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Nucleic acid therapeutics are used for silencing, expressing or editing genes in vivo. However, their systemic stability and targeted delivery to bone marrow resident cells remains a challenge. In this study we present a nanotechnology platform based on natural lipoproteins, designed for delivering small interfering RNA (siRNA), antisense oligonucleotides and messenger RNA to myeloid cells and haematopoietic stem and progenitor cells in the bone marrow. We developed a prototype apolipoprotein nanoparticle (aNP) that stably incorporates siRNA into its core. We then created a comprehensive library of aNP formulations and extensively characterized their physicochemical properties and in vitro performance. From this library, we selected eight representative aNP-siRNA formulations and evaluated their ability to silence lysosomal-associated membrane protein 1 (Lamp1) expression in immune cell subsets in mice after intravenous administration. Using the most effective aNP identified from the screening process, we tested the platform's potential for therapeutic gene silencing in a syngeneic murine tumour model. We also demonstrated the aNP platform's suitability for splice-switching with antisense oligonucleotides and for protein production with messenger RNA by myeloid progenitor cells in the bone marrow. Our data indicate that the aNP platform holds translational potential for delivering various types of nucleic acid therapeutics to myeloid cells and their progenitors., Competing Interests: Competing interests: M.G.N. and W.J.M.M. are scientific co-founders of and have equity in Trained Therapeutix Discovery. W.J.M.M. is CSO of Trained Therapeutix Discovery. M.G.N. and W.J.M.M. are scientific co-founders of and have equity in BioTrip. S.R.J.H., T.A., R.Z., H.M.J., P.M.F., W.J.M.M., E.K., and R.v.d.M. are listed as inventors on patent application WO2022268913A1 related to this manuscript. The other authors declare no competing interests., (© 2025. The Author(s).)

Industrial and household products, such as paints, inks and cosmetics usually consist of mixtures of macromolecules that are disperse in composition, in size and in monomer sequence. Identifying structure-function relationships for these systems is complicated, as particular macromolecular components cannot be investigated individually. For this study, we have addressed this issue, and have synthesized a series of five sequence-defined polyurethanes (PUs): one neutral-hydrophobic, one single-charged hydrophilic, one single-charged hydrophobic and two double-charged amphiphilic PUs (one symmetric and one asymmetric). These novel precision PUs - that were prepared by using stepwise coupling-deprotection synthetic protocols - have a defined composition, size and monomer sequence, where the chosen sequences were inspired by those that are abundantly formed in the production of industrial waterborne PU dispersions. By performing dynamic light scattering experiments (DLS), self-consistent field (SCF) computations and cryogenic transmission electron microscopy (cryo-TEM), we have elucidated the behavior in aqueous solution of the individual precision PUs, as well as of binary and ternary mixtures of the PU sequences. The double-charged PU sequences ('hosts') were sufficiently amphiphilic to yield single-component micellar solutions, whereas the two more hydrophobic sequences did not micellize on their own, and gave precipitates or ill-defined larger aggregates. Both the neutral-hydrophobic PU and the hydrophilic single-charged PU were successfully incorporated in the host micelles as guests, respectively increasing and reducing the micelle radius upon incorporation. SCF computations indicated that double-charged symmetric PUs stretch whilst double-charged asymmetric PUs are expelled from the core to accommodate hydrophobic PU guests within the micelles. For the ternary mixture of the double-charged symmetric and asymmetric hosts and the neutral-hydrophobic guest we have found an improved colloidal stability, as compared to those for binary mixtures of either host and hydrophobic guest. In another ternary mixture of precision PUs, with all three components not capable of forming micelles on their own, we see that the ensemble of molecules produces stable micellar solutions. Taken together, we find that the interplay between PU-molecules in aqueous dispersions promotes the formation of stable micellar hydrocolloids., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

While the impact of compositional parameters such as block length and ionic content on the micellization of (polymeric) amphiphiles is widely investigated, the influence of monomer sequence has received far less attention until recently. Here, we report the synthesis of two sequence-controlled polyurethane ionomers (PUIs) prepared via a stepwise coupling-deprotection strategy, and compare their solution association in aqueous-organic mixtures. The two PUIs are highly similar in mass and overall composition, yet differ markedly in the sequence of building blocks. PUI-A2 comprises a polytetrahydrofuran (pTHF) block connected to an alternation of isophorone diamine (IPDA) and dimethylolpropionic acid (DMPA) units that together are also arranged in a blockwise manner. The result is a macromolecular structure with a comparatively hydrophobic tail (pTHF) and a hydrophilic headgroup, which structure is reminiscent of those of traditional surfactants, albeit much larger in size. PUI-S2 instead resembles a bolaamphiphilic architecture with a pTHF midblock connected on either end to a singly charged segment comprising DMPA and IPDA. We detect micellization below a threshold cosolvent volume fraction (φ solv ) of 0.4 in aqueous-organic mixtures with tetrahydrofuran (THF), ethanol, and isopropyl alcohol. We use scattering tools to compare the aggregation number ( N agg ) and hydrodynamic radius ( R h ) of PUI-S2 and PUI-A2 micelles. Irrespective of the solvent composition, we observe in the micellar window of φ solv < 0.4, lower N agg for PUI-S2 micelles compared to PUI-A2 , which we attribute to packing restraints associated with its bolaamphiphilic architecture. The increase in micellar size with increasing φ solv is much more pronounced for PUI-S2 than for PUI-A2 . The micellar mass decreases with increasing φ solv for both PUIs; the effect is modest for PUI-S2 compared to PUI-A2 and is not observed in the most apolar cosolvent studied (THF). Upon the approach of the micellization boundary φ solv ≈ 0.4, both types of PUI micelles become less compact in structure, as (in most cases) PUIs are released and as micellar dimensions increase., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

The core of micelles self-assembled from amphiphiles is hydrophobic and contains little water, whereas complex coacervate core micelles co-assembled from oppositely charged hydrophilic polymers have a hydrophilic core with a high water content. Co-assembly of ionic surfactants with ionic-neutral copolymers yields surfactant-copolymer complexes known to be capable of solubilizing both hydrophilic and hydrophobic cargo within the mixed core composed of a coacervate phase with polyelectrolyte-decorated surfactant micelles. Here we formed such complexes from asymmetric ( PUI-A2 ) and symmetric ( PUI-S2 ), sequence-controlled polyurethane ionomers and poly( N -methyl-2-vinylpyridinium iodide) 29 - b -poly(ethylene oxide) 204 copolymers. The complexes with PUI-S2 were 1.3-fold larger in mass and 1.8-fold larger in radius of gyration than the PUI-A2 complexes. Small-angle X-ray scattering revealed differences in the packing of the similarly sized PUI micelles within the core of the complexes. The PUI-A2 micelles were arranged in a more ordered fashion and were spaced further apart from each other (10 nm vs. 6 nm) than the PUI-S2 micelles. Hence, this work shows that the monomer sequence of amphiphiles can be varied to alter the internal structure of surfactant-copolymer complexes. Since the structure of the micellar core may affect both the cargo loading and release, our findings suggest that these properties may be tuned through control of the monomer sequence of the micellar constituents.

Background: Agonist positron emission tomography (PET) tracers for dopamine D2/3 receptors (D2/3Rs) offer greater sensitivity to changes in endogenous dopamine levels than D2/3R antagonist tracers. D2/3R agonist tracers currently available for clinical research are labeled with the short-lived isotope carbon-11, which limits their use. We aimed to develop high-affinity D2R agonists amenable for labeling with the longer-living fluorine-18. Here, we report the evaluation as potential PET tracers of two homologous series of [(18)F]fluorinated tracers based on the 2-aminomethylchroman-7-ol (AMC) scaffold: (R)-2-((4-(2-fluoroalkoxy)benzylamino)methyl)chroman-7-ols (AMC13 homologues) and (R)-2-((2-(4-(4-(fluoroalkoxy)phenyl)piperazin-1-yl)ethylamino)methyl)chroman-7-ols (AMC15 homologues). We varied the length of the (18)F-fluoroalkyl chain in these structures to balance brain penetration and non-specific binding of the radioligands by adjusting their lipophilicity., Methods: The tracers were evaluated in brain slices of Sprague-Dawley rats by in vitro autoradiography and in living rats by microPET imaging and ex vivo autoradiography. PET data were analyzed with one- and two-tissue compartmental models (1TCM/2TCM), simplified reference tissue model (SRTM), and Logan graphical analysis. Specificity of binding was tested by blocking D2/3R with raclopride., Results: Homologues with a shorter fluoroalkyl chain consistently showed greater D2/3R-specific-to-total binding ratios in the striatum than those with longer chains. The fluoroethoxy homologue of AMC13 ([(18)F]FEt-AMC13) demonstrated the highest degree of D2/3R-specific binding among the evaluated tracers: mean striatum-to-cerebellum uptake ratio reached 4.4 in vitro and 2.1/2.8 in vivo/ex vivo (PET/autoradiography). Striatal binding potential (BPND) relative to cerebellum was 0.51-0.63 depending on the estimation method. Radiometabolites of [(18)F]FEt-AMC13 did not enter the brain. In vitro, application of 10 μmol/L raclopride reduced D2/3R-specific binding of [(18)F]FEt-AMC13 in the striatum by 81 %. In vivo, pre-treatment with 1 mg/kg (2.9 μmol/kg) raclopride led to 17-39 % decrease in D2/3R-specific binding in the striatum., Conclusions: Varying the length of the [(18)F]fluoroalkyl chain helped improve the characteristics of the original candidate tracers. Further modifications of the current lead [(18)F]FEt-AMC13 can provide an agonist radiopharmaceutical suitable for D2/3R imaging by PET.

Unlabelled: Dopamine D(2/3) receptor (D(2/3)R) agonist PET tracers are better suited for the imaging of synaptic dopaminergic neurotransmission than D(2/3)R antagonists and may also offer the opportunity to study in vivo the high-affinity state of D(2/3)R (D(2/3)RHigh). With the aim to develop (18)F-labeled D2/3R agonists suitable for widespread clinical application, we report here on the synthesis and in vitro and in vivo evaluation of a D(2/3)R agonist ligand from the aminomethyl chromane (AMC) class-(R)-2-[(4-(18)F-fluorobenzylamino)methyl]chroman-7-ol ((18)F- AMC20: )., Methods: In vitro affinities of AMC20: toward dopaminergic receptor subtypes were measured in membrane homogenates prepared from HEK293 cells expressing human dopamine receptors. Agonism of AMC20: was assessed in the arrestin recruitment assay in Chinese hamster ovary-K(1) cells expressing the long isoform of D(2)R (D(2)RLong). D(2/3)R-specific binding of (18)F- AMC20: was evaluated in brain slices of Sprague-Dawley rats by in vitro autoradiography and in living rats by in vivo small-animal PET imaging and ex vivo autoradiography. PET data were analyzed with 1- and 2-tissue compartmental models, the simplified reference tissue model, and Logan graphical analysis. Specificity of binding was tested by blocking D(2/3)R with raclopride (coincubation with 10 μM in vitro, administration of 1.0 mg/kg in vivo)., Results: In membrane homogenates, AMC20: demonstrated picomolar affinity at D(2)RHigh (mean inhibition constant [K(i)] = 85 pM) and excellent selectivity against the low-affinity state of D(2)R (D(2)RLow) (mean K(i) = 84 nM, 988-fold selectivity) and D(1)-like receptors (mean K(i) = 5,062 nM at D1R). The efficacy of AMC20: was 90% of that of dopamine in the arrestin recruitment assay. Up to 70% of total binding of (18)F- AMC20: in the D2/3R-rich striatum in rat brain slices was D(2/3)R-specific; in living rats, the uptake ratio between the striatum and the D(2/3)R-poor cerebellum reached 2.0-2.5, depending on the measurement method. Radiometabolites of (18)F- AMC20: did not enter the brain. Striatal binding potential of (18)F- AMC20: varied between 0.49 and 0.59 depending on the estimation method. Pretreatment with 1 mg of raclopride per kilogram reduced the apparent specific binding of (18)F- AMC20: in the striatum., Conclusion: (18)F- AMC20: shows specific binding to D(2/3)R in the striatum of living rats. Further optimization of the chemical structure of (18)F- AMC20: can lead to (18)F-labeled D(2/3) agonist PET tracers more suitable for in vivo clinical application., (© 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Imaging of dopamine D2/3 receptors (D2/3R) can shed light on the nature of several neuropsychiatric disorders in which dysregulation of D2/3R signaling is involved. Agonist D2/3 tracers for PET/SPECT imaging are considered to be superior to antagonists because they are more sensitive to dopamine concentrations and may selectively label the high-affinity receptor state. Carbon-11-labeled D2/3R agonists have been developed, but these short-lived tracers can be used only in centers with a cyclotron. Here, we report the development of a series of novel D2R agonist compounds based on the 2-aminomethylchromane (AMC) scaffold that provides ample opportunities for the introduction of longer-lived [(18)F] or [(123)I]. Binding experiments showed that several AMC compounds have a high affinity and selectivity for D2/3R and act as agonists. Two fluorine-containing compounds were [(18)F]-labeled, and both displayed specific binding to striatal D2/3R in rat brain slices in vitro. These findings encourage further in vivo evaluations.

For imaging of dopamine D2/3 receptors, agonist tracers are favoured over antagonists because they are more sensitive to detection of dopamine release and because they may selectively label the high-affinity receptor state. We have developed novel D2/3 receptor selective agonists that can be radiolabelled with [(123)I], which label is advantageous over most other labels, such as carbon-11, as it has a longer half-life. Particularly, we considered (R) N-[7-hydroxychroman-2-yl]-methyl 4-iodobenzyl amine (compound 1) as an attractive candidate for development as it shows high binding affinity to D2/3 receptors in vitro, and here we report on the characterization of this first [(123)I]-labelled D2/3 receptor agonist radiopharmaceutical intended for SPECT imaging. The appropriate tin precursor for [(123)I]-1 was developed and was successfully radiolabelled with iodine-123 giving a moderate yield (30-35%) and a good purity (>95%) for [(123)I]-1. In biodistribution experiments in Wistar rats intravenous injection of [(123)I]-1 resulted in a fast brain uptake, where the observed binding in the D2/3 receptor-rich striatum was slightly higher than that in the cerebellum 30 min to 4 h p.i. Storage phosphor imaging experiments, however, did not show specific D2/3 receptor binding. In conclusion, despite promising in vitro data for 1, neither specific ex vivo binding nor high signal-to-noise ratios were found in rodents for [(123)I]-1.

The major bottleneck in gene therapy remains the issue of delivery. In this work, various modified poly(propylene imine) (PPI) dendrimers are introduced as gene transfection agents. Commercially available PPI-dendrimers have been modified (i) at the exterior primary amines with acetyl groups or glycol gallate (PEG-like) groups, and (ii) at the interior tertiary amines with methyl iodide (MeI) or MeCl to produce multiple quaternized cationic sites in the core of the dendrimer. The prepared materials have been tested with respect to their binding capabilities to DNA, their toxicity in cell cultures, their in vitro transfection efficiency and their in vivo delivery possibilities. In all cases, a 33-mer oligonucleotide (DNAzyme) was used. Polyacrylamide gel electrophoresis (PAGE) studies have demonstrated strong but reversible binding, where the quarternized and higher generation dendrimer species have shown more potent binding. Typically, for the modified fourth PPI-dendrimers, binding is observed at a concentration of about 4 microM DNA and a dendrimer-DNA charge ratio of around 2:1-1:1. All the tested PPI-dendrimers display a low cellular toxicity, especially when higher serum contents are used in the culture medium. For example, most of the prepared fourth generation PPI-dendrimers are not or hardly toxic up to at least 20 microM in 20% serum. An in vitro characterization has revealed a high dendrimer-mediated intracellular uptake of the DNAzyme: all the tested fourth generation PPI-dendrimers display transfection efficiencies close to or exceeding 80%, even when the concentration of serum in the medium is increased from 10 to 40%. Finally, the potential of using modified PPI-dendrimers for in vivo gene therapy experiments is demonstrated. Injecting a G4-PEG(MeI)-ssDNA complex intravenously into Nude mice has resulted in a high nuclear uptake as confirmed by co-localization studies.