Cutting-Edge HEK293T Protein-Integrated Lipid Nanostructures: Boosting Biocompatibility and Efficacy

Abstract

Recently, artificial exosomes have been developed to overcome the challenges of natural exosomes, such as production scalability and stability. In the production of artificial exosomes, the incorporation of membrane proteins into lipid nanostructures is emerging as a notable approach for enhancing biocompatibility and treatment efficacy. This study focuses on incorporating HEK293T cell-derived membrane proteins into liposomes to create membrane-protein-bound liposomes (MPLCs), with the goal of improving their effectiveness as anticancer therapeutics. MPLCs were generated by combining two key elements: lipid components that are identical to those in conventional liposomes (CLs) and membrane protein components uniquely derived from HEK293T cells. An extensive comparison of CLs and MPLCs was conducted across multiple in vitro and in vivo cancer models, employing advanced techniques such as cryo-TEM (tramsmission electron microscopy) imaging and FT-IR (fourier transform infrared spectroscopy). MPLCs displayed superior membrane fusion capabilities in cancer cell lines, with significantly higher cellular uptake. Additionally, MPLCs maintained their morphology and size better than CLs when exposed to FBS (fetal bovine serum), suggesting enhanced serum stability. In a xenograft mouse model using HeLa and ASPC cancer cells, intravenous administration of MPLCs MPLCs accumulated more in tumor tissues, highlighting their potential for targeted cancer therapy. Overall, these results indicate that MPLCs have superior tumor-targeting properties, possibly attributable to their membrane protein composition, offering promising prospects for enhancing drug delivery efficiency in cancer treatments. This research could offer new clinical application opportunities, as it uses MPLCs with membrane proteins from HEK293T cells, which are known for their efficient production and compatibility with GMP (good manufacturing practice) standards.

Keywords: HEK293T cells; anticancer therapeutics; artificial exosomes; lipid nanostructures; membrane proteins.

Figure 1

Development of membrane-protein-bound lipid complexes (MPLCs). (A) Schematic illustration of full process of generating MPLCs. It begins with the extraction of membrane proteins from HEK293T cells, followed by their combination with a lipid mixture (DSPC, DSPE, and cholesterol) through a microfluidic method. The mixture then undergoes a 15-cycle extrusion process using a 100 nm pore-sized cellulose acetate membrane, resulting in the formation of MPLCs. (B) Cell viability assay for the determination of optimal protein-to-lipid ratio (PLR). Different PLRs ranging from 1:50 to 1:10,000 were tested using RAW264.7 macrophage cells. The assay results indicate that a PLR of 1:300 results in the lowest cell viability, suggesting the highest biocompatibility for MPLCs. Values are presented as mean ± standard deviation of three independent experiments. * p < 0.05.

Figure 2

Determination of physical characteristics of MPLCs. (A) ZetavVew analysis of nanoparticles. MPLCs have a larger average diameter, lower PDI, and reduced zeta potential compared to CLs, suggesting enhanced stability and potential for more precise targeting. (B) Morphological comparison using cryo-TEM. CLs exhibit a uniform spherical shape with well-defined bilayers, whereas MPLCs display a diverse morphology with surface-bound proteins, indicating improved cellular interaction capabilities. (C) Fourier Transform Infrared (FT-IR) spectroscopy, highlighting the amide bond peak in MPLCs absent in CLs. This peak indicates the presence of proteins in MPLCs, distinguishing them from the protein-free lipid profile of CLs. (D) Differential Scanning Calorimetry (DSC) demonstrating thermal properties of nanoparticles. MPLCs exhibit a unique endothermic peak, suggesting altered thermotropic behavior of their lipid bilayers, potentially impacting membrane fluidity and stability. (E) Comparison of membrane thickness. Cryo-TEM images, analyzed with ImageJ, reveal a significant increase in membrane thickness in MPLCs compared to CLs. This increased thickness supports the successful integration of proteins into the MPLC structure. Values are presented as mean ± standard deviation of three independent experiments. * p < 0.05.

Figure 3

Immunogenicity and biocompatibility of MPLCs. (A) Phagocytosis assay, assessing the immunogenicity of MPLCs in comparison to CLs. Green fluorescence indicates the uptake of zymosan particles by immune cells in the assay. MPLCs exhibit a lower phagocytosis rate in macrophages than CLs, implying reduced immunogenicity, which is favorable for applications requiring minimal immune response. (B) Hemocompatibility analysis. Human RBCs were incubated with CLs and MPLCs at 37 °C for 2 h, followed by centrifugation to separate the serum for analysis using ELISA kit. MPLCs had significantly lower levels of C5a (a component of the complement system) and F1 + 2 (Prothrombin Fragment 1 + 2, indicative of prothrombin breakdown), indicating better biocompatibility (p < 0.05). Other biomarkers like PF4, CD11b, and hemoglobin release were similar between MPLCs and CLs. Values are presented as mean ± standard deviation of three independent experiments. * p < 0.05.

Figure 4

Enhanced intracellular delivery and serum stability of MPLCs. (A) Assessment of intracellular delivery mechanisms through fluorescence microscopy. Each cancer cell line was treated with aptamer-incorporated CLs and MPLCs, respectively, and the subsequent uptake was measured by fluorescence microscopy. Aptamers were dual-labeled with Cy3 dye, which emits red fluorescence for endocytosis tracking, and with FAM dye, emitting green fluorescence for membrane fusion tracking (left). (A) (right) presents representative fluorescence microscopy images of ASPC1 cells following treatment with aptamer-labeled nanoparticles. Following incubation with ASPC1, SKBR3, and MCF-7 cancer cell lines, fluorescence microscopy analysis showed that MPLCs significantly favored fusion-mediated internalization over CLs in all cell lines, with notably higher fusion rates (bottom). (B) Evaluation of nanoparticle stability in fetal bovine serum (FBS), a simulated in vivo environment. TEM images demonstrate that, unlike CLs, MPLCs preserved their structural integrity and dimensional uniformity, suggesting enhanced serum stability, likely due to their protein coating. ZetaView analysis showed a significant increase in particle size for CLs after FBS treatment (p < 0.05), while MPLCs maintained consistent particle sizes, indicating their stability. Values are presented as mean ± standard deviation of three independent experiments. * p < 0.05.

Figure 5

Enhanced cellular uptake of MPLCs in cancer cell lines. (A) Cellular uptake in HeLa cervical cancer cells. Fluorescence micrographs were acquired following the treatment of HeLa cells with DiL-labeled CLs and MPLCs, respectively. The HeLa cells, transfected with green fluorescent protein (GFP), demonstrated significantly higher uptake of MPLCs (p < 0.05) than CLs, evidenced by the intense red fluorescence from the DiL dye, indicating enhanced internalization. (B) Cellular uptake in ASPC1 pancreatic cancer cells. This panel shows uptake patterns consistent with those observed in HeLa cervical cancer cells. Values are presented as mean ± standard deviation of three independent experiments. * p < 0.05.

Figure 6

In vivo determination of targetability and intracellular delivery of MPLCs. (A) Sequential whole-body fluorescence imaging after individual treatments. BALB/c nude mice bearing HeLa (left) and ASPC1 (right) cell xenografts received administrations of DiR-labeled CLs and MPLCs, respectively. MPLCs demonstrated a higher accumulation in near-tumor areas compared to CLs, signifying enhanced tumor-targeting efficacy. The tumor region was delineated with red dotted lines. (B) Fluorescence imaging of the excised tumor tissues from the xenograft mice. MPLCs showed a pronounced preferential accumulation in tumor tissue over CLs. (C) Assessment of intracellular delivery mechanisms through nanoparticle labeling. Mice received injections of nanoparticles (either CLs or MPLCs) tagged with pHrodo or BCECF dyes. In environments like the lysosome, where the pH is acidic (around pH 4–5), pHrodo dye exhibits red fluorescence, whereas BCECF dye emits green fluorescence in other conditions. Subsequent fluorescence microscopic analysis of the excised livers indicated that CLs predominantly displayed red fluorescence, suggesting endocytosis, whereas MPLCs predominantly showed green fluorescence, signifying uptake predominantly via membrane fusion. * p < 0.05.

Figure 6

In vivo determination of targetability and intracellular delivery of MPLCs. (A) Sequential whole-body fluorescence imaging after individual treatments. BALB/c nude mice bearing HeLa (left) and ASPC1 (right) cell xenografts received administrations of DiR-labeled CLs and MPLCs, respectively. MPLCs demonstrated a higher accumulation in near-tumor areas compared to CLs, signifying enhanced tumor-targeting efficacy. The tumor region was delineated with red dotted lines. (B) Fluorescence imaging of the excised tumor tissues from the xenograft mice. MPLCs showed a pronounced preferential accumulation in tumor tissue over CLs. (C) Assessment of intracellular delivery mechanisms through nanoparticle labeling. Mice received injections of nanoparticles (either CLs or MPLCs) tagged with pHrodo or BCECF dyes. In environments like the lysosome, where the pH is acidic (around pH 4–5), pHrodo dye exhibits red fluorescence, whereas BCECF dye emits green fluorescence in other conditions. Subsequent fluorescence microscopic analysis of the excised livers indicated that CLs predominantly displayed red fluorescence, suggesting endocytosis, whereas MPLCs predominantly showed green fluorescence, signifying uptake predominantly via membrane fusion. * p < 0.05.