Use of lipid vesicles to transfer biomolecules into cells

Large unilamellar vesicles (LUV), made of egg yolk lecithin (EYL), were demonstrated to be endocytosable by Chinese hamster V79 cells and therefore could be used as lysosomotropic agents for drug or enzyme delivery. Two cytochemical techniques were developed to provide evidence to support endocytosis of these vesicles.

When V79 cells were allowed to incubate with LUV containing ¹²⁵I-radiolabeled lysozyme, the lipid uptake was twice as fast as the vesicle-content uptake. Among the trapped-content uptake, 22.3% were inhibited by cytochalasin B or a combination of NaN₃ and deoxyglucose. Chloroquine did not inhibit the uptake. These results suggested that about 22.3% of the trapped-content uptake might be due to endocytosis.

When cells were treated with LUV containing horseradish peroxidase (HRP), cytochemical studies revealed that peroxidase-positive particles were enclosed in vacuoles inside the cells. The morphology of the deposited reaction product was distinctly different from that resulted from pinocytosis of free HRP. These observations indicated that lipid vesicles were endocytosed by the cells.

When cells were treated with LUV containing 5-Br, 4-Cl, 3-indolyl phosphate (BCIP), a substrate for lysosomal acid phosphatase, electron microscopy revealed precipitated granules inside the cells. This precipitate formation was inhibited when chloroquine or NaN₃ together with deoxyglucose were included in the incubation medium. The formation of the electron-dense reaction product located in the lysosomes would specifically indicate the breakdown of BCIP after the vesicles had been endocytosed and delivered to the lysosomes.

In summary, endocytosis took place when Chinese hamster V79 cells were incubated with LUV. HRP and BCIP are suitable entrapped markers for the endocytotic activity. LUV that have high trapping capacity can be potentially used as carriers to deliver bioactive molecules into cells by an endocytotic mechanism.

Acetylcholine receptor (AchR) from Torpedo californica was attempted to be transferred to L cell membrane by lipid vesicles. Cholate solubilized AchR were reconstituted with or without the Sendai virus envelope proteins into azolectin lipid vesicles by Sephadex G50 column chromatography. When the suspended L929 cells were incubated with vesicles containing only AchR (AchR vesicles), about 2.8 x 10⁴ AchR molecules/cell became associated with the cells. A two fold increase was observed when cells were treated with vesicles containing AchR and Sendai virus envelope proteins (AchR-VEP vesicles). The turnover of the AchR on the cell surface were rapid in both cases. Indirect immunofluorescent data showed distinct difference in the distribution of AchR on the cell surfaces. Every cell that was treated with AchR-VEP vesicles could still be labeled after 40 hr of culture, while most AchR vesicletreated cells could not. Immunoferritin EM studies suggested that AchR-VEP vesicles fused with cells resulting in the insertion of AchR into the cell membrane, while AchR vesicles only adsorbed on the cell surface. Complement-mediated cytotoxicity data also supported these results. AchR-VEP vesicle treatment resulted in 12% of the cell lyzed after antibody-complement fixation by anti-Sendai antibody, which was comparable to those treated with native Sendai virus. When anti-AchR antibody was used for the assay, the cytotoxicity for AchR-VEP vesicle-treated cells was 2.87 fold higher than those treated with AchR vesicles. Since the viability of the cells will only be affected by specific antibody binding to antigens in the membrane, the cytotoxicity data could be interpreted as that AchR molecules were introduced into cell membrane more efficiently by AchR-VEP vesicles than by AchR vesicles.

In summary, lipid vesicles containing Sendai envelope proteins can be used as carriers to introduce AchR into the plasma membrane of L cells. Possibilities of further studies of the functional properties of AchR in L cells were discussed.