CN103142469A - Application of using amphiphilic N-long-chain alkyl-N-arginine chitosan as solubilizing and absorption prompting carrier of gambogic acid - Google Patents

Abstract

The invention discloses a natural and biodegradable N-long-chain alkyl-N-arginine chitosan derivative as well as a micellar preparation method thereof and application of using the natural and biodegradable N-long-chain alkyl-N-arginine chitosan derivative as a solubilizing and absorption prompting nanometer carrier of gambogic acid which is difficult to dissolve by being taken orally and difficult to absorb.

Description

Application of Amphiphilic N-Long Chain Alkyl-N-Arginine Chitosan as Carrier for Solubilization and Absorption of Gambogic Acid

technical field

The invention relates to the use of amphiphilic N-long-chain alkyl-N-arginine chitosan derivatives (OACS) as solubilization and absorption-promoting nano-carriers of insoluble drug gambogic acid (GA), to prepare a nano-carrier with high solubility , Nano-drugs with good absorption of GA-OACS.

Background technique

Chitosan is the only alkaline polysaccharide in nature. It is a cationic polymer with bioadhesiveness, which can prolong the residence time of drugs on the surface of biological mucosa. Negatively charged cell membranes interact to enhance membrane permeability, thereby enhancing the absorption of drugs by the mucosa. The permeability-enhancing effect of chitosan is likely to be due to its significant influence on membrane proteins. The positive charge interacts with the open structure of the tight junction between cells, making the cytoskeleton protein change from filamentous to spherical, thus making macromolecular drugs Transmembrane absorption can be carried out through the paracellular pathway (van der Lubben IM, VerhoefJC, Borchard G, et al. Chitosan and its derivatives in mucosal drug and vaccine delivery [J]. Eur JPharm Sci. 2001, 14 (3): 201-207; Wang Zhiying, Zhang Qiang. Mechanism of penetration enhancer on pulmonary penetration of peptide drugs. Acta Pharmaceutica Sinica, 2003, 38(12): 957-961; Liu Mei, Chen Xiaoping. Chitosan and its derivatives in peptides Application of similar oral drug delivery system. Foreign Medical Pharmacy, 2000, 27(6): 351-354).

Gambogic acid (Gambogic Acid, GA) is an active ingredient extracted from the traditional Chinese medicine Gambogic. The experimental results show that GA can inhibit many common human tumors such as lung cancer, liver cancer, breast cancer, gastric cancer, melanoma, glioma, prostate cancer and leukemia, and has high selectivity for tumor cells within the effective dose range, and has high selectivity for normal animals. The hematopoietic system and immune function are not significantly affected, and the toxic and side effects are low. However, due to the low solubility of GA (<1 μg/mL), there is currently a patent to use L-arginine (GA-Arg) and gambogic acid to form a complex to prepare an aqueous solution of gambogic acid for intravenous injection (Dai, J.G. (2003 ).The preparation of a kind of gambogic acid injection.CHN Patent CN03131511.9.; Jin, B., Dong, H., and Qiao, L.(2003).The preparation of a kind of gambogic acid injection.CHN Patent CN02124510 .X.; You, Q.D., Guo, Q.L., Ke, X., Xiao, W., Dai, L.L., and Lin, Y. (2003).The preparations of gambogic acid and its compound.CHN Patent CN03132386.3.) .

In recent years, there have been many studies on the promotion of oral absorption of biomacromolecules and gene transfection by penetrating peptides. Membrane-penetrating peptides usually refer to a class of short peptides that have the ability to penetrate cell membranes and transport drugs into cells, usually less than 30 amino acid residues. These peptides have a common feature that they are all positively charged, that is, generally rich in alkali amino acids such as arginine or lysine. The guanidine group of arginine and the phospholipid of the lipid bilayer can form hydrogen bonds. The formation of hydrogen bonds and the strong basicity (pKa～12.5) of the guanidine group play a very important role in the control of cell membrane transport, but arginine The detailed permeation mechanism of acid is still unclear (Umezawa N, Gelman M A, Haigis M C, et al.Translocation of a beta-peptide across cell membranes.J Am ChemSoc, 2002,124(3):368-369; Dun Wan, Zhang Hailing, Bai Jingen, et al. Study on improving gene transfection efficiency by arginine-modified chitosan. Science Bulletin, 2007, 52: 2199-2205).

At present, polymer micelles are a hotspot in pharmacy research, which can be biodegradable and form drug nanocarriers. Polymer micelles are molecular ordered aggregates with hydrophobic groups as the inner core and hydrophilic groups as the outer shell. It can solubilize hydrophobic drugs, and the design of the particle size and surface characteristics of polymer micelles can help Avoid the recognition of the reticuloendothelial system and prolong the time of systemic circulation; some charged polymer micelles can protect the activity of genes, vaccines and protein drugs, and can be used as an oral delivery system for biomacromolecular drugs.

With reference to the above research results, if a certain amount of arginine can be introduced into chitosan as hydrophilic and a certain amount of hydrophobic groups can be introduced at the same time to form polymer micelles with a certain particle size and drug loading capacity, due to its The hydrophilic group is arginine which has excellent affinity and membrane penetration with phospholipid bilayers, and it has been reported in patents that arginine is often used to solubilize gambogic acid, so the prepared N-long Compared with traditional polymer micelles, alkanyl-N-arginine chitosan derivatives (OACS) nano drug-loaded micelles must be able to increase the solubility of gambogic acid in water to a greater extent, and can also utilize fine The transmembrane effect of amino acid improves the absorption effect of gambogic acid and achieves better therapeutic effect.

Contents of the invention

The technical problem to be solved in the present invention is: use biodegradable chitosan of natural origin as raw material, carry out chemical structure modification, make it form the polymer molecule with amphiphilicity, namely one end contains hydrophilic group, and this hydrophilic group The water base is arginine with good affinity and penetrability to the cell membrane; the other end contains a lipophilic group and is biodegradable polymer in vivo, which is suitable for encapsulating drugs to form nano-polymer micelles.

In order to solve the above technical problems, the present invention provides technical solutions as follows.

Amphiphilic N-long chain alkyl-N-arginine chitosan derivatives represented by the general formula:

In the structural formula, k, m, n and i are integers greater than zero, n>k≥m, and i is an integer between 5 and 11. Described amphiphilic N-long chain alkyl N-arginine chitosan derivative is characterized in that: chitosan in amphiphilic N-long chain alkyl-N-arginine chitosan derivative The molecular weight of the sugar is between 5,000-50,0000, the substitution degree of arginine is 1%-50%, and the substitution degree of long-chain alkyl is 1%-50%.

The preparation method of described amphiphilic N-long-chain alkyl-N-arginine chitosan derivative is characterized in that it is prepared using the following method:

(1) Add chitosan to acetic acid aqueous solution, stir at room temperature, add long-chain alkylaldehyde with 6 to 12 carbons, react for 3 to 6 hours, add NaBH ₄ aqueous solution, and continue to stir for 8 to 14 hours to obtain N -Long-chain alkyl chitosan; the reaction solution that generates N-long-chain alkyl chitosan is neutralized with sodium hydroxide solution, suction filtered, washed with ethanol and water, and dried;

(2) Take N-long-chain alkyl chitosan and add it to aqueous acetic acid solution, stir at room temperature, add arginine, NHS and EDC in turn, and react for 42 to 50 hours to generate N-long-chain alkyl-N-arginine Acid chitosan, the reaction solution was dialyzed with a dialysis bag (MWCO3500), and freeze-dried to obtain amphiphilic N-long chain alkyl-N-arginine chitosan powder.

(3) Take N-long-chain alkyl chitosan and add it to acetic acid aqueous solution, stir at room temperature, add BOC anhydride-protected arginine, NHS and EDC in turn, and react for 42 to 50 hours to generate N-long-chain alkyl- N-arginine chitosan, add more than 5% trifluoroacetic acid for deprotection, the reaction solution is dialyzed with a dialysis bag (MWCO3500), freeze-dried to obtain amphiphilic N-long chain alkyl-N-arginine Chitosan powder.

The use of the amphiphilic N-long chain alkyl-N-arginine chitosan derivatives is to be able to entrap drugs to form micelles, and at the same time improve the bioavailability of oral refractory drugs, so as to achieve the required for clinical treatment.

The preparation method of the described amphiphilic N-long-chain alkyl-N-arginine chitosan drug-loaded micelles is characterized in that: after dissolving the oral difficult-to-absorb drug with a pharmaceutically acceptable solvent, and claim The N-long-chain alkyl-N-arginine chitosan described in 1-3 is mixed, subjected to room temperature ultrasonic treatment, and freeze-dried to obtain a polymer micelle containing a therapeutically effective amount of medicine with a particle size of 50-800nm freeze-dried powder.

The described amphiphilic N-long-chain alkyl-N-arginine chitosan drug-loaded micelles is characterized in that the drug-loaded polymer micelles freeze-dried powder is directly filled in ordinary gelatin capsules or enteric-coated Capsules prepared in capsules.

The described amphiphilic N-long-chain alkyl-N-arginine chitosan drug-loaded micelle is characterized in that after the lyophilized powder of the drug-loaded polymer micelle is added with pharmaceutically acceptable excipients, it is filled in Capsules prepared in ordinary gelatin capsules or enteric-coated capsules.

The described amphiphilic N-long-chain alkyl-N-arginine chitosan drug-loaded micelle is characterized in that after the lyophilized powder of the drug-loaded polymer micelle is added with pharmaceutically acceptable excipients, it is directly compressed to form into tablets.

The described amphiphilic N-long-chain alkyl-N-arginine chitosan drug-loaded micelle is characterized in that the lyophilized powder of the drug-loaded polymer micelle is added into pharmaceutically acceptable adjuvants and then compressed into granules. into tablets.

Graphical synthesis route of N-long-chain alkyl-N-arginine chitosan:

The nano-micelle is characterized in that it is mainly composed of gambogic acid and amphiphilic N-long chain alkyl-N-arginine chitosan derivatives, and the amphiphilic N-long Alkyl-N-arginine chitosan derivatives are represented by the following general formula:

In the structural formula, n1, n2, and n3 are integers greater than zero, A is L-arginine or its pharmaceutically acceptable salt, and B is a long-chain alkyl group with 6-12 carbons.

The described gambogic acid nano-micelle is characterized in that it is mainly derived from 1-100 parts of gambogic acid and 1-100 parts of amphiphilic N-long chain alkyl N-arginine chitosan composed of things.

The nanomicelle is characterized in that: the molecular weight of chitosan in the amphiphilic N-long chain alkyl-N-arginine chitosan derivative is between 5000-500000, and the substitution degree of arginine is 1% to 50%, and the long-chain alkyl substitution degree is 1% to 50%.

The gambogic acid nano-micelle is characterized in that: the gambogic acid accounts for 1-50% of the weight of the preparation.

The preparation method of described gambogic acid nano-micelle is characterized in that, it comprises the steps:

(1) Weigh 1-50 parts of amphiphilic N-long-chain alkyl-N-arginine chitosan derivatives according to the weight-to-volume ratio, add 1-100 parts of water, and stir in a water bath at 20-70°C for 1-120min , made into a carrier solution;

(2) Add 1-50 parts of gambogic acid to the carrier solution according to the weight-to-volume ratio, sonicate the probe for 1-100 min in an ice bath, dialyze with double-distilled water, and filter to obtain the drug-loaded micellar solution.

The preparation method of described gambogic acid nano-micelle is characterized in that, it comprises the steps:

(1) Weigh 1-50 parts of amphiphilic N-long-chain alkyl N-arginine chitosan derivatives according to the weight-to-volume ratio, add 1-100 parts of water, stir in a water bath at 20-70°C for 1-120min, Make carrier solution;

(2) Take 1-50 parts of gambogic acid by weight to volume ratio, be dissolved in an organic solvent, and make concentration be the organic solution of the gambogic acid of 0.1-20g/L;

(3) Add the organic solution of gambogic acid obtained in step (2) dropwise to the carrier solution, sonicate the probe in an ice bath, stir overnight at room temperature, evaporate the organic solvent, and filter to obtain the drug-loaded micellar solution .

The preparation method of described gambogic acid nano-micelle is characterized in that, it comprises the steps:

(1) Weigh 1-50 parts of amphiphilic N-long-chain alkyl-N-arginine chitosan derivatives according to the weight-to-volume ratio, add 1-100 parts of water, and stir in a water bath at 20-70°C for 1-120min , made into a carrier solution;

(2) Take 1-50 parts of gambogic acid by weight to volume ratio, dissolve it in ethanol, and make a gambogic acid ethanol solution whose concentration is 0.1-20g/L;

(3) Add the ethanol solution of gambogic acid obtained in step (2) dropwise into the carrier solution until the prescription ratio is reached, and dialyze with double-distilled water overnight to obtain the drug-loaded micellar solution.

The preparation method of described gambogic acid nano-micelle is characterized in that, it comprises the steps:

(1) Weigh 1-50 parts of gambogic acid according to the weight volume ratio, put it in a round bottom flask, add ethanol to make a gambogic acid ethanol solution with a concentration of 0.1-20g/L, and rotate it at 20-70°C Evaporate for 1-100min to dry the ethanol to obtain a dry and transparent drug film skeleton;

(2) Take 1-100 parts of amphiphilic N-long chain alkyl-N-arginine chitosan derivatives by weight to volume ratio, add 1-100 parts of water;

(3) The solution obtained in step (2) is then added to the drug film skeleton obtained in step (1) until the prescription ratio is reached, hydrated at 20-70°C, and membrane filtered to obtain a transparent drug-loaded micellar solution.

The preparation made of nano micelles is characterized in that the preparation includes freeze-dried injections, injections, transdermal preparations, aerosols, oral liquids, solutions, ophthalmic preparations, tablets or capsules.

Described nano-micelle is characterized in that, described amphipathic N-long chain alkyl-N-arginine chitosan derivative is represented by following general formula:

In the structural formula, n1, n2, and n3 are integers greater than zero, and n is a long-chain alkyl group with 5 to 11 carbons.

Description of drawings

Detailed ways

The following examples are used to illustrate the specific implementation of the technical solutions of the present invention, but are not intended to limit the protection scope of the present invention.

Add 1g of chitosan (Mw100000) into 100ml of 1% acetic acid aqueous solution, stir at room temperature, add n-octylaldehyde (0.5g), react for 4 hours, add 1.8ml of NaBH4 (0.18g) aqueous solution, continue to stir for 12 hours, wash with 1M NaOH Adjust the pH to 7, filter with suction, wash with ethanol and water alternately for 4 times, and dry under vacuum at room temperature to obtain 0.84 g of milky white powder N-octyl chitosan; take N-octyl chitosan (0.5 g) and add 50 ml of 1% acetic acid In the aqueous solution, stir at room temperature, add a certain amount of arginine (3.00g), NHS (1.94) and EDC (7.8g) in sequence, react for 48 hours, dialyze with a dialysis bag (MWCO3500), freeze-dry, and obtain milky white flocs after drying Thin 0.15g, namely N-octyl-N-arginine chitosan.

The preparation method of gambogic acid-N-octyl-N-arginine chitosan (GA-OACS):

The present invention also provides four preparation methods of GA-OACS, comprising the following steps:

Embodiment 1: direct dissolution method:

(1) Weigh 1 gram of OACS, add 50ml of water, and stir in a water bath at 50°C for 30 minutes to make a carrier solution;

(2) Add 1 g of gambogic acid to the carrier solution, sonicate the probe for 30 minutes in an ice bath, dialyze with double distilled water, and filter to obtain the drug-loaded micellar solution.

Example 2: Solvent emulsification and volatilization method: Weigh 1 gram of OACS, add 50 ml of water, stir in a water bath at 50°C for 30 minutes, weigh 1 gram of gambogic acid, dissolve it in methylene chloride solution, and make 10 g/L of gambogic acid The dichloromethane solution was added dropwise to the carrier solution, the probe was sonicated for 10 minutes in an ice bath, the dichloromethane was evaporated with open stirring at room temperature overnight, and the drug-loaded micelle solution was obtained by filtering with a 0.45 μm filter membrane.

Example 3: Dialysis method: Weigh 1 gram of OACS, add 50ml of water, stir in a water bath at 50°C for 30 minutes, weigh 1 gram of GA and dissolve it in an appropriate amount of ethanol to make the concentration 10g/L, and add the ethanol solution of GA to the carrier drop by drop In the solution, the probe was sonicated for 10 minutes in an ice bath, dialyzed in double distilled water overnight, and filtered through a 0.45 μm filter membrane to obtain a drug-loaded micellar solution.

Example 4: Thin-film hydration method: Weigh 1 gram of GA, place it in a round-bottomed flask, add ethanol to make the concentration 10g/L, evaporate the ethanol to dryness by rotary evaporation at 50°C for 30 minutes, and obtain a dry and transparent medicine Membrane skeleton, then weigh 1 gram of OACS, add 50ml of water to dissolve OACS, add the solution of OACS to the membrane skeleton, hydrate at 50°C, and filter through a 0.45 μm filter membrane to obtain a transparent GA-OACS micellar solution.

The GA-OACS described in the present invention can also be produced according to the micelles preparation method known in the pharmaceutical industry.

In the GA-OACS produced by the invention, the encapsulation efficiency and drug loading capacity of GA can reach more than 80% and 30%, the particle diameter of GA-OACS is 160nm, and the solubility in water can reach 3.16mg/ml.

The GA-OACS nanomicelle of the present invention overcomes the disadvantages of low solubility of gambogic acid in water and low oral bioavailability, and successfully prepares a high drug-loading, high encapsulation efficiency, and high solubility gambogic acid with a particle size of 160nm. The GA-OACS nano-preparation greatly improves the bioavailability of gambogic acid. In addition, the technology is simple, the process requirements are low, and the slow-release material is cheap and low in cost.

Claims (10)

1. the nano-micelle of a gamlogic acid, is characterized in that, it mainly is comprised of gamlogic acid and amphipathic N-chain alkyl-N-arginine chitosan derivant, and described amphipathic N-chain alkyl-N-arginine chitosan derivant is represented by following general formula:

N1 in structural formula, n2, n3 are the integer greater than zero, and A is L-arginine or its pharmaceutical salts, and B is the chain alkyl of 6～12 carbon.

2. gamlogic acid nano-micelle claimed in claim 1, is characterized in that, it mainly is comprised of the gamlogic acid of 1-100 part and the amphipathic N-chain alkyl of 1-100 part-N-arginine chitosan derivant.

3. the described nano-micelle of claim 1-2 any one, it is characterized in that: in described amphipathic N-chain alkyl-N-arginine chitosan derivant, the molecular weight of chitosan is between 5000-500000, the arginine substitution value is 1%～50%, and the chain alkyl substitution value is 1%～50%.

4. the described gamlogic acid nano-micelle of claim 1 any one, it is characterized in that: described gamlogic acid accounts for 1～50% of weight of formulation.

5. the preparation method of the described gamlogic acid nano-micelle of claim 1-4 any one, is characterized in that, it comprises the steps:

(1) volume ratio takes amphipathic N-chain alkyl N-arginine chitosan derivant 1-50 part by weight, adds entry 1-100 part, and 20-70 ℃ of stirring in water bath 1-120min makes carrier solution;

(2) volume ratio adds gamlogic acid 1-50 part to carrier solution by weight, Probe Ultrasonic Searching 1-100min under ice bath, and the redistilled water dialysis is filtered, and namely gets polypeptide drug-loaded micelle solution.

6. the preparation method of the described gamlogic acid nano-micelle of claim 1-4 any one, is characterized in that, it comprises the steps:

(1) volume ratio takes amphipathic N-chain alkyl-N-arginine chitosan derivant 1-50 part by weight, adds entry 1-100 part, and 20-70 ℃ of stirring in water bath 1-120min makes carrier solution;

(2) volume ratio takes gamlogic acid 1-50 part by weight, is dissolved in organic solvent, makes the organic solution that concentration is the gamlogic acid of 0.1-20g/L;

(3) organic solution of gamlogic acid that dropwise drips step (2) gained in carrier solution, Probe Ultrasonic Searching under ice bath, the uncovered stirring of room temperature is spent the night, and volatilizes organic solvent, filters, and namely gets polypeptide drug-loaded micelle solution.

7. the preparation method of the described gamlogic acid nano-micelle of claim 1-4 any one, is characterized in that, it comprises the steps:

(1) volume ratio takes amphipathic N-chain alkyl-N-arginine chitosan derivant 1-50 part by weight, adds entry 1-100 part, and 20-70 ℃ of stirring in water bath 1-120min makes carrier solution;

(2) volume ratio takes gamlogic acid 1-50 part by weight, is dissolved in ethanol, and making concentration is the gamlogic acid alcoholic solution of 0.1-20g/L;

(3) the gamlogic acid alcoholic solution that dropwise drips step (2) gained is in carrier solution, until reach the prescription ratio, the redistilled water dialysed overnight namely gets polypeptide drug-loaded micelle solution.

8. the preparation method of the described gamlogic acid nano-micelle of claim 1-4 any one, is characterized in that, it comprises the steps:

(1) volume ratio takes gamlogic acid 1-50 part by weight, is placed in round-bottomed flask, adds ethanol, and making concentration is the gamlogic acid alcoholic solution of 0.1-20g/L, with the ethanol evaporate to dryness, gets dry transparent medicine membrane bone frame at 20-70 ℃ of lower rotary evaporation 1-100min;

(2) volume ratio takes amphipathic N-chain alkyl-N-arginine chitosan derivant 1-100 part by weight, adds entry 1-100 part;

(3) then step (2) gained solution joined in step (1) gained medicine membrane bone frame, until reach the prescription ratio, 20-70 ℃ of lower aquation, membrane filtration obtains transparent polypeptide drug-loaded micelle solution.

9. the preparation made of the described gamlogic acid nano-micelle of claim 1-4 any one, is characterized in that, described preparation comprises lyophilized injection, injection, preparation capable of permeating skin, aerosol, oral liquid, solution, ophthalmic preparation, tablet or capsule.

10. nano-micelle claimed in claim 1, is characterized in that, described amphipathic N-chain alkyl-N-arginine chitosan derivant is represented by following general formula:

N1 in structural formula, n2, n3 are the integer greater than zero, and n is the chain alkyl of 5～11 carbon.