Carboxylated Nanoparticle Surfaces Enhance Association with Mucoid Pseudomonas aeruginosa Biofilms.

Pseudomonas aeruginosa biofilms comprise three main polysaccharides: alginate, psl, and pel, which all imbue tolerance against exogenous antimicrobials. Nanoparticles (NPs) are an exciting new strategy to overcome the biofilm matrix for therapeutic delivery applications; however, zero existing FDA approvals for biofilm-specific NP formulations can be attributed to the complex interplay of physiochemical forces at the biofilm-NP interface. Here, we leverage a set of inducible, polysaccharide-specific, expressing isogenic P. aeruginosa mutants coupled with an assembled layer-by-layer NP (LbL NP) panel to characterize biofilm-NP interactions. When investigating these interactions using confocal microscopy, alginate-layered NPs associated more than dextran-sulfate-layered NPs with biofilms that had increased alginate production, including biofilms produced by mucoid P. aeruginosa isolates from people with cystic fibrosis. These differences were further confirmed in LbL NPs layered with polysaccharide- or hydrocarbon-based polymers with pendent carboxylate or sulfate functional groups. These data suggest carboxylated NP surfaces have enhanced interactions specifically with mucoid biofilms as compared to sulfated surfaces and lay the foundation for their inclusion as a design element for increasing biofilm-NP interactions and efficacious drug delivery.

Electrostatically assembled wound dressings deliver pro-angiogenic anti-miRs preferentially to endothelial cells.

Chronic non-healing wounds occur frequently in individuals affected by diabetes, yet standard-of-care treatment leaves many patients inadequately treated or with recurring wounds. MicroRNA (miR) expression is dysregulated in diabetic wounds and drives an anti-angiogenic phenotype, but miRs can be inhibited with short, chemically-modified RNA oligonucleotides (anti-miRs). Clinical translation of anti-miRs is hindered by delivery challenges such as rapid clearance and uptake by off-target cells, requiring repeated injections, excessively large doses, and bolus dosing mismatched to the dynamics of the wound healing process. To address these limitations, we engineered electrostatically assembled wound dressings that locally release anti-miR-92a, as miR-92a is implicated in angiogenesis and wound repair. In vitro, anti-miR-92a released from these dressings was taken up by cells and inhibited its target. An in vivo cellular biodistribution study in murine diabetic wounds revealed that endothelial cells, which play a critical role in angiogenesis, exhibit higher uptake of anti-miR eluted from coated dressings than other cell types involved in the wound healing process. In a proof-of-concept efficacy study in the same wound model, anti-miR targeting anti-angiogenic miR-92a de-repressed target genes, increased gross wound closure, and induced a sex-dependent increase in vascularization. Overall, this proof-of-concept study demonstrates a facile, translational materials approach for modulating gene expression in ulcer endothelial cells to promote angiogenesis and wound healing. Furthermore, we highlight the importance of probing cellular interactions between the drug delivery system and the target cells to drive therapeutic efficacy.

pH-Responsive, Charge-Reversing Layer-by-Layer Nanoparticle Surfaces Enhance Biofilm Penetration and Eradication.

Microbes entrenched within biofilms can withstand 1000-fold higher concentrations of antibiotics, in part due to the viscous extracellular matrix that sequesters and attenuates antimicrobial activity. Nanoparticle (NP)-based therapeutics can aid in delivering higher local concentrations throughout biofilms as compared to free drugs alone, thereby enhancing the efficacy. Canonical design criteria dictate that positively charged nanoparticles can multivalently bind to anionic biofilm components and increase biofilm penetration. However, cationic particles are toxic and are rapidly cleared from circulation in vivo, limiting their use. Therefore, we sought to design pH-responsive NPs that change their surface charge from negative to positive in response to the reduced biofilm pH microenvironment. We synthesized a family of pH-dependent, hydrolyzable polymers and employed the layer-by-layer (LbL) electrostatic assembly method to fabricate biocompatible NPs with these polymers as the outermost surface. The NP charge conversion rate, dictated by polymer hydrophilicity and the side-chain structure, ranged from hours to undetectable within the experimental timeframe. LbL NPs with an increasingly fast charge conversion rate more effectively penetrated through, and accumulated throughout, wildtype (PAO1) and mutant overexpressing biomass (ΔwspF) Pseudomonas aeruginosa biofilms. Finally, tobramycin, an antibiotic known to be trapped by anionic biofilm components, was loaded into the final layer of the LbL NP. There was a 3.2-fold reduction in ΔwspF colony forming units for the fastest charge-converting NP as compared to both the slowest charge converter and free tobramycin. These studies provide a framework for the design of biofilm-penetrating NPs that respond to matrix interactions, ultimately increasing the efficacious delivery of antimicrobials.

Surface Presentation of Hyaluronic Acid Modulates Nanoparticle-Cell Association.

Nanoparticle (NP) drug carriers have revolutionized medicine and increased patient quality of life. Clinically approved formulations typically succeed because of reduced off-target toxicity of the cargo. However, increasing carrier accumulation at disease sites through precise targeting remains one of the biggest challenges in the field. Novel multivalent ligand presentations and self-assembled constructs can enhance cell association, but an inability to draw direct comparisons across formulations has hindered progress. Furthermore, how nanoparticle structure influences function often is unclear. In this report, we leverage the well-characterized hyaluronic acid (HA)-CD44 binding pair to investigate how the surface architecture of modified NPs impacts their association with ovarian cancer cells that overexpress CD44. We functionalized anionic liposomes with 5 kDa HA by either covalent conjugation via surface coupling or electrostatic self-assembly using the layer-by-layer (LbL) adsorption method. Comparing these two methods, we observed a consistent enhancement of NP-cell association with the self-assembly LbL technique, particularly with higher molecular weight (≥10 kDa) HA. To further optimize association, we increased the surface-available HA. We synthesized a bottlebrush glycopolymer composed of a polynorbornene backbone and pendant 5 kDa HA and layered this macromolecule onto NPs. Flow cytometry revealed that the LbL HA bottlebrush NP outperformed the LbL linear display of HA. Cellular visualization by deconvolution optical microscopy corroborated results from all three constructs. Using exogenous HA to block NP-CD44 interactions, we found the LbL HA bottlebrush NP had a 4-fold higher binding avidity than the best-performing LbL linear HA NP. We further observed that decreasing the density of HA bottlebrush side chains to 75% had minimal impact on LbL NP stability or cell association, though we did see a reduction in binding avidity with this side-chain-modified NP. Our studies indicate that LbL surfaces are highly effective for multivalent displays, and the mode in which they present a targeting ligand can be optimized for NP cell targeting.

Tuning Nanoparticle Interactions with Ovarian Cancer through Layer-by-Layer Modification of Surface Chemistry.

Nanoparticle surface chemistry is a fundamental engineering parameter that governs tumor-targeting activity. Electrostatic assembly generates controlled polyelectrolyte complexes through the process of adsorption and charge overcompensation utilizing synthetic polyions and natural biomacromolecules; it can yield films with distinctive hydration, charge, and presentation of functional groups. Here, we used electrostatic layer-by-layer (LbL) assembly to screen 10 different surface chemistries for their ability to preferentially target human ovarian cancer in vitro. Our screen identified that poly-l-aspartate, poly-l-glutamate, and hyaluronate-coated LbL nanoparticles have striking specificity for ovarian cancer, while sulfated poly(ß-cyclodextrin) nanoparticles target noncancerous stromal cells. We validated top candidates for tumor-homing ability with a murine model of metastatic disease and with patient-derived ovarian cancer spheroids. Nanoparticle surface chemistry also influenced subcellular trafficking, indicating strategies to target the cell membrane, caveolae, and perinuclear vesicles. Our results confirm LbL is a powerful tool to systematically engineer nanoparticles and achieve specific targeting.

Solution Conditions Tune and Optimize Loading of Therapeutic Polyelectrolytes into Layer-by-Layer Functionalized Liposomes.

Layer-by-layer (LbL) nanoparticles offer great potential to the field of drug delivery, where these nanocomposites have been studied for their ability to deliver chemotherapeutic agents, small molecule inhibitors, and nucleic acids. Most exciting is their ability to encapsulate multiple functional elements, which allow nanocarriers to deliver complex combination therapies with staged release. However, relative to planar LbL constructs, colloidal LbL systems have not undergone extensive systematic studies that outline critical synthetic solution conditions needed for robust and efficient assembly. The multistaged process of adsorbing a series of materials onto a nanoscopic template is inherently complex, and facilitating the self-assembly of these materials depends on identifying proper solution conditions for each synthetic step and adsorbed material. Here, we focus on addressing some of the fundamental questions that must be answered in order to obtain a reliable and robust synthesis of nucleic acid-containing LbL liposomes. This includes a study of solution conditions, such as pH, ionic strength, salt composition, and valency, and their impact on the preparation of LbL nanoparticles. Our results provide insight into the selection of solution conditions to control the degree of ionization and the electrostatic screening length to suit the adsorption of nucleic acids and synthetic polypeptides. The optimization of these parameters led to a roughly 8-fold improvement in nucleic acid loading in LbL liposomes, indicating the importance of optimizing solution conditions in the preparation of therapeutic LbL nanoparticles. These results highlight the benefits of defining principles for constructing highly effective nanoparticle systems.

Water Dynamics from the Surface to the Interior of a Supramolecular Nanostructure.

Water within and surrounding the structure of a biological system adopts context-specific dynamics that mediate virtually all of the events involved in the inner workings of a cell. These events range from protein folding and molecular recognition to the formation of hierarchical structures. Water dynamics are mediated by the chemistry and geometry of interfaces where water and biomolecules meet. Here we investigate experimentally and computationally the translational dynamics of vicinal water molecules within the volume of a supramolecular peptide nanofiber measuring 6.7 nm in diameter. Using Overhauser dynamic nuclear polarization relaxometry, we show that drastic differences exist in water motion within a distance of about one nanometer from the surface, with rapid diffusion in the hydrophobic interior and immobilized water on the nanofiber surface. These results demonstrate that water associated with materials designed at the nanoscale is not simply a solvent, but rather an integral part of their structure and potential functions.

Synthetic high-density lipoproteins as targeted monotherapy for chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) remains incurable despite the introduction of new drugs. Therapies targeting receptors and pathways active specifically in malignant B cells might provide better treatment options. For instance, in B cell lymphoma, our group has previously shown that scavenger receptor type B-1 (SR-B1), the high-affinity receptor for cholesterol-rich high-density lipoproteins (HDL), is a therapeutic target. As evidence suggests that targeting cholesterol metabolism in CLL cells may have therapeutic benefit, we examined SR-B1 expression in primary CLL cells from patients. Unlike normal B cells that do not express SR-B1, CLL cells express the receptor. As a result, we evaluated cholesterol-poor synthetic HDL nanoparticles (HDL NP), known for targeting SR-B1, as a therapy for CLL. HDL NPs potently and selectively induce apoptotic cell death in primary CLL cells. HDL NPs had no effect on normal peripheral blood mononuclear cells from healthy individuals or patients with CLL. These data implicate SR-B1 as a target in CLL and HDL NPs as targeted monotherapy for CLL.