Lipo-Oligomer Nanoformulations for Targeted Intracellular Protein Delivery

Abstract

Here, we report novel lipo-oligoaminoamide nanoformulations for targeted intracellular protein delivery. Formulations are generated by first bioreversibly conjugating a sequence-defined amphiphilic lipo-oligomer 728 to the cargo protein via disulfide bonds, followed by formulation of the formed 728-SS-protein conjugate with different helper lipids in various compositions. The triblock oligoaminoamide 728 contains cysteines for reversible covalent protein conjugation and cross-link-stabilization of formed nanoparticles, polyethylene glycol (PEG) for shielding, and providing a hydrophilic domain, eight cationizable succinoyl tetraethylene pentamine (Stp) repeats for endosomal buffering and escape into the cytosol, and a tetra-oleic acid block for hydrophobic stabilization. The added helper lipids are supposed to enhance serum stability of the nanoparticles and provide targeting by lipid-anchored folic acid (FA)-PEG. The optimized protein nanoparticles, including 728, DOPS, cholesterol, DMPE-PEG2000, and the FA-PEG conjugated lipid 1042, presented a high colloidal stability without significant size increase in 72 h. Using cytotoxic ribonuclease A (RNase A) as cargo protein, FA-728-DOPS-DMPE-RNase A nanoformulation could be identified with highest potency of targeted RNase A-mediated folate-receptor-positive KB carcinoma cell killing among all tested formulations, resulting in 85% KB cell killing at a low concentration of 2 μM. These approximately 50 nm sized nanoparticles induced superior 70% KB cell killing even in the presence of 20% serum. Efficient targeted cytosolic delivery by coformulation with helper lipids was also demonstrated by FA-728-DOPS-DMPE-nlsEGFP nanoformulation using enhanced green fluorescent protein (EGFP) as cargo. Furthermore, partial nlsEGFP was imported into the nuclei of KB cells, validating effective endosomal escape, and following nuclear transport mediated by nuclear localization signal on nlsEGFP. As demonstrated, the screening and optimization of nanoformulations with helper lipids and coformulation agents is considered to be an important and rational next step in the development of intracellular biopharmaceuticals, following initial protein conjugate synthesis.

Microfluidics‐Assisted Engineering of pH/Enzyme Dual‐Activatable ZIF@Polymer Nanosystem for Co‐Delivery of Proteins and Chemotherapeutics with Enhanced Deep‐Tumor Penetration

“…[7][8][9][10][11] Nonetheless, the therapeutic applications of nanocarriers engineered to deliver protein drugs are still limited due to their rapid clearance from the peripheral circulation after systemic administration or limited penetration into solid tumors. [12][13][14][15] Furthermore, compared to protein-based therapeutics alone, combination cancer therapy with protein and smallmolecule drugs holds great promise, which has been confirmed by previous observations of greater therapeutic effects while avoiding side effects in clinical settings. [16,17] However, co-delivering proteins and chemotherapeutic drugs entails increased difficulty when implementing antitumor treatment strategies, since both types of therapeutics had substantially different molecular weights and physicochemical properties, making it difficult to combine them into a single drug-delivery system.…”

Non-viral delivery of the CRISPR/Cas system: DNAversusRNAversusRNP

“…Therefore, the development of generic strategies for protein delivery is sophisticated, and in many cases individual carrier design and optimization are required. Despite physical entrapment in liposomes, conjugation of cell penetrating peptides (CPPs) or protein transduction domains (PTDs) [34][35][36] as well as nanomicelles, [37][38][39] engineered nanocarriers 40,41 and other strategies have been used for cellular protein delivery. Since classic nucleic acid transfecting agents, cationic polymers or lipids, generally only work with negatively charged proteins, a very flexible strategy is rep-resented by charge-conversion.…”

Transduction Methods for Cytosolic Delivery of Proteins and Bioconjugates into Living Cells

“…[124] Further sophistication of this technique includes formulating lipid-oligomer nanoparticle complexes, where the added helper lipids enhance the serum stability of nanoparticles. [125]…”

“…In principle, this nanoparticle strategy may allow whole organism protein targeting. [125] The chemistry of the shell surrounded protein nanocapsules was further investigated by Yan et al (Figure 10A). To control the formation of the shell around each single-protein, several types of vinyl monomers were condensed onto lysine side chains of proteins by amide bond formation.…”

“…These particles were effectively internalized into cells, releasing the nanobody into the cytosol, where it bound both GFP and GFP‐fused proteins. In principle, this nanoparticle strategy may allow whole organism protein targeting …”