Finalidad de los Impuestos Ambientales.

In this thesis, we have described our experiments for the bottom-up design and assembly of a fully biodegradable/compatible polymersomal nanomotor system. In 2012, our lab reported the bottom-up approach to construct supramolecular nanomotors, which is based on the self-assembly of poly(ethylene glycol)-polystyrene (PEG-PS) block copolymers into polymersomal structures.1 Under osmotic stress, these spherical polymersomes underwent shape transformation forming unique bowl shaped architectures, known as stomatocytes. Performing this shape transformation process in presence of platinum nanoparticles allowed their encapsulation within the cavity of the stomatocytes via “artificial endocytosis”. Platinum is a known catalyst for the decomposition reaction of hydrogen peroxide into water and oxygen. In presence of hydrogen peroxide, the encapsulated platinum nanoparticles produced a rapid discharge of oxygen, after which the latter escaped the stomatocytes through their opening, propelling the structures forward. This work has been the main inspiration of this thesis, where we developed the system further to make the nanomotors biodegradable and compatible, with future biomedical applications as a major motivation.

Shape-characterization of nanoscale polymeric particles usually requires costly and time-consuming methods, such as cryo-TEM. Therefore, in chapter 2 we investigated the possibility to characterize the shape of various PEG-PS polymersome morphologies by means of light scattering techniques (both static and dynamic). We first presented theoretical descriptions for the possible shape factors (Rg/Rh) of various polymersomal shapes (spheres,

discs and rods). We then supported our theoretical descriptions of polymersome morphologies with light scattering and cryo-TEM microscopy experiments.

In Chapter 3, we described our first attempt to form a hybrid nanomotor system, replacing the previously used platinum with enzymes, using glucose as a natural fuel for propulsion. We integrated catalase or its combination with glucose oxidase (GOx) in the cavity of the PEG-PS stomatocytes to form a hybrid polymeric supramolecular nanomotor. These motors were able to decompose H2O2 - either when added directly or as side product

of the breakdown of glucose - into oxygen that escapes the stomatocyte through its tight, almost closed neck, resulting in nanomotor propulsion. Previously, platinum was encapsulated in presence of a large amount of organic solvents (50 % vol/vol) in a time- consuming process, which might lead to denaturation of most proteins. Therefore, shape transformation procedures were modified for enzyme encapsulation to prevent enzyme denaturation.Enzymes were first mixed with opened-neck stomatocytes and subsequently,

the neck was closed by adding only 150 L of organic solvent over a period of 30 minutes. The presence of enzymes within the stomatocytes cavity was confirmed by means of light scattering techniques and energy dispersive X-ray. Retention of activity was also confirmed after performing standard enzyme activity assays.

Autonomous movement of catalase- and GOx/catalase-filled stomatocytes was tested with various fuel types, H2O2 and glucose, respectively, and a range of concentrations

thereof using the nanoparticle-tracking analysis (NTA) technique. Catalase-filled stomatocytes were propelled with average speeds of 15 m s-1 and a remarkable high speed of 60 m s-1 _{at 11 mM and 111 mM of H}

2O2, respectively. Stomatocytes encapsulating

GOx/catalase showed propulsion at biologically relevant glucose concentration (5 mM). In presence of trypsin, a proteolytic enzyme known to inhibit enzyme activity and usually present in biological settings, stomatocytes did not stop and continued moving. This experiment showed the ability of stomatocytes to confine and protect enzymes against deactivating macromolecules that are usually present in biological environments.

Chapter 4 builds on chapter 3 by replacing the simple GOx/catalase cascade to a

functional out-of-equilibrium enzymatic network for sustained autonomous movement. The rationally designed enzymatic reaction network was able to convert naturally present substrates into molecular oxygen. This enzymatic network consisted of four metabolic modules, working together for a tunable and sustained output. The first module, the “activation module”, was based on hexokinase (HK) and pyruvate kinase (PK), which scavenged the phosphate donor phosphoenolpyruvate (PEP) to activate ATP necessary for glucose uptake and consequent network activation. Conversion of PEP into pyruvate triggered activation of the pyruvate–L-lactate cycle. In this cycle, two opposing reactions took place: the consumption and production of pyruvate by the action of lactate dehydrogenase (LDH) and lactate oxidase (LO), respectively. The concentration of pyruvate built up till the point that it became inhibitory towards LDH (feed-forward inhibition). Production of L-lactate from pyruvate by the action of LDH occurred when β-NADH was present, which was regenerated by the action of the second cycle (conversion of glucose-6- phosphate (G-6-P) to 6-glucosephosphogluconolactone (6-p-g) by the action of glucose-6- phosphate dehydrogenase (G6PDH)). The net product of these previous three modules was hydrogen peroxide. The last module depicts a functional cycle, which decomposed H2O2

into molecular oxygen.

This enzymatic reaction network was encapsulated in the cavity of PEG-PS stomatocytes and their motion was further analyzed in the presence of glucose as fuel. At 10

mM glucose concentration, the speed of the nanomotors was approximately 7 m s-1. After 180 min, the speed output remained constant, highlighting the ability of this network to sustain its output over extended periods of time by regulating its fuel consumption. The network output was also tuned by changing the ATP concentration. ATP determines the concentration of β-NADH and the consequent lactate and H2O2 production. Varying ATP

concentrations from 0.25 mM to 1 mM resulted in an increase of speed by approx. 40 % over this ATP regime. The ability of this enzymatic network to maintain a fixed nanomotor speed during glucose consumption and the possibility of tuning this output by controlling the speed of certain cycles in the network is unique and advantageous.

In this case, the protective element and the confinement effect of the stomatocyte nanomotor was highlighted by showing its functioning in complex media, human blood serum (HBS). HBS contains many different proteins and enzymes, amongst which catalase. When hydrogen peroxide was not produced in the cavity, but in bulk, it was converted by free catalase present in the medium. As a result, the hydrogen peroxide concentration was lowered to such an extent that the entrapped catalase could not induce any propulsion anymore. In HBS, motion of stomatocytes was maintained and unaffected by the medium. This aspect is important when considering the use of these systems in a biological context.

In the following chapters, we aimed to exchange the polystyrene hydrophobic material with a biodegradable and biocompatible one. In chapter 5, we show the construction of poly(ethylene glycol)-b-poly(D,L-lactide) (PEG-PDLLA) polymeric vesicles and their shape transformation. PEG-PDLLA is readily dissolved in organic solvents and its self-assembly into spherical polymersomes was promoted by the addition of water. Interestingly, introducing an osmotic shock to the flexible construct, allowed its shape transformation into nanotubes instead of stomatocytes. A closer look into the physical origin of PEG-PDLLA shape transformation shows that the main determinant in this process is the bending energy (Eb), which is described by 𝐸_𝑏 =

𝑘

2∮(2𝐶 − 𝐶0)

2_{𝑑𝐴, where k is bending}

rigidity (depends on material properties), C is mean surface curvature and Co is the

spontaneous surface curvature. When inner and outer surfaces of a flexible membrane are exposed to distinct solvent environments, the contribution of Co is not significant inducing

positive surface curvature and thus, formation of tubes is favored as it follows lower Eb

energy profile. Increasing the osmotic pressure during this shape transformation resulted in volume reduction and subsequent elongation of the final construct. We have further demonstrated the capacity of these nanotubes to be loaded with enzymes and drugs after their formation, which is important for future biomedical applications.

Although PEG-PDLLA polymersomal system is biodegradable and biocompatible, its formation requires large amounts of organic solvent, which is not suitable when encapsulation and compartmentalization of enzymes is considered. This notion led us to consider a different biodegradable and biocompatible polymersomal system that can be formed via a biocompatible method. In chapter 6, we describe our results in that direction. Inspired by previously reported poly(ε-caprolactone) (PCL) polymersomal systems, we set out to synthesize a copolymer comprising a blend of PCL and poly(trimethylene carbonate) PTMC in the hydrophobic block and PEG in the hydrophilic block. Applying the biocompatible direct hydration method of self-assembly on PEG-b-P(CL-g-TMC) promotes its self-assembly into polymersomes. We show that polymer composition and the formulation conditions used for self-assembly are of great importance to produce polymersomes reproducibly. Self-assembly of CL-rich polymers revealed formation of both polymersomes and worm-like micelles, while TMC rich polymers resulted in the formation of micelles. Copolymers with equal amounts (by mass) of CL and TMC consistently yielded polymersomes. The membrane thickness of PEG-b-P(CL-g-TMC) polymersomes could be easily tuned by changing the molecular weight of the copolymers, whereas a copolymer with

Mw ≈ 7.6 KDa yielded a ~ 14 nm thick polymersomal membrane and polymer with Mw ≈ 4.2

KDa resulted in a ~ 7 nm thick polymersomal membrane. The degradation behavior of these polymersomes was found to be dependent on their membrane thickness. 7 nm thick membrane polymersomes were fully degraded in human blood serum in a course of 3 hours, while the thicker ones (15 nm membrane) were stable even after 24 hours. These polymersomes were able to entrap active enzymes and to perform reactions within one polymersome or across two polymersomes due to the semipermeable nature of the PEG-b- P(CL-g-TMC) membrane.