Colloidal drug probe : method development and validation for adhesion force measurement using atomic force microscopy

This study demonstrates a novel technique of preparing drug colloid probes to determine the adhesion force between the drug salbutamol sulphate (SS) and the surfaces of polymer microparticles to be used as carriers for the dispersion of drug particles from a dry powder inhaler (DPI) formulation. Initially model silica probes of approximately 4 μm size, similar to a drug particle used in DPI formulations, were coated with a saturated SS solution with the aid of capillary forces acting between the silica probe and the drug solution. The developed method of ensuring a smooth and uniform layer of SS on the silica probe was validated using X-Ray Photoelectron Spectroscopy (XPS) and Scanning Electron Microscopy (SEM). Using the same technique, silica microspheres preattached on the AFM cantilever were coated with SS. The adhesion forces between the silica probe and drug coated silica (drug probe) and polymer surfaces (hydrophilic and hydrophobic) were determined. Our experimental results showed that the technique for preparing the drug probe was robust and can be used to determine the adhesion force between hydrophilic/hydrophobic drug probe and carrier surfaces to gain a better understanding on drug carrier adhesion forces in DPI formulations.

Colloidal drug probe : Method development and validation for adhesion force measurement using Atomic Force Microscopy

This study demonstrates a novel technique of preparing drug colloid probes to determine the adhesion force between a model drug salbutamol sulphate (SS) and the surfaces of polymer microparticles to be used as carriers for the dispersion of drug particles from dry powder inhaler (DPI) formulations. Model silica probes of approximately 4 lm size, similar to a drug particle used in DPI formulations, were coated with a saturated SS solution with the aid of capillary forces acting between the silica probe and the drug solution. The developed method of ensuring a smooth and uniform layer of SS on the silica probe was validated using X-ray Photoelectron Spectroscopy (XPS) and Scanning Electron Microscopy (SEM). Using the same technique, silica microspheres pre-attached on the AFM cantilever were coated with SS. The adhesion forces between the silica probe and drug coated silica (drug probe) and polymer surfaces (hydrophilic and hydrophobic) were determined. Our experimental results showed that the technique for preparing the drug probe was robust and can be used to determine the adhesion force between hydrophilic/ hydrophobic drug probe and carrier surfaces to gain a better understanding on drug carrier adhesion forces in DPI formulations.

Adhesion and dispersion of salmeterol xinofoate from lactose interactive mixtures for inhalation - can surface roughness of carriers determine performance

The objective was to understand the influence of the surface roughness of lactose carriers on the adhesion and dispersion of salmeterol xinafoate (SX) from interactive mixtures. The surface roughness of lactose carriers was determined by confocal microscopy. Particle images and adhesion forces between SX and lactose particles were determined by Atomic Force Microscopy. The dispersion of SX (2.5%) from interactive mixtures with lactose was determined using a twin-stage impinger (TSI) with a Rotahaler® at an airflow rate of 60L/min. SX was analysed using a validated HPLC assay. The RMS Rq of lactose carriers ranged from 0.93-2.84μm, the Fine Particle Fraction (FPF) of SX ranged between 4 and 24 percent and average adhesion force between a SX and lactose particles ranged between 49 and 134 nN. No direct correlation was observed between the RMS Rq of lactose carriers and either the FPF of SX for the interactive mixtures or the adhesion force of a SX on the lactose particles; however, the presence of fine lactose associated with the carrier surface increased the FPF of SX. Dispersion through direct SX detachment from the carrier surface was not consistent with the poor correlations described and was more likely to occur through complex particulate interactions involving fine lactose.

Single-cell force spectroscopy, an emerging tool to quantify cell adhesion to biomaterials

Cell adhesion receptors play a central role in sensing and integrating signals provided by the cellular environment. Thus, understanding adhesive interactions at the cell-biomaterial interface is essential to improve the design of implants that should emulate certain characteristics of the cell's natural environment. Numerous cell adhesion assays have been developed; among these, atomic force microscopy-based single-cell force spectroscopy (AFM-SCFS) provides a versatile tool to quantify cell adhesion at physiological conditions. Here we discuss how AFM-SCFS can be used to quantify the adhesion of living cells to biomaterials and give examples of using AFM-SCFS in tissue engineering and regenerative medicine. We anticipate that in the near future, AFM-SCFS will be established in the biomaterial field as an important technique to quantify cell-biomaterial interactions and thereby will contribute to the optimization of implants, scaffolds, and medical devices.

The effect of unlocking RGD-motifs in collagen I on pre-osteoblast adhesion and differentiation

Denaturation of extracellular matrix proteins exposes cryptic binding sites. It is hypothesized that binding of cell adhesion receptors to these cryptic binding sites regulates cellular behaviour during tissue repair and regeneration. To test this hypothesis, we quantify the adhesion of pre-osteoblastic cells to native (Col) and partially-denatured (pdCol) collagen I using single-cell force spectroscopy. During early stages of cell attachment (≤180 s) pre-osteoblasts (MC3T3-E1) adhered significantly stronger to pdCol compared to Col. RGD (Arg-Gly-Asp)-containing peptides suppressed this elevated cell adhesion. We show that the RGD-binding α5β1- and αv-integrins mediated pre-osteoblast adhesion to pdCol, but not to Col. On pdCol pre-osteoblasts had a higher focal adhesion kinase tyrosine-phosphorylation level that correlated with enhanced spreading and motility. Moreover, pre-osteoblasts cultured on pdCol showed a pronounced matrix mineralization activity. Our data suggest that partially-denatured collagen exposes RGD-motifs that trigger binding of α5β1- and αv-integrins. These integrins initiate cellular processes that stimulate osteoblast adhesion, spreading, motility and differentiation. Taken together, these quantitative insights reveal an approach for the development of alternative collagen I- based surfaces for tissue engineering applications.

Tunable “Nano-Shearing” : A physical mechanism to displace nonspecific cell adhesion during rare cell detection

We report a tunable alternating current electrohydrodynamic (ac-EHD) force which drives lateran fluid motion within a few nanometers of an electrode surface. Because the magnitude of this fluid shear force can be tuned externally (e.g., via the application of an ac electric field), it provides a new capability to physically displace weakly (nonspecifically) bound cellular analytes. To demonstrate the utility of the tunable nanoshearing phenomenon, we present data on purpose-built microfluidic devices that employ ac-EHD force to remove nonspecific adsorption of molecular and cellular species. Here, we show that an ac-EHD device containing asymmetric planar and microtip electrode pairs resulted in a 4-fold reduction in nonspecific adsorption of blood cells and also captured breast cancer cells in blood, with high efficiency (approximately 87%) and specificity. We therefore feel that this new capability of externally tuning and manipulating fluid flow could have wide applications as an innovative approach to enhance the specific capture of rare cells such as cancer cells in blood.

Targeting and transport : how microtubules control focal adhesion dynamics

Directional cell migration requires force generation that relies on the coordinated remodeling of interactions with the extracellular matrix (ECM), which is mediated by integrin-based focal adhesions (FAs). Normal FA turnover requires dynamic microtubules, and three members of the diverse group of microtubule plus-end-tracking proteins are principally involved in mediating microtubule interactions with FAs. Microtubules also alter the assembly state of FAs by modulating Rho GTPase signaling, and recent evidence suggests that microtubule-mediated clathrin-dependent and -independent endocytosis regulates FA dynamics. In addition, FA-associated microtubules may provide a polarized microtubule track for localized secretion of matrix metalloproteases (MMPs). Thus, different aspects of the molecular mechanisms by which microtubules control FA turnover in migrating cells are beginning to emerge.

Enhanced human bone marrow stromal cell affinity for modified poly(L-lactide) surfaces by the upregulation of adhesion molecular genes

To enhance and regulate cell affinity for poly (l-lactic acid) (PLLA) based materials, two hydrophilic ligands, poly (ethylene glycol) (PEG) and poly (l-lysine) (PLL), were used to develop triblock copolymers: methoxy-terminated poly (ethylene glycol)-block-poly (l-lactide)-block-poly (l-lysine) (MPEG-b-PLLA-b-PLL) in order to regulate protein absorption and cell adhesion. Bone marrow stromal cells (BMSCs) were cultured on different composition of MPEG-b-PLLA-b-PLL copolymer films to determine the effect of modified polymer surfaces on BMSC attachment. To understand the molecular mechanism governing the initial cell adhesion on difference polymer surfaces, the mRNA expression of 84 human extracellular matrix (ECM) and adhesion molecules was analysed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). It was found that down regulation of adhesion molecules was responsible for the impaired BMSC attachment on PLLA surface. MPEG-b-PLLA-b-PLL copolymer films improved significantly the cell adhesion and cytoskeleton expression by upregulation of relevant molecule genes significantly. Six adhesion genes (CDH1, ITGL, NCAM1, SGCE, COL16A1, and LAMA3) were most significantly influenced by the modified PLLA surfaces. In summary, polymer surfaces altered adhesion molecule gene expression of BMSCs, which consequently regulated cell initial attachment on modified PLLA surfaces.

Biomechanical force displacement analysis of strength of fixation of tracker holding devices to bone in computer aided joint replacement

Computer aided joint replacement surgery has become very popular during recent years and is being done in increasing numbers all over the world. The accuracy of the system depends to a major extent, on accurate registration and immobility of the tracker attachment devices to the bone. This study was designed to asses the forces needed to displace the tracker attachment devices in the bone simulators. Bone simulators were used to maintain the uniformity of the bone structure during the study. The fixation devices tested were 3mm diameter self drilling, self tapping threaded pin, 4mm diameter self tapping cortical threaded pin, 5mm diameter self tapping cancellous threaded pin and a triplanar fixation device ‘ortholock’ used with three 3mm pins. All the devices were tested for pull out, translational and rotational forces in unicortical and bicortical fixation modes. Also tested was the normal bang strength and forces generated by leaning on the devices. The forces required to produce translation increased with the increasing diameter of the pins. These were 105N, 185N, and 225N for the unicortical fixations and 130N, 200N, 225N for the bicortical fixations for 3mm, 4mm and 5mm diameter pins respectively. The forces required to pull out the pins were 1475N, 1650N, 2050N for the unicortical, 1020N, 3044N and 3042N for the bicortical fixated 3mm, 4mm and 5mm diameter pins. The ortholock translational and pull out strength was tested to 900N and 920N respectively and still it did not fail. Rotatory forces required to displace the tracker on pins was to the magnitude of 30N before failure. The ortholock device had rotational forces applied up to 135N and still did not fail. The manual leaning forces and the sudden bang forces generated were of the magnitude of 210N and 150N respectively. The strength of the fixation pins increases with increasing diameter from three to five mm for the translational forces. There is no significant difference in pull out forces of four mm and five mm diameter pins though it is more that the three mm diameter pins. This is because of the failure of material at that stage rather than the fixation device. The rotatory forces required to displace the tracker are very small and much less that that can be produced by the surgeon or assistants in single pins. Although the ortholock device was tested to 135N in rotation without failing, one has to be very careful not to put any forces during the operation on the tracker devices to ensure the accuracy of the procedure.

(-)-CGP12177 increases contractile force through beta1l-adrenoceptors but not through beta3-adrenoceptors in human right atrial myocardium

(-)-CGP12177 is a non-conventional partial agonist that causes modest and transient increases of contractile force in human atrial trabeculae (Kaumann and Molenaar, 2008). These effects are markedly increased and maintained by inhibition of phosphodiesterase PDE3. As verified with recombinant receptors, the cardiostimulant effect of (-)-CGP12177 is mediated through a site at the beta1-adrenoceptor with lower affinity (beta1LAR) compared to the site through which (-)-CGP12177 antagonizes the effects of catecholamines (beta1HAR). However, in a recent report it was proposed that the positive inotropic effects of CGP12177 are mediated through beta3-adrenoceptors (Skeberdis et al 2008). We therefore investigated whether the effects of (-)-CGP12177 on human atrial trabeculae are antagonized by the beta3-adrenoceptor-selective antagonist L-748,337 (1 microM). (-)-CGP12177 (200 nM) caused a stable increase in force which was significantly reduced by the addition of (-)-bupranolol (1 microM), P = 0.002, (basal 4.45 ± 0.78 mN, IBMX (PDE inhibitor) 5.47 ± 1.01 mN, (-)-CGP12177 9.34 ± 1.33 mN, (-)-bupranolol 5.79 ± 1.08 mN, n = 6) but not affected by the addition of L-748,337 (1 microM), P = 0.12, (basal 4.48 ± 1.32 mN, IBMX 7.15 ± 2.28 mN, (-)-CGP12177 12.51 ± 3.71 mN, L-748,337 10.90 ± 3.49 mN, n = 6). Cumulative concentration-effect curves for (-)-CGP12177 were not shifted to the right by L-748,337 (1 microM). The –logEC50M values of (-)-CGP12177 in the absence and presence of L-748,337 were 7.21±0.09 and 7.41±0.13, respectively (data from 25 trabeculae from 8 patients, P=0.2) The positive inotropic effects of (-)-CGP12177 (IBMX present) were not antagonized by L-748,337 but were blunted by (-)-bupranolol (1 microM). The results rule out an involvement of beta3-adrenoceptors in the positive inotropic effects (-)-CGP12177 in human right atrial myocardium and are consistent with mediation through beta1LAR. Kaumann A and Molenaar P (2008) Pharmacol Ther 118, 303-336 Skeberdis VA et al (2008) J Clin Invest, 118, 3219-3227