RO126041A2 - Process for the synthesis of a magnetic composite - Google Patents

Abstract

The invention relates to a process for preparing a magnetic composite meant to be used in analytical or clinical bio-medical applications. According to the invention, the process consists in the co-polymerization of poly(succinimide) and poly(ethyleneglycol) having a molecular weight of 4000, 10000 or 20000, in a gravimetric ratio of 2 : 1, in dimethylformamide in a ratio of 6.5 mol % in relation to the polymer, in the presence of ferrite, in a ratio of 3.73% in relation to the total amount of polymer, as a stabilized ferro-fluid in a concentration of 3.205%, while stirring continuously at 140°C, under nitrogen atmosphere, in the presence of an aqueous catalyst solution Mn(CHCOO).4HO in a ratio of 1.5% in relation to the polymer, admixed by dropping for 60 min, the reaction being continued for 6 h, at a temperature of 140°C, with water removal, the reaction medium being then cooled down and the resulting copolymer being settled from methanol, washed and dried, to obtain a product having combined hydrophylic and hydrophobic character, with encapsulated ferrite.

Description

The invention relates to a process for the synthesis of a magnetic composite in the form of microplate copolymer diblock with combined characteristics of hydrophilicity and hydrophobia with ferrite encapsulated in the polymeric matrix, applicable as a therapeutic instrument in detoxifying human blood or as a magnetic carrier for bioactive substances for transport. at the target and the controlled release of drugs.

The specialty literature presents polymeric microparticles with magnetic characteristics with both technical applications (separation, fluid transport, toners) and biomedical analytical or clinical, such as: detoxification of human blood by selective and rapid removal of biological or chemical contaminants, purification of nucleic acids and of proteins, cell separation, DNA extraction, enzyme immobilization, transport to a specific drug target, or as a magnetic probe with the ability to increase signal or contrast in magnetic resonance imaging.

Such particles can be functionalized in order to interact with the biological entity of interest. Due to the magnetic characteristics, they can be manipulated by an external magnetic field as practiced in hypothermia, magnetic cell separation, targeted transport of bioactive principles (drugs). Biomedical applications require biocompatibility, biodegradability, absence of immune reactions and toxicity. Also, hydrophilicity of the surface and relatively small size are required so that it can escape from the reticuloendothelial tissue.

Methods of multiphase preparation of polymeric particles with magnetic characteristics have been developed by the layer-by-layer deposition technique or by heterogeneous phase polymerization processes (emulsion, suspension, dispersion, micro- and miniemulsion). The resulting composite material has one of the structures: (1) magnetic material as core encapsulated in the polymer shell, (2) magnetic material dispersed in the polymer matrix after polymerization, and (3) magnetic shell material adsorbed in the pores of the pre-formed polymer as core.

Poly (aminic) acids of the poly (aspartic acid) type and their copolymers have been intensively studied as polymeric carriers for bioactive substances in controlled release systems.

_t x »ΐ D Ο 9 - Ο Ο 6 61 - 2 8 -08- 2009 of medicines. Amino acid-based polymers such as poly (aspartic acid) have the advantage of a polypeptide that provides specific binding to tissues and the presence of two functional groups (amino- and hydroxyl-) for functionalization with specific receptors such as radionuclear chelators or mononuclear antibodies.

In particular, poly (succinimide) as an intermediate product in the synthesis of the polyartic acid aspartic) from D, L-aspartic acid, due to its biocompatibility, biodegradability and lack of toxicity, is one of the most promising macromolecular carriers of drugs because it possesses structural and physical characteristics. chemicals corresponding to the stated purpose. The combination of poly (ethylene glycol) and poly (succinimide) hydrophobicity characteristics in the same macromolecule is important due to the potential biological and pharmaceutical applications that require solubilization in aqueous environment and the ability to create sites for finialization along the macromol. The presence of the magnetic component offers sensitivity in the presence of an external magnetic field.

U.S. Patent Bed. 4,582,622 (1986) presents the preparation of magnetic particles of gelatin, water soluble polysaccharide, sodium phosphate and magnetic substances. U.S. Patents Bed. 4,628,037 (1986) and 4,554,088 (1985) describe microparticles with magnetic metal oxide core encapsulated in siloxane polymer shell. in U.S. Pat. Bed. 4,452,773 (1984) particles of colloidal size with a ferromagnetic core are coated with a water soluble polysaccharide or a derivative thereof with functional groups. U.S. Patent Bed. 4,695,392 (1987) describes magnetically responsive ferrite core particles surrounded by a siloxane shell, to which a variety of organic and / or biological molecules can be coupled.

In U.S. Patents Bed. 4,554,088 (1985) and 4,628,037 (1986) a superparamagnetic metal oxide core is surrounded by a siloxane polymer; The synthesized magnetic microparticles have a large surface area and are applied in immunity and purification tests. Due to their small size, they are difficult to remove from the cell suspension and often non-specific binding to normal cells is excessive.

The use of magnetic polymer microparticles as a solid phase in various types of assays has been disclosed in US Pat. Bed. 4,152,210 (1979) and 4,343,901 (1982) for enzyme immobilization; U.S. Bed. 3,970,518 (1976), 4,230,685 (1980) and 4,672,040 (1987) for cell and U.S. separations. Bed. 4,554,088 (1985), 4,628,037 (1986), 3,933,997 (1976) for immunity tests.

It is further described in US Pat. 4,339,337 (1982) the preparation of magnetic polymeric microparticles by dispersing magnetic particles in the solution of a polymer dissolved in a polymerizable vinyl aromatic compound and subsequent polymerization of the mixture. In the patent

-ο 0 6 6 1 - 2 8 -08- 2009

US Pat. 4,358,388 (1982) magnetic nanoparticles are dispersed in an organic phase containing the initiator dissolved and a vinyl aromatic monomer. The organic phase is subsequently emulsified and polymerized to give a latex.

Glutaraldehyde-based magnetic microspheres are synthesized by its polymerization in the presence of magnetic nanoparticles (US Pat 4,267,234 - 1981 and 4,267,235 - 1981) and based on poly (acrolein) by polymerization of m-sites of acrolein (US Pat 4,438 239 - 1984).

The synthesis of composite metal oxide particles encapsulated in vinyl polymer is disclosed in U.S. Pat. Bed. 5,834,121, 1998. in U.S. Pat. Pat..6.204.033 from 2001 presents the synthesis of magnetic particles encapsulated in poly (vinyl alcohol), with the ability of functionalization in hydroxyl groups to be coupled with biomolecules.

In US Pat. No. 7,547,473 of 2005, microparticles of magnetic character are prepared by depositing a carboxylated polysaccharide with different functionalities as. Chelating agents or ion exchangers, on the surface of magnetic nanoparticles, Microparticles can be applied for adsorption of metal cations, various anionic species, drug molecules or biomolecules, acting as adsorbents for wastewater or biochemical separation, carrier with targeting function or drug genes, a contrast agent for magnetic resonance imaging.

Magnetic particles are used as reagents for purification and extraction of nucleic acids. U.S. Patents Bed. 5,750,338 from 1998 and 5,898,071 from 1999 use magnetic particles encapsulated in siloxane polymer in the presence of poly (ethylene glycol) for the capture, extraction and targeted release of a nucleic acid in the detection of specific DNA sequences. Also, U.S. Pat. Bed. 6 027,945 of 2000 describes the application of siloxane polymeric particles with a magnetic character and a diameter of 4 to 7 microns for the extraction and purification of nucleic acids. The processes and products described in said patents have one thing in common, namely that the magnetic particles are prepared by precipitating a solution of salts with a certain molar ratio of Fe (Il) and FE (III) in the presence of complexing agents, or during synthesis. polymeric coatings. This process causes a common disadvantage: microparticles resulting from polymers with encapsulated magnetic nanoparticles have a wide size distribution in the range of 100 Â - 100 μ and an amorphous, oval, non-spherical geometric structure. Small size favors non-specific binding to normal cells or to the walls of the storage container. Also the content of magnetic oxide varies.

The problem solved by the invention is the creation of a magnetic composite in the form of microplate copolymer diblock with combined characteristics of hydrophilicity and hydrophobia and in * 2 0 0 9 - 0 0 6 6 1 - - 2 * ϊ 8-08-2009 encapsulated ferrite in the polymeric matrix during synthesis, by copolensation in organic solvent medium of poly (succinimide) and poly (ethylene glycol), with biomedical applications. The process of synthesizing the magnetic composite according to the invention, removes the disadvantages mentioned by the fact that it is co-condensed in a nitrogen atmosphere and in an organic environment of dimethylformamide, at a temperature of 140 ⁰ C, for 7 hours, a mixture of poly (succinimide) and poly ( ethylene glycol) with a molecular weight of: 4000, 10,000 or 20000, in a gravimetric ratio of 2: 1, in the presence of ferrite in the form of stabilized ferrofluid (with a concentration of 3,205%), in a proportion of 3.73% ferrite relative to the total quantity of the two polymers and of the catalyst Mn (CH ₃ COO) 2 4 H ₂ O in a proportion of 1,5% compared to the total quantity of the two polymers.

The process according to the invention has the following advantages:

• It is an ecological process, again toxic emissions, simple to apply and safe in operation, with a reduced number of technological phases.

• The synthesized diblock copolymer has functional groups that allow it to be solubilized in water as well as functionalization with a wide variety of functional groups for the purpose of covalent coupling or conjugation with bioactive substances.

• Good control of the size of the microparticles and their narrow distribution range are achieved.

• The content of metal oxide can be varied according to the application.

• Storage and operational stability in the bloodstream.

An example of the invention is given below.

EXAMPLE

In a well-known laboratory facility, composed of a 50 ml round-bottomed vessel and four necks, equipped with a shaker, Dean-Stark hatch with reflux condenser, thermometer, nitrogen connection and oil bath for heating, it is fed with strong stirring poly (succinimide) (PSI) (4g, correspondingly 0.040 mole succinimide units), poly (ethylene glycol) (PEG) (2g, corresponding 0.032 mole units ethylene glycol) with the molecular weight of 4000, 10,000 or 20,000, dissolved in 30 ml dimethylformamide (0.39 mol, correspondingly 6.5 mol% compared to the polymer) and ferrite in the form of stabilized ferrofluid with a concentration of 3.205% (6g, corresponding 3.73% relative to the polymer). Heat the system to 140 ° C, keeping under stirring and bubbling a stream of nitrogen. When the reaction temperature reaches 140 ° C, the amount of 0.09 g Mn (CH3 COO) 2 4H ₂ O (0.367 mmol, 1.5% polymer) dissolved in 5 is added dropwise over 60 minutes. ml of water.

9 \ · ΐΰ0 9 - ο ο6.61 - 2 8 -08- 2009

Continue the reaction for another 6 hours. During this time a low vacuum is applied to remove about 10 ml of water. Cool the reaction medium by stirring. The dispersion resulting from the block copolymer with ferrite synthesized by synthesis, is filtered and washed several times with excess methanol to remove unreacted reactants. Dry at 45 ° C under reduced pressure. Spectroscopy does not detect residual poly (succinimide).

As the synthesized encapsulated ferrite diblock copolymer is hydrophilic, the small molecule impurities (catalyst, solvent) can be removed by dialysis of the reaction mixture for 2 weeks, resulting in a final purity of over 98%. The water is removed by vacuum drying, methanol washing and drying.

The poly (succinimide) -b-poly (ethylene glycol) block copolymer with encapsulated ferrite synthesized as a therapeutic tool in detoxifying human blood or as a magnetic carrier of bioactive substances for target transport and controlled drug release, has been characterized in terms of composition. chemical, particle size, thermal stability and magnetization. Table 1 presents the data obtained by near-infrared spectroscopy with NIR-CI chemical analysis and the size distribution by laser light diffusion, and in table 2 the temperatures characteristic of the thermal decomposition process, the weight loss corresponding to the maximum decomposition temperature and the magnetic susceptibility are recorded. determined for a certain intensity of the magnetic field.

Table 1

NIR-C1 analysis and size distribution of co-partitions (PSl-b-PEG) with encapsulated ferrite

Sample Composition prediction by N1R-CI Copolymer / ferrite spectroscopic analysis Particle size distribution Microparticle diameter, μ Dvl0 ^1} Dv50 ²⁾ Dv90 ³⁾ 4000 0.33 / 0.67 1269 5883 17.154 10000 0.50 / 0.50 1/717 11.311 20.503 20000 0.59 / 0.41 1929 7418 18.851 1) DvlO - the value of the diameter for which 10% of the sample volume has particles with a diameter smaller than this value 2) Dv50 - the value of the diameter for which 50% of the sample volume has particles with a diameter greater than this value and 50% of the sample volume has particles with a diameter smaller than this value. 3) Dv90 - the value of the diameter for which 10% of the sample volume has particles with a diameter smaller than this value.

CV-2 Ο Ο 9 - Ο Ο 6 6 1 - 2 8 -08- 2009

Table 2

Thermogravimetric analysis and magnetization of co-microparticles (PSI-b-PEG) with encapsulated ferrite

Sample Thermogravimetric analysis magnetization VC T _max , ° c T _f , ° C AW,% Magnetic susceptibility, uem / g Magnetic field intensity, Oe 4000 75 100 360 600 10 20 20.3 19 048 10000 75 100 350 600 10 18 8.8 19042 20000 90 120 340 600 22 30 4.6 19032 Tj (° C) - the starting temperature of the decomposition Tmax (° C) - the maximum decomposition temperature Tf (° C) - the final temperature of the decomposition AW - Weight loss during the interval T, (° C) - T _raax (° C) x) - the unblocked copolymer with the encapsulated ferrite has two decomposition peaks, represented by the two values for Tmax and AW

The results indicate that the microparticles have a narrow size distribution ranging from 1 to 20 microns. Magnetic measurements show a decrease in saturation magnetization from 20.3 to 4.6 µm / g with increasing molecular weight of poly (ethylene glycol) from 4,000 to 20,000.

Claims (1)

CLAIM Process for the synthesis of a magnetic composite in the form of microplate copolymer diblock with combined characteristics of hydrophilicity and hydrophobicity and ferrite encapsulated in the polymeric matrix during synthesis, characterized in that it is copoly-condensed in a nitrogen atmosphere and in an organic environment of dimethylformamide 6.5 mole% relative to the polymer, in the presence of ferrite in proportion of 3.73% compared to the total amount of polymer, added as stabilized ferrofluid with a concentration of 3.205%, poly (succinimide) and poly (ethylene glycol) with molecular weight of: 4000, 10,000 or 20000, in a gravimetric ratio of 2: 1, at 140 ° C, under continuous stirring and nitrogen bubbling, in the presence of catalyst Mn (CH3 COO) ₂ 4 H ₂ O in aqueous solution, in proportion of 1, 5% compared to the total amount of polymer, added dropwise for 60 minutes and continuing the reaction for 6 hours at the same temperature, eliminating an amount of 10 ml water, cooling, washing with methanol, drying at 45 ° C and reduced pressure.