Correction to: EpCAM aptamer mediated cancer cell specific delivery of EpCAM siRNA using polymeric nanocomplex.

An amendment to this paper has been published and can be accessed via the original article.

Correction to: Iron-free and iron-saturated bovine lactoferrin inhibit survivin expression and differentially modulate apoptosis in breast cancer.

After publication of the original article [1], the authors found that Fig. 3 contained an incorrect version of Fig. 3c. This does not affect the Figure legend, results and conclusions of the article.

Brain targeted PLGA nanocarriers alleviating amyloid-Β expression and preserving basal survivin in degenerating mice model.

The chronic systemic administration of d-Galactose in C57BL/6J mice showed a relatively high oxidative stress, amyloid-ß expression and neuronal cell death. Enhanced expression of pyknotic nuclei, caspase-3 and reduced expression of neuronal integrity markers further confirmed the aforesaid insults. However, concomitant treatment with the recombinant protein (SurR9-C84A) and the anti-transferrin receptor antibody conjugated SurR9-C84A (SurR9+TFN) nanocarriers showed a significant improvement in the disease status and neuronal health. The beauty of this study is that the biodegradable Food and Drug Administration (FDA) approved poly(lactic-co-glycolic acid) (PLGA) nanocarriers enhanced the biological half-life and the efficacy of the treatments. The nanocarriers were effective in lowering the amyloid-ß expression, enhancing the neuronal integrity markers and maintaining the basal levels of endogenous survivin that is essential for evading the caspase activation and apoptosis. The current study herein reports for the first time that the brain targeted SurR9-C84A nanocarriers alleviated the d-Galactose induced neuronal insults and has potential for future brain targeted nanomedicine application.

A Study of Gene Expression of Survivin, its Antiapoptotic Variants, and Targeting Survivin In Vitro for Therapy in Retinoblastoma.

Apoptosis is a natural process regulated by apoptotic and antiapoptotic molecules. We investigated mRNA expression of survivin and its splice variants, along with B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax), in a cohort of 20 retinoblastoma (RB) tumors by real-time polymerase chain reaction. We hypothesized a correlation between the Bcl-2/Bax and survivin splice variants and also that expression of these would be associated with clinicopathologic features of tumors. The Bcl-2 expression was significantly higher (P<0.001) in RB, and Bcl-2/Bax ratio was remarkably higher in poorly differentiated tumors. A statistically significant higher expression of Survivin-WT (wild type) compared with its variant Survivin-2ß (P<0.05) was observed. Bcl-2 did not exhibit positive correlation with any of the survivin variants except Survivin-2ß, whereas Bax exhibited significant (P<0.05) correlation with the variants. Thus, it could be suggested that a superior player out of a likely interaction between the variants and Bcl-2/Bax uses its activity for the progression of RB. Silencing of Survivin-WT in the Y79 cell line was studied by siRNA technology and cell-permeable dominant negative survivin (SurR9-C84A). siRNA showed higher proapoptotic effects and increased caspase 3/7 activity in Y79 cells. Effective internalization of SurR9-C84A in Y79 cells induced cytotoxic effects. Thus, the current study confirms survivin as a promising target for therapy.

Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization.

Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1-D3)-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities.

Iron-free and iron-saturated bovine lactoferrin inhibit survivin expression and differentially modulate apoptosis in breast cancer.

BACKGROUND: Iron binding, naturally occurring protein bovine lactoferrin (bLf) has attracted attention as a safe anti-cancer agent capable of inducing apoptosis. Naturally, bLf exists partially saturated (15-20%) with Fe(3+) however, it has been demonstrated that manipulating the saturation state can enhance bLf's anti-cancer activities. METHODS: Apo-bLf (Fe(3+) free) and Fe-bLf (>90% Fe(3+) Saturated) were therefore, tested in MDA-MB-231 and MCF-7 human breast cancer cells in terms of cytotoxicity, proliferation, migration and invasion. Annexin-V Fluos staining was also employed in addition to apoptotic protein arrays and Western blotting to determine the specific mechanism of bLf-induced apoptosis with a key focus on p53 and inhibitor of apoptosis proteins (IAP), specifically survivin. RESULTS: Apo-bLf induced significantly greater cytotoxicity and reduction in cell proliferation in both cancer cells showing a time and dose dependent effect. Importantly, no cytotoxicity was detected in normal MCF-10-2A cells. Both forms of bLf significantly reduced cell invasion in cancer cells. Key apoptotic molecules including p53, Bcl-2 family proteins, IAP members and their inhibitors were significantly modulated by both forms of bLf, though differentially in each cell line. Most interestingly, both Apo-bLf and Fe-bLf completely inhibited the expression of survivin protein (key IAP), after 48 h at 30 and 40 nM in cancer cells. CONCLUSIONS: The capacity of these forms of bLf to target survivin expression and modulation of apoptosis demonstrates an exciting potential for bLf as an anti-cancer therapeutic in the existing void of survivin inhibitors, with a lack of successful inhibitors in the clinical management of cancer.

EpCAM aptamer mediated cancer cell specific delivery of EpCAM siRNA using polymeric nanocomplex.

BACKGROUND: Epithelial cell adhesion molecule (EpCAM) is overexpressed in solid tumors and regarded as a putative cancer stem cell marker. Here, we report that employing EpCAM aptamer (EpApt) and EpCAM siRNA (SiEp) dual approach, for the targeted delivery of siRNA to EpCAM positive cancer cells, efficiently inhibits cancer cell proliferation. RESULTS: Targeted delivery of siRNA using polyethyleneimine is one of the efficient methods for gene delivery, and thus, we developed a novel aptamer-PEI-siRNA nanocomplex for EpCAM targeting. PEI nanocomplex synthesized with EpCAM aptamer (EpApt) and EpCAM siRNA (SiEp) showed 198 nm diameter sized particles by dynamic light scattering, spherical shaped particles, of 151 ± 11 nm size by TEM. The surface charge of the nanoparticles was -30.0 mV using zeta potential measurements. Gel retardation assay confirmed the PEI-EpApt-SiEp nanoparticles formation. The difference in size observed by DLS and TEM could be due to coating of aptamer and siRNA on PEI nanocore. Flow cytometry analysis revealed that PEI-EpApt-SiEp has superior binding to cancer cells compared to EpApt or scramble aptamer (ScrApt) or PEI-ScrApt-SiEp. PEI-EpApt-SiEp downregulated EpCAM and inhibited selectively the cell proliferation of MCF-7 and WERI-Rb1 cells. CONCLUSIONS: The PEI nanocomplex fabricated with EpApt and siEp was able to target EpCAM tumor cells, deliver the siRNA and silence the target gene. This nanocomplex exhibited decreased cell proliferation than the scrambled aptamer loaded nanocomplex in the EpCAM expressing cancer cells and may have potential for EpCAM targeting in vivo.

Nanoformulated Mutant SurR9-C84A: a Possible Key for Alzheimer's and its Associated Inflammation.

PURPOSE: Alzheimer's disease (AD) is one of the untreatable neurodegenerative diseases characterised by the pathologic amyloid plaque deposition and inflammation. The aim of this study is to evaluate the neuroprotective effects of nanoformulated SurR9-C84A, a survivin mutant belonging to the inhibitors of the apoptosis (IAP) protein family. The effect of SurR9-C84A was studied against the ß-amyloid toxicity and various inflammatory insults in the differentiated SK-N-SH neurons. METHOD: SurR9-C84A loaded poly(lactic-co-glycolic acid) nanoparticles were prepared following the modified double emulsion technique. The neuroprotective effect of SurR9-C84A was evaluated against the amyloid-ß (Aß) peptide fragment, N-methyl-D-aspartate (NMDA) toxicity and the inflammatory assaults. To mimic the in vivo situation, a co-culture of neurons and microglia was also studied to validate these results. RESULTS: SurR9-C84A treatments showed improved neuronal health following Aß, and NMDA toxicity in addition to inflammatory insults induced in mono and co-cultures. The neuroprotective effect was evident with the reduced neuronal death, accelerated expression of neuronal integrity markers (neurofilaments, beta-tubulin III etc.,) and the neuroprotective ERK/MAPK signalling. CONCLUSION: The current results demonstrated that the SurR9-C84A nanoformulation was very effective in rescuing the neurons and holds a potential future application against AD.

Multifunctional iron bound lactoferrin and nanomedicinal approaches to enhance its bioactive functions.

Lactoferrin (Lf), an iron-binding protein from the transferrin family has been reported to have numerous functions. Even though Lf was first isolated from milk, it is also found in most exocrine secretions and in the secondary granules of neutrophils. Antimicrobial and anti-inflammatory activity reports on lactoferrin identified its significance in host defense against infection and extreme inflammation. Anticarcinogenic reports on lactoferrin make this protein even more valuable. This review is focused on the structural configuration of iron-containing and iron-free forms of lactoferrin obtained from different sources such as goat, camel and bovine. Apart for emphasizing on the specific beneficial properties of lactoferrin from each of these sources, the general antimicrobial, immunomodulatory and anticancer activities of lactoferrin are discussed here. Implementation of nanomedicinial strategies that enhance the bioactive function of lactoferrin are also discussed, along with information on lactoferrin in clinical trials.

Chimeric aptamers in cancer cell-targeted drug delivery.

Aptamers are single-stranded structured oligonucleotides (DNA or RNA) that can bind to a wide range of targets ("apatopes") with high affinity and specificity. These nucleic acid ligands, generated from pools of random-sequence by an in vitro selection process referred to as systematic evolution of ligands by exponential enrichment (SELEX), have now been identified as excellent tools for chemical biology, therapeutic delivery, diagnosis, research, and monitoring therapy in real-time imaging. Today, aptamers represent an interesting class of modern pharmaceuticals which with their low immunogenic potential mimic extend many of the properties of monoclonal antibodies in diagnostics, research, and therapeutics. More recently, chimeric aptamer approach employing many different possible types of chimerization strategies has generated more stable and efficient chimeric aptamers with aptamer-aptamer, aptamer-nonaptamer biomacromolecules (siRNAs, proteins) and aptamer-nanoparticle chimeras. These chimeric aptamers when conjugated with various biomacromolecules like locked nucleic acid (LNA) to potentiate their stability, biodistribution, and targeting efficiency, have facilitated the accurate targeting in preclinical trials. We developed LNA-aptamer (anti-nucleolin and EpCAM) complexes which were loaded in iron-saturated bovine lactofeerin (Fe-blf)-coated dopamine modified surface of superparamagnetic iron oxide (Fe(3)O(4)) nanoparticles (SPIONs). This complex was used to deliver the specific aptamers in tumor cells in a co-culture model of normal and cancer cells. This review focuses on the chimeric aptamers, currently in development that are likely to find future practical applications in concert with other therapeutic molecules and modalities.

Survivin signaling in clinical oncology: a multifaceted dragon.

Survivin is an inhibitor of apoptosis protein (IAP) family member preferentially expressed in a myriad of clinical cancers. The complex functional mechanism and regulatory roles of survivin in cell division and cell death has hindered current therapeutic regimes from decoding its diagnostic, prognostic, and therapeutic significance in the area of translational oncology. Pharmacological modulation of survivin was tagged with its evolving functional complexity associated with various cell-signaling cascades including PI3K/AKT, mammalian target of rapamycin (mTOR), extracellular signal-regulated kinases (ERK), mitogen-activated protein kinases (MAPK), signal transducer and activator of transcription (STAT), hypoxia-inducible factor-1α (HIF-1α), heat-shock protein 90 (HSP90), p53, B-cell lymphoma 2 (Bcl2), epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF) etc. The present review provides a multifaceted role of survivin and its mechanistic action in an array of clinical cancers. Furthermore, the utilization of novel nanotechnology-based drug delivery systems for target-specific hurling of tumors enabling contemporaneous detection, treatment, and therapeutic imaging in cancer therapy are discussed.

Survivin: a target from brain cancer to neurodegenerative disease.

Apoptosis is an important contributing factor during neuronal death in a variety of neurodegenerative disorders, including multiple sclerosis, Parkinson's disease and sciatic nerve injury. Whereas several clinical and preclinical studies have focused on the neuroprotective effects of caspase inhibitors, their clinical benefits are still unclear. Here, we discuss novel alternative strategies to protect neuronal cells from apoptotic death using members of the inhibitors of apoptosis (IAP) family. We specifically review the different roles of survivin, which is an important member of the IAP family that serves a dual role in the inhibition of apoptosis as well as a vital role in mitosis and cell division. Due to the various roles of survivin during cell division and apoptosis, targeting this protein illustrates a new therapeutic window for the treatment of neurodegenerative diseases.

Target-specific delivery of doxorubicin to retinoblastoma using epithelial cell adhesion molecule aptamer.

PURPOSE: To study target-specific delivery of doxorubicin (Dox) using an RNA aptamer against epithelial cell adhesion molecule (EpCAM) in retinoblastoma (RB) cells. METHODS: The binding affinity of the EpCAM aptamer to RB primary tumor cells, Y79 and WERI-Rb1 cells, and Müller glial cell lines were evaluated with flow cytometry. Formation of physical conjugates of aptamer and Dox was monitored with spectrofluorimetry. Cellular uptake of aptamer-Dox conjugates was monitored through fluorescent microscopy. Drug efficacy was monitored with cell proliferation assay. RESULTS: The EpCAM aptamer (EpDT3) but not the scrambled aptamer (Scr-EpDT3) bound to RB tumor cells, the Y79 and WERI-Rb1 cells. However, the EpCAM aptamer and the scrambled aptamer did not bind to the noncancerous Müller glial cells. The chimeric EpCAM aptamer Dox conjugate (EpDT3-Dox) and the scrambled aptamer Dox conjugate (Scr-EpDT3-Dox) were synthesized and tested on the Y79, WERI-Rb1, and Müller glial cells. The targeted uptake of the EpDT3-Dox aptamer caused cytotoxicity in the Y79 and WERI-Rb1 cells but not in the Müller glial cells. There was no significant binding or consequent cytotoxicity by the Scr-EpDT3-Dox in either cell line. The EpCAM aptamer alone did not cause cytotoxicity in either cell line. CONCLUSIONS: The results show that the EpCAM aptamer-Dox conjugate can selectively deliver the drug to the RB cells there by inhibiting cellular proliferation and not to the noncancerous Müller glial cells. As EpCAM is a cancer stem cell marker, this aptamer-based targeted drug delivery will prevent the undesired effects of non-specific drug activity and will kill cancer stem cells precisely in RB.

Cell-penetrating properties of the transactivator of transcription and polyarginine (R9) peptides, their conjugative effect on nanoparticles and the prospect of conjugation with arsenic trioxide.

Cell-penetrating peptides (CPPs) are short chains of amino acids with the distinct ability to cross cell plasma membranes. They are usually between seven and 30 residues in length. The mechanism of action is still a highly debated subject among researchers; it seems that a commonality between all CPPs is the presence of positively charged residues within the amino acid chain. Polyarginine and the transactivator of transcription peptide are two widely used CPPs. One distinct application of these CPPs is the ability to further enhance the therapeutic properties of a range of different agents. One group of agents of particular importance are nanoparticles (NPs). Most NPs have no mechanism for cellular uptake. Hence, by conjugating CPPs to NPs, the amount of NPs taken up by cells can be increased, and therefore, the therapeutic benefits can be maximized. Some examples of this will be explored further in this review. In addition to CPPs, the concept of conjugation with the anticancer drug arsenic trioxide is reviewed and the prospect of transactivator of transcription-conjugated arsenic trioxide albumin microspheres is also discussed. Recent locked nucleic acid technology to stabilize nucleotides (RNA or DNA) aptamer complexes able to target cancer cells more specifically and selectively to kill tumour cells and spare normal body cells. NPs tagged with modified locked nucleic acid-aptamers have the potential to kill cancer cells more specifically and effectively while sparing normal cells.

Nanoparticles in the treatment and diagnosis of neurological disorders: untamed dragon with fire power to heal.

The incidence of neurological diseases of unknown etiology is increasing, including well-studied diseases such as Alzhiemer's, Parkinson's, and multiple sclerosis. The blood-brain barrier provides protection for the brain but also hinders the treatment and diagnosis of these neurological diseases, because the drugs must cross the blood-brain barrier to reach the lesions. Thus, attention has turned to developing novel and effective delivery systems that are capable of carrying drug and that provide good bioavailability in the brain. Nanoneurotechnology, particularly application of nanoparticles in drug delivery, has provided promising answers to some of these issues in recent years. Here we review the recent advances in the understanding of several common forms of neurological diseases and particularly the applications of nanoparticles to treat and diagnose them. In addition, we discuss the integration of bioinformatics and modern genomic approaches in the development of nanoparticles. FROM THE CLINICAL EDITOR: In this review paper, applications of nanotechnology-based diagnostic methods and therapeutic modalities are discussed addressing a variety of neurological disorders, with special attention to blood-brain barrier delivery methods. These novel nanomedicine approaches are expected to revolutionize several aspects of clinical neurology.

Gut health immunomodulatory and anti-inflammatory functions of gut enzyme digested high protein micro-nutrient dietary supplement-Enprocal.

BACKGROUND: Enprocal is a high-protein micro-nutrient rich formulated supplementary food designed to meet the nutritional needs of the frail elderly and be delivered to them in every day foods. We studied the potential of Enprocal to improve gut and immune health using simple and robust bioassays for gut cell proliferation, intestinal integrity/permeability, immunomodulatory, anti-inflammatory and anti-oxidative activities. Effects of Enprocal were compared with whey protein concentrate 80 (WPC), heat treated skim milk powder, and other commercially available milk derived products. RESULTS: Enprocal (undigested) and digested (Enprocal D) selectively enhanced cell proliferation in normal human intestinal epithelial cells (FHs74-Int) and showed no cytotoxicity. In a dose dependent manner Enprocal induced cell death in Caco-2 cells (human colon adencarcinoma epithelial cells). Digested Enprocal (Enprocal D: gut enzyme cocktail treated) maintained the intestinal integrity in transepithelial resistance (TEER) assay, increased the permeability of horseradish peroxidase (HRP) and did not induce oxidative stress to the gut epithelial cells. Enprocal D upregulated the surface expression of co-stimulatory (CD40, CD86, CD80), MHC I and MHC II molecules on PMA differentiated THP-1 macrophages in coculture transwell model, and inhibited the monocyte/lymphocyte (THP-1/Jurkat E6-1 cells)-epithelial cell adhesion. In cytokine secretion analyses, Enprocal D down-regulated the secretion of proinflammatory cytokines (IL-1beta and TNF-alpha) and up-regulated IFN-gamma, IL-2 and IL-10. CONCLUSION: Our results indicate that Enprocal creates neither oxidative injury nor cytotoxicity, stimulates normal gut cell proliferation, up regulates immune cell activation markers and may aid in the production of antibodies. Furthermore, through downregulation of proinflammatory cytokines, Enprocal appears to be beneficial in reducing the effects of chronic gut inflammatory diseases such as inflammatory bowel disease (IBD). Stimulation of normal human fetal intestinal cell proliferation without cell cytotoxicity indicates it may also be given as infant food particularly for premature babies.

Author Correction: Doxorubicin Conjugated to Immunomodulatory Anticancer Lactoferrin Displays Improved Cytotoxicity Overcoming Prostate Cancer Chemo resistance and Inhibits Tumour Development in TRAMP Mice.

A correction has been published and is appended to both the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Correction: Survivin Mutant Protects Differentiated Dopaminergic SK-N-SH Cells Against Oxidative Stress.

[This corrects the article DOI: 10.1371/journal.pone.0015865.].

Corrigendum: Topical Ophthalmic Formulation of Trichostatin A and SurR9-C84A for Quick Recovery Post-alkali Burn of Corneal Haze.

[This corrects the article DOI: 10.3389/fphar.2017.00223.].

Aged macular degeneration: current therapeutics for management and promising new drug candidates.

In elderly aged related macular degeneration (AMD) is the common eye disease which impairs the vision and most of the time it creates permanent vision loss. Because elderly population constitute the larger percentage among society, visual loss due to AMD has become a growing problem. Despite the advances made in developing therapeutics, there is still no satisfactory treatment. The limitations of the available treatments are due to the absence of potent, non-invasive therapy. Furthermore, part of the available drugs targets angiogenesis and create a hypoxic environment that augment further angiogenesis. Therefore, it is reasonable to consider eye integrity and the correlation between hypoxia and angiogenesis before developing successful drugs. This review highlighted issues regarding the available therapeutic strategies and explored whether AMD can be managed by employing specific nanoformulations.