CN110420335B - Preparation and application of nano immune preparation based on porous calcium carbonate - Google Patents
Preparation and application of nano immune preparation based on porous calcium carbonate Download PDFInfo
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- CN110420335B CN110420335B CN201810890136.2A CN201810890136A CN110420335B CN 110420335 B CN110420335 B CN 110420335B CN 201810890136 A CN201810890136 A CN 201810890136A CN 110420335 B CN110420335 B CN 110420335B
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- 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
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
- A61K33/10—Carbonates; Bicarbonates
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- 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
- A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6905—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
- A61K47/6911—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
- A61K47/6915—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome the form being a liposome with polymerisable or polymerized bilayer-forming substances, e.g. polymersomes
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Abstract
The invention belongs to the field of pharmaceutical preparations, and particularly relates to a nano immune preparation based on porous calcium carbonate, which is porous CaCO3The nano-particles are used as carriers, and an immune blocking inhibitor IDOi is loaded in the pore canal of the nano-particles and is coated by a liposome layer, and the surface of the liposome layer carries immune regulatory nucleic acid CpG ODNs. The nano immune preparation synthesized by the invention has good biocompatibility and high safety, has good effects of treating mouse breast cancer and preventing cancer recurrence, is simple and easy to realize in the preparation process, and has good application prospect when being applied to the field of antitumor drug preparation.
Description
Technical Field
The invention belongs to the field of pharmaceutical preparations, and particularly relates to a nano immune preparation which is porous CaCO3The nano-particles are used as carriers, immune blocking inhibitors IDOi are loaded in the pore canals of the nano-particles, immune regulatory nucleic acids CpG ODNs are carried on the surfaces of the nano-particles, and the synthesis method and the application thereof in the field of enhancing the curative effect of breast cancer and preventing the recurrence of the breast cancer are provided.
Background
Cancer is a malignant tumor that seriously threatens human health, and recurrence after treatment of a primary tumor is the leading cause of death in most cancer patients. The clinical routine treatment methods of tumors at present are mainly surgery, radiotherapy and chemotherapy, but the body still has residual cancer cells (i.e. micro-lesions) after the treatment by the treatment methods, and the micro-lesions are the most main reason of cancer recurrence. Therefore, the development of low-toxicity, highly effective therapies to treat cancer while destroying microscopic lesions and preventing recurrence of cancer is an urgent problem to be solved.
Immunotherapy of tumors is rapidly developing as a new generation of tumor treatment strategies and shows great potential in practical clinical applications. Can treat the tumor by adjusting or stimulating the body's own immune system, and can effectively prevent the recurrence of the tumor by utilizing the long-term memory effect of the body's own immune system. In recent years, various tumor vaccines have been developed to suppress tumors by activating patients' autoimmunity. The research shows that the prior commonly used immunization vaccine method has the following problems: first, the immune response induced by an immune vaccine is limited, and usually an immune adjuvant is added to enhance the immune response, but the immune adjuvant (such as aluminum hydroxide) has high toxicity. Secondly, when a tumor develops into a malignant state, most of the tumor-associated antigens are usually eliminated, which is a so-called tumor immune escape mechanism. And the immunosuppressive microenvironment at the tumor tissue is often difficult to break, which can easily lead to failure of tumor immunotherapy. Therefore, it is necessary and challenging to develop a safe and effective method to induce self-strong immune response, break the immunosuppressive microenvironment of tumors, improve the efficiency of cancer immunotherapy, and further prevent the recurrence of cancer by using the memory effect.
Disclosure of Invention
In order to overcome the defects of the clinical conventional cancer treatment method, the invention aims to provide a nano immune preparation based on porous calcium carbonate, and CaCO is used when the nano immune preparation circulates to a tumor site in blood3The nano material can be specifically dissolved in a weak acid environment, and CpG ODNs, IDOi and Ca are released at a tumor part2+The three components have in-situ synergistic effect to enhance the immune response of the organism, ensure the sustained occurrence of the immune response of tumor tissues and maintain the memory effect. The novel calcium carbonate nano preparation is applied to biological treatment, and has the effects of enhancing the efficiency of immunotherapy of cancers and preventing relapse of the cancers.
In a first aspect of the invention, a nano immune preparation based on porous calcium carbonate is provided, and the nano immune preparation comprises porous calcium carbonate nanoparticles, an immune blocking inhibitor IDOi, a liposome layer and immune regulatory nucleic acid CpG ODNs.
Preferably, in the immune preparation, the porous calcium carbonate nanoparticles are used as a drug carrier, the immune blocking inhibitor IDOi is loaded inside the pore channel of the particles, the porous calcium carbonate nanoparticles loaded with the immune preparation are coated by a liposome layer, and the surface of the liposome layer is modified by immune regulatory nucleic acids CpG ODNs.
Preferably, the liposome layer is composed of DPPC, cholesterol and DSPE-PEG.
In a second aspect of the present invention, a method for preparing a nano immune preparation based on porous calcium carbonate is provided, which comprises the following steps:
(1) synthesizing porous calcium carbonate nano-particles through gas diffusion reaction, placing the prepared porous calcium carbonate nano-particles into absolute ethyl alcohol to load IDOi to obtain CaCO3@IDOi；
(2) Modification of CaCO obtained in step (1) with the Liposomal layer3@ IDOi to CaCO3@ IDOi @ PEG @ PEI; the obtained CaCO3@ IDOi @ PEG @ PEI and CpG ODN are stirred in an aqueous solution to obtain the nano preparation.
Preferably, the preparation method comprises the following steps:
(1) adding CaCl2·H2O and NH4HCO3Simultaneously, placing the system in a vacuum drying chamber, keeping the whole system in a vacuum environment, and finally performing centrifugal separation to obtain calcium carbonate nano-particles; placing the calcium carbonate nano-particles obtained by separation into an absolute ethyl alcohol solution, stirring overnight, loading IDOi to obtain CaCO3@IDOi；
(2) CaCO prepared in the step (1)3Mixing ethanol solution of @ IDOi with DOPA, adding DPPC, cholesterol and DSPE-PEG to the above solution, spin-drying the organic solvent in the solution, dissolving the prepared nanoparticles in the aqueous solution, adding PEI, and stirring to prepare CaCO3@ IDOi @ PEG @ PEI; the obtained CaCO3@ IDOi @ PEG @ PEI and CpG ODN are stirred in an aqueous solution to obtain the nano preparation.
Further preferably, CaCl is added in step (1)2·H2O and NH4HCO3And simultaneously, placing the system in a vacuum drying chamber, setting the temperature to be 25-30 ℃, keeping the whole system in a vacuum environment for 20-30h, and then performing centrifugal separation at 10000-15000rpm to obtain the calcium carbonate nanoparticles.
Further preferably, the calcium carbonate obtained by separation in step (1)The nano particles are placed in absolute ethyl alcohol solution to load IDOi to obtain CaCO3@ IDOi the specific procedure is to stir calcium carbonate with an ethanolic solution of IDOi for at least 24 hours.
Further preferably, CaCl is used in step (1)2·H2O：NH4HCO3The mass ratio is (0.5-2): (30-50) CaCO3And the mass ratio of IDOi is (35-45): (0.5-2.5); more preferably, CaCl2·H2O：NH4HCO3The mass ratio of CaCO to CaCO is 1:403And IDOi in a mass ratio of 40: 1.
Further preferably, CaCO in step (2)3@ IDOi: DOPA: DPPC: cholesterol: DSPE-PEG: PEI: the CpG ODN mass ratio is (35-45): (12-18): (12-18): (6-10): (30-35): (0.2-1); further, is (38-42): (14-16): (14-16): (7-9): (31-33): (0.3-0.5); specifically, (40-41): 16: 16: 8: 32: 0.48.
the third aspect of the invention provides application of the nano immune preparation based on porous calcium carbonate in the field of preparing medicaments for preventing and treating tumors.
Preferably, the tumor prevention and treatment drug is a breast cancer prevention and treatment drug.
Preferably, the tumor prevention and treatment drug is a drug in the field of enhancing the curative effect of breast cancer and preventing tumor recurrence.
The invention has the beneficial effects
1. The porous calcium carbonate nano immune preparation has good treatment effect, low toxicity and high immune activation efficiency, can eliminate residual micro focus in the traditional operation treatment method, improves the immune treatment efficiency of breast cancer, effectively prevents the recurrence of the breast cancer, and can be used for organism treatment.
2. The preparation of the invention has simple synthesis method, no toxic reagent is introduced, the prepared nano preparation has no toxic reagent residue problem, and the safety is high. The preparation process does not need complex operations such as high-temperature heating and the like, and is suitable for large-scale production.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application.
FIG. 1 is a graph of tumor volume change and body weight change in mice;
FIG. 2 is an image of a live body of an experimental mouse for preventing tumor recurrence in example 2.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific examples and comparative examples.
Example 1 preparation of a Nanoparticulation based on porous calcium carbonate
(1) Synthesis of porous CaCO by gas diffusion reaction3Nano-particles: adding 220 mg of CaCl2·H2O and 8 g NH4HCO3Simultaneously placing in a vacuum drying chamber, controlling the temperature at 25 deg.C, maintaining the whole system in vacuum environment for 24 hr to obtain CaCO3The nanoparticles were separated by centrifugation at 12000 rpm. Then CaCO in absolute ethyl alcohol3Loading IDOi into the channels to obtain CaCO3@ IDOi (abbreviated CaI).
(2) The nanoparticles are modified with liposomes. First, 20 mg of CaI in ethanol and 2 mg of DOPA were mixed. Then the DPPC is put into the reactor,cholesterol and DSPE-PEG were added to the above solution at a ratio of 4:4:2M, the solvent was spun off using a rotary evaporator, and the resulting nanoparticles were dissolved in an aqueous solution for further use. Then, the obtained nanoparticles were stirred with 200 mg of PEI to prepare CaCO3@ IDOi @ PEG @ PEI (abbreviated CaIP).
(3) The CaIP described above was stirred with 12OD CpG ODN in aqueous solution to give the final product, abbreviated CaIPC.
Comparative example 1
As a contrast, the invention synthesizes a nano immune preparation (LIPC) only taking liposome as a carrier, IDOi is loaded into the liposome, DPPC, cholesterol and DSPE-PEG are mixed according to the proportion of 4:4:2M, added into an organic solution, stirred, then dried in a rotary mode, and then 12OD CpG ODN is modified, the prepared nano immune preparation is centrifuged for 10 minutes at 8000rpm and dispersed in physiological saline, and the prepared nano immune preparation is prepared into LIPC stock solution to be stored for later use.
Example 2 mouse model building experiment
2.1 in vivo assay
Selecting female Balb/c mice as a research object, preparing a tumor-bearing mouse model by injecting mouse breast cancer cells subcutaneously until the tumor grows to 50mm3Administration is initiated. The administration groups were CaIPC (experimental group), LIPC, and physiological saline, respectively. Tumor-bearing mice were dosed intravenously, and tumor volumes were measured and weighed every other day during the experimental period. Experimental results show that the growth speed of the mouse tumor treated by CaIPC in example 1 is the slowest, and the invention can effectively inhibit the tumor growth in a mouse model. In contrast, the LIPC group showed not very excellent tumor-inhibiting effect compared to the saline group, and this result indicates that only the group treated by the present invention showed the optimal tumor-treating effect. Body weight is an important parameter for assessing the systemic toxicity of the material to the body, and the body weight of mice in all groups does not change significantly with time, which means that the treatment with the present invention does not cause significant side effects on the mice, and is suitable for tumor treatment at the living body level (fig. 1).
2.2 experiment on prevention of tumor recurrence
In order to study the effect of the invention on preventing tumor recurrence after actual surgical treatment, the invention establishes a tumor postoperative recurrence model. First, mouse breast cancer cells were inoculated into the axilla of female Balb/c mice, and mice tumors were surgically excised leaving residual tumor lesions. The material of the invention is injected into vein and tumor side after operation for further treatment after operation, and the tumor recurrence condition of the mouse is observed. The experimental results show that the mice in the saline group had recurrent tumors. The CaIPC experimental group has no tumor recurrence by intravenous injection or paraneoplastic injection, which shows that the invention has the capacity of obviously inhibiting the tumor recurrence (figure 2).
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (11)
1. The nano immune preparation based on the porous calcium carbonate is characterized by comprising porous calcium carbonate nanoparticles, an immune blocking inhibitor IDOi, a liposome layer and immune regulatory nucleic acid CpG ODNs for modification;
the preparation method of the nano immune preparation based on porous calcium carbonate comprises the following steps:
(1) adding CaCl2·H2O and NH4HCO3Meanwhile, the calcium carbonate nano particles are placed in a vacuum drying chamber, the whole system is kept in a vacuum environment for a period of time, and then the calcium carbonate nano particles are prepared through centrifugal separation; putting the calcium carbonate nano-particles obtained by separation into an absolute ethyl alcohol solution for loading IDOi to obtain CaCO3@IDOi；
(2) CaCO prepared in the step (1)3Mixing ethanol solution of @ IDOi with DOPA, adding DPPC, cholesterol and DSPE-PEG to the above solution, spin-drying the organic solvent in the solution, dissolving the prepared nanoparticles in the aqueous solution, adding PEI, and stirring to prepare CaCO3@ IDOi @ PEG @ PEI; the CaCO is3@ IDOi @ PEG @ PEI and CpG ODN are stirred in aqueous solution to obtain the productA nano-formulation;
in the step (1), the CaCl2·H2O：NH4HCO3The mass ratio is (0.5-2): (3-5) CaCO3And the mass ratio of IDoi is (35-45): (0.5-2.5);
CaCO in step (2)3@ IDOi: DOPA: DPPC: cholesterol: DSPE-PEG: the mass ratio of PEI is (35-45): (12-18): (12-18): (6-10): (30-35): (0.2-1).
2. The nano-immune preparation as claimed in claim 1, wherein the immune preparation uses porous calcium carbonate nano-particles as carriers, the immune blocking inhibitor IDOi is loaded inside the pore channels of the porous calcium carbonate nano-particles, and after the porous calcium carbonate nano-particles are coated by the liposome layer, the immune regulatory nucleic acids CpG ODNs are modified on the surface of the liposome layer.
3. The nanoimmune formulation of claim 1, wherein the liposome layer consists of DPPC, cholesterol and DSPE-PEG.
4. A preparation method of a nano immune preparation based on porous calcium carbonate is characterized by comprising the following steps:
(1) adding CaCl2·H2O and NH4HCO3Meanwhile, the calcium carbonate nano particles are placed in a vacuum drying chamber, the whole system is kept in a vacuum environment for a period of time, and then the calcium carbonate nano particles are prepared through centrifugal separation; putting the calcium carbonate nano-particles obtained by separation into an absolute ethyl alcohol solution for loading IDOi to obtain CaCO3@IDOi；
(2) CaCO prepared in the step (1)3Mixing ethanol solution of @ IDOi with DOPA, adding DPPC, cholesterol and DSPE-PEG to the above solution, spin-drying the organic solvent in the solution, dissolving the prepared nanoparticles in the aqueous solution, adding PEI, and stirring to prepare CaCO3@ IDOi @ PEG @ PEI; the CaCO is3@ IDOi @ PEG @ PEI and CpG ODN are stirred in an aqueous solution to obtain the nano preparation;
in the step (1), the step (c),the CaCl is2·H2O：NH4HCO3The mass ratio is (0.5-2): (3-5) CaCO3And the mass ratio of IDoi is (35-45): (0.5-2.5);
CaCO in step (2)3@ IDOi: DOPA: DPPC: cholesterol: DSPE-PEG: the mass ratio of PEI is (35-45): (12-18): (12-18): (6-10): (30-35): (0.2-1).
5. The method according to claim 4, wherein the step (1) comprises the following steps: adding CaCl2·H2O and NH4HCO3And simultaneously placing the system in a vacuum drying chamber, setting the temperature to be 25 ℃, keeping the whole system in a vacuum environment for 24 hours, and then performing centrifugal separation at 10000-15000rpm to obtain the calcium carbonate nanoparticles.
6. The method of claim 4, wherein the calcium carbonate nanoparticles obtained by the separation in step (1) are loaded with IDOi in an absolute ethanol solution to obtain CaCO3The specific steps of @ IDOi are as follows: the calcium carbonate was stirred with an ethanolic solution of IDOi for at least 24 hours.
7. The method of claim 4, wherein the CaCl is prepared by2·H2O：NH4HCO3The mass ratio is 1:4, CaCO3And IDOi in a mass ratio of 40: 1.
8. The method of claim 4, wherein the reaction is performed using CaCO3@ IDOi: DOPA: and (2) DPPC: cholesterol: DSPE-PEG: the mass ratio of the PEI is (38-42): (14-16): (14:16): (7-9): (31-33): (0.3-0.5).
9. Use of the nano immune preparation based on porous calcium carbonate according to any one of claims 1 to 3 in the field of preparing medicaments for preventing and treating tumors.
10. The use of claim 9, wherein the anti-neoplastic agent is an anti-breast cancer agent.
11. The use of claim 9, wherein the tumor prevention and treatment drug is a drug in the field of enhancing the efficacy of breast cancer and preventing tumor recurrence.
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