Smart biomimetic nanosystems for stimuli-responsive drug delivery carriers

Abstract

The research conducted in this thesis finds its purpose in the development of cell-derived biomimetic nanosystems, aiming to further broaden nanomedicine´s tools towards advanced therapeutics in the drug delivery field. Nanotechnology offers a plethora of opportunities to design nanodelivery platfoms capable of crossing biological barriers and achieving specific targeting. Nowadays, nanoparticles (NPs) have gained widespread attention as drug delivery vehicles. Thanks to their physical and chemical properties, NPs can be tuned with multiple features, which makes them promising candidates for personalized nanomedicines. Despite the remarkable progress of the last decades in the field, multi-ligand strategies for targeted NPs delivery still suffer from poor in vivo efficacy caused by inadequate interfacing of synthetic NPs with biological environments. The main drawbacks are low specificity in vivo, fast renal clearance, short permanence time in blood torrent and the activation of the immune response. An innovative approach that exploits the natural biological membrane of living cells has been proven to be a successful strategy to improve the in vivo behavior of synthetic nanocarriers. Designing nanostructures by replicating the natural (bio)-physical properties and highly complex functionalities of the cell surface provides delivery nanosystems with effective biointerfacing, preserving the key functionalities of the origin cells. Depending on the type of the cell source, specific functionalities can be exploited for the development of tailored systems with different therapeutic outcomes, which regulate signaling, transport process and immune responses. Specific targeting of cancer cells represents an important challenge for nanomedicine to promote tumor regression while reducing the side effects of anti-cancer drugs. Potentially, cancer cell-derived NPs can exploit their homotypic affinity towards cancer cells from which they are extracted. It is known that cancer cells shown altered expressions of adhesion molecules on their surface that promote changes in their adhesive properties. In this thesis, cancer cells were used to obtain biomimetic membrane based nanosystems, with the aim of exploiting natural cell membrane features to enhanced biocompatibility, obtain high specific targeting efficacy and improve immune evading capability. The first part of this work was aimed to design and develop bio-synthetic cell-derived NPs (named cellsomes, CSMs) using plasmatic membranes of cancer cells. An easily scalable bottom-up process was optimized to obtain CSMs from different cell lines. Their physicochemical and biological properties were comprehensively characterized. To this end, a large variety of techniques were used to study the physiochemical properties of CSMs such as their morphology, hydrodynamic size, colloidal stability in different media, and concentration, among others. The presence of cell membrane components and specific surface biomarkers inherited from the cell source was assessed. The interaction between CSMs and different cell lines was investigated in vitro. Selective targeting capabilities of CSMs for the homotypic tumor cells were demonstrated across several tumoral and nontumoral cell lines. Hybrid CSMs composed of tumoral and non-tumoral cell membranes fragments proved the possibility of translating the targeting capabilities of the tumoral derived CSMs into other non-cancer cell-derived CSMs, thereby expanding their versatility and potential applications. To study the possibility of applying biomimetic coatings to synthetic materials, solid NPs (i.e., polystyrene NPs) were coated with CSMs membranes. The biomimetic effect on the biointerfacing behaviour of coreshell NPs was studied in 2D and 3D cell culture models. Specifically, it was observed that the CSMs-coated NPs inherently mimic the surface properties of the source cells and thus: (i) acquire homotypic targeting ability; (ii) decreased uptake by macrophage cells and; (iii) present a more efficient penetration into 3D tumor model. Remarkably, the possibility to escape from the immune system by avoiding the rapid recognition by the RES, confers an enhanced therapeutic efficacy to synthetic NPs. Based on the studies on their biomimetic properties and their exciting capability to interact with cells, the CSM carrier proved to be a promising candidate for the development of an effective drug delivery nanosystem. Two kinds of smart CSMs-based nanocarriers were set up in this study, focusing on the development of innovative strategies to achieve the intracellular delivery of different cargoes that otherwise would not overcome the cell membrane barrier or would be trapped in endo/lysosome vesicles. The first nanocarrier design was based on a hybrid stimuli-responsive drug delivery nanostructure. The CSMs were combined with photoresponsive NPs (gold nanorods, GNRs), which upon near infrared (NIR) activation, rapidly convert the absorbed energy into thermal energy, mediating the heating of the local environment. The GNRs-tagged CSMs presented several advantages inherited from the cell-membrane nature, such as the ability to target specific cell populations (homotypic targeting) and preventing cargo´s degradation. On the other hand, due to the thermoplasmonic properties of plasmonic GNRs, a spatiotemporal-controlled intracellular release of cargoes into the cytosol of living cells was obtained under NIR stimulation. NIR-triggered cargo release was demonstrated either at the level of single cells (micrometer NIR spot) and at the level of thousands of cells (energy-homogeneous collimated NIR excitation area of ∼0.33 cm2 ; circular spot with a diameter of ~0.65 cm). Through these procedures, photo-controlled intracellular delivery of non-permeant antibodies (anti-Tubulin antibody as a proof of concept) was achieved without compromising cell viability nor the antibody´s function. These results set the stage for the development of photoactive cell-derived nanocarriers, which in addition to cell specific functions, promise straightforward access to spatiotemporal-controlled intracellular delivery of antibodies for application in different immunotherapies. A second alternative strategy to achieve intracellular cargo delivery, escaping the lysosomal entrapping, was successfully developed by surfaceengineering the cell-derived nanocarrier to obtain fusogenic CSMs (FCSMs). The direct fusion of the nanocarrier with the plasma membrane of the cells is a straightforward strategy to achieve the intracellular release of the carried cargos into the cytosol. To this aim, the lipidic composition of CSMs was modified by integrating an optimal combination of cationic and dye-labeled neutral lipids (DOTAP and DOPE-Atto) without compromising the cellderived membrane features. FCSMs were efficiently loaded with manifold types of cargo, from small hydrophobic molecules such the bisbenzimide compound Hoechst H 33258 (HOE) to large molecular mass macromolecules such dye-labeled phalloidin, dextran polymers or polystyrene beads. Upon entering in contact with living cells, in contrast to non-fusogenic CSMs, FCSMs were proved to be able to fuse with the cell membrane of living cells, thereby leading to the release of the encapsulated compounds into the cytosol of cells, avoiding the endocytosis pathway and lysosomal entrapping of the cargo. This technology could represent a powerful tool for fast cytoplasmatic delivery of sensitive drugs, especially proteins and nucleic acids, enabling of designing a new generation of carriers for nanovaccines. The results of the research conducted in this thesis underline the importance of the biomimetic cell-derived coating technology, which offers a versatile tool for developing improved drug delivery nanovectors by easily recreating natural scenarios at the bionano-interface. These biomimetic interfaces have emerged to overcome some of the main drawbacks inherent to synthetic nanomaterials by translating specific complex functionalities from the cell surfaces that remain complex to replicate synthetically.