Current intracellular protein delivery strategies face the challenge of endosomal entrapment and consequent degradation of protein cargo. Methods to efficiently deliver proteins directly to the cytosol have the potential to overcome this hurdle. Here, we report the use of a straightforward approach of protein modification using citraconic anhydride to impart an overall negative charge on the proteins, enabling them to assemble with positively charged nano vectors. This strategy uses anhydride-modified proteins to electrostatically form polymer–protein nanocomposites with a cationic guanidinium-functionalized polymer. These supramolecular self-assemblies demonstrated the efficient cytosolic delivery of modified proteins through a membrane fusion-like mechanism. This approach was validated on five cell lines and seven proteins as cargo. Retention of protein function was confirmed through efficient cell killing via the intracellular enzymatic activity of RNase A. This platform provides a versatile, straightforward, and single-step method of protein modification and efficient direct cytosolic protein delivery.

Macrophages are multifunctional immune cells essential for both innate and adaptive immune responses. Tumor-associated macrophages (TAMs) often adopt a tumor-promoting M2-like phenotype, aiding tumor progression and immune evasion. Reprogramming TAMs to a tumoricidal M1-like phenotype is an emerging target for cancer immunotherapy. Resiquimod, a TLR7/8 agonist, can repolarize macrophages from the M2- to M1-like phenotype but is limited by poor solubility. We developed a gelatin nanoemulsion for the loading and delivery of resiquimod, utilizing eugenol oil as the liquid phase and riboflavin-crosslinked gelatin as a scaffold. These nanoemulsions showed high stability, low toxicity, and effective macrophage repolarization, significantly enhancing pro-inflammatory markers and anticancer activity in co-culture models.

Bio-orthogonal chemistry provides a powerful tool for drug delivery systems due to its ability to generate therapeutic agents in situ, minimizing off-target effects. Bio-orthogonal transition metal catalysts (TMCs) with stimuli-responsive properties offer possibilities for controllable catalysis due to their spatial-, temporal-, and dosage-controllable properties. In this paper, we fabricated a stimuli-responsive bio-orthogonal catalysis system based on an enhanced green fluorescent protein (EGFP)–nanozyme (NZ) complex (EGFP-NZ). Regulation of the catalytic properties of the EGFP-NZ complex was directly achieved by modulating the ionic strength of the solution. The dielectric screening introduced by salt ions allows the dissociation of the EGFP-NZ complex, increasing the access of substrate to the active site of the NZs and concomitantly increasing nanozyme activity. The change in catalytic rate of the NZ/EGFP = 1:1 complex was positively correlated with salt concentration from 0 mM to 150 mM.

Transition-metal catalysts (TMCs) can be encapsulated into nanomaterials to form bioorthogonal “nanozymes”. This encapsulation extends the lifetime of the catalyst, enhances water solubility, and can improve its catalytic performance. Several nanomaterials can act as host scaffolds. The choice of host nanomaterial impacts the properties of the respective nanozyme and provides modularity for engineering enhanced circulation time and targeting ability. This review examines various bioorthogonal nanozymes and their respective TMCs, discussing their advantages and disadvantages for bioorthogonal catalysis, and providing an outlook on their biomedical applications.

SiRNA delivery to target cellular processes is an appealing area in lung diseases such as asthma. To realize this potential requires efficient, targeted cytosolic delivery without cellular toxicity or siRNA degradation. We demonstrate the efficacy of PONI-Guan nanoparticles in accessing airway epithelial and smooth muscle cells with specific siRNA targeting of mRNAs of interest while maintaining barrier integrity and avoiding cellular toxicity.