Trajectory analysis quantifies transcriptional plasticity during macrophage polarization.

In recent years, macrophages have been shown to be tremendously plastic in both in vitro and in vivo settings; however, it remains unclear whether macrophages retain any persistent memory of past polarization states which may then impact their future repolarization to new states. Here, we perform deep transcriptomic profiling at high temporal resolution as macrophages are polarized with cytokines that drive them into "M1" and "M2" molecular states. We find through trajectory analysis of their global transcriptomic profiles that macrophages which are first polarized to M1 or M2 and then subsequently repolarized demonstrate little to no memory of their polarization history. We observe complete repolarization both from M1 to M2 and vice versa, and we find that macrophage transcriptional phenotypes are defined by the current cell microenvironment, rather than an amalgamation of past and present states.

The Cancer Microbiome: Distinguishing Direct and Indirect Effects Requires a Systemic View.

The collection of microbes that live in and on the human body - the human microbiome - can impact on cancer initiation, progression, and response to therapy, including cancer immunotherapy. The mechanisms by which microbiomes impact on cancers can yield new diagnostics and treatments, but much remains unknown. The interactions between microbes, diet, host factors, drugs, and cell-cell interactions within the cancer itself likely involve intricate feedbacks, and no single component can explain all the behavior of the system. Understanding the role of host-associated microbial communities in cancer systems will require a multidisciplinary approach combining microbial ecology, immunology, cancer cell biology, and computational biology - a systems biology approach.

Current state of in vivo panning technologies: Designing specificity and affinity into the future of drug targeting.

Targeting ligands are used in drug delivery to improve drug distribution to desired cells or tissues and to facilitate cellular entry. In vivo biopanning, whereby billions of potential ligand sequences are screened in biologically-relevant and complex conditions, is a powerful method for identification of novel target ligands. This tool has impacted drug delivery technologies and expanded our arsenal of therapeutics and diagnostics. Within this review we will discuss current in vivo panning technologies and ways that these technologies can be improved to advance next-generation drug delivery strategies.

Serum Nuclease Susceptibility of mRNA Cargo in Condensed Polyplexes.

Messenger RNA (mRNA) is a biomolecule with a wide range of promising clinical applications. However, the unstable nature of mRNA and its susceptibility to degradation by ribonucleases (RNases) necessitate the use of specialized formulations for delivery. Polycations are an emerging class of synthetic carriers capable of packaging nucleic acids, and may serve as suitable RNase-resistant formulations for mRNA administration. Here, we explore the application of VIPER and sunflower polycations, two polycations previously synthesized by our group, for the delivery of mRNA in comparison to branched poly(ethylenimine); all three polycations have been shown to efficiently deliver plasmid DNA (pDNA) to cultured cells. Despite successful mRNA condensation and packaging, transfection studies reveal that these three polycations can only efficiently deliver mRNA under serum-free conditions, while pDNA delivery is achieved even in the presence of serum. RNase resistance studies confirm that nuclease degradation of mRNA cargo remains a significant barrier to mRNA delivery using these polycations. These results emphasize the need for additional strategies for nuclease protection of mRNA cargo beyond electrostatic complexation with polycation.

Reversibly Switchable, pH-Dependent Peptide Ligand Binding via 3,5-Diiodotyrosine Substitutions.

Cell type-specific targeting ligands utilized in drug delivery applications typically recognize receptors that are overexpressed on the cells of interest. Nonetheless, these receptors may also be expressed, to varying extents, on off-target cells, contributing to unintended side effects. For the selectivity profile of targeting ligands in cancer therapy to be improved, stimuli-responsive masking of these ligands with acid-, redox-, or enzyme-cleavable molecules has been reported, whereby the targeting ligands are exposed in specific environments, e.g., acidic tumor hypoxia. One possible drawback of these systems lies in their one-time, permanent trigger, which enables the "demasked" ligands to bind off-target cells if released back into the systemic circulation. A promising strategy to address the aforementioned problem is to design ligands that show selective binding based on ionization state, which may be microenvironment-dependent. In this study, we report a systematic strategy to engineer low pH-selective targeting peptides using an M2 macrophage-targeting peptide (M2pep) as an example. 3,5-Diiodotyrosine mutagenesis into native tyrosine residues of M2pep confers pH-dependent binding behavior specific to acidic environment (pH 6) when the amino acid is protonated into the native tyrosine-like state. At physiological pH of 7.4, the hydroxyl group of 3,5-diiodotyrosine on the peptide is deprotonated leading to interruption of the peptide native binding property. Our engineered pH-responsive M2pep (Ac-Y-Î-Î) binds target M2 macrophages more selectively at pH 6 than at pH 7.4. In addition, 3,5-diiodotyrosine substitutions also improve serum stability of the peptide. Finally, we demonstrate pH-dependent reversibility in target binding via a postbinding peptide elution study. The strategy presented here should be applicable for engineering pH-dependent functionality of other targeting peptides with potential applications in physiology-dependent in vivo targeting applications (e.g., targeting hypoxic tumor/inflammation) or in in vitro receptor identification.

Evolution of a designed protein assembly encapsulating its own RNA genome.

The challenges of evolution in a complex biochemical environment, coupling genotype to phenotype and protecting the genetic material, are solved elegantly in biological systems by the encapsulation of nucleic acids. In the simplest examples, viruses use capsids to surround their genomes. Although these naturally occurring systems have been modified to change their tropism and to display proteins or peptides, billions of years of evolution have favoured efficiency at the expense of modularity, making viral capsids difficult to engineer. Synthetic systems composed of non-viral proteins could provide a 'blank slate' to evolve desired properties for drug delivery and other biomedical applications, while avoiding the safety risks and engineering challenges associated with viruses. Here we create synthetic nucleocapsids, which are computationally designed icosahedral protein assemblies with positively charged inner surfaces that can package their own full-length mRNA genomes. We explore the ability of these nucleocapsids to evolve virus-like properties by generating diversified populations using Escherichia coli as an expression host. Several generations of evolution resulted in markedly improved genome packaging (more than 133-fold), stability in blood (from less than 3.7% to 71% of packaged RNA protected after 6 hours of treatment), and in vivo circulation time (from less than 5 minutes to approximately 4.5 hours). The resulting synthetic nucleocapsids package one full-length RNA genome for every 11 icosahedral assemblies, similar to the best recombinant adeno-associated virus vectors. Our results show that there are simple evolutionary paths through which protein assemblies can acquire virus-like genome packaging and protection. Considerable effort has been directed at 'top-down' modification of viruses to be safe and effective for drug delivery and vaccine applications; the ability to design synthetic nanomaterials computationally and to optimize them through evolution now enables a complementary 'bottom-up' approach with considerable advantages in programmability and control.

A Facile Cyclization Method Improves Peptide Serum Stability and Confers Intrinsic Fluorescence.

Peptides are a growing class of macromolecules used in pharmaceutics. The path toward the clinical use of candidate peptides involves sequence optimization and cyclization for stability and affinity. For internalized peptides, tagging is also often required for intracellular trafficking studies, although fluorophore conjugation has an impact on peptide binding, permeability, and localization. Herein, a strategy based on cysteine arylation with tetrafluoroterephthalonitrile (4F-2CN), which simultaneously cyclizes peptides and imparts fluorescence, is reported. The 4F-2CN cyclization of an M2 macrophage-targeting peptide yields, in a single step, a peptide with improved serum stability, intrinsic fluorescence, and increased binding affinity. In a murine breast cancer model, it is demonstrated that the intrinsic fluorescence from the cyclized peptide is sufficient for monitoring biodistribution by whole-organ fluorescence imaging and cell internalization by flow cytometry.

Progress in tumor-associated macrophage (TAM)-targeted therapeutics.

As an essential innate immune population for maintaining body homeostasis and warding off foreign pathogens, macrophages display high plasticity and perform diverse supportive functions specialized to different tissue compartments. Consequently, aberrance in macrophage functions contributes substantially to progression of several diseases including cancer, fibrosis, and diabetes. In the context of cancer, tumor-associated macrophages (TAMs) in tumor microenvironment (TME) typically promote cancer cell proliferation, immunosuppression, and angiogenesis in support of tumor growth and metastasis. Oftentimes, the abundance of TAMs in tumor is correlated with poor disease prognosis. Hence, significant attention has been drawn towards development of cancer immunotherapies targeting these TAMs; either depleting them from tumor, blocking their pro-tumoral functions, or restoring their immunostimulatory/tumoricidal properties. This review aims to introduce readers to various aspects in development and evaluation of TAM-targeted therapeutics in pre-clinical and clinical stages.

Serum Stability and Affinity Optimization of an M2 Macrophage-Targeting Peptide (M2pep).

Tumor associated macrophages (TAMs) are a major stromal component of the tumor microenvironment in several cancers. TAMs are a potential target for adjuvant cancer therapies due to their established roles in promoting proliferation of cancer cells, angiogenesis, and metastasis. We previously discovered an M2 macrophage-targeting peptide (M2pep) which was successfully used to target and deliver a pro-apoptotic KLA peptide to M2-like TAMs in a CT-26 colon carcinoma model. However, the effectiveness of in vivo TAM-targeting using M2pep is limited by its poor serum stability and low binding affinity. In this study, we synthesized M2pep derivatives with the goals of increasing serum stability and binding affinity. Serum stability evaluation of M2pepBiotin confirmed its rapid degradation attributed to exolytic cleavage from the N-terminus and endolytic cleavages at the W10/W11 and S16/K17 sites. N-terminal acetylation of M2pepBiotin protected the peptide against the exolytic degradation while W10w and K(17,18,19)k substitutions were able to effectively protect endolytic degradation at their respective cleavage sites. However, no tested amino acid changes at the W10 position resulted in both protease resistance at that site and retention of binding activity. Therefore, cyclization of M2pep was investigated. Cyclized M2pep better resisted serum degradation without compromising binding activity to M2 macrophages. During the serum stability optimization process, we also discovered that K9R and W10Y substitutions significantly enhanced binding affinity of M2pep. In an in vitro binding study of different M2pep analogs pre-incubated in mouse serum, cyclic M2pep with K9R and W10Y modifications (cyclic M2pep(RY)) retained the highest binding activity to M2 macrophages over time due to its improved serum stability. Finally, we evaluated the in vivo accumulation of sulfo-Cy5-labeled M2pep and cyclic M2pep(RY) in both the CT-26 and 4T1 breast carcinoma models. Cyclic M2pep(RY) outperformed M2pep in both tumor localization and selective accumulation in M2-like TAMs. In conclusion, we report cyclic M2pep(RY) as our lead M2pep analog with improved serum stability and M2 macrophage-binding activity. Its enhanced utility as an in vivo M2-like-TAM-targeting agent was demonstrated in two tumor models, and is expected to be applicable for other tumor models or in models of M2 macrophage-related diseases.