Surface-Charge Tuned Polymeric Nanoemulsions for Carvacrol Delivery in Interkingdom Biofilms.

Biofilms, intricate microbial communities entrenched in extracellular polymeric substance (EPS) matrices, pose formidable challenges in infectious disease treatment, especially in the context of interkingdom biofilms prevalent in the oral environment. This study investigates the potential of carvacrol-loaded biodegradable nanoemulsions (NEs) with systematically varied surface charges─cationic guanidinium (GMT-NE) and anionic carboxylate (CMT-NE). Zeta potentials of +25 mV (GMT-NE) and -33 mV (CMT-NE) underscore successful nanoemulsion fabrication (∼250 nm). Fluorescent labeling and dynamic tracking across three dimensions expose GMT-NE's superior diffusion into oral biofilms, yielding a robust antimicrobial effect with 99.99% killing for both streptococcal and Candida species and marked reductions in bacterial cell viability compared to CMT-NE (∼4-log reduction). Oral mucosa tissue cultures affirm the biocompatibility of both NEs with no morphological or structural changes, showcasing their potential for combating intractable biofilm infections in oral environment. This study advances our understanding of NE surface charges and their interactions within interkingdom biofilms, providing insights crucial for addressing complex infections involving bacteria and fungi in the demanding oral context.

Non-viral vaccination through cationic guanidium polymer-pDNA polyplex mediated gene transfer.

Vaccination through cellular transfection of nucleotide-based vaccines is a powerful approach to combatting disease. Plasmid DNA (pDNA) vaccines are particularly promising vectors for non-viral immunomodulation that afford high degrees of potency and flexibility. Versatile guanidinium-functionalized poly(oxanorbornene)imide (PONI-Guan) homopolymers were used to facilitate non-disruptive pDNA condensation into discrete polyplexes, enabling efficient in vitro transfection of endothelial cells and HD-11 macrophages. Translation of these vectors for vaccination of white leghorn chickens against Newcastle disease virus (NDV) elicited strong humoral immune responses against the virus. This approach presents a highly versatile method for targeted immunomodulation in vivo, with the potential for translatability as a non-viral vaccine platform.

Engineered Polymer-siRNA Polyplexes Provide Effective Treatment of Lung Inflammation.

Uncontrolled inflammation is responsible for acute and chronic diseases in the lung. Regulating expression of pro-inflammatory genes in pulmonary tissue using small interfering RNA (siRNA) is a promising approach to combatting respiratory diseases. However, siRNA therapeutics are generally hindered at the cellular level by endosomal entrapment of delivered cargo and at the organismal level by inefficient localization in pulmonary tissue. Here we report efficient anti-inflammatory activity in vitro and in vivo using polyplexes of siRNA and an engineered cationic polymer (PONI-Guan). PONI-Guan/siRNA polyplexes efficiently deliver siRNA cargo to the cytosol for highly efficient gene knockdown. Significantly, these polyplexes exhibit inherent targeting to inflamed lung tissue following intravenous administration in vivo. This strategy achieved effective (>70%) knockdown of gene expression in vitro and efficient (>80%) silencing of TNF-α expression in lipopolysaccharide (LPS)-challenged mice using a low (0.28 mg/kg) siRNA dosage.

Direct Cytosolic Delivery of Citraconylated Proteins.

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.

Cytosolic Protein Delivery Using Modular Biotin-Streptavidin Assembly of Nanocomposites.

Current strategies for the delivery of proteins into cells face general challenges of endosomal entrapment and concomitant degradation of protein cargo. Efficient delivery directly to the cytosol overcomes this obstacle: we report here the use of biotin-streptavidin tethering to provide a modular approach to the generation of nanovectors capable of a cytosolic delivery of biotinylated proteins. This strategy uses streptavidin to organize biotinylated protein and biotinylated oligo(glutamate) peptide into modular complexes that are then electrostatically self-assembled with a cationic guanidinium-functionalized polymer. The resulting polymer-protein nanocomposites demonstrate efficient cytosolic delivery of six biotinylated protein cargos of varying size, charge, and quaternary structure. Retention of protein function was established through efficient cell killing via delivery of the chemotherapeutic enzyme granzyme A. This platform represents a versatile and modular approach to intracellular delivery through the noncovalent tethering of multiple components into a single delivery vector.

Direct Cytosolic Delivery of Proteins Using Lyophilized and Reconstituted Polymer-Protein Assemblies.

PURPOSE: Cytosolic delivery of proteins accesses intracellular targets for chemotherapy and immunomodulation. Current delivery systems utilize inefficient endosomal pathways of uptake and escape that lead to degradation of delivered cargo. Cationic poly(oxanorbornene)imide (PONI) polymers enable highly efficient cytosolic delivery of co-engineered proteins, but aggregation and denaturation in solution limits shelf life. In the present study we evaluate polymer-protein nanocomposite vehicles as candidates for lyophilization and point-of-care resuspension to provide a transferrable technology for cytosolic protein delivery. METHODS: Self-assembled nanocomposites of engineered poly(glutamate)-tagged (E-tagged) proteins and guanidinium-functionalized PONI homopolymers were generated, lyophilized, and stored for 2 weeks. After reconstitution and delivery, cytosolic access of E-tagged GFP cargo (GFPE15) was assessed through diffuse cytosolic and nuclear fluorescence, and cell killing with chemotherapeutic enzyme Granzyme A (GrAE10). Efficiency was quantified between freshly prepared and lyophilized samples. RESULTS: Reconstituted nanocomposites retained key structural features of freshly prepared assemblies, with minimal loss of material. Cytosolic delivery (> 80% efficiency of freshly prepared nanocomposites) of GFPE15 was validated in several cell lines, with intracellular access validated and quantified through diffusion into the nucleus. Delivery of GrAE10 elicited significant tumorigenic cell death. Intracellular access of cytotoxic protein was validated through cell viability. CONCLUSION: Reconstituted nanocomposites achieved efficient cytosolic delivery of protein cargo and demonstrated therapeutic applicability with delivery of GrAE10. Overall, this strategy represents a versatile and highly translatable method for cytosolic delivery of proteins.

Protein Delivery: If Your GFP (or Other Small Protein) Is in the Cytosol, It Will Also Be in the Nucleus.

Intracellular protein delivery is a transformative tool for biologics research and medicine. Delivery into the cytosol allows proteins to diffuse throughout the cell and access subcellular organelles. Inefficient delivery caused by endosomal entrapment is often misidentified as cytosolic delivery. This inaccuracy muddles what should be a key checkpoint in assessing delivery efficiency. Green fluorescent protein (GFP) is a robust cargo small enough to passively diffuse from the cytosol into the nucleus. Fluorescence of GFP in the nucleus is a direct readout for cytosolic access and effective delivery. Here, we highlight recent examples from the literature for the accurate assessment of cytosolic protein delivery using GFP fluorescence in the cytosol and nucleus.

Accessing Intracellular Targets through Nanocarrier-Mediated Cytosolic Protein Delivery.

Protein-based therapeutics have unique therapeutic potential due to their specificity, potency, and low toxicity. The vast majority of intracellular applications of proteins require access to the cytosol. Direct entry to the cytosol is challenging due to the impermeability of the cell membrane to proteins. As a result, multiple strategies have focused on endocytic uptake of proteins. Endosomally entrapped cargo, however, can have very low escape efficiency, with protein degradation occurring in acidic endolysosomal compartments. In this review, we briefly discuss endosomal escape strategies and review the strategy of cell membrane fusion, a recent strategy for direct delivery of proteins into the cell cytoplasm.

Delivery of drugs, proteins, and nucleic acids using inorganic nanoparticles.

Inorganic nanoparticles provide multipurpose platforms for a broad range of delivery applications. Intrinsic nanoscopic properties provide access to unique magnetic and optical properties. Equally importantly, the structural and functional diversity of gold, silica, iron oxide, and lanthanide-based nanocarriers provide unrivalled control of nanostructural properties for effective transport of therapeutic cargos, overcoming biobarriers on the cellular and organismal level. Taken together, inorganic nanoparticles provide a key addition to the arsenal of delivery vectors for fighting disease and improving human health.

In Vivo Editing of Macrophages through Systemic Delivery of CRISPR-Cas9-Ribonucleoprotein-Nanoparticle Nanoassemblies.

Macrophages are key effectors of host defense and metabolism, making them promising targets for transient genetic therapy. Gene editing through delivery of the Cas9-ribonucleoprotein (RNP) provides multiple advantages over gene delivery-based strategies for introducing CRISPR machinery to the cell. There are, however, significant physiological, cellular, and intracellular barriers to the effective delivery of the Cas9 protein and guide RNA (sgRNA) that have to date, restricted in vivo Cas9 protein-based approaches to local/topical delivery applications. Herein we describe a new nanoassembled platform featuring co-engineered nanoparticles and Cas9 protein that has been developed to provide efficient Cas9-sgRNA delivery and concomitant CRISPR editing through systemic tail-vein injection into mice, achieving >8% gene editing efficiency in macrophages of the liver and spleen.