Limited Impact of the Protein Corona on the Cellular Uptake of PEGylated Zein Micelles by Melanoma Cancer Cells.

The formation of a protein layer "corona" on the nanoparticle surface upon entry into a biological environment was shown to strongly influence the interactions with cells, especially affecting the uptake of nanomedicines. In this work, we present the impact of the protein corona on the uptake of PEGylated zein micelles by cancer cells, macrophages, and dendritic cells. Zein was successfully conjugated with poly(ethylene glycol) (PEG) of varying chain lengths (5K and 10K) and assembled into micelles. Our results demonstrate that PEGylation conferred stealth effects to the zein micelles. The presence of human plasma did not impact the uptake levels of the micelles by melanoma cancer cells, regardless of the PEG chain length used. In contrast, it decreased the uptake by macrophages and dendritic cells. These results therefore make PEGylated zein micelles promising as potential drug delivery systems for cancer therapy.

Multiple myeloma: therapeutic delivery of antibodies and aptamers.

Multiple myeloma is the second most common hematological malignancy in adults, accounting for 2% of all cancer-related deaths in the UK. Current chemotherapy-based regimes are insufficient, as most patients relapse and develop therapy resistance. This review focuses on current novel antibody- and aptamer-based therapies aiming to overcome current therapy limitations, as well as their respective limitations and areas of improvement. The use of computer modeling methods, as a tool to study and improve ligand-receptor alignments for the use of novel therapy development will also be discussed, as it has become a rapid, reliable and comparatively inexpensive method of investigation.

siRNA Delivery to Melanoma Cells with Cationic Niosomes.

RNA interference (RNAi) is a posttranscriptional regulatory mechanism that employs siRNA. It typically results in the degradation of a target mRNA that encodes a particular protein. Treatment with siRNA therapeutics requires the use of an effective drug delivery system to assist in delivering these therapeutics into the cytoplasm of the transfected cells. Here we describe the transfection of melanoma cancer cells with siRNA using cationic niosome nanoparticles as a delivery system. The method of niosome preparation is first introduced and is followed by complex formation with siRNA and the transfection method.

Use of Nanoparticles in Delivery of Nucleic Acids for Melanoma Treatment.

Melanoma accounts for 4% of all skin cancer malignancies, with only 14% of diagnosed patients surviving for more than 5 years after diagnosis. Until now, there is no clear understanding of the detailed molecular contributors of melanoma pathogenesis. Accordingly, more research is needed to understand melanoma development and prognosis.All the treatment approaches that are currently applied have several significant limitations that prevent effective use in melanoma. One major limitation in the treatment of cancer is the acquisition of multidrug resistance (MDR). The MDR results in significant treatment failure and poor clinical outcomes in several cancers, including skin cancer. Treatment of melanoma is especially retarded by MDR. Despite the current advances in targeted and immune-mediated therapy, treatment arms of melanoma are severely limited and stand as a significant clinical challenge. Further, the poor pharmacokinetic profile of currently used chemotherapeutic agents is another reason for treatment failure. Therefore, more research is needed to develop novel drugs and carrier tools for more effective and targeted treatment.Nucleic acid therapy is based on nucleic acids or chemical compounds that are closely related, such as antisense oligonucleotides, aptamers, and small-interfering RNAs that are usually used in situations when a specific gene implicated in a disorder is deemed a therapeutically beneficial target for inhibition. However, the proper application for nucleic acid therapies is hampered by the development of an effective delivery system that can maintain their stability in the systemic circulation and enhance their uptake by the target cells. In this chapter, the prognosis of the different types of melanoma along with the currently used medications is highlighted, and the different types of nucleic acids along with the currently available nanoparticle systems for delivering these nucleic acids into melanoma cells are discussed. We also discuss recently conducted research on the use of different types of nanoparticles for nucleic acid delivery into melanoma cells and highlight the most significant outcomes.

Niosome-encapsulated balanocarpol: compound isolation, characterisation, and cytotoxicity evaluation against human breast and ovarian cancer cell lines.

Natural products have been successfully used to treat various ailments since ancient times and currently several anticancer agents based on natural products are used as the main therapy to treat cancer patients, or as a complimentary treatment to chemotherapy or radiation. Balanocarpol, which is a promising natural product that has been isolated from Hopea dryobalanoides, has been studied as a potential anticancer agent but its application is limited due to its high toxicity, low water solubility, and poor bioavailability. Therefore, the aim of this study is to improve the characteristics of balanocarpol and increase its anticancer activity through its encapsulation in a bilayer structure of a lipid-based nanoparticle drug delivery system where the application of nanotechnology can help improve the limitations of balanocarpol. The compound was first extracted and isolated from H. dryobalanoides. Niosome nanoparticles composed of Span 80 (SP80) and cholesterol were formulated through an innovative microfluidic mixing method for the encapsulation and delivery of balanocarpol. The prepared particles were spherical, small, and uniform with an average particles size and polydispersity index ∼175 nm and 0.088, respectively. The encapsulation of balanocarpol into the SP80 niosomes resulted in an encapsulation efficiency of ∼40%. The niosomes formulation loaded with balanocarpol showed a superior anticancer effect over the free compound when tested in vitro on human ovarian carcinoma (A2780) and human breast carcinoma (ZR-75-1). This is the first study to report the use of SP80 niosomes for the successful encapsulation and delivery of balanocarpol into cancer cells.

Metabolomic Profiling of the Immune Stimulatory Effect of Eicosenoids on PMA-Differentiated THP-1 Cells.

Honey bee venom has been established to have significant effect in immunotherapy. In the present study, (Z)-11-eicosenol-a major constituent of bee venom, along with its derivations methyl cis-11-eicosenoate and cis-11-eicosenoic acid, were synthesised to investigate their immune stimulatory effect and possible use as vaccine adjuvants. Stimuli that prime and activate the immune system have exerted profound effects on immune cells, particularly macrophages; however, the effectiveness of bee venom constituents as immune stimulants has not yet been established. Here, the abilities of these compounds to act as pro-inflammatory stimuli were assessed, either alone or in combination with lipopolysaccharide (LPS), by examining the secretion of tumour necrosis factor-α (TNF-α) and the cytokines interleukin-1ß (IL-1ß), IL-6 and IL-10 by THP-1 macrophages. The compounds clearly increased the levels of IL-1ß and decreased IL-10, whereas a decrease in IL-6 levels suggested a complex mechanism of action. A more in-depth profile of macrophage behaviour was therefore obtained by comprehensive untargeted metabolic profiling of the cells using liquid chromatography mass spectrometry (LC-MS) to confirm the ability of the eicosanoids to trigger the immune system. The level of 358 polar and 315 non-polar metabolites were changed significantly (p < 0.05) by all treatments. The LPS-stimulated production of most of the inflammatory metabolite biomarkers in glycolysis, the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway, purine, pyrimidine and fatty acids metabolism were significantly enhanced by all three compounds, and particularly by methyl cis-11-eicosenoate and cis-11-eicosenoic acid. These findings support the proposed actions of (Z)-11-eicosenol, methyl cis-11-eicosenoate and cis-11-eicosenoic acid as immune system stimulators.

Microfluidic manufacturing of different niosomes nanoparticles for curcumin encapsulation: Physical characteristics, encapsulation efficacy, and drug release.

Curcumin, a natural chemical compound found in Curcuma longa that has been used in antitumor and anti-inflammation applications, exhibits very limited water solubility and rapid in vivo degradation, which limits its clinical application. To overcome these limitations, niosome nanoparticles were prepared by microfluidic mixing for curcumin encapsulation. Niosome nanoparticles are lipid-based, and composed of non-ionic surfactants with cholesterol orientated into a membrane bilayer structure. Two different non-ionic surfactants were used and the mixing parameters were varied to optimize the characteristics of the prepared niosomes. The prepared niosomes had an average particle size of 70-230 nm depending on the type of non-ionic surfactant used and the mixing parameter. Moreover, all prepared niosomes were monodisperse with an average polydispersity index ranging from 0.07 to 0.3. All prepared niosomes were spherical as demonstrated by transmission electron microscopy. Curcumin was encapsulated with a maximum encapsulation efficiency of around 60% using Tween 85 as the non-ionic surfactant. Niosomes prepared by microfluidic mixing provided a controlled release of curcumin, as indicated by the release profile of curcumin, improving its therapeutic capability. These results demonstrate that niosomes prepared by microfluidic mixing to encapsulate curcumin are a promising delivery system to reach target cells.

Propolis Exerts an Anti-Inflammatory Effect on PMA-Differentiated THP-1 Cells via Inhibition of Purine Nucleoside Phosphorylase.

Previous research has shown that propolis has immunomodulatory activity. Propolis extracts from different geographic origins were assessed for their anti-inflammatory activities by investigating their ability to alter the production of tumour necrosis factor-α (TNF-α) and the cytokines interleukin-1ß (IL-1ß), IL-6 and IL-10 in THP-1-derived macrophage cells co-stimulated with lipopolysaccharide (LPS). All the propolis extracts suppressed the TNF-α and IL-6 LPS-stimulated levels. Similar suppression effects were detected for IL-1ß, but the release of this cytokine was synergised by propolis samples from Ghana and Indonesia when compared with LPS. Overall, the Cameroonian propolis extract (P-C) was the most active and this was evaluated for its effects on the metabolic profile of unstimulated macrophages or macrophages activated by LPS. The levels of 81 polar metabolites were identified by liquid chromatography (LC) coupled with mass spectrometry (MS) on a ZIC-pHILIC column. LPS altered the energy, amino acid and nucleotide metabolism in THP-1 cells, and interpretation of the metabolic pathways showed that P-C reversed some of the effects of LPS. Overall, the results showed that propolis extracts exert an anti-inflammatory effect by inhibition of pro-inflammatory cytokines and by metabolic reprogramming of LPS activity in macrophage cells, suggesting an immunomodulatory effect.

Exosomes: fighting cancer with cancer.

Exosomes are nanovesicles secreted by many cells, including cancer cells. Extensive research has been carried out to validate potential applications of exosomes and to evaluate their efficiency in a wide range of diseases, including cancer. The current knowledge on the origin, biogenesis and composition of exosomes is described. This review then focuses on the use of exosomes in cancer diagnostics and therapeutics.

Proof of concept studies for siRNA delivery by nonionic surfactant vesicles: in vitro and in vivo evaluation of protein knockdown.

RNA interference is an effective and naturally occurring post-transcriptional gene regulatory mechanism. This mechanism involves the degradation of a target messenger RNA (mRNA) through the introduction of short interfering RNA (siRNA) that is complementary to the target mRNA. The application of siRNA-based therapeutics is limited by the development of an effective delivery system, as naked siRNA is unstable and cannot penetrate the cell membrane. In this study, we investigated the use of cationic niosomes (CN) prepared by microfluidic mixing for siRNA delivery. In an in vitro model, these vesicles were able to deliver anti-luciferase siRNA and effectively suppress luciferase expression in B16-F10 mouse melanoma cells. More importantly, in an in vivo mouse model, intratumoral administration of CN-carrying anti-luciferase siRNA led to significant suppression of luciferase expression compared with naked siRNA. Thus, we have established a novel and effective system for the delivery of siRNA both in vitro and in vivo, which shows high potential for future application of gene therapeutics.

Rationalising drug delivery using nanoparticles: a combined simulation and immunology study of GnRH adsorbed to silica nanoparticles.

Silica nanoparticles (SiNPs) have been shown to have significant potential for drug delivery and as adjuvants for vaccines. We have simulated the adsorption of GnRH-I (gonadotrophin releasing hormone I) and a cysteine-tagged modification (cys-GnRH-I) to model silica surfaces, as well as its conjugation to the widely-used carrier protein bovine serum albumin (BSA). Our subsequent immunological studies revealed no significant antibody production was caused by the peptide-SiNP systems, indicating that the treatment was not effective. However, the testosterone response with the native peptide-SiNPs indicated a drug effect not found with cys-GnRH-I-SiNPs; this behaviour is explained by the specific orientation of the peptides at the silica surface found in the simulations. With the BSA systems, we found significant testosterone reduction, particularly for the BSA-native conjugates, and an antibody response that was notably higher with the SiNPs acting as an adjuvant; this behaviour again correlates well with the epitope presentation predicted by the simulations. The range of immunological and hormone response can therefore be interpreted and understood by the simulation results and the presentation of the peptides to solution, paving the way for the future rational design of drug delivery and vaccine systems guided by biomolecular simulation.

Effect of Melittin on Metabolomic Profile and Cytokine Production in PMA-Differentiated THP-1 Cells.

Melittin, the major active peptide of honeybee venom (BV), has potential for use in adjuvant immunotherapy. The immune system response to different stimuli depends on the secretion of different metabolites from macrophages. One potent stimulus is lipopolysaccharide (LPS), a component isolated from gram-negative bacteria, which induces the secretion of pro-inflammatory cytokines in macrophage cell cultures. This secretion is amplified when LPS is combined with melittin. In the present study, pure melittin was isolated from whole BV by flash chromatography to obtain pure melittin. The ability of melittin to enhance the release of tumour necrosis factor-α (TNF-α), Interleukin (IL-1ß, IL-6, and IL-10) cytokines from a macrophage cell line (THP-1) was then assessed. The response to melittin and LPS, applied alone or in combination, was characterised by metabolic profiling, and the metabolomics results were used to evaluate the potential of melittin as an immune adjuvant therapy. The addition of melittin enhanced the release of inflammatory cytokines induced by LPS. Effective chromatographic separation of metabolites was obtained by liquid chromatography-mass spectrometry (LC-MS) using a ZIC-pHILIC column and an ACE C4 column. The levels of 108 polar and non-polar metabolites were significantly changed (p ˂ 0.05) following cell activation by the combination of LPS and melittin when compared to untreated control cells. Overall, the findings of this study suggested that melittin might have a potential application as a vaccine adjuvant.

Formulation of Nonionic Surfactant Vesicles (NISV) Prepared by Microfluidics for Therapeutic Delivery of siRNA into Cancer Cells.

Small interfering RNAs (siRNA) have a broad potential as therapeutic agents to reversibly silence any target gene of interest. The clinical application of siRNA requires the use of safe and effective delivery systems. In this study, we investigated the use of nonionic surfactant vesicles (NISV) for the delivery of siRNA. Different types of NISV formulations were synthesized by microfluidic mixing and then evaluated for their physiochemical properties and cytotoxicity. The ability of the NISV to carry and transfect siRNA targeting green fluorescent protein (GFP) into A549 that stably express GFP (copGFP-A549) was evaluated. Flow cytometry and Western blotting were used to study the GFP expression knockdown, and significant knockdown was observed as a result of siRNA delivery to the cells by NISV. This occurred in particular when using Tween 85, which was able to achieve more than 70% GFP knockdown. NISV were thus demonstrated to provide a promising and effective platform for therapeutic delivery of siRNA.

Metabolomic Profiling of the Synergistic Effects of Melittin in Combination with Cisplatin on Ovarian Cancer Cells.

Melittin, the main peptide present in bee venom, has been proposed as having potential for anticancer therapy; the addition of melittin to cisplatin, a first line treatment for ovarian cancer, may increase the therapeutic response in cancer treatment via synergy, resulting in improved tolerability, reduced relapse, and decreased drug resistance. Thus, this study was designed to compare the metabolomic effects of melittin in combination with cisplatin in cisplatin-sensitive (A2780) and resistant (A2780CR) ovarian cancer cells. Liquid chromatography (LC) coupled with mass spectrometry (MS) was applied to identify metabolic changes in A2780 (combination treatment 5 µg/mL melittin + 2 µg/mL cisplatin) and A2780CR (combination treatment 2 µg/mL melittin + 10 µg/mL cisplatin) cells. Principal components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) multivariate data analysis models were produced using SIMCA-P software. All models displayed good separation between experimental groups and high-quality goodness of fit (R²) and goodness of prediction (Q²), respectively. The combination treatment induced significant changes in both cell lines involving reduction in the levels of metabolites in the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, purine and pyrimidine metabolism, and the arginine/proline pathway. The combination of melittin with cisplatin that targets these pathways had a synergistic effect. The melittin-cisplatin combination had a stronger effect on the A2780 cell line in comparison with the A2780CR cell line. The metabolic effects of melittin and cisplatin in combination were very different from those of each agent alone.

Comparison of the physical characteristics of monodisperse non-ionic surfactant vesicles (NISV) prepared using different manufacturing methods.

Non-ionic surfactant vesicles (NISV) are synthetic membrane vesicles formed by self-assembly of a non-ionic surfactant, often in a mixture with cholesterol and a charged chemical species. Different methods can be used to manufacture NISV, with the majority of these requiring bulk mixing of two phases. This mixing process is time-consuming and leads to the preparation of large and highly dispersed vesicles, which affects the consistency of the final product and could hinder subsequent regulatory approval. In this study, we have compared the physical characteristics of NISV prepared using two conventional methods (thin-film hydration method and heating method) with a recently introduced microfluidic method. The resulting particles from these methods were assessed for their physical characteristics and in vitro cytotoxicity. Through microfluidics, nano-sized NISV were prepared in seconds, through rapid and controlled mixing of two miscible phases (lipids dissolved in alcohol and an aqueous medium) in a microchannel, without the need of a size reduction step, as required for the conventional methods. Stability studies over two months showed the particles were stable regardless of the method of preparation and there were no differences in terms of EC50 on A375 and A2780 cell lines. However, this work demonstrates the flexibility and ease of applying lab-on-chip microfluidics for the preparation of NISV that could be used to significantly improve formulation research and development, by enabling the rapid manufacture of a consistent end-product, under controlled conditions.

Metabolomic Profiling of the Effects of Melittin on Cisplatin Resistant and Cisplatin Sensitive Ovarian Cancer Cells Using Mass Spectrometry and Biolog Microarray Technology.

In the present study, liquid chromatography-mass spectrometry (LC-MS) was employed to characterise the metabolic profiles of two human ovarian cancer cell lines A2780 (cisplatin-sensitive) and A2780CR (cisplatin-resistant) in response to their exposure to melittin, a cytotoxic peptide from bee venom. In addition, the metabolomics data were supported by application of Biolog microarray technology to examine the utilisation of carbon sources by the two cell lines. Data extraction with MZmine 2.14 and database searching were applied to provide metabolite lists. Principal component analysis (PCA) gave clear separation between the cisplatin-sensitive and resistant strains and their respective controls. The cisplatin-resistant cells were slightly more sensitive to melittin than the sensitive cells with IC50 values of 4.5 and 6.8 µg/mL respectively, although the latter cell line exhibited the greatest metabolic perturbation upon treatment. The changes induced by melittin in the cisplatin-sensitive cells led mostly to reduced levels of amino acids in the proline/glutamine/arginine pathway, as well as to decreased levels of carnitines, polyamines, adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD+). The effects on energy metabolism were supported by the data from the Biolog assays. The lipid compositions of the two cell lines were quite different with the A2780 cells having higher levels of several ether lipids than the A2780CR cells. Melittin also had some effect on the lipid composition of the cells. Overall, this study suggests that melittin might have some potential as an adjuvant therapy in cancer treatment.

Assessment of the antigen-specific antibody response induced by mucosal administration of a GnRH conjugate entrapped in lipid nanoparticles.

Vaccines administered parenterally have been developed against gonadotrophin-releasing hormone (GnRH) for anti-fertility and anti-cancer purposes. The aim of this study was to demonstrate whether mucosal delivery using GnRH immunogens entrapped in lipid nanoparticles (LNP) could induce anti-GnRH antibody titers. Immunogens consisting of KLH (keyhole limpet hemocyanin) conjugated to either GnRH-I or GnRH-III analogues were entrapped in LNP. Loaded non-ionic surfactant vesicles (NISVs) were administered subcutaneously, while nasal delivery was achieved using NISV in xanthan gum and oral delivery using NISV containing deoxycholate (bilosomes). NISV and bilosomes had similar properties: they were spherical, in the nanometre size range, with a slightly negative zeta potential and surface properties that changed with protein loading and inclusion of xanthan gum. Following immunization in female BALB/c mice, systemic antibody responses were similar for both GnRH-I and GnRH-III immunization. Only nasal delivery proved to be successful in terms of producing systemic and mucosal antibodies. FROM THE CLINICAL EDITOR: The main research question addressed in this study was whether mucosal delivery using gonadotrophin-releasing hormone immunogens entrapped in lipid nanoparticles could induce anti-GnRH antibody titers. Only nasal delivery proved to be successful in terms of producing systemic and mucosal antibodies with this approach.

Reproductive component vaccine developments for contraceptive and non-contraceptive uses.

INTRODUCTION: There has been no novel contraceptive development since 'the Pill', 50 years ago. Despite the subsequent steady increase in the use of contraceptives, the contraceptive needs of a significant proportion of the world population have not yet been met. The key need is for novel, effective, practical, long-lasting, affordable, non-steroidal contraceptives. Immunocontraception, based on vaccination against components of the reproductive system that do not affect other physiological systems, fulfils most of the criteria of such a contraceptive. To date, immunocontraceptives have been developed for animal use and the application to human contraception is an exciting proposition. In addition, immunocontraceptive research has provided a greater understanding of the vaccination against 'self-antigens' and has led to non-contraceptive developments for these vaccines. AREAS COVERED: This review provides an understanding of the historic context of immunocontraceptives and the progress that has been made. In some cases, the contraceptive aspect has been abandoned, but the knowledge gained has enabled other therapeutic advances. EXPERT OPINION: Reproductive research is still an important area and innovations continue to arise, which offer hope for new therapeutics in reproduction and related fields.

Phytochemicals from Flemingia vestita (Fabaceae) and Stephania glabra (Menispermeaceae) alter cGMP concentration in the cestode Raillietina echinobothrida.

Cyclic GMP (cGMP) mediates various physiological functions of nitric oxide (NO) synthesized by nitric oxide synthase (NOS). A crude peel extract and purified fraction of Flemingia vestita, as well as a crude rhizome extract of Stephania glabra and fractions were tested with respect to the activity of NOS, NO efflux and cGMP concentration in the cestode Raillietina echinobothrida in order to find out the possible mode of anthelmintic action of these plant-derived components. For comparison purposes, the parasites were also treated with pure genistein, sodium nitroprusside (SNP-a known NO donor), and the reference drug, praziquantel (PZQ). At the time of onset of paralysis in the parasites, a significant increase (32%-87%) in the NOS activity and a two to three fold increase of NO efflux into the incubation medium were observed in the treated parasites in comparison to their respective controls. The cGMP concentration in the treated parasites' tissue was also increased by 44%-103%. However, in the presence of NG-nitro-L-arginine methyl ester, a potent inhibitor of NOS, there was no increase in the cGMP concentration in the parasite tissue. This study indicates that the phytochemicals, in particular genistein and tetrahydropalmatine, from F. vestita and S. glabra, respectively, disturb the downstream signalling pathway of NO, as indicated by the change in cGMP concentration in the parasite tissue.

Immunisation with a plasmid DNA vaccine encoding gonadotrophin releasing hormone (GnRH-I) and T-helper epitopes in saline suppresses rodent fertility.

Research into active immunisation against gonadotrophin releasing hormone (GnRH-I) has gained widespread acceptance as a means of controlling reproduction and behaviour of farm, companion and wild animals. Many studies describe the use of multiple copies of the self-peptide in linear alignment and conjugation with a large carrier protein to increase the immune response to the peptide. However, problems resulting from carrier protein epitope suppression have seen a diversion of interest into the use of genetic materials to elicit an optimum immune response. In this study, a 533-bp long DNA vaccine was constructed in pcDNAV5-HisB coding for 18.871 kDa GnRH-I-T-helper-V5 epitopes fusion protein. COS1 cells transfected with the vaccine construct were found to release fusion protein into culture supernatant. The vaccine construct (100 microg/mice) in saline solution administered into the anterior quadriceps muscle of ICR male and female mice stimulated antigen-specific IgG antibody responses. Testosterone levels in the vaccinated male mice were significantly (p = 0.021) reduced. A significant reduction in uterine implants were noted following mating between immunised males and control females (p = 0.028), as well as between immunised females and control males (p = 0.004). Histological examination of both the male and female gonads in study week 13 showed atrophy of the seminiferous epithelium and suppression of folliculogenesis.