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.

The average magnetic anisotropy of polystyrene in polymersomes self-assembled from poly(ethylene glycol)-b-polystyrene.

Using the diamagnetic anisotropy of polymers for the characterization of polymers and polymer aggregates is a relatively new approach in the field of soft-matter and polymer research. So far, a good and thorough quantitative description of these diamagnetic properties has been lacking. Using a simple equation that links the magnetic properties of an average polymer repeating unit to those of the polymer vesicle of any shape, we measured, using magnetic birefringence, the average diamagnetic anisotropy of a polystyrene (PS) repeating unit, ΔχPS, inside a poly(ethylene glycol)-polystyrene (PEG-PS) polymersome membrane as a function of the PS-length and as a function of the preparation method. All obtained values of ΔχPS have a negative sign which results in polymers tending to align perpendicular to an applied magnetic field. Combined, the same order of magnitude of ΔχPS (10-12 m3 mol-1) for all polymersome shapes proves that the individual polymers are organized similarly regardless of the PS length and polymersome shape. Furthermore, the value found is only a fraction (∼1%) of what it can maximally be due to the random coiling of the polymers. We, therefore, predict that further ordering of the polymers within the membrane could lead to similar responses at much lower magnetic fields, possibly obtainable with permanent magnets, which would be highly advantageous for practical applications.

Polymer Vesicles with Integrated Photothermal Responsiveness.

Functionalized polymer vesicles have been proven to be highly promising in biomedical applications due to their good biocompatibility, easy processability, and multifunctional responsive capacities. However, photothermal-responsive polymer vesicles triggered by near-infrared (NIR) light have not been widely reported until now. Herein, we propose a new strategy for designing NIR light-mediated photothermal polymer vesicles. A small molecule (PTA) with NIR-triggered photothermal features was synthesized by combining a D-D'-A-D'-D configuration framework with a molecular rotor function (TPE). The feasibility of the design strategy was demonstrated through density functional theory calculations. PTA moieties were introduced in the hydrophobic segment of a poly(ethylene glycol)-poly(trimethylene carbonate) block copolymer, of which the carbonate monomers were modified in the side chain with an active ester group. The amphiphilic block copolymers (PEG44-PTA2) were then used as building blocks for the self-assembly of photothermal-responsive polymer vesicles. The new class of functionalized polymer vesicles inherited the NIR-mediated high photothermal performance of the photothermal agent (PTA). After NIR laser irradiation for 10 min, the temperature of the PTA-Ps aqueous solution was raised to 56 °C. The photothermal properties and bilayer structure of PTA-Ps after laser irradiation were still intact, which demonstrated that they could be applied as a robust platform in photothermal therapy. Besides their photothermal performance, the loading capacity of PTA-Ps was investigated as well. Hydrophobic cargo (Cy7) and hydrophilic cargo (Sulfo-Cy5) were successfully encapsulated in the PTA-Ps. These properties make this new class of functionalized polymer vesicles an interesting platform for synergistic therapy in anticancer treatment.

Two-photon nanoprobes based on bioorganic nanoarchitectonics with a photo-oxidation enhanced emission mechanism.

Two-photon absorption (TPA) fluorescence imaging holds great promise in diagnostics and biomedicine owing to its unparalleled spatiotemporal resolution. However, the adaptability and applicability of currently available TPA probes, which act as a critical element for determining the imaging contrast effect, is severely challenged by limited photo-luminescence in vivo. This is particularly a result of uncontrollable aggregation that causes fluorescence quenching, and inevitable photo-oxidation in harsh physiological milieu, which normally leads to bleaching of the dye. Herein, we describe the remarkably enhanced TPA fluorescence imaging capacity of self-assembling near-infrared (NIR) cyanine dye-based nanoprobes (NPs), which can be explained by a photo-oxidation enhanced emission mechanism. Singlet oxygen generated during photo-oxidation enables chromophore dimerization to form TPA intermediates responsible for enhanced TPA fluorescence emission. The resulting NPs possess uniform size distribution, excellent stability, more favorable TPA cross-section and anti-bleaching ability than a popular TPA probe rhodamine B (RhB). These properties of cyanine dye-based TPA NPs promote their applications in visualizing blood circulation and tumoral accumulation in real-time, even to cellular imaging in vivo. The photo-oxidation enhanced emission mechanism observed in these near-infrared cyanine dye-based nanoaggregates opens an avenue for design and development of more advanced TPA fluorescence probes.

Inherently Fluorescent Peanut-Shaped Polymersomes for Active Cargo Transportation.

Nanomotors have been extensively explored for various applications in nanomedicine, especially in cargo transportation. Motile properties enable them to deliver pharmaceutical ingredients more efficiently to the targeted site. However, it still remains a challenge to design motor systems that are therapeutically active and can also be effectively traced when taken up by cells. Here, we designed a nanomotor with integrated fluorescence and therapeutic potential based on biodegradable polymersomes equipped with aggregation-induced emission (AIE) agents. The AIE segments provided the polymersomes with autofluorescence, facilitating the visualization of cell uptake. Furthermore, the membrane structure enabled the reshaping of the AIE polymersomes into asymmetric, peanut-shaped polymersomes. Upon laser irradiation, these peanut polymersomes not only displayed fluorescence, but also produced reactive oxygen species (ROS). Because of their specific shape, the ROS gradient induced motility in these particles. As ROS is also used for cancer cell treatment, the peanut polymersomes not only acted as delivery vehicles but also as therapeutic agents. As an integrated platform, these peanut polymersomes therefore represent an interesting delivery system with biomedical potential.

Complex Coacervate Materials as Artificial Cells.

Cells have evolved to be self-sustaining compartmentalized systems that consist of many thousands of biomolecules and metabolites interacting in complex cycles and reaction networks. Numerous subtle intricacies of these self-assembled structures are still largely unknown. The importance of liquid-liquid phase separation (both membraneless and membrane bound) is, however, recognized as playing an important role in achieving biological function that is controlled in time and space. Reconstituting biochemical reactions in vitro has been a success of the last decades, for example, establishment of the minimal set of enzymes and nutrients able to replicate cellular activities like the in vitro transcription translation of genes to proteins. Further than this though, artificial cell research has the aim of combining synthetic materials and nonliving macromolecules into ordered assemblies with the ability to carry out more complex and ambitious cell-like functions. These activities can provide insights into fundamental cell processes in simplified and idealized systems but could also have an applied impact in synthetic biology and biotechnology in the future. To date, strategies for the bottom-up fabrication of micrometer scale life-like artificial cells have included stabilized water-in-oil droplets, giant unilamellar vesicles (GUV's), hydrogels, and complex coacervates. Water-in-oil droplets are a valuable and easy to produce model system for studying cell-like processes; however, the lack of a crowded interior can limit these artificial cells in mimicking life more closely. Similarly membrane stabilized vesicles, such as GUV's, have the additional membrane feature of cells but still lack a macromolecularly crowded cytoplasm. Hydrogel-based artificial cells have a macromolecularly dense interior (although cross-linked) that better mimics cells, in addition to mechanical properties more similar to the viscoelasticity seen in cells but could be seen as being not dynamic in nature and limiting to the diffusion of biomolecules. On the other hand, liquid-liquid phase separated complex coacervates are an ideal platform for artificial cells as they can most accurately mimic the crowded, viscous, highly charged nature of the eukaryotic cytoplasm. Other important key features that researchers in the field target include stabilizing semipermeable membranes, compartmentalization, information transfer/communication, motility, and metabolism/growth. In this Account, we will briefly cover aspects of coacervation theory and then outline key cases of synthetic coacervate materials used as artificial cells (ranging from polypeptides, modified polysaccharides, polyacrylates, and polymethacrylates, and allyl polymers), finishing with envisioned opportunities and potential applications for coacervate artificial cells moving forward.

Enzymatic Regulation of Protein-Protein Interactions in Artificial Cells.

Membraneless organelles are important for spatial organization of proteins and regulation of intracellular processes. Proteins can be recruited to these condensates by specific protein-protein or protein-nucleic acid interactions, which are often regulated by post-translational modifications. However, the mechanisms behind these dynamic, affinity-based protein recruitment events are not well understood. Here, a coacervate system that incorporates the 14-3-3 scaffold protein to study enzymatically regulated recruitment of 14-3-3-binding proteins is presented, which mostly bind in a phosphorylation-dependent manner. Synthetic coacervates are efficiently loaded with 14-3-3, and phosphorylated binding partners, such as the c-Raf pS233/pS259 peptide (c-Raf), show 14-3-3-dependent sequestration with up to 161-fold increase in local concentration. The c-Raf domain is fused to green fluorescent protein (GFP-c-Raf) to demonstrate recruitment of proteins. In situ phosphorylation of GFP-c-Raf by a kinase leads to enzymatically regulated uptake. The introduction of a phosphatase into coacervates preloaded with the phosphorylated 14-3-3-GFP-c-Raf complex results in a significant cargo efflux mediated by dephosphorylation. Finally, the general applicability of this platform to study protein-protein interactions is demonstrated by the phosphorylation-dependent and 14-3-3-mediated active reconstitution of a split-luciferase inside artificial cells. This work presents an approach to study dynamically regulated protein recruitment in condensates, using native interaction domains.

Light-Responsive Elastin-Like Peptide-Based Targeted Nanoparticles for Enhanced Spheroid Penetration.

We describe here a near infrared light-responsive elastin-like peptide (ELP)-based targeted nanoparticle (NP) that can rapidly switch its size from 120 to 25 nm upon photo-irradiation. Interestingly, the targeting function, which is crucial for effective cargo delivery, is preserved after transformation. The NPs are assembled from (targeted) diblock ELP micelles encapsulating photosensitizer TT1-monoblock ELP conjugates. Methionine residues in this monoblock are photo-oxidized by singlet oxygen generated from TT1, turning the ELPs hydrophilic and thus trigger NP dissociation. Phenylalanine residues from the diblocks then interact with TT1 via π-π stacking, inducing the re-formation of smaller NPs. Due to their small size and targeting function, the NPs penetrate deeper in spheroids and kill cancer cells more efficiently compared to the larger ones. This work could contribute to the design of "smart" nanomedicines with deeper penetration capacity for effective anticancer therapies.

Cargo-loading of hybrid cowpea chlorotic mottle virus capsids via a co-expression approach.

Capsids of the cowpea chlorotic mottle virus (CCMV) are great candidates for the development into in vivo catalytic or therapeutic nanocarriers. However, due to their limited intrinsic stability at physiological pH, thus far no methods exist for incorporating cargo into these nanoparticles in cellulo. Here, we employ a stabilized VW1-VW8 ELP-CCMV variant for the development of a co-expression-based cargo-loading approach. Co-expression of the non-functionalized VW1-VW8 ELP-CCMV coat protein with fusion proteins with enhanced green fluorescent protein (mEGFP) and pyrrolysine synthase D (PylD) in E. coli enabled the purification of cargo-loaded capsids from the bacteria directly either via affinity chromatography or PEG-precipitation and subsequent size exclusion chromatography. Microscopy results indicated that the co-expression does not harm the E. coli cells and that proper folding of the mEGFP domain is not hampered by the co-assembly. Our co-expression strategy is thus a suitable approach to produce cargo-loaded CCMV nanoparticles.

The Dynamics of Viruslike Capsid Assembly and Disassembly.

Cowpea chlorotic mottle virus (CCMV) is a widely used model for virus replication studies. A major challenge lies in distinguishing between the roles of the interaction between coat proteins and that between the coat proteins and the viral RNA in assembly and disassembly processes. Here, we report on the spontaneous and reversible size conversion of the empty capsids of a CCMV capsid protein functionalized with a hydrophobic elastin-like polypeptide which occurs following a pH jump. We monitor the concentrations of T = 3 and T = 1 capsids as a function of time and show that the time evolution of the conversion from one T number to another is not symmetric: The conversion from T = 1 to T = 3 is a factor of 10 slower than that of T = 3 to T = 1. We explain our experimental findings using a simple model based on classical nucleation theory applied to virus capsids, in which we account for the change in the free protein concentration, as the different types of shells assemble and disassemble by shedding or absorbing single protein subunits. As far as we are aware, this is the first study confirming that both the assembly and disassembly of viruslike shells can be explained through classical nucleation theory, reproducing quantitatively results from time-resolved experiments.

Injectable alginate hydrogels for synergistic tumor combination therapy through repolarization of tumor-associated macrophages.

Locally administered drug delivery systems are promising as they allow to circumvent the side effects associated with systematic administration. In this study, we constructed multifunctional hydrogels by simply mixing commercial alginate (ALG) sols with glucose oxidase (GOx)-conjugated polyacrylic acid-stabilized iron oxide nanoparticles (GPI NPs) and Toll-like receptor 7/8 agonist resiquimod (R848). The injectable sols were able to transform into hydrogels (GPI/R848@ALG) by the ionic cross-linking between ALG and physiological Ca2+ to trap the therapeutic components within the hydrogel framework. Upon intratumoral injection, the hydrogels were employed for starvation therapy, promoted chemodynamic therapy and tumor-associated macrophages (TAMs) repolarization. The energy supply was blocked by consuming the intratumoral glucose via the GOx-catalyzed conversion of glucose into gluconic acid and hydrogen peroxide (H2O2).In vitro results showed that the generated H2O2 could be further converted into highly cytotoxic hydroxyl radicals (·HO) by the Fenton reaction to induce enhanced chemodynamic therapy. The TAMs repolarization studies in vitro exhibited that the GPI/R848@ALG hydrogels up-regulated the expression of CD86 by 63% and down-regulated the proportion of CD206 by 14% with a synergistic effect of the presence of Fe3O4 and R848, suggesting that the multifunctional hydrogels exert functions to direct the remodeling of TAMs from the tumor supportive M2-like phenotype to the tumor destructive M1-like phenotype to further contribute to the antitumor effect. Moreover, both in vitro and in vivo experiments demonstrate that the multifunctional hydrogels exhibit admirable antitumor performance towards 4T1 tumors. This work thereby provides a promising multifunctional nanoplatform for synergistic cancer starvation therapy, chemodynamic therapy and TAMs repolarization.

Peptide-based supramolecular assembly drugs toward cancer theranostics.

INTRODUCTION: Peptide-based supramolecular self-assembly has been demonstrated to be a flexible approach for the fabrication of programmable de novo nanodrugs by employing synergistic or reciprocal intermolecular non-covalent interactions. This class of nanomaterials holds significant promise for clinical translation, especially as cancer theranostics. AREAS COVERED: In this review, we describe the concept of cancer theranostic drug assembly by employing non-covalent interactions. That is, molecular drugs are formulated into nanoscale and even microscale architectures by peptide-modulated self-assembly. A series of peptide-based supramolecular assembly drugs are discussed, with an emphasis on the relation between structural feature and theranostic performance. EXPERT OPINION: Molecular design, manipulation of non-covalent interactions, and elucidation of structure-function relationships not only facilitate the implementation of supramolecular self-assembly principles in drug development, but also provide a new means for advancing anticancer nanostructured drugs toward clinical application.

Therapeutic Stomatocytes with Aggregation Induced Emission for Intracellular Delivery.

Bowl-shaped biodegradable polymersomes, or stomatocytes, have much potential as drug delivery systems, due to their intriguing properties, such as controllable size, programmable morphology, and versatile cargo encapsulation capability. In this contribution, we developed well-defined therapeutically active stomatocytes with aggregation-induced emission (AIE) features by self-assembly of biodegradable amphiphilic block copolymers, comprising poly(ethylene glycol) (PEG) and AIEgenic poly(trimethylene carbonate) (PTMC) moieties. The presence of the AIEgens endowed the as-prepared stomatocytes with intrinsic fluorescence, which was employed for imaging of cellular uptake of the particles. It simultaneously enabled the photo-mediated generation of reactive oxygen species (ROS) for photodynamic therapy. The potential of the therapeutic stomatocytes as cargo carriers was demonstrated by loading enzymes (catalase and glucose oxidase) in the nanocavity, followed by a cross-linking reaction to achieve stable encapsulation. This provided the particles with a robust motile function, which further strengthened their therapeutic effect. With these unique features, enzyme-loaded AIEgenic stomatocytes are an attractive platform to be exploited in the field of nanomedicine.

Refining the Design of Diblock Elastin-Like Polypeptides for Self-Assembly into Nanoparticles.

Diblock copolymers based-on elastin-like polypeptide (ELP) have the potential to undergo specific phase transitions when thermally stimulated. This ability is especially suitable to form carriers, micellar structures for instance, for delivering active cargo molecules. Here, we report the design and study of an ELP diblock library based on ELP-[M1V3-i]-[I-j]. First, ELP-[M1V3-i]-[I-j] (i = 20, 40, 60; j = 20, 90) that showed a similar self-assembly propensity (unimer-to-aggregate transition) as their related monoblocks ELP-[M1V3-i] and ELP-[I-j]. By selectively oxidizing methionines of ELP-[M1V3-i] within the different diblocks structures, we have been able to access a thermal phase transition with three distinct regimes (unimers, micelles, aggregates) characteristic of well-defined ELP diblocks.

Self-Assembly or Coassembly of Multiresponsive Histidine-Containing Elastin-Like Polypeptide Block Copolymers.

In this study a histidine containing elastin-like polypeptide (ELP) diblock copolymer is described with multiresponsive assembly behavior. Self-assembly into micelles is examined by two methods. First, the self-assembly is triggered by the addition of divalent metal ions, with Zn2+ being the most suitable one. Increasing the Zn2+ concentration stabilizes the nanoparticles over a large temperature window (4-45 °C). This diblock exhibits furthermore pH-responsiveness, and particles disassemble under mildly acidic conditions. Second, the coassembly of this ELP with a diblock ELP is examined, which is not responsive to pH and metal ions. Coassembly is triggered by heating the ELPs quickly above the transition temperature of the less hydrophobic block, which results in stable nanoparticles without the need to add metal ions. This novel ELP system offers a versatile modular nanocarrier platform that can respond to different stimuli and can be tuned effectively.

Biodegradable Polymersomes with Structure Inherent Fluorescence and Targeting Capacity for Enhanced Photo-Dynamic Therapy.

Biodegradable nanostructures displaying aggregation-induced emission (AIE) are desirable from a biomedical point of view, due to the advantageous features of loading capacity, emission brightness, and fluorescence stability. Herein, biodegradable polymers comprising poly (ethylene glycol)-block-poly(caprolactone-gradient-trimethylene carbonate) (PEG-P(CLgTMC)), with tetraphenylethylene pyridinium-TMC (PAIE) side chains have been developed, which self-assembled into well-defined polymersomes. The resultant AIEgenic polymersomes are intrinsically fluorescent delivery vehicles. The presence of the pyridinium moiety endows the polymersomes with mitochondrial targeting ability, which improves the efficiency of co-encapsulated photosensitizers and improves therapeutic index against cancer cells both in vitro and in vivo. This contribution showcases the ability to engineer AIEgenic polymersomes with structure inherent fluorescence and targeting capacity for enhanced photodynamic therapy.

Dual Site-Selective Presentation of Functional Handles on Protein-Engineered Cowpea Chlorotic Mottle Virus-Like Particles.

Protein cages hold much promise as carrier systems in nanomedicine, due to their well-defined size, cargo-loading capacity, and inherent biodegradability. In order to make them suitable for drug delivery, they have to be stable under physiological conditions. In addition, often surface modifications are required, for example, to improve cell targeting or reduce the particle immunogenicity by PEGylation. For this purpose, we investigated the functionalization capacity of the capsid of cowpea chlorotic mottle virus (CCMV), modified at the interior with a stabilizing elastin-like polypeptide (ELP) tag, by employing a combination of protein engineering and bio-orthogonal chemistry. We first demonstrated the accessibility of the native cysteine residue in ELP-CCMV as a site-selective surface-exposed functional handle, which was not available in the native CCMV capsid. An additional bio-orthogonal functional handle was introduced by incorporation of the noncanonical amino acid, azido-phenylalanine (AzF), using the amber suppression mechanism. Dual site-selective presentation of both a cell-penetrating TAT peptide and a fluorophore to track the particles was demonstrated successfully in HeLa cell uptake studies.

Photoactivated nanomotors via aggregation induced emission for enhanced phototherapy.

Aggregation-induced emission (AIE) has, since its discovery, become a valuable tool in the field of nanoscience. AIEgenic molecules, which display highly stable fluorescence in an assembled state, have applications in various biomedical fields-including photodynamic therapy. Engineering structure-inherent, AIEgenic nanomaterials with motile properties is, however, still an unexplored frontier in the evolution of this potent technology. Here, we present phototactic/phototherapeutic nanomotors where biodegradable block copolymers decorated with AIE motifs can transduce radiant energy into motion and enhance thermophoretic motility driven by an asymmetric Au nanoshell. The hybrid nanomotors can harness two photon near-infrared radiation, triggering autonomous propulsion and simultaneous phototherapeutic generation of reactive oxygen species. The potential of these nanomotors to be applied in photodynamic therapy is demonstrated in vitro, where near-infrared light directed motion and reactive oxygen species induction synergistically enhance efficacy with a high level of spatial control.

Pathway-Dependent Co-Assembly of Elastin-Like Polypeptides.

In natural systems, temperature-induced assembly of biomolecules can lead to the formation of distinct assembly states, created out of the same set of starting compounds, based on the heating trajectory followed. Until now it has been difficult to achieve similar behavior in synthetic polymer mixtures. Here, a novel pathway-dependent assembly based on stimulus-responsive polymers is shown. When a mixture of mono- and diblock copolymers, based on elastin-like polypeptides, is heated with a critical heating rate co-assembled particles are created that are monodisperse, stable, and have tunable hydrodynamic radii between 20 and 120 nm. Below this critical heating rate, the constituents separately form polymer assemblies. This process is kinetically driven and reversible in thermodynamically closed systems. Using the co-assembly pathway, fluorescent proteins and bioluminescent enzymes are encapsulated with high efficiency. Encapsulated cargo shows unperturbed function even after delivery into cells. The pathway-dependent co-assembly of elastin-like polypeptides is not only of fundamental interest from a materials science perspective, allowing the formation of multiple distinct assemblies from the same starting compounds, which can be interconverted by going back to the molecularly dissolved states. It also enables a versatile way for constructing highly effective vehicles for the cellular delivery of biomolecular cargo.