Molecular Pharmaceutics最新文献

Nonviral vectors hold great promise for mRNA (mRNA)-based therapeutics. However, achieving organ-selective mRNA delivery after systemic administration remains a major challenge. Herein, we engineered hydrophobized polymeric nanoparticles (PNPs) derived from arylated polyethylenimine (PEI) for efficient and organ-selective mRNA delivery. Nine new hydrophobized polymers were synthesized by grafting 2-phenylethyl acrylate (PA) onto PEI with three different molecular weights of PEI and three PA-to-PEI feeding molar ratios. The resulting PNPs were systematically evaluated for mRNA delivery both in vitro and in vivo. Through in vitro transfection screening, three optimal candidates (6PP3-PNP, 12PP6-PNP, and 24PP12-PNP) were selected for systemic mRNA delivery studies in mice. Remarkably, 6PP3-PNP preferentially delivered mRNA to the spleen, in contrast to the liver tropism observed with 12PP6-PNP and 24PP12-PNP. Moreover, 6PP3-PNP exhibited excellent biocompatibility both in vitro and in vivo. These findings elucidate the structure–function relationship of hydrophobized PEI in mRNA delivery and demonstrate a tunable strategy for developing organ-selective carriers, thereby expanding the potential of mRNA therapeutics for immunotherapy.

Purinergic receptor P2X7 has been considered as a potential new target for detecting and treating high-risk plaque. Nanobodies are the smallest antibody fragments with high antigen binding ability and specificity, which are well-suited for radionuclide imaging. The present study aimed to develop a novel P2X7-targeted nanobody SPECT tracer and to investigate its potential for identification of atherosclerotic plaque (AP). The anti-P2X7 nanobody 1c81 was site-specifically conjugated with [^99mTc]Tc-HYNIC-GGGC via sortase A-mediated transpeptidation to prepare [^99mTc]Tc-1c81. Saturation binding experiments and cell-binding assays were performed to evaluate their affinity and specificity. Biodistribution studies in C57 mice were conducted at 0.5, 1, and 2 h postinjection (p.i.), and SPECT/CT imaging was performed in ApoE^–/– (high-fat diet) and C57 mice (normal diet) at 2 h p.i, respectively. Specific binding was validated by blocking studies (coinjection of [^99mTc]Tc-1c81 with excess unlabeled 1c81) in ApoE^–/– mice. Target-to-background ratio (TBR) was calculated for AP at aortic arch. The harvested aortas were analyzed by autoradiography, Oil Red O lipid staining, and immunofluorescence staining (CD68, P2X7) to correlate tracer uptake with AP characteristics. To further validate the clinical relevance, human coronary endarterectomy (CE) specimens were analyzed for P2X7 and CD68 expression using immunohistochemistry. [^99mTc]Tc-1c81 was synthesized with 53.77 ± 0.06% radiochemical yield, > 95% purity, 11.13 ± 2.78 MBq/nmol molar activity, and a binding dissociation constant of 6.38 nM. Biodistribution showed rapid clearance from the blood and normal organs except the kidneys. SPECT imaging at 2 h p.i. revealed clear aortic arch visualization, with significantly higher TBR in ApoE^–/– mice compared to both C57 and blocking groups (4.49 ± 1.88 vs 0.96 ± 0.64, P = 0.012; 4.49 ± 1.88 vs 1.40 ± 0.28, P = 0.017). Autoradiography further confirmed specific tracer accumulation in APs, colocalizing with Oil Red O-positive lipid-rich regions. Immunofluorescence and immunohistochemical staining validated high P2X7 receptor expression in both mouse AP aortic valve sections and human CE specimens, which was colocalized with CD68⁺ inflammatory cells, confirming the clinical relevance of P2X7 as an imaging target for inflammation of AP. [^99mTc]Tc-1c81 exhibited specific binding to the P2X7 receptor in AP in vivo. It may serve as a novel P2X7-targeted SPECT tracer to detect AP, with promising applications in clinical risk stratification and treatment response monitoring.

Highly overexpressed somatostatin receptors (SSTRs), especially SSTR2, are frequently characterized in neuroendocrine tumors (NETs) and serve as a target for imaging and peptide receptor radionuclide therapy (PRRT). However, the SSTR2 expression decreases in higher-grade NETs, while the expression level of the integrin receptor α_Vβ₃ was increased. A reported heterodimer, [⁶⁸Ga]Ga-NOTA-3P-TATE-RGD, which targets both SSTR2 and integrin α_Vβ₃, has shown potential clinical value, but the high renal uptake limited its further development. The aim of this study is to evaluate [⁶⁸Ga]Ga/[¹⁷⁷Lu]Lu-DOTATATE-RGD ([⁶⁸Ga]Ga/[¹⁷⁷Lu]Lu-DTR) and [⁶⁸Ga]Ga-NOTA-TOC-RGD ([⁶⁸Ga]Ga-NCR) as potential SSTR and integrin α_vβ₃ heterodimer agents. Radiolabeling of both Ga-68 and Lu-177 was effectively accomplished with high yields and radiochemical purities. The three agents showed higher cell uptake compared with monomeric [⁶⁸Ga]Ga-RGD but reduced uptake compared with [⁶⁸Ga]Ga-DOTATATE. Results of biodistribution and small-animal positron emission tomography (PET) studies showed that the renal uptake of [⁶⁸Ga]Ga/[¹⁷⁷Lu]Lu-DTR decreased without affecting the tumor-binding ability compared to [⁶⁸Ga]Ga-NOTA-3P-TATE-RGD, but the tumor-to-kidney ratio of [⁶⁸Ga]Ga-NCR did not increase as desired. In initial clinical trials, [⁶⁸Ga]Ga-DTR PET/CT exhibited a lower uptake in the kidney and spleen than that of [⁶⁸Ga]Ga-NOTA-3P-TATE-RGD PET/CT (SUV_mean: 10.88 ± 3.27 vs 29.02 ± 8.89 and 3.41 ± 1.18 vs 8.16 ± 4.38, respectively), indicating that [⁶⁸Ga]Ga/[¹⁷⁷Lu]Lu-DTR may serve as a useful candidate ligand for theranostic agents in NET patients with a lower uptake of normal organs.

Although polymers can prevent the crystallization of glassy drugs in amorphous solid dispersions, their stabilization mechanism requires further clarification for an efficient formulation design. This study examined the impact of adding trace amounts (2 or 5 w/w %) of vinylpyrrolidone–vinyl acetate copolymer (PVPVA) on the physical stability of celecoxib (CEL) glass using differential scanning calorimetry and broadband dielectric spectroscopy. Long-term isothermal crystallization studies from 35 to 60 °C revealed that CEL glass was significantly stabilized by the addition of trace amounts of PVPVA. Its stabilization was attributed to the effect of PVPVA on the nucleation process rather than on crystal growth. The addition of PVPVA slowed down the α-relaxation of CEL, whereas it accelerated Johari–Goldstein relaxation. Moreover, the addition of PVPVA effectively slowed down γ- and δ-relaxations. Of these, suppression of γ-relaxation mobility had the most important effect, as it is related to the formation of hydrogen bonding between CEL and PVPVA molecules to inhibit nucleation. Moreover, the change in molecular cooperativity of the CEL glass upon adding PVPVA contributed to the inhibition of nuclei formation due to the decreased nucleation temperature. This study provides detailed insights into the physical stabilization mechanisms of glass using polymeric excipients.

Sphingosine-1-phosphate (S1P) and its receptors (S1PRs) are pivotal regulators of immune cell trafficking, vascular integrity, and various physiological processes, playing key roles in the pathogenesis of neuroinflammatory and immune-related disorders. Among these, multiple sclerosis (MS) is the most common chronic inflammatory condition affecting the central nervous system (CNS), marked by autoimmune-induced neurodegeneration, inflammation, and ongoing demyelination. FTY720, known as fingolimod or Gilenya, is an immunomodulatory medication that was approved in 2010 as the first oral therapy for relapsing-remitting MS. Upon phosphorylation, FTY720 mimics S1P and binds selectively to all S1PR subtypes, except S1PR2, underscoring the therapeutic possibilities of focusing on the S1P–S1PR signaling axis for neuroinflammatory conditions. This success emphasizes the relevance of S1P-mediated pathways in both disease mechanisms and treatment strategies. Emerging precision medicine approaches emphasize the importance of noninvasive imaging to elucidate molecular mechanisms in vivo. Positron emission tomography (PET) imaging–utilizing suitable radioactive tracers to probe biological targets and processes in vivo–offers a transformative approach to quantifying receptor expression, thereby delineating crucial insights into disease diagnosis, therapy monitoring, and therapeutic drug development. As such, S1PR-specific PET imaging provides a promising approach to explore the pivotal role of S1PRs in MS and other immune-mediated diseases. This review offers a comprehensive overview of the development and clinical applications of S1PR-targeted PET radiopharmaceuticals, illustrating their potential to transform therapeutic strategies. Further, recent advances in radiopharmaceutical design have yielded S1PR-targeted PET probes with high specificity, improved metabolic stability, and enhanced blood–brain barrier penetration, addressing key challenges in imaging neuroinflammation. Additionally, it critically discusses future directions for S1PR-targeted PET imaging in advancing our understanding of disease mechanisms, improving patient outcomes, and contributing to the broader vision of precision medicine.

As an important tumor-associated carbohydrate antigen, the Thomsen–Friedenreich (T or TF) antigen has become an attractive target for tumor diagnosis and treatment. However, there has been very limited success in developing peptide- and small-molecule-based radiopharmaceuticals for this important target. Currently, only ⁶⁴Cu-NO2A-TFpep has been reported as a radiolabeled peptide targeting the TF antigen, and it shows a low tumor-to-liver ratio due to ⁶⁴Cu retention in the liver. In this study, a novel PET probe targeting the TF antigen (DOTA-TFpep) was synthesized using 2,2′,2″,2‴-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) chelator for increasing the hydrophilic property and for radiolabeling with different radionuclides. DOTA-TFpep was then radiolabeled with ⁶⁸Ga for positron emission tomography (PET) imaging of breast tumors expressing the TF antigen. ⁶⁸Ga-DOTA-TFpep was confirmed by radio-HPLC with a purity greater than 98% and high stability in PBS. Immunofluorescence analysis confirmed the TF antigen expression of 4T1 and K1 cell lines. Cell uptake studies confirmed its targeting specificity. Further in vivo biodistribution studies in high (4T1) and low (K1) TF antigen-expression xenograft models demonstrated the favorable pharmacokinetics property of the probe. PET imaging and biodistribution showed that ⁶⁸Ga-DOTA-TFpep exhibited specific tumor uptake. Moreover, compared with the widely studied ⁶⁴Cu-NO2A-TFpep, ⁶⁸Ga-DOTA-TFpep showed lower liver uptake and a higher tumor-to-liver ratio (1.31 ± 0.20 for ⁶⁴Cu-NO2A-TFpep and 0.48 ± 0.15 for ⁶⁸Ga-DOTA-TFpep at 120 min after injection). In summary, this study demonstrates the synthesis and evaluation of the TF antigen-targeting probe ⁶⁸Ga-DOTA-TFpep. It shows favorable in vivo tumor imaging properties, highlighting it as a promising molecular probe targeting the TF antigen.

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.