PEGylated human serum albumin (HSA) nanoparticles: preparation, characterization and quantification of the PEGylation extent.

Modification with poly(ethylene glycol) (PEG) is a widely used method for the prolongation of plasma half-life of colloidal carrier systems such as nanoparticles prepared from human serum albumin (HSA). However, the quantification of the PEGylation extent is still challenging. Moreover, the influence of different PEG derivatives, which are commonly used for nanoparticle conjugation, has not been investigated so far. The objective of the present study is to develop a method for the quantification of PEG and to monitor the influence of diverse PEG reagents on the amount of PEG linked to the surface of HSA nanoparticles. A size exclusion chromatography method with refractive index detection was established which enabled the quantification of unreacted PEG in the supernatant. The achieved results were confirmed using a fluorescent PEG derivative, which was detected by photometry and fluorimetry. Additionally, PEGylated HSA nanoparticles were enzymatically digested and the linked amount of fluorescently active PEG was directly determined. All the analytical methods confirmed that under optimized PEGylation conditions a PEGylation efficiency of up to 0.5 mg PEG per mg nanoparticle could be achieved. Model calculations made a 'brush' conformation of the PEG chains on the particle surface very likely. By incubating the nanoparticles with fetal bovine serum the reduced adsorption of serum proteins on PEGylated HSA nanoparticles compared to non-PEGylated HSA nanoparticles was demonstrated using sodium dodecylsulfate polyacrylamide gel electrophoresis. Finally, the positive effect of PEGylation on plasma half-life was demonstrated in an in vivo study in mice. Compared to unmodified nanoparticles the PEGylation led to a four times larger plasma half-life.

Albumin nanoparticles with predictable size by desolvation procedure.

Nanoparticles prepared of human serum albumin (HSA) represent promising carriers for drug delivery. Desolvating procedure is a common method to obtain protein-based particles. However process parameters have a great influence on size, size distribution and particle yield of the resulting formulation. Therefore, it is crucial to investigate their effects on particle preparation. This study is focused on the influence of different desolvating agents on the resulting particle characteristics and cytotoxicity in cell culture. Another aspect of the study was the influence of different stirring rates, pre-stirring of the HSA solution and the continuous addition of desolvating agent during the preparation process. The results indicate that it is possible to predict the particle size depending on dielectric constant of the desolvation medium. Remarkably, combination of methanol and ethanol used as desolvating agent was able to produce very small spherical HSA nanoparticles in a size range between 50 and 80 nm.

Lecithin coating as universal stabilization and functionalization strategy for nanosized drug carriers to overcome the blood-brain barrier.

Lecithin coated cholesteryl oleate (ChOl) based nanoparticles (NPs) imitating natural lipoproteins represent a new and promising drug carrier strategy to cross the blood-brain barrier (BBB). In such systems lecithin serves as stabilizing as well as functionalizing agent and enables the adsorptive binding of apolipoprotein E3 (ApoE) as potential drug targeting ligand. The present work is focused on the effect of size reduction on the lecithin coating and ApoE binding. Furthermore, the transferability of this lecithin coating strategy to other NP cores, namely polylactic-co-glycolic acid (PLGA) and polylactic acid (PLA), is investigated in order to provide a universal strategy for a wide range of cores to overcome the BBB. The ChOl NPs' size was successfully reduced from 100 nm to 70 nm. Varying the core size of ChOl NPs illustrated, that the at least needed lecithin amount for sufficient stabilization could be calculated surface area dependently. However, the size reduction led to reduced dye loading per NP and increased ApoE need per NP mass. These effects turned out as huge disadvantages of smaller NPs by weakening the observed ApoE mediated effects. Nevertheless, the extended understanding of the lecithin coating could be used to transfer the concept to other core materials. PLGA and PLA NPs were investigated as alternative core materials for lecithin coating. PLGA was found to be unsuitable, whereas in the case of PLA sufficient stabilization and 100% adsorptive binding efficiency to ApoE could be achieved. The ApoE mediated effects of transcytosis at an in vitro BBB model by bypassing lysosomes were reproduced in even stronger quantities than with a ChOl core, proving lecithin coating as transferable strategy to disguise various NPs with a certain lipophilicity as lipoproteins.

Conversion of indomethacin nanosuspensions into solid dosage forms via fluid bed granulation and compaction.

Preparation of pharmaceutical nanosuspensions is a popular technique to increase the dissolution velocity of poorly water-soluble drugs. Subsequent drying into a compaction-ready powder or granule is a critical process due to possible adverse solid characteristics and the risk of growth of nanoparticles. This work evaluated the drying of nanosuspensions via fluid bed granulation with focus on the binder selection and used concentrations, as well as the parameters spray rate and atomization pressure. Design of experiments was used to identify significant parameters. Indomethacin nanosuspensions were prepared by wet media milling and dried on a carrier consisting of lactose, microcrystalline cellulose, and crospovidone with and without additional binder during granulation. Resulting granules were compacted into tablets and their in vitro dissolution performances were characterized. A higher content of binder PVP and a higher spray rate led to less growth of resuspended nanoparticles. Finally, indomethacin nanoparticle tablets showed a superior dissolution performance in contrast to raw indomethacin tablets.

Lipoprotein imitating nanoparticles: Lecithin coating binds ApoE and mediates non-lysosomal uptake leading to transcytosis over the blood-brain barrier.

Lipoproteins are naturally occurring nano sized transport vehicles in the human body. Therefore, lipoproteins could be applied as a drug carrier system. Additionally, several reports of apolipoprotein mediated blood-brain barrier (BBB) crossing suggest lipoprotein mimicking nanoparticles (NPs) as possible drug delivery vehicles to the brain. This could extend the therapy opportunities of various diseases of the central nervous system. A lipoprotein imitating NP system, consisting of a lecithin coated lipophilic cholesteryl oleate core with embedded fluorescent dye and adsorbed apolipoprotein E3 (ApoE) has been established using a two-step solvent injection method. Lecithin coating was proven to stabilize the NPs in isotonic saline solution and to bind ApoE in a highly efficient way. Fluorescent dye load (as model drug) and ApoE amount were varied, obtaining 100 nm sized, monodisperse NPs. The NPs' interaction with the BBB formed by primary porcine brain capillary endothelial cells (PBCEC) was investigated by fluorescence microscopy observing that ApoE mediated a lysosome bypassing uptake mechanism. Using this in vitro BBB model, ApoE concentration dependent permeation over the cell layer could be proven in both directions. An ApoE mediated transcytosis could be achieved, as it had been observed earlier for low-density lipoproteins. These results show that the newly developed NP system successfully mimics endogenous lipoproteins. An ApoE dependent penetration of the BBB was confirmed and provided an indication of apolipoprotein mediated transcytosis, avoiding lysosomal degradation.

Immunological effects of stress in psoriasis.

BACKGROUND: Psychological stress causes phenotypic changes in circulating lymphocytes and is regarded as an important trigger of the Th1-polarized inflammatory skin disease psoriasis. OBJECTIVE: To study the effects of psychological stress on immunological parameters, i.e. membrane molecules relevant to the pathophysiology of psoriasis, especially cutaneous lymphocyte-associated antigens (CLA) involved in T and natural killer (NK) cells homing in on the skin. METHODS: The severity of psoriasis was assessed in patients using the Psoriasis Area and Severity Index. Patients with psoriasis (n=15) and healthy volunteers (n=15) were exposed to brief psychological stress in the laboratory. In vitro analyses were conducted 1 h before, immediately following and 1 h after stress exposure. Peripheral T- and NK-cell subsets including CD8+ T lymphocytes, CLA+ lymphocytes and lymphocyte function-associated antigen type 1 (LFA-1)+ lymphocytes were analysed by flow cytometry. RESULTS: We found a significant stress-induced increase of CD3+ T lymphocytes in patients with psoriasis only. Analyses of T-cell subsets revealed that this increase was observable for cytotoxic CD8+ T lymphocytes and CLA+ CD3+ lymphocytes. The total number of circulating NK cells (CD16+, CD56+) increased immediately after stress in both groups whereas only patients with psoriasis showed a significant increase in CLA+ NK cells. CONCLUSIONS: A higher stress-induced increase of CLA+ T and CLA+ NK cells in the circulation of patients with psoriasis might point to an increased ability of T and NK cells in the presence of psoriasis to home in on the skin during mental stress. Further studies are needed to verify these relationships in more detail and to investigate the time point at which these cells accumulate within lesional skin, and whether or not psychotherapy improves the quality of life of patients with psoriasis and influences stress-dependent parameters.

Continuous subcutaneous insulin infusion leads to immediate, stable and long-term changes in metabolic control.

BACKGROUND: Evaluations of continuous subcutaneous insulin infusion (CSII) usually focus on one pre- and one post-CSII measurement to assess metabolic therapy outcome. AIM: Extending this research, the aim of the present study was to provide a more fine-grained analysis of achieved glycaemic control. METHODS: In 52 patients with type 1 diabetes (mean age of 37.85 years at CSII begin; s.d. +/- 12.41), haemoglobin A(1c) (HbA(1c)) levels were assessed every 3 months over a period of 5 years (1 year before and 4 years after the introduction of CSII). Mixed models were utilized to describe changes in glycaemic control. RESULTS: The pre-post course showed that already in the first quarter, a statistically significant lower HbA(1c) level was obtained [7.30%, in contrast to 8.21% at the last quarter with intensified conventional therapy (ICT)]. In the following 15 quarters, the mean HbA(1c) levels remained constantly lower than that with ICT. Overall, the aggregated mean HbA(1c) level of patients with CSII therapy was 7.19%, in contrast to 8.08% with ICT; thus, an overall decrease by 11% was achieved. In addition, individual differences in blood glucose level and age of diabetes onset as a predictor for therapy success were analysed. CONCLUSIONS: The data show an immediate, stable and long-term effect of CSII on HbA(1c). In addition, a significant relationship between metabolic control and age of diabetes onset was found, as well as a reduction of variance in HbA(1c) levels between subjects after change to CSII.

Lung injury following thoracic aortic occlusion: comparison of sevoflurane and propofol anaesthesia.

BACKGROUND: Halogenated anaesthetics have been shown to reduce ischaemia-reperfusion injuries in various organs due to pre- and post-conditioning mechanisms. We compared volatile and total intravenous anaesthesia with regard to their effect on remote pulmonary injury after thoracic aortic occlusion and reperfusion. METHODS: Eighteen pigs were randomized after sternotomy and laparotomy (fentanyl-midazolam anaesthesia) to receive either sevoflurane or propofol in an investigator-blinded fashion. Ninety minutes of thoracic aortic occlusion was induced by a balloon catheter. During reperfusion, a goal-directed resuscitation protocol was performed. After 120 min of reperfusion, the anaesthetic regimen was changed to fentanyl-midazolam again for another 180 min. The oxygenation index and intra-pulmonary shunt fractions were calculated. After 5 h of reperfusion, a bronchoalveolar lavage was performed. The total protein content and lactate dehydrogenase activity were measured in epithelial lining fluid (ELF). Alveolar macrophage oxidative burst was analysed. The wet to dry ratio was calculated and tissue injury was graded using a semi-quantitative score. Ten animals (n=5 for each anaesthetic) without aortic occlusion served as time controls. RESULTS: The oxygenation index decreased and the intra-pulmonary shunt fraction increased significantly in both occlusion groups. There were no significant differences between sevoflurane and propofol with respect to the oxygenation index, ELF composition, morphologic lung damage, wet to dry ratio and alveolar macrophage burst activity. Differences were, however, seen in terms of systemic haemodynamic stability, where catecholamine requirements were less pronounced with sevoflurane. CONCLUSION: We conclude that the severity of remote lung injury was not different between sevoflurane and propofol anaesthesia in this porcine model of severe lower-body ischaemia and reperfusion injury.

Human serum albumin (HSA) nanoparticles: reproducibility of preparation process and kinetics of enzymatic degradation.

Nanoparticles prepared from human serum albumin (HSA) are versatile carrier systems for drug delivery and can be prepared by an established desolvation process. A reproducible process with a low batch-to-batch variability is required for transfer from the lab to an industrial production. In the present study the batch-to-batch variability of the starting material HSA on the preparation of nanoparticles was investigated. HSA can build dimers and higher aggregates because of a free thiol group present in the molecule. Therefore, the quality of different HSA batches was analysed by size exclusion chromatography (SEC) and analytical ultracentrifugation (AUC). The amount of dimerised HSA detected by SEC did not affect particle preparation. Higher aggregates of the protein detected in two batches by AUC disturbed nanoparticle formation at pH values below 8.0. At pH 8.0 and above monodisperse particles between 200 and 300 nm could be prepared with all batches, with higher pH values leading to smaller particles. Besides human derived albumin a particle preparation was also feasible based on recombinant human serum albumin (rHSA). Under comparable preparation conditions monodisperse nanoparticles could be achieved and the same effects of protein aggregates on particle formation were observed. For nanoparticulate drug delivery systems the enzymatic degradation is a crucial parameter for the release of an embedded drug. For this reason, besides the particle preparation process, particle degradation in the presence of different enzymes was studied. Under acidic conditions HSA as well as rHSA nanoparticles could be digested by pepsin and cathepsin B. At neutral pH trypsin, proteinase K, and protease were suitable for particle degradation. It could be shown that the kinetics of particle degradation was dependent on the degree of particle stabilisation. Therefore, the degree of particle stabilisation will influence drug release after cellular accumulation of HSA nanoparticles.

A new preparation strategy for surface modified PLA nanoparticles to enhance uptake by endothelial cells.

Nanoparticles are promising drug delivery systems to overcome physiological barriers such as the blood-brain barrier. In this respect nanoparticle uptake into endothelial or epithelial cells is the first necessary step to overcome these obstacles. Therefore, a new strategy for the covalent attachment of drug targeting ligands on poly(lactic acid) (PLA) nanoparticles was developed and the influence of the resulting surface properties on the uptake behaviour in cerebral endothelial cells was investigated. PLA nanoparticles were modified on their surface by apolipoprotein E, penetratin, or ovalbumin using a newly developed vinyl sulfone-modified poly(vinyl alcohol)-derivative (VS-PVA) as steric stabilizer. With this approach an easy option for ligand coupling reactions to PVA-stabilized nanoparticles was achieved. All obtained formulations showed a favourable behaviour concerning cytotoxic effects on endothelial cells, not compromising their viability. Furthermore, a clear relation between cellular uptake and surface coupled functional ligand could be determined: Penetratin- and apolipoprotein E-modified nanoparticles showed a distinct higher cellular uptake than ovalbumin-modified or unmodified nanoparticles, which both can be explained by mechanistic reasons. Overall the use of the reactive VS-PVA as stabilizer for nanoparticle preparation is an universal and effective approach to couple several functional ligands to the particles' surface for targeting applications.

Preparation, characterisation and maintenance of drug efficacy of doxorubicin-loaded human serum albumin (HSA) nanoparticles.

Human serum albumin (HSA) nanoparticles represent promising drug carrier systems. Binding of cytostatics to HSA nanoparticles may diminish their toxicity, optimise their body distribution and/or may overcome multidrug resistance. In the present study, doxorubicin-loaded HSA nanoparticle preparations were prepared. Doxorubicin was loaded to the HSA nanoparticles either by adsorption to the nanoparticles' surfaces or by incorporation into the particle matrix. Both loading strategies resulted in HSA nanoparticles of a size range between 150nm and 500nm with a loading efficiency of 70-95%. The influence on cell viability of the resulting nanoparticles was investigated in two different neuroblastoma cell lines. The anti-cancer effects of the drug-loaded nanoparticles were increased in comparison to doxorubicin solution. Based on these result a standard protocol for the preparation of doxorubicin-loaded HSA nanoparticles for further antitumoural studies was established.

Influence of the formulation on the tolerance profile of nanoparticle-bound doxorubicin in healthy rats: focus on cardio- and testicular toxicity.

A toxicological study of doxorubicin bound to poly(butyl cyanoacrylate) or human serum albumin nanoparticles coated with polysorbate 80 was performed in healthy rats. The doxorubicin formulations were injected at a therapeutic dose regimen (3 x 1.5 mg/kg with a 72 h interval), and the animals were followed up for 15 or 30 days. The overall result of this study suggests that the surfactant-coated nanoparticle formulations of doxorubicin have favorable toxicological profiles. Specifically, these formulations display a considerably reduced cardio- and testicular toxicity, as compared to a free drug.

Barrier function of the gut and multiple organ dysfunction after cardiac surgery.

We studied the influence of sequential involvement of the gastrointestinal (GI) tract on the development of multiple organ dysfunction (MOD) after cardiopulmonary bypass (CPB). One hundred and forty-six patients undergoing elective cardiac surgery were included in this prospective observational study. Standardized oral inert-sugar tests (sucrose, lactulose, mannitol, sucralose) were performed before and after CPB in different patients. Enzyme-linked immunosorbent assay of plasma levels of endotoxin core antibodies (EndoCAb) were performed peri-operatively. The functional mucosal surface was calculated from the amount of mannitol absorbed from the GI tract. Lower urine concentrations of absorbed mannitol were observed pre-operatively in patients developing MOD. In binary logistic regression this was an independent parameter. Decreased plasma concentrations of EndoCAb after surgery were seen in every patient, but were more significant in patients developing MOD. A reduced pre-operative functional mucosal surface may predict the early occurrence of MOD after surgery.

Modifying plasmid-loaded HSA-nanoparticles with cell penetrating peptides - Cellular uptake and enhanced gene delivery.

Gene therapy bears great potential for the cure of a multitude of human diseases. Research efforts focussed on the use of viral delivery vectors in the past decades, neglecting non-viral gene therapies of physical or chemical origin due to low transfection efficiency. However, side effects such as activation of oncogenes and inflammatory reactions upon immune cell activation are major obstacles impeding the clinical applicability of viral gene therapy vectors. The aim of this study was the development of a non-viral gene delivery system based on plasmid-loaded human serum albumin nanoparticles, which are biocompatible, biodegradable, and non-toxic in relevant concentrations. The surface of said nanoparticles was modified with different cell penetrating peptides, namely Tat, nona-arginine R9, and the penetratin analogue EB1. We hypothesise that the surface modified nanoparticles can effectively enter HEK 293T cells based on the cell penetrating properties of the different peptides attached. A variety of inhibitors were used targeting distinct uptake pathways in an effort to understand the mechanisms utilized by the various cell penetrating peptides on the surface of the nanoparticles. A significant increase in transfection efficiency compared to free DNA or polyplexes was seen for these novel delivery vectors.

Asymmetrical flow field-flow fractionation for the analysis of PEG-asparaginase.

Monomethoxypolyethylene glycol L-asparaginase (PEG-ASNASE) is the PEGylated version of the enzyme L-asparaginase (ASNASE). Both are used for remission induction in acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). The treatment control is generally carried out by performing activity assays, though methods to determine the actual enzyme rather than its activity are rare. Using asymmetrical flow field-flow fractionation (AF4) offered the chance to develop a method capable of simultaneously measuring PEG-ASNASE and PEG. A method validation was performed in accordance with FDA guidelines for PEG-ASNASE from non-biological solutions. The method unfolded a linearity of 15-750 U/mL with coefficients of correlation of r(2)>0.99. The coefficients of variation (CV) for within-run and between-run variability were 1.18-10.15% and 2.43-8.73%, respectively. Furthermore, the method was used to perform stability tests of the product Oncaspar® (PEG-ASNASE) and estimation of the molecular weight by multi-angle light scattering (MALS) of stressed samples to correlate them with the corresponding activity. The findings indicate that Oncaspar® stock solution should not be stored any longer than 24 h at room temperature and cannot be frozen in pure aqueous media. The validated method might be useful for the pharmaceutical industry and its quality control of PEG-ASNASE production.

A microfluidic device for real-time monitoring of Bacillus subtilis bacterial spores during germination based on non-specific physicochemical interactions on the nanoscale level.

A microfluidic device for studies on the germination of bacterial spores (e.g. Bacillus subtilis) based on non-specific interactions on the nanoscale is presented. A decrease in the population of spores during germination followed by the appearance of transition forms and an increase in the number of vegetative cells can be registered directly and simultaneously by using the microfluidic device, which is equipped with a conductive polymer layer (polyaniline) in the form of a nano-network. The lab-on-a-chip-type device, operating in a continuous flow regime, allows monitoring of germination of bacterial spores and analysis of the process in detail. The procedure is fast and accurate enough for quantitative real-time monitoring of the main steps of germination, including final transformation of the spores into vegetative cells. All of this is done without the use of biomarkers or any bio-specific materials, such as enzymes, antibodies and aptamers, and is simply based on an analysis of physicochemical interactions on the nanoscale level.

The polymorphism of type 1 collagen (COL1A1) gene does not correlate with an increased risk of foot ulcers in patients with diabetes mellitus.

AIM: Diabetic foot syndrome (DFS) is a multifactorial debilitating complication of diabetes mellitus (DM). The identification of markers for predicting the risk of developing DFS could help and direct the efforts in the prevention to the highest risk patients. Type I collagen α1 (COL1A1) is the main component of type I collagen, the most abundant structural protein of the extracellular matrix of subcutaneous tissue. COL1A1 polymorphism has been previously investigated with regard to many clinical conditions affecting the bone or the skin. In this prospective study, we have assessed COL1A1 polymorphism in patients without and with DFS. PATIENTS AND METHODS: 202 DM patients without and 103 patients with DFS have been recruited. COL1A1 polymorphism, due to a mutation affecting the zinc-finger transcription factor specific protein, has been investigated. The most relevant clinical data (HbA1c, vascular risk factors, insulin treatment) have been collected and analyzed. RESULTS: No statistically significant difference in the distribution of the 3 genotypes constituting COL1A1 polymorphism between patients without and with DFS has been observed. Almost all DFS patients had at least one vascular risk factor, with a high rate of arterial hypertension and dyslipidemia. CONCLUSION: A multifaceted set of factors is involved in the development of DFS and only a combination of them may lead to such occurrence. In our DM patient population, COL1A1 polymorphism does not correlate with the occurrence of DFS, which appears to depend mostly on the presence of vascular risk factors. However, the impact of genetic factors affecting other components of the subcutaneous tissue cannot be excluded.