Synthesis and evaluation of polymer nanoparticles for delivery in RPE cells

Ocular pathologies are among the most debilitating medical conditions affecting all segments of the population. Traditional treatment options are often ineffective, and gene therapy has the potential to become an alternative approach for the treatment of several pathologies. Methacrylate polymers have been described as highly biocompatible and are successfully used in medical applications. Due to their cationic nature, these polymers can be used to form polyplexes with DNA for its delivery. This work aims to study the potential of PDMAEMA (poly(2-(N,N’-dimethylamino)ethyl methacrylate)) as a non viral gene delivery system to the retina. The first part of this work aimed to study the potential for gene delivery of a previously synthesized PDMAEMA polymer of high molecular weight (354kDa). In the second part, we synthesized by RAFT a PDMAEMA with a lower molecular weight (103.3kDa) and similarly, evaluated its ability to act as a gene delivery vehicle. PDMAEMA/DNA polyplexes were prepared at 5, 7.5, 10, 12.5 and 20 nitrogen/phosphorous (N/P) ratio for the 354kDa PDMAEMA and at 5 and 7.5 for the 103.3kDa PDMAEMA. Dynamic light scattering and zeta potential measurements confirmed the nanosize and positive charge of polyplexes for all ratios and for both polymers. Both high and low Mw PDMAEMA were able to efficiently complex and protect DNA from DNase I degradation. Their cytotoxicity was evaluated using a non-retinal cell line (HEK293) and a retinal pigment epithelium (RPE) cell line (D407). We have found that cytotoxicity of the free polymer is concentration and time dependent, as expected, and negligible for all the concentrations of the PDMAEMA-DNA polyplexes. Furthermore, for the concentrations to be used in vivo, the 354kDa PDMAEMA showed no signs of inflammation upon injection in the intravitreal space of C57BL/6 mice. The transfection efficiency, as evaluated by fluorescence microscopy and flow cytometry, showed that the D407 retinal cells were transfected by polyplexes of both high and low Mw PDMAEMA, but with varied efficiency, which was dependent on the N/P ratio. Althogether, these results suggest that PDMAEMA is a feasible candidate for non-viral gene delivery to the retina, and this work constitutes the basis of further studies to elucidate the bottleneck in transfection and further optimization of the material.

Evaluation of the antioxidant activity and antitumor activity of marine invertebrates extracts

Dissertação de Mestrado, Biologia Marinha, Faculdade de Ciências e Tecnologias, Universidade do Algarve, 2014

An alternative methodology for fitting selectivity curves to pre-defined distributions

A non-linear least-squares methodology for simultaneously estimating parameters of selectivity curves with a pre-defined functional form, across size classes and mesh sizes, using catch size frequency distributions, was developed based on the model of Kirkwood and Walker [Kirkwood, G.P., Walker, T.L, 1986. Gill net selectivities for gummy shark, Mustelus antarcticus Gunther, taken in south-eastern Australian waters. Aust. J. Mar. Freshw. Res. 37, 689-697] and [Wulff, A., 1986. Mathematical model for selectivity of gill nets. Arch. Fish Wiss. 37, 101-106]. Observed catches of fish of size class I in mesh m are modeled as a function of the estimated numbers of fish of that size class in the population and the corresponding selectivities. A comparison was made with the maximum likelihood methodology of [Kirkwood, G.P., Walker, T.I., 1986. Gill net selectivities for gummy shark, Mustelus antarcticus Gunther, taken in south-eastern Australian waters. Aust. J. Mar. Freshw. Res. 37, 689-697] and [Wulff, A., 1986. Mathematical model for selectivity of gill nets. Arch. Fish Wiss; 37, 101-106], using simulated catch data with known selectivity curve parameters, and two published data sets. The estimated parameters and selectivity curves were generally consistent for both methods, with smaller standard errors for parameters estimated by non-linear least-squares. The proposed methodology is a useful and accessible alternative which can be used to model selectivity in situations where the parameters of a pre-defined model can be assumed to be functions of gear size; facilitating statistical evaluation of different models and of goodness of fit. (C) 1998 Elsevier Science B.V.