Selective recognition of cancer-associated proteins (CAPs) by antibodies, followed by their delivery into the intracellular organelle, the lysosome, results in targeted degradation of CAPs and suppresses the growth of cancer cells. Translocating the antibody-CAP complex across the plasma membrane is, however, nontrivial. Phase-separating molecules are known to form membrane-translocating coacervates that can encapsulate proteins and transport them into the cytoplasm. Nevertheless, these coacervates generally lack the ability to guide the cargo to the lysosome. Here, we seal this gap and develop lysosome-targeting coacervates by tailoring a tetrapeptide into a phase-separating, coacervate-forming peptide. In the aqueous solution, the peptide derivative forms microdroplets, or coacervates, through liquid-liquid phase separation (LLPS), which spontaneously enter cells and colocalize with the lysosome; hence, these coacervates are referred to as Lysosome-Sorting Peptide Coacervates or LSP-Coa. We show that LSP-Coa can encapsulate proteins, facilitate the translocation of antibody-CAP complexes to the lysosome, and enable the degradation of membrane-bound CAPs - a mechanism we call Coacervate-mediated Lysosome-targeting Protein Degradation, or CoaLPD. Using the CoaLPD technology, we successfully degraded HER2 and EGFR in cancer cells and in tumor-bearing mice, showcasing its potential use as an anticancer treatment. The LSP-Coa system also increases the efficacy of PROTAC degradation through enhanced lysosomal uptake. Taken together, we present the design of lysosomal-targeting coacervates and demonstrate their use as vehicles for lysosome-specific antibody delivery and for the selective degradation of CAPs, thereby validating the CoaLPD strategy as a potential anticancer treatment.

Photothermal therapy (PTT) has garnered considerable attention for its noninvasive and localized treatment advantages. However, in response to PTT-induced hyperthermia, cancer cells increase the expression level of heat shock proteins (HSPs) and activate thermoresistance to shield themselves from heat-induced damage, thereby diminishing the efficacy of PTT. To overcome thermoresistance, here we have developed an on-demand responsive proteoliposome (PL) system. This system consists of PLs formed by a phospholipid conjugate of an elastin-like polypeptide (ELP) with vanadium oxide nanozymes (VOx NZs) incorporated in the lumen, referred to as VOx@ELP-PL. Upon photoirradiation, the enclosed VOx NZs generate a photothermal effect, inducing hyperthermia and enhancing HSP expression in cancer cells. Concurrently, as the temperature surpasses a critical threshold, ELP-PL undergoes liquid-liquid phase separation (LLPS) in situ, transitioning from a liposome state to ELP coacervate droplets. In the hyperthermic cancer cells, ELP coacervate droplets sequester and insulate the up-regulated HSPs, disrupting the thermoprotective response of thermoresistant cancer cells. Moreover, VOx@ELP-PL combines peroxidase-catalyzed generation of toxic hydroxyl radicals with coacervate droplet-mediated sequestration of HSPs, leading to potentiated immunogenic cell death both in vitro and in vivo. In a mouse model of colon cancer, intravenously injected VOx@ELP-PL showed marked tumor enrichment and resulted in highly effective cancer treatment. Altogether, this system presents a novel strategy to counteract thermoresistance by sequestering HSPs via LLPS of ELP-PL, thereby augmenting the effectiveness of PTT in cancer therapy.

The significant challenges pose by the high recurrence and metastasis rates of colorectal cancer (CRC) persist in its diagnosis and treatment. Activating innate immunity in CRC treatment has the potential to reduce drug resistance and side effects. Here, we develop a biomimetic platform by utilizing antimicrobial peptide-functionalized CRC cell membranes to encapsulate a cobalt-based metal-organic framework (C), hereby called peptide-functionalized camouflage C (PfCC). When injected into tumour-bearing mice, PfCC will degrade under the acidic condition of the tumour microenvironment and release cobalt ions, which react with endogenous H2S to generate black stellate precipitates with good photothermal properties, recruiting NK cells and mitigates the immunosuppressive tumour-microenvironment. Simultaneously, the degradation of PfCC will release structure-protected antimicrobial peptides, inhibiting harmful bacteria, such as Desulfovibrio, and reducing H2S production. The abovementioned synergistic top-down regulation of H2S promote the polarization of macrophages and further activates the innate immune response. Moreover, experiments including the convex hull algorithm from AI deep learning of the segment anything model indicate that PfCC exhibites the most effective therapeutic effect compared with the single H2S-regulated therapeutic modality. Taken together, PfCC represents a potential anti-cancer therapy for CRC with the combined effect of immune-regulation and the regulation of the gut flora.

Rationale: Synthetic molecules, meticulously designed according to the "sticker-and-spacer model", tend to form coacervates via liquid-liquid phase separation (LLPS), thereby acquiring properties beyond their discrete and soluble states. However, natural compounds, such as those from traditional Chinese medicines (TCMs), are not known to undergo phase separation. In this study, we demonstrate that curcumin, the active ingredient in the spice turmeric, forms phase-separated fluorescent coacervates when diluted from a concentrated organic-solvent solution into an aqueous solution. Methods: Curcumin coacervates were formed by diluting a concentrated stock solution in organic solvents into the aqueous solution. We utilized the coacervate droplets to encapsulate and transport various biomacromolecules, such as proteins and nucleic acids, across the plasma membrane into the cell. Supramolecular interaction between β-cyclodextrin (β-CD) and curcumin disassembles curcumin coacervates, leading to cargo release in the cytosol. Results: Intravenously injected curcumin coacervates spontaneously enrich in the tumor tissue in tumor-bearing BALB/c mice. Subsequent intratumoral injection of β-CD significantly enhances anticancer effects in mice, demonstrating the efficacy of coacervate-mediated siRNA drug delivery and supramolecular-interaction-responsive intracellular release in vivo. Conclusions: Taken together, we report here the coacervate-forming properties of the natural TCM compound curcumin, presenting a unique strategy for controlling coacervate states through supramolecular interactions with β-cyclodextrin in vitro and in vivo, along with the unexplored potential of curcumin coacervate-mediated siRNA delivery to enhance pyroptosis.

Proficient mismatch repair/microsatellite-stable colorectal cancer (pMMR/MSS CRC), which represents the majority of clinical cases, exhibits low tumor mutational burden and minimal responsiveness to immune checkpoint blockade. Disrupting the MMR system has been shown to resensitize pMMR/MSS CRC to immune surveillance; however, direct MMR inhibitors are currently unavailable. Here, we bridge this gap by developing small interfering RNA lipid nanoparticles (siMMR@LNPs) capable of silencing key MMR genes, including MLH1, MSH2, and MSH6, achieving over 70

ABSTRACT Many of the membraneless organelles inside cells are multiphasic condensates with complex structural organizations driven by the demixing of phase‐separating proteins. Tailoring the structures of multiphasic condensates by controlling their demixing states is a challenge. Here, we employ two proteins with distinctly different features, including thermal responsiveness, hydrophobicity, and charges: a positively charged RGGRGG protein, which forms phase‐separated condensates below an upper critical solution temperature, and a protein based on an elastin‐like polypeptide, which forms condensates above a lower critical solution temperature. These two proteins demix to form multiphasic condensates with nested and core‐shell structures under variable conditions, which can be tailored by altering the physical and chemical environments. The demixed multiphasic condensates can also be constructed inside Escherichia coli cells, recapitulating the properties of membraneless organelles. We also show that nucleic acids preferentially enrich in the positively charged segment of the multiphasic condensates. Lastly, multiphasic condensates can deliver nucleic acids across the plasma membrane into mammalian cells, enabling cell transfection.

The immunosuppressive nature of the tumor microenvironment (TME) remains a primary obstacle to effective cancer therapy. Emerging evidence indicates that intratumoral bacteria (ITB), notably Staphylococcus aureus (S. aureus), establish a pro-tumorigenic symbiosis in melanoma by secreting lipoteichoic acid (LTA) to trigger the TLR2/4–MyD88–NF-κB pathway. This process upregulates PD-L1 expression and recruits immunosuppressive cells, directly correlating with therapeutic resistance in clinical non-responders. To break this cancer–S. aureus symbiosis, we synthesize a multilayered nanotherapeutic, Cu-CpG@PDA@HA (CCPH), via a stepwise assembly process. The Cu-CpG core is fabricated via metal–DNA coordination-driven supramolecular self-assembly between antibacterial copper ions and CpG oligodeoxynucleotides (ODNs), then encapsulated by an in situ polymerized polydopamine (PDA) shell and a hyaluronic acid (HA) targeting layer. Physicochemical characterization confirms that CCPH nanoparticles have a uniform nanoscale size of approximately 123 nm, efficient photothermal conversion (46 °C within 5 min), and pH-responsive copper release (>80% at pH 5.0). HA-mediated CD44 targeting increases cellular uptake 2-fold and enables precise colocalization with intracellular S. aureus, resulting in 90% inhibition of both bacteria and melanoma cells. In vivo biodistribution analysis reveals a nearly 2-fold higher tumor accumulation for CCPH compared to non-targeted nanoparticles. In an intracellular bacteria-colonized melanoma mouse model, we show that CCPH treatment, combined with NIR irradiation, significantly promotes dendritic cell (DC) maturation, drives tumor-associated macrophage repolarization from an immunosuppressive M2 to an anti-tumor M1 phenotype, reduces regulatory T cells (Tregs), upregulates pro-inflammatory cytokines (TNF-α, IFN-γ, and IL-6), downregulates immunosuppressive factors (IL-10 and TGF-β), and stimulates cytotoxic T-cell activation. Collectively, this work introduces a highly efficient multilayered nanoplatform that integrates intracellular antibacterial action, photothermal therapy, and immunotherapy, offering a promising strategy for treating bacteria-associated malignancies. Significance statement 1.This study represents the first development of a multilayered nanoplatform specifically engineered to break the cancer cell–Staphylococcus aureus symbiosis, providing definitive evidence that integrating intracellular antibacterial action with photothermal therapy significantly enhances melanoma therapy.2.It first systematically demonstrates that the targeted eradication of intratumoral bacteria, coupled with CpG-mediated immune activation, achieves comprehensive remodeling of the tumor immune microenvironment, as evidenced by enhanced dendritic cell maturation and systemic cytotoxic T-cell infiltration.

Extracellular vesicles (EVs) are promising drug-delivery vehicles owing to their biocompatibility and low immunogenicity. Genetic engineering of a membrane-bound EV-sorting scaffold protein empowers EVs by installing targeting moieties on the surface and enriching therapeutic cargo in the lumen. However, the choice of scaffold proteins with simple structures and short sequences is limited. Here, we conduct mass spectrometry-based proteomic studies and identify ENPP1 as a superior scaffold protein. Furthermore, we show that a truncated 144-amino acid variant, EN144, efficiently loads diverse therapeutic cargoes and outperforms conventional scaffolds. By fusing EN144 to the IL-6 decoy receptor gp130, we create engineered decoy EVs that potently inhibit inflammatory IL-6 trans-signaling. In mouse models, these EVs reduce inflammation, improve survival in sepsis, and, when targeted to cartilage, alleviate tissue damage in osteoarthritis. Our work establishes EN144 as a minimal, high-performance scaffold for EV engineering and demonstrates its broad therapeutic potential for inflammatory diseases.

Objective Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint pain, swelling, and stiffness. Although smoking is a well-established risk factor for RA, the role of occupational inhalants in RA development is less well recognized. This study aimed to systematically review and synthesize existing evidence on the association between occupational inhalants and the risk of developing RA.Methods We conducted a systematic review and meta-analysis following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, searching MEDLINE, Embase, and Web of Science from database inception to November 20, 2024. Eligible studies were cross-sectional, were case-control and cohort designs, were population-based, reported original data on occupational inhalant exposures and RA, measured exposures, included a comparison group, and used reliable RA ascertainment methods. Studies relying solely on self-reported RA or focusing on treatment, prognosis, sick leave, or death were excluded. Two reviewers independently conducted literature screening, data extraction, and risk-of-bias assessment using the Newcastle-Ottawa Scale. Random-effects meta-analyses with relative risk were performed for cohort and case-control studies, and heterogeneity was assessed using the I2 statistic.Results In total, 31 studies met inclusion criteria, and 25 were included in meta-analyses across 10 types of occupational inhalants. Significant associations with RA risk were observed for exposure to silica, asbestos, solvents, pesticides, fertilizers, animal dust, and engine exhaust (relative risks ranging from 1.25 to 1.49). Moderate-to-high heterogeneity was observed in seven meta-analyses.Conclusion Occupational inhalants are associated with increased RA risk, underscoring the importance of workplace prevention strategies and further research into biologic mechanisms.

BACKGROUND:The pathogenesis of osteoarthritis (OA) involves the degradation of the extracellular matrix (ECM) of cartilage, which is mainly mediated by matrix metalloproteinases (MMPs). Small interfering RNA (siRNA)-based gene therapy has shown promise in treating various diseases, including OA, but its effectiveness is hindered by poor stability and low uptake efficiency. Additionally, the avascular nature of cartilage poses a challenge for drug delivery. This study aims to develop a novel plant-derived nanoparticle system for targeted, efficient delivery of siRNA to intra-articular chondrocytes, thereby alleviating OA progression. METHODS:We designed lipid nanoparticles (LNPs) based on lipids extracted from grapefruit extracellular vesicles (GEVs), which were functionalized with an ECM-binding peptide derived from placenta growth factor-2 (PIGF-2). siRNA targeting MMP13 was loaded, and the therapeutic efficacy and safety were evaluated in both the cellular and rat OA models. RESULTS:These PIGF2-LNPs delivering siRNA MMP13, termed PIGF2-siRNA MMP13-LNPs, were uniform in size (196 nm) and stable with a zeta potential of -9.96 ± 0.49 mV. The system delivered siRNA targeting MMP13 to chondrocytes. The PIGF2-modified LNPs exhibited high biocompatibility and low toxicity, and the PIGF2 peptide conferred specific chondrocyte-targeting ability, significantly enhancing cellular uptake. The system enhanced the siRNA-mediated gene silencing of MMP13, a key enzyme involved in cartilage degradation. The PIGF2-siRNA MMP13-LNPs effectively suppressed MMP13 protein expression (P <0.001) and increased collagen II protein expression (P <0.05) and lowered the Osteoarthritis Research Society International (OARSI) score (P <0.001) in rat chondrocytes. CONCLUSIONS:This study developed biocompatible grapefruit-derived LNPs for targeted intra-articular siRNA delivery. The peptide-functionalized LNPs effectively silenced MMP-13 in chondrocytes. The system attenuated the hydrolytic degradation of ECM proteins in cartilage and mitigated OA progression in a rat model, with minimal toxicity, suggesting a promising plant-based, cell-free, targeted therapy for OA.

This study aims to investigate the independent and combined associations of nocturnal sleep duration, sleep midpoint, and sleep onset latency with global cognitive function in older Chinese adults. Our cross-sectional study included 4601 community-dwelling cognitively unimpaired adults aged 60 years or older from the West China Health and Aging Cohort Study. Sleep characteristics were assessed using the Pittsburgh Sleep Quality Index, and global cognitive function was evaluated using the Mini-Mental State Examination (MMSE). Multivariable linear regression models were employed, adjusting for an extensive set of demographic, lifestyle, and comorbidity factors. Subgroup analyses were performed based on sex, age, and genetic risk profiles for cognitive performance. The mean age of participants was 69.0 ± 5.53 years, with 52.1

Background: While machine learning has advanced in medicine, its widespread use in clinical applications, especially in predicting breast cancer metastasis, is still limited. We have been dedicated to constructing a deep feed-forward neural network (DFNN) model to predict breast cancer metastasis n years in advance. However, the challenge lies in efficiently identifying optimal hyperparameter values through grid search, given the constraints of time and resources. Issues such as the infinite possibilities for continuous hyperparameters like L1 and L2, as well as the time-consuming and costly process, further complicate the task. Methods: To address these challenges, we developed the Single-Hyperparameter Grid Search (SHGS) strategy, serving as a preselection method before grid search. Our experiments with SHGS applied to DFNN models for breast cancer metastasis prediction focused on analyzing eight target hyperparameters (epochs, batch size, dropout, L1, L2, learning rate, decay, and momentum). Results: We created three figures, each depicting the experimental results obtained from three LSM-I-10+-year datasets. These figures illustrate the relationship between model performance and the target hyperparameter values. Our experiments achieved maximum test AUC scores of 0.770, 0.762, and 0.886 for the 10-year, 12-year, and 15-year datasets, respectively. For each hyperparameter, we analyzed whether changes in this hyperparameter would affect model performance, examined whether there were specific patterns, and explored how to choose values for the hyperparameter. Conclusions: Our experimental findings reveal that the optimal value of a hyperparameter is not only dependent on the dataset but is also significantly influenced by the settings of other hyperparameters. Additionally, our experiments suggest a reduced range of values for a target hyperparameter, which may be helpful for “low-budget” grid search. This approach serves as a foundation for the subsequent use of grid search to enhance model performance.

Liquid-liquid phase separation (LLPS)-driven coacervate droplets, formed by the self-assembly of phase-separating molecules, have emerged as a new platform for the intracellular delivery of macromolecular therapeutics such as antibodies, plasmids, and mRNA. Their appeal lies in their high loading capacity, low cytotoxicity, and high cellular uptake efficiency. Beyond traditional polymer and protein systems, recent advances have demonstrated that low-molecular-weight compounds, including peptides and small molecules, can also form functional coacervates. In this perspective, we will discuss and elucidate the possible mechanisms underlying coacervate cellular uptake and highlight their applications in macromolecular delivery and disease therapy. We also provide our perspective on future research directions and translational opportunities. By critically evaluating these aspects, we aim to bridge fundamental insights with translational potential while providing a promising strategy in disease treatment.

Androgenetic alopecia (AGA) is the most prevalent form of hair loss worldwide. Growth factors have been used to treat hair loss, but their intradermal delivery remains challenging. Type XVII collagen (COL17) has been reported to regulate the aging process of hair follicles (HFs). We reason that combining the therapeutic efficacy of growth factors and collagen biomaterials will provide maximal hair regeneration. Here, we design a microenvironment-responsive recombinant human COL17 microneedle (MRrhCOL17 MN) system for the transdermal delivery of the insulin-like growth factor-1 (IGF-1) to stimulate hair growth. We load IGF-1 into mesoporous polydopamine nanoparticles (MPDAs) to allow for continuous release of the growth factor. When applied to the skin, the composite MNs penetrate the skin, release IGF-1 and rhCOL17 in response to the alteration of the microenvironment and photothermal effect, and stimulate hair growth in a mouse model of AGA. Compared with the clinical drug minoxidil, our MN system more effectively enhances neovascularization, alleviates tissue inflammatory responses, and promotes hair regeneration in AGA mice. These therapeutic effects have been linked to the activation of the VEGF/VEGFR and Src/p38 MAPK signaling pathways. Taken together, the composite MRrhCOL17 MN thereby offers a new option for intractable AGA patients. STATEMENT OF SIGNIFICANCE: Growth factors hold the potential to effectively stimulate the growth of hair follicles; however, their transdermal delivery remains a formidable challenge. In this study, recombinant human type XVII collagen (rhCOL17) is employed as the primary scaffolding material to fabricate microneedles (MNs) for the delivery of insulin-like growth factor 1 (IGF-1), with the aim of promoting hair follicle regeneration. Additionally, the concept of microenvironmental responsiveness is integrated to enable the controlled release of IGF-1 from the MNs. Moreover, the low-temperature photothermal effect of nanoparticles is harnessed to optimize the process of hair regeneration, thereby maximizing the outcome of hair follicle rejuvenation.

Low-molecular-weight compounds of certain structural features may form coacervates through liquid-liquid phase separation (LLPS). These coacervates can enter mammalian cells and affect cellular physiology. Controlling the properties of the coacervates inside cells, however, is a challenge. Here, we report photochemical reactions of spiropyran (SP)-based coacervates with two wavelengths of light, in vitro, in the cell, and in animals, generating reactive oxygen species (ROS) for photo-controlled cell killing. We identify an SP-containing compound, SP-PEG8-SP, that forms coacervates (SP-C) in the aqueous solution. Photo illumination by a UV light triggers the isomerization of SP to merocyanine (MC), switching SP-C to the fluorescent coacervates MC-C. A visible light converts MC-C back to SP-C and induces ROS generation. Notably, coacervate formation increases the compound's ROS generation efficiency. The SP-C/MC-C coacervate system (collectively called spiropyran coacervates) can spontaneously enter cells, and a dual-wavelength-controlled reversible on/off switch and spatiotemporal-resolved ROS production is realized within the cytoplasm. Light-induced ROS generation leads to cytotoxicity to cancer cells, tumor organoids, and tumors in vivo, supporting spiropyran coacervates' potential use as coacervate photosensitizers in photodynamic therapies.

Apopotin is a small protein that can specifically induce apoptosis in tumor cells, thereby selectively killing a variety of human cancer cells. However, penetrating the cell membrane is often challenging and exosome is sometimes leveraged to deliver functional molecules into cells. As the expression of fusion lysosomal-associated membrane protein 2b (LAMP-2B) can integrate exogenous proteins into the exosome membrane, we investigated the ability of apoptin-armed exosomes to penetrate tumor cells and induce apoptosis. In this study, the recombinant plasmid Apoptin-EGFP-Lamp2b was constructed and transfected into COS-7 cells. The Apoptin-EGFP-Lamp2b recombinant fusion protein was expressed and rendered apoptin was displayed on the surface of exosomes. Our study showed that the engineered apoptin-containing exosomes could deliver apopotin across the cell membrane into the nucleus. CCK8 cytotoxicity tests demonstrated that apoptin- containing exosomes could specifically target and kill cancer cells. In addition, Hoechst33342 staining and Annexin-V-FITC/PI method were used to observe the ability of exosome mediated apoptin to induce apoptosis. These results showed that the engineered exosome could successfully deliver apoptin into cancer cells and induce apoptosis, indicating that exosomes could be used as a new type of protein delivery platform.This study developed a powerful apoptin- anchored exosome as a potential for tumor-specific therapy.

The propensity for controlled liquid–liquid phase separation and subsequent directed phase transition are crucial for the coacervation-mediated assembly of extracellular matrix (ECM). This spatiotemporally controlled ECM assembly can be used to develop coacervate-based polymer assembly strategies to generate biomimetic materials that can emulate the complex structures and biophysical cues of the ECM. Inspired by the tropoelastin structure, here we develop a designer minimalistic model consisting of alternating hydrophobic moieties and covalent crosslinking domains. By increasing the valence and enhancing the interaction strength of the hydrophobic moieties, we can control the degree of the assembly to enhance the propensity for phase separation and thus emulate the extracellular coacervation process of tropoelastin, including droplet formation, coalescence and maturation. The subsequent covalent-bonding-triggered coacervate–hydrogel transition with enhanced assembly order stabilizes the phase-separated structure in the form of a heterogeneous hydrogel, thereby mimicking covalent crosslinking-derived elastin fibrillation. Furthermore, the heterogeneous hydrogel network establishes a biomimetic matrix that can effectively promote the mechanosensing of adherent stem cells. The extracellular matrix (ECM) is assembled through liquid–liquid phase separation and directed phase transition. Now—inspired by tropoelastin—a designer minimalistic model incorporating alternating hydrophobic moieties and crosslinking domains can template the assembly of a biomimetic matrix that mimics the ECM, promoting the mechanosensing of stem cells.

Bacterial membrane vesicles (bMVs) are nanosized proteoliposomes naturally and ubiquitously shed by most bacterial species from their outer membranes or cell membranes, playing a pivotal role in microbe-microbe and microbe-host interactions. Owing to their distinctive attributes, such as non-replicative nature, cell-targeting ability, and versatility, bMVs have emerged as promising candidates for various biomedical applications, including vaccination, cancer immunotherapy, drug delivery, anti-infective therapy, and diagnostics. Recent studies in bioengineering and nanotechnology have facilitated the engineering of bMVs to augment their physiological activities and therapeutic functions. This review provides a comprehensive overview of bMVs, encompassing their composition, function, and mechanism of generation. Furthermore, it delves into the diverse methodologies for bMV engineering, encompassing genetic engineering and chemical and physical modification techniques, along with their applications in immunotherapy, infectious diseases, and tumor therapy, among other areas. Ultimately, this review addresses prevailing limitations and challenges associated with the biomedical utilization of bMVs. Engineered bMVs represent a compelling opportunity for developing effective disease treatments and alternative therapies.