Abstract Small interfering RNA (siRNA) is an emerging therapeutic modality for a variety of diseases, including cancer, as siRNA can silence target genes in a sequence-specific manner. The effective delivery of siRNA remains a major challenge due to rapid clearance by macrophages in the systemic environment. Nonspecific interactions with the serum proteins in the bloodstream contribute to macrophage uptake, limiting circulation time, thereby reducing the effective delivery of siRNA to the target site. Here, we report the efficient delivery of siRNA to cancer cells using hyaluronic acid (HA)-coated cationic polymeric nanovector (PONI-Guan)/siRNA polyplexes. The guanidinium-functionalized polymers self-assemble with siRNA and enable cytosolic delivery. HA serves as a noninteracting protective shield on the polyplexes that prevent macrophage uptake in vitro . These nanovectors facilitate efficient siRNA delivery to 4T1 triple-negative breast cancer cells in vitro , with a 4:1 selectivity relative to macrophages. Further, HA-coated polyplexes demonstrated efficient STAT3 gene knockdown (~50%) in 4T1 cells. Intravenous administration of HA-coated polyplexes in 4T1 tumor-bearing mice showed significantly (~50%) decreased accumulation in clearance organs, in comparison to the PONI-Guan polyplexes. Collectively, HA-coated polyplexes provide an effective strategy for selective siRNA delivery to tumor cells while avoiding macrophage uptake.

Small interfering RNAs (siRNAs) are powerful tools to target cellular protein expression, making them promising candidates for therapeutic applications. siRNA-based approaches to target detrimental mechanisms in airway diseases such as asthma and chronic obstructive pulmonary disease are highly appealing. However, such delivery systems must be nontoxic, protect siRNAs from degradation, and enable intracellular uptake that accesses cytosolic RNA machinery. The present study examines the mechanisms by which guanidinium-functionalized poly(oxanorbornene)imide polymer (PONI-Guan) nanoparticles can effectively and safely deliver siRNA in human bronchial epithelial (BEC) and airway smooth muscle cells (ASM). PONI-Guan polymers were engineered to self-assemble with siRNA through electrostatic interactions. Primary BEC and ASM cells were preincubated with methyl-β-cyclodextrin, dynasore, dansylcadaverine chlorpromazine, latrunculin B, or cytochalasin D followed by incubation with nanoparticles at a single concentration but different guanidinium/phosphate ratios (G/P). BEC and ASM treated with methyl-β-cyclodextrin and dynasore demonstrated significant decrease in nanoparticle uptake. In addition, BEC showed decreased uptake with latrunculin B. Minimal BEC toxicity was observed with 20, 30, and 40 G/P ratios; ASM showed some toxicity with 40 G/P. Transepithelial electrical resistance readings were stable with 20 and 30 G/P, whereas 40 G/P showed significant but transient changes, with barrier integrity restored in ∼6 h. Furthermore, 30 G/P did not induce markers of necrosis, apoptosis, or inflammation in BEC or ASM. Transfection of BDNF siRNA in ASM and Arginase 1 and 2 siRNA in BEC showed significant decrease in corresponding mRNA and protein expression. Overall, these data indicate that PONI-Guan polymers can deliver siRNA safely and effectively in bronchial cells, primarily through caveolar or macropinocytosis uptake, offering a promising tool for future siRNA-based therapies.NEW & NOTEWORTHY 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.

Bioorthogonal nanozymes offer in situ activation of pro-dyes and prodrugs using abiotic chemical transformations. Bacterial infections, especially biofilm-associated infections, are extremely difficult to treat due to obstacles such as poor antibiotic penetration and the rising threat of antibiotic resistance. Spatiotemporal control of bioorthogonal catalysis provides a strategy for "on-demand" generation of therapeutics, effectively localizing therapeutic action and minimizing side effects. Here, we present the fabrication of visible-light-responsive alginate hydrogel beads embedded with bioorthogonal polyzymes (PZs). Exposure to a 405 nm light induces the reduction of Fe(III) to Fe(II), triggering the dissolution of the PZ-gel beads with concomitant release and activation of the polyzyme. This approach enabled the selective activation of a prodrug of Linezolid, a last-in-line antibiotic for Gram-positive bacterial infections, enabling the targeted eradication of multidrug-resistantStaphylococcus aureus biofilms. Overall, the use of alginate biomaterial along with noninvasive visible light offers a nontoxic platform for spatiotemporal release of antibiotics through bioorthogonal activation.

Rationale : Airway remodeling in asthma involves increased airway smooth muscle (ASM) mass, changes to bronchial epithelial cell (BEC) layer, and increased fibrosis. ASM and BECs actively contribute by secreting growth factors and cytokines, regulating extracellular matrix (ECM) composition and enhancing cell proliferation/migration. Altering mechanisms which modulate remodeling presents a key to novel therapeutic strategies. BDNF increases ASM [Ca2+]i and contractility, and potentiates the effects of inflammation. Importantly, studies show that ASM is not only a target, but also a source of BDNF, raising the possibility of local autocrine/paracrine effects. Arginase 1 and 2 are expressed by BECs, but their roles in asthma are still under investigation with studies showing arginase 1 inhibition blunting epithelial remodeling and ASM hyperplasia. Knockdown of appealing targets such as ASM BDNF and epithelial arginases using small interfering RNA (siRNA) is a promising approach to combating inflammatory lung remodeling. Polymer based nanoparticles have been constructed which demonstrate cell-type specificity and high efficiency in delivering siRNA. Thus, we hypothesize that BDNF and arginases are central to airway remodeling and can be targeted via nanopartiacles.Methods: Primary human ASM and BEC cells from non-asthmatic vs. asthmatic individuals were obtained post-surgery from lung tissues after approval Mayo Clinic IRB. PONI-Guan NP were generated using block copolymer engineering. PCR, immunoblotting and ELISA were performed using standard techniques. Contractility studies were conducted in adult mouse precision cut lung slices. In vivo lung function studies were conducted in 6-8 week old mice, with or without mixed allergen (MA) treatment and Scireq Flexivent system. Results: PONI-Guan NP uptake demonstrates cell-type specificity in BEC/ASM co-culture and mouse lung. Arginase I/II and ECM expression is upregulated in BECs when exposed to BDNF and cytokines. In ASM, BDNF is increased in asthmatics compared to non-asthmatic controls; ECM expression is increased in response to BDNF and TNF-α. In BEC, PONI-Guan NP/siRNA inhibits Arg I/II expression, even in the presence of TNF-α. In ASM, TNF-α increases in BDNF secretion and ECM deposition are blunted by NP-delivered siRNA. PCLS and mouse lung function studies show contractility to MCh: effects suppressed by NP-delivered BDNF and Arg I siRNA. Conclusions: Our studies show the importance of BDNF/Arginase pathways in airway remodeling and efficacy of PONI-based siRNA delivery vectors in blunting remodeling processes.

siRNA enables highly specific and targeted gene silencing, offering potential treatment for a range of diseases. Cytosolic access of siRNA is essential for efficacy; Current delivery systems generally use endosomal uptake pathways, leading to siRNA degradation due to inefficient escape. Guanidinium functionalized poly(oxanorbornene)imide (PONI) polymers facilitate direct cytosolic siRNA delivery with excellent gene knockdown efficacy in vitro and in vivo. The use of lyophilization to generate stable powders that retain excellent delivery and knockdown activity when reconstituted is demonstrated, providing a key tool for translation. PONI-Guan polymers were mixed with siRNA to form PONI-Guan/siRNA polyplexes. The generated polyplexes were lyophilized and stored at varying temperature conditions for a total duration of 4 weeks. After reconstitution and delivery, cytosolic access of siRNA was assessed through confocal laser scanning microscopy. Knockdown efficacy was assessed in GFP expressing reporter deGFP HEK 293 T cell line using flow cytometry. Efficacy of reconstituted PONI-Guan/si_STAT3 in 4T1 breast cancer cells was evaluated by quantifying gene expression levels (qRT-PCR) and cell growth inhibition (Alamar blue assay). Delivery and therapeutic efficiency were compared between lyophilized and freshly made polyplexes. Lyophilized polyplexes retained critical functional features of freshly made polyplexes. Resuspended polyplexes facilitated effective cytosolic delivery siRNA and showed therapeutic relevance through the delivery of siRNA targeting STAT-3 gene in 4T1 cells with successful cell growth inhibition ( 70

Uncontrolled inflammation is the driver of numerous lung diseases. Current treatments, including corticosteroids and bronchodilators, can be effective. However, they often come with notable side effects. siRNA is a promising therapeutic modality for immune regulation. However, effective delivery of siRNA is challenged by issues related to cellular uptake and localization within tissues. This study investigates a series of guanidinium-functionalized polymers (Cn-Guan) designed to explore the effects of amphiphilicity on siRNA complexation and efficiency in vitro and in vivo. Nine polymers with varying side chain lengths (C3, C5, C7) and molecular weights (17 kDa, 30 kDa, 65 kDa) were synthesized, forming polyplexes with siRNA. Characterization revealed that C7-Guan/si_scr polymers exhibited the smallest polyplex sizes and the tightest complexation with siRNA. In vitro studies showed that 65 kDa polymers had the highest gene knockdown efficiency, with C3 and C5-Guan/si_TNF-α achieving ∼70 % knockdown, while C7-Guan/si_TNF-α achieved ∼30 %. In vivo, C7-Guan/Cy5-siRNA demonstrated the highest lung accumulation, and all polymers showed ∼70 % TNF-α knockdown with a low siRNA dosage (0.14 mg/kg) in a murine lung inflammation model. C7-Guan polymers, despite lower in vitro efficiency, were quite effective in vivo, potentially due to enhanced serum stability. These findings demonstrate that Cn-Guan/siRNA polyplexes are effective and safe for attenuating pulmonary inflammation and provide important insights for the development of future siRNA delivery vectors for lung disease treatment.

Multidrug-resistant bacterial infections present a serious global public health threat. This threat is exacerbated by biofilm-forming bacteria that have greater intrinsic resistance to various classes of currently available antibiotics. This study reports the fabrication of copper nanoparticles (CuNPs) by using a cell-free lysate of Aspergillus niger (A. niger). A library of CuNPs was generated by varying different parameters including salt (CuSO4) concentration, fungal lysate-to-salt ratio, pH, temperature, and exposure time and screened based on yield, size, and charge. The minimum inhibitory concentrations (MICs) of CuNPs were 4-fold lower for Gram-negative as compared to Gram-positive bacteria. CuNPs were able to effectively penetrate the dense structure of biofilms leading to the disruption of the bacterial cell membrane integrity as confirmed by confocal microscopy. CuNPs exhibited enhanced selectivity toward biofilms of Gram-negative bacteria, with low mammalian cell toxicity. CuNPs generated from A. niger lysate provided an effective option for the treatment of biofilm infections, addressing the critical need for combating drug-resistant bacteria.

Proteins and nucleic acid therapeutics represent a significant and growing share of the pharmaceutical landscape. The majority of biological and therapeutic applications of these biomolecules require access to the cytosol. Delivery of biologics directly to the cytosol is made difficult by the impermeability of the cell membrane. As a result, most delivery strategies have utilized endocytic uptake pathways to deliver biologics into the cell. However, endosomally entrapped cargo often faces limited escape efficiency and is prone to degradation within endo/lysosomal compartments. The emergence of delivery vehicles capable of bypassing endocytosis and directly traversing the cell membrane offers a promising approach to improve the cytosolic delivery efficiency of biomolecules. Here, we highlight recent developments in endocytosis-independent delivery systems for biologics and ways to accurately assess cytosolic delivery of biologics. Strategies employing covalent and non-covalent modification of biomolecules will be reviewed, along with strategies incorporating both covalent and supramolecular processes.

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.

Synthetic polymer scaffolds can encapsulate transition metal catalysts (TMCs) to provide bioorthogonal nanocatalysts. These "polyzymes" catalyze the in situ generation of therapeutic agents without disrupting native biological processes. The design and modification of polymer scaffolds in these polyzymes can enhance the catalytic performance of TMCs in biological environments. In this study, we explore the hydrophobic design space of an oxanorborneneimide-based polymer by varying the length of its carbon side chain to engineer bioorthogonal polyzymes. Activity studies indicate that modulating the hydrophobicity of the polymer scaffold can be used to enhance the catalyst loading efficacy, catalytic activity, and serum stability of polyzymes. These findings provide insight into the structural elements contributing to improving polymeric nanocatalysts for a variety of applications. image

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.

Self-assembly of siRNA with a block copolymer featuring guanidinium and zwitterion functionalized blocks generates core-shell-like nanovectors that provide cytosolic access to siRNA and efficiently evade phagocytic clearance. The guanidinium-functionalized inner block complexes siRNA in the nanovector interior and enables cytosolic delivery. The zwitterionic outer block provides a non-interacting shell on the nanovectors that reduces macrophage uptake in vitro and phagocytic clearance and enhances tumor localization in vivo. These nanovectors were used to treat a 4T1 (murine) model of triple-negative breast cancer (TNBC). The nanovectors deliver siRNA efficiently to 4T1 triple-negative breast cancer cells in vitro, with high selectivity relative to macrophages. This efficiency and selectivity translate into in vivo efficacy: diblock nanovectors evaded phagocytic clearance and efficiently localized in an aggressive murine 4T1 orthotopic model, with a ~3-fold increase of vector residing in the tumor compared to the homopolymer nanovectors. This increased localization efficiently knocked down STAT3 (~80%) and provided tumorostasis (100% growth inhibition) at a low dose of 0.14 mg/kg. The in vitro and in vivo efficacy of these nanovectors demonstrate the potential of engineered polymer architectures to generate effective self-assembled siRNA therapeutics that avoid phagocytic clearance for the treatment of diseases requiring systemic administration.

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.

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.

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.

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.

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. 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. 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. 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.

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.

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.