Molecular Pharmaceutics最新文献

Doxorubicin (DOX) faces significant challenges in oral chemotherapy due to low intestinal permeability and extensive first-pass metabolism. We developed microfluidics-prepared RGD-modified solid lipid nanoparticles (MF-SLNs) to enhance oral anticancer efficacy and investigate their impact on gut microbiota. In vitro analysis showed that MF-SLNs exhibited a smaller particle size (∼120 nm) and a more stable zeta potential (∼20 mV). They also showed high encapsulation efficiency (EE, EE > 80%). Particle size distribution from dynamic light scattering (DLS) and transmission electron microscopy (TEM) further confirmed the improved homogeneity of MF-SLNs (PDI of 0.073). DOX was released from MF-SLNs in a slow and sustained manner, indicating its potential for controlled delivery into the gastrointestinal tract. MF-SLNs showed good stability in simulated gastric and intestinal fluids. Confocal microscopy revealed that MF-SLNs significantly enhanced the transcellular transport of DOX across the FAE monolayer and subsequent uptake by MDA-MB-231 breast cancer cells. In vitro apoptosis in MDA-MB-231 breast cancer cells was assessed by using flow cytometry, revealing an increased percentage of apoptotic cells following MF-SLNs treatment. In vivo studies in nude mice demonstrated enhanced tumor inhibition and improved survival rates. Histopathological analysis, organ weight measurements, and echocardiography detection indicated favorable outcomes, complemented by assessments of tissue damage markers. Furthermore, 16S rRNA sequencing revealed a significant increase in beneficial gut bacteria, including Faecalibacterium and Bacillus, following MF-SLNs treatment. Collectively, MF-SLNs enhance antitumor efficacy and promote healthier gut microbiota, suggesting advantages over traditional DOX formulations. Further studies are needed to optimize this delivery system for breast cancer therapies.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer, characterized by rapid progression and poor prognosis. Silibinin (SIL), the main active constituent of milk thistle, inhibits proliferation, induces apoptosis, and suppresses metastasis of HCC. However, its clinical use is limited by poor water solubility and low oral bioavailability. Nanoencapsulation offers an effective strategy to overcome these drawbacks, enabling selective targeting of tumor cells. This work aimed to design, develop, and characterize silibinin-loaded PLGA nanoparticles coated with phenylalanine (Phe-SIL-Nps) to enhance SIL delivery to HCC cells. An L4 Taguchi design was used to optimize the formulation. PVA concentration was the most influential factor, significantly affecting particle size, drug loading, and encapsulation efficiency, while sonication time had a statistically significant effect on the PDI. The optimized formulation (SIL-Nps), prepared with 3% PVA, a sonication time of 8 min, and a sonicator amplitude of 75%, exhibited a particle size ≈250 nm, a PDI ≈0.2, a zeta potential of -26 mV, a drug loading of ≈450 μg SIL/10 mg Nps, and a high encapsulation efficiency (≈96%). Phenylalanine coating increased particle size up to 275 nm and shifted the zeta potential to more negative values (-35 mV). Both SIL-Nps and Phe-SIL-Nps showed a spherical shape and exhibited a controlled release profile for 7 days. Phe-SIL-Nps displayed higher cytotoxicity than free SIL and SIL-Nps, as well as greater ROS production in Hep3B cells. This enhanced effect is attributed to their higher internalization via LAT transporters, which are overexpressed in HCC cells. These results suggest that LAT-targeted nanoparticles represent a promising technological approach to enhance the antitumor efficacy of antineoplastic agents in hepatocellular carcinoma.

Adeno-associated virus serotype 2 (AAV2) is widely used as a gene therapy vector due to its favorable safety profile and broader transduction capabilities. However, pre-existing immunity poses a significant barrier to its therapeutic applications. In this study, we employed coarse-grained elastic network molecular dynamics simulations to investigate the structural and conformational dynamics of the wild-type AAV2 capsid and its six capsid variants (Q263A, S264A, S384A, Q385A, V708A, and V708K) upon binding to a mouse monoclonal antibody (A20), a robustly used AAV2-specific antibody. Notably, A20 recognizes a few immunodominant epitopes that can be utilized to design AAV2 mutants with robust resistance to human neutralizing sera. Our analysis revealed that the involvement of three different symmetry-related subunits of the AAV2 capsid is critical in mediating interactions with A20, particularly through its heavy-chain complementarity-determining regions (CDRs). Per-residue energy decomposition analysis identified key interaction hotspots, which are in agreement with the experimental neutralization data for escape mutants. Structural descriptors, such as root-mean-square deviation (RMSD), radius of gyration (R_g), solvent-accessible surface area (SASA), center-of-mass (COM) distances, and contact probabilities, were well correlated with experimental A20 binding data. A predictive model was developed using multiple linear regression (R_{CrossValidation}² = 0.949), successfully capturing the relationship between mutation-induced structural changes in AAV2 and fold reduction in A20 binding affinities. This integrative approach provides mechanistic insights into capsid-antibody recognition and offers a structure-guided, rational framework for designing AAV2 variants with reduced immunogenicity, thereby advancing the development of next-generation gene therapy vectors.

Efficient determination of developable protein drug candidates and stable solution conditions is a key challenge in industrial drug development. Protein aggregation is difficult to predict and can lead to challenges in manufacturing, storage, and patient safety. In this work, stability of four monoclonal antibodies (MAbs) were studied at a wide range of solution conditions and incubation temperatures intended to systematically evaluate attributes that can influence aggregation rates. The studies were conducted as a function of pH, ionic strength, MAb concentrations, and incubation temperatures that were representative of industrial stability studies. Results were analyzed in the contexts of conformational stability and net self-interactions. Interpretable machine learning models were applied to parse and quantify the phenomena relevant to high-concentration aggregation rates, with emphasis on refrigerated conditions representative of common storage conditions for MAb products. The results indicated that the aggregation rates were non-Arrhenius, and stability studies at 30 to 50 °C were broadly misleading with respect to the stability rankings of the different formulations and MAbs in comparison to the stability rankings at refrigerated storage conditions. For this set of MAbs and formulation conditions, the net valence was the most significant predictor of aggregation rates at refrigerated storage conditions.

We report a modified bicomponent solid-state form of tadalafil (TDF) and finasteride (FNS), prepared at a 1:1 molar stoichiometric ratio, corresponding to the USFDA-approved combination marketed under the trade name Entadfi, for the treatment of benign prostatic hyperplasia. Individually, both constituent drugs suffer from the limitation of low aqueous solubility, belonging to class-II of the biopharmaceutical classification system. A drug-drug coamorphous system of TDF and FNS was prepared by mechanochemical synthesis. Characterization of this novel phase was carried out by powder X-ray diffraction, thermal analysis, and FT-IR spectroscopy. Particle characteristics and morphological features of the coamorphous system were studied by scanning electron microscopy and 3D-laser scanning microscopy. Possible intermolecular interactions between TDF and FNS, facilitating the formation of the coamorphous phase, as indicated by spectroscopic analysis, were validated by the computational study employing density functional theory. Interestingly, in vitro dissolution studies showcased significant improvement in the dissolution profile of the coamorphous system compared with the physical mixture, which was successfully translated to the in vivo study in SD rats. Physical stability of the developed coamorphous system evaluated under accelerated as well as long-term stability conditions indicated reasonable stability for potential drug product usage. Considering its industrial applicability due to obvious benefits, viz., single solid phase, improved solubility, dissolution, and better pharmacokinetic parameters leading to higher bioavailability, the developed coamorphous system could prove to be a better therapeutic alternative over the marketed physical mixture.

Doxorubicin (dox) has been used for the treatments of many cancers for more than 50 years since its discovery. Currently, the treatment with dox is often limited by cardiotoxicity and the development of drug resistance. Doxazolidine (doxaz) is a dox-formaldehyde conjugate discovered in the 1990s. It bears an extra carbon, linking its daunosamine hydroxyl to that adjacent amino substituent to create an oxazolidine ring. In contrast to dox, which is a topoisomerase inhibitor, doxaz cross-links DNA to nonspecifically inhibit cell growth. Doxaz is significantly more cytotoxic than dox, even against the dox-resistant cancer cells, and in spite of its 3-minute half-life for hydrolysis to dox. Doxaz has been studied since its discovery, but not clinically, due to its cytotoxicity and unsuccessful attempts to generate the prodrugs of doxaz that are activated solely in cancer cells without damaging healthy normal cells. Here, we report an ROS-activatable prodrug of doxaz, named Doxaz-BA, formulated as a nanoparticle. We synthesized Doxaz-BA and its derivatives and tested them as nanoparticle formulations in vitro in cell cultures and in vivo in mouse xenografts. This technology provides a highly sought-after cancer therapy that kills only cancer cells, while toxicity to normal tissues is minimal. Doxaz-BA is effective against drug-resistant cancer cells, and the safety assessments showed no toxicity in mouse models. Therefore, this technology offers a possible solution for the clinical translation of Doxaz in treating drug-resistant cancers, which are often incurable in standard clinical settings.

Polysorbates (PSs) are frequently used as excipients in protein drug formulations. However, recent findings suggest that lipases and esterases can degrade PSs even at very low concentrations, thereby resulting in biotherapeutic instability and particle formation during stability assessments. Acid ceramidase, a lipase frequently present in drug formulations, was previously believed not to be a PS degrading enzyme. ( J. Pharm. Sci. 2024, 114, 1002-1009. J. Pharm. Sci. 2023, 112, (5), 1351-1363.) However, our study shows that acid ceramidase can be activated during purification process on an aged hydrophobic interaction chromatography column that subjected to multiple use cycles without effective regeneration procedures. After activation, low-abundance acid ceramidase degrades PS. Furthermore, the enzyme saposin D increases the lipase activity of activated acid ceramidase, thus accelerating PS degradation. Our study also demonstrated that effective regeneration of the HIC column can prevent acid ceramidase activation, and the cofactor saposin D can be eliminated by Ultrafiltration/Diafiltration (UF/DF) filtration through a 50 kDa membrane. Consequently, the rapid PS degradation by activated acid ceramidase and its cofactor observed herein is less likely to occur in drug products purified according to standard protocols and guidelines.

The protein corona formed upon systemic administration critically modulates the pharmacokinetics, biodistribution, and therapeutic efficacy of the nanomedicines. While emerging evidence links obesity to heightened chemosensitivity, the underlying nanobio-interfacial mechanisms remain poorly understood. Herein, we demonstrate that pegylated liposomal doxorubicin (PLD) exhibits significantly enhanced antitumor and antimetastatic efficacy in obese breast tumor-bearing mice compared to normal controls. Mechanistic investigations reveal that obesity confers PLD with prolonged systemic circulation and improved tumor accumulation. Notably, preincubation of PLD with plasma from obese mice reduces macrophage uptake while promoting internalization by breast cancer cells compared to that from normal mice. Genetic ablation of apolipoprotein E (ApoE) in obese mice abolishes obesity-associated improvements in PLD blood circulation, tumor accumulation, and uptake by cancer cells. Conversely, supplementation with recombinant ApoE restores these effects in ApoE-deficient mice and potentiates PLD's antitumor efficacy. Collectively, our findings demonstrate obesity-induced ApoE as a pivotal regulator of the protein corona that actively enhances tumor-targeted delivery of PLD, which offers a rational strategy for engineering protein-corona-mediated tumor-targeted nanomedicines.

Individuals diagnosed with multiple sclerosis (MS), an autoimmune disease, experience both physical and cognitive impairments due to the degeneration of the myelin sheath surrounding neurons triggered by autoreactive T cells and antibodies. Current MS therapies based on immunosuppressants often result in systemic immune suppression, known as immune tolerance, which leads to side effects including risk of infection and malignancy. Previously, we reported bifunctional peptide inhibitors (BPIs) that can induce antigen-specific immune tolerance and suppress disease progression in a mouse MS model of experimental autoimmune encephalomyelitis (EAE). While BPIs showed similar effectiveness via different routes of administration including intravenous (IV), subcutaneous (SC), and intraperitoneal (IP), whether the route of administration, specifically, mediates immunosuppressive mechanisms used to suppress EAE has not yet been studied. In this study, we prepared BPIs based on the model antigen ovalbumin (OVA₃₃₉-BPI) and myelin proteolipid protein (PLP-BPI) to evaluate immunomodulatory effects on splenocytes and the PLP-induced EAE model, respectively. The efficacy of BPIs inducing an immunomodulatory response was determined by measuring cytokine production as well as transcription factor and costimulatory molecule expression levels in microglia cells and splenocytes. In this study, PLP-BPI suppressed EAE in IV and SC treatment groups, with clinical scores reaching 0 (no clinical symptoms) by day 19. In contrast, the IP-treated group showed no suppression, with clinical scores similar to those of the EAE + no treatment group. Interestingly, IV and SC employed distinct immunomodulatory mechanisms: IV primarily reduces proinflammatory markers in microglia, while SC treatment increases the anti-inflammatory markers in microglia and transcription factor Foxp3⁺ in splenocytes. These results suggest that the route of BPI administration can determine the in vivo efficacy by differentially modulating the frequency and activity of immunosuppressive cell populations.