A polymer nanogel-based therapeutic nanovaccine for prophylaxis and direct treatment of tumors via a full-cycle immunomodulation.

Construction of a cancer nanovaccine that can simultaneously activate immune cells and exert efficient tumor treatment still remains a challenge. Herein, we showcase a proof-of-concept demonstration of an advanced therapeutic nanovaccine formulation based on poly(N-vinylcaprolactam) nanogels (NGs) which were loaded with manganese dioxide (MnO2), the sonosensitizer chlorin e6 (Ce6), and the immune adjuvant cyclic GMP-AMP (cGAMP). The gels were furthermore coated with apoptotic cancer cell membranes (AM). On the one hand, the AM promoted the recognition of NGs by antigen presenting cells (APCs) in lymph nodes due to their enhanced immunogenicity, then the loaded Mn and cGAMP could mature APCs via stimulator of interferon genes (STING) activation for triggering immunity to prevent tumor growth. On the other hand, the NGs could selectively release Mn2+ for hydroxyl radical production and Ce6 to generate single oxygen under ultrasound irradiation of tumors, respectively, thereby exerting local chemodynamic/sonodynamic therapy to induce immunogenic cell death (ICD). Moreover, the Mn2+ could also activate STING in tumors to synergize with ICD for potentiated immune responses. Overall, the biomimetic NG-based therapeutic nanovaccine could directly evoke immune system, and also conduct local tumor treatment to further activate ICD, thus realizing a full-cycle immunomodulation (tumor killing for ICD/antigen production, and tumor cells/APCs immune activation) to tackle bilateral tumor growth.

Engineering Functional Particles to Modulate T Cell Responses.

T cells play a critical role in adaptive immune responses. They work with other immune cells such as B cells to protect our bodies when the first line of defense, the innate immune system, is overcome by certain infectious diseases or cancers. Studying and regulating the responses of T cells, such as activation, proliferation, and differentiation, helps us understand not only their behavior in vivo but also their translation and application in the field of immunotherapy, such as adoptive T cell therapy and immune checkpoint therapy, the situations in which T cells cannot fight cancer alone and require external engineering regulation to help them. Nano- to micrometer-sized particulate biomaterials have achieved great progress in the assistance of T cell-based immunomodulation. For example, various types of microparticles decorated with T cell recognition and activation signals to mimic native antigen-presenting cells have shown successful ex vivo expansion of primary T cells and have been approved for clinical use in adoptive T cell therapy. Functional particles can also serve as vehicles for transporting cargos including small molecule drugs, cytokines, and antibodies. Especially for cargos with limited bioavailability and high repeat-dose toxicity, systemic administration in their free form is difficult. By using particle-assisted systems, the delivery can be tailored on demand, of which targeting and controlled release are two typical examples, ultimately aiding in the regulation of T cell responses. Furthermore, when T cells become overactive and behave in ways that contradict our expectations, such as attacking our own cells or innocuous foreign molecules, this can lead to a breakdown of immune tolerance. In such cases, particles to help reprogram those overactive T cells or suppress their activity are appreciated in vivo. The urgent need to introduce immune stimulation into the treatment of cancers, infectious diseases, and autoimmune diseases has driven recent advances in the engineering of functional particulate biomaterials that regulate T cell responses. In this Account, we will first cover a brief overview of the process of T cell-based immunomodulation from principle to development. It then outlines critical points in the design of functional particle platforms, including materials, size, morphology, surface engineering, and delivery of cargos, to modulate the features of T cells, and introduces selected work from our and other research groups with a focus on three major therapeutic applications: adoptive T cell therapy, immune checkpoint therapy, and immune tolerance restoration. Current challenges and future opportunities are also discussed.

Enzymatically-induced dynamic assemblies from surface functional stomatocyte nanoreactors.

Collective behavior has become a recent topic of investigation in systems chemistry. In pursuing this phenomenon, we present polymersome stomatocytes loaded with the enzyme urease, which show basic stigmergy-based communication and are capable of signal production, reception, and response by clustering with surface complementary binding partners. The collective behavior is transient and based on the widely known pH-sensitive non-covalent interactions between nitrilotriacetic acid (NTA) and histidine (His) moieties attached to the surface of urease-loaded and empty stomacytes, respectively. Upon the addition of the substrate urea, the urease stomatocytes are able to increase the environmental pH, allowing the NTA units to interact with the surface histidines on the complementary species, triggering the formation of transient clusters. The stomatocytes display a maximum clustering interaction at a pH between 6.3 and 7.3, and interparticle repulsive behavior outside this range. This leads to oscillating behavior, as the aggregates disassemble when the pH increases due to high local urease activity. After bulk pH conditions are restored, clustering can take place again. Within the detectable region of dynamic light scattering, individual stomatocytes can aggregate to agglomerates with 10 times their volume. Understanding and designing population behavior of active colloids can facilitate the execution of cooperative tasks, which are not feasible for individual colloids.

Assembly-enhanced indocyanine green nanoparticles for fluorescence imaging-guided photothermal therapy.

The development of theranostic agents that offer complete biocompatibility, coupled with enhanced diagnostic and therapeutic performance, is crucial for fluorescence imaging-guided photothermal therapy in anti-tumor applications. However, the fabrication of nanotheranostics meeting the aforementioned requirements is challenged by concerns regarding biosafety and limited control over construction. Herein, we reported a class of fluorescence imaging-guided photothermal theranostic nanomaterials that are composed of amino acid derivatives and clinically used small photoactive indocyanine green molecules. Through manipulation of noncovalent interactions, these binary building blocks can co-assemble into nanoparticles in a tunable manner. Significantly, such construction not only maintained the fluorescence properties of photoactive molecules, but also enhanced their stability to overcome barriers from photodegradation and complex physiological conditions. These collective features integrated their precise anti-tumor applications, including fluorescence imaging diagnosis and photothermal ablation therapy. This study reported a class of nanotheranostics characterized by biocompatibility, adjustable construction, and robust stability, which are beneficial for the clinical translation of fluorescence imaging-guided photothermal therapy against tumors.

Spatiotemporal Control Over Protein Release from Artificial Cells via a Light-Activatable Protease.

The regulation of protein uptake and secretion by cells is paramount for intercellular signaling and complex multicellular behavior. Mimicking protein-mediated communication in artificial cells holds great promise to elucidate the underlying working principles, but remains challenging without the stimulus-responsive regulatory machinery of living cells. Therefore, systems to precisely control when and where protein release occurs should be incorporated in artificial cells. Here, a light-activatable TEV protease (LaTEV) is presented that enables spatiotemporal control over protein release from a coacervate-based artificial cell platform. Due to the presence of Ni2+-nitrilotriacetic acid moieties within the coacervates, His-tagged proteins are effectively sequestered into the coacervates. LaTEV is first photocaged, effectively blocking its activity. Upon activation by irradiation with 365 nm light, LaTEV cleaves the His-tags from sequestered cargo proteins, resulting in their release. The successful blocking and activation of LaTEV provides control over protein release rate and triggerable protein release from specific coacervates via selective irradiation. Furthermore, light-activated directional transfer of proteins between two artificial cell populations is demonstrated. Overall, this system opens up avenues to engineer light-responsive protein-mediated communication in artificial cell context, which can advance the probing of intercellular signaling and the development of protein delivery platforms.

Spatiotemporal Communication in Artificial Cell Consortia for Dynamic Control of DNA Nanostructures.

The spatiotemporal orchestration of cellular processes is a ubiquitous phenomenon in pluricellular organisms and bacterial communities, where sender cells secrete chemical signals that activate specific pathways in distant receivers. Despite its importance, the engineering and investigation of spatiotemporal communication in artificial cell consortia remains underexplored. In this study, we present spatiotemporal communication between cellular-scale entities acting as both senders and receivers. The transmitted signals are leveraged to elicit conformational alterations within compartmentalized DNA structures. Specifically, sender entities control and generate diffusive chemical signals, namely, variations in pH, through the conversion of biomolecular inputs. In the receiver population, compartmentalized DNA nanostructures exhibit changes in conformation, transitioning between triplex and duplex assemblies, in response to this pH variation. We demonstrate the temporal regulation of activated DNA nanostructures through the coordinated action of two antagonistic sender populations. Furthermore, we illustrate the transient distance-dependent activation of the receivers, facilitated by sender populations situated at defined spatial locations. Collectively, our findings provide novel avenues for the design of artificial cell consortia endowed with programmable spatiotemporal dynamics through chemical communication.

Advancing Artificial Cells with Functional Compartmentalized Polymeric Systems - In Honor of Wolfgang Meier.

The fundamental building block of living organisms is the cell, which is the universal biological base of all living entities. This micrometric mass of cytoplasm and the membrane border have fascinated scientists due to the highly complex and multicompartmentalized structure. This specific organization enables numerous metabolic reactions to occur simultaneously and in segregated spaces, without disturbing each other, but with a promotion of inter- and intracellular communication of biomolecules. At present, artificial nano- and microcompartments, whether as single components or self-organized in multicompartment architectures, hold significant value in the study of life development and advanced functional materials and in the fabrication of molecular devices for medical applications. These artificial compartments also possess the properties to encapsulate, protect, and control the release of bio(macro)molecules through selective transport processes, and they are capable of embedding or being connected with other types of compartments. The self-assembly mechanism of specific synthetic compartments and thus the fabrication of a simulated organelle membrane are some of the major aspects to gain insight. Considerable efforts have now been devoted to design various nano- and microcompartments and understand their functionality for precise control over properties. Of particular interest is the use of polymeric vesicles for communication in synthetic cells and colloidal systems to reinitiate chemical and biological communication and thus close the gap toward biological functions. Multicompartment systems can now be effectively created with a high level of hierarchical control. In this way, these structures can not only be explored to deepen our understanding of the functional organization of living cells, but also pave the way for many more exciting developments in the biomedical field.

Poly(2-oxazoline)-Based Thermoresponsive Stomatocytes.

The design of biocompatible and biodegradable nanostructures with controlled morphological features remains a predominant challenge in medical research. Stimuli-responsive vesicles offer significant advantages in drug delivery, biomedical applications, and diagnostic techniques. The combination of poly(2-oxazoline)s with biodegradable polymers could provide exceptional biocompatibility properties and be proposed as a versatile platform for the development of new medicines. Therefore, poly(2-ethyl-2-oxazoline) (PEtOx) and poly(2-isopropyl-2-oxazoline) (PiPrOx) possessing a hydroxy terminal group that acts as an initiator for the ring-opening polymerization of d,l-lactide (DLLA) have been utilized in this study. The resulting amphiphilic block polymers were used to create polymersomes, which undergo solvent-dependent reorganization into bowl-shaped vesicles or stomatocytes. By blending PEtOx-b-PDLLA and PiPrOx-b-PDLLA copolymers, a thermoresponsive stomatocyte was generated, where the opening narrowed and irreversibly closed with a slight increase in the temperature. Detailed transmission electron microscopy analysis reveals the formation of both closed and fused stomatocytes upon heating the sample above the critical solution temperature of PiPrOx.

Polymersomes with splenic avidity target red pulp myeloid cells for cancer immunotherapy.

Regulating innate immunity is an emerging approach to improve cancer immunotherapy. Such regulation requires engaging myeloid cells by delivering immunomodulatory compounds to hematopoietic organs, including the spleen. Here we present a polymersome-based nanocarrier with splenic avidity and propensity for red pulp myeloid cell uptake. We characterized the in vivo behaviour of four chemically identical yet topologically different polymersomes by in vivo positron emission tomography imaging and innovative flow and mass cytometry techniques. Upon intravenous administration, relatively large and spherical polymersomes accumulated rapidly in the spleen and efficiently targeted myeloid cells in the splenic red pulp. When loaded with ß-glucan, intravenously administered polymersomes significantly reduced tumour growth in a mouse melanoma model. We initiated our nanotherapeutic's clinical translation with a biodistribution study in non-human primates, which revealed that the platform's splenic avidity is preserved across species.

Nanogel-based nitric oxide-driven nanomotor for deep tissue penetration and enhanced tumor therapy.

Antitumor agents often lack effective penetration and accumulation to achieve high therapeutic efficacy in treating solid tumors. Nanomotor-based nanomaterials offer a potential solution to address this obstacle. Among them, nitric oxide (NO) based nanomotors have garnered attention for their potential applications in nanomedicine. However, there widespread clinical adoption has been hindered by their complex preparation processes. To address this limitation, we have developed a NO-driven nanomotor utilizing a convenient and scalable nanogel preparation procedure. These nanomotors, loaded with the fluorescent probe / sonosensitizer chlorin e6 (Ce6), were specifically engineered for sonodynamic therapy. Through comprehensive in vitro investigations using both 2D and 3D cell models, as well as in vivo analysis of Ce6 fluorescent signal distribution in solid tumor models, we observed that the self-propulsion of these nanomotors significantly enhances cellular uptake and tumor penetration, particularly in solid tumors. This phenomenon enables efficient access to challenging tumor regions and, in some cases, results in complete tumor coverage. Notably, our nanomotors have demonstrated long-term in vivo biosafety. This study presents an effective approach to enhancing drug penetration and improving therapeutic efficacy in tumor treatment, with potential clinical relevance for future applications.

Mechanism-based and data-driven modeling in cell-free synthetic biology.

Cell-free systems have emerged as a versatile platform in synthetic biology, finding applications in various areas such as prototyping synthetic circuits, biosensor development, and biomanufacturing. To streamline the prototyping process, cell-free systems often incorporate a modeling step that predicts the outcomes of various experimental scenarios, providing a deeper insight into the underlying mechanisms and functions. There are two recognized approaches for modeling these systems: mechanism-based modeling, which models the underlying reaction mechanisms; and data-driven modeling, which makes predictions based on data without preconceived interactions between system components. In this highlight, we focus on the latest advancements in both modeling approaches for cell-free systems, exploring their potential for the design and optimization of synthetic genetic circuits.

Ultrafast light-activated polymeric nanomotors.

Synthetic micro/nanomotors have been extensively exploited over the past decade to achieve active transportation. This interest is a result of their broad range of potential applications, from environmental remediation to nanomedicine. Nevertheless, it still remains a challenge to build a fast-moving biodegradable polymeric nanomotor. Here we present a light-propelled nanomotor by introducing gold nanoparticles (Au NP) onto biodegradable bowl-shaped polymersomes (stomatocytes) via electrostatic and hydrogen bond interactions. These biodegradable nanomotors show controllable motion and remarkable velocities of up to 125 µm s-1. This unique behavior is explained via a thorough three-dimensional characterization of the nanomotor, particularly the size and the spatial distribution of Au NP, with cryogenic transmission electron microscopy (cryo-TEM) and cryo-electron tomography (cryo-ET). Our in-depth quantitative 3D analysis reveals that the motile features of these nanomotors are caused by the nonuniform distribution of Au NPs on the outer surface of the stomatocyte along the z-axial direction. Their excellent motile features are exploited for active cargo delivery into living cells. This study provides a new approach to develop robust, biodegradable soft nanomotors with application potential in biomedicine.

Competitive protein recruitment in artificial cells.

Living cells can modulate their response to environmental cues by changing their sensitivities for molecular signals. Artificial cells are promising model platforms to study intercellular communication, but populations with such differentiated behavior remain underexplored. Here, we show the affinity-regulated exchange of proteins in distinct populations of coacervate-based artificial cells via protein-protein interactions (PPI) of the hub protein 14-3-3. By loading different coacervates with different isoforms of 14-3-3, featuring varying PPI affinities, a client peptide is directed to the more strongly recruiting coacervates. By switching affinity of client proteins through phosphorylation, weaker binding partners can be outcompeted for their 14-3-3 binding, inducing their release from artificial cells. Combined, a communication system between coacervates is constructed, which leads to the transport of client proteins from strongly recruiting coacervates to weakly recruiting ones. The results demonstrate that affinity engineering and competitive binding can provide directed protein uptake and exchange between artificial cells.

Positively Charged Biodegradable Polymersomes with Structure Inherent Fluorescence as Artificial Organelles.

Polymersomes, nanosized polymeric vesicles, have attracted significant interest in the areas of artificial cells and nanomedicine. Given their size, their visualization via confocal microscopy techniques is often achieved through the physical incorporation of fluorescent dyes, which however present challenges due to potential leaching. A promising alternative is the incorporation of molecules with aggregation-induced emission (AIE) behavior that are capable of fluorescing exclusively in their assembled state. Here, we report on the use of AIE polymersomes as artificial organelles, which are capable of undertaking enzymatic reactions in vitro. The ability of our polymersome-based artificial organelles to provide additional functionality to living cells was evaluated by encapsulating catalytic enzymes such as a combination of glucose oxidase/horseradish peroxidase (GOx/HRP) or ß-galactosidase (ß-gal). Via the additional incorporation of a pyridinium functionality, not only the cellular uptake is improved at low concentrations but also our platform's potential to specifically target mitochondria expands.

Targeted pH-Activated Peptide-Based Nanomaterials for Combined Photodynamic Therapy with Immunotherapy.

Photodynamic therapy (PDT) has demonstrated efficacy in eliminating local tumors, yet its effectiveness against metastasis is constrained. While immunotherapy has exhibited promise in a clinical context, its capacity to elicit significant systemic antitumor responses across diverse cancers is often limited by the insufficient activation of the host immune system. Consequently, the combination of PDT and immunotherapy has garnered considerable attention. In this study, we developed pH-responsive porphyrin-peptide nanosheets with tumor-targeting capabilities (PRGD) that were loaded with the IDO inhibitor NLG919 for a dual application involving PDT and immunotherapy (PRGD/NLG919). In vitro experiments revealed the heightened cellular uptake of PRGD/NLG919 nanosheets in tumor cells overexpressing αvß3 integrins. The pH-responsive PRGD/NLG919 nanosheets demonstrated remarkable singlet oxygen generation and photocytotoxicity in HeLa cells in an acidic tumor microenvironment. When treating HeLa cells with PRGD/NLG919 nanosheets followed by laser irradiation, a more robust adaptive immune response occurred, leading to a substantial proliferation of CD3+CD8+ T cells and CD3+CD4+ T cells compared to control groups. Our pH-responsive targeted PRGD/NLG919 nanosheets therefore represent a promising nanosystem for combination therapy, offering effective PDT and an enhanced host immune response.

Dampened Transient Actuation of Hydrogels Autonomously Controlled by pH-Responsive Bicontinuous Nanospheres.

The fabrication of a soft actuator with a dampened actuation response is presented. This was achieved via the incorporation into an actuating hydrogel of urease-loaded pH-responsive bicontinuous nanospheres (BCNs), whose membrane was able to regulate the permeability and thus conversion of fuel urea into ammonia. The dampened response of these nanoreactors to the enzymatically induced pH change was translated to a pH-responsive soft actuator. In hydrogels composed of a pH-responsive and nonresponsive layer, the transient pH gradient yielded an asymmetric swelling behavior, which induced a bending response. The transient actuation profile could be controlled by varying the external fuel concentrations. Furthermore, we showed that the spatial organization of the BCNs within the actuator had a great influence on the actuation response. Embedding the urease-loaded nanoreactors within the active, pH-responsive layer resulted in a reduced response due to local substrate conversion in comparison to embedding them within the passive layer of the bilayer hydrogel. Finally, we were able to induce transient actuation in a hydrogel comprising two identical active layers by the immobilization of the BCNs within one specific layer. Upon addition of urea, a local pH gradient was generated, which caused accelerated swelling in the BCN layer and transient bending of the device before the pH gradient was attenuated over time.

Models of Chemical Communication for Micro/Nanoparticles.

Engineering chemical communication between micro/nanosystems (via the exchange of chemical messengers) is receiving increasing attention from the scientific community. Although a number of micro- and nanodevices (e.g., drug carriers, sensors, and artificial cells) have been developed in the last decades, engineering communication at the micro/nanoscale is a recent emergent topic. In fact, most of the studies in this research area have been published within the last 10 years. Inspired by nature─where information is exchanged by means of molecules─the development of chemical communication strategies holds wide implications as it may provide breakthroughs in many areas including nanotechnology, artificial cell research, biomedicine, biotechnology, and ICT. Published examples rely on nanotechnology and synthetic biology for the creation of micro- and nanodevices that can communicate. Communication enables the construction of new complex systems capable of performing advanced coordinated tasks that go beyond those carried out by individual entities. In addition, the possibility to communicate between synthetic and living systems can further advance our understanding of biochemical processes and provide completely new tailored therapeutic and diagnostic strategies, ways to tune cellular behavior, and new biotechnological tools. In this Account, we summarize advances by our laboratories (and others) in the engineering of chemical communication of micro- and nanoparticles. This Account is structured to provide researchers from different fields with general strategies and common ground for the rational design of future communication networks at the micro/nanoscale. First, we cover the basis of and describe enabling technologies to engineer particles with communication capabilities. Next, we rationalize general models of chemical communication. These models vary from simple linear communication (transmission of information between two points) to more complex pathways such as interactive communication and multicomponent communication (involving several entities). Using illustrative experimental designs, we demonstrate the realization of these models which involve communication not only between engineered micro/nanoparticles but also between particles and living systems. Finally, we discuss the current state of the topic and the future challenges to be addressed.

Artificial cells with viscoadaptive behavior based on hydrogel-loaded giant unilamellar vesicles.

Viscoadaptation is an essential process in natural cells, where supramolecular interactions between cytosolic components drive adaptation of the cellular mechanical features to regulate metabolic function. This important relationship between mechanical properties and function has until now been underexplored in artificial cell research. Here, we have created an artificial cell platform that exploits internal supramolecular interactions to display viscoadaptive behavior. As supramolecular material to mimic the cytosolic component of these artificial cells, we employed a pH-switchable hydrogelator based on poly(ethylene glycol) coupled to ureido-pyrimidinone units. The hydrogelator was membranized in its sol state in giant unilamellar lipid vesicles to include a cell-membrane mimetic component. The resulting hydrogelator-loaded giant unilamellar vesicles (designated as HL-GUVs) displayed reversible pH-switchable sol-gel behavior through multiple cycles. Furthermore, incorporation of the regulatory enzyme urease enabled us to increase the cytosolic pH upon conversion of its substrate urea. The system was able to switch between a high viscosity (at neutral pH) and a low viscosity (at basic pH) state upon addition of substrate. Finally, viscoadaptation was achieved via the incorporation of a second enzyme of which the activity was governed by the viscosity of the artificial cell. This work represents a new approach to install functional self-regulation in artificial cells, and opens new possibilities for the creation of complex artificial cells that mimic the structural and functional interplay found in biological systems.

Tuning of Cationic Polymer Functionality in Complex Coacervate Artificial Cells for Optimized Enzyme Activity.

Complex coacervates are a versatile platform to mimic the structure of living cells. In both living systems and artificial cells, a macromolecularly crowded condensate phase has been shown to be able to modulate enzyme activity. Yet, how enzyme activity is affected by interactions (particularly with cationic charges) inside coacervates is not well studied. Here, we synthesized a series of amino-functional polymers to investigate the effect of the type of amine and charge density on coacervate formation, stability, protein partitioning, and enzyme function. The polymers were prepared by RAFT polymerization using as monomers aminoethyl methacrylate (AEAM), 2-(dimethylamino)ethyl methacrylate (DMAEMA), imidazolepropyl methacrylamide (IPMAm), and [2-(methacryloyloxy)ethyl] trimethylammonium chloride (TMAEMA). Membranized complex coacervate artificial cells were formed with these polycations and an anionic amylose derivative. Results show that polycations with reduced charge density result in higher protein mobility in the condensates and also higher enzyme activity. Insights described here could help guide the use of coacervate artificial cells in applications such as sensing, catalysis, and therapeutic formulations.