Synthesis and characterization of curcumin loaded polymer/lipid based nanoparticles and evaluation of their antitumor effects on MCF-7 cells

Background

Hybrid materials are synthesized using hydrophilic polymer and lipids which ensure their long term systemic circulation through intravenous administration and enhance loading of hydrophobic drugs. The purpose of this study is to prepare, characterize and evaluate the in vitro efficacy of curcumin loaded poly-hydroxyethyl methacrylate/stearic acid nanoparticles in MCF-7.

Methods

C-PSA-NPs, prepared using the emulsification–solvent evaporation method were characterized by dynamic laser scattering, SEM, AFM, FT-IR, X-ray diffraction, and TGA. The in vitro release behavior was observed in PBS pH 7.4, the anticancer potential was analyzed by MTT assay, cell cycle and apoptosis studies were performed through flow cytometry. C-PSA-NPs drug localization and cancer cell morphological changes were analyzed in MCF-7 cell line.

Results

C-PSA-NPs exhibited the mean particle size in the range of 184 nm with no aggregation. The surface charge of the material was around − 29.3 mV. Thermal studies (TGA) and surface chemistry studies (FT-IR, XRD) showed the existence of drug curcumin in C-PSA-NPs. The MTT assay indicated higher anticancer properties and flow cytometry studies revealed that there were better apoptotic activity and maximum localization of C-PSA-NPs than curcumin.

Conclusions

Polymer lipid based drug delivery appeared as one of the advancements in drug delivery systems. Through the present study, a novel polymer lipid based nanocarrier delivery system loaded with curcumin was demonstrated as an effective and potential alternative method for tumor treatment in MCF-7 cell line.

General significance

C-PSA-NPs exhibited potent anticancer activity in MCF-7 cell line and it indicates that C-PSA-NPs are a suitable carrier for curcumin.     

Collagen adhesin–nanoparticle interaction impairs adhesin's ligand binding mechanism

Background

Pathogenic bacteria specifically recognize extracellular matrix (ECM) molecules of the host (e.g. collagen, fibrinogen and fibronectin) through their surface proteins known as MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules) and initiate colonization. On implantation, biomaterials easily get coated with these ECM molecules and the MSCRAMMs mediate bacterial adherence to biomaterials. With the rapid rise in antibiotic resistance, designing alternative strategies to reduce/eliminate bacterial colonization is absolutely essential.

Methods

The Rhusiopathiae surface protein B (RspB) is a collagen‐binding MSCRAMM of Erysipelothrix rhusiopathiae. It also binds to abiotic surfaces. The crystal structure of the collagen‐binding region of RspB (rRspB_31–348) reported here revealed that RspB also binds collagen by a unique ligand binding mechanism called “Collagen Hug” which is a common theme for collagen‐binding MSCRAMMs of many Gram-positive bacteria. Here, we report the interaction studies between rRspB_31–348 and silver nanoparticles using methods like gel shift assay, gel permeation chromatography and circular dichroism spectroscopy.

Results

The “Collagen Hug” mechanism was inhibited in the presence of silver nanoparticles as rRspB_31–348 was unable to bind to collagen. The total loss of binding was likely because of rRspB_31–348 and silver nanoparticle protein corona formation and not due to the loss of the structural integrity of rRspB_31–348 on binding with nanoparticles as observed from circular dichroism experiments.

General significance

Interaction of rRspB_31–348 with silver nanoparticle impaired its ligand binding mechanism. Details of this inhibition mechanism may be useful for the development of antimicrobial materials and antiadhesion drugs.     

Studying the enhancement of programmed cell death by combined AG1024 and paclitaxel in a model of chronic myelogenous leukemia

Aims

Chronic myelogenous leukemia is a clonal malignancy of the pluripotent hematopoietic stem cells that is characterized by the uncontrolled proliferation and expansion of myeloid progenitors. Myeloid progenitors express the fusion oncogene BCR–ABL, which has uncontrollable activity in malignant cells and prevents the cell apoptosis caused by some antineoplastic agents, such as paclitaxel. Targeting these abnormalities by blocking the tyrosine kinase enzymes of BCR–ABL is a promising approach for chronic myelogenous leukemia therapy.

Main methods

Conventional Liu's staining is an auxiliary technique used in microscopy to enhance the contrast in microscopic images, aiding the observation of cell morphology. The MTT assay, flow cytometry of the sub-G1 analysis and the TUNEL assay were applied to estimate the apoptosis levels. RT-PCR and western blot methods were used to evaluate the key molecules conferring anti-cell-death properties.

Key findings

The effects of the tyrosine kinase inhibitor AG1024 were evaluated with regard to the regulation of BCR–ABL expression, inhibition of cell proliferation, and enhanced paclitaxel-induced apoptosis in BCR–ABL-expressing K562 cell lines. AG1024 downregulated the expression of BCR–ABL and anti-apoptosis factors, such as Bcl-2 and Bcl-xL, which were present in K562 cells. Moreover, the combination of AG1024 with paclitaxel inhibited cell proliferation and enhanced paclitaxel-induced apoptosis within 24 h.

Significance

In summary, the present study shows that the combination of AG1024 with paclitaxel inhibited model cancer cell proliferation, suggesting a new use of paclitaxel-based chemotherapy for cancer control.     

Bioelectronics meets nanomedicine for cardiovascular implants: PEDOT-based nanocoatings for tissue regeneration

Background

An exciting direction in nanomedicine would be to analyze how living cells respond to conducting polymers. Their application for tissue regeneration may advance the performance of drug eluting stents by addressing the delayed stent re-endothelialization and late stent thrombosis.

Methods

The suitability of poly (3, 4-ethylenedioxythiophene) (PEDOT) thin films for stents to promote cell adhesion and proliferation is tested in correlation with doping and physicochemical properties. PEDOT doped either with poly (styrenesulfonate) (PSS) or tosylate anion (TOS) was used for films' fabrication by spin coating and vapor phase polymerization respectively. PEGylation of PEDOT: TOS for reduced immunogenicity and biofunctionalization of PEDOT: PSS with RGD peptides for induced cell proliferation was further applied. Atomic Force Microscopy and Spectroscopic Ellipsometry were implemented for nanotopographical, structural, optical and conductivity measurements in parallel with wettability and protein adsorption studies. Direct and extract testing of cell viability and proliferation of L929 fibroblasts on PEDOT samples by MTT assay in line with SEM studies follow.

Results

All PEDOT thin films are cytocompatible and promote human serum albumin adsorption. PEDOT:TOS films were found superior regarding cell adhesion as compared to controls. Their nanotopography and hydrophilicity are significant factors that influence cytocompatibility. PEGylation of PEDOT:TOS increases their conductivity and hydrophilicity with similar results on cell viability with bare PEDOT:TOS. The biofunctionalized PEDOT:PSS thin films show enhanced cell proliferation.

Conclusions

The application of PEDOT polymers has evolved as a new perspective to advance stents.

General significance

In this work, nanomedicine involving nanotools and novel nanomaterials merges with bioelectronics to stimulate tissue regeneration for cardiovascular implants. This article is part of a Special Issue entitled Organic Bioelectronics — Novel Applications in Biomedicine.     

Collagen based magnetic nanobiocomposite as MRI contrast agent and for targeted delivery in cancer therapy

Background

In this study, an attempt has been made with the advent of technology to prepare a multifunctional nanobiocomposite (NBC) for targeted drug delivery in cancer therapy.

Methods

Collagen (C) was fabricated as nanofibers with multifunctional moieties viz. CFeAb*D by incorporating iron oxide nanoparticles (Fe), coupling with fluorescein isothiocyanate (FITC) labeled antibody (Ab*) and loading an anticancer gemcitabine drug (D). This NBC was characterized by conventional methods and evaluated for its biological activities.

Results

The UV–vis and FTIR spectroscopic studies revealed the fluorescein to protein ratio and revealed the presence of iron oxide nanoparticles and their interaction with the collagen molecules, respectively. While SDS-PAGE showed the proteinaceous nature of collagen, VSM and TEM studies revealed magnetic saturation as 54.97 emu/g and a magnetic nanoparticle with a diameter in the range of 10–30 nm and the dimension of nanofiber ranging from 97 to 270 nm. A MRI scan has shown a super paramagnetic effect, which reveals that the prepared NBC can be used as a MRI contrast agent. The MTT assay has shown biocompatibility and an apoptotic effect while phase contrast microscopy exhibited receptor mediated uptake of endocytosis.

Conclusion

The novelty in the prepared NBC lies in the collagen nanofibers, which have a higher penetrating property without causing much cell damage, biocompatibility and multifunctional properties and is able to carry multifunctional agents.

General significance

The study has demonstrated the possible use of CFeAb*D as a multifunctional NBC for biomedical applications.     

Dicer knockdown induces fibronectin-1 expression in HEK293T cells via induction of Egr1

Background

Dicer is a multidomain ribonuclease III enzyme involved in the biogenesis of microRNAs (miRNAs) and small interfering RNAs (siRNAs); depletion of Dicer was found to impair the migration of endothelial cells.

Methods

siRNA transfection, cell migration assay, real-time RT–PCR, chromatin immunoprecipitation, Western blotting, ELISA, caspase-3 activity assay, and annexin-V–FITC assay were utilized.

Results

Knockdown of Dicer impairs the migratory capacity of HEK293T cells and induces fibronectin-1. The upregulation of fibronectin-1 is dependent on Egr1. Fibronectin-1/Dicer double-knockdown cells showed a marked increase in apoptosis compared with fibronectin-1 single knockdown cells.

Conclusions

Decreased Dicer expression induces fibronectin-1 expression via an Egr1-dependent manner.

General significance

Our data suggest that upregulation of fibronectin-1 protects Dicer knockdown HEK293T cells against apoptosis.     

PEP-1–metallothionein-III protein ameliorates the oxidative stress-induced neuronal cell death and brain ischemic insults

Background

Oxidative stress is considered to be involved in a number of human diseases including ischemia. Metallothioneins (MT)-III can protect neuronal cells from the cytotoxicity of reactive oxygen species (ROS). However, MT-III proteins biological function is unclear in ischemia. Thus, we examined the protective effects of MT-III proteins on oxidative stress-induced neuronal cell death and brain ischemic insult.

Methods

A human MT-III gene was fused with a protein transduction domain, PEP-1 peptide, to construct a cell permeable PEP-1–MT-III protein. PEP-1–MT-III protein was purified using affinity chromatograph. Transduced PEP-1–MT-III proteins were detected by Western blotting and immunoflourescence. Cell viability and DNA fragmentation were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) assay and terminal dexoynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, respectively. Brain ischemic injury was detected with immunohistochemistry.

Results

Purified PEP-1–MT-III proteins transduced into astrocytes in a time- and dose-dependent manner and protected against oxidative stress-induced cell death. Also, transduced PEP-1–MT-III proteins efficiently protected cells against DNA fragmentation. Furthermore, immunohistochemical analysis revealed that PEP-1–MT-III prevented neuronal cell death in the CA1 region of the hippocampus induced by transient forebrain ischemia. We demonstrated that transduced PEP-1–MT-III protein protects against oxidative stress induced cell death in vitro and in vivo.

General significance

Transduced PEP-1–MT-III protein has neuroprotective roles as an antioxidant in vitro and in vivo. PEP-1–MT-III protein is a potential therapeutic agent for various human brain diseases such as stroke, Alzheimer's disease, and Parkinson's disease.     

Detection and identification of cancerous murine fibroblasts,transformed by murine sarcoma virus in culture,using Raman spectroscopy and advanced statistical methods

Background

Cancer is one of the leading worldwide causes of death. It may be induced by a variety of factors, including carcinogens, radiation, genetic factors, or DNA and RNA viruses. The early detection of cancer is critical for its successful therapy, which can result in complete recovery from some types of cancer.

Methods

Raman spectroscopy has been widely used in medicine and biology. It is a noninvasive, nondestructive, and water-insensitive technique that can detect changes in cells and tissues that are caused by different disorders, such as cancer.In this study, Raman spectroscopy was used for the identification and characterization of murine fibroblast cell lines (NIH/3T3) and malignant fibroblast cells transformed by murine sarcoma virus (NIH-MuSV) cells.

Results

Using principal component analysis and LDA it was possible to differentiate between the NIH/3T3 and NIH-MuSV cells with an 80–85% success rate based on their Raman shift spectra.

Conclusions

The best results for differentiation were achieved from spectra that were obtained from the rich membrane sites.

General significance

Because of its homogeneity and complete control of most factors affecting its growth, cell culture is a preferred model for the detection and identification of specific biomarkers related to cancer transformation or other cellular modifications.     

Overexpression of human Argonaute2 inhibits cell and tumor growth

Background

Argonaute (Ago) proteins are essential for the biogenesis and function of ~ 20–30 nucleotide long RNAs such as microRNAs (miRNAs). Ago expression increases or decreases under various physiological conditions, although the functional consequences are unknown. In addition, while reduced global miRNA production was shown to enhance cellular transformation and tumorigenesis, how Ago proteins contribute to human diseases has not been reported.

Method

Ago2, an essential Ago isoform in mammals, was stably expressed in 293 T, the human embryonic kidney cell line, and H1299, the human lung adenocarcinoma cell line. miRNA and mRNA expression was investigated by quantitative PCR and microarray profiling. Cell proliferation and migration was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and scratch assay in the cell cultures, respectively. How Ago2 affected cell growth in vivo was determined by H1299 xenograft tumor growth in mice. Changes in Ago2 expression in human lung cancer samples were investigated by quantitative PCR and immunohistochemistry.

Results

Stable Ago2 overexpression elicited specific changes in miRNA and mRNA expression in both 293 T and H1299 cells. It also inhibited cell proliferation and migration in cell cultures as well as xenograft tumor growth in nude mice. Ago2 expression was lower in human lung adenocarcinomas than in the paired, non-cancerous tissues.

General significance

We concluded that changes in Ago2 expression might have significant physiological and pathological consequences in vivo.     

A novel inhibitor, 16-hydroxy-cleroda-3,13-dien-16,15-olide,blocks the autophosphorylation site of focal adhesion kinase (Y397) by molecular docking

Background

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine plays an important role in a number of cell signaling pathways, including cell migration, proliferation, and cell survival. This study was aimed to identify novel and specific inhibitors from natural compounds via molecular docking of FAK (Y397).

Methods

The 3D structure of FAK (PDB ID: 2AL6) was used for docking 109 natural compounds. Based on high affinity and energy interaction, four of ten candidate compounds, 16-hydroxy-cleroda-3,13-dien-16,15-olide (HCD), curcumin, quercetin, and catechin hydrate, were hit, and the inhibitory activity against FAK was validated in these compounds in C6 glioma and N18 neuroblastoma cell lines.

Results

HCD showed a potential effect on cell viability by MTT assay and cell arrest in the G0–G1 phase, and a TUNEL assay confirmed further apoptosis. Treatment with HCD decreased anti-apoptotic proteins and increased pro-apoptotic proteins. Atomic force microscopy data depicted that the formation of filopodia on the intracellular surface decreased in treated cells compared with the control. Zymography showed that HCD inhibited the activity of MMP-2 and MMP-9. The protein levels of FAK, pFAK, Rac1 and Cdc42, which are the key regulators for the formation of filopodia, were decreased. Additionally, HCD regulated the expression of epithelial mesenchymal transition proteins.

Conclusions

HCD effectively interacted at the autophosphorylation site of FAK and interaction analysis indicated an H-bond with the Arg 86 and Arg 125 residues.

General significance

This study suggests that HCD could be a potential inhibitor of FAK and could be used for anti-tumorigenesis and anti-metastasis treatments.     

Purification,characterization and cloning of a ricin B-like lectin from mushroom Clitocybe nebularis with antiproliferative activity against human leukemic T cells

Background

Lectins are a diverse group of carbohydrate-binding proteins exhibiting numerous biological activities and functions.

Methods

Two-step serial carbohydrate affinity chromatography was used to isolate a lectin from the edible mushroom clouded agaric (Clitocybe nebularis). It was characterized biochemically, its gene and cDNA cloned and the deduced amino acid sequence analyzed. Its activity was tested by hemagglutination assay and carbohydrate-binding specificity determined by glycan microarray analysis. Its effect on proliferation of several human cell lines was determined by MTS assay.

Results

A homodimeric lectin with 15.9-kDa subunits agglutinates human group A, followed by B, O, and bovine erythrocytes. Hemagglutination was inhibited by glycoprotein asialofetuin and lactose. Glycan microarray analysis revealed that the lectin recognizes human blood group A determinant GalNAcα1–3(Fucα1–2)Galβ-containing carbohydrates, and GalNAcβ1–4GlcNAc (N,N'-diacetyllactosediamine). The lectin exerts antiproliferative activity specific to human leukemic T cells.

Conclusions

The protein belongs to the ricin B-like lectin superfamily, and has been designated as C. nebularis lectin (CNL). Its antiproliferative effect appears to be elicited by binding to carbohydrate receptors on human leukemic T cells.

General significance

CNL is one of the few mushroom ricin B-like lectins that have been identified and the only one so far shown to possess immunomodulatory properties.     

A rubber transfer gasket to improve the throughput of liquid–liquid extraction in 96-well plates: Application to vitamin D testing

Background

The unmitigated rise in demand for the assessment of vitamin D status has taxed the ability of clinical mass spectrometry laboratories to preserve turn-around times. We aimed to improve the throughput of liquid–liquid extraction of plasma/serum for the assay of 25-hydroxy vitamin D.

Methods

We designed and fabricated a flexible rubber gasket that seals two 96-well plates together to quantitatively transfer the contents of one plate to another. Using the transfer gasket and a dry-ice acetone bath to freeze the aqueous infranatant, we developed a novel liquid–liquid extraction workflow in a 96-well plate format. We applied the technology to the mass spectrometric quantification of 25-hydroxy vitamin D.

Results

Cross-contamination between wells was ≤0.13%. The interassay imprecision over 132 days of clinical implementation was less than 10%. The method compared favorably to a standard liquid–liquid extraction in glass tubes (Deming slope = 1.018, S_x|y = 0.022). The accuracy of the assay was 102–105% as assessed with the recently released control materials from NIST.

Conclusions

The development of a plate-sealing gasket permits the liquid–liquid extraction of clinical specimens in a moderate-throughput workflow and the reliable assay of vitamin D status. In the future, the gasket may also prove useful in other sample preparation techniques for HPLC or mass spectrometry.     

A method to describe enzyme-catalyzed reactions by combining steady state and time course enzyme kinetic parameters

Background

Complete analysis of single substrate enzyme-catalyzed reactions has required a separate use of two distinct approaches. Steady state approximations are employed to obtain substrate affinity and initial velocity information. Alternatively, first order exponential decay models permit simulation of the time course data for the reactions. Attempts to use integrals of steady state equations to describe reaction time courses have so far met with little success.

Methods

Here we use equations based on steady state approximations to directly model time course plots.

Results

Testing these expressions with the enzyme β-galactosidase, which adheres to classical Michaelis–Menten kinetics, produced a good fit between observed and calculated values.

General significance

This study indicates that, in addition to providing information on initial kinetic parameters, steady state approximations can be employed to directly model time course kinetics.Integrated forms of the Michaelis–Menten equation have previously been reported in the literature. Here we describe a method to directly apply steady state approximations to time course analysis for predicting product formation and simultaneously obtain multiple kinetic parameters.     

Salinomycin induces apoptosis and senescence in breast cancer: Upregulation of p21, downregulation of survivin and histone H3 and H4 hyperacetylation

Background

In the present study, we investigated the effect of Salinomycin on the survival of three human breast cancer cell lines MCF-7, T47D and MDA-MB-231 grown in adherent culture conditions.

Methods

Cell viability was measured by CellTiter-Glo and Trypan blue exclusion assay. Apoptosis was determined by caspase 3/7 activation, PARP cleavage and Annexin V staining. Cell cycle distribution was assessed by propidium iodide flow cytometry. Senescence was confirmed by measuring the senescence-associated β-galactosidase activity. Changes in protein expression and histone hyperacetylation was determined by western blot and confirmed by immunofluorescence assay.

Results

Salinomycinwas able to inhibit the growth of the three cell lines in time- and concentration-dependent manners. We showed that depending on the concentrations used, Salinomycin elicits different effects on theMDA-MB-231 cells. High concentrations of Salinomycin induced a G2 arrest, downregulation of survivin and triggered apoptosis. Interestingly, treatment with low concentrations of Salinomycin induced a transient G1 arrest at earlier time point and G2 arrest at later point and senescence associatedwith enlarged cellmorphology, upregulation of p21 protein, increase in histone H3 and H4 hyperacetylation and expression of SA-β-Gal activity. Furthermore, we found that Salinomycin was able to potentiate the killing of the MCF-7 and MDA-MB-231 cells, by the chemotherapeutic agents, 4-Hydroxytamoxifen and frondoside A, respectively.

Conclusion

Our data are the first to link senescence and histone modifications to Salinomycin.

Significance

This study provides a new insight to better understand the mechanism of action of Salinomycin, at least in breast cancer cells.     

Chemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion

Background

Chronic inflammation in lung diseases contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated proline–glycine–proline (N-ac-PGP). In the current study, we investigate whether N-ac-PGP influences β₂-integrin activation and function in neutrophilic firm adhesion to endothelium.

Methods

Human polymorphonuclear leukocytes (PMNs) were isolated from fresh human blood. Subsequently, a transmigration assay was performed to evaluate the active migration of PMNs towards N-ac-PGP. Furthermore, the effect of the tripeptide on β₂-integrin activation was assessed by performing the adhesion assay using fibrinogen as a ligand. To determine whether this effect was due to conformational change of β₂-integrins, antibodies against CD11b and CD18 were used in the adhesion assay and the expression pattern of CD11b was determined.

Results

Human neutrophils transmigrated through an endothelial cell layer in response to basolateral N-ac-PGP. N-ac-PGP induced also a neutrophil adherence to fibrinogen. Using functional blocking antibodies against CD11b and CD18, it was demonstrated that CD11b/CD18 (Mac-1) was responsible for the N-ac-PGP-induced firm adhesion of neutrophils to fibrinogen. Pertussis toxin decreased the Mac-1 activation indicating the involvement of G-proteins. N-ac-PGP most likely activated Mac-1 by initiating a conformational change, since the expression pattern of Mac-1 on the cell surface did not change significantly.

Conclusions

Chemo-attractant N-acetyl proline–glycine–proline induces CD11b/CD18-dependent neutrophil adhesion.

General significance

This is the first study to describe that the chemo-attractant N-ac-PGP also activates Mac-1 on the surface of neutrophils, which can additionally contribute to neutrophilic transmigration into the lung tissue during lung inflammation.     

An antifungal peptide from Coffea canephora seeds with sequence homology to glycine-rich proteins exerts membrane permeabilization and nuclear localization in fungi

Background

The superfamily of glycine-rich proteins (GRPs) corresponds to a large and complex group of plant proteins that may be involved in many developmental and physiological processes such as RNA biogenesis, stress tolerance, pollen hydration and plant-pathogen interactions, showing defensive activity against fungi, bacteria and viruses.

Methods

In this study, the peptides from Coffea canephora seeds were extracted according to the methods of Egorov et al. (2005). The purified peptide was submitted for amino acid sequencing and antimicrobial activity measurement.

Results

The purified peptide with a molecular weight of 7 kDa, named Cc-GRP, was observed to display homology to GRPs. The Cc-GRP–fungi interaction led to morphological changes and membrane permeability, including the formation of pseudohyphae, which were visualized with the aid of SYTOX green dye. Additionally, Cc-GRP also prevented colony formation by yeasts. Antifungal assays of Fusarium oxysporum and Colletotrichum lindemuthianum, observed by light microscopy, showed that the two molds exhibited morphological changes after the growth assay. Cc-GRP coupled to FITC and its subsequent treatment with DAPI revealed the presence of the peptide in the cell wall, cell surface and nucleus of F. oxysporum.

Conclusions and general significance

In this work we purified, characterized and evaluated the in vitro effect on fungi of a new peptide from coffee, named Cc-GRP, which is involved in the plant defense system against pathogens by acting through a membrane permeabilization mechanism and localized in the nuclei of fungal cells. We also showed, for the first time, the intracellular localization of Cc-GRP during antimicrobial assay.