Novel lysine-based reducible copolymers for intracellular gene delivery

The development of biodegradable gene delivery systems, which have the ability to effectively deliver therapeutic DNA to a target tissue, is paramount to the success of nonviral gene delivery. One approach to developing biodegradable polymers is to introduce disulfide bonds along the backbone of the polymers to ensure release of the DNA in the reductive environment of the cytoplasm, whilst simultaneously reducing the molecular weight of the polymers. There is a crucial need to develop biocompatible and biodegradable polymers, which have low cytotoxicities so as to maintain cell viability and hence increase transfection efficiencies. Therefore, to produce a biocompatible gene delivery system, we have designed and synthesized novel reducible copolymers of the type (AB)n, which consist of repeating units of the natural amino acid, L-lysine and cystamine bisacrylamide (CBA). These novel reducible linear L-lysine copolymers (LLCs) were then modified with ethylenediamine so as to introduce primary amines for efficient DNA condensation. The molecular weight (MW) of the copolymers was found to be ~3.2 kDa with a polydispersity index of ~1.2. Gel retardation assays showed complete condensation of DNA at N/P ratios greater than 20/1 and exceptional LLC/pDNA polyplex stability during incubation with DNase I. To investigate the mechanism of DNA release from the polymer/pDNA complexes, fluorescence spectroscopy studies were performed with 1,4-dithio-DL-threitol (DTT). These data showed a significant reduction in fluorescence intensity following the addition of LLCs to DNA. After the addition of DTT, there was a 95 % increase in fluorescence intensity, which indicated the reduction of the disulfide bonds and the release of the DNA from the complexes. The particle sizes of LLC/pDNA polyplexes were found to be between 100-231 nm with surface charges of 0.8-17 mV respectively. The transfection efficiencies of the polyplexes as determined with a luciferase assay showed that LLC polyplexes produced five times higher transfection efficiencies in HDF cells, three times higher transfection efficiencies in MCF-7 cells, and four times higher transfection efficiencies in MA cells as compared to the optimal PLL control. The LLC/pDNA polyplexes showed significantly lower cytotoxicities as compared to the PLL/pDNA control in HDF, MCF-7, and MA cells at certain N/P ratios. Finally, in an exvivo study, LLCs were used as a nonviral gene carrier system to generate genetically modified stem cells to produce sufficient amounts of the angiogenic cytokine, vascular endothelial growth factor (VEGF165). These genetically modified stem cells were used to promote revascularization of an infarcted region of the heart, which can reduce myocardial damage and scar formation. A myocardial infarction model was generated in SCID mice deficient in T and B cells by permanent ligation of the left anterior descending coronary (LAD) artery. Cardiac hemodynamics, H&E staining and immunohistostaining results from this ex vivo study presented improved cardiac contractility, potential differentiation of hMSCs, new blood vessel formation, and a reduction in infarct size after treatment with the LLC genetically modified stem cells compared to the control animals. In conclusion, these results suggest that these novel LLCs are efficient, reducible and biocompatible polymers for nonviral gene delivery. Moreover, LLCs, as a nonviral gene carrier vector, hold great potential for the treatment of myocardial infarction in conjunction with stem cell therapy. Finally, adoption of novel nano-therapeutics strategies and techniques combining gene and cell therapies together could open the gate towards endless possibilities in the future of therapeutics and medicine.Pharmacological and Pharmaceutical Sciences, Department o

Is active targeting of brain metastases of breast cancer superior to passive targeting?

Brain metastasis is a major cause of death in patients with solid cancers. Breast cancer cells have high tendency to migrate towards brain. Cancer cells within brain are characterized by severe aggressiveness and inaccessibility. Currently, breast cancer and its metastasis are the second leading cause of death among women. Tumor microenvironment and blood brain barrier (BBB) represent great obstacles in targeting breast cancer and its metastasis. Chemotherapy is a safer treatment modality for brain metastasis compared with risky surgical resection and brain radiotherapy. Unfortunately, conventional chemotherapy lack penetration of BBB and suffer from multiple resistance mechanisms. Current treatment technologies for brain metastases of breast cancer have limited long-term success and numerous side effects, illustrating the urgent need for novel smart strategies. Various novel drug entities and nanosystems have been employed to improve diagnosis and targeted treatment of breast cancer and its metastasis. Immunotherapy agents and small tyrosine kinase inhibitors have been shown to reduce tumor size and increase survival in patients with breast cancer, but still poorly penetrate BBB. Tailored sized nanoparticles to some extent crossed brain tumor barrier and enhanced drug accumulation in tumors by taking advantage of enhanced permeability and retention. Furthermore, various active targeting strategies have been adopted to improve accessibility to brain malignancies. Therefore, to achieve enhanced antitumor therapy against breast cancer and its brain metastasis, multi-talented delivery systems are urgently needed for optimal treatment. This review focuses on the various active and passive targeting technologies for the treatment of breast cancer brain metastases in the past decade. A comprehensive summary and examples along with pros and cons of each system will be discussed. Different treatment modalities and nanotechnology facilities will be demonstrated to aid in designing the optimal smart, safe, targeted and effective systems to combat brain metastases of breast cancer

Adulteration and Counterfeiting of Online Nutraceutical Formulations in the United States: Time for Intervention?

<p>Global prevalence of nutraceuticals is noticeably high. The American market is flooded with nutraceuticals claiming to be of natural origin and sold with a therapeutic claim by major online retail stores such as Amazon and eBay. The objective of this commentary is to highlight the possible problems of online-sold nutraceuticals in the United States with respect to claim, adulterants, and safety. Furthermore, there is a lack of strict regulatory laws governing the sales, manufacturing, marketing, and label claims of nutraceutical formulations currently sold in the U.S. market. Major online retail stores and Internet pharmacies aid the widespread sale of nutraceuticals. Finally, according to the literature, many of these products were found to be either counterfeit or adulterated with active pharmaceutical ingredients (API) and mislabeled as being safe and natural. Therefore, regulatory authorities along with the research community should intervene to draw attention to these products and their possible effects.</p

P-glycoprotein Mediated Efflux Limits Substrate and Drug Uptake in a Preclinical Brain Metastases of Breast Cancer Model

The blood-brain barrier (BBB) is a specialized vascular interface that restricts the entry of many compounds into brain. This is accomplished through the sealing of vascular endothelial cells together with tight junction proteins to prevent paracellular diffusion. In addition, the BBB has a high degree of expression of numerous efflux transporters which actively extrude compounds back into blood. However, when a metastatic lesion develops in brain the vasculature is typically compromised with increases in passive permeability (blood-tumor barrier; BTB). What is not well documented is to what degree active efflux retains function at the BTB despite the changes observed in passive permeability. In addition, there have been previous reports documenting both increased and decreased expression of P-gp in lesion vasculature. Herein, we simultaneously administer a passive diffusion marker (14C-AIB) and a tracer subject to P-gp efflux (rhodamine 123) into a murine preclinical model of brain metastases of breast cancer. We observed that the metastatic lesions had similar expression (p&gt;0.05; n=756-1214 vessels evaluated) at the BBB and the BTB. Moreover, tissue distribution of R123 was not significantly (p&gt;0.05) different between normal brain and the metastatic lesion. It is possible that the similar expression of P-gp on the BBB and the BTB contribute to this phenomenon. Additionally we observed P-gp expression at the metastatic cancer cells adjacent to the vasculature which may also contribute to reduced R123 uptake into the lesion. The data suggest that despite the disrupted integrity of the BTB, efflux mechanisms appear to be intact, and may be functionally comparable to the normal BBB. The BTB is a significant hurdle to delivering drugs to brain metastasis