Preparation method of prune exosome and application of prune exosome in regulation and control of pigment metabolism and skin cell inflammatory response

Abstract

The invention discloses a preparation method of prune exosomes and application of the prune exosomes in regulation and control of pigment metabolism and skin cell inflammatory response, and the preparation method comprises the following steps: taking prune, washing, peeling, dicing, juicing, adding a compound enzyme preparation, centrifuging at 2,000 g for 20 min, carrying out trehalose-assisted ultracentrifugation, centrifuging at 12,000 g for 30 min, centrifuging at 40,000 g for 60 min, centrifuging at 120,000 g for 70 min, collecting precipitate PBS, re-suspending, and filtering to obtain the high-purity prune exosomes. It is proved for the first time that the prune exosome dose-dependently inhibits the melanin content and tyrosinase activity in B16F10 melanoma cells, the mRNA expression level of key inflammatory factors TNF-alpha, IL-1beta and IL-6 in TNF-alpha-induced Hacat cells is remarkably reduced, in addition, the inhibition effect of melanin can be amplified through an STAT3 channel by combining the prune exosome with luteolin, and the application of the prune exosome to the preparation of the anti-inflammatory drug is promoted. The polypeptide can be used for preparing a preparation for regulating pigment anabolism and skin-related inflammatory response, and a potential biological application value is exerted.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction and cosmetic raw material preparation technology, specifically involving a method for preparing plum exosomes and their application in regulating pigment metabolism and skin cell inflammatory response. Background Technology

[0002] Exosomes are a class of heterogeneous, endogenous nanoscale cellular secretory vesicles. Recent studies have found that exosomes isolated and purified from edible fruits, vegetables, and other plants have become important research subjects in the fields of medical health, tissue engineering, and cosmetic technology due to their unique biological characteristics. Compared to mammalian exosomes, plant exosomes exhibit significant advantages in terms of biosafety, preparation cost, storage stability, immunogenicity, and pharmacological activity, opening new avenues for their application in biomedical and cosmetic fields.

[0003] Prunes are rich in vitamin C and polyphenols (such as anthocyanins and flavonoids), which can neutralize free radicals, reduce UV-induced oxidative damage, and exert a whitening effect by inhibiting melanin formation. They interfere with tyrosinase activity, block the oxidation of dopaquinone into melanin, and reduce the deposition of dark oxidized pigments in the skin. They convert existing dark oxidized melanin into lighter reduced pigments, thus brightening the complexion. The polyphenols in prunes have antioxidant effects, reducing UV-induced inflammation and collagen degradation, indirectly delaying skin aging and dullness. The natural fruit acids in prunes gently exfoliate dead skin cells, reducing stratum corneum buildup and indirectly improving rough and dull skin. By accelerating epidermal renewal, they help metabolize melanin deposited on the surface, improving superficial sun-induced pigmentation. Therefore, prunes are a hot topic in the cosmetics industry. Furthermore, plant exosomes (such as kudzu root exosomes) have been shown to regulate inflammatory responses by delivering bioactive molecules. For example, studies have shown that miRNAs carried by plant exosomes can regulate gene expression in host cells and inhibit the release of inflammatory factors (such as TNF-α and IL-6), thereby alleviating inflammatory responses. Currently, the whitening and anti-inflammatory properties of prune exosomes have not been directly verified experimentally, but based on the active ingredients of their parent plant, mechanistic studies of other plant exosomes, and the versatility of exosome delivery systems, prune exosomes possess significant development potential.

[0004] Current research on exosomes largely focuses on animal and human cells, while research on plant exosomes is still in its early stages. Many extraction process parameters are unclear, leading to poor product reproducibility and low yields. Direct research on prune exosomes is limited, and no direct studies have clearly demonstrated that prune exosomes affect melanin metabolism or regulate inflammatory responses. Furthermore, there are no reports on combining luteolin, an existing melanin-reducing drug, with prune exosomes to explore their synergistic effects. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing plum exosomes.

[0008] To solve the above-mentioned technical problems, the present invention provides a method for preparing prune exosomes, comprising,

[0009] Wash and peel the plums, cut them into pieces, add pure water and juice them, filter to remove the pulp, add sodium citrate buffer to the initial filtrate, add compound enzyme preparation, shake and incubate at 37℃ for 30 minutes, and immediately stop the reaction in an ice bath.

[0010] The enzymatic hydrolysate was centrifuged at 2,000g for 20 minutes to remove large particles and plant fibers, resulting in supernatant a. Trehalose was added and the pH was adjusted.

[0011] Centrifuge the supernatant a with added trehalose and adjusted pH at 12,000g for 30 min to remove cell debris and obtain supernatant b.

[0012] Centrifuge supernatant b at 40,000g for 60 min to remove large vesicles and obtain supernatant c;

[0013] Centrifuge the supernatant c at 120,000g for 70 min, collect the precipitate, resuspend it in PBS, and filter it through a filter membrane to obtain plum exosomes.

[0014] As a preferred embodiment of the preparation method of the present invention, the following steps are included: the filtration to remove fruit residue is performed using a 100-200 mesh nylon filter; the concentration of trehalose added is 0.1M, and the pH is adjusted to 5.0 after mixing.

[0015] In a preferred embodiment of the preparation method described in this invention, the temperature during the centrifugation process is 4°C.

[0016] In a preferred embodiment of the preparation method described in this invention, the composite enzyme preparation comprises pectinase and cellulase, wherein the content of pectinase is 0.5 U / mL and the content of cellulase is 0.2 U / mL.

[0017] In a preferred embodiment of the preparation method described in this invention, the filter membrane is used for filtration, wherein the filter membrane has a size of 0.22 μm.

[0018] Another objective of this invention is to overcome the shortcomings of the prior art and provide a prune exosome, wherein the natural prune exosome has a particle size of 149.3±10.6 nm and a protein concentration of 4913.3 μg / ml.

[0019] Another objective of this invention is to overcome the shortcomings of the prior art and provide a prune exosome that can dose-dependently inhibit the melanin content and tyrosinase (TYR) activity in B16F10 melanoma cells, while also providing a combination of prune exosomes and luteolin to enhance the inhibitory effect on melanin.

[0020] Another objective of this invention is to overcome the shortcomings of the prior art and provide a plum exosome that can significantly downregulate the mRNA expression levels of key inflammatory factors TNF-α, IL-1β and IL-6 in TNF-α-induced Hacat cells.

[0021] Beneficial effects of this invention:

[0022] (1) This invention uses plum processing residue as raw material, which reduces production costs by 30%-50%, and conforms to the concept of circular economy.

[0023] (2) The present invention uses a unique optimized process in the preparation process, including precise centrifugation parameters, mild centrifugation conditions, and eliminates flocculent precipitation by enzymatically degrading the polysaccharide network, simplifying the centrifugation process, increasing the product concentration, and adding 0.1M trehalose to the centrifugation system before ultracentrifugation to form an exosome membrane protective layer, reducing membrane rupture caused by high-speed centrifugation.

[0024] (3) The natural exosomes of plum extracted by S1-S4 in this invention have a particle size of 149.3±10.6nm. The vesicle morphology and particle size are consistent as confirmed by transmission electron microscopy (TEM). The yield of exosome protein in plum is high as determined by BCA method. The extracted exosomes can be used for other applications. It is environmentally friendly and pollution-free.

[0025] (4) This invention reveals for the first time that the extracted natural exosomes of plum have good biological activity, affect the melanin synthesis metabolic pathway of B16F10 cells, successfully inhibit tyrosinase (TYR) activity, regulate the immune response of skin keratinocytes (Hacat) under inflammatory stimulation, and the combined use of plum exosomes and luteolin may synergistically block the upstream signal STAT3 of melanin synthesis and improve bioavailability. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0027] Figure 1 This is a flowchart of the method for preparing plum exosomes according to the present invention.

[0028] Figure 2 This is Example 1 of the present invention. Figure 2 a) and Example 2 ( Figure 2 b) Determination of particle size distribution of plum exosomes prepared by extraction method.

[0029] Figure 3 This is Example 1 of the present invention. Figure 3 a) and Example 2 ( Figure 3 b) Transmission electron micrograph of the prune exosome precipitate obtained after centrifugation.

[0030] Figure 4 This is a standard curve for determining the concentration of plum exosome protein in Example 2 of the present invention using BCA assay.

[0031] Figure 5 This is a schematic diagram illustrating the biological function of prune exosomes affecting pigment metabolism in B16F10 cells, as shown in Example 2 of this invention.

[0032] Figure 6 This is a schematic diagram illustrating the biological function of the combined use of prune exosomes and luteolin in the cytochrome metabolism of B16F10 cells, as described in Example 2 of this invention.

[0033] Figure 7 Example 2 of this invention describes the effect of prune exosomes on the levels of TNF-α, IL-1β, and IL-6 in Hacat cells. Detailed Implementation

[0034] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0035] In this embodiment of the invention, plums were purchased from Beijing Weier Technology Co., Ltd.; pectinase and cellulase were purchased from Yuanye Company; BCA protein concentration assay kit was purchased from Beyotime Company; B16F10 mouse melanoma cells and human immortalized keratinocytes (Hacat) were purchased from Pronosei Company, all of which are commercially available products.

[0036] Example 1

[0037] This invention provides a method for preparing natural exosomes from prunes, employing an optimized differential centrifugation combined with ultracentrifugation, specifically including the following steps:

[0038] (1) Take fresh plums, wash them, peel them, cut them into pieces, add about 200ml of pure water to soak and juice them, filter them with gauze to remove the pulp, centrifuge 2,000g for 20 minutes to remove large particles and plant fibers.

[0039] (2) Take 20,000g of the supernatant and centrifuge for 60min to remove cell debris;

[0040] (3) Take 40,000g of the supernatant and centrifuge for 60min to remove large vesicles;

[0041] (4) Take 120,000g of the supernatant and centrifuge for 130min. The final precipitate is resuspended in 500μl PBS. If flocculent matter is not completely dissolved, it needs to be placed in a 4℃ low-temperature shaker overnight to fully dissolve it. Filter through a 0.22μm filter membrane to obtain the plum exosome suspension.

[0042] Example 2

[0043] like Figure 1 As shown, this invention provides a method for preparing natural exosomes from prunes, employing an optimized enzyme-assisted preparation method, specifically including the following steps:

[0044] (1) Take fresh plums, wash them, peel them, cut them into pieces, add about 200ml of pure water to soak them and squeeze out the juice. Filter the juice through gauze to remove the pulp and obtain the initial filtrate.

[0045] (2) Enzymatic pretreatment: Add sodium citrate buffer (pH 5.0) to the initial filtrate to a total volume of 250 mL, add compound enzyme preparations: pectinase (final concentration 0.5 U / mL) and cellulase (final concentration 0.2 U / mL), incubate at 37 °C with shaking for 30 min, and immediately stop the reaction by placing it in an ice bath.

[0046] (3) Centrifuge the enzymatic hydrolysate at 2,000g for 20min to remove large particles and plant fibers, add 0.1M trehalose and adjust the pH to 5.0;

[0047] (4) Take 12,000g of the supernatant and centrifuge for 30 minutes to remove cell debris;

[0048] (5) Take 40,000g of the supernatant and centrifuge for 60min to remove large vesicles;

[0049] (6) Take 120,000g of the supernatant and centrifuge for 70min. The final precipitate is resuspended in 500μl of PBS and filtered directly through a 0.22μm filter membrane to obtain a high-purity plum exosome suspension.

[0050] Example 3

[0051] The prune exosomes extracted in Examples 1 and 2 were validated. The particle size of the plant exosomes was determined using a Malvern Nano ZS instrument with DLS: samples were diluted in PBS, transferred to dedicated quartz cuvettes, and each sample was measured three times at room temperature. The particle size was measured in at least three independent batches, and the mean ± standard deviation was calculated. Figure 2 The extracted natural exosomes from prunes had particle sizes of 192.4±14.18 nm (a) and 149.3±10.6 nm (b), respectively.

[0052] Observation was performed using a JEOL Jem-1400plus transmission electron microscope (TEM). 5 μL of sample was taken, and a carbon film was placed over the sample droplet for 1 min to allow adsorption. A copper mesh was then placed over the phosphotungstic acid staining solution for 30 s, followed by drying at room temperature for several minutes. Electron microscopy at 100 kV revealed that the prune exosomes exhibited a concave, cup-shaped appearance under the TEM. Figure 3 And the size is consistent with the DLS results.

[0053] Total protein concentration was determined using the BCA protein concentration assay kit: 2 mg / mL of BSA protein standard solution from the kit was diluted in a 96-well plate according to the table below.

[0054] Vial Diluent volume (μg / ml) BSA standard volume (μl) Final BSA concentration (μg / ml) A 0 100 2000 B 25 75 1500 C 50 50 1000 D 83 50 750 E 75 25 500 F 140 20 250 G 150 10 125 H 158 2 25 I 100 0 0 = Blank

[0055] Prepare BCA working solution with a volume of 200 μL per well. Mix solution A and solution B at a ratio of 50:1, mix thoroughly, and store in a sealed container at room temperature for 24 hours to ensure stability.

[0056] Take 10 μL of standard and test sample (the test sample is diluted 10 times) and add them to a 96-well plate. Add 200 μL of LCA working solution to each well, mix thoroughly by pipetting, incubate at 37°C for 30 min, cool to room temperature, and detect the absorbance at 562 nm wavelength on a microplate reader.

[0057] Based on the absorbance of the BSA standard (subtracting the OD value of the blank well in the standard to obtain the final reading), a standard curve was plotted (X - protein concentration μg / mL; Y - final OD 562 nm). The protein concentration of plum exosomes was calculated based on the standard curve and the dilution factor of the sample.

[0058] Standard curve such as Figure 4 The final concentration of prune exosome protein obtained from Example 2 was 4913.3 μg / ml.

[0059] Example 4

[0060] This embodiment provides the application of the plum exosomes prepared in Example 2 above. Cell experiments were conducted on the exosomes to detect their inhibitory effect on melanin and tyrosinase in B16F10 cells, in order to verify the effectiveness of the exosomes in affecting the pigment synthesis metabolic pathway of B16F10 cells.

[0061] Experimental methods:

[0062] Mouse melanoma B16F10 cells were cultured in high-glucose DMEM medium, with all media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution, and cultured in a humid environment at 37°C with 5% CO2.

[0063] Experimental groups: blank group (B16F10 cells + culture medium); positive control group (B16F10 cells + arbutin); drug treatment group: B16F10 cells + different concentrations of prune exosomes.

[0064] B16F10 cells were loaded at a rate of 2 × 10⁻⁶ 4 Cells were seeded at a density of 50-60% in 6-well plates. After the cells adhered and grew to 50-60%, different concentrations of prune exosomes (20, 60, 100 μg / ml) and Arbutin were added for incubation. Each group had three replicates.

[0065] Melanin detection:

[0066] The cells were then incubated for 48 hours. After 48 hours, the old culture medium in the wells was removed, and the cells were washed with cold PBS. 200 μL of trypsin was added to each well, and the cells were digested at 37°C for two minutes. The cell suspension was then transferred to EP tubes, mixed, and an additional 200 μL of cells was transferred to another EP tube for later use. The cells were centrifuged at 5000 rpm for 5 minutes, and the supernatant was discarded. Then, 500 μL of 1N NaOH containing 10% DMSO was added to the cell pellet, and the cells were lysed at 90°C for 1 hour. The total melanin content was measured using an enzyme-labeled immunosorbent assay (ELISA) at 450 nm.

[0067] Using the absorbance value of the blank group as a control, the relative content of melanin in the cells of each group was calculated. Cells were counted in all samples in 200 μL EP tubes, and the melanin content was normalized based on the cell count.

[0068]

[0069] Tyrosinase detection:

[0070] After incubation for 48 hours, the old culture medium in the wells was aspirated into EP tubes. Cells were then washed with cold PBS, and 200 μL of trypsin was added to each well for digestion at 37°C for two minutes. The cell suspension was then transferred to EP tubes, mixed, and an additional 200 μL of cells was transferred to another EP tube for later use. The cells were centrifuged at 5000 rpm for 5 minutes, and the supernatant was discarded. Then, 500 μL of 1% Triton X-100 solution was added to the cell pellet, and the centrifuge tubes were incubated at -80°C for 20-30 minutes, followed by thawing and lysis at room temperature. This process was repeated twice, centrifuging at 12,000 rpm for 10 minutes at 4°C. Subsequently, 100 μL of supernatant and 100 μL of 4 mM L-DOPA were added to 96-well plates, repeated 3-4 times. The plates were incubated at 37°C for 1 hour, and the absorbance was measured at 490 nm. This absorbance represents the activity of tyrosinase. The calculation method was the same as for melanin.

[0071] The results are as follows Figure 5 As shown, compared with the blank control group, with the increase of prune exosome concentration, the melanin content and tyrosinase activity in B16F10 cells decreased significantly in a dose-dependent manner, and at high concentrations, prune exosomes showed stronger inhibitory activity on melanin production in cells than the positive control drug Arbutin.

[0072] In addition, following the methods described above for detecting melanin, we investigated the synergistic effect of prune exosomes and luteolin on melanin regulation.

[0073] B16F10 cells were divided into four groups: control group, exosome group (60 μg / ml prune exosomes), luteolin group (10 μM), and combination group (30 μg / ml exosomes + 5 μM). To strictly distinguish between the synergistic effect and the simple dose-addition effect of prune exosomes and luteolin, this study adopted a constant total drug dosage design, achieved by normalizing the dosage of each component to a percentage of its total single dose. The specific implementation is as follows:

[0074] The effective dose used alone (60 μg / ml prune exosomes or 10 μM luteolin) is considered as a 100% dose unit.

[0075] While maintaining a total drug dosage of 100%, combination groups with different mass ratios were established. The combination group consisted of exosomes and luteolin at a ratio of 50%:50% = 30 μg / ml exosomes + 5 μM luteolin. Results were as follows... Figure 6 As shown, the combined use of prune exosomes and luteolin can amplify the inhibitory effect on melanin.

[0076] The above results demonstrate that the natural prune exosomes prepared in Example 2 successfully induced a decrease in melanin content and tyrosinase activity in B16F10 cells, and the melanin-reducing ability increased with increasing concentration. Previous literature reports that the core function of luteolin is to inhibit the STAT3 signaling pathway. By directly binding to the Src protein kinase domain, it inhibits phosphorylation, blocks STAT3 activation, and promotes STAT3 degradation via the ubiquitin-proteasome pathway, thereby downregulating the expression of TYR and microphthalmia-associated transcription factor (MITF). Simultaneously, the polyphenols carried by the exosomes enhance the solubility and cellular uptake efficiency of luteolin, improving its bioavailability. By directly inhibiting TYR enzyme activity and downregulating MITF mRNA expression, luteolin synergistically blocks upstream signals in melanin synthesis.

[0077] Example 5

[0078] This embodiment provides the application of the prune exosomes obtained in Example 2 above. The qRT-PCR detection verifies the role of the exosomes in inflammation-induced cells, in order to verify the effect of the extracted natural prune exosomes in anti-inflammatory applications.

[0079] Experimental methods:

[0080] Human immortalized keratinocytes (Hacat) were cultured in high-glucose DMEM medium. All media were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution and cultured in a humid environment at 37°C with 5% CO2.

[0081] Experimental groups: control group (culture medium); stimulation group (culture medium containing 20 ng / ml TNF-α); positive control group (culture medium containing 10 μM dexamethasone (Dex) + 20 ng / ml TNF-α); drug administration group (culture medium containing different concentrations of prune exosomes + 20 ng / ml TNF-α).

[0082] Hacat cells were loaded at 5 × 10 5 Cells were seeded at a density of 50-60% in 6-well plates. After the cells adhered and grew to 50-60%, different concentrations of prune exosomes (20, 60, 100 μg / ml) and Dex were added and incubated. Each group had three replicates.

[0083] After incubation for 24 hours, TNF-α was added to each well for 6 hours of stimulation, except for the control group. RNA was then extracted using an RNA extraction kit. RNA was reverse transcribed using the kit. The expression levels of inflammation-related genes were detected using qRT-PCR, such as... Figure 6 The results showed that natural prune exosomes could downregulate the mRNA expression of TNF-α, IL-1β, and IL-6, further confirming the anti-inflammatory activity of the prune exosomes of this invention.

[0084] In summary, this invention provides an optimized preparation process for high-purity prune exosomes. Through an optimized enzyme- and trehalose-assisted preparation method, combined with differential centrifugation and ultracentrifugation, the dual-enzyme synergistic targeted cell disruption allows for shorter centrifugation time. Trehalose protects the integrity of the exosome membrane structure, reducing the risk of rupture. This refined process improves yield and stability. Characterization was performed using dynamic light scattering (DLS), transmission electron microscopy (TEM), and protein detection, providing a reference for improving plant exosome databases. As plant-derived nanoparticles, prune exosomes effectively reduce melanin synthesis and inhibit tyrosinase activity in low doses. Furthermore, the combined use of prune exosomes and luteolin amplifies the melanin-inhibiting effect through the STAT3 pathway. In addition, the exosomes prepared by this invention can effectively inhibit the release of inflammatory factors from skin keratinocytes. With its natural source and environmental advantages, prune exosomes possess anti-inflammatory, whitening, and barrier repair functions, making them suitable for photoaging care and sensitive skin repair, and potentially providing a new strategy for the development of naturally derived cosmetic raw materials.

[0085] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. A method for preparing prune exosomes, characterized in that: include, Wash and peel the plums, cut them into pieces, add pure water and juice them, filter to remove the pulp, add sodium citrate buffer to the initial filtrate, add compound enzyme preparation, shake and incubate at 37℃ for 30 minutes, and immediately stop the reaction in an ice bath. The enzymatic hydrolysate was centrifuged at 2,000g for 20 minutes to remove large particles and plant fibers, resulting in supernatant a. Trehalose was added and the pH was adjusted. Centrifuge the supernatant a with added trehalose and adjusted pH at 12,000g for 30 min to remove cell debris and obtain supernatant b. Centrifuge supernatant b at 40,000g for 60 min to remove large vesicles and obtain supernatant c; Centrifuge the supernatant c at 120,000g for 70 min, collect the precipitate, resuspend it in PBS, and filter it through a filter membrane to obtain plum exosomes.

2. The preparation method according to claim 1, characterized in that: The filtration process removes fruit residue, and a 100-200 mesh nylon filter is used for filtration. The concentration of added trehalose is 0.1M, and the pH is adjusted to 5.0 after mixing.

3. The preparation method according to claim 1 or 2, characterized in that: The temperature during the centrifugation process is 4℃.

4. The preparation method according to claim 1, characterized in that: The compound enzyme preparation consists of pectinase and cellulase, with the pectinase content being 0.5 U / mL and the cellulase content being 0.2 U / mL.

5. The preparation method according to claim 3, characterized in that: The filter membrane is used for filtration, wherein the filter membrane has a size of 0.22 μm.

6. Prunus exosomes prepared by any one of the preparation methods described in claims 1 to 5.

7. The prune exosome as described in claim 7, characterized in that: The natural exosomes from the plum had a particle size of 149.3 ± 10.6 nm and a protein concentration of 4913.3 μg / ml.

8. The use of the prune exosomes according to claim 7 in the preparation of a formulation having the effect of influencing melanin metabolism.

9. The use of the prune exosomes of claim 7 in combination with luteolin in the preparation of a formulation that amplifies melanin metabolism.

10. The use of the prune exosomes of claim 7 in the preparation of agents that modulate inflammatory cellular immune responses.

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