RU2561062C2 - Application of chitosan derivative for selective delivery of medications and biologically active complexes - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, veterinary and food industry and consists in the application of a chitosan derivative, modified by a residue of fatty or amino acid and anhydride of succinic acid as a part of the composition, which contains a medication or a biologically active complex, intended for the delivery of the said medication or the said biologically active complex into an organism. The invention also relates to the composition, which contains the medication or the biologically active complex, containing the said chitosan derivative.

EFFECT: invention makes it possible to provide the medication or the biologically active complex, which contains the chitosan derivative, with high trasndermal and membrane permeability, increased sorption capacity, soluble in a wide pH range.

40 cl, 7 dwg, 1 tbl, 2 ex

Description

The invention relates to the use of a chitosan derivative for the selective delivery of drugs and biologically active complexes and may find application in medicine, veterinary medicine, cosmetology, and the food industry.

BACKGROUND

It is known from the prior art that chitosan is used as a matrix carrier in a pharmaceutical composition for the controlled release of terbutaline sulfate (EA 013969 B1, TE YORDANIEN PHARMACEUTICAL MENUFEKCHURING KO., 30.08.2010).

The use of chitosan in a composition for intranasal administration for regulating the absorption rate of fentanyl is known (EA 008500 B1, ARKIMIDIZ DEVELOPMENT LIMITED, 06/29/2007).

It is known to use chitosan as a drug stabilizer for the photodynamic therapy of cancer to reduce general toxicity and increase the specific properties of the drug (EA 002318 B1, RESEARCH AND PRODUCTION REPUBLICAN UNITARY ENTERPRISE "DIALEC", 04/25/2002).

The use of a crosslinkable chitosan composition for delivering an active agent is known (RU 2482133 C2, VISKOGEL AB, 05.20.2013).

The use of chitosan succinate and dioxidine in an antibacterial complex with chlorhexidine is known (RU 2471477 C1, FSBEI HPE "VSU", 10.01.2013).

The use of carbodiimide-modified chitosan succinate is known to increase bactericidal activity (RU 2269542 C1, INTERNATIONAL INSTITUTE OF ECOLOGICAL AND TECHNOLOGICAL PROBLEMS, February 10, 2006).

It is known to use thiolated chitosan and N-trimethyl quaternized chitosan (TMS) as a carrier for the delivery of macromolecular polymer drugs (WO 2010041279 A2, VB MEDICARE PVT LTD, 04/15/2010).

The use of chitosan and its derivatives is known, including those precipitating in the pH range of 5.0-7.5, in the form of a conjugate with a drug for the prolonged release of a drug from a pharmaceutical composition (WO 2006087028 A1, JORDANIAN PHARMACEUTICAL MFG, 24.06. 2008).

It is known to use chitosan functionalized with succinate as a biocompatible polymer matrix for immobilizing a bioactive agent (US 2007077305 A1, LE TIEN C, 04/05/2007).

It is known to use an amphiphilic chitosan derivative in the form of a macromolecule for drug loading (US 2012153520 A1, LIU DEAN-MO, 06/21/2012).

It is known to use an amphiphilic chitosan derivative for the preparation of drug-loaded micelles (CN 102961322 A, HANGZHOU PUSHKANG BIOLOG SCIENCE & TECHNOLOGY CO LTD, 03/13/2013).

It is known to use an amphiphilic chitosan derivative for the manufacture of a medicament for targeting tumor cells (CN 102924625 A, UNIV CHINA PHARMA, 02.13.2013).

It is known to use an amphiphilic chitosan derivative for drug loading (CN 102775515 A, CHINESE ACAD INST CHEMISTRY, 11/14/2012).

It is known to use a macromolecular prodrug based on an amphiphilic chitosan derivative for targeted delivery to tumor cells (CN 102688498 A, UNIV SHANDONG, 09/26/2012).

It is known to use an amphiphilic chitosan derivative to produce a targeted contrast agent and anticancer drug (KR 20080019507 A, KOREA INST SCI & TECH, 03/04/2008).

The present invention is directed to the development of a new chitosan derivative for the delivery of drugs and biologically active complexes, preferably selective delivery.

SUMMARY OF THE INVENTION

Recently, considerable interest has been shown in nanostructured polymers. Due to the large specific surface, nanoparticles exhibit properties different from ordinary polymers and a significant increase in their characteristic. This opens up great opportunities for using them in various fields of science and technology, in particular when creating medicines. Such structures have a large affinity for the cell membrane, and their small dimensions make it easy to penetrate the cell nucleus.

Nanoparticles are mainly of interest as a vector for delivering biologically active material to a cell. Chitosan derivatives based nanoparticles meet the requirements for such systems. Due to the high charge density on the surface, they actively interact with the cell membrane. Particles are biocompatible with living body tissues, as do not cause allergic reactions and rejection, gradually biodegradable under the action of enzymes on amino sugars, which are completely absorbed in a living organism. They are non-toxic and are easily eliminated from the body without causing competitive adverse reactions. The effectiveness of most biologically active substances is often limited due to the inability to approach the target organ (the place of therapeutic action) while maintaining their original properties. The use of chitosan-based nanoparticles as a substrate for biologically active substances should ensure their targeted delivery, reduce the dose used, prolong the effectiveness of the action and provide the possibility of using for oral or nasal administration, as the most convenient ways for introduction into a living organism. It is known that the introduction of hydrophobic groups into the chitosan molecule leads to an increase in the stability of colloidal systems obtained on their basis.

The present invention is to obtain a new nanostructured derivative of chitosan oligosaccharide with high transdermal and membrane permeability, increased sorption capacity, containing a hydrophobic and hydrophilic ligand, as well as soluble in a wide pH range in which the chitosan fragment has the general formula (I), and its use for drug delivery and biologically active complexes, mainly selective delivery.

Where

R is the residue of a fatty or amino acid,

n is from about 12 to about 25% relative to the amount of chitosan monosaccharide residues,

m is from about 30 to about 60% relative to the amount of chitosan monosaccharide residues.

The problem is solved by nanostructured chitosan, modified fatty acid residues and succinic anhydride having a working area (soluble in a wide pH range) pH from about 2 to about 9, preferably from about 4 to about 9, more preferably from about 6 to about 9, most preferably from about 7 to about 8, for example, about 7.4, where chitosan is represented by a spherical structure with a particle size of from about 1 nm to about 2000 nm, preferably from about 1 nm to about 1500 nm, more preferably from about 1 nm to about 900 nm, still b more preferably from about 1 nm to about 400 nm, most preferably from about 1 nm to about 100 nm.

The problem is also solved by nanostructured chitosan, a modified amino acid and succinic acid anhydride having a working area (soluble in a wide pH range) pH from about 2 to about 9, preferably from about 4 to about 9, more preferably from about 6 to about 9, most preferably from about 7 to about 8, for example about 7.4, where chitosan is represented by a spherical structure with a particle size of from about 1 nm to about 2000 nm, preferably from about 1 nm to about 1500 nm, more preferably from about 1 nm to about 900 nm, more preferably from about 1 nm to about 400 nm, most preferably from about 1 nm to about 100 nm.

To obtain an amphiphilic chitosan derivative having a hydrophilic ligand content of from about 12% to about 25%, preferably from about 15% to about 25%, more preferably from about 18% to about 25%, most preferably from about 20% to about 25% and the hydrophobic ligand content is from about 30% to about 60%, preferably from about 35% to about 55%, more preferably from about 40% to about 50%, most preferably from about 45% to about 50%, the starting chitosan with molecular weight (MM) from about 5 kDa to about 100 kDa, preferably from about a count of 7 kDa to about 80 kDa, more preferably from about 10 kDa to about 60 kDa, most preferably from about 30 kDa to about 50 kDa, for example, about 23 kDa.

As a hydrophilic ligand, various chemical compounds with a specific set of properties and their derivatives can be used.

Examples of fatty acids are caprylic, capric (decanoic), palmitic (saturated fatty acids) and oleic (unsaturated fatty acid).

To give the desired properties, two ligands are sequentially attached to the polysaccharide: a fatty or amino acid residue to ensure the bearing properties and binding affinity for the living cell membrane, and succinic acid residue to ensure solubility in a wider pH range, including physiological values (pH about 7.4 )

Thus, the present invention relates to the use of a chitosan derivative in which the chitosan fragment has the general formula (I)

Where

R is the residue of a fatty or amino acid,

n for a hydrophilic ligand is from about 12 to about 25% relative to the amount of chitosan monosaccharide residues,

m for a hydrophobic ligand is from about 30 to about 60% relative to the amount of monosaccharide residues of chitosan,

for the manufacture of a medicament or biologically active complex.

The drug is intended for selective delivery to the body.

The drug is intended for selective delivery to the human body.

The biologically active complex is intended for selective delivery to the body.

The biologically active complex is intended for selective delivery to the human body.

The specified fatty acid residue is a caprylic acid residue.

The specified amino acid residue is a valine residue.

The specified derivative is nanostructured.

The specified derivative has transdermal and / or membrane permeability.

The specified derivative has an increased sorption capacity.

The specified derivative is soluble at a pH of from about 2 to about 9, preferably from about 4 to about 9, more preferably from about 6 to about 9, most preferably from about 7 to about 8.

The specified derivative is soluble at a pH of about 7.4.

The specified derivative has a spherical structure with a particle size of from about 1 nm to about 2000 nm, preferably from about 1 nm to about 1500 nm, more preferably from about 1 nm to about 900 nm, even more preferably from about 1 nm to about 400 nm, most preferably from about 1 nm to about 100 nm.

Said derivative has a hydrophilic ligand content of preferably from about 15% to about 25%, more preferably from about 18% to about 25%, most preferably from about 20% to about 25%.

Said derivative has a hydrophobic ligand content of preferably from about 35% to about 55%, more preferably from about 40% to about 50%, most preferably from about 45% to about 50%.

The starting chitosan of said derivative has a molecular weight of from about 5 kDa to about 100 kDa, preferably from about 7 kDa to about 80 kDa, more preferably from about 10 kDa to about 60 kDa, most preferably from about 30 kDa to about 50 kDa.

The starting chitosan of said derivative has a molecular weight of about 23 kDa.

The starting chitosan of said derivative has a deacetylation degree of 85 + 2%.

The specified derivative is associated with the cell.

The specified derivative is biodegradable.

Thus, the present invention also relates to a composition of a medicament or biologically active complex containing a chitosan derivative in which the chitosan fragment has the general formula (I)

Where

R is the residue of a fatty or amino acid,

n for a hydrophilic ligand is from about 12 to about 25% relative to the amount of chitosan monosaccharide residues,

m for a hydrophobic ligand is from about 30 to about 60% relative to the amount of chitosan monosaccharide residues.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 (1-2). The component composition of the samples used in experimental tests.

FIG. 2 (3-4). The component composition of the samples used in experimental tests.

Figure 3. Analysis of the biological aspects of the obtained chitosan nanoparticles for biocompatibility, cell binding and biodegradation.

Figure 4. Analysis of the biological aspects of the obtained chitosan nanoparticles for biocompatibility, cell binding and biodegradation.

Figure 5. Analysis of the biological aspects of the obtained chitosan nanoparticles for biocompatibility, cell binding and biodegradation.

6. Analysis of the biological aspects of the obtained chitosan nanoparticles for biocompatibility, cell binding and biodegradation.

7. Analysis of the biological aspects of the obtained chitosan nanoparticles for biocompatibility, cell binding and biodegradation.

DETAILED DESCRIPTION OF THE INVENTION

In the presented examples, a chitosan oligosaccharide fraction with an average molecular mass of about 23 kDa and a deacetylation degree of 85 ± 2% (manufactured by Sigma Co.) was used.

A method of producing a chitosan derivative comprises the following steps:

1. The interaction of a fatty acid with N-hydroxysuccinimide in the presence of N, N′-dicyclohexylcarbodiimide to produce an N-oxuccinimide ester of a fatty acid.

N-Oxisuccinimide was dissolved in 10.5 ml of dry zyl acetate, a solution of CDI (N, N′-dicyclohexylcarbodiimide) in 6.3 ml of ethyl acetate and fatty acid were added. The solution was stirred at RT (room temperature) for 15 hours. The formed urea was separated by filtration, ethyl acetate was evaporated on a rotary evaporator, and N-oxysuccinimide fatty acid ester was obtained as an oil, which was stored in a refrigerator.

2. Acylation of (25/100 MO (molecular residues)) of chitosan with N-oxysuccinimide fatty acid ester.

Chitosan was dissolved in 1.05 ml of 0.1% acetic acid with the addition of 0.1 ml of 0.5% acetic acid and 2 ml of methanol. A solution of a fatty acid N-oxysuccinimide ester in 0.53 ml of methanol was added to the solution. The solution was stirred at RT for 12 hours. Methanol was evaporated on a rotary evaporator. The product was air dried.

3. Carboxy-acylation (30/100) with chitosan succinic anhydride, acylated with (25/100 MO) N-oxysuccinimide fatty acid ester.

Chitosan, acylated (25/100 MO) with N-oxysuccinimide fatty acid ester, was dissolved in 1 ml of 0.1% acetic acid with the addition of 4 ml of methanol. Succinic anhydride was added to the solution. The solution was stirred at RT for 12 hours. Upon precipitation with acetone, an almost instantaneous precipitation of a reddish precipitate was observed. The product was separated in a centrifuge. Air dried

Alternatively, when using an amino acid as a hydrophobic ligand, the method comprises the following steps:

1. Obtaining N-oxysuccinimide ester of an amino acid by reacting the amino acid with HBTU (N, N, N ′, N′-tetramethyl-O- (1H-benzotriazol-1-yl) uronium hexafluorophosphate - N, N, N ′, N′-tetramethyl -O- (1H-benzotriazol-1-yl) uronium hexafluorophosphate)

Interactions with HOBt (hydroxybenzotriazole)

N-Oxisuccinimide was dissolved in 10.5 ml of dry ethyl acetate, a solution of CDI in 6.3 ml of ethyl acetate and an amino acid were added. The solution was stirred at RT for 15 hours. The resulting urea was filtered off, ethyl acetate was evaporated on a rotary evaporator to give the amino acid N-oxysuccinimide ester as an oil, which was stored in a refrigerator.

2. Acylation of (25/100 MO) chitosan with an amino acid N-oxysuccinimide ester.

Chitosan was dissolved in 1.05 ml of 0.1% acetic acid with the addition of 0.1 ml of 0.5% acetic acid and 2 ml of methanol. A solution of an amino acid N-oxysuccinimide ester in 0.53 ml of methanol was added to the solution. The solution was stirred at RT for 12 hours. Methanol was evaporated on rotary evaporators. The product was air dried.

3. Carboxy-acylation (30/100) with chitosan succinic anhydride, acylated (25/100 MO) with an amino acid N-oxysuccinimide ester.

The amino acids chitosan acylated (25/100 MO) with N-oxysuccinimide ester were dissolved in 3 ml of 0.1% acetic acid with the addition of 4 ml of methanol. Succinic anhydride was added to the solution. The solution was stirred at RT for 12 hours. During precipitation with acetone, an almost instantaneous precipitation of a white precipitate was observed.

Removal of the Fmoc-protecting group is carried out by standard methods under standard conditions (T = 40 minutes in DMF: piperidine 1: 1 in a water bath at 40 ° C); the product is washed with acetone and freeze-dried.

Table 1. The composition of chitosan matrices. Cipher The content of fatty acid residues per 100 monosaccharide residues Succinic acid content per 100 monosaccharide residues R1 25 thirty R2 12 thirty R3 25 60

The matrices were evaluated using a 90 Plus S / N dynamic light scattering device, Brookhaven Instruments Corporation, USA.

The automatic function 90 Plus / BI-MAS was used to determine the particle size (diameter), and ZetaPlus and 90 Plus were used to determine the zeta potential (total charge on the particle surface).

The measurements were carried out at a solution pH of 5.0 and t = 25 ° C

The particle size (diameter) was determined for samples R2 and R3 (see above) in accordance with FIGS. 1 (1-2) and 2 (3-4), respectively.

Building a model of the resulting particles.

From the beginning of the study, chitosan of molecular weight of about 25 kDa was used to obtain particles. It was estimated that the number of monosaccharide units in a molecule of this weight is about 156. Using spatial molecular modeling programs, it was measured that the length in space of a chitosan polymer of this length would be about 70 nm.

Accordingly, for sample R2, with an experimental particle diameter of 458 nm according to the formula l = 2 pr, where l is the circumference and r is the radius of the sphere of the nanoparticle, l = 1438 nm. Thus, the circumference of these spherical nanoparticles, on average, is equal to 1438 nm. A spherical particle of 25 kDa polymer chitosan used here with such a circle can exist, and is a sphere with a circle formed by, on average, 21 polymer chitosan chains.

We do not doubt that the matrices R2 form precisely the sphere or a polygon closely approximated by the sphere. The spherical shape is an energetically low form of existence in solution, and there are no structural features of the R2 matrices that strongly change the energy preferences in their formation of a sphere in the solution.

For R2, recalculating the NMR data obtained for 100 monosaccharides (Fig. 1 (1-2)) for the studied polymer of 156 monosaccharides, we find that each of such 21 polymer chains of the circumference of the nanoparticle contains 18 chains of fatty caprylic acid inward of the nanoparticle and 45 succinyl chains facing the solution.

Similarly, calculations were performed for nanoparticles of sample R3. Particles of chitosan MM 25 kDa and experimentally obtained radius of 314 nm can also exist. Moreover, for this sample, the circumference of the nanoparticle-sphere is 986 nm, and it is formed by 14 chitosan polymers of the indicated MM. One chitosan polymer (156 monosaccharides) of sample R3 contains about 38 caprylic residues and 90 succinyl residues.

The results of measurements of the zeta potential (on the example of sample R3) showed that succinyl chains, as expected, go to the outer surface of the particle, contributing a negative charge to the total surface charge and ensuring the solubility of the particles at physiological pH 7.4. According to the iterations, the results on the surface charge of particles as -7, -5, -6, -1 mV were obtained. The surface charge of chitosan nanoparticles not modified with succinic acid is in the range from +25 to +54 mV [11] or from +22 to +28 mV (in this study, below). All the given zeta potentials were measured for solutions of nanoparticles pH 5.0-5.5.

Analysis of the biological aspects of the obtained chitosan nanoparticles: biocompatibility, cell binding and biodegradation.

Preparation of cells for analysis of biological aspects.

We used A431 human epidermoid carcinoma cell lines, NaCaT human keratinocytes, and BT-474 human breast carcinomas. The NaCaT and BT-474 cell lines were cultured on DMEM. The remaining cell lines were cultured on RPMI-1640 medium. Fetal bovine serum (FBS) 8%, L-glutamine 300 μg / ml, antibiotics ampicillin / streptomycin 50 μg / ml, 2-mercaptoethanol 5 × 10E-5M were added to the media. Cells were cultured in a CO2 incubator.

Indices of inhibition of cell proliferation, II, increasing titrations of nanoparticles on cell lines HEK-T, K562 and Wnt1204 are shown in Figure 3. Concentrations (titration; along the abscissa) of the reagent particles are measured in μg / ml.

Analysis of nanoparticles for binding to cells and biodegradation.

Binding and biodegradation were observed using confocal microscopy using an Eclipse E2000 confocal microscope from Nikon (Japan). The microscope is equipped with 405, 488 and 514 nm lasers.

To observe chitosan nanoparticles and, for comparison, molecular chitosan using confocal microscopy, we labeled them with the FITC fluorescent dye (fluorescein derivative - isothiocyanate). During the reaction, FITC is attached to molecular chitosan or as part of nanoparticles. The reaction of 50-100 μg / ml of chitosan or chitosan particles and 500 μg / ml of FITC was carried out in an acidic medium for 3 hours at room temperature in the dark. After the reaction, the preparations were dialyzed overnight in a 0.1% acetic acid solution to remove unbound FITC. After dialysis, the preparations were centrifuged for 10 minutes. at 2000 rpm to remove the formed insoluble precipitate. The supernatant was transferred to a new tube and used for experiments with cells.

The selected FITC ratio for chitosan and particles was used so that the luminescence of chitosan and particles on a confocal microscope was approximately adequate to their size and there was no excess fluorescence in the eye. With this ratio, the addition of FITC molecules occurs for every 2nd chitosan monosaccharide in the polymer. The software used by the confocal microscope has a so-called built-in ruler with which you can measure the size of particles or agglomerates. It must be said that with all the relative accuracy of this method in determining the particle size, the sizes we observed with a confocal microscope were the same as those described by light scattering, described above.

After trypsinization, the cells were transferred to 6-well plates with sterile coverslips embedded in them. Cells were grown to confluent monolayer in a nutrient medium. Preparations of molecular or chitosan nanoparticles were introduced into the culture medium and incubated at 37 ° C for 1 to 48 hours. For the last 30 minutes, the nuclear (blue) dye H33342 (Invitrogen) 10 μg / ml was added. After that, 4% paraformaldehyde was added to the culture medium to a final concentration of 1% to fix the cells. Cells were fixed for 30 min at 37 ° C, after which the medium was washed 2 times in phosphate buffer for 5 minutes. The glasses were dried and glued onto a Mowiol 4.88 resin on slides with the cells down.

An hourly incubation of cells (with chitosan nanoparticles of type R2: with 12 mol% residues of fatty caprylic acid and 30 mol% residues of succinic acid) showed that they already penetrate into the cell in 1 hour in accordance with Figure 5. The diameter of the nanoparticles, estimated using the built-in rulers: 0.4 μm, i.e. 4 00 nm, i.e. same as measured using the light scattering method (see above).

Visualization of the binding of FITC-labeled nanoparticles to A431 cells is shown in FIG. 4.

The cell nucleus fluoresces blue. It can be seen that the particles penetrated into the cell and are grouped around the cell nucleus. Magnification 2000x and 5000x and 15000x. Particle sizes 0.3-0.4 microns.

For the analysis of biodegradation of nanoparticles, a prolonged 24- and 48-hour incubation of FITC-labeled preparations with A431, BT-474 and NaCaT cells was used. After 24 hours, the glow decreased significantly, and after 48 hours it practically disappeared.

Depending on the chitosan matrices R1, R2 or R3 used in their composition (see Table 1). According to the pictures from the confocal microscope, in accordance with this, we came to the preliminary conclusion that penetration into the cell, and, accordingly, then biodegradation in the cell for R3 matrices takes place in a slightly shorter time interval than for R1 and R2 matrices.

After 4-hour incubation (Figure 5, Visualization of the binding to NaCaT cells after 4-hour incubation: left of the R3 matrices, right of the R2 matrices. 4000x magnification), it is seen that biodegradation is already taking place for R3 (left). The FITC glow is scattered rather than compactly in nanoparticles. Polymer chitosan is split into individual monosaccharides with an attached FITC fluorescent molecule to each, which are evenly distributed throughout the cell cavity. And for R2 (right) nanostructures are still clearly visible.

After an 8-hour incubation (Fig. 6, Visualization of binding to CHO-RFP cells after an 8-hour incubation: on the left of the R3 matrices, on the right of the R1 matrices. 1000x magnification), it can be seen that for R3 (left) biodegradation has already passed (only glow artifact FITC molecules that did not contact the test), and for R1 (right), biodegradation of chitosan particles continues - the green FITC glow is distributed around the cell nuclei (blue glow).

Figures 7 respectively (Visualization of binding to CHO-RFP cells after 8-hour incubation: left of R1 matrices, right of R2 matrices. 2000x magnification) are shown to show that after 8 hours of incubation, R1 or R2 particles still continue to biodegrade equally from R3 whose biodegradation is over.

EXAMPLES

EXAMPLE 1

Obtaining N-oxysuccinimide ester, caprylic acid.

N-Oxisuccinimide (367 mg, 3.18 mmol) was dissolved in 10.5 ml of dry ethyl acetate, a solution of CDI (639 mg, 3.18 mmol) in 6.3 ml of ethyl acetate and caprylic acid (458 mg, 3.18 mmol) were added. ) The solution was stirred at RT for 15 hours. The urea formed was filtered off, ethyl acetate was evaporated on a rotary evaporator to give caprylic acid N-oxysuccinimide ester as an oil, which was stored in a refrigerator.

Acylation of (25/100 MO) chitosan with N-oxysuccinimide ester of caprylic acid.

Chitosan (23 mg, 1 mmol) was dissolved in 1.05 ml of 0.1% acetic acid with the addition of 0.1 ml of 0.5% acetic acid and 2 ml of methanol. A solution of caprylic acid N-oxysuccinimide ester (7.3, mg, 30 mmol) in 0.53 ml of methanol was poured into the solution. The solution was stirred at RT for 12 hours. Methanol was evaporated on rotary evaporators. The product was air dried.

Carboxy-acylation (30/100) with chitosan succinic anhydride (25/100 MO) acylated N-oxysuccinimide ester of caprylic acid.

Chitosan acylated (25/100 MO) with caprylic acid N-oxysuccinimide ester (29.7 mg, 0.7 mmol) was dissolved in 1 ml of 0.1% acetic acid with the addition of 4 ml of methanol. Succinic anhydride (3.7 mg, 36.9 mmol) was added to the solution. The solution was stirred at RT for 12 hours. Upon precipitation with acetone, an almost instantaneous precipitation of a reddish precipitate was observed. The product was separated in a centrifuge. Dried in the air.

EXAMPLE 2

Obtaining N-oxysuccinimide ester of Nα-tert-butoxycarbonyl-L-valine.

N-Oxisuccinimide (367 mg, 3.18 mmol) was dissolved in 10.5 ml of dry ethyl acetate, a solution of CDI (639 mg, 3.18 mmol) in 6.3 ml of ethyl acetate and Nα-tert-butoxycarbonyl-L-valine ( 690 mg, 3.18 mmol). The solution was stirred at RT for 15 h. The urea formed was filtered off, ethyl acetate was evaporated on a rotary evaporator, and Nα-tert-butoxycarbonyl-L-valine N-oxysuccinimide ester was obtained as an oil, which was stored in a refrigerator.

Acylation (25/100 MO) of chitosan with N-oxysuccinimide ester of Nα-tert-butoxycarbonyl-L-valine.

Chitosan (23 mg, 1 mmol) was dissolved in 1.05 ml of 0.1% acetic acid with the addition of 0.1 ml of 0.5% acetic acid and 2 ml of methanol. A solution of Nα-tert-butoxycarbonyl-L-valine N-oxysuccinimide ester (9.48 mg, 30 mmol) in 0.53 ml of methanol was added to the solution. The solution was stirred at RT for 12 hours. Methanol was evaporated on rotary evaporators. The product was air dried.

Carboxy acylation (30/100) with chitosan succinic anhydride of acylated (25/100 MO) N-oxysuccinimide ester of Nα-tert-butoxycarbonyl-L-valine

Chitosan acylated (25/100 MO) N-oxysuccinimide Nα-tert-butoxycarbonyl-L-valine (20.7 mg, 0.7 mmol) was dissolved in 3 ml of 0.1% acetic acid with the addition of 4 ml of methanol. Succinic anhydride (3.7 mg, 36.9 mmol) was added to the solution. The solution was stirred at KT for 12 hours. During precipitation with acetone, an almost instantaneous precipitation of a white precipitate was observed. The product was separated in a centrifuge. Dried in the air.

Claims (40)

1. The use of a derivative of chitosan, in which the chitosan fragment has the General formula (I)

Where
R is the residue of a fatty or amino acid,
n for a hydrophilic ligand is from about 12 to about 25% per 100 monosaccharide chitosan residues in the chain,
m for a hydrophobic ligand is from about 30 to about 60% per 100 monosaccharide chitosan residues in the chain,
in the composition containing a drug or biologically active complex, intended for delivery to the body of the specified medicinal product or the specified biologically active complex. 2. The use according to claim 1, characterized in that the drug is intended for selective delivery to the body. 3. The use according to claim 2, characterized in that the drug is intended for selective delivery into the human body. 4. The use according to claim 1, characterized in that the biologically active complex is intended for selective delivery to the body. 5. The use according to claim 4, characterized in that the biologically active complex is intended for selective delivery into the human body. 6. The use according to claim 1, characterized in that said fatty acid residue is a caprylic acid residue. 7. The use according to claim 1, characterized in that said amino acid residue is a valine residue. 8. The use according to claim 1, characterized in that said derivative is nanostructured. 9. The use according to claim 1, characterized in that said derivative has transdermal and / or membrane permeability. 10. The use according to claim 1, characterized in that said derivative has an increased sorption capacity. 11. The use of claim 1, wherein said derivative is soluble at a pH of from about 2 to about 9, preferably from about 4 to about 9, more preferably from about 6 to about 9, most preferably from about 7 to about 8. 12. The use according to claim 11, characterized in that said derivative is soluble at Ph about 7.4. 13. The use according to claim 1, characterized in that said derivative has a spherical structure with a particle size of from about 1 nm to about 2000 nm, preferably from about 1 nm to about 1500 nm, more preferably from about 1 nm to about 900 nm, even more preferably from about 1 nm to about 400 nm, most preferably from about 1 nm to about 100 nm. 14. The use according to claim 1, characterized in that said derivative has a hydrophilic ligand content of preferably from about 15% to about 25% per 100 chitosan monosaccharide residues in a chain, more preferably from about 18% to about 25% per 100 chitosan monosaccharide residues in the chain, most preferably from about 20% to about 25% per 100 monosaccharide residues of chitosan in the chain. 15. The use according to claim 1, characterized in that said derivative has a hydrophobic ligand content of preferably from about 35% to about 55% per 100 chitosan monosaccharide residues in the chain, more preferably from about 40% to about 50% per 100 chitosan monosaccharide residues in the chain, most preferably from about 45% to about 50% per 100 monosaccharide residues of chitosan in the chain. 16. The use according to claim 1, characterized in that the starting chitosan of said derivative has a molecular weight of from about 5 kDa to about 100 kDa, preferably from about 7 kDa to about 80 kDa, more preferably from about 10 kDa to about 60 kDa, most preferably from about 30 kDa to about 50 kDa. 17. The use according to claim 16, characterized in that the starting chitosan of said derivative has a molecular weight of about 23 kDa. 18. The use according to claim 1, characterized in that the starting chitosan of said derivative has a deacetylation degree of 85 ± 2%. 19. The use according to claim 1, characterized in that said derivative is binding to the cell. 20. The use according to claim 1, characterized in that said derivative is biodegradable. 21. A composition for delivering to the body a drug or biologically active complex containing a chitosan derivative in which the chitosan fragment has the general formula (I)

Where
R is the residue of a fatty or amino acid,
n for a hydrophilic ligand is from about 12 to about 25% per 100 monosaccharide chitosan residues in the chain,
m for a hydrophobic ligand is from about 30 to about 60% per 100 monosaccharide chitosan residues in the chain,
and said drug or said biologically active complex. 22. The composition according to p. 21, characterized in that the drug is intended for selective delivery to the body. 23. The composition according to p. 22, characterized in that the drug is intended for selective delivery into the human body. 24. The composition according to p. 21, characterized in that the biologically active complex is intended for selective delivery to the body. 25. The composition according to p. 24, characterized in that the biologically active complex is intended for selective delivery into the human body. 26. The composition according to p. 21, characterized in that said fatty acid residue is a caprylic acid residue. 27. The composition of claim 21, wherein said amino acid residue is a valine residue. 28. The composition according to p. 21, characterized in that said derivative is nanostructured. 29. The composition according to p. 21, characterized in that said derivative has transdermal and / or membrane permeability. 30. The composition according to p. 21, characterized in that said derivative has an increased sorption capacity. 31. The composition according to p. 21, characterized in that the derivative is soluble at a pH of from about 2 to about 9, preferably from about 4 to about 9, more preferably from about 6 to about 21, most preferably from about 7 to about 8. 32. The composition according to p. 31, characterized in that the derivative is soluble at a pH of about 7.4. 33. The composition according to p. 21, characterized in that the derivative has a spherical structure with a particle size of from about 1 nm to about 2000 nm, preferably from about 1 nm to about 1500 nm, more preferably from about 1 nm to about 900 nm, even more preferably from about 1 nm to about 400 nm, most preferably from about 1 nm to about 100 nm. 34. The composition according to p. 21, characterized in that the derivative has a hydrophilic ligand content of preferably from about 15% to about 25% per 100 monosaccharide residues of chitosan in the chain, more preferably from about 18% to about 25% per 100 monosaccharide residues of chitosan in the chain, most preferably from about 20% to about 25% per 100 monosaccharide residues of chitosan in the chain. 35. The composition according to p. 21, characterized in that the derivative has a hydrophobic ligand content, preferably from about 35% to about 55% per 100 monosaccharide residues of chitosan in the chain, more preferably from about 40% to about 50% per 100 monosaccharide residues of chitosan in the chain, most preferably from about 45% to about 50% per 100 monosaccharide residues of chitosan in the chain. 36. The composition according to p. 21, characterized in that the starting chitosan of said derivative has a molecular weight of from about 5 kDa to about 100 kDa, preferably from about 7 kDa to about 80 kDa, more preferably from about 10 kDa to about 60 kDa, most preferably from about 30 kDa to about 50 kDa. 37. The composition according to p. 36, characterized in that the starting chitosan of said derivative has a molecular weight of about 23 kDa. 38. The composition according to p. 21, characterized in that the starting chitosan of said derivative has a deacetylation degree of 85 ± 2%. 39. The composition according to p. 21, characterized in that the derivative is associated with the cell. 40. The composition according to p. 21, characterized in that the said derivative is biodegradable.

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