Rapid crystallization of an active pharmaceutical ingredient can suppress its observable supersaturation and negate this potential advantage of the amorphous form. Excipients added to formulations can prolong supersaturation, but reduce drug loading and obscure the intrinsic dissolution behavior of the drug in testing. Here, we describe spring-and-parachute dissolution behavior in fasted-state simulated intestinal fluid of an archetypal poorly soluble and strongly crystallizing drug, griseofulvin (GSF), in the form of excipient-free, pure, amorphous nanopowders generated using a novel, single-step, solvent-free technique of organic vapor jet desublimation. Using experiments and modeling, we establish how initial dissolution rate, peak concentration, and area under the concentration-time curve scale with dose, distinguishing between dose regimes in which supersaturation behavior is monotonic and nonmonotonic. The roles of local surface crystallization, particle aggregation, and nonequilibrium crystallization at high supersaturation are elucidated, providing a framework for attaining excipient-free supersaturation and guiding the development of improved dissolution models.

The design and development of suitable dosage forms for pediatric patients face myriad challenges. Compared to other dosage forms, oral dosage forms are widely used and accepted in pediatric patients. However, oral dosage forms have their own challenges, which include the requirement for dose flexibility, limited bioavailability due to the poor solubility of the drug, safety concerns regarding excipients, and drug administration challenges for pediatric patients. The importance of research in drug delivery of anticancer drugs has increased due to poor drug solubility and the limited availability of pharmacokinetic data in pediatric patients. The poor solubility of anticancer drugs necessitates higher doses, leading to increased episodes of adverse effects. Furthermore, administering these drugs in the fed state results in bioavailability variations compared to the fasting state, which becomes more critical for pediatric patients. Therefore, it is essential to screen formulation strategies for these drugs to improve their bioavailability in the early stages of development. This review outlines key concerns of pediatric patients receiving long-term cancer drug therapy, including physiological and pharmacological challenges in the pediatric population, the impact of food on drug efficacy, and potential adverse effects associated with it. It also highlights various formulation strategies aimed at mitigating food-related variability through bioavailability enhancement. Furthermore, the review addresses the safety levels of excipients and development of age-appropriate dosage forms for the pediatric population.

Polymeric excipients are generally used for physical stabilization of amorphous drugs. However, a more strategic design of amorphous formulations is needed to reduce the amount of excipients, for which the use of mesoporous materials can be a promising option. In this study, mesoporous silica (MS) with various particle and pore sizes were added to amorphous acetaminophen (AAP) at various mixing ratios to systematically clarify their stabilization mechanisms. In particular, how the presence of MS affects the stability, at mixing ratios where AAP is present in excess relative to the pore volume, was focused. Six types of MS, with pore and particle sizes ranging from 2.5 to 21 nm and from 3 to 9 μm, respectively, were used in the study. Differential scanning calorimetry (DSC) analysis suggested the presence of three molecular fractions of amorphous AAP with different molecular mobilities: free, intermediate, and rigid fractions. The free fraction behaved similarly to pure AAP, whereas the intermediate fraction appeared to be relatively more stable. The rigid fraction had limited molecular mobility and was expected to strongly interact with the MS surface. AAP molecules mainly existed as the free fraction in the presence of 10% MS, regardless of the MS type. The free fraction disappeared to allow domination by the intermediate fraction with 50% MS having 17 or 21 nm pores, whereas a significant amount of the free fraction remained with MS having 2.5 nm pores. Broadband dielectric spectroscopy (BDS) analysis revealed that the addition of MS both increased and decreased the molecular mobility of amorphous AAP. The presence of all types of 10% MS slightly accelerated the γ-relaxation of amorphous AAP without affecting the α-relaxation. The addition of 50% MS with 17 or 21 nm pores slowed down the a-relaxation time of amorphous AAP, whereas the effect was marginal for MS with 2.5 nm pores. Isothermal crystallization of amorphous AAP at 60 °C was accelerated in the presence of MS with 2.5 nm pores at mixing ratios of both 10 and 50%. In contrast, 50% MS with 17 or 21 nm pores exhibited a strong stabilization effect. The accelerated crystallization in the presence of MS with 2.5 nm pores was explained by an increase in local molecular mobility. Mixtures with MS with 17 or 21 nm pores exhibited higher stability, which was explained by the large portion of the intermediate fraction with slower motion relative to that of pure AAP. The intermediate fraction was assumed to move inside and outside the pores, and the ease of molecular exchange was explained by the size of the pores and characteristic length of the cooperatively rearranging region of amorphous AAP. These observations provide guidance for the selection of MS materials for the stabilization of large amounts of pharmaceutical glasses and the scientific significance of the molecular dynamics of glasses in the presence of porous materials.

In our effort to design an effective ultralow-dose attenuated whole sporozoite (SPZ) vaccine, we explored the use of chemical adjuvants to increase the immunogenicity of sporozoites. Critical to the potency of attenuated sporozoites is their ability to migrate to and infect the liver. We thus used an in vivo mouse challenge model to compare the effects of external and internal chemical modifications to sporozoites. We loaded sporozoites with CL307 using two strategies: a supramolecular surface modification adjuvant system (SPZ-SAS(CL307)) and a mitochondrial membrane potential system (SPZ-MMP(CL307)). SPZ-SAS(CL307) contained 200 times more CL307 as compared to SPZ-MMP(CL307). However, SPZ-SAS(CL307) could not establish a liver infection after intravenous injection (25 000 sporozoites), suggesting loss of function of the sporozoites. On the contrary, SPZ-MMP(CL307) could establish an equivalent liver infection to the control already at a dose of 5000 sporozoites. When the immunized mice were challenged, SPZ-MMP(CL307) equaled the protective efficacy of control sporozoites. Our findings indicate that the MMP system can be used to chemically augment live cells, such as sporozoites, with adjuvants without negatively affecting their infectivity and protective efficacy in vivo. For malaria, further testing of different types of adjuvants at different concentrations is needed to ultimately develop vaccines with increased potency. However, in essence, this platform provides a proof-of-concept for a Trojan horse adjuvanting or drug loading concept for any whole-cell vaccine or therapy, within the infectious diseases space or beyond in oncological applications.

The high expression of transferrin receptors on the surface of cancer cells makes them a potential target for drug internalization, which has become a golden opportunity in targeted cancer therapy. Carvacrol, a naturally occurring phenolic compound present in essential oils, has attracted considerable attention for its pronounced anticancer activity against diverse human malignancies. Nevertheless, its therapeutic application in vivo is constrained by intrinsic physicochemical and pharmacokinetic limitations, including poor water solubility, suboptimal bioavailability, and rapid systemic elimination. To address these challenges, the present study highlights the fabrication of transferrin-targeted TPGS-conjugated carvacrol (CVC)-loaded polydopamine nanoparticles (CVC-PDA-TPGS-Tf NPs), with exploration of the antitumor efficacy of nanocarrier-mediated carvacrol, deploying a murine triple-negative breast cancer (TNBC) tumor xenograft model and a human TNBC three-dimensional (3D) spheroid model, as 3D cell culture technologies more thoroughly bear a resemblance to in vivo cell environments to accommodate improved precision in drug discovery. This work demonstrates the successful creation of nanoconjugate CVC-PDA-TPGS-Tf NPs (∼112 nm in size) and their thorough physicochemical characterization after synthesis. Intraperitoneal administration of CVC-PDA-TPGS-Tf NPs substantially inhibited tumor growth in the 4T1 xenograft model. Carvacrol effectively disintegrates MDA-MB-231 spheroids, illustrating a proportionately higher uptake of carvacrol nanoconjugates by these spheroids. The molecular docking studies also pinpointed the interaction of potential cancer biotargets with carvacrol in estrogen- and progesterone-receptor-positive breast cancer cells, in addition to its mentioned therapeutic efficiency against TNBC. In conclusion, these findings support CVC-PDA-TPGS-Tf as a promising platform for TNBC and potentially hormone receptor-positive breast cancers, warranting further mechanistic and translational studies.

Noninvasive detection of microglia is crucial for monitoring their spatiotemporal dynamics and facilitating early medical intervention during the prodromal stages of these neuroinflammatory diseases. Colony-stimulating factor 1 receptor (CSF1R) is a promising imaging biomarker for microglia in neuroinflammation. However, currently developed CSF1R-targeted probes lack [18F]-labeled variants, and their stability, sensitivity, and specificity are all suboptimal to some extent. In our study, based on pyrrolopyrimidines(a distinct structure of CSF1R inhibitors), we developed the [18F]-labeled CSF1R-targeted probe-18F-FPPA (6-(4-(2-(2-(2-(fluoro-18F)ethoxy)ethoxy)ethoxy)phenyl)-N-methyl-N-(3-methylbenzyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine).18F-FPPA was synthesized with a radiochemical yield of 4-7%, molar activity of 30-70 GBq/μmol, and radiochemical purity exceeding 99%, which possesses excellent physicochemical properties, rapid blood pharmacokinetics (T1/2α = 2.50 min, T1/2β = 32.4 min), favorable invitro and in vivo stability, and a clear metabolic pathway via dual hepatobiliary and renal excretion. A marked uptake of 18F-FPPA was observed at the LPS injection site in the LPS group at 30-40 min postinjection. A decreased uptake also was observed in cold 19F-FPPA, BLZ945, and PLX3397 blocking groups (P < 0.05), and no obvious increase in the group of intraperitoneal injection of cyclosporine A (P > 0.05). The in vivo imaging and immunohistochemical results confirmed the specific uptake of 18F-FPPA by targeting CSF1R on microglia in inflamed tissues. Overall, our findings demonstrated that 18F-FPPA had great potential as a promising tracer for the imaging of neuroinflammatory diseases, while its unique binding profile also provides insights into CSF1R-targeted ligand development.

The use of natural killer (NK) cells for the treatment of solid tumors such as cholangiocarcinoma has been hindered by challenges related to cell production, persistence and trafficking, as well as by reduced efficacy within an immunosuppressive tumor microenvironment. Antitumor immunity can be reduced by the interaction of NK cell expressed programmed cell death protein 1 (PD1) with ligands on tumor and other cells within the tumor microenvironment. The antitumor effect of NK cells is enhanced in cells engineered to express a truncated PD1 without an intracellular domain. The cytolytic potential and therapeutic efficacy of extracellular vesicles (EVs) derived from human NK cells expressing truncated PD1 was evaluated as a cell-free therapeutic to overcome some limitations faced by cell therapies. We demonstrate the feasibility of efficient expansion, production and isolation of therapeutic vesicles from human NK cell lines and from engineered NK92 cells expressing truncated PD1. The cytotoxic efficacy of these therapeutic vesicles was validated in both monolayer tumor cell cultures and in multicellular tumor spheroids. Concomitant administration of gemcitabine upregulated NKG2D ligands on tumor cells and increased susceptibility to NK cell and to NK cell derived EV mediated killing. These findings position engineered NK cell derived-EVs as a scalable and promising cell-free immunotherapeutic for cholangiocarcinoma.

Nectin-4-targeted imaging has emerged as a promising strategy for patient stratification in urothelial carcinoma, yet currently available radiotracers remain limited by rapid renal clearance, urinary background activity, or potential pharmacologic confounding associated with antibody-drug conjugate (ADC)-based constructs. To overcome some of these limitations, we developed a payload-free immunoPET radiotracer based on the native enfortumab antibody conjugated with the bifunctional chelator DFO*-sq and radiolabeled with zirconium-89 (89Zr). The resulting [89Zr]Zr-DFO*-sq-Enfortumab preserved Nectin-4-specific targeting while demonstrating high radiochemical stability in human serum (>99% through Day 12). In vitro studies showed efficient antigen-specific cellular internalization (80-85%), prolonged intracellular retention, and evidence consistent with partial recycling of antibody-associated species. In vivo, [89Zr]Zr-DFO*-sq-Enfortumab exhibited sustained tumor uptake reaching 17.4 ± 2.8% ID/g in high Nectin-4-expressing xenografts and correlated with differential Nectin-4 expression across multiple bladder cancer models. Importantly, the full-length antibody platform enabled high-contrast visualization of pulmonary metastatic lesions and orthotopic bladder tumors despite some urinary background activity. Together, these findings demonstrate that payload-free [89Zr]Zr-DFO*-sq-Enfortumab provides a biologically relevant immunoPET platform for noninvasive assessment of variable Nectin-4 expression and support its further evaluation as a companion imaging strategy for Enfortumab Vedotin-based therapies.

After intravenous administration, some heptamethine cyanine dyes bind to serum albumin and accumulate in tumors via the enhanced permeability and retention effect. Additionally, asymmetric cyanine dyes have two indole moieties which allow conjugation of a low-molecular-weight anticancer agent to one of the N-substituent side chains. Therefore, asymmetric heptamethine cyanine dyes have been studied as possible platforms for tumor-targeted drug delivery. However, the effects of functional groups on the opposite side chains on their in vivo behavior have not been explored in detail. Therefore, in this study, we aimed to clarify these effects by designing three model asymmetric cyanine dyes. For each dye, the side chain opposite the drug-conjugation site was modified with either an alkyl chain, a carboxyl group, or a sulfonyl group. A 125I atom was introduced on the other side of the molecule to enable quantitative biodistribution analysis. The resulting compounds are referred to as [125I]Bu, [125I]COOH, and [125I]SO3H. The albumin binding affinities (Kb values) for the corresponding nonradioactive compounds, Bu, COOH, and SO3H, were 0.82, 1.17, and 1.18 × 106 L/mol, respectively. In biodistribution studies, [125I]COOH showed higher radioactivity in the blood and tumors than [125I]Bu, whereas [125I]SO3H showed lower radioactivity levels. Notably, these differences did not correlate with their Kb values. Fluorescence analyses of the compounds incubated in serum-containing medium demonstrated that the fluorescence of COOH and Bu plateaued after approximately 1 and 2 h, respectively, whereas that of SO3H continued to change beyond 3 h, consistent with a slower time-dependent change in the protein-associated state of SO3H. Moreover, longer preincubation times of the radiolabeled asymmetric cyanine dyes in serum-containing medium decreased their transfer from the medium to the cellular side, with the greatest decrease observed for [125I]COOH. These results suggest that functional groups affect albumin-binding affinity, serum protein-associated states, and subsequent transfer to the cellular side, thereby influencing blood retention and tumor accumulation. Our findings provide a basis for the rational optimization of asymmetric cyanine dyes by modulating albumin interactions that influence their in vivo behavior.

Human serum albumin (HSA) plays a key role in regulating the transport and bioavailability of therapeutic molecules, making protein-ligand interaction studies essential for assessing biomedical compatibility. In this work, the interaction of cotarnine and its derivatives with HSA was systematically investigated using spectroscopic, thermodynamic, and molecular docking approaches, along with evaluation of antibacterial, wound healing, and hemostatic activities. Spectroscopic analyses confirmed efficient binding of the derivatives to HSA without disrupting its native secondary structure, while thermodynamic studies revealed spontaneous, enthalpy-driven interactions dominated by noncovalent forces. Docking studies identified favorable binding orientations within HSA binding pockets, complementing the experimental results. All derivatives of cotarnine show greater binding and biological activity, such as wound healing and hemostatic, than cotarnine. Among the derivatives, L2 exhibited the strongest binding affinity and superior biological performance, which can be attributed to enhanced hydrophobic interactions arising from its chloro-substituted aromatic moiety. Overall, the combined biophysical, computational, and biological findings highlight cotarnine derivatives, particularly L2, as promising multifunctional candidates for wound-healing and related biomedical applications.

Gastrin-releasing peptide receptor (GRPR) shows elevated expression in several malignancies and represents an attractive target in cancer imaging and radioligand therapy. However, improving in vivo stability without compromising high tumor uptake and low pancreatic uptake remains a key challenge for the development of GRPR-targeted radiopharmaceuticals. In the present study, we investigated whether incorporating unnatural amino acid substitutions at potential cleavage sites of the previously reported GRPR-targeted agonist [68Ga]Ga-SP01011 (Ga-DOTA-Pip-d-Phe-Gln7-Trp8-Ala9-Val10-Gly11-His12-Leu13-Ac6c14-NH2; Ac6c: 1-amino-1-cyclohexanecarboxylic acid) could enhance in vivo stability without compromising tumor-targeting capability. SP01012, SP01013, and SP01019 incorporating NMe-His12, Ala(tBu)13 and Tle10/NMe-His12 substitutions, respectively, were synthesized by Fmoc solid-phase peptide synthesis and obtained in 22-60% yield. Their corresponding nonradioactive Ga complexes were obtained in 58-89% yield by reacting the DOTA-conjugated precursors with excess GaCl3 in acetate buffer, and the products were subsequently purified by HPLC. In vitro competition binding assays demonstrated that the Ki values of Ga-SP01012, Ga-SP01013 and Ga-SP01019 were 1.36 ± 0.12, 11.5 ± 0.89 and 2.29 ± 0.77 nM, respectively. All three ligands retained agonistic characteristics as confirmed by the calcium release assay. Their 68Ga-labeled analogs were obtained by reacting the precursors with [68Ga]GaCl3 in HEPES buffer with microwave heating (100 °C, 1 min). Following HPLC purification, 68Ga-labeled tracers were obtained in 33-47% decay-corrected radiochemical yield with radiochemical purities >95%. PET/CT imaging together with ex vivo biodistribution analyses conducted at 1 h postinjection in PC-3 tumor-bearing mice demonstrated clear tumor visualization and predominantly renal excretion for all three tracers. Among them, [68Ga]Ga-SP01012 containing the NMe-His12 substitution alone, had the most favorable profile, with the highest tumor uptake (15.8 ± 0.65% ID/g), low pancreas uptake (1.27 ± 0.32% ID/g), and improved in vivo plasma stability (78.1 ± 0.83% intact tracer at 15 min postinjection compared to 63.5 ± 6.24% for [68Ga]Ga-SP01011). In contrast, [68Ga]Ga-SP01019, containing both Tle10 and NMe-His12 substitutions, showed the highest plasma stability (88.3 ± 2.90% intact tracer at 15 min postinjection), but this was accompanied by lower tumor uptake (12.4 ± 1.54% ID/g) and markedly increased pancreas uptake (23.7 ± 3.47% ID/g). [68Ga]Ga-SP01013, containing the Ala(tBu)13 substitution, showed reduced GRPR binding affinity and lower tumor uptake (8.33 ± 1.41% ID/g). Our findings indicate that [68Ga]Ga-SP01012 is a potential PET tracer for visualizing GRPR-expressing tumors, and the NMe-His12 substitution is a favorable modification for the design of GRPR-targeted agonist-based radiopharmaceuticals.

Synthetic trehalose glycolipids (TGLs) that modulate the Macrophage Inducible C-Type Lectin receptor (Mincle) are attractive targets for adjuvant development. However, natural ligands such as trehalose dimycolate (TDM), as well as existing synthetic mimetics, are limited by poor physicochemical properties and reactogenicity, motivating the development of rationally designed synthetic TGLs. Herein, we investigated the biophysical properties of amphiphilic TGLs featuring hydrophobic, aromatic moieties attached to the polar trehalose headgroup via ester, amide, or triazole linkages. The formation of homogeneous and colloidally stable aqueous nanodispersions was readily achieved through focused ultrasonication, eliminating the need for cosolvents and/or surfactants that might interfere with the self-assembling and biological properties of these compounds. These self-assembled particles were characterized using DLS, cryo-TEM, and synchrotron small-angle X-ray scattering to gain insight into their particle morphology and liquid crystalline phase behavior. Additionally, molecular dynamics (MD) simulations were conducted to evaluate the critical packing parameter (CPP), a predictive descriptor of the supramolecular structures formed by self-assembling amphiphiles, under conditions that closely mimic experimental environments. The synthetic Mincle ligands formed lyotropic liquid crystals, including cubosomal or hexagonal phases as well as lamellar phases. In vitro and in vivo testing of these self-assemblies revealed that all tested TGLs exhibited measurable immunological activity. Analogues forming cubic phases were associated with comparatively stronger cytokine production and enhanced antigen-specific humoral and cell-mediated immune responses within this compound set. Taken together, the data support a model in which both molecular structure and supramolecular organization contribute to immunostimulatory activity, although these effects cannot be fully decoupled in the present study. Additionally, we developed an in silico model capable of predicting supramolecular assembly, providing insight into the self-assembly tendencies of trehalose-based amphiphiles, and offering broader relevance to other biologically active, self-assembling systems.

The present study demonstrates the integration of the linear solvation energy relationship (LSER) concept with machine learning (ML) methodologies to improve the predictive and interpretative capabilities of chromatographic retention modeling. Immobilized artificial membrane (IAM) chromatography was employed as a model biochromatographic system, and an in-house library of 993 structurally diverse compounds, with experimentally determined chromatographic hydrophobicity index of IAM (CHIIAM), was used to train LSER-ML models. LSER descriptors were calculated using Absolv and extended with ionization-state descriptors to evaluate the applicability of the LSER framework for realistic in silico virtual screening scenarios. Several regression algorithms were tested, including linear, neighborhood-based, kernel-based, and ensemble tree-based models. Among them, the support vector regression with the radial basis function kernel (SVR-RBF) demonstrated the most balanced performance across all validation metrics of R2train = 0.884, R2test = 0.853, and Q2cv = 0.811, achieving predictive errors (RMSEtrain = 5.546, RMSEtest = 4.609, and RMSEcv = 6.989) close to the analytical uncertainty. Model interpretability was achieved using SHapley Additive exPlanations (SHAP), which confirmed the mechanistic relevance of the Abraham descriptors and the dominant contribution of hydrophobic volume and hydrogen-bonding properties to IAM retention. Applicability domain was verified with a Williams plot (±3 standardized residuals and leverage threshold h*). The results indicate that the proposed LSER-ML approach provides an interpretable, robust, and generalizable tool for modeling membrane-mimetic chromatographic systems and can be effectively applied in virtual screening and property-based molecular design.

Polyethylene glycol (PEG) is widely utilized in pharmaceuticals and cosmetics as a safe and less- or nonimmunogenic synthesized biocompatible polymer. However, the increasing detection of pre-existing anti-PEG antibodies in untreated individuals has raised concerns that daily exposure to PEG derivatives through cosmetics could induce immune responses against PEG. We have shown that daily topical applications of this type of cosmetic product cause anti-PEG IgM production in normal mice, and the efficacy of PEGylated therapeutics was suppressed by this induced anti-PEG IgM. In this study, the mechanism underlying anti-PEG IgM production caused by topical applications of a cosmetic product containing PEG derivatives, which are yet to be identified, was investigated in mice. Daily applications of such a cosmetic product to the compromised skin of mice elicited anti-PEG IgM production. Splenectomy, performed before the topical applications, markedly suppressed the anti-PEG response. In addition, when splenectomy was carried out after 14 days of daily topical applications, there was a remarkable decline in the anti-PEG IgM response that had already been induced. In this study, however, splenectomy completely diminished neither anti-PEG IgM production nor pre-existing anti-PEG IgM titers. Although immunodeficient C.B-17/Icr-scid/scid (SCID) mice caused no anti-PEG IgM production, the adoptive transfer of splenocytes obtained from normal mice to SCID mice restored the anti-PEG response to daily topical applications of this cosmetic product. Furthermore, anti-PEG IgM production was observed in T cell-deficient BALB/cAJcl-nu/nu (BALB/c-nu) mice. In the lymph node-deficient ALY/NscJcl-aly/aly (aly/aly) mice, daily topical applications of this cosmetic product also caused anti-PEG IgM production, but the titer was lower than that in normal mice. The results of this study show the spleen to be the primary contributor, while lymph nodes play a partial role, in the production of anti-PEG IgM via daily topical applications of this cosmetic product in a T cell-independent manner in mice. This study provides mechanistic insight into how daily topical exposure to PEG could lead to the formation of pre-existing anti-PEG IgM, which in turn would alter the pharmacokinetics and therapeutic efficacy of PEGylated therapeutics.