Protein conjugation with PAMAM nanoparticles: Microscopic and thermodynamic analysis.

PAMAM dendrimers form strong protein conjugates that are used in drug delivery systems. We report the thermodynamic and binding analysis of polyamidoamine (PAMAM-G4) conjugation with human serum albumin (HSA), bovine serum albumin (BSA) and milk beta-lactoglobulin (b-LG) in aqueous solution at physiological pH. Hydrophobicity played a major role in PAMAM-protein interactions with more hydrophobic b-LG forming stronger polymer-protein conjugates. Thermodynamic parameters showed PAMAM-protein bindings occur via hydrophobic and H-bonding contacts for b-LG, while van der waals and H-bonding interactions prevail in HSA and BSA-polymer conjugates. The protein loading efficacy was 45-55%. PAMAM complexation induced major alterations of protein conformation. TEM images show major polymer morphological changes upon protein conjugation.

Incidence of bleeding in 8172 percutaneous ultrasound-guided intraabdominal diagnostic and therapeutic interventions - results of the prospective multicenter DEGUM interventional ultrasound study (PIUS study).

PURPOSE: To analyse the incidence of bleeding after percutaneous ultrasound guided diagnostic and therapeutic intraabdominal interventions in a prospective multicentre study (DEGUM percutaneous interventional ultrasound study). MATERIALS AND METHODS: Within a time period of 2 years diagnostic and therapeutic intraabdominal interventions (with the exclusion of ascites paracentesis) performed percutaneously under continuous ultrasound (US) guidance were prospectively assessed using a pseudonymized standardized web site entry form. Number and type of intervention, operator experience, patient characteristics, medication, lab data as well as technical aspects of the procedure and bleeding complications were analysed according to the interventional radiology standards. RESULTS: 8172 US-guided intraabdominal interventions (liver n = 5903; pancreas n = 501, kidney n = 434, lymph node = 272, biliary system n = 153, spleen n = 63, other abdominal organs and extra-organic targets n = 999) were analysed in 30 hospitals. The majority were diagnostic biopsies including 1780 liver parenchyma, 3400 focal liver lesions and 404 pancreatic lesions. 7525 interventions (92.1 %) were performed in hospitalized patients (mean age 62.6 years). Most operators were highly experienced in US-guided interventions (> 500 interventions prior to the study n = 5729; 70.1 %). Sedation was administered in 1131 patients (13.8 %). Needle diameter was ≥ 1 mm in 7162 punctures (87.9 %) with main focus on core needle biopsies (18 G, n = 4185). Clinically relevant bleeding complications with need of transfusion (0.4 %), surgical bleeding control (0.1 %) and radiological coiling (0.05 %) were very rare. Bleeding complications with fatal outcome occurred in four patients (0.05 %). The frequency of major bleeding complications was significantly higher in patients with an INR > 1.5 (p < 0.001) and patients taking a medication potentially interfering with platelet function or plasmatic coagulation (p < 0.0333). CONCLUSION: This prospective multicentre study confirms the broad spectrum of percutaneous US-guided intraabdominal interventions. However diagnostic liver biopsies dominate with the use of core needle biopsies (18 G). Percutaneous US-guided interventions performed by experienced sonographers are associated with a low bleeding risk. Major bleeding complications are very rare. A pre-interventional INR < 1.5 and individual medication risk assessment are recommended.

Evaluation of prognostic scoring systems for spinal metastases in 196 patients treated during 2005-2010.

PURPOSE: Estimating the survival time of patients with spinal metastases based on pre-treatment parameters is important for the best choice of therapy. Following two previous studies, this sequel analyzes possible changes in the impact of various parameters and scoring systems and includes a comparison to the previous dataset for the purpose to find the most predictive parameters and scores for this patient group. METHODS: Included were 196 patients retrospectively with confirmed spinal metastases treated between 2005 and 2010 (35% surgery, 65% conservative). Possible prognostic factors [primary tumor, Karnofsky Performance Scale (KPS), visceral metastases, number of bone metastases, pathological fracture and neurologic status] and six scoring systems (Tokuhashi original/revised, Tomita, van der Linden, Bauer original and modified) were analyzed using Kaplan-Meier curves and Cox-regression models. RESULTS: Median overall survival was 7 months with 9% of all patients alive at the time of analysis. Stepwise multivariate analysis showed significant influence on survival for visceral metastases (p<0.0001), primary tumor (p<0.0001), KPS (p<0.0001) and number of spinal metastases (p=0.0271). All scoring systems significantly predicted longer survival at a better score (absolute scores, p<0.001) in this dataset. Significant differentiation between the prognostic groups was seen only for the Tokuhashi original, the Bauer original and modified scores (p<0.001). In comparison to the previous dataset with varying age, gender and primary tumor distribution, the Bauer original and modified scores were the least influenced by the different patient collectives. CONCLUSIONS: The Bauer modified score has shown consistent impact on predicting the remaining survival in patients with spinal metastases and is simultaneously simple in clinical use.

Effect of PEG and mPEG-anthracene on tRNA aggregation and particle formation.

Poly(ethylene glycol) (PEG) and its derivatives are synthetic polymers with major applications in gene and drug delivery systems. Synthetic polymers are also used to transport miRNA and siRNA in vitro. We studied the interaction of tRNA with several PEGs of different compositions, such as PEG 3350, PEG 6000, and mPEG-anthracene under physiological conditions. FTIR, UV-visible, CD, and fluorescence spectroscopic methods as well as atomic force microscopy (AFM) were used to analyze the PEG binding mode, the binding constant, and the effects of polymer complexation on tRNA stability, aggregation, and particle formation. Structural analysis showed that PEG-tRNA interaction occurs via RNA bases and the backbone phosphate group with both hydrophilic and hydrophobic contacts. The overall binding constants of K(PEG 3350-tRNA)= 1.9 (±0.5) × 10(4) M(-1), K(PEG 6000-tRNA) = 8.9 (±1) × 10(4) M(-1), and K(mPEG-anthracene)= 1.2 (±0.40) × 10(3) M(-1) show stronger polymer-RNA complexation by PEG 6000 and by PEG 3350 than the mPEG-anthracene. AFM imaging showed that PEG complexes contain on average one tRNA with PEG 3350, five tRNA with PEG 6000, and ten tRNA molecules with mPEG-anthracene. tRNA aggregation and particle formation occurred at high polymer concentrations, whereas it remains in A-family structure.

PEG and mPEG-anthracene induce DNA condensation and particle formation.

In this study, we investigated the binding of DNA with poly(ethylene glycol) (PEG) of different sizes and compositions such as PEG 3350, PEG 6000, and mPEG-anthracene in aqueous solution at physiological conditions. The effects of size and composition on DNA aggregation and condensation as well as conformation were determined using Fourier transform infrared (FTIR), UV-visible, CD, fluorescence spectroscopic methods and atomic force microscopy (AFM). Structural analysis showed moderate complex formation for PEG 3350 and PEG 6000 and weaker interaction for mPE-anthracene-DNA adducts with both hydrophilic and hydrophobic contacts. The order of ± stability of the complexes formed is K(PEG 6000) = 1.5 (±0.4) × 10(4) M(-1) > K(PEG 3350) = 7.9 (±1) × 10(3) M(-1) > K(m(PEG-anthracene))= 3.6 (±0.8) × 10(3) M(-1) with nearly 1 bound PEG molecule per DNA. No B-DNA conformational changes were observed, while DNA condensation and particle formation occurred at high PEG concentration.

Bundling and aggregation of DNA by cationic dendrimers.

Dendrimers are unique synthetic macromolecules of nanometer dimensions with a highly branched structure and globular shape. Among dendrimers, polyamidoamine (PAMAM) have received most attention as potential transfection agents for gene delivery, because these macromolecules bind DNA at physiological pH. The aim of this study was to examine the interaction of calf-thymus DNA with several dendrimers of different compositions, such as mPEG-PAMAM (G3), mPEG-PAMAM (G4), and PAMAM (G4) at physiological conditions, using constant DNA concentration and various dendrimer contents. FTIR, UV-visible, and CD spectroscopic methods, as well as atomic force microscopy (AFM), were used to analyze the macromolecule binding mode, the binding constant, and the effects of dendrimer complexation on DNA stability, aggregation, condensation, and conformation. Structural analysis showed a strong dendrimer-DNA interaction via major and minor grooves and the backbone phosphate group with overall binding constants of K(mPEG-G3) = 1.5 (±0.5) × 10(3) M(-1), K(mPEG-G4) = 3.4 (±0.80) × 10(3) M(-1), and K(PAMAM-G4) = 8.2 (±0.90) × 10(4) M(-1). The order of stability of polymer-DNA complexation is PAMAM-G4 > mPEG-G4 > mPEG-G3. Both hydrophilic and hydrophobic interactions were observed for dendrimer-DNA complexes. DNA remained in the B-family structure, while biopolymer particle formation and condensation occurred at high dendrimer concentrations.

Dendrimers bind human serum albumin.

Dendrimers are synthetic, highly branched, spherical macromolecules with nanometer dimensions and potential applications in DNA and drug delivery systems. Human serum albumin (HSA) is a major transporter for delivering several endogenous compounds and drugs in vivo. The aim of this study was to examine the interaction of human serum albumin with several dendrimers such as mPEG-PAMAM (G3), mPEG-PAMAM (G4), and PAMAM (G4) at physiological conditions, using constant protein concentration and various dendrimer compositions. FTIR, UV-visible, CD, and fluorescence spectroscopic methods were used to analyze macromolecule binding mode, the binding constant and the effects of dendrimers complexation on HSA stability and conformation. Structural analysis showed that dendrimers bind HSA via polypeptide polar groups (hydrophilic) with number of bound polymer (n) 1.08 (mPEG-PAMAM-G3), 1.50 (mPEG-PAMAM-G4), and 0.96 (PAMAM-G4). The overall binding constants estimated were of KmPEG-G3=1.3 (+/-0.2)x10(4) M(-1), KmPEG-G4=2.2 (+/-0.4)x10(4) M(-1), and KPAMAM-G4=2.6 (+/-0.5)x10(4) M(-1). HSA conformation was altered by dendrimers with a major reduction of alpha-helix and increase in random coil and turn structures suggesting a partial protein unfolding.

Adjustment of the myelin sheath to axonal atrophy in the rat spinal root by the formation of infolded myelin loops.

Atrophy of the L4 dorsal and ventral spinal roots was experimentally induced by unilateral sciatic neurectomy in groups of young (2 and 4 months) and older (12 months) albino rats. During the 4 months following neurectomy, the occurrence of infolded myelin loops (IMLs) was quantitatively examined in transverse sections prepared using perfusion fixation with glutaraldehyde and embedding in epoxy resin. The number of IMLs was higher on the operated side and increased with the time of survival and the age of the animals. The formation of IMLs is a characteristic early response of a large-caliber myelin sheath to axonal atrophy, probably reflecting the presence of redundant myelin.

Evaluation of skin carcinogenicity of technical 2,2-bis-(p-glycidyloxyphenyl)-propane in CF1 mice.

The carcinogenic potential of a technical-grade epoxy resin, Araldite GY 250, of which the diglycidyl ether of bisphenol A (DGEBPA) is the main component, was investigated in CF1 mice. Groups of 50 male and 50 female mice were treated for 2 yr by repeated epidermal application of a 1 or 10% (v/v) solution in acetone. The controls, 50 mice of each sex, were treated with acetone alone. The treatment had no effect on survival and no excess incidence of skin tumours occurred. A positive control group of 50 male and 50 female CF1 mice was treated by epidermal application of 2% (v/v) beta-propiolactone in acetone. In this group there was a high incidence of malignant skin tumours at the site of application and, consequently, increased mortality. Treatment with neither DGEBPA technical nor beta-propiolactone induced systemic neoplasia.