Targeting Somatostatin Receptors with Peptide-functionalized Silica Nanoparticles

ABSTRACT Nano-sized synthetic drug carriers comprise a valuable addition to oncology armaments, opening new avenues for improved delivery of anticancer therapies. Nanocarriers designed to bind surface receptors of tumor cells by virtue of bearing cognate high affinity ligands, also called as actively-targeted nanocarriers, have drawn a lot of attention during the last decade, promising to enable selective tumor accumulation via ligand-receptor interactions. The present work encompasses development and early in vitro evaluation efforts with a particular type of such a targeted nanocarrier [nanoparticles of mesoporous silica (SiO2), MSN], functionalized with short peptide ligands of somatostatin receptors (SSTR), frequently abundant in tumors. The synthesized targeted MSN, as well as control inactive peptide–functionalized counterparts, were characterized by physicochemical means and evaluated for their ability to bind to SSTR and enter living cells in vitro. We sequentially studied MSN in protein-depleted and serumenriched media – the latter condition involved adsorption of proteins to MSN surface (formation of the so-called protein corona), which invariably happens in vivo and affects cellular interactions of nanomaterials. We demonstrate that the targeted MSN can bind SSTR not only under the rectified conditions, but also in the presence of protein adsorption. Ultimately, we show that SSTR targeting leads to a decreased cellular accumulation of MSN with protein corona, which highlights the complexity of nanobiointeractions and urges to re-consider the applicability of the conventional model of tumor receptor targeting with nanocarriers to somatostatin receptors. The work covers a number of chemical (MSN design, synthesis and surface functionalization) and molecular biology (in vitro targetability evaluation and generation of relevant tools, receptor signaling, endocytosis) aspects of high relevance for development and characterization of targeted nanocarriers and as such should be of interest to the broad community working with nanomedicines and targeted delivery. The discussion of the original experimental evidence is preceded by a critical literature review, introducing the current concepts of nanocarriermediated delivery in oncology. KEYWORDS: nanocarrier, tumor receptor targeting, ligand-receptor interaction, nanoparticle corona, mesoporous silica nanoparticle, somatostatin receptor, cAMP.TIIVISTELMÄ Nanokokoiset lääkkeiden kantajat avaavat uusia keinoja syöpähoitojen parantamiseksi. Nanokantajat, joiden pinnalla olevat korkean affiniteetin ligandit ovat suunniteltu sitomaan tuumorisolujen pintareseptoreita (ns. aktiivisesti kohdennetut nanokantajat), mahdollistavat nanohiukkasten selektiivisen kertymisen syöpäkasvaimeen ligandi-reseptori-vuorovaikutusten kautta. Tämä työ kattaa kehityksen ja varhaiset in vitro-kokeet kohdennetulla nanokantajahiukkasella [mesohuokoisen piidioksidin (SiO2) nanohiukkaset, MSN], jotka on funktionalisoitu somatostatiinireseptorien lyhyillä peptidiligandeilla (SSTR), joita esiintyy usein runsaasti kasvaimissa. Syntetisoidut kohdennetut MSN-hiukkaset sekä kontrollina käytetyt inaktiiviset peptidifunktionaaliset vastineet karakterisoitiin fysikaaliskemiallisilla menetelmillä sekä arvioitiin niiden kykyä sitoutua SSTR:ään ja päästä eläviin soluihin in vitro. Tutkimus suoritettiin MSN-hiukkasilla sekä proteiinipitoisessa että seerumilla rikastetussa ravintoliuoksessa – jälkimmäisessä tapauksessa proteiinit kiinnittyivät MSN:n pinnalle (ns. proteiinikoronan muodostuminen), joka tapahtuu in vivo ja vaikuttaa nanomateriaalin ja solun väliseen vuorovaikutukseen. Osoitamme, että kohdennettu MSN voi sitoa SSTR:ää paitsi puhdistetuissa olosuhteissa, myös proteiinikoronan läsnäollessa. Lopuksi osoitamme, että SSTR-kohdennuksessa proteiinikorona vähentää MSN:n kertymistä soluihin, mikä korostaa nanobiovuorovaikutusten monimutkaisuutta, ja kyseenalaistaa nanokantajien kanssa käytetyn tavanomaisen kasvainreseptorikohdennusmallin soveltuvuutta somatostatiinireseptoreihin. Työssä käytettiin kemiallisia (MSN-suunnittelu, synteesi ja pinnan funktionalisointi) ja molekyylibiologisia (kohdennettavuuden arviointi in vitro, työkalujen luominen, reseptorisignalointi ja endosytoosi) menetelmiä, jotka ovat olennaisia kohdennettujen nanokantajien kehitykselle ja karakterisoinnille. Tulosten oletetaan kiinnostavan laajaa yhteisöä, joka työskentelee nanolääkkeiden ja niiden kohdentamisen parissa. Kokeellista osuutta edeltää kirjallisuuskatsaus, jossa esitellään nanokantaja-välitteisen kuljetuksen nykyiset käsitteet syöpäbiologiassa. AVAINSANAT: nanokantaja, kasvainreseptorikohdistus, ligandi-reseptorivuorovaikutus, nanohiukkasen korona, mesohuokoinen piidioksidi-nanopartikkeli, somatostatiinireseptori, cAMP

IGIST - A Kinetic Bioassay for Pertussis Toxin Based on Its Effect on Inhibitory GPCR Signaling

Detection of pertussis toxin (PTX) activity is instrumental for the development and manufacturing of pertussis vaccines. These quality and safety measures require thousands of mice annually. Here, we describe Interference in Gαi-mediated Signal Transduction (iGIST), an animal-free kinetic bioassay for detection of PTX, by measuring its effect on inhibitory G protein-coupled receptor (GPCR) signaling. PTX ADP-ribosylates inhibitory α-subunits of the heterotrimeric G proteins, thereby perturbing the inhibitory GPCR signaling. iGIST is based on HEK293 cells coexpressing a somatostatin receptor 2 (SSTR2), which is an inhibitory GPCR controllable by a high-affinity agonist octreotide; and a luminescent 3′5′-cyclic adenosine monophosphate (cAMP) probe. iGIST has a low sensitivity threshold in the pg/mL range of PTX, surpassing by 100-fold in a parallel analysis the currently used in vitro end-point technique to detect PTX, the cluster formation assay (CFA) in Chinese hamster ovary cells. iGIST also detects PTX in complex samples, i.e., a commercial PTX-toxoid-containing pertussis vaccine that was spiked with an active PTX. iGIST has an objective digital readout and is observer independent, offering prospects for automation. iGIST emerges as a promising animal-free alternative to detect PTX activity in the development and manufacturing of pertussis vaccines. iGIST is also expected to facilitate basic PTX research, including identification and characterization of novel compounds interfering with PTX.</p

In vitro Targetability Validation of Peptide-Functionalized Mesoporous Silica Nanoparticles in the Presence of Serum Proteins

Demonstration of receptor-mediated targeting of nanoparticles to specific organs and/or cell types is an integral aim in many bionanomedicine development projects. However, engagement of targeted receptors with ligands on nanocarriers, which is the cornerstone of the active targeting concept, is challenging to study under biologically relevant conditions and thus often stays overlooked. In this work, we utilize an in-house established bioassay for in vitro targetability validation of mesoporous silica nanoparticles (MSNs), functionalized with high-affinity peptide ligands to somatostatin receptors via protective group chemistry, ensuring the correct orientation of the peptide's pharmacophore. We demonstrate that targeted nanoparticles, but not scrambled peptide-decorated counterparts, specifically engage the targeted receptors in living cells in culture media containing serum protein. The importance of being able to exclude false positives originating from the premature detachment of targeting peptides from the MSNs is highlighted

Targeting Somatostatin Receptors By Functionalized Mesoporous Silica Nanoparticles - Are We Striking Home?

The concept of delivering nanoformulations to desired tissues by means of targeting membrane receptors of high local abundance by ligands anchored to the nanocarrier has gained a lot of attention over the last decade. Currently, there is no unanimous opinion on whether surface functionalization of nanocarriers by targeting ligands translates into any real benefit in terms of pharmacokinetics or treatment outcomes. Having examined the published nanocarriers designed to engage with somatostatin receptors, we realized that in the majority of cases targetability claims were not supported by solid evidence of targeting ligand-targeted receptor coupling, which is the very crux of a targetability concept. Here, we present an approach to characterize targetability of mesoporous silica-based nanocarriers functionalized with ligands of somatostatin receptors. The targetability proof in our case comes from a functional assay based on a genetically-encoded cAMP probe, which allows for real-time capture of receptor activation in living cells, triggered by targeting ligands on nanoparticles. We elaborate on the development and validation of the assay, highlighting the power of proper functional tests in the characterization pipeline of targeted nanoformulations

PIM-induced phosphorylation of Notch3 promotes breast cancer tumorigenicity in a CSL-independent fashion

Dysregulation of the developmentally important Notch signaling pathway is implicated in several types of cancer, including breast cancer. However, the specific roles and regulation of the four different Notch receptors have remained elusive. We have previously reported that the oncogenic PIM kinases phosphorylate Notch1 and Notch3. Phosphorylation of Notch1 within the second nuclear localization sequence of its intracellular domain (ICD) enhances its transcriptional activity and tumorigenicity. In this study, we analyzed Notch3 phosphorylation and its functional impact. Unexpectedly, we observed that the PIM target sites are not conserved between Notch1 and Notch3. Notch3 ICD (N3ICD) is phosphorylated within a domain, which is essential for formation of a transcriptionally active complex with the DNA-binding protein CSL. Through molecular modeling, X-ray crystallography, and isothermal titration calorimetry, we demonstrate that phosphorylation of N3ICD sterically hinders its interaction with CSL and thereby inhibits its CSL-dependent transcriptional activity. Surprisingly however, phosphorylated N3ICD still maintains tumorigenic potential in breast cancer cells under estrogenic conditions, which support PIM expression. Taken together, our data indicate that PIM kinases modulate the signaling output of different Notch paralogs by targeting distinct protein domains and thereby promote breast cancer tumorigenesis via both CSL-dependent and CSL-independent mechanisms.</p