Ny forskningsrapport från GlycoImaging

Igår publicerades en ny studie från Glyco-gänget. Jag tolkar studien som en säkerhetsstudie mest troligt som svar till EU:s patentverk. De som krånglat med patentgodkännandet. Studien ifråga har undersökt om MIP`sarna har nån påverkan på kroppens immunförsvar. En udda frågeställning,men troligtvis det EU vill ha svar på innan de godkänner patentet. Svaret de kommer få är ett rungande NEJ. 

Professor Anette Gjörloff-Wingren med team visar med studien att denna farhåga kan uteslutas.

In conclusion, SA-MIPs have a low degree of inflammatory properties, are not cytotoxic, and can be applicable for future in vivo studies.

Översatt : Sammanfattningsvis har SA-MIPs en låg grad av inflammatoriska egenskaper, är inte cytotoxiska och kan användas för framtida in vivo-studier. (in-vivo = inne i kroppen)

 

För att verkligen komma till botten med denna fråga har teamet varit flitiga i att använda HoloMonitor.

digital holographic cytometry (DHC) = HoloMonitor

Men till studien som är betitlad :

Molecularly Imprinted Polymers Exhibit Low Cytotoxic and Inflammatory Properties in Macrophages In Vitro

Published: 15 June 2022

Abstract

Molecularly imprinted polymers (MIPs) against sialic acid (SA) have been developed as a detection tool to target cancer cells. Before proceeding to in vivo studies, a better knowledge of the overall effects of MIPs on the innate immune system is needed. The aim of this study thus was to exemplarily assess whether SA-MIPs lead to inflammatory and/or cytotoxic responses when administered to phagocytosing cells in the innate immune system. The response of monocytic/macrophage cell lines to two different reference particles, Alhydrogel and PLGA, was compared to their response to SA-MIPs. In vitro culture showed a cellular association of SA-MIPs and Alhydrogel, as analyzed by flow cytometry. The reference particle Alhydrogel induced secretion of IL-1β from the monocytic cell line THP-1, whereas almost no secretion was provoked for SA-MIPs. A reduced number of both THP-1 and RAW 264.7 cells were observed after incubation with SA-MIPs and this was not caused by cytotoxicity. Digital holographic cytometry showed that SA-MIP treatment affected cell division, with much fewer cells dividing. Thus, the reduced number of cells after SA-MIP treatment was not linked to SA-MIPs cytotoxicity. In conclusion, SA-MIPs have a low degree of inflammatory properties, are not cytotoxic, and can be applicable for future in vivo studies.

Keywords: molecularly imprinted polymers; digital holographic cytometry; cytotoxicity; pro-inflammatory cytokines

1. Introduction

Nanoparticle-assisted cancer detection and monitoring have the potential for wider use in tumor diagnostics and treatment. We and others have previously investigated molecularly imprinted polymers (MIPs) that target different structures, such as glycans, peptides or proteins on the cell surfaces. Specifically, the monosaccharide sialic acid (SA) has been the focus of many studies, due to its role as a potential cancer biomarker. SA-MIPs have been demonstrated to target different cell lines in vitro, as shown by flow cytometry and cell imaging experiments. MIPs have also been frequently used and investigated in vivo, with promising results for both targeting and drug delivery. However, to continue using MIPs in vivo, further information regarding the overall effects of MIPs on the innate immune system is needed.

It is well documented that the physicochemical properties of nano- and submicron-sized particles are likely to influence their biological fate and actions of the particles in vivo. Nanoparticles often aggregate under physiological conditions, and it is known that aggregates of nanoparticles of an intermediate size (1–5 µm) are phagocytosed more readily than smaller or larger particles. 

At physiological conditions, phagocytosis of synthetic particles by monocytes and macrophage-derived cells plays a significant role in determining the fate of delivery systems utilizing nanoparticles. Phagocytosis is responsible for the failure of many drug delivery strategies using nanoparticles in vivo due to undesirable immune cell targeting. Moreover, protein adsorption to the surface due to the surface charge of the particles affects the interaction with the cells. If particles are removed by macrophages before reaching their intended destination in vivo they can induce an inflammatory response due to their interaction with phagocytosing cells. Therefore, understanding and monitoring nanoparticle phagocytosis by immune cells is critical for developing these particles for successful use in vivo.

 

Most leukocytes, including monocytes and macrophages express SA, and thereby SA-MIPs will target SA on those cells. The aim was to study the general impact of MIPs on the innate immune system regarding phagocytosing cells, using SA-MIPs. To study the impact of SA-MIPs on phagocytic cells, the monocytic/macrophage cell lines THP-1 and RAW 264.7 were chosen as classical phagocytosing cells. THP-1 and RAW 264.7 cells have both been extensively used to study macrophage functions, mechanisms, signaling pathways, and drug transport. SA-MIPs show the potential to interact with the cells, thus it is relevant to use SA-MIPs together with two different and well-characterized reference particles; aluminum adjuvant in the form of Alhydrogel^®  and poly(lactic-co-glycolic acid) (PLGA), for interaction studies with phagocytosing cells.

Inflammatory responses, elicited by nanoparticles, in particular, are important to control, since constant or hyperstimulation can result in chronic diseases. Macrophages are central mediators of innate immunity linked to chronic inflammation including anti-oxidative and pro-inflammatory responses as well as cell death. The reference particles Alhydrogel and PLGA were used to compare the possible inflammatory response and cytotoxicity caused by the SA-MIPs. To evaluate any inflammatory response by the SA-MIPs and the reference particles, induction and secretion of the pro-inflammatory cytokines IL-1β, TNF-α and IL-6 were investigated.

Cell proliferation upon culture in the presence of SA-MIPs was scrutinized using digital holographic cytometry (DHC). DHC is a non-phototoxic quantitative phase imaging technique that enables the monitoring of living cells. The DHC technique allows for long time monitoring of the cells, acquiring high time resolution images, which can be used for longitudinal tracking of individual cells. Tracking of individual cells using DHC demonstrated that the SA-MIPs had an impact on cell proliferation and cell division, but the reduction in cell numbers could not be attributed to SA-MIPs cytotoxicity.

Materials and Methods

2.13. Digital Holographic Cytometry (DHC) and Cell Tracking

RAW 264.7 cells were seeded in a Sarstedt lumox^® 96-well plate (Sarstedt, Germany) with 2 × 10⁴ cells per well and incubated overnight at 37 °C with 5% CO₂ allowing the cells to adhere. The medium in each well was renewed with either 40 µg/mL SA-MIPs, for treated cells, or culture medium for untreated cells. The lid was changed to Hololids for 96-well plates (PHIAB, Lund, Sweden), which enable cell imaging by the HoloMonitor M4 digital holographic cytometer (DHC) (PHIAB). The plates were placed on the motorized stages of HoloMonitor M4 in a standard 37 °C with 5% CO₂ incubator. Triplicated experiments were performed using 2 wells of treated and untreated cells in each experiment. Five positions in each well were chosen and images captured every 15 min for 48 h. Time-lapse imaging, image processing, segmentation and analysis were conducted with the App Suite software package (v3.5.0, PHIAB, Lund, Sweden) based on absolute values of refractive indexes for cells and culture medium. As the laser intensity is approximately 10 W/m² during imaging, and exposure time is less than 10 ms, it is assumed that the laser irradiation has only minimal effect on the physiological functions of the cells. App Suite was used for the tracking of individual cells. For each capturing position, cells were selected at time-point 0 h, which is the first image acquired in the 48 h time-lapse. When the cells divided, the two daughter cells were selected for further analysis and connected to the mother cell. This enables the calculation of the time of each cell division for individual cells. Based on known time-points for cell divisions and from which cell the daughter cells originated, a cell family tree was drawn for each cell selected at time-point 0 h using RStudio software (v.2021.09.1, RStudio, Boston, MA, USA).

Real-time holographic images were captured every 15 min during 48 h of RAW 264.7 cells cultured in the presence or absence of SA-MIPs. Examples of holographic images of cells incubated with 0 µg/mL SA-MIPs and 40 µg/mL SA-MIPs from a randomly selected area in the culture well at the first and last time-point of the time-lapse are shown (Figure 6a, b). SA-MIPs (40 µg/mL) were cultured together with RAW 264.7 cells for up to 48 h and the cell number was determined using DHC (Figure 6c). More than twice as many cells were identified in samples with cells grown in a culture medium only after 48 h compared to cells cultured in the presence of SA-MIPs.

Figure 6. DHC images in 2D and cell proliferation. Representative images from DHC time-lapse at time-point 0 h and 48 h of RAW 264.7 cells incubated with medium (a) or 40 µg/mL SA-MIPs (b). The SA-MIPs were added immediately before the start of the time-lapse and are seen as small dots surrounding the cells. The horizontal scale bars at the top in the images represent 300 µm and the vertical scale bars to the left represent the optical thickness −0.4 to 16 µm. Average number of cells visible in time-lapse frames over 48 h (c) of three independent experiments with ±SD. RAW 264.7 cells were seeded with medium for 24 h. Time-lapse imaging was initiated when 40 µg/mL SA-MIPs was added 24 h after cell seeding. This corresponds to time 0 in (c).

Figure 7. Schematic cell family trees for RAW-264.7 cells. RAW 264.7 cells were incubated with culture medium only (a) or with 40 µg/mL SA-MIP (b) for 48 h. Holographic images were captured every 15 min at several positions, thus creating 48 h time-lapse movies. For every capture position, cells in the first image (0 h) were selected and tracked until division. In the schematic cell family trees, each forking of the line indicates a cell division. The resulting daughter cells of each cell division were identified, and tracking continued throughout the 48 h time-lapse. In total 109 control cell family trees and 91 SA-MIPs treated cell family trees were analyzed.

5. Conclusions

We have demonstrated that SA-MIPs induced low induction and secretion of inflammatory cytokines that were comparable to phagocytosing cells cultured in the presence of the reference particles Alhydrogel and PLGA. The reduced number of cells upon incubation with SA-MIPs was not due to cytotoxicity but due to an attenuated cell cycle caused by the presence of the SA-MIP. The effects on the cell cycle were observed using a longitudinal investigation of individual cells and the results support the hypothesis that phagosomes also have regulatory functions with regard to the cell.

In summary, this suggests that synthesized MIPs would be applicable for future in vivo studies based on the low in vitro cytotoxicity and mild inflammatory properties of the SA-MIPs used in this study.

Min kommentar

Om mitt antagande stämmer (är 99% säker) har nu EU:s patentverk fått svar på de frågeställningar som var hindret för ett patentgodkännande.Med detta antagande kan vi se fram mot ett snyggt paketerat PM inom kort. Sannolikt kommer även Kina ge sitt godkännande samtidigt. 

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Tack till Oscar som hittade denna info och var vänlig att mejla över den.

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