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.

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.

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.

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.

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.

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.

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.

Ultra-small gold nanoclusters assembled on plasma polymer-modified zeolites: a multifunctional nanohybrid with anti-haemorrhagic and anti-inflammatory properties.

Hemostatic agents are pivotal for managing clinical and traumatic bleeding during emergency and domestic circumstances. Herein, a novel functional hybrid nanocomposite material consisting of plasma polymer-modified zeolite 13X and ultra-small gold nanoclusters (AuNCs) was fabricated as an efficient hemostatic agent. The surface of zeolite 13X was functionalised with amine groups which served as binding sites for carboxylate terminated AuNCs. Protein corona studies revealed the enhanced adsorption of two proteins, namely, coagulation factors and plasminogen as a result of AuNCs immobilization on the zeolite surface. The immune response studies showed that the hybrid nanocomposites are effective in reducing inflammation, which combined with a greater attachment of vitronectin, may promote wound healing. The hemostatic potential of the nanocomposite could be directly correlated with their immunomodulatory and anti-haemorrhagic properties. Together, the hybrid nanoengineered material developed in this work could provide a new avenue to tackle life-threatening injuries in civilian and other emergencies.

Encapsulation of polyprodrugs enables an efficient and controlled release of dexamethasone.

Water-soluble low molecular weight drugs, such as the synthetic glucocorticoid dexamethasone (DXM), can easily leak out of nanocarriers after encapsulation due to their hydrophilic nature and small size. This can lead to a reduced therapeutic efficacy and therefore to unwanted adverse effects on healthy tissue. Targeting DXM to inflammatory cells of the liver like Kupffer cells or macrophages is a promising approach to minimize typical side effects. Therefore, a controlled transport to the cells of interest and selective on-site release is crucial. Aim of this study was the development of a DXM-phosphate-based polyprodrug and the encapsulation in silica nanocontainers (SiO2 NCs) for the reduction of inflammatory responses in liver cells. DXM was copolymerized with a linker molecule introducing pH-cleavable hydrazone bonds in the backbone and obtaining polyprodrugs (PDXM). Encapsulation of PDXMs into SiO2 NCs provided a stable confinement avoiding uncontrolled leakage. PDXMs were degraded under acidic conditions and subsequently released out of SiO2 NCs. Biological studies showed significantly enhanced anti-inflammatory capacity of the polyprodrug nanoformulations over non-encapsulated DXM or soluble polyprodrugs. These results demonstrate the advantage of combining the polyprodrug strategy with nanocarrier-mediated delivery for enhanced control of the delivery of water-soluble low molecular weight drugs.

The conjugation strategy affects antibody orientation and targeting properties of nanocarriers.

Antibody-modified drug delivery systems in the nano-range have the ability to overcome current challenges for treating diseases due to their high specificity towards the targeted body region. However, no antibody-bound nanocarrier has been clinically approved to date. This missing clinical approval may be a result of the conjugation strategy that influences the spatial orientation of the attached antibody on the nanocarriers' surface. What is not missing, however, is a diverse selection of antibody to nanocarrier conjugation strategies that determine the success of an antibody functionalized drug delivery system. In this paper, two antibody conjugation strategies were compared by conjugating the surface of cross-linked starch iron oxide nanocarriers with specifically modified CD11c monoclonal antibodies. The antibody nanocarrier conjugates, synthesized either by the chemistry of thiol-maleimide coupling or copper-free click chemistry, were analyzed by flow cytometry to determine their binding affinity towards a murine dendritic cell line (DC2.4). In the cell uptake, different antibody amounts on the nanocarrier could induce a dendritic cell uptake for both conjugation strategies. However, blocking experiments further highlighted the importance of the orientation of the antibody on to the nanocarriers' surface. While the antibodies which were attached via the copper-free click chemistry were oriented, maleimide synthesized conjugates presented their antibodies randomly on the surface. Lastly, to evaluate the in vivo properties of the antibody modified nanocarriers, targeting experiments with mouse plasma were performed, and it was proven that the biomolecular corona does not diminish the targeting efficiency.

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.

Unraveling the In Vivo Protein Corona.

Understanding the behavior of nanoparticles upon contact with a physiological environment is of urgent need in order to improve their properties for a successful therapeutic application. Most commonly, the interaction of nanoparticles with plasma proteins are studied under in vitro conditions. However, this has been shown to not reflect the complex situation after in vivo administration. Therefore, here we focused on the investigation of magnetic nanoparticles with blood proteins under in vivo conditions. Importantly, we observed a radically different proteome in vivo in comparison to the in vitro situation underlining the significance of in vivo protein corona studies. Next to this, we found that the in vivo corona profile does not significantly change over time. To mimic the in vivo situation, we established an approach, which we termed "ex vivo" as it uses whole blood freshly prepared from an animal. Overall, we present a comprehensive analysis focusing on the interaction between nanoparticles and blood proteins under in vivo conditions and how to mimic this situation with our ex vivo approach. This knowledge is needed to characterize the true biological identity of nanoparticles.

Polyphosphoester surfactants as general stealth coatings for polymeric nanocarriers.

Opsonization of nanocarriers is one of the most important biological barriers for controlled drug delivery. The typical way to prevent such unspecific protein adsorption and thus fast clearance by the immune system is the covalent modification of drug delivery vehicles with poly(ethylene glycol) (PEG), so-called PEGylation. Recently, polyphosphoesters (PPEs) were identified as adequate PEG substitutes, however with the benefits of controllable hydrophilicity, additional chemical functionality, or biodegradability. Here, we present a general strategy by non-covalent adsorption of different nonionic PPE-surfactants to nanocarriers with stealth properties. Polyphosphoester surfactants with different binding motifs were synthesized by anionic ring-opening polymerization of cyclic phosphates or phosphonates and well-defined polymers were obtained. They were evaluated with regard to their cytotoxicity, protein interactions, and corona formation and their cellular uptake. We proved that all PPE-surfactants have lower cytotoxicity as the common PEG-based surfactant (Lutensol® AT 50) and that their hydrolysis is controlled by their chemical structure. Two polymeric nanocarriers, namely polystyrene and poly(methyl methacrylate), and bio-based and potentially biodegradable hydroxyethyl starch nanocarriers were coated with the PPE-surfactants. All nanocarriers exhibited reduced protein adsorption after coating with PPE-surfactants and a strongly reduced interaction with macrophages. This general strategy allows the transformation of polymeric nanocarriers into camouflaged nanocarriers and by the chemical versatility of PPEs will allow the attachment of additional moieties for advanced drug delivery.

Silica Nanocapsules with Different Sizes and Physicochemical Properties as Suitable Nanocarriers for Uptake in T-Cells.

INTRODUCTION: Adoptive T-cell immunotherapy emerged as a powerful and promising cancer therapy, as the problem regarding the immuno-reaction between different donors and recipients can be avoided. However, this approach is challenging. After long cultivation and expansion under laboratory media conditions, T-cells are losing their viability and function due to immune checkpoint proteins, leading to decreased efficiency in killing cancer cells. Therefore, a new strategy to improve T-cell survival and function is needed. With the advantages of nanotechnology and the biocompatibility of silica-based material, silica nanocapsules (SiNCs) provide an ideal delivery system to transport therapeutic biomolecules to T-cells. Up to now, there is a lack of cellular uptake studies of nanocarriers towards T-cells. METHODS: We systematically studied the influence of various physicochemical properties such as sizes, core hydrophobicities, surface charges, and surface functionalities of SiNC for their impact on cellular uptake and toxicity in CD8+ T-cells by flow cytometry and confocal laser scanning microscopy. Cytokine secretion assay was performed using the enzyme-linked immunosorbent assay. To identify suitable uptake conditions for SiNCs into CD8+ T-cells, the impact of human serum in cell culture medium was also investigated. RESULTS: The major impact on cellular uptake and toxicity was found to be size- and dose-dependent. Smaller sizes of SiNCs than 100 nm caused significant toxicity to the cells. It was found that the formed protein corona reduced the toxicity of the SiNCs. However, it also inhibited their uptake. CONCLUSION: Overall, we present a set of different criteria for a suitable design of nanocarriers and cell culture conditions, which need to be carefully considered for T-cell immunotherapy in vitro to facilitate uptake while avoiding toxicity.

Nanovaccine impact on dendritic cells: transcriptome analysis enables new insights into antigen and adjuvant effects.

Aim: For vaccines the combination between an antigen and adjuvants are both crucially important to trigger an effective immune response in dendritic cells. Innovative adjuvants like resiquimod or muramyldipeptide have their target protein inside the cell. Materials & methods: Up/downregulation and proteome expression was investigated for the adjuvant combination resiquimod and muramyldipeptide in a soluble form versus encapsulated into a nanocarrier. Results: We found that 1225 genes were upregulated after nanocarrier treatment while 478 genes were downregulated. Most prominent were interferon-stimulated genes with more than 25-times higher expression after nanocarrier treatment, for example RSAD2 and ISG15, which were recently found to have antiviral or antitumor effects. Conclusion: Encapsulation gives a more effective upregulation of vaccine-related genes.

Amphiphilic dendrimers control protein binding and corona formation on liposome nanocarriers.

Amphiphilic polyphenylene dendrimers (PPDs) with distinct lipophilic and positively or negatively charged surface groups were adsorbed onto liposomes and their impact on protein adsorption in blood plasma was studied. The PPD corona reduced binding of specific opsonins and increased the adsorption of proteins controlling cellular uptake based on their surface patches.