Biodegradable polyphosphoester micelles act as both background-free 31P magnetic resonance imaging agents and drug nanocarriers.

In vivo monitoring of polymers is crucial for drug delivery and tissue regeneration. Magnetic resonance imaging (MRI) is a whole-body imaging technique, and heteronuclear MRI allows quantitative imaging. However, MRI agents can result in environmental pollution and organ accumulation. To address this, we introduce biocompatible and biodegradable polyphosphoesters, as MRI-traceable polymers using the 31P centers in the polymer backbone. We overcome challenges in 31P MRI, including background interference and low sensitivity, by modifying the molecular environment of 31P, assembling polymers into colloids, and tailoring the polymers' microstructure to adjust MRI-relaxation times. Specifically, gradient-type polyphosphonate-copolymers demonstrate improved MRI-relaxation times compared to homo- and block copolymers, making them suitable for imaging. We validate background-free imaging and biodegradation in vivo using Manduca sexta. Furthermore, encapsulating the potent drug PROTAC allows using these amphiphilic copolymers to simultaneously deliver drugs, enabling theranostics. This first report paves the way for polyphosphoesters as background-free MRI-traceable polymers for theranostic applications.

Construction of viral protein-based hybrid nanomaterials mediated by a macromolecular glue.

A generic strategy to construct virus protein-based hybrid nanomaterials is reported by using a macromolecular glue inspired by mussel adhesion. Commercially available poly(isobutylene-alt-maleic anhydride) (PiBMA) modified with dopamine (PiBMAD) is designed as this macromolecular glue, which serves as a universal adhesive material for the construction of multicomponent hybrid nanomaterials. As a proof of concept, gold nanorods (AuNRs) and single-walled carbon nanotubes (SWCNTs) are initially coated with PiBMAD. Subsequently, viral capsid proteins from the Cowpea Chlorotic Mottle Virus (CCMV) assemble around the nano-objects templated by the negative charges of the glue. With virtually unchanged properties of the rods and tubes, the hybrid materials might show improved biocompatibility and can be used in future studies toward cell uptake and delivery.

Single-chain polymer nanoparticles in biomedical applications.

Single-chain polymer nanoparticles (SCNPs) are a well-defined and uniquely sized class of polymer nanoparticles. The advances in polymer science over the past decades have enabled the development of a variety of intramolecular crosslinking systems, leading to particles in the 5-20 nm size regime. Which is aligned with the size regime of proteins and therefore making SCNPs an interesting class of NPs for biomedical applications. The high modularity of SCNP design and the ease of their functionalization have led to growing research interest. In this review, we describe different crosslinking systems, as well as the preparation of functional SCNPs and the variety of biomedical applications that have been explored.

Single-Chain Polymer Nanoparticles Targeting the Ookinete Stage of Malaria Parasites.

Malaria is an infectious disease transmitted by mosquitos, whose control is hampered by drug resistance evolution in the causing agent, protist parasites of the genus Plasmodium, as well as by the resistance of the mosquito to insecticides. New approaches to fight this disease are, therefore, needed. Research into targeted drug delivery is expanding as this strategy increases treatment efficacies. Alternatively, targeting the parasite in humans, here we use single-chain polymer nanoparticles (SCNPs) to target the parasite at the ookinete stage, which is one of the stages in the mosquito. This nanocarrier system provides uniquely sized and monodispersed particles of 5-20 nm, via thiol-Michael addition. The conjugation of succinic anhydride to the SCNP surface provides negative surface charges that have been shown to increase the targeting ability of SCNPs to Plasmodium berghei ookinetes. The biodistribution of SCNPs in mosquitos was studied, showing the presence of SCNPs in mosquito midguts. The presented results demonstrate the potential of anionic SCNPs for the targeting of malaria parasites in mosquitos and may lead to progress in the fight against malaria.

Enhancing Cellular Internalization of Single-Chain Polymer Nanoparticles via Polyplex Formation.

Intracellular delivery of nanoparticles is crucial in nanomedicine to reach optimal delivery of therapeutics and imaging agents. Single-chain polymer nanoparticles (SCNPs) are an interesting class of nanoparticles due to their unique site range of 5-20 nm. The intracellular delivery of SCNPs can be enhanced by using delivery agents. Here, a positive polymer is used to form polyplexes with SCNPs, similar to the strategy of protein and gene delivery. The size and surface charge of the polyplexes were evaluated. The cellular uptake showed rapid uptake of SCNPs via polyplex formation, and the cytosolic delivery of the SCNPs was presented by confocal microscopy. The ability of SCNPs to act as nanocarriers was further explored by conjugation of doxorubicin.

Uniquely sized nanogels via crosslinking polymerization.

Nanogels are very promising carriers for nanomedicine, as they can be prepared in the favorable nanometer size regime, can be functionalized with targeting agents and are responsive to stimuli, i.e. temperature and pH. This induces shrinking or swelling, resulting in controlled release of a therapeutic cargo. Our interest lies in the controlled synthesis of functional nanogels, such as those containing epoxide moieties, that can be subsequently functionalized. Co-polymerization of glycidyl methacrylate and a bifunctional methacrylate crosslinker under dilute conditions gives rise to well-defined epoxide-functional nanogels, of which the sizes are controlled by the degree of polymerization. Nanogels with well-defined sizes (polydispersity of 0.2) ranging from 38 nm to 95 nm were prepared by means of controlled radical polymerization. The nanogels were characterized in detail by FT-IR, DLS, size exclusion chromatography, NMR spectroscopy, AFM and TEM. Nucleophilic attack with functional thiols or amines on the least hindered carbon of the epoxide provides water-soluble nanogels, without altering the backbone structure, while reaction with sodium azide provides handles for further functionalization via click chemistry.

Microscopic Proof of Photoluminescence from Mechanochemically Synthesized 1-Octene-Capped Quantum-Confined Silicon Nanoparticles: Implications for Light-Emission Applications.

Silicon nanoparticles (SiNPs) have been explored intensively for their use in applications requiring efficient fluorescence for LEDs, lasers, displays, photovoltaic spectral-shifting filters, and biomedical applications. High radiative rates are essential for such applications, and theoretically these could be achieved via quantum confinement and/or straining. Wet-chemical methods used to synthesize SiNPs are under scrutiny because of reported contamination by fluorescent carbon species. To develop a cleaner method, we utilize a specially designed attritor type high-energy ball-mill and use a high-purity (99.999%) Si microparticle precursor. The mechanochemical process is used under a continuous nitrogen gas atmosphere to avoid oxidation of the particles. We confirm the presence of quantum-confined NPs (<5 nm) using atomic force microscopy (AFM). Microphotoluminescence (PL) spectroscopy coupled to AFM confirms quantum-confined tunable red/near-infrared PL emission in SiNPs capped with an organic ligand (1-octene). Using micro-Raman-PL spectroscopy, we confirm SiNPs as the origin of the emission. These results demonstrate a facile and potentially scalable mechanochemical method of synthesis for contamination-free SiNPs.

Linear Poly(ethylenimine-propylenimine) Random Copolymers for Gene Delivery: From Polymer Synthesis to Efficient Transfection with High Serum Tolerance.

Naturally occurring oligoamines, such as spermine, spermidine, and putrescine, are well-known regulators of gene expression. These oligoamines frequently have short alkyl spacers with varying lengths between the amines. Linear polyethylenimine (PEI) is a polyamine that has been widely applied as a gene vector, with various formulations currently in clinical trials. In order to emulate natural oligoamine gene regulators, linear random copolymers containing both PEI and polypropylenimine (PPI) repeat units were designed as novel gene delivery agents. In general, statistical copolymerization of 2-oxazolines and 2-oxazines leads to the formation of gradient copolymers. In this study, however, we describe for the first time the synthesis of near-ideal random 2-oxazoline/2-oxazine copolymers through careful tuning of the monomer structures and reactivity as well as polymerization conditions. These copolymers were then transformed into near-random PEI-PPI copolymers by controlled side-chain hydrolysis. The prepared PEI-PPI copolymers formed stable polyplexes with GFP-encoding plasmid DNA, as validated by dynamic light scattering. Furthermore, the cytotoxicity and transfection efficiency of polyplexes were evaluated in C2C12 mouse myoblasts. While the polymer chain length did not significantly increase the toxicity, a higher PPI content was associated with increased toxicity and also lowered the amount of polymers needed to achieve efficient transfection. The transfection efficiency was significantly influenced by the degree of polymerization of PEI-PPI, whereby longer polymers resulted in more transfected cells. Copolymers with 60% or lower PPI content exhibited a good balance between high plasmid-DNA transfection efficiency and low toxicity. Interestingly, these novel PEI-PPI copolymers revealed exceptional serum tolerance, whereby transfection efficiencies of up to 53% of transfected cells were achieved even under 50% serum conditions. These copolymers, especially PEI-PPI with DP500 and a 1:1 PEI/PPI ratio, were identified as promising transfection agents for plasmid DNA.

Polymeric Nanoparticles Properties and Brain Delivery.

Safe and reliable entry to the brain is essential for successful diagnosis and treatment of diseases, but it still poses major challenges. As a result, many therapeutic approaches to treating disorders associated with the central nervous system (CNS) still only show limited success. Nano-sized systems are being explored as drug carriers and show great improvements in the delivery of many therapeutics. The systemic delivery of nanoparticles (NPs) or nanocarriers (NCs) to the brain involves reaching the neurovascular unit (NVU), being transported across the blood-brain barrier, (BBB) and accumulating in the brain. Each of these steps can benefit from specifically controlled properties of NPs. Here, we discuss how brain delivery by NPs can benefit from careful design of the NP properties. Properties such as size, charge, shape, and ligand functionalization are commonly addressed in the literature; however, properties such as ligand density, linker length, avidity, protein corona, and stiffness are insufficiently discussed. This is unfortunate since they present great value against multiple barriers encountered by the NPs before reaching the brain, particularly the BBB. We further highlight important examples utilizing targeting ligands and how functionalization parameters, e.g., ligand density and ligand properties, can affect the success of the nano-based delivery system.

Introduction of Surface Loops as a Tool for Encapsulin Functionalization.

Encapsulin-based protein cages are nanoparticles with potential biomedical applications, such as targeted drug delivery or imaging. These particles are biocompatible and can be produced in bacteria, allowing large-scale production and protein engineering. In order to use these bacterial nanocages in different applications, it is important to further explore their surface modification and optimize their production. In this study, we design and show new surface modifications of Thermotoga maritima (Tm) and Brevibacterium linens (Bl) encapsulins. Two new loops on the Tm encapsulin with a His-tag insertion after residue 64 and residue 127 and the modification of the C-terminus on the Bl encapsulin are reported. The multimodification of the Tm encapsulin enables up to 240 functionalities on the cage surface, resulting from four potential modifications per protein subunit. We further report an improved production protocol giving a better stability and good production yield of the cages. Finally, we tested the stability of different encapsulin variants over a year, and the results show a difference in stability arising from the tag insertion position. These first insights in the structure-property relationship of encapsulins, with respect to the position of a functional loop, allow for further study of the use of these protein nanocages in biomedical applications.

Dynamic Nuclear Polarization of Silicon Carbide Micro- and Nanoparticles.

Two dominant crystalline phases of silicon carbide (SiC): α-SiC and ß-SiC, differing in size and chemical composition, were investigated regarding their potential for dynamic nuclear polarization (DNP). 29Si nuclei in α-SiC micro- and nanoparticles with sizes ranging from 650 nm to 2.2 µm and minimal oxidation were successfully hyperpolarized without the use of free radicals, while ß-SiC samples did not display appreciable degrees of polarization under the same polarization conditions. Long T1 relaxation times in α-SiC of up to 1600 s (∼27 min) were recorded for the 29Si nuclei after 1 h of polarization at a temperature of 4 K. Interestingly, these promising α-SiC particles allowed for direct hyperpolarization of both 29Si and 13C nuclei, resulting in comparably strong signal amplifications. Moreover, the T1 relaxation time of 13C nuclei in 750 nm-sized α-SiC particles was over 33 min, which far exceeds T1 times of conventional 13C DNP probes with values in the order of 1-2 min. The present work demonstrates the feasibility of DNP on SiC micro- and nanoparticles and highlights their potential as hyperpolarized magnetic resonance imaging agents.

Stabilin-1 is required for the endothelial clearance of small anionic nanoparticles.

Clearance of nanoparticles (NPs) after intravenous injection - mainly by the liver - is a critical barrier for the clinical translation of nanomaterials. Physicochemical properties of NPs are known to influence their distribution through cell-specific interactions; however, the molecular mechanisms responsible for liver cellular NP uptake are poorly understood. Liver sinusoidal endothelial cells and Kupffer cells are critical participants in this clearance process. Here we use a zebrafish model for liver-NP interaction to identify the endothelial scavenger receptor Stabilin-1 as a non-redundant receptor for the clearance of small anionic NPs. Furthermore, we show that physiologically, Stabilin-1 is required for the removal of bacterial lipopolysaccharide (LPS/endotoxin) from circulation and that Stabilin-1 cooperates with its homolog Stabilin-2 in the clearance of larger (~100 nm) anionic NPs. Our findings allow optimization of anionic nanomedicine biodistribution and targeting therapies that use Stabilin-1 and -2 for liver endothelium-specific delivery.

Development of an In Vitro Airway Epithelial-Endothelial Cell Culture Model on a Flexible Porous Poly(Trimethylene Carbonate) Membrane Based on Calu-3 Airway Epithelial Cells and Lung Microvascular Endothelial Cells.

Due to the continuing high impact of lung diseases on society and the emergence of new respiratory viruses, such as SARS-CoV-2, there is a great need for in vitro lung models that more accurately recapitulate the in vivo situation than current models based on lung epithelial cell cultures on stiff membranes. Therefore, we developed an in vitro airway epithelial-endothelial cell culture model based on Calu-3 human lung epithelial cells and human lung microvascular endothelial cells (LMVECs), cultured on opposite sides of flexible porous poly(trimethylene carbonate) (PTMC) membranes. Calu-3 cells, cultured for two weeks at an air-liquid interface (ALI), showed good expression of the tight junction (TJ) protein Zonula Occludens 1 (ZO-1). LMVECs cultured submerged for three weeks were CD31-positive, but the expression was diffuse and not localized at the cell membrane. Barrier functions of the Calu-3 cell cultures and the co-cultures with LMVECs were good, as determined by electrical resistance measurements and fluorescein isothiocyanate-dextran (FITC-dextran) permeability assays. Importantly, the Calu-3/LMVEC co-cultures showed better cell viability and barrier function than mono-cultures. Moreover, there was no evidence for epithelial- and endothelial-to-mesenchymal transition (EMT and EndoMT, respectively) based on staining for the mesenchymal markers vimentin and α-SMA, respectively. These results indicate the potential of this new airway epithelial-endothelial model for lung research. In addition, since the PTMC membrane is flexible, the model can be expanded by introducing cyclic stretch for enabling mechanical stimulation of the cells. Furthermore, the model can form the basis for biomimetic airway epithelial-endothelial and alveolar-endothelial models with primary lung epithelial cells.

Cytosolic Delivery of Single-Chain Polymer Nanoparticles.

Cytosolic delivery of therapeutic agents is key to improving their efficacy, as the therapeutics are primarily active in specific organelles. Single-chain polymer nanoparticles (SCNPs) are a promising nanocarrier platform in biomedical applications due to their unique size range of 5-20 nm, modularity, and ease of functionalization. However, cytosolic delivery of SCNPs remains challenging. Here, we report the synthesis of active ester-functional SCNPs of approximately 10 nm via intramolecular thiol-Michael addition cross-linking and their functionalization with increasing amounts of tertiary amines 0 to 60 mol % to obtain SCNPs with increasing positive surface charges. No significant cytotoxicity was detected in bEND.3 cells for the SCNPs, except when SCNPs with high amounts of tertiary amines were incubated over prolonged periods of time at high concentrations. Cellular uptake of the SCNPs was analyzed, presenting different uptake behavior depending on the degree of functionalization. Confocal microscopy revealed successful cytosolic delivery of SCNPs with high degrees of functionalization (45%, 60%), while SCNPs with low amounts (0% to 30%) of tertiary amines showed high degrees of colocalization with lysosomes. This work presents a strategy to direct the intracellular location of SCNPs by controlled surface modification to improve intracellular targeting for biomedical applications.

Critical assessment of wet-chemical oxidation synthesis of silicon quantum dots.

Wet-chemical synthetic procedures are powerful strategies to afford fluorescent silicon quantum dots (Si QDs) in a versatile and scalable manner. However, development of Si QDs is still hampered by a lack of control over photoluminescence emission, in addition to synthesis and characterization complexities. The wet-chemical Si QD synthesis by oxidation of magnesium silicide (Mg2Si) with bromine (Br2) was revisited and a control reaction was carried out where the silicon source was omitted. Both reaction conditions resulted in substantial quantities of fluorescent material. Moreover, a comparative analysis of their optical properties (UV-vis/fluorescence) revealed no apparent differences. Other characterization techniques also confirmed the resemblance of the two materials as 1H NMR, FTIR and XPS spectra were nearly identical for both samples. Elemental analysis revealed the presence of only 2 wt% silicon in the Si QD sample. No evidence was found for the formation of significant amounts of Si QDs via this wet-chemical procedure.

Glucose Single-Chain Polymer Nanoparticles for Cellular Targeting.

Naturally occurring glycoconjugates possess carbohydrate moieties that fulfill essential roles in many biological functions. Through conjugation of carbohydrates to therapeutics or imaging agents, naturally occurring glycoconjugates are mimicked and efficient targeting or increased cellular uptake of glycoconjugated macromolecules is achieved. In this work, linear and cyclic glucose moieties were functionalized with methacrylates via enzymatic synthesis and used as building blocks for intramolecular cross-linked single-chain glycopolymer nanoparticles (glyco-SCNPs). A set of water-soluble sub-10 nm-sized glyco-SCNPs was prepared by thiol-Michael addition cross-linking in water. Bioactivity of various glucose-conjugated glycopolymers and glyco-SCNPs was evaluated in binding studies with the glucose-specific lectin Concanavalin A and by comparing their cellular uptake efficiency in HeLa cells. Cytotoxicity studies did not reveal discernible cytotoxic effects, making these SCNPs promising candidates for ligand-based targeted imaging and drug delivery.

Single-chain polymer nanoparticles in controlled drug delivery and targeted imaging.

As a relatively new class of materials, single-chain polymer nanoparticles (SCNPs) just entered the field of (biomedical) applications, with recent advances in polymer science enabling the formation of bio-inspired nanosized architectures. Exclusive intramolecular collapse of individual polymer chains results in individual nanoparticles. With sizes an order of magnitude smaller than conventional polymer nanoparticles, SCNPs are in the size regime of many proteins and viruses (1-20 nm). Multifaceted syntheses and design strategies give access to a wide set of highly modular SCNP materials. This review describes how SCNPs have been rendered water-soluble and highlights ongoing research efforts towards biocompatible SCNPs with tunable properties for controlled drug delivery, targeted imaging and protein mimicry.

Biocompatible Single-Chain Polymer Nanoparticles for Drug Delivery-A Dual Approach.

Single-chain polymer nanoparticles (SCNPs) are protein-inspired materials based on intramolecularly cross-linked polymer chains. We report here the development of SCNPs as uniquely sized nanocarriers that are capable of drug encapsulation independent of the polarity of the employed medium. Synthetic routes are presented for SCNP preparation in both organic and aqueous environments. Importantly, the SCNPs in organic media were successfully rendered water soluble, resulting in two complementary pathways toward water-soluble SCNPs with comparable resultant physicochemical characteristics. The solvatochromic dye Nile red was successfully encapsulated inside the SCNPs following both pathways, enabling probing of the SCNP interior. Moreover, the antibiotic rifampicin was encapsulated in organic medium, the loaded nanocarriers were rendered water soluble, and a controlled release of rifampicin was evidenced. The absence of discernible cytotoxic effects and promising cellular uptake behavior bode well for the application of SCNPs in controlled therapeutics delivery.

Responsive crosslinked polymer nanogels for imaging and therapeutics delivery.

Water-soluble, nano-sized crosslinked polymer networks, or nanogels, are delivery vehicles, which have highly interesting properties for therapeutic delivery and imaging. Nanogels may also possess responsive properties, depending on the employed polymers, allowing controlled release of therapeutics or image contrast generation upon exposure to physical or (bio)chemical cues. In this review, polymer nanogels are explored for application in imaging as well as for controlled drug and gene delivery. Moreover, nanogels are explored as responsive biomaterials and future applications are highlighted.