SI21222A - Method for preparation of nanoparticles - Google Patents

Abstract

The submitted invention refers to a new method used in preparation of nanoparticles. In particular, it relates to a method in which a water-in-oil-in-water (W/O/W) double emulsion technique applied under low-level energy conditions is used to prepare biologically degradable nanoparticles from copolymers of lactic and glycol acids. The technique includes simultaneous processes of mixing and sonification with nanoparticles containing one or more active compounds while biological activity of the active compound involved is preserved.

Description

Process for preparing nanoparticles

Technical field of the invention

The present invention relates to a novel process for the preparation of nanoparticles. The invention relates in particular to a process for the preparation of biodegradable nanoparticles of lactic and glycolic acid copolymers containing one or more active substances, using a water-in-oil-in-water (V / O / V) double emulsion process.

BACKGROUND OF THE INVENTION

In the past, as a result of extensive advances in biotechnology and genetic engineering, many new protein drugs have been developed that are believed to have high potential in the treatment of various diseases. For example, many proteins have been proposed as potential anticancer agents for the treatment of cancer, with the ability to attenuate tumor growth, invasion, and metastasis. These proteins also include protease inhibitors such as TIMPs, tissue plasminogen activator inhibitors and cystatins.

One of the drawbacks of protein drugs is their instability in the physiological environment, their propensity for enzymatic degradation, and thus their shorter half-life in vivo. Furthermore, proteins are, in most cases, large molecules that are difficult to pass through biological membranes, significantly reducing their usefulness.

To overcome some of the above problems, carrier systems for active substances have been developed that improve the half-life and bioavailability of such substances. Among such systems, nanoparticles represent a very promising approach as their properties of active substance protection and controlled release are very useful. In particular, it has been found that nanoparticles allow the transport of active substances through biological membranes and specific targeting with potential surface modification (Lamprecht A., Ubrich N.,

Perez M. H., Lehr C. M., Hoffman M., Maincent P., Biodegradable monodispersed nanoparticles prepared by pressure homogenization-emulsification, Int. J. Pharm. 184 (1999) 97-105).

The main advantage of nanoparticles is the possibility of their different use. They provide parenteral and non-parenteral routes of administration. They can be injected intravenously and since the protein is incorporated within the polymer matrix and thus protected against circulating enzymes, the plasma half-life of the protein involved is prolonged compared to the injected protein solution. The release of protein from nanoparticles is also delayed, resulting in prolonged protein activity. Furthermore, by selecting the appropriate polymer, the nanoparticles can facilitate absorption through biological membranes. Oral administration is also possible. The decisive parameter is the particle size that determines the absorption from the gastrointestinal tract. Smaller nanoparticles are usually absorbed on a larger scale.

So far, several encapsulation techniques have been developed for the preparation of nanoparticles using two main principles, the single (I / O) and the double (I / O / V) emulsion method.

Thus, US 4,177,177 describes a process for the direct emulsification of a solution of water-insoluble polymers in suitable organic solvents into an aqueous solution containing at least one emulsifier of the nonionic, anionic or cationic type. Ultrasound (sonication) was used to reduce the size of the emulsion droplets to achieve a particle size of less than 0.5 pm.

EP 0 052 510 B2 describes a phase separation technique using co-preservatives or non-solvents such as mineral oils and vegetable oils. The active substance, e.g. the polypeptide is first dissolved in the aqueous phase of the water-in-oil emulsion. The polymer is precipitated around water droplets by the addition of a non-solvent polymer such as silicone oil. A curing agent is then added to extract the organic solvent from the micro or nanoparticles.

However, one of the disadvantages of this process is the high consumption of organic solvents required for extraction and washing.

US 5,019,400 describes another process employing a spray drying technique and / or a spray coating technique For spray drying, the polymer and the active substance are dispersed in a polymer solvent. The solvent is then evaporated by spraying the solution, resulting in the formation of polymer droplets containing the active substance. However, sensitive substances such as proteins can become inactive during the process due to the elevated temperatures used and the exposure to the boundary surfaces of the organic solvent / air.

US 5,407,609 describes a process using solvent evaporation. The solvent evaporation technique (oil-in-water (O / V) process) involves the dissolution of the polymer and the active substance in an organic solvent, after which the solution is added to a water-immiscible, water-immiscible polymer phase. The aqueous outer phase usually contains surfactants that stabilize the oil-in-water emulsion and prevent agglomeration. The emulsifier used is usually polyvinyl alcohol. The organic solvent is then evaporated for several hours or more, with the polymer precipitated to form a polymer matrix.

A so-called water-in-oil-in-water (V / O / V) double emulsion method has also been developed, which is useful especially when using water-soluble peptides that are difficult to incorporate in the conventional oil process -in-leads (O / V) (EP 0 190 833 BI). The main advantage of this process is that during the nanoparticle formation process, the protein remains in the internal aqueous phase, thereby reducing direct contact between the protein and the organic solvent. This process involves dissolving the protein in water, adding an aqueous solution of the protein while stirring to an organic polymer solution, and forming the first I / O emulsion. This V / O emulsion is then poured into an aqueous phase containing the emulsifier, forming a double V / O / V emulsion when stirred. The organic solvent is subsequently removed by evaporation or extraction.

However, during the preparation of this type of nanoparticle, characteristic destructive interactions can occur between the solvent and the protein, partly due to the organic solvents used, the shear forces during mixing and sonication, all of which reduce the biological activity of the incorporated active substance.

The decisive step in the preparation of micro- and nanoparticles is emulsification, which can generally be performed by sonication, high-pressure homogenization or high-shear homogenization. Mixing combined with subsequent sonication for one hour usually results in nanoparticles larger than 1400 nm in diameter. (Ferdous A. J., Stembridge N. Y., Singh M. Role of monensin PLGA polymer nanoparticles and liposomes as a potentiator of ricin A immunotoxins in vitro, J. Control. Rel. 50, 1998: 71-78). Higher energy is required to obtain particles in the nanometer range, which often results in a loss of protein biological activity. When they used an oil-in-water (O / V) solvent evaporation process and combined all three processes, i.e. the sample was homogenized (20,000 rpm, 20 minutes), followed by simultaneous stirring (500 rpm) and sonication for 1 hour to give particles smaller than 200 nm in size.

Solve a technical problem with implementation examples

The object of the present invention is therefore to solve the drawbacks of the prior art and to provide a new process for the preparation of nanoparticles which does not have a significant adverse effect on the active substances which we wish to include.

In the extensive studies leading to the present invention, the inventors have found that a combination of different measures, namely mixing and sonication, both at a level at which each individual action as such does not result in sufficiently intense emulsification to result in a small size nanoparticle, in which the incorporated active substance expresses enhanced biological activity.

The present invention therefore relates, in one aspect, to a process for the preparation of nanoparticles with incorporated one or more active substances, employing an emulsion method whereby mixing and sonication are carried out simultaneously, each at an energy level which is not sufficient for the formation of the nanoparticles, and which maintains the biological activity of the incorporated active substance.

According to the invention, mixing and sonification are carried out simultaneously and under moderate conditions. In a preferred embodiment, the mixing speed is in the range from 4000 to 15000 rpm, preferably from 5000 to 10000 rpm and more preferably from 5000 to 7000 rpm

Sonification is preferably performed at a frequency of 20 kHz to 70 kHz.

The process of the invention particularly comprises:

I) dissolving the biodegradable polymer in a suitable organic solvent (preferably in a water-immiscible organic solvent),

II) emulsification, in which mixing and sonification are carried out simultaneously, each at an energy level which is not sufficient for the formation of the nanoparticles, comprising:

a) emulsifying the active substance (preferably a polypeptide or peptide) dissolved in water or a suitable aqueous solvent (e.g., in buffer) into the organic solution obtained in step I) to ensure the formation of the primary emulsion with the active substance in the internal aqueous phase and

b) emulsifying the primary emulsion obtained in step Ha) into the aqueous solution of the emulsifier as a continuous phase (preferably with an aqueous solution of polyvinyl alcohol) to obtain nanoparticles in which the active substance is incorporated; and

III) isolation and drying of the nanoparticles in a known manner (preferably drying is carried out by lyophilization, whereby the active substance in step Ha is dissolved in a suitable aqueous solvent containing cryoprotectants).

In the process of the invention, a new appropriately modified emulsification technique is used, which simultaneously utilizes high shear homogenization at low rpm / sonication. The resulting primary emulsion is then emulsified into the emulsifier solution to obtain a double emulsion (V / O / V). The double emulsion is diluted with excess water to allow the removal of the organic solvent, and the mixture is stirred to allow the solvent to evaporate, thereby inducing the precipitation of the polymer and thereby the formation of solid nanoparticles with the incorporated active substance. The particles were isolated by centrifugation or filtration and washed several times with distilled water or appropriate aqueous buffers to remove excess emulsifier from the surfaces. The particles are then dried by conventional means, for example in vacuo, by blowing with nitrogen gas or airflow, by lyophilization or by spray drying.

The organic solvent used in the dissolution step of the biodegradable polymer may be any solvent capable of forming an emulsion with a given amount of aqueous emulsifier solution and which can be removed from the emulsion droplets by the addition of an excess aqueous emulsifier solution used and further having the ability to dissolve biodegradable polymers. In other words, the solvent must not be miscible with or substantially miscible with water, but is partially soluble in said aqueous emulsifier solution. Examples of organic solvents that can be used to dissolve a polymer are chloroform, benzene, dichloromethane, chloroethane, dichloroethane, trichloroethane, carbon tetrachloride, ethyl ether, cyclohexane, n-hexane, toluene, more preferably ethyl acetate, methylene chloride or a mixture of methylene chloride and a mixture of methylene chloride acetone, most preferably ethyl acetate. Surprisingly, we have found that the use of ethyl acetate limits the loss of biological activity of proteins such as cystatin and provides smaller particles compared to other solvents. Without wishing to be limited by any theory, we believe that this may be due to the higher solubility of ethyl acetate in water, which leads to faster diffusion of ethyl acetate into the outer aqueous phase, leaving behind insoluble polymer particles with the protein involved.

As emulsions are thermodynamically unstable systems, the use of an emulsifier is essential. The emulsifier serves several purposes: it helps to obtain the correct size distribution of the emulsion droplets, stabilizes the I / O / V emulsion to avoid coalescence of the droplets and prevent the precipitated nanoparticles from sticking to each other. Preferred examples of emulsifiers are anionic surfactants (eg, sodium oleate, sodium stearate, sodium lauryl sulfate, etc.), nonionic surfactants (eg esters of polyoxyethylene sorbitan and fatty acids (Tween 80, Tvveen 60, etc.)), polyoxysuccinyl ore derivatives, polyoxytin ethylene derivatives pyrrolidone, polyvinyl alcohol, carboxymethylcellulose, lecithin, gelatin, etc., more preferably polyvinyl alcohol. Such emulsifiers can be used either alone or in combination.

A variety of polymeric materials, such as lactic and glycolic acid polyesters, polylactic acid, poly-β-hydroxybutyric acid, polyhydroxyvaleric acid, polycaprolactone, polyesteramides, polycyanoacrylates, poly (amino acids), poly (amino acids), can be used to incorporate the active substance whereby lactic and glycolic acid polyester are preferably preferred. The (poly) lactic acid / (poly) glycolic acid by weight ratio is preferably from about 99/1 to 36/65, more preferably 95/5 to 50/50, with the precise composition of the polymer being selected by one skilled in the art and depending on of the desired release kinetics. Microspheres and nanoparticles of lactic and glycolic acid (PLGA) copolymers are biocompatible, break down upon the formation of non-toxic monomers, and the polymer matrix perfectly protects proteins and peptides from destructive environmental conditions, especially when administered orally. By varying the molecular weight and the ratio of PLGA monomers, the kinetics of the release of the incorporated active substances can also be controlled.

Any active substance, in particular substances having a short half-life in the body, such as proteins and / or peptides, can be used as the active ingredient to be incorporated in the present invention. Bioactive macromolecules such as interferons, interleukins, colonial stimulating factors, tumor necrosis factors, other immune modulators, growth factors, transforming growth factors, erythropoietin, albumin, blood proteins, hormones, vaccines, viruses, toxins, antibodies, antibodies, fragments can be used enzymes, enzyme inhibitors including cystatin.

In addition to the active substance, other substances such as stability control agents and, if desired, solubility control agents of the biologically active substance may be included. Such agents may be pH control agents, preservatives, and stabilizers and cryoprotectants, which may include glycols, albumin, gelatin, amino acids, ethylenediamine tetraacetic acid, dimethyl sulfoxide, citric acid, dextrin, sucrose, fructose, mannose, trehalose, trehalose. their combinations.

Another aspect of the present invention is the nanoparticles obtained by the process of the present invention, preferably having a size of from about 100 to about 800 nm. These nanoparticles give surprisingly increased biological activity and stability to the active substances incorporated into them.

The present invention is essentially based on the observation that the rotation of the high-speed homogenizer (15000 rpm) normally required for the production of nanoparticles can be compensated for at a lower speed if the system is simultaneously exposed to low-energy ultrasound, the biological activity and bioavailability of the active substance incorporated improves compared to the nanoparticles produced by conventional techniques of self-mixing or sonication, or a sequential combination of both.

We also found that shorter homogenization time (2 minutes versus 7 minutes) retains protein activity to a greater extent. This may at least in part be due to a decrease in the time available for the protein to contact the organic solvent. Short production time can also lead to greater efficiency of the embedded protein within the nanoparticles, since it impedes the diffusion of the protein from the inner aqueous phase to the outer bulk phase.

The nanoparticles of the invention are suitable for the manufacture of a medicament for parenteral, nasal, pulmonary, oral, oral, transdermal or rectal administration of the active substance.

The pictures show:

FIG. 1 shows the size of the nanoparticles obtained by the process of the present invention in comparison with the prior art nanoparticles.

FIG. 2 shows cystatin activity under different experimental conditions.

FIG. 3 shows the activity of cystatin during storage when incorporated in the nanoparticles (3a) obtained according to the present invention as compared to the cystatin solution (3b).

The following examples illustrate the invention without being limited thereto.

Example la

The following procedure was used to prepare blank nanoparticles from lactic and glycolic acid (PLGA) copolymers (nanoparticles without protein included). We conducted a study to evaluate how experimental constraints affect nanoparticle size.

The polymer solution was first prepared by dissolving 50 mg of PLGA (Resomer RG 503H, Boehringer Ingelheim) in 1 ml of ethyl acetate in a test tube. Then, 200 μΐ of water was added to the polymer solution and the mixture was emulsified homogeneously with a rotor-stator homogenizer (Omni Lab run, Omni International, USA) for two minutes. We used two different rotation speeds, 15000 oz., On two different samples. 12500 rpm After one minute of homogenization, 4 ml of 5% aqueous PVA (polyvinyl alcohol) solution was added to the homogenized mixtures to form a stable double emulsion (V / O / V). When the homogenization was complete, the resulting double emulsion was slowly poured onto 100 ml of 0.1% aqueous PVA solution. The resulting mixture was then homogenized for 5 minutes at 5000 rpm.

The dispersion was then ultracentrifuged at 15,000 rpm for 15 minutes using an Sorvall RC 5C plus ultracentrifuge, SS 34 rotor, USA. After separation, the nanoparticles were washed three times with distilled water (20 ml) and collected by centrifugation under the above conditions.

The particles obtained had a size in the range of 600 nm to 700 nm, reaching a minimum size in the formulation prepared at the highest rotational speed.

-1010

Example lb

The following study was performed to determine the loss of cystatin activity during homogenization under different conditions and to identify the maximum rotational speed still possible to maintain cystatin activity. Cystatin, a cysteine proteinase inhibitor, was isolated from chicken egg proteins as described (Kos, J., Dolinar, M., Turk, V.: Isolation and characterization of chicken L- and H-kininogens and their interaction with chicken cysteine proteinases and papain, Agends and Actions 38, 331-339, 1992). The biological activity of cystatin was determined on plant proteinase papain using BANA (α-N-benzoyl-DL-arginine-3-naphthylamide) as a substrate. The effect of different homogenization conditions on cystatin activity was observed on an aqueous cystatin solution. We performed the same sequence of individual stages of the process as in the case of la. Therefore, in case of lb, the PLGA polymer was not used, 1 ml of ethyl acetate, 4 ml of 5% PVA aqueous solution and 100 ml of 0.1% PVA aqueous solution were replaced with distilled water. A protein solution was used instead of 200 μΐ of water (2 mg cystatin was dissolved in 200 μΐ 0.1 M phosphate buffer, pH = 6.0). The same conditions were used as in the case of 1a and the effect of rotational speed on the inhibitory activity of cystatin was determined as described above.

It was found that homogenization at 15000, 12500 and 10000 rpm resulted in a loss of cystatin biological activity of 82%, 66% and. 30%.

We found that the required rotational speed was reduced to 5000 rpm to maintain cystatin activity at about 98%. However, such a reduction in energy input does not allow the production of nanoparticles.

Example 2a

To enable the nanoparticles to be prepared even at lower rotational speeds, we performed the same procedure as in the case of la, except that ultrasound was used simultaneously with homogenization (10000, 7500, and 5000 rpm). The resulting nanoparticles had a size of 320, 350 and respectively. 360 nm.

-1111

This suggests that we can more efficiently utilize energy input by performing different types (processes) of emulsification simultaneously, e.g. homogenization and ultra-sonification. Therefore, the synergistic effect of mechanical and ultrasonic energy enables the production of nanoparticles at lower rpm (5000 rpm) and mild ultrasonic waves.

Example 2b

The following test was performed to determine the synergistic effect of emulsification (Example 2a) on cystatin activity.

We performed the same procedure as in the case of lb, except that ultrasound was used simultaneously with homogenization at 5000 rpm. The loss of cystatin biological activity was only 20%. This suggests that this process is a good compromise between maintaining a high level of protein activity and forming the desired size nanoparticles.

Example 3a

The following study was performed to determine the stability of a protein within nanoparticles under simulated physiological conditions.

The nanoparticles were manufactured by the process of the present invention. We performed the same procedure as in the case of la, except that instead of using 200 μΐ of water, we used 200 μΐ of cystatin solution in water, and instead of using high-speed homogenization, homogenization was performed at 5000 rpm with simultaneous use of ultrasound.

The resultant nanoparticles were washed, centrifuged and collected as in the case of 1a and finally dispersed in 5 ml of 0.12 M phosphate buffer (PBS) with a pH of 7.2-7.4 and incubated for 19 days at 37 ° C with stirring on magnetic mixer (2 rpm).

At different time intervals (3 hours, 1, 2, 5, 7, 12, 14, 16 and 19 days), 300 μΐ of the sample was taken, then the samples were centrifuged (15 minutes at 16000 rpm) and determined

-1212 cystatin activity in the supernatant by enzymatic spectrophotometric assay (BANA test). The remaining nanoparticles were returned to the incubation dispersion.

Example 3b

Further, cystatin-incorporated nanoparticles were prepared according to the present invention except that instead of water, cystatin was dissolved in a solution of 2% albumin, 300mM trehalose, 300mM mannose, 300mM fructose and 100mM sucrose (cryoprotectant). After preparation of the nanoparticles, as in the case of la, these were centrifuged for 15 min at 7000 rpm using an Sorvall RC 5C plus ultracentrifuge, SS 34 rotor, USA. The sediment was resuspended in water by ultrasound and dried by lyophilization. Cystatin activity in nanoparticle dispersion after lyophilization accounted for 90% of that before lyophilization. When cystatin nanoparticles were prepared as in lb and lyophilized, cystatin activity was only 17% of that before lyophilization.

Example 3c

In order to compare the stability of the protein released from the nanoparticles with the normal protein solution, a control sample was prepared and treated under the same conditions as the nanoparticle dispersion in Example 3a.

The control sample was prepared by dissolving an appropriate amount of cystatin in 5 ml of PBS (approximately the same concentration relative to the protein in the released medium of Example 3 a).

The same procedure as in Example 3a was performed and stability of the incubated protein was obtained for different time intervals.

Claims (14)

Patent claims A process for the preparation of nanoparticles incorporating one or more active substances, characterized in that an emulsion method is used whereby mixing and sonication are carried out simultaneously, each at an energy level which is not sufficient for the formation of the nanoparticles and which retains the biological activity of the incorporated active substances. A method according to claim 1, characterized in that it comprises: I) dissolving the biodegradable polymer in a suitable organic solvent, II) emulsification, in which mixing and sonification are carried out simultaneously, each at an energy level which is not sufficient for the formation of the nanoparticles, comprising: a) emulsifying the active substance dissolved in water or a suitable aqueous solvent into the organic solution obtained in step I) to ensure the formation of the primary emulsion with the active substance in the internal aqueous phase, and b) emulsifying the primary emulsion obtained in step Ha) into the aqueous solution of the emulsifier as a continuous phase to obtain nanoparticles in which the active substance is incorporated; ter III) isolation and drying of nanoparticles in a known manner. Method according to claim 1 or 2, characterized in that the mixing speed is in the range of 4000 to 15000 rpm. A method according to any one of claims 1 to 3, characterized in that sonification is carried out at 20 kHz to 70 kHz. A process according to any one of claims 2 to 4, characterized in that the water-miscible organic solvent, preferably chloroform, benzene, dichloromethane, chloroethane, dichloroethane, trichloroethane, carbon tetrachloride, ethyl ether, cyclohexane, is used as the organic solvent. n-hexane, toluene, more preferably ethyl acetate, methylene chloride or a mixture of methylene chloride and acetone, most preferably ethyl acetate. -1414 Process according to any one of claims 2 to 5, characterized in that the anionic surfactants, nonionic surfactants, polyoxyethylene castor oil derivatives, polyvinyl pyrrolidone, polyvinyl alcohol, carboxymethylcellulose, lecithin, gelatin or any combination of alcohol are used as emulsifiers. Process according to any one of claims 2 to 6, characterized in that the polymer is lactic and glycolic acid polyester, polylactic acid, ροΐΐ-β-hydroxybutyric acid, polyhydroxyvaleric acid, polycaprolactone, polyesteramide, polycyanoacrylate, poly (amino acid) , a polyanhydride, a biodegradable polymer, preferably a lactic and glycolic acid copolymer. Method according to any one of the preceding claims, characterized in that the active substance is a protein and / or peptide and / or biologically active macromolecule such as interferons, interleukins, colonial stimulating factors, tumor necrosis factors, other immune modulators, growth factors, transforming growth factors, erythropoietin, albumin, blood proteins, hormones, vaccines, viruses, toxins, antibodies, antibody fragments, enzymes, enzyme inhibitors including cystatin. Method according to any one of the preceding claims, characterized in that the nanoparticles, in addition to one or more active substances, also include stability control agents and / or solubility control agents for the active substance, preferably pH control agents, preservatives, stabilizers and cryoprotectants . Process according to any one of claims 2 to 9, characterized in that the drying of the nanoparticles is carried out by lyophilization, the active substance being dissolved in a suitable aqueous solvent containing cryoprotectants. Process according to claim 10, characterized in that the cryoprotectants may be sugars, glycols, albumin, gelatin, amino acids, dimethyl sulfoxide or their various -1515 combinations, preferably albumin, sucrose, fructose, mannose, trehalose or combinations thereof. 12. Nanoparticles, characterized in that they are obtained by the process according to any one of claims 1 to 11. 13. Nanoparticles according to claim 12, characterized in that they have an average diameter of from about 100 nm to about 800 nm. Use of nanoparticles obtained by the method according to any of claims 1 to 11 for the manufacture of a medicament for parenteral, nasal, pulmonary, oral, oral, transdermal or rectal administration of the active substance.