Biomedical subjects

Clive J Roberts

Publications and source records attributed to Clive J Roberts.

At least 19 recordsLinked to original sources

PubMed2006

Elastic modulus measurements from individual lactose particles using atomic force microscopy.

The elastic modulus of pharmaceutical materials affects a number of pharmaceutical processes and subsequently formulation performance and is currently assessed by bulk methods, such as beam bending of compacts. Here we demonstrate the accurate measurement of the elastic modulus of alpha monohydrate lactose from the dominant (011) face of single crystals using atomic force microscopy (AFM) as 3.45+/-0.90GPa. The criteria to ensure this data is recorded within the elastic limit and can be modelled using Hertzian theory are established. We compare and contrast this AFM method to a permanent indentation technique based upon a much larger Berkovich pyramidal indenter on a lactose compact and the wider literature. Finally the AFM was utilized to study the elastic response of amorphous lactose, demonstrating that the physical state of the amorphous material changes under repeated loading and behaves in a more crystalline manner under repeated force measurements, suggesting a pressure induced phase transition. The AFM based approach demonstrated has the significant advantages of requiring minimal sample, no need for producing a compact, being non-destructive in that no permanent indent is required and providing a technique capable of detecting variations in material properties across a single particle or a number of particles.

Chemistry, Pharmaceutical↗

PubMed2006

Single particle friction on blister packaging materials used in dry powder inhalers.

Using atomic force microscopy (AFM) the adhesion and sliding friction behaviour of single lactose particles attached directly to AFM cantilevers has been studied. Measurements were made on the two sides of a blister packaging material used in dry powder inhalers (DPI). Although no significant differences in adhesion were observed, clear differences in particle friction were evident, where one side offers consistently greater friction across the range of loads studied here. The packaging samples were characterised by time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS) and found to have different surface chemistries. The observed difference in friction behaviour is discussed in the context of the differences seen in surface chemistry, topography and hardness. It is reasoned that in this case hardness has the largest influence, and on one sample soft surface layers are displaced by the particle. A clear relationship between friction and load was only observed with one of the three particles tested; this was attributed to multiple asperities being brought into contact, illustrating the important role of nanoscale contact geometry in determining friction behaviour.

Drug Packaging↗

PubMed2006

Influence of collagen denaturation on the nanoscale organization of adsorbed layers.

Adsorption (at 37 degrees C) of type I collagen, in native and heat-denatured (30 min at 40 and 90 degrees C) forms, on polystyrene was studied using quartz crystal microbalance with energy dissipation monitoring (QCM-D), atomic force microscopy (AFM) in tapping mode and X-ray photoelectron spectroscopy (XPS). The significance of the parameters deduced from QCM-D data was examined by comparing different approaches. The adsorbed layer of native collagen has a complex organization consisting of a thin mat of molecules near the surface, in which fibrils develop depending on concentration and time, and of a thicker overlayer containing protruding molecules or bundles which modify noticeably the local viscosity. As a result of drastic denaturation, the ability of collagen to assemble into fibrils in the adsorbed phase is lost and the protrusion of molecules into the aqueous phase is much less pronounced. The adsorbed layer of denatured collagen appears essentially as a monolayer of flattened coils. At low concentration, this is easily displaced upon drying, leading to particular dewetting figures; at high concentration, aggregates add to the first layer. Moderate denaturation leads to an adsorbed phase which shows properties intermediate between those observed with native and extensively denatured collagen, regarding the ability to form fibrillar structures and the adlayer thickness and viscosity.

Adsorption↗

PubMed2006

A nanoscale study of particle friction in a pharmaceutical system.

Studies of single particle interactions in dry powder inhaler (DPI) formulations using atomic force microscopy (AFM) have recently grown in popularity. Currently, these experiments are all based on measuring particle adhesion forces. We broaden this approach by presenting a novel AFM friction study of single particles in a pharmaceutical system, to examine forces acting parallel to a surface. The sliding friction signal of lactose particles attached to AFM cantilevers was recorded in lateral force (LF) mode over 5 microm x 5 microm areas on five different surfaces chosen to represent both relevant inter-particle and particle-surface interactions. A ranking of friction forces was obtained as follows: glass approximately equal to zanamivir >zanamivir-magnesium stearate (99.5%/0.5%, w/w) blend approximately equal to magnesium stearate approximately equal to PTFE. The addition of magnesium stearate to the zanamivir surface dominated and significantly reduced the friction (Kruskal-Wallis test, P<0.001). AFM images of the contacting asperities of the lactose particles show changes in contact morphology due to two processes. Firstly the asperity wears flat due to abrasion and secondly small magnesium stearate particles transfer onto the asperity. It is proposed that in combination with AFM particle adhesion measurements, this method could be used to screen new formulations and the effectiveness of tertiary components in modifying carrier-drug interactions.

Drug Combinations↗

PubMed2006

A comparison of morphology and surface energy characteristics of sulfathiazole polymorphs based upon single particle studies.

The morphological, adhesion and surface energetic properties of three sulfathiazole polymorphs (III, IV and polymorph I prepared from both acetone and methanol, designated I-ace and I-met, respectively) produced using Nektar supercritical fluid (SCF) technology have been characterized using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Surface roughness values for each polymorph were determined at different length scales. At sample sizes less than 1micromx1microm the polymorphs rank in terms of roughness as follows: I-met>I-ace approximately equal to IV>III. At the larger scales the polymorphs rank in terms of roughness as follows: I-met>III>I-ace approximately equal to IV. The surface energies for polymorphs determined against graphite (HOPG) and particles of the same polymorph were, respectively, I-met: 0.99mJm(-2) (S.D. 1.25mJm(-2)), 3.09mJm(-2) (S.D. 2.67mJm(-2)); I-ace: 309mJm(-2) (S.D. 329mJm(-2)), 16mJm(-2) (S.D. 11mJm(-2)); III: 1.17mJm(-2) (S.D. 1.5mJm(-2)), 5.4mJm(-2) (S.D. 3.6mJm(-2)); IV: 20.35mJm(-2) (S.D. 28.5mJm(-2)), 16.8mJm(-2) (S.D. 9.6mJm(-2)). In terms of surface energies the polymorphs hence rank I-ace>IV>III approximately equal to I-met (HOPG adhesion measurements) and IV approximately equal to I-ace>III>I-met (particle cohesion measurements). Consideration of contacting asperities and surface roughness was shown to have limited effect on the surface energies, and instead the differences were ascribed to variations in the surface chemistry as a result of changes in crystallization mechanisms.

Acetone↗

PubMed2006

Determination of the surface free energy of crystalline and amorphous lactose by atomic force microscopy adhesion measurement.

PURPOSE: This study was conducted to accurately measure the dispersive surface free energy of lactose solids in ordered and disordered states. METHODS: Atomic force microscopy (AFM) was used to determine the contact adhesion force between an AFM tip and lactose under low humidity (ca. 1% RH). The geometry of the tip contacting apex was characterized by scanning a porous aluminum film with ultrasharp spikes (radius 2-3 nm). A sphere vs. flat surface model was employed to relate the adhesion force determined to the surface energy based upon the Johnson-Kendal-Roberts theory. Spray-dried amorphous lactose in a compressed-disk form and single crystals of alpha-lactose monohydrate were prepared as model samples. RESULTS: The condition of the smooth sample surface and sphere-shaped tip used was shown to be appropriate to the application of the JKR model. The surface energy of crystalline [(0,-1,-1) face] and amorphous lactose was determined to be 23.3 +/- 2.3 and 57.4 +/- 7.9 mJ m(-2), respectively. CONCLUSIONS: We have demonstrated the capability of AFM to measure the dispersive surface free energy of pharmaceutical materials directly through a blank probe at the nanometer scale. These data, although consistent with results from more traditional methods, illustrate some unique attributes of this approach, namely, surface energies are directly derived from solid-solid interactions, measurements may be made on specific crystalline faces, and the potential exists to identify the submicron heterogeneity of organic solids in terms of their molecular energy states (such as ordered and disordered lactose).

Algorithms↗

PubMed2006

The DNA-remodelling activity of DnaD is the sum of oligomerization and DNA-binding activities on separate domains.

The Bacillus subtilis DnaD protein is an essential protein that has been implicated in the primosomal step of DNA replication, and recently in global DNA remodelling. Here we show that DnaD consists of two domains with distinct activities; an N-terminal domain (Nd) with oligomerization activity, and a C-terminal domain (Cd) with DNA-binding activity and a second DNA-induced oligomerization activity. Although Cd can bind to DNA and form large nucleoprotein complexes, it does not exhibit global DNA-remodelling activity. The presence of separate Nd does not restore this activity. Our data suggest that the global DNA-remodelling activity of DnaD is the sum of three separate oligomerization and DNA-binding activities residing on two distinct but linked domains.

Amino Acid Motifs↗

PubMed2006

The Bacillus subtilis primosomal protein DnaD untwists supercoiled DNA.

The essential Bacillus subtilis DnaD and DnaB proteins have been implicated in the initiation of DNA replication. Recently, DNA remodeling activities associated with both proteins were discovered that could provide a link between global or local nucleoid remodeling and initiation of replication. DnaD forms scaffolds and opens up supercoiled plasmids without nicking to form open circular complexes, while DnaB acts as a lateral compaction protein. Here we show that DnaD-mediated opening of supercoiled plasmids is accompanied by significant untwisting of DNA. The net result is the conversion of writhe (Wr) into negative twist (Tw), thus maintaining the linking number (Lk) constant. These changes in supercoiling will reduce the considerable energy required to open up closed circular plectonemic DNA and may be significant in the priming of DNA replication. By comparison, DnaB does not affect significantly the supercoiling of plasmids. Binding of the DnaD C-terminal domain (Cd) to DNA is not sufficient to convert Wr into negative Tw, implying that the formation of scaffolds is essential for duplex untwisting. Overall, our data suggest that the topological effects of the two proteins on supercoiled DNA are different; DnaD opens up, untwists and converts plectonemic DNA to a more paranemic form, whereas DnaB does not affect supercoiling significantly and condenses DNA only via its lateral compaction activity. The significance of these findings in the initiation of DNA replication is discussed.

Bacillus subtilis↗

PubMed2005

Resolution of the vertical and horizontal heterogeneity of adsorbed collagen layers by combination of QCM-D and AFM.

Collagen (type I from calf skin) adsorption on polystyrene (PS) and plasma-oxidized polystyrene (PSox) was studied, using a quartz crystal microbalance with energy dissipation measurements (QCM-D) and atomic force microscopy (AFM) in tapping mode. Radio-labeled collagen was used to measure the adsorbed amount and the ability of adsorbed collagen to exchange with molecules in the solution. The results show that the collagen adlayer consists of two parts: a dense and thin sheet in which fibrils are formed (directly observed by AFM) and an overlying thick layer (up to 200 nm) containing protruding molecules or bundles which are in very low concentration but modify noticeably the local viscosity. The thickness and viscosity of the semi-liquid adlayer both increase with adsorption time and collagen concentration. Fibril formation near the surface also increases with time and collagen concentration and occurs more readily on PS compared to PSox. Radiochemical measurements show that this may be related to the larger mobility of molecules adsorbed on PS, presumably owing to a smaller number of binding points.

Animals↗

PubMed2005

The Bacillus subtilis DnaD and DnaB proteins exhibit different DNA remodelling activities.

Primosomal protein cascades load the replicative helicase onto DNA. In Bacillus subtilis a putative primosomal cascade involving the DnaD-DnaB-DnaI proteins has been suggested to participate in both the DnaA and PriA-dependent loading of the replicative helicase DnaC onto the DNA. Recently we discovered that DnaD has a global remodelling DNA activity suggesting a more widespread role in bacterial nucleoid architecture. Here, we show that DnaB forms a "square-like" tetramer with a hole in the centre and suggest a model for its interaction with DNA. It has a global DNA remodelling activity that is different from that of DnaD. Whereas DnaD opens up supercoiled DNA, DnaB acts as a lateral compaction protein. The two competing activities can act together on a supercoiled plasmid forming two topologically distinct poles; one compacted with DnaB and the other open with DnaD. We propose that the primary roles of DnaB and DnaD are in bacterial nucleoid architecture control and modulation, and their effects on the initiation of DNA replication are a secondary role resulting from architectural perturbations of chromosomal DNA.

Bacillus subtilis↗

PubMed2005

Characterization of drug particle surface energetics and young's modulus by atomic force microscopy and inverse gas chromatography.

PURPOSE: Particulate interactions are dominated by aspects such as surface topography, exposed chemical moieties, environmental conditions, and thermodynamic properties such as surface free energy (gamma). The absolute value and relative magnitude of surface energies of a drug and excipients within a formulation can significantly influence manufacture, processing, and use. This study utilizes and compares the potentially complementary analytical techniques of atomic force microscopy (AFM) and inverse gas chromatography (IGC) in the quantitative determination of the surface energy of drug (budesonide) particles (micronized and unmilled) relevant to inhaled delivery. In addition, the study investigates with AFM another important parameter in determining material interactions, the local mechanical properties of the drug. METHODS: AFM was used to acquire force of adhesion (Fadh) and related work of adhesion (WA) and surface energy values between individual mironized drug particles and also model substrates (graphite and mica). In addition, AFM probes were used to interrogate the surface energy of unmilled drug particles. Measurement with AFM probes also yielded localized measurements of Young's modulus for the unmilled drug. IGC was also used to probe the surface characteristics of the bulk drug material. RESULTS: The average values for surface energies acquired from budesonide micronized particle interactions with graphite, mica, and drug particles of the same substance were found to range from 35 to 175, 5 to 40, and 10 to 32 mJ m(-2), respectively. The unmilled material displayed a range of values of 39-88 mJ m(-2) with an average of 60 mJ m(-2). The IGC result for the surface energy of the micronized material was 68.47 +/- 1.60 mJ m(-2). The variability in surface energy from AFM, a feature particularly apparent for the micronized material was attributed to two factors, intrinsic material variations within a single particle and assumptions present within the contact mechanics model used. Here we provide a detailed description of these factors to go some way to rationalize the results. The Young's modulus of the unmilled drug was determined to be approximately 10 GPa. CONCLUSION: The range of determined surface energies between the AFM measurement on graphite, mica, and the drug is proposed to reflect the different chemistries displayed by the drug at the single particle level. The maximum values of these ranges can be related to the sites most likely to be involved in adhesion. AFM and IGC yield surface energy estimates in approximate agreement, but clearly are interrogating surfaces in different fashions. This raises questions as to the nature of the measurement being made by these approaches and to the most appropriate time to use these methods in terms of a direct relation to formulation design, manufacture, and drug delivery. Finally, we demonstrate a novel method for assessing the Young's modulus of a drug from a single particle.

Administration, Inhalation↗

PubMed2005

Identifying and mapping surface amorphous domains.

PURPOSE: Undesirable amorphous material generation during formulation is implicated in a growing number of pharmaceutical problems. Due to the importance of interfacial properties in many drug delivery systems, it seems that surface amorphous material is particularly significant. Consequently, this study investigates a range of methods capable of detecting and mapping surface amorphous material. METHODS: A micron-sized localized surface domain of amorphous sorbitol is generated using a novel localized heating method. The domain is subsequently investigated using atomic force microscopy (AFM) imaging, nanomechanical measurements, and Raman microscopy 3-D profiling. RESULTS: AFM phase and height images reveal nanoscale-order variations within both crystalline and amorphous sorbitol domains. Nanomechanical measurements are able to quantitatively distinguish the amorphous and crystalline domains through local Young's modulus measurements. Raman microscopy also distinguishes the amorphous and crystalline sorbitol through variations in peak width. This is shown to allow mapping of the 3-D distribution of the amorphous phase and is hence complementary to the more surface sensitive AFM measurements. CONCLUSIONS: AFM and Raman microscopy map the distribution of amorphous material at the surface of a sorbitol crystal with submicron spatial resolution, demonstrating surface analysis methods for characterizing semicrystalline solids generated during pharmaceutical processing.

Chemistry, Pharmaceutical↗

PubMed2005

Preferential liver gene expression with polypropylenimine dendrimers.

Previously, the lower generation (DAB 8-generation 2 and DAB 16-generation 3) polypropylenimine dendrimers have been shown to be effective gene delivery systems in vitro. In the current work, we sought to: (a) test the effect of the strength of the carrier, DNA electrostatic interaction on gene transfer and (b) to study the in vivo gene transfer activity of these low molecular weight (<1687 Da) non-amphiphilic plain and quaternary ammonium gene carriers. Towards this aim, methyl quaternary ammonium derivatives of DAB 4 (generation 1), DAB 8, DAB 16 and DAB 32 (generation 4) were synthesised to give Q4, Q8, Q16 and Q32, respectively. Quaternisation of DAB 8 proved to be critical in improving DNA binding, as evidenced by data from the ethidium bromide exclusion assay and dendrimer-DNA colloidal stability data. This improved colloidal stability had a major effect on vector tolerability, as Q8-DNA formulations were well tolerated on intravenous injection while a similar DAB 8-DNA dose was lethally toxic by the same route. Quaternisation also improved the in vitro cell biocompatibility of DAB 16-DNA and DAB 32-DNA dendrimer complexes by about 4-fold but not that of the lower generation DAB 4-DNA and DAB 8-DNA formulations. In contrast to previous reports with non-viral gene delivery systems, the intravenous administration of DAB 16-DNA and Q8-DNA formulations resulted in liver targeted gene expression as opposed to the lung targeted gene expression obtained with the control polymer-Exgen 500 [linear poly(ethylenimine)] and a lung avoidance hypothesis is postulated. We conclude that the polypropylenimine dendrimers are promising gene delivery systems which may be used to target the liver and avoid the lung and also that molecular modifications conferring colloidal stability on gene delivery formulations have a profound effect on their tolerability on intravenous administration.

Animals↗

PubMed2005

What can we learn from atomic force microscopy adhesion measurements with single drug particles?

Frequently solid dosage form formulation manufacture and delivery depend critically on the control and exploitation of interparticulate interactions. Traditional approaches to understand such interactions rely on indirect assessments of adhesion or consider the behaviour of large numbers of particles. In recent years, the possibility of characterizing and perhaps quantifying forces of adhesion between individual micron and sub-micron sized particles has become viable using the atomic force microscope. This has significant potential in formulation development, particularly in the optimization of inhalation and other solid-dosage form based therapies. However, before a widespread acceptance of this approach by pharmaceutical scientists and industry can proceed a number of issues remain to be considered. These include how can single particle events be mapped on to bulk behaviour, the need to understand the sometimes wide variations in adhesion data observed and can formulations be compared quantitatively and perhaps be screened by this approach?

Adhesiveness↗

PubMed2005

Atomic force microscopy study of human amylin (20-29) fibrils.

Here we present atomic force microscopy images of the fibrils formed by human amylin(20-29). This peptide is a fragment of the polypeptide amylin, the major proteinaceous component of amyloid deposits found in cases of type-II diabetes mellitus. Our results demonstrate that the amylin(20-29) peptide fragment forms amyloid-like fibrils that display polymorphic structures. Twisting along the axis of fibrils was often observed in fibrils aged for 6 hours but disappeared in mature fibrils aged for longer time periods.

Amino Acid Sequence↗

PubMed2004

Surface morphology and chemistry of Prunus laurocerasus L. leaves: a study using X-ray photoelectron spectroscopy, time-of-flight secondary-ion mass spectrometry, atomic-force microscopy and scanning-electron microscopy.

The surface properties of the plant cuticle play a crucial role in plant-pathogen interactions and the retention and penetration of agriculturally important chemicals. This paper describes the use of X-ray photoelectron spectroscopy (XPS), time-of-flight secondary-ion mass spectrometry (ToF-SIMS), tapping-mode atomic force microscopy (TM-AFM) and scanning electron microscopy (SEM) to determine surface-specific chemical and material properties of the adaxial surface of Prunus laurocerasus L. leaves. XPS data, derived from the uppermost few nanometres (< 10 nm) of the leaf surface, were consistent with the wax components and functionality known to be present within the waxes. ToF-SIMS provided molecular speciation from the outermost monolayer of the leaf surface, indicating the importance of a family of acetates with chain lengths ranging from C20 to C34. The presence of alkanes with C29 and C31 chain lengths was also confirmed. SEM and TM-AFM topography images revealed a textured granular surface, while simultaneously recorded AFM phase images revealed heterogeneous material properties at the nanoscale. The relevance of these data to plant cuticle development, allelochemistry and agrochemical delivery is discussed.

Microscopy, Atomic Force↗

PubMed2004

The Bacillus subtilis DnaD protein: a putative link between DNA remodeling and initiation of DNA replication.

The Bacillus subtilis DnaD protein is an essential protein and a component of the oriC and PriA primosomal cascades, which are responsible for loading the main replicative ring helicase DnaC onto DNA. We present evidence that DnaD also has a global DNA architectural activity, assembling into large nucleoprotein complexes on a plasmid and counteracting plasmid compaction in a manner analogous to that recently seen for the histone-like Escherichia coli HU proteins. This DNA-remodeling role may be an essential function for initiation of DNA replication in the Gram +ve B. subtilis, thus highlighting DnaD as the link between bacterial nucleoid reorganization and initiation of DNA replication.

Bacillus subtilis↗

PubMed2004

pH-dependent behavior of surface-immobilized artificial leucine zipper proteins.

The coiled-coil protein motif occurs in over 200 proteins and has generated interest for a range of applications requiring surface immobilization of the constituent peptides. This paper describes an investigation of the environment-responsive behavior of a monolayer of surface-immobilized artificial proteins, which are known to assemble to form coiled-coil structures in bulk solution. An extended version of the quartz crystal microbalance (QCM-D) and surface plasmon resonance (SPR) are independently employed to characterize the adsorption of the proteins to a gold surface. The data suggest that the molecules arrange in a closely packed layer orientated perpendicular to the surface. QCM-D measurements are also employed to measure pH-induced changes in the resonant frequency (f) and the energy dissipation factor (D) of a gold-coated quartz crystal functionalized with the formed monolayer. Exposure of the protein monolayer to a pH 4.5 solution results in a shift of 43 Hz in f and a shift of -0.7 x 10(-6) in D as compared to pH 7.4. In contrast, increasing the pH to 11.2, results in f and D shifts of -17 Hz and 0.6 x 10(-6), respectively. The magnitude of the observed shifts suggests that the proteins form a rigid layer at low pH that can be hydrated to a fluid layer as the pH is increased. These observations correlate with spectroscopic changes that indicate a reduction in the helical content of the protein in bulk solutions of high pH.

Adsorption↗