Mucopenetrating Janus Nanoparticles For Field-Coverage Oral Cancer Chemoprevention.

INTRODUCTION: Oral squamous cell carcinoma (OSCC), is associated with high morbidity and mortality. Preemptive interventions have been postulated to provide superior therapeutic options, but their implementation has been restricted by the availability of broadly applicable local delivery systems. METHODS: We address this challenge by engineering a delivery vehicle, Janus nanoparticles (JNP), that combine the dual mucoadhesive properties of a first cationic chitosan compartment with a second hydrophobic poly(lactide-co-glycolide) release compartment. JNP are designed to avoid rapid mucus clearance while ensuring stable loading and controlled release of the IL-6 receptor antagonist, tocilizumab (TCZ). RESULTS: The JNP featured defined and monodispersed sizes with an average diameter of 327 nm and a PDI of 0.245, high circularities above 0.90 and supported controlled release of TCZ and effective internalization by oral keratinocytes. TCZ released from JNP retained its biological activity and effectively reduced both, soluble and membrane-bound IL-6Rα (71% and 50%). In full-thickness oral mucosal explants, 76% of the JNP breached the stratum corneum and in 41% were observed in the basal cell layer indicating excellent mucopenetrating properties. When tested in an aggressive OSCC xenograft model, TCZ-loaded JNP showed high levels of xenograft inhibition and outperformed all control groups with respect to inhibition of tumor cell proliferation, reduction in tumor size and reduced expression of the proto-oncogene ERG. CONCLUSION: By combining critically required, yet orthogonal properties within the same nanoparticle design, the JNP in this study, demonstrate promise as precision delivery platforms for intraoral field-coverage chemoprevention, a vastly under-researched area of high clinical importance.

Nanoparticle Properties Influence Transendothelial Migration of Monocytes.

Nanoparticle-based delivery of therapeutics to the brain has had limited clinical impact due to challenges crossing the blood-brain barrier (BBB). Certain cells, such as monocytes, possess the ability to migrate across the BBB, making them attractive candidates for cell-based brain delivery strategies. In this work, we explore nanoparticle design parameters that impact both monocyte association and monocyte-mediated BBB transport. We use electrohydrodynamic jetting to prepare nanoparticles of varying sizes, compositions, and elasticity to address their impact on uptake by THP-1 monocytes and permeation across the BBB. An in vitro human BBB model is developed using human cerebral microvascular endothelial cells (hCMEC/D3) for the assessment of migration. We compare monocyte uptake of both polymeric and synthetic protein nanoparticles (SPNPs) of various sizes, as well as their effect on cell migration. SPNPs (human serum albumin/HSA or human transferrin/TF) are shown to promote increased monocyte-mediated transport across the BBB over polymeric nanoparticles. TF SPNPs (200 nm) associate readily, with an average uptake of 138 particles/cell. Nanoparticle loading is shown to influence the migration of THP-1 monocytes. The migration of monocytes loaded with 200 nm TF and 200 nm HSA SPNPs was 2.3-fold and 2.1-fold higher than that of an untreated control. RNA-seq analysis after TF SPNP treatment suggests that the upregulation of several migration genes may be implicated in increased monocyte migration (ex. integrin subunits α M and α L). Integrin ß 2 chain combines with either integrin subunit α M chain or integrin subunit α L chain to form macrophage antigen 1 and lymphocyte function-associated antigen 1 integrins. Both products play a pivotal role in the transendothelial migration cascade. Our findings highlight the potential of SPNPs as drug and/or gene delivery platforms for monocyte-mediated BBB transport, especially where conventional polymer nanoparticles are ineffective or otherwise not desirable.

Multifunctional Synthetic Protein Nanoparticles via Reactive Electrojetting.

Protein nanoparticles are a promising approach for nanotherapeutics, as proteins combine versatile chemical and biological function with controlled biodegradability. In this work, the development of an adaptable synthesis method is presented for synthetic protein nanoparticles (SPNPs) based on reactive electrojetting. In contrast to past work with electrohydrodynamic cojetting using inert polymers, the jetting solutions are comprised of proteins and chemically activated macromers, designed to react with each other during the processing step, to form insoluble nanogel particles. SPNPs made from a variety of different proteins, such as transferrin, insulin, or hemoglobin, are stable and uniform under physiological conditions and maintain monodisperse sizes of around 200 nm. SPNPs comprised of transferrin and a disulfide containing macromer are stimuli-responsive, and serve as markers of oxidative stress within HeLa cells. Beyond isotropic SPNPs, bicompartmental nanoparticles containing human serum albumin and transferrin in two distinct hemispheres are prepared via reactive electrojetting. This novel platform provides access to a novel class of versatile protein particles with nanoscale architectures that i) can be made from a variety of proteins and macromers, ii) have tunable biological responses, and iii) can be multicompartmental, a prerequisite for controlled release of multiple drugs.

Highly Luminescent Linear Complex Arrays of up to Eight Cuprous Centers.

Linearly arranged metal atoms that are embedded in discrete molecules have fascinated scientists across various disciplines for decades; this is attributed to their potential use in microelectronic devices on a submicroscopic scale. Luminescent oligonuclear Group 11 metal complexes are of particular interest for applications in molecular light-emitting devices. Herein, we describe the synthesis and characterization of a rare, homoleptic, and neutral linearly arranged tetranuclear Cu(I) complex that is helically bent, thus representing a molecular coil in the solid state. This tetracuprous arrangement dimerizes into a unique octanuclear assembly bearing a linear array of six Cu(I) centers with two additional bridging cuprous ions that constitute a central pseudo-rhombic Cu(I) 4 cluster. The crystal structure determinations of both complexes reveal close d(10) ⋅⋅⋅d(10) contacts between all cuprous ions that are adjacent to each other. The dynamic behavior in solution, DFT calculations, and the luminescence properties of these remarkable complexes are also discussed.

Improving paclitaxel delivery: in vitro and in vivo characterization of PEGylated polyphosphoester-based nanocarriers.

Nanomaterials have great potential to offer effective treatment against devastating diseases by providing sustained release of high concentrations of therapeutic agents locally, especially when the route of administration allows for direct access to the diseased tissues. Biodegradable polyphosphoester-based polymeric micelles and shell cross-linked knedel-like nanoparticles (SCKs) have been designed from amphiphilic block-graft terpolymers, PEBP-b-PBYP-g-PEG, which effectively incorporate high concentrations of paclitaxel (PTX). Well-dispersed nanoparticles physically loaded with PTX were prepared, exhibiting desirable physiochemical characteristics. Encapsulation of 10 wt% PTX, into either micelles or SCKs, allowed for aqueous suspension of PTX at concentrations up to 4.8 mg/mL, as compared to <2.0 µg/mL for the aqueous solubility of the drug alone. Drug release studies indicated that PTX released from these nanostructures was defined through a structure-function relationship, whereby the half-life of sustained PTX release was doubled through cross-linking of the micellar structure to form SCKs. In vitro, physically loaded micellar and SCK nanotherapeutics demonstrated IC50 values against osteosarcoma cell lines, known to metastasize to the lungs (CCH-OS-O and SJSA), similar to the pharmaceutical Taxol formulation. Evaluation of these materials in vivo has provided an understanding of the effects of nanoparticle structure-function relationships on intratracheal delivery and related biodistribution and pharmacokinetics. Overall, we have demonstrated the potential of these novel nanotherapeutics toward future sustained release treatments via administration directly to the sites of lung metastases of osteosarcoma.

Supramolecularly knitted tethered oligopeptide/single-walled carbon nanotube organogels.

A facile polymerization of an allyl-functionalized N-carboxyanhydride (NCA) monomer is utilized to construct an A-B-A-type triblock structure containing ß-sheet-rich oligomeric peptide segments tethered by a poly(ethylene oxide) chain, which are capable of dispersing and gelating single-walled carbon nanotubes (SWCNTs) noncovalently in organic solvents, resulting in significant enhancement of the mechanical properties of polypeptide-based organogels.

Holistic assessment of covalently labeled core-shell polymeric nanoparticles with fluorescent contrast agents for theranostic applications.

The successful development of degradable polymeric nanostructures as optical probes for use in nanotheranostic applications requires the intelligent design of materials such that their surface response, degradation, drug delivery, and imaging properties are all optimized. In the case of imaging, optimization must result in materials that allow differentiation between unbound optical contrast agents and labeled polymeric materials as they undergo degradation. In this study, we have shown that use of traditional electrophoretic gel-plate assays for the determination of the purity of dye-conjugated degradable nanoparticles is limited by polymer degradation characteristics. To overcome these limitations, we have outlined a holistic approach to evaluating dye and peptide-polymer nanoparticle conjugation by utilizing steady-state fluorescence, anisotropy, and emission and anisotropy lifetime decay profiles, through which nanoparticle-dye binding can be assessed independently of perturbations, such as those presented during the execution of electrolyte gel-based assays. This approach has been demonstrated to provide an overall understanding of the spectral signature-structure-function relationship, ascertaining key information on interactions between the fluorophore, polymer, and solvent components that have a direct and measurable impact on the emissive properties of the optical probe. The use of these powerful techniques provides feedback that can be utilized to improve nanotheranostics by evaluating dye emissivity in degradable nanotheranostic systems, which has become increasingly important as modern platforms transition to architectures intentionally reliant on degradation and built-in environmental responses.

Conjugation-induced fluorescent labeling of proteins and polymers using dithiomaleimides.

Dithiomaleimides (DTMs) with alkyl substituents are shown to be a novel class of highly emissive fluorophores. Variable solubility and further functionalization can easily be tailored through the choice of N and S substituents. Inclusion of a DTM unit into a ROP/RAFT initiator or insertion into the disulfide bond of salmon calcitonin (sCT) demonstrates the utility for fluorescent labeling of polymers and proteins. Simultaneous PEGylation and fluorescent labeling of sCT is also demonstrated, using the DTM unit as both a linker and a fluorophore. It is anticipated that DTMs will offer an attractive alternative to commonly used bulky, planar fluorophores.

New functional handle for use as a self-reporting contrast and delivery agent in nanomedicine.

The synthesis and photophysical characterization of a chromophore-bridged block copolymer system is presented. This system is based on a dithiomaleimide (DTM) functional group as a highly emissive functionality which can readily be incorporated into polymeric scaffolds. A key advantage of this new reporter group is its versatile chemistry, ease of further functionalization, and notably small size, which allows for ready incorporation without affecting or disrupting the self-assembly process critical to the formation of core-shell polymeric contrast and drug delivery agents. We demonstrate the potential of this functionality with a diblock system which has been shown to be appropriate for micellization and, when in the micellar state, does not self-quench. The block copolymer is shown to be significantly more emissive than the lone dye, with a concentration-independent emission and anisotropy profile from 1.5 mM to 0.15 µM. An emission lifetime and anisotropy decay comparison of the block copolymer to its micelle displays that time-domain fluorescence lifetime imaging (FLIM) is able to rapidly resolve differences in the supramolecular state of this block-dye-block polymer system. Furthermore, the ability to resolve these differences in the supramolecular state means that the DTM micelles are capable of self-reporting when disassembly occurs, simply by monitoring with FLIM. We demonstrate the great potential for in vitro applications that this system provides by using FLIM to observe micelle disassembly in different vascular components of rat hippocampal tissue. In total this system represents a new class of in-chain emitter which is appropriate for application in quantitative imaging and the tracking of particle degradation/disassembly events in biological environments.

Responsive organogels formed by supramolecular self assembly of PEG-block-allyl-functionalized racemic polypeptides into ß-sheet-driven polymeric ribbons.

A chemically reactive hybrid diblock polypeptide gelator poly(ethylene glycol)-block-poly(dl-allylglycine) (PEG-b-PDLAG) is an exceptional material, due to the characteristics of thermo-reversible organogel formation driven by the combination of a hydrophilic polymer chain linked to a racemic oligomeric homopeptide segment in a range of organic solvents. One-dimensional stacking of the block copolymers is demonstrated by ATR-FTIR spectroscopy, wide-angle X-ray scattering to be driven by the supramolecular assembly of ß-sheets in peptide blocks to afford well-defined fiber-like structures, resulting in gelation. These supramolecular interactions are sufficiently strong to achieve ultra low critical gelation concentrations (ca. 0.1 wt%) in N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and methanol. The critical gel transition temperature was directly proportional to the polymer concentration, so that at low concentrations, thermoreversibility of gelation was observed. Dynamic mechanical analysis studies were employed to determine the organogel mechanical properties, having storage moduli of ca. 15.1 kPa at room temperature.

The steep road to nonviral nanomedicines: Frequent challenges and culprits in designing nanoparticles for gene therapy.

The potential of therapeutically loaded nanoparticles (NPs) has been successfully demonstrated during the last decade, with NP-mediated nonviral gene delivery gathering significant attention as highlighted by the broad clinical acceptance of mRNA-based COVID-19 vaccines. A significant barrier to progress in this emerging area is the wild variability of approaches reported in published literature regarding nanoparticle characterizations. Here, we provide a brief overview of the current status and outline important concerns regarding the need for standardized protocols to evaluate NP uptake, NP transfection efficacy, drug dose determination, and variability of nonviral gene delivery systems. Based on these concerns, we propose wide adherence to multimodal, multiparameter, and multistudy analysis of NP systems. Adoption of these proposed approaches will ensure improved transparency, provide a better basis for interlaboratory comparisons, and will simplify judging the significance of new findings in a broader context, all critical requirements for advancing the field of nonviral gene delivery.

Systematic studies into uniform synthetic protein nanoparticles.

Nanoparticles are frequently pursued as drug delivery carriers due to their potential to alter the pharmacological profiles of drugs, but their broader utility in nanomedicine hinges upon exquisite control of critical nanoparticle properties, such as shape, size, or monodispersity. Electrohydrodynamic (EHD) jetting is a probate method to formulate synthetic protein nanoparticles (SPNPs), but a systematic understanding of the influence of crucial processing parameters, such as protein composition, on nanoparticle morphologies is still missing. Here, we address this knowledge gap by evaluating formulation trends in SPNPs prepared by EHD jetting based on a series of carrier proteins and protein blends (hemoglobin, transferrin, mucin, or insulin). In general, blended SPNPs presented uniform populations with minimum diameters between 43 and 65 nm. Size distributions of as-jetted SPNPs approached monodispersity as indicated by polydispersity indices (PDISEM) ranging from 0.11-0.19. Geometric factor analysis revealed high circularities (0.82-0.90), low anisotropy (<1.45) and excellent roundness (0.76-0.89) for all SPNPs prepared via EHD jetting. Tentatively, blended SPNPs displayed higher circularity and lower anisotropy, as compared to single-protein SPNPs. Secondary statistical analysis indicated that blended SPNPs generally present combined features of their constituents, with some properties driven by the dominant protein constituent. Our study suggests SPNPs made from blended proteins can serve as a promising drug delivery carrier owing to the ease of production, the composition versatility, and the control over their size, shape and dispersity.

Synthesis and two-photon absorption enhancement of porphyrin macrocycles.

Improving the nonlinear optical properties of organic materials through transition to macromolecular architectures of highly conjugated chromophores has been shown to be a viable strategy for generating materials suitable for TPA applications. In this study we display a simple and elegant method by which to synthesize macrocycles of porphyrin dimers. Two-photon absorption studies show 2 orders of magnitude enhancement of cross-section of the material, giving a maximum delta(2) of 10(6) GM.

Fluorescent Block Copolymer Micelles That Can Self-Report on Their Assembly and Small Molecule Encapsulation.

Block copolymer micelles have been prepared with a dithiomaleimide (DTM) fluorophore located in either the core or shell. Poly(triethylene glycol acrylate)-b-poly(tert-butyl acrylate) (P(TEGA)-b-P(tBA)) was synthesized by RAFT polymerization, with a DTM-functional acrylate monomer copolymerized into either the core forming P(tBA) block or the shell forming P(TEGA) block. Self-assembly by direct dissolution afforded spherical micelles with Rh of ca. 35 nm. Core-labeled micelles (CLMs) displayed bright emission (Φf = 17%) due to good protection of the fluorophore, whereas shell-labeled micelles (SLMs) had lower efficiency emission due to collisional quenching in the solvated corona. The transition from micelles to polymer unimers upon dilution could be detected by measuring the emission intensity of the solutions. For the core-labeled micelles, the fluorescence lifetime was also responsive to the supramolecular state, the lifetime being significantly longer for the micelles (τAv,I = 19 ns) than for the polymer unimers (τAv,I = 9 ns). The core-labeled micelles could also self-report on the presence of a fluorescent hydrophobic guest molecule (Nile Red) as a result of Förster resonance energy transfer (FRET) between the DTM fluorophore and the guest. The sensitivity of the DTM fluorophore to its environment therefore provides a simple handle to obtain detailed structural information for the labeled polymer micelles. A case will also be made for the application superiority of core-labeled micelles over shell-labeled micelles for the DTM fluorophore.

Protein resistance efficacy of PEO-silane amphiphiles: Dependence on PEO-segment length and concentration.

UNLABELLED: In contrast to modification with conventional PEO-silanes (i.e. no siloxane tether), silicones with dramatically enhanced protein resistance have been previously achieved via bulk-modification with poly(ethylene oxide) (PEO)-silane amphiphiles α-(EtO)3Si(CH2)2-oligodimethylsiloxane13-block-PEOn-OCH3 when n=8 and 16 but not when n=3. In this work, their efficacy was evaluated in terms of optimal PEO-segment length and minimum concentration required in silicone. For each PEO-silane amphiphile (n=3, 8, and 16), five concentrations (5, 10, 25, 50, and 100µmol per 1g silicone) were evaluated. Efficacy was quantified in terms of the modified silicones' abilities to undergo rapid, water-driven surface restructuring to form hydrophilic surfaces as well as resistance to fibrinogen adsorption. Only n=8 and 16 were effective, with a lower minimum concentration in silicone required for n=8 (10µmol per 1g silicone) versus n=16 (25µmol per 1g silicone). STATEMENT OF SIGNIFICANCE: Silicone is commonly used for implantable medical devices, but its hydrophobic surface promotes protein adsorption which leads to thrombosis and infection. Typical methods to incorporate poly(ethylene oxide) (PEO) into silicones have not been effective due to the poor migration of PEO to the surface-biological interface. In this work, PEO-silane amphiphiles - comprised of a siloxane tether (m=13) and variable PEO segment lengths (n=3, 8, 16) - were blended into silicone to improve its protein resistance. The efficacy of the amphiphiles was determined to be dependent on PEO length. With the intermediate PEO length (n=8), water-driven surface restructuring and resulting protein resistance was achieved with a concentration of only 1.7wt%.

Tungsten-loaded SMP foam nanocomposites with inherent radiopacity and tunable thermo-mechanical properties.

Shape memory polymer (SMP) foams have been developed for use in neurovascular occlusion applications. These materials are predominantly polyurethanes that are known for their biocompatibility and tunable properties. However, these polymers inherently lack X-ray visibility, which is a significant challenge for their use as implantable materials. Herein, low density, highly porous shape memory polyurethane foams were developed with tungsten nanoparticles dispersed into the foam matrix, at increasing concentrations, to serve as a radiopaque agent. Utilizing X-ray fluoroscopy sufficient visibility of the foams at small geometries was observed. Thermal characterization of the foams indicated altered thermal response and delayed foam actuation with increasing nanoparticle loading (because of restricted network mobility). Mechanical testing indicated decreased toughness and strength for higher loading because of disruption of the SMP matrix. Overall, filler addition imparted x-ray visibility to the SMP foams and allowed for tuned control of the transition temperature and actuation kinetics for the material.

In vivo fate tracking of degradable nanoparticles for lung gene transfer using PET and Cerenkov imaging.

Nanoparticles (NPs) play expanding roles in biomedical applications including imaging and therapy, however, their long-term fate and clearance profiles have yet to be fully characterized in vivo. NP delivery via the airway is particularly challenging, as the clearance may be inefficient and lung immune responses complex. Thus, specific material design is required for cargo delivery and quantitative, noninvasive methods are needed to characterize NP pharmacokinetics. Here, biocompatible poly(acrylamidoethylamine)-b-poly(dl-lactide) block copolymer-based degradable, cationic, shell-cross-linked knedel-like NPs (Dg-cSCKs) were employed to transfect plasmid DNA. Radioactive and optical beacons were attached to monitor biodistribution and imaging. The preferential release of cargo in acidic conditions provided enhanced transfection efficiency compared to non-degradable counterparts. In vivo gene transfer to the lung was correlated with NP pharmacokinetics by radiolabeling Dg-cSCKs and performing quantitative biodistribution with parallel positron emission tomography and Cerenkov imaging. Quantitation of imaging over 14 days corresponded with the pharmacokinetics of NP movement from the lung to gastrointestinal and renal routes, consistent with predicted degradation and excretion. This ability to noninvasively and accurately track NP fate highlights the advantage of incorporating multifunctionality into particle design.

Examination of radio-opacity enhancing additives in shape memory polyurethane foams.

Three microparticle additives, tungsten (W), zirconium oxide (ZrO2), and barium sulfate (BaSO4) were selected to enhance the radio-opacity in shape memory polymer (SMP) foam biomaterials. The addition of filler causes no significant alterations of glass transition temperatures, density of the materials increases, pore diameter decreases, and total volume recovery decreases from approximately 70 times in unfilled foams to 20 times (4% W and 10% ZrO2). The addition of W increases time to recovery; ZrO2 causes little variation in time to shape recovery; BaSO4 increases the time to recovery. On a 2.00 mean X-ray density (mean X.D.) scale, a GDC coil standard has a mean X.D. of 0.62; 4% W enhances the mean X.D. to 1.89, 10% ZrO2 to 1.39 and 4% BaSO4 to 0.74. Radio-opacity enhancing additives could be used to produce SMP foams with controlled shape memory kinetics, low density, and enhanced X-ray opacity for medical materials.

Facile Synthesis of a Phosphorylcholine-Based Zwitterionic Amphiphilic Copolymer for Anti-Biofouling Coatings.

An antibiofouling polymer coating, combined with both zwitterionic and amphiphilic features, is engineered by a two-step modification of a commodity polymer. The surface properties of the resultant polymer coating can be easily tuned by varying the extent of cross-linking in the network. Higher antibiofouling efficiency was observed for these surfaces vs. an elastomeric polydimethylsiloxane standard (Sylgard 184) against the adsorption of biomacromolecules and a marine fouling organism (Ulva zoospores) has been demonstrated. This design establishes a platform for the achievement of functionalized amphiphilic zwitterionic copolymers from relatively inexpensive starting materials via simple chemical manipulations.