Rapid Generation of Therapeutic Nanoparticles Using Cell-Free Expression Systems

Abstract

The surface modification of membrane-based nanoparticles, such as liposomes, polymersomes, and lipid nanoparticles, with targeting molecules, such as binding proteins, is an important step in the design of therapeutic materials. However, this modification can be costly and time-consuming, requiring cellular hosts for protein expression and lengthy purification and conjugation steps to attach proteins to the surface of nanocarriers, which ultimately limits the development of effective protein-conjugated nanocarriers. Here, the use of cell-free protein synthesis systems to rapidly create protein-conjugated membrane-based nanocarriers is demonstrated. Using this approach, multiple types of functional binding proteins, including affibodies, computationally designed proteins, and scFvs, can be cell-free expressed and conjugated to liposomes in one-pot. The technique can be expanded further to other nanoparticles, including polymersomes and lipid nanoparticles, and is amenable to multiple conjugation strategies, including surface attachment to and integration into nanoparticle membranes. Leveraging these methods, rapid design of bispecific artificial antigen presenting cells and enhanced delivery of lipid nanoparticle cargo in vitro is demonstrated. It is envisioned that this workflow will enable the rapid generation of membrane-based delivery systems and bolster our ability to create cell-mimetic therapeutics.

Keywords: cell-free expression; functionalization; lipid nanoparticles; liposomes; vesicles.

Figure 1.

Cell-free expressed proteins can be conjugated to liposomes. A) Proteins can be expressed and conjugated to liposomes via a self-conjugating enzyme. By adding 0.1 mol% 18:1 PE PEG2000 benzylguanine to the membrane and fusing a SNAP tag to mEGFP, SNAP-mEGFP forms a covalent bond with the benzylguanine-labeled membrane. (B, C) SNAP-mEGFP conjugation to rhodamine-labeled liposomes is confirmed via size exclusion chromatography. mEGFP elutes with liposomes when fused to a SNAP tag and 18:1 PE PEG2000 benzylguanine lipid is present in liposome membranes (blue box) and free mEGFP elutes later (grey box). D) 62% of the benzylguanine lipid is bound to mEGFP after conjugation, or a ratio of 6E-4 mol protein to 1 mol of lipid, as determined by comparing Rhodamine lipid and mEGFP fluorescence in elution fractions to known standards. E) Liposome diameter before and after mEGFP conjugation, as measured by dynamic light scattering. Liposomes were composed of 68.9 mol% DOPC, 30 mol% cholesterol, 0.9 mol% 18:0 PEG2000, 0.1 mol% 18:1 PE PEG2000 benzylguanine, and 0.1 mol% 18:1 Rhodamine PE unless otherwise specified. All experiments were performed n=2, error bars represent the S.E.M.

Figure 2.

Cell-free expressed proteins can bind and localize lipid-based particles to cells. Binding of A) mEGFP, B) αHER2 affibody, C) computationally designed αCD3 miniprotein, and D) αCD3 scFv to Jurkat, MDA-MB-231, and SKBR3 cells in soluble and liposome-conjugated forms was assessed. Soluble proteins were labeled with fluorophore SNAP547 and liposomes were labeled with lipid-conjugated rhodamine. Binding of proteins was assessed via flow cytometry. Reported is the fold change in fluorescence of each condition relative to the fluorescence of unstained cells. E) Ratio of fold change of liposomes conjugated to protein to soluble protein shows vesicle conjugation enhances binding in most conditions. Conditions which should be positive for protein binding are outlined in red. Liposomes were composed of 68.9 mol% DOPC, 30 mol% cholesterol, 0.9 mol% 18:0 PEG2000, 0.1 mol% 18:1 PE PEG2000 benzylguanine, and 0.1 mol% 18:1 Rhodamine PE. All experiments were performed n=3, error bars represent the S.E.M.

Figure 3.

Functionalization is indiscriminate to functionalization strategy and particle type. SNAP-αHER2 affibody was conjugated to A) polymersomes and B) lipid nanoparticles (LNPs) via 18:1 PE PEG2000 benzylguanine. Particle binding to HER2-expressing SKBR3 cells was assessed via flow cytometry. C) The αHER2 affibody can be functionalized to liposomes via genetic-fusion to a transmembrane domain. Plots report the fold change in median fluorescence intensity (MFI) over an unstained control. Polymersomes were composed of 99.8 mol% poly(ethylene oxide)-b-poly butadiene, 0.1 mol% 18:1 PE PEG2000 benzylguanine, and 0.1 mol% 18:1 rhodamine. LNPs were composed of 50 mol% D-Lin-MC3-DMA, 10 mol% DSPC, 38.9 mol% cholesterol, 0.9 mol% 18:0 PEG2000, 0.1 mol% 18:1 PE PEG2000 benzylguanine, and 0.1 mol% 18:1 Rhodamine PE. Liposomes used in transmembrane domain conjugation experiments were composed of 69.9 mol% DOPC, 30 mol% cholesterol, and 0.1 mol% 18:1 Rhodamine PE. All experiments were performed n=3, error bars represent the S.E.M.

Figure 4.

Protein functionalized liposomes as artificial antigen presenting cells (aAPCs) to activate T cells. A) A T cell line which expresses GFP upon activation was used to assess cell-free protein-conjugated liposomes ability to interact with and activate T cells. B) NFAT GFP-Jurkat cells express GFP when incubated with liposomes conjugated to αCD3 scFv but not when incubated with only liposomes or only protein. Increasing the C) liposome dose and D) the amount of protein conjugated to liposomes enhances the amount of GFP expressed. Red point corresponds to condition in (B). E) Functionalizing liposomes with αCD3 and αCD2 scFv enhances activation above αCD3 alone. All experiments were performed n=3, error bars represent the S.E.M.

Figure 5.

Protein functionalized lipid nanoparticles can deliver mRNA cargo to T cells. A) The ability of lipid nanoparticles functionalized with cell-free expressed proteins to functionally deliver mRNA to T cells was assessed. B) Microscopy images of T cells treated with lipid nanoparticles encapsulating eGFP mRNA functionalized with no protein or αCD3 scFv after 24 hours. Scale bars are 100 μm. C) Lipid nanoparticles encapsulating Fluc mRNA were then functionalized with no protein, αCD3 scFv, αCD2 scFv, αCD3 scFv and αCD2 scFv, or αCD3 scFv and αHER2 affibody. Luciferase expression, as a result of mRNA delivery, was assessed 24 hours after dosing Jurkat cells with 1000 ng of mRNA. Lipid nanoparticles were composed of 50 mol% D-Lin-MC3-DMA, 10 mol% DSPC, 39 mol% cholesterol, 0.9 mol% 18:0 PEG2000, and 0.1 mol% 18:1 PE PEG2000 benzylguanine. Experiments were performed n=3, error bars represent the S.E.M.

Schematic 1.

Cell-free expressed proteins can enhance the functionality of membrane-based materials. Cell-free expressed protein and membrane-based nanoparticles can be produced in parallel and then conjugated to produce functionalized nanoparticles within a single day. These nanoparticles are amenable to a wide array of downstream therapeutic applications.