Synthetic routes to nanoconjugates of anthracyclines.

Anthracyclines (Anth) are widely used in the treatment of various types of cancer. Unfortunately, they exhibit serious adverse effects, such as hematopoietic depression and cardiotoxicity, leading to heart failure. In this review, we focus on recently developed conjugates of anthracyclines with a range of nanocarriers, such as polymers, peptides, DNA or inorganic systems. Manipulation of the composition, size and shape of chemical entities at the nanometer scale makes possible the design and development of a range of prodrugs. In this review we concentrate on synthetic chemistry in the long process leading to the introduction of novel therapeutic products.

Molecular Mechanisms of Antitumor Activity of PAMAM Dendrimer Conjugates with Anticancer Drugs and a Monoclonal Antibody.

Taxanes are considered fundamental drugs in the treatment of breast cancer, but despite the similarities, docetaxel (doc) and paclitaxel (ptx) work differently. For this reason, it is interesting to identify mechanisms of antitumor activity of PAMAM dendrimer conjugates that carry docetaxel or paclitaxel and monoclonal antibody trastuzumab, specifically targeted to cells which overexpressed HER-2. For this purpose, the impact on the level of reactive oxygen species, the mitochondrial membrane potential, cell cycle distribution and the activity of caspases-3/7, -8 and -9 of PAMAM-doc-trastuzumab and PAMAM-ptx-trastuzumab conjugates was determined and compared with free docetaxel and paclitaxel toward HER-2-positive (SKBR-3) and negative (MCF-7) human breast cancer cell lines. Moreover, apoptosis and necrosis were studied using flow cytometry and confocal microscopy, respectively. Our studies show the complexity of the potential mechanism of cytotoxic action of PAMAM-drug-trastuzumab conjugates that should be sought as a resultant of oxidative stress, mitochondrial activation of the caspase cascade and the HER-2 receptor blockade.

Multicomponent Conjugates of Anticancer Drugs and Monoclonal Antibody with PAMAM Dendrimers to Increase Efficacy of HER-2 Positive Breast Cancer Therapy.

PURPOSE: Conjugation of nanocarriers with antibodies that bind to specific membrane receptors that are overexpressed in cancer cells enables targeted delivery. In the present study, we developed and synthesised two PAMAM dendrimer-trastuzumab conjugates that carried docetaxel or paclitaxel, specifically targeted to cells which overexpressed HER-2. METHODS: The 1H NMR, 13C NMR, FTIR and RP-HPLC were used to analyse the characteristics of the products and assess their purity. The toxicity of PAMAM-trastuzumab, PAMAM-doc-trastuzumab and PAMAM-ptx-trastuzumab conjugates was determined using MTT assay and compared with free trastuzumab, docetaxel and paclitaxel toward HER-2-positive (SKBR-3) and negative (MCF-7) human breast cancer cell lines. The cellular uptake and internal localisation were studied using flow cytometry and confocal microscopy, respectively. RESULTS: The PAMAM-drug-trastuzumab conjugates in particular showed extremely high toxicity toward the HER-2-positive SKBR-3 cells and very low toxicity towards to HER-2-negative MCF-7 cells. As expected, the HER-2-positive SKBR-3 cell line accumulated trastuzumab from both conjugates rapidly; but surprisingly, although a large amount of PAMAM-ptx-trastuzumab conjugate was observed in the HER-2-negative MCF-7 cells. Confocal microscopy confirmed the intracellular localisation of analysed compounds. The key result of fluorescent imaging was the identification of strong selective binding of the PAMAM-doc-trastuzumab conjugate with HER-2-positive SKBR-3 cells only. CONCLUSIONS: Our results confirm the high selectivity of PAMAM-doc-trastuzumab and PAMAM-ptx-trastuzumab conjugates for HER-2-positive cells, and demonstrate the utility of trastuzumab as a targeting agent. Therefore, the analysed conjugates present an promising approach for the improvement of efficacy of targeted delivery of anticancer drugs such as docetaxel or paclitaxel.

Sugar Modification Enhances Cytotoxic Activity of PAMAM-Doxorubicin Conjugate in Glucose-Deprived MCF-7 Cells - Possible Role of GLUT1 Transporter.

PURPOSE: In order to overcome the obstacles and side effects of classical chemotherapy, numerous studies have been performed to develop the treatment based on targeted transport of active compounds directly to the site of action. Since tumor cells are featured with intensified glucose metabolism, we set out to develop innovative, glucose-modified PAMAM dendrimer for the delivery of doxorubicin to breast cancer cells. METHODS: PAMAM-dox-glc conjugate was synthesized and characterized by 1H NMR, FT-IR, size and zeta potential measurements. The drug release rate from conjugate was evaluated by dialysis under different pH conditions. The expression level of GLUT family receptors in cells cultured in full and glucose-deprived medium was evaluated by quantitative real-time RT-PCR and flow cytometry. The cytotoxicity of conjugate in presence or absence of GLUT1 inhibitors was determined by MTT assay. RESULTS: We showed that PAMAM-dox-glc conjugate exhibits pH-dependent drug release and increased cytotoxic activity compared to free drug in cells cultured in medium without glucose. Further, we proved that these cells overexpress transporters of GLUT family. The toxic effect of conjugate was eliminated by the application of specific GLUT1 inhibitors. CONCLUSION: Our findings revealed that the glucose moiety plays a crucial role in the recognition of cells with high expression of GLUT receptors. By selectively blocking GLUT1 transporter we showed its importance for the cytotoxic activity of PAMAM-dox-glc conjugate. These results suggest that PAMAM-glucose formulations may constitute an efficient platform for the specific delivery of anticancer drugs to tumor cells overexpressing transporters of GLUT family.

Synthetic routes to nanomaterials containing anthracyclines: noncovalent systems.

Chemotherapy still constitutes a basic treatment for various types of cancer. Anthracyclines are effective antineoplastic drugs that are widely used in clinical practice. Unfortunately, they are characterized by high systemic toxicity and lack of tumour selectivity. A promising way to enhance treatment effectiveness and reduce toxicity is the synthesis of systems containing anthracyclines either in the form of complexes for the encapsulation of active drugs or their covalent conjugates with inert carriers. In this respect nanotechnology offers an extensive spectrum of possible solutions. In this review, we discuss recent advances in the development of anthracycline prodrugs based on nanocarriers such as copolymers, lipids, DNA, and inorganic systems. The review focuses on the chemical architecture of the noncovalent nanocarrier-drug systems.

Conjugate of PAMAM Dendrimer, Doxorubicin and Monoclonal Antibody-Trastuzumab: The New Approach of a Well-Known Strategy.

The strategy utilizing trastuzumab, a humanized monoclonal antibody against human epidermal growth receptor 2 (HER-2), as a therapeutic agent in HER-2 positive breast cancer therapy seems to have advantage over traditional chemotherapy, especially when given in combination with anticancer drugs. However, the effectiveness of single antibody or antibody conjugated with chemotherapeutics is still far from ideal. Antibody⁻dendrimer conjugates hold the potential to improve the targeting and release of active substance at the tumor site. In the present study, we developed and synthesized PAMAM dendrimer⁻trastuzumab conjugates carrying doxorubicin (dox) specifically to cells overexpressing HER-2. ¹HNMR, FTIR and RP-HPLC were used to characterize the products and analyze their purity. Toxicity of PAMAM⁻trastuzumab and PAMAM⁻dox⁻trastuzumab conjugates compared with free trastuzumab and doxorubicin towards HER-2 positive (SKBR-3) and negative (MCF-7) human breast cancer cell lines was determined using MTT assay. Furthermore, the cellular uptake and cellular localization were studied by flow cytometry and confocal microscopy, respectively. A cytotoxicity profile of above mentioned compounds indicated that conjugate PAMAM⁻dox⁻trastuzumab was more effective when compared to free drug or the conjugate PAMAM⁻trastuzumab. Moreover, these results reveal that trastuzumab can be used as a targeting agent in PAMAM⁻dox⁻trastuzumab conjugate. Therefore PAMAM⁻dox⁻trastuzumab conjugate might be an interesting proposition which could lead to improvements in the effectiveness of drug delivery systems for tumors that overexpress HER-2.

Unusual Enhancement of Doxorubicin Activity on Co-Delivery with Polyhedral Oligomeric Silsesquioxane (POSS).

Polyhedral oligomeric silsesquioxane (POSS), bearing eight 3-chloroammoniumpropyl substituents, was studied as a potential nanocarrier in co-delivery systems with doxorubicin (DOX). The toxicity of doxorubicin and POSS:DOX complexes at four different molar ratios (1:1; 1:2, 1:4, 1:8) towards microvascular endothelial cells (HMEC-1), breast cancer cells (MCF-7), and human cervical cancer endothelial cells (HeLa) was determined. The rate of penetration of the components into the cells, their cellular localization and the hydrodynamic diameter of the complexes was also determined. A cytotoxicity profile of POSS:DOX complexes indicated that the POSS:DOX system at the molar ratio of 1:8 was more effective than free DOX. Confocal images showed that DOX co-delivery with POSS allowed for more effective penetration of doxorubicin through the cell membrane. Taking all the results into account, it can be claimed that the polyhedral oligomeric silsesquioxane (T8-POSS) is a promising, complex nanocarrier for doxorubicin delivery.

[Trastuzumab - a monoclonal antibody - and dendrimers in a targeted therapy for breast cancer]. / Przeciwcialo monoklonalne trastuzumab i dendrymery w terapii celowanej raka piersi.

Breast cancer is the most frequently occurring cancer in women. It has been confirmed that approximately 30% of patients have overexpression of human epidermal growth factor 2 (HER2) on the surface of tumor cells. Trastuzumab - a recombinant, humanized monoclonal antibody - is directed against this receptor. Its use in traditional chemotherapy (with anthracyclines or taxanes) causes an increase of therapy efficiency. However, the systemic toxicity of the anticancer drugs is still a serious problem. Therefore, new solutions are sought, especially in the field of selective drug transport to tumor cells. Dendrimers are composed of a core and branches. They are the bestknown group of nanoparticles. A lot of publications have shown that they can be used as carriers of various types of molecules, including anticancer drugs. The branched structure provides effective protection against premature release of the drug into the circulatory system. It gives a chance to reduce the dose while maintaining a therapeutic effect, and to reduce the toxicity of the drug for normal cells. Furthermore, the surface of dendrimers can be modified by a monoclonal antibody to achieve a targeted therapy. For that reason synthesis of conjugates of trastuzumab, dendrimers, and anticancer drugs is so crucial. This paper presents an overview of publications about the use of trastuzumab in in vitro, in vivo and clinical studies, as well as the latest developments of biology and chemistry, whose goal is to create the perfect, targeted carrier.

Humans cannot consciously generate random numbers sequences: Polemic study.

It is widely believed, that randomness exists in Nature. In fact such an assumption underlies many scientific theories and is embedded in the foundations of quantum mechanics. Assuming that this hypothesis is valid one can use natural phenomena, like radioactive decay, to generate random numbers. Today, computers are capable of generating the so-called pseudorandom numbers. Such series of numbers are only seemingly random (bias in the randomness quality can be observed). Question whether people can produce random numbers, has been investigated by many scientists in the recent years. The paper "Humans can consciously generate random numbers sequences..." published recently in Medical Hypotheses made claims that were in many ways contrary to state of art; it also stated far-reaching hypotheses. So, we decided to repeat the experiments reported, with special care being taken of proper laboratory procedures. Here, we present the results and discuss possible implications in computer and other sciences.

Numerical model of heat exchange in perfused tissues.

A vascular model of the bioheat transfer for perfused tissues is described, along with its numerical implementation. The method consists in creating a vascular tree model within the tissue region of interest and formulating a numerical scheme that couples the temperatures of the tissue and of the arterial and venous blood flowing through it. The necessary equations are derived and cast in finite difference form. The numerical implementation of the method is then used to obtain results for a specific example. The presented method allows modelling of the effect of individual blood vessels present in the tissue domain on the heat transfer. It is significantly more expensive computationally than continuum models. At the same time, it is numerically cheaper than most vascular models known in the literature. The present implementation can be extended to cover 3D geometry and thermoregulation effects (vasoconstriction and vasodilation) at moderate cost.

Discrete vessel heat transfer in perfused tissue--model comparison.

The aim of this paper is to compare two methods of calculating heat transfer in perfused biological tissue using a discrete vessel description. The methods differ in two important aspects: the representation of the vascular system and the algorithm for calculating the heat flux between tissue and blood vessels. The first method was developed at the University of Utrecht between 1994 and 1998 and has been used in several clinical applications. The second method has been proposed by the first author. The methods are briefly described, their assumptions and limitations are discussed. Finally, the test simulation is introduced and the results produced by both methods are compared. The test indicates that the simpler, and less computationally intensive method proposed by the present author for calculating 2D problems containing countercurrent blood vessel systems can reproduce quite well some features of the solution obtained by the more complex 3D method. The observed discrepancies could be explained on physical grounds.