Method for loading a carrier with a substance
By applying ultrasound stimulation to exosomes and useful substances and co-culturing them, and optimizing the ultrasound treatment conditions, the problem of low loading efficiency in existing technologies was solved, and the effect of efficient loading and delivery to target cells was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- STROMON BIOTECH CO LTD
- Filing Date
- 2024-07-10
- Publication Date
- 2026-07-24
AI Technical Summary
In existing technologies, the efficiency of loading useful substances into exosomes via ultrasound is less than 30%, and there is a need to improve loading efficiency to maximize its application in cosmetics, health functional foods, or medical devices.
By applying ultrasonic stimulation to a carrier containing a phospholipid layer and a useful substance, and then culturing the mixture of the ultrasonically stimulated carrier and the useful substance for a certain period of time, the ultrasonic treatment conditions were optimized to improve loading efficiency.
It significantly improved the loading efficiency of useful substances in exosomes, reaching over 60%, reduced damage to useful substances, and achieved effective delivery to target cells.
Smart Images
Figure CN122458968A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for loading a useful substance onto a carrier. Background Technology
[0002] Numerous attempts have been made to load various substances such as nucleic acids, proteins, and drugs into vectors such as exosomes. Representative methods include incubation, electroporation, extrusion, freeze-thaw cycle, chimeric exosome method, endogenous loading, and sonication (ultrasound treatment).
[0003] According to the survey, the loading efficiency of protein catalase via ultrasound was approximately 26±1.2%, which is slightly higher than that of extrusion (22±3.1%), surfactant treatment (18.5±1.3%), freeze-thaw treatment (14.7±1.1%), and incubation (4.9±0.5%). Furthermore, the loading efficiency of chemotherapeutic agent via sonication (28.29±1.38%) was also higher than that via electrophoresis (5.30±0.48%). Gold nanomaterials could also be loaded with high efficiency via sonication (19.3±10%), while the loading efficiency of incubation was 13.7±9.9%.
[0004] However, while the efficiency of loading useful substances into exosomes via ultrasound is approximately 19.3%–28.3%, higher than other methods, it is still below 30%, thus necessitating further improvement in loading efficiency. Improving loading efficiency not only brings significant advantages in subsequent processes and costs, but also maximizes the effect on recipient cells even with only a small amount of carrier. Therefore, research is currently underway on optimizing ultrasound loading methods and maximizing loading efficiency, so that these methods can be utilized as useful proteins in cosmetics, health foods, or medical devices. Summary of the Invention
[0005] Technical issues
[0006] The technical problem to be solved by the present invention is to provide a method for loading useful substances onto a carrier.
[0007] The technical problems to be solved by the present invention are not limited to the above-mentioned technical problems. Those skilled in the art can also clearly understand other technical problems not explicitly mentioned based on the following description.
[0008] Problem-solving methods
[0009] To address the aforementioned technical problems, one embodiment of the present invention provides a method for loading a useful substance onto a carrier, comprising: applying ultrasonic stimulation to the carrier containing a phospholipid layer and the useful substance respectively; and culturing the mixture of the ultrasonically stimulated carrier and the useful substance for a certain period of time.
[0010] In embodiments of the present invention, the carrier may be an exosome.
[0011] In embodiments of the present invention, the carrier may be an exosome derived from human skin fibroblasts (HDF).
[0012] In an embodiment of the present invention, the exosomes can be prepared by the following steps: applying ultrasound stimulation to cells derived from human skin fibroblasts and applying ultrasound stimulation to a cell-free culture medium; culturing a mixture formed by mixing the ultrasound-stimulated human skin fibroblast-derived cells with the ultrasound-stimulated culture medium for a certain period of time; and isolating exosomes from the mixture.
[0013] In embodiments of the present invention, the useful substance may be nucleic acid, protein, peptide, nanomaterial, plasmid DNA, low molecular weight compound, drug, hormone, enzyme or neurotransmitter.
[0014] In an embodiment of the present invention, the step of applying ultrasonic stimulation to the carrier can be performed for 1 to 10 seconds at an amplitude of 20% to 40% of AMP.
[0015] In embodiments of the present invention, the step of applying ultrasonic stimulation to the useful substance can be performed for 0.1 to 5 seconds at an amplitude of 15% to 30% of AMP.
[0016] In an embodiment of the present invention, when the useful substance is plasmid DNA, the ultrasonic stimulation can be performed for 0.1 to 3 seconds at an amplitude of 15% to 25% of AMP.
[0017] In embodiments of the present invention, when the useful substance is a protein, nucleic acid, or nanomaterial, the ultrasonic stimulation can be performed for 1 to 5 seconds at an amplitude of 20% to 30% of AMP.
[0018] In an embodiment of the present invention, the step of culturing the mixture can be carried out at 30°C to 40°C for 30 minutes to 2 hours.
[0019] The effects of the invention
[0020] This invention relates to a method for loading useful substances onto a carrier. By utilizing ultrasound, the loading efficiency of useful substances in carriers such as exosomes can be maximized. Therefore, it can be applied to the technical field of effectively delivering various useful substances such as deoxyribonucleic acid, proteins, and nanomaterials to target cells.
[0021] The effects of the present invention are not limited to those described above, but should be understood to include all effects that can be derived from the technical solutions described in the present invention specification or claims. Attached Figure Description
[0022] Figure 1 This is a schematic diagram showing the results of comparing the loading efficiency of a mixture of ultrasound-treated exosomes and useful substances under different culture conditions.
[0023] Figure 2 This is a graph showing the loading efficiency of useful material when ultrasonic treatment is performed under the conditions of 1 hour of incubation at 37°C and 5 seconds of sonication at 30% amplitude (AMP).
[0024] Figure 3 This figure shows the loading efficiency of useful substances when sonicated at 4°C for 1 hour and with an amplitude of 20% (AMP) for 1 second. The sonication conditions (useful substance: AMP 20%, 1 second, 1 time; exosomes: AMP 30%, 5 seconds, 1 time), exosome particle number: 2 × 10^8 particles / sample, and useful substance loading are shown in Table 1.
[0025] Figure 4 This figure shows the loading efficiency of useful substances when sonicated at 37°C for 1 hour and with an amplitude of 20% (AMP) for 1 second. The sonication conditions (useful substance: AMP 20%, 1 second, 1 time; exosomes: AMP 30%, 5 seconds, 1 time), exosome particle number: 2 × 10^8 particles / sample, and useful substance loading are shown in Table 1.
[0026] Figure 5 This is a graph comparing the loading efficiency of useful material (Q-dot) based on whether or not sonication was performed. Exosome particle number: 2×10^8 particles / sample, Q-dot 705 loading: 1 nmole (0.01 μL), sonication conditions: AMP 30%, 5 seconds, 1 time, culture conditions: 37℃, 1 hour, DiD staining: 37℃, 30 minutes.
[0027] Figure 6 and Figure 7This is a result diagram confirming whether the useful substances (IgG and Cy3-DNA) are damaged according to the ultrasound processing conditions.
[0028] Figure 8 This is a diagram showing the results of confirming whether exosomes contain plasmid DNA.
[0029] Figure 9 This is a diagram confirming whether plasmid DNA is damaged under ultrasound conditions.
[0030] Figures 10 to 13 This is a diagram showing the results confirming the delivery of useful substances loaded in exosomes to cells. Detailed Implementation
[0031] The present invention will now be described in detail.
[0032] This invention relates to a method for loading a useful substance onto a carrier.
[0033] The present invention includes the steps of applying ultrasonic stimulation to a carrier containing a phospholipid layer and a useful substance respectively; and the step of culturing the mixture of the ultrasonically stimulated carrier and the useful substance for a certain period of time.
[0034] The carrier comprises a phospholipid layer, and its type and morphology are unrestricted as long as a core can be formed within the phospholipid layer and useful substances can be contained (loaded) within the core. For example, it can be an exosome; more specifically, it can be an exosome derived from human skin fibroblasts (HDF).
[0035] The exosomes are exosomes isolated from cells and can be isolated using known methods and conditions. For example, they can be prepared by: applying ultrasound stimulation to cells derived from human skin fibroblasts (HDF) and applying ultrasound stimulation to a cell-free culture medium; culturing a mixture formed by mixing the ultrasound-stimulated HDF cells with the ultrasound-stimulated culture medium for a certain period of time; and isolating exosomes from the mixture.
[0036] Applying ultrasound stimulation to the HDF-derived cells can be done by directly sonicating the cells, or by using only a minimal amount of initial culture medium just enough to cover the cells. In this case, the initial culture medium is a conventional medium used to maintain the health of the cells, such as DMEM medium containing antibiotics and serum.
[0037] There are no particular restrictions on the type of cell-free culture medium, such as any one of embryonic stem cell culture medium, neural stem cell culture medium, cardiac stem cell culture medium, dermal papilla cell culture medium, mesenchymal stem cell culture medium, osteoblast culture medium, myoblast culture medium, hematopoietic stem cell culture medium, neuron culture medium, astrocyte culture medium, oligodendrocyte culture medium, hepatocyte culture medium, adipocyte culture medium, myocyte culture medium, vascular endothelial cell culture medium, pancreatic β-cell culture medium, or cardiomyocyte culture medium.
[0038] The ultrasound stimulation applied to the cells can be performed for 1 to 10 seconds at 10 to 30 kHz and 0.5 to 3 W / cm², for example; preferably, it can be performed for 3 to 7 seconds at 15 to 25 kHz and 0.5 to 1.5 W / cm².
[0039] The ultrasonic stimulation applied to the cell-free culture medium can be performed for 1 to 20 minutes at 10 to 30 kHz and 1 to 20 W / cm², for example; preferably, it can be performed for 5 to 15 minutes at 15 to 25 kHz and 1 to 10 W / cm².
[0040] The mixture of the ultrasound-stimulated human skin fibroblast-derived cells and the ultrasound-stimulated culture medium can be cultured using known methods and conditions. For example, the culture can be carried out for 1 to 10 days, 1 to 6 days, or 1 to 2 days. Exosomes secrete the highest amount on the first day after ultrasound treatment, and their secretion gradually decreases over time; therefore, the culture time can be appropriately selected within the above range.
[0041] The step of separating exosomes from the mixture can be performed using known means and under known methods and conditions. For example, it may include: centrifuging the cultured mixture to obtain a supernatant; filtering the supernatant through a filter to obtain a filtrate; and concentrating the filtrate.
[0042] The centrifugation is performed to remove cell debris and dead cells, and is preferably carried out for 10 to 60 minutes at 1000×g to 5000×g.
[0043] The step of filtering the supernatant through a filter is to further remove cell debris and retain only particles smaller than a certain size, wherein the filter is preferably a syringe filter.
[0044] The step of concentrating the filtrate can preferably be performed using a centrifugal filter. Using a centrifugal filter allows for the removal of particles smaller than a certain size while concentrating the filtrate.
[0045] If necessary, the step of separating exosomes may further include storing the supernatant at below 4°C for 7 days to 3 months before filtering it through a filter. The storage is preferably carried out at below 4°C for no more than 7 days; more preferably at below -20°C for no more than 1 month; and most preferably at below -80°C for no more than 3 months.
[0046] The useful substance is a physiologically active substance, which may include any substance that promotes or inhibits biological function. For example, it may include nucleic acids, proteins, peptides, nanomaterials, plasmid DNA, low molecular weight compounds, drugs, hormones, enzymes, or neurotransmitters.
[0047] The step of applying ultrasonic stimulation to the carrier can, for example, be performed for 1 to 10 seconds at an amplitude of 20% to 40% of the AMP. Preferably, it can be performed for 3 to 7 seconds at an amplitude of 25% to 35% of the AMP.
[0048] The step of applying ultrasonic stimulation to the useful substance can, for example, be performed for 0.1 to 5 seconds at an amplitude of 15% to 30% of the AMP, or for 1 to 5 seconds at an amplitude of 20% to 30% of the AMP.
[0049] More specifically, when the useful substance is plasmid DNA, the ultrasound stimulation can be performed for 0.1 to 3 seconds at an amplitude of 15% to 25% of AMP. Preferably, it can be performed for 0.5 to 2 seconds at an amplitude of 18% to 22% of AMP. Additionally, when the useful substance is protein, nucleic acid, or nanomaterial, the ultrasound stimulation can be performed for 1 to 5 seconds at an amplitude of 20% to 30% of AMP.
[0050] Although the conditions for ultrasonic stimulation are not limited to the above range, when performed within the above range, damage to useful materials can be minimized and loading efficiency can be improved.
[0051] The step of culturing the mixture of the ultrasonically stimulated carrier and the useful substance can be carried out using known means and according to known methods and conditions. For example, it can be carried out at 30°C to 40°C for 30 minutes to 2 hours. Preferably, it can be carried out at 35°C to 40°C for 50 minutes to 70 minutes.
[0052] The present invention will be described in more detail below through examples.
[0053] [Example 1] Preparation of exosomes derived from human skin fibroblasts (HDF)
[0054] Ultrasound stimulation at 20 kHz and 1.0 W / cm² was applied directly to 1×10^6 human dermal fibroblasts (HDF) for 5 seconds using UltraRepro 1001 (STEMON Inc., Seoul, Republic of Korea). (Hereinafter, the ultrasound-stimulated HDF will be referred to as UHDF).
[0055] Two × 10^5 UHDFs were seeded in 35 mm culture dishes and co-cultured for one day with embryonic stem cell culture medium [DMEM / F12, 15% FBS, 2 mM GlutaMAX, 0.1% NEAA, 0.1% penicillin / streptomycin, 0.1 mM β-mercaptoethanol, 1000 units / mL leukemia inhibitory factor (LIF)] that had been sonicated (20 kHz, 5.0 W / cm², 10 min).
[0056] Exosomes were then isolated from the culture medium containing UHDF using the following method:
[0057] Centrifuge the culture medium at 3,000×g for 20 minutes to remove cell debris and dead cells;
[0058] The resulting supernatant was passed through a 0.22 μm filter (Minisart® Syringe Filter, Sartorius, Goettingen, Germany).
[0059] The filtered culture medium was placed in an Amicon® Ultra-15 100,000 kDa device (Millipore, Billerica, MA, USA) and centrifuged at 14,000×g for 20 minutes to concentrate the exosomes.
[0060] [Example 2] Loading of useful substances into exosomes
[0061] The exosomes prepared in Example 1 were aliquoted into 1.5 mL centrifuge tubes according to the required amount, and DPBS was added to bring the final volume to 100 μL.
[0062] According to Table 1, the useful substances to be loaded were aliquoted into 1.5 mL centrifuge tubes, and DPBS was added to bring the final volume to 100 μL.
[0063] Table 1
[0064]
[0065] Exosomes were sonicated once for 5 seconds at 30% AMP amplitude, and the loaded useful material was sonicated once for 5 seconds at 30% AMP amplitude, or once for 1 second at 20% AMP amplitude. The sonicated exosomes were then thoroughly mixed with the useful material and incubated at 37°C or 4°C for 30 minutes or 2 hours.
[0066] [Experimental Example 1] Comparison of loading efficiency based on culture conditions of sonicated exosomes and mixtures of useful substances
[0067] The results showed that culturing at 37°C was more beneficial for improving loading efficiency compared to culturing at 4°C; and that under culturing conditions ranging from 30 minutes to 2 hours, loading efficiency increased with increasing culturing time. However, considering production efficiency, the culturing time was ultimately determined to be 1 hour. Figure 1 ).
[0068] Furthermore, when using AMP with 30% amplitude for a single 5-second ultrasonic treatment, the loading efficiency is approximately 60%. Figure 2 When using AMP with 20% amplitude for a single 1-second ultrasonic treatment, the loading efficiency is approximately 70%. Figure 3 and Figure 4 ).
[0069] [Experimental Example 2] Comparing loading efficiency based on ultrasonically processed objects
[0070] Loading efficiencies were compared when only the useful material was sonicated, when only exosomes were sonicated, and when both the useful material and exosomes were sonicated separately. The sonication conditions were one 5-second sonication cycle using an AMP at 30% amplitude.
[0071] The results showed that the loading efficiency was 13.04% without sonication; 25.2% when only the useful substance (Q-dot) was sonicated before loading; 42.07% when only exosomes were sonicated before loading; and 59.68% when both the useful substance and exosomes were sonicated.
[0072] This demonstrates that when the ultrasonic processing technology of this invention is used, the loading efficiency of the material reaches its maximum. Figure 5 ).
[0073] [Experiment Example 3] Confirm whether ultrasonic treatment causes damage to useful materials
[0074] To confirm whether sonication would damage useful substances, useful substances (IgG and Cy3-DNA) were sonicated once for 5 seconds at 30% AMP amplitude or once for 1 second at 20% AMP amplitude. The results were then analyzed by SDS-PAGE (with immunoblotting) on 10% acrylamide gel and by 2% agarose gel electrophoresis.
[0075] The results showed that when a single 5-second sonication was performed using 30% of the AMP amplitude, damage to the useful material was confirmed, with the damage level being approximately 50% of that without sonication. On the other hand, when a single 1-second sonication was performed using 20% of the AMP amplitude, it was confirmed that only about 25% of the damage was induced. Figure 6 and Figure 7 ).
[0076] Therefore, since ultrasonic treatment can damage the useful material, it is important to load the material under appropriate ultrasonic treatment conditions. It has also been confirmed that the ultrasonic treatment technique of the present invention can reduce material damage and improve loading efficiency.
[0077] [Experiment Example 4] Plasmid DNA Loading Experiment
[0078] Plasmid DNA was loaded into exosomes and then processed into cells to confirm whether the substances loaded in the exosomes could be delivered to the cells and perform their functions properly.
[0079] (1) Confirm whether Plasmid DNA is loaded in exosomes.
[0080] To confirm whether sonication could promote the loading of Plasmid DNA into exosomes, a single 5-second sonication session was performed using AMP at 30% amplitude, with the following conditions: 1) sonication of exosomes only; 2) sonication of Plasmid DNA only; and 3) sonication of both exosomes and Plasmid DNA simultaneously (negative control: no sonication).
[0081] Subsequently, Plasmid DNA from the exosomes was purified using a mini prep kit, and the purified Plasmid DNA was used for PCR analysis.
[0082] The results showed that Plasmid DNA was partially loaded into exosomes even without sonication; however, when only exosomes were sonicated (i.e., sonication promoted loading), a greater amount of Plasmid DNA was loaded into the exosomes. On the other hand, when both exosomes and Plasmid DNA were sonicated simultaneously, the loading rate decreased. This phenomenon is presumably due to damage to the Plasmid DNA caused by sonication. Figure 8 ).
[0083] Therefore, the following experiments were conducted to confirm the Plasmid DNA damage caused by sonication.
[0084] (2) Confirm Plasmid DNA damage caused by ultrasound treatment
[0085] Similar to the previous experiments, to confirm the damage to Plasmid DNA caused by sonication, a 5-second sonication was performed at 30% AMP amplitude and a 1-second sonication was performed at 20% AMP amplitude. The results were then analyzed by 1% agarose gel electrophoresis. Figure 9 ).
[0086] The results showed that, similar to other useful substances identified above, Plasmid DNA was more severely damaged (approximately 90%) under a single 5-second sonication treatment with 30% AMP amplitude; while under a single 1-second sonication treatment with 20% AMP amplitude, the degree of damage was observed to decrease (approximately 50%).
[0087] Based on the above experimental results, it can be confirmed that the loading efficiency and damage degree obtained are as shown in Table 2, depending on the different ultrasonic treatment conditions.
[0088] That is, from the perspective of loading efficiency, the loading efficiency of the present invention is significantly improved compared to the loading efficiency of about 20% of the prior art; and when the degree of damage to the useful substance is controlled at about 50%, it is considered suitable for loading the useful substance into exosomes.
[0089] Table 2
[0090]
[0091] (3) Delivery of useful substances loaded in exosomes to cells
[0092] Based on the above-mentioned useful substance damage confirmation experiment, Q-dot, IgG488 or Plasmid DNA were loaded into exosomes using a method of one 1-second sonication with 20% amplitude of AMP.
[0093] Subsequently, HDF cells were seeded at 1 × 10^5 cells / well in 24-well cell culture plates, and exosomes loaded with Q-dot or IgG488 were added to the cells after 16 hours. After 24 hours, fluorescence analysis was used to confirm whether the useful substances were delivered to the cells.
[0094] The results showed that, compared with untreated HDF cells, the fluorescence values increased significantly after the addition of exosomes loaded with useful substances to the cells. This confirms that exosomes loaded with useful substances can be delivered to cells and transport the useful substances into the cellular interior. Figure 10 ).
[0095] To confirm whether Plasmid DNA loaded in exosomes could be delivered to cells and perform its function, 1 × 10^9 particles of exosomes were sonicated once for 5 seconds at 30% AMP amplitude, and Plasmid DNA was sonicated once for 1 second at 20% AMP amplitude before being loaded into exosomes. The exosomes were then processed into cells. Subsequently, the delivery into cells was confirmed directly or indirectly by analyzing the antibiotic resistance encoded by the plasmid, luciferase activity, and PCR.
[0096] To process exosomes loaded with Plasmid DNA, cells were seeded at 1 × 10^5 cells / well in 24-well cell culture plates. Antibiotics were added 24 hours after the addition of exosomes loaded with Plasmid DNA, and cell resistance to the antibiotics was confirmed 24 hours thereafter.
[0097] The results showed that cell death was observed when neomycin was added to cells without Plasmid DNA (HDF, Reprosome, DNA only); however, when exosomes loaded with Plasmid DNA were added to the cells, Neomycin-induced cell death was reduced. Figure 11 ).
[0098] For the luciferase assay, HDF cells were seeded at 1 × 10^5 cells / well in 24-well cell culture plates, and exosomes loaded with Plasmid DNA were added 16 hours later. The luciferase assay was then performed 24 hours later.
[0099] The results showed that increased Luciferase activity was observed only in cells treated with exosomes loaded with Plasmid DNA. Figure 12 ).
[0100] Finally, to confirm intracellular delivery of substances via exosomes (Plasmid DNA delivery) using PCR via cDNA synthesized from intracellular mRNA, HDF cells were seeded at 1 × 10^5 cells / well in 24-well cell culture plates, and exosomes loaded with Plasmid DNA were added 16 hours later. Luciferase RNA was isolated 24 hours later, cDNA was synthesized, and PCR analysis was performed. Figure 13 ).
[0101] The results showed that PCR bands were detected in cells treated with exosomes loaded with Plasmid DNA (lane 4), and the amplification signal was also confirmed by qPCR. Lane 1 represented only cells; lane 2 represented exosome treatment; lane 3 represented plasmid DNA treatment only; lane 5 represented plasmid DNA purified from exosomes; and PC was the positive control (original plasmid DNA).
Claims
1. A method for loading a useful substance onto a carrier, characterized in that, Includes the following steps: Ultrasonic stimulation was applied to the carrier containing the phospholipid layer and the useful substance, respectively; and The mixture of the ultrasonically stimulated carrier and the useful substance is cultured for a certain period of time.
2. The method according to claim 1, characterized in that, The carrier is an exosome.
3. The method according to claim 1, characterized in that, The carrier is an exosome derived from HDF of human skin fibroblasts.
4. The method according to claim 3, characterized in that, The exosomes were prepared by the following steps: Ultrasonic stimulation was applied to cells derived from human skin fibroblasts and to cell-free culture medium. The ultrasound-stimulated cells derived from human skin fibroblasts were mixed with the ultrasound-stimulated culture medium, and the resulting mixture was cultured for a certain period of time; and Exosomes were isolated from the mixture.
5. The method according to claim 1, characterized in that, The useful substances are nucleic acids, proteins, peptides, nanomaterials, plasmid DNA, low molecular weight compounds, drugs, hormones, enzymes, or neurotransmitters.
6. The method according to claim 1, characterized in that, The step of applying ultrasonic stimulation to the carrier is performed for 1 to 10 seconds at an amplitude of 20% to 40% of AMP.
7. The method according to claim 1, characterized in that, The step of applying ultrasonic stimulation to the useful substance is performed for 0.1 to 5 seconds at an amplitude of 15% to 30% of AMP.
8. The method according to claim 7, characterized in that, When the useful substance is plasmid DNA, the ultrasound stimulation is performed for 0.1 to 3 seconds at an amplitude of 15% to 25% of AMP.
9. The method according to claim 7, characterized in that, When the useful substance is a protein, nucleic acid, or nanomaterial, the ultrasound stimulation is performed for 1 to 5 seconds at an amplitude of 20% to 30% of AMP.
10. The method according to claim 1, characterized in that, The step of culturing the mixture is carried out at 30°C to 40°C for 30 minutes to 2 hours.