Preparation of dual enzyme-responsive nanomicelles

Abstract

The application mainly relates to the field of tumor research and treatment, and particularly relates to a preparation method of a double-enzyme response intelligent anti-cancer nanomicelle. 3350 Preparation of TPGS 3350 - GPLGVR-DOX; (3) preparation of FA-DEVD; (4) preparation of FA-DEVD-DOX; (5) preparation of TPGS 3350 - GPLGVR-DOX & FA-DEVD-DOX micelles by a dialysis method, and finally, the double-enzyme response anti-cancer nanomicelle is obtained. The method has the advantages of low requirement for equipment, simple operation, short production cycle and the like. The carrier prepared by the method is a new generation of intelligent nanomicelle which simultaneously has active targeting, in-vivo long circulation, low toxic and side effects and rapid drug release in and out of cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of tumor research and treatment, and particularly relates to a preparation method of a dual-enzyme response intelligent anti-cancer nanomicelle. BACKGROUND

[0002] In recent years, with the increasing trend of global population aging, the increasingly deteriorating living environment, and people's growing material needs and gradually unhealthy daily habits, and many other factors, the incidence of malignant tumors has increased year by year. According to the latest statistics of the WHO, due to the destruction of various homeostasis, cancer has now surpassed cardiovascular and cerebrovascular diseases and become the largest cause of human death. Traditional chemotherapy drugs and traditional nanomedicines generally have many shortcomings, resulting in poor treatment effect, and greater toxic and side effects on normal cell tissues, greatly reducing the quality of life of patients. In order to achieve better tumor treatment effect, people have designed and developed new nanomedicine delivery systems, which can help overcome the shortcomings of traditional chemotherapy drugs such as short in vivo half-life, large toxic and side effects, poor selectivity, etc., and can achieve the purposes of increasing drug stability, controlling drug release, and more gathering in tumor sites, thereby reducing the amount of drug administration, improving the bioavailability of drugs in the human body, helping more drugs to play a curative effect, while reducing the generation of side effects. The present application designs a drug-loaded nanomicelle according to the high expression of MMP2 / 9 enzyme sensitivity in the extracellular matrix of tumor cells and the Caspase-3 enzyme sensitivity in tumor cells. The nanosystem selects D-alpha-tocopherol succinate polyethylene glycol (TPGS 3350 ) as a carrier, connects the drug doxorubicin (DOX) and TPGS 3350 with the MMP2 / 9 enzyme response polypeptide GPLGVR, and selects folate as a carrier, connects the drug DOX and folate with the Caspase-3 enzyme response polypeptide DEVD, and synthesizes TPGS 3350 -GPLGVR-DOX and FA-DEVD-DOX two materials. Among them, TPGS 3350 is a water-soluble polymer, which has been approved by the US Food and Drug Administration (FDA) and is used as a solubilizer, absorption enhancer and drug delivery system.

[0003] The present application utilizes the two materials of TPGS 3350 -GPLGVR-DOX and FA-DEVD-DOX which are responsive to MMP2 / 9 enzymes and Caspase-3 enzymes, and successfully prepares a dual-enzyme response TPGS 3350-GPLGVR-DOX & FA-DEVD-DOX nanomicelle. The nanomicelle integrates multiple functional modules into a system, enabling the nanomicelle to respond differently to different stages of drug delivery to meet the multiple biological barriers that DOX needs to overcome during in vivo delivery. Under normal physiological conditions, the PEG 3350 outer layer of TPGS 3350 can prolong the circulation time of the nanomicelle in the blood and achieve enrichment at tumor tissues through the EPR effect. The polypeptide GPLGVR in the structure has MMP2 / 9 enzyme sensitivity, and the TPGS 3350 outer layer is removed through MMP2 / 9 enzyme response, exposing the targeting molecule folic acid, and then the micelle enters the cell through the folic acid receptor-mediated endocytosis pathway. After entering the interior of the tumor cell, part of the physically loaded naked drug DOX in the micelle is released, inducing tumor cell apoptosis and activating the activity of Caspase-3 in the cell, causing the polypeptide DEVD to break, accelerating the release of the drug in the cell, and enhancing the anti-tumor effect.

[0004] The dual enzyme-responsive nanomicelle in the application not only improves the drug loading capacity, but is also very stable, has long circulation in the body, active targeting, rapid release in and out of the cell, reduces toxic side effects, penetrates and aggregates at the tumor site, and improves the preparation of multifunctional intelligent nanomicelles for chemotherapy, providing a broad application prospect for targeted tumor drugs. SUMMARY

[0005] The purpose of the application is to overcome the shortcomings of the prior art and design a dual enzyme-responsive nanomicelle that has long circulation in the body, active targeting, rapid release in and out of the cell, and less toxic side effects.

[0006] The second purpose of the application is to provide a preparation method of TPGS 3350 -GPLGVR-DOX & FA-DEVD-DOX nanomicelle.

[0007] The purpose of the application is achieved by the following technical solutions.

[0008] Step 1, preparation of TPGS 3350 -GPLGVR

[0009] 1) Weigh 1 mmol of TPGS 3350 -COOH, 1.2 mmol of N-hydroxysuccinimide (NHS) and 1.2 mmol of N,N-dicyclohexyl carbodiimide (DCC) into a round-bottom flask, and stir under nitrogen at room temperature for 4-6 hours; then filter with a 0.45 μm organic filter membrane to obtain TPGS 3350 -NHS;

[0010] 2) The solution obtained in step 1) was filtered with 0.45 μm organic filter membrane to remove the by-product N, N-dicyclohexylurea (DCU) ; 3350 -NHS (1 mmol) and polypeptide GPLGVR (1.2 mmol) were dissolved in a certain amount of anhydrous DMF, and the reaction was stirred magnetically at room temperature for 24 h;

[0011] 3) The solution in step 2) was filtered with 0.45 μm organic filter membrane, and the filtrate was transferred to a dialysis bag (MWCO 3500), dialyzed against DMF for 24 h, and then against distilled water for 48 h, with the water being changed every 4 h;

[0012] 4) The filtrate in the dialysis bag in step 3) was transferred to a culture dish, sealed with plastic wrap, and punctured with a syringe. After being frozen in a refrigerator, it was placed in a freeze dryer to obtain dry and pure white solid TPGS 3350 -GPLGVR.

[0013] Step 2, preparation of TPGS 3350 -GPLGVR-DOX

[0014] 1) TPGS (1 mmol) was weighed and dissolved in 5 mL DMF, and the solution was placed in a round-bottom flask and stirred magnetically at room temperature for 4-6 h under nitrogen; 3350 -GPLGVR (1 mmol), NHS (1.2 mmol), and DCC (1.2 mmol) were dissolved in 5 mL DMF, and the solution was placed in a round-bottom flask and stirred magnetically at room temperature for 4-6 h under nitrogen;

[0015] 2) The solution in step 1) was filtered with 0.45 μm organic filter membrane to remove the by-product N, N-dicyclohexylurea (DCU) ;

[0016] 3) Doxorubicin hydrochloride (1.2 mmol) and TEA (6 mmol) (molar ratio 1:5) were weighed and dissolved in a certain amount of DMF, and then added to the filtrate in step 2). The solution was first stirred at 50°C in the dark for 4-6 h, and then stirred magnetically overnight;

[0017] 4) The solution in step 3) was filtered with 0.45 μm organic filter membrane, and the filtrate was transferred to a dialysis bag (MWCO 3500), dialyzed against DMF for 24 h, and then against distilled water for 48 h, with the water being changed every 4 h;

[0018] 5) The filtrate in the dialysis bag in step 4) was transferred to a culture dish, sealed with plastic wrap, and punctured with a syringe. After being frozen in a refrigerator, it was placed in a freeze dryer to obtain the final product TPGS 3350 -GPLGVR-DOX.

[0019] Step 3, preparation of FA-DEVD

[0020] 1) Folic acid activation: weigh folic acid (1 mmol), NHS (1.2 mmol), DCC (1.2 mmol), dissolve in 10 mL dimethyl sulfoxide (DMSO), place in a round-bottom flask, react at 50°C for 6 h in the dark;

[0021] 2) Filter the solution in step 1) with a 0.45 μm organic filter membrane to remove the by-product DCU after the reaction; add the polypeptide DEVD dissolved in DMSO (molar ratio 1:1.2) to the filtrate, and stir at room temperature for 48 h in the dark;

[0022] 3) Filter the solution in step 2) with a 0.45 μm organic filter membrane, and transfer the filtrate to a dialysis bag (MWCO 500), dialyze for 24 h using DMSO as the dialysis liquid, and then dialyze for 24 h using distilled water as the dialysis liquid, changing the water every 4 h;

[0023] 4) Transfer the filtrate in the dialysis bag in step 3) to a culture dish, seal with plastic wrap, and puncture a small hole with a syringe, then place in a refrigerator to freeze, and then place in a freeze dryer to obtain the product, yellow powder FA-DEVD.

[0024] Step 4, preparation of FA-DEVD-DOX

[0025] 1) Weigh FA-DEVD (1 mmol), NHS (1.2 mmol), DCC (1.2 mmol), dissolve in 5 mL DMSO, place in a round-bottom flask, and stir at room temperature for 12 h;

[0026] 2) Filter the solution in step 1) with a 0.45 μm organic filter membrane to remove the by-product DCU;

[0027] 3) Weigh doxorubicin hydrochloride (1.5 mmol), TEA (7.5 mmol) (molar ratio 1:5), dissolve in a certain amount of DMSO, then add to the filtrate in step 2), stir at room temperature for 24 h in the dark;

[0028] 4) Filter the solution in step 3) with a 0.45 μm organic filter membrane, and transfer the filtrate to a dialysis bag (MWCO 1000), dialyze for 24 h using DMSO as the dialysis liquid, and then dialyze for 24 h using distilled water as the dialysis liquid, changing the water every 4 h;

[0029] 5) Transfer the filtrate in the dialysis bag in step 4) to a culture dish, seal with plastic wrap, and puncture a small hole with a syringe, then place in a refrigerator to freeze, and then place in a freeze dryer to obtain the product, red powder FA-DEVD-DOX.

[0030] Step 5, micellar TPGS 3350Preparation of -GPLGVR-DOX & FA-DEVD-DOX

[0031] 1) Weigh 10 mg of TPGS using a balance under light-protected conditions. 3350 -GPLGVR-DOX, 2mg FA-DEVD-DOX and 1mg DOX (mass ratio of 10:2:1) were placed in a 5mL round-bottom flask, and 2-3mL of DMSO were added. The mixture was then magnetically stirred at room temperature for 3 hours to ensure complete dissolution.

[0032] 2) Add 4 mL of deionized water to a 25 mL round-bottom flask, then use a syringe to draw up the solution from step 1) and slowly add it dropwise to the vigorously stirred round-bottom flask. Stir magnetically at room temperature for 1-2 hours.

[0033] 3) Filter the solution from step 2) using a 0.45 μm inorganic filter membrane, transfer the filtrate to a dialysis bag (MWCO5000), and dialyze with deionized water for 48 hours, changing the water every 4 hours; after dialysis, freeze-dry to obtain DOX-loaded TPGS. 3350 -GPLGVR-DOX&FA-DEVD-DOX nanomicelles.

[0034] The two-segment enzyme-responsive material of this invention can self-assemble into micelles in water, improving the solubility and stability of doxorubicin in water. This invention constructs a novel polymer nanoprodrug with dual enzyme responses to the tumor microenvironment and surrounding environment, overcoming many disadvantages of traditional nanocarriers in in vivo delivery, thereby improving the efficacy of chemotherapy while reducing its toxic side effects and achieving rapid intracellular and extracellular release. Attached Figure Description

[0035] Figure 1 TPGS 3350 -Schematic diagram of the structure of GPLGVR-DOX;

[0036] Figure 2 Schematic diagram of FA-DEVD-DOX;

[0037] Figure 3 TPGS 3350 Transmission electron microscopy (TEM) images of -GPLGVR-DOX & FA-DEVD-DOX nanomicelles Detailed Implementation

[0038] The invention will be further illustrated below with reference to examples.

[0039] Example 1

[0040] TPGS 3350 - Preparation of GPLGVR

[0041] 1) Weigh 1 mmol TPGS 3350 -COOH, 1.2 mmol N-hydroxysuccinimide (NHS) and 1.2 mmol N,N-dicyclohexyl carbodiimide (DCC) are dissolved in 5 mL N,N-dimethylformamide (DMF) in a round bottom flask, and stirred magnetically under nitrogen at room temperature for 4-6 hours; then filtered with 0.45 μm organic filter membrane to obtain TPGS 3350 -NHS;

[0042] 2) The TPGS obtained in step 1) is dissolved in a certain amount of anhydrous DMF with 1 mmol NHS and 1.2 mmol polypeptide GPLGVR, and stirred magnetically at room temperature for 24 hours; 3350 -NHS (1 mmol) and polypeptide GPLGVR (1.2 mmol) are dissolved in a certain amount of anhydrous DMF, and stirred magnetically at room temperature for 24 hours;

[0043] 3) The solution in step 2) is filtered with 0.45 μm organic filter membrane, and the filtrate is transferred to a dialysis bag (MWCO 3500), dialyzed in DMF for 24 hours, and then dialyzed in distilled water for 48 hours, with water changed every 4 hours;

[0044] 4) The filtrate in the dialysis bag in step 3) is transferred to a petri dish, sealed with plastic wrap, and punctured with a syringe, and then placed in a refrigerator for freezing, and then placed in a freeze dryer to obtain dry and pure white solid TPGS 3350 -GPLGVR.

[0045] Example 2

[0046] TPGS 3350 -GPLGVR-DOX

[0047] 1) Weigh TPGS 3350 -GPLGVR (1 mmol), NHS (1.2 mmol), DCC (1.2 mmol) are dissolved in 5 mL DMF in a round bottom flask, and stirred magnetically under nitrogen at room temperature for 4-6 hours;

[0048] 2) The solution in step 1) is filtered with 0.45 μm organic filter membrane to remove by-product N,N-dicyclohexyl urea (DCU);

[0049] 3) Weigh doxorubicin hydrochloride (1.2 mmol) and TEA (6 mmol) (molar ratio 1:5) in a certain amount of DMF, and then added to the filtrate in step 2), and stirred at 50°C in the dark for 4-6 hours, and then stirred magnetically overnight;

[0050] 4) The solution in step 3) was filtered with 0.45 μm organic filter membrane, and the filtrate was transferred into dialysis bag (MWCO 3500), dialyzed with DMF for 24 h, and then dialyzed with distilled water for 48 h, and the water was changed every 4 h;

[0051] 5) The filtrate in the dialysis bag in step 4) was transferred into a culture dish, sealed with plastic wrap, and the small hole was pierced with a syringe, and then the culture dish was put into a refrigerator for freezing, and then put into a freeze dryer to obtain the final product TPGS. 3350 -GPLGVR-DOX.

[0052] Example 3

[0053] Preparation of FA-DEVD

[0054] 1) Folic acid activation: folic acid (1 mmol), NHS (1.2 mmol), DCC (1.2 mmol) were weighed and dissolved in 10 mL dimethyl sulfoxide (DMSO) in a round-bottom flask, and reacted at 50°C in the dark for 6 h;

[0055] 2) The solution in step 1) was filtered with 0.45 μm organic filter membrane to remove the by-product DCU after reaction; the polypeptide DEVD (molar ratio 1:1.2) dissolved in DMSO was added to the filtrate, and the reaction was stirred at room temperature in the dark for 48 h;

[0056] 3) The solution in step 2) was filtered with 0.45 μm organic filter membrane, and the filtrate was transferred into a dialysis bag (MWCO 500), dialyzed with DMSO for 24 h, and then dialyzed with distilled water for 24 h, and the water was changed every 4 h;

[0057] 4) The filtrate in the dialysis bag in step 3) was transferred into a culture dish, sealed with plastic wrap, and the small hole was pierced with a syringe, and then the culture dish was put into a refrigerator for freezing, and then put into a freeze dryer to obtain the product yellow powder FA-DEVD.

[0058] Example 4

[0059] Preparation of FA-DEVD-DOX

[0060] 1) FA-DEVD (1 mmol), NHS (1.2 mmol), DCC (1.2 mmol) were weighed and dissolved in 5 mL DMSO in a round-bottom flask, and the reaction was stirred at room temperature for 12 h;

[0061] 2) The solution in step 1) was filtered with 0.45 μm organic filter membrane to remove the by-product DCU;

[0062] 3) Take doxorubicin hydrochloride (1.5 mmol), TEA (7.5 mmol) (molar ratio 1:5) and dissolve in a certain amount of DMSO, then add to the filtrate of step 2) and stir at room temperature for 24 h in the dark;

[0063] 4) The solution in step 3) is filtered with a 0.45 μm organic filter membrane, and the filtrate is transferred to a dialysis bag (MWCO 1000), dialyzed with DMSO for 24 h, and then dialyzed with distilled water for 24 h, and the water is changed every 4 h;

[0064] 5) The filtrate in the dialysis bag in step 4) is transferred to a culture dish, sealed with plastic wrap, and a small hole is made with a syringe, then placed in the refrigerator for freezing, and then placed in a freeze dryer to obtain the product red powder FA-DEVD-DOX.

[0065] Example 5

[0066] Micellar TPGS 3350 Preparation of GPLGVR-DOX & FA-DEVD-DOX

[0067] 1) Under the condition of light, 10 mg TPGS 3350 GPLGVR-DOX, 2 mg FA-DEVD-DOX and 1 mg DOX (mass ratio 10:2:1) are placed in a 5 mL round-bottom flask, 2-3 mL of DMSO is added, and it is fully dissolved by magnetic stirring at room temperature for 3 h;

[0068] 2) In a 25 mL round-bottom flask, add 4 mL of deionized water, then use a syringe to suck the solution in step 1), slowly drop into the round-bottom flask with vigorous stirring, and stir at room temperature for 1-2 h;

[0069] 3) The solution in step 2) is filtered with a 0.45 μm inorganic filter membrane, and the filtrate is transferred to a dialysis bag (MWCO 5000), dialyzed with deionized water for 48 h, and the water is changed every 4 h; after dialysis, freeze-drying to obtain DOX-loaded TPGS 3350 GPLGVR-DOX & FA-DEVD-DOX nanomicelles.

Claims

1. A method for preparing dual-enzyme-responsive anticancer nanomicelles, comprising the following steps: Step 1, TPGS 3350 - Preparation of GPLGVR 1) Weigh 1 mmol TPGS 3350 -COOH, 1.2 mmol N-hydroxysuccinimide (NHS), and 1.2 mmol N,N-dicyclohexylcarbodiimide (DCC) were dissolved in 5 mL N,N-dimethylformamide (DMF) and placed in a round-bottom flask. The mixture was then magnetically stirred under nitrogen at room temperature for 4–6 h. The solution was then filtered through a 0.45 μm organic filter membrane to obtain TPGS. 3350 -NHS; 2) Take the 1 mmol TPGS obtained in step 1) 3350 -NHS and 1.2 mmol GPLGVR of peptide were dissolved in a certain amount of anhydrous DMF and reacted magnetically at room temperature for 24 h. 3) Filter the solution from step 2) using a 0.45μm organic filter membrane, transfer the filtrate to a MWCO 3500 dialysis bag, dialyze for 24 hours with DMF as the dialysate, and then dialyze for 48 hours with distilled water as the dialysate, changing the water every 4 hours. 4) Transfer the filtrate from the dialysis bag in step 3) to a petri dish, seal it with plastic wrap, poke a small hole with a syringe, freeze it, and then freeze-dry it to obtain a dry, pure white solid TPGS. 3350 -GPLGVR; Step 2, TPGS 3350 Preparation of -GPLGVR-DOX 1) Weigh 1 mmol TPGS 3350 - GPLGVR, 1.2 mmol NHS, and 1.2 mmol DCC were dissolved in 5 mL of LDMF and placed in a round-bottom flask. Nitrogen gas was introduced and the mixture was magnetically stirred at room temperature for 4–6 hours. 2) Filter the solution from step 1) using a 0.45 μm organic filter membrane to remove the byproduct N,N-dicyclohexyl saline (DCU); 3) Weigh 1.2 mmol of doxorubicin hydrochloride and 6 mmol of TEA in a molar ratio of 1:5 and dissolve them in a certain amount of DMF. Then add them to the filtrate from step 2). First, stir the mixture at 50°C in the dark for 4-6 hours, and then stir it magnetically overnight. 4) Filter the solution from step 3) using a 0.45 μm organic filter membrane, transfer the filtrate to a MWCO 3500 dialysis bag, dialyze for 24 h with DMF as the dialysate, and then dialyze for 48 h with distilled water as the dialysate, changing the water every 4 h. 5) Transfer the filtrate from the dialysis bag in step 4) to a petri dish, seal it with plastic wrap, poke a small hole with a syringe, freeze it, and then freeze-dry it to obtain the final product TPGS. 3350 -GPLGVR-DOX; Step 3: Preparation of FA-DEVD 1) Folic acid activation: Weigh 1 mmol folic acid, 1.2 mmol NHS and 1.2 mmol DCC, dissolve in 10 mL dimethyl sulfoxide (DMSO), place in a round-bottom flask, and react at 50 °C in the dark for 6 h. 2) Filter the solution from step 1) using a 0.45 μm organic filter membrane to remove the byproduct DCU after the reaction; add the peptide DEVD dissolved in DMSO to the filtrate at a molar ratio of 1:1.2, and stir the mixture at room temperature in the dark for 48 h. 3) Filter the solution from step 2) using a 0.45μm organic filter membrane, transfer the filtrate to a MWCO 500 dialysis bag, dialyze for 24 hours with DMSO as the dialysate, and then dialyze for 24 hours with distilled water as the dialysate, changing the water every 4 hours. 4) Transfer the filtrate from the dialysis bag in step 3) to a petri dish, seal it with plastic wrap, poke a small hole with a syringe, freeze it in a refrigerator, and then freeze it in a freeze dryer to obtain the product, yellow powder FA-DEVD. Step 4: Preparation of FA-DEVD-DOX 1) Weigh 1 mmol FA-DEVD, 1.2 mmol NHS, and 1.2 mmol DCC and dissolve them in 5 mL DMSO. Place the solution in a round-bottom flask and stir at room temperature for 12 h. 2) Filter the solution from step 1) using a 0.45 μm organic filter membrane to remove the byproduct DCU; 3) Weigh 1.5 mmol of doxorubicin hydrochloride and 7.5 mmol of TEA, dissolve them in a certain amount of DMSO at a molar ratio of 1:5, and then add them to the filtrate of step 2). Stir and react at room temperature in the dark for 24 hours. 4) Filter the solution from step 3) using a 0.45 μm organic filter membrane, transfer the filtrate to a MWCO 1000 dialysis bag, dialyze for 24 h with DMSO as the dialysate, and then dialyze for 24 h with distilled water as the dialysate, changing the water every 4 h. 5) Transfer the filtrate from the dialysis bag in step 4) to a petri dish, seal it with plastic wrap, poke a small hole with a syringe, freeze it in a refrigerator, and then freeze it in a freeze dryer to obtain the product, a red powder FA-DEVD-DOX. Step 5, micelle TPGS 3350 Preparation of -GPLGVR-DOX & FA-DEVD-DOX 1) Weigh 10 mg of TPGS using a balance under light-protected conditions. 3350 GPLGVR-DOX, 2 mg FA-DEVD-DOX and 1 mg DOX in a mass ratio of 10:2:1 were placed in a 5 mL round-bottom flask, and 2-3 mL of DMSO were added. The mixture was then magnetically stirred at room temperature for 3 hours to ensure complete dissolution. 2) Add 4 mL of deionized water to a 25 mL round-bottom flask, then use a syringe to draw up the solution from step 1) and slowly add it dropwise to the vigorously stirred round-bottom flask. Stir magnetically at room temperature for 1-2 hours. 3) Filter the solution from step 2) using a 0.45 μm inorganic filter membrane, transfer the filtrate to a MWCO 5000 dialysis bag, and dialyze for 48 hours using deionized water as the dialysate, changing the water every 4 hours; after dialysis, freeze-dry to obtain DOX-loaded TPGS. 3350 -GPLGVR-DOX&FA-DEVD-DOX nanomicelles.

2. The preparation method according to claim 1, characterized in that: TPGS 3350 -COOH, N-hydroxysuccinimide NHS and N,N-dicyclohexylcarbodiimide DCC, in a molar ratio of 1:1.2:1.

2.

3. The preparation method according to claim 1, characterized in that: Folic acid, N-hydroxysuccinimide (NHS), and N,N-dicyclohexylcarbodiimide (DCC) were present in a molar ratio of 1:1.2:1.

2.

4. The preparation method according to claim 1, characterized in that: TPGS 3350 -GPLGVR-DOX, FA-DEVD-DOX and DOX were mixed in a mass ratio of 10:2:1 and prepared by dialysis to obtain nanomicelle particles.

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