Bioreducible Amphiphilic Hyperbranched Polymer-Drug Conjugate for Intracellular Drug Delivery.

This paper reports synthesis of a bioreducible hyperbranched (HB) polymer by A2+B3 approach from commercially available dithiothreitol (DTT) (A2) and an easily accessible trifunctional monomer (B3) containing three reactive pyridyl-disulfide groups. Highly efficient thiol-activated disulfide exchange reaction leads to the formation of the HB polymer (Mw = 21000; D = 2.3) with bioreducible disulfide linkages in the backbone and two different functional groups, namely, hydroxyl and pyridyl-disulfide in the core and periphery, respectively, of the HB-polymer. Postpolymerization functionalization of the hydroxyl-groups with camptothecin (CPT), a topoisomerase inhibitor and known anticancer drug, followed by replacing the terminal pyridyl-disulfide groups with oligo-oxyethylene-thiol resulted in easy access to an amphiphilic HB polydisulfide-CPT conjugate (P1) with a very high drug loading content of ∼40%. P1 aggregated in water (above ∼10 µg/mL) producing drug-loaded nanoparticles (Dh ∼ 135 nm), which showed highly efficient glutathione (GSH)-triggered release of the active CPT. Mass spectrometry analysis of the GSH-treated P1 showed the presence of the active CPT drug as well as a cyclic monothiocarbonate product, which underpins the cascade-degradation mechanism involving GSH-triggered cleavage of the labile disulfide linkage, followed by intramolecular nucleophilic attack by the in situ generated thiol to the neighboring carbonate linkage, resulting in release of the active CPT drug. The P1 nanoparticle showed excellent cellular uptake as tested by confocal fluorescence microscopy in HeLa cells by predominantly endocytosis mechanism, resulting in highly efficient cell killing (IC50 ∼ 0.6 µg/mL) as evident from the results of the MTT assay, as well as the apoptosis assay. Comparative studies with an analogous linear polymer-CPT conjugate showed much superior intracellular drug delivery potency of the hyperbranched polymer.

Antibody Polymer Conjugates (APCs) for Active Targeted Therapeutic Delivery.

Antibody drug conjugates (ADCs) are poised to have an enormous impact on targeted nanomedicine, especially in many cancer pathologies. The reach of the current format of ADCs is limited by their low drug-to-antibody ratio (DAR) because of the associated physiochemical instabilities. Here, we design antibody polymer conjugates (APCs) as a modular strategy to utilize polymers to address ADC's shortcomings. We show here that conjugation of polymer-based therapeutic molecules to antibodies helps increase the DAR, owing to the hydrophilic comonomer in the polymer that helps in masking the increased hydrophobicity caused by high drug loading. We show that the platform exhibits cell targetability and selective cell killing in multiple cell lines expressing disease-relevant antigens, viz., HER2 and EGFR. The ability to use different functionalities in the drug as the handle for polymer attachment further demonstrates the platform nature of APCs. The findings here could serve as an alternative design strategy for the next generation of active targeted nanomedicine.

Thiol-Disulfide Exchange as a Route for Endosomal Escape of Polymeric Nanoparticles.

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.

What's Next after Lipid Nanoparticles? A Perspective on Enablers of Nucleic Acid Therapeutics.

Recent success of mRNA-based COVID-19 vaccines have bolstered the strength of nucleic acids as a therapeutic platform. The number of new clinical trial candidates is skyrocketing with the potential to address many unmet clinical needs. Despite advancements in other aspects, the systemic delivery of nucleic acids to target sites remains a major challenge. Thus, nucleic acid based therapy has yet to reach its full potential. In this review, we shed light on a select few prospective technologies that exhibit substantial potential over traditional nanocarrier designs for nucleic acid delivery. We critically analyze these systems with specific attention to the possibilities for clinical translation.

Evaluation of Cellular Targeting by Fab' vs Full-Length Antibodies in Antibody-Nanoparticle Conjugates (ANCs) Using CD4 T-cells.

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 CD4high 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 CD4high 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 nanoparticle-conjugate format. Fab'-NCs are equally capable of achieving targeted drug delivery to CD4high T-cells as ANCs and are a versatile alternative to ANCs by offering site-selective modification strategy while retaining their advantages.

Influence of Polymer Structure and Architecture on Drug Loading and Redox-Triggered Release.

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.

1,6-heptadiynes based cyclopolymerization functionalized with mannose by post polymer modification for protein interaction.

Carbohydrate functionalized polymers or Glycopolymers have earned a great deal of interest in recent times for their potential biomedical applications. In the present study, a mannose containing glycopolymer was synthesized by cyclopolymerization of malonic acid derivative using second generation Hoveyda Grubbs' catalyst. Post-polymerization modification was done to install a propargyl moiety. Finally, functionalization of the propargylated polymer with 2-azidoethyl mannoside using azide-alkyne "click chemistry" furnished the target glycopolymer which was successfully characterized using NMR, FT-IR, mass spectroscopy and advanced polymer chromatography. The glycopolymer was found to self-assemble into capsule and spherical shape in water and DMSO respectively and these morphologies were observed through SEM and TEM. Upon interaction with Con A, the mannose containing glycopolymer showed an increment in aggregation induced fluorescence with increasing concentration of the lectin. In vitro cytotoxicity studies on MCF 7 cell line showed 90% cell viability up to glycopolymer concentration of 500 µg/mL.

Evaluating Endosomal Escape of Caspase-3-Containing Nanomaterials Using Split GFP.

The ability for biologics to access intracellular targets hinges on the translocation of active, unmodified proteins. This is often achieved using nanoscale formulations, which enter cells through endocytosis. This uptake mechanism often limits the therapeutic potential of the biologics, as the propensity of the nanocarrier to escape the endosome becomes the key determinant. To appropriately evaluate and compare competing delivery systems of disparate compositions, it is therefore critical to assess endosomal escape efficiencies. Unfortunately, quantitative tools to assess endosomal escape are lacking, and standard approaches often lead to an erroneous interpretation of cytosolic localization. In this study we use a split-complementation endosomal escape (SEE) assay to evaluate levels of cytosolic caspase-3 following delivery by polymer nanogels and mesoporous silica nanoparticles. In particular, we use SEE as a means to enable the systematic investigation of the effect of polymer composition, polymer architecture (random vs block), hydrophobicity, and surface functionality. Although polymer structure had little influence on endosomal escape, nanogel functionalization with cationic and pH-sensitive peptides significantly enhanced endosomal escape levels and, further, significantly increased the amount of nanogel per endosome. This work serves as a guide for developing an optimal caspase-3 delivery system, as this caspase-3 variant can be easily substituted for a therapeutic caspase-3 cargo in any system that results in cytosolic accumulation and cargo release. In addition, these data provide a framework that can be readily applied to a wide variety of protein cargos to assess the independent contributions of both uptake and endosomal escape of a wide range of protein delivery vehicles.

Charge-Conversion Strategies for Nucleic Acid Delivery.

Nucleic acids are now considered as one of the most potent therapeutic modalities, as their roles go beyond storing genetic information and chemical energy or as signal transducer. Attenuation or expression of desired genes through nucleic acids have profound implications in gene therapy, gene editing and even in vaccine development for immunomodulation. Although nucleic acid therapeutics bring in overwhelming possibilities towards the development of molecular medicines, there are significant loopholes in designing and effective translation of these drugs into the clinic. One of the major pitfalls lies in the traditional design concepts for nucleic acid drug carriers, viz. cationic charge induced cytotoxicity in delivery pathway. Targeting this bottleneck, several pioneering research efforts have been devoted to design innovative carriers through 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 interesting approaches.

Tracking exogenous intracellular casp-3 using split GFP.

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. endosomally-entrapped 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.

Synergistic Interplay of Covalent and Non-Covalent Interactions in Reactive Polymer Nanoassembly Facilitates Intracellular Delivery of Antibodies.

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. To address this, we outline an electrostatics-enhanced covalent self-assembly strategy to generate polymer-protein/antibody nanoassemblies. Through structure-activity studies, we down-select the best performing self-immolative pentafluorophenyl containing activated carbonate polymer for bioconjugation. With the help of an electrostatics-aided covalent self-assembly approach, we demonstrate efficient encapsulation of medium to large proteins (HRP, 44 kDa and ß-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 towards binding intracellular protein epitopes and inducing apoptosis.