Pelin Mutlu

Synthesis of Doxorubicin loaded magnetic chitosan nanoparticles for pH responsive targeted drug delivery

Targeted drug delivery is a promising alternative to overcome the limitations of classical chemotherapy. In an ideal targeted drug delivery system carrier nanoparticles would be directed to the tumor tissue and selectively release therapeutic molecules. As a novel approach, chitosan coated magnetic nanoparticles (CS MNPs) maintain a pH dependent drug delivery which provides targeting of drugs to the tumor site under a magnetic field. Among various materials, chitosan has a great importance as a pH sensitive, natural, biodegradable, biocompatible and bioadhesive polymer. The aim of this study was to obtain an effective targeted delivery system for Doxorubicin, using chitosan coated MNPs. Different sized CS MNPs were produced by in situ synthesis method. The anti-cancer agent Doxorubicin was loaded onto CS MNPs which were characterized previously. Doxorubicin loading was confirmed by FTIR. Drug loading and release characteristics, and stability of the nanoparticles were investigated. Our results showed that the CS MNPs have pH responsive release characteristics. The cellular internalization of Doxorubicin loaded CS MNPs were visualized by fluorescent microscopy. Doxorubicin loaded CS MNPs are efficiently taken up by MCF-7 (MCF-7/S) and Doxorubicin resistant MCF-7 (MCF-7/1 μM) breast cancer cells, which increases the efficacy of drug and also maintains overcoming the resistance of Doxorubicin in MCF-7/Dox cells. Consequently, CS MNPs synthesized at various sizes can be effectively used for the pH dependent release of Doxorubicin in cancer cells. Results of this study can provide new insights in the development of pH responsive targeted drug delivery systems to overcome the side effects of conventional chemotherapy.

Bioapplications of poly(amidoamine) (PAMAM) dendrimers in nanomedicine

Poly(amidoamine) (PAMAM) dendrimers are a novel class of spherical, well-designed branching polymers with interior cavities and abundant terminal groups on the surface which can form stable complexes with drugs, plasmid DNA, oligonucleotides, and antibodies. Amine‐terminated PAMAM dendrimers are able to solubilize different families of hydrophobic drugs, but the cationic charges on dendrimer surface may disturb the cell membrane. Therefore, surface modification by PEGylation, acetylation, glycosylation, and amino acid functionalization is a convenient strategy to neutralize the peripheral amine groups and improve dendrimer biocompatibility. Anticancer agents can be either encapsulated in or conjugated to dendrimer and be delivered to the tumor via enhanced permeability and retention (EPR) effect of the nanoparticle and/or with the help of a targeting moiety such as antibody, peptides, vitamins, and hormones. Biodegradability, non-toxicity, non-immunogenicity, and multifunctionality of PAMAM dendrimer are the key factors which facilitate steady increase of its application in drug delivery, gene transfection, tumor therapy, and diagnostics applications with precision and selectivity. This review deals with the major topics of PAMAM dendrimers including structure, synthesis, toxicity, surface modification, and also possible new applications of these spherical polymers in biomedical fields as dendrimer-based nanomedicine.

Idarubicin-loaded folic acid conjugated magnetic nanoparticles as a targetable drug delivery system for breast cancer.

Conventional cancer chemotherapies cannot differentiate between healthy and cancer cells, and lead to severe side effects and systemic toxicity. Another major problem is the drug resistance development before or during the treatment. In the last decades, different kinds of controlled drug delivery systems have been developed to overcome these shortcomings. The studies aim targeted drug delivery to tumor site. Magnetic nanoparticles (MNP) are potentially important in cancer treatment since they can be targeted to tumor site by an externally applied magnetic field. In this study, MNPs were synthesized, covered with biocompatible polyethylene glycol (PEG) and conjugated with folic acid. Then, anti-cancer drug idarubicin was loaded onto the nanoparticles. Shape, size, crystal and chemical structures, and magnetic properties of synthesized nanoparticles were characterized. The characterization of synthesized nanoparticles was performed by dynamic light scattering (DLS), Fourier transform-infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), scanning electron microscopy (SEM) analyses. Internalization and accumulation of MNPs in MCF-7 cells were illustrated by light and confocal microscopy. Empty MNPs did not have any toxicity in the concentration ranges of 0-500μg/mL on MCF-7 cells, while drug-loaded nanoparticles led to significant toxicity in a concentration-dependent manner. Besides, idarubicin-loaded MNPs exhibited higher toxicity compared to free idarubicin. The results are promising for improvement in cancer chemotherapy.

Chitosan magnetic nanoparticles for pH responsive Bortezomib release in cancer therapy.

The use of nanotechnology in cancer treatment offers exciting opportunities, including the possibility of destroying tumors with minimal damage to healthy tissue by novel targeted drug delivery systems. pH differences between healthy and tumor microenvironment provide pH responsive release of drugs at tumor site via smart nanoparticles. In this study, chitosan coated superparamagnetic iron oxide nanoparticles (CS MNPs) were in situ synthesized by ionic crosslinking method as nanocarrier systems and loaded with the drug Bortezomib (Velcade(®)). The drug loading capacity, drug release and stability of CS MNPs were analyzed. CS MNPs were visualized inside the cells by fluorescence microscopy. The cytotoxicity of Bortezomib, CS MNPs and Bortezomib loaded CS MNPs were tested by XTT analyses in vitro. Gene expression analyses revealed that pro-apoptotic PUMA and NOXA genes were upregulated while anti-apoptotic BCL-2, SURVIVIN and cIAP-2 genes were downregulated at Bortezomib loaded CS MNP treated cells. Immunocytochemical analyses demonstrated an increase in p53 tumor suppressor protein levels at treated cells, which supports the upregulation of PUMA and NOXA genes, while Survivin protein level did not significantly change. This study points out that the pH responsive magnetic targeting of Bortezomib is more efficacious than free drug treatment. Moreover, targeted delivery of Bortezomib would reduce the frequency of drug administration by lowering the required amount of drug dose.

Multidrug Resistance Mediated by MRP1 Gene Overexpression in Breast Cancer Patients

Multidrug resistance (MDR) is a serious handicap towards the effective treatment of breast cancer patients. One of the most prevalent MDR mechanisms is through the overexpression of genes coding the proteins called Multidrug Resistance-associated Proteins (MRPs). The aim of this study was to investigate the expression of MRP1 in tumor tissues from breast cancer patients. In this study, a semi-quantitative RT-PCR approach was utilized. Our results suggest that MRP1 overexpression can mediate MDR in patients. Pre-evaluation of the level of such MDR mediators before chemotherapy can increase the efficacy of the treatment.

Loading of Gemcitabine on chitosan magnetic nanoparticles increases the anti-cancer efficacy of the drug.

Targeted delivery of anti-cancer drugs increase the efficacy, while decreasing adverse effects. Among various delivery systems, chitosan coated iron oxide nanoparticles (CsMNPs) gained attention with their biocompatibility, biodegradability, low toxicity and targetability under magnetic field. This study aimed to increase the cellular uptake and efficacy of Gemcitabine. CsMNPs were synthesized by in situ co-precipitation and Gemcitabine was loaded onto the nanoparticles. Nanoparticle characterization was performed by TEM, FTIR, XPS, and zeta potential. Gemcitabine release and stability was analyzed. The cellular uptake was shown. Cytotoxicity of free-Gemcitabine and Gem-CsMNPs were examined on SKBR and MCF-7 breast cancer cells by XTT assay. Gemcitabine loading was optimized as 30µM by spectrophotometric analyses. Drug release was highest (65%) at pH 4.2, while it was 8% at pH 7.2. This is a desired release characteristic since pH of tumor-tissue and endosomes are acidic, while the blood-stream and healthy-tissues are neutral. Peaks reflecting the presence of Gemcitabine were observed in FTIR and XPS. At neutral pH, zeta potential increased after Gemcitabine loading. TEM images displayed, Gem-CsMNPs were 4nm with uniform size-distribution and have spherical shape. The cellular uptake and targetability of CsMNPs was studied on MCF-7 breast cancer cell lines. IC50 value of Gem-CsMNPs was 1.4 fold and 2.6 fold lower than free-Gem on SKBR-3 and MCF-7 cell lines respectively, indicating the increased efficacy of Gemcitabine when loaded onto nanoparticles. Targetability by magnetic field, stability, size distribution, cellular uptake and toxicity characteristics of CsMNPs in this study provides a useful targeted delivery system for Gemcitabine in cancer therapy.

Effect of cobalt-60 (γ radiation) on multidrug-resistant multiple myeloma cell lines

Emergence of resistance to chemotherapy and radiotherapy is a major obstacle for the successful treatment of MM (multiple myeloma). Prednisone, vincristine and melphalan are commonly used chemotherapeutic agents for the treatment of MM. In the current study, we examined the presence of possible cross‐resistance between these drugs and gamma (γ) radiation. Prednisone, vincristine and melphalan resistant RPMI‐8226 and U‐266 MM cells were generated by stepwise increasing concentrations of the drugs. The sensitive and resistant cells were exposed to 200‐ and 800 cGy γ radiation, and proliferation was examined by XTT {2,3‐bis(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐5‐[(phenylamino)carbonyl]‐2H‐tetrazolium hydroxide} assay. The results showed that Prednisone‐ and melphalan‐resistant RPMI‐8226 cells were also cross‐resistant to 200 and 800 cGy γ radiation application, while vincristine‐resistant cells did not show resistance. On the other hand, Prednisone‐, vincristine‐ and melphalan‐resistant U‐266 cells showed cross‐resistance to 200‐ and 800 cGy γ radiation application. These results demonstrated that MM cells resistant to anticancer agents respond to radiation in different levels. These findings may be important in the clinical applications of radiation therapy in the treatment of vincristine resistant MM.

Half generations magnetic PAMAM dendrimers as an effective system for targeted gemcitabine delivery.

Tumor-specific delivery of anticancer drugs by magnetic nanoparticles will maximize the efficacy of the drug and minimize side effects, and reduce systemic toxicity. The magnetic core of these nanoparticles provides an advantage for selective drug targeting as they can be targeted to the tumor site and accumulated in cancer cells by means of an external magnetic field. Magnetic nanoparticles can be coated with Polyamidoamine (PAMAM) dendrimer and loaded with drugs. However, biomedical applications of PAMAM dendrimers are limited due to their toxicity associated with their multiple cationic charges due to terminal NH2 groups. Modifying the positively charged end groups with negatively charged COOH groups, is a satisfactory strategy for obtaining less toxic PAMAM dendrimers. Gemcitabine being an analogue of deoxycytidine, is an effective anticancer drug. However, clinical benefits of Gemcitabine are limited due to its short biological half-life. The aim of this study was to obtain an effective, less toxic targeted delivery system for Gemcitabine. Half generations, between G4.5 and G7.5, of PAMAM dendrimer coated magnetic nanoparticles (DcMNPs) were synthesized and conjugated with Gemcitabine. TEM images showed nanoscale size (12-14nm) of the nanoparticles. The zeta-potential analysis indicated a decreased negativity of surface charge in drug bound dendrimer compared to the empty nanoparticles. Gemcitabine was effectively conjugated successfully onto the surface of half-generations of PAMAM DcMNPs. It was observed Gemcitabine did not effectively bind to Generations G4 and G5. The highest drug loading was obtained for DcMNPs with Generation 5.5. Empty nanoparticles showed no significant cytotoxicity on SKBR-3 and MCF-7 cells. On the other hand, Gemcitabine loaded nanoparticles were 6.0 fold more toxic on SKBR-3 and 3.0 fold more toxic on MCF-7 cells compared to free Gemcitabine. Gemcitabine loaded on Generation 5.5 DcMNPs showed a higher stability than free Gemcitabine. About 94% of the drug was retained over 6 weeks period, at pH 7.2. Due to their targetability under magnetic field, stability, size distribution, cellular uptake and toxicity characteristics the dendrimeric nanoparticles obtained in this study can be useful a delivery system for Gemcitabine in cancer therapy.

An update on molecular biology and drug resistance mechanisms of multiple myeloma

Multiple myeloma (MM), a neoplasm of plasma cells, is the second most common hematological malignancy. Incidance rates increase after age 40. MM is most commonly seen in men and African-American population. There are several factors to this, such as obesity, environmental factors, family history, genetic factors and monoclonal gammopathies of undetermined significance (MGUS) that have been implicated as potentially etiologic. Development of MM involves a series of complex molecular events, including chromosomal abnormalities, oncogene activation and growth factor dysregulation. Chemotherapy is the most commonly used treatment strategy in MM. However, MM is a difficult disease to treat because of its marked resistance to chemotherapy. MM has been shown to be commonly multidrug resistance (MDR)-negative at diagnosis and associated with a high incidence of MDR expression at relapse. This review deals with the molecular aspects of MM, drug resistance mechanisms during treatment and also possible new applications for overcoming drug resistance.

Differential gene expression analysis related to extracellular matrix components in drug-resistant RPMI-8226 cell line.

Drug resistance remains a major obstacle to the successful use of chemotherapeutic drugs for many types of cancers including multiple myeloma. It is becoming increasingly apparent that tumor microenvironment could provide a shelter to malignant plasma cells that allow their survival after initial drug exposure. This study demonstrates alterations in gene expression levels of several extracellular matrix (ECM) components in prednisone, vincristine and melphalan-resistant RPMI-8226 myeloma cells. Resistant RPMI-8226 cells were developed through stepwise selection of cells by increasing concentrations of drugs. Microarray analysis was carried out and genes up- or downregulated more than two-folds were considered as significant. Different types of ECM components were altered in different drug resistant RPMI-8226 sublines. ITGAL and ITGB2 were both overexpressed in vincristine resistant cell line whereas they were both downregulated in prednisone resistant subline. On the other hand, LAMC1 gene was drastically overexpressed in prednisone resistant subline whereas it was downregulated in its melphalan resistant variant. FN1 gene was only upregulated in vincristine resistant cells. However, COL21A1 which is an ECM component of the blood vessel walls, was drastically downregulated in all of the drug resistant RPMI-8226 sublines. ADAM17 gene was upregulated in melphalan resistant subline. This report provides a preliminary in vitro study to the relationship between drug resistance and ECM components in multiple myeloma. Since in vitro developed drug-resistant multiple myeloma sublines do not have similar microenvironment of tumor cells, correlation of ECM proteins with drug resistance requires further analysis.