Uptake of plasmid-loaded nanoparticles in breast cancer cells and effect on Plk1 expression.

The development of nucleic acid-based drugs for cancer therapeutic application has shown promising results in the past. However the delivery of these drugs to target cells is one problem which remains to be resolved. Nanoparticles have been described as promising strategies to deliver drugs into target cells. Human serum albumin (HSA) nanoparticles conjugated to trastuzumab for a cell type-specific targeting of human epidermal growth factor receptor 2 (HER2)-overexpressing cells were developed with incorporated expression plasmids for small hairpin RNAs (shRNAs) targeting polo-like kinase 1 (Plk1). Plk1 is a promising target for such an approach because it is overexpressed in all known cancer types and is a negative prognostic factor. Receptor-mediated uptake of the trastuzumab-modified nanoparticles into HER2-positive cells could be observed leading to reduced Plk1 expression. Taken together, HSA nanoparticles represent promising tools to deliver expression plasmids for shRNAs into target cells and should be further evaluated with regard to a therapeutic application of RNA interference in cancer therapy.

Effect of trastuzumab-modified antisense oligonucleotide-loaded human serum albumin nanoparticles prepared by heat denaturation.

Nanoparticles represent a promising tool for targeted drug delivery to tumour cells and are able to protect drugs against degradation. In our present study we developed targeted nanoparticles loaded with antisense oligonucleotides (ASOs) against Plk1 (polo-like kinase 1) prepared by heat denaturation instead of using glutaraldehyde. Glutaraldehyde can lead to an inactivation of ASOs through chemical crosslinking and is a toxic entity. We examined the ideal preparation conditions and characterised the resulting particles in terms of physico-chemical properties, ASO recovery after enzymatic degradation and stability. Stable monodisperse nanoparticles with an ASO recovery of more than 80% could be prepared at a temperature of 105 degrees C for 10 min. Furthermore we performed quantitative real-time PCR and Western blot to detect an ASO-mediated effect on Plk1 in BT-474 cells. We observed a significant reduction of Plk1 mRNA and protein expression. Thus, this is the first report of ASO-loaded HSA nanoparticles prepared by heat denaturation, where an impact on gene expression could be observed. The data provide the basis for the further development of carrier systems for ASOs to reduce off-target effects evoked by systemically administered ASOs and to achieve a better penetration into primary and metastatic target cells.

Downregulation of Plk1 expression by receptor-mediated uptake of antisense oligonucleotide-loaded nanoparticles.

Human serum albumin (HSA) nanoparticles represent a promising tool for targeted drug delivery to tumor cells. The coupling of the antibody trastuzumab to nanoparticles uses the capability of human epidermal growth factor receptor 2 (HER2)-positive cells to incorporate agents linked to HER2. In our present study, we developed targeted nanoparticles loaded with antisense oligonucleotides (ASOs) against polo-like kinase 1 (Plk1). We evaluated the receptor-mediated uptake into HER2-positive and -negative breast cancer and murine cell lines. We performed quantitative real-time PCR and Western blot analyses to monitor the impact on Plk1 expression in HER2-positive breast cancer cells. Antibody-conjugated nanoparticles showed a specific targeting to HER2-overexpressing cells with cellular uptake by receptor-mediated endocytosis and a release into HER2-positive BT-474 cells. We observed a significant reduction of Plk1 mRNA and protein expression and increased activation of Caspase 3/7. Thus, this is the first report about ASO-loaded HSA nanoparticles, where an impact on gene expression could be observed. The data provide the basis for the further development of carrier systems for Plk1-specific ASOs to reduce off-target effects evoked by systemically administered ASOs and to achieve a better penetration into primary and metastatic target cells. Treatment of tumors using trastuzumab-conjugated ASO-loaded HSA nanoparticles could be a promising approach to reach this goal.

Trastuzumab-modified nanoparticles: optimisation of preparation and uptake in cancer cells.

Nanoparticles consisting of human serum albumin (HSA) represent a promising strategy for targeted drug delivery to tumour cells. The coupling of the antibody trastuzumab to HSA nanoparticles takes advantage of the capability of HER2-positive cells to incorporate substances binding to HER2. In our present study, we developed nanoparticles based on HSA which were covalently modified on their surface with thiolated trastuzumab. A special focus was on the optimisation of the thiolation procedure of the antibody under the aspect of an effective binding to particle surfaces. Different thiolation conditions were evaluated and the degree of antibody dimerisation was determined. We analysed the thiolated antibody by size exclusion chromatography (SEC) and identified the best thiolation procedure for the preparation of trastuzumab-conjugated nanoparticles. Over a storage period of 6 weeks the resulting particles were stable and physico-chemical properties such as size and zetapotential did not show any changes. Biological activity was confirmed under cell culture conditions: antibody-conjugated nanoparticles showed a specific targeting to HER2-overexpressing cells with cellular uptake by receptor-mediated endocytosis. These data provide the basis for the development of stable and biological active carrier systems for directed targeting of tumour cells using trastuzumab-conjugated HSA nanoparticles.