PNA-ATP aptamer-capped doxorubicin-loaded silica nanoparticles for targeted cancer therapy.

Nanomaterial-based drug delivery has opened new horizons in cancer therapy. This study aimed to investigate the in vitro and in vivo anti-cancer effects of a hyaluronic acid (HA)-targeted nanocarrier based on hollow silica nanoparticles (HSNPs), gated with peptide nucleic acid (PNA) and ATP aptamer (ATPApt) and loaded with doxorubicin (DOX). After formulation of a smart drug delivery nanosystem (HSNPs/DOX/ATPApt/PNA/HA), drug release, cytotoxicity, uptake, and in vivo anti-tumor properties were studied. Drug release test showed the controlled release of encapsulated DOX in response to ATP content. MTT and flow cytometry indicated that HA could improve both cytotoxicity and cellular uptake of the formulation. Moreover, HA-targeted formulation enhanced both the survival rate and tumor inhibition in the tumor-bearing mice compared with free DOX (P < 0.05). Our findings confirmed that HA-targeted nanoformulation, gated with PNA/aptamer and loaded with DOX can provide a novel therapeutic platform with great potential for cancer therapy.

Differentiation of human adipose-derived mesenchymal stem cells toward tenocyte by platelet-derived growth factor-BB and growth differentiation factor-6.

Mesenchymal Stem Cells (MSCs) are important in regenerative medicine and tissue engineering and will be a very sensible choice for repair and regeneration of tendon. New biological practices, such as cellular therapy using stem cells, are promising for facilitating or expediting tendon therapy. Before using these cells clinically, it is best to check and confirm the optimal conditions for differentiation of these cells in the laboratory. Hence, in the present study, the impacts of PDGF-BB and GDF-6 supplementation on adipose-derived MSCs (ASCs) culture were studied. The frozen ASC were recovered and expanded in basic culture medium (DMEM with 10%FBS). The cells after passage five (P5) were treated with basic medium containing L-Prolin, Ascorbic Acid and only PDGF-BB or GDF-6 (20 ng/ml) or both of them (mix) as 3 groups for 14 days to investigate efficiency of ASCs differentiation towards tenocytes. The cells culturing in basic medium were used as control group. To validate tenogenic differentiation, H&E and Sirius Red staining were used to assess cell morphology and collagen production, respectively. In addition, mRNA levels of collagen I and III, Scleraxis and Tenomodulin as tenogenic markers were analyzed using qPCR. In all test groups, cells appeared slenderer, elongated cytoplasmic attributes compared to the control cells. The intensity of Sirius Red staining was significantly higher in GDF-6, PDGF-BB alone, than in group without supplements. The optical density was higher in the GDF-6 than PDGF-BB and mix-group. QPCR results showed that Col I and III gene expression was increased in all groups compared to the control. SCX expression was significantly increased only in the PDGF-BB group. TNMD mRNA expression was not significant among groups. In this study, we have corroborated that human ASCs are reactionary to tenogenic induction by GDF-6 and PDGF-BB alone or in combination. These outcomes will help greater insight into GDF-6 and PDGF-BB driven tenogenesis of ASCs and new directions of discovery in the design of ASC-based treatments for tendon healing.

Smac peptide and doxorubicin-encapsulated nanoparticles: design, preparation, computational molecular approach and in vitro studies on cancer cells.

The N-terminal sequence of the Smac (second-mitochondria derived activator) protein is known to be involved in binding to the BIR3 (Baculovirus IAP repeat) domain of the IAPs (inhibitors of apoptosis proteins), and antagonized their function. Short peptides derived from N-terminal residues of Smac have shown to sensitize cancer cells to chemotherapeutic agents. In this regard, small library including 6-mer peptides were designed using docking to the BIR3 domain of cIAP1 in silico. Molecular dynamics simulation studies were also done on top-scored hits (SmacAQ, SmacIQ) using Desmond 2017-2 for 150 ns simulation time. These two peptides were conveniently synthesized using solid phase peptide synthesis on Fmoc-Gln (Trt)-Wang resin. Furthermore, we encapsulated DOX (doxorubicin) and synthesized peptides in PLGA: PLGA-PEG (9:1) NPs (nanoparticles) followed by MD (molecular dynamic) studies to understand the NP structure and the interactions between either DOX or peptide with polymeric nanoparticles during 100 ns simulation. Finally, the cytotoxic activity of these peptides in combination with DOX against two cancer cell lines including MCF7 and C26 were investigated. As a result, we found that DOX or peptide-loaded NPs had stable structure during the simulation. MD simulation also showed that alanine at N-terminal of Smac could be replaced with isoleucine without alternation of biological activity which was in agreement with in vitro experiments. Moreover, NPs-SmacIQ and NPs-SmacAQ significantly enhanced the cytotoxicity effect of NPs-DOX in vitro (p < 0.001).Communicated by Ramaswamy H. Sarma.

Self-assembly of an aptamer-decorated chimeric peptide nanocarrier for targeted cancer gene delivery.

In this study, we developed a peptide-based non-viral carrier decorated with aptamer to overcome the specific gene delivery barriers. The carrier (KLN/Apt) was designed to contain multiple functional segments, including 1) two tandem repeating units of low molecular weight protamine (LMWP) to condense DNA into stable nanosize particles and protect it from enzymatic digestion, 2) AS1411 aptamer as targeting moiety to target nucleolin and promote carrier internalization, 3) a synthetic pH-sensitive fusogenic peptide (KALA) for disrupting endosomal membranes and enhancing cytosol escape of the nanoparticles, and 4) a nuclear localization signal (NLS) for active cytoplasmic trafficking and nuclear delivery of DNA. The obtained results revealed the developed carrier capacity in terms of specific cell targeting, overcoming cellular gene delivery barriers, and mediating efficient gene transfection. The KLN/pDNA/aptamer nanoparticles offer remarkable potential for the conceptual design and formation of promising multi-functionalized carriers towards the most demanding therapeutic applications.

Ladder-like targeted and gated doxorubicin delivery using bivalent aptamer in vitro and in vivo.

Regarding side effects of commonly used chemotherapeutic drugs on normal tissues, researchers introduced smart delivery and on-demand release systems. Herein, we applied a bivalent aptamer composed of ATP and AS1411 aptamers for separate targeting and gating of mesoporous silica nanoparticles in a ladder like structure with one bifunctional molecule. First part of the apatmer, AS1411, direct the delivery system to the desired site while the second part, ATP aptamer, opens the pores and release the drug just after penetrance to the cytoplasm ensuring delivery of DOX into the tumor cells. This approach faced the previous challenge of coincident targeting and gating with one aptamer. Our results demonstrated that the proposed nano-system remarkably accumulated in cancer tissue and released the drug in a sustained pattern in cancer cells. It was notably effective for inducing apoptosis in cancer cells and tumor growth inhibition without any significant side effect on normal cells and organs. Moreover, Si-cs-DOX-AAapt improved the mice survival time compared with free doxorubicin and there was no significant change in weight of mice administered with the targeted formulation. This report may open new insight for providing smart delivery systems for successful cancer treatment by introducing separate gating and targeting property by a bivalent aptamer to increase the control over drug release.

Induction of tenogenic differentiation of equine adipose-derived mesenchymal stem cells by platelet-derived growth factor-BB and growth differentiation factor-6.

Managing tendon healing process is complicated mainly due to the limited regeneration capacity of tendon tissue. Mesenchymal stem cells (MSCs) have potential applications in regenerative medicine and have been considered for tendon repair and regeneration. This study aimed to evaluate the capacity of equine adipose tissue-derived cells (eASCs) to differentiate into tenocytes in response to platelet-derived growth factor-BB (PDGF-BB) and growth differentiation factor-6 (GDF-6) in vitro. Frozen characterized eASCS of 3 mares were thawed and the cells were expanded in basic culture medium (DMEM supplemented with 10% FBS). The cells at passage 5 were treated for 14 days in different conditions including: (1) control group in basic culture medium (CM), (2) induction medium as IM (CM containing L-prolin, and ascorbic acid (AA)) supplemented with PDGF-BB (20 ng/ml), (3) IM supplemented with GDF-6 (20 ng/ml), and (4) IM supplemented with PDGF-BB and GDF-6. At the end of culture period (14th day), tenogenic differentiation was evaluated. Sirius Red staining was used to assess collagen production, and H&E was used for assessing cell morphology. mRNA levels of collagen type 1 (colI), scleraxis (SCX), and Mohawk (MKX), as tenogenic markers, were analyzed using real-time reverse-transcription polymerase chain reaction (qPCR). H&E staining showed a stretching and spindle shape (tenocyte-like) cells in all treated groups compared to unchanged from of cells in control groups. Also, Sirius red staining data showed a significant increase in collagen production in all treated groups compared with the control group. MKX expression was significantly increased in PDGF-BB and mixed groups and COLI expression was significantly increased only in PDGF-BB group. In conclusion, our results showed that PDGF-BB and GDF-6 combination could induce tenogenic differentiation in eASCs. These in vitro findings could be useful for cell therapy in equine regenerative medicine.

Combination therapy using Smac peptide and doxorubicin-encapsulated MUC 1-targeted polymeric nanoparticles to sensitize cancer cells to chemotherapy: An in vitro and in vivo study.

Targeting inhibitors of apoptosis proteins (IAPs) family comprising high level expression in many cancer cells, could sensitize tumor cells to conventional chemotherapies. In the present study, we designed both doxorubicin and SmacN6 (an antagonist of the IAPs) encapsulated polymeric nanoparticles (NPs) and investigated their synergistic effect of combination therapy in vitro and in vivo. According to the results, NPs-SmacN6 significantly enhanced the cytotoxicity effect of NPs-DOX and reduced its IC50 in MCF-7, 4T1 and C26 cancer cells. Western blot analysis confirmed mechanism of cell apoptosis via caspase activation through intrinsic and also extrinsic pathways. Moreover, 5TR1 aptamer-modified NPs could effectively deliver DOXor SmacN6 to C26 cancer cells (MUC1 positive) in comparison with the non-targeted one (p < 0.001). However, they could not be efficiently internalized into CHO cells (MUC1 negative), showing less cytotoxicity in this cell line. In vivo experiments in BALB/c mice bearing C26 tumor indicated that Apt-NPs-DOX in combination with Apt-NPs-SmacN6 had significant tumor growth inhibition in comparison with mice receiving either free DOX or Apt-NPs-DOX with p < 0.0001 and p < 0.05, respectively. Our results revealed that combination therapy of DOX and SmacN6 via Apt-modified nanoparticles can lead to improvement of therapeutic index of DOX in MUC1 positive cancer cells.

Design, Synthesis, and In Vitro Evaluation of Low Molecular Weight Protamine (LMWP)-Based Amphiphilic Conjugates as Gene Delivery Carriers.

Development of efficient non-viral carriers is one of the major challenges of gene delivery. In the current study, we designed, synthesized, and evaluated the in vitro gene delivery efficiency of novel amphiphilic constructs composed of cholesterol and low molecular weight protamine (LMWP: VSRRRRRRGGRRRR) peptide. Vectors having both hydrophobic and hydrophilic moieties were evaluated in terms of particle size and charge, DNA condensation ability, cytotoxicity, and gene transfection efficiency. The prepared vectors spontaneity self-assembled into the liposome-like particles with a high local positive density. The nano-vehicle A (H5-LMWP-Cholestrol) and nano-vehicle B (LMWP-Cholesterol) could form micelles at concentrations above 50 µg/mL and 65 µg/mL, respectively. The gel retardation assay showed that nano-vehicles A and B could condense pDNA more efficiently than the corresponding unconjugated peptides. The mean of size and zeta potential of complexed nano-vehicle A at N/P ratios of 5, 15, and 30 were 151 nm and 23 mv, and those of nano-vehicle B were 224 nm and 19 mv, respectively. In terms of transfection efficiency, the designed nano-vehicles showed almost two-fold higher gene expression level compared to PEI 25 kDa at optimal N/P ratios, and also exhibited negligible cytotoxicity on a model cancer cell, Neuro 2a. The findings of the present study revealed that these cationic micelles can be promising candidates as non-viral gene delivery vehicles.

Design, synthesis and biological evaluation of novel benzo- and tetrahydrobenzo-[h]quinoline derivatives as potential DNA-intercalating antitumor agents.

A new series of benzo- and tetrahydro benzo-[h]quinoline bearing a flexible (dimethylamino)ethylcarboxamide side chain was designed and synthesized as DNA-intercalating antitumor agents. The cytotoxic activity of the synthesized compounds was evaluated against four human cancer cell lines including MCF-7, A2780, C26 and A549. In general, saturated quinolines (tetrahydrobenzo[h]quinolines) exhibited more cytotoxicity compared to their corresponding unsaturated quinolines (benzo[h]quinolines). Compound 6e showed significant cytotoxicity against all four human cancer cell lines with IC50 values ranging from 1.86 to 3.91 µM. The interaction of the selected compounds showed significant cytotoxicity (6b, 6e, 6i and 6j) with calf thymus DNA (CT-DNA) was studied by UV and florescent spectroscopy. In general, benzo[h]quinolines showed higher interacting effect with DNA than their corresponding saturated tetrahydrobenzo[h]quinolines. Compound 6i exhibited the most DNA intercalating effects among the series. The apoptotic induction potential of the most cytotoxic compounds (6e, 6b and 6i) in A549 cells was studied using Annexin V-FITC/Propidium iodide staining assay. Compound 6e which showed the most cytotoxic effect against A549 cancer cells also exhibited stronger apoptotic induction activity in comparison with 6b and 6i.The docking was performed in order to study the DNA interaction properties of these compounds. According to the computational data, these compounds can interact with DNA as DNA-intercalating agents.

DNA protection, antioxidant and xanthine oxidase inhibition activities of polyphenol-enriched fraction of Berberis integerrima Bunge fruits.

OBJECTIVES: The aim of this study was to prepare fraction and determine the biological activities of the polyphenol-enriched fraction of Berberis integerrima Bunge fruits. MATERIALS AND METHODS: In this assay fraction was extracted by column chromatography, using Amberlite column as the stationary phase. Phenol and flavonoids in the extract and fraction were analyzed by high performance liquid chromatography (HPLC). DNA protection ability, antioxidant and xanthine oxidase inhibition capacities of this fraction were also examined. RESULTS: Phenol and flavonoid content measurement and HPLC analyses of this fraction confirmed that phenol and flavonoids were increased in fraction in comparison to extract (before using Amberlite column). In antioxidant measurement assay, the trolox equivalent values were 1.05± 0.04 and 0.8±0.11 in oxygen radical absorbance capacity (ORAC) and the EC50 values for cellular antioxidant activity were 55.51±0.21 and 95.67±0.13 µg/ml for quercetin and the fraction, respectively. The xanthine oxidase inhibition percentages were 97.6±0.003 and 90.2 6±0.003 in 100 µg/ml concentration of fraction and vitamin C respectively. Comet assay analysis showed that this fraction protects human lymphocytes against H2O2-induced DNA damages at 12.5 to 100 µg/ml concentrations. CONCLUSION: This study suggests that Amberlite column as the stationary phase help to improve phenolic compound in separating fractions. The results showed that B. integerrima fruits are rich in phenolic compounds and they are potent antioxidants with protective effects on oxidative damages. They might be used as functional ingredients in food and supplements.

Aptamer-based biosensors and nanosensors for the detection of vascular endothelial growth factor (VEGF): A review.

Vascular endothelial growth factor (VEGF) is a key regulator of vascular formation and a predominant protein biomarker in cancer angiogenesis. Owing to its crucial roles in the cancer metastasis, VEGF detection and quantification is of great importance in clinical diagnostics. Today, there exist a wide variety of detection strategies for identifying many types of disease biomarkers, especially for VEGF. As artificial single-stranded DNA or RNA oligonucleotides with catalytic and receptor properties, aptamers have drawn lots of attention to be applied in biosensing platforms due to their target-induced conformational changes as well as high stability and target versatility. So far, various sensitivity-enhancement techniques in combination with a broad range of smart nanomaterials have integrated into the design of novel aptasensors to improve detection limit and sensitivity of analyte detection. This review article provides a brief classification and description of the research progresses of aptamer-based biosensors and nanobiosensors for the detection and quantitative determination of VEGF based on optical and electrochemical platforms.

MUC1 aptamer-targeted DNA micelles for dual tumor therapy using doxorubicin and KLA peptide.

Targeted delivery of DNA nanoparticles is a promising approach in cancer therapy. Using aptamers, target specific delivery of DNA nanoparticles can be achieved. Further, aptamers can indirectly improve drug encapsulation efficiency of DNA nanoparticles for drugs intercalated within nucleic acid base pairs. Using DNA blocks, a micellar hybrid nanoparticle was prepared for the targeted co-delivery of doxorubicin and a pro-apoptotic peptide, KLA to tumor cells. Results demonstrated that anti-MUC1 aptamer could specifically deliver the synthesized DNA micelle into MCF-7 cells by improving its cellular uptake. Additionally, co-delivery of doxorubicin and KLA could significantly enhance the therapeutic efficacy of the construct resulting in reduction of required dose of doxorubicin that is a pivotal point in reducing chemotherapeutics side effects. Moreover, DOX-KLA-anti-MUC1-micelle remarkably inhibited tumor growth of tumor-bearing mice when compared with free drug. DOX-KLA-anti-MUC1-micelle also reduced toxic effect of free doxorubicin as determined by percent of body weight loss and survival rate in vivo.

Comparison study of the effect of alkyl-modified and unmodified PAMAM and PPI dendrimers on solubility and antitumor activity of crocetin.

Poor water solubility of hydrophobic drugs is one of the major problems in pharmaceutical sciences. Herein, in order to address the poor solubility of crocetin, the 30% of primary amines of PAMAM G4 and PPI G4 were alkylated and evaluated as drug delivery vectors. According to the results, this modification could improve the drug delivery efficiency in term of drug solubility, release pattern and cytotoxicity; however, the encapsulation yield was decreased. Accordingly, it can be concluded that the designed alkylated dendrimers are able to improve the drug delivery efficiency, may be through improving the solubility and cellular uptake of crocetin.

Protective effects of flavonoids against microbes and toxins: The cases of hesperidin and hesperetin.

Many plants produce flavonoids as secondary metabolites. These organic compounds may be involved in the defense against plant-threatening factors, such as microbes and toxins. Certain flavonoids protect their origin source against plant pathogens, but they also exhibit potential healthy properties in human organisms. Hesperidin (Hsd) and its aglycone, hesperetin (Hst), are two flavonoids from the Citrus species that exhibit various biological properties, including antioxidant, antiinflammatory and anticancer effects. Recent studies indicated that Hst and Hsd possess antimicrobial activity. Although the exact mechanisms behind their antimicrobial properties are not fully understood, several mechanisms such as the activation of the host immune system, bacterial membrane disruption, and interference with microbial enzymes, have been proposed. Hsd and Hst may also have protective effects against toxicity induced by various agents. These natural substances may contribute to the protection of cells and tissues through their antioxidant and radical scavenging activities. This review discusses the protective activities of Hsd and Hst against microbes and several toxicities induced by oxidants, chemicals, toxins, chemotherapy and radiotherapy agents, which were reported in vitro and in vivo. Furthermore, the probable mechanisms behind these activities are discussed.

Molecular mechanisms behind the biological effects of hesperidin and hesperetin for the prevention of cancer and cardiovascular diseases.

Hesperidin (Hsd) and its aglycone, hesperetin (Hst), are two flavonoids from citrus species that have various biological properties, particularly those for the prevention of cancer and cardiovascular diseases. Studies have shown both anti-cancer and cancer chemopreventive effects for Hsd and Hst. Cancer chemopreventive properties of Hsd and Hst are mainly associated with their antioxidant, radical scavenging and anti-inflammatory activities. In addition, Hsd and Hst interfere at different stages of cancer. Unlike conventional anti-cancer drugs, Hsd and Hst inhibit tumor growth by targeting multiple cellular protein targets at the same time, including caspases, Bcl-2 (B-cell lymphoma 2) and Bax (Bcl-2 associated X protein) for the induction of apoptosis, and COX-2 (cyclooxygenase-2), MMP-2 (matrix metalloproteinase-2) and MMP-9 for the inhibition of angiogenesis and metastasis. The results of the recent basic and clinical studies revealed the beneficial effects for Hst, Hsd and their derivatives in the treatment of heart failure and cardiac remodeling, myocardial ischemia and infarction, and hypertension. In addition, the valuable effects of Hst and Hsd in the treatment of diabetes and dyslipidemia with their anti-platelet and anticoagulant effects make them good candidates in the treatment of various cardiovascular diseases. In this review, new findings regarding the molecular targets of Hsd and Hst, animal studies and clinical trials are discussed.

Development of a novel histone H1-based recombinant fusion peptide for targeted non-viral gene delivery.

In this study a new multifunctional recombinant gene delivery system (vector) was developed for targeted gene delivery to ZR-75-1 breast cancer cells. The vector backbone contained multiple domains including: (1) two tandem repeating units of truncated histone H1 to condense pDNA, (2) a model cell targeting peptide to target ZR-75-1 cells, (3) a pH-responsive synthetic fusogenic peptide, KALA, to destabilize endosomal membrane, and (4) a nuclear localization signal from human immunodeficiency virus to enhance translocation of pDNA toward the cell nucleus. The vectors were cloned and expressed in Escherichia coli BL21 (DE3) followed by purification with Ni-NTA affinity chromatography. They were then characterized using physicochemical and in vitro biological methods to evaluate the gene transfer efficiency and vector multifunctionality. The results demonstrated that the recombinant vector bearing all four functional domains had the highest rate of gene transfection efficiency as compared to the vectors which lacked one or more functional motifs. Beside the ability to target, the developed multifunctional vector was able to disrupt endosomal membranes, reach cell nucleus by utilizing microtubules and transfect efficiently while showing no detectable toxicity.

Auraptene from Ferula szowitsiana protects human peripheral lymphocytes against oxidative stress.

The antigenotoxicity effects of auraptene on DNA damage in human peripheral lymphocytes were studied using alkaline single cell gel electrophoresis. Auraptene at concentrations of 5, 10, 25, 50, 100, 200 and 400 microM was tested under simultaneous treatment with 25 microM H(2)O(2). The data are expressed as % tail DNA and compared with ascorbic acid at concentrations of 25, 50, 100, 200 and 400 microM. Auraptene significantly reduced the genotoxicity of H(2)O(2 )at concentrations higher than 25 microM (p < 0.001). Interestingly, the antigenotoxicity activity of auraptene was higher than ascorbic acid (p < 0.01), however, at some concentrations (25, 50 and 200 microM) there was no significant difference between auraptene and ascorbic acid (p > 0.05). It seems that the significant antigenotoxicity effects of auraptene may be due to the prenyl moiety and also the suppression of superoxide anion (O(2) (-)) generation. This study suggests that the antigenotoxic property of auraptene is of great pharmacological importance and might be beneficial for cancer prevention.

Antigenotoxic effects of the disulfide compound persicasulfide A (PSA) on rat lymphocytes exposed to oxidative stress.

The antigenotoxic effect of persicasulfide A (PSA) from Ferula persica on DNA damage induced by hydrogen peroxide (H2O2) was evaluated using single cell gel electrophoresis (SCGE). PSA was extracted from F. persica, characterized by NMR and its antioxidant/antigenotoxic effects were investigated. The antigenotoxic effect of solutions containing either PSA (1, 10, 50, 100, 200, 300, 400 and 500 microM) or ascorbic acid (250, 500, 750 and 1000 microM) alone, or in the presence of H2O2 (25, 50, 100 and 200 microM) were tested on lymphocytes derived from the blood of healthy male Wistar rats (250-300 g) by using the comet assay. The degree of damage to DNA after exposure to different solutions was calculated based on the amount of DNA present in the tail compared to the total amounts of lymphocyte DNA. PSA did not show genotoxicity and caused a 50% reduction in DNA damage induced by H2O (EC50:476.47+/-67.46 microM). Compared to the EC50 for ascorbic acid (1399.23+/-205.21 microM), it was deduced that PSA was more effective than ascorbic acid in the prevention of oxidative damage to DNA.