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.

Regulating Chemokine-Receptor Interactions through the Site-Specific Bioorthogonal Conjugation of Photoresponsive DNA Strands.

Oligonucleotide conjugation has emerged as a versatile molecular tool for regulating protein activity. A state-of-the-art labeling strategy includes the site-specific conjugation of DNA, by employing bioorthogonal groups genetically incorporated in proteins through unnatural amino acids (UAAs). The incorporation of UAAs in chemokines has to date, however, remained underexplored, probably due to their sometimes poor stability following recombinant expression. In this work, we designed a fluorescent stromal-derived factor-1ß (SDF-1ß) chemokine fusion protein with a bioorthogonal functionality amenable for click reactions. Using amber stop codon suppression, p-azido-L-phenylalanine was site-specifically incorporated in the fluorescent N-terminal fusion partner, superfolder green fluorescent protein (sfGFP). Conjugation to single-stranded DNAs (ssDNA), modified with a photocleavable spacer and a reactive bicyclononyne moiety, was performed to create a DNA-caged species that blocked the receptor binding ability. This inhibition was completely reversible by means of photocleavage of the ssDNA strands. The results described herein provide a versatile new direction for spatiotemporally regulating chemokine-receptor interactions, which is promising for tissue engineering purposes.

Biodegradable Grubbs-Loaded Artificial Organelles for Endosomal Ring-Closing Metathesis.

The application of transition-metal catalysts in living cells presents a promising approach to facilitate reactions that otherwise would not occur in nature. However, the usage of metal complexes is often restricted by their limited biocompatibility, toxicity, and susceptibility to inactivation and loss of activity by the cell's defensive mechanisms. This is especially relevant for ruthenium-mediated reactions, such as ring-closing metathesis. In order to address these issues, we have incorporated the second-generation Hoveyda-Grubbs catalyst (HGII) into polymeric vesicles (polymersomes), which were composed of biodegradable poly(ethylene glycol)-b-poly(caprolactone-g-trimethylene carbonate) [PEG-b-P(CL-g-TMC)] block copolymers. The catalyst was either covalently or non-covalently introduced into the polymersome membrane. These polymersomes were able to act as artificial organelles that promote endosomal ring-closing metathesis for the intracellular generation of a fluorescent dye. This is the first example of the use of a polymersome-based artificial organelle with an active ruthenium catalyst for carbon-carbon bond formation.

Responsive Peptide Nanofibers with Theranostic and Prognostic Capacity.

Photodynamic therapy (PDT) is a highly promising therapeutic modality for cancer treatment. The development of stimuli-responsive photosensitizer nanomaterials overcomes certain limitations in clinical PDT. Herein, we report the rational design of a highly sensitive PEGylated photosensitizer-peptide nanofiber (termed PHHPEG 6 NF) that selectively aggregates in the acidic tumor and lysosomal microenvironment. These nanofibers exhibit acid-induced enhanced singlet oxygen generation, cellular uptake, and PDT efficacy in vitro , as well as fast tumor accumulation, long-term tumor imaging capacity and effective PDT in vivo . Moreover, based on the prolonged presence of the fluorescent signal at the tumor site, we demonstrate that PHHPEG 6 NFs can also be applied for prognostic monitoring of the efficacy of PDT in vivo , which would potentially guide cancer treatment. Therefore, these multifunctional PHHPEG 6 NFs allow control over the entire PDT process, from visualization of photosensitizer accumulation, via actual PDT to the assessment of the efficacy of the treatment.

DNA-Mediated Protein Shuttling between Coacervate-Based Artificial Cells.

The regulation of protein uptake and secretion is crucial for (inter)cellular signaling. Mimicking these molecular events is essential when engineering synthetic cellular systems. A first step towards achieving this goal is obtaining control over the uptake and release of proteins from synthetic cells in response to an external trigger. Herein, we have developed an artificial cell that sequesters and releases proteinaceous cargo upon addition of a coded chemical signal: single-stranded DNA oligos (ssDNA) were employed to independently control the localization of a set of three different ssDNA-modified proteins. The molecular coded signal allows for multiple iterations of triggered uptake and release, regulation of the amount and rate of protein release and the sequential release of the three different proteins. This signaling concept was furthermore used to directionally transfer a protein between two artificial cell populations, providing novel directions for engineering lifelike communication pathways inside higher order (proto)cellular structures.

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.

Engineering of Biocompatible Coacervate-Based Synthetic Cells.

Polymer-stabilized complex coacervate microdroplets have emerged as a robust platform for synthetic cell research. Their unique core-shell properties enable the sequestration of high concentrations of biologically relevant macromolecules and their subsequent release through the semipermeable membrane. These unique properties render the synthetic cell platform highly suitable for a range of biomedical applications, as long as its biocompatibility upon interaction with biological cells is ensured. The purpose of this study is to investigate how the structure and formulation of these coacervate-based synthetic cells impact the viability of several different cell lines. Through careful examination of the individual synthetic cell components, it became evident that the presence of free polycation and membrane-forming polymer had to be prevented to ensure cell viability. After closely examining the structure-toxicity relationship, a set of conditions could be found whereby no detrimental effects were observed, when the artificial cells were cocultured with RAW264.7 cells. This opens up a range of possibilities to use this modular system for biomedical applications and creates design rules for the next generation of coacervate-based, biomedically relevant particles.

Programmed spatial organization of biomacromolecules into discrete, coacervate-based protocells.

The cell cytosol is crowded with high concentrations of many different biomacromolecules, which is difficult to mimic in bottom-up synthetic cell research and limits the functionality of existing protocellular platforms. There is thus a clear need for a general, biocompatible, and accessible tool to more accurately emulate this environment. Herein, we describe the development of a discrete, membrane-bound coacervate-based protocellular platform that utilizes the well-known binding motif between Ni2+-nitrilotriacetic acid and His-tagged proteins to exercise a high level of control over the loading of biologically relevant macromolecules. This platform can accrete proteins in a controlled, efficient, and benign manner, culminating in the enhancement of an encapsulated two-enzyme cascade and protease-mediated cargo secretion, highlighting the potency of this methodology. This versatile approach for programmed spatial organization of biologically relevant proteins expands the protocellular toolbox, and paves the way for the development of the next generation of complex yet well-regulated synthetic cells.

Molecular Repolarisation of Tumour-Associated Macrophages.

The tumour microenvironment (TME) is composed of extracellular matrix and non-mutated cells supporting tumour growth and development. Tumour-associated macrophages (TAMs) are among the most abundant immune cells in the TME and are responsible for the onset of a smouldering inflammation. TAMs play a pivotal role in oncogenic processes as tumour proliferation, angiogenesis and metastasis, and they provide a barrier against the cytotoxic effector function of T lymphocytes and natural killer (NK) cells. However, TAMs are highly plastic cells that can adopt either pro- or anti-inflammatory roles in response to environmental cues. Consequently, TAMs represent an attractive target to recalibrate immune responses in the TME. Initial TAM-targeted strategies, such as macrophage depletion or disruption of TAM recruitment, have shown beneficial effects in preclinical models and clinical trials. Alternatively, reprogramming TAMs towards a proinflammatory and tumouricidal phenotype has become an attractive strategy in immunotherapy. This work summarises the molecular wheelwork of macrophage biology and presents an overview of molecular strategies to repolarise TAMs in immunotherapy.