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1.
J Control Release ; 132(3): 260-6, 2008 Dec 18.
Article in English | MEDLINE | ID: mdl-18662730

ABSTRACT

The number one cause of mortality in the US is cardiovascular related disease. Future predictions do not see a reduction in this rate especially with the continued rise in obesity [P. Poirier, et al., Obesity and cardiovascular disease: pathophysiology, evaluation, and effect of weight loss, Arterioscler Thromb Vasc Biol. 26(5), (2006) 968-976.; K. Obunai, S. Jani, G.D. Dangas, Cardiovascular morbidity and mortality of the metabolic syndrome, Med.Clin. North Am., 91(6), (2007) 1169-1184]. Even so, potential molecular therapeutic targets for cardiac gene delivery are in no short supply thanks to continuing advances in molecular cardiology. However, efficient and safe delivery remains a bottleneck in clinical gene therapy [O.J. Muller, H.A. Katus, R. Bekeredjian, Targeting the heart with gene therapy-optimized gene delivery methods, Cardiovasc Res, 73(3), (2007) 453-462]. Viral vectors are looked upon favorably for their high transduction efficiency, although their ability to elicit toxic immune responses remains [C.F. McTiernan, et al., Myocarditis following adeno-associated viral gene expression of human soluble TNF receptor (TNFRII-Fc) in baboon hearts, Gene Ther, 14(23), (2007) 1613-1622]. However, this high transduction does not necessarily translate into improved efficacy [X. Hao, et al., Myocardial angiogenesis after plasmid or adenoviral VEGF-A(165) gene transfer in rat myocardial infarction model, Cardiovasc Res., 73(3), (2007) 481-487]. Naked DNA remains the preferred method of DNA delivery to cardiac myocardium and has been explored extensively in clinical trials. The results from these trials have demonstrated efficacy in regard to secondary end-points of reduced symptomatology and perfusion, but have failed to establish significant angiogenesis or an increase in myocardial function [P.B. Shah, D.W. Losordo, Non-viral vectors for gene therapy: clinical trials in cardiovascular disease, Adv Genet, 54, (2005) 339-361]. This may be due in part to reduced transfection efficiency but can also be attributed to use of suboptimal candidate genes. Currently, polymeric non-viral gene delivery to cardiac myocardium remains underrepresented. In the past decade several advances in non-viral vector development has demonstrated increased transfection efficiency [O.J. Muller, H.A. Katus, R. Bekeredjian, Targeting the heart with gene therapy-optimized gene delivery methods, Cardiovasc Res, 73(3), (2007) 453-462]. Of these polymers, those that employ lipid modifications to improve transfection or target cardiovascular tissues have proven themselves to be extremely beneficial. Water-soluble lipopolymer (WSLP) consists of a low molecular weight branched PEI (1800) and cholesterol. The cholesterol moiety adds extra condensation by forming stable micellular complexes and was later employed for myocardial gene therapy to exploit the high expression of lipoprotein lipase found within cardiac tissue. Use of WSLP to deliver hypoxia-responsive driven expression of hVEGF to ischemic rabbit myocardium has proven to provide for even better expression in cardiovascular cells than Terplex and has demonstrated a significant reduction in infarct size (13+/-4%, p<0.001) over constitutive VEGF expression (32+/-7%, p=0.007) and sham-injected controls (48+/-7%). A significant reduction in apoptotic values and an increase in capillary growth were also seen in surrounding tissue. Recently, investigations have begun using bioreducible polymers made of poly(amido polyethylenimines) (SS-PAEI). SS-PAEIs breakdown within the cytoplasm through inherent redox mechanisms and provide for high transfection efficiencies (upwards to 60% in cardiovascular cell types) with little to no demonstrable toxicity. In vivo transfections in normoxic and hypoxic rabbit myocardium have proven to exceed those results of WSLP transfections by 2-5 fold [L.V. Christensen, et al., Reducible poly(amido ethylenediamine) for hypoxia-inducible VEGF delivery, J Control Release, 118(2), (2007) 254-261]. This new breed of polymer(s) may allow for decreased doses and use of new molecular mechanisms not previously available due to low transfection efficiencies. Little development has been seen in the use of new gene agents for treatment of myocardial ischemia and infarction. Current treatment consists of using mitogenic factors, described decades earlier, alone or in combination to spur angiogenesis or modulating intracellular Ca2+ homeostasis through SERCA2a but to date, failed to demonstrate clinical efficacy. Recent data suggests that axonal guidance cues also act on vasculature neo-genesis and provide a new means of investigation for treatment.


Subject(s)
DNA/metabolism , Gene Transfer Techniques , Genetic Therapy/methods , Myocardial Infarction/therapy , Myocardial Ischemia/therapy , Myocardium/metabolism , Neovascularization, Physiologic/genetics , Polymers/chemistry , Animals , DNA/chemistry , Disease Models, Animal , Humans , Myocardial Infarction/genetics , Myocardial Infarction/metabolism , Myocardial Infarction/physiopathology , Myocardial Ischemia/genetics , Myocardial Ischemia/metabolism , Myocardial Ischemia/physiopathology , Polyethyleneimine/chemistry
2.
Biophys J ; 88(5): 3689-98, 2005 May.
Article in English | MEDLINE | ID: mdl-15722433

ABSTRACT

The relationship between the mechanical properties of cells and their molecular architecture has been the focus of extensive research for decades. The cytoskeleton, an internal polymer network, in particular determines a cell's mechanical strength and morphology. This cytoskeleton evolves during the normal differentiation of cells, is involved in many cellular functions, and is characteristically altered in many diseases, including cancer. Here we examine this hypothesized link between function and elasticity, enabling the distinction between different cells, by using a microfluidic optical stretcher, a two-beam laser trap optimized to serially deform single suspended cells by optically induced surface forces. In contrast to previous cell elasticity measurement techniques, statistically relevant numbers of single cells can be measured in rapid succession through microfluidic delivery, without any modification or contact. We find that optical deformability is sensitive enough to monitor the subtle changes during the progression of mouse fibroblasts and human breast epithelial cells from normal to cancerous and even metastatic state. The surprisingly low numbers of cells required for this distinction reflect the tight regulation of the cytoskeleton by the cell. This suggests using optical deformability as an inherent cell marker for basic cell biological investigation and diagnosis of disease.


Subject(s)
Biomarkers, Tumor , Cell Transformation, Neoplastic , 3T3 Cells , Actins/metabolism , Animals , Biomarkers , Breast Neoplasms/diagnosis , Breast Neoplasms/pathology , Cell Line , Cell Line, Tumor , Cell Shape , Cytoskeleton/metabolism , Disease Progression , Elasticity , Epithelial Cells/metabolism , Fibroblasts/metabolism , Humans , Lasers , Mice , Mice, Inbred BALB C , Microscopy, Confocal , Microscopy, Fluorescence , Neoplasm Metastasis
3.
Cytometry A ; 59(2): 203-9, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15170599

ABSTRACT

BACKGROUND: Elasticity of cells is determined by their cytoskeleton. Changes in cellular function are reflected in the amount of cytoskeletal proteins and their associated networks. Drastic examples are diseases such as cancer, in which the altered cytoskeleton is even diagnostic. This connection between cellular function and cytoskeletal mechanical properties suggests using the deformability of cells as a novel inherent cell marker. METHODS: The optical stretcher is a new laser tool capable of measuring cellular deformability. A unique feature of this deformation technique is its potential for high throughput, with the incorporation of a microfluidic delivery of cells. RESULTS: Rudimentary implementation of the microfluidic optical stretcher has been used to measure optical deformability of several normal and cancerous cell types. A drastic difference has been seen between the response of red blood cells and polymorphonuclear cells for a given optically induced stress. MCF-10, MCF-7, and modMCF-7 cells were also measured, showing that while cancer cells stretched significantly more (five times) than normal cells, optical deformability could even be used to distinguish metastatic cancer cells from nonmetastatic cancer cells. This trimodal distribution was apparent after measuring a mere 83 cells, which shows optical deformability to be a highly regulated cell marker. CONCLUSIONS: Preliminary work suggests a deformability-based cell sorter similar to current fluorescence-based flow cytometry without the need for specific labeling. This could be used for the diagnosis of all diseases, and the investigation of all cellular processes, that affect the cytoskeleton.


Subject(s)
Breast Neoplasms/pathology , Erythrocyte Deformability , Erythrocytes/cytology , Erythrocytes/radiation effects , Flow Cytometry/methods , Cell Line , Cell Size/radiation effects , Cytoskeleton/radiation effects , Female , Flow Cytometry/instrumentation , Humans , Image Processing, Computer-Assisted/methods , Lasers , Predictive Value of Tests
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