FR2515960A1 - Biodegradable nano:capsules contg. biologically active cpd. - having envelope of polymerised alkyl cyanoacrylate - Google Patents

Abstract

Biodegradable nanocapsules comprise an envelope made by polymerising an alkyl cyanoacrylate (I) and an internal cavity contg. a soln. or oily suspension of a biologically active substance (A). Pref. the diam. of the capsules is below 1 micron. The capsules are made by first dissolving (e.g. in ethanol) an oily prod. (and opt. (A)) and (I). This soln. is then injected into distilled or deionised water, opt. contg. a nonionic surfactant, then if necessary the solvent is eliminated and the resulting suspension conc. Partic. (I) is isobutyl 2-cyanoacrylate (Ia) and the soln. is introduced into the water (contg. 0.5-1% 'Pluronic F 68' (RTM)) slowly with vigorous stirring. In this stage the heat of mixing is sufficient to cause polymerisation. The capsules can contain pharmaceuticals (for human or veterinary use), allergens, enzymes, diagnostic prods. etc.; specifically 'ultra-fluid lipidol' (radio-opaque material contg. 48 g iodine/100 ml); lomustine; progesterone or insulin. They can be administered orally, subcutaneously, intradermally, intramuscularly or intravenously, and are stable against autoclaving, centrifuging, freezing and storage. (I) are polymerised under milder conditions than other acrylic derivs. and nanocapsules can be prepd. at any pH.

Description

 The present invention relates to biodegradable nanocapsules or nanoparticles formed of a polymerized alkyl cyanoacrylate and containing a biologically active substance, their preparation and their application.

 The nanocapsules according to the invention, under the transmission electron microscope, are presented after negative staining with phosphotungstic acid in the form of a non-contrasting particle senssiblelllent round.

Their diameter is homogeneous and the proposed method of preparation makes it possible to control it.

 Depending on the operating conditions it is possible to obtain nanocapsules whose diameter ranges from a few tens to several hundred nanometers. The outer shell of the nanocapsules containing an oil or an active substance dispersed in an oil appears on the same microscope after successive, positive double staining with the osmic acid which contrasts inside the nanocapsules and negative with the phosphotungstic acid.

The oil and the active ingredient are completely coated by a wall of very regular thickness of the order of a few nanometers. The wall is formed by the polymerization of an alkyl cyanoacrylate and more particularly isobutyl-2 cyanoacrylate.

 Using the scanning electron microscope the nanocapsules after washing to remove the oil and after shading with a layer of gold, appear as spherical particles having approximately the same size.

 The encapsulation rate is always very high in the case of lipophilic products. 1 ml of monomer makes it possible, for example, to encapsulate 8 ml of iodinated ultrafluid oil (lipiodol) at 48% iodine w / v or 400 mg of a lomustine antitumor product (CCNU) and 4 ml of an oil used. as support.

 The nanocapsules containing an oil such as ultra-fluid lipiodol are stable for autoclaving for more than 15 minutes at 120 OC under a pressure of 1 bar.

 The nanocapsules are stable to ultracentrifugation. After ultracentrifugation at 220,000 g for 1 hour, most of the nanocapsules can be easily redispersed in water.

 The nanocapsules are stable to freezing.The average size remains the same after freezing | at - 52 Oc.

 The nanocapsules are stable over time. The average size of nanocapsules containing 20% lipiodol ultra-fluid remains unchanged after 6 months of storage at room temperature or 50 OC.

 The nanocapsules ensure the stability of active ingredients that are usually unstable in an aqueous medium. Lomustine, for example, whose half-life is of the order of 20 to 200 minutes in water at 37 OC and at pH 7 remains stable in the nanocapsules.

 The above results can only be explained by the presence of a wall enclosing the encapsulated material and only by a complete encapsulation of the active ingredient.

 The biologically active substance contained in the nanocapsules of the invention may be, for example, a medicinal substance for human or veterinary use or a product for diagnosis. As a drug substance, there are more particularly chemicals with pharmacological properties and, for example, antimitotic or antindoplastic substances such as lomustine (UNFC), methotrexate, actinomycin D, adriamycin, daunorubicin, bleomycin and vincristine or antibiotic substances such as penicillins, cephalosporins and nalidixic acid, antibiotics of the aminoglycoside type and those of the virginiamycin family and hormonal substances, in particular stromal hormones, such as progesterone. These medicinal substances may be especially high molecular weight chemical compounds such as insulin and heparin and the expression drug substances, also includes biological products such as antigens, allergens, enzymes, proteins, viruses or constituents of viruses, bacteria or cells. The nanocapsules according to the invention may also contain a product for diagnosis, in particular lipophilic radiopaque products such as iodinated oils.

 In human or veterinary medicine the submicroscopic particles of the invention can be administered with a suitable oral, subcutaneous, intradermal intramuscular or intravenous excipient and their diffusion into the tissues makes them particularly useful for systemic treatments.

 Particles with a diameter of less than 500 nanometers formed of a polymerised material, as well as their preparation and their application, in particular as a support for a biologically active substance, are already known.

 Thus, Belgian Patents N 808,034 and 839,748 describe submicroscopic particles formed of polymerizable materials such as derivatives of acrylic or methacrylic acid, for example methyl methacrylate, butyl methacrylate, methacrylamide or a mixture of these compounds. The submicroscopic particles formed by micellar polymerization of these different monomers have the property of completely or partially coating the biologically active substance and the property of forming aqueous colloidal solutions which allow parenteral administration of these particles so loaded with biologically active.

 However, the polymers of the acrylic or methacrylic acid derivatives described in these patents for the preparation of submicroscopic particles containing a biologically active substance are substantially stable so that they persist for a long time as such in the tissues or in the cavity. where they have been administered and this is a disadvantage, especially in the case of parenteral administration in human medicine.

Belgian Patent No. 869,107 discloses this disadvantage by describing biodegradable nanoparticles containing a biologically active substance. The material used consists of alkyl cyanoacrylate polymers already used in surgery as tissue adhesives and known for their biodegradability. Its major disadvantage lies in the structure of the particles obtained and the mode of incorporation of the active substance. The described operating mode only makes it possible to prepare particles formed of a very dense polymer network. The active substance can only be incorporated by adsorption and the fixed level is still relatively low.

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<SEP> Quantity <SEP> dc <SEP> monomer <SEP> Amount <SEP> of <SEP> adsorption principle <SEP> active <SEP> Z <SEP>
<tb><SEP> used <SEP> setting <SEP> in <SEP> work <SEP>
<tb> 0.83ml <SEP> Methyloyanoacryl <SEP> 1 <SEP> mg <SEP> Actinomycin <SEP> D <SEP> 90 <SEP>% <SEP>
<tb> 0.83ml <SEP> ate <SEP> 1 <SEP> mg <SEP> = <SEP> 90%
<tb> 0.83 <SEP> m1 <SEP> I <SEP> I <SEP> mg <SEP> w <SEP> 90 <SEP> Z
<tb> 0.6 <SEP> ml <SEP> Ethylcyanoacryl- <SEP> 10 <SEP> mg <SEP> Fluorescein <SEP> 65 <SEP> - <SEP> 70 <SEP> Z
<tb><SEP> ate
<tb> 0.83 <SEP> ml <SEP> Methyloyanoacryl <SEP> 5 <SEP> mg <SEP> Methotrexate <SEP> 25 <SEP> + <SEP> 5 <SEP> Z <SEP>
<tb> 0.6 <SEP> ml <SEP> - <SEP> ate <SEP> 10 <SEP> mg <SEP> Daunorubicin <SEP> 85 <SE> Z <SEP>
<Tb>
The nanocapsules which are the subject of the present invention are constituted as the previous nanoparticles of polyalkylcyanoacrylates. They therefore have the advantage of being biodegradable. Their administration is followed by a release of the biologically active substance, and it is possible to obtain a biodegradability, suitably programmed by using polyalkylcyanoacrylates differing from each other by the nature of the alkyl chain.

 The nanocapsules of the present invention also have the advantage of allowing a significantly higher incorporation rate. Unlike nanoparticles formed of a very dense network they consist of a wall surrounding a hollow cavity and the presence of the latter makes it possible to encapsulate much larger quantities of active substances.

 Another advantage of alkyl cyanoacrylates over the other acrylic derivatives previously used for the preparation of submicroscopic particles containing a biologically active substance lies in the process that can be used for their polymerization. Indeed, unlike other acrylic derivatives whose polymerization requires an energy supply likely to affect the stability of the adsorbed active ingredient, alkyl cyanoacrylates po0ynrise particularly easily without such a contribution.

 With the process of the present invention it is possible to nanoencapsulate all vegetable or mineral oils and oily substances that are soluble or that form a sufficiently fine emulsion in absolute ethanol, or in any solvent that can play the same role. As solvents may be used in particular all those missiblcs water and solubiliser can oil and monomer.

 In the case of an oil or an oily active substance, the principle of preparing the nanocapsules by taking absolute ethanol (absolute alcohol) as an example of a solvent is as follows.

a) The oil or the oily substance and the monomer are solubilized in
absolute alcohol.

b) The solution previously prepared with the aid of a
appropriate device in demineralized or demineralized water
and distilled, possibly containing 0.5 X to 1% of a surfactant
nonionic, for example Pluronic F 68 under magnetic stirring.

encapsulation is done immediately. The solution becomes white,
milky and presents the characteristic Tyndall effect of colloidal solutions.

c) The alcohol is removed and the nanocapsules are concentrated. In an eva
suitable porator and under slight depression the alcohol is evaporated
and a large part of the water can be eliminated. It is possible
thus cocentrate the nanocapsules.

Theory of encapsulation:
In the solvent for example in absolute ethanol, the oily substance and the monomer form a true solution. The monomer has surfactant properties due to its particular structure. The oil is completely lipophilic. The important stage of the preparation is the slow injection of the solution containing the oil and the monomer into the aqueous phase. Absolute alcohol has a considerable affinity for water, and at the moment of contact, it rapidly mixes with the latter by releasing a certain amount of heat (called heat of the mixture). The oily substance precipitates by forming small particles of the order of a nanometer. At the same time, the molecules of the monomer orient themselves thanks to their polarity by forming a film which completely coats the nanoparticles of oil. In the presence of water, the film consisting of the monomer polymerizes on the surface of the nanoparticles, at least 30 seconds. The role of the surfactant used is pls or less important. It is possible to obtain nanocapsules in water without surfactit.However the nanocapsules thus obtained agglomerate quickly in the case where it is interesting to concentrate, forming aggregates difficult to redisperscr.

 The described method also allows nanoencapsules any non-oily active substance soluble in absolute ethanol and insoluble in water using an appropriate oil carrier. In this case the operational mode is analogous. It is sufficient to solubilize the active substance with the oily carrier and the monomer in absolute ethanol. This method can be used for example to prepare lomustinc nanocapsules (C.C.N.U) or progesterone.

 The active substances insoluble in absolute ethanol, and soluble in water can also be encapsulated. It suffices that they have a certain affinity for the lipophilic phase. The process for preparing the nanocapsules according to the invention is similar. It is sufficient to solubilize the active substance in the aqueous phase. The degree of encapsulation depends on the coefficient of partition of the active substance and its lipophilicity.

 In the case of an active substance which is insoluble in water and in absolute ethylol, it is possible to obtain encapsulation by preparing a suspension of the active substance in a suitable oily carrier. It is then necessary that the particle size of the active substance in suspension is related to the desired diameter for the nanocapsules.

 It is also possible to prepare with this process nanoparticles (nanospheres) whose structure is similar to that of the nanoparticles described in Belgian Patent No. 869,107. In order to prepare these nanoparticles according to the process that is the subject of this invention, it is sufficient not to use an oily support during the preparation. The process then leads to biodegradable nanoparticles.

 The advantage of this new process over the method described in Belgian Patent No. 869,107 is that it eliminates the constraints of PH.

The method described in the Belgian patent makes it necessary to prepare the nanoparticles between PH 2 and 3. With the method forming the subject of the present invention, the nanoparticles can be prepared at any pH. It is not necessary to operate in an acid medium. and this advantage is important for example in the case of unstable products or which crystallize in such a medium. Another advantage of the preparation of the nanoparticles according to the process which is the subject of this invention is the possibility that it offers to fix on the nanoparticles substances that are insoluble in water. The third advantage of this new method of preparation of the nanoparticles is that it makes it possible to obtain nanoparticles of very small average size. It is possible, for example, to prepare nanoparticles containing lomustine or the progesturoyl, which is a medium-sized 80 lumens

 A study was made to evaluate the influence on nanocapsule size and nanoparticle size of the pllase a concentration of surfactant concentration, pH of the aqueous phase, and absolute ethanol concentration. The study was done with nanocapsules prepared with ultra-fluid lipiodol and 2-isobucylynacrylate, which showed that the size increases with temperature.

 The influence of surfactant concentration and that of Pll are limited, however, at low levels the polymerization reaction is excessively fast. The most important factor is the concentration of absolute ethanol.

When the latter is increased the average size of the nanocapsules is significantly reduced.

 The invention is illustrated by the following examples which do not limit the scope thereof.

Example 1
In 4 ml of ultra-fluid lipiodol (ethyl esters of iodinated fatty acids of flaxseed oil at 48 g of iodine per 100 ml) 0.5 ml of 2-isobutyl-cyanoacrylate monomer (IBC 2 ETHICON) and solubilized in 30 ml of absolute ethanol.

 0.5 g of Pluroaic F 68 (mixed polymer of ethylene oxide and polypropylene glycol), non-ionic surfactant, are dissolved in 50 ml of deionized water. The alcoholic solution is then slowly injected with a suitable device into the aqueous phase, with vigorous magnetic stirring.

 The spontaneous polymerization first makes the suspension opalescent and then gives it a milky appearance. The size of the nanocapsules is measured immediately after the reaction with a "Nano-Sizer".

The nanocapsules have an average size of 265 nm with a polydispersion of 0.75.

The suspension of the nanocapsules is then concentrated under low vacuum and low temperature to a volume of 20 ml to remove absolute ethanol and have a suitable iodine level. The nanocapsules are then filtered on sintered glass whose pores have a diameter of between 9 and 15 micrometers. The size of the nanocapsules is measured again at "Nano
Sizer "The average size and the polydispersion remained unchanged: part of the nanocapsule suspension is centrifuged at 220,000 g for 1 hr / 2 The pellet consisting of white nanocapsules can be redissolved in water by simply stirring (upper part ).

 Redispersion can only be explained by the existence of solid nanocapsules.

Examination of the nanocapsules with a transmission electron microscope reveals that they have a spherical shape of very uniform average diameter
Example 2
The procedure is as described in Example 1, but without the monomer. It is found that 99% of the amount of oil used is separated at the bottom of the beaker.This test shows the fundamental role of the moflollère
Example 3
The procedure described in Example 1 is carried out and the concentrated nanocapsules are used in an autoclave at 120 ° C. for 15 minutes.
Examination of the nanocapsules under the electron microscope before and after sterilization shows no change. the average size of the nanocapsules before and after the sterilization measured with the Nano-Sizer is substantially the same
Example 4
The procedure is as described in Example 1, but the following amounts are used: 8 ml of lipiodol, 1 ml of isobutyl-2-cyanoacrylate, 100 ml of absolute ethanol and 2 g of Pluronic F 68 solubilized in 200 ml of Demineralized Water . Three batches of nanocapsules are prepared according to the general method. The three batches are mixed and concentrated until a lipiodol concentration of 20% V / V is obtained. The suspension of nanocapsules thus prepared is distributed in penicillin-type bottles. The bottles are closed with rubber stoppers set with an aluminum ring.

The nanocapsules thus prepared are sterilized by autoclaving at 120 OC for 15 minutes.
Part of the flasks is maintained at room temperature, the average size of the nanocapsules (256 nm) has not changed after 6 months of storage
Example 5
The procedure is as described in Example 4, but the amount of surfactant is decreased. 1 g of solubilized Pluronic F 68 is used in 200 ml of demineralized water. The stability of these nanocapsules at ambient temperature and at 50 OC is studied. The study shows that nanocapsules are very stable in both conditions
Example 6
The procedure is as described in Example 1, but 4 ml of Labrafil M 1944 (polyoxyethylenated oleic glycerides) are used and are polymerized in 50 ml of demineralized water in the presence of 0.5% of Pluronic F 68. Nanocapsules are obtained. With an average diameter of the order of 260 nm, the nanocapsules under an electron microscope have the usual appearance
Example 7
The procedure is as described in Example 6, but 4 ml of neutralized flax oil is used. The oil is not soluble in absolute ethanol but, in the presence of the monomer, it forms a micellar dispersion by means of magnetic stirring.

 With this oil are obtained nanocapsules having a mean diameter of the order of 250 nm.

 In the electron microscope the morphological aspect is the usual one.

Example 8
The procedure is as described in Example 6, but 4 ml of
Miglyol 812 -Oil comprises a mixture of saturated and fractionated coco fatty acid triglycerides with a C8-C10 diamine length. The average size of the nanocapsules obtained after concentration up to 20Z V / V is 261 nm.

Example 9
The procedure is as described in Example 6, but using 4 ml of a perfume, Bouquet 4-5312 oil and polymerized in 200 ml of demineralized water at 0.5% Pluronic F68. The average size of the nanocapsules is 221 nm, directly after the reaction, and without concentration.

 The suspension of nanocapsules can be easily diluted without modifying the characteristics of the nanocapsules. After application the odor persists longer than with the unencapsulated product.

Example 10
The procedure is as described in Example 6, but using 4 ml of castor oil, and polymerization in 100 ml of demineralized water in the presence of 0.5% Pluronic F68. The average size of the nanocapsules after concentration at 20Z V / V is 217 nm.

Example
The procedure is as described in Example 10, but 4 ml of peanut oil, which is insoluble in absolute ethanol, is used. Nanocapsules having an average size of 261 nm are obtained.

Example 12
The procedure is as described in Example 10, but 4 ml of Niaouli oil is used. The average size of the nanocapsules obtained is 186 nm.

Example 13
200 mg of lomustine (UNFC) and 1 ml of Miglyol 812 and 0.5 ml of isobutylcyanoacrylate are dissolved in 50 ml of absolute ethanol. It is polymerized in 100 ml of demineralized water in the presence of 0.5 of Pluronic F68. Nanocapsules with an average size of about 184 nm are obtained, after concentration up to 250 ml.
After 24 h, crystals of (CCNU) are observed on the walls of the flasks
Example 14
The procedure is as described in Example 13, but using 2 ml of
Miglyol, and is polymerized in 100 ml of demineralized water in the presence of 1Z of luronic F 68, nanocapsules average diameter is obtained by 201 nm, after concentration up to 20 ml.Les nanocapsules obtained are distributed in Penicillin-type vials, no crystallization of (CCNU) nc occurs. The flasks are kept at room temperature. The nanocapsules thus preserved undergo identical control every 10 days, the (CCNU) is assayed in the filtrate after filtration through a millipore membrane whose pore diameters is equal to 50 nm under pression. The total (CCNU) is also assayed in the suspension of the nanocapsules, and the size is measured with the Nano-Sizer. The assay of the (CCNU) is carried out in UV at 230 nm. It shows that 100% of the quantity used is encapsulated. It also shows that the encapsulated (CCNU) remained intact after 40 days of storage at the writing date of this patent. The size of the nanocapsules also remains unchanged
Examination under the electron microscope after double staining of the nanocapsules shows the presence of a casing completely surrounding the encapsulated product. The thickness of the envelope is estimated at about 6 nm.

Example 15
The procedure is as described in Example 13, but without oily support (without Miglyol). Most of the (CCNU) crystallizes and remains on the filter (9-15 micrometer). The filtrate has the bluish reflection very characteristic of colloidal solutions. This time we obtain nanoparticles and not nanocapsules. The average size at the Nano-Sizer is 110 nm. The fixation rate is very low
Example 16
200 mg of progesterone, 2 ml of Miglyol and 0.5 ml of isobutyl 2 cyanoacrylate are dissolved in 50 ml of absolute ethanol. It is polymerized in 100 ml of demineralized water in the presence of 1Z Pluronic F 68.Nanoocapsules of average diameter equal to 220 nm are obtained
The determination of progesterone in UV at 241 nm in the filtrate (millipore 50 nm) and total progesterone shows that the degree of encapsulation is equal to 100 Z
Example 17
The procedure is as described in Example 16, but without Miglyol. Nanoparticles with an average size of 83 nm are obtained. The adsorption rate is less than 1 Z
Example 18
2 ml of Miglyol and 0.5 ml of isobutylcyanoacrylate are dissolved in 50 ml of absolute ethanol. 200 mg of eosin (substance very soluble in water and in absolute methanol) are dissolved in 100 ml of demineralized water in the presence of 1 × of Pluronic F 68. The alcoholic solution is injected into the aqueous phase. . Nanocapsules of one L. The average is obtained at 230 nm. During filtration through a Millipore membrane (50 nm). Part of the dye is attached by the nanocapsules, while the other part passes with the filtrate. The fixed part resists Zi a wash by the water

Claims (9)

 1. Biodegradable nanocapsules formed from an envelope resulting from the polymerization of an alkyl cyanoacrylate and a central cavity containing an oily solution or suspension of a biologically active substance.  2. Biodegradable nanocapsules according to claim 1, characterized in that their diameter is less than 1 micrometer  3. Process for manufacturing the nanocapsules defined according to any one of claims 1 and 2, characterized in that it consists of:  a) to dissolve in an appropriate solvent, absolute ethanol or any other solvent that can play the same role, an oily product with or without a biologically active substance, and an alkyl cyanoacrylate  b) forming nanocapsules by injecting the preceding solution into demineralised or distilled water containing or not containing a nonionic surfactant.  c) if necessary, remove the solvent and concentrate the suspension of the nanocapsules  4. A process for producing biodegradable nanoparticles (nanosplers) formed of a more or less dense polymeric network and containing a biologically active substance characterized in that it consists of:  a) dissolving in a suitable solvent (absolute ethanol or any other solvent which can play the same role) a biologically active substance and an alkyl cyanoacrylate  b) forming nanoparticles by injecting the preceding solution in demineralized or demineralized distilled water containing or not a nonionic surfactant.  c) if necessary, to remove the solvent and to concentrate the suspention of nanoparticles  5. Nanocapsules or biodegradable nanoparticles according to one of claims 1 to 4, characterized in that the biologically active substance is a product endowed with antimitotic, antineoplastic, antibiotic or hormonal properties, a virus, a constituent of a virus, bacterium or bacterium. cell, antigen, allergen or enzyme or product for diagnosis  6. Nanocapsules or biodegradable nanoparticles according to any one of claims 1 to 5, characterized in that the encapsulated active substance is ultra-fluid lipiodol radiopaque product 48% g of iodine / 100ml.  7. Nanocapsules or biodegradable nanoparticles according to any one of claims 1 to 6, characterized in that the encapsulated active substance is lolllustine (C.C.N.U), or progesterone or insulin.  8. Nanocapsules or biodegradable nanoparticles according to any one of claims 1 to 7, characterized in that the biologically active substance at the time of preparation can be dissolved or suspended in the alcoholic or aqueous phase  A pharmaceutical composition containing biodegradable nanocapsules or nanoparticles as claimed in any one of claims 1 to 8, with an expestant for oral, subcutaneous, intradermal, intramuscular or intravenous administration.