Connectosomes for Direct Molecular Delivery to the Cellular Cytoplasm

Abstract

Transport of biomolecules, drugs, and other reagents across the cell's plasma membrane barrier is an inefficient and poorly controlled process, despite its fundamental importance to biotechnology, cell biology, and pharmaceutics. In particular, insufficient membrane permeability frequently limits the accumulation of drugs and reagents in the cytoplasm, undermining their efficacy. While encapsulating drugs in particles increases uptake by cells, inefficient release of drugs from these particles into the cytoplasm ultimately limits drug efficacy. In contrast, gap junctions provide a direct route to the cytoplasm that bypasses the plasma membrane. As transmembrane channels that physically connect the cytoplasm of adjacent cells, gap junctions permit transport of a diverse range of molecules, from ions and metabolites to siRNA, peptides, and chemotherapeutics. To utilize gap junctions for molecular delivery we have developed Connectosomes, cell-derived lipid vesicles that contain functional gap junction channels and encapsulate molecular cargos. Here we show that these vesicles form gap junction channels with cells, opening a direct and efficient route for the delivery of molecular cargo to the cellular cytoplasm. Specifically, we demonstrate that using gap junctions to deliver the chemotherapeutic doxorubicin reduces the therapeutically effective dose of the drug by more than an order of magnitude. Delivering drugs through gap junctions has the potential to boost the effectiveness of existing drugs such as chemotherapeutics, while simultaneously enabling the delivery of membrane-impermeable drugs and reagents.

Bioinspired Molecular Factories with Architecture and In Vivo Functionalities as Cell Mimics

“…In order to obtain E‐GPMVs with a modified membrane and inner cavity, we simultaneously equipped them with a small hydrophilic fluorophore (CellTracker Deep Red Dye, referred herein as CTDR) and Chol‐PEG5000‐FITC polymer. First, the fluorescence intensity associated with CTDR alone indicates its successful transfer to the inside of the E‐GPMVs in a concentration dependent manner, in the range 1 × 10 −6 to 10 × 10 −6 m (Figure S6, Supporting Information), and is in agreement with the previously reported transfer of low molecular weight compounds present in the cytoplasm of the donor cells . However, we observed that this process is not linear, but exponential, and thus by controlling the number of desired molecules in the donor cell cytosol, it is possible to control their final amount inside E‐GPMVs, which is a necessary step when reactions have to be implemented inside MFs.…”

“…In order to establish the size distribution of E‐GPMVs, we supplement them with a fluorescent model membrane protein LcK tyrosine kinase‐GFP (Lck‐GFP) by genetically engineering HepG2 cells to overexpress this protein using a baculovirus gene transfer (BacMam 2.0) . When Lck‐GFP is successfully inserted, it preserves its fluorescence property, and does not affect the integrity of the membrane compartment (Figure S1, Supporting Information), in agreement with previous reports about the insertion of gap junction proteins in the membrane of giant plasma membrane vesicles . E‐GPMVs are isolated with a size range of 2–20 µm, as demonstrated by confocal laser scanning microscopy (CLSM) and flow cytometry data ( Figure A–C).…”

“…The exact number of E‐GPMVs injected could neither be fixed nor determined, due to the sedimentation process when E‐GPMVs were aspirated into the microinjection needle and subsequent needle fixation in the micro injector (10–15 min). As both GPMVs and PMOXA‐PDMS‐PMOXA polymersomes have been reported as noncytotoxic, we did not expect toxic effects for their combination as E‐GPMVs or MFs. Indeed, we did not observe acute toxicity, change of behavior, or heart failure in the injected ZFE.…”

“…Artificial cells that have previously been reported only showed functionality in vitro where they interacted with their environment, among themselves, or with other eukaryotic/prokaryotic cells in culture or solid tumor models . In order to study whether our MFs preserve their integrity and functionality and are nontoxic when injected in vivo, we selected zebrafish embryos (ZFEs) as a vertebrate animal model, which has previously been used to bridge the gap between in vitro studies and studies in mammalian organisms (e.g., rodents) .…”

“…The membrane and internal composition directly mirror the composition of the cells from which they originated, except for larger cellular organelles and intracellular structures (e.g., nuclei, Golgi apparatus, vesicles) . Although this is very promising for situations requiring a complex composition of the inner cavity of the compartment, efforts to date have been mainly devoted to their supplementation with small molecular weight compounds, or with biomolecules, such as lipids, or proteins, and only one study has involved the internal encapsulation of nanoparticles . There are currently no reports of the creation of functional artificial cells with the outer membrane complexity of a cell, and an internal cytoplasm.…”