase functionalized vesicles demonstrated diminished enzymatic activity compared to vesicle formulations in which acid phosphatase was mixed with vesicle solutions but had similar activity to acid phosphatase that was mixed with Triton X-100 enriched vesicles. The acid phosphatase concentration in each sample is 30 μg/mL. Negative control samples (samples containing the substrate but no acid phosphatase) were used to normalize absorbance values.

Figure 4:. DLS size distributions of Triton X-100 enriched vesicles and acid phosphatase functionalized vesicles.

Sample key: TX SVs: Triton X-100 enriched vesicles; AP vesicles: acid phosphatase functionalized vesicles (prepared by mixing acid phosphatase with esterified Triton X-100 enriched vesicles and then purifying by SEC). Data is from a single, representative DLS measurement from each sample. The increase in size is attributed to proteins attached to the surface.