Non-labelled surface sensitive techniques as platforms for pharmaceutical nanotechnology research

Insufficient delivery of drugs to the target sites like tumors and cells has been a barrier for achieving satisfying therapeutic effects in many diseases. Distribution and exposure of drugs to normal and healthy tissues may enhance the possibility of side effects and toxicity in vivo. Nanoparticle (NP) drug delivery systems have been developed to enable targeting of drugs to target sites and at the same time also reduce or even eliminate the distribution and exposure of drugs to non-targeted sites (normal and healthy tissues). The interactions of ligand attached NPs with specific receptors on the cell surface enable intracellular delivery of drugs. Knowledge of the molecular mechanisms (kinetics and affinity) of specific NP surface interactions is vital for designing and optimizing NPs based targeted drug delivery systems. Biophysical non-labelled surface sensitive detection techniques allow the characterization of the specific NP-cell interactions in vitro at the molecular levels. In this work, surface sensitive non-labelled surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) biosensors were optimized, utilized and further developed as platforms for in vitro characterization and evaluation of the targeting of NP drug delivery systems. A multi-parameter SPR (MP-SPR) prototype was modified, improved and optimized for characterizing molecular surface interactions and phospholipid based thin film properties. The methodologies to extract simultaneously the thickness and the optical properties of thin films were developed by using the multi-wavelength SPR technique. The methodologies were extended to cover the film thickness from few nanometers to micrometers by combining the SPR wavelength and the waveguide mode analysis. These methods were successfully utilized for analyzing LB mono- and multilayers and further for the polyelectrolyte multilayer films. In order to enable the combined use of SPR and QCM techniques for drug and NP interaction studies, these two devices were synchronized to achieve consistent hydrodynamic conditions in the flow channels by computational fluid dynamics (CFD) modelling. The flow channels and the device synchronization were verified by the streptavidin-biotin and liposome-surface interactions. The synchronized SPR and QCM devices were further utilized for the examination of the targeting properties via the streptavidin-biotin liposome interactions under different shear flows. The effect of the flow rate and shear stress on the targeted liposome with the target surface was investigated. The results from SPR and QCM measurements were compared, showing that the binding of the targeted liposome was flow rate and shear stress regulated. According to the SPR measurements, high flow rates improved the binding of liposomes to the target surface. However, the results obtained from the QCM measurements were somehow different. They gave additional information about the liposome binding behavior, indicating deformation or rupture of the bound liposomes at high flow rates and shear stresses. In conclusion, SPR and QCM, the two label free surface sensitive techniques, are excellent platforms for pharmaceutical nanotechnology research. These allow for both the nanoparticle interaction studies and the characterization of nanoscale thin films. Especially, the combined use of the synchronized SPR and QCM techniques forms a powerful platform for the qualitative and quantitative characterization of NP-surface interactions for obtaining in-depth understanding of the targeting behavior of NP drug delivery systems. The results obtained provides the basis for developing new complementary in vitro platforms to traditional cell based in vitro assays for optimizing and screening of NP based targeted drug delivery systems.Lääkeannostelu on keskeinen alue lääketutkimuksessa. Lääkeaineiden riittämätön jakelu kohteeseen estää monien sairauksien kohdalla tyydyttävän terapeuttisen vaikutuksen saavuttamisen. Lääkeaineiden leviäminen ja vaikutus terveisiin kudoksiin saattaa lisätä sivuvaikutuksien ja toksisuuden mahdollisuutta in vivo. On kehitetty nanopartikkelipohjaisia lääkeannostelumenetelmiä, jotta lääkeaineita voitaisiin kohdentaa paremmin ja samalla vähentää tai jopa kokonaan välttää lääkeaineiden leviämistä ja vaikutusta kohdealueen ulkopuolelle. Ligandilla funktionalisoitujen nanopartikkeleiden vuorovaikutus solupinnalla olevien spesifisten reseptorien kanssa mahdollistaa lääkeaineiden kuljettamisen solun sisään. Spesifisten nanopartikkeleiden pintavuorovaikutukset ja niiden molekulaaristen mekanismien tuntemus (kinetiikka ja affiniteetti) ovat keskeisiä kehitettäessä ja optimoitaessa nanopartikkelipohjaisia lääkeannostelumenetelmiä. Biofysikaaliset pintaherkät mittausmenetelmät tarjoavat mahdollisuuden karakterisoida in vitro spesifisiä nanopartikkelin ja solun välisiä vuorovaikutuksia molekyylitasolla ilman leimoja. Pintaherkät ja leimattomat pintaplasmonresonanssi- (SPR) ja kvartsikidemikrovaaka- (QCM) biosensorit optimoitiin ja niitä hyödynnettiin ja kehitettiin edelleen alustoiksi kohdennettujen nanopartikkelipohjaisten lääkeannostelumenetelmien in vitro -karakterisointia ja arviointia varten. Moniparametristä SPR- (MP-SPR) prototyyppilaitetta muokattiin, paranneltiin ja optimoitiin molekulaaristen pintavuorovaikutusten ja fosfolipidipohjaisten ohutkalvojen karakterisointia varten. Moniaallonpituus-SPR-menetelmää hyödyntäen kehitettiin metodologia, jolla voi samanaikaisesti määrittää ohutkalvojen paksuuden ja sen optiset ominaisuudet. Lisäksi tämä metodologia laajennettiin kattamaan kalvojen paksuudet muutamasta nanometristä mikrometreihin yhdistämällä SPR-aallonpituus- ja aaltojohdinanalyysit. SPR- ja QCM-laitteiden hydrodynaamiset ominaisuudet synkronisoitiin virtauslaskentamallinusten (CFD) kautta, mikä mahdollisti SPR- ja QCM-laitteiden yhteiskäytön lääkeaineiden ja nanopartikkeleiden vuorovaikutuksien tutkimiseen. Virtauskanavien ja laitteiden synkronisointi todennettiin tutkimalla biomolekulaarista vuorovaikutusta ja nanopartikkeli-pinta-vuorovaikutusta. Synkronoituja SPR- ja QCM-laitteita hyödynnettiin edelleen kohdennuksen ominaisuuksien tutkimiseen mittaamalla streptavidin-biotinyloitujen liposomien vuorovaikutuksia eri virtausnopeuksilla. Virtausnopeuden ja leikkausjännitteen vaikutus kohdennetun liposomin käyttäytymiseen kohdepinnan kanssa tutkittiin. SPR- ja QCM-mittauksia verrattiin keskenään. Mittaukset osoittivat, että kohdennetun liposomin sitoutumista pintaan säätelivät virtausnopeus ja leikkausjännite. QCM antoi lisätietoa liposomien sitoutumiskäyttäytymisestä ja osoitti, että pinnalle sitoutuneet liposomit muuttivat muotoaan tai hajosivat korkeissa virtaunopeuksissa tai leikkausjänitteissä. Päätelmänä voidaan todeta, että, SPR ja QCM ovat erinomaisia menetelmiä farmaseuttisen nanoteknologian tutkimukseen. Nämä menetelmät mahdollistavat nanopartikkeleiden vuorovaikutuksien tutkimisen sekä ohutkalvojen karakterisoimisen. Synkronoitujen SPR- ja QCM-menetelmien yhteiskäyttö muodostaa tehokkaan alustan kvalitatiiviselle ja kvantitatiiviselle nanopartikkeli-pinta-vuorovaikutuksien karakterisoinnille, joka auttaa paremmin ymmärtämään nanopartikkelipohjaisten lääkeannostelumenetelmien kohdennuskäyttäytymistä. Tulokset tarjoavat perustan uusien täydentävien in vitro -alustojen kehittämiselle perinteisten solupohjaisten in vitro -menetelmien rinnalle nanopartikkelipohjaisten lääkeannostelumenetelmien optimointiin ja seulontaan

Global transcriptional profiles of beating clusters derived from human induced pluripotent stem cells and embryonic stem cells are highly similar

<p>Abstract</p> <p>Background</p> <p>Functional and molecular integrity of cardiomyocytes (CMs) derived from induced pluripotent stem (iPS) cells is essential for their use in tissue repair, disease modelling and drug screening. In this study we compared global transcriptomes of beating clusters (BCs) microdissected from differentiating human iPS cells and embryonic stem (ES) cells.</p> <p>Results</p> <p>Hierarchical clustering and principal component analysis revealed that iPS-BCs and ES-BCs cluster together, are similarly enriched for cardiospecific genes and differ in expression of only 1.9% of present transcripts. Similarly, sarcomeric organization, electrophysiological properties and calcium handling of iPS-CMs were indistinguishable from those of ES-CMs. Gene ontology analysis revealed that among 204 genes that were upregulated in iPS-BCs vs ES-BCs the processes related to extracellular matrix, cell adhesion and tissue development were overrepresented. Interestingly, 47 of 106 genes that were upregulated in undifferentiated iPS vs ES cells remained enriched in iPS-BCs vs ES-BCs. Most of these genes were found to be highly expressed in fibroblasts used for reprogramming and 34% overlapped with the recently reported iPS cell-enriched genes.</p> <p>Conclusions</p> <p>These data suggest that iPS-BCs are transcriptionally highly similar to ES-BCs. However, iPS-BCs appear to share some somatic cell signature with undifferentiated iPS cells. Thus, iPS-BCs may not be perfectly identical to ES-BCs. These minor differences in the expression profiles may occur due to differential cellular composition of iPS-BCs and ES-BCs, due to retention of some genetic profile of somatic cells in differentiated iPS cell-derivatives, or both.</p

Global transcriptome analysis of murine embryonic stem cell-derived cardiomyocytes

Microarray analysis reveals that the specific pattern of gene expression in cardiomyocytes derived from embryonic stem cells reflects the biological, physiological and functional processes occurring in mature cardiomyocytes

Effect of grazing types on community-weighted mean functional traits and ecosystem functions on Inner Mongolian steppe, China

The relationships between community-weighted mean (CWM) functional traits and ecosystem functions have been extensively studied. However, how CWM traits and ecosystem functions respond to grazing types and whether the relationships between CWM traits and ecosystem functions mediate the response of ecosystem functions to grazing types remains controversial. In the present study, we set up a seven-year grazing experiment with four grazing types: no grazing (NG), cattle grazing (CG), sheep grazing (SG), and mixed grazing by sheep and cattle (MG) on Inner Mongolia grassland. Nine functional traits of dominant species and five ecosystem functions under different grazing types were determined, and the relationships between CWM traits and ecosystem functions were analyzed. The results showed that the CWM height decreased after grazing, while the CWM nitrogen and phosphorus contents increased after CG. SG caused a greater decrease in aboveground biomass (AGB) and a greater increase in the net ecosystem CO2 exchange (NEE) of grassland ecosystems than did CG. This result may be partially because the CWM nutrient content and NEE were more negatively related after CG; and the increase in the CWM nitrogen and phosphorus contents suppressed NEE after CG. Therefore, to protect the sustainability of grassland ecosystem functions, SG should be reduced. Additionally, our work emphasizes that the relationships between plant functional traits and ecosystem functions may mediate the response of ecosystem functions to grazing types

Single grazing is more detrimental to grasslands than mixed grazing : evidence from the response of functional traits of dominant plants to grazing systems

Plant functional traits reflect species ecological strategies and determine how plants respond to environmental changes, Plant functional traits reflect species ecological strategies and determine how plants respond to environmental changes, however, how dominant species in the Inner Mongolia grassland adapt to different grazing systems by changing their functional traits has not been systematically investigated. In this study, we established four 7-year grazing treatments in the Inner Mongolia grassland: cattle grazing, sheep grazing, mixed sheep and cattle grazing, and no grazing. Fourteen functional traits of three dominant species (Stipa grandis, Leymus chinensis, and Cleistogenes squarrosa) were measured under the different grazing treatments. We found convergences of plant functional traits that indicate herbivory avoidance or tolerance. Plants reduced their vegetative height (VH) and stem: leaf ratio (SLR) to avoid grazing; increased their ability to acquire resources by increasing their specific leaf area (SLA), leaf nitrogen concentration (LNC), and leaf phosphorus concentration (LPC); and reduced their leaf dry matter content (LDMC) to tolerate grazing. Moreover, plants may adapt to grazing by increasing the intraspecific variability of their functional traits. Sheep-only grazing adversely affected L. chinensis growth and survival, while cattle-only grazing hindered S. grandis growth and increased the intraspecific variability of its plant functional traits. Our study emphasizes that intraspecific variability is an important indicator of the responses of plant functional traits to grazing. Since single grazing is more detrimental to the functional traits of dominant plants, we suggest that mixed cattle and sheep grazing may be a more environmentally friendly and sustainable practice for the Inner Mongolia grassland than single grazing

Prevalence and Characterization of Staphylococcus aureus Isolated From Women and Children in Guangzhou, China

The prevalent Staphylococcus aureus clones and antibiotic susceptibility profiles are known to change dynamically and geographically; however, recent S. aureus strains causing infections in women and children in China have not been characterized. In this study, we analyzed the molecular epidemiology and antimicrobial resistance of S. aureus isolated from patients in four centers for women and children in Guangzhou, China. In total, 131 S. aureus isolates (100 from children and 31 from women) were analyzed by spa typing, multi-locus sequence typing, virulence gene and antimicrobial resistance profiling, staphylococcal chromosomal cassette mec typing, and mutation analyses of rpoB. A total of 58 spa types, 27 sequence types (STs), and 10 clonal complexes (CCs) were identified. While CC59 (ST59-IV, 48.8%; ST338-III, 35.7%) and CC45 (ST45-IV, 100%) were the major clones (84.4%) among MRSA isolates, CC5 (ST188, 24.3%; ST1, 21.6%) and CC398 (ST398, 70%) were the major ones (70.1%) among MSSA isolates. ST338-MRSA-III mostly found in pus but hardly in respiratory tract samples while ST45-MRSA-IV was on the opposite, even though they both found in blood and cerebrospinal fluid sample frequently. Staphylococcal enterotoxin genes seb-seq-sek were strongly associated with ST59 and ST338, while sec was associated with ST45, ST121, ST22, and ST30. All ST338, ST1232, and SCCmec III isolates carried lukF/S-PV genes. A total of 80% of ST338 isolates were resistant to erythromycin, clindamycin, and tetracycline. All ST45 isolates exhibited intermediate or complete resistance to rifampicin. In total, 481 HIS/ASN mutations in rpoB were found in rifampicin-resistant or intermediate-resistant isolates. ST338-III and ST45-IV emerged as two of three major clones in MRSA isolates from women and children in Guangzhou, China, though ST59-MRSA-IV remained the most prevalent MRSA clone. Clonal distribution of S. aureus varied, depending on the specimen source. Virulence genes and antibiograms were closely associated with the clonal lineage. These results clarified the molecular epidemiology of S. aureus from women and children in Guangzhou, China, and provide critical information for the control and treatment of S. aureus infections

Pre-Targeting and Direct Immunotargeting of Liposomal Drug Carriers to Ovarian Carcinoma

Peer reviewe