Brush Conformation of Polyethylene Glycol Determines the Stealth Effect of Nanocarriers in the Low Protein Adsorption Regime.

For nanocarriers with low protein affinity, we show that the interaction of nanocarriers with cells is mainly affected by the density, the molecular weight, and the conformation of polyethylene glycol (PEG) chains bound to the nanocarrier surface. We achieve a reduction of nonspecific uptake of ovalbumin nanocarriers by dendritic cells using densely packed PEG chains with a "brush" conformation instead of the collapsed "mushroom" conformation. We also control to a minor extent the dysopsonin adsorption by tailoring the conformation of attached PEG on the nanocarriers. The brush conformation of PEG leads to a stealth behavior of the nanocarriers with inhibited uptake by phagocytic cells, which is a prerequisite for successful in vivo translation of nanomedicine to achieve long blood circulation and targeted delivery. We can clearly correlate the brush conformation of PEG with inhibited phagocytic uptake of the nanocarriers. This study shows that, in addition to the surface's chemistry, the conformation of polymers controls cellular interactions of the nanocarriers.

The Influence of Nanoparticle Shape on Protein Corona Formation.

Nanoparticles have become an important utility in many areas of medical treatment such as targeted drug and treatment delivery as well as imaging and diagnostics. These advances require a complete understanding of nanoparticles' fate once placed in the body. Upon exposure to blood, proteins adsorb onto the nanoparticles surface and form a protein corona, which determines the particles' biological fate. This study reports on the protein corona formation from blood serum and plasma on spherical and rod-shaped nanoparticles. These two types of mesoporous silica nanoparticles have identical chemistry, porosity, surface potential, and size in the y-dimension, one being a sphere and the other a rod shape. The results show a significantly larger amount of protein attaching from both plasma and serum on the rod-like particles compared to the spheres. Interrogation of the protein corona by liquid chromatography-mass spectrometry reveals shape-dependent differences in the adsorption of immunoglobulins and albumin proteins from both plasma and serum. This study points to the need for taking nanoparticle shape into consideration because it can have a significant impact on the fate and therapeutic potential of nanoparticles when placed in the body.

Revisability of polyetheretherketone suture anchors utilised in primary and revision Bankart repair.

BACKGROUND: Polyetheretherketone (PEEK) suture anchors are frequently used in Bankart shoulder stabilisation. This study analyzed the primary stability and revisability of PEEK anchors in-vitro in case of primary Bankart repair and revision Bankart repair after failed primary repair. METHODS: To simulate primary Bankart repair, 12 anchors (Arthrex PEEK PushLock® 3.5 mm) were implanted in 1, 3, 5, 7, 9 and 11 o'clock positions in cadaveric human glenoids and then cyclically tested. To simulate revision Bankart repair, 12 anchors were implanted in the same manner, over-drilled and 12 new anchors of the same diameter were implanted into the same bone socket as the primary anchors and then cyclically tested. The maximum failure loads (Fmax), system displacements, force at clinical failure and modes of failure were recorded. RESULTS: One primary anchor failed prematurely due to a technical problem. Three out of 12 revision anchors (25%) dislocated while setting the 25 N preload. The Fmax, the displacement and clinical failure of the remaining 9 revision anchors were non-significant when compared to the 11 primary repair anchors. The main mode of failure in the primary and revision Bankart surgery group was suture slippage. Anchor dislocations were observed four times in the primary and once in the revision repair groups. CONCLUSIONS: Revision Bankart repair using PEEK anchors of the same diameter in a pre-existing bone socket is possible but bears high risk of premature anchor failure and can jeopardize the reconstruction. PEEK suture anchor in revision Bankart surgery should be implanted in a new bone socket if possible.

Amphiphilic Polyphenylene Dendron Conjugates for Surface Remodeling of Adenovirus†5.

Amphiphilic surface groups play an important role in many biological processes. The synthesis of amphiphilic polyphenylene dendrimer branches (dendrons), providing alternating hydrophilic and lipophilic surface groups and one reactive ethynyl group at the core is reported. The amphiphilic surface groups serve as biorecognition units that bind to the surface of adenovirus 5 (Ad5), which is a common vector in gene therapy. The Ad5/dendron complexes showed high gene transduction efficiencies in coxsackie-adenovirus receptor (CAR)-negative cells. Moreover, the dendrons offer incorporation of new functions at the dendron core by in situ post-modifications, even when bound to the Ad5 surface. Surfaces coated with these dendrons were analyzed for their blood-protein binding capacity, which is essential to predict their performance in the blood stream. A new platform for introducing bioactive groups to the Ad5 surface without chemically modifying the virus particles is provided.

Timing of Heparin Addition to the Biomolecular Corona Influences the Cellular Uptake of Nanocarriers.

Few studies have considered the interaction of nanocarriers with drugs and the implications for their individual efficiency. Here, we demonstrate that heparin, a common anticoagulant, interacts with nanocarriers. Hence, nanocarriers, precoated with heparin and plasma in different conditions, were incubated with cancer cells, as well as primary cells from human blood. The relation between the timing of the heparin's addition to the nanocarrier and the cellular uptake extent was assessed by flow cytometry. Through proteomics the effect of heparin on the biomolecular corona composition was determined. We found that HeLa cells, monocytes and macrophages reacted differently to the presence of heparin: the uptake of the precoated nanocarriers decreased for HeLa and primary monocytes, while it increased for macrophages. Heparin induced no obvious change in the protein corona composition; thus, we suggest that heparin itself, through its adsorption on the nanocarrier, was responsible for the change of uptake.

Functionalization of Liposomes with Hydrophilic Polymers Results in Macrophage Uptake Independent of the Protein Corona.

Liposomes are established drug carriers that are employed to transport and deliver hydrophilic drugs in the body. To minimize unspecific cellular uptake, nanocarriers are commonly modified with poly(ethylene glycol) (PEG), which is known to minimize unspecific protein adsorption. However, to date, it has not been studied whether this is an intrinsic and specific property of PEG or if it can be transferred to hyperbranched polyglycerol (hbPG) as well. Additionally, it remains unclear if the reduction of unspecific cell uptake is independent of the "basic" carrier at which a surface functionalization with polymers is usually applied. Therefore, we studied the protein corona of differently functionalized liposomes (unfunctionalized vs PEG or hbPG-functionalized) using PEGylated and PGylated lipids. Their cellular uptake in macrophages was compared. For all three liposomal samples, rather similar protein corona compositions were found, and also-more importantly-the total amount of proteins adsorbed was very low compared to other nanoparticles. Interestingly, the cellular uptake was then significantly changed by the surface functionalization itself, despite the adsorption of a small amount of proteins: although the PEGylation of liposomes resulted in the abovementioned decreased cell uptake, functionalization with hbPG lead to enhanced macrophage interaction-both in the media with and without proteins. In comparison to other nanocarrier systems, this seems to be a liposome-specific effect related to the low amount of adsorbed proteins.

The Transferability from Animal Models to Humans: Challenges Regarding Aggregation and Protein Corona Formation of Nanoparticles.

Nanomaterials are interesting candidates for applications in medicine as drug delivery or diagnostic agents. For safe application, they have to be evaluated in in vitro and in vivo models to finally be translated to human clinical trials. However, often those transfer processes fail, and it is not completely understood whether in vitro models leading to these animal models can reliably be compared to the situation in humans. In particular, the interaction of nanomaterials with components from different blood plasma sources is difficult to compare, and the outcomes of those interactions with respect to body distribution and cell uptake are unclear. Therefore, we investigated the interactions of differently functionalized polymeric and inorganic nanoparticles with human, mouse, rabbit, and sheep plasma. The focus was put on the determination of aggregation events of the nanoparticles occurring in concentrated plasma and the correlation with the respectively formed protein coronas. Both the stability in plasma as well as the types of adsorbed proteins were found to strongly depend on the plasma source. Thus, we suggest evaluating the potential use of nanocarriers always in the plasma source of the chosen animal model for in vitro studies as well as in human plasma to pin down differences and eventually enable transfer into clinical trials in humans.

Denaturation via Surfactants Changes Composition of Protein Corona.

The use of nanocarriers as drug delivery vehicles brings them into contact with blood plasma proteins. Polymeric nanocarriers require some sort of surfactant to ensure colloidal stability. Formation of the protein corona is therefore determined not only by the intrinsic properties of the nanocarrier itself but also by the accompanying surfactant. Although it is well-known that surfactants have an impact on protein structure, only few studies were conducted on the specific effect of surfactants on the composition of protein corona of nanocarriers. Therefore, we analyzed the composition of the protein corona on "stealth" nanoparticles with additional surfactant (cetyltrimethylammonium chloride, CTMA-Cl) after plasma incubation. Additional CTMA-Cl led to an enrichment of apolipoprotein-A1 and vitronectin in the corona, while less clusterin could be found. Further, the structural stability of apolipoprotein-A1 and clusterin was monitored for a wide range of CTMA-Cl concentrations. Clusterin turned out to be more sensitive to CTMA-Cl, with denaturation occurring at lower concentrations.

Hydrophilicity Regulates the Stealth Properties of Polyphosphoester-Coated Nanocarriers.

Increasing the plasma half-life is an important goal in the development of drug carriers, and can be effectively achieved through the attachment of polymers, in particular poly(ethylene glycol) (PEG). While the increased plasma half-life has been suggested to be a result of decreased overall protein adsorption on the hydrophilic surface in combination with the adsorption of specific proteins, the molecular reasons for the success of PEG and other hydrophilic polymers are still widely unknown. We prepared polyphosphoester-coated nanocarriers with defined hydrophilicity to control the stealth properties of the polymer shell. We found that the log P value of the copolymer controls the composition of the protein corona and the cell interaction. Upon a significant change in hydrophilicity, the overall amount of blood proteins adsorbed on the nanocarrier remained unchanged, while the protein composition varied. This result underlines the importance of the protein type for the protein corona and cellular uptake.

Reversible Self-Assembly of Degradable Polymersomes with Upper Critical Solution Temperature in Water.

Temperature-induced self-assembly of block copolymers allows the formation of smart nanodimensional structures. Mostly, nondegradable lower critical solution temperature (LCST) segments are applied to prepare such dynamic aggregates. However, degradable upper critical phase separation (UCST) block copolymers that would allow the swelling or disassembly at elevated temperatures with eventual backbone hydrolysis have not been reported to date. We present the first well-defined degradable poly(phosphonate)s with adjustable UCST. The organocatalytic anionic ring-opening copolymerization of 2-alkyl-2-oxo-1,3,2-dioxaphospholanes provided functional polymers with excellent control over molecular weight and copolymer composition. The prepolymers were turned into thermoresponsive polymers by thiol-ene modification to introduce pendant carboxylic acids. By this means, non cell-toxic, degradable polymers exhibiting UCST behavior in water between 43 and 71 °C were produced. Block copolymers with PEG as a nonresponsive water-soluble block can self-assemble into well-defined polymersomes with narrow size distribution. Depending on the responsive block, these structures either swell or disassemble completely upon an increased temperature.

Analytical techniques for the characterization of nanoparticles for mRNA delivery.

Nanotechnology-assisted RNA delivery has gotten a tremendous boost over the last decade and made a significant impact in the development of life-changing vaccines and therapeutics. With increasing numbers of emerging lipid- and polymer-based RNA nanoparticles progressing towards the clinic, it has become apparent that the safety and efficacy of these medications depend on the comprehensive understanding of their critical quality attributes (CQAs). However, despite the rapid advancements in the field, the identification and reliable quantification of CQAs remain a significant challenge. To support these efforts, this review aims to summarize the present knowledge on CQAs based on the regulatory guidelines and to provide insights into the available analytical characterization techniques for RNA-loaded nanoparticles. In this context, routine and emerging analytical techniques are categorized and discussed, focusing on the operation principle, strengths, and potential limitations. Furthermore, the importance of complementary and orthogonal techniques for the measurement of CQAs is discussed in order to ensure the quality and consistency of analytical methods used, and address potential technique-based differences.

Ten characteristics of high-quality planetary health education-Results from a qualitative study with educators, students as educators and study deans at medical schools in Germany.

Aim: The climate and ecological crises are considered fundamental threats to human health. Healthcare workers in general and doctors in particular can contribute as change agents in mitigation and adaptation. Planetary health education (PHE) aims to harness this potential. This study explores perspectives among stakeholders involved in PHE at German medical schools on the characteristics of high-quality PHE and compares them to existing PHE frameworks. Methods: In 2021, we conducted a qualitative interview study with stakeholders from German medical schools involved in PHE. Three different groups were eligible: faculty members, medical students actively involved in PHE, and study deans of medical schools. Recruitment was performed through national PHE networks and snowball sampling. Thematic qualitative text analysis according to Kuckartz was used for the analysis. Results were systematically compared to three existing PHE frameworks. Results: A total of 20 participants (13 female) from 15 different medical schools were interviewed. Participants covered a wide range of professional backgrounds and experience in PHE education. The analysis revealed ten key themes: (1) Complexity and systems thinking, (2) inter- and transdisciplinarity, (3) ethical dimension, (4) responsibility of health professionals, (5) transformative competencies including practical skills, (6) space for reflection and resilience building, (7) special role of students, (8) need for curricular integration, (9) innovative and proven didactic methods, and (10) education as a driver of innovation. Six of our themes showed substantial overlap with existing PHE frameworks. Two of our themes were only mentioned in one of the frameworks, and two others were not explicitly mentioned. Few important elements of the frameworks did not emerge from our data. Conclusions: In the light of increased attention regarding the connections of the climate and ecological crises and health, our results can be useful for anyone working toward the integration of planetary health into medical schools' and any health professions' curricula and should be considered when designing and implementing new educational activities.

A new methodology combining QCM-D and proteomic profiling enables characterization of protein adsorption on 2D surfaces.

One of the critical features of biomedical material design is controlling the plasma protein adsorption to modulate the material behavior in biological media. Protein adsorption is highly influenced by the material surfaces and the proteins present in the biological medium. Thus, it is necessary to study protein-surface interactions that eventually take place on nanomaterials introduced into the body by the use of human plasma. However, very little information is available about human plasma interaction with planar surfaces under physiological conditions. Due to the limitation of the current characterization techniques to investigate the complicated interaction between the complex milieu of plasma proteins and planar materials, most efforts have focused on single proteins. To face this challenge, we have developed a new methodology based on the combination of quartz crystal microbalance with dissipation monitoring (QCM-D) and liquid chromatography coupled with mass spectrometry (LC-MS) to obtain information about protein-surface interactions on planar surfaces. First, QCM-D allowed us to determine the adsorbed protein mass and layer thickness. After detaching the proteins by a surfactant treatment, LC-MS analysis revealed the proteomic profile. Here, we have investigated three base materials, polystyrene (PS), gold (Au), and silica (SiO2) with or without precoating and compared the protein profiles.

The Effect of Smoking on the Outcome of Matrix-Based Autologous Chondrocyte Implantation: Data from the German Cartilage Registry.

Smoking is known to have various deleterious effects on health. However, it is not clear whether smoking negatively affects the postoperative outcome following matrix-based autologous cartilage implantation (MACI) in the knee. The purpose of this study was to evaluate the effect of smoking on the outcome of MACI in the knee. A total of 281 patients receiving MACI in the knee between 2015 and 2018 were registered in the German Cartilage Database. The cohort was divided into ex-smokers, smokers, and nonsmokers. Data regarding the Knee Injury and Osteoarthritis Outcome Score (KOOS), the numeric rating scale (NRS) for pain, and satisfaction with the outcome were analyzed and compared. Follow-ups were performed at 6, 12, and 24 months after surgery. Of the 281 patients, 225 (80.1%) were nonsmokers, 43 (15.3%) were smokers, and 13 (4.6%) were ex-smokers. The three groups were comparable with respect to age, sex, body mass index (BMI), height, defect size, the need for additional reconstruction of the subchondral bone defect, number of previous knee surgeries, and defect location. However, nonsmokers had a significantly lower weight as compared with smokers. Besides a significantly lower preoperative NRS of nonsmokers as compared with smokers, there were no significant differences between the three groups with respect to KOOS, NRS, and satisfaction at 6, 12, and 24 months of follow-ups. The present study of data retrieved from the German Cartilage Registry suggests that the smoking status does not influence the outcome of MACI in the knee.

Initiatives promoting planetary health education in Germany: An overview.

Planetary health education focuses on the climate and ecological crises and their adverse health effects. Given the acceleration of these crises, nationwide integration of planetary health education into undergraduate and graduate education, postgraduate training and continuing education for all health professionals has repeatedly been called for. Since 2019, planetary health education has been promoted by several national initiatives in Germany that are summarized in this commentary: 1. National Working Group Planetary Health Education, 2. Manual for planetary health education, 3. Catalog of National Planetary Health Learning Objectives in the National Competency-Based Catalog of Learning Objectives for Medical Education, 4. Working Group Climate, Environment and Health Impact Assessment at the Institute for Medical and Pharmaceutical Examinations, 5. Planetary Health Report Card, and 6. PlanetMedEd study: planetary health education in medical schools in Germany. We hope these initiatives promote collaboration across institutions involved in educating and training health professionals, inter-professional cooperation as well as rapid implementation of planetary health education.

Systematic modulation of the lipid composition enables the tuning of liposome cellular uptake.

As liposomes have been widely explored as drug delivery carriers over the past decades, they are one of the most promising platforms due to their biocompatibility and versatility for surface functionalization. However, to improve the specific design of liposomes for future biomedical applications such as nanovaccines, it is necessary to understand how these systems interact with cell membranes, as most of their potential applications require them to be internalized by cells. Even though several investigations on the cellular uptake of liposomes were conducted, the effect of the liposome membrane properties on internalization in different cell lines remains unclear. Here, we demonstrate how the cellular uptake behavior of liposomes can be driven towards preferential interaction with dendritic cells (DC2.4) as compared to macrophages (RAW264.7) by tuning the lipid composition with varied molar ratios of the lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Cellular internalization efficiency was analyzed by flow cytometry, as well as liposome-cell membrane co-localization by confocal laser scanning microscopy. The corresponding proteomic analysis of the protein corona was performed in order to unravel the possible effect on the internalization. The obtained results of this work reveal that it is possible to modulate the cellular uptake towards enhanced internalization by dendritic cells just by modifying the applied lipids and, thus, mainly the physico-chemical properties of the liposomes. STATEMENT OF SIGNIFICANCE: In the field of nanomedicine, it is of key importance to develop new specific and efficient drug carriers. In this sense, liposomes are one of the most widely known carrier types and used in clinics with good results. However, the exact interaction mechanisms of liposomes with cells remain unclear, which is of great importance for the design of new drug delivery platforms. Therefore, in this work we demonstrate that cellular uptake depends on the lipid composition. We are able to enhance the uptake in a specific cell type just by tuning the content of a lipid in the liposome membrane. This finding could be a step towards the selective design of liposomes to be internalized by specific cells with promising applications in biomedicine.

Endosomal sorting results in a selective separation of the protein corona from nanoparticles.

The formation of the protein corona is a well-known effect when nanoparticles (NP) are exposed to biological environments. The protein corona is the most important factor, which determines the rate and route of endocytosis, and decisively impacts cellular processes and even the release of the active pharmaceutical ingredient from the nanoparticles. While many studies concentrate on the effect of the protein corona formation extracellularly or the uptake consequences, little is known about the fate of the protein corona inside of cells. Here, we reconstruct for the first time the separation of the protein corona from the NPs by the cell and their further fate. Ultimately, the NPs and protein corona are separated from each other and end up in morphologically different cellular compartments. The cell directs the NPs towards recycling endosomes, whereas the protein corona gathers in multivesicular bodies. From this, we conclude that the NPs are prepared for subsequent exocytosis, while the protein corona remains in the cell and is finally metabolized there.

Antibacterial Nanostructured Surfaces Modulate Protein Adsorption, Inflammatory Responses, and Fibrous Capsule Formation.

The present study interrogates the interaction of highly efficient antibacterial surfaces containing sharp nanostructures with blood proteins and the subsequent immunological consequences, processes that are of key importance for the fate of every implantable biomaterial. Studies with human serum and plasma pointed to significant differences in the composition of the protein corona that formed on control and nanostructured surfaces. Quantitative analysis using liquid chromatography-mass spectrometry demonstrated that the nanostructured surface attracted more vitronectin and less complement proteins compared to the untreated control. In turn, the protein corona composition modulated the adhesion and cytokine expression by immune cells. Monocytes produced lower amounts of pro-inflammatory cytokines and expressed more anti-inflammatory factors on the nanostructured surface. Studies using an in vivo subcutaneous mouse model showed reduced fibrous capsule thickness which could be a consequence of the attenuated inflammatory response. The results from this work suggest that antibacterial surface modification with sharp spike-like nanostructures may not only lead to the reduction of inflammation but also more favorable foreign body response and enhanced healing, processes that are beneficial for most medical devices implanted in patients.

Antibody-Functionalized Carnauba Wax Nanoparticles to Target Breast Cancer Cells.

Development of safer nanomedicines for drug delivery applications requires immense efforts to improve clinical outcomes. Targeting a specific cell, biocompatibility and biodegradability are vital properties of a nanoparticle to fulfill the safety criteria in medical applications. Herein, we fabricate antibody-functionalized carnauba wax nanoparticles encapsulated a hydrophobic drug mimetic, which is potentially interesting for clinical use due to the inert and nontoxic properties of natural waxes. The nanoparticles are synthesized applying miniemulsion methods by solidifying molten wax droplets and further evaporating the solvent from the dispersion. The pH-selective adsorption of antibodies (IgG1, immunoglobulin G1, and CD340, an antihuman HER2 antibody) onto the nanoparticle surface is performed for practical and effective functionalization, which assists to overcome the complexity in chemical modification of carnauba wax. The adsorption behavior of the antibodies is studied using isothermal titration calorimetry (ITC), which gives thermodynamic parameters including the enthalpy, association constant, and stoichiometry of the functionalization process. Both antibodies exhibit strong binding at pH 2.7. The CD340-decorated wax nanoparticles show specific cell interaction toward BT474 breast cancer cells and retain the targeting function even after 6 months of storage period.

Proteomics reveals differential adsorption of angiogenic platelet lysate proteins on calcium phosphate bone substitute materials.

Protein adsorption on biomaterials for bone substitution, such as calcium phosphates (CaP), evokes biological responses and shapes the interactions of biomaterials with the surrounding biological environment. Proteins adsorb when CaP materials are combined with growth factor-rich hemoderivatives prior to implantation to achieve enhanced angiogenesis and stimulate new bone formation. However, the identification of the adsorbed proteins and their angiogenic effect on bone homeostasis remain incompletely investigated. In this study, we analyzed the adsorbed complex protein composition on CaP surfaces when using the hemoderivatives plasma, platelet lysate in plasma (PL), and washed platelet lysate proteins (wPL). We detected highly abundant, non-regenerative proteins and anti-angiogenic proteins adsorbed on CaP surfaces after incubation with PL and wPL by liquid chromatography and mass spectrometry (LC-MS) proteomics. Additionally, we measured a decreased amount of adsorbed pro-angiogenic growth factors. Tube formation assays with human umbilical endothelial cells demonstrated that the CaP surfaces only stimulate an angiogenic response when kept in the hemoderivative medium but not after washing with PBS. Our results highlight the necessity to correlate biomaterial surfaces with complex adsorbed protein compositions to tailor the biomaterial surface toward an enrichment of pro-angiogenic factors.