DE102022103893A1 - Membrane vesicles containing a spectroscopically detectable indicator and their use in a method for detecting phagocytic function in biological cells - Google Patents

Abstract

The invention relates to a method for detecting the phagocytosis function in biological cells in vitro, which comprises at least the following steps: - providing membrane vesicles which contain a spectroscopically detectable indicator; - contacting the membrane vesicles with target cells whose phagocytosis function is to be tested ;- At least partial incorporation of the membrane vesicles by phagocytosis in those target cells which are capable of phagocytosis;- Detection of the incorporation of membrane vesicles by observing a spectroscopically detectable property of the indicator and, if necessary, determining a change in this property in indicators located within target cells as compared to indicators in unincorporated membrane vesicles. Preferably, these membrane vesicles are lipid vesicles and the spectroscopically detectable property of the indicator is fluorescence -value inducing the presence and/or concentration of certain molecules or ions, preferably Ca2+, or the presence of certain enzymes.Further aspects of the invention relate to membrane vesicles containing a spectroscopically detectable indicator and their use in detecting phagocytic function in biological cells and tissues, and kits comprising such membrane vesicles.

Description

Background of the Invention

Phagocytosis is a central function for various cell types. These include epithelial cells of the digestive tract, scavenger cells of the immune system and other cell types that absorb substances and particles from their environment and metabolize them further. During phagocytosis, external particles are internalized by invagination and constriction of the cell membrane via actin polymerization. The phagocytosis is specifically triggered by signal molecules (markers) on the external particles. However, the cell can also absorb non-specific particles and liquid (pinocytosis) from its environment.

The investigation of phagocytosis with regard to extent, speed, signal cascade and further fate of the internalized phagosomes plays an important role in many areas of biology, pharmacy and medicine.

A typical example is the functional proof of a correctly differentiated retinal pigment epithelium (RPE). A main function of this epithelium is the uptake and metabolic processing of so-called photoreceptor gut segments (POS), approximately 1 µm in size, membrane-covered constrictions of the photoreceptors in the retina. These POS contain cellular waste products and spent visual pigment. The constricted POS are specifically recognized by the RPE and taken up by phagocytosis. Since there is great therapeutic interest in RPEs and these can be generated from primary cells, embryonic stem cells and induced pluripotent stem cells, meaningful proof of function is very important.

Currently, phagocytosis assays are mostly used as evidence of the central function using fluorescent polymer particles (see e.g. Tsuboi S. and Meerloo J. (2007) in J. Biol. Chem. 282(47):34194-203) or fluorescently labeled POS ( obtained from animal retinas e.g., Westenskov et al (2012) Invest Ophthalmol Vis Sci 53:6282-6290).

Analogous proof of function in cells of the immune system (macrophages) is achieved with the help of inactivated fluorescently labeled bacteria (e.g. Berger et al. (2010), Nature Immunol. 11:920-927).

However, these approaches to the detection of phagocytic function have serious disadvantages. The use of bacteria cannot be applied to all biological questions and polymer beads do not allow differentiation from non-specific uptake, which all cells show to a certain extent. To detect specific, marker-driven phagocytosis, the markers in question would have to be brought to the surface of the polymer particles via chemical coupling reactions. Once internalized, the polymer beads would remain unchanged inside the cell. Since the cell membrane is only about 6 nm thick, an optical microscope cannot tell whether a particle is located on the outside or inside of the membrane. Their resolution limit is in the range of a few hundred nm. The information content of such phagocytosis assays is therefore low. Your advantage is the simple implementation.

The advantage of phagocytosis assays based on biogenic POS is their close proximity to physiological reality. In contrast, the cost of their production is immense. Performing a phagocytosis assay on a 33 mm ² artificial RPE first requires the processing of 80 porcine retinas, which must be dissected from the eyes of slaughtered animals. The POS still have to be purified and functionalized with fluorescent molecules before they can be used in the assay. This resource-binding path is not feasible for routine applications and the ultracentrifuges required for this are not available in every laboratory. In addition, standardization can hardly be achieved with particles from a biogenic source, which also react very sensitively to environmental conditions (especially light). Furthermore, without cell-consuming analysis steps, it is also not possible to distinguish between POS on the outside and inside of the RPE cells. Since the POS are released from the photoreceptors for phagocytosis in the RPE, they are specifically internalized very efficiently. Unfortunately, it is now no longer possible to use POS to determine non-specific uptake, since there are no marker-free POS.

Against this background, a main object of the invention is to provide new and improved means for detecting the phagocytosis function in biological cells, with which the above disadvantages of the prior art can be largely or completely avoided. In particular, the detection of the phagocytosis function should be as simple as possible and also allow a differentiation between specific and non-specific phagocytosis.

This object is achieved according to the invention by the method according to claim 1 using membrane vesicles which contain a spectroscopically detectable indicator, and by the membrane vesicles according to claim 15. Further aspects and advantageous embodiments of the invention result from the further claims chen and are explained in more detail in the following description.

Description of the invention

• • es kann sowohl eine spezifische (mit Oberflächenmarkern) als auch eine unspezifische Phagozytose (ohne Oberflächenmarker) von Membranvesikeln veranlasst und unterschieden werden
• • es ist weder eine Fixierung von Gewebe erforderlich, noch wird sonst die Zellphysiologie mittelfristig beeinflusst; der Assay könnte daher mehrfach am selben Gewebe durchgeführt werden
• • die Verfahrensparameter bzw. eingesetzten Partikel entsprechen hinsichtlich Größe, Chemie und Mechanik weitgehend physiologischen Bedingungen
• • die Phagozytose, d.h. Internalisierung der Membranvesikel, wird durch ein einfaches, typischerweise nicht bildgebendes Verfahren festgestellt
• • es kann eine ratiometrische Größe als Read-Out benutzt werden, welches ermöglicht, von äußeren Faktoren unbeeinflusst zu messen
• • es sind keine überkomplexen Präparationsroutinen erforderlich
• • das erfindungsgemäße Verfahren ermöglicht einen weitergehenden Nachweis der Funktionalität des zu untersuchenden Gewebes als Nachweisverfahren des Standes der Technik, da neben den reinen Phagozytoseschritten auch noch die korrekte Sekretion von funktionellen Faktoren und die lysosomale Metabolisierung belegt werden können.

With the present invention, a particle platform and a method for the functional routine investigation of phagocytosis in biological cells, in particular in RPE cells and macrophages, is provided, which is characterized by a large number of advantageous properties:

• • Both a specific (with surface markers) and a non-specific phagocytosis (without surface markers) of membrane vesicles can be induced and differentiated
• • It is not necessary to fix tissue, nor is the cell physiology influenced in the medium term; the assay could therefore be performed multiple times on the same tissue
• • The process parameters and particles used largely correspond to physiological conditions in terms of size, chemistry and mechanics
• • The phagocytosis, ie internalization of the membrane vesicles, is determined by a simple, typically non-imaging method
• • A ratiometric value can be used as a read-out, which allows measurements to be made without being influenced by external factors
• • no overly complex preparation routines are required
• • the method according to the invention enables a more extensive detection of the functionality of the tissue to be examined as a detection method of the prior art, since in addition to the pure phagocytosis steps the correct secretion of functional factors and the lysosomal metabolism can also be proven.

• - Bereitstellen von Membranvesikeln, die einen spektroskopisch nachweisbaren Indikator beinhalten;
• - Kontaktieren der Membranvesikel mit Targetzellen, deren Phagozytose-Funktion getestet werden soll;
• - mindestens teilweise Inkorporation der Membranvesikel durch Phagozytose in solche Targetzellen, die zur Phagozytose in der Lage sind;
• - Nachweis der Inkorporation von Membranvesikeln durch Beobachtung einer spektroskopisch nachweisbaren Eigenschaft des Indikators und gegebenenfalls (d.h. falls eine Inkorporation stattfindet) Feststellung einer Änderung dieser Eigenschaft bei Indikatoren, die sich innerhalb von Targetzellen befinden, im Vergleich zu Indikatoren in nicht-inkorporierten Membranvesikeln.

The method according to the invention for detecting the phagocytosis function in biological cells in vitro comprises at least the following steps:

• - Providing membrane vesicles containing a spectroscopically detectable indicator;
• - Contacting the membrane vesicles with target cells whose phagocytic function is to be tested;
• - at least partial incorporation of the membrane vesicles by phagocytosis into target cells capable of phagocytosis;
• - Detection of the incorporation of membrane vesicles by observing a spectroscopically detectable property of the indicator and optionally (ie if incorporation takes place) determining a change in this property in indicators located within target cells compared to indicators in non-incorporated membrane vesicles.

The membrane vesicles used according to the invention are not particularly limited in principle, but are advantageously as close to physiology as possible (in terms of size, deformability, membrane structure and composition), and are preferably selected from the group comprising lipid vesicles, including liposomes and other unilamellar or multilamellar vesicles.

Artificial vesicles of different sizes can be easily produced from natural lipids and are referred to as liposomes, large unilamellar vesicles (LUV) or giant unilamellar vesicles (GUV), depending on their size. To differentiate between specific and non-specific phagocytosis, these can be provided with surface markers that trigger specific phagocytosis.

The sizes of these membrane vesicles are typically in a range from 50 nm to 50 μm, preferably from 300 nm to 2 μm.

The spectroscopically detectable property of the indicator is typically luminescence, especially fluorescence. Preferably, both the substrate and the product of the indicator are luminescent or fluorescent. In this case, the measurement can be carried out ratiometrically, i.e. by dividing the product fluorescence by the substrate fluorescence. Ratiometric assays are insensitive to fluctuations in excitation and detection efficiencies.

A preferred embodiment of the method according to the invention is characterized in that the change in the spectroscopically detectable property of the indicator is caused by a specific parameter of the cell interior.

In more specific embodiments, the specific parameter of the cell interior is selected from the group consisting of pH, the presence and/or concentration of certain molecules or ions, for example Ca ²⁺ , K ⁺ , Cl ^- , preferably Ca ²⁺ , the presence of certain Includes enzymes, in particular phosphatases and esterases, inside the cell.

Accordingly, the change in the spectroscopically detectable property of the indicator can be caused, for example, by the difference in the pH value or in the Ca ²⁺ concentration inside the cell compared to the pH value or the Ca ²⁺ concentration outside the cell or by an enzymatic conversion of the indicator be initiated inside the cell.

In other more specific embodiments of the invention, the indicator is selected from the group consisting of pH indicators, in particular fluorescence-based pH indicators, fluorescent esterase substrates, fluorescent phosphatase substrates, cell-specific enzyme substrates, in particular fluorescence-based cell-specific enzyme substrates, Ca ²⁺ indicators, in particular fluorescent based Ca ²⁺ indicators.

An embodiment of the present invention is characterized in that all or part of the membrane vesicles are provided with surface markers which trigger specific phagocytosis.

In principle, these surface markers are not particularly restricted, but are preferably selected from the group comprising lipids, in particular phosphatidylserine, sugars, in particular mannose, proteins, in particular transferrin and galectin-3, and lipid-anchored markers, in particular integrins, tetraspanins, glycans.

In an advantageous embodiment of the invention, phagocytosis is initiated by the lipid phosphatidylserine.

In further more specific embodiments of the invention, vesicles with surface markers that trigger specific phagocytosis, e.g. phosphatidylserine, and vesicles without surface markers are used to quantify specific, marker-mediated phagocytosis against the non-specific background.

The membrane vesicles used according to the invention can also be provided with additional components, in particular transmembrane proteins and/or an embedding matrix for the indicator.

In principle, the biological cells to be examined are not particularly restricted. However, the biological target cells are preferably epithelial cells, in particular cells of the retinal pigment epithelium (RPE) or macrophages.

In the case of RPE cells (and macrophages), the architecture of the indicator particles (i.e. the membrane vesicles) is particularly simple, since the phagocytosis-triggering marker is not a (glyco)protein attached to the lipid membrane, but the presence of the lipid phosphatidylserine in the outer side of the lipid bilayer. Functional RPE and macrophages secrete (among others) the factors MFG-E8 and PROS-1 into the apical medium, which bind to the phosphatidylserines of the POS and the indicator particles. These in turn couple to the MerTK and αVβ5 integrin receptors, which are presented on the surface of RPE and macrophages.

The coupling that has taken place then triggers the phagocytosis of the particle. Once the particle/membrane vesicle has been ingested, it fuses with lysosomes, which contain digestive enzymes and lower the pH inside the vesicle. If this pH value change or the enzymatic conversion is linked to a spectral change in the fluorescence indicator, the recording can be monitored spectroscopically. This approach typically no longer requires microscopic examination, but makes phagocytosis amenable to measurement with fluorescence spectrometers (and plate readers).

In a specific embodiment, the detection method according to the invention is characterized in that it is a quantitative detection method and the change in a spectroscopically detectable property of the indicator is determined by measuring this property before incorporation into target cells and after incorporation into target cells and the ratio of these two values is determined.

Further related aspects of the present invention relate to the use of membrane vesicles containing a spectroscopically detectable indicator for the detection of phagocytic function in biological cells and multicellular systems, in particular tissues, and kits with essential components for performing a detection of phagocytic function in biological Cells in vitro comprising membrane vesicles containing a spectroscopically detectable indicator, preferably aliquoted into ready-to-use portions of the dispersion. Optionally, further components can be added, in particular those components whose presence enables or ensures a negative control (no uptake of the vesicles whatsoever) or a positive control (complete uptake of all vesicles that come into contact with the cell membrane).

Such a kit can include, in particular, vesicles with surface markers, which trigger specific phagocytosis, eg phosphatidylserine, and vesicles without surface markers, for quantifying specific, marker-mediated phagocytosis against the non-specific background.

A still further aspect of the invention relates to membrane vesicles which contain a spectroscopically detectable indicator and are characterized in that they are lipid vesicles and the indicator is selected from the group consisting of pH indicators, in particular fluorescence-based pH indicators, fluorescent esterase substrates , fluorescent phosphatase substrates, cell-specific enzyme substrates, in particular fluorescence-based cell-specific enzyme substrates, Ca ²⁺ indicators, in particular fluorescence-based Ca ²⁺ indicators.

character list

• 1 shows schematically a specific embodiment of the phagocytosis assay according to the invention: (a) secretion of MFG-E8 and PROS-1, (b) binding of MFG-E8 and PROS-1 to the phosphatidyserine of the lipid bilayer of the vesicle, (c) coupling of the vesicle to the Cell membrane via MerTK (on MFG-E8) and/or αVβ5 integrin (on PROS-1), (d) initiation of phagocytosis, (e) incorporation of the vesicle, (f) metabolic conversion of the fluorescent tracer with concomitant change in its fluorescence spectrum.
• 2 shows inverted fluorescence images of incorporated liposomal-encapsulated pH indicators in intact RPE cultures.

The following examples are intended to explain the invention in more detail, but without restricting it to the specific parameters and conditions in each case.

EXAMPLE 1

Detection of phagocytosis in RPE cells

As a qualitative proof of concept, liposomes with and without phosphatidylserine (PS) were prepared in two different diameters and loaded with a fluorescent pH indicator (Dyomics DY344IN; transition point around pH 5, isosbestic excitation at approx. 363 nm, fluorescence maximum of the basic form at 453 nm, the acidic form at 535 nm). The standard filter combinations of plate readers do not match the spectral characteristics of this indicator, so the read-out was performed on a confocal microscope. This too could only detect the acidic, long-wave form of the indicator. The results of this measurement are therefore absolute, not ratiometric. Commercial kits for carrying out the assay should preferably be equipped with a fluorescent tracer that allows long-wavelength excitation in the visible spectral range and has emissions for acidic and basic forms bathochromic to cellular autofluorescence.

The results of a phagocytosis assay using a 42-day-old RPE-UKBi-P3 culture on a Transwell ^® insert are in 2 shown. The CLSM images were converted to grayscale and inverted for better reception.

1. 1. Bereitstellung von vier liposomalen Formulierungen des verkapselten Indikators in isotonischer NaCl-Lösung:
   1. a. Cholesterin 50%, Phosphatidylcholin (PC) 50%, d = 400 nm (Kontrolle)
   2. b. Cholesterin 50%, PC 50%, d = 1000 nm (Kontrolle)
   3. c. Cholesterin 47%, PC 47%, PS 6%, d = 400 nm
   4. d. Cholesterin 47%, PC 47%, PS 6%, d = 1000 nm
2. 2. Einstellung der Liposomenkonzentrationen auf 5×10⁸ ml^-1
3. 3. Medium von den Zellen entfernen. Zugabe Kulturmedium + 30 % Serum, Inkubation für 1 h bei 37 °C. Volumen im Transwell^®: Apikal: 40 µl, Basal: 200 µl
4. 4. Zugabe von 10 µl Liposomendispersion; Eingesetzte Menge/well: 5×10⁶ (^~10 Partikel/Zelle)
5. 5. Inkubation für 3 h bei 37 °C
6. 6. Read-Out am konfokalen Fluoreszenzmikroskop:
   1. a. Abziehen und mit Zellseite zum Objektiv in Tropfen Medium auf ein Deckglas legen
   2. b. Bildaufnahme mit Anregung bei 405 nm und Emission von 475 - 650 nm

The protocol of the assay is:

1. 1. Provision of four liposomal formulations of the encapsulated indicator in isotonic NaCl solution:
   1. a. cholesterol 50%, phosphatidylcholine (PC) 50%, d = 400 nm (control)
   2. b. Cholesterol 50%, PC 50%, d = 1000 nm (control)
   3. c. Cholesterol 47%, PC 47%, PS 6%, d = 400 nm
   4. i.e. Cholesterol 47%, PC 47%, PS 6%, d=1000nm
2. 2. Adjustment of the liposome concentrations to 5×10 ⁸ ml ^-1
3. 3. Remove medium from cells. Addition of culture medium + 30% serum, incubation for 1 h at 37 °C. Volume in the Transwell ^® : apical: 40 µl, basal: 200 µl
4. 4. Addition of 10 µl liposome dispersion; Amount used/well: 5×10 ⁶ ( ^~ 10 particles/cell)
5. 5. Incubation for 3 h at 37°C
6. 6. Read-Out on the confocal fluorescence microscope:
   1. a. Peel off and place in drops of medium on a coverslip with the cell side toward the objective
   2. b. Image acquisition with excitation at 405 nm and emission from 475 - 650 nm

Claims (18)

Method for detecting the phagocytic function in biological cells in vitro, characterized in that it comprises at least the following steps: - providing membrane vesicles containing a spectroscopically detectable indicator; - Contacting the membrane vesicles with target cells whose phagocytic function is to be tested; - at least partial incorporation of the membrane vesicles by phagocytosis into target cells capable of phagocytosis; - Detection of the incorporation of membrane vesicles by observing a spectroscopically detectable property of the indicator and, if appropriate, determining a change in this property in indicators located within target cells compared to indicators in non-incorporated membrane vesicles. procedure after claim 1 , characterized in that the membrane vesicles are selected from the group consisting of lipid vesicles including liposomes as well as other unilamellar or multilamellar vesicles. Procedure according to one of Claims 1 - 2 , characterized in that the spectroscopically detectable property of the indicator is luminescence, in particular fluorescence. Procedure according to one of Claims 1 - 3 , characterized in that the change in the spectroscopically detectable property of the indicator is caused by a specific parameter of the cell interior. procedure after claim 4 , characterized in that the specific parameter of the cell interior is selected from the group which the pH, the presence and / or concentration of certain molecules or ions, preferably Ca ²⁺ , and the presence of certain enzymes, in particular phosphatases and esterases, im Includes cell interior. procedure after claim 4 or 5 , characterized in that the change in the spectroscopically detectable property of the indicator by the difference in the pH value or in the Ca ²⁺ concentration inside the cell compared to the pH value or the Ca ²⁺ concentration outside the cell or by an enzymatic reaction of the indicator inside the cell. Procedure according to one of Claims 1 - 6 , characterized in that the indicator is selected from the group consisting of pH indicators, in particular fluorescence-based pH indicators, fluorescent esterase substrates, fluorescent phosphatase substrates, cell-specific enzyme substrates, in particular fluorescence-based cell-specific enzyme substrates, Ca ²⁺ indicators, in particular fluorescence-based Ca ²⁺ indicators. Procedure according to one of Claims 1 - 7 , characterized in that all or part of the membrane vesicles are provided with surface markers which trigger specific phagocytosis. procedure after claim 8 , characterized in that the surface markers are selected from the group comprising lipids, in particular phosphatidylserine, sugars, in particular mannose, proteins, in particular transferrin and galectin-3, and lipid-anchored markers, in particular integrins, tetraspanins, glycans. procedure after claim 8 or 9 , characterized in that phagocytosis is triggered by the lipid phosphatidylserine. Procedure according to one of Claims 8 - 10 , characterized in that vesicles with surface markers, which trigger a specific phagocytosis, eg phosphatidylserine, and vesicles without surface markers are used to quantify specific, marker-mediated phagocytosis against the non-specific background. Procedure according to one of Claims 1 - 11 , characterized in that the membrane vesicles are also provided with additional components, in particular transmembrane proteins and/or an embedding matrix for the indicator. Procedure according to one of Claims 1 - 12 , characterized in that the target cells are epithelial cells, in particular cells of the retinal pigment epithelium (RPE), or macrophages. Procedure according to one of Claims 1 - 13 , characterized in that it is a quantitative detection method and the change in a spectroscopically detectable property of the indicator is determined by measuring this property before incorporation into target cells and after incorporation into target cells and determining the ratio of these two values . Membrane vesicles containing a spectroscopically detectable indicator, characterized in that they are lipid vesicles and the indicator is selected from the group consisting of pH indicators, in particular fluorescence-based pH indicators, fluorescent esterase substrates, fluorescent phosphatase substrates, cell-specific enzyme substrates, in particular fluorescence-based cell-specific enzyme substrates, Ca ²⁺ indicators, in particular fluorescence-based Ca ²⁺ indicators. Use of membrane vesicles containing a spectroscopically detectable indicator to detect phagocytic function in biological cells and multicellular systems, especially tissues. Kit for performing a detection of the phagocytosis function in biological cells in vitro, comprising membrane vesicles containing a spectroscopically detectable indicator, preferably aliquoted into ready-to-use portions of the dispersion, and optionally further components, in particular those components whose presence is a negative control (no inclusion of the vesicles) or a positive control (complete uptake of all vesicles that come into contact with the cell membrane) enables or ensures. kit after Claim 17 , comprising vesicles with surface markers that trigger specific phagocytosis, eg phosphatidylserine, and vesicles without surface markers, for quantifying specific, marker-mediated phagocytosis against the non-specific background.