Labeling of DOTA-conjugated HPMA-based polymers with trivalent metallic radionuclides for molecular imaging

Background In this work, the in vitro and in vivo stabilities and the pharmacology of HPMA-made homopolymers were studied by means of radiometal-labeled derivatives. Aiming to identify the fewer amount and the optimal DOTA-linker structure that provides quantitative labeling yields, diverse DOTA-linker systems were conjugated in different amounts to HPMA homopolymers to coordinate trivalent radiometals Me(III)* = gallium-68, scandium-44, and lutetium-177. Results Short linkers and as low as 1.6% DOTA were enough to obtain labeling yields > 90%. Alkoxy linkers generally exhibited lower labeling yields than alkane analogues despite of similar chain length and DOTA incorporation rate. High stability of the radiolabel in all examined solutions was observed for all conjugates. Labeling with scandium-44 allowed for in vivo PET imaging and ex vivo measurements of organ distribution for up to 24 h. Conclusions This study confirms the principle applicability of DOTA-HPMA conjugates for labeling with different trivalent metallic radionuclides allowing for diagnosis and therapy

N1-acetylspermidine is a determinant of hair follicle stem cell fate

Stem cell differentiation is accompanied by increased mRNA translation. The rate of protein biosynthesis is influenced by the polyamines putrescine, spermidine and spermine, which are essential for cell growth and stem cell maintenance. However, the role of polyamines as endogenous effectors of stem cell fate and whether they act through translational control remains obscure. Here, we investigate the function of polyamines in stem cell fate decisions using hair follicle stem cell (HFSC) organoids. Compared to progenitor cells, HFSCs showed lower translation rates, correlating with reduced polyamine levels. Surprisingly, overall polyamine depletion decreased translation but did not affect cell fate. In contrast, specific depletion of natural polyamines mediated by spermidine/spermine N1-acetyltransferase (SSAT; also known as SAT1) activation did not reduce translation but enhanced stemness. These results suggest a translation-independent role of polyamines in cell fate regulation. Indeed, we identified N1-acetylspermidine as a determinant of cell fate that acted through increasing self-renewal, and observed elevated N1-acetylspermidine levels upon depilation-mediated HFSC proliferation and differentiation in vivo. Overall, this study delineates the diverse routes of polyamine metabolism-mediated regulation of stem cell fate decisions. This article has an associated First Person interview with the first author of the paper.Peer reviewe

Metabolic control of hair follicle stem cell fate decisions

Aging is the main risk factor for the manifestation of numerous serious diseases, including cancer, cardiovascular diseases and neurodegeneration. Thus, the growing proportion of elderly people in the population poses a major challenge to the health care systems worldwide. Tissue homeostasis declines with age and its maintenance late in life has been intimately linked to prolonged healthspan. Stem cells act as drivers of tissue homeostasis through the interplay of self-renewal and differentiation. Therefore, it is of utmost importance to identify stem cell fate determinants to be able to delay stem cell exhaustion in the elderly. Of note, metabolic changes have been described to influence function and maintenance of stem cell populations. However, whether manipulation of metabolism affects cell fate decisions and stem cell maintenance remains poorly understood. Here, I focus on three metabolic pathways that have been described to become dysregulated with age, and to affect stemness: the hexosamine pathway (HP), aerobic sugar metabolism, and the polyamine metabolism. I assess their influence on cell fate decisions in an in vitro organoid culture system, which allows for the manipulation of hair follicle stem cells (HFSCs) and their direct progeny. First, I activate the HP by GlcNAc supplementation and through genetic manipulation of the rate-limiting enzyme GFAT1. While pathway flux and downstream metabolites increase, HFSC fate is not affected by these interventions. Second, I investigate the effect of sugar supplementation on cell fate decisions. Both, D-glucose and D-mannose, enhance stemness. While D-glucose supplementation boosts glycolysis, D-mannose addition specifically increases the acetylated polyamines N1-acetylspermidine (N1-AcSpd) and N1-acetylspermine without elevating glycolytic flux. Third, I elucidate how polyamine levels influence cell fate decisions. I confirm that low translation rates mark the stem cell state and that a forced decrease in translation is sufficient to elevate stemness. Surprisingly, I demonstrate that reducing translation by changes in polyamine availability does not correlate with increased stemness in the organoids. I identify N1-AcSpd as novel regulator of HFSC fate decisions, accelerating cell cycle progression. Finally, I demonstrate that HFSC activation by depilation results in an elevation of the acetylated polyamines, suggesting a functional role of N1-AcSpd in cell fate decisions in vivo. Overall, my results suggest that manipulation of metabolism is an effective means to control cell fate decisions, delay stem cell exhaustion, and improve tissue homeostasis in the elderly

From defined polymer architectures to structure-property relationships in vivo

Makromolekulare Wirkstoffträgersysteme sind von starkem Interesse bezüglich der klinischen Anwendung chemotherapeutischer Agenzien. Um ihr klinisches Potential zu untersuchen ist es von besonderer Bedeutung das pharmakokinetische Profil in vivo zu bestimmen. Jede Veränderung der Polymerstruktur beeinflusst die Körperverteilung des entsprechenden Makromoleküls. Aufgrund dessen benötigt man detailliertes Wissen über Struktur-Eigenschaftsbeziehungen im lebenden Organismus, um das Nanocarrier System für zukünftige Anwendungen einzustellen. In dieser Beziehung stellt das präklinische Screening mittels radioaktiver Markierung und Positronen-Emissions-Tomographie eine nützliche Methode für schnelle sowie quantitative Beobachtung von Wirkstoffträgerkandidaten dar. Insbesondere poly(HPMA) und PEG sind im Arbeitsgebiet Polymer-basierter Therapeutika stark verbreitet und von ihnen abgeleitete Strukturen könnten neue Generationen in diesem Forschungsbereich bieten.rnDie vorliegende Arbeit beschreibt die erfolgreiche Synthese verschiedener HPMA und PEG basierter Polymer-Architekturen – Homopolymere, Statistische und Block copolymere – die mittels RAFT und Reaktivesterchemie durchgeführt wurde. Des Weiteren wurden die genannten Polymere mit Fluor-18 und Iod-131 radioaktiv markiert und mit Hilfe von microPET und ex vivo Biodistributionsstudien in tumortragenden Ratten biologisch evaluiert. Die Variation in Polymer-Architektur und darauffolgende Analyse in vivo resultierte in wichtige Schlussfolgerungen. Das hydrophile / lipophile Gleichgewicht hatte einen bedeutenden Einfluss auf das pharmakokinetische Profil, mit besten in vivo Eigenschaften (geringe Aufnahme in Leber und Milz sowie verlängerte Blutzirkulationszeit) für statistische HPMA-LMA copolymere mit steigendem hydrophoben Anteil. Außerdem zeigten Langzeitstudien mit Iod-131 eine verstärkte Retention von hochmolekularen, HPMA basierten statistischen Copolymeren im Tumorgewebe. Diese Beobachtung bestätigte den bekannten EPR-Effekt. Hinzukommend stellen Überstrukturbildung und damit Polymergröße Schlüsselfaktoren für effizientes Tumor-Targeting dar, da Polymerstrukturen über 200 nm in Durchmesser schnell vom MPS erkannt und vom Blutkreislauf eliminiert werden. Aufgrund dessen wurden die hier synthetisierten HPMA Block copolymere mit PEG Seitengruppen chemisch modifiziert, um eine Verminderung in Größe sowie eine Reduktion in Blutausscheidung zu induzieren. Dieser Ansatz führte zu einer erhöhten Tumoranreicherung im Walker 256 Karzinom Modell. Generell wird die Körperverteilung von HPMA und PEG basierten Polymeren stark durch die Polymer-Architektur sowie das Molekulargewicht beeinflusst. Außerdem hängt ihre Effizienz hinsichtlich Tumorbehandlung deutlich von den individuellen Charakteristika des einzelnen Tumors ab. Aufgrund dieser Beobachtungen betont die hier vorgestellte Dissertation die Notwendigkeit einer detaillierten Polymer-Charakterisierung, kombiniert mit präklinischem Screening, um polymere Wirkstoffträgersysteme für individualisierte Patienten-Therapie in der Zukunft maßzuschneidern.rnMacromolecular based drug delivery systems are of emerging interest regarding the clinical administration of (chemo) therapeutic agents. In order to investigate their clinical potential, it is crucial to determine the pharmacokinetic profile in vivo. Each alteration in polymer structure is influencing the body distribution of the respective macromolecule and thus deepening knowledge about structure-property relationships in the living organism is needed to adjust the nanocarrier system for future applications. In this regard, pre-clinical screening via radiolabeling combined with Positron Emission Tomography (PET) constitutes a useful tool for fast and quantitative monitoring of drug delivery candidates in vivo. Particularly p(HPMA) and PEG are widely exploited in the field of polymer based therapeutics and thus derivatized structures might provide new generations in this research area.rnThis work describes the successful synthesis of diverse HPMA and PEG based polymer architectures - including homopolymers, random and block copolymers – accomplished via RAFT polymerization and reactive ester chemistry. Furthermore, their radioactively labeling and biological evaluation was carried out by means of 18F and 131I combined with µPET imaging and ex vivo biodistribution in tumor bearing rats. The variation in polymer architecture and subsequent analysis in vivo resulted in some major conclusions. First, the hydrophilic / lipophilic balance had a major influence on the pharmacokinetic profile, demonstrating most favorable in vivo characteristics (low hepatic and spleenic uptake as well as prolonged blood circulation times) for HPMA-ran-LMA copolymers with increasing content of hydrophobic moiety. Second, long term biodistribution studies with iodine-131 demonstrated enhanced retention in the tumor tissue for high molecular weight HPMA based random copolymers thus proofing the well-known EPR-effect. Third, superstructure formation and hence polymer size are key factors for efficient tumor targeting since polymer structures above 200 nm in diameter are rapidly recognized by the MPS and cleared from the bloodstream. In this regard, the herein synthesized HPMA block copolymers were chemically modified with PEG side chains to induce a decrease in size as well as reduced blood clearance, yielding enhanced tumor accumulation in the Walker 256 mammary carcinoma model. Forth, the overall polymer architecture as well as molecular weight are strongly influencing the body distribution of HPMA / PEG based polymers and their efficacy of tumor treatment is significantly depending on the properties of each individual tumor. Due to this observation, the present work is underlining the necessity of a precise polymer characterization in combination with pre-clinical screening to tailor polymeric carrier systems for individualized patients’ (chemo) therapy in the future.r

The RATIOnal Role of Polyamines in Epidermal Differentiation

Polyamines have been implicated in skin tumorigenesis; however, their role in epidermal homeostasis remains obscure. In a new article in the Journal of Investigative Dermatology, Rahim et al. (2021) report that keratinocyte differentiation requires a shift in polyamine ratios that is mediated by AMD1. Results suggest that targeting polyamine availability might be useful in the treatment of hyperproliferative skin disorders