Targeted delivery and endosomal cellular uptake of DARPin-siRNA bioconjugates: Influence of linker stability on gene silencing

Specific cell targeting and efficient intracellular delivery are major hurdles for the widespread therapeutic use of nucleic acid technologies, particularly siRNA mediated gene silencing. To enable receptor-mediated cell-specific targeting, we designed a synthesis scheme that can be generically used to engineer Designed Ankyrin Repeat Protein (DARPin)-siRNA bioconjugates. Different linkers, including labile disulfide-, and more stable thiol-maleimide- and triazole- (click chemistry) tethers were employed. Crosslinkers were first attached to a 3’-terminal aminohexyl chain on the siRNA sense strands. On the protein side thiols of a C-terminal cysteine were used as anchoring site for disulfide- and thiol-maleimide conjugate formation, while strain-promoted azido-alkyne cycloadditions were carried out at a metabolically introduced N-terminal azidohomoalanine. After establishing efficient purification methods, highly pure products were obtained. Bioconjugates of EpCAM-targeted DARPins with siRNA directed at the luciferase gene were evaluated for cell-specific binding, uptake and gene silencing. As shown by flow cytometry and fluorescence microscopy, all constructs retained the highly specific and high-affinity antigen recognition properties of the native DARPin. As expected, internalization was observed only in EpCAM-positive cell lines, and predominantly endolysosomal localization was detected. Disulfide linked conjugates showed lower serum stability against cleavage at the linker and thus lower internalization into endosomes compared to thiol-maleimide- and triazole-linked conjugates, yet induced more pronounced gene silencing. This indicates that the siRNA payload needs to be liberated from the protein in the endosome. Our data confirm the promise of DARPin-siRNA bioconjugates for tumor targeting, but also identified endosomal retention and limited cytosolic escape of the siRNA as the rate-limiting step for more efficient gene silencing

Das Problem des Nocebo-Effekts beim Einsatz von Generika im Osten Österreichs

Ein Generikum ist ein Arzneimittel, das die gleiche qualitative und quantitative Zusammensetzung aus Wirkstoffen sowie die gleiche Darreichungsform wie das Referenzarzneimittel aufweist. Die Qualitätsanforderungen sind für Generikahersteller gleich wie für alle anderen Arzneimittelproduzenten. Bei den meisten Arzneimitteln ist ein Austausch zwischen Generikum und Originalpräparat unproblematisch. Der Nocebo-Effekt von Arzneimitteln ist nicht auf Grund der Zusammensetzung oder Eigenschaften einer Substanz und Behandlung erklärbar. Er kann den Gesundheitszustand der Patienten, meist begründet durch deren negative Erwartungshaltung, schädigen. Es wurde eine Literaturrecherche und eine Umfrage von Patienten und Apothekern in drei Apotheken (Großstadt, Stadt und Gemeinde) durchgeführt um mehr über den Zusammenhang von Generika und Nocebo-Effekt zu erfahren. Literatur aus den USA, Irland und Deutschland verdeutlicht die Skepsis vieler Patienten, Ärzte und Apotheker. Vor allem bei Austausch von Präparaten kommt es zu Problemen und häufig schlechterer Compliance. Außerdem wird die Wichtigkeit der Aufklärung der Patienten durch das medizinische Fachpersonal betont, um Nocebo-Effekte zu vermeiden. Die Patienten-Umfrage zeigt, dass es doch einige Patienten gibt, die gegenüber Generika skeptisch sind. Vor allem in der Gemeinde-Apotheke ist dies deutlich. Interessant ist, dass aber gerade dort die wenigsten Personen über vermehrte Nebenwirkungen klagen. Bei den Apothekern herrscht teilweise unterschiedliche Meinung über die Gleichwertigkeit von Generika. Allerdings sind nur wenige Pharmazeuten befragt worden und Rückschlüsse daher schwierig. Ob der Nocebo-Effekt wirklich beteiligt an vermehrten Nebenwirkungen ist und welche Rolle er spielt, ist schwer zu sagen. Jedenfalls zeigt die Arbeit, dass viele Patienten unsicher sind. Eine umfangreiche Aufklärung, unter anderem durch Arzt und Apotheker, stellt daher eine gute Möglichkeit dar das Auftreten eines Nocebo-Effekts zu vermeiden.A generic drug is a medicinal product which consists of the same qualitative and quantitative composition of active substances and has the same application form as the reference product. The quality standards are the same for producers of generics as for producers of any medicinal product. Normally the substitution with a generic instead of the original medicinal product is unproblematic. The nocebo-effect of drugs cannot be explained due to composition or characteristics of a substance or treatment. It can affect the health state of the patient in a bad way, mostly because of the patient‘s negative expectations. Literature research and a survey of patients and pharmacists in three different pharmacies (major city, city and village) were carried out to find out more about the relation of generics and the nocebo-effect. Literature from the USA, Ireland and Germany shows that a lot of patients, physicians and pharmacists are skeptical. Above all the most problems occur due to the exchange of the medicinal products. They can even lead to poor compliance. Furthermore it is shown that the education of patients through medicinal professionals plays an important role to avoid a nocebo-effect. The patient-survey shows that there are indeed some patients who are skeptical in matters of generics. Most of them were seen in the pharmacy in the village. But it is interesting that also there the fewest persons complain about additional adverse effects. The opinions of the pharmacists differ related to the equivalence of generics. However, very few pharmacists were interviewed. So it is not easy to make a proper conclusion. It is difficult to say if the nocebo-effect really causes increased adverse effects and what role it plays. Anyway the work shows that a lot of patients are insecure. So the right information through physicians and pharmacists is a great possibility to avoid the occurrence of a nocebo-effect