FI106723B - Process for the preparation of nanoparticles - Google Patents

Abstract

Process for the preparation of nanoparticles by solubilising a polylactic polyethylene oxide and/or polypropylene oxide copolymer in an organic solvent and then forming nanoparticles by mixing the solution containing the polymer with an aqueous solution by precipitation without employing any additional colloidal protective agent or by microfluidisation and solvent evaporation.

Description

1 106723

A process for preparing nanoparticles

The present invention relates to a novel process for the production of spherical particles having small dimensions, often less than 500 nm, capable of transporting or targeting the active ingredient. These particles, also referred to as nanoparticles, have the advantage of being able to circulate in the bloodstream without a measurement problem at the level of the capillaries. The invention also relates to the novel particles obtained and their use in human or veterinary pharmaceutical preparation.

According to the state of the art described in FR-A-2 608 988, particles of less than 500 nm in size can be made by at least three process types.

According to the first type of method, the monomer is polymerized in solution to obtain a micellar dispersion of the polymer in the solution. This type of process is limited to the monomers to be polymerized in solution, it being necessary to use after the polymerisation step to remove the polymerization catalyst, low molecular weight oligomers, monomers and polymers. surfactants necessary for swallowing. Sent by. the polymer shows a random molecular weight distribution • · · 25 countries.

According to the second and third type of processes, the preformed polymers are dissolved in a solvent and a precipitate or dispersion is formed from the solution of these polymers and non-solvent followed by evaporation of the solvent to recover the nano- · · · particles in the form of a colloidal slurry. The solubilizing solution is usually an organic solution of the polymer, a non-soluble solution ·· # .1. is often an aqueous solution.

According to another type of process, the polymer is dissolved. 35 in a water miscible organic solvent. Upon mixing this solution into an aqueous phase mixture, the aqueous phase / organic solvent insoluble polymer precipitates in the form of nanoparticles.

According to a third type of process, the non-water miscible organic solvent containing the polymer is emulsified in the aqueous phase, after which the organic solvent is evaporated off.

Formation of a precipitate or emulsion requires the presence of a surfactant far from the unlabeled one. However, during subsequent evaporation, it is very difficult to remove the surfactant remaining in the resulting colloidal slurry of nanoparticles, and this agent is often undesirable for good biocompatibility. The last two techniques are thus not useful for preparing biocompatible nanoparticles due to the presence of a colloidal preservative.

The aforementioned FR patent application 2,608,988 relates to a process for the preparation of dispersible colloidal systems in the form of nanoparticles of a size less than 500 nm. These are nanoparticles based on a substance which can be polymer and / or *: 1 without distinction. active ingredient obtained by another of the above methods. . ·. by. The resulting polylactic acid polymer-based nanoparticles contain, for the most part, examples of surface tension reducing agents, corresponding to the amount of polymer · 1.2 · 1.2. In this single application, in one single example (Example 4), the inventor claims to obtain polymeric acid nanoparticles without surface tension reducing agent. We have repeated this experiment, whereby we obtained • »· Polymeric Acid Polymer Nanoparticles from · 3: Extremely * · · .1 of Poly-Lactic Acid Solution and Water. in low yields, which were always below 10%. This »1 · technique is thus not useful for the preparation of polylactic acid nano-4 · 2 •« 3 • 3 106723 particles in the absence of surface tension reducing agent.

The present invention has made it possible to obtain nano-particles from polymers consisting predominantly of degradable constituents in the absence of a surface tension reducing agent. Accordingly, nanoparticles are prepared starting from a polyethylene oxide and / or polypropylene oxide-polylactic acid copolymer in the absence of added surface tension reducing agent or colloid protective agent.

According to this, a copolymer is used which consists mainly of polylactic acid moieties which have been modified by the inclusion of polyethylene oxide and / or polypropylene oxide moieties. The majority of the constituents of this copolymer 15 preferably corresponds to the following formula (I):

R '- O-CHU-CH --- O-CO-CH - OH

1 I (I) R CH, L JnL -5 Jm 20 wherein R in each alkylene oxide moiety is: an identical or different group selected from hydrogen and: a methyl group, 25 n is an integer from 20 to 1000,? represents hydrogen or an alkyl group containing from 1 to 4 carbon atoms, preferably a methyl group, '··· m being an integer from 10 to 1500.

The polylactic acid moiety of this copolymer preferably has a molecular weight of 700 to 100,000, while the molecular weight of the polyethylene oxide moiety is preferably 1000 to 100,000. Still more: 1 ·, · more preferably, the polymeric polylactic acid moiety has a molecular weight of 1,000 to 60,000, the polyethylene oxide and / or 9 · 99 9 · 9 9 9 9 9 • 106723 polypropylene oxide moiety has a molecular weight of 1,000-6,000.

Finally, preferably, the polylactic acid polymer is a polymer containing 50% of the configuration D-lactic acid-5 drill (PLA50) and the polyoxide is polyethylene oxide.

These polymers are preferably in the form of two groups, i.e., according to a practical embodiment, starting from commercial monofunctional polyethylene and / or polypropylene glycol wherein R 'is preferably a methyl group and having a molecular weight of between 1000 and 40 000, or further comprising 20-1000 ethylene or propylene oxide moieties, and preferably comprising 20-150 ethylene oxide moieties or 20-100 propylene oxide moieties, to which lactide moieties are added until the desired molecular weight of the polylactic acid chain is obtained, such as a catalyst, in the presence of.

It may be specified that about 10-1000 lactide moieties are desirable to obtain polylactic acid groups having a molecular weight of 1000-60,000. Particularly preferred are polyethylene and / or polypropylene oxide poly lactic acid copolymers having chain V containing 10 to 150 lactide moieties.

* · 1. . ·. Still more preferably, the commerce is started. «·« Y '.' 25 polyethylene glycol comprising 48 ethylene µl oxide moieties having a molecular weight of 2,100 and reacting with 40-150 lactic acid moieties.

The polymers obtained after the polymerization in solution are purified so that the catalyst and the remaining: the lactide is completely removed. This purification is accomplished by precipitation of the polymer with a non-solvent or by gel-chromatography. The copolymer is then dried and stored in powdered form.

»· · •« · • • • • • · 106 1063

According to the first method, for preparing the nanoparticles of the invention, the desired polyethylene and / or polypropylene oxide polylactic acid polymer is dissolved in a solvent or mixture of solvents, after which the organic solution is poured into an aqueous solution to cause precipitation of the nanoparticles. This method does not use an added colloidal preservative. Colloidal refers to substances that include surface tension reducing agents that promote colloid formation.

The copolymer-solubilizing solvent or solvent mixture is selected from ketones such as acetone, cyclic ethers such as tetrahydrofuran, dioxanes; nitriles such as acetonitrile. Acetone is preferably used. The copolymer must have a solubility in these solvents of at least 10 g / liter.

The volume ratio of the aqueous solution to the copolymer solution is preferably 0.5 to 10, and more particularly 1 to 10. The amount of copolymer introduced into the solvent will naturally depend on the solubility of the copolymer, but according to the most preferred embodiment of the invention. 10 to 50 mg / ml is preferred.

. . ·. According to another method, for the preparation of nanoparticles, the polyethylene and / or polypropylene oxide polypropylene oxide polymer is dissolved in an ester, preferably * 1.1 ethyl acetate, and then the organic solution is poured into an aqueous solution. The nanoparticles are formed using a microfluidizer.

The solvent of the copolymer is then evaporated off by heating the · · · - colloidal solution of the nanoparticles above the boiling point of the solvent in the case of -. is performed at or below atmospheric pressure

• M

I to the temperature if evaporation is carried out under reduced pressure.

. After the solvent has been removed, the slurry of nanoparticles in water is filtered as a filter with a pore diameter of about 1 µm to remove aggregates and large particles. The yield of the obtained nanoparticles is generally more than 50%.

Formation of the nanoparticles may be carried out in the presence of a pharmaceutically active ingredient which may be incorporated in either the copolymer solvent or the precipitating solvent, preferably being soluble in the polymer solvent and insoluble in water, although it is always possible to form nanoparticles in the decreases.

The resulting nanoparticles, which also form part of the present invention, contain no more than 15 polymers of formula (I) and optionally an active ingredient if precipitation is carried out in the presence of the active ingredient. They have an average diameter of 50 to 500 nm and preferably an average diameter of 50 to 250 nm.

20 The resulting nanoparticles are used in numerous applications such as agrochemical, reprographic paper, photographic paper, but their fully biocompatible * r. by their very nature, these nanoparticles are particularly suitable for the pharmaceutical industry, which is very well linked to humans or animals. These products can be injected intramuscularly, subcutaneously, subcutaneously, intravenously, intravenously, intravenously, intravitreally or intravitreally without the risk of a hypersensitivity reaction.

The invention will be more fully described by the following examples, which are not to be construed as limiting the invention.

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Example 1

Preparation of Polyethylene Glycol Poly-Lactic Acid Copolymers 1.1) Polymer PLA 2 ° -PEG2100 5 in a 250 ml three-necked flask equipped with a wing stirrer and a dry nitrogen rotary evaporator, heated with a regulated oil bath: 3 g tin (2) octate 0.256 g distilled toluene 335 g

The lactide is recrystallized the previous night from ethyl acetate, and then washed the same day with ethyl ether. It is dried under vacuum. All reagents are charged, followed by gentle reflux (110-114 ° C) for 5.5 hours. The solvent is then removed in vacuo using a rotary evaporator (40 mmHg, 100 ° C).

226.3 g of concentrate are obtained.

Purification of the copolymer is carried out as follows:

Let's focus: 215 g of concentrate «• · 1. 25 dichloromethane 280 g i · t w • · ·. ·. ·. Mix until a homogeneous solution is obtained.

j '# 1 # Slowly pour this solution into 900 ml of hexane in a cooler. The polymer precipitates in the form of a paste which is separated by decantation. The polymerization catalyst is removed to the hexane • 1 · * · 1 1 30 niphase. After separation, the polymer is placed in a vacuum oven at 40 ° C to dry. 188.4 g of copolymer are obtained, the mass of which is analyzed by nuclear magnetic resonance, the mass of polyethylene glycol being 2 100 and the mass of polylactic acid is 2 900, which corresponds to 40 parts of lactic acid ani and 48 ethylene oxide moieties.

• · · • · • • # «8 106723 1.2) Polymer PLä |

Example 1.1 is repeated using the following compounds: dl-lactide 48.6 g 5 polyethylene glycol 10 g tin (2) octoate 0.085 g distilled toluene 90 g

After the reaction, 63.6 g of a concentrate are obtained, which is purified by the following procedure: 40 g of concentrate 10 are dissolved in 200 g of dichloromethane until a homogeneous solution is obtained. This solution is slowly poured into 800 ml of water maintained at 55-60 ° C. The polymer precipitates and the dichloromethane is evaporated off, the unreacted lactide is left in the aqueous solution, the polymer is centrifuged off, then dried in a vacuum oven at 40 ° C.

35 g of polymer are obtained, the analysis of which by nuclear magnetic resonance enables the determination of the molecular weight. The latter is 9,600 for the lactic acid chain and 2,100 for the polyethylene oxide chain, which corresponds to 20,133 lactic acid constituents and 48 ethylene oxide constituents.

1.3) Polymer PLA ^ 000 in a 1 liter reactor heated with an oil bath and equipped with an anchor-shaped agitator. with a reflux condenser and maintained in a nitrogen filter: measure 180 g of xylene distilled before use and 0,180 g of tin octoate, heat to:. followed by the addition of 120 g of Boehringer dl-S-lactide, pre-recrystallized from ethyl acetate and washed with a solution of sulfuric acid in ether.

The mixture is allowed to react for 5 hours at 140 ° C and is quenched rapidly at the end of the reaction, then part of the xylene is removed in vacuo. The polymer is dissolved in dichloromethane and precipitated with methanol. Se. ·. : dried in a vacuum oven at 65 ° C.

♦ · • · • · * i »• · 1 · · 9 106723

Example 2

Preparation of Nanoparticles Starting From These Polymers 50 mg of the copoly-5 polymer prepared in 1.1, dissolved in 0.5; 2.5; To 5 and 10 ml of acetone. Nanoparticles are prepared by precipitating by slowly pouring this volume into 5 ml of water. The resulting colloidal slurry is evaporated for 30 minutes in a rotary evaporator at ambient temperature and 3 mm Hg. The slurry is then dated as a 1.2 µm filter to remove large particles and aggregates. The particle diameter as well as the particle yield of the polymer used are given in the table below.

Water / Acetone 10/1 2/1 1/1 0.5 / 1

Yield 75% 76% 92% 79%

Diameter (nm) 67 ± 36 63 ± 30 50 ± 21 38 ± 10

Control Example according to Example 4 of FR-A-2 608 988 20 The same procedure but with the lactic acid polymer of Example 1.3 having a molecular weight of 40,000 is used. Nanoparticles are prepared by precipitating an acetone solution in 5 ml of water. The acetone is evaporated off; 25 rotary loudspeakers in 30 minutes. During this phase ·· 1. the polymer precipitates and adheres to the walls. The sludge is taken • ♦:. and filtered as a filter with a diameter of · · · 1.2 µm. The filtrate is completely clear. The polymer content is • · * .. 1 too low to measure.

• · · * · 1 1 30 Example 3

Preparation of the nanoparticle containing the active ingredient • · · * ... 1 · Dissolve 40 mg of spiramycin in 200 ml of acetone and 200 mg of the copolymer prepared according to 1.2 above. »· · •« 10 106723 ri. The nanoparticles are prepared by precipitating the acetone solution in 20 ml of 0.1 M phosphate buffer, pH 7.4.

The acetone is evaporated off in a rotary evaporator for 30 minutes, after which the solution is filtered with a 1.2 µm filter. Spiramycin and copolymer are obtained in the form of female particles having a diameter of 74 nm.

Example 4

Preparation of Nanoparticles by Microfluidization Without Surfactant 10 Polymer (100 mg) is dissolved in 1 ml of ethyl acetate. This organic solution is poured into 10 ml of water with stirring on an Ultraturrax. After stirring for 30 seconds, the emulsion is homogenized and triturated for 2 minutes with a microfluidizer 15 (Microfluidics 110 S). The ethyl acetate is removed by evaporation under partial vacuum for 45 minutes, after which the nanoparticles are filtered through a Sartorius or Millipore filter with a pore size of 1.2 µm.

20 Total Yield of Polymer

Nature of experiment (nm) (%)

PLA-PEG

1 2900/2100 65 71 2 9600/2100 100 85 25 3 8000/4000 60 87 .V. 4 16000/4000 120 61.2 • · · j ’·% 5 30000/2000 280 no rate.

i: Y 6 30000/5000 150 65.5 • 1 · * .. 1 Cl 52000/0 not feasible; · · · * · 1 1 30 polymer immediately precipitates ··· · · · · · · · · · · · · · · · · · · · : In Example 1, starting from the following amounts of polyethylene • · 1 | 35 glycol, d, 1-lactide, toluene and tin octoate.

• · ·· »<» · • «·« »11 106723

Experience PEG d, l- Toluene Tin-

Character Weight lactide octate 3 4000 13 g 26 g 40 g 0.05 g 5 4 4000 10 g 40 g 60 g 0.08 g 5 2000 2.5 g 37.5 g 60 g 0.08 g 6 5000 5 g 30 g 45 g 0.06 g (methyl ester) • · »• • • * •

* I I

• · · «M · • • • • • • • • • · · · · · · · · · · · · ·

• I I

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Claims (12)

A process for the preparation of nanoparticles, characterized in that solubilizing a polyethylene oxide and / or polypropylene oxide polymer lactic acid copolymer comprising a predominant moiety of structural moieties having the formula: R '- O-CH 2 -CH --- O-CO CH - OH 10 1 1 (I) R CH, 11 JnL J Jm whereby: R in each alkylene oxide structural portion represents an identical or different group selected from hydrogen and a methyl group, R 'represents hydrogen or an alkyl group having 1- 4 carbon atoms, n is an integer of 20-1,000, 20 m is an integer of 10-1,500, in an organic solvent to form the nanoparticles by mixing the polymer-containing solution with an aqueous solution without using an aqueous solution. * * added colloidal protective agent. 2. A process according to claim 1, characterized in that in the formula (I) n is 20 - 150 and: m is 10 - 1000. • ·· · Γ **: 3. A method according to claim 2, characterized in - characterized in that in formula (I) R is a methyl group, R 'is a methyl group and n is 20 - 100. A process according to claim 3, characterized in - that in formula (I). · · *! * R is a hydrogen atom, • * ·. * ·: R 'is a methyl group, ·! * ·: 35 n is 48 and m is 40 - 150. «« t · · »·» · »CO 16 106723 5. A process according to claim 1, characterized in that the polyethylene oxide and / or polypropylene oxide polymer lactic acid polymer solvent is selected from ketones, ethers, dioxanes and nitriles. 6. Process according to claim 5, characterized in that the solvent is acetone. 7. Process according to claim 1, characterized in that the volume ratio between the aqueous solution and the organic solution of the copolymer is 0.5 - 10. 8. Process according to claim 1, characterized in that the concentration of the copolymer in the organic solution is higher or equal to 10 g / l. Method according to any of the preceding claims, characterized in that in one of the solutions a pharmaceutically active ingredient is added. 10. Nanoparticles, characterized in that they consist mainly of a copolymer of the formula: 20 Γ 1 Γ Ί R1--0-CHo-CH --- O-CO-CH - OH 2. In dl R CH, JnL J Jm • (* · where: • tl 25. in each alkylene oxide structural portion denotes an • · *. *. * Identical or different group, selected from hydrogen and a methyl: group, • R represents hydrogen or an alkyl group of 1-4: T: carbon atoms, n is an integer of 20-1,000, m is an integer of 10-1,500, · · * ·· ♦. ···, and that they do not contain any colloidal agent. 11. Nanoparticles according to claim 10, characterized in that they have an average size of between 50 and 500 nm. • «•» · • · • · • 4 · 17 106723 12. Nanoparticles according to claim 11, characterized in that they have an average size between 50 and 250 runs. • · «• · • · · • · ·«. · ♦ · «· · · · · · · · · · · ♦ ♦ · · · · · ♦ ♦ · · · · · ♦ ♦ · · · · · ♦ · · • · · • • «« · · · 1 4 · · • · I '«4« * ot> · • «1» • · «·