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.

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.

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.

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.

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.

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.

Noncovalent Targeting of Nanocarriers to Immune Cells with Polyphosphoester-Based Surfactants in Human Blood Plasma.

Dendritic cells (DCs) are part of the immune system and can internalize pathogens by carbohydrate receptors. The uptake induces maturation and migration of the DCs resulting in an adaptive immune response by presenting antigens to T-cells. Thus, targeted delivery to DCs is a powerful tool for immunotherapy. However, in blood, specific targeting is challenging as blood proteins adsorb to the nanocarriers and mask the targeting molecules. Additionally, covalent coupling of targeting groups to nanocarriers requires new chemistry for each nanocarrier, while a general strategy is missing. A general protocol by noncovalent adsorption of mannosylated polyphosphoesters (PPEs) on the nanocarriers' surface resulting in specific uptake into DCs combined with low protein adsorption of PPEs is presented. PPEs with hydrophobic anchors and multiple mannose units are reported and adsorbed to different model nanocarriers. Their protein corona remain similar to pure stealth nanocarriers and prove only low uptake into nontargeted cells (monocytes). Due to the "stealth" properties of PPEs, a high specific uptake into DCs is achieved after incubation in human blood plasma, proving an efficient combination of "stealth" and targeting after simple adsorption of the PPEs. This strategy can transform any nanocarrier into DC-targeting by noncovalent adsorption of PPEs and will aid in developing novel immunotherapies.

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.

Patchy Amphiphilic Dendrimers Bind Adenovirus and Control Its Host Interactions and in Vivo Distribution.

The surface of proteins is heterogeneous with sophisticated but precise hydrophobic and hydrophilic patches, which is essential for their diverse biological functions. To emulate such distinct surface patterns on macromolecules, we used rigid spherical synthetic dendrimers (polyphenylene dendrimers) to provide controlled amphiphilic surface patches with molecular precision. We identified an optimal spatial arrangement of these patches on certain dendrimers that enabled their interaction with human adenovirus 5 (Ad5). Patchy dendrimers bound to the surface of Ad5 formed a synthetic polymer corona that greatly altered various host interactions of Ad5 as well as in vivo distribution. The dendrimer corona (1) improved the ability of Ad5-derived gene transfer vectors to transduce cells deficient for the primary Ad5 cell membrane receptor and (2) modulated the binding of Ad5 to blood coagulation factor X, one of the most critical virus-host interactions in the bloodstream. It significantly enhanced the transduction efficiency of Ad5 while also protecting it from neutralization by natural antibodies and the complement system in human whole blood. Ad5 with a synthetic dendrimer corona revealed profoundly altered in vivo distribution, improved transduction of heart, and dampened vector sequestration by liver and spleen. We propose the design of bioactive polymers that bind protein surfaces solely based on their amphiphilic surface patches and protect against a naturally occurring protein corona, which is highly attractive to improve Ad5-based in vivo gene therapy applications.

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 Protein Corona as a Confounding Variable of Nanoparticle-Mediated Targeted Vaccine Delivery.

Nanocarriers (NC) are very promising tools for cancer immunotherapy. Whereas conventional vaccines are based on the administration of an antigen and an adjuvant in an independent fashion, nanovaccines can facilitate cell-specific co-delivery of antigen and adjuvant. Furthermore, nanovaccines can be decorated on their surface with molecules that facilitate target-specific antigen delivery to certain antigen-presenting cell types or tumor cells. However, the target cell-specific uptake of nanovaccines is highly dependent on the modifications of the nanocarrier itself. One of these is the formation of a protein corona around NC after in vivo administration, which may potently affect cell-specific targeting and uptake of the NC. Understanding the formation and composition of the protein corona is, therefore, of major importance for the use of nanocarriers in vaccine approaches. This Mini Review will give a short overview of potential non-specific interactions of NC with body fluids or cell surfaces that need to be considered for the design of NC vaccines for immunotherapy of cancer.

How Low Can You Go? Low Densities of Poly(ethylene glycol) Surfactants Attract Stealth Proteins.

It is now well-established that the surface chemistry and "stealth" surface functionalities such as poly(ethylene glycol) (PEG) chains of nanocarriers play an important role to decrease unspecific protein adsorption of opsonizing proteins, to increase the enrichment of specific stealth proteins, and to prolong the circulation times of the nanocarriers. At the same time, PEG chains are used to provide colloidal stability for the nanoparticles. However, it is not clear how the chain length and density influence the unspecific and specific protein adsorption keeping at the same time the stability of the nanoparticles in a biological environment. Therefore, this study aims at characterizing the protein adsorption patterns depending on PEG chain length and density to define limits for the amount of PEG needed for a stealth effect by selective protein adsorption as well as colloidal stability during cell experiments. PEG chains are introduced using the PEGylated Lutensol AT surfactants, which allow easy modification of the nanoparticle surface. These findings indicate that a specific enrichment of stealth proteins already occurs at low PEG concentrations; for the decrease of unspecific protein adsorption and finally the colloidal stability a full surface coverage is advised.

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.

Highly Loaded Semipermeable Nanocapsules for Magnetic Resonance Imaging.

Magnetic resonance imaging has become an essential tool in medicine for the investigation of physiological processes. The key issues related to contrast agents, i.e., substances that are injected in the body for imaging, are the efficient enhancement of contrast, their low toxicity, and their defined biodistribution. Polyurea nanocapsules containing the gadolinium complex Gadobutrol as a contrast agent in high local concentration and high relaxivity up to 40 s-1 mmol-1 L are described. A high concentration of the contrast agent inside the nanocapsules can be ensured by increasing the crystallinity in the shell of the nanocapsules. Nanocapsules from aliphatic polyurea are found to display higher crystallinity and higher relaxivity at an initial Gadobutrol concentration of 0.1 m than aromatic polyurea nanocapsules. The nanocapsules and the contrast agent are clearly identified in cells. After injection, the nanocarriers containing the contrast agent are mostly found in the liver and in the spleen, which allow for a significant contrast enhancement in magnetic resonance imaging.

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.

Fully degradable protein nanocarriers by orthogonal photoclick tetrazole-ene chemistry for the encapsulation and release.

The encapsulation of sensitive drugs into nanocarriers retaining their bioactivity and achieving selective release is a challenging topic in current drug delivery design. Established protocols rely on metal-catalyzed or unspecific reactions to build the (mostly synthetic) vehicles which may inhibit the drug's function. Triggered by light, the mild tetrazole-ene cycloaddition enables us to prepare protein nanocarriers (PNCs) preserving at the same time the bioactivity of the sensitive antitumor and antiviral cargo Resiquimod (R848). This catalyst-free reaction was designed to take place at the interface of aqueous nanodroplets in miniemulsion to produce core-shell PNCs with over 90% encapsulation efficiency and no unwanted drug release over storage for several months. Albumins used herein are major constituents of blood and thus ideal biodegradable natural polymers for the production of such nanocarriers. These protein carriers were taken up by dendritic cells and the intracellular drug release by enzymatic degradation of the protein shell material was proven. Together with the thorough colloidal analysis of the PNCs, their stability in human blood plasma and the detailed protein corona composition, these results underline the high potential of such naturally derived drug delivery vehicles.