A drug carrier system and production method thereof

Abstract

The present invention relates to a drug carrier system in the form of nanocapsules and nanospheres loaded with luteolin and oleuropein, which have a counteracting effect on tumour cell growth, invasion and angiogenesis, and to a production method (100) of this drug carrier system.

Description

[0001] DESCRIPTION

[0002] A DRUG CARRIER SYSTEM AND PRODUCTION METHOD THEREOF

[0003] Technical Field

[0004] The present invention relates to a drug carrier system in the form of nanocapsules and nanospheres loaded with luteolin and oleuropein, which have a counteracting effect on tumour cell growth, invasion and angiogenesis, and to a production method of this drug carrier system.

[0005] Background of the Invention

[0006] Oleuropein is a substance that belongs to the secoiridoid family and is abundant in olive fruit and leaves. In addition to its anti-inflammatory activity, its cardioprotective, antiarrhythmic, hypotensive and spasmolytic effects have been shown in various studies. Furthermore, its counteracting effects on tumour cell growth, invasion and angiogenesis have also been proven by in vitro and in vivo studies. Luteolin is a polyphenolic compound, which is an important flavonoid found naturally in various plant species and is known for its anti-inflammatory, antioxidant and apoptosis-inducing effects. However, a drug carrier system formulation containing a combination of these two substances is not known in the current technique. On the other hand, it is known that the water solubility and oral bioavailability of both substances are low.

[0007] For this reason, there is a need to develop a new structure in which these two substances will be contained in a drug carrier system in order to increase the oral bioavailability and efficiency of both substances.

[0008] The Chinese patent document no. CN111388534, an application included in the state of the art, discloses a preparation method of an olive leaf extract. A dry olive leaf is added into an ethanol aqueous solution for heating and reflux extraction, an extracting solution is placed for cooling after recovery of a solvent and then filtered, and a concentrated extracting solution is obtained; a macroporous resin column is used for adsorbing effective constituents in the concentrated extracting solution; after adsorption, the resin column is washed with water for removal of impurities, then desorption is conducted with an ethanol aqueous solution, and a desorption solution is collected; and in the end, the desorption solution is treated with solvent recovery and dried, so that the olive leaf extract is obtained. Results of high- performance liquid chromatography testing show the olive leaf extract prepared contains the following effective constituents: 40%-65% of oleuropein, 3%-7% of ligustrum glycoside, 2%-8% of luteolin-7-O-beta-D glucoside, and 0.1%-1% of hydroxytyrosol. An animal experiment shows the olive leaf extract prepared has a high hypoglycaemic activity, so that the olive leaf can be used as an active constituent in preparation of hypoglycaemic drugs.

[0009] Summary of the Invention

[0010] An object of the present invention is to realize a drug carrier system in the form of nanocapsule and nanosphere formulation loaded with luteolin and oleuropein, which have a counteracting effect on tumour cell growth, invasion and angiogenesis, and a production method of this drug carrier system.

[0011] Another object of the present invention is to realize a drug carrier system which increases the water solubility and bioavailability of luteolin and oleuropein and thus enables them to get into circulation by easily passing through the veins, a the production method of this drug carrier system.

[0012] A further object of the present invention is to realize a drug carrier system which can be targeted to a diseased area or to a designated cell, tissue or organ, and a production method of this drug carrier system. Detailed Description of the Invention

[0013] “A Drug Carrier System and Production Method Thereof’ realized to fulfil the objectives of the present invention is shown in the figures attached, in which:

[0014] Figure 1 is a flowchart of the inventive method.

[0015] Figure 2 is a graph of spectrophotometric absorbance values versus concentration of luteolin.

[0016] Figure 3 is a graph of spectrophotometric absorbance values versus concentration of oleuropein.

[0017] The inventive drug carrier system with counteracting effect against tumor cell growth, invasion and angiogenesis is nanosphere and nanocapsule formulation which is obtained by loading oleuropein and / or luteolin into polylactic acid (PLA), polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA) polymer mixture by nanoprecipitation method.

[0018] 100. Method

[0019] The production method (100) of the inventive drug carrier system comprises the steps of preparing stock solution for the PLGA, PCL and PLA polymers (101); preparing stock solutions for the oleuropein and / or luteolin drugs (102); mixing the stock solutions prepared for polymer and drug (103); and obtaining nanospheres and nanocapsules by adding the mixture to the water phase (104).

[0020] In the step of preparing stock solution for the PLGA, PCL and PLA polymers (101) of the inventive method (100), stock solutions are prepared separately in such a way that their concentrations will be 1-200 mg / mL by weighing 0.1-10 mg of each of PLGA, PCL and PLA and adding them separately into 0.1-20 mL of acetone, which is an organic solvent.

[0021] In the step of preparing stock solutions for the oleuropein and / or luteolin drugs (102) of the inventive method (100), stock solutions of 1-200 mg / mL are prepared by weighing 0.1-10 mg of each of oleuropein and luteolin and dissolving them in acetone.

[0022] In the step of mixing the stock solutions prepared for polymer and drug (103) of the inventive method (100), polymer and drug solutions are combined in such a way that the content of the organic phase will consist of 0.1-200 mg / mL polymer and 0.1-200 mg / mL drug by mixing in a magnetic stirrer at 300-600 rpm at room temperature in order to prepare formulation and the organic phase is obtained.

[0023] In the step of obtaining nanospheres and nanocapsules by adding the mixture to the water phase (104) of the inventive method (100), a 0.1-10 mL organic phase is added dropwise to a 0.1-20 mL water phase under a magnetic stirrer at 500-900 rpm, preferably by using a Gilson pipette. After mixing for 5-60 minutes, the drug carrier systems with oleuropein and / or luteolin-loaded nanosphere and nanocapsule formulations are obtained by evaporating the organic phase under pressure at 25- 40° C in rotavapor.

[0024] With the inventive drug carrier system, the bioavailability and efficiency of oleuropein and / or luteolin are increased by loading them into nanostructures. These structures can easily pass through the veins and get into circulation due to their nanoparticle size. As the surface area increases, their solubility also increases. Increased solubility as a result of the preparation of nanoparticle formulations increases the absorption of the particle and consequently increases its bioavailability. Nanoparticles can be targeted to a diseased area or to a designated cell, tissue or organ. More than one active substance and targeting molecule can be attached to a nanocarrier. The average particle size and zeta potentials of the obtained inventive drug carrier systems were measured by dynamic light scattering (DLS) method. When the results indicated in Table 1 are examined, the average particle size of oleuropein- loaded nanocapsule formulations was in the range of 200-250 nm, while the particle size of oleuropein-loaded nanosphere formulations was in the range of 85-115 nm. The average particle size of luteolin-loaded nanocapsule formulations was in the range of 200-230 nm, while the particle size of luteolin-loaded nanosphere formulations was in the range of 80-165 nm.

[0025] On the other hand, in Table 2 and Table 3, the results of nanoparticles in which oleuropein and luteolin were loaded together are shown. While the average particle size for nanosphere formulations was found in the range of 85-140 nm, the average particle size for nanocapsule formulations was found in the range of 250-360 nm.

[0026] Table 1. Average particle size, polydispersity index and zeta potential results of nanocapsule / nanosphere formulations containing oleuropein or luteolin (n=3), (OLE: Oleuropein, LUT: Luteolin, O-L-POLYMER NP: Oleuropein- and Luteolin- Loaded Nanoparticles)

[0027] Table 2. Average particle size, poly dispersity index and zeta potential results of nanosphere formulations containing oleuropein and luteolin (n=3)

[0028] Table 3. Average particle size, poly dispersity index and zeta potential results of nanocapsule formulations containing oleuropein and luteolin (n=3)

[0029]

[0030] When the results are examined, it is considered that each nanoparticle can reach the targeted tissues by passive targeting by means of enhanced permeability and retention effect (EPR) as each is below 300 nm.

[0031] First, a spectrophotometric method was developed for both substances for quantitation during the loading activity studies. Each substance was dissolved in acetone at different concentrations and calibration line and equation were obtained by determining absorption values against concentration. While luteolin was studied at 347 nm wavelength (Table 4, Figure 1), oleuropein was studied at 282 nm wavelength (Table 5, Figure 2).

[0032] Table 4. Spectrophotometric absorbance values of luteolin versus concentration

[0033] The equation obtained for luteolin quantitation is shown by equation (1). y= 0,00508x + 0,00929 . (1) r2= 0,997 Table 5. Spectrophotometric absorbance values of oleuropein versus concentration

[0034] The equation obtained for oleuropein quantitation is shown by equation (2). y= 0,00333x + 0,00273 . (2) r2= 0,999

[0035] Then, nanocapsules were centrifuged at 13500 rpm for 1 hour and nanospheres for 30 minutes for loading activity study. After 48 hours of lyophilization process, the nanoparticles were dispersed by DCM:DMS0 (1:1 (v / v)). The dispersion medium is water.

[0036] When the obtained results were examined, as indicated in Table 6, while the loading activity for oleuropein-loaded nanocapsules was found to be 55-85%, the loading activity for oleuropein-loaded nanospheres was found to be in the range of 80- 110%. On the other hand, in Table 7, loading activity results for luteolin-loaded nanoparticles are given. While the loading activity for luteolin-loaded nanocapsules was found to be 35-70%, the loading activity for luteolin-loaded nanospheres was found to be in the range of 90-100%.

[0037] Table 6. % loading activity results of oleuropein-loaded nanoparticles

[0038] Table 7. % loading activity results of luteolin-loaded nanoparticles Within these basic concepts; it is possible to develop various embodiments of the inventive “A Drug Carrier System and Production Method (100) Thereof’; the invention cannot be limited to examples disclosed herein and it is essentially according to claims.

Claims

CLAIMS1. A drug carrier system characterized in that it has a nanosphere and nanocapsule form obtained by loading oleuropein and / or luteolin into a polymer mixture of polylactic acid (PLA), polycaprolactone (PCL) and poly(lactic-co- glycolic acid) (PLGA) by nanoprecipitation method, which has counteracting effects against tumor cell growth, invasion and angiogenesis.

2. A production method (100) of the inventive drug carrier system characterized in that it comprises the steps of preparing stock solution for the PLGA, PCL and PLA polymers (101); preparing stock solutions for the oleuropein and / or luteolin drugs (102); mixing the stock solutions prepared for polymer and drug (103); and obtaining nanospheres and nanocapsules by adding the mixture to the water phase (104).

3. A method (100) according to Claim 2; characterized in that at step of preparing stock solution for the PLGA, PCL and PLA polymers (101), stock solutions are prepared separately in such a way that their concentrations will be 1- 200 mg / mL by weighing 0.1-10 mg of each of PLGA, PCL and PLA and adding them separately into 0.1-20 mL of acetone, which is an organic solvent.

4. A method (100) according to Claim 2 or 3; characterized in that at step of preparing stock solutions for the oleuropein and / or luteolin drugs (102), stock solutions of 1-200 mg / mL are prepared by weighing 0.1-10 mg of each of oleuropein and luteolin and dissolving them in acetone.

5. A method (100) according to Claim 2 to 4; characterized in that at step of mixing the stock solutions prepared for polymer and drug (103), polymer and drug solutions are combined in such a way that the content of the organic phase will consist of 0.1-200 mg / mL polymer and 0.1-200 mg / mL drug by mixing in amagnetic stirrer at 300-600 rpm at room temperature in order to prepare formulation and the organic phase is obtained.

6. A method (100) according to any one of the Claim 2 to 5; characterized in that at step of obtaining nanospheres and nanocapsules by adding the mixture to the water phase (104), a 0.1-10 mL organic phase is added dropwise to a 0.1-20 mL water phase under a magnetic stirrer at 500-900 rpm.

7. A method (100) according to any one of the Claim 2 to 6; characterized in that at step of obtaining nanospheres and nanocapsules by adding the mixture to the water phase (104), after mixing for 5-60 minutes, the drug carrier systems with oleuropein and / or luteolin-loaded nanosphere and nanocapsule formulations are obtained by evaporating the organic phase under pressure at 25-40° C in rotavapor.

Images