Eduardo Fernandez-Megia

Bioinspired synthetic cells are rapidly transforming the way we interrogate the principles of cellular life and the development of bioengineering and medical applications. However, despite significant progress in modeling cell-like behavior, material engineering remains a time-consuming and often behind-the-scenes endeavor when optimizing cytomimetic functions. Here, we describe how dynamic covalent chemistry can be used to bypass this bottleneck using membranized coacervate microdroplets (MCM) as synthetic cell models. Specifically, the potential of dynamic covalent boronate chemistry for the in situ formation, interfacial stabilization, and adaptive cytomimetic optimization of MCM is presented. Simultaneous addition of cationic and anionic catechols to a polymeric boronic acid (BA) generates dynamic zwitterionic polyboronates that spontaneously phase separate into microdroplets, which can then be interfacially stabilized as MCM with a BA-functionalized block copolymer. The cytomimetic properties, membranization, internal dynamics, and enzymatic activity within the MCM can be modulated in situ using dynamic covalent libraries to fine-tune material properties (either by adjusting the charge ratio between oppositely charged catechols, varying the catechol-to-BA ratio, or introducing auxiliary catechol dopants) without the need to synthesize, isolate, purify, and characterize new polymeric materials. Application of this technology to other catechols, multivalent BA, and synthetic cell architectures holds promise for optimizing diverse biomimetic functions and providing programmable synthetic cells with emerging properties.

Synthetic cells are emerging as cornerstone in our understanding of prebiotic forms of early life and the development of therapeutics. Although several types of vesicles have been proposed for this purpose, their development is often hampered by limited membrane permeability. On the other hand, polyion complex vesicles (PICsomes) with a high permeability for small molecules suffer from a small size (typically sub-200 nm) and low encapsulation efficiency of enzymes (less than 4%). Herein, we describe the peripheral charge density of dendrimers and the ionic strength of the medium as powerful tools in the size tuning of PICsomes via a dendrimer-to-PIC hierarchical transfer of structural information. PICsomes beyond the micron range were readily obtained from a single dendrimer generation (G) and their ability to emulate life-like technologies explored through chemical communication. As opposed to the low protein encapsulation in the lumen of classical PICsomes, a selective enzyme embedding in the PIC membrane was revealed with efficiencies up to 85%. Notably, membrane-embedded enzymes retain high catalytic activity (85% relative to free enzymes), even in the presence of proteases, enabling fast enzymatic cascades between synthetic cell populations.

Bottom-up synthetic biology seeks to construct artificial cells with biomimetic or novel functionalities to uncover the fundamental principles of cellular evolution and drive advances in medicine and bioengineering. Among them, membranized coacervate microdroplets (MCM) uniquely combine a molecularly crowded aqueous interior with a surrounding membrane, both hallmarks of eukaryotic cells. Replicating cellular functions requires synthetic cells to remain structurally stable in biological environments, where ionic strength presents a significant threat to the integrity of complex coacervates. By leveraging the globular and rigid architecture of dendrimers, MCM, composed of oppositely charged small dendrimers and polypeptides─further stabilized by a charged PEG-dendritic copolymer assembled at the periphery─exhibits a critical salt concentration more than twice that of coacervates formed from polypeptides or branched polyelectrolytes with significantly higher degrees of polymerization. This highlights the enhanced robustness of dendritic MCM under physiological conditions and their suitability as synthetic cells in biological media. By mimicking key cell-like behavior such as efficient enzyme encapsulation (irrespective of the isoelectric point), fast internal dynamics, and chemical communication, dendritic MCM emerge as a promising synthetic cell platform for the selective delivery of therapeutic enzymes. In addition, their ability to engage in signal transduction pathways within synthetic-natural cell consortia, enabling responses to extracellular cues via chemical signaling, paves their way in tissue engineering and regenerative medicine.

Multivalency enables interactions with higher affinities and specificities than monovalent interactions. The strategy exploited by nature to modulate biorecognition has inspired the design of multivalent conjugates with therapeutic properties. However, chemical functionalization often requires coupling agents, additives, or metal catalysts that complicate isolation and purification. Herein, azide-alkyne cycloaddition (AAC) with alkynyl dicarbamates (Alk-R) is presented as a flexible, reliable, atom-economical, and user-friendly strategy for the multivalent functionalization of polymeric scaffolds. Alk-R functionalized with biologically relevant ligands have been prepared and used for the multivalent AAC functionalization of azide-bearing dendrimers and block copolymers. The resulting polymers with double multivalency reveal a platform for the development of bioinspired functional systems with promising applications in drug delivery: block copolymer micelles and multifunctional nanocarriers with synergistically integrated probes-ligands-drugs. The extension of this strategy to other ligands and scaffolds is expected to open up a wide range of therapeutic and diagnostic opportunities.

Gene therapies represent promising new therapeutic options for a variety of indications. However, despite several approved drugs, its potential remains untapped. For polymeric gene delivery, endosomal escape represents a bottleneck. SO1861, a naturally occurring triterpene saponin with endosomal escape properties isolated from Saponaria officinalis L., has been described as additive agent to enhance transfection efficiency (sapofection). However, the challenge to synchronize the saponin and gene delivery system in vivo imposes limitations. Herein, we address this issue by conjugating SO1861 to a peptide-based gene vector using a pH-sensitive hydrazone linker programmed to release SO1861 at the acidic pH of the endosome. Nanoplexes formulated with SO1861-equipped peptides were investigated for transfection efficiency and tolerability in vitro and in vivo. In all investigated cell lines, SO1861-conjugated nanoplexes have shown superior transfection efficiency and cell viability over supplementation of transfection medium with free SO1861. Targeted SO1861-equipped nanoplexes incorporating a targeting peptide were tested in vitro and in vivo in an aggressively growing neuroblastoma allograft model in mice. Using a suicide gene vector encoding the cytotoxic protein saporin, a slowed tumor growth and improved survival rate were observed for targeted SO1861-equipped nanoplexes compared to vehicle control.

Gene therapy provides exciting new therapeutic opportunities beyond the reach of traditional treatments. Despite the tremendous progress of viral vectors, their high cost, complex manufacturing, and side effects have encouraged the development of nonviral alternatives, including cationic polymers. However, these are less efficient in overcoming cellular barriers, resulting in lower transfection efficiencies. Although the exquisite structural tunability of polymers might be envisaged as a versatile tool for improving transfection, the need to fine-tune several structural parameters represents a bottleneck in current screening technologies. By taking advantage of the fast-forming and strong boronate ester bond, an archetypal example of dynamic covalent chemistry, a highly adaptable gene delivery platform is presented, in which the polycation synthesis and pDNA complexation occur in situ. The robustness of the strategy entitles the simultaneous evaluation of several structural parameters at will, enabling the accelerated screening and adaptive optimization of lead polymeric vectors using dynamic covalent libraries.

Linear-dendritic block copolymers assemble in solution due to differences in the solubility or charge properties of the blocks. The monodispersity and multivalency of the dendritic block provide unparalleled control for the design of drug delivery systems when incorporating poly(ethylene glycol) (PEG) as a linear block. An accelerated synthesis of PEG-dendritic block copolymers based on the click and green chemistry pillars is described. The tandem composed of the thermal azide-alkyne cycloaddition with internal alkynes and azide substitution is revealed as a flexible, reliable, atom-economical, and user-friendly strategy for the synthesis and functionalization of biodegradable (polyester) PEG-dendritic block copolymers. The high orthogonality of the sequence has been exploited for the preparation of heterolayered copolymers with terminal alkenes and alkynes, which are amenable for subsequent functionalization by thiol-ene and thiol-yne click reactions. Copolymers with tunable solubility and charge were so obtained for the preparation of various types of nanoassemblies with promising applications in drug delivery.

Tumor-associated macrophages (TAMs), a class of immune cells that play a key role in tumor immunosuppression, are recognized as important targets to improve cancer prognosis and treatment. Consequently, the engineering of drug delivery nanocarriers that can reach TAMs has acquired special relevance. This work describes the development and biological evaluation of a panel of hyaluronic acid (HA) nanocapsules (NCs), with different compositions and prepared by different techniques, designed to target macrophages. The results showed that plain HA NCs did not significantly influence the polarization of M0 and M2-like macrophages towards an M1-like pro-inflammatory phenotype; however, the chemical functionalization of HA with mannose (HA-Man) led to a significant increase of NCs uptake by M2 macrophages in vitro and to an improved biodistribution in a MN/MNCA1 fibrosarcoma mouse model with high infiltration of TAMs. These functionalized HA-Man NCs showed a higher accumulation in the tumor compared to non-modified HA NCs. Finally, the pre-administration of the liposomal liver occupying agent Nanoprimer™ further increased the accumulation of the HA-Man NCs in the tumor. This work highlights the promise shown by the HA-Man NCs to target TAMs and thus provides new options for the development of nanomedicine and immunotherapy-based cancer treatments. Graphical abstract

Herein, we describe the synthesis of an aptadendrimer by covalent bioconjugation of a gallic acid–triethylene glycol (GATG) dendrimer with the G-quadruplex (G4) AT11 aptamer (a modified version of AS1411) at the surface. We evaluated the loading and interaction of an acridine orange ligand, termed C_8, that acts as an anticancer drug and binder/stabilizer of the G4 structure of AT11. Dynamic light scattering experiments demonstrated that the aptadendrimer was approximately 3.1 nm in diameter. Both steady-state and time-resolved fluorescence anisotropy evidenced the interaction between the aptadendrimer and C₈. Additionally, we demonstrated that the iodine atom of the C₈ ligand acts as an effective intramolecular quencher in solution, while upon complexation with the aptadendrimer, it adopts a more extended conformation. Docking studies support this conclusion. Release experiments show a delivery of C₈ after 4 h. The aptadendrimers tend to localize in the cytoplasm of various cell lines studied as demonstrated by confocal microscopy. The internalization of the aptadendrimers is not nucleolin-mediated or by passive diffusion, but via endocytosis. MTT studies with prostate cancer cells and non-malignant cells evidenced high cytotoxicity mainly due to the C₈ ligand. The rapid internalization of the aptadendrimers and the fluorescence properties make them attractive for the development of potential nanocarriers.