Reaching the tumour: nanoscopy study of nanoparticles biological interactions

Abstract

The precise delivery of therapeutic agents to their specific site of action is a big challenge in cancer treatment, which would enhance the efficacy and reduce the side effects of drugs. In this framework, nanotechnology can greatly contribute to the development of novel drug delivery systems. Nowadays, a large number of nanoparticles, differing in chemical nature, have been synthesized and evaluated for their therapeutic performances. However, most of the newly developed delivery systems are ineffective in the clinic because they don’t reach the specific cancer cells. One of the main reasons of this failure is the lack of knowledge about the interactions between the designed nanosystems and the biological media before getting to the targeted cells, the so-called nanobiointeractions. In particular, undesired interactions with blood proteins and other molecules in vasculature are often responsible for the poor performance of nanocarriers. Further research about the biological interactions occurring in the blood vessels is needed in order to design novel and improved therapeutic nanoparticles. We believe that the understanding of these critical steps together with an in-depth study of the structural composition of nanoparticles will guide a rational design of systems, increasing their applicability and performance in the clinic. To accomplish a comprehensive study, in this thesis, we propose the use of advanced optical microscopy techniques to investigate the chemical and biological identity of nanomaterials and to understand their role with a nanometric precision.

One of the first biological barriers nanoparticles encounter when introduced intravenously to the body are proteins which travel through the blood stream. These molecules form the so-called protein corona: a shell of proteins attached to the surface of the nanoparticle. One of the main drawbacks caused by protein corona formation is the hindering of the nanoparticle’s surface, reducing the specific interactions of nanosystems with the cancer cells they are targeting. Protein corona formation is mainly studied using ensemble techniques which give only an approximate idea of the molecules interacting with the surface of the nanoparticles. In chapter two, STORM imaging of corona is presented as a new methodology to obtain an in situ characterization of protein corona on individual nanoparticles. This study reveals a high interparticle heterogeneity regarding the number of proteins per nanoparticle, which may be one of the causes of their poor clinical performance.

Protein interactions are not only responsible of reducing specific interactions but can dramatically affect the stability of nanosized delivery systems. Therefore, it is important to study their stability in the blood complex environment. Polyplexes are nanocarriers characterized by the electrostatic interactions between the carrier and the nucleic acid. These systems need to be fully complexed during their circulation in the blood vessels in order to protect their cargo from degradation. Up to now, the challenges in characterizing the molecular distribution of the individual components have limited the rational design of nanosystems. In the third chapter, dSTORM imaging is used to visualize the exact molecular composition of polyplexes. dSTORM imaging unveiled the differences in the stoichiometry of individual systems, revealing a heterogeneity inside the same population. Once the system is fully characterized, his complexation can be followed under different blood-like conditions thanks to the molecular resolution of the technique. This new

method allows to determine the real molecular stability of the system in contact with serum proteins and provides mechanistic insights into the disassembly process.

The stability in complex biological media is a determining factor of the good performance of drug delivery systems, especially in the use of supramolecular structures, due to their dynamic nature. Therefore, it is necessary to understand the behavior of self-assembled nanoparticles in conditions close to the ones they would confront in vivo. Serum proteins can prematurely disassemble the system, as seen in the previous chapter, and lead to non-selective release of the cargo in healthy tissues. Another critical issue of self-assembling systems is the strong dilution they undergo when injected in blood, which may severely affect the supramolecular stability. Hence, it is of crucial importance to investigate the effect of dilution in biologically relevant media. These issues are often overlooked in the literature, most likely due to the difficulties of studying supramolecular assemblies in complex biological media. In chapter four, micelles that change their fluorescent properties upon disassembly are characterized under different blood- like conditions using a combination of fluorescence spectroscopy and microscopy techniques, allowing to predict the system with the best properties.

A last critical step nanoparticles face when injected into the blood vessels is the flow, which may also affect the stability of the system. Moreover, their efficiency is directly proportional to the ability of extravasation from the blood vessel across the tight endothelial layer before reaching the cancer cells. In each of these barriers, the stability of supramolecular systems may be compromised. In chapter five a microfluidic chip mimicking the vascular tumor microenvironment is optimized to study the ability and stability of supramolecular structures during extravasation. A monolayer of human umbilical vein endothelial cells are grown in the microfluidic device forming a blood-vessel-like channel. Moreover, the chip contains a second channel of tumorigenic cells to test the stability of the nanocarrier in the extracellular matrix close to cancer cells after extravasation. This device allows to screen the behavior of the different delivery systems and predicts the most stable and promising system thus optimizing and reducing the pre-clinical and clinical testing.

La nanomedicina pot contribuir en la millora dels tractaments de càncer gràcies al ús de nanopartícules per el lliurament controlat de fàrmacs. En els últims anys s’ha dut a terme un gran nombre d’investigacions però fins ara la majoria de nanopartícules investigades són ineficients en pacients ja que no arriben a les cèl·lules específiques del càncer. Una de les raons principals d’aquest fracàs és la manca de coneixement sobre les interaccions entre les nanopartícules i els medis biològics amb els quals es troben dins el cos. En particular, les interaccions no desitjades amb les proteïnes de la sang i altres molècules en els vasos sanguínies són sovint responsables del seu mal funcionament. La comprensió d’aquests passos crítics juntament amb un estudi en profunditat de la composició estructural de les nanopartícules guiarà un disseny racional dels sistemes, augmentant la seva traducció a les clíniques. Actualment, una de les limitacions principals d’aquestes investigacions és la manca de tècniques experimentals capaces de proporcionar informació del comportament de les nanopartícules a escala nano, així com, que es puguin realitzar en medis biològics complexes. L'objectiu principal d'aquesta tesi és l'ús de noves tècniques de microscòpia òptica avançades per donar a conèixer les interaccions biològiques de les nanopartícules en medis biològics complexos.

Per a aquest propòsit, el capítol 2, descriu l'ús de la imatge dSTORM com a nova metodologia per obtenir una caracterització in situ de la corona proteica. En conjunt, revela una alta heterogeneïtat pel que fa al nombre de proteïnes per nanopartícula, que pot ser una de les causes del seu mal rendiment clínic. En el capítol 3, la tècnica dSTORM s'utilitza per visualitzar la composició molecular exacta de poliplexes. Una vegada caracteritzats aquests sistemes es pot seguir la seva complexació en sèrum per estudiar la seva estabilitat. En el capítol 4, micel·les, que canvien la seva fluorescència en funció de si estan intactes o no, es caracteritzen en diferents condicions similars a la sang mitjançant una combinació d’espectroscòpia de fluorescència i tècniques de microscòpia espectral. En aquest capítol es mostra una relació entre l'estabilitat de les micel·les, i la seva internalització cel·lular. Finalment, el capítol 5 té com a objectiu desenvolupar un xip microfluídic imitant els vasos sanguinis tumorals per estudiar la capacitat i l'estabilitat de les nanopartícules durant l'extravasació mitjançant tècniques de microscòpia espectral.