Molecular determinants and signaling effects of PKA RIα phase separation.

Spatiotemporal regulation of intracellular signaling molecules, such as the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), ensures proper cellular function. Liquid-liquid phase separation (LLPS) of the ubiquitous PKA regulatory subunit RIα promotes cAMP compartmentation and signaling specificity. However, the molecular determinants of RIα LLPS remain unclear. Here, we reveal that two separate dimerization interfaces, combined with the cAMP-induced unleashing of the PKA catalytic subunit (PKA-C) from the pseudosubstrate inhibitory sequence, drive RIα condensate formation in the cytosol of mammalian cells, which is antagonized by docking to A-kinase anchoring proteins. Strikingly, we find that the RIα pseudosubstrate region is critically involved in forming a non-canonical R:C complex, which recruits active PKA-C to RIα condensates to maintain low basal PKA activity in the cytosol. Our results suggest that RIα LLPS not only facilitates cAMP compartmentation but also spatially restrains active PKA-C, thus highlighting the functional versatility of biomolecular condensates in driving signaling specificity.

Local and Targeted Delivery of Immune Checkpoint Blockade Therapeutics.

Immune checkpoint blockade (ICB) therapy elicits antitumor response by inhibiting immune suppressor components, including programmed cell death protein 1 and its ligand (PD-1/PD-L1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4). Despite improved therapeutic efficacy, the clinical response rate is still unsatisfactory as revealed by the fact that only a minority of patients experience durable benefits. Additionally, "off-target" effects after systemic administration remain challenging for ICB treatment. To this end, the local and targeted delivery of ICB agents instead could be a potential solution to maximize the therapeutic outcomes while minimizing the side effects.In this Account, our recent studies directed at the development of different strategies for the local and targeted delivery of ICB agents are discussed. For example, transdermal microneedle patches loaded with anti-programmed death-1 antibody (aPD1) and anti-CTLA4 were developed to facilitate sustained release of ICB agents at the diseased sites. Triggered release could also be achieved by various stimuli within the tumor microenvironment, including low pH and abnormally expressed enzymes. Recently, the combination of an anti-programmed death-ligand 1 antibody (aPD-L1) loaded hollow-structured microneedle patch with cold atmospheric plasma (CAP) therapy was also reported. Microneedles provided microchannels to facilitate the transdermal transport of CAP and further induce immunogenic tumor cell death, which could be synergized by the local release of aPD-L1. In addition, in situ formed injectable or sprayable hydrogels were tailored to deliver immunomodulatory antibodies to the surgical bed to inhibit tumor recurrence after primary tumor resection. In paralell, inspired by the unique targeting ability of platelets toward the inflammatory sites, we engineered natural platelets decorated with aPD-L1 for targeted delivery after tumor resection to inhibit tumor recurrence. We further constructed a cell-cell combination delivery platform based on conjugates of platelets and hematopoietic stem cells (HSCs) for leukemia treatment. With the homing ability of HSCs to the bone marrow, the HSC-platelet-aPD1 assembly could effectively deliver aPD1 in an acute myeloid leukemia mouse model. Besides living cells, we also leveraged HEK293T-derived vesicles with PD1 receptors on their surfaces to disrupt the PD-1/PD-L1 immune inhibitory pathway. Moreover, the inner space of the vesicles allowed the packaging of an indoleamine 2,3-dioxygenase inhibitor, further reinforcing the therapeutic efficacy. A similar approach has also been demonstrated by genetically engineering platelets overexpressing PD1 receptor for postsurgical treatment. We hope the local and targeted ICB agent delivery methods introduced in this collection would further inspire the development of advanced drug delivery strategies to improve the efficiency of cancer treatment while alleviating side effects.

Molecular Determinants and Signaling Effects of PKA RIα Phase Separation.

Spatiotemporal regulation of intracellular signaling molecules, such as the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), ensures the specific execution of various cellular functions. Liquid-liquid phase separation (LLPS) of the ubiquitously expressed PKA regulatory subunit RIα was recently identified as a major driver of cAMP compartmentation and signaling specificity. However, the molecular determinants of RIα LLPS remain unclear. Here, we reveal that two separate dimerization interfaces combined with the cAMP-induced release of the PKA catalytic subunit (PKA-C) from the pseudosubstrate inhibitory sequence are required to drive RIα condensate formation in cytosol, which is antagonized by docking to A-kinase anchoring proteins. Strikingly, we find that the RIα pseudosubstrate region is critically involved in the formation of a non-canonical R:C complex, which serves to maintain low basal PKA activity in the cytosol by enabling the recruitment of active PKA-C to RIα condensates. Our results suggest that RIα LLPS not only facilitates cAMP compartmentation but also spatially restrains active PKA-C, thus highlighting the functional versatility of biomolecular condensates in driving signaling specificity.

Injectable Biodegradable Polymeric Complex for Glucose-Responsive Insulin Delivery.

Insulin therapy is the central component of treatment for type 1 and advanced type 2 diabetes; however, its narrow therapeutic window is associated with a risk of severe hypoglycemia. A glucose-responsive carrier that demonstrates consistent and slow basal insulin release under a normoglycemic condition and accelerated insulin release in response to hyperglycemia in real-time could offer effective blood glucose regulation with reduced risk of hypoglycemia. Here, we describe a poly(l-lysine)-derived biodegradable glucose-responsive cationic polymer for constructing polymer-insulin complexes for glucose-stimulated insulin delivery. The effects of the modification degree of arylboronic acid in the synthesized cationic polymer and polymer-to-insulin ratio on the glucose-dependent equilibrated free insulin level and the associated insulin release kinetics have been studied. In addition, the blood glucose regulation ability of these complexes and the associated glucose challenge-triggered insulin release are evaluated in type 1 diabetic mice.

Advances in engineering local drug delivery systems for cancer immunotherapy.

Cancer immunotherapy aims to leverage the immune system to suppress the growth of tumors and to inhibit metastasis. The recent promising clinical outcomes associated with cancer immunotherapy have prompted research and development efforts towards enhancing the efficacy of immune checkpoint blockade, cancer vaccines, cytokine therapy, and adoptive T cell therapy. Advancements in biomaterials, nanomedicine, and micro-/nano-technology have facilitated the development of enhanced local delivery systems for cancer immunotherapy, which can enhance treatment efficacy while minimizing toxicity. Furthermore, locally administered cancer therapies that combine immunotherapy with chemotherapy, radiotherapy, or phototherapy have the potential to achieve synergistic antitumor effects. Herein, the latest studies on local delivery systems for cancer immunotherapy are surveyed, with an emphasis on the therapeutic benefits associated with the design of biomaterials and nanomedicines. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.