Modeling the effect of intercalative particle binding on the overstretching transition of double-stranded DNA

DNA is best known from the perspective of genetics, but its mechanical properties are also an interesting and important field of study. These mechanical properties play an important role in cellular processes such as replication and transcription. To characterize the mechanics of DNA, physicists measure force-extension curves of individual double-stranded DNA molecules. They observe that the DNA molecule cooperatively overstretches to a length 1.7 times longer than B-DNA at a well-defined force of about 65 pN: the overstretching transition. To visualize these DNA mechanics, fluorescent molecules are bound to the DNA via the process of intercalation. However, these intercalators are known to perturb the DNA structure and thus change the features of the force-extension curve. In particular, the overstretching transition is found experimentally to shift to higher forces than 65 pN, as a function of intercalator concentration. In this work, we develop multi-state freely jointed chain models to gain an understanding of the physical principles behind the effect of intercalative particle binding on the overstretching transition of double-stranded DNA. We show that a freely jointed chain like model with three possible segment lengths reproduces experimental force-extension curves, and that this model captures the physical principles behind the effect of intercalation on the force-extension curve. The three segment lengths represent B-DNA, overstretched DNA, and intercalated DNA. Moreover, our model agrees quantitatively with the experimentally found linear dependence of the overstretching force on the intercalator concentration. Finally, our theory predicts a further elongation to twice the length of B-DNA, induced by intercalative binding at every base pair, in the force-regime beyond the overstretching transition. DNA is best known from the perspective of genetics, but its mechanical properties are also an interesting and important field of study. These mechanical properties play an important role in cellular processes such as replication and transcription. To characterize the mechanics of DNA, physicists measure force-extension curves of individual double-stranded DNA molecules. They observe that the DNA molecule cooperatively overstretches to a length 1.7 times longer than B-DNA at a well-defined force of about 65 pN: the overstretching transition. To visualize these DNA mechanics, fluorescent molecules are bound to the DNA via the process of intercalation. However, these intercalators are known to perturb the DNA structure and thus change the features of the force-extension curve. In particular, the overstretching transition is found experimentally to shift to higher forces than 65 pN, as a function of intercalator concentration. In this work, we develop multi-state freely jointed chain models to gain an understanding of the physical principles behind the effect of intercalative particle binding on the overstretching transition of double-stranded DNA. We show that a freely jointed chain like model with three possible segment lengths reproduces experimental force-extension curves, and that this model captures the physical principles behind the effect of intercalation on the force-extension curve. The three segment lengths represent B-DNA, overstretched DNA, and intercalated DNA. Moreover, our model agrees quantitatively with the experimentally found linear dependence of the overstretching force on the intercalator concentration. Finally, our theory predicts a further elongation to twice the length of B-DNA, induced by intercalative binding at every base pair, in the force-regime beyond the overstretching transition

Enzymatic activity toward poly(L-lactic acid) implants

Tissue reactions toward biodegradable poly(L-lactic acid) implants were monitored by studying the activity pattern of seven enzymes as a function of time: alkaline phosphatase, acid phosphatase, -naphthyl acetyl esterase, -glucuronidase, ATP-ase, NADH-reductase, and lactate dehydrogenase. Cell types were identified by their specific enzyme patterns, their morphology and location. Special attention was paid to the enzyme patterns of macrophages, fibroblasts and polymorphonuclear granulocytes (PMNs), being involved in foreign body reactions or inflammatory responses. One day after implantation, an influx of neutrophilic and eosinophilic granulocytes was observed, coinciding with activity of alkaline phosphatase (PMN's) and -glucuronidase (eosinophils). From day 3 on, macrophages containing ATP-ase, acid phosphatase and esterase could be observed. From day 7 on, lactate dehydrogenase, the enzyme normally involved in the conversion of lactic acid, and its coenzyme NADH-reductase were observed in macrophages and fibroblasts. These two enzymes demonstrated more activity than expected on basis of wound-healing reactions upon implantation of a nonbiodegradable, inert biomaterial (as, e.g., Teflon). It is concluded that the biodegradable poly (L-lactic acid) used in these implantation studies is tissue compatible, and evokes a foreign body reaction with minor macrophage and giant cell activity, as observed during this 3-week implantation period. Most enzyme patterns were simply due to a wound-healing reaction. The slightly increased levels of LDH and NADH suggest the release of lactic acid from the implant, and thus confirms the biodegradable nature of this polymer

The effect of phagocytosis of poly(L-lactic acid) fragments on cellular morphology and viability

The aim of this study was to investigate the effect of phagocytosed poly(L-lactic acid) particles on the morpholgy and viability of phagocytes, mainly macrophages. Therefore, predegraded poly(L-lactic acid) (P-PLLA) and nontreated PLLA (N-PLLA) particles, both having diameters not exceeding 38 µm, were injected intraperitoneally in mice. P-PLLA particles were obtained by 25 kGy γ-irradiation of N-PLLA particles. N-PLLA and P-PLLA particles were injected using an 0.3% ethanol/0.9% saline solution intraperitoneally to the mice. We also studied the release of the absorbed ethanol as a possible model for the release of low molecular weight, potentially toxic products. As control, nondegradable polytetrafluoroethylene (PTFE) particles and the carrier solution were used. After 1, 2, 3, 4, 5, and 7 days, the cells of the abdominal cavity were harvested to study the effect of phagocytosis of polymer particles on phagocytic cell morphology and viability. Studies with transmission electron microscopy indicated that, upon injection of particles in the peritoneal cavity, macrophages demonstrated signs of cell damage, cell death, and cell lysis due to phagocytosis of a large amount of P-PLLA particles. The morphology of the cells that had phagocytosed the N-PLLA and PTFE particles did not differ substantially from those of control animals in which only the solution was injected. Also, in the controls, hardly any cell death and no debris was observed. When the PLLA particles were injected as a suspension in a 0.3% ethanol/0.9% saline solution, no difference was observed between N-PLLA and P-PLLA. After phagocytosis, both cause cell damage, sometimes leading to cell death. The highest numbers of necrotic cells were observed on day 2. The effects could be caused by the (peak) release of degradation products from P-PLLA fragments or by the release of the absorbed ethanol when the 0.3 ethanol/0.9 saline solution was used to administer the particles. In conclusion, it can be stated that cell damage, sometimes leading to cell death, may be caused by phagocytosed poly(L-lactic acid) particles

Biodegradable hollow fibres for the controlled release of drugs

Biodegradable hollow fibres of poly-l-lactic acid (PLLA) filled with a suspension of the contraceptive hormone levonorgestrel in castor oil were implanted subcutaneously in rats to study the rate of drug release, rate of biodegradation and tissue reaction caused by the implant. The in vivo drug release was compared with the release in vitro using different release media. Fibres, disinfected with alcohol showed a zero-order release, both in vitro and in vivo, for over 6 months. Fibres, either γ-sterilized or disinfected with alcohol were harvested at time intervals ranging from 1 d to 6 months after implantation. Molecular weights of PLLA, tensile strengths, and remaining amounts of drug were determined as a function of time.\ud \ud The tissue reaction can be described as a very moderate foreign body reaction with the initial presence of macrophages, which are gradually replaced by fibroblasts which form a collagen capsule. Molecular weight determinations of PLLA showed a decrease from an initial Mw of 1.59x10 5 to 5.5 × 10 4 in 4 months (after alcohol sterilization). A gradual decrease in fibre strength with time was observed which did not significantly impair the release rate of levonorgestrel

Modeling the effect of intercalators on the high-force stretching behavior of DNA

DNA is structurally and mechanically altered by the binding of intercalator molecules. Intercalation strongly affects the force-extension behavior of DNA, in particular the overstretching transition. We present a statistical model that captures all relevant findings of recent force-extension experiments. Two predictions from our model are presented. The first suggests the existence of a novel hyper-stretching regime in the presence of intercalators and the second, a linear dependence of the overstretching force on intercalator concentration, is verified by re-analyzing available experimental data. Our model pins down the physical principles that govern intercalated DNA mechanics, providing a predictive understanding of its limitations and possibilities.Comment: 5 pages, 4 figure