Graphene Nanoribbons as a Drug Delivery Agent for Lucanthone Mediated Therapy of Glioblastoma Multiforme

We report use of PEG-DSPE coated oxidized graphene nanoribbons (O-GNR-PEG-DSPE) as agent for delivery of anti-tumor drug Lucanthone (Luc) into Glioblastoma Multiformae (GBM) cells targeting base excision repair enzyme APE-1 (Apurinic endonuclease-1). Lucanthone, an endonuclease inhibitor of APE-1, was loaded onto O-GNR-PEG-DSPEs using a simple non-covalent method. We found its uptake by GBM cell line U251 exceeding 67% and 60% in APE-1-overexpressing U251, post 24 h. However, their uptake was ~ 38% and 29% by MCF-7 and rat glial progenitor cells (CG-4), respectively. TEM analysis of U251 showed large aggregates of O-GNR-PEG-DSPE in vesicles. Luc-O-GNR-PEG-DSPE was significantly toxic to U251 but showed little/no toxicity when exposed to MCF-7/CG-4 cells. This differential uptake effect can be exploited to use O-GNR-PEG-DSPEs as a vehicle for Luc delivery to GBM, while reducing nonspecific cytotoxicity to the surrounding healthy tissue. Cell death in U251 was necrotic, probably due to oxidative degradation of APE-1.

Graphical abstract

We used O-GNR-PEG-DSPE as a reliable, non-toxic vehicle for delivery of APE-1 inhibiting Lucanthone into GBM tumor cell lines. LUC-O-GNR-PEG-DSPE particles showed 60% or more uptake by CMV/U251 and A1-5/CMV/U251 where as the uptake by MCF7 and normal CG4 glial cells was much lower (38% and 29% respectively). Different concentrations of Luc (5–80 μM) loaded onto O-GNR-PEG-DSPE showed lower toxicity in the exposed cells compared to the free drug, due to possible slow release of the drug from this particle, which ensures minimum non-specific release of the drug from the particle once it is injected in vivo.
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Development and characterization of novel vehicles for topical drug delivery

Tese de doutoramento, Farmácia (Tecnologia Farmacêutica), Universidade de Lisboa, Faculdade de Farmácia, 2016

'Rushing behind the wheel' : investigating the prevalence of 'drug driving' among club and rave patrons in Melbourne, Australia

The paper reports the findings of survey research recently completed in Melbourne, Australia, among a sample of 455 club and rave festival patrons. This research aims to provide a clearer account of the prevalence of drug driving within such settings in Melbourne, as well as identifying relevant ‘predictors’ of this drug driving. Just under half of the sample (48%) indicated that they had driven a motor vehicle within four hours of consuming an illicit substance at least once in the past year; 22% of respondents reported driving while ‘knowingly intoxicated’ in the previous year. Fifteen percent reported such behaviour ‘several times’ or more in this time. Relatively permissive attitudes towards drug driving, coupled with higher than average prevalence of drug driving in one's peer group, were found to be significant predictors of drug driving. So too was the reported frequency of the use of cannabis and ecstasy, and the propensity to use these substances in ‘rave’ and ‘party’ settings. Given the levels of drug driving revealed in this study, the paper closes with a series of recommendations regarding the design and delivery of more effective anti-drug-driving strategies within rave and club settings.

Graphene/tri-block copolymer composites prepared via RAFT polymerizations for dual controlled drug delivery via pH stimulation and biodegradation

A novel tri-block copolymer poly(oxopentanoate ethyl methacrylate)-block-poly(pyridyl disulfide ethyl acrylate)-block-poly(ethylene glycol acrylate) [poly(OEMA-b-PDEA-b-PEGA)], retaining active keto groups and pyridyl disulfide (PDS) side functionalities, was synthesized as a drug delivery vehicle using reversible addition-fragmentation chain transfer (RAFT) polymerization method. One mimic drug pyridine-2-thione (PT) was introduced into the monomer, PDEA for copolymerization. The other mimic drug O-benzylhydroxylamine (BHA) was conjugated with tri-block copolymer via efficient oxime coupling chemistry, followed by the attachment onto graphene via π-π stacking interaction to obtain a graphene/tri-block copolymer composite. 1H NMR, UV-vis absorption spectroscopy, fluorescence spectroscopy, gel permeation chromatography (GPC), atomic force microscope (AFM) and transmission electron microscope (TEM) were used to verify the successful step-wise preparation of the tri-block copolymer and drug loaded composite. In vitro release behaviors of BHA and PT from graphene/tri-block copolymer composite via dual drug release mechanisms were investigated. BHA can be released under acid environment, while PT will be released in the presence of reducing agents, such as dithiothreitol (DTT) or glutathione (GSH). It can be envisioned that this novel composite could be exploited as a novel intracellular drug delivery system via dual release mechanisms.

Cell-responsive nanogels for anticancer drug delivery

One of the main goals in Nanomedicine is to create innovative drug delivery systems (DDS) capable of delivering drugs into a specific location with high efficiency. In the development of DDS, some essential properties are desired, such as biocompatibility and biodegradability. Furthermore, an ideal DDS should be able to deliver a drug in a controlled manner and minimize its side effects. These two objectives are still a challenge for researchers all around the world. Nanogels are an excellent vehicle to use in drug delivery and several other applications due to their biocompatibility. They are polymer-based networks, chemically or physically crosslinked, with at least 80-90% water in their composition. Their properties can be tuned, like the nanogel size, multifunctionality and degradability. Nanogels are capable of carrying in their interior bioactive molecules and deliver them into cells. The main objective of this project was to produce nanogels for the delivery of anticancer drugs with the ability of responding to existent stimuli inside cells (cellresponsiveness nanogels) and/or of controlled drug delivery. The nanogels were mainly based on alginate (AG), a natural biopolymer, and prepared using emulsion approaches. After their synthesis, they were used to encapsulate doxorubicin (Dox) which was chosen as a model drug. In the first part of the experimental work, disulfide-linked AG nanogels were prepared and, as expected, were redox-sensitive to a reducing environment like the intracellular medium. In the second part, AG nanogels crosslinked with both calcium ions and cationic poly(amidoamine) dendrimers were developed with improved sustained drug delivery. The prepared nanogels were characterized in terms of size, chemical composition, morphology, and drug delivery behavior (under redox/pH stimuli). The in vitro cytotoxicity of the nanogels was also tested against CAL-72 cells (an osteosarcoma cell line).

Nasal administration of liquid crystal precursor mucoadhesive vehicle as an alternative antiretroviral therapy

The purpose of this study was to develop a mucoadhesive stimuli-sensitive drug delivery system for nasal administration of zidovudine (AZT). The system was prepared by formulating a low viscosity precursor of a liquid crystal phase, taking advantage of its lyotropic phase behavior. Flow rheology measurements showed that the formulation composed of PPG-5-CETETH-20, oleic acid and water (55, 30, 15% w/w), denominated P, has Newtonian flow behavior. Polarized light microscopy (PLM) revealed that formulation P is isotropic, whereas its 1:1 (w/w) dilution with artificial nasal mucus (ANM) changed the system to an anisotropic lamellar phase (PD). Oscillatory frequency sweep analysis showed that PD has a high storage modulus (G′) at nasal temperatures. Measurement of the mucoadhesive force against excised porcine nasal mucosa or a mucin disk proved that the transition to the lamellar phase tripled the work of mucoadhesion. Ex vivo permeation studies across porcine nasal mucosa exhibited an 18-fold rise in the permeability of AZT from the formulation. The Weibull mathematical model suggested that the AZT is released by Fickian diffusion mechanisms. Hence, the physicochemical characterization, combined with ex vivo studies, revealed that the PPG-5-CETETH-20, oleic acid, and water formulation could form a mucoadhesive matrix in contact with nasal mucus that promoted nasal absorption of the AZT. For an in vivo assessment, the plasma concentrations of AZT in rats were determined by HPLC method following intravenous and intranasal administration of AZT-loaded P formulation (PA) and AZT solution, respectively, at a dose of 8 mg/kg. The intranasal administration of PA resulted in a fast absorption process (Tmax = 6.7 min). Therefore, a liquid crystal precursor formulation administered by the nasal route might represent a promising novel tool for the systemic delivery of AZT and other antiretroviral drugs. In the present study, the uptake of AZT absorption in the nasal mucosa was demonstrated, providing new foundations for clinical trials in patients with AIDS. © 2012 Elsevier B.V. All rights reserved.

Development and characterization of biocompatible isotropic and anisotropic oil-in-water colloidal dispersions as a new delivery system for methyl dihydrojasmonate antitumor drug

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Oil-in-water biocompatible microemulsion as a carrier for the antitumor drug compound methyl dihydrojasmonate

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

The Use of Nanotechnology in Cosmetic Formulations: The Influence of Vehicle in the Vitamin A Skin Penetration

Despite the efficacy of topical retinoic acid, skin reactions have limited its acceptance by patients. Other retinoids, like Retinyl Palmitate (RP), are considerably less irritating, but they are also less effective. In order to enhance the performance of retinoids, in this work RP has been added to cosmetic formulations such as nanoemulsions, which can provide better penetration of this active substance. Because the vehicle can directly influence the skin penetration and the effectiveness of RP, two skin care products containing 5000 UI RP have been developed and investigated, namely a nanoemulsifying system and a classic gel cream. In vitro penetration tests were conducted by using Franz diffusion cells and placing porcine ear skin and iso-propanol in the receptor compartment. The RP concentration in the skin layers was analyzed by high performance liquid chromatography, and a Zeta-Sizer system was employed for measurement of the the particle size distribution. The penetration tests revealed a large difference between the vehicles in terms of the RP concentrations in each skin layer. The classic gel cream furnished better RP penetration in both the stratum corneum and the epidermis without stratum corneum + dermis, as compared to the self-nanoemulsifying system. The two vehicles displayed the same particle size (between 100 and 200 nm). Better understanding of RP skin delivery using different vehicles has been acquired, and the importance of evaluating the efficacy of nanocosmetics. Results from the present study should also contribute to the assessment of commercial self-nanoemulsifying systems with potential application in the facile production of nanoemulsions.

Scale Up of PLA Nanoparticle Synthesis by Solvent Displacement and Functionalization of the Particles with a Silica Shell for Targeted Drug Delivery Applications

Biodegradable polymer nanoparticles have the properties necessary to address many of the issues associated with current drug delivery techniques including targeted and controlled delivery. A novel drug delivery vehicle is proposed consisting of a poly(lactic acid) nanoparticle core, with a functionalized, mesoporous silica shell. In this study, the production of PLA nanoparticles is investigated using solvent displacement in both a batch and continuous manner, and the effects of various system parameters are examined. Using Pluronic F-127 as the stabilization agent throughout the study, PLA nanoparticles are produced through solvent displacement with diameters ranging from 200 to 250 nm using two different methods: dropwise addition and in an impinging jet mixer. The impinging jet mixer allows for easy scale-up of particle production. The concentration of surfactant and volume of quench solution is found to have minimal impact on particle diameter; however, the concentration of PLA is found to significantly impact the diameter mean and polydispersity. In addition, the stability of the PLA nanoparticles is observed to increase as residual THF is evaporated. Lastly, the isolated PLA nanoparticles are coated with a silica shell using the Stöber Process. It is found that functionalizing the silica with a phosphonic silane in the presence of excess Pluronic F-127 decreases coalescence of the particles during the coating process. Future work should be conducted to fine-tune the PLA nanoparticle synthesis process by understanding the effect of other system parameters and in synthesizing mesoporous silica shells.

Development and formulation of wax-based transdermal drug delivery systems

Topical and transdermal formulations are promising platforms for the delivery of drugs. A unit dose topical or transdermal drug delivery system that optimises the solubility of drugs within the vehicle provides a novel dosage form for efficacious delivery that also offers a simple manufacture technique is desirable. This study used Witepsol® H15 wax as a abase for the delivery system. One aspect of this project involved determination of the solubility of ibuprofen, flurbiprofen and naproxen in the was using microscopy, Higuchi release kinetics, HyperDSC and mathematical modelling techniques. Correlations between the results obtained via these techniques were noted with additional merits such as provision of valuable information on drug release kinetics and possible interactions between the drug and excipients. A second aspect of this project involved the incorporation of additional excipients: Tween 20 (T), Carbopol®971 (C) and menthol (M) to the wax formulation. On in vitro permeation through porcine skin, the preferred formulations were: ibuprofen (5% w/w) within Witepsol®H15 + 1% w/w T; flurbiprofen (10% w/w) within Witepsol®H15 + 1% w/w T; naproxen (5% w/w) within Witepsol®H15 + 1% w/w T + 1% C and sodium diclofenac (10% w/w) within Witepsol®H15 + 1% w/w T + 1% w/w T + 1% w/w C + 5% w/w M. Unit dose transdermal tablets containing ibuprofen and diclofenac were produced with improved flux compared to marketed products; Voltarol Emugel® demonstrated flux of 1.68x10-3 cm/h compared to 123 x 10-3 cm/h for the optimised product as detailed above; Ibugel Forte® demonstrated a permeation coefficient value of 7.65 x 10-3 cm/h compared to 8.69 x 10-3 cm/h for the optimised product as described above.

Microspheres as a vehicle for biomolecule delivery to neural stem cells

Neural stem cells (NSC) are a valuable model system for understanding the intrinsic and extrinsic controls for self-renewal and differentiation choice. They also offer a platform for drug screening and neurotoxicity studies, and hold promise for cell replacement therapies for the treatment of neurodegenerative diseases. Fully exploiting the potential of this experimental tool often requires the manipulation of intrinsic cues of interest using transfection methods, to which NSC are relatively resistant. In this paper, we show that mouse and human NSC readily take up polystyrene-based microspheres which can be loaded with a range of chemical or biological cargoes. This uptake can take place in the undifferentiated stage without affecting NSC proliferation and their capacity to give rise to neurons and glia. We demonstrate that ß-galactosidase-loaded microspheres could be efficiently introduced into NSC with no apparent toxic effect, thus providing proof-of-concept for the use of microspheres as an alternative biomolecule delivery system.

Phytochemical-loaded mesoporous silica nanoparticles for nose-to-brain olfactory drug delivery

Central nervous system (CNS) drug delivery is often hampered due to the insidious nature of the blood-brain barrier (BBB). Nose-to-brain delivery via olfactory pathways have become a target of attention for drug delivery due to bypassing of the BBB. The antioxidant properties of phytochemicals make them promising as CNS active agents but possess poor water solubility and limited BBB penetration. The primary aim of this study was the development of mesoporous silica nanoparticles (MSNs) loaded with the poorly water-soluble phytochemicals curcumin and chrysin which could be utilised for nose-to-brain delivery. We formulated spherical MSNP using a templating approach resulting in ∼220nm particles with a high surface porosity. Curcumin and chrysin were successfully loaded into MSNP and confirmed through Fourier transformation infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and HPLC approaches with a loading of 11-14% for curcumin and chrysin. Release was pH dependant with curcumin demonstrating increased chemical stability at a lower pH (5.5) with a release of 53.2%±2.2% over 24h and 9.4±0.6% for chrysin. MSNP were demonstrated to be non-toxic to olfactory neuroblastoma cells OBGF400, with chrysin (100μM) demonstrating a decrease in cell viability to 58.2±8.5% and curcumin an IC50 of 33±0.18μM. Furthermore confocal microscopy demonstrated nanoparticles of <500nm were able to accumulate within cells with FITC-loaded MSNP showing membrane localised and cytoplasmic accumulation following a 2h incubation. MSNP are useful carriers for poorly soluble phytochemicals and provide a novel vehicle to target and deliver drugs into the CNS and bypass the BBB through olfactory drug delivery.

Enhancement of a novel gene therapy approach for Sandhoff disease through complimentary drug therapy

GM2 gangliosidoses is a family of severe, neurodegenerative disorders resulting from a deficiency in the β-hexosaminidase A (Hex A) enzyme. This disorder is typically caused by a mutation to either the HEXA gene, causing Tay Sachs disease, or a mutation to the HEXB gene, causing Sandhoff disease. The HEXA and HEXB genes are required to produce the α and β subunits of the Hex A enzyme respectively. Using a Sandhoff disease (SD) mouse model (Hexb-/-) we tested the potential of a low dose of systemically delivered single stranded adeno-associated virus 9 (ssAAV9) expressing human HEXB and human HEXA cDNA under the control of a single promoter through the use of a bicistronic vector design with a P2A linker to correct the neurological phenotype. Neonatal mice were injected with either this ssAAV9-HexB-P2A-HexA vector (HexB-HexA) or a vehicle solution via the superficial temporal vein. HexB-HexA treatment alone conferred an increase in survival of 56% compared to vehicle-injected controls and biochemical analysis of the brain tissue and serum revealed an increase in HexA activity and a decrease in brain GM2 ganglioside buildup. Additionally, treatments with the non-steroidal anti-inflammatory drug indomethacin (Indo), the histone deactylase inhibitor ITF2357 (ITF) and the pharmacological chaperone pyrimethamine (Pyr) were tested. The anti-inflammatory treatments of Indo and ITF conferred an increase in survival of 12% and 8% respectively while causing no alteration in the HexA activity or GM2 ganglioside buildup. Pyr had no observable effect on disease progression. Lastly HexB-HexA treatment was tested in conjunction with Indo, ITF and Pyr individually. Additive increases in survival and behavioural testing results were observed with Indo and ITF treatments while no additional benefit to HexA activity or GM2 ganglioside levels in the brain tissue was observed. This indicates the two treatments slowed the progression of the disease through a different mechanism than the reduction of the GM2 ganglioside substrate. Pyr treatment was shown to have no effect when combined with HexB-HexA treatment. This study demonstrates the potential amelioration of SD with a novel AAV9 gene therapy approach as well as helped to identify the additive potential of anti-inflammatory treatments in gene therapy of GM2 gangliosidoses.

Development of polymeric drug delivery vehicles for targeted cancer therapy

The aim of my Ph. D. thesis is to generalize a method for targeted anti-cancer drug delivery. Hydrophilic polymer-drug conjugates involve complicated synthesis; drug-encapsulated polymeric nanoparticles limit the loading capability of payloads. This thesis introduces the concept of nanoconjugates to overcome difficulties in synthesis and formulation. Drugs with hydroxyl group are able to initiate polyester synthesis in a regio- and chemo- selective way, with the mediation of ligand-tunable Zinc catalyst. Herein, three anti-cancer drugs are presented to demonstrate the high efficiency and selectivity in the method (Chapter 2-4). The obtained particles are stable in salt solution, releasing drugs over weeks in controlled manner. With the conjugation of aptamer, particles are capable to target prostate cancer cells in vitro. These results open the gateway to evaluate the in vivo efficacy of nanoconjugates for target cancer therapy (Chapter 5). Mechanism study of the polymerization leads to the discovery of chemosite selective synthesis of prodrugs with acrylate functional groups. Functional copolymer-drug conjugates will expand the scope of nanoconjugates (Chapter 6). Liposome-aptamer targeting drug delivery vehicle is well studied to achieve reversible cell-specific delivery of non-hydoxyl drugs e.g. cisplatin (Chapter 7). New monomers and polymerization mechanisms are explored for polyester in order to synthesize nanoconjugates with variety on properties (Chapter 8). Initial efforts to apply this type of prodrugs will be focused on the preparation of hydrogels for stem cell research (Chapter 9).