Influence of the orthodontic bonding procedure on biofilm formation.

INTRODUCTION: In orthodontics, white spot lesions are a persistent and widespread problem caused by the demineralization of buccal tooth surfaces around bonded brackets. The remaining adhesive around the brackets leads to surface roughness, which might contribute to demineralization. The present in vitro study aimed to compare a conventional and a modern adhesive system (APC Flash-Free technology) for orthodontic brackets with regard to the adhesion of Streptococcus sobrinus, a leading caries pathogen. METHODS: This in vitro study included 20 premolar teeth and compared 10 APC Flash-Free adhesive-coated ceramic brackets (FF)with 10 conventionally bonded (CB) ceramic clarity brackets. Specimens were incubated in an S. sobrinus suspension for 3 h. To evaluate the bacterial formation, samples were analysed with a scanning electron microscope (SEM). Imaging software was used to quantify and statistically compare percentage values of colonization (PVC) in both groups' adhesion and transition areas. RESULTS: We found a significant difference in biofilm formation between the groups for the adhesive and transition areas. PVC in the adhesive area was approximately 10.3-fold greater for the CB group compared with the FF group (median: 3.2 vs 0.31; P < 0.0001). For the transition area, median PVC was approximately 2.4-fold greater for the CB group compared with the FF group (median: 53.17 vs 22.11; P < 0.01). CONCLUSIONS: There was a significantly lower level of S. sobrinus formation around the FF bracket system than there was surrounding the conventionally bonded group. This study suggests that the FF adhesive bracket system can help reduce the occurrence of bacterial growth around orthodontic brackets.

Effects of reducing excess dental adhesive on bacterial adhesion in the bracket periphery.

OBJECTIVES: White spot lesions are one of the most common side effects of orthodontic therapy with a multibracket appliance and may indicate a preliminary stage of caries, also known as initial caries. Several approaches may be utilized to prevent these lesions, such as reducing bacterial adhesion in the area surrounding the bracket. This bacterial colonization can be adversely affected by a number of local characteristics. In this context, the effects of excess dental adhesive in the bracket periphery were investigated by comparing a conventional bracket system with the APC flash-free bracket system. MATERIALS AND METHODS: Both bracket systems were applied to 24 extracted human premolars, and bacterial adhesion with Streptoccocus sobrinus (S. sobrinus) was performed for 24 h, 48 h, 7 d, and 14 d. After incubation, bacterial colonization was examined in specific areas by electron microscopy. RESULTS: Overall, significantly fewer bacterial colonies were found in the adhesive area around the APC flash-free brackets (n = 507 ± 13 bacteria) than the conventionally bonded bracket systems (n = 850 ± 56 bacteria). This is a significant difference (**p = 0.004). However, APC flash-free brackets tend to create marginal gaps with more bacterial adhesion in this area than conventional bracket systems (n = 265 ± 31 bacteria). This bacterial accumulation in the marginal-gap area is also significant (*p = 0.029). CONCLUSION: A smooth adhesive surface with minimal adhesive excess is beneficial for reducing bacterial adhesion but also poses a risk of marginal gap formation with subsequent bacterial colonization, which can potentially trigger carious lesions. CLINICAL RELEVANCE: To reduce bacterial adhesion, the APC flash-free bracket adhesive system with low adhesive excess might be beneficial. APC flash-free brackets reduce the bacterial colonization in the bracket environment. A lower number of bacteria can minimize white spot lesions in the bracket environment. APC flash-free brackets tend to form marginal gaps between the bracket adhesive and the tooth.

Extracellular and Intracellular Lanthanide Accumulation in the Methylotrophic Beijerinckiaceae Bacterium RH AL1.

Recent work with Methylorubrum extorquens AM1 identified intracellular, cytoplasmic lanthanide storage in an organism that harnesses these metals for its metabolism. Here, we describe the extracellular and intracellular accumulation of lanthanides in the Beijerinckiaceae bacterium RH AL1, a newly isolated and recently characterized methylotroph. Using ultrathin-section transmission electron microscopy (TEM), freeze fracture TEM (FFTEM), and energy-dispersive X-ray spectroscopy, we demonstrated that strain RH AL1 accumulates lanthanides extracellularly at outer membrane vesicles (OMVs) and stores them in the periplasm. High-resolution elemental analyses of biomass samples revealed that strain RH AL1 can accumulate ions of different lanthanide species, with a preference for heavier lanthanides. Its methanol oxidation machinery is supposedly adapted to light lanthanides, and their selective uptake is mediated by dedicated uptake mechanisms. Based on transcriptome sequencing (RNA-seq) analysis, these presumably include the previously characterized TonB-ABC transport system encoded by the lut cluster but potentially also a type VI secretion system. A high level of constitutive expression of genes coding for lanthanide-dependent enzymes suggested that strain RH AL1 maintains a stable transcript pool to flexibly respond to changing lanthanide availability. Genes coding for lanthanide-dependent enzymes are broadly distributed taxonomically. Our results support the hypothesis that central aspects of lanthanide-dependent metabolism partially differ between the various taxa. IMPORTANCE Although multiple pieces of evidence have been added to the puzzle of lanthanide-dependent metabolism, we are still far from understanding the physiological role of lanthanides. Given how widespread lanthanide-dependent enzymes are, only limited information is available with respect to how lanthanides are taken up and stored in an organism. Our research complements work with commonly studied model organisms and showed the localized storage of lanthanides in the periplasm. This storage occurred at comparably low concentrations. Strain RH AL1 is able to accumulate lanthanide ions extracellularly and to selectively utilize lighter lanthanides. The Beijerinckiaceae bacterium RH AL1 might be an attractive target for developing biorecovery strategies to obtain these economically highly demanded metals in environmentally friendly ways.

Internal Structure of Nanometer-Sized Droplets Prepared by Antisolvent Precipitation.

Antisolvent precipitation (AP) is a low-cost and less-invasive preparation alternative for organic nanoparticles compared to top-down methods such as high-pressure homogenization or milling. Here we report on particularly small organic nanoparticles (NPs) prepared by AP. It has been found for various materials that these NPs in their liquid state exhibit a significant degree of molecular order at their interface toward the dispersion medium including ubiquinones (coenzyme Q10), triglycerides (trimyristin, tripalmitin), and alkanes (tetracosane). This finding is independent of the use of a stabilizer in the formulation. While this is obviously a quite general interfacial structuring effect, the respective structural details of specific NPs systems might differ. Here, a detailed structural characterization of very small liquid coenzyme Q10 (Q10) NPs is presented as a particular example for this phenomenon. The Q10 NPs have been prepared by AP in the presence of two different stabilizers, sodium dodecyl sulfate (SDS) and pentaethylene glycol monododecyl ether (C12E5), respectively, and without any stabilizer. The NPs' size is initially analyzed by photon correlation spectroscopy (PCS). The SDS-stabilized Q10 NPs have been studied further by differential scanning calorimetry (DSC), small-angle X-ray and neutron scattering (SAXS, SANS), wide-angle X-ray scattering (WAXS), and cryogenic transmission electron microscopy (CryoTEM). A simultaneous analysis of SAXS and contrast variation SANS studies revealed the molecular arrangement within the interface between the NPs and the dispersion medium. The Q10 NPs stabilized by SDS and C12E5, respectively, are small (down to 19.9 nm) and stable (for at least 16 months) even when no stabilizer is used. The SDS-stabilized Q10 NPs reported here, are therewith, to the best of our knowledge, the smallest organic NPs which have been reported to be prepared by AP so far. In particular, these NPs exhibit a core-shell structure consisting of an amorphous Q10 core and a surrounding shell, which is mainly composed of oriented Q10 molecules and aligned SDS molecules. This structure suggests a significant amphiphilic behavior and a rather unexpected stabilizing role of Q10 molecules.

Structural Characterization of Self-Assembling Hybrid Nanoparticles for Bisphosphonate Delivery in Tumors.

Hybrid self-assembling nanoparticles (hsaNPs) encapsulating bisphosphonates (BPs) recently showed very promising results in preclinic experiments for the treatment of brain tumor. However, the poor knowledge on the architecture of hybrid nanovectors is certainly one of the main reasons hampering further clinical and industrial development of these technologies. Here we propose to combine different techniques, that is, small angle neutron scattering (SANS) and X-ray Sscattering (SAXS), with cryo-electron transmission microscopy (cryo-TEM) to study the architecture of the final hsaNPs as well as of the four components before the assembling process. Data analysis based on SANS and SAXS experiments suggested a multiple compartment architecture of the final product, consisting of two bilayers sourrounding a core. Structures consisting of two shells surrounding an internal core were also observed in the cryo-TEM analysis. Such high resolution insight, also combined with size distribution and zeta potential of the NPs, provides exhaustive characterization of hsaNPs encapsulating BPs, and it is aimed at supporting further their clinical and industrial development.

Endoglin based in vivo near-infrared fluorescence imaging of tumor models in mice using activatable liposomes.

BACKGROUND: Endoglin (CD105) is overexpressed on tumor cells and tumor vasculatures, making it a potential target for diagnostic imaging and therapy of different neoplasms. Therefore, studies on nanocarrier systems designed for endoglin-directed diagnostic and drug delivery purposes would expose the feasibility of targeting endoglin with therapeutics. METHODS: Liposomes carrying high concentrations of a near-infrared fluorescent dye in the aqueous interior were prepared by the lipid film hydration and extrusion procedure, then conjugated to single chain antibody fragments either selective for murine endoglin (termed mEnd-IL) or directed towards human endoglin (termed hEnd-IL). A combination of Dynamic Light Scattering, electron microscopy, cell binding and uptake assays, confocal microscopy and in vivo fluorescence imaging of mice bearing xenografted human breast cancer and human fibrosarcoma models were implemented to elucidate the potentials of the liposomes. RESULTS: The mEnd-IL and hEnd-IL were highly selective for the respective murine- and human endoglin expressing cells in vitro and in vivo. Hence, the hEnd-IL bound distinctly to the tumor cells and enabled suitable fluorescence imaging of the tumors, whereas the mEnd-IL bound the tumor vasculature, but also to the liver, kidney and lung vasculature of mice. CONCLUSIONS: The work highlights key differences between targeting vascular (murine) and neoplastic (human) endoglin in animal studies, and suggests that the hEnd-IL can serve as a delivery system that targets human endoglin overexpressed in pathological conditions. GENERAL SIGNIFICANCE: The endoglin-targeting liposomes presented herewith represent strategic tools for the future implementation of endoglin-directed neoplastic and anti-angiogenic therapies.

Lipid Architectonics for Superior Oral Bioavailability of Nelfinavir Mesylate: Comparative in vitro and in vivo Assessment.

Nelfinavir mesylate (NFV), a human immunodeficiency virus (HIV) protease inhibitor, is an integral component of highly active anti retro viral therapy (HAART) for management of AIDS. NFV possesses pH-dependent solubility and has low and variable bioavailability hampering its use in therapeutics. Lipid-based particulates have shown to improve solubility of poorly water soluble drugs and oral absorption, thereby aiding in improved bioavailability. The current study compares potential of vesicular and solid lipid nanocarriers of NFV with drug nanocrystallites and microvesicular systems like cochleates in improving bioavailability of NFV. The paper outlines investigation of systems using in vitro models like in vitro lipolysis, in vitro release, and permeation through cell lines to predict the in vivo potential of nanocarriers. Finally, in vivo pharmacokinetic study is reported which provided proof of concept in sync with results from in vitro studies. Graphical Abstract ᅟ.

Vesicle aggregates as a model for primitive cellular assemblies.

Primitive cell models help to understand the role that compartmentalization plays in origin of life scenarios. Here we present a combined experimental and modeling approach towards the construction of simple model systems for primitive cellular assemblies. Charged lipid vesicles aggregate in the presence of oppositely charged biopolymers, such as nucleic acids or polypeptides. Based on zeta potential measurements, dynamic light scattering and cryo-transmission electron-microscopy, we have characterized the behavior of empty and ferritin-filled large unilamellar POPC vesicles, doped with different amounts of cationic (DDAB, CTAB) and anionic (sodium oleate) surfactants, and their aggregation upon the addition of anionic (tRNA, poly-l-glutamic acid) and cationic (poly-l-arginine) biopolymers, respectively. The experimental results are rationalized by a phenomenological modeling approach that predicts the average size of the vesicle aggregates as function of the amount of added biopolymers. In addition, we discuss the mechanism of vesicle aggregation induced by oppositely charged biopolymers. Our study complements previous reports about the formation of giant vesicle clusters and thus provides a general vista on primitive cell systems, based on the association of vesicles into compartmentalized aggregates.

Micro-spherical cochleate composites: method development for monodispersed cochleate system.

Cochleates have been of increasing interest in pharmaceutical research due to their extraordinary stability. However the existing techniques used in the production of cochleates still need significant improvements to achieve sufficiently monodispersed formulations. In this study, we report a simple method for the production of spherical composite microparticles (3-5 µm in diameter) made up of nanocochleates from phosphatidylserine and calcium (as binding agent). Formulations obtained from the proposed method were evaluated using electron microscopy and small angle X-ray scattering and were compared with conventional cochleate preparation techniques. In this new method, an ethanolic lipid solution and aqueous solution of a binding agent is subjected to rapid and uniform mixing with a microfluidic device. The presence of high concentration of organic solvent promotes the formation of composite microparticles made of nanocochleates. This simple methodology eliminates elaborate preparation methods, while providing a monodisperse cochleate system with analogous quality.

Understanding cochleate formation: insights into structural development.

Understanding the structure and the self-assembly process of cochleates has become increasingly necessary considering the advances of this drug delivery system towards the pharmaceutical industry. It is well known that the addition of cations like calcium to a dispersion of anionic lipids such as phosphatidylserines results in stable, multilamellar cochleates through a spontaneous assembly. In the current investigation we have studied the intermediate structures generated during this self-assembly of cochleates. To achieve this, we have varied the process temperature for altering the rate of cochleate formation. Our findings from electron microscopy studies showed the formation of ribbonlike structures, which with proceeding interaction associate to form lipid stacks, networks and eventually cochleates. We also observed that the variation in lipid acyl chains did not make a remarkable difference to the type of structure evolved during the formation of cochleates. More generally, our observations provide a new insight into the self-assembly process of cochleates based on which we have proposed a pathway for cochleate formation from phosphatidylserine and calcium. This knowledge could be employed in using cochleates for a variety of possible biomedical applications in the future.

Filter-extruded liposomes revisited: a study into size distributions and morphologies in relation to lipid-composition and process parameters.

Filter-extrusion is a widely used technique for down-sizing of phospholipid vesicles. In order to gain a detailed insight into size and size distributions of filter-extruded vesicles composed of egg phosphatidyl-choline (with varying fractions of cholesterol)--in relation to extrusion-parameters (pore-size, number of filter passages, and flow-rate), flow field-flow fractionation in conjunction with multi-angle laser light scattering (AF4-MALLS, Wyatt Technology Corp., Santa Barbara, CA) was employed. Liposome size-distributions determined by AF4-MALLS were compared with those of dynamic light scattering and correlated with cryo-transmission electron microscopy and (31)P-NMR-analysis of lamellarity. Both the mean size of liposome and the width of size distribution were found to decrease with sequential extrusion through smaller pore size filters, starting at a size range of ≈70-415 nm upon repeated extrusion through 400 nm pore-filters, eventually ending with a size range from ≈30 to 85 nm upon extrusion through 30 nm pore size filters. While for small pores sizes (50 nm), increased flow rates resulted in smaller vesicles, no significant influence of flow rate on mean vesicle size was seen with larger pores. Cholesterol at increasing mol fractions up to 0.45 yielded bigger vesicles (at identical process conditions). For a cholesterol mol fraction of 0.5 in combination with small filter pore size, a bimodal size distribution was seen indicating cholesterol micro-crystallites. Finally, a protocol is suggested to prepare large (∼ 300 nm) liposomes with rather narrow size distribution, based on the filter extrusion at defined flow-rates in combination with freeze-/thaw-cycling and bench-top centrifugation.

Analysis of the structure of nanocomposites of triglyceride platelets and DNA.

DNA-complexes with platelet-like, cationically modified lipid nanoparticles (cLNPs) are studied with regard to the formation of nanocomposite structures with a sandwich-like arrangement of the DNA and platelets. For this purpose suspensions of platelet-like triglyceride nanocrystals, stabilized by a mixture of two nonionic (lecithin plus polysorbate 80 or poloxamer 188) and one cationic stabilizer dimethyldioctadecylammonium (DODAB), are used. The structure of the platelets in the native suspensions and their DNA-complexes, ranging from the sub-nano to the micron scale, is investigated with small- and wide-angle scattering (SAXS, SANS, WAXS), calorimetry, photon correlation spectroscopy, transmission electron microscopy and computer simulations. The appearance of strong, lamellarly ordered peaks in the SAXS patterns of the DNA-complexes suggests a stacked arrangement of the nanocrystals, with the DNA being partially condensed between the platelets. This finding is supported with computer simulated small-angle scattering patterns of nanocrystal stacks, which can reproduce the measured small-angle scattering patterns on an absolute scale. The influence of the choice of the nonionic stabilizers and the amount of the cationic stabilizer DODAB on the structure of the native suspensions and the inner structure of their DNA-complexes is studied, too. Using high amounts of DODAB, lecithins with saturated acyl chains and polysorbate 80 instead of poloxamer 188 produces thinner nanocrystals, and thus decreases their repeat distances in the nanocomposites. Such nanocomposites could be of interest as DNA carriers, where the triglyceride platelets protect the sandwiched DNA from degradation.

Immuno-LipoTRAIL: Targeted delivery of TRAIL-functionalized liposomal nanoparticles.

The TNF-related apoptosis-inducing ligand (TRAIL) is a powerful inducer of apoptosis in tumor cells; however, clinical studies with recombinant soluble TRAIL were rather disappointing. Here, we developed TRAIL-functionalized liposomes (LipoTRAIL, LT) to mimic membrane-displayed TRAIL for efficient activation of death receptors DR4 and DR5 and enhanced induction of apoptosis, which were combined with an anti-EGFR single-chain Fv fragment (scFv) for targeted delivery to EGFR-positive tumor cells. These immuno-LipoTRAILs (ILTs) bound specifically to EGFR-expressing cells (Colo205) and exhibited increased cytotoxicity compared with that of nontargeted LTs. Compared to that of the soluble TRAIL, the plasma half-life of the functionalized liposomes was strongly extended, and increased antitumor activity of LT and ILT was demonstrated in a xenograft tumor model. Thus, we established a multifunctional liposomal TRAIL formulation (ILT) with improved pharmacokinetic and pharmacodynamic behavior, characterized by targeted delivery and increased induction of apoptosis due to multivalent TRAIL presentation.

Electron microscopy and theoretical modeling of cochleates.

Cochleates are self-assembled cylindrical condensates that consist of large rolled-up lipid bilayer sheets and represent a novel platform for oral and systemic delivery of therapeutically active medicinal agents. With few preceding investigations, the physical basis of cochleate formation has remained largely unexplored. We address the structure and stability of cochleates in a combined experimental/theoretical approach. Employing different electron microscopy methods, we provide evidence for cochleates consisting of phosphatidylserine and calcium to be hollow tubelike structures with a well-defined constant lamellar repeat distance and statistically varying inner and outer radii. To rationalize the relation between inner and outer radii, we propose a theoretical model. Based on the minimization of a phenomenological free energy expression containing a bending, adhesion, and frustration contribution, we predict the optimal tube dimensions of a cochleate and estimate ratios of material constants for cochleates consisting of phosphatidylserines with varied hydrocarbon chain structures. Knowing and understanding these ratios will ultimately benefit the successful formulation of cochleates for drug delivery applications.

Asymmetrical flow field-flow fractionation with on-line detection for drug transfer studies: a feasibility study.

Knowledge about drug retention within colloidal carriers is of uppermost importance particularly if drug targeting is anticipated. The aim of the present study was to evaluate asymmetrical flow field-flow fractionation (AF4) with on-line UV/VIS drug quantification for its suitability to determine both release and transfer of drug from liposomal carriers to a model acceptor phase consisting of large liposomes. The hydrophobic porphyrin 5,10,15,20-tetrakis(4-hydroxyphenyl)21H,23H-porphine (p-THPP), a fluorescent dye with an absorbance maximum in the visible range and structural similarity to the clinically used photosensitizer temoporfin, was used as a model drug, and two types of large liposomes were studied as a potential model acceptor phase. Efficiency of separation of small donor from large acceptor liposomes by AF4 was evaluated in dependence on the injected lipid mass using two different channel geometries. Drug quantification by on-line absorbance measurements was established by comprehensive evaluation of the size-dependent turbidity contribution in on-line UV/VIS detection and by comparison with off-line results obtained for the respective dye-loaded donor formulations (dissolved in methanol). Due to distinct differences in size, the acceptor liposomes (mean diameters ∼300-400 nm) could efficiently be separated from the donor liposomes (mean diameter ∼70 nm) with less than 4 % of p-THPP detected in the overlap region between both vesicle populations. Whereas p-THPP could accurately be determined in the fraction of small vesicles, on-line quantification was impaired in the fraction of the large acceptor liposomes due to the pronounced contribution of turbidity (about 80 % of total UV/VIS extinction signal). The AF4-based release/transfer approach suggested here was found repeatable and robust. The employed combination of AF4 with multi-angle laser light scattering furthermore provided detailed size information of the eluting sample and thus allowed to detect instabilities and/or interactions between the donor and acceptor liposomes. Drug quantification by on-line absorbance measurements was found feasible for the chosen model drug, but careful (re-)evaluation of turbidity effects is crucial for other drug and carrier combinations.

Formation of liquid crystalline phases in aqueous suspensions of platelet-like tripalmitin nanoparticles.

Suspensions of platelet-like shaped tripalmitin nanocrystals stabilized by the pure lecithin DLPC and the lecithin blend S100, respectively, have been studied by small-angle x-ray scattering (SAXS) and optical observation of their birefringence at different tripalmitin (PPP) concentrations φ(PPP). It could be demonstrated that the platelets of these potential drug delivery systems start to form a liquid crystalline phase already at pharmaceutically relevant concentrations φ(PPP) of less than 10 wt. %. The details of this liquid crystalline phase are described here for the first time. As in a previous study [A. Illing et al., Pharm. Res. 21, 592 (2004)] some platelets are found to self-assemble into lamellar stacks above a critical tripalmitin concentration φ(PPP)(st) of 4 wt. %. In this study another critical concentration φ(PPP)(lc) ≈ 7 wt. % for DLPC and φ(PPP)(lc) ≈ 9 wt. % for S100 stabilized dispersions, respectively, has been observed. φ(PPP)(lc) describes the transition from a phase of randomly oriented stacked lamellae and remaining non-assembled individual platelets to a phase in which the stacks and non-assembled platelets exhibit an overall preferred orientation. A careful analysis of the experimental data indicates that for concentrations above φ(PPP)(lc) the stacked lamellae start to coalesce to rather small liquid crystalline domains of nematically ordered stacks. These liquid crystalline domains can be individually very differently oriented but possess an overall preferred orientation over macroscopic length scales which becomes successively more expressed when further increasing φ(PPP). The lower critical concentration for the formation of liquid crystalline domains of the DLPC-stabilized suspension compared to φ(PPP)(lc) of the S100-stabilized suspension can be explained by a larger aspect ratio of the corresponding tripalmitin platelets. A geometrical model based on the excluded volumes of individual platelets and stacked lamellae has been developed and successfully applied to reproduce the critical volume fractions for both, the onset of stack formation and the appearance of the liquid crystalline phase.

[FeFe]-hydrogenase models assembled into vesicular structures.

Compartmentalization is a major prerequisite for the origin of life on earth according to Wächtershäuser "Iron-Sulfur-World". The hypothesis is mainly based on an autocatalytic inorganic energy reproducing redox system consisting of iron and sulfur as requirement for the subsequent synthesis of complex organic structures. Here, we modified [FeFe]-hydrogenase models by means of covalent coupling to either oleic acid or the amphiphilic block copolymer polybutadiene-polyethyleneoxide (PB-PEO) and incorporated those into the membranes of vesicles composed of phospholipids (liposomes) or the unmodified amphiphilic polymer (polymersomes). We employed a [2Fe-2S] cluster as a hydrogenase model, since these structures are known to be suitable catalysts for the generation of H2 in the presence of weak acids. Successful incorporation was confirmed by spectrophotometric iron quantification and the vesicles formed were characterized by size determination (photon correlation spectroscopy (PCS)), and zeta potential as well as by cryo-transmission electron microscopy (Cryo-TEM). The modified models could be incorporated into liposomes or polymersomes up to molar proportions of 3.15% and 28%, respectively. Due to the immobilization in vesicular bilayers the [FeFe]-hydrogenase models can even exhibit catalytic action under the particular conditions of the intravesicular microenvironment. Our results suggest that the vesicular systems described may be applied as a nanoreactor for the reduction of encapsulated substances by generating hydrogen and thus as a minimal cell model.

Mesoscopic Structure of Lipid Nanoparticle Formulations for mRNA Drug Delivery: Comirnaty and Drug-Free Dispersions.

Lipid nanoparticles (LNPs) produced by antisolvent precipitation (ASP) are used in formulations for mRNA drug delivery. The mesoscopic structure of such complex multicomponent and polydisperse nanoparticulate systems is most relevant for their drug delivery properties, medical efficiency, shelf life, and possible side effects. However, the knowledge on the structural details of such formulations is very limited. Essentially no such information is publicly available for pharmaceutical dispersions approved by numerous medicine agencies for the use in humans and loaded with mRNA encoding a mimic of the spike protein of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) as, e.g., the Comirnaty formulation (BioNTech/Pfizer). Here, we present a simple preparation method to mimic the Comirnaty drug-free LNPs including a comparison of their structural properties with those of Comirnaty. Strong evidence for the liquid state of the LNPs in both systems is found in contrast to the designation of the LNPs as solid lipid nanoparticles by BioNTech. An exceptionally detailed and reliable structural model for the LNPs i.a. revealing their unexpected narrow size distribution will be presented based on a combined small-angle X-ray scattering and photon correlation spectroscopy (SAXS/PCS) evaluation method. The results from this experimental approach are supported by light microscopy, 1H NMR spectroscopy, Raman spectroscopy, cryogenic electron microscopy (cryoTEM), and simultaneous SAXS/SANS studies. The presented results do not provide direct insights on particle formation or dispersion stability but should contribute significantly to better understanding the LNP drug delivery process, enhancing their medical benefit, and reducing side effects.

Exploring Microemulsion Systems for the Incorporation of Glucocorticoids into Bacterial Cellulose: A Novel Approach for Anti-Inflammatory Wound Dressings.

The effective pharmacological treatment of inflamed wounds such as pyoderma gangraenosum remains challenging, as the systemic application of suitable drugs such as glucocorticoids is compromised by severe side effects and the inherent difficulties of wounds as drug targets. Furthermore, conventional semi-solid formulations are not suitable for direct application to open wounds. Thus, the treatment of inflamed wounds could considerably benefit from the development of active wound dressings for the topical administration of anti-inflammatory drugs. Although bacterial cellulose appears to be an ideal candidate for this purpose due to its known suitability for advanced wound care and as a drug delivery system, the incorporation of poorly water-soluble compounds into the hydrophilic material still poses a problem. The use of microemulsions could solve that open issue. The present study therefore explores their use as a novel approach to incorporate poorly water-soluble glucocorticoids into bacterial cellulose. Five microemulsion formulations were loaded with hydrocortisone or dexamethasone and characterized in detail, demonstrating their regular microstructure, biocompatibility and shelf-life stability. Bacterial cellulose was successfully loaded with the formulations as confirmed by transmission electron microscopy and surprisingly showed homogenous incorporation, even of w/o type microemulsions. High and controllable drug permeation through Strat-M® membranes was observed, and the anti-inflammatory activity for permeated glucocorticoids was confirmed in vitro. This study presents a novel approach for the development of anti-inflammatory wound dressings using bacterial cellulose in combination with microemulsions.

Supramolecular assembly of micellar aggregates is the basis of low endotoxin recovery (LER) in a drug formulation that can be resolved by a whole blood assay.

Lipopolysaccharide (LPS, endotoxin) is ubiquitous and represents a harmful contaminant of pharmaceutical compounds, recombinant biologicals and drug products. The pyrogen can induce severe immune responses and pathology in vitro and in vivo. Health authorities require strict control of endotoxin in parenteral drugs. However, for research and pre-clinical compound analysis, endotoxin testing is not a required quality control, which may cause potential drawbacks in the translational pipeline. Endotoxin testing is usually performed by the Limulus amebocyte lysate (LAL) assay, which is hampered by the so-called low endotoxin recovery (LER) effect when certain drug formulations are tested. A comprehensive study including structural, biophysical, and biological analyses was conducted to identify LER root cause for phosphate- and polysorbate-containing parenteral drug products. LPS in water showed extended ribbon-like aggregate structures. In placebo (formulation buffer without drug) and in drug product (drug in formulation buffer), a reaggregation of LPS into a network of interlinked micelles with hidden head group charges, and a strong reduction of the negative surface potential was observed. The non-accessibility of the LPS backbone has a direct impact leading (i) to a loss of activation of the LAL-cascade, (ii) reduced activation of the TLR4/MD-2 receptor system, and (iii) increased survival in a mouse model of endotoxemia. These data provide a structure-based explanation of the LER-underlying mechanisms. A human whole blood assay is shown to resolve LER and detect the pyrogenic activity of endotoxin with high sensitivity. This may open new test options to improve quality control in drug development and drug safety.