Acrylamides with hydrolytically labile carbonate ester side chains as versatile building blocks for well-defined block copolymer micelles via RAFT polymerization

En route towards improved delivery systems for targeted chemotherapy, we propose a straightforward approach for the hydrophobic modification of the acrylamide N-(2-Hydroxyethyl) acrylamide (HEAm). An ethyl or benzyl group was introduced via a hydrolytically sensitive carbonate ester yielding HEAm-EC and HEAm-BC, respectively. Block copolymers of HEAm, respectively PEG and HEAm-EC or HEAm-BC were successfully synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization, obtaining a library of well-defined block copolymers with different degrees of polymerization (DP). To further explore the versatility of our approach in terms of polymer synthesis, self-assembly, drug solubilization and in vitro cell interaction, polyethylene glycol (PEG) and polyHEAm as hydrophilic polymer blocks were compared. The block copolymers formed micellar nanoparticles (10-100 nm) in PBS and could efficiently solubilize hydrophobic dyes and anti-cancer drugs. Benzyl carbonate ester side chains increased micellar stability and drug loading capacity. Moreover, PEG as hydrophilic block showed in comparison to HEAm more promising results concerning both colloidal stability and drug loading capacity. Confocal microscopy showed that the micelles could efficiently deliver a hydrophobic dye inside the cells. Finally, we also demonstrated efficient formulation of the anti-cancer drug paclitaxel with an in vitro cancer cell killing performance comparable or even better than the two commercial PTX nano-formulations Abraxane and Genexol-PM at equal drug dose. In conclusion, modification of HEAm through carbonate linkages offers a versatile platform for the design of degradable polymers with potential for biomedical applications

Control over imidazoquinoline immune stimulation by pH-degradable poly(norbornene) nanogels

The reactivation of the innate immune system by toll-like receptor (TLR) agonists holds promise for anticancer immunotherapy. Severe side effects caused by unspecific and systemic activation of the immune system upon intravenous injection prevent the use of small-molecule TLR agonists for such purposes. However, a covalent attachment of small-molecule imidazoquinoline (IMDQ) TLR7/8 agonists to pH-degradable polymeric nanogels could be shown to drastically reduce the systemic inflammation but retain the activity to tumoral tissues and their draining lymph nodes. Here, we introduce the synthesis of poly(norbornene)-based, acid-degradable nanogels for the covalent ligation of IMDQs. While the intact nanogels trigger sufficient TLR7/8 receptor stimulation, their degraded version of soluble, IMDQ-conjugated poly(norbornene) chains hardly activates TLR7/8. This renders their clinical safety profile, as degradation products are obtained, which would not only circumvent nanoparticle accumulation in the body but also provide nonactive, polymer-bound IMDQ species. Their immunologically silent behavior guarantees both spatial and temporal control over immune activity and, thus, holds promise for improved clinical applications

Squaric Ester-Based, pH-Degradable Nanogels:Modular Nanocarriers for Safe, Systemic Administration of Toll-like Receptor 7/8 Agonistic Immune Modulators

Small-molecular Toll-like receptor 7/8 (TLR7/8) agonists hold promise as immune modulators for a variety of immune therapeutic purposes including cancer therapy or vaccination. However, due to their rapid systemic distribution causing difficult-to-control inflammatory off-target effects, their application is still problematic, in particular systemically. To address this problem, we designed and robustly fabricated pH-responsive nanogels serving as versatile immunodrug nanocarriers for safe delivery of TLR7/8-stimulating imidazoquinolines after intravenous administration. To this aim, a primary amine-reactive methacrylamide monomer bearing a pendant squaric ester amide is introduced, which is polymerized under controlled RAFT polymerization conditions. Corresponding PEG-derived squaric ester amide block copolymers self-assemble into precursor micelles in polar protic solvents. Their cores are amine-reactive and can sequentially be transformed by acid-sensitive cross-linkers, dyes, and imidazoquinolines. Remaining squaric ester amides are hydrophilized affording fully hydrophilic nanogels with profound stability in human plasma but stimuli-responsive degradation upon exposure to endolysosomal pH conditions. The immunomodulatory behavior of the imidazoquinolines alone or conjugated to the nanogels was demonstrated by macrophages in vitro. In vivo, however, we observed a remarkable impact of the nanogel: After intravenous injection, a spatially controlled immunostimulatory activity was evident in the spleen, whereas systemic off-target inflammatory responses triggered by the small-molecular imidazoquinoline analogue were absent. These findings underline the potential of squaric ester-based, pH-degradable nanogels as a promising platform to permit intravenous administration routes of small-molecular TLR7/8 agonists and, thus, the opportunity to explore their adjuvant potency for systemic vaccination or cancer immunotherapy purposes.</p

Hyperprolactinemia in adults with Prader-Willi syndrome

Prader-Willi syndrome (PWS) is a rare neurodevelopmental genetic disorder typically characterized by body composition abnormalities, hyperphagia, behavioural challenges, cognitive dysfunction, and hypogonadism. Psychotic illness is common, particularly in patients with maternal uniparental disomy (mUPD), and antipsychotic medications can result in hyperprolactinemia. Information about hyperprolactinemia and its potential clinical consequences in PWS is sparse. Here, we present data from an international, observational study of 45 adults with PWS and hyperprolactinemia. Estimated prevalence of hyperprolactinemia in a subset of centres with available data was 22%, with 66% of those related to medication and 55% due to antipsychotics. Thirty-three patients were men, 12 women. Median age was 29 years, median BMI 29.8 kg/m2, 13 had mUPD. Median prolactin was 680 mIU/L (range 329–5702). Prolactin levels were higher in women and patients with mUPD, with only 3 patients having severe hyperprolactinemia. Thyroid function tests were normal, 24 were treated with growth hormone, 29 with sex steroids, and 20 with antipsychotic medications. One patient had kidney insufficiency, and one a microprolactinoma. In conclusion, severe hyperprolactinemia was rare, and the most common aetiology of hyperprolactinemia was treatment with antipsychotic medications. Although significant clinical consequences could not be determined, potential negative long-term effects of moderate or severe hyperprolactinemia cannot be excluded. Our results suggest including measurements of prolactin in the follow-up of adults with PWS, especially in those on treatment with antipsychotic

Polymer- and lipid-based nanomaterials and conjugates for cancer therapy

During the last few decades, remarkable progress has been made towards the understanding of cancer biology. However, cancer remains a major health concern and the treatment of cancer continues to be very challenging. Current cancer treatment modalities include surgery, radiotherapy, immunotherapy and chemotherapy. A primary limitation of chemotherapeutic drugs is their lack of specificity, as the drugs not only target cancerous cells, but also other rapid proliferating healthy cells. Accordingly, this may result in severe and/or life-threatening side effects. Nanomedicine offers great potential to address and alleviate some of the significant limitations of traditional chemotherapy. Their most striking feature is their ability to enclose or bind thousands of therapeutic drug molecules and specifically target the drugs to the tumor site via active and/or passive targeting strategies. Moreover, nanocarriers provide a platform to overcome solubility, stability and resistance issues.The main aim of this dissertation was to improve the selective delivery of chemotherapeutic agents to tumors by means of degradable targeted nanoparticle delivery systems and hence overcome some of the main limitations of cytostatic drugs. Furthermore, the concept of immunogenic cell death (ICD) was explored, which harnesses the immunogenic properties of chemotherapeutic drugs. Immunochemotherapy, which involves the combination of immunotherapy and chemotherapy, was investigated with the purpose to further enhance clinical efficacy and broaden therapy-responsive patient population

Covalent cell surface conjugation of nanoparticles by a combination of metabolic labeling and click chemistry

Conjugation of nanoparticles (NP) to the surface of living cells is of interest in the context of exploiting the tissue homing properties of ex vivo engineered T cells for tumor-targeted delivery of drugs loaded into NP. Cell surface conjugation requires either a covalent or non-covalent reaction. Non-covalent conjugation with ligand-decorated NP (LNP) is challenging and involves a dynamic equilibrium between the bound and unbound state. Covalent NP conjugation results in a permanently bound state of NP, but the current routes for cell surface conjugation face slow reaction kinetics and random conjugation to proteins in the glycocalyx. To address the unmet need for alternative bioorthogonal strategies that allow for efficient covalent cell surface conjugation, we developed a 2-step click conjugation sequence in which cells are first metabolically labeled with azides followed by reaction with sulfo-6-methyl-tetrazine-dibenzyl cyclooctyne (Tz-DBCO) by SPAAC, and subsequent IEDDA with trans-cyclooctene (TCO) functionalized NP. In contrast to using only metabolic azide labeling and subsequent conjugation of DBCO-NP, our 2-step method yields a highly specific cell surface conjugation of LNP, with very low non-specific background binding

Unexpected reactivity switch in the statistical copolymerization of 2-oxazolines and 2-oxazines enabling the one-step synthesis of amphiphilic gradient copolymers

Poly(2-oxazoline)s and, more recently, also poly(2-oxazine)s represent an emerging class of polymers with a broad range of applications. Surprisingly, to date, the statistical copolymerization of these two cyclic imino ether monomers has not yet been reported. Herein, we demonstrate that the statistical copolymerization of 2-oxazines with 2-oxazolines can lead to the formation of amphiphilic gradient copolymers in a single step. These gradient copolymers combine the high structural modularity of poly(2-oxazoline)s with the excellent biological properties of poly(2-oxazine)s, especially poly(2-methyl-2-oxazine). The copolymerization was found to proceed in a nonexpected way with the relative incorporation rates of the monomers being opposite to the reactivity observed for the corresponding homopolymerizations. In fact, the statistical copolymerizations lead to faster incorporation of the 2-oxazine followed by a gradual transition toward the 2-oxazoline. The self-assembly properties of the prepared amphiphilic poly[(2-methyl-2-oxazine)-grad-(2-butyl-2-oxazoline)] (PMeOzi-grad-PBuOx) as well as the thermoresponsive poly[(2-methyl-2-oxazine)-grad-(2-propyl-2-oxazoline)] (PMeOzigrad-PPrOx) confirmed their potential as stimuli-responsive nonionic surfactants for various applications. Finally, the noncytotoxic character and cellular uptake of PMeOzi-grad-PBuOx copolymers was confirmed in vitro in SKOV3 cells

Acrylamides with hydrolytically labile carbonate ester side chains as versatile building blocks for well-defined block copolymer micelles via RAFT polymerization

En route towards improved delivery systems for targeted chemotherapy, we propose a straightforward approach for the hydrophobic modification of the acrylamide N-(2-Hydroxyethyl)acrylamide (HEAm). An ethyl or benzyl group was introduced via a hydrolytically sensitive carbonate ester yielding HEAm-EC and HEAm-BC, respectively. Block copolymers of HEAm, respectively PEG and HEAm-EC or HEAm-BC were successfully synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization, obtaining a library of well-defined block copolymers with different degrees of polymerization (DP). To further explore the versatility of our approach in terms of polymer synthesis, self-assembly, drug solubilization and in vitro cell interaction, polyethylene glycol (PEG) and polyHEAm as hydrophilic polymer blocks were compared. The block copolymers formed micellar nanoparticles (10-100 nm) in PBS and could efficiently solubilize hydrophobic dyes and anti-cancer drugs. Benzyl carbonate ester side chains increased micellar stability and drug loading capacity. Moreover, PEG as hydrophilic block showed in comparison to HEAm more promising results concerning both colloidal stability and drug loading capacity. Confocal microscopy showed that the micelles could efficiently deliver a hydrophobic dye inside the cells. Finally, we also demonstrated efficient formulation of the anti-cancer drug paclitaxel with an in vitro cancer cell killing performance comparable or even better than the two commercial PTX nanoformulations Abraxane and Genexol-PM at equal drug dose. In conclusion, modification of HEAm through carbonate linkages offers a versatile platform for the design of degradable polymers with potential for biomedical applications

Thermoplastic polyurethane-based intravaginal rings for prophylaxis and treatment of (recurrent) bacterial vaginosis

The aim of the present study was to develop thermoplastic polyurethane (TPU)-based intravaginal rings (IVRs) for prophylaxis and treatment of bacterial vaginosis via hot melt extrusion/injection molding. Therefore, different TPU grades were processed in combination with lactic acid or metronidazole, targeting a sustained lactic acid release over a 28 day-period and sustained metronidazole release over 47 days. Hot melt extrusion of lactic acid/TPU combinations required a lower extrusion temperature due to the plasticizing properties of lactic acid, evidenced by the lower glass transition temperature (T-g) and cross-over point (T-tan delta (=) (1)) values. NIR-chemical imaging data showed a homogenous distribution of lactic acid in TPU matrices at drug loads up to 30% (w/w). The addition of metronidazole did not lower processing temperatures, as the active pharmaceutical ingredient remained crystalline in the TPU matrix. Hydrophobic TPUs with a low ratio between the soft and hard segments (SS/HS ratio) in the polymer structure were suitable carriers for the lactic acid-eluting device over a 28-day period, while hydrophilic TPUs were needed to achieve the required release rate of metronidazole-eluting IVRs. IVRs manufactured with a TPU grade having a higher SS/HS ratio and lactic acid/TPU ratio exhibited a more elastic behavior. The addition of 25% (w/w) metronidazole did not affect the mechanical properties of the IVRs. Hydrophilic TPUs were most prone to biofilm formation by Candida albicans and Staphylococcus aureus, but the incorporation of metronidazole in the device prevented biofilm formation. Based on the drug eluting performance and mechanical tests, a mixture of lactic acid and Tecoflex (TM) EG-93A (20/80, w/w) and a combination of metronidazole and Tecophilic (TM) SP-93A-100 (25/75, w/w) were selected to design IVRs for the prophylaxis and treatment of bacterial vaginosis, respectively. Slug mucosal irritation tests predicted low irritation potency for both devices. (C) 2017 Elsevier B.V. All rights reserved