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.

Melanoma, as the most aggressive and treatment-resistant skin malignancy, is responsible for about 80% of all skin cancer mortalities. Prone to invade into the dermis and form distant metastases significantly reduce the patient survival rate. Therefore, early treatment of the melanoma in situ or timely blocking the deterioration of metastases is critical. In this study, a sulfur dioxide (SO 2 ) polymer prodrug was designed as both an intracellular glutathione (GSH)-responsive SO 2 generator and a carrier of doxorubicin (DOX), and used for the treatment of subcutaneous and metastatic melanoma. Firstly, chemical conjugation of 4- N -(2,4-dinitrobenzenesulfonyl)-imino-1-butyric acid (DIBA) onto the side chains of methoxy poly (ethylene glycol) grafted dextran (mPEG- g -Dex) resulted in the synthesis of the amphiphilic polymer prodrug of SO 2 , mPEG- g -Dex (DIBA). The obtained mPEG- g -Dex (DIBA) could self-assemble into stable micellar nanoparticles and exhibited a glutathione-responsive SO 2 release behavior. Subsequently, DOX was encapsulated into the core of mPEG-g-Dex (DIBA) micelles to form DOX-loaded nanoparticles (PDDN-DOX). The formed PDDN-DOX could be internalized by B16F10 cells and synchronously release DOX and SO2 into the tumor cells. As a result, PDDN-DOX exerted synergistic anti-tumor effects in B16F10 melanoma cells because of the oxidative damage properties of SO 2 and toxic effects of DOX. Furthermore, in vivo experiments verified that PDDN-DOX had great potential for the treatment of subcutaneous and metastasis melanoma. Collectively, our present work demonstrates that the combination of SO 2 -based gas therapy and chemotherapeutics offers a new avenue for inhibiting melanoma progression and metastases.