Micro- and macro-viscosity relations in high concentration antibody solutions.

Molecular crowding in highly concentrated monoclonal antibody (mAb) solutions results in significant increases in viscosity, which complicates fill-finish steps and patient administration by subcutaneous injection. As viscosity measurements for optimization of the mAb formulation require significant amounts of material not always available in early development, fluorescence correlation spectroscopy (FCS) is evaluated as a potential ultra-low volume technique for viscosity measurement of high concentration protein solutions assuming the Generalised Stokes Einstein relation (GSE) remains valid. Using like-charge fluorescent tracers of different sizes, FCS provided measurements of microviscosities which were compared to the macroviscosity. After parametrising the protein concentration dependence of the viscosity by the exponential coefficient (k) of a simple exponential model, FCS derived k-values of like-size tracer to the crowder followed the same ordering as the macroviscosity derived k-values with respect to solvent conditions. Furthermore, k and the diffusion-derived protein-protein interaction parameter, kD, are linked, and, attractive conditions for mAbs result in a stronger concentration dependence of the viscosity. For tracers and crowders of like-size, a key result is negative deviations from the GSE relation are observed in presence of strong attractive interactions between crowder molecules. These data demonstrate that FCS has application to the screening of high concentration mAb solutions for formulation selection.

Individual microflora beget unique oral microcosms.

AIMS: To examine the efficacy of the multiple Sorbarod device (MSD) for the reproduction of inter-individual variations in oral microbiotas. The MSD supports sessile growth on parallel cellulose filters, perfused with artificial saliva. This enables biofilms (BF) to be grown and sampled, together with released cells in eluted medium (perfusates, PAs). METHODS AND RESULTS: Two sets of triplicate MSDs were established. One set was inoculated using fresh saliva from three separate volunteers; the second set was inoculated from one saliva donor. Both were incubated in an anaerobic cabinet. BF and PA were analysed at 24-h intervals by PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rDNA. Hierarchical dendrograms were constructed in order to sort community fingerprints over time, based on community relatedness. The MSD supported complex oral communities, as evidenced by DGGE (>20 distinct DGGE bands) and confocal scanning laser microscopy. DGGE band sequencing revealed bacteriological diversity and a high incidence of anaerobic species, including Prevotella sp. Dendrograms demonstrated marked inter-individual variation in the relative species abundance within salivary inocula from different volunteers (DV) and each associated MSD (all >45%, majority c. 85% concordance). Less variation was shown between triplicate models established using saliva from a single volunteer (SV) (all >58%; majority c. 95% concordance). PAs clustered together with the associated biofilms and inocula in the majority of cases for the DV MSDs whilst SV MSD community profiles clustered between replicate MSDs. CONCLUSIONS: Data indicate that marked inter-individual variations in human salivary composition can be partially replicated in individualized MSD microcosms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the in vitro reproduction of individual oral microbiotas and suggests that taking inter-individual variability into account will increase the relevance of microcosm studies.

Comparison of IgG diffusion and extracellular matrix composition in rhabdomyosarcomas grown in mice versus in vitro as spheroids reveals the role of host stromal cells.

The tumour extracellular matrix acts as a barrier to the delivery of therapeutic agents. To test the hypothesis that extracellular matrix composition governs the penetration rate of macromolecules in tumour tissue, we measured the diffusion coefficient of nonspecific IgG in three rhabdomyosarcoma subclones growing as multicellular spheroids in vitro or as subcutaneous tumours in dorsal windows in vivo. In subcutaneous tumours, the diffusion coefficient decreased with increasing content of collagen and sulphated glycosaminoglycans. When grown as multicellular spheroids, no differences in either extracellular matrix composition or diffusion coefficient were found. Comparison of in vitro vs in vivo results suggests an over-riding role of host stromal cells in extracellular matrix production subjected to modulation by tumour cells. Penetration of therapeutic macromolecules through tumour extracellular matrix might thus be largely determined by the host organ. Hence, caution must be exercised in extrapolating drug penetrability from spheroids and multilayer cellular sandwiches consisting of only tumour cells to tumours in vivo.

Solid stress facilitates spheroid formation: potential involvement of hyaluronan.

When neoplastic cells grow in confined spaces in vivo, they exert a finite force on the surrounding tissue resulting in the generation of solid stress. By growing multicellular spheroids in agarose gels of defined mechanical properties, we have recently shown that solid stress inhibits the growth of spheroids and that this growth-inhibiting stress ranges from 45 to 120 mmHg. Here we show that solid stress facilitates the formation of spheroids in the highly metastatic Dunning R3327 rat prostate carcinoma AT3.1 cells, which predominantly do not grow as spheroids in free suspension. The maximum size and the growth rate of the resulting spheroids decreased with increasing stress. Relieving solid stress by enzymatic digestion of gels resulted in gradual loss of spheroidal morphology in 8 days. In contrast, the low metastatic variant AT2.1 cells, which grow as spheroids in free suspension as well as in the gels, maintained their spheroidal morphology even after stress removal. Histological examination revealed that most cells in AT2.1 spheroids are in close apposition whereas a regular matrix separates the cells in the AT3.1 gel spheroids. Staining with the hyaluronan binding protein revealed that the matrix between AT3.1 cells in agarose contained hyaluronan, while AT3.1 cells had negligible or no hyaluronan when grown in free suspension. Hyaluronan was found to be present in both free suspensions and agarose gel spheroids of AT2.1. We suggest that cell-cell adhesion may be adequate for spheroid formation, whereas solid stress may be required to form spheroids when cell-matrix adhesion is predominant. These findings have significant implications for tumour growth, invasion and metastasis.

Role of tumor-host interactions in interstitial diffusion of macromolecules: cranial vs. subcutaneous tumors.

The large size of many novel therapeutics impairs their transport through the tumor extracellular matrix and thus limits their therapeutic effectiveness. We propose that extracellular matrix composition, structure, and distribution determine the transport properties in tumors. Furthermore, because the characteristics of the extracellular matrix largely depend on the tumor-host interactions, we postulate that diffusion of macromolecules will vary with tumor type as well as anatomical location. Diffusion coefficients of macromolecules and liposomes in tumors growing in cranial windows (CWs) and dorsal chambers (DCs) were measured by fluorescence recovery after photobleaching. For the same tumor types, diffusion of large molecules was significantly faster in CW than in DC tumors. The greater diffusional hindrance in DC tumors was correlated with higher levels of collagen type I and its organization into fibrils. For molecules with diameters comparable to the interfibrillar space the diffusion was 5- to 10-fold slower in DC than in CW tumors. The slower diffusion in DC tumors was associated with a higher density of host stromal cells that synthesize and organize collagen type I. Our results point to the necessity of developing site-specific drug carriers to improve the delivery of molecular medicine to solid tumors.

Diffusion of macromolecules in agarose gels: comparison of linear and globular configurations.

The diffusion coefficients (D) of different types of macromolecules (proteins, dextrans, polymer beads, and DNA) were measured by fluorescence recovery after photobleaching (FRAP) both in solution and in 2% agarose gels to compare transport properties of these macromolecules. Diffusion measurements were conducted with concentrations low enough to avoid macromolecular interactions. For gel measurements, diffusion data were fitted according to different theories: polymer chains and spherical macromolecules were analyzed separately. As chain length increases, diffusion coefficients of DNA show a clear shift from a Rouse-like behavior (DG congruent with N0-0.5) to a reptational behavior (DG congruent with N0-2.0). The pore size, a, of a 2% agarose gel cast in a 0.1 M PBS solution was estimated. Diffusion coefficients of the proteins and the polymer beads were analyzed with the Ogston model and the effective medium model permitting the estimation of an agarose gel fiber radius and hydraulic permeability of the gels. Not only did flexible macromolecules exhibit greater mobility in the gel than did comparable-size rigid spherical particles, they also proved to be a more useful probe of available space between fibers.

Migration of single-stranded DNA in polyacrylamide gels during electrophoresis.

By using fluorescence recovery after photobleaching (FRAP) and electric birefringence, the migration of single-stranded DNA in polyacrylamide gels and orientation as a response to an electric pulse were investigated. Electrophoretic mobility is in good agreement with the model of biased reptation including fluctuations. The determination of the electrophoretic mobility in solution, mu0, allows an estimation of the gel pore diameter seen by the molecule. As previously observed for double-stranded DNA, the electric birefringence results from two processes: the alignment of the molecule along the electric field and the elongation of the primitive path in the gel, for long single-stranded DNA (>2000 bases). The combination of results obtained with the two techniques allows us to propose experimental conditions to improve the separation of single-stranded DNA with pulsed field techniques.

Band broadening in gel electrophoresis: scaling laws for the dispersion coefficient measured by FRAP.

We determined quantitatively the band broadening effect during gel electrophoresis by measuring the longitudinal dispersion coefficient Dx, with a fluorescence recovery after photobleaching setup, coupled to an electrophoretic cell. We carried out measurements as a function of the electric field, the average pore size, and the molecular length of DNA fragments. Our results are in good agreement with the predictions of the biased reptation model with fluctuations described by T. A. Duke et al. [(1992) Physics Review Letters, vol. 69, pp. 3260-3263]. This agreement is observed on single-stranded DNA [persistence length approximately equal to 4 nm; B. Tinland et al. (1997) Macromolecules, vol. 30, pp. 5763-5765] in polyacrylamide gels and on double-stranded DNA (persistence length approximately equal to 50 nm) in agarose gels, two systems where the ratio between the average pore size and the Kuhn length is larger than 1.

Dynamics of single-stranded DNA in polyacrylamide gels during pulsed field gel electrophoresis. A birefringence study.

The study of the orientation of single-stranded DNA in polyacrylamide gels in denaturing conditions has been undertaken by electric birefringence in order to determine the mechanism involved in the electrophoretic transport. The presence of an overshoot in the birefringence signal, when applying the electric field, and the study of the influences of the electric field and of the gel concentration on the dynamics show that a mechanism of reptation with elongation of the molecule occurs in polyacrylamide gels with low T values. Therefore it is suggested that the use of pulsed fields in sequencing electrophoresis is possible and can lead to a large increase of the length of the fragments that can be sequenced in one single run.