Tesi etd-08282025-180819
Link copiato negli appunti
Tipo di tesi
Dottorato
Autore
ROBERTI, ELISA
URN
etd-08282025-180819
Titolo
CELL-INSPIRED MICROROBOTS FOR SENSING PH GRADIENTS
Settore scientifico disciplinare
ING-IND/34
Corso di studi
Istituto di Biorobotica - PHD IN BIOROBOTICA
Relatori
relatore Prof. PALAGI, STEFANO
Parole chiave
- microrobots
- GUVs
- bioinspiration
- cancer
Data inizio appello
06/02/2026;
Disponibilità
completa
Riassunto analitico
Cancer is the second leading cause of death worldwide, and one of the central challenges in oncology is the development of therapies that selectively target tumors while minimizing collateral damage to healthy tissues. Despite advances in the specificity of chemotherapeutic agents, tumor heterogeneity and the emergence of drug resistance continue to undermine therapeutic efficacy. Nanoparticle-based drug delivery systems, which rely on passive accumulation and ligand-mediated targeting, have shown to be a promising approach, but typically achieve delivery of less than 1% of the administered drug to the tumor site. To overcome these limitations, researchers have begun exploring microrobotics: actively controlled microscale systems capable of navigating complex biological environments and administering therapeutic agents with high spatial precision. While still in the early stages of development, microrobots offer significant potential for improving targeting accuracy. However, challenges such as real-time control, biocompatibility, and efficient navigation through tissues are still substantial obstacles.
This thesis is part of the European project CELLOIDS, focused on the development of cell-inspired microrobots, which we named celloids. They are inspired by leukocytes, whose interstitial migration through complex human body tissues is enabled by highly adaptable ameboid motion. Similarly, celloids are intended to move autonomously, sense external cues, and reach specific targets – such as solid tumors – for the localized administration of therapeutic agents. Structurally, they are designed with a liquid active core (the motion system) enclosed within a thin membrane. The membrane is a key component of the celloid microrobots, as it both guarantees the physical integrity of the microrobots and enables their mechanical and chemical interaction with the external environment. The primary focus of this thesis is thus on engineering the microrobot’s membrane to achieve three essential characteristics: (i) morphological adaptation, allowing celloids to overcome narrow gaps and to achieve body-shape changes and motion driven by the active core; (ii) selective permeability, enabling molecular exchange and communication with the surrounding environment while preserving the internal core (and potential cargo); and (iii) perception of external environment and directional response, which in our case means the ability to locally deform or change mechanical properties in response to chemical stimuli such as pH changes.
As an initial approach, we investigated hydrogel-based core-shell structure for the microrobots’ body. Specifically, we developed a novel fabrication method that exploits the distinct gelation mechanisms of alginate and agarose under mild fabrication conditions. Using this method, we successfully obtained alginate shells with tunable thickness, modulated by CaCl₂ concentration, and a semi-liquid core derived from sacrificial agarose templates. However, our investigation revealed that such structures were excessively permeable and not sufficiently deformable to serve as effective celloids membranes. Furthermore, the presence of a not fully liquid core posed an additional limitation, as it could hinder the action of the core components.
To address these limitations, we shifted our focus to Giant Unilamellar Vesicles (GUVs), which are well-established biomimetic models widely used in synthetic biology. GUVs are used to study membrane dynamics or to develop artificial cells, particularly because they replicate the selective permeability of natural membranes, allowing chemical reactions to remain confined. This property is crucial for our design, as it enables the containment of active microparticles, which are easily perturbed by the presence of other molecules (i.e. ions). Moreover, the nanometrically thin and fluid membrane of GUVs is composed of a lipid bilayer closely mimicking natural cell membranes, thereby providing a more suitable platform for the development of cell-like deformable microrobots.
In the first phase, we optimized the droplet transfer method for GUVs preparation and modified the membrane composition to tune both mechanical and chemical properties. Cholesterol is a crucial element in biological cell membranes. Among its functions, cholesterol plays a central role in regulating membrane deformability. Given its importance and considering that its impact on synthetic membranes depends both on its fraction and on the phospholipid composition, we investigated its effect in the microrobots’ membranes. Specifically, cholesterol was incorporated into the lipid bilayers to investigate its effect on vesicle size, temporal stability, and membrane rigidity. Our results indicate that cholesterol increased vesicle diameter and, under the experimental conditions tested, did not substantially alter overall deformability.
As the tumor microenvironment has a lower pH than the surrounding tissues, we aim at making the microrobots able to perceive and move along pH gradients. To this aim, we incorporated linoleic acid, a pH-responsive fatty acid, into the vesicle membrane and evaluated its influence on the physicochemical properties of the GUVs. In particular, we assessed changes in vesicle size, membrane zeta potential, and deformability under both neutral and acidic conditions. We observed the incorporation of linoleic acid into the membrane affected vesicle properties, including size, surface charge, and deformability. However, given the limited number of vesicles analyzed, these findings should be considered preliminary. In parallel, we investigated the possibility of inducing microrobots' deformability by incorporating β-lactoglobulin (β-Lg), a whey protein known to undergo gelation at mildly acidic pH (~5). The working hypothesis was that, upon acidification, β-Lg dispersed on GUV membranes would aggregate, exerting mechanical forces on adjacent membranes and potentially inducing local deformations. To promote this interaction, we introduced DOTAP, a cationic phospholipid, into the GUV formulation, thereby generating positively charged vesicle membranes capable of enhancing electrostatic binding with negatively charged β-Lg. Positively charged GUVs were successfully developed, and the gelation properties of β-Lg were systematically characterized using established biophysical techniques (e.g. circular dichroism and turbidity assays). Furthermore, we evaluated the behavior of both the vesicles and the protein under acidic conditions to assess their potential responsiveness.
These findings suggest GUVs as a promising chassis for celloid microrobots, providing a versatile platform with tunable mechanical properties and chemical responsiveness. Looking ahead, their ability to deform and respond to stimuli could be harnessed to navigate the dense extracellular matrix of tumors and selectively target the acidic tumor microenvironment. In this way, celloids could enable localized, in-tumor administration of anticancer therapies, sparing healthy cells, thus overcoming key limitations of current therapies and drug delivery systems.
This thesis is part of the European project CELLOIDS, focused on the development of cell-inspired microrobots, which we named celloids. They are inspired by leukocytes, whose interstitial migration through complex human body tissues is enabled by highly adaptable ameboid motion. Similarly, celloids are intended to move autonomously, sense external cues, and reach specific targets – such as solid tumors – for the localized administration of therapeutic agents. Structurally, they are designed with a liquid active core (the motion system) enclosed within a thin membrane. The membrane is a key component of the celloid microrobots, as it both guarantees the physical integrity of the microrobots and enables their mechanical and chemical interaction with the external environment. The primary focus of this thesis is thus on engineering the microrobot’s membrane to achieve three essential characteristics: (i) morphological adaptation, allowing celloids to overcome narrow gaps and to achieve body-shape changes and motion driven by the active core; (ii) selective permeability, enabling molecular exchange and communication with the surrounding environment while preserving the internal core (and potential cargo); and (iii) perception of external environment and directional response, which in our case means the ability to locally deform or change mechanical properties in response to chemical stimuli such as pH changes.
As an initial approach, we investigated hydrogel-based core-shell structure for the microrobots’ body. Specifically, we developed a novel fabrication method that exploits the distinct gelation mechanisms of alginate and agarose under mild fabrication conditions. Using this method, we successfully obtained alginate shells with tunable thickness, modulated by CaCl₂ concentration, and a semi-liquid core derived from sacrificial agarose templates. However, our investigation revealed that such structures were excessively permeable and not sufficiently deformable to serve as effective celloids membranes. Furthermore, the presence of a not fully liquid core posed an additional limitation, as it could hinder the action of the core components.
To address these limitations, we shifted our focus to Giant Unilamellar Vesicles (GUVs), which are well-established biomimetic models widely used in synthetic biology. GUVs are used to study membrane dynamics or to develop artificial cells, particularly because they replicate the selective permeability of natural membranes, allowing chemical reactions to remain confined. This property is crucial for our design, as it enables the containment of active microparticles, which are easily perturbed by the presence of other molecules (i.e. ions). Moreover, the nanometrically thin and fluid membrane of GUVs is composed of a lipid bilayer closely mimicking natural cell membranes, thereby providing a more suitable platform for the development of cell-like deformable microrobots.
In the first phase, we optimized the droplet transfer method for GUVs preparation and modified the membrane composition to tune both mechanical and chemical properties. Cholesterol is a crucial element in biological cell membranes. Among its functions, cholesterol plays a central role in regulating membrane deformability. Given its importance and considering that its impact on synthetic membranes depends both on its fraction and on the phospholipid composition, we investigated its effect in the microrobots’ membranes. Specifically, cholesterol was incorporated into the lipid bilayers to investigate its effect on vesicle size, temporal stability, and membrane rigidity. Our results indicate that cholesterol increased vesicle diameter and, under the experimental conditions tested, did not substantially alter overall deformability.
As the tumor microenvironment has a lower pH than the surrounding tissues, we aim at making the microrobots able to perceive and move along pH gradients. To this aim, we incorporated linoleic acid, a pH-responsive fatty acid, into the vesicle membrane and evaluated its influence on the physicochemical properties of the GUVs. In particular, we assessed changes in vesicle size, membrane zeta potential, and deformability under both neutral and acidic conditions. We observed the incorporation of linoleic acid into the membrane affected vesicle properties, including size, surface charge, and deformability. However, given the limited number of vesicles analyzed, these findings should be considered preliminary. In parallel, we investigated the possibility of inducing microrobots' deformability by incorporating β-lactoglobulin (β-Lg), a whey protein known to undergo gelation at mildly acidic pH (~5). The working hypothesis was that, upon acidification, β-Lg dispersed on GUV membranes would aggregate, exerting mechanical forces on adjacent membranes and potentially inducing local deformations. To promote this interaction, we introduced DOTAP, a cationic phospholipid, into the GUV formulation, thereby generating positively charged vesicle membranes capable of enhancing electrostatic binding with negatively charged β-Lg. Positively charged GUVs were successfully developed, and the gelation properties of β-Lg were systematically characterized using established biophysical techniques (e.g. circular dichroism and turbidity assays). Furthermore, we evaluated the behavior of both the vesicles and the protein under acidic conditions to assess their potential responsiveness.
These findings suggest GUVs as a promising chassis for celloid microrobots, providing a versatile platform with tunable mechanical properties and chemical responsiveness. Looking ahead, their ability to deform and respond to stimuli could be harnessed to navigate the dense extracellular matrix of tumors and selectively target the acidic tumor microenvironment. In this way, celloids could enable localized, in-tumor administration of anticancer therapies, sparing healthy cells, thus overcoming key limitations of current therapies and drug delivery systems.
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