The primary impediments in developing large antibodies as drugs against intracellular targets involve their low transfection efficiency and suitable reversible encapsulation strategies for intracellular delivery with retention of biological activity.T oa ddress this,w eo utline an electrostatics-enhanced covalent self-assembly strategy to generate polymer-protein/antibody nanoassemblies.Through structureactivity studies,w ed own-select the best performing selfimmolative pentafluorophenyl containing activated carbonate polymer for bioconjugation. With the help of an electrostaticsaided covalent self-assembly approach,w ed emonstrate efficient encapsulation of medium to large proteins (HRP,44kDa and b-gal, 465 kDa) and antibodies (ca. 150 kDa). The designed polymeric nanoassemblies are shown to successfully traffic functional antibodies (anti-NPC and anti-pAkt) to cytosol to elicit their bioactivity towardsb inding intracellular protein epitopes and inducing apoptosis.

The newly developed polymeric nanocarrier could open a new avenue for cancer therapy, due to its unique design as well as, most importantly, its biocompatible and biodegradable nature.

Nucleic acids are considered as one of the most potent therapeutic modalities, as their roles go beyond storing genetic information and chemical energy or as signal transducers. Attenuation or expression of desired genes through nucleic acids have profound implications in gene therapy, gene editing, and even in vaccine development. Although nucleic acid therapeutics bring in overwhelming possibilities toward the development of molecular medicines, there are significant loopholes in their effective clinical translation. One of the major pitfalls lies in the traditional design concepts of nucleic acid drug carriers, namely, cationic charge induced cytotoxicity. Targeting this bottleneck, several innovative carrier designs have been proposed accommodating charge‐conversion approaches, whereby built‐in functionalities convert from cationic to neutral or anionic, or even from anionic to cationic enabling the carrier to overcome several critical barriers for therapeutics delivery, such as serum deactivation, instability in circulation, low transfection, and poor endosomal escape. This review will critically analyze various molecular designs of charge‐converting nanocarriers in a classified approach for the successful delivery of nucleic acids. Accompanied by the narrative on recent clinical nucleic acid candidates, the review concludes with a discussion on the pitfalls and scope of these emerging approaches.

Cytosolic protein delivery promises diverse applications from therapeutics, to genetic modification and precision research tools. To achieve effective cellular and subcellular delivery, approaches that allow protein visualization and accurate localization with greater sensitivity are essential. Fluorescently tagging proteins allows detection, tracking and visualization in cellulo. However, undesired consequences from fluorophores or fluorescent protein tags, such as nonspecific interactions and high background or perturbation to native protein's size and structure, are frequently observed, or more troublingly, overlooked. Distinguishing cytosolically released molecules from those that are endosomally entrapped upon cellular uptake is particularly challenging and is often complicated by the inherent pH-sensitive and hydrophobic properties of the fluorophore. Monitoring localization is more complex in delivery of proteins with inherent protein-modifying activities like proteases, transacetylases, kinases, etc. Proteases are among the toughest cargos due to their inherent propensity for self-proteolysis. To implement a reliable, but functionally silent, tagging technology in a protease, we have developed a caspase-3 variant tagged with the 11th strand of GFP that retains both enzymatic activity and structural characteristics of wild-type caspase-3. Only in the presence of cytosolic GFP strands 1-10 will the tagged caspase-3 generate fluorescence to signal a non-endosomal location. This methodology facilitates easy screening of cytosolic vs. endosomallyentrapped proteins due to low probabilities for false positive results, and further, allows tracking of the resultant cargo's translocation. The development of this tagged casp-3 cytosolic reporter lays the foundation to probe caspase therapeutic properties, charge-property relationships governing successful escape, and the precise number of caspases required for apoptotic cell death.

Disulfide cross-linked nanoassemblies have attracted considerable attention as a drug delivery vehicle due to their responsiveness to the natural redox gradient in biology. Fundamentally understanding the factors that influence the drug loading capacity, encapsulation stability, and precise control of the liberation of encapsulated cargo would be profoundly beneficial to redox-responsive materials. Reported herein are block copolymer (BCP)-based self-cross-linked nanogels, which exhibit high drug loading capacity, high encapsulation stability, and controllable release kinetics. BCP nanogels show considerably higher loading capacity and better encapsulation stability than the random copolymer nanogels at micromolar glutathione concentrations. By partially substituting thiol-reactive pyridyl disulfide into the unreactive benzyl or butyl group, we observed opposite effects on the cross-linking process of BCP nanogels. We further studied the redox-responsive cytotoxicity of our drug-encapsulated nanogels in various cancer cell lines.

Targeted delivery of chemotherapeutic drugs can improve their therapeutic efficiency by localizing their toxic effects at the diseased site. This is often achieved either by direct conjugation of drugs to antibodies targeting overexpressed receptors on cancer cells (antibody−drug conjugates/ADCs) or by conjugating antibodies to nanoparticles bearing drugs (antibody−nanoparticle conjugates/ANCs). Here, we report a platform for utilizing hinge cysteines on antigen-binding fragment (Fab′) of an anti-CD4 antibody for site-specific conjugation to nanoparticles giving rise to anti-CD4 Fab′-nanoparticle conjugates (Fab′-NCs). We demonstrate a convenient route for obtaining functional anti-CD4 Fab′ from full-length antibody and examine the targeted delivery efficiencies of anti-CD4 Fab′-NCs vs ANCs for selective delivery to CD4 high mT-ALL cells. Our results indicate that higher avidity of full-length anti-CD4 antibody, i.e., protein alone translated to higher binding ability to CD4 high mT-ALL cells in comparison with anti-CD4 Fab′ alone. However, the targeted delivery efficiency of anti-CD4 Fab′-NCs was comparable to ANCs indicating that the avidity of Fab′ is restored in a nanoparticleconjugate format. Fab′-NCs are equally capable of achieving targeted drug delivery to CD4 high T-cells as ANCs and are a versatile alternative to ANCs by offering site-selective modification strategy while retaining their advantages.

Endosomal entrapment has remained the major bottleneck for cytosolic delivery of nanoparticle‐based delivery systems. Uncovering fundamentally new pathways for endosomal escape is therefore highly sought. Herein, we report that disulfide bonds can enhance endosomal escape through contacts with cellular exofacial thiols, in addition to facilitating cellular uptake. Our results are supported through comparative analysis of polymeric nanogels with variable accessibility to disulfide bonds by placing these functionalities at the core or the shell of the nanogels. The findings here inform future chemical design of delivery vehicles.

The primary impediments in developing large antibodies as drugs against intracellular targets involve their low transfection efficiency and suitable reversible encapsulation strategies for intracellular delivery with retention of biological activity.T oa ddress this,w eo utline an electrostatics-enhanced covalent self-assembly strategy to generate polymer-protein/antibody nanoassemblies.Through structureactivity studies,w ed own-select the best performing selfimmolative pentafluorophenyl containing activated carbonate polymer for bioconjugation. With the help of an electrostaticsaided covalent self-assembly approach,w ed emonstrate efficient encapsulation of medium to large proteins (HRP,44kDa and b-gal, 465 kDa) and antibodies (ca. 150 kDa). The designed polymeric nanoassemblies are shown to successfully traffic functional antibodies (anti-NPC and anti-pAkt) to cytosol to elicit their bioactivity towardsb inding intracellular protein epitopes and inducing apoptosis.