CN111803468B - Nasal cavity film spraying agent for targeting, slow-release, film-forming and virus and bacteria inhibiting and preparation method thereof - Google Patents
Nasal cavity film spraying agent for targeting, slow-release, film-forming and virus and bacteria inhibiting and preparation method thereof Download PDFInfo
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- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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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
- A61K33/18—Iodine; Compounds thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/61—Myrtaceae (Myrtle family), e.g. teatree or eucalyptus
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- 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
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
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- 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
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
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- A—HUMAN NECESSITIES
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- 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
- A61K47/38—Cellulose; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0043—Nose
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
- A61K9/7015—Drug-containing film-forming compositions, e.g. spray-on
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
A nasal cavity spray agent for targeting, slow release, film forming and virus and bacteria inhibition and a preparation method thereof relate to the field of biology. The invention aims to solve the problems that the prior common film spraying agent: the targeted inhibition and killing of virus and bacteria can not be realized, the action time is short, the living environment of the virus can not be effectively isolated for a long time, and the virus replication inhibition efficiency is low. The invention adopts PVP to form a layer of soluble protective film and utilizes the slow release effect of CMC-NA. The living environment of the virus and the bacteria is effectively destroyed through a physical and biological double antibacterial mechanism, and the effects of resisting inflammation, inhibiting bacteria, isolating and killing virus microorganisms (including bacteria, viruses and the like) are achieved. By targeting agents of angiotensin 2 inhibitor and EDTA2Na, the dual advantages of a process prescription and a component mechanism combining targeting and sustained-release drug delivery are realized. The invention is applied to the field of medicine.
Description
Technical Field
The invention relates to the field of biology, in particular to a nasal cavity film spraying agent for targeting, slow release and film formation for inhibiting viruses and bacteria and a preparation method thereof.
Background
Viruses are microorganisms composed of a single nucleic acid molecule (DNA or RNA) and proteins, which have no cellular structure but genetic, replicative, etc. vital characteristics. The virus is characterized in that: (1) the volume is small, the diameter is 20-200 nm, and the bacteria can pass through a bacteria filter when the observation is carried out by an electron microscope; (2) the structure is simple, the virus is a non-cell type microorganism, the virus has no cell wall, cell membrane or even no cell nucleus, and only a layer of protein capsid is coated outside the genetic material RNA or DNA, and the capsid has the functions of (1) having antigenicity; (2) protecting the nucleic acid; (3) mediating binding of the virus to the host cell; (3) strict intracellular parasitism cannot be copied, most viruses are in a dormant state, and once meeting cells, protease is used for dissolving cell walls or cell membranes to directly enter the cells; (4) the virus is replicated and propagated by using self nucleic acid as a template, and the infection and mass propagation of host cells are completed through 5 stages of adsorption, penetration, shelling, biosynthesis, assembly and release after encountering the host cells. Viruses are of a wide variety, and the international committee for viral classification (ICT V) classifies all known viruses into several broad classes, based on nucleic acid type, DNA viruses, RNA viruses, DNA and RNA retroviruses. Viruses are widely distributed in nature, and can infect bacteria, fungi, plants, animals and humans, often causing host morbidity.
SARS-CoV-2 is a new type coronavirus belonging to beta genus, which has envelope, and the granule is round or oval, usually polymorphic, and has a diameter of 60-140 nm. The gene characteristics are obviously different from SARS-CoV and MERS-CoV. The present research shows that the homology with bat SARS-like coronavirus (bat-SL-CoVZC 45) is up to above 85%, and based on the present epidemiological investigation, the latent period is generally 3-7 d, and the longest period is not more than 14d. The new coronavirus is infectious in latent stage, acute pneumonia can be caused by infection, and clinical manifestations (1) fever and/or respiratory symptoms; (2) having the above-mentioned pneumonitis imaging characteristics; (3) The total number of leukocytes is normal or reduced, or the lymphocyte count is reduced, in the early stages of disease. The population is generally susceptible, the disease is serious after the infection of the old, the immunocompromised and the patients with basic diseases, and the disease of a few patients is critical. Death cases are often seen in the elderly and in patients with chronic underlying disease.
At present, the first stage of effective protection and lasting fighting is to shield and isolate new coronavirus from entering human body, the nasal cavity and the oral cavity are the first door for the virus to enter the human body, particularly the nasal cavity is the first cavity organ for the human body to store dirt and the oral cavity, and the data shows that the influenza virus is spread by coughing or sneezing in people. These coughs and sneezes can eject 10 ten thousand saliva droplets at a time, we count that each droplet contains roughly one thousand viruses, if i sneeze, i can eject 10000 viruses, and these droplets advance in the air at an excessive speed of 160 km per hour, which can advance 40 meters. What is this a concept? Unfortunately if I catch a cold today, I sneeze through it, all people in the room may become infected. A droplet of fluid in a sneeze carries thousands of intruders who pass through the weak points of the body and the functional orifices nasal and oral cavities that regularly feed and breathe, which are very delicate and fatal machines. For example, adenovirus is one of the 20 viruses that infect humans and can cause a variety of diseases, from the common cold to pneumonia. This is why, among many precautions, the physical barrier and barrier of the "respirator" is particularly important, i.e. when coughing or sneezing, the nasal cavity is shielded by paper towels or clothes to reduce the spread of germs.
The proliferation of the virus, which first recognizes a specific site attached to the surface of the host cell, may result in selective invasion of a specific cell by recognizing the same or a specific cell surface protein by a different virus. It was found that SARS-CoV-2 virus binds to the angiotensin converting enzyme 2 (ACE 2) receptor on human cells via its spike protein. Whereas TMPRSS2 enzyme helps to activate the spike protein of the new coronavirus, thereby helping the virus to enter the host cell, far less than 10% of respiratory and intestinal cells in humans produce ACE2 and TMPRSS2 simultaneously, and these cells are divided into three types: mucus secreting goblet cells (goblet cells) in the nasal cavity, type II alveolar cells (type II pneumocytes) in the lung and absorptive intestinal epithelial cells. Therefore, SARS-CoV-2 attacks weak nasal mucosa cells and respiratory system cells specially, so the first step of preventing the invasion of fatal viruses is to protect the nasal mucosa of people and isolate the invasion of viruses, and the nasal cavity film spraying agent which is a novel targeted, sustained-release and film-forming agent for inhibiting viruses (bacteria) is one of favorable weapons which can isolate the viruses from directly invading the nasal mucosa cells, destroy the living environment of the viruses, further stick the viruses to prevent the viruses from approaching cell surface proteins and further cannot enter cells for replication.
Disclosure of Invention
The invention aims to solve the problems that the prior common film spraying agent: the film-spraying agent has the advantages of no target inhibition and killing of viruses and bacteria, short action time, no long-term effective isolation of the virus living environment and low virus replication inhibition efficiency, and forms a novel target-targeted slow-release film-forming agent which can isolate viruses, protect mucosa, stick the viruses to make the viruses incapable of replicating and destroy the virus living for a long time.
The invention relates to a nasal cavity spray film agent for targeting, slow release and film formation for inhibiting viruses and bacteria, which is prepared from A, B and a C phase component, wherein the three-phase volume ratio is A: B: C = (4-6): (0.5-1.5): (4-6);
wherein, phase A comprises benzalkonium bromide, borneolum Syntheticum, mentholum, tween-80, 95% ethanol and 0.9% sodium chloride;
phase B is eucalyptus oil;
phase C comprises polyvinylpyrrolidone, polyvinylpyrrolidone iodine, sodium carboxymethylcellulose, angiotensin II inhibitor, disodium edetate and 0.9% sodium chloride.
Furthermore, every 500mL of phase A contains benzalkonium bromide 0.1-0.3g, borneolum 0.5-2g, mentholum 0.2-0.6g, tween-80 15-24g, 95% ethanol 10mL, and sodium chloride 0.9% in balance.
Furthermore, every 500mL of phase A contains benzalkonium bromide 0.2-0.3g, borneolum Syntheticum 1-2g, mentholum 0.3-0.5g, tween-80 18-22g, 95% ethanol 10mL, and sodium chloride 0.9% in balance.
Furthermore, each 500mL of the C phase contains 30-70g of polyvinylpyrrolidone, 0.5-1g of polyvinylpyrrolidone iodine, 5-10g of sodium carboxymethylcellulose, 20-40mg of angiotensin II inhibitor, 10-30mg of disodium ethylene diamine tetraacetate and the balance of 0.9% of sodium chloride.
Furthermore, every 500mL of the C phase contains 40-60g of polyvinylpyrrolidone, 0.8-1g of polyvinylpyrrolidone iodine, 8-10g of sodium carboxymethylcellulose, 30-40mg of angiotensin II inhibitor, 20-30mg of disodium ethylenediamine tetraacetic acid and the balance of 0.9% sodium chloride.
The invention relates to a method for targeting, slowly releasing and forming a film to inhibit viruses and bacteria by a nasal cavity film spraying agent, which is carried out according to the following steps:
1. preparation of phase A
1) Dissolving Borneolum Syntheticum and Mentholum in 95% ethanol, adding Tween-80, and mixing to obtain solution A;
2) Adding benzalkonium bromide into the solution A for uniform dispersion;
3) Finally, dissolving with 0.9% sodium chloride, stirring and mixing uniformly by magnetic force, and subpackaging to obtain phase A;
2. preparation of phase C
4) Completely dissolving polyvinylpyrrolidone and sodium carboxymethylcellulose in 0.9% sodium chloride to obtain solution B;
5) Adding polyvinylpyrrolidone iodine, an angiotensin II inhibitor and disodium ethylene diamine tetraacetate into the solution B, and uniformly mixing by magnetic stirring;
6) Finally, uniformly mixing with 500mL of 0.9% sodium chloride solution, and subpackaging to obtain a phase C;
3. preparation of film spraying agent
7) Adding the phase B into the phase A while stirring, and completely homogenizing the liquid by using a homogenizer after the phase B is completely added to form a water-in-oil suspension;
8) Adding phase C into the water-in-oil suspension, stirring, homogenizing, microemulsifying, and degassing to obtain water-in-oil-in-water uniform nasal spray;
wherein the volume ratio of the phase A to the phase B to the phase C is (4-6): (0.5-1.5): (4-6); phase B is eucalyptus oil.
Furthermore, every 500mL of phase A contains benzalkonium bromide 0.1-0.3g, borneolum 0.5-2g, mentholum 0.2-0.6g, tween-80 15-24g, 95% ethanol 10mL, and sodium chloride 0.9% in balance.
Furthermore, every 500mL of phase A contains benzalkonium bromide 0.2-0.3g, borneolum Syntheticum 1-2g, mentholum 0.3-0.5g, tween-80 18-22g, 95% ethanol 10mL, and sodium chloride 0.9% in balance.
Further, every 500mL of the C phase contains 30-70g of polyvinylpyrrolidone, 0.5-1g of polyvinylpyrrolidone iodine, 5-10g of sodium carboxymethylcellulose, 20-40mg of angiotensin II inhibitor, 10-30mg of disodium ethylenediamine tetraacetic acid and the balance of 0.9% sodium chloride.
Furthermore, every 500mL of the C phase contains 40-60g of polyvinylpyrrolidone, 0.8-1g of polyvinylpyrrolidone iodine, 8-10g of sodium carboxymethylcellulose, 30-40mg of angiotensin II inhibitor, 20-30mg of disodium ethylenediamine tetraacetic acid and the balance of 0.9% sodium chloride.
The technical mechanism of the invention is as follows:
the nasal administration film spraying agent is a combination of the advantages of film agents and spraying agents, is prepared into spray and is sprayed on skin mucous membranes, and a layer of soluble protective film can be formed on the surfaces of the nasal mucosa due to the action mechanism of a polyvinylpyrrolidone (PVP) film forming agent in the formula, so that mucosal cells are isolated and protected from being attacked by viruses and bacteria, and the viruses are stuck and cannot be copied; because the slow release action of the slow release film-forming agent CMC-NA in the prescription can prolong the retention time of the medicine in the mucous membrane, relieve the release and improve the medicine effect, effectively destroys the living environment of virus and bacteria through a physical and biological double antibacterial mechanism, plays roles of resisting inflammation, inhibiting bacteria, isolating and killing virus and microorganism (including bacteria, viruses and the like), and forms a biological molecular membrane which is a medicine carrier, can play the protection and barrier roles through film formation, can reduce water evaporation, promotes the skin hydration and other efficacies, has quantitative output, good compliance and improves the medicine bioavailability; due to scientific dispensing of the angiotensin 2 inhibitor and EDTA2Na serving as targeting agents in the prescription, the dual advantages of a process prescription and a component mechanism combining targeting and sustained-release administration are realized. 1) broad-spectrum isolation is different from the common traditional film spraying agent, but the target can not carry out isolation, inhibition and killing on certain viruses; 2) The action time is short, and the living environment of the virus cannot be effectively isolated for a long time; 3) The virus inhibiting component is complex, the inhibiting efficiency is not high, the effect on human bodies can be achieved only by high concentration, but the use frequency is high, and the possible accumulative side effect of the human bodies is increased.
The invention has the following beneficial effects:
1. the targeted technology is introduced: the components contain a targeted ACE2 inhibitor, and the targeted ACE2 positive cells are inhibited to be combined with virus S protein, so that the lethal viruses such as new coronavirus, SARs, MERS and the like are prevented from being directly combined with ACE2 receptors on the surfaces of nasal mucosa cells;
2. the slow release technology is applied as follows: the long-acting film-forming agent contains sustained-release agent sodium carboxymethyl cellulose, polyvinylpyrrolidone, eucalyptus oil and the like, and further destroys the living environment of viruses and bacteria and isolates the invasion mucosal cells of germs while preserving moisture;
3. film formation protection and gas permeation coexistence: the antivirus and bacterial medicaments are ensured to enter nasal mucosa and permeate into nasal cells, and the invasion of external microorganisms can be isolated;
4. the multi-component synergistic effect is as follows: has the synergistic effect of abundant antiviral, antibacterial, resuscitation inducing drugs and herbal essence (Borneolum Syntheticum, menthol, oleum Eucalypti, and vinylpyrrolidone iodine) for killing virus and bacteria.
5. The preparation process of the invention has the characteristics that: the innovation is taken as a base point to break through the prior art of a plurality of preparations such as the traditional process, integrated targeting, slow release and the like, and the preparation process of the product is compounded and assisted by adopting a plurality of international advanced preparation technologies such as advanced differential dissolution, fractional phase-splitting fixed dissolution, high-speed magnetic molecular shearing, high-efficiency homogeneous mixing and the like.
Drawings
FIG. 1 is a flow chart of the process for preparing the nasal cavity administration film spraying agent of the invention;
FIG. 2 is a photograph of the nasal cavity administration film spray prepared in example 1; wherein, A is a diagram (50X) of the nasal cavity film spraying agent film, B is a diagram (50X) of purified water;
FIG. 3 is a photograph of a bacteriostatic experiment; wherein, the left plate is a test sample, the middle plate is a test sample control group, and the right plate is positive;
FIG. 4 is a graph showing the effect of virus inhibition; wherein, A: cell status (white light, 100 ×) of 293T cells infected in culture systems without membrane spray agent, B: cell status (fluorescence, 100 ×) of 293T cells after infection in culture system without membrane spray, C: cell status (white light, 100 ×) of 293T cells infected in culture system containing film spray agent, D: a graph of the cell state (fluorescence, 100 x) of 293T cells infected in a culture system containing a film spraying agent;
FIG. 5 is a graph of the release rate of the nasal spray prepared in example 1;
FIG. 6 is a graph of the viral titer of a nasal delivery spray prepared in example 1;
FIG. 7 is a graph showing the antiviral results of the nasal spray film prepared in example 1; wherein, A blank group, state of BEAS2B cells without virus infection (100X), state of B cells with virus infection (100X), state of C cells with virus infection (100X), state of D cells with virus infection (100X) with virus infection (5H BEAS 2B), state of D cells with virus infection (100X) with virus injection (2H BEAS 2B) after the spray agent of example 1 is killed, and state of E cells with virus injection (5H BEAS 2B) after the spray agent of example 1 is killed (100X);
FIG. 8 is a graph showing the oxygen passage of nasal administration spray.
Detailed Description
It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples for carrying out the invention, and that various changes in form and details may be made therein without departing from the spirit and scope of the invention in practice.
To make the objects, aspects and advantages of the embodiments of the present invention more apparent, the following detailed description clearly illustrates the spirit of the disclosure, and any person skilled in the art, after understanding the embodiments of the disclosure, may make changes and modifications to the technology taught by the disclosure without departing from the spirit and scope of the disclosure.
The exemplary embodiments of the present invention and the description thereof are provided to explain the present invention and not to limit the present invention.
Example 1
The preparation process of the nasal cavity spray agent for targeting, slow release and film formation for inhibiting viruses and bacteria comprises the following steps:
1. the phase A component preparation method comprises the following steps:
1) Dissolving Borneolum Syntheticum and Mentholum in 95% ethanol, and adding Tween-80 to obtain solution A;
2) Adding the benzalkonium bromide with the prescription amount into the solution A and uniformly dispersing the benzalkonium bromide into the solution A;
3) And finally, shearing, stirring and mixing uniformly by using 500mL of high-speed magnetic force of 0.9% sodium chloride solution, and subpackaging to obtain the phase A component of the subentry solution.
2. The preparation method of the phase C component comprises the following steps:
4) Completely dissolving polyvinylpyrrolidone and CMC-Na by using 0.9 percent of sodium chloride to prepare a solution B;
5) Adding prescribed amounts of povidone iodine, angiotensin inhibitor 2 and EDTA2Na into the solution B, shearing, stirring and mixing uniformly at a high speed by magnetic force;
6) And finally, uniformly mixing with 500mL of 0.9% sodium chloride solution, and subpackaging to obtain the itemized and dissolved C phase component.
3. And (3) forming of a film spraying agent:
7) Adding the component B into the phase A component which is subjected to item determination and dissolution while stirring;
8) After the addition is completed, thoroughly homogenizing the liquid by a high-speed homogenizer to form water-in-oil suspension;
9) Adding C phase component dissolved according to terms into the suspension, stirring at high speed, homogenizing, micro-emulsifying, and degassing to obtain w/o/w (water-in-oil-in-water) homogeneous three-phase nasal spray.
In the nasal spray agent of the present example, the volume ratio of the phase a, the phase B and the phase C is a ratio of B: C =5:1:5.
phase A component:
| serial number | Name (R) | Amount of prescription | Function of |
| 1 | Benzalkonium bromide | 0.2g | Antiviral and bacteriostatic agent |
| 2 | Borneol (borneol) | 1g | Antibacterial, antiinflammatory, and resuscitation inducing effects |
| 3 | Menthol crystal | 0.4g | Anti-inflammatory and flavoring agent |
| 4 | Earth temperature-80 | 20g | Cosolvent |
| 5 | 95% ethanol | 10mL | Disinfecting and dissolving agent |
| 6 | 0.9% sodium chloride | Blending to 500mL | Solvent(s) |
B phase component:
c phase component
| Serial number | Name (R) | Amount of prescription | Function of |
| 1 | Polyvinylpyrrolidone (PVP) | 50g | Film-forming agent |
| 2 | Povidone iodine | 0.8g | Sterilizing and killing virus |
| 3 | CMC-Na | 7.5g | Sustained release agent |
| 4 | Angiotensin II inhibitors | 30mg | Targeted inhibition of ACE2 activity |
| 5 | EDTA2Na | 20mg | Targeted inhibition of ACE2 activity |
| 6 | 0.9% sodium chloride | Blending to 500mL | Solvent(s) |
The effect of the nasal cavity spray agent for targeting, slow release and film formation for inhibiting viruses and bacteria prepared in the example 1 is verified:
1. and (3) film forming effect verification:
the nasal cavity film spraying agent is sprayed on a slide glass with the size of 25.4mm multiplied by 76.2mm, the slide glass is placed in an incubator with the temperature of 30 ℃ to be dried, then the slide glass is placed under a Leica positive microscope, the objective lens is 5 times, the ocular lens is 10 times, the aperture and the focal length are adjusted, and the picture analysis is carried out on the expression morphological structure, and the result is shown in figures 2 and 3.
Fig. 2 shows the state of the nasal cavity spray film agent of the present embodiment with regular edge, uniform and dense film formation, and no uneven distribution of visible substances in the film, wherein arrow a in fig. 2 is the film formation edge of the nasal cavity spray film agent of the present embodiment, and arrow B in fig. 2 is the state of non-smooth edge.
2. Nasal cavity spray film agent stability test:
the nasal cavity spray film agent is placed in several environments of 4 degrees, 20 degrees, 37 degrees and 40 degrees for 24 hours, a centrifugal acceleration test is carried out for 10 minutes at corresponding temperature, after the test, the sample layering condition and precipitates are analyzed, and the condition of a key ingredient benzalkonium bromide in the nasal cavity spray film agent is quantitatively analyzed by adopting chromatography, wherein the condition is shown in table 1.
TABLE 1 analysis of benzalkonium bromide in nasal spray
The results in Table 1 show that the film-forming compositions of this example are stable.
3. The bacteriostatic effect test:
microorganism experiment comparison method:
1) And (3) testing the sample: taking 0.5mL of the product to a sterile plate with a diameter of 90mm, preparing 2 plates in parallel, adding tryptone soy peptone agar medium with a temperature not higher than 45 ℃ respectively, placing in a 33 ℃ incubator, and culturing for 3 days.
2) Test article control group: mixing 0.5mL of the product and 1mL of golden yellow grape ball into sterile plates with diameter of 90mm, preparing 2 plates in parallel, adding tryptone soy peptone agar medium with temperature not higher than 45 ℃, placing in an incubator with temperature of 33 ℃, and culturing for 3 days.
3) Positive: taking 1mL of golden yellow grape ball (the bacterial content is less than 100 cfu), putting the golden yellow grape ball into a sterile plate with the diameter of 90mm, preparing 2 plates in parallel, respectively adding trypticase soy peptone agar culture medium with the temperature not more than 45 ℃, putting the mixture into a 33 ℃ incubator, and culturing for 3 days.
The results are shown in FIG. 3. As shown in FIG. 3, the nasal cavity spray agent can inhibit the growth of bacteria.
4. The film spray of example 1 significantly inhibited infection of cells by viruses:
inoculating cells: 293T cells with good growth status were selected, counted and counted at 1X 10 5 Density per mL, seeded in 6cm dishes. Normal medium (90% DMEM +0% FBS) as control group, and 10% film-spraying agent as test group.
Cell infection: 10uL of lentivirus empty vector stock solution with EGFP fluorescent marker is added into two groups of culture media and cultured for 1 day at 37 ℃. And observing the invasion condition of the cells under a fluorescence microscope. The results are shown in FIG. 4. In fig. 4, a: cell status after infection of 293T cells in a culture system without a membrane spray (white light, 100 ×); b: cell status after infection of 293T cells in culture system without membrane spray (fluorescence, 100 ×); c: cell status (white light, 100 ×) after 293T cells in the culture system containing the film spraying agent are infected; d: cell status after infection of 293T cells in the culture system with the membrane spray (fluorescence, 100X). It can be seen that the cell infection ability of the virus to the cells is significantly reduced by the treatment of the film spraying agent of the embodiment.
5. Measurement of Release degree
Randomly selecting 3 batches of nasal cavity spray film agent, standing for 10min, 30 min, 60min, 90 min and 120min, and taking distilled water as test solution. The absorbance was measured at a wavelength of 256nm with an ultraviolet-visible spectrophotometer, respectively. And respectively calculating the slow-release degrees at different times. The results are shown in FIG. 5.
6. Nasal mucosa protection test
WDVE-6MeV linear accelerator 8Gy is selected to irradiate the nasal cavity of the animal, and a nasal mucosa injury model is established. Rats were divided into 3 groups, blank (no treatment), model (nasal mucosa injury), and spray (nasal mucosa injury after nasal spray). After 24h, the nasal cavity condition of the animals is observed, the animals are killed, and blood and nasal cavity tissues are collected.
Test results
1) And (3) animal observation: after the model group animals are irradiated, the nasal mucosa has the conditions of congestion and the cilia of the nasal cavity drop, and the appetite is poor and the animals are not good. Animals in the blank and spray groups were not abnormal.
2) Change of physiological index
7. After the film spraying agent is isolated, the virus titer is obviously reduced:
inoculating cells: 293T cells with good growth status were selected, counted and counted at 1X 10 5 Density per mL, seeded in 6cm dishes. Put in 37℃，5％CO 2 Culturing in an incubator.
Preparing a film spraying agent: spraying film-spraying agent into three holes and spraying normal saline into three holes in a 24-well plate, drying in a 37-degree incubator, and forming a film. After 0.5mL of lentivirus suspension was added to each well and allowed to act in the well plate for 24 hours, the virus suspensions were collected separately.
Preparing a virus suspension diluent: the virus suspension collected above was diluted in EP tubes with 293T cell complete medium in 10-fold gradient, 10 dilutions in series, as follows: for each virus, 10 1.5mL EP tubes were prepared, 90. Mu.l of the culture medium was added to each tube, 10. Mu.l of the virus stock solution was added to the first tube, and after mixing, 10. Mu.l of the virus stock solution was aspirated and added to the second tube. And so on, namely 10 -1 ～10 -10 。
Cell infection: the diluted virus dilutions of each group were replaced on cultured 293T cells, each titer was 3 replicates, after 2h of infection, the virus supernatant was aspirated with a sterile pasteur pipette, 4mL agarose overlay was added, and the agarose was allowed to solidify at room temperature for 10-20 min. Each plate was sealed with Parafilm (to prevent drying) and incubated at 37 ℃ for 5 days.
Calculating the virus titer: a trypan blue cover was prepared and poured 1mL into a dish with good plaque formation cultured for 4 days. Incubate the plates overnight at 37 ℃ to allow the dye to diffuse into the dead cells. The number of blue plaques was counted and the virus titer was determined.
Titer was calculated as titer (TU/mL) = (X + Y × 10) × 1000/2/X well of virus content (μ l). Wherein the X-well is the number of plaques for the well in which the number of plaques can be noted, and the Y-well is the number of plaques for the ten times diluted multiple well of the X-well. The results of the titer detection after the control treatment of physiological saline are shown in the following table
| Parallel 1 hole | Parallel 2 holes | Parallel 3 holes | Mean value of | |
| X hole (10) -7 ) | 119 | 105 | 158 | 127 |
| Y hole (10) -8 ) | 25 | 17 | 34 | 25 |
Calculating according to the formula; the viral titer (TU/mL) was 1.9X 10 12 。
The titer detection results after the action of the film-forming agent are shown in the table
| Parallel 1 hole | Parallel 2 holes | In parallel3 holes | Mean value of | |
| X hole (10) -6 ) | 94 | 62 | 70 | 75 |
| Y hole (10) -7 ) | 12 | 9 | 15 | 12 |
Calculating according to the formula; the viral titer (TU/mL) was 9.75X 10 10 。
The virus titer results are shown in figure 6. As can be seen from FIG. 6, after the film spraying agent interacts with the virus, the virus is inhibited by the film spraying agent, and is further killed, and the number of the virus is obviously reduced.
8. And (3) verification of the effect of resisting the new coronavirus:
and (3) packaging the virus:
1) Reagents/plasmids
Reagent: lip2000 transfection kit, opti-MEM Medium
Plasmid: a) pCDH-CMV-3F-HnCoV-S-EF1-copGFP (New coronavirus S protein-containing) plasmid (purchased from Shanghai Ji Haoge Biotech, inc.); b) plasmid pMD2. G; c) plasmid psPAX2
2) And (3) packaging the virus:
a) Cell preparation: when the density of 293T cells is 50%, replacing the non-resistant culture medium for culturing for 24h; opti-MEM medium was replaced before transfection.
b) Preparation of transfection sample: to 50u of optiti-MEM medium, 3 kinds of plasmids were added and mixed gently. Before use, the lip2000 was gently mixed, and then added to the opti-MEM medium, gently mixed, and then allowed to stand at room temperature for 5min. The two dilutions 1:1 were mixed, gently mixed and allowed to stand at room temperature for 20min.
c) And circularly dripping the mixed solution into cells, uniformly mixing, culturing for 6h, and then replacing with a normal culture medium.
d) Collecting culture medium supernatant every 24h, collecting for 3 times, and temporarily storing at 4 deg.C. After collection, the virus was concentrated using an ultrafiltration tube, and the virus titer was measured and stored for a long period at-80 ℃.
3) Dosage of virus package
Inoculating cells: human ACE 2-positive lung epithelial BEAS2B cells with good growth status were selected, counted and counted at 1X 10 5 Density per mL, seeded in 6cm dishes.
Virus dilution: selection of appropriate dilution concentrations based on titer test (selection 10) -7 )。
Spraying and killing of the film spraying agent: and spraying contrast normal saline and the film spraying agent of the invention to the bottom of the culture dish containing the new coronavirus S protein, standing for 60min, and collecting viruses at the bottom of the culture dish respectively.
And (3) verifying the killing effect: the cell state was observed in 3 groups, 1) blank group (without any treatment of BEAS2B cells), 2) virus infection control group (with addition of virus collection liquid after normal saline spray treatment to BEAS 2B), and disinfection virus infection control group (with addition of virus collection liquid after spray treatment to BEAS 2B), respectively at 2h and 5h of infection.
The results are shown in fig. 7, where a in fig. 7 is a blank group, the status of the virus-uninfected BEAS2B cells (100 ×), and it can be seen from a in fig. 7 that the BEAS2B cells are adherent, fully expanded in morphology, and plump in morphology. FIG. 7 shows the cell status of B-infecting virus 2h BEAS2B (100X), and FIG. 7 shows the cell status of C-infecting virus 5h BEAS2B (100X).
In the process of infecting BEAS2B cells by viruses, normal saline cannot inhibit the infection effect of new coronavirus on the cells, and the BEAS2B cells become round in shape and reduced in stereoscopic impression along with the increase of time after the virus infection, are paved on the wall, are easy to fall off, increase in particles and increase in impurities.
FIG. 7D status of cells infected with virus 2h BEAS2B (100X) after killing with spray agent of example 1;
FIG. 7E status of virus infection of 5h BEAS2B cells (100 ×) after killing of spray agent in example 1;
as can be seen from D and E in FIG. 7, after the film spraying agent of the embodiment 1 is killed, the state of BEAS2B cells is good, no matter 2h or 5h, and is basically similar to the state of uninfected cells, so that the film spraying agent of the embodiment 1 can play a role in inhibiting new coronavirus for a long time and prevent the coronavirus from entering lung and mucosal cells.
9. Experimental verification of absorption time of film spraying agent
The male rats were anesthetized, depilated with scissors, the back skin was peeled off, the subcutaneous adipose tissue was separated, and washed with physiological saline. Fixing rat skin at one end of a diffusion cell, enabling the skin cuticle to face a dosing chamber, uniformly spraying 0.5mL of film spraying agent on the skin cuticle, filling the device, enabling the skin to be in contact with a receiving solution (the receiving solution is 150mL of physiological saline, stirring at a constant temperature of 37 ℃, and the speed is 50 r/min), taking the contact solution 1 time every 1 hour, taking 8mL each time, taking 8 times, taking the contact solution and simultaneously supplementing the contact solution with the same volume. And detecting the concentration of the sampled sample, and calculating the concentration of a key component benzalkonium bromide, thereby calculating the accumulated transdermal quantity and the transdermal rate.
Cumulative transdermal quantity and transdermal rate of benzalkonium bromide in different time
| Time (h) | Cumulative transdermal volume (ug/mL) | Cumulative skin penetration (%) |
| 1 | 1.19±0.12 | 0.66±0.16 |
| 2 | 3.81±0.45 | 2.12±0.51 |
| 3 | 6.22±0.67 | 3.46±0.78 |
| 4 | 10.03±1.14 | 5.57±1.12 |
| 5 | 12.29±1.48 | 6.83±1.39 |
| 6 | 15.64±2.63 | 8.69±1.58 |
| 7 | 17.48±2.20 | 9.71±2.04 |
| 8 | 20.07±3.19 | 11.15±2.98 |
10. Verification of air permeability effect of film spraying agent
Taking 6 blocks of 1.5cm 2 The artificial skin of (1) was randomly divided into 2 groups, wherein 1 group was sprayed with the film forming agent and the other group was not sprayed with the film forming agent, and the oxygen transmission rate was measured using an oxygen transmission rate measuring system.
Oxygen passage scale for each group
| Film spraying forming agent group cm 3 /(cm 3 *24h) | Group cm without spraying film-forming agent 3 /(cm 3 *24h) |
| 18522.38±355.22 | 19189.04±225.48 |
The results of the permeability effects of the film spraying agents are shown in fig. 8, and it can be seen from the graph that the oxygen permeability of the film spraying agent group and the film non-spraying agent group has no obvious difference.
Claims (4)
1. A nasal cavity film spraying agent for targeting, slow release, film forming and virus and bacteria inhibition is characterized by being prepared from A, B and a C phase component, wherein the three-phase volume ratio is A: B: C = (4-6): (0.5-1.5): (4-6);
wherein, phase A comprises benzalkonium bromide, borneolum Syntheticum, mentholum, tween-80, 95% ethanol and 0.9% sodium chloride;
phase B is eucalyptus oil;
phase C comprises polyvinylpyrrolidone, polyvinylpyrrolidone iodine, sodium carboxymethylcellulose, angiotensin II inhibitor, disodium edetate and 0.9% sodium chloride;
every 500mL of the phase A contains 0.1-0.3g benzalkonium bromide, 0.5-2g borneol, 0.2-0.6g menthol, 15-24g Tween-80, 10mL of 95% ethanol and the balance of 0.9% sodium chloride;
and each 500mL of the phase C contains 30-70g of polyvinylpyrrolidone, 0.5-1g of polyvinylpyrrolidone iodine, 5-10g of sodium carboxymethylcellulose, 20-40mg of angiotensin II inhibitor, 10-30mg of disodium ethylene diamine tetraacetate and the balance of 0.9% of sodium chloride.
2. The nasal cavity film spraying agent for targeting, slow release and film formation for inhibiting viruses and bacteria according to claim 1, wherein each 500mL of phase A contains 0.2-0.3g of benzalkonium bromide, 1-2g of borneol, 0.3-0.5g of menthol, 18-22g of Tween-80, 10mL of 95% ethanol and the balance of 0.9% sodium chloride.
3. The nasal cavity film spraying agent for targeting, slow release and film forming to inhibit viruses and bacteria as claimed in claim 1, wherein each 500mL of C phase contains 40-60g of polyvinylpyrrolidone, 0.8-1g of polyvinylpyrrolidone iodine, 8-10g of sodium carboxymethylcellulose, 30-40mg of angiotensin II inhibitor, 20-30mg of disodium edetate and the balance of 0.9% sodium chloride.
4. The method for preparing the nasal cavity film spraying agent for targeting, slowly releasing and forming films to inhibit viruses and bacteria, which is characterized by comprising the following steps:
1. preparation of phase A
1) Dissolving Borneolum Syntheticum and Mentholum in 95% ethanol, adding Tween-80, and mixing to obtain solution A;
2) Adding benzalkonium bromide into the solution A for uniform dispersion;
3) Finally, dissolving the mixture in 0.9% sodium chloride solution, uniformly mixing the solution by magnetic stirring, and subpackaging the mixture to obtain a phase A;
2. preparation of phase C
4) Completely dissolving polyvinylpyrrolidone and sodium carboxymethyl cellulose by using 0.9 percent sodium chloride to obtain a solution B;
5) Adding polyvinylpyrrolidone iodine, an angiotensin II inhibitor and disodium ethylene diamine tetraacetate into the solution B, and uniformly mixing by magnetic stirring;
6) Finally, uniformly mixing with 500mL of 0.9% sodium chloride solution, and subpackaging to obtain a phase C;
3. preparation of film spraying agent
7) Adding the phase B into the phase A while stirring, and completely homogenizing the liquid by using a homogenizer after the phase B is completely added to form a water-in-oil suspension;
8) Adding phase C into the water-in-oil suspension, stirring, homogenizing, microemulsifying, and degassing to obtain water-in-oil-in-water uniform nasal spray;
wherein the volume ratio of the phase A to the phase B to the phase C is (4-6): (0.5-1.5): (4-6); phase B is eucalyptus oil.
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| US20100272769A1 (en) * | 2005-08-03 | 2010-10-28 | Amcol International | Virus-, Bacteria-, and Fungi-Interacting Layered Phyllosilicates and Methods of Use |
| EP1837020A1 (en) * | 2006-03-24 | 2007-09-26 | Bioalliance Pharma | Mucosal bioadhesive slow release carrier for delivering active principles |
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| CN104619345A (en) * | 2012-05-18 | 2015-05-13 | 罗达制药有限公司 | Liquid formulation |
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| CN106361816A (en) * | 2016-08-27 | 2017-02-01 | 广东罗浮山国药股份有限公司 | Wound disinfecting spraying film agent and preparation method thereof |
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