Apoptosis induction activity and molecular docking studies of survivin siRNA carried by Fe3O4-PEG-LAC-chitosan-PEI nanoparticles in MCF-7 human breast cancer cells.

Delivery of small interfering RNAs (siRNAs) into cells still remains a challenge in gene delivery studies. Here, we investigated the ability of synthesized Fe3O4-PEG-LAC-chitosan-PEI nanoparticles for siRNA delivery of survivin as the model gene into cells. The cellular uptake of survivin siRNA carried by synthesized nanoparticles into MCF-7 breast cancer cell line was evaluated by florescent microscopy and flowcytometry, both proving the efficacy of nanoparticles in delivery of up to 64.7% in comparison with lipofectamine 2000. Furthermore, the delivery of survivin siRNA by the nanoparticles (nanoplex) induced apoptosis that was assessed through DAPI staining and Annexin V/PI assays. In addition, we evaluated the efficacy of treatment with nanoplexes in the presence of mitoxantrone, as a chemotherapeutic agent. Our data indicated that inhibition of survivin expression increased the cell sensitivity to mitoxantrone. Real-time PCR and western blotting analysis revealed a significant reduction in mRNA and protein levels of survivin upon delivery of siRNA. Molecular docking studies showed that nanoparticles can bind to centeral BIR domain of survivin, exactly above zinc ion location with high affinity (ΔG: -10.3Kcal/mol). Also, thermodynamic studies proved the experimental results theoretically, revealing that the siRNA-loaded nanoparticles have a suppressing effect on survivin mRNA. Therefore, delivery of survivin siRNA into MCF-7 cells using Fe3O4-PEG-LAC-chitosan-PEI nanoparticles as a carrier enhances the cell death.

Multifunctional Superparamagnetic Nanoparticles: From Synthesis to siRNA Delivery.

BACKGROUND: Targeted delivery of small interfering RNA (siRNA) to the specific tumor tissues and cells is the key challenge in the development of RNA interference as a therapeutic application. METHODS: To target breast cancer, we developed a cationic nanoparticle as a therapeutic delivery system. The successful synthesis of the magnetic nanoparticles modified by polyaspartate (PAA) and polyethyleneimine (PEI) was confirmed using fourier transform infrared (FT-IR) measurements. The designed nanoparticle has been characterized evaluating its size and charge before and after nanoplex formation with siRNA. RESULTS: The designed nanoparticle could effectively form nanoplex with siRNA in 2:1 w/w ratio. Survivin siRNA was used to suppress the antiapoptotic gene, survivin, in MCF-7 cells. According to the importance of combinational therapy, Mitoxantrone (MTX) was used as a chemotherapeutic agent as well. The multifunctional nanoparticles have been successfully entered into about 63% of the MCF-7 cells shown via microscopic and flowcytometric methods. This effective cellular uptake led to the cell apoptosis. Down regulation of survivin was determined in mRNA and protein levels using Real Time PCR and western blotting, respectively. CONCLUSION: Gathering all obtained data, it was concluded that Fe3O4-PAA-PEI nanoparticles can deliver siRNA effectively into the cytoplasm of the MCF-7 breast cancer cells and induce apoptosis.

Novel polyacrylate-based cationic nanoparticles for survivin siRNA delivery combined with mitoxantrone for treatment of breast cancer.

As a gene delivery method in breast cancer therapy, knocking down the undesired genes in the cancerous cells would be promising. Inhibitors of Apoptosis Protein (IAP) family genes are some of the genes whose responsibility is inhibition of apoptosis in cells. Silencing these genes seems to be helpful directing the tumor cells to death. siRNA sequence designed against survivin anti-apoptotic gene can play this role if carried to the cytoplasm. Here we prepared a positive charged biocompatible nano-sized particle made up of a Fe3O4 core covered respectively by polyacrylate (PA) and polyethyleneimine (PEI) layer, which could successfully deliver the siRNA into the MCF-7 cells. The particle structure was checked and having less than 50 nm diameter in size, positive charge and, safety towards MCF-7 cells besides being able to form nanoplexes with the siRNA strand helps it entering into the biologic assays part. The siRNA delivery evaluated via flowcytometry. Apoptosis induction was determined by DAPI staining. The efficiency of survivin gene knockdown was evaluated in mRNA and protein levels using Real time PCR and western blotting methods. Overall, the Fe3O4-PA-PEI nanoparticles can deliver siRNA effectively into the cytoplasm of the MCF-7 breast cancer cells and induce apoptosis.

Evaluation of the cytotoxic, apoptosis inducing activity and molecular docking of spiroquinazolinone benzamide derivatives in MCF-7 breast cancer cells.

Previous studies have suggested that quinazolinone derivatives are potent apoptosis-inducing agents in various cancer cell lines. In the present study, we have investigated cytotoxic, apoptosis induction, and molecular docking activities of the spiroquinazolinone benzamide derivatives family on MCF-7 human breast cancer cells. The MTT cytotoxicity assays and docking studies showed that 4t-CHQB was the most active compound among the prepared spiroquinazolinone benzamide compounds with IC50 of 50 ± 1.2 µM and was selected for further assessments. Apoptosis, as the mechanism of cell death, was assessed morphologically by acridine orange/ethidium bromide (AO/EtBr) double staining, evaluation of the cell surface phosphatidylserine (PS) expression through annexin V/PI technique and, the formation of DNA ladder. Down regulation of survivin was evaluated in protein level after cell treatment with 4t-CHQB using western blotting method. Molecular modeling experiments involving 4t-CHQB binding site of survivin showed several strong hydrogen bonds and hydrophobic interactions between many important amino acid residues. Overall, the obtained data suggest that the assessed spiroquinazolinone benzamide compounds may provide a novel therapeutic approach for further evaluation, as an effective chemotherapeutic family acting through down regulation of survivin and apoptosis induction in breast cancer.

pH-sensitive, polymer modified, plasma stable niosomes: promising carriers for anti-cancer drugs.

The aim of this study was the design and evaluation of a novel plasma stable, pH-sensitive niosomal formulation of Mitoxantrone by a modified ethanol injection method. Cholesterol hemisuccinate was added instead of cholesterol in order to produce pH-sensitivity property and using PEG-Poly (monomethyl itaconate)-CholC6 (PEG-PMMI-CholC6) copolymer introduced simultaneously pH-sensitivity and plasma stability properties in prepared niosomes. The pH-sensitivity and cytotoxicity of Mitoxantrone niosomes were evaluated in vitro in phosphate buffer with different pHs as well as using human ovarian cancer cell line (OVCAR-3), human breast cancer cell line (MCF-7) and human umbilical vein endothelial cells (HUVEC). Results showed that both cholesterol derivatives bearing formulations had pH-sensitive property and were found to release their contents under mild acidic conditions rapidly. In addition, the PEG-PMMI-CholC6-based niosomes could reserve the pH-sensitivity after incubation in plasma. Both Mitoxantrone-loaded pH-sensitive niosomes showed higher cytotoxicity than the conventional niosomes on OVCAR-3 and MCF-7 cell lines. However, both pH-sensitive niosomes exhibited lower cytotoxic effect on HUVEC cell line. Plasma stable, pH-sensitive niosomes could improve the cytotoxic effect and reduce the side effects of anti-tumor drugs.

Pioglitazone prevents morphine antinociceptive tolerance via ameliorating neuroinflammation in rat cerebral cortex.

BACKGROUND: Opioid induced neuroinflammation is shown to be implicated in opioid analgesic tolerance development. In the present study the effect of pioglitazone on morphine-induced tolerance and neuroinflammation in the cerebral cortex of the rat was investigated. MATERIALS AND METHODS: Various groups of rats received morphine (10mg/kg; ip) and vehicle (po), or morphine (10mg/kg) and pioglitazone (20 or 40 mg/kg; po) once a day for 17 days. In order to determine the possible involvement of PPAR-γ in the pioglitazone effect, one group of rats received PPAR-γ antagonist, GW-9662 (2mg/kg; sc), and pioglitazone (40 mg/kg) and morphine once daily for 17 days. Nociception was assessed using a tail flick apparatus and the percentage of the maximal possible effect was calculated as well. On 18th day, 2h after the last morphine injection, the cerebral cortex of the animals were harvested and the tissue levels of tumour necrosis factor alpha, interleukin-1beta, interleukin-6, interleukin-10 and nuclear factor-kappa B activity were determined. RESULTS: Co-administration of pioglitazone (40 mg/kg) with morphine not only attenuated morphine-induced tolerance, but also prevented the up-regulation of pro-inflammatory cytokines (tumour necrosis factor alpha, interleukin-1beta, interleukin-6) and nuclear factor-kappa B activity in the rat cerebral cortex. Moreover, GW-9662 (2mg/kg) administration 30 min before pioglitazone, antagonized the above mentioned pioglitazone-induced effects. CONCLUSION: It is concluded that oral administration of pioglitazone attenuates morphine-induced tolerance. This effect of pioglitazone may be, at least in part, due to its anti-inflammatory property which suppressed the cortical pro-inflammatory cytokine and inhibited of nuclear factor-kappa B activity.

Nonionic surfactant-based vesicular system for transdermal drug delivery.

OBJECTIVE: The objective of this study was to formulate and evaluate the Ibuprofen niosomal formulation as a transdermal drug delivery system. MATERIALS AND METHODS: Niosomes were prepared by a modified ethanol injection method, using Span 60, Tween 60 and Tween 65 as well as cholesterol with various cholesterol:surfactant molar ratios. The prepared vesicles were characterized for entrapment efficiency (EE), particle size, zeta potential and in vitro release study. Skin permeation studies were conducted using modified Franz diffusion cell, and excised rat skin was treated with niosomal, liposomal and conventional Carbopol 914 gel of Ibuprofen. RESULTS AND DISCUSSION: The results showed that the type of surfactant and molar ratio of cholesterol:surfactant altered the EE, size and in vitro drug release of niosomes. Higher EE was obtained with the niosomes prepared with cholesterol and Span 60 at molar ratio of 0.5:1. It has been observed that both niosomal and liposomal formulations enhanced the drug permeation and the percentage of accumulated dose in the skin compared to control conventional gel formulation. However, niosomes prepared by Span 60 and Tween 65 exhibited higher permeation and retention of Ibuprofen, respectively. CONCLUSION: Our results suggested that niosomal formulations could be used as a promising carrier for the Ibuprofen transdermal delivery system.

Plasma stable, pH-sensitive non-ionic surfactant vesicles simultaneously enhance antiproliferative effect and selectivity of Sirolimus.

OBJECTIVE: The purpose of the present investigation was to prepare a plasma stable, pH-sensitive niosomal formulation to enhance Sirolimus efficacy and selectivity. MATERIALS AND METHODS: pH-sensitive niosomal formulations bearing PEG-Poly (monomethyl itaconate)-CholC6 (PEG-PMMI-CholC6) copolymers and cholesteryl hemisuccinate (CHEMS) were prepared by a modified ethanol injection method and characterized with regard to pH-responsiveness and stability in human serum. The ability of pH-sensitive niosomes to enhance the Sirolimus cytotoxicity was evaluated in vitro using human erythromyeloblastoid leukemia cell line (K562) and compared with cytotoxicity effect on human umbilical vein endothelial cells (HUVEC). RESULTS AND DISCUSSION: This study showed that both formulations can be rendered pH-sensitive property and were found to rapidly release their contents under mildly acidic conditions. However, the CHEMS-based niosomes lost their pH-sensitivity after incubation in plasma, whereas, PEG-PMMI-CholC6 niosomes preserved their ability to respond to pH change. Sirolimus encapsulated in pH-sensitive niosomes exhibited a higher cytotoxicity than the control conventional formulation on K562 cell line. On the other hand, both pH-sensitive niosomes showed lower antiproliferative effect on HUVEC cells. CONCLUSION: Plasma stable, pH-sensitive PEG-PMMI-CholC6-based niosomes can improve the in vitro efficiency and also reduce the side effects of Sirolimus.

Fusogenic pH sensitive liposomal formulation for rapamycin: improvement of antiproliferative effect.

CONTEXT: Liposomes are increasingly employed to deliver chemotherapeutic agents, antisense oligonucleotides, and genes to various therapeutic targets. OBJECTIVE: The present investigation evaluates the ability of fusogenic pH-sensitive liposomes of rapamycin in increasing its antiproliferative effect on human breast adenocarcinoma (MCF-7) cell line. MATERIALS AND METHODS: Cholesterol (Chol) and dipalmitoylphosphatidylcholine (DPPC) (DPPC:Chol, 7:3) were used to prepare conventional rapamycin liposomes by a modified ethanol injection method. Dioleoylphosphatidylethanolamine (DOPE) was used to produce fusogenic and pH-sensitive properties in liposomes simultaneously (DPPC:Chol:DOPE, 7:3:4.2). The prepared liposomes were characterized by their size, zeta potential, encapsulation efficiency percent (EE%), and chemical stability during 6 months. The antiproliferative effects of both types of rapamycin liposomes (10, 25, and 50 nmol/L) with optimized formulations were assessed on MCF-7 cells, as cancerous cells, and human umbilical vein endothelial cells (HUVEC), as healthy cells, employing the diphenyltetrazolium bromide (MTT) assay for 72 h. RESULTS AND DISCUSSION: The particle size, zeta potential, and EE% of the liposomes were 165 ± 12.3 and 178 ± 15.4 nm, -39.6 ± 1.3, and -41.2 ± 2.1 mV as well as 76.9 ± 2.6 and 76.9 ± 2.6% in conventional and fusogenic pH-sensitive liposomes, respectively. Physicochemical stability results indicated that both liposome types were relatively stable at 4 °C than 25 °C. In vitro antiproliferative evaluation showed that fusogenic pH-sensitive liposomes had better antiproliferative effects on MCF-7 cells compared to the conventional liposomes. Conversely, fusogenic pH-sensitive liposomes had less cytotoxicity on HUVEC cell line.

Improvement of the antiproliferative effect of rapamycin on tumor cell lines by poly (monomethylitaconate)-based pH-sensitive, plasma stable liposomes.

pH-responsive polymers produce liposomes with pH-sensitive property which can release their encapsulated drug under mild acidic conditions found inside the cellular endosomes, inflammatory tissues and cancerous cells. The aim of this study was preparing pH-sensitive and plasma stable liposomes in order to enhance the selectivity and antiproliferative effect of Rapamycin. In the present study we used PEG-poly (monomethylitaconate)-CholC6 (PEG-PMMI-CholC6) copolymer and Oleic acid (OA) to induce pH-sensitive property in Rapamycin liposomes. pH-sensitive liposomal formulations bearing copolymer PEG-PMMI-CholC6 and OA were characterized in regard to physicochemical stability, pH-responsiveness and stability in human plasma. The ability of pH-sensitive liposomes in enhancing the cytotoxicity of Rapamycin was evaluated in vitro by using colon cancer cell line (HT-29) and compared with its cytotoxicity on human umbilical vein endothelial cell (HUVEC) line. Both formulations were found to release their contents under mild acidic conditions rapidly. However, unlike OA-based liposomes, the PEG-PMMI-CholC6 bearing liposomes preserved their pH-sensitivity in plasma. Both types of pH-sensitive Rapamycin-loaded liposomes exhibited high physicochemical stability and could deliver antiproliferative agent into HT-29 cells much more efficiently in comparison with conventional liposomes. Conversely, the antiproliferative effect of pH-sensitive liposomes on HUVEC cell line was less than conventional liposomes. This study showed that both OA and PEG-PMMI-CholC6-based vesicles could submit pH-sensitive property, however, only PEG-PMMI-CholC6-based liposomes could preserve pH-sensitive property after incubation in plasma. As a result pH-sensitive PEG-PMMI-CholC6-based liposomal formulation can improve the selectivity, stability and antiproliferative effect of Rapamycin.

Potential applications of nanoshells in biomedical sciences.

Nanotechnology is an emerging, disruptive technology with significant impact and application in cancer research. Nanoshells are the one such gift by this technology which is a dielectric core material made up of silica covered by thin metals especially gold. To achieve more effective and better diagnostic and/or therapeutic goals, nanoshells can be conjugated to antibodies, oligonucleotides, fluorophores, targeting ligands, polymers, therapeutic agents, and radioisotope. Nanoshells (and especially gold nanoshells) show promise application in biomedical imaging, target therapy, gene delivery, tissue welding, drug delivery systems, therapeutic applications in general and cancer imaging and treatment in particular. They find place in the field of medicine for such applications because of their safety, biocompatibility, stability, bioavailability, optically tunable, and photoluminescent ability as well as high ability to attach to many therapeutic materials. In this review, we comprehensively discuss the potential biomedical applications of different type of nanoshells. This review is briefly outlined in .

The effects of folate-conjugated gold nanorods in combination with plasmonic photothermal therapy on mouth epidermal carcinoma cells.

The use of lasers has emerged to be highly promising for cancer therapy modalities, most commonly, the photothermal therapy method. Unfortunately, the most common disadvantage of laser therapy is its nonselectivity and requirement of high power density. The use of plasmonic nanoparticles as highly enhanced photoabsorbing agents has thus introduced a much more selective and efficient cancer therapy strategy. In this study, we aimed to demonstrate the selective targeting and destruction of mouth epidermal carcinoma cells (KB cells) using the photothermal therapy of folate-conjugated gold nanorods (F-GNRs). Considering the beneficial characteristics of GNRs and overexpression of the folate receptor by KB cells, we selected F-GNRs as a targeted photothermal therapy agent. Cell viability was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was determined by flow cytometry using an annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit. No cell damage or cytotoxicity from the individual treatment of laser light or F-GNRs was observed. However, a 56% cell lethality was achieved for KB cells using combined plasmonic photothermal therapy of 20 µM F-GNRs with seven pulses of laser light and 6-h incubation periods. Cell lethality strongly depends on the concentration of F-GNRs and the incubation period that is mainly due to the induction of apoptosis. This targeted damage is due to the F-GNRs present in the cancer cells strongly absorbing near-infrared laser light and rapidly converting it to heat. This new therapeutic avenue for cancer therapy merits further investigation using in vivo models for application in humans.

Plasma stable, pH-sensitive fusogenic polymer-modified liposomes: A promising carrier for mitoxantrone.

pH-sensitive liposomes are designed to undergo acid-triggered destabilization. In the present study, we prepared polymer-modified, plasma stable, pH-sensitive fusogenic mitoxantrone liposomes to increase efficacy and selectivity on cancer cell lines. Conventional liposomes were prepared using cholesterol and dipalmitoyl-sn-glycero-3-phosphatidylethanolamine. Dioleoylphosphatidylethanolamine and a cholesteryl derivative, poly(monomethylitaconate)-co-poly(N,N-dimethylaminoethyl methacrylate) (PMMI-co-PDMAEMA), were used for the preparation of pH-sensitive fusogenic liposomes. Using polyethylene glycol (PEG)-poly(monomethylitaconate)-CholC6 (PEG-PMMI-CholC6) copolymers instead of cholesterol introduced pH-sensitive and plasma stability properties simultaneously in prepared liposomes. All formulations were prepared by thin film hydration method and subsequently, pH-sensitivity and stability in human serum were evaluated. The ability of pH-sensitive fusogenic liposomes to enhance the mitoxantrone cytotoxicity and selectivity in cancerous cell lines was assessed in vitro compared to normal cell line using human breast cancer cell line (MCF-7), human prostate cancer cell line (PC-3), and human umbilical vein endothelial cells line. Results revealed that both PMMI-co-PDMAEMA and PEG-PMMI-CholC6-based formulations showed pH-sensitive property and were found to rapidly release mitoxantrone under mildly acidic conditions. Nevertheless, only the PEG-PMMI-CholC6-based liposomes preserved pH-sensitivity after incubation in plasma. Mitoxantrone loaded-pH-sensitive fusogenic liposomes exhibited a higher cytotoxicity than the control conventional liposomes on MCF-7 and PC-3 cell lines. On the contrary, both pH-sensitive fusogenic liposomes showed lower cytotoxic effect on human umbilical vein endothelial cell line. Plasma stable, pH-sensitive fusogenic liposomes are promising carriers for enhancing the efficiency and selectivity, besides reduction of the side effects of anticancer agents.

Enhanced transdermal delivery of diclofenac sodium via conventional liposomes, ethosomes, and transfersomes.

The aim of this study was to improve the transdermal permeation of Diclofenac sodium, a poorly water-soluble drug, employing conventional liposomes, ethosomes, and transfersomes. The prepared formulations had been characterized for the loaded drug amount and vesicle size. The prepared vesicular systems were incorporated into 1% Carbopol 914 gel, and a survey of in vitro drug release and drug retention into rat skin has been done on them using a modified Franz diffusion cell. The cumulative amount of drug permeated after 24 h, flux, and permeability coefficient were assessed. Stability studies were performed for three months. The size of vesicles ranged from 145 to 202 nm, and the encapsulation efficiency of the Diclofenac sodium was obtained between 42.61% and 51.72%. The transfersomes and ethosomes provided a significantly higher amount of cumulative permeation, steady state flux, permeability coefficient, and residual drug into skin compared to the conventional liposomes, conventional gel, or hydroethanolic solution. The in vitro release data of all vesicular systems were well fit into Higuchi model (RSD > 0.99). Stability tests indicated that the vesicular formulations were stable over three months. Results revealed that both ethosome and transfersome formulations can act as drug reservoir in skin and extend the pharmacologic effects of Diclofenac sodium.

The radioprotective effects of Origanum vulgare extract against genotoxicity induced by (131)I in human blood lymphocyte.

Radioiodine ((131)I) has been widely used for the treatment of patients with thyroid diseases. However, there is a persisting concern about the induction of second tumor and genetic damage after (131)I therapy. The purpose of this study was to investigate the radioprotective effects of Origanum vulgare extract against genotoxicity induced by (131)I in human lymphocytes. Whole blood samples from human volunteers were incubated with origanum extract at doses of 12.5, 25, 50 and 100 µg/mL. After 1 hour of incubation, the lymphocytes were incubated with (131)I (20 µCi/mL) for 1 hour. The lymphocytes were then cultured with a mitogenic stimulant to evaluate micronucleus formation in cytokinesis-blocked binucleated cells. Incubation of lymphocytes with (131)I induced additional genotoxicity and shown by increases in micronuclei (MN) frequency in human lymphocytes. Origanum at three last doses significantly reduced the MN frequency in cultured lymphocytes. The maximum protective effect and the maximum decrease in the frequency of MN were observed at 100 µg/mL of origanum, which caused a reduction of 70% (p<0.0001). Origanum extract also exhibited an excellent and dose-dependent radical-scavenging activity against 1,1-diphenyl-2-picrylhydrazyl-free radicals. This study has important implications for patients undergoing nuclear medicine procedures. The results indicate a protective role for origanum extract against the genetic damage induced by radiopharmaceutical administration.