Thiol‐Click Based Polyglycerol Hydrogels as Biosensing Platform with In Situ Encapsulated Streptavidin Probes

An in situ streptavidin‐encapsulated hydrogel based on dendritic polyglycerol (dPG) which is functionalized with either an acrylate, allyl or acrylamide group and dithiolated polyethylene glycol (PEG) is constructed via a thiol‐click chemistry approach and is investigated for biosensing applications. The hydrogel platform is screened for the encapsulation and release efficiencies of the model protein streptavidin under varying physicochemical conditions, for example, crosslinking chemistry reactions, the molar ratio between the two gel components, macromonomer concentrations or pH‐values. By that, tailor‐made hydrogels can be developed, which are able to encapsulate or release the model protein for several days based on its modality. Furthermore, the accessible binding site of encapsulated streptavidin or in other words, the biotin‐binding performance is quantified, and the stability of the various hydrogel types is studied by rheology measurements, 1H NMR, gel permeation chromatography (GPC), and mass loss experiments

Tunable Polyglycerol-Based Redox-Responsive Nanogels for Efficient Cytochrome C Delivery

The sensitivity of therapeutic proteins is a challenge for their use in biomedical applications, as they are prone to degradation and opsonization, thus limiting their potential. This demands for the development of drug delivery systems shielding proteins and releasing them at the site of action. Here, we describe the synthesis of novel polyglycerol-based redox-responsive nanogels and report on their potential as nanocarrier systems for the delivery of cytochrome C (CC). This system is based on an encapsulation protocol of the therapeutic protein into the polymer network. NGs were formed via inverse nanoprecipitation using inverse electron-demand Diels–Alder cyclizations (iEDDA) between methyl tetrazines and norbornenes. Coprecipitation of CC led to high encapsulation efficiencies. Applying physiological reductive conditions of l-glutathione (GSH) led to degradation of the nanogel network, releasing 80% of the loaded CC within 48 h while maintaining protein functionality. Cytotoxicity measurements revealed high potency of CC-loaded NGs for various cancer cell lines with low IC50 values (up to 30 μg·mL−1), whereas free polymer was well tolerated up to a concentration of 1.50 mg·mL−1. Confocal laser scanning microscopy (CLSM) was used to monitor internalization of free and CC-loaded NGs and demonstrate the protein cargo’s release into the cytosol

Dendritic Glycerol-Cholesterol Amphiphiles as Drug Delivery Systems: A Comparison between Monomeric and Polymeric Structures

The application of micelles as drug delivery systems has gained a great deal of attention as a means to overcome the current several drawbacks present in conventional cancer treatments. In this work, we highlight the comparison of polymeric and monomeric amphiphilic systems with a similar hydrophilic–lipophilic balance (HLB) in terms of their biocompatibility, aggregation behavior in aqueous solution, and potential in solubilizing hydrophobic compounds. The polymeric system consists of non-ionic polymeric amphiphiles synthesized via sequential RAFT polymerization of polyglycerol first-generation [G1] dendron methacrylate and cholesterol methacrylate to obtain poly(G1-polyglycerol dendron methacrylate)-block-poly(cholesterol methacrylate) (pG1MA-b-pCMA). The monomeric system is a polyglycerol second-generation [G2] dendron end-capped to a cholesterol unit. Both amphiphiles form spherical micellar aggregations in aqueous solution, with differences in size and the morphology in which hydrophobic molecules can be encapsulated. The polymeric and monomeric micelles showed a low critical micelle concentration (CMC) of 0.2 and 17 μg/mL, respectively. The results of our cytotoxicity assays showed that the polymeric system has significantly higher cell viability compared to that of the monomeric amphiphiles. The polymeric micelles were implemented as drug delivery systems by encapsulation of the hydrophobic small molecule doxorubicin, achieving a loading capacity of 4%. In summary, the results of this study reveal that using cholesterol as a building block for polymer synthesis is a promising method of preparation for efficient drug delivery systems while improving the cell viability of monomeric cholesterol

Gram Scale Synthesis of Dual-Responsive Dendritic Polyglycerol Sulfate as Drug Delivery System

Biocompatible polymers with the ability to load and release a cargo at the site of action in a smart response to stimuli have attracted great attention in the field of drug delivery and cancer therapy. In this work, we synthesize a dual-responsive dendritic polyglycerol sulfate (DR-dPGS) drug delivery system by copolymerization of glycidol, ε-caprolactone and an epoxide monomer bearing a disulfide bond (SSG), followed by sulfation of terminal hydroxyl groups of the copolymer. The effect of different catalysts, including Lewis acids and organic bases, on the molecular weight, monomer content and polymer structure was investigated. The degradation of the polymer backbone was proven in presence of reducing agents and candida antarctica Lipase B (CALB) enzyme, which results in the cleavage of the disulfides and ester bonds, respectively. The hydrophobic anticancer drug Doxorubicin (DOX) was loaded in the polymer and the kinetic assessment showed an enhanced drug release with glutathione (GSH) or CALB as compared to controls and a synergistic effect of a combination of both stimuli. Cell uptake was studied by using confocal laser scanning microscopy with HeLa cells and showed the uptake of the Dox-loaded carriers and the release of the drug into the nucleus. Cytotoxicity tests with three different cancer cell lines showed good tolerability of the polymers of as high concentrations as 1 mg mL−1, while cancer cell growth was efficiently inhibited by DR-dPGS@Dox

Chemical Approaches to Synthetic Drug Delivery Systems for Systemic Applications

Poor water solubility and low bioavailability of active pharmaceutical ingredients (APIs) are major causes of friction in the pharmaceutical industry and represent a formidable hurdle for pharmaceutical drug development. Drug delivery remains the major challenge for the application of new small-molecule drugs as well as biopharmaceuticals. The three challenges for synthetic delivery systems are: (i) controlling drug distribution and clearance in the blood; (ii) solubilizing poorly water-soluble agents, and (iii) selectively targeting specific tissues. Although several polymer-based systems have addressed the first two demands and have been translated into clinical practice, no targeted synthetic drug delivery system has reached the market. This Review is designed to provide a background on the challenges and requirements for the design and translation of new polymer-based delivery systems. This report will focus on chemical approaches to drug delivery for systemic applications

Dendritic polyglycerolsulfate-SS-poly(ester amide) micelles for the systemic delivery of docetaxel: pushing the limits of stability through the insertion of π–π interactions

Insufficient stability of micellar drug delivery systems is still the major limitation to their systematic application in chemotherapy. This work demonstrates novel π-electron stabilized polyelectrolyte block copolymer micelles based on dendritic polyglycerolsulfate-cystamine-block-poly(4-benzoyl-1,4-oxazepan-7-one)-pyrene (dPGS-SS-POxPPh-Py) presenting a very low critical micelle concentration (CMC) of 0.3 mg L−1 (18 nM), 55-fold lower than that of conventional amphiphilic block copolymer micelles. The drug loading capacities of up to 13 wt% allow the efficient encapsulation of the chemotherapeutic Docetaxel (DTX). The spherical morphology of the micelles was proven by cryogenic electron microscopy (cryo-EM). Gaussian Analysis revealed well-defined sizes of 57 nm and 80 nm in the unloaded/loaded state, respectively. Experiments by dynamic light scattering (DLS), ultraviolet-visible spectroscopy (UV-VIS), fluorescence spectroscopy, and cross-polarization solid-state 13C NMR studied the π–π interactions between the core-forming block segment of dPGS-SS-POxPPh-Py and DTX. The findings point to a substantial contribution of these noncovalent interactions to the system's high stability. By confocal laser scanning microscopy (CLSM), the cellular uptake of fluorescein-labelled FITC-dPGS-SS-POxPPh-Py micelles was monitored after one day displaying the successful cell insertion of the cargo-loaded systems. To ensure the drug release in cancerous cells, the disassembly of the micellar DTX-formulations was achieved by reductive and enzymatic degradation studied by light scattering and GPC experiments. Further, no size increase nor disassembly in the presence of human serum proteins after four days was detected. The precise in vitro drug release was also given by the high potency of inhibiting cancer cell growth, finding half-maximal inhibitory concentrations (IC50) efficiently reduced to 68 nM coming along with high viabilities of the empty polymer materials tested on tumor-derived HeLa, A549, and McF-7 cell lines after two days. This study highlights the substantial potential of micelles tailored through the combination of π-electron stabilization with dendritic polyglycerolsulfate for targeted drug delivery systems, enabling them to have a significant foothold in the clinical treatment of cancer

Esterase-Responsive Polyglycerol-Based Nanogels for Intracellular Drug Delivery in Rare Gastrointestinal Stromal Tumors

Rare gastrointestinal stromal tumors (GISTs) are caused by mutations in the KIT and PDGFRA genes. Avapritinib (BLU-285) is a targeted selective inhibitor for mutated KIT and PDGFRA receptors that can be used to treat these tumors. However, there are subtypes of GISTs that exhibit resistance against BLU-285 and thus require other treatment strategies. This can be addressed by employing a drug delivery system that transports a combination of drugs with distinct cell targets. In this work, we present the synthesis of esterase-responsive polyglycerol-based nanogels (NGs) to overcome drug resistance in rare GISTs. Using inverse nanoprecipitation mediated with inverse electron-demand Diels–Alder cyclizations (iEDDA) between dPG-methyl tetrazine and dPG-norbornene, multi-drug-loaded NGs were formed based on a surfactant-free encapsulation protocol. The obtained NGs displayed great stability in the presence of fetal bovine serum (FBS) and did not trigger hemolysis in red blood cells over a period of 24 h. Exposing the NGs to Candida Antarctica Lipase B (CALB) led to the degradation of the NG network, indicating the capability of targeted drug release. The bioactivity of the loaded NGs was tested in vitro on various cell lines of the GIST-T1 family, which exhibit different drug resistances. Cell internalization with comparable uptake kinetics of the NGs could be confirmed by confocal laser scanning microscopy (CLSM) and flow cytometry for all cell lines. Cell viability and live cell imaging studies revealed that the loaded NGs are capable of intracellular drug release by showing similar IC50 values to those of the free drugs. Furthermore, multi-drug-loaded NGs were capable of overcoming BLU-285 resistance in T1-α-D842V + G680R cells, demonstrating the utility of this carrier system

Chemische Ansätze für synthetische Wirkstofftransportsysteme für systemische Anwendungen

Schlechte Wasserlöslichkeit und geringe Bioverfügbarkeit von pharmazeutischen Wirkstoffen (APIs) sind die Hauptursache für Verzögerungen in der pharmazeutischen Industrie und stellen eine große Hürde für die Entwicklung neuer Arzneimittel dar. Der Transport von Arzneimitteln ist nach wie vor die größte Herausforderung für die Anwendung niedermolekularer Medikamente und Biopharmazeutika. Die drei Herausforderungen für synthetische Transportsysteme sind: (i) Kontrolle über die Wirkstoffverteilung und Clearance im Blut, (ii) Solubilisierung schlecht wasserlöslicher Wirkstoffe und (iii) selektive Akkumulation in bestimmten Geweben. Obwohl viele Polymer-basierte Systeme die ersten beiden Anforderungen erfüllen und in die klinische Praxis umgesetzt wurden, hat bisher noch kein zielgerichtetes, synthetisches Abgabesystem den Markt erreicht. Dieser Aufsatz soll einen Überblick über die Herausforderungen und Anforderungen zur Entwicklung und Umsetzung neuer Polymer-basierter Darreichungssysteme geben. Hauptaugenmerk liegt hierbei auf den chemischen Ansätzen für die Darreichung von Wirkstoffen für systemische Anwendungen

Stimulus-sensitive Nanocarrier für Diagnostik und Therapie

In this thesis, the overall goal was to develop novel stimuli-responsive nanocarriers which are able to overcome biological barriers and furtherly transport and release various therapeutic cargos to the target site. Furthermore, the aim was to develop a nanomedicine platform that shows the ability to protect the sensitive cargo and use natural stimuli for a controlled release. Besides therapy, the tailor-made nanocarriers are targeted to be adaptable for diagnostic approaches. In the first project of this thesis, a pH degradable dPG-amine nanocarrier for the delivery of genetic material was developed. Due to the introduction of stimuli-responsive acetal groups the nanocarrier could be cleaved at acidic pH values which occur in the endosomal compartments and remained stable at neutral pH which appearrs in the extracellular matrix. Modified benzacetal moieties realized a fine-tune in the cleavage kinetics. The complexation and release of DNA out of nanocarriers was studied by various methods. The pH triggered cleavage of the outer amine shell results in the degradation of the multivalent amine architecture. By that, the ability to bind genetic material is reduced and a controlled release of DNA can occur. In vitro transfection demonstrated that pH cleavable dPG-amines could transfect HeLa cells with GFP-DNA and resulted in cell-compatible cleavage products with a significantly reduced toxicity compared to the non- degradable gold standard PEI. The synthesis of these degradable core-shell polymers realizes a new carrier platform for the complexation and controlled release of genetic material at the target site by the loss of multivalent interactions. Additionally, a novel approach for the synthesis of dendritic polyglycerol based nanogels was developed. The Thiol–Michael nanoprecipitation method which operates under mild conditions and did not require any catalyst or surfactant was used to develop tailor-made nanogels in the size range of 100-1000 nm. The biocompatible nanogels were used for diagnostic as well as therapy for topical skin application. Dye-labeled nanogels were used for the first time as pH-nanosensors to determine the pH of the hair follicle (HF) at different depth in an ex vivo porcine ear model. The non-toxic nanogels showed a high potential for the penetration via the follicular uptake route. An automated analysis of confocal laser scanning microscopy (CLSM) images helped to accurately determine the pH inside the HF. The pH gradient ranged from 6.5 on the skin surface to 7.4 in deeper areas of the HF with a sharp pH increase over the first 300 μm. This finding provides a clear direction for the development of pH responsive DDS for follicular drug delivery. Furthermore, functional nanogel-peptide conjugates were developed which could complex and release hydrophobic drugs into the skin. The conjugation of the tailor-made peptide chains to the polyglycerol based nanogel increased the loading capacity and binding specificity of the targeted therapeutic model drug significantly. Skin penetration tests showed efficient dermal delivery and release of a photosensitizer which can be used for photo-dynamic therapy against cancer. Moreover, cationic nanogels based on dendritic polyglycerol and low molecular weight PEI were used for the delivery of siRNA. The genetic material could be encapsulated by the above-mentioned Thiol-Michael nanoprecipitation. The mild and bioorthogonal reaction enables the in-situ binding of GFP-siRNA into the nanogel matrix. Acetal moieties which were incorporated inside the nanogel realize a pH dependent degradation of the sub-100 nm carrier. In vitro transfection demonstrated that the pH cleavable nanogels could successfully silence GFP processing HeLa cells and were significantly less toxic compared to non-cleavable PEI. The cationic nanogel- platform will be investigated further for the delivery of anti-inflammatory siRNA for topical skin application. Additionally, formation and impact of the protein corona on the dPG-based nanogels with a varied surface charge was analyzed. Here, nanogels with a higher surface charge showed an increased binding of the blood protein human serum albumin (HSA) which resulted in an increased hydrodynamic radius and reduced surface charge. In the future, the nanogel manufacturing process could be upscaled to produce nanocarriers with a standard operation protocol in a one batch procedure to ensure further development to a nanomedicine platform. Recently, nanoparticles were prepared by nanoprecipitation process in a microfluidic device.[219] In general, this technique is used for the production of microparticles[220] which are useful for advanced drug delivery (e.g. the encapsulation of biological cargos like living cells[221]) and may be adapted to produce stimuli responsive nanogels in a continuous process in high quantity. Furthermore, the nanogel-platform could be used to encapsulate various therapeutic proteins or therapeutic hydrophobic drugs. Due to the flexible nanogel size range the application area is widely spread. Smaller nanogels in the sub-100 nm area could be used for the intracellular delivery of encapsulated therapeutic cargo. In contrast, bigger nanogels could be applied to transport their freight into deeper areas of the hair follicle for therapeutic dermal applications. Incorporated stimuli-responsive groups would realize a controlled release and the degradation to biocompatible residues.Das übergeordnete Ziel dieser Arbeit war es neuartige Stimulus empfängliche Nanotransporter zu entwickeln, die biologische Barrieren überwinden und zusätzlich therapeutische Fracht transportieren und freisetzen können. Weiterhin sollte eine nano-medizinische Plattform hergestellt werden, der es möglich ist die sensible Fracht zu schützen und durch natürliche Stimuli kontrolliert am Zielort freizugeben. Neben therapeutischen Aspekten sollen die maßgeschneiderten Nanotransporter auch für diagnostische Ansätze genutzt werden. Im ersten Projekt dieser Thesis wurde ein pH spaltbarer Nanotransporter entwickelt, der genetisches Material befördern kann. Auf Basis der Einführung von Stimulus empfänglichen Acetalgruppen kann der Nanotransporter in saurem pH Milieu gespalten werden. Diese treten in den endosomalen Kompartimenten auf und bleiben bei neutralem pH-Wert, wie er in einer extrazellularen Matrix auftritt, stabil. Modifizierte Benzacetal-gruppen ermöglichen dabei einen Feinschliff der Spaltkinetik. Die Komplexierung und die Freisetzung der DNA aus Nanotransportern wurde mit verschiedenen Ansätzen analysiert. Die pH-getriggerte Spaltung der äußeren Aminschale resultiert in einem Zerfall der multivalenten Amin-Architektur. Aufgrund dessen ist die Fähigkeit der Bindung von genetischen Material reduziert. Dies ermöglicht eine kotrollierte Freisetzung der DNA. Mit Hilfe von in vitro Transfektions-Tests konnte gezeigt werden, dass pH-spaltbare dPG-Amine HeLa Zellen mit GFP-DNA transfizieren können. Die Spaltprodukte des Nanotransporters weisen im Vergleich zum abbauresistenten Goldstandard PEI eine signifikant reduzierte Toxizität auf. Die Synthese dieser spaltbaren Kern-Schale-Polymere ermöglicht eine neue Transportplattform für die Komplexierung sowie kontrollierter Freisetzung von genetischem Material, die durch den Verlust multivalenter Interaktionen genau am Zielort greifen kann. Zusätzlich wurde ein neuer Ansatz für die Synthese von Nanogelen entwickelt, die auf dendritischen Polyglycerin basieren. Die Methode der Thiol-Michael Nanofällung, die unter milden Reaktionsbedingungen durchgeführt wurde, benötigt weder einen Katalysator noch Tenside, um maßgefertigte Nanogele im Größenbereich von 100-1000 nm herzustellen. Die biokompatiblen Nanogele wurden sowohl für die Diagnostik als auch für topische Hautanwendungen benutzt. Zum ersten Mal wurden farbstoffmarkierte Nanogele als pH-Nanonsensoren genutzt, um den pH-Wert von Haarfollikeln (HF) in verschiedenen Tiefen in einem ex vivo Schweineohrmodell zu bestimmen. Die nicht-toxischen Nanogele wiesen ein hohes Potenzial für das Eindringungsvermögen über den follikulären Aufnahmeweg auf. Mit Hilfe einer automatisierten Analyse durch konfokale Laser-Scanning-Mikroskopie konnte der pH-Wert innerhalb des HFs genau bestimmt werden. Der pH Gradient erstreckte sich von Werten von 6,5 auf der Hautoberfläche bis hin zu Werten von 7,4 in tieferen Arealen des HFs mit einem starken pH-Anstieg über die ersten 300 µm. Diese Erkenntnis gibt eine klare Richtung für die Entwicklung von pH- responsiven DDS für den follikulären Wirkstofftransport. Weiterhin wurden funktionelle Nanogel-Peptid Konjugate entwickelt, die hydrophobe Wirkstoffe komplexieren und in der Haut freigeben können. Die Konjugation der maßgeschneiderten Peptidketten an Polyglycerin basierten Nanogelen konnte die Ladungskapazität sowie die Bindungsspezifität im anvisierten therapeutischen Modell-wirkstoff signifikant erhöhen. Tests zur Hautpenetration zeigten einen effizienten dermalen Wirkstofftransport sowie die Freigabe eines Photo- Sensibilisators der in der photo-dynamischen Krebstherapie eingesetzt werden kann. Darüber hinaus wurden kationische Nanogele, basierend auf dendritischen Polyglycerin und niedermolekularem PEI, für den Transport von siRNA verwendet. Das genetische Material wurde dabei durch die oben bereits erwähnte Thiol- Michael Reaktion eingekapselt. Die milde, bioorthogonale Reaktion befähigt die in situ Bindung von GFP-siRNA in die Nanogelmatrix. Mittels Acetalgruppen, die in das Nanogel eingebettet wurden, konnte ein pH-abhängiger Abbau der sub-100 nm Träger realisiert werden. Mittels in vitro Transfektion wurde gezeigt, dass pH-spaltbare Nanogele erfolgreich die GFP Bildung von HeLa Zellen unterdrücken können. Zusätzlich zeigten sich diese als weniger toxisch verglichen mit nichtspaltbarem PEI. Die kationische Nanogelplattform wird weiter für den Transport anti-entzündlicher siRNA für topische Hautanwendungen untersucht werden. Zusätzlich wurde die Bildung sowie der Einfluss der Proteinkorona auf dPG-basierte Nanogele mit unterschiedlichen Oberflächenladungen untersucht. Dabei zeigte sich, dass Nanogele mit einer höheren Oberflächenladung eine erhöhte Bindung des Blutproteins Humanalbumin aufweisen. Dies resultierte in einem erhöhten hydrodynamischen Radius sowie einer reduzierten Oberflächenladung des Nanogels. In Zukunft wäre ein Upscale des Herstellungsprozesses von Nanogelen denkbar, um Nanotransporter mittels eines Standardprotokolls in einer ein-Batch Prozedur herzustellen, um die Entwicklung zu einer nanomedizinischen Plattform zu ermöglichen. Erst kürzlich wurden Nanopartikel im Nanofällungsprozess mittels Mikrofluidik hergestellt.[219] Generell wird diese Technik für die Produktion von Mikropartikeln benutzt.[220] Diese sind besonders für die Anwendung des fortgeschrittenen Wirkstofftransports (z.B. die Einkapselung von biologischer Fracht wie lebenden Zellen[221]) nützlich. Außerdem besteht die Möglichkeit dieses Verfahren zu adaptieren, um Stimuli-sensitive Nanogele in einem kontinuierlichen Prozess in hohen Mengen herzustellen. Weiterhin ist es möglich die Nanogelplattform zu nutzen, um verschiedene therapeutische Proteine oder hydrophobe Wirkstoffe einzukapseln. Aufgrund des weiten Größenbereichs der Nanogele ist das Anwendungsspektrum sehr breit. Kleinere Nanogele im sub-100 nm Bereich könnten für den intrazellulären Transport eines eingekapselten therapeutischen Wirkstoffes genutzt werden. Im Gegensatz dazu könnten größere Nanogele dazu genutzt werden, um ihre Fracht in tiefere Areale des Haarfollikels für therapeutische dermale Anwendungen zu transportieren. In das Nanogel eingebaute, Stimuli-sensitive Gruppen würden eine kontrollierte Freigabe und die Zersetzung zu biokompatiblen Abbauprodukten realisieren

Automated Solvent-Free Polymerization of Hyperbranched Polyglycerol with Tailored Molecular Weight by Online Torque Detection

Polymerization processes with high reproducibility, traceability, and nontoxic compounds are required for biomedical applications. Here an automated solvent-free polymerization of hyperbranched polyglycerol has been established on a multiple-hundred gram scale. Performed is an anionic ring-opening multibranching (ROMB) polymerization with slow addition of glycidol. The solvent-free approach avoids commonly used organic solvents during the polymerization and work-up. Due to the automation of the polymerization process a high reproducibility and traceability is accomplished. The used reactor is equipped with an anchor stirrer and stirrer control, which measures the applied torque. A linear correlation of the increasing torque and the degree of polymerization is observed, which can be used to monitor the molecular weight in situ during the polymerization