Subject-matter of the invention is a medium for introducing macromolecules into cells, especially mammalian cells. Another subject-matter of the invention is a method for introducing macromolecules into cells, especially mammalian cells, using the phenomenon of pinocytosis. The invention finds its application in all biomedical studies where it is necessary to cross the cell membrane barrier to perform the experiment. Among the possible applications one could mention: studies on molecular targets of new drugs, protein functions, gene functions, intracellular nanoparticle distribution, as well as labeling of whole cells to study their migration in tissues.

From Canadian patent application CA2430087C a method for introducing a molecule into the cytosol of a cell is known, in which the cell is contacted with a photosensitizing agent, the cell is irradiated with light, o a wavelength effective to activate the photosensitizing agent, and substantially at the same time or after irradiation, the cell is contacted with the molecule to be introduced, particularly for use in cancer treatment, gene therapy and vaccination.

European patent application EP1637597A1 describes a method for nucleic acid infusion, comprising the step (a) of bringing a nucleic acid, a hypertonic solution and cells into contact with each other and the step (b) of lowering the osmotic pressure of the hypertonic solution after the step (a). There is further provided a reagent for nucleic acid infusion, comprising as an ingredient at least one substance belonging to the category of oligosaccharides or a polyhydric alcohol (including polyethylene glycol having a molar mass of not more than 2000 g/mol).

A commercial product called Influx™ Pinocytic Cell-Loading Reagent is known from the prior art. It is a product based on the work of: Okada and Rechsteiner, "Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles" (Cell 29 (1), 33-41(1982)). In this work, a hypotonic solution composition was proposed in the form of: PEG1000 polymer at a concentration of 24% w/w and sucrose sugar at a concentration of 1.2 M. In the Influx™ composition, the polymer used is too small and its concentration is too low, and hence its application range is limited. According to the cited work, the introduction takes place in three steps:

(1) Placing the cell in a hypertonic (high concentration) solution containing an osmotic agent and macromolecules to be introduced into the cell. During this time, the following take place: (a) equalization of the entire system osmotic pressure by the efflux of water from the cell and (b) continuous pinocytosis - a spontaneous pulling of vesicles containing components of the hypertonic solution into the cell. (2) Sudden transfer of the cell into a hypotonic (low concentration) solution causing water to flow into the cell and dilute its contents. At the same time, there is an intense influx of water into the pinocytic vesicles, which causes rupture of their membranes and release of their contents.

(3) Placing the cell back into an isotonic solution. Equalization of concentrations to the initial state takes place. The macromolecule is present in the cell.

Macromolecules are introduced into cultured mammalian cells based on osmotic lysis of the pinocytic vesicles. Cells are first incubated in culture medium containing 0.5 M sucrose, 10% polyethylene glycol 1000, and the macromolecule to be transferred. The cells are then placed in medium diluted with 0.33 parts water. Most pinocytic vesicles formed in the presence of sucrose burst in hypotonic medium, thus releasing the enclosed macromolecule. L929 cells remain fully viable after a single hypertonic sucrose treatment, and most survive four consecutive cycles of osmotic lysis. This procedure, termed osmotic lysis of pinosomes, has been used in the work cited above to transfer substantial amounts of horseradish peroxidase, antiricin antibodies, and 70kDa dextran into the cytosol of L929 cells.

The means and methods described in the prior art enable, in certain cases, to introduce proteins, dextrans and nucleic acids into mammalian cells. However, these solutions are not universal. At least two cases, where the known methods failed, were identified: non-invasive introduction into mammalian cells probes with radii greater than 20 nm, or introduction of any probes, regardless of their size, both small - of the order of 1 nm and much larger - up to about 100 nm, into cancerous cells. Moreover, the solutions known from prior art do not guarantee that the cells after probe introduction will be characterized by survival rate not less than 80%.

The first subject-matter of the invention is a hypertonic solution for introducing macromolecules into mammalian cells, comprising a component for generating osmotic pressure and a solvent, characterized in that the component for generating osmotic pressure is a polymer selected from the group that includes: polyethylene glycol, glucose polymer, sucrose-epichlorohydrin copolymer; with a concentration not less than the concentration limit, where the polyethylene glycol concentration limit is not less than 8 g/L, the glucose polymer concentration limit is not less than 37 g/L, and the sucrose-epichlorohydrin copolymer concentration limit is not less than 81 g/L, wherein the concentration limit of the component for generating osmotic pressure is the concentration at which the polymer in hypertonic solution is in the form of entangled chains. In a preferred embodiment of the invention, the average molar mass of polyethylene glycol is 20 kg/mol to 218 kg/mol, the average molar mass of glucose polymer is 10 kg/mol to 2000 kg/mol, the average molar mass of sucrose-epichlorohydrin copolymer is 20 kg/mol to 218 kg/mol.

In a further preferred embodiment of the invention, the glucose polymer is dextran.

In a still further preferred embodiment of the invention, the sucrose-epichlorohydrin copolymer is Ficoll.

In another preferred embodiment of the invention, the entangled polymer has a radius of gyration of 4 nm to 38 nm.

In a further preferred embodiment of the invention, the solvent is selected from the group that includes: DMEM-type culture medium, MEM-type culture medium, IMDM-type culture medium, RPMI-1640-type culture medium, other mammalian cell culture media, PBS. The indicated selection of culture media in no way limits the scope of the invention. The culture medium, which could be used as a solvent, should meet several conditions: It should be a sterile, non-toxic aqueous solution with a concentration of components which ensures isotonicity, i.e., with an osmotic pressure ranging from 280 to 300 mOsm/l.

A second subject-matter of the invention is a method for introducing macromolecules into mammalian cells, comprising steps of: a) preparing a hypertonic solution, b) preparing a working solution, c) preparing a hypotonic solution, d) introducing macromolecules into cells, characterized in that in step b) the working solution is prepared by dissolving a macromolecule constituting probe in a hypertonic solution as defined in the first subject-matter of the invention, then in step c) the hypotonic solution is prepared by mixing DMEM-type culture medium with deionized water in a volume ratio of 6:4, in step d) the culture medium and hypotonic medium are heated, the old culture medium is removed from above the cells, the working solution obtained in step b) is added and the cells are incubated for 10 min, then the hypotonic medium is added and left for 2 min, after which the media mixture is removed, the culture medium is added and the cells are transferred to an incubator and incubated for not less than 15 min.

In a preferred embodiment of the invention, the macromolecule constituting the probe is selected from the group that includes: polyethylene glycol-coated silica nanospheres, tetramethylrhodamine- modified dextran, or deoxyribonucleic acid. In a further preferred embodiment of the invention, the macromolecule has a hydrodynamic diameter of 1.3 nm to about 100 nm.

In a still further preferred embodiment of the invention, the hydrodynamic radius of the polyethylene glycol-coated silica nanospheres is 3.8 nm to 33.5 nm.

In another preferred embodiment of the invention, the hydrodynamic radius of the tetramethylrhodamine-modified dextran is 1.3 nm to 8.6 nm.

In still another preferred embodiment of the invention, the hydrodynamic radius of the deoxyribonucleic acid is 2 nm to about 100 nm.

In yet another further preferred embodiment of the invention, the molar mass of the tetramethylrhodamine-modified dextran is 4.4 kg/mol to 155 kg/mol.

In another further preferred embodiment of the invention, the molar mass of the deoxyribonucleic acid is 2 kg/mol to 3800 kg/mol.

In yet another further preferred embodiment of the invention, the deoxyribonucleic acid is a plasmid deoxyribonucleic acid.

In a preferred embodiment of the invention, the macromolecule concentration is 61 nM to 10 mM if the macromolecule is a macromolecule selected from the group that includes: polyethylene glycol- coated silica nanospheres or tetramethylrhodamine-modified dextran.

In still another preferred embodiment of the invention, the macromolecule concentration is from 12.5 ng/mL if the macromolecule is a deoxyribonucleic acid.

In a preferred embodiment of the invention, the culture medium and the hypotonic medium are heated to 37°C.

In another further preferred embodiment of the invention, the cells are selected from the group that includes cancer cells, preferably they are selected from the group that includes cervical cancer cells, lung cancer cells, breast cancer, or normal cells, preferably the normal cells are cells of connective tissue proper, more preferably fibroblasts.

In another further preferred embodiment of the invention, the deionized water is sterile.

In a still further preferred embodiment of the invention, the silica nanospheres are coated with polyethylene oxide, preferably with polyethylene oxide having chains of 6-9 monomers.

In another preferred embodiment of the invention, the incubation in step c) is carried out for up to 24 hours.

In the present invention, by osmotic polymer (abbreviated as OA, Osmotic Agent) is meant a component of the hypertonic medium that determines the successful introduction of macromolecules into the cells. The type and concentration of OA will be described in the embodiment of the present invention.

In the present invention, by macromolecule, which constitutes the probe, is meant a particle to be introduced into the cell and whose presence can be detected. Typically, these are fluorescently labeled dextrans or PEG-coated silica nanoparticles. Experiments have also been performed in which the probe constituted DNA encoding fluorescent proteins.

The present invention has a variety of advantages. It allows carrying out the introduction of probe macromolecules (PEG-modified nanospheres, modified dextran, DNA) of various sizes (1.3 nm to about 100 nm) in less than 30 min. Moreover, after completing the procedure, the cells with macromolecules introduced are characterized by high viability: in experiments the viability of over 85% for HeLa cells, over 90% for A549 cells, and 90% for MDA-MB-231 cells in Alamar Blue metabolic activity assay was obtained. In addition, the method according to the invention is characterized by a higher effectiveness in comparison with the procedure known from the prior art (based on our own qualitative studies, Fig. 6).

The embodiments of the invention are illustrated in the drawings, where: Fig. 1 shows a mechanism for introducing probes into the cell cytoplasm using hypertonic medium according to the prior art; Fig. 2 shows exemplary FCS autocorrelation curves obtained for the cytoplasm of cells subjected to the fluorescent probe introduction procedure: 2A - high noise and no autocorrelation indicate no freely diffusing fluorescent probe in the sample - low probe introduction effectiveness; 2B - correct autocorrelation curve, well fitted with the physical model, indicates the presence of freely diffusing probe in the cytoplasm - high probe introduction effectiveness; parameters for fitting the physical model to experimental data are the number of molecules in the sample (concentration, basis for effectiveness quantification) and the probe diffusion time (characteristic value, helps in unambiguous probe identification); Fig. 3 shows the comparison of the osmotic polymer molecule size and the concentration regime used in the procedure with the effectiveness of introducing the probe into the cell cytoplasm; Fig. 4 shows juxtaposition of the effectiveness of introducing particles into cells with the concentration regime of the polymer used and its size; concentrations higher than the regime limit constitute a regime characterized by polymer chain entanglement (according to the lUPAC nomenclature); Fig. 5 shows confocal microscopy images for evaluation the effectiveness of introducing probes by the developed method: A - signal accumulated in the bright spots indicates a large number of pinocytic vesicles that have not released their contents into the cytoplasm (low effectiveness of probe introduction); B - signal distributed evenly in the cell cytoplasm indicates a predominance of probe released into the cytosol (high effectiveness of probe introduction). Whereas, Fig. 6 shows a comparison of the performance of the hypertonic medium based on the solution known from the prior art and the work of Okada, (Cell 1982, - the medium contains polyethylene oxide with molecular weight of 1 kg/mol, and sucrose) with the exemplary hypertonic medium according to the invention, taking into account the regime of OA chain entanglement (dextran with molecular weight of 70 kg/mol); Fig. 7 shows distribution of pinocytic vesicles with probes (TRITC-dextran) in a HeLa line cancer cell - qualitative image; Fig. 8 shows distribution of pinocytic vesicles with probes (TRITC-dextran) in HeLa line cancer cell - quantitative curve; Fig. 9 shows distribution of pinocytic vesicles with probes (S1O2 nanospheres coated with PEG(6-9) - polyethylene oxide with chains of 6-9 monomers) in MDA-MB-231 line cancer cell - qualitative image; Fig. 10 shows distribution of pinocytic vesicles with probes (PEG(6-9)-coated S1O2 nanospheres) w MDA-MB-231 line cancer cell - quantitative curve; Fig. 11 shows distribution of pinocytic vesicles with probes (plasmid DNA) in MDA-MB-231 line cancer cell - qualitative image; Fig. 12 shows distribution of pinocytic vesicles with probes (plasmid DNA) in MDA-MB-231 line cancer cell - quantitative curve; Fig. 13 shows distribution of pinocytic vesicles with probes (TRITC- dextran) in a normal cell (fibroblasts) - qualitative image; Fig. 14 shows distribution of pinocytic vesicles with probes (TRITC-dextran) in a normal cell (fibroblasts) - quantitative curve; Fig. 15 shows distribution of pinocytic vesicles with probes having hydrodynamic diameter greater than 21 nm (PEG(6-9)-coated S1O2 nanospheres) in MDA-MB-231 line cancer cell - qualitative image; Fig. 16 shows distribution of pinocytic vesicles with probes having hydrodynamic diameter greater than 21 nm (PEG(6-9)-coated S1O2 nanospheres) in MDA-MB-231 line cancer cell - quantitative curve; Fig. 17 shows distribution of pinocytic vesicles with probes having hydrodynamic diameter greater than 3 nm (TRITC-dextran) in MDA-MB-231 line cancer cell - qualitative image; Fig. 18 shows distribution of pinocytic vesicles with probes having hydrodynamic diameter greater than 3 nm (TRITC-dextran) in MDA-MB-231 line cancer cell - quantitative curve; Fig. 19 shows distribution of pinocytic vesicles with probes having hydrodynamic diameter greater than 3 nm (TRITC-dextran) in A549 line cancer cell - qualitative image; Fig. 20 shows distribution of pinocytic vesicles with probes having hydrodynamic diameter greater than 3 nm (TRITC-dextran) in A549 line cancer cell - quantitative curve.

Example 1

Various fluorescent probes were utilized for the cells in culture. A range of hypertonic media was used, differing in OA, i.e., the polymer type, its molecular weight, concentration. The hypotonic medium and protocol (time scales of the process) were fixed: they included 10 min of hypertonic medium treatment, and 90 seconds of hypotonic medium treatment (cell culture medium was diluted with 0.66 parts water). The effectiveness of a given variant (defined by OA type, size, and concentration) of the hypertonic medium was determined by two methods:

(1) qualitatively - by confocal imaging: the number and integrity of pinocytic vesicles and the presence of the probe in the cytoplasm of the cell were assessed (Fig. 5);

(2) quantitatively - by fluorescence correlation spectroscopy: the presence of the fluorescent probe in the cytoplasm was identified and its concentration was measured (Figs. 2A-2B).

The hypertonic medium variants tested, with sucrose and without, along with the observed effectiveness of probe introduction are presented in Table 1. A solution without OA, containing only sucrose, was used as a comparison solution. It was observed that the presence (or absence) of sucrose has no apparent effect on the effectiveness of the method. In contrast, significant differences in effectiveness were noted between the different OAs.

Table 1. Method optimization results: osmotic agents. The concentration of polymers in the hypertonic medium ranged from 120 to 640 g/L. The introduction effectiveness was examined using fluorescent probes. The osmotic pressure is denoted by AP _OSm .

The osmotic pressure of hypertonic solutions (containing OA) was calculated based on the formula:

RT

M [5], where c is the polymer concentration [kg/m ³ ], M is the polymer molar mass [kg/mol], A is the second virial coefficient [m ⁵ /s ² kg]. The A parameter is determined empirically for each polymer and takes the following values for the tested OAs: PEG 35 kg/mol - 5.63; dextran 10 kg/mol - 5.76 [6]; dextran 40 kg/mol - 2.5, dextran 70 kg/mol - 1.69, dextran 500 kg/mol - 0.43 [5], On the other hand, the osmotic pressure of sucrose solution was calculated based on van Hoffs law: AP _osm = RT c, where c is the sucrose concentration [mol/dm ³ ].

A comparison of the method effectiveness with polymer particle size and concentration regime is shown in Fig. 3. It is noted that the procedure was effective with OA having particles larger than 6 nm (radii of gyration). In addition, all hypertonic medium variants that were effective in the probe introduction had concentrations in the range of chain entanglement regime (i.e., the polymer tangles snagged on each other) [4], Lower polymer concentrations (untangled regime) resulted in retention of pinocytic vesicle contents - there was no probe release into the cytoplasm (Fig. 5).

To judge the importance of the chain entanglement regime in the effectiveness of macromolecule introduction into the cells, additional experiments were performed: the same polymers were used in different concentration regimes. The results are presented in Fig. 4. On the basis of the results shown it can be concluded that for the method to work properly the necessary condition is the hypertonic medium containing polymer in the chain entanglement regime. Whereas an additional factor improving the method effectiveness was the use of polymers with larger particles, i.e., with the radii of gyration larger than 6 nm.

Materials Tested

During experimental work the utility of the invention for introducing different probes and into different cell types was demonstrated (Table 2). The usefulness of the method was presented for probes of different types (polymers, nanoparticles, nucleic acids), with a wide range of sizes (diameters 2-100 nm). Detailed characteristics of the probes introduced into the cells are presented in Table 3.

Table 2. Summary of applications of the method for introducing macromolecules into the living cells (the list will be extended). The table cells present the probes introduced in a given variant.

Table 3. Characteristics of probes introduced into cells by the method according to the present invention. a - Plasmid DNA is a structure in constant conformational change, from supercoiled to relaxed and vice versa. It was not possible to measure which of these forms was predominating during the process of introduction into cells, hence the approximate value of the hydrodynamic radius given.

Table 4. Examples of concentrations of osmotic polymers in hypertonic medium. The table shows concentrations constituting the limit of the regime that allows polymer chain entanglement (column 4), and examples of concentrations higher than this limit (column 5).

The values given in column 4 (Table 4) were calculated based on the equations presented in the work [4], The concentration limit for the polymer chain entanglement regime was taken to be such a value of the concentration (C) that satisfies the following equation: , where M _w is the molecular weight of the polymer, RH and R _g are the hydrodynamic radius and radius of gyration of the polymer molecule, respectively, and N _A is the Avogadro number. Whereas the

-v b exponent b depends on Flory's exponent (v) as in: 1 ~ 3V . The Flory's exponent is a quantity characterizing the movement of a given polymer in a given solvent and takes the values: VPE _G = 0.60, 0.42, 0.44. Mechanism of Action

Based on the conducted experiments, the following mechanism for introducing probes into cells was proposed (Fig. 1):

(1) Pinocytosis occurs in a hypertonic medium. Pinocytic vesicles contain components of the hypertonic medium: both small molecules (sucrose and/or probe) and macromolecules (polymer and/or probe).

(2) After transferring cell to a hypotonic medium, the cytoplasm - due to the properties of the cell membrane - also becomes a hypotonic solution.

(3) At the pinocytic vesicle-cytoplasm interface an osmotic pressure difference is generated. As a result of this difference, there is a water influx into the vesicle. In the first phase of this process, loosening of the phospholipid bilayer structure takes place - small molecules contained in the vesicle flow into the cytoplasm. This is the crucial stage for the effectiveness of the method.

(4) If the macromolecules (including OA) remaining in the vesicle maintain a sufficiently high osmotic pressure, further water influx into the vesicle results in membrane rupture and release of the entire contents into the cytoplasm.

The presented mechanism indicates that the limiting factor for the method effectiveness is the presence of large OAs in the pinocytic vesicle after vesicle membrane loosening (step (3)). Thus, it is reasonable to correlate the effectiveness with the size/entanglement of the osmotic polymers - the larger the macromolecule, the greater the probability that it will remain in the vesicle until the rupture of its membrane (step (4)), and a lower concentration is required to achieve the chain- entangling regime.

Example 2. Introduction of Probes (TRITC-dextran) into HeLa Line Cancer Cells

Purpose of the Experiment

Investigation of HeLa cell cytoplasm viscosity for probes with a hydrodynamic radius of 8.6 nm. Biological Material

The experiment was performed using HeLa cells (ATCC) routinely grown in a culture medium containing DMEM base (Dulbecco's Modified Eagle’s Medium, Institute of Immunology and Experimental Therapy, Wroclaw, Poland) supplemented with 10% _v/v fetal bovine serum (FBS, Gibco), 1% L-glutamine (Sigma-Aldrich), and 1% antibiotics (Penicillin/Streptomycin, Sigma-Aldrich). On the day before the experiment, the cells were seeded into an 8-well glass bottom plate (Cellvis) and allowed to adhere to the surface overnight (1 cmVwell). 70% confluence was achieved.

Preparation of Reagents

The hypertonic medium was obtained by dissolving dextran with a molecular weight of 70 kg/mol (Sigma-Aldrich) at a concentration of 11 ,6% _w /v in RPMI-1640 medium (Sigma-Aldrich). The working solution was obtained by dissolving the probe, i.e., the rhodamine-labeled dextran with a molecular weight of 155 kg/mol (TRITC-dextran 155 kDa, Sigma-Aldrich) in a hypertonic medium to a final concentration of 10 mM. The hypotonic medium was obtained by mixing DMEM medium with sterile deionized water at a volume ratio of 6:4.

Probe Introduction into the Cytoplasm

Prior to the procedure, pure culture medium and hypotonic medium were heated to 37°C. In a culture plate, the old culture medium was removed from above the cells in a 1 cm ² culture well. Then, 8 mI_ of the working solution was added and allowed to incubate for 10 min. After this time, 250 mI_ of the hypotonic medium was added and left for 2 min. After this time, the hypotonic medium was removed from above the cells and 300 mI_ of culture medium was added. The cells were transferred to an incubator for at least 15 min.

Cytoplasmic Viscosity Measurements

Measurements were performed using fluorescence correlation spectroscopy (FCS). The FCS equipment was calibrated prior to the measurements to determine the confocal volume (for more information about the calibration see: T. Kalwarczyk et a/., J. Phys. Chem. B, 121, 9831, 2017). Then, using a confocal microscope, the confocal spot was positioned in the cytoplasm of a selected living cell. Eight measurements of fluorescence fluctuations at the selected spot were performed, each lasting 60 s. The probe concentration in the cytoplasm was about 3 orders of magnitude lower than in the working solution, i.e., about 10 nM. This was the concentration that provided about 1 molecule in the confocal spot, the optimal concentration for the FCS method. An autocorrelation of the recorded signal was performed, a one-component anomalous diffusion model was fitted, and based on this, the diffusion time (TD) of the probe in the confocal spot was determined. Using the equation the diffusion coefficient of 155 kDa dextran in the cytoplasm of HeLa cells was calculated. Based on the ratio (resulting from the Smoluchowski-Einstein equation), where Do, ho refer to measurements in water, the cytoplasmic viscosity q _cyt0 can be determined.

Confirmation of Effectiveness

The effectiveness was confirmed by two methods:

(1) qualitatively - by confocal imaging: the number and integrity of pinocytic vesicles and the presence of the probe in the cytoplasm of the cell were assessed (Fig. 7);

(2) quantitatively - by fluorescence correlation spectroscopy: the presence of the fluorescent probe in the cytoplasm was identified based on the diffusion time (Fig. 8).

Example 3. Introduction of Probes (PEG-coated Si0 ₂ Nanospheres) into MDA-MB-231 Line Cancer Cells

Purpose of the Experiment

Investigation of MDA-MB-231 cell cytoplasmic viscosity for probes with a hydrodynamic radius of 33.5 nm.

Biological Material

The experiment was performed using MDA-MB-231 cells (ATCC) routinely grown in a culture medium containing RPMI 1640 with sodium bicarbonate (Sigma-Aldrich) supplemented with 10% _v/v fetal bovine serum (FBS, Gibco), 1% L-glutamine (Sigma-Aldrich), and 1% antibiotics (Penicillin/Streptomycin, Sigma-Aldrich). On the day before the experiment, cells were seeded into an 8-well glass bottom plate (ibidi) and allowed to adhere to the surface overnight (1 cmVwell). 70% confluence was achieved.

Preparation of Reagents

The hypertonic medium was obtained by dissolving dextran with a molecular weight of 70 kg/mol (Sigma-Aldrich) at a concentration of 11 ,6% _w /v in the culture medium. The working solution was obtained by dissolving probe i.e., the polyethylene glycol-coated rhodamine-labeled silica nanospheres (Siliquan) in the hypertonic medium to a final concentration of 100 nM. The hypotonic medium was obtained by mixing DMEM medium with sterile deionized water at a volume ratio of 6:4.

Probe Introduction into the Cytoplasm

Prior to the procedure, pure culture medium and hypotonic medium were heated to 37°C. In a culture plate, the old culture medium was removed from above the cells in a 1 cm ² culture well. Then, 8 mI_ of the working solution was added and allowed to incubate for 10 min. After this time, 250 mI_ of the hypotonic medium was added and left for 2 min. After this time, the hypotonic medium was removed from above the cells and 300 mI_ of culture medium was added. The cells were transferred to an incubator for at least 15 min.

Cytoplasmic Viscosity Measurements

Measurements were performed using fluorescence correlation spectroscopy (FCS). The FCS equipment was calibrated prior to the measurements to determine the confocal volume (for more information about the calibration see: T. Kalwarczyk et a/., J. Phys. Chem. B, 121, 9831, 2017). Then, using a confocal microscope, the confocal spot was positioned in the cytoplasm of a selected living cell. Eight measurements of fluorescence fluctuations at the selected spot were performed, each lasting 90 s. The probe concentration in the cytoplasm was about 3 orders of magnitude lower than in the working solution, i.e., about 10 nM. This was the concentration that provided about 1 molecule in the confocal spot, the optimal concentration for the FCS method. An autocorrelation of the recorded signal was performed, a one-component anomalous diffusion model was fitted, and based on this, the diffusion time (TD) of the probe in the confocal spot was determined. Using the equation coefficient of nanoparticles with a radius of 33.5 nm in the cytoplasm of MDA-MB-231 cells was calculated. Based on the ratio (resulting from the Smoluchowski-Einstein equation), where Do, ho refer to measurements in water, the cytoplasmic viscosity q _cyto can be determined.

Confirmation of the Effectiveness of the Invention

The effectiveness of the invention was confirmed by two methods:

(1) qualitatively - by confocal imaging: the number and integrity of pinocytic vesicles and the presence of the probe in the cytoplasm of the cell were assessed (Fig. 9);

(2) quantitatively - by fluorescence correlation spectroscopy: the presence of the fluorescent probe in the cytoplasm was identified based on the diffusion time (Fig. 10). Example 4. Introduction of Probes (Plasmid DNA) into MDA-MB-231 Line Cancer Cells

Purpose of the Experiment

Investigation of MDA-MB-231 cell cytoplasmic viscosity for plasmid DNA with a radius of about 100 nm.

Biological Material

The experiment was performed using MDA-MB-231 cells (ATCC) routinely grown in a culture medium containing RPMI 1640 with sodium bicarbonate (Sigma-Aldrich) supplemented with 10% _v/v fetal bovine serum (FBS, Gibco), 1% L-glutamine (Sigma-Aldrich), and 1% antibiotics (Penicillin/Streptomycin, Sigma-Aldrich). On the day before the experiment, cells were seeded into an 8-well glass bottom plate (Lab-Tek) and allowed to adhere to the surface overnight (1 cmVwell). 70% confluence was achieved.

Preparation of Reagents

The hypertonic medium was obtained by dissolving dextran with a molecular weight of 70 kg/mol (Sigma-Aldrich) at a concentration of 11 ,6% _w /v in the culture medium. The working solution was obtained by dissolving the probe i.e., the plasmid DNA encoding EGFP (Addgene) in the hypertonic medium to a final concentration of 12.5 ng/ml. The working solution was incubated for 5 min at room temperature before being added to the cells. The hypotonic medium was obtained by mixing DMEM medium with sterile deionized water at a volume ratio of 6:4.

Probe Introduction into the Cytoplasm

Before the procedure, pure culture medium and hypotonic medium were heated to 37°C. In a culture plate, the old medium was removed from above the cells in a 1 cm ² culture well. Then, 8 mI_ of the working solution was added and allowed to incubate for 10 min. After this time, 250 mI_ of the hypotonic medium was added and left for 2 min. After this time, the hypotonic medium was removed from above the cells and 300 mI_ of culture medium was added. The cells were transferred to an incubator for 24 hours. After 24 hours, the measurements began.

Cytoplasmic Viscosity Measurements

Measurements were performed using fluorescence correlation spectroscopy (FCS). The FCS equipment was calibrated prior to the measurements to determine the confocal volume (for more information about the calibration see: T. Kalwarczyk et a/., J. Phys. Chem. B, 121, 9831, 2017). Then, using a confocal microscope, the confocal spot was positioned in the cytoplasm of a selected living cell. Eight measurements of fluorescence fluctuations at the selected spot were performed, each lasting 30 s. The probe concentration in the cytoplasm was about 3 orders of magnitude lower than in the working solution, i.e., about 10 nM. This was the concentration that provided about 1 molecule in the confocal spot, the optimal concentration for the FCS method. An autocorrelation of the recorded signal was performed, a one-component anomalous diffusion model was fitted, and based on this, the diffusion time (TD) of the probe in the confocal spot was determined. Using the equation the diffusion coefficient of EGFP in the cytoplasm of MDA-MB-231

^cyt D cells was calculated. Based on the ®cyto ratio (resulting from the Smoluchowski-Einstein equation), where Do, ho refer to measurements in water, the cytoplasmic viscosity q _cyt0 can be determined.

Confirmation of the Effectiveness of the Invention

The effectiveness of the invention was confirmed by two methods:

(1) qualitatively - by confocal imaging: the number and integrity of pinocytic vesicles and the presence of the probe in the cytoplasm of the cell were assessed (Fig. 11);

(2) quantitatively - by fluorescence correlation spectroscopy: the presence of the fluorescent probe in the cytoplasm was identified based on the diffusion time (Fig. 1 ).

Example 5. Introduction of Probes (TRITC-dextran) into Normal Cells (Fibroblasts)

Purpose of the Experiment

Investigation of the cytoplasmic viscosity of normal cells (fibroblasts) for a probe with a hydrodynamic radius of 5.57 nm.

Biological Material

The experiment was performed using fibroblast cells (Coriell Institute) routinely grown in a culture medium containing DMEM (Sigma-Aldrich) with sodium bicarbonate (Sigma-Aldrich) supplemented with 10% _v/v fetal bovine serum (FBS, Gibco), 1% L-glutamine (Sigma-Aldrich), and 1% antibiotics (Penicillin/Streptomycin, Sigma-Aldrich). On the day before the experiment, cells were seeded into an 8-well glass bottom plate (Lab-Tek) and allowed to adhere to the surface overnight (1 cm ² /well). 70% confluence was achieved. Preparation of Reagents

The hypertonic medium was obtained by dissolving dextran with a molecular weight of 70 kg/mol (Sigma-Aldrich) at a concentration of 11.6% _w/v in the culture medium. The working solution was obtained by dissolving the probe i.e., the rhodamine-labeled dextran with a molecular weight of 75 kg/mol (TRITC-dextran 75 kDa, Sigma-Aldrich) in a hypertonic medium to a final concentration of 10 mM. The hypotonic medium was obtained by mixing DMEM medium with sterile deionized water at a volume ratio of 6:4.

Probe Introduction into the Cytoplasm

Before the procedure, pure culture medium and hypotonic medium were heated to 37°C. In a culture plate, the old medium was removed from above the cells in a 1 cm ² culture well. Then, 8 mI_ of the working solution was added and allowed to incubate for 10 min. After this time, 250 mI_ of the hypotonic medium was added and left for 2 min. After this time, the hypotonic medium was removed from above the cells and 300 mI_ of culture medium was added. The cells were transferred to an incubator for at least 15 min.

Cytoplasmic Viscosity Measurements

Measurements were performed using fluorescence correlation spectroscopy (FCS). The FCS equipment was calibrated prior to the measurements to determine the confocal volume (for more information about the calibration see: T. Kalwarczyk et a/., J. Phys. Chem. B, 121, 9831, 2017). Then, using a confocal microscope, the confocal spot was positioned in the cytoplasm of a selected living cell. Eight measurements of fluorescence fluctuations at the selected spot were performed, each lasting 60 s. The probe concentration in the cytoplasm was about 3 orders of magnitude lower than in the working solution, i.e., about 10 nM. This was the concentration that provided about 1 molecule in the confocal spot, the optimal concentration for the FCS method. An autocorrelation of the recorded signal was performed, a one-component anomalous diffusion model was fitted, and based on this, the diffusion time (TD) of the probe in the confocal spot was determined. Using the equation the diffusion coefficient of 75 kDa dextran in the cytoplasm of fibroblast cells was calculated. Based on the ratio (resulting from the Smoluchowski-

Einstein equation), where Do, ho refer to measurements in water, the cytoplasmic viscosity q _cyt0 can be determined. Confirmation of the Effectiveness of the Invention

The effectiveness of the invention was confirmed by two methods:

(1) qualitatively - by confocal imaging: the number and integrity of pinocytic vesicles and the presence of the probe in the cytoplasm of the cell were assessed (Fig. 13);

(2) quantitatively - by fluorescence correlation spectroscopy: the presence of the fluorescent probe in the cytoplasm was identified based on the diffusion time (Fig. 14).

Example 6. Introduction of Probes (PEG(6-9)-coated Si0 ₂ nanospheres) into MDA- MB-231 Line Cancer Cells

Purpose of the Experiment

Investigation of MDA-MB-231 cell cytoplasmic viscosity for probes with a hydrodynamic radius of 21 nm.

Biological Material

The experiment was performed using MDA-MB-231 cells (ATCC) routinely grown in a culture medium containing RPMI 1640 with sodium bicarbonate (Sigma-Aldrich) supplemented with 10% _v/v fetal bovine serum (FBS, Gibco), 1% L-glutamine (Sigma-Aldrich), and 1% antibiotics (Penicillin/Streptomycin, Sigma-Aldrich). On the day before the experiment, cells were seeded into an 8-well glass bottom plate (Lab-Tek) and allowed to adhere to the surface overnight (1 cmVwell). 70% confluence was achieved.

Preparation of Reagents

The hypertonic medium was obtained by dissolving dextran with a molecular weight of 40 kg/mol (Sigma-Aldrich) at a concentration of 11 ,6% _w /v in the culture medium. The working solution was obtained by dissolving probe i.e., the polyethylene glycol (PEG(6-9))-coated rhodamine-labeled silica nanospheres (Siliquan) at a concentration of 550 nM in hypertonic medium. The hypotonic medium was obtained by mixing DMEM medium with sterile deionized water at a volume ratio of 6:4.

Probe Introduction into the Cytoplasm

Before the procedure, pure culture medium and hypotonic medium were heated to 37°C. In a culture plate, the old medium was removed from above the cells in a 1 cm ² culture well. Then, 8 mI_ of the working solution was added and allowed to incubate for 10 min. After this time, 250 mI_ of the hypotonic medium was added and left for 2 min. After this time, the hypotonic medium was removed from above the cells and 300 mI_ of culture medium was added. The cells were transferred to an incubator for at least 15 min.

Cytoplasmic Viscosity Measurements

Measurements were performed using fluorescence correlation spectroscopy (FCS). The FCS equipment was calibrated prior to the measurements to determine the confocal volume (for more information about the calibration see: T. Kalwarczyk et a/., J. Phys. Chem. B, 121, 9831, 2017). Then, using a confocal microscope, the confocal spot was positioned in the cytoplasm of a selected living cell. Eight measurements of fluorescence fluctuations at the selected spot were performed, each lasting 90 s. The probe concentration in the cytoplasm was about 3 orders of magnitude lower than in the working solution, i.e., about 10 nM. This was the concentration that provided about 1 molecule in the confocal spot, the optimal concentration for the FCS method. An autocorrelation of the recorded signal was performed, a one-component anomalous diffusion model was fitted, and based on this, the diffusion time (TD) of the probe in the confocal spot was determined. Using the equation the diffusion coefficient of nanoparticles with a radius of 21 nm in the tfcyta __ ¾ cytoplasm of MDA-MB-231 cells was calculated. Based on the ¾ ^cyto _ra tio (resulting from the Smoluchowski-Einstein equation), where Do, ho refer to measurements in water, the cytoplasmic viscosity q _cyt0 can be determined.

Confirmation of the Effectiveness

The effectiveness was confirmed by two methods:

(1) qualitatively - by confocal imaging: the number and integrity of pinocytic vesicles and the presence of the probe in the cytoplasm of the cell were assessed (Fig. 15);

(2) quantitatively - by fluorescence correlation spectroscopy: the presence of the fluorescent probe in the cytoplasm was identified based on the diffusion time (Fig. 16).

Example 7. Introduction of Probes (TRITC-dextran) into MDA-MB-231 Line Cancer Cells

Purpose of the Experiment

Investigation of MDA-MB-231 cell cytoplasmic viscosity for a probe with a hydrodynamic radius of 3.75 nm. Biological Material

The experiment was performed using MDA-MB-231 cells (ATCC) routinely grown in a culture medium containing RPMI 1640 with sodium bicarbonate (Sigma-Aldrich) supplemented with 10% _v/v fetal bovine serum (FBS, Gibco), 1% L-glutamine (Sigma-Aldrich), and 1% antibiotics (Penicillin/Streptomycin, Sigma-Aldrich). On the day before the experiment, cells were seeded into an 8-well glass bottom plate (Lab-Tek) and allowed to adhere to the surface overnight (1 cmVwell). 70% confluence was achieved.

Preparation of Reagents

The hypertonic medium was obtained by dissolving 400 kg/mol molecular weight sucrose- epichlorohydrin copolymer - Ficoll (Sigma-Aldrich) at a concentration of 11.6% _w/v in the culture medium. The working solution was obtained by dissolving the probe i.e., the rhodamine-labeled dextran with a molecular weight of 20 kg/mol (TRITC-dextran 20 kDa, Sigma-Aldrich) in a hypertonic medium to a final concentration of 10 mM. The hypotonic medium was obtained by mixing DMEM medium with sterile deionized water at a volume ratio of 6:4.

Probe Introduction into the Cytoplasm

Before the procedure, pure culture medium and hypotonic medium were heated to 37°C. In a culture plate, the old medium was removed from above the cells in a 1 cm ² culture well. Then, 8 mI_ of the working solution was added and allowed to incubate for 10 min. After this time, 250 mI_ of the hypotonic medium was added and left for 2 min. After this time, the hypotonic medium was removed from above the cells and 300 mI_ of culture medium was added. The cells were transferred to an incubator for at least 15 min.

Cytoplasmic Viscosity Measurements

Measurements were performed using fluorescence correlation spectroscopy (FCS). The FCS equipment was calibrated prior to the measurements to determine the confocal volume (for more information about the calibration see: T. Kalwarczyk et a/., J. Phys. Chem. B, 121, 9831, 2017). Then, using a confocal microscope, the confocal spot was positioned in the cytoplasm of a selected living cell. Eight measurements of fluorescence fluctuations at the selected spot were performed, each lasting 60 s. The probe concentration in the cytoplasm was about 3 orders of magnitude lower than in the working solution, i.e., about 10 nM. This was the concentration that provided about 1 molecule in the confocal spot, the optimal concentration for the FCS method. An autocorrelation of the recorded signal was performed, a one-component anomalous diffusion model was fitted, and based on this, the diffusion time (TD) of the probe in the confocal spot was determined. Using the equation the diffusion coefficient of 20 kDa dextran in the cytoplasm of MDA- __

7)

MB-231 cells was calculated. Based on the ¾* ⁱ "cyt ratio (resulting from the Smoluchowski-

Einstein equation), where Do, ho refer to measurements in water, the cytoplasmic viscosity q _cyt0 can be determined.

Confirmation of the Effectiveness of the Invention

The effectiveness of the invention was confirmed by two methods:

(1) qualitatively - by confocal imaging: the number and integrity of pinocytic vesicles and the presence of the probe in the cytoplasm of the cell were assessed (Fig. 17);

(2) quantitatively - by fluorescence correlation spectroscopy: the presence of the fluorescent probe in the cytoplasm was identified based on the diffusion time (Fig. 18).

Example 8. Introduction of Probes (TRITC-dextran) into A549 Line Cancer Cells (Lung Cancer)

Purpose of the Experiment

Investigation of A549 cell cytoplasmic viscosity for a probe with a hydrodynamic radius of 5.57 nm. Biological Material

The experiment was performed using A549 cells (ATCC) routinely grown in a culture medium containing DMEM (Dulbecco's Modified Eagle’s Medium, Institute of Immunology and Experimental Therapy, Wroclaw, Poland) supplemented with 10% _v/v fetal bovine serum (FBS, Gibco), 1% L- glutamine (Sigma-Aldrich), and 1% antibiotics (Penicillin/Streptomycin, Sigma-Aldrich). On the day before the experiment, the cells were seeded into an 8-well glass bottom plate (Ibidi ^® ) and allowed to adhere to the surface overnight (1 cmVwell). 70% confluence was achieved.

Preparation of Reagents

The hypertonic medium was obtained by dissolving dextran with a molecular weight of 70 kg/mol (Sigma-Aldrich) at a concentration of 11 ,6% _w /v in the culture medium. The working solution was obtained by dissolving the probe, i.e., the rhodamine-labeled dextran with a molecular weight of 75 kg/mol (TRITC-dextran 75 kDa, Sigma-Aldrich) in a hypertonic medium to a final concentration of 10 mM. The hypotonic medium was obtained by mixing DMEM medium with sterile deionized water at a volume ratio of 6:4.

Probe Introduction into the Cytoplasm

Prior to the procedure, the culture medium and hypotonic medium were heated to 37°C. The medium was removed from above the cells in a 1 cm ² culture well. Then, 8 mI_ of the working solution was added and allowed to incubate for 10 min. After this time, 250 mI_ of the hypotonic medium was added and left for 2 min. After this time, the medium was removed from above the cells and 300 mI_ of culture medium was added. The cells were transferred to an incubator for at least 15 min.

Cytoplasmic Viscosity Measurements

Measurements were performed using fluorescence correlation spectroscopy (FCS). The FCS equipment was calibrated prior to the measurements to determine the confocal volume (for more information about the calibration see: T. Kalwarczyk et a/., J. Phys. Chem. B, 121, 9831, 2017). Then, using a confocal microscope, the confocal spot was positioned in the cytoplasm of a selected living cell. Eight measurements of fluorescence fluctuations at the selected spot were performed, each lasting 60 s. The probe concentration in the cytoplasm was about 3 orders of magnitude lower than in the working solution, i.e., about 10 nM. This was the concentration that provided about 1 molecule in the confocal spot, the optimal concentration for the FCS method. An autocorrelation of the recorded signal was performed, a one-component anomalous diffusion model was fitted, and based on this, the diffusion time (TD) of the probe in the confocal spot was determined. Using the

_ „ **¾¾? / equation *>'*<> /4¾ the diffusion coefficient of 75 kDa dextran in the cytoplasm of A549 cells was calculated. Based on the ratio (resulting from the Smoluchowski-Einstein equation), where Do, ho refer to measurements in water, the cytoplasmic viscosity q _cyt0 can be determined.

Confirmation of the Effectiveness of the Invention

The effectiveness of the invention was confirmed by two methods:

(1) qualitatively - by confocal imaging: the number and integrity of pinocytic vesicles and the presence of the probe in the cytoplasm of the cell were assessed (Fig. 19); (2) quantitatively - by fluorescence correlation spectroscopy: the presence of the fluorescent probe in the cytoplasm was identified based on the diffusion time (Fig. 20).

References

[1] Okada and Rechsteiner, “Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles”, Cell 29 (1), 33-41 (1982)

[2] Kalwarczyk et al., “Apparent Anomalous Diffusion in the Cytoplasm of Human Cells: The Effect of Probes’ Polydispersity”, J. Phys. Chem. B 121 (42), 9831-9837 (2017)

[3] Kwapiszewska et al., “Nanoscale Viscosity of Cytoplasm Is Conserved in Human Cell Lines”, J.

Phys. Chem. Lett. 11 (16), 6914-6920 (2020)

[4] Wisniewska et al., “Scaling of activation energy for macroscopic flow in polyethylene glycol) solutions: entangled-non-entangled crossover ”, Polymer 55 (18), 4651-4657 (2014)

[5] S. Rad, J. Gao, et al. „Depletion of high molecular weight dextran from the red cell surface measured by particle electrophoresis”, Electrophoresis 30, 450-456 (2009)

[6] J. Li, M. Turesson, et al. “ Equation of state of PEG/PEO in good solvent. Comparison between a one-parameter EOS and experiments”, Polymer 80, 205-213 (2015)