CN111228212A - Drug-loaded injectable implantation in-situ hydrogel - Google Patents
Drug-loaded injectable implantation in-situ hydrogel Download PDFInfo
- Publication number
- CN111228212A CN111228212A CN202010045805.3A CN202010045805A CN111228212A CN 111228212 A CN111228212 A CN 111228212A CN 202010045805 A CN202010045805 A CN 202010045805A CN 111228212 A CN111228212 A CN 111228212A
- Authority
- CN
- China
- Prior art keywords
- drug
- situ hydrogel
- injectable
- situ
- precursor material
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Neurosurgery (AREA)
- Dermatology (AREA)
- General Chemical & Material Sciences (AREA)
- Inorganic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention discloses a drug-loaded injectable implantable in-situ hydrogel which is characterized in that the hydrogel is formed in situ at an injection part by a chemical bond crosslinking mode based on Schiff base reaction, the hydrogel has the self-adaptability of the shape of an injection cavity, the gel has long detention time in the cavity, and the drug can be controlled to release. The gel is used for filling holes after tumor operation, can quickly fill a cavity by utilizing the fluidity of the in-situ hydrogel, is in close contact with the edge tissue after tumor resection, better eliminates residual tumor cells, and has the effect of remarkably preventing tumor recurrence.
Description
Technical Field
The invention relates to the field of medical implant preparations, in particular to a drug-loaded injectable implant in-situ hydrogel.
Background
Cancer (malignant tumor) is a serious disease that seriously threatens human health, with high morbidity and mortality. The current clinical treatment means mainly comprise surgery, chemotherapy and radiotherapy. Surgery is often used as a first line treatment regimen, depending on the size of the tumor and the stage of pathology. After the tumor is removed by operation, the in-situ recurrence and metastasis rate of the tumor is high, and about 70 percent of patients finally fail to treat the tumor due to the recurrence and metastasis, so the methods of radiotherapy, chemotherapy and the like are often adopted to inhibit the recurrence and metastasis of the tumor after the operation. Radiotherapy and general chemotherapy generally cause serious toxic and side effects, reducing the quality of life of patients. Therefore, a new administration mode is needed to improve the curative effect of the medicine and reduce the toxic and side effects.
Compared with the systemic administration mode, the local administration at the focus part can improve the drug concentration at the focus part and reduce the systemic distribution of the drug, thereby improving the curative effect and reducing the toxic and side effects. The local administration can lead the drug to be retained at the focus part for a long time, can release the drug and take effect for a long time after once implantation, avoids frequent administration and improves the tolerance of patients. The topical administration mainly comprises film agent, spinning, foam, hydrogel, etc. The hydrogel generally presents a three-dimensional network structure, has good biocompatibility, is suitable for loading and releasing medicaments, and is suitable for wide medicament types including small molecular medicaments, proteins, nucleic acids and the like. In-situ gels are gels in which a flowing liquid is converted to a non-flowing liquid under certain conditions, and generally include temperature-sensitive in-situ gels and ion-sensitive in-situ gels. However, these environmentally sensitive native gels have a slow rate of gel transition from liquid to gel, typically several minutes; at higher volumes, the overall gelation rate may be less.
Disclosure of Invention
The invention discloses a drug-loaded injectable implantation in-situ hydrogel which is characterized in that a chemical bond crosslinking mode is adopted to form in situ at an injection part, the injection cavity has the shape adaptability, the gel has long detention time in the cavity, and the drug can be controlled to release.
The chemical bond crosslinking mode adopted by the invention is based on Schiff base reaction. The in-situ hydrogel formed by the reaction has multiple advantages, including (1) the reaction condition of Schiff base is mild, the reaction can be carried out in the physiological state of human body, no additional catalyst is needed, and the reaction product only generates water except the gel, which is a green chemical reaction; (2) schiff base has pH sensitivity, is unstable under weak acid conditions such as tumor and inflammation, and is favorable for drug release after being degraded; (3) the Schiff base is an unstable covalent bond, presents dynamic balance of fracture and formation, enables the gel to have self-healing property, and can prolong the retention time at an injection site.
In the preparation process of the drug-loaded injectable implantable in-situ hydrogel, the reaction speed and the gel strength of the Schiff base can be controlled by the content of aldehyde groups and amino groups in the gel precursor material and the concentration of the gel precursor material, wherein the concentration of the gel precursor material is selected from 1-20%, preferably 2-10%.
The invention relates to a drug-loaded injectable implantation in-situ hydrogel, which is prepared by the following steps:
(1) the gel precursor material comprises aldehyde precursor material and amination precursor material;
(2) loading the drug or cell by covalent or non-covalent bonding;
(3) mixing two or more gel precursors carrying drugs to obtain the drug-carrying injectable in-situ hydrogel.
In the above step, the starting material of the gel precursor material is a high molecular compound selected from synthetic high molecular compounds, polysaccharide high molecular compounds, and protein high molecular compounds, preferably polysaccharide high molecular compounds. The synthetic high molecular compound is selected from polyethylene glycol PEG, polylactic acid PLA, polycaprolactone PCL, polylactic acid-glycolic acid copolymer PLGA, polyvinyl alcohol PVA, polyetherimide PEI and polyacrylamide PAAM. The polysaccharide polymer is selected from hyaluronic acid, chitosan, sodium alginate, starch, dextran, cellulose, and chondroitin sulfate, preferably hyaluronic acid, chitosan, and sodium alginate. The protein polymer compound is selected from fibrin, collagen, fibroin, globulin, and gelatin.
When the polysaccharide macromolecular compound is used as the initial material of the gel precursor material, aldehyde group modification and amination modification are carried out to obtain the gel precursor material, and the gel precursor material is gelled to form stable gel after mixing. The gel time can be adjusted by the modification degree of aldehyde group and amino group and the concentration of the precursor solution.
The gel precursor material of the invention is preferably aldehyde polysaccharide, and the preparation method is selected from aldehyde compound direct modification method and sodium periodate oxidation method. The direct aldehyde compound modification method is to directly mix and react the aldehyde compound and polysaccharide to obtain the product. The aldehyde compound is selected from p-aminobenzaldehyde, p-carboxytolualdehyde, p-hydroxybenzaldehyde, m-aminobenzaldehyde, m-bromobenzaldehyde, 2, 4-hydroxybenzaldehyde, 3, 4-hydroxybenzaldehyde, o-aminobenzaldehyde, 1-cyanobenzaldehyde, decenal, 4-iodophenylacetaldehyde, p-aminobenzaldehyde, aminoacetaldehyde, 3-aminofuran-2-formaldehyde, 4-hydroxypentanal, preferably from p-aminobenzaldehyde, p-carboxytolualdehyde, p-hydroxybenzaldehyde. The sodium periodate oxidation method is to mix sodium periodate and polysaccharide in solution, the sodium periodate oxidizes vicinal diol groups in polysaccharide molecules to obtain dialdehyde polysaccharide; the aldehyde group modification degree of dialdehyde polysaccharide is related to the concentration of sodium periodate, the molar ratio of the sodium periodate to the adjacent diol groups, the reaction temperature and the oxidation time. In the sodium periodate oxidation method, the concentration of periodic acid is selected from 0.2 to 2 percent, preferably from 0.5 to 1 percent; the molar ratio of the sodium periodate to the vicinal diol groups is selected from 0.5 to 5, preferably from 0.5 to 2; the reaction temperature is 20-40 ℃, preferably 25-30 ℃; the oxidation time is from 2 hours to 24 hours, preferably from 6 hours to 12 hours.
The gel precursor material of the invention is preferably aminated polysaccharide, and the preparation method is selected from amino compound modification method, hydrazine compound modification method and natural aminopolysaccharide derivative. The amino compound modification method comprises connecting amino compound and polysaccharide by acylation reaction to obtain aminated polysaccharide, wherein the amino compound is selected from ethylenediamine, hexamethylenediamine, dodecanediamine, 2-aminobutyric acid, diaminobenzoic acid, diaminocaproic acid, and 6-amino acid, preferably selected from ethylenediamine, hexamethylenediamine, and 2-aminobutyric acid. The hydrazine compound modification method is to connect hydrazine compounds with polysaccharide by acylation reaction to obtain aminated polysaccharide, wherein the hydrazine compounds are selected from adipic dihydrazide, succinic dihydrazide, 4-hydrazinobenzoic acid hydrazine and dihydrazinoethane, and preferably adipic dihydrazide. The natural aminopolysaccharide derivative is selected from chitosan, glycosaminoglycan, chitosan derivative, and glycosaminoglycan derivative. The chitosan is water-soluble chitosan. The chitosan derivative is selected from carboxymethyl chitosan, carboxyethyl chitosan, chitosan hydrochloride, chitosan quaternary ammonium salt, chitosan lactate, chitosan glutamate, and chitosan sulfate, preferably from carboxymethyl chitosan and carboxyethyl chitosan. The glycosaminoglycan derivative is selected from deacetylated hyaluronic acid, deacetylated chondroitin sulfate, deacetylated dermatan sulfate, and deacetylated keratan sulfate.
When the starting material of the gel precursor material is a polysaccharide macromolecular compound, the molecular weight of the polysaccharide macromolecular compound is selected from 100kDa to 1600kDa, preferably 500kDa to 1000 kDa. The gel precursor material in the molecular weight range has proper fluidity, so that the injection and mixing are convenient, and the gel formed by crosslinking the gel precursor material has proper strength and is easy to retain in a cavity in a body.
The drug-loaded injectable implant in-situ hydrogel provided by the invention is loaded with drugs or cells, is used for injection of various parts in vivo, and is used for disease treatment, prevention and regeneration. The application range is selected from but not limited to treatment and prevention of osteoarthritis, tumor treatment, prevention of in-situ recurrence after tumor operation, tuberculosis treatment, oral disease treatment, ophthalmic disease treatment, neurological disease treatment, urological disease treatment, cardiovascular disease treatment, tissue regeneration and nerve regeneration.
The drug-loaded injectable implantation in-situ hydrogel provided by the invention can be various clinically used drugs or drugs which are determined to have activity but are not clinically used. The type of the drug is not limited, and can be specifically selected from antibacterial drugs, antifungal drugs, antitubercular drugs, antitumor drugs, antipyretic and analgesic drugs, central nervous system drugs, and biotechnological drugs. The antibacterial drug is selected from penicillin, ampicillin, amoxicillin, piperacillin sodium, ticarcillin, sultamicin, mezlocillin sodium, azlocillin sodium, carbenicillin sodium, sulcillin sodium, furacilin sodium, nevucillin, dicloxacillin, methicillin, pimecrillin, cephalexin, ceftizolid, cefadroxil, cefradine, cefuroxime sodium, cefuroxime axetil, cefaclor, ceftriaxone sodium, cefoperazone sodium, ceftazidime, cefmetazole, cefdinir, cefoxitin sodium, cefotiam, cefonicid sodium, cefradine lysine salt, cefsulodin sodium, cefpiramide sodium, cefixadine, cefbuperazone sodium, aztreonam, meropenem, kanamycin, amikacin, neomycin, livemycin, paromomycin, tetracycline, lincomycin, oxytetracycline, doxycycline, minocycline, and doxycycline, Aureomycin, josamycin, milbemycin, rokitamycin, acetylspiramycin, norvancomycin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin, ciprofloxacin, fleroxacin, moxifloxacin, metronidazole and tinidazole. The antifungal agent is selected from amphotericin B, miconazole, ketoconazole, itraconazole, fluconazole, clotrimazole, econazole, terbinafine, griseofulvin, ciclopirox olamine, nystatin, and netitifen. The antituberculotic is selected from isoniazid, sodium para-aminosalicylate, rifampicin, rifapentine, rifamycin, streptomycin, ethambutol, ethionamide, and pyrazinamide. The antineoplastic agent is selected from paclitaxel, docetaxel, adriamycin, daunorubicin, epirubicin, mitoxantrone, cisplatin, carboplatin, oxaliplatin, camptothecin, hydroxycamptothecin, etoposide, teniposide, irinotecan, gemcitabine, carmustine, nimustine, cyclophosphamide, mitomycin, bleomycin, methotrexate, 5-fluorouracil, cytarabine, mercaptopurine, gefitinib, erlotinib, trastuzumab, rituximab, and cetuximab. The antipyretic analgesic and antiinflammatory drug is selected from acetylsalicylic acid, acetaminophen, indomethacin, piroxicam, meloxicam, nimesulide, diclofenac sodium, naproxen, ibuprofen, ketoprofen, fenbufen, and loxoprofen. The analgesic is selected from morphine, fentanyl, methadone, tramadol, dezocine, rotundine, and capsaicin. The biotechnology medicine is selected from polypeptide protein medicines, nucleic acid medicines and vaccines, and specifically comprises cytokines, monoclonal antibodies, siRNA, microRNA, oligonucleotides and antisense nucleic acids. The cytokine is selected from granulocyte colony stimulating factor G-CSF, nerve growth factor, fibroblast growth factor, epidermal growth factor, interferon, and interleukin. The medicines can also be prepared into preparations such as cyclodextrin inclusion compound, liposome, nanoparticle, emulsion and the like according to the properties of the medicines and then added into the gel precursor.
The corresponding drug injectable implantation in-situ hydrogel is prepared from the drugs, such as penicillin injectable implantation in-situ hydrogel, cefuroxime axetil injectable implantation in-situ hydrogel, meropenem injectable implantation in-situ hydrogel, kanamycin injectable implantation in-situ hydrogel, amikacin injectable implantation in-situ hydrogel, terramycin injectable implantation in-situ hydrogel, ciprofloxacin injectable implantation in-situ hydrogel, moxifloxacin injectable implantation in-situ hydrogel, metronidazole injectable implantation in-situ hydrogel, amphotericin B injectable implantation in-situ hydrogel, fluconazole injectable implantation in-situ hydrogel, terbinafine injectable implantation in-situ hydrogel, griseofulvin injectable implantation in-situ hydrogel, isoniazid injectable implantation in-situ hydrogel, rifampicin injectable implantation in-situ hydrogel, flutriaxin injectable implantation in-situ hydrogel, Rifamycin injectable implantation in-situ hydrogel, streptomycin injectable implantation in-situ hydrogel, paclitaxel injectable implantation in-situ hydrogel, docetaxel injectable implantation in-situ hydrogel, doxorubicin injectable implantation in-situ hydrogel, daunorubicin injectable implantation in-situ hydrogel, cisplatin injectable implantation in-situ hydrogel, hydroxycamptothecin injectable implantation in-situ hydrogel, etoposide injectable implantation in-situ hydrogel, methotrexate injectable implantation in-situ hydrogel, 5-fluorouracil injectable implantation in-situ hydrogel, cytarabine injectable implantation in-situ hydrogel, gefitinib injectable implantation in-situ hydrogel, trastuzumab injectable implantation in-situ hydrogel, acetylsalicylic acid injectable implantation in-situ hydrogel, indomethacin injectable implantation in-situ hydrogel, ibuprofen injectable implantation in-situ hydrogel, morphine injectable implantation in-situ hydrogel, paclitaxel injectable implantation in-situ hydrogel, docetaxel injectable implantation in-situ hydrogel, Tramadol can be injected into the in-situ hydrogel, nerve growth factor can be injected into the in-situ hydrogel, and interferon can be injected into the in-situ hydrogel.
The drug-loaded injectable implantable in-situ hydrogel provided by the invention can be used for loading various stem cells.
The drug-loaded injectable implantable in-situ hydrogel comprises a covalent bond combination mode and a physical packaging mode, wherein covalent bonds related to the covalent bond combination mode are selected from carbonate bonds, phosphate bonds, amide bonds, imine bonds, disulfide bonds, thioether, thioester, oxime and phenylboronic acid ester bonds, and are preferably selected from ester bonds, amide bonds, imine bonds, oxime and disulfide bonds.
The in-vivo implantation mode of the drug-loaded injectable implantation in-situ hydrogel is that a gel precursor material is directly injected into the body for implantation, and the injection mode is selected from rapid injection after the gel precursor material is mixed and direct injection by a double-tube injection device.
The drug-loaded injectable implantable in-situ hydrogel is used for filling holes after tumor operation, has the effect of remarkably preventing tumor recurrence, and has small trauma to a body. Because the cavity after the body part tissue is cut off by the operation is irregular, the cavity can be quickly filled by utilizing the fluidity of the in-situ hydrogel and is tightly contacted with the marginal tissue after the tumor is cut off, and the residual tumor cells are better eliminated.
The invention is further illustrated by the following examples and experimental examples.
Drawings
FIG. 1 is a scheme for synthesis of aldehyde-modified hyaluronic acid.
FIG. 2 shows nuclear magnetic hydrogen spectrum of aldehyde-based hyaluronic acid
FIG. 3 shows an IR spectrum of aldehyde hyaluronic acid, HA means hyaluronic acid, and HA-CHO means aldehyde hyaluronic acid.
Figure 4. gelling process of drug loaded injectable implant in situ hydrogel. After the two gel precursor materials (the aldehyde hyaluronic acid and the carboxymethyl chitosan) are mixed, the gelation and solidification can be rapidly carried out.
FIG. 5 scanning electron micrograph of in situ hydrogel after freezing and breaking and freeze-drying for drug-loaded injectable implantation
FIG. 6. inhibitory Effect of doxorubicin and gemcitabine and the combination of the two drugs on 4T1 cell proliferation.
FIG. 7 is a schematic diagram of the in vivo drug effect investigation process
FIG. 8 in vivo imaging System investigation of post-operative, treatment conditions
FIG. 9 shows the change in tumor volume after the recurrence of the tumor in mice, the tumor volume exceeded 20mm3I.e. the tumor is considered to have recurred.
FIG. 10 bioluminescence after treatment completion on day 24 tumor lung metastasis
FIG. 11 immunohistochemical and pathological examination of anatomical tumor and cardiopulmonary tissues after the end of treatment, staining of tumor tissues Ki67 and TUNEL, and staining of lung and cardiac HE.
Detailed Description
Example 1 Isoniazid injectable implantable in situ hydrogels
Dissolving 1g of sodium hyaluronate with the molecular weight of 500kDa in 100mL of water, adding sodium periodate with equal molar weight, stirring for 2h at room temperature in the dark, then adding 1mL of ethylene glycol, continuing to stir for 1h, dialyzing the reaction solution with water for 3 days, obtaining aldehyde-based hyaluronic acid after freeze-drying, dissolving with phosphate buffer solution to obtain 20mg/mL of aldehyde-based hyaluronic acid, adding 1g of isoniazide, uniformly stirring to obtain an aldehyde-based precursor material, taking a carboxymethyl chitosan solution with the same volume as 20mg/mL of an amination precursor material, mixing the two precursor materials, and rapidly injecting into a cavity in vivo to obtain the isoniazide injectable implantation in-situ hydrogel.
Fig. 1 is a scheme diagram of synthesis of aldehyde-based hyaluronic acid, fig. 2 is nuclear magnetic hydrogen spectrum of aldehyde-based hyaluronic acid, fig. 3 is infrared spectrum of aldehyde-based hyaluronic acid, fig. 4 is gelation process of injectable implant in-situ hydrogel, and fig. 5 is scanning electron microscope photograph of injectable implant in-situ hydrogel after freezing and brittle fracture and freeze-drying.
Example 2 injectable Moxisaxin Implantation of in situ hydrogels
Connecting one end of adipic dihydrazide with hyaluronic acid through an amide bond by using an EDC/NHS catalysis method, dialyzing, purifying, and freeze-drying to obtain aminated hyaluronic acid as an aminated precursor material, preparing aldehyde-based hyaluronic acid as an aldehyde-based precursor material by using a sodium periodate oxidation method, mixing the aldehyde-based hyaluronic acid with the concentration of 20mg/mL and a phosphate buffer solution of the aminated hyaluronic acid in equal volumes, adding moxifloxacin to obtain a mixed solution containing 10mg/mL of moxifloxacin, and quickly injecting the mixed solution into a cavity in vivo to obtain the moxifloxacin injectable implantation in-situ hydrogel.
Example 3 injectable paclitaxel Implantation in situ hydrogels
Preparing aldehyde sodium alginate serving as an aldehyde precursor material by using a sodium periodate oxidation method, dialyzing, purifying, freeze-drying, adding a phosphate buffer solution to prepare a 30mg/mL aldehyde sodium alginate solution, adding paclitaxel, uniformly stirring to obtain a 10mg/mL aldehyde sodium alginate solution containing the paclitaxel, and injecting the 10mg/mL aldehyde sodium alginate solution and an isovolumetric 20mg/mL carboxyethyl chitosan phosphate buffer solution into a cavity in a body by using two injectors simultaneously to obtain the paclitaxel injectable implantable in-situ hydrogel.
Example 4 injectable Implantation of nerve growth factor in situ hydrogels
Connecting para aminobenzaldehyde and hyaluronic acid through an amide bond by using an EDC/NHS catalytic method to obtain aldehyde-based hyaluronic acid as an aldehyde-based precursor material, performing dialysis and purification, then freeze-drying, adding a phosphate buffer solution to prepare 20mg/mL aldehyde-based hyaluronic acid solution, adding a nerve growth factor, uniformly stirring to obtain the aldehyde-based hyaluronic acid solution containing 0.1mg/mL nerve growth factor, and simultaneously injecting the aldehyde-based hyaluronic acid solution and an isometric 20mg/mL carboxyethyl chitosan phosphate buffer solution into a cavity in a body by using two injectors to obtain the nerve growth factor injectable implantable in-situ hydrogel.
Example 5 Adriamycin/Gemcitabine injectable Implantation in situ hydrogels
Using aldehydic hyaluronic acid obtained by oxidizing sodium periodate as an aldehydic precursor material, dissolving with phosphate buffer solution to obtain 80mg/mL aldehydic hyaluronic acid solution, dripping 8mg/mL adriamycin solution into the aldehydic hyaluronic acid solution under the stirring condition, and stirring for 12 hours at 37 ℃ in a dark place to obtain hyaluronic acid-adriamycin copolymer solution; and dissolving gemcitabine hydrochloride and carboxymethyl chitosan by using a phosphate buffer solution to obtain a solution containing 2mg/mL gemcitabine and 20mg/mL carboxymethyl chitosan, and simultaneously injecting the two solutions with equal volume into the cavity of the body by using two injectors to obtain the adriamycin/gemcitabine injectable implantation in-situ hydrogel.
Experimental example 1 Adriamycin/Gemcitabine injectable Implantation in situ hydrogel anti-tumor recurrence Effect
The following experimental examples demonstrate that doxorubicin and gemcitabine have synergistic effects on cell killing, and the doxorubicin/gemcitabine injectable implantable in situ hydrogel of example 5 can inhibit postoperative recurrence of tumors.
Sample preparation: doxorubicin, gemcitabine, the doxorubicin/gemcitabine injectable implant in situ hydrogel of example 5 (referred to simply as doxorubicin/gemcitabine gel), the drug-free blank gel prepared according to the procedure of example 5, the doxorubicin-only doxorubicin injectable implant in situ hydrogel prepared according to the procedure of example 5 (referred to simply as doxorubicin gel), the gemcitabine-only gemcitabine injectable implant in situ hydrogel prepared according to the procedure of example 5 (referred to simply as gemcitabine gel).
(1) Adriamycin and gemcitabine inhibiting effect on cell in vitro
The experimental process comprises the following steps: the effect of doxorubicin and gemcitabine in inhibiting tumor cells in vitro was demonstrated by 4T1 cells. Collecting 4T1 cells in logarithmic growth phase, inoculating the cells in a 96-well plate at a density of 3000 cells per well, after the cells are cultured and attached to the wall, independently or jointly administering doxorubicin and gemcitabine with a series of concentration gradients, continuously culturing for 48 hours, adding 10% of CCK-8 solution into the culture plate, continuously incubating for 2 hours, detecting absorbance at 450nm by using an enzyme-labeling instrument, and calculating cell viability and joint administration indexes.
The experimental results are as follows: through calculation, the adriamycin and the gemcitabine can obviously inhibit the growth of 4T1 cells, are concentration-dependent, have a combined treatment index of less than 1 and show obvious synergistic effect. FIG. 6 is a graph of the inhibition of 4T1 cell proliferation by doxorubicin and gemcitabine in combination with two agents.
(2) Inhibition effect of doxorubicin/gemcitabine injectable implantation in-situ hydrogel on postoperative recurrence of breast cancer in mice
The experimental process comprises the following steps: luciferase-labelled 4T1 cells (4T1-luc) were cultured in vitro at 5X 10 cells each5Inoculating each cell to Balb/c female mice subcutaneously on the back, and growing the tumor to 100mm3Left and rightThe side surface skin opening of the tumor position sharply strips the tumor, the skin around the tumor is reserved to ensure that residual tumor cells cause tumor recurrence, the residual tumor cells are verified through live imaging of the small animal, and a tumor postoperative recurrence model is successfully established. Mice were randomly divided into 6 groups: a model group; a blank gel group; gemcitabine gel group (5 mg/kg); doxorubicin gel group (10 mg/kg); doxorubicin/gemcitabine gel group (gemcitabine 5mg/kg, doxorubicin 10 mg/kg); doxorubicin/gemcitabine solution group. The treatment is carried out the next day after tumor resection, the gel preparation group is injected and administered in situ in the tumor surgery cavity, the solution group is injected and administered through tail vein, and the tumor recurrence condition is observed after one treatment. The body weight was weighed every three days, and whether there was tumor recurrence was observed and the volume of the tumor that had recurred was measured, i.e., long diameter x short diameter2X 0.5. Tumor recurrence and lung metastasis were examined by in vivo imaging on days 12 and 24 post-treatment. The experiment was terminated 24 days after treatment, mice were sacrificed after anesthesia, tumor tissues and major organs were dissected out, fixed in formalin, dehydrated to make wax blocks, sectioned and stained with HE, Ki67 and TUNEL to examine tumor inhibition. Fig. 7 is a schematic diagram of the in vivo efficacy study procedure.
The experimental results are as follows: 12 days after 4T1-luc cell inoculation, tumors grew to 100mm3And performing surgical excision, and verifying the existence of residual tumor cells after surgery through a small animal living body imaging experiment, thereby indicating the successful establishment of a mouse postoperative recurrence model. On day 12 post-treatment, tumor recurrence was observed in all groups except the doxorubicin/gemcitabine gel group by in vivo small animal imaging. On the 24 th day after treatment, two mice in the model group died, and the fluorescence value of the tumor-removed part of the adriamycin/gemcitabine gel group is consistent with the background value of the skin on the back of the mice, so that no recurrence phenomenon occurs, which indicates that the adriamycin/gemcitabine gel group completely inhibits the recurrence of the tumor, and the recurrence rate is 0%. Compared with the fluorescence value of residual cells after surgical resection, the tumor recurrence rate higher than the fluorescence value is considered, the tumor recurrence rate lower than the fluorescence value is considered, and the results show that the recurrence rate of the model group and the blank gel group is 100%, the recurrence rate of the gemcitabine gel group is 67%, the recurrence rate of the adriamycin gel group is 33%, and the recurrence rate of the adriamycin/gemcitabine is 33%The relapse rate of the shore solution group was 83%. After the adriamycin/gemcitabine gel is injected into the tumor surgery cavity, the medicine can be directly released around the residual tumor cells, the medicine concentration is improved and is slowly released for a long time, and the killing of the tumor cells is increased due to the synergistic effect of the two medicines, so that the adriamycin/gemcitabine gel completely inhibits the recurrence of the tumor. FIG. 8 shows the post-operative, treatment scenario of the in vivo imaging system.
Tumor volume can also be used as a criterion for postoperative recurrence when the recurrence tumor volume exceeds 20mm3After that, the tumor is considered to have recurred. The tumor recurrence rate determined from tumor volume was consistent with that determined from fluorescence standards. In addition, the model group and blank gel group had faster growth of the recurrent tumors, indicating that the tumors grew rapidly without drug treatment. The body weight of the mice has no obvious change, which indicates the safety of the drug-loaded gel. Distal metastasis of tumors is also a critical issue in tumor therapy, and the lung was found to be the major organ for tumor metastasis by in vivo imaging of mice. Except for the doxorubicin/gemcitabine gel group, the other groups had different degrees of lung metastasis, 4 of the model groups had lung metastasis, 3 of the blank gel group had lung metastasis, 2 of the gemcitabine gel group had lung metastasis, 1 of the doxorubicin group had lung metastasis, 2 of the doxorubicin/gemcitabine solution group had lung metastasis, and the doxorubicin/gemcitabine gel completely inhibited lung metastasis of tumors. FIG. 9 shows the change in tumor volume after the recurrence of the tumor in mice, the tumor volume was more than 20mm3I.e. the tumor is considered to have recurred. Fig. 10 shows tumor lung metastasis observed by bioluminescence after 24 days of treatment.
Pathological and immunohistochemical staining also showed therapeutic effects of different drugs, since doxorubicin/gemcitabine gel group had no tumor recurrence and was not able to stain tumor tissues. Ki67, TUNEL staining all proves that the adriamycin gel group has stronger effect of inhibiting tumor growth. FIG. 11 shows immunohistochemical and pathological examination of anatomic tumor and cardiopulmonary tissues after treatment.
In conclusion, the adriamycin/gemcitabine gel completely inhibits postoperative recurrence and lung metastasis of breast cancer, and the reason for the adriamycin/gemcitabine gel is that the concentration of the drug in an operation cavity can be improved by locally implanting the drug-loaded injectable implantation in-situ hydrogel, the killing of residual tumor cells is improved by the synergistic effect of the two drugs, and in addition, the hyaluronic acid-drug copolymer released after the gel degradation increases the phagocytosis of the drug by the tumor cells, and the drug effect is improved.
Claims (10)
1. A medicine-carrying injectable implantation in-situ hydrogel is characterized in that a chemical bond crosslinking mode is adopted to form in situ at an injection site.
2. The drug-loaded injectable implantable in situ hydrogel of claim 1, wherein the chemical bond crosslinking is based on Schiff base reaction.
3. The drug-loaded injectable implantable in-situ hydrogel according to claim 1, wherein the chemical bond crosslinking manner is based on Schiff base reaction, and the speed and gel strength of the Schiff base reaction can be controlled by the content of aldehyde group and amino group in the gel precursor material and the concentration of the gel precursor material, wherein the concentration of the gel precursor material is selected from 1-20%.
4. The drug-loaded injectable implantable in situ hydrogel of claim 1 prepared by the steps of:
(1) the gel precursor material comprises aldehyde precursor material and amination precursor material;
(2) loading the drug or cell by covalent or non-covalent bonding;
(3) mixing two or more gel precursors carrying drugs to obtain the drug-carrying injectable in-situ hydrogel.
In the above step, the starting material of the gel precursor material is a polymer compound selected from a synthetic polymer compound, a polysaccharide polymer compound, and a protein polymer compound.
5. The drug-loaded injectable implantable in-situ hydrogel according to claim 4, wherein the gel precursor material is prepared from polysaccharide high molecular compounds through aldehyde modification and amination modification, and is mixed and gelled to form stable gel.
6. The drug-loaded injectable implantable in-situ hydrogel according to claims 4 and 5, wherein the gel precursor material is a polysaccharide high molecular compound with a molecular weight selected from 100kDa to 1600 kDa.
7. The drug-loaded injectable implantable in situ hydrogel of claim 1 loaded with drugs or cells for injection at various sites in the body for disease treatment, prevention and regeneration.
8. The drug-loaded injectable implantable in situ hydrogel of claim 1, wherein the drug loading means comprises covalent bonding means and physical encapsulation means.
9. The drug-loaded injectable implantable in-situ hydrogel according to claim 1, wherein the in-vivo implantation mode is an implantation mode of directly injecting the gel precursor material into the body, and the injection mode is selected from rapid injection after the gel precursor material is mixed and direct injection by a double-tube injection device.
10. The pre-loaded injectable implantable in situ hydrogel of claim 1, which is an doxorubicin/gemcitabine injectable implantable in situ hydrogel prepared as follows: using aldehydic hyaluronic acid obtained by oxidizing sodium periodate as an aldehydic precursor material, dissolving with phosphate buffer solution to obtain 80mg/mL aldehydic hyaluronic acid solution, dripping 8mg/mL adriamycin solution into the aldehydic hyaluronic acid solution under the stirring condition, and stirring for 12 hours at 37 ℃ in a dark place to obtain hyaluronic acid-adriamycin copolymer solution: and dissolving gemcitabine hydrochloride and carboxymethyl chitosan by using a phosphate buffer solution to obtain a solution containing 2mg/mL gemcitabine and 20mg/mL carboxymethyl chitosan, and simultaneously injecting the two solutions with equal volume into the cavity of the body by using two injectors to obtain the adriamycin/gemcitabine injectable implantation in-situ hydrogel.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010045805.3A CN111228212A (en) | 2020-01-16 | 2020-01-16 | Drug-loaded injectable implantation in-situ hydrogel |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010045805.3A CN111228212A (en) | 2020-01-16 | 2020-01-16 | Drug-loaded injectable implantation in-situ hydrogel |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111228212A true CN111228212A (en) | 2020-06-05 |
Family
ID=70876127
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010045805.3A Pending CN111228212A (en) | 2020-01-16 | 2020-01-16 | Drug-loaded injectable implantation in-situ hydrogel |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111228212A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112472666A (en) * | 2020-12-10 | 2021-03-12 | 北京大学第三医院（北京大学第三临床医学院） | Hydrogel drug loading system |
| CN113813443A (en) * | 2021-10-19 | 2021-12-21 | 蓝科医美科学技术(吉林)有限公司 | Glucan-based hydrogel dressing and preparation method thereof |
| CN114377139A (en) * | 2022-03-11 | 2022-04-22 | 四川省医学科学院·四川省人民医院 | Carrier, drug delivery system and application thereof |
| CN115350144A (en) * | 2022-07-28 | 2022-11-18 | 苏州弘森药业股份有限公司 | Metronidazole hydrogel and preparation method thereof |
| WO2023148351A1 (en) * | 2022-02-03 | 2023-08-10 | Gcs Institut De Cancerologie Strasbourg Europe | Pharmaceutical formulation for subcutaneous administration of proteins |
| CN117430831A (en) * | 2023-10-25 | 2024-01-23 | 吉林农业大学 | Gastrodia elata polysaccharide-based composite hydrogel and preparation method and application thereof |
| WO2024120364A1 (en) * | 2022-12-07 | 2024-06-13 | 青岛蓝谷多肽生物医药科技有限公司 | Hydrogel compound and use thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104479150A (en) * | 2014-10-29 | 2015-04-01 | 上海大学 | Preparation method of multiple cross-linked polysaccharide injectable hydrogel |
| CN108926714A (en) * | 2018-06-28 | 2018-12-04 | 哈尔滨医科大学 | A kind of high-molecular gel and preparation method thereof for irrigation of bladder targeting bladder cancer delivering pharmacological active substance |
-
2020
- 2020-01-16 CN CN202010045805.3A patent/CN111228212A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104479150A (en) * | 2014-10-29 | 2015-04-01 | 上海大学 | Preparation method of multiple cross-linked polysaccharide injectable hydrogel |
| CN108926714A (en) * | 2018-06-28 | 2018-12-04 | 哈尔滨医科大学 | A kind of high-molecular gel and preparation method thereof for irrigation of bladder targeting bladder cancer delivering pharmacological active substance |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112472666A (en) * | 2020-12-10 | 2021-03-12 | 北京大学第三医院（北京大学第三临床医学院） | Hydrogel drug loading system |
| CN113813443A (en) * | 2021-10-19 | 2021-12-21 | 蓝科医美科学技术(吉林)有限公司 | Glucan-based hydrogel dressing and preparation method thereof |
| WO2023148351A1 (en) * | 2022-02-03 | 2023-08-10 | Gcs Institut De Cancerologie Strasbourg Europe | Pharmaceutical formulation for subcutaneous administration of proteins |
| CN114377139A (en) * | 2022-03-11 | 2022-04-22 | 四川省医学科学院·四川省人民医院 | Carrier, drug delivery system and application thereof |
| CN114377139B (en) * | 2022-03-11 | 2023-07-04 | 四川省医学科学院·四川省人民医院 | Carrier, drug delivery system and application thereof |
| CN115350144A (en) * | 2022-07-28 | 2022-11-18 | 苏州弘森药业股份有限公司 | Metronidazole hydrogel and preparation method thereof |
| WO2024120364A1 (en) * | 2022-12-07 | 2024-06-13 | 青岛蓝谷多肽生物医药科技有限公司 | Hydrogel compound and use thereof |
| CN117430831A (en) * | 2023-10-25 | 2024-01-23 | 吉林农业大学 | Gastrodia elata polysaccharide-based composite hydrogel and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111228212A (en) | Drug-loaded injectable implantation in-situ hydrogel | |
| Li et al. | Advances in biodegradable and injectable hydrogels for biomedical applications | |
| US11672756B2 (en) | Temperature sensitive hydrogel composition including nucleic acid and chitosan | |
| JP3094074B2 (en) | Polysaccharide gel composition | |
| US9089512B2 (en) | Active scaffolds for on-demand drug and cell delivery | |
| US8771734B2 (en) | Sustained-release hydrogel preparation | |
| Zhan et al. | An injectable hydrogel with pH-sensitive and self-healing properties based on 4armPEGDA and N-carboxyethyl chitosan for local treatment of hepatocellular carcinoma | |
| CN107708675A (en) | The composition and kit of pseudoplastic behavior microgel matrix | |
| EP1891941A1 (en) | Aqueous gels comprising microspheres | |
| US20200164079A1 (en) | Anti-bacterial substance and composition thereof, medical and non-medical uses using the same, and products comprising said substance and compositions | |
| JP2008295885A (en) | Two-reaction agent type medical lyogel forming agent and hyaluronic acid gel obtained from the same | |
| Wu et al. | Polysaccharide-based tumor microenvironment-responsive drug delivery systems for cancer therapy | |
| CN110251457B (en) | Anti-tumor sustained-release implant with strong adhesion and hemostasis functions and preparation method thereof | |
| Qian et al. | Dual-modal imaging and synergistic spinal tumor therapy enabled by hierarchical-structured nanofibers with cascade release and postoperative anti-adhesion | |
| US20120035531A1 (en) | On-demand and reversible drug release by external cue | |
| US20230248642A1 (en) | Injectable high-drug-loaded nanocomposite gels and process for making the same | |
| WO2009116556A1 (en) | Pharmaceutical composition for injection | |
| WO2023071102A1 (en) | Cisplatin cross-linked protein hydrogel and preparation method | |
| CN111686075B (en) | In-situ hydrogel composition taking nano-micelle as cross-linking agent and application thereof | |
| JP6339062B2 (en) | Radiation / chemotherapy sensitizer for intratumoral injection that releases hydrogen peroxide slowly using hydrogel as carrier | |
| CN107019806B (en) | Double-drug-loading self-healing hydrogel system and preparation method thereof | |
| CN110951072A (en) | Compound with cell calcification inducing capability, preparation method and application thereof | |
| Goyal et al. | Hydrogel-bound cytotoxic drug delivery system for breast cancer | |
| Rostami | Chitosan‐based nanoparticles for drug delivery | |
| CN108992399B (en) | Preparation method and application of EPR effect enhanced combined drug system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20200605 |