Molecular Pharmaceutics最新文献

Efficient cryopreservation of Natural Killer (NK) cells is fundamental to manufacturing NK cell-based immunotherapy products, but it remains challenging due to irreversible cryoinjuries to cell structures (e.g., the membrane system). Herein, inspired by the damage repair mechanism of natural organisms, recombinant human MG53 (rhMG53) protein, known as a membrane repair protein, is reported as a functional additive for improving the NK cell cryopreservation efficacy. The results showed that incubating NK cells with 30 μg/mL rhMG53 protein could preserve the integrity of membranes subjected to mechanical injury, reactive oxygen species damage, and dimethyl sulfoxide (DMSO)-induced toxic injury. During the cryopreservation process, incorporating 30 μg/mL rhMG53 protein into the conventional cryopreservation solution (10% DMSO) led to a 10% increase in cell viability immediately after thawing and an 11% increase in cell viability 72 h after thawing. Besides, during the post-thaw culture process, additional supplementation of 30 μg/mL rhMG53 protein to the culture medium resulted in a 6% decrease in delayed onset cell death and promoted cell proliferation. Meanwhile, post-thawed NK cells also exhibited enhanced immune functionality compared to those cryopreserved with DMSO. Notably, RNA sequencing analysis demonstrated that post-thawed NK cells with our developed cryopreservation method showed fewer changes in transcript profiles compared to fresh cells than those in the DMSO group. This work may highlight the crucial role of membrane protection in NK cell cryopreservation and promote translation of NK cell therapy.

Current imaging techniques (ultrasonography, CT) have limited sensitivity for detecting occult lymph node metastases in thyroid cancers. This first-in-human study assesses Trop2-targeted immunoPET/CT for preoperative differentiation of benign and malignant thyroid nodules and for identifying lymph node metastases to aid surgical planning. Sixteen patients with thyroid cancer underwent preoperative Trop2-targeted immunoPET/CT imaging. Tracer uptake (SUVmax) in tumors and lymph nodes was measured, pathology was confirmed through histopathology, and Trop2 expression was validated by immunohistochemistry. Malignant thyroid cancers had higher Trop2 tracer uptake than benign (38.46 ± 8.04 vs 8.97 ± 4.80, P = 0.046), and PTC had a 6.2-fold increase over adjacent normal tissue (46.65 ± 7.54 vs 7.53 ± 1.71, P = 0.007). The uptake value of the Trop2 tracer was strongly correlated to PTC tumor size (Spearman correlation coefficient ρ = 0.81, R² = 0.66). Trop2 immunoPET/CT effectively differentiates metastatic lymph nodes (LN (+): 25.15 ± 5.16 vs LN (-): 3.34 ± 1.27, P = 0.00017), with an AUC of 0.89 (95% CI: 0.78-1.00). Diagnosis of lymph node metastasis achieved an accuracy of 91.3%. Both metastatic lymph nodes and PTC show high Trop2 tracer uptake, but there was no statistically significant difference between them (46.65 ± 7.54 vs 25.15 ± 5.16, P = 0.252). Trop2 tumor proportion score and SUVmax were strongly correlated (Spearman correlation coefficient ρ = 0.88; R² = 0.64). Consequently, the following conclusions can be drawn: Trop2 immunoPET/CT offers superior diagnostic accuracy for PTC and metastatic lymph nodes, enhances surgical planning, and may revolutionize the clinical management of PTCs.

Prefilled syringes (PFSs) serve as primary containers for therapeutic peptides and proteins in combination drug products. Silicone oil, a lubricant in PFSs to facilitate plunger movement during injection, and the headspace from syringe fillings, have been reported to induce protein aggregation and particle formation as a result of the associated interfacial stress. However, their impact on the stability of peptide drugs is underexplored. In this study, we employed biophysical techniques, including size exclusion chromatography (SEC), circular dichroism (CD) spectroscopy, and thioflavin T fluorescence (ThT), to investigate aggregate formation, secondary structural changes, and fibrillation following physical stress on liraglutide in the presence of varying silicone oil concentrations, headspace, and agitation strength. In contrast to the minimal structural changes or formation of higher-molecular-weight aggregates observed under mild agitation or limited headspace, enhanced headspace and agitation promote fibrillation of liraglutide. Furthermore, we utilized advanced techniques to probe silicone-oil-liraglutide interactions and interfacial stress, including high-resolution label-free stimulated Raman scattering (SRS) for chemical imaging and nuclear magnetic resonance (NMR) spectroscopy for structural characterization. We observed, for the first time, the peptide adsorption on the silicone oil surface at submicrometer resolution by SRS. ¹H NMR showed line broadening and signal loss, consistent with peptide aggregation or surface adsorption, but no chemical shift changes, supporting the absence of strong, specific interactions. Therefore, our data suggest that the dual air–water and silicone-oil–water interfacial stress, rather than either alone, plays a significant role in liraglutide aggregation. These findings emphasize the interactive roles of several stress parameters, including silicone oil concentration, silicone oil interface, headspace, and agitation strength, on the stability of peptide combination drug products, and highlight the critical role of interfacial stress-induced biophysical instability.

The limited efficacy of programmed death-ligand-1 (PD-L1) monoclonal antibodies (aPD-L1) in triple-negative breast cancer (TNBC) is largely attributable to the immunosuppressive tumor microenvironment (TME). Notably, the abundance of cancer-associated adipocytes (CAAs) constitutes a distinctive feature of the TNBC microenvironment, contributing significantly to its immunosuppressive nature. CAAs upregulate cluster of differentiation 36 (CD36), a fatty acid translocase on tumor cells, thereby promoting excessive fatty acids (FAs) uptake and lipid droplet (LD) accumulation, which starve immune cells and reinforce immunosuppression through this metabolic adaptation. Berberine (BBR), a bioactive alkaloid derived from Rhizoma coptidis, has previously been shown to ameliorate lipid metabolism disorders through downregulation of CD36 in metabolic diseases such as hepatic steatosis. We therefore hypothesize that BBR inhibits CD36-mediated FAs uptake and reduces LD accumulation in tumor cells, representing a novel mechanism that remains unexplored in the context of TNBC. In this study, we demonstrated that BBR counteracts the tumor-promoting effects of CAAs in 4T1 cells by inhibiting CD36 upregulation and its mediated FAs uptake, thereby reducing CAA-induced LD accumulation and ultimately suppressing tumor cell proliferation. Furthermore, BBR remodeled the TME by enhancing CD8⁺ T cell recruitment and activity, while reducing immunosuppressive factors. In order to improve the sustained release of BBR at the tumor site and overcome its poor aqueous solubility, we created a thermosensitive hydrogel-based nanoparticle system (BBR-NPs-GEL). This injectable hydrogel demonstrated favorable thermosensitive gelation and shear-thinning behavior, making it suitable for localized administration. It exhibited a gelation temperature of 35.3 ± 0.2 °C and a sustained release profile with 52% of BBR released within 48 h. In 4T1^Fluc tumor-bearing mice, BBR-NPs-GEL significantly suppressed tumor growth and remodeled the TME, as evidenced by increased infiltration of CD8⁺ T cells (+8.49%), activation of dendritic cells (+8.39%), and a shift toward M1-macrophages (+39.9%), accompanied by a reduction in M2-macrophages (−19.13%). Importantly, when combined with aPD-L1 therapy, the treatment elicited synergistic antitumor effects, resulting in enhanced tumor regression. This combination strategy effectively overcame metabolic immunosuppression and reversed immune resistance in TNBC.

Triple-negative breast cancer (TNBC) is characterized by poor prognosis, limited therapeutic options, and low survival rates, underscoring the urgent need for novel and effective treatment strategies. This study aimed to investigate the potential of CDH3 as a theranostic target for TNBC and to develop a CDH3-based targeted radionuclide theranostic approach. Flow cytometry was used to analyze CDH3 expression, and a CDH3-overexpressing HCC1806 xenograft mouse model was established. The performance of the dual-functional probe ⁸⁹Zr/¹⁷⁷Lu-11E10C11 was systematically evaluated through radioligand binding, internalization, PET/CT imaging, and biodistribution assays. The anti-CDH3 monoclonal antibody 11E10C11 was labeled with either ⁸⁹Zr or ¹⁷⁷Lu to separately assess diagnostic capability and targeted antitumor efficacy. Results demonstrated that CDH3 was highly and specifically expressed in TNBC (HCC1806), with [⁸⁹Zr]Zr-DFO-11E10C11 clearly delineating tumor lesions (72 h SUVmax, 2.11 ± 0.31) and [¹⁷⁷Lu]Lu-DOTA-11E10C11 significantly inhibiting tumor growth (TGI, 74.94%) without inducing hematologic toxicity or damage to normal organs, indicating favorable safety. In conclusion, CDH3 is a promising theranostic target for TNBC, and the ⁸⁹Zr/¹⁷⁷Lu-11E10C11 dual-functional probe successfully achieved an integrated “diagnosis–therapy” strategy, offering a novel approach with translational potential for precision management of TNBC.

Photothermal therapy (PTT) in the near-infrared II window (NIR-II, 1000–1700 nm) is favored for noninvasive cancer treatment due to its deep tissue penetration, reduced background interference, and better biosecurity. However, excessive reactive oxygen species (ROS) and inflammatory cytokines during localized hyperthermia limit its therapeutic efficiency. In this work, lactoferrin-functionalized copper sulfide nanoparticles (Lf-CuS NPs) with a hydrodynamic size of 31.2 nm were synthesized via “one-step” biomineralization for NIR-II-guided photothermal therapy. The Lf-CuS NPs exhibited efficient NIR-II light absorption at 1064 nm, high photostability for effective PTT, and desirable ROS scavenging capability for inflammation-resolving therapy. Following a 1064 nm laser irradiation of 1.0 W/cm², 4T1 tumors were efficiently ablated with no recurrence over a 14 day period. Remarkably, Lf-CuS NPs possessed good photoacoustic imaging (PAI) capabilities, enabling real-time monitoring of their accumulation at the target site after intravenous administration. This ensures precise timing for PTT, optimizing therapeutic outcomes while reducing potential damage to the surrounding skin and tissues. Furthermore, the low toxicity of Lf-CuS NPs was confirmed through extensive in vitro and in vivo experiments, highlighting their potential as a promising photothermal agent. In conclusion, this research presents a promising strategy for tumor-targeted therapy and highlights lactoferrin as an inflammation alleviating agent in antitumor application.

Hepatocellular carcinoma (HCC) demands advanced multimodal therapies to overcome heterogeneity-driven treatment resistance. Although aptamers exhibit superior targeting advantages over antibodies, their clinical translation remains severely hindered by ambiguous target identification and the limited drug-loading capacity of direct aptamer–drug conjugates. To address this, we first identified vascular endothelial growth factor receptor 2 (VEGFR2) as the target of the HCC-specific aptamer JHIT2e using an integrated approach combining 3D photo-cross-linking chip technology, surface plasmon resonance (SPR), and liquid chromatography–mass spectrometry (LC–MS). Building on this, we engineered a multifunctional DNA nanotrain (¹³¹I-NTs-Dox, namely, ¹³¹I-labeled DNA nanotrains loaded with doxorubicin) comprising three key components: the JHIT2e aptamer as a navigation module for tumor-specific targeting, hybridization chain reaction (HCR)-assembled dsDNA duplexes as “carriages” intercalated with doxorubicin (Dox), and carboxyfluorescein (FAM)-terminated termini radiolabeled with iodine-131 (¹³¹I) via an optimized chloramine T method. Notably, this FAM-mediated labeling strategy significantly enhanced radiolabeling efficiency to 93%, surpassing conventional tyrosine-based methods (70–85%). Each nanotrain entity achieved an unprecedented payload capacity of 50 Dox molecules and 80 ¹³¹I atoms. In vitro studies demonstrated that ¹³¹I-NTs-Dox exhibits specific affinity for HepG2 cells, rapid lysosomal endocytosis, and enhanced tumor accumulation. Furthermore, the platform synergistically inhibited HCC cell viability through dual chemoradiotherapeutic mechanisms: Dox-induced DNA repair inhibition and ¹³¹I-mediated DNA damage. Collectively, ¹³¹I-NTs-Dox represents a scalable nanoplatform with high-precision HCC targeting, dual-modal therapeutic efficacy, and promising clinical potential for chemoradiotherapy.

Antigen presentation to autoreactive T cells in the pancreatic islets by dendritic cells and B lymphocytes is paramount to the pathogenesis of Type 1 diabetes (T1D). Immunosuppressive therapies, such as rituximab (anti-CD20) and teplizumab (anti-CD3), can delay T1D onset; however, risks associated with B or T cell depletion, such as an increased susceptibility to infections, are considerable. Antigen-specific immunotherapy (ASIT) offers a more targeted approach to suppress or delete the autoreactive cells, specifically recognizing autoantigens associated with disease. Soluble antigen arrays (SAgAs) are a platform consisting of a hyaluronic acid (HA) backbone and multiple copies of the autoantigen to selectively suppress autoreactive B cells and T cells. Here, a new SAgA was developed by conjugating a mutated proinsulin antigen (proinsulin(F25D)) to HA (SAgA_M-PI(F25D)) to avoid glycemic activity while retaining the ability to target anti-insulin antibodies and B cells. SAgA_M-PI(F25D) had a pronounced reduction in insulin receptor-β activity and lacked glycemic activity in vivo. Furthermore, this SAgA successfully bound a disease-relevant monoclonal anti-insulin antibody with low nanomolar binding affinity and murine transgenic (Tg125) B cells specific for human insulin. This collection of data demonstrated the avoidance of glycemic effects of proinsulin SAgAs while retaining specificity to anti-insulin antibody and insulin-binding B cells, thus highlighting the potential as a candidate for intervening in T1D as an ASIT.

Enhancing antibody exposure within the central nervous system (CNS) is critical for developing therapeutic monoclonal antibodies (Mabs) to treat CNS disorders. However, limited antibody penetration across the blood–brain barrier (BBB) and rapid efflux from the cerebrospinal fluid hinder effective CNS delivery. While most research efforts focus on enhancing the permeation of antibodies across the BBB, here we present an alternative strategy to enhance antibody retention by directly binding hyaluronic acid (HA) in the brain’s extracellular space. To accomplish this, we fused the G1 domain of versican (VG1), a proteoglycan known for HA binding, with nontargeting Fab and Mab (IgG) antibodies. Using single-photon emission computed tomography/X-ray computed tomography imaging, we tracked the biodistribution of ¹²⁵I-labeled Fab- and Mab-(VG1)₂ fusion constructs, as well as their parent antibodies, after direct infusion into the lateral ventricles of cannulated mice. Over 96 h, both VG1 constructs significantly enhanced antibody exposure in the brain and spine compared to the parent antibodies. Fab-VG1 exhibited a more widespread brain distribution, while Mab-(VG1)₂ was localized around the administration site. Fluorescence microscopy demonstrated periventricular distribution of antibody with a greater depth of infiltration for Fab-VG1 across all ventricles at 1 and 96 h time points compared to Mab-(VG1)₂. Escalating the dose of Fab-VG1 enhanced tissue penetration distance. These findings demonstrate the feasibility of using HA-binding technology to enhance antibody exposure in the CNS, offering potential for the development of more effective antibody-based therapies for CNS disorders.

Prefilled syringes (PFS) and autoinjectors are increasingly used to deliver biologic drug products, particularly monoclonal antibodies (mAbs), to improve convenience, compliance, and dosing accuracy. However, these systems face performance and quality challenges such as needle clogging, particularly for high-concentration, viscous, and aggregation-prone formulations. The origins and mechanisms by which device-derived leachables influence clogging and protein stability remain poorly understood. In this study, we investigated zinc (Zn) leaching from rigid needle shields (RNS) and its interactions with a high-concentration dupilumab formulation and common excipients. Inductively coupled plasma-mass spectrometry (ICP-MS) quantification shows that Zn was the predominant metal in RNS batches, and its extraction kinetics depended strongly on time, temperature, and the presence of a routinely utilized surfactant, polysorbate 80 (PS-80). Stressing RNS materials in the dupilumab formulation at 40 °C for 14 days yielded up to 550 μg/mL Zn(II), roughly 100-fold above typical specifications. Isothermal titration calorimetry (ITC) revealed millimolar Zn(II) binding to PS-80 and weaker interaction with buffer components (histidine, arginine, and acetate), which together promote Zn release. Structural modeling identified surface-exposed regions of dupilumab enriched in histidine-, sulfur-, and carboxylic-acid-containing residues that are geometrically arranged to chelate Zn(II), highlighting likely Zn(II) binding motifs. These protein-metal and excipient-metal interactions, along with PS-80 degradation catalyzed by Zn, correlate with increased solution viscosity and the formation of high-molecular-weight species. Zn leaching from RNS and its synergistic interactions with PS-80, buffer components, and the mAb can drive PS-80 degradation, increase viscosity, and promote higher-order protein aggregation, factors that plausibly contribute to needle clogging. Overall, Zn(II) can simultaneously interact with proteins, increasing their propensity to aggregate while degrading the excipients intended to stabilize them against aggregation. Understanding these mechanisms can inform candidate selection, formulation design, and device choice to mitigate protein aggregation and syringe clogging and improve product reliability and therapeutic outcomes.