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Dithiolanes are used to obtain dynamic and reversible crosslinks between polymer chains. Copolymers of two different dithiolane-containing cyclic carbonate monomers and ϵ-caprolactone (CL) were synthesized by ring-opening polymerization using a methoxy-poly(ethylene glycol) (mPEG) initiator and different catalysts (diphenyl phosphate (DPP), methanesulfonic acid (MSA), 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD), or Sn(Oct)2). Each catalyst required a different temperature, which had a pronounced influence on the reactivity ratio of the monomers and occurrence of transesterification reactions and, therefore, the monomer sequence. Self-crosslinkable copolymers were obtained when the dithiolane units were connected closely to the polymer backbone, whereas the presence of a linker unit between the dithiolane and the backbone prevented self-crosslinking. The former amphiphilic PEGylated block copolymers formed micelles by nanoprecipitation in the aqueous environment and crosslinked spontaneously by disulfide exchange during subsequent dialysis. These dithiolane-crosslinked micelles showed reduction-responsive dissociation in the presence of 10 mM glutathione, making them promising drug delivery systems for the intracellularly triggered cargo release.

The aim of this study was to get insight into the internalization and transport of PEGylat-ed mixed micelles loaded by vitamin K, as mediated by Scavenger Receptor B1 (SR-B1) that is abundantly expressed by intestinal epithelium cells as well as by differentiated Caco-2 cells. Inhibition of SR-B1 reduced endocytosis and transport of vitamin-K-loaded 0%, 30% and 50% PEGylated mixed micelles and decreased colocalization of the micelles with SR-B1. Confocal fluorescence microscopy, fluorescence-activated cell sorting (FACS) analysis, and surface plasmon resonance (SPR) were used to study the interaction between the mixed micelles of different compositions (varying vitamin K loading and PEG content) and SR-B1. Interaction of PEGylated micelles was independent of the vitamin K content, indicating that the PEG shell prevented vitamin K exposure at the surface of the micelles and binding with the receptor and that the PEG took over the micelles’ ability to bind to the receptor. Molecular docking calculations corroborated the dual binding of both vita-min K and PEG with the binding domain of SR-B1. In conclusion, the improved colloidal stability of PEGylated mixed micelles did not compromise their cellular uptake and transport due to the affinity of PEG for SR-B1. SR-B1 is able to interact with PEGylated nanoparticles and mediates their subsequent internalization and transport.

Background CriPec technology enables the generation of drug-entrapped biodegradable core-crosslinked polymeric micelles (CCPM) with high drug loading capacity, tailorable size, and drug release kinetics. Docetaxel (DTX)-entrapped CCPM, also referred to as CPC634, have demonstrated favorable pharmacokinetics, tolerability, and enhanced tumor uptake in patients. Clinical efficacy evaluation is ongoing. CPC634 is currently stored (shelf life > 5 years) and shipped as a frozen aqueous dispersion at temperatures below -60°C, in order to prevent premature release of DTX and hydrolysis of the core-crosslinks. Consequently, like other aqueous nanomedicine formulations, CPC634 relies on cold chain supply, which is unfavorable for commercialization. Lyophilization can help to bypass this issue. Methods and results Freeze-drying methodology for CCPM was developed by employing CPC634 as a model formulation, and sucrose and trehalose as cryoprotectants. We studied the residual moisture content and reconstitution behavior of the CPC634 freeze-dried cake, as well as the size, polydispersity index, morphology, drug retention, and release kinetics of reconstituted CPC634. Subsequently, the freeze-drying methodology was validated in an industrial setting, yielding a CPC634 freeze-dried cake with a moisture content of less than 0.1 wt%. It was found that trehalose-cryoprotected CPC634 could be rapidly reconstituted in less than 5 min at room temperature. Critical quality attributes such as size, morphology, drug retention, and release kinetics of trehalose-cryoprotected freeze-dried CPC634 upon reconstitution were identical to those of non-freeze-dried CPC634. Conclusion Our findings provide proof-of-concept for the lyophilization of drug-containing CCPM and our methodology is readily translatable to large-scale manufacturing for future commercialization.

Active self-encapsulation (ASE) is a recently developed post-loading method based on absorption of (positively charged) proteins in microporous PLGA microspheres loaded with negatively charged polysaccharides (trapping agents). The aim of this study was to investigate ASE for simultaneous loading and controlled release of multiple growth factors. For this purpose, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and insulin-like growth factor (IGF) were loaded in microspheres containing high molecular weight dextran sulfate (HDS) as trapping agent; loading was performed in a concentrated growth factor solution of low ionic strength and of pH 5 under conditions at which the proteins are positively charged. Subsequent pore closure was induced by incubation of the growth factor-loaded microspheres at 42.5 °C, i.e. above the Tg of (hydrated) PLGA (~30 °C). A 1:1:1 combination of VEGF, FGF and IGF was loaded with high loading (4.3%) and loading efficiency (91%). The in vitro release kinetics and bioactivity of loaded growth factors were studied for 4 weeks using ELISA and an endothelial cell proliferation assay, respectively. While IGF was released quickly, VEGF and FGF were continuously released for 4 weeks in their bioactive form, whereby a growth factor combination had a synergistic angiogenic effect. Therefore, ASE is a suitable method for co-loading growth factors which can provide sustained release profiles of bioactive growth factors, which is attractive for vascularization of biomaterial implants.

In this study, a new type of injectable hydrogel called "HyMic" that can convert into core cross-linked (CCL) micelles upon exposure to matrix metalloproteinases (MMP's), was designed and developed for drug delivery applications. HyMic is composed of CCL micelles connected via an enzyme cleavable linker. To this end, two complementary ABA block copolymers with polyethylene glycol (PEG) as B block were synthesized using atom transfer radical polymerization (ATRP). The A blocks were composed of a random copolymer of N-isopropylacrylamide (NIPAM) and either N-(2-hydroxypropyl)methacrylamide-cysteine (HPMA-Cys) or N-(2-hydroxypropyl) methacrylamide-ethylthioglycolate succinic acid (HPMA-ETSA). Mixing the aqueous solutions of the obtained polymers and rising the temperature above the cloud point of the PNIPAM block resulted in the self-assembly of these polymers into flower-like micelles composed of a hydrophilic PEG shell and hydrophobic core. The micellar core was cross-linked by native chemical ligation between the cysteine (in HPMA-Cys) and thioester (in HPMA-ETSA) functionalities. A slight excess of thioester to cysteine groups (molar ratio 3:2) was used to allow further chemical reactions exploiting the unreacted thioester groups. The obtained micelles displayed a Z-average diameter of 80 ± 1 nm (PDI 0.1), and ζ-potential of -4.2 ± 0.4 mV and were linked using two types of pentablock copolymers of P(NIPAM-co-HPMA-Cys)-PEG-peptide-PEG-P(NIPAM-co-HPMA-Cys) (Pep-NC) to yield hydrogels. The pentablock copolymers were synthesized using a PEG-peptide-PEG ATRP macroinitiator and the peptide midblock (lysine-glycine-proline-glutamine-isoleucine-phenylalanine-glycine-glutamine-lysine (Lys-Gly-Pro-Gln-Gly-Ile-Phe-Gly-Gln-Lys)) consisted of either l- or d-amino acids (l-Pep-NC or d-Pep-NC), of which the l-amino acid sequence is a substrate for matrix metalloproteases 2 and 9 (MMPs 2 and 9). Upon mixing of the CCL micelles and the linker (l/d-Pep-NC), the cysteine functionalities of the l/d-Pep-NC reacted with remaining thioester moieties in the micellar core via native chemical ligation yielding a hydrogel within 160 min as demonstrated by rheological measurements. As anticipated, the gel cross-linked with l-Pep-NC was degraded in 7-45 days upon exposure to metalloproteases in a concentration-dependent manner, while the gel cross-linked with the d-Pep-NC remained intact even after 2 months. Dynamic light scattering analysis of the release medium revealed the presence of nanoparticles with a Z-average diameter of ∼120 nm (PDI < 0.3) and ζ-potential of ∼-3 mV, indicating release of core cross-linked micelles upon HyMic exposure to metalloproteases. An in vitro study demonstrated that the released CCL micelles were taken up by HeLa cells. Therefore, HyMic as an injectable and enzyme degradable hydrogel displaying controlled and on-demand release of CCL micelles has potential for intracellular drug delivery in tissues with upregulation of MMPs, for example, in cancer tissues.

Journal of controlled release 328, 970-984 (2020). doi:10.1016/j.jconrel.2020.09.013 special issue: "Special Issue Henry Kopecek / edited by Wim Hennink, Raymond Schiffelers", Published by Elsevier, New York, NY [u.a.]

One of the complications of bronchotracheal cancer is obstruction of the upper airways. Local tumor resection in combination with an airway stent can suppress intraluminal tumor (re)growth. We have investigated a novel drug-eluting stent coating for local release of the anticancer drug gefitinib. A polyurethane (PU) sandwich construct was prepared by a spray coating method in which gefitinib was embedded between a PU support layer of 200 μm and a PU top layer of 50–200 μm. Gefitinib was either embedded in the construct as small crystals or as gefitinib-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres (MSP). The drug was incorporated in the PU constructs with high recovery (83–93%), and the spray coating procedure did not affect the morphologies of the embedded microspheres as demonstrated by scanning electron microscopy (SEM), confocal laser scanning microscopy and fluorescence microscopy analysis. PU constructs loaded with gefitinib crystals released the drug for 7–21 days and showed diffusion based release kinetics. Importantly, directional release of the drug towards the top layer, which is supposed to face the tumor mass, was controlled by the thicknesses of the PU top layer. PU constructs loaded with gefitinib microspheres released the drug in a sustained manner for > 6 months indicating that drug release from the microspheres became the rate limiting step. In conclusion, the sandwich structure of drug-loaded PLGA microspheres in PU coating is a promising coating for airway stents that release anticancer drugs locally for a prolonged time.

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.

For realization of a wearable artificial kidney based on regeneration of a small volume of dialysate, efficient urea removal from dialysate is a major challenge. Here a potentially suitable polymeric sorbent based on phenylglyoxaldehyde (PGA), able to covalently bind urea under physiological conditions, is described. Sorbent beads containing PGA groups were obtained by suspension polymerization of either styrene or vinylphenylethan-1-one (VPE), followed by modification of the aromatic groups of poly(styrene) and poly(VPE) into PGA. It was found that PGA-functionalized sorbent beads had maximum urea binding capacities of 1.4–2.2 mmol/g and removed ∼0.6 mmol urea/g in 8 h at 37 °C under static conditions from urea-enriched phosphate-buffered saline, conditions representative of dialysate regeneration. This means that the daily urea production of a dialysis patient can be removed with a few hundred grams of this sorbent which, is an important step forward in the development of a wearable artificial kidney.

Poly(n-butyl cyanoacrylate) microbubbles (PBCA-MB) are extensively employed for functional and molecular ultrasound (US) imaging, as well as for US-mediated drug delivery. To facilitate the use of PBCA-MB as a commercial platform for biomedical applications, it is important to systematically study and improve their stability and shelf-life. In this context, lyophilization (freeze drying) is widely used to increase shelf-life and promote product development. Here, we set out to analyze the stability of standard and rhodamine-B loaded PBCA-MB at three different temperatures (4 &deg, C, 25 &deg, C, and 37 &deg, C), for a period of time of up to 20 weeks. In addition, using sucrose, glucose, polyvinylpyrrolidone (PVP), and polyethylene glycol (PEG) as cryoprotectants, we investigated if PBCA-MB can be lyophilized without affecting their size, concentration, US signal generation properties, and dye retention. Stability assessment showed that PBCA-MB remain largely intact for three and four weeks at 4 &deg, C and 25 &deg, C, respectively, while they disintegrate within one to two weeks at 37 &deg, C, thereby compromising their acoustic properties. Lyophilization analyses demonstrated that PBCA-MB can be efficiently freeze-dried with 5% sucrose and 5% PVP, without changing their size, concentration, and US signal generation properties. Experiments involving rhodamine-B loaded MB indicated that significant dye leakage from the polymeric shell takes place within two to four weeks in case of non-lyophilized PBCA-MB. Lyophilization of rhodamine-loaded PBCA-MB with sucrose and PVP showed that the presence of the dye does not affect the efficiency of freeze-drying, and that the dye is efficiently retained upon MB lyophilization. These findings contribute to the development of PBCA-MB as pharmaceutical products for preclinical and clinical applications.

The aim of the study is to investigate the uptake by and transport through Caco-2 cells of two mixed micelle formulations (based on egg phosphatidylcholine and glycocholic acid) of vitamin K, i.e., with and without DSPE-PEG2000. The uptake of vitamin K and fluorescently labeled mixed micelles with and without PEG coating showed similar kinetics and their uptake ratio remained constant over time. Together with the fact that an inhibitor of scavenger receptor B1 (BLT-1) decreased cellular uptake of vitamin K by ∼80% compared to the uptake in the absence of this inhibitor, we conclude that both types of micelles loaded with vitamin K can be taken up intactly by Caco-2 cells via this scavenger receptor. The amount of vitamin K in chylomicrons fraction from Caco-2 cell monolayers further indicates that mixed micelles (with or without PEGylation) are likely packed into chylomicrons after internalization by Caco-2 cells. Uptake of vitamin K from PEGylated mixed micelles increased four- to five-fold at simulated gastrointestinal conditions. In conclusion, PEGylated mixed micelles are stable upon exposure to simulated gastric conditions, and as a result, they do show overall a higher cellular uptake efficiency of vitamin K as compared to mixed micelles without PEG coating.

Styrene-maleic acid (SMA) copolymers have attracted interest in membrane research because they allow the solubilization and purification of membrane-spanning proteins from biological membranes in the form of native-like nanodisks. However, our understanding of the underlying SMA-lipid interactions is hampered by the fact that SMA preparations are very polydisperse. Here, we obtained fractions of the two most commonly used SMA preparations: SMA 2:1 and SMA 3:1 (both with specified M(w) ∼10 kD), with different number-average molecular weight (M(n)) and styrene content. The fractionation is based on the differential solubility of styrene-maleic anhydride (SMAnh) in hexane and acetone mixtures. SMAnh fractions were hydrolyzed to SMA and added to lipid self-assemblies. It was found that SMA fractions inserted in monolayers and solubilized vesicles to a different extent, with the highest efficiency being observed for low-M(n) SMA polymers. Electron microscopy and dynamic light scattering size analyses confirmed the presence of nanodisks independent of the M(n) of the SMA polymers forming the belt, and it was shown that the nanodisks all have approximately the same size. However, nanodisks bounded by high-M(n) SMA polymers were more stable than those bounded by low-M(n) polymers, as indicated by a better retention of the native lipid thermotropic properties and by slower exchange rates of lipids between nanodisks. In conclusion, we here present a simple method to separate SMAnh molecules based on their M(n) from commercial SMAnh blends, which allowed us to obtain insights into the importance of SMA length for polymer-lipid interactions.

The aim of the present study was to develop folic acid (FA) conjugates which can deliver the kinase inhibitor dactolisib to the kidneys via folate receptor-mediated uptake in tubular epithelial cells. Dactolisib is a dual inhibitor of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) and is considered an attractive agent for treatment of polycystic kidney disease. The ethylenediamine platinum(II) linker, herein called Lx, was employed to couple dactolisib via coordination chemistry to thiol-containing FA-spacer adducts to yield FA-Lx-dactolisib conjugates. The dye lissamine was coupled via similar linker chemistry to folate to yield fluorescent FA-Lx-lissamine conjugates. Three different spacers (PEG(5)-Cys, PEG(27)-Cys or an Asp-Arg-Asp-Asp-Cys peptide spacer) were used to compare the influence of hydrophilicity and charged groups in the spacer on interaction with target cells and in vivo organ distribution of the final conjugates. The purity and identity of the final products were confirmed by UPLC and LC-MS analysis, respectively. FA-Lx-dactolisib conjugates were stable in serum and culture medium, while dactolisib was released from the conjugates in the presence of glutathione. All three type of conjugates were internalized efficiently by HK-2 cells and uptake could be blocked by an excess of folic acid in the medium, demonstrating FR mediated uptake. FA-Lx-dactolisib conjugates showed nanomolar inhibition of the PI3K pathway (Akt phosphorylation) and mTOR pathway (S6 phosphorylation) in cultured kidney epithelial cells (HK-2 cells). After intraperitoneal administration, all three types conjugates accumulated extensively in kidneys of iKsp-Pkd1(del) mice with polycystic kidney disease. In conclusion, folate conjugates were successfully prepared by platinum(II) coordination chemistry and accumulated in a target-specific manner in kidney cells and polycystic kidneys. The folate conjugate of dactolisib thus may have potential for targeted therapy of polycystic kidney disease.

Electrospun membranes based on biodegradable polymers are promising materials to be used for guided bone regeneration (GBR) therapy. The incorporation of osteostimulatory compounds can improve the biofunctionality of those membranes, making them active players in bone regeneration. Simvastatin has been shown to promote osteogenic differentiation both in vitro and in vivo. However, in most of these systems, the drug was quickly released, not matching the pace of bone regeneration. The aim of this study was to develop poly(L-lactic acid) (PLLA) membranes containing simvastatin (SV) that have a prolonged drug release rate, compatible with GBR applications. To this end, SV was mixed with PLLA and electrospun. The membranes were subjected to a thermal treatment in order to increase the crystallinity of PLLA. Morphological, structural and chemical properties of the electrospun membranes were characterized. The effect of the thermal treatment on the release profile of SV was evaluated by near physiological release experiments at 37 °C. The osteostimulatory potential was determined by in vitro culture of the membranes with rat bone marrow stromal cells (rBMSCs). The results confirmed that the thermal treatment led to an increase in polymer crystallinity and a more sustained release of SV. In vitro assays demonstrate cellular proliferation over time for all the membranes and a significant increase in osteogenic differentiation for the membranes containing SV subjected to thermal treatment.