Fabrication and Characterization of Mucin Nanoparticles for Drug Delivery Applications.

Mucin glycoproteins are ideal biomacromolecules for drug delivery applications since they naturally offer a plethora of different functional groups that can engage in specific and unspecific binding interactions with cargo molecules. However, to fabricate drug carrier objects from mucins, suitable stabilization mechanisms have to be implemented into the nanoparticle preparation procedure that allow for drug release profiles that match the requirements of the selected cargo molecule and its particular mode of action. Here, we describe two different methods to prepare crosslinked mucin nanoparticles that can release their cargo either on-demand or in a sustained manner. This method chapter includes a description of the preparation and characterization of mucin nanoparticles (stabilized either with synthetic DNA strands or with covalent crosslinks generated by free radical polymerization), as well as protocols to quantify the release of a model drug from those nanoparticles.

DNA Crosslinked Mucin Hydrogels Allow for On-Demand Gel Disintegration and Triggered Particle Release.

Whereas hydrogels created from synthetic polymers offer a high level of control over their stability and mechanical properties, their biomedical activity is typically limited. In contrast, biopolymers have evolved over billions of years to integrate a broad range of functionalities into a single design. Thus, biopolymeric hydrogels can show remarkable capabilities such as regulatory behavior, selective barrier properties, or antimicrobial effects. Still, despite their widespread use in numerous biomedical applications, achieving a meticulous control over the physical properties of macroscopic biopolymeric networks remains a challenge. Here, a macroscopic, DNA-crosslinked mucin hydrogel with tunable viscoelastic properties that responds to two types of triggers: temperature alterations and DNA displacement strands, is presented. As confirmed with bulk rheology and single particle tracking, the hybridized base pairs governing the stability of the hydrogel can be opened, thus allowing for a precise control over the hydrogel stiffness and even enabling a full gel-to-sol transition. As those DNA-crosslinked mucin hydrogels possess tunable mechanical properties and can be disintegrated on demand, they can not only be considered for controlled cargo release but may also serve as a role model for the development of smart biomedical materials in applications such as tissue engineering and wound healing.

Photocatalytic CO2 -to-Syngas Evolution with Molecular Catalyst Metal-Organic Framework Nanozymes.

Syngas, a mixture of CO and H2 , is a high-priority intermediate for producing several commodity chemicals, e.g., ammonia, methanol, and synthetic hydrocarbon fuels. Accordingly, parallel sunlight-driven catalytic conversion of CO2 and protons to syngas is a key step toward a sustainable energy cycle. State-of-the-art catalytic systems and materials often fall short as application-oriented concurrent CO and H2 evolution requires challenging reaction conditions which can hamper stability, selectivity, and efficiency. Here a light-harvesting metal-organic framework hosting two molecular catalysts is engineered to yield colloidal, water-stable, versatile nanoreactors for photocatalytic syngas generation with highly controllable product ratios. In-depth fluorescence, X-ray, and microscopic studies paired with kinetic analysis show that the host delivers energy efficiently to active sites, conceptually yielding nanozymes. This unlocked sustained CO2 reduction and H2 evolution with benchmark turnover numbers and record incident photon conversions up to 36%, showcasing a highly active and durable all-in-one material toward application in solar energy-driven syngas generation.

Tailored mechanosensitive nanogels release drugs upon exposure to different levels of stenosis.

Owing to the unhealthy lifestyle and genetic susceptibility of today's population, atherosclerosis is one of the global leading causes of life-threatening cardiovascular diseases. Although a rapid intervention is required for severe blood vessel constrictions, a systemic administration of anticoagulant drugs is not the preferred method of choice as the associated risk of bleeding complications is high. In this study, we present mechanosensitive nanogels that exhibit tunable degrees of disintegration upon exposure to different levels of stenosis. Those nanogels can be further functionalized to encapsulate charged drug molecules such as heparin, and they efficiently release their cargo when passing stenotic constrictions; however, passive drug leakage in the absence of mechanical shear stress is very low. Furthermore, heparin molecules liberated from those mechanosensitive nanogels show a similar blood clot lysis efficiency as the free drug molecules, which demonstrates that drug encapsulation into those nanogels does not interfere with the functionality of the cargo. Thus, the hemocompatible and mechanoresponsive nanogels developed here represent a smart and efficient drug delivery platform that can offer safer solutions for vascular therapy.

A pH-stable, mucin based nanoparticle system for the co-delivery of hydrophobic and hydrophilic drugs.

Biopolymer-based drug carriers are commonly used for the development of safe delivery systems. However, biopolymer-based systems are often highly sensitive to the acidic pH levels in the stomach and release most of their cargo before they have reached their point of destination. Such premature drug release combined with the resulting high dose requirements is not cost-efficient and comes with the risk of unwanted side effects on non-target tissues/organs. This problem can be mitigated by the mucin-based drug carriers developed here, which exhibit good stability at acidic pH levels as proven by dynamic light scattering and enzymatic degradation tests with pepsin. In addition, the mucin-based particles can deliver hydrophobic and hydrophilic drugs simultaneously, which is demonstrated both with experiments performed under in vitro sink conditions and with drug transport tests involving eukaryotic cells as targets. As photo-induced cross-links covalently stabilize those particles, they can release their payload over time in a sustained manner. The drug carrier system introduced here combines good stability with high drug encapsulation efficiency and very good biocompatibility and thus may be valuable for a broad spectrum of applications in biological settings.

Bio-based and bio-inspired adhesives from animals and plants for biomedical applications.

With the "many-headed" slime mold Physarum polycelphalum having been voted the unicellular organism of the year 2021 by the German Society of Protozoology, we are reminded that a large part of nature's huge variety of life forms is easily overlooked - both by the general public and researchers alike. Indeed, whereas several animals such as mussels or spiders have already inspired many scientists to create novel materials with glue-like properties, there is much more to discover in the flora and fauna. Here, we provide an overview of naturally occurring slimy substances with adhesive properties and categorize them in terms of the main chemical motifs that convey their stickiness, i.e., carbohydrate-, protein-, and glycoprotein-based biological glues. Furthermore, we highlight selected recent developments in the area of material design and functionalization that aim at making use of such biological compounds for novel applications in medicine - either by conjugating adhesive motifs found in nature to biological or synthetic macromolecules or by synthetically creating (multi-)functional materials, which combine adhesive properties with additional, problem-specific (and sometimes tunable) features.

Purified mucins in drug delivery research.

One of the main challenges in the field of drug delivery remains the development of strategies to efficiently transport pharmaceuticals across mucus barriers, which regulate the passage and retention of molecules and particles in all luminal spaces of the body. A thorough understanding of the molecular mechanisms, which govern such selective permeability, is key for achieving efficient translocation of drugs and drug carriers. For this purpose, model systems based on purified mucins can contribute valuable information. In this review, we summarize advances that were made in the field of drug delivery research with such mucin-based model systems: First, we give an overview of mucin purification procedures and discuss the suitability of model systems reconstituted from purified mucins to mimic native mucus. Then, we summarize techniques to study mucin binding. Finally, we highlight approaches that made use of mucins as building blocks for drug delivery platforms or employ mucins as active compounds.

Biopolymer-based nanoparticles with tunable mucoadhesivity efficiently deliver therapeutics across the corneal barrier.

To overcome the natural barriers of the ocular system that limit the topical delivery of therapeutically active molecules to the posterior eye, nanoscale drug carriers can be used to improve transcorneal drug transport. So far, using mucoadhesive drug carriers has been put forward as the most promising strategy to optimize drug transport. However, if the mucoadhesivity of a drug carrier is too high, this might limit the diffusive entry of molecules/drug carriers into the vitreous. In this study, we show how modulating the net charge of biopolymer-based drug carrier particles alters not only their mucoadhesivity but also other important properties, e.g., their stability, drug loading capacity and drug release profiles. Compared to simple aqueous solutions of free drug molecules as used in current treatments, nanoparticulate drug carriers with intermediate mucoadhesivity show improved drug transport across the corneal barrier. Therefore, our study shows that mucoadhesion of drug carrier particles is a feature that needs to be considered with great care - not only for ocular delivery attempts but for all drug delivery approaches dealing with mucosal barriers.

DNA Strands Trigger the Intracellular Release of Drugs from Mucin-Based Nanocarriers.

Gaining control over the delivery of therapeutics to a specific disease site is still very challenging. However, especially when cytotoxic drugs such as chemotherapeutics are used, the importance of a control mechanism that can differentiate "sick" target cells from the surrounding healthy tissue is pivotal. Here, we designed a nanoparticle-based drug delivery process, which releases an active agent only in the presence of a specific trigger DNA sequence. With this strategy, we are able to initiate the release of therapeutics into the cytosol with high efficiency. Furthermore, we demonstrate how an endogenous marker (e.g., a specific miRNA sequence) that is overexpressed in the initial phases of certain cancer types can be used as a stimulus to autonomously initiate intracellular drug release-and only in cells where this pathophysiological marker is present. We expect that this precisely controlled delivery mechanism can facilitate the design of site-specific treatments for such diseases, where an overexpression of signature oligonucleotide sequences has been identified.

The effect of biomimetic coating and cuttlebone microparticle reinforcement on the osteoconductive properties of cellulose-based scaffolds.

Polymer-based scaffolds have already gained popularity in many biomedical applications due to convenient routes for fabrication and favourable structural, physicochemical and functional characteristics. However, polymeric scaffolds lack osteoconductivity and some synthetic polymers carry the risk of inflammatory response caused by degradation by-products. Those facts limit their practical use in bone tissue engineering. In this study, three-dimensional (3D) porous scaffolds from naturally derived polymer, namely regenerated cellulose, were prepared using a non-hydrolytic sol-gel and lyophilization techniques. To induce osteoconductive properties of the polymeric scaffolds, cuttlebone microparticles were immobilized and the surface coating was achieved via in vitro mineralization using 10-fold concentrated simulated body fluid (10x SBF). Biogenic activity of cuttlebone is explained by its chemical composition, which includes polysaccharide ß-chitin and macro-, micro- and trace elements favourable for mineralization. Parallel the scaffolds were examined during long-term (24 weeks) in vitro mineralization in 1x SBF for the purpose to investigate apatite-forming ability of the scaffolds. A nice cauliflower-like structures and needle-like dents of the spherical aggregates, which are characteristic to hydroxyapatite precursors, were observed on the surface of cellulose/cuttlebone scaffolds by SEM. 10x SBF coating enhanced cell attachment to the scaffolds because SBF elements are known to increase bioactivity by inducing re-deposition of carbonate apatite crystallites on scaffold surface. Additionally, calcium and phosphate depositions were clearly observed on the developed scaffolds using von Kossa and Alizarin Red S staining. Proliferative and osteoconductive effects on the osteoblast-like MG-63 cells demonstrate the cellulose/cuttlebone scaffolds soaked in 10x SBF as a favourable material for bone tissue engineering.

Molecular micromanagement: DNA nanotechnology establishes spatio-temporal control for precision medicine.

Current advances in DNA nanotechnology pinpoint exciting perspectives for the design of customized, patient-specific treatments. This advance is made possible by the exceptionally high precision and specificity that are typical for DNA base pairing on the one hand and our growing ability to harness those features in synthetic, DNA-based constructs on the other hand. Modern medicine may soon benefit from recent developments in this field, especially regarding the targeted delivery of drugs and the rational interference of synthetic DNA strands with cellular oligonucleotides. In this Review, we summarize selected examples from the area of DNA nanotechnology, where the development of precisely controlled, advanced functional mechanisms was achieved. To demonstrate the high versatility of these rationally designed structures, we categorize the dynamic DNA-based materials suggested for precision medicine according to four fundamental tasks: "hold & release," "heal," "detect & measure," as well as "guide & direct." In all the biomedical applications we highlight, DNA strands not only constitute structural building blocks but allow for creating stimuli-responsive objects, serve as an active cargo, or act as molecular control/guidance tools. Moreover, we discuss several issues that need to be considered when DNA-based structures are designed for applications in the field of precision medicine. Even though the majority of DNA-based objects have not been used in clinical settings yet, recent progress regarding the stability, specificity, and control over the dynamic behavior of synthetic DNA structures has advanced greatly. Thus, medical applications of those nanoscopic objects should be feasible in the near future.

Bioactive diatomite and POSS silica cage reinforced chitosan/Na-carboxymethyl cellulose polyelectrolyte scaffolds for hard tissue regeneration.

Recently, natural polymers are reinforced with silica particles for hard tissue engineering applications to induce bone regeneration. In this study, as two novel bioactive agents, effects of diatomite and polyhedral oligomeric silsesquioxanes (POSS) on chitosan (CS)/Na-carboxymethylcellulose (Na-CMC) polymer blend scaffolds are examined. In addition, the effect of silica reinforcements was compared with Si-substituted nano-hydroxyapatite (Si-Hap) particles. The morphology, physical and chemical structures of the scaffolds were characterized with SEM, liquid displacement, FT-IR, mechanical analysis, swelling and degradation studies. The particle size and the crystal structure of diatomite, POSS and Si-Hap particles were determined with DLS and XRD analyses. In vitro studies were performed to figure out the cytotoxicity, proliferation, ALP activity, osteocalcin production and biomineralization to demonstrate the promising use of natural silica particles in bone regeneration. Freeze-dried scaffolds showed 190-307 µm pore size range and 61-70% porosity. Both inorganic reinforcements increased the mechanical strength, enhanced the water uptake capacity and fastened the degradation rate. The nanocomposite scaffolds did not show any cytotoxic effect and enhanced the surface mineralization in osteogenic medium. Thus, diatomite and POSS cage structures can be potential reinforcements for nanocomposite design in hard tissue engineering applications.

Engineering an orchestrated release avalanche from hydrogels using DNA-nanotechnology.

Most medical therapies require repeated, sequential administration of therapeutic agents in well-defined intervals and over extended time windows. Typically, the patient is in charge of applying the individual drug doses, and insufficient patient compliance reduces the efficiency of the treatment. Therefore, the development of a smart delivery mechanism releasing therapeutic agents in a pre-defined, time-controlled fashion would be beneficial for many medical treatments. Here, we present a DNA-mediated release cascade which allows for precisely controlling the sequential delivery of several different nanoparticles. By using complementary DNA sequences, nanoparticle aggregates are created, embedded into distinct layers of a hydrogel and released by triggering aggregate dispersal. This mechanism is compatible with physiological conditions as the release cascade is initiated by exposing the nanoparticle-loaded gel to physiological salt concentrations. Moreover, we show that the reservoir hydrogel can be enriched with biopolymers to receive charge-selective release properties towards small molecules - without interfering with the DNA-based release cascade. Owing to the excellent reproducibility, precision and effectiveness of the presented mechanism, a similar DNA-mediated release avalanche may lead to the development of autonomous and robust delivery systems, which minimize the possibility of pharmaceutical therapy failure due to patient non-compliance.

Chitosan/montmorillonite composite nanospheres for sustained antibiotic delivery at post-implantation bone infection treatment.

Despite the advancements in bone transplantation operations, inflammation is still a serious problem that threatens human health at the post-implantation period. Conventional antibiotic therapy methods may lead to some side effects such as ototoxicity and nephrotoxicity, especially when applied in high doses. Therefore, local drug delivery systems play a vital role in bone disorders due to the elimination of the disadvantages introduced by conventional methods. In the presented study, it was aimed to develop Vancomycin (VC) and Gentamicin (GC) loaded chitosan-montmorillonite nanoclay composites (CS/MMT) to provide required antibiotic doses to combat post-implantation infection. CS/MMT nanocomposite formation was supplied by microfluidizer homogenization and spherical drug carrier nanoparticles were obtained by electrospraying technique. Three factors; voltage, distance and flowrate were varied to fabricate spherical nanoparticles with uniform size. Emprical model was developed to predict nanosphere size by altering process variables. Nanospheres were characterized in terms of morphology, hydrodynamic size, zeta potential, drug encapsulation efficiency and release profile. Drug loaded nanospheres have been successfully produced with a size range of 180-350 nm. Nanocomposite drug carriers showed high encapsulation efficiency (80%-95%) and prolonged release period when compared to bare chitosan nanospheres. The drug release from nanocomposite carriers was monitored by diffusion mechanism up to 30 d. The in vitro release medium of nanospheres showed strong antimicrobial activity against gram-positive S. aureus and gram-negative E. coli bacteria. Furthermore, it was found that the nanospheres did not show any cytotoxic effect to fibroblast (NIH/3T3) and osteoblast (SaOS-2) cell lines. The results demonstrated that the prepared composite nanospheres can be a promising option for bone infection prevention at the post implantation period.

Osteoconductive 3D porous composite scaffold from regenerated cellulose and cuttlebone-derived hydroxyapatite.

Recently, usage of marine-derived materials in biomedical field has come into prominence due to their promising characteristics such as biocompatibility, low immunogenicity and wide accessibility. Among these marine sources, cuttlebone has been used as a valuable component with its trace elemental composition in traditional medicine. Recent studies have focused on the use of cuttlebone as a bioactive agent for tissue engineering applications. In this study, hydroxyapatite particles were obtained by hydrothermal synthesis of cuttlebone and incorporated to cellulose scaffolds to fabricate an osteoconductive composite scaffold for bone regeneration. Elemental analysis of raw cuttlebone material from different coastal zones and cuttlebone-derived HAp showed that various macro-, micro- and trace elements - Ca, P, Na, Mg, Cu, Sr, Cl, K, S, Br, Fe and Zn were found in a very similar amount. Moreover, biologically unfavorable heavy metals, such as Ag, Cd, Pb or V, were not detected in any cuttlebone specimen. Carbonated hydroxyapatite particle was further synthesized from cuttlebone microparticles via hydrothermal treatment and used as a mineral filler for the preparation of cellulose-based composite scaffolds. Interconnected highly porous structure of the scaffolds was confirmed by micro-computed tomography. The mean pore size of the scaffolds was 510 µm with a porosity of 85%. The scaffolds were mechanically characterized with a compression test and cuttlebone-derived HAp incorporation enhanced the mechanical properties of cellulose scaffolds. In vitro cell culture studies indicated that MG-63 cells proliferated well on scaffolds. In addition, cuttlebone-derived hydroxyapatite significantly induced the ALP activity and osteocalcin secretion. Besides, HAp incorporation increased the surface mineralization which is the major step for bone tissue regeneration.

Novel zein-based multilayer wound dressing membranes with controlled release of gentamicin.

Recently, functional multilayer scaffolds with controlled drug release ability come into prominence for wound healing applications to mimic the layered structure of skin tissue and prevent the possible infections at the defect site. In this study, controlled antibiotic releasing zein bilayer membranes were fabricated for treatment of acute skin infections. Gentamicin loaded fibers were prepared by electrospinning on the membrane surface. Membranes were characterized with scanning electron microscope, atomic force microscopy, Fourier transform infrared spectroscopy, contact angle, mechanical analysis, swelling, degradation, and water vapor permeability studies. In vitro cytotoxicity, cell attachment, and proliferation were investigated. Cell attachment on fiber layer was observed with fluorescence imaging. Fabricated fibers showed structural similarity to the skin tissue layers with a fiber diameter range of 350-425 nm and film thickness in the range of 311-361 µm. Mechanical properties were found compatible with the skin tissue. In addition, membranes showed antimicrobial activity against Staphylococcus aureus and Escherichia coli. The sustained release was achieved with a cumulative release of 94%. Membranes did not show any cytotoxic effect. NIH/3T3 and HS2 cell lines were proliferated on each layer mimicking the multilayer skin tissue. Hence, zein-based bilayer membrane showed promising properties to be used as a potential antimicrobial wound dressing for skin tissue regeneration. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2057-2070, 2019.