CRISPR-based designer nucleases can facilitate genome engineering targeting almost any genomic locus. However, safe and efficient methods for delivering gene editors into primary human cells and tissues remain a central challenge. In this study, we employed a PepFect14 (PF14) analog, PF14-K, to deliver high-fidelity Cas9-ribonucleoproteins and non-viral repair templates into primary human skin cells to mediate gene editing and repair targeting genes underlying the group of genetic skin blistering disorders epidermolysis bullosa (EB). Peptide-RNP nanoparticles enabled consistent gene editing of >70% in primary wild type fibroblasts and >50% in primary wild type keratinocytes. In more difficult-to-transfect primary EB skin cells, this strategy facilitated up to 68% exon deletion-mediated reframing targeting COL7A1 and 37% precise homology-directed repair of a prevalent LAMB3 mutation. Compared to electroporation, the gold standard for ex vivo delivery, PF14-K enabled similar total yields of edited cells. Deliverable PF14-K nanoparticles are highly cost-effective, as they can be formed on the benchtop through a simple mix-and-incubate approach, with future potential to deliver base and prime editors.
Therapeutic challenges in endometrial carcinoma (EC) arise from the limited efficacy and toxicity of current treatments. Although exosome-based RNA interference shows promise, its clinical translation is hindered by inefficient cargo loading, low yields, and poor tumor targeting. We have engineered an exosome platform (cRGD-ExoM) that integrates the following innovations: Firstly, RNA motifs enable the enrichment of shRNA loading by over 80-fold for targeting of ferroptosis regulators (glutathione peroxidase 4/ferroptosis suppressor protein 1/ferritin heavy chain [GPX4/FSP1/FTH]). Secondly, Rab4 silencing amplifies exosome biogenesis via dysregulated endosomal recycling, enhancing tumor cell uptake by impairing endosome maturation-a dual-action mechanism that boosts both production and delivery. Thirdly, cRGD peptides confer αvβ3-integrin-specific targeting. cRGD-ExoM induces potent ferroptosis by increasing lipid peroxidation and downregulating GPX4/FSP1/FTH, significantly suppressing EC tumor growth in vivo without causing systemic toxicity. The platform's modular design allows for spatiotemporal control of loading, production, and targeting, demonstrating its scalability. This study provides new insights into the precision treatment of endometrial cancer by developing engineered, multifunctional, exosome-based therapeutic drugs that combine mechanism precision and translational feasibility in tumor treatment.
Neutrophils are the primary immune cell type present within the blood and the first to arrive at sites of inflammation. As such, neutrophils set the stage for how the inflammatory response progresses and resolves. In some cases of non-resolving inflammation, excessive neutrophil accumulation can cause tissue damage. Accordingly, maintaining tight control over their recruitment and behavior at sites of inflammation is paramount. Historically, corticosteroids have been used to modulate inflammation, but they are associated with systemic side effects that have limited their use. Here, we developed an iron-dexamethasone nanoparticle (DexOx NP) therapeutic that modulates neutrophil behavior and reroutes neutrophils away from sites of inflammation. DexOx NPs inhibit neutrophil activation, as indicated by prevention of L-selectin shedding and reduced neutrophil extracellular trap formation. Furthermore, treatment with DexOx NPs in a murine acute lung injury model significantly reduced the total number of neutrophils in the bronchoalveolar lavage fluid while avoiding the side effects of systemic soluble dexamethasone phosphate delivery. Thus, DexOx NPs represent a new option to control non-resolving neutrophilic inflammation.
Brain organoids provide three-dimensional human cellular systems that can reproduce selected features of early neural development, regional patterning, cellular diversification, and emerging neural activity more effectively than conventional two-dimensional cultures. However, their translational value depends not only on morphological resemblance to brain tissue, but also on whether construction strategies, functional validation, reproducibility, and application-specific model fitness are appropriately aligned. This structured narrative review synthesizes representative engineering strategies for brain organoid construction and examines how cell source, embryoid body formation, extracellular matrix support, patterning strategy, culture platform, vascularization, and cellular complexity influence functional validation and translational applicability. We further organize functional assessment into a hierarchical validation framework that includes morphology and growth, lineage and regional identity, tissue viability, synaptic maturation, electrophysiological activity, neurochemical signaling, BBB-like function, and omics-based benchmarking. These advances support the use of brain organoids in developmental biology, neurological disease modeling, drug screening, neurovascular research, and exploratory biohybrid interfaces, although their interpretation remains constrained by immature cellular states, incomplete vascular perfusion, batch variability, and limited standardization. Overall, this review reframes brain organoids as engineered biological platforms whose value should be judged by the alignment among construction strategy, biological benchmark, functional readout, and intended translational application. The emphasis is comparative conceptual synthesis of engineering strategies and multi-layer functional validation rather than systematic quantitative meta-analysis.
DNA and RNA methylation are key epigenetic and epitranscriptomic modifications involved in gene regulation, genome stability, RNA metabolism, and disease progression. Aberrant methylation patterns in cell-free DNA and RNA have emerged as valuable biomarkers for cancer detection, disease monitoring, and therapeutic stratification. However, conventional methods such as bisulfite sequencing, methylation-specific PCR, MeRIP-seq, SCARLET, and LC-MS/MS often require harsh processing, high sample input, complex instrumentation, or lack site-specific resolution, limiting their clinical and point-of-care applications. CRISPR-based diagnostics provide a promising alternative by combining programmable nucleic acid recognition with collateral cleavage-mediated signal amplification. This review summarizes recent CRISPR strategies for detecting DNA and RNA methylation, including chemical conversion-assisted assays, restriction enzyme-mediated detection, direct amplification-free sensing based on methylation-modulated Cas activity, detection of oxidized cytosine derivatives, reverse transcription-mediated Cas12 detection of m6A, and structure-sensitive Cas13 sensing. We highlight how methylation-dependent sequence conversion, enzyme accessibility, polymerase behavior, and nucleic acid structure can be translated into CRISPR-readable signals. Finally, we discuss current translational challenges and emerging opportunities in point-of-care methylation diagnostics, integrated DNA-RNA profiling, engineered Cas effectors, AI-guided assay design, and CRISPR-compatible methylome analysis.
The endoplasmic reticulum (ER) is an indispensable organelle responsible for the synthesis and transport of proteins and membrane lipids, playing a critical role in numerous physiological and pathological processes. Leveraging the properties of the ER, we developed novel quinoxalinone-based, ER-targeting photosensitizer nanoparticles (Qui-PS NPs) by conjugating an ER-specific targeting peptide(RACR) and evaluated their photodynamic therapy (PDT) efficacy. The size, morphology, cellular uptake, ER targeting capability, cell viability, biodistribution, and antitumor efficacy were assessed using dynamic light scattering, transmission electron microscopy, confocal microscopy, CCK-8 assay, ICP-Mass spectrometry, and tumor volume measurements, respectively. The results demonstrated that the synthesized Qui-PS NPs possessed an average diameter of 79.74 ± 9.4 nm, a polydispersity index (PDI) of 0.23 ± 0.02 , and a Zeta potential of - 11.63 ± 2.86 mV. These nanoparticles exhibited near-infrared fluorescence emission centered at 830 nm and demonstrated superior singlet oxygen (1O2) generation capability. The NPs were readily internalized by MCF-7 cells, displayed specific ER targeting, and induced cytotoxic effects upon light irradiation, with an I C 50 value of 3.2 ± 0.06 μg/mL. In tumor-bearing mice, Qui-PS NPs preferentially accumulated in tumor tissue and significantly suppressed tumor progression under light irradiation, with minimal impact on body weight. These findings suggest that these ER-targeted NPs represent a promising nanoplatform for potential application in tumor PDT.
Cardiovascular diseases remain the leading cause of global mortality, yet traditional preclinical models fail to accurately capture the physiological and genetic complexity of the human heart, hindering the development of targeted therapies. Cardiac microphysiological systems (cardiac MPS), including self-organizing human cardiac organoids and engineered cardiac tissue models, have emerged as promising human-relevant platforms for recapitulating selected aspects of cardiac development, tissue organization, and function. This review evaluates current strategies for the construction of these cardiac microphysiological systems through a systematic comparison of two major approaches: development-driven self-organization based on intrinsic stem-cell programs, and engineering-driven assembly supported by bioactive materials, 3D bioprinting, and microfluidic technologies. To address key bottlenecks limiting translational utility, we outline a multidimensional maturity assessment framework encompassing sarcomeric ultrastructural organization, the fidelity of electromechanical coupling, and metabolic reprogramming toward fatty acid β-oxidation. Furthermore, we discuss the translational applications of cardiac microphysiological systems in elucidating early cardiogenesis, modeling complex genetic and ischemic cardiovascular diseases, and enabling high-throughput cardiotoxicity screening. Despite persistent challenges in building perfusable multi-scale vascular networks, reducing batch-to-batch variability, and modeling multi-organ crosstalk, the integration of cardiac microphysiological systems with spatial multi-omics, next-generation biomaterials, and artificial intelligence-assisted culture systems may enhance their translational relevance, provided that these approaches are supported by rigorous benchmarking and cross-laboratory validation.
Chronic kidney disease (CKD) has become one of the major diseases threatening global health, with its increasing incidence and mortality rates. Early identification of chronic kidney disease is crucial for accurate disease staging, timely intervention, and improved prognosis. However, the commonly used clinical diagnostic indicators, such as creatinine, albumin level and glomerular filtration rate, can only identify advanced CKD. Imaging examinations, such as computed tomography and magnetic resonance imaging, have limitations such as high costs and high risk of complications, and are not suitable for large-scale screening of high-risk populations. Renal biopsy is the gold standard for diagnosing renal fibrosis, but it is invasive. Previous studies have shown that capillary rarefaction is an early event of renal fibrosis, occurring earlier than tubular atrophy and interstitial collagen deposition. However, there is a lack of a minimally invasive, low-cost, high-resolution, dynamic and real-time monitoring method for renal microcirculation that is suitable for large-scale screening. There is an urgent need for reliable methods to screen early-stage CKD patients. In this study, we have for the first time combined real-time and wide-field laser speckle imaging (RFLSI), near-infrared II imaging (NIR-II), duplex ultrasound (DUS) and Contrast-Enhanced Ultrasound (CEUS) to monitor vascular changes and evaluate the progression of renal fibrosis. The combination of four imaging methods enabled the early monitoring of CKD progression by monitoring microcirculation. Among them, CEUS can detect changes in the renal microcirculation at the early stage of renal fibrosis, even earlier than the pathological damage of the kidneys. Through visualization and quantification of the progression of renal fibrosis and validation through histopathology, this study utilizes preclinical imaging to supplement clinical imaging of renal fibrosis, providing a more comprehensive method for monitoring microcirculation, which is helpful for guiding clinical decisions and providing insights into disease progression.
Current glioblastoma (GBM) models often rely on chemically undefined exogenous extracellular matrices (ECMs) and serums that limit understanding of autonomous cellular behaviors driving tumor progression. Here, we present an exogenous ECM-free biomanufacturing process for GBM organoids (GBOs) that recapitulate pathological development relative to size and spatial distribution. These GBOs exhibit a conserved glioma signature established from The Cancer Genome Atlas clinical datasets. Trajectory analyses of neurovascular unit (NVU) zonation, cell-type differentiation, and basement membrane converged with clinical benchmarks highlighting a critical developmental milestone at the 2 mm diameter stage. Immunofluorescence revealed a matrix-priming cascade of tenascin-C (TNC) at 1 mm diameter and fibronectin (FN) at 1.5 mm diameter to provide the necessary biochemical cues and biophysical assembly for endothelial cells at 2 mm diameter. Lastly, the 2 mm diameter GBOs displayed a distinct vascular-like area characterized by capillary-scale lumen distributions (5-15 μm) and peak junctional complexity, similar to in vivo capillary beds. However, transcriptional divergences in arterial and proteoglycan markers suggest biochemical cues and morphology are insufficient for full physiological maturation without confirmed internal hemodynamic shear stress. Despite these results, our GBOs serve as a high-fidelity structural framework for modeling GBM NVU-like regions that may be used as a translatable platform for TNC- and FN-targeted therapies designed to enhance drug delivery across the NVU.
Nucleus pulposus (NP) cell quiescence maintains intervertebral disc homeostasis, while mTORC1 regulates autophagy-lysosomal function and inflammatory secretion to preserve quiescence-rapamycin specifically targets mTORC1. Herein, we fabricated rapamycin-nanoliposomes (rapa-lipos) via ultrasonic dispersion, thin-film dispersion, and filtration to improve rapamycin bioavailability, investigating their role in inhibiting the senescence phenotype of NP cells through β-gal staining, lysosomal staining, transmission electron microscopy, ELISA, and cell cycle inhibitors. The mechanistic effects of rapa-lipos on mTORC1, NLRP3/Caspase-1 pathway (NCP) and autophagy-lysosomal pathway (ALP) were also analyzed by western blotting, immunofluorescence (IF), Si-RNA (raptor), and PCR. In vivo, rapa-lipos were injected into rat intervertebral disc with IL-1β-induced degeneration, assessed via HE staining, x-ray, MRI, and IF. Rapa-lipos exhibited high encapsulation efficiency, favorable drug loading, uniform particle size, and controlled release, suppressing NP cell senescence-related phenotypes (morphological changes, elevated IL-1β/TNF-α secretion, increased β-gal activity, lysosomal dysfunction, upregulated P21/P16 and reduced P27 expression). Mechanistically, rapa-lipos targeted-inhibited mTORC1, then blocked NCP and activated ALP to maintain NP cell quiescence. In vivo, x-ray, MRI and histological evaluation confirmed rapa-lipos mitigated intervertebral disc degeneration. Collectively, rapa-lipos target mTORC1-mediated NCP and ALP to inhibit NP cell senescence, offering a promising strategy for intervertebral disc degeneration prevention.
Autoimmune diseases-including systemic sclerosis (SSc), systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA)-are increasingly understood as programmable microenvironmental states, wherein evolving changes in matrix mechanics, barrier integrity, interferon and cytokine networks, immune-complex deposition, and stromal-immune reciprocity progressively reshape tissue behavior. These shifting axes generate nonlinear trajectories of fibrosis, vascular injury, and joint destruction that static or reductionist in vitro systems fail to recapitulate. Organoid and organ-on-a-chip platforms now allow controlled reconstruction of these dynamic microenvironments in human-derived systems. By integrating iPSC-derived epithelial, endothelial, stromal, and immune lineages with tunable extracellular matrix (ECM) stiffness and viscoelasticity, perfusable microvasculature, and modular innate and adaptive immune components, these systems reproduce key autoimmune phenomena. These include stiffness-driven fibroblast activation and endothelial-to-mesenchymal transition (EndoMT), interferon-conditioned barrier collapse, immune-complex-mediated injury, and cytokine-dependent stromal invasion. Patient-specific induced pluripotent stem cell (iPSC), CRISPR-based editing of risk alleles, and controlled exposure to sera or autoantibody repertoires provide genetic and immunologic personalization, while multi-omic profiling, spatial imaging, and machine-learning analytics enable quantitative alignment between organoid states and patient tissues. By translating pathogenic microenvironmental logic into controllable 3D systems, these platforms establish autoimmune organoids as programmable, human-relevant tools for mechanistic discovery, therapeutic interrogation, and precision modeling-laying the groundwork for next-generation, potentially animal-free pipelines and advancing personalized immunology across SSc, SLE, and RA.
Abstract Heart failure remains a leading cause of global morbidity and mortality, yet routine clinical indices often miss the regional biomechanical disturbances that drive progression and shape treatment response. This State‐of‐the‐Art review examines how finite‐element (FE) modeling, additive manufacturing, and artificial intelligence (AI) are converging to improve the diagnosis, phenotyping, procedural planning, and prognostic assessment of heart failure (HF). Although these technologies have matured in structural heart disease and transcatheter intervention research, their greatest translational potential may lie in HF, where patient‐specific ventricular remodeling, myocardial stress–strain heterogeneity, valve‐ventricular coupling, and device‐tissue interaction are incompletely captured by conventional clinical indices. We synthesize translational and clinical literature published from 2015 to 2026 on imaging‐derived FE models, multimaterial 3D‐printed cardiovascular phantoms, and AI‐based analytic pipelines relevant to HF populations or HF‐related decision pathways. FE approaches provide mechanistic estimates of regional myocardial deformation, wall stress, and remodeling trajectories; 3D‐printed phantoms enable bench‐top validation, device rehearsal, and hemodynamic replication; and AI supports segmentation, phenotyping, multimodal integration, and risk prediction. Across all three domains, however, the evidence base remains dominated by retrospective studies, bench validation, and small translational cohorts, with limited prospective outcome validation and inconsistent reporting of reproducibility. We therefore propose an evidence‐aware, HF‐centered framework for integrating computational mechanics, physical phantoms, and data‐driven models into digital‐physical twin workflows. From a clinical perspective, the near‐term opportunity is not autonomous decision‐making, but better mechanistic phenotyping, more transparent procedural planning, and more rigorous testing of patient‐specific hypotheses before intervention. Standardized reporting, external validation, careful materials characterization, and multicenter endpoint‐linked studies will be essential for clinical translation.
Abstract Ocular drug delivery faces tremendous challenges in clinical practice. The eyeball possesses sophisticated anatomical and physiological characteristics that uniquely influence the pharmacokinetics of delivered drugs. This complexity necessitates innovative solutions to ensure effective drug delivery. Conventional ocular drug administration routes (topical, intravitreal, and systemic) each pose unique limitations. With the advancements of biomaterials science and medical engineering technology, nanofiber hydrogels have garnered significant attention, primarily represented by self‐assembled peptide‐based hydrogels and cellulose nanofiber‐based hydrogels. They can be tailored to have precise physical and chemical properties, enabling controlled release of drugs and enhanced biocompatibility. Furthermore, their nanofibrous structure mimics the extracellular matrix, promoting cell adhesion and tissue regeneration. This review introduces advanced manufacturing techniques which are capable of precisely modifying the properties of nanofiber hydrogels to meet specific therapeutic needs. The distinctive advantages of nanofiber hydrogels are elaborated in detail, including their ability to enhance drug penetration, provide sustained release, and reduce systemic toxicity. We also delve into the therapeutic applications, potential limitations, and developmental perspectives of nanofiber hydrogels. Preclinical studies have validated their efficacy in treating ophthalmologic conditions such as age‐related macular degeneration, bacterial keratitis, ocular alkali burns, and non‐infectious uveitis. However, translating these laboratory findings into clinical applications remains limited, primarily due to significant challenges in human trials, including species‐specific responses, the complexity of human biology, and the safety of nanofiber hydrogels. Future refinements in fabrication techniques and rigorous safety assessments are necessary to revolutionize the clinical application of nanofiber hydrogels.
Abstract Tumor‐associated macrophages (TAMs) shape the tumor microenvironment through plastic transitions between pro‐inflammatory M1‐like and immunosuppressive M2‐like states, yet clinical drug therapies are limited by toxicity, resistance, and delivery barriers. This review explains how non‐invasive physical stimulation (NIPS) reprograms TAMs via defined couplings between physical inputs and signaling pathways. Hypoxia‐tolerant photodynamic strategies and mild photothermal heating reset hypoxia‐ and lactate‐driven programs; cavitation‐dominant ultrasound and sonodynamic therapy trigger danger signaling and reactive oxygen species; ultrasound microbubble destruction provides endothelial repair cues; nanosecond pulsed electric fields activate cyclic GMP‐AMP synthase–stimulator of interferon genes (cGAS–STING) pathway; piezoelectric materials convert mechanical input into calcium‐dependent transcription; and appropriately dosed radiotherapy elicits immune‐active responses while avoiding hypoxia‐driven M2 recruitment. Across models, these regimens promote pro‐inflammatory reprogramming, normalize aberrant vasculature, and strengthen antitumor immunity while restraining immunosuppression. We synthesize parameter windows, delivery options, and combination strategies with checkpoint blockade and macrophage‐directed agents to guide the translation of NIPS into precise, low‐toxicity TAM‐targeted immunotherapy.
Abstract Cervical cancer, driven mainly by human papillomavirus (HPV) infection, remains one of the most common malignant tumors among women worldwide, posing significant challenges in treatment and drug development. Traditional two‐dimensional (2D) cell culture models fail to accurately replicate the in vivo tumor microenvironment (TME), especially HPV‐driven oncogenic signaling, immune contexture, and stromal interactions unique to cervical cancer, limiting their predictive value for therapeutic efficacy (Y. Liu, H. Ai. Comprehensive insights into human papillomavirus and cervical cancer: pathophysiology, screening, and vaccination strategies. Biochim Biophys Acta Rev Cancer. 2024;1879(6):189192). Hydrogels have emerged as promising biomaterials for constructing three‐dimensional (3D) tumor models due to their tunable physicochemical properties, excellent biocompatibility, and ability to mimic the extracellular matrix. This review focuses on hydrogel applications in 3D cervical cancer TME modeling, with an emphasis on recapitulating HPV‐driven biology, immune‐stromal crosstalk, and stromal interactions, emphasizing their role in simulating key aspects of tumor biology such as cell–cell and cell–matrix interactions, hypoxia, and drug resistance. Recent advances in hydrogel‐based 3D models for high‐throughput drug screening are critically analyzed, highlighting their potential to improve the precision of cervical cancer treatment and accelerate novel drug discovery. However, critical challenges including high cost, limited industrial scalability, technical complexity, and strict regulatory constraints remain to be addressed to realize their full translational potential. By integrating current research findings, this review aims to provide a theoretical framework and technical guidance for future studies focused on enhancing the physiological relevance of in vitro cervical cancer models and optimizing therapeutic strategies.
Rapid diagnosis of infectious diseases is of paramount importance to prevent or control outbreaks and pandemics. Detection of bacteria is commonly performed using culture-based and molecular detection methods, which cannot address the need for quick, specific and cheap diagnostics. Bacteriophage-based assays rely on the rapidity, specificity, contaminant-tolerance and effectiveness of phage-host interactions and can be engineered with fluorescence or luminescence-based reporters. Previous attempts, however, required transcription and translation of reporter genes, leading to long assays and restrictive protocols. Here, we shortened the signal generation time by detecting the injection of a phage protein, thereby circumventing the need for gene expression altogether. In our model diagnostic assay, we demonstrate that injection of the N-terminal fragment of the split nanoluciferase protein of Oplophorus gracilirostris, fused to the products of genes g6.7 or g14 of phage K1F, is detectable upon injection into an Escherichia coli cell as early as 3 min after phage addition. The engineered phages generate a signal upon exposure to cognate K1-but not to non-cognate K5 capsule-enclosed E. coli cells, confirming the specificity of our system. The early luminescent signal and the ability to detect as few as 104 bacteria may open the way to the development of a rapid diagnostic tool based on phage-mediated protein injection.
Abstract Chimeric antigen receptor (CAR) technology has propelled CAR‐T cells to transformative success in hematologic malignancies, yet translation to solid tumors remains limited, motivating exploration of alternative CAR‐engineered immune effectors. Macrophages, sentinels of the innate immune system, are abundantly recruited to solid tumors, making them compelling candidates. Preclinical studies show that CAR‐engineered macrophages (CAR‐M) exhibit precise tumor homing, potent antigen‐directed phagocytosis, and the capacity to remodel immunosuppressive tumor microenvironments (TMEs). Notably, early‐phase clinical investigations indicate a favorable safety profile, strengthening confidence in their therapeutic potential against solid cancers. In this review, we synthesize design principles for CAR‐M constructs, with a particular focus on emerging engineering strategies for next‐generation CAR‐M. We further discuss their applications in oncology and emerging non‐oncologic indications, and summarize the current clinical trial landscape. We further propose a “4S framework” (specificity, switchability, synergy, and safety) to guide next‐generation CAR‐M development. Collectively, these advances support CAR‐M as a new paradigm for cancer therapy and beyond.
The recent availability of a technology providing real-time, seconds-resolved in vivo drug concentration measurements has opened the door to performing fully autonomous, closed-loop feedback control over drug dosing. The controllers employed in prior demonstrations of such dosing, however, were designed and optimized using population-based pharmacokinetic models. In the face of individual pharmacokinetic variation (between subjects or even within a single subject over time as their physiology varies), these controllers must be set rather conservatively so as to avoid potentially dangerous overshooting. This, however, slows the speed with which they achieve the desired set point and opening the possibility of their nevertheless still overshooting if the response of a subject differs too much from that of the "average" subject. To address these issues, here we have developed an adaptive feedback-control system that, rather than employing population-pharmacokinetic information, instead uses real-time drug concentration measurements to individualize drug delivery to the specific subject, "on the fly." To achieve this, the system estimates the pharmacokinetics of the individual subject during the initial stages of the infusion, and then continuously updates this subject-specific pharmacokinetic model to maintain effective controller performance even in the face of physiological variations brought on, for example, by changing health status. Using this approach, we then demonstrate a feedback controller that rapidly (20-30 min) achieves and accurately (5%-12% root-mean-squared deviations, though this also includes sensor noise) maintains pre-defined concentrations of the anesthetic procaine in the ventricles of live rats, an application that, due to the delays associated with intracranial drug transport, represents a particular challenge for feedback-controlled intravenous drug delivery. Given the precision and accuracy it achieves in our rat animal model, we believe that the use of adaptive feedback control will ultimately enable safer, more precise drug dosing in humans.
This study introduces hollow sponge Haliclona sp. spicules (oSHS) as an innovative microneedle technology for enhancing the skin delivery of biomacromolecules. oSHS were developed by etching SHS in 2 mol/L NaOH for 48 h, optimizing hollow channel formation while preserving structural integrity. In vitro experiments demonstrated that oSHS significantly enhanced drug absorption efficiency compared to SHS, microneedles, roller microneedles, and iontophoresis alone. Combining oSHS with iontophoresis yielded a synergistic effect, increasing biomacromolecule delivery by up to 35.09% (p <0.05). In vivo skin delivery of ovalbumin (OVA) via oSHS elicited immune responses comparable to subcutaneous injection, with elevated OVA-specific IgE and IL-4 levels. A mathematical model was developed to describe oSHS-mediated permeation, revealing dose-dependent delivery and uniform distribution of biomacromolecules in deep skin layers, contrasting with localized accumulation from injections. Safety assessments confirmed minimal and reversible skin irritation, resolving within 72 h. These findings highlight oSHS as a safe, efficient, and scalable strategy for skin delivery of proteins, nucleic acids, and other macromolecules, offering significant potential for non-invasive therapeutic applications.
Energy-based minimally invasive cancer focal therapies (FTs), including thermal ablation (Heat), cryoablation (Cryo), and irreversible electroporation (IRE), can induce immunomodulatory effects and can synergize with immunotherapy to engage the immune system for systemic and long-term antitumor response. However, the immunogenicity of each FT can vary based on treatment specifics, and the mechanisms underlying these differences are not fully understood. We employed a model antigen (ovalbumin, OVA) and an in vitro platform to investigate the upstream processing steps that initiate immune priming. This platform enabled quantitative assessment of antigen release, antigen presentation, dendritic cell (DC) activation, antigen-specific T cell proliferation and the generation of damage-associated molecular patterns (DAMPs), cytokines and chemokines. Both antigen-specific signaling and T cell response are highly dependent on the focal therapeutic conditions applied to the cancer cells. Cryo resulted in the highest release of protein, OVA antigen, ATP, and HMGB1, followed by IRE and Heat. Despite not yielding the most antigen, IRE-treated B16-OVA elicited the most DC activation and OVA-specific T cell proliferation. Dose-response analysis using OVA protein and SIINFEKL peptide confirmed a positive correlation between antigen availability and T cell response. We also observed substantial adjuvant effects that enhance the immune processing and T cell response by stimulus in cell lysates independently of antigen quantity. Specifically, IRE-treated B16 cell lysates induced the most significant improvement in dendritic cell (DC) activation and T cell proliferation compared to Heat and Cryo when paired with equal amounts of antigen. Our findings position IRE as the most immunogenic FT condition in our system. This study highlights the critical interplay between antigen release, adjuvant signals, and immune processing in shaping FT-driven antitumor immunity.
