Oleh Taratula

Co‐delivery of Doxorubicin and Bcl‐2 siRNA by Mesoporous Silica Nanoparticles Enhances the Efficacy of Chemotherapy in Multidrug‐Resistant Cancer Cells

Development of multidrug resistance in cancer cells and adverse side effects are the major obstacles for effective cancer chemotherapy.[1-3] Therapeutic strategies to overcome drug resistance and specific tumor targeting with minimal premature drug release should have a great impact on the treatment of cancer. The term multidrug resistance (MDR) is used to define a resistance phenotype where cancer cells become resistant simultaneously to multiple drugs with no obvious structural resemblance and with different molecular targets.[4, 5] The multidrug resistance can be divided into two distinct classes, pump and nonpump resistance.[3] The pump resistance is caused by certain proteins that form membrane-bound ATP-dependent active drug efflux pumps, which significantly decrease the intracellular concentration of the drug and thereby the efficacy of the treatment. Membrane proteins, P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP) have been shown to be the main players for pump resistance to a broad range of structurally and functionally distinct cytotoxic agents.[6] The main mechanism of nonpump resistance is an activation of cellular antiapoptotic defense, mainly by Bcl-2 protein. Most of the anticancer drugs trigger apoptosis and simultaneously activate both pump and nonpump cellular defense of multidrug resistance, which prevents cell death. Therefore, to effectively suppress the overall resistance to chemotherapy, it is essential to simultaneously inhibit both pump and nonpump mechanisms of cellular resistance by targeting all the intracellular molecular targets.[3, 7-9]

Surface-Engineered Targeted PPI Dendrimer for Efficient Intracellular and Intratumoral siRNA Delivery

Low penetration ability of Small Interfering RNA (siRNA) through the cellular plasma membrane combined with its limited stability in blood, limits the effectiveness of the systemic delivery of siRNA. In order to overcome such difficulties, we constructed a nanocarrier-based delivery system by taking advantage of the lessons learned from the problems in the delivery of DNA. In the present study, siRNA nanoparticles were first formulated with Poly(Propyleneimine) (PPI) dendrimers. To provide lateral and steric stability to withstand the aggressive environment in the blood stream, the formed siRNA nanoparticles were caged with a dithiol containing cross-linker molecules followed by coating them with Poly(Ethylene Glycol) (PEG) polymer. A synthetic analog of Luteinizing Hormone-Releasing Hormone (LHRH) peptide was conjugated to the distal end of PEG polymer to direct the siRNA nanoparticles specifically to the cancer cells. Our results demonstrated that this layer-by-layer modification and targeting approach confers the siRNA nanoparticles stability in plasma and intracellular bioavailability, provides for their specific uptake by tumor cells, accumulation of siRNA in the cytoplasm of cancer cells, and efficient gene silencing. In addition, in vivo body distribution data confirmed high specificity of the proposed targeting delivery approach which created the basis for the prevention of adverse side effects of the treatment on healthy organs.

Tumor targeted quantum dot-mucin 1 aptamer-doxorubicin conjugate for imaging and treatment of cancer.

In this study, we report the design and delivery of a tumor-targeted, pH-responsive quantum dot-mucin1 aptamer-doxorubicin (QD-MUC1-DOX) conjugate for the chemotherapy of ovarian cancer. To achieve active cancer targeting, QD was conjugated with a DNA aptamer specific for mutated MUC1 mucin overexpressed in many cancer cells including ovarian carcinoma. DOX was attached to QD via a pH-sensitive hydrazone bond in order to provide the stability of the complex in systemic circulation and drug release in acidic environment inside cancer cells. The data show that this bond is stable at neutral and slightly basic pH and undergoes rapid hydrolysis in mildly acidic pH. Confocal microscopy and in vivo imaging studies show that the developed QD-MUC1-DOX conjugate had higher cytotoxicity than free DOX in multidrug resistant cancer cells and preferentially accumulated in ovarian tumor. Data obtained demonstrate a high potential of the proposed conjugate in treatment of multidrug resistant ovarian cancer.

Surface-Modified and Internally Cationic Polyamidoamine Dendrimers for Efficient siRNA Delivery

A novel internally quaternized and surface-acetylated poly(amidoamine) generation four dendrimer (QPAMAM-NHAc) was synthesized and evaluated for intracellular delivery of siRNA. The proposed dendrimer as a nanocarrier possesses the following advantages: (1) modified neutral surface of the dendrimer for low cytotoxicity and enhanced cellular internalization; (2) existence of cationic charges inside the dendrimer (not on the outer surface) resulting in highly organized compact nanoparticles, which can potentially protect nucleic acids from degradation. The properties of this dendrimer were compared with PAMAM-NH 2 dendrimer, possessing surface charges, and with an internally quaternized charged and hydroxyl-terminated QPAMAM-OH dendrimer. Atomic force microscopy studies revealed that internally charged and surface neutral dendrimers, QPAMAM-OH and QPAMAM-NHAc, formed well-condensed, spherical particles (polyplexes) with siRNA, while PAMAM-NH 2 resulted in the formation of nanofibers. The modification of surface amine groups to amide significantly reduced cytotoxicity of dendrimers with QPAMAM-NHAc dendrimer showing the lowest toxicity. Confocal microscopy demonstrated enhanced cellular uptake and homogeneous intracellular distribution of siRNA delivered by the proposed QPAMAM-NHAc nanocarrier. The results clearly demonstrated distinct advantages of developed QPAMAM-NHAc/siRNA polyplexes over the existing nucleic acid dendrimeric carriers.

Nanostructured Lipid Carriers as Multifunctional Nanomedicine Platform for Pulmonary Co-Delivery of Anticancer Drugs and siRNA

We developed, synthesized, and tested a multifunctional nanostructured lipid nanocarrier-based system (NLCS) for efficient delivery of an anticancer drug and siRNA directly into the lungs by inhalation. The system contains: (1) nanostructured lipid carriers (NLC); (2) anticancer drug (doxorubicin or paclitaxel); (3) siRNA targeted to MRP1 mRNA as a suppressor of pump drug resistance; (4) siRNA targeted to BCL2 mRNA as a suppressor of nonpump cellular resistance and (5) a modified synthetic analog of luteinizing hormone-releasing hormone (LHRH) as a targeting moiety specific to the receptors that are overexpressed in the plasma membrane of lung cancer cells. The NLCS was tested in vitro using human lung cancer cells and in vivo utilizing mouse orthotopic model of human lung cancer. After inhalation, the proposed NLCS effectively delivered its payload into lung cancer cells leaving healthy lung tissues intact and also significantly decreasing the exposure of healthy organs when compared with intravenous injection. The NLCS showed enhanced antitumor activity when compared with intravenous treatment. The data obtained demonstrated high efficiency of proposed NLCS for tumor-targeted local delivery by inhalation of anticancer drugs and mixture of siRNAs specifically to lung cancer cells and, as a result, efficient suppression of tumor growth and prevention of adverse side effects on healthy organs.

Internally Cationic Polyamidoamine PAMAM-OH Dendrimers for siRNA Delivery: Effect of the Degree of Quaternization and Cancer Targeting

A novel cancer targeted, internally cationic and surface neutral polyamidoamine (PAMAM) dendrimer, was designed, synthesized, and evaluated as a nanocarrier for the targeted intracellular delivery of siRNA. The dendrimer contained a synthetic analog of Luteinizing hormone-releasing hormone as cancer targeting moiety. The proposed delivery system possesses the following advantages: (1) internal cationic charges for complexation with siRNA and enhanced siRNA protection; (2) low cytotoxicity; (3) lesser degree of quaternization offering free tertiary amines for potential proton sponge effect; and (4) targeting specifically to cancer cells for enhancing siRNA uptake and efficiency and potential limitation of adverse side effects of chemotherapy on healthy organs. Both nontargeted and targeted dendrimer-siRNA complexes formed compact nanometer size spherical particles, exhibited very low cytotoxicity even at the higher concentration, and efficiently penetrated cancer cells in vitro. However, only the targeted dendrimer-siRNA complex was able to substantially decrease the expression of a targeted BCL2 gene.

DNA and carbon nanotubes as medicine

The identification of disease-related genes and their complete nucleotide sequence through the human genome project provides us with a remarkable opportunity to combat a large number of diseases with designed genes as medicine. However, gene therapy relies on the efficient and nontoxic transport of therapeutic genetic medicine through the cell membranes, and this process is very inefficient. Carbon nanotubes, due to their large surface areas, unique surface properties, and needle-like shape, can deliver a large amount of therapeutic agents, including DNA and siRNAs, to the target disease sites. In addition, due to their unparalleled optical and electrical properties, carbon nanotubes can deliver DNA/siRNA not only into cells, which include difficult transfecting primary-immune cells and bacteria, they can also lead to controlled release of DNA/siRNA for targeted gene therapy. Furthermore, due to their wire shaped structure with a diameter matching with that of DNA/siRNA and their remarkable flexibility, carbon nanotubes can impact on the conformational structure and the transient conformational change of DNA/RNA, which can further enhance the therapeutic effects of DNA/siRNA. Synergistic combination of the multiple capabilities of carbon nanotubes to deliver DNA/siRNAs will lead to the development of powerful multifunctional nanomedicine to treat cancer or other difficult diseases. In this review, we summarized the current studies in using CNT as unique vehicles in the field of gene therapy.

Anti-HER2 IgY antibody-functionalized single-walled carbon nanotubes for detection and selective destruction of breast cancer cells.

BackgroundNanocarrier-based antibody targeting is a promising modality in therapeutic and diagnostic oncology. Single-walled carbon nanotubes (SWNTs) exhibit two unique optical properties that can be exploited for these applications, strong Raman signal for cancer cell detection and near-infrared (NIR) absorbance for selective photothermal ablation of tumors. In the present study, we constructed a HER2 IgY-SWNT complex and demonstrated its dual functionality for both detection and selective destruction of cancer cells in an in vitro model consisting of HER2-expressing SK-BR-3 cells and HER2-negative MCF-7 cells.MethodsThe complex was constructed by covalently conjugating carboxylated SWNTs with anti-HER2 chicken IgY antibody, which is more specific and sensitive than mammalian IgGs. Raman signals were recorded on Raman spectrometers with a laser excitation at 785 nm. NIR irradiation was performed using a diode laser system, and cells with or without nanotube treatment were irradiated by 808 nm laser at 5 W/cm2 for 2 min. Cell viability was examined by the calcein AM/ethidium homodimer-1 (EthD-1) staining.ResultsUsing a Raman optical microscope, we found the Raman signal collected at single-cell level from the complex-treated SK-BR-3 cells was significantly greater than that from various control cells. NIR irradiation selectively destroyed the complex-targeted breast cancer cells without harming receptor-free cells. The cell death was effectuated without the need of internalization of SWNTs by the cancer cells, a finding that has not been reported previously.ConclusionWe have demonstrated that the HER2 IgY-SWNT complex specifically targeted HER2-expressing SK-BR-3 cells but not receptor-negative MCF-7 cells. The complex can be potentially used for both detection and selective photothermal ablation of receptor-positive breast cancer cells without the need of internalization by the cells. Thus, the unique intrinsic properties of SWNTs combined with high specificity and sensitivity of IgY antibodies can lead to new strategies for cancer detection and therapy.

Innovative strategy for treatment of lung cancer: targeted nanotechnology-based inhalation co-delivery of anticancer drugs and siRNA

A tumor targeted mesoporous silica nanoparticles (MSN)-based drug delivery system (DDS) was developed for inhalation treatment of lung cancer. The system was capable of effectively delivering inside cancer cells anticancer drugs (doxorubicin and cisplatin) combined with two types of siRNA targeted to MRP1 and BCL2 mRNA for suppression of pump and nonpump cellular resistance in non-small cell lung carcinoma, respectively. Targeting of MSN to cancer cells was achieved by the conjugation of LHRH peptide on the surface of MSN via poly(ethylene glycol) spacer. The delivered anticancer drugs and siRNA preserved their specific activity leading to the cell death induction and inhibition of targeted mRNA. Suppression of cellular resistance by siRNA effectively delivered inside cancer cells and substantially enhanced the cytotoxicity of anticancer drugs. Local delivery of MSN by inhalation led to the preferential accumulation of nanoparticles in the mouse lungs, prevented the escape of MSN into the systemic circulation, and limited their accumulation in other organs. The experimental data confirm that the developed DDS satisfies the major prerequisites for effective treatment of non-small cell lung carcinoma. Therefore, the proposed cancer-targeted MSN-based system for complex delivery of drugs and siRNA has high potential in the effective treatment of lung cancer.

Multifunctional nanomedicine platform for cancer specific delivery of siRNA by superparamagnetic iron oxide nanoparticles-dendrimer complexes.

The ability of Superparamagnetic Iron Oxide (SPIO) nanoparticles and Poly(Propyleneimine) generation 5 dendrimers (PPI G5) to cooperatively provoke siRNA complexation was investigated in order to develop a targeted, multifunctional siRNA delivery system for cancer therapy. Poly(ethylene glycol) (PEG) coating and cancer specific targeting moiety (LHRH peptide) have been incorporated into SPIO-PPI G5-siRNA complexes to enhance serum stability and selective internalization by cancer cells. Such a modification of siRNA nanoparticles enhanced its internalization into cancer cells and increased the efficiency of targeted gene suppression in vitro. Moreover, the developed siRNA delivery system was capable of sufficiently enhancing in vivo antitumor activity of an anticancer drug (Cisplatin). The proposed approach demonstrates potential for the creation of targeted multifunctional nanomedicine platforms with the ability to deliver therapeutic siRNA specifically to cancer cells in order to prevent severe adverse side effects on healthy tissues and in situ monitoring of the therapeutic outcome using clinically relevant imaging techniques.