A comparative metabolomics investigation of flavonoid variation in faba bean flowers.

INTRODUCTION: Faba bean (Vicia faba L.) flowers are edible and used as garnishes because of their aroma, sweet flavor and attractive colors. Anthocyanins are the common plant pigments that give flowers their vivid colors, whereas non-anthocyanin flavonoids can serve as co-pigments that can modify the color intensity of flowers. OBJECTIVES: To explore the polyphenol diversity and differences in standard and wing petals of faba bean flowers; and identify glycosylated flavonoids that contribute to flower color. METHODS: Flower standard and wing petals from 30 faba bean genotypes (eight color groups with a total of 60 samples) were used for polyphenol extraction. Samples were analyzed using a targeted method and a semi-untargeted analysis using liquid chromatography-high resolution mass spectrometry (LC-HRMS) combined with photodiode array (PDA) detection. Compound Discoverer software was used for polyphenol identification and multivariate analysis. RESULTS: The semi-untargeted analysis guided by the PDA detected 90 flavonoid metabolites present in faba bean flower petals. Ten anthocyanins largely influenced the flower colors, but other flavonoids (63 flavonols and 12 flavones) found with variable levels in different flower color groups appeared to also influence color, especially in mixed colors. CONCLUSION: Analysis of the different colored faba bean flowers confirmed that the color variation between the flowers was mainly controlled by anthocyanins in brown, red and purple-red flowers. Of the other flavonoids, multiglycosylated kaempferols were abundant in white and brown flowers, monoglycosylated kaempferols were common in red and purple-red flowers, and quercetin and apigenin glycosides were abundant co-pigments in purple-red flowers.

Mass Spectrometry-Based Untargeted Metabolomics Reveals the Importance of Glycosylated Flavones in Patterned Lentil Seed Coats.

Lentil seed coats are rich in antioxidant polyphenols that are important for plant defense and have potential as valorized byproducts. Although biochemical differences among lentil seed coat colors have been previously studied, differences among seed coat patterns remain largely unexplored. This study used mass spectrometry-based untargeted metabolomics to investigate polyphenol differences among lentil seed coat patterns to search for biochemical pathways potentially responsible for seed coat pattern differences. Comparing patterned with non-patterned green lentil seed coats, 28 significantly upregulated metabolites were found in patterned seed coats; 19 of them were identified as flavones. Flavones were virtually absent in non-patterned seed coats, thereby strongly suggesting a blockage in their flavone biosynthetic pathway. Although the black pattern is not readily discernible on black seed coats, many of the same flavones found in green marbled seed coats were also found in black seed coats, indicating that black seed coats likely have a marbled pattern.

Development of a liquid chromatography-tandem mass spectrometry method for the analysis of docetaxel-loaded Poly(lactic-co-glycolic acid) nanoparticles.

Docetaxel is among the most effective chemotherapeutic agents used for the treatment of solid tumors, such as breast cancer. Targeting docetaxel to the tumor site would increase the safety and efficacy of the treatment. The focus of this work was to develop an efficient liquid chromatography tandem mass spectrometry (LC-MS/MS) method to quantify docetaxel entrapped in optimized poly lactic-co-glycolic acid (PLGA) nanoparticles. Several nanoparticle formulations were prepared to optimize the nanoparticles based on their size and yield percentage using a modified solvent evaporation technique. The MS/MS fingerprints of docetaxel and paclitaxel (as internal standard) were used to identify diagnostic product ion for developing a multiple reaction monitoring (MRM) LC-MS/MS method for the quantification of docetaxel in the PLGA nanoparticles. A triple quadrupole linear ion trap instrument (AB Sciex 4000 QTRAP) equipped with electrospray ionization was used. The optimized nanoparticles had a zeta potential of -23.2 ± 1.4 mV and mean particle sizes of 202.2 ± 4.7 nm and 251.7 ± 8.2 nm before and after freeze-drying, respectively. Polydispersity index values of the nanoparticles confirmed their uniform size distribution. The developed LC-MS/MS method could quantify docetaxel in the PLGA matrix with accuracy and precision covering a broad linear range of 15.6-4000 ng/mL. Method validation was conducted using the regulatory guidelines of the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) and showed acceptable values for all the tested criteria. The developed LC-MS/MS method with the novelty of using a phenyl column will be beneficial for future analysis of docetaxel loaded polymeric nano-delivery systems.

Determination of phytosterol oxidation products in pharmaceutical liposomal formulations and plant vegetable oil extracts using novel fast liquid chromatography - Tandem mass spectrometric methods.

Phytosterol oxidation products (POPs) formed by the auto-oxidation of phytosterols can lead to negative health consequences. New liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantitative and qualitative approaches were developed. For quantification, sixteen phytosterol oxidation products (POPs) in liposomal formulations; namely 7-keto, 7-hydroxy, 5,6-epoxy, and 5,6-dihydroxy derivatives of brassicasterol, campesterol, stigmasterol, and ß-sitosterol were quantified. The method has a short run time of 5 min, achieved on a poroshell C18 column, using isocratic elution. To the best of our knowledge, this is the shortest run time among reported methods for the quantitative analysis of POPs. Atmospheric pressure chemical ionization (APCI) was used, and the mobile phase was composed of acetonitrile/methanol (99:1 v/v). The quantitative method was validated as per the FDA guidelines for linearity, accuracy, precision, selectivity, sensitivity, matrix effect, dilution integrity, and stability. The method was applied for the quantification of POPs in liposomal phytosterol formulations prepared with and without tocopherols, as antioxidants. The formulation process had little impact on the formation of POPs as only 7-ketobrassicasterol was quantified in tested samples. The quantified value of POPs in liposomal samples was insignificant to impart any toxicological effects. Other degradation products such as 7-hydroxy, 5,6-epoxy and 5,6-dihydroxy derivatives of brassicasterol, campesterol and ß-sitosterol were below the lower limit of quantification. Phytosterol-containing formulations were then assessed for their oxidative stability after microwave exposure for 5 min. The incorporation of tocopherols significantly increased the stability of phytosterols in the liposomal formulations. Finally, LC-MS/MS qualitative identification of phytosterols obtained from extra virgin olive oil was performed. New POPs, namely 7-ketoavenasterol, and 7-ketomethylenecycloartenol were putatively identified, illustrating the applicability of the method to identify POPs with varying structures present in various phytosterol sources. In fact, it is the first time that 7-ketomethylenecycloartenol is reported as a POP.

Development and validation of patient-community pharmacist encounter toolkit regarding substance misuse: Delphi procedure.

BACKGROUND: Pharmacists' roles and services for patients with substance use are not well defined and inconsistent from site to site. Several barriers have been identified that hinder pharmacists' care for people who use substances, such as a lack of training and resources. Clinical practice tools can aid in transferring evidence-based approaches to the practice sphere. OBJECTIVES: The aim of the study was to develop a substance misuse management toolkit for community pharmacists to help them manage their encounters with people who use substances. METHODS: A focused literature review was conducted and 2 needs assessment studies, one for community pharmacists and one for patients informed the development of the toolkit. The toolkit is an adaption of the screening, brief intervention, and referral to treatment (SBIRT) approach, which is one of the most well-defined and effective strategies for substance use management. However, SBIRT is a novel care model in community pharmacy settings. Therefore, a substance misuse management toolkit with 20 items was created for community pharmacists incorporating evidence-based strategies and clinical algorithms. Delphi procedure was used to validate the toolkit. RESULTS: Two rounds of questions were sent to experts in the field of substance misuse, some of whom were pharmacists. In both rounds, these experts were asked to rate the appropriateness and clarity of items in the toolkit and provide comments and suggestions. Items with a median rating of 7 or more out of 10 were included in the toolkit. In the second round, the experts were asked to rerate the revised version and provide additional feedback. After the second round, agreement was reached for almost all items of the toolkit. CONCLUSION: A Delphi procedure was successfully used to provide evidence of the validity of the new guiding toolkit for community pharmacists. The toolkit will be implemented and evaluated to provide additional evidence of validity in practice.

An Untargeted Metabolomics Approach for Correlating Pulse Crop Seed Coat Polyphenol Profiles with Antioxidant Capacity and Iron Chelation Ability.

Pulse crop seed coats are a sustainable source of antioxidant polyphenols, but are typically treated as low-value products, partly because some polyphenols reduce iron bioavailability in humans. This study correlates antioxidant/iron chelation capabilities of diverse seed coat types from five major pulse crops (common bean, lentil, pea, chickpea and faba bean) with polyphenol composition using mass spectrometry. Untargeted metabolomics was used to identify key differences and a hierarchical analysis revealed that common beans had the most diverse polyphenol profiles among these pulse crops. The highest antioxidant capacities were found in seed coats of black bean and all tannin lentils, followed by maple pea, however, tannin lentils showed much lower iron chelation among these seed coats. Thus, tannin lentils are more desirable sources as natural antioxidants in food applications, whereas black bean and maple pea are more suitable sources for industrial applications. Regardless of pulse crop, proanthocyanidins were primary contributors to antioxidant capacity, and to a lesser extent, anthocyanins and flavan-3-ols, whereas glycosylated flavonols contributed minimally. Higher iron chelation was primarily attributed to proanthocyanidin composition, and also myricetin 3-O-glucoside in black bean. Seed coats having proanthocyanidins that are primarily prodelphinidins show higher iron chelation compared with those containing procyanidins and/or propelargonidins.

Establishment of the tandem mass spectrometric fingerprints of taxane-based anticancer compounds.

RATIONALE: Compounds in the taxane drug family are among the most successful and effective chemotherapeutic agents used in the treatment of solid tumors, such as breast, ovarian, and prostate cancers. The tandem mass spectrometric (MS/MS) fragmentation behavior of these compounds is described in detail, and a generalized MS/MS fingerprint is established for the first time. METHODS: Five compounds, namely paclitaxel, docetaxel, cabazitaxel, cephalomannine, and baccatin III, were evaluated. A hybrid quadrupole orthogonal time-of-flight (Q-TOF) mass spectrometer was used to obtain accurate mass measurements, whereas MS/MS and second-generation MS/MS (MS3 ) analyses were performed using a triple quadrupole-linear ion trap mass spectrometer. Both instruments were equipped with an electrospray ionization source operated in the positive ion mode. RESULTS: All taxanes showed an abundant singly charged [M + H]+ species in the single-stage analysis with mass accuracies less than 3 ppm. The evaluated compounds exhibited common fragmentation behavior in their MS/MS analysis, which allowed for the production of a universal fragmentation pattern. MS3 experiments confirmed the genesis of the various product ions proposed in the fragmentation pathway. In addition, diagnostic product ions were originated from a cleavage in the ester bond between the core diterpene ring structure and the side chain. CONCLUSIONS: Varying functional groups present in these compounds resulted in unique product ions that are specific to each structure. The established MS/MS fingerprints will be used in the near future for identification and for the development of multiple reaction monitoring liquid chromatography-MS/MS quantification methods.

Mass Spectrometric Detection and Characterization of Metabolites of Gemini Surfactants Used as Gene Delivery Vectors.

Gemini surfactants are a class of lipid molecules that have been successfully used in vitro and in vivo as nonviral gene delivery vectors. However, the biological fate of gemini surfactants has not been well investigated. In particular, the metabolism of gemini surfactants after they enter cells as gene delivery vehicles is unknown. In this work, we used a high-resolution quadrupole-Orbitrap mass spectrometry (Q-Exactive) instrument to detect the metabolites of three model gemini surfactants, namely, (a) unsubstituted (16-3-16), (b) with pyridinium head groups (16(Py)-S-2-S-16(Py)), and (c) substituted with a glycyl-lysine di-peptide (16-7N(GK)-16). The metabolites were characterized, and structures were proposed, based on accurate masses and characteristic product ions. The metabolism of the three gemini surfactants was very different as 16-3-16 was not metabolized in PAM 212 cells, whereas 16(Py)-S-2-S-16(Py) was metabolized primarily via phase I reactions, including oxidation and dealkylation, producing metabolites that could be linked to its observed high toxicity. The third gemini surfactant 16-7N(GK)-16 was metabolized mainly via phase II reactions, including methylation, acetylation, glucose conjugation, palmityl conjugation, and stearyl conjugation. The metabolism of gemini surfactants provides insight for future directions in the design and development of more effective gemini surfactants with lower toxicity. The reported approach can also be applied to study the metabolism of other structurally related gemini surfactants.

Tandem mass spectrometric analysis of novel caffeine scaffold-based bifunctional compounds for Parkinson's disease.

RATIONALE: Novel bifunctional compounds composed of a caffeine scaffold attached to nicotine (C8 -6-N), 1-aminoindan (C8 -6-I), or caffeine (C8 -6-C8 ) were designed as therapeutics or diagnostics for Parkinson's disease (PD). In order to probe their pharmacological and toxicological profile, an appropriate analytical method is required. The goal of this study is to establish a tandem mass spectrometric fingerprint for the development of quantitative and qualitative methods that will aid future assessment of the in vitro and in vivo absorption, distribution, metabolism, excretion (ADME) and pharmacokinetic properties of these lead bifunctional compounds for PD. METHODS: Accurate mass measurement was performed using a hybrid quadrupole orthogonal time-of-flight mass spectrometer while multistage MS/MS and MS3 analyses were conducted using a triple quadrupole linear ion trap mass spectrometer. Both instruments are equipped with an electrospray ionization (ESI) source and were operated in the positive ion mode. The source and compound parameters were optimized for all three tested bifunctional compounds. RESULTS: The MS/MS analysis indicates that the fragmentation of C8 -6-N and C8 -6-I is driven by the dissociation of the nicotine and 1-aminoindan moieties, respectively, but not caffeine. A significant observation in the MS/MS fragmentation of C8 -6-C8 suggests that a previously reported loss of acetaldehyde during caffeine dissociation is instead a loss of CO2 . CONCLUSIONS: The collision-induced tandem mass spectrometry (CID-MS/MS) analysis of these novel bifunctional compounds revealed compound-specific diagnostic product ions and neutral losses for all three tested bifunctional compounds. The established MS/MS fingerprint will be applied to the future development of qualitative and quantitative methods.

Peptide-Modified Gemini Surfactants: Preparation and Characterization for Gene Delivery.

Diquaternary ammonium-based gemini surfactants have been investigated widely as nonviral gene delivery systems. These unique cationic lipids have versatility in their chemical structure, show relatively low toxicity, are able to compact genetic material (pDNA, RNA) into nano-sized lipoplexes, and can be easily produced. In addition, the gemini surfactants show significant improvement in the transfection activity and biocompatibility compared to other cationic lipids used as nonviral gene delivery agents. The successful applications of gemini surfactant-based lipoplexes as topical gene delivery systems in animal models indicate their potential as noninvasive carriers for genetic immunization, theranostic agents, and in other gene therapy treatments. Detailed physicochemical characterization of gemini surfactant lipoplexes is a key factor in terms of formulation optimization and elucidation of the cellular uptake and stability of the lipoplexes system. In this chapter, we describe in detail different formulation methods to prepare gemini surfactant lipoplexes and comprehensive physicochemical characterization. In addition, we illustrate general protocols for in vitro evaluations.

Development and Characterization of Liposomal Formulations Containing Phytosterols Extracted from Canola Oil Deodorizer Distillate along with Tocopherols as Food Additives.

Phytosterols are plant sterols recommended as adjuvant therapy for hypercholesterolemia and tocopherols are well-established anti-oxidants. However, thermo-sensitivity, lipophilicity and formulation-dependent efficacy bring challenges in the development of functional foods, enriched with phytosterols and tocopherols. To address this, we developed liposomes containing brassicasterol, campesterol and ß-sitosterol obtained from canola oil deodorizer distillate, along with alpha, gamma and delta tocopherol. Three approaches; thin film hydration-homogenization, thin film hydration-ultrasonication and Mozafari method were used for formulation. Validated liquid chromatographic tandem mass spectrometry (LC-MS/MS) was utilized to determine the entrapment efficiency of bioactives. Stability studies of liposomal formulations were conducted before and after pasteurization using high temperature short time (HTST) technique for a month. Vesicle size after homogenization and ultrasonication (<200 nm) was significantly lower than by Mozafari method (>200 nm). However, zeta potential (-9 to -14 mV) was comparable which was adequate for colloidal stability. Entrapment efficiencies were greater than 89% for all the phytosterols and tocopherols formulated by all three methods. Liposomes with optimum particle size and zeta potential were incorporated in model orange juice, showing adequate stability after pasteurization (72 °C for 15 s) for a month. Liposomes containing phytosterols obtained from canola waste along with tocopherols were developed and successfully applied as a food additive using model orange juice.

The determination of gemini surfactants used as gene delivery agents in cellular matrix using validated tandem mass spectrometric method.

A simple, reliable flow injection analysis (FIA)-tandem mass spectrometric (MS/MS) method was developed for the determination of gemini surfactants, designated as 16-3-16, 16(Py)-S-2-S-(Py)16 and 16-7N(GK)-16, as gene delivery agents in cellular matrix. 16-3-16 is a conventional gemini surfactant bearing two quaternary amines, linked by a 3-carbon spacer region, 16(Py)-S-2-S-(Py)16 contains two pyridinium head groups, while 16-7N(GK)-16 bears a glycine-lysine di-peptide in the space region. The method was fully validated according to USFDA guidelines. It is the first time that FIA-MS/MS method was developed for the quantification of gemini surfactants, belonging to different structural families. The method was superior to existing liquid chromatographic (LC)-MS/MS methods in terms of sensitivity and time of analysis. Positive electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode were used on a triple quadrupole-linear ion trap (4000 QTRAP®) instrument. Deuterated internal standards were used to correct for matrix effects and variations in ionization within the ESI source. Isotope dilution standard curves were established in cellular matrix, with a linear range of 10 nM-1000 nM for 16-3-16 and 16(Py)-S-2-S-(Py)16, and 20 nM-2000 nM for 16-7N(GK)-16. The precision, accuracy, recovery and stability were all within the acceptable ranges as per the USFDA guidelines. The method was successfully applied for the quantification of target gemini surfactants in the nuclear fraction of PAM 212 keratinocyte cells treated with nanoparticles, which varied significantly and may explain differences in the observed efficiency and/or toxicity of these gemini surfactants in gene delivery.

Molecular Engineering as an Approach To Modulate Gene Delivery Efficiency of Peptide-Modified Gemini Surfactants.

The unique molecular structure confers the diquaternary ammonium gemini surfactants with enhanced nucleic acid complexation ability, bottom-up design flexibility, and relatively low cytotoxicity. To capitalize on their potential as gene delivery vectors, novel structural modifications should be explored. In this work, 22 novel peptide-modified gemini surfactants with various alkyl tails and peptide spacer modifications were evaluated. This work represents the first report of dendrimer-like gemini surfactants and first evaluation of the impact of incorporating a hydrocarbon linker into the peptide chain. Our aim was to establish a structure activity relationship of the peptide-modified gemini surfactants and to identify the fundamental architectural requirements needed for the ultimate gene delivery systems. In vitro assessment revealed that the highest transfection efficiency and lowest cytotoxicity were associated with the glycyl-lysine modified gemini surfactants having the hexadecyl tail, 16-7N(G-K)-16. In fact, it showed an 8-fold increase in secreted protein with 20% increase in cell viability relative to the first-generation unsubstituted gemini surfactants. Further increase in the size of the attached peptides resulted in a decrease in the transfection efficiency and cell viability. Whereas the incorporation of a hydrocarbon linker into the peptide chain decreased the transfection efficiency of compounds with dipeptides, it increased the transfection efficiency of compounds with larger peptide chains. Such an increase was more prominent with the incorporation of a longer hydrocarbon linker. We conclude that a balance between the hydrophilic and hydrophobic characteristics of the compound is necessary since it results in physicochemical parameters conducive to the gene delivery process.

The development of simple flow injection analysis tandem mass spectrometric methods for the cutaneous determination of peptide-modified cationic gemini surfactants used as gene delivery vectors.

Diquaternary ammonium gemini surfactants are a class of non-viral gene delivery vectors, primarily studied for their dermal applications. However, their biological fate has rarely been investigated. In this work, we developed simple flow injection analysis tandem mass spectrometric methods, (FIA)-MS/MS, to understand the fate and biodistribution of topically applied gemini surfactant-based therapeutics in an ex-vivo skin model. Three peptide-modified gemini surfactants with varied structures and transfection efficiencies were evaluated. For each compound, two methods were developed to quantify their presence in skin tissue and in phosphate buffered saline (PBS). The methods were developed using single-point calibration mode. Skin penetration was assessed on CD1 mice dorsal skin tissue mounted in a Franz diffusion cell after extraction. Amongst the five evaluated liquid-liquid extraction protocols, the Folch method provides the highest extraction efficiency for all compounds. Weak cationic exchange solid phase extraction was also used to further isolate gemini surfactants from endogenous skin lipids. FIA-MS/MS analysis of the skin revealed that all compounds were detected in the skin with minimal partition into the PBS compartment, which represents circulation. Interestingly, the detected amounts of gemini lipids in the skin were correlated with their transfection efficiencies.

Rapid and simple flow injection analysis tandem mass spectrometric method for the quantification of melphalan in a lipid-based drug delivery system.

RATIONALE: The use of the anticancer drug melphalan is limited due to its poor water solubility. To address this limitation, it is incorporated within a novel delivery system using ß-cyclodextrin-gemini surfactants (18:1ßCDg). METHODS: Herein, two fast and simple flow injection analysis/tandem mass spectrometric (FIA-MS/MS) methods are developed for the quantification of melphalan (Mel) within the drug delivery system so that the solubilization efficiency of the system can be assessed. FIA-MS/MS methods are developed using a triple quadrupole linear ion trap mass spectrometer, equipped with electrospray ionization (ESI) in the positive ion mode. A deuterated form of melphalan (melphalan-d8) was used as an internal standard (IS). The methods were validated according to the FDA guidance. RESULTS: A linearity in the range of 2-100 ng/mL and accuracy and precision below 15% were observed for all standard points and quality control samples. The intra- and inter-day variations and freeze-thaw stability were within the acceptable range according to the criteria set by regulatory guidelines. On the other hand, other stability measures, such as room temperature stability and long-term stability, did not meet the required guidelines in some cases, indicating the need for quick sample analysis upon preparation. Such a fact could have been overlooked if full method validation had not been performed. CONCLUSIONS: The developed methods were applied to determine the encapsulation/solubilization of the [18:1ßCDg/Mel] delivery system. 18:1ßCDg enhances the aqueous solubility of melphalan without the need for co-solvent. The highest melphalan solubility was observed at a melphalan18:1ßCDg/Mel complex molar ratio of 2:1. This study demonstrated that a fast analysis for the purpose of quantifying a chemically unstable drug, such as melphalan, is feasible and important for the development of commercial dosage forms.

Design and evaluation of cyclodextrin-based delivery systems to incorporate poorly soluble curcumin analogs for the treatment of melanoma.

Various analogs of curcumin show high in vitro cytotoxic activity and are potential candidates for treating a deadly skin disease, melanoma. Due to the low solubility of the drugs, a new delivery agent, namely a cationic gemini surfactant-conjugated ß-cyclodextrin, was designed to incorporate novel drug candidates of the 1,5-diaryl-3-oxo-1,4-pentadienyl family. Based on physicochemical parameters, such as particle size and zeta potential, a schematic model for the potential interaction of the drug with the delivery agent was developed. The drug formulations were highly efficient in inhibiting the growth of melanoma cells, with IC(50) values significantly lower than melphalan, the drug currently used for the treatment of in-transit melanoma. CDgemini formulations showed excellent cellular selectivity, triggering apoptosis in the A375 cell line while showing no cytotoxicity to healthy human epidermal keratinocytes. The goal is to develop this novel nanoparticle approach into a non-invasive therapy for in-transit melanoma metastasis that lacks adequate treatment to date.

Proteomics in the diagnosis of hepatocellular carcinoma: focus on high risk hepatitis B and C patients.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. The high morbidity rate associated with this cancer is mainly linked to the late diagnosis, when therapy is no longer effective and this is particularly true for high risk patients, such as hepatitis B and C infected individuals. A biomarker can be defined as a substance, found in an increased amount in the body fluids, such as blood, which can indicate the presence of liver cancer. Current screening methodologies for liver cancer in at-risk patients rely on measuring the serum level of alpha-fetoprotein (AFP), a biomarker, as well as ultrasound imaging. AFP's sensitivity is very limited since many other liver diseases can result in a very high blood level of AFP similar to that observed in HCC. In addition, AFP is not always elevated in the early stages of cancer development, when therapy is mostly effective. Imaging, on the other hand, depends to a large extent on the operator. Therefore, better diagnostic methods are needed to increase the survival rate in liver cancer patients. Proteomics can be simply defined as the protein expression of the genome; and protein expression can vary depending on the biological state. Antibody microarrays can scan for multiple targets (antigens) within the tissue or in the circulation. This technology is still in its infancy and has great potential as a diagnostic tool for hepatitis liver cancer patients. Another proteomic approach is mass spectrometry, which can detect proteins and present them as charged species (ions). The mass spectrometric technique termed SELDI (surface enhanced laser desorption ionization), releases proteins in a sample from a capturing surface that can specifically bind groups of proteins which share common features (hydrophobic, negatively charged, etc.) and the expression of thousands of proteins can be monitored simultaneously. Proteomic profiles of hepatitis patients, liver cancer patients and healthy individuals can be established and evaluated for diagnosis. Elevated proteins can further be isolated and identified using the well-established mass spectrometric protein identification methods. In this article, the technological SELDI mass spectrometry and antibody microarrays are presented at the basic level. In addition, the current state of the novel liver cancer diagnostic methods (and biomarkers) that have been evaluated with focus on high risk hepatitis B and C patients using proteomic approaches are reviewed and highlighted.

Current strategies in cancer gene therapy.

Cancer gene therapy is the most studied application of gene therapy. Many genetic alterations are involved in the transformation of a normal cell into a neoplastic one. The two main gene groups involved in cancer development are oncogenes and tumor suppressor genes. While the latter eliminates cancerous cells via apoptosis, the former enhances cell proliferation. Therefore, apoptotic genes and anti-oncogenes are widely used in cancer gene therapy. In addition to oncogenes and tumor suppressor genes, chemotherapy and gene therapy can be combined through suicide gene strategy. A suicide gene encodes for a non-mammalian enzyme; this enzyme is used to convert a non-toxic prodrug into its active cytotoxic metabolite within the cancerous cells. Tumor suppressor genes, anti-oncogenes and suicide genes target cancer cells on the molecular level. On the other hand, cancer is immunogenic in nature; therefore, it can also be targeted on the immunological level. Boosting the immune response against cancerous cells is usually achieved via genes encoding for cytokines. Interleukin-12 gene, for example, is one of the most studied cytokine genes for cancer gene therapy applications. DNA vaccines are also used after conventional treatments to eliminate remnant malignant cells. All these therapeutic strategies and other strategies namely anti-angiogenesis and drug resistant genes are briefly reviewed and highlighted in this article.

An overview of current delivery systems in cancer gene therapy.

The main objective in gene therapy is the development of efficient, non-toxic gene carriers that can encapsulate and deliver foreign genetic materials into specific cell types such as cancerous cells. During the past two decades, enormous research in the area of gene delivery has been conducted worldwide, in particular for cancer gene therapy application. Viral vectors are biological systems derived from naturally evolved viruses capable of transferring their genetic materials into the host cells. Many viruses including retrovirus, adenovirus, herpes simplex virus (HSV), adeno-associated virus (AAV) and pox virus have been modified to eliminate their toxicity and maintain their high gene transfer capability. The limitations associated with viral vectors, however, in terms of their safety, particularly immunogenicity, and in terms of their limited capacity of transgenic materials, have encouraged researchers to increasingly focus on non-viral vectors as an alternative to viral vectors. Non-viral vectors are generally cationic in nature. They include cationic polymers such as poly(ethylenimine) (PEI) and poly(L-lysine) (PLL), cationic peptides and cationic liposomes. The newly described liposomal preparation LPD (liposomes/protamine/DNA), for example, has shown superiority over conventional liposomes/DNA complexes (lipoplexes). Although non-viral vectors are less efficient than viral ones, they have the advantages of safety, simplicity of preparation and high gene encapsulation capability. This article reviews the most recent studies highlighting the advantages and the limitations of various types of gene delivery systems used in cancer gene therapy.