Preparation and evaluation of amino acid-bearing polymers for enhanced gene expression in tumours

This thesis was previously held under moratorium from 26th July 2011 until 26th July 2013.Non-viral gene delivery is emerging as potential safer alternative to the use of viral vectors for the treatment of various gene related diseases including cancer. Although non-viral vectors may not be as effective as the viral ones, the continuous research on rationally designing multifunctional non-viral polymeric gene delivery carriers resulted in improved delivery. In this study, our aim was to develop polyethyleneimine (PEI) and generation 3 diaminobutyric polypropyleneimine (DAB) polyplex formulations that could efficiently deliver the therapeutic genes to tumours after intravenous administration through the use of three different amino acids. Conjugation of amino acids such as arginine, lysine and leucine seems an attractive tool in virtue of their excellent cell penetration properties, which results in enhancing DNA transportation into cells and improving the transfection efficacy. The conjugation of arginine, lysine and leucine with PEI led to an increase of the in vitro anti-proliferative activity in A431 cells, respectively by 34-fold, 51-fold and 40-fold compared to the unmodified polyplex. In T98G cells, the conjugation of the amino acids to PEI improved the anti-proliferative activity of the polyplex, by 2-fold for PEI-Arg, 89-fold for PEI-Lys and 269-fold for PEI-Leu. The intravenous administration of arginine-, lysine- and Leucine-bearing PEI polyplexes led to a significant increase of gene expression in the tumour, with a β-galactosidase expression at least 3-fold higher than that obtained after treatment with unmodified polyethyleneimine polyplex. The grafting of arginine, lysine and leucine to DAB led to a significant increase of in vitro anti-proliferative activity in A431 cells, respectively by 30-fold, 22-fold and 20-fold compared to the unmodified DAB polyplex. In T98G cells, the conjugation of the amino acids to DAB improved the anti-proliferative activity of the polyplex, by 43-fold for DAB-Arg, by 47-fold for DAB-Lys and by 35-fold for DAB-Leu compared to the unmodified DAB polyplex. In vivo, the intravenous administration of amino acid-bearing DAB polyplexes resulted in an improved tumour gene expression, with the highest gene expression level observed after treatment with DAB-Lys polyplex. This work corresponds to the first evaluation of gene expression of amino acid bearing non-viral delivery nanosystems in tumours following intravenous administration. In conclusion, these results, together with the lack of toxicity, make arginine-, lysine- and leucine-bearing polyethyleneimine and generation 3 polypropyleneimine polymers highly promising gene delivery systems.Non-viral gene delivery is emerging as potential safer alternative to the use of viral vectors for the treatment of various gene related diseases including cancer. Although non-viral vectors may not be as effective as the viral ones, the continuous research on rationally designing multifunctional non-viral polymeric gene delivery carriers resulted in improved delivery. In this study, our aim was to develop polyethyleneimine (PEI) and generation 3 diaminobutyric polypropyleneimine (DAB) polyplex formulations that could efficiently deliver the therapeutic genes to tumours after intravenous administration through the use of three different amino acids. Conjugation of amino acids such as arginine, lysine and leucine seems an attractive tool in virtue of their excellent cell penetration properties, which results in enhancing DNA transportation into cells and improving the transfection efficacy. The conjugation of arginine, lysine and leucine with PEI led to an increase of the in vitro anti-proliferative activity in A431 cells, respectively by 34-fold, 51-fold and 40-fold compared to the unmodified polyplex. In T98G cells, the conjugation of the amino acids to PEI improved the anti-proliferative activity of the polyplex, by 2-fold for PEI-Arg, 89-fold for PEI-Lys and 269-fold for PEI-Leu. The intravenous administration of arginine-, lysine- and Leucine-bearing PEI polyplexes led to a significant increase of gene expression in the tumour, with a β-galactosidase expression at least 3-fold higher than that obtained after treatment with unmodified polyethyleneimine polyplex. The grafting of arginine, lysine and leucine to DAB led to a significant increase of in vitro anti-proliferative activity in A431 cells, respectively by 30-fold, 22-fold and 20-fold compared to the unmodified DAB polyplex. In T98G cells, the conjugation of the amino acids to DAB improved the anti-proliferative activity of the polyplex, by 43-fold for DAB-Arg, by 47-fold for DAB-Lys and by 35-fold for DAB-Leu compared to the unmodified DAB polyplex. In vivo, the intravenous administration of amino acid-bearing DAB polyplexes resulted in an improved tumour gene expression, with the highest gene expression level observed after treatment with DAB-Lys polyplex. This work corresponds to the first evaluation of gene expression of amino acid bearing non-viral delivery nanosystems in tumours following intravenous administration. In conclusion, these results, together with the lack of toxicity, make arginine-, lysine- and leucine-bearing polyethyleneimine and generation 3 polypropyleneimine polymers highly promising gene delivery systems

Potentiation of raloxifene cytotoxicity against MCF-7 breast cancer cell lines via transdermal delivery and loading on self-emulsifying nanoemulsions

Purpose: To enhance raloxifene (RLX) delivery and cytotoxicity against breast cancer (MCF-7) cell lines. Methods: This was a solubility study of RLX in different oils, surfactants, and co-surfactants. Twelve formulae were tested to reach the smallest  globular size, and hydroxypropyl methylcellulose, (HPMC), and Carbopol 947 polymers were tested for formation of transdermal films. The formula  with the lowest size was compared with raw RLX in diffusion studies using a Franz diffusion cell. Finally, a cytotoxicity study against MCF-7 breast cancer cell lines was conducted. Results: The maximum solubility of RLX was in Tween 80, peppermint oil, and PEG 200; therefore, these were the main components of the 12 formulations. The release of RLX loaded on the selfnanoemulsion drug delivery system (SNEDDS) was increased 3-fold compared with raw RLX.Cytotoxicity results revealed that RLX SNEDDs decreased MCF-7 cell survival by approximately 40 %, compared with raw RLX (control), which augmented the RLX suppression of breast cancer cell lines. Conclusion: Improvement in RLX cytotoxicity is a novel strategy to suppress breast cancer. Keywords: Raloxifene, Osteoporosis, Bioavailability, Nanoemulsion, Nanoparticle

Potential Use of C

The photosensitizing ability of C60/2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) nanoparticles under visible light irradiation was studied by electron spin resonance (ESR) and phototoxicity on cancer cells. In addition, the photoinduced antitumor effect to the tumor-bearing mice was evaluated. C60 nanoparticles were prepared by grinding a mixture of HP-β-CyD. The resulting C60/HP-β-CyD nanoparticles were highly-sensitive to visible light and generated higher levels of 1O2 than protoporphyrin IX (PpIX). C60/HP-β-CyD reduced the viability of cancer cells (HeLa cells and A549 cells) in response to irradiation by visible light in a dose-dependent manner. The IC50 values of the C60/HP-β-CyD nanoparticles was 10 μM for HeLa cells and 60 μM for A549 cells at an irradiation level of 35 mW/cm2. The photodynamic effect of C60/HP-β-CyD nanoparticles on the in vivo growth of mouse sarcoma S-180 cells was evaluated after intratumor injection. The outcome of PDT by C60/HP-β-CyD was directly dependent on the dose of irradiated light. Treatment with C60/HP-β-CyD nanoparticles at a C60 dose of 2.0 mg/kg under visible light irradiation at 350 mW/cm2 (63 J/cm2) markedly suppressed tumor growth, whereas that at 30 J/cm2 was less effective. These findings suggest that C60/HP-β-CyD nanoparticles represent a promising candidate for use in cancer treatment by PDT

Evaluation of Hepatoprotective Activity of Adansonia digitata

The methanol extract of the fruit pulp of Adansonia digitata L. (Malvaceae) was examined for its hepatoprotective activity against liver damage induced by acetaminophen in rats. The principle depends on the fact that administration of acetaminophen will be associated with development of oxidative stress. In addition, hepatospecific serum markers will be disturbed. Treatment of the rats with the methanol extract of the fruit pulp of Adansonia digitata L. prior to administration of acetaminophen significantly reduced the disturbance in liver function. Liver functions were measured by assessment of total protein, total bilirubin, ALP, ALT, and AST. Oxidative stress parameter and antioxidant markers were also evaluated. Moreover, histopathological evaluation was performed in order to assess liver case regarding inflammatory infiltration or necrosis. Animals were observed for any symptoms of toxicity after administration of extract of the fruit pulp of Adansonia digitata L. to ensure safety of the fruit extract

Anticonvulsant and Neuroprotective Activities of Phragmanthera austroarabica

Anticonvulsant and neuroprotective activity of Phragmanthera austroarabica extract were tested in pentylenetetrazole-kindled mice. All the chemical constituents of the plant extract were identified. Additionally, the extract was standardized and proved to contain total phenolic contents equal to 379.92±1.32 mg gallic acid equivalents/g dry plant extract. Induction of kindling was achieved by repeated intraperitoneal administration of pentylenetetrazole (35 mg/kg) twice weekly. Male albino mice were given P. austroarabica extract (200, 400, or 800 mg/kg). The two higher doses (400 or 800 mg/kg) of the extract significantly caused notable reduction in seizure activity and hippocampal malondialdehyde level compared to pentylenetetrazole control group. The highest dose enhanced cortical GSH level and showed intact DNA in the laddering assay. Upon studying the neuroprotective effect, mice treated with the higher dose of the extract demonstrated an improvement in the percent of surviving neurons in the cortex and hippocampus. We concluded that P. austroarabica extract ameliorated seizure activity and protected cortical and hippocampal neurons against pentylenetetrazole-induced kindling in mice

Evaluation of Hepatoprotective Activity of Adansonia digitata Extract on Acetaminophen-Induced Hepatotoxicity in Rats

The methanol extract of the fruit pulp of Adansonia digitata L. (Malvaceae) was examined for its hepatoprotective activity against liver damage induced by acetaminophen in rats. The principle depends on the fact that administration of acetaminophen will be associated with development of oxidative stress. In addition, hepatospecific serum markers will be disturbed. Treatment of the rats with the methanol extract of the fruit pulp of Adansonia digitata L. prior to administration of acetaminophen significantly reduced the disturbance in liver function. Liver functions were measured by assessment of total protein, total bilirubin, ALP, ALT, and AST. Oxidative stress parameter and antioxidant markers were also evaluated. Moreover, histopathological evaluation was performed in order to assess liver case regarding inflammatory infiltration or necrosis. Animals were observed for any symptoms of toxicity after administration of extract of the fruit pulp of Adansonia digitata L. to ensure safety of the fruit extract

Hepatoprotective effects of bioactive compounds from traditional herb Tulsi (Ocimum sanctum Linn) against galactosamine-induced hepatotoxicity in rats

Ocimum sanctum L. (Tulsi; Family: libiaceae), also known as “The Queen of herbs” or “Holy Basil,” is an omnipresent, multipurpose plant that has been used in folk medicine of many countries as a remedy against several pathological conditions, including anticancer, antidiabetic, cardio-protective, antispasmodic, diaphoretic, and adaptogenic actions. This study aims to assess O. sanctum L.’s hepatoprotective potential against galactosamine-induced toxicity, as well as investigate bioactive compounds in each extract and identify serum metabolites. The extraction of O. sanctum L as per Ayurveda was simultaneously standardized and quantified for biochemical markers: rutin, ellagic acid, kaempferol, caffeic acid, quercetin, and epicatechin by HPTLC. Hepatotoxicity was induced albino adult rats by intra-peritoneal injection of galactosamine (400 mg/kg). The quantified hydroalcoholic and alcoholic extract of O. sanctum L (100 and 200 mg/kg body weight/day) were compared for evaluation of hepatoprotective potential, which were assessed in terms of reduction in histological damage, change in serum enzymes such as AST, ALT, ALP and increase TBARS. Twenty chemical constituents of serum metabolites of O. sanctum were identified and characterized based on matching recorded mass spectra by GC-MS with those obtained from the library-Wiley/NIST. We evaluated the hepatoprotective activity of various fractions of hydroalcoholic extracts based on the polarity and investigated the activity at each phase (hexane, chloroform, and ethyl acetate) in vitro to determine how they affected the toxicity of CCL4 (40 mM) toward Chang liver cells. The ethyl acetate fraction of the selected plants had a higher hepatoprotective activity than the other fractions, so it was used in vacuum liquid chromatography (VLC). The ethyl acetate fraction contains high amounts of rutin (0.34% w/w), ellagic acid (2.32% w/w), kaempferol (0.017% w/w), caffeic acid (0.005% w/w), quercetin (0.038% w/w), and epicatechin (0.057% w/w) which are responsible for hepatoprotection. In comparison to standard silymarin, isolated bioactive molecules displayed the most significant hepatoprotective activity in Chang liver cells treated to CCl4 toxicity. The significant high hepatoprotection provided by standard silymarin ranged from 77.6% at 100 μg/ml to 83.95% at 200 μg/ml, purified ellagic acid ranged from 70% at 100 μg/ml to 81.33% at 200 μg/ml, purified rutin ranged from 63.4% at 100 μg/ml to 76.34% at 200 μg/ml purified quercetin ranged from 54.33% at 100 μg/ml to 60.64% at 200 μg/ml, purified epicatechin ranged from 53.22% at 100 μg/ml to 65.6% at 200 μg/ml, and purified kaempferol ranged from 52.17% at 100 μg/ml to 60.34% at 200 μg/ml. These findings suggest that the bioactive compounds in O. sanctum L. have significant protective effects against galactosamine-induced hepatotoxicity

Optimized D-α-tocopherol polyethylene glycol succinate/phospholipid self-assembled mixed micelles: A promising lipid-based nanoplatform for augmenting the antifungal activity of fluconazole

Fluconazole (FLZ) is the most widely used antifungal agent for treating cutaneous candidiasis. Although oral FLZ has been proved to be effective, the incidence of side effects necessitates the development of an effective formulation that could surpass the pitfalls associated with systemic availability. Accordingly, this research aimed at developing a self-assembled mixed micelles topical delivery system to enhance the topical delivery of the drug. Self-assembled mixed micelles were developed using D-α-tocopheryl polyethylene glycol 1000 succinate and phospholipids and optimized using Box-Behnken design. The optimized formulation with minimized size was then tested in vivo for the antifungal activity against C. albicans in immunocompromised mice. Treatment with the optimized formulation led to decreased peripheral erythema as well as lesions due to fungal infection in comparison to raw FLZ loaded gel. Therefore, the developed formulation was found to be a promising vehicle for the treatment of cutaneous candidiasis

Optimized Chitosan/Anion Polyelectrolyte Complex Based Inserts for Vaginal Delivery of Fluconazole: In Vitro/In Vivo Evaluation

(1) Background: Fluconazole, used orally for vaginal candidiasis, has reported gastrointestinal side effects. Therefore, researchers directed towards the drug vaginal delivery. However, vaginal delivery is limited by poor retention and leakage. Thus, this work aimed at exploring chitosan/anion polyelectrolyte complex (PEC) for the formulation of fluconazole vaginal inserts with controlled release and appreciable mucoadhesion. (2) Methods: PECs were prepared and assessed for interactions. Fluconazole PEC based vaginal inserts were prepared by lyophilization using mannitol. 3151 factorial design was applied to investigate the effect of the anion type and Chitosan/anion ratio on the inserts mucoadhesion and release properties. The optimized insert [based on 5:5 chitosan: anionic polymer (sodium alginate)] release was modulated by the release retardant; Compritol® 888. The selected formulation was subjected to microbiological and histological evaluation. (3) Results: Fluconazole inserts showed satisfactory drug content, acceptable friability percentages and highest swelling indices at six hours. Statistical analysis showed significant effect of the studied factors on detachment force and release properties. Microbiological assays revealed significantly higher antifungal activity of inserts compared to fluconazole solution. Reduced inflammatory cells were confirmed by histological evaluation. (4) Conclusion: CH/Alg based vaginal insert could be a promising platform for vaginal delivery of antifungal drugs used for vaginal candidiasis treatment

Formulation and Optimization of Avanafil Biodegradable Polymeric Nanoparticles: A Single-Dose Clinical Pharmacokinetic Evaluation

Avanafil (AVA) is a second-generation phosphodiesterase-5 (PDE5) inhibitor. AVA shows high selectivity to penile tissues and fast absorption, but has a bioavailability of about 36%. The aim was to formulate and optimize AVA-biodegradable nanoparticles (NPs) to enhance AVA bioavailability. To assess the impact of variables, the Box–Behnken design was utilized to investigate and optimize the formulation process variables: the AVA:poly (lactic-co-glycolic acid) (PLGA) ratio (w/w, X1); sonication time (min, X2); and polyvinyl alcohol (PVA) concentration (%, X3). Particle size (nm, Y1) and EE% (%, Y2) were the responses. The optimized NPs were characterized for surface morphology and permeation. Furthermore, a single-oral dose (50 mg AVA) pharmacokinetic investigation on healthy volunteers was carried out. Statistical analysis revealed that all the investigated factors exhibited a significant effect on the particle size. Furthermore, the entrapment efficiency (Y2) was significantly affected by both the AVA:PLGA ratio (X1) and PVA concentration (X3). Pharmacokinetic data showed a significant increase in the area under the curve (1.68 folds) and plasma maximum concentration (1.3-fold) for the AVA NPs when compared with raw AVA. The optimization and formulation of AVA as biodegradable NPs prepared using solvent evaporation (SE) proves a successful way to enhance AVA bioavailability