Molecular Pharmaceutics最新文献

The stimulator of interferon (IFN) genes (STING) pathway has garnered considerable attention due to its significant potential in anti-cancer effect. However, cyclic dinucleotides (CDNs), which are natural agonists or their derivatives for STING, are often hindered by their inherent properties of poor transmembrane permeability and potential systemic toxicity. Herein, we employed polyethylenimine (PEI)-functionalized spiky TiO₂ microparticles (SP-PEI) for the efficient delivery of CDNs into antigen-presenting cells (APCs). This delivery system leverages the electrostatic interaction between PEI and CDNs, allowing for the absorption of CDNs onto the spiky microparticle (SP) structure. Furthermore, the optimized microstructure of SP-PEI facilitates preferential phagocytosis by APCs, thereby enabling effective intracellular delivery of CDNs. Our findings demonstrated that SP-PEI significantly promoted CDNs phagocytosis by dendritic cells (DCs), leading to robust activation of the STING pathway and subsequent induction of type I interferons, which in turn fostered DC maturation. Furthermore, in MC38 tumor models, SP-PEI-CDN was observed to significantly delay the progression of both primary and distant tumors. Finally, the co-administration of SP-PEI-CDN with the chemotherapeutic agent doxorubicin (DOX) achieved excellent therapeutic efficacy against melanoma in both primary and distant tumor sites, underscoring the broad effectiveness of our delivery strategy.

Gastric cancer is a highly prevalent malignant tumor of the digestive system worldwide. Although andrographolide (AG) and coixol (CX) possess potential anti-gastric cancer effects, their clinical application is restricted by low bioavailability and poor targeting ability. Based on the meridian tropism theory of traditional Chinese medicine and the concept of “integrated drug and carrier”, this study constructed Bletilla striata polysaccharide-vitamin E succinate (BSP-VES) polymeric micelles co-loaded with AG and CX (AG&CX@BSP-VES) for synergistic targeted therapy of gastric cancer. In this study, AG&CX@BSP-VES was first systematically characterized in vitro to clarify its physicochemical properties, drug release behavior and hemolysis profile. Subsequently, using subcutaneous and orthotopic gastric cancer mouse models as research objects, in vitro cellular uptake and in vivo tumor targeting ability efficacy and biological safety of this drug delivery system were further evaluated. Results showed that the micelles were regularly spherical with a particle size of approximately 105 nm, exhibited favorable biological safety and asynchronous differential release characteristics, and could be specifically internalized by cells in vitro (which could be blocked by free BSP). In vivo, the accumulation of micelles in tumor masses was significantly higher than that in normal gastric tissues. Moreover, the cytotoxicity of the micelles against SGC-7901 tumor cells was remarkably higher than that of free AG or CX alone, which was attributed to enhanced cellular drug uptake and the synergistic effect between AG and CX. This study provides novel insights for the precise delivery of active ingredients from traditional Chinese medicine and individualized therapy of gastric cancer.

18β-Glycyrrhetinic acid (GA) exhibits excellent anti-inflammatory and antioxidant activities, and has attracted considerable attention in the field of cerebral ischemia−reperfusion (I/R) injury therapy. However, it is plagued by problems of low bioavailability and dose-dependent toxicity. As a drug delivery platform for the treatment of ischemic stroke, microenvironment-responsive liposomes can cross the impaired blood−brain barrier, efficiently deliver drugs to the lesion sites, and improve therapeutic efficacy. Herein, to address the challenges posed by the highly oxidative-stress microenvironment of I/R and the intrinsic physicochemical properties of GA, we encapsulated GA into liposomes and incorporated thioketal (TK) linkages into the phospholipid bilayer to fabricate GA@TK-Lip. GA@TK-Lip can effectively target damaged neurons, attenuate neuroinflammation, scavenge excessive ROS, ameliorate cellular oxidative stress via the Nrf2/HO-1 pathway, reduce neuronal apoptosis and cerebral infarct volume to 43.2% and 23.3% of that in the MCAO group, and restore neurological function. Collectively, this study provides novel insights into the development of promising therapeutic strategies for ischemic stroke.

The absence of estrogen receptor, progesterone receptor, and HER2 expression limits the utility of targeted therapies in triple-negative breast cancer (TNBC). Chemotherapy is the standard of care for advanced TNBC but offers limited responses and an overall poor prognosis. We have developed several tumor-selective alkylphosphocholine analogs (NM600) for radiopharmaceutical therapy (RPT) of TNBC. Herein, we investigate the preclinical [²²⁵Ac]Ac-NM600 therapeutic efficacy and safety in two murine models of TNBC. NM600 was radiolabeled with ²²⁵Ac following previous methods. In vitro cytotoxicity studies were performed in metastatic (4T1) and non-metastatic (4T07) murine TNBC cells. Monte Carlo-based ²²⁵Ac dosimetry estimates enabled viability and clonogenic survival measurements in cells treated with ²²⁵Ac (0.03−1 Gy). Longitudinal ex vivo biodistribution studies were performed in BALB/c mice bearing 4T1 or 4T07 tumor grafts injected with 9.25 kBq [²²⁵Ac]Ac-NM600 at 24, 72, 120, and 144 h post-injection (p.i.), and dosimetry estimation was performed using a MIRD formalism and a standard mouse model. Therapeutically, three groups of mice bearing 4T1 or 4T07 grafts (n ⦥5) received IV injections of vehicle, or 9.25 or 18.5 kBq [²²⁵Ac]Ac-NM600. Tumor volume and overall survival were monitored thrice weekly. Toxicity was evaluated in separate groups of BALB/c mice (n = 3) at days 7, 30, 60, and 120 p.i. through complete blood count (CBC), serum chemistry, and histopathological analysis. [²²⁵Ac]Ac-NM600 was produced with high purity and serum stability. Clonogenic assays showed comparable dose-dependent ²²⁵Ac cytotoxicity in 4T1 and 4T07 cells, with an RBE of 5 compared to EBRT. Ex vivo biodistribution exhibited elevated and persistent tumor uptake, resulting in absorbed dose coefficients of 46 Gy/μCi and 42 Gy/μCi. Due to the hepatobiliary excretion of [²²⁵Ac]Ac-NM600, distribution in normal tissues was most prominent in the liver, with absorbed doses of 57 Gy/μCi and 63 Gy/μCi for mice bearing 4T1 and 4T07 tumors, respectively. Among other relevant tissues, blood and lungs were initially elevated but rapidly declined over time. [²²⁵Ac]Ac-NM600-treated mice exhibited significant tumor growth inhibition (p ≤ 0.015), with median survival of 21, 49, and 32 days, or 30, 77, and 64 days, for the vehicle, 9.25 kBq, and 18.5 kBq groups, in mice bearing 4T1 or 4T07 tumors, respectively. Only mild effects and no treatment-related mortality were observed in animals given [²²⁵Ac]Ac-NM600 9.25 or 18.5 kBq. Our findings underscore the potential of [²²⁵Ac]Ac-NM600 as a promising therapeutic agent for TNBC, warranting further assessments of efficacy and potential toxicities in additional TNBC animal models.

Quorum sensing (QS) is a bacterial communication mechanism that regulates collective behaviors such as virulence, motility, and biofilm formation. Consequently, materials capable of interacting with molecules associated with QS signaling pathways are of increasing interest as potential tools for studying and modulating bacterial communication processes. In this study, molecularly imprinted polymers (MIPs) were designed and synthesized using (R/S)-2,2-dimethyl-1,3-dioxolane-4-methanamine, an autoinducer-2 (AI-2)-inspired surrogate template to investigate molecular recognition of structures relevant to bacterial signaling systems. Five bulk MIPs were prepared by thermal radical polymerization using methacrylic acid, 4-vinylbenzoic acid, itaconic acid, trifluoromethacrylic acid, and acrylic acid as functional monomers. The resulting polymers were evaluated in terms of adsorption capacity, imprinting factor, and selectivity toward the template and structurally related compounds using liquid chromatography coupled with mass spectrometry (LC−MS)-based binding studies. Among the synthesized materials, the polymer prepared from methacrylic acid exhibited the highest specificity, with an imprinting factor of 3.10 at 50 μg L⁻¹ and up to 19.14 at 2 μg L⁻¹ of template concentrations, indicating strong concentration-dependent recognition. Computational modeling using density functional theory, molecular mechanics, and molecular dynamics simulations was employed to elucidate the molecular interactions governing template recognition and cavity formation. Theoretical analyses revealed that multiple hydrogen bonding, hydrophobic interactions, and electrostatic contacts contribute to stable analyte-polymer binding, supporting the experimentally observed selectivity. Selectivity experiments demonstrated preferential adsorption of the template and its closest structural analogue relative to several structurally related amine-containing compounds. Biological evaluation using the Chromobacterium violaceum model revealed that both molecularly imprinted and non-imprinted polymers reduced violacein production to varying degrees, indicating modulation of a QS-regulated phenotype under the experimental conditions employed. Collectively, these findings demonstrate the feasibility of using molecular imprinting strategies to generate polymeric materials capable of recognizing an AI-2-inspired surrogate template and influencing a QS-regulated biological response, highlighting their potential for development as innovative materials for antivirulence strategies against bacterial infections.

Intracellular infections impose fundamental physicochemical barriers that can disrupt antibiotic synergy observed under well-mixed in vitro conditions. Mycobacterium abscessus, a macrophage-resident pathogen, exemplifies this challenge, as co-administered drugs must traverse multiple membrane barriers and achieve coordinated intracellular exposure. The combination of clofazimine (CFZ) and amikacin (AMK) exhibits robust, time-dependent synergy in broth, yet their divergent physicochemical properties (lipophilic versus hydrophilic) predict differential intracellular access. Here, we evaluated whether formulation architecture can mitigate this spatial and temporal separation. CFZ and AMK were engineered either as physical mixtures of micronized powders or as spray-dried composite particles across multiple drug ratios and compared in terms of aerosol performance and intracellular efficacy in a THP-1 macrophage infection model. Neither drug alone achieved meaningful intracellular killing despite activity in broth. In contrast, combination efficacy was strongly formulation-dependent and exhibited a non-monotonic relationship with composition. Physical mixtures were most effective at CFZ-rich ratios, whereas spray-dried composites achieved superior killing at an intermediate mass ratio (1:4 CFZ/AMK) and displayed a delayed-onset kinetic profile consistent with architecture-dependent temporal drug availability. Aerosol studies showed that composite particles generally improved dispersibility and reduced bulk aerodynamic fractionation between the two drugs relative to physical mixtures, although this effect was condition-dependent. Together, these findings indicate that intracellular efficacy is governed not solely by drug identity or dose, but by formulation-controlled spatial and temporal drug presentation. This work establishes formulation architecture as a critical design variable for translating antibiotic synergy into biologically constrained environments.

Visible particles were observed for a therapeutic monoclonal antibody at pH 5.5. This pH was considered optimal for this antibody during preclinical development. To minimize the particle formation, the pH of the final drug substance was increased to 6.5. There were no visible particles at pH 6.5 initially, but visible particles started to form after 2 months. To minimize impact to the clinical program, a lyophilized formulation presentation was chosen. Subsequently, due to the desire for patient administration through subcutaneous injection for this antibody, a stable, high concentration solution formulation was needed. Thus, the pH was further increased to 7.2 to mitigate the visible particle formation. LC-MS-based approaches, including reduced LC−MS analysis and cathepsin D-targeted LC−MS Lys-C peptide map analysis, were developed to identify the root cause of the visible particle formation of this antibody at pH 5.5 to ensure the successful development of a stable solution formulation at pH 7.2. Results from studies supported by LC-MS-based analyses indicated that visible particles observed in the antibody protein A capture main pool sample at pH 5.0 were associated with cathepsin D clipping of the antibody. In contrast, visible particles observed in the purified antibody drug substance samples at pH 5.5 and pH 6.5 and in the antibody in-process samples (from the low pH viral inactivation, anion exchange chromatography, hydrophobic interaction chromatography, and tangential flow filtration unit operations) were associated with succinimide intermediate formation at an Asp residue with an Asp−Ser sequence motif in one of the complementarity-determining regions of the light chain. Understanding the root cause of the particle formation of this antibody at pH 5.5 enabled us to perform targeted experiments to rapidly develop a stable high concentration solution formulation for this antibody and meet project timelines.

Dysregulated wnt/β-catenin signaling cascades drive self-renewal, proliferation, differentiation, and therapeutic resistance in cancer stem cells (CSCs), thus contributing to their tumor aggression and poor clinical outcomes. Earlier, we demonstrated that a glucocorticoid receptor (GR)-targeted liposomal formulation carrying dexamethasone (Dex) and the wnt/β-catenin inhibitor FH535 (D1XFH) selectively sensitizes colorectal cancer (CRC) cells and suppresses tumor growth. Herein, we investigate the immuno-therapeutic potential of D1XFH, especially within colorectal CSCs and against tumor recurrence. Our results reveal that D1XFH significantly downregulates CD133 and stemness-associated transcription factors Nanog, Sox2, and Oct4 in CSCs. D1XFH induces selective cytotoxicity by enhancing ROS generation, triggering apoptosis, reducing clonogenic potential, inhibiting cellular migration, and reversing multidrug resistance in CSCs. Co-administration of D1XFH-engineered CSCs and D1XFH into subcutaneous tumor-bearing mice resulted in pronounced tumor regression, accompanied by inhibition of wnt/β-catenin signaling, reversal of EMT, and antitumorigenic immune responses, while improving long-term survival. Additionally, D1XFH treatments post-tumor resection resulted in near-zero tumor recurrence, while zero recurrence was observed even after tumor re-challenge. We observed 250 day long-term survivability with possible resurgence of immune-memory response within tumor-resected, D1XFH-treated mice. Collectively, dual targeting of GR and wnt/β-catenin-driven CSC engineering and immunomodulation is a promising strategy for colorectal cancer therapy.

Rational design of PEGylated protein therapeutics requires mechanistic understanding of how coupling chemistry and PEG size jointly determine product homogeneity, receptor engagement, and structural integritythe properties that collectively govern whether a conjugate is a viable candidate for further development. Anakinra (recombinant interleukin-1 receptor antagonist, IL-1Ra) is an approved anti-inflammatory biologic whose short systemic residence necessitates daily subcutaneous injection and is associated with steep peak−trough exposure fluctuations that, in chronic high-dose regimens, have been linked to IL-1Ra-derived systemic amyloidosis. PEGylation offers a chemically established route to increase the apparent molecular size of IL-1Ra and thereby reduce renal filtration and smooth exposure, but the structural and functional consequences of conjugation have never been characterized at residue resolution for this targeta gap that limits informed design choices between available chemistries. Here, we address this gap by establishing a head-to-head characterization framework comparing site-specific thiol-maleimide conjugation and random amino-coupling on a uniformly ¹⁵N-labeled recombinant IL-1Ra variant (M143V) that preserves all native conjugation sites and is potency-matched to Anakinra. Across a 5−20 kDa PEG ladder at a substoichiometric 0.5:1 PEG:protein feed, site-specific thiol-maleimide coupling yielded substantially higher conversion (∼81%) and a homogeneous single-species di-PEGylated (DoP 2) conjugate, whereas random amino-coupling produced heterogeneous mixtures across all PEG sizes (∼33−36% conversion, multiple degrees of polymerization). Purified single-species conjugates were benchmarked under a fixed IL-1β EC₈₀ challenge in HEK-Blue reporter cells; all retained full maximal efficacy but exhibited chemistry- and size-dependent right-shifts in IC₅₀, indicating steric modulation of receptor engagement rather than loss of function. To directly establish whether PEG attachment perturbs structural integrity, 2D ¹H−¹⁵N HSQC NMR on the ¹⁵N-labeled thiol-PEG10 conjugate demonstrated a preserved global fold with localized chemical-shift perturbations near cysteine-proximal surface regions, confirming site-selective modification and intact binding scaffold. Together, these data establish the conjugation quality, antagonist activity, and structural integrity of the lead conjugate as the prerequisite characterization foundation for its further development and provide a transferable analytical framework for the rational design of PEGylated cytokine therapeutics.