M. Lúcio

Tunable pDNA/DODAB:MO lipoplexes : the effect of incubation temperature on pDNA/DODAB:MO lipoplexes structure and transfection efficiency

Dioctadecyldimethylammonium bromide (DODAB):1-monooleoyl-rac-glycerol (MO) cationic liposomes were reported as a promising alternative to common transfection agents, showing superior effectiveness on the transfection of the 293T mammalian cell line with pSV-β-gal plasmid DNA. The study of DODAB:MO aggregates in the absence of DNA has indicated that their morphology depends on the balance between DODABs tendency to form bilayer structures and MOs propensity to form inverted non-lamellar structures. Other parameters, such as the temperature have proved to be crucial in the definition of the morphology of the developed nanocarrier. Therefore, in this work, a step forward to the current gene carrier system will be given by studying the effect of the tunable parameters (incubation temperature and MO content) on the structure of pDNA:DODAB:MO lipoplexes. More importantly, the implications that these tunable parameters could have in terms of lipoplex transfection efficiency will be investigated. Dynamic light scattering (DLS), zeta (ζ) potential, cryo-transmission electron microscopy (cryo-TEM) and ethidium bromide (EtBr) exclusion were used to assess the formation, structure and destabilization of pDNA:DODAB:MO lipoplexes at DODAB molar fractions of (1:1) and above equimolarity (2:1, 4:1) prepared at incubation temperatures from 25 to 50°C. Experimental results indicate that pDNA:DODAB:MOs structure is sensitive to the lipoplex incubation temperature, resulting in particles of distinct size, superficial charge and structure. These variations are also visible on the complexation dynamics of pDNA, and subsequent release upon incubation with the model proteoglycan heparin (HEP), at 25 and 50°C. Increase in temperature leads to re-organization of DODAB and MO molecules within the liposomal formulation, causing a positive charge re-localization in the lipoplex surface, which not only alters its structure but also its transfection efficiency. Altogether, these results confirm that in the DODAB:MO carriers, an increase in the incubation temperature has a similar effect on aggregate morphology as the observed with an increase in MO content. This conclusion is extended to the pDNA:DODAB:MO lipoplexes morphology and subsequent transfection efficiency defining new strategies in lipoplexes preparation that could be used to modulate the properties of other lipid formulations for nonviral gene delivery applications.

Structural dynamics and physicochemical properties of pDNA/DODAB:MO lipoplexes: effect of pH and anionic lipids in inverted non-lamellar phases versus lamellar phases.

Dioctadecyldimethylammonium bromide (DODAB):Monoolein (MO) lipoplexes have mainly been studied within the range of high molar ratios of DODAB, with noticeable transfection efficiencies in the Human Embryonic Kidney (HEK, a.k.a. 293T) cell line. In this work, we intend to study the effect of high MO content on the structure and physicochemical properties of pDNA/DODAB:MO lipoplexes to achieve some correlation with their transfection efficiency. Static/Dynamic Light Scattering and Cryo-TEM imaging were used to characterize the size/morphology of DNA/DODAB:MO lipoplexes at different DODAB:MO contents (2:1, 1:1, 1:2) and charge ratios (CRs) (+/-). Nile Red fluorescence emission was performed to detect changes in microviscosity, hydration and polarity of DNA/DODAB:MO systems. Lipoplexes stability at physiological pH values and in the presence of anionic lipids was evaluated by Förster Resonance Energy Transfer (FRET). Physicochemical/structural data were complemented with transfection studies in HEK cells using the β-galactosidase reporter gene activity assay. This work reports the coexistence of multilamellar and non-lamellar inverted phases in MO-richer lipoplexes (DODAB:MO 1:2 and 1:4), leading to transfection efficiencies comparable to those of multilamellar (DODAB-richer) lipoplexes, but at higher charge ratios [CR (+/-)=6.0] and without dose-effect response. These results may be related to the structural changes of lipoplexes promoted by high MO content.

Role of counter-ion and helper lipid content in the design and properties of nanocarrier systems: a biophysical study in 2D and 3D lipid assemblies

There is a direct correlation between the physicochemical properties of nanocarrier systems and their biological performance, including stability under physiological conditions, cellular internalization and transfection efficiency. Therefore, understanding the biophysical aspects that affect self-assembled nanocarriers is determinant for a rational design of efficient formulations. In this study, a comprehensive evaluation of the effects of each component on the molecular organization of aggregates formed by the cationic lipids dioctadecyldimethylammonium bromide and chloride (DODAB and DODAC) and the neutral lipid monoolein (MO) was made. Specifically, the effects of the helper lipid content (MO) and the role of the counter-ion of the cationic lipids were evaluated in 2D and 3D assemblies by Langmuir surface pressure–molecular area (π–A) isotherms, Brewster Angle Microscopy (BAM), infrared reflection absorption spectroscopy (IRRAS), confocal Raman microscopy, and Small Angle X-ray Scattering (SAXS). The results show that MO has a different distribution on the DODAC and DODAB bilayers, and a fluidizing effect dependent on the MO content. For low MO molar ratios, the fluidizing effect was more pronounced in DODAC : MO mixtures, indicating a more homogeneous distribution of MO in DODAC than in DODAB bilayers. For high MO molar ratios, packing of membranes was similar for both cationic lipids, and the effect of the counter-ion is attenuated. The distribution of MO in the two cationic systems is closely related with the efficiency of the counter-ions in the screening of the charged group.

How Multi-Step versus One-Step Preparation Method Affects the Physicochemical Properties and Transfection Efficiency of DNA/DODAB:MO Lipoplexes

The consequences for the transfection efficiencies of different lipoplexes preparation methods, largely remain to be explored, but the knowledge of how different experimental approaches can affect the physicochemical properties and transfection efficiency is essential for a proper tailoring of transfection complexes to particular applications. Therefore, the influence of the number of mixing steps (one-step addition versus multi-step addition of liposomes to plasmid DNA (pDNA)) and lipoplex incubation temperature on the final physicochemical properties and transfection efficiency of pDNA/ Dioctadecyldimethylammonium Bromide (DODAB):1-monooleoyl-rac-glycerol (MO) complexes was studied in three distinct DODAB:MO molar ratios: 4:1, 2:1 and 1:1. Dynamic Light Scattering (DLS), Zeta (I¶) Potential, Ethidium Bromide (EtBr) exclusion assays were used to assess the formation, structure and destabilization of the lipoplexes, whereas in vitro transfection assays with pSV-I²-gal plasmid DNA were performed to evaluate their transfection efficiency on the 293T mammalian cell line. Results indicate that the morphology of pDNA/DODAB:MO complexes is dependent on the lipoplex preparation method, resulting in particles of distinct size, surface charge and membrane fluidity. These variations are visible during the complexation dynamics of pDNA and continue throughout the profile of pDNA release from pDNA/DODAB:MO lipoplexes upon incubation with Heparin (HEP), as well as in the in vitro transfection assays. The stepwise addition of DODAB:MO vesicles to pDNA decreases the transfection efficiency of the lipoplexes, while the effect of the lipoplex preparation methods is dependent on the MO content.

Lipid-based colloidal carriers for topical application of antiviral drugs

Abstract Herpes infections are very frequent, affecting both men and women, especially those whose immune system is weakened, and causing manifestations in face, lips, oral, nasal and genital mucosa. Herpes can also spread to other parts of the body such as the central nervous system and leave severe squeals in about 80% of cases. The current topical therapies to treat herpes infections are based on formulations containing an antiviral agent, but whose effectiveness is limited due to low skin penetration of the actives. For this reason, the development of topical formulations containing lipid-based colloidal carriers is a promising approach to promote more effective penetration through the skin and to reduce the frequency of application. This chapter provides an overview of the current lipid-based colloidal carriers developed for topical application of antiviral drugs, and summarizes the most important challenges and strategies that researchers will find when developing formulations with this purpose.

The influence of nanocrystal size on optical second harmonic generation by para-nitroanaline embedded in electro-spun polymeric fibers

Poly(methyl methacrylate) electro-spun fibers with embedded nanocrystals of the paradigmatic donor–acceptor nonlinear chromophore para-nitroaniline have been recently demonstrated to be efficient generators of second harmonic light. To understand the influence of the size and local strain experienced by the embedded para-nitroaniline nanocrystals, a Williamson−Hall analysis was carried out on the X-ray diffraction intensity. Both the mean crystal size and strain can be tuned by simple changes in the deposition parameters of flow rate and applied voltage. The observed second harmonic signal is well correlated with the ratio of the fiber diameter to the mean para-nitroaniline crystal size suggesting that surface effects are the main source of the strong nonlinear optical response. Adjusting the electro-spinning deposition parameters when producing polymeric fibers doped with strong nonlinear organic chromophores with high dipole moments has the potential to provide a versatile and efficient method for developing second-order nonlinear optical materials.

The nanotherapy revolution: Three different applications of lipid nanosystems in therapeutics

T purpose of this work is to evaluate and quantify the impact of calculation of electrons transport on dose distribution and radiobiological predictions for lung radiotherapy. The dose was calculated using Modified Batho (PB-MB) method and Anisotropic Analytical Algorithm (AAA). Dose parameters derived from DVH for target and lung were compared. To compare dose distribution, 2D gamma (γ) index was applied. The radiobiological indices, TCP and NTCP, were also compared using EUD model. Spearman’s rank test was used to explore the best correlation coefficient (r) predicting the dose difference. The bootstrap method was used to estimate the 95% confidence intervals. Wilcoxon paired test was used to calculate p-values. For the same prescribed dose to the PTV, the plans generated with AAA predicted less dose in the target and a more heterogeneous dose distribution inside the target with p<0.05. However, MB predicted a better coverage of the target. The γ analysis showed that the difference between MB and AAA could reach up to ±10%. The MB overestimated the TCP while underestimating the NTCP with p<0.05. The data showed a good correlation between TCP and D95%, as well as NTCP with mean dose, V20 and V30 with r>0.7. The electrons’ transport taken into account by calculation algorithm as AAA showed a significant impact on delivered dose, dose distribution and TCP/NTCP. Readjusting the prescribed dose and a better optimization to protect the organs at risks should be considered in order to obtain the best clinical outcome when using AAA.

Polymeric versus lipid nanoparticles: comparative study of nanoparticulate systems as indomethacin carriers

This work was supported by FEDER through POFC – COMPETE and by national funds from FCT through the projects PEst-C/FIS/UI0607/2013 (CFUM). Marlene Lucio holds a position of Researcher FCT with the reference IF/00498/2012. We acknowledge NanoDelivery-I&D em Bionanotecnologia, Lda for access to their equipment.