A METHOD FOR DELIVERING A SUBSTANCE INTO CELLS

Abstract

A method for delivering a substance into cells, wherein the method comprises the following steps: a) providing the substance, wherein the substance comprises a cell-penetrating compound comprising a basic amino functional group, b) providing cells; and c) contacting the substance with the cells; wherein the method comprises the additional step ofadjusting a p H in an extracellular fluid to a p H of at least 7.7.

Claims

1. A method for delivering a substance into cells, wherein the method comprises the following steps: a) providing the substance, wherein the substance comprises a cell-penetrating compound comprising a basic amino functional group, b) providing cells; and c) contacting the substance with the cells; wherein the method comprises the additional step of adjusting a pH in an extracellular fluid to a pH of at least 7.7.

2. The method according to claim 1, wherein the cell-penetrating compound is selected from the group comprising peptides, proteins, carbohydrates, lipids and nucleic acids.

3. The method according to claim 1, wherein the cells are chosen from the group comprising mammalian cells, plant cells, bacterial cells or insect cells.

4. The method according to claim 1, wherein in step b) providing cells is consisting of providing a suspension of cells, and wherein in step c) contacting the substance with the cells is consisting of creating a cell-substance-suspension by combining the substance with the suspension of cells, and wherein the pH in an extracellular fluid is an extracellular pH in the cell-substance-suspension.

5. The method according to claim 1, further comprising in step a) providing a suspension or solution of the substance in a pH buffer, particularly in an extracellular fluid being a pH buffer.

6. The method according to claim 1, wherein the method further comprises adding to the substance and/or to an extracellular fluid and/or the cell-substance-suspension, a mediator compound comprising at least one carboxyl functional group or carboxylate functional group coupled to a hydrophobic residue.

7. The method according to claim 1, wherein the basic amino functional group of the cell-penetrating compound are part of a guanidinium functional group.

8. The method according to claim 1, wherein the cell-penetrating compound is a peptide or a protein comprising a basic amino acid selected from arginine and lysine.

9. The method according to claim 1, wherein the cell-penetrating compound is a peptide or a protein and the method further comprises the step of adding a capping agent comprising a guanidinium group.

10. The method according to claim 1, wherein the cell-penetrating compound comprises a DNA repair enzyme comprising a basic amino functional group.

11. The method according to claim 1, wherein the cell-penetrating compound comprises a linker moiety.

12. The method according to claim 11, wherein the linker moiety is a peptide linker or a nucleotide linker.

13. The method according to claim 1, wherein an additional agent is covalently or non-covalently conjugated to the cell-penetrating compound, the additional agent being selected from small molecules, nucleic acids, peptide nucleic acids, peptides, proteins, nucleotides, oligonucleotides, inorganic particles and liposomes.

14. The method according to claim 13, wherein the additional agent is selected from the group comprising biomarker, drug, medically, pharmaceutically and cosmetically active substance.

15. The method according to claim 13, wherein the additional agent comprises at least one carboxyl functional group or carboxylate functional group.

16. The method according to claim 15, wherein the additional agent is a peptide or a protein comprising an acidic amino acid selected from aspartate/aspartic acid and glutamate/glutamic acid.

17. The method according to claim 1, wherein the mediator compound is selected from the group consisting of saturated and non-saturated fatty acids, saturated and non-saturated hydroxy fatty acids, dicarboxy acids, aromatic acids, and salts thereof.

18. The method according to claim 1, wherein the cells and/or the substance are pre-incubated with the mediator compound before the cell-substance-suspension is created and/or before in step c) the substance is contacted with the cells.

19. The method according to claim 1, wherein step c) of contacting the substance with the cells, comprises delivering the substance into the cell.

20. The composition according to claim 19, wherein the cell-penetrating compound is selected from the group comprising peptides, proteins, carbohydrates, lipids and nucleic acids.

21. The composition according to claim 19, wherein the composition comprises a suspension of the substance in pH-buffer, particularly in an extracellular fluid being a pH buffer.

22. The composition according to claim 19, wherein the composition comprises a mediator compound comprising at least one carboxyl functional group or carboxylate functional group coupled to a hydrophobic residue.

23. The composition according to claim 19, wherein the composition is in a form of a cream, a gel, an unguent, a lotion, a spray, an aerosol, a solution, in an emulsion, in liposomes or in microcapsules, wherein preferably the composition is for skin treatment.

24. The composition according to claim 19, further comprising a pharmaceutical acceptable carrier, filler, bulking agent, disintegrant, stabilizer, binder, humectant, extender, emulsifying agent, dissolution retarder, absorption enhancer, preservative, antioxidant, wetting agent, adsorbent, lubricant or a combination thereof.

25. Use of the composition according to claim 31 for treatment and/or prevention of a disease, diagnosis of diseases, as a research tool, as a targeting system, as a pharmaceutical composition or as a cosmetic composition.

26. Use of the composition according to claim 31 in the preparation of a medicament for treatment, prevention and/or diagnosis of a disease.

27. A method for manufacturing the composition according to claim 31, comprising at least the following steps: providing a substance comprising a cell-penetrating compound comprising a basic amino functional group, and adjusting the pH of the composition to at least 7.7.

28. A kit comprising in (a) suitable container(s) at least a composition according to claim 31, and optionally a package insert.

29. The kit according to claim 28, wherein the composition is contained in a ready-to-use form.

30. The method according to claim 1, further comprising a step of applying the substance to a subject, wherein the method is a cosmetic or therapeutic method.

31. A composition comprising a substance to be delivered to cells, wherein the substance comprises a cell-penetrating compound comprising a basic amino functional group, and wherein the pH of the composition is at least 7.7.

Description

EXAMPLES

Example 1: Uptake of TAMRA Labeled HIV 1 TAT-Peptide in HeLa Living Cells

[0062] HeLa cells where seeded at 60% confluence in a tissue culture treated 6 channel -Slide VI (from Ibidi GmbH, Germany) 24 hr before peptide treatment. The uptake imaging at different pHs was done by washing two times with HEPES buffers (140 mM NaCl, 2.5 mM KCl, 5 mM HEPES, 5 mM glycine, pH adjusted with NaOH or HCl) at the pH of interest. The substance, here the HIV 1 TAT-peptide was provided in HEPES buffer. The buffer solution was replaced with said suspension, comprising the HEPES buffer with the peptide added at a final concentration of 2 M, and adjusted to the desired pH value. The sample was taken to the microscope and imaged at equally spaced intervals of 2 min. This was done simultaneously for pH 6, 7.5 and 9 to compare the relative peptide uptake side by side. HeLa cells were imaged by swapping at each time point between two objectives, an 60 and an 20 immersion oil. This was done to be able to perform two types of time-lapse analysis over the same sample to compute the cellular uptake of the TAT peptide. With the 20 objective is possible to simultaneously visualize several cells but is not easy to separate the fluorescence intensity from internalized peptide from the fluorescent intensity of membrane bound peptide. With the 60 objective a fewer number of cells are visualized but the relative intensity of free peptide can be computed by measuring the fluorescence intensity of peptides accumulated at the nucleolus, since peptide bound to the cell plasma membrane or trapped in endosomes cannot reach the nucleolus. Using the 20 images, the uptake was computed by measuring the average background fluorescence intensity in an area of the images without cells and subtracting this value from the average intensity of the whole image. Using the 60 images, the uptake was computed by measuring the average background fluorescence intensity in an area without cells and subtracting this value from the average intensity in the nucleus. The nucleus area was obtained using the DIC channel. This experiment was repeated 3 times and the average and the standard error plotted. In each experiment, after 30 min the cells were washed with DMEM cell culture media and calcein was added to detect cell viability at a final concentration of 5 M. In live cells, the non-fluorescent calcein AM is converted to green-fluorescent calcein, after acetoxymethyl ester hydrolysis by intracellular esterases. This was incubated for 30 min and then imaged. Cell viability was also assessed using the DIC images used to detect the cell morphology along the experiments. To further evaluate the viability of cells after uptake of the TAT at pH 9, cell division was monitored for 16 hours.

[0063] The results are shown in FIG. 1. FIG. 1 shows the fluorescence intensity of TAT (2 M) uptake in living cells at pH 6, 7.5 and 9 vs. time. Accordingly, at the concentration of 2 M of the TAT peptide there is no uptake at pH 6 and 7.5, most cells kept at pH 9 display a significant uptake within this time interval (30 min).

Example 2: Comparison of Different Mediator Compounds on the Uptake Amino-Rich and Guanidinium-Rich Cell-Penetrating Compounds in Living Cells

[0064] A comparative test of cellular penetration of different cell-penetrating compounds was performed in the presence and absence of different mediator compounds at low and high pH. The cell-penetrating compounds (2 M) and the mediator compounds (6 M) were mixed in Hepes buffers at low pH (6.5) and high pH (8.5).

[0065] The cell media (DMEM) was removed and exchanged by these solutions and incubated for 30 min. Then the solutions were removed and the cells were embedded back in cell culture media (DMEM) and imaged. The efficiency of uptake in the different conditions was determined.

[0066] The results are shown in Table 1 below. The determined efficacy is depicted with increasing efficacy from no uptake detectable, + low efficiency of uptake detectable, ++ moderate efficiency of uptake detectable, +++ strong de efficiency of uptake detectable, ++++ very strong efficiency of uptake detectable.

TABLE-US-00001 TABLE 1 Efficiency of uptake in different conditions. Mediator 1: Mediator 2: no mediator Curcumin Levothyroxine low pH high pH low pH high pH low pH high pH of 6.5 of 8.5 of 6.5 of 8.5 of 6.5 of 8.5 Peptide 1: TAT peptide + ++ ++ labeled with TAMRA Peptide 2: R10 + +++ +++ labeled with FITC cyclic Peptide: cR10 ++ ++++ ++++ labeled with FITC DNA repair enzyme ++ ++ Nanoparticle with amino + ++ + +++ + +++ functional groups: Cube octameric silsesquioxanes (COSS) labeled with FITC. Comparative example peptide without amino functional groups: TAMRA-Ahx- LGQQQPFPPQQPY

Example 3: Cellular Uptake in Fatty Acid Enriched Cells

[0067] A HEPES buffer at pH 7.5 was mixed with 0.2% volume of oleic acid. Cells were washed twice and incubated for 15 min with this buffer. Next, cells were washed once with a HEPES buffer at pH 7.5 (without oleic acid) and the TAT peptide mixed with this last buffer was added at different concentrations. Cells were washed after 5 min with DMEM cell culture media two times, media plus calcein was added to monitor for enzymatic activity and the cells were imaged.

[0068] The results are shown in FIG. 5. It may immediately taken therefrom that fatty acid enriched cells display a much higher uptake efficiency than the control cells and most cells are viable as indicated by their morphology and their enzymatic activity. Therefore, enriching the plasma membrane with fatty acids enhances the binding and uptake of arginine-rich peptides.

Example 4: Peptides Absorption into a Hydrophobic Phase as a Function of pH

[0069] TAMRA labeled TAT peptides were diluted in HEPES buffer (140 mM NaCl, 2.5 mM KCl, 5 mM HEPES, 5 mM glycine, pH adjusted with NaOH or HCl) to a final concentration of 10 M at each pH. 150 l of the buffer-peptide mix was added to 146 l of octanol plus 4 l of a compound selected from octanol, oleic acid, acetic acid, mono-N-dodecylphosphate, sodium dodecylsulfate, lithocholic acid, sunflower oil, castor oil and olive oil. Each pH mix was vortexed for 5 min and centrifuged for 2 min with a centripetal force of 2200 g to separate the octanol from aqueous phases. The octanol phase in contact with each pH buffer was then extracted with a pipette and mixed with a buffer at pH 4, which was also vortexed and centrifuged. In this way the fraction of peptide previously absorbed in the octanol phase was reabsorbed in the pH 4 buffer and in this case the octanol phase (with little or no traces of the TAT peptides) was again removed with a pipette and discarded. The peptide in each buffer solution was measured using a fluorescent spectrometer, using as a reference a buffer solution of 10 M peptide at pH 4, measuring the relative fluorescent intensity emission between the reference and solution and the solution of interest, exciting with a laser wavelength of 543 nm and measuring the emission wavelength of 575 nm. Similarly the peptides that remained in the aqueous phase were compared to a reference solution of 10 M peptide at each pH without having been in contact with the octanol phase.

[0070] The results are shown in FIG. 2. In the left column of FIG. 2 are shown snapshots of microcentrifuge tubes containing the different hydrophobic phases in contact with the aqueous buffers at different pHs. FIG. 2a shows that in the absence of oleic acid as well and in the presence of acetic acid TAT does not enter the hydrophobic phase. FIG. 2b shows that phosphate and sulfur groups containing compounds remain bounded to the TAT peptide and partition into the hydrophobic phase at every pH. Lithocholic acid displays a similar behavior as oleic acid although the deprotonation in this case is shifted to a higher pH. FIG. 2c shows partition of the TAT peptide into three distinct types of natural vegetable oils: sunflower oil, castor oil and olive oil. Sunflower oil displays a behavior consistent with a composition of only triglycerides displaying no absorption of the TAT peptide in the hydrophobic phase. Castor oil behaves as having also free fatty acids showing an absorption behavior similar to oleic acid. Olive oil displays absorption of the TAT peptide at the interface for every pH revealing the presence of phospholipids.

[0071] The results shown in FIG. 2c open a new approach for detecting if natural oil has expired or if it has been adulterated by mixing it with a different oil.

[0072] It is well known that oxidized or aged olive oil can be detected by determining the content and nature of free fatty acids. Therefore, the absorption of a peptide at high pHs into a hydrophobic phase would change.

[0073] An olive oil that is adulterated with tea tree oil can be detected by two significant changes depending on the percentage of tea tree oil added. Firstly, the absorption of peptides at the interface between the oil and the aqueous buffer changes, since the concentration of phospholipids naturally present in olive oil changes, and secondly, the amount of peptide absorbed at high pH also changes, since the mix reduces the amount of free fatty acids.

[0074] Using a proper calibration, the above methods offer a simple, immediate and cheap way to detect oil that has been adulterated or that is expired. Particularly, the present application in a further aspect is related to a method for determining the state, particularly the quality, of an oil. Such method particularly comprises at least the following steps: providing a sample of the oil, applying an aqueous buffer to the oil sample, adding a peptide according to the present invention to the sample, and determining the amount of peptide absorbed at high pH and/or determining the absorption of peptides at the interface between the oil and the aqueous buffer. The absorption of the peptides in olive oil and canola oil or other oils is very different. Therefore, knowing how the peptide is absorbed at different pHs allows a person skilled in the art to conclude on the quality of the oil, and even if the oil has been adulterated.

[0075] As apparent from FIG. 2, the peptides can be easily used to detect different kind of oils and compositions within the oils.

Example 5: Efficacy of the Composition of the Present Invention as a Skin Treatment

[0076] For determining the efficacy of the composition of the present invention, a composition was manufactured comprising a buffer solution at a pH higher than 7.8 an RRP at a concentration of 10 micro Molars labeled with a fluorescent die.

[0077] The composition is tested in a clinical study, wherein volunteers donate 30 mm.sup.2 of skin extracted by a punch biopsy. The skin sample is placed in a tube with the stratum corneum exposed to air while the other kin layers are submerged into cell culture media. A drop of the buffer solution containing the cell-penetrating compound is applied on top of the stratum corneum for 1 hr. Then the area is washed 3 times with PBS, the skin is sectioned with a scalp and imaged at a confocal microscope. It can be seen that the cell-penetrating substance is able to cross the stratum corneum and get inserted in cells within several layers below the stratum corneum.

[0078] The efficacy can be clearly seen as the fluorescently labeled guanidinium-rich peptide is able to cross the stratum corneoum and reach the interior of cells. In the cells the nucleolus is also labeled indicating the peptide is not entrapped in endosomes and is consequently immediately bioavailable.

Example 6: Uptake of Compounds at Different pHs

[0079] FIG. 3 shows the uptake of other compounds at different pHs, showing that the pH enhances penetration of guanidinium and amino groups into the cells.

[0080] It can be immediately taken therefrom that increasing the extracellular pH consistently increases the transduction efficiency of arginine rich peptides with different structures, lengths and chirality. Fluorescence images show the uptake of the peptides listed in Table 1 (2 M) in living cells (HeLa) at pH 6, 7.5 and 9 after 30 min. For each pH is shown the fluorescent confocal image of the peptides (green) and an image composed of an overlay of the DIC, the peptides and the nucleus (red). Cells permanently expressing Proliferating Cell Nuclear Antigen (PCNA) labeled with Cherry or GFP were used to facilitate the detection and visualization of confocal planes across the nucleus. This helped to easily classify and count cells containing transduced peptides from the cells that only contain membrane bound peptides. Although, this is clear when the peptides are in D form (R10 or cR10) since they are stable and distinctly label the nucleolus, the peptides in L form are being actively degraded and the signal quickly redistributes more homogeneously within the cell making more challenging to distinguish membrane bound peptides form internalized peptides. Cells were counted as positive when the peptide signal co-localized with the PCNA signal. The percentage of cells counted with intracellular peptide at pH 9 was consistently larger relative to pH 6 and 7.5 for all arginine rich peptides, while the poly-lysine peptide K10 displays no uptake at all pHs. The uptake efficiency also increases with the number of arginine amino acids and by cyclization. The images where acquired with a 60 objective magnification. Each experiment was repeated 3 times and the percentage represents the average over more than 400 cells in each case. Scale bar 15 m.

Example 7: Delivery of an Arginine and Amino Rich Peptide

[0081] FIG. 4 shows the delivery of an arginine and amino rich peptide, here the TAT peptide, transporting a florescent molecule across the skin.

[0082] A skin sample was obtained from a healthy individual and a drop of the compound in a buffer at pH 9 mixed with ricinoliec acid (the compound and the fatty acid at a concentration of 20 M) was immediately applied on the skin surface for 60 min. The dermis was kept permanently in contact with cell media supplemented with nutrients to keep the tissue alive. The living sample was then washed, fractioned with a scalp and imaged with a confocal microscope specially designed to image living tissue.

[0083] It can be immediately taken from FIG. 4 that the fluorescently labeled molecule is transported across the stratum corneum and reaches the epidermis and dermis layers and the cells interior (distributed in the cytosol and the cell nucleus). The different layers are schematically shown as a reference. The distribution gradient of the drug accumulation is immediately evident, as it goes deeper into the skin layers, naturally with higher concentration in the upper layers.

[0084] One with ordinary skill in the art will recognize from the provided description and examples that modifications and changes can be made to the various embodiments of the invention without departing from the scope of the invention defined by the claims and their equivalents.

[0085] The features of the present invention disclosed in the specification, the claims, examples and/or the figures may both separately and in any combination thereof be material for realizing the invention in various forms thereof.