Lipid nanoparticles (LNPs) are clinically established carriers for nucleic acid therapeutics, most notably messenger RNA (mRNA). Beyond mRNA, LNPs can be used to deliver alternative gene-expressing nucleic acids with distinct biological properties, offering opportunities to modulate expression kinetics. Here, we compared LNPs formulated with firefly luciferase (FLuc)-encoding mRNA, self-amplifying RNA (saRNA), and plasmid DNA (pDNA), as well as defined combinations of these payloads, to assess how payload identity and composition influence expression in vitro and in vivo. Using a constant ALC-0315-based lipid composition and microfluidic formulation process, single-payload LNPs displayed distinct expression profiles following intramuscular and intravenous administration in mice: mRNA produced high, transient expression levels, saRNA produced delayed and sustained expression, and pDNA enabled prolonged low-level expression. Combined-payload formulations, prepared either by mixing the payloads before LNP formulation or by mixing separately formulated LNPs, exhibited expression profiles consistent with the proportional contributions of their constituent payloads, providing proof-of-concept evidence of additive behaviour under the conditions tested, with no clear evidence of synergistic or antagonistic interactions. Characterisation of LNP physicochemical properties, including mean hydrodynamic diameter, polydispersity, zeta potential, and encapsulation efficiency, showed minimal differences between formulations. These findings indicate that, within the common formulation system investigated, differences in expression profiles were associated with payload identity and composition. Overall, this study shows that distinct expression kinetics can be observed when different nucleic acid payloads are delivered using a common LNP formulation and that combined-payload preparations can exhibit additive expression behaviour under defined experimental conditions.

Persistent inflammation and oxidative stress are central barriers to effective diabetic wound repair. Successful healing requires restoration of a coordinated local microenvironment, yet achieving this remains a major clinical challenge. Herein, a composite ginsenoside Rd-functionalized hydrogel (Gel@Rd) with dynamic and static dual networks was fabricated using extracellular matrix-mimicking biomaterials. Gel@Rd exhibited favorable biocompatibility, self-healing capacity, and mechanical stability, facilitating prolonged retention and release of Rd at the wound site. In diabetic wounds, Gel@Rd attenuated oxidative stress and inflammatory signaling, promoted angiogenesis and collagen deposition, and reshaped a regeneration-favorable immune microenvironment by driving macrophage polarization toward the reparative M2-like phenotype. Transcriptomic and proteomic analyses indicated that Rd exerted inflammation-regulatory effects potentially via modulating the TLR4-NF-κB cascade and the TNF-α signaling pathway, and also participated in regulating pathways associated with metabolism and tissue repair. These multifaceted effects translated into accelerated wound closure and improved tissue regeneration in diabetic mice. Collectively, this work establishes an integrated natural product-based hydrogel platform for diabetic wound therapy and provides a rational strategy for combining sustained drug delivery with microenvironmental reprogramming to enhance healing.

Graft-versus-host disease (GVHD) is a major immune-mediated complication of allogeneic hematopoietic stem cell transplantation and remains the leading cause of non-relapse mortality. Glucocorticosteroids are the first-line treatment; however, there is a critical need for more effective and less toxic alternatives. Nanomedicine constitutes a promising approach by enabling a more tailored and safer drug delivery, although their use in GVHD remains very scarce. This review aims to systematically analyse the existing literature on nanomaterial-based approaches for the diagnosis, prevention and treatment of GVHD. Encouraging results have been reported across major nanomaterial subtypes, suggesting nanomedicine as a viable and versatile platform to improve GVHD outcomes compared with conventional approaches. However, this review also highlights the heterogeneity and frailty of the available data, and the urgent need for clinical translation and deeper exploration of alternative formulations. Finally, we advance as final remarks innovative encapsulation and targeting rationales, supported by an exploration of novel cellular and molecular mechanisms underlying GVHD, to increase therapeutic efficacy, reduce systemic toxicity, and eventually uncouple GVHD from the beneficial graft-versus-leukemia effect.

The effectiveness of intratumoral and locoregional therapies depends on achieving and maintaining therapeutic drug concentrations within tumors. Intratumoral therapy refers to direct injection into tumor tissue, locoregional therapy refers to delivery confined to a tumor-bearing region, and retention refers to persistence of the administered agent within the tumor over time. Retention is distinguished from initial delivery, accumulation, penetration, spatial distribution, and therapeutic efficacy. We conducted a PRISMA-ScR-guided scoping review to map strategies that enhance local retention or exposure after intratumoral or locoregional administration and classify them by principal mechanism. Two investigators independently screened records; 125 records were identified, 100 remained after duplicate removal, 84 full-text reports were assessed, and 73 studies were included. The finalized charting fields were citation, model/population, intervention strategy, principal evidence group, key retention/local-exposure finding, translational stage, and evidence classification. Studies were assigned to vascular modulation (n = 10), depot/formulation strategies (n = 23), ECM/stromal approaches (n = 17), ultrasound- or lymphatic-related approaches (n = 8), intratumoral immunotherapy (n = 11), or combination/multimodal strategies (n = 4). The evidence was predominantly preclinical (66/73; 90.4%) and used heterogeneous endpoints, precluding direct quantitative comparison. Current evidence supports several mechanistically distinct approaches to prolong local exposure, but comparative effectiveness and clinical translation remain uncertain. Future studies should standardize retention endpoints and integrate tumor persistence, systemic exposure, and safety.

Osteosarcoma (OS) is the most prevalent primary bone malignancy in children and adolescents; however, therapeutic outcomes remain suboptimal due to tumor heterogeneity, chemoresistance, and inadequate immune activation. Doxorubicin (Dox), the standard therapy that induces immunogenic cell death, has its efficacy compromised by the immunosuppressive tumor microenvironment (TME). While interleukin-12 (IL-12) can activate and recruit various immune cells, making it an attractive combination partner, its systemic delivery is severely limited by dose-limiting toxicity. We have previously reported that intravenous injection of A3 collagen binding domain (CBD) of von Willebrand Factor preferentially accumulates into the TME of various tumor models enriched in collagen I and III. Furthermore, CBD-fused IL-12 (CBD-IL-12) demonstrated superior therapeutic effects against various cancer models compared to unmodified IL-12 due to its collagen-targeted delivery and the resulting tumor-localized inflammation. Given that the OS TME also exhibits higher collagen I and III expression compared to normal bone, we hypothesized that a CBD-IL-12 fusion protein could showcase potent anti-tumor efficacy in OS via tumor-specific accumulation. Here, we demonstrated that CBD-IL-12 exhibited 4-fold enhanced tumor accumulation compared to unmodified IL-12 and increased cytotoxic T cell infiltration by 2.2-fold within the immune-cold microenvironment in a mouse model of OS. The combination of CBD-IL-12 with Dox significantly prolonged median survival in two independent murine OS models. This coordinated approach utilizing Dox coupled with precision-targeted IL-12 immunotherapy represents a clinically translatable strategy that overcomes the inherent limitations of single-agent treatments for OS.

A major bottleneck in radio-immunotherapy is the inability to precisely couple the localized stimulus of radiotherapy (RT) with the systemic action of immunotherapeutics. To bridge this gap, we developed a chemically engineered RT-ignited nanoplatform that productively harnesses the biochemical consequences of RT to precisely activate immunotherapy locally, thereby amplifying its benefits while minimizing systemic exposure. The platform co-delivers a hydrogen sulfide (H2S)-responsive prodrug of toll-like Toll-like receptor 7/8 (TLR7/8) agonist imiquimod (IMQ) using a reactive oxygen species (ROS)-sensitive, targeted nanocarrier. RT ignites a biochemical sequence: ROS bursts disassemble the carrier, and the subsequent H2S upregulation cleaves the prodrug, ensuring tumor-localized drug release. This RT-primed, sequentially activated therapy profoundly enhances antitumor immunity by inducing immunogenic cell death (ICD), activating antigen-presenting cells (APCs), and overcoming systemic immunosuppression, culminating in durable tumor regression and immunological memory. This study highlights a chemistry-driven approach to spatially and temporally confine immune activation to the tumor site, effectively broadening the therapeutic window and transforming RT-induced biochemical changes into a precise trigger for systemic immunity.

Engineered bacterial biotherapeutics have shown increasingly promising potential for cancer treatment, yet they are hindered by unresolved challenges regarding off-target toxicity and suboptimal therapeutic efficacy. In this study, we report the first systematic screening of strong constitutive anaerobic promoters in the probiotic Escherichia coli Nissle 1917 (EcN). Via RNA-seq analysis, we initially identified forty candidate promoters from EcN. Subsequent functional characterization using a firefly luciferase reporter assay yielded six robust constitutive anaerobic promoters, whose activity was 1.18- to 1.85-fold higher than that of Ptac-a strong constitutive promoter traditionally leveraged for therapeutic protein expression in EcN-based cancer biotherapeutics. Further functional validation confirmed that these promoters enable efficient production of immune checkpoint nanobodies, PD-L1nb and CTLA-4nb, in EcN under anaerobic conditions. Notably, in MC38 murine tumor models, EcN-based therapy incorporating these potent constitutive anaerobic promoters achieved significantly enhanced antitumor efficacy compared to conventional promoter-based systems. This not only reinforces the safety profile of engineered EcN but also boosts its therapeutic potency when employed as a tumor-targeted delivery platform in cancer immunotherapy. Collectively, our work establishes a precision delivery framework for potent yet highly toxic anticancer agents, facilitating the development of bacterial biotherapeutics.

Lyophilization has the potential to enhance mRNA-LNP stability at elevated temperatures. The present study aimed to comprehensively evaluate the influence of lyoprotectants on critical quality attributes (CQAs) and function of mRNA-LNPs post-lyophilization and upon storage at 4 °C, 25 °C, and 40 °C. Twelve formulations containing sucrose, trehalose, and polyvinylpyrrolidone (PVP) and their combinations at three total lipid-to-lyoprotectant weight ratios (1:5, 1:10, and 1:20) were studied. The CQAs of lyophilized mRNA-LNPs depended on the lyoprotectant composition and the lipid-to-lyoprotectant weight ratio, with the 1:20 ratio performing best. At this ratio, mRNA-LNPs containing sucrose, sucrose: trehalose 1:1, and sucrose: trehalose: PVP 1:1:1 showed limited change in particle size, mRNA encapsulation efficiency, and in-vitro transfection efficiency compared to other formulations. Interestingly, a lipid-to-sucrose 1:10 ratio provided stabilization of particle size and encapsulation post-lyophilization but resulted in reduced transfection efficiency relative to the 1:20 ratio. Thus, similarities in physicochemical properties did not assure equivalent biological function. After one month of storage at up to 40 °C, lyophilization with sucrose: trehalose: PVP = 1:1:1 at a 1:20 ratio enabled mRNA-LNPs to maintain particle size distribution, encapsulation efficiency, mRNA integrity, and in-vitro transfection. The enhanced stability of the formulation is likely due to its higher glass transition temperature of the lyoprotectant combination. In contrast, at 40 °C, when sucrose was used as the sole lyoprotectant it crystallized, reducing its protective effect. The results highlight a strategy for high-temperature stability of mRNA-LNPs as a reference for formulation development.

The positively charged surface underpins the biointerface activity of cationic liposomes, enabling electrostatic membrane engagement, enhanced cellular internalization, and efficient intracellular delivery. However, its premature exposure during circulation compromises delivery efficiency and increases systemic toxicity risk. Conventional PEGylation improves circulation stability by masking the cationic surface, but persistent shielding suppresses biointerface functions, and repeated dosing may provoke accelerated blood clearance and hypersensitivity reactions. Herein, we developed a physiologically adaptive prodrug liposomal nanoplatform that exploits endogenous carboxylesterase (CES) as a physiological trigger to remove polyethylene glycol (PEG) shielding and restore biointerface functions, including sialic acid-Siglec-E axis-mediated active recognition and cationic lipid-enhanced electrostatic anchoring. This physiologically triggered restoration of biointerface functionality enhanced targeted delivery to myeloid-derived suppressor cells (MDSCs), a key suppressive hub underlying poor immunotherapy responsiveness in cold tumors, while intracellular prodrug activation translated this delivery advantage into pharmacological regulation. The nanoplatform reduced MDSC burden across peripheral and tumor-localized immunity, reactivated systemic antitumor immunity, and attenuated tumor stemness. In Panc02 tumors poorly responsive to immune checkpoint blockade therapy, it markedly potentiated immunotherapy, achieving tumor eradication, durable antitumor immune memory against homologous tumor rechallenge and broader systemic antitumor protection against heterologous tumor challenge. Overall, this work establishes a physiologically adaptive nanotherapeutic strategy that couples programmable biointerface switching with intracellular prodrug activation to potentiate the efficacy and durability of ICB in cold tumors.

Gemcitabine (Gem) is an effective anticancer drug used for the treatment of various cancers. Nevertheless, its clinical benefit is limited owing to its very short plasma half-life, rapid enzymatic inactivation, and systemic toxicity. Here, we report the design, synthesis, and comprehensive biological evaluation of four N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-bound Gem nanomedicines differing in their linker structure and thereby drug release kinetics. The appropriate design enabled us to prepare polymer nanomedicines with highly prolonged Gem circulation and tumor accumulation. All conjugates exerted cytostatic and pro-apoptotic activity in multiple mouse and human cancer cell lines in vitro, with efficacy inversely correlated with Gem release rate. The nanomedicine with the slowest drug release in plasma showed remarkably prolonged systemic exposure up to 96 h versus 2 h for free Gem upon intravenous administration. The prolonged systemic exposure showed superior therapeutic efficacy across multiple mouse and human tumor models, including significantly inhibited tumor growth, considerably extended survival with complete long-term tumor remission, and reduced lung metastatic burden. Mechanistic studies revealed sustained pro-apoptotic activity of the nanomedicine with transcriptional reprogramming of tumor cells, and profound immunomodulatory effects. Optimization of Gem release rate improved pharmacokinetics and antitumor efficacy as well as favorably reshaped the tumor immune microenvironment. Our findings establish Gem release kinetics as a key determinant of HPMA copolymer-Gem conjugate performance and highlight the translational potential of these nanomedicines for overcoming limitations of conventional Gem therapy.

Whereas antifouling hydration is a general property shared by several classes of zwitterionic materials, amino acid-derived interfaces offer the conditional possibility of coupling such interfacial hydration with recognition by endogenous nutrient transporters. When appropriately presented, amino acid-derived motifs may reduce nonspecific interactions during circulation while promoting transporter-dependent interactions with cancer cells, tumor-associated endothelium, or brain capillary endothelial cells. However, these two functions are mechanistically distinct and require independent experimental validation. This review critically examines amino acid transporter-targeted prodrugs, polymer conjugates, and nanoparticle systems, with emphasis on ligand identity and conjugation, multivalency, ligand density, spacer architecture, conditional ligand presentation, and off-target transporter expression. EPR- and ATR-related processes are treated as potentially coexisting delivery steps rather than mutually exclusive mechanisms. We further propose standardized reporting frameworks for ligand presentation and stealth behavior and a graded evidence hierarchy for transporter involvement. Representative applications in cancer therapy, brain delivery, and oral administration illustrate both the potential and the current mechanistic limitations of the field. This design- and evidence-oriented framework aims to support more reproducible development and interpretation of amino acid transporter-targeted DDSs.

Messenger RNA (mRNA) therapeutics enable in situ production of immunoregulatory proteins. However, cellular stress induced during delivery remains an underappreciated yet critical determinant of immune outcomes. Strategies to mitigate this stress have so far been limited. Here, we harnessed the intrinsic antioxidant and anti-inflammatory activities of curcumin by conjugating it to the cationic polymer block via pH-responsive boronate ester bonds, yielding stress-buffered mRNA nanocarriers that mitigate delivery-induced cellular perturbations. The nanocarriers remain stable in serum and enable efficient cytosolic mRNA release while reducing reactive oxygen species, suppressing stress-induced cytokine expression and avoiding activation-associated immune bias. Systemic delivery of interleukin-2 (IL-2) mRNA with these nanocarriers selectively expanded regulatory T cells, spared effector T-cell activation, and reversed established colitis in mice. In contrast, delivery with conventional carriers induced cellular stress, blunted the anti-inflammatory effects of IL-2 and failed to relieve the disease. These findings establish stress buffering as a strategy to correct the delivery-induced immune bias and enable context-specific mRNA immunotherapies.

While targeted therapies for hepatocellular carcinoma (HCC) have advanced, their clinical benefits remain constrained by limited efficacy, off-target toxicity, and the frequent emergence of drug resistance and recurrence. To address these challenges, we developed a multivalent DNA nanoflower-based dual-targeting LYTAC (lysosome-targeting chimera) platform tailored for HCC. This NanoLYTAC integrates two key functional elements: (i) a high-density array of bispecific aptamers that simultaneously recognize glypican-3 (GPC-3)-a highly HCC-specific surface marker-and vascular endothelial growth factor receptor (VEGFR), and (ii) galactose (Gal) modifications that enable liver-specific targeting via the asialoglycoprotein receptor (ASGPR). The construct is preferentially internalized by HCC cells, where it promotes the ASGPR-mediated lysosomal degradation of both VEGFR and GPC-3, leading to potent suppression of cell proliferation and migration, and induction of apoptotic death. In vivo, the NanoLYTAC exhibits pronounced tumor-specific accumulation and achieves significant tumor growth inhibition without inducing detectable systemic toxicity. Together, this work establishes a targeted, efficient, and well-tolerated dual-protein degradation strategy, offering a promising therapeutic avenue for HCC.

Human serum albumin (HSA) undergoes post-translational modifications that alter its receptor recognition and biodistribution. While HSA receptors such as gp18, gp30, cubilin, and megalin regulate HSA distribution, the relationship between HSA structural changes and organ-specific targeting remains poorly understood. In this study, we systematically prepared five types of modified HSA (acid-treated, base-treated, oxidized, fatty acid-modified, and heat-treated) and characterized their structural alterations using circular dichroism, fluorescence spectroscopy, and site-specific fluorescent probe-binding assays. Comprehensive radar chart analysis revealed that structural changes could be classified into three distinct categories based on their conformational profiles. Biodistribution studies in normal mice demonstrated treatment-specific organ accumulation patterns: heat-treated HSA showed 2-fold increased liver and spleen accumulation; oxidized and fatty acid-modified HSA exhibited 2-fold increased liver and kidney accumulation; while acid-treated and base-treated HSA showed minimal changes compared to untreated HSA. Notably, in tumor-bearing mice, acid-treated HSA which exhibited structural similarity to Abraxane-derived HSA demonstrated 2.5-fold higher tumor accumulation than untreated HSA and comparable or superior tumor targeting versus Abraxane. These findings establish a systematic framework correlating albumin structural modifications with biodistribution patterns and suggest that acid-treated HSA warrants further investigation as a tumor-targeting drug carrier. The mechanism underlying enhanced tumor accumulation requires elucidation in future receptor-specific studies.