• Biophysics
• Dendrimers
• Fluorescence Microscopy
• Single-Molecule

Biophysics is a relatively new approach to the Life Sciences, founded on a strongly multidisciplinary background of Biology, Chemistry and Physics. It focuses on the biochemical processes that characterize the carbon-based life, and aims to explain them as fully as possible trying to frame them in a theoretical model. In order to reach this goal, fluorescence microscopy and spectroscopy, paticularly in time-resolved variants, are establishing as primary research tools. Some of the most recent and promising applications of Biophysics are in the biomedical field. One of the scientists’ ultimate purpose would be realizing nano-devices capable of diagnosing and healing the single diseased cells, without involving the healthy ones. It is evident how nano-medicine would be much less invasive and much more accurate than its macroscopic counterpart. To achieve this goal, it is desirable to develop and create a multivalent dispositive with the following characteristics: a scaffold that should be biocompatible and metabolizable by the body once its job is finished, cell-penetrating and able to carry targeting agents (peptides, antibodies, small ligands), sensing and/or imaging moieties (fluorophores or probes of other kind) and actuators (drugs or similar). In this view, many macromolecules have been proposed as building blocks for this nanotool, like nanoparticles, nanotubes and dendrimers.

Dendrimers are highly branched synthetic polymeric molecules, with all bonds emanating radially from a central core with highly reproducible composition. They have revealed a considerable potential in several biological and biomedical applications; one of the interesting features of these macromolecules is that they have surface groups that can be successfully functionalized in a controlled way, e.g. with drugs, fluorophores, or other contrasting or sensing agents, in order to serve as biosensors in the cellular environment or as drug carriers once they are internalized in living cells or organisms ([1], [2]).

This emerging scenario motivates the present thesis work. Indeed, while there exist many papers and works regarding (or even exploiting) fluorophore-functionalized dendrimers ([3], [4]), a careful analysis of the impact of dendrimers on the photophysics of those fluorophores is still missing. In the work described in this thesis, several of the fluorescence techniques most widely used in Biophysics have been employed to study the physical-chemical properties of Polyamidoamine (PAMAM) dendrimers functionalized with Carboxyfluorescein N-hydroxy succinimide ester (5(6)-FAM SE or NHS-carboxyfluorescein) fluorescent dyes. In particular, the dendrimers used are of Generation 4 (G4) and have an ethylendiamine core. Beyond the classical spectroscopic techniques aimed at investigating absorption and fluorescence properties of the samples, three specific fluorescence microscopy techniques have been exploited: Fluorescence Lifetime Imaging Microscopy (FLIM), Fluorescence Correlation Spectroscopy (FCS) and Single Molecule Detection (SMD) ([5]).

The ultimate goal of this project is to study how the optical and mechanical properties of the samples may vary with respect to the dye alone by tuning relevant parameters, such as the charge on the surface of the dendrimer (i.e. by acetylating the usually positively charged amino end groups), the number of fluorophores on the surface, the pH, and other significant quantities that may affect the internalization, diffusion and the general behavior of dendrimers in cells. In order to reach this goal, molar extinction coefficients of the charged samples and quantum yields of either charged and acetylated samples were determined; these values were compared with the known ones of the dye alone (also confirmed by measurements I performed firsthand). We found that the direct linkage to the dendrimer surface affects the optical properties of the dye by a similar extent for charged and neutral samples: in particular, we observed a strong decrease in its average quantum yield in a range of about 80 − 90% for samples with different numbers of linked fluorophores close to physiological pH (7.4). Secondly, absorption and emission spectra at various pH were recorded for all of the charged samples and for two of the neutral ones: we noted differences in the shape and peak wavelength of the spectra with respect to those of the non-conjugated fluorophore, which we
interpreted as caused by interactions between the dye(s) and the dendrimer local environment. Finally, measurements performed by means of fluorescence microscopy (FLIM, FCS) and Single-Molecule techniques allowed us to better elucidate the results obtained with spectroscopy methods, reaching the following conclusions: there are interactions between NHS-carboxyfluorescein fluorophores bound directly to the surface of PAMAM dendrimers with the dendrimers themselves, which affect significantly the photophysical properties of the dyes; there are different configurations which cause different brightnesses for the dyes; these configurations seem to evolve dynamically in the single dendrimer-dye(s) systems, probably according to the ever-changing local conformation of these complexes.

This thesis is organized as follows:

• Chapter 1 focuses on dendrimers, with particular attention to their chemical structure, the unique features
of the dendritic architecture with respect to the linear one, and their biological applications.

• Chapter 2 starts with a brief overview of the general theory that underlies the interactions between light
and matter. In particular, I describe the processes of absorption and emission of radiation, the mechanism of
fluorescence and its typical characteristics, such as lifetime, quantum yield and quenching. Then, the main
spectroscopic and fluorescence microscopy techniques exploited during the experimental part of this project
are illustrated, with particular emphasis on the three mentioned above.

• In Chapter 3, after a brief outline of the chemical and optical properties of fluorescein and its derivative
dyes, the materials and methods are listed, and the obtained results reported, for each technique employed.

• Chapter 4 reports the main experimental results of this thesis work. In this chapter (and in the conclusions) a qualitative model of interpretation for the peculiar behavior of the system dendrimer-fluorophores is proposed and the evidences observed in the experiments are enclosed in a global descriptive frame. Moreover, as an example of future perspectives of this thesis work, I report a comparison with an improved sample developed at NEST Laboratories, with a spacer inserted between the dendrimer surface and the fluorophore in order to stabilize its optical properties by reducing the interactions with the dendrimer.

References

[1] C.C. Lee , J.A. MacKay, J.M. Frechet, F.C. Szoka Designing Dendrimers for Biological Applications. Nat.
Biotechnol. 2005, 23 (12) 1517-26.

[2] J.M. Oliveira, A.J. Salgadoc, J.F. Manoa and R.L. Reisa Dendrimers and Derivatives as a Potential Therapeutic Tool in Regenerative Medicine Strategies - A Review. Progress in Polymer Science, 35, 2010, 1163-1194.

[3] L. Albertazzi, M. Serresi, A. Albanese and F. Beltram Dendrimer Internalization and Intracellular Trafficking in Living Cells. Mol. Pharmaceutics, 2010, 7 (3), pp 680-688.

[4] A. Saovapackhiran, A. D’Emanuele, D. Attwood and J. Penny Surface Modification of PAMAM Dendrimers Modulates the Mechanism of Cellular Internalization. Bioconjugate Chem., 2009, 20, 693-701.

[5] J. R. Lakowicz Principles of Fluorescence Spectroscopy, Third Edition, Chapters 22, 23, 24. Springer, 2006.